Preservation of Surfactant Formulations

Preservation of Surfactant
Formulations

Edited by

F.F. MORPETH
Zeneca Biocides
Wilmington
Delaware

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V

First edition 1995
© 1995 Springer Science+Business Media Dordrecht
Originally published by Chapman & Hali in 1995
Softcover reprint ofthe hardcover Ist edition 1995

Typeset in 10/12pt Times by Cambrian Typesetters, Frimley, Surrey

ISBN 978-94-010-4272-7 ISBN 978-94-011-0621-4 (eBook)

DOI 10.1007/978-94-011-0621-4
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Preface

Microbes are known to live in an enormous range of environments. Their

ability to survive and proliferate in diverse industrial systems is often a
surprise to those not exposed to these problems in their work. These
systems contain a range of potential carbon sources, one common theme
being surfactants. Surfactants are often not the components most prone to
spoilage since some systems contain highly susceptible natural components,
such as starch and xanthum gum, but the surfactant is a key part of the
formulation, and its extensive breakdown usually means that the material
is beyond recovery.
The aim of this book is to describe in detail all aspects of the
preservation of surfactant containing materials.
The book should be viewed as being in three discrete sections.
• chapters 1-5 deal with and summarise essential background information
• chapters 6-11 discuss in detail various end use applications
• chapters 12-15 outline the regulatory and toxicology implication
associated with the safe handling of preservatives
Given the format of the book there is inevitably some duplication of
information in the middle section with different authors describing
essentially the same phenomena but on different substrates. I hope the
reader will find that although different chapters touch on the same topics
the information around these areas is sufficiently different to justify their
inclusion in this book and to be of interest. It should also demonstrate what
can be the most useful source of information, the hard practical experience
of the authors.
Much of the expertise in this area is based in industry. This is reflected in
the spread of authors and indeed much of the information presented here
has been gained from direct experience and is not found anywhere else.
As might be imagined from the comments above, the literature around
the area covered in this book is sparse. Thus any comments on the content
of this book or information about the subject will be gratefully received.
Finally I would like to thank Maria, Ross and Lawrence for their
understanding during the long hours spent pulling this manuscript
together.

F.F.M
Contributors

P.W. Austin Zeneca Specialties Research Centre, Zeneca Specialties,

Blackley, Manchester, M9 3DA, UK
D.K. Brannan Abilene Christian University, ACU Box 7721, Abilene,
Texas 37134, USA
M.E. Burt Zeneca Biocides, 1800 Concord Pike, FOC 2W,
Wilmington, Delaware 19897, USA
M.A. Cresswell Vinamul Ltd, Mill Lane, Carshalton, Surrey SM2 2JU,
UK
H. Gibson Campden FDRA, Chipping Campden, Gloucestershire,
GL55 6LD, UK
J.T. Holah Campden FDRA, Chipping Campden, Gloucestershire,
GL55 6LD, UK
K. Holland National Starch & Chemical Ltd., Galvin Road, Slough,
Berkshire, SLl 4DF, UK
D.A. Knowles FORM-AK, 10 The Forstal, Hadlow, Tonbridge, Kent,
TNll ORT, UK
P.S.K. Lee Zeneca Biocides, 1800 Concord Pike, CR & DL 208,
Wilmington, Delaware 19897, USA
J.J. Lewis ICI Surfactants, PO Box 90, Wilton Centre,
Middlesborough, Cleveland, TS90 8JE, UK
G. Lloyd Albright & Wilson Ltd., 210-222 Hagley Road West,
Oldbury, West Midlands, B68 ONN, UK
J. Martin Calgon Corp. PO Box 1346, Pittsburgh, Pennsylvania
15230-1346, USA
F.F. Morpeth Zeneca Biocides, 1800 Concord Pike, Wilmington,
Delaware 19897, USA
D.N. Munro Zeneca FCMO, North of England Works, PO Box A38,
Leeds Road, Huddersfield, Yorkshire, HD2 IFF, UK
J.A. Pierce Zeneca Biocides, 1800 Concord Pike, CR & DL 248,
Wilmington, Delaware 19897, USA
Vlll CONTRIBUTORS

P.M. Prichard ECC International Technology Centre, PO Box 471,

Kaolin Road, Sandersville, Georgia 31082, USA
R.A. Rodford Unilever Research, Environmental Safety Laboratory,
Colworth House, Sharnbrook, Bedford, MK44 1LQ,
UK
R.D. Yore Zeneca Biocides, 1800 Concord Pike, CR & DL 248,
Wilmington, Delaware 19897, USA
G.F. White School of Molecular and Medical Biosciences,
Biochemistry Unit, University of Wales, College of
Cardiff, PO Box 911, Cardiff, CF1 3US, UK
Contents

1 An introduction to microbial spoilage 1

F.F. MORPETH
1.1 Growth of spoilage microorganisms 1
1.1.1 pH 1
1.1.2 Temperature 1
1.1.3 Oxidation-reduction potential 2
1.1.4 Water activity 2
1.2 Presence of a suitable carbon source 3
1.3 Presence of antimicrobial components 3
1.4 Consequences of microbial spoilage 3
1.4.1 Buildup of gas and foul odors in paints, adhesives and
related systems 3
1.4.2 Damage or loss of performance of processing equipment 4
1.4.3 Thinning and phase separation 4
1.4.4 Discoloration 4
References 5

2 Chemical preservatives 6
F.F. MORPETH and P.W. AUSTIN
2.1 Isothiazolinones 6
2.1.1 Toxicology 11
2.1.2 Mode of action 11
2.2 Other nitrogen-sulphur compounds 13
2.3 Organobromine compounds 15
2.4 Thiocyanates 20
2.5 Dithiocarbamates 21
2.6 Organoiodine compounds 21
2.7 Aldehydes and aldehyde-release agents 23
2.8 Phenols 27
2.9 Organic acids and salts 28
2.10 Mercury and other inorganic elements 28
References 28

3 Control of microbes through plant hygiene 30

H. GIBSON and J. T. HOLAH

3.1 Introduction 30
3.2 Design and construction of the premises 31
3.2.1 Site selection 31
3.2.2 Factory structure 31
3.2.3 Production area 32
3.3 Control of product contact surfaces 34
3.3.1 Hygienic design 34
3.3.2 Cleaning and disinfection 37
X CONTENTS

3.4 Control of production environment air 45

3.4.1 Aim 45
3.4.2 Sources of air-borne contamination 46
3.4.3 Control of air-borne contamination 47
3.4.4 Methods of reduction of air-borne contamination 49
3.4.5 Monitoring of air quality 49
3.5 Control of the personnel route of contamination 50
Further reading 51
References 51

4 An introduction to surfactants 53
J.J. LEWIS

4.1 Why are surfactants of interest? 53

4.2 How surfactants work 54
4.2.1 Molecular interactions and interfacial tension 54
4.2.2 What does a surfactant do? 55
4.2.3 Structure of an idealised surfactant and its interfacial activity 56
4.2.4 Surfactancy and the reduction of interfacial tension 58
4.2.5 MicelIisation 58
4.3 Surfactant phenomena and effects 60
4.3.1 Emulsification 60
4.3.2 Foams 63
4.3.3 Wetting 64
4.3.4 Solid dispersion in liquids 65
4.3.5 Solubilisation 66
4.3.6 Microemulsions 66
4.3.7 Detergency 67
4.4 Chemistry of surfactants 69
4.4.1 Oleochemicals 69
4.4.2 Petrochemicals 72
4.4.3 Major hydrophilising technologies 73
4.4.4 Cationics 74
4.4.5 A fuJI range of functionality 75
4.4.6 Understanding surfactant chemistry 76
4.5 Formulating with surfactants 77
4.5.1 The hydrophile-lipophile balance (HLB) 77
4.5.2 Synergy 79
4.5.3 Physical and chemical compatibility 80
4.5.4 Cost 81
4.5.5 Other considerations 81
4.6 Some final thoughts 81
Further reading 82

5 Biodegradation of surfactants 83
G.F. WHITE

5.1 Introduction 83
5.1.1 Background 83
5.1.2 Surfactants and bacterial nutrition 84
5.2 Biodegradation of anionic surfactants 86
5.2.1 Alkyl sulphates 86
5.2.2 Alkyl phosphates 87
5.2.3 Dialkyl sulphosuccinates 88
5.2.4 Alkylethoxy sulphates 89
5.2.5 Alkane sui phonates 92
5.2.6 Fatty acid ester sulphonates 94
 CONTENTS Xl

5.2.7 Linear alkylbenzene sulphonates 94

5.2.8 Fatty acid alkanolamide sulphates 97
5.2.9 Sulphonated esters and amides of fatty acids 98
5.3 Biodegradation of nonionic surfactants 100
5.3.1 Polyethylene glycol (ethoxylate) chains as bacterial nutrients 100
5.3.2 Linear alcohol ethoxylates 100
5.3.3 Branched alcohol ethoxylates 101
5.3.4 Alkylphenol ethoxylates 103
5.3.5 Polyglycols 104
5.3.6 Fatty acid alkanolamides and ethoxylated fatty amines 106
5.4 Biodegradation of cationic surfactants 106
5.4.1 Adsorption 107
5.4.2 Acclimation 108
5.4.3 Biodegradability and metabolic pathways 109
5.5 Relevance to biodeterioration 110
References III

6 Preservation of agrochemicals 118

D.A. KNOWLES

6.1 Introduction 118

6.2 Agrochemical formulations 120
6.2.1 Conventional formulations 122
6.2.2 New generation formulations 127
6.3 Surfactants for agrochemicals 134
6.3.1 Conventional surfactants 136
6.3.2 Recent surfactant developments 139
6.4 Preservatives for agrochemicals 140
6.4.1 Spoilage microorganisms 141
6.4.2 Types of preservatives 142
6.4.3 Testing and regulatory protocols 144
References 146

7 Preservation of personal care products 147

D.K. BRANNAN

7.1 Introduction 147

7.1.1 History of personal care products and preservation concerns 147
7.1.2 A look at the future 151
7.2 Importance of preservation 151
7.2.1 Types of personal care products needing preservation 151
7.2.2 Surfactants used 153
7.2.3 Consequences of not adding a preservative 153
7.2.4 Microorganisms encountered 154
7.2.5 Considerations for preservation 155
7.3 Preservative efficacy testing 157
7.3.1 General considerations of preservative efficacy testing (PET) 157
7.3.2 Common issues shared by the PET methods 159
7.3.3 Other PET methods 172
7.4 Preservation available for use 174
7.4.1 Mode of action of preservatives 174
7.4.2 Selection of preservative 176
7.4.3 Safety considerations of preservatives 176
7.5 Conclusion 178
Acknowledgements 178
References 178
xu CONTENTS

8 Preservation of paint formulations 185

R.D. YORE and J.A. PIERCE

8.1 Introduction 185

8.2 The chemistry and manufacture of paint 185
8.3 Consequences of microbial spoilage 187
8.4 Requirements for microbial growth 192
8.5 Typical flora associated with paint spoilage 193
8.5.1 Bacteria 193
8.5.2 Yeasts 194
8.5.3 Fungi 194
8.6 The problem with solvent free systems: spoilage 195
8.7 The chemistry of industrial preservatives 196
8.7.1 Biocide terminology 196
8.7.2 Mercurials 196
8.7.3 Desirable properties of biocides 196
8.7.4 Biocides presently available 197
8.7.5 The future of biocides 199
8.8 Methods to establish the correct biocide level for preservation 199
8.8.1 In-can preservation 199
8.9 Implementing the laboratory report in practice 201
8.10 Plant sanitation and hygiene audits 203
8.10.1 Cleaning equipment 204
8.10.2 Treating wash/rinse water 205
8.10.3 Hygiene audits 205
8.11 When spoilage occurs 207
8.11.1 Handling contaminated product 208
8.11.2 Treating contaminated product 208
8.12 Implementing a HACCP program 209
8.13 Summary 209
References 210

9 Preservation of aqueous-based synthetic polymer emulsions and

adhesive formulations 212
M.A. CRESSWELL and K. HOLLAND

9.1 General introduction 212

9.2 Aqueous-based synthetic polymer emulsions 214
9.2.1 Applications and uses of polymer emulsions 214
9.2.2 Range and chemical types of polymer emulsions 214
9.2.3 The chemical nature of polymer emulsions 216
9.2.4 Manufacture of synthetic polymer emulsions 222
9.3 Adhesive formulations 223
9.3.1 Applications and uses of adhesives 223
9.3.2 Range and chemicaJtypes ofadhesive 225
9.3.3 The key properties and tests on adhesives 228
9.3.4 Chemical nature and formulation of aqueous-based adhesives
and their raw materials 230
9.4 Microbial spoilage of polymer emulsions and adhesives 231
9.4.1 Consequences and effects of microbial spoilage 232
9.4.2 Types of spoilage microorganisms encountered 235
9.4.3 Sources of microbial contamination 239
9.5 Prevention and control of microbial spoilage in polymer emulsions
and adhesives 243
9.5.1 Manufacturing plant hygiene, cleaning and sanitisation
methods 245
 CONTENTS xiii

9.5.2Preservation of polymer emulsions and adhesives with

antimicrobial agents 246
9.5.3 Chemistry of active ingredients as antimicrobial agents 248
9.5.4 Physical and chemical factors influencing the choice of
preservative strategies 248
9.5.5 Test methodology for the selection of biocides and analytical
methods for their quantification 251
9.5.6 Regulatory issues governing the choice and selection of
antimicrobial agents and biocides 253
9.6 Preservation strategies and related issues affecting polymer emulsions
and adhesives for the next millennium 256
9.6.1 Safety, health and environmental issues 256
9.7 Concluding remarks 257
References 258
Further reading 261

10 Preservation of inorganic systems 262

P.M. PRICHARD and J. MARTIN

10.1 Introduction 262

10.1.1 Kaolin and calcium carbonate 263
10.1.2 Titanium dioxide 263
10.1.3 Processing kaolin and calcium carbonate 264
10.1.4 Uses 265
10.2 Contamination sources 266
10.2.1 Types of organism found in kaolin aqueous manufacturing
processes 266
10.3 Consequences of microbiological contamination 267
10.3.1 Kaolin 267
10.3.2 Calcium carbonate 267
10.3.3 General 268
10.3.4 Paper 268
10.4 Sampling 268
10.5 Equipment design 269
10.6 Exponential growth 269
10.7 Microbiological test methods 270
10.7.1 Standard plate count procedure 271
10.7.2 Dip slide method 271
10.7.3 Assessment of bacterial populations using dip slides 272
10.8 Anaerobic contamination 273
10.8.1 Anaerobic microbial analysis 273
10.9 Microbiological control 274
10.9.1 Cleaninglhousekeeping 274
10.9.2 Radiation 275
10.9.3 Oxidants 275
10.10 Biocides 275
10.10.1 FDA approval 276
10.10.2 Approval outside the United States 276
10.10.3 Typesofbiocide 277
10.11 Biocide performance testing 281
10 .12 Biocide addition systems 281
10.13 Residual biocide testing 282
10.13.1 Thione 282
10.13.2 Glutaraldehyde 282
10.13.3 Benzisothiazoline 282
10.13.4 Kathon 283
10 .14 Working with the external customer 283
10.15 Slurry made from dry material and slurry storage 283
xiv CONTENTS

11 Preservation of metalworking fluids 284

P.S.K. LEE

11.1 Introduction to metalworking fluids 284

11. 2 Conseq uences of metalworking fluids failure 284
11.3 Ranges and types of metalworking fluid 285
11.3.1 Straight or neat oils 285
11.3.2 Soluble oils 286
11.3.3 Semi-synthetic metalworking fluids 286
11.3.4 Synthetic metalworking fluids 287
11.4 Major surfactant types used in metalworking fluids 288
11.4.1 Anionics 288
11.4.2 Nonionics 289
11.4.3 Cationics and amphoterics 291
11. 4.4 Bioresistant surfactants 291
11.5 Types of microorganism in metalworking fluids 292
11.5.1 Aerobic bacteria 293
11.5.2 Anaerobic bacteria 293
11.5.3 Fungi 293
11.6 Types of preservative used in metalworking fluids 294
11.6.1 ANGUS Chemical Company 295
11.6.2 Buckman Laboratories, Inc. 296
11. 6. 3 The Dow Chemical Company 296
11.6.4 Olin Corporation 297
11.6.5 Rohm and Haas Company 298
11.6.6 R.T. Vanderbilt Company, Inc. 298
11.6.7 Stepan Company 299
11.6.8 Union Carbide Corporation 299
11.6.9 US Professional Laboratories 299
11.6.10 ZENECABiocides 299
11. 6.11 Potentiation of preservatives 300
11.7 Testing protocols used in metalworking fluid preservative selection 300
11.7.1 Laboratory testing of metalworking fluid preservatives 301
11.7.2 Field testing of metalworking preservatives 302
11.7.3 Monitoring for microbial contamination in metalworking
fluid systems 302
11.8 Summary 305
References 305

12 Toxicology of preservatives 311

R.A. RODFORD

12.1 Introduction 311

12.2 Preservatives in general use 312
12.2.1 Organic acids 313
12.2.2 Phenolics 313
12.2.3 Quaternary ammonium salts 314
12.2.4 Mercury salts 314
12.2.5 Ureas 314
12.2.6 Isothiazolinones 314
12.2.7 Hydantoins (formaldehyde donors) 315
12.2.8 Miscellaneous organics 315
12.3 Acute toxicity 317
12.3.1 Formaldehyde 317
12.3.2 Isothiazolinones 317
 CONTENTS XV

12.3.3 2-Bromo-2-nitropropane-1,3-diol 318

12.3.4 4,4-Dimethyl-1,3-oxazolidine and 1-azo-3,7-diox-5-ethylbicyclo
(3.3.0) octane 318
12.3.5 Glutaraldehyde 318
12.4 Skin and eye irritation 319
12.4.1 Formaldehyde 319
12.4.2 Isothiazolinones 319
12.4.3 Bronopol 320
12.4.4 Oxabans A and E 320
12.4.5 Glutaraldehyde 320
12.5 Skin and respiratory sensitization 321
12.5.1 Formaldehyde 321
12.5.2 Isothiazolinones 321
12.5.3 Bronopol 322
12.5.4 OxabansAandE 322
12.5.5 Glutaraldehyde 323
12.6 Subacute and chronic toxicity including carcinogenicity 323
12.6.1 Formaldehyde 323
12.6.2 Kathon CG 324
12.6.3 Bronopol 324
12.6.4 Oxaban A 325
12.6.5 Glutaraldehyde 325
12.7 Genetictoxicology 325
12.7.1 Formaldehyde 325
12.7.2 Isothiazolinones 326
12.7.3 Bronopol 326
12.7.4 OxabansAandE 326
12.7.5 Glutaraldehyde 327
12.8 Reproductive and developmental effects 327
12.8.1 Formaldehyde 327
12.8.2 Isothiazolinones 327
12.8.3 Bronopol 327
12.8.4 Glutaraldehyde 328
12.9 Environmental considerations 328
12.10 Conclusion 329
References 331

13 The safe use of preservatives 337

D.N. MUNRO

References 348

14 Regulatiou of preservatives in the USA 350

M.E. BURT

14.1 Introduction 350

14.2 Industrial preservatives 350
14.3 Indirect food additives 353
14.4 Cosmetic preservatives 354
14.5 Summary 356
References 357
XVI CONTENTS

15 European preservative legislation 358

G. LLOYD

15.1 Introduction 358

15.2 Preservatives in the oil industry 358
15.3 Preservatives in the paper industry 362
15.4 The proposed Biocidal Products Directory (93/239/03) 362
References 363
Appendix 1- Countries with registration procedures for preservatives 364
Appendix 2 - Typical data requirements for preservatives use din the
offshore oil drilling and production industry 365

Index 367
1 An introduction to microbial spoilage
F.F. MORPETH

1.1 Growth of spoilage microorganisms

Microbes can survive and flourish in a range of aqueous systems. The

dominant organisms vary with the nature of the components and
parameters such as pH, temperature, oxidation-reduction potential and
water activity. The influence of these parameters of microbial growth is
considered below.

1.1.1 pH
Most microorganisms grow best at around neutral pH (6.5-7.5). Few thrive
below pH 4 or above pH 9 although microbes can survive from less than
pH 1 to greater than pH 11. In general bacteria tend to be more fastidious
about pH than yeasts or fungi which can predominate in acidic systems less
than pH 6, such as acidic lattices and some dyes. The pH of most of the
systems described in this book is fixed to within about 0.2 to 0.3 of a pH
unit. However, it can be possible to limit the susceptibility of a system by
taking away from neutrality by 1 pH unit or more. This can be especially
useful in those cases where a single organism has infected the manu-
facturing plant and is proving resistant to killing. Tipping the balance
against the organism in this situation can sometimes have just enough of a
detrimental effect on the microbe to enable the biocide to re-establish
control.

1.1.2 Temperature
Specialized microorganisms have been reported to be able to grow from
less than -35 to greater than 90°C. However, most spoilage microbes tend
to grow best between 15 and 40°C, though significant growth can occur
between 7 and 60°C. Thus, many manufacturing facilities suffer spoilage
problems only during the summer months. Indeed some companies even
change their product specification between May and September to increase
the amount of biocide incorporated.
2 PRESERVATION OF SURFACTANT FORMULATIONS

1.1.3 Oxidation-reduction potential

Microorganisms display a varying degree of sensitivity to the oxidation-
reduction potential of their environment. The most significant example of
this is how the organisms respond to the presence of oxygen. Bacteria may
be classified as:
- Aerobes (able to grow only in the presence of oxygen).
- Facultative anaerobes (able to grow in the presence or absence of
oxygen).
- Anaerobes (only able to grow in the absence of oxygen).
- In addition, low oxygen tension favors certain bacteria, known as
microaerophiles.
- Fungi and yeast tend to be aerobes though there are examples of
facultative anaerobic fungi and many facultative and anaerobic yeasts.
There is a natural order in which microorganisms infect and grow in a
newly prepared bulk industrial material not protected by a biocide. In
general the initial growth tends to be that of vigorous aerobes usually the
pseudomonads. However if a bulk storage tank is not agitated then these
organisms quickly deplete the oxygen from the system. This allows the
development of an insidious threat, the sulphate-reducing bacteria. Once
established these organisms can cause significant pitting corrosion to any
system. 1 Thus when the recovery is attempted of a particularly badly
contaminated system it is often sensible to vigorously aerate the system
prior to biocide addition and continue this agitation throughout the
recovery process. This has two effects. First, it vents off any toxic hydrogen
sulphide accumulated via the action of the sulphate reducing bacteria
(which can also inactivate many commonly-used preservatives). Second,
it inhibits further growth of the sulphate-reducing bacteria. Thus the effect
of oxygen can be seen easily in a single bulk storage tank with
pseudomonads predominating when sampled from the top and sulphate
reducers predominating when sampled at the bottom.
In some cases the actual redox potential of a system also can affect pro-
foundly the ability of microbes to survive and grow. However, this is rare.

1.1. 4 Water activity

Water activity Aw is a measure of the moisture content of a system. It may
be defined as the ratio of the vapor pressure of a system to that of water at
the same temperature. Most spoilage bacteria require an Aw of at least
0.91; yeast can usually grow down to 0.88 and fungi can grow at 0.8.
However, there are examples of microbes able to grow in an Aw as low as
0.6. Water activity is generally not used as a means of controlling microbial
growth in industrial systems. Indeed it is a parameter which is seldom
measured or used. This could be a missed opportunity.
 MICROBIAL SPOILAGE 3

1.2 Presence of a suitable carbon source

Various microorganisms are able to utilize a wide range of carbon

compounds to support their growth. Both the concentration and type of
carbon source will, to a large degree, determine which organisms are able
to survive in any given industrial system. Examples of carbon sources able
to support vigorous microbial growth, making them particularly prone to
spoilage are soluble cellulose thickeners; starch derivatives; casein and
other protein based materials; and biodegradable sufactants.

1.3 Presence of antimicrobial components

Though many components of aqueous industrial systems can serve as

carbon sources able to support microbial growth, some will actually have
an antimicrobial activity. This can either lower the possibility that the
system will become spoiled or it can select for spoilage by those organisms
not susceptible to these components. Examples are methanol commonly
present in polyacrylic acid and the unreacted monomers in a latex system.
Thus, the system to be preserved will have a profound influence on the
range of organisms able to grow in it. Those organisms able to establish
themselves, in turn determine what spoilage problems are encountered.
Most wet-state spoilage microbes are bacteria. Among those identified
in the technical laboratories of ZENECA Biocides are members of the
genera Aeromonas sp., Alcaligenes sp., Bacillus sp., Flavobacterium sp.,
Pseudomonas sp. and Serratia sp. with Pseudomonas sp. accounting for at
least half the cases of spoilage.
Fungi can also contaminate some systems. Most common are Fusarium
sp., Penicillium sp. and Geotrichum sp.

1.4 Consequences of microbial spoilage

Examples of the types of problem which may be encountered by different

industries are buildup of gas and foul odors, loss of performance of
processing equipment, thinning and phase separation and discoloration.

1.4.1 Buildup of gas and foul odors in paints, adhesives and related
systems
Bacterial growth can, if uncontrolled especially in hot climates, generate
sufficient gas to distend and/or blow the top off paint and latex containers.
These problems are particularly insidious since they are usually the result
4 PRESERVATION OF SURFACTANT FORMULATIONS

of long term microbial action and are only apparent when the final user
opens the product. Occurrences such as this lead to a serious loss of
reputation for the producing company. They can also lead to damaging
claims for compensation against any company who supplied contaminated
materials to the end user.

1.4.2 Damage or loss of performance of processing equipment

Failure to protect aqueous systems can lead to long term damage of
manufacturing equipment. For example, it has been estimated that
something like 10% of all corrosion is of microbial origin. This is mainly
caused by cathodic depolarization and the in situ production of hydrogen
sulphide by sulphate-reducing bacteria.
Less extreme, but more widespread, is the buildup of microbial biofilms
in processing lines and on heat exchangers. Biofilms should probably be
regarded as the 'natural' state of bacteria outside the laboratory. 2 They
consist of mixed populations of bacteria encased in a polysaccharide
(usually alginate) gel which is immobilized on a surface. Biofilms can be
anything from one layer of cells to inches thick. Indeed they can be thick
enough to block process pipes and significantly affect the efficiency of heat
exchangers. In addition to this, they represent a reservoir of microbial
contamination. A system not protected by a biocide which suffers a biofilm
buildup cannot usually be cleared by chemical means alone. It will
normally require physical cleaning. This can be an expensive, time-
consuming process, involving considerable downtime for a production
facility.

1.4.3 Thinning and phase separation

These are particularly difficult problems since they are brought about by
enzymes secreted by microbes which persist after the organism has been
killed. Thus, if biocide is added too late in a process, it will stop microbial
growth but will not necessarily prevent further spoilage. This is a particular
problem in the paint and latex industries but can occur in any system where
cellulose ethers or surfactants are major components.

1.4.4 Discoloration
Discoloration can occur either through growth of a colored microorganism
or through the microbial modification of colored components in a dye or
paint. Sulphide precipitates can also occur when sulphate-reducing
bacteria are present in a system.
 MICROBIAL SPOILAGE 5

References

1. Postgate, J.R. (1979) The Sulphate Reducing Bacteria, Cambridge University Press,
Cambridge-London-New York-Melbourne.
2. Costerton, J.W., Cheng, K.J., Geesey, G.G., Ladd, T.I., Nickel, J.C., Dasgupta, M. and
Marrie, T.J. (1987) Bacterial Biofilms in Nature and Disease. Ann. Rev. Microbiol., 41,
435-464.
2 Chemical preservatives
F.F. MORPETH and P.W. AUSTIN

A wide range of chemical agents is available to preserve surfactant-

containing formulations. The criteria for the selection of the preservative
of choice in each area are dealt with in subsequent chapters. The purpose
of this chapter is to document the available preservatives and summarize
their chemical, physical and antimicrobial properties. There have been
some excellent books on preservatives which have covered in detail many
of the more mature products. 1 ,2 In general many of these agents are in
decline for safety and environmental reasons. Thus, this chapter will
concentrate on the more 'modern' preservatives. Other agents will be
referred to in passing and sufficient references given to enable the
interested reader to pursue the subject. The reader is also referred to a
recent book by Paulus which offers additional information to that given in
this chapter. 3

2.1 Isothiazolinones

The isothiazolinones as a class are probably the most commonly used

industrial, agrochemical and cosmetic preservatives. The structures of
those isothiazolinones in common use as preservatives are shown in Figure
2.1.

