Engineering Tools for Environmental

Risk Management – 2
Engineering Tools for Environmental
Risk Management – 2

Environmental Toxicology

Editors

Katalin Gruiz
Department of Applied Biotechnology and Food Science, Budapest University
of Technology and Economics, Budapest, Hungary

Tamás Meggyes
Berlin, Germany

Éva Fenyvesi
Cyclolab, Budapest, Hungary
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British Library Cataloging in Publication Data
A catalogue record for this book is available from the British Library
Library of Congress Cataloging-in-Publication Data
Environmental toxicology (CRC Press)
Environmental toxicology / editors Katalin Gruiz, Department of Applied
Biotechnology and Food Science, Budapest University of Technology and
Economics, Budapest, Hungary,Tamás Meggyes, Berlin, Germany, Éva
Fenyvesi, Cyclolab, Budapest, Hungary.
pages cm. – (Engineering tools for environmental risk management ; 2)
Includes bibliographical references and index.
ISBN 978-1-138-00155-8 (hardback : alk. paper) – ISBN 978-1-315-77877-8 (ebook)
1. Environmental toxicology. 2. Pollutants–Toxicity testing. I. Gruiz, Katalin.
II. Meggyes,T. (Tamás) III. Fenyvesi, Éva. IV. Title.
RA1226.E56855 2015
615.9’02–dc23
2014048002
Published by: CRC Press/Balkema
P.O. Box 11320, 2301 EH Leiden,The Netherlands
e-mail: [email protected]
www.crcpress.com – www.taylorandfrancis.com
ISBN: 978-1-138-00155-8 (Hardback)
ISBN: 978-1-315-77877-8 (eBook PDF)
Table of contents

Preface xvii
List of abbreviations xix
About the editors xxix

1 Environmental toxicology – A general overview 1

K. GRUIZ
1 Introduction, basic definitions 1
1.1 Toxicology and its role 3
1.2 Regulatory toxicology for chemical substances and
contaminated land 9
1.3 Future of environmental toxicology 12
1.3.1 Molecular technologies 13
1.3.2 Cell-based technologies 15
1.3.3 Computational toxicology 15
1.4 What environment means in the context of toxicology 16
1.5 Environmental toxicology versus human toxicology 17
1.6 Animal studies 18
1.7 In vitro contra in vivo: alternative test methods 20
1.8 Evidence-based toxicology 23
2 Adverse effects to be measured by environmental toxicology 23
2.1 Hazardous effects of chemical substances 24
2.2 Toxic effects of chemical substances 25
2.3 Carcinogenic effects 31
2.4 Mutagenic effects 32
2.5 Reprotoxicity 32
2.6 Persistent and very persistent substances 33
2.7 Bioaccumulative and very bioaccumulative substances 33
2.8 Emerging pollutants 34
3 Interaction of a chemical substance with living organisms 36
3.1 Dose–response relationship 39
3.2 Test end points: the results of the environmental toxicity test 42
3.3 Classification of environmental toxicological tests 44
3.3.1 Test type according to the aim of the test 44
3.3.2 Test organisms 45
vi Table of contents

3.3.3 Test design 48

3.3.4 Most commonly measured end points 49
3.3.5 Environmental compartments and phases to test 50
3.3.6 Aims of environmental toxicity tests 51
3.4 Environmental toxicology in relation to hazard and
risk assessment 51
3.4.1 Testing hazard or risk? 51
3.4.2 Standardized or customized test methods? 53
3.4.3 Testing or modeling? – QSAR and
environmental toxicology 55
3.5 Statistical evaluation of ecotoxicological tests 60
3.5.1 Evaluation of acute toxicity tests 60
3.5.2 Data analysis for chronic toxicity tests 62
3.5.3 Data analysis of multispecies toxicity tests 62
3.6 Standardization and international acceptance of newly
developed toxicity tests 63

2 Fate and behavior of chemical substances in

the environment 71
K. GRUIZ, M. MOLNÁR, ZS. M. NAGY & CS. HAJDU
1 Introduction 71
2 Interaction of the contaminants with environmental phases 74
2.1 Transport and partitioning 75
2.1.1 Partitioning between air and water 75
2.1.2 Partitioning between solid and water 76
2.1.3 Transport models 77
2.2 Chemical interactions between chemical substances and
the environment 81
2.2.1 Photolysis 81
2.2.2 Hydrolysis 82
2.2.3 Chemical oxidation and reduction 83
3 Interactions of chemical substances – with the biota 84
3.1 Biodegradation and biotransformation 84
3.1.1 Classification of environmental fate of chemicals for
regulatory purposes 84
3.1.2 Biodegradation – definitions 86
3.1.3 Biodegradation – the process 87
3.1.4 QSAR for biodegradation 88
3.1.5 Aims of testing biodegradation 90
3.1.6 Measurement end points for characterizing
biodegradation 91
3.1.7 Standardized biodegradability test methods for
chemical substances 93
3.1.8 Measuring biodegradation in soil 94
3.1.9 Soil respiration, biodegradative activity of the
soil – problem-specific applications 95
 Table of contents vii

3.2 Bioaccumulation 102

3.2.1 Definitions 102
3.2.2 Bioaccumulative potential of chemicals 104
3.2.3 QSAR for bioaccumulation 106
3.2.4 Testing bioaccumulation 107
3.2.5 Standardized tests for measuring bioaccumulation 109
3.2.6 Field determination of bioaccumulation 112
3.3 Bioleaching 113
4 Availability of contaminants for environmental actors 114
5 Utilizing fate properties of chemicals to reduce their risk
in the environment 117
5.1 Environmental transport and fate processes change
contaminant risk 117

3 Human toxicology 125

K. GRUIZ
1 Introduction 125
1.1 Adverse effects of chemicals on humans 127
1.2 Testing the adverse effects of chemicals on humans 130
2 Test organisms for human toxicology purposes 132
2.1 Microorganisms used in human toxicity testing 132
2.2 Isolated cells, tissue cultures in human toxicology 132
2.3 Lower animals in human toxicology 133
2.4 Birds 133
2.5 Mammals 134
2.6 3R in animal testing 135
3 Toxicity end points and methods 136
3.1 Acute toxicity 136
3.1.1 Animal tests for acute systemic toxicity 137
3.1.2 Non-animal, in vitro tests for acute systemic toxicity 137
3.2 Repeated-dose and organ toxicity testing 138
3.2.1 Animal test methods for repeated-dose and
organ toxicity 139
3.2.2 Alternative methods for repeated-dose and organ
toxicity testing 140
3.3 Genotoxicity 141
3.3.1 In vivo animal tests for assessing potential heritable
genotoxicity 141
3.3.2 OECD test guidelines for in vitro genotoxicity and
mutagenicity testing 142
3.3.3 New in vivo genotoxicity tests 142
3.3.4 QSAR for genotoxicity and genotoxic carcinogenicity 145
3.4 Chronic toxicity 145
3.4.1 Chronic toxicity testing methods on animals 146
3.5 Carcinogenicity 146
3.5.1 Animal methods for carcinogenicity testing 147
3.5.2 Non-animal testing of carcinogenicity 147
viii Table of contents

3.6 Reproductive and developmental toxicity 148

3.6.1 Animal tests for reproductive and developmental
toxicity 149
3.6.2 In vitro methods for reproductive and developmental
toxicity 149
3.7 Dermal penetration 151
3.7.1 Animal testing of dermal penetration 151
3.7.2 In vitro testing of dermal penetration 151
3.8 Skin irritation and corrosion 152
3.8.1 Animal testing of skin irritation and corrosion 152
3.8.2 Alternative, non-animal test methods for skin irritation
and corrosion 152
3.9 Skin sensitization 154
3.9.1 Skin sensitization: animal tests for regulatory
requirements 154
3.9.2 Non-animal alternative methods 154
3.10 Eye irritation and corrosion 155
3.10.1 Animal testing of eye irritation and corrosion
on rabbits 155
3.10.2 Non-animal alternative methods for evaluating eye
irritation and corrosion 156
3.11 Toxicokinetics, pharmacokinetics and metabolism 156
3.11.1 Testing of toxicokinetics, pharmacokinetics and
metabolism on animals 158
3.11.2 In vitro dermal testing 159
3.12 Neurotoxicity 159
3.12.1 Animal testing of neurotoxicity 160
3.12.2 In vitro models for neurotoxicology studies and testing 160
3.13 Endocrine toxicity and disruption 161
3.13.1 Animal tests for screening endocrine disruption 161
3.13.2 Validated non-animal alternatives for endocrine
disruptor activity 161
3.13.3 The US EPA endocrine disruptor screening program 162
3.14 Phototoxicity 164

4 Aquatic toxicology 171

K. GRUIZ & M. MOLNÁR
1 Introduction to aquatic toxicology 171
2 Human and ecosystem exposure to aquatic hazards 174
3 Some commonly used aquatic test organisms for testing
adverse effects 180
3.1 Microorganisms: bacteria, algae and protozoa 180
3.2 Fresh-water macroplants 184
3.3 Fresh-water invertebrates 185
3.4 Aquatic vertebrates 190
3.5 Sediment-dwelling organisms 192
 Table of contents ix

4 Measuring adverse effects of chemical substances on the

aquatic ecosystem 194
5 Some commonly used aquatic test methods 196
5.1 OECD guidelines for testing chemicals in aquatic environment:
water, sediment, wastewater 196
5.2 Water-testing methods standardized by the International
Organization for Standardization 198
5.2.1 Standardized bacterial tests for toxicity testing of water
and waste-water 199
5.2.2 Standardized algal and plant tests for waters 199
5.2.3 Invertebrates using standard methods for testing water 201
5.2.4 Standardized fish tests for water and waste-water 201
5.2.5 Ecological assessment of surface waters 201
6 Non-animal testing of aquatic toxicity 203
7 Testing sediment 203
8 Sewage and sewage sludge tests 208
9 Testing waste using an ‘Ecotox’ test battery 209
10 Non-standardized bioassays and other innovative test methods 212
11 Multispecies and microcosm test methods for aquatic toxicity 217
12 Description of Tetrahymena pyriformis bioassay 220
12.1 Experimental 221
12.2 Evaluation and interpretation of the results 222

5 Terrestrial toxicology 229

K. GRUIZ, M. MOLNÁR, V. FEIGL, CS. HAJDU, ZS. M. NAGY, O. KLEBERCZ,
I. FEKETE-KERTÉSZ, É. UJACZKI & M. TOLNER
1 Introduction 229
2 Terrestrial test organisms 237
2.1 Soil-living bacteria and fungi as test organisms 238
2.2 Terrestrial plants for soil toxicity testing 242
2.3 Soil fauna members as test organisms 246
3 Measuring terrestrial toxicity: end points and methods 254
3.1 Soil biodiversity 255
3.2 Evolutionary convergence phenomenon 258
3.3 Terrestrial bioassays for testing chemical substances and
contaminated soil 259
4 Standardized and non-standardized test methods 260
4.1 OECD standards for testing chemical substances in soil and
dung with terrestrial organisms 260
4.2 ISO and other standards for testing soil and sediment 260
4.3 Testing waste: a terrestrial test battery for solid waste 263
5 Non-standard terrestrial toxicity test methods 263
5.1 Some aspects of problem-oriented and site-specific soil testing 264
5.1.1 Soil community response 265
5.1.2 Concepts for characterizing soil functioning and health 266
5.1.3 Aims of testing whole soil response 266
x Table of contents

5.1.4 Consequences of the effect of soil matrix on the test

methodology 267
5.1.5 Field assessment or laboratory testing? 271
5.2 Ecological assessment: field testing of habitat quality, diversity
of species and abundance of indicator organisms 272
5.2.1 Abundance and diversity of soil microbiota 272
5.2.2 The use of carbon substrate utilization patterns for
ecotoxicity testing 273
5.2.3 Dung-dwelling organisms, a not yet standardized
field study 273
5.2.4 Effects of pollutants on earthworms in field situations:
avoidance 274
5.3 Non-standardized contact bioassays: description of some tests 275
5.3.1 Single species bacterial contact tests 275
5.3.2 Single species animal contact tests 278
5.3.3 Plant tests 279
5.3.4 Soil as a test organism 280
6 Multispecies terrestrial tests 282
6.1 Classification of multispecies soil tests 282
6.1.1 Terrestrial microcosm system for measuring
respiration 283
6.1.2 Terrestrial microcosm for substrate-induced respiration
technique (SIR) 283
6.1.3 Terrestrial model ecosystems (TME) 284
6.1.4 The cotton strip assay 285
6.1.5 Soil litter bag 285
6.1.6 Pitfall traps 286
6.1.7 Bait lamina 286
6.1.8 Soil in jar 287
6.1.9 Soil lysimeters 288
6.2 Characteristics of multispecies toxicity tests 290
6.3 Evaluation and monitoring of microcosms 290
7 Microcalorimetry – a sensitive method for soil toxicity testing 291
7.1 Background of microcalorimetric heat production by living
organisms 291
7.2 Experimental setup 292
7.3 Heat response of Folsomia candida to the effect of
diesel oil 293
7.4 Heat response of Panagrellus redivivus on contaminated soil 294
7.5 Heat response of Sinapis alba to the effect of toxicants
in soil 296
7.6 Heat production response of Azomonas agilis to toxicants 296
7.7 Evaluation and interpretation of the microcalorimetric heat
production results 299
7.8 Summary of microcalorimetric toxicity testing: experiences and
outlook 299
7.9 Acknowledgement to microcalorimetry research 301
 Table of contents xi

6 Advanced methods for chemical characterization of

soil pollutants 311
GY. ZÁRAY & I. VARGA
1 Introduction 311
2 Analytical methods for the determination of inorganic compounds 313
2.1 ICP-based analytical methods 313
2.1.1 Sample preparation 313
2.1.2 Inductively coupled plasma as photon and ion source 313
2.1.3 Analytical figures of merit 316
2.2 X-ray fluorescence spectrometry 318
2.2.1 Sample preparation 318
2.2.2 Basic equipment and set-up for XRF analysis 319
2.2.3 X-ray sources 319
2.2.4 Detectors 320
2.2.5 Quantification 322
2.2.6 Analytical figures of merit 322
2.2.7 Comparison of XRF and ICP-based analytical
techniques 323
3 Analytical methods for analysis of organic pollutants 323
3.1 Sample pretreatment 324
3.2 Extraction of analytes from soil samples 324
3.2.1 Supercritical fluid extraction (SFE) 324
3.2.2 Microwave assisted extraction (MAE) 325
3.2.3 Pressurized liquid extraction (PLE) 326
3.2.4 Ultrasonic assisted extraction (UAE) 327
3.3 Cleanup process 328
3.4 Preconcentration/enrichment of analytes 328
3.5 Separation and detection techniques 328
3.6 Applications 329
3.6.1 Pesticide analysis 329
3.6.2 Veterinary pharmaceuticals 330
3.6.3 Petroleum hydrocarbons 330
3.7 Recent developments and future trends 331

7 Bioaccessibility and bioavailability in risk assessment 337

CS. HAJDU & K. GRUIZ
1 Introduction 337
2 Managing bioaccessibility and bioavailability of contaminants in the
environment 341
2.1 Mobility, bioaccessibility, bioavailability and risk
assessment 343
2.2 Risk reduction in view of mobility and bioavailability 344
3 Bioavailability and bioaccessibility – definitions 345
3.1 Definitions and mechanisms 346
3.2 Contaminants’ location and form in soil and the related
accessibility and availability 348
xii Table of contents

4 Assessing bioavailability of contaminants 351

4.1 Bioaccessibility and bioavailability assessment methods 352
5 Mathematical models for contaminant bioavailability in soil 354
6 Chemical models for contaminant mobility and availability in soil 355
6.1 Partition between n-octanol and water to predict accessibility
of organic contaminants 355
6.2 Solid phase and membrane-based extractions – chemical
bioavailability models 356
6.3 Liquid-phase extractions to predict accessibility of toxic metals 358
7 Complex models 362
7.1 Interactive laboratory tests 362
7.2 Dynamic testing 363
7.3 Integrated evaluation 366
8 Examples of interactive testing of bioavailability in soil 367
8.1 Toxic metal bioavailability in mine tailings – the chemical
time bomb 368
8.2 Decreased bioavailability, lower toxicity – a soil
remediation tool 369
8.3 Correlation of chemical analytical and bioassay results 372
8.4 Bioavailability and biodegradation of organic soil contaminants 374
9 Worst-case and realistic worst-case simulation 378
9.1 Realistic worst-case models for dynamic testing of
bioavailability 378
9.2 Effect of soil sorption capacity on bioavailability 381
10 Bioaccessibility and bioavailability of contaminants for humans 381
10.1 Mathematical models for calculation of bioaccessibility- and
bioavailability-dependent human risk 383
10.2 Chemical models for estimating accessibility of contaminants
for humans 384
10.2.1 Human bioaccessibility of toxic metals 385
10.2.2 Bioaccessibility of organic compounds in humans 389
10.2.3 Chemical models combined with biological models –
measuring toxic effects after digestion 390
11 Conclusions 391

8 Microcosm models and technological experiments 401

K. GRUIZ, M. MOLNÁR, V. FEIGL, E. VASZITA & O. KLEBERCZ
1 Introduction 401
2 Aquatic microcosms for screening chemical substances
and technologies 402
3 Soil micro- and mesocosms for modeling environmental processes
in bio- and ecotechnologies 407
3.1 Testing the effects of environmental and anthropogenic
interventions in a small volume 412
3.2 Testing biodegradation and bioavailability 412
3.3 Testing long-term pollution processes in the environment 413
 Table of contents xiii

3.4 Testing microbial activity and plant growth in

contaminated soil 413
3.5 Technological pre-experiments 414
4 Biodegradation and biodegradation-based remediation studies
in soil microcosms 416
4.1 Testing natural and enhanced biodegradation 416
4.2 Integrated monitoring and evaluation of the biodegradation
experiments 418
4.3 Scaled-up technological micro- and mesocosms 421
4.4 Summary of biodegradation testing for technological purposes 422
5 Testing technologies based on contaminant stabilization 422
5.1 Experiment design 423
5.2 Microcosm set-up and implementation 424
5.3 Monitoring of the microcosms 425
5.4 Evaluation, interpretation and use of the stabilization
microcosm results 427
5.5 Summary and conclusions of stabilization microcosm
application 427
6 Testing and utilizing the complex leaching process 428
6.1 Flow-through soil microcosm for studying bioleaching 430
6.2 Microcosm set-up 431
6.3 Monitoring the leaching microcosms 432
6.4 Evaluation and interpretation of the results 432
6.5 Summary and conclusions about leaching microcosm
application 433
7 Transport processes studied in soil columns 434
7.1 Test set-up 435
7.2 Monitoring the soil column microcosm 436
7.3 Evaluation 437
7.4 Summary 437
8 Modeling secondary sodification 438
8.1 Modeling sodification in microcosms 438
8.2 Sodification microcosm set-up 439
8.3 Technological microcosms for reducing risk of sodification 440
8.4 Evaluation and interpretation of results 440
8.5 Summary of sodification modeling 440

9 Data evaluation and interpretation in environmental

toxicology 445
K. GRUIZ, CS. HAJDU & T. MEGGYES
1 Introduction 446
2 Inhibition rate 451
3 Concentration/dose–response relationship 453
4 Evaluation of the response based on the growth curves of
cultured organisms 456
xiv Table of contents

5 Evaluation of the effect of contaminants on heat production:

A special case 458
6 Evaluation of biodegradation of chemicals in water and soil 460
6.1 Monitoring the depletion of the chemical substance 461
6.2 Evaluation of biodegradation based on CO2 production 461
6.3 Substrate induction 462
7 Attenuation rate method for environmental samples 464
8 Toxic equivalency of contaminated environmental samples for
exploration and screening 467
8.1 Toxic equivalency for organic and inorganic contaminants 469
8.2 Graphical determination of equivalent toxic concentrations
from measured data 470
8.3 Numerical determination of the toxicity equivalent
concentration 472
8.4 Equivalent toxicity of contaminated water: examples
and validation 473
8.4.1 4CP equivalent of selected organic contaminants
in water: examples 475
8.4.2 Copper equivalent of cadmium-contaminated water 477
8.5 Toxicity equivalent of soil: examples and validation 477
8.5.1 4CP equivalent of selected organic contaminants in soil:
examples 478
8.5.2 Copper equivalent of soils contaminated with cadmium
and a mixture of metals 480
9 Statistical evaluation of toxicity data 484
9.1 Statistics in general 484
9.2 Statistical evaluation and analysis in environmental toxicology 490
9.3 Hypothesis testing 492
9.3.1 Hypothesis testing for the determination of NOEC 493
9.3.2 Reporting hypothesis testing 497
9.4 Regression and regression analysis 497
9.4.1 The use of regression and regression analysis in
toxicology 498
9.4.2 Evaluation of quantal data 500
9.4.3 Choice of the models 500
9.4.4 Evaluation of continuous data 501
9.4.5 Choice of the models 501
9.4.6 Reporting regression statistics 503
9.5 A comparative study on statistical evaluation of dose–response
data 504
9.6 Biology-based methods 505
9.6.1 Parameters 507
9.7 IT tools for statistical evaluation 508
10 Environmental hazard and risk assessment using toxicity data 513
10.1 Extrapolation 513
10.2 Hazard assessment 515
 Table of contents xv

10.2.1 Hazard identification 515

10.2.2 Hazard quantification 516
10.3 Validation of toxicity tests 522
10.4 Exposure assessment 524
10.5 Risk assessment 525
10.6 Summary comments on risk assessment and risk management
based on toxicity data 531
11 Conclusions 533

Subject index 545

Preface

This is the second volume of the five-volume book series “Engineering Tools for
Environmental Risk Management’’. The book series deals with the following topics:

1 Environmental deterioration and pollution, management of environmental

problems
2 Environmental toxicology – a tool for managing chemical substances and contam-
inated environment
3 Assessment and monitoring tools, risk assessment
4 Risk reduction measures and technologies
5 Case studies for demonstration of the application of engineering tools

The authors aim to describe interactions and options in risk management by

• providing a broad scientific overview of the environment, its human uses and the
associated local, regional and global environmental problems;
• interpreting the holistic approach used in solving environmental protection issues;
• striking a balance between nature’s needs and engineering capabilities;
• understanding interactions between regulation, management and engineering;
• obtaining information about novel technologies and innovative scientific and
engineering tools;
• providing a broader perspective for engineers and explaining engineering solutions
to environmental managers, owners and other decision makers

This second volume provides an overview on environmental toxicology, the main

concepts, methods and applications, focusing on environmental knowledge and its
conscious and structured application in environmental engineering, management and
decision making.
The main topics of this second volume include

• the legal and managerial context of environmental toxicology;

• the chemical and biological as well as in silico models for toxicology;
• fate and behavior of chemical substances in the environment and their influence
on the actual effects;
• human, aquatic and terrestrial toxicology – test organisms and test methods;
• ecotoxicology and ecological assessment;
xviii Preface

• alternative test methods to reduce the use of animals in research and testing;
• new trends in environmental analytics;
• bioaccessibility and bioavailability of chemical substances;
• interactive and dynamic testing of environmental solid phases;
• developments of soil ecotoxicology;
• microcosm models and experiments;
• evaluation and interpretation of environmental fate and effect data;
• statistics of environmental toxicology.
Abbreviations

2-D GC – two-dimensional gas chromatography

3R – replacement, reduction and refinement in animal testing
4CP – 4-chlorophenol, a chlorinated pesticide/biocide
AA-EQS – annual average environmental quality criterion
ADE – absorption, distribution and elimination
ADI – acceptable daily intake
ADME – absorption, distribution, metabolism, and elimination
AF – assessment factor
AMD – acid mine drainage
ANCOVA – analysis of covariance
ANOVA – analysis of variance
APHA – American Public Health Association
ARD – acid rock drainage
ATO – aquatic test organism
ATP – adenosine triphosphate
AS – allometric scaling factor
ASTM – American Society for Testing and Materials
ASTM – International the new name of ASTM
AVS – acid-volatile sulfide
AWWA – American Water Works Association
BAF – bioaccumulation factor
BAFK – kinetic bioaccumulation factor
BALB/c 3T3 – albino mouse fibroblast cell line for cytotoxicity testing;
the standard fibroblast cell line
BAT – best available technology
BCC – bioaccumulative chemicals of concern
BCF – bioconcentration factor
BCOP – bovine corneal opacity and permeability test
BCR – Commission of the European Communities Bureau of Reference
BCR – extraction sequential extraction method for soil/sediment
Bhas42 – immortalized rodent cells line
BIOWIN – biodegradation probability program for estimating aerobic and
anaerobic biodegradability of organic chemicals
BLOBs – binary large objects
BMD – benchmark dose
xx Abbreviations

BMD10 – benchmark dose, associated with a 10% response in

animal tests
BMDL – lower limit of BMD
BMDL10 – lower limit of the benchmark dose expected to produce
a 10% reduction in the response
BMF – biomagnification factor
BMR – benchmark response
BOD – biological oxygen demand
BPA – bisphenol-A
BSAF – biota-sediment accumulation factor
BTEX – benzene, toluene, xylenes, alkyl benzenes
CA – correspondence analysis
Caco-2 – human epithelial colorectal adenocarcinoma cells
CAS – Chemical Abstracts Service
Cb – background concentration
CB – closed-bottle test
CBR – critical body residue (theory)
cDNA – DNA sequences synthetized in vitro from messenger RNA
CDT – cyclodextrin technology (cyclodextrin-enhanced
bioremediation of contaminated soil)
CE – capillary electrophoresis
CEC – cation exchange capacity
CECs – contaminants of emerging concern
CEN – European Committee for Standardization
CFU – colony-forming units
C3H – a mouse strain used as an animal model for research
cl – confidence limits
CLH – European counterpart of Globally Harmonized System of
Classification and Labeling of Chemicals (GHS)
CLP – European regulation on classification, labeling and
packaging of substances and mixtures
CMR – chemical substances with carcinogenic, mutagenic and
reprotoxic effects
CR – cancer risk
CSA – chemical safety assessment
CSIRO – Commonwealth Scientific and Industrial Research
Organization, Australia
CTA – cell transformation assay
Cx – the concentration causing x% reduction in the response
CZE – capillary zone electrophoresis
DBALM – DataBase service on ALternative Methods to animal
experimentation
DBNPA – 2,2-dibromo-3-nitril-propionamide, a biocide
DBP – dibutyl phthalate, an industrial chemical
D – dose
DBNPA – 2,2-dibromo-3-nitrilopropionamide, a quick-kill biocide
DDE – dichlorodiphenyldichloroethane, a toxic breakdown
product of DDT
 Abbreviations xxi

DDT – dichlorodiphenyltrichloroethane, a persistent CMR pesticide

DEB – dynamic energy budget
DEH – dehydrogenase enzyme activity
DGT – diffusive gradients in thin films
DNA – deoxyribonucleic acid
DNA-SIP – stable isotope probing, a PCR method utilizing 13 C isotopes
DNAPL – dense non-aqueous phase liquides
DMEL – derived minimal effect level
DNEL – derived noeffect level
DOC – dissolved organic carbon
DOM – dissolved organic matter
DSD – Dangerous Substance Directive in Europe
DT50 – half-life of a chemical substance
DTA – direct toxicity testing
EAMD – Ecological Assessment Methods Database, USA
EBTC – Evidence-Based Toxicology Collaboration
EC – effective concentration
EC – European Commission
ECHA – European Chemicals Agency
ECVAM – European Centre for the Validation of Alternative Methods
ECx – effective concentrations that cause x% decrease in the response
EC20 – effective concentrations that cause 20% decrease in the response
EC50 – effective concentrations that cause 50% decrease in the response
EDx – effective dose that causes x% decrease in the response
ED20 – effective dose that causes 20% decrease in the response
ED50 – effective dose that causes 50% decrease in the response
ED – effective dose
EDCs – endocrine disrupting chemicals
EDSP – endocrine disruptor screening program
EDTA – ethylenediaminetetraacetic acid, a chelating agent
ED-XRF – energy dispersive X-ray fluorescence
EFDB – environmental fate database
EFSA – European Food Safety Authority
EPH – extractable petroleum hydrocarbons
EPH GC – extractable petroleum hydrocarbon content measured by GC-FID
EQC – environmental quality criteria
EQS – Environmental Quality Standards
ERA – environmental risk assessment
ERAPharm – environmental risk assessment of pharmaceuticals
Er C20 – an effective concentration causing 20% reduction in the growth rate,
calculated from the slope of the growth curve
Er C50 – an effective concentration causing 50% reduction in the growth rate,
calculated from the slope of the growth curve
ERDC – Engineer Research and Development Center, US Army
EROD – ethoxyresorufin-O-deethylase, a biomarker for chemical exposure
ESAC – ECVAM’s Scientific Advisory Committee
ESIS – European Chemical Substances Information System
EsD – effective sample dose
EsD20 – effective sample dose causing 20% decrease in the measured response
xxii Abbreviations

EsD50 – effective sample dose causing 50% decrease in the measured

response
EsMx – effective sample mass, causing x% decrease in the
measured response
EsVx – effective sample volume causing x% decrease in the
measured response
ETV – electrothermal vaporization
EU – European Union
EURL ECVAM – EU Reference Laboratory for Alternatives to Animal Testing
FA – fly ash
FAME – factorial extrapolation method
FDA – Food and Drug Administration (US)
FETAX – frog embryo teratogenesis assay on Xenopus
FGETS – food and gill exchange of toxic substances
FID – flame ionization detection
FIFRA – Federal Insecticide, Fungicide, and Rodenticide Act (US)
FQAI – floristic quality assessment index
FRAME – Fund for the Replacement of Animals in Medical Experiments
in the UK
GC – gas chromatography
GC-FID – gas chromatography with flame ionization detector
GC-MS – gas chromatography with mass spectrometry detector
GEE – generalized estimating equations
GFE – gastric fluid extraction methods
GFP – green fluorescent protein
GHS – Globally Harmonized System of Classification and Labeling
of Chemicals, UN
GIS – geographic information system
GJIC – gap junction intercellular communication
GLM – generalized linear models for logistic regression
GPL – general public license
GUI – graphical user interface
H – dimensionless Henry’s law constant: the ratio of the
concentration of a chemical substance in water to the
concentration in the equilibrium gas phase (caq /cgas )
Henry – Henry’s law constant with the dimension of L × Pa/mol: the
ratio of the partial pressure of gas above the solution (in Pa) to
the concentration (molarity) of gas in solution (in mol/L)
H% – inhibition rate of the luminescent light emission
H – null hypothesis in hypothesis testing
HA – alternative hypothesis in hypothesis testing
HAC – hierarchical agglomerative clustering
HDPE – high density polyethylene
HEH GC – hydrocarbons extracted by aqueous hydroxypropyl
beta-cyclodextrin solution and measured by GC-FID
HEP – habitat evaluation procedure
HepG2 – liver cell line (hepatoma) for testing cell growth and cytotoxicity
HET-CAM – hen’s egg test – chorioallantoic membrane assay
HHPN – hydraulic high pressure nebulizers
 Abbreviations xxiii

HL-60 – human acute promyelocytic leukemia cell line

HPLC-MS – high-performance liquid chromatograph–mass spectrometer
HQ – human hazard quotient
HSDB – hazardous substances database
HTS – high throughput screening
HxCDD – 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin, persistent organic toxicant
I% – inhibition rate: difference in response to the effect of a toxicant
compared to the control, given in the % of the control
IBI – index of biological integrity
ICCVAM – Interagency Coordinating Committee on the Validation of
Alternative Methods
ICE – isolated chicken eye assay
ICP-AES – inductively coupled plasma atomic emission spectroscopy
ICP-OES – inductively coupled plasma optical emission spectrometry
ICP-MS – inductively coupled plasma with mass spectrometry
IHCP – Institute for Health and Consumer Protection, JRC, EC
INT – 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride,
an indicator dye for microbial respiration bioassays
IOBC – International Organization for Biological Control
IPCS – International Programme on Chemical Safety
IPPC – Integrated Pollution Prevention and Control, EU Directive
IR – infrared light
IRE – isolated rabbit eye assay
IRIS – Integrated Risk Information System US EPA
ISCO – in-situ chemical oxidation
ISO – International Organization for Standardization
IT – information technology
IVG – in vitro gastrointestinal digestion model
JaCVAM – Japanese Center for the Validation of Alternative Methods
JRC – Joint Research Centre, EC
Kd – equilibrium partitioning of charged molecules / contaminants
between water and solid phases
Kliquid-gas – equilibrium partitioning of the chemical substance between
liquid and gas
Koc – equilibrium partitioning of the contaminant between the soil’s organic
and water content
Kow – octanol-water partition coefficient, the ratio of the concentration
of a chemical in octanol to the concentration in water (co /cw )
at equilibrium
Kp – equilibrium partitioning of neutral contaminants between solid and
water phases
LAD – least sum of absolute deviations
LC20 – lethal concentrations that cause 20% mortality of the test organisms
LC50 – lethal concentrations that cause 50% mortality of the test organisms
LD20 – lethal doses that cause 20% mortality of test organisms
LD50 – lethal doses that cause 50% mortality of test organisms
LDPE – low-density polyethylene
LE – solution of ammonium lactate and acetic acid applied first by
Lakanen & Erviö for soil extraction to imitate plants’ uptake
xxiv Abbreviations

LLC-PK1 – kidney proximal tubule cell line for testing cell damage
LNAPL – light non-aqueous phase liquids
LOEAsD – lowest effective sample dose calculated from the area under
the growth curve
LOEC – lowest observed effect concentration
LOEL – lowest observed effect level
LOErsD – growth ratebased lowest effect sample dose calculated from
the slope of the growth curve
LOEsD – lowest tested sample dose showing an effect
LOEsV – lowest tested sample volume or mass showing an effect
MAC – maximum allowable concentration
MAE – microwave assisted extraction
MANCOVA – multivariate analysis of covariance
MANOVA – multivariate analysis of variance
MATC – maximum allowable toxicant concentration
MAWI – multi-scale assessment of watershed integrity, ERDC,
US Army
MCA – multiple correspondence analysis
MDCK – (Madin-Darby) canine kidney epithelial cells
MDGC – multidimensional gas chromatography
METI – Japanese Ministry of Economy, Trade and Industry, before
2001: MITI
MFC – mixed-flask culture mesocosm
MI – maturity index
MINISSA – Michigan-lsrael-Nijmegen lntegrated Smallest Space Analysis
MITI – Japanese Ministry of International Trade and Industry,
from 2001: METI
ML – maximum likelihood
MML – marginal maximum likelihood
MNA – mean number of class attributes
MNT – mammalian cell micronucleus test
MoA – mode of action
MoE – margin of exposure
MPA – maximum permissible addition
MPC – maximum permissible concentration
MPN – most probable number, a statistical method
mRNA – messenger RNA
MS – mass spectrometry
MTD – maximum tolerated dose
MW – microwave
NADPH – the reduced form of nicotinamide adenine dinucleotide
phosphate
NCBI – National Center for Biotechnology Information, US
NEC – no-effect concentration the concentration of a substance
that will not adversely affect the species or the community
exposed to it
NHK – normal human keratinocyte cells
NICEATM NTP – Interagency Center for the Evaluation of Alternative
Toxicological Methods, National Toxicology Program,
National Institutes of Health, US
 Abbreviations xxv

NIH – National institute of Health, US

NIST – National Institute for Standardization and Technology US
NIST/SEMATECH – Working Group Elaborating an E-Handbook for Engineering
Statistics
NGO – non-governmental organization
NOAEC – no observed adverse effect concentration
NOAEL – no observed adverse effect level
NOEC – no-observed effect concentration
NOEL – no observed effect level
NOEAsD – no observed effect sample dose, based on the area under the
growth curve
NOEsV – highest tested sample volume with no observed effect on
the response
NOEsD – highest tested sample dose with no observed effect on
the response
NPDS – National Pollutant Discharge Elimination System, US
NRU – neutral red uptake test
NSAID – non-steroidal anti-inflammatory drug
NTP – National Toxicology Program, US
OECD – Organization for Economic Cooperation and Development
PAHs – polycyclic aromatic hydrocarbons, frequent environmental
CMR pollutants
PBASE – sequential extraction of metals modeling the potential
bioavailability
PBET – physiologically based extraction test for predicting metals
bioavailability
PBPKs – physiologically based pharmacokinetic models
PBTs – persistent, bioaccumulative and toxic chemicals
PCA – principal components analysis
PCBs – polychlorinated biphenyls
PCP – pentachlorophenol
PCR – polymerase chain reaction, a technique to amplify a piece
of DNA
PEC – predicted environmental concentration
PICT – pollution-induced community tolerance
PID – photoionization detector
PLE – pressurized liquid extraction
PLFA – phospholipid fatty acid analysis
PNEC – predicted no-effect concentration
POM – polyoxymethylene membrane
POPs – persistent organic pollutants
PPCPs – pharmaceuticals and personal care products
PPY – proteose peptone yeast extract
PSM – primary biodegradation model
psRNAs – putative sRNAs
PVC – polyvinylchloride
QSAR – quantitative structure–activity relationship
xxvi Abbreviations

QSPR – quantitative structure–permeability relationship

(Q)SAR – both methods of structure–activity relationship and
quantitative structure–activity relationship
RAMEB – randomly methylated beta-cyclodextrin
RBALP – relative bioaccessibility leaching procedure
RBA – relative bioavailability
RBP – rapid bioassessment procedure
RC – primary biodegradability
RCR – risk characterization ratio
RDA – redundancy analysis
REACH – Registration, Evaluation, Authorization and Restriction of Chemicals,
EU regulation
REH GC – hydrocarbons extracted by aqueous RAMEB solution and measured
by GC-FID
RF – radiofrequency
RfD – human oral reference dose
RHE – reconstructed human epidermis
RIVM – Dutch National Institute for Public Health and the Environment
RNA – ribonucleic acid
ROC – rat keratinocyte organotypic culture
RQ – risk quotient
RS – remote sensing
SA – solubilizing agent
SAM – standardized aquatic microcosm
SAR – structure–activity relationship
SAS – Statistical Analysis System a software package
SB – soil biota
SBET – simplified bioaccessibility extraction test
SCGE – single cell gel electrophoresis
sD – sample dose
s.e. – standard error
SEM – solvent extractable mass
SETAC – Society of Environmental Toxicology and Chemistry
SFE – supercritical fluid extraction
SHE – Syrian hamster embryo cell line
SHIME – simulator of the human intestinal microbial ecosystem
SIFT – mouse skin integrity function test
SIN – substrateinduced nitrification
SIR – substrate-induced respiration technique
SIRC – rabbit corneal cells
SIT – skin irritation test
SME – multi-step sequential extraction method
SOM – self-organizing map
SPE – solid–liquid extraction
SPMD – semi-permeable membrane device
SPSS – Statistical Package from IBM
SQT – sediment quality triad
sRNAs – small RNAs
SS – sum of squares method for regression analysis
 Abbreviations xxvii

SSC – soil screening concentration

SSD – species sensitivity distribution
SSE – selective sequential extraction
STT – soil testing triad
STTA – stably transfected transactivation assay
T25 – carcinogenicity potency estimate the chronic dose rate which will
give tumors to 25% of the animals
TAM – thermo activity monitor
TCDD – 2,3,7,8-tetrachlorodibenzo-p-dioxin, persistent organic toxicant
TCE – trichloroethylene, a toxic solvent
TD50 – 50% toxic dose
TDI – tolerable daily intake
TECAM – triolein-embedded cellulose acetate membrane
TEF – toxicant equivalent factor
Tenax TA beads – porous polymer resin based on 2,6-diphenylene oxide
TEQ4CP – toxicity-equivalent value expressed in mg 4-chlorophenol/kg soil
TEQCu – toxicity-equivalent value expressed in mg Cu/kg soil
TER – toxicity/exposure ratio
TG – test guideline
TGD – technical guidance document
THQ – target hazard quotient
TIE – toxicity identification evaluation for sediments
TIM – TNO intestinal model
TMC – terrestrial microcosm chamber
TME – terrestrial model ecosystem
TO – transformer oil
ToA – Treaty of Amsterdam
TOFMS – time-of-flight mass spectrometer
TPF – triphenylformazan
TPH – total petroleum hydrocarbons
TTC – 2,3,5-triphenyl-tetrazolium-chloride, an indicator dye, used
for microbial respiration bioassays
TTO – terrestrial test organism(s)
UAE – ultrasonic assisted extraction
UBA – Umweltbundesamt, the Federal Environment Agency in Germany
UBM – unified bioaccessibility method
UCLA – University of California, Los Angeles
UDS – unscheduled DNA synthesis test
UMAM – uniform mitigation assessment method
UNECE – United Nations Economic Commission for Europe
US EPA – Environmental Protection Agency of the United States
USM – ultimate biodegradation model
UV-VIS – ultraviolet and visible light
vPvBs – very persistent and very bioaccumulative chemical substances
VARMINT – variables for assessing reasonable mitigation in
new transportation
VMPs – veterinary medicinal products
xxviii Abbreviations

WD-XRF – wavelength dispersive X-ray fluorescence

WHAP – wildlife habitat appraisal procedure
WET – whole effluent toxicity, direct toxicity testing of effluents
WFD – Water Framework Directive, EU Directive
WoE – weight of evidence
WPCF – Water Pollution Control Federation
WVA – wetland value assessment methodology
XAD – crosslinked polystyrene copolymer resin adsorbent
XAFS – X-ray absorption fine structure
XANES – X-ray absorption near edge structure
XRF – X-ray fluorescence
YES – yeast estrogen screen assay
About the editors

Katalin Gruiz graduated in chemical engineering at

Budapest University of Technology and Economics in
1975, received her doctorate in bioengineering and
her Ph.D. in environmental engineering. Her main
fields of activities are: teaching, consulting, research
and development of engineering tools for risk-based
environmental management, development and use of
innovative technologies such as special environmental
toxicity assays, integrated monitoring methods, biolog-
ical and ecological remediation technologies for soils
and waters, both for regulatory and engineering pur-
poses. Prof. Gruiz has published 35 papers, 25 book
chapters, more than hundred conference papers, edited
6 books and a special journal edition. She has coor-
dinated a number of Hungarian research projects and
participated in European ones. Gruiz is a member of the REACH Risk Assessment
Committee of the European Chemicals Agency. She is a full time associate professor
at Budapest University of Technology and Economics and heads the research group of
Environmental Microbiology and Biotechnology.

Tamás Meggyes is a research co-ordinator specialising

in research and book projects in environmental engi-
neering. His work focuses on fluid mechanics, hydraulic
transport of solids, jet devices, landfill engineering,
groundwater remediation, tailings facilities and risk-
based environmental management. He contributed to
and organised several international conferences and
national and European integrated research projects in
Hungary, Germany, United Kingdom and USA. Tamás
Meggyes was Europe editor of the Land Contamination
and Reclamation journal in the UK and a reviewer of
several environmental journals. He was invited by the
EU as an expert evaluator to assess research applications
and by Samarco Mining Company, Brazil, as a tailings
management expert. In 2007, he was named Visiting Professor of Built Environment
Sustainability at the University of Wolverhampton, UK. He has published 130 papers
xxx About the editors

including eleven books and holds a doctor’s title in fluid mechanics and a Ph.D. degree
in landfill engineering from Miskolc University, Hungary.

Éva Fenyvesi is a senior scientist, a founding member

of CycloLab Cyclodextrin Research and Development
Ltd. She graduated as a chemist and received her PhD
in chemical technology at Eötvös University of Natural
Sciences, Budapest. She is experienced in the prepa-
ration and application of cyclodextrin polymers, in
environmental application of cyclodextrins and in gas
chromatography. She participated in several national
and international research projects, in the develop-
ment of various environmental technologies applying
cyclodextrins. She is author or co-author of over 50
scientific papers, 3 chapters in monographs, over 50
conference presentations and 14 patents. She is an edi-
tor of the Cyclodextrin News, the monthly periodical on
cyclodextrins.
Chapter 1

Environmental toxicology – A general

overview
K. Gruiz
Department of Applied Biotechnology and Food Science,
Budapest University of Technology and Economics, Budapest, Hungary

ABSTRACT

The core issues of this book are chemical substances and their adverse effects on the
environment and humans. Every aspect of environmental management is linked to the
topic of chemical pollution. Millions of people die and millions of hectares of land are
degraded every year because of chemical pollution of the environment, thus dealing
with pollution is one of the most important global challenges.
Environmental toxicology – capable of directly measuring the adverse effects of
chemicals on individual organisms or complex communities – is the scientific tool of
sustainable environmental management and is a rapidly developing field in science and
engineering. Now that it has been found that chemical models exhibit numerous short-
comings in the description of the complex system of the environment, environmental
toxicology has moved to the forefront of attention.
This chapter summarizes the basics of environmental toxicology, the science of
measuring and using data on adverse effects from the point of view of environmental
management such as legislation, monitoring, risk assessment and risk reduction.
The test organisms and test methods used for assessing the adverse effects of chemi-
cal substances or contaminated land on the environment and humans will be discussed.
Humans are considered as part of the environment, being in direct contact with the
atmosphere, the water and soils as well as in indirect contact through the food chain.
The topics will range from the basic theory to engineering practice, including legislative
toxicology, standardization and site-specific tools.
Traditional methods will be discussed extensively, but special emphasis will be
placed on innovative procedures, for example, the alternatives to animal testing, rapid
and cost-saving bioassays and molecular methods such as in vitro and in silico models
and the tiered approach to ensure time and cost efficiency, to save animal lives and
avoid overextending the use of laboratory equipment and materials.

1 INTRODUCTION, BASIC DEFINITIONS

Millions of chemical substances are produced and used world-wide and different scales
of precaution and prevention are applied to them, depending on region, country or
substance type. According to the CAS Registry Number – an identifier for chemical
2 Engineering Tools for Environmental Risk Management – 2

Figure 1.1 Distribution of man-made chemical substances in the environment.

substances assigned by the Chemical Abstracts Service (CAS, 2013) – 309 000 invento-
ried/regulated substances and 65 million commercially available organic and inorganic
chemicals were listed in the registry by May 2014 (CAS, 2014). In 2011, the number
of inventoried substances had been 282 000 and 48 million commercial substances
were available.
The last century showed that all chemical substances that are produced and used
are emitted into the environment, and, depending on their fate and behavior, they
reach the ecosystem and affect humans sooner or later. Some others such as pesticides
are produced especially for killing insects and are spread directly into the environment.
Figure 1.1 shows the sources and transport pathways of chemical substances
produced and used for industrial, agricultural and household purposes.
Many of the chemical substances produced and used in large volumes are so-
called xenobiotics which differ from natural molecules and have structures alien to
living organisms. Other chemical substances are of natural origin, but they occur in
the wrong place and in an abnormal concentration.
The cause of the risk due to chemicals in the environment is their hazardousness;
including their behavior in the environment and their adverse effects on the ecosystem
and humans.
The adverse effects of hazardous chemical substances can be observed and mea-
sured in the environment or tested in the laboratory. The probable damage to the
 Environmental toxicology – A general overview 3

natural or built environment including workplaces and to users of the environment

can be forecast based on the type and extent of the adverse effects and the chemicals’
fate and behavior. The probability of an adverse effect multiplied with the extent of
future damage is called risk, in this case environmental risk.
Adverse effects of contaminated environmental compartments, i.e. air, water and
soil can be measured directly in the environment, in situ or on environmental samples,
ex situ. For example, one can put well-controlled test organisms into the surface water
(mussels in a cage) or deliver water samples to the lab and test them on the same mus-
sels or on fish or daphnids. Besides direct toxicity testing of environmental samples,
another possibility is the prediction of the adverse effects of a chemical substance (e.g.
on aquatic ecosystems), either based on the concentration (measured by chemical ana-
lytical methods) or the toxicity (measured by standardized toxicity testing methods) of
the pure chemical. The difference between the two is that the hazardous chemical sub-
stance in the environment always occurs together with other substances, whether they
are contaminants or the natural constituents of the environmental medium. The inter-
actions between the contaminant and other physical, chemical and biological agents
influence its adverse effect on a large scale. The environmental relevance of chemi-
cal models based on concentration or biological models based on the toxicity of the
pure chemical is generally poor. On the other hand, their reproducibility and precision
are much better than that of environmental assessments. Some other models such as
microcosms and mesocosms give priority to environmental reality at the expense of
reproducibility and precision. The concept and design of the environmental assessment
should determine the right set of models and test methods to fulfill the requirements
of the assessment goal and tier.
The results of the testing of adverse effects can be used to calculate the risk of
the contaminated environment, more precisely the risk which the chemical in the
environment poses to the users (receptors) of the contaminated environment. Envi-
ronmental toxicology equally applies mathematical models, chemical analyses and a
wide range of biomarkers in biochemical, physiological, organism-, population- and
community-level tests.
Environmental risk assessment of chemicals integrates chemical and toxicological
methodologies for the identification of the presence, fate and adverse effects of chemical
substances in the environment (Figure 1.2).

1.1 Toxicology and its role

Toxicology involves all aspects of the adverse effects of chemicals on living systems.
Adverse effects are those effects which are damaging to either the survival or normal
function of living organisms or their communities.
Environmental toxicology is the science and engineering of the adverse effects –
mainly of chemicals and other man-made agents – in the environment and through the
environment. Both the ecosystem and humans may be the targeted receptors. Envi-
ronmental toxicology also includes studying the chemical substances – which may be
potential and real contaminants – polluting air, water, soil and food, their impacts
upon the structure and function of ecological systems, including people as well as the
use of these results for decision making and environmental management.
4 Engineering Tools for Environmental Risk Management – 2

Figure 1.2 The role of chemical and biological methods in environmental risk assessment.

The results of environmental toxicology are mainly used to predict hazard and risk
due to certain chemical substances or the contaminated environment at local, regional
and global scales. The key function of environmental toxicology is to support deci-
sion making in environmental management and policy by setting risk-based priorities,
establishing environmental quality criteria, designing monitoring systems, selecting
risk reduction measures, establishing specific land use target values and performing
risk-based management activities. Environmental toxicity results are suitable for direct
decision making: decisions are based only on the type and scale of adverse effects. The
use of fate and hazard data and information will be discussed and demonstrated in
Chapter 2.
Chemical substances are materials with a specified chemical composition. Based
on this specification, they can be pure chemicals, mixtures of chemicals, or products
with a known chemical composition. The production and use of chemical substances
are likely to create the main sources of risk in the civilized world. The hazard of chemi-
cal substances derives from their structure, their intrinsic physico-chemical, biological
and environmental fate properties, but foremost their potential to adversely affect liv-
ing organisms, the members of the ecosystem and humans. The actual impact is based
on their interaction with the other party – the properties of the environment and the
living organisms. When preparing a forecast one can be faced with a known or as
yet unknown environment and receptors. When the target environment is unknown
(e.g. the chemical product is not on the market yet), only the chemicals’ potential
 Environmental toxicology – A general overview 5

behavior and effect data are given: a hazard assessment can be done. One can calcu-
late the environmental risk when the target environment and its users, their sensitivity
and resistance are known and can be taken into account. The influence of the exposed
party on the appearance of the effect can also be estimated. The proper management of
chemical substances demands scientific and engineering tools, which are only available
to a limited extent. Managers and politicians have to understand that the scientific and
engineering basis of their activities and decisions is in constant development and must
be further developed and refined. The long-term effects, non-dose-related effects, accu-
mulation, changes and interactions in the environment or in the bodies of organisms
are not yet fully understood and quantitatively characterized. In addition to all of these
aspects, data acquisition, evaluation and interpretation must be standardized so as to
be able to utilize the available measured data for decision making and management of
the environment; otherwise, they are useless. Understanding and using the mathemati-
cal and statistical evaluation tools, dealing with uncertainties, validation of the results
and verification of the methods and technologies are essential for environmental man-
agement, legislation and political decision making. Managing the dynamic system of
the environment in combination with the dynamic system of knowledge – necessary
for good decision making – cannot be reduced to a mechanical choice between two
alternatives.
Xenobiotics are substances foreign to a biological system. They are artificial sub-
stances which had not existed in nature before being synthesized by humans. The term
stems from Greek and means foreigner, stranger. Xenobiotic substances can mimic nat-
ural molecules and in this way, they may partly or fully substitute biotic molecules in
the metabolic pathways. They can be degraded, modified or utilized by microorgan-
isms or higher organisms. This false metabolism sometimes leads to the production
of more hazardous metabolites and secondary effects such as the destruction of the
endocrine or immune systems.
Ecological system is defined as a complex system ranging from the molecular level
through individual organisms and communities to the whole ecosystem. An ecosystem
comprises the organisms and their habitat; it means all of the organisms living in a
particular area and the non-living, physical components of the environment with which
the organisms interact, i.e. air, water, soil, sediment and other environmental elements
such as sunlight, wind, precipitation, temperature, elevation and so on.
Aquatic and terrestrial ecosystems differ to a great extent, and a more or less clear
line of division can be drawn between these two habitats. The size and form of ecosys-
tems is subjective, it depends on our interest; it may be a microecosystem within a drop
of water, a flock of sewage sludge or the rhizosphere of a plant, the local ecosystem of
a contaminated site, smaller or larger regions such as the Mátra Hill in Hungary, the
watershed of the Danube in Europe, or the entire earth (global ecosystem). When the
deterioration of an ecosystem is measured, the habitat, the components of the ecosys-
tem and the ecosystem services such as regulating (climate, nutrient and water balance),
provisioning (water, food) and cultural (science, spiritual, ceremonial, recreation,
aesthetic) services are assessed and evaluated by the environmental manager.
Environment itself is a complex entity: the environmental compartments – in spite
of the differences in the methods needed for monitoring and managing their risks –
should be handled in an integrated way because environmental compartments are in
close connection, interaction and interrelationship with each other. The state of the
6 Engineering Tools for Environmental Risk Management – 2

earth’s ecosystem and its capacity can only be estimated if the global environmen-
tal/ecological trends are understood. These trends and the risky changes necessitate
monitoring, predictions and forecast. Scientific knowledge and engineering tools
are available to support correct short-term environmental risk management and the
connecting decision making. A typical problem is that short-term socio-economic rea-
soning overrides environmental interest. The lack of knowledge about long-term global
trends results in locally justifiable decisions and environmental management measures
that often fail to harmonize with the needs of regions or the entire earth. Hydroelectric
power plants can solve the energy problems of a small country but may cause huge
problems for the downstream environment of another country. Opening a mine pro-
vides social advantage for the local community but causes environmental damage to the
whole of the watershed. If we were positive about global warming as a natural trend,
we would not spend huge financial resources on preventing it. Moving ‘dirty’ indus-
trial and mining activities from industrialized countries to third-world countries with
less stringent environmental criteria is extremely harmful from a global perspective.
The ‘result’ is that the global state of the environment will suffer.
The IPPC Directive of the EU (Integrated Pollution Prevention and Control,
2008/1/EC) (IPPC, 2008) reflects the scientific and managerial experience concern-
ing the fact that individual environmental compartments cannot be managed on their
own: preventing only water from being polluted at the expense of sediments and soil,
treating waste water without taking into account the treatment or utilization of sewage
sludge, or cleaning the local environment without dealing with its neighborhood do
not qualify as sustainable management of the environment.
Environmental toxicology needs an integrated approach and requires a multidis-
ciplinary scientific basis to a variety of specialist fields as listed here:

– Analytical chemistry
– Biology
– Biochemistry
– Biometrics
– Chemistry, chemical engineering
– DNA and molecular techniques
– Ecology
– Evolutionary biology
– Limnology
– Marine biology and oceanography
– Mathematical and computer modeling
– Meteorology
– Microbiology
– Molecular biology and genetics
– Pharmacokinetics
– Physiology
– Population biology
– Risk assessment
– Risk management
– Statistics
– Toxicology
– Toxicokinetics.
 Environmental toxicology – A general overview 7

The application of toxicology is widespread. Apart from the management of the

environment, many other areas of human activity manage risk based on toxicological
results, such as human healthcare, drug development and occupational health and
safety as well as legislation.
Occupational toxicology – also called industrial toxicology – deals with (poten-
tial) toxic effects at workplaces on workers. Occupational toxicology serves the aims
of occupational risk management to protect workers from physical agents and chem-
ical substances and to make their work environment safe. Occupational hazard and
risk assessment rely on human and environmental toxicology, but the environment is
restricted to the workplace in this case: the air inside an industrial plant, the risk of
dermal or eye contact with chemical substances at work, as well as the development
of occupational diseases in association with the chemical substances used or produced
in the technologies.
Regulatory toxicology gathers and evaluates existing toxicological information,
develops uniform, standardized, comparable methods for testing and evaluating not
only the effects but also the fate and behavior of chemical substances both in the envi-
ronment and in organisms. Regulatory toxicology means the interpretation and use of
toxicity results for the establishment of effect-based quality criteria for food, drinking
water, other water uses (e.g. irrigation), animal feed, for all environmental compart-
ments such as air, surface waters, sediments, subsurface waters, soils, depending on
their use and users (land use) as well as for waste utilization (e.g. sewage sludge utiliza-
tion on soil). Regulatory toxicology tries to control hazardous substances and materials
in a safe manner, ensuring an acceptable risk level, or in other words, a safe exposure,
an exposure level which does not pose unacceptable risk to the ecosystem and humans.
It supports the authorization, restriction or ban of chemicals, licensing of technolo-
gies using chemicals, and ensures a globally harmonized system for classification and
labeling of chemicals for their safe use.
Food toxicology aims at providing a supply of safe and edible food to consumers,
assessing poisonous or allergenic food components and the adverse effects of food
processing additives and pesticide residues.
Air toxicology measures indoor and outdoor air quality and compares it to quality
criteria. It also deals with the atmosphere as a source of contaminants that reach soil
and water.
Aquatic toxicology is based on the response of the aquatic ecosystem (marine
and freshwater), the response of ecosystem diversity or the response of representative
ecosystem components in laboratory tests. Exploring the fate and behavior of chemical
substances in water is an important part of aquatic toxicology.
Terrestrial toxicology concentrates on the terrestrial ecosystem as well as on the
fate and behavior of chemicals in soil. The terrestrial ecosystem is understood to a lesser
degree than the aquatic one. Diversity of terrestrial species, first of all soil microflora
and fauna is difficult to investigate using traditional methods. In the case of soil and
other solid phase compartments such as sediments, solid wastes and sludge, the mobil-
ity, partitioning, accessibility and bioavailability of toxic substances play an extremely
important role in producing adverse effects and posing an actual risk to the ecosystem
and humans.
Clinical toxicology is concerned with diseases and illnesses associated with short-
or long-term exposure to toxic chemicals.
8 Engineering Tools for Environmental Risk Management – 2

Forensic toxicology is the science that looks into the cause and effect relationships
between exposures to a drug or chemical substance and the resulting toxic or lethal
effects.
Epidemiology is closely related to toxicology and its key aim is finding connections
between human health and contamination in food or drinking water or the environ-
ment. Epidemiology is the study of patterns of health and illness and associated factors
at the population level. It supports evidence-based medicine with proper statistical
data on risk factors for diseases including the risk of environmental pollution or the
accidental exposure to toxicants. Epidemiology is based on good study design, data
collection, statistical analysis and documentation. The methods and concepts of epi-
demiology provide suitable tools for finding relationships between GIS-based pollution
maps and the health quality of human, animal or plant populations in the future. It is
very important all over Europe and the world to harmonize and utilize the collection
and processing of available data.
Human toxicology aims to specify the dose–response relationship between haz-
ardous chemical substances and human responses. Since these relationships cannot be
tested on a well-designed, statistically relevant human population (the human toxicity
of chemical substances is mainly based on the results of animal toxicity tests), the extent
of toxic effects on humans is usually an estimate. Extrapolation from animal data to
humans is possible assuming that the animal species has been properly selected, i.e. its
response is analogous to the human body’s response and the test method and scenario
applied truly model real human exposure. The main extrapolation methodology – for
example from rat to man – applies a safety factor based on experience. The default
value for the interspecies safety factor is ED50 (human)/ED50 (animal) = 0.1 because
drugs and toxic chemical substances are generally 10 times more potent in humans as
indicated by available pharmacological and toxicological data, therefore a 10-times
smaller dose suffices to cause the same effect.
Animal data are suitable for establishing the dose or concentration of the chemical
substance that would cause an adverse effect, damage or death to 10, 20, 50 or 90%
of the treated animals, or for determining the lowest effect and the highest no-effect
concentrations or doses. These end points represent manageable limit values.
Animal tests have many subclasses discerned according to the applied animal taxon
(fish, bird, mouse, rat, dog, monkey, etc.), type of exposure (acute, repeated or chronic
exposure), exposure routes (inhalation, oral, cutaneous and mixed routes) and aim of
the test (testing of toxicity, mutagenicity, reprotoxicity, neurotoxicity, sensitization
and irritation, endocrine disruption, immunotoxicity, phototoxicity or photoallergy).
Depending on end points, tests can be based on death or minor deterioration of the
whole organism such as immobilization of daphnids, or behavioral changes (e.g. con-
taminated soil avoidance by insects), irritation or corrosion of the skin or eye, changes
in the metabolism. The end point can be

– organ-specific: cardiac, ophthalmic, cutaneous, muscle, bone- or hepatotoxic, etc.

– cellular: cell death, mitochondrial or peroxisome irregularities, cellular tight
junctions, etc. and
– molecular: reactive oxygen species, glutathione and glutathione transferase,
metabolomics, DNA changes, chemokines and other genomics.
 Environmental toxicology – A general overview 9

Impairments in the reproduction can be studied through fertility, quantitative and

qualitative characterization of the offsprings and contaminant analysis of the milk of
mammals.
Pharmacology is the closest related branch of applied science to human toxicol-
ogy, whose aim is the study of drug actions. The methodology is very similar to human
toxicity assessment, the difference is that drugs are applied for their beneficial effects
on human or other organisms, and the main effect has a special target, e.g. to kill
pathogens or harmful cells. More specifically, pharmacology is the study of the inter-
actions that occur between a living organism and the chemical substance, i.e. the drug
that affects certain biochemical function(s). If the substances have medicinal properties,
they are considered pharmaceuticals and the assessment is qualified as a pharmacolog-
ical assessment. If the drug is a toxicant, it is toxicology that deals with its effects. As
all drugs are toxicants, even though they have a dose range which leads to medicinal
applications, the methodologies are very closely related. It is mainly the target that
distinguishes pharmacology from toxicology.
Ecotoxicology and human toxicology represent the two main pillars of envi-
ronmental toxicology. The subject of the study of ecotoxicology is the potential for
physical, chemical and biological agents – also called stressors – to affect ecosystems.
Such stressors might disrupt natural genetics, biochemistry, physiology, behavior, struc-
ture, form and interactions of living organisms that comprise the ecosystem if they
occur in a concentration, level or density which causes adverse effects on microbial,
plant, animal and human constituents as well as the complex structure they form in
the ecosystem.
Truhaut coined the term ecotoxicology in 1969 defining it as ‘the branch of toxicol-
ogy concerned with the study of toxic effects, caused by natural or synthetic pollutants,
to the constituents of ecosystems, animal (including human), vegetable and microbial,
in an integral context’ (Truhaut, 1977). Ecotoxicology covers not only the constituents
of the ecosystem but also their forms and structures.
Ecotoxicology is the integration of toxicology and ecology or, as Chapman (2002)
suggested, ‘ecology in the presence of toxicants’. It aims to quantify the effects of stres-
sors upon natural populations, communities, or ecosystems. Chapman (2002) tries to
distinguish ecotoxicity and environmental toxicity, saying that environmental toxicity
is more anthropocentric, but Landis and Yu (1999) consider environmental toxic-
ity as impacts of chemicals and other stressors on ecological systems, their function
and structure, including man. Our approach in this book, similarly to the develop-
ment of environmental toxicology over the last few decades, corresponds to the latter:
environmental toxicology includes both human toxicology and ecotoxicology.

1.2 Regulatory toxicology for chemical substances and

contaminated land
Regulations are often discussed in this book in the context of environmental toxicology
and environmental risk management of chemicals.
Regulatory toxicology is the study of the adverse effects of chemicals, not just
to humans, but also to all living organisms including plants, animals or fungi. The
integration of metabolism, toxicity, pathology and effect mechanisms plays a much
greater role today than ever before. A better understanding of the interactions and
10 Engineering Tools for Environmental Risk Management – 2

relationships is essential for proper regulation of chemical substances and drugs and
every other material, product or waste which contains hazardous chemical substances.
It should be emphasized that the origin of chemical risk is not only the hazard of
a substance, but also the abnormal concentration or presence of a chemical substance
in the wrong place at the wrong time. A large number of major pollution events
are caused, for example, by petroleum products, metals, mine wastes, agricultural
nutrients and various waste materials, which are natural products such as organic
waste, but emerge in the improper quantity and quality or in the wrong place.
The complex approach of regulatory toxicology has the advantage that the results
of environmental toxicity can be utilized for the regulation of activities which pose
a risk to the environment. It is of utmost importance for regulation to be based on
science and the decisions of environmental management should be effect-based. What
does this mean? This means that decision making, risk management, environmental
monitoring and the implementation of risk reducing measures should be based on sci-
entific evidence concerning the real adverse effects of the pollutant in a generic area
or on a real site. Science-based regulation reflects the state of the art of scientific and
engineering knowledge, the continuous development in science and technology and
in environmental risk management. This kind of dynamism in the regulation pro-
cess makes possible the integration of new scientific knowledge and innovative risk
management tools, which provide improvement vis-à-vis former BATs (Best Available
Technologies).
Another aspect of regulatory toxicology is that regulations require information,
data, methodology, standardized analytical and test methods as well as interpretations.
Environmental toxicology has to fulfill the needs of regulation and fill gaps in our
knowledge.
There is a close interaction between the two sides: environmental toxicology should
serve regulation, while scientists are supposed to know the concepts of regulation
and the way to fill the gaps with new methodologies and information. Science should
provide advice about the need for the integration of scientific knowledge into regulatory
decision making.
The concepts and problems involved with the generation, evaluation and inter-
pretation of experimental ecotoxicological, animal and human data should be placed
by regulatory toxicology in the wider perspective of societal considerations towards
protecting human health and the environment.
Regulatory toxicology and risk management are closely related, and both of them
should continuously include new scientific developments to slowly reduce or eliminate
methodological shortcomings and data gaps impairing the quality of decisions and
regulations. Even after upgrading the scientific grounds, many uncertainties will remain
due to natural variabilities, statistical uncertainties and subjective errors and bias.
The importance of regulatory toxicology is well characterized by the regulations on
pesticides, biocides, food additives, cosmetics and the regulation of hazardous chemical
substances and materials all over the world. In Europe, hazardous chemical substances
and materials are regulated by the REACH (Registration, Evaluation, Authorization
and Restriction of Chemicals, EC No 1907/2006) (REACH, 2006) and CLP (Classifi-
cation, Labelling and Packaging, 1272/2008) (CLP, 2008a) regulations. Additionally,
specific regulations exist for specific families of products such as pesticides, biocides,
fertilizers, detergents, explosives, pyrotechnic articles, and drug precursors.
 Environmental toxicology – A general overview 11

In principle, REACH applies to all chemical substances: not only chemicals used in
industrial processes but also those in our daily lives, for example, in cleaning products,
paints and in articles such as clothes, furniture and electrical appliances.
The aims of REACH are:

– improving the protection of human health and the environment from the risks
posed by chemicals;
– enhancing the competitiveness of the EU chemical industry, a key sector of the
EU’s economy;
– promoting alternative methods for the assessment of hazards of substances;
– ensuring the free circulation of substances on the EU’s internal market.

Further details can be found in Chapter 2 in Volume 1 not only about REACH and
CLP but other European and world-wide regulations related to chemical substances,
such as the Globally Harmonized System (GHS, 2007), which ensures the uniform clas-
sification and labeling of chemical substances to alert the users of chemicals in industry,
commerce, transport or households to be cautious and apply preventive measures or
means.
Currently, chemical substances are classified according to GHS into the following
15 categories:

– Explosive substances and preparations;

– Oxidizing substances and preparations;
– Extremely flammable substances and preparations;
– Highly flammable substances and preparations;
– Flammable substances and preparations;
– Very toxic substances and preparations;
– Toxic substances and preparations;
– Harmful substances and preparations;
– Corrosive substances and preparations;
– Irritant substances and preparations;
– Sensitizing substances and preparations;
– Carcinogenic substances and preparations;
– Mutagenic substances and preparations;
– Substances and preparations which are toxic for reproduction;
– Substances and preparations which are dangerous for the environment.

REACH, CLP and GHS regulations need a uniform and standardized analysis
and testing methodology as well as globally harmonized evaluation, interpretation
of the results such as the classification and labeling of chemical substances and the
physico-chemical, ecotoxicological and toxicological methods. A standard and uni-
form methodology is a fundamental principle of regulatory toxicology, otherwise the
results would not be comparable, and differences due to methodology, laboratory,
country, etc. would limit the efficiency of regulation to a large extent.
The European Council regulation No 440/2008 of 30 May 2008 (EC, 2008) lays
down the test methods pursuant to REACH.
12 Engineering Tools for Environmental Risk Management – 2

OECD (OECD, 2013a) (Organisation for Economic Cooperation and Devel-

opment) has standardized the guidelines in support of the regulation and uniform
management of chemical substances (OECD, 2013b). OECD developed, validated
and published a collection of the most relevant internationally agreed test methods
used by government, industry and independent laboratories to determine the adverse
effects, the hazards and safety of chemicals and chemical preparations, including pesti-
cides and industrial chemicals. They cover tests for the physical-chemical properties of
chemicals, human health effects, environmental effects, degradation and accumulation
in the environment.
The European Union (EU) and OECD initiated the development of alternative test
methods which either do not use animals at all, or use only a much smaller number of
animals compared to traditional toxicological tests.
The European Centre for the Validation of Alternative Methods (ECVAM, 1991)
was created by a ‘Communication from the Commission to the Council and the
Parliament’ in October 1991 (COM, 1991), pointing to a requirement in Directive
86/609/EEC (EEC, 1986) on the protection of animals used for experimental and
other scientific purposes, which requires that the Commission and Member States
should actively support the development, validation and acceptance of methods which
could reduce, refine or replace the use of laboratory animals.
ECVAM was established in 1992 at the Joint Research Centre (JRC, 2013), and
is now the part of the Institute for Health and Consumer Protection (IHCP, 2013)
located in Ispra, Italy. Its mission is to:

– Promote the scientific and regulatory acceptance of non-animal tests that are
of importance to biomedical sciences, through research, test development and
validation and the establishment of a specialized database service;
– Coordinate at the European level the independent evaluation of the relevance and
reliability of tests for specific purposes, so that chemicals and products of various
kinds, including medicines, vaccines, medical devices, cosmetics, household prod-
ucts and agricultural products, can be manufactured, transported and used more
economically and more safely, whilst the current reliance on animal test procedures
is progressively reduced.

1.3 Future of environmental toxicology

The protection of test animals and animal welfare is not the only reason for searching
for alternatives, but also because of the developing trend in toxicology to find the
molecular fundamentals of the effects of chemical substances on an organism’s body,
organ, cell or molecule. The methodologies slowly changed from the whole body level
and drastic end points such as lethality, to earlier and milder indicators of the same
effect. Both the protection of animals and a refined understanding of the mechanisms
of effects guided events in the same direction: the use of molecular tools, metabolites,
enzymes, RNA and DNA molecules as indicators or the heat production (Gruiz et al.,
2010) of living organisms. MacGregor (2003) summarized the future possibilities of
environmental toxicology, emphasizing that the molecular biology revolution and the
development of genomic and proteomic technologies support the transformations in
toxicological and clinical practice. Traditional biomarkers of cellular integrity, cell and
 Environmental toxicology – A general overview 13

tissue homeostasis and morphological alterations that stem from cell damage or death
can be supplemented or replaced with DNA or other molecular end points and can
become a realistic alternative.
Molecular and cell-based technologies are discussed in detail in the following sec-
tions; the application of QSAR-models – aiming at predicting the physicochemical and
biological properties of molecules based on their structure and their application in
environmental toxicology – was introduced in Chapter 1 in Volume 1.

1.3.1 Molecular technologies

Molecular technologies permit simultaneous monitoring of many hundreds or thou-
sands of molecules (OMICS, 2009) and promise to allow functional monitoring of
key cellular pathways simultaneously. New biomarkers based on molecular responses
to functional perturbations and cellular damage are foreseen. Responses that can be
monitored directly in humans should provide bridging biomarkers that may eliminate
much of the current uncertainty in extrapolating from laboratory models to human
outcome. Another aspect of genomics is the currently enhanced ability to associate
DNA sequence variations with biological outcomes and individual sensitivity. Genetic
approaches, for example, the correlation of genetic variants with human diseases and
adverse reactions from exposure to toxicants proved that individual variations play
an important role in sensitivity to environmental agents, disease susceptibility, and
therapeutic responses. Regulatory toxicological practice is likely to be shaped in the
future by the combination of conventional pathology, toxicology, molecular genet-
ics, biochemistry, cell biology and computational bio-informatics – resulting in the
broad application of molecular approaches to monitoring functional disturbances.
The forecast of MacGregor (2003) has become a reality in the past few years.
The AltTox (2009) definition of omics is as follows: omics refers to the collective
technologies used to explore the roles, relationships and actions of the various types of
molecules that make up the cells of an organism (OMICS, 2009). Omics technologies
are increasingly used in different fields of science, mainly in system biology, which is
defined as biology focusing on complex interactions in biological systems, and using
holism instead of reducing the scope of investigation, which might be particularly
important in environmental sciences and management.
The main users of omics-based technologies are:

– Systems biology;
– Computational systems biology;
– Medicinal systems biology;
– Cell signaling and networking;
– Drug discovery and development;
– Synthetic biological systems;
– Environmental toxicology.

Omics technologies include

– Genomics: the study of genes and their function such as in the Human Genome
Project (HUGO, 2012; DOE, 2003). The first of the “omics’’ technologies to
be developed, genomics has resulted in massive amounts of DNA sequence data
14 Engineering Tools for Environmental Risk Management – 2

requiring great amounts of computer capacity. Genomics has progressed beyond

sequencing of organisms (structural genomics) to identifying the function of the
encoded genes (functional genomics). As well as functional genomics, other areas
include nutrigenomics, gene expression profiling, micro-array technologies, drug
metabolism and toxicology.
– Transcriptomics: the study of the mRNA as in genomics.
– Proteomics: the study of proteins, characterizing the identity, function, regulation,
and interaction of all of the cellular proteins of an organism (the proteome). With
proteomics, it is possible to identify the changes in cells and tissues exposed to
toxic materials, and to understand the mechanisms of toxicity. Data collected in
databases can be utilized in a predictive in silico tool in environmental toxicology.
The most advanced approaches and technologies are: functional and computa-
tional genomics, molecular and cellular proteomics, gel-based proteome profiling,
mass spectrometry-based and gel electrophoresis-based proteomics, and protein
arrays.
– Metabolomics: the study of molecules involved in cellular metabolism. It provides
information on the metabolic state of a cell, organ or organism to identify bio-
chemical changes that are characteristic of environmental stresses such as exposure
to chemical substances or environmental contaminants, which alter the metabolic
pathways in cells, and result in a specific metabolite profile suitable for assessing
and identifying toxic exposures and consequent responses.
– Glycomics: the study of cellular carbohydrates, using methods such as glycan array
technology, structural and chemical glycobiology and microbial glycomics.
– Lipomics: the study of cellular lipids, such as cellular and membrane lipidomics.

The molecular profiles can vary with cell or tissue exposure to chemicals or drugs
and thus have potential use in toxicological assessments. These new methods have
already facilitated significant advances in our understanding of the molecular responses
to cell and tissue damage, and of perturbations in functional cellular systems (Aardema
& MacGregor, 2002).
The large amount of data provided by molecular methodologies should be man-
aged and analyzed by bioinformatics which uses advanced computing techniques.
Bioinformatics tools include computational tools that can mine information from large
databases of biological data such as genomics or proteomics data.
The integrative approach of generating maps of cellular and physiological
pathways and responses using bioinformatics and molecular databases is called
computational biology.
The aim of computational system biology is the creation of computational models
of the functioning of the cell, multicellular systems, and ultimately the whole organism.
These in silico models will provide virtual test systems for evaluating the toxic responses
of cells, tissues, and organisms to the effect of toxic chemical substances.
Innovative molecular methods such as microarrays have been developed in the last
decade. Microarrays consist of DNA or protein fragments placed as small spots onto a
slide, which are then used as ‘miniaturized chemical reaction areas’ (DOE, 2003). The
studies typically involve looking for changes in gene or protein expression patterns
from the effect of drugs or environmental chemicals. Thousands of genes or proteins
 Environmental toxicology – A general overview 15

can be investigated simultaneously. The evaluation by a robotic system, called HTS,

i.e. High Throughput Screening, is another innovation.
Toxicogenomics investigates the genes expressed in organisms that have been
exposed to a chemical substance, a drug, or a toxin and the results are compared to
unexposed (untreated) organisms (negative controls). The goal of toxicogenomics is to
identify patterns of gene expression related to specific chemical substances or chemical
classes of substances to use these expression patterns as end points for toxicity assess-
ments. Until today, toxicogenomics has been useful in refining animal experiments
and identifying mechanisms of toxicity in laboratory tests. The new toxicogenomics
approach can be utilized for cell cultures exposed to toxicants, and to develop a
predictive tool for in vivo toxicity testing (Aardema & MacGregor, 2002).

1.3.2 Cell-based technologies

Cultured animal and human cells in so-called ‘cell cultures’ are widely used for screen-
ing and deeper exploration of the effects, including the toxic effects, of chemical
substances. Many alternative test methods have been developed to replace animals
for testing of chemical substances or reduce their number as much as possible. Replac-
ing animal organisms with cellular models has many advantages, for example, creating
a ‘clear’ model with good reproducibility by concentrating on the main exposure routes
and end points. These in vitro cellular models are very useful in regulatory toxicol-
ogy where uniformity and reproducibility have priority in generic risk assessment. A
further advantage may be that the individual- or species-specific complexity of the
answer can be excluded from the model. However, when the realistic response of a
specific organism is required, a simplified model can hardly simulate the organism’s
complexity and its interactions with the real environment. The (general) model may
be a disadvantage when a certain species, strain or individual is the target of testing.
In this case simulation models or microcosms should be applied to approach reality as
much as possible.
Simplified cellular models such as single layers of cells living on a nutrient medium
in a culturing vessel are too much reduced, and they are not able to simulate living
organisms. Part of the new developments aims to increase the complexity of the cellular
models, for example, by three-dimensional (3D) cellular or tissue models that can
improve replication of the in vivo tissue.

1.3.3 Computational toxicology

A vast quantity of measured toxicological data is available in databases because molec-
ular toxicology, microarrays and their robotic evaluation disgorge abundant data. New
mathematical tools have to be developed and introduced for the efficient evaluation
and interpretation of these data (Kavlock et al., 2005). Computational toxicology is
the overall term for these tasks which deal with:

– The construction and curation of large-scale data repositories necessary to save

the information from new technologies (Richard, 2006).
16 Engineering Tools for Environmental Risk Management – 2

– The introduction of virtual and laboratory-based high-throughput screening (HTS)

assays on hundreds to thousands of chemicals per day and the development of high-
content assays with hundreds to thousands of biological end points per sample for
the identification of toxicity pathways (Inglese et al., 2006; Dix et al., 2007).
– The latest advances in computational modeling that provide the tools needed to
integrate information across multiple levels of biological organization for the char-
acterization of chemical hazard and risk to individuals and populations (Di Ventura
et al., 2006).

The mathematical, molecular and cellular models are often integrated into test
systems or test batteries and used for risk assessment and decision making in envi-
ronmental risk management. No doubt, in the future, these innovative molecular and
cellular methods will play an important role in predictive and regulatory toxicology
and in toxicological research, not only by replacing animals, but also because of the
opportunity to increase the number of tests and experiments, which are highly limited
when laboratory animals are used.

1.4 What environment means in the context of toxicology

Environment is much more than just natural environment: it includes – according to
a holistic view – all those parts of the earth that create and influence the habitats of
ecosystem members and human beings and those parts of the ecosystem that provide
services for them. This means that waste in the earth’s stratosphere, deep-sea oilfields,
workplaces and technological rooms are part of our environment due to their direct
or potential contacts with other parts of the ecosystem or living organisms!
Environment includes air, waters, both subsurface and surface waters as well as
their sediments, rocks and soils. These compartments and their three main physical
phases (gaseous, liquid, and solid) are in close connection and interaction with each
other; soil gas with soil solid, pore water (partition) and atmosphere (diffusion); sedi-
ment with free water and with soil (flood); soil with sediment (erosion); groundwater
with surface waters (convective transport in both directions), precipitation with all
compartments (seepage, runoff), etc. One compartment additional to the non-living
ones is the biota and its local habitat, which can be differentiated as a water-based
environmental compartment and others, where the solid phase is dominant (biofilms,
rhizosphere, activated sludge, soil and sediment etc.). These habitats are locations with
intensive interactions between all environmental compartments and phases.
The ecosystem itself is difficult to define: it is a widespread view that the ecosys-
tem is limited to the natural ecosystem, but in our context ‘natural’, agricultural,
recreational and fully artificial ecosystems cannot be handled separately because of
interactions and free transfer and also because of the participation of humans. Chem-
ical substances in this complex system may have a very invasive effect, even though
they are natural substances but occurring in abnormal concentrations, or xenobiotics
which are ab ovo xenon, i.e. alien to the environment. As they are unknown invaders,
their behavior and interactions with ecosystems or humans are not fully predictable.
The best example is the problem of pesticides: producers, users and regulators are
well aware of the target effect of pesticides. It was realized very soon that exploring
 Environmental toxicology – A general overview 17

and measuring the non-target effects on non-target ecosystem constituents is also nec-
essary and it has become an obligation to minimize the adverse effects of pesticides
on non-target ecosystem members. Today, we know that many of the pesticides have
long-term effects on the endocrine system causing hormone and immune system dis-
ruption as well as neurotoxic effects. It is a painful fact that science is not ready to
measure these emerging effects and to manage the problem of the so-called emerging
pollutants.

1.5 Environmental toxicology versus human toxicology

The aim of human and environmental toxicologists was very different in the begin-
ning: human toxicologists intended to characterize the acute risk to the human body
due to chemicals through the usual exposure routes: inhalation, digestion or dermal
contact and using the traditional pharmacological tools. As the epidemiological data
sets are typically not complete, the quality of existing epidemiological information
is still questionable and individual differences between the members of the human
population may be significant. The toxicologists usually rely on animal tests whose
results are then extrapolated to humans. Animal models have a number of advantages
but even more disadvantages: similarities and differences in metabolic pathways, high
deviation in references, and the small number of animals tested and, as a consequence,
low statistical power and high cost. In addition, there are ethical problems because
of welfare concerns of the experimental animals. End points of animal tests have
also changed in the last 20 years: lethality and the number of progeny as end points
have been refined by molecular and cellular indicators and metabolomics or genetic
analyses. New branches of science were born such as neurotoxicology, toxicokinetics,
toxicogenomics, hormone and immune system responses and many other sub-areas.
On the other hand, ecotoxicology, which was the sole concern in the beginning
and dealt with changes in the structure and constituents of the ecosystem and their
comparison with healthy ecosystems, has also changed: the extrapolation from indi-
vidual species to the whole environment has become more widespread in comparison
to more complex ecosystem assessments (which remains a simplification, in spite of
going into details) due to its easier implementation and better statistical power. The use
of microorganisms and other small organisms made it possible to increase the numbers
of tested organisms, simultaneous tests and repetitions. Increased sensitivity of the test
methods and early indication have moved to the fore as prime metabolic end points
instead of lethality. Metabolomics and DNA markers have become part of the practice
of ecotoxicity testing.
Finally, the importance and significant effects of interactions between human
receptors and the ecosystem through food chains, agroecosystems and commonly used
air and water have made it clear that environmental toxicology must find a common
denominator between human toxicology and ecotoxicology.
In the end, a significant part of both ecotoxicology and toxicology can be reduced
to the same cellular and molecular methods. Although toxicity and ecotoxicity tests
may use the same methods and model systems, the extrapolation to human or
ecosystem is different. Many of the omics are the same or very similar in biologi-
cal systems. The genetic code (genomics), the enzymes (proteomics), the metabolites
(metabolomics), the stress mechanisms and end products need the same methodology
18 Engineering Tools for Environmental Risk Management – 2

whether they are daphnids, fish, rats, human cells or organs. Uniform molecular meth-
ods which, of course, cannot estimate the response of a whole organism, can indicate
almost all of the adverse effects. A suitable combination of a number of molecular
markers can give a refined picture of the possible adverse effects and the mechanisms.
At the other extreme in vivo tests on live animals, e.g. rats are also models with
limitations: a rat can never replicate the complex response of humans. An efficient
assessment strategy, preferably a tiered assessment, has to be applied to obtain the
correct answer concerning the extent and quality of adverse effects and the subsequent
risks. An efficient assessment based on optimized algorithms can provide a better and
more precise answer at lower cost compared to traditional tests. For example, applying
non-expensive and sensitive screening tests prior to animal tests makes it possible to
exclude negative substances from further testing. In silico QSAR models (Quantitative
Structure–Activity Relationship) are advantageous in tiered assessments as a screening
tool both in human- and ecotoxicology. Toxicology and ecotoxicology are ready to
fertilize each other, and their methodological tools are available to establish the uni-
form science of environmental toxicology. However, traditions, current routines and
the different trends of the professional branches may raise obstacles which must be
dealt with.

1.6 Animal studies

Animal experiments are common in physiological studies for the development of new
chemicals or medicines, for studying environmental effects or testing new food addi-
tives. The protection and welfare of animals is covered by a number of international
treaties and other legal instruments. These refer to wildlife, zoo and farm animals,
animals in transport and also to animals used in scientific experiments. EU legislation
on the protection of animals used for experimental and other scientific purposes is
covered by Directive 86/609/EEC (EEC, 1986).
The Treaty of Amsterdam (ToA, 1997) requires the EU and its Member States
to take animal welfare considerations into account in a number of policy areas and
also in practice by developing and using alternative test methods instead of animal
experiments.
US legislation on laboratory animals is based to a large extent on the international
guidelines about the use of humans in research and testing. The basis of these guidelines
is the Nuremburg Code, a list of criteria developed for judging if the tools applied by
the Nazis in World War Two constitute crimes ‘against humanity’.
The most pragmatic approach to reduce experiments on animals is the replacement
of animal tests with non-animal ones. Whenever replacement is not possible, all efforts
should be made to apply those methods which use a smaller number of animals and
cause least harm to them. Animal tests can be replaced by in vitro methods which use
living cells or tissue cultures, or with mathematical models such as QSAR.
The EU started a program in 2010 for the minimization and possible exclusion of
higher animals from toxicity and other adverse-effect tests and for the development of
alternative test methods, using microbes, single-cell animals, tissue cultures, isolated
organs from slaughterhouse animals, or applying in vitro techniques and in silico
methodologies. Many of these methods are identical to or very closely related to the
methods used in environmental toxicology.
 Environmental toxicology – A general overview 19

The European Chemicals Agency (ECHA, 2013) prepared a Practical Guideline

entitled How to avoid unnecessary testing on animals (ECHA, 2010). Clearly, it takes
time to replace all animal tests with new alternative tests which completely avoid the
use of animals. According to the Practical Guideline, there are some other options
which can reduce animal testing for the purposes of regulation of chemicals in the
transitional period:

– Data sharing: the REACH test data can be shared between registrants to avoid
unnecessary tests on animals. This means that use of studies conducted by one
registrant on vertebrate animals can be shared by all registrants for that substance.
– Gathering information before test planning, looking for existing data in all pos-
sible sources, acquiring data on analogous substances if ‘read across’ is possible,
using QSAR and SAR estimates; weight-of-evidence approach to fill data gaps for
particular end points if this is appropriate.
– Strategies to avoid unnecessary tests on animals:

◦ In vitro methods: in vitro tests performed in a controlled environment such

as a test tube or Petri dish, without using a higher living organism. A test
performed in vivo is one that is carried out on a living organism. Results
obtained from suitable in vitro methods may indicate the presence of certain
dangerous properties or may be important in relation to understanding the
mode of action of the substance. In this context, ‘suitable’ means sufficiently
well-developed according to internationally agreed test development criteria.
◦ Grouping of substances and read across: animal tests on a substance can be
avoided if there is enough evidence on similar substances and can be read
across between the two substances. Substances whose physicochemical, tox-
icological and ecotoxicological properties are likely to be similar or follow
a regular pattern as a result of structural similarity, may be considered as a
group, or category of substances. Applying the group concept means that the
physicochemical properties, human health effects and environmental effects
or environmental fate may be predicted from data for one substance within the
group by interpolation to other substances in the group; this is the read-across
approach. This avoids the need to test every substance in the group for every
hazard end point. Preferably, a category should include all similar substances.
◦ Quantitative Structure–Activity Relationship (QSAR) models: animal tests can
be avoided if the hazardous properties of a substance can be predicted using
computer models. The (QSAR and SAR approach seeks to predict the intrin-
sic properties of chemicals by using various databases and theoretical models,
instead of conducting tests. Based on knowledge of the chemical structure,
QSAR quantitatively relates the characteristics of the chemical to a mea-
sure of a particular activity. QSAR should be distinguished from SAR, which
makes qualitative conclusions about the presence or absence of a property of
a substance, based on a structural feature of the substance.
◦ Weight-of-evidence approach: animal tests can be avoided if there is a weight
of evidence which points to the likely properties of a substance. This approach
may be applied if there is sufficient information from several independent
sources leading to the conclusion that a substance does or does not exhibit a
20 Engineering Tools for Environmental Risk Management – 2

particular dangerous property, while the information from each single source
alone is regarded as insufficient to support an assertion.
– Another possibility to reduce animal testing is eliminating duplication and utilizing
scientific evidence from other sources. An obvious negative case is where no irrita-
tion or corrosion has been observed in an acute dermal toxicity test. In such cases
no additional dermal irritation/corrosion test should be carried out. The positive
evidence can also be utilized; a known irritant/corrosive substance, e.g. a strong
acid or base, or a substance flammable in air at room temperature should not be
tested for skin irritation/corrosion, but classified without animal testing. Low Kow
organic substances can also be classified as non-bioaccumulative without animal
testing.

The NTP Interagency Center for the Evaluation of Alternative Toxicological Methods
(NICEATM, 2013) and the Interagency Coordinating Committee on the Validation
of Alternative Methods (ICCVAM, 2013) define the alternatives to animal tests in
a simpler way, which is called the 3R approach, including Reducing, Refining and
Replacing animal use in toxicity testing (NICEATM/ICCVAM, 2013):

– Reducing the number of animals used to the minimum number required to obtain
scientifically valid data.
– Refining procedures to lessen or eliminate animal pain and distress.
– Replacing animals with non-animal systems or one animal species with a less highly
developed one (for example, replacing a mouse with a fish).

1.7 In vitro contra in vivo: alternative test methods

ACuteTox (2005–2010) was an integrated project in the EU between 2005 and 2010,
which aimed to develop and pre-validate a simple and robust in vitro test strategy for
the prediction of acute human toxicity.
The main objectives – as indicated on the website of the project (ACuteTox, 2005–
2010) – are ‘the compilation, evaluation and generation of high-quality in vitro and
in vivo data on a set of reference chemicals for comparative analyses, and the identi-
fication of factors that influence the correlation between in vitro (concentration) and
in vivo (dose) toxicity, particularly taking into consideration biokinetics, metabolism
and organ toxicity (liver, central nervous system and kidney). Moreover, innovative
tools (such as cytomics) and new cellular systems for anticipating animal and human
toxicity were explored. Ultimately, the goal was to design a simple, robust and reli-
able in vitro test strategy amendable for robotic testing, associated with the prediction
models for acute oral toxicity.’
Collected data and studies by ACuteTox have shown good correlation between in
vitro basal cytotoxicity data and rodent LD50 values and, in addition, a good corre-
lation (around 70%) was found between in vitro basal cytotoxicity data and human
lethal blood concentrations. Based on these results, the ACuteTox project is divided
into the following scientific work packages (Anonymous, 2006):

– Generation of high quality in vivo and in vitro databases (which currently contain
LD50 values from 2206 animal studies and human data from 2902 case reports);
 Environmental toxicology – A general overview 21

– Iterative amendment of the test strategy, including adapting various cell lines to
commercially available high-throughput robotic platforms;
– New cell systems and new end points, such as in vitro production of cytokines in
whole human blood cultures;
– Role of kinetics (ADE = absorption, distribution and elimination), including fur-
ther evaluation of in vitro and in silico models for gut and blood–brain-barrier
passage;
– Role of metabolism, including further evaluation of a variety of metabolically
active cell systems;
– Role of target organ toxicity, with an emphasis on neuro-, nephro- and hepato-
toxicities;
– Technical optimization of the amended test strategy;
– Pre-validation of the test strategy.

At the same time, the pharmaceutical acute toxicity working group expressed its
opinion on acute (animal) toxicity results: ‘the information obtained from conventional
acute toxicity studies is of little or no value in the pharmaceutical development process’.
Based on this, the ACuteTox project completed quantitative surveys and summarized
the required expert opinion (Seidle, 2007; Chapman & Robinson, 2007; NC3Rs,
2007):

– 100% of respondents found data from acute toxicity studies of little or no use
and only used the information in dose setting for other studies in exceptional
circumstances;
– 100% of respondents agreed that they would not carry out acute toxicity testing
if it were not a regulatory requirement;
– 100% of respondents agreed that acute toxicity studies were not used to identify
target organs;
– 100% of respondents never use acute toxicity data to help set the starting dose in
man;
– 81% of respondents thought the data obtained from acute toxicity studies was of
no use to regulators or clinicians.

In addition to the above opinions, the time requirement, the costs and the ethical
issues together urge the scientific community to find a solution for the replacement of
animal testing with in vitro and in silico methods, and develop such a testing strategy
where animal tests can be reduced to a minimum. Part of this work should be education
and development and convincing experts that the traditional toxicity tests, in spite of
their long history, are not the best possible methods for toxicity and, in particular, not
for acute toxicity testing.
AltTox summarized arguments against animal tests as follows (AltTox, 2009):

– Testing methods have not kept pace with scientific progress: today, there has been a
revolution in biology and biotechnology and these developments and opportunities
are not reflected by the traditional animal testing methods. Emerging technologies
such as bioinformatics, genomics, proteomics, metabolomics, systems biology, and
in silico (computer-based) systems offer more potential alternatives to animal use.
22 Engineering Tools for Environmental Risk Management – 2

In vitro methods are recommended to use cells, cell lines, or cellular components,
in the case of human toxicology, preferably of human origin. The new approach
would generate more relevant data, expand the number of chemicals that could
be scrutinized, and reduce time, money, and animals involved in testing.
– Questionable reliability and relevance of current testing methods, using animals. It
is recognized that different species may respond differently to the same substance.
It is impossible to know whether the results of testing on rodents, rabbits, or dogs
will provide an accurate prediction of toxic effects in humans. There is also much
debate concerning the relevance of extrapolating from high doses administered to
animals to realistic human or environmental exposure levels. The chronic testing,
which goes on partly on elderly animals, may lead to an undue overestimate of
toxicity. Despite efforts to standardize procedures, the results of some animal tests
can be highly variable and difficult to reproduce.
– Animal welfare considerations also play an important role when stressing alter-
natives. Some conventional toxicity test methods consume hundreds or thousands
of animals per substance examined. Toxicity testing causes pain, mainly because
evaluation is to be done near death.
– Time and cost of conventional tests. These take months or years to conduct and
analyze (4–5 years, in the case of carcinogenicity studies) at a huge cost.
– Legal obligations prohibit testing on animals where alternative methods are
reasonably and practicably available (EEC, 1986).

Computational toxicology is a realistic means; its validity depends on the number

and quality of existing data. A number of (quantitative) structure–activity relationships
(QSAR and SARs), expert systems, and other in silico models have been developed for
the prediction of various toxicity values. For example, the TOPKAT (Accelrys Inc.,
Cambridge, UK) models of oral LD50 and inhalation LC50 in rats and mice have been
constructed based on published experimental toxicity data for thousands of substances.
Regulatory authorities in Canada, Denmark and the US have reported extensive use
of QSAR and SAR models as a basis for interim regulatory classifications of exist-
ing and/or new chemicals (Zeeman et al., 1995; Danish EPA, 2001; Cronin et al.,
2003; Meek, 2005). The Danish EPA has published statistical results on the degree of
accuracy of QSARs of 70–85%, depending on the model used (Danish EPA, 2001).
The OECD also has a project on QSAR development, for estimating the properties
of a chemical from its molecular structure and has the potential to provide information
on hazards of chemicals, while reducing the time, monetary cost and animal testing
currently needed. The aim of the project is to facilitate practical application of QSAR
and SAR approaches in regulatory contexts and to improve their regulatory accep-
tance. The OECD QSAR and SAR project has developed various outcomes such as the
principles for the validation of QSAR and SAR models, guidance documents as well
as the QSAR Toolbox.
The QSAR Toolbox is a software application designed for governments, industry
and other stakeholders to fill the gaps in (eco)toxicity data needed for assessing the
hazards of chemicals. This new document specifically provides guidance on how to use
the Toolbox to group chemicals to fill data gaps for genotoxicity and carcinogenicity
of chemicals (OECD Assessment of Chemicals, 2013c).
 Environmental toxicology – A general overview 23

1.8 Evidence-based toxicology

The new and quick alternative test methods result in numerous toxicological data
in addition to the already existing huge amount. A toolbox for the validation of
toxicological methods and their results is essential for further development to reach
a more dynamic toxicity measuring tool both in human and environmental fields
(Hartung, 2010; 2012). This validation concept is also called evidence-based toxi-
cology (along the lines of evidence-based medicine) and may be defined as all available
data and information is collected from different sources and evaluated and reviewed in
a systematic way. It makes possible both the retrospective validation of traditional ani-
mal studies and innovative non-animal test methods (OECD, 2005). It makes testing
much quicker and less expensive than the traditionally used methodology for measuring
and evaluating toxicity. Evidence-based toxicology can be applied for regulatory pur-
poses and for the evaluation of actual environmental exposures endangering humans
and the ecosystem.
The first summary of evidence-based toxicology was established at a work-
shop titled “21st Century Validation for 21st Century Tools’’ (Rudacille, 2010).
It was followed by the formation of the Evidence-Based Toxicology Collaboration
(EBTC, 2014). The EBTC’s EU branch was officially opened during the 2012 Eurotox
conference (Hoffmann, 2012).

2 ADVERSE EFFECTS TO BE MEASURED BY

ENVIRONMENTAL TOXICOLOGY

Adverse effects of chemical substances are effects which endanger the health and
integrity of living organisms and their communities over the short or long term.
Environmental toxicology is the main pillar of environmental policy, and regulates
the production, transport and use of any hazardous chemical substances all over the
world, whether they are industrial, agricultural or pharmaceutical substances, pesti-
cides, cosmetics or food additives. But adverse effects are much more numerous and
complex than those that are covered by regulations today.
Chemical substances with a specified acute effect – which directly affects the mem-
bers of an ecosystem – may have the same or different chronic effects arising only over
the long term. It can appear in the same member of the ecosystem, in other members
of the ecosystem or in the whole ecosystem, including humans. This means that quan-
titative and qualitative changes are going on in the ecosystem: change in the structure,
in species diversity, in food chains or food webs. The final output can be the change
in the function and productivity of the whole ecosystem, influencing the environment
regionally or globally.
Which of the many complex, often unknown ecosystem characteristics should
be measured, which changes should be monitored? How can the most characteristic
ones be found? Who knows the key points? The answer is that our knowledge is very
moderate in this field, the situation is somewhat similar to climate change: we do not
understand the system in its totality and the changes over the long term. We cannot
decide whether or not short-term or longer-term trends have the same direction, are
they part of the normal global ecological trends, and, in addition to the normal trends,
24 Engineering Tools for Environmental Risk Management – 2

does the scale of the changes endanger global equilibrium? Which changes are ‘good’
and which are ‘bad’? On which time scale? And for whom? Human beings tend to
place themselves on the top as the most important species, but the local and short-term
interest of humans and mankind is not the same as the long-term interest of the world,
the habitat of humans and ecosystem. The answer to these questions is as complicated
as finding the answer to the main question of humanity: why are humans on the earth?
We can only hope that we do not destroy it.

2.1 Hazardous effects of chemical substances

In spite of the European REACH and all other regulations such as pesticide, biocide,
cosmetics, and pharmaceuticals regulation all over the world, there are many chemical
substances used in large quantities without any control. None of the petroleum prod-
ucts are managed using a risk-based approach, and in most of the relevant regulations
and standards, no composition is specified for oils, fuels and other petroleum-based
products in terms of their chemical composition and hazardous properties.
Wastes are also managed without knowing their chemical composition, in spite of
the well-known fact that adverse effects originate from certain chemical substances.
Industrial waste is managed in view of its origin, without taking into consideration
its exact composition. A good example is the red-mud catastrophe in Hungary in
2010: red mud, the residue of bauxite processing was not considered hazardous waste
because there was no differentiation between wet storage of the highly alkaline sodium
hydroxide (NaOH) solution and dry storage after neutralization and desiccation. If
the chemical composition had been the basis of evaluation, red mud should have been
considered as a mixture of sodium hydroxide, a highly corrosive chemical substance
and the residue from aluminum ore, with a specific, in many cases harmless, mineral
composition.
Household waste is also misjudged when treated as chemically neutral and handled
as something with low chemical hazard (i.e. only its hygienic hazard is being consid-
ered). What a misunderstanding! Batteries, paints, metal tools, electronic equipment,
plastics and everything which is collected non-selectively is accumulated in house-
hold garbage. The best recycling performance from households is 30% in the most
environmentally conscious countries.
From the unduly underestimated risk of non-regulated or not risk-based regulated
hazardous substances, we jump now to substances with known hazards. The necessary
use of hazardous substances needs adequate control to manage and reduce their risk
to an acceptable level.
The hazards of chemical substances due to their fate and behavior in the environ-
ment and their adverse effects on ecosystems and humans will be summarized here.
Hazardous chemical substances and their environmental risks have been reviewed in
Chapter 2 in Volume 1.
Pesticides and biocides, aimed at killing a target organism, and cosmetics,
which are in regular and intensive contact with humans, will be discussed from the
point of view of environmental risk. Food ingredients and additives are also impor-
tant chemical substances and regulated outside REACH by food-centered European
regulations.
 Environmental toxicology – A general overview 25

GHS classifies the adverse effects into 15 classes whose most general and typical
toxic effects in or through the environment will be discussed here:

– Acute toxicity;
– Skin corrosion;
– Skin irritation;
– Eye effects: irritation and corrosion;
– Sensitization;
– Mutagenicity;
– Carcinogenicity;
– Reproductive toxicity;
– Target organ systemic toxicity: single exposure and repeated exposure;
– Aspiration toxicity;
– Hazardous to the aquatic environment;
– Acute aquatic toxicity;
– Chronic aquatic toxicity.

REACH and GHS do not aggregate all existing hazards due to the adverse effects of
chemical substances. Some others, which may play an important role in environmental
and human health risk, are listed here:

– Acute toxicity on the terrestrial ecosystem;

– Chronic toxicity on the terrestrial ecosystem;
– All kinds of toxicity on a single species of wildlife;
– Phototoxicity;
– Endocrine disruption;
– Immune system disruption;
– Nervous system disruption.

2.2 Toxic effects of chemical substances

Toxicity or toxic effect is an adverse effect on the health of humans, wildlife or entire
ecosystems. Chemical substances with adverse effects may be synthetic xenobiotics or
natural chemical substances of mineral, organic or living origin.
The term toxic is used in a broad sense for most of the adverse effects, includ-
ing acute and chronic toxicity, reprotoxicity, immunotoxicity, neurotoxicity, evolving
through inhalation, digestion, dermal contact or whole body contact.
Human toxicity (discussed in Chapter 3) is traditionally based on animal tests.
Ecotoxicity deals with the effects of toxic chemicals on ecosystem health and on
single ecosystem members. Environmental toxicity integrates both human and ecotox-
icity characterized by qualitative and quantitative tools and considers the interactions
between the two.
According to Paracelsus’ theory, toxicity is a dose-related phenomenon, and up to
the present time, we define toxicity and the scale of toxicity based on limit doses or
concentrations.
The consequences of classification of a chemical substance as hazardous are greater
than just a hazard label; it also has a direct effect on the management of associated risks.
26 Engineering Tools for Environmental Risk Management – 2

Table 1.1 Toxic classes according to ED50 and EC50 upper limit values (GHS, 2007).

Exposure route Category 1 Category 2 Category 3 Category 4 Category 5

Oral: LD50 mg substance/kg 5 50 300 2000 5000

of bw
Dermal: LD50 mg substance/kg 50 200 1000 2000 5000
of bw
Gas inhalation: LC50 mg/kg 100 500 2500 20,000 No
(in volume)
Vapor inhalation: 0.5 2.0 10 20 No
LC50 mg/L
Dust and mite inhalation: 0.05 0.5 1.0 5.0 No
LC50 mg/L

No: no classification; bw: body mass

Classification and labeling of chemicals is regulated globally based on their hazard, in

a harmonized way (GHS, 2007).
According to the definition of REACH, a substance fulfills the toxicity criterion
when:

– an acute lethal effect may arise following oral, dermal or inhalation exposure, and
is classified as toxic;
– a substance is classified as carcinogenic, mutagenic, or toxic for reproduction;
– there is evidence of chronic toxicity; its short-term or long-term toxicity for
marine or freshwater organisms is under the threshold of EC50 = 0.1 mg/L and
the NOEC = 0.01 mg/L respectively.

Table 1.1 shows the doses and concentrations of toxic substances, which cause an
LD50 or LC50 value, namely the dose or concentration that kills half of the affected
organisms. Table 1.2 shows the toxicity criteria for the water ecosystem.
As can be seen in Table 1.1, toxicity is split into five categories of severity where
Category 1 requires the least amount of exposure and Category 5 requires the greatest
exposure to be lethal, making it the least dangerous category.
EC50 is determined from short-term aquatic toxicity data of key indicator species
such as fish, crustaceans and algae or aquatic macroplants, and the lowest-effect con-
centration is selected and compared with the concentrations in Table 1.2. Fish are
exposed for 96 h and crustaceans for 48 h and the results for algae are given in Er C50 ,
based on the reproduction (growth) curve of the algal culture. NOEC or EC10 val-
ues from long-term toxicity tests are used to determine chronic aquatic toxicity for
classification purposes.
The European CLP criteria take also degradability into account when categorizing
aquatic toxicity: chronic exposure to a readily biodegradable substance is categorized
less strictly, but for those substances for which adequate chronic toxicity data are
not available the categorization is stricter: EC50 ≤ 1 mg/L belongs to Cat.1 (similar to
categorization based on acute toxicity).
Photodegradation and hydrolysis are generally known physicochemical phenom-
ena, but biodegradability should be measured in standardized test methods. Substances
 Environmental toxicology – A general overview 27

Table 1.2 Toxic to aquatic ecosystem: categories based on EC50 , Er C50 or NOEC values (CLP, 2008).

Exposure Category 1 Category 2 Category 3

Acute (mg/L) ≤1.0 – –

Chronic, non-rapidly degradable (mg/L) ≤0.1 ≤1.0 ≤10.0
Chronic, rapidly degradable (mg/L) ≤0.01 ≤0.1 ≤1.0
Chronic, adequate chronic toxicity data are not ≤1.0 ≤10 ≤100
available (mg/L): non-rapidly degradable, BCF > 500,
acute toxicity results are available

are considered rapidly degradable in the environment if at least the following level of
degradation is achieved within 10 days of the start of degradation in a 28-day ready
biodegradation study:

– 70% in tests based on dissolved organic carbon;

– 60% of the theoretical maximum in tests based on oxygen depletion or carbon
dioxide generation.

Bioaccumulation of substances within aquatic organisms can give rise to toxic

effects over longer time scales even when actual water concentrations are low. For
organic substances the potential for bioaccumulation will normally be determined
by using the octanol–water partition coefficient, Kow . A cut-off value of log Kow ≥ 4
identifies the substances with a real potential to bioconcentrate. Of course an experi-
mentally determined BCF provides a better measure. A BCF of ≥500 in fish is indicative
for classification purposes.
The European CLP introduces a “safety net’’ classification (category Chronic 4)
for use when available data do not allow classification but there are some grounds for
concern.
Category 1 substances with acute or chronic aquatic toxicity contribute as com-
ponents of a mixture to the toxicity of the mixture significantly. This increased weight
is considered by a multiplying factor, called M-factor.
LC50 and NOEC values of chemical substances and their hazard-based classifi-
cation can be used for the risk management of chemicals when their environmental
concentration is also known.
When testing environmental samples and not pure chemical substances, the quali-
tatively measured toxicity directly indicates the extent of the risk of the environmental
sample to the test organism, even if the chemical composition and concentration of the
contaminants is not known. Extrapolation from single species to the whole ecosystem
is also possible by using uniform testing and extrapolation methodologies.
Although the actual contaminants have not been identified and their concentra-
tion in environmental samples is not known, the no-effect dose or ED50 value can be
determined for water, soil or sediment samples, which gives the amount of sample in
grams or liters that caused 50% lethality or a 10%, 20%, or 50% decrease in any mea-
sured end points. This measured effective or no effect dose is methodology-dependent
because only 1 g of sample is used in one test, but 5 or 50 g is used in another. These
28 Engineering Tools for Environmental Risk Management – 2

kinds of test results need further interpretation for decision making. The basis of inter-
pretation can be a kind of ‘calibration’ of the test method by comparing the toxicity
of the environmental sample with the toxicity of known substances. As a result of this
kind of quantitative calibration, the effective water or soil dose (ED) can be expressed
as a substance-equivalent toxicity in concentration and as such, can better fit into a
conventional risk assessment procedure. For example, the toxicity of soil contaminated
with a mixture of metals is given in copper equivalents as if copper caused the entire
toxicity. Another type of interpretation for toxicity results of environmental samples
is verbal characterization of toxicity according to the scale of inhibition, for example,
very toxic, toxic, moderately toxic and non-toxic. A third plausible mode for char-
acterizing the measured toxicity of environmental samples is the necessary rate for
achieving a “no effect’’ dilution. This interpretation is a good indication for the scale
of the necessary risk reduction to achieve a “no risk’’ situation in the environment.
Xenobiotics are substances foreign to an entire biological system. Their interaction
with living organisms may lead to the impairment of the organisms’ normal function
and health. Xenobiotics can be metabolized and secreted from the body, but they
may be bound to essential molecules, enzymes or receptors of the cells and organs,
resulting in damage to the genetic, biochemical, physiological, or behavioral soundness
of the exposed organism. Organisms can adapt to xenobiotics; they (first of all the
microbes) can learn to utilize, eliminate, tolerate or resist xenobiotics. The mechanism
of xenobiotic metabolism is important both in medical practice and in the environment,
and indicators such as glutathione S-transferases can be used for the exploration of
both drug effect mechanisms and pesticide resistance.
Mixtures of chemical substances may interact with each other either in the environ-
ment or in the body. Their interaction may result in changes in the common adverse
effect which may be additive, synergic or antagonistic. When estimating the risk of
mixtures, it is necessary to know the type of interaction of the chemicals present.
Not only the effect of different chemicals but also the intakes through different
exposure routes may be added together. A person who is exposed to contaminated
air, contaminated water and soil at the same time, may inhale contaminating chemical
substances, take them in orally or through his skin. Terrestrial and aquatic plants
and animals live in the contaminated environmental matrix and are in contact with it
through their whole body, digestive system and inhalation. In this case the response
includes all exposures via different routes.
The details of the realization of the toxic effect are explored by toxicology,
ecotoxicology and toxicokinetics.
Toxicokinetics studies the uptake and the further fate and interactions of the toxic
substances in the organism’s body, organs or cells, while toxicology mainly deals with
the result, the end point of adverse effects and the dose–response relation. The steps of
toxicokinetics are absorption, distribution, biotransformation and excretion. Absorp-
tion describes the entrance of the chemical substance into the body, through the air,
water, food, or soil. Once a chemical is inside a body, it is distributed to other areas
of the body through diffusion or biological transport. The chemical substance may be
biotransformed into other chemicals; these are metabolites. These metabolites can be
more toxic than the parent compound. After biotransformation, the metabolites may
leave the body, may be transformed into other compounds, or continue to be stored
in the body compartments. In many cases, the presence of the toxic substance is not
 Environmental toxicology – A general overview 29

a simple storage, but it may act biologically, evoking a false biological response of
the endocrine, immune or nervous systems. Toxicokinetics tries to find relationships
between exposures in animal toxicity tests and the corresponding exposures in humans.
Some other branches of toxicology, such as genetic toxicology, reproductive, endocrine,
immune and neurotoxicology will be investigated in detail under human toxicology.
Toxicogenomics is a new branch of toxicology, dealing with the collection, inter-
pretation, and storage of information about the response of genes and proteins within
the cell, tissue or organ of an organism when affected by a toxic substance. It com-
bines toxicology with genomics, and other closely related new molecular technologies
such as transcriptomics, proteomics and metabolomics. Toxicogenomics endeavors to
explore the molecular mechanisms of toxic effects and to find connections between
toxic effects and molecular biomarkers, which would indicate toxic effects at an early
stage, making early warning possible in both medical and environmental practice.
Ecotoxicogenomics is the challenge of integrating genomics into aquatic and
terrestrial ecotoxicology. Snape et al. (2004) formulated ecotoxicogenomics and its
importance in understanding the response of ecosystems and proposed the term eco-
toxicogenomics to describe the integration of genomics (transcriptomics, proteomics
and metabolomics) into ecotoxicology. Ecotoxicogenomics is defined as the study of
gene and protein expression in non-target organisms that is important in responses to
environmental toxicant exposures. Instead of whole-organism responses (e.g. mortal-
ity, growth, reproduction), we can use a much more sensitive indicator for identifying
chemicals of potential toxicants. To find the proper molecule to indicate the toxic
potential of contaminating substances, we have to understand the effect mechanisms
and the interactions between ecological levels and in the population. Snape et al.
(2004) hope that ecotoxicogenomic tools may provide us with a better mechanistic
understanding of aquatic ecotoxicology.
Microbial ecology and handling the microflora of water, soil, sediment or sludge as
a community with a metagenome may help in understanding and following the differ-
ences and changes due to environmental conditions and differentiate spatial, climatic
and seasonal changes from the adverse effects of contaminants.
Microbial species and communities have specific functions in ecosystems, in
nutrient cycling and contaminant elimination. Identification of microorganisms by tra-
ditional microbiological methods is often difficult because only a minority of the species
within the system will grow on artificial media. DNA techniques help to overcome this
problem and to extend our knowledge on species diversity because non-cultivable
species are also detected.
Soil microbial ecology has already found some interesting relationships, for exam-
ple, the relative stability of the metagenome of soil microflora was found to be unrelated
to temperature or latitude, which strongly determines plant or animal diversity. The
most influential factors in soil are pH, redox potential, available energy substrates and
contaminants (Jeffery et al., 2010).
The study of the metagenome needs special molecular techniques, particularly
DNA techniques, an adequate amount of historical or background data and a suitable
statistical tool for the evaluation.
Metagenomics, in short, is the science of biological diversity. Technically, it is the
study of nucleotide sequences, structure, regulation and function within the complex
ecosystem. An environmental system such as the soil can be characterized by the DNA
30 Engineering Tools for Environmental Risk Management – 2

from all of the organisms living in the community. We can extract total DNA and
study it directly or after cloning it into a host cell, for example Escherichia coli, to
express the information coded in the cloned metagenome in the form of DNA (dur-
ing propagation of E. coli) or in the form of RNAs and proteins (during the life of
the cells). We can create a genetic library from the metagenome of the soil, and ana-
lyze it as a whole (characteristic DNA fingerprint), based on similarity (comparison
with known fingerprints) or for the identification of specific genes, their presence and
frequency.
Metagenomics represents a powerful tool to qualify and quantify the biodiversity
of native environmental samples. It can characterize the genetic diversity in samples
regardless of the availability of laboratory culturing techniques, or knowledge of the
microorganisms present. The metagenomic DNA from the library, prepared for exam-
ple from soil, can be analyzed by PCR techniques, using DNA probes and DNA chips or
by the newly developed technique of DNA-SIP (Stable Isotope Probing). The problem
of evaluation and interpretation may be the same as in the case of diversity assessments,
namely what should be considered as reference. The advantage of metagenomics is that
the number of tests is not limited, so one can generate temporal and spatial series for
identifying concentration-dependence, trends and gradients.
The DNA–SIP method is based on the utilization of a substrate labeled with
stable isotope of carbon (13 C). Those cells that are active and able to utilize the
13
C-labeled substrate and proliferate, build the carbon isotope into their DNA, which
is easily detectable after isolating the metagenome. The food chains or other trophic
communities can also be mapped by this method.
Metatranscriptomics are the gene products of the metagenome, namely DNAs or
RNAs synthesized from genes and intergenetic regions of DNA. Metatranscriptomics
offers the opportunity to reach beyond the community’s genomic potential as assessed
in DNA-based methods, towards its in situ activity, meaning not only the presence but
also the activity (transcription) of the genes.
Analyses by Shi et al. (2009) showed that metatranscriptomic data sets can
reveal new information about the diversity, taxonomic distribution and abundance of
small RNAs (sRNAs) in naturally occurring microbial communities and indicate their
involvement in environmentally relevant processes including carbon metabolism and
nutrient acquisition. In their published research, a large fraction of cDNA sequences
(DNA synthetized in vitro from messenger RNA) were detected, which are partly
identical to well-known sRNA, and partly to new groups of previously unrecognized
putative sRNAs (psRNAs). These psRNAs mapped specifically to intergenic regions of
microbial genomes recovered from similar habitats, displayed characteristic conserved
secondary structures and were frequently flanked by genes that indicated potential reg-
ulatory functions. Depth-dependent variation of psRNAs generally reflected known
depth distributions of broad taxonomic groups, but fine-scale differences in the psR-
NAs within closely related populations indicated potential roles in niche adaptation.
Metaproteomics, also called environmental proteomics, or community proteoge-
nomics, is based on the analyses of expressed proteins by the metagenome. The proteins
are extracted from the soil, then analyzed by mass spectrometry after electrophoretic
or chromatographic separation. Metaproteomics supports the understanding of the
microbial communities and the relation of community composition to its function.
Schulze et al. (2005) applied mass spectrometry-based proteomics to analyze pro-
teins isolated from Dissolved Organic Matter (DOM) from soil. The focal question
 Environmental toxicology – A general overview 31

was to identify extracellular enzymes important in the carbon cycle. Proteins were
classified using the National Center for Biotechnology Information (NCBI, 2013) pro-
tein and taxonomy database. They found that 78% of proteins in a lake but less than
50% in forest soil DOM originated from bacteria. In a deciduous forest, the number of
identified proteins decreased from 75 to 28 with increasing soil depth and decreasing
total soil organic carbon content. The number of identified proteins and taxonomic
groups was 50% higher in winter than in summer. Cellulases and laccases were found
among proteins extracted from soil particles, indicating that degradation of soil organic
matter takes place in biofilms on particle surfaces. These results demonstrate that the
‘proteomic fingerprint’ is suitable to prove the presence and activity of organisms in
an ecosystem.

2.3 Carcinogenic effects

CMR substances are chemical substances with carcinogenic, mutagenic or toxic effects
to reproduction. The abbreviation CMR is used for the characterization of chemical
substances with these properties within the REACH regulation.
A carcinogenic effect may be caused by a substance or a mixture of substances
that induces cancer or increases its incidence and/or malignancy, or shortens the time
to tumor occurrence. Cancer may stem from the ability to damage the genome or to
disrupt the cellular metabolic processes. Carcinogenic chemicals are conventionally
divided into two categories according to the assumed mode of action. Non-genotoxic
modes of action include epigenetic changes, i.e. effects that do not involve alterations
in DNA but which may influence gene expression, altered cell–cell communication, or
other factors involved in the carcinogenic process.
Cancer is a disorder of the cells, characterized by the lack of programmed cell
death, which is responsible for destroying damaged cells. If the pathway is damaged,
the cell cannot prevent itself from becoming a cancer cell. Carcinogens induce the
uncontrolled, malignant division of cells, ultimately leading to the formation of tumors.
The objective of investigating the carcinogenicity of chemicals is to identify potential
human carcinogens, their mode(s) of action, and their potency.
Once a chemical has been identified as a carcinogen, there is a need to elucidate
the underlying mode of action, i.e. whether the chemical is directly genotoxic or not.
For genotoxic carcinogens, it is assumed that usually there is no discernible threshold
and that any level of exposure carries a risk. For non-genotoxic carcinogens, no-effect
thresholds are assumed to exist and to be discernible. Human studies are generally
not available to make a distinction between the above-mentioned modes of action,
and a conclusion on this, in fact, depends on the outcome of mutagenicity testing and
other mechanistic studies. In addition to this, animal studies may also inform on the
underlying mode of carcinogenic action.
GHS classifies carcinogens into two categories, of which the first may be divided
again into subcategories if so desired by the competent regulatory authority:

– Category 1: known or presumed to have carcinogenic potential for humans;

◦ Category 1A: the assessment is based primarily on human evidence;
◦ Category 1B: the assessment is based primarily on animal evidence;
– Category 2: suspected human carcinogens.
32 Engineering Tools for Environmental Risk Management – 2

The cancer hazard and mode of action may also be highly dependent on expo-
sure conditions such as the route of exposure. Therefore, all relevant effect data and
information on human exposure conditions are evaluated.
A list of carcinogenic substances is given in Chapter 1.2 in Volume 1.

2.4 Mutagenic effects

Mutagenic substances are those which induce mutations in living cells. Mutagenicity
refers to the induction of permanent transmissible changes in the amount or structure
of the genetic material of cells or organisms. These changes may involve a single gene
or gene segment, a block of genes or chromosomes.
Alterations to the genetic material of cells may occur spontaneously or be induced
as a result of exposure to ionizing or ultraviolet radiation or genotoxic chemical sub-
stances. In principle, human exposure to substances that are mutagens may result in
increased frequencies of mutations above the baseline. Heritable damage to offspring,
and possibly to subsequent generations, of parents exposed to substances that are
mutagens may follow if mutations are induced in parental germ cells (reproduction
cells). Mutations in somatic cells (cells other than reproduction cells) may be lethal
or may be transferred to daughter cells with deleterious consequences for the affected
organism. There is considerable evidence of a positive correlation between the muta-
genicity of substances in vivo and their carcinogenicity in long-term studies on animals.
The aims of testing for mutagenicity are to assess the potential of substances to induce
effects, which may cause heritable damage in humans or lead to cancer.

2.5 Reprotoxicity
Reprotoxics are chemical substances which may cause reproductive toxicity. Reproduc-
tive toxicity by the definition of REACH includes adverse effects on sexual function and
fertility in adult females and males as well as developmental toxicity in the offspring:

– Adverse effects on sexual function and fertility;

– Adverse effects on development;
– Adverse effects on or via lactation.

Reproductive toxicity is clearly of great concern because the continuance of the

human species is dependent on the integrity of the reproductive cycle. It is characterized
by multiple diverse end points, such as impairment of female and male reproduc-
tive functions or capacity (fertility), induction of non-heritable harmful effects on the
progeny (developmental toxicity) and effects on or mediated via lactation.
The objectives of assessing reproductive toxicity are to establish:

– Whether exposure of humans to the substance of interest has been associated with
reproductive toxicity;
– Whether, on the basis of information other than human data, it can be predicted
that the substance will cause reproductive toxicity in humans;
– Whether the pregnant female is potentially more susceptible to general toxicity;
– The dose–response relationship for any adverse effects on reproduction.
 Environmental toxicology – A general overview 33

A complete list of the EU-classified substances is given in Annex VI of the CLP

regulation (CLP, 2008b).

2.6 Persistent and very persistent substances

Persistent and very persistent substances are substances that persist in the environment
for a long time. The reason for persistence is that the substance cannot be degraded
by light or other radiation, heat, oxygen, water or moisture or by biological effects.
Many of the xenobiotics are designed to be persistent in the environment (similar to
drugs in the body), otherwise the amount to be applied and, as a consequence, the cost
of the substance would be very high, and the efficiency, due to too short a contact time
and unstable concentration, would be limited.
According to the REACH regulations, persistent substances fulfill the following
criteria:

– The half-life in marine water is higher than 60 days or

– the half-life in fresh- or estuarine water is higher than 40 days or
– the half-life in marine sediment is higher than 180 days or
– the half-life in fresh- or estuarine water sediment is higher than 120 days or
– the half-life in soil is higher than 120 days.

Very persistent substances are those chemical substances that fulfill the following
criteria:

– The half-life in marine, fresh- or estuarine water is higher than 60 days or

– the half-life in marine, fresh- or estuarine water sediment is higher than 180 days or
– the half-life in soil is higher than 180.

Hazard and persistency together increase the risk of the substance, as ecosystems
or humans are exposed for longer times to the substance, so it has higher chance to
cause harm.

2.7 Bioaccumulative and very bioaccumulative substances

Bioaccumulative and very bioaccumulative substances are those which are able to con-
centrate in the body of living organisms of microbial cells, plants or animals, including
man. Bioconcentration is measured relative to the environment and is quantitatively
characterized by the bioconcentration factor (BCF), which is the ratio of two con-
centrations, the concentration in the organism or organ and the concentration in the
environmental compartment.
BCF plant is Cplant /Csoil , and BCF fish is Cfish /Cwater . Bioaccumulation of certain
substances, e.g. hydrophobic organic substances in liver or fat tissue leads to the cor-
ruption of the food chain. Bioaccumulation of inorganic substances such as toxic
metals – lead, cadmium, or mercury – in plant shoots and leaves poisons the con-
sumers, and may cause biomagnification along the food chain. The mode and rate
of bioaccumulation along food chains and within food webs depends not only on the
characteristics and environmental concentration of the chemical substance, but also on
34 Engineering Tools for Environmental Risk Management – 2

the genetic potential and the biochemical mechanism developed by the living organisms
mainly as a defensive reaction (low accessibility deposits outside the cell membrane,
in the cell membrane or inside the cell).
According to REACH, a substance fulfills the bioaccumulative criterion when its
bioconcentration factor (BCF) is greater than 2000, and very bioaccumulative when
greater than 5000. According to the REACH Regulations, the assessment of bioac-
cumulation shall be based on measured data on bioconcentration in aquatic species.
Data from freshwater as well as marine water species can be used. In CLP regulation, a
cut-off value of log Kow ≥ 4 is intended to identify only substances with a real potential
to bioconcentrate. If a BCF value e.g. in fish is available, a BCF ≥500 is indicative for
classification purposes. Relationships can be observed between chronic toxicity and
bioaccumulation potential, as toxicity is related to body burden.
It can be seen from the hazard classes that chemical substances are not classified
according to their chemical structure and properties, but according to their effects.
Nevertheless, the grouping of hazardous chemical substances in environmental science
and practice is a mixed system: sometimes the hazard has priority such as in the REACH
regulations, which is a hazard- and risk-based system, but in other cases, physico-
chemical character and behavior are more important, so that some groupings are based
on chemistry (chlorinated solvents, PAH, PCB, etc.), origin (petroleum products, non-
ferrous ore mining, etc.) or on the use of the chemical substance (plant protection
products).

2.8 Emerging pollutants

Emerging pollutants are hazardous chemical substances that have recently been dis-
covered in the environment, such as endocrine or immune disruptors, sensitizers and
allergic compounds. Strictly speaking these are contaminants of emerging concern,
also abbreviated as CECs. Their presence, frequency of occurrence, and sources are
generally not known. Many pharmaceuticals, personal care products (PPCPs) and
well-known contaminants are amongst them due to newly recognized adverse impacts
different to their target effects. Emerging pollutants are chemicals or materials of
interest that can be characterized by:

– Perceived or real threat to human health or the environment;

– Lack of a clear dose–response relationship;
– Significant individual differences in sensitivity and the realization of effects;
– Their effects depend on age, life-stage, season and other environmental conditions;
– Lack of published health standards or an evolving standard;
– Contaminants may also be ‘emerging’ because of the discovery of a new source, a
new pathway to humans, or a new detection method or technology.

These chemical substances are discussed in detail in Chapter 2 of Volume 1, only

a summary on the more frequent endocrine disruptors is given here:

– An endocrine disruptor interferes with the synthesis, secretion, transport, binding,

action, or elimination of natural hormones in the body, which are responsible for
the maintenance of homeostasis, reproduction, development and/or behavior.
 Environmental toxicology – A general overview 35

Table 1.3 Some ‘emerging pollutants’ in the Hungarian course of the Danube: screening based on
substance quantities and hazards (Molnár et al., 2013).

Ranking Chemical substance Hazard-based score Type of substance

1 Bisphenol A 57 Industrial chemical

2 Sulfamethoxazole 55 Antibiotic drug
3 Metamizole (sodium salt) 53 NSAID
4 Diuron 52 Pesticide
5 Nonylphenol 51 Surfactant
6 Urethane/ethyl carbamate 51 Fermentation product
7 Carbamazepine 51 Antidepressant
8 Doxorubicin 50 Anticancer antibiotic
9 Bis(tributyltin) oxide 50 Pesticide
10 Paracetamol 50 NSAID
11 Aminophenazone 49 NSAID
12 Nicotine/cotinine 49 Luxury drug
13 Verapamil 46 Calcium-antagonist drug
14 Carboplatin 46 Anticancer drug
15 Gemfibrozil 46 Anti-cholesterol and
triglyceride lowering drug
16 Diclofenac 45 NSAID
17 17ß-estradiol 44 Hormone: contraceptive
and therapeutic agent
18 Ethinylestradiol 44 Hormone: contraceptive
and therapeutic agent
19 Di(2-ethylhexyl)-phthalate 42 Plasticizer
20 Dibutyl phthalate 42 Plasticizer
21 Trifluralin 41 Pesticide
22 Progesteron 40 Hormone: therapeutic agent
23 Ketoprofen 39 NSAID
24 Naproxene 38 NSAID
25 Ibuprofen 36 NSAID

NSAID: non-steroidal anti-inflammatory drug

– Immune disruptors affect the immune system via endocrine disruption or

directly.
– Sensitizers and allergens are chemical substances that may develop an allergic reac-
tion in normal tissue generally after repeated exposure. Sensitization is a causeless
immune response.

Table 1.3 shows the results of a preliminary score-based risk assessment on emerg-
ing pollutants in the Hungarian course of the Danube. The risk score was created
based on produced and used amounts; physical, chemical and environmental fate
properties (partitioning, (bio)degradability, bioaccumulative potential); known or sus-
pected adverse effects on the environment and human health (toxicity, mutagenicity,
reprotoxicity, endocrine and immune disruption) as well as occurrence/measured con-
centrations in the Danube or in the waste water discharged to Danube. The available
quantitative results were converted into scores by assigning ranges/classes created by
experts.
36 Engineering Tools for Environmental Risk Management – 2

Nearly one hundred suspected chemicals were assessed and ranked by the scores
created from mixed information. The second tier, the quantitative environmental risk
assessment of the priority chemicals modified the picture. For example, the risk posed
on the Danube ecosystem (RCRaq ) by bisphenol A is 0.009, far below the threshold of
RCR = 1. On the other hand diclofenac, based on several sampling, chemical analysis
and ecotoxicity tests carried out by the authors, has an aquatic RCRaq = 1–5, indicating
high ecological risk.

3 INTERACTION OF A CHEMICAL SUBSTANCE

WITH LIVING ORGANISMS

The effect of a chemical substance and its measurement is based on the interaction
between the chemical substance and the living organism.
Testing in the lab makes the test conditions controllable, i.e. quality and quantity
of the materials, time and temperature. The test can be performed at an equilibrium
state or the changes can be followed over time.
Field assessment or monitoring is accompanied by uncertainties due to environ-
mental circumstances which are uncontrolled and uncontrollable by the assessor, for
example, the continuous transport and fate processes, the interactions between

– a few different contaminants;

– the contaminant and the matrix of the medium;
– the contaminant and the biota.

Non-equilibrium conditions in the environment make the evaluation indeterminate

because the instantaneously measured values cannot be placed on the time curve of
the ongoing processes. It must be decided whether laboratory testing under controlled
conditions but low relevance for the environment or environmental testing with high
relevance but low reproducibility and low interpretability fit better with the actual case
or problem. A suitable micro- or mesocosm can unify the advantages of the two test
concepts.
Regardless of the testing scenario used – laboratory bioassay, microcosm or field
testing – the cause of the effect of a chemical substance to a living organism is the
same: the interaction between the chemical substance and the test organism. The con-
sequences of this interaction yield the end points needed for environmental toxicity
testing, as shown in Figure 1.3.
The first stage in the process is the fate, transport and distribution of the mate-
rial in the environment after its emission into the environment and before meeting
the organism. The degradation or other modification of the chemical structure influ-
ences the effect which arises later on when meeting the living organism. Photolysis,
hydrolysis, or enzymatic degradation of the chemical substance may take place in the
environment without or before reaching the ecosystem members. Xenobiotics often
mimic natural substances, which leads to their biodegradation in the environment by
water-, soil- or sediment-living microorganisms.
 Environmental toxicology – A general overview 37

Figure 1.3 Ecosystem effects and indicators/biomarkers of environmental toxicology.

The second stage is when the chemical substance reaches the site of action, i.e.
a certain part of the living organism: a molecule, a receptor site or any biochemical
compartment of the organism.
The last stage is the consequence of the second, and this is the interaction between
the chemical substance and the site of action. The impact of this interaction can mate-
rialize at molecular, cellular, organ, organism, population, community or ecosystem
levels.
The indicators of the interaction between chemical substances and the biota are
summarized using the system of Landis and Yu (1999). These indicators can be used
both for biomarkers and end point measurement when a toxicity test is being designed.
Indicators of the interaction between a chemical substance and the ecosystem:

– Biotransformation after introduction of the chemical substance/xenobiotic into

the environment, and before meeting the site of action:
◦ Induction of hydrolase enzymes;
◦ Oxidation in the presence of mixed-function oxidases;
◦ DNA repair enzymes;
◦ Biotransformation;
◦ Biodegradation;
◦ Bioaccumulation.
38 Engineering Tools for Environmental Risk Management – 2

– Interaction with the site of action: this is the molecular-level interaction (bonding)
of the chemical substance with the receptor site of a DNA molecule, a membrane,
a nerve synapsis, specific receptors of hormones or antigens.
◦ DNA/RNA;
◦ Membrane receptors;
◦ Key enzymes.
– Biochemical parameters:
◦ Stress proteins such as adrenalin, noradrenalin;
◦ Metabolic indicators such as respiration, heat production, substrate utiliza-
tion;
◦ Acetylcholine esterase inhibition and other molecules of the nervous system;
◦ Metallothionein production;
◦ Immune suppression or any other influence on the immune system, e.g.
sensitization or allergy.
– Physiological and behavioral characteristics reflect the healthy state of the popu-
lation. Tumors, developmental failures and lesions are typical markers showing
the poor health conditions of the population.
◦ Mutagenic effect indicators;
◦ Chromosomal damage;
◦ Carcinogenicity indicators;
◦ Reproductive success and reprotoxicity;
◦ Developmental toxicity indicators;
◦ Lesion and necrosis;
◦ Mortality;
◦ Behavioral alterations, movement;
◦ Compensatory behaviors, migration, escape.
– Population parameters such as size and structure, including age-structure of the
population are closely related to environmental conditions and stress.
◦ Population density;
◦ Productivity;
◦ Mating success;
◦ Alterations in genetic structure measured by DNA techniques;
◦ Competitive alterations, e.g. benefit from accepting a xenobiotic substance as
a substrate.
– Community parameters, particularly species composition and community func-
tioning, are characterized and compared. The problem in community charac-
terization is that we do not know what is optimal for a certain locality and
time.
◦ Community structure;
◦ Diversity: number of species and their relative abundance characterized by
diversity indices;
◦ Existence and role of minor components of the community;
 Environmental toxicology – A general overview 39

◦ Energy transfer efficiency;

◦ Dynamic character of the community;
◦ Stability;
◦ State of succession;
◦ Chemical parameters.

All of these parameters together constitute the effect of the chemical substance
on the ecosystem as a whole. The combined effect of chemical substances or other
stress factors changes the composition and metabolism of an ecosystem and results
in the endangerment and extinction of species and consequently the complete change
of food chains and food webs, or the dominance of certain species, as in the case or
eutrophication, acid rain or oil spills. These factors together with climate change and
the accelerated evolution of the microflora in the environment undeniably increase
the rate of ageing of the earth’s ecosystem. Many of the changes in the ecosystem
are irreversible and the constantly moving target of the ecosystem towards reaching
equilibrium and maintaining homeostasis makes the effects non-proportional to the
causes, and the system cannot be recognized in full detail. This is the reason for the
simplification of ecotoxicity to the toxicity of hazardous substances on certain repre-
sentative organisms or on a well-defined microcosm system. The extrapolation from
this result to the whole ecosystem may not be able to truly model and predict the
long-term history of the ecosystem in detail, but is no worse from the point of view of
statistics than a field assessment and could be a useful tool for the management of the
environment and hazardous chemicals.

3.1 Dose–response relationship

The well-known and widespread chemical models used for the management of the
environment are based on the connection between the dose or concentration of the
chemical substance and its effect. An increasing dose mixed into an experimental rat’s
feed causes an increase in the effect. An increasing concentration series of a tested
substance in water shows gradually stronger effect on fish, plant or crustacean living
in that water.
Paracelsus (1493–1541), the renaissance physician, botanist, alchemist, astrologer,
and general occultist, published his experimental results that every chemical substance
is poison, and the ‘dosage makes the distinction between poison and remedy’. Based
on this knowledge, he used low concentrations of mercury to cure syphilis. The same
is true for many of the environmental contaminants, e.g. metals, semimetals and other
natural chemical compounds, and man-made pharmaceutical and biocide substances.
Plotting any of the biomarkers, relevant for the actual measurable effect, against
the dose or concentration, an S-shaped sigmoid is obtained in each case, as shown in
Figure 1.4.
In Figure 1.4, the measured end point is the luminescent light of the test organism,
the light emitting bacterium, Vibrio fischeri, which gives a concentration-dependent
response by emitting less light in the presence of toxic chemicals than normal. In the
experiment shown in the graph, the toxic chemical substance is copper, and the light
emission of the bacterial suspension was measured in the presence of different copper
concentrations between 5 and 200 mg/kg.
40 Engineering Tools for Environmental Risk Management – 2

Figure 1.4 Concentration–response curve: inhibition of light emission (H%) of luminobacterium as a

function of Cu concentration in a dilution series.

The end point of the measurement – depending on the test organism and its
response – can be any of the formerly listed biochemical, physiological, behavioral,
population or community parameters. In the turning point (inflection point) on the
curve, the response changes from a proportionally increasing phase to a saturation
phase, where the concentration increase of the toxic agent cannot trigger a propor-
tional response, but only a smaller one due to the damage (inactivation, death) of the
test organism.
If the test organism is a bacterial strain or a plant or small insect, our goal is rarely
to protect this particular species based on the results, but instead to try to extrapolate
from the single species test results to the entire, more complex, ecosystem. Based on
experience and known correlations, the rule is that the test results from species at a
minimum of three trophic levels should be used, and depending on the number of
tested species, test type and duration, safety factors or a probabilistic estimation from
species sensitivity distribution (SSD) should be applied. The prediction of the effect (or
no-effect) concentrations of the chemical substances for the ecosystem in question can
be used for decision making or as an environmental quality criterion. The predicted
no-effect concentration (PNEC) of a chemical substance e.g. for an aquatic ecosystem
can be estimated based on the result of one algal, one crustacean and one fish species
using factorial extrapolation. No-effect estimates for terrestrial ecosystems require
test results on bacterial and plant species, and on the members of soil-living micro- or
mesofauna. The value of the assessment factor depends on the type and duration of
the tests: having three acute test results the assessment factor is 1000, but having one,
two or three chronic test results instead of acute ones, the factor goes down to 100,
50 and 10, respectively.
 Environmental toxicology – A general overview 41

Figure 1.5 SSD curve drawn from nonylphenol NOEC data (Nonylphenol, 2013).

The species sensitivity distribution (%) can be plotted if results from a minimum of
6–8 (preferably more) representative species are available: one should plot the propor-
tion of species affected as a function of the logarithm of stressor concentration or dose
(Posthuma et al., 2002; Solomon, 2008; SSD, 2013). The log–normal distribution of
the measured ecotoxicological end points is a prerequisite for this kind of extrapola-
tion. The predicted no-effect, or protective concentrations (arbitrary protective level
of 95%, 90%, 50%, etc.), can be read from the SSD curve as shown by the example
of nonylphenol aquatic toxicity in Figure 1.5.
SSD curves are generally plotted based on EC50 , a rather strong end point. NOEC
or LOEC values would serve the protective concept better. Laboratory ecotoxicity tests
are based on the deterioration – on a response of the adversely affected organisms –
while a real ecosystem assessment is based on the survival/presence of species. Species
actually endangered and lowered in number cannot be accounted for by a field assess-
ment. To harmonize the laboratory-based SSDs with the results of field assessments,
for the latter case one should derive the difference of the expected (reference) and the
counted species numbers and their distribution. This difference is comparable with
the “affected proportion’’ of species read from the SSD curve based on laboratory test
results. An SSD curve prepared from 10 chronic end points of nonylphenol NOEC is
shown in Figure 1.5. One can read from the curve the desired target concentration
42 Engineering Tools for Environmental Risk Management – 2

assigned to 50, 90 or 95% protection of the species distribution. HC5 , the concentra-
tion hazardous for 5% of the species is 2.93 µg/L for nonylphenol, calculated from
the log NOEC of 0.47 µg/L. An assessment factor between 5–10 is still necessary to
compensate for uncertainties when using SSDs (Posthuma et al., 2001).
If the test organism is a rat and the aim of the test is not the protection of the rat, but
of humans, the results must be extrapolated from rats to humans. Extrapolation uses a
mathematical model based on the known differences between human and rat genetics,
biochemistry, physiology and behavior. Extrapolation from animal study results to
man applies safety factors: generally a safety factor of 10, unless the exact quantitative
relation between human and rat sensitivity is known.

3.2 Test end points: the results of the environmental toxicity test
In the test in Figure 1.4, the dilutions in seven test vessels were contacted with the
luminobacterium, including one untreated control. Light emission was measured from
every dilution and the test end point was read from the concentration–response (light
emission) curve.
Light emission did not change with increasing concentration for a while; no inhi-
bition was measured in the vessel containing 5 mg/kg of copper; 5% inhibition was
measured at 10 mg/kg, 10% at 20 mg/kg, and full inhibition, i.e. no light emission at
all, when 80 mg/kg copper was present in the test vessel. After plotting the individual
measured results of the vessels, the curve was fitted to the measured points with the
help of a statistical method, using software that can find the statistical optimum of
the fitting. Having this curve, the following so-called test end points can be read from
the concentration–response curve:

– EC20 and EC50 : the concentrations that have the effect of a 20% and 50% decrease
in the measured end points: luminescence inhibition in the example, but it can be
respiration rate, enzyme activity, etc. in other cases. The abbreviation EC stands
for effective concentration. These values are always estimated using graphical or
computational means.
– ED20 and ED50 : the doses that have the effect of a 20% and 50% decrease in
the measured end point value. The abbreviation ED stands for effective dose. The
difference from EC is that the amount of the effective material is given as a dose.
This is the case when animal tests are used and the toxicant administered to the
test animal (rat, mouse, rabbit) is measured, used and plotted on the graph in
mass units such as µg, mg or g. Dose is also used when the amount or even the
identity of the contaminants in an environmental sample are not known. In this
case, the mass of water or soil which results in 20% or 50% inhibition in growth,
respiration, light emission, etc. is determined.
– LC20 and LC50 : the concentrations that cause 20% and 50% mortality in the test
organisms – estimated by graphical or computational means. The difference from
the previously introduced EC20 and EC50 is that the measured end point is not
optional, but fixed: it is lethality and LC stands for lethal concentration.
– LD20 and LD50 : the doses that cause 20% and 50% mortality in test organisms –
estimated by graphical or computational means. Lethal dose is the amount of the
 Environmental toxicology – A general overview 43

effective chemical substance or environmental sample given in mass units (µg, mg

or g).
– NOEC and NOEL: No Observed Effects Concentration and Level. This is the
highest applied concentration or dose in the test, which did not show any effect
when tested, compared to the non-treated control.
– NOAEC/NOAEL: No Observed Adverse Effects Concentration or Level, the high-
est applied concentration or dose in the test that did not show an adverse effect. It
has to be emphasized that stimulation as an effect is excluded from the evaluation.
– LOEC and LOEL: Lowest Observed Effects Concentration or Level, the lowest
applied concentration that has caused an effect. When our concentration or dose
series is of too large a scale, the difference between LOEC and NOEC can be
significant.
– MATC: Maximum Allowable Toxicant Concentration, determined by graphical
or statistical methods from NOEC and LOEC: NOEC < MATC < LOEC.

These end points of the test are uniformly used for the quantification of adverse
effects in environmental toxicology. These are objective measures, and can be directly
used in environmental management and applied in decision making.
It is necessary to clarify again: dose, being an amount of mass or volume, is mainly
used in the practice of toxicity testing when the exposure routes of ingestion, injection
or dermal contact are applied to the test animals. The concept is that the only exposure
to the toxicant is the controlled intake and from this input dose, knowing the body
mass of the test organism, the body burden can be calculated, which is very useful
when extrapolating from test animal results to humans.
Concentration is the input parameter when the representative ecosystem members
are tested in water or in soil/sediment. The concept is that the entire body of these
organisms is in contact with water or soil and, consequently, more simultaneous expo-
sure routes are responsible for interacting and taking up contaminants from the habitat
medium.
Animal testing of exposures from air also applies the concentration of the toxicant
in the air as an input parameter.
When contaminated land, site-specific effects or the quality of water and soil are
tested, we do not know whether contaminants are present or not. Even if we know
from clear signals that contaminants are present, they are not identified and quantified,
so that the only thing we can test is the dilution rate or mass (dose) of the tested envi-
ronmental sample (water or soil), which does not affect the test organisms adversely
under standard conditions. The interpretation of the results given in dilution rates or
sample doses is based on the experience of the assessors. This kind of interpretation
may be different according to the test organism and test method.
A more sophisticated solution is the application of the ‘calibration’ method and
giving the scale of effect not as a dilution rate or dose of the tested water or soil sample,
but as a toxicity equivalent value, compared to a well-known and easy-to-test chemical
substance. An example is introduced in Chapter 9.
The toxicity test end points are functions of the duration of the test. Acute (short-
term) tests use the end points of EC/ED and LC/LD causing 20%, 50%, sometimes
10% or 90% inhibition.
44 Engineering Tools for Environmental Risk Management – 2

NOEC/NOEL and LOEC/LOEL are applied for long-term (chronic) tests. Dura-
tion of the tests is discussed in Section 3.3 below, dealing with the classification of the
test methods.

3.3 Classification of environmental toxicological tests

Environmental toxicology is the science and practice of the adverse effects – mainly of
chemicals and other man-made agents – in and through the environment. The targeted
receptors of these adverse effects may be both ecosystems and humans.
According to this definition, we consider physico-chemical analyses, biological
tests and DNA techniques as environmental toxicity tests, which are capable of char-
acterizing the fate and transport of the chemical substance, and its effect on living
organisms, and their organizational systems, including man. As part of the environ-
mental toxicity measuring tool battery, all those model test systems are considered
that are feasible using extrapolation from the measured effect on individual organisms,
ecosystem and man.

3.3.1 Test type according to the aim of the test

Depending on the aim and targeted end point of the test, the types of environmental
toxicological tests vary from simple biotests to ecosystem assessments.

– Bioassays are simple, single species laboratory methods for testing:

◦ acute toxicity;
◦ chronic toxicity;
◦ mutagenicity;
◦ carcinogenicity;
◦ teratogenicity;
◦ reprotoxicity.
– Bioassays cannot simulate reality in detail, but they represent one single organism,
they work with one single chemical substance or a known mixture. Environmental
samples can be tested by bioassays: the samples may derive from one physical phase
(filtrated water, soil gas, etc.) or from a complex compartment of the environment
(whole soil with three phases). Bioassays need small amounts of sample, but still
are statistically relevant. Their controllability is high, so that they are easy to
standardize. Multiple numbers of replicates may be tested at the same time; they
are reproducible and comparable.
– Microcosms and mesocosms are multispecies toxicity tests modeling the real
ecosystem. Micro- and mesocosms are described in detail in Chapter 9 in Vol-
ume 3. Their main characteristics are environmental reality and their own history
based on their individual evolution.
– In situ biomonitoring means the observation of indicator organisms existing natu-
rally (passive biomonitoring) or placed by the assessor into the environment (active
monitoring). Natural organisms show large deviation in size, age, sex, etc., causing
large scale uncertainty in the results. Homogeneous (e.g. synchronized cultures)
and well controlled, lab-grown test organisms may improve test statistics. The
measured end points from molecular to population level are optional.
 Environmental toxicology – A general overview 45

– Diversity is the statistics of species, chiefly species richness and relative abundance
of the species in the ecosystem. The observed species richness in an ecosystem is
usually referred to as species density. The species evenness is the relative abundance
or proportion of individuals among the species. By statistical means, we can obtain
an index of diversity, such as species richness, Shannon index, Simpson’s index or
the Berger-Parker index.
– Biodegradation tests can measure the biodegradability of a chemical substance
using a standardized biodegradation test. Real biodegradation in a certain envi-
ronment can be tested by biodegradation tests modeling the real, site-specific
conditions (see also Chapter 2).
– Bioaccumulation tests provide information on the bioaccumulative potential of a
chemical substance or about the real biodegradation under site-specific conditions.

3.3.2 Test organisms

The type of test organisms can be chosen from a wide range of selections. Practically all
organism types can be used when they fulfill the criteria for organisms for environmen-
tal toxicity testing. They should be suitable and feasible for providing a useful answer
in the form of the organism’s response to the questions of environmental toxicologists,
decision makers, managers and regulators.
General requirements toward the test organism are summarized below:

– Availability: the test organism should be widely available in the nature or in

commerce.
◦ Laboratory cultures are the most widely used test organisms because, under
controlled conditions, their stability and good quality can be ensured. The
culturing lab can be the same as the testing one or it can be a specialized lab
for culturing the test organisms and guaranteeing high quality.
◦ Other culture facilities may also be used, e.g. hatcheries for crustaceans, fish,
clams or water plants. Test organs, tissues and blood can be collected from
slaughterhouses.
◦ Collection from the field may also be a good solution, mainly in those cases
where test organisms are not easy to culture or maintain in the labora-
tory: marine organisms, plankton, freshwater clams and higher water plants,
terrestrial species such as insects, or mites.
– Maintenance of the test cultures in the laboratory can be successful when the
species requirements relating to food, space and stress are well known. It is very
important to save the sensitivity and all of the required properties of the test
organism during cultivation and maintenance, and have a sufficient supply for
testing.
– Genetics of the test organism and history of the culture are essential to be able
to follow the changes and reach the required statistical quality of the test. The
genomes of Escherichia coli, Saccharomyces yeasts, Vibrio fischeri luminobac-
terium, Tetrahymena the single-cell animal, Drosophila or some of the Nematoda
have been fully mapped. The culture collections describe the main genetic and
physiological characteristics of the organism species, subspecies or strain. This
46 Engineering Tools for Environmental Risk Management – 2

controlled origin may ensure the conservation of these characteristics over the
long term by going back to the original culture any time. Amongst the higher
test organisms, there are many species/subspecies which have been used for a long
time and are well known from the genetic and physiologic point of view: rat, mice,
guinea pig, birds or rabbits.
– Sensitivity of the test organism is closely related to the aim of the tests and this is
why it is a significant parameter.