Benzisothiazolin-3-one. Benzisothiazolin-3-one (BIT) is an excellent

broad spectrum antimicrobial. It is most commonly encountered in the
Proxel range of preservatives by ZENECA Biocides. As such it is supplied
either as a solution in caustic and glycol or as a neutral dispersion. The
recommended use levels for BIT are in the range of 100 to 500 ppm. The
physical-chemical properties of BIT are summarized in Table 2.1.

Some of the advantages of BIT are:

- good broadspectrum antimicrobial activity.
- excellent temperature stability.
- compatible with a wide range of industrial systems.
- excellent stability to both acid and alkaline pH.
- fully compatible with amines.
 CHEMICAL PRESERVATIVES 7

o
~N-H
~S)
Benzisothiazolin-3-one

2-Methyl-isothiazolin-3-one

5-Chloro-2-methyl-isothiazolin-3-one

2-Methyl-4,5-trimethyleneisothiazolin-3-one

2-0ctylisothiazolin-3-one

Figure 2.1 Structures of isothiazolinones in common use as preservatives.

Table 2.1 Physical-chemical properties of BIT

Property Result

Colour Colourless, although formulations may be straw

coloured to brown
Melting point 157-158°C
Dissociation constant pKa = 7.5 at 25°C
Vapour pressure 5.8 X 10-5 Pa at 20°C
Solubility Soluble in hot water. Forms water-soluble salts with alkalis
and amines. Soluble in most organic solvents except
petroleum ether.
Partition coefficient Log P = 1.3 at 25°C
Density 1.45 g ml- 1 at 20°C
Stability Good heat resistance. Stable over a wide pH range.
8 PRESERVATION OF SURFACTANT FORMULATIONS

- chlorine/halogen free.
- comparatively low toxicity to higher forms of life.
- biodegradable by soil organisms, resulting in lack of accumulation in
the environment.
The weaknesses of BIT tend to be common to all isothiazolinones. They
are:
- inactivation by simple thiols. This is an inevitable consequence of its
mode of action as a broad spectrum thiol reagent.4-7
- inactivation by oxidation/reducing agents.

BIT may be readily analysed by HPLC. Alternately in those systems

where there are no other competing chromophores BIT's near UV
spectrum (Emax = 318 nm) may be used to estimate its concentration. A
rapid semi-quantitative method which is often used in production
environments to confirm that BIT has been added to a material is to look
for BIT fluorescence on irradiation with a fluorescence lamp. A key factor
in the analysis of BIT in complex systems such as a polymer emulsion or a
paint is the initial separation of the BIT. This may be done either by
separating the system by centrifugation or by removing the BIT by dialysis.
In both procedures the extraction is optimized by converting BIT into its
highly soluble ammonium salt via the addition of 3M ammonia prior to
centrifugation or dialysing against ammonia.

Mixture of 5-chloro-2-methyl-isothiazolin-3-one (eMIT) and 2-methyl-3-

isothiazolin-3-one (MIT). Kathon is the tradename for a range of
preservatives based on these actives and sold by Rohm and Haas Ltd. It is
supplied in many formulations but mainly as either a 15% or a 1.5%
solution. Rohm and Haas also supply the material in several grades of
varying purity and containing various additives. Generic copies of this
product are available in most countries (but not the USA) via a number of
other manufacturers and formulators. The ratio of the two active
components in Kathon is typically 3:1 in favour of the chlorinated
component. Indeed most of the antimicrobial activity of Kathon can be
ascribed to the chlorinated component which is about four to five hundred
times more active than its unchlorinated analogue. 6 Physical-chemical
properties of CMIT/MIT are summarized in Table 2.2
The main strengths of the CMIT/MIT mixture of biocides are:
- its extremely high activity
- excellent compatibility with many classes of commonly used industrial
chemicals
- low VOC
- water-based formulation
 CHEMICAL PRESERVATIVES 9

Table 2.2 Physical-chemical properties of CMITIBIT

Property MIT CMIT

Appearance Colourless, extremely Slightly yellow crystals,

hygroscopic crystals darkening on storage
Melting point 50-51°C 54-55°C
Boiling point 93°C at 0.03 mm
Solubility 30 g 1-1 in H 2 0. Soluble in a Approximately 5 g 1-1 in H 20.
variety of organic solvents Soluble in a wide variety of
organic solvents
Stability The free base is converted The free base is not very stable
almost instantaneously into an on storage, tending to go dark
oil on exposure to moist air. and gummy. The hydrochloride
The hydrochloride is much less is more stable, but not
deliquescent. completely so.

Decomposition on heating starts Decomposition on heating starts

at 55°C. at 5SOC.

Reacts rapidly with sodium Reacts rapidly with sodium

bisulphite, and readily with bisulphite and readily with
oxidizing agents, thiols and thiols, amines and alkali.
(more slowly) amines. Sensitive to oxidizing agents,
but less so than most other
isothiazolinones.

- increased stability to most oxidizing agents compared with other

currently available isothiazolinones
Drawbacks are:
high inherent toxicity, especially the 15% material
- instability at alkali pH. 5-Chloro-2-methyl-isothiazolinone tends to
degrade rapidly above pH 9 though problems can occur if long term
stability is required at a pH of 8.5 or even less. 8
lack of temperature stability. At temperatures above 45°C 5-chloro-2-
methyl-isothiazolinone will slowly degrade over the course of several
days.9 Above 60°C this inactivation becomes quite rapid.
inactivation by some nitrogen-containing compounds. Amine bases
such as ammonia, triethanolamine, diethanolamine, mono ethanolamine
and some amine dispersing agents are able to react, in some cases
rapidly, and inactivate 5-chloro-2-methyl isothiazolinone. 7 •10
inactivation by some thiols. Simple thiols such as 2-mercaptoethanol
and dodecyl mercaptan 7 are able to react with and inactivate CMIT/
MIT (and indeed all isothiazolinones).
inactivation by reducing agents. CMIT/MIT are rapidly inactivated by
reducing agents such as sodium metabisulphite.
10 PRESERVATION OF SURFACTANT FORMULATIONS

- a requirement for a stabilizer. This is required for many outlets for

CMIT/MIT and is usually the nitrate of a divalent metal ion such as
magnesium, copper or calcium though in the past formaldehyde has
been used. There is an extensive patent literature on various stabilizers
for CMIT/MIT.

2-Methyl-4,5-trimethyleneisothiazolin-3-one. 2-Methyl-4,5-trimethylene-
isothiazolin-3-one (MTI) is a new antimicrobial agent recently introduced
by ZENECA Biocides under the tradename of Promexal X50. A summary
of the physical-chemical properties of MTI is given in Table 2.3.
MTI's main strengths are:
- high activity
- excellent compatibility with many classes of commonly-used industrial
chemicals
- stable at alkaline pH
- good temperature stability
- water-based product
- no stabilizer required
- readily lost from the environment by a combination of biochemical and
photochemical degradation
- chlorine free.
The drawbacks of MTI are essentially the same as those of benziso-
thiazolin-3-one.

2-0ctyl-isothazolin-3-one. 2-0ctyl-isothiazolin-3-one (OIT) differs from

the other isothiazolinones in common use in that it is predominantly a
fungicide. Its physical-chemical properties are outlined in Table 2.4.
Its main use is as a dry film fungicide for paint and plastic dry films.
However, it is occasionally used either by itself or in a mixture with a
suitable bactericide in those systems (usually low pH and high solids) which
are particularly susceptible to fungal spoilage.

Table 2.3 Physical-chemical properties of MTI

Property Result

Appearance Colourless, crystalline solid

Melting point 122-123°C
Solubility Very soluble in water. Soluble in the majority of organic
solvents.
Thermal stability Decomposition starts at about 216°C
Chemical stability Reacts with reducing agents such a bisulphite and oxidizing
agents. Also reacts with thiols and amines
 CHEMICAL PRESERVATIVES 11

Table 2.4 Physical-chemical properties of OIT

Property Result

Appearance Colourless to pale yellow oil

Boiling point ~125°C at 0.2 mm
Solubility 480 ppm in water. High solubility in the majority of organic
solvents.
Partition coefficient log P 3.35
Vapour pressure 2.7 X 10-7 Pa at 20°C
Stability Inactivation by ammonia, primary and secondary amines
increasing with increasing pH. Reacts with reducing agents
such as bisulphite and thiols and oxidizing agents.

2.1.1 Toxicology
All isothiazolinones are irritants and sensitizers. I I The potential to cause
allergic dermatitis is probably the biggest drawback of this class of
molecule. The occurrence of sensitization due to eMIT is particularly well
documented due to its widespread use in personal care products. 12

2.1.2 Mode of action

The mechanism of action of BIT against one bacterium, Staphylococcus
aureus, was investigated by Fuller and coworkers. 4 Briefly they found that
BIT was a non-specific thiol reagent which reacted with a range of thiol-
dependent processes associated with the cytoplasmic membrane. The
general reaction of BIT with thiols is illustrated in Scheme 2.1.

o
~N-H
~sj
+ RSH

1l
o
o o ~NH2 + RSSR

~NH2 + ~N-H ~s,s'()

~SH ~s) H2NyJ
o
(+ RSSR)

Scheme 2.1
12 PRESERVATION OF SURFACTANT FORMULATIONS

BIT was shown to react rapidly with thiols, setting up a complex series of
equilibria, depending on the concentrations involved. The initial reaction
is to form an unsymmetrical disulphide, which slowly rearranges to the two
symmetrical disulphides. In the presence of excess thiol, the unsymmetrical
disulphide reacts further to give 2-mercaptobenzamide and the disulphide.
The symmetrical disulphide from BIT is in equilibrium with BIT and 2-
mercaptobenzamide, the reaction being particularly favoured at alkaline
pH. When Staphylococcus aureus was challenged with sub-MIC levels of
BIT, almost all of the BIT was detected as 2-mercaptobenzamide in the
supernatant liquors, thus indicating that this was the preferred route for
detoxification by the bacteria under these conditions.
This work was taken up and developed with other isothiazolinones,
namely 2-methyl-isothiazolin-3-one and 5-chloro-2-methyl-isothiazolin-3-
one. 5- 7 These studies suggested a fundamental difference in the mode of
action of 5-chloro-2-methyl-isothiazolin-3-one able to explain its much
greater efficacy. The basis of this difference is shown in Scheme 2.2.

o o
~N-CH3
c,)Ls;
+ RSH
~-----------------~ f'<NH-CHs
a)L~
SR

· RSH11
o o
~NH-CHs f'<NH-CH3
a As' a)Ls~
+ RSSR
Scheme 2.2

Reaction of the isothiazolinone with thiols is thought to proceed initially

as for BIT with the formation of the mixed disulphide, although these
compounds are poorly characterized (except in the case of the hindered t-
butylmercaptan) and the reversibility of the reaction is less well known.
Further reaction with thiols initially produces the mercaptoacrylamide
which rapidly tautomerizes to the thermodynamically more stable yet
highly reactive thioacyl chloride. This highly reactive species is thought to
be able to react rapidly and irreversibly with nucleophilic species such as
amines, water, alcohols and thiols. 5- 7 This suggests that the greater
antimicrobial activity of CMIT depends not so much on the greater activity
 CHEMICAL PRESERVATIVES 13

with thiols but rather on the extreme activity of one of its breakdown
products. Thus, while the mercaptoamide structure in Scheme 2.2 is an
innocuous breakdown product with BIT or 2-methyl-isothiazolin-3-one,
with 5-chloro-N-methyl-isothiazolin-3-one it is a precursor of a highly
active intermediate, explaining both the increased antimicrobial activity
and its increased toxicity.

2.2 Other nitrogen-sulpbur compounds

Dithio-2,2' -bis(benzmethylamide). This agent is structurally related to

the isothiazolinones being the two-electron reduction product of 2-methyl
BIT. Dithio-2,2'-bis(benzmethylamide) is sold by ZENECA Biocides as a
neutral 25% dispersion under the trade name of Densil P. Densil P is an
unusual preservative in that it is effective against both bacteria and fungi;
its physical-chemical properties are summarized in Table 2.5.

Typical use levels are 100--500 ppm active ingredient (AI). Its main
drawbacks are its lack of solubility which is more pronounced at neutral to
alkali pH and its lack of key regulatory clearances.

2-Mercaptopyridine N-oxide. The structures of the two commercially

available forms of 2-mercaptopyridine N-oxide, (pyrithione) the sodium
salt and the zinc complex, of this agent are shown at the top of page 14.

Table 2.5 Physical-chemical properties of Dervil P

Property Result

Appearance Colourless, crystalline solid

Melting point 220-22l"C
Solubility 200 ppm in water. 1000 ppm in methyl ethyl ketone (MEK).
10 ppm in white spirit.
Heat stability Very good.
Chemical stability Hydrolyses in alkaline medium to N-methylbenzisothiazolinone
and N-methylthiosalicylamide. Readily oxidized by peracids
at alkaline pH, more slowly at pH 6. Reacts with bisulphite.
14 PRESERVATION OF SURFACTANT FORMULATIONS

Cl N S"Na+
"'~f----l.~ o
llN~s
6- 6- Na+

Zinc Pyrithione
Sodium Pyrithione

Both forms of this agent are broad spectrum antimicrobials displaying

activity against bacteria and fungi. However, they are usually considered to
be fungicides with a significant antibacterial activity. Their physical-
chemical properties are outlined in Table 2.6.

Table 2.6 Physical-chemical properties of sodium pyrithione and zinc pyrithione

Property Sodium pyrithione Zinc pyrithione

Appearance White to pale yellow crystalline White to pale yellow crystalline

solid with a slight odour solid with a slight characteristic
odour
Melting point 250°C (decomposition) 240°C (decomposition)
Solubility >500 g 1-1 in water. Soluble in Very low solubility in water
ethanol, ethylene, glycol, (-20 ppm), but solubility
dimethyl sulphoxide. increased by the addition of
amines such as diethylene
triamine. Moderately soluble in
dimethyl formamide and
dimethyl sulphoxide.
pH of 2% aqueous 8 Insoluble
solution
Stability Solid is stable although can go At acid pH the zinc is lost to give
yellowish on prolonged pyrithione which is unstable.
exposure to light. Aqueous Under alkaline conditions it
solutions are stable between reacts similarly to sodium
pH 4.5 to 9.5. At lower pHs pyrithione, although the very
converted to free pyrithione low solubility undoubtedly
which is unstable to air helps to protect it. Oxidizing
(oxidation to the disulphide) and reducing agents affect it as
and light. At more alkaline pHs for sodium pyrithione. The zinc
slowly converts to the sodium atom is readily replaced by
salt of pyrithione sulphonic other transition metal ions that
acid. Oxidizing agents convert form stronger complexes, and
it via the disulphide to the these are usually strongly
sulphinylsulphide, sulphonyl coloured.
sulphide and eventually the
sulphonic acid. Strong reducing
agents reduce the N-oxide to
give 2-mercaptopyridine.
Forms highly coloured
complexes with transition metal
ions.
 CHEMICAL PRESERVATIVES 15

The main difference between the two forms of pyrithione is their

aqueous solubility and stability. That is, zinc pyrithione is stable and
insoluble while sodium pyrithione is soluble but unstable particularly in the
presence of light or oxygen. Sodium and zinc pyrithione are sold under the
trade name, Omadine, by Olin Corporation.
The major use of sodium pyrithione is in the preservation of metal
working fluids. Zinc pyrithione is predominantly sold as a personal care
preservative though it is also used as a tank-side preservative for metal
working fluids. Sodium pyrithione has only limited use in cosmetics due to
concerns over irreversible effects on eyes.
Apart from the concerns over eye damage due to pyrithione, sodium and
zinc pyrithione have a reasonably good toxicology profile. Both are skin
irritants but are not sensitizers. Zinc pyrithione may be used over the range
pH 4.5 to 9.5. Outside this range it breaks down to yield free pyrithione.
However, at pH 9.5 pyrithione is fully ionized and is rapidly converted to
the corresponding sulphinic acid which has no antimicrobial activity. At
neutral pH zinc pyrithione is an extremely heat stable molecule tolerating
temperatures of lOO°C for 120 h and only decomposing at 240°C.

2.3 Organobromine compounds

2-Bromo-2-nitropropane-I,3-diol. 2-Bromo-2-nitropropane-1 ,3-diol com-

monly known as Bronopol is a formaldehyde condensate but it is con-
sidered separately here since it is known to kill microbes by a mechanism
not related to its formaldehyde-release properties. The main suppliers of
Bronopol are Boots and Angus.

HO~N02
HO-/"Br

The physical-chemical properties of Bronopol are given in Table 2.7.

Traditionally Bronopol's main use has been as a preservative for
personal care products. However, in recent years it has found an increasing
application as a preservative in acidic polymer emulsions and adhesives.
Bronopol's use as a preservative in more alkali systems is limited since, as
discussed below, above pH 8 it can break down to give formaldehyde
losing activity in the process. The decomposition of Bronopol in solution at
elevated pH has been studied by various authors. In a recent study13 a
reaction scheme (Scheme 2.3) was proposed to account for the many
observed decomposition products.
16 PRESERVATION OF SURFACTANT FORMULATIONS

Table 2.7 Physical·chemical properties of Bronopol

Property Result

Appearance Colourless, odourless crystalline solid

Melting point 130°C
Vapour pressure at 20°C 1.68 X 10-5 hPa
Solubility 250 g 1-1 in water. Soluble in alcohols. Very low solubility in
petroleum ether
Stability Stable as the solid. Aqueous solutions are relatively stable on
the acid side but increasing tendency to decompose (releasing
formaldehyde and many other products) as the pH is
increased.
Discoloration and corrosion can occur when aqueous solutions
of Bronopol are in contact with iron and aluminium.
Alcoholic solutions of bronopol are photochemically very
unstable, readily going yellow to brown when exposed to
light.

It can be seen that the decomposition of Bronopol is quite complicated,

but the significant points are the release of formaldehyde and nitrite, the
latter giving rise to the possibility of nitrosamine formation in the presence
of amines. 14 •15

2.3.1 Mode of action

Various studies 16 ,17 have indicated that the antimicrobial activity of
Bronopol is primarily due to its interaction with essential thiols within the

• HN02 HCHO HC02H

• HCHO _
+

HO~No,
HO-./"Br
HCHO.. [ OHC,,}o, No2
HO~OHJ -HO~OH
1 OH
l.

Scheme 2.3
 CHEMICAL PRESERVATIVES 17

cell, although there is a secondary action on the cell membrane which is not
thought to be very significant. Although Bronopol can release up to two
moles of formaldehyde, this plays at best only a minor role in its
antimicrobial activity, formaldehyde-resistant and formaldehyde-sensitive
strains of Pseudomonas aeruginosa being equally sensitive to the action of
Bronop01.18 Although Bronopol is thus essentially a thiol reagent, it differs
in some respects from the isothiazolinones considered earlier, and there
have been several studies on the interaction of gem-bromonitro compounds
with thiols or thiolate anions. 17 ,19,ZO Several different reaction types can
take place, as can be seen from the accompanying schemes (Reaction paths
1-4) in which RzC(Br)NO z is a generalized gem-bromonitro compound.

R2 C(Br)N0 2 + R'S- • R2 C(Br)NO; + R'S'

R2 C(Br)NO;
• R2 CN0 2 + Br-
Reaction path 1
2 R'S' • R'SSR'

2 R2 CNOi • R2 C(N0 2 )-C(N0 2 )R 2

Scheme 2.4

Reaction path 1 (Scheme 2.4) involves the initial attack of the thiolate
anion to form a radical and a radical anion and is reported to give almost
quantitative yields of the dimeric products and bromide ion when
performed anaerobically under argon.

R2 C(Br)N0 2

2 R'S'
+ R'S- .. R2 C(Br)NO;

R'SSR'
+ R'S'
Reaction path 2

R2 C(Br)NO; + O2 , >

Scheme 2.S

Reaction path 2 (Scheme 2.5) is thought to be important under oxic

conditions where the initially formed radical anion of Bronopol reacts with
oxygen to generate superoxide and regenerate Bronopol.

R2C(Br)N02 + R'S
• R2C(Br)NO; + R'S' (initiation)

• + Br-

I
R2C(Br)N0 2 R2 CNOi
Reaction
R2CNO, + R'S- • R C(SR')NO;
2 (propagation) path 3

R2C(SR')NO; + .. R2 C(SR')NQ2 +
R2C(Br)N0 2 R2C(Br)N02

Scheme 2.6
18 PRESERVATION OF SURFACTANT FORMULATIONS

Reaction path 3 (Scheme 2.6) is the SRN I reaction (radical chain

nucleophilic substitution) where the initially formed radical anion takes
part in chain propagation r«actions. Termination is via radical-radical
dimerizations.

R2 C(Br)N0 2 + R1 S- - - - - -....~ R2 CNO; + R1 SBr

R1 SBr + R1 S- - - - - -....~ R1 SSR 1 + Br- Reaction path 4

R2 CNO; + - - - - . . . . ,..~ R2 C(N0 2 )-C(N0 2 )R 2

R2 C(Br)N0 2 + Br-

Scheme 2.7

Reaction path 4 (Scheme 2.7) is the so-called 'halogenophile' reaction

and does not involve a radical anion.

1,2-Dibromo-2,4-dicyanobutane. This agent is predominantly sold as the

'Tektamer 38' range of preservatives by Calgon Corporation and is
available only as a racemic mixture of the two enantiomers (it is not known
if there is any difference in activity between the two enantiomers). The
main forms are Tektamer 38 Powder, a 98% active agent powder, which is
recommended for addition to systems which require vigorous agitation and
Tektamer 38 A.D a 25% dispersion. Probably the major use of 1,2-
dibromo-2,4-dicyanobutane is in Europe as a personal care preservative.
The major suppliers in this market are Schuelke and Mayer who sell 1,2-
dibromo-2,4-dicyanobutane as a solution in 2-phenoxyethanol under the
tradename of Euxkyl K400. 1,2-Dibromodicyanobutane is also widely used
as a preservative for adhesives, slurries, polymer emulsions and some
metal working fluids. Physical-chemical properties of this molecule are
given in Table 2.8.
,Br
Br ~
NC
•
eN
The key advantage of 1,2-dibromo-2,4-dicyanobutane is its good
toxicology profile especially with regard to its low skin-sensitizing
potential. The widespread use of this agent in cosmetics has resulted in
some reports of sensitization but much fewer than competing materials. It
also shows good compatibility with many industrial components particularly
oxidation and reduction chemicals.
The weaknesses of 1,2-dibromo-2,4-dicyanobutane are its instability in
the presence of amines and high pH. It is recommended that above pH 8 its
stability in various systems is always checked. The breakdown products are
not well characterized, but are presumed to be due to attack of
nucleophiles present in the solution at elevated pH. Elimination of the
 CHEMICAL PRESERVATIVES 19

Table 2.8 Physical-chemical properties of 1,2-dibromodicyanobutane

Property Result

Appearance White to yellow crystalline powder

Melting point 51-53°C
Solubility 3.8 g 1-1 in water. Soluble in most organic solvents
Stability Reacts readily with a variety of nucleophiles. However,
aqueous solutions are sufficiently stable up to pH 8.
Relatively stable to oxidizing and reducing agents

tertiary bromine substituent is also expected to be a favoured reaction and

the resulting substituted acrylonitrile should be susceptible to Michael
addition reactions and would also activate the primary bromine substituent.
'Nu' represents a nucleophile (Scheme 2.8).

CN
l NC,.,Br . Nu NC,.,Br .
~CN
Br ............... .. Br~CN ~ NU~CN
NUr--___~u ______

cN ~ CN ~
Br~CNNU NU~CN
Nu-... CN ~
Br~CN
NU

Scheme 2.8

Of course, where water is the nucleophile, the initially-formed alcohols

could react further with unreacted bromo-compounds to give ethers etc.
1,2-Dibromo-2,4-dicyanobutane rapidly breaks down above SO°c. In
solution, the breakdown pathway would presumably be similar to the
scheme above. Under anhydrous conditions the thermal degradation
pathway probably starts with the loss of HBr and goes on to give further
degradation products (see Ref. 21) which comments on the thermal
instability of the corresponding dichloro compound on attempted distilla-
tion at 12 Torr. At elevated levels these breakdown products can cause
significant discoloration of many industrial products.
Little has been published in the open literature on the mode of
antimicrobial action of 1,2-dibromodicyanobutane. However, based on the
known chemistry of this class of molecules it most probably reacts as an
alkylating agent. Thus it would be expected to react similarly to other
alkylating agents such as chloroacetamide etc. with available cellular
nucleophiles (see Scheme 2.8).
20 PRESERVATION OF SURFACTANT FORMULATIONS

2.4 Thiocyanates

While there are many examples of antimicrobial thiocyanates, two

compounds containing this active group are used as industrial anti-
microbials. They are methylene bisthiocyanate (MBT) and 2-(thiocyano-
methylthio)-benzothiazole (TCMTB). The structures of these agents are
shown below.

~N
~s,>-S'-SCN
MBT
TCMTB

MBT and TCMTB are potent broad spectrum antimicrobials. 1 A

summary of their physical properties is given in Table 2.9.
The thiocyanates find only limited use as wet-state preservatives the
main use of MBT being as a water treatment chemical and TCMTB as a
fungicide for timber and leather.
Organic thiocyanates are potent sulphydryl reagents and two reaction
pathways (Scheme 2.9) have been characterized, depending on whether
cyanide or thiocyanate is the leaving group.

RSCN + R'S- -------l.~ CN- + RSSR'

I
Reaction path 1

RSSR
+
+ R'SSR'
RSCN + R'S- _ _ _ _ _ _......~ SCW + RSR' Reaction path 2

Scheme 2.9

Table 2.9 Physical-chemical properties of MBT and TCMTB

Property MBT TCMTB

Appearance Yellow crystalline solid with a Yellow oil (or low melting solid)
characteristic odour with a characteristic smell
Density 2.0 g ml- 1 1.38 g ml- I
Melting point 105°C
Vapour pressure at 4.75 X 1~ hPa
25°C
Solubility 5 g 1-1 in water. Soluble in most 0.033 g 1-1 in water. Soluble in
organic solvents acetone, DMF etc.
Stability Stable in acidic systems at Decomposition in alkaline
ambient temperatures. media (>pH 8) and on heating
Decomposes in alkaline (>60°C)
solution above pH 7.5.
Unstable above 100°C
 CHEMICAL PRESERVATIVES 21

It is considered that the first pathway is more important for aliphatic

thiocyanates while the second becomes significant for aromatic thiocanates
which are activated by electron-withdrawing groups.

2.5 Dithiocarbamates

Dithiocarbamates have the general structures shown below.

Where Me + is a cation, if the cation is polyvalent, then there will, of

course, be more than one dithiocarbamate anion present to satisfy overall
neutrality. If the metal is a transition metal, then neutral complexes may be
formed, although these may be polymeric.
As a class the dithiocarbamates have been largely superseded by more
modern preservatives such as the isothiazolinones. They are predominantly
fungicides and their main non-agrochemical use has been in water
treatment. However, they are occasionally used as in can preservatives, in
systems prone to fungal spoilage, where their main disadvantage is a
tendency to discolour some systems. Dithiocarbamates are believed to kill
microorganisms via their thiol-modifying activity (in the case of those
where one of RbR2 is hydrogen, where they decompose to generate
isothiocyanates) or via their metal-complexing abilities.

2.6 Organoiodine compounds

3-Iodopropargyl-N-butylcarbamate. 3-Iodopropargyl-N-butylcarbamate
is sold almost exclusively as Troysan Polyphase by the Troy Corporation.