◦ Relative sensitivity means that a test organism shows different sensitivity to

different toxicants/contaminants. The user should have this information, oth-
erwise any additional chemical substance, its metabolites or contaminants may
cause an effect which is comparable to the main toxicant’s effect, and makes
the response of the test organism disproportionately strong and non-linear in
terms of the concentration. Another reason why information on sensitivity is
needed is that the test organism must match the problem or the substance to
be tested.
◦ Special sensitivity towards one or a few toxicants.
◦ Sensitivity towards a broad number of toxicants.

– Representative information about the ecosystem or ecosystem constituents is

needed for devising the environmental tests.
– Sensitivity should be representative for a class or phyla to protect certain taxa,
in this case, additional information is needed as to which families or phyla are
represented by the test organism.

◦ The most sensitive ecosystem member must be used for early warning.
◦ The results of tests on organisms with higher (but not much higher) sensitivity
can be integrated into a conservative risk assessment/management system.
◦ Average sensitivity is the best for risk managers since it provides a response that
is characteristic of the entire ecosystem, without implementing complicated
and costly monitoring.
◦ Lower sensitivity than that of the average test organisms can be used for
screening hot spots or the most risky elements of a complex system.
◦ Some families or phyla, or minor components of a complex ecosystem are
generally not represented by any of the test organism types.

– Concentration/dose–response relationship has multiple requirements:

◦ Connection between the amount of the toxicant/contaminant and the response

of the test organism.
◦ Proportional response to the concentration/dose of the toxicant.
◦ The effective concentration/dose range should be as broad as possible.

Reproducibility, statistics: it is one of the most important requirements when

environmental testing should be integrated into a quantitative risk assessment proce-
dure where objective and quantitative data are needed, and evaluated together with
physico-chemical analytical data. When this integrated application is understood, it
becomes evident that environmental toxicity data cannot be of lower quality than
 Environmental toxicology – A general overview 47

physico-chemical data, otherwise poor statistics and validity dominate the entire pro-
cedure. The reproducibility and statistics of a test method also depend on the practice
of the laboratory. Toxicity measuring laboratories prepare their own statistics and
give the historical averages and deviations of control and reference tests as additional
information to the test results.
Organisms that give some kind of response to toxicants/contaminants are numer-
ous. The selection of the test organism is often determined by the historical expertise
of the laboratory or organization. DNA, microbiology, veterinary labs or human tox-
icologists generally select those test organisms which they have the methods, tools
and equipment to work with and the knowledge needed to truly interpret the results.
Accordingly, all possible organisms and living systems of organisms, which are or may
be used for environmental tests, can be listed:

– Bacterial cells;
– Algae;
– Fungi;
– Plants: micro- and macrophytes;
– Animals from single-cell through few-cell and micro-size animals to macro-size
animals, viz. crustaceans, fish, clams, insects, rodents, or other mammals;
– Multispecies systems such as soil microflora, sewage sludge, rhizosphere, prey–
predator systems, food chains or food webs;
– The whole ecosystem can also be tested in microcosms or mesocosms or in the
field. Methodologies of testing aquatic and terrestrial ecosystems are distinguished
owing to practical reasons and differences in test arrangements.

Number of species used in the test is a basic design parameter and the evaluation
and interpretation needs different methodologies.

– Single-species tests apply one single species for testing the effect of chemical
substances. These species are well-known organisms, deriving from controlled cul-
tures. Single species are used in most of the laboratory bioassays and toxicological
tests.
– Multispecies tests involve more species in the same test. In the field of microbiology,
the competition test of two bacterial species uses the competing bacterial strains
grown together in the test medium. A special relationship is tested in the prey–
predator tests. Food-chain effects can be tested using the members of the food
chain. Microcosms and mesocosms are multispecies tests where – like the real
ecosystem – environmentally relevant species in representative numbers and dis-
tribution are tested together. In these models any ecosystem characteristic can be
measured or monitored: the number of organisms, number of species, relative
distribution of species, respiration or any other metabolic activity of the whole
microcosm, independent of the contribution of the individual species or organisms.
Mesocosms are larger in size and longer in time than microcosms, consequently,
sampling and monitoring have fewer limitations than is the case for microcosms,
therefore diversity and its changes, food chain and food web characteristics can
also be traced and measured.
48 Engineering Tools for Environmental Risk Management – 2

3.3.3 Test design

Exposure scenario, test duration and arrangement are manifold and their combination
results in endless options in test design.
Exposure Scenarios describe the conditions how a toxicant comes into contact
with the test organisms’ body. Depending on the circumstances, the following exposure
routes can be distinguished:

– Full-body test is when the test organism is immersed in the tested water, sediment
or soil and there is direct contact between the contaminant/contaminated environ-
ment and the test organism. In such cases, the whole body, the skin and all dermal
surfaces, eye, gill, hair, etc. are exposed and in many cases, the internal mucosal
surfaces in the mouth, (trachea, digestive system, etc.) as well.
– Feeding studies aim at testing the eating of food or toxicants mixed into food or
drinking water to model the uptake and effect through the digestive system. The
problem with this kind of testing is food intake; when the test animals feel the
presence of toxic material in the food, they eat much less or not at all. In proper
tests, the amount of water drunk and food eaten are measured individually. This
needs special drinkers and feeders.
– Placement of a controlled amount into the stomach by a tube (gavage) makes the
amount of food or water delivered more precise and controlled.
– Injection of a controlled amount (intramuscular, intravenous) of toxicant into
body-tissues or blood forces contact with cell membranes and uptake by the
metabolic apparatus of the cells.

Test Duration

– Short-term: acute tests cover a relatively short time period compared to the life
span of the test organism. 48-h Daphnia tests or 70-h fish tests are acute tests, and
the result is given as EC20 /ED20 or EC50 /ED50 values. The testing time for animals
with longer life spans (dog, monkey) is only a small portion of their whole life.
– Long-term: chronic tests may cover one or more generations. The duration of
chronic tests on test organisms with longer life span takes a significant proportion
of their life, including the gestational period of females and spermatogene-
sis of male test organisms. Chronic test results are given as NOEC/NOEL or
LOEC/LOEL values.
– Growth tests of microorganisms are special cases because of their generation time,
which is very short. The growth curve provides the best method for the character-
ization of the adverse effects of the chemicals, inhibiting metabolism and growth
of the population. In growth tests, the microbes are cultured in a series of growth
media containing increasing concentrations of the chemical substance to be tested.
The number of cells or the biomass at a certain time as well as the whole growth
curve of the culture is plotted and evaluated. Any parameter of the growth curve
such as the length of the lag phase, the rise of the growth curve, or the cell concen-
tration at the point of inflexion can be used as a response. In practice, the ErC20
and ErC50 values are applied for the characterization of toxicity where the growth
rate can be read from the curve and used for drawing the concentration–response
curve.
 Environmental toxicology – A general overview 49

Test arrangement guarantees a constant toxicant concentration, nutrient supply

and other parameters in the test medium. Test arrangement, size, medium, material
fluxes, should be fitted to the concept and aim of testing.

– Static tests: the test medium which contains the chemical substance to be tested
is the same solution in the same vessel during the whole test. The problem with
this method is that, during the test, gradients in aerobicity, pH, distribution of the
toxicant and concentration of metabolites develop both from normal substrates
and the toxicant. Biodegradation of the tested toxicant may also be a problem
which must be solved during testing. Most of the acute tests and microcosms are
static tests.
– Static renewal tests: in this type of test, the medium is renewed from time to time.
This can hinder the decrease or depletion of toxicants in the test solution and
the accumulation of harmful or otherwise effective metabolites, thus abating their
influence on the test results. Static renewal tests are often used for chronic aquatic
tests.
– Recirculation may ensure a continuous toxicant supply, the removal of metabolites
and keeping the whole test system in a steady state. This arrangement needs more
sophisticated equipment and the costs are also higher compared to the static or the
renewal tests. It may have certain disadvantages and technical problems which are
caused by the limitations of the techniques used for the treatment of the recycled
water: selective filters, supplies, additives, etc.
– Flow-through tests continuously ensure fresh test medium with the same nutrient
concentration and unchanged toxicants. In addition to pumps and flow meters, it
needs a mixing apparatus which refreshes the test medium. Of course this is the
most expensive, but the most reliable test arrangement.
– Flow-through test chambers can be placed in the environment to establish a mon-
itoring system. Flow-through chambers for Daphnia, fish, and clams are placed
in rivers, lakes, and inlets of sensitive surface waters for effluent monitoring.

3.3.4 Most commonly measured end points

The measurement end points detected can be the characteristics of an organism, the
components of the test medium, or any substrate, product or metabolite resulting from
the activity of the test organism.

– Toxicity test end points may be:

◦ Growth, in terms of cell number, mass production, root lengths, chlorophyll
content, nitrogen content of the cell mass, reproduction, etc.;
◦ Survival or mortality, sometimes immobilization;
◦ Respiration by monitoring O2 consumption, CO2 production or measuring
the enzyme activities of the respiratory (electron transport) chain, as well as
ATP production;
◦ Luminescence;
◦ Other enzyme activities as well as the decrease in the substrate of the enzyme
or increase in the product concentration;
50 Engineering Tools for Environmental Risk Management – 2

◦ Metabolites of biochemical processes;

◦ Gene products such as RNAs and proteins.
– Mutagenicity test end points are:
◦ Number of mutants;
◦ Number of revertants;
◦ Chromosome abnormalities.
– Carcinogenicity end points are mainly tumors.
– Reprotoxicity can be quantitatively characterized by the following end points:
◦ Reproductive success;
◦ Cytogenetic characteristics of the offspring;
◦ Morphological characteristics of the offspring.
– Biodegradation tests generally use the end points of:
◦ O2 consumption;
◦ Substrate consumption;
◦ Production of end products such as CO2 or metabolites from the tested
substance.
– Evaluation of bioaccumulation tests is based on the chemical analysis of the qual-
ity and quantity of accumulated substances and the medium tested (water, soil,
food/feed, etc.).

3.3.5 Environmental compartments and phases to test

The design of the test methods for gaseous, liquid or solid aggregates is different
depending on the tested environmental compartments and phases. Laboratory bioas-
says for the testing of chemical substances are mainly carried out in dissolved form in
the liquid phase, but in some cases in artificial or real sludge or soil. Samples taken
from the environment can be tested in their original form or as an extract or leachate.
The most frequently tested environmental samples are:

– Liquid samples
◦ water from freshwater or marine environment;
◦ subsurface waters, runoffs and groundwater;
◦ pore water from soil and sediment;
◦ leachate or seepage from soil or waste;
◦ waste waters;
◦ liquid phase extracts, eluate or leachate from solid samples.
– Solid phase samples
◦ whole soil;
◦ whole sediment;
◦ solid waste.
– Slurries and sludges can also be tested: mainly waste sludge, including sewage
sludge; liquid and solid phase can be separated before testing.
 Environmental toxicology – A general overview 51

3.3.6 Aims of environmental toxicity tests

The aims of testing chemicals for their environmental toxicity are numerous. Reg-
ulation and managing chemical substances, contaminated land and waste needs the
quantitative information on adverse effects listed below as a basis for decision making
and managing the environment.

– Characterizing the environmental fate and behavior of chemical substances;

– Measuring toxicity, mutagenicity, carcinogenicity and reprotoxicity of single
chemicals;
– Measuring toxicity, mutagenicity and reprotoxicity of mixtures;
– Establishing effect-based environmental quality criteria;
– Environmental monitoring via biomonitoring and/or integrated monitoring;
– Early warning within environmental monitoring and management;
– Measuring toxicity, mutagenicity, carcinogenicity and reprotoxicity of environ-
mental samples;
– Assessing and monitoring the ecosystem;
– Measuring toxicity, mutagenicity and reprotoxicity of waste materials, leachates
of wastes;
– Direct, effect-based decision making to support environmental management.

3.4 Environmental toxicology in relation to hazard

and risk assessment
Hazard is the intrinsic character of a chemical substance, its hazardousness (flamma-
bility, toxicity, mutagenicity, etc.) is anchored in its chemical structure. It is the basis
of QSAR (Quantitative Structure-Activity Relationship) and the fundament of hazard-
based legislation and management of chemical substances. Nevertheless, hazard is only
an indicator for environmental risk. Hazardous chemical compounds are risky to the
environment when they are produced and used in such an amount which, on enter-
ing the environment, adversely effects humans and the ecosystem or members of the
ecosystem. This means that risk depends on the amount of the substance produced,
used and emitted, as well as on the potential adverse effects and the exposed receptors.
Hazardous substances can be controlled, handled and used in a safe way when their
risk is reduced to an acceptable level, in spite of being hazardous.

3.4.1 Testing hazard or risk?

According to the above explained difference between hazard and risk, environmental
toxicity tests can also be divided into hazard-testing and risk-testing methods. When
assessing hazard, the test focuses on the toxicant, while the test organism and the test
method are standardized. Testing with the aim of characterizing risk, focuses on the
exposed receptor and tries to simulate the real environmental conditions of the impact.
One part of the test methods is well standardized, with good statistics both at the intra-
and interlaboratory level, suitable for legislation of chemicals and regional or global
decision making. In these tests, the environment plays a secondary role, environmental
specialties are integrated in a generic way as standard test conditions; the tests are not
52 Engineering Tools for Environmental Risk Management – 2

Figure 1.6 Environmental realism of the assessment models.

intended for modeling the environment, rather ensuring a stable test scenario. Another
part of the test methods is designed in such a way that they have close contact with the
real environment and they can model the environment or at least its most important
characteristics. If environmental reality of the tests were put on a scale, then the scale
of realism of the test designs can be ‘measured’ (Figure 1.6).
The distinction between hazard and risk, their assessment and integration of one
of them into an environmental management system with a certain target is the key
feature of sustainable management of the environment.
A good example is the difference between degradability of a chemical substance
and the degradation of the same substance as a pollutant in a generic or in a site-
specific environment. Degradability is an intrinsic property of a chemical substance
and depends on its chemical structure. However, one can speak about degradation
in the context of interaction with the environment. Degradation may be estimated
under theoretical or generic environmental conditions (average annual temperature,
water flux, redox and pH conditions, average sediment characteristics, humidity, soil
type and conditions in the soil, etc.). Degradation or especially biodegradation of
the same contaminant in a contaminated site needs a site-specific approach, either by
using site-specific environmental characteristics or by testing the biodegradation of the
contaminant in the contaminated soil or water in situ or in a sample taken from the site
in question. The prediction of the fate and behavior, e.g. degradation of a substance in
the environment is a key issue in risk assessment because readily degradable substances
have obviously less chance to meet receptors than persistent ones.
Photodegradability, readiness for hydrolysis and biodegradability of a sub-
stance can be calculated from its physico-chemical properties using physico-chemical
 Environmental toxicology – A general overview 53

functions or QSAR equations. There are test methods available for measuring degrad-
ability and these methods represent a uniform generic test environment. Certain
generic environmental parameters such as radiation/light conditions, temperature,
redox potential, pH, etc., which may significantly influence degradation depending on
regions and climate, can be considered in the calculation or measurement of degrad-
ability of a substance. However, one must be aware of the fact that actual, site-specific
degradation of the same substance can be higher or lower, and in some cases, signifi-
cantly different from the calculated or measured degradability. Photodegradation of a
photodegradable compound in particular can be zero in deep waters or in sediments,
or in a northern region with little sunshine, but significant on a shallow seashore in
the sunny south.
Biodegradability tests have been standardized by OECD. All regulations on
hazardous chemical substances (REACH, pesticide, biocide, food, cosmetics, etc. reg-
ulations) require test results. Reproducibility and comparability of the test method is
more important for regulatory purposes than its environmental reality, partly because
the target environment, where the substance will be used and emitted, is not known
beforehand. This is the reason why the potential biodegradability is tested in a stan-
dard test medium by using a standardized (?) biodegrading microflora. A key aspect,
which weakens this concept, can be identified here: it is hardly possible to prepare,
maintain and use a standard but still non-selective or non-adapted microflora for car-
rying out biodegradation tests. One or a few microorganisms are not good enough,
they might not be able to degrade even a readily biodegradable substance or they may
accidentally degrade something which is not easy to degrade in the environment. The
use of a bacterial community, e.g. stemming from sewage sludge, may also be uncer-
tain because its microbial composition depends on the history of the sewage sludge,
its past opportunity of having met the same or similar substance and having been
adapted to it or not. Experts should be aware that the microbiota of the sewage sludge
are highly adapted to different, locally occurring contaminants and cannot represent
a natural community in natural waters or sediments. The same applies to contam-
inated sites: biodegradability in general and the real biodegradation at a long-term
acclimated site with a very likely adapted microflora may differ greatly for the same
contaminant.
Due to the large-scale deviations biodegradability test results are loaded with high
uncertainty, and, even though one would like to, this test cannot be made independent
of the environment. In hazard assessment cases, it may be better to apply a mathemat-
ical model, especially if abundant historical data are available which the mathematical
function or the QSAR equation can be based on.
The other extreme option is the site-specific assessment of biodegradation at a
real site, at the source of a contaminant in a micro- or mesocosm, simulating the
existing conditions. Managing risk at a contaminated site and making decisions on
biodegradation-based risk reduction cannot be supported by biodegradability results;
it can only be done with measured site-specific biodegradation results.

3.4.2 Standardized or customized test methods?

The dilemma of selecting the right test method, a standardized or a site- and problem-
specific one for measuring adverse effects is severe and frequent. Standardized test
54 Engineering Tools for Environmental Risk Management – 2

methods have many advantages; they are uniform, their results are comparable, and
less skilled personnel are capable of carrying them out. All test conditions are defined
in the test description. Data collection, evaluation, interpretation and the necessary
documentation are also specified. In most cases, the optional species or subspecies are
listed as test organisms, from which the laboratory can select the proper one.
Before choosing and using the standardized methods, the key decision point, and
the question to be answered by the test must be clearly identified. This also means
that all standardized test options and the user’s requirements towards the results must
be known to be able to correctly select the best suiting method. In plain English: a
non-suitable standard method will fail to provide the correct answer. Sometimes none
of the standardized methods can provide the proper answer; in this case, a problem-
specific test should be developed for the actual case. Standardized tests answer the
most frequently asked questions, but it does not make any sense to develop standard
methods for rarely occurring or very special problems.
Some regulations, e.g. REACH specify the required test method for all decision
points and criteria of the regulation. The tests which serve the regulation can yield
comparable results, and, based on them, priorities can be set. Another concept of
decision making is to compare test results with legal criteria. Most of these standardized
tests can measure the hazard due to adverse effects but not the actual risk. In order to
obtain a risk-related answer from a test, a model must be created for the real situation,
which represents the most important (even if not all) parameters and factors in an
environmental problem, which contribute to risk generation.
When a contaminant is immobile in the environment, strongly bound to the solid
matrix of an environmental compartment, e.g. soil, and the question to be answered
is, how much threat this contaminant poses to the soil ecosystem (plants and soil-
dwelling organisms), the proper way to assess the effect-based risk of the soil is not
analyzing or testing the extract from this soil, contrary to the wrong practice. Rather,
the representative terrestrial/soil-living test organism should be placed in the contam-
inated soil, let them interact with each other, simulating real situations. The response
measured in such a direct contact scenario has high environmental relevance. But if
the groundwater is the receptor, i.e. the assessment’s target, a leachate from the soil
column irrigated with real or model precipitation should be tested to be able to decide
whether or not the groundwater is endangered by the immobile contaminant.
Characterizing site-specific risk by effect testing, the test design should simulate
or model the real situation. The test design must be able to rely on proper knowledge
of the site and the risk components. A conceptual risk model must be created which
characterizes the source(s), transport routes, the contaminated environmental com-
partments and their dimensions, as well as the land uses and the users of the polluted
environment.
Standardized tests, even complex ones, for example, long-term pesticide testing
mesocosms (EPA, 1996/2008), are not capable of answering other questions related to,
for example, sediments of special texture, of extreme organic or inorganic content, or to
the presence of species other than specified by the standard. In conclusion, a simplified
rule is that standardized tests give proper results for hazard-based or generic risk-based,
mainly regulatory environmental management. On the other hand, site- and problem-
specific simulations and models are useful in solving concrete risk management tasks.
 Environmental toxicology – A general overview 55

3.4.3 Testing or modeling? – QSAR and environmental toxicology

The large number of existing chemicals makes the detailed testing of every single
case impossible. Every year, several thousand new chemicals are added to the about
100,000 chemical substances already produced and used. This vast amount of sub-
stances, regardless of the quantities produced and used and whether or not they are
hazardous, cannot be tested alone due to the work and tremendous costs involved.
Additionally, the low environmental reality of many of the tests must be considered
since the forecast for the real environment has a very poor statistical value.
Toxicity data are not available for all kinds of chemicals. Missing ecotoxicity data
for existing chemicals cannot be reinstated by myriads of tests, but proper mathemati-
cal models enable the environmental toxicologist to estimate the toxicity of non-tested
chemicals based on their chemical structure or any intrinsic physico-chemical charac-
teristics, which are associated with chemical structures such as melting point, volatility,
water solubility and octanol–water partitioning. Modeling is a valuable method and
the proper place of modeling in environmental management must be found. However,
even long-term monitoring or environmental measurements and tests performed over
many years are just models, perhaps more realistic ones than mathematical models, but
still models, which greatly simplify the real environment. A detailed ecological survey
is another model because only a small part of the environment, which is considered rep-
resentative, is being assessed. The acceptance of monitoring results as ‘true’ values may
be misleading. One must interpolate and/or extrapolate from the information obtained
from measurements at discrete points, which is tantamount to considering the collected
information as a model. The results of both modeling and testing/monitoring should be
validated. Table 1.4 displays a comparison between modeling and testing/monitoring
to help assess their advantages and disadvantages.

Creating QSAR models

The QSAR (Quantitative Structure–Activity Relationship) is based on the fact that
chemically similar substances have similar physico-chemical properties (solubility,
partition between physical phases, mobility), environmental fate and behavior (degra-
dation, concentration and accumulation) and biological effects such as toxicity,
mutagenicity, carcinogenicity, reprotoxicity, neurotoxicity, hormone- and immune-
disrupting, sensitizing or corrosive effects.
Based on the connection between chemical structure, behavior and effects of chem-
ical substances and assuming that abundant data on the toxicity of chemically similar
substances are available, the relationship between structure and toxic effect can be
described using mathematical equations. In order to formulate the proper equation, a
QSAR model must be established.
Data of good quality can be collected from the literature and existing databases,
and a frame of reference with the coordinates of a certain toxic effect (e.g. fish or
Daphnia toxicity) and the structure of any related physico-chemical property of the
chemical substance can be created. Environmental behavior and biological/toxic effects
of chemicals are closely associated with their Kow value (octanol–water partition coeffi-
cient). In our example, plotting literature results of fish/Daphnia toxicity as a function
of Kow value of many substances, a linear relationship can be found between a large
number of points, thus a straight line can be fitted to the data. The linear equation
56 Engineering Tools for Environmental Risk Management – 2

Table 1.4 Comparison of modeling and testing/monitoring (ECOFRAM, 2011).

Modeling Testing/monitoring

Advantages
Cost effective Provides an actual measurement of chemical
Time requirement: days to months concentration, hydrologic response, etc.
Simple model scenario: what happens if … Avoids multiple conservatism due to compounding
Able to measure the effect of risk conservative assumptions
reduction measures Accounts for the inherent heterogeneity of the system
Able to predict concentrations in a There is a greater acceptance of measured data
continuum over space and time There is public confidence in monitoring data
Able to simulate concentrations below
the analytical limits of quantification
Comparative assessments are possible:
both exposures and effects
Disadvantages
Environmental reality: low Costly
Uncertainty in model predictions Time requirement: weeks to years
Input of data may be uncertain and Sampling difficulties: planning, selection of sampling
not feasible points, statistics, interpolation and extrapolation
Simplifications required Requires a long time to evaluate effectiveness
General public reluctance to accept Handling non-detects is difficult
predicted data Sampling represents discrete points
Needs calibration to see how closely Sample represents one unique combination of
predicted values match reality conditions
Constrained by analytical precision and the level of detail
Results can be misleading (1 event occurring in 100 years)
Difficult to interpret results in a probabilistic fashion
Cause and effect may be difficult to assign

of the relationship can be used for the calculation of fish/Daphnia toxicity from Kow .
Most of the data will be close to the line, their deviation from the line shows how close
the correlation is between Kow and fish toxicity. Some of the literature data will be far
from the line, and in these cases, the reason for the variance must be investigated: it
is highly probable that the chemical type of the substance should also be taken into
account, and different equations must be established for aromatic, aliphatic, chlori-
nated, polycyclic or other chemicals. There are some individual substances that have
special modes of action, and, as a consequence, do not fit to any of the group-specific
lines. These identified outliers can be expunged from the training set of data used for
creating the model, but an acceptable and scientifically reasonable explanation should
be given for their removal.
A good-quality QSAR model needs several hundreds or thousands of data, their
quality must be screened and they must be grouped according to chemical structure,
molecular size, mode of action, etc. Series within the structure may fit well to the line
within a certain range, but outside this range, the equation may not fit any more, which
means that QSAR is applicable only under certain conditions and within certain ranges.
It is therefore very important to publish the accuracy together with the equation.The
definition of some terms, based on the Technical Guidance Document applied by the
 Environmental toxicology – A general overview 57

REACH regulation (EU-TGD, 2003), should be given here for a better understanding
of QSAR and the methodology:

– QSAR method is the theory underlying a QSAR, including the adequacy of the
descriptor variables, the form of the model and the description of the activity
represented by the model.
– QSAR model is the quantification of the QSAR method, for example, through the
derivation of a mathematical equation describing the activity for a specific class
of substances.
– Domain of a QSAR is the group of substances for which the model is valid. This
group of substances can be defined by structural rules, mechanistic information
and/or parameter ranges.
– A QSAR is considered reproducible if it can be applied by all assessors indepen-
dently and leads to the same results.
– Training set is the set of data used to construct a QSAR model.
– Validation set is the set of data used to validate the QSAR model. The data in this
set should not be included in the training set and should be chosen in the domain
of the model, but independently of the training set (EU-TGD, 2003).
– Accuracy: it should be checked whether the correlation coefficient and the overall
validity and accuracy of the model have been given. These statistics should include
the estimated standard deviation of the prediction errors, the significance of the
model as a whole, its variables and parameters.

The Technical Guidance Document (TGD) of the EC prepared for the risk assess-
ment of new and existing chemical substances (EU-TGD, 2003) recommends the QSAR
methodology for the prediction of environmental fate, behavior and effects of chemical
substances on aquatic ecosystem members:

– Henry’s Law Constant (H);

– Octanol–water partition coefficient (log Kow );
– Partitioning between soil/sediment organic matter and water (Koc );
– Photolysis (kdeg air );
– Hydrolysis (khydr water );
– Biodegradation (not-ready biodegradability);
– Acute toxicity to fish (96-h LC50 );
– Acute toxicity to Daphnia (48-h EC50 );
– Acute toxicity to algae (72–96-h EC50 );
– Long-term toxicity to fish (NOEC, 28-day study);
– Long-term toxicity to Daphnia (NOEC, 21-day study);
– Bioconcentration of fish and worms.

This branch of QSARs also underlines the fact that any adverse effect of chemicals
depends to a large extent on their fate and behavior in the environment. This means that
the environmental effect of a chemical substance can only be reliably predicted based
on laboratory tests if additional data on partitioning, degradation and accumulation
of the substance in the environment are gathered, from which the actual or forecasted
environmental concentration of the substance can be established. The adverse effect
58 Engineering Tools for Environmental Risk Management – 2

of this environmental concentration, estimated as described, is the basis of the risk

assessment.
Available QSARs
In the case of aquatic toxicity, the guideline differentiates between polar and non-
polar toxic substances as they cannot be handled by the same QSAR equation due to
their different behavior and effect mechanisms in the organisms: polar substances are
hydrophilic, while non-polar ones are hydrophobic. This is why biological availability
of the latter is poor, mainly over the short term, but if their accessibility increases due
to environmental effects, e.g. in the presence of co-solvents or tensides, their adverse
effects can be triggered. Organism-specific active transport may occur and as a result,
the non-polar substance can reach adipose tissues, nerves or the liver, where it can
exert its toxic effect or accumulate. If no specific toxic mechanisms occur, the internal
effective concentrations are almost constant.
Currently, reliable QSARs are available for chemicals that act through a non-
specific mode of action such as non-polar narcosis (caused by inert compounds) as
well as polar narcosis (caused by substituted phenols, anilines, pyridines and mononi-
trobenzenes). Regarding non-polar narcosis, QSARs are recommended for fish (short
and long term), Daphnia (short and long term) and algae (short term). With respect to
polar narcosis, QSARs are recommended for fish (short term) and Daphnia (short
term). No QSARs have been recommended for substances that act through more
specific modes of action.
Some typical applications of QSAR models should be introduced here (EU TGD,
2003). Many of the models recommended today have been selected and recalculated
for ecotoxicity estimation by Verhaar et al. (1995) (n is the number of data, r2 is the
correlation coefficient, Q2 is the cross-validated r2 and s.e. is the standard error of
estimate).
Biodegradability of chemical substances
Primary biodegradability of phthalate esters
RC = −24.308 · log Kow + 394.84
RC = primary biodegradability, n = 12, r2 = 0.87 (Boethling, 1986)
Biological oxygen demand of alcohols, phenols, ketones, carboxylic acids, ethers,
sulfonates
BOD = 1105 ·
/δ/x−y + 1.906
BOD = Biological Oxygen Demand,
/δ/x–y = difference in the modulus of atomic
charge across a functional group bond x–y
n = 112, r2 = 0.98 (Dearden & Nicholson, 1987)
Biological oxygen demand of alcohols
BOD = 0.093 · SE − 3.163
SE = electrophilic superdelocalizability
n = 19, r2 = 0.96 (Dearden & Nicholson, 1987)
Adverse effects
Fish toxicity of non-polar substances
Pimephales promelas 96-h LC50 , mol/L
 Environmental toxicology – A general overview 59

log LC50 = − 0.85 · log Kow − 1.39

n = 58, r2 = 0.94, Q2 = 0.93, s.e. = 0.36 (Verhaar et al., 1995)
Brachydanio rerio and P. promelas 28- to 32-day NOEC, ELS test, mol/L
log NOEC = −0.90 · log Kow − 2.30
n = 27, r2 = 0.92, Q2 = 0.91, s.e. = 0.33 (Verhaar et al., 1995)
Daphnia toxicity of non-polar substances
Daphnia magna 48-h EC50 immobilization test, mol/L
log EC50 = −0.95 · log Kow − 1.32
n = 49, r2 = 0.95, Q2 = 0.94, s.e. = 0.34 (Verhaar et al., 1995)
Daphnia magna 16-day NOEC, growth, reproduction, mol/L
log NOEC = − 1.05 · log Kow − 1.85
n = 10, r2 = 0.97, Q2 = 0.95, s.e. = 0.39 (Verhaar et al., 1995)
Algae toxicity of non-polar substances
Selenastrum capricornutum 72–96-h EC50 growth, mol/L
log EC50 = −1.00 · log Kow − 1.23
n = 10, r2 = 0.93, Q2 = n.d., s.e. = 0.17 (Van Leeuwen et al., 1992)
Fish toxicity of polar substances
Pimephales promelas 96-h LC50 , mol/L
log LC50 = −0.73 · log Kow − 2.16
n = 86, r2 = 0.90, Q2 = 0.90, s.e. = 0.33 (Verhaar et al., 1995)
Daphnia toxicity of polar substances
Daphnia magna 48-h EC50 immobilization test, mol/L
log EC50 = −0.56 · log Kow − 2.79
n = 37, r2 = 0.77, Q2 = 0.73, s.e. = 0.37 (Verhaar et al., 1995)
Bioconcentration
QSAR for BCF of substances with log Kow < 6
log BCF = 0.85 · log Kow − 0.70
n = 55, r2 = 0.90 (Veith et al., 1979)
QSAR for BCF of substances with log Kow > 6
a) Polynomial equation log Kow > 6
log BCF = 6.9 · 10−3 · (log Kow )4 − 1.85 · 10−1 · (log Kow )3 + 1.55 · (log Kow )2 − 4.18
· log Kow + 4.79n = 45, r2 = n.a. (Connell & Hawker, 1988)
b) Parabolic equation log Kow > 6
log BCF = − 0.20 · (log Kow )2 + 2.74 · log Kow − 4.72
n = 43, r2 = 0.78 (recalculated from Connell & Hawker, 1988)
Bioconcentration of aromatic compounds by Daphnia magna
log BCF = 0.898 · log Kow − 1.315 (Calow, 1994)
Bioconcentration in earthworms
log BCF = 1.0 · log Kow − 0.6
n = 100, r2 = 0.91 (Connell & Markwell, 1990)
The toxicity or other Kow -dependent parameters of a chemical substance can be
calculated using a good QSAR, but this is still inaccurate compared to its environmental
60 Engineering Tools for Environmental Risk Management – 2

fate and behavior as well as the effects brought about in the real environment, in surface
waters and sediments, in soils and sludges, and in the organism of individuals. In the
real environment and individual bodies, interactions between the chemical substances
and matrix materials, partition between physical phases, interactions of contaminants
with each other and with members of the biota, and, after reaching the receptor,
with the individuals’ biochemical system will to a large extent modify the measurable
behavior, characteristics and effects of the substance. One should be aware that QSAR
and other models, e.g. chemical models, are suitable for the estimation and prediction
of behavior and effect of chemicals in the case of incomplete data, but the uncertainty
of these estimates may be high.

3.5 Statistical evaluation of ecotoxicological tests

When testing the adverse effects of chemicals, finding a good compromise between
statistical values and practical factors such as time requirement, workload and cost
is a key task of risk managers. Apart from expense, labor and time requirements,
an excessive number of replicates may cause a time-shift, and thus the replicates are
processed at different times, corrupting the statistics.
To be able to select the proper statistical tool, the environmental problem must
be understood, the conceptual risk model devised and then the most suitable test
methodologies and statistical tools must be found.
In the statistics of an acute test – given that the EC50 point is on the steepest part
of the concentration–response curve – much smaller errors are expected than is the
case for NOEC or LOEC values where the dose–response curve just starts increasing
and this increase is slow, related to the applied concentrations.
Statistical methods are supported by software tools, which are available as free or
commercial products.

3.5.1 Evaluation of acute toxicity tests

Evaluation of an acute toxicity test implies the reading of the test end point from the
dose–response curve.
Graphical interpolation requires plotting the concentration–response curve and
reading the concentration which causes 50% or 20% (in special cases 10% or 90%)
inhibition. From the plotted curve, the experienced environmental toxicologist knows
whether the shape of the curve is typical or atypical. If the curve is atypical, the
effect mechanism behind it is probably not a simple one, and the evaluation cannot be
performed mechanically. This kind of subjectivity is both an advantage and a disad-
vantage. The best way to utilize the advantage is to use graphical interpolation as an
exploratory or control means. The disadvantage is that the confidence interval cannot
be calculated.
The probit method is the most popular statistical tool for the evaluation of typical
S-shaped concentration–response curves. The original data are processed by probit-
transformation. Probit is a binary response model that employs a probit link function.
This model is most often estimated using a standard maximum likelihood procedure,
such an estimation being called a probit regression. In the probit model, the inverse
standard normal distribution of the probability is modeled as a linear combination of
the predictors (PROBIT, 2013).
 Environmental toxicology – A general overview 61

The confidence interval can be easily calculated using the probit method for the
evaluation of acute toxicity test-end points. The disadvantage is that it needs two sets
of partial kills, which differs from 0 and 100%.
A great number of computerized programs are available such as

– STATA-PROBIT (Data Analyses and Statistical Software) (STATA, 2013);

– SAS-PROBIT (Statistical Analysis System) (SAS, 2013);
– STATISTICA (STATSOFT, 2013), SPSS-PROBIT (original name: Statistical Pack-
age for the Social Sciences, but today SPSS is used without any explanation)
(SPSS-PROBIT, 2013);
– Mplus-PROBIT (Mplus, 2013);
– R-PROBIT (R-project for Statistical Computing) (R-PROBIT, 2013).

Some specified methods are recommended for toxicity evaluation such as

– ASTM-PROBIT (developed by ASTM International, originally called: American

Society for Testing and Materials) (ASTM, 2013);
– TOXSTAT (TOXSTAT, 2013);
– DULUTH-TOX (US-EPA), etc.

Roberts (1988) compared five readily available computer programs for probit
analysis. The methods were evaluated for input/output options and reliability of
output.

– DULUTH-TOX by Charles Stephan of the Environmental Protection Agency’s

Environmental Research Laboratory, Duluth;
– ASTM-PROBIT from the ASTM Guide for Probit Analysis, Draft 2;
– UG-PROBIT, which was written by statisticians at the University of Guelph,
Canada;
– SPSSx as different from the commercially available software;
– SAS, part of the SAS software package.

According to Roberts’ (1988) results, except for UG-PROBIT, the programs

yielded essentially identical median lethal concentration (LC50 ) values for the 20 data
sets tested. DULUTH-TOX and ASTM-PROBIT include an objective evaluation of
the validity of input data and calculated results, but they lack graphical output. SAS-
PROBIT provides graphical output superior to that from SPSSx-PROBIT, which yields
inappropriate fiducial limits in some cases.
UG-PROBIT, SPSSx-PROBIT and SAS-PROBIT purport to consider control or
natural responses. Only SPSSx-PROBIT and SAS-PROBIT actually adjusted observed
treatment responses for the control response.
DULUTH-TOX and ASTM-PROBIT, in all implementations, have a major advan-
tage that objective tests are included to determine the validity of input data and to guide
the interpretation of output. The commercial statistical programs have the advantages
of graphical output and a method for handling control mortality (Roberts, 1988).
Logit transformation of data and fitting the curve based on the maximal likelihood
method can also be applied for calculating EC or LD values. Similar to the probit
62 Engineering Tools for Environmental Risk Management – 2

method, in the case of lack of partial kill data, some assumptions are required for the
calculations.
The moving average method is similar to graphical interpolation, but the
concentration–response curve should be properly linearized. The confidence interval
can be established only when partial kill data are available.

3.5.2 Data analysis for chronic toxicity tests

The standard method for analyzing chronic toxicity data is ANOVA, which is the
abbreviation of Analysis of Variance. This determines the concentrations that are
significantly different in effect from the untreated control.
In ANOVA, the observed variance in a particular variable is partitioned into com-
ponents attributable to different sources of variation. In its simplest form, ANOVA
provides a statistical test of whether or not the means of several groups are all equal,
and therefore generalizes the t-test to more than two groups.
The fixed-effects model of variance analysis applies to situations in which the
experimenter applies one or more treatments to the subjects of the experiment to see
if the response variable values change. This allows the experimenter to estimate the
ranges of response variable values that the treatment would generate in the population
as a whole.
In the case of chronic toxicity, the equivalence of the control and treated is tested.
Analysis of variance is performed on the treatment group. By multiple comparisons
between the treatment groups, those groups can be identified which are different from
control.
In the first step, ANOVA calculates the distance between all treated and control
groups. If the F-score is not statistically significant, the treatment has the same effect,
there is no difference between the groups. If the F-score is significant, data are exam-
ined in a second step to find the groups different from the control. Using multiple
comparisons, the groups different from each other can also be identified.
The goal of the ANOVA-evaluation is to find the LOEC or NOEC values, namely
the lowest contaminant concentration which is different from control showing a sig-
nificant adverse effect or the highest no-effect concentration which is identical to the
control but different from LOEC. This is a so-called hypothesis-testing model.

3.5.3 Data analysis of multispecies toxicity tests

Multispecies tests need multivariate techniques for the exploration of patterns within
ecological data sets and microcosm and mesocosm data.
Multispecies toxicity tests – as discussed in detail in Chapter 8:

– They are similar to natural ecological systems in complexity and

– In the consequences of being historical: having their own evolution (the parallel
tests may also differ from each other);
– They include trophic structures, food chains or food webs;
– Environmental parameters such as sunshine, precipitation, nutrient supply, inter-
action with and between environmental compartments and phases play an
important role.
 Environmental toxicology – A general overview 63

The main paradox of multispecies testing is the loss of statistical power with
increasing ecological relevance.
In view of the complexity and unique history of micro- and mesocosms as well
as natural structures and their dynamic nature, the evaluation and interpretation of
measured data and the correspondence of changes to the treatments require carefully
planned and statistically evaluated test design and monitoring. When designing the
number of replicas, the unique history of these simultaneous microcosms must be
taken into account, and the detected indicators should be multivariate and associated
with the treatments as much as possible. The suitable statistical methods are also
multivariate because univariate methods such as simple ANOVA cannot handle the
temporal dependence of variables or the community level changes (which cannot be
characterized by single species responses) and, as a consequence, the risk of the false
zero hypothesis increases.
Evaluations of these multivariate tests need a concept that lets the variables explore
the ecological patterns. Multivariate statistics is able to examine all data, but it is the
test designer’s responsibility to decide which data should be measured or gathered.
Multivariate statistical methods differ from each other to a large extent: the evalua-
tors must know the relationships among variables and find the most suitable statistical
means. Multivariate statistical evaluation should be coupled with association analyses
to identify the result of the treatments. Multivariate statistical techniques commonly
used for the evaluation of ecological structures are:

– PCA: principal components analysis (assumption: linearity);

– DPC: detrended principal components (a polynomial is used to remove non-
linearity);
– NMDS: non-metric, multidimensional scaling (non-linearity is considered using
ranks);
– RDA: PCA coupled with redundancy analysis;
– Clustering: grouping by similarities: algorithm has no knowledge about treatment
groups;
– Divergence between treatment groups;
– NCAA: non-metric clustering and association analysis: a multivariate derivative
of artificial intelligence (Landis & Yu, 1999).

3.6 Standardization and international acceptance of newly

developed toxicity tests
After finding the relationship between the concentration or dose of a hazardous chemi-
cal substance and a measurable end point, there are many tasks until an internationally
agreed, standardized test method is established.

– Research & development: exploring effect mechanisms and developing technical

details.
– Prevalidation: an approximately 2-year process that aims to standardize and opti-
mize the test protocol and evaluate within-lab variability and define a ‘prediction
model’ or ‘data interpretation procedure’, which articulates the process by which
test results can be used to predict toxicological end points in vivo.
64 Engineering Tools for Environmental Risk Management – 2

– Validation: an approximately 1-year process which means the evaluation of the

test’s transferability to other laboratories and measuring between-labs variability
and reproducibility (minimum four external laboratories).
– Peer reviewing by independent peer review body (ECVAM Scientific Advisory
Committee, or ESAC).
– Regulatory acceptance in Europe means ESAC endorsement, in the US, ICC-
VAM formulates recommendations. This process can take 2 years or more at
the national/regional level and longer in the case of international consensus-
driven bodies such as OECD (OECD, 2013a), the International Conference on
Harmonization (ICH, 1991), and the Veterinary International Co-operation on
Harmonization (VICH, 1996).

Environmental toxicology, this increasingly important tool in environmental deci-

sion making, should be better understood and fully integrated into the tool batteries
of environmental management. The integration of direct toxicity testing of our envi-
ronment and the application of their full models in the form of micro- and mesocosms
bring environment (nature) and its inhabitants closer to the increasingly mechanically
thinking and functioning humanity, which is one of the main obstacles in evolving
a holistic approach necessary for really efficient environmental management. Envi-
ronmental toxicology demonstrates and helps to understand and solve many of the
pitfalls of environmental management and policy: e.g. handling uncertainties due to
the variability of natural systems and the wide range of errors in sampling, measuring,
evaluating and interpreting the acquired information, the importance of the references
and benchmarks, the temporal and spatial extension of the impacts and the objective
and subjective factors behind uncertainties. Understanding the dynamic nature of the
environment and adapting the management concept to it, is a basic requirement. A
future vision of the most efficient environmental management is a simple preventive
tool, just based on a high level of respect for the environment. But before reaching
this level of harmony with the environment, one has to learn more about the envi-
ronmental responses to anthropogenic exposures by measuring and evaluating these
impacts and responses. The general overview in this Chapter of the aims, approaches
and developments in environmental toxicology has provided the basis to introduce
the methodological details of human, aquatic and terrestrial toxicology in Chapters
2–6, and the methods for acquiring information from environmental toxicity data in
Chapter 9.

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Chapter 2

Fate and behavior of chemical

substances in the environment
K. Gruiz, M. Molnár, Zs. M. Nagy & Cs. Hajdu
Department of Applied Biotechnology and Food Science,
Budapest University of Technology and Economics, Budapest, Hungary

ABSTRACT

The production and use of chemical substances is accompanied with certain emissions
to the environment. Depending on the physico-chemical characteristics of contami-
nants they are primarily emitted to air, water or the soil. The fate and behavior of
chemicals in the environment is determined in addition to their physico-chemical char-
acteristics, to their interactions with the ecosystem, with the living and non-living
components as well as the users of the environment. The fate and behavior of chemi-
cal substances are well known under ‘in vitro’ (laboratory) conditions; however, our
knowledge about the interactions between chemical substances and environmental
compartments, phases and their inhabitants is limited. The assessment, refinement
and the pitfalls of the characterization of the behavior of chemicals in the environ-
ment is the topic of this chapter to support understanding the role and the use of fate
properties in hazard and risk assessment, as well as in the risk-based management of
chemicals.

1 INTRODUCTION

Environmental behavior, fate, mobility, availability and degradability of a contaminant

or a mixture of contaminants in the environment have a strong influence on their actual
toxicity and environmental risk. Furthermore, information about the contaminants’
behavior and fate such as extreme partitioning between physical phases, photodegra-
dation, hydrolysis or biodegradation may provide key data for the remediation of
contaminated land.
Environmental risk assessment attributes a high priority to environmental fate
characteristics for the assessment of chemical substances and contaminated sites. The
real toxicity of chemical substances is determined by their effective concentration,
accessibility and availability to the potentially impacted receptors. A rapidly hydrolyz-
ing compound does not pose a high risk to the aquatic ecosystem because it has limited
opportunity, i.e. a very short time period, to meet and interact with the receptors. A
highly sorbable toxic element or molecule is bound to the solid phase of the sediment
and does not interfere with aquatic organisms. However, sediment-dwelling organisms
may be affected by the same sorbed substances. These examples explain the difference
72 Engineering Tools for Environmental Risk Management – 2

between ‘actual’ and ‘potential’ adverse impacts, or more generally, the hazard and
the risk of chemical substances. The potential occurrence of chemical substances in the
environment and their potential adverse effects are associated with their production,
use and emission to the environment as well as with their intrinsic material properties.
The hazard of a substance can be characterized based on intrinsic properties. Risk
is based on the real interaction of the chemical substance and the actors of the envi-
ronment: it is the actualization of the potential. The potential is not closely related
to existence in the environment: a molecule is known as hazardous already on the
design board. In addition to hazard and risk, regulators created a third category, called
generic risk, indicating the adverse impact of chemicals under generic environmental
and land-use conditions.
Along the parallel lines of hazard and risk, one can speak of environmental fate
properties and actual environmental behavior, about toxicity of chemical substances
and actual adverse/toxic effect of the same toxicant under certain conditions. For exam-
ple, toxicity in freshwater in general differs from the toxic effect in the river Danube;
the adverse effect of a toxic metal in soil in general may greatly differ from the same
effect in a tropical sandy soil in the desert, or the same soil in a kindergarten, etc. Soil
type, land use and the users of the land influence the realization of the contaminant’s
toxic potential.
The effect of the chemical substance can be measured in standardized water or soil
samples by standard bioassays, using standard test organisms, but the actual impact
should be measured in situ or in an artificial test system, which works with a repre-
sentative sample and truly simulates the environmental conditions of the case being
assessed. The extrapolation from hazard to environmental risk is possible, but is loaded
with multiple uncertainties. For example, the absence of short-term risk in an acute test
does not mean that no risk will occur over the long term in the presence of hazardous
substances in the environment; some negative aquatic toxicity tests do not imply that
the substance is harmless in the natural environment.
Similar to actual toxicity, ‘actual concentration’ is also an estimate based gener-
ally on the simplified model of a dynamic process. The potentially emitted amount
of a chemical substance and its potential partitioning among physical phases in the
environment can be calculated with relatively low uncertainties. On the other hand,
real interactions with the living and non-living components of the environment mov-
ing away from the source in time and space are less and less predictable because of
the contaminant properties. The concentration of the contaminant depends on its fate
and transport in the environment, and the instantaneous concentration at a specific
time and location highly depends on the environmental conditions (climatic, sea-
sonal, meteorological parameters, hydrological and hydrogeological properties, soil
type, geochemistry, heterogeneities, etc.). The effective concentration at the site of
action, which can produce an effect on the receptor, is determined by the accessibility
and availability of the contaminant for the targeted receptor.
Environmental risk reduction should involve and utilize the environmental fate
characteristics of pollutants. Volatile substances should be volatilized in order to
remove them, water-soluble pollutants should be flushed out, and the risk of
sorbable substances should be reduced by a technique using absorption or adsorp-
tion. Technology can handle the sorbent in a separate reactor volume, which can be
a proper engineering device or a specified environmental volume (reactive soil zone).
 Fate and behavior of chemical substances in the environment 73

Strong sorption, without removing the pollutant, may decrease the risk by reducing
the contaminants’ mobility and availability to receptors (see also Chapter 2.4 in
Volume 4). If the contaminant is biodegradable or highly reactive to chemical compo-
nents or other reactive agents of the environment, this property is worth being utilized
for its elimination from the environment.
Natural attenuation of contaminants in the environment – their transport per-
formance, degradation or transformation – may serve as a basis for site remediation.
Bioaccumulation can also be the basis of remediation, and in this case, the technologist
should separate the accumulating organisms from the biologically ‘extracted’ medium,
e.g. by filtrating microorganisms from water or by harvesting plants from soil. Block-
ing or changing the transport pathway itself provides an easy and commonly used
solution for risk reduction of water and soil pollution, for example, collecting leachate
from the contaminant-containing wastes instead of letting it enter surface waters or
infiltrate into soil. A natural cap may reduce contaminant discharge into the air and
water, etc.
The suggestion to consider environmental fate in risk assessment and utilize it for
environmental risk reduction appears self-evident. However, the history of environ-
mental management shows the opposite: environmental risk assessment is still being
performed based on emitted amounts or measured concentrations of chemicals without
taking into account degradation, elimination or low bioavailability. Limiting values
are often based on pure chemical models, and not on a risk value which, for exam-
ple, would take into account the strong bonding capability of certain elements and
compounds to environmental matrices, where their adverse effects cannot be activated
because they are not accessible/available for the receptors.
Risk reduction by remediation, the other main task of risk management, does not
utilize sufficiently the information about transport and fate of contaminants either.
‘Washing out’ contaminants from soil by groundwater, pumping them to the surface
and treating them (pump and treat) is still practiced at a poor technology level. It has
been applied for decades at very high cost and little success, e.g. for solid-bound con-
taminants with limited water solubility or subsurface lenses of dense non-aqueous
phase liquids (DNAPLs) which are a long-term source for dissolved contaminant
plumes.
These examples elucidate how important the environmental fate and behavior of
chemical substances are and how important it is that environmental managers acquire
and use this information. Ignorance of fate characteristics deteriorates the efficiency
of risk management and leads to risk overestimation, resulting in unnecessary costs.
Site-specific risk primarily depends on the environmental fate of contaminants,
highly influenced by the quality of the environment. The transport and behavior of the
same contaminant are different in a sandy or loamy soil to those in a river or a lake.
The true characterization of the actual fate and effects as well as the consequent risk of
a contaminant requires an integrated approach, which includes a joint assessment of
the contaminant’s physico-chemical and environmental fate properties as well as the
characteristics of the environment.
The two main facets of the environmental fate of chemicals – partitioning among
physical phases and biodegradation – have not been extensively studied by chemical
science. The need for this kind of information emerged with the development of envi-
ronmental toxicology and environmental risk assessment. The hazard of chemicals
74 Engineering Tools for Environmental Risk Management – 2

mainly depends on their own characteristics and their predicted interaction with the
environment. The behavior of the chemicals under standardized conditions is mea-
sured and put into databases as transport and fate characteristics such as partitioning
between physical phases, degradability, or their (bio)accumulative potential.
The mobility and bioavailability of a contaminant depend on its intrinsic proper-
ties and represent its potential to move on and affect biological entities. Mobility and
availability can only be interpreted when interactions with other environmental agents
and matrices are considered. Mobility of a chemical only makes sense when its interac-
tion with the medium is taken into account. Availability should reflect the interactions
with radiation/light, water, or the members of the ecosystem. The characteristics of the
environment determine the receiving capability, the resistance toward the behavior and
impacts of the contaminant. For example, transport of the contaminant in soil will be
determined by its mobility and the retention capacity of the soil. Retention capacity of
the soil depends on its texture, i.e. sandy, loamy sandy, loamy or clayey. The sorption
capacity of the solid phase and the partitioning of the contaminant between physical
phases determines the scale of volatilization, leaching, desorption or sorption, and
highly influences the contaminant’s availability for water and living organisms. Mag-
nitude of the toxic effect depends both on the ecotoxicity of the chemical compound
and the sensitivity of the organisms living on the site of exposure, as well as the mode
or interactions between them. Transport and availability of contaminants, e.g. in soils
can properly be characterized only by the integration of chemical analytical, biological
and ecotoxicological information.
Partitioning of toxicity in soil among the solid, aqueous and gaseous phases results
in risk to groundwater, soil air and, as a consequence, to the surrounding atmospheric
air and surface waters. Partitioning in sediments between the solid phase and pore
water determines water quality. Strong binding and low biodegradability may lead to
the development of a chemical time bomb, i.e. the latent presence of a contaminant
without any symptoms for a while, and the potential for a sudden risk increase due
to a change in environmental conditions such as redox potential, pH, temperature or
pressure, chemical composition or any other factors influencing mobility and toxicity.
When measuring the toxicity of solid environmental matrices, actual toxicity can
be better characterized by contact tests that simulate a realistic scenario, where mutual
interactions occur. The results of interactive bioassays include the results of the possi-
ble interactions between all participants: contaminant, contaminated medium and test
organism.

2 INTERACTION OF THE CONTAMINANTS WITH

ENVIRONMENTAL PHASES

Interactions between chemical substances and environmental compartments can be

classified as physico-chemical or biological processes. Another classification of inter-
actions is based on the compartment: interactions within the atmosphere, in surface
waters and sediments or soils. The biota is considered as an additional compartment
linked to aquatic and terrestrial ecosystems. All compartments have three physical
phases, but one phase generally dominates: gas in the atmosphere, liquid in aquatic
compartments and solid in soils and sediments. Other phases are always present such
 Fate and behavior of chemical substances in the environment 75

as liquid in the form of vapor and solid in the form of dust in air, suspended solid
and dissolved gas in waters, pore water in soil and sediment, and soil gas in the three
phases of soil. We shall discuss the interactions of chemicals with the environment in
the following three groups:

– Transport and fate processes, including partitioning;

– Physico-chemical interactions;
– Biological interaction, primarily biodegradation and bioaccumulation.

2.1 Transport and partitioning

Transport processes and partitioning of a chemical substance between the phases play
a major role in the contaminant’s spread in the environment. These transport pro-
cesses alter the distribution and concentration of contaminants, but do not change the
absolute amount present in the environment.
Transport refers to the way chemicals move in the environment or in organisms
to their ultimate destination and also the way they reach their final target. Transport
of chemicals in the environment integrates advection, diffusion, sorption and desorp-
tion, solution and dissolution, infiltration, drainage, dilution, partitioning, etc. of the
contaminant on the pathway from the source to the receptor.
Partitioning is a complex transport process which includes the distribution of a
chemical substance among the environmental phases. The two main partitioning sce-
narios occur between air and water, and water and solid. In these situations opposing
processes seek to balance evaporation and condensation; dissolution and precipita-
tion; sorption and desorption; uptake and release, integration and disintegration,
mobilization and immobilization, etc.
Environmental transport and fate – this term covers all physical, chemical and
biological processes linked to the movement (changing position and phases) and
transformation (physical alteration, chemical and biological reactions, including degra-
dation processes) of the chemical agent after it enters the environment. The fate of
contaminants in the environment allows for prediction of impacts and risk calculations
for the ecosystem and humans.
Equilibrium partitioning of a chemical substance between physical phases can
be calculated from the basic physico-chemical parameters of the chemical substance
(vapor pressure, water solubility, etc.) and the environmental parameters (temperature,
pH, etc.). Given that there is never an equilibrium state in the environment, measured
distribution – preferably as a function of time – should be used to reflect the real
situation. In the following paragraphs the calculation of equilibrium partitioning will
be introduced.

2.1.1 Partitioning between air and water

Partitioning between air and water is characterized by Henry’s law constant quantita-
tively, giving the equilibrium ratio of the concentration of the chemical substance in
air to that in water.

Henry = p · M/c,
76 Engineering Tools for Environmental Risk Management – 2

where
– Henry: Henry’s law constant (Pa·m3 /mol);
– p: vapor pressure (Pa);
– M: molecular mass (g/mol);
– c: solubility in water (mg/L).
Partitioning between any liquid phase and gaseous phase can be characterized by
the dimensionless Henry’s law constant:
Kliquid-gas = Henry/R · T,
where
– Kliquid-gas : partitioning of the chemical substance between liquid and gas;
– R: gas constant (Pa·m3 /mol·K);
– T: temperature (K).

2.1.2 Partitioning between solid and water

Partitioning between solid and water is quantified by the ratio of the concentration in
the soil and sediment solid to that in the liquid phase. When calculating partitioning,
one should distinguish between organic and inorganic contaminants. The equilibrium
partitioning can be expressed as
Kp = Csolid /Cwater
The value of Kp for organic substances depends on the organic matter content in
the solid phase, given that organic contaminants are bound to the organic matrix (e.g.
the humus fraction of the soil)
Kp = Foc · Koc ,
where
– Foc : the organic fraction in the soil or sediment;
– Koc : partitioning of the contaminant between the soil’s organic carbon content and
water content (i.e. pore water or soil moisture).

Koc = a · Kow /1000,

where
– a: a constant (a = 0.411);
– Kow : octanol–water partition coefficient.
Cations, the mobile inorganic atoms or molecules with an electrical charge, are
distributed between liquid and solid phases in the soil. Kd , the equilibrium constant,
characterizes their partitioning. The easiest way to determine this value is the analytical
measurement of the cations in both phases after equilibrium setting:
Kd = Ccation water /Ccation solid
The value of Kd depends on the size and strength of the cation and the sorption
capacity of soil. When using these mathematical relationships, it has to be considered
 Fate and behavior of chemical substances in the environment 77

that there is no equilibrium (or steady state) in the environment, in spite of the fact that
the processes tend towards equilibrium. This means, for example, when contaminants
are transported by groundwater which flows through various volumes of the solid soil,
a part of the contaminant is sorbed on the solid soil depending on contact time, but
the concentration in the water is always higher than the equilibrium concentration.
Using equilibrium concentration in the calculations may lead to underestimation of
the risk.

2.1.3 Transport models

For the purpose of generic risk assessment, for example, authorization or restriction
of chemicals, the European REACH regulation proposes the use of transport models,
which should uniformly be used. The models indicate all possible transport routes
and the risk assessor should decide about the priority risk components and adapt
the generic model to the specific chemical substance and problem. The same transport
models can be used for site-specific purposes, but only after a careful study and creation
of the conceptual risk model for the site identifying the key transport routes and risk
components.
The main difference between the generic and site-specific use of transport and fate
models is that, for regulatory purposes, so-called generic environmental parameters are
included as default values, which are valid for the whole of Europe (i.e. for no particular
site). In the site-specific version, one has to apply the characteristic parameters of the
particular site, which are not readily available and should be gathered or measured by
the user of the transport model.
Currently, many fate and transport models are available for chemicals in the
environment. When using a transport model to estimate the predicted environmental
concentration (PEC) of a chemical substance, the environmental concentration in all
environmental compartments must be determined, taking into consideration partition-
ing, degradation and other physical, chemical and biological changes and interactions
(Figure 2.1). The measured and calculated parameters and the pre-chosen data have
to be discussed and documented in the risk assessment study.
Figure 2.1 shows the scheme of transport from the source of chemical substance to
environmental compartments on single or multiple transport pathways. Environmen-
tal compartments themselves are complex systems with up to three physical phases.
They also contain organic and inorganic constituents in different chemical forms and
the community of the members of the ecosystem. The transport itself includes every
kind of displacement or movement of the contaminant by precipitation, infiltration,
surface runoff, leaching, diffusion, dilution, erosion, sedimentation, partition, chem-
ical reactions (photodegradation, hydrolysis, oxidation and reduction), interaction
with the biota (biotransformation, uptake and biodegradation), etc. The end points of
the transport are the environmental compartments which are themselves endangered
by the contaminating chemical substance, in addition to their ecosystem and the land
users.
Generally, the following parameters are given as input data in exposure models:

– Physical-chemical characterization of the substance:

◦ molecular mass;
◦ vapor pressure;
78 Engineering Tools for Environmental Risk Management – 2

Figure 2.1 Transport from source to environmental compartments.

◦ boiling point;
◦ Henry or Henry’s law constant
◦ octanol–water partition coefficient;
◦ water solubility.
– Known amounts or fluxes of the chemical substance, taking into consideration
their use patterns:
◦ amount produced;
◦ amount used;
◦ amount emitted;
◦ measured or calculated flux in the environment.
– Environmental parameters:
◦ environmental compartments concerned;
◦ environmental phases;
◦ geochemical properties;
◦ hydrogeological properties, etc.
The concentration of substances found in the environment can be construed on
local (PEClocal ), regional (PECregional ) and continental (PECcontinental ) scales. These PEC
values have an effect on each other because the continental area contains the regional
scale and the regional scale contains the local area. The boundaries of the site to be
modeled should be exactly defined and delineated.
 Fate and behavior of chemical substances in the environment 79

Figure 2.2 Simple Box concept: environmental compartments and transport routes.

The most frequently used conceptual model for environmental purposes is the
Simple Box model (Mackay et al., 1992; Van de Meent, 1993; Brandes et al., 1996)
which enables the calculation of both regional and local exposure concentrations in all
environmental compartments. The main facet of a box model is that it does not resolve
the spatial heterogeneities and the distribution of the contaminant concentration inside
the box. It is assumed that the box is “well-mixed’’ and from the difference of input
and output contaminant fluxes one can calculate a removal rate or residence time.
The Simple Box model incorporates the fate properties of direct and indirect emis-
sions, biotic and abiotic degradation in all compartments, diffusive transport, advective
transport and partitioning between phases. Substance input to the model is considered
continuous and equivalent to continuous diffuse emission. The results provided by
the model are steady-state concentrations which can be considered long-term average
exposure values. In the model, the substance released is distributed among the com-
partments according to the properties of the substance and the modeled environment.
The concentration of a substance at the border of the modeled region must be taken
into account as background concentration.
The Simple Box model (Figure 2.2) can be tailored to concrete site-specific uses,
modified or simplified by excluding any of the environmental compartments, phases
and non-typical processes in a certain case, for example, evaporation and partitioning
between air and water can be excluded when the transport of a non-volatile chemical
substance is modeled in a two-phase system of solid and water.
80 Engineering Tools for Environmental Risk Management – 2

Another type of model, used for generic purposes, such as regulation of chemi-
cals or catchment-scale environmental management, having an important role in the
estimation of environmental concentrations of chemical substances in aquatic systems,
describes wastewater treatment. The basis of these models is the SimpleTreat model
shown in Figure 2.3 (Struijs et al., 1991) which provides a quantitative description of
the processes that take place in an average-sized sewage treatment plant based on aer-
obic degradation using activated sludge. The SimpleTreat model enables computation
of the steady-state concentrations in a sewage treatment plant.
The SimpleTreat model provides information about the amounts of chemical sub-
stances entering and leaving a sewage treatment plant. The removal of a substance is
influenced by the physical-chemical and biological characteristics of the substance and
the operational conditions of the sewage treatment plant.
The revised version of the SimpleTreat model (Mikkelsen, 1995) incorporates an
improved process formulation for volatilization from the aeration tank. More specific
information on the biodegradation of a substance may be available in the modified
version of the SimpleTreat model at a higher tier of the risk assessment process. The
following scenarios are optional:

– Temperature dependence of biodegradation;

– Degradation kinetics according to the Monod equation, created for the description
of biological reactions, such as enzyme reactions, microbial growth rate, etc.;

Figure 2.3 SimpleTreat model for characterizing the fate and transport of contaminants in a wastewater
treatment plant using activated sludge.
 Fate and behavior of chemical substances in the environment 81

– Degradation of the substance in the adsorbed phase;

– Variation in sludge retention time;
– Exclusion of a primary settler.

The Simple box model is a strong simplification suitable for local transport model-
ing with local parameters or for regional transport modeling with generic parameters,
supposing homogeneous environmental distribution in the “box’’. A different concept
is required for site-specific transport modeling, e.g. for the calculation of flux or access
time, for example from the contamination source of an industrial facility to the next
drinking water well. This kind of calculation needs (partly) numerical models, includ-
ing the site-specific environmental parameters, their distribution, heterogeneities and
interactions between the contaminant and the medium. When regional, e.g. watershed-
scale transport is to be modeled by real parameters or well established generic ones,
a GIS-based approach should be applied, using meteorological, topographical, hydro-
logical, geological, geochemical, soil typological parameters, surface coverage, etc. of
the area in the form of 3-dimensional maps (see also Chapter 10 in Volume 1).

2.2 Chemical interactions between chemical substances

and the environment
Physico-chemical interactions between contaminants and the environment take place
in the form of photolysis, hydrolysis, sorption, desorption and chemical reactions
such as oxidation, reduction, radical reactions and chemical modifications. Certain
interactions with air or water cause a very abrupt or very intensive reaction or produce
dangerous chemicals, for example, flammable solids, spontaneously combustible solids
(dangerous when wet) or explosives.
Some of the chemical reactions in the environment result in a decrease in toxicity
or other adverse effects of chemicals, but the opposite may also occur: a chemical
transformation may produce a more dangerous form (oxidation of sulfides into sulfu-
ric acid), or a more risky situation (desorption from soil or sediment particles and thus
contaminating sensitive waters). Risk-reducing chemical interactions, such as precip-
itation or reduction in an insoluble form can be utilized in risk-reduction measures
(waste (water) treatment, reduction of technological discharge, etc.) and in remedial
technologies applied to contaminated environments.

2.2.1 Photolysis
Photolysis, also called photodissociation or photodecomposition is a chemical reaction
in which a chemical compound is broken down by photons. Photodissociation is not
limited to visible light: electromagnetic waves or ultraviolet light, X-rays and gamma
rays are usually involved in these radical photoreactions, given that their energy is
higher than that of visible light.
Photolysis in the atmosphere plays an important role in eliminating many atmo-
spheric pollutants, such as hydrocarbons (e.g. methane) or nitrogen oxides. The
formation of the ozone layer in the stratosphere is also a result of a photoreaction
of oxygen.
82 Engineering Tools for Environmental Risk Management – 2

The degradation rate by photolysis in the atmosphere can be described by the

equation:

kdeg air = kOH × COH air × 24 × 3600,

where

– kOH : specific degradation rate constant with OH radicals (cm3 /molecule/s);

– COH air : concentration of OH radicals in the atmosphere (molecules/cm3 )1 ;
– kdeg air : pseudo first-order rate constant for degradation in air (1/day).

Photochemical degradation in water is important for such chemical substances

that are not degradable in any other way i.e. by hydrolysis or biodegradation. Pho-
tolysis in natural waters is strongly limited by the intensity of light (seasonal and
geographical differences) and the density of water (dissolved and suspended solids in
water) which absorbs most of the light.
The value of half-life for photolysis in water (if known) can be converted into a
pseudo first-order rate constant:
kphoto water = ln 2/DT50 photo water

where

– DT 50 photo water : half-life for photolysis in surface water (days);

– kphoto water : first-order rate constant for photolysis in surface water (1/day);
– ln: natural logarithm (to the base e).

2.2.2 Hydrolysis
Hydrolysis is the degradation of chemical compounds by water. It can be a chemical
process, including the hydrolysis of salts into acids and alkaline products, degradation
of starch or cellulose into sugars, and the hydrolysis of esters and fatty acids. For many
substances, the rate of hydrolysis depends to a large extent on the specific environmen-
tal pH and temperature and, in the case of soil, on moisture content as well. The rate
of hydrolysis always increases with increasing temperature.
In addition to chemical hydrolysis, enzymatic hydrolysis is also common in the
environment. Enzymatic hydrolysis can proceed in water and soil triggered by free
enzymes bound to soil or sediment particles or by enzymes within living organisms.
The free ones are exoenzymes produced and secreted by living organisms, or other-
wise derived from them, e.g. following their death and decomposition. Most of the
enzymatic processes are connected to or take place within living organisms.
Hydrolysis is quantified by the half-life (DT 50 ) of hydrolysable chemical sub-
stances. DT 50 or the degradation rate of chemical substances can be determined in
standardized tests. DT50 can be converted into degradation rate. The first-order rate
constant khydr water is:

1
The global annual average OH radical concentration can be assumed to be 5×105
molecules/cm3 (EU-TGD 2003).
 Fate and behavior of chemical substances in the environment 83

khydr water = ln2/DT50 hydr water ,

where
– DT 50 hydr water : half-life for hydrolysis in surface water (days);
– khydr water : first-order rate constant for hydrolysis in water (1/day);
– ln: natural logarithm (to the base e).

2.2.3 Chemical oxidation and reduction

Oxidation is the loss of electrons or an increase in oxidation number. Reduction is the
gain of electrons or a decrease in oxidation number.
Both oxidation and reduction may strongly influence the contaminant’s chemical
form and behavior in the environment. Oxidation or reduction of a substance in the
environment depends on its oxidation state and the redox potential of the environment.
The change in redox potential generally modifies the fate and transport properties of
chemical substances as well as their availability to and effects on living organisms.
For example, mercury is highly toxic in elemental or methylated forms, but not at all
in low redox-potential wetlands where it is present in the chemical form of sulfides.
Chromium VI loses its toxicity when reduced to chromium III, due to the alternative
respiration of facultative anaerobic microbes that lower redox potential in the soil.
Toxic organic compounds degrade and lose their toxicity because of the oxidizing
effect of chemicals or microorganisms. They can be degraded into CO2 and water
which are not toxic.
As there is a redox gradient proportional to depth both in water and soil, this
controls the form and rate of oxidation for the contaminants and other components
of the environment.
OECD has issued Test Guidelines for testing fate properties and REACH made
them obligatory and uniformly applicable. The key tests for the characterization of
transport, fate and behavior of chemical substances in the environment are shown in
Table 2.1.

Table 2.1 OECD guidelines for the measurement those physico-chemical properties of chemicals that
influence their environmental fate and behavior.

Test Guideline Test name

OECD TG 101 (1981) UV-VIS Absorption Spectra

OECD TG 104 (2006) Vapor Pressure
OECD TG 105 (1995) Water Solubility
OECD TG 106 (2000) Adsorption–Desorption Using a Batch Equilibrium Method
OECD TG 107 (1995) Partition Coefficient (n-octanol–water) – Shake Flask Method
OECD TG 108 (1981) Complex Formation Ability in Water
OECD TG 111 (2004) Hydrolysis as a Function of pH
OECD TG 112 (1981) Dissociation Constants in Water
OECD TG 113 (1981) Screening Test for Thermal Stability and Stability in Air
OECD TG 116 (1981) Fat Solubility of Solid and Liquid Substances
OECD TG 117 (2004) Partition Coefficient (n-octanol–water) – HPLC Method
OECD TG 120 (2000) Solution/Extraction Behavior of Polymers in Water
OECD TG 121 (2001) Estimation of the Adsorption Coefficient (K oc ) on Soil and on Sewage Sludge
using High Performance Liquid Chromatography (HPLC)
OECD TG 123 (2006) Partition Coefficient (n-octanol–water) – Slow-stirring Method
84 Engineering Tools for Environmental Risk Management – 2

3 INTERACTIONS OF CHEMICAL SUBSTANCES

WITH THE BIOTA

Interactions of chemicals with the biota reflect both the ability of the chemical sub-
stance to become available and accessible to living organisms in the environment and
the genetic, metabolic, physiological and behavioral characteristics of the living organ-
ism to respond to it. This response can be detrimental, lethal or adaptive, determining
the survival of the individual, the population or community.
Adverse effects such as toxic, mutagenic, reprotoxic, hormone and immune system
disrupting and sensitizing effects are discussed in detail in Chapter 3. In the course of
interactions with the contaminants, the sensitivity of an organism and the adaptive
behavior of a community in the environment are essential: for example, emergence
and dispersal of one single gene in a microbial community can reverse the response
from lethality to survival.
Adaptation, a change in the genome of individual organisms and its rapid spread
to the whole population and the community, is a basic property of microorganisms
which typically live in soils, sediments, slurries and sludges, in liquid and solid wastes.
Adaptation plays an essential role in environmental biodegradation, biotransforma-
tion, bioaccumulation, bioleaching and biostabilization in general, and, in particular,
related to contaminants and xenobiotics.
A chemical’s three specific properties used to describe its potential hazard to the
environment and to humans through the environment are summarized here.

– Toxicity and other adverse effects: the hazard posed by a substance to living organ-
isms, based on toxicity measured by aquatic and terrestrial organisms, bacteria,
fungi, plants, animals and humans.
– Degradability: persistence of the substance in the environment, based on its
molecular structure or test results.
– Bioaccumulation/bioconcentration: the accumulation of a substance in living
organisms, and toxication of humans and other top predators causing biomag-
nification through the food chain.

Toxicity and its testing are discussed in Chapters 3, 4 and 5, biodegradation and
bioaccumulation in the following two sections (3.1 and 3.2).

3.1 Biodegradation and biotransformation

Biodegradation of chemical substances, mainly xenobiotics, is a key process of the
elimination of chemicals from the environment both spontaneously and by engineering
solutions using biological and ecological technologies. The risk of a chemical substance
is primarily determined by its biodegradability which, together with its toxicity and
bioaccumulative potential, determines the type and scale of risk, the risk management
methods, the urgency of intervention and the legal obligations.

3.1.1 Classification of environmental fate of chemicals for

regulatory purposes
Biodegradation and bioaccumulation play key roles in the categorization of the
most dangerous chemicals, the so-called PBTs (Persistent, Bioaccumulative and Toxic
 Fate and behavior of chemical substances in the environment 85

chemicals). Classification is based on the combination of persistency, bioaccumula-

tive potential and toxicity within the REACH, and is described in Annex XIII of the
regulation (PBT REACH, 2008). The REACH criteria are the following:

– Persistent (P) if bioaccumulation half-life is:

◦ t1/2 > 60 days in marine water or
◦ t1/2 > 40 days in fresh- or estuarine water or
◦ t1/2 > 180 days in marine sediment or
◦ t1/2 > 120 days in fresh- or estuarine sediment or
◦ t1/2 > 120 days in soil.
– Bioaccumulative (B), characterized with a bioconcentration factor of
◦ BCF >2000 L/kg or Kow ≥ 4.
– Toxic (T), and fulfilling the following chronic toxicity criteria:
◦ NOEC (long-term) <0.01 mg/L;
◦ substance is classified as carcinogenic (category 1 or 2), mutagenic (category
1 or 2), or toxic for reproduction (category 1, 2 or 3) or
◦ there is other evidence of chronic toxicity, as identified by the classifications:
T, R48, or Xn, R48 according to Directive 67/548/EEC.

Another priority group within the REACH Regulation is formed by PBTs or vPvBs:

– Very persistent (vP) characterized by the biodegradation rate of:

◦ t1/2 > 60 days in marine, fresh- or estuarine water or
◦ t1/2 > 180 days in marine, fresh- or estuarine sediment or
◦ t1/2 > 80 days in soil.
– Very bioaccumulative (vB) is a substance which fulfills the biodegradation
criterion of:
◦ BCF > 5000 L/kg or Kow > 5.

Both PBTs and vPvBs are subjected to close scrutiny in the EU as they may have a
long-term impact on the environment.
Quantitative criteria for persistence, bioaccumulation and toxicity in other coun-
tries are also strictly regulated, in Canada, for example, the following applies (GM
CEPA, 1999):

– Persistence: half-life values expressed in days

◦ air ≥ 2
◦ water ≥ 182
◦ sediment ≥ 365
◦ soil ≥ 182
– Bioaccumulation:
◦ bioconcentration factor, BCF ≥ 5000 or
◦ log Kow ≥ 5
86 Engineering Tools for Environmental Risk Management – 2

– Toxicity (mg/L)
◦ acute hazardous effect L(E)C50 ≤ 1
◦ chronic hazardous effect NOEC ≤ 0.1.

The regulations also suggest that, in addition to toxicity, the environmental fate
properties, first of all biodegradability, determine the environmental risk of chemicals
and their categorization as PBT.

3.1.2 Biodegradation – definitions

Before introducing the biodegradation measurement methods, some terms must be
clarified in connection with biodegradation of chemicals.
Biodegradability or more precisely a chemical’s potential to be degraded by bio-
logical systems is an intrinsic material characteristic, which depends on the chemical
structure and molecular mass of the chemical substance.
Biodegradation is the biological decomposition or breakdown of a substance. The
term is mainly used for the action of environmental microorganisms such as bacteria
or fungi. Biodegradation is affected by both the chemical substance’s structure and
resistance to biodegradation and the biological activity of microorganisms, or the com-
munity of microorganisms in the environment, which can be highly diverse depending
on climate, season and the adaptive character and behavior of the degrading commu-
nity. The presence of other chemicals is another influencing factor. Mineralization is
distinguished by biodegradation: it denotes the natural process of complete biodegra-
dation in which organic matter is fully degraded under aerobic or anaerobic conditions.
As a result, gaseous or water-soluble inorganic residues of H2 or H2 O are produced
from all reduced organic molecules, C or CO2 from organic carbon, NH3 or NO− 3
−
from nitrogen-containing organic molecules, PO3− 4 from phosphorous- and S 2−
, SH
2−
or SO4 from sulfur-containing biomolecules, and positive or negative ions from all
kinds of metals, originally contained in living or dead organic matter. These inorganic
forms are available and can be taken up by plants and other primary producers in soil
and sediment. Mineralization is responsible for the biogeochemical element cycles i.e.
the transition from biologically controlled forms to abiotic forms in the Earth’s hydro-
sphere, lithosphere, and the atmosphere. Many of the xenobiotics are only partially
biodegraded, but not mineralized, so they become excluded from the closed element
cycles for a long time.
Primary biodegradation changes the identity of the parent chemical as a first step
toward more extensive mineralization, whereas ultimate biodegradation results in
the complete mineralization of the chemical to water, carbon dioxide and inorganic
compounds.
Biotransformation of chemical substances plays a role in higher-order organisms
so that they can be more readily eliminated whereas simpler organisms such as bacteria
and fungi are responsible for most of the extensive biodegradation and elimination of
substances from the environment as part of the mineralization process. Biotransforma-
tion can also be aimed to reach a stable form of the toxicant which is not available for
the organisms during their normal lives. The stable form may include reserve nutrients,
defender compounds or any kind of harmful material stored after translocation and
sequestration in organelles. These biochemically and genetically established protective
 Fate and behavior of chemical substances in the environment 87

mechanisms ensure reduced risk for the species, but not for consumers that come next
in the food chain.

3.1.3 Biodegradation – the process

Biodegradation of contaminants in the environment as part of the mineralization pro-
cess means that the community of microorganisms tries to utilize its existing genetic
and metabolic tools to produce energy from any form of a chemical substance that
contains energy (molecules in reduced form). If their existing metabolic potential is
not enough to acquire this chemically rapidly available energy, they try to adapt them-
selves to the ‘new’, less accessible, often toxic energy source by genetic modifications.
These include:
– Switching on formerly switched-off (reserve) genes;
– Increasing the mutagenic rate to multiply the number of mutations and selecting
the beneficial mutants for replication through normal reproduction;
– Creation of genetic recombinants by horizontal gene transfer, meaning the delivery
(handover) of genes between the cohabiting members of the population or the
whole community without any sexual event generating offspring.
For the mineralization of a chemical, several enzymes must act sequentially to
transform or break down the chemical substance to molecules that enter the pathways
of intermediary metabolism. Synthetic chemicals that are likely to be substrates for
enzyme(s) produced by microorganisms can enter these pathways and become miner-
alized. Xenobiotics, not being complementary to any existing enzymes, have no chance
of entering any of the active metabolic pathways and become degraded.
The rate of biodegradation in soil, as an example is characterized by the following
equation (EU-TGD, 2003):
kbiodeg soil = ln 2/D50 biodeg soil
where
– kbiodeg soil : first-order rate constant of biodegradation (1/day);
– DT 50 : half-life of the chemical substance (days);
– ln: natural logarithm (to the base e).
Biodegradation of chemical substances in water and soil is classified according to
their biodegradation rate. The classes proposed by the European guidance are shown
in Table 2.2.
The rate of biodegradation in surface water, soil and sediment is related to the
chemical structure and other environmental properties, such as Kow of substances,

Table 2.2 Biodegradation classes, relevant rate constants and half-lives.

Classification of biodegradation k (1/day) DT50 (days)

Readily biodegradable 4.7 × 10−2 15

Readily, but failing 10-day window 1.4 × 10−2 50
Inherently biodegradable 4.7 × 10−3 150
Not biodegradable 0 ∞
88 Engineering Tools for Environmental Risk Management – 2

microbial activity, and environmental conditions such as temperature, pH, redox

potential, moisture content, etc. These properties vary from site to site: the random
adaptation of the microbiota yields biodegradation of a site-specific character. There-
fore the best practical solution is to directly measure biodegradation as developed at
a certain location in soils, sediments or wastes.
For generic and regulatory purposes, the notion of ‘biodegradability’ of a chemical
substance is being used, which is a function based on quantitative structure–activity
relationship (QSAR) models or standard bioassays and is the intrinsic property of
a chemical substance. QSAR applies the knowledge of structure and biodegradability
relationships to create a mathematical model. The already existing information, i.e. test
results, are collected and statistically evaluated and a generally applicable QSAR equa-
tion is established, which can be used to calculate biodegradability of most chemical
substances when their chemical structure and/or Kow is known.
Laboratory bioassays measuring biodegradability are not well established because
the test cannot be applied to a stable, uniform, standardized microflora, given that such
a microflora does not and will not exist. Even if it were established, its stability could
not be ensured (unchanged metagenome) for a long time. It cannot be guaranteed either
that specific genes, responsible for certain biodegradation pathways, would not be
present, or would not appear suddenly. So, one must be aware of all these shortcomings
when applying a standardized biodegradability test for the characterization of the
chemical substances’ intrinsic ability to be biodegraded.
Precise estimation is hampered by the simplifications of the models that assume
that the kinetics of biodegradation is of pseudo first-order, which is strictly speaking
not true in the real environment. Another simplification is that the dissolved portion
of the substance is considered as available for biodegradation, which is of course
incorrect in a dynamic system (shift in balance, mobilization and recharge, interactions
with microorganisms) and is refuted by the results of those interactive test setups
which let the interactions develop between solid-matrix-bound contaminants and living
organisms. The microbes produce biotensides, exoenzymes and mucilage to control
mobility and increase biological access to contaminants of strong tendency to be bound
to soil matrix.
Site-specific biodegradation tests may provide a realistic and adequate answer to
the elimination rate of a contaminant at a certain locality under natural conditions.
Biodegradation reduces the concentration of chemicals in the environment and
influences their adverse effects. If the chemical substance is degradable via photolysis,
hydrolysis or biodegradation, the amount that reaches the receptors and is capable
of exerting an adverse effect decreases in time. Emissions from the same amount of a
readily biodegradable and a persistent chemical substance – even if they have the same
level of toxicity – result in very different environmental concentrations after a certain
time. For example, the biodegradable one will be completely eliminated within two
weeks reducing its concentration to 0%, while 100% of the persistent one will remain
in the environment.

3.1.4 QSAR for biodegradation

QSAR, based on a database that contains a number (n) of empirical data on biodegra-
dation, establishes the mathematical relationship between the structure of a chemical
 Fate and behavior of chemical substances in the environment 89

substance and its biodegradability. One set of results, serving as the basis of a QSAR
equation, should be derived from the same standardized test method.
Qualitative information is available for many biodegradation pathways. Major
sources of empirical data are the University of Minnesota Biocatalysis/Biodegradation
Database (Wackett & Ellis, 1999), Biodegradability Evaluation and Simulation System
(Punch et al., 1996a,b; BESS, 2013), the Syracuse Research Corporation’s BIODEG
database (BIODEG, 2013), which contains over 5800 records of experimental results
on biodegradation studies for approximately 800 chemicals, and the MITI database of
the Japanese Ministry of International Trade and Industry (METI, 2013). Most of these
data are derived from laboratory studies. These databases have been used in model
development and can also be used in generic risk assessment. However, the established
models are not considered directly applicable for estimating environmentally relevant
biodegradation rates for a wide range of chemicals.
Amongst the many software developed for the estimation of biodegradation in
nature and biodegradability of a chemical for regulatory purposes, we emphasize the
Biodegradation Probability Program (BIOWIN, 2013), which estimates the probability
for the rapid biodegradation of an organic chemical in the presence of mixed popula-
tions of environmental microorganisms (Boethling & Sabljic, 1989; Raymond et al.,
1999; Howard et al., 1992; Boethling et al., 1994, 2003; Tunkel et al., 2000; Howard
et al., 2005; Pavan & Worth, 2006).
BIOWIN was developed by the Syracuse Research Corporation (SRC, 2013) and
the US EPA as part of the Estimation Program Interface Suite (EPISUITETM , 2013)
model package and is freely available from the US EPA website (US EPA, 2004).
BIOWIN includes six different models:
– Linear probability BIODEG;
– Non-linear probability BIODEG;
– Expert survey ultimate biodegradation model (USM);
– Expert survey primary biodegradation model (PSM);
– Japanese MITI linear and
– Japanese MITI non-linear (Arnot et al., 2005).
These are generally referred to as BIOWIN 1–6. Estimates are based on fragment
constants and molecular mass and require only a chemical structure. BIOWIN models
have been developed and tested for a range of chemical substances for assessing the
biodegradation potential of chemicals by regulatory agencies to exclude the uncertainty
due to the natural variability and adaptive character of degrading microflora. So one
can directly estimate biodegradation from the structure of the chemical substance,
which is the cheapest way to obtain useful data.
Some QSAR equations are introduced here, for the biodegradability of selected
chemical substances using the octanol–water partition coefficient (Kow ) and the charge
of chemical bonds or electrophilicity of the molecules for the determination of
biodegradability.
– Phthalate esters:
RC = −24.308 × log Kow + 394.84
RC: primary biodegradability n = 12 r2 = 0.87 (Boethling, 1986);
log: common logarithm (to the base 10);
90 Engineering Tools for Environmental Risk Management – 2

– Alcohols, phenols, ketones, carboxylic acids, ethers, sulfonates:

BOD = 1105 ·
/δ/x−y + 1.906

BOD: Biological Oxygen Demand n = 112 r2 = 0.98 (Dearden & Nicholson,
1987)

/δ/x−y : differences in the modulus of atomic charge across a functional group
bond x−y;

– Alcohols:

BOD = 0.093 × SE − 3.163

SE : electrophilic superdelocalizability n = 19 r2 = 0.96 (Dearden & Nicholson,
1987).

3.1.5 Aims of testing biodegradation

The principle of biodegradability testing is the same in any applied test method: degra-
dation of a chemical substance as the only carbon source. In biodegradability tests a
standardized culture medium containing microflora or artificial/natural water or soil
is used to indicate and monitor biodegradation. The most widespread way is mea-
suring O2 depletion and/or CO2 production during an aerobic biodegradability test,
but any other actor of the energy production and the respiration chain in the cells
can be selected and detected, most frequently the substrate, metabolites or enzymes.
Using alternative electron acceptors to indicate electron transport – the concomitant
of energy production – makes the evaluation easy, i.e. by producing a proportionate
amount of simply detectable colored product. These types of tests reveal the tendency
of a chemical substance to become biodegraded as its intrinsic property. In spite of
careful standardization, the composition of the microorganisms used in these stan-
dardized tests cannot be controlled completely: this may cause high uncertainties and
makes biodegradability of chemicals a fiction.
The simulation of biodegradation of a certain chemical substance under water,
sediment or soil conditions is a test type which combines the chemicals’ intrinsic
biodegradability with typical environmental conditions existing in water, sediment
or soil. Testing biodegradability in soil, a standard soil type with constant texture,
organic matter content, soil moisture, bulk density, pH and redox potential should
be ensured. Simulation tests provide useful results for the evaluation of the generic
risk of chemicals on aquatic or terrestrial ecosystems, but the uncertainties are high
and the differences between the generic and the site-specific ecosystem may deviate –
theoretically in both directions jeopardizing the conservative (pessimistic) approach to
be applied in an environmental risk assessment.
When extrapolating from generic biodegradability test results to actual biodegra-
dation at a real site, deviation mainly occurs in favor of biodegradation in a real
environment, due to ecosystem flexibility. The real biodegradation rate may be much
higher compared to the results of biodegradability or simulation tests due to the adap-
tation of the biodegrading microbiota at a contaminated site, especially if it has been
contaminated for a long time.
Testing local biodegradation in contaminated water, sediment or soil reflects
the complex situation and the progress of degradation. The outcome of site-specific
 Fate and behavior of chemical substances in the environment 91

biodegradation tests includes the results of interactions between contaminants, con-

taminated matrices and the degrading community. The intrinsic biodegradability of
the chemical substance is combined with its actual accessibility and bioavailability,
and with the genetic and biochemical potential of the exposed microbiota. Based on
biodegradation test results, the site-specific risk can be precisely assessed and adequate
risk reduction measures can be implemented. The biodegradability measurement meth-
ods cannot be standardized similar to biodegradability or generic simulation tests: the
main rule in site-specific testing is to copy or mimic real conditions as much as possible,
e.g. by using microcosms or mesocosms.
The most frequently used test set-ups for measuring biodegradation are the closed
and open bottles and the flow-through systems. These test systems are suitable for
testing water and soil under aerobic, anoxic and fully anaerobic conditions with or
without additive injection, component extraction or sampling.

3.1.6 Measurement end points for characterizing biodegradation

Biodegradation tests may detect the following end points:

– Substrates used for energy consumption or co-metabolism;

– The degrading cells’ density and activity;
– Enzymes produced playing a role in the biodegradation process;
– The metabolites and end products of the biodegradation.

The chemical analysis methods which are based on chemical substance abatement
measure the residue of the chemical substance and its possible metabolites.
Figure 2.4 shows the enzymatic reaction of biodegradation with the components
suitable as test end points. Symbols of the equation are:

– S: substrate which produces energy by aerobic/anaerobic respiration: the chemical

substance to be degraded.
– E: enzyme or enzyme system: proteins produced by degrading microorganisms to
break down the substrate into smaller units and oxidize it for energy extraction,
including the enzymes of aerobic/anaerobic respiration and the electron transport
chain (terminal oxidation).
– In : intermediate, a temporary enriched product, whose utilization varies;
– P: product, end product, e.g. CO2 as end product of aerobic mineralization;
– Ep : a measurable or easy-to-detect consequence of the presence of the product, e.g.
pH of an acidic product.

Figure 2.4 Enzyme reaction of biodegradation and measurable endpoints.

92 Engineering Tools for Environmental Risk Management – 2

Figure 2.5 Enzymatic reaction applying an indicator substrate.

In addition to the natural players of the substrate utilizing reaction, one may
apply artificial alternative substrates or intermediates, which results in special products
utilizing the enzymes active in the biodegrading cells.
Figure 2.5 shows the concept of using an assistant molecule in the test to facili-
tate the detection of the selected measuring end point of Si , Ii or Pi . Symbols of the
equation are:

– Si : indicator substrate, added to the test in addition to the chemical substance

(S) for easy indication. It can be a labeled substance or an alternative electron
acceptor, producing a color reaction as the effect of the enzymes.
– Ii : Intermediate from the indicator substrate.
– Pi : product of Si , an easy-to-indicate substance due to the radioactive label or color
reaction.

Respiration-based methods work in such a way that the chemical substance to be

tested for biodegradability is added to the ‘standard’ degrading microflora in artificial
or natural water, sediment or soil. Another concept suggests testing biodegradation in
the contaminated environmental sample itself by measuring the biodegradation poten-
tial of its own microflora. End points to be measured can be O2 depletion or CO2
production for aerobic biodegradation, gaseous products of H2 , CH4 , NH3 , H2 S for
anaerobic degradation or any other molecule which plays a role in energy production
and the respiratory chain of the cells. The use of alternative electron acceptors is the
most widely applied solution to indicate energy production from the contaminant.
Isotope-labeled chemicals make the evaluation easy and selective.
The measurement of one or more enzyme activities in the respiratory chain can be
used as an index for the total oxidative activities of the cell; therefore, dehydrogenase
enzyme activity has been used as a measure for overall microbial activity (Alef &
Nannipieri, 1995). The assay of dehydrogenase can be used as a simple method to
investigate microbial activities and possible inhibitory effects of the contaminants on
the microorganisms. Nearly all microorganisms reduce 2,3,5-triphenyl-tetrazolium-
chloride (TTC), this artificial electron acceptor, to triphenyl formazan (TPF), which
can be measured colorimetrically.
The dehydrogenase enzyme activity test based on the estimation of the rate of TTC
reduction to TPF can be characterized and used as an indicator for overall microbial
activity of the soil (Molnár et al., 2009a). For this test, field-moist soil (5 g) is incubated
for 24 h at 28◦ C in TTC–tris–HCl buffer solution. TPF concentration is measured by
colorimetry after extraction with acetone at 546 nm. For the interpretation of the
dehydrogenase activity, the TPF concentration is given in µg TPF/g soil.
 Fate and behavior of chemical substances in the environment 93

Table 2.3 OECD Guidelines for testing the biodegradability of chemical substances.

Test guideline Test name

OECD TG 301 (1992) Ready Biodegradability

OECD TG 302A (1981) Inherent Biodegradability: Modified Semi-Continuous Activated Sludge
(SCAS) Test
OECD TG 302B (1992) Inherent Biodegradability: Zahn-Wellens/EVPA (EMPA) Test
OECD TG 302C (2009) Inherent Biodegradability: Modified MITI Test (II)
OECD TG 303 (2001) Simulation Test – Aerobic Sewage Treatment – A:Activated Sludge Units; B:
Biofilms
OECD TG 306 (1992) Biodegradability in Seawater
OECD TG 307 (2002) Aerobic and Anaerobic Transformation in Soil
OECD TG 308 (2002) Aerobic and Anaerobic Transformation in Aquatic Sediment Systems
OECD TG 309 (2004) Aerobic Mineralization in Surface Water – Simulation Biodegradation Test
OECD TG 310 (2006) Ready Biodegradability – CO2 in Sealed Vessels (Headspace Test)
OECD TG 311 (2006) Anaerobic Biodegradability of Organic Compounds in Digested Sludge: by
Measurement of Gas Production
OECD TG 314 (2008) Simulation Tests to Assess the Biodegradability of Chemicals Discharged in
Wastewater

Soil respiration can also be determined on the basis of CO2 production or O2

consumption in closed static jars and also in a dynamic flow-through system. CO2
is usually trapped in NaOH and determined by HCl titration or by measuring the
electrical conductivity of the NaOH solution. Manometric indication can be applied
in a closed apparatus, and a direct CO2 analysis in flow-through systems, e.g. by
infrared spectroscopy, due to the asymmetrical stretch of CO2 giving a strong band in
the IR spectrum at 2350 cm−1 .
As a microbiological end point of reproduction/growth of a microbial culture, the
number of cells or their measurable structural and functional compartments or prod-
ucts are suitable (cell number, growth curve, nitrogen content, chlorophyll content,
light emission, etc.), when directly associated with biodegradation (Gruiz et al., 2001;
Hyman & Dupont, 2001).

3.1.7 Standardized biodegradability test methods for chemical substances

Biodegradability tests aim at the generic characterization of the fate and behavior of
chemical substances in the environment, but their objective also includes determination
of the site-specific biodegradation rate.
The OECD test guidelines – prepared to measure the generic biodegradability
of chemical substances – are shown in Table 2.3. Simulating aquatic ecosystems or
wastewater, the test mainly applies to microorganisms from the environment or simply
natural water, sediment or wastewater.
The biodegradability of chemical substances provides priority information for
authorization, classification and restriction of chemicals in European or other regional
regulations. Based on the test results, decisions are taken throughout Europe and
biodegradation results of the listed OECD tests are applied as generic values. They
can be applied in the conservative approach for generic environmental risk assessment
94 Engineering Tools for Environmental Risk Management – 2

Table 2.4 Standardized ISO tests for testing biodegradation in the environment.

Test guideline Test name

ISO 7827:2010 Evaluation of the ‘ready’,‘ultimate’ aerobic biodegradability of organic compounds

in an aqueous medium – Method by analysis of dissolved organic carbon (DOC)
ISO 14592-1:2002 Evaluation of the aerobic biodegradability of organic compounds at low
concentrations – Part 1: Shake-flask batch test with surface water or surface
water/sediment suspensions
ISO 14593:1999 Evaluation of ultimate aerobic biodegradability of organic compounds in
aqueous medium – Method by analysis of inorganic carbon in sealed vessels (CO2
headspace test)
ISO 14592-2:2002 Evaluation of the aerobic biodegradability of organic compounds at low
concentrations – Part 2: Continuous flow river model with attached biomass
sealed vessels (CO2 headspace test)
ISO 9408:1999 Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous
medium by determination of oxygen demand in a closed respirometer
ISO 9439:1999 Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous
medium – Carbon dioxide evolution test
ISO 10634:1995 Guidance for the preparation and treatment of poorly water-soluble organic
compounds for the subsequent evaluation of their biodegradability in an
aqueous medium
ISO 10707:1994 Evaluation in an aqueous medium of the ‘ultimate’ aerobic biodegradability of
organic compounds – Method by analysis of biochemical oxygen demand
(closed-bottle test)
ISO 10708:1997 Evaluation in an aqueous medium of the ultimate aerobic biodegradability of
organic compounds – Determination of biochemical oxygen demand in a
two-phase closed-bottle test
ISO 16221:2001 Guidance for determination of biodegradability in the marine environment

because real biodegradation rates tend to be higher than those in the tests due to greater
diversity and adaptation in the environment.
Table 2.4 shows some ISO biodegradability test standards.

3.1.8 Measuring biodegradation in soil

Testing biodegradation in a defined environment needs in situ measurement or the
simulation of the real situation under laboratory conditions. This can be done by
standardized or non-standardized test methods. Some of the possibilities are introduced
here, with the focus on soil, where many of the contaminants are finally eliminated by
myriads of microorganisms.
The direct chemical analysis of contaminant residues in the soil or groundwater
determines overall losses and thus may provide equivocal evidence for biodegradation
(Crawford, 2002; Alvarez & Illman, 2006), provided that abiotic degradation does
not occur. A decrease in concentration or a change in composition may be a conse-
quence of abiotic mechanisms such as volatilization, leaching and complexation to soil
organic matter, or may be due to biotic processes such as biodegradation. A variety of
quantitative and qualitative techniques are available to measure organic contaminant
losses in the environment (in soil, sediment, groundwater). The choice of a particular
 Fate and behavior of chemical substances in the environment 95

method usually depends both on the type of the contaminant and the matrix. For
example, the biodegradation of a single and easy-to-analyze biodegradable contam-
inant should be tested by a substance-specific chemical analysis, but biological end
points (e.g. production of CO2 or degrading enzymes) should have priority for the
biodegradation of complex mixtures such as petroleum products (e.g. TPH or PAHs),
or mixed and unknown contaminants.
The analysis of the organic contaminants usually requires separation from the
matrix by extraction with organic solvents, assisted by ultrasound or microwave
digestion. Sometimes, further separation may be necessary before determining the con-
centration of the components of mixtures. The measured end point is a concentration
value in the extract. Solvents of different polarities (water, fluid state CO2 , DMSO,
hexane, acetone, n-pentane, dichloromethane, etc.) are used for extraction (separation
of the contaminant from the matrix) depending on the chemical nature of the contam-
inant to be analyzed. Mixtures of contaminants are further separated into components
or fractions before their quantitative determination using fractionated extraction, gas
chromatography, high performance liquid chromatography, capillary electrophoresis
or any other suitable separation method. The contaminant residues may be quantita-
tively determined by gravimetry, conventional chemical analysis, mass spectrometry
or atomic and molecular spectrometric methods, such as UV or visible light (UV-VIS),
infrared (IR) and fluorescence spectroscopy. FID (flame ionization detection), based on
the detection of ions formed during combustion of organic compounds in a hydrogen
flame, became a frequently used detector, mainly coupled with gas-chromatography.
Biological methods play an important role in measuring aerobic and anaerobic
biodegradation in the environment, in contaminated surface waters or wastewaters,
dredged or living sediments, or soils and groundwater.

3.1.9 Soil respiration, biodegradative activity of the soil –

problem-specific applications
Measuring soil respiration is one of the oldest and the most frequently used methods
for quantifying microbial activity in soils (King et al., 1992; Alef & Nannipieri, 1995;
Crawford, 2002; Alvarez & Illman, 2006).
Soil respiration in a ‘closed bottle’ is applied for (Molnár et al., 2009a):

– Testing the biodegradability of chemicals under standard conditions;

– Measuring the actual biodegradation in contaminated soil;
– Testing enhancement of biodegradation in soil and sediment;
– Testing toxicity in soil.

Manometric respirometry: the closed-bottle technique

Closed bottles filled with real soil can be considered as a special soil microcosm as
discussed in Chapter 9. Manometric respirometry carried out in a Sensomat system
applies a piezoresistive pressure sensor technology. Test conditions can be properly
standardized, and the soil used can be ‘generic’ or site-specific. Since the continuously
decreasing oxygen concentration is unrealistic, the method is of limited applicability
for the assessment of realistic biodegradability. However, proper interpretation of the
96 Engineering Tools for Environmental Risk Management – 2

Figure 2.6 Closed bottle for testing the respiration rate in soil based on manometry. The pressure
decrease is detected by the sensor in the cap and read remotely.

results can yield a useful and sound indicative tool, similar to BOD (biological oxygen
demand) used in wastewater tests.
The Sensomat System for respiration and biodegradation testing in contaminated
soil (Figure 2.6) was characterized and compared to other test methods by Molnár
et al. (2009a). The pressure that develops in the closed bottle filled with moist soil
due to microbial activity and gas exchange is measured and logged with the pressure
sensor on top of the jar. A vial containing NaOH is placed in each vessel to trap CO2 . If
oxygen is consumed in the closed vessels at a constant temperature, negative pressure
develops. The pressure difference (decrease) due to microbial activity in closed vessels
is generally measured for 5 days at 25◦ C and read remotely. For the interpretation of
the results, the linear part of the respiration curve (pressure–time) can be used from
which the respiration index can be determined by linear regression.
Advantages of the manometric method are:

– simplicity: no dilution of sample required, no seeding, no blank sample;

– direct reading of pressure, CO2 production or O2 consumption values;
– continuous display of any of the end point values at the current incubation time.

Figure 2.7 illustrates the biodegradation of crude oil in the closed-bottle system.
The blue curve shows the freshly contaminated soil’s respiration in the first 24 hours,
which is negligible, indicating no oxygen consumption, no biodegradation. Testing
after a two-day adaptation period – demonstrated by the pink curve – the soil micro-
biota was able to utilize the hydrocarbon mixture as an energy source, indicated by
the decline of the pink curve due to oxygen consumption. A steepening negative slope
of the curve indicates ongoing adaptation and increasing respiration.
As another example, Figure 2.8 shows the respiration curves of the biodegradation
of transformer (PCB-free) oil in soil. Transformer oil was added to an average-quality
forest soil in concentrations of 10, 20 and 30 g/kg. The pressure decrease (increase in
 Fate and behavior of chemical substances in the environment 97

Figure 2.7 Respiration curve of crude oil in soil immediately and 2 days after contamination (MOKKA
Project, 2004–2008).

Figure 2.8 Transformer oil biodegradation in soil; tested in a closed bottle by measuring the pressure
decrease due to oxygen consumption (MOKKA Project, 2004–2008).

the negative direction) is proportional to O2 consumption. Respiration rate is propor-

tional to biodegradation in the soil containing 10 g/kg contaminant, and is inhibited
after 35 and 21 hours in soils contaminated with 20 and 30 g/kg oil. The inhibition is
due to the decreasing oxygen content in the bottle.
The closed-bottle soil respiration test faces two problems: the continuously
decreasing oxygen concentration and the microbiota’s capability of switching from
98 Engineering Tools for Environmental Risk Management – 2

Figure 2.9 Flow-through system for measuring the respiration rate in soil. The inlet air is free of CO2 .
CO2 produced is measured in the outlet air.

aerobic to anoxic metabolism after the consumption of oxygen and producing pressure-
increasing gases, e.g. N2 and H2 , which makes impossible the evaluation based on
pressure decrease.

Flow-through soil-column
A simple flow-through system can be applied to measure the respiration rate of con-
taminated water or soil and the effect of various engineering measures to enhance
biodegradation rate (Gruiz et al., 2001; Molnár et al., 2005; 2009a). The evaluation
is performed by the continuous or frequent measurement of CO2 production and the
concentration of residual contaminants at the end of the experiment. Flow-through
column reactors of a volume of 1–20 L and a height of 0.5–2.5 m can be used in
laboratory biodegradation tests of petroleum hydrocarbons, PAHs, transformer oils,
creosote, pesticides and any other aerobically biodegradable organic contaminants in
soil or water.
The soil column in Figure 2.9 is filled with the control (uncontaminated) and
artificially contaminated soils, or soils derived from contaminated sites. The column
reactor is generally aerated for 2–24 h daily with different aeration rates. From expe-
rience, 24 h/day aeration does not make any difference compared to 2–3 × 1–2 h/day
in the case of soil. Two flow-through traps filled with NaOH ensure CO2 -free atmo-
spheric air. The CO2 -free air is sucked through the soil columns by a water jet pump,
ventilation or a small vacuum pump. Air flow is measured by a gas meter. The CO2
produced by the soil microorganisms during the aeration period is trapped in 1 N
NaOH solution and determined by HCl titration or measured by CO2 meters based
on infrared spectrography.
The cumulated CO2 production by the microorganisms in the soil column is pro-
portional to the biodegradation/mineralization. The contaminant to be tested is not the
only carbon source (unlike in water or in liquid phase test solutions). Therefore, CO2
production in the uncontaminated soil is simultaneously measured, and the biodegra-
dation rate of the tested contaminant is calculated from the difference. Figure 2.10
shows the CO2 production in two soils contaminated with 10,000 mg/kg diesel (blue)
and 10,000 mg/kg crude oil (brown), compared to the uncontaminated control soil
 Fate and behavior of chemical substances in the environment 99

Figure 2.10 Diesel oil and crude oil biodegradation in soil: CO2 production compared to control soil
(Molnár, 2006).

(green). The curves indicate that diesel oil started to degrade on the second day, while
crude oil only after the third. A certain fraction of diesel oil was rapidly degraded on
the second day, but the degradation rate from the third day onwards is similar to that of
the crude oil: the two curves rise in a parallel fashion. The residual contaminant mass
or the number of cells can be used to validate the test results of CO2 production. 82%
of diesel oil was removed by biodegradation, and 66% of crude oil. The cell number
of oil-degrading microorganisms on the 7th day was 3 × 107 in the soil contaminated
with diesel oil and 2.5 × 107 in the one contaminated with crude oil.
The use of radioactive isotope-labeled chemical substances in biodegradation stud-
ies and the application of radioactivity as the measured end point increase the specificity
and selectivity of the biodegradation tests, both in chemical biodegradability testing
and soil biodegradative activity testing. Labeled substrates can also be used as an
internal standard in the biodegradation studies.

Cell counting for contaminant-utilizing microorganisms

Microbial enumeration is an indirect method for characterizing microbial activity in the
environment; it provides data for the estimation of microbial vitality and forecasting
biodegradation potential. It is important to note that uncertainties may result in real
soil from other available energy sources out of the contaminant and by the isolation
and growth methods, which may only capture a small percentage of the total microbial
community. Cell counting by direct microscopy and viable plate counts of organisms
able to grow on agar media are enumeration methods that are widely applied.
The quantitative metrics of soil microbiota (cell concentration) may be closely
related to the biodegradation of a contaminant if it is the only substrate for energy
100 Engineering Tools for Environmental Risk Management – 2

production or biosynthesis. In average soils with high nutrient and humus content, the
numbers of living microbial cells in general are not closely associated with the micro-
bial utilization of a contaminant. To obtain a contaminant biodegradation-related cell
concentration, microbes from the soil should be grown on a selective nutrient medium
containing the contaminant as the only (or the main) source of energy and biosynthesis.
This may be done in a liquid medium for cell growth (counting cells as an end point) or
on agar medium for colony forming (counting colonies as an end point). The number
of colony-forming microorganisms is always smaller than their real number in the soil
so that the measured cell concentration values can only be used for comparison of the
differently treated samples or processes in time.
The aerobic heterotrophic bacterial cell concentration can be determined by colony
counting after cultivation of microorganisms in a soil suspension (in water) on peptone–
glucose–meat-extract nutrient agar plates in Petri dishes. When the aim is to count the
fungal cells, nutrient media containing carbohydrates (molasses, saccharose, etc.) and
yeast extract are applied for the cultivation of soil-living microfungi. The colonies are
counted at 3–5 different dilutions after 48 h. The calculated averages are presented as
colony-forming units (CFU/g soil) (Gruiz et al., 2001; Molnár, 2006; Molnár et al.,
2005; 2009b).
Population densities of specialized contaminant-degrading bacteria can be best
estimated by applying a serial dilution to the point of extinction in numbers of cells.
The method is called limiting dilution analysis and is used to measure the abundance of
cells present in a mixed population and able to perform a particular function: degrading
a specific contaminant in this case.
Performing 5–9 simultaneous tests, the statistical method of Most Probable Num-
ber (MPN) can be applied based on cell density or appearance of color of an alternative
electron acceptor in the wells, which is identical to the presence (+) or absence (–) of
microorganisms. The calculation of cell concentration is possible without an actual
count of single cells or colonies (Alef & Nannipieri, 1995). The selective nutrient
broth-containing wells are inoculated with aliquots of the serial dilution. For the prop-
agation (growth) of the contaminant-degrading cells, a nutrient medium is prepared
containing the organic contaminant (petroleum products, pesticides, etc.) as the only
energy source. The dilution series prepared from the contaminated soils (minimum
three replicates) contains all of the soil microorganisms (see Figure 2.11), but only
those that can utilize the contaminant as an energy source will be able to grow (propa-
gate) (Gruiz et al., 2001; Molnár et al., 2005). The liquid medium is supplemented with
inorganic salt solution, trace elements and with an artificial electron acceptor of 2-(p-
iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). The Most Proba-
ble Number (MPN) is calculated from the red color (+/−) using probability tables.
The example in Figure 2.12 demonstrates the correlation of respiration rate and oil
degrading cell concentration in a soil contaminated with transformer oil after treatment
with a bioavailability enhancing, solubilizing agent (SA). The transformer oil in the
soil is difficult to access for oil degrading microorganisms, so that it was treated with
the oil solubilizing agent (SA) in increasing concentrations (SA 0.1, 0.5 and 1.0%). The
additive increased pollutant bioavailability, which resulted in higher cell concentrations
and respiration rates in the flow-through reactors. Cell concentration was measured
in a nutrient medium containing transformer oil as the only carbon source, using the
above described limiting dilution method.
 Fate and behavior of chemical substances in the environment 101

Figure 2.11 Dilution series for determining the most probable cell concentration.

Figure 2.12 Increase in transformer oil-degrading cell concentration and respiration rate due to
treatment with a bioavailability-enhancing agent (Molnár, 2006).

ATP in the cells – a biochemical endpoint

The biodegradative activity of the biota can be quantitatively characterized by measur-
ing the adenosine triphosphate (ATP) concentration in the microorganisms. ATP is the
central reactant in both the energy-producing (catabolic) and biosynthetic (anabolic)
reactions within the cells and is proportional to energy production. One of the most
straightforward methods is based on use of the luciferin reaction (Paul & Clark, 1996).
The substrate luciferin can react with ATP and in the presence of Mg and O2 luciferase
breaks down to produce free adenosine monophosphate, inorganic P and light. This
emitted light can be measured by a photometer.
The prerequisite for using the ATP-proportional light for the characterization of
the biodegradation of chemical substances is that energy is produced from, and only
from, the chemical substance concerned in the test.
102 Engineering Tools for Environmental Risk Management – 2

Molecular techniques for measuring biodegradation potential

Genetic methods and molecular techniques are also powerful tools for characterizing
microbial communities and evaluating biodegradation processes in soil. For example,
current genetic techniques called fluorescent in situ hybridization using species-specific
oligonucleotides and competitive quantitative PCR have been used successfully for
counting microorganisms (Crawford, 2002). These methods can be used to enumerate
microorganisms without the need for a cultivation step. When detection of biodegrad-
ing microorganisms is the aim, the specific genes or related DNA sequences should be
identified and the hybridization probes for in situ hybridization and the primers for
PCR should be designed accordingly.

3.2 Bioaccumulation
Bioaccumulation, bioconcentration, and biomagnification are relatively new terms,
sometimes misused. Therefore, adequate definitions have been collected and posted
here.

3.2.1 Definitions
Bioaccumulation is the accumulation of contaminants in the tissue of organisms
through any route, including respiration, ingestion, or direct contact with contam-
inated air, water, soil, sediment, pore-water, or dredged material. It is the most general
process, including the uptake from food.
Bioaccumulative potential is the inherent potential of a substance to accumulate
in organisms. Its application in a general sense – for regulatory purposes – bears high
uncertainty due to the different pathways of the chemicals in different organisms,
depending on uptake, elimination kinetics, metabolism, organ-specific accumulation
and the level of bound residues.
Bioaccumulation in the environment depends on specific environmental scenarios,
the nature of the chemical substance, environmental conditions and the characteristics
of the receptor organisms. All of these play a role in the realizable bioaccumulation, in
the bioaccumulated amount and in the mode of bioaccumulation. We know of defense
mechanisms inhibiting uptake and/or accumulation, and hyperaccumulation, e.g. those
plants which are capable of extracting toxic metals from soil. Bioaccumulation is the
result of the following processes:

– Absorption: chemical transport across a biological membrane into the organism;

– Distribution after absorption by circulation throughout the body;
– Metabolism by enzymes (biotransformation);
– Excretion: elimination of the chemical.

Bioavailability greatly influences bioaccumulation. The bioavailable fraction of

the total concentration (e.g. in water or soil) is that which can be absorbed by the
organisms via a specific route of intake. It does not depend on the rate at which the
chemical is absorbed and accumulated.
Bioconcentration: this term has been monopolized for the process which pro-
duces a net accumulation of a chemical directly from water into aquatic organisms
resulting from simultaneous uptake (e.g. by gill or epithelial tissue) and elimination
 Fate and behavior of chemical substances in the environment 103

(Rand & Petrocelli, 1985; Rand, 1995). In a more general sense, the term bioaccumu-
lation is used for all kinds of uptake and accumulation of chemicals from air, water, soil
and sediment into the body of an organism. In the real environment the accumulation
via inhalation, oral and dermal routes are added together, resulting in an aggregated
body burden.
Bioconcentration factor (BCF) is used in the EU (EU-TGD, 2003), and is also
known as the bioaccumulation factor (BAF), mainly by the US EPA (Kravitz, 1998),
but both have the same meaning: the ratio of a substance’s concentration in the tissue
of an aquatic organism to its concentration in ambient water, in situations where the
organism is exposed to the substance through the water only, and the ratio does not
change substantially over time. This is the static bioconcentration factor, the ratio
of the concentration in the organism to the concentration in water in a steady-state
(dynamic equilibrium) situation. When uptake and depuration kinetics are measured,
the dynamic bioconcentration factor can be calculated from the quotient of the uptake
and depuration rate constants:
BCFfish = Cfish /Cwater = k1 /k2 ,
where
– Cfish : concentration in fish (mg/kg);
– Cwater : concentration in water (mg/L);
– k1 : uptake rate constant from water (L/kg/day);
– k2 : elimination rate constant (1/day);
– BCF fish : bioconcentration factor (L/kg).
Others than the equilibrium model based on Kow are also used for the determina-
tion/calculation of BCF. The most widespread BCF calculation models are:
– Equilibrium partitioning models (depending on physico-chemical properties of the
chemical substance).
– Correlation with Kow (see aquatic and terrestrial bioaccumulation).
– Correlation with water solubility or other chemical properties.
– Physiological models (relying on physico-chemical properties of the chemical and
biological attributes of the target animal).
– FGETS (Food and Gill Exchange of Toxic Substances) simulation model, for exam-
ple, uses fish’s gill and intestinal morphometry, the body weight of the fish, and
fractional aqueous, lipid, and structural organic composition (EPA Food Chain
Model, 2013).
– PBPKs, physiologically based pharmacokinetic models describe the chemical’s
kinetics in an animal species using literature and measured parameters. The model
can be applied for simulating time trends.
Body burden is the result of bioaccumulation from the point of view of the organ-
isms: it means the amount of chemicals which are not readily excreted and so can
remain for years in the blood, adipose (fat) tissue, semen, muscle, bone or brain tissue,
or in certain organs of the organisms.
Biomagnification is the result of the process of bioconcentration and bioaccu-
mulation by which tissue concentrations of bioaccumulated chemicals increase as the
chemical passes up through two or more trophic levels. The term implies an efficient
104 Engineering Tools for Environmental Risk Management – 2

transfer of a chemical from food to consumer, so that residue concentrations increase

systematically from one trophic level to the next, resulting in an increase in the internal
concentration in organisms at higher levels in the trophic chain.
Secondary poisoning is concerned with toxic effects in the higher members of
the food chain living either in the aquatic or terrestrial environment, and which
results from ingestion of organisms from lower trophic levels that contain accumulated
substances.
Biomagnification factor (BMF) is defined as the relative concentration in a
predatory animal compared to the concentration in its prey:
BMF = Cpredator /Cprey .
The concentrations used to derive and report BMF values should, where possible,
be lipid normalized.
Biota-sediment accumulation factor (BSAF) is the relative concentration of a
substance in the tissues of an organism compared to the concentration of the same
substance in the sediment (US EPA and US ACE, 1998).
Bioaccumulative chemicals of concern (BCC) in sediment: chemicals identified as
a concern for sediment quality assessment because of their ability to accumulate in the
tissue of organisms through any route, including respiration, ingestion, or direct con-
tact with contaminated water, sediment, pore-water, or dredged material (US EPA &
US ACE, 1998).

3.2.2 Bioaccumulative potential of chemicals

The most important and widely accepted indication of bioaccumulation potential is
a high value of the n-octanol–water partition coefficient. It is accepted that values
of log Kow greater than or equal to 3 indicate that the substance may bioaccumulate
(EU-TGD, 2003). For certain types of chemicals, for example, surface-active agents
and those which ionize in water, log Kow values may not be suitable for the calculation
of a BCF. Also, metal concentration cannot be calculated from partitioning, because
Kow models only the lipid fraction of organisms’ tissues.
The theoretical correlation between Kow and bioaccumulation is modified by
or completely changed by the active transport of the chemical in the organism,
metabolic pathway-specific interactions with tissue components, receptors or enzymes,
uptake and depuration kinetics. Further conditions influencing bioaccumulation are:
adsorption and uptake, hydrolysis, degradation, and the size of the molecule.

Aquatic bioaccumulation
For substances with a log Kow of 2–6, the following linear relationship can be used as
developed by Veith et al. (1979) for aquatic organisms:
log BCFfish = 0.85 × log Kow − 0.70,
where

– Kow : octanol–water partition coefficient (−);

– BCF fish : bioconcentration factor (L/kg wet fish);
– log: common logarithm (to the base 10).
 Fate and behavior of chemical substances in the environment 105

Table 2.5 Biomagnification factors (BMF) for organic substances

estimated from bioconcentration in fish.

Log Kow of substance BCFfish BMF

<4.5 <2000 1
4.5–5 2000–5000 2
5–8 >5000 10
8–9 2000–5000 3
>9 <2000 1

Knowing the BCF values, future bioaccumulation can be estimated from environ-
mental concentrations: Cfish = Cwater × BCF.
Factors influencing a substance accumulation have an impact on BCF both in the
environment and during tests:

– Slow uptake;
– Steric hindrance or other obstacles to transfer the chemical into the body;
– Reduced bioavailability of the chemical substance;
– Organism growth and other changes, e.g. in lipid content;
– Ongoing metabolism of the chemical substance;
– Variation in the concentration of the chemical substance in water over time;
– Solubilization and mobilization of the contaminant by biotensides or other
contaminating tensides.

The biomagnification factor (BMF) should ideally be based on measured data.

However, the availability of such data is at present limited. Therefore in simple cases,
biomagnification can be estimated from bioaccumulation which originates from Kow .
Table 2.5 shows the recommendation of the guidance of REACH regulation (EU-TGD,
2003).

Terrestrial bioaccumulation
Bioconcentration can be described as a hydrophobic partitioning between the pore
water and the phases inside the organism and can be modeled according to the
following equation as described by Jager (1998):

0.84 + 0.012Kow
BCFearthworm =
RHOearthworm

where

– RHOearthworm : earthworm density, by an average of 1 (kgwwt /L) can be assumed.

106 Engineering Tools for Environmental Risk Management – 2

Using this equation, the concentration in the full worm can be calculated as:
BCFearthworm × Cpore water + Csoil × Fgut × CONVsoil
Cearthworm = ,
1 + Fgut × CONVsoil
where
– Fgut : fraction of gut loading in worm (kgdwt /kgwwt );
– CONV soil : conversion factor for soil concentration wet-dry weight soil
(kgwwt /kgdwt );

RHOsoil
CONVsoil = ,
Fsolid × RHOsolid
where
– Fsolid : volume fraction of solids in soil (m3 /m3 );
– RHOsoil : bulk density of wet soil (kgwwt /m3 );
– RHOsolid : density of solid phase (kgdwt /m3 ).

3.2.3 QSAR for bioaccumulation

The QSAR models of bioaccumulation of organic contaminants in the cells and tissues
of organisms are based on the Kow value, which is proportional to the binding of the
chemicals to any organic, e.g. lipid phases within the organism (Mackay & Fraser,
2000). This simple relationship is written as:
BCF = a × Kow ,
When Kow is less than 6, the following linear equation is recommended by Veith
et al. (1979):
log BCF = 0.85 × log Kow − 0.70
where
– log: common logarithm (to the base 10),
– n = 55, r2 = 0.90
Meylan et al. (1999) refined the correlation between BCF and log Kow for nonionic
and ionic compounds as follows:

– Nonionic organic compounds:

◦ log Kow < 1 → log BCF = 0.50;
◦ log Kow 1–7 → log BCF = 0.77 log Kow − 0.70 + Fi ,
(Fi = structural correction factor);
◦ log Kow > 7 → log BCF = −1.37 log Kow + 14.4 + Fi ;
◦ log Kow > 10.5 → log BCF = 0.50.
– Ionic organic compounds:
◦ log Kow < 5 → log BCF = 0.50;
◦ log Kow 5–6 → log BCF = 0.75;
◦ log Kow 6–7 → log BCF = 1.75;
◦ log Kow 7–9 → log BCF = 1.00.
 Fate and behavior of chemical substances in the environment 107

Fu et al. (2009) recommended the following equation for ionizable chemicals:

◦ log Kow (ion) = log Kow (neutral) − 3.5

Isnard and Lambert (1988) found linear correlation between the logarithms of
BCF and water solubility, investigating 107 chemicals with diverse water solubility of
0.02–36 000 mg/L:

◦ log BCF = −0.47∗ log S + 2.02

The application of QSARs for the estimation of the bioaccumulative potential of
chemicals is essential; otherwise, laboratory tests and the accompanying chemical anal-
yses of the bioaccumulated contaminants in the organism make the determination very
expensive. In addition, the biological model is a simplified model of reality, simulating
the uptake by one or a few organisms only (Pavan et al., 2006). Of course QSARs also
have disadvantages: they cannot model a complex organism and complicated interac-
tions, e.g. biochemical mechanisms and in the case of special chemicals, e.g. at high
Kow , the model’s validity is questionable.

3.2.4 Testing bioaccumulation

Empirical determination of bioaccumulation is a simple way to collect information on
the bioaccumulation of chemicals in aquatic and terrestrial organisms. Direct determi-
nation can be conducted using either laboratory-exposed or field-collected organisms,
and generally this approach minimizes or eliminates many of the problems associated
with QSAR models. Nevertheless, the number of chemicals and the cost of tests, as well
as the ethics associated with particular animals used for testing, justify the importance
of QSAR models.
The goal of testing bioaccumulation is to monitor existing bioaccumulation or
to predict bioaccumulation under future exposure conditions. For the prediction of
bioaccumulative risk, a generic approach can be used which characterizes chemi-
cal substances in general, creating a default bioaccumulation value which depends
primarily on the chemical substance and secondarily on a default environment, e.g.
the ‘European environment’, or the prediction can be based on the bioaccumulation
of a chemical in a well-known locality of the environment. Bioaccumulation tests
can be:

– Laboratory testing of the bioaccumulation of chemical substances in standardized

tests for regulatory purposes, e.g. the OECD tests for the purposes of REACH
regulation in aquatic and terrestrial scenarios.
– Simulation tests for modeling specific environmental scenarios and bioaccumula-
tion under site-specific conditions. In this case, measured BCF is multiplied by an
appropriate factor (for example, food chain multiplier), reflecting the difference
between the laboratory organisms and the field organisms.
– A site-specific bioaccumulation rate (BCF) is the result of field testing. Field assess-
ment of bioaccumulation applies to plants and animals living in the local habitat.
The contaminants can be followed through the whole food chain, but the best
indicators are those animals which are at the top of the food chain. Passive mon-
itoring of bioaccumulation means the collection of adequately selected species
by sampling. We can sample plants and herbivorous insects, as well as predator
108 Engineering Tools for Environmental Risk Management – 2

birds or mammals from the terrestrial ecosystem of contaminated land. In the

aquatic ecosystem, algae, plankton, clams and fish as well as sediment-dwelling
organisms are suitable as bioaccumulation indicators. In addition to sampling
of indigenous organisms, adequately maintained and controlled cultures of test
organisms can be placed into the environment and the accumulated contaminants
in their body tissues can be measured after recollection. This method is called
active biomonitoring.

It must be borne in mind that laboratory testing of bioaccumulation represents only

one step in a complex process in the environment: the uptake of contaminants from the
environment by direct contact with water and soil by one species. This means that many
of the results of bioaccumulation tests need further extrapolation for the members of
the food chain and the whole ecosystem. For example, data on the bioaccumulation
of one fish species is only an indication for the secondary poisoning of other fish-
eating organisms. In the case of terrestrial plants, the situation is better because we
can compare their contaminant content to NOEC values or regulatory limit values for
food or feed.
When planning risk assessment and selecting data for estimation/prediction of
BCF or BMF, it is important to know the advantages and disadvantages of Kow -based
QSAR, laboratory tests and field assessments.

– Kow -based QSAR:

◦ Advantages: ready to apply, non-expensive;
◦ Disadvantages: depends on the accuracy of Kow data; metabolism and specific
physiology of the animals are not covered, some types of chemicals cannot be
properly modeled.
– Laboratory tests:
◦ Advantages: standardized tests with good reproducibility, large databases are
available on results, applicable for all kinds of chemicals and the mechanism
and kinetics can also be investigated.
◦ Disadvantages: use of animals is less and less acceptable, expensive, food-chain
and food-web effects are not covered.
– Field tests:
◦ Advantages: applicable for all type of chemicals in the presence of all envi-
ronmental phases, relevant for metabolism, kinetics, food-chain and food-web
situations, applicable for mixtures, are site specific, and include bioavailability
and other interactions, etc.
◦ Disadvantages: quality of data is often questionable, environmental concen-
tration of the chemical is uncertain, sediment–water distribution and presence
of other contaminants and surface active agents may influence the results, het-
erogeneities in environmental parameters and variability of the organisms in
size, age, gender, etc. increase uncertainties.
The best concept for the estimation of environmental risk due to bioaccumulation
is the tiered approach. The first tier is screening, using QSAR. In the case of a positive
indication of the bioaccumulative potential by QSAR, chemicals are laboratory tested
 Fate and behavior of chemical substances in the environment 109

for bioaccumulation. As a third step, the most expected members at the top of the
food chain are field sampled and chemically analyzed to validate the results of QSAR
and biotests.
Databases with generally sufficient amount of information are available to start
a first-tier prediction in the course of risk assessment. Some of the environmental fate
databases containing BCF information are listed below:

– Chemical Risk Information Platform Database, METI–NITE Japan (METI–

NITE–CHRIP, 2013)
– Hazardous Substances Database, US National Library of Medicine’s (HSDB –
Toxnet, 2013)
– Environmental Fate Data Base, Syracuse Research Corporation (EFDB–SRC,
2013)
– European Chemical Substances Information System, JRC (ESIS–JRC, 2013)

The worst situation for the risk manager is the problem of accumulation of multi-
ple chemicals from contaminated water, soil, sediment or waste because QSARs cannot
handle the interactions between contaminants (only additivity), and the chemical anal-
yses cannot help in the case of an unknown mixture. These tests would considerably
increase the cost of testing.
An interesting theory was suggested by Yoo et al. (2003) based on the critical
body residue (CBR) theory that non-polar organic contaminants acting via non-
polar narcosis (anesthesia) cause acute toxicity when the total tissue concentration
of all organic compounds exceeds 2–8 mmol/kg (McCarty & Mackay, 1993). This
approach assumes that the compounds acting together through non-polar narcosis
exert their effect synergistically. Using DDE (dichlorodiphenyldichloroethane) as a
challenging toxicant in addition to the already bioaccumulated chemicals provokes a
higher response than without preceding bioaccumulation. The difference between the
toxic effect of DDE without previous accumulation and after bioaccumulation gives
the rate of bioaccumulation and the toxicity in one test series.

3.2.5 Standardized tests for measuring bioaccumulation

Standardized bioassays for bioaccumulation of chemicals aim to indicate the bioac-
cumulation potential of chemical substances or contaminants in an environmental
sample.

Testing bioaccumulation of chemicals in water, sediment and soil

The OECD test guidelines are prepared to support the testing of chemicals for regu-
latory purposes such as for authorization, classification and restriction of chemicals
under REACH regulation. The same or slightly modified methodology can also be
applied for testing contaminated environmental samples for in situ assessment and
monitoring (Table 2.6).
Testing the bioaccumulation potential of chemicals uses artificially contaminated
water, sediment or soil. Sorption and binding pure chemicals after their addition to
the test medium can never truly copy the much more complex and generally long-term
environmental process. One can assume that the biological availability is greater in the
110 Engineering Tools for Environmental Risk Management – 2

Table 2.6 OECD Guidelines for testing bioaccumulation in aquatic and terrestrial organisms.

Test guideline Test name

OECD TG 305 (1996) Bioconcentration: Flow-through Fish Test

OECD TG 315 (2008) Bioaccumulation in Sediment-dwelling Benthic Oligochaetes
OECD TG 317 (2010) Bioaccumulation in Terrestrial Oligochaetes

soil or sediment artificially contaminated and the result of the test is not an underesti-
mate. Mobility/bioavailability-enhancing agents can ensure certain overestimation in
simplified tests.

Bioaccumulation testing by sediment-dwelling organisms

OECD 315 (2008) measures the bioaccumulation of sediment-associated chemicals
in endobenthic oligochaete worms. The test consists of two phases: uptake and
elimination phases.
During the uptake phase, worms are exposed to sediment spiked with the test
substance, topped up with reconstituted water and equilibrated as appropriate. Groups
of control worms are held under identical conditions. The duration of the uptake phase
is by default 28 days, unless a steady state has been reached before.
For the elimination phase, the worms are transferred to a sediment-water system
free of the test substance. This second phase is terminated when either the 10% level
of steady-state concentration, or of the concentration measured in the worms on day
28 of the uptake phase, is reached. Change of the concentration of the test substance
in/on the worms is monitored throughout both phases of the test. The uptake rate
constant (ks ), the elimination rate constant (ke ) and the kinetic bioaccumulation factor
(BAFK = ks /ke ) are calculated. Lumbriculus variegates, Tubifex tubifex or Branchiura
sowerbyi can be applied. The worms are weighed, and the accumulated chemical is
analyzed (OECD 315, 2008).
Other freshwater oligochaete species are recommended by Ingersoll et al.
(1995) for testing bioaccumulation from sediment, such as Stylodrilus heringianus,
Limnodrilus hoffmeisteri, and Pristina leidyi. Ingersoll et al. (1996) recommended the
use of the amphipod Hyalella azteca and the midge Chironomus tentans for testing the
bioavailability of contaminants in freshwater sediments.

Sediment testing by fish

Bioaccumulation testing using fish has a number of advantages. Certain fish species
resuspend sediments, thus increasing contaminant bioavailability. Fish can provide
adequate tissue mass for chemical analysis because they have higher lipid mass com-
pared to invertebrates. Fish can also experience sediment-associated contaminant
exposure through several routes, including direct ingestion and accumulation through
gills and skin, and their gills may act as a substrate for the dissociation of hydrophobic
chemicals from sediment particles. Fish can be used in laboratory exposure tests as
well as in in situ studies on caged or free-ranging animals for testing biomagnification
and estimating human exposure via secondary poisoning (OECD 305, 1996).
 Fate and behavior of chemical substances in the environment 111

Bioaccumulation and bioavailability

The fate and effects of persistent, bioaccumulative and toxic (PBT) pollutants in soils
and sediments need special attention, as also stated in the status report of US EPA
(2000). The document discusses the basics of bioaccumulation, the fate and nature
of PBTs and the factors that affect the bioavailability of sediment-associated con-
taminants. It also identifies how bioaccumulation data can be used for sediment
management decisions. The factors influencing bioavailability in sediment are grouped
under physical, chemical and biological factors.

– Physical factors:
◦ Rate of mixing;
◦ Rate of sedimentation;
◦ Diffusion;
◦ Resuspension.
– Chemical factors:
◦ Acid-volatile sulfide (AVS) concentrations for Cu, Cd, Pb, Ni, Zn, a possible
predictive tool for divalent metals toxicity (cold acid extraction of hydrogen
sulfide and iron sulfide);
◦ Redox conditions;
◦ pH;
◦ Interstitial water hardness;
◦ Sediment organic carbon content;
◦ Dissolved organic carbon content;
◦ Organic-water equilibration constants for organic compounds;
◦ Organic matter characteristics;
◦ Equilibration time with sediment.
– Biological factors:
◦ Biotransformation;
◦ Bioturbation;
◦ Organism size/age;
◦ Lipid content;
◦ Gender;
◦ Organism behavior;
◦ Diet, including sediment ingestion, feeding mechanism;
◦ Organism response to physicochemical conditions.

Laboratory testing of bioaccumulation in environmental samples

Bioaccumulation in the environment is much more complex than in the laboratory
because the environmental fate of chemical substances, food-chain and food-web
effects always produces a unique combination, which is not easy to model or sim-
ulate. Decision makers should be cautious when extrapolating from laboratory test
results to the real environment and take uncertainties into account.
Actual bioconcentration can be determined using simplified environmental models
such as direct contact tests and microcosms, or field testing based on active or passive
biomonitoring.
112 Engineering Tools for Environmental Risk Management – 2

Table 2.7 Rapid plant accumulation test: metal-containing mine waste before and after chemical
stabilization using different fly ashes (Feigl, 2012).

Bioaccumulated Mine Mine Mine Mine

metal waste waste + FA 1 waste + FA 2 waste + FA 3 QC forage

Cd (mg/kg) 10.6 5.7 0.7 0.4 1

Zn (mg/kg) 884 130 80 90 Non-existent
Pb (mg/kg) 96.1 9.1 8.3 4.7 10
As (mg/kg) 15.1 2.0 1.9 1.3 2

QC forage: Hungarian quality criteria for animal fodder (in mg/kg)

FA: fly ash

An important application of bioaccumulation tests is the determination of the

mobile and bioavailable fraction of chemically measurable contaminants in environ-
mental samples, needing an integrated approach in the application and evaluation of
physico-chemical and biological methods.
The fate of a chemical time bomb in an environmental compartment can be inves-
tigated by dynamic bioaccumulation tests applying a provoked increase in the mobility
of known or often unknown contaminants (see also Chapter 9).
The authors of this chapter have developed and applied a rapid bioaccumulation
test (Table 2.7) on plant seedling Sinapis alba (white mustard). In this test, 40 seeds
were placed on 5 g of soil wetted with water to its water holding capacity and incubated
at 20◦ C in darkness for 5 days. The soil was re-wetted on the third day. The seedlings
were harvested after 5 days, their roots and shoots were separated, washed and air-
dried. The metal content of the plants was analyzed by ICP-AES (inductively coupled
plasma atomic emission spectroscopy) after a HNO3 and H2 O2 (2.5:1) digestion at
105◦ C for 3 hours.

3.2.6 Field determination of bioaccumulation

To measure bioaccumulation in field experiments, first the appropriate test species has
to be selected, then it must be decided whether natural or transplanted populations
should be used. Finding a suitable reference site may also be a problem.
For bioaccumulation field tests, test animals can be taken from resident popula-
tions on the site of interest or they can be transplanted from other sources or locations.
Resident populations can be selected, collected, maintained and analyzed before carry-
ing out the field test on them. Those stemming from other sources may be commercial
or self-grown populations, maintained in a lab, on the original site or on the site to be
tested. This means that there are many variations for the selection of test organisms.
The concept and test assemblage may vary from caged test organisms through field
micro- or mesocosms to gathering and analyzing free living organisms. The advantages
and disadvantages of each approach should be determined in each case.
The US EPA (2000) summarized the advantages and disadvantages of alternative
bioaccumulation tests in connection with the testing of sediment quality. According
to their statement, the use of transplanted test animals confined to cages can simplify
the field assessment of bioaccumulation in a number of ways. Test animals can often
 Fate and behavior of chemical substances in the environment 113

Table 2.8 Metal accumulation in field-grown and laboratory plants in the same contaminated soil before
and after chemical stabilization.

Field grown Laboratory rapid test

Sudan grass Sorghum Maize Sinapis alba
Untreated Stabilized Untreated Stabilized Untreated Stabilized Untreated Stabilized
Accumulated
metals soil

Cd (mg/kg) 3.0 0.9 6.6 0.7 5.3 1.6 3.0 0.9

Zn (mg/kg) 348 104 503 108 665 301 743 218
Pb (mg/kg) 3.3 2.2 6.3 1.9 25.4 5.6 3.0 2.0
As (mg/kg) 0.8 0.4 0.5 0.5 4.7 1.0 0.6 0.4

be obtained from commercial vendors in large quantities or from uncontaminated

reference locations. Because of this, the variability associated with exposure conditions
and times for individual test animals can be eliminated. All test organisms can begin the
exposure period with the same, or similar, background contaminant concentrations
in their tissues, and they will receive essentially the same exposure during the test
period. Test animals can be selected based on size and age as well as sex. Studies can
also be devised so that water column and sediment exposures can be differentiated
and uptake kinetics studied in situ. Finally, and perhaps most importantly, exposure
concentrations or conditions can be accurately determined because the animals will
be confined to a small, well-defined area. The exposure period is controlled by the
investigator, permitting time to achieve steady state and other issues to be considered
when designing a transplant study on caged animals. Transplant studies provide a
balance between experimental control and environmental reality not usually attained
in either standard laboratory bioassays or assessment of resident populations.
Testing plant bioaccumulation from soil is less problematic, given that plants grow
naturally and in large quantities both on agricultural and natural areas. Plants used
for assessing bioaccumulation are those which are cultivated or are planned to be cul-
tivated on agricultural and natural land. Plants in contaminated areas are the primary
toxic actors in food chains causing “secondary poisoning’’ to consumers, including
man. Their accumulating capacity varies considerably. Using available information,
cultivation can be managed aiming to reduce risk as much as possible. The example
in Table 2.8 shows differences between plants growing in soil contaminated with met-
als and the effect of chemical metal stabilization in the same soil. Accumulation of
field-grown and laboratory test plants are also compared in the table (Feigl, 2012).

3.3 Bioleaching
Bioleaching is a type of leaching where the extraction of metal from solid minerals into
a solution is facilitated by the metabolism of certain microbes, e.g. thiobacilli, also
called miner bacteria. Bioleaching is a process described as ‘the use of microorganisms
to transform elements so that the elements can be extracted from a material when water
is filtered through it’ (BioMineWiki, 2013). It is a risky process in the environment but
it provides benefits under controlled circumstances, e.g. for mining.
114 Engineering Tools for Environmental Risk Management – 2

OECD prepared a guideline for the testing of the leaching behavior of chemical
substances – OECD TG 312 (2004) Leaching in Soil Column. The method described
in this Test Guideline is based on soil column chromatography (OECD 312, 2004).
Two types of experiments are performed:

1 Those on chemical substances for regulatory purposes: determining the leach-

ing potential of the test substance and the leaching potential of transformation
products in soils under controlled laboratory conditions.
2 The same test equipment is suitable for testing the leachates from disturbed
or undisturbed solid environmental samples. Based on the assumption that the
leachate flows into surface waters, the aquatic toxicity of the leachate is mea-
sured. When the target of the leachate in the environment is the groundwater, the
tests may be of generic use or may simulate a specific groundwater use.

The test design of the OECD test guideline – duplicate leaching columns, packed
with air-dried and sieved soil (<2 mm) up to a height of 30 cm, saturated and equili-
brated with an ‘artificial rain’ solution and allowed to drain – can be applied or fitted
to any environmental process related to the infiltration of precipitation and/or con-
taminants with the precipitation. The leachate can be collected in optional fractions.
The leached soil can also be analyzed as a whole or in layers.
The authors of this chapter used soil ‘minilysimeters’ to monitor the stabilization
of metals in microcosms because the sample volume was strongly limited. In a 31 mm
diameter glass column, 200 g air-dried and sieved (<2 mm) soil was placed on a ceramic
filter covered with a synthetic textile at the bottom of the column. The soil was wetted
up to its water holding capacity and irrigated with 50 mL fractions of model precip-
itation (0.16 mmol/L CaCl2 solution). The leachate was collected and analyzed for
metal content, pH and electrical conductivity (EC). In another version, the stabilizing
additive was layered between the ceramic filter and the soil. This provided a model for
permeable reactive barriers.
Other problem-specific methods for modeling and assessing leaching in micro-
cosms are described in Chapter 8.

4 AVAILABILITY OF CONTAMINANTS FOR

ENVIRONMENTAL ACTORS

The environmental behavior of contaminants is primarily determined by the physico-

chemical properties of the chemical substances, but their actual fate can be charac-
terized and interpreted only as an interaction with the environment. Sorption of a
contaminant to the soil’s solid matrix is determined by the chemical’s sorbability and
the soil particles’ sorption capacity. Biodegradation of a contaminant depends on its
biodegradability and its bioavailability to potentially degrading microorganisms, their
access to the chemical substance and their degrading activity. Partitioning between
liquid and solid in soil, and leaching depend on the access of water to the contaminant.
Moreover, the result of chemical analyses is determined by the access of the extract-
ing solvent to the contaminant, which can be bound either weakly or strongly to the
matrix of the environmental compartment. The availability of the contaminant from
 Fate and behavior of chemical substances in the environment 115

the point of view of an organic solvent is completely different to the availability for
water or for living cells in the soil. As a rough approximation we can say that an organic
solvent can extract much more, while water extracts much less from the organic con-
taminant than the fraction available for living organisms. The models and tools to
be used to assess a contaminated environment must take into account the differences
between the contaminants’ environmental behavior and their interactions with the
environmental matrix and other components such as water or microorganisms.
Two case studies are introduced here to explain contaminants’ availability for dif-
ferent actors in the environment and for the agents used by the assessors in analyses and
tests. Traditional extracting solvents can never truly mimic the “extraction’’ by biolog-
ical organisms: methods using organic solvents generally intend to extract “all’’ of the
contaminants (with moderate success). Risk assessment results based on total extracts
may be a massive overestimate. To bring a chemical model closer to the biological
response in the environment, one direction of the methodological developments is to
find so-called biomimetic extractants, which are able to mimic the access of organisms
or biological systems to certain contaminants.
One of the pollution cases presented involves organic, the other inorganic con-
taminants. These pollution cases were analyzed in detail with regard to contaminant
availability for different solvents, microorganisms and plants. The topic is discussed
in greater detail in Chapter 7.
Accessibilities of transformer oil (a PCB-free mixture of hydrocarbons of petroleum
origin) for different solvents (hexane–acetone and water dissolved cyclodextrins used
as solubilizing agents) as well as for soil organisms were compared and the correlation
of the results evaluated. Table 2.9 contains the values measured in the course of a soil
remediation case (biodegradation-based clean-up of a transformer oil-contaminated
soil) (Molnár et al., 2009a)
Abbreviations in Tables 2.9 and 2.10:

– TO: Transformer oil, a commercial product, namely TO 40A;

– SEM: Solvent extractable mass, TO accessible for hexane:acetone 3:1. Measured
by gravimetry;
– EPH GC: TO accessible for hexane:acetone = 3:1. Measured by gas chromato-
graphy;

Table 2.9 Analyses and tests of soil sample series from a remediation experiment: different solvents
and microorganisms under different conditions have different access to the contaminant.

Measurement end points 0 week 2 weeks 4 weeks 6 weeks

SEM (mg TO/kg soil) 34,000 33,400 30,500 26,200

EPH GC (mg TO/kg soil) 27,850 26,600 22,800 19,400
REH GC (mg TO/kg soil) 10,600 16,200 11,000 13,800
HEH GC (mg TO/kg soil) 7,400 11,200 3,500 2,600
CO2 (% in flow through air) 0.27 0.34 0.25 0.21
CB Respiration Index (1000*hPa/min) 24 36 18 17
DEH (µg TPF/g soil) 87 62 34 47
MPN of oil degrading cell conc. (cell/g soil) 210,000 350,000 15,000 23,000
116 Engineering Tools for Environmental Risk Management – 2

Table 2.10 Correlations between chemical analytical and biotest results; correlations highlighted in
bold are significant at p < 0.05.

Biological end points

MPN DEH RESP CB RESP AER

Chemical end points

SEM 0.844 0.793 0.706 0.820
p = 0.156 p = 0.207 p = 0.294 p = 0.180
EPH GC 0.902 0.864 0.711 0.826
p = 0.098 p = 0.136 p = 0.289 p = 0.174
REH GC 0.343 −0.306 0.681 0.540
p = 0.657 p = 0.694 p = 0.319 p = 0.460
HEH GC 0.966 0.609 0.961 0.990
p = 0.034 p = 0.391 p = 0.039 p = 0.010

– REH GC: TO accessible for randomly methylated beta-cyclodextrin, measured by

GC-FID;
– HEH GC: TO accessible for hydroxypropyl beta-cyclodextrin, measured by
GC-FID;
– GC-FID: Gas chromatography coupled with flame ionization detector;
– CB: Closed-bottle test;
– DEH: Dehydrogenase enzyme activity in contaminated soil;
– TPF: Triphenyl formazan (product of an alternative electron acceptor);
– MPN: Most probable number.

The correlation matrix in Table 2.10 includes the correlation coefficients and the
significance levels (p). The correlation between chemical analytical and soil microbio-
logical end points was investigated for transformer oil where the trends were correlated
to each other.
One can learn from the results that the access of organic solvents and micro-
organisms to the transformer oil in this soil differs greatly: SEM and EPH GC have no
good correlation with any of the biological end points. One of the two water-based
cyclodextrin solutions is able to mimic the access of the degrading microorganisms:
the HEH extract shows good correlation with the cell number and with the respiration
both in closed and flow-through systems. This leads to the conclusion that a model
can be created based on a hydroxypropyl-beta-cyclodextrin solution to mimic and
extrapolate to the biodegradability of transformer oil. However, this does not mean
that HEH can generally be applied for all kinds of contaminants.
In another case a toxic metal contaminant endangers agricultural soil at a former
mining site. The site was exposed to floods over many years and became contaminated
with cadmium and zinc. Table 2.11 shows the metal concentrations of the soil treated
with water, a mildly acetic acetate solution, aqua regia, and test plants. Access of
test plants to the metals in the soil was measured through plant toxicity (only the
available part is effective) and plant bioaccumulation (only the metal available for the
root is taken up and accumulated). For comparison, the same results for the chemically
stabilized soil are also shown in the table.
 Fate and behavior of chemical substances in the environment 117

Table 2.11 Availability of toxic metals in soil for different solvents and plants.

Test method Non-treated soil Stabilized by fly ash

Cd accessible to aqua regia (mg/kg) 5.2 5.2

Cd accessible to acetate solution* (mg/kg) 1.5 0.3
Cd accessible to water (mg/kg) 0.05 <0.004
Cd accumulated by plant (mg Cd/kg dry plant) 6.6 0.7
Cd extracted from soil by plant biomass (mg Cd/kg soil) 0.33 0.13
Zn accessible to aqua regia (mg/kg) 1102 1102
Zn accessible to acetate solution (mg/kg) 237 48
Zn accessible to water (mg/kg) 4.1 0.3
Zn accumulated in plant (mg Zn/kg dry plant) 503 108
Zn extracted from soil by plant biomass (mg Zn/kg soil) 25 19
Plant biomass in accumulation tests (g biomass/5 g soil) 0.25 0.9
Relative accessibility to plant (shoot growth in mm)** 20 32

*Acetate solution: an ammonium-acetate buffer solution of pH = 4.5

**These results cannot be converted into available metal concentrations

5 UTILIZING FATE PROPERTIES OF CHEMICALS TO REDUCE

THEIR RISK IN THE ENVIRONMENT

Many of the transport and fate processes in the environment may lead to a reduction
of environmental risk.
The term natural attenuation is generally used for spontaneous biodegradation of
organic contaminants in the groundwater. In this chapter, the term natural attenua-
tion will be used in a wider sense, integrating all spontaneously occurring natural risk
reducing processes such as in situ transformation and degradation processes (mainly
biodegradation), irreversible immobilization by sorption or by incorporation into the
solid matrix as well as dilution, dispersion and partitioning if they result in a decrease
in environmental risk. It is advisable not to use the term ‘decrease in the concentration
or amount of a contaminant’ instead of the decrease of risk because it is not necessarily
proportional to the concentration or amount of the contaminant, but is strongly influ-
enced by its physical and chemical forms as well by the characteristics of the matrix
and the receptors.
The continuous change of a contaminant’s risk due to natural processes in the
environment results in a site-specific risk profile and the change of risk as a function
of time. A continuous falling slope of the profile indicates natural attenuation of the
risk which may run parallel to the decrease in contaminant concentration, but it may
also be attributed to decreasing mobility and bioavailability. A short summary is given
here about the topic, a detailed discussion can be found in Volume 4.

5.1 Environmental transport and fate processes

change contaminant risk
Transport processes such as advection, dispersion, diffusion, dilution, volatilization
and partitioning change the flux of dissolved contaminants. The gravitational transport
118 Engineering Tools for Environmental Risk Management – 2

of precipitation, groundwater and surface waters results in dispersion, increasing the

area or volume of contaminated land (which may increase the risk), but at the same
time, decreasing concentration and risk due to sorption or filtering out and dilution
along the transport pathways.
Partitioning between air–water and solid–water may result in either increased or
decreased risk, depending on the source of the contaminant and the direction of the
process. Due to partitioning between air and water, the contaminant can be eliminated
from the water (to the air) or the water can absorb the contaminant from the air.
Contaminants transported by groundwater can be distributed by partitioning between
water and solid soil phases. This process is also called sorption of the contaminant on
solid particles or filtration of the contaminant by the solid fraction. The same amount
of a chemical is less risky when bound to the solid phase of the soil: there is a three
to four orders of magnitude difference between the effective concentrations of the
same contaminant in the soil/sediment solid phase and in soil moisture, pore-water
or groundwater. In which of the phases with the same amount of contaminant is the
risk higher depends on many factors, but chiefly on the volume contaminated, the
sensitivity and density of exposed receptors and exposure pathways.
The rearrangement of contaminants by partitioning may proceed between water
and the living ‘phase’ as well as between solid and the living ‘phase’. The harmful
nutrients (N, P) from water can be taken up by algae and water plants, which leads
to eutrophication when the processes are uncontrolled, but may be beneficial when
the plants are removed from the water system after take-up of nutrients and before
dying and nutrient recycling. Partitioning of metals between soil and plants may be
harmful to the food chain, but it can be beneficial for the soil if the bioaccumulating
plants are removed from the soil after the contaminant has been extracted. Natural
ecological systems, e.g. the plants of aerobic or semianaerobic wetlands are able to
filter nutrients and contaminants with the help of their rhizosphere. Bacteria settled
in the rhizosphere are ready to mineralize trapped nutrients and contaminants. This
is why the concentration of contaminants and their risk decrease in the water and soil
or even in wastewaters or leachates that reach the wetland.
When living organisms interact with the toxicant, active transport, not only simple
partitioning, may occur, and the contaminant may accumulate in the tissues of the
living organisms.
The equilibrium of natural process pairs such as mobilization–immobilization,
sorption–desorption, oxidation–reduction depends to a large extent on the environ-
mental conditions such as pH, temperature and redox potential.
Mobilization may support the elimination and removal of contaminants from soil
and sediment as well as solid waste, reducing the risk in the solid but increasing it in the
liquid/water phase. The solution is again the controlled and isolated handling of the
water. Immobilization may have a direct risk reducing effect: strong and irreversible
sorption in or on the solid phase of environmental compartments brings the transport
of water-soluble contaminants to a halt. This kind of stabilization lowers the risk in
waters, but over the long term, may result in the enrichment of contaminants in the
soil or sediment, which, after a while, will act as a chemical time bomb. Mobilization
first appears to be a risk-increasing phenomenon, but if the mobilized and transported
contaminant can be diluted and resorbed under less risky conditions (e.g. at a place
isolated from the environment or at a less sensitive location), the result of dilution is
 Fate and behavior of chemical substances in the environment 119

satisfactory. When only the front part of a subsurface contaminant plume has been
degraded by soil microbes, dispersion of the plume may intensify biodegradation but
increase the size of area affected.
There is a continuous redox gradient in nature, e.g. a decreasing redox potential
with depth in water and soils. The chemical form and, as a consequence, the mobility
of chemicals are determined by the redox potential. Certain contaminants, such as dis-
solved metals can be immobilized by higher pH and lower redox potential, depending
on the chemical species. Precipitation of metals in deeper soil layers, sediments and
wetlands in sulfide form produces a highly stable and biologically inaccessible form,
as long as the redox potential is low.
The pH is also responsible for chemical forms of different mobility, for example,
limestone in nature may convert ionic metals to hydroxide compounds, which are less
mobile. This means that the waters and the living organisms are protected from the
risk, but the metals are still there.
The most efficient and final reduction of the risk due to chemical contaminants is
their degradation and complete elimination from the environment. Hydrolysis, photol-
ysis, chemical degradation by oxidation or reduction and biodegradation can eliminate
organic contaminants. The utilization of risk reducing natural processes in remedial
technologies is highly recommended. The vast genetic and biochemical potential of the
microorganisms in the environment makes possible the elimination of any organic com-
pound, even xenobiotics. Mankind should be grateful for this natural risk-reducing
‘measure’. Of course, risk may also increase due to temporary toxic metabolites or
end products, but this is not a typical case. However, this possibility, too, has to be
checked and controlled, e.g. by using ecotoxicity tests for the monitoring of natural
attenuation. Inorganic compounds, chiefly metals differ in this respect from organic
substances: they can hardly be removed from the environment, so that the “back-
ground’’ concentration is continuously increasing. This gives all the more cause for
concern as global reserves of some metals are already close to depletion.
The above summarized environmental processes may serve as the basis for bio-
and ecotechnologies. Engineers can optimize the environmental conditions for the
biological catalyst, i.e. the living organisms which can reduce or eliminate risk.
Not only are these nature-based in situ technologies suitable for the remediation
of contaminated water, sediment and soil, but also for nature conservation and envi-
ronmental health protection, in one word for the sustainable management of waters
and land. The prevention of contamination and deterioration of the environment is
more important; it can be the key tool of long-term precautionary environmental
risk management in the future. These ‘soft’ technologies are eco-efficient, they can
harmonize the good environmental/ecological status with land use and ensure sus-
tainability. Ecoengineers have to select the beneficial, risk-reducing environmental
transport and fate processes of the chemical substances and utilize them. In terms
of engineering, there are no risk-increasing processes in the environment, only inad-
equate control and application! The same process can increase or reduce the risk,
depending on whether it is isolated from, or is in contact with the environment. A
properly selected technology, e.g. a simple ‘barrier’, which is able to isolate the process
of sorption, accumulation or leaching from the environment, can turn the processes
into beneficial ones. Another approach to increase efficacy by engineering tools is
the intensification of the beneficial risk-reducing processes such as biodegradation,
120 Engineering Tools for Environmental Risk Management – 2

bioprecipitation or bioleaching with the help of redox potential control and nutrient
circulation.
The risk profile of a contaminant due to naturally occurring processes provides
information about the direction and velocity of the changes in its risk. Technologists
may develop remediation technology on the existing risk profile without any change;
they can speed it up, or change the direction of the risk profile from an increasing
risk profile (in contact with the environment) into a decreasing risk profile by isolating
the process from the environment, e.g. collecting the leachate of high contaminant
concentration (see also Chapter 8 in Volume 4 on risk-reducing natural processes).

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Chapter 3

Human toxicology
K. Gruiz
Department of Applied Biotechnology and Food Science,
Budapest University of Technology and Economics, Budapest, Hungary

ABSTRACT

Humans are exposed to chemical hazards through inhalation, consumption of water

and food, and dermal contact. Human life today is highly non-natural, and it takes
place predominantly in a man-made (built) environment which is different from the
natural ecosystem. Human exposure is restricted to (mainly indoor) air, processed
foods and beverages and to minor dermal contact. Occupational health protection
gadgets and equipment prevent employees from absorbing hazardous chemical sub-
stances at the workplace. In addition to protective wear and equipment, the human
organism is highly adaptive and capable of self-defense. But all of this is no help when
people are highly exposed to chemical hazards, including new, emerging chemicals
whose effects are not (yet) fully known. These effects are different from those of tra-
ditional toxicants and are not proportional to their concentrations or doses, and they
cannot be measured using traditional toxicological methods.
This chapter gives an overview of traditional toxicology, including acute and
chronic toxicity, corrosivity, mutagenicity, carcinogenicity and reprotoxicity, as well as
newly recognized chemical hazards such as sensitization, allergization, and endocrine
and immune system disruption. In addition to traditional epidemiological studies
and animal tests, alternative methods using molecules, cells or tissues instead of
living animals will be discussed. Extrapolation methods from animal or alternative
end points to humans and the associated uncertainties are also discussed because
they are key factors in the utilization of human toxicity data for environmental
management.

1 INTRODUCTION

People are exposed to environmental chemicals when they are in contact with con-
taminated land. Exposure to air, surface waters and sediments, soil and groundwater
can take the form of residential, recreational, agricultural, and industrial land uses,
as illustrated in Figure 3.1. Humans are exposed to contaminants directly by inhala-
tion, ingestion and dermal contact or through the food chain. Exposure of humans
via plants, fish and livestock to environmental contaminants is also called secondary
126 Engineering Tools for Environmental Risk Management – 2

Deposition to ground
Gas, vapor and particulate matter
Deposition to crops

Em
iss

Inhalation
ion
int
Deposited oa
ir
material

Exp
osu
re to
ma depo
teria site
l d Plant

Liq
Irrigation

uid
Eating crops Drinking gro
und-

eff
water

lue
nt
Eating meat
ce
urfa
gs
kin ter
estion Drin wa
Ing

Surface
water

Figure 3.1 Human exposures to chemical substances in the environment.

poisoning. The two main sources of secondary poisoning are crops and fish. The most
sensitive exposure pathways are:

– The inhalation of air, vapors and dust;

– The consumption of water from subsurface or surface water resources;
– The consumption of agricultural products such as cereals, fruits, vegetables, animal
products (meat and milk);
– Eye and dermal contact with air, water (suspended solids) and soil.

The adverse effects of environmental chemicals on humans cannot be tested directly

on a statistically suitable number of test persons at different concentrations aim-
ing to find the no-effect concentration or dose. Theoretically, we could use existing
epidemiological information (mainly from occupational poisoning cases or chemical
accidents) for extrapolation to an average population or to children, but since epi-
demiological data are neither available for every chemical substance nor are the data
reliable, extrapolation is accompanied by a high level of uncertainty resulting in undue
overestimation.
A more realistic estimate may be obtained from animal tests, based on the
assumption that the applied test organism is a good model for humans. Traditional tox-
icological testing is largely based on the use of higher laboratory animals: rat, mouse,
rabbit, dog, etc. The drawbacks of this approach are low throughput, high cost, and
difficulties inherent to inter-species extrapolation.
Increasing knowledge of the differences between humans and the most widely
used test organisms suggests that more advanced methods are needed to extrapolate
e.g. from rat to man. Scientific toxicological knowledge and sophisticated tests are use-
less, if a safety factor of 10 must be applied to extrapolate from rat to man. Satisfactory
 Human toxicology 127

results are difficult to obtain and they are not proportional to the investments in animal
testing. Overestimates, which cannot always be justified, result in high risk reduction
costs. In addition, animal testing is ethically questionable and is of limited use in eval-
uating the very large number of chemicals with regard to inadequate toxicological
data.
These problems have led to a search for alternative test methods to replace lab-
oratory animal tests with in vitro or in silico methods. Current developments in
molecular biology and computer science open new possibilities to find the genes or
other key-molecules and mechanisms responsible for the interaction with toxicants
and effectuating toxicity of the chemical substances.
The tests must have clear aims and well-identifiable results in order to be able
to develop and apply alternative methods. Generic testing of the chemicals’ effects for
regulatory purposes has different requirements than site-, age- or gender-specific assess-
ments for managing specific, e.g. locally arising problems. Tests on pure chemicals or
water, soil and agricultural products also have their own specific aims and method-
ology. Every task and every case needs its own optimal test method that requires the
smallest number of animals, provides rapid results and is cheap yet informative. Tier-
ing may help integrate the rapid screening methods into the risk assessment procedure.
Rapid testing can exclude negative cases for a large number of chemicals or envi-
ronmental samples at as early a stage as possible, thus minimizing expenditure. The
expensive, detailed assessment with costly labor and animal requirements should only
be used for the most risky cases.

1.1 Adverse effects of chemicals on humans

Adverse effects of chemicals on humans are classified into groups according to the time
duration necessary for the appearance of an effect in the target of the impact:

– *1 Acute toxicity: adverse effects occurring following oral or dermal administration

of a single dose or repeated dose within a short time (24 h) of a substance, or
inhalation exposure (of 4 hours);
– *Specific target organ toxicity with single exposure via any of the exposure
routes. It is a specific, non-lethal toxicity arising from a single exposure result-
ing impaired function of any or the organs, reversible and irreversible, immediate
and/or delayed. See also specific target organ toxicity with repeated exposure.
– Neurotoxicity belongs to specific target organ toxicity, developmental neurotoxi-
city to reprotoxicity.
– Repeated dose toxicity studies characterize the toxicological profile of a toxicant
following repeated administration. The profile includes the identification of poten-
tial target organs, the exposure–response relationships and reversibility of toxic
effects. It may play a role in the determination of the maximal tolerated dose
(MTD).
– Chronic toxicity is the ability of a chemical substance to cause harm to an organism
as a result of repeated exposures for long periods of time, even a lifetime. Very

1
The effects marked with * are GHS classes as well.
128 Engineering Tools for Environmental Risk Management – 2

small chronic exposures to a potentially harmful chemical can be handled by the

exposed organisms, but increasing exposures cannot be fended off. The turning
point between no-effect and effect levels is the threshold for chronic exposures; it
should be the regulatory governing value for chronic toxicants.
– *Specific target organ toxicity which arises from a repeated exposure to a sub-
stance or mixture resulting in impaired function of any of the organs, reversible
and irreversible, immediate and/or delayed. Typical signs are: morphological
and functional damage of organs (necrosis, fibrosis or granuloma, cell death,
etc.), hematotoxicity, serious damage or morbidity due to bioaccumulation of
the substance, damage to the nervous system’s function, etc.
– Mutagenicity: the effect of mutagenic agents which are giving rise to an increased
occurrence of mutations in populations of cells and/or organisms, including
somatic cells and germ cell. The latter will lead to a heritable genetic change
and form generally a separate group called genotoxicity or germ cell mutagenicity.
In vivo mutagenic activity may indicate that a substance also has a potential for
carcinogenic effects.
– *Germ cell mutagenicity impacts the egg or sperm cells (germ cells) and therefore
can be transmitted to the next generation.
– Genotoxicity and genotoxic carcinogenicity mean alteration in the structure,
information content, or segregation of DNA, including the normal replication
processes. Genotoxicity test results are usually taken as indicators for mutagenic
or carcinogenic effects.
– *Carcinogenicity of a substance or a mixture of substances means that they induce
cancer or increase its incidence. Carcinogenicity is traceable to genotoxicity or
non-genotoxic origin.
– *Toxicity for reproduction, also called reprotoxicity, includes adverse effects on
sexual function and fertility of adults, the developmental toxicity on the fetus (also
called teratogenic effect), as well as adverse effects via lactation. Genetically based
heritable effects in the offspring are addressed in germ cell mutagenicity.
– *Irritation: production of reversible damage to the skin or reversible changes in
the eye;
– *Corrosiveness: the production of irreversible damage to the skin or causing
serious eye damage;
– *Sensitization: respiratory sensitizers lead to hypersensitivity of the airways fol-
lowing inhalation of the substance, skin sensitizer to an allergic response following
skin contact.
– Endocrine disruption is an adverse effect of endocrine disruptor chemicals, which
may interfere with the body’s endocrine system and produce adverse developmen-
tal, reproductive, neurological, and immune effects in both humans and wildlife.
It means that immune-system disruption and neurotoxicity may be a special case
of endocrine disruption.
– Immune-system disruption is a subcategory of endocrine disruption, meaning a
long term adverse effect resulting in allergic and autoimmune diseases and their
growing prevalence rate, e.g. in the case of type 1 diabetes.

These types of adverse effects are thoroughly defined and specific test methods are
developed for providing end points for the identification and characterization of these
 Human toxicology 129

groups of effects. The grouping includes overlaps in deterioration and the classification
point of views are also combined with each other: the definition of the single adverse
effects includes details on duration, routes of exposure, metabolism, reversibility and
other characteristics of the evolution and consequences of the effect.
There are many important issues to be refined and clarified, only a few from them
are mentioned below, just to draw the attention:

– Dose–response: all chemicals may pose adverse effects in abnormally large

doses/concentrations and toxicants can be harmless under a certain threshold.
– The threshold is a turning point between no-effect and effect levels/concentrations:
values measurable in tests and applicable as quality criteria in risk management.
– Threshold chemicals are those chemicals which do not show adverse effects
after a certain case of dilution, and the opposite: show an effect with growing
doses/concentrations. In contrast, some chemicals are thought to be non-threshold
chemicals because a certain no-effect value for them could not be determined.
Scientists do not agree, some of them state that all chemicals have a threshold
(including mutagens, carcinogens. teratogens, endocrine disruptors), some others
maintain that there are no thresholds e.g. for carcinogenic effects of chemicals,
meaning that one molecule of a carcinogen is capable of starting a cancer. There
is no scientific evidence for either the first or the second view.
– Bioaccumulation is a risk factor both in the ecosystem and in humans, but its
interpretation is often oversimplified by considering as a one-way process with
a static outcome. The storage of toxicants is a defense mechanism in the body,
and is a dynamic process: the depots are increasing when inflow is larger than the
outflow, and start to decrease when no resupply is coming in; it is a question of
equilibrium, which of course depends greatly on the partitioning of the chemical
substance between blood and the tissue of storage.
– Reversibility–irreversibility of effects may be an important issue when the risk
of the chemicals is calculated. Reversible effect means that the exposed tissue
recovers and the organism returns to normal, when exposure ceases, e.g. skin or
eye irritation and non-lethal acute narcotic effects. Irreversible effects are final and
permanent, no recovery takes place such as in the case of skin or eye corrosion,
tissue lesion, developmental/congenital disorder or cancer, etc.
– Mixtures of chemicals and additivity of effects: legislation is based predominantly
on hazard and risk of individual substances. In recent years several debates arose
due to the large number of chemicals present in the environment in low con-
centration (under their individual threshold), and the consequent co-exposure
of ecosystem and man. The exposure from several environmental compartments
and secondary poisoning from water and food cannot be quantified by currently
existing scientifically based methodologies. The additivity of the effects is also
questionable in most of the cases: even the effects of chemicals of similar effect-
mechanism are not additive because significant synergism or antagonism may
occur. The cumulated effect of mixtures and coexisting chemicals in the envi-
ronment should be considered, but a groundless overestimation by multiplying
highly conservative risk scenarios for each should be avoided.
– Weight of evidence (WoE) is a term scientifically not well-defined. It covers the
assessment of the relative weights of different pieces of the available information
130 Engineering Tools for Environmental Risk Management – 2

on the topic to decide on. The weight given to the available evidence can be for-
malized by pre-established factors which characterize the quality of the data, the
consistency of the results, the type and severity of the effect and the relevance
of the information. REACH Regulation recognized the WoE as a useful tool in
classification and labeling (CLP) as well as in the safety assessment of chemicals
(CSA). It is a sufficient weight of evidence for a chemical substance having, or not
having, dangerous properties when several independent sources of information
conclude the same (e.g. well-documented human cases), or when newly developed
test methods, not yet acknowledged by the regulations, demonstrate the effect or
prove the “no effect’’.
“Where sufficient weight of evidence for the presence or absence of a particular
dangerous property is available: i) further testing on vertebrate animals for that
property shall be omitted; ii) further testing not involving vertebrate animals may
be omitted. In all cases adequate and reliable documentation shall be provided’’
(REACH, 2006).

1.2 Testing the adverse effects of chemicals on humans

In vivo or ex vivo animal tests and in vitro non-animal models can be used to measure
the adverse effects of chemicals on humans. In silico models can be created for any of
the data sets that satisfies the statistical requirements. The most frequent human tox-
icity end points are dermal and eye irritation and corrosion, acute and repeated-dose
systemic toxicity, organ-specific toxicity, chronic toxicity, carcinogenicity, mutagenic-
ity, reprotoxicity, endocrine and immune toxicity, neurotoxicity, and sensitization. In
this chapter, test organisms, existing standardized animal test methods and in vitro
alternatives will be introduced.
The methods of extrapolating from test or model results to humans should be based
on knowledge, experience and statistical analysis. Extrapolation from mathematical (in
silico), molecular or biochemical and the more complex biological models bears high
uncertainty due to the great standard deviation of the test methods and the differences
between the models and the human body. Increasing knowledge on these similarities
and differences may reduce uncertainties, but intraspecies differences within the human
population still remain and represent a major unknown factor.
Figure 3.2 shows the relationship of models to the human population: the distance
(D) from humans (due to differences and complexity) and the angle α, β (characterizing
the error of the extrapolation method) together determine the deviation of the result of
the model from real human values. The dotted line around the models illustrates their
standard deviation. We can see that a smaller extrapolation error and smaller distance
of animal models compared to molecular models cause smaller deviation from current
human values, in spite of a higher standard deviation of animal testing. If an amended
molecular model could reduce the angle “β’’ to “α’’, i.e. to that of the animal tests,
the extrapolated value would be closer to real human values (small deviation). Experts
should choose the optimal tests to obtain the most reliable result with minimum effort.
Depending on the initial data set, in silico methods can be either very close or very far
from reality (human body or human populations). With an extremely large amount of
data available, data mining and processing methods may help develop mathematical
models which are perfectly fit for human risk assessment.
 Human toxicology 131

Figure 3.2 Modeling human exposures to hazardous chemical substances.

Nowadays the assessment of toxicological risk relies primarily on in vivo animal

experiments that were designed decades ago and cost about $3 billion/year world-wide
(Hartung, 2012). Uncertainties in animal representativeness (due to the differences in
animal and human toxicity responses) are handled by applying safety factors when
extrapolating from animal toxicity data to human risk (NRC, 2000; Kavlock et al.,
2009) which may lead to significant overestimates. Another shortcoming of animal
testing is the use of extremely high doses which are much higher than those adminis-
tered in reality. In these studies the highest dose gives mostly positive response which
entails undue measures in risk management (Hartung, 2009). FDA (2004) statistics on
drug development demonstrate that not only do overestimations occur, but more dan-
gerous underestimations do too: 20% of the developed drugs fail due to toxic effects
in humans not identified in pre-clinical animal testing (another 72% fails due to other
reasons, totaling 92%).
Validated in vitro test methods (OECD, 2005) and evidence-based toxicology may
help (see Chapter 1) by their systematic collection and validation of all existing data
including the results of traditional and innovative alternative toxicity measurement
methods, the knowledge on mechanisms and computer evaluations.
132 Engineering Tools for Environmental Risk Management – 2

Figure 3.3 Wistar rat (Stephens, 2014) and guinea pigs (Sandos, 2014), popular laboratory animals.

2 TEST ORGANISMS FOR HUMAN TOXICOLOGY PURPOSES

For human toxicology purposes, the main test organisms are rats and mice, but for a
reduction of the number of test animals, isolated organs from cadavers, tissue cultures
and isolated cells are also used in alternative tests. Some effects of chemicals on the
genome are modeled on microorganisms.

2.1 Microorganisms used in human toxicity testing

– Salmonella for Reverse Mutation Test (Ames Test) (Figure 3.3);
– Escherichia coli for Reverse Mutation Assay;
– Escherichia coli for UMU test;
– Saccharomyces cerevisiae for Gene Mutation Assay;
– Saccharomyces cerevisiae for Mitotic Recombination Assay;
– Saccharomyces cerevisiae with cloned estrogen receptor in YES Assay.

2.2 Isolated cells, tissue cultures in human toxicology

– BALB/c 3T3 – mouse fibroblast cell line for cytotoxicity testing;
– Normal human keratinocytes for cytotoxicity;
– LLC-PK1 kidney proximal tubule cell line for cell damage;
– MDCK dog kidney epithelial cell line for cell damage;
– HepG2 liver cell line (hepatoma) for cell growth and cytotoxicity;
– HL-60 human acute promyelocytic leukemia cell line for energy production and
metabolism;
– Granulocytes/macrophages for acute neutropenia;
– Mammalian chromosomes for in vitro Chromosome Aberration Test;
– Mammalian cells for in vitro Gene Mutation Test;
– Mammalian cells for in vitro Sister Chromatid Exchange Assay;
– Mammalian cells for testing DNA Damage and Repair;
 Human toxicology 133

– Mammalian cells for testing Unscheduled DNA Synthesis

– Mammalian erythrocytes for Micronucleus Test;
– Human peripheral blood lymphocytes for Micronucleus Test;
– Syrian Hamster Embryo (SHE) for Micronucleus Test;
– Cell lines CHO, V79, CHL/IU and L5178\Y for Micronucleus Test;
– Human reconstructed skin for Micronucleus Test.

2.3 Lower animals in human toxicology

– Drosophila melanogaster (Drosophilidae, flies, Diptera), the common fruit fly
or vinegar fly, is a historical species used in genetics and developmental research.
Originally, its giant chromosomes made this little fly popular. Its use is still common
due to easy maintenance, quick breeding, and their laying many eggs. It is used in
alternative genotoxicity test methods.
– Xenopus genus of aquatic frogs, also known collectively as African Clawed Frogs
or Platanna. They are a popular model for gene and protein expression and knock-
down studies. Xenopus oocytes, 1 mm in diameter, are very large cells, which are
easy for scientists to culture and use in experiments. Xenopus laevis is the most
commonly used species for developmental toxicology studies. For example, the
Frog Embryo Teratogenesis Assay on Xenopus (FETAX) is one such alternative
bioassay that can be effectively used for toxicity testing.
– Bufo marinus (Anura, Bufonidae) is a large, terrestrial true toad, native to Central
and South America. It is a popular species for toxicological studies of environ-
mental pollutants, but it is also used in pregnancy testing and other laboratory
research. It is easy and inexpensive to maintain and handle in the lab.
– Bull frog (Rana catesbeiana, Anura, Salientia) belonging to the tailless amphibians
such as frogs and toads. It is an aquatic frog, a popular laboratory animal.
– Mudpuppy (Necturus maculosus, Caudata, Urodeles), belonging to amphibians,
comprising newts and salamanders. Mudpuppies or waterdogs are aquatic sala-
manders of the family Proteidae. Their name originates from the misconception
that they make a dog-like barking sound.

2.4 Birds
– Domestic chickens can be easily bred and housed so that they are increasingly
used as experimental animals in many areas of scientific research and toxicology.
Their main fields of use are breeding and genetics, embryology, anatomy, health,
hygiene, toxicology and pharmacology, physiology, biochemistry, endocrinology
and neurobiology.
– Other domesticated and wild birds such as
◦ Dabbling duck, Anas platyrhynchos;
◦ Rock dove, Columba livia;
◦ Virginia quail, Colinus virginianus;
◦ Japanese quail, Coturnix japonica;
◦ Common pheasant, Phasianus colchicus;
◦ Red-legged partridge, Alectoris rufa.
134 Engineering Tools for Environmental Risk Management – 2

2.5 Mammals
Estimates of animals used globally for experiments range from tens of millions to 100
million or more, annually. In toxicology, the most popular mammal test organisms are
rodents.

– Norway rat (Rattus norvegicus, Rodentia, Muridae) is the species most commonly
used in research and human toxicology testing. In the USA alone, about 4 million
rats are used annually in different laboratories. The adult rat ranges in weight
from 250 to 500 g. Brown color is dominant and typical. The Wistar stock of
Rattus norvegicus was established in Chicago in the Wistar Institute during the
years 1906–1930. This lab began standardizing albino rat strains and today most
of the laboratory rats are white. The Norway rat is easily maintained and rela-
tively resistant to diseases. Rats are important in all kinds of toxicity testing and
behavioral research.
– Mice (Mus domesticus, Rodentia, Muridae) are the most common laboratory
animals. Laboratory strains of mice used today are descendants of the western
European house mouse, Mus domesticus, with some genes from Asian species.
Mus musculus is a composite taxonomic designation for several interbreeding
species. A yellow mutant is used in studies of pigmentation, implantation, obe-
sity, and sterility. BALB/c, the white laboratory mouse, is the most widely used
test animal; 14 million are used in the US alone annually, and the total number
used globally is close to 100 million. Mice are especially useful for cancer research
because of their high tumor incidence. Their small size and rapid reproduction
make them useful in all areas of research and risk management such as toxicity,
radiobiology, cancer research, behavior research, nutrition, and genetic studies
(FAU, 2013c).
– Guinea pig (Cavia porcellus, Rodentia, Caviidae) is used in antibody production,
tumor genesis, nutrition, genetics, radiation research, and dental studies. It was
an important species in the discovery of vitamin C and the diagnosis of tuberculo-
sis. The guinea pig has been widely employed in biomedical research since 1780.
Lavoisier used the cavy to measure heat production (FAU, 2013a).
– Hamsters (Syrian hamster, Mesocricetus auratus, Rodentia, Cricetidae) have a
unique feature. Their reversible cheek pouch provides a site for normal and abnor-
mal tissue transplants that have the virtue of visibility and ready access. In some
studies, a tumor maintained in one cheek pouch was exposed to an experimental
treatment while the control tumor was maintained in the opposite cheek pouch
(FAU, 2013b).
– Other rodents such as the white-footed mouse (Peromyscus leucopus, Rodentia,
Cricetidae), Mongolian gerbil (Meriones unguiculatus, Rodentia, Cricetidae) and
chinchilla (Chinchilla laniger, Rodentia, Chinchillidae) are also used as laboratory
animals in special cases, mainly for research.
– Rabbit (family Leporidae, order Lagomorpha) has been used in research studies
in genetics, nutrition, and toxicology, also in legislative toxicology, physiology,
immunology and reproduction. The pharmaceutical and cosmetics industries use
the rabbit widely to test the toxic effects of cosmetics and pharmaceuticals. It
is widely used for the production of antibodies and antiserums because of its
relatively large size (FAU, 2013d).
 Human toxicology 135

– Sheep, goat, cow, and pig are also used as laboratory animals, primarily as
transgenic animals or for immunological purposes; they have less importance in
toxicology. Ovis aries, the domestic sheep, has been used in experiments in such
diverse fields of study as endocrinology and reproductive physiology, cardiovascu-
lar physiology, fluid and electrolyte homeostasis, immunology, neurophysiology
and neuroanatomy, thermoregulation, hematology, ingestive behavior, nutrition
and gastrointestinal physiology. The first cloned mammal was also a sheep, called
Dolly.
– Dogs and cats are often used in laboratories. Although all breeds and mixes are
used, Beagles are the most popular test dogs because of their size and docile behav-
ior. Dogs have been used to study maternal deprivation, the effects of smoking,
chemical toxicity of many substances, and the effectiveness of medical devices.
The laboratory cat (Felis catus) is mainly used in reflex studies, exposure to chem-
ical stimuli, in neuropharmacology, particularly the testing of psychotropic drugs,
in behavioral studies, toxicology, oncology and for the study of chromosomal
abnormalities.
– Non-human (NH) primates: the best known laboratory primates are the squirrel
monkey (Saimiri sciureus), baboon (Papio hamadryas,), rhesus (Macaca mulatta),
Japanese or snow macaque (Macaca fuscata), cynomolgus or crab-eating macaque
(Macaca fascicularis) and the chimpanzee (Pan troglodytes). The most famous lab-
oratory monkeys are the Silver Spring macaque monkeys, having been used for
behavioral research for a long time, but becoming the subjects of the first animal
research case to reach the United States Supreme Court in 1991.

2.6 3R in animal testing

“3R’’ in animal testing means replacement, reduction and refinement. The ethical
issue has priority in animal testing. Ethics may play a role in deciding the dilemma of
testing on animals at all. The approach today is very much in favor of humanity and
because of the lack of alternative methods of the same quality. The other component
of the issue is how to keep and handle animals. In this topic, there is now a unified
approach regarding animal welfare, humanized methods, suitable space and food for
the animals, mitigating pain due to interventions and in the case of injury or illness,
and not to mistreat animals.
Non-profit organizations such as ‘Against Cruelty to Animals’ work for animal
welfare and against the use of animals in laboratories (Figure 3.3). Though animals
cannot be removed from tests right away, a number of traditional toxicological tests
have been replaced by alternative methods, and a stepwise reduction of animal use
has already been decided and practiced all over the world. In Europe, the protec-
tion of animals used for experimental and scientific purposes is regulated by Directive
2010/63/EU (EU, 2010). Several organizations and funds such as FRAME in the UK
(Fund for the Replacement of Animals in Medical Experiments) promote the develop-
ment and application of sound scientific principles and methodology which can lead to
the progressive reduction and replacement of laboratory animal procedures. They are
dealing with new concepts, more efficient experimental design, in vitro test methods
and animal welfare education. The 3R concept has been in use for over 50 years and is
still timely, but is unlikely to meet future requirements. The attitude toward, and the
136 Engineering Tools for Environmental Risk Management – 2

basic fundamentals of, toxicology should be changed, i.e. animal tests results should
be stripped of their golden standard role. Evidence-based toxicology is a step forward
in this direction.
Replacement refers to the preferred use of non-animal methods over animal
methods whenever it is possible to achieve the same scientific aim.
Reduction refers to methods that enable researchers to obtain comparable levels of
information from fewer animals, or to obtain more information from the same number
of animals using more adequate statistical methods.
Refinement refers to methods that alleviate or minimize potential pain, suffering
or distress, and enhance animal welfare for the animals still used.
AltTox (2014) is dedicated to reducing the numbers and suffering of animals used
in current toxicology assessments. Its website is designed to exchange information on
in vitro and in silico methods for all types of toxicity tests.
The use of alternatives to testing on animals is on the agenda of ECHA too.
ECHA published two comprehensive reports on alternative methods for the REACH
Regulation (ECHA, 2011 and 2014).

3 TOXICITY END POINTS AND METHODS

In this section, the well-known toxicity end points are discussed in line with the
regulatory requirements for chemical substances.

3.1 Acute toxicity

Acute toxicity is short-term toxicity, the adverse effects of chemical substances resulting
either from a single exposure or from multiple exposures in a short period of time. In
animal tests, toxicity is considered acute when the adverse effects occur within 14
days of administering the substance. In ecotoxicity tests, acute toxicity is defined as a
period of time shorter than the generation time of the test organism. The EC50 , LC50
or ED50 and LD50 values represent the end points used to quantitatively characterize
acute toxicity.
Acute toxicity is distinguished from chronic toxicity: it describes the adverse health
effects from repeated exposures, often at lower levels, to a substance over a longer time
period (months or years).
Acute Systemic Toxicity means adverse effects occurring within a short time after
administering a single, usually extremely high-dose substance via one or more of the
exposure routes of oral way, inhalation and dermal contact or injection into the blood-
stream or the muscles. Systemic effects require absorption and distribution of the
toxicant to a site distant from its entry point. Systemic toxicity does not cause a simi-
lar degree of toxicity in all organs, but usually to one or two organs, which are called
the toxic effect’s target organs.
Acute systemic toxicity testing is the estimation of the human hazard potential of
a substance by determining its systemic toxicity in an animal test system following an
acute exposure. Its assessment has traditionally been based on the median lethal dose
(LD50 ) value – an estimate of the dose of a test substance that kills 50% of the test
animals.
 Human toxicology 137

Table 3.1 OECD test guidelines (TG) for acute systemic testing (OECD, 2014).

Test guideline Test name

OECD TG 403 (2009) Acute Inhalation Toxicity

OECD TG 436 (2008) Acute Inhalation Toxicity – Acute Toxic Class Method
Draft OECD TG 433 (2004) Acute Inhalation Toxicity – Fixed Concentration Procedure
OECD TG 420 (2002) Acute Oral Toxicity – Fixed Dose Procedure
OECD TG 423 (2002) Acute Oral Toxicity – Acute Toxic Class Method
OECD TG 425 (2008) Acute Oral Toxicity: Up-and-Down Procedure
OECD TG 402 (1987) Acute Dermal Toxicity

3.1.1 Animal tests for acute systemic toxicity

In recent years, using death as an end point has been discouraged in all testing contexts,
so the use of the reduction alternatives is now mandatory for acute systemic toxicity
testing. Three reduction alternatives to the oral LD50 tests have been developed and
validated: the Fixed Dose Procedure, the Acute Toxic Class method, and the Up-and-
Down Procedure as summarized in Table 3.1.
As a non-animal alternative, the EU Reference Laboratory for Alternatives to Ani-
mal Testing (EURLECVAM, 2014) proposed a tiered testing strategy for acute systemic
toxicity (Siebert et al., 1996; ECVAM, 2002a). In this testing strategy, a substance
would be sequentially evaluated by (quantitative) structure–activity relationship and
in vitro assays in the following way: QSAR or SAR → cytotoxicity testing → computa-
tional model for metabolism → biotransformation assays → cell-specific toxicity tests.
When classified as very toxic at any step, the testing would end with the classification
of the substance. A limited in vivo acute toxicity test would be performed only if all
of the prior assessments indicated the substance to be not very toxic.

3.1.2 Non-animal, in vitro tests for acute systemic toxicity

Several organizations are involved in developing non-animal tests for human toxicity.
In the US the Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM, 2014), a permanent committee of the NIEHS under the National
Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological
Methods (NICEATM, 2014), in Europe, the European Union Reference Laboratory
for Alternatives to Animal Testing (EURL ECVAM, 2014) is responsible for the 3R in
animal testing. ECVAM DB-ALM (2014) is a searchable database containing in vitro
methods and QSAR models.
ICCVAM validated and endorsed two basic in vitro cytotoxicity assays: the Neutral
Red Uptake (NRU) test with 3T3 rodent cells and the NRU test with normal human
keratinocyte (NHK) cells as adjunct for determining starting doses. OECD established
a technical guidance (TG for 3T3 NRU (2010) and a draft TG for NHK NRU (2010).
EURL ECVAM also recommended the 3T3 NRU assay for supporting the identification
of substances that do not require classification for acute oral toxicity.
Table 3.2 shows validated, and Table 3.3 not yet validated, alternative test meth-
ods. A summary on validated and non-validated alternative test methods is presented
on the AltTox web site
138 Engineering Tools for Environmental Risk Management – 2

Table 3.2 Acute systemic toxicity testing by cell lines: validated in vitro test methods.

Test guideline Method Test purpose Validation

authority

OECD TG BALB/c 3T3 Neutral Red Uptake Adjunct to in vivo acute oral ICCVAM
(3T3 NRU, 2010) Assay toxicity tests for determining
starting doses
Draft OECD TG Normal Human Keratinocyte Adjunct to in vivo acute oral ICCVAM
(NHK NRU, 2010) (NHK) Neutral Red Uptake Assay toxicity tests for determining
starting doses
ECVAM DB-ALM Colony Forming Unit- Hematotoxicity test for ECVAM
Protocol No.101 Granulocyte/Macrophage Assay acute neutropenia DB-ALM
(CFU GM, 2006)

Table 3.3 Acute systemic toxicity testing by cell lines: some in vitro methods under development.

Method Test purpose End point

LLC-PK1 kidney proximal tubule Transepithelial resistance (TER) Barrier integrity/cell damage
cell line and paracellular permeability
MDCK dog kidney epithelial Transepithelial resistance (TER) Barrier integrity/cell damage
cell line and paracellular permeability
HepG2 liver cell line (hepatoma) Protein content Cell growth
HL-60 human acute promyelocytic Adenosine triphosphate (ATP) Energy production and
leukemia cell line content metabolism
Change of liver cell line Morphology change followed Cell growth/cytotoxicity
by pH change

AltTox (2014) is dedicated to advancing non-animal methods of toxicity testing

through online discussion and information exchange.

3.2 Repeated-dose and organ toxicity testing

General toxicological effects occur as a result of repeated daily exposure to a substance
(via oral, inhalation and/or dermal routes) for a portion of the expected life span (i.e.,
subacute or subchronic exposure) or for a major part of the life span (i.e., chronic
exposure) (REACH, 2006).
These general toxicological effects include effects on body weight and/or body
weight gain, absolute and/or relative organ and tissue weights, alterations in clinical
chemistry, urinalysis and/or hematological parameters, functional disturbances in the
nervous system as well as in organs and tissues in general, and pathological alterations
to organs and tissues examined macroscopically and microscopically. In addition to
this information on possible general toxicological effects, repeated-dose toxicity studies
may also provide other information on reproductive toxicity or carcinogenicity or may
identify specific manifestations of toxicity such as neurotoxicity, immunotoxicity or
endocrine-mediated effects (REACH, 2006).
 Human toxicology 139

Table 3.4 OECD test guidelines of animal test methods for repeated-dose and organ toxicity
(OECD, 2014).

Test guideline Test name

OECD TG 412 (2009) Repeated-Dose Inhalation Toxicity: 28-Day or 14-Day Study

OECD TG 413 (2009) Subchronic Inhalation Toxicity: 90-Day Study
OECD TG 408 (1998) Repeated-Dose 90-Day Oral Toxicity Study in Rodents
OECD TG 409 (1998) Repeated-Dose 90-Day Oral Toxicity Study in Non-Rodents
OECD TG 407 (2008) Repeated-Dose 28-Day Oral Toxicity Study in Rodents
OECD TG 410 (1981) Repeated-Dose Dermal Toxicity: 21/28-Day Study
OECD TG 411 (1981) Subchronic Dermal Toxicity: 90-Day Study
OECD TG 422 (1996) Combined Repeated-Dose Toxicity Study with the Reproduction/
Developmental Toxicity Screening Test

3.2.1 Animal test methods for repeated-dose and organ toxicity

Animal test methods are listed in Table 3.4.
OECD is updating TG 407, the repeated-dose 28-day oral toxicity test in rats,
to validate a protocol suitable for detecting the endocrine disruption potential of test
substances. OECD 422 makes a reduction in the number of animals possible.
The following tissues/organs can be studied histologically in a repeated-dose
toxicity study:

Adrenal glands Pituitary gland

Aorta Prostate
Blood smears Salivary glands
Brain Seminal vesicles (rodents)
Epididymides Skeletal muscle
Eyes and optic nerves Skin and subcutaneous tissue
Gall bladder Small intestines
Gross lesions Spinal cord
Heart Spleen
Joint with bone Sternebrae, femur or vertebrae
Kidneys and ureters Stomach
Large intestines Testes
Larynx Thymus
Liver Thyroid/Parathyroid glands
Lungs with bronchi and bronchioles Tissue masses of tumors
Lymph nodes Tongue
Mammary glands Trachea
Esophagus Urinary bladder
Ovaries Uterus with uterine cervix and oviducts
Pancreas Vagina
Peripheral nerves
140 Engineering Tools for Environmental Risk Management – 2

3.2.2 Alternative methods for repeated-dose and

organ toxicity testing
Potential non-animal alternatives for chronic toxicity testing include human and animal
perfused organs such as liver, kidney, lung, organ tissue slices, isolated, suspended cells,
primary cultured cells, cultured cell lines, genetically engineered cell lines, reaggregat-
ing cell cultures, three-dimensional cell cultures and co-cultures, and computational
systems for SAR and QSAR.
As yet, ICCVAM and ECVAM have not validated any non-animal methods for
assessing chronic toxicity end points or repeated exposure target organ toxicity, but
there are several potential candidates among the innovative in vitro test methods e.g.
in the DB-ALM database:

– Alginate entrapped primary hepatocytes: cryopreserved, immobilized three-

dimensional cell cultures, which can be used to study hepatotoxicity;
– Collagen-gel sandwich configuration culture of primary hepatocytes: a long-term
hepatocyte culture system used to perform cytotoxic and metabolism-mediated
toxicity studies;
– Culture of freshly isolated nonparenchymal liver cells and nonparenchymal cell
lines (Kupffer cells, biliary epithelial cells, stellate cells) is used to study the
mechanisms of hepatotoxicity;
– Genomics technologies are used to investigate hepatotoxicity in vitro, e.g. toxi-
cogenomics (mRNA analysis) and proteomics (gene product analysis) for screening
and mechanistic hepatotoxicity studies;
– Hepatocyte cultures on biosensor systems: direct examination of certain cellular
processes following exposure to xenobiotics;
– Monolayer culture of genetically engineered liver cell lines are designed to express
defined xenobiotic metabolizing enzymes. They may be used in short- and long-
term metabolism and hepatotoxicity studies;
– Monolayer culture of liver cell lines can be used for basal toxicity, hepatotoxicity,
genotoxicity and metabolism-mediated toxicity studies;
– Monolayer culture of primary hepatocytes is the most frequently used in vitro
system for cytotoxic, genotoxic and mechanistic studies of hepatotoxicity and
metabolism-mediated toxicity;
– Perfused two- and three-dimensional culture systems (bioreactors) with liver cells
improve the viability and functionality of liver cells in culture and allow for
short- and long-term studies of hepatotoxicity, metabolism-mediated toxicity and
genotoxicity;
– Suspension culture of freshly isolated or cryopreserved hepatocytes derived
from animals or humans are used for short-term studies on hepatotoxicity and
metabolism-mediated toxicity;
– Avian (hen, turkey, quail, duck) embryonic tissue are applied for studies on
metabolism and metabolism-mediated toxicity of chemicals in ovo;
– Freshly prepared or preserved liver slices from laboratory and domestic animals,
humans, fish, birds and amphibians can be used for short- or long-term studies on
hepatotoxicity;
– Stem or progenitor cell-derived hepatocyte-like cells may be used in studies on
hepatotoxicity and metabolism-mediated toxicity;
 Human toxicology 141

– Subcellular fractions of liver tissue (such as microsomes and mitochondria) and

liver homogenate can be used for short-term studies on hepatotoxicity and
metabolism-mediated toxicity studies;
– Isolated Rat Glomeruli and Proximal Tubules isolated from the kidney show cyto-
toxic effect of chemicals by cell glucose and/or fatty acid oxidation and de novo
protein synthesis.

3.3 Genotoxicity
Genotoxicity is chemically induced genetic mutations and/or other alterations of the
structure, information content, or segregation of genetic material. Genotoxic chemicals
are genotoxins, which cause heritable changes in the genetic material of spermatocytes
or oocytes. Compared to mutagens which cause mutations in DNA, genotoxins may
interact in a broader sense with DNA or non-DNA targets. Genotoxic carcinogens can
lead to DNA mutations with the potential to cause cancer. Distinguishing the point
at which exposure to a carcinogen increases mutation rates, is challenging. Currently,
there is a general agreement that, for genotoxic carcinogens, no specific threshold can
be identified. On the other hand, scientists have experienced in practice that a series
of mutation events are needed before malignancy occurs and a single, small exposure
may not result in disease. In addition, cells have their own defense mechanism to
counter the effects of mutagens. All in all, many research and scientific activities deal
with the question of thresholds for genotoxic chemicals, because a scientifically based
NOAEL may play an important role in regulation and risk management of genotoxic
and mutagenic substances (Greim, 2012).
A simple classification of the measurement end points for genotoxic substances
and genotoxic mutagens is given below:
– In vitro gene mutation – Ames mutagenicity;
– In vitro chromosomal mutation – micronucleus assay;
– In vivo chromosomal mutation – micronucleus assay;
– In vivo genotoxic carcinogenicity – rodent assay.
The term genotoxicity is used in general, without specifying the type of interaction or
the name of the test assay. There are in vivo and alternative in vitro test methods for
assessing the potential of heritable genotoxicity on germ cells, on somatic cells, and
on chromosomes, as the above list shows.

3.3.1 In vivo animal tests for assessing potential heritable genotoxicity

Table 3.5 shows the traditional animal test methods for genotoxicity.

Table 3.5 OECD test guidelines of animal test methods for genotoxicity (OECD, 2014).

Test guideline Test name

OECD TG 478 (1984) Genetic Toxicology: Rodent Dominan Lethal Test

OECD TG 479 (1986) Genetic Toxicology: In vitro Sister Chromatid Exchange Assay
in Mammalian Cells
OECD TG 484 (1986) Genetic Toxicology: Mouse Spot Test
OECD TG 485 (1986) Genetic Toxicology: Mouse Heritable Translocation Assay
OECD TG 483 (1997) Mammalian Spermatogonial Chromosome Aberration Test
OECD TG 475 (1997) Mammalian Bone Marrow Chromosome Aberration Test
142 Engineering Tools for Environmental Risk Management – 2

Table 3.6 OECD guidelines for non-animal genotoxicity and mutagenicity testing methods
(OECD, 2014).

Test guideline Test name

OECD TG 477 (1984) Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila
melanogaster
OECD TG 479 (1986) Genetic Toxicology: In vitro Sister Chromatid Exchange Assay
in Mammalian Cells
OECD TG 480 (1986) Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay
OECD TG 481 (1986) Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay
OECD TG 482 (1986) Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis
in Mammalian Cells In Vitro
OECD TG 471 (1997) Bacterial Reverse Mutation Test (Ames Test)
OECD TG 472 (1997) Genetic Toxicology: Escherichia coli, reverse assay
OECD TG 473 (1997) In vitro Mammalian Chromosome Aberration Test
OECD TG 474 (1997) Mammalian Erythrocyte Micronucleus Test
OECD TG 476 (1997) In vitro Mammalian Cell Gene Mutation Test
OECD TG 486 (1997) Unscheduled DNA Synthesis (UDS) Test with Mouse Liver Cells in vitro
OECD TG 487 (2010) In vitro Mammalian Cell Micronucleus Test (MNT) – Alternative to the
in vitro chromosome aberration assay for genotoxicity testing

3.3.2 OECD test guidelines for in vitro genotoxicity and

mutagenicity testing
The non-animal mutagenicity tests may use bacterial cells, yeasts or isolated mam-
malian cells. Most of them are used as screening assays in tiered mutagenicity testing.
Table 3.6 shows the OECD guidelines for in vitro genotoxicity test methods.
The Ames test is one of the most widely used screening tests for mutagenicity and
carcinogenicity, and uses several different strains of histidine auxotrophic Salmonella
typhimurium mutants that carry mutations in genes responsible for histidine synthesis,
and essential for growth. If histidine synthesis is absent in the bacterial cell, it cannot
grow on a histidine-free nutrient medium. Six strains are specially selected and mixed to
have both frameshift and point mutations in the genes required to synthesize histidine,
which allows for the detection of mutagens acting via different mechanisms.
A positive test indicates that the chemical substance might act as a mutagen or
carcinogen (Figure 3.4). A substance that proves positive in the Ames test is not nec-
essarily mutagenic or carcinogenic in tests performed on mammal cells or organisms.
As the Ames test overestimates mutagenic risk, it is appropriate to use it as a screening
method in a tiered assessment procedure.

3.3.3 New in vivo genotoxicity tests

The Comet Assay or single cell gel electrophoresis (SCGE) assay is a rapid, sensitive
and relatively simple method for detecting DNA damage at the level of individual cells
(Singh et al., 1988). It combines the simplicity of biochemical techniques for detecting
DNA single strand breaks (strand breaks and incomplete excision repair sites), alkali-
labile sites, and cross-linking, with the single cell approach typical of cytogenetic assays
(COMET, 2013).
 Human toxicology 143

Figure 3.4 Ames reverse mutation of Salmonella typhimurium: the number of revertants is proportional
to the mutagenicity of the tested chemical substance.

Depending on the origin of the sample cells with which the chemical substances
interact (toxic chemicals or drugs to be tested), the Comet Assay can be in vivo (cells
from an in vivo test subject) or in vitro (cells extracted from an in vitro cell culture).
As only a small numbers of cells are required for analysis, literally any tissue or organ
is amenable to investigation. The only requirement is that a sufficient number of single
cells (or nuclei) are obtained for analysis and that no or minimal damage is induced
during tissue processing (Tice et al., 2000).
The Comet assay is eventually gel electrophoresis of the DNA from whole cells
under alkaline (pH > 13) conditions, resulting in an image similar to a ‘comet’ with
a distinct head and tail (Figure 3.5). The head is composed of intact DNA, while the
tail consists of damaged and broken pieces of DNA with double strand breaks, single
strand breaks, alkali-labile sites, oxidative base damage, and DNA cross-linking with
DNA or protein. Both animal and plant cells can be investigated using this method.
The thin agarose-gel together with treated cells is poured onto a microscope slide
and cells are lysed by alkali, which lets the DNA unwind. Unwinding allows the bro-
ken and damaged DNA fragments to migrate away from the nucleus. Staining with
ethidium bromide or other DNA-specific dyes allows the head and tail length to be
evaluated based on fluorescence intensity. The extent of DNA liberated from the head
and forming the tail is proportional to the rate of DNA damage.
The international validation studies of both the in vitro and in vivo genetic toxicity
Comet Assays were ongoing in 2010, with the lead of JaCVAM (2013) – the Japanese
Center for the Validation of Alternative Methods – in collaboration with NICEATM-
ICCVAM and ECVAM.
144 Engineering Tools for Environmental Risk Management – 2

Figure 3.5 CometAssay: living cells are treated with the chemical substance to test, and then embedded
into agarose which is poured on a slide. After electrophoresis it is stained.

The in vivo transgenic mutation assay uses live animals. This is a well-established
assay employing transgenic rats or mice, which contain multiple copies of chromoso-
mally integrated plasmid or phage DNA that harbor reporter genes for the detection
of mutations. The mutations of the transgenes of lac repressor or lacZ (gene of beta-
galactosidase) are of no consequence for the rodents as they are genetically neutral.
This means that the mutations provide their host cell with neither an advantage nor dis-
advantage in either survival or proliferation. OECD concluded that there is more than
sufficient evidence of validation of the in vivo transgenic mutation assay to support
the establishment of a Test Guideline (Douglas, 2010).
An ECVAM panel proposed that total replacement of animal testing for genotoxi-
city/mutagenicity would require models for evaluating toxicokinetics and metabolism
(Maurici et al., 2005a). In vitro genotoxicity tests also need to be modified to use cell
lines relevant to the target organs of interest, which would require standardization and
validation of in vitro assays in mammalian germ cells for predicting heritable germ cell
damage. Some other genotoxicity and mutagenicity test protocols from the ECVAM
DB-ALM (2014) database include:

– Cytotoxicity and Genotoxicity in Primary Cultures of Human Hepatocytes of the

test compound by measuring cell viability, DNA damage and unscheduled DNA
synthesis (DB-ALM Protocol No.16);
– Stable Cell Lines Expressing Cytochromes CYP cDNA can be inserted into cell
lines such as V79 Chinese hamster cells, which are used for in vitro toxicity testing.
This means that the metabolites of xenobiotics are produced in the same cells in
 Human toxicology 145

which any toxic effect will be observed, thus negating the problems associated
with co-cultures and the use of subcellular fractions (DB-ALM Protocol No.107);
– DNA Binding in Bacteria may be used to elucidate primary genotoxic mecha-
nisms through the analysis of mutated bacterial DNA by high pressure liquid
chromatography (HPLC) (DB-ALM Protocol No.8);
– Alkaline Unwinding Genotoxicity Test applies to mouse lymphoma cells cul-
tured in the presence of test chemicals, with or without a metabolic activating
system, and resultant DNA-strand breaks detected by alkaline unwinding and
hydroxyapatite elution (DB-ALM Protocol No.19);
– Prostaglandin H Synthase (PHS) mediated Genotoxicity of Xenobiotics. This pro-
tocol describes the use of SEMV cells (a cell line derived from ram seminal vesicles)
for studying prostaglandin H synthase-mediated metabolism of xenobiotics in
intact cells (DB-ALM Protocol No.61).

3.3.4 QSAR for genotoxicity and genotoxic carcinogenicity

The QSAR approach is also applicable for genotoxic chemicals based on the covalent
DNA binding as the molecular initiating event of genotoxicity and genotoxic carcino-
genicity (Schultz, 2010). OECD prepared a QSAR Toolbox to support grouping of
genotoxic and carcinogenic substances, on the basis of a common molecular initiating
event, the ability of a chemical to bind covalently to DNA (the effect-mechanism of
non-genotoxic mutagens includes many other molecular initiating events). Out of the
chemical structure and other physico-chemical characteristics the following databases
were used by the OECD QSAR Toolbox (OECD, 2012):

– Bacterial mutagenicity ISSSTY: 7367 chemicals’ effect on gene mutation, measured

by in vitro Salmonella typhimurium test;
– Carcinogenic potency database CPDB: 1530 chemicals’ carcinogenic effect (TD50 ),
measured by in vivo animal tests (rats, mice, hamsters, dogs and non-human
primates);
– Carcinogenicity ISSCAN: 1150 chemicals’ carcinogenic effect (TD50 ), measured
in vivo on rats and mice;
– Cell Transformation Assay ISSCTA: 327 chemicals’ non-genotoxic carcinogenic-
ity, measured by in vitro cell lines (SHE cells, BALB/c 3T3, C3H/10HT1/2 and
Bhas42);
– Genotoxicity OASIS: 7500 chemicals’ gene mutation and chromosomal mutation,
both (yes/no), tested in vitro by Salmonella typhimurium and in vitro by Chinese
hamster lung cells and T-lymphoma cell lines;
– Micronucleus ISSMIC: 564 chemicals’ chromosomal mutation (yes/no), measured
in vivo by rats and mice;
– Micronucleus OASIS: 557 chemicals’ chromosomal mutation, measured in vivo.

3.4 Chronic toxicity

Chronic toxicity describes the adverse health effects due to repeated exposures to a
substance over a longer time period (months or years).
146 Engineering Tools for Environmental Risk Management – 2

Table 3.7 OECD guidelines for testing chronic toxicity

(OECD, 2014).

Test guideline Test name

OECD TG 452 (2009) Chronic Toxicity Studies

OECD TG 453 (2009) Combined Chronic Toxicity/
Carcinogenicity Studies

3.4.1 Chronic toxicity testing methods on animals

– The objectives of chronic toxicity studies include:
– The identification of the hazardous properties of a chemical substance
– The identification of target organs
– Characterization of the dose–response relationship
– Identification of a No Observed Adverse Effect Level (NOAEL) or point of
departure for establishment of a Benchmark Dose (BMD)
– The prediction of chronic toxicity effects at human exposure levels
– Provision of data to test hypotheses with regard to mode of action (OECD, 2008a).

Currently, two OECD guidelines are in existence for testing chronic toxicity
(Table 3.7). The updating of TG 452 has been carried out at the same time as the
revisions of the Test Guidelines 451 on Carcinogenicity Studies and TG 453, with the
objective of obtaining additional information from the animals used in the study and
providing further details on dose selection.
The majority of chronic toxicity studies are carried out in rodent species, therefore
Test Guideline 452 is intended to apply primarily to studies carried out on these species.
In a chronic test, the test substance is administered daily in graduated doses to
several groups of experimental animals for a period of 12 months, although longer or
shorter durations may also be chosen.
This duration is chosen to be sufficiently long to allow any effects of cumulative
toxicity to become manifest, without the confounding effects of geriatric changes.
Deviations from the exposure duration of 12 months must be justified, particularly
for shorter durations. The test substance is normally administered by the oral route
although testing by the inhalation or dermal routes may also be appropriate. The
study design may also include one or more interim kills, e.g. at 3 and 6 months,
and additional groups of animals may be included to accommodate this. During the
period of administration, the animals are observed closely for signs of toxicity (OECD,
2008a).

3.5 Carcinogenicity
Carcinogenicity is the character of a chemical substance, which is able to induce
cancer or is carcinogenic. This may occur through genotoxic (see Section 3.3)
or non-genotoxic mechanisms. While covalent DNA binding has been identified
as the molecular initiating event of genotoxicity and genotoxic carcinogenicity,
 Human toxicology 147

Table 3.8 OECD guidelines for testing carcinogenicity

(OECD, 2014).

Test guideline Test name

OECD TG 451 (2009) Carcinogenicity Studies

OECD TG 453 (2009) Combined Chronic Toxicity/
Carcinogenicity Studies

non-genotoxic carcinogenicity may be based on other molecular initiating events such

as protein binding, non-covalent interactions with protein receptors, intercalation
with DNA and the formation of free radicals. Carcinogenic substances are those that
induce benign or malignant tumors, increase their incidence or malignancy, or shorten
the time of tumor occurrence when they are inhaled, injected, dermally applied, or
ingested.

3.5.1 Animal methods for carcinogenicity testing

The conventional test for carcinogenicity is the long-term rodent carcinogenicity bioas-
say described in OECD TG 451 (Table 3.8). The objective of this test is to observe test
animals for a major portion of their life span for the development of neoplastic lesions
during or after exposure to various doses of a test substance by an appropriate route
of administration.
Two end points in animal bioassays, carcinogenicity and chronic toxicity, can be
combined to reduce animal use, as described in OECD TG 453. Another guidance
gives new methodological support for chronic toxicity and mutagenicity testing, for
example, with proper dose selection (OECD, 2009).

3.5.2 Non-animal testing of carcinogenicity

Non-animal methods include in vitro cell-based assays and computational prediction
models. The cell transformation assay (CTA) detects phenotypic changes induced by
chemicals in mammalian cell cultures (Maurici et al., 2005b). Different bioassays use
different cell cultures:

– Syrian hamster embryo (SHE) assay, detects the early steps of carcinogenesis
– Low-pH SHE assay, detects the early steps of carcinogenesis
– Balb/c 3T3 assay, detects later carcinogenic changes
– C3H/10T1/2 assay, detects later carcinogenic changes (OECD, 2006a).

The gap junction intercellular communication (GJIC) method is based on the

disruption of the intercellular exchange of low-molecular-weight molecules through
the gap junctions of adjacent cells; this disruption can result in abnormal cell growth
and behavior (Maurici et al., 2005b). The assay appears to be a good candidate for
screening for non-genotoxic carcinogens and tumor promoters, but it still needs to be
standardized and validated.
148 Engineering Tools for Environmental Risk Management – 2

Mutagenicity/genotoxicity assays are the most commonly used in vitro test sys-
tems to predict carcinogenicity. Mutagenicity refers to the induction of transmissible
changes in the structure of the genetic material of cells or organisms. Mutations may
involve a single gene or a group of genes. Genotoxicity is a broader term that refers to
changes to the structure or number of genes via chemical interaction with DNA and/or
non-DNA targets such as the spindle apparatus and topoisomerase enzymes. The term
genotoxicity is generally used unless a specific assay is being discussed. In use for over
30 years, genotoxicity assays are employed in a tier-testing approach that starts with
Tier I in vitro tests, followed by Tier II in vivo genotoxicity tests to determine the
biological relevance of chemicals that are positive in the in vitro tests. Common geno-
toxicity testing batteries include assays that measure mutations as well as structural
and numerical chromosome aberrations (Maurici et al., 2005a).
In vitro genotoxicity test methods have been adopted at the EU level with OECD
guidelines and, additionally, the old in vitro chromosome aberration assay has been
replaced with the in vitro micronucleus test for genotoxicity testing (see Table 3.6 in
Section 3.3.2).
Numerous other in vitro genotoxicity tests, including the in vitro Comet assay,
are being developed but are not yet validated. ECVAM DB-ALM Database, (2014)
comprises some protocols for carcinogenicity testing:

– Lucifer Yellow Intercellular Exchange assay for Tumor Promoters: the effect of the
test substance on the transfer of the dye lucifer yellow between SV-40-transformed
hamster fibroblasts is an indication of potential tumor-promoting activity (DB-
ALM Protocol No.65);
– DNA Binding Studies for Alkylating Compounds using isolated perfused rat liver.
This procedure uses an adaptation of the perfused rat liver technique to assess the
capacity of directly alkylating compounds to induce DNA-binding and therefore
mutagenicity (DB-ALM Protocol No.89);
– GreenScreen HCTM Genotoxicity test is a fast, quantitative genotoxicity assay
in vitro. The assay uses the DNA damage-inducible “Growth Arrest and DNA
Damage 45 alpha-Green Fluorescent Protein’’ reporter gene, expressed in p53-
competent TK6 cell line. The response to a chemical insult leads to an increase
in green fluorescence. GreenScreen HC + S9 allows for a detection of genotoxic
potential of the test compound with and without metabolic activation. It is
available in reagent kit form (DB-ALM Protocol No.132);
– In vitro Syrian Hamster Embryo Cell Transformation assay (SHE CTA) is a short-
term assay recommended as an alternative method for testing of the carcinogenic
potential of chemicals (both genotoxic and non-genotoxic). The assay is based on
the change of the phenotypic features of cell colonies undergoing the first steps of
the conversion from normal cells to neoplastic-like cells with oncogenic properties
(DB-ALM Protocol No.136).

3.6 Reproductive and developmental toxicity

Chemically induced adverse effects on sexual function, fertility, and/or normal off-
spring development (resulting in, for example, spontaneous abortion, premature
 Human toxicology 149

Table 3.9 OECD guidelines for reproductive and developmental toxicity (OECD, 2014).

Test guideline Test name

OECD TG 415 (1983) One-Generation Reproduction Toxicity Study, in Rats and Mice
OECD TG 421 (1995) Reproduction/Developmental Toxicity Screening Test, with male
and female rats, oral administration for 4–9 weeks
OECD TG 422 (1996) Combined Repeated Dose Toxicity Study with the Reproduction/
Developmental Toxicity Screening Test
OECD TG 414 (2001) Prenatal Development Toxicity Study, with female rats and rabbits
OECD TG 416 (2001) Two-Generation Reproduction Toxicity, by dosing offspring
OECD TG 426 (2007) Developmental Neurotoxicity Study
OECD TG 443 (2012) Extended One-Generation Reproductive Toxicity Study

delivery, and birth defects) are generally determined through the breeding of one or
more generations of offspring.
Reproductive toxicity includes the toxic effects of a substance on the reproductive
ability of an organism and the development of its offspring. It has been defined by
UNECE (2003a; 2009a) as any effect of chemicals that would interfere with reproduc-
tive ability or capacity, including effects on lactation. The definition of developmental
toxicity is very broad; GHS (Globally Harmonized System) considers the follow-
ing UNECE (2003a) definition sufficient for classification purposes: adverse effects
induced during pregnancy, or as a result of parental exposure, that can be manifested
at any point in the life span of the organism.

3.6.1 Animal tests for reproductive and developmental toxicity

OECD standardized five animal tests, including rat, mice and rabbit one- and two-
generation tests and developmental neurotoxicity. Animal tests are listed in Table 3.9.

3.6.2 In vitro methods for reproductive and developmental toxicity

The ECVAM Scientific Advisory Committee (ESAC) endorsed three in vitro methods
for embryotoxicity testing as scientifically validated (ECVAM, 2001):

– Embryonic stem cell test for embryotoxicity

– Micromass embryotoxicity assay
– Whole rat embryo for embryotoxicity assay.

An ICCVAM-NICEATM meeting (2000) reviewed the Frog Embryo Teratogenesis

Assay: Xenopus (FETAX) as a potential alternative for assessing developmental tox-
icants. The method was deemed not ready for validation, so recommendations were
made for its development.
FETAX is a 4-day, whole embryo-larval developmental toxicity screening assay,
which uses young embryos of the South African clawed frog, Xenopus laevis (Fig-
ure 3.6). The FETAX system is capable of monitoring acute, chronic, developmental,
and behavioral toxicity for ecological and human health hazard assessment. The first
150 Engineering Tools for Environmental Risk Management – 2

Figure 3.6 Albino Xenopus laevis (Kenpei, 2013) (left) and Xenopus laevis (Gratwitzke, 2012) (right).

Figure 3.7 Malformations of Xenopus embrios due to gene mutations (Hikasa et al., 2002).

96 h of embryonic development in Xenopus parallel many of the major processes of

human organogenesis.
FETAX is able to measure embryolethality, embryonic malformation, embryonic
growth reduction and embryonic developmental delay. Figure 3.7 shows the morpho-
logical consequences of the mutations of the globin gene and the Xror2 (timidin kinase
receptor) gene (Hikasa et al., 2002).
 Human toxicology 151

Table 3.10 OECD guidelines for in vivo and in vitro testing

of dermal penetration (OECD, 2014).

Test guideline Test name

OECD TG 427 (2004) Skin Absorption: in vivo method

OECD TG 428 (2004) Skin Absorption: in vitro method

As a screening test, a positive FETAX response would indicate a potential human

hazard while a negative FETAX response would not indicate the absence of a hazard.
In the role of a screening assay, a negative response would be followed by in vivo
mammalian testing, while a positive response would require no further testing unless
the investigator is concerned about a potential false positive response (ICCVAM,
2000).
Xenopus is a well-developed vertebrate model for biomedical research to under-
stand fundamental biological processes and toxicity testing. The knowledge on
Xenopus is collected and published in the bioinformatic database of Xenbase
(2014).

3.7 Dermal penetration

Dermal penetration, also called percutaneous penetration or skin absorption (dermal
uptake), measures the rate of penetration of a chemical substance through the dermal
barrier and getting into the systemic circulation. Two mechanisms may play a role in
the transport of chemicals: passive diffusion and biotransformation.

3.7.1 Animal testing of dermal penetration

Local or systemic effects on the rat, the test organism, follow dermal penetration as
described in OECD TG 427. Table 3.10 includes one in vivo and another in vitro
method standardized by OECD.

3.7.2 In vitro testing of dermal penetration

A variety of in vitro methods have been developed for dermal penetration testing.
The chemical substance is applied to the surface of a skin sample separating the
two chambers (a donor chamber and a receptor chamber) of a diffusion cell. Static
and flow-through diffusion cells are both acceptable. Most of these methods use full
or partial thickness human or animal skin mounted in a diffusion chamber (Figure
3.8). Although viable skin is preferred, non-viable skin can also be used. Pigskin is
commonly used.
Human cell-based or reconstituted human skin cell models such as EpiDerm,
EpiSkin, and SkinEthic and a rat keratinocyte culture model (ROC) are also being
used to evaluate dermal penetration. These models are considered to be metabolically
active, but in most cases, they are more permeable than in vitro human and animal
skin preparations.
152 Engineering Tools for Environmental Risk Management – 2

Figure 3.8 Testing skin penetration applying viable or non-viable skin.

3.8 Skin irritation and corrosion

Chemically induced skin damage may be reversible (called irritation) or irreversible
(called corrosion).
GHS defines skin irritation as the production of reversible damage to the skin
following the application of a test substance for up to 4 h. Skin corrosion is defined as
the production of irreversible damage to the skin; namely, visible necrosis through the
epidermis and into the dermis, following the application of a test substance for up to
4 h (UNECE, 2009c).

3.8.1 Animal testing of skin irritation and corrosion

One of the functions of the skin is to protect the body from environmental hazards
such as toxic or corrosive chemicals. When the skin is exposed to a chemical, spe-
cific immunological and histological responses can occur. Four OECD test guidelines
(Table 3.11) exist on acute, repeated-dose and subchronic dermal toxicity.
TG 402 Acute Dermal Toxicity test is also mentioned amongst acute systemic
toxicity tests, 410 and 411 as repeated-dose and organ-specific test methods.

3.8.2 Alternative, non-animal test methods for skin

irritation and corrosion
A number of alternative test methods have been developed for dermal irritation and
corrosion as shown in Table 3.12. Most of them are included in the European leg-
islation and used for classification and labeling (EU Test Methods, 2008; EU Test
 Human toxicology 153

Table 3.11 OECD guidelines for in vivo testing of skin irritation and corrosion
(OECD, 2014).

Test guideline Test name

OECD TG 402 (1987) Acute Dermal Toxicity

OECD TG 410 (1981) Repeated-Dose Dermal Toxicity: 21/28-Day Study
OECD TG 411 (1981) Subchronic Dermal Toxicity: 90-Day Study
OECD TG 404 (2002) Acute Dermal Irritation/Corrosion, 4 h exposure
and 14 day observation of exposed albino rabbit

Table 3.12 OECD guidelines for in vitro testing of skin irritation and corrosion (OECD, 2014).

Test guideline Test name

OECD TG 439 (2013) In vitro Skin Irritation – Reconstructed Human Epidermis Test Method
for hazard identification of irritant chemicals and to identify non-classified
chemicals
– EpiSkin SIT for dermal irritation
– EpiDerm SIT modified for dermal irritation
OECD TG 430 (2013) In vitro Skin Corrosion – Rat Skin Transcutaneous Electrical Resistance (TER)
OECD TG 431 (2013) In vitro Skin Corrosion – Reconstructed Human Epidermis (RHE)
Test Methods:
– EpiSkin – standard model
– EpiDerm – skin corrosivity
– SkinEthic – for distinguishing corrosive from non-corrosive substances
– Epidermal Skin Test epiCS for distinguishing corrosive from non-corrosive
substances
OECD TG 435 (2006) In vitro Membrane Barrier Test Method for Skin Corrosion – Corrositex –
non-cellular membrane

Methods; 2009, B.4, 2008; and B.46, 2008). These in vitro test methods generally use
in vitro grown human skin for testing. To make the penetration and response of the
skin more realistic, 3D models have also been established.
In vitro 3D skin models, or reconstructed human epidermis (RHE) models, consist
of human cells grown on a membrane at the air–liquid interface. This method of
culturing induces the cells to grow in multilayers and to form junctions between the
cells so that the cultures are similar to small pieces of human skin in the wells of a plate.

– EpiDerm Skin Irritation Test (SIT) (MatTek Corporation, 2014);

– EpiSkin SIT, Nice, France (EpiSkin, 2014);
– SkinEthic RHE, Nice, France (SkinEthic, 2014);
– LabCyte EPI-MODEL from J-TEC, Japan (Vitro-Life Skin, 2014);
– EST-1000 RHE (CellSystems, 2014).

The end points typically evaluated for skin irritation and corrosion testing are:

– Cytotoxicity (MTT assay) for both irritation and corrosion;

– Cytotoxicity (MTT assay) plus IL-1a (cytokine release) for irritation.
154 Engineering Tools for Environmental Risk Management – 2

Ex vivo models, or skin explants, consist of pieces of skin from humans or animals
for in vitro testing applications. These have been used in screening for skin irritants
but are more useful for testing skin corrosion or dermal absorption (skin penetration).
Ex vivo models for skin irritation, penetration and corrosion are:

– Mouse skin integrity function test (SIFT);

– Human cadaver skin;
– Human skin from surgery;
– Pig ear test (Zuang et al. 2005);
– Rat skin transcutaneous electrical resistance (TER) method has been validated for
skin corrosion testing.

A cell-free barrier model called Corrositex® has also been validated for skin cor-
rosion testing. The Corrositex assay is based on the time it takes for a chemical to
penetrate an artificial biobarrier. In 1999, an ICCVAM review of Corrositex (National
Toxicology Program, 1999) recommended its use as a stand-alone assay for evaluat-
ing acids, bases, and acid derivatives for the US Department of Transportation, and
otherwise, as part of a tiered testing strategy.

3.9 Skin sensitization

The induction of allergic contact dermatitis following exposure to a chemical agent is
the response of the immune system. This is the only validated immunotoxicity test that
measures chemically induced adverse effects on the immune system through the skin.
GHS defines a skin sensitizer as a substance that will induce an allergic response
following skin contact in a substantial number of persons or when there are posi-
tive results from an appropriate animal test (UNECE, 2003c). Human experiments
to evaluate the skin sensitization potential of a material are not generally conducted,
but human testing might be done to confirm a negative animal test result. Human
data could also be the result of an epidemiological study or documentation of allergic
contact dermatitis from more than one dermatology clinic (AltTox, 2008).

3.9.1 Skin sensitization: animal tests for regulatory requirements

The initial exposure is called the sensitization phase; it has no clinical symptoms. The
delayed skin response from a later exposure to the allergen is called the elicitation phase.
Clinical symptoms include erythema (redness), vesicles/bulla, papules, scaling, and
pruritus (itching). Common examples of substances that can induce a skin sensitization
reaction in certain individuals – also known as allergic contact dermatitis – include
metals in jewelry and chemicals in cosmetics or in latex gloves.
Table 3.13 shows the traditional rat and mice tests for skin sensitization. Local
Lymph Node Assay has two new versions, the non-radioactive and the reduced versions
of the Local Lymph Node Assay.

3.9.2 Non-animal alternative methods

There are currently no validated in vitro or in silico methods to replace animal testing
for the detection of skin sensitizers. However, many promising methods are in the
 Human toxicology 155

Table 3.13 OECD guidelines for in vivo testing of skin sensitization (OECD, 2014).

Test guideline Test name

OECD Test TG 406 (1992) Skin Sensitization – testing with rat and mice
OECD Test TG 429 (2010) Skin Sensitization – Local Lymph Node Assay (LLNA) with rat or mice
OECD Test TG 442A (2010) Skin Sensitization – a non-radiactive LLNA method, based on
chemiluminescence of ATP
OECD Test TG 442B (2010) Skin Sensitization – a non-radiactive LLNA method, based on
thymidine analogue of 5-bromo-2-deoxyuridine (BrdU) content

developmental stage. It is expected that a predictive method to totally replace animal

testing will be a test battery composed of molecular, cell-based, and/or computational
methods, as follows:

– Peptide reactivity (or depletion) assay measures binding to skin proteins.

– Cell-based assays with cultured cells to model the mechanism(s) of induction of
contact allergy in the skin.
– Quantitative structure–activity relationship for classifying allergens and non-
allergens on the basis of physico-chemical data and the reactivity parameters of
functional groups.
– Predictive Test Battery integrates SAR or QSAR and molecular and/or cell-based
assays to form predictive test batteries to identify human skin sensitizers and
replace animal testing.

None of these methods have been validated yet.

3.10 Eye irritation and corrosion

Eye irritation is defined as the production of changes in the eye following the appli-
cation of a test substance to the anterior surface of the eye, which are fully reversible
within 21 days of application (UNECE, 2003b; UNECE, 2009b). Eye corrosion (seri-
ous eye damage) is defined as the production of tissue damage in the eye, or serious
physical decay of vision, following application of a test substance to the anterior sur-
face of the eye, which is not fully reversible within 21 days of application (UNECE,
2003b).

3.10.1 Animal testing of eye irritation and corrosion on rabbits

The Draize rabbit eye irritation test still remains the standard method (Table 3.14)
for evaluating the ocular irritation/corrosion potential of a substance for regulatory
purposes (Draize et al., 1944). In this test, a material is instilled into one eye of albino
rabbits (the other eye serving as the negative control), and the response of the animals
is monitored using a standardized scoring system for injury to the cornea, conjunctiva,
and iris.
Apart from the ethical issues, the Draize rabbit eye test has other disadvantages,
for example, it overpredicts the human responses.
156 Engineering Tools for Environmental Risk Management – 2

Table 3.14 OECD guidelines for in vivo testing of eye

irritation and corrosion (OECD, 2014).

Test guideline Test name

OECD TG 405 (2012) Acute Eye Irritation/Corrosion

Table 3.15 In vitro ocular test methods considered valid for limited regulatory testing (OECD, 2014;
DB-ALM, 2014).

Test guideline Method Test purpose Validation authority

OECD TG 438 (2013) Isolated Chicken Eye (ICE) assay Eye corrosion/ ICCVAM;
severe irritation ECVAM
OECD TG 437 (2013) Bovine Corneal Opacity and Eye corrosion/ ICCVAM;
Permeability (BCOP) assay severe irritation ECVAM
DB-ALM Protocol No.85 Isolated Rabbit Eye (IRE) assay Eye corrosion Ongoing/CLP R41
DB-ALM Protocol No.96 Hen’s Egg Test – Chorioallantoic Eye corrosion Ongoing/CLP R41
Membrane assay (HET-CAM)

CLP R41: Although not formally endorsed as valid, positive outcomes can be used for classifying and labeling
substances (CLP) as severe eye irritants (R41) in the EU.

A number of non-animal test methods have been developed in the search for a
replacement for the Draize rabbit eye test (Table 3.15). These are not validated and
accepted test methods yet. The most interesting question is, if a negative in vitro test
result can be and will be accepted by regulatory agencies.

3.10.2 Non-animal alternative methods for evaluating

eye irritation and corrosion
The ECVAM Scientific Advisory Committee (ESAC) statement (ECVAM, 2007) on the
ICCVAM (2006) retrospective study of the four in vitro screening assays for ocular
corrosive/severe irritants endorsed the validity of the BCOP and ICE methods for
use in a tiered strategy as part of the weight of evidence approach. ESAC indicated
that further work is needed for the IRE and HET-CAM methods. In vitro ocular
test methods considered valid for regulatory application can be seen in Table 3.15.
OECD TG 438 was amended in 2013 for the identification of chemicals not requiring
classification for eye irritation or serious eye damage.
Although only two in vitro test methods have been endorsed with some conditions,
many new developments are ready to be validated and accepted for standardized and
uniform application (Table 3.16) (AltTox, 2007a).
Many of the raw materials for in vitro testing originate from slaughterhouses.

3.11 Toxicokinetics, pharmacokinetics and metabolism

The study of the absorption, distribution, metabolism, and elimination (usually
abbreviated as ADME) of chemicals in the body is also called toxicokinetics or
pharmacokinetics.
 Human toxicology 157

Table 3.16 Alternative in vitro methods for testing eye irritation and corrosion.

Test type Name of the test

Isolated eye assays Bovine Corneal Opacity and Permeability (BCOP)

Porcine Corneal Opacity and Permeability (PCOP)
Isolated Chicken Eye (ICE) assay
Isolated Rabbit Eye (IRE)
Isolated Mouse Eye
Human Cornea (discarded from eye banks)
Chicken egg membrane assays Chorioallantoic Membrane Vascularization Assay
(CAMVA)
Hen’s Egg Test – Chorioallantoic Membrane
(HET-CAM) assay
Reconstituted human cornea models Human corneal equivalent
Reconstituted rabbit cornea models 3D corneal tissue construct
Reconstituted bovine cornea models Epithelium and stroma
3D human corneal epithelial cell models HCE-T human corneal epithelial cell model
SkinEthic HCE model; CEPI
Coty corneal epithelial model
3D epithelial cell models EpiOcular
MDCK fluorescein leakage
Tissue equivalent assay
3D human conjunctival epithelial cell models Human conjunctival model
Monolayer epithelial cell cultures Human corneal epithelial cells
Rabbit corneal cells; SIRC cell line
Various cultured cells
Epithelial and fibroblast cell lines
Red blood cells Red cell hemolysis
Neural cell models (for detecting TRPV1-expressing neuroblastoma SH-SY5Y cells
neurogenic ocular pain)
Acellular models EYTEX/Irritection test
Hemoglobin denaturation
Computational models SAR/QSAR

Pharmacokinetics or toxicokinetics is defined as the study of the rates of absorp-

tion, distribution, metabolism, and excretion of toxic substances or substances under
toxicological study (OECD, 2006b). Pharmacokinetics/toxicokinetics testing involves
describing ‘the bioavailability of a substance and its kinetic and metabolic fate within
the body’. Pharmacokinetics is also the term used to describe the assessment of
absorption, distribution and metabolism in the context of drug preclinical testing.
Absorption is the process of active or passive transport of a substance from the
environment into the organism, across the cell membranes, through the lungs, the gas-
trointestinals or the skin. Absorbed dose of a substance is the mass of the substance
transported into the organs of the organism. Ratio of the absorbed amount of a sub-
stance to the administered amount can be characterized by the absorption coefficient.
Distribution is a dynamic process following absorption, in which a chemical
substance translocates throughout the body and reaches the site of action via blood
circulation. Bioavailability is the fraction of an administered dose entering the systemic
circulation. As the result of distribution, a chemical substance is able to reach the target
site and evolve its effect, it can be transformed, eliminated or stored in depots. The rate
158 Engineering Tools for Environmental Risk Management – 2

of distribution depends on uptake and elimination rates, the blood flow, the metabolic
activity of the organism, the affinity of the substance to specific tissues and its parti-
tioning among blood and the target tissue. The substance in the blood can be unbound
or bound to the blood cells or the plasma proteins. Some peripheral tissues for example
the adipose tissues are preferred by fat-soluble (high Kow ) organic substances, or bone
tissues by lead, cadmium or fluoride.
The metabolism of a substance distributed in the body (blood and peripheral
tissues) is an enzyme-mediated change in the physico-chemical properties, e.g. chemical
structure, size, configuration, polarity, reactivity of the toxicants and may result in:
i) the activation of the chemical substance making it able for a physiologic effect and
increased toxic potential at the target site of action; ii) deactivation/detoxication and
producing a harmless product; iii) storage by accumulation in special tissues with high
affinity to the substance; iv) elimination e.g. excretion from the body mainly via the
kidneys and the digestive tract, but also sweat and tears. Hydrophobic (lipophilic)
substances cannot partition into urine and faeces.
OECD has defined metabolism as all aspects of the fate of a substance in an organ-
ism; however, metabolism generally refers to the biotransformation of a substance (via
an enzymatic or non-enzymatic process) within the body to other molecular species
(metabolites). Two types of enzymes are involved in metabolism: phase 1 (cytochrome
P450 enzyme family) and phase 2 enzymes.
An understanding of the metabolism of a substance in the body is critical to
understanding its toxicity. For example biotransformation may result in a more toxic
substance, or the lack of metabolism of a substance may result in its bioaccumulation
in the body. Understanding a substance’s metabolism can also facilitate identification
of possible target organs and the route of clearance.
Pharmacokinetic/toxicokinetic data may be used to

– assist in the interpretation of other toxicological data,

– select doses for other toxicological studies, and/or
– extrapolate data from animals to humans (AltTox, 2007b).

Elimination is the disappearance of a substance in the body; it is the result of

metabolism, involving excretion from the body or transformation to other substances.
The rate of disappearance may be expressed by the elimination rate constant, bio-
logical half-time or residential time. Clearance is a measure of the body’s ability to
completely clear a toxicant from blood or plasma. Clearance is the rate of elimination
by all routes (blood, renal, enzyme activities) relative to the concentration in a systemic
biologic tissue.

3.11.1 Testing of toxicokinetics, pharmacokinetics and

metabolism on animals
OECD accepts one animal test (TG 417) to study toxicokinetics, shown in Table 3.17.
The dermal route of entry can play an important role in human exposure to chemicals,
typically for personal care products or agrochemicals. OECD worked out in vitro and
in vivo skin absorption models in 2004.
 Human toxicology 159

Table 3.17 OECD guidelines for in vivo and in vitro pharmacokinetic

and metabolic tests (OECD, 2014).

Test guideline Test name

OECD TG 427 (2004) Skin Absorption: in vivo method

OECD TG 428 (2004) Skin Absorption: in vitro method
OECD TG 417 (2010) Toxicokinetics using various animal species
and routes of administration

3.11.2 In vitro dermal testing

Dermal absorption methods are defined by an OECD Test Guideline TG 428, and
are accepted by some regulatory authorities as non-animal pharmacokinetics and
metabolism test methods.
ECVAM publications have reviewed in vitro and in silico approaches, includ-
ing tiered testing strategies, mathematical models such as SAR, QSAR, METEOR,
MetabolExpert, COMPACT, META or protein modeling and assessing metabolism by
using microsomes, suspended liver cells, cultured monolayer and 3D liver cells, and
precision-cut liver slice methods (ECVAM, 2002b; Coecke et al., 2005). Mathemati-
cal Models aim to predict percutaneous absorption of a test compound based on its
intrinsic properties (empirical models, using quantitative structure-permeability rela-
tionships, QSPR) or by describing skin structures and the flux of a chemical through
these structures using mechanistic models (physiological mass transfer and diffusion
models). Some protocols from the DB-ALM database are enlisted here:

– Permeability assay can be used to determine the permeability of the test compound
across an in vitro model of the human intestine Caco-2 cells. The permeability is
expressed as apparent permeability coefficient (Papp ) (DB-ALM Protocol No.142);
– Metabolic Stability assay can be used to rank the test compounds with respect
to their metabolic stability or biotransformation. The disappearance of the parent
compound is measured during an incubation with human and rat liver microsomal
fractions, S9 or hepatocytes (DB-ALM Protocol No.141);
– Plasma Protein Binding assay gives an estimate on the affinity of a test compound
for plasma proteins by measuring the fraction unbound in pooled human plasma
using equilibrium dialysis against an isotonic buffer (DB-ALM Protocol No.143).

3.12 Neurotoxicity
Neurotoxicology is the study of the adverse effects (such as deficits in learning or
sensory ability) by chemical, biological, and certain physical agents on the nervous sys-
tem (the brain, spinal cord, and/or peripheral nervous system) and/or behavior during
development and in maturity. Many common substances are neurotoxic, including
lead, mercury, some pesticides, and ethanol (Harry et al., 1998).
Neurotoxicity testing is used to identify potential neurotoxic substances. Neu-
rotoxicity is a major toxicity end point that must be evaluated for many regulatory
160 Engineering Tools for Environmental Risk Management – 2

Table 3.18 OECD guidelines for in vivo neurotoxicity testing (OECD, 2014).

Test guideline Test name

OECD TG 418 (1995) Delayed Neurotoxicity of Organophosphorus Substances Following Acute

(oral) Exposure on hens
OECD TG 419 (1995) Delayed Neurotoxicity of Organophosphorus Substances: 28-day
Repeated-Dose Study on hens
OECD TG 424 (1997) Neurotoxicity Study in Rodents, acute (28 d), subchronic (90 d) and
chronic (1 year)
OECD TG 426 (2007) Developmental Neurotoxicity Study, in utero and early postnatal
effects on offspring after exposure of pregnant rats

applications. Sometimes neurotoxicity testing is considered as a component of target

organ toxicity. Developmental neurotoxicity means in utero exposure to chemicals and
drugs exerting an adverse effect on the development of the nervous system. Common
exposure routes such as oral, dermal, or inhalation and both acute and repeated dosing
(chronic) may cause neurotoxicity. OECD accepted four animal-using test methods,
introduced in Table 3.18. None of the alternative methods have been validated yet.

3.12.1 Animal testing of neurotoxicity

The best alternatives encompass the most important neurotoxic end points organized
into test batteries (ECVAM, 2002c). The first test tier would distinguish neurotoxicants
from cytotoxic chemicals, and the second tier would consist of mechanism-specific
tests. It was proposed that a minimum battery might consist of methods for assessing
blood–brain barrier function, basal cytotoxicity (damage of the basic cellular function),
and energy metabolism.

3.12.2 In vitro models for neurotoxicology studies and testing

Test systems used for neurotoxicological testings are:

– Primary cells: neurons and glia (microglia, oligodendrocyte, astrocyte) from

different brain regions;
– Cell lines: neuroblastoma, astrocytoma, glioma, pheochromocytoma;
– Brain slices: hippocampus;
– Reaggregating neuronal and glial cell cultures;
– Organotypic (3D) cultures: usually co-cultures (more than one type of cell);
– Neural stem (progenitor) cells: primary cell cultures and cell lines.

ICCVAM and OECD have not reviewed or validated any non-animal methods or
alternative testing strategies for assessing neurotoxicity. Regulatory authorities have
not accepted any non-animal methods or alternative testing strategies for neurotoxicity
testing until now (AltTox, 2009). ECVAM DB-ALM established a protocol titled:
“Whole Rat Brain Reaggregate Spheroid Culture’’. This culture system (single cell
 Human toxicology 161

Table 3.19 In vivo testing of endocrine disrupting effect of chemical substances (OECD, 2014).

Test guideline Test name

OECD TG 440 (2007) Uterotrophic Bioassay in Rodents: a short-term screening test

for estrogenic properties
OECD TG 441 (2009) Hershberger Bioassay in Rats: a short-term screening assay
for (anti)androgenic properties
OECD TG 455 (2009) The Stably Transfected Human Estrogen Receptor-alpha Transcriptional
Activation Assay for Detection of Estrogenic Agonist-Activity of Chemicals

suspension) allows the testing of neurotoxic compounds during development, differ-

entiation and relative maturity of the brain reaggregate (DB-ALM Protocol No.11
(DB-ALM, 2014).

3.13 Endocrine toxicity and disruption

An endocrine disruptor is an exogenous agent that interferes with the synthesis, secre-
tion, transport, binding, action or elimination of natural hormones in the body
that are responsible for the maintenance of homeostasis, reproduction, development,
and/or behavior. We gave a detailed overview of endocrine disruption in Chapter 7 in
Volume 1. Here we discuss endocrine disruption only from the testing point of view.

3.13.1 Animal tests for screening endocrine disruption

The estrogenic and androgenic effects of chemical substances can be tested and
evaluated from the results of animal tests (Table 3.19).
The uterotrophic bioassay evaluates the ability of a chemical to elicit biological
activities consistent with agonists or antagonists of natural estrogens (e.g. 17β-
estradiol). It is based on the response of the uterus to estrogens: an increase in weight
due to water imbibition as an initial response, which is followed by a weight gain due
to tissue growth.
The Hershberger bioassay is based on the changes in weight of five androgen-
dependent tissues in the castrate-peripubertal male rat.

3.13.2 Validated non-animal alternatives for endocrine disruptor activity

Endocrine disruption is an emerging problem; screening and proving whether or not
chemical substances have an endocrine disrupting potential is an urgent task for devel-
opers of new chemicals, authorities, regulators, managers or other decision makers.
Fast and cheap in vitro methods are needed and a large number of such methods have
been developed in recent years. Some of them have been validated and accepted or are
under evaluation as screening methods for regulatory purposes. Table 3.20 contains
the validated in vitro methods.
OECD has already accepted the Stably Transfected Transactivation Assay (STTA)
using the hERα-HeLa-9903 cell line to detect estrogenic agonist activity for evaluating
the ability of a chemical substance to function as an estrogenic receptor-α ligand and
162 Engineering Tools for Environmental Risk Management – 2

Table 3.20 In vitro testing of endocrine disrupting potential of chemical substances (OECD, 2014;
OCSPP, 2009).

Test guideline Test name

OECD TG 456 (2011) Steroidogenesis Assay with H295R human cell line – Screening assay
OECD TG 455 (2012) Performance-Based Test Guideline for Stably Transfected Human Estrogen
Receptor-α Transcriptional Activation Assay for Estrogenic Agonist-
Activity – Screening assay
OECD TG 457 (2012) BG1Luc Estrogen Receptor Transactivation Test Method for Identifying
Estrogen Receptor Agonists and Antagonists
890.1150 (OCSPP, 2009) Androgen receptor binding assay (rat prostate) – Endocrine screen
890.1200 (OCSPP, 2009) Aromatase inhibition assay (human recombinant) – Endocrine screen
890.1250 (OCSPP, 2009) Estrogen receptor binding assay – Endocrine screen

activate an agonist response. It is used for screening and prioritization purposes but
can also provide mechanistic information that can be used in a weight of evidence
approach.
The Steroidogenesis Assay uses the H295R human carcinoma cell line to assess
the effects of chemicals on testosterone and 17β-estradiol production.

3.13.3 The US EPA endocrine disruptor screening program

The EPA methods shown in Table 3.21 are part of a battery for screening the endocrine
disruptor effect of chemicals in the Endocrine Disruptor Screening Program (EDSP,
2014). The Office of Chemical Safety and Pollution Prevention developed the harmo-
nized Endocrine Disruptor Screening Program Test Guidelines – Series 890 (OCSPP,
2009) for the tier 1 and tier 2 screening. Tier 2 guidelines are not issued yet. The vali-
dation status of the tests under development is shown in the Assay Status Table (EDSP
AST, 2014).
At the end of the in vivo tests, measurements are made that are reflective of the
status of the reproductive endocrine system, including male secondary sex character-
istics, gonadal histopathology, gonado-somatic index, and plasma concentrations of
vitellogenin.
The YES (Yeast Estrogen Screen) assay is one of the promising test methods under
development. It uses transgenic yeast to test the endocrine disrupting potential of
chemical substances. The transgene built into the yeast cells is the human estrogen
receptor.
Saccharomyces cerevisiae is chromosomally transformed by hER gene, coding the
human estrogen receptor protein, which binds estrogen or other xenoestrogens with
a complementary spatial structure. After binding to the estrogen responsive element
on the yeast plasmid, the complex of hER and the estrogenic compound initiates the
transcription of Lac-Z reporter gene, producing β-galactosidase, an enzyme capable of
hydrolyzing the sugar from chlorophenol red-β-D-galactosidase (used as an indicator
in the assay) and producing chlorophenol red with an easily detectable color. The
concept is shown in Figure 3.9 after Routledge and Sumpter (1996).
Instead of Saccharomyces cerevisiae, other yeast species, e.g. Blastobotrys
adeninivorans, have also been used for the construction of the YES assay with the
 Human toxicology 163

Table 3.21 Test guidelines for the Endocrine Disruptor Screening Program
of US EPA (OCSPP, 2014).

Tier/test number Test name

Tier 1 In vitro
890.1150* Androgen Receptor Binding – Rat Prostate
890.1200* Aromatase – Human Recombinant Microsomes
890.1250* Estrogen Receptor Binding – rat uterine cytosol
890.1300** Estrogen Receptor Transcriptional Activation –
Human Cell Line HeLa-9903
890.1550** Steroidogenesis – Human Cell Line H295R
Tier 1 In vivo
890.1600 15-Day Intact Adult Male Rat Assay
Uterotrophic (rat)
890.1400** Hershberger (rat)
890.1450 Female Pubertal (rat)
890.1500 Male Pubertal (rat)
890.1100** Amphibian Metamorphosis – frog
890.1350* Fish – Short Term Reproduction
Tier 2 In vivo
Amphibian 2-Generation Development, Reproduction
Avian 2-Generation
Fish Lifecycle
Invertebrate Lifecycle
Mammalian 2-Generation
In utero and through Lactation

*in vitro test, the same as in Table 3.20; **existing test methods as OECD TG.

Figure 3.9 Schematic explanation of the YES assay.

164 Engineering Tools for Environmental Risk Management – 2

reporter gene of phytase. Instead of estrogenic receptors, androgenic receptors can

also be built in.
The advantages of the method are:

– Easy to cultivate test microorganism;

– Unlimited number of cells to use in the test;
– Well-known reporter genes ensuring an easily detectable response;
– Short time duration of the test makes high throughput screening possible;
– Able to test bioavailability of substances with estrogenic effect;
– Possibility to use radioactive substances.

Disadvantages of the YES assay are:

– Yeast cells differ from animal cells in their membrane composition, first of all in
the sterol content and, as a consequence, in the binding of the tested chemical
substances;
– Different yeast strains give different test results;
– The YES assay may significantly overestimate the estrogen disrupting effect;
– The YES assay may give false negative results.

Further development and finding the most fitting yeast strain may result in a
fast and cheap in vitro method in the future for screening chemicals for endocrine
disruption.

3.14 Phototoxicity
Phototoxicity is an elicited or increased (at lower dose levels) toxic response to chem-
icals after subsequent exposure to light or induced by skin irradiation after systemic
administration of a substance (Table 3.22).
OECD TG 432 assays (Table 3.22) measure the viability of mouse Balb/c 3T3 cells
following their exposure to a chemical in the presence and absence of light. The test
identifies compounds that act in vivo phototoxic after systemic application, as well
as compounds, including UV filter chemicals, that act as photoirritants after topical
application and distribution to the skin.
Cytotoxicity (cell death) reduces the uptake of neutral red dye by the cells. The
concentration-dependent reduction in dye uptake in 24 h following the treatment, typi-
cally the IC50 value (the concentration of the test substance that reduces cell viability to
50% of the untreated control value), is used as the assay end point.

Table 3.22 Validated non-animal alternatives for testing phototoxicity

(OECD, 2004; OECD/OCDE, 2004).

Test guideline Test name

OECD TG 432 (2004) In vitro 3T3 Neutral Red Uptake

Phototoxicity Test (3T3 NRU PT)
 Human toxicology 165

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Chapter 4

Aquatic toxicology
K. Gruiz & M. Molnár
Department of Applied Biotechnology and Food Science,
Budapest University of Technology and Economics, Budapest, Hungary

ABSTRACT

Aquatic toxicology is the pioneer in environmental toxicology, but still very young as
an independent science. The very first developments and applications are dated back
to 1960–70 when surface waters were endangered by the then applied industrial and
agricultural practice.
This chapter will introduce the most important test organisms and test methods
used in aquatic toxicology for the measurement of adverse effects of chemical
substances to the members of the aquatic ecosystem.
Similar trends to those in human toxicology can also be identified in the field of
aquatic toxicology: the use of in silico methods such as QSAR (Quantitative Structure
Activity Relationship) is spreading and the tests on higher animals are less frequent.
Instead, molecular and cell-level biomarkers for assessment and monitoring are being
increasingly used.

1 INTRODUCTION TO AQUATIC TOXICOLOGY

The impacts of pollution on water quality and the conditions of aquatic habitats (sur-
face water systems of rivers, lakes, seas and oceans, coastal zones and estuaries) have
endangered the ecosystem and humanity over the last quarter of the twentieth century.
The deterioration of aquatic ecosystems has been studied in detail and a wide range of
engineering tools have been developed for assessing, monitoring and reducing the risk
of water pollution by chemicals.
The very first model applied for aquatic toxicity assessment was the chemical model
which tried to extrapolate from chemical analysis data to adverse effects. The chemical
approximation failed not only because insufficient information was available on the
effect of chemicals to aquatic organisms, but also because chemical models cannot
handle accessibility, bioavailability and multiple interactions between contaminants,
water and sediments as well as members of biota. Modern environmental management
uses an integrated approach in environmental assessment and monitoring: chemical,
biological and ecotoxicological methodologies are used jointly as a tool battery tailored
to the specific problem of contaminants, waters or sediments.
Aquatic toxicology aims to understand the stresses chemical substances exert on
aquatic ecosystems and to find the quantitative relationship between contaminant
172 Engineering Tools for Environmental Risk Management – 2

Figure 4.1 Contaminant sources endangering the aquatic ecosystem.

concentration and adverse effects. This knowledge supports forecasting the deteriora-
tion of the ecosystem and enables sustainable management of our waters by preventing
the contamination and deterioration and by the remediation of endangered ecosystems
and habitats.
Two different strategies may lead to achieving these aims:

– Monitoring the environment, following the trends of ecosystem changes and com-
paring them to the acceptable rate of changes. Monitoring data can be obtained
from the time series of aquatic ecosystem characterization. The “acceptable’’ rate
of changes is based on the knowledge of the ecosystem’s adaptive potential and
actual ecosystem quality and integrity. For example, an acceptable change of 5% in
the diversity of species means that 95% of ecosystem diversity remains unchanged.
It is an acceptable rate of change in most of the cases. Of course a refined knowledge
on the lost 5% may also be important, and may indicate unacceptable deteriora-
tion. It is typical that the roles particular species play in ecosystem quality and
function are not transparent; in light of this the loss of e.g. minor components of
an ecosystem may remain undetected. All these may cause large uncertainty in the
outcome of assessment and monitoring.
– Another strategy is testing the response of selected species with the aim of repre-
senting the whole of the ecosystem. From these (generally laboratory) experiments
it can be extrapolated to the whole ecosystem using factorial methods or relying on
known probabilistic distributions. Applying the factorial extrapolation method in
 Aquatic toxicology 173

effect assessment, representatives of aquatic ecosystems from a minimum of three

different trophic levels should be used and tested in laboratory bioassays, micro-
cosms or mesocosms. When results from 6–8 or more representative aquatic species
are available, the species sensitivity distribution can be derived from the laboratory
test result and the concentration belonging to the 5% of fraction affected (95%
non-affected) can be considered as a limit value for acceptable ecosystem change
(deterioration).

Ecotoxicity test results can be used in screening, monitoring and control and also
for direct decision making in water management. Depending on the nature of the
problem and the aim of the assessment, concepts and methods have a wide range of
selection. Original new scientific data may be acquired applying individual concepts
and innovative methods or by adapting existing methods to the scenario to be inves-
tigated. In other cases, e.g. for regulatory ecotoxicology purposes the stipulations of
regulations should be routinely fulfilled using the prescribed standard methods.
Direct decision making based on the measured adverse effects is an efficient way
of environmental management: it is practised in the USA and in some European and
Asian countries for the biomonitoring of industrial discharges on a pass–fail basis
(Thompson et al., 2005). In the USA the National Pollutant Discharge Elimination
System (US NPDS, 2004) has established aquatic toxicity bioassay criteria for effluents,
sediments, and oil spills.
Besides ecosystem assessments and bioassays, chemical models still dominate
environmental risk characterization. Advanced chemical analysis technologies have
enabled the determination of contaminants in concentrations at the picogram and
femtogram level, opening new perspectives in environmental toxicology. Although sen-
sitivity, selectivity and reproducibility analysis bears great potential and advantages,
the results of chemical analysis alone cannot truly model the fate, behavior, interactions
and effects of chemical substances in the environment. Adverse effects of chemical sub-
stances depend to a great extent on the presence of other chemicals – there is always a
mixture of chemicals in the environment – and the environmental compartment’s qual-
ity and physico-chemical properties such as temperature, pH, redox potential, density,
composition, exposed physical phases and their proportion. The effects are greatly
influenced by the interaction between the substance investigated and other substances,
matrices and members of the ecosystem over the short and long term. These interac-
tions depend on the chemicals’ mobility, biological accessibility and availability, which
may greatly differ from the chemical availability i.e. extractability. Chemical analy-
sis methods, even if they are chemical test batteries with complex interpretation (e.g.
sequential extraction), are not sufficient to predict actual effects of contaminants or
contaminant mixtures on a complex ecosystem and its members with different sen-
sitivity. Finally, the most problematic point in chemical analysis is that only those
chemicals can be analyzed which are included in an analysis program, assessment or
monitoring plan. This may work properly when the contaminants have already been
identified and the situation is uncomplicated, e.g. there is one contaminant without
metabolites requiring special analysis methods. However, in the case of complex situ-
ations involving many interacting chemicals, matrices and living organisms, which are
typical in the environment, an integrated approach is needed. This integrated approach
should include water and sediment characterization and monitoring and may comprise
174 Engineering Tools for Environmental Risk Management – 2

ecosystem assessment, laboratory testing of adverse effects and chemical analysis in

the form of a problem-specific test battery (Gruiz, 2009; Gruiz et al., 2009). Chapter 2
in Volume 3 deals with this topic in greater detail.

2 HUMAN AND ECOSYSTEM EXPOSURE TO

AQUATIC HAZARDS

People are not independent of aquatic ecosystems; there is a direct interconnection

between them through the food chain: bioconcentration and biomagnification of con-
taminants in aquatic species which are consumed by people, pose high risk to human
health. Man as a top-predator is exposed to the highest accumulated concentrations
of chemical substances such as mercury and other toxic metals, chlorinated pesticides
and POPs (Persistent Organic Pollutants). Deterioration of aquatic ecosystems occurs
as an indirect effect in the form of losing the ecosystem’s services, water quantity and
quality and water-dependent renewable resources.
The most important ecosystem services provided by fresh-water biodiversity are
the following:

– Delivery of clean water for people;

– Providing human food (rice, fish, crayfish);
– Providing goods for human use such as reeds, building materials;
– Flood attenuation in flood-prone areas;
– Attenuation of adverse anthropogenic impacts such as micropollutants, nutrients,
tensides, drugs;
– Suppression of waterborne diseases;
– Recreation;
– Inspiration, religion, cultural purposes.

Any of these functions and services can deteriorate when the intact ecosystem is
disturbed, when biodiversity is reduced or adversely changed and as a consequence,
the community is not able to fulfill its normal function.
Urbanization, compaction and sealing of surfaces, the lack of vegetation greatly
influence water cycling and transport routes and as a consequence aquatic ecosystem
services and aquatic habitats. Figure 4.2 shows the percentage distribution of precip-
itation between groundwater, surface runoff and evapotranspiration in a pre-urban
and an urban situation.
Aquatic biodiversity can be assessed by generally applicable methods such as the
US EPA Rapid Bioassessment Procedure (RBP) (Barbour et al., 1999) or by problem-
and site-specific ecological tools to characterize watersheds, streams or lakes. Based on
the physical, functional and biological results, the watersheds can be classified which
makes management easier and more uniform. The RBP method is recommended by US
EPA for aquatic habitat assessment and characterization. The most important parts of
the protocol are:

– Habitat assessment by physico-chemical parameters;

– Periphyton assessment using laboratory or field assessment protocols;
 Aquatic toxicology 175

Figure 4.2 Pre-urban and urban distribution of precipitation between groundwater, surface runoff and
evapotranspiration.

– Benthic macroinvertebrate sampling and assessment protocols;

– Fish protocols for collection, identification and metrics;
– Biological data analysis.

Site- and problem-specific ecological methods can be developed for any arising
problem in the habitats’ suitability, biological integrity or vulnerability. Assessment
protocols for some ecosystem-types already exist and are accessible on-line at the
Ecological Assessment Methods Database of the USA (EAMD, 2013).

– Wetland habitats;
– Riparian community;
– Coastal region habitat;
– Streams and small stream habitats;
– Tidal rivers;
– Special habitats such as peat land, reefs, glaciers;
– Index of Biological Integrity (IBI) for plants;
– IBI for invertebrates;
– IBI for fish;
– IBI for birds;
– Aquatic vertebrates;
– Benthic organisms: (meio-, macro-, phyto- and zoobenthos);
– Floristic assessment;
– Marsh bird community, etc.
176 Engineering Tools for Environmental Risk Management – 2

The ecological status of surface waters is associated with phytoplankton and zoo-
plankton diversity. One of the main problems of surface waters, primarily of shallow
lakes, is eutrophication world-wide. The EU Water Framework Directive (WFD, 2000)
specifies phytoplankton to be used in the assessment of the ecological status of surface
waters. Phytoplankton indices such as the Carlson Index (the most commonly used
trophic index) can characterize the trophic status of surface waters by measuring the
algal biomass. It is only applicable when suspended solid and rooted plants are in low
concentration in the water.
Zooplankton communities are also suitable for ecological assessment of lakes as
they are an important component of the pelagic food web. Caroni and Irvine (2010)
found that zooplankton can be a good indicator for acidity and overload by nutrients.
Acidic and oligotrophic lakes were typified by a high relative abundance of cladocerans,
but with some key taxa groups absent from the most acidified lakes. In spite of their
ability to indicate anthropogenic impact, monitoring of zooplanktons is not required
by the Water Framework Directive (WFD, 2000).
Macrozoobenthos is a characteristic group of animals in sediment. They are
defined as invertebrate bottom fauna living on or in the bottom. The size of these ani-
mals vary within a wide range: the “macro’’ fraction is retained on a sieve with a mesh
size of 1 mm × 1 mm. The group of smaller animals that pass through such a sieve are
called meiozoobenthos. Macrobenthic animals live in the sediment environment that
is affected by eutrophication, pollution, fisheries, or, in general, by intensive influx
of organic matter. Contaminants, mainly the persistent organic and inorganic ones,
accumulate in the sediment and are in close interaction with the sediment-dwelling
organisms. The aquatic habitats (lakes and rivers) have characteristic benthic fauna,
which makes possible the monitoring and the comparison of the time-points of time
series.
Indicators of ecological conditions other than species diversity may come from
lower levels than ecosystem or community, i.e. from population, organism, organ,
physiological, histological, biochemical or enzymatic as well as molecular levels
including DNA, RNA, proteins or other type of molecules (sugars, lipids, etc.).
A genomic screening tool for identifying gene expression patterns may indicate the
effect of contaminants on the activation of genes. For example the vitellogenin gene
expression in male fathead minnow is an indicator of exposure to endocrine disrupting
chemicals (EDCs, see in Chapter 2 in Volume 1) in an aquatic environment.
Many biochemical processes and biomolecules indicate pollution, stress and
anthropogenic impact on the water ecosystem: DNA damage, stress proteins such as
acetylcholine esterase, adrenaline, or oxidizers; detoxifying molecules such as antiox-
idant enzymes, bile metabolites, methallothioneins or immune supressors. Measuring
acetylcholine esterase inhibition in river snail (Sinotaia ingallsiana) was useful in deter-
mining pesticide contamination. The high level of adrenaline in fish or clam as a result
of the effect of chronic chemical or other stress can be an ecological indicator in aquatic
habitats.
Bacteria, other micro-size organisms and planktonic organisms are relatively
easy to sample and analyze, and additional stress due to catching, transporting and
investigating does not influence the result as in the case of higher animals (fish or birds).
Remote sensing technologies used in fresh-water biodiversity monitoring can char-
acterize aquatic habitats and special inhabitants and enable the identification of the
 Aquatic toxicology 177

size of water bodies, land uses around watersheds; they can measure the changes in
water level, temperature, water color and density. The application of remote sensing
and GIS together in monitoring water quality parameters such as suspended matter,
phytoplankton, turbidity, and dissolved organic matter is especially useful. The combi-
nation with hyperspectral analysis enables the selective monitoring of any characteristic
components of the aquatic environment.
Remote sensing will be a key tool in identifying priorities in water body man-
agement, e.g. conservation or risk reduction. Based on the remotely sensed data,
predictive modeling of fresh-water systems can identify the most susceptible sites that
need intervention or protection. US EPA carried out the “Development of Water Qual-
ity Indicators Using Remote Sensing’’ project (RS, 2013) to develop a library of water
quality indicators that can be measured remotely.
Management and legislation utilized the results of aquatic toxicology very soon
by creating effect-based quality criteria and quality objectives as well as monitoring
and characterizing the ecological status of surface waters. The introduction of the
European Water Framework Directive in 2000 (WFD, 2000) placed great emphasis on
the quality and uniform management of surface waters.
Measuring water quality using the integrated approach i.e. applying chemical anal-
ysis, ecological assessment and environmental toxicity testing methods in parallel made
it possible to draw a detailed picture on ecosystem health and water quality. Direct test-
ing of adverse effects instead of predictions based on chemical models made possible to
measure the effect of mixed contaminants and the results of their interactions in water.
Sediments, the formerly underestimated compartment of surface waters, became one
of the main risk components.
Developing DNA techniques to the level of being able to describe the metagenome
of a community created a new possibility for characterizing and monitoring the whole
ecosystem instead of single species.
Innovative technologies have been developed for testing adverse effects and
monitoring ecosystem characteristics and health:

– Early warning by the most sensitive species approach;

– Models for simulating aquatic ecosystems, e.g. micro- and mesocosms;
– Dynamic testing of waters and water–sediment systems;
– Measuring relative sensitivity and sensitivity distribution of species;
– Utilization of specific biological or biochemical indicators for chemical substances;
– Indicators for biological pollutants such as invasive species or pests.

There are some fields where the trend of new developments can be clearly
identified. Examples are: testing the effects of endocrine disruptors in water, using
DNA techniques and other molecular tools, e.g. the -omics (genomics, proteomics,
metabolomics, etc.) and other new indicators for measuring and monitoring stress and
multiple stressors. The aggregation and utilization of all available information from
different sources in an efficient and integrated way is becoming increasingly impor-
tant. Knowledge gained from case studies enhances our understanding of nature and
the mechanisms of the effect of chemicals and other relationships between pollutants
and aquatic deterioration. For example, regional and watershed-scale environmental
178 Engineering Tools for Environmental Risk Management – 2

Figure 4.3 Concentration–response curve: the main tool for bioassay evaluation.

management will benefit from mapped information on ecosystem quality based on

remote sensing and evaluated in correlation with the map of chemicals.
In spite of the impressive developments outlined above, there are still many prob-
lems to be solved in the near future. One of them is the extrapolation of uncertainty
from lab tests or even from micro- or mesocosm tests to the real environment. Another
not fully solved problem is the integration of the ecological assessment results (e.g.
diversity indices), into the risk assessment and risk management procedure.
Ecotoxicity – according to the common definition – involves the identification
of chemical hazards to the environment. US EPA definition (US EPA, 2007) is more
concrete: ecotoxicity studies measure the effects of chemicals on fish, wildlife, plants,
and other wild organisms as well as the complex community of wild organisms, the
whole of the ecosystem.
Ecotoxicity testing is another branch of the animal, plant or microorganism testing
methods to determine whether a chemical substance or an environmental sample has
an adverse effect. It uses laboratory bioassays (Figure 4.3), microcosms or mesocosms
as test designs and may select the end points starting from molecular level indicators
to the lethality of the test organism, or diversity of the community in the micro- or
mesocosm. As an example, Figure 4.3 shows the effect of a contaminant with growing
concentration on the inhibiton of Vibrio fischeri luminescence.
Ecological assessment and ecotoxicity testing methodologies have the same con-
clusion: one has to extrapolate from the test results to the real environment and this
information must be used for decision making in the course of the management of the
environment.
 Aquatic toxicology 179

Figure 4.4 Modeling adverse effects on the aquatic ecosystem.

One can extrapolate to the environment using chemical or mathematical models,

but it is important to know that they are much farther from reality than biologi-
cal or ecological models as indicated in Figure 4.4. The application of mathematical
and chemical models is reasonable if their good statistics and repeatability com-
pensate for the lack of environmental reality or if the goal of the assessment is
generic, e.g. European legislation, or watershed-scale management of chemicals, and
when heterogeneity, environmental parameters and interactions are handled as generic
characteristics, which can be simulated by standardized test methods.
The biological and ecological methods are highly diverse. Test species can be
selected to represent the average, 95% of the ecosystem members, the most sensi-
tive species, those less sensitive than the average, or those having specific sensitivity
to certain contaminants, etc. The first step is always to formulate the concept of risk
assessment and to test the adverse effects. The conceptual model of the case specifies
the goals, the spatial and time frames, the hot spots, transportation pathways and the
water-use-specific receptors. After establishing a detailed picture of the case, the best
fitting test type, set-up, exposure system, duration and test organisms to characterize
aquatic risks can be selected. There is a wide variety of test organisms and abundant
information on their capabilities. A complete “database’’ is not available today, but
one can find a large number of publications on species sensitivity and the so-called
Species Sensitivity Distribution (SSD), which is a relatively new area of environmental
toxicology. Test organisms of different sensitivity are used for risk assessment, estab-
lishing safe contaminant concentration in the environment, screening pollution and
ranking surface waters and pollutants. Appropriate aquatic indicator organisms can
be applied to any environmental changes, from deterioration due to global warming,
through eutrophication, to adverse effects emerging at local scale due to individual con-
taminants or hazardous biological agents. These indicators vary from community level
180 Engineering Tools for Environmental Risk Management – 2

down to organism and molecular levels and are used in traditional ecological methods
such as field assessments or mesocosms, microcosms, laboratory bioassays, as well
as specific biochemical and genetic methods. The latter ones apply traditional chemi-
cal analyses and innovative molecular methods, including genomics, transcriptomics,
proteomics, lipomics or any other metabolomics and their combinations.
One of the main challenges of aquatic toxicology is finding the suitable test battery
for emerging problems in aquatic ecosystem health. There are still open questions in
spite of a number of model organisms that can detect acute or chronic toxicity and
standardized methods that can provide comparable results. It is still not known how to
detect signals of changes in the chemically modulated behavior of aquatic organisms,
or those caused by the transportation of POPs (Persistent Organic Pollutants) in food
chains and webs for instance. Areas yet unexplored include chronic effects due to
acutely non-toxic, endocrine and immune disruptor chemicals. These chronic effects
are typically not proportional to their concentrations/doses, thus the use of information
from metagenome changes, bioinformatics and computational toxicology may help to
identify suitable indicators. Many fields of aquatic toxicology must still be developed
in order to exploit their full potential.

3 SOME COMMONLY USED AQUATIC TEST ORGANISMS

FOR TESTING ADVERSE EFFECTS

Hundreds of aquatic species are used in standardized and non-formalized test meth-
ods. Most of the OECD test guidelines describe one methodology, but give a list of the
test organism options, requiring the user to choose. The ISO tests use bacteria, algae,
water plants, invertebrates living in water and sediment, macrozoobenthos (sediment-
dwelling organisms) of the sediment and a great number of fish species. From among
the standardized tests, many organisms are used in tests as biomarkers or indicator
species, for example the members of the phyto- and zooplankton, special water plants,
clams, crustaceans and fishes. The adequate test organism should be selected accord-
ing to the aim of the test: i) specific effects require selective indicator organisms, for
example those extremely sensitive to Zn toxicity or photosynthesis inhibition, or able
to accumulate Hg, etc.; ii) the general quality of water can be characterized by the
aquatic/benthic species diversity or by test organisms with a wide-spectral sensitivity,
i.e. sensitive to most of the toxic metals and also to organic pollutants. In the fol-
lowing, we will list the most popular aquatic test organisms without attempting to be
comprehensive.

3.1 Microorganisms: bacteria, algae and protozoa

Bacteria
Bacteria are easy to handle and rapidly growing organisms, which enable test methods
of good reproducibility. They are considered to be able to represent their taxa in a
wide context, but they are also used as toxicological “reagents’’ independent of their
environmental role. Natural and laboratory strains, mutants, and genetically modified
bacteria are also used to test toxicity both of chemicals and environmental samples.
 Aquatic toxicology 181

Figure 4.5 Liquid culture ofVibrio fischeri in daylight and darkness producing luminescent light emission.

Laboratory strains, e.g. Vibrio fischeri or Azomonas agilis, are used as indicators to
detect adverse effects. Natural strains are also used as endangered species or sensitive
key actors of an ecosystem (responsible for nitrogen fixation or biodegradation of oil
spills and xenobiotics) or as the causes of a hazard (e.g. Salmonella or other pathogens).
Growth rate, metabolic activity and products can be measured as end points.
Vibrio fischeri is a Gram-negative rod-shaped, heterotrophic (saprotrophic) bac-
terium found globally in marine environments. It moves by means of flagella and has
bioluminescent properties, so it can emit bluish-green light (490 nm) due to a chemical
reaction between riboflavin-5 -phosphate, luciferin and molecular oxygen. Figure 4.5
shows the luminescent light emission from a liquid culture in daylight and darkness. An
enzyme called luciferase catalyzes the reaction. Vibrio fischeri is predominantly found
in symbiosis with various marine animals. It is a key research organism for examina-
tion of microbial bioluminescence, quorum sensing, and bacterial-animal symbiosis.
In environmental toxicity testing it is a generally used test organism, based on the
correlation between light emission and toxic chemicals present. The test using Vibrio
fischeri is standardized (ISO 11348, 2007) and this test organism – due to its wide-
range sensitivity and easy laboratory use – is applied not only as a marine species,
but also as a generic test organism for any environmental sample: water, wastewater,
leachate, soil, sediment, solid waste, etc.
Salmonella typhimurium is a Gram-negative, facultative anaerobic bacterium of
the family of Enterobacteriaceae, genus Salmonella. It is used in the Ames muta-
genicity assay, a short-term bacterial reverse mutation assay using histidine auxotroph
Salmonella, which carries a mutation in the genes of histidine synthesis.
Azomonas agilis is a Gram-negative bacteria, motile with peritrichous flagella,
found in water and wastewater, capable of fixing atmospheric nitrogen. It belongs
to the phylum Proteobacteria, family Pseudomonadaceae. It is a non-selective test
182 Engineering Tools for Environmental Risk Management – 2

organism, used in laboratory bioassays. Respiration can be followed by O2 consump-

tion, CO2 production, enzyme activity or ATP production. Both chemical substances
and environmental samples can be tested, applicable for polluted water and wastewater
toxicity assessment and for monitoring water treatment technologies. Toxic inhibition
of the dehydrogenase enzyme is caused by the deterioration of the electron transport
chain on the effects of contaminants. Decreased dehydrogenase activity is indicated
in the test by applying an alternative electron acceptor, which turns into red when
dehydrogenase is active but remains colorless when it is not.
Other bacteria, first of all, Escherichia coli and the coliforms as well as other
environmental strains from the genus Pseudomonas, Flavobacteria, Gammaproteobac-
teria, Bacillus, etc. are often used as test organisms in laboratory bioassays.
Adverse effects caused by bacteria also represent an environmental problem: in this
case one can detect the hazardous bacterial strain itself or specific molecules indicating
their presence. Amongst these hazardous bacteria there are human, animal and plant
pathogens, e.g. human pathogenic coliforms (Clostridium, Staphylococcus, etc.), bac-
teria causing macroalgal diseases, fish pathogens (Vibrio harveyi, Pasteurella skyesisn
or Chryseobacterium species), etc.

Phototrophic bacteria and algae

Phototrophic bacteria and algae are mainly used for the indication of toxic chemical
substances in water by showing reduced growth rates. Testing growth rate of bacterial
and algal strains is described in the OECD test guideline No. 201.
Synechococcus leopoliensis, the unicellular cyanobacterium, is a member of the
class Cyanophyceae and the family Synechococcaceae, used in the detection of phyto-
toxic pollutants such as herbicides and toxic metal cations. Found in oceans as well as
in fresh-water reservoirs and lakes. The test method OECD 201 (2011) of Fresh-water
Algae and Cyanobacteria, Growth Inhibition Test describes its use in toxicity testing
(see also Table 4.1).
Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
belongs to the algal taxonomical groups of Raphidocelis, specifically Selenastraceae.
Thalli (green shoots) unicellular or forming small colonies embedded in highly irregular
and structureless mucilage. Cells shape is lunate to sigmoidal, – as shown in Figure
4.6 – pointed at both ends with evenly granulated cell walls. Raphidocelis species
are planktonic in fresh water, and known only from central Europe. It is one of the
most sensitive algal strains, which often produces the lowest toxic concentrations in
environmental toxicity testing.
Scenedesmus quadricauda and Desmodesmus subspicatus (formerly known as
Scenedesmus subspicatus) are two very similar algal species. They can be single-celled
or colonial, forming 2- to 32-celled coenobia, cells arranged linearly. Scenedesmus
species are members of the class Chlorophyceae and the family Scenedesmaceae. They
are planktonic algae, mainly in eutrophic fresh-water ponds and lakes, rarely in
brackish water; reported worldwide in all climates.
Chlorella kessleri is a member of the class Trebouxiophyceae and the family
Chlorellaceae; it is a fresh-water algal species.
Stichococcus bacillaris is a member of the class Trebouxiophyceae and the family
Prasiolaceae, it is a fresh-water algal species.
 Aquatic toxicology 183

Figure 4.6 Microscopic view of Pseudokirchneriella subcapitata (2014), a Desmodesmus species (2014)
and Closterium ehrenbergii (2014).

Chlamydomonas reinhardtii is a member of the class Chlorophyceae and the

family Chlamydomonadaceae. It is a fresh-water algal species used in the detection of
phytotoxic pollutants.
Closterium ehrenbergii belongs to Charophyceae. Its sensitivity to nonionic and
anionic surfactants enables testing agricultural chemicals and heavy metals in an
aquatic environment. In laboratory bioassays morphological effects and ecotoxicity
of the chemicals are measured by algal growth and in zygospore inhibition tests using
Closterium ehrenbergii.

Protozoa
Protozoa are a diverse group of unicellular eukaryotic animal organisms. Most of
them are motile, moving by flagella or cilia or by pseudopodia (amoebas). Their size
ranges between 10–50 micrometers. Some of them are easy to handle and grow in the
laboratory. They may represent several trophic levels and their response to toxicants
can be characterized by animal nature.
Tetrahymena species are non-pathogenic free-living ciliate protozoa, belonging to
the order Hymenostomatida of phylum Ciliophora. Protozoans are real eukaryotic cells
and ubiquitous in the aquatic and terrestrial environment. Tetrahymena species used
as model organisms in ecotoxicology are Tetrahymena thermophila (PROTOXKIT F,
2013) and Tetrahymena pyriformis.
Among protozoa, Tetrahymena pyriformis is one of the most commonly used
model organisms in laboratory research. The body of Tetrahymena pyriformis – shown
in Figure 4.7 – is generally 50–60 µm long and 30 µm wide and is pear-shaped, a
characteristic from which the name of the species is derived. In Tetrahymena, the usual
cytoplasmic organelles are mitochondria, endoplasmic reticulum, Golgi complexes,
ribosome, peroxisome and lysosome. Their structures reflect the physiological state
of the cell. Tetrahymena species can grow very rapidly to high density due to their
short generation time, which allows easy cultivation in suitable laboratory conditions
(Sauvant et al., 1999).
Current research indicates the presence of a hormonal system in Tetrahymena that
includes receptors, hormones and signal transduction pathways as well as hormonal
interactions (Csaba et al., 2005).
184 Engineering Tools for Environmental Risk Management – 2

Figure 4.7 Tetrahymena pyriformis electronmicroscopic image (Robinson, 2006).

All these properties explain why so many studies have been performed on this
organism in physiological, biochemical, pharmacological and toxicological research
(Csaba et al., 2005; Leitgib et al., 2007; Mortimer et al., 2010; Novotny et al., 2006;
Stefanidou et al., 2008). The test protocol will be discussed in detail in this Chapter
(see Section 11).

3.2 Fresh-water macroplants

Lemna minor or common duckweed (Figure 4.8) has a subcosmopolitan distribution.
Its habitats are ponds, ditches, canals and slow parts of rivers and streams. It prefers
water with a high nutrient level and often becomes the dominant population. It is a
free-floating, monocotyledonous macrophyte with one to four fronds (leaves), each
with a single root hanging in the water (OECD 221, 2006).
Myriophyllum aquaticum or parrot feather is an emergent, dicotyledonous macro-
phyte (Figure 4.9). Because of its high regeneration potential after cutting the plant into
pieces, a lot of equal sprouts can easily be produced – to create good initial conditions
for biotests. The Parrot feather is native to South America.
 Aquatic toxicology 185

Figure 4.8 Lemna minor or common duckweed in the cultivating vessel, under the microscope (10x)
and the extracted chlorophyll content from a test series.

Figure 4.9 Myriophillum aquaticum (2011), Cabomba caroliniana and Ceratophyllum demersum (2009).

Cabomba caroliniana is an invasive aquatic plant species, commonly used as an

aquarium plant because of its delicate appearance. Its widespread occurrence and fast
growth make it suitable for testing chemicals and plant inhibitory effects (Figure 4.9).
Elodea canadensis or Canadian waterweed is a submergent monocotyledonous
macrophyte. It anchors with its roots in the sediment. Elodea canadensis can also
develop adventitious roots at the whole sprout. It is native to North America, but as
an invasive species, today it is distributed over the whole world. Elodea canadensis is
well known for its rapid growth and its high oxygen production (Figure 4.10).
Ceratophyllum demersum or rigid hornwort is a cosmopolitan, submergent,
dicotyledonous macrophyte. It likes a high nutrient level and summer water temper-
atures of 15–30◦ C. It anchors in the sediment without real roots and develops up to
3-m-long sprouts. It produces a lot of oxygen, so gas bubbles can be seen between its
leaves (MESOCOSM, 2013).

3.3 Fresh-water invertebrates

Rotifers
Brachionus calyciflorus is a fresh-water zooplankton belonging to the class Mono-
gononta and the order Plioma. Rotifers are ecologically very important members of
many aquatic communities. With copepod and cladoceran crustaceans they are the
186 Engineering Tools for Environmental Risk Management – 2

Figure 4.10 Elodea canadensis: photos with growing magnification, the microscopic image shows a 40x
magnification.

major constituents of fresh-water zooplankton with turnover rates higher than those
of crustaceans. Rotifers such as Brachionus calyciflorus are favored test animals in
aquatic toxicology because of their sensitivity to most toxicants, e.g. ROTOXKIT F
(2013).
Brachionus plicatilis is a euryhaline (tolerate a wide range of salinity) rotifer in
the family Brachionidae and is possibly the only commercially important rotifer, being
raised in the aquaculture industry as food for fish larvae. It has a broad distribution in
salt lakes around the world and has become a model system for studies in ecology and
evolution. Marine rotifer Brachionus plicatilis is used in the ROTOXKIT M bioassay
(2013).

Crustacea
Daphnia species or water fleas are small, planktonic crustaceans between 0.2 and
5 mm in length (Figure 4.11). Daphnia are members of the order Cladocera, and
belong to the several small aquatic crustaceans such as Daphnia magna (Figure 4.11),
Daphnia pulex, Daphnia pulicaria, Ceriodaphnia dubia, water fleas are counted
among the Crustacea and live in biotopes like big lakes and in small ponds with standing
water areas. These species are used in many aquatic toxicology test systems. Daphnia
is an indicator species of ecosystem health exhibiting consistent responses to toxins
according to OECD 202 (2004) and OECD 211 (2012).
Heterocypris incongruens or seed shrimp is a fresh-water ostracod, living in water
ponds and lakes. Ostracoda is a class of Crustacea, also known as seed shrimp because
of their morphology. Ostracods are small crustaceans, typically around 1 millimeter
in size; their bodies are flattened from side to side and protected by a bivalve-like,
chitinous or calcareous valve or shell (Figure 4.12). A distinction is made between the
valve (hard parts) and the body with its appendages (soft parts). It is a sensitive aquatic
test organism.
Thamnocephalus platyurus is a fresh-water crustacean belonging to the class
Branchiopoda and family Curculionoidea. Used in ecotoxicity tests, e.g. THAMNO-
TOXKIT F (2013).
Amphipoda or amphipods are important components of fresh-water ecosystems,
an order of malacostracan crustaceans. They have laterally compressed bodies and no
 Aquatic toxicology 187

Figure 4.11 Daphnia magna or water flea, the most popular aquatic test organism.

Figure 4.12 Heterocypris incongruens lives in a tiny shell.

carapace. Their name refers to the different form and size of appendages, in contrast
to isopods. They are very often used in ecotoxicology, particularly the fresh-water
amphipods Gammarus pulex, Gammarus lacustris, Gammarus fasciatu and Hyalella
azteca. Gammaridea is the largest suborder, 5500 of the 7000 amphipods belong to it.
The sensitivity to chemical substances shows a wide range, depending on the applied
species, subspecies and the age of the test organism.
Copepods are a group of small crustaceans found in the sea and nearly every
fresh-water habitat. Some of the species are planktonic (drifting in sea waters), others
benthic (living in bottom sediment). Planktonic copepods are important to food chain
and aquatic ecosystem health. They dominate zooplankton functioning as feed for
small fish, whales, seabirds and other crustaceans in the ocean and in fresh waters.
The calanoid copepod Eurytemora herdmani is applied as a test organism in short-term
toxicity testing of effluents and toxic chemicals in waters.
188 Engineering Tools for Environmental Risk Management – 2

Crayfish, crawfish, or crawdads, belong to orconetes, family Astacoidea. They are

fresh-water crustaceans, feed on living and dead animals and plants. They live in water
bodies that do not freeze to the bottom – mainly in brooks and streams – sheltering
them against predators. They breathe through feather-like gills. Most crayfish are very
sensitive to chemical contaminants in polluted water. Some invasive species however
are less sensitive to toxic chemical substances and crayfish diseases. Due to the presence
of these invasive species such as American crayfish (Orconetes limosus) and Louisiana
crayfish (Procambarus clarkii), native species declined dramatically in European fresh
waters. Pacifastacus species such as Pacifastacus leniusculus and Cambarus species are
typical crayfish in European fresh waters.
Plecoptera or stoneflies: their name comes from Greek and means braided wings.
This name properly characterizes their two pairs of wings which are membranous and
fold flat over the back. They deposit hundreds or thousands of eggs into water. After
two to three weeks the eggs start hatching if environmental conditions are suitable.
Stoneflies are generally not good fliers. Most of them are seasonally aquatic, but some
wingless species are exclusively aquatic from birth to death. Some species in Europe
such as Nemoura cinerea and Nemurella pictetii are specific indicators of ecosystem
health; some others can be used as laboratory test organisms, e.g. Pteronarcys species.
Ephemeroptera or mayflies can be characterized with a very short life span of the
adults. Mayfly is a fresh-water insect whose immature stage (called naiad or nymph)
usually lasts one year in water. Eggs are laid on the surface of lakes or streams, and sink
to the bottom. The naiads live primarily in streams under rocks, decaying vegetation,
or in the sediment. They feed on algae or diatoms. Some common species are Baetis
sp., Ephemerella sp., and Hexagenia limbata.
Trichoptera or caddisflies are small moth-like insects, having two pairs of hairy
membranous wings. The Greek name means hairy wing. They are also called sedge-
flies or rail-flies. The larvae of caddisflies are aquatic and they live in different types of
surface waters: streams, rivers, lakes, ponds, spring seeps, and temporary waters. The
species Dicosmoecus avripes is shown in Figure 4.13. Caddisflies prepare handsome
cases for their larvae. Depending on the caddisfly, these cases can be of a variety of
plant or mineral matter, such as snail and clam shell or gravel as shown in the picture.
Chironomidae or midges from the order Diptera, are little flies, similar to
mosquitoes, distributed globally. They are also called non-biting midges. Chirono-
mus midges are well known from early observed polytene (giant) chromosomes in
larval salina gland. Chironomids spend their larval stages in aquatic or semiaquatic
habitats. The approx. 1 cm long larvae are often red colored (Figure 4.13). They form
an important fraction of the macro zoobenthos of most fresh-water ecosystems. Their
presence is associated with degraded or low-biodiversity ecosystems, some very adap-
tive species are dominant in polluted waters. Eleven subfamilies and several hundreds
of species belong to the chironomids.
The total number and relative abundance of different taxa collected from water,
belonging to the orders Ephemeroptera (mayflies), Plecoptera (stoneflies), and Tri-
choptera (caddisflies) and Chironomidae (midges) are good indicators of aquatic
ecosystem health.
Nematoda or roundworms are probably the most diverse of all animals. Over
28,000 species have been described, but the estimate for the total species number
is one million. They are microscopic in size. Caenorhabditis elegans has become a
 Aquatic toxicology 189

Figure 4.13 Limnephilidae (Trichoptera) larvae and cases (Hodges, 2013) and Chirnomidae (Diptera)
larvae (Penrose, 2013).

Figure 4.14 The nematode Panagrellus redivivus.

laboratory model organism as it was the first multi-cellular organism to have its genome
completely sequenced. Nematodes are used in laboratory assays (lethality, motility) to
measure inhibitory effects of chemicals. Panagrellus redivivus is shown in Figure 4.14.
Planaria or flatworms are common to many parts of the world, living in both
saltwater and fresh-water ponds and rivers. The order of Tricladida plays an important
role in aquatic ecosystems and is used as a bioindicator. The 1–20 mm animals are able
to regenerate their body and re-grow split or missing parts. The famous experiment in
which a reflex was developed by an electric shock combined with light was carried out
in 1955 on a triclad. Later on it was possible to trigger the reaction when only light
was applied. Pieces of the animal exhibited the same knowledge after regeneration.
Small pieces also have transferred the knowledge into animals which were fed by parts
of the trained ones.
Another flatworm, Dugesia tigrina (Platyhelminthes, Turbellaria), is used as a
model for the study of regeneration (cell re-growth) in people and animals.
Oligochaeta or worms, is a subclass of Annelida, which is made up of many types
of aquatic and terrestrial worms. It contains fresh-water tubificids, pot worms and
190 Engineering Tools for Environmental Risk Management – 2

ice worms (Enchytraeidae), blackworms (Lumbriculidae) and several marine worms

living in the pore water of sediments.
Snails (Mollusca, Gastropoda). Many kinds of snails can be found in fresh waters,
in spite of the fact that marine species constitute the majority of snails. Snails may have
lungs or gills but this fact does not constrain them to living in water with lungs or with
gills as a terrestrial snail.
The purple snail or Plicopurpura pansa, a predator snail, naturally inhabits the
high intertidal of rocky shores exposed to the open sea with high impact waves,
used also as a laboratory snail. Biomphalaria glabrata is a species of air-breathing
fresh-water snail, intermediate host for the trematode Schistosoma mansoni, a human-
pathogen. As such, it is the subject of many laboratory research and tests. The river snail
Sinotaia ingallsiana is used for indicating pesticide contamination of waters via acetyl-
choline esterase inhibition in the snails. Some other species e.g. Physa integra, Physa
heterostropha, and Amnicola limosa are frequently subjects of laboratory research and
tests on aquatic ecosystem and environmental effects.
Physa integra is a small, left-handed or sinistral, air-breathing fresh-water snail,
it belongs to aquatic pulmonate gastropod molluscs in the family Physidae and feeds
on algae, diatoms and detritus.
Amnicola limosa populations are typically found in lentic environments, in slow-
moving rivers and swamps of the coastal plain, often on woody debris. They belong to
the class Gastropoda and genus Amnicola and appear to be sensitive to water chemistry,
especially hardness and pH.

3.4 Aquatic vertebrates

Fish, when considering environmental toxicity testing, represent the highest aquatic
trophic level. Arguments for the application of fish as priority aquatic test organism is
not only their metabolic capacities, e.g. in biotransformation competence for certain
chemical classes, but also the high pollution levels and frequencies of chemical spills,
fish frequently being the target. There is a wide range of selection in recommended fish
species which can be used for acute, prolonged and chronic toxicity and reprotoxicity
tests. Fish can be used in an early developmental stage such as embryo, hatchlings (sac
fry), juvenile, or as adult fish. The most popular fish species are introduced below.
Onchorhynchus kisutch, coho salmon or silver salmon is a species of anadromous
fish in the salmon family. The traditional range of the coho salmon runs from both
sides of the North Pacific Ocean, from Hokkaidô, Japan and eastern Russian, around
the Bering Sea to mainland Alaska, and south all the way to Monterey Bay, California.
Onchorhynchus mykiss, rainbow trout, salmon trout has been introduced for
food or sport to at least 45 countries, and every continent except Antarctica. In
some locations such as southern Europe, Australia and South America, they have
negatively impacted upland native fish species, either by eating them, outcompeting
them, transmitting contagious diseases, or hybridization with closely-related species
and subspecies that are native to western North America. They belong to the order
Salmoniformes and genus Oncorhynchus (Figure 4.15).
Salvelinus fontinalis, brook trout, speckled trout, squaretail is a species of fish in
the salmon family of order Salmoniformes. The brook trout is native to small streams,
 Aquatic toxicology 191

Figure 4.15 Onchorhynchus mykiss, rainbow trout (2013); Danio rerio, zebrafish and Lepomis macrochirus,
bluegill (2004).

creeks, lakes, and spring ponds. Some brook trout are anadromous. Salvelinus fonti-
nalis prefers clear waters of high purity and a narrow pH range in lakes, rivers, and
streams, being sensitive to poor oxygenation, pollution, and changes in pH caused by
environmental effects such as acid rain.
Carassius auratus, goldfish, is a fresh-water fish in the family Cyprinidae and
order Cypriniformes. It was one of the earliest fish to be domesticated, and is one of
the most commonly kept aquarium fish. A relatively small member of the carp, the
goldfish is a domesticated version of a less-colorful carp native to East Asia.
Pimephales promelas, fathead minnow is a species of temperate fresh-water fish
belonging to the Pimephales genus of the Cyprinid family. The natural geographic
range extends throughout much of North America, from central Canada south along
the Rockies to Texas, and east to Virginia and the Northeastern United States. The
fathead is quite tolerant to turbid, low-oxygenated water, and can be found in muddy
ponds and streams that might otherwise be inhospitable to other species of fish. It can
also be found in small rivers.
Ictalurus punctatus, channel catfish is North America’s most numerous catfish
species belonging to the Ictalurus genus of the Ictaluridae family. Channel catfish are
native to the Nearctic1 , being well distributed in Lower Canada and the eastern and
northern United States, as well as parts of northern Mexico. They thrive in small and
large rivers, reservoirs, natural lakes, and ponds. They are cavity nesters, meaning
they lay their eggs in crevices, hollows, or debris, in order to protect them from swift
currents.
Lepomis macrochirus, bluegill, bream, brim, copper nose is a species of fresh-
water fish belonging to the Lepomis genus of the Centrarchidae family. It is a member
of the sunfish family Centrarchidae of the order Perciformes. It is native to a wide area
of North America, from Québec to northern Mexico (Figure 4.15).
Lepomis cyanellus, green sunfish is a species of fresh-water fish in the family
Centrarchidae of the order Perciformes. It is native to a wide area of North America
east of the Rocky Mountains, from the Hudson Bay basin in Canada, to the Gulf
Coast in the United States, and northern Mexico. The species prefers vegetated areas

1
The Nearctic ecozone covers North America, Greenland and the highlands of Mexico.
192 Engineering Tools for Environmental Risk Management – 2

in sluggish backwaters, lakes, and ponds with gravel, sand, or bedrock bottoms. Its
diet may include insects, zooplankton, and other small invertebrates.
Danio rerio, zebrafish is a tropical fresh-water fish belonging to the minnow family
(Cyprinidae) of the order Cypriniformes. It is an important vertebrate model organism
in scientific research (Figure 4.15). It commonly inhabits streams, canals, ditches,
ponds, and slow-moving to stagnant water bodies, including rice fields. Danio rerio is
a common and useful model organism for studies of vertebrate development and gene
function. It has a fully-sequenced genome.
The requirement of REACH regulation and of other legislations on chemicals (pes-
ticides, biocides, etc.) to submit ecotoxicity studies to regulatory authorities to support
the registration and/or approval of the products increases the number of laboratory
test organisms for toxicity testing (Test methods for REACH, 2008). Both ethical issues
and the cost of testing motivates research and development to find new in silico and
in vitro molecular and cell-based techniques replacing and reducing the number of fish
in testing chemicals. For waters of environmental origin, living animals are still the
best models. The non-animal alternatives will be discussed in Chapter 1 in Volume 3.

3.5 Sediment-dwelling organisms

Species of the macrozoobenthos and their diversity are suitable to characterize the
environmental health of the sediment ecosystem in surface waters.
Macrozoobenthos are defined as invertebrate bottom fauna living on or in the
bottom, which are retained on a 1 mm × 1 mm mesh sieve. Several hundreds of species
should be identified from a carefully taken sample. Their quantitative and qualita-
tive parameters such as biomass, number of species or the relative abundance of the
species are determined to get information on the ecological status of a river, lake or
sea. The results of such an assessment are usually given in the form of diversity indices
such as species richness, species evenness, Simpson’s diversity index, Shannon index or
some indices which measure the lack of diversity. All these output results of ecological
assessment of macrozoobenthos can characterize spatial and seasonal changes, biotic
or abiotic adverse effects of environmental origin and the long-term trends when reg-
ular biomonitoring is applied. The most important fresh-water and marine organism
taxa to be evaluated when macrozoobenthos are assessed are listed below.
Mollusca: molluscs or mollusks are a large phylum of invertebrates, including
Gastropoda and Bivalvia. Cephalopod molluscs such as squid, cuttlefish and octopus
also belong here.
Bivalvia: bivalves have a shell consisting of two asymmetrically rounded halves
called valves that are mirror images of each other, joined at one edge by a flexible lig-
ament called the hinge (Figure 4.16). Their food is taken up by filter-feeding, differing
from most of the molluscs.
Gastropoda: snails and slugs, the major part of mollusca, univalves, show-
ing extraordinary diversification in habitats. They are also common individual test
organisms in toxicity testing.
Polychaeta: polychaetes or bristle worms are a class of annelid worms, having a
pair of fleshy protrusions called parapodia that bear many bristles, called chaetae on
each body segment.
 Aquatic toxicology 193

Figure 4.16 Bivalve molluscs Dreissenia polymorpha, Unio pictorum and Mytilus edulis.

Figure 4.17 Crab, shrimp, crayfish, and lobster.

Crustacea: group of arthropods, including crabs, lobsters, crayfish, shrimp, krill

and barnacles. Classes of Branchiopoda, Remipedia, Cephalocarida, Maxillopoda,
Ostracoda and Malacostraca belong to crustaceans. They are also used as individual
test organisms in environmental toxicity tests (Figure 4.17).
Amphipoda: is an order of crustaceans, having appendages of different size and
form. It includes the suborders of Gammaridea, Caprellidea or Corophiidea and Hyper-
iidea. Corophium volutator, one of the estuarine mud shrimps is a small (10 millimeters
long) amphipod from the family Corophiidae. It inhabits the upper layers of sand on
the coasts of the Netherlands, Germany, the United Kingdom and France. It has been
applied as sediment test-organism in a US EPA standardized method since 1998, but its
importance increased in the last years as a sediment-dwelling organism testing chronic
toxicity of contaminants associated with whole sediments (Scarlett et al., 2007).
Isopoda: isopods are an order of crustaceans, including well-known animals
such as woodlice and pill bugs. Isopods lack an obvious carapace; it is reduced to
a cephalic shield covering only the head. Respiration is carried out by specialized
gill-like pleopods.
194 Engineering Tools for Environmental Risk Management – 2

Echinodermata: echinoderms are found at every ocean depth, they are very impor-
tant taxa of sea and oceanic environment as part of the community and also due to
their ossified skeletons, which are major building blocks of the abiotic sedimentary
limestone formations. Two main subdivisions of echinoderms are mentioned here:
the motile Eleutherozoa, which encompasses Asteroidea or starfish, Ophiuroidea or
brittle stars, Echinoidea, sea urchins and sand dollars and Holothuroidea, known as
sea cucumbers and the sessile Pelmatazoa which consists of the crinoids, sea lilies or
feather-stars.

4 MEASURING ADVERSE EFFECTS OF CHEMICAL

SUBSTANCES ON THE AQUATIC ECOSYSTEM

The effects of chemical substances are studied to provide information for the regulatory
management of chemicals or for the environmental management of water systems.
This chapter will list the OECD (2014) guidelines which primarily serve the aims
of the REACH regulation in Europe (Test methods for REACH, 2008), i.e. testing
chemical substances for legislative purposes. The most important requirement of reg-
ulatory toxicology is comparability: comparison of measurement results to each other,
to references and to thresholds and limits; comparison between alternatives or addi-
tional options. It is possible only by using standard test organisms and standardized
test methods ensuring uniform application and the comparability of the results.
The same test organisms and test methods can be used with or without modifica-
tions to test environmental water, wastewater or diluted liquid waste samples. When
the test result supports the local assessment of waters and the risk-based decision on
the necessary measures, the test should reflect the local situation and simulate the
real environment to obtain as close an answer as possible to the response of the real
ecosystem. Many of the test methods mentioned below are standardized such as the
ISO Standards (2014) for environmental water samples, but some are developed for
site- and problem-specific uses. Tests methods for environmental waters and wastewa-
ters serve water monitoring and are important management tools in protecting aquatic
ecosystems against toxic effluents.
Several organizations are active in developing, collecting and publishing standard-
ized test methods to ensure harmonization world-wide. The following organizations
play a key role in the field of aquatic toxicity testing:

– American Public Health Association – APHA (2014)

– American Society for Testing and Materials – ASTM International (2014)
– American Water Works Association – AWWA (2014)
– CEN Water Analyses. Published Standards – CEN Standards (2014)
– Ecological Assessment Methods Database – EAMD (2014)
– Ecotoxicity database maintained by US EPA – Ecotox (2014)
– Environment Canada – EC (2014)
– European Committee for Standardization – (CEN, 2014)
– Non-animal Methods for Toxicity Testing – AltTox (2014)
– Organization for Economic Co-operation and Development – OECD (2014)
– Society of Environmental Toxicology and Chemistry – SETAC (2014)
 Aquatic toxicology 195

– Standard Methods for the Examination of Water and Wastewater, 2014

– United States Environmental Protection Agency – US EPA (2014)
– Water Bodies in Europe. Integrative Systems to Assess Ecological Status and
Recovery. Methods for assessing and restoring aquatic ecosystems – WIESER
(2014)
– Water Pollution Control Federation – WPCF (2014).

There are patented but not standardized tests, test kits and apparatus that have
reached a certain level of statistical quality, but not used for regulatory purposes.
The trophic levels of the selected test organism are important both in the case of
testing chemicals and environmental samples or when testing the environment directly,
because the ecosystem members from different trophic levels together can characterize
the state of the environment, the risk to food chains and to ecosystem health. Organisms
from a minimum of three trophic levels should be tested when the results are intended
to be used for risk assessment.
Test types, exposure scenarios and classification according to environmental
relevance are important characteristics of aquatic tests (see also Chapter 1).