Its main use has been as a fungicide for paint films and timber. However it
is also used, usually at the same time as an antibacterial, to provide
22 PRESERVATION OF SURFACTANT FORMULATIONS

protection to those systems which are particularly prone to fungal as well as

bacterial spoilage. It has the chemical-physical properties shown in Table
2.10.
Very little information has been published about the mode of action of 3-
iodopropargyl-N-butylcarbamate. The carbon-iodine bond is weak and
readily broken (this accounts for the characteristic yellowing that plagues
this class of compound). However, the antimicrobial activity (MIC) is
unaltered in the presence of radical scavengers suggesting that the release
of iodine radicals is not of major importance in its antimicrobial activity.
The iodopropargyl group will react with a number of thiols, oxidizing them
to disulphides and being reduced itself to the uniodinated prop argyl
derivative, thus acting as a source of 'positive iodine'. It must be
emphasized that the iodine substituent in iodopropargyl compounds is very
reluctant to act as a leaving group in nucleophilic substitution reactions.
2 R-=.-I + 2 R 1SH ~ 2 R-=-H + R 1SSRl + 21-
Diiodomethyl-p-tolylsulphone. Diiodomethyl-p-tolylsulphone is also pre-
dominantly a fungicide which like 3-iodopropargyl-N-butylcarbamate has
found application in boosting the antifungal activity of antibacterial
preservatives. The properties of diiodomethyl-p-tolylsulphone are
summarized in Table 2.11.

Diiodomethyl-p-tolylsulphone displays actlVlty predominantly against

fungi and yeast. It does have some activity versus Gram positive bacteria
but is almost inactive against Gram negative microorganisms.

Table 2.10 Physical-chemical properties of Troysan Polyphase

Property Result

Melting point
Solubility 156 ppm in water. Very soluble in most organic solvents.
Thermal stability Decomposes above 192°C
Chemical stability Formulations of Troysan need a stabilizer to prevent
disproportionation (giving mixtures of mono-, di- and
triiodoallylbutyl carbamate together with uniodinated
species). Photochemical loss of iodine.
 CHEMICAL PRESERVATIVES 23

Table 2.11 Physical-chemical properties of diiodomethyl-p-tolylsulphone

Property

Appearance Fine, tan-coloured powder

Melting point 157°C
Solubility -1 ppm in water. Soluble in many organic solvents such as acetone
Density 2.2 g ml- 1
Stability Stable between pH 4-10. Photochemical loss of iodine

There has been very little published information on the mode of action
of diiodomethylsulphones. In contrast to other aliphatic iodine compounds
such as iodoacetamide (where the iodine substituent acts as a leaving group
in nucleophilic substitution reactions) the iodine substituents in diiodo-
methylsulphones are virtually inert to normal substitution reactions and
can probably best be considered as a source of 'positive iodine' which can
act as an oxidizing agent, particularly for thiols (see section on the
iodopropargyl compounds).
2 ArS02CHI2 + 2RSH --~) 2 ArS02CH2I + RSSR + 21-

2.7 Aldehydes and aldehyde-release agents

Formaldehyde and formaldehyde-release agents. Formaldehyde in

aqueous/methanol solution (formalin) is one of the oldest most potent
antimicrobials available. It displays many desirable features, being
biodegradable and displaying a potent sporicidal activity. However,
formaldehyde is not suitable in most cases for use as a preservative since it
is rapidly lost from systems due to its volatility and unwanted side
reactions. Indeed since formaldehyde is a well known cross-linking reagent
its presence can cause gelling and severe compatibility problems in many
systems. In addition there are serious toxicology concerns around
formaldehyde and indeed free formaldehyde gas is significantly more toxic
than cyanide!
To overcome this, use has been made of the ability of formaldehyde
reversibly to form stable and in general safe adducts with itself and a very
wide variety of compounds possessing active hydrogen atoms (alcohols,
phenols, ketones, ammonia, amines, hydroxylamines, amides, hydantoins,
ureas, thiols, thioureas, amino acids, amino alcohols, aliphatic nitro
compounds etc.). The initially formed adducts can further react to give
more condensed molecules so that there is an almost limitless number of
24 PRESERVATION OF SURFACTANT FORMULATIONS

formaldehyde addition and condensate molecules potentially available.

These reaction types are shown in Scheme 2.10, but it must be emphasized
that these are only a small fraction of the potential starting materials and
reactions possible, and the reader is recommended to consult some of the
many excellent reviews available. 22
All these many addition and condensation reactions are potentially
reversible but the position of equilibrium and the speed of the reaction to
re-establish equilibrium once the equilibrium is disturbed (e.g. by
removing formaldehyde from the system by reacting with some biological
target) varies with the structure in question. Where the compound is very
stable, such as phenol-formaldehyde condensates, there is usually insuffi-
cient formaldehyde ever available to provide an antimicrobial effect. As a
general rule, unless the formaldehyde bound up within the molecule is
sufficiently available to be detected by the Tannenbaum reagent23 or the
Nash reagent 24 then the molecule is very unlikely to have significant
activity as a formaldehyde-release biocide, although of course there may
be another mode of action. A further characteristic of formaldehyde-
release biocides is their differing activities on formaldehyde-sensitive and
-resistant strains of the same bacteria, which should parallel that of
formaldehyde. 18 In view of the very large number of formaldehyde-release
biocides that have been developed, it is impossible to include them all.
Table 2.12 gives the structures of some of the more common ones, together
with the number of moles of formaldehyde/mole of biocide potentially
available (the potential formaldehyde being highlighted), and the release
rate (although this is described as 'fast' or 'slow', there is, of course, a
gradation between them all, and some compounds have both a fast and a
slow component of the formaldehyde release).
As shown above formaldehyde is capable of reacting rapidly with a range
of nucleophiles and this is un doubt ably its mechanism of action. A full
consideration of the toxicology of formaldehyde and its condensates may
be found in chapter 13.

Glutaraldehyde. Glutaraldehyde is a potent antimicrobial whose use is

increasing as formaldehyde and formaldehyde-release agents decline due
to regulatory and toxicology pressures. A summary of its physical-chemical
properties is given in Table 2.13.

The antimicrobial action of glutaraldehyde has been the subject of

several excellent reviews. 1,25,26 The main advantages of glutaraldehyde are
 CHEMICAL PRESERVATIVES 25

4. H2NC(R)(CH20H)2 + HCHO --~~ HOCH 2 NH(R)(CH20H)2

~O
01>. -H20 ~~:H. + HCHO q(OH
"'(or R = CH 2 0H) R R

+ HCHO~ ~OCH2)nOH H O XOH

Q l, b HO NO
vN......,.
X
+HCHO/ 2

CH2 0H HCHO H 0'

5. RCH2N02 + HCHO --~~ RCH( ~(F R-H~) HO
NO or - NO
2 2
o
-H2 0 II
~ R1 CCR 2=CH 2

Scheme 2.10
26 PRESERVATION OF SURFACTANT FORMULATIONS

Table 2.12 Structures of more common formaldehyde-release biocides

Structure Moles Ease of HCHO

HCHO/mole release
biocide

PhCH 20CH2 0H Fast

PhOCH 2CH 20CH20H Fast
02NC(CH 20Hh 3 Slow
1 and 2 Slow

6 Negligible at pH
above 6,
appreciable in
acid solution
6 Slow

4 Slow

~ NH-l'. .
[ol:J.oi,
3 Slow

gig

C!!:!gQH 2

3 Fast

HO 3 Fast
'-""'~~~OH
~~
OH

CH 2(OCHg)nOH 2+n Fast and slow

O~O
YNV
 CHEMICAL PRESERVATIVES 27

Table 2.12 continued

Structure Moles Ease of HCHO

HCHO/mole release
biocide

Fast. Mode of
action not
solely
dependent on
HCHO release
4 Slow

r OH2~
- "
HOH C
... l,H?,OH

- g - -?,-
CH OH
~+
2
-2
804

Table 2.13 Physical· chemical properties of glutaraldehyde

Property Result

Appearance Colourless oil, smell reminiscent of succindialdehyde

Boiling point 187-189°C (with decomposition) at atmospheric pressure,
106-108°C at 50 hPa, 83-85°C at 15 hPa.
1.43
Solubility Soluble in water with a tendency to polymerize, depending on
the pH. Soluble in most organic solvents
Stability Characteristic reactions of an aldehyde (reduction, oxidation,
condensation etc.). Solutions in water are stable at acid pH
but rapidly decompose as the pH is increased, particularly in
the presence of amines

its rapid rate of kill, broad spectrum of activity and sporicidal activity.
Disadvantages are its odour, unacceptable in many applications, and
instability at neutral to alkaline pH.

2.8 Phenols

The phenolics are an established class of antimicrobials with a long record

of use as disinfectants. They are also used, albeit to a limited degree, as
28 PRESERVATION OF SURFACTANT FORMULATIONS

preservatives in some systems. Chlorinated phenols such as pentachloro-

phenol are no longer in common usage due to environmental and
toxicology concerns.

2.9 Organic acids and salts

Used extensively in the food industry these agents are making a comeback
in some of the areas covered in this book. This is despite their much lower
efficacy than many of the other agents referred to in this chapter and
reflects their excellent toxicology profile. Their use overall and in
cosmetics is covered in chapter 7 of this book.

2.10 Mercury and other inorganic elements

For many years mercurials were among the most widely used industrial
preservatives due to their high activity, low cost, and lack of colour and
odour. While their use is declining steadily they are still popular in those
markets where they are allowed. A review of the history and use of
mercurials antimicrobial agents may be found in Ref 1. The two most
commonly used mercurials are phenylmercuric acetate in its various forms
and di(phenylmercury)dodecyl succinate. The main difference between the
two agents is that phenylmercuric acetate is soluble in organic solvents but
essentially insoluble in water while the converse is true of di(phenyl-
mercury)dodecyl succinate.
Many other inorganic agents have been used historically as preservatives,
for example tributyltinoxide, 10,10' -oxybisphenoxy arsine and barium
metaborate, and indeed they still continue to be used in other industrial
antimicrobial outlets. However, they do not come within the remit of this
volume, though interested readers may refer to several chapters in Block. 1

References

1. Block, S. (ed) (1991) Disinfection, Sterilization and Preservation, Lea and Febinger,
Philadelphia.
2. Albert, A. (1985) Selective Toxicity: Physical Chemical Basis of Therapy, Chapman &
Hall, London.
3. Paulus, W. (1993) Microbicides for the Protection of Materials, Chapman & Hall,
London.
4. Fuller, S.l., Denyer, S.P., Hugo, W.B., Pemberton, D., Woodcock, P.M. and Buckley,
A.I. (1985) The mode of action of 1,2-benzisothiazolin-3-one on Staphylococcus aureus.
Lett. Appl. Microbiol., 1, 13-15.
 CHEMICAL PRESERVATIVES 29

5. Collier, P.J., Austin, P.W. and Gilbert, P. (1990) Uptake and distribution of some
isothiazolone biocides into Escherichia coli A TCC 8739 and Schizosaccharomyces pombe
NCYC 1354. Internat. 1. Pharmaceut., 66, 201-206.
6. Collier, P.J., Ramsey, A.J., Austin, P.W. and Gilbert, P. (1990) Growth inhibitory and
biocidal activity of some isothiazolone biocides. 1. Appl. Bacteriol., 69, 569-577.
7. Collier, P.J., Ramsey, A.J., Waigh, R.D., Douglas, K.T., Austin, P.W. and Gilbert, P.
(1990) Chemical reactivity of some isothiazolone biocides. 1. Appl. Bacteriol., 69, 578-
584.
8. Barman, B.N., and Preston, H.G. (1992) The effects of pH on the degradation of
isothiazolone biocides. Tribology Internat., 25, 281-287.
9. Barman, B.N. (1994) Influence of temperature on the degradation of isothiazolinone
biocides in aqueous media and in a metal working fluid concentrate. Tribology Internat.,
50, 351-355.
10. Brown, S.A. personal communication.
11. Botham, P.A., Hilton, J., Evans, C.D., Lees, D. and Hall, T.J. (1991) Assessment of the
relative skin sensitising potency of three biocides using the murine local lymph node
assay. Contact Dermatitis, 25, 172-177.
12. DeGroot, A.C. and Herxheimer, A. (1989) Isothiazolinone preservative: cause of a
continuing epidemic of cosmetic dermatitis. The Lancet, 314-316.
13. Challis, B.C. and Yousaf, T.!. (1991) The reaction of geminal bromonitroalkanes with
nucleophiles. Part 1. The decomposition of 2-bromo-2-nitropropane-l ,3-diol ('Bronopol')
in aqueous base. 1. Chem. Soc. Perkin Trans., 2, 283-286.
14. Schmeltz, !. and Wenger, A. (1979) 2-Bromo-2-nitropropane-1,3-diol as a nitrosating
agent for diethanolamine: a model study. Food Cosmetic Toxicol., 17, 105-109.
15. Holland, V.R (1981) BNPD and nitrosamine formation. Cosmetic Technol., May Issue,
31-36.
16. Stretton, R.J. and Manson, T.W. (1973) Some aspects of the mode of action of the
antibacterial compound Bronopol (2-bromo-2-nitropropan-1,3-diol). 1. Appl. Bacteriol.,
36,61-76.
17. Shepherd, J.A., Waigh, RD. and Gilbert, P. (1988) Antibacterial action of 2-bromo-2-
nitropropane-1,3-diol. Antimicrob. Agents Chemother, 32,1693-1698.
18. Sondossi, M., Rossmoore, H.W. and Wireman, J.W. (1986) The effect offifteen biocides
on formaldehyde-resistant strains of Pseudomonas aeruginosa. 1. Ind. Microbiol., 1, 87-
96.
19. Bowman, W.R. and Richardson, G.D. (1981) Reactions of thiolate anions with 2-
substituted-2-nitropropanes. Tet. Lett., 22, 1551-1564.
20. Amrollah-Madjdabi, A., Begelmans, R. and Lechevalier, A. (1987) Sur la reactivite de
gem-bromonitroalcanes en presence de thiolates. Tet. Lett., 28, 4525-4528.
21. Fritz, H., Weis, C.D., Winkler, T. (1976) 20.Halogenierte pyridine die herstellung von 3-
halogenmethylpyridinen aus dimerem acrylnitril. Helv. Chim. Acta, 59, 179-190.
22. Rossmore, H.W. and Sordossi, M. (1988) Applications and mode of action of
formaldehyde condensate biocides. Adv. Appl. Microbiol., 33, 223-277.
23. Tannenbaum, M. and Bricker, C.E. (1951) Microdetermination of free formaldehyde.
Anal. Chem., 23, 354-357.
24. Nash, T. (1953) The colorimetric estimation of formaldehyde by means of the Hantzsch
reaction. Biochem. 1., 55, 416-421.
25. Snyder, R W. and Cheatle, E.L. (1965) Alkaline glutaraldehyde; an effective disinfectant.
Am. 1. Hosp. Disin., 22, 321-327.
26. Gorman, S.P. and Scott, E.M. (1980) The state of the glutaraldehyde molecule in
relation to its biocidal activity. 1. Pharm. Pharmacol., 32, 131-132.
3 Control of microbes through plant hygiene
H. GIBSON and J.T. HOLAH

3.1 Introduction

There is awareness in the food industry that control of factory hygiene is

essential in the prevention of microbial contamination. This chapter covers
the important considerations in controlling microbial contamination from
environmental sources in the processing environment, in an industry sector
in which hygiene is critical. The research and experience gained from the
food industry and presented in these guidelines are of relevance to the
manufacture of surfactant and surfactant based materials, particularly for
surfactants used in the food industry or liable to spoilage, and should
therefore be used in conjunction with risk assessments.
Contamination of the product may arise from four sources: the
constituent raw materals, surfaces, people (and other animals) and the air.
Control of the raw materials is addressed, for example, by specification,
positive release of materials after sampling, quality assessment/quality
control (QAlQC) procedures and supplier audits. This route of contamina-
tion is the only non-environmental route of contamination.
Water is often a raw material in many types of production, or may be
used for conveying and cooling. In these cases treatment is required to
produce water of an acceptable microbiological quality, for example by
chlorination, filtration or ozonation. 1 This article describes control of the
environmental routes of product contamination; discussion of water as a
source of contamination is beyond the scope of this chapter.
Microbes on product contact surfaces can contaminate product directly
whilst microorganisms on environmental surfaces can contaminate product
indirectly through transfer by vectors such as personnel, utensils, pests,
cleaning systems and air movement. The air of the processing environment
acts as both a source of contamination and a transport medium, moving
contamination from non-product contact surfaces to product contact
surfaces.
Knowledge of the sources, vectors and routes of contamination is
essential for the prevention of contamination. The environmental surfaces
are controlled through the design, construction and operation of the plant.
Control of contamination from product contact surfaces and equipment is
dependent upon the hygienic design of the equipment and the use of
 PLANT HYGIENE 31

effective cleaning and disinfection regimes. The personnel route of

contamination is controlled through training and specific entry procedures
for the production area. The production environment air is managed by air
control systems is conjunction with control of practices that generate air-
borne contamination. Finally management commitment is essential.

3.2 Design and construction of the premises

3.2.1 Site selection

Selection of a suitable site for new premises must involve consideration of
the availability of services, i.e. water supply and effluent disposal. The
premises should be located in areas which are not liable to flooding,
smoke, dust or odours. The immediate surroundings of the premises
should be well kept and free from refuse, rubbish, waste materials and
overgrown vegetation. Ideally the site should be surrounded with aim
wide hard surface. The yard surfaces and roads of the site must have a
suitable impervious surface with adequate drainage.
Activities such as agriculture, sewage treatment and rubbish tipping may
present potential sources of contamination. Consideration of the control of
birds, rodents and insects is important. In addition to preventing the access
of pests to the process area, it is important that the areas around the site
are kept free of rubbish and spilled product.

3.2.2 Factory structure

The types of external walls used include concrete, profiled metal sheeting,
brick and block cavity walls, used in conjunction with profiled metal
cladding and composite panels.
Pests may eat or contaminate ingredients, packaging materials and
finished product. In addition to the associated health hazards, rodents can
cause damage to buildings and electrical cabling, whilst birds can damage
mortar. Pest control is therefore an inseparable part of profitable and
hygienic production.
The major controls of pests are their exclusion from the premises and the
prevention of spread within the building. The building should be
constructed to prevent the entrance and harbouring of vermin, pests and
birds. This is achieved by using insect screens, insectocutors, screens on air
intakes and exits, air curtains or plastic strip barriers on doorways, and
traps on drains. In addition foundations should be constructed to prevent
rodents from burrowing underneath. Rodents are able to squeeze through
very small (6-10 mm) holes to gain access to buildings. Pest proofing of
32 PRESERVATION OF SURFACTANT FORMULATIONS

doors is difficult because of the requirement for movement of staff and

forklift trucks etc. However, all external doors should be self closing and fit
closely so that gaps should be less than 3 mm.

3.2.3 Production area

3.2.3.1 Walls. The type of finish to be applied will depend on the

particular area. In wet processing areas, the finish must be impervious to
moisture and withstand the water pressure and chemicals used in the
cleaning regime. In contrast, in dry and possibly dusty environments the
walls must have a hard, smooth antistatic surface. The European Council
Directive on the hygiene of foodstuffs gives general guidance for food
premises. 2 In addition relevant Directives or Regulations for specific
processing environments should be consulted for particular requirements.
The Meat Products Directive,3 for example, requires that walls must be
covered with a light coloured coating. A hygienic, durable and easily
cleanable coating is required for all processing areas.
The types of finishes available include liquid coatings, reinforced liquid
coatings, cladding sheet, composite panels and tiles.
There are a range of liquid coatings including emulsion paints, oil-based
one-pack epoxy and polyurethane paints, two-pack epoxy and polyurethane
paints, chlorinated rubber paints and fungicidal and mould resistant paints.
Emulsion paints are suitable for walls and ceilings in 'dry' areas and are
cleanable rather than washable. Oil-based (one-pack) epoxy and poly-
urethane paints can present taint and toxicity hazards. Chlorinated rubber
paints are particularly suitable for use in wet areas or the non-product
contact surfaces of equipment involved in wet processing. Fungicidal and
mould resistant paints are used in areas with high levels of humidity or
condensation. The non-leaching fungicidal paints are appropriate as the
leaching varieties may pose a potential taint hazard.
Reinforced liquid coatings which are based on glass fibres mixed with an
epoxy resin result in an easily cleaned smooth surface which is resistant to
impact damage, abrasion, a range of chemicals and heat.
Ceramic wall tiles are particularly suitable for areas where frequent
wash downs occur and abrasion and heat resistance are required. Tiles
should be fully vitrified glazed, light in colour and possess Group 1
classification for water absorption (for example in the UK in accordance
with BS6431 (1983)4 and any fixing or grouting material should be mould
resistant (for example meet the requirements of BS5980 (1980».5
Wall-to-floor stops are required to prevent doors damaging wall
surfaces, and wall corners should be protected by non-corrosive metal or
PVC angles. Galvanised steel or stainless steel rails should be used if
trolleys are likely to damage wall surfaces.
 PLANT HYGIENE 33

3.2.3.2 Floors. Floors should be durable, non-absorbent, antislip,

without crevices, capable of being effectively cleaned and should slope
sufficiently for liquids to drain. In certain cases, floors must also be
resistant to acids, grease and salts. Satisfactory drainage is obtained by
ensuring sufficient fall to drainage points. Normally a fall of 1 in 60 is
adequate, but this may vary depending on the surface texture of the
flooring and type of operation.
There are several types of flooring materials, but all are dependent on
the structural slab, the membrane, the screed and movement joints. The
types of flooring include concrete, ceramic tiles and resins. Concrete is not
generally suitable as although it is resistant to chemicals such as alkalis,
mineral oil and many salts, it is attacked by acids, vegetable and animal
oils, sugar solutions and some salts. In addition it is porous. The properties
of concrete can be improved by the addition of a polymer which improves
the resistance to liquid penetration.
Ceramic tiles are suitable as a durable and hygienic flooring material
with a range of antislip surfaces; however, it is important that they are
properly laid and grouted. Hygiene requirements dictate that only fully
vitrified tiles are used because of their very low water absorption.
Various resin-based systems are available for application to the concrete
substrate such as epoxy, polyurethane, polyester and methacrylate. These
systems provide hygienic seamless floors.

3.2.3.3 Ceilings. Ceilings may be either solid or suspended. Suspended

ceilings are preferable as all the supporting members and services are
physically separated from the processing area and services taken down
directly to the equipment. The ceiling should be smooth and easy to clean.
Special attention must be paid to the ceiling finish above equipment
generating steam so that mould growth is minimised.

3.2.3.4 Lighting. Suitable and sufficient lighting must be provided

throughout the premises including store rooms and passageways. A
lighting level of 500 Ix is suitable for work areas and 700 Ix in inspection
and quality control procedures. All glass tubes in the processing area must
be protected by covers to protect the glass and contain it in the event of
breakage.

3.2.3.5 Ventilation. In areas where large quantities of steam and moist

air are involved, adequate ventilation is required for the comfort of the
personnel and to reduce microbial growth and corrosion of surfaces.
Section 3.4 describes air control.

3.2.3.6 Factory layout. The factory layout should be such that processing
operations are as direct as possible because the straight line flow minimises
34 PRESERVATION OF SURFACTANT FORMULATIONS

the possibilities of contamination of processed or semi-processed product

by unprocessed or raw materials and is more efficient in terms of handling.
This also allows easy separation of different processing operations.
Premises should be designed so that there is physical separation of storage
and processing operations whilst maintaining efficient product flow.
Separation of processes reduces the risk of contamination. Separation of
personnel is an essential part of the separation of processes with respect to
the avoidance of cross contamination. Processing areas in which operations
of high microbiological risk are undertaken should be limited to essential
personnel.
Maintenance, hygiene and technical support, quality assurance and
management personnel, who are required to move between high and low
risk areas, must change their clothing and wash their hands.

3.2.3.7 Services. Services such as pipework should be installed in such a

way that they do not constitute a hygiene hazard. Pipes should not pass
over product lines, in order to prevent possible contamination from
condensation, leakage, lagging or dust. Pipework must be arranged so that
it can be cleaned by having adequate clearance from walls or ceilings. Pipe
insulation should be crevice free, with sealed joints, and have a durable
cleanable outer surface. Water supply pipelines should be free from 'dead
legs' which could allow stagnation and an increase in the number of
microorganisms.
Any drainage system must be of suitable construction for the particular
effluent, adequate in size, cleaned regularly and capable of operating
efficiently under the maximum load imposed. Effluent flow should always
be directed from high risk areas.

3.3 Control of product contact surfaces

3.3.1 Hygienic design

Processing equipment of poor hygienic design can result in contamination
incidents that can be both costly and damaging to the processor, but also
increase cleaning costs and downtime. Hygienic processing equipment
should be easy to maintain to ensure that it will perform as expected to
prevent microbiological problems. Equipment must be cleanable and
protect the product from contamination.
Hygienic design should be based on an appropriate combination of
mechanical, process and microbiological requirements. Hygienic require-
ments should be taken into account at the initial design stage as upgrading
existing designs to meet hygienic requirements may be prohibitively
expensive and possibly unsuccessful. In addition to maintaining food
quality and safety, compliance with hygienic design requirements may
 PLANT HYGIENE 35

increase the life expectancy of the equipment and reduce maintenance

needs and therefore reduce manufacturing costs.
In June 1989, a European Communities Directive relating to machinery
was adopted. 6 This is chiefly concerned with safety, but there is a section
concerning the essential health and safety requirements of agri-foodstuffs
machinery. This states that equipment intended to prepare and process
foodstuffs must be so designed and constructed as to avoid any risk of
infection, sickness or contagion. Several hygienic rules are listed that refer
to the materials of construction, surface finish, joints, fasteners, internal
angles, drainage, deadspaces, prevention of contamination by lubricants
etc. and installation. A harmonised European standard is being produced
as a consequence.
Some of the major food processors have recognised the importance of
hygienic design and produced their own standards and guidelines;
however, these documents have not been generally available. Various
organisations have, however, been producing guideline documents. The
UK Food Manufacturers Federation in conjunction with the Food
Machinery Association produced a booklet on the 'Hygienic Design of
Food Plant' with particular reference to tanks, pumps and pipework. In
addition, since 1982 the Campden Food and Drink Research Association
has been working with an industrial working party of manufacturers and
users of equipment to produce guidelines on hygienic design. Since 1992
the European Hygienic Equipment Design Group (EHEDG) has been
publishing guideline documents on the hygienic design and operation of
food plants and in particular a summary of the principle designations. 7

3.3.1.1 Surfaces. Surfaces must be cleanable and must not present a

toxicological hazard by leaching of components into the product. Product
contact surfaces must be inert, be made of non-absorbent materials and
must satisfy particular roughness requirements. All product contact
surfaces must be easily accessible for visual inspection and manual
cleaning, or it must be demonstrated that routine cleaning eliminates the
possibility of contamination from bacteria. If cleaning-in-place (CIP)
techniques are used it must be demonstrated that the results achieved
without dismantling are satisfactory.

3.3.1.2 Draining and layout. The exterior and interior of all equipment
and pipework should be self draining and easily cleanable. Horizontal
surfaces must be avoided to prevent harbouring of product soils and
cleaning liquids. Surfaces should slope so that any liquid would flow away
from the main product area.

3.3.1.3 Installation. In design, construction, installation and mainten-

ance it is important to avoid dead spaces which trap product, prevent
36 PRESERVATION OF SURFACTANT FORMULATIONS

effective cleaning and possibly allow microbial growth. Whenever possible,

the risk of condensation on equipment, pipework and the fabric of the
building should be avoided as the presence of condensation is particularly
associated with microbial growth on surfaces. Any clearance between
equipment and walls, floors and ceilings should be adequate for cleaning
and inspection (a minimum of at least 1 m).
Noise suppression may be required to provide acceptable working
conditions. It is important to select and install materials that are not prone
to microbiological or infestation problems.
The equipment should be designed, installed and maintained so that
foreign body contamination, e.g. by lagging material, loose nuts and bolts,
does not occur. The connection of different items of equipment to form a
line can cause major problems such as hold up of the product due to
unsatisfactory transfer which may subsequently re-enter the main product
flow. This can result in organoleptic or microbiological problems.

3.3.1.4 Construction materials. Materials used in the construction of

product contact surfaces must be inert to the product under operating
conditions, as well as to the detergents and disinfectants used in cleaning.
The materials must also be corrosion resistant, non-toxic, mechanically
stable and have a robust surface finish.
Austentic stainless steels are the most commonly used construction
material for food processing equipment. Type AISI 304 is most often used
in environments where fluids do not contain any chlorides. If chlorides are
present, the corrosion resistant-type AISI 316 steel which contains
molybdenum (or possibly titanium) is more suitable. Selection of the type
of stainless steel mainly depends on the corrosive properties of the process
and cleaning fluids as well as on welding requirements.
Hard plastics are also used as materials of construction but only certain
plastics are approved for food contact and are inert to cleaning chemicals.
Polypropylene is susceptible to attack by relatively low levels of sodium
hypochlorite. Plastics are not as abrasion resistant as stainless steel and
their clean ability is detrimentally affected. Polytetrafluoroethylene can be
used for gaskets and seals but has the disadvangage that it suffers from cold
flow under compression and is difficult to clean as it may not be
permanently impermeable to microorganisms. Conveyor belts are particu-
larly difficult to clean and can be a common source of contamination,
especially if the belting has a carcass of an absorbent material such as
canvas or cotton. As a consequence polyester is now widely used for the
carcass of belting because it is stable and non-absorbent.

3.3.1.5 Internal design. Of major importance in the hygienic design of

equipment is the internal design. The surface finish can affect the
clean ability as rougher surfaces can be more difficult to clean. A maximum
 PLANT HYGIENE 37

Table 3.1 Examples of surface treatments and roughness (Ra) of

stainless steel

Treatment Ra(!-\m)

Cold rolling 0.2-0.5

Hot rolling >4
Glass bead blasting 1.0-1.2
Bright-annealing 0.4-1.2
Electropolishing Dependent on the original finish

surface roughness Ra value of 0.8 !-lm is recommended for surface finish

unless there is evidence that a rougher surface is acceptable. Stainless steel
sheet, as supplied directly by the steel mill, typically has a roughness Ra
value of less than 0.5 !-lm. Electropolishing has relatively little effect on the
surface finish, although eletropolishing can improve cleanability. Table 3.1
lists several surface treatments and the associated surface roughness.
Metal-to-metal joints should not be used in product contact areas as they
are uncleanable and may retain high numbers of microorganisms which
may subsequently contaminate product. Permanent joints should instead
be continously welded and the weld ground and polished to a surface
roughness value similar to that of the surrounding material to avoid
cleaning problems. This may not always be possible, and in these cases a
gasket of a suitable material should be used; however, it is important that a
flush, continuous, crevice free surface is obtained. In certain cases non-
toxic solders may also be used but it is important to ensure their integrity
with respect to cleaning chemical action.
There should be no internal crevices or 'dead spaces' in which product
can be retained or from which product residue cannot be easily removed by
cleaning. To facilitate cleaning, the internal radii of tanks, hoppers etc.
should be as generous as technically possible.
Exposed screw threads, nuts, bolts, and rivets must be avoided where
possible as they are difficult to clean. In addition, any fastener used in or
above product areas must be secure so as to avoid the possibility of product
contamination.

3.3.2 Cleaning and disinfection

Provided that the process environment and production equipment have
been hygienically designed, cleaning and disinfection are the major
controls of the environmental routes to product contamination. Cleaning
and disinfection, when performed correctly, are cost effective, easy to
manage and can reduce the risk of microbial and foreign body contamina-
tion.
38 PRESERVATION OF SURFACTANT FORMULATIONS

3.3.2.1 Principles of cleaning and disinfection. The cleaning and dis-

infection procedure removes undesirable material (or soil) from the
surfaces, including microorganisms, product residues, foreign bodies and
cleaning chemicals. This soil may be derived from normal production,
spillages, line jams, maintenance of equipment, packaging or general
contamination from the environment (dust and dirt).
Product residues are normally easy to visualise and are characterised by
their chemical composition, e.g. fat, protein or carbohydrate. In addition,
the process or environmental factors may influence the ease of soil
removal. The moisture content of the soil, the soil temperature and the
time period before cleaning takes place affect the ease of removal.
Microorganisms may be present in the soil, or can attach to surfaces and
form biofilms. Holah and coworkers8 •9 described the attachment of
stainless steel coupons to production lines to assess the buildup of
microorganisms in a variety of processing environments and found levels of
103_107 cells cm-2 . Microorganisms are able to attach and grow on the
materials used in the factory environment; these include stainless steel,
aluminium, nylon, polypropylene, polycarbonate, PVC and Teflon.
Understanding the characteristics of the soil in terms of chemical and
microbial components is essential for a successful and economic cleaning
and disinfection programme. The cleaning and disinfection programme can
be divided into a number of phases: 10--12
1. Wetting and penetration of the soil and equipment surface by the
cleaning solution,
2. Reaction of the cleaning solution with the surface and soil. This
facilitates dissolution of soluble organics and minerals, emulsification of
fats, peptisation of organic materials and removal of solid soil particles
from the surface,
3. Prevention of redeposition of the soil removed from the surface,
4. Reaction of the disinfectant solution with the residual microbes, and
possibly removal of the cells by rinsing.
There are four factors involved in a cleaning and disinfection programme
which are used in combination to achieve the four phases described above.
These factors are:
• Chemical energy
• Mechanical or kinetic energy
• Temperature or thermal energy
• Time
Chemical energy is important for the cleaning and disinfection phases. In
the cleaning phase the chemicals break down the soils to facilitate removal
from the surface. In the disinfection phase, the chemicals reduce the
viability of the microbes remaining after cleaning.
 PLANT HYGIENE 39

Mechanical or kinetic energy is employed physically to remove soils

from the surface and may include manual brushing, scraping, automated
scrubbing, pressure jet washing or the circulation of fluid in clean-in-place
systems.
Temperature affects cleaning and disinfection in several ways. First, the
chemical effects increase linearly with temperature. Second, temperatures
above the melting points of fats and oils facilitate their removal.
Increasing the time component, for example by the use of soak tanks, or
foams and gels to extend the contact time, can increase the efficiency of
cleaning and disinfection.

3.3.2.2 Cleaning chemicals. The determination of the correct detergent

for any cleaning process is subject to a number of selection criteria. These
criteria include plant design, cleaning techniques available, the type of soil
present, the way the soil is formed, the nature of the production process
and the water chemical composition. There is no single cleaning agent that
fulfils all the requirements of an efficient cleaning programme, and for this
reason cleaning solutions or detergents are formulated to contain a number
of components with specific abilities. Typically a detergent may contain
some of the following components: water, surfactants, inorganic alkalis,
inorganic and organic acids and sequestering agents.
Water is the basic ingredient of most cleaning systems and provides a
cheap readily available transport medium for rinsing and dispersing soils.
Salts, sugars and other water-soluble components will be dissolved by
water, and at temperatures above the melting point of fats, water will aid
emulsification. Water is, however, a poor wetting agent and cannot
dissolve nonionic compounds.
Surfactants are widely used in detergent formulations for wetting soils,
soil penetration, soil suspension and to aid rinsing by reduction of surface
tension. Surfactants are composed of a long non-polar (hydrophobic) tail
and a polar (hydrophilic) head. They may be anonic, cationic or nonionic
depending on their ionic charge, but anonics and nonionics are most
common. If a surfactant is added to a drop of water the polar end of the
molecule interacts with the water droplet in such a way as to reduce the
surface tension of the droplet and thereby increase wettability and the
spread of the droplet. In the case of fats and oils, and non-polar tail
interacts with the oil and the molecule orientates with the hydrophilic part
around the fat micelle, consequently aiding the dispersion of fats from the
surface.
Alkalis (and particularly sodium hydroxide) are useful cleaning agents as
they are relatively cheap, saponify fats, break down proteins and may also
be biocidal at higher concentrations. Sodium hydroxide, a strong alkali,
exhibits high levels of saponification and protein disruption. This alkali is,
however, corrosive and hazardous to operatives. Weak alkalis may be used
40 PRESERVATION OF SURFACTANT FORMULATIONS

Table 3.2 Cleaning procedures and solubility characteristics recommended for a range of soil
types (modified from Elliot )20

Soil type Solubility characteristics Cleaning procedure

recommended

Sugars, organic acids, salt Water soluble Mildly alkaline detergent

High protein Water and alkali soluble Chlorinated alkaline
Slightly acid soluble detergent
Starchy foods Alkali soluble, slightly Mildly alkaline detergent
water soluble
Fatty acids Alkali soluble Mildly alkaline detergent
Water insoluble or strong alkali
Heat precipitated water Acid soluble Acid cleaner used on a
hardness, milk stone, Alkali and water insoluble periodic basis
protein scale

as they are less hazardous but also less effective. The major disadvantage
of alkalis is precipitation of hard water ions (calcium and magnesium).
Inorganic and organic acids are not used as frequently as alkalis;
however, they are effective at solubilizing mineral scales and are used in
CIP applications.
Sequestering or chelating agents are used to prevent the precipitation of
water hardness salts by affecting the physical structure of the precipitate or
complexing with the salts to form water-soluble complexes. Sequestering
agents may be organic or inorganic. Organic sequesterants are usually
based on polyphosphates, whilst inorganic chelating agents include the
sodium salts of ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic
acid (NTA).
The detergent chosen for a particular application will depend on the soil
to be removed and its solubility characteristics. Table 3.2 summarises the
recommended detergents for a range of soil types.

3.3.2.3 Disinfectants. The cleaning phase has been shown to result in

the most significant reduction in the numbers of bacteria present on the
surfaces;12 however, significant levels of viable cells are likely to remain
even after cleaning. The aim of the disinfection stage is to remove or
reduce the viability of these remaining microbes. Temperature is the ideal
disinfectant as it penetrates the surface, is non-corrosive, is non-selective in
terms of vegetative microorganism types and leaves no residue. High
temperatures are often successfully used as disinfectants in CIP systems;
the use of hot water on open surfaces is uneconomic, hazardous and
impractical. In these cases chemical biocides are used.
A number of factors must be considered when choosing a disinfectant
product:
1. Compatibility with other chemicals, such as any residual detergent that
may be left on the surface,
 PLANT HYGIENE 41

2. Compatibility with the construction materials,

3. Hard water and soil tolerance,
4. Non-tainting - disinfectants should not affect the product by causing
taints and therefore phenolics cannot be used in food applications,
5. Residual activity,
6. Rinsing ability,
7. Antimicrobial range,
8. Cost,
9. Toxicity, irritancy and environment impact.
There are two main types of disinfectants: oxidising and non-oxidising
biocides. The oxidising type of disinfectants include halogen-releasing
components (commonly chlorine), peroxy acids and hydrogen peroxide.
These disinfectants are generally fast acting, bactericidal products with a
wide spectrum of activity. The non-oxidising biocides include quaternary
ammonium compounds (Quats or QACs), amphoterics and biguanides,
and they are slower acting with a narrower spectrum of activity and may be
bacteriostatic at certain concentrations.
The most commonly used disinfectants are chlorine-releasing compon-
ents, QACs, iodine compounds, amphoterics and peracetic acid. Table 3.3
lists some of the characteristics of these products.

Table 3.3 Characteristics of the common disinfectants

Characteristic Chlorine QACs Iodophor Amphoteric Peracetic

acid

Microorganism control
Gram positive ++ ++ ++ ++ ++
Gram negative ++ + ++ ++ ++
Spores + +/- ++
Yeast ++ ++ ++ ++ ++
Resistance acquisition + +
Inactivation by
Organic matter ++ + + + +
Water hardness +
Detergency properties ++ + +
Surface activity ++ +1- ++
Foaming potential ++ + ++
Problems with
Taints +/- +/- +/-
Stability +/- +/-
Corrosion + +
Safety + + ++
Other chemicals + +
Potential environmental impact ++ -/+ -/+ -/+
Cost ++ + ++ +
Key: -, No effectlno problem; +, effect; ++, large effect.
42 PRESERVATION OF SURFACTANT FORMULATIONS

Chlorine is the cheapest disinfectant and is available as hypochlorite

(fast acting) or slow releasing forms such as chloramines and dichloro-
dimethylhydantoin. Quaternary ammonium compounds are amphipolar
cationic detergents derived from ammonium salts substituted with bromine
or chlorine. Iodophores are soluble complexes between elemental iodine
and nonionic surfactants. Amphoterics are based on the amino acid
glycine, frequently with an imidazole group.
Disinfection efficiency is controlled primarily by four factors: interfering
substances (such as organic matter), pH, concentration and contact time.
Any organic matter or cleaning chemical residues may protect the
microbes from disinfectant penetration or may react with the disinfectant
resulting in inactivation. For these reasons it is important to remove all
cleaning residues and soil before disinfection.
Biocides have optimum activity within certain pH ranges, depending
upon the disinfectant type.
Disinfectant concentration is important as the relationship between
microbial death and disinfectant concentration is sigmoidal. Consequently
at low concentrations the microbial population is difficult to kill but as the
disinfectant concentration is increased a point is reached where the
majority of the population is reduced. Above this concentration, a
proportion of the cells may survive regardless of increasing biocide
concentration. It is, therefore, essential to use the disinfectant at the
correct concentration as changes in concentration may significantly reduce
the activity, whilst increasing concentration may not produce any enhanced
effect.
To be effective, biocides must reach the bacterial cell, and bind to and
traverse the cell envelope to reach the target site.13 For this reason
sufficient contact time between the disinfectant and microbes is essential
for biocide efficiency.

3.3.2.4 Cleaning techniques. Cleaning of open surfaces can be under-

taken by hand using simple tools such as brushes, but specialist equipment
is usually required for large areas to dispense chemical and/or provide
mechanical energy.
Manual cleaning is limited due to the fact that only certain chemicals and
low temperatures can be used for operator safety. Small items of
equipment can be subjected to higher temperatures and chemical
concentrations by soaking in tanks for extended periods.
To clean large areas of open surface, equipment is used to disperse
chemicals and/or provide mechanical energy. Chemicals may be applied as
mists, foams or gels and the mechanical energy supplied by water jets or
scrubbing actions. The use of foams and gels prolongs the contact time
between the chemical and the soil on the surface.
Closed systems such as liquid processing equipment can be cleaned in
 PLANT HYGIENE 43

place (CIP). This involves circulation of water, detergents and disinfectants

through the processing equipment whilst assembled and the mechanical
energy is provided by the turbulent flow of the liquid through the
pipework. CIP systems are ideally used to clean process equipment that
has been specifically designed for CIP cleaning.

3.3.2.5 Cleaning and disinfection procedures. Cleaning and disinfection

regimes are designed to be efficient with water and chemicals, to allow use
of selected chemicals under optimum conditions, to operate safely, to be
easily managed and to reduce manual labour. The cleaning procedure
covers the stage at which cleaning is implemented and the sequence of
cleaning and disinfection.
The main phases in the cleaning and disinfection programme are:
1. Production period practices. Good housekeeping practices should
operate so that product is removed from production lines during breaks,
possibly followed by controlled manual cleaning. In addition sound
sanitation practices should be used for major product spillages during
production.
2. Preparation of equipment. Equipment should be dismantled as far as
is practical or necessary for cleaning, and dismantled equipment stored
on racks etc. Machinery should be isolated electrically and electrics etc.
covered for protection.
3. Removal of gross soil. Loosely adhered or gross product debris should
be removed by brushing, vacuum, scraping, etc. Soil on floors should be
picked up rather than rinsed to drain by hoses.
4. Pre-rinse. Loosely adhered small debris is removed with a low
pressure cold water rinse. The use of hot water aids the removal of fats,
but too high a temperature may coagulate proteins.
5. Cleaning. Cleaning chemicals, temperature and mechanical energy are
applied to the surface to remove the adhered soil.
6. Inter-rinse. Detached soil and cleaning chemicals should be rinsed
from the surfaces using low pressure cold water.
7. Disinfection. Chemical disinfectants are applied to reduce the
viability of the remaining microbes to a level deemed to pose no
significant risk.
8. Post-rinse. Disinfectants should be rinsed from the surface using low
pressure cold water, although some disinfectants are surface active and
are intended to be left on the surfaces.
9. lnterproduction cycles. Certain procedures may be undertaken to
prevent the growth of microorganisms on production contact surfaces
before the next production period; these include removal of excess
water and equipment drying. The process area may alternatively be
fogged.
 44 PRESERVATION OF SURFACTANT FORMULATIONS

The sequence of cleaning and disinfection determines the order in which

product contact surfaces (equipment) and environmental surfaces (walls,
floors, drains etc.) are cleaned and disinfected to ensure that once product
contact surfaces are disinfected they are not recontaminated. A typical
sequence would be as follows:
1. remove gross soil from production equipment
ll. remove gross soil from environmental surfaces
iii. rinse down environmental surfaces (from top to bottom)
IV. rinse down equipment (from top to bottom) and flush to drain
v. clean environmental surfaces
VI. rinse environmental surfaces
Vll. clean equipment
viii. rinse equipment
ix. disinfect equipment
x. fog

3.3.2.6 Monitoring the effectiveness of the cleaning and disinfection

system. Due to their role in the control of the environmental routes of
product contamination, cleaning and disinfection programmes require
regular monitoring as part of a structured quality assurance system. This
evaluation normally involves an immediate sensory evaluation and an
historical assessment of the programme's effectiveness by detecting
remaining microorganisms or chemicals.
Sensory evalution involves a visual inspection of the surfaces, smelling
for products or offensive odours and feeling for greasy or encrusted
surfaces.
Microbiological (or chemical) techniques may be applied if product
residues are not detected. Traditional microbiological techniques involve
sampling the microorganisms present on the surface using, for example,
sterile cotton or alginate swabs and sponges. The microorganisms are then
suspended in a suitable recovery or transport medium for dilution and
plating. Other sampling techniques include self prepared or commercial
agar contact plates that are pressed onto the surface. These methods give
an historical evaluation of the hygiene of the surfaces. Relatively recently,
rapid methods have been developed to assess surface hygiene, such as
epifluorescent microscopy and adenosine triphosphate (ATP) analysis.
These techniques give a result in a timescale relevant to process control such
that the cleaning and disinfection regime can be repeated if necessary.
For epifluorescence microscopy, microorganisms are sampled by
swabbing or rinsing and then collected onto filters; the microorganisms
present on the filter are enumerated using the direct epifluorescent filter
technique (DEFT).I4
A TP bioluminescence is based on detecting the levels of adenosine
 PLANT HYGIENE 45

triphosphate (ATP) present in plant, animal and microbial cells, using an

enzyme system that produces light in proportion to the amount of ATP
present. The light output can be measured in a luminometer in approxim-
ately 5 min. In the assessment of surface hygiene, analysis of total ATP is
most suitable as after cleaning and disinfection any residues, whether
microbial or product in origin, should have been removed.
Surface assessment methods only sample a very small proportion of the
production plant surface and are therefore analogous to 'end product
analysis' of a production batch. These assessment techniques should,
therefore, monitor the effectiveness of a structured hazard analysis critical
control point (HACCP) approach.12 In a similar manner to production
processes, critical control points (CCPs) require identification and control
and vary between processing areas and types, but typical cleaning CCPs
may include: detergent and disinfectant concentrations, chemical solution
temperature, chemical contact times, degree and time of application
procedures, equipment settings (i.e. pressure), cleaning equipment main-
tenance and chemical stock rotation. The use of HACCP in relation to
cleaning and disinfection would put emphasis on control of the programme
as it was happening and should ensure that an acceptable level of surface
hygiene was consistently achieved.

3.4 Control of production environment air

3.4.1 Aim
Control of the production environment air may be for several purposes.
• prevention of contamination or deterioration of the product
• temperature control
• control of relative humidity
• control of dust
• control of vapours.
Air control systems do not guarantee freedom from air-borne contamina-
tion as operations within the production area such as movement of
personnel, machinery and packaging, and cleaning will influence the level
of air-borne contamination.
Air-borne particles cover the range of sizes from sub micron to about
100 microns (Figure 3.1), i.e. from viruses to large spores. The size of
the air-borne particle has a direct effect upon its stability in aerosol form
and particles of 2-3 !-tm or less remain suspended in air almost indefinitely.
Larger particles sediment at rates dependent on their size and density.
46 PRESERVATION OF SURFACTANT FORMULATIONS

Particle diameter (microns)

0.001 0.01 0.1 1 10 100
viruses
bacteria

pollen

fo

nuclei

flour

Figure 3.1 Comparison of the sizes of air-borne particles.

3.4.2 Sources of air-borne contamination

Microorganisms may be introduced into the processing area air from a
number of sources and by several mechanisms. Contamination outside the
processing area may enter via:
• doors and windows
• ventilation systems (therefore high efficiency particulate air (HEPA)
filters should be fitted)
• wheels of trolleys and fork lift trucks, and shoes and clothing of
personnel
• surface of outer packaging material or on the raw materials.
Air-borne contamination may be created within the processing area by
the production practices.
• Movement of dry powders such as flour, spices, dried milk etc.,
generates significant levels of dust particles, some of which may have
microorganisms attached to them.
• Processes involving liquid handling such as vegetable washing are likely
to create aerosols.
• Cleaning systems and techniques such as high pressure/low volume
 PLANT HYGIENE 47

spraying, low pressure/high volume spraying, and floor scrubbers create

aerosols containing viable microorganisms over significant distances
from the site of use. IS
Table 3.4 shows the levels of organisms present in the air related to the
process taking place. Floors and drains have been shown to be a source
of high levels of air-borne microorganisms, particularly when flooded. 16
Figure 3.2 shows the effect of flooding on the levels of air-borne bacteria.
• Dusty surfaces also contribute to the amount of air-borne contamination.
Re-entrainment has been shown to occur at air velocities normally
existing in the processing environment, and is dependent on surface
roughness and air turbulence.
• Humans contribute to the presence of air-borne contamination. Skin
particles are constantly shed from the body and may carry viable
bacteriaY In addition hairs are lost from the head at a rate of 100 per
day18 and may also carry viable microorganisms. Speaking, coughing
and sneezing generate aerosols containing microorganisms.
• The use of compressed air and vacuum systems can redistribute dust and
microorganisms.

3.4.3 Control of air-borne contamination

Air control systems are mainly effective in controlling air-borne contamina-
tion entering the production area, but have little effect on contamination
arising from other sources. Air control systems are very effective at
controlling the air entering the area and can effectively combat condensa-
tion. Air filtration systems are also effective in controlling the contamina-
tion from compressed lines and exhausts from vacuum systems.

3.4.3.1 Air filtration. Filtration is the most commonly used technique

for controlling air-borne contamination. Frequently two or more filters will
be incorporated in the filter unit (e.g. primary, secondary and tertiary) to
filter progressively decreasing particle sizes.
Air filters are categorised on the basis of arrestance and efficiency on a
scale from 1 to 14 on the Eurovent 4/5 grading system. Arrestance is a
measure of the filter's ability to capture and retain a known weight of
synthetic dust fed into the filter at a specific rate to a final static pressure
drop. Efficiency is a measure of the discoloration to filter test discs up and
downstream of the filter. Table 3.5 summarises the grades of filters
available.
The air quality required is dependent on the type of production area. For
areas requiring good manufacturing practice (GMP) quality air, final
filtration to EU4-5 is sufficient. For medium care areas, final filtration to
EU5-7 would suffice, whereas high care areas require filtration to
EU7-1O.
 48 PRESERVATION OF SURFACTANT FORMULATIONS

Table 3.4 Mean levels of microorganisms per 60 I of air for different production processes

Process Mean cfu/6O I air

Total viable count YeastsIMould count

Vegetable freezing 552.5 (n = 103) 316.3 (n = 104)
Cleaning 366.1 (n = 226) 225.0 (n = 210)
Meat filling 295.3 (n = 59) 34.6 (n = 42)
Waste disposal 280.0 (n = 6)
Engineering areas 278.0 (n = 6) 1.4 (n = 5)
Conveyors total 207.8 (n = 26) 45.0 (n =26)
meat 282.0 (n = 26) 68.8 (n =16)
vegetable 146.7 (n = 6) 15.2 (n =6)
pizzas 3.3 (n = 4) 0.5 (n =4)
Slicing 187.5 (n = 112) 83.5 (n = 112)
Raw materials receipt 183.6 (n = 16) 128.0 (n = 11)
Tray washing 145.5 (n = 94) 85.5 (n = 86)
Aseptic filling 89.5 (n = 54) 20.1 (n = 48)
Primary packaging 47.2 (n = 239) 13.6 (n = 213)
Stores 35.9 (n = 18) 19.9 (n = 18)

cfu = colony forming units.

200

Air-bon Ie 15)
aut
3
(Q!lls per m 100
x10 -2)

O~----~----==~------T-----~

o 10 40
line (Mn.tes)
Figure 3.2 Effect of flooding at 10 min intervals on the air-borne bacteria counts above drains
in four food production areas (adapted from Heldman). 16
 PLANT HYGIENE 49

Table 3.5 Air filter types and their particle retention capabilities

EU grading General air filtration Approaching 100%

Eurovent 4/5 description retention of particles

EUl-EU4 (5) Primary filters >5 11m

EU6-EU7 Secondary filters >2 11m
EU8-EU9 Secondary filters >1 11m
EUlO--EU14 Semi REP A and REPA filters >0.5 11m

3.4.4 Methods of reduction of air-borne contamination

As aerosols that are produced in a food processing environment can
potentially remain air-borne indefinitely, methods are required to reduce
levels of air-borne microorganisms. The most widely used method to date
is disinfectant fogging. The spraying of a disinfectant mist into the air can
only be performed outside production hours due to the health risk to
workers and the possibility of contamination of product. Alternative
methods for the disinfection of air-borne particles include the use of
negative ions, ozone, ultraviolet light and electric fields.

3.4.5 Monitoring of air quality

Air velocity is measured using vane anemometers which require calibra-
tion. Air pressure is measured by electronic differential pressure gauges
and again calibration is required. Electronic particle counters are available
but these instruments are designed for 'clean room' -type facilities and the
sensors may become overwhelmed and fouled by processing environments.
There are a number of microbiological methods available for the
estimation of air-borne contamination, although different methods of
sampling will provide different estimates of air-borne contamination. The
microbiological techniques include sedimentation, impingement techniques
and filtration methods.
The sedimentation method involves exposing a standard Petri dish
containing non-selective agar for a defined exposure time. This method is
simple and easy, but does not measure the number of viable organisms per
unit volume of air. It relies on the deposition of particles over a period of
time and this can be greatly influenced by air currents.
Impingement methods involve purpose built samplers that draw a set
volume of air over an agar plate or strip. These samples are portable and
allow the sample volume to be adjusted.
Filtration methods involve placing a cellulose, glass fibre, or membrane
filter in a suitable holder and drawing a known volume of air through the
membrane using a vacuum pump. The membrane is then either placed
50 PRESERVATION OF SURFACTANT FORMULATIONS

directly on an agar plate, or the organisms are recovered from the filter and
plated out. The filtration method is most suitable for desiccation-resistant
mould and bacterial spores.

3.5 Control of the personnel route of contamination

People can be a source of contamination and/or can transfer contamination

from other sources to the product.
Bacteria found on the skin fall into two categories: transients, which are
organisms deposited onto the skin and do not multiply, and residents,
which readily mUltiply on the skin (e.g. Staphylococcus aureus). The
concentration of bacteria on the skin is normally 105_10 6 bacteria per cm 2
depending on the area of the body. Skin particles are constantly shed from
the body due to friction with clothing and drying of skin particles. During
undressing, for example, it is estimated that 5 X 105 scales become air-
borne, 5-10% of which may carry viable microbesY
Hands are the principal agents in transferring pathogens to product and
handling should be kept to a minimum. Microbiological examination of
food handlers often demonstrates the presence of a large number of
potentially pathogenic organisms. 19 Consequently regular and thorough
hand washing is essential.
Before any staff are allowed to commence work they should be advised
of their personal obligations with respect to hygiene and should complete a
detailed medical questionnaire. An experienced medical officer should
then decide whether a medical examination is required prior to employ-
ment. A high standard of personal hygiene is therefore required to
minimise the risk of contamination to product. A list of the basic personal
hygiene requirements is given below (for guidance only).
1. Protective clothing, footwear and headgear issued by the company
must be worn.
Protective clothing must be worn by all personnel in processing
areas, and is designed to protect the product from contamination and
the wearer's own clothing. The protective clothing should be in good
repair, laundered regularly and changed when soiled. The clothing
should be light coloured and may be colour coded if segregation of
personnel is required, for example in high and low risk operations. The
clothing must cover the operative from neck to knee, have tight cuffs
and no outside pockets.
A fine hairnet may be appropriate in addition to the protective
headgear, depending on the processing environment. Hairclips and
grips should not be worn.
2. Beards and moustaches must be kept short and trimmed and a
protective cover worn when appropriate.
 PLANT HYGIENE 51

3. Personnel must wash their hands thoroughly before commencing

work, after using toilets, after handling waste food, on returning to the
production area after leaving for any reason and as frequently as
necessary during the day. Hands should be washed in hot water (45°C)
with a suitable non-perfumed bactericidal soup using non-hand
operable sinks. The hands should then be dried effectively and in
certain circumstances an alcohol-based hand sanitiser should be used.
4. Fingernails should be kept short and clean. The wearing of make up,
false eyelashes, false nails and nail varnish should be prohibited.
5. Watches and jewellery must not be worn (except plain wedding bands
and possibly 'sleeper' earrings).
6. Personal items must be left in lockers outside the production area.
7. Food and drink must not be taken into or consumed in areas other
than the tea bars, restaurants etc. Sweets and chewing gum must not
be consumed in production areas.
8. Smoking and the taking of snuff must be prohibited. If smoking is
permitted at all, designated areas should be provided that are
separated physically from production areas.
9. Spitting is forbidden.
10. Superficial injuries such as cuts, grazes etc. must be covered by a
waterproof dressing that is distinctly coloured and metal detectable.
11. Personnel suffering from heavy colds, stomach disorders, diarrhoea,
skin conditions, etc. must report the illness to the medical officer or
supervisor. The person should be excluded from work involving food
handling until pronounced fit. Staff returning from foreign travel or
who have been in contact with infected persons should also inform the
medical officer.
Management is responsible for ensuring that all personnel are aware of
and understand the relevant factory regulations. Regular training pro-
grammes should be conducted to stress the importance of personal hygiene
and behaviour and to issue up-dates on legal obligations.