– Acute tests are short-term exposure tests with a duration of hours or days, generally
shorter than the test organism’s life span. The chemical substance to be tested is
used in a relatively high concentration to be able to provoke an immediate positive
response. Results are reported in terms of EC50 or EC20 .
– Chronic tests are long-term tests lasting for days, weeks or months, including a
minimum of two generations of the test organism. Continuous low concentra-
tions are applied and NOEC (no observed effects concentration) or LOEC (lowest
observed effects concentration) are the test’s end points.
– Early life stage tests use exposures for less than a complete reproductive life cycle
and include exposure during early, sensitive life stages of an organism. These are
named as embryo-, larval, or egg-fry tests.
– Biodegradation tests measure the existence, type and rate of biodegradation of
the chemical substance in water. Decrease in the concentration of the chemical
substance, biodegradative enzymes/activity of the test organisms and the concen-
tration of the metabolites and end products can be measured. Biodegradation
half-life is calculated from measured data.
– Bioaccumulation tests measure the concentration of high-Kow chemicals in the test
organism’s whole body or in its fatty tissue. Test end point is the bioconcentra-
tion factor (BCF), which is the ratio of the concentration of chemical substance
accumulated in the tissue to the concentration in the water.
– Effluent toxicity is characterized by so-called short-term chronic tests used to mon-
itor the quality of municipal wastewater treatment plant effluent with the goal to
ensure that the wastewater is not chronically toxic.
– Freshwater and saltwater tests apply different test organisms, test methods and
standards.
– Sediment tests have priority when the contaminants have a potential to accumulate
in sediment. The Sediment Quality Triad (SQT) involves an integrated sediment
test using chemical and toxicological methods and field assessment.
196 Engineering Tools for Environmental Risk Management – 2

– Aquatic microcosm tests are multispecies tests truly simulating generic exposure
scenarios or specific cases. The main differences are the size and, as a consequence,
geometry and species diversity.
– Mesocosm tests are ecosystems smaller than or a part of the real ecosystem. They
may be natural or artificial systems, which contain water and sediment. Sediment
can be fully natural or under fully controlled environmental conditions.

The exposure scenario in an aquatic study can be:

– A static test where the organism is exposed to still water containing the
contaminant;
– An arrangement with recirculation where the test solution is pumped through
a device to maintain water quality without reducing the concentration of the
toxicant;
– A renewal test where the test solution is periodically renewed by transferring the
test organism to a fresh test medium;
– A flow-through test exposes the organism to the toxicant by directing a flow into
the test chambers where water and contaminant flux is controlled.

5 SOME COMMONLY USED AQUATIC TEST METHODS

The test methods standardized by OECD, ISO, US EPA and other standardization
bodies are collected and listed in the following tables. These methods are established
for the testing of dissolved chemical substances, surface waters, wastewaters, leachate
and extracts or other waters of agricultural or industrial origin. They can be used easily
or in a modified form for the testing of special liquid/solution samples. The aims of
testing the adverse effect both of the chemicals dissolved in waters and of the waters
for different purposes may be fulfilled by the same test design and test organism.

5.1 OECD guidelines for testing chemicals in aquatic

environment: water, sediment, wastewater
OECD recommends validated test methods for characterizing the fate and behavior
as well as the effect of chemical substances for regulatory purposes. These are listed
in Table 4.1. Chemical substances are tested in a dissolved form in water. The same
methods can measure the effect of environmental water samples.
As an example Figure 4.18 shows the growth curve of Chlorella vulgaris green
alga, i.e. the result of the “Fresh-water algae growth inhibition test’’.
The growth curve is plotted for each dilution member of the concentration series
of the toxicant to be tested. The area under the curve (mathematically known as
integral) or the average specific growth rate (µ) or any characteristic parameter of
the growth curve can be used for the evaluation of the test. From the calculated
results–alternatively read from the growth curve of each treatment and the control
group, the final result of the test may be given as percentage of inhibition (I%) or as
Er C50 , Er C20 or NOEr C, read from the concentration–I% (the response in this case)
curve (Figure 4.19). Er Cx is interpreted as a concentration causing x% reduction in
 Aquatic toxicology 197

Table 4.1 OECD guidelines for testing the effects of chemical substances on aquatic organisms
(OECD, 2014).

Test guideline Test name

OECD TG 201 (2013) Fresh-water algae and cyanobacteria, growth inhibition test
OECD TG 221 (2006) Lemna sp., growth inhibition
OECD TG 202 (2004) Daphnia sp., acute immobilization test
OECD TG 211 (2012) Daphnia magna, reproduction test
OECD TG 231 (2009) Amphibian metamorphosis assay
OECD TG 219 (2004) Sediment-water Chironomid toxicity using spiked water
OECD TG 218 (2004) Sediment-water Chironomid toxicity using spiked sediment
OECD TG 225 (2007) Sediment-water Lumbriculus toxicity test using spiked sediment
OECD TG 203 (1992) Fish, acute toxicity test
OECD TG 204 (1984) Fish, prolonged toxicity test: 14-day study
OECD TG 210 (1992) Fish, early-life stage toxicity test
OECD TG 212 (1998) Fish, short-term toxicity test on embryo and sac-fry stages
OECD TG 215 (2000) Fish, juvenile growth test
OECD TG 229 (2009) Fish, short term reproduction assay
OECD TG 230 (2009) 21-day fish assay: short-term screening for estrogenic and androgenic activity,
and aromatase inhibition
OECD TG 209 (2010) Activated Sludge, Respiration Inhibition Test
OECD TG 224 (2007) Determination of the inhibition of the activity of anaerobic bacteria:
reduction of gas production from anaerobically digesting (sewage) sludge

Figure 4.18 Growth curve of Chlorella vulgaris (green algae).

198 Engineering Tools for Environmental Risk Management – 2

Figure 4.19 Concentration–response curve of Chlorella vulgaris grown in the presence of increasing
metazachlor (a herbicide) concentration.

growth (characterized by the area under the growth curve or the specific growth rate).
Figure 4.19 has been plotted based on 8 algal growth curves (control and seven different
metazachlor concentrations).

5.2 Water-testing methods standardized by the International

Organization for Standardization
The identification numbers and names of the ISO (2014) (International Organization
for Standardization) guidelines are collected in this paragraph. These have been devel-
oped primarily for testing the effect of environmental water samples (unlike OECD
tests established for pure chemical substances dissolved in water).
Water testing methods play a key role in integrated environmental monitoring, in
assessing and managing environmental risks related to water. Direct decision making
is also possible based on the results of effect testing methods. We can interconnect
the scale of measured adverse effects of waters and their safe use or the test results
of wastewaters and their free inlet into natural surface waters, or the ecological
classification of waters and the following obligations.
Toxicity of environmental contaminants adversely affects all members of the
ecosystem, the food chains and food webs as well as the number and abundance
 Aquatic toxicology 199

Table 4.2 ISO Guidelines for testing the effect of water and wastewater on bacteria (ISO, 2014).

Test guideline Test name

ISO 11348-1:2007 Determination of the inhibitory effect of water samples on the light emission
of Vibrio fischeri (Luminescent bacteria test) – Part 1: Method using freshly
prepared bacteria
ISO 11348-2:2007 Determination of the inhibitory effect of water samples on the light
emission of Vibrio fischeri (Luminescent bacteria test) – Part 2: Method using
liquid-dried bacteria
ISO 11348-3:2007 Determination of the inhibitory effect of water samples on the light
emission of Vibrio fischeri (Luminescent bacteria test) – Part 3: Method using
freeze-dried bacteria
ISO 10712:1995 Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication
inhibition test)
ISO/DIS 11350:2012 Determination of the genotoxicity of water and wastewater – Salmonella/
microsome fluctuation test (Ames fluctuation test)
ISO 16240:2005 Determination of the genotoxicity of water and wastewater – Salmonella/
microsome test (Ames test)
ISO 13829:2000 Determination of the genotoxicity of water and wastewater using the umu-test

of individual organisms and species. Laboratory tests on single species assume that
prediction is possible from the results of single species for the whole of the ecosystem.
Prediction from single species toxicity test results is possible by extrapolation from
at least three different trophic levels. In the case of aquatic ecosystems we generally
use algae or plants, crustaceans and fish as suitable representatives of the aquatic
ecosystem. Bacteria are also often used, mainly if biodegradation is a characteristic
process in a certain environment or if a tiered assessment strategy is used.

5.2.1 Standardized bacterial tests for toxicity testing of

water and waste-water
The bacterial tests standardized by ISO for water testing (in Table 4.2) are all laboratory
species, they have no relevance for the aquatic environment, but they are able to give a
sensitive response to chemicals with adverse effects. These tests are equally suitable for
testing of new and existing chemicals as well as samples from contaminated waters,
pore waters, leachates, waste extracts process-waters and wastewaters of agricultural
and industrial origin.

5.2.2 Standardized algal and plant tests for waters

Algae, micro- and meso-flora of the aquatic environment are highly sensitive and
widely applicable test-organisms in environmental tests. The same test methods fit to
the testing of water-dissolved chemicals, waters from aquatic habitats and from other
water uses, as well to assessing wastewaters. The Guidelines managed by the Inter-
national Standardization Organization (ISO) and using algal and plant test organisms
are gathered and listed in Table 4.3. These tests greatly overlap with the OECD tests,
specified for the testing of pure chemical substances (Section 5.1).
200 Engineering Tools for Environmental Risk Management – 2

Table 4.3 ISO Guidelines for testing the effect of water and wastewater on algae and plant (ISO
Standards, 2014).

Test guideline Test name

ISO/TR 11044:2008 Scientific and technical aspects of batch algae growth inhibition tests
ISO 14442:2006 Guidelines for algal growth inhibition tests with poorly soluble materials,
volatile compounds, metals and wastewater
ISO 8692:2004 Fresh-water algal growth inhibition test with unicellular green algae
ISO/DIS 8692:2010 Freshwater algal growth inhibition test with unicellular green algae
ISO 10253:2006 Marine algal growth inhibition test with Skeletonema costatum and
Phaeodactylum tricornutum
ISO/DIS 13308: 2010–12 Toxicity test based on reproduction inhibition of the green macroalga
Ulva pertusa
ISO 10710:2010 Growth inhibition test with the marine and brackish water macroalga
Ceramium tenuicorne
ISO 20079:2005 Determination of the toxic effect of water constituents and wastewater
on duckweed (Lemna minor) – Duckweed growth inhibition test

Figure 4.20 Growth curves of Lemna minor (duckweed).

Figure 4.20 shows the growth curve of Lemna minor or common duckweed. From
the number of fronds the doubling time (Td ) and from that, the average specific growth
rate (µ) can be calculated according to the formula of Td = ln 2/µ. Er C20 or Er C50 can
be determined based upon average specific growth rate values, resulted from testing
the concentration series.
 Aquatic toxicology 201

Table 4.4 ISO Guidelines for testing the effect of water and wastewater on invertebrates (ISO
Standards, 2014).

Test guideline Test name

ISO 6341:1996 Determination of the inhibition of the mobility of Daphnia magna Straus
(Cladocera, Crustacea) – Acute toxicity test
ISO 10706:2000 Determination of long term toxicity of substances to Daphnia magna Straus
(Cladocera, Crustacea)
ISO 20665:2008 Determination of chronic toxicity to Ceriodaphnia dubia
ISO/DIS 14371 Determination of fresh-water-sediment subchronic toxicity to Heterocypris
incongruens (Crustacea, Ostracoda)
ISO/DIS 14380 Determination of the acute toxicity to Thamnocephalus platyurus
(Crustacea,Anostraca)
ISO 14669:1999 Determination of acute lethal toxicity to marine copepods
(Copepoda, Crustacea)
ISO 16303:2013 Determination of toxicity of fresh water sediments using Hyalella azteca
ISO 16712:2005 Determination of acute toxicity of marine or estuarine sediment to amphipods
ISO 19493:2007 Guidance on marine biological surveys of hard-substrate communities
ISO 21427-1:2006 Evaluation of genotoxicity by measurement of the induction of micronuclei –
Part 1: Evaluation of genotoxicity using amphibian larvae

5.2.3 Invertebrates using standard methods for testing water

Amongst aquatic invertebrates, crustaceans and amphipods are the most popular
organisms in standardized ISO tests (Table 4.4), nevertheless all kind of arthropods,
aquatic insects, planktonic animals, ciliate protozoa, nematodes, amoebas could be
used for water testing. Numberless methods are existing and used for special purposes
both as indicators in the assessment of natural waters, in simple laboratory assays or
in more complex micro- and mesocosm tests.

5.2.4 Standardized fish tests for water and waste-water

Fish represents the highest-level vertebrate in water both from the taxonomic and
food-chain point of view. Acute, chronic, mutagenic or teratogenic effects as well
as toxicokinetics can be tested on different life stages and the test-design can also
vary widely (static, flow through, prolonged, etc.). The ISO standards are shown in
Table 4.5.
US EPA requires acute and chronic toxicity tests for freshwater and marine organ-
isms for testing and biomonitoring industrial discharges in the frame of the US NPDS
(2004). The water quality management system is based on the toxicity criteria shown
in Table 4.6 and introduced in the relevant US EPA Guidelines (2002a, b, c).

5.2.5 Ecological assessment of surface waters

Both fresh-water and marine water health can be characterized by the diversity of
the ecosystem and the assessed habitat. The most important element of the ecolog-
ical status, the biological one, is based on five groups of aquatic flora and fauna:
phytoplankton, phytobenthon, macrophytes, macroinvertebrates and fish. The most
202 Engineering Tools for Environmental Risk Management – 2

Table 4.5 ISO Guidelines for testing the effect of water and wastewater on fish (ISO Standards, 2014).

Test guideline Test name

ISO 7346-1:1996 Determination of the acute lethal toxicity of substances to a fresh-water fish
[Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] –
Part 1: Static method
ISO 7346-2:1996 Determination of the acute lethal toxicity of substances to a fresh-water fish
[Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] –
Part 2: Semi-static method
ISO 7346-3:1996 Determination of the acute lethal toxicity of substances to a fresh-water fish
[Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] –
Part 3: Flow-through method
ISO 10229:1994 Determination of the prolonged toxicity of substances to fresh-water fish –
Method for evaluating the effects of substances on the growth rate of rainbow
trout [Oncorhynchus mykiss Walbaum (Teleostei, Salmonidae) ]
ISO 20666:2008 Determination of the chronic toxicity to Brachionus calyciflorus in 48 h
ISO 23893-1:2007 Biochemical and physiological measurements on fish –
Part 1: Sampling of fish, handling and preservation of samples
ISO/TS 23893-2: Biochemical and physiological measurements on fish –
2007 Part 2: Determination of ethoxyresorufin-O-deethylase (EROD)
ISO 23893-3:2013 Biochemical and physiological measurements on fish –
Part 3: Determination of vitellogenin
ISO 12890:1999 Determination of toxicity to embryos and larvae of fresh-water fish –
Semi-static method
ISO 15088:2007 Determination of the acute toxicity of wastewater to zebrafish eggs (Danio rerio)

Table 4.6 Whole effluent toxicity (WET) methods and manuals for measuring acute toxicity to
freshwater/marine organisms (US NPDS, 2004).

US EPA
Guideline Method

Fresh water acute toxicity

2002.0 Daphnid, Ceriodaphnia dubia, acute toxicity
2021.0 Daphnid, Daphnia pulex and Daphnia magna, acute toxicity
2000.0 Fathead minnow, Pimephales promelas/bannerfin shiner, Cyprinella leedsi, acute toxicity
2019.0 Rainbow trout, Oncorhynchus mykiss and brook trout, Salvelinus fontinalis, acute toxicity
Marine water acute toxicity
2007.0 Mysid, Americamysis bahia, acute toxicity
2004.0 Sheepshead minnow, Cyprinodon variegatus, acute toxicity
2006.0 Silverside, Menidia beryllina, Menidia menidia and Menidia peninsulae, acute toxicity
Short-term methods for estimating chronic toxicity to fresh-water organisms
1000.0 Fathead minnow, Pimephales promelas, larval survival and growth
1001.0 Fathead minnow, Pimephales promelas, embryo-larval survival and teratogenicity
1002.0 Daphnia, Ceriodaphnia dubia, survival and reproduction
1003.0 Green alga, Selenastrum capricornutum, growth
Short-term methods for estimating chronic toxicity to marine/estuarine organisms
1004.0 Sheepshead minnow, Cyprinodon variegatus, larval survival and growth
1005.0 Sheepshead minnow, Cyprinodon variegatus, embryo-larval survival and teratogenicity
1006.0 Inland silverside, Menidia beryllina, larval survival and growth
1007.0 Mysid, Americamysis bahia, survival, growth and fecundity
1008.0 Sea urchin, Arbacia punctulata, fertilization
 Aquatic toxicology 203

Table 4.7 European water quality standards of biological classification of the aquatic environment
(CEN Standards, 2014).

EN 14184:2003 Water quality – Guidance standard for the surveying of aquatic macrophytes
in running waters.
EN 14407:2004 Guidance standard for the identification, enumeration and interpretation of
benthic diatom samples from running waters.
EN 15460:2006 Guidance standard for the surveying of aquatic macrophytes in lakes.
EN 15204.2007 Guidance for phytoplankton analysis using inverse microscopy (Utermöhlmethod).
EN 14996:2006 Guidance on assuring the quality of biological and ecological assessments in the
aquatic environment.

important indicator species and their abundance characterize the quality and risks
in living surface waters. The European Committee for Standardization (CEN, 2014)
established standards (CEN Standards, 2014) to survey key actors of aquatic ecosys-
tems of rivers, as shown in Table 4.7. Based on these results fresh waters can be typified
into classes based on EU EQS (2008) and WFD (2000).

6 NON-ANIMAL TESTING OF AQUATIC TOXICITY

To replace aquatic test organisms alternative testing methods such as cell-based assays,
toxogenomic microarrays, and SAR or QSAR models are used to predict toxic effects
to aquatic organisms. However, current in vitro methods for acute aquatic toxicity are
neither standardized nor validated. Current in vitro and in silico approaches replacing
animals in aquatic toxicity testing include (AltTox, 2007):
– Fish cell-based cytotoxicity assays;
– Fish cell-based assays with other mechanisms of toxicity as end points;
– Mammalian cell assays using cytotoxicity or other end points;
– Bacterial cell assays, usually based on luminescent reporter genes (primarily used
for detection);
– Fish embryo assays based on embryo survival and pathophysiological changes;
– In vitro endocrine disruptor assays;
– Genomic microarrays (toxicogenomics);
– (Q)SAR and other computational programs;
– Test batteries and/or tiered testing schemes incorporating the above types of assays.

7 TESTING SEDIMENT

Sediments are in close relationship with surface waters; the contaminants are dis-
tributed between the solid and liquid phases according to their partition coefficient.
Chemical substances with low water solubility and low mobility and high Kow
(octanol–water partition coefficient) are sorbed or bound to the solid phase of the
sediment; it is even filtrated out from the water by the suspended solid and later
on settled with the organic or inorganic particulate solid matter in the form of fine
particle-size bottom-sediment.
204 Engineering Tools for Environmental Risk Management – 2

Based on the partition of chemical substances between the physical phases of sed-
iments, the partition of toxicity or other adverse effects can also be measured, but
the partition of toxicity does not accurately follow the chemical partition, because the
biologically available fraction is much smaller than the total sorbed amount or the
exhaustively extracted chemical content in the sediment.
The partition of toxicity between solid and water can be followed by the parallel
testing of whole sediment and its pore water. Pore-water is the equilibrium state
interstitial water in the sediment which can be obtained by centrifugation or by vacuum
recovery. Integrated evaluation of chemically measured concentrations and toxicity
provides additional information on the mobility, bioaccessibility and bioavailability
and consequent risk of the contaminants bound to sediments. The most frequent cases
are the following:

– High contaminant concentration and high toxicity both in pore water and the
whole sediment indicates a mobile contaminant, which is toxic and available both
chemically and biologically.
– High contaminant concentration and toxicity in pore water but lower in the whole
sediment indicates highly mobile, water-soluble and bioavailable toxicants.
– Low concentration in pore water but high concentration in the whole sediment
and positive toxicity in parallel can be explained by a sorbed contaminant which
is not water-soluble or otherwise chemically available but is still bioavailable due
to special biological mobilization (e.g. biotensides, local pH or redox potential
changes) and uptake mechanisms (chelating agents, active transport). If a highly
toxic chemical substance is present in low concentration, the same combination
results. It may happen that the chemical analysis does not show anything because
the effective toxicant has not been included in the analysis program or cannot
be analyzed due to an immeasurable, low concentration or no suitable analytical
method is available.
– Chemical analyses show high concentration in the whole sediment but it has no
effect on test organisms because of very low mobility and bioavailability. This
combination suggests that the contaminant’s actual stability is high. In this case
one has to assess toxicity and the dependence of stability and/or toxicity from
environmental conditions (which refers to temperature, pH, redox potential and
hydrology). To assess the risk of such cases, one has to create a worst case scenario
in a dynamic test set-up to simulate the maximum mobilization potential of the
sorbed (bound) chemical substance and either prove or disprove its chemical time-
bomb behavior.

The ISO standardized tests methods concentrate on the field assessment of the
benthic fauna as a good indicator for surface-water health. Sampling and identifying
the members of the ecosystem of benthic habitat (Table 4.8) is standardized by ISO.
Some species of the zoobenthos are shown in Figure 4.21 and 4.22.
Benthos can be categorized according to size, the test-methods may use any of
them:

– Macrobenthos, size greater than one mm;

– Meiobenthos, size less than one mm but greater than 32 µm;
– Microbenthos, size less than 32 µm.
 Aquatic toxicology 205

Table 4.8 Guidelines for testing the effect of sediment (ISO Standards, 2014; CEN Standards, 2014).

Test guideline Test name

ISO/DIS 10870:2012 Guidelines for the selection of sampling methods and devices for benthic
macroinvertebrates in fresh waters
ISO 16665:2005 Guidelines for quantitative sampling and sample processing of marine
soft-bottom macrofauna.
(EN) ISO 8689-1:2000 Biological classification of rivers – Part 1: Guidance on the interpretation
of biological quality data from surveys of benthic macroinvertebrates.
(EN) ISO 8689-2:2000 Biological classification of rivers – Part 2: Guidance on the presentation
of biological quality data from surveys of benthic macroinvertebrates.
EN 13946:2004 Guidance standard for the routine sampling and pre-treatment of benthic
diatoms from rivers.
EN 14407:2004 Guidance standard for the identification, enumeration and interpretation
of benthic diatom samples from running waters.
ISO 8689-1:2000 Guidance on the interpretation of biological quality data from surveys
of benthic macroinvertebrates.
ISO 8689-2:2000 Biological classification of rivers – Part 2: Guidance on the presentation
of biological quality data from surveys of benthic macroinvertebrates.

Figure 4.21 Freshwater benthic animals: larva of the mosquito Aedes notoscriptus (2014), dragonfly larva,
gilled snail (2014), stonefly nymph (2014), damselfly nymph (2014), midgenymph (2014),
Hirudo medicinalis, medical leech (Gjertsen, 2007).

Sediment testing includes surveying sediment-dwelling organisms and their diver-

sity as well as laboratory bioassays on sensitive laboratory test organisms or
communities. The survey can be focused on

– Suspended solid (potential sediment) to determine the partition of the toxicity;

– Bottom sediment, as habitat (the top few centimeters of the sediment);
– The impact of dredging (deeper layers should also be assessed).
206 Engineering Tools for Environmental Risk Management – 2

Figure 4.22 Marine benthic animals: nematode Draconema (2014), Christmas tree worm, fire worm,
mussels on the shore, sea urchin, marine snail Nembrotha rutilans.

Sediment-disturbing and -reworking organisms have high impact on sediment and

contaminants in sediment, which should be considered when measuring sediment
toxicity in simulation tests, or microcosms.
Suspended solid, the potential sediment, should be sampled and tested to moni-
tor the chemical pollution of surface waters and the subsequent adverse effects. The
suspended solid is in close contact and interaction with water, thus the partition of the
contaminants between water and solid is close to equilibrium. Therefore, there is a high
probability that information obtained from adverse effects measured on suspended
solids will be valid for the whole aquatic ecosystem.
Another approach is the assessment and monitoring of benthic habitats including
bed sediment using an integrated chemical analytical and ecotoxicity testing method-
ology. When biological tools have indicated adverse effects, the next step is to identify
the cause of the effect, i.e. the responsible chemical substances. For decision mak-
ing on risk reduction measures (e.g. by restricting discharge or reducing the use of
certain chemicals or cleaning up the sediment), risk managers should identify the
contaminating chemical substance, determine its exposure level, identify the adverse
effects and characterize the risk: the extent of ecosystem deterioration and predictable
damage.
In the case of sediment – similar to soil – the adverse effect predictions from
chemically measured concentrations often differ from actual toxicity or other measured
adverse effect results. The main causes of the discrepancies are the following:

– Presence of mixtures of chemicals which interact with each other; they may be
synergistic, i.e. their joint effect is greater than their sum.
 Aquatic toxicology 207

– Due to a common source, a source-specific assemblage of contaminants may occur

making it impossible to assign the proper cause (one contaminant) to the adverse
effect observed.
– Thousands of chemicals are released and bound to the sediment, but only a few
are included into the analysis programs – generally those which have default limit
values in the relevant legislation. Environmental toxicity testing can detect all of
the contaminants’ adverse effects.
– Biological availability and accessibility of chemical contaminants bound to sedi-
ments are small, and their chemical form, mobilization, thus their actual (active)
concentration depends on a number of general and local environmental param-
eters from the outside temperature to the pore volume of the sediment and pH,
redox potential and chemical concentrations in the pore water.
A complex and dynamic approach is needed to characterize the complex system
of sediment. The Sediment Toxicity Identification Evaluation (TIE, 2007) system,
developed by US EPA is based on a biologically controlled fractionation and physi-
cal/chemical manipulation of sediment samples to find, isolate or change the different
groups of toxicants that are potentially present. The biological system, i.e. the test
organism is used as an “indicator’’ to determine whether the manipulation has changed
toxicity.
TIE comprises three phases: characterization, identification and confirmation:
– Characterization chiefly uses environmental toxicity tests (bioassays or other bio-
logical tools) and physical/chemical manipulations such as changes in partition
(sorption–desorption), redox potential (oxidative–reductive environment), and
pH (acidic–alkalic) to build a general “profile’’ of the causative toxicant(s). In
this phase general categories of toxicants can be determined such as volatile sub-
stances, water soluble (e.g. ionic metals), biodegradable organics, highly sorbable,
high-Kow , persistent substances (e.g. PBTs), etc.
– Identification means chemical characterization and analytical measurement of the
suspected toxicant.
– Confirming the presence of the identified contaminant in the water means con-
firming its origin, production or use which is needed for proper decision making
on risk reduction or other management measures.
The TIE system separately investigates whole sediment and its pore water, also
called interstitial water. Pore water is usually separated by centrifugation from the
solid phase and benthic test organisms are used, shown in Table 4.9. The results of
pore water tests give information on the mobile fraction of the pollutants, while tests
on whole sediment clarify intensive and long term interactions between benthic organ-
isms and the sediment sample. The tests look into the effect of the test organism on the
sediment-bound contaminant, which is by several orders of magnitude higher than in
the equilibrium pore water. Direct contact of the test organism with the whole sediment
may result in mobilization (e.g. by secreted acids or mucilage), dermal uptake, dietary
uptake and bioaccumulation over the long term. Whole sediment tests are pessimistic
models because sediment-dwelling organisms can escape from contaminated matrices,
but they cannot do so in the test. Another important issue is the disturbance of the sed-
iment sample: bedded sediment in the depths of rivers and lakes is highly undisturbed,
in an (or close to) equilibrium state, at stable and low redox potential. During sampling
208 Engineering Tools for Environmental Risk Management – 2

Table 4.9 Organisms used for pore water and whole sediment testing (TIE, 2007).

Pore water Whole sediment

Test organism testing testing

Fresh-water benthic
Chironomus dilutus, Chironomid – midge larvae + +
Hyalella azteca, Amphipod – scud + +
Lumbriculus variegatus, Oligochaete – worm + +
Gammarus pulex, Amphipod – crustacean + +
Marine benthic
Americamysis bahia, Mysid – shrimp + +
Ampelisca abdita, Amphipod (Atlantic) + +
Eohaustorius estuarius, Amphipod (Pacific) + +
Leptocheirus plumulosus, Amphipod (Atlantic) + +
Arbacia punctulata, Echinoderm – sea urchin +
Strongylocentrotus purpuratus, Echinoderm – purple urchin +
Mytilus galloprovincialis – mussel +
Corophium volutator, Amphipod – mud shrimp +
Mercenaria mercenaria – hard shell clam +
Mulinia lateralis – dwarf surf clam +
Microtox (Vibrio fischeri) – bacterium +
Fresh-water pelagic
Ceriodaphnia dubia, Cladoceran – water flea + +
Daphnia magna, Cladoceran – water flea + +
Daphnia pulex, Cladoceran – water flea + +

and testing however the samples are disturbed and a completely new equilibrium is
established, which may contribute to mobilization, oxidation or other risk-influencing
changes of the contaminants. In the case of bed sediments a typical change in redox
conditions may appear during sampling and transport: anaerobic or anoxic sediments
from deeper layers may get in contact with air and aerobic organisms.

8 SEWAGE AND SEWAGE SLUDGE TESTS

Wastewater (both treated and untreated) has a higher risk, higher adverse effects on the
ecosystem than receiving waters. This may be an argument to investigate wastewater
toxicity and other adverse effects before its release or utilization. Sewage sludge consists
of the surplus of microorganisms from wastewater treatment technology and sorbed
organic and inorganic matter. The use of treated wastewater on soil as fertilizer may
be an efficient solution when the toxicant content is low and the process is properly
controlled. But the risks associated with sewage sludge could be high, depending on
the wastewater’s hazardous material content. Both wastewater and sewage sludge can
be managed using environmental toxicological tools: wastewater can be discharged
into receiving waters and sewage sludge onto soil if they do not show toxicity or other
adverse effects in properly selected tests. Risk managers can rely on wastewater and
sewage sludge toxicity results for decision making.
Another argument for testing wastewater is the risk posed by untreated wastewa-
ter to the microbiota of the sewage-treatment plants. If a toxic wastewater enters the
biological treatment plant, it may kill the activated sludge and the living microorgan-
isms which biodegrade the (dangerous, toxic) components of the wastewater. If the
 Aquatic toxicology 209

Table 4.10 ISO guidelines for the testing of sewage and sewage sludge (ISO Standards, 2014).

Test guideline Test name

ISO 9888:1999 Evaluation of ultimate aerobic biodegradability of organic compounds in

aqueous medium – Static test (Zahn-Wellens method)
ISO 8192:2007 Test for inhibition of oxygen consumption by activated sludge for carbonaceous
and ammonium oxidation
ISO 9509:2006 Toxicity test for assessing the inhibition of nitrification of activated sludge
microorganisms
ISO 9887:1992 Evaluation of the aerobic biodegradability of organic compounds in an aqueous
medium – Semi-continuous activated sludge method (SCAS)
ISO 11733:2004 Determination of the elimination and biodegradability of organic compounds
in an aqueous medium – Activated sludge simulation test
ISO 11734:1995 Evaluation of the ‘ultimate’ anaerobic biodegradability of organic compounds
in digested sludge – Method by measurement of the biogas production
ISO 13641-1:2003 Determination of inhibition of gas production of anaerobic bacteria
– Part 1: General test
ISO 13641-2:2003 Determination of inhibition of gas production of anaerobic bacteria
– Part 2: Test for low biomass concentrations
ISO 15522:1999 Determination of the inhibitory effect of water constituents on the growth
of activated sludge microorganisms
ISO 18749:2004 Adsorption of substances on activated sludge – Batch test using specific
analysis methods

microflora are killed, they cannot fulfill their task, the wastewater treatment technol-
ogy is malfunctioning, and the organic contaminants in the water fail to be degraded.
Those contaminants that cannot be degraded are present in the effluent or are sorbed by
the flocks and other form of biofilms of the sewage sludge. The ISO tests in Table 4.10
are used to control or protect the sewage microflora. The same tests can be used for
any chemical substance, contaminated environmental or waste sample in the lab, if
a ‘standard’ or a well-known and reliable sewage sludge is available, as a complex
degrading microbiota.

9 TESTING WASTE USING AN ‘ECOTOX’ TEST BATTERY

The classification of chemical substances has been harmonized with GHS by the CLP
regulation that replaced the former Dangerous Substance Directive (DSD, 1967). But
in the field of waste regulation – in spite of having the category H14 “Ecotoxic’’
containing substances and preparations which (may) present immediate or delayed
risks to one or more sectors of the environment – the risk-based classification of wastes,
considering the chemical composition and adverse effects has not been carried out.
The ecotoxicological characterization of waste is part of its assessment as haz-
ardous or non-hazardous according to the European Waste List. However, as of 2007
no methodological recommendations have been provided to cover the hazard criterion
H14 “ecotoxicity’’. Therefore, a European interlaboratory comparison (or round robin
test) (“H14 EU RingTest’’) evaluated a biotest battery, which can be used to assess the
ecotoxicity of waste material and waste eluates (Sander et al., 2008). An interlabora-
tory comparison is a quality assurance program for a new or existing measurement
method. Usually a reference institute sends identical samples to different laboratories
and the results are statistically evaluated.
210 Engineering Tools for Environmental Risk Management – 2

In the European interlaboratory comparison 7 primary and 10 additional ecotox-

icity measurement methods were evaluated and verified for solid wastes and waste
eluates. The interlaboratory comparison has been executed in Europe in 2006–2007
and was managed by Umweltbundesamt or Federal Environment Agency (UBA, 2014),
Germany (Moser et al., 2011).
The ecotoxicological characterization of wastes is laid down in the European Stan-
dard CEN 14735 (2005), which describes the sample preparation and provides an
informative collection of appropriate test procedures for the investigation of wastes.
This collection of test procedures was condensed to the “basic test battery’’, contain-
ing 3 aquatic and 2 terrestrial methods, with two algal and two plant applications,
thus the final test battery contains 4 aquatic (Vibrio fischeri luminescence bacterium;
Desmodesmus and Pseudokirchneriella algae and Daphnia, the water flea) and 3

Table 4.11 Ecotoxicological characterization of waste: basic test battery.

Standard Test name Test description

ISO 11348- Acute test method with This test measures the inhibition of the luminescence
1/2/3:1998 the luminescent bacterium emitted by the marine bacterium Vibrio fischeri (NRRL
Vibrio fischeri. B-11177) using freshly prepared, liquid-dried or
Representative of marine freeze-dried bacteria.
bacteria
ISO Fresh-water algal growth The test measures the growth inhibition of the
8692:2004 inhibition test method unicellular green algae Pseudokirchneriella subcapitata by
with Pseudokirchneriella wastewater or waste extract. The method is
subcapitata. applicable for substances that are easily soluble in
Representative of algae water. With modifications to this method, as described
in ISO 14442 and ISO 5667-16, the inhibitory effects of
poorly soluble organic and inorganic materials, volatile
compounds, heavy metals can also be tested.
ISO Fresh-water algal growth The test measures the growth inhibition of the
8692:2004 inhibition test method unicellular green algae Desmodesmus subspicatus by
with Desmodesmus wastewater or waste extract. Desmodesmus is less
subspicatus. sensitive than Pseudokirchneriella.
Representative of algae
ISO Acute test method with A method for the determination of acute toxicity to
6341:1996 water flea Daphnia magna. Daphnia magna Straus of chemical substances that are
Representative of aquatic soluble under the conditions of test or can be
invertebrates maintained as a stable suspension or dispersion, treated
or untreated sewage effluents, surface or groundwaters.
ISO Seedling emergence and This method measures the toxic effects of solid or liquid
11269-2:1995 growth method with oats, form waste in soil environment, after mixing the waste
Avena sativa. to test into soil. The early stages of growth and
Representative of development of plant Avena sativa is tested. The waste
monocotyledone plants containing soil is compared to clean soil (without waste).
ISO Seedling emergence and This method measures the toxic effects of solid or
11269-2:1995 growth method with rape liquid form waste in soil environment, after mixing the
Brassica napus. waste to test into soil. The early stages of growth and
Representative of development of the plant Brassica napus is tested. The
dicotyledone plants waste containing soil is compared to clean soil
(without waste).
ISO Acute toxicity test Earthworm lethality is measured in artificial soil, which
11268-1:1993 method with earthworm is amended with the concentration series of the waste
Eisenia fetida or waste eluate to be tested.
 Aquatic toxicology 211

terrestrial procedures, two plants (Avena – oats and Brassica – rape) and one animal
test (earthworm) (Table 4.11).
The set of 10 additional ecotoxicity bioassay methods were selected in the inter-
laboratory comparison (European Ring Test of Ecotoxicity of Waste, 2013). Five
aquatic and five terrestrial tests were selected as being potentially appropriate for
the determination of ecotoxicity of waste (Table 4.12).

Table 4.12 Ecotoxicological characterization of waste: potentially appropriate tests.

Standard Test name Test description and remarks

ISO Genotoxicity method The umu-lux test is a genotoxicity test using the two
13829:2000 for water and genetically modified Salmonella typhimurium TA1535 strains
wastewater tested by (TL210 and TL210ctl) transformed with the luxC, D,A, B, E
Salmonella (luciferase gene and fatty acid reductase genes) of Vibrio
typhimurium TA1535 fischeri as a reporter gene. The TL210 strain detects
genotoxicants and the TL210ctl strain detects cytotoxicants.
ISO Bacterial growth A method for determining the inhibitory effect of surface,
10712:1995 inhibition test method ground and wastewater on Pseudomonas putida growth. Not
with Pseudomonas suitable for highly colored samples or samples containing
putida undissolved or volatile substances which react with the
nutrient solution or undergo changes such as biochemical
degradation. Also suitable for testing substances soluble
in water.
DIN Contact test with soil Bacteria and soil are in direct contact. Bacterial cell suspension
38412- bacterium Arthrobacter is spread into the agar-medium and soil blocks or disks are
48:2002 sp. Representative of put on the surface of the growth medium. The diameter of
soil bacteria the inhibition zone (the ring without bacterial growth around
the soil block) is measured.
ISO Duckweed growth A method for the determination of the growth-inhibiting
20079:2005 inhibition test method response of duckweed to substances and mixtures
with Lemna minor contained in water, treated municipal wastewater and
industrial effluents.
ISO/DIS Chronic toxicity test Practical but no higher sensitivity than Daphnia magna
20665:2007 with crustacean
Ceriodaphnia dubia
ISO/DIS Chronic toxicity test Practical but no higher sensitivity than Daphnia magna
13829:2007 method with rotifer
Brachionus calyciflorus
ISO/DIS Earthworm avoidance The short test period, the exposure scenario that is
17512- test method with similar to field conditions, high sensitivity and the use
1:2007 Eisenia fetida and of a taxonomically higher test organism are advantages
Eisenia andrei that confirm the utility of this test in waste
characterization. It can be utilized as a screening
tool. Further development is necessary.
ISO 11268- Earthworm A method based on placing adult earthworms in an artificial soil
2:1998 reproduction containing the test substance (waste or waste eluate) in
test method with different concentrations and determining the percent mortality
Eisenia fetida. after 7 days and 14 days. It is not applicable to volatile
Representative of substances, i.e. substances for which Henry’s constant or the
soil-dwelling air/water partition coefficient is greater than 1, or for which
worms the vapor pressure exceeds 0.0133 Pa at 25◦ C. It does not
take into account the possible degradation of the test
substance. It shows high sensitivity but rather long duration.

(continued)
212 Engineering Tools for Environmental Risk Management – 2

Table 4.12 Continued

Standard Test name Test description and remarks

ISO White worm A method for determining the effects of substances or

16387:2004 reproduction test contaminated soils on reproduction and on survival of the
method with worm Enchytraeus albidus (Enchytraeidae). The animals are
Enchytraeus albidus. exposed to the substance to test by dermal and alimentary
Representative of uptake using a defined artificial soil to which specified
soil-dwelling amounts of wastes or waste eluates are added.
annelid worms
ISO Collembolan (springtail) A long term test, in which Collembola is in contact with
11267:1999 reproduction test with soil containing a dilution series of waste and waste
Folsomia candida. eluates for 3 weeks. The effect on reproduction is quantified
Representative of soil by counting the members of the first and second
invertebrates generation and compared to waste-free soil.

Figure 4.23 Closed and open mussel.

10 NON-STANDARDIZED BIOASSAYS AND OTHER INNOVATIVE

TEST METHODS

In this chapter we introduce some non-standardized, innovative environmental toxicity

measurement methods, which are not widely known or used but they are expected
to spread due to their easy use and targeted application. It should be noted that in
addition to the new test methods, completely new approaches are coming up e.g. non-
testing approaches in ecotoxicology, molecular methods and the 3Rs of Replacement,
Reduction and Refinement of animal experiments in ecotoxicology.
Mussel monitor is a monitoring tool for (contaminated) surface waters; it can also
be used as an early warning system. It is based on the notion that when mussels meet
contaminated water, they close their shells (as opposed to their normal, open state) in
order to protect themselves from the contaminants and to shorten the time of exposure.
Figure 4.23 shows a mussel in closed and open state. In the mussel monitor, the mussel
itself is the sensor. The signals generated by the opening and closing of the shell – using
an inductive electromagnet – are forwarded in the form of an electric signal to the
data-processing unit, which can be several kilometers away. Analyzing the increased
frequency of the shell’s movement compared to the normal movement – by filtering the
 Aquatic toxicology 213

Figure 4.24 Tubifex tubifex, sludge worm or sewage worm, a common inhabitant in lake and river
sediments.

signal through an adequate statistical sensor system – one can obtain a warning signal
indicating that there is some problem. The cause of the problem cannot be identified
from this signal, the only information is that something has triggered the protective
mechanism in the mussels. The appearance of the signal initiates a series of actions
which lead to a risk-decreasing intervention after toxicological testing and/or chemical
analysis of the samples taken from the monitoring location, and after the source has
been identified (Musselmonitor, 2013; Kramer & Foekema, 2000).
Active biomonitoring with fish and Daphnia, which can also be used as bioindi-
cators by placing the specimen, which has been raised under supervision, into the river
or lake in a flow-through cell. The water to be tested should be continuously in contact
with the animals. Any of the animals’ suitable measuring end points can be used as
indicators: the number of living animals, their motility, behavior, proliferation, num-
ber and quality of offspring, etc. Some of the end points can be continuously observed
visually, for example by cameras, and the measurements can be evaluated using an
automatic evaluation system. After fitting the data to a statistical analysis, an auto-
matic warning signal will be obtained if the system detects an anomaly that exceeds
standard deviation. The frequency of the gill movement of the fish and the opening
and closing of the mussel’s shell are monitoring end points that are relatively easy to
detect.
Tubifex monitor is also based on remote signal processing. The members of the
Tubificidae family are aquatic organisms that often occur in waters contaminated by
organic substances. Half of their body burrows into the bottom sediment, while the
rest floats in the water. In the presence of certain contaminants, these organisms can
retract a large portion of their bodies, and burrow deeper into the bottom sediment.
This behavior is proportional to the concentration of the contaminating substance.
The retraction can be observed both visually and with the help of a camera. By using
digital image-analyzing systems, it can be quantitatively analyzed and evaluated. Based
on the measurements of Leynen et al. (1999) the conclusion can be drawn that the
movement/behavior of Tubifex worms shown in Figure 4.24 can be reproduced, and,
by tracing and evaluating their movement, a water monitoring and early warning
system can be developed.
214 Engineering Tools for Environmental Risk Management – 2

In situ toxicity assessment and other in situ biological testing strategies play an
increasingly important role in aquatic and sediment risk characterization and man-
agement. In situ testing has many advantages compared to laboratory testing of single
samples after storage and transportation into the lab. Many contaminants are reactive,
volatile and capable of being degraded and/or dissipating during transportation and
storage. The change in the pH and redox potential between the time of sampling and
testing may also influence the toxicity to a large extent.
In situ testing of adverse effects is especially important if the source of contam-
ination is ephemeral or when the exposure varies over time and space. The mussel
monitor, the tubifex monitor, and biosensors can provide continuous and integrating
signals from the environment during the life span of the test organism.
Another trend of the innovations and developments in the field of in situ testing
is the establishment of the mobile versions of laboratory bioassays to enable in situ
decision making during site characterization, screening and mapping of hazardous
contaminants, identification of hot spots, as well as in the determination of the place,
time and frequency of sampling during local risk assessment.
In situ measured toxic effects and bioaccumulation integrates all the environmental
effects of changing parameters such as temperature, water level, seasonal changes, etc.
which may greatly influence bioavailability and the realization of adverse effects. On
the other hand natural stressors together with the anthropogenic adverse effects may
result in a confusing picture.
As a summary, we can list the advantages of in situ assessment of toxicity and
bioaccumulation:

– Increased realism;
– Incorporation of spatial/temporal variability;
– Integration of multiple stressors;
– Reduced sample manipulation;
– Matrix-specific risk identification;
– In situ planning and decision making is possible.

There are some limitations too, which can be eliminated only partly by new
developments:

– No control over natural exposure factors;

– Ammonia–ammonium-ion ratio is pH dependent: ammonium increases with the
pH, and is more toxic, than the ionic form because the uptake of ammonia (NH3 )
is not inhibited by any cellular mechanisms;
– Hydrogen sulfide to sulfide ion ratio is also pH dependent; the sulfide ion is more
toxic and its ratio increases with pH decrease;
– Groundwater seeping into sediment influences the quality of habitat, mainly the
nutrients and micro-contaminants endanger the sediment ecosystem;
– Problems associated with caging of the test organism are significant: they are
not able to carry out normal behavior, e.g. they cannot escape. Cage design may
influence water flux and oxygen dissolution;
– Transportation, handling, and physical stress of the test organisms influences their
response;
 Aquatic toxicology 215

– Predation and competition of the indigenous test organisms with the native ones
may change the response;
– Need for appropriate controls and reference sites.

Some laboratory screening tests have already been modified and applied to in situ
testing. They are useful in quick mapping of the extent of contamination at contami-
nated sites. These types of tests are typically rapid and can be conducted at relatively
low cost and in large numbers. A quick turnaround time is useful for decision making
with respect to subsequent steps in the assessment of ecological risk at a site. Some of
them are introduced here in detail, based on the review of Rosen et al. (2009).

– Sea urchin fertilization tests: the fertilization success as end point refers to the
percentage of eggs that develop fertilization membranes following 20 minutes of
exposure to sperm that have been previously exposed to test samples (also for
20 minutes). These life stages are ecologically relevant because of their tendency
to be negatively buoyant, and therefore, are likely to be associated with surficial
sediment (Anderson et al., 1996). This test needs only 5–10 mL per replicate, less
than one hour exposure period, it is sensitive to a variety of contaminants, and
has high ecological relevance. It needs extensive preparation time and cost.
– Microtox® , using Vibrio fischeri luminescent bacterium is suitable for on site
use. It is moderately sensitive, its time and cost requirement is also average.
Environmental relevance is low if the marine species is used for fresh water.
– QwikLite is similar to Microtox, but using a higher animal, the planktonic bio-
luminescent dinoflagellate. It has high ecological relevance in marine ecosystems.
Dinoflagellates emit light only as an effect of mechanical stimulation, so that a
stirrer is built into the measuring chamber. Light emission is measured generally
after 24 hours exposure.
– Toxkits of ecologically relevant algal and rotifer species ensure easy-to-conduct and
easy-to-use test methods, with dehydrated organisms. Rehydrated Toxkit organ-
isms were as sensitive as laboratory species when compared to traditional standard
laboratory assays and in situ exposures. The Toxkits have the advantage of not
requiring culture facilities, they require little equipment and training, and the
test can be easily and quickly conducted under a wide range of environmental
conditions.
– Modification of short-term laboratory tests is a successful way to develop a
portable test setup. 10 mL pore water per replicate was enough for sea urchin
fertilization, mussel embryo, rotifer, and QwikLite standard tests methods after
the reduction of the normal chamber size.

In situ tests provide more realistic exposures. The integrated application of in situ
bioassays and laboratory testing may ensure optimal information, especially when
multiple stressors are involved and improve decision making with respect to manage-
ment decisions. Results from in situ studies will provide much greater confidence in
assessing true exposures and effects occurring at a particular site. This confidence is
critical when costly decisions and implications to remediate or not is at stake (Rosen
et al., 2009).
216 Engineering Tools for Environmental Risk Management – 2

The time requirement of in situ but sometimes also laboratory tests can significantly
be reduced by using conserved test organisms, e.g. lyophilized or gently dried ones. The
ready-made test-kits, containing preserved test organisms able to revive instantly, can
be used comfortably and quickly, without laboratory and professional preparedness
for the maintenance of the test-strain. Daphnids are typical organisms suitable for
long-term storage in dried form and being able to become active again when meeting
the test medium. Some standardized and ready for use kits with different crustaceans
are commercially available:

– DAPHTOXKIT F (2013) using Daphnia magna. This toxicity test adheres to

OECD and ISO Guidelines.
– DAPHTOXKIT F (2013) using Daphnia pulex. This toxicity test adheres to the
OECD Guideline.
– CERIODAPHTOXKIT F (2013) using Ceriodaphnia dubia. This toxicity test
adheres to the US EPA Test Guideline.

Microbial communities are the most important components of the ecosystem, and
therefore a number of microbiological methods are being developed which will be
suitable to characterize microbiological communities and individual species. These are
non-destructive, in situ, real-time measurement methods, which can observe details of
the microbial community in action because the measurements are carried out at the
sub-millimeter scale.
Biosensors and microprobes are analytical instruments created by alloying elec-
tronic and biological systems: they transform biological signals measured in the water
or in the soil to electrical signals. Genetic bioindicators (bioreporter genes) are genes
that can create easily detectable products; thus, because they work only under certain
conditions, they can enhance the selectivity and amplify the signal. The microprobes
and microelectrodes can measure the characteristics of the living area and the ecosys-
tem at the sub-millimeter scale in soils and other solid-phase samples. The measured
parameters include pH, temperature, chloride ion concentration and dissolved oxygen
concentration (Revsbech & Jorgensen, 1986). Microprobes are able to characterize
the redox system of the soil via the measurement of ammonium (De Beer & Van
den Heuvel, 1988), nitrate (Jensen et al., 1993) or oxygen (Revsbech, 1989). The
nitrification was monitored by means of a combined oxygen and nitrogen oxide micro-
probe by Christensen et al. (1989). Ramsing et al. (1993) applied a combination of
oligonucleotides and microelectrodes to detect sulfate reduction.
Visible or fluorescent light provides easy-to-measure end points; thus, the most
widespread bioreporter genes are those responsible for the emission of light and GFP
(green fluorescent protein), which is responsible for green fluorescence. By planting
these genes into selectively sensitive microorganisms, they will signal the damage in
real time as part of the photosensitive biosensor, through decreased emission of light.
The lux gene of Vibrio fischeri or Vibrio harveyi is only seven kilobases long. It can be
built into any selectively sensitive microorganism. The GFP gained from the jellyfish
species Aequorea victoria can be built either into a prokaryote or a eukaryote, and it
will result in an easily detectable, strong green light emitted by the host.
Aquatic and terrestrial ecosystems try to adapt to the environmental circum-
stances, climatic conditions, seasons and contaminating substances, thus showing
great flexibility. The changes in the species distribution are caused by the fact that
 Aquatic toxicology 217

those species that are sensitive to the contaminating substance will decline and eventu-
ally disappear, while those species that can tolerate or utilize the contaminant will gain
an advantage, proliferate and their relative numbers will increase within the commu-
nity. Since certain species have genes that are responsible for the coexistence with the
contaminating substance, these genes will naturally proliferate in the community in a
contaminated area, not just through an increase in the species’ population, but also due
to other mechanisms such as horizontal gene transfer between members of the commu-
nity. As a global ecological trend, the diversity of the microflora in water and the soil
is continuously growing, even if the contaminants exert a detrimental effect at a local
level, but all in all they will trigger the evolution of further genes in the metagenome,
and thus the quantity and information content in the genes will gradually increase.
Biological and molecular methods for characterizing the diversity and detection
of species have growing application.
There are two concepts used to characterize the ecosystem of an environmental
compartment, i.e. a complicated living area. The first concept suggests that we can
examine all of the genes, gene products or gene activities of a community, e.g. a soil
microflora, irrespective of which species’ genome it belongs to. This examination can
take place at the DNA level with the help of DNA chips, real-time PCRs, or through
the measurement of gene products (generally enzymes) and their metabolic activities
(patterns of the community’s substrate utilization, for example using the BIOLOG
(2013) method). The aim is to characterize the community, and statistical evaluation
is needed for the correct interpretation of the result (Dobler et al., 2001; Nagy et al.,
2010). It can be used as an early warning system if the harmful effect can be statistically
separated from seasonal and climatic anomalies.
Another concept is based on the notion that only the gene that selectively appears
as a direct consequence of the harmful effect, or this gene’s (metabolic) product or
the organism carrying the gene can be detected. In cases like these, the most selective
methods are used, for example DNA hybridization, fluorescent in situ hybridization,
or PCR. If that particular gene can be detected, the effect, too, is most likely the
consequence of the harmful substance/agent.
Molecular methods are selective and sensitive by themselves, and traceability can
be further enhanced using sensitivity- and detection-enhancing signals. One of the most
widespread techniques, PCR itself, is based on detection after signal multiplication.

11 MULTISPECIES AND MICROCOSM TEST METHODS

FOR AQUATIC TOXICITY

Multispecies test methods include microcosms and mesocosms. The volume of micro-
and mesocosms varies from 0.1 liter to thousands of liters, there is not limit in volume
or size, and it is rather their complexity than their size that distinguishes between
micro- and mesocosm.
The following are the main characteristics of micro- and mesocosms:
– They are historical, like the ecosystem itself, and irreversible in time.
– They have a trophic structure, which is sometimes very simple, sometimes close
to the real environment.
– Evolutionary events occur in micro- and mesocosms: for example biodegrading
ability or resistance to xenobiotics may arise and, as a consequence, the structure
218 Engineering Tools for Environmental Risk Management – 2

and diversity of the community changes by adaptation to the circumstances, e.g.

to the contaminant content.
– Evolution of new metabolic pathways for biodegradation (of e.g. pesticides or
xenobiotics) is possible. It can also be enforced by adding a xenobiotic stepwise.
– Microcosms and mesocosms are characterized by reduced complexity compared
to natural systems: the number of species is smaller.
– The organisms in a microcosm cannot move freely, they cannot leave the test vessel
or escape from the test volume.
– Dynamics of the ecosystem differs from the natural one: the enforced isolation
into a small scale causes changes in the dynamics of the whole system. The surface
to volume ratio in a microcosm differs to a large extent from the natural systems.
These changes should be distinguished from the effect of the toxicant.
– Since the solid phase (sediment) is disturbed, the natural equilibrium is not main-
tained, and a new equilibrium state is established in the microcosms after a
transient phase. This may influence the sampling concept and the results.
– Heterogeneity: in natural ecosystems spatial and temporal heterogeneity is the key
to species richness. Artificial test systems should not be heterogeneous or unique
and must not lose their statistical power.
– Multispecies tests are complex systems and, unlike simple-species tests or biochem-
ical assays, they cannot be repeated due to their dynamics and history as their past
is conserved in population dynamics down to the DNA sequence.

All this information should be considered when designing and evaluating micro-
cosm and mesocosm tests. The parallel tests indicate great differences, and certain
microcosms can show a completely different behavior than others due to individual
evolution.
Micro- and mesocosm tests have a number of goals: a mesocosm can be used
to address ecology and management of element cycles, nutrient load due to fertilizer
runoff, toxicity of natural and synthetic chemicals, and effects on or manipulation of
food chains (Dibble & Pelicice, 2010). Further goals are measurement of adverse effects
of biological agents and pests, biodegradation and bioaccumulation of contaminants,
the mechanism of the spread of invasive species, etc. The effects of protective measures
such as influencing microbial activity to enhance nutrient consumption, degrade con-
taminants or synthesize useful substances can be modeled. This can include simulating
adverse or beneficial effects of physical, chemical and biological agents and investi-
gating and manipulating the response of the benthic-pelagic ecosystem in micro- and
mesocosms. The simulations and manipulations are aimed at conserving ecosystems
and habitats as well as utilizing the ecosystem in green technologies for environmental
protection, waste treatment or production technologies.
We will now introduce the type and set-up of aquatic microcosms and mesocosms
based on the literature. These microcosms are mainly used for testing the effect of
chemical substances on aquatic diversity (Landis & Yu, 1999).

– Mixed-flask culture mesocosm (MFC) is a small-scale microcosm with a com-

plex community. It works in 1 liter volume for 12–14 weeks. The representative
community is separately grown and then added to the test vessel. The commu-
nity contains two single-cell algae or diatoms, one filamentous algal species,
one nitrogen-fixing blue-green alga, one grazing pelagic microinvertebrate, one
 Aquatic toxicology 219

benthic macroinvertebrate and several bacterial and protozoan species. The effect
of xenobiotics on the community diversity is measured.
– Standardized aquatic microcosm (SAM) is a 4 liter glass vessel with artificial sterile
sediment and synthetic water. 10 algal, 4 invertebrate and 1 bacterial species are
added to the water. Nutrients, pH, temperature and dissolved oxygen are measured
and controlled. Test material is added on the 7th day. Parallel tests of treated
and control vessels are performed and evaluated. Measured end points are pH,
dissolved oxygen, nutrient level, and cell/organism counts.
– Benthic-pelagic microcosms containing both sediment and water are suitable for
testing the normal functions of shallow waters or the effect of contaminated
water and sediments on benthic and pelagic organisms. Contaminant partition
and cycling between water and sediment, as well as sediment solid phase and pore
water can be monitored. The effect of pH, redox potential and the different forms
of the contaminants on benthic and pelagic organisms can be simulated. Natural
or artificial sediment can be placed into the test vessel and natural or separately
cultivated organisms into the microcosms.
– Eutrophication pelagic microcosm was developed for measuring chlorophyll syn-
thesis from nutrients. Gowen et al. (1992) proposed the use of the yield of
chlorophyll from dissolved nutrient to estimate the risk of marine eutrophication.
The yields of chlorophyll from nitrogen were further investigated, under more
controlled conditions, in microcosm experiments reported by Edwards (2001),
Edwards et al. (2003), Edwards et al. (2005). The apparatus used in these micro-
cosm experiments is a vessel containing seawater, phytoplankton, pelagic bacteria
and protozoa, filtered to exclude mesozooplankton. Seawater, filtered of all organ-
isms, and nutrient enriched, was placed in the reservoir tank and steadily pumped
into the reactor vessel, giving a dilution rate of about 0.2 per day. Chlorophyll
production was measured as an end point.
– Many kinds of static microcosms have been developed for authorization purposes
to test aquatic biodiversity and the effect of fungicides, herbicides or insecticide
on the abundance of pelagic and benthic species. After reaching a steady state,
a known number of phytoplankton, zooplankton, macrozoobenthos, crustaceans
and fish species are added to the benthic-pelagic microcosm and monitored over
short or long term (Figure 4.25).
– The US-standardized FIFRA mesocosms are test vessels with a surface area of
5 m2 , a depth of at least 1.25 m, volume at least 6 m3 and made of inert material.
Sediment obtained from an existing pond containing natural benthic community,
is placed onto the bottom of the mesocosm on trays, in a 5 cm-thick layer. Water
should derive from a healthy, ecologically active pond. The water level in the micro-
cosm should be controlled and regulated by adding or removing water. Weather
should be recorded and taken into account during the evaluation of the test
results. FIFRA mesocosm uses bluegill sunfish, fathead minnow, channel catfish,
phytoplankton, periphyton, zooplankton, emergent insects and benthic macroin-
vertebrates in the test system. The test protocol prescribes the size of organisms,
e.g. the biomass of fish added which should not exceed 2 g/m3 of water. Smaller
tanks can be used without fish.
A test substance is added after 6–8 weeks aging of the microcosm, by spraying
on the water surface, pouring into the water phase if dissolved in water, or put to
the bottom in the form of a soil slurry.
220 Engineering Tools for Environmental Risk Management – 2

Figure 4.25 Aquatic mesocosms (outdoor setup).

Sampling begins 2 weeks after the construction of the microcosm, it continues

for 2–3 months after the last treatment with the test material. Frequency of testing
depends upon characteristics of the test substance and on the treatment regime.
Dosage of pesticide level, frequency and number of replicates are determined based
on the objectives of the study. Measured end points are oxygen content, respiratory
activity, biomass, cell and organism numbers and distribution.
– Stream microcosms with a flow-through system can model streams.
– Pond microcosms are models of real ponds simulating the ecosystem of the original
pond.
– Compartmentalized lake means that a natural lake is divided into compartments.
A small compartment, which theoretically keeps all characteristics of the whole
lake is isolated and used for testing.
– Sediment core microcosms contain an undisturbed volume of natural sediments
trying to keep the original equilibrium between the solid phase and pore-water
phase of sediment to get a system as close to realty as possible.
– Waste treatment microcosms simulate ecological technologies designed for the
treatment of liquid and sludge-form wastes such as aerobic and anoxic ponds or
wetlands for wastewater treatment, rhizosphere filtering or living machines. (See
also Chapter 8).

12 DESCRIPTION OF TETRAHYMENA PYRIFORMIS BIOASSAY

M. Molnár
The experimental design and test parameters of the acute (short-term) and chronic
(based on reproduction) toxicity tests using Tetrahymena pyriformis, a ciliate proto-
zoon, are introduced here (Figure 4.26). This test is neither standardized nor widely
 Aquatic toxicology 221

Figure 4.26 Tetrahymena pyriformis: a ciliate protozoon.

used, but the authors of this book have experience in applying this sensitive organism
for testing toxicity of water, sediment and soil for generic and site-specific risk assess-
ment and decision-making purposes. It is an important organism because the animal
trophic level is the bottleneck and is generally represented only by Daphnia and fish in
ecotoxicity tests. As a single-cell organism, Tetrahymena pyriformis is easy to handle
and it grows relatively rapidly, so that we give priority to this organism to amend the
test batteries for measuring aquatic ecotoxicity. Single-cell animals are good models
for higher animals, inasmuch as their cells and metabolisms are identical with higher
animals from many points of view.
Tetrahymena pyriformis – due to its wide-range sensitivity – can be applied for
toxicity testing of chemical substances and environmental samples: water, wastewater,
leachate, soil, sediment and solid waste. It can also be used in contact tests.

12.1 Experimental
A dilution series should be prepared from the chemical substances or contaminated
environmental samples. At least five concentrations are to be tested simultaneously,
preferably arranged in a geometric series. The lowest concentration should have no
observed effect on the growth. Sterile distilled water is applied for dilution of liquid-
phase samples and as a control medium.
Tetrahymena pyriformis A-759-b is cultivated in proteose-peptone-yeast extract
medium (PPY), containing 1% proteose peptone and 0.1% yeast extract.
The Erlenmeyer flasks (100 mL) contain 600 µL of liquid-phase or 1.5 g of solid-
phase environmental samples supplemented with 30 mL of PPY medium and with
222 Engineering Tools for Environmental Risk Management – 2

penicillin, streptomycin and nystatin solutions (to protect Tetrahymena from harm-
ful bacteria) at final concentrations of 0.01 mg/mL, 0.1 mg/mL and 0.05 mg/mL,
respectively (Leitgib et al., 2007).
After vigorous mixing, 600 µL of exponentially growing culture of test organisms
(initial density 1 × 105 cells/mL) are added to the flasks.
Erlenmeyer flasks are incubated with shaking (125 rpm) in a dark chamber
(20 ± 2◦ C), and the concentration of cells is determined after 0, 24, 48, 72, 96 and
120-h exposure by direct counting in a Bürker counting chamber in a light microscope
or any other cell counting methods.

12.2 Evaluation and interpretation of the results

The mean cell-concentration values of the parallels of each dilution and the control
are log-transformed and plotted versus time to produce growth curves.
Average specific growth rate can be read from the exponential phase of the growth
curves and the inhibition rate (%) is calculated by comparing the treated cultures to the
untreated control. Inhibition percentage is plotted as a function of concentration of the
tested chemical substance or dose of the tested water, soil, sediment, etc. Dose–response
analysis using a logistic model by ORIGIN 8.0 software is applied to determine effective
concentration or dose values (Er Cx and Er Dx ) or the highest no-effect and/or the lowest
effect concentrations/doses (NOEr C/LOEr C or NOEr L/LOEr L) of the samples.
Testing pure chemical substances, the effective concentrations (Er C20 and Er C50 )
which cause 20% and 50% reduction in growth rate are derived statistically from a
plot of percentage inhibition against the logarithm value of the concentration of the
substance.
For environmental samples, Er D20 or Er D50 values were the end points of the tests,
where Er D20 and Er D50 are doses (mass or volume) of the sample which caused 20%
and 50% reduction in the growth rate, respectively. Instead of water dose, the rate
of dilution can also be used as an end point of the tests. This means that the result
can be given as a quantity of water or soil (depending on the quantity used by the test
method), or as a dilution rate which causes 20%, 50% inhibition or “no effect’’ on the
test organism. The toxicity of environmental samples can also be termed as toxicity
equivalent – as a comparison to a known toxicant. The results of direct toxicity tests
of environmental samples are discussed in Chapter 9 in this Volume and Chapter 2 in
Volume 3.

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PHOTOS

Aedes notoscriptus (2014) Aedes notoscriptus mosquito larva. [Online] Available from:
http://uq.edu.au/integrative-ecology/evolution-of-inducible-defences-in-mosquito-larvae.
[Accessed 6th November 2013].
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Bluegill_sc.jpg. [Accessed 6th November 2013].
Ceratophyllum demersum (2009) Aquarium fish. [Online] Available from: http://diszhal.info/nov
enyek/Ceratophyllum_demersum.php. [Accessed 6th November 2013].
228 Engineering Tools for Environmental Risk Management – 2

Closterium ehrenbergii (2014) Alga Resource Database, microscopic photo. [Online] Avail-
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6th November 2013].
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Damselnymph.jpeg from: Water Quality Macroorganism List. http://scioly.org/wiki/index.
php/Water_Quality/Macroorganism_List. [Accessed 6th November 2013].
Desmodesmus species (2014) Alga Resource Database, microscopic photo. [Online] Avail-
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2013].
Hodges, J.C. Jr. (2013) Trichoptera, Limnephilidae: Dicosmoecus avripes larva & case. [Online]
Available from: http://freshwater-science.org. http://freshwater-science.org/Education-and-
Outreach/Media-Galleries/Invertebrates.aspx?imagepath=%2fEducation-and-Outreach%2f
Media-Galleries%2fMedia%2fimages%2fmacro062-jpg. [Accessed 3rd November 2013].
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jpeg from: Water Quality Macroorganism List. http://scioly.org/wiki/index.php/Water_
Quality/Macroorganism_List. [Accessed 6th November 2013].
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novenyek/Myriophyllum_aquaticum.php. [Accessed 6th November 2013].
Penrose, D. (2013) Diptera, Chironomidae larvae. [Online] Available from: http://freshwater-
science.org, http://freshwater-science.org/education-and-outreach/media-galleries/inverteb
rates.aspx?imagepath=/Education-and-Outreach/Media-Galleries/Media/images/macro100-
jpg). [Accessed 6th November 2013].
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[Online] Available from: http://www.shigen.nig.ac.jp/algae/images/strainsimage/nies-0035.
jpg.[Accessed 6th November 2013].
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nocookie.net/__cb20130730132445/acnl/images/0/01/RainbowTroutIRL.jpg. [Accessed 6th
November 2013].
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nymph.jpeg from: http://scioly.org/wiki/index.php/Water_Quality/Macroorganism_List.
[Accessed 6th November 2013].
Pictures without reference are commercially available or prepared by the authors.
Chapter 5

Terrestrial toxicology
K. Gruiz, M. Molnár,V. Feigl, Cs. Hajdu, Zs. M. Nagy,
O. Klebercz, I. Fekete-Kertész, É. Ujaczki & M. Tolner
Department of Applied Biotechnology and Food Science,
Budapest University of Technology and Economics, Budapest, Hungary

ABSTRACT

Soil is the key compartment of the terrestrial ecosystem, the scene of element cycling,
the habitat of myriads of bacteria and other microorganisms, plants, fungi and animals.
The intensively interacting solid, liquid and gas phases of the soil together with the
complex microbiota ensure an enormous capacity of genetic, chemical, biochemical
and biological activities, storage and buffering capacity of the terrestrial habitat.
Exposure to chemical substances endangers the delicate equilibrium of the soil
ecosystem and leads to soil deterioration. Science still cannot precisely identify a
healthy soil structure and function, so assessment of deviations from a healthy state
and identification of the unacceptable scale of changes are difficult both in theory and
practice. Terrestrial toxicology may provide valuable information about chemical risk
in soil. Terrestrial toxicology seeks to understand the soil’s capability to buffer adverse
effects by capturing and releasing toxicants. The toxicity buffering capacity together
with air, water and nutrient management protects soil habitats and at the same time
serves the benefit of the environment as a whole. Simplified chemical, biological and
ecological models as well as complex field assessments are applied in terrestrial ecotox-
icology depending on the aim of the survey. Mobility, bioavailability, partition between
physical phases, degradation, biological uptake, bioaccumulation and any other out-
come of the interaction of the soil matrix and its inhabitants with the contaminant
play an important role in chemical substances exerting their effect.
This chapter describes the basic knowledge of test organisms and test methods
used in terrestrial toxicology. Microcalorimetry, which is introduced in detail, is not
a typical toxicity testing tool although heat production is directly related to all the
organism’s activities. The authors introduce the application of TAM (Thermo Activity
Monitor) for testing the effect of chemical substances in soil.

1 INTRODUCTION

Chemical contaminants may be present in the soil and sediments for a long time dur-
ing their life cycle. They often enter the soil via direct processes such as fertilization,
plant nutrient supply, pesticides application and disposal of wastes on or into the soil.
Atmospheric deposition from airborne contaminants, first of all dry particulate mat-
ter with adsorbed contaminants, acid rain and pollutants dissolved or suspended in
230 Engineering Tools for Environmental Risk Management – 2

precipitation also deliver considerable amount of organic and inorganic micropollu-

tants into the soil. Floods transport both suspended and dissolved matter onto the
soil, polluting the flood area. Contaminants of subsurface waters are filtrated and
concentrated by soil solids.
Contamination and soil deterioration endangers ecosystem services attributed to
soil. Most of the ecosystem services are based on good health and function of the soil,
and the activity of the terrestrial ecosystem. The most important ecosystem services
the soil provides are:
Supporting services:

– Nutrient cycling and primary production which underlie the delivery of all other
services but are not directly accessible to people.

Provisioning services:

– Groundwater for human uses;

– Food, crops, wild food, spices;
– Pharmaceuticals, biochemicals, industrial products;
– Energy (geothermal, biomass for fuel).

Regulating services:

– Carbon sequestration and climate regulation;

– Water cycling;
– Water and wastewater purification;
– Element cycling;
– Natural hazard, pest and disease control;
– Waste decomposition and detoxification;
– Contaminant elimination;
– Crop pollination.

Cultural services:

– Recreation and ecotourism service;

– Cultural, intellectual and spiritual inspiration;
– Scientific discovery.

The aim of soil toxicology is to understand how contaminants affect the soil ecosys-
tem and, as a consequence, the ecosystem services. Soil toxicology determines the
quantitative relationship between contaminant concentration and adverse effects. This
knowledge enables forecasting and managing the soil ecosystem in order to prevent or
stop soil deterioration. Risk levels acceptable over the long term ensure good-quality
and sustainable use of soil at local, regional and global levels.
Soil health, diversity and functions can be characterized by monitoring soil on
a regular basis, identifying the adverse trends and trying to reverse them. The main
global soil deterioration trends are the prime targets of soil monitoring, including:

– Loss of diversity;
– Loss of organic matter/humus content;
 Terrestrial toxicology 231

– Acidification;
– Sodification;
– Erosion;
– Compaction;
– Contamination by chronic and acute emissions from point and diffuse sources.

Adverse effects on the terrestrial environment include:

– Effects on soil functions and well balanced nutrient cycling, ensuring nutrients for
plants, microorganisms and soil invertebrates;
– Effects on plant biomass production and plant activities, such as participating in
nutrient cycling and ensuring equilibrium of gases in the atmosphere;
– Effects on invertebrates, which represent food for other organisms, and covers
essential roles as pollinators, detrivores, saprophages and pest controllers;
– Effects on terrestrial vertebrates, domestic and wild species;
– Accumulation of toxic compounds in food through the food chain, endangering
higher animals and humans;
– Effect on humans through cultivated and wild species, by the deterioration of the
landscape, and endangering recreation in the nature.

The extent of contamination is continuously growing world-wide. Its origin is

emission from point sources (production units, chemical plants) and diffuse deposition
from polluted air, diffuse pollution from mining and ore processing, from agriculture
using persistent chemicals (nutrients and pesticides) as well as legal or illegal waste
disposal on soils.
Humans are exposed to contaminated soil as shown in Figure 5.1:

– Directly (children play on the ground and sometimes eat soil; farmers and
gardeners are in intensive contact with soil);
– Via the use of groundwater and surface waters (drinking, bathing, irrigation, etc.);
– Through dust inhalation;
– By consuming plant and animal products contaminated through the food chain.

Ecosystem health can be characterized by ecological assessment methods, describ-

ing species diversity and their changes in soil, or ecotoxicological assessment using
laboratory bioassays, microcosms or mesocosms and their extrapolation to the real
terrestrial ecosystem.

– The trends of ecosystem changes in soil can only be properly interpreted if the
healthy soil characteristics and the acceptable rate of change are known. This
acceptable rate of change is almost impossible to specify because the soil biota
have an extremely high adaptive potential and their survival and activity do not
necessarily mean soil health. Species diversity and activity of up to 109 cells in
one gram of soil together with higher soil-dwelling organisms and plants can char-
acterize soil health. The limit of acceptable adverse changes is normally 5% in
terms of species diversity. Soil diversity assessment faces rather high uncertainties
due to spatial heterogeneity, climatic and seasonal changes and biological adapta-
tion. Our insufficient knowledge of species diversity in healthy soil, for example
232 Engineering Tools for Environmental Risk Management – 2

Figure 5.1 Humans endangered by contaminated soil.

the hidden minor components of the microbial community or the rate of healthy
natural adaptation and deviations may cause serious problems when assessing and
interpreting species diversity.
– Testing the response of certain species which can represent the soil ecosystem as a
whole, is another strategy in soil health management and risk assessment. It can
be extrapolated from soil toxicity tests on representative test organisms to the soil
ecosystem using safety factors or known distributions for extrapolation to the real
ecosystem. In practice we use soil-living microbes, plants and animals representing
three different trophic levels used to determine the limit concentrations of effect
and no effect – the lowest concentration which shows a measurable effect or the
highest tested concentration which fails to show any effect. These values are used
for risk management and decision making. The main uncertainty of this strategy
derives from the representativeness of the selected test organisms.

Soil ecotoxicity results can be used for screening, monitoring and control as well
as for direct decision making in management in general or for specific contaminated
sites.
Most European countries regulate contaminated site management by standard
requirements and methodologies. The basic theory suggests that contaminated site
management should be risk based, giving priority to environmental risks considering
(future) land use and including economic and social impacts. All contaminated sites and
 Terrestrial toxicology 233

soils are individual cases where non-identified mixtures of contaminants together with
their derivatives and metabolites typically occur. This situation together with local
environmental conditions results in a unique combination and requires site-specific
management. This means that one should assess locally relevant adverse effects on
local land users (ecosystem and humans) and make decisions based on the site-specific
risk which arises from the results of actual adverse effects.
Toxicity and other adverse effects typically can serve as information for direct
decision making in the first exploratory step of a tiered assessment and in recurrent
cases under a generic or constant environment where toxic effect is the only variable of
risk assessment. In the first assessment tier a yes or no answer on toxicity may decide
on the exclusion from or inclusion in further assessment. Quality and further use of
ex situ remedied soil (e.g. in a soil treatment plant) can be decided based on its toxicity.
Effect-based decisions can facilitate rapid and efficient risk management in many cases.
Direct decision making in situ, based on measured soil toxicity or other observed
adverse effects can be used to decide whether:

– The in-depth contaminated site assessment should be continued;

– Sampling and further analysis are needed;
– Contaminated soil should be removed from the environment, the fraction to be
removed should be delineated;
– Treated, remedied soils should be utilized;
– Plants should be applied to deteriorated, contaminated or remedied soils.

Figure 5.2 shows the different forms of contaminants and their binding to and
transport by the different components of the soil matrix:

– Volatilization;
– Dissolution in pore water, groundwater or runoff;
– Chemical reactions with the environmental constituents (air, water, components
of the matrix) and contaminants with each other;
– Biological transformation by microbial, plant and animal impact as well as by free
enzymes bound to soil particles;
– Binding to humus and soil organic matter;
– Binding to oxides and clay minerals;
– Sorption to roots;
– Uptake by microorganisms, plants or animals.

The combination of several contaminants and all the above-mentioned transport

and fate characteristics varied with the environmental conditions (temperature, pH,
redox potential, osmotic characteristics, etc.) are endless and hardly predictable.
Chemical models often fail in contaminated soil assessment due to the strong
interaction between contaminants and the complex solid matrix of the soil. The conse-
quence of the various interactions is that partition, chemical availability (by water, by
solvents or by other contaminants) and biological accessibility/availability of contami-
nants may significantly differ from each other. The interactions between contaminants,
the three phases of the soil matrix and soil-dwelling organisms greatly influence the
effect of contaminants on soil community. The same interactions influence the response
234 Engineering Tools for Environmental Risk Management – 2

Figure 5.2 Fate and behavior of a contaminant in soil.

of the test organisms in a bioassay or other tests for measuring ecotoxicity. Extrapo-
lation from chemical data to the risk of contaminants on soil ecosystem or soil-using
humans is a multi-step procedure handicapped by multiple uncertainties. There have
been many trials to refine the chemical models, e.g. by incorporating biomimetic chem-
ical reagents or sequential contaminant extraction, or parallel extraction by different
solvents (see Chapter 2.6). However, these complicated and rather expensive chemical
analytical methods still cannot integrate all the biological and genetic factors of the
ecosystem and simulate the current rate adverse effects of contaminants. In addition,
these complex chemical models based results are often difficult to interpret.
Complex environmental situations such as the soil itself with the three physical
phases, complicated matrix materials, milliards of living cells and mixtures of contami-
nants require an integrated approach for characterization and monitoring (Gruiz et al.,
2001; Gruiz, 2009; Gruiz et al., 2009). Integrated approach means the simultaneous
application of physico-chemical and biological, toxicological and ecological methods,
as well as the integrated evaluation and interpretation of their results.
The strong binding of pollutants to the solid matrix may lead to high contami-
nant concentrations in soil. At the same time, high sorption capacity of soil reduces
contaminant availability to water and biological systems. The buffering and filtering
behavior of the soil is beneficial for the waters and the ecosystem, but this “filter’’
is not separated, it is in close contact and dynamic interaction with the environment
thus the bound contaminants can be released again, depending on actual environmen-
tal conditions. Organic contaminants are strongly bound to the soil’s organic fraction
 Terrestrial toxicology 235

(humus) and inorganic contaminants both to clay minerals and humus, thus the trans-
port of contaminants by soil moisture and groundwater and their uptake by the biota
is restricted by their retention in soil-solids. The high concentration of contaminants
may turn the soil into a chemical time bomb, which may explode when environmental
conditions reduce the sorption and toxicity buffering capacity of the contaminated soil.
This is why direct toxicity testing of soils and dynamic test methods are so impor-
tant: they can measure actual toxicity and changes in contaminant mobility and
availability. The mobility and availability of a contaminant in soil is the result of
diffusion, dissolution, emulsification, suspension, solubilization in water, or in water-
based ionic (diluted acids or alkali) and organic solvents (chelating and the complexing
agents, biotensides). These are key processes in seeking material balances in soil and are
in continuous variation as the response of soil to external conditions, to which different
equilibria belong. Soil analysis and testing require a strict and problem-specific concept
for the measurement of the effects under conditions that are capable of truly modeling
the concrete problem. When the conceptual model does not fit to the problem, the
assessor can easily under- or overestimate the risk posed by soil contaminants.
Toxicity end points of terrestrial ecosystems can be selected within a wide range
according to the type of chemical substance, source, transport routes, land uses and
receptors. Any change in an organism, which can clearly be attributed to the adverse
effect of a contaminant, can be applied as an end point. Options are:

– Mineralization by soil microorganisms: any enzyme or metabolic pathway taking

part in biodegradation, such as
◦ hydrolyzing enzymes: cellulose, hemicellulose, carbohydrate, lipid and protein
hydrolyzing enzymes;
◦ oxidation and respiration: ATP production, methane oxidation, 14 C labeled
substrate oxidation, dehydrogenase activity, oxygen uptake, CO2 generation
or O2 consumption using endogenous or added substrates;
◦ specific xenobiotics-degrading enzymes and mechanisms;
◦ damage to microbial activities in hydrolyzing and denitrifying enzymes, in
nitrification or nitrogen fixation.
– Plant germination, growth and metabolism:
◦ decrease in photosynthesis rate;
◦ changes in the composition of chlorophyll or other photosynthetic pigments;
◦ damage to evapotranspiration;
◦ enzymatic alterations;
◦ effects on biochemical plant protection mechanisms, increased susceptibility
to pathogens (viruses, bacteria, fungi) or pests;
◦ ineffective pollination, reduction of gain of biomass, loss of leaves, mortality.
– Interactions between soil microbes and plants:
◦ loss of mycorrhiza, rhizoplane and root microflora;
◦ diminished nodulation of leguminous plants.
– Soil animals and their molecular, cellular, population or community level
responses:
◦ changes in enzyme synthesis
◦ endocrine and immune system disruption;
236 Engineering Tools for Environmental Risk Management – 2

◦ stress response by producing stress proteins such as metallothioneins or heat-

shock proteins;
◦ adverse effects on reproduction rates, natural sex ratio, increased susceptibility
to infectious diseases and mortality.
– Ecosystem structure, population densities and diversities:
◦ adverse changes in population dynamics, species diversity, yield, energy
transfer and food chain structure.
– Plant–animal interactions:
◦ influence of adversely affected plants on animals;
◦ influence of adversely affected animals on plants.

Toxicity bioassays may substantially diminish uncertainty regarding biological

availability, uptake, and biological toxicant effects, by ensuring completely controlled,
standard circumstances. Besides the strengths of single-species tests such as simpli-
fied and interpretable systems, there are also weaknesses, first of all the lack of
environmental realism.
Multispecies test systems such as terrestrial model systems (microcosms) offer
a potentially useful compromise between the simplicity of laboratory tests and the
complexity of field assessments. Inexpensive test systems using field microbiota
communities and soil microinvertebrates (nematodes, mites, springtails, and other
microarthropods) have been used to investigate the effects of toxicants in terrestrial
systems since the 1980s. Community-level adverse effects on soil microbiota and the
disruption of food web structures can be measured in microcosm systems. A shift in
bacterial community composition and, as a consequence, changes in soil activities can
be detected by biochemical tests, measuring enzyme activities in multiwell systems such
as BIOLOG (2013).
In situ testing and biomonitoring is the most realistic type of toxicity testing. In this
test organisms are exposed to contaminated soil in the field, wherein plants and animals
are subject to both realistic conditions and natural variations in exposure. This testing
protocol is best applicable to sessile organisms, for example plants, or caged animals,
but other disadvantages and unrealistic conditions, e.g. the lack of avoidance must
be taken into account. Biological monitoring may integrate and evaluate cumulative
impacts from a variety of natural and anthropogenic stressors.
Terrestrial toxicity is an adverse environmental condition for soil-living microbes,
soil-dwelling organisms, and all kind of plants, terrestrial wildlife and of course
humans, using terrestrial ecosystem services. Soil plays a role in the life cycles of water,
chemical elements and organic matter, including contaminants. When the biodegrad-
ability or the bioaccumulative potential of toxic substances is the question, biotesting
provides information on what happens when contaminated soil meets the organism.
Biodegradation of chemical substances in soil can be easily measured both in static
and dynamic test systems, and this is introduced in Chapters 3 and 8. Nevertheless,
predicting the environmental fate and behavior of chemicals in soil and their effect
on living organisms over the long term from measured results is burdened with uncer-
tainties due to heterogeneity and individual character of the soils of different areas.
For long-term predictions the analyst has to investigate and compare the uncertainties
 Terrestrial toxicology 237

of the methodological options in every individual case and determine which type of
methodology should be used for extrapolation: relatively well-reproducible and stan-
dardized chemical analytical methods, laboratory bioassays, microcosms, mesocosms
or the combination of any of these.

2 TERRESTRIAL TEST ORGANISMS

Individual members of the soil-living community, a well-defined group of the ecosys-

tem (e.g. plants), or soil biota as a whole can be used to test fate, behavior and adverse
effects of chemical substances in soil or to explore and observe the impacts of contam-
inants on the soil ecosystem. The organisms to be tested can belong to either micro-,
meso- or macro-fauna and -flora.
The proper selection of test species is a key issue in terrestrial ecotoxicity testing.
We have to choose species that are relevant to the aim of the assessment (screening,
monitoring, early warning, etc.), are representative for the functional roles played
by the community in question (e.g. mineralization, nitrogen-fixing, decontamination,
bioaccumulation), and are sensitive to the contaminants or other exposures to be
characterized. They have to fulfill some general requirements (ASTM, 1998; Laskowski
et al., 1998):

– Rapid life cycles;

– Uniform reproduction and growth;
– Ease of culturing and maintenance in the laboratory;
– Uniformity of population-wide phenotypic characteristics;
– Routes of exposure similar to that encountered in the field.

Cell density in soil can be measured directly as a cell concentration, or through

the activity of the living microbiota. The restrictions of traditional, cultivation-based
methods can be eliminated by DNA and omic techniques.
Species diversity in soil means the abundance and the relative distribution of
plant or soil-dwelling mesofauna members. Diversity assessment requires identifying
and counting relevant species or members after proper sampling. When diversity of
microorganisms of the soil is the issue, the task is more complicated. Billions of species
and cells live in one gram soil while only a few can be grown in the laboratory and
isolated on special growth media to identify them by using e.g. colony descriptors.
However, it is now known from the comparison of direct microscopic observation and
new DNA fingerprinting techniques that the organisms that can be grown in artificial
laboratory conditions represent a tiny percentage of the total number. It is possible to
extract DNA directly from soil and estimate the density of different species by analyzing
the complexity of this whole community DNA, the metagenome (see also Chapter 1).
Traditional and innovative methods are available for the complex characterization of
soil microbiota from the assessment of the totality of the genetic material (genome)
and proteins (proteome) to biochemical, physiological and behavioral characterization
of the whole microbiota of the soil.
238 Engineering Tools for Environmental Risk Management – 2

Traditionally, in practice, ecotoxicology is based on two main concepts:

– Use of specific indicator organisms to extrapolate to the whole soil biota and
function;
– Direct measurement of the soil’s biological, biochemical and enzymatic functions,
based on the aggregated activity of the microorganism community.

Species diversity cannot be determined because of the large number of viable but
nonculturable microorganisms in soil.
All microbiological characteristics of the soil can be studied using the modern
molecular biology techniques: determining presence, density and activity of individual
organisms, species diversity and the aggregated density and activities of the community.
Contaminated soil may show unusual characteristics, for example good bio-
chemical activities and enzyme functions, including contaminant biodegradation,
accompanied by completely unusual species distribution and missing indicator species.
It may be the result of long-term adaptation of the microbiota to the contaminant.
Another extreme situation is when the dominating species are not sensitive to the con-
taminant, but the whole community gradually becomes inactive. The reason for that
may be the disappearance of some sensitive minor components with an essential role
within the community. These kinds of changes cannot be handled as regular trends due
to too high variability in soil microbial diversity, only a case-by-case interpretation is
possible.
In this chapter some selected soil biota members are introduced, mainly those
which are used in practice as indicator organisms and/or laboratory species for soil
toxicity testing. The discussed species include microorganisms, terrestrial plants, and
some members of the micro-, meso- and macrofauna as well as higher members of the
terrestrial ecosystem.

2.1 Soil-living bacteria and fungi as test organisms

Using bacteria for soil testing has many advantages: it is representative for soil
microflora and active soils contain 109 bacteria/gram soil. Bacteria and fungi are
directly responsible for decomposition of organic matter in terrestrial ecosystems. They
fill a critical position in the soil food web and all nutrient cycling pathways and pro-
cesses. They are easy to maintain and grow, the time and cost requirement of the tests
is generally low, the number of replicates is arbitrary, and the statistics of the tests are
good. Microbial activities, population density and species distribution are extremely
sensitive to soil conditions, continuously changing parameters such as moisture con-
tent and temperature, as well as to seasonal alterations. This means in the practice that
the test strategy should be based on a comparison with a negative/untreated control
or reference instead of using the absolute values of microbial activities for measuring
adverse effects.
Pseudomonas species are Gram-negative rod-shaped bacteria, mainly living
in soil. Pseudomonas is a genus of gammaproteobacteria, belonging to the family
Pseudomonadaceae. There are 191 Pseudomonas species described in the litera-
ture. Most of them are very active in biodegradation and mineralization; and they
are responsible for the degradation of a number of organic soil contaminants of
 Terrestrial toxicology 239

petroleum origin. The best-known species are Pseudomonas fluorescens, Pseudomonas

putida and Pseudomonas syringae as biodegrading strains of xenobiotics.
Pseudomonas aeruginosa is increasingly recognized as an emerging opportunistic
pathogen, frequently occurring in petroleum-contaminated soils.
Pseudomonas putida is a saprotrophic species. It demonstrates a very diverse
metabolism, including the ability to degrade organic solvents such as toluene. It was
the ever first bacterium pioneering patenting of living organisms. It may play a key
role in contaminated soil bioremediation. ISO 10712:1995 standard method uses
Pseudomonas putida growth inhibition for measuring the toxic effects of chemicals
or contaminated environmental samples.
Azotobacter species are Gram-negative, aerobic soil-dwelling bacteria, belong-
ing to the Pseudomonadaceae family. There are around six species in the genus,
some of which are motile by means of peritrichous flagella while others are not.
They are typically polymorphic (different sizes and shapes depending on age and life
conditions). Old cells tend to form cysts; they have the ability to fix atmospheric
nitrogen by converting it to ammonia. Many species are used in soil toxicology
tests: Azotobacter chroococcum, Azotobacter beijerinckii, Azotobacter nigricans and
Azotobacter vinelandii. Azotobacter croococcum is applied in the direct-contact soil
block test.
Azomonas agilis found in soil and water is closely related to other species of the
Pseudomonas and Azotobacter genus in the same Pseudomonadaceae family. It is a
motile, Gram-negative bacterium, capable of fixing atmospheric nitrogen. The main
difference, compared to Azotobacter, is that Azomonas does not form cysts. It is a
widespread test organism for contaminated soil, eluates and waters. Its former name
is Azotobaceter agile.
Rhizobia fixing nitrogen in symbiosis with plants are sensitive to a wide range
of contaminants. Rhizobia form nodules on the plants’ roots, this is their habitat
as shown in Figure 5.3. Their nodule formation and count can be utilized as end
points.

Figure 5.3 Root nodules of a 4-week-old Medicago italica inoculated with Sinorhizobium meliloti
(Ninjatacoshell, 2009).
240 Engineering Tools for Environmental Risk Management – 2

Arthrobacter species from the Gram-positive bacterial genus Arthrobacter are

widely distributed in soil. They are able to metabolize and, as a consequence, degrade
a variety of toxic soil contaminants. Arthrobacter can grow in the presence of hex-
avalent chromium, and is able to reduce it to less toxic trivalent chromium. They
are able to biodegrade persistent pesticides including organophosphate insecticides.
Arthrobacter chlorophenolicus A6 can survive at extremely high concentrations of the
toxic pollutant 4-chlorophenol.
Arthrobacter globiformis forms small colonies on nutrient agar, ranging in color
from yellow to white and measuring 2 mm in diameter on average. It is used for soil
toxicity testing in standardized contact tests.
Bacillus genus of Gram-positive rod-shaped bacteria and a member of the division
Firmicutes is one of the best known soil-living microorganisms and the best understood
prokaryotes, in terms of molecular biology and cell biology. Bacillus species can be
obligate aerobes or facultative anaerobes. Under stressful environmental conditions,
the cells produce oval endospores that can stay dormant for extended periods. Bacillus
genus used in soil toxicity testing in many forms: e.g. Bacillus subtilis and Bacillus
pumilus in contact test, many different Bacillus species and strains in growth tests,
and enzyme-activity tests.
Bacillus subtilis, also called hay bacillus or grass bacillus is very commonly found
in soil. It can convert (decompose) natural organic compounds and contaminants into
simple organic or mineral products. A high number of exoenzymes produced enable
them to attack the big-size molecules of dead organic matter taking part actively in
mineralization and element cycling. B. subtilis is used in growth inhibition tests for
screening toxic metal cations in soil both in form of contact tests in solid soil and testing
soil extracts and eluates. The macroscopic view in Figure 5.4 shows the chain of cells
in a laboratory grown culture and the staining with 4 ,6-diamidino-2-phenylindole
(DAPI, a fluorescent stain) of the smear under fluorescence microscope.
Vibrios are Gram-negative bacteria with curved rod-shaped cells. Some of them
are pathogen s (e.g. Vibrio cholerae) which can cause foodborne infection, usually
from seafood. They are facultative anaerobes, motile with polar flagella. Vibrio fis-
cheri, Photobacterium phosphoreum, and Vibrio harveyi are able to ‘communicate’
by light emission, produced through bioluminescence and regulated by quorum sens-
ing. The lux gene from these natural strains has been transformed into other indicator
bacteria used for environmental analyses and testing.

Figure 5.4 Bacillus subtilis under the microscope: native and DAPI-stained cells.
 Terrestrial toxicology 241

Vibrio fischeri is a popular bacterial strain also used for soil testing, nonetheless
it is not a soil-living, but a marine species, but its wide range of sensitivity, the strain
availability and the standardized test method makes possible its use for the testing of
contaminated soil and sediment in tests using soil-extracts or leachates and whole soil.
Cellulose-degrading bacteria and fungi including actinomycetes degrade cellulose
when it is associated with pentosans such as xylans and mannans, and it undergoes
rapid decomposition. The best known cellulose-degrading bacteria are: Fibrobacter
succinogenes, Cellulomonas sp., Clostridium thermocellum.
Aerobic bacteria can be considered as a whole, characterized by density, activities
and diversity. The activity and response of soil-living bacteria as a whole can also be
utilized for the characterization of soil health, its dynamic adaptability and loadability.
Using the site-specific soil in the tests (sampled from the contaminated site) one can
directly measure the response of the targeted soil ecosystem, without extrapolation.
But to use whole soil for generic risk assessment of chemical substances is always
problematic, due to the unachievable requirement of an everlasting, unabated reference
soil with a standard response.
The growth and total activities of the soil microbiota as toxicity end points, (e.g.
respiration rate, consumption and conversion rate of substrates, production of metabo-
lites) can provide a quantitative picture on soil function. The rise of the response, the
time of adaptation, etc. can be investigated in a dynamic way but the qualitative
changes in diversity cannot be detected in growth tests. Cell numbers and activi-
ties should be amended with information on the microbial diversity in soil to obtain
complete information.
Zak et al. (1994) suggest that a sound interpretation of the pollution-induced
effects on the structure and function of soil microbial communities must be based
on measuring the integrating parameters such as litter decomposition or nutrient
mineralization (e.g. using the BIOLOG plate).
Pollution-induced Community Tolerance (PICT) is a toxicological tool used for
characterizing soil community tolerance to the contaminants present over a long time.
Community tolerance is formed by adaptation, using various mechanisms: switch-
ing on existing, but mute, genes responsible for tolerance, increasing the relative
abundance of the tolerant species, creating new gens by random mutation–selection
mechanism and spreading useful genes by horizontal gene transfer. Any of these pro-
cesses are good indicators to prove PICT, what is an indirect evidence for the presence
of contaminants.
Anaerobic bacteria dominate the habitat in deep and water-saturated soils. They
can be handled as a whole, characterized by density, activities and diversity. Anaerobic
soil has a microbiota highly different from aerobic soil, in spite of the fact that some
microbes are the same as aerobic ones; these are the facultative anaerobes, able to
switch their metabolism from aerobic to anaerobic, using alternative forms of respi-
ration, such as nitrate- or sulfate-respiration. Atmospheric oxygen is not available for
soil organisms under low redox potentials, so that they can utilize alternative electron
acceptors, such as nitrate, iron, manganese, sulfate, carbonate, and, in contaminated
soils, exotic pollutants such as chloride. Anaerobic metabolism of soil-microorganisms
occurs in normal soil, as shown by the redox potential gradient in every soil. Anaerobic
microbes and processes play a key role in the biodegradation of certain soil contami-
nants such as chlorinated contaminants degraded by reductive dechlorination. In spite
242 Engineering Tools for Environmental Risk Management – 2

of their reduced activity compared to aerobic ones, anaerobic bacteria play an impor-
tant role, mainly in the two-phase soil and groundwater and in the elimination of its
contaminants. The density and diversity of anaerobic microbes in deeper soils and
groundwater suitably characterize anaerobic soil habitats.
Microfungi as a separate group of microbes may characterize soil by their density
and activities. They are of special importance in agricultural soils where their roles and
species distribution is well known and utilized.
Soil enzymes of microbial origin and their activities are good indicators of micro-
bial activity, heterotrophy and mineralization in soil ecosystems. When evaluating the
absolute values of enzyme activity, results should be compared to the enzyme activity of
the healthy soil community. This requires monitoring data or time-series information.
The other concept to utilize enzyme activities of soils is the use of a standard-quality
test soil for generic testing of chemical substances or environmental samples.
Any of the enzymes of the respiration chain, i.e. energy production (biodegra-
dation, mineralization) as well as of biosynthesis can be used to test toxicity.
The commonly measured enzymes are beta-glucosidase, carboxymethylcellulase,
n-acetylglucosaminidase and acid/alkaline phosphatases.
Proteomics – referring to the study of proteome, the totality of proteins – has not yet
been adapted to soil because of the many problems of analysis which arise. However, it
can be applied in a complementary way to find a relationship between composition and
activity of soil microflora. Proteogenomics, the science of the metagenome responsible
for the diversity of proteins in soil microflora, is a promising tool to overcome the
problems of soil proteome analysis (Armengaud et al., 2013).

2.2 Terrestrial plants for soil toxicity testing

As primary producers, plants capture the solar energy in their structure, thus their
function influences the whole terrestrial ecosystem and modifies the landscape and land
uses. Measuring and/or predicting toxicant impacts on plants enables environmental
and agricultural managers to identify potential thresholds of contamination above
which floral establishment, growth, and reproduction may be compromised, and direct
mortality is a concern.
The general advantage of plants as test organisms is their ready availability, for
example seeds, which can be purchased in bulk, their easy activation by germination,
their relatively long life and usability of up to several months, and in all life stages.
Low costs and high reliability in site- and problem-specific applications makes their
application feasible. When using plants for soil toxicity testing, one must be aware of
the great variability in sensitivity between species (Fletcher et al., 1990).
The long list of terrestrial plants reflects the trials to find the best fitting plant
to the topic of testing. Available test plants should represent both dicotyledonous and
monocotyledonous species, the two major groups of plants. Traditionally, plants have
been used for

– testing toxicity of soil by inhibition of germination and plant growth;

– plant injury by destruction of cellular structures;
– effects of contaminants on plant metabolism, respiration and photosynthesis and
– for testing contaminant uptake.
 Terrestrial toxicology 243

Intact plants or plant tissue can be used for soil testing. ASTM methodology
for terrestrial plant toxicity testing lists nearly 100 plant taxa that have been used in
phytotoxicity testing (Davy et al., 2001). The most commonly used test plants for soil
testing are:

– Agrostis gigantea, redtop, a perennial grass from the monocotyledonous family

Poaceae;
– Allium cepa, onion, family Liliaceae, sensitive to herbicides;
– Amaranthus retroflexus, redroot or pigweed, Amaranthaceae family, a flowering
plant, sensitive for herbicides;
– Arabidopsis thaliana, thale cress, Brassicaceae family, sensitive response by
reproduction;
– Artemisia filifolia silver sagebrush or sand sage Asteraceae family, sensitive for
herbicides;
– Avena sativa, oat, monocot cereal from the family Poaceae;
– Beta vulgaris, sugar beet, a core eudicot for the family Chenopodiaceae, a
monoculture plant, sensitive to herbicides;
– Brassica napus, rape, close relative of cabbage and mustard, family Brassicaceae.
It is also used in standardized tests for measuring plant growth inhibition.
– Brassica rapa, turnip field mustard or turnip mustard is a plant widely cultivated
as a leaf vegetable, a root vegetable (see turnip), and an oilseed (rapeseed oil). It is
used for the study of whole life-cycle assessment and other biochemical research.
– Cucumis sativus, cucumber, an eudicot from family Cucurbitaceae;
– Glycine max, soybean, a eudicot from family Fabaceae, representative of nitrogen-
fixing plants in symbiosis with rhizobia;
– Helianthus annuus, sunflower, Asteraceae family, sensitive response by growth
and reproduction;
– Ipomoea purpurea, morning glory, flowering plant from the family Convolvu-
laceae;
– Lactuca sativa, lettuce, a temperate annual or biennial plant of the daisy family
Asteraceae. It is very sensitive to some herbicides.
– Lolium perenne, perennial ryegrass, family Poacea, sensitive to herbicides and
plays role in the wild food chain;
– Medicago sativa, alfalfa, family Fabacea;
– Phaseolus vulgaris, garden bean, family Fabaceae, sensitive plant;
– Pisum sativum, pea, an eudicot from family Fabaceae, sensitive to herbicides as a
non-target plant;
– Raphanus sativus, radish, an edible root vegetable of the Brassicaceae family
– Sinapis alba, white mustard, an eudicot from family Brassicaceae, the most
widespread test plant used for germination, root and shoot-growth testing as well
as for the testing of bioaccumulation (Figure 5.5).
– Solanum lycopersicum, tomato, family Solanaceae, sensitive to herbicides;
– Sorghum species, many of them, such as energy grasses, broom sorghum,
or broomcorn and sorghum for biofuels are products of industrial agriculture
(Figure 5.6). These may be cultivated on deteriorated or contaminated land under
controlled conditions.
– Trifolium repens, white clover, family Fabaceae;
244 Engineering Tools for Environmental Risk Management – 2

Figure 5.5 Sinapis alba seedlings, just germinated from seeds on soil and different plants grown on a
soil containing agar plate in a laboratory soil test.

Figure 5.6 Sorghum sudanense (energy grass), Sorghum vulgare (broomcorn) and Zea mays (maize) in a
contaminated soil remedial field experiment.

– Triticum spp., wheat, a monocot cereal from family Poaceae;

– Zea mays, maize or corn, a monocot from Poaceae family (Figure 5.6).

Markwiese (2001) gives four pieces of advice regarding the use of plants, their
seeds, standardized and non-standardized species:

– When purchasing seeds of non-standard test species from landscaping companies,

it is important to gather as much information as possible about the seed lot and
to buy enough seeds from the same lot for running the experiment.
– While collecting seeds or other plant material from the field, it is critical that only
seeds, seedlings or cuttings from the species of interest be collected. Details regard-
ing location, field conditions time in growing season, and potential confounding
factors (e.g., previous pesticide and/or herbicide use in the area) must be recorded
for the collection event.
– Selection of species for testing needs pretesting seed germination and early seedling
growth. The following recommendations should be considered:
 Terrestrial toxicology 245

◦ as a reference toxicant: an effective and nonselective biocide, e.g. boric acid,

◦ as control soils: artificial and soils collected from the field,
◦ as end points: mortality, shoot length, root length, shoot wet mass, root wet
mass, and shoot and root dry mass.
– Data evaluation and interpretation of phytotoxicity tests on relatively unstudied
species must be supported by detailed ecological characterization (ASTM, 1998) to
be able to distinguish ecotoxicological effects specific to the species. Tests should be
accomplished by running rigorous control tests with enough replicates to achieve
the desired statistical test power.

In addition to toxicity assessment, plant growth tests are applied to see if the
planned plant is able to grow and function on a certain deteriorated or contaminated
soil.
The following can be used as end points of plant tests:

– Germination rate;
– Root or shoot growth, root length and plant height;
– Biomass quantity: wet and dry mass;
– Photosynthetic activity, simplest way via chlorophyll content;
– Respiration rate;
– Enzyme activities;
– Metabolites specific for selected metabolic pathways;
– The concentration of the contaminating and accumulated chemical substances in
different parts or in the whole plant;
– Plant reproduction: both quantitative and qualitative indicators are useful end
points;
– Chromosome aberration;
– Biomarkers such as antioxidant enzyme activity can be sensitive indicators of
toxicant-related stress;
– Abnormal plant growth, morphology, flowering.

Plants may show a very specific response to environmental stress; this is why their
use as laboratory test organisms often fails: they do not represent the average of the
plant community, rather the opposite, they give unique and specific responses, not
suitable for generalization.
With the increasing information on plant functional genomics, the association
between ecological functions and molecular stress responses, genes or gene products
can be mapped and utilized as a basis for molecular ecotoxicology. The identification
of signaling substances, enzymes and genes in plants involved in any plant response
to contaminants or reaction against pathogens, xenobiotics or any other environ-
mental stressors increases the number of bioindicators of plant origin to be used in
environmental toxicology. Some of these are listed below:

– Detoxification: plant glutathione S-transferase enzyme family play a role in the

response on oxidative stress and detoxification. Glutathione and glutathione
S-transferase participate not only in antioxidative and detoxification reactions
246 Engineering Tools for Environmental Risk Management – 2

but also in redox regulation of the expression of protective genes in infected cells
(Edwards et al., 2000; Sandermann, 2004).
– Defense response: cross-talk between ethylene and jasmonate signaling pathways
determines the activation of a set of defense responses against pathogens and her-
bivores in plants (Lorenzo et al., 2003). Ethylene and jasmonate synthesis and
release from plants can be used as ecotoxicological end point.
– Phytosensing pathogens: plants possess defense mechanisms to protect against
pathogen attack. Inducible plant defense is controlled by signal transduction path-
ways, inducible promoters and cis-regulatory elements corresponding to key genes
involved in defense, and pathogen-specific responses. Identified inducible promot-
ers and cis-acting elements could be utilized in plant sentinels, or phytosensors,
by fusing these to reporter genes to produce plants with altered phenotypes in
response to the presence of pathogens. Pathogen-inducible genes as well as those
responsive to the plant defense signal molecules salicylic acid, jasmonic acid, and
ethylene are suitable stress indicators (Mazarei et al., 2008).
– Salicylic acid and nitric oxide play a role in programmed cell death and induced
resistance (Sandermann, 2004).
– Cis-regulatory elements (cis-elements are present on the same DNA molecule as
the gene they regulate) and transcription factors regulating gene promoters play a
role in response to environmental stress, e.g.
◦ Ozone-induced gene regulation (ethylene);
◦ Xenobiotic-responsive cis-elements;
◦ Activation of DNA sequences by heavy metal ions, etc.

2.3 Soil fauna members as test organisms

Soil fauna together with soil microorganisms are responsible for the harmonic
operation of mineralization and element cycling in the soil.
Invertebrates of the soil fauna regulate the decomposition process by preparatory
mincing of dead organic matter – also called detritus – before mineralization by soil
microorganisms. They play an important role in soil, in spite of the fact that soil
invertebrates count for less than 10% considering respiration and energy production
in terrestrial environments. In addition to initialing decomposition, they are essential
in predating and controlling the decomposer microbiota. Soil-dwelling invertebrates
together with the biodegrading microbiota are responsible for mineralization in soil
participating in the short turnover of nutrients in terrestrial systems.
The use of invertebrates for soil testing is dated for the beginning of the 1990s, as
it is indicated by the book of Løkke and Van Gestel (1998) on soil vertebrates toxicity.
Laskowski et al. (1998) selected the test organisms for testing in the same book.
Soil animals, suitable for adverse-effect testing may belong to the soil micro-, meso-
or macrofauna. The most important groups and some individuals are introduced in
the following.
Soil microfauna covers single-cell animals such as amoeba and ciliophora. They
are better known as inhabitants of waters but they also show high diversity in soils.
They contribute to mineralization and, as key members of the food web, to feed-
ing mineralizing soil microflora. Foissner et al. (2002, 2003) have found huge ciliate
diversity in Austrian forest soils as well in in arid areas of Namibia.
 Terrestrial toxicology 247

Tetrahymena, the best-known unicellular animal test organisms, have been used
for biochemical studies for many years. They fulfill all the requirements to be met by test
organisms: they are easy to maintain and grow, genetically stable and their genome
is well-known. They represent animal cells regarding biochemistry, physiology and
metabolism. The authors of this chapter have developed a soil toxicity test method
using the ciliate Tetrahymena pyriformis for soil extracts and for soil suspension with
direct contact between solid soil and Tetrahymena cells. They are described in Chapter
4 as the aquatic test organisms, but they can also be applied for soils and soil leachates.
Nematoda are the most frequent organisms on the earth, four of every five mul-
ticellular animals on the planet are nematodes. These organisms occupy any niche in
terrestrial environments that provides an available source of organic carbon (Bongers
& Ferris, 1999).
Nematodes are of high ecological relevance at contaminated sites because their
permeable cuticle provides direct contact with soil contaminants in their microenvi-
ronment (Bongers & Ferris, 1999). They occupy key positions in the soil food web; they
can be placed into four trophic groups: bacterivores, fungivores, omnivore predators,
and plant feeders (Zak & Freckman, 1991).
Acute nematode tests were developed by Vainio (1992) and Donkin and Dusen-
berry (1993) with the end point of survival and parasitism. Kammenga and van Koert
(1992) worked out a nematode reproduction test and calculated both EC50 and NOEC
values from the measured data. The nematode chronic toxicity test of Niemann and
Debus (1996) is based on the measured end point of abundance.
In addition to mortality, the ratio of adult to juvenile nematodes, termed the
maturity index (MI), is also useful for characterizing soil stressors (Bongers & Bongers,
1998). The MI has been shown to be a sensitive metric for monitoring natural and
anthropogenic stressors for certain nematode populations (Yeates & Bongers, 1999).
Caenorhabditis elegans is a free-living, transparent nematode, about 1 mm in
length. It lives in temperate soil environments, used extensively as a model organ-
ism. C. elegans was the first multicellular organism to have its genome completely
sequenced.
Panagrellus redivivus, sour paste nematode, a free-living species, is a tiny round-
worm about 50 µm in diameter and 1 mm in length as shown by the microscopic
photo in Figure 5.7. It is widely used in aquacultures as food for a variety of fish and
crustacean species. This microworm has also been used in genetic studies.
Pristionchus pacificus is similar to Caenorhabditis elegans in many points of view,
but differs in one important feature; similar to all species of the family Diplogastridae:
it has an embryonic molt.
Soil mesofauna consist of bigger size animal organisms than the microscopic
organisms of the microfauna. Four groups are discussed in detail: Tardigrada, Acari,
Collembola and Enchytraeidae.
Tardigrada is a phylum of 1–1.5 mm dwelling in water and soil and occurring over
the entire world. Tardigrades have a cylindrical body and eight legs, their movement is
typically slow, similar to that of the bear, this is why they are also called water bear or
moss piglets (Figure 5.8). They live on lichens and mosses, hunting nematodes, rotifers
and protozoans, but also consuming plants. They can pierce both animal and plant
cells by their sharp-edged stylets and suck out the cell fluid. They are highly adaptive
to extreme environmental conditions.
248 Engineering Tools for Environmental Risk Management – 2

Figure 5.7 Microscopic view of Panagrellus redivivus, a free-living nematode.

Figure 5.8 3D Tardigrade Echniscus granulatus, also called water bear or moss piglet (European Atlas of
Soil Biodiversity, 2010).

Acari, mites and ticks are microarthropods, a diverse group of arthropods which
are generally omnivorous and reduce the size of dead organic matter fragments.
There are fungal feeders like oribatids (moss mites or beetle mites) and fungiphagous
prostigmatid mites amongst them. Mites meet several requirements of good laboratory
 Terrestrial toxicology 249

Figure 5.9 Hypoaspis aculeifer, a new test organism in soil toxicity testing (CEH, 2013).

Figure 5.10 Microscopic mite from a bird’s nest, plant pathogen microscopic mite and velvet mite.

test species such as ease of handling, measurable reproductive output, and monotypic
genomic structure. Testing requires low cost, average laboratory and small space. Acute
toxicity and inhibition of reproduction can be measured on the soil mite, Hypoaspis
(Geolaelaps) aculeifer (Figure 5.9), and the predator mites of Typhlodromus pyri and
Phytoseiulus persimilis. Hypoaspis aculeifer is used in the standardized tests of OECD
TG 226 (2008). Oppia nitens, an herbivorous and fungivorous oribatid mite, lives in
Canadian soils and is used in soil tests on survival and reproduction. Figures 5.10 and
5.11 show some mite and tick species.
Van Gestel and Doornekamp (1998) used the oribatid mite of Platynothrus peltifer
in the test developed for soil testing. Survival, reproduction and feeding rate was
measured and the results given as LC50 , EC50 and NOEC. Krogh (1995) developed a
sublethal test using a predatory mite, and measured its survival and reproduction.
250 Engineering Tools for Environmental Risk Management – 2

Figure 5.11 Sheep tick (Bartz, 2009), common blood sucking tick well fed and hard deer tick.

Collembola or springtails are small, wingless microarthropods that live primarily

in the soil litter layer. Along with the mites, collembola make up the majority of
the soil litter arthropod fauna. Soil collembola are primarily detritivorous, living on
partially decomposed vegetation or on the soil microflora, especially fungi. Collembola
contribute to soil organic matter breakdown and nutrient cycling by shredding organic
matter into fragments, feeding on humus and feces in the litter, and feeding on decaying
roots (Bardgett & Chan, 1999).
Collembola shown in Figure 5.12 exhibit significant morphological differences.
Folsomia candida, the little white springtail, highly adapted to life below the ground:
no visual apparatus, no pigmentation, short appendages. Isotoma violacea is at a
lower level of adaptation: has developed visual apparatus, pigmentation and longer
appendages. Collembola have become popular test organisms in toxicity bioassays
because they are easy to maintain in laboratory cultures, have uniform genomic
structure (many species are parthenogenic), and have short generation times.
Folsomia candida has been selected as a test organism for soil chronic (reproduc-
tion) toxicity testing by OECD (OECD TG 232, 2009) and ISO (ISO 11267, 2014).
Folsomia candida reprotoxicity test is generally more sensitive than acute earthworm
mortality tests (Crouau et al., 1999). The OECD test guideline characterizes Folsomia
candida and Folsomia fimetaria as soil-dwelling. Collembola is an ecologically relevant
species for ecotoxicological testing. Their thin exoskeleton is greatly permeable to air
and water, and they represent an arthropod species with a different route and rate of
exposure compared to earthworms and enchytraeids (OECD TG 232, 2009).
Enchytraeidae, potwurm, iceworm, or whiteworm belongs to a group of tiny
worms popular as live food cultured by aquarists, also used as laboratory test
organisms in ecotoxicity testing.
Enchytraeus albidus or whiteworms are one of the most popular cultured worms.
Enchytraeus albidus Henle 1873 is the test organism of the OECD test guideline
(OECD TG 220, 2004). In addition to testing hardly dissolvable chemicals in arti-
ficial soil, ISO 16387 (2004) gives recommendations on the testing of soil quality
by the modification of the original method. Enchytraeus buchholzi is another species
applied to laboratory tests.
Soil macrofauna is the group of soil-living animals which are visually observable
such as earthworms, insects, spiders, etc.
 Terrestrial toxicology 251

Figure 5.12 Springtails Entomobrya nivalis (Duine, 2014), Isotoma viridis (Duine, 2011) and Isotomurus
unifasciatus (Duijne, 2013).

Figure 5.13 Allolobophora chlorotica (soil-living earthworm) and Eisenia veneta (compost earthworm)
(Earthworm, 2013).

Lumbricidae, earthworms are the priority animal test organisms for soil and
compost. In many countries soil ecotoxicity data are limited to earthworms. In the
beginning it was used for toxicity screening of hazardous waste sites and US EPA
endorsed this test with Eisenia foetida. It has been studied extensively, producing a
large data set on the toxicity and bioaccumulation of a number of compounds. Other
species used as test organisms in laboratory and field tests are Eisenia andrei and
Lumbricus terrestris. Figure 5.13 shows Allolobophora chlorotica, an endogeic
soil-living earthworm and Eisenia veneta, a compost earthworm (Earthworm, 2013).
Arthropods are widely applied in soil toxicity testing both in the laboratory and
field tests. Different phyla and classes belong to soil-living Arthropods such as

– Class Arachnida (Chelicerata) with spiders, mites, ticks, scorpions, allies

– Class Malacostraca (Crustacea), order: Isopoda
– Class Diplopoda, millipedes, (Myriapoda)
– Class Chilopoda or centipedes (Myriapoda)
– Class Insecta, insects (Hexapoda), with beetles, crickets, flies, bees and wasps,
ants and termites, etc.

Spiders play an important role in biotic interactions in terrestrial systems so that

they have been the focus of several toxicant impact assessments in recent years. In
a review of invertebrate ecotoxicological test systems, Van Gestel and Van Straalen
(1994) note that spiders seem to be a particularly sensitive group among arthropods.
252 Engineering Tools for Environmental Risk Management – 2

Pardosa, a large number of species of the wolf spider genus are used in standardized
tests by the International Organisation for Biological Control (IOBC, 2000) as non-
target organism affected by pesticides.
Beetles and their larvae play an essential role in soil by aerating the soil, pollinat-
ing blossoms, controling insect and plant pests and decompose dead materials. The
larva is usually the principal feeding stage of the beetle life cycle. Larvae tend to feed
voraciously once they emerge from their eggs. Some feed externally on plants, such as
those of certain leaf beetles, while others feed within their food sources.
Poecilus cupreus and other carabid beetles have been the focus of several toxico-
logical investigations (Heimbach et al., 1994; Heimbach & Baloch, 1994). Heimbach
and coworkers used carabid beetles to evaluate pesticide-contaminated soils by caging
the beetles in field enclosures. Staphylinid beetle Aleochara bilineata was used in the
survival and reproduction tests developed by Samsøe-Petersen (1992).
Dung beetles are the most abundant species among organisms forming a complex
food web in livestock dung (Hanski & Cambefort, 1991). Other members of the dung
community include mites, nematodes and annelids.
Aphodius constans and Onthophagus taurus dung beetles (Scarabaeidae) are
applied in a new OECD laboratory test, for veterinary pharmaceuticals, especially
parasiticides. The test organism is planned to be applied not only in laboratory but
also in field toxicity assessments and monitoring (OECD, 2008)
Diptera, flies and its larvae are used for toxicity and developmental toxicity testing
of chemicals substances and also for the dung itself originating from livestock treated
with the chemical.
Scathophaga stercoraria and Musca autumnalis dung flies are considered to be
suitable indicator species for estimating the developmental toxicity of parasiticides
on dung-dependent Diptera because the species cover a wide geographic range. Both
species are dung-dependent, multi-voltine, do not undergo obligate diapause and are
easy to culture and have a short life cycle which makes it possible to determine effects
on development and survival in the laboratory (OECD TG 228, 2008).
Cockroaches belong to Neoptera (Insecta) and are applied in non-standardized
tests in terrestrial toxicology. Blattella germanica is one of the best known household
species. It is relatively small, about 1.3 cm to 1.6 cm long; although it has wings, it is
unable to sustain flight. German cockroach is omnivorous and a scavenger.
Crickets are representatives of macroarthropod detrivores (harmonize with
p. 231) in the order Orthoptera and class Insecta. In fact, crickets are known to
exhibit a high degree of cannibalism (Crawford, 1991). Crickets are widespread in
most terrestrial systems. These organisms serve a valuable purpose by consuming and
processing plant litter and are prey for other animals. Acheta domesticus, house cricket
and Gryllus pennsylvanicus, field cricket belong to Gryllinae.
Social insects within the Hymenoptera order such as wasps, bees and ants are
popular test organisms both in ecological and contaminated site assessment and biocide
and pesticide hazard assessment.
Wasps play an important role in natural control of pest insects and are therefore
important in food chains. Parasitic wasps are applied in biological plant protection
mainly in foliage. Their sensitivity to pesticides is an important characteristic, so
that they are applied to testing the effect of pesticides as non-target insects. Aphidius
 Terrestrial toxicology 253

rhopalosiphi, Trichogramma cacoeciae and Encarsia formosa are popular parasitic

wasps, available in commerce.
Bees, for example the best-known Apis mellifera grown by beekeepers and api-
aries, are good sources of well-kept, homogenous populations of bees. For test
purposes, young adult worker bees of the same race should be used, from adequately
fed, healthy, as far as possible disease-free and queen-right colonies with known history
and physiological status. They could be collected in the morning of use or in the evening
before test and kept under test conditions until the next day. Bees treated with chemical
substances, such as antibiotics, anti-varroa, etc., should not be used for toxicity test
for four weeks from the time of the end of the last treatment (OECD TG 213, 1998).
Ants and termites from the Formicidae family are valuable bioindicators of
environmental quality. Relevant attributes of ants include:

– Active throughout most of the year;

– Relative abundance;
– High species richness;
– Many specialists;
– Occupation of higher trophic levels;
– Easily sampled;
– Easily identified (Lobry de Bruyn, 1999; Whitford et al., 1999).

Termites are generally the primary detritivores in warm desert systems, actively
consuming standing dead vegetation, plant litter and feces. In desert grasslands, ter-
mites consume 50 percent or more of all photosynthetically fixed carbon (Whitford,
1991). Termites also serve as an important food source for higher trophic levels. In less
humid soils the role of earthworms is taken over by termites, ants, and tenebrionids
(Van Gestel & Van Straalen, 1994).
Isopoda, crustaceans, include familiar animals such as woodlice and pill bugs.
They can consume in excess of 35% of their body weight in a single 24-hour period
in laboratory cultures. They are able to survive for up to 180 days without food
(Drobne & Hopkin, 1994). Isopods are considered to be good candidates as standard
test species because they are common, easy to handle, and generally respond quickly to
environmental contamination (Paoletti & Hassall, 1999). Several common species are
readily available from commercial marketers of biological educational material, and
gives ease of culturing with better-understood species e.g., Porcello scaber and Oniscus
asellus. Drobne et al. (2002) found the microflora of Porcello scaber as suitable end
point for toxicology testing. Living species such as Trachelipus rathkii and Armadil-
lidium nasatum can be studied in gardens and greenhouses as non-target organisms
of pesticides and biocides. Hornung et al. (1998) developed both a growth test with
the end points of survival, growth and feeding rate and a reproduction test with the
end points of survival, reproduction and oorsoption.
Diplopoda (millipede) exposed to high metal contamination accumulate toxic
metal in their intestine tissues. They have been studied as indicators of high risks
due to metals (Köhler et al., 1995).
Chilopoda (centipede) are able to accumulate not only metals but also persistent
pesticides and other persistent organic substances. The assimilation of zinc, cadmium,
254 Engineering Tools for Environmental Risk Management – 2

lead and copper by the centipede Lithobius variegatus was studied (Hopkin & Martin,
1984). Lithobius mutabilis lives in Central Europe; it plays an important role as a
dominant epigeic predator especially in woodlands.
Gastropoda (molluscs and slugs) are the biggest animals in size being in intensive
contact with soil through feeding and skin contact. They are used not only in toxicity
testing but also in behavior tests, such as avoidance and herbivory. Slug Arion rufus
L. proved itself to be sensitive in contaminated soil showing differences in herbivory
(form of predation in which plants are consumed) (Mench & Bes, 2009).
Vertebrates, the higher members of the terrestrial macrofauna, primarily birds and
mammals, have been used both for environmental and human toxicity assessments.
The results of the tests developed for the evaluation of human health effects can also be
used for the estimation of the environmental/ecological effects of industrial chemicals
and pesticides already tested. Properly selected mammalian end points can serve for
both human health and ecological risk assessment, and thus fulfill the requirement
for minimizing animal testing. Animal welfare consideration fosters non-destructive
techniques in vertebrate testing such as biochemical markers.
Birds represent both terrestrial and water ecosystems and food chains. Avian tests
are required for pesticide registration and play a role in food chain assessments and bird
species protection. They are easy to cultivate with the possibility to test reproduction
via the eggs. OECD TG 205 (1984) recommended more bird species for testing of
chemicals such as:

– Anas platyrhynchos, mallard duck;

– Colinus virginianus, bobwhite quail;
– Columba livia, pigeon;
– Coturnix coturnix japonica, Japanese quail;
– Phasianus colchicus, ring-necked pheasant;
– Alectoris rufa, redlegged partridge.

Mammals typically living in soil are mole and rodents, which are exposed via res-
piration, skin contact and nutrition, like all the other smaller organisms; nevertheless
they are able to escape from deteriorated or contaminated soil to the neighborhood
or to a farther distance. In some cases higher terrestrial wild animals are also targeted
receptors of pollution.
Mole, rodents and higher wild animals (Figure 5.14), for example small mam-
mals, which sensitively react to environmental stress (Elliott & Root, 2006; Torre et al.,
2004), are suitable organisms for field assessments and diversity and abundance eval-
uation of ecosystems. Ecological assessment methods (EAMD, 2013) also recommend
and assess mammals, including endangered species in areas of ecological value.

3 MEASURING TERRESTRIAL TOXICITY: END POINTS AND

METHODS

For testing fate, behavior and adverse effects of chemical substances, or to measure
the adverse effect of contaminated soil, many of the soil-living micro-, meso- and
macro-fauna and -flora members can be used.
 Terrestrial toxicology 255

Figure 5.14 Rat, mole (Hill, 2005) and prairie dog (Weiss, 2011).

Soil biodiversity and the changes in the density and distribution of species indicate
and characterize truly soils’ health or deterioration. On the other hand the complete
assessment of all species, their density and distribution in the soil is not possible. The
solution is that only some indicator species are assessed for their density and share and
by comparing these results to the diversity of the healthy soil, the scale of deteriora-
tion can be estimated. Further methodological simplification is the use of well-known,
healthy and uniform test organisms instead of naturally occurring endemic ecosystem
members, and controlled conditions instead of the non-controllable and heterogenic
natural circumstances. The use of bioassay may exclude many ‘natural’ uncertainties
which may overrule the loss in environmental reality of the bioassays compared to the
natural environment. The concept of characterizing soil species diversity in its com-
plexity can be fulfilled by studies on the metagenome and related molecules extracted
from soil. Through metagenome, metatrascriptome and other ome classes (proteome,
metabolome) arising from whole soil, the soil community can be considered as one
entire ‘organism’, a dynamic and adaptive biological system. With the development
and perfection of the molecular techniques the interpretation of the results will also
make a step forward, creating an efficient ecotoxicological tool.

3.1 Soil biodiversity

Soil biodiversity assessment is a practical tool that can characterize soil as a natural
habitat. Biodiversity assessment is based on mapping either the total ecosystem, or
part of it or only some characteristic bioindicator species.
Soil species diversity and their changes would be the most appropriate end point
for the characterization of environmental health, but our knowledge and our financial
and labor potential is not enough for regular and comprehensive ecosystem monitor-
ing. The correlation between spatial and temporal ecological diversity changes and the
production and use of chemicals could be the ideal tool for the risk assessment, identi-
fication and management of chemical substances, but only highly reduced surrogates
are available today.
Characterization of the total ecosystem – including soil-living microorganisms,
plants and animals – can hardly be achieved using traditional tools such as counting the
individual organisms, and calculating the species distribution after their identification.
Investigation of the metagenome (total DNA) of soil biota is an innovative molecular
256 Engineering Tools for Environmental Risk Management – 2

tool for the ecosystem as a whole and it is suitable for the characterization of following
changes at the DNA and RNA levels.
Characterization of soil biota and microbiota requires high expertise and experi-
ence. Reduction of the ecosystem to a part or even to some indicator organisms is a
major simplification, which may deform the picture as a whole. Another conceptual
problem is that a target is difficult to specify because it is hard to define what normal
is, what can be used to characterize the diversity of a healthy ecosystem. Of course
major changes, large-scale deteriorations can be easily observed, but not the early signs
which are important for efficient risk prevention.
Soil community has a strong impact on soil processes and on the changes of these
processes in time and space. The foodweb – the structure and interactions between
the groups of soil-living organisms – reflects both the dynamics of populations and
the dynamics in material, energy, and nutrient cycles but sustainability (stability and
biodiversity) cannot be characterized based on food-web models alone. The model that
truly characterizes soil sustainability must describe the interaction between the living
community and the soil’s function. It also must include functional redundancy i.e. the
soil’s capability to continue to function even if the species composition changes e.g. a
species disappears and another takes over its place in the food web.
Another problem is that the diversity index, the result of the complex ecologi-
cal assessment, cannot be integrated into the contaminant-specific quantitative risk
assessment scheme. The reason is that the results of biodiversity assessments are
not quantitative values, but indexes on a relative scale not in direct relation with
contaminants and their concentrations but with the aggregate of unidentified impacts.
Classification of soil biodiversity measurement methods:

– Direct observation and counting visually or by microscopy;

– Functional tests:
◦ Enzyme analysis, proteome and activities;
◦ Metabolic activity profile, metabolome analysis;
◦ Carbon-source utilization profiles;
◦ Protein analysis, proteome analysis using proteomics;
◦ Functional gene arrays.
– Taxonomic tests:
◦ Nucleic acid fingerprinting techniques, genomics and transcriptomics;
◦ Cloning;
◦ Gene arrays, hybridization;
◦ Massively parallel sequence analysis;
◦ Phospholipid fatty acid analysis (PLFA).

The biodiversity assessment based on counting and identification can be applied to

macroplants and the members of meso- and macrofauna but not to soil microorganisms
because most of the soil-living microorganisms cannot be isolated and cultivated under
laboratory circumstances. Molecular techniques such as the analysis of the genome
(DNA), the transcripted RNA, proteins, the active gene-products and any metabo-
lite of the biochemical processes of the organism can be applied without isolating the
organism. These methods can be used for individuals, for populations or for the whole
community without isolating any of the microbes from the soil, but analyzing the
whole genome, the whole RNA (transcriptome), the whole of proteins (proteome) or
 Terrestrial toxicology 257

Figure 5.15 Enviromics: the study of envirome, the total complement of environmental characteristics,
conditions, and processes required for life.

metabolites (metabolome) isolated directly from soil. Another possibility is the cloning
of the metagenome and the analysis of the gene products expressed by a host cell, e.g.
E. coli or yeast. Screening phylogenetic and functional marker genes certainly fulfills
the requirement of ecotoxicology. The “only’’ information still needed is the list and
combination of genes which corresponds to the functional or taxonomic indicator
characteristics. “Enviromics’’ is the special study of environmentally critical genes and
chemicals. It tries to collect all the environmental components and their genetic pairs
(Omics, 2014; Genomic Glossaries, 2014) and to reflect on the interaction between
the environment and the inhabiting community: how the genome dwells within the
environment, and how the genomic expression shapes and is shaped by the environ-
ment (Anthony, 2001). Figure 5.15 shows an overview of the soil “-omes’’ and the
omics techniques.
The soil microbiota is extremely complex and variable, and the detection of genes
and characterization of the genome and other “omes’’ such as transcriptome, pro-
teome, and metabolome in soil has increased our knowledge of soil microbiota. It
became possible to study unculturable microorganisms and their possible functions,
metabolic pathways, and the relationship between composition and activity of soil
microbiota. Therefore, omics application in soil sciences is advancing and spreading
rapidly in spite of the difficulties of DNA and other ome extraction from soils. The
new book by Nanniperi et al. (2014) gives a comprehensive overview of the state
258 Engineering Tools for Environmental Risk Management – 2

of the art in the science of the omes of soil and omics techniques suitable for soil,
focusing on:

– The potential applications and methodological problems;

– Identifying marker genes that play a role in soil microbial ecology,
– Gene expression in a particular environment and its product, the metatranscrip-
tome, the sum of the transcribed genes in contrast to metagenome (covering all
existing genes, including inactive ones);
– Soil volatilomics, dealing with the analysis of the multitude of volatile organic
compounds produced, stored or degraded in soils;
– Poteomics and proteogenomics for a better description of protein diversity in soil
microbiota;
– The MEtaGenome ANalyzer (MEGAN), a program for analyzing different ome
molecules and providing a deep insights into ecosystem functioning by aggregating
all the above-mentioned concepts and methods.
– All of the functional and taxonomic methods characterizing biodiversity need a
sophisticated statistical evaluation and a good concept to find or create a suitable
reference.

3.2 Evolutionary convergence phenomenon

An interesting and important relationship is described by the Evolutionary Conver-
gence Phenomenon, which helps to understand evolutionary processes that influence
soil diversity and the phenotypes of species. Traditionally, convergence means the inde-
pendent evolution of similar phenotypes by distant genealogical species. Convergence
can be explained by adaptation to similar environments by adjusting phenotype and
morphology. For example, if a species is divided or separated and then the separated
part adapts to aquatic or terrestrial conditions, its morphology will differ greatly from
the original after a period of time. The process of adaptation to living conditions in
soil may result in a high level of convergence. For example, the visual apparatus of
soil-dwelling organisms is reduced, pigmentation is lost and the size of appendages is
reduced, while special structures essential for life below the ground such as chemo-
and hydroreceptors are distributed diffusely on the body, not only in the oral region.
The scale of adaptation to the soil by losing and acquiring special morphological
and phenotypical markers indicates the extent of stability of the habitat. In a stable
soil environment diverse factors such as water, temperature and organic matter vary
only slightly over the short and medium term, as compared to large variations in
above-ground environments. There is obviously no light in soil at depths greater than
a few millimeters. As a result of all of these factors combined, edaphic (living in soil)
microarthropods are sensitive and unable to survive abrupt variations in environmental
factors. They are particularly sensitive to soil degradation and to the disturbances
caused, for example, by agricultural cultivation and trampling.
Because of the great variation and the difficulties in interpreting the biodiversity
assessment results of healthy or contaminated and deteriorated soils, the simplified
model of bioassays is a good alternative. Instead of a complete biodiversity assessment,
the results obtained on test organisms of a minimum of three trophic levels allow a pre-
diction for the whole soil ecosystem, although with high uncertainty. Bioassay results
 Terrestrial toxicology 259

are suitable for risk assessment and decision making about the necessary intervention
for a deteriorated or contaminated soil. In some cases, direct decision on risk reduction
or soil use can also be made.
Field assessment and bioassay application involve microcosms and mesocosms
which can simulate the most important characteristics of the real environment, but are
still easy to control and monitor. The microflora of the soil can be observed both by
traditional microbiological or by new molecular techniques such as metagenomics and
transcriptomics.

3.3 Terrestrial bioassays for testing chemical substances and

contaminated soil
Laboratory bioassay measures chemically induced adverse effects on soil organisms,
including microorganisms, soil-living micro-, meso- and macrofauna, birds or mam-
mals. The study designs include acute systemic, dietary, and reproductive (also known
as ‘life-cycle’) toxicity testing. Figure 5.16 shows the lethal (% death) effect by a soil
artificially contaminated with diesel oil on Collembola. In this test, diesel oil is added
to the soil, the test is run in a temporarily ventilated, closed system, where the actual
concentration of diesel oil can be controlled. But in the case of real contaminated soil,
neither the components of the complex pollutant, nor the selective changes of these
components are known. The end point is a certain amount (mass) of soil which caused
50% decrease in the number of surviving animals. In this case the result is given as gram
soil, – which has no information for the decision makers or managers, only for special
experts. The amount of soil which causes 50% lethality is different in every single test
type, according to the test setup. To be able to interpret the toxicity expressed in gram

Figure 5.16 Collembola’s response as a function of diesel oil concentration in soil.

260 Engineering Tools for Environmental Risk Management – 2

soil, toxicity limit values must be created in soil gram. Subsequently it must be decided
if the measured value is above or under this limit, and the exceedance rate should be
calculated. This value is proportional to the risk characterization ratio (RCR). Another
concept is the application of uniform equivalents, as described in Chapter 9.

4 STANDARDIZED AND NON-STANDARDIZED TEST METHODS

In this chapter we give an overview on the guidelines of soil toxicology test meth-
ods, recommended and standardized by OECD, ISO and other organizations for
standardization.

4.1 OECD standards for testing chemical substances in soil

and dung with terrestrial organisms
OECD guidelines were prepared to support the legislation of chemical substances.
Formerly the Dangerous Substance Directive (1967), nowadays REACH Regulation
(2006) requires the use of the OECD guidelines (Table 5.1).

Table 5.1 OECD guidelines for testing of chemicals by terrestrial tests.

Guideline number Test name

OECD No. 216 (2000) Soil microorganisms: nitrogen transformation test

OECD No. 217 (2000) Soil microorganisms: carbon transformation test
OECD No. 227 (2006) Terrestrial plant test: vegetative vigor test
OECD No. 208 (2003) Terrestrial plant test: seedling emergence and seedling growth test
OECD No. 207 (1984) Earthworm, acute toxicity tests
OECD No. 222 (2004) Earthworm reproduction test (Eisenia fetida/Eisenia andrei)
OECD No. 220 (2004) Enchytraeid reproduction test with several species
OECD No. 232 (2009) Collembolan reproduction test in soil
OECD No. 226 (2008) Predatory mite (Hypoaspis (Geolaelaps) aculeifer) reproduction test in soil
OECD No. 228 (2008) Dung flies (Scathophaga stercoraria, Musca autumnalis) laboratory tests
OECD (2010, draft) Dung beetle (Aphodius constans) laboratory test
OECD No. 213 (1998) Honeybees, acute oral toxicity test
OECD No. 214 (1998) Honeybees, acute contact toxicity test
OECD No. 205 (1984) Avian dietary toxicity test
OECD No. 206 (1984) Avian reproduction test
OECD No. 304A (1981) Inherent biodegradability in soil
OECD No. 307 (2002) Aerobic and anaerobic transformation in soil
OECD No. 312 (2004) Leaching in Soil Columns
OECD GD 54 (2006) Breakdown of organic matter in litter bags in the field**

**Field microcosms.

4.2 ISO and other standards for testing soil and sediment
Various international standardization organizations ensure uniform methods and com-
parable results in soil testing, similarly to other areas of environmental management
and practice. Table 5.2 summarizes the most well-known soil test standards, including
 Terrestrial toxicology 261

Table 5.2 ISO and other standardized test methods for testing soil quality and toxicity (ISO, 2014).

Standard Title of the standard method

ISO 10381-6:2009 Sampling – Part 6: Guidance on the collection, handling and storage
of soil under aerobic conditions for the assessment of microbiological
processes, biomass and diversity in the laboratory
ISO 23611-1-6:2006-12 Sampling of different groups of soil invertebrates
ISO 11063:2012 Method to directly extract DNA from soil samples
ISO/DIS 17601:2013 Estimation of abundance of selected microbial gene sequences by
quantitative real-time PCR from DNA directly extracted from soil
ISO/TS 29843-1:2010 Determination of soil microbial diversity – Part 1: Method by
phospholipid fatty acid analysis (PLFA) and phospholipid ether lipids
(PLEL) analysis
ISO/TS 29843-2:2011 Determination of soil microbial diversity – Part 2: Method by
phospholipid fatty acid analysis (PLFA) using the simple PLFA
extraction method
ISO 14240-1:1997 Determination of soil microbial biomass – Part 1: Substrate-induced
respiration method
ISO 14240-2:1997 Determination of soil microbial biomass – Part 2: Fumigation-extraction
method
ISO 23753-1:2005 Determination of dehydrogenase activity in soils – Part 1: Method using
triphenyltetrazolium chloride (TTC)
ISO 23753-2:2005 Determination of dehydrogenase activity in soils – Part 2: Method using
iodotetrazolium chloride (INT)
ISO/DIS 10871:2009 Determination of the inhibition of dehydrogenase activity of Arthrobacter
globiformis – Solid contact test using the redox dye resazurine
ISO 16072:2002 Laboratory methods for determination of microbial soil respiration
ISO 17155:2012 Determination of abundance and activity of soil microflora using
respiration curves
ISO 11266:1994 Guidance on laboratory testing for biodegradation of organic chemicals
in soil under aerobic conditions
ISO 14239:1997 Laboratory incubation systems for measuring the mineralization
of organic chemicals in soil under aerobic conditions
ISO 15473:2002 Guidance on laboratory testing for biodegradation of organic chemicals
in soil under anaerobic conditions
ISO 15685:2012 Determination of potential nitrification and inhibition of nitrification –
Rapid test by ammonium oxidation
ISO 14238:2012 Determination of nitrogen mineralization and nitrification in soils and
the influence of chemicals on these processes
ISO/TS 22939:2010 Measurement of enzyme activity patterns in soil samples using
fluorogenic substrates in micro-well plates
ISO 11269-1:2012 Determination of the effects of pollutants on soil flora. Part 1: Method
for the measurement of the inhibition on root growth
ISO 11269-2:2012 Determination of the effects of pollutants on soil flora. Part 2: Effects of
chemicals on the emergence and growth of higher plants
ISO 22030:2005 Chronic toxicity in higher plants

(continued)
262 Engineering Tools for Environmental Risk Management – 2

Table 5.2 Continued

Standard Title of the standard method

ISO 17126:2005 Determination of the effects of pollutants on soil flora – Screening test
for emergence of lettuce seedlings (Lactuca sativa L.)
ISO 29200:2013 Assessment of genotoxic effects on higher plants – Vicia faba
micronucleus test
ISO/TS 10832:2009 Effects of pollutants on mycorrhizal fungi – Glomus mosseae spore
germination test
ISO 11268-1:2012 Effects of pollutants on earthworms (Eisenia fetida) – Part 1:
Determination of acute toxicity using artificial soil substrate
ISO 11268-2:2012 Effects of pollutants on earthworms (Eisenia fetida) – Part 2:
Determination of effects on reproduction
ISO 11268-3:2014 Soil quality – Effects of pollutants on earthworms – Part 3: Guidance on
the determination of effects in field situations
ISO 16387:2014 Effects of pollutants on Enchytraeidae (Enchytraeus sp.). Determination
of effects on reproduction
ISO 17512-1:2008 Avoidance test for determining the quality of soils and effects of chemicals
on behavior – Part 1: Test with earthworms (Eisenia fetida and Eisenia
andrei)
ISO 11267:1999 Inhibition of reproduction of Collembola (Folsomia candida) by
soil pollutants
ISO 17512-2:2011 Avoidance test for determining the quality of soils and effects of chemicals
on behaviour – Part 2:Test with collembolans (Folsomia candida)
ISO 20963:2005 Effects of pollutants on insect larvae (Oxythyrea funesta) – Determination
of acute toxicity
IOBC (2000) Non-target arthropod acute and chronic laboratory tests (surface
(Candolfi et al., 2000) dwellers like Aleochara bilineata, Poecilus cupreus, Pardosa spec.)
ISO/DIS 18311:2013 Method for testing effects of soil contaminants on the feeding activity of
soil dwelling organisms — Bait-lamina test
ISO 15952:2006 Effects of pollutants on juvenile land snails (Helicidae) – Determination
of the effects on growth by soil contamination
ISO 10872:2010 Determination of the toxic effect of sediment and soil samples on growth,
fertility and reproduction of Caenorhabditis elegans (Nematoda)
ISO 16191:2013 Determination of the toxic effect of sediment and soil on the growth
behavior of Myriophyllum aquaticum – Growth test
ISO 21338:2010 Kinetic determination of the inhibitory effects of sediment, other solids
and colored samples on the light emission of Vibrio fischeri (kinetic
luminescent bacteria test)

ISO = International Organization for Standardization.

IOBC = International Organization for Biological Control.

new tests on DNA extraction from whole soil, the rapid nitrification test, the new
earthworm reproduction test, the Vicia faba micronucleus test and the simple PLFA
method. The standards cover sampling, chemical methods, including DNA and other
omics technologies, biological methods, laboratory bioassays, simulation methods and
 Terrestrial toxicology 263

field assessment. A special trend is testing soil as a whole using any of the chemical,
DNA or biological and ecological methods.
US EPA (2014) Chemical Safety and Pollution Prevention Harmonized Test Guide-
lines (OCSPP, 2014) has developed a series of harmonized test guidelines for use in the
testing of pesticides and toxic substances in soil and to provide uniform data for the
regulatory and management purposes. Some of them are presented in Table 5.3.

Table 5.3 Some selected soil tests from the US EPA harmonized test-guidelines.

Toxicity in soil

850.3200 (2012) Soil microbial community toxicity test

850.3040 (2012) Field testing for pollinators
850.4000 (2012) Background and special considerations – Tests with terrestrial
and aquatic plants, cyanobacteria, and terrestrial soil-core microcosms
850.4100 (2012) Seedling emergence and seedling growth
850.4150 (2012) Vegetative vigor
850.4230 (2012) Early seedling growth toxicity test
850.4300 (2012) Terrestrial plants field study
850.4600 (2012) Rhizobium-legume toxicity
850.4800 (2012) Plant uptake and translocation test
850.4900 (2012) Terrestrial soil-core microcosm test
850.2500 (2012) Field testing for terrestrial wildlife

Fate and behavior in soil

835.1210 (1998) Soil thin layer chromatography

835.1220 (1998) Sediment and soil adsorption/desorption isotherm
835.1230 (2008) Adsorption/desorption (batch equilibrium)
835.1240 (2008) Leaching studies
835.2410 (2008) Photodegradation in soil
835.3300 (1998) Soil biodegradation
835.4100 (2008) Aerobic soil metabolism
835.4200 (2008) Anaerobic soil metabolism
835.6100 (2008) Terrestrial field dissipation
835.6300 (2008) Forestry dissipation
835.8100 (2008) Field volatility

4.3 Testing waste: a terrestrial test battery for solid waste

Environmental toxicity and other adverse effects of wastes can be tested using three
terrestrial, three aquatic and one genotoxicity test according to the recommendation of
the European Ring Test (correctly: European Interlaboratory Comparison) (Römbke
et al., 2009). The terrestrial tests for wastes are shown in Table 5.4.

5 NON-STANDARD TERRESTRIAL TOXICITY TEST METHODS

Terrestrial habitats and soil differ from aquatic habitats mainly in their intrinsically
heterogenic character. The origin of this heterogeneity is based on the geochemical
264 Engineering Tools for Environmental Risk Management – 2

Table 5.4 Standardized terrestrial tests suitable for solid wastes.

Standard or guideline Title/name of the method

ISO 10871:2008 Solid-contact test using Arthrobacter globiformis ISO NWI – Dehydroge-
nase activity 20%
ISO 11269-2:2005 Determination of the effects of pollutants on soil flora. Part II: Effects of
chemicals on the emergence and growth of higher plants (Brassica napus)
– Biomass 30%
ISO 11268-1:1993 Effects of pollutants on earthworms (Eisenia fetida) Part I: Determination
of acute toxicity using artificial soil substrate – Mortality 20%
ISO 17512-1:2007a Avoidance test for determining the quality of soils and effects of chemicals
on behavior – Part 1: Test with earthworms (Eisenia fetida and Eisenia
andrei)

heterogeneity of the bed/host rock and differences in elevation, positions and conse-
quent exposure to sun, water and wind, etc. Heterogeneity of soil matrix is combined
with the heterogeneity of the communities and individuals using the soil as habitat.
All these mean that site- and problem-specific tests may play a prime role in terrestrial
habitat and soil assessment. Non-standard species may be used to more accurately
represent the functional roles played by local flora or fauna. Non-standard methods,
mainly site-specific microcosms and mesocosms can simulate certain soil situation or
terrestrial ecosystem.
The cost of tests on non-standard species can be higher than standardized ones
due to the additional tasks of defining optimal conditions for the organism’s growth,
establishing the organism’s sensitivity to site-related contaminants, and establishing
control condition responses. These additional costs may be justified by obtaining
surplus information about adverse effects on resident species.

5.1 Some aspects of problem-oriented and site-specific

soil testing
Most of the environmental problems are unique and have an individual risk profile,
which is reflected by the conceptual model. Standardized test methods often fail to
meet the needs of site-specific risk management requiring site-specific test organisms,
or models and test setups that suite to the problem. The problems may include an
area or a certain species to be monitored, contaminated sites to be assessed, or a
contaminant to be detected.
Before planning the assessment or monitoring of an environmental problem, the
aim of testing should be clarified, the main risks identified, the concept of testing, the
selection of field or laboratory methods decided. It should also be decided how to deal
with the soil matrix effects and the soil’s physical phases, whether to apply single- or
multispecies tests, etc. These aspects will be discussed below. Assignment of sampling
and monitoring points is also an important issue and it will be discussed in Chapters 3
and 4 in Volume 3.
 Terrestrial toxicology 265

5.1.1 Soil community response

Any parameter of the whole soil that characterizes soil in general may be suitable for
assessing and monitoring soil activity and health from the environmental point of view.
These can be:

– The concentrations of microbial cells or higher organisms, abundance and

diversity of
◦ aerobic heterotrophic cells;
◦ facultative and obligate anaerobes;
◦ cyanocteria;
◦ plants;
◦ microfungi and macrofungi;
◦ single-cell animals;
◦ multi-cell animals, such as micro- and meso- and macrofauna members:
nematodes, worms, collembolans, insects, snails, vertebrates.
– Characteristics of the metagenome, the chemical, biochemical, metabolic and
physiological parameters of the whole biota of the soil. These indicators use
innovative DNA and other molecular techniques such as specific microscopes,
genomics, proteomics, enzyme analysis, carbon-source utilization profiles, func-
tional gene arrays, phospholipid fatty acid analysis (PLFA), nucleic acid finger
printing techniques, cloning the metagenome, and identifying transcripts, gene
arrays, massively parallel sequence analysis, etc. The following chemical species
can be used as indicators:
◦ DNA, its quantity and quality, the guanine-cytosine (GC) content of DNA and
RNAs;
◦ Structural building chemicals such as structural proteins, muramic acid,
ergosterol, technoic acid, phospholipids, lipopolysaccharides, glucoseamine,
diaminopimelic acid;
◦ Functional proteins, mainly enzymes such as oxidases, glucosidase, sac-
charase, xilanase, proteases, lipases, urease, amidases, esterases (phosphor
mono-, di- and triesterases), cellulases, kitinase, arilsulfatase, etc.
– Functions of living soil, its energy production (ATP), respiration, denitrification,
biodegradation, biosynthesis.
– Dynamic functions: increase or decrease in any of the products or soil func-
tions further to the effect of environmental parameters or stresses such as habitat
deterioration or the presence of contaminants.

There are plenty of chemical analytical, enzymological and biological techniques

for extracting as well as selective methods for direct testing the relevant parameter in
the soil.
In addition to single tests, innovative test batteries and multi-well test systems
do exist, integrating the experience of experts and helping the design, evaluation and
interpretation of measured data.
The dynamic changes in soil activity and function as a response to the presence
of adversely affecting physical, chemical or biological agents can be followed on site
266 Engineering Tools for Environmental Risk Management – 2

or can be simulated or even provoked in microcosms, mesocosms or other type of

dynamic and interactive tests.
The above-listed potential end points can be used in all strategies of soil toxicity
testing.

5.1.2 Concepts for characterizing soil functioning and health

There are two main concepts for the characterization of soil health and its normal
functioning: monitoring absolute values of characteristic indicators, or testing on
representatives and extrapolate to the whole.