Further reading

Imholte, T.J. (1984) Engineering for Food Safety and Sanitation, Technical Institute Foods
Engineering, Crystal, Minnesota.
Sprenger, R.A. (1993) Hygiene for Management, Highfield Publications, Doncaster.

References

1. Troller, J.A. (1993) Sanitation in Food Processing, Academic Press Inc., San Diego,
California.
2. Council Directive, 93/431EEC (1993) on the hygiene of foodstuffs. Official J. European
Communities, L175, 1-11.
52 PRESERVATION OF SURFACTANT FORMULATIONS

3. Council Directive, 92/41EEC (1992) for the intra-community trade in meat products.
Official I. European Communities, L57, 1-14.
4. BS6431: Part 1 (1983) (EN87) Ceramic Floor and Wall Tiles, British Standards
Institution, London.
5. BS5980 (1980) Specifications for Adhesives for Use with Ceramic Tiles and Mosaics,
British Standards Institute, London.
6. Council Directive, 89/392/EEC (1989). Relating to machinery. Official I. European
Communities, L183, 9-26.
7. European Hygienic Equipment Design Group (1993). Hygiene equipment design
criteria. Trends Food Sci. Technol., 4, 225-229.
8. Holah, J.T., Betts, R.P. and Thorpe, R.H. (1989) The use of epifluorescent microscopy
to determine surface hygiene. Internat. Biodeterioration, 25 147-153.
9. Holah, J.T. and Kearney, L.R. (1992) Introduction to biofilms in the food industry. In
Biofilms: Science and Technology, Melo, L.F., Bott, T.R., Fletcher, M. and Capdeville,
B. (eds), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 395-402.
10. Jennings, W.G. (1965) Theory and practice of hard surface cleaning. Adv. Food Res.,
14, 325-459.
11. Koopal, L.K. (1985) Physico-chemical aspects of hard surface cleaning. Netherlands
Milk Dairy I., 39, 127-154.
12. Holah, J.T. (1992) Cleaning and disinfection. In Chilled Foods: A Comprehensive Guide,
Dennis, e. and Stringer, M. (eds), Ellis Horwood, London, pp. 319-341.
13. Klemperer, R. (1982) Tests for Disinfectants: Principles and Problems in Disinfectants:
Their Assessment and Industrial Use, Scientific Symposia Ltd, London.
14. Pettipher, G.L., Mansell, R. and McKinnon, C.M. (1980) Rapid membrane filtration
epifluorescent microscopy technique for direct enumeration of bacteria in raw milk.
Appl. Environ. Microbiol., 39, 423-429.
15. Holah, J.T., Taylor, J.H. and Holder, J.S. (1993) The Spread of Listeria by Cleaning
Systems, Part II. Technical Memorandum No. 673. Campden Food and Drink Research
Association.
16. Heldman, D.R. (1974) Factors influencing air borne contamination of foods: A review.
I. Food Sci., 39, 962-969.
17. Noble, w.e. (1981) Microbiology of human skin. Major Problems in Dermatology, 2,
5353-655.
18. Haynes, P.R. (1985) Food Microbiology and Hygiene, Elsevier Applied Science
Publishers, Barking, UK.
19. Kerr, K.G., Birkenhead, D., Seale, K., Major, J. and Hawkey, P.M. (1993) Prevalence
of Listeria spp on the hands of food workers. I. Food Protection, 56, 525-527.
20. Elliot, R.D. (1980) Cleaning and sanitation. In Principles of Food Processing Sanitation,
Katsuyama, A.M. (ed.) The Food Processors Institute, USA, pp. 91-129.
4 An introduction to surfactants
1.1. LEWIS

4.1 Why are surfactants of interest?

Use of surfactant chemicals to modify the surface or interface between two

phases leads to a number of very interesting effects. These include:
• The ability to form a stable emulsion of oil and water, which allows the
production of mayonnaise for salad dressing, hand creams and lotions
for personal grooming, or to emulsify bitumen for road laying.
• The ability to generate foam for use in bubble baths, toothpaste, or
polyurethane foam for refrigerator insulation.
• The ability to disperse solid particles in a liquid matrix, and so give rise
to cream cleansers for bathrooms, or emulsion paints.
• The ability to wet a surface not normally easy to wet, and so to provide
leaf penetration for insecticides, or in combination with the other
properties listed above, to provide detergency for the domestic washing
of clothes.
The above list of effects is by no means exhaustive, but is included to
show that there is a very wide variety of domestic and industrial uses for
these products. The major uses, however, are for systems which deal with
the interface between solid and liquid, liquid and liquid, and liquid and gas
phases. In general, water is one of the liquid phases for these pairs of
interactions.
This short introduction to surfactants seeks to illustrate the elegant
nature of these molecules in their applications. This will be done by
describing the reasons for their modes of action, the uses these actions can
be employed for, some of the chemical syntheses and composition of
commercially available materials and an approach to formulating with
these materials.
The intention of this chapter is to provide a first impression of the main
principles of surfactancy, and hopefully to provoke an interest in knowing
more about what is an exceedingly diverse field of chemistry and physical
phenomena. One of these physical phenomena is that of emulsification,
where surfactants can be used to combine materials such as oil and water
into a relatively stable, inhomogeneous mixture which may be difficult to
achieve otherwise.
54 PRESERVATION OF SURFACTANT FORMULATIONS

The question may be asked: What properties of surfactant molecules

allow this emulsification to take place readily? In order to answer this
question, it is worth asking another question first: Why are oil and water
not miscible?

4.2 How surfactants work

4.2.1 Molecular interactions and interfacial tension

Whether a substance is a solid, liquid or gas at a particular pressure and
temperature depends upon the strength of the intermolecular interactions
between molecules. If we consider the types of cohesive energies which are
operating in formulated products, the most significant of these are:
• Van der Waals' forces, either dispersion (arising from the relative
dissymmetry of electron clouds of two non-polar molecules in close
proximity) or permanent (arising from permanent dipoles in polar
molecules) .
• Hydrogen bonding, which arises by the formation of a 'hydrogen bridge'
between two electronegative atoms or molecules.
• Ionic forces, which arise due to permanent ionic charge interactions
between atoms or molecules.
The respective strengths of these three forces are of the order of 1:5:150
respectively, although this is system dependent.
For materials which only rely on weak molecular interactive forces, such
as propane or butane, the result is that at room temperature and pressure
these materials are gases. If the molecule is larger, such as pentane or
hexane, the sum of these small cohesive forces over a bigger compound is
enough to make these materials liquids. However, water is a much smaller
molecule, but due to its polar nature and shape, and consequent hydrogen
bonding, it is a liquid under the same conditions. Common salt, sodium
chloride, is an ionic material, solid at room temperature, and only melting
at 800°C. However, when salt is dissolved in water, the strong ionic
bonding is replaced by hydrogen bonding of the anion and the cation with
water. The combination of hydrogen bonding and electroneutrality in
solution allows the ionic lattice of the salt to be replaced by a multitude of
weaker forces.
If a paraffinic oil is mixed with water, a number of cohesive forces are in
operation. For oil-oil, or oil-water, only dispersion forces are present. For
water-water, the far stronger hydrogen bonding forces are in play. In
consequence, water preferentially bonds to water, and the oil collects as a
separate layer due to a difference in density. This immiscibility of oil and
water is therefore the most thermodynamically stable state.
 INTRODUCTION TO SURFACTANTS 55

A further phenomenon now arises. For molecules in the bulk of the

water, hydrogen bonding occurs in all the usual directions, and the net
forces on these molecules is zero. However, at the interface between oil
and water, water molecules are pulled towards the bulk by the hydrogen
bonding, but only pulled away by the weak dispersion forces. Consequently,
the entire interfacial area is subject to a force trying to contract it to the
smallest possible area. If a single drop of water were present in the oil, it
would be spherical. In the case of a water-air interface, the interfacial
tension is much greater, as the dispersion forces of air are very much
weaker than oil, due to the much lower density. The special case of
interfacial tension against air is known as surface tension.
Most commonly encountered room temperature liquids have surface
tensions against air or their vapours that lie in the range 10-80 dyn cm- 1
(Table 4.1). Water has a value at the high end ofthis range (72-73 dyn cm- 1),
while hydrocarbons are toward the lower end (20-30 dyn cm-1 ).
Of course, the above is a simple picture of what is really going on. The
interface is treated as a static entity, whereas there is actually a rapid
interchange of molecules. A water molecule may typically have an average
residence time at the interface of 3 !lS at room temperature! Not
surprisingly, temperature has a large effect on interfacial tension due to the
mobility of surface molecules.

4.2.2 What does a surfactant do?

A surfactant is a chemical species that is active, and has a preference for, a
surface or interface, which is the boundary between two phases. When the
surfactant is used as an emulsifier, the surfactant will lower the energy
differences between the two phases to allow a relatively stable mixture of
oil and water to form. It should be recognised at this stage that the most
thermodynamically stable state remains the separation of the emulsion or
foam, but that surfactants can maintain the metastable state required for
sufficient time in order to allow advantageous exploitation.
There are many compounds which, when dissolved in water, can

Table 4.1 Interfacial tension for different liquids

Liquid Interfacial tensions

(dyn em-I)

vs Air vs Water

Water 72.8
Octane 21.8 50.8
Benzene 28.9 35.0
n-Octanol 27.5 8.5
56 PRESERVATION OF SURFACTANT FORMULATIONS

Table 4.2 Effect of organic additions on surface tension

System Surface tension

(dyn cm- I )

Water 73
Acetone in water (20%) 42
Acetone in water (50%) 30

EAS in water (0.0004%) 48

EAS in water (0.004%) 31

decrease the interfacial tension, but surfactants are generally the most cost
effective and efficient way of achieving these effects. This is due to their
ability to concentrate at the interface, rather than to remain in the bulk of
the solution. Table 4.2 illustrates this point, comparing the reduction of
interfacial tension of an air/water system by acetone, and an ethoxylated
alcohol surfactant (EAS, lauryl alcohol plus 5 ethylene oxide).
Table 4.2 shows that the surfactant compound is 10 000 times more
capable of reducing the surface tension than the simple organic molecule!

4.2.3 Structure of an idealised surfactant and its interfacial activity

In simple terms, an idealised surfactant molecule is essentially two
chemicals in one; a hydrophilic portion, which tends to keep the molecule
in the aqueous phase, and a hydrophobic portion, which will prefer to be
expelled from the aqueous phase. There is therefore a dual ('confused')
nature to these materials. A wide range of commercially available
materials will be discussed below, but for now a common type of material
will be exemplified - soap.
Soaps are the most common and greatest volume surfactants in use
around the world. They are usually sodium salts of long alkyl chain
carboxylic acids, the alkyl chain being from 10--18 carbon atoms long
(Figure 4.1).
The long alkyl chain is comparatively bulky when compared to the ionic
part of the molecule, which is more frequently described as the headgroup.
The simplified view of a surfactant molecule is therefore to picture it as a
small matchstick, with an oily tail and a highly polar head.
Consider this molecule in the bulk of an aqueous phase. The polar head
will be solvated by polar bonding to the water. However, the oily chain will
be bound to the water molecules only by the much weaker dispersion
forces. As a consequence of this, the water will become much more
structured, as hydrogen bonding on one side is not balanced out by the
dispersion forces acting on the other (as occurs for interfacial tension). The
result of this imbalance is that an 'iceberg' of structured water is formed
 INTRODUCTION TO SURFACTANTS 57

around the alkyl chain. This increase in the structure of water is

en tropically disfavoured, as the system has become more, and not less,
ordered.
If the molecule migrates to an oil-water interface, it may orientate itself
such that the polar head remains in the aqueous phase, and the oily tail can
lose the ordered sheath of water molecules by transferring to the oil phase.
This is thermodynamically favoured.
Now consider the surfactant molecule in the bulk of the oil phase. Here,
the only forces operating are the weak dispersion type, and both the polar
head and the oily tail are unencumbered by solvation effects. But, should
the molecule migrate to the oil-water interface, it is energetically
favourable for the polar head to be solvated by the water molecules (as salt
readily dissolves in water). However, as above, there is an energy barrier
due to entropic effects which disfavours the easy passage of the molecule
fully into the aqueous phase.
In summary, then, it is thermodynamically favoured for the model
surfactant molecule to orientate itself at a water-oil interface such that the
polar head is preferentially in the water phase, and the oily tail is
preferentially in the oil phase. This is illustrated in Figure 4.2.
It should be noted that the ability of surfactants to migrate to an
interfacial region is driven from both sides by the strength of the hydrogen

(n = 1-9)

P<XXXXXXXx><xxxx><x><><><><><x3 No 8:1

Hydrophobic tail Hydrophilic head

Figure 4.1 Chemical structure of soap, and an idealised surfactant model.

Gain of hydrogen bonding to the

OydcopOm, heod / ~
Oil
o
....,~,
Water

.o~. X ,"eched WOlff

("iceberg") oround hydrophobic tail

,hel

Figure 4.2 Forces leading to the preference of surfactants for an interface.

58 PRESERVATION OF SURFACTANT FORMULATIONS

bonding interactions of the aqueous phase, and is not simply the result of
the polar head group preferring the polar environment, and the oily tail
preferring the oily phase. The surfactant emulsifier may therefore be
described as having a limited solubility in both the aqueous and oil phases.
In the case of a simple solute, such as acetone, this is clearly not the case.

4.2.4 Surfactancy and the reduction of interfacial tension

The above section has discussed why it is thermodynamically favourable
for surfactants to collect at an oil-water interface. This interface represents
the boundary between very polar, densely packed water molecules, and
less dense, weakly interacting oil compounds. The presence of the
surfactant markedly reduces the difference between these two environments
by appearing to be a surface demonstrating properties similar to the phase
from which it is viewed. The aqueous environment is bounded by the polar
heads of the surfactant, and the oily phase is bounded by the oily tails.
However, seen from the other side, the water appears oily (bounded by
the surfactant tails), and the oil appears highly polar (bounded by the polar
heads).
The result of this is that the water surface assumes the dispersion
characteristics of the oil, and the imbalance of forces between this 'skin'
and the true oil layer is dramatically reduced. The interfacial tension is
therefore markedly reduced, and the aqueous phase becomes much easier
to disperse into the oil. Conversely, the oil surface can assume the polar
characteristics of the aqueous phase, leading to ease of dispersion in water.
This argument can be extended to explain why surfactants have no effect
on the interfacial tension between oils and air; on both sides of the
interface only dispersion forces are in operation, so there is no driving
force to concentrate common surfactants at the surface, and no real energy
difference to result in a change in interfacial tension. The effect is, of
course, finite. The reduction in interfacial tension will continue as more
surfactant concentrates at the interface, until a complete monolayer is
formed. The interfacial tension will effectively reach a steady value, even
though surfactant concentration may continue to build up at the interface.

4.2.5 Micellisation
Once the amount of surfactant available has increased beyond the point at
which an interfacial monolayer has been formed, the interfacial tension
becomes relatively constant. In addition to this, a number of other solution
properties in the aqueous phase also change, such as conductivity and
detergency (Figure 4.3).
Although more surfactant molecules cannot adsorb at interfacial sites,
once the interface is saturated a further phenomenon occurs, which
 INTRODUCTION TO SURFACTANTS 59

explains the changes mentioned above. This is called micellisation. This

occurs in aqueous solution when the molecules aggregate together and
order themselves with their hydrocarbon tails towards each other, and
their hydrophilic groups outermost. In effect, a small spherical interface is
created inside the bulk of the solution (see Figure 4.4). Even though this
orientation is more structured than the monomeric forms of surfactant
dissolved in water, and is therefore disfavoured on entropy grounds, it is
not as disfavoured as the state in which all the individually dissolved
molecules carry around their own 'iceberg' shell of oriented water
molecules. This is due to the appearance of the micelle as a collection of

(JJ
:J
Solubilisotion
o
>
0'
c
CJl
o
~ Osmotic pressure
u
C

Surface tension

Interfacial tension

CMC Increasing concentration

Figure 4.3 Change in aqueous solution characteristics due to micellisation around the critical
micelle concentration (CMC).

Air

§
0
tllP

II
0

•
0
0
o 0
00 0
0

o 0
Water 0000

Figure 4.4 Formation of micelles in an aqueous medium after surface saturation.

60 PRESERVATION OF SURFACTANT FORMULATIONS

hydrophilic headgroups arranged in a sphere, allowing hydrogen bonding

with the surrounding water readily to take place.
Once again, the case for micellisation of surfactant in the oil phase is less
clear cut, as the driving mechanism for aggregation of molecules in the
non-polar environment is considerably less.
The concentration of surfactant at which the change in solution
properties takes place when micelles form is known as the critical micelle
concentration (CMC). The number of surfactant molecules needed for this
to happen ranges from 30-100 for ionic molecules, where micellisation is
countered by repulsion between the similar charges on the headgroups, to
several thousand for non-ionic molecules, where there is no charge
repulsion to oppose micellisation. Not surprisingly, this lower resistance to
forming a micelle means that non-ionics will form these structures at lower
concentration than ionics, typical values being at 0.002% and 0.1 %,
respectively.
Temperature, pH, other ions and organic solutes also affect the process.
However, as for surfactants concentrating at interfaces, it should be
remembered that micelles are dynamic entities, with individual members
joining and leaving at a very high rate. In addition, micelles need not be
simply spherical in shape, and have also been shown to occur as discs, rods,
and even in sheet structures.

4.3 Surfactaut phenomena and effects

Section 4.2 discussed the driving force for the concentration of surfactants
at an interface between a polar environment (usually water) and a second
phase (air, oil or solid). That driving force was identified as attributable to
the strongly polar molecular interactions of the polar phase (the hydrogen
bonding of water).
This section deals with some of the more useful applications of this
phenomenon (emulsification, foaming, wetting, solids dispersion, solubil-
isation, microemulsions and finally, detergency). Section 4.4 introduces
more of the chemical structure of surfactants and the processes used for
their production, with section 4.5 discussing the practical aspects of using
these materials.

4.3.1 Emulsification
An emulsion can be defined as the dispersion together of two immiscible
liquids in the presence of a third component, the emulsifier. It should be
recognised at the outset that an emulsion is a thermodynamically
metastable state, and that even in the presence of the emulsifier, this
mixture will only have a finite lifetime. This definition applies to
 INTRODUCTION TO SURFACTANTS 61

macroemulsions, which are defined as mixtures with particle sizes between

about 0.1 to 20 !Lm. For mixtures with particle sizes of less than 0.1 !Lm, the
term microemulsion is used (see section 4.3.6).
The most common emulsions are either oil in water, or water in oil. For
oil in water (O/W), such as milk or bitumen emulsions, the water is the
continuous phase. For water in oil (W/O) , such as butter or hydraulic
fluids, the continuous phase is the oil.
During the emulsification process, when one phase is dispersed in the
other, a massive increase in the interfacial area between the two phases
takes place. The difference in free energy between the two-phase state and
the emulsion is primarily dependent on the need to increase interfacial area
in the presence of interfacial tension between the two. Therefore, the
ability of the surfactant to lower this interfacial tension is key to the ease of
emulsification.
Two further points are worth noting. The first is that there is obviously
an entropy term working in favour of dispersing one phase into a multitude
of droplets. However, the overall thermodynamics of the process is
unfavourable, due to the large amount of energy (large enthalpy) required
to generate the extra area. Secondly, several orders of magnitude of energy
above that which may nominally be required to effect the emulsification
process are needed to produce the emulsion, as the process itself is usually
carried out with much agitation, and energy is lost as heat and material
motion.
Given that emulsions are only kinetically stable, the role of the
surfactant in slowing the breakdown processes is now considered. The
breakdown processes are illustrated in Figure 4.5.

4.3.1.1 Creaming or sedimentation. This is dependent upon the density

difference between the continuous phase and the dispersed phase. If it is
not possible to match the densities closer than those for the chosen system,
the addition of thickeners will help to increase the emulsion lifetime, as will
the generation of smaller particle sizes during processing. Surfactancy
changes have little effect here.

4.3.1.2 Flocculation. This is the process whereby droplets form clusters,

and is a reversible phenomenon. This low energy interaction is due to the
balance of a weak attractive force (Van der Waals') with a steric repulsion
between the two droplets. By choosing a surfactant which can increase the
steric repulsion further than the surfactant used for the initial emulsification,
flocculation may be prevented altogether. If we consider an O/W system
emulsified with an ethoxylated alcohol, then the hydrophilic ethoxylate
chain would be on the outside of the oil droplets in the water phase.
Increasing the ethoxylate chain length would force the droplets further
apart until the Van der Waals' attractive forces fade away. Should the
62 PRESERVATION OF SURFACTANT FORMULATIONS

Sedimentation

•. ..
Flocculation

~ ~~.

Creaming •• ·• •
• •• •.
•
Coalescence

II L
-----------...
---------
1

~
Phase Inversion
Ostwald Ripening

Figure 4.5 Emulsion breakdown mechanisms.

droplets form clusters which cannot be readily redispersed, the emulsion is

said to aggregate.

4.3.1.3 Ostwald ripening. As two emulsified liquids are seldom com-

pletely immiscible, there can be some transfer of material from one
dispersed droplet to another via the continuous phase. The driving force
for this transfer of material is partially dependent directly on the interfacial
tension, and also on the inverse of the droplet surface area, that is, smaller
droplets have a larger internal pressure than large droplets. Ostwald
ripening is therefore manifest when larger droplets grow at the expense of
smaller ones. A surfactant may be employed to counteract this method of
emulsion decay in two ways. The reduction in interfacial tension which will
occur by using a surfactant will reduce the driving force for the transfer of
material, and the adsorption at the interfacial boundary will lead to a steric
hindrance of the material being transferred.

4.3.1.4 Inversion. When one phase is dispersed in another, it is possible

to keep adding this first phase until it reaches a critical volume fraction of
the emulsion as a whole. At this critical volume, the emulsion may invert,
for example change from O/W to W/O. For most emulsions, this is usually
manifest by a sudden drop in viscosity of the mixture. Unfortunately,
although there are several empirical rules which describe this phenomenon,
it is not well understood quantitatively.
 INTRODUCTION TO SURFACTANTS 63

4.3.1.5 Coalescence and the Gibbs-Marangoni effect. Coalescence can

occur if two droplets come close enough together that the film of the
second phase which separates them ruptures. The effectiveness of a
surfactant adsorbed at the interface to prevent the collapse of the emulsion
can be seen as threefold.
1. It can reduce the overall potential energy of the system by reducing the
interfacial tension.
2. If the adsorbed surfactant carries an electric charge, repulsion between
approaching droplets is possible.
3. If the molecule adsorbed at the interface is sufficiently bulky, a steric
interaction may also prevent coalescence.

Beyond these effects, there are further phenomena which occur when
the film continues to thin prior to coalescence. This is the phenomenon of
self healing, or the Gibbs-Marangoni effect.
Consider an interface on a thin film, with an equilibrium saturation of
surfactant molecules. As the film is stretched (by the second phase
molecules moving out from between the two droplets of the first phase)
some surfactant is dragged out from the interface. Two things now take
place which oppose this disruption of the film.

1. A local increase in interfacial tension takes place, as there is not

sufficient surfactant at the interface. The increase in interfacial tension
opposes the force stretching and thinning the film. This is known as the
Gibbs effect.
2. As surfactant moves back towards the area of lower concentration, in
order to re-establish the equilibrium at the interface, molecules of the
second phase are dragged back with it, restoring the film thickness. This
is known as the Marangoni effect.

4.3.2 Foams
Foam phenomena can be seen as an extension of the attributes associated
with emulsions, but replacing the liquid-liquid with a liquid-gas mixture.
As for emulsions, a third element (e.g. surfactant) is required if a
kinetically stable foam is to be produced, as pure liquids will not allow
foams to be produced. As for emulsions, the primary role of the surfactant
is the ability to oppose film thinning and ultimate coalescence of the gas
phase trapped in the liquid. The Gibbs-Marangoni effects are therefore
crucial in this area too, and the driving force for film thinning that this
effect opposes is the draining of the film under gravity.
The optimum concentration of surfactant for maximum foaming is
usually around the CMC for a number of systems. The onset of micelle
formation can be seen as a way of providing a reservoir of surfactant
64 PRESERVATION OF SURFACTANT FORMULATIONS

monomers which can aid the Marangoni effect. However, if there is an

oversupply of surfactant within the foam, then there is a smaller driving
force for the restoration of the thinning film, as the need to transfer
surfactant (and associated solvent) to the site of thinning is now less.
In practice, foams tend to be much more difficult to study and to draw
conclusions from than emulsions, as most applications involve the
simultaneous production and decay of the foam. This rapid turnover makes
close study of the surfactant mechanisms for stabilisation and their role in
the ultimate destruction of a foam very difficult. The ability to control
foam is sometimes very important for particular applications. Some uses
(e.g. shampoos) require the generation of a thick and creamy foam for
performance or aesthetic reasons. However, should the same formulation
be used in a washing machine, undesirable consequences could easily
result!
Antifoaming agents generally work through a reverse Marangoni effect.
The rationale is that addition of a substance which displaces the surfactant
from the interfacial film not only decreases the surfactant content there,
but also drags solvent molecules away with it. This may be achieved by
displacing the surfactant with a more interfacially active material, but one
which interrupts the interfacial packing and stability of the original
surfactant film. Polyamides, EO-PO (ethylene oxide-propylene oxide)
copolymers and polydimethyl siloxanes are well-known examples of this
type of compound.

4.3.3 Wetting
If a drop of liquid is placed on a solid surface and spreads over it, it is said
to wet the solid. If it does not wet the surface, the liquid will remain as a
drop. For wetting to take place, the liquid must have a surface tension
below the critical surface tension (CST) which is a characteristic of the
solid. As water has a surface tension of about 72 mN m-I , it will not wet
polyethylene terephthalate or polystyrene, which have CSTs of 43 and
32 mN m-I , respectively. However, any surface with a CST above
72 mN m-I (e.g. glass) will wet easily. The addition of a surfactant to water
may allow the surface tension to be lowered beyond the CST of a substrate,
therefore promoting wetting on a surface normally hard to wet.
Substrates which readily wet with water are usually hydrophilic so that
direct polar interactions can allow the wetting to occur. However, with
non-polar substrates, water will again reject interaction with the non-polar
surface in favour of the stronger hydrogen bonding to itself. Following the
addition of a surfactant, rejection of the surfactant hydrophobic chains to
the solid interface will occur such that the hydrophilic head is orientated
towards the aqueous phase. As a consequence, the solid becomes wettable
by water.
 INTRODUCTION TO SURFACTANTS 65

In practice, the speed of wetting must also be taken into account. For
some applications (e.g. textiles), the processes require a fast wetting of a
fibre surface to take place in order to improve cycle times, and hence
overall economic efficiency. While it would seem that the best practical
surfactant for this application would be the material demonstrating the
greatest reduction in interfacial tension, this is not necessarily so. Most
interfacial tension measurements are carried out when an equilibrium has
been reached for a particular system, but the processing requirements may
require a kinetic effect. Consequently, the speed of migration must also be
taken into account. For a particular surfactant series, the expectation
would be that as the alkyl chain length increased, the surfactancy effect
would also increase. However, as the alkyl chain increases together with
the overall size of the molecule, the ability to migrate quickly to an
interface will fall away. As a consequence of this, there is usually an
optimum size of alkyl chain for a given headgroup of surfactant, and on
both sides of this optimum, wetting power will fall away due to either the
alkyl chain being too short or too long, both effects decreasing wetting
power.