– Measuring the absolute values of the selected soil characteristics, or a set of charac-
teristics, meaning the absolute value of cell concentrations, contents and activities.
These results can be compared to the healthy average or to the formerly recorded
results of the same soil. In the case of existing monitoring data the trends can be
characterized by these absolute values. When one gathers data from one single
time-point, the changes due to seasonal, climatic and incidental events are hardly
to be distinguished from the adverse effects of chemicals, therefore the statistical
evaluation of testing will be very weak.
– Testing selected members of the soil community and extrapolation from their
response to the entire soil. In this case organisms from a minimum of three different
trophic levels are selected and tested to exclude soil toxicity or to predict no-effect
values for risk assessment. In such type of soil testing one can select standard test
organisms, which are – with high probability – not relevant to the soil to be tested,
or can use non-standard test organisms well representing the biota of the soil in
question. Both site-specific and generally used or standard test organisms can be
included in test batteries according to the aim of the test. Based on the results of
the laboratory test batteries or of the tested remedied soil, one can decide whether
the soil or waste is hazardous or not.

5.1.3 Aims of testing whole soil response

The two main objectives of whole soil tests are: testing the fate and effect of chemical
substances in soil and testing the adverse effect of contaminated soil, sediment or solid
waste on representative organisms.
There are two main concepts for testing adverse effects of pure chemical substances,
as well as waste and soil contaminated with chemicals:

1 To use the formerly listed soil indicators for measuring the adverse effects of chem-
ical substances in soil environment, the chemical substance is added in different
concentrations into a well-defined and stable natural soil, containing the origi-
nal microbiota (the ‘test organism’). From the response of the spiked soils, one
can identify the highest no-effect concentration of the chemical substance in the
investigated soil (NOECin soil ), the lowest-effect concentration (LOECin soil ) or the
concentration resulting in 50% or any other percentage inhibition in the measured
soil characteristic.
 Terrestrial toxicology 267

Using this concept the huge adaptive and toxicity buffering capacity of the soil can
be experienced i.e. by comparing test results with chemical substances dissolved
in water.
2 Standard healthy soil may be suitable for testing solid waste or contaminated soil,
mainly in such cases when mixing waste or contaminated soil into healthy soil (e.g.
into agricultural soil) is a true model of a real scenario, e.g. waste disposal on soil
or soil amendment using compost, etc. In this concept contaminated soil/solid
waste is mixed into healthy soil in incremental concentrations and the effect of
the mixture on the relevant soil activity is measured as an end point. In this con-
cept soil is used as a ‘test organism’ and the test is evaluated similarly to single
organism bioassays, but the result is obtained as contaminated soil mass NOEL,
LOEL or ED50 , instead of contaminant concentration. These results cannot be
integrated into a chemical-substance-centered risk assessment concept, but can be
used for classification of wastes/contaminated soils and for direct decision making
on their use or disposal. Applying this concept, another problem arises: the dose–
response curve may be deformed due to possible positive interactions between test
soil and waste/contaminated soil. For example a waste with high organic matter
content may activate soil mineralization, or the microflora and other components
of the otherwise contaminated soil could substitute the missing components, the
bottleneck of the test soils.

5.1.4 Consequences of the effect of soil matrix on the test methodology

Testing soils is more difficult than testing water due to the three equivalent phases (gas,
liquid and solid), the partition of the contaminants between the phases and the limited
availability of test organisms (Sijm et al., 2000; Horvath et al., 2000; Fenyvesi et al.,
2002).
There are some important aspects, which have to be considered when deciding on
the concept of soil testing. Availability and accessibility should be considered according
to the differences between soil matrix and the test system’s matrix. The following
scenarios should be distinguished:

– Liquids, containing dissolved contaminants infiltrate the soil, and solid soil par-
ticles compete with living organisms for the dissolved chemicals. If the sorption
capacity of soil particles is very strong, biological access will be reduced.
– Contaminated soil (to be tested) is added to a water-based test system: desorp-
tion and dissolution are the limiting factors of biological availability for aquatic
organisms. Terrestrial organisms can interact and enhance bioavailability.
– Contaminated solid (contaminated soil, sediment or waste) is mixed into soil:
the contaminants have to redistribute between four phases of the mixture: soil
solid and solid waste, pore water of soil and moisture content of solid waste until
establishing a new equilibrium.
– Testing soil may involve nutrients together with contaminants (e.g. plant nutrients
in a plant test) in the test system modifying the response of the test organism.

Figure 5.17 shows the changes in soil toxicity during a remediation process based
on biodegradation. Toxicity increases in the beginning due to the biological mobiliza-
tion of the sorbed contaminants prior to biodegradation. Soil microorganisms have
268 Engineering Tools for Environmental Risk Management – 2

Figure 5.17 Time dependence of soil toxicity in a soil remediation process based on biodegradation.

their own set of tools (biotensides, complexing agents) to increase access to sorbed or
otherwise bound substances. The run of time-dependent toxicity shown by Figure 5.17
is typical in the case of aged, sorbable, but still biodegradable contaminants, e.g. most
of the petroleum-based hydrocarbon mixtures. After making the sorbed molecules bio-
logically available, the microbial community degrades the contaminants and lowers
toxicity of the soil.
Table 5.5 shows a matrix of the potential test scenarios including the most impor-
tant interactions between the test systems containing test organism(s) and the samples
to be tested. As Table 5.5 indicates, the following two cases represent different
situations:

– Testing pure chemicals mixed into groundwater or soil representing a generic sce-
nario to simulate their fate and behavior plus their adverse effects on soil ecosystem
members (representing generic or specific ecosystems), and
– Contaminated soils or interacting liquid phases (runoff, leachate, flood water)
containing various chemical substances (representing the contaminated site).

Nevertheless, the same tests are still often applied to both pure chemicals and
contaminated soil testing. In ISO 11268-3 standardized earthworm test – a guidance for
the determination of the effects of chemicals on earthworms in field situations – Kula
et al. (2006) recommended splitting the current guideline into two fields of application:
one for testing chemicals and pesticides (i.e. within the scope of OECD) and another
one for testing soil quality (i.e. within the scope of ISO). They indicate a major need for
guidance concerning the interpretation of effects determined in such complex field tests.
Table 5.5 Laboratory bioassays for testing chemicals in real contaminated soil.
Effect of chemical substance mixed into
Effect of a chemical substance Effect of contaminated environmental runoff, standard model soil or site-specific
Sample to be tested dissolved in water leachate, pore water, wastewater, sludge (with reference soil (with SB) Effect of contaminated soil or solid waste (with
Test organism Simulation test for chemicals dissolved unidentified mixture of contaminants and LB) Simulation tests for contaminants mixed un-identified mixture of contaminants and SB)
Test system in water Liquid phase environmental sample testing into the soil Solid phase environmental sample testing

Aquatic test organism(s) Water-based system, the model of Water-based system, the liquid phase Water-based slurry system modeling Water-based system modeling water erosion,
(ATO) in liquid medium groundwater contaminated by environmental sample is tested by ATO. water erosion, leaching, infiltration, leaching, infiltration, resuspension and the fate of
(generally sterilized*). dissolved chemicals. Bioassay is based on the interaction resuspension and the fate of the soil contaminant mixture when interacting with
The response of ATO is Bioassay is based on the between ATO and the mixture of dissolved chemicals during these. water.
measured. interaction between ATO and or otherwise solubilized contaminants and Bioassay is based on the interaction Interaction between ATO and mobilized,
dissolved (diluted) pure chemicals. other dissolved chemical substances (with between ATO and desorbed, mobilized, solubilized contaminants or other chemical
inhibitory or stimulatory effects). solubilized chemicals. constituents of soil.
Interaction of ATO with natural biota (LB) Soil texture, composition and Interaction between ATO and SB may be
maybe significant in non-sterilized samples. pre-incubation with the chemical significant.
(availability) are influential. Soil texture and composition is integrated into the
response.
Terrestrial test 3-phase wet soil or 2-phase slurry. 3-phase wet soil or 2-phase slurry. A) Chemical substance mixed into 3- or 2 phase soil.
organism(s) (TTO) in Modelling irrigation, flood, liquid Modelling effect of contaminated liquids standard soil or Direct contact and interaction of TTO with solid
standard/model soil nutrients or manure application on on soil ecosystem. B) site-specific reference soil. phase bound and partly desorbed mixture of
(may or may not be soil and the infiltration of dissolved Bioassay is based on the interaction of 3- or 2-phase soil. Modeling direct chemicals (contaminants, nutrients, etc.).
sterilized*). The response contaminants. TTO with partitioned (from dissolved to contamination of soil with chemicals. Nutrients, stimulants from soil are influential.
of TTO Bioassay is based on the interaction sorbed) contaminants or other chemicals Direct contact and close interaction Soil texture and composition as well as
is measured. of TTO with partitioned (from resulted from liquid sample. between TTO and the partitioned contaminant bounding is influential.
dissolved to sorbed) chemical. Interaction between indigenous biota (mainly sorbed, partly dissolved) Influence of TTO on contaminants may be (locally)
Interaction of TTO with SB may be (LB+SB) and TTO may be significant in chemical. significant. Avoidance is possible.
influential. non-sterilized soil, but also occurs in Interaction of TTO with living or dead
The influence of TTO on sterilized soil. SB may be significant and is
contaminants may be weak (due to The influence of the test organism on the unpredictable.
dilution of its biologically active contaminant may be weak. Influence of TTO on contaminants may
products, e.g. exoenzymes or be significant. Avoidance is possible.
exudates).

(continued)
Table 5.5 Continued
Effect of chemical substance mixed into
Effect of a chemical substance Effect of contaminated environmental runoff, standard model soil or site-specific
Sample to be tested dissolved in water leachate, pore water, wastewater, sludge (with reference soil (with SB) Effect of contaminated soil or solid waste (with
Test organism Simulation test for chemicals dissolved unidentified mixture of contaminants and LB) Simulation tests for contaminants mixed un-identified mixture of contaminants and SB)
Test system in water Liquid phase environmental sample testing into the soil Solid phase environmental sample testing

Real (site-specific or 3-phase wet soil or 2-phase slurry. 3-phase wet soil or 2-phase slurry 3- or 2-phase soil. 3- or 2-phase soil.
reference) soil with Simulating real soil. Simulating the effect of contaminated Modeling direct contamination of soil Based on the interaction of SB with the sorbed
indigenous microbiota The interaction of SB with the liquid phase compartments on real soil. with chemicals. mixture of contaminants and other chemicals.
(SB). partitioned (partly dissolved, partly Bioassay is based on the interaction of SB Bioassay is based on the interaction Interaction of SB with soil matrix is very strong.
The response of SB is sorbed) chemical is intensive. with the partitioned (dissolved, sorbed) between SB and the chemical mixed in The reaction of SB and its influence may be
measured. Strong interaction of SB with soil mixture of contaminants and other soil and partitioned between soil significant.
matrix. chemicals. phases. Soil used for receiving and diluting the
The influence of SB on the Interaction of SB with soil matrix is strong. Interaction of SB with soil matrix is contaminated soil or waste causes further
contaminant may be significant. SB interacts with LB. strong. The reaction of SB and its interactions between two soils or soil and waste:
The influence of SB on the contaminants influence on contaminant mobilization contaminants and nutrients, inhibitors and
may be significant. and degradation may be significant. stimulants in changing ratios along the dilution
Soil conditions and nutrient content series, with different equilibrium states.
may be synergic (strengthening) or
antagonistic (canceling) the effect of
the chemical substance to be tested.
Terrestrial test 3-phase wet soil or 2-phase slurry. 3-phase wet soil or 2-phase slurry. 3-phase wet soil or 2-phase slurry. 3- or 2-phase real soil.
organism(s) in real Modeling the fate and effect of a Simulating the effect of contaminated Simulating the fate of the chemical and Simulating the addition of solid phase soil/waste
contaminated soil. dissolved chemical in contaminated liquid phase compartments on its direct effect on contaminated soil into contaminated soil: the fate and effect of
TTO and SB response soil containing adapted SB. contaminated soil. containing adapted SB. contaminants can be followed.
together is measured in Bioassay is based on the Bioassay is based on the interaction of Bioassay is based on the interaction of Bioassay is based on the interaction of TTO and
real contaminated soil. interaction of TTO and SB with TTO, SB and LB with partitioned (partly TTO and SB with sorbed contaminants. adapted SB with the solid form (mainly sorbed)
This case is typical in soil partitioned (partly dissolved, partly dissolved, partly sorbed) mixture of Interaction of TTO with adapted SB. mixture of contaminants and other chemicals.
testing, because SB cannot sorbed) contaminant. contaminants and other chemicals. The influence of adapted SB on the Interaction of TTO with adapted SB.
be excluded, neither in the The influence of the adapted SB on Interaction of TTO with LB and SB. tested contaminant may be significant. The influence of TTO and SB on the contaminants
case of sterilized samples. the contaminant to be tested may The influence of adapted SB on the Avoidance is possible. in the tested waste may be significant.
be significant. contaminants of the tested liquid may be Close interaction of TTO with soil matrix.
significant. Avoidance is possible.
Soil used for receiving the contaminated soil or
waste and diluting the sample causes further
interactions between two soils or soil and waste:
contaminants and nutrients, inhibitors and
stimulants in changing ratios along the
dilution series with different equilibrium states.

ATO: aquatic test organism TTO: terrestrial test organism LB: biota of the liquid phase of environmental sample SB: soil biota.
*Effectivity of sterilization of environmental samples is questionable and may cause further problems due to chemical changes and dead biomass.
 Terrestrial toxicology 271

Table 5.5 summarizes the test scenarios, modeling or simulating the fate and effect
of pure chemicals and the tests for soil or solid waste, soil leachates and other liquid
phase environmental samples. The table contains information on the possible inter-
actions during testing (between soil matrix, contaminant(s), soil biota and the test
organism). Some other key factors are also highlighted. The table intends to cover
bacterial, plant and animal test organisms as well as test setups of bioassays, leach-
ing tests or microcosms. A dilution series for studying the dose–response function of
contaminated soil is usually difficult to produce. Dilution series of chemicals in soil
can be best prepared by adding exactly weighed masses of the chemical substance (e.g.
1.0 mg, 2.0 mg, 5 mg, 10 mg, etc.) into soil samples of the same mass (e.g. 50 g). When
mixing liquid samples to soil, the same volumes of a water-based dilution series should
be added to the soil. When contaminated solid matter (waste or contaminated soil)
is mixed into the receiving soil, the result of the mixing should be calculated (e.g. in
percentage), and the same amounts of the solid mixture should be tested.

5.1.5 Field assessment or laboratory testing?

This question can be answered after having defined the assessment target and the
conceptual risk model associated with the problem, the potential contaminants and
land uses.
The field assessment’s results may be representative for the site if the assess-
ment (monitoring) concept truly reflects the real distribution of risks and the key risk
components, for example the most sensitive species and those suffering the greatest
exposure.
On the other hand, the results of field assessment can only be quantified on a
relative scale, and the use of these relative results is restricted to the site in question,
cannot be generalized and used for other sites or different regions. The statistical
power of field assessment is generally weak due to non-repetitions and interactions
with unidentified co-existing strengthening or canceling effects.
Field microcosms may avoid unexpected and disturbing external effects as they
are partly isolated and better controlled than real nature. Of course if a small part of
the environment is isolated for test purposes, a great part environmental reality is lost,
but replicates can increase validity of the results.
Moving towards simpler models of the soil environment, the loss of environmental
relevance can be compensated for by using a good concept, for example, by selecting
the priority risk components and the most important participants.
When long-term processes such as biodegradation, bioaccumulation and food-
chain effects play a role in the quality and health of the soil, a test system should
be created which simulates the complex natural soil, where these long-term processes
can proceed and their effects can be monitored. Soil microcosms, even laboratory
microcosms can meet these requirements.
A test concept suited to soil contaminants which are easy to mobilize must ensure
the contact between the contaminated soil and the test organism. Direct contact tests –
also called solid contact or contact tests – are such tests. The role of contact tests is
to ensure the interaction between contaminated soil and the test organism, and let
the strongly bound contaminants be mobilized or otherwise influenced by the organ-
ism. Bioassays on soil extracts or leachates can model runoffs, infiltrates, drainage
272 Engineering Tools for Environmental Risk Management – 2

and leachates from the soil to monitor contaminant transport from solid soil to water.
Contaminated precipitation, flood or liquid waste disposal can be modeled by irrigat-
ing dissolved contaminants onto the ground. If the soil should be assessed as a habitat
or an object used by humans or by an ecosystem, soil as whole should be tested. When
the partition of the contaminant between solid and liquid phases plays a role in the
formation of risk, soil as a whole and pore water should be simultaneously tested.
Chemical models fulfill the requirement of being exact, reproducible and capable of
characterizing the risk of a potential soil contaminant; e.g. estimating the bioavailable
fraction from the extracted amount by biomimetic agents. In silico toxicology, i.e. the
use of mathematical models has growing importance in soil toxicology, but the number
of reliable mathematical models is yet small due to the relatively small amount of data
(e.g. compared to aquatic toxicology).

5.2 Ecological assessment: field testing of habitat quality,

diversity of species and abundance of indicator organisms
Site- and problem-specific ecological assessment methods can be created for charac-
terizing habitat quality and suitability as well as biological integrity or vulnerability
of habitats. Some protocols for terrestrial ecosystems have been standardized by US
EPA. These are accessible online from the Ecological Assessment Methods Database
of the USA.

– Floristic quality assessment index – FQAI;

– Habitat assessment model;
– Habitat evaluation procedure – HEP;
– Index of biological integrity (IBI) for birds;
– Index of biological integrity (IBI) for invertebrates;
– Index of biological integrity (IBI) for plants;
– GIS based wetland assessment (Montana Natural Heritage);
– Multi-scale assessment of watershed integrity – MAWI;
– Remotely-sensed indicators for monitoring condition of natural habitat in water-
sheds;
– Soil management assessment framework;
– Uniform mitigation assessment method – UMAM;
– Variables for assessing reasonable mitigation in new transportation – VARMINT;
– Wetland value assessment methodology – WVA;
– Wildlife habitat appraisal procedure – WHAP.

A Guidance Document has been established for terrestrial field studies by Fite et al.
(1988) for US EPA, ensuring a standardized protocol for the various assessments. This
area has been developed for terrestrial field dissipation studies for pesticides and a
guidance document has been established by several interested organizations under the
auspices of NAFTA (Corbin et al., 2006).

5.2.1 Abundance and diversity of soil microbiota

Measuring pollution-induced changes in soil microbiota diversity, metabolic activ-
ity (for example ability to biodegrade contaminants) or community tolerance
 Terrestrial toxicology 273

(PICT = Pollution-Induced Community Tolerance) is based on shifts in diversity

toward new metabolic pathways or tolerant microbial communities in the presence
of contaminants (Rutgers & Breure, 1999). In this case, microbial community shifts
may be characterized by carbon substrate utilization.

5.2.2 The use of carbon substrate utilization patterns for

ecotoxicity testing
In a normal carbon substrate utilization study the carbon substrates are contained
in a multi-well plate consisting of a freeze-dried medium, a standard set of carbon
substrates (up to 96 wells) and a redox dye for monitoring microbial activity. The
soil suspension that contains the soil microbial community is uniformly inoculated
in all wells of the plate and incubated under controlled laboratory conditions. When
the community in any of the wells is able to utilize the carbon source present in a
certain well, the redox dye (tetrazolium violet) turns purple, showing the oxidation of
the carbon substrate. Each substrate in the BiologTM plate represents a different test
system and provides information on the microbial oxidation reaction (binary 0 or 1,
rate constants, etc.) that is specific to the inoculated microbial community (Rutgers &
Breure, 1999). The wells together give a fingerprint on the metabolic activity of the
community.
The same system can be used for ecotoxicity testing purposes. In this case a
healthy microbial community is inoculated into the wells and single chemicals, extracts,
leachates or pore water from contaminated soil or contaminated soil itself is added to
the healthy microbial community of the wells.
The relative sensitivity of the microbial community to toxicant exposure is calcu-
lated as the toxicant concentration that causes a 50% decrease in activity (substrate
utilization) in comparison to the untreated control. When comparing an adapted soil
community to a non-adapted one, higher EC50 (ED50 ) will be measured in the case
of adaptation. This assessment concept can be applied for both tolerance assessment
(Rutgers et al., 1998) and the assessment of biodegrading potential of the soil commu-
nity (Nagy et al., 2010). Both are associated with the presence of contaminant in soil
over a long term.
Tolerance is manifested as an enhanced ability to use a substrate (higher EC50 ) rel-
ative to an unexposed population. The concept of PICT encompasses the phenomenon
that, given variation in species’ sensitivities, toxic effects will reduce the survival and
growth rate of the most sensitive organisms within a population, thus increasing the
average tolerance of the community.

5.2.3 Dung-dwelling organisms, a not yet standardized field study

To register veterinary medicinal products (VMPs) as parasiticides on pastured animals,
legislation in the European Union requires an environmental risk assessment to test
the potential non-target effects of fecal residues on dung-dwelling organisms. Products
showing adverse effects in single-species laboratory tests require further, higher-tier
studies to assess the extent of the adverse effects on entire communities of dung-
dwelling organisms under more realistic field or semi-field conditions. Currently, there
are no documents specifically written to assist researchers in conducting higher-tier
studies or to assist regulators in interpreting the results of such tests in an appropriate
274 Engineering Tools for Environmental Risk Management – 2

context. The members of the SETAC (2013) Advisory Group DOTTS (Dung Organism
Toxicity Testing Standardization) provide a description on dung fauna in Central and
Southern Europe, Canada, Australia, and South Africa. This document briefly reviews
the organisms that make up the dung community and their role in dung degradation,
identifies key considerations in the design and interpretation of experimental studies,
and makes recommendations on how to proceed (Jochmann et al., 2011).
The veterinary parasiticide ivermectin was selected as a case study compound
within the project ERAPharm (2013) (Environmental Risk Assessment of Pharma-
ceuticals). Current ERA clearly demonstrates unacceptable risks for all investigated
environmental compartments, and several gaps in the existing guidelines for ERA of
pharmaceuticals have been indicated and improvements suggested. In addition, guid-
ance is lacking for the assessment of effects at higher tiers of the ERA, e.g. for field
studies or a tiered effects assessment in the dung compartment (Liebig et al., 2010).

5.2.4 Effects of pollutants on earthworms in field situations: avoidance

The ability of organisms to avoid contaminated soils can act as an indicator of toxic
potential in a particular soil. Based on the escape response of earthworms and collem-
bolan, avoidance tests with these soil organisms have great potential as early screening
tools in site-specific assessment. These tests are becoming more common in soil eco-
toxicology because they are ecologically relevant and have a shorter duration time
compared to standardized soil toxicity tests. The avoidance of soil-dwelling inverte-
brates, however, can be influenced by the soil properties (e.g. organic matter content
and texture) that affect the behavior of the test species in the exposure matrix.
Earthworms are suitable organisms for measuring avoidance and for the evalua-
tion of hazardous wastes sites. Avoidance tests show higher sensitivity compared to
acute toxicity tests based on the deterioration because organisms generally exhibit
behavioral responses at lower levels of stress than those that acute toxicity tests can
detect. Avoidance is an ecologically relevant end point that can potentially indicate
sub-lethal stress within a short period of time, the test is easy to perform in a soil
matrix, and an avoidance test has the potential of specialized applications for soil
testing (Natal-da-Luz et al., 2008).
‘Dual-control’ test data have shown that, in the absence of a toxicant, worms did
not congregate, instead distributed themselves fairly randomly with respect to the two
sides of the test chambers, that is, they did not display behavior that might be mistaken
for avoidance. In tests on artificial soil spiked with reference toxicants and hazardous
site soils, worms avoided soils that contained toxic chemicals. Avoidance behavior
proved in most cases a more sensitive indicator of chemical contamination than acute
tests. Determination of avoidance was possible in 1 to 2 days, much less than the
current duration of acute and sub-lethal earthworm tests (Yardley et al., 1996).
This means that the earthworm avoidance test for soil assessments is an efficient
alternative to acute and reproduction tests (Hund-Rinke & Wiechering, 2001) and is
a sensitive screening method. Currently, two test designs, a two-chamber system and
a six-chamber system, are in the standardization process.
Other animals than earthworms have also been used in avoidance tests: the spring-
tail Folsomia candida, other springtails and the white-worm Enchytraeus albidus
(Enchytraeidae). All of them are ecologically relevant soil species and are commonly
 Terrestrial toxicology 275

used in standardized toxicity tests. Their rapid reaction to a chemical exposure can be
used as a toxicological measurement end point that assesses the avoidance behavior
(Amorim et al., 2008).

5.3 Non-standardized contact bioassays: description of

some tests
Tests of contaminated soil as well as potential contaminants in the soil environment
should be adjusted to the risk scenario to be modeled by the test. Table 5.5 explains
that extracts and leachate made from soils mainly represent the risk of soil posed to
subsurface or surface waters. If we assess soil as a habitat, direct contact between
soil and test organism is an important condition, which ensures realistic and dynamic
interactions between contaminants, soil matrix components and the organisms present.
In order for the test results to show this integrated effect, representing actual risk, we
have to make the actors to interact with each other in a more or less realistic way.
In the case of generic testing, this kind of soil-specific realism is a disturbing con-
dition, which should be excluded and ‘abstract models’ with very low environmental
relevance but high reproducibility and precision should be preferred.
In the following, we shall introduce some of the useful contact tests the authors
have had successful experience with.

5.3.1 Single species bacterial contact tests

Vibrio fischeri is generally used as a test organism for testing liquid samples, but it
can also be used in soil suspensions. The problem of detection is that the soil particles
absorb a part of the luminescent light the bacterium emits. When the control soil differs
in texture and composition from the tested soil, this is another problem because the
light absorption of the two differs. As a solution, soil sample is compared to itself by
measuring light emission immediately after soil addition and 30 minutes later. This
experimental setup assumes that the measurable light intensity decreases immediately
after soil addition only due to the presence of light-absorbing solid particles of the soil.
Within 30 minutes, the effect of the toxic chemical is effectuated by further decreasing
light emission. The difference in light intensity of these two time points represents the
real inhibitory effect of the contaminated soil on bacterial luminescence.
The tests need 2 grams of soil, which is suspended in 2 mL of 2% NaCl solution.
A minimum of five-step (seven is better) dilution series is prepared from the contam-
inated soil. After measurement of the reference luminescence intensity, 50 µL of each
member of the dilution series is added to the test medium that contains the luminescent
test organism. The light production of the test bacterium in these tests is measured by
a luminometer. Figure 5.18 introduces the luminescent bacterial culture, the sample
preparation and the portable equipment.
Azomonas agilis bioassay is based on the dehydrogenase activity inhibition caused
by the toxic effect of the contaminated soil to be tested. 100 mL sterile medium is
supplemented with 1 mL 1% 2,3,5-triphenyl tetrazolium chloride (TTC) as an arti-
ficial electron acceptor and with the test bacteria previously incubated on a rotary
shaker at 28◦ C for 72 h. The stock solution is injected into the tubes that contain the
dilution series (dilution factor 2) of the contaminated soil. After incubation in dark,
276 Engineering Tools for Environmental Risk Management – 2

Figure 5.18 Luminescence inhibition of contaminated soil measured by a portable luminometer.

the red-colored formasan (produced by the reduction of TTC) is determined visu-

ally (semi-quantitative method) or after extraction by organic solvents (quantitative
determination).
The inhibited growth and metabolic activities of several sensitive bacterial or
microfungal strains can indicate toxicity. Therefore these activities can be used as end
points for testing contaminated soil. When growing in suspension, density or color as a
result of microbial growth cannot be measured directly from the suspension due to the
disturbance of soil particles. Plating and counting of colonies grown from living cells is
feasible to measure growth. The metabolic activity of the microorganisms can be mon-
itored by the microplate technique (Figure 5.19) from the soil/sediment suspension.
Substrate and indicator is placed into the microwells (ready-made or home-prepared)
and the suspended soil sample with the cells is poured on that. The developed color
of the indicator after incubation proves the metabolic activity. The type and number
of substrates and parallels is optional. The properly compiled metabolic fingerprint
can show activity and adaptation compared to reference or to baseline when testing
time series. It is important to note that soil suspension differs from three-phase soil
significantly, it can be considered as a model of water eroded soil, flooded soil or
re-suspended sediment (see also Table 5.5).
Bacterial species of E. coli, Pseudomonas, Bacillus, Azomonas, Azotobacter,
Arthrobacter, yeasts such as Saccharomyces cerevisiae, Rhodotorula, Torula,
Candida species and filamentous fungi such as Penicillium, Aspergillus, Trichoderma,
etc. have been successfully used by the authors for soil suspension and sediment
slurry contact testing.
Bacillus subtilis and Azotobacter croococcum growth test using an agar-diffusion
method and soil discs is an efficient innovative test method developed by Gruiz
 Terrestrial toxicology 277

Figure 5.19 Soil suspension and positive metabolic activity shown by coloration in the microplate.

Figure 5.20 Agar-diffusion test on metal sensitive Bacillus subtilis.

et al. (2001) and applied to screening of metal-contaminated soil at watershed-scale

(Figure 5.20).
Soil is fixed with plain agarose gel and diskettes cut out from this gelled soil are
placed on the surface of a relatively high-density bacterial culture containing agar
medium. The agar-based soil disks and the nutrient agar-medium are prepared from
the same agar-agar, ensuring similar transport character and capacity. The transport
between the two media is free, the interaction between bacterial exudates and soil-
contaminants can take place. The agar-medium is poured into 20-cm-diameter round
or 30 × 30 cm rectangular trays, where 20–40 samples can be tested in parallel. The
inhibition zone without bacterial growth around the soil diskette is measured in mm.
278 Engineering Tools for Environmental Risk Management – 2

Inhibition is compared to the concentrations of the identified or supposed contaminat-

ing substances (mixed into soil) or to any well-known contaminant, when the chemicals
responsible for soil toxicity have not been identified. This fast and simple test method
is suitable for screening a great number of soil and sediment samples and for toxicity
mapping of large sites.
The Bacillus species which has medium sensitivity, and as such is suitable for
screening and finding the hot spots at an extended contaminated area, were selected by
the authors specifically for the purpose of screening a small watershed with unidentified
contaminants (Gruiz, 2005). Hot spots were identified by 500 soil samples using this
rapid bioassay. In a second step, contaminants have been identified from the hot spot
samples using chemical analysis.

5.3.2 Single species animal contact tests

The protozoon Tetrahymena pyriformis reproduction inhibition test characterizes the
toxic effect of the contaminated soils on a primary consumer in the food chain. T. pyri-
formis stock is grown in a proteose peptone yeast extract medium (PPY), containing
1% proteose peptone and 0.1% yeast extract. The test goes on in a soil suspension
of 0.25 g of soil in 5 mL of PPY medium supplemented with penicillin-, streptomycin-
and nystatin (antibiotics) solutions to prevent the infectious damage of the test organ-
ism when contacting soil. 100 µL of six-day-old test organisms (about 1000 cells/mL)
are used for the inoculation of the test tubes. Incubation period is 3–4 days, in the
course of which sampling and counting of the cells is carried out 6–7 times. A growth
curve can be drawn from the cell counts and Er C50 , NOEC or LOEC is read from
the curve.
Tetrahymena tests on real contaminated soils showed great differences between
the results of contact tests in soil suspension and on extracts from the same soils. A
soil historically contaminated with organic pollutants and another one with extremely
high toxic metal content failed to exhibit toxicity when the extract was tested, but
showed high toxicity after being in direct contact with the test organism for a period
of time. A third soil with low contaminant concentration but high water-soluble metal
content showed toxic effect measured both from extract and in direct contact (Leitgib
et al., 2007).
Folsomia candida, a collembolan, is a popular test organism as it is easy to main-
tain and handle in tests. An OECD guideline exists for testing reproduction, including
mortality as a range-finding test. The test requires a synchronized culture and its prepa-
ration needs some experience. Otherwise it is very easy to perform: a two-fold dilution
series is prepared from the contaminated soil (OECD soil or healthy reference soil)
at final concentrations of 6.25%. Ten to twenty pieces of twenty-day-old springtails
are gently delivered from the gypsum block (1) into the trap by weak air flow. A syn-
chronized culture is used for acute testing. The animals are transferred from the trap
into 250-mL test flasks (2) containing 20 g of the soil mixtures with equilibrium mois-
ture (3). Test flasks are incubated at 20◦ C in the dark for 14 days. At the end of the
incubation period, each soil in the test flask is flooded with distilled water and the
survivor animals, floating on the surface (4) are counted. Figure 5.21 shows the test
steps described above.
 Terrestrial toxicology 279

Figure 5.21 Folsomia candida test in jar: placing 10–20 animals into the jar and counting the survivors.

The avoidance end point is more sensitive than lethality in acute toxicity tests. In
avoidance tests, two containers have to be used with a path between the two, easily
available for the little animals.

5.3.3 Plant tests

Plant tests were only used for testing waste extracts over a long period of time. ISO has
standardized chronic plant tests with two species: a rapid-cycling variant of turnip rape
(Brassica rapa CrGC syn. Rbr) and oat (Avena sativa). The standardized tests are rec-
ommended for both contaminated soil and pure chemicals in soil (ISO 22030, 2005).
White mustard, Sinapis alba also enjoys a widespread use for acute and chronic
toxicity assessment in laboratory bioassays. Based on a Hungarian Standard (MSZ
21976-17, 1993) for waste leachate, germination and root and shoot growth test
was developed by Gruiz et al. (2001) for soil toxicity. A two-fold dilution series was
prepared by mixing the contaminated soil with OECD standard or healthy reference
soil. 5 g wet mass of the soil mixtures is put into 10-cm diameter Petri dishes and
brought to equal moisture content. 20 seeds of high viability (germination ability
>90%) are arranged on the soil surface. The test dishes are kept in the dark at 20◦ C
and plant growth is evaluated after 72 h (Figure 5.22). The number of germinated
seeds and the length of the grown root and shoot of the seedlings are measured as it is
shown in Figure 5.23.
Sinapis alba growth test is a useful screening and assessment test, but root growth
may give false negative results due to the roots avoiding contaminated spots in het-
erogeneous soils. Therefore the inhibition of shoot length as an end point has higher
relevance for toxicity. Another problematic point of this test is that artificial soil used
for dilution may have a too low nutrient content, while the contaminated soil has a
high content. The adverse effect (growth inhibition) of the mixture (of the contami-
nated and diluting artificial soils) may be lower due to high nutrient content in spite
of the presence of toxicants, compared to the artificial reference soil.
Sinapis alba, similar to any other plants, can be used for chronic testing by
evaluating the biomass produced in long-term experiments.
280 Engineering Tools for Environmental Risk Management – 2

Figure 5.22 Plant growth test in Petri dishes: a dilution series from soil extracts.

Figure 5.23 Root and shoot length of a seedling.

A third application of Sinapis alba is its utilization in rapid bioaccumulation tests.

Feigl et al. (2009) developed a 5-day test for measuring the metal bioaccumulation in
the seedlings grown in soils contaminated with metals. The rapid bioaccumulation test
proved to be suitable for monitoring of remediation of metal-contaminated soil and
waste rock by in situ soils remedied by chemical stabilization (Feigl et al., 2009).

5.3.4 Soil as a test organism

Soil itself can be used as ‘a test organism’ in ecotoxicity tests. In this concept healthy
soil – with the active microbiota – is the ‘test organism’ and contaminated soil or pure
 Terrestrial toxicology 281

chemicals are added to it in increasing concentrations. Any relevant soil characteris-

tics, microbiological indicators or soil activities, including respiration, nitrification,
cellulase enzyme activity, etc. can be measured as end points.
It should be noted that the chemical substance or contaminated soil can inhibit or
stimulate the microbiota. Toxic chemicals will decrease soil activities such as respira-
tion and enzyme activities while biodegradable contaminants may increase metabolic
activities. On the other hand, toxic chemicals may inhibit respiration immediately after
their addition to the soil, but may stimulate it later on, after a period of adaptation.
Changes in diversity, e.g. in the community’s species diversity or in the genome diver-
sity of the metagenome may explain the changes in the activities. The background is
that suppressed genes or minor species may come to the fore, or completely new genes
can emerge.
In these types of toxicity tests, the reduced respiration of the test soil (CO2 produc-
tion or O2 consumption) is the response to the contaminant’s presence. The respiration
rates of the concentration series prepared from the contaminant (in the healthy test-
soil) is evaluated one by one and the contaminant-concentration–response (respiration)
curve an EC50 or a NOEC value is read. If a contaminated soil (with unidentified con-
taminant content) is tested, it should be added to the healthy test soil in increasing
ratio and the amount of the contaminated soil which caused 50% (90, 20 or 10%)
inhibition in the respiration of the test soil can be read from the soil dose–response
curve. Another feasible test end point is the highest amount which has not affected the
respiration (No Observed Effect Dose).
Soil respiration can be tested under aerobic or anaerobic conditions. Open and
aerated test systems with three-phase soil create a realistic model of aerobic conditions
for a soil community. Two-phase soil (solid and pore water) in a closed system can
ensure anaerobic conditions. Closed-bottle soil tests are between the two conditions
that is why the results are often difficult to interpret. It is similar to wastewater BOI
determination: continuously decreasing atmospheric oxygen is available for the soil
microbes. Soil microbes in the closed bottle have to switch from aerobic to anoxic
or anaerobic metabolism after a certain decrease of available oxygen. These changes
make the evaluation rather complicated, given that the pressure drop in the closed
bottle is generally the end point measured, based on the assumption that pressure
drop and O2 consumption are in close relationship as CO2 is absorbed by alkali. In
addition to the appearance of alternative respiration forms, soil microbes can produce
different volatile organic compounds and gases, for example, H2 and N2 , which greatly
influence pressure in the bottle.
Soil nitrification is also a suitable indicator for soil health and toxicity. Nitrifi-
cation activity inhibition tests measure the potential ammonium oxidation activity of
the soil. The method has been developed by Leitgib et al. (2007) based on the original
method of Berg and Rosswall (1985).
An uncontaminated garden soil ensures a 100% nitrification activity and this high
activity is then decreased by the added contaminant or contaminated soil. A minimum
five-step dilution series is prepared from contaminated soil. 5 g of soil mixture is sus-
pended in 0.1 mL NaClO3 (1.5 M) and 20 mL (NH4 )2 SO4 (1 mM) solutions. Test flasks
are incubated on a rotary shaker (220 rpm) at 28◦ C for 5 hours. To determine the initial
concentration of nitrite, the soil is suspended in distilled water – instead of (NH4 )2 SO4 –
at 20◦ C for 5 hours. At the end of the incubation period, the nitrification process is
282 Engineering Tools for Environmental Risk Management – 2

terminated by the addition of 5 mL of 2 M KCl. After centrifuging (4500 rpm, 2 s)

samples are sieved on an N-free filter. The nitrite content is measured by photometry
at 538 nm.
Similar to respiration and nitrification inhibition in healthy whole soil due to chem-
ical substances or contaminated environmental samples, toxicity measuring methods
based on other enzymes or enzyme systems can be created. The end point can be the
residual substrate, the enzyme and the metabolites, all measuring substrate utilization.
Depending on the test setup, it can be measured one by one or in a multiwell system
such as in the BIOLOG system described earlier (Figure 5.19).
The application of these tests in the practice of contaminated soil characterization,
risk assessment and risk reduction is described in Chapter 3 in Volume 3.

6 MULTISPECIES TERRESTRIAL TESTS

Multispecies tests are most widespread in soil testing due to the extremely high micro-
bial activity and the complexity of the microbiotic system in soils, giving its intrinsic
entity and making an artificial simulation impossible. Another argument for using mul-
tispecies systems in soil tests is the complexity of interactions between the soil’s physical
phases, strong matrix effects, multispecies microbiota, higher organisms and single or
complex contaminants. The best solution in many cases is the application of real soil
with a complex matrix and microbiota. Some multispecies test methods apply not only
soil microorganisms, but also plants and soil-dwelling animals. Several authors pub-
lished their results in the 1990s on successful applications of terrestrial microcosms for
ecotoxicity testing, for example Mothes-Wagner et al. (1992), Morgan and Knacker
(1994), Edwards et al. (1997), Sheppard (1997) and Olesen and Weeks (1998).

6.1 Classification of multispecies soil tests

Soil micro- or mesocosms are typical test setups for multispecies studies. Their design
and structure are determined by the aim of the study, which can be the fate and behavior
of the contaminants, their effects on the diversity of the soil and on the food web, or
long-term multiple effects. In multispecies tests the community of soil-living organisms
integrates all primary and secondary effects of a contaminant and can aggregate the
adverse effects of several contaminants. The interaction between the members of the
microbial or animal community can also be observed and the result utilized for risk
assessment (Förster et al., 1996). A few published test setups are listed below:

– Terrestrial microcosm system for measuring respiration in closed bottles;

– Terrestrial microcosm system for measuring respiration in flow-through system;
– Terrestrial microcosm for substrate-induced respiration technique (SIR);
– Terrestrial model ecosystem (TME) (Knacker 1998; Förster et al., 1999);
– Soil litter bags;
– Cotton-strip method (Kratz, 1996);
– Bait-lamina test (ISO, 2014);
– Root microcosm system for the investigation of root growth, root exudates and
the mycorrhizosphere;
– Soil core microcosms (EPA, 1987; ASTM, 1993);
 Terrestrial toxicology 283

Figure 5.24 Closed-bottle system and respiration curve with and without substrate addition.

– Soil in jar;
– Terrestrial microcosm chamber (TMC): a model ecosystem with real or synthetic
soil medium with invertebrates, rodents and agricultural crops. Sometimes also
voles (e.g. gray-tailed Microtus canicaudus) are called into the testing;
– Versacore;
– Soil lysimeters.

Many of the published studies confirmed that microcosm tests have many advan-
tages, are more sensitive and realistic than laboratory bioassays (Teuben & Verhoef,
1992; Vink & Van Straalen, 1999). Some of these microcosms are introduced in detail
in the following paragraphs.

6.1.1 Terrestrial microcosm system for measuring respiration

Respiration is generally measured in soil-filled columns or other type of closed con-
tainers with or without controlled air flow. The pressure continuously decreases in
the closed bottle without air flow when CO2 is eliminated by an alkaline absorber.
Palmborg and Nordgren published respiration test setups, results and evaluation for
soil toxicity assessment in the book of Torstensson (1993).
Figure 5.24 shows the respiration curve measured in the closed-bottle system with
and without substrate induction. When using a flow through a system, alkali absorbers
treat the air before entering the soil system to eliminate CO2 (Figure 5.25). The air
leaving the column is generally analyzed for CO2 or O2 . The produced CO2 and
the consumed O2 is proportional to soil respiration. In such microcosms, we can
measure the respiration rate of soil contaminated with a series of growing concentration
of chemical substances or contaminated soil. The results can be used to draw the
concentration-response curve and read NOEC, LOEC or EC50 values.

6.1.2 Terrestrial microcosm for substrate-induced respiration

technique (SIR)
The terrestrial microcosm introduced above can be supplemented with injection. After
reaching steady state, the substrate is injected as a short impulse. The type and size of
284 Engineering Tools for Environmental Risk Management – 2

Figure 5.25 Soil respiration measurement in flow-through columns.

Figure 5.26 Soil respiration curve with substrate addition.

the response on the suddenly introduced energy source may be characteristic for the
soil, depending on its activity and adaptability. Figure 5.26 shows the respiration curve
of a high-quality forest soil: CO2 production increases suddenly after the injection of
the energy substrate. On the other hand, having a high-quality, active and adaptable
soil enables the effect of chemical substances on soil respiration to be measured. Its
time-dependence graph can be drawn based on the changes in the rate of respiration,
and time, intensity and size of the response can be read from this curve.

6.1.3 Terrestrial model ecosystems (TME)

TMEs are semi-field tests for studying effects and the fate of chemicals in soil. There are
both indoor and outdoor protocols for these tests. TME uses the litter layer of the soil
for testing soil function. The litter layer is the uppermost soil layer consisting of plant
 Terrestrial toxicology 285

residues in a relatively non-decomposed form. This layer is the source of energy for
mineralizing soil biota and also for humus formation from the residue of mineralized
organic matter.
TME is also recommended to be used for testing pesticides in soil because specific
exposure to plant protection products takes place in the soil litter layer, including
direct exposure, uptake via food and food web transfer (biomagnification). It is an
important energy resource in soil and thus of vital importance for maintaining organism
communities and their great biodiversity. Not protecting the natural processes and
organisms in litter will fail the EU’s objectives regarding biodiversity, soil erosion,
organic matter decline, and the integrity of soil and soil biota. Therefore, the litter
layer should be taken into consideration in the environmental risk assessment of plant
protection products. It was the summary of the European Food Safety Authority (EFSA,
2014) in an overview on the composition of litter in an agricultural context (EFSA
Opinion, 2010), the underlying processes which play a role in litter decomposition
and an outline of how to consider the litter layer in the environmental risk assessment
of plant protection products (Shäffer et al., 2011).

6.1.4 The cotton strip assay

The cotton strip assay uses the loss of tensile strength in a standardized cotton material
as a parameter. It needs an equipment to measure tensile strength. A major shortcoming
of this method is that the degradation of pure cotton compared to natural litter is an
unacceptable simplification (Kratz, 1996). This is the main reason why this test has
been substituted by the soil litter bag test.

6.1.5 Soil litter bag

Toxicant effects on carbon mineralization can be quantified in several ways. One of
the simplest techniques encloses pre-weighed plant litter in a mesh bag, bury it, and
collect and weigh the bag’s contents after a period of time, and compare the mass loss
relative to similarly bagged litter in reference soil (Figure 5.27).
OECD has issued the Guidance Document (OECD No 56, 2006) to identify pos-
sible and suitable approaches to the evaluation of chemical impacts, and in particular
the impact of plant protection products on soil organic matter breakdown. Methods
identified in this document, and specifically the litter bag test method, add important
tools to a battery of existing standardized protocols for assessing chemical impacts
on the soil biota communities. Procedures outlined in this Guidance Document are
primarily intended for the evaluation of agricultural chemicals but they can also be
applied to ecological risk assessment at contaminated sites as well as to laboratory
chemical toxicity testing (Kula & Römbke, 1998; Römbke et al., 2003; Schäffer et al.,
2011).
Heath et al. (1964) already described in 1964 that the litter-bag method offers
a simple and efficient tool for measuring decomposition in terrestrial systems. This
method is of high ecological relevance because the actual rates of decomposition with
(presumably) native material can be measured in situ, providing real-time data for
toxicant impacts on decomposition at a study site. In addition, various functional and
taxonomic groups can be chemically restricted (e.g., with fungicides, bactericides, and
pesticides) or physically restricted (e.g. the mesh size of the bag can exclude certain
286 Engineering Tools for Environmental Risk Management – 2

Figure 5.27 Soil litter bag (left) and pitfall trap (right).

size groups of microarthropods) in order to evaluate their contribution to the process.

An aspect of the litter-bag method that detracts from its ecological relevance is simply
that the litter is not in direct contact with contaminated soil, and thus it presents a
microenvironment different from buried litter in native surroundings (De Jong, 1998).

6.1.6 Pitfall traps

The pitfall trap – shown in Figure 5.27 – is a relatively simple field assessment tool
for collecting arthropods, mainly insects such as ground beetles, crickets, etc. from
contaminated land. Pitfall traps are placed at the contaminated site or the landfill and
next to a reference site. Abundance, diversity and bioaccumulation of the animals
fallen into the pit can be determined in comparison with the reference.

6.1.7 Bait lamina

Bait lamina is an alternative to the litter-bag method, also concentrating on the litter
layer of the soil and its biological activity. Small bait portions (standard mixture of
cellulose, bran flakes and active coal, in a ratio of 70:27:3) are fixed in holes pierced
in PVC strips (Figure 5.28) that are then exposed to the soil’s biological decomposi-
tion activity. Soil invertebrates and soil microorganisms progressively degrade the bait
placed in the soil substrate. It is assumed that the disappearance of the bait material
is directly associated with the feeding activity of soil invertebrates, even if microbial
processes and microbiogenic metabolism may play a minor role (von Törne, 1990;
Kratz, 1998).
The general requirement for the bait material is that a mixture of natural (pow-
dered) materials are used that can be eaten by soil-dwelling animals and have enough
consistency, elasticity and stability to be placed easily in moist, fine and coarse soil
without any damage. The bait material mixture, proposed by the terra protecta GmbH
company, consists of cellulose, bran flakes and active coal. Materials from field sites
 Terrestrial toxicology 287

Figure 5.28 Bait sticks or laminaTM are suitable for laboratory and field testing of metabolic activity
in soil.

such as powdered leaves of birch (Betula pendula L.) or different grass species (e.g.
Calamagrostig epigaeios L.), have already been used.
The bait-lamina strips are left in the soil/substrate until about 10–40% of the
baits are perforated. Since the necessary exposure time depends on the site and on
the moisture content of the soil, feeding activity assessment can require an exposure
between 7 (in soils with good moisture conditions) and 20 days (dryer soils). The most
favorable conditions are easily obtained by a short pre-test.
The exposed baits are evaluated after washing the strips carefully under flowing
tap water and examining the strips on a lighted bench. Differentiation is made only
between ‘bait eaten’ (“1’’, light falls through the bait) and ‘bait not eaten’ (“0’’, light
does not fall through the bait). Utilized bait-lamina strips can be reused if refilled after
soaking and cleaning in water: the company selling it offers a cleaning and refilling
service of used strips (terra-protecta, 2013).

6.1.8 Soil in jar

Soil in jar is a simple test method for soil monitoring under controlled conditions in
open flowerpots or locker jars with a volume of a few mL to 10–50 liters. Both 3-phase
or 2-phase soils can be modelled in jars; layers of different texture and drainage can
be established on the bottom. Soil or contaminated environmental samples of soil,
sediment or water can be treated or mixed with chemical substances. In addition to
soil microbes, soil invertebrates and plants grown on the soil surface can be added.
288 Engineering Tools for Environmental Risk Management – 2

Figure 5.29 Soil in pot (left) and soil in jar (right).

The measured end point can be abundance, diversity, or any physico-chemical change
in the soil. Soil can be analyzed after careful sampling.
Soil-in-jar type microcosms (Figures 5.29) were applied to model soil contamina-
tion by flood that was transporting mine waste as sediment and mixing sediment to soil
or placing a sediment layer on the top of the soil. Flood microcosms clearly showed an
enhanced weathering process that occurs when putting sediment on soil surface and
interacting with soil (Gruiz, 2005).
Soil-in-jar type microcosms were efficiently applied to mobilization and immobi-
lization laboratory tests dealing with toxic metals. The tests included different chemical
treatments and incubation (followed by physico-chemical analyses), analysis of the
emerged toxic effects on soil microbiota, plants and animals (ecotoxicological tests),
and the plant uptake of toxic metals (bioaccumulation test) (Feigl et al., 2008).

6.1.9 Soil lysimeters

Lysimeters can model the infiltration of precipitation into the soil and the results of
this complex transport process over the short and long term. Lysimeter sizes range
from laboratory microlysimeters of 50 mL through 200, 500, and 1000 mL to field
lysimeters of a few cubic meters.
Regardless of the size, the concept shown in Figures 5.30 and 5.31 is always the
same: the infiltrating precipitation flows through the system and dissolves, extracts,
leaches substances from the soil and washes them into deeper layers or removes them
from the soil-filled column (Vaszita et al., 2009). In situ placed lysimeters need a
sampling installation at the outflow level of the experimental soil column.
 Terrestrial toxicology 289

Figure 5.30 Field lysimeter filled with soil: buried below the surface.

Figure 5.31 Lysimeters filled with soil above the surface with built-in sensors.

Figure 5.30 shows a simple underground lysimeter and an adjacent pit for sampling
the outflow. Figure 5.31 illustrates a more sophisticated setup: open-air lysimeters
installed on the ground. They can operate as either open or closed columns. Observing
and sampling the outflow is easier than with the underground lysimeters. The pictured
large-size lysimeters are equipped with special sensors integrating temperature, pH and
290 Engineering Tools for Environmental Risk Management – 2

conductivity measurement devices to monitor the transport of ions in soils which have
been deteriorated or endangered by acidification, sodification or contamination.

6.2 Characteristics of multispecies toxicity tests

The most important characteristics of multispecies toxicity tests are summarized in the
next paragraph.

– Micro- and mesocosms are multispecies test methods: depending on the aim of the
testing, ‘only’ soil microbiota or also invertebrates or vertebrates are involved.
– Size varies from 0.1 liter to a few cubic meters.
– They are ‘historical’: like the ecosystem itself they are irreversible in time.
– They have a trophic structure, with trophic levels, sometimes very simple,
sometimes close to the real environment.
– Evolutionary events occur in the micro- and mesocosms: strains, able to use
contaminants as energy sources or resistant to xenobiotics, arise.
– New metabolic pathways for biodegradation (of pesticides, xenobiotics) can
develop spontaneously or under an external effect.
– Reduced complexity compared to the field: the number of species is generally
smaller than in natural systems.
– Dynamics of the ecosystem: the enforced isolation into a small scale results in
changes in the dynamics of the soil. These changes should be distinguished from
the toxicant’s effect.
– Heterogeneity: in natural ecosystems spatial and temporal heterogeneity is the key
to species richness and adaptability. Artificial test systems are less heterogeneous,
less unique; this ensures their statistical power as opposed to field cases.
– Multispecies tests are complex systems, with dynamics and history, so they are not
repeatable unlike simple species tests or biochemical assays. The past is conserved
in population dynamics down to the DNA sequence.

All this information should be considered in designing and using micro- and
mesocosms for environmental toxicity testing. These specific characteristics make the
evaluation rather complex; therefore the usual statistical methods do not work for
micro- and mesocosms.

6.3 Evaluation and monitoring of microcosms

In order to monitor micro- or mesocosms, these separate ecosystems, biotic and abiotic
components need to be observed.

– Biotic factors refer to all living organisms present in an ecosystem such as microbes,
plants and animals, etc. They can be monitored by biological methods and tools.
– Abiotic factors refer to all non-living or physical factors present in an ecosystem:
soil texture, geochemistry, oxygen, elements and salts, temperature, moisture,
water-forms, pH, redox potential, etc. These parameters can be monitored using
physico-chemical analysis methods and tools.
– Micro- and mesocosms are (should be) stable and self-sustaining systems, meaning
inside cycling of energy, water and elements as well as their balance. The most
 Terrestrial toxicology 291

important cycles in these individual systems are the water cycle, energy cycle,
organic matter mineralization and humus formation, carbon and nitrogen cycles,
sulfur, phosphor, iron and microelement cycles.
– Limiting factors may change the survival, metabolism and adaptation of the micro-
biota in micro- and mesocosms. Such limiting factors are oxygen or alternative
electron donors, nutrient source, and water.
– Any component of these self-sustaining mini-ecosystems can be monitored: biotic
and abiotic components and their interactions and relationships.

Micro- and mesocosms can be monitored by both built-in measurement systems

(electrodes to continuously measure pH, redox potential, conductivity, CO2 , etc.) and
by sampling the soil to analyze its biotic and abiotic components in the laboratory.
The time series of the results will provide information about the behavior of the micro-
or mesocosm, the changes in the qualitative and quantitative characteristics and the
physico-chemical properties of the soil.
Similar to the real environment, an integrated methodology comprising the
combination of physico-chemical analyses, biological-ecological assessment and tox-
icological testing is the most appropriate tool to monitor micro- and mesocosms.
Simultaneously acquired physico-chemical and biological-ecotoxicological informa-
tion should be evaluated and compared to obtain a true picture of the complex
environment and a reliable prognosis on its characteristics and responses.
Statistical evaluation of micro- and mesocosms should be adjusted to the acquired
data. The evaluation of these data requires multivariate techniques, see Chapter 8.

7 MICROCALORIMETRY – A SENSITIVE METHOD FOR SOIL

TOXICITY TESTING

K. Gruiz,V. Feigl, Cs. Hajdu & M. Tolner

Microcalorimetry is not a typical method for measuring ecotoxicity. It is chiefly used
to characterize physico-chemical reactions and for biological studies in pharmacology.
In this chapter the heat production by soil living microorganisms, plants and animals
is used as end point for soil toxicity characterization.

7.1 Background of microcalorimetric heat production

by living organisms
All chemical, physical and biological processes trigger a net flow of heat. The response
of an organism to adverse effects is accompanied by increased or decreased heat pro-
duction. Microcalorimeters can detect very small heat flows, for example ±50 nW
(0.5 × 10−6◦ C) with the help of TAM (Thermo Activity Monitor) (Figure 5.32). This
means that heat production and its change can be a sensitive end point of a bioassay
using a microcalorimeter. The selective response to a chemical substance or to a con-
taminated environmental sample can be measured and compared to the response of an
untreated control soil.
292 Engineering Tools for Environmental Risk Management – 2

The reaction of bacteria, fungi, whole microbial communities, small animals

and plants to certain environmental parameters and conditions such as temperature
(Querioz et al., 2000; Meissner & Schaarschmidt, 2000; Burger & Qian, 2008) or
O2 and CO2 concentration in air (Zhou et al., 2000) has already been investigated
using TAM.
Sparling (1983) and Critter et al. (2002a,b, 2004a,b) compared respirometry,
microorganism counting and calorimetric heat production of microbial biomass and
found significant correlations. Barros et al. (1995, 1997) found that the total heat
production of a soil microbial community significantly correlated with soil moisture
content and soil organic matter content.
An overview on calorimetry of soil (Rong et al., 2007) summarizes the applications
on soil, mainly measuring microbial activity (Barros, 2007; Wadsö, 2009). However,
ecotoxicological applications similar to those we introduce here are not yet available.
The first application of microcalorimetry in soil toxicity testing published by Gruiz
et al. in 2010 drew the attention to a possible new tool to increase sensitivity and
selectivity in toxicity testing as well as to study the effect mechanism. The concept
and test design of toxicity measurement by TAM was similar to other toxicity tests
based on measuring the relation between concentration/dose and response of soil-living
organisms or their community.
A TAM was used to measure the heat production of the whole soil (including
its microbiota) or that of the applied test organism in a direct contact scenario. This
means that the organisms are placed into the soil thus ensuring a close interaction
with soil through respiration of soil air, ingestion of dissolved and solid-bond matter
as well as having dermal contact with all three soil phases and the contaminants.
Well-known bacterial (Azomonas agilis), plant (Sinapis alba) and animal (Folsomia
candida) organisms were tested for heat production and compared with traditional
ecotoxicological end points. The heat response to toxic metals (Cu and Zn) and to
organic pollutant exposure in contaminated soils was measured. PCP (pentachloro-
phenol), a pesticide and disinfectant (its sodium-salt dissolves easily in water); DBNPA
(2,2-dibromo-3-nitril-propionamide) a quick-kill biocide (easily hydrolyzes under both
acidic and alkaline conditions) and diesel oil (a mixture of hydrocarbons) were used
as model organic contaminants (Gruiz et al., 2010). Some results are introduced here.

7.2 Experimental setup

Experiments in the microcalorimeter are carried out in a perfectly closed sterile glass
ampoule of 5 mL or 20 mL volume.
0.5–2.5 g air-dried, grained, sieved (2 mm sieve), sterilized soil is placed into the
ampoule and is wetted with distilled water or growth media one day before the mea-
surement and incubated at 25◦ C. After the incubation period the test organisms are
placed into the ampoules:
– Azomonas agilis (soil bacterium) 48 h culture, 2 × 108 cells/200 µL);
– Sinapis alba (white mustard) 10 seeds from a controlled order (10 seeds);
– Panagrellus redivivus (nematode) 500 µL from the 5-fold dilution of a standard-
ized culture;
– Folsomia candida (collembolan) 50 insects from a 14-day-old synchronized
culture.
 Terrestrial toxicology 293

Console for
electronic modules

Control panel
Ampoule
Digital display lifter

Holes for
additional
calorimetric
units
Heat exchanger
Calorimetric coil (for
unit flow mode)

Water bath
Ampoule
Water bath
circulating
pump Sample
Reference
measuring
measuring cup
Water bath cup
temperature Thermopile
control unit arrays
Heat sink
Calibration Intermediate
heater heat sink

Exploded view of the system... ... and a calorimetric unit

Figure 5.32 TAM Microcalorimeter with four test vessels (red caps), its inner design and the
calorimetric unit (LKB Bromma, 2013).

Samples from contaminated soil or artificially contaminated solid samples can be

tested. As an alternative no test organism is added to the soil, but the heat production
of the soil’s own microbiota is detected. In this case the heat production of the soil’s
own microbiota is measured.
Heat production is measured by a TAM 2277 (Thermal Activity Monitor by LKB
Bromma) device (Figure 5.32). In the TAM microcalorimeter the thermal energy differ-
ence between the reference and the test-ampoule is converted into electric energy and is
measured in µW units by a pair of Peltier elements. Recent/new TAM models are able
to measure simultaneously 24 or 48 samples. The equipment shown in Figure 5.32
contains only 4 microcalorimetric test vessels.
Microcalorimetric results are evaluated using specific software, Digitam for Win-
dows v. 4.0. The power-time curves obtained during the real-time measurements were
used for reading the characteristic parameters of the curve: the initial slope (m) of
the curve (after the equilibration between the ampoule and the thermal bath of the
microcalorimeter), maximum power (Pmax ) and the time of the appearance of the max-
imum (tmax ). The authors created an S×Pmax /tmax factor to magnify the trends, as S and
Pmax usually decrease and tmax increases as functions of contaminant concentration.
EC20 and EC50 values were calculated from the data obtained from dilution series
of the contaminants in soil. The results were compared to the results of traditional
toxicity measuring methods, evaluated in the conventional way (see also Chapter 9).

7.3 Heat response of Folsomia candida to the effect of diesel oil

Folsomia candida is a soil-dwelling springtail, extremely sensitive to volatile soil con-
taminants because its main exposure route is the cutaneous respiration, thus it is highly
exposed to soil air and solid sorbed volatile contaminants. Collembola placed onto the
294 Engineering Tools for Environmental Risk Management – 2

P (µw)
120 0 mg/kg diesel oil
5,000 mg/kg diesel oil
20,000 mg/kg diesel oil
90

60

30

0 44 47 50 53
t (hour)

Figure 5.33 The effect of diesel oil on heat production by Folsomia candida.

contaminated soil in the calorimetric ampoule increased its heat production propor-
tionally to the diesel oil concentration. Maximum heat production was observed at
the beginning – 108 µW for the 20,000 mg/kg and 95 µW for the 5000 mg/kg diesel
contaminated soil. This enhanced heat production gradually decreased during the test.
Figure 5.33 shows the decreasing heat production as a function of time due to the
effect of the diesel oil in contaminated and untreated control soil. At a certain time
point the decrease of heat production declines steeply (shown by the arrows) until it
reached zero. This moment was: 45.2 h and 102 µW for 20,000 mg/kg and 48.2 h and
75 µW for 5000 mg/kg diesel oil concentration. The uncontaminated control showed
no change in heat production over 53 hours.
The number of animals survived in the closed ampoule was counted at the end of
the measurement. The results were: 0 animals in soil contaminated with 20,000 and
5000 mg/kg diesel oil and 48 (from 50) animals in the unpolluted control soil. The
traditional test method in an open jar provided similar results: 0 animals alive in soil
contaminated with 20,000 and 5000 mg/kg diesel oil and 9 (from 10) animals alive
(after 1 week) in the non-polluted control soil.
Heat production used as an end point can help differentiate between 5,000 and
20,000 mg/kg diesel oil contamination, since the time of decline in heat production
(probable time of death) is different. Heat production instead of lethality provides the
benefit of a much shorter time required: results are available in 2–3 days instead of
the one week in the lethality test. Measuring several concentrations between 0 and
5000 mg/kg, the highest no-effect concentration can be precisely identified.

7.4 Heat response of Panagrellus redivivus on contaminated soil

The traditional test on the nematode Panagrellus redivivus is a 2-week-long repro-
duction test. It is moderately sensitive to most of the organic soil contaminants,
 Terrestrial toxicology 295

P (µW)

400

350
15 000 mg/kg transformer
oil contaminated soil
300

250
Non-contaminated soil
200

150

100

50

0
0 2 4 6 8 10 12 14 16 18 20

t (hour)

Figure 5.34 Heat production curve of Panagrellus redivivus in soil contaminated with transformer oil.

15,000 mg/kg transformer oil (not containing polychlorinated biphenyls) or 250 mg/kg
phenanthrene (a polycyclic aromatic hydrocarbon) do not show any inhibition in the
reproduction of this little animal.
The heat production by the nematode in a soil contaminated with 15,000 mg/kg
transformer oil (Figure 5.34) increased by 50% compared to the non-contaminated
soil indicating an increased metabolic activity. As a consequence, increased energy
production (increased respiration rate), i.e. the decline in the curve occurs 50% earlier
than in the non-contaminated soil. Decline of heat production is caused by the death
of the animals, due to poisoning and/or oxygen depletion in the closed vessel. Sim-
ilar trend has been measured by the nematode in soil contaminated with 250 mg/kg
phenanthrene (Figure 5.35).
The response of elevated heat production can be measured within three hours,
which is a very quick response compared to the two-week time requirement of the
reproduction test. In the microcalorimeter, P. redivivus does not propagate, just lives,
feeds, and tries to survive during the 16–18 hours of the test. Increased heat produc-
tion is characteristic of those concentrations that are not strongly inhibitory or lethal,
against which an increased metabolic activity may act successfully for some time. At
higher concentrations where increased activity does not help survival, heat production
decreases, similarly to conventional end points. The produced heat is less in 500 mg/kg
phenanthrene-contaminated soil than in the non-contaminated control soil.
Sensitivity of the heat-response-based measurement is much greater than the
sensitivity of lethality or the reproduction end points, meaning that much smaller
contaminant concentration is sufficient for the heat response.
296 Engineering Tools for Environmental Risk Management – 2

P (µW)
250 mg/kg phenanthrene
350
contaminated soil
300
Non-contaminated soil
250
500 mg/kg phenanthrene
200 contaminated soil
150
100
50
0
0 2 4 6 8 10 12 14 16 18
T (hour)

Figure 5.35 Heat production curve of Panagrellus redivivus in soil contaminated with phenanthrene.

7.5 Heat response of Sinapis alba to the effect of toxicants in soil

Plants in the microcalorimeter are continuously growing during the test. Germinated
seeds are placed into the relatively large test vessel where they grow and metabolize in
the closed system.
The effect of Cu- and Zn-contaminated soils on Sinapis alba (white mustard) is
shown in Figure 5.36.
The higher the Cu and Zn concentration, the smaller the maximum of the power–
time curve (Pmax ) and the greater its delay in time (tmax ). The slope of the curve (µW/h)
is inversely proportional to the concentration.
The lowest concentrations in the experiments were 40 mg/kg Cu and 50 mg/kg
Zn, which are much less than the soil screening values (CuEUcountries : 100–500 mg/kg,
ZnEUcountries : 200–1000 mg/kg). 40 mg/kg Cu concentration caused 26% inhibition,
50 mg/kg Zn caused 55% inhibition compared to non-contaminated soil. EC50 of
156 mg/kg Cu soil was calculated for copper based on heat production, whose order
of magnitude is similar to EC50 based on shoot growth. EC50 of Zn based on heat
production is about 46 mg/kg Zn soil. The same Zn concentration did not cause
any inhibition in traditional growth-based tests and is 4–20 times less than the
environmental threshold in European countries.

7.6 Heat production response of Azomonas agilis to toxicants

Azomonas agilis is a typical soil-dwelling bacterium, a priority test organism used
for soil toxicity tests. The bacterium placed in the microcalorimeter ampoule shows
metabolism and growth (propagation) during the 24-hour test. The result of these
metabolic activities is heat production.
Figure 5.37 shows some of the power-time curves characterizing heat production of
the bacterium in soil contaminated with DBNPA (2,2-dibromo-3-nitrilopropionamide,
 Terrestrial toxicology 297

Figure 5.36 Heat production by Sinapis alba in copper- and zinc-contaminated soils.

a biocide). The concentration series cover only 0–2 mg/kg, however a very large
drop can be seen between 0.02 and 0.2 mg/kg where 22% inhibition (relative to
non-contaminated soil) drops to 98% (see also Table 5.5).
Table 5.6 contains the characteristic parameters of the power–time curves of
Azomonas agilis affected by DBNPA contaminated soil. Inhibition in %, EC20 and
EC50 values can be read from the concentration–response curve. The higher the
concentration of DBNPA in the soil, the smaller the slope (S = slope in µW/h) and
the maximum (Pmax ) of the curve, indicating reduced heat production. There is a
shift in the power maximum in time (tmax ) too. The values of EC20 = 0.008 and
298 Engineering Tools for Environmental Risk Management – 2

0.00 mg/kg DBNPA

0.002 mg/kg DBNPA
180 0.02 mg/kg DBNPA
0.20 mg/kg DBNPA
160 2.00 mg/kg DBNPA

140

120
P (µW)

100

80

60

40
0 5 10 15 20 25
t (hour)

Figure 5.37 Power–time curves of Azomonas agilis due to the effect of DBNPA.

Table 5.6 Effect of DBNPA-polluted soils on heat production by Azomonas agilis.

DBNPA
concentration Slope tmax Pmax S × Pmax /tmax Inhibition* EC20soil * EC50soil * LC50aq **
(mg/kg) (µW/h) (h) (µW) (µW2 /h2 ) (%) (mg/kg) (mg/kg) reference

0.000 13.9 9.6 178 258 0

0.002 11.2 8.6 155 203 22
0.02 13.5 9.0 117 176 32 0.008 0.038 1.0–1.5 mg/L
0.2 1.3 >22.8 >73 ∼ 4.1 ∼ 98
2.0 1.1 >22.8 >60 ∼ 2.9 ∼ 99

*Calculated from S × Pmax /tmax .

**LC50 aquatic (fish, Daphnia) from MSDS DBNPA, 2013.

EC50 = 0.038 mg DBNPA/kg soil have been determined from S×Pmax /tmax , calculated
from the slope, while tmax and Pmax have been read from the curve.
Traditional Azomonas agilis growth inhibition parallel tests on the same soil and
at the same DBNPA concentrations did not show any toxicity.
No soil screening value exists for DBNPA, only aquatic LC50 values are available
(MSDS, 2013), which are 1.5 orders of magnitude higher than the EC50 measured in
soil by microcalorimetry (generally, toxicity in soil is 2–4 orders of magnitude smaller
than in water). This means that the sensitivity of the microcalorimetric test using
Azotobacter agilis to DBNPA is much greater than that of the aquatic test organisms
of Daphnia and fish.
 Terrestrial toxicology 299

7.7 Evaluation and interpretation of the microcalorimetric

heat production results
The first exploratory results shown by the graphs demonstrate that the heat response
of a test organism to contaminated soil can be a new and sensitive end point
in environmental toxicology and risk assessment of chemicals and contaminated
environmental samples.
The response to low concentrations of contaminants in soil is due to the increased
activity of the test organisms. Higher concentrations of the contaminants cause inhi-
bition in heat production. A concentration/dilution series has to be tested to be able to
distinguish between the responses to low and high concentrations of the toxicant and
to plot the concentration–response curve. Microcalorimetric testing of serial concen-
trations (dilutions) from chemical substances or contaminated environmental samples
will identify the borderline between the initial activation and the following inhibition.
At low concentrations of Zn (20 and 200 mg/kg) and Cu (4 and 40 mg/kg),
increased heat production was observed both in animal (nematode and collembolan)
and bacterial (Azomonas) test organisms. This may have been caused by real stimula-
tion or fight for survival and switching on the test organism’s defense mechanism. The
initial period of increased heat production may yield important information for “ome’’
research (genome, proteome or metabolome) and the development of new techniques
in the field of genomics, proteomics, metabolomics, since heat production is part of
the complex transcriptional and translational processes associated with the response
to toxicants or other biologically active substances.
Later in time, heat production (P) becomes inversely proportional to the contam-
inant concentration even in those cases when an increase occurred at the beginning.
The time (tmax ) and the maximal intensity (Pmax ) together with the slope of the curve
(S) can be a good and easy-to-calculate inhibition indicator. The best indicator would
be the integrated amount of the produced heat (the surface area under the power–time
curve), but this has not been solved yet. The usual test end points were applied: inhibi-
tion in percentage (I%) was calculated compared to a control and ECx was read from
the concentration–response curve.
A comparison of traditional and microcalorimetric test methods shows that
microcalorimetry is more sensitive and much faster. With the traditional test methods
no change has been found in some cases, however microcalorimetry enabled a response
to be measured. For example based on the traditional method one would conclude that
500 mg/kg Zn has no adverse effect on Sinapis alba, but the microcalorimetry indicated
92% inhibition in heat production at this concentration. Also the EC20 and EC50 and
mainly the NOEC (No Observed Effect Concentration) value is much lower in the case
of the microcalorimetric test compared to the traditional growth inhibition test. This
means that the heat response can be utilized as an early indicator of adverse effects.

7.8 Summary of microcalorimetric toxicity testing:

experiences and outlook
Microcalorimetry in soil ecotoxicity is a potential useful new tool both for quantify-
ing the hazard of chemicals and direct toxicity testing of environmental samples. It
was concluded that heat production of the test organisms is inversely proportional to
300 Engineering Tools for Environmental Risk Management – 2

the concentration of toxicants in soil. Compared to traditional test methods (growth

and lethality), microcalorimetrical detection of heat production provides an extremely
sensitive and selective end point in soil ecotoxicity testing, which can indicate minor
changes in the test organisms’ energy production and general metabolism. Heat pro-
duction can be selectively detected and it does not encounter the difficulties of direct
contact tests where microbes or little animals are counted in the soil or the length of
roots grown in soil is measured.
Metabolic activities that follow the exposure of an organism to toxic agents are
always accompanied by heat flow, usually by additional heat production at the begin-
ning. Heat production decreases later on simultaneously with the upcoming traditional
responses. This means that by measuring heat production a primary metabolic signal
is used as a toxicity test end point, which can be detected in the early phase of the
long metabolic pathway leading to the traditionally measured dramatic end points
(growth inhibition, death rate or reprotoxic effect). Innovative toxicity end points
such as genome, proteome or metabolome may also act as early indicators, but the
metabolic pathways and mechanisms should be clarified and the key point identified.
Heat production studies can be of great help in such developments.
Heat production is an integrative signal, it includes all metabolic changes accom-
panied by energy production. This may be an advantage compared to single receptors,
which cannot represent the organism or the culture as a whole.
Three major areas of application can be envisaged for microcalorimetry:

– Research into the mechanism of the organisms’ response to toxic agents and sup-
plying information for other test developments, for example, testing genome and
other “omes’’;
– Identifying the real NOEC (No Observable Adverse Effect Concentration) and
LOEC (Lowest Observable Adverse Effect Concentration) values;
– Spreading direct contact soil and sediment tests by creating an end point which is
easy to measure and is exempt from matrix effects and other disturbing influences.

The main advantages are:

– Broad-spectral, generic;
– Highly sensitive;
– Rapid;
– Dynamic.

Applicability:

– For testing chemical substances in water or in soil;

– For testing environmental samples;
– The response of soils’ own microbiota can also be tested;
– Any kind of small organism or mixture of organisms can be used in the test;
– Static or growing organisms or cultures can be tested.

Disadvantages:

– No widespread or routine methodology;

– Expensive equipment.
 Terrestrial toxicology 301

Proposed developments:

– Use of multichannel equipment;

– Evaluation by integration of the produced heat;
– Identifying metabolites parallel to increased heat production;
– Preparation of a standardized protocol.

It can be concluded from the results that heat production is an early indicator
showing what cannot be measured and seen using conventional methods, in other
words, it shows the ‘no effect’. That is to say it measures dynamic changes within
the concentration range of contaminants considered non-harmful. It may be true that
the risk at these concentrations is low enough to be accepted, but it must be borne in
mind that the calm on the surface conceals sensitive responses against contaminants
and increased energy demand to implement early responses.

7.9 Acknowledgement to microcalorimetry research

The authors thank Prof. Wolfgang Sand and Dr. Thore Rohwerder for the opportunity
to carry out the measurements at the Biofilm Center, University of Duisburg-Essen.
The measurements were financed by the Hungarian-German TéT project (Hungarian
National Office for Research and Technology), the LOKKOCK project (GVOP-3.0-
0257-04, National Competitiveness Program of Hungary) and the MOKKA project
(NKFP-3-020/05, Hungarian National R&D Fund).

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Chapter 6

Advanced methods for chemical

characterization of soil pollutants
Gy. Záray & I. Varga
Cooperative Research Centre of Environmental Sciences,
Eötvös Loránd University, Budapest, Hungary

ABSTRACT

The chemical model and chemical analyses are dominant in current environmental risk
management of chemical substances and contaminated land. Management based on
the chemical model characterizes the state of the environment based on the prognosed
or measured environmental concentrations, and manage the risk mechanically with
the aim to reach a chemically based threshold.
Advanced chemical analytical methods used in various stages of environmental
management starting from site assessment, through technology monitoring to evalua-
tion of the risk and the outcome of risk reduction measures are briefly introduced. The
methods aiming to determine inorganic and organic pollutants in soil and sediment
are the focus of this chapter.
Inductively coupled plasma optical emission and mass spectrometry, and X-
ray fluorescence spectrometry are used for the analysis of inorganic compounds,
while organic compounds are investigated using the entire scope of analytical pro-
cedure (sample pretreatment, extraction, cleanup, preconcentration, separation and
detection techniques). Analytical capabilities of these methods (e.g. detection limits,
dynamic range, time demand of analysis) and some typical examples, literature on
the analysis of pesticides, veterinary pharmaceuticals and petroleum hydrocarbons are
reviewed.
The selection of the adequate chemical analytical method that suits the manage-
ment task in terms of sensitivity, speed and precision is equally important. This chapter
concentrates on the analytical techniques and the quality of the results obtained.
Advice on their adequate applications, possible combinations and integration into
targeted tool batteries for different environmental management activities can be found
in Volume 3.

1 INTRODUCTION

Chemical analysis was believed to be the main tool for environmental toxicology and
the management of chemicals and contaminated land. The chemical approach is still
312 Engineering Tools for Environmental Risk Management – 2

considered a major tool in environmental management, mainly in regulatory risk man-

agement. The chemical model may ease the handling of the environment in a uniform
and standardized way, but chemical analyses alone cannot provide a true picture of the
environment. They must be part of an integrated methodology in combination with
biological and (eco)toxicological methods.
Uncertainties in environmental fate and behavior of chemicals and their effective
proportions are accounted for by using additional transport and fate models, bioavail-
ability models and refined target values. Environmental variables in sensitivity and
adaptation are usually handled by extrapolations, applying assessment or uncertainty
factors (often in the range of 10 to 100). If this is the case, the benefits of chemi-
cal analysis precision are lost. On the other hand, if ecological or human damage is
prognosed, observed or demonstrated, precise chemical analysis is required to detect
and quantitatively determine the contaminants. High-sensitivity analytical methods
are also needed for chemicals that are hazardous in very small concentrations over
the long term. Effect mechanism and toxicokinetic studies require advanced analyti-
cal methods to search for target molecules that play a role in the resulting responses
and molecular mechanisms. Environmental risk management requires an integrated
approach, balanced and joint application of chemical analyses and a study into the
adverse effects. The adequate chemical analytical methods and their suitability to the
environmental management tool battery are important for efficiency of environmental
monitoring and risk assessment.
The determination of the chemical composition of soils is a sophisticated analytical
task. Depending on the chemical information required, the analysis is focused on
the investigation of inorganic or organic compounds of natural origin or those being
pollutants due to different anthropogenic activities. Inductively coupled plasma optical
emission spectrometry (ICP-OES) (Nölte, 2003), inductively coupled plasma mass
spectrometry (ICP-MS) (Thomas, 2008) and X-ray fluorescence (XRF) spectrometry
(Beckhoff et al., 2006) are currently the most widely used analytical techniques for
elemental analysis due to their multi-elemental capabilities and large dynamic ranges
for characterizing different soils. These methods can also be applied to measure uptake
and distribution of various toxic elements such as arsenic and lead by the vegetables
grown on the soil (McBride, 2013) or copper zinc and zinc oxide nanoparticles by free
living nematodes (Sávoly 2014).
The determination of organic compounds requires a proper extraction procedure
to separate organic pollutants from the inorganic matrix (Raynie, 2006). Efficient
extraction methods result in exhaustive extraction of the soil samples which makes it
possible to measure the total contaminant concentration, while less efficient extrac-
tion methods might give an estimate of the easily available (bioavailable) fraction of
the contaminants. For details see Chapter 7. After the cleanup and preconcentration
steps, the extracts are investigated by a powerful gas chromatograph-mass spectrome-
ter (GC-MS) (Hubschmann, 2009) or a high-performance liquid chromatograph-mass
spectrometer (HPLC-MS) (Barcelo, 1996) system to separate, identify and quan-
tify different organic analytes. In this chapter, the determination of some typical
organic pollutants (pesticides and pharmaceutical residues, petroleum hydrocarbons)
is demonstrated.
 Advanced methods for chemical characterization of soil pollutants 313

2 ANALYTICAL METHODS FOR THE DETERMINATION OF

INORGANIC COMPOUNDS

2.1 ICP-based analytical methods

2.1.1 Sample preparation
Since nebulization of sample solutions is the traditional method of introduction into
the ICP (Nölte, 2003; Thomas, 2008), the soil samples must be dissolved. Dried
and homogenized or size-fractionated soil samples can be fully dissolved in a mix-
ture of HF+HCl+HNO3 , or HF+HNO3 using a pressure and temperature controlled
microwave assisted digestion system (Kingston & Jassie, 1988). However, the dissolu-
tion of silicates can often be avoided. Microwave (MW)-assisted acidic soil extraction
using aqua regia or nitric acid is usually sufficient to receive reliable analytical infor-
mation about the inorganic pollutants, e.g. toxic heavy metals. It should be noted
that the total salt concentration of sample solutions to be nebulized is limited. The
recommended concentrations for ICP-OES and ICP-MS generally are 1.0 and 0.1%,
respectively.

2.1.2 Inductively coupled plasma as photon and ion source

Inductively coupled plasma is an electrodeless discharge in a gas (mostly argon) at
atmospheric pressure, generated by an electromagnetic field at a frequency of 27.12
or 40.7 MHz. A water-cooled coupling coil arranged around the quartz plasma torch
(Figure 6.1) is connected to a radio frequency generator. The torch consists of three
coaxial tubes with gas entering tangentially by a side tube and creating a vertical flow.
The outer gas flow, termed the coolant flow, protects the tube walls and acts as the
main plasma support gas, usually of 10–15 L min−1 . The small diameter (∼1.5 mm)
of the injector tube is instrumental in producing a high velocity gas jet by which the
sample aerosol can be introduced into the plasma to form a cooler axial channel.
To initiate the discharge, free electrons are generated by spark from a Tesla coil,
and the electrical energy applied to the coil is converted into kinetic energy of electrons.
The free electron path prior to the collision with an argon or analyte atom, to which
its energy is transferred, is only about 1 µm thus, the plasma is heated forming a bright
discharge. Due to the skin effect, the plasma has a toroidal form; therefore, wet or dry
aerosol particles can be introduced into the plasma with a good efficiency resulting in
a self-absorption-free source for the atomic emission spectrometry. The temperature
in the induction region amounts to 10,000 K. However, the gas kinetic temperature
varies between 5,000 and 7,000 K in the central channel above the mouth of the torch.
This temperature is high enough to atomize the aerosol particles and to excite and
ionize the analyte atoms.
For the introduction of liquid samples into the ICP, the solutions are nebulized
by pneumatic, ultrasonic or hydraulic high pressure nebulizers (HHPN) combined
with spray chambers. The cut-off diameter of spray chambers generally is 5 µm. The
nebulization efficiency (volume of solution transported to the ICP/volume of solu-
tion transported to the nebulizer) is relatively poor (1–3%) for pneumatic nebulizers
314 Engineering Tools for Environmental Risk Management – 2

Figure 6.1 Schematic structure of ICP torch.

(Meinhard, cross flow, glass frit), better for ultrasonic nebulizers (∼15%) and the
highest for the HHPN (∼50%). In the last two cases, a desolvation unit is necessary
to eliminate the loading of the plasma with a significant amount of solvents.
Instead of dissolving soil samples, the soil particles can be transported into the ICP
by nebulization of soil suspension with a concentration of 0.5–1.0% using Babington-
type nebulizers (Ebdon & Goodall, 1992). This method is applicable if the soil sample
is finely ground (d < 5 µm) and homogeneous. The uncertainty of analytical data can
be reduced by addition of surfactants (e.g. Triton-X) to the slurry or by selecting the
optimal pH range (Varga et al., 1996). The transport efficiency can be increased by
desolvation of slurry aerosols (Hartley et al., 1993).
Another way to transport the analytes of soil samples to the plasma is generating
dry aerosols by electrothermal vaporization (ETV) of highly volatile elements (As, Cd,
Pb, Zn) (Záray & Kántor, 1995). Figure 6.2 demonstrates the set-up of this unit. 1–
10 mg dried and uniformly ground soil sample in a graphite boat can be introduced into
the graphite furnace. Following the drying (105◦ C) and the ashing steps (to remove
organic compounds) at 500–600◦ C, the temperature of the graphite boat is rapidly
increased to 1,600◦ C resulting in hot vapors of volatile elements which are mixed
with cool Ar gas forming a well-transportable dry aerosol. In order to eliminate the
 Advanced methods for chemical characterization of soil pollutants 315

Figure 6.2 Setup of electrothermal vaporization unit for ICP-OES or ICP-MS investigations.

transport losses between the ETV unit and the ICP, it is recommended to apply chemical
modifiers, e.g. sodium selenite. In this case, the formation of dry aerosols is also
supported by the chemical condensation of vapors. During the last decade, the ETV-
ICP-MS ultrasonic slurry sampling method was mostly applied to determine highly
volatile elements in soils and sediments (Dias et al., 2005; Tseng et al., 2007). For the
determination of mercury, the cold vapor generation technique coupled with ICP-MS
offers a powerful way. The detection limit is 6 ng/g and 300 mg samples are used for
analysis (Picoloto et al., 2012).
Collisions of analyte atoms with high energy electrons or metastable argon atoms
are primarily responsible for the excitation and ionization processes in the analytical
channel of ICP. Analytical data about the elements can be collected by measuring the
emitted photons at the elements’ characteristic wavelengths or by detecting the ions
of stable isotopes through application of quadrupole, time-of-flight or high-resolution
mass spectrometers. The ions are sampled from the plasma through a water-cooled
interface consisting of a sampler and a skimmer cone with holes of 1.0 and 0.8 mm
diameter. These cones are generally made of nickel; however, platinum-iridium cones
are preferred for the investigation of reactive samples. Figure 6.3 shows the schematic
structures of the interface unit located between the plasma and the mass spectrometer.
316 Engineering Tools for Environmental Risk Management – 2

Figure 6.3 Schematic structure of ICP-MS interface.

Independently of the detection system (optical emission or mass spectrometer),

there are different spectral interferences which cause errors in the analytical data. In the
case of ICP-OES, overlapping of spectral lines of different elements is the most critical
phenomenon. Therefore, it is recommended to apply a monochromator to check the
spectral interferences at the wavelengths of the analytical lines in the multichannel
optical emission spectrometers or a multiple line detection for all elements investigated.
Applying mass spectrometers, the ionized stable isotopes of analyte elements are
detected at different m/z values. Unfortunately, this technique also suffers from spectral
interferences originating from different sources. The first one is the isobaric overlap.
This phenomenon occurs if two elements have stable isotopes of essentially the same
mass. Fortunately, all elements have a minimum of one interference-free isotope. How-
ever, their abundance is not the highest in many cases. Doubly charged ions provide the
second source of spectral interferences. They generate a number of isotopic overlaps
at half of the mass of the parent elements. The third group includes polyatomic ions
formed of the atoms of the plasma gas, the solvents (mostly water and acids) and the
analyte elements. A few characteristic polyatomic ions and the disturbed elements are
listed in Table 6.1.
There are various methods to eliminate these spectral interferences: application
of high-resolution mass spectrometers (R = 10,000); use of collision cells to destroy
the polyatomic ions; mathematical correction; modification of plasma parameters (e.g.
application of helium instead of argon); or removal of disturbing matrix elements prior
to ICP-MS measurements.

2.1.3 Analytical figures of merit

The ICP-OES or ICP-MS techniques can determine about 70–75 elements simulta-
neously and the quantification limits for solutions are in the ppb and ppt range,
 Advanced methods for chemical characterization of soil pollutants 317

Table 6.1 Spectral interferences caused by polyatomic ions.

Mass Polyatomic ion Disturbed stable isotope

12 +
24 C2 Mg
25 12
C2 H, 12 C13 C+ Mg
12 14 +
26 C N Mg
28 14
N+ 12 16 +
2, C O Si
29 14
N2 H + Si
14 16 +
30 N O Si
31 N OH+
14 16
P
16 +
32 O2 S
33 16
O 2 H+ S
39 38
ArH+ K
41 40
ArH+ Ca
44 12
C16 O+ 2 Ca
45 12
C16 O2 H+ , 13 C16 O+2 Sc
32 14 +
46 S N Ti
31 16 +
47 P O Ti
48 P OH+ , 32 S16 O+
31 16
Ti
49 S OH+
32 16
Ti
34 16 +
50 S O Ti,V
51 Cl O, 34 S16 OH+
35 16
V
52 40
Ar12 C+ , 36Ar16 O+ , 35 Cl16 OH+ , 40Ar16 C+ Cr
37 16 +
53 Cl O Cr
54 40
Ar14 N+ , 37 Cl16 OH+ Fe, Cr
55 40
Ar14 NH+ Mn
56 40
Ar16 O+ Fe
57 40
Ar16 OH+ Fe
31 16 +
63 P O2 Cu
32 + 32 16 +
64 S2 , S O 2 Zn
34 16 +
66 S O2 Zn
35 16 +
67 Cl O2 Zn
37 16 +
69 Cl O2 Ga
70 38
Ar32 S+ Ge
71 40
Ar31 P+ Ge
72 40
Ar32 S+ , 38Ar34 S+ Ge
74 40
Ar34 S+ Ge
75 40
Ar35 Cl+ As
76 40
Ar36Ar+ Se
77 40
Ar37 Cl+ Se
78 40
Ar38Ar+ Se
80 40
Ar+ 2 Se

respectively. Considering the dilution factors, the quantification limits of inorganic

compounds of soil samples are in the ppm and ppb range. It should be emphasized
that the dynamic ranges of ICP-OES and ICP-MS methods amount to 4–5 and 7–8
orders of magnitudes, respectively. This means that ICP-MS makes it possible to
determine major, minor and trace elements simultaneously.
318 Engineering Tools for Environmental Risk Management – 2

2.2 X-ray fluorescence spectrometry

XRF spectrometry is a well-established method of elemental analysis. The determina-
tion of chemical composition through the measurement of XRF spectra has become a
routine analytical method, employed in a wide variety of research areas ranging from
material research to biomedical and environmental sciences. XRF is a non-destructive
method, for which only simple sample preparation is necessary. The elements which can
be analyzed by commercial laboratory-scale equipment range between Z = 11 (Na) and
Z = 92 (U). The analysis of elements below Na requires specific conditions: a detector
with a thin entrance window, measurements in vacuum, special sources for excitation
(e.g. synchrotron radiation or special X-ray tubes with low Z anodes).
The basic form of XRF spectrometry consists of two steps: the first is the ionization
process in the K shell caused by high-energy photons (photoelectric effect) and the
second is the neutralization of the K-shell by an electron removed from another atomic
shell having lower binding energy such as L, M, N, etc. The difference of the two
binding energy values (K and L, M, N, etc.) will be carried by a fluorescence photon
that is emitted during the relaxation process.
Due to the quantified binding energies of the different electron shells in the atoms,
the emitted-fluorescence photons have a well-defined energy at a given wavelength. By
measuring the energy of the photons, information can be gained on the two atomic
shells involved in this process. The emitted photons are called characteristic radiation
because they exactly identify the two shells between which the process takes place.
The energy spectrum of these photons is a line spectrum, characteristic of the atomic
number of the atom. These characteristic lines in the X-ray spectra provide information
on the elements contained in the analyzed material.
In each X-ray emission spectrum, along with the characteristic lines, there is also
a continuous component of X-ray radiation. This part of the spectrum originates from
the electrons decelerated by the Coulomb field of the target material’s nucleus. The
electrons do not excite the X-ray energy levels of the atoms during the deceleration
process, but they lose their kinetic energy on their way between the nuclei of the target
material, and emit electromagnetic radiation at different wavelengths and different
angles from the original direction. One portion of this radiation is in an X-ray region
called Bremsstrahlung. Figure 6.4 shows a typical X-ray spectrum demonstrating the
intensity distribution of the characteristic X-ray lines emitted by the major, minor and
trace elements of a calcareous loam soil certified reference material (BCR No. 141).

2.2.1 Sample preparation

Quick and easy sample preparation is one of the reasons why XRF-spectrometry has
become a popular analytical method for soil analysis. However, the very sample prepa-
ration is a major source of analytical error. First of all, matrix effects including particle
size, uniformity and homogeneity are responsible for errors. To eliminate or reduce
matrix effects, two groups of sample preparation procedures have been developed:

– Fusion-based methods: dry and ground (d < 2 mm) soil samples are mixed with
Li2 B4 O7 in a ratio of 1:2–1:6 and heated to 900–1,100◦ C in graphite or plat-
inum crucibles applying muffle furnace or RF-coil (Sweileh & Vanpeteghem, 1995;
Johnson et al., 1999);
 Advanced methods for chemical characterization of soil pollutants 319

Figure 6.4 XRF spectrum of calcareous loam soil certified reference material (BCR141).

– Pelleting techniques: dry and finely ground (d < 60 µm) soil samples are mixed
and homogenized with different binding materials e.g. boric acid (dos Anjos et al.,
2000), cellulose (Hayumbu et al., 1995), polyvinyl alcohol (Anda et al., 2009),
phenolic resin (Longerich, 1995), and pressed to pellets applying a pressure of
200–300 MPa.

2.2.2 Basic equipment and set-up for XRF analysis

Several XRF technical set-ups and experimental approaches have been developed and
used routinely for the quantitative characterization of a great variety of materials.
In general, any type of XRF spectrometer consists of an X-ray source producing the
excitation beam, focusing devices for controlling shape and size of the X-ray beam, and
the detection system. Figure 6.5 shows the simple set-up of the PANalytical MiniPal 2
equipment.

2.2.3 X-ray sources

There are various possible approaches, e.g. X-ray tubes, radioactive sources, syn-
chrotron facilities, ion accelerators, and electron sources used to generate X-rays. The
operation principle of each type of X-ray tubes is based on the interactions between
electrons and atoms in the anode material. In an X-ray tube a cathode emits elec-
trons that are accelerated by an electric field. After reaching the surface of the anode,
they interact with the anode atoms (van Grieken & Markowitz, 2004) resulting in
the deceleration of free electrons and excitation of atomic shell electrons. Finally, this
procedure yields characteristic X-ray photons and Bremsstrahlung radiation, as men-
tioned above. The energy of both types of radiations originates from the energy loss
320 Engineering Tools for Environmental Risk Management – 2

Samples

Sample changer
Measuring
position

Filters
Si-PIN
detector

X-ray
tube

Figure 6.5 Set-up of X-ray fluorescence spectrometer.

of the electrons. The high voltage used for accelerating the electrons is set between
20–300 kV depending on the type of X-ray tube, spectrometer, and atomic number of
the element being tested.
For special XRF analysis such as in-situ or in-field type measurements, sometimes
radioactive isotopes (57 Co, 109 Cd, 241 Am) are used as X-ray sources. The advantage
of these types of X-ray sources is that the application is very simple, and the anal-
ysis is often less expensive than that using X-ray tubes. The use of radioisotopes as
X-ray sources is based on the emission of characteristic X-ray lines from a selected
radioactive element. In this decay process the nucleus of the atom captures one elec-
tron on the K-shell. After the decay process, the atomic number of the daughter
atom is decreased by one unit. Since one K-electron is missing, the excited atom
relaxes, and emits characteristic photons which transport the excitation energy of
the atom.

2.2.4 Detectors
The detection of X-rays is one of the most important steps in an X-ray-based study
which gathers the information coded in the emitted characteristic and scattered X-rays
on the atomic and molecular structure of the irradiated material. X-ray detectors can
be divided into two main groups, (Van Grieken & Markowicz, 2004):

i) Detectors used in Wavelength Dispersive X-Ray Fluorescence (WD-XRF) analy-

sis, which are based on the Bragg reflection of X-rays on a crystal surface;
ii) The Energy Dispersive X-Ray Fluorescence (ED-XRF) detectors.

Wavelength dispersive X-ray detectors measure the X-ray at a specific wavelength

diffracted by a perfect single crystal. The wavelength of the impinging X-ray and the
 Advanced methods for chemical characterization of soil pollutants 321

crystal lattice spacing are related by Bragg’s law. The basic difference between the
wavelength- and the energy-dispersive detection mode is that the WD-XRF detec-
tor only reads counts at a selected wavelength and it does not produce a broad
spectrum.
The operation principle of the second group of detectors is the interaction between
X-ray photons and semiconductor materials. The photons that enter a semiconductor
single crystal (Si, Ge) excite some electrons of the crystal atoms, and they pass their
energy on to the crystal during this process. Due to the additional energy received from
the absorbed photons, the excited electrons will not bind to the crystal atoms; therefore,
they will be able to move freely in the whole semiconductor crystal. When applying
voltage to this crystal, the free electrons are forced to move out of the semiconductor
detector and the sum of their electric charge can be measured. The electric charge is to
the absorbed energy in the crystal. The X-ray detectors act similarly to a calorimeter.
The deposited energy delivered by the absorbed photons is transformed into free electric
charge.
The most frequently used semiconductor detector material uses single-crystal Si
for detection of soft (E < 25–30 keV) X-rays and high-purity Ge (HPGe), GaAs or
CdZnTe for hard (E > 25–30 keV) X-rays, and γ rays. Conventional Si detectors such
as Si(Li), essentially have a semiconductor diode structure, which is made up of three
semiconductor layers that have different conductive properties. In the Si(Li) detectors
a high resistivity intrinsic layer is sandwiched between n- and p-type layers. The thick-
ness of the central layer doped by Li amounts to 2–3 mm. Due to random electron
generation, the amount of collected charges greatly depends on the temperature of the
detector crystal.
Since the X-ray radiation does not hit the middle part of the detector (the so-
called depleted region), it is free of electric charge. If an X-ray photon is absorbed
in the depleted region, free charges will be generated, i.e. electrons and holes of elec-
trons with positive charge. Because of the relatively strong electric field in the depleted
region, the free charges are collected separately by two electrode layers located on the
surface of the semiconductor detector. With increasing temperature, the number of the
free electrons becomes greater, resulting in a temperature-dependent constant electric
current (leakage current) in the detector output. In order to reduce the thermally gen-
erated leakage current and to keep its value as low as possible, the detector must be
cooled down to the temperature of liquid nitrogen (≈77 K). Without cryogen cooling,
this type of X-ray sensor would only continuously detect intensive noise. The necessity
of intensive cryogen cooling mode limits the possible application of semiconductor
detectors due to the complicated technical conditions for producing and storing liquid
nitrogen. In order to avoid this limitation, new types of Si detectors were developed and
introduced for use in X-ray spectroscopy in the last two decades. These devices need a
higher operating temperature than those mentioned above (220–240 K) whereas some
of them, e.g. the silicon drift detector (SDD), can operate at room temperature. Due
to the absence of the cryogenic cooling unit, the size of the high-temperature semicon-
ductor detector decreased dramatically, which allowed portable X-ray spectroscopic
devices to be manufactured.
The SDD detectors have remarkable, advantageous spectroscopic properties, such
as: i) very fast signal detection (about 105 cps) whereas the detection capability of
conventional Si(Li) detectors is no more than 104 cps; ii) low noise, with energy
322 Engineering Tools for Environmental Risk Management – 2

resolution similar to that of Si(Li) detectors (120–140 eV at 5.89 keV). However, it

should be noted that their detection efficiency is much lower than that of the Si(Li)
detectors.

2.2.5 Quantification
The quantification methods and algorithms can be divided into two major groups
(Jenkins et al., 1995): i) empirical methods, when the quantification procedure is
based on the use of standard samples that have a composition similar to that of
the material analyzed; ii) fundamental parameter methods based on physical and
mathematical models which describe the physical processes occurring between the
sample atoms and the excitation X-ray beam. A mathematical relationship is obtained
between the elemental compositions of the sample material and the characteristic
X-ray intensities as detected by the X-ray technique applied (Lachance & Claisse,
1994).
The key problem is that the attenuation of the sample at the X-ray excitation
energy and at the energy of the characteristic lines is unknown; therefore, the experi-
mental determination of these values is rather complicated. In order to circumvent this
problem, the most effective and simplest solution is offered by an empirical method
known as the standard addition procedure. The method fits X-ray analysis of environ-
mental samples well because this procedure is primarily suitable for the determination
of trace and minor elements. The principle of the method is to add a known amount of
the element under test to a given mass amount of the sample material. The procedure is
repeated 5–8 times using different masses. A set of samples is eventually available, all
having the same matrix composition, but with known different concentration of the
investigated element. This allows a calibration curve to be constructed for the element
being tested. If the sample contains only a trace amount of the analyte, the calibration
curve is linear. For most environmental samples this assumption is acceptable. On the
other hand, if the sample contains high concentrations of the element to be analyzed,
the first step is to dilute the matrix so that the diluted sample can be used as the working
matrix for the standard addition method.
The Fundamental Parameter Method (FPM) is based on the physical description of
fundamental atomic processes between the excitation X-ray beam and the atoms of the
sample. The theory gives an exact mathematical relationship between the concentration
of the sample elements and their characteristic X-ray fluorescence intensities (K, L, and
M lines).

2.2.6 Analytical figures of merit

Laboratory-scale XRF-spectrometers working with X-ray tubes offer quantification
limits for different elements from Na to U in the ppm concentration range. Portable
XRF-devices have higher quantification limits, and the analytical results are only qual-
itative or semi-quantitative due to the matrix effect. Since the calibration curves are
linear in a concentration range of 3–4 orders of magnitude, the XRF spectrometry is a
powerful and rapid method to provide multi-elemental information about major and
minor inorganic compounds in soils and sediments.
 Advanced methods for chemical characterization of soil pollutants 323

2.2.7 Comparison of XRF and ICP-based analytical techniques

Comparing the analytical figures of merit, time demand and cost of analysis, as well
as the “green character’’ of these multi-elemental analytical methods, the following
conclusions can be drawn:

– ICP-based techniques offer lower detection limits and larger dynamic range than
XRF spectrometry, therefore the ICP-based techniques can be recommended for
trace analysis.
– Calibration can be performed more reliably for the ICP-based techniques com-
pared to XRF spectrometry where the matrix effect has a strong influence on the
analytical signal. Therefore, this analytical method needs standard samples with
the same or similar matrix composition to that of the sample to be analyzed.
– Due to the time-consuming sample preparation, ICP-based techniques need longer
time for producing analytical data than XRF-spectrometry.
– The cost of analysis for the ICP-based techniques is considerably higher than that
for XRF spectrometry due to the consumption of high-purity argon and high-purity
chemicals used for sample digestion.
– Due to the chemical-free sample preparation procedure, the XRF-spectrometry is
a “green’’ analytical technique.

In addition to this theoretical comparison of ICP-based techniques and XRF spec-

trometry, it should be mentioned that it is useful to experimentally compare the
efficiency of acidic digestion or extraction of different analytes from soil samples.
Some authors demonstrated that special attention should be paid to chromium which
had a low recovery in MW assisted digestion with aqua regia (Amorosi & Sammartino,
2011) or HNO3 -HF-H2 O2 (Congiu et al., 2013).

3 ANALYTICAL METHODS FOR ANALYSIS OF ORGANIC

POLLUTANTS

The presence and bioavailability of organic pollutants and their metabolites in soil can
adversely affect animal and human health, plants and soil microorganisms. The most
important organic pollutants in soil can be classified into the following groups (a more
detailed grouping of the soil contaminants can be found in Volume 1, Chapter 2):

– Pesticides, including herbicides, fungicides, bactericides, rodenticides, repellents,

insecticides applied in agriculture;
– Veterinary and human pharmaceutical residues (antibiotics, anti-inflammatories,
steroids, hormones, analgesics) and their metabolites originating chiefly from
veterinary medicine;
– Polychlorinated biphenyls, terphenyls, naphtalenes, paraffins;
– Polybrominated biphenyls and diphenyl ethers; hexabromocyclododecane applied
as flame retardants;
– Petroleum hydrocarbons.
324 Engineering Tools for Environmental Risk Management – 2

Their identification and quantitative determination in soils and sediments need a

multi-step analytical procedure:

– Sample pretreatment (drying and grounding);

– Extraction of analytes from the soil matrix;
– Cleanup of extracts in order to remove the interfering matrix compounds;
– Preconcentration/enrichment of analytes;
– Separation and detection of analytes using GC-MS or LC-MS techniques.

3.1 Sample pretreatment

The ideal soil sample for extraction is dry, and the particle size is smaller than 0.5 mm.
Air-drying, oven-drying or freeze-drying (lyophilization) can be applied to remove
water. The latter two methods provide a lower volatile compound recovery due to
the possible losses. Another problem in soil analysis might be that the soil particles
can form aggregates preventing an efficient extraction. Therefore, it is recommended
to mix the soil sample with inert material such as sand (Crescenzi et al., 2000) or
diatomaceous earth (Eskilsson & Bjorklund, 2000).

3.2 Extraction of analytes from soil samples

The aim of the extraction step is the quantitative transfer of a small quantity of organic
pollutants from a relatively large amount of soil sample into a liquid organic phase.
Polar (e.g. methanol, acetonitrile) or nonpolar (e.g. benzene, n-hexane) organic sol-
vents can be applied for extraction depending on the chemical properties of the organic
pollutants and the nature of the soil sample.
The relatively old, but simple and economic Soxhlet extraction technique (Figure
6.6) is widely used in laboratory practice. 50–100 g soil samples are usually placed into
the thimble chamber. The solvent vapor travels up through a distillation branch, and
floods the chamber housing the thimble of soil. A condenser cools the solvent vapor,
which drips back onto the soil sample. The desired compounds will then dissolve in the
warm solvent. Due to the repetition of vaporization, condensation and extraction steps
the desired compounds are concentrated in the distillation flask. After extraction the
solvent is removed, typically by means of a rotary evaporator, yielding the extracted
compounds. The non-soluble portion of the extracted solid remains in the thimble,
and is usually discarded. The efficiency of this extraction technique is high, but its
time demand is 8–36 hours.
Modern extraction techniques (Raynie, 2006) were developed to facilitate sam-
ple preparation and to reduce analysis time, exposure of laboratory personal to toxic
chemicals and the cost of sample preparation. The improved extraction methods need
instrumental systems, and the enhanced efficiency of these methods is the result of ele-
vated solvent temperature. The higher temperature leads to favorable kinetic, diffusion
and solubility parameters.

3.2.1 Supercritical fluid extraction (SFE)

Gases can be converted to a supercritical fluid state above a critical temperature and
pressure. For example, CO2 exists in a supercritical fluid state above T = 304.2 K and
 Advanced methods for chemical characterization of soil pollutants 325

Figure 6.6 Soxhlet extraction system.

72.8 bar where its density is closer to that of the liquid state, while its viscosity and
diffusion capability are near to those of the gaseous state. Therefore, this material is
an excellent solvent. The efficiency of SFE is similar to the Soxhlet technique, however,
extraction takes only 10–30 minutes (Smith, 1999; Rissato et al., 2005).

3.2.2 Microwave assisted extraction (MAE)

MW devices applied in the laboratory practice operate at the frequency of 2.45 GHz
and in the power range of 100–1000 W. Microwave energy can be transferred to kinetic
326 Engineering Tools for Environmental Risk Management – 2

energy of molecules with high dipole momentum or to ions, based on dipole-rotation

and ionic-conductance effect, respectively. In most applications the solvent is selected
as the medium to absorb microwave energy and transform it to heat in the soil sample
(Padron-Sanz et al., 2005; Fuentes et al., 2006; Esteve-Turillas et al., 2006; Azzouz &
Ballesteros, 2012; Perez et al., 2012).

3.2.3 Pressurized liquid extraction (PLE)

Pressurized liquid extraction (PLE) offers an efficient way to achieve quantitative
extraction of organic pollutants from soil samples. This technique was initially called
accelerated solvent extraction (ASE), patented for a commercial device by Dionex. In
the literature there are several alternative names such as subcritical solvent extraction
(SSE), pressurized hot solvent extraction (PHSE), pressurized fluid extraction (PFE),
and high pressure solvent extraction (HPSE). Conventional liquid solvents and elevated
temperatures can considerably reduce extraction time and the amounts of organic sol-
vent applied. At high temperature, viscosity and surface tension are less than those at
room temperature. In addition, diffusion rate and mass transfer are higher resulting in
higher solubility (Richter, 2000; Konda et al., 2002).
The basic experimental set-up is illustrated in Figure 6.7. Most PLE applications
operate within the temperature range of 50–150◦ C in which there is a compromise
between the degradation of target molecules and extraction efficiency. The pressures
applied in the PLE process are higher than the threshold value required to keep the
solvents in their liquid state. Generally 6.5–10 MPa is the optimal range. PLE can be
carried out in static or dynamic mode. In static mode, a fixed volume is used and
the efficiency of PLE depends on the analyte mass-transfer equilibrium between the
solvent and the matrix. In the dynamic mode, the solvent is passed through the soil
sample at a fixed flow rate. The time demand of PLE depends on the experimental
parameters of the extractor as well as the chemical properties of analytes and the

Figure 6.7 Pressurized liquid extraction equipment.

 Advanced methods for chemical characterization of soil pollutants 327

selected solvents. Approximately 5–15 minutes are in most cases sufficient to achieve
extraction efficiency above 90%.

3.2.4 Ultrasonic assisted extraction (UAE)

Sound waves are mechanical vibrations in gases or solids. The frequencies of ultra-
sonic waves applied in practice vary from 20 kHz to 1–2 GHz. If sound waves are
traveling through a medium, they generate expansion and compression cycles. In a
liquid the expansion cycle results in negative pressure creating bubbles or cavities. The
process by which vapor bubbles form, grow and undergo implosive collapse known as
“cavitation’’. Rapid adiabatic compression of gases and vapors within the bubbles or
cavities produces extremely high temperatures in these hot spots (about 5,000◦ C), and
the pressure amounts to roughly 1,000 bar (Suslick, 1994). Since the size of bubbles is
very small compared to the total liquid volume, the heat is rapidly dissipated resulting
in no remarkable change in the environmental conditions. Due to these processes, the
ultrasound assisted leaching for extraction of different compounds from soil matrices
is an efficient method (Luque-Garcia & Luque de Castro, 2003; Ho et al., 2012).
The efficiency of extraction depends on the polarity of the solvent, the homogene-
ity of the matrix and the ultrasonic treatment time (Babic et al., 1998; Goncalves &
Alpendurada, 2005).
The advantages and disadvantages of the extraction methods discussed above are
summarized in Table 6.2.

Table 6.2 Comparison of advantages and disadvantages of different extraction techniques.

Advantages Disadvantages

Soxhlet Simple, inexpensive equipment Large amount of solvent

Matrix neutral High time demand (up to 8–36 h)
Large size of sample
Filtration not required
SFE Small amount of solvent Expensive equipment
Time demand 30–60 min Matrix dependent
Filtration not required Small size of sample
CO2 is environmentally friendly
PLE Matrix independent Expensive equipment
Small amount of solvent Cleanup necessary
Large amount of sample
Time demand 15–20 min
Filtration not required
MAE Matrix independent Expensive equipment
Small amount of solvent Filtration required
Large size of sample Cleanup necessary
Time demand 15–20 min Polar solvent needed
Degradation possible
UAE Simple, inexpensive equipment Filtration required
Any solvent can be used Cleanup necessary
Small amount of solvent
Time demand 5–20 min
328 Engineering Tools for Environmental Risk Management – 2

3.3 Cleanup process

Since the extract generally contains co-extracted compounds causing interferences dur-
ing quantification, they must be removed. For example, the co-extractives can change
the characteristic of the chromatography column, or halogen impurities may result
in interference in the case of electron capture detection. Adsorption methods, solvent
partition, gel or ion-exchange chromatography and distillation are used as cleanup
techniques. The selection of adsorbent depends on the chemical properties of the co-
extracted compounds. A short column of alumina, charcoal, silica gel or Florisil is
sufficient to remove disturbing compounds (Dobor et al., 2010; Varga et al., 2010). The
adsorbents can be activated by pretreatment with bases, acids, organic solvents and/or
strong heating depending on their physico-chemical properties. It is recommended to
check regularly the activity of the adsorbents by recovery studies.

3.4 Preconcentration/enrichment of analytes

Enrichment of the analyte is required in some cases before the separation and detection
processes. The choice of enrichment techniques depends on the volatility and solubility
of the contaminant, e.g. pesticide residues, to be measured, the degree of concentration
required and the nature of the analytical technique to be applied. There are two options
that meet these requirements. One is the solvent removal by lyophilization, freeze
concentration, distillation, ultrafiltration and reverse osmosis. The second possibility
is the isolation of the analyte, e.g. pesticide residues mostly by solid-liquid extraction
(SPE) using polymeric adsorbents, activated carbon, polyurethane foam plugs, etc.

3.5 Separation and detection techniques

Prior to the separation step, the derivatization of analytes is recommended in most
cases in order to improve specificity, selectivity and sensitivity resulting in lower quan-
tification limits (Toys’oka, 1999; Görög, 2004). In the case of gas chromatography, the
aim of derivatization is to increase the volatility and thermal stability of analytes and
to eliminate the overlapping of analytical signals. For these purposes, silylation, acyla-
tion and alkylation/esterification reactions are generally used. Table 6.3 contains the
list of the most common reagents and the target functional groups. The derivatization
should be made prior to the injection of the sample into the GC unit.

Table 6.3 Commonly used derivatization reagents and target functional groups.

Reaction type Target functional group Reagent

Silylation –COOH, –OH, acidic and N,O-bis-trimethylsilyl-trifluoroacetamide,

basic –NH groups N-methyl-N-trimethylsilyl-trifluoroacetamide
Alkylation –COOH, acidic –OH, Diazomethane, (Perfluoro)alkyl halides,
–NH groups Quaternary ammonium/sulfonium salts
Esterification –COOH Alcohols, (perfluoro)alkyl chloroformates
Acylation –OH, basic –NH groups (Perfluoro)acyl halides, (perfluoro)acyl anhydrides
 Advanced methods for chemical characterization of soil pollutants 329

In the case of liquid chromatography, sensitivity of UV-VIS and fluorescence detec-

tions can be increased by formation of chromophore groups on the target molecules
or by a reaction with derivatization reagents (e.g. o-phthalaldehyde, benzoxadiazole,
5-dimethylaminonaftil-1-sulfonil, 9-fluorenil-methyloxycarbonylchlorid) resulting in
molecules with high fluorescence yield. It is advantageous in mass spectrometric
detection if the fragmentation only leads to a few fragments with high intensity. Triflu-
oroacetyl (CF3 CO− ) or t-butyl-dimethylsilyl derivatives are usually produced for this
purpose before the mobile phases enter the detector unit. Due to this sequence, the
separation and detection can be optimized separately.
For identification and quantitation of different organic pollutants, the capillary GC
and HPLC coupled with mass spectrometric detection are the most powerful analyt-
ical techniques. Their application makes the multiresidue analysis of pesticides (Paya
et al., 2007; Sanchez Brunete et al., 2004) or pharmaceutical pollutants (Jacobsen,
2004; Sebők, 2008) possible. However, it should be noted that reliable analytical
information can also be achieved by the application of simpler detection systems,
e.g. flame ionization and photoionization detectors for measurements of hydrocar-
bon compounds, or electron capture detector for chlorinated pesticides using GC or
fluorescence detector for HPLC.

3.6 Applications
3.6.1 Pesticide analysis
Nowadays there is a need to regularly undertake systematic surveillance and to mon-
itor pesticide residues in soil, surface and groundwater, and food commodities, etc.
in order to keep pesticidal pollution within a safe level. Amongst these environmental
compartments, soil is the largest reservoir of pesticides. Therefore the investigation
of agricultural soils is a key issue in the environmental chemistry studying the fate
and impact of pesticides residues on the living systems. A handbook gives an excel-
lent overview about the analytical methods applied for the determination of pesticide
residues in the environment (Nollet & Rathore, 2010).
A review of the literature suggests that acetone is the most preferred extracting
solvent (Babic et al., 1998; Vigh et al., 2001) for the isolation of pesticides from
soil. A mixture of acetone with hexane (Suri & Joia, 1996; Sharma et al., 2006) and
dichloromethane (Babic et al., 1998; George et al., 2007) is also favored. A variety of
other solvents e.g. hexane (Fuentes et al., 2006) or ethyl acetate (Castro et al., 2001;
Sanchez Brunete et al., 2004) are also used. GC (George et al., 2007), HPLC (Kang
et al., 2007), ultra performance liquid chromatography (Kovalczuk et al., 2008), and
capillary electrophoresis (Cooper et al., 2000) are well adaptable for separation. The
EPA method for pesticide determination in water, soil, sediment, biosolids, and tissue
applies high resolution GC-MS (EPA, 2007). Single or tandem MS equipment offer the
best capability for detecting pesticides. Due to the high number of chemical compounds
which belong to this group of organic pollutants, several authors developed analytical
methods for multiresidue analysis of pesticides (Bao et al., 1996; Castro et al., 2001;
Sanchez Brunete et al., 2004; Rissato et al., 2005). The detection limits are in the
range of 0.1–10 µg/kg depending on the target molecules and simultaneously 200–300
pesticides can be identified and quantified.
330 Engineering Tools for Environmental Risk Management – 2

3.6.2 Veterinary pharmaceuticals

Veterinary pharmaceuticals have been in use for many years as feed additives, and for
prophylactic, metaphylactic and therapeutic purposes. Because of the persistence of
different veterinary pharmaceuticals in liquid manure, these compounds reach the soil
after the fertilization process and have already been shown to persist in this environ-
mental compartment. The typical organic pollutants from this group are the antibiotics,
antiparasitic agents and the coccidiostats. The most frequently used substance classes
are listed below (Boxall et al., 2004):

– Aminoglycosides (gentamicin, neomycin B, streptomycin);

– β-lactams (amoxicillin, ampicillin, benzylpenicillin, cefazolin);
– Macrolides (oleandomycin, tylosin);
– Sulfonamides (sulfachloropyridazine, sulfadicizine, sulfadimethoxine, sulfamet-
hazine, sulfathiazole);
– Tetracyclines (chlorotetracycline, doxycycline, oxytetracycline).

Several authors developed analytical methods for their identification and quan-
titative determination. For example, persistent tetracycline residues in soil fertilized
with liquid manure were determined by a LC-MS/MS system applying electrospray
ionization following an extraction with ethyl acetate and citrate buffer. The detec-
tion limits for oxytetracycline and chlorotetracycline were 1 and 2 µg/kg, respectively
(Hamscher et al., 2002). Simultaneous extraction of tetracycline, macrolide and sulfon-
amide antibiotics from agricultural soil was carried out using pressurized liquid extrac-
tion, followed by solid-phase extraction and LC-MS/MS investigation. Detection limits
were in the concentration range of 1–5 µg/kg (Jacobsen et al., 2004). A rapid technique
based on dynamic microwave–assisted extraction coupled on-line with solid-phase
extraction was developed for the determination of sulfonamides including sulfadiazine,
sulfameter, sulfamonomethoxine and sulfaquinoxaline in soil by applying LC-MS/MS
equipment. The detection limits amounted to 1.4–4.8 µg/kg (Chen et al., 2009). A
rapid analytical method was developed based on ultrasonic extraction, solid-phase
extraction and LC-MS/MS measurement for simultaneous determination of veterinary
antibiotics and hormones in broiler manure, soil and manure compost (Ho et al., 2012).

3.6.3 Petroleum hydrocarbons

For total petroleum hydrocarbon (TPH) analysis three methods – based on gas chro-
matography, infrared spectrometry and gravimetry – have become popular in the
practice. Currently the GC-based methods are preferred since they detect a broad range
of hydrocarbons with appropriate sensitivity and selectivity, and they can be used for
identification and quantification of TPH. Chromatographic columns are commonly
used to determine TPH compounds, approximately in the order of their boiling points.
The analytes between C6 and C25 or C36 can be detected with a flame ionization detec-
tor (FID). The highly volatile compounds (below C6 ) usually cannot be quantitatively
detected due to the interference caused by the solvent peak. The detection limits are in
the range of 5–10 µg/g soil. By changing the FID to a photoionization detector (PID),
the aromatics can be selectively detected. The typical detection limit for light aromatics
(benzene, ethylbenzene, toluene and xylene, BTEX) is 5 µg/g.
 Advanced methods for chemical characterization of soil pollutants 331

GC-MS systems are used to measure concentrations of target volatile and

semivolatile petroleum constituents. The advantage of this technique is the high selec-
tivity and the ability to confirm compounds by identifying them through retention
time and unique spectral pattern. The detection limits for volatile and semivolatile
analytes in soil are 20 ng/g and 50 ng/g, respectively. It should be noted that the total
ion chromatogram of petroleum hydrocarbons is similar to the GC-FID signals.
In the field of petroleum hydrocarbon analysis focused on soil or sediment matri-
ces, numerous papers have been published in the past years. In order to demonstrate
the typical analytical procedures, the following four publications are discussed: soils
contaminated with hydrocarbons were investigated by GC-FID following an accel-
erated solvent extraction with dichloromethane-acetone (1:1) at the temperature of
175◦ C for 5 minutes (Richter, 2000). Aliphatic hydrocarbons (C9 -C27 including pris-
tane and phytane) were determined applying continuous microwave-assisted extraction
coupled on-line with liquid-liquid extraction for cleanup purposes and GC-MS tech-
nique. The limits of detection values were 0.1–0.2 µg/g (Serrano & Gallego, 2006). For
the determination of PAH compounds in sediments, a microwave-assisted extraction
was applied using 1M KOH/methanol in combination with solid-phase extraction and
GC-MS detection (Itoh et al., 2008). A novel microwave-assisted extraction procedure
using ionic liquid (1-hexadecyl-3-methyldimidazolium bromide) extractant and HPLC
with fluorescence detector offers an environmentally friendly way for the analysis of
PAH compounds (Pino et al., 2008).

3.7 Recent developments and future trends

In order to improve the isolation and identification of volatile and semivolatile organic
compounds present in complex mixtures, the multidimensional gas chromatography
(MDGC) has been developed. The fundamentals and new applications are summa-
rized in a valuable review paper (Seeley & Seeley, 2013). This analytical technique
employs two or more gas chromatographic separations in a sequential fashion. Since
the majority of MDGC separations use two columns, they are called two-dimensional
gas chromatography (2-D GC). The material transport between the two columns that
have substantially different selectivity can be regulated by special interfaces. In the case
of the heart-cutting 2-D GC equipment only the selected analytes and disturbing com-
pounds that coeluate on the primary column are subjected to a secondary separation
step. The comprehensive 2-D GC (GCxGC) technique subjects each sample component
to both separation stages. The primary separation is similar to a temperature-ramped
single-column GC-separation, whereas the secondary separations are essentially a
series of fast isothermal analyses conducted with an increasing temperature. Since
the peaks that enter the detector after the GCxGC separation have widths in the order
of magnitude of 100 ms, the detector must have a fast response. For this purpose the
time-of-flight mass spectrometers (TOFMS) offer the best analytical capabilities (Silva
et al., 2012). The usefulness of the GCxGC-TOFMS analytical system in soil analy-
sis was recently demonstrated for dioxins and polychlorinated dibenzofurans (de Vos
et al., 2011), and for PAHs (Pena-Abaurrea et al., 2012; Manzano et al., 2012).
Another important research area is the development of a rapid, simple and portable
micro-GC (µGC) system for separation and detection of analytes with various volatili-
ties and polarities within a few minutes. The multi-point on column detection applying
332 Engineering Tools for Environmental Risk Management – 2

optofluidic ring resonators (Sun et al., 2010) or Fabry-Pérot cavity sensors (Liu et al.,
2010) makes it possible to perform field measurements to localize polluted areas or
monitor the migration of organic pollutants.

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Chapter 7

Bioaccessibility and bioavailability in

risk assessment
Cs. Hajdu & K. Gruiz
Department of Applied Biotechnology and Food Science
Budapest University of Technology and Economics, Budapest, Hungary

ABSTRACT

Contaminants in the environment occur in various physical and chemical forms and in
close interaction with the environmental phases. The combination of environmental
and contaminant characteristics is responsible for this interaction, which determines
the contaminant’s accessibility. This interaction may be extremely strong in soil, sed-
iment and other solid phase compartments. Mobility, i.e. volatility, water solubility
and sorbability of a contaminant molecule and the bonding/sorption capacity of solid
matrices together determine bioaccessibility, i.e. the probability of encountering and
interacting with living organisms. Potential adverse effects on an ecosystem member
are determined by another interaction between the environmental contaminant and
the organism. A contaminant that can be taken up by an organism is called ‘bioavail-
able’. Bioavailability or biological availability determines the potential of a chemical
substance to be absorbed by an organism. The potential of the organism to absorb,
distribute and metabolize the chemical substance plays an important role in environ-
mental risk. As far as humans are concerned, the gastrointestinal tract belongs to the
‘environment’, and digestion modifies the contaminant’s bioaccessibility by liberating
the bond contaminant from the matrix.
Low bioavailability results in low probability of being absorbed by living organ-
isms and lower risk compared to a bioavailable substance with the same rate of toxicity.
Bioavailability is limited by bioaccessibility: an organism which has no access to a sub-
stance neither encounters nor interacts with it. Environmental risk management of
chemicals should place great emphasis on the chemicals’ mobility and bioavailability
in the environment because they can overwrite the risk determined by known adverse
effects.
This chapter aims to summarize the importance of mobility and bioavailability
of chemicals in risk management and introduces some studies and methods to use
bioavailability as a dynamic tool for obtaining experimental results. These results
directly relate to the environmental risk posed by chemicals simulating worst-case
situations and mimicking interactions between contaminants, environmental matrices
and living organisms.

1 INTRODUCTION

Environmental compartments function as complex and dynamic systems incorporating

three physical phases and several physical, chemical and biological interactions among
338 Engineering Tools for Environmental Risk Management – 2

the physical phases, the living and non-living compartments of the environment at
molecular, cellular and organism levels. When a substance, an agent or any living
entity, or simply energy enters this dynamic system, it changes the rate and direction
of the ongoing processes. No static balance exists in nature, and the outcome of the
changes is determined by a hypothetical balance, which, in turn, is determined by the
interactions between the living and non-living actors.
Soil is the compartment most exposed to the interactions between physical phases
because gaseous, liquid and solid phases play equally important roles in terms of both
its structure and function. The soil microbiota represents a fourth, biological ‘phase’
which is extremely important. Soil microbiota and their habitat are a microcosm within
the soil with special micro-surfaces and biofilms that significantly influence bioacces-
sibility and bioavailability of soil components and contaminants. This is the reason
why this chapter mainly deals with soil, but the same bioavailability problems are
of course present in sediments, water and air. Bioaccessibility is the prerequisite of
bioavailability, which arises only when the organisms have access to the chemical sub-
stance. Bioaccessibility and bioavailability that influence environmental interactions
are discussed with respect to i) soil as the habitat of the terrestrial ecosystem and ii)
humans exposed to soil and contaminants in soil via inhalation, ingestion and skin
contact.
Figure 7.1 shows the direct interactions of an organic contaminant with the most
relevant soil phases, and its indirect interactions with soil organisms. Contaminated
soil gas is inhaled by microbes and animals, and pore water is used by facultative and
obligate anaerobic microorganisms living in the saturated (two-phase) soil. Plant roots
may also utilize pore water. Soil moisture in the micro- and mesopores of the vadose
zone ensures the basic condition for soil life. Microorganisms and the food web relying
on them live in the pores of the three-phase soil in biofilms, and in the root hair of
the plants. The unsaturated soil structure includes a mixture of inorganic and organic
matter. The organic matter – mainly colloidal humus components – plays a major
role in the interactions with the organic contaminants while the clay minerals interact
with the inorganic ones. The main process pairs that play a role in the actual mobility
and bioaccessibility of chemical substances (both nutrients and contaminants) are the
following:

– volatilization–condensation;
– solubilization–precipitation;
– sorption–desorption;
– oxidation–reduction;
– weathering–structure formation;
– loss of soil and soil components–gain of soil and components.

Each of these processes have further types and components, e.g. solubilization
may include dissolution in water or acids, emulsification by biotensides, chelation,
complexation, enzymatic transformation, partial biodegradation, suspension, etc. The
process pairs are active both at the interface between the contaminant and physical
soil phases and at the interface between living organisms and physical soil phases. The
mutual influence between contaminants and living organisms is covered by the term
bioavailability.
 Bioaccessibility and bioavailability in risk assessment 339

Figure 7.1 Main interactions of an organic contaminant with soil and organisms living in soil.

Figure 7.2 demonstrates the interactions between inorganic substances and soil
components. The variability is fairly large due to the variable affinity of inorganic
substances to different inorganic and organic soil particles. The same processes play
a role in nutrient and contaminant bioaccessibility: this makes soil health and fertility
difficult to reconcile with soil which is contaminated with inorganics.
Soil contaminants are generally mixtures of chemicals, and the interactions with
soil phases, including the biota, may result in an infinite number of combinations.
Changing environmental conditions or contamination events may trigger continu-
ous changes in the physical, geochemical, nutrient and habitat status of the soil as
well as in the density and diversity of the soil microbiota. The continuously chang-
ing non-equilibrium system is able to adapt to these conditions up to a certain
limit.
Age of soil contamination is a crucial characteristic that influences accessibility
and availability and, as a consequence, the risk of the contaminant. Sorption of the
contaminants to solid surfaces and the type of chemical bonds continuously change.
The direction of these changes depends on the type and evolutionary phase of the soil
as well as on the environmental conditions. When humus formation is the dominant
340 Engineering Tools for Environmental Risk Management – 2

Figure 7.2 Main interactions of an inorganic chemical substance with soil components.

process in a soil, persistent organic contaminants may stably be built into the structure
of the newly formed humus colloids. When clay minerals formation is the dominant
process, metal ions are integrated into the clay structures. When sulfides are formed
in anaerobic sediments, metal ions or oxides will be transformed into metal sulfides,
even in crystalline forms. These processes result in biologically inaccessible forms of
the contaminants. These kinds of stabilization processes occur in healthy soils and
sediments, but these relatively stable forms are also part of the dynamic equilibrium
system, meaning not completely irreversible binding. Regressive soil evolution pro-
cesses, i.e. most soil degradation processes such as humus and clay disintegration or
acidification, may mobilize the contaminants. The result in these cases is an increased
risk not only in the soil, but also in the subsurface and surface waters in contact with
the contaminated soil.
The effect of the environmental conditions on the soil contaminants’ accessibility
may also be significant. Organic contaminants go through oxidization, condensation,
aggregation, and may form molecules of large size and low accessibility. In the case
of metals the combinations of pH and redox potential (Eh) may result in completely
different ratios of metal species such as ionic forms, oxides or hydroxides of different
metal valences and sulfides. The stable mineral forms of metals in aqueous media are
described by the so-called Eh–pH diagrams (also called Pourbaix diagrams after the
name of its originator). These diagrams are constructed from calculations based on
the Nernst equation and equilibrium solubility data of the metal in question and its
species. The stability regions (read from the diagrams) are good for indication, but the
actual, non-equilibrium values may differ from them given that metals always occur
together with other molecules in the environment. Research is in progress on the Eh–pH
diagrams of metals in natural aquatic systems and soils. Brookins (1988) developed
 Bioaccessibility and bioavailability in risk assessment 341

Eh–pH relationships based on thermodynamic data describing interactions between

copper and potential inorganic ligands (oxides, hydroxides, carbonates, and sulfides) in
natural aquatic systems (wetlands). Lin et al. (2011) characterized the stability of iron
and manganese in saturated and unsaturated soils and groundwater by plotting Eh–
pH diagrams. More knowledge on these relations would support hazard assessment
of chemicals and upgrade the screening phase of environmental risk assessment of
contaminated sites.

2 MANAGING BIOACCESSIBILITY AND BIOAVAILABILITY OF

CONTAMINANTS IN THE ENVIRONMENT

The chemical approach in environmental risk management means that the risk is
calculated based on generic environmental parameters and environmental fate char-
acteristics of the chemical substances (regulatory risk management) or on the results
of analytical measurements (contaminated sites). Analytical methodologies seek the
exhaustive extraction of contaminants from environmental samples or try to differen-
tiate between chemical species and extractability of the contaminating chemicals by
partial and sequential extractions. Those chemical models which intend to model the
“extraction’’ by biological systems (bioavailability) are called biomimetic extraction
methods.
It has increasingly become evident that adverse effects of contaminants in the envi-
ronment are not proportional to their concentrations measured by chemical analysis.
When water and groundwater are the focus of environmental management, the water-
extractable fraction of soil, sediment or solid waste represents the toxic fraction that
poses risk to the aquatic ecosystem and water-consuming receptors. In contrast, water-
extractable and biologically available fractions of the soil are not identical and both
represent smaller portions than the total contaminant content in the soil as illustrated
in Figure 7.3. The water-extractable fraction is the result of interactions between soil,
contaminant and water. Water competes with soil solid to acquire the contaminant. The
bioavailable fraction results from the interaction of the biological organization/system
with the contaminant partitioned between solid and liquid phases of the soil. The bio-
logical system actively participates in this interaction by producing chelating agents,
biotensides, degrading enzymes, etc. Both water extractability, bioaccessibility and bio-
logical availability are influenced by environmental conditions and the relative ratio
of the participants.
The risk posed by hazardous contaminants in soil, sediment and solid waste is
redefined by modeling or measuring the bioavailable fraction of contaminants. Test
methods characterizing fate and behavior as well as adverse effects of chemicals in
the environment are continuously developed and disseminated. The main aim of these
tests is the correct estimation of the actual risk originating from a direct contact and
material transport between contaminated environmental matrices and receptor organ-
isms. A part of a contaminant is mobile and has the ability to be dispersed in the
environment and exert an adverse effect, while another part is immobile and has no
impact on the biotic and abiotic environment due to its stable chemical form and strong
bond to matrices. This condition may be easy to modify or may be very stable. The
dynamic behavior of contaminants in the real environment must be studied in order
342 Engineering Tools for Environmental Risk Management – 2

Figure 7.3 Relationship between total, extractable, bioaccessible and bioavailable proportions of a soil
contaminant.

to be able to differentiate between mobile, mobilizable and irreversibly immobile con-

taminants. Based on this information, bioaccessibility can be determined for hazard
assessment purposes by “only’’ considering the chemical substance, the matrix and the
environmental conditions but not the entire biological system.
Furthermore, soil remediation can be efficient in reducing risk when contami-
nant mobility and the resulting bioaccessibility in soil are hindered or eliminated.
In such cases the immobilized contaminant is still present in the environment but
does not pose any risk because it is not accessible, i.e. is not in contact with living
organisms due to stable physical and chemical forms and not available, i.e. it can-
not be taken up by living organisms. At the same time the physically or chemically
stable/immobilized contaminants are not available to water, which lowers the risk of
contaminant transport by water from soil. The risk-based approach considers contami-
nant stabilization/immobilization as an efficient risk reduction method. Unfortunately,
many countries and authorities prefer the chemical model based approach, and do not
accept immobilization as a full-value remedial measure. The crucial feature of contam-
inant immobilization in soil is the scale of reversibility: highly irreversible stabilization
is a full-value solution for risk reduction.
The management of a complex and dynamic system of contaminated soil requires
complex and dynamic engineering tools such as environmental monitoring, risk
assessment methods and remediation technologies. When assessing the actual risk
posed by contaminated soil, the mobility and bioavailability of the contaminants
 Bioaccessibility and bioavailability in risk assessment 343

should be taken into consideration because the actual adverse effects of the contami-
nants greatly depend on their bioavailability. As risk is the probability of damage, it is
clear that managing risk on the basis of a contaminant property such as the dynam-
ically changing bioaccessibility in the environment can be a problem and it is not
manageable mechanically. How should bioaccessibility and bioavailability be deter-
mined and taken into consideration to increase the certainty of risk assessment? There
is no general answer, various modes can be and have been applied to assess the ‘true’
bioavailability as will be discussed below.
Risk may be overestimated if mobility and availability of environmental contam-
inants are not taken into consideration, thus leading to unnecessary expenditure on
risk reduction. Underestimation due to momentary and short-term non-availability
of contaminants is even more detrimental as it can result in a ‘chemical time bomb’,
ready to ‘explode’ at any time. Risk management should maneuver between these two
extremes to find the optimal conservative solution.
Risk and consequently the cost of risk reduction should reflect the real situation,
i.e. the actual risk. For example, the risk posed by toxic metals in soil should not be
evaluated based on analysis results from an aqua regia extract, but as a better option,
based on an eluate or leachate resulting from the strongest possible acid rain, which
is still a pessimistic scenario. This concept reflects the fact that environmental condi-
tions influencing accessibility cannot vary within infinite limits, but within a certain
range.
Bioaccessibility can be modeled by mathematical or chemical models and the
results used for hazard assessment or contaminated site risk assessment in the screening
phase. Bioavailability should be determined based on the results of tests that enable the
interaction between contaminated soil and exposed organisms. Instead of ecotoxicity
or toxicity tests one can apply mathematical models based on existing test results or
biomimetic chemical tests, which give an answer close to the biological aspect. These
results can be used for generic or site-specific risk assessment.

2.1 Mobility, bioaccessibility, bioavailability and

risk assessment
When assessing a contaminated site, the results of direct chemical analyses carried
out on the relevant samples may show poor correlation with actual effects. Chemical
models based on direct sample analysis have little relevance to the real environment
when the contaminant’s bioavailability is limited. An integrated approach is needed
to obtain a more realistic picture about the risk posed by pollutants in soil. This
approach comprises complementary ecological or biological (ecotoxicity, mutagenicity,
reprotoxicity, food-chain effects, diversity, etc.) tests and physico-chemical analysis of
the contaminant and the soil.
Another possibility to move closer to environmental reality is dynamizing the
chemical model system and combining it with the testing of adverse effects. Dynamiza-
tion means in this context that the model should simulate the dynamic processes
expected in the soil, e.g. possible changes in humidity, temperature, decrease or increase
in pH and redox potential, changes in the nutrient supply, etc. Dynamization may
involve gradual changes or impulse-like intervention (a push) to establish a realistic
worst-case scenario in the test. The aim is to simulate the changing conditions and the
344 Engineering Tools for Environmental Risk Management – 2

maximum risk which may occur in the environment, but not more: a realistic max-
imum should be achieved and simulated by changing the conditions. Dynamic tests
can be implemented in the laboratory or on the field. Monitoring of changes provides
information for the evaluation and characterization of the probable worst case. In
a dynamic test system, one can investigate the speed, intensity and duration of the
response of the soil and the soil-living organisms. Dynamic testing and using its results
for risk assessment is a tool which makes risk management optimally efficient both
environmentally and economically. The results of dynamic tests may contribute to a
correct risk assessment and to the planning of risk reduction.

2.2 Risk reduction in view of mobility and

bioavailability
Reduction of the risk posed by soil contaminants may be based on physical, chemical,
thermal or biological mobilization and removal of the contaminant or on the oppo-
site: immobilization, stabilization and hindrance of its availability to water and living
organisms. In the latter case, the contaminant remains in the soil or sediment, but in
a stable and immobile form.
Reduced mobility and bioavailability of contaminants in the soil are equivalent
to reduced short-term risk, but only dynamic tests can determine the contaminant’s
stability (irreversibility of stabilization) under varying environmental conditions. Tem-
perature, pH, redox potential, humidity, ion concentrations, microbial activity and
the equilibrium state of the soil processes influence the participating molecules’ mobil-
ity. Over the long term, increased bioavailability may lead to biotransformation and
biodegradation, which are generally risk-reducing processes.
The selection of the proper risk management measures depends to a large extent
on mobility/bioavailability of the contaminants in soil:

– If mobility and bioaccessibility of the contaminants are irreversibly reduced, both

short- and long-term risks are low, and safety monitoring is the only necessary risk
management measure.
– If mobility and bioaccessibility are only reduced over the short term, the risk is
temporarily reduced; therefore continuous monitoring and a final risk reduction
measure are needed.
– If the contaminants’ mobility has not been and cannot be reduced, the risk is high;
immediate risk reduction is necessary using remediation technologies other than
stabilization.

In those cases when the goal is the removal of toxic contaminants, efficiency
of remediation based on contaminant biodegradation is often limited by the con-
taminants‘ low bioavailability. Bioremediation of soils contaminated with high
molecular-weight hydrocarbons such as polycyclic aromatic hydrocarbons (PAHs),
chlorinated aliphatic and aromatic hydrocarbons, persistent pesticides or polychlori-
nated biphenyls (PCBs) may be slow, despite the presence of the microorganisms able
to degrade these contaminants. This is one of the reasons why the more expensive and
drastic chemical and thermal methods are preferred in their elimination. Limited bioac-
cessibility and bioavailability can be mitigated by pollutant-solubilizing (mobilizing)
 Bioaccessibility and bioavailability in risk assessment 345

additives applied in the bioremediation technology. Several innovative technologies are

based on the use of surfactants, cosolvents, nanomaterials and other mobilizing agents
using molecular encapsulation and micelle formation. In general, these additives have
proved to be effective bioaccessibility and bioavailability enhancing agents, but they
may also be recalcitrant to biodegradation in the amended soils and toxic to higher
soil-living organisms in charge of pollutant degradation. Fava et al. (2010) summed
up the results of a number of studies documenting the advantages related to the use of
commercially available biogenic products such as technical mixtures of hydroxypropyl
beta-cyclodextrins, randomly methylated beta-cyclodextrins, soya lecithins or humic
substances as pollutant bioavailability enhancing natural or naturally friendly agents
in the bioremediation of soils historically contaminated by hydrophobic organic pol-
lutants. Fava pointed out that cyclodextrins and saponins can be used as biological
agents without causing additional environmental risk.

3 BIOAVAILABILITY AND BIOACCESSIBILITY – DEFINITIONS

Before giving a precise definition, the topic discussed above will be recapitulated. The
fate and effect of toxic contaminants in soil and sediment are greatly influenced by their
partitioning between physical phases, their physical and chemical forms, as well as by
the environmental conditions. These together determine the pollutants’ bioaccessibility.
Bioavailability assumes the interaction with organisms. The attention of environmental
scientists has been focused on these two terms in the last 15 years; but a clear definition
and interpretation has still not been given and no uniform evaluation method has been
established despite the fact that bioaccessibility and bioavailability of contaminants in
soil and sediment constitute key parameters of their environmental risk.
For humans, pharmacology and toxicology have had a long-standing definition for
the bioavailable part of a drug that reaches the circulatory system, while the bioaccessi-
ble fraction is the part that has the potential to reach the circulatory system once taken
up by the organism/body. This definition can be modified/adapted to a case where a
person swallows contaminated soil instead of a pure drug in a controlled matrix of
the vehicle1 . The problem is compounded if a mixture of contaminants or different
chemical forms of the same contaminant are present in the soil.
In the context of environmental pollution a molecule is said to be bioavailable
when it is ready to cross an organism’s cellular membrane from the environment. It
can only happen if the organism has access to the chemical (Kirk et al., 2004), and is
able to absorb it. The organism has access to those soil contaminants which are not
very strongly bound to the solid matrix and are not located in a confined inclusion,
or do not form a non-aqueous liquid phase. It depends on the physical and chemical
form of the chemical substances as well as on the characteristics of the soil matrix and
on the environmental conditions.
The contaminant in the soil is bioavailable when terrestrial organisms are able
to take it up: the biophysical and biochemical interactions between the substance
and the organism are able to ensure it. Bioavailability is organism-specific: what is

1
A substance of no therapeutic value which conveys an administered drug.
346 Engineering Tools for Environmental Risk Management – 2

available for a microorganism, is not necessarily available for a worm or a plant

root and the opposite. When microbial processes are in the focus, e.g. biodegradation
and soil microbiota are considered as a whole biodegrading entity, bioaccessibility
and bioavailability may be overlapping to a large extent. Not, because knowledge is
not enough to differentiate, but also because of the already mentioned flexibility and
adaptability of the soil microbiota: when the contaminant is in an accessible form, it
is highly probable that there are community members which can interact with it.
The topic can further be complicated by distinguishing between the ‘ability’, the
potential access and the actual access to, as well as the potential availability and the
actual uptake of a contaminant. For example large-size or non-motile microorganisms
or soil-dwelling animals have no access to contaminants in very narrow soil capillaries,
even if the contaminant is accessible (in terms of its chemical form and sorption)
and available to bacteria (in terms of the existence of the interaction between the
contaminant and microorganism). Plant roots which have access to and can take up
available toxic metals, can also avoid metal-contaminated soil particles by chemotaxis,
resulting in no actual uptake. Soil-living animals can alter soil structure and, as a result,
the accessibility of contaminants. The same is valid for sediment-dwelling organisms,
which actively contribute to changing the chemical forms of sediment contaminants
by resuspending, mixing (bioturbation) and increasing the redox potential in surficial,
sometimes also in the buried sediment layers.
Bioaccessibility and bioavailability in soil is a limiting factor in the removal of
toxic substances by biodegradation, or in reducing the risk by advantageous biotrans-
formation. Plant nutrient accessibility and bioavailability play an important role in
crop production: hindered or blocked nutrient uptake, e.g. phosphorus deficiency can
be traced back to soil acidification and the accompanying precipitation of phosphates
in non-soluble chemical forms.

3.1 Definitions and mechanisms

Bioaccessibility can be defined as the potential of a chemical substance to encounter
and interact with an organism. It is the characteristic of the substance, and depends
on its physical and chemical properties in interaction with the environmental matrix
and environmental conditions. Matrix effects can significantly decrease the bioac-
cessibility of a soil- or sediment-bound contaminant. It is not actively influenced by
biological organisms. The actual access of specific organisms is mainly determined by
physical conditions such as depths or pore size. Bioaccessibility is a precondition to
bioavailability. Bioaccessibility can be determined and studied by chemical models or
chemistry-based mathematical models. The bioaccessible part of contaminants is never
less (maximum equal) than the bioavailable part.
Bioavailability specifies the amount of a chemical substance which reaches the
site of physiological activity after adsorption/uptake. In this relationship the living
organism and its physiology and anatomy as well as the exposure route and the
pharmacokinetics play an important role. The actual adsorption is influenced by
bioaccessibility and the organism’s conditions and behavior. Bioavailability can be
determined and studied by biological or biomimetic (chemical) models or mathematical
statistical models based on them.
 Bioaccessibility and bioavailability in risk assessment 347

Figure 7.4 Bioaccessibility and bioavailability of soil contaminants.

The bioaccessible fraction can be considered as the potentially bioavailable pool

of the pollutant. In soils and sediments the difference between the bioaccessible and
bioavailable amount can be significant, so that bioaccessibility is a preferred term in soil
science, while aquatic ecotoxicology mainly uses the term of bioavailability assuming
that a large part of the contaminant is in dissolved form (which may not be true when
suspended solid matter is also present).
Several definitions have been established, mainly in pharmacological, but also in
environmental context, e.g. Wiktionary (2014) or the IUPAC Glossary (2014). The
selection introduced here may clarify the proper meaning and the differences between
these terms. In soil, the bioavailable fraction of the contaminating compound is free to
penetrate the organism’s cell membrane at a given time (Semple et al., 2004), and can
be transferred into the circulatory system or other tissues where it has the ability to
develop a biological response (Ruby et al., 1993). Bioaccessibility is the integration of
(i) desorption of the contaminant from the soil and (ii) its transportation to a biologi-
cal membrane (Semenzin et al., 2007; Puglisi et al., 2009), called external availability
by Caussy (2003). The final step, (iii), i.e. the uptake through the biological mem-
brane is jointly determined by the bioavailability and by the previous step that limits
bioaccessibility (see also Figure 7.4).
In the case of soil-living microorganisms, the three steps of the uptake process are
not differentiated, and the situation is simplified considering the bioaccessible and the
348 Engineering Tools for Environmental Risk Management – 2

bioavailable fractions to be the same. In turn, soil-living organisms have a significant

impact on the mobility of the contaminant in both directions: they are able to further
mobilize the contaminants by acidic exudates, biotenzides and specific exoenzymes,
or can do the opposite and immobilize the toxic contaminants in order to be protected
by hindering uptake. Another simplification in soil and sediment ecotoxicology is that
the exposure routes of the organisms are not exactly known. That is why they are not
taken into account, but it is assumed that there is a direct contact between external
and internal surfaces of the organism and the soil (similar to Daphnia or small fish
and water). From the toxicity testing point of view it is still important to know the
exposure routes of the test organism, otherwise the proper measurement end point
cannot be selected. For example, worms digest soil but springtails do not, i.e. oral
administration is negligible for springtails.
For higher organisms, including humans, the first step is the ingestion or inhalation
(bioaccessibility), which, as a result of the digestion steps, provides the bioavailable
fraction with the ability of passing through the cell membrane and spreading in the
cell or tissue. Assessing the risk of inhaled and ingested soil starts with the problem of
the amount ingested. Children, the most exposed members of the human population,
ingest, inhale and dermally adsorb a significant amount of soil and dust. Chemicals
which are inhaled, dermally adsorbed or ingested and mobilized in the gut fluid are
bioaccessible (Caussy et al., 2003; Peijnenburg & Jager, 2003; Ren et al., 2006).
This definition does not hold for soil microorganisms, so, it is worth distinguish-
ing between organisms contacting soil with their external surface and those which
ingest the contaminated soil. One can expect, in both cases, a significant impact
of the organism on contaminated soil and contaminants. For example, root exu-
dates are able to dissolve non-ionic metals or exchange metal ions with hydrogen
ions. Bacterial tenzides can form micelles from water-insoluble large-Kow substances
and mobilize them. Higher animals ingest soil and digest it with a series of acidic,
alkaline and enzyme-containing digestives. One could hardly create a chemical model
that includes all these details, but well-chosen biotests or test batteries can incorpo-
rate all these availability-dependent data into the biological response of soil-dwelling
organisms.
Further related terms are availability and mobility which mean similar behav-
ior but in a broader sense and context: availability in the environment means that
the chemical is available not only for living organisms but also for air or water and
the term mobility considers the same fate characteristic from the chemical substance’s
point of view. Availability of a chemical substance means the extent to which this
substance becomes soluble, disaggregates and becomes mobile via air, water or biolog-
ical organisms. For metal availability, the extent to which the metal ion portion can
disaggregate from the rest of the compound is the main influencing factor. Availability
is not a prerequisite for bioavailability (GHS, 2011).

3.2 Contaminants’ location and form in soil and the related

accessibility and availability
Contaminants in soil represent a wide range of physical and chemical forms. The
following overview in Table 7.1 summarizes the different occurrences, their bioacces-
sibility and bioavailability.
Table 7.1 Bioaccessibility and bioavailability of different type of soil contaminants.