4.3.4 Solid dispersion in liquids

Dispersions of solids in liquids are usually made by the nucleation of
materials from solution, or by grinding up of large particulate solid into
finer particles followed by their suspension in a liquid. Whichever method
is used for their generation, the important aspects of the resultant mixture
is the large surface area of the solid-liquid interface. If the particles are
small enough to prevent rapid settling out to either the top or bottom of the
liquid, then there are two main mechanisms available for the stabilisation
of the resultant slurry; electrostatic and steric. Use of these two methods of
stabilisation is normally necessary, as fine particles will tend to attract and
collide with each other due to the similar nature of their inherent molecular
properties, as they are the same type of solid.
Assuming again that the liquid phase is water, the two types of solid
particle that may be dispersed in it are polar and non-polar solids.
Addition of an ionic surfactant to a suspension of a polar solid in water
may result in adsorption of the polar headgroup onto the solid surface. The
non-polar surfactant tails now present the outward disposition of the solid
particles, and hence approach and aggregation of the solid is disfavoured
on steric grounds when the alkyl groups begin to overlap.
Addition of an ionic or non-ionic surfactant into a suspension of a non-
polar solid in water may result in the adsorption of the non-polar tails onto
the solid surface. Stabilisation in this case can be provided via the mutual
repulsion of the similarly charged headgroups in the case of ionic
surfactants, or by the steric repulsion between the long hydrophilic heads
66 PRESERVATION OF SURFACTANT FORMULATIONS.

in the case of the non ionic surfactants (usually polyoxyethylene

chains).

4.3.5 Solubilisation
One consequence of the formation of micelles in aqueous solution is the
production of a zone inside the water which is essentially an oily droplet.
The presence of hydrophilic heads on the outside of the assembled
surfactant molecules ensures that this microscopic oil droplet is a relatively
stable structure in water. This structure allows organic materials, which are
usually sparingly soluble in water, to give clear solutions, within the ability
of the micelles to absorb organics. For example, ethyl benzene can be
solubilised at levels of up to one mole of organic compound for each mole
of soap in solution. The resultant mixture is clear and stable. Not
surprisingly, the ability to solubilise organic compounds depends upon the
factors which affect micellar formation.
The effect is not limited to non-polar organics. Micelles formed from
surfactants containing polyoxyethylene (POE) units have a core of alkyl
chains, and this is surrounded by a polyether layer of chains into which
water is essentially excluded due to close steric packing of the units.
Materials which are not easily soluble in the alkyl region (ethers and esters)
may be absorbed preferentially into this region (usually known as the
pallisades layer).
This phenomenon can be used to advantage when pharmaceutical or
pesticide formulations are made up. Many of the active materials in such
formulations are not water soluble, and yet only a small amount of these
usually potent materials are required to give the desired effects. Not only is
solubilisation conferred upon the material, but the surfactant may also aid
penetration of the material into the host organism, e.g. by promoting
wetting. Solubilisation is also beginning to be used to remediate land
contaminated with undesirable organic chemicals (e.g. chlorinated bi-
phenyls, PCBs) which may simply not be washed out with water
treatments.

4.3.6 Microemulsions
Between simple micellar solubilisation and normal macroemulsions, a
third state of oil and water mixtures can be defined. This state is called a
micro emulsion , and is a swollen version of the solubilising micelles in
solution. Microemulsions share some of the properties of the other two
states. Like macroemulsions, there is less surfactant than that necessary to
solubilise the organic phase, but like solubilisation, the mixture is still clear
and thermodynamically stable. This combination of properties can be
extremely useful when the other states cannot deliver the desired effects.
 INTRODUCTION TO SURFACTANTS 67

The mixture of oil, water and surfactant is still as before, but addition of
a short or medium chain alcohol as a cosurfactant can aid the formation of
the microemulsion. This is due to the hydroxyl headgroup being able to
moderate the charges on ionic surfactants, and the steric hindrance of long
chain nonionics by packing at the surface of the microsphere and spacing
out the primary surfactant.
Applications for microemulsions are based on the properties which allow
micro emulsions to carry more solubilised organics than a simple swollen
micelle, but still to retain their thermodynamic stability.
A microemulsion can be the final form of the product, or can be used to
allow physical processing or as a special reaction medium in its own right.
One of the earliest uses was in wax polishes, where the small size of the
wax particles formed from molten microemulsions not only allowed easy
processing, but also imparted a ready shine as the particles were less than
the wavelength of visible light.

4.3.7 Detergency
Detergency is discussed as an illustration of the principal properties of
surfactants because detergents probably need all the processes associated
with surfactants to operate efficiently. Detergents also account for the use
of about 70% of all surfactants manufactured in the world!
Detergency is the removal of solid or liquid material from a surface in
preparation for some further treatment, or as an end in itself. Domestic
detergency involves the cleaning of fabric fibres of polar or non-polar
solids and liquids for aesthetic and hygienic effects. Initial replacement of
the fibre-air interface with a fibre-water interface will naturally allow
ready removal of polar materials from the cloth. However, this leaves non-
polar oils and solids which must be removed and separated from the fibre
environment and disposed to drain. When considering these processes, it is
easy to see how the principal properties of surfactants will be beneficial in
this application.
Micellisation will not only provide reservoirs of surfactant monomers,
but will also allow solubilisation to occur and micro- and macro-emulsions
to form. The surfactant monomers in solution will aid wetting of the fabric
and the impurity interface, particularly for fabrics with low critical surface
tensions such as polyester. Controlled foaming properties will aid
suspension and transport of the dirt.
Although the general properties of surfactants account in a gross way for
cleaning power, two further phenomena have been observed for detergency
operations with regard to the removal of non-polar oils and solids.

4.3.7.1 The roll-up mechanism. For non-polar oils spread over a

relatively non-polar surface such as polyester, dispersion forces act to keep
68 PRESERVATION OF SURFACTANT FORMULATIONS

the two adhering substances together. Replacement of the fibre and oil to
air interfaces with aqueous interfaces raises the potential energy of the
system due to the high fibre-water and oil-water interfacial tensions.
Addition of a surfactant to the aqueous phase then allows reduction in the
overall potential energy of the system to take place as surfactant adsorbs at
the appropriate interfaces.
Should the surfactant be nonionic in composition, it is possible for it
slowly to adsorb at the oil-water interface. When this occurs at the edges of
the oil which is spread on the fibre, the surfactant may migrate to the fibre-
oil interface. It is then possible for the surfactant to form a continuous
layer under the oil extending out to the fibre-water region. A reduction in
the potential energy of the system as a whole is then possible if the polar
surfactant head is absorbed into the aqueous layer rather than the oily
layer. This preference for hydrogen bonding with water makes the oil
retreat from the fibre. By absorbing more surfactant at the oil-water
interface, the potential energy of the system is again reduced.
Overall, the oil will retreat from the fibre and 'roll up' into a ball, which
can then be shaken loose from the fibre (see Figure 4.6). Microscopic
examination of soiled clothing during the cleaning process with nonionic
surfactants has revealed these globules of oil as part of the washing
cycle.

4.3.7.2 Particulate dispersion. With a non-polar solid attached to a non-

polar fibre, adsorption of the typical anionic present in most detergent
formulations onto both surfaces will be such that the hydrophobic tail is
anchored, and the charged head will be outermost. Repulsion between the
like charges on both the solid and fibre, and the reduction in potential
energy between the water and both solids allows the soil to be easily
detached from the fibre.
Although this mechanism is difficult to detect or prove, it is quite clear
that addition of doubly charged cations such as calcium or magnesium ions
have been shown to have a very deleterious effect on detergency, and such
ions, present in hard water, usually cause a soap scum of insoluble salts to
form.

f ~J 0=

~{~~fi~~

Figure 4.6 The 'roll-up' mechanism of detergency.

 INTRODUCTION TO SURFACTANTS 69

4.4 Chemistry of surfactants

In this chapter so far, the chemical nature of a surfactant has been confined
to a vague description of a hydrophobic tail and a hydrophilic head, which
looks something like a matchstick. The real situation is much more
complex, but an understanding of the broad principles of their origins and
composition is necessary before useful selection and employment of
materials for a target application is possible, particularly in view of the vast
range of commercially available products.
The correct balance of hydrophobe and hydrophile are important in
order to maximise the desired effect. For example, if we consider common
soap, which has an alkali metal salt of a carboxylic acid function as the
headgroup, it is possible for the alkyl chain to be too short (less than 8
carbon atoms), which will result in the material being too water soluble to
act as a surfactant (it will not be rejected from an interface). However, if
the carbon chain is too long (more than 18 carbon atoms), then the
material is usually too water insoluble, and may behave much more like an
oil or fat itself.
Let us first consider the origin of the hydrophobe in more detail, before
going on to the major technologies surrounding the production of
hydrophilic functionalities. There are two sources of hydrophobe used to
generate commercially available surfactants; plant- and animal-derived
oleochemicals, and petrochemicals (see Figure 4.7).

4.4.1 Oleochemicals
Oleo chemicals are derived from oil-containing plants (e.g. coconut or palm
kernels), and from beef tallow fat (a byproduct of the beef industry).
Saponification of these esters is easily carried out to give either the fatty
acid feedstocks for further transformation, or alkali metal salts (soaps).
The ease of production of soaps has allowed the use of these surfactants in
one form or another for almost 2000 years. Some 30% of western Europe
and about 70% of all worldwide surfactant usage is accounted for by soaps.
There are three important characteristics to be noted about the use of
oleo chemical feedstocks for surfactant synthesis. The first is that the alkyl
chains built up in nature give only linear materials, and principally even
numbers of carbon atoms, due to their biological origin. A small number of
odd-numbered alkyl chains are detected, but branching of the alkyl chain is
very rare. Secondly, splitting oleo chemical esters gives fatty acid materials
which are mixtures of isomers, as Table 4.3 shows. Use of mixed isomer
materials is usually preferred, not only from an economical point of view
(efficient use of all material produced), but also because mixtures almost
invariably perform better than single isomer products (a happy coincid-
ence!).
 Oleochemicals

Benzene. Ethylene
. - - . a-olefin Fatty Esters
Propylene acids
1 Ph"ol Ethylene
oxide Fatty
+
Propylene alcohols
Alkyl
benzene
! oxide
(EO)
•
(EO)
).. ~
(EO) - ;! ~
.. S· '"
!} ~
t>l ~ ~
~ ~ So ;:;.. "g.
~ t>:I ~ ~ "" {5
"" ~
'B" So ;! ;! 'B" ~ 'B"
'"
;:- ~ <:> 'B" ~...
;:,
;:-
""'"
;:- 1} ~ ;:-
i:l I:l ;! I:l
<:> ;:, ~ ;:;-
~ ~ '"2· '"2· ;:, 5' 5' ~. ~
I:l 5" I:l I:l I:l ;:, ;:, 1:i 2 I:l
g, g, g, g, &.
<:>
§ ;:, ,;:,S' ;:, ;:,
s' ;:,
5'
;:,
~. ;:, ~
,Ir

Linear alk Alcohol and Primary! Sorbitan!

EOIPO olef:
benzene alcohol ether tertiary Soaps sucrose
copolymers sulp honlates
sulphonat, ~s sulphates fatty amines esters
J
Alkyl Poly
E~ Fatty
r--
Fattyacid Amine oxide Fatty acid
phenol ethylene alcohol + etthanol ethoxylates ester
ethoxylates glycols ethoxylates ami,des quaternaries sulphonates
'------

Figure 4.7 Chemical origin of surfactants.

 INTRODUCTION TO SURFACTANTS 71

Table 4.3 Composition of oleochemical fatty acids

Alkyl chain Fatty acid name Coconut Tallow Commercial grades

length oil fat
Systematic Common Stearic Oleic

C-lO Decanoic Capric 6

C-12 Dodecanoic Lauric 57
C-14 Tetradecanoic Myristic 19 I 3
C-16 Hexadecanoic Palmitic 11 26 45 4
C-18 Octadecanoic Stearic 2 26 52 7
C-18' 9c-Octadecanoic Oleic 5 43 71
C-18" 9c,12c- Linoleic 4 9
Octadecadienoic

C-18' = Carbon chain with one double bond. C-18" = Carbon chain with two double bonds.

The third point to note is that not all fatty alkyl groups derived from
oleo chemicalsare fully saturated. For example, a C-18 fully saturated alkyl
group is known as a stearic functionality. With one double bond between
the C-9 and C-lO position in a cis arrangement, the alkyl group is known as
an oleic chain. Linoleic acid is the fatty carboxylic acid with a C-18 chain
and two unsaturated linkages. Each different oleochemical source will
have its own blend of fatty acid types following the above general rules.
Although soaps remain the major tonnage products in this area, chemical
transformation of this monoalkyl fatty carboxylic acid gives rise to a
number of industrially important materials.

4.4.1.1 Condensation with alcohols or polyols. This gives other ester

products, examples being the reaction of myristic acid (C-14 saturated)
with isopropanol to form isopropyl myristate for personal care applications,
or condensation of fatty acids with sorbitol to form the sorbitan esters
which have wide uses in many industrial and personal care areas.

4.4.1.2 Condensation of the fatty acids with primary or secondary

amines. This gives rise to amide groups. Reaction with diethanolamine,
for example, gives rise to diethanolamides, which are used as foam
boosting agents for bubble baths and dishwashing liquids. Reaction with
amines derived from reductive amination of glucose gives rise to fatty alkyl
glucamides.

4.4.1.3 Reduction of the carboxylate group with hydrogen. This gives

fatty alcohols, which may be ethoxylated and sulphated as described
below.

4.4.1.4 Reductive animation. This can be carried out in several chemical

steps to give fatty amine products. These are more fully described under
72 PRESERVATION OF SURFACTANT FORMULATIONS

cationics (section 4.4.4). In summary, therefore, a great deal of chemistry

can be carried out on the oleochemical fatty acids, leading to a wide variety
of interesting materials.

4.4.2 Petrochemicals
The rise of the petrochemical industry over the last 100 years has provided
an enormous range of cheap building blocks for industry. These building
blocks can be put together in such a way that surfactant hydrophobes can
be produced easily on a large scale. Ethylene can be polymerised to give
a-olefins, or the appropriate fatty alcohols, depending on the technology
used. Olefins can be joined to either benzene or phenol in an alkylation
reaction, and even paraffins can be oxidised to fatty acids. In this way,
hydrophobes are built up which have the same basic properties as the
oleochemical fatty groups, that is, they can be built up to give the same
amount of water insolubility as the C-IO-C-18 fatty oleochemical groups.
This is done by adjusting the hydrophobe length to suit the surfactant type.
For a-olefins, which may be reacted to give the sulphonate via reaction of
sulphur trioxide directly with the olefin double bond, the chain length
needs to be C-IO-C-18, which is also true for the fatty alcohols. However,
for an alkyl benzene or an alkyl phenol, the alkyl group need only be C-6-
C-9 to achieve the same effect. The main technologies for the hydro-
philisation of these feedstocks are ethoxylation or sulphation, described in
section 4.4.3. Again, it should be remembered that even the simple
descriptions of the types of petrochemical hydrophobe given above do not
give a complete picture.
Mixtures of chemical entities are again the rule, rather than the
exception. For example, consider the chemistry that takes place during the
production of a linear alkyl benzene hydrophobe. Alkylation of benzene
with a linear olefin will generally result in the production of all different
isomers of the secondary alkyl benzene. This is in spite of even starting
with a single pure olefin, as rearrangement to a more randomised mixture
can take place under the conditions of the reaction itself.
These petrochemical processes give products which are complementary
to the oleochemical types, and use of material from all of these production
processes depends upon the final combination of effects required, how best
to match chemical structure to them and the cost of each potential solution.
For oleochemicals, the hydrophobic part is defined by which oil or fat is
sourced to provide the feedstock, and the chemistry is more concentrated
around the transformation of the head carboxylate group prior to its final
derivatisation. For petrochemicals, the chemical transformation element is
evident over the whole production pathway.
 INTRODUCTION TO SURFACTANTS 73

4.4.3 Major hydrophilising technologies

Two technologies have become common in order to convert hydro-
phobic materials into water-soluble surfactant products-sulphation and
ethoxylation.

4.4.3.1 Sulphation or sulphonation. Reaction of sulphur trioxide with a

number of different substrates will form a sulphate or sulphonate group in
its acid form, which may revert easily to starting materials when the
sulphating medium is removed. In order to prevent this happening,
sulphated products are neutralised with base, for example sodium
hydroxide, and this then prevents their reversion from taking place. If the
reaction is carried out on feedstock containing hydroxyl functions, a
sulphate group (-OS03Na) is formed. If the reaction is carried out on an
alkyl benzene, a-olefin or an alkyl methyl ester, the sulphur trioxide
bonds directly to a carbon, forming a sulphonate group (-S03Na). The
reaction is usually very exothermic, and as sulphur trioxide is easily
generated from sulphur, a large range of hydrophobes can be easily
converted to families of surfactant molecules at low cost.
These groups of materials are structurally similar to soap, in that the
head group is an alkali metal ionic form of an oxyacid. However, there is a
crucial difference with regard to their performance characteristics which
has allowed this technology to grow in importance over the years. The
detergency property of soaps falls off markedly when calcium or
magnesium doubly charged cations are present in wash water, as these
cations precipitate the soaps into an insoluble scum. Sulphates or
sulphonates are markedly less sensitive to concentrations of these ions, and
these have been the standard basic ingredients of detergent formulations.
As these compounds have this performance advantage in the major
tonnage outlet for surfactants, it is not surprising that this technology has
become so important.

4.4.3.2 Ethoxylation. Reaction of sulphur trioxide with a hydrophobe is

a simple way of producing a step change in hydrophilic character.
However, if different degrees of hydrophilisation are required in order to
try to match product structure to a needed performance, ethoxylation
offers a much more controlled way of doing this.
Ethylene oxide (EO) is a highly reactive molecule, as it is a three-
membered ring under considerable strain. Opening of the ring under acid
or basic conditions is highly favoured as it releases this strain energy. If the
ethylene oxide is reacted with an alcohol, the product from the reaction is
also an alcohol, and so further reaction to give poly(oxyethylene) chains is
easily achieved (Figure 4.8).
As the poly( oxyethylene) chain is built up, the hydrophobic starting
74 PRESERVATION OF SURFACTANT FORMULATIONS

(EO) (EO)
R-OH R-OCH 2CHPH - R-(OCH 2CH 2)n -OH

Figure 4.8 Ethoxylation of an alcohol functionality.

material becomes more and more water soluble. It is therefore a relatively

simple matter to control the ratio of ethylene oxide to feedstock to obtain
the degree of solubility of surfactancy required. As the ethylene oxide
reagent itself is used on a large tonnage scale for other business areas, the
material is an economically viable feedstock for surfactant production.
One important point should be noted about this process. Addition of a
certain number of moles of EO to a material containing a reactive
hydrogen site (e.g. -OH, -NH, -COOH) does not give a single
compound. As the addition takes place, for example between a fatty
alcohol and two moles of EO, a spread of molecules is formed, as the
hydroxyl end of one homologue will react similarly to the hydroxyl end of
another homologue. A spread of isomers will build up which will range
from the starting alcohol itself, with no EO added to it, through molecules
with 1, 2, 3, 4 and 5 EO, up to substantial quantities of material with six
ethoxylate groups attached. Although ethoxylation is an imprecise way of
generating a required molecular species, it appears that it is more often the
case than not that this spread of products is actually more useful in most
applications than a single homologue would be, and this is probably an
example of the synergistic effects discussed in section 4.5.2.
Reactions can also be carried out by polymerising propylene oxide, but
this is much less common, as propylene oxide tends to add hydrophobicity
rather than hydrophilicity.

4.4.4 Cationics
As described above, 70% of all surfactant production is accounted for by a
few product families. These include anionics (soap, sulphates and
sulphonates) and nonionics (principally ethoxylates). Cleaning is the major
use for these materials.
It is also possible to create families of cationic compounds which are
derived essentially from oleochemical fatty acids via reductive ami nation
processes requiring several steps. These nitrogen-based materials display
an ability to bond particularly strongly with some substrates, a feature
known as substantivity. Many materials in nature display a surface negative
charge, especially when wet, and a positively charged surfactant will bind
strongly to this material, imparting a hydrophobic effect. If the surface is
textile fibres, the hydrophobic effect allows the fibres to slide over one
 INTRODUCTION TO SURFACTANTS 75

another, and to untangle easily. This offsets the usual matting of the
material, and the fabric becomes soft to the touch. In the same way as
fabrics are softened by this mechanism, hair can also be conditioned.
Cationics also display interesting biocidal properties which have been
related to their ability to disrupt bacterial cell walls, a property not
displayed by anionics or nonionics.

4.4.5 A full range of functionality

The surfactant families given in Table 4.4 are a first introduction to the
main chemical processes used by industry. An exhaustive list is not offered
and the reader is referred to more detailed texts for this information
and to the literature of surfactant-producing companies for an apparently
bewildering array of commercially available materials. However, the table
does give a good preliminary idea of some of the major elements which are
combined to give surfactant molecules.
In addition to the product families shown in Figure 4.7, a few more types
of compound have been illustrated. Phosphates have been added and
extended anionics are possible, as any reaction which can be carried out on
a fatty alcohol can also be carried out on the appropriate ethoxylate.
Reaction of an ethoxylate with chloroacetic acid gives the ether carboxylate,
while reaction of the same reagent with a tertiary amine gives a betaine
structure (two charge centres in the same molecule).

Table 4.4 Selection of hydrophobes and hydrophiles

Main hydrophobes:
Oleochemical Linear alkyl (acids, alcohols, esters)
Petrochemical Linear alkyl benzene
Alkyl phenol
a-olefin

Main hydrophiles:
Anionic - COONa Soaps
- S03Na Sulphonates
- OS03Na Sulphates
- OP0 3 Na Phosphates
- CH(S03Na)CO zCH 3 Ester sulphonates

Extended anionic - (EO)nS03Na Ether sulphate

- (EO)nP03Na Ether phosphate
- (EO)nCH2 C0 2 Na Ether carboxylate

Nonionic - (EO)nH Ethoxylates

- CON(CH2 CH2 0Hh Ethanolamides
- N(CH3h-O Amine oxides

Cationic - N+(RhX- Quaternaries

N+(CH 3hCH 2 C0 2 - Betaines
76 PRESERVATION OF SURFACTANT FORMULATIONS

Each combination of hydrophilic groups adds to the possible range of

uses that these molecules can display. For example, an ether sulphate
displays a higher degree of hard water tolerance and lower skin irritation
when compared to the parent alcohol sulphate. Many other product
families have not been mentioned due to the nature of this article, but most
will be found in the Further reading section.

4.4.6 Understanding surfactant chemistry

The major theme that runs through this short introduction to the chemistry
of surfactants is that all of the commercially available products are
mixtures. Although this is usually an advantage in terms of performance, it
is something of a handicap when a simple picture of structure and effect is
required.
One of the major themes for the pharmaceutical industry is the idea of
designing high purity and highly specific molecules, to act at a particular
site to create one specific end effect for the patient. This concept is one of
key and lock, and the search consists in trying to find a specific key to tum
in the lock and alleviate the illness without creating other complications
(turning other locks).
Understanding surfactant effects is usually approached in terms of
simplified models, and this simplification process can mean that much of
the real problem becomes lost in the modelling process. This is not always a
bad thing, trying to extend current understanding with carefully worked
out theories, but it is essential that a good grasp of product composition
should be a part of this process.
Consider the likely chemical composition of a fatty alcohol which has
been reacted with ethylene oxide. The product displays three main
compositional elements.
1. The alkyl group will be composed of a mixture of alkyl chains which will
depend upon the fat or oil from which it was sourced.
2. The ethoxylation process will give a spread of ethoxylate chains for each
of the individual alkyl chains, creating a matrix of ethoxylate products.
3. If the amount of ethoxylation is low, the free alcohol itself (with no
ethylene oxide added to it) will be a large part of the product. This is
because the alcohol hydroxyl group reacts slower than the ethoxylate
hydroxyl, with the result that ethoxylates continue growing in preference
to the alcohol reacting.
Surfactancy therefore cannot be seen as lock and key. It is a teamwork of
molecules and their end effects. To achieve the best results, a single key
will not give the answer. The lock is usually a combination lock.
 INTRODUCTION TO SURFACTANTS 77

4.5 Formulating with surfactants

This chapter has so far introduced three concepts; that surfactant

molecules are essentially driven to an interface by forces arising from a
polar medium, that this phenomenon can lead to a series of useful effects,
and that commercial grades of surfactants, although similar in structure to
the idealised molecules originally used to explain these effects, are in fact
gross mixtures of many chemical entities. Given the complexity of
available products, some helpful guidelines are required in order to
demonstrate that practical formulating is not as random as section 4.4 may
suggest.

4.5.1 The hydrophile-lipophile balance (HLB)

Surfactants have been widely studied in liquid-liquid emulsions, as the
production and destruction of emulsions are fundamental to many
commercial processes and their longevity has allowed detailed studies to be
carried out. Consequently, a wealth of data has built up with time. In an
attempt to convert this knowledge into a practical guide, Griffin proposed
a system in 1949 which sought to classify the surfactant nature of a
particular product. This he called the HLB of the surfactant (the
hydrophile-lipophile balance). Lipophile here means essentially the same
as the term hydrophobe. The proposal was to regard the emulsification of
an oil into an aqueous system as requiring a certain balance of hydrophobe
and hydrophile in order to allow the formulation of one in the other. That
is, only materials with the right balance will enable the formulation to be
achieved efficiently.

4.5.1.1 The HLB scale. Griffin defined the HLB scale as having
numerical values between 1 and 20. Surfactants which are hydrophilic
would lie towards the top end of the scale, and hydrophobic materials
would lie towards the bottom end. The HLB of an ethoxylated nonionic
surfactant could be calculated using the equation:
HLB = molar % EO groups/5
The idea was extended by Rideal and Davies, and attempts were made
to place numerical values on each functional part of the surfactant
molecule and to link these together with the following equation:
HLB = 7 + sum (hydrophilic numbers) - sum (hydrophobic numbers)
Some typical numbers for various groups are listed in Table 4.5.
For example, let us take cetyl alcohol plus 10 ethylene oxide units. The
measured HLB value for this surfactant is 12.9.
78 PRESERVATION OF SURFACTANT FORMULATIONS

Table 4.5 Typical HLB numbers for common functional

groups

Hydrophilic Hydrophobic
-S04Na 38.7 -CH2- 0.48
-COzNa 21.1 -CH 3 0.48
-C0 2H 2.1 -CHzCH- 0.15
-OH 1.9 I
-CH2 CH 2 O- 0.33 CH3

• From Griffin, the percentage of the molecule composed of hydrophilic

groups is 66%. The HLB is (66/5) = 13.2.
• From Rideal and Davies, the HLB = 7 plus 10 EO groups, plus one OH
group = 7 + 3.3 + 1.9 = 12.2
The above example illustrates the usefulness of such simple rules. It does
not give anything other than a rough guide, however, as it makes no
allowance for changes in the conditions of use, the nature of the oil and
water phases, or the presence of other ingredients.