Physical, Bio- Bio-

Contaminant Location in soil chemical form accessibility availability Organism Remarks

Volatile organic Soil gas Gas and ++ ++ Soil-living and terrestrial Transport to atmospheric air is a risk (CO2 ,
chemicals and vapor organisms methane, chlorinated and fluorinated organics, etc.).
metabolic products
Organic Groundwater Water- ++ ++ Facultative and obligate Transport to surface waters and drinking water
chemicals pore water soluble anaerobic microbes, is a risk. Food chains/webs are endangered.
plant roots, food web
Organic Soil moisture Water- ++ ++ Aerobic and facultative Precipitation infiltrate or capillary upload from
chemicals capillary waters soluble anaerobic microbes, groundwater may differ. Significant adverse effect
plants, soil-dwelling on food chains/webs.
animals, food web
Organic Biofilm Soluble ++ +++ Biofilm organisms: Strong interaction between contaminant and
chemicals bacteria and microbial biofilm organisms in the biofilm and efficient
food chain co-operation of the organisms may enhance
biodegradation.
Organic The surface of Adsorbed ++ ++ Microorganisms, Become bioavailable after desorption.
chemicals organic matter animals Microorganisms have versatile tools to
particles increase substances’ bioavailability (biotensides,
complexing agents, enzymes, mucous fluids, etc.).
Both in two- and three-phase soils.
Organic The matrix of Absorbed, (+) + Soil ingesting animals Become accessible by weathering. Become
chemicals soil organic covalently bioavailable as a result of animal digestion. Mainly
matter particles built in in three-phase soil.
LD NALP Surface Free layer ++ ++ Aerobic, facultative The plume moves at the interface of the two- and
plume anaerobic and obligate the three-phase soil. Microbial degradation may
anaerobic microorganisms, occur on both sides of the plume. Soil-dwelling
plant roots, (animals) animals and some plant roots are able to avoid it.
LD NALP Inside Free layer − (+) Physically not accessible, The inside of contaminant plumes is highly persistent
plume although the chemical itself due to no access by the microbiota or to water,
may be bioavailable oxygen/electron acceptors, nutrients, etc.
HD NALP lens Surface Free layer/ + + Mainly anaerobic The lens is stable on the surface of the first or
lens microorganisms second impermeable layer (confining bed).

(continued)
Table 7.1 Continued

Physical, Bio- Bio-

Contaminant Location in soil chemical form accessibility availability Organism Remarks

HD NALP lens Inside Free layer/ − (+) Physically not accessible, The inside of the contaminant lens is highly
lens although the chemical itself
persistent due to no access by the microbiota, or
may be bioavailable to water, electron acceptors, nutrients, etc. Risk
of penetration of the water-impermeable layer.
Organic Soil organic Mixed in (+) + Only the surface is It becomes accessible as a result of weathering.
particulate matrix particle, physically accessible, the Weathering of the organic matter desintegrates/
matter part of soil inside not, although the disaggregates the formerly hardly accessible particle.
texture chemical itself maybe It becomes bioavailable as a result of animal digestion.
bioavailable Both in the two- and three-phase soil.
Volatile metal Soil gas Gas or vapor ++ ++ Soil-living and terrestrial Transport to atmospheric air is a risk (mercury,
species organisms methyl mercury)
Metal ions Groundwater, Dissolved ++ ++ Microorganisms, plant Transport to surface waters and drinking water is a
pore water roots, animals, food web risk. Food chains are endangered.
Metal ions Soil moisture, Dissolved ++ ++ Microorganisms, plant Precipitation infiltrate or capillary upload from
capillary waters roots, animals, food web groundwater may differ. Significant adverse effects on
food chains/webs may occur.
Metal ions Biofilm Dissolved ++ ++ Biofilm organisms Cooperation between biofilm organisms ensures
efficient protection (resistance).
Metal ions Surface of clay Adsorbed + + Plant roots, Desorption by ion exchange assisted and enhanced
minerals and microorganisms, animals by interacting organisms: e.g. acidic root exudates,
oxides microbial complexing agents, etc. Both in two- and
three-phase soil.
Metals (ions, Matrix of clay Absorbed or (−) + Specialized microorganisms Physically not accessible before weathering, soil
oxides, silicates, minerals and built in deterioration or significant changes in environmental
sulfides) oxides conditions (e.g. metal sulfides come to the surface
by dredging of bottom sediment or mine-excavation
of sulfide ores). May act as a chemical time bomb.
Metals/inorganic Soil texture Mixed in (+) + The surface is physically It becomes accessible due to weathering.
particulate matter particle, accessible, the inside not. Desintegration and disaggregation increase
part of soil The chemical itself maybe accessibility and availability. Mainly in
texture bioavailable. two-phase soil.
∗ LD NALP: low density non-aqueous liquid phase
∗∗ HD NALP: high density non-aqueous liquid phase
 Bioaccessibility and bioavailability in risk assessment 351

4 ASSESSING BIOAVAILABILITY OF CONTAMINANTS

Risk assessment should include accessibility and availability of the contaminants in

soil because they have significant influence on the adverse effects and risks. Test meth-
ods which are expected to be environmentally realistic should ensure direct contact
between the contaminant and the biological system or the chosen test organism. If the
test concept cannot assure direct contact, availability should be accounted for by an
empirical factor due to the accessibility of the substance.
Bioavailability suggests that the chemical substance spreads in the cytoplasm and
reacts with the living cell. Organisms take up the bioavailable fraction of the contam-
inant and, depending on the metabolic potential of the organism, the contaminant
can be

– accumulated in the organism with or without modification;

– secreted from the organism without or after modification;
– transformed or degraded by the organism’s metabolic pathways.

The residence time of the contaminant in the organism’s cell or body and the
interactions with the morphological or functional part, i.e. receptor sites or molecules
of the organism may result in changes in metabolism (enzyme activities) or adverse
effects (toxicity, neurotoxicity, genotoxicity, reprotoxicity, etc.) in the body. The most
pronounced changes as a result of the contaminant’s effects can be used as measured
end points in a toxicity test.
While bioavailability depends on both the chemical substance and the organisms
e.g. a particular species exposed to the contaminant (Stokes et al., 2006), the size of the
bioaccessible fraction depends on the chemical substance and the environmental con-
ditions (Hamelink et al., 1994). If the requirement is to simulate the real environment,
the test should ensure that the test organism and the environment do interact.
Bioavailability cannot be considered as physico-chemical partitioning like water
extractability from soil, which is in direct relation to the soil–water partition coef-
ficient and Kow of the chemical substance. The bioavailable fraction depends on the
structure and activity of the living participant that interacts with the chemical sub-
stance. These living organisms with a dynamic adaptive behavior and active membrane
systems which influence metabolic and physiological characteristics have a signifi-
cant impact on the fate of chemicals in the environment. Standardized bioavailability
tests on ‘standardized’ test organisms are suitable for the comparison of chemicals,
but real bioavailability is always case-specific. Mathematical models or standardized
simulation tests are used in general to determine ‘bioavailability of a contaminant’.
In simulation tests the pure chemical is ‘mixed’ into a standard or reference soil, sed-
iment or waste before being tested. These simulated environments are considerably
different from the real ones. However, real environmental bioavailability of a contam-
inant can only be tested via measurable adverse effects or other activities. However, it
should be borne in mind that the results of a toxicity test include bioavailability in a
non-distinguishable form from the adverse effect.
Bioavailability, biodegradability or the bioaccumulation potential of pure chem-
icals are fictions, their tests create a paradoxical situation: one tries to characterize
an interaction-specific chemical property without the interacting partner or by a
352 Engineering Tools for Environmental Risk Management – 2

standardized substitute in these tests. It is clear that the results of such tests have a
limited value, but they still provide useful information in a generic or screening step of
a tiered risk assessment procedure. Ahlf et al. (2009) studied the incorporation of metal
bioavailability into the European regulatory framework. They proposed the use of the
biotic ligand model (BLM) to predict the bioavailability-dependent ecotoxicological
effect of metals in the environment.

4.1 Bioaccessibility and bioavailability assessment methods

The methods for assessing bioaccessibility- and bioavailability-dependent adverse
effects – similar to any other environment assessment methods – are models of the
real environment. Some of them are very distant models, while others are closer to the
environment. The basic methods for testing mobility and bioavailability of toxicants
as well as the terms used are explained here.
Mathematical models must be based on a large number of data. Depending on
the quality and quantity of data, they may truly represent reality and are very useful
tools in regulatory and predictive risk assessment. When modeling bioaccessibility, the
chemical speciation of the contaminant, its binding to the components of the soil matrix
and the environmental conditions must be considered. In the context of bioavailability,
geochemical conditions, contaminant and matrix interaction as well as the organisms’
characteristics and activity must be taken into consideration.
Physico-chemical models are mainly based on the empirical correlation between
the physico-chemical characteristics of a molecule and its fate and behavior in the
environment. The most plausible example is establishing a link between the concen-
tration of a chemical substance and the extent of its effect. A correlation measured
in vitro between concentration and response of pure chemicals is not valid for the soil.
In the context of bioaccessibility, one can mention the AVS concept developed for the
bioavailability of metals in sediments: sulfide-bound metals are described by the ratio
of simultaneously extracted metals and acid volatile sulfide. In their BLM model, Ahlf
et al. (2009) considered the dissolved phase, metal complexes, dietary, and particle-
bound metals. This model determines bioaccessible metal fraction, and based on that,
predicts the probable toxicity in the environment.
Simple biological models are based on the contaminant’s bonds to receptors or
membranes, transport through the skin or the cell membrane, on the toxicity, bioaccu-
mulation or biodegradation by single species. Biotests do not measure bioavailability
directly, but the uptake or the effects, assuming that the degraded or bioaccumulated
contaminant that shows an adverse effect, has been available to the test organism. If
the response is positive, it is obvious that the effect has occurred, proving that the
contaminant has been bioavailable. But if the response of the test organism is neg-
ative, it is hard to decide whether the contaminant is not available or the effect has
not materialized due to the organism’s characteristics. The test organism may have a
protective tool and may not be sensitive to the toxicant. In the case of biodegrada-
tion, the test organism may have no enzymes for degrading the otherwise ‘available’
contaminant. Organisms secreting contaminants instead of accumulating them are not
suitable for measuring bioavailability through testing their bioaccumulation. Summing
up, confirming bioavailability by testing the fate and effect of the contaminants after
interaction with the test organism(s) is a simple task. But the opposite, demonstration
 Bioaccessibility and bioavailability in risk assessment 353

of the lack of bioavailability needs an integrated approach, e.g. i) testing several dif-
ferent toxicity end points on a number of test organisms or ii) the application of
dynamic tests for the simulation of an increased or maximized (highest realistic scale)
bioavailability. iii) Another way to obtain a clear picture is the stepwise application of a
physico-chemical, e.g. BLM model for metals and the biological testing of the sample’s
toxic effect. An integrated evaluation can provide a larger number of combinations
and refine the information on the assessed environment:

– bioavailable contaminants are toxic to the ecosystem;

– bioavailable contaminants are non-toxic due to the ecosystem’s (or its members’)
low sensitivity or resistance;
– non-bioavailable contaminants are non-toxic to the ecosystem (members);
– non-bioavailable contaminants are not irreversibly immobile, and toxicity emerges
after a certain time
◦ as a result of interactions with the ecosystem, e.g. the mobilizing effect of
plants by root exudates or
◦ as a result of changes in environmental conditions and following uniden-
tified transformations, e.g. acidification and consequent solubilization of
precipitates, or resuspension and oxidization of anaerobic sediments, etc.

The conclusion is that biological models are closer to the real environment than
other models but still cannot perfectly model the interactions in a complex system and
to distinguish bioavailability from uptake and the effects of contaminants. To avoid
misinterpreting the negative biological test results, and, as a consequence, underesti-
mating risk, the actual state of the contaminated soil/sediment must be evaluated in
an integrated way using joint physicochemical analyses and ecotoxicity tests.
The triad approach is an environmental characterization and monitoring tool
that applies the combination of physico-chemical, biological/ecological and toxicolog-
ical assessment tools and evaluates their results in an integrated way, relative to each
other. Application of the ‘triad’ approach has several advantages in comparison to
only chemical or only biological assessments. The integrated evaluation using the soil
testing triad will be introduced for the interactive tests and the dynamic simulation
tests in Section 7.6.
Testing bioavailability or bioavailability-dependent toxicity requires the applica-
tion of interactive tests – also called contact or direct contact tests – which allow for the
test organism to interact with the soil matrix and the contaminants. The test organism
exerts an impact on the soil and on the contaminant by the produced acids, and other
exudates, exoenzymes, biotensides, etc.
Simulation tests try to simulate real conditions, integrating all important compo-
nents and actors of the environmental process in which environmental contaminants
encounter and affect living organisms. A good example is the multi-step digestion of
contaminated soil before human toxicity testing.
Dynamic simulation of contaminant bioavailability and bioaccessibility means
creating test conditions which increase availability and accessibility compared to the
original situation. The aim is to achieve maximum realistic mobilization/solubilization
of contaminants under conditions totally different from normal natural conditions.
This can be achieved by changing temperature, pH and redox conditions, quantity
354 Engineering Tools for Environmental Risk Management – 2

and quality of precipitation, flood on floodplains, exposure to light and the presence
of organic matter in the soil or groundwater, etc.
Ensuring real-scale or greater bioavailability and bioaccessibility during the test-
ing of adverse effects is an essential requirement, otherwise the effect measured in
the test may become smaller than in reality leading to risk underestimation. Due to
uncertainties, some overestimation is generally desirable meaning that accessibility and
availability of contaminants should represent a realistic maximum, which is the real-
istic worst case from the point of view of risk assessment. Exhaustive extraction of soil
contaminants represents a worst case which would never occur in the real environment
and it would result an extreme overestimate in environmental toxicity and risk.
The same concept can be used to forecast the mobilization of a contaminant dur-
ing soil remediation, e.g. soil washing, remediation based on chemical oxidation or
biodegradation with the aim to enhance the removal of the contaminant from the soil
using physical, chemical or biological technologies.
To model the realistic environmental scenario, the concept should include both the
environment and the body of an organism as reactive media since both can change the
contaminant and its availability. The environmental parameters such as temperature,
pH, redox potential, the presence of other contaminants and the components of the
solid matrix can change the physical and chemical form of the contaminant and, as a
result, its mobility, accessibility and availability. The body of the organisms exposed
to contaminated soil is even more reactive, able to switch to generic and contaminant-
specific metabolic and transport mechanisms (interactions by the gastrointestinal tract,
active transport through skin or inner membranes, secretion in a chemically mobi-
lized form or accumulation in a chemically bound, immobile form) or create specific
responses (toxicity or immune response). When measuring the potential adverse effects
of a contaminant in soil, the test method should let the risk-increasing changes take
place. A moderately pessimistic test concept simulates the realistic worst case. The
results of such tests can fit into a tiered and iterative risk assessment procedure and
used for decision making.
For environmental risk assessment, the test organism is placed into the con-
taminated soil and the response of the organism is measured. The response can be
lethality, change in energy production or any other metabolic and enzyme activity,
the absorption, accumulation or biodegradation of the contaminant (earthworm or
plant bioaccumulation). Biodegradation is an advantageous interaction between soil
microbiota and the contaminant which leads to risk reduction. Those soil contami-
nants are available which can reach the inside of the biodegrading cells, and those
are biodegradable which are accepted by any of the cell’s degrading enzymes and for-
warded on a metabolic pathway. Bioavailability and biodegradation of contaminants
in soil are strongly linked to each other; bioavailability may limit biodegradation.
Microbial biodegradability assumes bioavailability, so that these two tests are often
used and considered as alternatives.

5 MATHEMATICAL MODELS FOR CONTAMINANT

BIOAVAILABILITY IN SOIL

Mathematical equations properly estimate toxicity or bioaccumulation when suit-

able quality and amount of data are available to create the QSAR (Quantitative
 Bioaccessibility and bioavailability in risk assessment 355

Structure-Activity Relationship) between chemical characteristics, e.g. Kow and the

toxicity to a certain test organism (EU-TGD, 2003). Physico-chemical properties of
chemical substances determine their water solubility, partitioning between phases,
including biological phases and their affinity to air, water or solid environmental
phases. The mathematical models use chemical properties and predict mobility and
fate of the chemicals in a standard test scenario to calculate bioavailability or bioac-
cessibility of the compound. These calculated results are valid for pure chemicals.
Some of the mathematical models are freely available in the form of software pack-
ages. EPI Suite™ (2014) for example needs only some easily available input data e.g. the
contaminant’s molecular weight, chemical structure, melting point and boiling point,
from which the partition coefficients between physical phases such as octanol–water
(Kow ), organic carbon–water (Koc ) and sediment–water (Kp ) partition coefficient, the
biodegradation half-time or even EC50 or EC20 values for fish species can be calculated.

6 CHEMICAL MODELS FOR CONTAMINANT MOBILITY AND

AVAILABILITY IN SOIL

The target organisms in the environment are degrading microbes, plants or animals.
The chemical substance in the soil is transformed physically and chemically before
it reaches the target organism’s active site, i.e. a receptor molecule, and becomes
ready to act. The type and rate of contaminants’ transformation depends on the
environmental conditions. Risk management should focus on partitioning of chem-
icals between environmental phases and the balance of the process pairs determining
the direction of contaminant transport in evaporation–condensation, dissolution–
precipitation, suspension–sedimentation, sorption–desorption, oxidation–reduction,
etc. These process pairs and their balance in a steady state specify the availability of
chemical substances in the environment. Not only availability to the living organisms,
but in a wider sense availability to air and water, interacting with chemicals and ‘taking
up’ and transporting chemicals in space and time should be considered.
Special chemical extraction methods have been created to mimic the uptake by a
participant of the environment or the leaching of the chemical substance from matri-
ces. Some of these chemical extraction methods are extremely conservative tools for
the calculation of environmental risk (hexane–acetone extraction of organic contami-
nants or aqua regia extraction of toxic metals), some others are closer to the realistic
worst-case estimates (extraction of metals from soil by organic acids, or of organic
contaminants by tenside-containing water).

6.1 Partition between n-octanol and water to predict

accessibility of organic contaminants
The octanol-water partition coefficient (Kow ) of organic chemicals, an intrinsic molec-
ular characteristic, correlates with their mobility and bioavailability. Each phase (step)
of the interaction between the organism and the contaminant can be characterized by
the Kow : desorption from the soil material; transportation to the cell through the water
phase of the soil and uptake by the cell.
356 Engineering Tools for Environmental Risk Management – 2

As bioavailability suggests, the uptake of contaminants, measurement of toxic

effects, biodegradation and bioaccumulation are suitable tools to predict bioavailabil-
ity of contaminants. By now it has been proved that log Kow is in linear relationship
with toxicity, biodegradation and bioaccumulation characterized by the following end
points:

– ECx , EC50 or LC50 , (concentration having x% inhibitory effect, especially 50%

effect, or causing lethality in 50% of the test organisms);
– half-life (DT50 ) or biodegraded fraction (Sbi ) of the contaminant;
– BCF, bioconcentration factor, the rate of contaminant concentration in the
organism or in the environment.

Based on the same relationship, chemical methods can be used to predict the
bioavailable fraction of contaminants in soil such as liquid–solvent extractions, solid
phase and membrane-based devices. Some of the chemical agents which produce
similar ‘uptake’ as a living organism are called biomimetic extractants or sorbents.
The distribution of organic contaminants between soil, water and living organ-
isms can be modeled by moderately polar organic solvents e.g. butanol, methanol (1%,
50% methanol in water or 100% methanol), n-propanol, Tween 80, ethyl acetate and
the aqueous solution of cyclodextrin (CD) (Kelsey et al., 1997; Tang & Alexander,
1999). These extraction methods are generally followed by the determination of the
concentration using chemical analysis of the extract or the mass by gravimetry after
evaporating the solvent. The basis of the model is the correlation found between the
simultaneously measured amount of extracted contaminant and the bioaccumulated
or biodegraded amount of contaminant. For example, the amount of an organic con-
taminant extracted from the soil by cyclodextrin solution was found to correlate with
the biodegraded amount in the same soil measured by a biodegradability test (Reid
et al., 2000; Stokes et al., 2005; Semple et al., 2007; Fenyvesi et al., 2008). Con-
taminant amounts extracted by cyclodextrin and butanol were compared to amounts
accumulated by Eisenia fetida and Lolium florum from deuterated PAH (Gomez-Eyles
et al., 2011) and correlation was found. For more details see Chapter 9, Volume 3.
Chemical models can be applied in predictive risk assessment but their results
cannot guarantee that the contaminant will really be biodegraded, for example it may
kill the degrading organisms if it is very toxic before biodegradation could commence.
Nevertheless, these chemical models fit well to chemistry-based risk management and
chemists alone can assess the risk, thus avoiding tests on living organisms.

6.2 Solid phase and membrane-based extractions – chemical

bioavailability models
Solid phase and membrane-based extractions are chemical models for bioavailability
estimation of organic contaminants. The solid phase and membrane-based systems
move towards cell modeling: semi-permeable membranes or/and polymers inside of
the devices are dedicated to mimicking the cell membrane (Huckins et al., 1990), able
to simulate saturation and time-dependency of material uptake. These biomimetic
sorbents were tested for soil contaminants such as PAHs, PCP and PCBs and compared
to earthworm and plant root accumulation (Table 7.2) and the contaminant’s log Kow .
 Bioaccessibility and bioavailability in risk assessment 357

Table 7.2 Solid phase and membrane-based extraction methods compared to solvent extraction.

Extraction method Bioassay or chemical Contaminant

and device model for comparison matrix Result Reference

Butanol Earthworm and PAHs Butanol was the best Gomez-Eyles

Cyclodextrin rye grass root from soil for earthworm. et al., 2011
Tenax TA beads accumulation All of them
extractions correlated well for
rye grass roots
POM-55 log Kow PCB POM-55–water Cornellissen
55 µm thick POM in sediment partition coefficient et al., 2008,
membrane and water correlates with 2009
extraction log Kow
TECAM Kow PAHs in two Good correlation Tao et al.,
TECAM extraction Earthworm phase soil betweenTECAM–water 2008, 2009
accumulation partition and both Kow
and earthworm
accumulation
SPMD extraction Earthworm PAH SPMD absorbs higher Bergknut
HPBCD accumulation in soil proportion of small PAH et al., 2007
extraction using molecules than
hydroxypropyl- earthworms.
beta-cyclodextrin HPBCD correlates well
with earthworm
accumulation.
TECAM Carrot root DDT TECAM after 6 hours Yang et al.,
triolein-embedded accumulation contaminated shows good correlation 2010
cellulose acetate soil with carrot
membrane accumulation
XAD-2 CaCl2 , butanol, PCP XAD-2 correlates with Wen et al.,
polystyrene dichloromethane in soil both, using soil 2009
non-ionic beads sequential extraction contaminated for a
earthworm long time.
accumulation
POM-55 Earthworm PAH POM overestimates Barthe et al.,
55 µm thick accumulation contaminated the bioaccumulation of 2008
polyoxymethylene sediment PAHs compared with
membrane the bioassay

PAH: polycyclic aromatic hydrocarbons; PCB: polychlorinated biphenyls;

DDT: dichlorodiphenyltrichloroethane; PCP: pentachlorophenol;
Tenax TA beads: porous polymer resin based on 2,6-diphenylene oxide.

Another artificial cell model is the semi-permeable membrane device (SPMD) con-
sisting of an external LDPE (low-density polyethylene) layer which can be filled with
triolein, isooctane, trimethylpentane or with an ionic liquid e.g. organic salts (Esteve-
Turillas et al., 2008). SPMDs are widely used for sampling air and water, but a more
permanent external layer is required for soil. Table 7.2 shows a few SPMD applica-
tions together with other solid phase and membrane-based extraction methods using
TECAM, triolein-embedded cellulose acetate (Figure 7.5); XAD, polystyrene, POM,
or polyoxymethylene membranes.
358 Engineering Tools for Environmental Risk Management – 2

Figure 7.5 Schematic diagram of TECAM accumulation of PAHs from soil (Tao et al., 2008).

6.3 Liquid-phase extractions to predict accessibility of

toxic metals
It has been accepted that neither the water-soluble, nor the total concentration of
toxic metals in soil is in correlation with their accessibility and bioavailability and
they are not suitable for the characterization of environmental risk. In addition, the
chemical forms of toxic metals largely influence their toxicity, fate and transport, as
well as partitioning between the soil phases. Various partial extraction methods such
as extraction with salt solutions, acidic and alkaline solutes, chelating agents, etc. have
been studied and routinely used to model toxic metal bioavailability. Extractions with
solutes of various pH and ion concentrations were applied to mimic living organisms
in interaction with the contaminated soil. The extractions are followed by chemical
analysis, and the result is handled as the bioavailable fraction of the total metal content
of the soil. Numerous studies proved that EDTA (ethylenediaminetetraacetic acid) is
truly mimicking the metal accumulation by plants (Gupta & Sinha, 2007; Zhang
et al., 2010) and the metal uptake by benthic invertebrates (Tack & Verloo, 1995).
The solution of ammonium lactate and acetic acid (LE) is also considered by some
authors as a good imitator of plants (Lakanen & Erviö, 1971), but some others found
that LE greatly overestimates plant uptake (Feigl, 2011). There is no generally suitable
extractant, some solvents may correlate with certain plants in a certain soil for a certain
element such as, in the case of phosphorous, 0.01 M CaCl2 solution proved to be a true
extractant for modeling plant-available phosphorous in soil (Hylander et al., 1995).
According to a more conservative approach, when simulation of a realistic worst case
is the goal instead of true mimicking, other type of solvents such as diluted nitric acid
can be used for environmental risk estimation.
An important conceptual question may arise when extracting contaminants from
soil by different extractants if an environmental process is mimicked or a standardized
 Bioaccessibility and bioavailability in risk assessment 359

Table 7.3 List of sequential multiple metal extraction methods and the separated fractions.

Extraction method Fractions Reference

Tessier method 1 Exchangeable Tessier et al., 1979

2 Associated with carbonates
3 Associated with Fe–Mn oxides
4 Associated with organic matter
5 Residual
Three-level extraction 1 Mobile Gupta et al., 1996
2 Immobile Aten & Gupta, 1996
3 Pseudo total metal fraction
BCR method 1 Acid-extractable fraction Geebelen, 2003
2 Reducible fraction
3 Oxidizable fraction
4 Residual
Selective sequential 1 Water-soluble Becquer et al., 2005
extraction (SSE) procedure 2 Exchangeable
3 Acid-soluble
4 Bound to Mn oxides
5 Bound to amorphous Fe oxides
6 Bound to crystalline Fe oxides
7 Oxidizable fraction
8 Residual
Chemometric Identification Increasing strengths of simple mineral acids as Cave et al., 2004
and Substrate and Element the extractant, followed by chemometric data Wragg & Cave, 2012
Distribution (CISED) processing of the resulting multi-element data Appleton et al., 2012
obtained from the extract analysis.
Extracts approximating water Parallel extraction with water Gruiz et al., 1998,
availability, plant availability Artificial acid-rain acetate-buffer solution Gruiz, 2000
and total metal content (pH = 4.0 or 4.5) ammonium acetate 0.5 M Feigl et al., 2009b
and EDTE (0.02 M) (pH = 4.65) aqua regia
AVS and SEM procedure for Parallel determination of acid volatile sulfide US EPA, 1991
anoxic sediments (AVS) simultaneously extracted metals (SEM)
SEM (µmol/g)/AVS (µmol/g)
Bioaccessibility of lead Parallel determination of acidic glycine US EPA, 2012
extractable and total extractable Pb and
calculating their ratio

chemical extraction proceeds. In the first case a geometrically valid leaching test is the
best option with realistic infiltrating water quantity, no adjustment of pH and other
conditions. In the second case the extractant should be buffered and standardized
conditions are necessary during the extraction. The environmental trueness of such an
extraction is very low, but it is useful for comparative studies.
Another widely used type of extraction is the multi-step sequential extraction
(SME) method. The sequential extraction separates the metals into five fractions:
exchangeable, bound to carbonates, bound to Fe-Mn oxides, bound to organic mat-
ter, and residual (Tessier et al., 1979). Simplified methods of three or four sequential
steps were also developed as introduced in Table 7.3. The extracts are generally ana-
lyzed by multi-element analysis methods such as ICP-AES (Inductively Coupled Plasma
with Atomic Emission Spectroscopy), ICP-MS (Inductively Coupled Plasma with Mass
Spectrometry) and XRF (X-ray fluorescence analysis). The results of SME enable the
360 Engineering Tools for Environmental Risk Management – 2

Table 7.4 In situ measurements and devices for the dynamic assessment of labile metal species.

Method Measured end point Reference

In situ DGT device Measuring the diffusive Davison et al., 1994;

gradients in thin films Zhang et al., 1995a and 1995b
Gel integrated microelectrode array Dynamic electroanalytical Buffle & Tercier, 2005
(GIME) with voltammetric detection metal speciation
Gel integrated stripping Dynamic electroanalytical Town, 1998;
chronopotentiometry (GISCP) metal speciation van Leeuwen & Town, 2002;
Town & van Leeuwen, 2004
Hollow fiber permeation liquid Buffle et al., 2000;
membrane Salaun & Buffle, 2004
Donnan membrane technique Can be used as equilibrium Temminghoff et al., 2000;
or as dynamic technique Weng et al., 2005; Kalis et al.,
2006
Competitive ligand exchange/ Can be used as equilibrium Xue & Sigg, 2002
adsorption stripping voltammetry or as dynamic technique

estimation of mobility, bioaccessibility and leaching from soil’s solid to soil’s water
phase and, to some extent, toxicity.
Anoxic sediments may contain a large amount of toxic metals in a non-mobile
chemical form. As sulfide controls the mobility of metals in anoxic sediments, AVS,
the acid volatile sulfide content of sediments (H2 S is released when treated with strong
acid) is important information on the amount of the sulfide-form metals. The relative
amount of SEM (SEM = simultaneously extracted metals, which are solubilized in
the acid-treatment step: Cd, Cu, Hg, Ni, Pb, and Zn) and AVS can be used for the
prediction of metal bioavailability. If the molar ratio of SEM for bivalent metal ions
to AVS exceeds one (>1), the metals in the sample are potentially bioavailable.
The US EPA (2012) method is typically applicable for the characterization of lead
bioaccessibility in soil. It is intended to be used as reference for developing site-specific
Quality Assurance Project Plans (QAPPs) and Sampling and Analysis Plans (SAPs).
The amounts of lead in 0.4 molar glycine (pH = 1.5) extract and in the so-called total
extract (both received from the same amount of soil) are compared to each other and
it is called the ‘in vitro bioaccessibility’ of Pb in the soil or solid waste:

Lead in vitro bioaccessibility = Pbext × Vext × 100/Pbtotal × Msoil ,

where
– Pbext – lead concentration in the acidic glycine extract;
– Vext – volume of the extract;
– Pbtotal – Pb concentration in the solid sample;
– Msoil – mass of the soil sample analyzed.

The in situ DGT device (Table 7.4), based on measuring the diffusive gradients
in thin films was developed by Davison and Zhang between 1990 and 1995 (Davison
et al., 1994; Zhang et al., 1995a; 1995b). It has become a popular tool in the last ten
years for measuring ‘labile’ metals in water, sediments and soils (Han et al., 2013).
 Bioaccessibility and bioavailability in risk assessment 361

In addition to ready-for-use measurement cells, separate components allowing self-

assembly are supplied. The plastic device with a 2.0 cm diameter window is loaded
with a three-layer system of i) a restricted pore size resin-impregnated hydrogel; ii)
an open pore diffusive gel layer (polyacrylamide) and iii) a membrane filter contact-
ing the environmental matrix. Specific gels are available for metals (Chelex gel), for
phosphorus (Fe oxide gel), for cesium and for sulfides. It can be applied for in situ
measurements (single measurement or time averaged concentrations), for measuring
fluxes in sediments and soils, for the determination of kinetic and thermodynamic
constants. Its pH tolerance is limited: it can work in the range of pH = 5–9.
DGT directly measures the mean flux of mobile/labile species (including free and
kinetically labile metal species) to the device during the deployment. Where supply from
sediment particles to solution is rapid, the interfacial concentration is the same as the
concentration of metal in water or bulk pore water. For a given device and deployment
time, the interfacial concentration can be related directly to the concentration of labile
metals (Zhang et al., 2001 and Zhang & Davison, 2001).
In the interpretation of DGT Research (2014) this concentration represents the
supply of metal to any sink, be it DGT or an organism that comes from both dif-
fusion in solution and release from the solid phase. It is proven by comparative
studies that this concentration is suitable to approximate bioaccessibility, so it can be
a simple surrogate measurement of the biologically potentially effective concentration
(Søndergaard et al., 2014). It supports well the presently accepted Free-Ion Activity
Model (FIAM) which stipulates that the biological response of organisms to metals
in water-based systems is proportional to the free-ion activity of the metals and not
their total or dissolved concentrations (Zhang et al., 2002). DGT–bioassay correlation
experiments conducted demonstrate clearly that DGT provides a more representa-
tive measure of metal bioavailability, and hence toxicity, than conventional water
quality parameters (i.e., total and/or dissolved metal concentrations). Specifically, the
results indicate that metal uptake by DGT and aquatic biota (e.g., Daphnia magna and
rainbow trout) are both reduced in the presence of metal-complexing ligands (INAP,
2002; Senila et al., 2012). It means that metal-ligand complexes that are unavailable
to aquatic biota are also undetected by DGT.
The theoretical background of the TGD method to using DGT in sediments and
saturated soils can be found in the publications of Davison et al. (2000) and Zhang et al.
(2001). A detailed description on the application and the mode of calculations of the
DGT measured concentrations, the bioavailable concentration in the liquid medium
and kinetic parameters is also written in the DGT handbook in details (DGT Research,
2014). The DGT measured concentration can be calculated as shown by the equation
below:

CDGT = M ×
g/(D × t × A),

where
– CDGT – DGT measured concentration;
– M – metal mass accumulated in the resin gel;
–
g – thickness of the diffusive gel;
– D – diffusion coefficient of the metal in the gel;
– t – time of depletion;
– A – exposure area.
362 Engineering Tools for Environmental Risk Management – 2

The real concentration in sediment or soil pore water depends on the rate of
resupply (R) of solutes to the pore water. The resupply can be determined as the ratio
of CDTG to C, the real concentration of labile metal forms in the pore water. R is the
measure of the dynamic ability of the solid phase to resupply the pore water in response
to the depletion induced by the DGT sink.
The in situ electroanalytical techniques combined with gel- and membrane-
separation shown in Table 7.4 are mostly based on the application of microelectrodes
immersed into gels or combined with membranes. The gels and membranes separate
free metals from a complex matrix, which detect the electrochemically active forms,
i.e. the charged metal ions from the electrodes. The electrodes should be calibrated
before their in situ environmental application. Sigg et al. (2006) published a compar-
ative study on six electrochemical methods applied to natural surface waters. They
concluded that the free ion activity model, which makes the free ions responsible for
biological activities and adverse effects, is a highly simplified model of a dynamic and
complex situation, and the relative time scales and the rates of complex dissociations
should also be taken into consideration, even in the case of water. The fast devel-
opment in electroanalytical methods hopefully makes this tool suitable to contribute
to the necessary dynamic tool battery, which will be able to produce reliable predic-
tors for bioavailability, biological uptake and ecotoxicological risk in natural waters,
sediments and soils.

7 COMPLEX MODELS

In environmental management, bioavailability and bioaccessibility must be determined

when the actual risk posed by hazardous contaminants present in the environment
(soil/sediment) should be evaluated or model experiments and field implementation of a
remediation technology should be designed and monitored. One has to consider in both
cases that the dynamic interaction of contaminated soil and the microbiota may change
contaminant bioavailability. The detection of the interactions between contaminated
soil and soil-living organisms require interactive tests, dynamic microcosms and their
integrated monitoring using physico-chemical, biological and toxicological methods.
Microcosms established for testing bioavailability and changes in bioavailability should
model realistic average or worst-case scenarios. A laboratory lysimeter, for example,
can be irrigated i) with average precipitation, ii) with an amount of precipitation
representing the rainy season, or iii) with an artificially prepared acidic precipitation,
simulating a worst case from the point of view of e.g. Zn and Cd mobilization.

7.1 Interactive laboratory tests

Interactive soil and sediment tests ensure direct contact between contaminated
soil/sediment and test organisms, thus simulating a real environmental situation.
Generic tests required by legislation to assess the hazards posed by chemicals create
a standard test environment, while problem-specific tests intend to model site-specific
transport and land-use-specific exposure scenarios. Testing the biological effects of
contaminated soil can, if necessary, be achieved by performing chemical analysis of
the concentration and composition of soil contaminants.
 Bioaccessibility and bioavailability in risk assessment 363

Most of the interactive tests measuring either toxicity, biodegradation or bioac-

cumulation as an end point are in strong relationship with bioavailability. The effect
can only be measured when the contaminant is really available to the test organism:
an interaction is established between the test organism and the contaminant and the
contaminant is adsorbed by the test organism.
The following direct-contact laboratory tests can be used to characterize con-
taminant bioavailability in general and in real contaminated soil. The results of
direct-contact test methods integrate the degree of mobility, bioavailability and the
consequent effects of the contaminated soil:

– Biodegradation of a chemical substance mixed into a real soil with indigenous

microflora in closed-bottle or aerated microcosms;
– Ongoing biodegradation of contaminants in a contaminated soil sample using
indigenous soil microflora in closed-bottle or aerated microcosms;
– Bioleaching of the contaminant from contaminated soil using soil indigenous
microflora in laboratory lysimeters;
– Bioaccumulation of the contaminants from contaminated environmental samples
(soil, sediment, waste) using test plants or earthworms: pot experiments;
– Measuring acute and chronic toxicity of contaminated soil, sediment, waste or
slurries by direct-contact bioassays using bacterial, fungal, animal or plant test
organisms;
– Acute and chronic toxicity of contaminated soil extracts and leachates: liquid-
phase bioassays using bacterial, fungal, animal or plant test organisms;
– Mutagenicity of contaminated soil, sediment, solid waste direct-contact muta-
genicity assay using bacterial test organisms;
– Any other adverse effects (reprotoxicity, endocrine disruption, etc.) of the
contaminated environmental samples.

In addition to laboratory tests, field ecosystem assessment, bioaccumulation of

field-grown organisms, gene expression profile of the soil microbiota, and many other
indicators of biodegradation, bioaccumulation or toxicity can be applied as an end
point for the demonstration of the bioavailability of the contaminant occurring in the
real environment.

7.2 Dynamic testing

A single measurement of a contaminant’s mobility and bioavailability in a soil sample
yields static information about one time point. However, no data are available about
the future rate and direction of changes or any response to external e.g. environmental
or climatic impacts. To obtain information on all these dynamic parameters, the micro-
cosm must be monitored: sampling the microcosm regularly, analyzing the samples by
parallel chemical analyses and biotesting, evaluating and plotting the results as a func-
tion of time as illustrated by the microcosm biodegradation example (Figure 7.6). The
three cases shown in the graph are:

1. the contaminant’s mobilization rate is equal to the biodegradation rate;

2. the mobilization rate is greater than the biodegradation rate;
364 Engineering Tools for Environmental Risk Management – 2

Figure 7.6 Change in bioavailability in the initial phase of biodegradation. 100% = initially bioavailable
amount.

3. the mobilization rate is lower than the biodegradation rate. In the latter case (red
line), bioavailability is the limiting factor of biodegradation, and the limitation
can be eliminated by enhancing bioavailability.

The impact of changes in external parameters on the contaminant’s mobility and

bioavailability in soil can dynamically be tested in several ways. i) When the starting
point is an equilibrium-state environmental soil sample under realistic/average envi-
ronmental conditions, the study may apply significantly different test conditions and
monitor the behavior of the soil in the microcosm until it reaches the new equilibrium.
ii) If the soil is not in equilibrium, first a controlled steady state has to be reached in
the batch or flow-through soil microcosm. After the steady state has been achieved,
the environmental parameters (temperature, pH, redox potential and humidity/water
content, etc.) should be changed at a realistic or a slightly accelerated rate. The changes
are monitored until the soil adapts to the new environmental conditions and reaches
a new steady state which is different from the original one (Figure 7.7). The reaction
of the soil to the changed environmental conditions can be:

1. increased (blue line) or decreased (not shown in the graph) mobility and
bioavailability, and a new steady state;
2. temporary increase in mobility and bioavailability, then return to the original or
a slightly different new steady state;
 Bioaccessibility and bioavailability in risk assessment 365

Figure 7.7 Bioavailability testing: typical changes in the soil microcosm between two different steady
states.

3. temporary decrease in bioavailability, then return to the original or a slightly

different new steady state.

A microcosm or a field test can be made dynamic by stepwise adjusting new envi-
ronmental conditions or applying a one-off, impulse-like impact, a push. The push
can be changing pH, irrigation with ‘acid rain’, adding nutrients, injecting solubilizing
agents, introducing more and special microorganisms, suddenly increasing temper-
ature, or changing the redox potential by aeration, oxygen removal or injection of
electron-donor molecules, etc. The microcosms should be in a steady state before the
application of the push, and the response of the soil to the impulse should be moni-
tored, e.g. by measuring the changes in the chemical forms, concentrations, partitions,
mobility and bioavailability of the contaminants, respiration rate, metabolites, activity
of the test organisms (Figure 7.8). The types of responses from the point of view of the
contaminant’s mobility and bioavailability are:

1. Sudden increase or decrease (not shown in the graph) in mobility and bioavail-
ability of the contaminant in soil and after a fast response, then reverting to the
original steady state;
2. Slow increase and very slow attenuation, returning slowly or not returning at all
to the original steady state;
3. Sudden increase, then slow decrease and return to a new steady state.

The effect of impulse-like technological interventions on the soil, on the contami-

nant and on the soil microbiota can be observed in the field by measuring air, water and
366 Engineering Tools for Environmental Risk Management – 2

Figure 7.8 Bioavailability testing: typical changes in the soil microcosm due to the effects of an impulse-
like impact.

contaminant flows, contaminant concentrations in soil gas or groundwater, ion concen-

trations in soil water and soil moisture, and conductivity of soil water and soil moisture,
or measuring the products of respiration and the biodegradation of microorganisms.
The direct-push soil analysis technologies (see Volume 3) allow targeted studies also
in deep soil layers (in 30 m or even 150 m depths).

7.3 Integrated evaluation

Both the interactive and dynamic tests should be evaluated by measuring the effect
on single or multiple test organisms or by observing the process in a microcosm using
integrated physico-chemical and biological monitoring: measuring the effects (lethality,
biological and biochemical activity, changes in diversity, etc.) and chemical concentra-
tions as a function of time. The physico-chemical, biological and ecotoxicity results
are evaluated together in relation to each other. The consistency or the rate and type
of inconsistency between physico-chemical and biological–ecotoxicological data give
extensive information about the character of the risk, e.g. the biological availability of
the contaminant; the presence of a chemical time bomb; the interaction between single
contaminants (synergism or antagonism) and draws our attention to chemically not
measured or non-measurable, but still dangerous components via their adverse effects
(Gruiz et al., 1999; Gruiz, 2005).
The integrated test methodology for contaminated soil has been called by the
authors Soil Testing Triad (STT). It contains physico-chemical analytical, biologi-
cal/ecological assessment methods and toxicity tests (Figure 7.9).
Physico-chemical analysis, biological (ecological) and toxicity tests are of similar
significance. The methods are complementary. They provide information about the
 Bioaccessibility and bioavailability in risk assessment 367

Figure 7.9 Composition of the Soil Testing Triad (STT).

quality and quantity of the contaminant; the characteristics and the biological status
of the soil; the activity, vitality and adaptive behavior of the soil-ecosystem; the effects,
mobility, bioavailability and biodegradability of the contaminant; and the response of
the soil to external effects.
The target of physico-chemical analyses may be the soil, the contaminant or the
members of the ecosystem. The biological and ecological characteristics are used to
determine the diversity and quality of the ecosystem and the dynamic nature of soil,
e.g. its response to certain natural or provoked external effects. The answer gives
information about the buffering capacity and the adaptive behavior of the soil and its
biological system. The third party in the triad is toxicity testing: it provides information
about the adverse effects of the contaminant or the contaminated soil, and thus the
hazards posed to non-adapted ecosystems or humans. The STT plays a central role in
site assessment, environmental risk assessment and environmental monitoring. Envi-
ronmental toxicity testing indicates the actual adverse effect that is directly associated
with the environmental risk posed by the soil. This value often differs from the results
of chemical analyses.

8 EXAMPLES OF INTERACTIVE TESTING OF BIOAVAILABILITY

IN SOIL

Simultaneously to chemical analysis, soil samples polluted with inorganic and organic
contaminants have been directly tested using different test organisms to find the
bioavailable portion. This was compared to the metal content which is available to
368 Engineering Tools for Environmental Risk Management – 2

Figure 7.10 Grey flotation tailings covered by a thin soil layer: extensive water erosion.

Table 7.5 Toxicity of flotation tailings and cover soil tested by bacteria and plants.

Azotobacter Sinapis alba root Vibrio fischeri

Test method agile dehydrogenase and shoot growth luminescence

Black soil layer Very toxic Toxic Very toxic

Grey flotation tailings Non-toxic Slightly toxic Non-toxic

chemical extractants. A comparative evaluation of the results of physico-chemical anal-

yses and toxicity tests enables conclusions on bioavailability, which can be used to
calculate the risk posed by the polluted soil.

8.1 Toxic metal bioavailability in mine tailings – the

chemical time bomb
A flotation tailings dump at an abandoned mining site in Hungary has been covered
by a thin soil layer without isolating the waste from the soil to accelerate plant growth
and improve the aesthetic value of the 4-million-tonne deposit located near a village.
The pictures in Figure 7.10 were taken five years after placing the cover. The cover and
the tailings were eroded by wind and water and, as Table 7.5 indicates, the soil became
highly toxic. The soil cover could not fulfill its role: erosion, leaching and acidification
as well as metal uptake by plants were in progress (Gruiz, 2000; Dobler et al., 2001).
The metal content of the soil and of the tailings layers was analyzed using ICP–AES
after aqua regia extraction. The results of the ‘total metal content’ were in sharp con-
trast to the results of toxicity. When artificial rainwater was used for extraction, it was
found that the metal content of the tailings was not extractable by water (Table 7.6).
In spite of the relatively low metal content of the soil cover (two- to three-fold of
the soil quality criteria) the metals were extractable by water and available to the test
organisms. The metal content of the soil cover was water soluble, therefore the metals
were taken up and easily transported into the food chain. Low water permeability
of the tailings and the acidic character of the top layer resulted in continuous acidic
 Bioaccessibility and bioavailability in risk assessment 369

Table 7.6 Comparison of the total and the water-soluble metal contents in soil and tailings.

Total metal content Rain-water-soluble

(mg/kg) metal content (mg/kg)

Layer pH Zn Pb Cu Zn Pb Cu

Black soil layer 4.7–5.2 603 186 72 42.2 1.9 0.5

Grey flotation tailings 7.0–8.0 31,858 4,971 2,450 3.4 1.2 0.6

Table 7.7 Metal content of some plants on the tailings dump.

Zn Cu Cd
Species (mg/kg dry plant) (mg/kg dry plant) (mg/kg dry plant)

Achillea millefolium 255.4 17.0 2.4

Agrostis sp. 409.8 31.9 6.3
Carex sp. 354.6 55.0 3.0
Echium vulgare 607.5 45.3 5.0
Phalaris canadiensis 144.9 4.1 0.5
Phragmites australis 767.5 40.5 0.7
Populus sp. 1158.5 12.9 19.5
Silene alba 693.5 50.4 2.6
Silene vulgaris 505.5 20.7 4.6
Tussilago falifara 568.5 38.9 8.8
Maximum limit for forage plants∗ /vegetable∗∗ 150–200/20 15–50/10 1.0/0.5
∗ 17/1999 EüM; ∗∗ 44/2003 FVM (Hungarian regulations).

extraction of the metals from the tailings and their capillary transport into the soil
cover, i.e. the habitat of the local ecosystem.
The plants that grow on the surface of the soil overlaying the tailings are highly
contaminated with accumulated metals (Table 7.7) (Dobler et al., 2001).
Flotation tailings represent a typical chemical time bomb: their metal content is
non-available until precipitation, acid rain, acidic infiltrates and plant root exudates
are not in contact with the waste. When metals are extracted, leached or otherwise
mobilized, they are transported by capillary forces into the top soil layer. Tailings in
direct contact with the soil layer represent an infinite contaminant source. The long-
term risk and the chemical time-bomb fate of these materials can only be confirmed
by the integrated evaluation of the results of chemical analyses and toxicity tests. If
this seemingly neutral waste material (no short-term toxicity was measured and the
water extract showed low contamination due to the high pH) is diffusely dispersed
by erosion, the sudden increase in its specific surface area accelerates weathering,
acidification, leaching and mobilization of its toxic metal content.

8.2 Decreased bioavailability, lower toxicity – a soil

remediation tool
Availability of toxic metals to water and plants is responsible both for the short-
term risks to aquatic and terrestrial ecosystem, as well as for the long-term human
370 Engineering Tools for Environmental Risk Management – 2

Figure 7.11 Root and shoot elongation of Sinapis alba in metal-contaminated soil with and without
chemical stabilization by fly ash.

and ecological risks posed by contaminated soil through direct exposure and food
chains. Weathering and leaching of the tailings material further increases the risk due
to increased bioavailability of toxic metals. Mobility and bioavailability of toxic met-
als should be decreased to reduce the risk. Availability of metals in soil can be reduced
by increasing their sorption rate, strengthening their bonds or conversion into sta-
ble chemical forms: e.g. from ionic to metal-hydroxide, -oxide or -silicate compounds.
These transformations are controlled by chemical and mineralogical processes resulting
new mineral-formation over the long term, considered the opposite of weathering.
Figures 7.11 and 7.12 illustrate the measured plant toxicity and plant-uptake
results of contaminated soil before and after chemical stabilization by fly ash to
decrease mobility and bioavailability of toxic metals (Feigl et al., 2007, 2009b). Plant
toxicity decreased significantly: root growth increased by 50% and shoot growth by
150%. Metal uptake of the plants decreased from Zn = 740 mg/kg and Cd = 3 mg/kg
plant by up to 70%.
The total metal content of the soil with and without stabilization is the same: the
fly-ash treatment has not reduced the total concentration but only the bioavailable
fraction.
Water and ammonium acetate (pH = 4.5) extracts were analyzed using ICP–AES
to obtain chemical data about the changes. The toxic metal contents of water and
acetate extracts are shown in Figures 7.13 and 7.14.
Water can extract 180 mg/kg Zn from the soil, while acetate solution extracted
300 mg/kg Zn from the same soil, which contains a ‘total’ of 1800 mg/kg Zn
concentration (extracted by aqua regia). Plants (Sinapis alba) ‘extracted’ 120 mg Zn
per kg wet plant, which is equivalent to 740 mg/kg dry plant biomass.
Fly ash (2% and 5%) reduced Zn concentration in the water-extract from 180 to
10 and ∼0 mg/kg whereas Cd concentration decreased from 1.1 to 0.2 and 0 mg/kg,
respectively. The plant-accumulated amount in leaves decreased from 740 mg/kg to
 Bioaccessibility and bioavailability in risk assessment 371

Figure 7.12 Decrease in metal uptake by plants (Sinapis alba) after soil stabilization with fly ash.

Figure 7.13 Zinc concentration in the water extract of the soil before and after stabilization.

220 mg/kg plant dry biomass. Plant toxicity (Sinapis alba) decreased to only 50% of
the initial value. The decrease in toxicity is closer to the metal concentration reduc-
tion in the acetate extract (30–50%) than to the water extract (approx. 90%). The
availability of the metals to plants is higher than to water due to acidic root exudates
mobilizing metals locally. A comparison of the decrease in plant-accumulated and
acetate-extracted amounts shows a maximum 50% decrease in the latter in contrast
to 70% decrease in plant bioaccumulation.
The results of this bioavailability mitigation experiment showed that acetate
extraction slightly overestimates the risk compared to plant bioassays. The rapid plant
372 Engineering Tools for Environmental Risk Management – 2

Figure 7.14 Zinc concentration of ammonium acetate extract before and after soil stabilization.

accumulation test, developed by the authors, is in itself a pessimistic biological model

(using a type of seedling that tends to concentrate toxins), which overestimates the
true metal uptake by plants.
It is worth noting that a moderate overestimate fits well a conservative risk assess-
ment approach, and that the best possible set of tests for assessment and monitoring
requires a wide selection of biological and chemical methods.

8.3 Correlation of chemical analytical and bioassay results

River sediments contaminated with mine waste have led to high toxic metal contents
in the soil of flooded allotments in the Toka Valley, a former mining area in north-
ern Hungary (Feigl et al., 2009a; Gruiz et al., 2009; Vaszita et al., 2009). Chemical
analysis of soils and wastes indicated that high metal content often failed to lead to
toxicity (cf. Tables 7.4 and 7.5) and sometimes the opposite, i.e. low concentration was
accompanied by high toxicity. The authors carried out several parallel assessments and
comparisons between the results of toxicity bioassay and chemical analysis of differ-
ent extracts from metal-contaminated soils. The results demonstrated that the flooded
gardens in the relatively small Toka Valley show large variations and different con-
tamination patterns. The results of chemical analyses and toxicity tests show a range
between very good correlation and no correlation at all, depending on the type, origin,
age and the proportion of the contaminating material and the characteristics of the
recipient plot and its soil.
Figures 7.15 and 7.16 illustrate two extreme cases. Figure 7.15 shows the test
results of soil samples from an allotment (Plot 1) homogeneously polluted by a regular
(annual) high water level. The flooded soil is morphologically homogeneous, and
plant toxicity and chemically measured metal concentration from an acetate buffer
 Bioaccessibility and bioavailability in risk assessment 373

Figure 7.15 Plant toxicity is proportional to the concentration of the acetate-extracted toxic metals.
Plot 1 is exposed to regular annual floods (HM: sum of all extractable metals As, Cu, Cd,
Hg, Pb and Zn).

Figure 7.16 Plant toxicity is not proportional to the chemically measured mobile metal concentration.
Plot 2 was exposed to sudden runoffs (HM: sum of all extractable metals As, Cu, Cd, Hg,
Pb and Zn).
374 Engineering Tools for Environmental Risk Management – 2

correlate well. The gradient visibly decreases with the distance from the creek. The
proportionality is illustrated by the toxicity as a function of acetate-extractable metal
content.
Figure 7.16 demonstrates another scenario where Plot 2 was exposed to sudden
runoffs and subsequent long-term high water levels. There is no correlation between
measured plant toxicity and chemical analysis results as demonstrated by Figure 7.16.
A clear discrepancy can be seen between plant toxicity and chemical analytical data.
The sudden heavy runoffs and subsequent long-term high water levels have disposed
of eroded material and creek sediment of different origin (eroded soil and parent
rock, waste rock, different mine wastes, other waste from illegal disposals upstream,
etc.) and morphology (yellowish, grayish, stone-like, flocculent-like, etc.). In spite of
the contradiction between metal content and toxicity, good explanation can be given
for the differences found between the sediment-polluted soil samples. High chemical
content and low plant toxicity indicate non-weathered waste rock or ore. Flotation tail-
ings – as seen in Section 8.1 – also belong to this kind of mine waste with non-available
metal content, representing a serious chemical time bomb.
On the other hand, dissolved metals transported by water and sorbed by the
flooded soil particles exhibit high bioavailability, similar to the very fine rock and
mine waste which show advanced weathering. Former anoxic bed sediments, with
stable metal forms are easily transformed into labile, e.g. water-soluble ionic forms by
chemical and microbiological oxidation under oxidative conditions once they reach
the surface. This is why soil samples with low metal concentration may exhibit high
toxicity and the opposite: low toxicity with high metal content.
The examples of the two flooded plots demonstrate the necessity of integrated
application of the chemical and toxicological assessment methods when bioavailabil-
ity has a significant impact on the risk of soil contaminants. The chemical results are
sufficient to estimate the adverse effects of similar quality soil-polluting sediment or
waste (Figure 7.15), but not when the soil-polluting material is of different origin, type
and age (Figure 7.16). Generally speaking, the chemical model is suitable for compar-
ison when only one parameter changes within a system. Direct toxicity testing is the
method which has a direct link to the risk posed by contaminated soil. Bioavailability
can only be characterized by an integrated application of chemical analyses and the
testing of the adverse effects or bioaccumulation to determine the effective, bioavail-
able proportion of soil contaminants. The difference between the chemical analytical
and biological results refers to the unavailable, strongly sorbed fraction of the soil con-
taminant. This difference – explained from the soil’s point of view – can be assigned
to the soil’s toxicity buffering capacity.

8.4 Bioavailability and biodegradation of organic soil

contaminants
Monitoring of bioremediation provides valuable information on bioavailability and
biodegradation when chemical analyses and biological tests are applied in an inte-
grated way. Only bioavailable contaminants can be biodegraded, but bioavailability
and biodegradation are hard to distinguish. It is evident that the biodegraded fraction
of the contaminant has become biologically available before the degrading attack by
 Bioaccessibility and bioavailability in risk assessment 375

the microorganisms. But the opposite is not necessarily true: not all bioavailable con-
taminants are biodegraded. In addition, if the contaminant is biodegradable and only
moderately bioavailable, limited bioavailability will in general be the bottleneck in the
biodegradation-based soil treatment process. In this case, mobilization of the contam-
inants using solubilizing, mobilizing or bioavailability-enhancing agents provides the
solution. Enhanced biodegradation confirms the effect of bioavailability-enhancing
agents (Volumes 4 and 5).
Multiple interactions can make contaminants bioavailable in soil: the initial
bioavailability – especially if it is not high enough to fulfill the substrate need of the
soil microbiota (waste, wastewater, etc.) – can be increased microbiologically. Soil
microorganisms can utilize the less available substrates such as litter and large organic
molecules. Making these large molecules bioavailable is part of their metabolic activ-
ity and the necessary enzymes, biotensides, complexing agents are part of their tool
battery. A complex community of microorganisms, specialized in ‘common dining’,
so-called commensalism, has the task to mobilize organic molecules and make them
bioavailable in soil. Under normal circumstances the process of producing bioavailable
substrates and biodegrading them is harmonized to a great extent. However, in the case
of a disturbed environment e.g. when unnatural substrates, i.e. xenobiotics or mixtures
of various persistent organic pollutants are present, the natural harmonization within
the soil community is not always successful.
In the following examples the temporary accumulation of the mobilized, but not
yet degraded portion of the soil pollutant mixture will be detected by toxicity tests.
Mobilization of substrates or biodegradable contaminants is a natural process, but
it often does not take place in contaminated soils; therefore many soil remediation
technologies apply mobilizing agents.
Figure 7.17 shows the typical trend in soil toxicity for transformer oil, a moder-
ately biodegradable hydrocarbon mixture. Soil samples from the field were artificially
contaminated with 30,000 mg/kg transformer oil and aged for two months, then stud-
ied in biodegradability studies in laboratory microcosms. Two different types, a sandy
and a clayey soil were biovented (R0 = biovented only) and treated by the chemical
mobilizing agent RAMEB, randomly methylated cyclodextrin (R1 = biovented and
treated by RAMEB). Toxicity was measured by bacterial, protozoan, insect and plant
test organisms. Figure 7.7 shows the bioluminescence inhibition of Vibrio fischeri dur-
ing the study. The toxic equivalency (TEQ) is given in 4CP (see Section 7.9): the risk is
acceptable under TEQ = 10 mg/kg, while a TEQ less than 5 mg/kg represents ‘no risk’.
The sandy soil’s initial toxicity is low and the clayey soil is not toxic at all in spite of their
contaminant content. After the beginning of remediation, the contaminant’s mobility
and bioavailability increased, as indicated by the extremely high toxicity after a one-
week treatment. The contaminant must become available to the microorganisms for it
is the prerequisite of biodegradation. This can be achieved spontaneously (activating
mobilizing microorganisms) or artificially (applying mobilizing agents).
In the example shown, the concentration and toxicity decreased proportionally
in both soils during biodegradation. Starting from the second week, the decrease in
toxicity was in agreement with the chemical results but the sudden increase in the first
week was hardly (sandy soil) or not detectable (clayey soil) using gas-chromatography.
If the contaminant consists of a wide range of compounds such as in the case of
mineral oil products, mobilization–degradation may occur in several consecutive steps.
376 Engineering Tools for Environmental Risk Management – 2

Figure 7.17 Increased toxicity caused by the soil microbiota in the initial phase of biodegradation.

Figure 7.17 shows the first key step of the bioremediation model experiment on the
soils contaminated with transformer oil.
Comparing the sandy and clayey soils, Figure 7.17 demonstrates well that the
pollutant is less strongly sorbed and easier to mobilize in sandy soil (higher toxicity)
while clayey soil is capable to buffer toxicity. As this clayey soil was a more suitable
habitat for the degrading microorganisms than the sandy one, cell numbers were higher
and the contaminant content lower after six weeks: remaining hydrocarbon content is
15,000 mg/kg in the sandy and 9,000 mg/kg in the clayey soil. A field demonstration
of the same technology in a 10 × 20 × 5 m soil volume resulted in 90% contaminant
depletion, and no toxicity after 10 months of treatment.
Figure 7.18 shows another example, the changes in toxicity during a slurry
phase bioremediation of an aged coal-tar polluted soil-like waste. Bioavailability and
biodegradability of coal tar is known to be poor, but the results shown here do
not support this belief. In the experiments presented, the hexane-acetone extractable
and gravimetrically determined initial hydrocarbon content (total extractable: EPH)
was 20,000 mg/kg and the gaschromatographically measurable content (GPH) only
8,000 mg/kg. The treatment covered aeration (M0 = only aeration) and the applica-
tion of a self-made microbial inoculant to enhance mobilization and biodegradation
(M1 = aeration and inoculation). According to the gaschromatographic analyses,
about 50% of GPH was eliminated in 6 weeks and 80% in 10 weeks treatment. Toxic-
ity shows a different picture: from the initial medium scale toxicity, it increases until the
6th week, reaching extremely high toxicity (measured by the highly sensitive marine
luminobacterium), then it starts to decrease, until reaching a close-to-acceptable level of
TEQ = 15 without a microbial inoculant and TEQ = 10 with the inoculant. Increased
 Bioaccessibility and bioavailability in risk assessment 377

Figure 7.18 Toxicity (TEQ) and concentration (EPH), in bioremediation of coal-tar contaminated soil.

biological activity in itself goes parallel to the increase of biological availability of

relatively stable contaminants in an active soil. Certain members of the community pro-
duce biotensides and complexing agents which help all the other community members
acquire the strongly sorbed contaminants (and other nutrients). The highest increase
in toxicity was in the beginning, and in the presence of the inoculant. The residue, in
spite of its low toxicity, was still present in a high concentration (GPH: 1800 mg/kg
378 Engineering Tools for Environmental Risk Management – 2

in M0 and 1000 mg/kg in M1 soil). After 20 weeks (not shown in the graphs), an
insignificant further decrease was measured in the contaminant content, and toxicity
remained under TEQ = 5, which indicates a ‘no risk’ situation. This means that the
approx. 1000 mg/kg residual contaminant was biologically unavailable, and could be
considered recalcitrant.

9 WORST-CASE AND REALISTIC WORST-CASE SIMULATION

Characterization of environmental risk adopts a conservative approach to avoid

underestimation due to uncertainties. Different tools are used to avoid ‘intentional
overestimation’, for example applying uncertainty factors. Every step of the serial
extrapolation is accompanied by a certain level of uncertainty, that is why an uncer-
tainty or safety factor is applied at every step. The aggregation of these safety factors
may result in a multiple overestimate. Another solution is based on stochastic models
which do not use concrete values, but intervals, probable ranges and fuzzy values. Com-
plex uncertainties, typical in the real environment, may cause undue overestimates and
corresponding costs. Direct toxicity tests under provoked worst-case conditions may
produce results which fulfill the data requirements of a conservative risk assessment
and can be used directly for decision making without risk posed by underestimation
or failing to detect the problem (e.g. failing to include the toxic contaminants into the
analytical program-based model). The application of pessimistic biological models will
show – even in the most overestimated scenario – less risk than the chemical model (e.g.
based on hexane-acetone extracted contaminant concentration). Chemical models can-
not measure the biologically unavailable portion and cannot characterize the dynamic
changes based on the interaction between the contaminants and the organisms.

9.1 Realistic worst-case models for dynamic testing of

bioavailability
The difference between the actual and potential adverse effects, the short-term and
long-term risks and the potential under- or overestimation of environmental risk
posed by contaminants in soil, sediment or solid waste is in close relation with
the bioavailability of contaminants and the possible changes in their mobility and
availability.
Organic contaminants’ bioavailability is in close correlation with their Kow value
and with the corresponding partition among soil physical phases or their solubility in
pore water. The interaction with the soil microbiota is an equally important influenc-
ing factor. Toxic metals’ bioavailability depends on the chemical form of the metal,
the pH, composition, sorption capacity of the soil and on the effect of plant root exu-
dates and other biological agents. A pessimistic, also called realistic worst-case test
scenario, changes the influencing factors to reach the worst case in the still living and
actively working soil. One can induce mobilization, increased bioavailability of the
contaminants before or during the test by lowering the Kow with tensides, cosolvents
or complexing agents, and increasing or decreasing the pH. One may apply a series of
tests with an increasing level of provoked changes, measuring the trend of the probable
 Bioaccessibility and bioavailability in risk assessment 379

Table 7.8 Toxicity results of pessimistic models for toxic metal contaminated soil.

EDTA +
No Lime Acid EDTA acidic
Test scenario Soil additive additive rain addition rain

Growth inhibition of Non-contaminated soil 0 9 9 0 0

Tetrahymena pyriformis (%) Soil contaminated with a 45 97 88 0 76
mixture of toxic metals
Luminescence inhibition of Non-contaminated soil 0 0 0 0 30
Vibrio fischeri (%) Soil contaminated with a 64 42 64 34 90
mixture of toxic metals
Reverse mutation of Non-contaminated soil 3 6 8 3 4
Salmonella typhimurium Soil contaminated with a 39 20 28 2 73
TA 1535 (No. of revertant mixture of toxic metals
colonies/Petri dish)

risk increase, and thereby making the test more dynamic and predictive. The influenc-
ing factor can be applied as a one-off or a repeated impact and the effect can be static
or impulse-like according to the test set-up.
The basic test set-up is a small-scale microcosm with direct contact between the soil
and the test organism (Gruiz et. al., 2001). Toxic metal mobility and bioavailability in
the contaminated soils were increased by changing the pH (by applying lime and ‘acid
rain’) and by adding EDTA, a chelate-complex-forming agent to the soil. The indicators
of bioavailability in these demonstration cases were toxicity and mutagenicity. The
results are shown in Table 7.8.
Acid rain alone has increased metal mobility, as shown by the effect on the unicel-
lular animal, Tetrahymena pyriformis. Lime reduced both toxicity and mutagenicity
measured by microorganisms, but surprisingly increased the toxicity to Tetrahymena.
An EDTA and acid rain combination increased dramatically both toxic and mutagenic
effects (point mutation on Salmonella typhimurium TA 1535). However, EDTA alone
rather hides the toxic effects by chelate complexing. The results indicate high risk
potential for the tested soil: both an increase and a decrease in the pH increased the
toxicity in some of the indicator organisms. This is because the soil contained high
concentrations of Cd, Zn, Cu, Pb and As although in a relatively stable form. Not
only did these models increase the scale of response, they made a better differentiation
possible.
Kow was changed by the addition of RAMEB (randomly methylated beta-
cyclodextrin) – an inclusion-complex-forming solubilizing agent – to the contaminated
soil. Changes in the bioavailability of pentachlorophenol in contaminated soil was
investigated in a matrix experiment where increasing PCP concentrations and increas-
ing RAMEB concentrations were applied and the changes in the availability were
indicated by direct contact toxicity and mutagenicity tests (Table 7.9).
An interesting trend can be observed in tests on Tetrahymena: both RAMEB and
the test organism interact with the toxic molecule (they compete with each other). 1%
RAMEB increased the bioavailability of PCP, but higher RAMEB concentrations were
able to decrease toxicity by encapsulating most of the PCP molecules and make them
380 Engineering Tools for Environmental Risk Management – 2

Table 7.9 Results from the pessimistic models of increased bioavailability of PCP in soil.

Soil 1% 2.5% 5% 10%

PCP No RAMEB RAMEB RAMEB RAMEB
Test concentration additive addition addition addition addition

Growth inhibition Non-contaminated soil 0 0 0 0 0

of Tetrahymena 25 mg/kg PCP 13 53 6 0 0
pyriformis (%) 50 mg/kg PCP 17 83 49 30 41
100 mg/kg PCP 79 100 69 56 69
200 mg/kg PCP 99 100 100 100 100
Luminescence Non-contaminated soil 0 0 0 0 0
inhibition 25 mg/kg PCP 5 0 0 0 0
of Vibrio 50 mg/kg PCP 46 29 29 11 0
fischeri (%) 100 mg/kg PCP 81 69 42 32 22
200 mg/kg PCP 88 84 81 71 57
Reverse mutation Non-contaminated soil 2 ND 3 3 2
of Salmonella 25 mg/kg PCP 12 ND 18 16 11
typhimurium TA 1535 50 mg/kg PCP 0 ND 29 47 81
(No. of revertant 100 mg/kg PCP 2 ND 42 219 115
colonies/Petri dish) 200 mg/kg PCP 1 ND 517 411 396

less available to the test organisms. In terms of bacterial toxicity (Vibrio fischeri),
RAMEB reduces toxicity by complexing and making PCP less available to the test
bacterium proportionally to the applied concentration.
A mutagenicity study of PCP is particularly interesting because recently published
results reveal a number of contradictions: some authors reported PCP as a positive,
others as a negative mutagen (Seiler, 1991; Gopalaswamy & Nair, 1992; Sekine et al.,
1997). In some studies, Ames reverse-mutation assay proved PCP to be negative, either
with or without metabolic activation. In this case the frameshift mutation occurred on
Salmonella typhimurium TA 1538 after complex formation with RAMEB, which indi-
cates that the contaminant’s low availability may be the reason for its being negative.
Other mechanisms may also play a role, but these have not been explored yet.
Artificially modified bioavailability indicates that the tested soil has the potential
to increase its adverse effects when the environmental conditions enhance the contam-
inant’s mobility and bioavailability. Contaminants are often in a non-available form
in abandoned, undisturbed soils because the soil and soil microbiota itself provide
protection against toxic contaminants. In other cases, mine waste, for example, has
a mineral composition which binds contaminants strongly into the mineral structure.
These stable forms are not always irreversible over the long term, but may be ready for
degradation e.g. by natural weathering. This way the built-in hazardous metals can be
mobilized. The ‘chemical time bomb’ phenomenon covers the presence and potential
remobilization of latent, immobile and non-bioavailable contaminants. The study of
toxicity or mutagenicity under the circumstances of enhanced bioavailability (e.g. due
to additives) provides a realistic worst-case scenario, and the results can be used for
decision making.
 Bioaccessibility and bioavailability in risk assessment 381

9.2 Effect of soil sorption capacity on bioavailability

Mobile forms of inorganic contaminants such as toxic metal ions have the affinity to
bond to oxides and hydroxides or to the large specific surface of clay minerals. Later,
during soil formation, they can be built into the molecular grid of the newly formed
minerals. When the mineral structure of the soil is deteriorated, these metals can be
mobilized again.
The mobility of organic contaminants in soil mainly depends on the organic mate-
rial content (OM) of the soil, given that organic contaminants are sorbed onto the
surface of the particles or built into the structure of stable (large size) humus molecules.
Sorption and incorporation of the contaminants into the humus takes place during the
long-term humus formation. The opposite process, humus degradation may also occur.
Both humus formation and humus degradation largely influence the bioavailability of
organic contaminants in the soil. This may be the key factor in the case of inherited
polluted sites where these changes have already taken place.
Regardless of time and humus formation, humus influences the organic contam-
inants’ mobility and bioavailability. Some regulatory screening values are higher for
humic soil, which shows the rationality in risk management.
Dependence of OM on the bioavailability of soil contaminants was demonstrated
on contaminated soils with different OM contents. Two different sandy soil types
with 0.45% humus content and a brown forest soil with 1.3% humus content were
contaminated with 4CP (log Kow = 2.39; Toxnet, 2014). Bioavailability was detected
by toxicity tests: Vibrio fischeri bioluminescence inhibition, Tetrahymena pyriformis
growth inhibition and Folsomia candida mortality tests.
A soil correction factor fsoil/water is used to characterize the difference between
toxicities in soil and water. This factor gives the ratio of a contaminant’s toxicity in
soil to the toxicity of the same concentration of the same toxicant in water (Eq. 7.1).
It is calculated from the EC50 values of 4CP in soils with different OM and in water.
Figure 7.19 shows the toxicity results depending on soil type.
 

 EC mg
L soil50 g
fsoil/water =   (7.1)
g ECwater50 mg
L

The fsoil/water values (>1 L/g) show how many times the toxicity of the same con-
taminant amount in soil is less than in water. A lower fsoil/water for sandy soil compared
to forest soil indicates that the toxicity in water is closer to sandy soil than to forest
soil as forest soil with a higher OM content has greater sorption capacity which results
in lower bioavailability.

10 BIOACCESSIBILITY AND BIOAVAILABILITY OF

CONTAMINANTS FOR HUMANS

Bioaccessibility and bioavailability of environmental contaminants may be a dominant

factor in exposures and human health risk. Microorganisms or other small animals
interact with the contaminants through their entire inner and outer surfaces as they
382 Engineering Tools for Environmental Risk Management – 2

Figure 7.19 4CP toxicity in sandy and humic forest soils: soil toxicity compared to water.

live in and are in direct contact with the environmental matrices. It such cases bioac-
cessibility and bioavailability may be very close to each other. Human exposures are
different: humans’ interaction is restricted to a limited interface by inhalation, inges-
tion and skin contact during a limited time period and with a limited proportion of
contaminants.
The most radical exposure from the viewpoint of bioavailability is the oral expo-
sure, which endangers mostly children because of uncontrolled uptake and high
sensitivity. Contaminants taken orally from soil are considered to be equal to the
bioavailable fraction absorbed by digestion. Dermal uptake can be modeled in vitro
by active animal skin, and the biologically available fraction of the contaminant
which penetrates the skin can be measured (see Chapter 3). Bioavailability tests using
respiration are mainly based on animal experiments: the large surface area of the
lung and repeated exposures are assessed. Human oral bioavailability is the ratio
of the internal (effective) dose to the orally administered dose. The orally adminis-
tered dose is quantitatively characterized compared to the intravenously administered
dose.
The applied mathematical, physico-chemical and biological models and their
application in the assessment of human health risk via environment are discussed
below.
 Bioaccessibility and bioavailability in risk assessment 383

10.1 Mathematical models for calculation of bioaccessibility- and

bioavailability-dependent human risk
The pathway of chemical substances within the human body, into the blood and the
organs is precisely described, but the pathway from the external environment into
the human body is still not sufficiently known because of the large variety of scenar-
ios. Human exposure models for pharmacological and toxicological purposes focus
on the absorption and mobility of contaminants inside the human body: this specific
scenario provides a practical model for the risk assessment of drugs. The mathemat-
ical models based on biochemical and physiological information are routinely used
in human health and safety management and in the pharmaceutical industry. For the
environment and for soil in particular, the only existing model is the gastrointestinal
model. Absorption of a contaminant (toxicant) from the environment into the circula-
tory system is the critical kinetic component of bioavailability and environmental risk
assessment of hazardous environmental contaminants. Statistical studies which model
skin permeability showed that the efficacy of dermal absorption is related to the log
Kow value (Cleek & Bunge, 1993; Roberts et al., 1995; Vecchia & Bunge, 2002), and
availability via inhalation depends on the vapor pressure, molecular weight, and log
Kow of organic contaminants. Calculation of dust inhalation is based on the bonds to
and the size of soil dust particles. The size of the airborne particles is a crucial factor;
fractions under 2.5 µm in diameter are completely absorbed by the lung tissue.
The most complex process occurs in the gastrointestinal tract after ingestion of soil.
Toxicity of soil contaminants depends upon their absorption from the gut. Informa-
tion on how well a contaminant is absorbed from the gut is important in determining
how much of a contaminant humans can be exposed to before health effects occur.
The difference between ingested and absorbed amounts is generally significant for
soil contaminants due to their limited accessibility and availability. The subcellular
mechanisms of absorption, and the influencing factors, as well as the transport from
the gastrointestinal tract to the blood should be properly understood for individual
contaminants Not only the amount, but also the rate of uptake is important, partic-
ularly under acute or subacute exposure conditions. The dose-dependent absorption
of lead, for example, is influenced by several factors, which together result in the rate
of absorption (Maddaloni et al., 1996; Ruby et al., 1996; US EPA, 1994 and US EPA,
2003):

– interactions with other inorganic and organic substances, particularly such

nutrients as calcium, iron, phosphate, vitamin D, fats, etc.
– metal species and particle size may influence the solubility and because of that, the
accessibility and bioavailability of lead;
– physico-chemical complexity of the environmental matrices;
– lead uptake is markedly lower when consumption is high than under fasting
conditions;
– children absorb more lead than adults do.

Several dynamic mechanisms can be identified behind these findings, which make
the modeling of bioavailability difficult and the result uncertain. Chemical models
and statistical evaluation of literature data can provide more relevant and practical
384 Engineering Tools for Environmental Risk Management – 2

estimates. The US EPA, for example, characterizes human risk of contaminated soil by
the relative bioavailability (RBA) which gives the ratio of the amount of a contaminant
absorbed from soil to the amount of that contaminant absorbed from food or water.
In the case of lead absorption in humans, relative bioavailability in soil compared to
water or food is about 60% on average (US EPA, 2009).
In vivo swine model was applied by Juhász et al. (2007, 2008) to establish the in
vitro digestion model for arsenic by comparing adipose tissue, blood plasma concentra-
tions and liver enzyme activities. As arsenic was studied by hundreds of studies, later on
Juhász et al. (2011) worked out a method for predicting relative arsenic bioavailability
in contaminated soils, using meta analysis and relative bioavailability–bioaccessibility
regression models. They found that arsenic RBA from the source of chromated copper
arsenate (CCA) posts, herbicide/pesticide, mining/smelting and weathered rock/iron
cap2 was 78.0, 78.4, 67.0 and 23.7%, respectively.

10.2 Chemical models for estimating accessibility of

contaminants for humans
Contaminants in soil experience the most radical changes and mobilization effects
when they enter the human gastrointestinal tract where extremely low pH prevails
especially in the stomach, and the bile salts have solubilizing effects on compounds
with high Kow .
Two types of models are mainly used for bioaccessibility testing: chemical
models (separation/speciation methods, such as extraction, membrane separation,
complexation, etc.) and in vitro gastrointestinal digestion models.
Chemical analysis methods such as sequential extractions using extractants of dif-
ferent affinity to the metal species and growing extraction strength target the different
solid matrices (humus, oxides, clay minerals) in the soil (Table 7.3). The interpretation
of the results is not always clear, but the aim is always to find the bioaccessible fraction
correspondent extract(s), e.g. Juhász et al. (2011).
There are more sophisticated analytical methods too, which can draw a true pic-
ture of chemical species and of the distribution of metals between different solid soil
constituents: these are the synchrothron-based X-ray absorption fine structure (XAFS)
and X-ray absorption near edge structure (XANEX) methods. Unfortunately they are
extremely costly, time-consuming and of limited availability.
Numerous in vitro digestion methods can be found in the literature mainly origi-
nating from the pharmaceutical industry. In environmental science, the most relevant
transformation of the contaminants is the focus when developing chemical/biochemical
simulation models for digestion. These digestion models are well-studied and compared
to real bioavailability data for the purpose of risk assessment. For example, play-
ground soils contaminated with arsenic and PAH were extensively investigated using
in vitro gastrointestinal models to assess the exposure of children. The results of the
gastrointestinal model extraction from lead-contaminated soil were compared to the
contaminant concentration in the blood serum of children playing on the playground
by Ren et al. (2006). They have found a linear relationship between the bioaccessible

2
Intensely oxidized, weathered or decomposed rock, usually the upper and exposed part of an
ore deposit or mineral vein.
 Bioaccessibility and bioavailability in risk assessment 385

fraction mobilized from soil into the ingestion juice and the metal contamination of
the ingested soil. Lead in the blood serum was also linearly related to the bioaccessible
amount.
Determination of the bioaccessible fraction from contaminated soil is a major
research topic all over the world. In the US the Solubility/Bioavailability Research
Consortium (SBRC) worked out the SBET, also called SBRC method for testing lead
and arsenic bioaccessibility and determining relative bioaccessibility.
The Bioaccessibility Research Group of Europe (BARGE, 2014a) is a network of
European organizations for studying human bioaccessibility of priority contaminants
in soil and disseminating oral data, mainly for regulatory purposes (BARGE, 2014b).
The Canadian working group for Bioaccessibility Research (BARC, 2014) has
studied inorganic and organic contaminant bioaccessibility in soils on contaminated
sites in Canada. A priority objective of BARC is to advance the scientific basis for
incorporating bioaccessibility testing into site specific human health risk assessments.
Health Canada (2007) collected existing data from in vivo and in vitro bioaccessi-
bility research and compared them to physico-chemical soil parameters that influence
bioavailability. Some results on As, Pb and Cd are presented in this section.
A useful guide has been prepared for US defense facilities (Metals Bioavailability,
2003) to incorporate bioavailability adjustments into human health and ecological risk
assessments. It has gathered current information on metals bioavailability, it explains
concepts and identifies types of data necessary for assessing bioavailability and incor-
porating them into risk assessment. The first part of the guide gives a brief description
on test methods suitable for human health and ecological risk assessment and deals with
the acceptability of the results. In the second part, the technical background for metals
bioavailability assessment, types of studies and connected protocols are discussed in
details for every relevant metal.

10.2.1 Human bioaccessibility of toxic metals

Metal availability in soil is mainly determined by the dissolution–precipitation process
pair. The same processes are present and influence human health effects by inorganic
mineral components in soil. Dissolution kinetics controls the availability of metal ions,
and basic rock minerals undergo continuous dissolution in natural soil. Other pro-
cess pairs such as oxidation–reduction, sorption–desorption and ion exchange also
play a crucial role in bioaccessibility of metals. Soil inhaled or ingested by humans
contains metal species of different bioaccessibility. The basic bioaccessibility, valid in
natural soil, will dramatically change as a result of new equilibrium of dissolution–
precipitation, sorption–desorption, oxidation–reduction processes in the human body.
The two together yield the actual metals bioaccessibility in the human body. This is
compounded by the changing environment of the digestive system. The conditions and
biochemistry of the organism (humans) determine the final bioavailable and taken-up
proportion of the metal.
Bioaccessibility in the context of oral exposures means that contaminants are
released from soil particles within the gastrointestinal tract during digestion. In addi-
tion to the chemical speciation of the contaminants, e.g. the chemical formula of a
toxic metal, the co-occurring metals and metal species, their bonds to the soil’s matrix
386 Engineering Tools for Environmental Risk Management – 2

are of crucial importance for bioaccessibility and in human and environmental toxicity
(Figure 7.2).
For example arsenic may occur in the form of Ca3 (AsO4 )2 , Mg3 (AsO4 )2 , As2 O5 in
aerobic soil and as As or As2 S3 in anoxic or anaerobic soil. Lead occurs as PbO, PbCO3
or Pb3 (CO3 )(OH)2 under aerobic and as Pb or PbS under anaerobic conditions. The
chemical transformations during digestion increase bioaccessibility, depending on the
original mineral speciation, chemical bonds and the actual parameters pH and redox
potential of the digestive system.
The above described partial and limited description makes clear that modeling of
metal bioaccessibility in the human body is accompanied by high uncertainty.
The chemical model of PBASE (Basta & Gradwohl, 2000) models the potentially
bioavailable sequential extraction. It is based on the assumption that the acidic juice of
the stomach (pH = 1–2 at fasting and pH = 2–4 when fed) is the main factor responsible
for making metals accessible during digestion. PBASE extraction for metals applies a
four-step extraction method using
1. 0.5 M Ca(NO3 )2 for the exchangeable, readily soluble
2. 1 M NaOAc (pH = 5) for weak acid-soluble weak surface complexes
3. 0.1 M Na2 EDTA (pH = 7) for surface complexes and precipitates and
4. 4 M HNO3 for very insoluble species.
The method of Mercier et al. (2002) is an even more simplified model based on
the extraction of the soil samples at 37◦ C in HCl at pH = 2. This method was used to
determine oral availability of antimony, arsenic, barium, cadmium, chromium, lead,
mercury and selenium. In comparison to other bioaccessibility and bioavailability data,
it resulted in a slight overestimate.
IVG (In Vitro Gastrointestinal) digestion method (Schroder et al., 2004) is based
on a leaching procedure using i) a gastric extraction fluid (NaCl and pepsin) at pH
values of 1.5, 2.0, or 2.5 and ii) an intestinal fluid of pancreatin and bile extract
(Rodrigues & Basta, 1999).
Total Migratable Screening, European Standard (EN 71-3, 2013) – also uses HCl
to obtain the migratable portion of a tested object. The aim of the standard is a pre-
liminary screening that looks at the total migration from a toy (Safety Toys Directive,
2009), but it is also useful for soil. The principle of the procedure is the extraction of
soluble elements from solid materials under the conditions which simulate the material
remaining in contact with stomach acid for a period of time after being swallowed. The
following heavy metals are measured: aluminum (Al), antimony (Sb), arsenic (As), bar-
ium (Ba), boron (B), cadmium (Cd), chromium III (Cr3+ ), chromium VI (Cr6+ ), cobalt
(Co), copper (Cu), lead (Pb), manganese (Mn), mercury (Hg), nickel (Ni), selenium
(Se), strontium (Sr), tin (Sn), organic tin and zinc (Zn).
There are several digestion fluids in the gastrointestinal tract during digestion such
as gastric juice with low pH and the intestinal fluids that can mobilize or immobilize
toxic metals, which can also be bound to a protein molecule or accumulated in liver
or fat tissues. Gastrointestinal models simulate one or several steps from the com-
plex human digestion system applying a realistic residential time and using artificial
biofluids.
– Oral cavity: residential time: seconds–minutes, pH = 6.5;
– Stomach when fasting residential time: 8–15 minutes, pH = 1−2; when fed
residential time: 0.5–3 h, pH = 2−5;
 Bioaccessibility and bioavailability in risk assessment 387

– Small intestine has further sections:

◦ duodenum: 0.5–0.75 h, pH = 4–5.5

◦ jejunum: 1.5–2.0 h, pH = 5.5–7
◦ ileum: 5–7 h, pH = 7–7.5

– Colon 15–60 h, pH = 6–7.5.

SBET – Simplified Bioaccessibility Extraction Test (Ruby et al., 1996; Juhász et al.,
2008; Lamb et al., 2009; Mingot et al., 2011) is a routinely used rapid method which
only comprises the gastric phase extraction.
PBET – Physiologically Based Extraction Test (Ooemen et al., 2003) simulates
the leaching of a solid matrix in the human gastrointestinal tract, and determines the
bioaccessibility of a particular element that is available for adsorption during transit
through the small intestine. The applied gastric component consists of pepsin, citrate,
malate, lactic acid and acetic acid, and the intestinal component comprises pancreatin
and bile salts.
Ruby et al. (1996) and Rodriguez et al. (1999) claimed in their studies that the
PBET method showed a decrease in the bioaccessibility of lead due to the high pH in the
intestinal phase where lead precipitated. Contradictory results were found on the bioac-
cessibility of arsenic, which became more accessible at high pH (Juhász et al., 2008;
Cave et al., 2002). Juhász et al. (2008) compared the results of the five-step extraction
method (Teisser method) (Wenzel et al., 2001), those of the SBET digestion model and
blood plasma from in vivo swine tests on animals kept on arsenic-contaminated soil.
The comparison resulted in good correlation between the results of the SBET method
and the in vivo model.
RBALP – The Relative Bioaccessibility Leaching Protocol was specifically
developed for lead in soil. It uses physiologically relevant pH of the stom-
ach but uses a glycine buffer as the extraction medium (Drexler & Brattin,
2007).
RIVM – Method of the Dutch National Institute for Public Health and the Envi-
ronment. It mimics the chemical environment of the human gastrointestinal system
using the mixture of pepsin, mucin and BSA as a gastric fluid and bile, duodenal juice,
chime, pancreatin and lipase as intestinal secretion components (Versantvoort et al.,
2004).
SHIME – Simulator of the Human Intestinal Microbial Ecosystem. This in vitro
model was developed according to De Boever et al. (2000) and Laird et al. (2007). The
artificial gastric biofluid contains pectin, mucin, cellobiose, proteose peptone, starch
and the intestinal one comprises pancreatin. This maintains a microbial community
representative of that found in the human intestine. The SHIME is operated under
anaerobic conditions with human gastrointestinal microorganisms in the colon stage
since these microbes may influence metals bioaccessibility (Laird, 2010a and Laird,
2010b).
TIM – TNO Intestinal Model: it is a dynamic simulation for dissolution and
bioaccessibility in the human gastrointestinal tract. It is used both for drugs, food
and environmental samples, mainly soil. It is a multi-compartment method using
lipase, pepsin as gastric fluid and bile, porcine, pancreatin as the intestinal component
(Bosso & Enzweiler, 2008; Bosso et al., 2008).
388 Engineering Tools for Environmental Risk Management – 2

Figure 7.20 Unified Bioaccessibility Method (UBM) gastrointestinal model (BARGE, 2014b).

GFE – Gastric Fluid Extraction methods is based on the PBET (Ruby et al., 1996)
and the IVG methods (Rodriguez et al., 1999) using a liquid to solid ratio of 100:1,
gastric pH of 1.8 and intestinal pH of 7.0. It applies only gastric secretion components:
pepsin, citrate, malate and acetic acid.
UBM – Unified Bioaccessibility Method (BARGE, 2014b; Broadway et al. 2010) –
is based on the Oomen et al. (2003) methodology and is recommended for inorganic
substances. UBM contains four main digestion juices (saliva, gastric and duodenal
juices as well as bile) and the samples are analyzed both after the gastric and intestinal
treatment phase (Figure 7.20) to see the difference between the two.
According to the results found in the literature, the bioaccessibility of several
toxic metals such as lead, cadmium (Tang et al., 2006a), zinc or chromium (VI) is
increased by gastric ingestion due to a low pH. The bioaccessibility peak of arsenic
occurred after the duodenal juice treatment at a higher pH. The significantly more
toxic and carcinogenic As(III) and As(V) are present in various ratios in the subsequent
gastrointestinal tracts. That is why arsenic toxicity is hard to predict in the human body.
Further time- and cost-intensive research is needed to explain and model the influence
of the processes in the mouth on the bioaccessibility of toxic metals.
 Bioaccessibility and bioavailability in risk assessment 389

A comprehensive comparative study prepared by Health Canada (2007) yielded

interesting results. Arsenic and lead content and their bioavailablity/bioaccessibility
results were collected from several hundred soil samples, measured both by in vivo
animal tests and in vitro applied artificial biofluids such as SBET, PBET, IVG, SHIME,
TIM and GFE. Some important lessons learned are summarized below.
In vivo study results for arsenic-contaminated soil:
The average arsenic concentration of 55 soil samples was 1950 ± 3760 mg/kg, of which
23 ± 20% was bioavailable. Arsenic bioavailability was shown to be less than 5% in
13 of the 55 soils tested in in vivo studies. However, there were four soils where
bioavailability was greater than 50%. Based on these results, Health Canada (2007)
recommended the use of 25% as bioavailable for uptake into the bloodstream from
the whole of soil arsenic in human health risk assessments. Statistical analysis indicates
that the bioavailable percentage is independent of the total arsenic concentration in
the soil samples. Rats contained significantly less bioavailable arsenic than other test
species (monkey, rabbit, swine and rat were studied).
In vitro study results for arsenic-contaminated soil:
The average arsenic concentration of 102 soil samples was 1204 ± 2775 mg/kg. The
bioaccessible percentage was 25.6 ± 19.0% from a total (100%) of arsenic. Therefore,
based on in vitro studies, on average less than 26% of arsenic can be considered bioac-
cessible and available for uptake into the bloodstream. Given that soluble arsenic is
almost 100% absorbed into the bloodstream, arsenic bioaccessibility and bioavailabil-
ity (absorbed into the blood stream) are equal. An acidic medium (pH of 4.0) resulted
in significantly lower arsenic bioaccessibility. An evaluation of accessible arsenic and
liquid–solid ratio in the test indicated that there was no statistical difference across the
varying ratios. The accessible fraction is independent of the total arsenic concentration
in the soil sample.
In vivo study results for lead-contaminated soil:
soil samples were analyzed: the average lead concentration in the soils was
6067 ± 3521 mg/kg and the bioavailable percent was 46 ± 27%. This means that on
average less than 50% of total lead can be considered available for uptake into the
bloodstream. The bioavailable portion is independent of the total lead concentration in
the soil. Unlike arsenic, only 50% of soluble lead is absorbed across the intestinal wall.
In vitro study results for lead-contaminated soil:
Based on 86 discrete test results on soil lead bioaccessibility, the average lead concen-
tration in soil was 3180 ± 3285 mg/kg (N = 86). The bioaccessible percentage in the
stomach phase was 51 ± 26% and 4.2 ± 5.0% (n = 45) in the intestinal phase. There
was some weak correlation between bioaccessible and total lead concentrations and
with the pH of the soil.
The results introduced above based on the Health Canada survey can be utilized
in generic predictions. Site-specific risk assessments need a different approach; site-
specific bioaccessibility and bioavailability should be assessed case by case because the
standard deviations are large due to soil types and environmental conditions.

10.2.2 Bioaccessibility of organic compounds in humans

While metals mobility is mainly influenced by the changing pH along the gastroin-
testinal tract, organic compounds’ accessibility is determined by the mobilizing effects
390 Engineering Tools for Environmental Risk Management – 2

Figure 7.21 Equipment of an in vitro digestion test (Tang et al., 2006b).

of bile salts. Among organic substances, PAH bioaccessibility is mostly studied by

in vitro gastrointestinal models. Tang et al. (2006b) found increased bioaccessibility
in the small intestinal phase, confirmed both by chemical analysis and effect testing.
Figure 7.21 shows the set-up for the digestion test.
Otherwise negative phenanthrene toxicity occurred after intestinal digestion due
to micelle formation with bile salts. In vivo bioavailability of dioxins and furans was
determined in orally exposed minipigs and rats. Tissue sampling and analysis showed
concentration in liver and adipose tissue (Wittsiepe et al., 2007; Budinsky et al., 2008).
Chemical analysis failed to provide adequate information as to degradation products
of organic contaminants are more toxic than the original substance.

10.2.3 Chemical models combined with biological models – measuring

toxic effects after digestion
Pre-treatment before toxicity assessment applying a human digestion simulation model
may result in a realistic scenario similar to the natural process. Theoretically, the tox-
icity (mutagenicity, reprotoxicity) tests with in vitro digested samples should provide
highly realistic effect results similar to the actual effects in humans. On the other hand,
toxicity tests may have several disadvantages, for example extensive dilution of in vitro
digested samples. The digestion fluid (saliva:gastric:duodenal:bile = 1:1.5:3:1) has to
be applied in an extremely high ratio to the soil – a minimum of 98:1 – to ensure the
dissolution of metals (Ruby et al., 1996). Before toxicity testing, further extraction
and concentration of the digested sample is necessary.
 Bioaccessibility and bioavailability in risk assessment 391

In the case of organic contaminants, the detection of toxic metabolites in the

digested fluid presents a problem unresolved as yet because tracing various unidentified
metabolites is a difficult task for chemical analysis.
There are some developments to eliminate the disadvantages which occur in
toxicity testing of contaminated soil and modeling maximum bioavailability:
1. For organic contaminants, soil spiking with radioactive labeled contaminants
can be a solution before in vitro digestion. It helps monitor the various
transformations of the labeled compounds during digestion.
2. The S9 enzyme mix represents a fairly simple gastrointestinal model. It includes
the addition of the commercially available S9 enzyme mix that contains 5 mM
glucose-6-phosphate, 4 mM NADPH and S9 from Spargue-Dawley rat liver
oxidative enzymes (Maron & Ames, 1983). This method is suitable for further
testing e.g. using the Ames test to examine mutagenic effect of soil (Hajdu et al.,
2008).
3. Another promising but not yet practiced method for soils applies Caco-2 cell
lines, which is a monolayered cell line originating from the small intestine and
is routinely used in pharmacokinetics. It is suitable to estimate the absorption of
pharmaceuticals and to test cytotoxicity by measuring cell layer integrity, enzyme
activities or contaminant concentration in the harvested cells (Laparra et al.,
2005). As Caco-2 epithelial cells originate from the human large intestine, this
in vitro test system can provide one of the most realistic absorption models of
in vitro digested contaminants.

11 CONCLUSIONS

Several methods are available for modeling and predicting bioavailability and bioacces-
sibility of hazardous environmental contaminants. The less environmentally relevant
but simplest method is the determination of log Kow or various partial extractions
and solvent distributions of the contaminants. These methods are often the cheap-
est but the results are only hazard estimates that fail to provide information on the
actual environment and the target ecosystem or human receptors. The developments of
bioavailability measurement methods as summarized in this chapter reflect the various
concepts and introduce the mathematical, chemical and combined models that try to
describe the phenomenon of bioavailability which itself has not yet been fully defined.
The first step to successfully manage this problem is to identify those points of
the conceptual risk model (Figure 1.1 in Volume 1) where bioavailability may have an
impact on the quantity or quality of risk. A thorough investigation of the environmen-
tal fate and behavior of contaminants should include the interactions with the living
part of the ecosystems. Earthworm models, plant accumulation tests, in vivo swine
experiments, and epidemiological data serve to address the contaminant–matrix–biota
interactions and the representation of these processes in toxicity, mutagenicity, repro-
toxicity tests. Another uncertainty is caused by the environmental conditions, which is
handled by the ‘pessimistic testing’ concept introduced here or by realistic worst-case
methods that can directly test the adverse effects exerted by contaminated matrices.
Since our knowledge lags behind what is required to understand contaminants’
bioaccessibility and bioavailability in the environment, it is clear that this is one of
392 Engineering Tools for Environmental Risk Management – 2

the fields of environmental management that needs further development, innovative

approaches, a uniform assessment and improved interpretation tools. These tools
are necessary for efficient assessment methods and for treating uncertainties asso-
ciated with bioavailability such that risk is neither unnecessarily nor unacceptably
overestimated.

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Zhang, H., Davison, W., Mortimer, R.J.G., Krom, M.D., Hayes, P.J. & Davies, I.M. (2002).
Localised remobilisation of metals in a marine sediment. Science of the Total Environment,
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ability of heavy metals in polluted soils to rice communications in soil. Science and Plant
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Chapter 8

Microcosm models and technological

experiments
K. Gruiz, M. Molnár,V. Feigl, E. Vaszita & O. Klebercz
Department of Applied Biotechnology and Food Science,
Budapest University of Technology and Economics, Budapest, Hungary

ABSTRACT

Microcosms are easily controllable, small-size models of the environment. In spite

of some shortcomings deriving from high surface/volume ratio and reduced diversity,
microcosms provide highly realistic results, suitable for decision making. Microcosms
are useful tools for learning and exploring nature, the water and soil ecosystems, for
simulating natural or anthropogenic impacts on the ecosystems, for measuring toxicity
by a multispecies, dynamic system and for the small-scale modeling of technologies.
This chapter introduces the practice and implementation of microcosm testing and
modeling via examples from the authors’ experience. The examples include micro-
cosms used for modeling the fate and behavior of contaminants in the environment,
their biodegradation and bioavailability, and microcosms for modeling bioleaching
and stabilization. In addition to the experimental set-up of the small-scale models, this
chapter discusses the integrated monitoring of microcosms in a general way by com-
bining physico-chemical analysis and biological testing. It also discusses the evaluation
and interpretation of the results’ utilization in risk management and decision making.
Applications and results of various microcosms are discussed in Chapters 4 and 5.

1 INTRODUCTION

In environmental toxicology, microcosm is defined as a small representative part of the

real environment selected for the study of ecosystem response to environmental con-
ditions or chemical contaminants (Chapters 4 and 5). Microcosm studies are usually
performed in the laboratory, sometimes in the field.
Microcosms are also used as small-scale technological models for water treat-
ment or soil remediation. These microcosms can simulate those natural environmental
processes which are responsible for the hazard and risk posed by environmen-
tal contaminants or can provide a suitable basis for remediation and engineered
interventions.
In both applications, microcosms simulate the real ecosystem, measure the effects
of physical, chemical or environmental parameters and those of the pollutants on the
environmental compartments, phases and, on the complex multispecies ecosystem at
different trophic levels.
402 Engineering Tools for Environmental Risk Management – 2

Microcosm and mesocosm studies can be considered as intermediates between

bioassays and ecosystem studies. Fate processes and behavior of chemicals and
biological changes on population, community or ecosystem level can be monitored
by all kinds of physical, chemical, biological, ecological or toxicological tools and
their combinations.
Microcosm in general means a ‘little world’, a ‘world in miniature’, for exam-
ple a part of the environment, natural water, sediment or soil that is regarded as a
miniaturized copy of the natural system. There is a wide range in the naturalness or
artificiality of microcosms, from undisturbed natural aliquots (extracted from the real
environment) under controlled conditions to a fully artificial controlled and regulated
set-up of a system with desired complexity. It is a highly realistic model, but – since
every single microcosm has its own history and evolution – the statistical evaluation
of the changes in space and time needs special tools (Gruiz et al., 2001).
The microcosm experiments do not only model the natural system but also allow
for investigation of external interventions into the natural processes, thus challeng-
ing the system artificially. Microcosms enable the monitoring and control of natural
processes and provide basic information for the design of biological and ecological
technologies. The outcomes of technological processes and their complex effects on
the physical, chemical and biological properties and activities can be measured and
monitored in time. The series of technological parameters can be tested in order to
find their optimum.
Microcosm applications can cover both water and soil ecosystems, and they can
vary in size, duration and complexity (Gruiz et al., 2001). They are key engineer-
ing tools in modern environmental management for both risk assessment and risk
reduction and the results provided by them can be used for direct decision making.
The essence of microcosm testing – when modeling either natural or technologi-
cal processes – is the appropriate selection of the determining factors and influential
parameters. Once there is knowledge on or familiarity with the environment, the con-
cept of the model should be created by transforming the complex environment into a
simplified model. The main target of a microcosm concept is to maintain as much of
the important and influencing environmental factors, and the diversity and activity of
the community as possible. This means that the principle and the concept have priority
when planning the microcosms, but the set-up and the implementation can be fairly
basic. The concept of a microcosm needs an accurate conceptual risk model.

2 AQUATIC MICROCOSMS FOR SCREENING CHEMICAL

SUBSTANCES AND TECHNOLOGIES

Open or closed batch reactors or flow-through set-ups in single or cascade arrange-

ment can be used for water, wastewater, leachates and other liquid-form environmental
matter. Living ecosystems can be compressed into a small-size ecosystem model or into
a water-treatment technology model. Batch-type microcosms work with or without
mixing, with inlets and outlets suitable for recycling or for injection of air, chemi-
cals, nutrients, regulators and other chemical or biological additives as well as for the
extracting of samples or aliquots. Physical factors such as heat or radiation can also
be included in these microcosms. The aim of manipulations in the microcosm is the
 Microcosm models and technological experiments 403

study of the contaminant and/or the community as well as the community’s response
to natural or simulated impacts.
Flow-through column and tube reactors are suitable for the simulation of natural
transport behavior of chemicals, transport pathways in the water column, depths and
the relevant redox potential as well as water treatment processes such as stripping,
adsorption and absorption, filtration, flotation, various chemical reactions, or the
intensification of community activity. They are popular set-ups for aquatic model
systems and also for the simulation of water treatment including wastewater treatment
technologies.
Realistic aquatic micro- and mesocosms contain sediment too, and can be used
for testing the fate and the behavior of the chemical substance in the water–sediment
system, the response of aquatic and benthic ecosystems to chemical substances. In addi-
tion, they can be used for general or targeted ecosystem studies and for the simulation
of technological interventions in aquatic systems.
The American Society for Testing and Materials (ASTM International, 2014)
standardized aquatic and terrestrial microcosms for regulatory purposes:

– Standard Practice for Standardized Aquatic Microcosms, fresh water (ASTM

E1366, 2011)
– Standard Guide for Conducting a Terrestrial Soil-Core Microcosm Test (ASTM
E1197, 2012)

United States Environmental Protection Agency (US EPA, 2014) uses standardized
microcosms for generic and site-specific risk assessment:

– Generic Freshwater Microcosm Test (1996) is used for assessing the potential
hazard of a chemical substance to freshwater ecosystems by acquiring data on
the chemical fate and/or ecological effects of chemical substances and mixtures.
Standardized aquatic microcosms, naturally derived mixed-flask culture micro-
cosms, or naturally derived pond microcosms are used with and without sediment.
The microcosms contain freshwater algae and zooplankton with an assortment of
unidentified bacteria and fungi.
– Site-Specific Aquatic Microcosm Test (1996) is performed in a microcosm made
of an indigenous water column and sediment core. This test system is capable of
evaluating organic chemical substances, either soluble or insoluble, which may
form either air–water surface films or aggregates which sink to bottom sediments.
The acquired data can support hazard assessment of a chemical substance to a
particular natural aquatic system.
– Sediment/Water Microcosm Biodegradation Test (1998) has been developed for
testing pesticides, biocides and other toxic substances. Sediment and water with
a representative sample of the natural microbial community from a test site of
interest are required in this test.

Some uses of standardized microcosms and the results are:

– Use of microcosm data for regulatory decisions: fate and behavior of chemicals as
well as the extent of their adverse effects at several trophic levels on competitive
species, spontaneous detoxification and system recovery, etc.
404 Engineering Tools for Environmental Risk Management – 2

– Use of microcosm data for generic and site-specific risk management: quality
and quantity of risk, effect of risk management options and risk reduction using
technologies.
– Determination of safe levels of chemical exposure.
– Study of community changes and ecosystem functional complexity.
– Study of community behavior after chemical stress, how ecosystems recover to
resemble the reference state.
– Measurement of microbial population and community dynamics, density, diver-
sity, biochemical/enzyme activities, biodegradation and mineralization of toxicant
molecules under various environmental conditions such as pH, redox potential,
salinity, temperature and sediment/water interface conditions, etc.

The aim and size of aquatic micro-and mesocosms show a wide scope from a few
milliliter jar or column through laboratory aquariums or water columns, to open air
ponds, artificial lakes, koi ponds or huge-size aquariums or aquacultures.
Aquatic mesocosms integrate the experience of ecology, aquarium science, aqua-
cultures and wastewater treatment. MESOCOSM (2014) is an information hub of
mesocosm facilities in aquatic ecosystems world-wide, provided by the MESOAQUA
project. The aim of most research on aquatic mesocosms is to better understand the
ecological behavior of the aquatic component of the ecosystem and its utilization in
ecological technologies such as

– artificial ponds and wetlands for the treatment of contaminated runoff waters and
leachates;
– passive artificial systems for the neutralization and clean-up of acidic and/or
contaminated mine drainage or leachates;
– active subsurface soil zones for the filtration or detoxification of seepage, drainage
or leachates;
– rhizofiltration for the elimination of nutrients or other contaminants from runoff
waters;
– living machines for the remediation of contaminated lakes and reservoirs or just
for the conservation of natural water bodies as well as for the treatment of
wastewaters;
– all possible ecotechnologies applied to conservation or sustainable use of waters.

From the point of view of evaluation and interpretation of the results it is important
to understand the limitations of micro- and mesocosms due to the compression of the
real environment into a small volume (Landis & Yu, 1999; Adey & Loveland, 1999).
The aim of testing and manipulating the ecosystem and the bio- or ecotechnologies
determine the type and the set-up of the micro- and mesocosms. Aquatic micro- and
mesocosms are supplied and available in a wide range for teaching and research pur-
poses as well as for koi ponds or water gardens: these may be suitable for technological
purposes, too. But if the ready-made ones do not fit the necessary set-up, they must be
designed and built by ourselves.
The most common form of an aquatic mesocosm is a pond that contains nat-
ural fresh or marine water and sediment, and a community consisting of at least
micro-organisms, plankton, macrophytes, macroinvertebrates and, if necessary, fish.
The mesocosm may be the perfect tool for studying impacts on an ecosystem under
 Microcosm models and technological experiments 405

field-relevant conditions. Technological mesocosms measure the impact of technolog-

ical parameters such as flow rate, temperature, pH, redox potential, aeration, ion
concentration, nutrient content, the effect of additives, special nutrients, chemicals of
beneficial or adverse effects on the aquatic ecosystems, diversity and activity of the
community, as well as element and energy cycling of the micro- or mesocosm system.
Based on the results of technological studies, the real environmental system can be
manipulated and controlled by engineering tools applying the technological parame-
ters that proved to be optimal in the micro- and mesocosm experiment. The purpose
of these engineering manipulations may only be to maintain the healthy state of our
waters, i.e. conservation, or to remediate the unhealthy environment by reducing the
risk of deterioration and reversing disadvantageous changes. Our natural environ-
ment can only be engineered in an eco-efficient and sustainable way if the actions
are based on a deep insight into the structure and function of ecosystems, natural
processes, trends and acceptable risk levels. Micro- and mesocosms greatly support
the acquisition of such knowledge on natural diversity and processes such as element
cycling, eutrophication, and the fate and behavior of contaminants in the environment,
e.g. their biodegradation.
Figure 8.1 shows a simple laboratory aquatic microcosm vessel equipped with
inlet and outlet fittings for aeration, water pumping (subtraction, flow-through or
recycling), injection and sampling. The size can vary from a few milliliters to hundreds
of liters.
In Figure 8.2 parallel aquatic microcosms are shown in a water bath for tempera-
ture control. The open-top columns are provided with plugs at the bottom and filled

Figure 8.1 Open aquatic microcosm.

406 Engineering Tools for Environmental Risk Management – 2

Figure 8.2 Open laboratory microcosms with undisturbed sediment cores placed into a thermostat
for testing biodegradation, eutrofication or toxicity.

with disturbed or undisturbed sediment cores. Continuous aeration and water cycling
are applied by plastic tubes.
Figure 8.3 and 8.4 show open-air aquatic microcosms for research and testing
of changes in aquatic ecosystems. The number of units enables repetitions and serial
investigations. The experimental devices shown in Figure 8.3 can study the impact
of warming on an aquatic ecosystem (Yvon-Durocher et al., 2011). Figure 8.4 shows
open-air microcosms used for ecotoxicological tests. The microcosms have a water
depth of 1 m, a diameter of 3.9 m, and a volume of about 12,000 L. They are open to
the elements, e.g. rain, light, temperature (University of Guelph, 2013).
Figure 8.5 shows a sediment microcosm set-up. Sediment microcosms can be run
using a homogenized sediment or undisturbed sediment core, which can simulate
original layers and real redox potentials to test aerobic, anoxic or fully anaerobic
processes.
Plants are important parts of the aquatic ecosystem and can be utilized in natural
water treatment technologies such as artificial ponds, wetlands, living machines or
reactive zones on the surface or partly subsurface. The set-up in Figure 8.6 is suitable
for modeling and testing the transforming, biodegrading and cleaning capacity of a
plant-based aquatic system.
Figure 8.7 shows the laboratory treatment of contaminated water in a fluidiza-
tion column. Different sorbents can be fluidized in the contaminated water to ensure
 Microcosm models and technological experiments 407

Figure 8.3 Microcosms for investigating the impact of warming on a freshwater ecosystem (Yvon-
Durocher, 2013).

intensive contact between the dissolved contaminant molecules and the sorbent. The
same set-up was applied for the molecular encapsulation and removal of bisphenol-A
using polymerized cyclodextrin beads (Gruiz et al., 2010a).

3 SOIL MICRO- AND MESOCOSMS FOR MODELING

ENVIRONMENTAL PROCESSES IN BIO- AND
ECOTECHNOLOGIES

Soil micro- and mesocosms provide information under controlled conditions on

the fate, natural changes and evolution within an actual representative part of the
contaminated soil, and can model typical natural processes and remediation scenarios.
408 Engineering Tools for Environmental Risk Management – 2

Figure 8.4 Aquatic ecotoxicology microcosms (University of Guelph, 2013).

Figure 8.5 Closed sediment microcosms.

 Microcosm models and technological experiments 409

Figure 8.6 Aquatic microcosm with sediment, water and vegetation.

Figure 8.7 Microcosm for modeling wastewater fluidization.

General laboratory microcosms are being increasingly used for treatability testing
of soil and groundwater. Microcosms are essential tools for pre-testing soil to help
technology selection and planning because experience and routine cannot substitute
site-specific information. Every site is unique and the number and combinations of
biotic and abiotic parameters are so high that the impact of an additive or a technology
410 Engineering Tools for Environmental Risk Management – 2

Figure 8.8 Batch and column testing on contaminated groundwater (Adventus, 2013).

cannot be forecasted without testing, even if a thorough assessment has been carried
out. This is of particular importance when new additives and innovative technologies
are being developed.
Figure 8.8 shows flow-through columns to mimic groundwater flow conditions.
The columns contain soil from a contaminated site and a special inoculum for enhanc-
ing biodegradation. Groundwater is pumped through the column in an up-flow
manner. The 2-liter glass jars filled with soil ensure longer residence time for natu-
ral attenuation of the contaminated groundwater by biodegrading the contaminants.
Column and batch systems can be combined as shown in the photo: the outflow of the
column is treated in the jars. The outflow from the jars is analyzed. The test system
 Microcosm models and technological experiments 411

in the picture is used by the Laboratory Treatability Testing Services of the Adventus
Company (Adventus, 2013).
The model ecosystems, or laboratory microcosms introduced here have become a
key research tool in soil ecology and soil remediation. Due to the speed, statistical
power and mechanistic insights attainable by laboratory-based microcosm experi-
ments, they have made considerable contribution to today’s knowledge about soil
and the behavior of the soil microbiological community. Soil science and remediation
have proved the value and importance of microcosm testing, but at the same time
they drew attention to the fact that, given the small scale and artificiality, microcosm
results must be validated by field model ecosystems (mesocosms), field tests or pilot
applications to enable low-uncertainty extrapolation to field dimensions.
Various soil microcosm configurations/types have been developed to study soils
contaminated with organic and inorganic pollutants.

– Open static (batch) reactors can be used to study the progress of biological and
chemical processes, biodegradation, in situ natural attenuation, metal mobilization
and immobilization (stabilization).
– Closed-reactor systems have been used for modeling anoxic conditions or for
comparative studies of aerobic and anaerobic conditions and their effects on
biodegradation or toxicity.
– Flow-through soil microcosms can be arranged horizontally or vertically and are
used to study site-specific transport processes and fate, and the effects of risk
reduction measures/interventions such as stabilization, filtering or the application
of permeable reactive barriers.

Disadvantages of the microcosms are their small size and large surface–volume
ratio as well as the lack of interaction with the natural environment. Their gradual
application from the simplest set-up to the most complicated one supports the decision
e.g. on the application of serial technological parameters (for example, aeration for one
hour, 2, 6, 12 and 24 hours a day, or the application of 1%, 2%, 5% and 10% from
an additive, etc.) and provides benefits or compensates for deficiencies. The possibility
of repetitions and the ’unlimited’ number of combinations or variations made it an
essential tool for technology pre-experiments.
The following is an introduction to soil microcosms constructed and applied to
monitoring natural or anthropogenic processes in the environment and to the mea-
suring of the effect of contaminants and the results of remedial activities. Prior to the
selection, planning and field application of an environmental technology, the techno-
logical parameters (pH, temperature, aeration, additives, etc.) must be examined and
their applicable range determined. Technological microcosms proved to be the ideal
tools for selecting and planning soil remedial technologies, as well as for the moni-
toring of the remediation process. Biodegradability of a mixture of soil contaminants,
the effect and the proper amount of additives, the changes of mobility and the adverse
effects of the contaminants, and the adaptive behavior of soil microbiota were suc-
cessfully tested in microcosms by the authors of this chapter. These applications will
be introduced below and their use demonstrated in Volumes 4 and 5 in the context
of field applications (Molnár et al., 2009a; Hajdu et al., 2009; Molnár et al., 2009b;
Vaszita et al., 2009a,b,c; Feigl et al., 2009a).
412 Engineering Tools for Environmental Risk Management – 2

Figure 8.9 Test series for screening biodegradation in soil and the effect of enhancing additives.

3.1 Testing the effects of environmental and anthropogenic

interventions in a small volume
50–500 g soil in closed, open or flow-through laboratory systems can be used for
testing
– the effect of natural environmental and technological parameters (pH, tempera-
ture, moisture content, etc.) on contaminants’ risk and microbial response;
– the adverse effects of pollutants and their different chemical forms;
– any adverse or beneficial effects on the natural soil biota or on controlled organisms
placed into the soil.
The number, activity and diversity of the organisms or other end points characteristic
of the soil ecosystem or test organisms are measured once (at the end) or at regular
time intervals (Gruiz, 2005; Leitgib et al., 2007). A large number of microcosms can
be studied in parallel, and a series of variable technological parameters can be applied
to ensure great statistical power and low uncertainty in deciding whether a parameter
has an effect or not.
The soil sample can be placed into simple jars, pots, plastic containers (Figure 8.9)
or more sophisticated tanks with built-in sensors, injectors or sampling devices.
The experimental pattern is based on the comparison of untreated and treated
soils: the difference is measured and evaluated using statistical tools.

3.2 Testing biodegradation and bioavailability

Closed, open and flow-through reactors filled with soil (50–2000 g) are used to measure
soil-specific biodegradation and bioavailability of pollutants. Contaminated soil is used
 Microcosm models and technological experiments 413

or the substance to be tested is mixed with or infiltrated into the soil (Molnár, 2006;
Molnár et al., 2009a; Molnár et al., 2009b). The simplest set-up is a pot or a jar filled
with soil. The end points may be

– the decreasing amount/concentration or mobility of the substance,

– the increasing amount/concentration of the biodegradation products or
– any indicator of the metabolic activity of the soil microbiota.

Short- or long-term biodegradation, intensified and accelerated processes can be

tested in static and dynamic modes. Dynamic mode in this case requires that the
critical parameter is applied in an impulse mode and the response of the soil is
monitored.

3.3 Testing long-term pollution processes in the environment

When studying soil pollution by simulating a polluting process, e.g. floods, precipita-
tion infiltration or soil pollution caused by deposited contaminated sediment (Gruiz,
2005), it is important to understand those natural processes which influence the scale
of risk and can serve as a basis for risk reduction. Transport and dissipation, spon-
taneous stabilization, physical, chemical or biological degradation may lead to rapid
removal of or reduction in the active form of the contaminant. If the risk of a con-
taminant is reduced by transport in one place, but increased in another, the risk
management measure should separate the first place from the other by controlling
the transport. A typical environmental process is leaching, which can be very risky
when it goes on in an uncontrolled way, but may serve as a biotechnology when the
leachate is collected and treated. Complex leaching of mine waste stockpiled in the
environment (Vaszita et al., 2009a) was modeled in a long-term (3–4 years) experi-
ment, observing and monitoring the processes in 2000–4000 g batch or flow-through
microcosm vessels equipped with solid and leachate sampling possibilities. The results
were integrated into the risk management plan of an abandoned mining site (see also
Volume 5).

3.4 Testing microbial activity and plant growth in

contaminated soil
Activating soil microorganisms and growing plants on contaminated soil may reduce
risk due to dusting, runoff waters, leachates and erosion. Vegetation normalizes water
balance, reduces or stops deflation and erosion, and increases organic matter content
and microbial activity of the soil.
In the example shown in Figure 8.10 the soils in the pots were contaminated with
incremental red mud doses/amounts. Plant growth (tolerance) was tested as a function
of red mud content. The aim was to find those candidate plants which can grow on
soils contaminated with various amounts of red mud and to find the upper red mud
level candidate plants can tolerate. These simple tests give an answer within 1–2 weeks
to questions which neither scientists nor practitioners can answer since nobody has
experienced the special problem of an alkali red mud flood on agricultural soil that
happened in the Ajka, Hungary, red mud disaster in 2010.
414 Engineering Tools for Environmental Risk Management – 2

Figure 8.10 Serial tests in static microcosms: soil contaminated with increasing concentrations of
red mud.

3.5 Technological pre-experiments

Technology screening as part of remediation planning and scale-up prior to technol-
ogy selection (Molnár et al., 2009a; Feigl et al., 2009a) provides useful information
on the effect of engineering manipulations such as aeration, nutrient supply by injec-
tion or other ways, introduction of additives, chemically or biologically active agents,
etc. A good example is the development of a CDT technology for biologically non-
available soil pollutants (Molnár et al., 2009a). Various technological versions were
tested in 0.5–40 kg microcosms to determine the scale of aeration, nutrient addition and
cyclodextrin application. Cyclodextrin is a bioavailability-enhancing agent: its mode
of introduction and the necessary amount were also exactly specified in the small-scale
microcosms. Another example for the technological application of soil microcosms
is the development of in situ chemical stabilization of a soil contaminated with toxic
metals. The optimum stabilizing agent and its necessary concentration range was iden-
tified in short- and long-term laboratory experiments in open-batch soil microcosms
(Feigl et al., 2009a). The findings of the microcosm experiment were validated in field
experiments (see also Volume 5).
Some microcosm types are introduced below from the practical point of view
and with regard to the aim of the microcosm application. The microcosm set-up as
detailed in the next chapters accommodates the testing of three technologies based on
the predominant natural processes:

– soil remediation based on natural attenuation and biodegradation;

– natural stabilization of metals in the soil and the application of combined chemical-
and phytostabilization;
 Microcosm models and technological experiments 415

Figure 8.11 Solubilization of groundwater contaminants using tensides and cosolvents in increasing
concentration from left to right.

– natural mobilization of metals in soil or waste rock and its utilization for
bioleaching-based technologies;
– simulation of a red-mud spill on agricultural soils;
– simulation of secondary sodification due to inadequate irrigation and alkali
intervention.

In addition to natural soil processes and technologies based on natural and

biological activities, microcosms also enable the measurement of the result of physico-
chemical treatments. Soil washing, injection of additives, chemical reactions such as
ISCO (in-situ chemical oxidation) can be tested at small size to support the decision on
treatability of contaminated soil and groundwater. Figures 8.11 and 8.12 show the very
first experiment on treatability of contaminated groundwater using tensides and cosol-
vents to increase solubility of water-insoluble contaminants (Figure 8.11) and the appli-
cation of oxidizing agents such as peroxide, persulfate or permanganate (Figure 8.12).
The tests on groundwater contaminated with trichloroethylene (TCE), shown in
Figure 8.11, investigated the rate of solubilization, biodegradation and chemical oxi-
dation at increasing cosolvent concentration. The test bottles contain a TCE lens which
functions as an inexhaustible contaminant source and simulates the underground
situation. The more dissolved the contaminants, the denser the solute is. Chemical
analyses provide more details on quantities.
Laboratory batch experiments modeling ISCO with potassium permanganate are
shown in Figure 8.12. The tests were set up in 250 mL glass vessels containing
groundwater contaminated with 1000 mg/L TCE. The oxidizing agent was potas-
sium permanganate (from left to right: control without KMnO4 , 1.2 g/L KMnO4 and
6.0 g/L KMnO4 ). Large amounts of precipitated MnO2 can be seen in the reactor
vessels. The permanganate-based ISCO was effective at laboratory scale, and the high
416 Engineering Tools for Environmental Risk Management – 2

Figure 8.12 Chemical oxidation of dissolved contaminants in groundwater using potassium perman-
ganate.

TCE (1000 mg/L) concentration of the groundwater dropped significantly. The applied
1.2 g/L KMnO4 removed 92% of TCE within 24 hours. A disadvantage of the tech-
nology is the insoluble precipitate. A considerable amount of MnO2 precipitated in
the reactors and the same can be expected in the real environment.

4 BIODEGRADATION AND BIODEGRADATION-BASED

REMEDIATION STUDIES IN SOIL MICROCOSMS

The planning of soil bioremediation needs reliable methods for assessing and monitor-
ing soil microbiota and its activity. Testing site-specific biodegradation of the pollutant
in soil microcosms is a prerequisite for a successful technology selection and planning.
Microcosm studies for testing natural biodegradation and performance assessment of
bioremediation are frequently used to investigate natural processes in contaminated
soil and to examine the adverse effects of organic contaminants and the beneficial
changes due to remedial activities (Alvarez & Illman, 2006; Fernández et al., 2005;
Gruiz et al., 2001; Leitgib et al., 2007; Molnár et al., 2005; Molnár et al., 2009a;
Salminen et al., 2002).

4.1 Testing natural and enhanced biodegradation

Static and dynamic microcosm systems were set up with three-phase soils artificially
contaminated with petroleum hydrocarbons to study the bioavailability and natural
biodegradation of the contaminants in a soil from a polluted industrial site (Molnár
et al., 2009a). Experiments on contaminated soil spiked with diesel oil and transformer
 Microcosm models and technological experiments 417

Figure 8.13 Soil biodegradation microcosms in jars to test enhanced biodegradation.

oil at 30,000 mg/kg concentration and reference soil were run for 10 weeks. Biodegra-
dation and the activity of the indigenous soil microorganisms were characterized by
chemical and biological methods at the very beginning (after spiking the soil), after the
adaptation period (4 weeks), and after 6, 8 and 10 weeks.
Uncontaminated reference soil was used as a control to compare the effects of
contaminants. The soils were amended with inorganic nutrients ((NH4 )2 SO4 , KNO3 ,
KH2 PO4 ) to reach a final C:N:P ratio of 100:10:1, and were incubated at 25 ± 2◦ C.

Static soil-microcosms for screening/checking natural biodegradation

Static open batch reactors containing 0.1–2.0 kg of contaminated and reference soils
were incubated under atmospheric (aerobic) conditions between 10–30 ± 2◦ C. Optimal
humidity (10–15% by mass) was maintained throughout the experiments. Duration
varied typically from one week to one year. The number of tests can be large due to
the small space requirement and low cost.
These simple test vessels filled with soil proved suitable for testing the biodegra-
dation of aged petroleum hydrocarbons, transformer oil, mazout, pesticides, solvents
or any organic soil contaminant of industrial origin. The influence of soil type, soil
microbiology as well as of the physical phases present (two- and three-phase soils and
soil slurries) can be tested. The aim of the tests is to decide whether biodegradation
takes place, and whether the biodegradation process can be enhanced with additives
or by changing environmental parameters.
To follow the processes, the initial and final states are evaluated and compared to
reference or control. The evaluation needs an integrated approach applying physico-
chemical, biological and toxicological methods and the comparison of their results.
The influence of cyclodextrin on enhancing bioavailability of contaminants was
tested at different concentrations in three tests each: control, 0.1% cyclodextrin and
0.5% cyclodextrin from left to right in Figure 8.13.

Static soil-microcosms for measuring biodegradation rate and finding

the optimal scale of technological parameters
Technological studies can be performed in the laboratory in well-controlled static
microcosms (batch reactors: solid phase and mixed slurry) or flow-through soil micro-
cosms, where the mobile phases of air and/or water can ‘flow through’. The size of
these technological experiments may vary between 0.5–100 kg soil or beyond. Dura-
tion of the experiments may vary between two weeks or two years. The number of tests
is defined by the statistical method used and its power. Generally, a combination of
418 Engineering Tools for Environmental Risk Management – 2

two or three previously selected parameters is tested and evaluated in comparison with
each other and the reference.
Microcosms are easy tools not only to evaluate the “yes’’ or “no’’ responses of
the soil microbiota but also to find a detailed explanation about the mechanism and
rate of biodegradation. One can measure the effects of serially varying parameters
of environmental technologies such as aeration, nutrient addition and the application
of physical, chemical or biological additives (e.g. electron acceptors and agents that
adjust the soil redox potential and influence contaminant mobility and bioavailability).
Regular sampling, integrated analysis and testing and integrated data evaluation help
monitor and assess the processes. The number of samples to be taken influences the
microcosms’ size to a large extent.
In the cyclodextrin-enhanced soil bioremediation (CDT) case study, four- to ten-
week-long microcosm experiments were performed and evaluated to study the progress
of biodegradation and to find the optimum parameters for aeration, nutrient and
RAMEB (randomly methylated beta cyclodextrin) applications. Aeration of three dif-
ferent intensities, nutrient addition and cyclodextrin (type and amount) application
were evaluated. The microcosms simulating real situations showed that a 2 × 1 hours-
per-day aeration results in equally high biodegradation as one for 12 or 24 hours, and
that bioavailability and biodegradation cannot be further increased by the addition of
more than 0.5% CD. These findings have been validated later in the field.

Dynamic soil microcosms for monitoring biodegradation and the effects

of interventions
Laboratory flow-through column reactors were designed for the problem-specific
dynamic testing of the effects of technological parameters such as aeration and nutrient
supply on soil remediation. Columns of a volume of 1–3 liters filled with 0.5–2 kg of
contaminated soil were used for the experiments at 15–25 ± 2◦ C (Figure 8.14). The
reactors filled with the contaminated soils were aerated for 2 hours daily at a rate of
10 L/h air. Two flow-through traps filled with NaOH ensured a CO2 -free atmospheric
air inflow. The CO2 -free air was sucked through the soil columns by vacuum produced
by a water jet pump or ventilator. The humidity was maintained at between 10–15%
by mass throughout the experiment. The microbial activities in the soil were character-
ized by measuring CO2 production and O2 consumption. The same microcosm set-up
is suitable for the qualitative and quantitative characterization of biodegradability,
bioavailability or toxic effects of organic contaminants. The effects of technological
parameters, e.g. temperature and aeration rate, and those of additives such as nutrients
and tensides or oxidizing, reducing, etc. agents were also measured.

4.2 Integrated monitoring and evaluation of the biodegradation

experiments
An integrated methodology including physico-chemical analyses and biological tests is
the most useful tool to evaluate soil microcosms because soil has a high potential
to influence contaminant mobility and bioavailability. There are several physico-
chemical and biological end points that can detect the biodegradation processes in
air, water (moisture) and soil samples taken from the microcosm or by applying in situ
sensors.
 Microcosm models and technological experiments 419

Figure 8.14 Dynamic soil microcosms with flow-through column reactors.

Chemical analyses can determine the quality and quantity of the residual con-
taminants or the biodegradation product (metabolite) measured by in situ sensors, by
using labeled substrates or complete or selective extraction. Water-based complex-
ing agents or organic solvents can be used as extractants. The analytical method
identifying the contaminant or contaminant mixture should be substance-specific.
After extraction a separation or fractionation technique is applied such as ion chro-
matography, gas chromatography (GC), high performance liquid chromatography
(HPLC) or capillary electrophoresis (CE, CZE), and gravimetry, flame-ionization
detection (FID), mass spectrometry (MS), etc. for the detection of the contaminants
depending on the material properties of the contaminating and biodegrading chemical
substances.
Chemical methods alone cannot give a complete picture of the biodegradation pro-
cess, in particular, for complex contaminant mixtures, aged and low-bioavailability
contaminants. Chemical analysis should be supported by biological (the presence
of biodegrading microorganisms or enzymes plays a role in the degradation pro-
cess) and toxicological (decreasing toxicity or other adverse effects) methods. If
chemical and biological/toxicological results are contradictory, the cause of this con-
tradiction must be found. For example: the toxic substance disappears from the
extract (it cannot be detected by chemical methods), but toxicity does not. The
answer will be given by microbiological and toxicological test results. Is a more toxic
metabolite – produced by the microbes – responsible for the still existing toxicity, or
have the chemical properties of the original contaminants changed and is this why the
applied analytical technique is not working any more, or something else? One can
measure the concentration and the metabolic activities of the contaminant-degrading
cells in the soil through enzyme activities such as the dehydrogenase enzyme activ-
ity and the changes in substrate specificity or diversity. The respiration rate can be
420 Engineering Tools for Environmental Risk Management – 2

Figure 8.15 Integrated methodology for the monitoring of biodegradation experiments.

continuously measured in the microcosm test vessel or on separate samples taken from
the microcosm.
Dynamic testing of soil makes it possible to measure the intensity and emer-
gence rate of the response to sudden changes in environmental conditions and to
technological interventions.
Laboratory microcosms can be equipped with automatic precision pumps and
dispensers which ensure that parallel tests are completely identical. The precise timing
and scaling of the technological measures can also be ensured; thus the quality of the
microcosm study results is similar to standardized biotests or chemical analyses. There
is no guarantee that the communities will remain identical in the microcosms during
the study.
The integrated monitoring of the micro- and mesocosms is illustrated by an exam-
ple used for the remediation of a soil contaminated with transformer oil in an old
inherited industrial site (Figure 8.15).
 Microcosm models and technological experiments 421

Figure 8.16 Scale-up of laboratory soil microcosms.

4.3 Scaled-up technological micro- and mesocosms

Soil microcosms of different scales and configurations can be applied to testing natu-
ral and enhanced biodegradation with the aim to establish and develop innovative soil
bioremediation technologies. Large-size (scaled-up) microcosms can be used to model
the intensification of remediation technologies based on natural biodegradation by soil
venting (bioventing), by the addition of nutrients, electron acceptors, bioavailability-
enhancing agents such as cyclodextrins, cosolvents or tensides and microbes with
special abilities (bioaugmentation).
Figure 8.16 shows that a large number of small-scale microcosms are performed
and evaluated to find out which technological intervention has an effect at all. In
the next step, the optimal value of technological parameters and their combinations
should be determined by serially testing the effect of aeration, duration, nutrient or
additive supplement such as the RAMEB in the innovative cyclodextrin technology
(CDT), which was successfully used to enhance the biodegradation of a soil polluted
with transformer oil (Fenyvesi et al., 2003; Gruiz et al., 1996; Leitgib et al., 2003;
Molnár et al., 2003; Molnár et al., 2005; Molnár et al., 2009b).
The microcosm experiments with an incrementally increasing scale from
laboratory- to field-scale (Figure 8.17) included four CDT development steps: small-
scale laboratory screening tests, small- and large-scale laboratory microcosms for
testing technological parameters and field tests (demonstration) on an inherited site
contaminated with transformer oil at a transformer station. According to the expe-
rience of the technology developers, biodegradation and bioremediation tests in soil
422 Engineering Tools for Environmental Risk Management – 2

Figure 8.17 Stabilization microcosm set-up.

microcosms under controlled conditions provided valuable information on the micro-

biological processes in contaminated soil, and they supported decision making about
the risk reduction options.

4.4 Summary of biodegradation testing for technological

purposes
Biodegradation and biodegradation-based soil remediation tests in micro- and meso-
cosm type experimental systems proved to be suitable for studying natural and
enhanced biodegradation, and for finding the optimal technological parameters that
support the development of scientifically established innovative technologies. In
the case of already demonstrated technologies, their site-specific application and
technological efficacy can be verified before new field applications.

5 TESTING TECHNOLOGIES BASED ON CONTAMINANT

STABILIZATION

Microcosm models can be applied to technological pre-experiments, as part of the

remediation scale-up procedure prior to technology selection and planning. In the
 Microcosm models and technological experiments 423

case of in situ chemical stabilization or combined chemical and phytostabilization

(see Volume 4), microcosm experiments provide useful information on the effect of
the introduction of additives and plants to the soil (Gruiz & Vaszita, 2009).
Microcosm experiments are often used to assess the suitability and efficacy of
chemical stabilizers applied for immobilizing soil contaminants. Other fields of appli-
cation are effect and efficacy studies on stabilization-improving additives and process
reversibility (Kiikkila et al., 2002; Geebelen et al., 2006; Feigl et al., 2007). Micro-
cosm assessments may be used to investigate the joint stabilizing effect of stabilizers
and plants, which is a common solution in practice (Chlopecka & Adriano, 1997;
Lombi et al., 2002; Brown et al., 2005).
Stabilization microcosm experiments comprise four steps:

• planning the experimental set-up and parameter combinations,

• assembling the microcosms and performing the experiment,
• monitoring and sampling individual microcosms,
• comparative evaluation of the results.

5.1 Experiment design

Stabilization microcosms were applied as the first tier of technological experiments
to choose the best chemical stabilizer for soils contaminated with toxic metals and
mine wastes in the Gyöngyösoroszi, Hungary, mining area and to identify its optimal
concentration range (Volume 5). Table 8.1 shows the combinations of additives, soil
and mine waste to be stabilized (Feigl et al., 2009a, b; 2010a, b).
The microcosms that model toxic metal stabilization in soil and mine waste are
arranged in as many parallel specimens as required or in a serial arrangement. ‘Treated’
microcosms are compared to a ‘non-treated’ control. The stabilization process is mon-
itored via time-serial samples using an integrated methodology focused on the two
main risk components, i.e. mobility and bioavailability of the toxic metals.
Another soil stabilization microcosm application assesses the stabilizing potential
of lignite (Uzinger & Anton, 2008; Uzinger, 2010). A mathematical model provid-
ing information about a multidimensional response function has been applied to test
samples that were influenced by a number of environmental and technological factors
(Biczók et al., 1994). The effects of four additives (lignite, Pb, Cr and Zn; the latter
ones in the form of Pb(NO3 )2 , Cr(NO3 )3 ·9H2 O and ZnSO4 ·7H2 O) were tested in
three specimens. Five levels of each of the four factors considered were arranged in an
orthogonal pattern based on the normalized matrix plan (Table 8.2). The plan enables
the investigation of linear and quadratic effects and interactions of pairs of variables.
Temperature and soil moisture were constant during the incubation period (4 weeks).
The mathematical formula for the model is the following:

y = B0 + B1 ∗ L + B2 ∗ Pb + B3 ∗ Zn + B4 ∗ Cr + B5 ∗ L ∗ Pb + B6 ∗ L ∗ Zn + B7 ∗ L
∗ Cr + B8 ∗ Zn ∗ Pb + B9 ∗ Cr ∗ Pb + B10 ∗ Cr ∗ Zn + B11 ∗ L2 + B12 ∗ Pb2 + B13 ∗ Cr2 ,

where:
– y = dependent variable;
– B0 to B13 = parameters of the model;
424 Engineering Tools for Environmental Risk Management – 2

Table 8.1 Experimental matrix for chemical stabilization: combination of metal-contaminated soils,
additives and mine waste.

Waste rock
Contaminated from the Acidic mine
Pot agricultural soil mine waste
size
Stabilizing additive Start date (kg) Amount of additive (% by mass)

Fly ash A (Oroszlány) 15.09.2003 2 1 2 5

Fly ash B (Oroszlány) 15.09.2003 2 1 2 5
Hydrated lime 10.05.2004 2 1
Raw phosphate 10.05.2004 2 1
Alginite 10.05.2004 2 1.5
Lignite 10.05.2004 2 10
Lime + phosphate + 10.05.2004 2 1 + 1 + 1.5 + 10
alginite + lignite
Fly ash from Tata 17.02.2006 1.5 2 5 2 5
Red mud 17.02.2006 1.5 2 5 2 5
Water treatment residue 1 17.02.2006 1.5 2 5 2 5
Water treatment residue 2 17.02.2006 1.5 2 5 2 5
Fly ash from Tata (T) 23.02.2007 1.5 5 5
Fly ash from Visonta (V) 23.02.2007 1.5 5 5
Fly ash from Tata + lime 23.02.2007 1.5 2.5 + 2 5 + 2 2.5 + 2 5 + 2
Fly ash T +V + lime 23.02.2007 1.5 2.5 + 2.5 + 2.0 2.5 + 2.5 + 2.0
Lime 03.06.2008 1.5 2 2
Lime + steel shots 03.06.2008 1.5 2+1 2+1
Fly ash T 03.06.2008 1.5 5 5
Fly ash T + steel shots 03.06.2008 1.5 5+1 5+1
Fly ash T +V + lime 03.06.2008 1.5 2.5 + 2.5 + 1 2.5 + 2.5 + 1
Fly ash T +V + lime + 03.06.2008 1.5 2.5 + 2.5 + 2 + 1 2.5 + 2.5 + 2 + 1
steel shots

– L = lignite concentration in %;
– Pb, Zn, Cr = toxic metal concentration in mg/kg.

This kind of experimental arrangement ensures high statistical power when look-
ing for the relationship between simultaneous effects such as pH, three metals and
lignite additives.

5.2 Microcosm set-up and implementation

The stabilization microcosms are simple, open batch reactors with a mass of 0–20 kg,
placed into a thermostat to ensure controlled temperature and humidity. The authors
applied such vessels for contaminated soil and mine waste stabilization. The vessels
were filled with 1–2 kg soil or mine waste, mixed with different stabilizers at 1–10% by
mass and were incubated at 25◦ C for 2–3 years. They were mixed and watered to 60%
of their water holding capacity every month. The soil was sampled and analyzed at
certain intervals to check the complex physico-chemical and microbiological processes
in the microcosms (Figure 8.17) (Feigl et al., 2009a).
 Microcosm models and technological experiments 425

Table 8.2 Experimental matrix for the assessment of

the stabilizing potential of lignite.

Sample Lignite Pb Zn Cr
code (%) (mg/kg) (mg/kg) (mg/kg)

Control 0 0 0 0
1 7.5 1125 1125 1125
2 2.5 1125 1125 1125
3 7.5 375 1125 1125
4 2.5 375 1125 1125
5 7.5 1125 375 1125
6 2.5 1125 375 1125
7 7.5 375 375 1125
8 2.5 375 375 1125
9 7.5 1125 1125 375
10 2.5 1125 1125 375
11 7.5 375 1125 375
12 2.5 375 1125 375
13 7.5 1125 375 375
14 2.5 1125 375 375
15 7.5 375 375 375
16 2.5 375 375 375
17 10 750 750 750
18 0 750 750 750
19 5 1500 750 750
20 5 0 750 750
21 5 750 1500 750
22 5 750 0 750
23 5 750 750 1500
24 5 750 750 0
25 5 750 750 750
26 5 750 750 750
27 5 750 750 750
28 5 750 750 750
29 5 750 750 750
30 5 750 750 750
31 5 750 750 750
32 5 750 750 750
33 5 750 750 750
34 5 750 750 750
35 5 750 750 750
36 5 750 750 750

5.3 Monitoring of the microcosms

An integrated methodology was developed to monitor stabilization experiments which
combined physico-chemical analysis with biological and ecotoxicity testing. Its purpose
was to provide a realistic view of the risk posed by metal contaminants in the soil (Gruiz
et al., 2009). The outcome is that the risk of contaminating metals can be reduced by
changing their mobility and accessibility, but not by removing them from the soil.
Chemical stabilization can be based on
426 Engineering Tools for Environmental Risk Management – 2

Figure 8.18 Integrated methodology for the monitoring of chemical stabilization in soil microcosms.

– suppressing ionic metal forms,

– reducing dissolution by supporting precipitation,
– reducing desorption in favor of sorption,
– changing redox state in order to obtain a less water-accessible and bioavailable
form,
– enhancing mineral formation by reducing weathering.

The risk posed by metal species of ionic–non-ionic, dissolved–precipitated, sorbed–

desorbed, oxidized–reduced and incorporated minerals or free metal forms cannot be
characterized using chemical analyses alone. The difference between adverse effects of
the soil before and after stabilization is directly related to the metals’ risk determined
by mobility, biological availability and actual adverse effects, and thus biological and
ecotoxicological methods would be preferred for technology monitoring.
The chemical analyses as part of the integrated methodology for the monitoring of
chemical stabilization (shown in Figure 8.18) developed by Feigl et al. (2007) included
the application of soil extractants with increasing acidity: i) distilled water, ii) aqueous
solution of ammonium-acetate (pH = 4.5), iii) ammonium-acetate + EDTA (ethylene-
diaminetetraacetic acid) and iv) aqua regia. The mobility of As, Cr-VI, Mo and Se was
 Microcosm models and technological experiments 427

analyzed using an alkaline extract since these metal and metalloid species pose a threat
in an alkaline environment. The metal content of the acidic and alkaline extracts was
determined by ICP-AES (Inductively Coupled Plasma–Atomic Emission Spectrometry).
The metal content of these extracts characterizes the extractability i.e. the ‘chemical
availability’ of the metals. Chemical availability was not only measured in extracts
but also in leachates using a dynamic leaching method in microlysimeter columns
(developed for this purpose) at the end of the long-term stabilization experiments. The
mini-lysimeters are also microcosms: they are filled with stabilized soil, average pre-
cipitation is applied and a realistic seepage or drainage is simulated in a soil column
of optional height.
Besides these experiments other soil characteristics such as mechanical compo-
sition, texture, pH, EC, CaCO3 content, salt content, organic matter content and
nutrients (ammonium-acetate extractable P and K, total N) were also determined.
According to the aim of the experiment, in addition to the above parameters, one can
analyze for example the natural redox indicators such as oxygen, NO− − 2−
3 , NO2 , SO4 ,
SO2−3 , FeII, FeIII, MnII, MnIII, CH4 , or artificial redox indicators added to the soil
such as thionine, toluidine-blue or cresyl violet in dissolved or immobilized forms. The
results help better understand and interpret the redox-potential-dependent biological
and chemical processes.
The actual risk of various metal species can be best characterized by direct effect
assessment (Gruiz, 2005). Biological indicators and their changes can be monitored
and used for the evaluation of the efficiency of remediation. Soil activity was char-
acterized in the stabilization study by the concentration of the aerobic heterotrophic
cells in the soil. The diversity and activities of the microbiota provides a huge variety
of bioindicators. Soil toxicity measurements in the stabilization experiments included
direct contact tests with test organisms from three trophic levels: bacteria (Vibrio fish-
eri luminescence inhibition test), plant (Sinapis alba root and shoot growth inhibition
test) and animal (Tetrahymena pyriformis single cell animal reproduction inhibition
test). A rapid (five-day) bioaccumulation test on Sinapis alba developed by the authors
was used to obtain direct information on the suitability of the chemical stabilizer from
the point of view of the phytostabilization process following chemical stabilization.

5.4 Evaluation, interpretation and use of the stabilization

microcosm results
The results of the chemical analyses and toxicity measurements have to be evaluated
together in order to obtain a detailed picture on the effect of stabilizing agents. The
application of the integrated monitoring and evaluation methodology enables choos-
ing the best stabilizing agent that should be applied in the large-scale technological
experiments (Figure 8.19).

5.5 Summary and conclusions of stabilization microcosm

application
The authors used the simple batch or lysimeter type microcosm to develop a technology
based on chemical stabilization for the remediation of mine waste containing toxic
metals. In addition to the effects of additives, long-term processes and the effects
428 Engineering Tools for Environmental Risk Management – 2

1. Microcosm 2. Lysimeter 3. Field experiment

Plant collection Plant collection

Soil sampling
and analysis and analysis
and analysis
90 m3
Soil and additives
0.35 m3
Soil and
additives
Water collection
and analysis

Thermostat 25°C
Soil and
additives
Water collection
and analysis
Soil sampling
2 kg and analysis

Figure 8.19 Scaled-up technological experiments for combined chemical and phytostabilization.

of vegetation can successfully be modeled. The results of microcosm and mesocosm

experiments were validated by field demonstration of the developed technology.

6 TESTING AND UTILIZING THE COMPLEX LEACHING PROCESS

Microcosm models and experiments can simulate site-specific conditions and provide
information about parameters needed to improve risk assessment and enhance risk
reduction (Gruiz et al., 2001). For most applications the soil–groundwater pathway is
considered, therefore the leaching behavior of contaminated soil and solid waste is of
great importance (Kalbe et al., 2008).
Complex leaching in the environment is the result of physico-chemical and bio-
logical processes playing a role in the weathering of rock and the formation of soil. In
the case of contaminated rock and soil or uncontrolled waste deposits, the complex
leaching process and its environmental risks can be studied in leaching microcosms.
The technological aim is either to reduce the leachate amount and its contaminant
concentration, or to remove the contaminant from the soil, rock or solid waste by
leaching. Also, bioleaching as a basic process of biomining, which recovers valuable
metals from ores or mine waste (biomining of copper, gold or uranium), can be studied
in microcosms.
Laboratory leaching microcosms are lysimeters (Figure 8.20) which can be used
for source characterization and pollution transport determination which serves as a
 Microcosm models and technological experiments 429

Figure 8.20 Principle of the lysimeter: water filtrating through the soil is collected and analyzed.

basis for risk assessment. Leaching experiments in laboratory microcosms provide

information about the production and chemical composition of solutions derived for
example from sulfide rocks present in a watershed (Munk et al., 2006). The lysimeter’s
principle is that water filtrates through the three-phase soil sample, is collected at the
bottom and then analyzed (Figure 8.20). The water balance of a site can be estimated
with the help of a lysimeter. The amount of precipitation that the area receives and
the amount lost through the soil are recorded (leachate measured in the lysimeter).
The difference is the amount of water remaining in the soil and the proportion lost to
evapotranspiration (Figure 8.21).
Lysimeters are used to collect leachates from contaminated soil and to measure the
contaminant concentration in the leachate and thus the mobile or mobilizable contami-
nant fraction can be measured. The difference between the contaminant concentrations
in soil and in leachate characterizes the interaction between contaminant and soil (solid
phase) from which the retention of a contaminant or the sorption capacity of the soil
can be estimated. Both are basic characteristics responsible for environmental risk of
soil contaminants.
430 Engineering Tools for Environmental Risk Management – 2

Figure 8.21 In situ lysimeter set-up with natural precipitation and an underground collector system.

Mine wastes containing metal sulfides are oxidized due to the weathering process
which is accelerated by naturally occurring sulfur- and iron-oxidizing bacteria. This
phenomenon is recognized as bioleaching and it results in acid mine (AMD) or acid
rock drainage (ARD) (Bosecker, 2001). AMD/ARD is characterized by acidic, metal-
rich waters and red-orange iron-bearing solids formed by the weathering of pyrite
(FeS2 ) and subsequent oxidation of soluble ferrous iron (Fe[II]) to insoluble ferric iron
(Fe[III]). Iron-oxidizing bacteria present in AMD/ARD environments can accelerate
the rate of ferrous oxidation by almost 106 compared to inorganic mechanisms. Thus,
iron-oxidizing bacteria can be responsible for nearly uninterrupted generation of AMD.
Acid mine/rock drainage is the biggest environmental threat facing the mining industry.
The environmental impact of AMD and ARD can be severe in both abandoned and
active mining areas. The generation of low pH drainage enhances the dissolution of
heavy metals in water. The dissolved metals contaminate soil, ground- and surface
waters and can pose high risk to the ecosystem and human health (Gruiz et al., 2007;
Sipter et al., 2008).
Interdisciplinary studies have resulted in substantial progress in understanding the
processes that control the release of metals and acidic water from inactive mines and
mineralized areas, the transport of metals and acidic water to streams (Gruiz et al.,
2009), and the fate and effect of metals and acidity on downstream ecosystems (Sipter
et al., 2008).

6.1 Flow-through soil microcosm for studying bioleaching

Vaszita et al. (2009a) conducted microcosm experiments in four flow-through soil
microcosms which were operated for more than five years to model bioleaching of
toxic metals from the Gyöngyösoroszi, Hungary, mine waste material in contact with
rainwater. Two scenarios were modeled: 1. bioleaching within a large waste dump
 Microcosm models and technological experiments 431

Figure 8.22 Simple leaching microcosms.

in microcosms containing only mine waste (M1, M2); and 2. bioleaching in a waste
dump in contact with the surrounding soil in microcosms containing mine waste on
top of a soil layer (T1, T2).

6.2 Microcosm set-up

Microcosm set-ups and monitoring depend on the aim of the test that can simulate

– natural leaching processes and the amount and quality of ARD;

– leachate production and reduction;
– the effect of permeable reactive barriers with different reactive fillings;
– an ore-processing technology applied to mine waste;
– soil remediation based on complex bioleaching of contaminated soil.