4.5.1.2 The required HLB. It has been shown that it is possible to

generate a numerical value which gives an approximate idea of the
character of the surfactant being considered. However, the other essential
element in the use of the HLB concept is the idea that for each
emulsification carried out between a particular oil and an aqueous system,
a certain balance of hydrophobe and hydrophile is required to make this
work. For example, the required HLB for the generation of an oil-in-water
emulsion of isopropyl myristate (IPM) is 11.
In order to find an HLB suitable for the duty required, the following
procedure can be adopted.
• Make up small samples of a mixture of oil and water and add into each
sample a set amount of surfactant of known HLB. Agitate the samples,
and observe them for the ease of formation of an emulsion. By using
different surfactant combinations of known HLB, it is possible that a
particular part of this range will be obviously better than other parts.
• If all of the samples emulsify satisfactorily, repeat the experiments but
use a lower level of surfactant until an optimum is reached.
• If none of the samples emulsifies well, repeat the experiment but increase
the level of emulsifier in each.
A table of typical blends of Span (sorbitan esters) and Tween
(polysorbate products) which give unitary HLB values are included in
Table 4.6.
As a general rule of thumb, the HLBs for particular applications are as
shown in Table 4.7.
 INTRODUCTION TO SURFACTANTS 79

Table 4.6 Surfactant combinations giving unitary HLB numbers

HLB Composition

2 Span 80 8% Span 85 92%

4 Span 80 88% Span 85 12%
6 Span 80 83% Tween 80 17%
8 Span 80 65% Tween 80 35%
10 Span 80 46% Tween 80 54%
12 Span 80 28% Tween 80 72%
14 Span 80 9% Tween 80 91%
16 Tween 20 60% Tween 80 40%

(Span and Tween are registered trademarks of ICI surfactants)

Table 4.7 Typical HLB numbers for different applications

HLB range Use

4-6 Water-in-oil (W/O) emulsifiers

7-9 Wetting agents
8-18 Oil-in-water (O/W) emulsifiers
10-18 Solubilisers
13-15 Detergents

4.5.2 Synergy
While the above is a good starting point, the possibility of achieving a
better end effect by the blending of two surfactants should be considered.
This is for a number of reasons, such as:
• Ease of matching the required HLB by blending.
• A denser packing may be achieved at the interface between dissimilar
alkyl and headgroup functionalities.
• The higher packing will result in an even lower interfacial tension.
• Electrostatic repulsion between similar headgroups (e.g. sulphates) may
be mitigated by the use of nonionics (e.g. ethoxylates).
An example of this effect is the synergism that exists between the Span-
and Tween-type products. Spans are low HLB materials formed by the
condensation of fatty acids and sorbitol. Tweens are produced by
ethoxylation of unreacted hydroxyl groups in the Spans, giving high HLB
materials. Blends of these two materials can be seen to produce two
additive effects. The Span is oil soluble and will approach the interface
from the oil side. The Tweens are water soluble and approach the interface
from the water side. Packing at the interface is therefore increased by the
two surfactants essentially keeping the bulk of their structures on different
sides of the interface.
80 PRESERVATION OF SURFACTANT FORMULATIONS

4.5.3 Physical and chemical compatibility

For a successful formulated product, it is likely that the correct blend of
surfactants and other chemicals will be required. It is not enough to arrive
at a correct HLB, as many other considerations must be taken into
account.

4.5.3.1 Viscosity. The final product may be needed with a particular

viscosity. Hard surface cleaners and disinfectants may be clear liquids,
creams, gells or suspensions, depending upon the consumer preference for
performance or perception. However, it is not enough to manufacture the
best product for the outlet, if after a month on the supermarket shelf the
clear solution clouds, the cream splits into two layers, the gells collapse
into a thin liquid, or the solid either falls out of solution or sets the mixture
solid. Viscosity over time is a very problematical area, and although
accelerated ageing tests can be carried out, either by storing at temperatures
above normal (40o q, or by centrifugation, there is yet no easy way of
predicting emulsion or suspension lifetime.

4.5.3.2 pH Stability. If the product must necessarily be of a low pH,

materials such as soaps would be of little use, as they would be present in
their protonated forms, rather than as their alkali salts. Compare the
hydrophilicity of the alkali form with the protonated form in Table 4.5 (21
versus 2). However, if the product is strongly alkali itself, there would be
little sense in adding an ester surfactant, as this would break down to give a
fatty acid salt (a soap) and a non-surfactant polyol.

4.5.3.3 Temperature of use. Anionics such as sodium lauryl sulphate

tend to crystallise out of solution below certain temperatures, as the alkyl
groups reach or go below their paraffinic melting points. This general loss
of utility for anionics is known as the Krafft point. In contrast, nonionics
such as ethoxylates tend to lose their water of hydration (the ordered
hydrogen bonding of water neighbours which gives them their aqueous
solubility) at higher temperatures, and so can separate out of aqueous
solution as a result of this. The temperature is generally referred to as the
cloud point, as a clear solution of surfactant clouds when the solubility is
lost upon warming. Cooling down will return the clear solution. Surfactancy
effects seem to be at a maximum at or around the cloud point, falling away
on both sides of this. A complete understanding of this observation has yet
to be advanced.

4.5.3.4 Chemical compatibility. The easiest way to illustrate this is to

refer to the most well known of all surfactant systems; the washing and
conditioning of clothing. The major surfactant types used in washing are
 INTRODUCTION TO SURFACTANTS 81

the anionics. However, after washing, softening of the garment is

commonly carried out by the addition of a cationic. These two operations
are usually carried out in a two-step process, rather than both together, for
the simple reason that blending the two together will neutralise the effects
needed from each! This is one of the most obvious examples where there is
a need to consider the formulation as part of a total process, rather than as
an isolated exercise in its own right.

4.5.4 Cost
Once the primary and secondary effects have been clearly identified and
the physical and chemical compatibilities have been established, the next
consideration when choosing a surfactant is cost. While there are a large
number of readily available, high tonnage products at relatively cheap
prices, it is not, however, always the best advice to eliminate higher cost
surfactants at too early a stage in the screening process. Obviously, if a
simple sulphate or alkoxylate is required and this performs as wanted, then
that is all that needs to be considered. However, if by use of the higher cost
surfactant, a further advantage can also be gained in processing or in final
product performance, this may justify its use for that application.

4.5.5 Other considerations

If the primary screening exercise is complete and some technically
successful candidates have been identified, this is not the end of the
process. Depending upon the final use, other considerations may need to
be met. Detergents may need to fulfil certain biodegradability criteria. A
skin care formulation must not corrode or sensitise the treated area. The
more specialised the end effect required, the more likely it is that
commodity surfactants will not be adequate performers, or that a special
grade may be required to suit the application.

4.6 Some final thoughts

In section 4.1, the intention was outlined to provide the reader with a first
impression of the main principles of surfactancy. What has been
adumbrated has been a necessarily simplified snapshot of a very complex
field. For example, surfactants have been described as essentially medium
molecular weight species, with a hydrophobic portion containing 10-18
carbons, and an appropriate hydrophilic head. However, many polymeric
surfactant types are known, with molecular weights of 3000-5000 being
common. It is clearly not sufficient to describe such molecules in terms of a
simple matchstick model! In addition, the speed with which such large
82 PRESERVATION OF SURFACTANT FORMULATIONS

mole~ules migrate to an interface will dictate how they may be used to

advantage for desired effects.
Similarly, while Figure 4.7 seeks to described common technologies and
product types, each field of surfactant application will use other product
types which will be common to that area. Sulphosuccinates are known to
be mild surfactants, used in the personal care area, and alkyl poly-
saccharides are used as adjuvants in agrochemical formulations. The
reader may wish to determine for themselves where these products would
fit into the scheme of Figure 4.7.
Accordingly, the texts listed in the following section have been selected
as books which take on themes of surfactancy in an easily readable format,
or in a way in which the themes can provide a good understanding of
particular essential aspects. These provide some of the broader background
which it has not been possible to include here, and which has in turn led to
the necessary qualifications and generalisations of this short introduction.

Further reading

Clint, J.H. (1992) Surfactant Aggregation, Blackie, Glasgow.

Falbe, J. (1987) Surfactants in Consumer Products, Springer-Verlag, Heidelberg.
Meyers, D. (1992) Surfactant Science and Technology (2nd edn), VCH, New York.
Porter, M.R. (1991) Handbook of Surfactants, Blackie, Glasgow.
Schick, M.J. (1987) Nonionic Surfactants, Physical Chemistry, Vol. 23, Surfactant Science
Series, Dekker, New York.
Tadros, Th.F. (1984) Surfactants, Academic Press, London.
5 Biodegradation of surfactants
G.F. WHITE

5.1 Introduction

5.1.1 Background
The purpose of this chapter is to outline the principles and pathways
involved in the biodegradation of synthetic surfactants. Biodegradation
may be defined concisely as a biologically catalysed reduction in
complexity of chemicals. 1 Although higher organisms metabolize surfact-
ants?,3 we are concerned here with biodegradation by microorganisms, of
which the most versatile and important contributors to surfactant bio-
degradation are the bacteria.
Restricted availability of animal fats for soap making during the 1914-18
and 1939-45 World Wars led to the production of alternative synthetic
surfactants based on petrochemical feedstocks. Subsequent diversification
within the synthetic surfactant industry spawned a range of surfactants with
properties suitable for the more varied applications evident in the
accompanying chapters. The growth in the use of synthetic surfactants in
detergent formulations faltered in the 1960s when significant pollution
episodes became apparent. 4,5 Investigations in Europe and the USA
enabled the detergent industry to trace the cause of the resistance of some
surfactants, especially highly branched alkylbenzene sulphonates, to
biodegradation by bacteria. This period saw the beginning of much work
on the biodegradation of surfactants which continues to this day, so that
synthetic surfactants are probably the most thoroughly studied group of
compounds in terms of their biodegradability by bacteria. Necessarily,
therefore, this account can only attempt to distil an essence of the
extensive literature that exists on biodegradation of surfactants. More
detailed accounts and compilations of data can be found in Cain,6,7 Karsa
and Porter,8 Swisher,s White and Russe1l 9 ,10 and White. 11
What we know about surfactant biodegradation is largely a reflection of
the environmental pollution background against which the work has
developed. Thus most work has focused on the biodegradation of spent/
waste surfactants in relatively dilute solution (e.g. effluent streams, river
water) using environmental isolates from activated sewage sludge, river
water and sediments, or soils. Moreover, the perspective usually taken has
84 PRESERVATION OF SURFACTANT FORMULATIONS

been that of seeking to achieve maximum biodegradation (extent and

rates) in order to minimize environmental impact. In contrast, in the
present context we are concerned not so much with the biodegradation of
spent or waste surfactant formulations (ultimately desirable though that
may be), but rather with minimizing the undesirable biodeterioration of
surfactants before they have served their intended purpose. Unfortunately,
there are very few published studies on the microbial processes and
mechanisms involved in the biodeterioration of surfactants in formulations,
so our metabolic and mechanistic knowledge must, at least for now, be
based on studies of the biodegradation process in microbes in the
environmental context.

5.1.2 Surfactants and bacterial nutrition

To provide a framework in which to describe the how and why of
surfactant degradation in microbes, we need first to appreciate some
aspects of environmental microbiology.
Although there is an anthropocentric distinction between the desirable
biodegradation of waste surfactant and the undesirable spoilage of
surfactant formulations, from a microbial perspective the difference all but
vanishes because in both situations opportunistic microorganisms are
exploiting potential sources of nutrition. Microbes do not collude to spoil
our formulations, any more than they are altruistic in clearing up our
waste! Bacterial life is characterized by periods of rapid growth of the
population by cell division when nutrients are plentiful, interspersed with
periods of starvation. Successful proliferation of bacteria means that
nutrients are depleted rapidly so that for much of the time bacteria exist in
low nutrient conditions. Consequently microorganisms have evolved
several adaptations to survive such widespread privation. A central
requirement for the survival of microbial cells is that they must be able to
generate a minimum metabolic energy in order to maintain their structural
and functional integrity. The vast majority of microorganisms are chemo-
organotrophs, i.e. they obtain their energy by breaking down organic
nutrients (catabolism) and oxidizing the reduced carbon/hydrogen that is
present. Consequently for many microorganisms, survival depends on
scavenging for reduced organic carbon compounds in their environment
and organisms which develop catabolic diversity enabling them to utilize a
wider range of chemicals as potential nutrients will have an advantage. 12
This brings us back to synthetic surfactants.
In recent times on the evolutionary timescale, microbial survival has
been assisted by human mobilization of organic carbon in fossil deposits.
Hitherto, this organic carbon has been of relatively restricted accessibility
to microorganisms because of the localized nature of such deposits.
However, the exploitation of fossil carbon for feedstocks in the chemical
 BIODEGRADATION OF SURFACTANTS 85

industry has mobilized this resource into a much wider environment. For
example, many synthetic surfactants derive their hydrophobic chains from
paraffin fractions in crude oil via intermediates such as olefins and
alcohols. 13 In addition, agriculture provides animal and vegetable oils such
as tallow, coconut oil and palm-kernel oil, the lipids which have been
traditional starting materials for the soap industry and are now also used as
raw materials for the production of 'synthetic' (i.e. non-soap) surfactants.
The key step in production of most surfactants is covalent attachment of
the hydrophobic alkyl chains found in petrochemical and agricultural lipid
feedstocks, to a hydrophilic group, commonly for example the -S03-
group either as a sulphonate or a sulphate ester. This clearly alters the
properties of the molecule to human advantage in the intended application,
but it also potentially occludes the molecule from the normal catabolic
pathways which would otherwise have been involved in its biodegradation
by bacteria. Thus, the biodegradation of surfactants needs to be viewed in
terms of how microorganisms exploit their catabolic versatility to meet the
challenge of accessing the carbon in these synthetic compounds.
Broadly speaking, microorganisms can use two general strategies to gain
access to the reduced carbon in the hydrophobic chains of surfactants: 10,14
either by separating the hydrophilic group from the hydrophobic group to
leave an alkyl chain with a readily metabolizable functional group such as
-CHzOH or -COOH, or by direct attack at the w-position of the intact
molecule with the hydrophilic group still in place. In some surfactants the
hydrophilic moiety is itself comprised of significant amounts of polymeric
carbon, for example in the form of polyethylene glycols. In such cases a
third strategy presents itself, namely progressive destruction of the
hydrophilic group by successive removal of glycol units. All three strategies
(separation of hydrophile from hydrophobe, or the erosion of either of
these groups from the ends of the molecule) will result in the loss of the
surfactant properties of the molecule. This phase of the process, which is
easily assessed (for example by loss of foaming, increase in surface tension,
or specific dye-based assays for the parent surfactant), is termed primary
biodegradation. Subsequent bacterial metabolism of the products of
primary biodegradation leads ultimately, at least for the more readily
degradable surfactants, to the production of COz, HzO and compounds
which are the 'normal' metabolic intermediates of bacterial cells. This
phase is referred to as ultimate biodegradation. There is evidence that for
some industrial compounds, the products of primary biodegradation are
more toxic to aquatic and other organisms than are the parent compounds
(e.g. alkylphenols from alkylphenol ethoxylates, see section 5.3.4). Thus
in the environmental context, it is important to assess the fates of all parts
of the surfactant molecule, i.e. both primary and ultimate biodegradation.
However in terms of microbial spoilage of surfactant-containing formula-
tions, the primary steps are particularly important. In succeeding sections,
86 PRESERVATION OF SURFACTANT FORMULATIONS

both aspects will be addressed but with particular emphasis on primary

biodegradation.

5.2 Biodegradation of anionic surfactants

5.2.1 Alkyl sulphates

Alkyl sulphate (alcohol sulphate) surfactants contain ester bonds between
long chain alcohols and sulphuric acid. Primary biodegradation of these
compounds is achieved in bacteria by enzymic hydrolysis of the sulphate
group to liberate the parent alcohol 15- 17 which then undergoes oxidation
via the alkanal to the fatty carboxylic acid (Figure 5.1). Fatty acids either
undergo elongation to C 16 and C I8 homologues and incorporation into
phospholipids, or they enter central metabolic pathways via fl-oxidation to
acetyl-coenzyme A, thus achieving ultimate biodegradation. fl-Oxidation is
an ubiquitous catabolic process in living cells which leads to the
degradation of carboxylated aliphatic chains by sequential removal, from
the carboxyl terminus, of 2-carbon acetyl fragments in the form of acetyl-
coenzyme A.Is Secondary alkyl sulphates such as decyl-4-sulphate follow a
similar biodegradation pathway, except that the liberated secondary
alcohols loop out of the pathway to undergo C-C bond scission I9 at the
substituted carbon atom via a dione intermediate (Figure 5.1). The
resulting alkyl chain fragments are alkanals and alkanoic acids which then
re-enter the central metabolic pathways to yield acetyl-CoA.
Alkylsulphatase enzymes catalysing the hydrolysis of alkyl sulphates in
Pseudomonas spp. have been purified and well characterized in terms of
their substrate specificity and kinetics and the mechanisms of action (for
reviews see Dodgson et al. ;20 Dodgson and White;21 Cain, 1987;7 Whit.e
and Russell lO). Bacteria which contain alkylsulphatases are commonly
found in natural environments. 22- 24 Naturally-occurring alkyl sulphates are
equally ubiquitous;2o examples include the sulphated lipids and chloro-
sulpholipids present in freshwater algae,25.26 and bile salt sulphates in the
gastrointestinal tract of animals.27 These compounds may be natural
substrates for bacterial alkylsulphatases. Thus, evolution of the bio-
degradative capacity towards alkyl sulphate surfactants is probably not a
recent response to synthetic compounds, but dates from much earlier
encounters with natural analogues. Some enteric microorganisms develop
a resistance to high concentrations of surfactant through mechanisms not
involving biodegradation. 28 Presumably this results from exposure in the
gut to natural bile-salt surfactants, microbial biodegradation of which
might compromise the microorganism's symbiotic relationship with the
host.
Most of the work described in the foregoing paragraphs relates to
 BIODEGRADATION OF SURFACTANTS 87

Primary alkyl sulphates Secondary alkyl sulphates

V0Nv0so ;

V\l'COOH

I ~ '- P_O-~d'-tion,- - - '

:: cellular components
and CO2

Figure 5.1 Metabolic pathways for bacterial biodegradation of primary and secondary alkyl
sulphates. Open arrows show the extra reactions involved in C-C bond cleavage for
secondary alcohols. Adapted from White and Russell.1O

aerobic systems. Alkyl sulphates are also readily degradable in anaerobic

waste treatment plants,29,3o and several non-fermentative denitrifying
bacteria have been isolated from river sediment which can grow anaerobic-
ally on sodium dodecyl sulphate. 31 Extracts of cells grown aerobically or
anaerobically contained alkylsulphatase activity, implying that the hydro-
lytic mechanism operates under both conditions. Sulphatase-mediated
hydrolysis also occurs in the anaerobic degradation of naturally-occurring
sulphated bile-salt surfactants by intestinal bacteria. 32 ,33

5.2.2 Alkyl phosphates

Alkyl phosphates (alcohol phosphates) are analogous to the alkyl sulphates
in so far as they are alcoholic esters of a mineral oxyacid, except of course
that the tribasic nature of phosphoric acid permits both monoalkyl and
dialkyl esters (ROP0 32- and (RO)zP0 2-) to serve as anionic surfactants.
Every living cell produces a plethora of phosphate esters such as
phospholipids, DNA, RNA, ATP and many other intermediary meta-
bolites. Phosphatase enzymes which are important for the turnover of these
88 PRESERVATION OF SURFACTANT FORMULATIONS

compounds are equally ubiquitous. In contrast, industrial production of

alkyl phosphates occurs on a relatively small scale. The consequent lack of
interest in the biodegradability, together with natural and ubiquitous
occurrence of many phosphatases, has led to the tacit assumption that
these enzymes act also on the synthetic long chain alkyl phosphates. Such
data as is available indicates that linear primary alkyl phosphates and
ethoxylate phosphates34 ,35 are readily biodegradable. Moreover the
diminution of phosphate ester linkages accompanying the loss of foaming
and increase in surface tension during biodegradation suggests that
phosphate ester hydrolysis may indeed be the initiating step (d. alklyl
sulphates, section 5.2.1).

5.2.3 Dialkyl sulphosuccinates

Like the alkyl sulphates and phosphates, dialkyl sulphosuccinate (DASS)
surfactants also contain ester bonds, but in this case the acid components
are the carboxylic acid groups of succinic acid rather than mineral acid. Not
surprisingly, hydrolytic scission of the alcohol from ester linkage is again
the mechanism 36 that allows bacteria to gain access to the long chain
alcohol. Removal of the first alkyl group yields monoalkyl sulphosuccinate
(MONAS) and depending upon which alcohol is removed first, both a-
and fi-isomers may accumulate transiently (Figure 5.2). Removal of the
alkyl chain from either a- or fi-MONAS completes the primary bio-
degradation process, yielding sulphosuccinate. To achieve ultimate bio-
degradation, this hydrophilic residue containing a -S03- unit still in
organic linkage with the succinate moiety must be further biodegraded.
Recent work has shown that sulphosuccinate is catabolized by a Pseudo-
monas sp. under aerobic conditions via a monooxygenation pathway that
first introduces an oxygen atom from O 2 at the sulphonated carbon to
produce a hydroxyl group. The resulting structure (2-sulphomalate, see
Figure 5.2) is equivalent to the bisulphite adduct of a carbonyl group and
undergoes spontaneous decomposition to eliminate bisulphite and form
the common intermediary metabolite, oxaloacetate. 37 Under anaerobic
conditions, mixed cultures accomplish complete primary biodegradation of
DASS but there was no catabolism of the sulphosuccinate group.36 This is
consistent with hydrolytic removal of alkyl chains and subsequent inability
to attack the sulphosuccinate in the absence of O 2 ,
As with the alkylsulphatases, esterase reactions involving natural
analogues 38 can be readily identified in lipid metabolism and so the
capacity to attack DASS is likely to be widely distributed. Monooxygenation
of sulphosuccinate on the other hand, may be a more specialized
capability.
In alkyl sulphates, alkyl phosphates and dialkyl sulphosuccinates, a long
chain alcohol is linked to an acidic hydrophilic group by an ester bond.
 BIODEGRADATION OF SURFACTANTS 89
Dioctyl sulphosuccinate

2-Sulphomalate Oxaloacetate

Figure 5.2 Metabolic pathway for bacterial biodegradation of dioctylsulphosuccinate.

Consequently a simple hydrolytic mechanism is all that is needed to access

the alkyl chain. This has advantages for bacteria because enzymic
hydrolysis proceeds without the need for any energetically expensive
cofactors to be consumed.

5.2.4 Alkylethoxy sulphates

Alkylethoxy sulphate (alcohol ethoxylate sulphate) surfactants may be
considered as alkyl sulphate analogues in which a polyethylene glycol
moiety has been inserted between an alkyl chain and the sulphate group,
and the hydrocarbon chain is attached via an ether bond to polyethylene
glycol (e.g. sodium dodecyltriethoxy sulphate, Figure 5.3). This structure
provides several points of attack for primary biodegradation in bacteria, all
of which are known to occur.
90 PRESERVATION OF SURFACTANT FORMULATIONS

Strain SC25A Strain TES5

82
86

Figure 5.3 Relative contribution of different routes to the primary biodegradation of

dodecyltriethoxy sulphate in four strains of Pseudomonas. Arrows marked sand m indicate
sulphatase-mediated hydrolysis and m/ft-oxidation, respectively. Other arrows indicate ether
fission. Lengths of arrows are proportional to the percentages of surfactant (quoted at the
base of each arrow) which undergo primary biodegradation at the indicated site. Adapted
from White and Russel1. 9

First, all Pseudomonas spp. so far isolated by enrichment culture on this

surfactant as growth substrate, are capable of breaking the alkyl-glycol
ether bond (sometimes called central ether cleavage) in order to gain
access to the alkyl chain (Figure 5.3). Thus, separation of hydrophobic
from hydrophilic groups is an important route of primary biodegradation
for this type of surfactant. In some bacteria,39 the responsible 'etherase'
enzyme is very specific for this bond (e.g. strains SC25A and TESS, Figure
5.3), whereas in other isolates the enzyme(s) are relatively non-specific in
so far as they will also attack other ether bonds within the polyethylene
glycol (stains DESl and Cl2B).
Secondly, alkylethoxy sulphate surfactants are also substrates for
enzymic hydrolysis of the sulphate group (Figure 5.3), d. alkyl sulphates,
yielding alcohol ethoxylates (Figure 5.4). Thirdly, direct attack on the alkyl
chain of the intact alkylethoxy sulphate surfactant by m/ft-oxidation has also
been demonstrated for strain TESS (Figure 5.3). m-Oxidation involves
hydroxylation of the terminal methyl group of alkyl chain by mono-
oxygenase which inserts oxygen from O 2 . The resulting hydroxylic group is
subsequently oxidized via aldehyde to carboxylic acid which opens the
chain to sequential removal of C 2 units by the ubiquitous ft-oxidation
pathway for catabolism of fatty acid chains. This relatively minor route in
 BIODEGRADATION OF SURFACTANTS 91

alkyl ethoxysulphates assumes much greater importance m the bio-

degradation of some sulphonated surfactants (see below).
Ether-bond scission of sodium dodecyltriethoxy sulphate by Pseudo-
monas spp., produces metabolites from the hydrophilic end of the
molecule 4o ,41 in which the anionic S03- group remains attached to ethylene
glycol residues (Figure 5.4). These metabolites constitute a homologous
series of sulphated glycols containing mono-, di- or tri-ethylene glycol
units, and they are susceptible to oxidation to the corresponding carboxylic

Dodecyl triethoxy
sulphate fVV\NV'o'\P";o'V°s o;

,so; .....
. _ - - - - - - - - _ __
°<DH
._------1----1
,so; .....
~
He>°

Glycol
oxidation

1
L -_ _ _
D I h t·
t
e_su_p_a_lo_n_ _
I_
---lr-
Glycollate and -----
PEG carboxylates ----- Biomass and C02

Figure 5.4 Metabolic pathways for bacterial biodegradation of dodecyltriethoxy sulphate in

Pseudomonas spp.
92 PRESERVATION OF SURFACTANT FORMULATIONS

acids. Not all these metabolites are formed in single organisms, but in
mixed environmental cultures, all three glycol sulphates and the three
corresponding carboxylates have been detected. 41 These compounds
clearly contain nutritionally useful organic carbon (and sulphur). In sewage
populations the glycol sulphates are fully mineralized to inorganic sulphate
but the mechanism(s) of biodegradation have yet to be established. These
sulphate esters are probably not degraded by the surfactant-degrading
alkylsulphatases (section 5.2.1) because these enzymes are specific for long
chain (>C5) surface-active analogues. to Bacterial metabolism of some
short chain (Cr C 7 ) alkyl sulphates is initiated by hydrolase enzymes 42 .43
but others, e.g. monomethyl sulphate44 .45 and isopropyl sulphate,46.47
involve oxygenation reactions in the initial biodegradation steps.
The main hydrophobic coproducts of etherase and sulphatase actions on
dodecyltriethoxy sulphate (Figure 5.4) are the nonionic surfactants mono-,
di-, and tri-ethylene glycol dodecyl ethers (see section 5.3.2), together with
dodecanol and the four corresponding oxidation products in which the
terminal hydroxyls are converted to carboxylic acids. 48 The corresponding
long chain alkanals have also been detected but there are no clear
indications as to whether the alcohols or the alkanals are the primary
cleavage products. These products may serve as substrates in further
rounds of ether cleavage48 until the alkyl chain is made available.