The microcosm set-up was arranged to simulate the natural leaching processes
(dominated by bioleaching) of an acidic mine waste and the filtering ability and capacity
of the soil layer beneath. The set-up included four 6-liter HDPE containers (Figure 8.22)
filled with 4.5 kg homogenized, crushed metal-sulfide-containing mine waste placed
on top of a 5-cm gravel layer to allow for the artificial precipitation to filtrate through.
6-mm diameter holes were drilled in the bottom of the HDPE containers to collect the
resulting leachate in the underlying HDPE tray. In addition, the T1 and T2 microcosms
contained 1 kg of single-layer control soil between the mine waste and the gravel layer
(Figure 8.23). This uniform 8-cm soil layer was wrapped in a polyamide membrane to
prevent the soil particles from being washed out by the water flowing through.
The microcosms were manually irrigated at pre-arranged time intervals simulating
four low-intensity events and one heavy rain per month. The irrigation rate was based
on an annual rainfall rate of 756 mm/m2 /year (OMSZ 2002) with an allowance for
432 Engineering Tools for Environmental Risk Management – 2

Figure 8.23 Leaching microcosm containing mine waste on top of a thin soil layer and mine waste only.

evaporation. The low-intensity rain event was simulated by spraying 50–100 mL tap
water on the surface of the soil microcosm. The heavy-rain event comprised about
400 mL of leachant flowing through the microcosm over twelve hours resulting in an
output leachate.
A similar configuration can model and test the reactive filling of permeable reactive
barriers and active alkali soil zones for the treatment of different leachate types.

6.3 Monitoring the leaching microcosms

Monitoring of the microcosms was based on physical, chemical and microbiological
methodologies as shown in Figure 8.24.
As metal mobilization in the solid material is the focus of these experiments,
metal content of the waste and soil was analyzed using ICP atomic emission spec-
troscopy (ICP-AES). The total metal content was determined from an aqua regia extract
and the mobile fraction from an ammonium acetate + acetic acid + EDTA extract
(Lakanen & Erviö, 1971). Cell concentration of the sulfide-oxidizing bacteria was
measured according to Sand et al. 2007.

6.4 Evaluation and interpretation of the results

The evaluation of the results was focused on simulating various conditions of the
process:

– Initial phase within which the steady-state equilibrium of the process can be
reached and the annual water input is equal to the average annual rain.
– Dry condition phase within which water input is 1/3 of the average annual rain.
– Final phase within which the microcosms are depleted of metal sulfides, and the
water input is ∼2/3 of the average annual rain.
– Simulation of strong (5x average) precipitation and flood.
 Microcosm models and technological experiments 433

Figure 8.24 Integrated methodology for the monitoring of the leaching microcosm.

The simultaneously measured physical, chemical and biological results were

evaluated in an integrated way.
When modeling natural processes, the input parameters considered are the
quantity and quality of the contamination source, the amount and distribution of
precipitation, the quality and capacity of the soil layer. When the microcosm simulates
a technology based on bioleaching, the input parameters considered can be the water
flow, the geometry of the container and the additives. The results of the soil microcosm
experiment were evaluated based on the following end points: input/output leachate
amount, leachate pH, metal concentration of the leachate, amount of leached metal,
leaching efficiency versus total and mobile metal content.

6.5 Summary and conclusions about leaching microcosm

application
The laboratory soil microcosm experiment modeled, at laboratory level, complex
leaching of mine waste that contained base-metal sulfides and a site-specific process
non-measurable under field conditions. It also simulated the contact between the
434 Engineering Tools for Environmental Risk Management – 2

produced leachate and the soil, a typical pollutant transport pathway in an environ-
ment affected by acid-rock/mine drainage. The process was evaluated in a controlled
environment allowing for the use of replicates and the modeling of three conditions
(average rain, dry conditions and metal depletion of the microcosms). The microcosm
experiment simulated the site-specific conditions to provide information about the
parameters needed for risk assessment and risk reduction. The microcosm parame-
ters helped to estimate long-term metal emissions from the sources at watershed level
(Gruiz et al., 2006; Vaszita et al., 2009b), to determine the borders of the polluted
areas and to calculate the environmental risk in the area with regard to the nature and
fate of metal emissions.
The risk of mine waste can be characterized by the specific leaching efficiency,
i.e. the percentage (%) of the total metal amount leached annually from the mine
waste in the microcosms. It is identical to the emitted metal amount from uncontrolled
waste disposal. The leaching efficiency calculated from the microcosm study enables
a conservative prediction of the long-term metal supply in the studied area. ‘Con-
servative’ in this context means that a highly contaminated mine waste is leached in
the experiment. Leachates under average rain conditions, dry conditions and heavy
rain conditions were measured quantitatively and qualitatively to model weather vari-
ables. In addition to the initial high metal content, metal-depleted conditions were also
studied to estimate long-term leaching efficacy.
The results of the microcosm experiment showed significant differences depending
on the rain conditions and on the initial metal content of the waste. For example, under
average rain conditions, based on the specific leaching efficiency, the total cadmium
supply of the waste material would be fully exhausted in 8.5 years, zinc in 11.8 years
and lead in 3,287 years. Taking leaching efficiency under dry conditions, the cadmium
content of the waste would be fully exhausted in 24 years, zinc in 26 years and lead
only in 24,657 years.
The results of laboratory lysimeter tests enable dynamic characterization of the
environmental risk as a function of time, waste type and the weathering scale of
the waste rock. Advanced weathering, for example, slowed down significantly the
bioleaching process. The calculated time for full exhaustion of cadmium and zinc in
the microcosms containing highly weathered waste material was 3–4-fold of the less
weathered waste rock, but it was shorter for lead.

7 TRANSPORT PROCESSES STUDIED IN SOIL COLUMNS

Both 3- and 2-phase soils can be modeled in disturbed or undisturbed natural soil
columns or in artificially assembled soil profiles. This model enables us to measure the
infiltration rate and the effects of precipitation- and irrigation-transported ions, nutri-
ents, and other agrochemical additives, liquid- and solid-phase-related contaminants
and the physico-chemical and biological processes at various depths of the soil profile.
As a next level, technological interventions can also be studied to modify natural
or contamination processes and reduce the risk. This kind of soil column is an ideal
set-up to model technologies in combination with depths, infiltration and drainage.
Soil columns with a height from 20 cm to 3 meters are described in the literature. Soil
 Microcosm models and technological experiments 435

columns can be equipped with electrodes (measuring the changes in situ) and sampling
ports and devices.

7.1 Test set-up

An assemblage designed for measuring the effect of red mud on agricultural soils is
presented here to support decision making on remedial measures.
As a consequence of the Ajka, Hungary, aluminum factory disaster (October
2010), red mud flooded more than 800 hectares. In many areas the red mud layer
remained on the surface for more than 3 months after the flood. Soils have not
been sampled during the first risk management phase when the mitigation of the
human health risk and of the damage to the residential area was the highest prior-
ity. Therefore field samples were not available that could have helped to unravel the
infiltration process and adverse effects of the slurry containing NaOH. A column
microcosm was designed to identify and possibly quantify the rate of infiltration and
the depth-dependent changes of soil properties in a more abstract system compared to
uncontrollable field situations. One of the main goals was to detect the mobilization
and potential enrichment of toxic metals (especially As, Co, Cr, Ni, Pb), other poten-
tially toxic elements (B, Ba, Cd, Cu, Hg, Mo, Se, Sn, V and Zn) and Na in the soil
profile and the changes in the mobility of ions that play a role in plant nutrition. An
integrated methodology consisting of physico-chemical, microbiological and ecotoxi-
cological methods was applied for monitoring the transport and effects of red mud in
the soil profile.
The columns – shown in Figure 8.25 – were 100-cm-long PVC tubes with a diam-
eter of 15 cm, but similar 2.5 m long columns of 30 cm diameter were also used. The
bottom of the columns was closed with a polyethylene textile. The soil filled into the
column was neither completely mixed, nor undisturbed. Four characteristic layers were
identified and separately removed from their original place in the soil and, after hav-
ing been dried and homogenized, were filled into the column according to the original
in situ sequence of the layers:

– 0–30 cm: sandy loam with gravel,

– 30–50 cm: sand with higher gravel content,
– 50–80 cm: sand with gravel and a coherent gravel layer at 60 cm,
– 80–100 cm: sand with gravel.

The bulk density of each soil layer in the columns was adjusted to the same value
and the weight of the column was measured after every 5 cm soil layer. For this pur-
pose, the soil had to be dried before filling it into the columns. Three periods were
investigated: 30, 60 and 120 days. Each treatment had three repetitions, thus the test
arrangement comprised nine columns. At the end of the given time period, the columns
were sectioned into four pieces according to the soil layers to be analyzed.
Before the application of 10 cm red mud layer on the top, the soils were saturated
up to the maximum water holding capacity from the bottom of the column to simulate
the situation before the disaster. The water content of red mud (originating from the
area of the disaster) was set at 38% according to the original value. During the time
436 Engineering Tools for Environmental Risk Management – 2

Figure 8.25 Soil column experiment on red mud.

of the experiment, the columns were irrigated according to the precipitation observed
in the disaster area.

7.2 Monitoring the soil column microcosm

Integrated monitoring was applied to observe the processes in the soil column (Figure
8.26). The effluent water was collected at the bottom of the column and analyzed at
the end of each time period when the following parameters were measured from the
soil samples:

– Microbiology: plate count test, microbial biomass, FDA analysis (fluorescein

diacetate assay to estimate general microbial activity);
– Soil chemistry: pH, organic matter content and quality, CaCO3 content, salt con-
tent, acid buffering capacity, cation exchange capacity (CEC), base saturation,
partial extraction (aqua regia soluble element fraction; acetate buffer soluble frac-
tion; water soluble fraction for modeling soil solution and leaching; ammonium
acetate + EDTA soluble fraction for modeling plant availability);
– Soil physical properties: particle size distribution, upper limit of plasticity, water
retention capacity, stability of soil aggregates, dispersity factor;
 Microcosm models and technological experiments 437

Figure 8.26 Integrated monitoring of the red mud column experiments.

– Ecotoxicology tests: Vibrio fischeri luminescence inhibition assay, Sinapis alba

germination, root and shoot growth inhibition assay, Folsomia candida mortal-
ity test.
– The pH and element composition were measured also from the collected leachates.

7.3 Evaluation
Data were analyzed for treatment effects using one-way variance analysis (ANOVA).
Variance was related to the main parameters (time, different layers). Significant differ-
ences among the parameter means were calculated by Duncan’s post hoc test and by
the LSD (least significant difference) test at p < 0.05.

7.4 Summary
The three- or two-phase laboratory soil column enables the investigation of depth-
depending physico-chemical and biological processes. Undisturbed, completely mixed
or semi-disturbed systems can be established: the red-mud column employed the
438 Engineering Tools for Environmental Risk Management – 2

semi-disturbed version. Natural and contaminating processes such as floods, water

infiltration and desiccation, contaminant intake (deposition, infiltration, mixing into
surface soil layers) and in situ applied technologies can be detected and modeled in these
long-column microcosms, which are able to simulate depth-dependent characteristics,
close to reality.

8 MODELING SECONDARY SODIFICATION

Salinization is the accumulation of water-soluble salts in the soil. These salts include
potassium (K+ ), magnesium (Mg2+ ), calcium (Ca2+ ), chloride (Cl− ), sulfate (SO2− 4 ),
2− − +
carbonate (CO3 ), bicarbonate (HCO3 ) and sodium (Na ) ions. Sodium has a key
role in salinization, also called sodification.
Salts dissolve and move around with water. When water evaporates, salts are left
behind and an excess of sodium destructs the soil structure, which, due to the lack
of oxygen, becomes incapable of sustaining either plant growth or animal life (SoCo,
2009).
Primary salinization involves salt accumulation through natural processes due to
a high salt content of the parent material or in groundwater. Secondary salinization is
caused by human interventions such as inappropriate irrigation practices, for example,
using salt-rich irrigation water and/or insufficient drainage.
Sodification is a process of exchange between sodium present in the soil water
and divalent cations (mainly Ca2+ ) adsorbed on the exchange complex of the soil.
It occurs when sodium is present in higher concentration than the divalent cations.
The exchangeable sodium content of the soil increases and some minerals precipitate
in the order of their own solubility from the less soluble ones (e.g. calcite, CaCO3 ) to
the most soluble ones (e.g. NaCl).

8.1 Modeling sodification in microcosms

Its dependence on geochemistry, hydrogeology, precipitation, groundwater flow, soil
texture, soil water balance and several other environmental contextual parameters sets
limits to the extent the complex sodification process can be modeled.
Since sodification is based on the transport by precipitation/rain water/runoff or
groundwater flow and accumulation of metal ions (most frequently Na+ ), the soil’s
water balance is the key aspect to be considered when setting up the sodification
microcosm.
Secondary sodification is generally caused by relatively short-term anthropogenic
effects such as irrigation with salty or alkali water or by inadequate agrotechnolo-
gies. The authors of this chapter looked into the topic of secondary sodification in the
context of the red-mud flood problem in Hungary where Na intake of the soil was
extremely high.
Three different microcosm set-ups containing 5 kg soil in 50–100 cm high columns
were assembled to model worst-case scenarios (Figure 8.27). The microcosm tests
were also aimed at screening the sodification potential of various soils and developing
techniques to prevent this disadvantageous process by additives or water regulation.
Because the natural sodification process is fairly slow, saline-sodic soils develop during
 Microcosm models and technological experiments 439

Figure 8.27 Water balance scenarios in microcosms used for modeling sodification.

several years. The process tested in the soil microcosm was accelerated by increasing
the evaporation rate (applying air flow and heating). Thus a 1–2-year-long sodifi-
cation process was modeled within one–two months. Sodification microcosm study
results may fill a gap because the direct effect of NaOH on soil has not yet been stud-
ied, therefore our knowledge on the effect of Na+ ions originating from NaOH is
limited.

8.2 Sodification microcosm set-up

The microcosms were aimed at testing the sodification of red-mud flooded soils. The
scenario of tilling (mixing) residual red-mud into the soil was modeled. The reason
behind this technology option was that the removal of the thin (1–2 cm) residual red
mud deposits and of the upper contaminated soil layer after the red mud spill was not
feasible due to the large affected area, therefore it was tilled into the agricultural soil
(see also Chapter 5).
The red-mud sodification microcosms modeled three cases in which the distance
of the groundwater level to the soil layer was different:

– Case 1 models a deep groundwater level scenario where the soil’s water balance is
not affected by the groundwater. In this case the ion-loaded infiltrated water flows
vertically toward deeper layers. This causes sodification only if the surface layers
are rich in salts or the soil is irrigated with salt-containing water.
– Case 2 models a shallow 3-phase soil scenario where the water table is at medium
depth and only rises occasionally when capillary forces transport water and ions
from the deeper layers to the surface. This type of sodification occurs if the ground-
water itself or the deep soil layers contain an abundant stock of Na+ or other ions.
440 Engineering Tools for Environmental Risk Management – 2

– Case 3 models a high, near-surface groundwater level scenario where neither

precipitation nor capillary forces have a considerable effect on Na+ ion trans-
port. Instead, ion transport is chiefly determined by the groundwater flow, and
if ion-containing precipitation occurs, groundwater flow may dilute the ion con-
centration. However, if the groundwater is contaminated with ions, the capillary
forces transport the ions continuously to the surface where they are concentrated.

It should be noted that any of these three cases rarely occur solely in nature. Since
groundwater level and rainfall rates are always fluctuating, ion flows also change with
time, and eventually the sodification process is determined by the superposition of all
of these processes.

8.3 Technological microcosms for reducing risk of sodification

The same extreme microcosm set-ups can support technological experiments aimed
at preventing or reversing sodification in different soils, and at assessing the effect of
different soil additives to reduce sodification.

8.4 Evaluation and interpretation of results

The changes can be monitored by analyzing separately the vertical layers of the micro-
cosms. The most important indicators of sodification are Na+ and other exchangeable
ions. Soil geochemistry and water balance indicators such as soil texture, porosity
and rate of capillary rise can show the direct effect of ion accumulation on soil, while
biological and toxicological results (diversity of soil microflora, plant growth) can
complete the monitoring methodology.

8.5 Summary of sodification modeling

In spite of the simplification and problems of modeling, the design introduced above
was able to distinguish between the effects of different NaOH concentrations simulat-
ing a worst-case scenario. It was possible to draw attention to the risk of sodification
when NaOH containing red mud remains on the soil surface or is mixed into the soil
at more than 5% by mass.

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Chapter 9

Data evaluation and interpretation in

environmental toxicology
K. Gruiz, Cs. Hajdu & T. Meggyes
Department of Applied Biotechnology and Food Science,
Budapest University of Technology and Economics, Budapest, Hungary

ABSTRACT

Adverse effects of pure chemicals are usually characterized by the concentration–

response or dose–response curve. The effective concentration or effective dose can
be read from this curve, given that the concentration, mass or volume of the chemical
substance used in the study is exactly known. The test results indicate the hazard posed
by the chemical substance. When the environmental concentration is known, the risk
can be calculated from the scale of the hazardous effect.
The assessment of adverse effects of environmental samples is different because
the concentration of the contaminants in water or soil samples is not known, and
in most cases an unidentified mixture of contaminants is present. Studies, tests,
bioassays or microcosms can measure the toxicity quantitatively based on the actual
adverse effects of environmental samples. The actual adverse effect is in direct relation
to risk.
Direct toxicity assessment (DTA) ensures high environmental relevance repre-
senting all possible interactions between contaminants, ecosystem members and soil
phases aggregating the effect of all contaminants present in the sample. In addi-
tion to this, DTA can simulate different water and soil uses and real, multiple
exposures.
On the other hand, directly measured toxicity of environmental samples cannot
be expressed in concentration thus it does not fit the chemical risk assessment model
and concentration-based screening values applied to pure chemicals.
DTA provides the effective or non-effective soil doses or dilutions as test end point,
read from the dose–response curve. This is a shortcoming not only from the regula-
tory point of view but also for most environmental professionals who think and act
mechanically, according to the chemical model. The authors have introduced an option
to bridge direct toxicity assessing methods with the chemical model of environmental
risk assessment. The equivalency methodology applies the copper equivalency toxicity
to waters and soils contaminated with inorganic and the 4-chlorophenol equivalent to
organic chemicals.
A separate section is devoted to statistics in toxicology. An overview of statis-
tical methods, the most frequently applied methods and the relevant IT tools are
discussed in detail. A short summary is given on the use of toxicity results in risk
assessment.
446 Engineering Tools for Environmental Risk Management – 2

1 INTRODUCTION

The use of chemical models in environmental toxicology, monitoring, risk assess-

ment and risk management resulted in the chemical concentration-based assessment of
environmental risk, threshold concentration values and other environmental quality
criteria, expressed in concentration. However, if a mechanical way of thinking focuses
attention only on concentration values in the environment, this handicaps the develop-
ment of a more complex and realistic evaluation method including the site-specific fate
of a contaminant and the scale of measured adverse effects which can truly characterize
the actual risk posed by chemicals to the environment.
A “hazard’’ refers both to harmful properties of the contaminants, generalized
by the term “toxicity’’, and the effective concentration of the chemical measured by
standard toxicity tests (e.g. using fish, rats, etc.). The hazard scale is proportional to
the hazardous properties and the amount/concentration of contaminants from which
the hazardous levels can be determined. However, the risk scale is influenced by many
other environmental factors and the individual sensitivities of the targeted ecosystem
and human receptors. Environmental factors are responsible for the interactions and
processes which determine the fate and effects of a contaminant in the real environment,
e.g. temperature, moisture content, redox potential, microbial activity, bioavailability
and accessibility. All the interactions are part of a dynamic system with spatial and
temporal variations. Mathematical models and simulation tests can transform hazard
to risk: from the potential of a chemical substance to damage into the actual probability
to cause damage. A chemical substance is already hazardous in theory, but it is risky
only in the environment. Generic risk lies between the two, given a regular environment
and an average receptor organism (e.g. humans) in the models or in the tests.
Generic risk posed by a contaminant can be characterized by using standard matri-
ces and standardized test conditions. These types of tests simulate the interactions
between the pure contaminant, the artificial test medium and the properly controlled
test organism. These test conditions are highly abstracted as they represent the intrinsic
hazard of pure chemical substances and the environment is limited to a test medium.
Due to the constancy of the experimental ‘environment’, the reproducibility of the
measurement is good and the generic fate and behavior of the chemical substance and
its environmental hazardousness based on the effects caused can be predicted from
the measured results. In addition, a mathematical model can be established for each
standardized test based on measured data. For instance the QSAR models are based on
the quantitative relationship between the chemical structure and the activities, taking
the form of environmental behavior and adverse effects.
The risk posed by the contaminated environment cannot be characterized by
the contaminants alone. The adverse effects of environmental samples integrate the
characteristics of the contaminants, the physicochemical characteristics as well as
sensitivity and vulnerability of the environment and the interactions between con-
taminants, matrix compounds and ecosystem members. Measuring the toxicity of
environmental samples is called direct toxicity assessment. It can be done in situ on
site or in the laboratory. Based on the results, the risk can be quantitatively character-
ized without chemically identifying the contaminants. Unlike pure chemical substances,
environmental samples may contain a mixture of contaminants interacting with each
other, with the components of the environmental matrices, the microbiota and higher
 Data evaluation and interpretation in environmental toxicology 447

members of the ecosystem. During direct toxicity assessments neither the contaminant
components, nor the matrix components, nor the microbiota members are known to
or can be controlled by the assessor. It is only the response of some selected representa-
tives of the ecosystem that is known. As a consequence, the contaminant content and
the impact of the interactions cannot be expressed in terms of contaminant concentra-
tion. Instead, they can be specified as inhibition, lethality or abnormal behavior of the
affected organism or community.
Direct toxicity testing (DTA) provides information for direct decision making
based on the measured scale of adverse effects, because DTA

– measures the toxicity of the entire effluent;

– characterizes sediment and soil toxicity with a more realistic representation of the
environment;
– accounts for the aggregated effect of chemical mixtures;
– provides results that include a combination of different exposure routes and effects;
– measures a response proportional to the bioavailable fraction;
– accounts for the effects of unknown chemicals and those for which the toxicity is
not well known;
– increases safety, given that complex industrial effluents, industrially contaminated
soils and sediments may remain toxic despite complying with chemical-based
limits.

As described in Chapter 1 the test end points quantify environmental fate prop-
erties and adverse effects of chemicals. The test end points (a.k.a. study end points)
may vary from the simplest inhibition rate, through dose–response function and phar-
macokinetic characteristics to complex field assessment results of species density and
diversity. The most widespread test end points for DTA are (i) the inhibition rate –
calculated from the response scale of the test organism in the sample compared to the
negative control or reference – and (ii) the dilution-series-based toxicity end points
giving the volume or mass of the sample causing a certain percentage of inhibition, or
the highest measured level without affect (NOEL), or the lowest one found to affect
the test organism (LOEL).
In DTA the sample dose–response curve describes the dose-dependent response of
the test organism or the test system. The measured effect values are plotted against
the sample doses (mass or volume) represented by the dilutions of the ‘dilution series’
prepared from environmental samples of contaminated water, wastewater, sediment or
soil. The test end points may be the amount or the dilution rate of the sample causing an
arbitrary level (e.g. 10%, 20%, 50% or 90%) of inhibition of the measured response
or the highest no-effect amounts read from the dose–response curve. In DTA the ’dose’
of the environmental sample is the amount (volume or mass) of the original liquid
or solid sample in the tested dilution. The sample dose in DTA is not the same as
in animal tests (the amount administered by the test organisms) but the amount of
the environmental sample(s) in the test medium. ‘sD’ stands for ‘sample dose’ in this
chapter, to distinguish it from consumed or administered dose (D). The dose of the
environmental sample corresponding to EsD20 or EsD50 and the no-effect or lowest-
effect data can be calculated from the amount of the sample (in mL or g) used in the
test and the rate of dilution. The sample amount (in mL or g) depends on the test
448 Engineering Tools for Environmental Risk Management – 2

methodology; there are microbiological tests using only 200 µg samples while other
ones use 50 or 100 grams. As the effective amount defined above of an environmental
sample is test-dependent, it cannot be used in general for the characterization of toxicity
in this form. Instead, a comparable and convertible interpretation is needed, such as
the equivalence method introduced by the authors in Section 9.
The dilution rate is a useful value that refers to a generally applied outcome of envi-
ronmental toxicity testing, as it is in direct relation both to the sample and to the risk.
Based on the chemical definition, the risk characterization ratio, RCR = PEC/PNEC,
reflects how many times the predictable environmental concentration is greater than
the likely acceptable concentration of a contaminant. In this sense, the dilution rate
shows how many times the adverse effect of an environmental sample is higher than
the no-effect dilution, the dilution of the sample not causing any adverse effects. This
no-effect dilution is closely related to the acceptable exposure in the environment.
The no-effect dilution or the dilution needed to achieve a predetermined percent
inhibition (e.g. less than 10%) seems to be a clear and easy end point, but there is
still the problem of inconsistency with regulation and environmental quality crite-
ria expressed as contaminant concentrations. Most of the decisions in environmental
management are based on concentration values and on the comparison of chemically
measured concentrations to threshold values. Since the disadvantages of this kind of
simplification have been realized by practitioners and by regulators, a refined chemical
model of differentiated limiting values has been introduced at the regulatory level, for
example, special soil quality criteria for clayey, loamy and sandy soils, or aerobic and
anaerobic sediments. Environmental regulation and management rarely apply dilution
rates to define thresholds and obligations such as: ‘The risk of the soil is unacceptable
and should be reduced if the dilution rate necessary to reach the no-effect point is higher
than 5 for any of three terrestrial test organisms representing three trophic levels’. This
would be a correct but completely unusual criterion and hardly understandable for
environmental professionals who grew up using chemical models. Despite all these
some practical cases are known where environmental criteria are based on the results of
direct toxicity assessment, e.g. treated wastewater discharge into specific surface water
or remedied soil reuse for purposes of different intensity and sensitivity. In these cases
‘the absence of toxicity’ is the screening factor. Unfortunately, the results of direct toxi-
city assessment are not used when managing risk and planning risk reduction measures
of complex pollution cases, although the scale of reduction or attenuation of adverse
effects to reach a no-effect level could be equivalent to the scale of the necessary risk
reduction regardless of the actual contaminants present. In current risk management
practice, the directly measured adverse effects are only used for indication, and, based
on positive toxicity screening results, chemical tools are involved to determine a con-
centration value. In spite of the limitations of the chemical approach – for example in
the case of inherited mixed pollution, when the contaminants and metabolites present
are highly uncertain – most of the decisions are made based on the measured concen-
trations of arbitrarily selected or regulated priority contaminants. The authors of this
chapter prefer direct toxicity assessment as a routine tool in risk management in all
cases when a mixture of contaminants is present, when bioavailability has great influ-
ence and causes uncertainties in exposure and when risk is reduced by biodegrading
communities such as in the biological waste-water treatment and soil bioremediation
processes. It is also highly recommended as a screening tool in the first step of a tiered
 Data evaluation and interpretation in environmental toxicology 449

site assessment and as an approval or verifying tool for risk reduction activities, as
well as a general environmental monitoring tool.
It is a common experience that environmental toxicity results only make sense
for eco-toxicologists because toxicity data are hard to understand for practitioners
brought up on chemical models. This is one of the reasons for the actual gap between
availability and practical application of environmental toxicology in contaminated
land management, although everyone who manages the risk posed by environmental
contamination knows that the adverse effects and their scale are and should be the core
information supporting environmental decision making. Decisions based on the results
of direct toxicity assessment could be an effective tool in environmental management,
but today only a few examples can be given on the direct use of environmental toxicity
data in regulation or decision making.
Another possibility for toxicity data interpretation is the verbal characterization
of the measured toxicity. It is based on the creation of inhibition ranges for each
test, assigning verbal characterization such as ‘very toxic’, ‘moderately toxic’, ‘slightly
toxic’ or ‘non toxic’. The interpretation is test-specific, which means that, for example,
different ranges are applied to a bacterial and a plant toxicity test given that the shape
of the dose–response curve is different for these two test organisms. When creating
these ranges, the quantitative and differential character of the test results is lost.
A similar approach is applied to the regulatory classification and labeling of
chemicals: based on detailed hazard assessment, the chemical substance, product or
waste will be labeled and handled mechanically according to the ‘label’ or the group
into which it has been classified. This makes the situation understandable for non-
professionals and simplifies the management of chemicals, wastes and contaminated
land, but it also leads to a loss of information. For example, it may happen that two
environmental toxicants are in the same category and obtain the same label with an
EC50 of 11 mg/L and 99 mg/L because they fall into the same hazard category.
To make the adverse-effect results of bioassays better understandable and more
applicable, we introduce the “equivalent evaluation and interpretation’’ and “equiv-
alent calibration’’ tools for organic and inorganic contaminants. The essence of this
evaluation and interpretation methodology is that the inhibition of environmental sam-
ples is compared to the toxicity of a reference (calibration) compound, which is the
same for each measuring set, and finally the inhibition of samples is expressed as an
equivalent toxicity value for the selected reference substance. These methods use a
“calibration curve’’ for the conversion of the measured end point to the concentration
of the calibrating (reference) substance (Gruiz, 2009). The toxicity equivalent is a tech-
nical tool which may bridge the chemical and biological models in risk management
of contaminated land. It makes possible the comparison of the character of the impact
of different contaminants with each other and of the contaminated environmental
compartment with pure chemicals and other environmental samples.
The different concepts of environmental risk management are illustrated in
Figure 9.1 that shows the application options of chemical, biological and ecosystem
models and their relation to the risk management measure. Chemical models interpret
ecosystem response as a chemical characteristic, typically as an effective concentra-
tion, then compare this concentration to a chemically based threshold value. Finally,
the ecosystem response/characteristic is estimated based on the difference between the
actual environmental and the threshold values. Chemical models reduce ecosystem
450 Engineering Tools for Environmental Risk Management – 2

Figure 9.1 The concept of environmental risk management applying chemical, biological and
ecosystem models.

complexity to one or very few chemical characteristics. Consequently, they entail

extreme uncertainties due to the discrepancy of the model from the real environment.
These uncertainties are partly compensated for by a low measurement error. Biological
models measuring toxicity directly from environmental samples are much closer to the
real environmental situation, reflecting correctly the microenvironment and the envi-
ronmental matrices, but not the whole environment. Measurement errors of toxicity
tests are higher than those of the chemical analysis methods, but they can be reduced
by well-established and precisely executed methods. Field assessment vindicates the
role of closely modeling the environment, but the subset of the environment assessed
(assessable) by the assessor corresponds only to a sample, which may or may not
produce a sufficiently true value with low uncertainty.
The selection of the measurement end points and the evaluation methodology
depend on test goals, the type of test, the sensitivity of the test organisms and on the
quality and quantity of the toxic hazard. Expected capabilities of a proper measurement
end point are summarized as follows:

– A proper measurement end point serves the purpose of the assessment. The end
point of the assessment should have a diagnostic value and should be in close
relationship with the hazardous effect and risk;
– The measured end point should be consistent with the main goal of the study in
terms of preciseness, exhaustiveness and qualitativeness (e.g. sensitivity of the test
organisms);
– The end point allows for the measurement of direct and indirect effects such as
genetic, metabolic (consumption and production), reproductive, growth and lethal
effects;
 Data evaluation and interpretation in environmental toxicology 451

– The measured end points should represent adequate sensitivity (average, higher or
lower than average) and the response time is as short as possible;
– The measurement end point should provide a high level of the desired signal
compared to the level of background, i.e. high signal-to-noise ratio;
– The implementation, evaluation and interpretation of the measurement should be
easy and practical.

When testing contaminated soil, the selection of an adequate reference soil rep-
resenting the uncontaminated soil is always a challenge. Artificial soils as reference
such as the OECD-recommended mixture (5% peat, 20% clay with high kaolinite
content and 75% sand with a dominant fraction of 50–200 µm particle size) need
consistent material properties to be able to maintain constant quality. When using the
same artificial soil for dilution, nonlinear changes may occur in the different soil char-
acteristics influencing soil toxicity (OECD soil may inhibit growth due to low nutrient
content, overcompensating for the toxicity based inhibition of a nutrient reaching the
contaminated soil). If a natural soil is selected as reference, its storage, maintaining
its equilibrium and microbial activity may be difficult. The best reference for contam-
inated soils taken from the field is an uncontaminated aliquot from the same or a very
similar site.
The most frequently used evaluation methods applied in environmental toxic-
ity assessments are introduced as follows. The evaluation of toxicity results of pure
chemicals and environmental samples are discussed in parallel.

2 INHIBITION RATE

The rate of inhibition is the most popular measured value for laboratory bioassays,
given that toxicity of a chemical substance or an environmental sample is generally
compared to an uncontaminated reference, a negative control or blank, and the result
is expressed as percentage.
The inhibition rate of pure chemicals dissolved in water results in a regular satura-
tion curve (such as the dose–response curve) when plotted against concentration. Pure
chemicals dissolved in organic solvents create a more complicated situation. The sol-
vents may have (and generally do have) an effect on the test organism, so the dissolved
chemical should be considered as a mixture with additive or non-additive effects. Pure
chemicals mixed in soil in growing concentration for generating a concentration series
create an even more complicated situation due to continuously changing contaminant
speciation and non-equilibrium partitioning between three physical phases as well as
to concentration-dependent bioavailability. The best practice for preparing a concen-
tration series is mixing an exactly known mass (dose) of contaminant into every single
aliquot of the standard soil. The inhibition percentages are plotted as a function of the
contaminant concentration.
Contaminated soils and sediments are the most difficult target for toxicity assess-
ment because the original sample should be diluted to get the mass dependent response
and reach the ’no inhibition’ point. The dilution of solid environmental samples raises
a number of questions: what to dilute with, how to ensure equilibrium conditions, etc.
Mixing two different soils is the beginning of the development of a new equilibrium
452 Engineering Tools for Environmental Risk Management – 2

Figure 9.2 Plant root and shoot growth inhibition is proportional to toxicity.

for all soil processes. A further common problem is that the reference or artificial soil
used for the dilution of the contaminated soil may (and definitely does) generate fur-
ther inhibitory or stimulatory effects, which would render the results ambiguous. The
selection of the proper reference soil may be a problem in itself: it can be a site ref-
erence (similar soil from the same place but definitely not contaminated) or a general
reference (artificial soil or laboratory reference). The result of a test carried out with
contaminated soil is always the effective amount (mass) of soil causing a certain scale
of inhibition (5%, 10%, 20%, 50%, 90%) or no inhibition (NOEC).
In soil studies, e.g. when measuring plant growth in contaminated soil (Figure 9.2),
it is common that results from a complete dilution series or more than two effective
concentrations are not available because toxicity disappears rapidly with the dilution
of the contaminated soil. Another problem is that the uncontaminated reference soil
or artificial soil used for the dilution of the contaminated soil generates additional
inhibitory or stimulatory effects, which makes the evaluation ambiguous. Therefore,
plant toxicity is often expressed just in the percentage of a relative inhibition compared
to the negative control.
Inhibition of root and shoot growth is given as compared to the control, the growth
on uncontaminated soil. Inhibition rate can be calculated in % by Equation 9.1:

XL (%) = 100 ∗ (LC − LT )/LC , where (9.1)

– XL = relative growth of root/shoot compared to control, in %;

– L = seedlings’ root or shoot length in millimeters;
– C = control, grown on reference soil and
– T = tested, grown on contaminated soil.
The rate of inhibition may consider not only the length but also the produced
biomass (Equation 9.2).
XM (%) = 100 ∗ (MC − MT )/MC , where (9.2)

– XM = relative growth in plant mass compared to control, in %;

– M = seedlings’ mass in g or kg;
 Data evaluation and interpretation in environmental toxicology 453

– C = control, grown on reference soil and

– T = tested, grown on contaminated soil.

Inhibition values can be assessed for the whole dilution series of the contaminated
soil (or any other environmental sample) and the soil dose–response curve can be
plotted from the inhibition rates: inhibition percentage as a function of the amount of
the contaminated soil.
For statistical evaluation of the inhibition results, the most appropriate is to use
the Student’s t statistic to test whether the means of the treated and the untreated
control are different. The independent two-sample t-test can be applied if the sample
sizes (the number of parallels) are equal and the two distributions have the same type
of variance, e.g. if they are normal distributions.

3 CONCENTRATION/DOSE—RESPONSE RELATIONSHIP

The concentration–response curves of pure chemicals are sigmoid functions reflecting

the fact that the response does not rise linearly as a function of the concentration, but a
certain level is needed for triggering an effect and after a certain toxicant concentration
the response cannot grow proportionally. The increasing concentration results in a
growing response up to the inflection point, after which the response inhibition starts
decreasing with the number of test organisms or with their activity in the test vessel.
Concentration–response curves are plotted from primary (measured) results or
from derived values such as the inhibition rate (calculated from measured end points)
or more complex derived values, such as the area under a growth curve, or any other
significant characteristics of the response curve, e.g. the maximal response or the slope.
Figure 9.3 shows a typical concentration–response curve: the effects of the mem-
bers of the concentration series made of PCP dissolved in water. The best sigmoid is
fitted to the measurement points by using statistics-based methods supported by suit-
able software tools and the ECx , NOEC and LOEC values are read from the curve.
Statistical evaluation will be discussed in the next subchapter.
It is important to understand that there is a difference between the measured NOEC
value, which means the highest measured concentration of the tested concentration
series, which does not show a significant adverse effect, and the hypothetical NOEC,
which is likely to be a larger value than the measured one. The difference between
the measured and the hypothetical NOEC depends on the scale and spacing of the
concentration series prepared for testing. This explains also the rule that the design of
the study should be fitted to the goal, the tested end point and other requirements of
the study.
In assessing the adverse effect (inhibition rate) of contaminated environmental
samples, the above described method results in a sample dose–response sigmoid. The
contaminant composition and concentration in the sample are not known. Instead of
the contaminants’ concentration, the sample mass or volume is applied as a variable.
As an example, Figure 9.4 shows the dose–response curve of the Vibrio fischeri
culture measured with a serially increasing proportion of contaminated water (in ref-
erence water), given in mL. In other cases, e.g. when testing contaminated soil or
454 Engineering Tools for Environmental Risk Management – 2

Figure 9.3 Test end points on the concentration–response curve of pentachlorophenol (PCP) dissolved
in water.
Legend to Figure 9.3:
Inhibition of the luminescent light emission (H%), a specific type of inhibition for the lumi-
nobacterium Vibrio fischeri
EC90 : concentration resulting in 90% inhibition
EC50 : concentration resulting in 50% inhibition
LOEC: lowest tested concentration exerting an effect
NOEC: highest tested concentration giving a no-effect response.

sediment the sample mass (in g) is used instead of the sample volume. Dilution rate
can also be applied as an alternative to mass/volume.
The inhibition rate of H% for luminescence is a special case of inhibition. H% is
a more complex value than I% in the plant example (Section 2), integrating a time-
correction factor based on the luminescent light measurement methodology (Equations
9.3–9.5):

H% = 100 ∗ (Icalc − I30 )/Icalc , where (9.3)

Icalc = f ∗ I0 (9.4)

f = Ic30 /Ic0 , with (9.5)

– I0 = initial luminescence of the bacterial suspension without sample;

– I30 = luminescence of the test suspension 30 minutes after sample addition;
– Ic0 = initial luminescence of the negative control;
– Ic30 = luminescence of the negative control after 30 minutes;
– f = the ratio of Ic30 to Ic0 , i.e. the negative control at the beginning and after
30 minutes;
 Data evaluation and interpretation in environmental toxicology 455

H%
100

90

80

70

60

50

40

30

20

10

0.01 0.1 LOEsV 1 EsV50 10 100

NOEsV EsV20 EsV90 Sample mL

Figure 9.4 Test end points on the sample volume–response curve of environmental water samples
containing unidentified contaminants.
Legend to Figure 9.4:
H%: inhibition of the luminescent light emission (a specific type of inhibition)
EsV90 : sample volume resulting in 90% inhibition
EsV50 : sample volume resulting in 50% inhibition
EsV20 : sample volume resulting in 20% inhibition
LOEsV: lowest tested sample volume showing an effect
NOEsV: highest tested sample volume giving a no-effect response
s: sample whose toxicity is measured.

– Icalc = calculated light intensity, which would be emitted by the test suspension
without toxic sample addition;
– H% = luminescence inhibition of the sample.

The evaluation is based on the initial and the 30 minutes luminescence. As an

alternative, one can continuously measure luminescent light emission for each sample
and plot the time-dependent changes in the emitted light. After measuring the light
emission–time curves, one has to characterize the typical shape of the curve and select
the most characteristic features, which adequately characterize the response of the
tested bacterial culture, and use this feature (slope, area under the curve, maximum of
light emission, and so on) for the calculation of the inhibition rate.
A concentration/dose–response curve can be plotted from any measured end point
which is proportional to the concentration/dose of the stressor and is able to quantify
the response and thus differentiate it from a no-effect response or from effects of signif-
icantly lesser or greater values. Both directly measured end points (lethality, activities,
456 Engineering Tools for Environmental Risk Management – 2

reproduction) or derived values (inhibition rate, slope of the activity–time curve, etc.)
can be utilized for characterizing the adverse effect of chemical compounds or con-
taminated environmental samples. The derived values may be the maximum value of
the response and the time of its appearance, the slope of and the area under the curve,
and other characteristic features of the time-curves for respiration, luminescence, any
enzyme activities, heat production, growth and movement of the test-organism. Some
examples for the use of derived end points are introduced in Section 5.

4 EVALUATION OF THE RESPONSE BASED ON THE GROWTH

CURVES OF CULTURED ORGANISMS

Reproduction is the most adequate end point for rapidly growing microorganisms,
bacteria, fungi, algae or single-cell animals. The microbial growth process includes
consecutive reproduction cycles of microorganisms by repeated cell division according
to the power of two, resulting in an exponentially increasing cell number and biomass.
The initial cell divisions are slower (lag phase) due to adaptation to the growth sub-
strate. The speed then increases steadily to a maximum (exponential phase) after which
it slows down due to the lack of nutrients (assuming propagation in batch). Time
dependence of the number of cells is shown by the growth curve in Figure 9.5).
Tetrahymena is a sensitive protozoan (single-cell animal) (light microscopic and
electron microscopic images can be seen in Chapter 4). Its response to contaminants
and contaminated environmental samples is proportional to the concentration of the
contaminant or the amount of the contaminated environmental sample.
All phases and special points of the growth curve – the length of the lag-phase,
the slope of the exponential phase or the maximum of the curve and the time of
rise – are suitable indicators for the growth and growth inhibition. The integrative
indicator of the area below the growth curve can be calculated by any mathematical
tool, for example by approximate calculation, assuming that two evaluation points
can be connected by a straight line (which is not entirely true), and the area under the
growth curve can be mapped as right-angled trapezoids, e.g. using the formula below
(Equation 9.6):

A = t1 ∗ (X1 − X0 )/2 + (t2 − t1 ) ∗ ((X1 − X0 ) + (X2 − X0 ))/2 + · · · + (tn+1 − tn )

∗ ((Xn − X0 ) + (Xn+1 − X0 ))/2 (9.6)

– A = area
– X0 = nominal number of cells/mL at the beginning of test;
– X1 = measured number of cells/mL at t1 ;
– Xn = measured number of cells/mL at time tn ;
– t1 = time of first measurement after the beginning of test;
– tn = time of nth measurement after the beginning of test.

From A, as an indicator of cell reproduction (biomass), the inhibition values (I)

can be calculated in percent, comparing the treated to the control. Then the individual
I% values can be compared to each other or the serial I% values can be plotted vs.
contaminant concentration or environmental sample mass/volume.
 Data evaluation and interpretation in environmental toxicology 457

Figure 9.5 Growth curves of Tetrahymena pyriformis at various contaminant concentrations.

The effect of pure chemicals can be measured this way and the EA C50 , the NOEA C
or LOEA C values (the “A’’ index indicates that the result is based on the area under the
growth curve) can be read from the I%–concentration curve plotted from the inhibition
values of the concentration series.
Environmental water and soil samples can also be characterized based on the
growth curves by the effective dose of the environmental sample (the volume of the
water or the mass of the soil). The sample amount causing a certain percentage of
inhibition and the highest no-effect and lowest effect sample doses are symbolized by
the following abbreviations for results obtained from the area under the curve:

– EA sDx : effective dose for a specified percentage of inhibition rate, where x may
range from 0% to 100%;
– NOEA sD: no-effect sample dose;
– LOEA sD: lowest effect sample dose.

Another possibility to evaluate the growth curves is the comparison of the growth
rates. The average specific growth rate (µ) for exponentially growing cultures can
be read from the slope of the growth curve, plotted in the form of lnX vs. time, or
calculated by the following formula:

– µ = (ln Xn − ln X1 )/(tn − t1 ), where

◦ µ = average specific growth rate for exponentially growing cultures (logarith-
mic increase of biomass during the test period, expressed in day−1 );
◦ tn − t1 = time interval within the exponential growth phase.
458 Engineering Tools for Environmental Risk Management – 2

From the growth rates of the toxicant treated cultures in comparison with the untreated
control one can create inhibition percentage values (I%):

– I (%) = (µc − µt ) ∗ 100/µc , where

◦ µc = specific growth rate of the control culture
◦ µt = specific growth rate of the treated culture

Growth rates of cultures grown on culture media containing serial concen-

trations/doses of a toxicant result in the growth rate–contaminant concentration
function. The Er Cx or Er sDx values as well as NOEr C/LOEr C for pure chemi-
cals and NOEr sD/LOEr sD for environmental samples can be read from this growth
rate–contaminant concentration sigmoid.
Using this marking, the end points of the toxicity tests based on average specific
growth rate (slope of the exponential part of the growth curves) of the cultivated test
organism may be the following:

– Er Cx , NOEr C, LOEr C – for pure chemicals;

– Er sDx, NOEr SD, LOEr SD – for water of soil samples (“s’’ stands for sample).

The index “r’’ indicates that the evaluation is based on the growth rate.
The yield (Y), the biomass increase during the test periods can also be used as a
measurement end point. Its calculation is: biomass at the end of the exposure period
minus biomass at the beginning.
Biomass and cell count may correlate with each other well but they can differ
on a significant scale due to the deviations in cell size, which is why cell count is
considered by many of the standardized methods to be a surrogate method compared
to cell biomass analysis.
The same type of evaluation is used for any microbial and algal growth tests.
The ‘test organism’ is a culture, the quasi-homogeneous culture of the microorganisms
where the individual cells are not considered. The response of such a system depends
mainly on the experimental parameters and is described typically by the log-normal
or normal distributions. It is worth using all the data of the replicates (instead of
the means). Data should be fitted to a functional equation by non-linear regression.
Selection of the appropriate functional equation can be a problem: if it is not possible
to fit to the whole of the curve, it is advisable to choose and fit different equations to
the two distinct parts of the growth curve. From the fitted equation the characteristic
point estimates of Er Cx values can be determined. More detailed discussion on statistics
is found in the next subchapter.

5 EVALUATION OF THE EFFECT OF CONTAMINANTS ON

HEAT PRODUCTION: A SPECIAL CASE

Toxicity evaluation based on the response–time function makes the tests and evaluation
more complicated, but the information behind the numbers may be more important
and necessary for making a good decision based on toxicity assessment.
 Data evaluation and interpretation in environmental toxicology 459

0 ppm PCP
P (µW)
1 ppm PCP
5 ppm PCP

180 10 ppm PCP

20 ppm PCP

120

60

5 10 15 20
Time (h)

Figure 9.6 Microcalorimetric power–time curve of Azomonas agilis in PCP-polluted soil.

An example for the evaluation of the heat production curve for quantitative toxi-
city assessment is explained here. Contaminated soil was tested in a microcalorimeter
(see Chapter 5) adding an Azomonas agilis bacterial strain to the sterilized soil sample.
The concentration series of PCP (pentachlorophenol, a pesticide and disinfectant) and
DBNPA (2,2-dibromo-3-nitrilopropionamide, a quick-kill biocide) were assessed and
the power–time curve was plotted.
The power-time curve of PCP-contaminated soil is shown in Figure 9.6. Theoret-
ically, the area under the curve is proportional to the total heat produced during the
test. This may be a good end point, but less capable of distinguishing among cases
than some other characteristics of the curve. The results indicate that the higher the
concentration of PCP in the soil, the smaller the initial rise (Ri ) and the maximum
(Pmax ) of the curve. A shift in time in the power maximum can be observed (tmax ).
In the special case shown in Figure 9.6, instead of the total heat production (area
under the curve), the most significant indicators of Ri , Pmax and tmax were combined to
establish the Ri × Pmax /tmax factor, denoted Ah . Ah integrates and magnifies the effects
being in reciprocal relation with PCP concentration in the tested soil. The Ri × Pmax /tmax
value (Ah ), the inhibition rate (I) and the EC20 and EC50 values are summarized in
Table 9.1.
EC20 and EC50 are read from the concentration vs. Ah (response) sigmoid although
the EC20 and EC50 values can be determined from any of the end points (R, P, t, RxP/t)
read from the curve, which is useful for making comparison. Other end points can
also be read from the sigmoid such as NOEC or LOEC.
460 Engineering Tools for Environmental Risk Management – 2

Table 9.1 Effect of PCP- and DBNPA-polluted soil on the heat production of Azomonas agile.

PCP conc. Ri t max P max Ah = Ri × P m /t max IA EC 20 EC 50

(mg/kg) (µW/h) (h) (µW) (µW 2 /h2 ) (%) (mg/kg) (mg/kg)

0 36.4 9.3 219 856 0 0.7 3.9

1 25.1 9.6 208 544 36
5 19.7 11.2 198 351 59
10 18.8 11.4 203 334 61
20 8.9 17.4 157 80 91

Legend to Table 9.1:

Ri (µW/h): initial rise of the power–time curve;
tmax (h): time of occurrence of the power maximum (Pmax );
Pmax (µW): the maximum power;
Ah = Ri × Pm /tm : an indicator for the magnification of the positive responses, shortly called ‘Ah ’;
IA (%): inhibition based on IA = (AhControl − AhPCP )/AhControl *100.

6 EVALUATION OF BIODEGRADATION OF CHEMICALS IN

WATER AND SOIL

Testing biodegradability and actual biodegradation in water, sediment, soil and

wastewater or solid waste is based on similar methodologies: to utilize contaminants as
energy substrates and to measure the consumption of the substrate or the production of
inorganic end products or any metabolite or by-products of the degrading microbiota.
The biodegradation rate is the measure of the contaminants’ tendency to be
degraded or their effect on biodegrading organisms. Its measurement has the follow-
ing aims:

– Characterizing the biodegradability of pure chemical substances or contaminants

or mixtures of contaminants in the environment;
– Characterizing the biodegradation potential of the soil or wastewater community;
– Measuring the toxicity of a chemical substance or contaminated environmental
samples on the microbiota or on other degrading members of the ecosystem of
water, sediment, soil, or wastewater. The inhibition of biodegradation is measured
in this case.

The evaluation of the biodegradation of chemicals is based on the mass balance,

which is described by the reaction equation. A generic equation for organic compounds
consisting of C (carbon), H (hydrogen) and N (nitrogen), i.e. the complete aerobic
biodegradation (mineralization) in the presence of microorganisms and O2 , neglecting
cell mass growth, can be described as (Equation 9.7):

CX HY NZ + (X + Y/2 + 3Z/2)O2 → microorganisms →

XCO2 + Y/2H2 O + ZNO3 + biomass (9.7)

One can measure the decrease in the mass of the chemical substance being
degraded, the oxygen consumed and the CO2 or the NO3 produced and calculate
 Data evaluation and interpretation in environmental toxicology 461

all the others based on the equation. If the microorganisms have the opportunity (e.g.
time) to grow and propagate, the increase in cell mass should be added to the right
side of the equation. If the product is different to CO2 , one has to use the molecular
weight of the identified product in the calculation.
Depending on the naturally occurring degradation process other types of
equations can be used for partial degradation and for anoxic degradations and arti-
ficial indicators (radioactive labels or color-markers) can also be applied to facilitate
evaluation.

6.1 Monitoring the depletion of the chemical substance

Degradation of the substance is generally followed by chemical analyses. Sampling
and analysis should be performed at least at the beginning and the end of the tests, but
recording several time points may be beneficial. Isotope-labeled substances are easier
to follow based on their activity. The result based on mass values is compared to the
initial mass and given in percentage as a biodegraded proportion.
Toxic compounds may inhibit normal biodegradation in water and soil. In this case
the biodegraded proportion should be evaluated in comparison with uncontaminated
control water or soil to calculate inhibition rate in percent. The effect of a series of
contaminant concentrations or of the contaminated wastewater volumes on the healthy
biodegradation can also be measured. Based on the results, the dose/concentration–
response curve can be drawn and the no-effect and various effect values can be read
from the curve.

6.2 Evaluation of biodegradation based on

CO2 production
Biodegradability is most widely tested based on CO2 production because the test
equipment and set-up ensures that the only degradable substrate is the chemical
substance/contaminant to be tested. The produced mass of CO2 is equivalent to its
degraded C-content. Soil without contaminant (without substrate) is used as a reference
(negative control).
As an alternative methodology, impulse-like addition of the biodegradable sub-
stance to the steady-state aerated test system makes the test more dynamic. In this case
the scale of the response and its time-dependent occurrence can be evaluated.
If the kinetics of biodegradation is known, the half-life time can be the end point
of the biodegradability test.
The biodegradable fraction of the contaminants in wastewater or contaminated
soil can be determined based on the produced CO2 , equivalent to BOD (biological
oxygen demand) according to the carbon transformation formula (Equation 9.7).
Figures 9.7 and 9.8 show the respiration curves of artificially contaminated soil
with growing concentrations of a mixture of toxic metals (Cd, Cu, Hg, Ni and Zn).
The concentration series represents 0, 1x, 2x, 4x, 8x and 16x of the soil screening
concentrations (SSC). Similar serial curves are created by contaminated soils added to
healthy soil in growing percentage.
462 Engineering Tools for Environmental Risk Management – 2

Figure 9.7 Decreasing CO2 production by the soil with increasing toxic metal concentration.

Figure 9.8 The concentration–respiration inhibition curve from the 24h respiration rate.

6.3 Substrate induction

To characterize soil health, activity and its ability to biodegrade, induction of ready-
to-biodegrade additives such as glucose is a usual test (Torstensson, 1994). The same
 Data evaluation and interpretation in environmental toxicology 463

Figure 9.9 Glucose-induced respiration rates for contaminated and uncontaminated soils.
Legend to Figure 9.9:
B: Soil respiration baseline value, before glucose addition
– B2 : uncontaminated soil: intensive respiration rate
– B1 : contaminated soil: low respiration rate
I: Soil respiration rate after the addition of easy-to-utilize glucose
– I2 : uncontaminated soil: higher increase
– I1 : contaminated soil: lower increase
L: Lag phase: adaptation to the new situation, before the exponential phase
– L2 : uncontaminated soil: short lag phase
– L1 : contaminated soil: long lag phase
E: Exponential phase with constant respiration rate
– E2 : uncontaminated soil: steep slope
– E1 : contaminated soil: less steep slope.

test is suitable to test inhibitory or adverse effects of contaminants based on the

decreased biodegrading activity due to poisoning of the water or soil microbiota.
Substrate-induced respiration (SIR) as well as substrate-induced nitrification (SIN)
bioassays can measure a response proportional to adverse effects using standardized
and non-standardized test methods. Measuring the glucose-induced respiration rate (or
that of any other adequate substrate) after the incubation of the soil and the suspected
contaminating substance, the response to the easy-to-degrade substance (glucose) will
be inversely proportional to the toxicity.
Using biodegradation measurements as an ecotoxicity test method, the effects of
a series of concentrations (of the chemical substance or the contaminated water/soil)
on carbon transformation can be determined. The data from these tests are used to
prepare a dose–response curve and to calculate ECx or EsDx values.
Adding glucose to the soil, a sudden increase in respiration rate can be measured
(Figure 9.9). The increase (I) is proportional to soil health, adaptive behavior, and
the presence (if any) of contaminants with inhibitory effects. Test design and data
464 Engineering Tools for Environmental Risk Management – 2

evaluation and interpretation depend on the aim of testing: biodegradability of con-

taminants or any other substrates to degrade, respiration rate and dynamics of the
wastewater/soil or ecotoxicity of contaminants in water or soil. Evaluation can pro-
vide relative values when the measurement results are compared to an uncontaminated,
untreated or reference water/soil, or absolute values when a series is tested and the
response is measured as a function of growing concentration or amount.
The biodegradation rate in substrate-induced respiration studies can be used for
toxicity testing. The concentration–response sigmoid can be plotted from any of the
characteristics of the respiration curve as a function of the applied toxicant con-
centrations or, alternatively, of the doses (mass or volume) or the dilutions of the
contaminated sample. The height of the increase after induction (I), the length of the
lag phase (L) or the slope factor (E) of the exponential phase, as well as the combina-
tions of these end points can be used for the evaluation. Similarly to the evaluation of
the growth curves (see Section 4) one can integrate the area under the curve and use
the integrated CO2 production values as “response’’ as a function of the contaminant
concentration to calculate ECX or LOEC/NOEC. Alternatively, in the case of contam-
inated water/soil samples, the mass or the dilution of the sample enables to determine
EsDx and LOEsD/NOEsD.

7 ATTENUATION RATE METHOD FOR

ENVIRONMENTAL SAMPLES

From the dose/concentration–response curve, amongst others NOEC, NOEsV (no-

effect volume of the water sample) or NOEsD (no-effect mass of the soil sample)
values can be read. They are the highest concentration and the sample volume or mass
which do not cause any adverse effect. This refers to the no-risk state of the water
or soil in contaminated land management and can be considered as the target of risk
reduction. The management measure should ensure a risk reduction rate needed to
achieve the no-risk situation in the field. This is supposed to be equivalent to the no-
effect dilution of the DTA sample in terms of toxicity. It is reasonable to introduce a
safety factor based on the uncertainties accompanying the assessment.
The tested serial volume or mass of environmental samples is created during the
test by diluting the original sample with uncontaminated water or soil. In these cases
the dilution rate to reach the no-effect point on the dose–response curve is identical
with the necessary risk reduction rate. This does not mean that the risk is reduced by a
dilution process in the real environment; it means rather the attenuation due to natural
or technological processes responsible for reaching an acceptable risk value.
Liquid-phase wastes (i.e. wastewaters) contain unidentifiable contaminants, as
hundreds and thousands of toxicants enter the wastewater treatment plants and a
chemical analysis is not practiced as a daily routine. Residual organic matter and
toxicity can be measured easily and the decision on when wastewater can be discharged
into effluents can be based on measured toxicity results. The toxicity of the treated
wastewater poses a risk to the ecosystem of the recipient water body; the wastewater
should not be discharged from the treatment plant, but further treated. The water
of inadequate quality is stored in a buffer pool until recycling or further treatment.
The ecosystem of the recipient water body in question can be represented by selected
 Data evaluation and interpretation in environmental toxicology 465

Figure 9.10 Graphic determination of the dilution rate of a contaminated soil sample to reach the
no-effect volume measured by the collembolan test.

aquatic organisms and can be used as site-specific test organisms. If the measured
ecotoxicity exceeds the NOEsV values, it requires interventions by the operator, e.g.
changes in the technological parameters, use of additives or any other treatment to
reach the required lower toxicity. For properly representing an aquatic ecosystem, a
minimum of three organisms from different trophic levels should be tested and the
results used for decision making in a way that can take uncertainties into account.
When using measured ecotoxicity data for direct decision making, managing uncer-
tainties should have priority. Uncertainty derives from several details: the selection of
the test organisms, their representativeness, their rate of sensitivity, the composition
of the wastewater, adaptation of the ecosystem, etc. Depending on the scale of the
expected uncertainties, more test organisms and safety factors should be applied.
The dilution rate read from the dose–response curve should be considered as an
attenuation rate from the technological point of view which should be reached by the
waste water treatment technology.
If the contaminated groundwater or leachate shows 12 times higher toxicity than
the no-effect volume of the groundwater or leachate, the applied risk reduction measure
(treatment in ponds, artificial wetlands or permeable reactive barriers, etc.) should
ensure a minimum of 12-fold reduction in toxicity according to the uncertainty (safety)
factor used.
From Figure 9.10 the initial toxicity of the sample and the no-effect value can
be read in dilution or in gram units because a sample of known mass is used in the
test (100 g in the case shown). Dilution of the original sample is 1 and it caused 100%
466 Engineering Tools for Environmental Risk Management – 2

inhibition. The original sample needed 200-fold dilution to reach the first measurement
point without any adverse effect (NOEsD = 0.5 g soil). This approach can further
be refined by increasing the resolution of the curve between NOEsD and LOEsD or
calculating their average. A safety factor may also be applied. After refining the system,
the necessary dilution rate decreased to 150. This way, the risk characterization ratio
of a contaminated soil can be defined as RCR = the dilution rate that produces no
adverse effect (150) = mass of the sample tested (100 g)/mass of the sample with no
effect (0.66 g).
There are practical cases when decision making in environmental management is
based on toxicity, for example if it should be decided whether or not

– the treated wastewater can be released into natural waters,

– the treated contaminated soils can be re-used for certain purposes,
– the application of fertilizers, nutrients, organic wastes or any other waste materials
on soils is acceptable from the point of view of toxic risk.

Using the attenuation-rate approach, one can make a decision not only by saying
‘yes’ or ‘no’ but by determining the allowable quantity of the discharged water or
disposed waste. The same approach can be used to create and control remedial target
values based on toxicity.
A stepwise approach is recommended to determine the target of remediation: after
a rapid screening, the extent and distribution of toxicity at a contaminated site can be
evaluated. If the screening tests indicate either mortality or a sub-lethal effect that is
greater than a certain percentage (for example 20% or 25%) of that of the reference
soil, the contaminated soils toxicity should be investigated in more detail by applying
standardized or site-specific test batteries. The test battery for soil should include plants
(monocot and dicot), soil-dwelling animals (arthropods and earthworms) and/or soil
microorganisms and their activities such as respiration or nitrification rates. The last
two integrate several microbial species’ activity, representing the soil as a whole.
Results acquired from laboratory bioassays have shortcomings similar to those
expected from any oversimplified model: their environmental reality is generally poor.
A larger number of tests on numerous test organisms can provide a better estimation.
Monitoring the species distribution in microcosms, mesocosms or field tests may serve
as a scientifically established basis for decision making. Unfortunately, utilization of
the results of species distribution to calculate a risk characterization ratio (RCR) and to
integrate it into a rapid decision support tool is not yet practiced, mainly due to the time
requirement. Instead of measuring species distribution, the application of more test
organisms and end points is the solution for a statistically correct and still rapid decision
making. The selection of the most restrictive responses or other statistically based
criteria may increase efficiency and lower time requirement. The “omics’’ technologies
may improve the situation in this field too.
In tests on non-site-specific, but generally applicable and high-sensitivity test
organisms such as Vibrio fischeri, i.e. luminobacterium, the comparative strategy may
help to characterize the tested soil in comparison to an uncontaminated local soil. In
this case the target toxicity may be the same as the reference, more precisely ‘statisti-
cally the same’: it must not exceed the toxicity of the reference soil by more than e.g.
10 or 20%.
 Data evaluation and interpretation in environmental toxicology 467

Another aspect of the decision is that, in addition to toxicity, other environmental

parameters should also be considered in the decision making: pH and redox potential
of the recipient environment or the intensity of light in the case of emission of pho-
todegradable contaminants, etc. Bioavailability and its inclusion in risk assessment is
a recurring problem to be dealt with. Validation of toxicity based on the results of
long term monitoring of the recipient’s quality is necessary to prove that there is no
increased risk in the environment due to additional toxicity load.

8 TOXIC EQUIVALENCY OF CONTAMINATED ENVIRONMENTAL

SAMPLES FOR EXPLORATION AND SCREENING

The toxic equivalency method is not unknown in environmental practice: it is used to

quantitatively characterize the toxicity of mixtures of similar chemical substances. The
first and best known application was developed for dioxins and dioxin-like compounds
in the context of regulatory toxicology. The aggregated adverse effect of mixtures can
be expressed in the form of toxic equivalency (TEQ) for a similar toxicant with additive
effects. TEQ is based on the TEF value of individual dioxin-type chemical substances in
the mixture. TEF quantifies the toxicity compared to that of the most toxic dioxin com-
pound, the 2,3,7,8-TCDD (tetrachlorodibenzo-p-dioxin, van den Berg et al., 2006).
For example the toxicity of the 1,2,3,7,8,9-HxCDD (hexachlorodibenzo-p-dioxin) is
0.1 because it is ten times less than the toxicity of the 2,3,7,8-TCDD. Based on the
chemical composition of a mixture, one can calculate its aggregated toxicity from the
TEFs and the proportions of the components. A similar scheme has been developed
for PCBs. Toxic equivalency exclusively uses chemical data which supports risk assess-
ment and risk management. Within this context, the risk of complicated mixtures of
different dioxin components (with a different size of effects) can be determined, as
with single contaminants. This model can be applied in those cases where dioxin-
like compounds are the only contaminants. With regard to dioxins, the equivalency
method solely applies the chemical model based on chemical analytical data and fits
well into regulatory risk management which is also based on the chemical model. The
latter relies on environmental quality criteria (expressed in concentrations) and envi-
ronmental exposures (also expressed in concentrations). The TEF values are constant
factors.
The authors applied the equivalency approach in an inverse fashion and used the
effect results of direct toxicity assessment of environmental samples. DTA characterizes
the effects of unknown contaminants, which are complicated mixtures of chemically
and toxicologically different contaminants, with unknown speciation, bioavailability
and interactions, as typical in the environment.
Given that the measured toxicity values of environmental samples cannot be fitted
directly to the chemical model-based ERA, the equivalency method was used to convert
toxicity values to concentrations of environmental samples with unknown composi-
tion, speciation and bioavailability. This method required only the measurement of
the effects and not that of the concentrations or the modeling of the environmental
fate processes. This is not a very precise method because the assessor cannot decide
whether or not the components are additive, whether their dose–response curves run
parallel (as will be seen, they often do not), or if they have the same or different effect
468 Engineering Tools for Environmental Risk Management – 2

mechanism since the contaminants have not yet been identified. Nevertheless, toxic
equivalency can be very useful in a tiered risk assessment to screen large areas with
similar contaminants and select and classify a great number of environmental samples
tested by different tests and test organisms. An additional benefit is that the equivalency
method enables the results of (eco)toxicity studies to be translated into the language
of chemistry and make them understandable for non-ecotoxicologists.
The authors have almost 20 years’ experience in the use of toxic equivalency (TEQ)
for quantitative characterization of environmental sample toxicity (Gruiz et al., 2001,
Molnár et al., 2005; 2009; Leitgib et al., 2007; Feigl et al., 2007). This approach
was spontaneously applied along the line of ‘calibration’ useful in chemical analytical
practice. It was used for many years (first in 1992) for the evaluation of soils in a large
mining area contaminated with various metal mixtures (Gruiz & Vodicska, 1992;
1993). At that time multi-component analytical methods were not as widespread as
today and the owners and the authorities selected two out of approximately 20 metals
that contaminated a former mining site. The risk management of the site was based
on the concentration of the two metals. No logical correlation between the observed
toxicity and the measured metal concentrations was found; therefore, soil, sediment
and leachate toxicity was measured on the abandoned mining site. Copper was selected
by the authors for this purpose to control the sensitivity of a special test bacterium and
thus it was logical to express toxicity (EC50 , EC20 , NOEC) in copper equivalent – as
though copper had only caused toxicity. Toxicity of the samples tested on various test
organisms was expressed in copper equivalent concentration, which helped evaluation,
interpretation and communication with different stakeholders, owners, municipalities,
authorities, NGOs and the mine’s management. Later on the copper equivalent was
also used to monitor the efficacy of bioremediation technologies (Molnár et al., 2005;
2009; Leitgib et al., 2008; Feigl et al., 2007). It proved to be useful in explaining
and interpreting toxicity test results and integrating them into the quantitative risk
assessment procedure (Molnár et al., 2007; Leitgib et al., 2007) during the initial
phases (screening phase and preliminary risk assessment). The basic concept and the
creation and interpretation of the toxic equivalency method and toxic equivalency
factor are introduced below in this chapter.
Equivalent toxicity, expressed for example in mg Cu/kg soil (TEQCu ), integrates the
effects and the actual bioavailability of all metals present in the environmental sample,
the interactions between the metals present and the partition of contaminants between
soil phases, as well as interactions between metals and metal forms that influence effects
and bioavailability. TEQ is able to handle the differences between bioassay methodolo-
gies and the sensitivity of the test organism to a contaminant, resulting in a generally
applicable equivalent value, a common benchmark. Copper (Cu) was selected as the
reference substance for all metals toxic to the environment because it is toxic to many
of the ecosystem members (including the test organisms studied) and because its salts
dissolve in water but are not particularly toxic to humans under laboratory conditions.
Toxicity of soils contaminated with organic compounds, per analogiam, is com-
pared to 4-chlorophenol (4CP) toxicity and expressed in 4CP equivalent: TEQ4CP (mg
4CP/kg soil). Measured toxicity values of a 4CP dilution series serve as a calibration
curve, and the toxicity of soil contaminated with an organic chemical substance can
be expressed as if it was caused by 4CP. The risk management is based on this toxicity.
However, this does not mean that 4CP can model other characteristics of the substance
 Data evaluation and interpretation in environmental toxicology 469

than toxicity. The reason why 4CP was chosen, similar to copper, is that it is toxic to
all of the most widely used test organisms, it is easy to work with (it is not volatile, it is
stable and has adequate solubility in water), and the risks to humans can be managed
under laboratory circumstances (protective equipment, ventilation).
To calculate TEQ, 4CP or Cu calibration series and the environmental sample with
unknown contaminants are prepared and measured at the same time. This calibration
series also serves to control sensitivity and behavior of test organisms. The calibration
curve has to be measured in water when water samples are tested. When dealing with
soils, it is important to use uncontaminated soil which is the same as or similar to the
soil tested for toxicity. The calibration curve should be measured at the same time as
contaminated environmental samples are measured. The different dilutions belonging
to equal effect can be read from the curves and the sample dose is converted into
copper or 4CP concentration. Any special point of the concentration–response curves,
e.g. EC20 , EC50 , NOEC or LOEC, can be used. The selection depends on the aim
and concept of risk management. Depending on the calibration concept, the biological
response to any environmental sample can be converted to a hypothetical concentration
and risk management methods based on a chemical model can be applied, e.g. the result
can be compared to environmental quality criteria expressed in concentration.
Equivalency tools have been employed by the authors in their everyday practice,
and experience shows that standardized calibration tools are able to make toxicity
testing more practicable and independent of the actual measurement conditions. Not
only can test organisms be controlled, their sensitivity to different contaminants can be
better characterized. Different test organisms can also be compared with one another.
The equivalency tool is similar to those used by many chemical analytical methods that
need a calibration between a measured end point (e.g. color) and the concentration
of a chemical substance. It should be noted that 4CP or copper might not be present
in the sample, but handling the results in concentration units makes sense in practical
toxicity management.

8.1 Toxic equivalency for organic and inorganic contaminants

Bioassays measuring toxicity and the calculation of TEQs are introduced below as an
example of a direct-contact soil bioassay using:

– Vibrio fischeri bacterial luminescence inhibition test;

– Tetrahymena pyriformis protozoan growth inhibition test;
– Folsomia candida, collembolan mortality test;
– Sinapis alba plant root and shoot growth test and
– Daphnia magna, water flea immobilization test.

The main benefits of a TEQ value lie in its potential to characterize the toxicity and
risk of a contaminated environment without specifying the actual contaminants, i.e.
in the case of unknown contaminant and of contaminant mixtures. Copper and 4-CP
‘calibration series’ have other benefits as well: due to their positive control in toxicity
tests, they can confirm test repeatability and good biological status of test organisms.
4CP or Cu equivalents of toxicity of known (specified) contaminants enable a quan-
titative comparison between the two toxicants and the relative sensitivity of the test
470 Engineering Tools for Environmental Risk Management – 2

Figure 9.11 Calibration curve of 4CP and the dose–response curve of contaminated soils (mass) and
water (volume).

organism. Comparing the adverse effects (to the same test organism) of different chem-
ical substances or mixed contaminants in soil, the contaminant can be characterized by
a toxic equivalency factor (TEF) with regard to 4CP (organic toxicants) or Cu (toxic
metals). This shows how many more times it is toxic compared to 4CP or Cu. TEF can
help in toxicant ranking and risk evaluation. TEF can be calculated from different test
end points and it enables a dynamic comparative characterization and the aggregation
of the results of different studies.

8.2 Graphical determination of equivalent toxic concentrations

from measured data
The response of the test organism is plotted against the 4CP or Cu concentration series,
and the sigmoid is fitted to the points providing the concentration–response curve. The
same can be done to the dilution series of contaminated soil or water and the corre-
sponding contaminated soil mass (M in mg) or contaminated water volume (V in mL)
can be read from the curves obtained. The sigmoid is fitted to the measurement points
(Figures 9.11 and 9.12) using statistical software (Origin 8.0 or Statistica 6.0). Any
common end point (EC50 , EC20 , LOEC, NOEC, etc.) and the equivalencing chemical
substances in terms of sample volume, sample mass, 4CP or Cu concentration, respec-
tively, can be read from these curves of the samples. This is the basis for creating the
 Data evaluation and interpretation in environmental toxicology 471

Figure 9.12 Calibration curves of Cu equivalent and dose–response curves of Cd-contaminated soil
(mass) and water (volume) and mixed metals-contaminated soil.

equivalent value, i.e. ‘calibrating’ the unknown contaminant. The same method can
be applied for samples in dissolved form or in solid matrices.
The volume of the sample and the concentration–response curve of 4CP or Cu (Cu
mg/L or 4CP mg/L) are plotted based on liquid sample tests. The value read from the
sigmoid at a default effect (highest no-effect, lowest effective inhibition or the effects
causing 5, 10, 20, 50, 90% inhibition) is the volume or mass of the sample equivalent
to the amount of 4CP or Cu causing the same effect. The effective mass of the sample
is labeled by the abbreviation sM = sample mass/dose, or sV = sample volume. EsMx
(effective sample dose causing x% inhibition) values can be read from the curves and
the TEQx values calculated from the known 4CP or Cu values. The calibration curve
examples of pure and mixed organic soil contaminants such as PCP and hydrocarbons
are shown in Figure 9.11 and those of soils contaminated with toxic metals using the
Cu equivalent in Figure 9.12. Both figures show the dose–response curve of Vibrio
fischeri luminescence inhibition and the graphical determination of EC50 .
The graphically determined EC50 values e.g. in Figure 9.12 can be directly inter-
preted as follows: 6 µL contaminated water causes 50% inhibition, which is the same
level of toxicity as the one caused by a copper solution of 8.2 mg/L concentration.
6 µL from the unknown is equivalent (based on EC50 ) to 8.2 mg/L copper. Further
possible calculations: 6 µL (causing 50% inhibition) is the 41.66-fold dilution of the
original sample (250 µL), consequently the original sample’s toxicity (based on EC50 )
is equivalent to 341.7 mg/L copper. In reality, the artificially contaminated Cd solution
472 Engineering Tools for Environmental Risk Management – 2

had 500 mg/L Cd concentration, the copper calibrating solution 400 mg/L Cu. Taking
these into account, Cd is less toxic for Vibrio fischeri than Cu, the ratio of Cd/Cu
toxicity is 0.68 based on the EC50 value of Vibrio fischeri (See also Table 9.4).

8.3 Numerical determination of the toxicity

equivalent concentration
The rate of mortality or inhibition is plotted against the volume/mass of the tested
water/soil contaminated with unidentified contaminants: this gives the sigmoid of the
sample dose–response curve. Simultaneously, the artificially contaminated water or
soil with the reference contaminant (4CP or Cu) is also tested and its concentration–
response curve is drawn. Both the reference chemicals’ concentration (EC50 ) and the
tested water or soil dose (EsV50 or EsM50 ) that cause 50% inhibition can be read from
the two curves, and these two values are considered as equivalent values causing the
same level of toxicity (50%). The toxicity equivalent is the amount of the unknown
water or soil expressed in the concentration of the reference contaminants: 4CP or
Cu. The result means the equivalent EC50 of the tested water or soil as though it were
caused by the 4CP or Cu reference contaminants. By analogy of the dilution point
causing 50% inhibition (EC50 ), the same can be done using NOEC or LOEC.
The method is presented using the example of 4CP equivalent; the same can be
used for calculating Cu equivalent.
If the liquid-phase sample is tested and the adverse effect is plotted against dilution
in the graph, the effective volume can be calculated from the dilution rate read from
the graph and from the test-specific sample volume. The equivalent toxicity of the
liquid-phase sample can be calculated by equation (Equation 9.8). A similar equation
is valid when no-effect volume (NOEsV) or lowest effect volume (LOEsV) are used as
end points.


mg 4CP EC50(4CP) [mg 4CP/L] · Vsample [µL or mL]
TEQ4CP(EC50) = (9.8)
L EsV50(sample) [µL or mL sample]

where

– TEQ4CP(EC50) : 4CP equivalent concentration of the sample with unknown contam-

inant(s) causing 50% inhibition (alternatively TEQ4CP(LOEC) and TEQ4CP(NOEC) );
– EC50(4CP) : effective concentration of 4CP solution causing 50% inhibition;
– EsV50(sample) : effective volume of the liquid sample causing 50% inhibition;
– Vsample : tested volume according to the test protocol, e.g. the volume of the tested
sample is 0.25 mL for Vibrio fischeri luminescence inhibition;
– Vsample /EsV50(sample) : dilution rate of the unknown sample until reaching the 50%
inhibition point in the graph.

If solid-phase sample such as soil or solid waste is tested, the equation should
be modified according to (Equation 9.9). Similar equations are valid for LOEC and
NOEC values when the basis of equivalencing is LOEC or NOEC.

    
mg 4CP EC50(4CP) mg4CP/kg · Msample mg or g
TEQ4CP(EC50) =   (9.9)
kg EsM50(sample) mg or g sample
 Data evaluation and interpretation in environmental toxicology 473

where

– TEQ4CP(EC50) : 4CP-equivalent concentration in soil of an unknown contaminant

or mixture of contaminants causing 50% inhibition;
– EC50(4CP) : effective concentration of 4CP solution causing 50% inhibition, lowest
effect or no effect;
– EsM50 : effective mass of the solid sample causing 50% inhibition, lowest effect or
no effect;
– Msample : test mass for e. g. Vibrio fischeri luminescence inhibition; the volume of
the tested sample is 0.25 mL;
– Msample /EsM50(sample) : dilution rate of the unknown sample up to reaching dilution
resulting in 50% inhibition.

TEQ can be calculated for the no-effect and lowest effect points, too; in this case
the volume or mass corresponding to no effect or lowest effect is used for the calculation
by analogy. The toxicity of soil can be equivalenced compared to the calibrating water
solution if the partition of the toxicity between soil and water is known or has been
measured (See also Chapter 7).

8.4 Equivalent toxicity of contaminated water: examples

and validation
The use of the equivalency tool in the case of water contaminated by single chemicals
and mixtures of contaminants is introduced below by some examples. The unknown
contaminant’s toxicity is expressed in 4CP-equivalent toxicity based on the EsV50
(TEQ4CP(EC50) ) or EsVLOEL or EsVNOEL and the relevant TEQ values. EsV50 means the
volume of contaminated water which causes 50% inhibition or 50% lethality; EsVLOEL
is the lowest tested volume causing an observable effect, EsVNOEL , the highest tested
volume of contaminated water not causing measurable adverse effect.
The toxicity of the unknown samples expressed as if it were 4CP or Cu makes
possible the comparison of the results to the screening values or other effect-based envi-
ronmental quality criteria established for 4CP and Cu. Some of the existing screening,
intervention, threshold limit and other guideline values are listed here:

– 4CP groundwater quality criterion: 1 µg/L (Hungarian soil regulation, 2000)

– Italian 4CP limit value for groundwater: 0.5 µg/L (Carlon et al., 2007);
– 4CP soil quality criterion: 0.01 mg/kg (Hungarian soil regulation, 2000);
– 4CP intervention value for soil: 1–3 mg/kg (Hungarian soil regulation, 2000);
– 4CP soil intervention value: 10 mg/kg (Swartjes, 1999);
– Finnish 4CP soil guideline values for soil: lower: 10 mg/kg, upper: 40 mg/kg
(Carlon et al., 2007);
– Italian 4CP limit for public parks: 0.01 mg/kg, for industrial areas: 5 mg/kg (Carlon
et al., 2007);
– Cu groundwater quality criterion: 200 µg/L (Hungarian soil regulation, 2000);
– Italian Cu limit value for groundwater: 1000 µg/L (Carlon et al., 2007);
– Cu groundwater intervention: 300–500 µg/L (Hungarian soil regulation, 2000);
– Cu soil quality criterion: 75 mg/kg (Hungarian soil regulation, 2000);
474 Engineering Tools for Environmental Risk Management – 2

Table 9.2 4CP-equivalent toxicity of contaminated water based on 50% inhibition.

Measured Calculated Tested Analysis

EsV 50 TEQ4CP(EC50) V sample result EC 50 TEF 4CPEC50
Contaminants and tests mL sample mg 4CP/L mL mg/L mg/L –

PCP-contaminated water
Luminescence inhibition of Vibrio 0.0417 57.79 0.25 1.8 0.30 32.1
fischeri
Growth inhibition of Tetrahymena 0.91 362.76 31.5 144 4.17 2.5
pyriformis
Immobilization of Daphnia magna 1.11 50.45 100 1.8 0.02 28.0
BPA-contaminated water
Luminescence inhibition of Vibrio 0.0205 117.55 0.25 156 12.76 0.76
fischeri
Immobilization of Daphnia magna 0.69 81.16 100 1.07 0.52
DBP-contaminated water
Luminescence inhibition of Vibrio 0.0233 103.43 0.25 30 000 2800 0.003
fischeri
Immobilization of Daphnia magna >10* N.A. 100 30 000 N.A. N.A.
Mix of NSAIDs in water
Luminescence inhibition of Vibrio >0.050* N.A. 0.25 7.5 N.A. N.A.
fischeri
Immobilization of Daphnia magna 9.10 6.16 100 7.5 0.68 0.8
Nicotine in water
Immobilization of Daphnia magna 0.40 140.35 100 20 000 79.8 0.007
4CP calibration series
Luminescence inhibition of Vibrio 0.0121 200 0.25 200 9.64 1.0
fischeri
Growth inhibition of Tetrahymena 0.66 500 31.5 500 10.48 1.0
pyriformis
Immobilization of Daphnia magna 0.28 200 100 200 0.56 1.0

*Sample toxicity failed to reach the 50% inhibition level

N.A. not applicable

– Cu soil intervention: 200–300 mg/kg (Hungarian soil regulation, 2000);

– Cu soil intervention value: 100 mg/kg (Swartjes, 1999);
– Finnish Cu soil guideline values for soil: lower: 150 mg/kg, upper: 200 mg/kg,
(Carlon et al., 2007);
– Italian Cu limit for public parks: 120 mg/kg, industrial areas 600 mg/kg (Carlon
et al., 2007).

Table 9.2 shows the calculation details and the resulted TEQ4CP values of selected
organic contaminants for Vibrio fischeri, Tetrahymena pyriformis and Daphnia
magna. The basis of the TEQ calculation was the 50% inhibition. The following
values are listed in the head of the table:

– The EsV50 , (EsVLOEC and EsVNOEC also can be used) is the volume of the contami-
nated water representing the theoretical point (extrapolated by fitting the sigmoid)
of the serial dilution resulting in 50% inhibition. Its unit of measurement is ml
sample.
 Data evaluation and interpretation in environmental toxicology 475

– TEQ4CP is the calculated 4CP equivalent of the tested contaminated water: as if

the water were only contaminated by 4CP. Its unit of measurement is mg 4CP/L.
– Sample volume is the volume prescribed/required by the test protocol (this data is
necessary for the calculation of EC50 and TEF);
– Result of the chemical analysis (only for identified contaminants) of the samples
for the purpose of validating the toxic equivalency and calculating TEF) given in
mg contaminant in 1 liter sample.
– EC50 is the calculated toxicity, i.e. the concentration of the contaminated water
causing 50% inhibition expressed in mg/L sample and determined from the
analysis result and the measured dilution.
– TEF (Toxic Equivalency Factor) describes how many mg 4CP is equivalent to 1 mg
contaminant. It is the equivalent mass of the contaminant causing the same toxicity,
compared to 4CP. TEF = 1: equally toxic; TEF > 1: more toxic; TEF < 1: less toxic
than 4CP. It can only be calculated when the contaminating substance has been
identified and quantified by chemical analysis. The TEF is calculated from 50%
inhibition, the NOEC or LOEC values are generally different due to the different
shape of the concentration–response curve of the tested contaminant and 4CP.

8.4.1 4CP equivalent of selected organic contaminants

in water: examples
In the demonstration cases, real or artificially contaminated water samples were used
and their contaminant content was analyzed during the test for the validation of
the equivalency method. The toxic equivalency factor (TEF) was calculated for con-
crete (identified) contaminants for comparison. Chemical contaminants tested and
whose toxicities were expressed as 4CP equivalents, were pentachlorophenol (PCP),
bisphenol-A (BPA), dibutyl phthalate (DBP) and nicotine. As a mixture, an artifi-
cial mix of NSAIDs (non-steroidal anti-inflammatory drugs) was prepared, containing
diclofenac (645 mg/L), ketoprofen (456 mg/L), naproxen (438 mg/L) and ibuprofen
(362 mg/L). The Cu-calibration has been demonstrated on cadmium-contaminated
water and soil.
The dose–response curves in Figure 9.10 show that the 41.67 µL sample of PCP-
contaminated water caused 50% inhibition in the Vibrio fischeri test, while only
12.1 µL of the 4CP solution with 200 mg/L concentration caused the same level of
toxicity which corresponds to 9.64 mg/L 4CP in terms of concentration. Calculat-
ing back to the original solutions using Equation 9.8, the PCP solution is equivalent
to a 57.79 mg/L 4CP solution. The TEF factor, which tells how many times PCP is
more toxic to Vibrio fischeri than 4CP, can also be calculated. The concentration of
the calibrating 4CP solution (200 mg/L) is a priori known and the unknown water
sample contaminated with PCP has been analyzed: the result was a PCP concentra-
tion of 1.8 mg/L. The table shows that PCP is 32.1 times more toxic when EC50 is
the basis of the calculation (9.64/0.30). Daphnia magna shows a ratio similar to
that shown by Vibrio fischeri, but Tetrahymena found PCP only 2.5-times as toxic
as 4CP.
BPA based on its EC50 value is less toxic than 4CP both to Vibrio and Daphnia,
but a different TEF is obtained based on the NOEC values (Table 9.3) because Vibrio
is sensitive to small BPA concentrations as well, while Daphnia is not.
Table 9.3 4CP-equivalent toxicity of contaminated water based on NOEC and LOEC values.

Measured Calculated Measured Calculated

EsV LOEC TEQ4CP(LOEC) LOEC TEF LOEC EsV NOEC TEQ4CPNOEC NOEC TEF 4CPNOEC
Contaminants and test mL mg/L mg/L – mL mg/L mg/L –

PCP-contaminated water
Luminescence inhibition of Vibrio fischeri 0.020 50.00 0.144 27.8 0.0100 50.0 0.072 27.8
Growth inhibition of Tetrahymena 0.134 559.48 0.61 3.9 0.067 559.5 0.30 3.9
Immobilization of Daphnia magna 0.25 100.00 0.0045 55.6 0.05 100.0 0.0009 55.6
BPA-contaminated water
Luminescence inhibition of Vibrio fischeri 0.00032 3,125 0.20 20.0 0.00008 6,250 0.05 40.1
Immobilization of Daphnia magna 0.75 33.33 1.17 0.2 0.5 10.0 0.78 0.06
DBP-contaminated water
Luminescence inhibition of Vibrio fischeri 0.0167 60,000 2.00 2.0 0.0000083 60,000 1.00 2.0
Immobilization of Daphnia magna 0.0167 15,000 0.50 0.5 0.000067 75,000 0.02 2.5
Mix of NSAIDs
Luminescence inhibition of Vibrio fischeri 0.0125 80.00 0.375 10.7 0.005 100.0 0.15 13.3
Immobilization of Daphnia magna 0.67 37.50 0.05 5.0 0.067 75.0 0.005 10.0
Nicotine contaminated water
Immobilisation of Daphnia magna 0.0125 2,000 2.5 1.0 0.00005 100,000 0.01 5.0
4CP calibration series
Luminescence inhibition of Vibrio fischeri 0.0050 200 4.00 1.0 0.0025 200 2.00 1.0
Growth inhibition of Tetrahymena 0.15 500 2.38 1.0 0.075 500 1.19 1.0
Immobilization of Daphnia magna 0.125 200 0.25 1.0 0.025 200 0.05 1.0
 Data evaluation and interpretation in environmental toxicology 477

The effect of nicotine is also interesting from this point of view: TEF = 0.007
based on EC50 , but TEF = 1 when calculated from LOEC and TEF = 5.0 from NOEC,
characterizing nicotine as a slightly toxic substance (not causing lethality) in high
concentrations, but also toxic in very low concentration ranges.
The toxicity based on EC50 and LOEC or NOEC may be significantly different for
a chemical substance, as we can see in Tables 9.2 and 9.3. It is best demonstrated by the
TEQ4CP and TEF4CP values calculated from the different end points. The differences
in TEQ and TEF calculated from EC50 , LOEC and NOEC are characteristic of the
contaminants’ interaction with the test organism and of the effect mechanisms. Grow-
ing TEF toward LOEC and NOEC indicates adverse effects of the chemical substance
at very low concentrations or high dilution rates. This means that the dose–response
curve declines slightly and does not reach zero inhibition.
A comparison of the NOEC, LOEC and EC50 values with each other reflects the
differences in the shape of the concentration–response curves. Equivalencing means
that the contaminants’ curve is compared to the curve of 4CP. 4CP gives a response
proportional to the dilution (e.g. EC50 : 9.64; LOEC: 4.0; NOEC: 2.0) for all of the test
organisms. The mixture of NSAIDs is a good example to demonstrate the differences
in the shape of the dose–response curve: it hardly reaches the 50% inhibition, but
a very high dilution rate is still needed to reach the no-effect point. It results in a
surprisingly high TEQ value when calculating it from NOEC and LOEC. TEFNOEC
of 10.7 (10 times more toxic than 4CP) draws attention to the importance of the less
drastic adverse effects of low contaminant concentrations. Thus NSAIDs are typically
non-killing agents, but they may be inhibitory or disrupting for the endocrine and
immune system in small concentrations.

8.4.2 Copper equivalent of cadmium-contaminated water

To demonstrate the equivalent method for inorganic contaminants, the copper equiv-
alent of water artificially contaminated with cadmium (Cd) is introduced here.
Cu-calibration series were used as a comparison and the adverse effects of the water
are characterized by the copper equivalent toxicity (as if the toxicity were only caused
by Cu). Table 9.4 contains the equivalents based on EC50 . Table 9.5 shows the LOEC
and NOEC values.
Cadmium appears to be less toxic to the two bacteria and the plant than Cu. All
TEF from LOEC and NOEC are <1.

8.5 Toxicity equivalent of soil: examples and validation

Soil, sediment and solid waste toxicity is difficult to model therefore direct toxicity
assessment of solid-phase samples provide useful information for decision making. The
interaction of contaminants with each other, with the solid matrix and with thousands
of living organisms in soil, sediments and waste greatly influence the actual adverse
effect of a hazardous chemical or the aggregated effect of mixtures of chemicals. Direct
toxicity tests on samples containing solid phase integrate the interactions within the
solid matrix, the partition, mobility, bioavailability etc. Direct toxicity tests on soil,
in spite of providing highly realistic results, are still rarely used in contaminated land
management practice because latter is still based on the chemical model.
478 Engineering Tools for Environmental Risk Management – 2

Table 9.4 Copper-equivalent toxicity of cadmium-contaminated water based on EC50 .

Measured Tested Analysis

EsV 50 TEQCu(EC50) V sample results EC 50 TEF Cu(EC50)
Contaminants and tests mL mg/L mL mg/L mg/L –

Cd-contaminated water
Luminescence inhibition of 0.006 341.7 0.25 500 12.0 0.68
Vibrio fischeri
Dehydrogenase activity of 0.09 80.0 2.5 18.0 0.16
Azomonas agilis
Shoot growth of Sinapis alba 2.19 285 5.0 219.0 0.57
Root growth of Sinapis alba 1.44 433 5.0 144 0.86
Cu calibration series
Luminescence inhibition of 0.005 400 0.25 400 8.2 1.0
Vibrio fischeri
Dehydrogenase activity of 0.018 400 2.5 2.9 1.0
Azomonas agilis
Shoot growth of Sinapis alba 1.56 400 5.0 125 1.0
Root growth of Sinapis alba 1.56 400 5.0 125 1.0

8.5.1 4CP equivalent of selected organic contaminants in soil: examples

To demonstrate the use of 4CP equivalent, we compared the toxicity of soil con-
taminated with pentachlorophenol (PCP) of two transformer oils and unknown soils
contaminated with benzenes, alkyl benzenes (BTEX), polycyclic aromatic hydrocar-
bons (PAHs) and extractable petroleum hydrocarbons (EPH). For demonstration
purposes and for verifying the equivalent toxicity quantification method, the con-
taminant content was determined by chemical analysis and the 4CP equivalent (TEF)
was calculated. In the case of PCP contamination, uncontaminated reference soil (from
the same site) spiked with 4CP was used for comparison.
In this experiment series, soil was tested using the luminescent bacterium, the
protozoan and Folsomia candida, the soil-dwelling springtail. Similar to water samples,
PCP showed higher toxicity compared to 4CP in the case of microorganisms, but the
collembolan did ‘feel’ PCP less toxic than 4CP; TEF = 0.77 (Table 9.6). This can be
explained by the difference between 4CP and PCP in volatility and availability for the
springtail, which is mainly impacted via respiration.
Soils polluted with hydrocarbon mixtures demonstrate that the chemical analysis
(fulfilling regulatory obligations, but still only analyzing selected components) cannot
accurately reflect the risk of contaminated soils. The analytically measured 780 and
7,600 mg/kg EPH (extractable petroleum hydrocarbons) content of soil samples con-
taminated with (PCB-free) transformer oil – differs significantly, almost tenfold. With
respect to toxicity results, the difference between B and A soils is only sevenfold.
Soil contaminated with the mixture of non-volatile contaminants is characterized
by 277, 143 and 100 (mg 4CP/L) of TEQ expressed as 4CP for the three test organisms.
These TEQ values are 27-, 14- and 10-fold greater than the intervention value of 4CP
(10 mg/kg) while the analyzed concentration of the three contaminant groups is signif-
icantly under the lowest Hungarian intervention value (EPH: 300; BTEX: 0.5 mg/kg;
PAH: 5 mg/kg) (HU soil regulation, 2000). Trusting the response of the test organisms
rather than the chemical model, this soil should be further assessed and remedied.
Table 9.5 Cu-equivalent toxicity of metal-contaminated water based on NOEC and LOEC values.

Measured Calculated Measured Calculated

EsV LOEC TEQCuLOEC LOEC TEF Cu(LOEC) EsV NOEC TEQCu NOEC NOEC TECu(NOEC)
Contaminants and tests mL mg/L mg/L – mL mg/L mg/L –

Cd-contaminated water
Luminescence inhibition/Vibrio fischeri 0.000625 32.00 1.25 0.06 0.0000625 40 0.125 0.08
Dehydrogenase activity/Azomonas agilis 0.050 50.00 10.00 0.10 0.025 20 5.0 0.04
Shoot growth of Sinapis alba 1.0 250.00 100.00 0.50 0.50 100 50 0.2
Root growth of Sinapis alba 0.2 250.00 20.00 0.50 0.10 250 10.0 0.5
Cu calibration series
Luminescence inhibition/Vibrio fischeri 0.00005 400 0.08 1.0 0.0000062 400 0.01 1.0
Dehydrogenase activity/Azomonas agilis 0.0125 400 2.00 1.0 0.00125 400 0.20 1.0
Shoot growth of Sinapis alba 0.625 400 50.00 1.0 0.125 400 10.0 1.0
Root growth of Sinapis alba 0.125 400 10.00 1.0 0.0625 400 5.0 1.0
480 Engineering Tools for Environmental Risk Management – 2

Table 9.6 4CP-equivalent toxicity of contaminated soil samples based on EC50 values.

Measured Calculated Tested Result of

EsM50 TEQ4CP50 Msample the analysis EC 50 TEF 4CPEC50
Contaminants and tests g mg/kg g mg/kg mg/kg –

PCP-contaminated soil
Luminescence inhibition of 0.0049 981 0.25 191 3.74 5.1
Vibrio fischeri
Growth inhibition of 0.14 1,537 31.5 0.85 3.2
Tetrahymena pyriformis
Mortality of Folsomia candida 8.0 146 20 76.4 0.77
Transformer oil contaminated
soil A
Luminescence inh. of Vibrio fischeri 0.025 194 0.25 EPH:780 77.5 0.24
Growth inhibition of 1.0 215 31.5 24.6 0.27
Tetrahymena pyriformis
Mortality of Folsomia candida 2.54 461 20 99.0 0.59
Transformer oil contaminated
soil B
Luminescence inhibition of 0.0036 1,353 0.25 EPH: 7,600 107.7 0.18
Vibrio fischeri
Growth inhibition of 0.35 615 31.5 84.7 0.08
Tetrahymena pyriformis
Mortality of Folsomia candida 1.1 1,060 20 419 0.14
Non-volatile hydrocarbon-
contaminated soil
Luminescence inhibition of 0.0173 277 0.25 EPH: 120 N.A. N.A.
Vibrio fischeri
Growth inhibition of 1.5 143 31.5 BTEX: 0.51 N.A. N.A.
Tetrahymena pyriformis
Mortality of Folsomia candida 11.6 100 20 PAH: 1.68 N.A. N.A.
4CP calibration series
Luminescence inhibition of 0.024 200 0.25 200 19.20 1.0
Vibrio fischeri
Growth inhibition of 0.43 500 31.5 500 6.83 1.0
Tetrahymena pyriformis
Mortality of Folsomia candida 5.85 200 20 200 58.50 1.0

N.A.: not applicable.

Using LOEC and NOEC for equivalencing a completely different impression is

obtained compared to the EC50 -based evaluation (Table 9.7).
Hydrocarbons of petroleum origin are non-proportionately losing their toxicity
when diluted, compared to 4CP. The explanation may be that they are – strictly
speaking – natural compounds.

8.5.2 Copper equivalent of soils contaminated with cadmium

and a mixture of metals
Mixtures of metals are typical soil contaminants for example in industrially contami-
nated sites. The interactions in the soil contaminated with metal mixtures are enhanced
Table 9.7 4CP-equivalent toxicity of contaminated soil samples based on NOEC and LOEC values.

Measured Measured
EsMLOEC TEQ4CP(LOEC) LOEC TEF 4CP(LOEC) EsMNOEC TEQ4CP(NOEC NOEC TEF 4CP(NOEC)
Contaminants and tests g mg/kg mg/kg – g mg/kg mg/kg –

PCP-contaminated forest soil

Luminescence inh. of Vibrio fischeri 0.000625 4.8 0.48 0.025 0.0003125 2 0.24 0.01
Growth inh. of Tetrahymena pyriformis 0.012 1625 0.072 8.6 0.006 1583 0.036 8.3
Mortality of Folsomia candida 2.5 100 23.88 0.52 1.25 100 11.94 0.52
Transformer oil-contaminated soil A
Luminescence inh. of Vibrio fischeri 0.0200 1.5 62.56 0.0019 0.0100 0.0625 31.28 0.00008
Growth inh. of Tetrahymena pyriformis 1.00 19.5 24.80 0.025 0.50 18.9 12.40 0.0242
Mortality of Folsomia candida 10.00 25 391 0.032 5.00 25 195.5 0.032
Transformer oil-contaminated soil B
Luminescence inh. of Vibrio fischeri 0.0005 6 15.2 0.0013 0.00025 2.5 7.59 0.000329
Growth inh. of Tetrahymena pyriformis 0.12 162.5 28.90 0.021 0.06 158 14.50 0.02
Mortality of Folsomia candida 2.0 125 760 0.0165 1.00 125 380 0.0165
Non-volatile hydrocarbons-contaminated soil
Luminescence inh. of Vibrio fischeri 0.003125 0.96 N.A. N.A. 0.0016125 0.39 N.A. N.A.
Growth inh. of Tetrahymena pyriformis 1.0 19.5 N.A. N.A. 0.50 18.9 N.A. N.A.
Mortality of Folsomia candida 2.5 100 N.A. N.A. 1.00 125 N.A. N.A.
4CP calibration series
Luminescence inh. of Vibrio fischeri 0.000015 200 0.012 1.0 0.0000031 200 0.0025 1.0
Growth inh. of Tetrahymena pyriformis 0.039 500 0.62 1.0 0.019 500 0.30 1.0
Mortality of Folsomia candida 1.25 200 12.5 1.0 0.625 200 6.25 1.0

N.A.: not applicable.

482 Engineering Tools for Environmental Risk Management – 2

Table 9.8 Copper-equivalent toxicity of metal-contaminated soil based on EC50 values.

Measured V sample Analysis

EsM50 TEQCu 50 g or result EC 50 TEF CuEC50
Contaminants and tests g mg/kg mL mg/kg mg/kg –

Cd-contaminated soil 500

Luminescence inhibition of Vibrio fischeri 0.021 202 0.25 42 0.41
Dehydrogenase enzyme activity of 0.325 86 2.5 65 0.17
Azomonas agilis
Shoot growth of Sinapis alba >5 >139 5 >500 <0.28
Root growth of Sinapis alba 2.13 366 5 213 0.73
Soil contaminated with a mixture As: 228
of toxic metals
Luminescence inhibition of Vibrio fischeri 0.020 212.5 0.25 Cd: 0.84 266 0.064
Dehydrogenase enzyme activity of 0.363 77.1 2.5 Cu: 54.7 482 0.023
Azomonas agilis
Shoot growth of Sinapis alba 3.78 184 5 Pb: 2748 2511 0.055
Root growth of Sinapis alba 5.0 156 5 Zn: 294 3325 0.047
Cu calibration series 400
Luminescence inhibition of Vibrio fischeri 0.0106 400 0.25 17 1.0
Dehydrogenase enzyme activity of 0.07 400 2.5 11.2 1.0
Azomonas agilis
Shoot growth of Sinapis alba 1.74 400 5 139 1.0
Root growth of Sinapis alba 1.95 400 5 156 1.0

by the variety of their chemical forms (chemical speciation) and changing environ-
mental conditions. In such situations actual toxicity is essential information and direct
toxicity assessment is a tool to fill the knowledge gap.
In this example soils artificially contaminated with cadmium and a mixture of
metals stemming from contaminated land are tested and subjected to equivalencing.
The validation after chemical analyses and TEF calculation helps to understand the
results.
Similar to the previous cases, the results in Table 9.8 confirm that cadmium is less
toxic to microorganisms and plants than copper; TEF is less than one in all tested cases.
The mixture of arsenic, cadmium, copper, lead and zinc in a soil from an abandoned
mining site with extremely high lead content showed somewhat less than 400 mg/kg
copper in the soil. Considering the analyzed lead content itself (2,748 mg/kg), or total
metal content together (3,325 mg/kg), this soil has a TEF of 0.02–0.06 compared to
Cu. It can be explained by the immobile (stable) form of the metals in this soil and
their limited biological availability.
As the above tables (Table 9.8 and 9.9) show, it often occurs that EsM50 cannot
be detected (Cd-contaminated soil root growth inhibition) because the inhibition does
not reach 50%. In these cases long-term LOEC and NOEC values provide information
about the adverse effects.
The TEQ values can interpret the differences due to matrix effects occurring in
solid environmental samples and due to the differences in the test organisms’ responses.
They may help to understand the nature of organic contaminant toxicity in soil and
integrate the toxicity of different and unknown contaminating substances and their
interactions with the soil matrix and soil organisms.
Table 9.9 Equivalent toxicity of metal-contaminated soil based on NOEC and LOEC values.

Measured Measured
EsMLOEC TEQCu(LOEC) LOEC TEF Cu(LOEC) EsMNOEC NOEC TEQCu(NOEC)
Contaminants and tests g mg/kg mg/kg – g mg/kg mg/kg

Cadmium-contaminated soil
Luminescence inhibition of Vibrio fischeri 0.0025 250 5.0 0.5 0.001 2.0 250
Dehydrogenase enzyme act. of A. agilis 0.10 100 20 0.2 0.05 10 100
Shoot growth of Sinapis alba 5.0 100 500 0.2 2.50 250 100
Root growth of Sinapis alba 0.02 25,000 2.0 50 0.01 1.0 25,000
Toxic metal mixture-contaminated soil
Luminescence inhibition of Vibrio fischeri 0.00625 100 83.13 0.03 0.003125 41.56 80
Dehydrogenase enzyme act. of A. agilis 0.0625 160 83.14 0.05 0.03125 41.57 160
Shoot growth of Sinapis alba 1.25 400 831 0.12 0.625 415 400
Root growth of Sinapis alba 2.5 200 1663 0.06 1.25 831 200
Cu calibration series
Luminescence inhibition of Vibrio fischeri 0.00156 400 2.5 1.0 0.000625 1.0 400
Dehydrogenase enzyme act. of A. agilis 0.025 400 4.0 1.0 0.0125 2.0 400
Shoot growth of Sinapis alba 1.25 400 100 1.0 0.625 50 400
Root growth of Sinapis alba 1.25 400 100 1.0 0.625 50 400
484 Engineering Tools for Environmental Risk Management – 2

9 STATISTICAL EVALUATION OF TOXICITY DATA

Uncertainties in toxicology should be managed by statistical evaluation and analysis

of the dose–response function and in the risk assessment procedure, i.e. quantifying
the probability and size of an adverse effect and determining the safe exposure lim-
its. Biostatistics has been developed for the analysis of toxicological data, but it is
equally important in the planning and execution of studies and in the analysis and
interpretation of the results.
Statistical evaluation of toxicity data generally entails the problem of selecting the
proper mathematical model and the most appropriate software. It is typical that several
different statistical tools can be used for the evaluation of an experiment. The quality
of the resulting test end point e.g. NOEC or ECx does not primarily depend on the
statistical method but on the design and precision of the study. There are two pragmatic
ways to select the proper evaluation method: (i) accepting the recommendation of a
qualified statistician, or (ii) understanding the capacity of all potential statistical tools
and selecting the most appropriate one for the purpose of evaluation and the dataset to
be evaluated. When using a standard test organism and a standard test method, verified
statistical methods can be applied as a routine, but the mathematical–statistical model
should be chosen after thorough preparatory work and cautious circumspection in all
other cases. To support the method selection, some general information on statistics
and the models and methods applied in environmental toxicology are discussed below.
Dose and concentration are used as interchangeable terms in this chapter.

9.1 Statistics in general

The general objectives and functions of statistical analysis are summarized below under
the keywords of describing, identifying, comparing, predicting and explaining, based
on the plausible overview of a professional blogger (Stats with cats, 2014):
– Describing the characteristics of biological entities, ecosystems, samples, areas
etc. using default qualitative or quantitative descriptors, statistical intervals,
correlation coefficients, graphics or maps;
– Identifying or classifying a known or hypothesized entity or group of entities
using descriptive statistics; statistical intervals and tests, graphics and multivariate
techniques such as cluster analysis;
– Comparing and detecting differences between statistical populations or reference
values using simple hypothesis tests, and analysis of variance and covariance;
– Predicting measurements using regression and neural networks, forecasting using
time-series modeling techniques, interpolating spatial data;
– Explaining latent aspects of phenomena using regression, cluster analysis, discrim-
inant analysis, factor analysis, and data mining techniques.
The basic statistical parameters are as follows (Larson et al., 2001):
– Statistics are numerical values used to summarize and compare sets of data.
– The following three statistics are measures of central tendency:
◦ The mean, or average, of n numbers is the sum of the numbers divided by
n. The mean is denoted by x, which is read as “x-bar’’. For the data, x1 ,
x2 , . . . , xn , the mean is x = x1 + x2 n+ ...+ xn .
 Data evaluation and interpretation in environmental toxicology 485

◦ The median of n numbers is the middle number when the numbers are written
in order. If n is even, the median is the mean of the two middle numbers.
◦ The mode of n numbers is the number or numbers that occur most frequently.
There may be one mode, no mode, or more than one mode.
– Measures of dispersion tell how spread out the data are:
◦ The range is the difference between the greatest and least data values.
◦ The standard deviation describes the typical difference (or deviation) between
the mean and a data value. The standard deviation σ of x1 , x2 , …, xn is:

(x1 − x)2 + (x2 − x)2 + · · · + (xn − x)2
σ= .
n

◦ Variance also describes the difference between the mean and a data value. It
can be considered the square of standard deviation:

(x1 − x)2 + (x2 − x)2 + · · · + (x − x)2

V = σ2 = .
n

– Statistical graphs
◦ Box-and-whisker plot: The “box’’ encloses the middle half of the data set and
the “whiskers’’ extend to the minimum and maximum data values. The plot
itself comprises a rectangle (the “box’’) between the lower quartile and the
upper quartile, and a straight line (the “whiskers’’) between the minimum and
maximum data values, running through the box. The median divides the data
set into two halves. The lower quartile is the median of the lower half, and
the upper quartile is the median of the upper half.
◦ In a histogram data are grouped into intervals of equal width. The number of
data values in each interval is the frequency of the interval.

A number of web pages (e.g. idre, 2014; Statistics, 2014; MathWorks, 2014;
StatTrek, 2014; UDEL, 2014; Statpages, 2014) give advice for finding the best fitting
statistical method for the above-mentioned cases in the form of lists, algorithms and
tables, but all these cannot help in choosing the proper statistics if the evaluator/
assessor does not understand the aim, the data type and its variations. The priority
information needed for proper statistical method selection comprises:

– The objective of the evaluation;

– The number of variables;
– The scale of the variables;
– Whether the variable is dependent and/or independent;
– If the samples are autocorrelated and
– if the data are quantal or continuous.

The difference between quantal and continuous data is one of the main issues in
toxicity evaluation. Quantal response data reflect incidences. Frequently used quantal
end points are lethality (how many test organisms die or survive), immobilization (how
486 Engineering Tools for Environmental Risk Management – 2

many daphnids are mobile or immobile) or genetic anomalies. Continuous data are
growth, enzyme activities, etc. data, which can take a continuously changing value
during the study (body or organ weight, respiration or enzyme activities etc.). In tox-
icity studies the route and duration of the exposure are essential information and
should be comparable to those occurring in nature. These two independent variables
get less emphasis when standardized test evaluations are discussed. The statistics of
time-dependence, exposure routes and exposure repetition deals with cases when more
species are tested together or other interactions are expected. However, the biological
changes under the impact of the assessed stressor (e.g. resistance, adaptation), as well
as the extrapolation from laboratory to field are not discussed in this chapter, only
attention is drawn to the importance of these aspects.
The mathematical basis of the statistics in toxicology and other biological studies
goes back to the fifties and has not changed much (Brown, 1978), but new possibili-
ties opened up with the expansion of information technologies. Nowadays universities
and statistics courses offer descriptions and helpdesks for self-made statisticians and
several software are available for free or for small subscription fees (StatPages, 2014;
Unistat, 2014; Usablestat, 2014; Statistics, 2014; Statistical Solutions, 2014 and the
large software providers such as BMDP, 2014; SAS, 2014 and SPSS, 2014; PROAST,
2014; Slob, 2003; etc.). Some of the complex software tools offer tool batteries
for toxicity tests and bioassay evaluation (Unistat – Bioassay, 2014; Origin, 2014;
Toxstat, 2014). Several high-quality tutorials/lectures are also available on YouTube
as presentations of highly qualified professionals and from the software providers, e.g.
the STATISTICA user guide (StatSoft, Getting started, 2014) or the excel introduction
to XLSTAT (XLSTAT Introduction, 2014).
Study design in environmental toxicology plays a crucial role, for it is more impor-
tant than the analysis itself. Design and analysis mutually determine each other: design
of a study governs the necessary data analysis, e.g. ensuring satisfactory power or con-
fidence interval may influence study design. When using standardized test methods,
one should not deal with the design of the study only if input and output data fulfil the
prerequisites of the standard method. The same is true for the test duration: duration,
treatment and sampling schedule are key factors which strongly influence statistics in
newly designed (not standardized) i.e. problem-specific studies. The majority of envi-
ronmental toxicology studies are so-called prospective studies, applying randomized
controlled trial and covering one or more treatments and one control. The importance
of randomization is that the known and unknown prognostic factors are completely
balanced in the different treatment groups. Treatment and control should occur in the
same period of time.
Some well-known statistical tests and procedures are listed below. Most of them
have importance in toxicology. For the definitions and explanations below, the fol-
lowing sources have been used: Berkeley (2014); Doncaster and Davey (2007); OECD
(2006); Statistics (2014); Upton and Cook (2014) and Wikipedia (2014).

– Akaike information criterion characterizes the relative quality of a statistical model

for a given set of data, so it is suitable for model selection.
– Analysis of variance (ANOVA): differences between group means and their vari-
ation among and between groups. The ANOVA model can be described as:
 Data evaluation and interpretation in environmental toxicology 487

Response = Factor(s) + ε where the response refers to the data that require explain-
ing, the factor or factors are the putative explanatory variables contributing to the
observed pattern of variation in the response, and ε is the residual variation in
the response left unexplained by the factor(s). For the confirmation of the model
the residuals should be analyzed. Follow-up testing is necessary in order to assess
which groups are different from which other groups or to test other hypotheses.
If the test method applies a control, Dunnett’s test (a modification of the t-test)
can be applied for saying whether each of the other treatment groups has the same
mean as the control.
– Anderson–Darling test is a powerful tool for detecting most departures from
normality or deciding if the normal distribution adequately describes a set of data.
– Bartlett test is used to test if the samples are from populations with equal variances,
called homogeneity of variances.
– Bonferroni correction is used in multiple comparisons to control the family-wise
error rate.
– Bootstrapping measures accuracy to sample estimates. This technique allows esti-
mation of the sampling distribution of almost any statistic. It uses simple methods
such as resampling. The same technique can be used for the validation of the
regression model by bootstrapping the residuals.
– Box-Cox transformation is often used for stabilizing variance and transform-
ing data to normality, or normal distribution-like data. It is a rank-preserving
transformation of data using power functions.
– Chi-squared test: a hypothesis test in which the sampling distribution of the test
statistic is a chi-squared distribution when the null hypothesis (in toxicology, it is
typically the assumption that the treated does not differ from the control) is true.
– Cramér–von Mises criterion is used for judging the goodness of fit of a cumulative
distribution function compared to a given empirical distribution function, or for
comparing two empirical distributions.
– Correlation: any kind of statistical relationships involving dependence.
– Dunnett’s test is a multiple comparison procedure to compare each of a number of
treatments with a single control. In contrast to the Bonferroni correction it exploits
the correlation between the test statistics.
– Factor analysis: describes variability among observed and correlated variables in
terms of a potentially lower number of unobserved variables called factors.
– F-test: is used for testing F-distribution under the null hypothesis to find the best
fit. Exact “F-test’’ is used when the model has been fitted to the data using least
squares.
– Hosmer–Lemeshow test is applied for goodness of fit for logistic regression models.
– Kolmogorov–Smirnov test is a non-parametric test of the equality of continuous,
one-dimensional probability distributions.
– Mann–Whitney U-test: a non-parametric test of the null hypothesis that two pop-
ulations are the same against an alternative hypothesis, e.g. that a particular
population tends to have larger/smaller values than the other.
– Multivariate analysis of variance (MANOVA) is used when there is more than one
response variable. In this case simultaneous observation and analysis of more than
one outcome variable is necessary. The application of multivariate statistics is mul-
tivariate analysis. Multivariate statistics is concerned with multivariate probability
488 Engineering Tools for Environmental Risk Management – 2

distributions of the observed/measured data and the interpretation of the results.

Several types of multivariate analysis are known and applied such as: multivariate
analysis of covariance (MANCOVA), multivariate regression analysis, principal
components analysis (PCA), canonical correlation analysis and redundancy analy-
sis (RDA), correspondence analysis (CA), multidimensional scaling, discriminant
analysis and clustering. Artificial neural networks extend regression and cluster-
ing methods to non-linear multivariate models. Statistical graphics can be used to
explore multivariate data.
– Pearson’s chi-squared test or chi-squared goodness-of-fit test is used to assess two
types of comparison, tests of goodness of fit and tests of independence. It is for
studying/assessing any type of correlation with an arbitrary model. It has priority
for discrete variables. If data are continuous, it is worthwhile to transform them
into a discrete form and then apply the Pearson’s chi-squared test. It applies for
non-parametric models.
– Pearson product–moment correlation coefficient is the measure of the linear cor-
relation between two variables, giving a value between +1 and −1. The value of
1 means total positive correlation, 0 means no correlation and −1 corresponds to
total negative correlation. It is obtained by dividing the covariance of the two vari-
ables by the product of their standard deviations. It is for characterizing the degree
of linear dependence between two variables. Permutation tests or bootstrapping
can be used for constructing confidence intervals for Pearson correlation coeffi-
cients. To evaluate the distribution of the coefficient one can calculate the Student’s
distribution or the real distribution.
– Permutation test is for testing the hypothesis that two or more samples might
belong to the same population. It combines the observations from all the samples
first, than shuffles them and redistributes and resamples them 2–3 times (using the
same resample sizes as the original samples). Finally it determines how often the
resampled statistic gives the same values as the originally observed value.
– Regression analysis is the statistical evaluation of the regression and estimates the
relationships among variables. The focus is on the relationship between a depen-
dent variable and one or more independent variables. It demonstrates how the
typical value of the dependent variable changes when any one of the independent
variables is varied, while the other independent variables are held fixed. For val-
idation of the model, confirmation of the goodness of fit one can use R-squared,
the analysis of residuals or hypothesis testing. For checking statistical significance
an F-test (for the fit), and t-tests (for individual parameters) are recommended.
– Shapiro–Wilk’s test utilizes the null hypothesis principle to check whether a sample
came from a normally distributed population.
– Spearman’s rank correlation coefficient is a non-parametric test used to measure
the strength of association between two variables where the value r = 1 means
a perfect positive correlation and r = −1 a perfect negative correlation. It can be
applied not only to data with normal distribution, but also to unevenly distributed
data. It is an additional possibility when ANOVA and similar probes fail due to
normality and homogeneity issues.
– Student’s t-test is used to determine if two sets of data are significantly different
from each other, and is most commonly used when the test statistic would follow
a normal distribution.
 Data evaluation and interpretation in environmental toxicology 489

Some statistics-related terms are defined as follow (Berkeley, 2014; Doncaster &
Davey, 2007; OECD, 2006; Statistics, 2014; Upton & Cook, 2014; Wikipedia, 2014):

– Accuracy is a measure of how close the estimate is to the (unknown) true value of
a parameter.
– Precision is a measure of the amount of variability in the estimate. This estimate
is quantified by the standard error or the confidence interval. Precision can be
increased by increasing the number of samples and by decreasing experimental
variation.
– Confidence interval is an interval of parameter values that covers the true value
of the estimated parameter with a certain % of confidence. In environmental
toxicology 90% or 95% confidence level is typically used.
– Correlation or correlation coefficient, a numeric measure of the strength of linear
relationship between two random variables, e.g. the Pearson product-moment
correlation coefficient. Independent variables have a correlation of 0.
– Data types may be quantal (binary), continuous or discrete. Quantal data are
recorded as yes/present or no/absent (also denoted by all or nothing), or dead
or alive when lethality is the end point in toxicology. Quantal data can change
between 0 and the total number of test organisms. Continuous data can take any
value in an open interval, e.g. size of the organism, body weight and activity.
Continuous data should be measured and usually have a dimension. The range in
practice can be specified. Discrete data (to be counted such as number of certain
developmental failures, number of heart rate, etc.) may be nominal, ordinal or
interval. Categorical data – data sorted or divided into categories – are always
nominal. If a variable can take on any value between two specified values, it is a
continuous variable and the values follow a continuous distribution. However, if
the value can only take on a finite number of values, the values follow a discrete
(Bernoulli, binomial or Poisson) distribution.
– Dependent variable is substituted by response variable (as the pair of an explana-
tory variable), regressand, measured variable, explained variable, outcome
variable, experimental variable or output variable.
– Independent variable in statistics is also named as a predictor variable, regressor,
controlled variable, explanatory variable, exposure variable, risk factor, input vari-
able. Explanatory variable is preferred when the quantities treated as “independent
variables’’ may not be statistically independent.
– Null hypothesis as a general statement or default position refers to no relationship
between two measured phenomena. In toxicology it means that the treated sample
does not differ from the control. Rejecting null hypothesis is equal to concluding
that there is a relationship between two phenomena or a potential treatment has
a measurable effect. Both Type I and Type II error (see below) may occur.
– Outliers are inconsistent or questionable data points, out from the general trends.
Statistical evaluation of the data is recommended to be executed both with and
without the outliers.
– Parameter i.e. statistical parameter can be a population parameter, a distribution
parameter, an unobserved parameter and is often a quantity to be estimated.
– Non-parametric statistics and methods are not based on parameterized (by
mean or variance) probability distributions, so non-parametric methods make no
490 Engineering Tools for Environmental Risk Management – 2

assumptions about the probability distributions of the variables being assessed.

A non-parametric model is based on weak assumptions. This model is chosen
when the assessor is not willing to assume any distribution of the parameters and
it is based on the rank order of the observations and used mainly to test the null
hypothesis. It is mainly used for quantal and discrete data in toxicology. With the
application of non-parametric methods the problem of failing the prerequisites of
the chosen probe can be excluded.
– Parametric statistics means that data have come from a type of probability distri-
bution and make inferences about the parameters of the distribution. The model is
fully specified, except the value of the parameter. Most of the statistical methods
are parametric, and parametric methods are easier to handle than non-parametric
ones. For parametric methods the population is generally known as approxi-
mately normal. Parametric methods are primarily applied to continuous data in
toxicology.
– Population in statistics is a complete set of items that share at least one property
in common that is the subject of a statistical analysis.
– Randomization ensures statistical independence among observations.
– Statistical significance is the low probability that an observed effect would have
occurred due to chance.
– Type I error in statistics means false positives, occurring when the null hypothesis
is the truth but the hypothesis test results in a rejection of the null hypothesis in
favor of the alternative hypothesis. The probability of the occurrence of a Type I
error is referred to as α and generally specified at 0.05, or 5%.
– Type II errors are false negatives occurring when the alternative hypothesis is true
but the test fails to reject the null hypothesis (insufficient evidence to support the
alternative hypothesis). The probability of making a Type II error is referred to as
β (1–power).
– Power is the probability of rejecting the null hypothesis (H0 ) in favor of the alter-
native hypothesis (HA ), given that the alternative hypothesis is the true one. The
power of a test varies with sample size, with the variance of the measured response
and the size of the detectable effect. Power to detect differences can be increased
by increasing the sample size and reducing variation in the measured responses
(minimize experimental residual error) and using powerful statistics.

9.2 Statistical evaluation and analysis in environmental

toxicology
Evaluation of the measured response on a minimum of two or a series of concentrations
or doses may apply hypothesis testing, regression models or biology-based meth-
ods to the determination of the targeted test end points of traditional environmental
toxicology such as I%, ECx , (BMD), LOEC and NOEC.
Three main statistical methods will be discussed in detail in this chapter:

– hypothesis testing,
– concentration–response modeling with regression methods and
– biology-based methods.
 Data evaluation and interpretation in environmental toxicology 491

The various statistics and statistical analyses include general tasks and rules. The
toxicology-related steps are summarized in the following:

– Experimental design of the independent variables such as the concentration of

the substance to test, the duration of exposure, number of replicates to control
experimental variation, times of observation, etc. The goal of the test can be to
determine I%, ECx , LOEC and NOEC, and according to these test end points the
study design will differ in demands. ECx needs sufficient number of concentrations
for good curve fitting, NOEC needs warranted statistical power.
– Randomization ensures independence of errors from the statistical point of view.
When carrying out a test, randomization may include experimental materials,
test conditions, test organisms, treatments, choosing the place of containers and,
finally, measuring the responses (e.g. the measurement order of the units).
– Replication works against noise and increases statistical power in hypothesis
testing and confidence limits of parameter estimates.
– Multiple controls are included in the experimental design in those cases when the
chemical substance to be tested is not water soluble and a solvent/vehicle should
be used for its solubilization. In this case two controls belong to the study design:
one without and another one with solvent. If the two do not differ, the results can
be averaged and used as one control. If the difference is statistically significant,
the solvent-free control should be eliminated from the evaluation. General rule is
not to use solvent/vehicles if possible.
– Process of data analysis always follows the same pattern:

◦ Data inspection and elimination of outliers.

◦ Data inspection and assumptions about the general pattern of the scatter of
data to decide if variance is homogeneous or heterogeneous. Heterogeneity
of variances may be eliminated by the right transformation, which is able to
approximate/convert data to normal distribution.
◦ Transformation of data aims at stabilizing the variance so that the stan-
dard ANOVA tests or regression analysis allow for application. Non-normal
distribution can be transformed e.g. by Box-Cox transformation.
◦ Parametric and non-parametric methods: if the scatter is symmetric and homo-
geneous after transformation, parametric methods based on normality can
be used for the analysis. If the normality is violated, generalized lineariza-
tion model (GLM) or non-parametric method based on rank order of the
observation should be used.
◦ Quantal and continuous data should be distinguished. Quantal data are
categorical data – in toxicology the end points of mortality/survival or immo-
bilization are such – denoting the facts of dead or alive as well as mobile or
immobile with a yes or no characterization. Continuous test end points such
as weight, growth, activities, light emission, etc. are based on responses taking
values on a continuous scale, e.g. time-scale. The values can be monotonic or
non-monotonic. The statistical method should be selected according to these
data characteristics as shown later in Table 9.10.
◦ Pre-treatment of data other than transformation for model fitting: e.g. logit
and probit transformation, which should be applied with caution, due to the
492 Engineering Tools for Environmental Risk Management – 2

problem of the logarithm of a zero concentration. To apply a very small value

instead of zero is not recommended.
◦ Model fitting follows a general principle: the model contains specific param-
eters, and the goal of data analysis is to estimate these parameters. The
parameters are estimated by fitting the model to the data.
◦ Model checking and validation covers the evaluation of the appropriateness
of the fitted model, controlling if the data indeed comply with the model
assumptions. It is done by evaluation of residuals for checking if the variances
are homogenous, if the deviations from the fitted model are systematic, and if
the experimental factors deviate or not.
◦ Reporting the results in a statistical analysis of toxicological results should
cover not only the ECx and NOEC values and the summary report of the
statistics, but all details which are critical and may influence the results: start-
ing with the experimental design, the tests used for hypothesis validation and
model fitting. A detailed list of the required information in the report will be
given at the end of the chapter on statistical methods.

9.3 Hypothesis testing

Hypothesis testing is the statistical evaluation of the hypothesis on the distribution of
variables. It is also called confirmatory data analysis: checking if the hypothesis was
correct. It is a method for identifying statistically significant study results which are
predicted as unlikely to have occurred by chance alone. Significance is judged according
to a pre-determined threshold probability. It is based on distinguishing null hypoth-
esis (i.e. no difference between control and treated) and the alternative hypothesis
(the treated significantly differs from the control). Hypothesis testing includes several
methods which should be selected from among the most suitable ones for the concept
of the study to be evaluated. Parametric, sometimes non-parametric, one-sided and
multiple comparison tests are generally recommended for the evaluation of toxicity
study results. Multiple comparisons refer to simultaneous statistical inferences e.g. in
the case of Dunnett’s test (1955) or the Williams method (1971, 1972) for comparing
a number of treatments to a single control group.
Multiple comparison procedures are commonly used in an analysis of variance
after obtaining a significant test result. A significant ANOVA result suggests rejecting
the global null hypothesis that the means are the same across the groups being com-
pared. Multiple comparison procedures determine the means differing. A one-way
ANOVA involves pair-wise comparisons: having 4 treatments including a control, the
number of these pair-wise comparisons is 4*3/2; having n groups, the combination of
treatments gives n*(n–1)/2 pairs.
Non-parametric methods can also be used for the evaluation of concentration–
response data, e.g. the Kruskal–Wallis test (1952) or the Jonckheere–Terpstra test
(Jonckheere, 1954; Terpstra, 1952), which are non-parametric alternatives to ANOVA.
Multiple comparisons can be done using pair-wise comparisons and using a correction –
for example a Bonferroni correction (Abdi, 2007) – to determine if the post-hoc
comparison tests are significant.
Post-hoc comparison tests are applied in the second step of ANOVA or MANOVA,
after the null-hypothesis has been rejected. The testing procedures may be the
 Data evaluation and interpretation in environmental toxicology 493

Bonferroni adjustment or the Dunn test. When the hypothesis has been rejected, post
hoc is still able to find (otherwise existing and visible) associations. This is possible
because ANOVA is sometimes too stringent for biological data. A post-hoc test gives
one more chance for clarification and objective evaluation.
ANOVA has no precise definition because it is the combination of ideas, serving
very different purposes and adapted to the analyses of a wide variety of experimen-
tal designs, for example for prospective evaluation without knowledge of effect sizes
(Doncaster et al., 2013).
Multiple comparisons ANOVA F-test in one-way analysis of variance is the
standard statistical tool to determine those concentrations which exert an effect sig-
nificantly different from the untreated control. The null hypothesis is that the means
of the responses on the effect of different concentrations are the same. The ANOVA
F-test is for assessing whether any of the concentrations caused an effect on average
superior, or inferior to the others versus the null hypothesis. F-test is able to perform
multiple comparisons instead of the comparison of the responses on single concentra-
tions to each other, using several t-tests. When only two groups are evaluated by a
one-way ANOVA F-test, F = t 2 where t is Student’s t statistic. The definition of F is:
F = between-group variance/within-group variance.
Considering the concentration–response curve, LOEC, EC20 and EC50 are signif-
icantly different from the control, and NOEC is the same as the control within the
confidence interval.
Hypothesis testing has many different uses in ecotoxicology, ranging from detect-
ing whether there is a significant difference in the measured response between the
control and a given concentration to establishing a LOEC or NOEC value. In the
OECD guidelines for testing the effect of pure chemicals and determination of NOEC,
hypothesis testing is the recommended priority method (OECD, 2006).
Despite being the main statistical tool, hypothesis testing may have significant
limitations in toxicology, e.g. it does not consider mechanisms of the toxicant and the
biology of the organism. If the goal is the determination of a NOEC, one will face
further shortcomings:

– No confidence interval can be assessed for NOEC since it does not estimate a
model parameter.
– NOEC is not a tested but a hypothetical highest concentration with no significant
effect; any tested concentrations may be different to NOEC.
– In the case of low statistical power the biologically important differences between
the control and treatment groups may not be identified as significantly different.
If the power is high, it may occur that biologically unimportant differences are
found to be statistically significantly different.
9.3.1 Hypothesis testing for the determination of NOEC
NOEC can be determined by using single-concentration (yes/no or pass/failed) toxicity
test results or multi-concentration test results.

– The steps for the statistical evaluation of single-concentration test results are:
◦ transformation of response data to get the proportion survived (log, square
root, arc sine transformations);
494 Engineering Tools for Environmental Risk Management – 2

◦ testing normality assumption (e.g. Shapiro-Wilk’s test);

◦ testing the homogeneity of variance (F-test) and a t-test for significance. In the
case of failure (unequal variance) a modified t-test should be used;
◦ is the difference compared to control significant (e.g. in survival)? Yes or no?
– The steps for the statistical evaluation of multi-concentration test results are:
◦ transformation of data to proportion survived (log, square root, arc sine
transformations);
◦ testing normality assumption (e.g. Shapiro-Wilk’s test);
◦ testing the homogeneity of variance (e.g. Bartlett’s test);
• yes: t-test with Bonferroni adjustment (equal number of replicates) or
Dunnett’s test (not equal number of replicates),
• no: Steels many-one (equal number of replicates) or Wilcoxon rank sum
tests (non-equal number of replicates).
◦ NOEC estimation.

The OECD (2006) guideline gives detailed explanation and instructions for the
experimental design and statistical evaluation of toxicity studies and for the selection of
the proper statistical method. The scope of the guideline is “restricted’’ to the testing
of pure chemicals by standard test methods, so the user of the guidance document
cannot provide a recipe for every type of experiment, yet the guideline is very useful
for understanding the statistics, statistical analyses, and the importance of consistency
in statistics as well as the design of toxicity measuring experiments. Hypothesis testing
in toxicology is summarized below based on the OECD guideline.
The NOEC is defined as the highest test concentration without significant effect
below the lowest concentration that did result in a significant effect in a toxicity
study. The hypothesis that is tested when determining the NOEC reflects the question:
“Which is the concentration that is likely to have no effect on the test organism?’’ The
true answer depends on several test-specific conditions; some typical ones are listed:

– Has solvent been used? Have both solvent and non-solvent controls been tested?
– Is the study a dose–response type test?
– Is the dose–response function monotonic?
– Are there more than two concentrations tested?
– Are data measured normally distributed and homogeneous?

Having all this information one can choose the proper statistical method.
The one-sided hypothesis – also called one-tailed test – is appropriate when NOEC
is a concern in one direction only:

– µ0 > µi , where µ0 = the mean of the control; µi = the mean of the treated samples
– Null hypothesis of H0 : µ0 = µ1 = µ2 = · · · = µk
– Alternative hypothesis of H1 : µ0 > µi , for at least one i where
◦ µ0 denotes the mean of the control
◦ µi is the mean of the test populations, and i = 0, 1, 2, 3, . . . , k.

A two-sided form of the hypothesis symbolized by µ0

= µi requires a two-sided
trend or pair-wise test. In toxicity studies the model may assume monotonicity because
 Data evaluation and interpretation in environmental toxicology 495

the treatment group only differs in the exposure concentration/dose from the control,
and growing exposure will tend to increase the effect, which shows up as an increase
or a decrease in the measured end point:

– µ0 ≥ µ1 ≥ µ2 ≥ µ3 ≥ · · · ≥ µk
– Null hypothesis: H0∗ : µ0 = µ1 = µ2 = · · · = µk
– Alternative hypothesis: H1∗ : µ0 ≥ µ1 ≥ µ2 ≥ µ3 ≥ · · · ≥ µk , with µ0 > µk .

The trend test is recommended for dose–response experiments where a monotonic

response is expected and when data distribution is normal and the variance is homoge-
neous. If the one-sided approach also fulfils the requirement of the assessor, it should
be preferred, given that the statistical tests of a one-sided hypothesis are more powerful
than tests of the two-sided hypothesis.
Comparing single-step (pair-wise comparisons) and step-down trend tests to deter-
mine the NOEC, the step-down trend test should be given priority if monotonicity is
biologically justifiable: e.g. in the case of plant tests hormesis1 may appear i.e. positive
effect of the toxicants at low concentration, so the response will not be monotonic.
Monotonicity can be accepted as a rule or tested formally: in the latter case either
monotonicity or non-monotonicity can be tested.
The power of a toxicity test can be calculated if the size of the effect to be detected,
the variability of the end point measured, the number of treatment groups and the
number of replicates in each treatment group are known. Power is important for the
selection of a process, but it is not the only condition. The main goal should be finding
the most appropriate method for the data and the end results: statistically insignificant
concentrations, which are large enough to cause damage, must not be overlooked and,
on the other hand, minor concentrations must not be characterized as statistically
significant. Statistical significance should agree with biological significance or with
regulatory thresholds.
Experimental design and evaluation is based on the initial variance estimates
which are calculated based on historical control data. Post-hoc power should be com-
pared to the design power and reported. Other important experimental factors are
the number and spacing of exposure levels (concentrations, doses), the number of
test subjects in each group, and the type of subgroups if adequate. These factors
will determine the power which should be designed to be able to identify the bio-
logically important effect levels. Substance concentration is the most important issue
from this point of view. It should be close to the NOEC when the aim is NOEC
determination. The number of the organisms tested may also be crucial. According
to the allocation rule of Dunnett (1955), the number of organisms in the control
group (n0 ) should be calculated by the square-root rule to get a test design optimiz-
ing the power of Dunnett’s test. By this rule, the value of n0 is the solution of the
equation √ √
N = k n + n k, and n0 = N − kn → n0 = n k, where

– N = the total number of subjects tested;

– k = the total number of treatments;

1
Adaptive response of cells and organisms to a moderate (usually intermittent) stress.
496 Engineering Tools for Environmental Risk Management – 2

Table 9.10 Statistical methods for hypothesis testing in environmental toxicology (OECD, 2006).

For quantal data Parametric methods Non-parametric methods

Single-step (pair-wise) Dunnet Mann-Whitney with Bonferroni-Holm

adjustments
Poisson comparisons Chi-squared with Bonferroni-Holm
adjustment
Steel’s Many-to-One
Fisher’s exact test with
Bonferroni-Holm adjustment
Step-down (trend based) Poisson Trend Williams Cochran-Armitage
Bartholomew Jonckheere-Terpstra test
Welch Mantel-Haenszel
Brown-Forsythe
Sequences of linear contrasts

For continuous data Parametric methods Non-parametric methods

Single-step (pair-wise) Dunnett Dunn

Tamhane-Dunnett Mann-Whitney with Bonferroni
correction
Step-down (trend based) Williams Jonckheere-Terpstra
Bartholomew Shirley
Welch trend
Brown-Forsythe trend
Sequences of linear contrasts

– n = equal number of subjects in all treatment groups;

– n0 = number of subjects in the control group.

The argument for having more tested subjects in the control group is that the same
control is applied for comparison in every single treatment group.
Covariates that influence the conclusion should be included in the evaluation,
and the analysis should be adjusted to these covariates such as age and size or other
characteristics of the test organism at the beginning of the test. For continuous,
normally distributed responses with homogeneous variances, analysis of covariance
(ANCOVA) is well developed. For continuous responses that do not meet the normality
or homogeneity requirements, non-parametric ANCOVA is available.
In Table 9.10 some OECD (2006) recommended statistical methods are given for
hypothesis testing of toxicity results as the combination of stepwise and trend-based,
as well as parametric and non-parametric cases, with quantal and continuous data
types distinguished.
The problem of outliers can be solved by a simple procedure: the first step is visual
observation of dose–response data. Experience of professionals is an adequate basis
for a personal judgment and decision making about outliers that should be excluded
from the evaluation. For continuous data, the individual responses can be plotted
in addition to the group means as a function of dose. For quantal data, the observed
frequencies of response as a function of dose may be conclusive. Several outlier rules can
be used for identifying undesirable data such as Tukey’s rule (Tukey, 1977). It is worth
 Data evaluation and interpretation in environmental toxicology 497

Table 9.11 Statistics-related information recommended by OECD (2006) to be included in the toxicity
study report.

NOEC: quantal study result NOEC: continuous study result

Description of the statistical methods used

Test end point assessed Test end point assessed
Number of test groups Number of test groups
Number of subgroups Number of subgroups within each group/way of
within each group/way of handling handling
Identification of the experimental unit Identification of the experimental unit
Nominal and measured concentrations Nominal and measured concentrations (if available)
(if available) for each test group for each test group
Number of those exposed in each The concentration/dose metric used (actual, log,
treatment group equally spaced scores)
Number of those affected in each Number of those exposed in each treatment group
treatment group (or subgroup)
Proportion affected in each treatment Group means (median, if a non-parametric test was
group used) and standard deviations
Confidence interval for the percent Confidence interval for the percentage effect at the
effect at the NOEC, provided that the NOEC, provided that the basis for the calculation
basis for the calculation is consistent is consistent with the distribution of observed
with the distribution of observed responses responses
P value for test of homogeneity if performed The NOEC value
Name of the statistical method used to P value at the LOEC (if applicable)
determine the NOEC
The concentration/dose metric used (actual, Results of power analysis
log, equally spaced scores)
The NOEC Plot of response versus concentration
P value at the LOEC
Design power of the test to detect an effect of
biological importance based on historical
control background and variability

distinguishing between outliers relating to entire treatment groups and individuals

because the first one is typical when experimental factors are different in the group,
which should be excluded by proper design and randomization.
9.3.2 Reporting hypothesis testing
The information required in the study report by the OECD (2006) methodology for
testing pure chemicals is summarized as an example in Table 9.11, both for quantal
and continuous data.

9.4 Regression and regression analysis

In regression analysis the estimation target is a function, a relationship between a
dependent variable and one or more independent variables. When using standardized
test methods, regression analysis helps to understand how the response – measured
end point – changes when the concentration or dose of the contaminant is varied (in
standardized tests, duration and uncontrolled interactions are excluded from the inde-
pendent variables). The variation of the dependent variable in the regression function
can be described by a probability distribution.
498 Engineering Tools for Environmental Risk Management – 2

The most frequently applied regression models are as follows:

– Linear interpolation of means is the estimation of an intermediate value of the inde-

pendent variable of a function. This may be achieved by curve fitting or regression
analysis.
– Linear regression is modeling the relationship between a scalar dependent variable
and one or more explanatory variables. In linear regression, data are modeled using
linear predictor functions, and unknown model parameters are estimated from the
data.
– Polynomial regression is the generalization of linear regression in which the rela-
tionship between the independent variable and the dependent variable is modeled
by a polynomial.
– Logit or logistic regression is a type of probabilistic statistical classification model
for binary response variables. The probabilities describing the possible outcomes
of a single trial are modeled, as a function of the explanatory (predictor) variables
using a logistic function.
– Probit model is a binary response model, employing a probit link function and uses
the standard maximum likelihood procedure. It treats the same set of problems as
logistic regression does.
– Spearman-Karber is a non-parametric method, which does not require the assump-
tion of normality, compared to Probit. It is known as a method, resulting in close
estimates to EC50 even in the case of small datasets. It is only applicable for EC50 ,
and not for other end points.
– Weibull is a proportional hazard model which means that the effect of an inde-
pendent variable on the hazard rate is assumed to be multiplicative. It is mainly
used for modeling survival data in toxicology.

9.4.1 The use of regression and regression analysis in toxicology

Regression models are especially useful in concentration/dose–response curve fitting
and in the determination of ECx . F-test is suitable for studying the hypothesis that a
proposed regression model fits the data well based on the comparison of two fits and
the determination of the significantly better fit to the measured data. The larger the
F-statistic, the more useful the model is.
The OECD (2006) guidance on statistical tools describes the theory and practice
as well as the steps of curve fitting to determine Cx , the concentration causing x%
effect in the measured end point, the latter typically being lethality or inhibition of any
biological function. Several suitable functions are able to describe measured data prop-
erly, but there are favored mathematical models accepted widely and used frequently.
Regression methods cover the analyses for quantal data and continuous data as well as
parametric approaches (when a specific underlying distribution is assumed) and some
non-parametric ones (not typical in toxicity). They are for estimating the arbitrary x%
effect in the biological response variable and the associated confidence bounds (limits).
The concentration/dose–response curve can be symbolized by the function of y = f
(x), where

– x is the independent variable: concentration or dose, and

– y is the function of x, i.e. the dependent variable or response.
 Data evaluation and interpretation in environmental toxicology 499

Other independent variables are not included in a standardized test with strict
conditions and a one-off evaluation. Otherwise duration of the exposure is typically
an important determinant: even a simple dose results in a growing cumulative value
when measuring the response (e.g. the number of responding subjects) in several time
points.
Dose–response data are handled by regression statistics as a whole, not point by
point. The variables of the function are determined by the measured data. The number
of parameters is proportional to the flexibility of the model: the larger the number
of parameters, the more flexible the model is. On the other hand, rationality of the
number of the parameters says that inclusion of an additional parameter into the model
is recommended only if it leads to a significantly better fit.
The model can be fitted to the data in various ways. The sum of squares method
(SS), for example, optimizes the fit by minimizing the sum of squared residuals. Resid-
uals in this context are the distances between the data and the model. The fit may
involve the maximization of the likelihood based on the assumed distribution of data:
normal or log-normal for continuous data, binomial distribution for quantal data and
Poisson distribution for count data.
The term power of the statistics is only used in hypothesis testing, in regression
models the confidence interval is used for characterizing the goodness of the model.
Sample size and variation in the response are important characteristics: small sample
sizes and large variability in the response within groups will increase the width of
the confidence interval of the parameters, and the fitted model may not reflect the
true concentration–response relationship. The number of replicates, the location and
number of concentrations can be increased for better statistics (smaller confidence
interval). The design of the experiment depends on the x value of Cx because C90 , C50
and C05 require different designs, mainly in the number and spacing of concentrations.
Basic assumptions in regression for toxicology are as follows:

– In the case of continuous data, the number of dose groups showing significantly
different response levels is – by experience – a minimum of four including the
control. It can be greater than four in practice;
– In the case of quantal data, the minimum number of partial responses should
be two: almost complete survival and complete mortality. The test design should
exclude the inappropriate extrapolation for determining the desired ECx ;
– A monotonic concentration–response relationship;
– The fitted curve is close to the true concentration–response relationship;
– There are weak assumptions about the mechanisms of the toxicant or the biology
of the organism and interaction between more stressors and more species.

Some further limitations of concentration–response modeling should be

mentioned:

– Extrapolated ECx values (outside the measured concentration range) are rather
uncertain;
– Too large gaps between consecutive response levels lead to uncertain interpolation
and make the fitting uncertain (allowing many different models to be fitted).
500 Engineering Tools for Environmental Risk Management – 2

Quantal and continuous data are evaluated along the same steps:

– Fitting various models;

– Evaluating the fit in comparison;
– If more or all models fit well and result in the same ECx , the determination of ECx
has come to an end. If some models do not fit, more additional models should be
applied and outliers and precision of the test-method should be checked.
– The goodness of the regression is investigated through the confidence intervals
among ECx estimates and the lowest confidence bounds are chosen.

9.4.2 Evaluation of quantal data

As OECD (2006) describes in its guideline for standardized tests, the choice of the
model is crucial, and is governed by the data. For quantal data the dose–response
function ranges between 0 and 1 (0% and 100%), the response normally is mono-
tone. Cumulative distribution functions (normal, logistic, Weibull) are the primary
candidates for dose-response modeling. Cumulative distribution functions are pop-
ular because they can be interpreted as individual tolerance distributions within the
population. Plotting the tolerance distribution cumulatively results in the quantal dose–
response relationship. A predicted response of 20% lethality at a concentration of
100 mg/L can be interpreted as 20% of the individuals having a tolerance lower than
100 mg/L. Tolerance distribution of the individuals in the population is equal to the
probability of a response from each individual, which means that the chance of each
individual is 1:5 to give response to the 100 mg/L concentration.
The dose–response model for quantal data is a function of the concentration
y = f(x) where y is the true quantal response and x is the concentration. The func-
tion f(x) cannot strictly equal zero at concentration zero, so the model should include
a background incidence parameter (a):
y = f(x) = a + (1 − a)g(x), where

– a denotes the true background probability of response;

– g(x) is a function increasing from 0 to 1 when x increases from zero to infinity.
– In this formulation the response at infinite concentration is g(x) = 1.

9.4.3 Choice of the models

– The probit model is the cumulative normal distribution function, usually applied
to the log-concentrations, implying that a lognormal tolerance distribution is
assumed.
– y = a + (1 − a) ∗ pnorm(b ∗ log 10(x/EC50 ))
– The logit model is the cumulative logistic distribution function. The logit model
is also applied usually to the log-concentrations.
– y = a + (1 − a)/1 + exp(b ∗ log10(EC50 /x))
– Both the probit and the logit model have two parameters: the EC50 and the slope
(b), all other ECx values (the quantal response of y) are determined based on these
two parameters. ECx should be adjusted to the background value. When using
the additional risk model, the background value (let say 3%) will be added to
 Data evaluation and interpretation in environmental toxicology 501

x%, when determining ECx (i.e. EC20 should be read at 20 + 3 = 23%) from the
dose–response curve.
– The Weibull distribution is not necessarily symmetrical, and is usually applied to
the concentrations themselves and not their logs. EC50 is related to two parameters
in Weibull: the location and the slope.

y = a + (1 − a) ∗ (1 − exp(−(x/b)c))

– Simplified multi-stage models are often used for describing tumor dose–response
data.

9.4.4 Evaluation of continuous data

A continuous response has a certain amount of scatter, depending on the homogeneity
of the treatment group. This scatter follows a certain distribution such as normal,
lognormal or Poisson distribution.
ECx in continuous responses relates to the change in the degree of the effect. This
interpretation differs from the one for quantal responses where ECx relates to a change
in response rate, such as growth rate, respiration rate, enzyme activity rate and any
kind and any percentage of inhibition.
The function is formally the same as for quantal data
y = f(x), where

– y covers here continuous data and

– x% = 100 ((y × ECx /y0 ) − 1)% the degree of effect.

9.4.5 Choice of the models

The observed/measured dose–response outcomes are smoothened by the regression
model and analyzed for estimating the most correct ECx and assessing the confidence
intervals. As the regression model has no meaning in biological sense (it has no bio-
logical interpretation), and the selection of the available models is wide, the choice of
model is largely arbitrary; the most popular families of models are as follows:

– Linear regression models may be nonlinear in respect to the independent variable,

they can be higher-order polynomial or quadratic. Nested models can easily be
modified regarding the incorporated parameters or turning the linear model into
quadratic and evaluate the fit of these modified models (applying F-test) until
getting the best fit. The function is:
◦ y = a + bx or y = a + bx + cx2 or y = a + bx + cx2 + dx3 ,
– Threshold models contain an additional parameter reflecting a dose threshold,
below which the change in the end point is zero. The function describing this
situation is
◦ y=a if x < c, where c = the threshold concentration and
◦ y = a + f(x − c) if x > c − for any function
◦ y = a + b(x − c) if x > c − for linear function.
502 Engineering Tools for Environmental Risk Management – 2

– Additive vs. multiplicative models are supposed to solve the problem of back-
ground noise, meaning that the response is not zero when the exposition is zero.
The background level of a can be incorporated into the model as an additive or
as a multiplicative value. Priority is given to the latter one because in addition to
the logical solution that the additional value should be a percentage similar to the
response, it is very useful in the case of testing more populations with different
background levels. The two alternative formulae are:
◦ f(x) = a + g(x) − additive way of incorporating background level;
◦ f(x) = a × g(x) – multiplicative way of incorporating background level.
– Models based on “quantal’’ models adjust continuous data applicable for quantal
models.
– Nested nonlinear models were proposed by Slob (2002) including 5 models:
◦ model 1 y = a a > 0;
◦ model 2 y = a × exp(x/b) a > 0;
◦ model 3 y = a × exp(±(x/b) ∗ d) a > 0, b > 0, d ≥ 1;
◦ model 4 y = a × [c – (c–1) exp(–x/b)] a > 0, b > 0, c > 0;
◦ model 5 y = a × [c – (c–1) exp(–(x/b) ∗ d )] a > 0, b > 0, c > 0, d ≥ 1 where
• y = any continuous end point
• x = concentration or dose.
– Hill model dates back to 1910 and was elaborated for receptor binding and enzyme
kinetics. The value of c=1 in the following function results in the Michealis-Menten
equation.
◦ y = a × xc /b + xc , where
◦ c is the Hill parameter
– The Michaelis-Menten equation has a widely known form:
◦ v = Vmax × [S]/Km + [S], where
• v = reaction rate
• Vmax = maximum rate
• [S] = substrate concentration
• Km = is the substrate concentration, at which the reaction rate is half of
the maximum.

The model is fitted to the dose–response data and the parameters estimated by
applying the suitable software. It is necessary to be aware of the assumptions under-
lying the fit algorithm. An iterative algorithm tries to find better parameter values in
an evaluation process if the fit can be improved by changing the parameter values.
The solution is to find the maximum likelihood or minimum SS in the function. The
iteration stops when the software has found a clear maximum in the log-likelihood
function, or in a minimum in the SS.
Several assumptions are used in choosing and fitting the model such as exper-
imental design ensuring randomized test conditions, no dependence between tested
organisms, no systematic difference between dose groups, exactly known con-
centration/dose, normal distribution of the data (before or after transformation),
 Data evaluation and interpretation in environmental toxicology 503

homogeneity of variance and goodness of the fit. These assumptions should be tested
by normality tests, goodness of fit tests, etc. and a confidence interval should be
determined. Confidence may be assessed by:

– The delta method: plus or minus twice the standard error as estimated by the
second derivative of the likelihood function;
– Based on the profile of the log-likelihood function, using the Chi-square approxi-
mation of the log-likelihood;
– Bootstrap methods;
– Bayesian methods, having preliminary knowledge on the range of the parameter
value(s).

When can the fitted model be accepted? The fit is good when the fitted model
incorporates all the measurement points and gives a true estimate for all not measured
responses to all not applied concentrations/doses between two measurement points.
Statistical evaluation should cover both: the measured and the estimated responses.
The best way to check the goodness of fit is the visual check. Absolute and relative
tests can simultaneously be executed to characterize the goodness of fit. Absolute tests
are able to quantify the deviation of data from the model curve; relative tests compare
the different test results with each other. The most comforting answer is when different
models (e.g. logit, probit and Weibull) result in the same or very close parameters and
the same ECx values. Good-quality measured data make it suitable to apply different
models, but excessively high uncertainties and imprecise test implementation, lack of
randomization or outlier exclusion etc. violate the initial assumptions and may cause
disagreement of the model with the data and the incorrect rejection of the model. But
the opposite may also happen: the goodness of fit looks perfect by standard testing
because the test execution is very precise. However, the data do not follow the chosen
model e.g. due to biological reasons.

9.4.6 Reporting regression statistics

Table 9.12 shows the summary of reporting regression statistics.

Table 9.12 Regression statistics-related information recommended by OECD (2006) to be included

in the toxicity study report.

Quantal data Continuous data

Test end point assessed Test end point assessed

Number of test groups Number of test groups
Number of subgroups within Number of subgroups within each group (if applicable)
each group (if applicable)
Identification of the Identification of the experimental unit
experimental unit
Nominal and measured Nominal and measured concentrations (if available) for each
concentrations (if available) test group
for each test group
Number exposed in each Number exposed in each treatment group (or subgroup
treatment group (or subgroup if appropriate)
if appropriate)

(continued)
504 Engineering Tools for Environmental Risk Management – 2

Table 9.12 Continued

Quantal data Continuous data

Number affected in each

treatment group (or subgroup
if appropriate)
Proportion affected in each Arithmetic group means and standard deviations, but
treatment group (or subgroup geometric group means and standard deviation if
if appropriate) lognormality was assumed
The dose metric used The dose metric used
The model function chosen for The model function chosen for deriving the EC
deriving the EC50 (ECx )
Plot of dose-response data with Plot of dose-response data with fitted model, including the
fitted model, including the point point estimates of the model parameters and the
estimates of the model parameters log-likelihood (or residual SS)
and the log-likelihood (or residual SS)
Fit criteria for other fitted models Fit criteria for other fitted models
The EC50 together with its The ECx (CED) together with its 90%-confidence interval
90%-confidence interval
If required: the ECx together with its
90%-confidence interval
Method used for deriving confidence Method used for deriving confidence intervals
intervals

9.5 A comparative study on statistical evaluation of

dose–response data
Statistical models are becoming more common with the spread of IT techniques and
their importance has greatly increased in toxicology. OECD recommended the appli-
cation of regression models for the evaluation of response vs. concentration/dose data
and also decided to phase out NOEC as a chronic end point being uncertain due to the
arbitrary selection of the lowest tested concentration and the difference between two
adjacent concentration points. The research group of Isnard et al. (2001) prepared
a comparative study on the statistical evaluation of 27 chronic ecotoxicity datasets
of algae, daphnia and fish. The same study results were evaluated by ANOVA-type
hypothesis testing (Dunnett, Williams and Jonkheere-Terpstra) and several differ-
ent regression models (linear interpolation, polynomial regression, logit, probit and
Weibull models). The confidence interval was estimated using the bootstrap resampling
technique. In addition to a general comparison their aim was to find the best regression
model and to substitute NOEC with an ECx close to NOEC, obviously EC05 .
The most important conclusions from the comparative study are:

– Different hypothesis testing methods lead to different results from the same dataset.
– Both EC50 and EC05 values were very close by linear interpolation, polynomial
regression and logit, in the case of good quality data and good fitting.
– Probit and Weibull models were excluded in an early stage of the study, proven
not to be useful in the evaluation of the selected datasets.
– Bootstrap-simulated confidence intervals were found better and more realistic than
asymptotic calculations.
 Data evaluation and interpretation in environmental toxicology 505

– The paired comparison of NOEC and ECx gave the result that there is no signif-
icant difference between NOEC and EC05 , allowing the pragmatic approach of
substituting NOEC with EC05 .
– Logit provided suitable fitting in most studied cases, but probit did not converge
in all cases.
– Experimental design and precise test implementation are crucial prerequisites.
– Fisher test is recommended for testing both ANOVA and regression models.

Kooijman (1993 and 1996) proposed a threshold model based on Dynamic Energy
Budget (DEB), giving directly a no-effect result, but it is rather complicated and not
validated yet. The DEB model looks useful also in biology based toxicology and
toxicokinetics (Kooijman et al., 2009).

9.6 Biology-based methods

Biology-based methods aim to explain not only the result itself, but the underlying
processes. Human toxicology focusing on toxicokinetics and reproduction (Kooijman,
1996) specifies a “response surface’’ which is a function of concentration and exposure
time and includes the chemistry and biology of the system. Several parameters deter-
mine the response surface. After estimating these parameters from data, the same
parameters can be used for calculating the time dependence of ECx , the effect of
changing concentrations, the effect on growing populations or other, more complex
changes in the field. Biology-based models enable to consider the time-dependence of
the internal concentration (increasing in time), or the changing concentration in the test
medium which directly determines the effect (Kooijman, 1983; Gerritsen, 1997; Péry
et al., 2002). The changes in the slope in time can be modeled by the response surface
of biology-based methods. Biology-based methods are able to include more datasets,
such as activity, lethality and the changes of internal concentration (accumulation in
and elimination from the body) in time. The effective concentration in complex orga-
nizations depends on the internal concentration, and the target parameter is assumed
to be linear to this. In single-cell microorganisms or very small organisms the internal
and external concentration (in the medium) can be considered equal.
Parameter estimation of biology-based methods apply Maximum Likelihood (ML)
tests e.g. the least squared deviation method in cases when the scatter is independently
normally distributed and has a constant variance. This means that the deviation is inde-
pendent for the different data sets such as various toxicity end points and the internal
concentrations. For different datasets a composite likelihood function is used that
contains all parameters for all models and can handle different types of distributions.
The profile likelihood function is used to obtain confidence intervals for parameters
such as the No-Effect Concentration (NEC). Biology-based methods use NEC as a free
parameter.
The eco-physiological model differentiates three steps:

– Change in the internal concentration: from the local environment to the concen-
tration in the test organism.
– Change in a physiological target parameter: from an internal concentration to a
hazard rate such as assimilation rate, specific maintenance rate, etc.
506 Engineering Tools for Environmental Risk Management – 2

– Change in an end point: the step from the hazard rate to a change in an end
point such as reproduction rate, total number of offspring during an exposure
period, etc.
The model applies three concentration ranges from where it is obvious that the
occurrence of the no-effect case is very common:
– Effects due to shortage: in this range the chemical may function as a stimulant;
– No-effect range: activities are seemingly independent of the concentration;
– Toxic effects: inhibition arises in this range not only for toxicants but also for
substances with a wide range of no effect, e.g. glucose.
The toxico-kinetic model is based on the accumulation flux, which is proportional
to the concentration in the external environment and the elimination flux, which is pro-
portional to the internal concentration (inside the organism). This result is a first-order
kinetic model. The growth of the organism during the experiment causes deviation
from first order kinetics. This deviation can be predicted and taken into consideration.
The Dynamic Energy Budget model (DEB) is not only based on the material fluxes
which are described by the external and the internal dynamic concentrations (result-
ing from biaccumulation and elimination), but also includes energy fluxes. The DEB
theory unifies the commonalities between organisms as prescribed by the implications
of energetics which link different levels of biological organization i.e. cells, organ-
isms and populations (DEB, 2014). The theory presents simple mechanistic rules that
describe the uptake and use of energy and nutrients (substrates, food, light) and the
consequences for the physiological organization throughout an organism’s life cycle,
including the relationships of energetics with aging and effects of toxicants. The model
of Kooijman (1993, 1996, 2010) introduces the factor of reserve (food is converted to
feces and reserve), and the reserve is used for maintenance and synthesis (energy) and
allocated to structures known as growth; to maturity as development and to gametes as
reproduction. The rate of the reserve use is the catabolic rate. The DEB model describes
the full material balance, which can be very useful in biology-based modeling.
The DEB model specifies how changes in one or more target parameters translate
into changes in a specified end point. Reproduction rates for example depend on age
(the first few offsprings contribute much more to population growth than later off-
springs). There is no need to study all ages of the test organism once its DEB parameters
are known. The use of the DEB models are detailed in the OECD Guideline (OECD,
2006), a short summary is given here.
– The DEB survival model: the effects on the survival probability of individuals
are specified via the hazard rate. The hazard rate (probability per time) is also
known as the instantaneous death rate. The hazard rate h(t) relates to the survival
probability q(t) as:
h(t) = −q(t)−1 d/dt q(t).

– The DEB body growth model allows for three routes affecting body growth: (i)
decrease in the assimilation rate; (ii) increase in the somatic maintenance costs; (iii)
increase in the specific costs for growth. In the case of fish growth, as an example,
the growth is described by the equation below (applied mainly in fisheries science):
Lt = L∞ − (L∞ − L0 )exp{−rb × t}, where
 Data evaluation and interpretation in environmental toxicology 507

◦ L(t) is the length at time t,

◦ L0 is the initial length,
◦ L∞ is the ultimate length (the asymptotic length at which growth is zero)
◦ rb is the von Bertalanffy growth rate.

Effects on growth are determined by an increase of the maintenance costs and by

a decrease of assimilation in the DEB model. This equation can be applied if, for
example, the fish follow a von Bertalanffy (1969) growth curve in the control and
the chemical substance follows first order kinetics. Further preconditions are that
the toxicokinetic parameters (assimilation, maintenance, cost for growth) should
increase linearly with the internal concentration and the concentration of the test
compound is constant.
– Reproduction allows indirect routes similar to those for growth (i) decreased assim-
ilation rate; (ii) increased maintenance rate; and (iii) increase in cost for growth. In
addition, direct routes are specified: (iv) an increase in the costs per offspring (an
effect on the transformation from reserves of the mother to that of the embryo)
and (v) death of early embryos which are not counted.
– Population growth is a complex process, but in the case of algal or duckweed
growth it can be simplified (under certain assumptions) and considered as growth
and division (one cell into two) in the context of the DEB model.

9.6.1 Parameters
– NEC, the no-effect concentration (internal concentration of the chemical sub-
stance), a 0% effect level at very long exposure times;
– Killing rate: effect on survival or tolerance concentration (external concentration
of the chemical substance);
– Elimination rate (a dynamic parameter);
– The hazard rate = hazard rate of the control + killing rate × (internal concentra-
tion/BCF – NEC);
– Stress value = 1/tolerance value × (internal concentration/BCF – NEC);
– Eco-physiological parameters: are test-species-specific values and should be mea-
sured in advance for the DEB model. These are for growth and reproduction:
rb = von Bertalanffy growth rate; L0 = initial body length; scaled length of puberty
and energy investment ratio. For population growth: inoculum size and specific
population growth rate.

The fitted data should be checked by using goodness-of-fit methods. The DEBtox
software includes these functions. Experimental design and the applied statistics should
always be harmonized. DEBtox has been designed for the analysis of the results of
specified OECD standard test methods: so, when using these ones, no harmonization
is necessary from the users’ point of view.
The IT tools necessary for the evaluation of the above-mentioned biology-based
tests are the DEBtox and DEBtool packages. The latest versions can be downloaded
free of charge from the electronic DEB laboratory (2014). The Hill parameters can
be estimated using IBM SPSS nonlinear regression software and others (see next
section).
508 Engineering Tools for Environmental Risk Management – 2

9.7 IT tools for statistical evaluation

The development of information technology made mathematical calculation easy and
eliminated the barrier of manual work and time requirement. Several pieces of soft-
ware are available as mathematical tools for statistical evaluation using hypothesis
testing or regression analysis. On the other hand, the wide selection of ready-made
statistical methods and software make the correct decision difficult. Except for some
routine cases advice from professionals is necessary when choosing the most appropri-
ate statistics. On-line support is also available, e.g. the website of UCLA (2014). These
support materials should not be considered as strict rules, but as general guidelines since
data can be analyzed in multiple ways and most of them may yield a correct answer.
These web-based decision support tools are categorized according to the nature of the
independent variables and the number and nature (nominal or categorical, ordinal,
interval) of the dependent variables. The type of data distribution also determines the
choice of statistical methods. The same statistical methods are available from different
sources in the form of free or paid software. Software packages are offered by the big
providers such as BMDP, SAS, SPSS, R, STATA, OriginLab, etc.
For education and information on statistics see Statistics (2014), The Institute for
Statistics Education. Statistics software information is also available from Statistics
(2014). A broad overview can be found on the website of StatPages (2014), providing
information on:

– General Packages: support a wide variety of statistical analyses;

– Subset Packages: deal with a specific area of analysis or a limited set of tests;
– Curve Fitting and Modeling: handling complex, nonlinear models and systems;
– Biostatistics and Epidemiology: especially useful in life sciences;
– Surveys, Testing and Measurement: especially useful in business and social
sciences;
– Excel Spreadsheets and Add-ins: recent version of Excel is needed (see later in
details);
– Programming Languages and Subroutine Libraries;
– Scripts and Macros: for scriptable packages such as SAS, SPSS, R, etc.;
– Other collections of links to free software.

GNU (2014) General Public License (GPL) applies to most of the Free Software
Foundation’s (2014) software and to any other program whose authors are committed
to using it. GNU gives its users complete freedom. Free Software Foundation is a
nonprofit organization with a world-wide mission to promote computer user freedom
and to defend the rights of all free software users.
Some well-known statistics tools and web-pages also useful in toxicology are
gathered and listed below in alphabetic order:
Benchmark Dose (BMD 2014) software, developed by US EPA is the IT support of
the benchmark dose approach for deriving a ‘Point of Departure’ for risk assessment.
It is a scientifically more advanced method compared to the No Observed Adverse
Effect Level (NOAEL). The BMD method pre-defines a specific effect, referred to
as the Benchmark Response (BMR) and estimates the dose (BMD) associated with
the specified effect. The BMD is estimated from the complete dose response dataset by
 Data evaluation and interpretation in environmental toxicology 509

fitting dose response models. Statistical uncertainties in the data are taken into account
by the confidence interval around the BMD, the lower limit of which (denoted by
BMDL) is the Point of Departure for deriving exposure limits. The software package is
suitable for dose-response analysis and deriving a BMDL from dose-response data. The
software is available from the Integrated Risk Information System (IRIS, 2014) website.
BMDP (2014) is a statistical package developed in 1965 at UCLA. Based on the
older BIMED program, developed in 1960 for biomedical applications, it used key-
word parameters in the input instead of fixed-format cards, so the letter P was added
to the letters BMD, although the name was later defined as being an abbreviation
for Biomedical Package. BMDP was originally distributed free of charge. It is now
marketed by Statistical Solutions. The software package of BMDP is distributed by
Statistical Solutions (2014).
IBM SPSS Statistics (2014) is a software package used for statistical analysis. Long
produced by SPSS Inc., it was acquired by IBM in 2009. The current versions (2014)
are officially named IBM SPSS Statistics which stands for Statistical Package for the
Social Sciences (SPSS) reflecting the original market, although the software is now
popular in other fields as well, including health sciences and marketing:

– Descriptive statistics: cross tabulation, frequencies, descriptives, descriptive ratio

statistics;
– Bivariate statistics: means, t-test, ANOVA, correlation (bivariate, partial, dis-
tances), non-parametric tests;
– Prediction for numerical outcomes: linear regression;
– Prediction for identifying groups: factor analysis, cluster analysis (two-step,
K-means, hierarchical), discriminant;
– Companion products in the same family are used for survey authoring and
deployment (IBM SPSS Data Collection), data mining (IBM SPSS Modeler).

JMP (2014) the Statistical Discovery software from SAS is an easy-to-use data
analysis and graphics tool. The statistical analysis is linked with interactive graphics,
in memory and on the desktop.
Microsoft Excel Add-ins (2014) provides several categories and a free trial version
is also available (XLSTAT, 2014). Statistical tools package is part of the latest EXCEL
version, but it is not included in the basic functions, but it should be downloaded
from Excel by using Options within the File menu, and selecting Add Ins and Analysis
ToolPak. Detailed information can be found on YouTube: How to Get Excel 2010
data analysis tool (Excel data analysis tool, 2014). A statistical functions list (Excel
statistical functions, 2014) and detailed description about the statistical analysis tools
is also available (Excel statistical analysis tools, 2014).
MINITAB (2014) is a general statistical and graphical analysis package. It can do
various general analyses including time series. An interactive Assistant helps through
every step of the analysis.
Origin (2014) is graphing and analysis software that includes regression and curve
fitting tools for linear, polynomial, and nonlinear curve fitting along with validation
and goodness-of-fit tests.
PROAST (2014) is a software package that has been developed by the Dutch
National Institute for Public Health and the Environment for the statistical analysis of
510 Engineering Tools for Environmental Risk Management – 2

dose–response or concentration–response data and nonlinear regression. It can be used

for (i) dose–response modeling, (ii) deriving a BMD in human risk assessment, and (iii)
deriving an effect concentration in ecotoxicological risk assessment. It is suitable for
an in depth analysis of a single dataset, but also for a quick (automated) analysis of
a whole series of response end points, which may be useful for analyzing complete
studies. It allows for comparing dose–responses among various subgroups, e.g. among
sexes, study durations or among replicate studies. PROAST not only indicates if the
various dose–response relationships differ among the subgroups, but gives information
if the difference is in the background response, in sensitivity to the chemical, or in
dose-response shape. The present version (38.9) has the possibility to run PROAST in
a user-friendly Graphical User Interface (GUI) for standard applications. The GUI was
developed in collaboration with the Health and Safety Laboratory (2014) in Buxton,
United Kingdom.
R (2014) is a free software environment for statistical computing and graphics.
It compiles and runs on a wide variety of UNIX platforms, Windows and MacOS.
Free software can be obtained from the R Project for Statistical Computing website.
It does basic statistics, resampling, regression, logistic regression, GLM (generalized
linear models for logistic regression) and GEE (generalized estimating equations, taking
into account correlation between measurements at multiple time points). User-written
routines are also available.
SAS (2014) Statistical Analysis System is a software suite developed by SAS Insti-
tute for advanced analytics, business intelligence, data management, and predictive
analytics. The compartments of SAS are:

– Base SAS – Basic procedures and data management;

– SAS/STAT – Statistical analysis;
– SAS/GRAPH – Graphics and presentation;
– SAS/OR – Operations research;
– SAS/ETS – Econometrics and Time Series Analysis;
– SAS/INSIGHT – Data mining;
– SAS/PH – Clinical trial analysis.

SAS University (2014) is a special edition for students and universities. It needs a
virtual machine that is a file or folder that contains an entire computer with operating
system and program in software. A virtual machine player is needed to access the SAS
virtual machine. Two recommended virtual machine players are Oracle’s VirtualBox
(runs on Windows, Mac or Linux) and VMWare’s VMWare Player (runs on Windows
and Linux). Both are free of charge.
Stata 13 (2014) Statistical package designed for researchers of all discipline. Appli-
cable for treatment of effects, multilevel mixed-effects GLM, power and sample size,
multilevel SEM (structural equation modeling) with generalized outcomes, forecasting,
long strings and BLOBs (binary large objects). A trial copy of Stata contains standard
statistics, resampling, time series, regression, logistic regression, GLM and GEE.
StatCrunch (2014) is statistical software available online that allows users to per-
form complex analyses, share data sets, and generate compelling reports of their data.
Interactive graphics help users understand statistical concepts and are available for
export to enrich reports with visual representations of data.
 Data evaluation and interpretation in environmental toxicology 511

STATGRAPHICS (2014) is an easy-to-learn, easy-to-use personal computer soft-

ware package designed for experts and non-experts alike. There is no need to download
any software since all calculations are done on a remote server and the statistical
analysis is done through the web browser. Figures are handled and images and area
calculations are based on pixels. It provides simple and multiple regressions.
STATISTICA (2014) is a statistics and analytics software package developed by
StatSoft. STATISTICA provides data analysis, data management, statistics, data min-
ing, and data visualization procedures. Its techniques include the widest selection
of predictive modeling, clustering, classification, and exploratory techniques in one
software platform. It offers a free STATISTICA trial (2014) version.
Several pieces of free statistical software are available online or can be downloaded.
Free Software (2014) offers several free IT tools. On the page of StatPages free statistical
software with short descriptions and links to the sources are introduced (StatPages
free, 2014). In the following, a list of free trials or completely free statistical software
is shown:

– AM (2014) serves analyzing data from complex samples, especially large-scale

assessments, as well as non-assessment survey data. It has sophisticated stats, easy
drag & drop interface, and an integrated help system that explains the statistics
as well as how to use the system. It can estimate models via marginal maximum
likelihood (MML) and automatically provides appropriate standard errors for
complex samples via Taylor-series approximation, jackknife & other replication
techniques.
– Dataplot (2014) is a tool for scientific visualization, statistical analysis and
non-linear modeling. It has extensive mathematical and graphical capabilities.
It is closely integrated with the Engineering Statistics Handbook (2014) from
NIST/SEMATECH.
– Develve (2014) a stats package for fast and easy interpretation of experimental
data: statistical testing, design of experiments and sample size calculation modes
are available. Everything is directly accessible and the results are directly visible
with no hidden menus.
– Epi InfoVersion 7 (2014) Classic public health and epidemiology application, with
free download, developed by Centers for Disease Control and Prevention (CDC,
2014) in Atlanta, Georgia (USA). The program allows for data entry and anal-
ysis including t-tests, ANOVA, non-parametric statistics, cross tabulations and
stratification with estimates of odds ratios, risk ratios, and risk differences, logis-
tic regression, survival analysis and analysis of complex survey data. See also
OpenEpi.
– ezANOVA (2014) is a free program for analyzing data. It has been developed
for statistics courses. It is not a particularly powerful tool, but it is useful for
illustrating the basics of the Analysis of Variance (ANOVA).
– GNU PSPP (2014) is a program for statistical analysis of sampled data. It is a free
replacement for the proprietary program SPSS, and appears very similar to it with
a few exceptions. Its main characteristics are:

◦ Choice of terminal or graphical user interface;

◦ Choice of text, postscript or html output formats;
512 Engineering Tools for Environmental Risk Management – 2

◦ Inter-operates with Gnumeric, OpenOffice.org and other free software;

◦ Easy data import from spreadsheets, text files and database sources;
◦ Fast statistical procedures, even on very large data sets;
◦ No license fees; no expiration period; no unethical “end user license agree-
ments’’;
◦ Fully indexed user manual;
◦ Cross platform;
◦ Runs on many different computers and many different operating systems.

– ICRISTAT (2014) serves data management and basic statistical analysis of experi-
mental data. It is primarily used for analysis of agricultural field trials. It includes:
data management with a spreadsheet, text editor, ANOVA, regression, genotype
& environment interaction analysis, quantitative trait analysis, single site analysis,
pattern analysis, graphics, utilities for randomization and layout, general factorial
EMS (Expected Means Squares) and orthogonal polynomials.
– MicrOsiris (2014) statistical and data management package for Windows, derived
from the OSIRIS IV package, developed at the University of Michigan. It pro-
vides extensive statistics: univariate, scatter plot, cross-tabs, ANOVA/MANOVA,
log-linear, correlation/regression MCA (multiple correspondence analysis), MNA
(Mean Number of Class Attributes), binary segmentation, cluster, factor, MINISSA
(smallest space analysis), item analysis, survival analysis, internal consistency.
– MIX (2014) Meta-analysis with Interactive eXplanations is a statistical add-in for
Excel. Recommended for learning meta-analysis.
– OpenEpi (2014) provides a number of epidemiologic and statistical tools.
– OpenStat (2014) a general stats package for all Windows versions and for Linux
systems. Developed by Bill Miller with a very broad range of data manipulation
and analysis capabilities and an SPSS-like user interface.
– PAST (2014) an easy-to-use data analysis package: common statistical, plotting
and modelling functions, curve fitting, significance tests (F, t, permutation t,
Chi-squared w. permutation test, Kolmogorov-Smirnov, Mann-Whitney, Shapiro-
Wilk, Spearman’s Rho and Kendall’s Tau tests, correlation, covariance, con-
tingency tables, one-way ANOVA, Kruskal-Wallis test), diversity and similarity
indices & profiles, abundance model fitting, multivariate statistics, time series
analysis, geometrical analysis, parsimony analysis (cladistics), and biostratigraphy.
– SalStat-2 (2014) provides tools for data management, statistical calculations
such as descriptive summaries, probability functions, chi-square, t-tests, 1-way
ANOVA, regression, correlation, non-parametric tests, Six-Sigma and graphic
system (inherited from matplotlib).
– SISA (2014) is a Simple Interactive Statistical Analysis tool for PC (DOS), a collec-
tion of individual DOS modules for several statistical calculations including some
analyses not readily available elsewhere.
– SOFA (2014) Statistics Open For All is a user-friendly, open-source statistics,
analysis, and reporting package.
– Statext (2014) has basic statistical tests such as rearrange, transpose, tabulate
and count data; random sample; basic descriptives; text-plots for dot, box-
and-whiskers, stem-and-leaf, histogram, scatterplot; find z-values, confidence
interval for means, one- and two-group and paired t-test; one- and two-way
 Data evaluation and interpretation in environmental toxicology 513

ANOVA; Pearson, Spearman and Kendall correlation; linear regression, Chi-

square goodness-of-fit test and independence tests; sign test, Mann-Whitney U
and Kruskal-Wallis H tests, probability tables (z, t, Chi-square, F, U); random
number generator; Central Limit Theorem, Chi-square distribution.
– Statist (2014) is a compact, portable program that provides most basic statisti-
cal capabilities: data manipulation (recoding, transforming, selecting), descriptive
stats (including histograms, box & whisker plots), correlation & regression, and
the common significance tests (chi-square, t-test, etc.).
– Statistical Software (2014) by Paul W. Mielke Jr. is a large collection of executable
DOS programs (and Fortran source). It includes: matrix occupancy, exact g-sample
empirical coverage test, interactions of exact analyses, spectral decomposition
analysis, exact mrpp (multi-response permutation procedure) and exact mrbp
(analysis of multivariate data for the randomized block design, based on permu-
tation procedures), Fisher’s exact test for cross-classification and goodness-of-fit,
Fisher’s combined p-values (meta analysis), largest part’s proportion, Pearson-
Zelterman, Greenwood-Moran and Kendall-Sherman goodness-of-fit, runs tests,
multivariate Hotelling’s test, least-absolute-deviation regression, sequential per-
mutation procedures, LAD (least sum of absolute deviations) regression, prin-
cipal component analysis, matched pair permutation, contingency tables and
Jonkheere-Terpstra.
– STPLAN (2014) performs power, sample size, calculations needed to study design.
Includes binomial, Poisson, normal and log-normal distributions, survival times
and correlation factors.
– Tanagra (2014) includes data mining, descriptive statistics (cross-tab, ANOVA,
correlation), instance selection (sampling, stratified), regression (multiple linear),
factorial analysis (PCA, MCA), clustering (kMeans, self-organizing map=SOM,
hierarchical agglomerative clustering=HAC).
– ViSta (2014), the Visual Statistics System, features highly dynamic and interactive
statistical visualizations.
– WinIDAMS (2014) is suitable for numerical information processing and statis-
tical analysis from UNESCO. Provides classical and advanced statistical tech-
niques including interactive construction of multidimensional tables, graphical
exploration of data, time series analysis, and a large number of multivariate
techniques.

10 ENVIRONMENTAL HAZARD AND RISK ASSESSMENT

USING TOXICITY DATA

Statistically evaluated toxicity test results can be used for generating environmen-
tal hazard and risk values that form the basis of decision making in environmental
risk management. Figure 9.13 shows the pathway of the measured toxicity data from
acquisition to their use in risk assessment.

10.1 Extrapolation
Available or measured physico-chemical and toxicity information/data (or those of
other adverse effects) should be extrapolated to estimate the extent and calculate
514 Engineering Tools for Environmental Risk Management – 2

Figure 9.13 The pathway of measured effect-data from acquisition to application in risk assessment.

the probability of future adverse environmental impacts. The extrapolation may

include:

– from chemical properties to environmental fate and behavior of a contaminant,

e.g.,
◦ from Kow to partition among physical phases;
◦ from Kow to degradability;
◦ from Kow to the bioaccumulative potential,
– from chemical properties to biological effects:
◦ from Kow to toxicity;
◦ from the chemistry of mixtures to the effects of mixtures;
– from one response end point to another:
◦ from EC20 to EC50 ;
◦ from acute to chronic toxicity end points;
◦ from effect doses to no-effect doses;
– from taxa to taxa:
◦ from the tested species to another, e.g. an endangered species;
◦ from the tested species to humans;
◦ from more tested species to entire ecosystems;
◦ from micro- and mesocosms to the field;
◦ from a tested field to the real field, etc.;
 Data evaluation and interpretation in environmental toxicology 515

– from species level to higher organizational levels:

◦ from species abundance to food chains or food webs;
◦ from short term damage of one or several species to the function of the entire
the ecosystem (deterioration or recovery/sensitivity or resistance);
◦ from direct effects to indirect effects and
◦ from indirect effects to direct effects;
– spatial and temporal scales:
◦ from one habitat to another;
◦ from one region to another;
◦ from an e.g. 10-year-old information to the current one, etc.

Study end points are calculated from measured end points taking into account
uncertainties by applying a suitable statistics for evaluation (Section 9). Extrapolation
steps are needed from the study end points of individual test organisms such as rats or
mice in human toxicology or aquatic/terrestrial species in ecotoxicology. The extrap-
olation method is based on type, quality and quantity of the initial and target data.
Most frequently factorial extrapolation is used with assessment factors, also called
uncertainty or safety factors, but probabilistic methods based on known or estimated
distributions are also widespread.

10.2 Hazard assessment

Hazardousness of a chemical substance is the result of its potential to pose an adverse
effect on living organisms or non-living entities. Hazardousness is an inherent prop-
erty of chemicals due to their molecular structure. Researchers know as early as in
the planning phase, when the molecules do not yet exist, that the material will be
hazardous. Environmental hazard is more extensive because the hazard is evaluated in
an environmental context using information on the fate and behavior of the substance
under environmental conditions and its effects occurring via the environment by air
inhalation, water drinking, food consumption and dermal contact with environmental
compartment. Toxicity data are used for hazard identification and hazard assessment.

10.2.1 Hazard identification

Hazard identification denotes the chemical substance’s potential adverse effects (tox-
icity, mutagenicity, reprotoxicity) posed to organisms (humans, aquatic and terrestrial
species, etc.), as well as the type and nature of the adverse effect. Toxicity is not the
only threat which constitutes a hazard: several physical, chemical, biological hazards
also play a role and act together. Integrated management of different hazards has
not been fully achieved in practice. Hazard characterization is the qualitative and,
wherever possible, quantitative description of the inherent property of the substance
and/or the situation which has the potential to cause adverse effects. Hazard assess-
ment includes the quantification of the hazard, a process which is required to determine
the size and describe the characteristics of the possible adverse effects of a chemical
substance and/or the situation to which an organism, population or the ecosystem may
516 Engineering Tools for Environmental Risk Management – 2

be exposed. These three steps are also parts of risk assessment, together with exposure
assessment.
Several environmental management and regulatory tools are based on hazard iden-
tification and/or assessment, thus differing from risk assessment in its lack of exposure
assessment. Regulation of chemical substances is largely based on their toxic hazard
along with their environmental fate and behavior (degradation, transformation, accu-
mulation, etc.). Most of the regulations integrate lists of chemicals of high concern,
based only on the identified hazard. The Globally Harmonized System of Classifi-
cation and Labeling of Chemicals (GHS, 2011) and its European counterpart, the
CLH, under the regulation of Classification, Labeling and Packaging of Substances
and Mixtures (CLP, 2008) are based on hazard identification only. Quality standards
and environmental quality criteria (EQC) are created based on the quantified hazards
of chemicals, e.g. the European Quality Standards in the Field of Water Policy (EQS
Water, 2008).
Priority lists of chemicals or the lists of chemicals to be monitored are created
mainly based on their hazard. Taking the substance’s production volume into account
is a step toward risk assessment. Tiered assessments apply hazard assessment as a
preliminary tool.

10.2.2 Hazard quantification

The quantified hazardous effect may relate to individual organisms as a toxicity end
point (NOEC, EC50 , etc.) or as an estimate from toxicity results for complete ecosys-
tems. Individual no-effect concentrations can be used as a basis for the protection
of the individual species. Several extrapolation methods are known to estimate the
ecosystem’s protective level from the measured toxicities posed to individual species.
Two of the methods are explained here in detail: the factorial extrapolation and the
species sensitivity distribution methods.
Extrapolation from test results to humans
Extrapolating from animal test end points to humans: interspecies and intraspecies as
well as a duration factor can be applied as default, but information from literature or
factual databases can also be used instead of default assessment factors. The frequency
distributions of the assessment factors found can/should be evaluated by statistical
methods.

– An allometric scaling factor (AS) is necessary because the greater size and life
span of humans relative to experimental animals have a significant impact on
the amount of chemical intake needed to provoke the same level of response: the
human dose is 7-fold smaller than a mouse and 4-fold smaller than a rat dose.
These AS factors are applied in toxicological studies often as a default.
– Another type of assessment factor is the default duration extrapolation factor: (i)
factor 2 for sub-chronic to chronic; (ii) factor 6 for subacute to chronic; and (iii)
factor 3 for subacute to sub-chronic.
– Interspecies differences in the human population show great variability so that
a default factor of 10 is recommended for the protection of the most sensitive
 Data evaluation and interpretation in environmental toxicology 517

Table 9.13 Assessment factors to derive a predicted concentration not affecting the ecosystem
(PNEC).

Extrapolation
Aquatic data set Terrestrial data set factor

At least one short-term L(E)C50 from L(E)C50 from short-term toxicity test(s) 1000–100
each of three trophic levels of the base-set on plants, earthworms, or
of fish, Daphnia and algae microorganisms
One long-term NOEC, either fish or NOEC for one long-term toxicity test 100
Daphnia (e.g. plant)
Two long-term NOECs from species NOEC for additional long-term toxicity 50
representing two trophic levels such tests of two trophic levels
as fish and/or Daphnia and/or algae
Long-term NOECs from at least three NOEC for additional long-term toxicity 10
species (normally fish, Daphnia and algae) tests for three species of three trophic
representing three trophic levels levels
Species sensitivity distribution (SSD Species sensitivity distribution (SSD 5–1
method) method)
Field data or model ecosystems Field data/data of model ecosystems case-by-case
basis

individuals. The assessor can depart from that when he has concrete information
on deviations.

Extrapolation from test results to the ecosystem

In the extrapolation from single species tests to the ecosystem, the factorial extrapola-
tion method (FAME) has priority, and factors between 1000 and 1 are used depending
on study type and duration. The study end point is generally the EC50 or the no-effect
concentration and, in a prognosis, the predicted no-effect concentration. A similar
system is recommended for aquatic and terrestrial ecosystems (see Table 9.13).
Another example for factorial extrapolation is the secondary poisoning of birds or
mammals. The recommended extrapolation factor by CSTEE (2000) decreases with
the duration of the test as follows:

– LC50 for birds, factor: 1000

– NOEC (28-day repeated dose test), factor: 100
– NOEC (90-day repeated dose test), factor: 30
– NOEC for chronic studies, factor: 10.

Toxicity study end points such as NOEC, EC50 , etc. reflect the responses of the test
organism on the effect – generally a serial concentration or dose of a toxicant. Study
end point is the result of statistical evaluation of hypothesis testing, regression analysis
or other biology-based statistical methods (Section 9). Large enough uncertainties in
the study results are superposed by the uncertainties of the extrapolation steps along
the pathway of hazard or risk assessment, which ultimately produces the information
that is used for environmental decision making. Risk managers should be aware of the
scale of uncertainty; otherwise they may make the wrong decision.
518 Engineering Tools for Environmental Risk Management – 2

Incorporation of the receptor-specific no-effect value into a hazard assessment

procedure may yield environmental quality criteria (EQCs): PNECaquatic is the basis for
water quality criteria and PNECterrestrial should be used for the generation of soil EQCs.
These criteria form the basis of legally binding effect-based or risk-based environmental
quality standards (EQS).
PNEC derivation from acute and chronic toxicity data
Uniformly accepted methodologies are used for the derivation of predicted no-effect
concentration (PNEC) on the respective ecosystem from effect data measured by sin-
gle species tests. The most widespread tool is the factorial extrapolation method, the
application of assessment or safety factors to compensate uncertainties (Garber et al.,
2010; EQS Water, 2008; ECHA, 2008). Assessment factors recommended by the EU-
TGD (2003) for the derivation of freshwater PNECaquatic and terrestrial ecosystems’
PNECsoil values are shown in Table 9.10. If only water toxicity data are available,
PNECsoil and PNECsediment can be calculated from PNECwater by the equilibrium parti-
tioning method, assuming that adverse effects are distributed between solid and water
according to the equilibrium-partitioning coefficient Ksoil−water and Ksusp−water .
PNEC derivation from species sensitivity distribution
Instead of factorial extrapolation using fixed assessment factors, the species sensitivity
distribution (SSD) can also be applied for extrapolation from single species to the entire
ecosystems.
Sensitivity distribution can be defined as the distribution of sensitivity of different
taxa of the aquatic or terrestrial ecosystem to the same contaminant. There are species
with average or low sensitivity among the ecosystem members. Species highly resis-
tant due to adaptation to contaminants should also be counted. The sensitivity of the
selected test organisms should be fit to the aim of testing if only one or a few species
are used. For example, an early indicator should be selected from the very sensitive
ones. A low-sensitivity species can be used as a screening tool for hot spot identifica-
tion. An average sensitivity may be used as a representative of the entire ecosystem.
One organism can never represent the ecosystem (properly) so that species sensitivity
distribution (SSD) has become the priority tool for this kind of representation. The
level of representativeness of an ecosystem has been designated as 95%, i.e. 95% of
the species should be represented. Not only species but higher taxonomic groups can
also be targeted by sensitivity distribution if appropriate.
The SSD method is based on collecting ecotoxicity results (from literature and other
data sources) of different test organisms from different taxa and fitting continuous
distribution to the orderly structured toxicity data. One can read the concentration
where the theoretical percentage of species exposed above their NOEC or EC50 is less
than e.g. five percent from the curve of fraction-affected vs. effective concentration
(arbitrary end points such as NOEC or EC50 ).
SSD may cover acute and chronic effects and the relevant test end points. The
SSD curve will be plotted from ECx or LCx data for acute effects, the chronic one is
obtained from NOEC, LOEC or MATC results. Both the measured and QSAR data
can be applied for SSD assessment.
Creation of a SSD curve:
 Data evaluation and interpretation in environmental toxicology 519

– As a first step, toxicity data are collected and selected.

– The minimum requirement for regulatory purposes is 10 toxicity data from 8
taxonomic groups;
– The toxicity data (ECx or NOEC, LOEC, etc.) are log-transformed;
– HCx is estimated by ranking the species sensitivities and choosing the appropriate
concentration. This non-parametric approach is less preferred than a parametric
approach which applies a distribution fitted to the cumulative species sensitivities.
The word “fit’’ means estimating the parameters of the distribution using statistical
tools.
– The log-transformed toxicity data allow a cumulative distribution to be derived.
Sensitivities are ranked from the most sensitive to the least sensitive.
– Several distribution functions have been recommended by different authors
(OECD, 2005), e.g. log–triangular function, log–logistic and log–normal func-
tion. Aldenberg and Slob (1993) refined the way to estimate the uncertainty of
the 95th percentile by introducing confidence levels, which was further refined
by Aldenberg and Jaworska (2000). Placing confidence limits (cl) around the esti-
mated HC5 can be noted as follows: HCcl 5 , where “cl’’ denotes the confidence with
which the estimated HC5 is not higher than the true HC5 .

Input data requirement of SSD for regulatory purposes:

The evaluation should include as much EC50 or NOECs as available from chronic/long-
term studies, preferably on full life cycle or multi-generation studies. The minimum
species requirements, for example according to the guidance for chemical safety
assessment in the EU, are as follows (ECHA, 2008):

– Fish (salmonids, minnows, bluegill sunfish, channel catfish, etc.);

– A second family in the phylum Chordata (fish, amphibian, etc.);
– A crustacean (cladoceran, copepod, ostracod, isopod, amphipod, crayfish etc.);
– An insect (mayfly, dragonfly, damselfly, stonefly, caddisfly, mosquito, midge, etc.);
– A family in a phylum other than Arthropoda or Chordata (Rotifera, Annelida,
Mollusca, etc.);
– A family in any order of insects or any phylum not already represented;
– Algae;
– Higher plants.

Minimal sample size (number of data), considering the statistical extrapolation

methods necessary to apply when a PNEC is derived, is at least 10 different species’
NOEC values (or more, preferably 15) covering at least 8 taxonomic groups (ECHA,
2008). Minimal requirements can also be specified otherwise; principally they should
be based on statistical calculations. According to Newman et al. (2000) approximate
optimal sample sizes based on statistical evaluation for HC5 estimation ranged from
15 to 55 with a median of 30 species-sensitivity values. Similar sample sizes were
needed for HC10 and HC20 estimation: estimates ranged from 10 to 75. No differ-
ence was apparent in ranges for EC50 –LC50 or NOEC data. The study says that the
520 Engineering Tools for Environmental Risk Management – 2

statistically based sample sizes are much higher than those recommended as acceptable
for regulatory purposes (i.e. four to eight species in different regulations).
Derivation of PNEC from the SSD curve:

– The HC5 or any other concentration belonging to arbitrary percentiles can be read
from the SSD curve. The HC5 concentration is the most frequently used hazard
concentration for regulatory purposes.
– PNECaquatic can be created from HC5 by dividing it by the assessment factor (AF):
PNECaquatic = HC5 /AF
– AF depends on the overall quality of the database (collected ecotoxicological data
for SSD) and the diversity of the taxonomic groups covered by the database (organ-
isms belonging to a minimum of 8 taxonomic groups). The recommended AF is
generally 5, which can be lowered when results from much greater number of taxa
and species are available than required as the minimum. An AF value less than
2 is only accepted in extraordinary cases. An AF of 2 was found most adequate,
for example, for nickel (TGD EQS, 2011; Nickel EQS, 2011; Nickel RAR, 2008)
because:
◦ The large size of the acute and long-term aquatic database covers all sensitive
life stages.
◦ Acute aquatic toxicity results came from 65 freshwater and 21 marine species
(together 86) of 12 taxonomic groups; exceeding the minimal requirements of
8 species from 8 taxonomic groups when employing the SSD approach.
◦ Chronic toxicity results were obtained from 31 freshwater and 15 marine
species of 9 and 6 taxonomic groups, respectively.
◦ The representativeness of the taxonomic groups was based on 12 vertebrate,
7 invertebrate, one algal and one higher aquatic plant species.

Figure 9.14 shows the example of the graph taken from the nickel EQS dossier
(Nickel EQS, 2011). Acute data were analyzed using ETX 2.0 (2014) from RIVM
and Burrlioz (2014) software from CSIRO for deriving SSDs. The HC5 using the
ETX software was 0.065 mg/L (90% confidence interval = 0.0282–0.1289 mg/L). The
result of Burrlioz of 0.069 mg/L is very close to ETX. An assessment factor of 2 yields
0.067 mg/L: 2 = 0.0335 mg/L ≈ 0.034 mg/L for the maximum allowable concentration
(MAC) in the marine and freshwater ecosystem. The proposed MAC of 0.034 mg/L
was below individual EC50 values for the majority of tested species and below the most
sensitive fish, invertebrate and plant species. Chronic marine toxicity input data and an
AF of 2 provided a PNECmarine value of 8.6 µg Ni/L. It is used as an AA-EQS (annual
average environmental quality criterion). Proposed AA-EQSbioavailable for freshwater
ecosystem is 2 µg/L.
Creation of PNEC, MAC, or other EQC is based on statistical evaluation. Several
statistical tools are recommended and compared by scientists and authorities. For
example a CCME (2006) project evaluated several potential statistical models for
freshwater SSD

(i) for distribution: normal, logistic, lognormal, Burr-type, Weibull, and extreme
value;
 Data evaluation and interpretation in environmental toxicology 521

1
Fraction affected

0.9

0.8

0.7
Alga
Annelid
0.6 Cnidarian
Crustacean
0.5 Echinoderm
Fish
Insect
0.4 Macrophyte
Mollusc
Platyhelminth
0.3 Rotifer

0.2

0.1

0
–4 –3 –2 –1 0 1 2 3 4 5
Log LC50 and EC50

Figure 9.14 SSD graph of nickel from 86 freshwater and marine toxicity data, taken over from the EQS
dossier (Nickel EQS, 2011).

(ii) for goodness of fit: Anderson-Darling, chi-square, Kolmogorov-Smirnov,

Shapiro-Wilk’s tests and several graphical techniques and
(iii) for choosing the adequate statistical tool and software.

It is becoming more and more widespread to estimate HC5 values for environmen-
tal risk assessment and regulatory applications of sensitivity distributions. Decision
makers hope that this will refine information on the environment and that a less con-
servative risk value, and consequently less expensive risk reduction will be possible
compared to EC50 or the highly uncertain NOEC.
The end point of hazard assessment is DNEL or ADI in human toxicology and
PNEC for the ecosystem. These predicted/derived no-effect values can be applied to:

– Hazard identification and assessment;

– Risk assessment by comparing DNEL/PNEC to the environmental concentration;
– Establishing environmental quality criteria (screening values, thresholds).

Generation of measures for adverse effects from other than toxicity values should
also be included in an integrated risk assessment and decision-making procedure:
522 Engineering Tools for Environmental Risk Management – 2

interactions between species, bioavailability, bioaccumulation and other food-chain

and food-web effects as well as the (bio)degradation of the contaminants.

– Bioavailability and biodegradation belong to the exposure side of risk assessment:

both interactions reduce the amount of the contaminant in the environment. In
contrast to the logic of risk assessment, hazard assessment and hazard classifica-
tion also take into account biodegradation and bioavailability. The reason for this
is that ready biodegradability leads to a short life time and, as a result, low chronic
toxicities. Low bioavailability leads to reduced adsorption and low inner concen-
trations, and, as a consequence, decreases adverse effects and hazard. A tiered
concept and risk assessment should decide how bioavailability and biodegrada-
tion shall be taken into consideration by the hazard and risk values. Failure to
do so when assessing hazard results in an overestimate because the effect is deter-
mined as though the contaminant were fully available and not degraded at all.
Once incorporated into the hazard value, it is questionable if they can be included
in the transport and fate model used for PEC calculation.
– Bioaccumulation and other secondary poisoning processes along the food chain or
in the food web could be considered as an increased environmental concentration
on the side of PEC, but it should be integrated as a factor (bioaccumulation factor)
in hazard assessment and used similarly to the assessment factors: the toxicological
result should be divided by it when calculating a NOEC.
– Method-specific individual solutions are needed to establish a PNEC or similar
threshold values since no standardized or recommended uniform methods are
available for using micro- or mesocosms or field assessment results.

10.3 Validation of toxicity tests

In addition to data evaluation using statistical methods, evaluation and validation of
the test methods are equally important for decision making. Risk assessments should be
based on the most reliable information available. Validation of the applied test meth-
ods is a science-based evaluation at the interface between test method development,
application and acceptance for risk management or for regulatory purposes.
The statistics of toxicity data section demonstrated the number of uncertainties
in effect and hazard assessment. Use of the most reliable and scientifically sound
information on the effects of the chemical substances can reduce the extent of these
uncertainties.
Standardized test guidelines primarily from US EPA, EU REACH and OECD, as
well as quality assurance, quality control and good laboratory practice (GLP) are nec-
essary to ensure safety of chemical substances and products. A standardized procedure
for data evaluation will guarantee consistency and transparency of risk assessment
which are key factors in public acceptance. The risk assessor needs a validation method
to provide data of sufficient quality to underlie the hazard and risk assessments (Küster
et al., 2009; McCarty et al., 2012).
Several organizations have developed validation tools such as ICCVAM and
US EPA. The Klimish tool should also be mentioned as one of the most popular
toxicological evaluation tools.
 Data evaluation and interpretation in environmental toxicology 523

The ICCVAM criteria for test validation, including alternative test methods, are
as follows (ICCVAM, 1997, 2003, 2014):
– Clear statement of the proposed use of the test results;
– The relationship of the test method’s end point(s) to the biologic effect of interest;
– A detailed protocol of the test method;
– The extent of within-test variability, and reproducibility among laboratories;
– Proven performance of reference chemicals and representative test agents;
– Comparative data with competing or to be substituted tests;
– The limitations of the method;
– Reporting data to support the validity of a test method in accordance with Good
Laboratory Practices (GLPs);
– Availability of data to support the assessment of the validity of the test method.
The main assessment criteria applied generally by US EPA are formulated as:

– Soundness: reasonable for the intended purpose;

– Applicability and utility;
– Clarity and completeness;
– Uncertainty and variability;
– Evaluation and review.

Klimish et al. (1977) elaborated an evaluation tool for assessing the reliability of
toxicological studies, mainly for regulatory purposes. The Klimish score based on a
systematic approach became a standard and uniform evaluation system in Europe and
a software tool has been developed relying on it. It assesses reliability, relevance, and
adequacy of data. The Klimish scoring method assigns studies to one of four reliability
categories as follows:

– Reliable without restriction: “Studies or data generated according to generally

valid and/or internationally accepted testing guidelines (preferably performed
according to GLP) or in which the test parameters documented are based on a spe-
cific (national) testing guideline, or in which all parameters described are closely
related/comparable to a guideline method.’’
– Reliable with restriction: “Studies or data (mostly not performed according to
GLP), in which the test parameters documented do not totally comply with the
specific testing guideline, but are sufficient to accept the data or in which inves-
tigations are described which cannot be subsumed under a testing guideline, but
which are nevertheless well documented and scientifically acceptable.’’
– Not reliable: “Studies or data, in which there were interferences between the mea-
suring system and the test substance, or in which organisms/test systems were used
which are not relevant in relation to the exposure (e.g., unphysiologic pathways of
application) or which were carried out or generated according to a method which
is not acceptable, the documentation of which is not sufficient for assessment and
which is not convincing for an expert judgment.’’
– Not assignable: “Studies or data which do not give sufficient experimental details
and which are only listed in short abstracts or secondary literature (books, reviews,
etc.).
524 Engineering Tools for Environmental Risk Management – 2

All the main criteria are split into numerous very detailed items in the form of
simple questions, which can be answered with yes or no when evaluating the study.
For example the reliability of ecotoxicological studies is evaluated according to the
following aspects:

– Clear description of the test procedure (complete documentation);

– Specification of the test substance (purity, by-products);
– Data on the test species and the number of individuals tested;
– Data on the measured parameters (including definitions);
– Data on exposure period;
– Use of solubilizers/emulgeators;
– Data on concentration control analysis;
– Data on neutralization of samples;
– Data on physical and chemical test conditions (pH value, conductivity, light
intensity, temperature, hardness of water);
– Determined effect concentrations (EC/LC/NOEC/ LOEC);
– Data on the statistical evaluations (including the method);
– Data on dosing the test substance (static, semistatic, flow-through system);
– Additional items in the case of chronic studies: information about the investigated
period of the life cycle of the test animals and data on feeding of test animals.

Relevance/adequacy of ecotoxicological studies is evaluated based on criteria

such as:

– Testing strategy (organism, exposure scenario) should agree with the occurrence
and the persistence of the test substance in the environment (target compartment);
– Data obtained from experiments on non-standard organisms (specialist, spread)
should be converted into useful ecotoxicological information;
– Physico-chemical properties of the test substance (stability, volatility, solubility,
sorbability) should be considered when planning the test.

Klimisch categories 1, 2 or 3 can be determined by the newly developed software

tool, called ToxRTool (2014) (Schneider et al., 2009). It is applicable to various types
of experimental data, end points and studies to assess reliability of toxicological data.
The tool consists of two parts, one to evaluate in vivo and another in vitro data. The
final version, ToxRTool, is publicly available for tests and use (ToxRTool Instruction,
2014; ToxRTool Download, 2014).

10.4 Exposure assessment

Generic or site-specific exposure assessment is the next step in the approach toward
risk assessment as shown by the scheme in Figure 9.13. The risk, as it is defined,
depends on the probability of occurrence and the extent of the damage, which, in
turn, depends on the adverse effects of the chemical(s) and the probability of any
interaction with the receptors, e.g. their uptake. Probability of uptake is a function
of the contaminant’s biologically available proportion and the uptake mechanism of
the receptor organism. The driving force of the potentially effective proportions is the
 Data evaluation and interpretation in environmental toxicology 525

concentration of the chemical substance in the environment, i.e. the exposure (PEC).
Fully perspective risk assessment can calculate exposure relying on the planned volumes
of production and use and the transport and fate modeling in a generic environment e.g.
for authorization of a future product. The generic model parameters for transport and
fate modeling are default values and are different from region to region. Site-specific
transport parameters may differ from the generic parameters in both directions. The
other terminal of exposure assessment may be a fully assessed contaminated site where
both the measured chemical concentrations and the directly measured toxicity data are
available. The prediction is restricted in this case to the contaminants’ further spread
and the time dependence of their transport and fate.
The result of the exposure assessment is a concentration both for a generic and a
targeted case:

– It is a conservative estimate (due to model uncertainties and the variables in the

environmental characteristics) in the generic situation, which may be applicable
for the whole region (e.g. EU), but probably overestimated in every single local
case. Generic PEC is useful for regulation but not for handling local problems.
A local PEC may deviate from the generic PEC value in both positive and negative
directions.
– A targeted, i.e. problem- or site-specific concentration may be in good correlation
with the actual acute risk of the specific case. Long-term risks bear high uncertainty
and additional physical, chemical and biological risks posed to the site may increase
the uncertainties.

10.5 Risk assessment

Environmental risk is defined as the product of the probability (probable frequency)
and the probable extent of damage due to adverse effects on humans and/or ecosystems.
It is the occurrence of an uncertain event with a specific probability in general, which
is determined by the extent of damage and the probability of the occurrence. Both
can be quantified with large uncertainty, e.g. the scale and cost of ecosystem damage.
This probability is expressed as the ratio of the predicted environmental concentra-
tion and the predicted highest no-effect concentration (on humans or the ecosystem),
often called RCR or risk characterization ratio. This ratio can characterize the risk
of chemicals in the environment by a quantitative value. RCR defines the probable
concentration present in the environment and accessible by the users of the environ-
ment, and it determines if PEC is small or great, and how many times more than the
acceptable (no-effect) concentration. Risk estimates may be greatly uncertain because
not only can the value or the damage not be precisely estimated but the temporal and
spatial extension is also uncertain.
Environmental risk assessment is split into two main branches, according to its
integration into environmental management: (i) generic risk assessment and (ii) tar-
geted, i.e. problem- or site-specific risk assessment. Generic risk assessment studies
the hazardous chemical in a generic environment (e.g. Europe) for average receptors.
Problem- or site-specific risk assessment looks into the chemicals in a certain environ-
ment (e.g. the Po River Delta or a garden close to a mine, etc.) and problems caused
to local users of this environment.
526 Engineering Tools for Environmental Risk Management – 2

Regulatory risk assessment and management mainly applies generic tools while
targeted or problem-specific risk management relies on a tiered approach including
both generic and specific information.
Both hazard and risk management include the steps of identification, analysis,
assessment, control, and risk reduction by avoidance, minimization, or elimination
of unacceptable hazard/risks. Environmental risk assessment relies on environmen-
tal monitoring, evaluation and interpretation of the acquired data and is governed by
environmental law mainly concerning the fields of chemical substances, human health,
water and soil, ecosystem/nature, waste and contaminated land. Terminology and def-
initions in risk assessment have been more or less harmonized in the last 10–15 years.
IPCS (2004) collected and organized the relevant terms in a harmonization project in
the context of chemicals regulatory management (i.e., notification/authorization, reg-
istration, and classification). Their guidelines serve as basis for the definitions in this
chapter.
Risk characterization is the qualitative and, wherever possible, quantitative deter-
mination of the probability of occurrence of known and potential adverse effects of
a chemical substance in a given organism, population or the ecosystem under defined
exposure conditions. It includes uncertainties, and the TER (toxicity/exposure ratio:
toxicity/PEC) and RCR (risk characterization ratio: PEC/PNEC) values are used (see
below) for its characterization.
Risk assessment is the process intended to calculate/estimate the risk to a given
target organism, population or system. It includes the exposure and the hazard, as well
as uncertainties. The risk assessment process includes four steps: hazard identification,
hazard characterization, exposure assessment and risk characterization.
Risk evaluation is a further step toward establishing the relationship between risks
and benefits of exposure to a chemical substance or other agents. It is synonymous with
risk–benefit evaluation.
Validation of the outcomes of the assessment is the most important final step in risk
management. Reliability refers to the reproducibility of the outcome; and relevance
can be defined as the meaningfulness and usefulness of the assessment for the defined
purposes of environmental management.
Harmonized and standardized risk assessment methodologies are prescribed by
several guidelines all over the world; in Europe EU-TGD (2003) is the basic Techni-
cal Guidance defining and laying down protocols and uniform tools for generic risk
assessment of chemicals (industrial chemicals, pesticides and biocides) for regulatory
purposes. Several other types of guidance, e.g. on REACH, CLP and biocides legisla-
tion are published on the website of ECHA (ECHA Guidance, 2014). US EPA (2014)
developed guidance and tools (databases and models), for the assessment of human
risks (US EPA – Human Risk, 2014), including developmental, carcinogenic, muta-
genic, reproductive, neurotoxic risks; ecological risk (US EPA Eco Risk, 2014), as well
as microbiological risks and the risk assessment of mixtures.
Generic risk assessment works with the assumption of a generic environment, e.g.
a generic Europe, and is mainly used for regulatory purposes. It is a fully prospec-
tive activity forecasting a fully theoretical situation, e.g. the generic risk of a not yet
notified and produced chemical substance in Europe. The generic risk assessment is
based on the adverse effects of pure chemicals (PNECgeneric ) and the results of trans-
port modeling of the produced and used amount of the chemicals in the environment
 Data evaluation and interpretation in environmental toxicology 527

of a certain region. Study of the effects of pure chemicals and dissolved forms gives
a well reproducible test situation. The standard study results are extrapolated to the
hypothetical generic environment by relying on defaults and averages as well as on
general experience. On the other hand, this ‘sterile’ system and the results of generic
RA may greatly differ from reality. The application of uncertainty factors may reflect
and handle the differences in space and time, but may also lead to undue overesti-
mates. Simulation studies are advantageous in properly modeling multiple interactions
and bioavailability, but have the same shortcomings as generic models: the spa-
tial and temporal variations in the environment cause excessive uncertainty which
should be compensated with large safety factors resulting in undue overestimation.
An overestimate is accepted by the precautionary approaches (go beyond safety when
scientific evidence on the safe conditions is lacking: “better safe than sorry’’), but a
feedback provided by environmental monitoring is always necessary. An additional
uncertainty is that the theoretical environment may be generic, but the test organisms
are not and thus the protection level of species or ecosystems remains a source of
uncertainty.
Generic risk values are mainly used for decision making in risk management of
chemicals, pesticides, biocides, waste, air, water and soil. The relevant national and
European regulations such as the REACH regulation, pesticide and biocide directives,
water framework and waste directives apply this tool for notification, authorizations,
restrictions and for enacting generic risk reduction measures. Generic risk assessment
can be placed between hazard assessment and targeted risk assessment. From many
points of view its result is closer to the hazard than to the real risk values: actually the
hazard values are evaluated in comparison with the same generic environment, so the
generic risk values are hazards on the same denominator. The chapter on bioavailability
(Chapter 7) introduces several examples where highly toxic substances have the same
hazard, but taking the different environments into consideration, their risk will be
small due to immobile and unavailable chemical forms or rapid biodegradation but
may be significantly large due to complete availability and harmful transformations.
Adaptation of the ecosystem, resistant species, and modified food webs may also
contribute to a small risk situation even in the presence of significantly hazardous
chemicals. In a workplace situation the risk of hazardous compounds can be reduced
to a very small value by applying protective equipment.
Targeted, i.e. problem- and site-specific risk values are locally relevant and make
decision making and risk management of contaminated sites, waste disposal, safe
workplaces and other targeted tasks highly efficient within a certain time interval.
Targeted risk assessment incorporates the past, the present and future. A retrospective
assessment can be based on historical information covering the past up to the present,
and a prospective assessment serves to predict the future situation. In this respect, risk
assessment of a contaminated site will start with the collection of data on former and
ongoing land uses, activities and all kinds of historical information on the chemicals
used, measured concentrations and damage, and the adverse effects observed on human
health and the ecosystem. Collection and evaluation of historical information will
be followed by the acquisition of necessary new information and the evaluation of
measured data on chemicals and adverse effects. The last step is the assessment of the
present and the prediction of the future state of the environment, assuming that no
risk reduction measures have been taken. Long-term data enable to identify the trends
528 Engineering Tools for Environmental Risk Management – 2

and increase the validity of the risk assessment. This topic is discussed in Volume 3 of
the book series.
Biology- and ecology-based risk assessment/monitoring observes pharmacokinetic
characteristics and epidemiological results for the human population and key species
(individuals and populations), food chains, and species diversity for the ecosystem to
find the point where changes are unacceptable. These assessment types are applied
for cases when the adverse impact is already known. The time series of assessments
(monitoring data) make the trends visible and enable a good prognosis. Biology-based
assessment covers cumulative effects, so it is applicable to mixtures of chemicals and
the combinations of chemical impact with other, e.g. meteorological or microbiological
impacts. It may integrate the biomarkers approach, and early indication of potential
damage. Using biomarkers in risk assessment means that the forecast is based on a
nature-like biological and/or pharmacokinetic model and not on mathematical and
chemical models only. The biology-based ecological risk assessment can also be tiered,
applying methods from simple DTA to complete ecosystem assessments.
The comparison between measured contaminant concentrations and specific
assessment criteria is the simplest way practiced in site-specific risk assessment that
can decide whether a significant risk to human or ecosystem health (may) exist. The
assessment criteria may be generic (laid down in national or regional regulations) or
site-specific. A large number of studies and guidelines have been published in the last 20
years in connection with contaminated land management and remediation programs.
These methodologies recommend a tiered assessment approach based on a concep-
tual risk model that integrates transport and exposure models, following the pathway
of contaminants from the source to the receptors. The tiers of the assessment are as
follows:

– First tier: a preliminary, largely desk-based assessment step to decide whether or

not there is a potential exposure;
– Second tier: if the potential exposure cannot be excluded, a generic-type risk
assessment is undertaken, which means a site-specific exposure assessment and
comparison to generic no-effect criteria;
– Third tier: a refined risk assessment is executed using site-specific assessment
criteria, i.e. site-specific target values;
– Fourth tier: site-specific risk management which includes a risk–benefit assessment
and a comprehensive risk communication.

Quantification of the risk

There are several terms to be defined and quantified concerning the risk posed by
chemicals to humans and the environment.
The risk posed to individual organisms can be characterized by the ratio of
TER = Toxicity/Exposure. The ratio of the effect on a living organism to the envi-
ronmental exposure can be calculated as PEC using any of the toxicological end points
(LC50 , LD50 or NOEC/NOEL). TER quantifies the risk in terms of possibility of
occurrence of an acute or toxic adverse effect (EC50 or NOEC) posed to a certain
organism.
The risk posed by a contaminant to the aquatic or terrestrial ecosystem (as a
whole) can be expressed by a simplified risk assessment as a risk characterization
 Data evaluation and interpretation in environmental toxicology 529

ratio, abbreviated as RCR, which is the ratio of the predicted environmental expo-
sure to the receptor-specific predicted effect or no-effect value (PEC/PNEC). Exposure
is an estimate based on transport and fate modeling. The no-effect value is an esti-
mate based on single species toxicity tests of the chemical substance or environmental
samples, as well as results acquired from microcosms and field assessments. With
the exception of the latter, all other estimates are expressed in concentration (or
dose) and can be integrated into the RCR based risk assessment approach. RCR is
widely used for screening of environmental chemicals and contaminated sites. The
RCR approach is a strongly conservative one, which works with worst-case assump-
tions for both the exposure and the effects. It is applied by regulations (e.g. the
European REACH) and regional risk assessment tools where generic environmental
parameters are applied. Mathematical (QSAR, generic transport models), chemical
(environmental fate and concentrations of chemicals, no-effect concentrations) and
biological models (adverse effects) are applied in a problem-specific combination. The
problem-specific method gradually refined generally reduces the conservatism of the
assessment.
The human pendant of RCR is also called HQ, human hazard quotient designed
by US EPA for the characterization of human risks posed by chemicals as the ratio
between the ingested dose (D) calculated from measured or predicted environmental
concentration and a human oral reference dose (RfD), both measured in mg/kg/day.
The safe level, called target hazard quotient (THQ), is 1, a higher value indicates health
risk. RfD is generated from NOEL, NOAEL or BMD (benchmark dose), e.g. BMD10 or
BMDL10 of animal tests divided by the uncertainty factors. Instead of RfD, European
legislation prefers DNEL. The derived no-effect level DNEL includes a NOAEL-specific
uncertainty while TDI (tolerable daily intake) is calculated from the same animal test
results by constant assessment factors. Instead of TDI, the term ADI (acceptable daily
intake) is also used having the same meaning.

Definitions
– BMD, benchmark dose, is defined as the dose of a substance that is expected to
result in a pre-specified level of effect, for example BMD10 means the dose causing
an inhibition rate of 10%.
– BMDL is the lower 95% confidence limit of BMD (the result of statistical
evaluation).
– DNEL, the derived no-effect level defined by REACH (ECHA, 2008) as a
human health-based limit value for threshold substances. The NOAEL dose
descriptor may be used as a reference point. DNEL = (NOAEL or BMD)/(AF1 ×
AF2 × · · ·× AFn ).
– RfD in human risk characterization is the daily oral exposure to the human popu-
lation that is likely to be without an appreciable risk of deleterious effects during
a lifetime. It is used by US EPA.
– TDI, ADI: tolerable/acceptable daily intake of a chemical substance, an amount
that can be ingested (orally) on a daily basis over a lifetime without any appreciable
health risk. Acceptable daily intake (ADI) was initially used for food additives,
whereas the term TDI is preferred for contaminants. Their values are based on
530 Engineering Tools for Environmental Risk Management – 2

animal experiments, and they are calculated as NOAEL divided by a multiple

assessment factor of 100.

Human health risk professionals fight with the problem of non-threshold chemicals
and their effects when assessing genotoxicity, carcinogenicity and endocrine disruption.
The relevant board in the UK (IGHRC, 2003) and US EPA (2002) have recommended
taking a common approach to risk assessment for both threshold and non-threshold
types of substances (IOM, 2012), but other professionals still manage threshold and
non-threshold chemicals differently. Many genotoxic agents and genotoxic carcinogens
have theoretically no thresholds because there is a linear relationship between the dose
and the number of DNA damage and even one molecule can trigger gene mutation.
If the molecular mechanism is not a direct one, as in the case of gene mutations, a
threshold should exist even theoretically because there is a minimum of damage to the
mediating molecule (enzyme, hormone, etc.), which triggers the effect in an organism.
There are two semiquantitative methods available for the estimation of a no-effect
level (US EPA, 1995; ECETOC, 2002; US EPA, 2005; EFSA, 2005; SCHER, 2009;
ECHA, 2010; EFSA, 2012) for non-threshold genotoxic and carcinogenic toxicants:

– The “linear dose–response’’ approach is based on a linear extrapolation from high

to low doses, for example a straight line is drawn from the point of departure
(typically the BMDL10 or T25 ) to the origin (zero dose with a zero response), and
the slope of this line is used for estimation of cancer risk (CR = exposure ∗ slope
factor). The generated DMEL is compared to an acceptable cancer risk which is
between 10−5 and 10−6 in different legislations. It is considered as the upper limit
of risk;
– The large extrapolation factor, also called margin of exposure (MOE) approach
divides the estimated human exposure by the value of the reference point, usually
the BMDL10 , but T25 can also be used. EFSA considers a MOE of 10,000 or
higher, based on BDML10 , as a value of low concern.

Definitions
– DMEL is the derived minimal effect level used for non-threshold substances
(ECHA, 2008). Both the T25 and BMDL10 dose descriptors may be used as
reference points.

◦ DMEL at 10−5 (or 10−6 ) risk = BMD10 /AF ∗ 10,000 for workers
◦ BMD10 /AF ∗ 100,000 for general population
◦ T25 /AF ∗ 25,000 for workers
◦ T25 /AF ∗ 250,000 for general population.

– MOE is the margin of exposure. MOE approach is useful for assessing exposures
of genotoxic carcinogens and mutagens when carcinogenicity data are lacking.
Both T25 and BMDL10 may be used as a reference point. MOE = BMDL10 /human
exposure. The larger the MOE, the smaller the risk of exposure.
– T25 is a carcinogenicity potency estimate that is defined as the chronic dose rate
which will cause tumors in 25% of the animals at a specific tissue site, after
correction for spontaneous incidence, within the standard lifetime of that species.
 Data evaluation and interpretation in environmental toxicology 531

– TD50 is the standardized measure of carcinogenic potency, the daily dose rate in
mg/kg body weight/day to induce tumors in half of the test animals that would
have remained tumor-free at zero dose. Whenever there is more than one positive
experiment in a species, the reported TD50 value is a harmonic mean calculated
using the TD50 value from the most potent target site in each positive experiment.

The risk characterization ratio alone is not enough for decision making and effi-
cient risk management. The problem of mixtures and the impact of the background
concentrations should also be solved. Crommentuijn et al. (1997, 2001) suggested a
Maximum Permissible Concentration (MPC) based on the addition of a background
concentration (Cb) to a derived Maximum Permissible Addition (MPA). This way
MPC = Cb + MPA. Long-term trends and wider scopes, as well as life cycles of con-
nected chemicals, elements and energies should also be considered. A widespread
database would be necessary to compare alternatives and substitute options of chemi-
cals and technologies for the preparation of decisions. In addition to environmental and
human health risks, the benefits of chemicals applications should also be considered
and quantitatively assessed.

10.6 Summary comments on risk assessment and risk

management based on toxicity data
The three risk assessment concepts – generic, targeted and biology-based – can be
considered and applied as consecutive steps of a tiered methodology, and a lower-
ranked step can be validated by using the higher-ranked one. PNEC can be refined or
validated by a probabilistic no-effect value, and field assessment of actual impacts can
validate risk modeling. Validation or other kind of comparison between the results of
the different tiers needs careful analysis to understand discrepancies. For example one
can include extremely sensitive species for SSD (instead of treating them as outliers).
Another example is where one cannot distinguish between the environmental impacts
of chemical contaminants and other simultaneous adverse effects, e.g., a virus infection,
or micronutrient depletion.
The extension of risk in time and space is essential information in risk manage-
ment: the assessment and the measure taken should cover the extension of the risk.
Typically several different scopes should be managed at the same time so that scoping
(determination of the scope or scopes) should always be the first step of the assessment
and management activities.
Tiered risk assessment is fast and efficient; mathematical models can be widely
applied, which reduces assessment costs by excluding negative cases at early tiers.
A risk overestimate may lead to an unreasonable increase in risk reduction costs.
Tiered assessment with a refined and increasingly precise assessment can minimize
overestimation. Tiering can be applied to every risk-based management task, including
planning the monitoring, generic and targeted risk characterization and assessment as
well as the creation of environmental quality criteria and risk reduction target values.
The management steps in Figure 9.15 demonstrate the order of activities and the
supportive relationship of environmental law (regulation) and monitoring to the man-
agement and the connected decision making. Hazard identification, quantification,
532 Engineering Tools for Environmental Risk Management – 2

Figure 9.15 Environmental risk management of environmental contaminants.

and generic and targeted risk assessment may be the consecutive steps of environmen-
tal risk assessment, but the results of the steps have their own usability in fulfilling legal
requirements or special targets of environmental management. The reciprocal relation-
ships between environmental risk assessment and monitoring indicate that monitoring
data can be used for risk assessment, and monitoring design should be based on the
chemicals’ risk values (priority risk substances should be monitored). Environmental
management should fulfill legal requirements and follow the relevant guidance, but
experience and new knowledge from risk assessment and monitoring as well as from
the application of risk reduction measures should be incorporated into the legal tool
battery.
The most common applications of the assessment of environmental risk posed by
adverse effects of chemicals to human and ecosystem health are as follows:

– Regulatory ERM of chemical substances (pesticides, biocides, industrial chemi-

cals);
– Regulatory ERM of waters and soils;
– Regulatory ERM of wastes, waste disposal sites;
– Creation of generic environmental quality criteria;
– ERM of contaminated sites;
– Creation of site-specific EQC;
– Monitoring design based on risk values.
 Data evaluation and interpretation in environmental toxicology 533

The environmental impact of chemical substances in air, water and soil has reached
enormous global proportions. This impact cannot continuously be observed and
assessed because of its large-scale dimensions and lack of knowledge about the healthy
(non-deteriorated) environment, ideal references and the dynamic environmental
processes. The risk of chemicals posed to the environment alone – even if the ecosys-
tem/people of a subarea were perfectly known – could not generally be used for manag-
ing the regional or global environment. This is because several other risks and impacts
(mixture of chemicals, hazardous effects other than toxicity, social and economic risks,
etc.) must be taken into account and managed (see also Volume 1, Chapter 8).
The extrapolation steps are accompanied by large uncertainties depending on the
type and quality of the information/data, and the variability within and the relation-
ship between the entities from and to which it has been extrapolated. On the other
hand, excessive uncertainty can result from the environment itself, meteorological and
climatic conditions, changes of habitats, behavior of natural ecosystems, communities,
ecosystem members, dynamic equilibrium processes, etc. The natural variability may
create extreme uncertainties.
The consequences are that statistical evaluation and analysis should be applied not
only to the determination of the true values of the toxicological end points (Section
9), but along the whole procedure of risk assessment and risk management. A wide
selection of statistical tools can be applied in practice from uncertainty (extrapolation)
factors through the analysis of distributions to the regression methods.
The quality of the extrapolated results depends on:

– The quality of the acquired data;

– The quantity of the available data;
– The proper selection of the test organism: does it represent the receptor ecosystem
truly if not, what is the difference?
– The proper selection of the test type, the tested system and the end points;
– The realism of the model applied;
– The complexity of the situation, e.g. mixtures of contaminants, natural land
loaded by anthropogenic impacts, climatic changes, unexpected meteorological
conditions, extremely sensitive ecosystems, etc.
– The fit of the conceptual model to the scope and the aim of the management.

That is why impact prediction methods have been intensively developed in the last
25 years. Potential impacts, i.e. environmental risk posed by chemical substances to
the ecosystem and man can be assessed using mathematical models, chemical analysis,
biological and ecological testing and their combinations.

11 CONCLUSIONS

This chapter provides an overview of toxicity data evaluation and interpretation.

Toxicity data should be used for risk assessment and decision making in risk man-
agement. The generic risk assessment of existing and new chemicals and the targeted
risk assessment of contaminated environment are discussed, covering both human and
ecological risks. Definitions of the most frequently applied terms are also given. The
534 Engineering Tools for Environmental Risk Management – 2

flow of toxicity data starts at data acquisition followed by data transformations and
evaluations up to the use of the derived data in risk management.
A test end point can be determined by mathematical statistical methods from the
measurement end points (e.g. lethality, inhibition rate, slope of the growth curve, etc.).
The selection of the suitable test end point and the further evaluation and interpretation
of the results depend on the aim and concept of the assessment. The test end points such
as ECx or NOEC can be further extrapolated to other taxa or the entire ecosystem:
from laboratory microcosms to field, from field to field, to other temporal or spatial
conditions or to special cases of ecosystem adaptation or recovery.
Examples are used to introduce typical measurement and test end points and
explain the evaluation and use of the toxicity results. Uncertainties as intrinsic part
of hazard and risk and the essential statistical tools for their management are empha-
sized and discussed in detail. The extrapolation steps for generating hazard values for
human populations and the ecosystem are explained and demonstrated by examples.
In addition to hazard and risk assessment based on the chemical model which
measures the contaminant concentration and predicts the adverse effects from this
value, direct toxicity assessment and the use of DTA results for decision making are
introduced. Arguments are listed on decision making that is based on toxicity values
measured directly on environmental samples. This helps avoid high interpretational
uncertainty of chemical analysis.
The direct toxicity-based approach adopted nowadays is hard to integrate into the
chemical-model-based environmental management theory and practice that uses con-
centrations, threshold values and other EQCs expressed in terms of concentration as
well as monitoring by chemical analysis, etc. Direct toxicity assessment (laboratory or
field tests) should have priority when the cumulative effect of several toxicants (includ-
ing metabolites or degradation products) has to be measured and when the impact
of the environmental matrices and their living part is significant. The complexity of
the response of an ecosystem needs site-specific effect assessments; the puzzle of the
environment cannot be compiled only from models and generic data. The relationship
between a certain area’s character and generic characteristics is also important informa-
tion for environmental decision making. Response of key species must be known when
identifying representative indicator organisms. Proper indicator organisms ensure effi-
cient environmental monitoring from the point of view of information and expenses.
Specific screening values or other quality criteria can be established based on the key
species.
The calibration curve, i.e. any test end point plotted as a function of 4CP or Cu
and the calculated toxicity equivalent (an environmental concentration expressed in
terms of the equivalencing compounds 4CP and Cu) help to understand and inter-
pret the toxicity-based characterization of environmental contaminants. On the other
hand, the same test can provide the dilution rate necessary to reach the no-effect dose
or concentration of an environmental sample. This supports decision making on the
necessity and rate of environmental risk reduction.
According to the results discussed in this chapter, the calibration tool can character-
ize and integrate the effects of different contaminants and mixtures on test organisms
of different sensitivity. Unidentified or unidentifiable contaminants can be handled
with the help of this tool. Direct toxicity assessment can compare the response curves
of the equivalencing chemical with that of the unknown contaminant and provide
 Data evaluation and interpretation in environmental toxicology 535

information about its nature and effect mechanism. The toxicity equivalent method
enables the different bioassay results to be compared with one another and simultane-
ously evaluated. TEQ results can be used for quantitative risk assessment and decision
making by calculating the quantitative risk and the target concentration.
Even if equivalent toxicity, risk value or target toxicity values do not contain more
information than the measured toxicity data, information in the form of TEQ may be
valuable. Comparability and aggregability enable the uniform use of directly measured
toxicity results, similar to the effect data of known components. The understanding
and handling of toxicity of environmental samples as an effective concentration (EC)
value allow the integration of toxicity into the quantitative risk assessment procedure
which is based on the PEC/PNEC of the contaminant. The equivalencing approach
bridges the gap between the chemistry-based environmental management and the direct
toxicity-testing-based approach and may support professionals in integrating scientific
and practical/engineering knowledge.
This chapter provides the fundamentals of evaluation and use of toxicity data
for environmental risk management. Best management practice can be achieved when
decision makers harmonize the effect-based assessment with the management concept
and select the best fitting test methods and evaluation tools.

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XLSTAT Introduction (2014) [Online] Available from: http://www.youtube.com/watch?
v= 4AZ8G_MqyiM&list=PLFAD0C5745D4A1F60. [Accessed 4th August 2014].
Subject index

1,2,3,7,8,9-hexachlorodibenzo-p-dioxin adrenaline 177

(HxCDD) 467 adsorption 72, 83, 104, 208, 263, 264, 328,
2,2-dibromo-3-nitril-propionamide (DBNPA) 346, 360, 383, 387, 403, 522
293, 298, 459 adverse effects 1–23, 23–34, 41–44, 48, 51,
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 53–54, 57–62, 71–73, 81, 84, 88,
467 126–132, 136, 145–146, 148–149, 154,
3R in animal testing 20, 135, 137 159–160, 171–174, 177, 179, 180–222,
4CP (4-chlorophenol) equivalent 375, 381, 229–238, 246, 255, 259, 261, 264,
382, 468–481, 534 266–269, 274, 280, 283, 292, 300, 312,
absorption 21, 28, 72, 83, 102, 136, 151, 323, 337, 341, 343, 349–354, 362–363,
154, 156, 157, 158–159, 275, 313, 354, 366–367, 374, 378, 380, 391, 403, 405,
383–384, 391, 403 411–412, 419, 426, 435, 445–449, 453,
abundance 176, 188, 192, 198, 203, 219, 456, 463–467, 470, 472, 473, 477, 482,
237, 241, 253, 255, 262, 265, 272, 273, 484, 508, 513–518, 521–522, 524,
287, 288, 316, 512, 515 525–529, 531–534
Acari (mites and ticks) 45, 236, 247, 248, aerobic (bio)degradability 90, 93, 94, 209
249, 251 aerobic (bio)degradation 80, 86, 91–95, 98,
accumulation 5, 12, 49, 55, 57, 102–113, 261, 262, 264, 411, 417, 460
116, 117, 119, 231, 354, 357, 371–375, aerobic/anaerobic respiration 91, 281
438, 440, 505, 516 aerobic heterotrophic bacteria/cells 100, 265,
acetylcholinesterase 38, 177, 190 427
acid mine drainage 430 aerobic mineralization 91
acid rain 39, 191, 229, 343, 359, 365, 369, aerobic organisms/bacteria 208, 239, 241,
379, 349
acid rock drainage 430 aerobic soil 241, 264, 386
acute toxicity 21, 25–27, 44, 57, 60–61, 109, aerobic wetland/sediment 118, 220, 448
127, 136–138, 197, 201–202, 210, 249, facultative anaerobic microorganisms 349
261–264, 274, 279, obligate anaerobic microorganisms 349
actinomycetes 241 agarose 143–144, 277
activated sludge 16, 80, 93, 197, 208–209 agricultural chemicals 183, 286
acute systemic toxicity 136, 137, 138, 152 agricultural ecosystem/agroecosystems 16, 17
acute toxicity 21, 25, 26, 44, 57, 60, 61, agricultural land use 125
109, 127, 136, 137, 197, 201, 202, 210, agricultural nutrients 10
249, 261, 263, 264, 274, 279 agricultural soil 116, 242, 267, 329, 330,
adaptation 31, 84, 88, 90, 94, 96, 148, 218, 413, 415, 424, 435, 439
231, 232, 238, 241, 250, 258–259, 273, agriculture 231, 243, 324
277, 280, 291, 312, 417, 456, 463, 465, agrotechnologies 439
486, 518, 527, 534 airborne particles/contaminants 229, 383
546 Subject index

air toxicology 7 aquatic toxicity tests 49, 72, 195, 196–222,

Ajka 413, 435 261, 299
algae 26, 47, 57–59, 108, 118, 180–182, aquatic vertebrates 175, 190
188, 190, 197–200, 210, 218, 403, 456, arachnids (spiders, mites, ticks, scorpions)
504, 517, 519 251–252
alkylating compounds 148 arsenic 312, 384–389, 482
alkylation 328 Arthrobacter 211, 240, 262, 264, 277
alkyl benzenes 478 arthropods 193, 201, 248, 250–252
allergens/allergic compounds 34, 35, 155 aspiration toxicity 25
allergic response/diseases 128, 154 assessment factor 40, 42, 515–518, 520–522,
allergization 125 529
alternative test methods 12, 15, 18, 20, 23, atmospheric deposition 229
127, 137, 152, 523 ATP 49, 101, 138, 155, 182, 235, 266
Ames mutagenicity test 132, 141–143, 181, attenuation rate 464–466
380, 391, availability 30, 45, 58, 71–74, 83, 105, 109,
Ames fluctuation test 199 114–117, 173, 207, 233–236, 241–242,
amphibians 133, 140 267–268, 270, 337, 339, 341, 343, 344,
346–350, 353–354, 355–362, 366,
amphipods 110, 186–187, 193, 201, 208,
369–371, 379, 380, 383–386, 426–427,
519
436, 449, 478, 482, 522, 527
anaerobic (bio)degradation 86, 91–95, 98,
avoidance 8, 211, 236, 254, 263, 270–271,
209, 261, 262, 264, 411
274–275, 279, 526
anaerobic microbes/bacteria 83, 182, 197,
Azomonas agilis 180–181, 239, 276–277,
209, 241, 338, 349
293, 298–299, 459–460, 479, 481
anaerobic sediment 208, 340, 353,406, 448
Azotobacter 239, 277, 299, 368
anaerobic soil 241, 242, 264, 386
animal studies 18, 20, 23, 31 baboon (Papio hamadryas) 135 Bacillus 182,
animal testing 1, 19, 20, 21, 22, 43, 127, 240, 277–278
130, 131, 135, 137, 144, 151, 154, 155, BAF (bioaccumulation factor) 103
160, 254 BALB/c 3T3 – mouse fibroblast cell line 132,
ANOVA 62–63, 437, 486–488, 491–493, 138, 145, 147, 164
503, 505, 509, 511–513 bauxite 2
antibiotics 253, 278, 323, 330 BCF (bioconcentration factor) 33–34, 59, 85,
ants 252, 253, 103–105
aquatic bioaccumulation 103, 104–105 beetles 248, 252, 261, 286
aquatic ecosystem 3, 5, 7, 27, 40, 47, 57, 71, bees 252–253, 261
74, 80, 90, 93, 108, 171–174, 177, benthos 175, 204
187–190, 194, 199, 203, 206, 216, 340, best management practice 535
341, 361, 369, 403–412, 465, 517–518, bioaccessibility 204, 337–400
528 bioaccumulation 27, 33–34, 37, 45, 50, 73,
aquatic (eco)toxicity/(eco)toxicology 7, 75, 84–85, 102–113, 116, 117, 128, 129,
25–29, 41, 58, 64, 114, 171–228, 273, 158, 195, 207, 214, 218, 229, 237, 243,
347, 520 251, 272, 280, 287, 289, 351, 352, 354,
aquatic hazards 174 356–358, 363, 371–375, 427, 506, 522
aquatic mesocosm 220, 402–412 bioaccumulative chemicals of concern
aquatic microcosm 196, 218, 402–412, (BCC)/bioaccumulative substances 33–34,
aquatic organism/species 27, 28, 34, 71, 84, 84–85, 104, 111
102, 105, 107, 110, 133, 171, 176, 180, bioaccumulative potential of chemicals 35,
185–193, 196–222, 247, 264, 268, 270, 45, 84–85, 102, 104–108, 236, 514
299, 465, 515, 520 bioassay 1, 36, 44, 47, 50, 72, 74, 88, 109,
aquatic test organisms 180, 203, 247, 299 113, 133, 147, 161, 173, 178, 180, 182,
 Subject index 547

183, 186, 205, 207, 211, 212–215, biosensors 214, 216–217

220–222, 231, 234, 236, 237, 250, biostabilization 84
255–264, 267, 269, 270–272, 275–285, biosynthesis 100, 242, 266
292, 357, 361, 363, 371, 372–374, 402, biota 16, 36, 37, 60, 74, 77, 84, 101, 171,
445, 449, 451, 463, 466, 467–468, 486, 231, 235–238, 256, 265–267, 269–271,
535 285, 296, 339, 361, 391, 412
bioaugmentation 421 biota-sediment accumulation factor (BSAF)
bioavailability 7, 73, 74, 91, 100–106, 108, 104
110, 111, 114, 117, 157, 164, 171, 204, biotensides 88, 105, 204, 235, 268, 338,
214, 229, 268, 312, 323, 337–400, 401, 341, 349, 353, 375, 377
412, 414, 416–418, 520, 423, 446, 448, biotransformation 28, 37, 77, 84–87, 102,
451, 467–468, 477, 522, 527 111, 137, 151, 158–159, 190, 344, 346
bioavailability models 312, 356–358, bioturbation 111, 346
bioconcentration 33–34, 57–59, 84, 102, bioventing 421
103–105, 110–111, 174 birds 46, 108, 133, 140, 175, 176, 187, 254,
bioconcentration factor (BCF) 33–34, 59, 85, 259, 272, 517
103–105, 195, 356 bisphenol A 35–36, 407, 475
biodegradability 26, 45, 52, 53, 58, 73, 74, Bivalvia (molluscs) 192
84, 84–102, 114, 116, 207, 209, 236, 261, BMF (biomagnification factor) 104–105
273, 274, 280, 351, 354, 356, 374–378, BOD (biological/biochemical oxygen
411, 418, 460–464, 522 demand) 58, 90, 94, 96, 461
biodegradation 27, 36, 37, 45, 49, 52, 53, boric acid 245, 319
58, 71, 73–75, 77, 80, 82, 84–102, 114, Bragg reflection 320
115, 117, 119, 181, 195, 199, 208, 218, Bragg’s law 321
235, 236, 238, 240–242, 246, 262, 264, Bremsstrahlung radiation 318–319
266, 268, 272, 290, 338, 344–346, 349, BTEX 330, 478, 480
352, 354–356, 363–367, 374–378, Bufo marinus (terrestrial toad) 133
401–406, 410–422, 448, 460–464, 522, bull frog (Rana catesbeiana) 133
527
biodegradation-based remediation 416 Caco-2 cells 159, 391
biodegradation-based soil remediation 422 cancer 31–32, 35, 128, 129, 134, 141, 146,
biodiversity 30, 174, 176, 188, 219, 255, 530
256–259, 285, carbon substrate 273
bioindicator 189, 213, 216, 245, 253, 256, carbon substrate utilization patterns 273–274
427 carboxymethylcellulase 242
bioleaching 84, 113–114, 120, 363, 401, carcinogenic effects 31, 128, 129, 145
415, 428–438 carcinogenic risk 526
biological/biochemical oxygen demand carcinogenicity 22, 25, 31–32, 38, 44, 50,
(BOD) 58, 90, 94, 96, 461 51, 55, 125, 128, 130, 138, 141, 142, 145,
biology-based methods 490, 505–507 146–148, 530
bioluminescence 181, 240, 375, 381 carcinogens 11, 26, 31–32, 85, 129, 141,
biomagnification 33, 84, 102, 104–105, 110, 142, 145, 147, 388, 530
174, 285 Carlson Index 176
biomagnification factor (BMF) 104–105 cats 135
biomass 48, 94, 117, 176, 192, 209, 219, cell-based technologies 13, 15
220, 230, 231, 235, 245, 262, 271, 280, cellulase 31, 242, 266, 280
292, 371, 436, 452, 456–460 cellulose-degrading bacteria 241
biomonitoring 44, 51, 108, 111, 173, 192, chemical time bomb 343, 350, 366,
201, 213, 236 368–369, 374, 380
bioprecipitation 120 chemiluminescence 155
bioreporter genes 216 cephalopod molluscs 192
548 Subject index

Ceriodaphnia dubia 186, 201–202, 208, cornea 155, 157

211, 216 corneal opacity 156–157
chemical models 1, 3, 39, 60, 73, 171, 173, corrosivity (skin or eye) 8, 20, 24, 25, 55,
177, 179, 233–234, 272, 341, 343, 346, 125, 128, 129, 152–155, 155–157,
352, 355–362, 378, 383, 384–391, 446, cosmetics 10, 12, 23, 24, 53, 134, 154
448–449, 528 crickets 252–253, 286
chemical oxidation and reduction 83 crustacea (crustaceans) 26, 39, 40, 45, 47,
Chilopoda (centipedes) 252–254 180, 185–188, 193, 199, 201, 208, 211,
chimpanzee (Pan troglodytes) 135 216, 219, 247, 251, 253, 519, 521
Chironomidae (midges) 110, 188, 197, 208 customized test methods 53–54
Chlamydomonas reinhardtii 183 cyanobacteria 182, 197, 264, 265
Chlorella kessleri 182 cyclodextrin, 345, 356–357, 375, 379, 380,
Chlorella vulgaris 196–198 407, 414, 417–418, 421
chlorophyll 49, 93, 185, 219, 235, 245 cytotoxicity 20, 132, 137, 138, 144, 153,
chromium 83, 240, 323, 386, 383 160, 164, 203, 391
chromosome 32, 50132–133, 141–142, 148,
188, 245
Dabbling duck 133
chromosomal damage 38
Dangerous Substance Directive (DSD) 209,
chronic toxicity 25–27, 34, 44, 62, 85, 125,
260
127, 130, 136, 139–140, 145–147, 180,
Danio rerio (zebrafish) 191, 192, 202
190, 193, 201–202, 211, 247, 262, 279,
Daphnia 48, 49, 55–59, 186–187, 197, 201,
363, 514, 518, 520
202, 208, 209–217, 221, 299, 348, 361,
cis-regulatory elements 246
469, 474–476, 503, 517
cladocerans 176
Daphnia magna 59, 186–187, 197, 201–202,
clinical toxicology 7
208, 210, 216, 361, 469, 474–476
closed-bottle test/technique 94, 95, 97, 116,
Daphnia pulex 186, 202, 208, 216
283, 284, 363
closed reactor systems 411 Daphnia pulicaria 186
cockroaches 252 data analysis 62, 175, 486, 491–492,
collembolans 212, 247, 250, 259–261, 263, 508–535
265, 274, 279, 293–294, 299, 465, 469, DBNPA
478 (2,2-dibromo-3-nitril–propionamide) 293,
Comet assay 142–144, 148 298, 459
compaction 174, 231 DDE (dichlorodiphenyldichloroethane) 109
computational toxicology DDT (dichlorodiphenyltrichloroethane) 357
concentration–response relationship/curve dechlorination 241,
40, 42, 48, 60, 62, 178, 198, 281, 284, decision makers 111, 161, 259, 521
298–299, 445, 453–454, 461, 464, decision making 173, 178, 198, 206–208,
469–472, 475, 477, 490, 492–493, 499, 214–216, 221, 232–233, 259, 267, 354,
510 378, 380, 401, 402, 422, 435, 447, 449,
conceptual model 79, 179, 235, 265, 533 465–467, 478, 498, 513, 517, 521, 522,
conjunctiva 155, 157 527, 531, 533–535
conjunctival cell model 157 decision-support tools 508
contact bioassays 275–282, 363 dehydrogenase 92, 116, 182, 235, 262, 264,
contaminated land 1, 9, 43, 51, 71, 108, 276, 367, 419, 478–479, 482–483
118, 125, 243, 287, 311, 449, 464, 477, denitrification 266
482, 526, 528 dense non-aqueous phase liquids (DNAPL)
copepods (crayfish) 185, 187, 201, 519 73, 345, 350
copper 39, 42, 254, 297, 312, 341, 384, 386, derivatization 328–329
428, 468, 482 dermal absorption/uptake 151, 207, 212,
copper equivalent 28, 445, 468–483, 534 348, 382–383
 Subject index 549

dermal contact 7, 17, 25, 43, 48, 103, 287, 288, 339, 343, 366–367, 401–405,
125–127, 136, 147, 158, 293, 515 412, 419, 427, 440, 447, 512, 520, 528
dermal irritation/corrosion 20, 25, 128–130, diversity assessment 30, 237, 257, 259
152–154 diversity index 38, 45, 178, 192
dermal penetration 151, 154 DNA 6, 8, 12–14, 17, 29–31, 37–38, 44, 47,
dermal toxicity 20, 26, 130, 137–139, 146, 102, 128, 132–133, 141–148, 176–177,
152, 153, 159–160 217–218, 237–238, 246, 256–257,
dermatitis 154 260–262, 265, 291, 530
Desmodesmus subspicatus (former name DNA damage and repair 132, 142–144, 148,
Scenedesmus subspicatus) 182–183, 210 176, 530
desorption 74, 75, 81, 83, 119, 207, 264, DNA markers 17
268, 338, 347, 349, 350, 355, 385, 426 DNA-SIP method 30
detergents 10 dogs 22, 135, 145
detritus 190, 246 dose–response relationship/curve 2, 28, 32,
developmental disabilities/disorder/failures 34, 47, 60, 129, 146, 222, 267, 269, 281,
38, 129, 489 445, 447, 449, 451, 453–456, 463–467,
developmental toxicity 32, 38, 128, 133, 470–473, 475, 477, 484, 494–501,
139, 148–150, 160, 252, 526 503–504, 508–510, 530
diaminopimelic acid 265 drinking water 7, 8, 48, 81, 126, 349
dibenzofurans 331 Drosophila 45, 133, 142
dibutylphthalate 35, 475 Drosophila melanogaster (common fruit fly)
dichlorodiphenyldichloroethane (DDE) 109 45, 133, 142
dichlorodiphenyltrichloroethane (DDT) 357 drugs 7–10, 13–15, 28, 33, 35, 131, 135,
dichloromethane 95, 329, 331, 357 143, 157, 160, 174, 345, 383, 387, 475
diclofenac 35, 36, 475 DSD (EU Dangerous Substance Directive)
dicotyledonous 184, 185, 242 209, 260
di-(2-ethylhexyl)phthalate 35 DTA (direct toxicity assessment/testing) 3,
diesel 98–99, 259–260, 293–295, 416 64, 222, 235, 300, 374, 378, 445–449,
digestion 17, 25, 95, 112, 312, 323, 337, 467, 477, 482, 534–535
348–350, 354, 382, 384–387, 391 duck 133, 140, 254
digestion model/test 384–391 duckweed 184–185, 200, 211, 507
dilution 28, 42, 75, 77, 96, 100, 117–118, dung beetles 252, 261
129, 221–222, 280, 293, 299, 390, 445, dung flies 261
447, 448, 451, 454, 464, 469, 471, 474, dung-dwelling organisms 274
475 Duncan post hoc test 437
dilution factor 317 dye 143, 148, 164, 262, 273
dilution rate 43, 219, 222, 447, 448, 454, dynamic testing 177, 344, 363–366,
464–466, 472–473, 534 378–380, 418, 420
dilution series 40, 100, 101, 196, 212, 221,
269–271, 276, 279–282, 294, 299, 447, early warning 29, 46, 51, 177, 212, 213,
452, 453, 468, 470, 477 217, 237
dioxins 331, 390, 467 earthworms 59, 105–106, 210–211,
Diplopoda (millipedes) 252, 254 250–252, 254, 260–261, 263–264, 269,
Diptera (flies) 133, 188–189, 252 274–275, 354, 356–357, 363, 391, 466,
direct toxicity assessment/testing (DTA) 3, 517
64, 222, 235, 300, 374, 378, 445–449, Echinodermata 194, 208, 521
467, 477, 482, 534–535 E. coli 30, 45, 132, 142, 182, 257, 277
diversity 7, 23, 29, 38, 45, 47, 94, 172, 174, ecological assessment 149–150, 176–178,
176, 180, 188, 192, 196, 201, 205, 192, 194, 201–203, 231, 245, 253,
217–219, 230–232, 236–238, 241–242, 254–256, 272–274, 286, 291, 353, 366,
246, 255–262, 265, 272–273, 282, 285, 385, 528, 533
550 Subject index

ecological functions 245 environmental compartments 3–15, 33, 50,

ecological indicator 176 54, 62, 71, 74, 77–78, 112, 114, 118, 129,
ecological levels 29 173, 217, 233, 274, 329, 330, 401, 449,
ecological methods/technologies/tools 84, 515
174, 179–180, 220, 234, 261, 343, 402, environmental concentration 27, 34, 57, 58,
404 77, 80, 88, 105, 108, 311, 445, 448, 521,
ecological model 179, 229 522, 525, 529, 534
ecological relevance 63, 215, 247, 250, 274, environmental effects 12, 18–19, 58, 190,
286 191, 214
ecological risk 36, 215, 254, 286, 370, 385, environmental fate 19, 35, 51, 55, 57, 59,
526, 528, 533 71–73, 75, 83, 84–120, 236, 312, 341,
ecological survey 55 391, 447, 467, 514, 516, 529
ecological status 176–177, 192, 195 environmental impacts 514, 531
ecological system 3, 5, 9, 62, 118 environmental interventions 412
ecosystem services 5, 174, 230, 236 environmental (risk) management 1–64,
ecotechnologies 119, 404, 407 71–120, 125, 171, 173, 178, 194, 260,
ecotoxicity 9, 17, 25, 36, 39, 41, 55, 58, 74, 311–312, 337, 341, 362, 392, 402,
119, 136, 173, 178, 183, 186, 192, 195, 448–450, 466, 516, 525, 526, 531, 534,
206, 209–211, 221, 232, 234, 237, 535
250–251, 273–274, 280, 282, 292, 300, environmental monitoring 51, 171, 198, 342,
343, 353, 366, 425, 463–465, 503, 518 367, 449, 526, 527, 534
ecotoxicogenomics 29 environmental pollution 7, 133, 345
ecotoxicology 9, 17–18, 28, 29, 173, 183, environmental phases 74–81, 108, 337, 355,
187, 212, 229, 238, 245, 257, 274, 347, 401
348, 408, 437, 493, 515 environmental quality criteria 4, 40, 51, 446,
EDTA (ethylenediaminetetraacetic acid) 358, 448, 467, 469, 473, 516, 518, 520, 521,
379, 386, 426, 432, 436 531, 532
Eisenia foetida 210–211, 251, 261, 263–264, environmental reality 53, 55, 56, 113, 179,
356 236, 255, 272, 343, 466
electron acceptors 90–92, 100, 116, 182, environmental relevance 3, 30, 54, 89, 195,
241, 276, 349, 350, 418, 421 215, 272, 275, 391, 445
electron capture detection 328–329 environmental risk 3–64, 71, 84, 108, 117,
electron donors 291, 365 119, 173, 198, 232, 337, 345, 355, 358,
electron source 319 367, 378, 428–429, 434, 446, 525,
electron transport 49, 83, 90–92, 182 531–533
Elodea canadensis 185–186 environmental risk assessment 3, 4, 36,
embryotoxicity 149 71–73, 90, 93, 274, 285, 341, 354, 367,
emerging pollutants 17, 34–35 383, 445, 521, 513–535
endocrine disruption 8, 25, 35, 125, environmental technology 411, 418
128–130, 139, 161–164, 176, 235, 363, environmental stress/stressors 245–246, 254
530 environmental toxicity/toxicology 1–64,
endocrine disruptor 34–35, 128, 161–164, 71–120, 171–222, 229–301, 311, 354,
177, 180, 201, 367, 386, 401, 445–535
endocrine disruptor screening program environmental transport 75, 77–78, 117–120
162–164 enzyme 12, 17, 28, 31, 37, 38, 42, 49, 80,
endocrine screen 162 82, 87, 90–92, 95, 102, 104, 116, 140,
endocrine system 5, 17, 29, 128, 164, 235, 148, 158, 162, 176, 181, 182, 195, 217,
477 233, 235, 236, 238, 240, 242, 245, 256,
endocrine toxicity 161–164 262, 265, 266, 280, 282, 341, 348, 349,
enhanced biodegradation 375, 416–418, 421 351, 352, 354, 375, 384, 391, 404, 419,
environmental assessment 3, 171 456, 482, 483, 486, 501, 502, 530
 Subject index 551

enzymatic (bio)degradation 36, 91, 338 125–126, 141, 163, 174–176, 178, 180,
enzymatic hydrolysis 82 182, 186, 188, 190–194, 197, 199,
Ephemeroptera (mayflies) 188 201–203, 213, 216, 219, 221, 247, 299,
epidemiology 8, 508, 511 348, 355, 404, 427, 446, 496, 503,
equivalent toxicity 28, 449, 468, 472, 505–507, 513, 517, 519–521
473–483, 535 flammable substances 11, 81
ergosterol 265 flotation 368–369, 374, 403
erosion 16, 77, 231, 270, 285, 368, 369, 413 Folsomia candida 212, 250, 263, 275, 279,
erythema 154 293, 294–295, 381, 437, 469, 478,
Escherichia coli 30, 45, 132, 142, 182, 257, 480–481
277 food additives 10, 18, 23, 25, 529
esterase 38, 176, 190, 266 food chain 1, 17, 23, 30, 33, 39, 47, 62, 84,
estrogen 132, 161–164, 197 87, 103, 104, 107–109, 111, 113, 118,
ethidium bromide 144 125, 174, 180, 187, 195, 198, 201, 218,
ethylene 246 231, 236, 243, 253, 254, 272, 278, 343,
ethylenediaminetetraacetic acid (EDTA) 358, 349–350, 368, 370, 515, 522, 528
379, 386, 426, 432, 436 food crops
eukaryotes 183, 216 food toxicology 7food web 23, 39, 47, 62,
eukaryotic cells 183 108, 111, 176, 180, 198, 236, 238, 246,
eudicot 243 247, 252, 256, 282, 285, 338, 349–350,
eutrophication 39, 118, 176, 179, 182, 219, 368, 515, 522, 527
405 forensic toxicology 8
EU Dangerous Substance Directive (DSD) fresh-water invertebrates 185–190
209, 260 fresh-water macroplants 184–185
EU waste directive 527 frog 133, 149, 163
evapotranspiration 174–175, 235, 429 frog embryo teratogenesis assay (FETAX)
evidence-based toxicology 23, 131, 136 133, 149
evolutionary convergence phenomenon fuels 24, 230, 243
258–259 fungicides 219, 286, 323
excitation 313–315, 318–322
exoenzyme 82, 88, 240, 270, 348, 353 gammaproteobacteria 182, 238
expert systems 22 gamma rays 81
explosives 10–11, 81 Gammarus 188, 208
exposure assessment 524 gastrointestinal microorganisms 388
eye corrosion 8, 25, 55, 125, 128, 129, gastrointestinal models 383–391
155–157 gastrointestinal tract 135, 157, 337, 354,
eye irritation 25, 128–130, 155–157 383–390
Gastropoda (snails and slugs) 190, 192, 254
fertility 9, 32, 128, 148, 263, 339 gene arrays 256–257, 265
fertilizers 10, 208, 218, 466 gene expression 14, 15, 29, 31, 176, 257
FETAX 133, 149–151 genetic bioindicators 216
field assessment 36, 39, 41, 107, 108, 112, genetic diversity 30
174, 180, 195, 204, 229, 236, 255, 259, genetic methods/techniques 102, 180
261, 269–272, 286, 447, 450, 522, 529, gene/genetic mutation 132, 142, 145, 150,
531 181, 486
field testing/tests 36, 107, 108, 111, gene mutation assay 132, 142
112–113, 251, 264, 269, 272–275, 284, genetic potential 34, 91, 119
287, 365, 411, 421, 466, 534 genetics 7, 9, 13, 28, 30, 38, 42, 45, 84, 87,
filtration 118, 327, 403, 404 128, 133, 134, 148, 229, 234, 247
fish 3, 8, 18, 20, 26–27, 33–34, 39, 40, 45, genetic toxicity/toxicant/toxicology 29,
47–49, 55–59, 103–105, 108, 110, 141–143, 14
552 Subject index

gene transfer 87, 217, 241 herbicides 182, 219, 243

genome 30, 31, 45, 84, 132, 189, 192, 217, heritable genotoxicity 141–142
237, 247, 249, 250, 257, 281, 299, 300 Heterocypris incongruens 186–187, 201
genomics 8, 13–14, 17, 21, 29, 140, 176, hl-60 human acute promyelocytic leukemia
177, 180, 245, 257, 265, 299 cell line 132
genotoxicity 141 histidine 142, 181
germ cell 2, 128, 141, 144 homeostasis 13, 34, 39, 135, 161
germ cell mutagenicity 128 hormone 17, 34, 35, 38, 55, 84, 161, 183,
germination 235, 242–245, 263, 279, 280, 323, 330, 530
296, 437 HPBCD (hydroxypropyl beta-cyclodextrins)
germination rate 245 116, 345
global warming 6, 179 human bioaccessibility 385–389
glucose 100, 141, 462–463, 506 human carcinogens 30–31
glucoseamine 265 human carcinoma cell line H295R 162
glutathione 8, 245 human cornea 157
glutathione transferase 8, 28, 245 human exposure 8, 32, 110, 125–126, 131,
glycomics 14 146, 158, 174–180, 231–232, 338,
gold 428 382–385, 530
goldfish 191
human health 8, 10–12, 19, 22, 34, 35, 149,
greenhouse 254 254, 323, 381–385, 389, 430, 435, 526,
groundwater 2, 50, 54, 73, 74, 77, 94, 95, 527–534
114, 117, 118, 125, 126, 174, 175, 210,
human health risk 25, 381, 382, 385, 389,
214, 230–235, 242, 269–270, 329, 341,
435, 530, 531
349–350, 354, 366, 409–410, 415–416,
human health risk assessment 130, 385, 389,
428, 438–440, 465, 473
510, 529
growth curves 198, 200, 222, 456–458, 464
human hepatocytes 144
guinea pig (Cavia porcellus) 46, 132, 134
human intestine Caco-2 cells 159
Gyöngyösoroszi 423, 430
human keratinocytes (NHK) 132, 137
habitat quality 272–276 human lymphocytes 133
hamster 133, 134, 144, 145, 147, 148 human reconstructed skin 133
hazard assessment 5, 7, 51–53, 71, 149, 153, human skin/epidermis cell model 151, 153
209, 253, 300, 341–343, 403, 449, human pathogen 189–190
513–522, 527, 531 human risk 131, 383, 526, 529
hazard categories/classes 34, 449 human toxicology/toxicity 7, 8–9, 17–18,
hazard criterion H14 209 20–22, 25, 29, 47, 125–164, 171, 254,
hazard identification 153, 515–516 353, 386, 505, 515, 521
hazard quantification 516 humic substances 345
hazardous bacteria 182 humic soil 381–382
hazardous biological agents 179, 182 humus 76, 100, 230, 233, m235, 250, 285,
hazardous chemical substances 2, 3, 7–12, 291, 338–340, 381, 384
23, 24, 34, 51, 53, 63, 72, 109, 125, 131, HxCDD
403, 446, 477, 515, 525, 527 (1,2,3,7,8,9-hexachlorodibenzo-p-dioxin)
hazardous waste 251, 274 467
hazard rate 498, 505–507 hybridization 102, 190, 217, 257
heat production 12, 38, 134, 229, 292–301, hydrocarbon 81, 96, 98, 115, 268, 293, 295,
456, 458–460 311, 312, 323, 329–331, 344, 357,
heat response 292–301 375–376, 416–417, 471, 478–481
heavy metals 183, 210, 313, 386, 430 hydrogen sulfide 111, 214
Henry’s law constant 57, 75–76, 78, 211 hydrolysis 26, 36, 52, 57, 71, 77, 81, 82–83,
hepg2 liver cell line (hepatoma) 132, 138 88, 104, 119
 Subject index 553

hydroxypropyl beta-cyclodextrins (HPBCD) in vitro sister chromatid exchange assay 132,

116, 345 141, 142
hyperspectral analysis 177 indicator organisms 44, 179, 180, 238, 256,
hypothesis testing 492–497 272–275, 379, 534
inhibition rate 222, 447, 451–461, 529, 534
ibuprofen 35, 475 insects (Hexapoda) 2, 8, 40, 45, 47, 107,
immobilization 75, 117, 118, 197, 289, 342, 188, 192, 201, 219, 250, 252–253, 263,
344, 411, 469, 474, 476, 485, 491 265, 286, 293, 375, 519, 521
immune disruptors 34, 55, 180, 235 integrated evaluation 204, 234, 355, 366,
immune effects 125 369
immune suppression 38, 176 integrated monitoring 51, 362, 401,
immune system 5, 17, 25, 29, 38, 84, 125, 418–421, 427, 436–437
128, 154, 477 International Organization for
immunology 134–135, 152 Standardization (ISO) 199–203, 263
immunotoxicity 8, 25, 29, 130, 138, 154 IPPC Directive (Integrated Pollution
infiltration 75, 77, 114, 270, 289, 413, Prevention and Control) 6
434–438 iris 155
inhalation 8, 17, 22, 25, 26, 28, 103,
IRIS (Integrated Risk Information System)
125–128, 136, 137–139, 146, 160, 231,
509
338, 348, 382, 383, 515
iron-bearing solid 430
Integrated Risk Information System (IRIS)
iron-oxidizing bacteria 430
509
innovation 15, 214 irritation 8, 20, 128–130, 152–154, 155–157
inorganic compounds 2, 33, 86, 94, 119, isolated cells 132–133
210, 311, 312, 313–323, 339–340, 383 isopods 187, 193, 251, 253, 519
inorganic contaminants 76, 115, 235, 311, IT tools 445, 507, 508–514
367, 381, 385, 388, 411, 445, 449, 469, ivermectin 274
477
inorganic matter 203, 208, 338, 350 Japanese quail (Coturnix japonica) 133, 254
inorganic micropollutants 230 jasmonate 246
insecticides 219, 240, 323 Joint Research Centre (JRC) 12, 109
insects 219, 240, 250, 252–253, 263, 265,
286, 293, 323, 375, 519, 521
ketoprofen 35, 475
in situ biomonitoring 44, 236
kitinase 266
in situ chemical oxidation 415
Klimish tool 522–523
in situ chemical stabilization 414, 423
in situ hybridization 102, 217 Kow (octanol – water partition coefficient) 20,
in situ lysimeter 430 27, 34, 55–59, 76, 85, 87–89, 103–108,
in situ sensors 418–419 158, 195, 203, 207, 348, 351, 355–357,
in situ toxicity assessment 214–217 378–379, 381, 383, 384, 391, 514
Integrated Pollution Prevention and Control
(IPPC) Directive 6 leachate 50, 51, 54, 73, 113, 118, 181, 196,
intermediates 92, 402 199, 221, 241, 247, 269, 270, 272, 279,
interspecies safety factor 8, 343, 363, 402, 404, 413, 427, 428–438,
invertebrate 110, 163, 175–176, 180, 185, 465, 4687
192, 201, 205, 212, 218–220, 231, 236, leaching 74, 77, 94, 114, 119, 261, 264, 269,
246, 252, 262, 272, 274, 283, 287–288, 270, 273, 275, 327, 355, 359, 368–370,
290, 358, 404, 520 386, 387, 413, 428–438
in vitro chromosome aberration test 132 leaching microcosm 428, 431–434
in vitro dermal testing 159 lead 33, 158, 159, 254, 359–360, 383–389,
in vitro gene mutation test 132 434, 482
554 Subject index

legislation 1, 5, 7, 18, 51, 129, 152, 177, mercury 33, 39, 83, 159, 174, 315, 350, 386
179, 192, 207, 260, 274, 362, 526, 529, mesocosm 3, 36, 44, 47, 53, 54, 62, 63, 64,
530 91, 112, 173, 177, 178, 180, 196, 201,
Lemna minor 184–185, 197, 200, 211 217–220, 231, 237, 259, 263, 2665, 282,
Lepomis cyanellus (green sunfish) 191 290–291, 402–405, 407–416, 420,
Lepomis macrochirus (bluegill) 191 421–422, 428, 466, 514, 522
life-cycle assessment 243 metabolism 5, 8, 9, 14, 20, 21, 28, 30, 39,
lime 379, 424 48, 87, 91, 98, 102, 105, 108, 113, 129,
limestone 119, 194 132, 137–140, 144, 145, 156–160, 221,
lipase 266, 387 235, 239, 241–242, 247, 264, 281, 287,
lipidomics/lipomics 14 291, 298, 300, 351
LLC-PK1 kidney proximal tubule cell line metabolism-mediated toxicity 140
132, 138 metabolites 5, 12, 17, 28, 46, 49, 50, 90, 91,
LOEC 41, 43, 44, 48, 60, 62, 195, 267, 278, 119, 144, 158, 173, 176, 195, 233, 241,
283, 300, 453, 454, 459, 464, 469, 245, 257, 282, 301, 323, 365, 391, 448,
470–483, 490–497, 518, 519, 524 534
luminescence 42, 49, 178, 210, 276, 368, metabolomics 8, 14, 17, 21, 29, 177, 180,
379, 380, 427, 437, 454–456, 469, 299
471–474, 476, 478–483 metagenome/metagenomics 29–30, 88, 177,
Lumbricidae (earthworms) 251 180, 217, 237, 242, 255, 256, 257, 259,
lysimeter 283, 289–290, 362, 363, 427–430 265, 281
metals 10, 24, 28, 39, 72, 86, 102, 104,
macaca monkeys 135 111–117, 118–119, 154, 174, 180, 182,
macro(zoo)benthos 176, 180, 192, 204, 219 183, 200, 207, 210, 240, 246, 254,
macroinvertebrate 175, 201, 205, 219, 404 277–278, 280, 289, 293, 313, 340, 343,
macrofauna 205, 238, 246, 250, 254, 257, 346, 348, 350, 352, 353, 355, 358–362,
259, 265 367, 368–374, 378–389, 390, 411, 415,
macrophyte 47, 184, 185, 201, 203, 404, 423–438, 462, 468, 470, 471, 479–483
521 metallothionein 38, 176, 236
macroplants 26, 184–186, 257 metaproteomics 30
macrozoobenthos 176, 180, 192, 219!! metatranscriptomics 30
magnesium 438 methane 81, 235, 349
mammals 9, 47, 108, 134, 135, 254, 255, mice (Mus domesticus) 22, 46, 132, 134,
259, 517 144, 145, 149, 154, 155, 515
mammalian cells 132–133, 141–142, 144, monocotyledonous 184, 185, 242, 243
147, 163, 203 microarrays 14, 15, 203
management options 404 microarthropod 236, 248, 250, 259, 286
mapping 241–215, 256, 278 micro(zoo)benthos 204
mathematical models 3, 18, 55, 130, 159, microbial activity 88, 92, 95, 99, 218, 235,
179, 272, 322, 343, 346, 351, 352, 238, 242, 273, 282, 293, 344, 413, 418,
354–355, 383–384, 446, 498, 531, 533 436, 446, 451
material balance 235, 506 microbial biomass 262, 292, 436
maturity index 247 microbial cells 33, 93, 100, 265
mazout 417 microbial community 30, 84, 99, 102, 216,
mdck dog kidney epithelial cell line 132, 138, 232, 241, 264, 268, 273, 283, 292, 293,
157 387, 403
medicines, drugs 7–9, 12, 14, 15, 18, 28, 33, microbial complexing agents 350
35, 131, 135, 143, 157, 160, 174, 345, microbial (bio)degradation 349, 354
383, 387, 475 microbial diversity 238, 241, 262
meio(zoo)benthos 176, 204 microbial ecology 29, 257
membrane-based extractions 356–358 microbial ecosystem 9, 387
 Subject index 555

microbial genome 30 337, 338, 342, 343–344, 348, 352, 354,

microbial growth 80, 276, 456, 458 355–370, 375, 378–383, 389, 411, 413,
microbial inoculant 376 418, 423, 425, 426, 435, 477
microbial oxidation 273 mobilization 8, 49, 59, 75, 88, 105, 118,
microbial soil respiration 262 204, 207, 208, 268, 271, 289, 344, 354,
microbiota 53, 88, 90, 91, 96, 97, 99, 208, 362–364, 369, 375, 376, 378, 380, 384,
209, 229, 236–238, 241, 246, 256, 257, 411, 415, 432, 435
267, 271, 273, 280, 282, 289, 290,291, molecular initiating event 145–147
293, 301, 338, 339, 345, 349, 350, 354, molecular methods 1, 14, 17, 18, 180, 212,
362, 363, 365, 375, 376, 378, 380, 411, 217
413, 416, 418, 427, 446, 447, 460, 463 molecular technologies 13–15, 29
microcalorimetry 229, 292–301 molluscs 192–193, 254, 519, 521
microcosm 3, 15, 16, 39, 44, 47, 49, 62, 63, monitoring 1, 4, 5, 6, 10, 13, 36, 44, 46, 47,
91, 95, 111, 114, 173, 178, 180, 196, 206, 49, 51, 55, 56, 63, 107, 109, 119, 149,
217–220, 231, 236, 237, 259, 263–264, 171–177, 182, 194, 198, 206, 212–213,
266, 269, 272, 282–291, 362–366, 375, 230, 232, 234, 236, 237, 242, 247, 252,
379, 401–440, 4455, 466, 529, 534 256, 265, 266, 269, 273, 280, 288, 291,
microcosm models 401–444 311, 312342, 344, 353, 362, 366, 367,
microcosm set-up 406, 414, 418, 422, 372, 374, 401, 402, 411, 413, 416,
424–425, 431–432, 438, 439–440 418–421, 423, 425–427, 431, 432–433,
microcosm test 196, 217–220, 264, 283, 435, 436–437, 440, 446, 449, 461, 466,
401–403, 411, 420, 439–440 467, 526–528, 531–534
microflora 7, 29, 39, 47, 53, 88–90, 92, 209, most probable number 100, 116
217, 235, 238, 242, 246, 250, 253, 259, mudpuppy 133
262, 267, 363, 440 multispecies tests 47, 63–64, 196, 218, 265,
microfungi 100, 242, 265 282, 283, 290–291
microinvertebrate 218, 236 multispecies toxicity tests 63–64, 290–291
microlysimeter/minilysimeter 114, 289, 427 multi-step sequential extraction 359
micronucleus test 133, 141, 142, 145, 148, muramic acid 265
260, 263 mussels 3, 206, 208, 212–214, 215
micropollutants 174, 230 mussel monitor 212
microprobes 216 Myriophyllum aquaticum 184, 263
microsomes 140, 159, 163 mutagenicity 8, 25, 31, 32, 35, 44, 50, 51,
microwave 95 55, 125, 128, 130, 141–148, 181, 343,
microwave assisted extraction 313, 325–326, 363, 379, 380, 390, 391, 515
330, 331 mutagenic effect/risk 31, 32, 38, 84, 87, 128,
microzoobenthos 204 201, 379, 391, 526
mineralization 86–87, 91, 93, 98, 235, 237, mutagens 11, 26, 32, 85, 129, 141, 142, 145,
238, 240, 241, 242, 246, 262, 267, 287, 380, 530
291, 404, 460 mutants 50, 87, 134, 142, 180
mineral oils 375 mycorrhiza 235, 263
mine excavation 350
mine drainage 404, 434, 404, 430, 434 naproxen 35, 475
miner bacteria 113 natural attenuation 73, 117, 119, 410, 411,
mine tailing 368 414
mine waste 10, 112, 288, 372, 374, 380, natural biodegradation 416–417, 421
413, 423–424, 427–434 nebulization 313–314
minilysimeter/microlysimeter 114, 289, 427 Nematoda/nematodes (roundworms) 45,
mites 26, 45, 236, 248–252, 261 188, 247, 263
mobility 7, 55, 71, 73, 74, 88, 110, 112, neurobiology 133
117, 119, 173, 201, 203, 204, 229, 235, neuroblastoma cells 157, 160
556 Subject index

neuropharmacology 135 occupational risk/hazard 7

neurophysiology 135 occupational toxicology 7
neurotoxic agents/substances 159 octanol – water partition coefficient (Kow ) 20,
neurotoxic effects/risk 17, 526 27, 34, 55–59, 76, 85, 87–89, 103–108,
neurotoxicity/neurotoxicology 8, 17, 21, 29, 158, 195, 203, 207, 348, 351, 355–357,
25, 55, 127, 128, 130, 138, 149, 159–161, 378–379, 381, 383, 384, 391, 514
351 OECD guidelines 83, 93, 110, 142, 146–151,
nervous system 20, 29, 38, 128, 138, 159, 153–160, 196–198, 260–261, 493
160
Oligochaeta (worms) 110, 189–190, 208
nickel 315, 386, 520, 521
nicotine 35, 474–477 Onchorhynchus kisutch (coho salmon) 190
nitrates 216, 241 Onchorhynchus mykiss (rainbow trout)
nitrate respiration 241 190–191
nitric oxide 246 Oniscus asellus (woodlouse) 253
nitrification 209, 216, 235, 260, 280, 282, organic carbon 27, 31, 76, 86, 111, 247, 355
463, 466 organic contaminants 76, 94, 95, 98, 100,
nitrogen oxide 216 106, 109, 115, 117, 119, 174, 180, 209,
nitrogen fixation 181, 218, 235, 237, 239, 234, 238, 278, 293, 295, 311, 312, 324–
243 332, 338, 340, 345, 355–358, 367, 374,
NOAEC 43 375, 381, 383, 385, 390, 391, 411, 418,
NOAEL 43, 141, 146, 509, 529 445, 449, 469, 471, 474, 475, 478, 482
NOEC 43, 44, 48, 57, 59, 60, 62, 85, 86, organic matter 57, 76, 86, 90, 94, 111, 176,
108, 195, 247, 249, 267, 278, 281, 283, 177, 203, 208, 230, 233, 236, 238, 240,
300, 452, 453, 454, 459, 464, 468–484, 246, 248, 250, 259, 261, 267, 274, 285,
490–497, 503–504, 516–524, 529, 534 286, 291, 293, 338–339, 349, 354, 359,
NOEL 43, 44, 48, 267, 447, 473, 528, 529 413, 416, 417, 427, 436, 464
“no effect’’ dilution 28, 448 organic micropollutants 230
non-animal tests 12, 23, 137, 147, 152, 156,
organic substances 20, 25, 27, 33, 76, 83, 89,
203
93, 94, 105, 106, 109, 119, 158, 209, 210,
non-aqueous phase liquids (DNAPL) 73,
213, 240, 254, 257, 262, 282, 311, 312,
345, 350
349, 383, 389, 390, 403, 460, 468, 471
non-human primates 145
non-steroidal anti-inflammatory drugs organic waste 10, 466
(NSAID) 35, 474–477 organophosphorous substances 160
nonylphenol 40–42 organ toxicity testing 138–141
noradrenalin 38 oxidation 37, 77, 81, 83, 91, 92, 118, 119,
norway rat (Rattus norvegicus) 134 141, 143, 208–209, 235, 262, 273, 282,
NSAID (non-steroidal anti-inflammatory 338, 340, 353–355, 374, 385, 415–416,
drugs) 35, 474–477 430
Nuremburg Code 18 oxidase 37, 266
nutrient oxidative conditions 374
5, 10, 15, 29, 30, 49, 62, 86, 100, 118, oxidative enzymes 391
120, 142, 174, 176, 184, 185, 211, 214,
oxidative-reductive environment 207
218, 219, 229, 230, 231, 238, 240, 241,
246, 250, 256, 268, 270–271, 277, 280, oxidative stress 245
291, 338, 339, 343, 346, 349, 350, 365, oxide 35, 81, 86, 94, 216, 233, 246, 312,
377, 383, 402, 404, 405, 414, 417, 418, 340, 341, 350, 356, 359, 361, 370, 381,
421, 427, 434, 451, 456, 466, 506, 531 384
oxidizer/oxidizing agents/substances 11, 176,
occupational diseases 7, 126 415, 418
occupational health 7, 125 ozone 81, 246
 Subject index 557

PAH (polycyclic aromatic hydrocarbons) 34, photodegradability 52, 467

95, 98, 331, 344, 356–358, 384, 390, 478, photoelectric effect 318
480 photoionization 329, 330
Pardosa (wolf spiders) 252, 263 photoirritants 164
partition 7, 16, 27, 35, 55, 57, 60, 62, photolysis 36, 57, 81–82, 88, 119
71–74, 75–78, 83, 89, 103–105, 114, photon source 313
117–118, 129, 158, 203–207, 211, 219, photosynthesis 180, 235, 242, 245, 253
229, 233, 267, 270–272, 328, 341, 345, photosynthetic activity 245
351, 355–357, 358, 365, 378, 451, 468, phototoxicity 8, 25, 164
473, 477, 514, 518 phototrophic bacteria 182
pathogens 9, 181–183, 190, 235, 239, 240, phthalate 35, 58, 89, 475
245, 246, 249 phytane 331
partial extraction 358, 391, 436 phytobenthos 201
particulate matter 126, 203, 204, 229, 350 phytoplankton 176–177, 180, 201, 203, 219
partitioning 7, 35, 55, 57, 71–81, 103–105, phytosensor 246
114, 117–118, 129, 158, 345, 351, 355, phytostabilisation 414, 423, 427, 428
358, 451, 518 phytotoxic pollutants 182, 183
PCBs 34, 344, 356–357, 467 phytotoxicity 243, 245
PCB-free 96, 115, 478 pill bugs 193, 253
PCP (pentachlorophenol) 293, 357, 379, Pimephales promelas (fathead minnow) 58,
454, 459, 475, 478 59, 191, 202
PEC 448, 526, 529, 535 Planaria 189
PNEC 41, 448, 517, 518–522, 526, 529, plant (bio)accumulation 112–117, 354, 356,
531, 535 358, 371–372, 391
pentachlorophenol (PCP) 293, 357, 379, plant biomass 231, 370
454, 459, 475, 478 plant cells 143, 247
periphyton 174, 219 plant germination 235
persistent substances 33, 207 plant growth 235, 242, 243, 245, 280, 368,
pessimistic approach 90, 354 413–428, 438, 440
pessimistic models 207, 372, 378, 379, 380, plant pathogens 182, 249
391 plant protection products 34, 285, 286
pesticides 2, 7, 10, 12, 16, 17, 23, 24, 28, 35, plant root 338, 346, 349–350, 356, 369,
53, 54, 98, 100, 159, 174, 176, 190, 192, 378, 452
218, 220, 229, 231, 240, 244, 252, 253, plant tests 199–203, 245, 261–264, 279,
254, 261, 269, 273, 285, 286, 290, 293, 349–350, 363, 372, 375, 391, 495, 517
311, 312, 323, 328, 329, 344, 384, 403, plant tissue 243
417, 459, 526, 527, 532 plant toxicity 116, 199–203, 242–246,
petroleum hydrocarbons 98, 311, 312, 323, 261–264, 368, 370–375, 449, 452
330–331, 416, 417, 478 Plecoptera (stoneflies) 188
pharmaceutical residues/pollutants 312, 323, pleopods 193
329 plume 73, 119, 349
pharmaceuticals 9, 23, 34, 39, 134, 230, Poecilus cupreus (carabid beetle) 252, 263
274, 391 pollination 230, 235
pharmacology 9, 133, 292, 345 Pollution-induced Community Tolerance
pharmacokinetics 6, 157–159, 346, 391 (PICT) 241, 273
pheasant (Phasianus colchicus) 133, 254 polybrominated biphenyls 323
phenanthrene 295–296, 390 Polychaeta (marine worms) 192
phenols 58, 90 polycyclic aromatic compounds 56, 295,
phosphorus 346, 361 344, 357, 478
Photobacterium phosphoreum 240 polychlorinated biphenyls (PCBs) 295, 323,
photodegradation 26, 53, 71, 77, 264 344, 357
558 Subject index

polychlorinated dibenzofuranes 331 randomly methylated beta-cyclodextrins

population biology 6 (RAMEB) 116, 345, 375, 379
population density 38, 100, 236, 238, 404 RCR (risk characterization ratio) 36, 259,
population dynamics 218, 236, 256, 291, 448, 466, 525, 526, 529
404 REACH (EU regulation on Registration,
population growth 506, 507 Evaluation, Authorization and
population parameters 38, 40, 489 Restriction of Chemicals) 10, 11, 19, 24–26,
Porcello scaber (rough woodlouse) 253 31–34, 53–54, 57, 77, 83, 85, 105, 107,
pore water 16, 50, 74, 75, 76, 102, 104–106, 109, 130, 138, 192, 194, 260, 522, 526,
118, 190, 199, 204, 207–208, 216, 219, 527, 529
220, 233, 268, 270, 272, 273, 281, 338, reactive barriers 114, 411, 431–432, 465
350, 361, 362, 378 reactive oxygen species 8
potassium 415, 416, 438 reactive soil zone 72, 406
predators 84, 188, 247 realistic worst case 343, 354, 355, 358,
pressurized fluid extraction (PFE) 326 378–381, 391
pressurized hot solvent extraction (PHSE) receptor 3, 4, 17, 28, 37, 38, 44, 51, 52, 54,
326 60, 71–75, 88, 102, 104, 117, 118, 132,
pressurized liquid extraction (PLE) 326, 330 147, 150, 151, 161–164, 179, 183, 235,
prevention 119, 162, 172, 256, 261, 440, 254, 300, 341, 351, 352, 355, 391, 446,
511 502, 518, 524, 525, 528, 529, 533
preventive tools 64 recycling 24, 118, 402, 405, 464
priority risk components 77, 272 red mud 24, 413–415, 424, 435–440
priority risk substances 532 reductive dechlorination 241
pristane 331 reductive environment 207
probit 60–61, 491, 498, 500, 503, 504 redox conditions 52, 111, 208, 353
prokaryotes 216, 240 redox dye 262, 273
protease 266 redox gradient 119
proteogenomics 30, 242, 257 redox indictors 427
proteomics 14, 17, 21, 29, 30, 140, 177, redox potential 29, 53, 74, 83, 88, 90, 118,
180, 242, 256, 265, 299 119, 120, 173, 204, 207, 214, 219, 233,
protozoa 180, 183, 201, 219, 247, 375, 456, 241, 291, 340, 343, 344, 346, 354, 364,
469, 478 365, 386, 403–406, 418, 427, 446, 467
Pseudokirchneriella subcapitata (former redox regulation 246
name Selenastrum capricornutum) regression 60, 96, 384, 458, 484, 487, 488,
182–183, 210 491, 497–503
Pseudomonas 182, 199, 211, 238–239, 277 regression analysis 488, 491, 497–503
Pseudomonas putida 199, 211, 239 regulatory toxicology 7, 9–12, 15, 16, 194,
psRNA (putative small RNA) 30 467
pyridines 58 remedial field experiment 244
remedial target values 466
QSAR (quantitative structure-activity remedial technologies 81, 119, 412
relationship) 13, 18, 19, 22, 51, 53, 55–60, remediation 71, 73, 115, 119, 120, 172, 268,
88–89, 106–109, 137, 140, 145, 155, 157, 280, 342, 344, 354, 362, 369, 375, 401,
159, 171, 203, 354, 446, 518, 529 404, 407, 411, 414, 416, 418, 420–422,
quantification limit 316–317, 322, 328 427, 431, 466, 528
quorum sensing 181 remote sensing 176–178
repeated-dose toxicity 127, 138–139, 149
rabbit 22, 42, 46, 126, 134, 149, 153, reproductive toxicity 25, 32–33, 138, 149
155–157, 389 reprotoxicity/reprotoxic effects 25, 32, 35,
RAMEB 116, 345, 375, 379 38, 44, 50, 51, 55, 84, 125, 127, 128, 130,
 Subject index 559

190, 250, 300, 343, 351, 363, 390, 391, S9 enzyme 391
515 Saccharomyces 45, 132, 142, 162, 277
resident population 112, 113 Saccharomyces cerevisiae 132, 142, 162,
respiration 38, 42, 47, 49, 83, 90–93, 277
95–101, 102, 104, 116, 182, 193, 197, safety factor 8, 40, 42, 126, 131, 232, 378,
235, 241, 242, 246, 254, 262, 266, 464–4656, 515, 518, 527
280–285, 293, 294, 366, 382, 456, 461, safety toys directive 386
463, 478, 486 salicylic acid 246
respiration index 115 salinization 438
respiration rate 42, 100, 241, 245, 296, 365, Salmonella 132, 142, 143, 145, 181, 199,
419, 462–464, 466, 501 211, 379, 380
respiratory activity 220 sample pretreatment 311, 324
respiratory chain 49, 92 saponins 345
respiratory sensitizers 128 Scenedesmus quadricauda 182
revertants 50, 143 SDD (silicon drift detector) 322
rhesus (Macaca mulatta) 135 sediment 5–7, 16,27, 29, 33, 36, 43, 48, 50,
rhizoplane 235 52, 53, 54, 57, 60, 71, 74–76, 81, 82,
rhizosphere 5, 16, 47, 118, 220, 283 84–95, 102–104, 108–113, 118–119, 125,
Rhizobia 243, 264 171, 173, 176, 177, 180, 181, 185, 187,
rhizofiltration 404 188, 190, 192–197, 201, 203–208,
risk assessment 1–7, 15, 16, 28, 35–36, 46, 213–215, 218–222, 229, 241, 260,
51–60, 71, 73, 77, 80, 89, 90, 93, 108, 263–264, 267–268, 277–278, 288–289,
109, 115, 127, 130, 178, 179, 195, 214, 300, 311, 315, 322, 324, 329, 331,
221, 232, 233, 241, 254, 256, 259, 266, 337–338, 340, 341, 344–355, 357,
267, 274, 282–286, 299, 312, 337–392, 359–363, 372, 374, 378, 402–409, 411,
402, 403, 428, 429, 434, 445, 446, 467, 447–448, 451, 454, 460, 468,
468, 484, 508, 510, 513–535 477, 518
risk characterization ratio (RCR) 36, 259, sediment-dwelling organisms 192–194, 205,
448, 466, 525, 526, 529 207, 346
risk communication 528 sediment quality triad (SQT) 196
risk profile 117, 120, 265 sedimentation 77, 11
risk reduction 1, 4, 28, 53, 72, 73, 81, 91, selective sequential extraction 359
127, 177, 206, 207, 259, 282, 311, semiconductor detector 321
342–345, 354, 402, 404, 411, 413, 422, semimetals 39
428, 434, 448, 449, 464, 465, 521, 526, semivolatile organics 331
527, 531–535 semivolatile petroleum constituents 331
rodents 22, 47, 134, 139, 144, 160, 161, sensitization 8, 25, 35, 38, 125, 128, 130,
254, 283 154–155
rodenticides 323 sensitizers 34, 35, 128, 154–155
root accumulation 356–357 sensitizing effect 55, 84
root exudates 283, 348, 353, 369, 371, 378 separation 30, 95, 311, 324, 328–329, 331,
root length 49, 245, 300 362, 384, 419
root microcosm 283 sequential extraction 174, 341, 357, 359,
root microflora 235 384, 386
root and shoot growth 243, 245, 262, 279, sewage sludge 5–7, 47, 50, 53, 83, 197,
280–281, 283, 368, 370, 427, 437, 452, 208–209
469, 478, 479, 482–483 sewage/wastewater treatment plant 80, 195,
rotifers 185–186, 211, 215, 247, 519, 208, 465
521 sex ratio 236
runoff 16, 50, 77, 174, 175, 218, 233, 269, sheep 135
270, 272, 373, 374, 404, 413, 438 silicates 313, 350, 370
560 Subject index

silicon drift detector (SDD) 322 soil-living microfungi 100

silver salmon 191 soil-living organisms 36, 40, 54, 232, 235,
silverside 202 236, 238, 241, 246, 261, 268, 323, 344,
Simple Box model 79–81 345, 348, 362
SimpleTreat model 80 soil-living worms 211, 212, 251
simulation 15, 54, 89–91, 93–94, 103, 107, soil macrofauna 250
206, 209, 218, 260, 270, 282, 351, 353, soil microbial ecology 29, 257
358, 378–380, 384, 387, 390, 403, 415, soil microcosm 95, 272, 282, 283, 364–366,
432, 446, 527 404, 407–440
Sinapis alba (white mustard) 112–113, soil microflora/microbiota 8, 29, 47, 96, 98,
243–244, 279–280, 293, 296–297, 300, 99, 100, 119, 217, 236, 237, 238, 241,
368, 370–371, 427, 437, 469, 478–479, 242, 246, 257, 262, 273, 282, 287, 289,
482–483 290, 338, 339, 346, 348, 363, 365, 375,
site-specific risk 54, 73, 91, 117, 221, 233, 376, 378, 380, 411, 413, 416, 417, 418,
265, 343, 389, 403, 404, 525, 528 440, 463
skin/dermal corrosion/corrosivity 8, 20, 24, soil microinvertebrates 236
25, 55, 125, 128, 129, 152–155 soil organic matter 31, 233, 250, 286
skin/dermal irritation 20, 25, 128–130, soil phases 118, 338–339, 358, 445, 468
152–154 soil pollution 73, 413
skin sensitization 154–155 soil quality 250, 262, 269
sludge 5–7, 29, 47, 50, 53, 60, 80, 81, 83, soil quality criteria 368, 448, 473
84, 93, 197, 208–209, 213, 220, 270 soil respiration 93, 95–98, 262, 269,
small RNA (sRNA) 30 283–285, 463
snails 190, 192, 263, 265 soil sorption 109, 381
social insects 253 soil structure 229, 338, 346, 350, 438, 440
sodification 231, 290, 415, 438–440 soil testing triad 353, 366–367
sodic soil 438 soil texture 270, 291, 440
sodification 231, 290, 415, 438–440 soil toxicology/toxicity 230, 232, 233, 238,
sodium 24, 35, 293, 315, 438 239, 240, 242, 247, 249, 251, 260, 266,
soil acidification 346 268, 273, 278, 279, 292–301, 374–375,
soil biota 231, 237, 238, 256, 269, 285, 412 427, 447, 451
soil biodiversity 256–258 solid-phase extraction 330, 331
soil biodegradation 264, 417 sorption 72, 74, 75, 76, 81, 109, 114,
soil bioremediation 239, 416, 418, 421, 448 117–119, 136, 207, 233–235, 268,
soil column 54, 98, 114, 283, 418, 427, 337–339, 346, 355, 370, 381, 385, 426,
434–438 429
soil correction factor 381 sorption capacity 74, 76, 114, 234, 268, 337,
soil degradation 259, 340 378, 381, 429
soil deterioration 229–230, 350 Soxhlet extraction 324–325
soil diversity 258, 262 soya lecithins 345
soil-dwelling organisms 54, 231, 233, 236, spiders 250–252
239, 258, 263, 346, 348, 466, 478 species sensitivity distribution (SSD) 40–41,
soil ecosystem 54, 229, 230, 232, 234, 237, 173, 177, 179
241, 242, 259, 269, 367, 401, 412 spectral interferences 316–317
soil ecotoxicity 232, 251, 273, 464 sperm (cells) 128, 215
soil fauna 246, 255, 259 spermatocytes 141
soil functions 231, 241, 256, 266, 284 spermatogenesis 48
soil invertebrates 231, 246, 262, 274, 287, spider 250–252
289 springtail 212, 236, 250–251, 275, 279, 294,
soil-living microorganisms 240, 256, 257, 348, 478
347 SQT (sediment quality triad) 196
 Subject index 561

squirrel monkey (Saimiri sciureus) 135 Synechococcus leopoliensis (cyanobacterium)

sRNA (small RNA) 30 182
SSD (species sensitivity distribution) 40–41, syrian hamster embryo (SHE) 133, 147, 148
173, 177, 179 synthetic biological systems 14
stabilization-based soil remediation 422 synthetic chemicals 87, 218
standard addition 322 synthetic pollutants 9
standardization 1, 63–64, 90, 144, 194, 196, synthetic soil 283
198–203„ 260–261, 263, 274–275 synthetic water 219
standardized test methods 3, 10–12, 26, 45, synthetic xenobiotics 25
51, 53–54, 63–64, 72, 82, 88–91, 93–94,
95, 107, 108, 109–112, 130, 149, 151,
tailings 368–370, 374
155, 156, 179–181, 193–196, 197–203,
Tardigrada (slow stepper) 247–248
204, 216, 219, 237, 240, 241, 243, 244,
249, 252, 260–264, 265, 269, 272–275, target effects 34
286, 301, 312, 351, 352, 358–359, 403, targeted receptors 3, 44, 72, 254
420, 446, 458, 463, 466, 469, 486, 497, target hazard quotient (THQ) 529
499, 500, 522 target organ toxicity 21, 127–128, 140, 160
statistical evaluation 5, 60–63, 217, 258, targeted risk assessment 527, 531, 533
266, 291, 383, 402, 453, 484–513, 517, TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)
519, 520, 524, 529, 533 467
statistical evaluation of toxicity data 60–63, technological experiments 401–440
484–513 technology monitoring 311, 426
statistics 39, 44–49, 51, 55, 57, 60, 63, 131, TEF (toxic equivalency factor) 467, 470,
179, 238, 445, 453, 458, 484–489, 474–483
503–504, 507–515, 522 TEQ (toxic equivalency) 375–378, 467–483,
stressor 9, 41, 177, 214–215, 236, 245, 247, 535
455, 486, 499 teratogenic effects 128, 201
stripping in metal analysis 360 teratogenicity 44, 202
stripping in groundwater treatment 403 termites 252–253
substrate induction 283, 462–464 terrestrial bioassays 259–260
subsurface waters 50, 230 terrestrial bioaccumulation 103–114
sulfate 241, 438 terrestrial ecosystem 5, 7, 25, 40, 47,
sulfate reduction 216 74, 90, 108, 216, 229–231, 235–236,
sulfate respiration 241 238, 242, 263, 272, 338, 369, 517–518,
sulfamethoxazole 35 528
sulfide 81, 83, 111, 119, 214, 340–341, 350, terrestrial ecotoxicology 29, 229, 237
352, 359–361, 429, 430–433 terrestrial microcosm 282–295, 403
sulfonamides 330 terrestrial toxicology 7, 64, 229–301
sulfonates 58, 90 test design 48–49, 52, 54, 63, 114, 178, 196,
sulfur 86, 291, 430 201, 275, 293, 463, 495, 499
sulfuric acid 81 test organisms 1, 3, 42–44, 45–47, 48, 54,
supercritical fluid extraction 324 72, 108, 112–113, 126, 130, 132–136,
surface waters 7, 16, 49, 73–74, 95, 114, 171, 179, 180–194, 195, 199, 203–207,
118, 125, 171, 176–177, 179, 188, 192, 214–216, 222, 229, 232, 234, 236,
196, 198, 201, 203, 206, 212, 231, 275, 237–255, 259, 265–269, 278, 293, 299,
340, 349–350, 362, 430 300, 351, 353, 356, 362, 363, 365–368,
surfactant 35, 183, 314, 345 375, 380, 412, 427, 447–450, 453,
sustainable environmental management 1, 6, 465–469, 477, 478, 482, 485, 489, 491,
52, 119, 172 515, 518, 527, 534
sustainability 119, 256 tetrachlorodibenzo-p-dioxin 467
562 Subject index

Tetrahymena 45, 183–184, 220–222, 247, tubifex monitor 213–214

278, 379–381, 427, 456–457, 469,
474–475, 480–481 ultrafiltration 329
Tetrahymena pyriformis 45, 183–184, ultrasonic assisted extraction 327
220–222, 247, 278–279, 379–381, 427, uncertainty 13, 44, 53, 56, 60, 89, 102, 126,
456–457, 469, 474–476, 480–481 130, 173, 178, 232, 236, 259, 312, 314,
Thamnocephalus platyurus (freshwater 378, 386, 391, 411, 412, 450, 465, 515,
crustacean) 186, 201 517, 519, 523, 525, 527, 529, 533, 534
thiobacilli 113 uncertainty factor 312, 378, 465, 515, 527,
tissue cultures 18, 132 529
thermo activity monitor (TAM) 229, unscheduled DNA synthesis 133, 142, 144
292–301 uranium 428
thermophilic bacteria 183 urbanization 174
THQ (target hazard quotient) 529 uterotrophic 161, 163
tissue culture 18, 132–133
toluene 239, 330 validation 5, 12, 20–23, 57, 63–64, 131,
topoisomerase 148 137, 138, 143–144, 149, 156, 162, 467,
toxic equivalency 375–378, 467–483, 535 473, 477–483, 487–488, 492, 509,
total ion chromatogram 331 522–524, 526, 531
toxic metals 33, 102, 117, 174, 180, 289, vapor pressure 75–77, 83, 211, 383
293, 343, 346, 355, 358, 360, 369, 370, very bioaccumulative substances 33–34, 85
373, 378, 379, 385–389, 414, 423, 427, very persistent substances33, 85
435, 461, 468, 470, 471, 482 vertebrates 175, 190–192, 231, 246, 254,
toxicogenomics 15, 17, 29, 140, 203 265, 290
toxicokinetics 6, 17, 28–29, 144, 157–159, veterinary pharmaceuticals 252, 311, 330
201, 505 Vibrio fischeri 39, 45, 178, 180–181, 199,
TPH (total petroleum hydrocarbons) 95, 330 208, 210–211, 215–216, 240, 241, 263,
transcriptomics 14, 29–30, 180, 257, 259 275, 368, 375, 379–381, 437, 453–454,
transformer oil 96–98, 100–101, 115–116, 466, 469–483
295, 375–376, 417, 420–421, 478, vibrios 240
480–481 virginia quail (Colinus virginianus) 133, 254
transport2, 11, 12, 16, 18, 23, 28, 34, 36, vitellogenin 162, 176, 202
44, 49, 54, 58, 72–74, 75–81, 83, 90–91, volatile organic compounds 257, 282, 331
102, 104, 117–120, 151, 157, 161, 174, volatilization 74, 80, 94, 117, 233, 338
179, 180, 182, 204, 208, 233, 235, 272,
277, 289, 290, 312, 314, 315, 320, 331, wasps 252–253
341, 342, 349–350, 352, 354, 355, 358, Waste Directive 527
362, 369, 383, 403, 411, 413, 428, 430, waste disposal 231, 267, 272, 434, 527, 532
434–440, 522, 525–529 waste dump 430–431
transport model 77, 81, 526, 529 wastewater/sewage treatment plant 80, 195,
Treaty of Amsterdam 18 208, 465
triad 196, 353, 366–367 Water Framework Directive (WFD) 176, 527
tributyltin 35 water solubility 55, 73, 75, 78, 83, 107, 203,
Trichoptera (caddisflies) 188–189 337, 355
triton-X 314 wetlands 83, 118–119, 220, 341, 404, 406,
trophic chain 104 465
trophic communities 30 WFD (Water Framework Directive) 176, 527
trophic index 176 wild animals 254
trophic levels 41, 104, 173, 183, 190, 195, woodlice 193, 253
199, 221, 232, 253, 259, 266, 290, 401, worst case 204, 337, 343–344, 354–355,
403, 427, 448, 465, 517 358, 362, 378–381, 391, 438, 440, 529
 Subject index 563

xenobiotics 2, 5, 16, 25, 28, 33, 36, 84–87, zinc 116, 254, 297, 312, 371–372, 386, 388,
119, 140, 144–145, 181, 217–219, 235, 434, 482
239, 245, 290, 375 zoobenthos 188, 204
Xenopus (African clawed frogs) 133, zooplankton 176, 180, 185–187, 192, 219,
149–151 403
X-ray 81, 312, 318–323, 359, 384
X-ray fluorescence spectrometry 312, Zea mays 244
318–323, 359

YES assay 132, 162–164