5.2.5 Alkane sulphonates

Alkyl sulphates are formed by the sulphation of an alcohol with S03 or
close relatives such as oleum or chlorosulphonic acid,49 a process which is
readily reversible by hydrolysis. Alkane sulphonates on the other hand are
produced by sulpho-oxidation of paraffins with S02 plus O 2 to give
secondary isomers, or addition of bisulphite to olefins in the presence of O 2
to yield primary isomers. 5o These processes are less easily reversed so that
alkane suI phonates are much more stable to hydrolysis than are sulphate
esters;51 in the presence of hot dilute acids, the latter hydrolyse in minutes
or hours but sulphonates remain stable for days. Not surprisingly then,
primary biodegradation of alkane sulphonates in bacteria does not occur
by simple hydrolysis. Early work 52 suggesting that bacterial desulphonation
was occurring to yield sulphite and a long chain alkanal was later
confirmed53 ,54 by the demonstration that the desulphonation in cell-free
bacterial extracts was dependent on the presence of molecular oxygen and
NAD(P)H. The postulated mechanism (Figure 5.5) involves the operation
of a monooxygenase enzyme to introduce an oxygen atom from O 2 at the
sulphonated carbon; the hydroxylated product is equivalent to an
aldehyde-bisulphite adduct which spontaneously loses sulphite to yield the
alkanal. Secondary alkane sulphonates are also readily biodegradable5
probably by formation and subsequent hydrolysis of an analogous keto-
 BIODEGRADATION OF SURFACTANTS 93

H+ so~- Na+
Na+ +
NADH NAD

f
o~l~N"
-
O=CH

~
H/U-

-
H2O
°2
Dodecane- 1-Hydroxydodecane- Dodecanal
1-sulphonate 1-sulphonate

Figure 5.5 Metabolic pathway for bacterial biodegradation of primary alkane-1-sulphonate

by hydrophilelhydrophobe separation. Reproduced with permission from White and Russell. 9

bisulphite adduct. 54 These pathways are analogous to the catabolism ofthe

secondary sulphonate, sulphosuccinate (section 5.2.3).
In addition to the oxidative hydrophile-hydrophobe separation route,
there is also evidence to implicate the m/ft-oxidation pathway in the
biodegradation of alkane sulphonates. 55 This is based on studies of the
biodegradation of dodecane sulphonate in mixed cultures in which
disappearance of the parent surfactant preceded the appearance of any
inorganic sulphate. No direct information is available to determine how far
along the alkyl chain ft-oxidation can progress, but by analogy with the
bacterial m/ft-oxidation of other surfactants (see section 5.2.7), and with
m/ft-oxidation of alkane sulphonates in mammalian systems56 the process
is unlikely to progress closer than 4-5 carbon atoms from the sulphonate
group. In view of the requirement for molecular O 2 in both desulphonation
and m/ft-oxidation pathways, it is not surprising that alkane sulphonates are
resistant to anaerobic biodegradation. 30
Thus for alkane sulphonates in which hydrophile/hydrophobe separation
by hydrolysis is more difficult than for surfactants containing ester linkages
(e.g. alkyl sulphates, alkyl phosphates, dialkyl sulphosuccinates or alkyl-
ethoxy sulphates), biodegradation mechanisms become more complex and
place significantly greater demands on cellular resources such as energetic-
ally expensive cofactors. In those surfactants for which alkyl chain
separation becomes even more difficult, m/ft-oxidation appears to be the
only route yet observed for bacteria to assimilate carbon for growth. This is
the case for the linear alkylbenzene sulphonates (LAS) such as 2-(p-
sulphophenyl) dodecane (section 5.2.7).
Primary biodegradation of hydroxy alkane and alkene (a-olefin)
sulphonates is rapid and complete57- 59 but the mechanisms are unknown.
By analogy with the structurally similar primary alkane sulphonates, both
the hydroxylation/sulphite-elimination pathway and the m/ft-oxidation
pathway may operate. It is also possible that other mechanisms involving
the hydroxyl or olefinic groups already present, may exist.
94 PRESERVATION OF SURFACTANT FORMULATIONS

5.2.6 Fatty acid ester suIphonates

a-Sulphofatty acid methyl esters are known to undergo both primary and
ultimate biodegradation in mixed environmental cultures55 ,60.61 but little is
known about the enzymic mechanisms involved. During investigations on
the biodegradation of a-sulphofatty acid methyl esters in a model sewage
treatment plant, Steber and Wierich 62 detected short chain sulphonated
intermediates that had retained the ester methyl group. These compounds,
analogous to sulphosuccinate, were presumably formed by w/ft-oxidation of
the aliphatic chain. Separation of the sulphonate group must therefore
occur at a late stage, presumably by mechanisms similar to that for
sulphosuccinate. Fatty acid ester sulphonates are resistant to biodegrada-
tion under anaerobic conditions,62 in keeping with the requirement for O 2
in w-oxidation.

5.2.7 Linear alkylbenzene sulphonates

The chemical stability of the aromatic carbon-sulphur bond, compared
with, for example sulphate esters, makes linear alkylbenzene sulphonates
(LAS) relatively resistant to biodegradation by hydrophile removal. Under
these circumstances, the alternative strategy of direct attack on the alkyl
chain without removal of the hydrophilic group becomes the easiest way by
which bacteria can access the alkyl chain. Bacteria achieve this feat by a
combination of w-oxidation and ft-oxidation. As we have seen, ft-oxidation
is a universal pathway for the degradation of carboxylated aliphatic chains
by sequential removal, from the carboxyl terminus, of 2-carbon acetyl
fragments in the form of acetyl-coenzyme A.18 In order to biodegrade the
alkyl chain in a surfactant like LAS by ft-oxidation, it is first necessary to
introduce a carboxylic acid functional group at the end of the chain. The
capacity for terminal oxidation to yield long chain carboxylic acids is widely
distributed in the microbial world, and is important for example in
initiating the metabolism of alkanes. 63 When a linear aliphatic chain is
already substituted at one end, terminal oxidation at the other end is
referred to as w-oxidation. This is the usual case for surfactants because one
terminus is often occupied by the hydrophilic group. Insertion of an oxygen
atom at the w-carbon is achieved by a hydroxylation reaction (usually
catalysed by a monooxygenase, Figure 5.6) analogous to that used to
labilize the sulphonate group in alkane sulphonates (Figure 5.5). The
resulting primary hydroxyl group is oxidized subsequently via alkanal to
carboxylic acid which then is the starting point for ft-oxidation.
At first sight, w/ft-oxidation seems the most direct route for bacteria to
assimilate carbon from the alkyl chain. However, the initiating mono-
oxygenation is NADH dependent and thus requires an initial investment of
metabolic energy. Consequently, the initiation of alkyl-chain biodegrada-
tion through w-?_xidation is energetically more demanding than is hydrolytic
 ":'( ]0._
YVWVCH,~ CHPH CHO 0::1
'? _ + 02 H20 ot:1
803 Na t!1
803 Na
r l r S03 Na o
~
t:1
~
o
Z
o.."
CIl
C
CO H CDDH CDDH '~"
: (
803 Na 803 Na 803 Na 803 Na
~
~
r t ~~DH T r T r CH 3CO-8CoA CH 3CO-8CoA CH 3CO-8CoA

Figure 5.6 Metabolic pathways for bacterial biodegradation of linear alkylbenzene sulphonate. The upper row of reactions achieved w-oxidation of the
alkyl chain and the lower row shows sequential release of C 2 units as acetyl coenzyme A via fl-oxidation. Adapted from White and Russell. 9

\0
VI
96 PRESERVATION OF SURFACTANT FORMULATIONS

separation of hydrophile from hydrophobe. Therefore, w-oxidation is not

normally observed where facile separation of hydrophile is possible,
e.g. in microbial biodegradation of alkyl sulphates. However, w/fi-oxidation
does occur for those compounds in which separation of hydrophile is more
difficult, for example the alkane sulphonates and alkyl ethoxysulphates,
and it is the only route so far observed for assimilation of carbon from
LAS.
The fi-oxidative enzymes which are used in surfactant biodegradation
seem to be those of the normal fatty acid oxidation pathway7 which
requires an unsubstituted CH2 group at the fi-position (two carbons away
from the carboxyl group) and at least one proton on the a-carbon. This
pathway can cope with single methyl branches on the a-carbon, but
substitution at the fi-carbon or gem-dimethyl branching anywhere in the
chain prevents further progress in oxidation of the alkyl chain. Those
alkyl benzene sulphonates produced and marketed in significant quantities
during the 1950s were based on tetrapropylene alkylbenzene feedstocks
which produced quaternary substitution in the alkyl chains with gem-
dimethyl branching. Such highly branched alkyl chains are particularly
resistant to fi-oxidation which led to environmental persistence of these
surfactants, and eventual prohibition of their inclusion in domestic
detergent formulations.
fi-Oxidation proceeds until the site of oxidation approaches the aromatic
ring, usually to within 4-6 alkyl carbon atoms. 7 Further metabolism of the
resulting short chain sulphophenyl alkanoates such as 5-(p-sulphophenyl)
hexanoate then involves desulphonation and ring cleavage, usually64-{;6
but apparently not always67 by other microorganisms constituting a
consortium.
Biodegradation of the alkyl chain in linear alkylbenzene sulphonates by
w/fi-oxidation yields short chain sulphophenyl alkanoates. Ultimate bio-
degradation of these intermediates may be viewed as two distinct
processes, namely desulphonation and ring opening. The current balance
of opinion is that these events occur in the order given,68-71 but it must be
remembered that this conclusion is based on studies not with sulphophenyl
alkanoates but with structural analogues. Thus toluene sulphonic acid is
metabolized first by monooxygenation at the methyl group to produce 4-
sulphobenzoate which in turn undergoes a dioxygenation reaction in which
both oxygen atoms of O 2 are introduced on adjacent carbon atoms to
destabilize the aromatic nucleus (Figure 5.7). The resulting sulphono-
dihydrodiol intermediate is the equivalent of the bisulphite adduct of an
a-hydroxycyclic ketone which is sufficiently unstable that it breaks
down spontaneously with the elimination of sulphite and an electronic
rearrangement to produce 3,4-dihydroxybenzoic acid (pr()tocatechuate).
Subsequent biodegradation of 3,4-dihydroxybenzoic acid via the so-called
meta-pathway is well established from studies on the biodegradation of
 BIODEGRADATION OF SURFACTANTS 97

9¥ 0Yt0"
+ ° - OH
1Si)1Ij'

ceO"
NADH NAD

Spontaneous
I H
,~
//
HSes- °2
CO2- °2
C~
4-Sulphobenzoate Protocatechuate

Figure 5.7 Desulphonation of 4-sulphobenzoate by a dioxygenase. Reproduced with

permission from White and Russell. 10

aromatic compounds in general. 72 ,73 Thus, removal of the highly polar

sulphonate group before ring opening enables competent organisms to
biodegrade the catechol products using the widely distributed ring-
cleavage enzymes which bacteria have evolved to assimilate carbon from
the numerous aromatic compounds that occur in nature.
Bacteria which attack the alkyl chain in LAS, do so because they are
carbon-limited and they need to acquire further supplies of carbon for
energy and growth. Recently Kertesz et al. 74 have shown that when a
mixed culture derived from a sewage treatment plant was grown with
abundant carbon and nitrogen but under conditions of sulphur limitation,
primary biodegradation occurred by direct desulphonation of intact LAS.
Thus environmental conditions can significantly affect biodegradation
patterns.
The requirement for O 2 in m/ft-oxidation and in the desulphonation
of arylsulphonates precludes biodegradation under anaerobic condi-
tions 30 ,75-77 cf. the alkane sulphonates.

5.2.8 Fatty acid alkanolamide sulphates

The sulphate esters of stearic and oleic fatty monoethanolamides, in which
the ethanolamine OH is sulphated (e.g. Figure 5.8a), undergo very rapid
and complete primary biodegradation (loss of surfactant property) but in
fact there is scant evidence on which to base any mechanistic proposals. In
the presence of sewage mixed microorganisms, significant amounts of
carbon (25-50%) remain as undegraded intermediates. No inorganic
sulphate was detectable,S showing that primary biodegradation was not
achieved by sulphatase action (cf. alkyl sulphates, section 5.2.1). Amide
hydrolysis seems improbable because this would liberate most of the
carbon (>80%) in the molecule as readily degradable fatty acids (cf.
ethoxylated fatty amide nonionics, section 5.3.6). Of the remaining
possible sites of primary attack (deamination of the ethanolamine units and
m/ft-oxidation of the acyl chain), the latter emerges as the most likely
mechanism, because the CO 2 yields were consistent with degradation of
 98 PRESERVATION OF SURFACTANT FORMULATIONS

(c) 0

~N-CH-CH_-SO- I 2 "2 3
Na+

CH3

Figure 5.8 Examples of anionic surfactants based on fatty acid amides and esters. (a) Stearic
acid monoethanolamide sulphate ester, (b) arylsulphamido stearic acid, (c) stearic amide of
N-methyl taurine, (d) stearyl isethionate.

the fatty acyl chain to within a few carbons of the amide bond which is
typical of that observed in other wlj3-oxidations of surfactants (section
5.2.5).

5.2.9 Sulphonated esters and amides of fatty acids

Sulphoaryl amides of fatty acids (arylsulphamido carboxylic acids; fatty
acyl sulphanilates, Figure 5.8b) are degraded by Escherichia coli in accord
with the distance principle78 which states5 that the greater the distance
between the sulphonate group and the free terminus of the alkyl chain, the
faster the biodegradation. This principle was derived from data for LAS
and other wlj3-oxidized surfactants, thus implying that the arylsulphamido
carboxylic acids also suffer the same fate. Disinclination to hydrophile-
hydrophobe separation (here by central fission of the amide bond) may be
caused by the presence of the aryl nucleus and this is also consistent with
parallel observations for alkylphenol ethoxylates (section 5.3.4). Bio-
degradation data for the closely related sulphoalkyl ami des of fatty acids
(alkylsulphamido carboxylic acids, Igepon T, Figure 5.8c) and sulphoalkyl
esters of fatty acids (Igepon A, Figure 5.8d), are equally scanty but
nevertheless indicate rapid and extensive biodegradation in mixed cultures.
To account for this, Sawyer and colleagues79 ,8o have speculated that an
initial hydrolytic attack liberates free fatty acids which are then rapidly
assimilated by central metabolic pathways present in all bacteria (d.
nonionic fatty alkanolamides, section 5.3.6). Unfortunately there is no
direct experimental evidence with pure cultures to support this claim, and
in fact it conflicts with available data for the analogous fatty acid
alkanolamide sulphates (section 5.2.8) and for the sulphoaryl amides of
fatty acids just described.
Whatever the initiating pathways, it seems likely that biodegradation of
sulphoalkyl esters and ami des of fatty acids will lead to the formation of
 BIODEGRADATION OF SURFACTANTS 99

isethionate and (methyl-substituted) taurines. Taurine is produced naturally

in abundance in animal tissues where it serves many functions including
conjugation with toxic compounds as a preliminary to excretion. Con-
sequently large amounts are deposited in the environment where bacteria
have evolved to exploit what, for them, is a nutritionally useful source of
carbon, nitrogen and sulphur. Extracts of a taurine-degrading species of
Agrobacterium isolated from river mud81 degraded taurine with the
consumption of O 2 and the release, first, of ammonia, then sulphite
(Figure 5.9) which was subsequently converted to sulphate. 81 The
occurrence of the implied oxidative deamination prior to desulphonation
was later confirmed by the detection of a taurine dehydrogenase which
catalysed the conversion of taurine to sulphoacetaldehyde. 82 .83 De-
sulphonation of sulphoacetaldehyde to acetate and sulphite 84 was catalysed
by a specific sulpholyase enzyme 85 which required thiamine pyrophosphate
and magnesium ions for activity.86 Although the structurally related
isethionate (HO-CH 2-CH2·S03-) served as a carbon source for growth of
the same organism (a Gram-negative rod from sewage), the sulpho-
acetaldehyde sulpholyase (Figure 5.9) was inactive on this compound
which required prior conversion to sulphoacetaldehyde through catalysis
by a specific dehydrogenase before desulphonation by the sulpholyase
could occur. In Pseudomonas aeruginosa, conversion of taurine to
sulphoacetaldehyde is accomplished by transamination with pyruvate
rather than oxidative deamination,87 before sulpholyase-catalysed de-
sulphonation. 88 Other Pseudomonas and Achromobacter species also use
aminotransferase enzymes to achieve taurine deamination via this
route. 89 ,90

(a)
Oxidative deamination
or transamination

O;CH-CHz-SO; Na +

Taurine Sulphoacetaldehyde

(b)

Sulphoacetaldehyde
O;CH-CHz-S03 Na
- +
r
Thiamine pyro-
OH
I
?H-CHz-S03 Na

TPP
_ +
T _
OH
I
?=CHz -
TPP
°II
?-
TPP
C~ -
C~COO-

TPP
HS0 3
phosphate, TPP
+
Na

Figure 5.9 Bacterial biodegradation of taurine showing (a) overall pathway; the sequential
release of ammonia then sulphite, and (b) the thiamine pyrophosphate-dependent
desulphonation of sulphoacetaldehyde.
100 PRESERVATION OF SURFACTANT FORMULATIONS

5.3 Biodegradation of nonionic surfactants

5.3.1 Polyethylene glycol (ethoxylate) chains as bacterial nutrients

Anionic groups such as -S03- are highly polar and heavily solvated in
aqueous solution, thus making these groups highly hydrophilic. In
contrast, sufficient hydrophilicity in ethoxylate nonionic surfactants is
achieved only by combination of many units (typically 5-25) of uncharged
ethylene glycol units in the form of a polyethylene glycol (PEG) moiety.
Thus the hydrophilic group in ethoxylated surfactants contains abundant
carbon, often more than in the hydrophobic alkyl chain. These moieties
are therefore potential sources of carbon for bacterial growth. Consequently
the two strategies of bacterial attack on surfactants considered so far
(hydrophile separation and wl.f3-oxidation) are now supplemented with a
third,l1 namely destruction of the hydrophilic group by successive removal
of glycol units from the terminus. Evidence for this contribution to
biodegradation is available for a number of nonionic surfactants as will
now emerge.

5.3.2 Linear alcohol ethoxylates

A major route for biodegradation of linear alcohol ethoxylates is via a
hydrophile/hydrophobe separation pathway, in this case a scission at the
central ether link, evidence for which has been collated in earlier reviews 4,6
and more recently by Swisher. 5 From studies with laboratory die-away tests,
Patterson et al. 91 first proposed that the cleavage sites specifically included
the alkyl-ether (central ether) bond and subsequent studies, also with
mixed cultures but in other laboratories92- 96 have confirmed this view.
Radiotracer experiments97 with a pure bacterium Pseudomonas sp. SC25A
able to grow on alcohol ethoxylates, showed that [1-14C]dodecyl decaeth-
oxylate was degraded with a very rapid production of 14C02 and
simultaneous liberation of decaethoxylate (Figure 5.10). These observa-
tions strongly suggested scission of the central ether bond to liberate the
decyl chain (as dodecanol, dodecanal or dodecanoic acid) from the
decaethoxylate, followed by oxidation of the decyl chain to acetyl-
coenzyme A, thence to 14C02 via .f3-oxidation and the tricarboxylic acid
cycle, respectively.
In addition to the central ether cleavage pathway, there is plenty of
evidence demonstrating the occurrence of the wl.f3-oxidation route in the
biodegradation of alcohol ethoxylates. Thus TLC, IR and NMR analyses 91
have demonstrated the conversion of alcohol ethoxylates to metabolites
containing carboxylic acids and ethoxylated material, via pathway(s)
additional to, but independent of the central ether cleavage route. By
 BIODEGRADATION OF SURFACTANTS 101

Central-ether Pseudomonas sp. SC25A

fission

Dodecyl decaethoxylate

*CO:!
Decaethoxylate

Figure 5.10 Primary biodegradation of dodecyl decaethoxylate via central ether cleavage in a
Pseudomonas sp.; an example of hydrophile/hydrophobe separation in nonionics. The
asterisk indicates the carbon radiolabelled with 14C. Reproduced with permission from
WhiteY

combining measurements of rates of 14C02 production froni alkyl-labelled

and ethoxylate-Iabelled octadecyl ethoxylate, with IR and NMR analysis of
metabolites, Nooi et al. 98 also concluded that biodegradation by mixed
microbial populations from activated sewage sludge begins with 00113-
oxidation of the alkyl chain of the intact surfactant. The dominance of
these two distinct primary biodegradation pathways (ether cleavage and
oolf3-oxidation) has been confirmed more recently for biodegradation of
linear octadecyl ethoxylate. 96
Alcaligenes faecalis var. denitrificans grew anaerobically on linear alkyl
ethoxylates under denitrifying conditions. 99 Although little is known about
this process, at least some degree of anaerobic degradation of these non-
ionics is not surprising because their PEG cousins succumb readily under
anaerobic conditions (section 5.3.5).

5.3.3 Branched alcohol ethoxylates

Oxo alcohols produced by Zeigler synthesis are mixtures of linear and 2-
methyl-branched primary alcohols. Alcohol ethoxylates derived from them
thus contain a proportion of molecules with a-methyl branches close to
the central ether bond. Swisher5 has collated information from several
sources which indicates that biodegradation of oxo alcohol ethoxylates by
mixed bacterial cultures yields mixtures of metabolites, mainly PEGs plus
some carboxylated PEGs. Collectively these results were consistent with
most of the surfactant (probably the linear components) being biodegraded
via central fission to give PEGs, and the remainder (the branched isomers)
undergoing oolf3-oxidation. Confirmation that C-2 alkyl-branched ethoxyl-
ates are inhibited from central cleavage came from studies on biodegrada-
tion of 2-ethyl[3- 14C)decyl decaethoxylate in mixed bacterial cultures. 97 No
102 PRESERVATION OF SURFACTANT FORMULATIONS

PEGs were formed, but radioactive intermediates were detected, probably

arising from oo/j3-oxidation of alkyl chain. These results are consistent with
earlier findingslOO that extensive (95%) primary biodegradation of linear
secondary alcohol ethoxylates in sewage treatment tests was accompanied
by the accumulation of compounds of the type (HOOChCH-
(OCH2CH2)nOH arising from oo/j3-oxidation of the two alkyl chains
attached to the ether-linked carbon atom. Evidently central cleavage was
prevented by substitution at the ether-linked carbon. The pattern was thus
emerging that branching of the alkyl chain in the vicinity of the central
ether bond hindered central ether cleavage, thus allowing the alternative
mechanism of oo/j3-oxidation to playa more significant role.
Later work showed that biodegradation of the oxo alcohol ethoxylates
(linear and branched mixture) in mixed cultures 101 ,102 also yielded
metabolites which were partly oo/j3-oxidized, partly ethoxylate shortened
(17-25%) but also 6--7% of ethoxylate shortened molecules with the alkyl
chain still intact (Figure 5.11a). It appears that, by restricting central
scission, the presence of light branching in the alkyl chain makes
ethoxylate shortening, as well as oo/j3-oxidation, more significant.
The trend towards ethoxylate shortening without oxidation of the alkyl
chain would be expected to become more pronounced with increased

(a)

Mixed HOOC (CH2)3_7 L/\ \OH + \lv\lVV'y'{01~:

culture 17-25% \V '14 6-7% CH 3

+ PEGs

Mixed
No PEGs
culture

(c)

Mixed
No PEGs
culture

(d)

Mixed ~ / \ 0 COOH No PEGs

____~.~ 0 V V
culture

Figure 5.11 Evidence for the occurrence of ethoxylate shortening during the biodegradation
of alcohol ethoxylates and alkylphenol ethoxylates. Reproduced with permission from
White.lO (a) Linear oxo alcohol ethoxylates; (b) tetrapropylene ethoxylates; (c) linear
alkylphenol ethoxylates; (d) branched alkylphenol ethoxylates.
 BIODEGRADATION OF SURFACTANTS 103

branching of the alkyl chain which would inhibit not only central ether
cleavage but also w/f3-oxidation. The highly branched tetrapropylene alkyl
chains are renowned for their recalcitrance in surfactants such as the now
notorious tetrapropylene benzene sulphonates which produced numerous
environmental pollution problems during the 1950s and 1960s. Use of these
groups as hydrophobes in ethoxylate nonionics does indeed produce a
marked shift in the biodegradation pattern. 102 Thus biodegradation of
tetrapropylene ethoxylate was incomplete even after 7 weeks, PEGs were
almost undetectable and the only detectable metabolites were a homologous
series of shorter ethoxylates with the alkyl chain intact which could only
have arisen from progressive ethoxylate shortening (Figure 5.11 b).

5.3.4 Alkylphenol ethoxylates

Biodegradation of linear (primary or secondary) alkylphenol ethoxylates in
mixed culture invariably fails to produce PEGs from central scission.103
The presence of the aryl nucleus probably provides sufficient steric
hindrance to prevent central scission; in effect the presence of the aromatic
nucleus is equivalent to branching adjacent to the central ether bond.
Instead, w/f3-oxidation and ethoxylate shortening combine to produce the
metabolites shown in Figure 5.11c. If in addition to the presence of an
aryl nucleus, the alkyl chain is made highly branched or shortened, w/f3-
oxidation of the hydrophobe would also be precluded so that in mixed
culture the dominant biodegradation mechanism expected is ethoxylate
shortening (Figure 5.11d). Positive identification of nonylphenol and its
mono- and di-ethoxylates in the environment 104 ,105 and nonylphenol
diethoxylate as major accumulating intermediates in the biodegradation of
nonylphenol decaethoxylate,106-108 together with the detection of several
lower ethoxylate homologues during biodegradation of 4-n-butylphenol
hexaethoxylate,109 clearly demonstrate that sequential degradation of the
PEG chain is indeed the major biodegradative route. Environmental
accumulation of these biodegradation products is giving cause for concern
because not only are they toxic, but they also accumulate in aquatic
organisms. 105 Moreover, they are suspected of contributing to 'hormone
pollution,ll0 because alkylphenols have been shown to cause changes in
gender characteristics in developing male trout at very low concentrations.
To summarize, three routes of attack are available in principle by which
bateria may gain access to utilizable carbon in ethoxylate surfactants, viz,
central ether scission, w/f3-oxidation and ethoxylate shortening. All three
operate in the biodegradation of linear alkyl ethoxylates, thus enabling
these compounds to undergo rapid primary, and complete ultimate,
biodegradation, Introduction of alkyl-chain branching or an aryl group
next to the central ether bond restricts central scission, so that biodegrada-
tion at the alkyl and ethoxylate extremities of the surfactants becomes
104 PRESERVATION OF SURFACTANT FORMULATIONS

relatively more significant. Extensive branching of the alkyl chain further

precludes ro/.f3-degradation of the alkyl chain, leaving ethoxylate shortening
as the dominant pathway. Ethoxylate shortening is probably operative for
all types but is more readily apparent for those surfactants in which the
other routes are restricted.

5.3.5 Polyglycols
We have already encountered polyethylene glycols (PEGs) as hydrophilic
components in alcohol ethoxylates, and in free form as the products of
central ether cleavage of those surfactants. PEGs and the analogous
polypropylene glycols (PPGs) are industrially important in their own right
in, for example, pharmaceuticals, emolients and lubricants.
There is ample evidence that PEGs are readily degradable aerobically by
soil and aquatic bacteria (for reviews, see COX;111 Kawai 112), and
depolymerization of low molecular weight PEGs l13- 115 and higher
polymers116.117 in pure culture is well documented. The occurrence of
anaerobic degradation is also firmly established,99.118,119 and for both
aerobic 116 and anaerobic 119 microorganisms, extracellular depolymerizing
enzymes have been reported which break down high molecular weight
PEGs to smaller oligomers more easily assimilated into the cells. On the
other hand, intracellular enzymes are usually involved in the assimilation
and degradation of low molecular weights PEGs and their metabolites.
Cain6 has identified four possible mechanistic strategies that micro-
organisms might use to depolymerize PEGs: (i) oxygenative cleavage by
monooxygenases, (ii) oxidation of the carbon atom a to the ether bond
followed by hydrolysis of the resulting hemiacetal or ester, (iii) direct
hydrolysis of the C-O-C ether bond, (iv) carbon-oxygen lyase-mediated
cleavage. Of these, the commonest mechanisms for exo-cleavage of PEGs
(i.e. sequential removal of terminal glycol units) which is emerging,
involves the enzymic formation, then spontaneous hydrolysis, of a
hemiacetal intermediate. However, there is considerable variety in the
routes to formation of the hemiacetal. For example, in the anaerobic
Pelobacterium venetianus an intracellular diol dehydratase removes H 20
from the terminal glycol to yield a vinyl intermediate which is then
rehydrated in the reverse orientation to produce a hemiacetal (Figure
5.12a) carrying a terminal methyl group. The hemiacetal is easily
hydrolysed to produce acetaldehyde and a PEG now shortened by one
glycol unit, which can undergo further rounds of exo_cleavage. 118 ,120,121 A
similar non-oxidative mechanism has been proposed for an obligately
aerobic strain of Acinetobacter. 122 In a Pseudomonas sp. capable of aerobic
growth on a range of PEGs, an intracellular PEG-dehydrogenase is
believed to extract a hydrogen atom from each methylene in the terminal
glycol unit to produce an enol which then hydrates to the hemiacetal
 BIODEGRADATION OF SURFACTANTS 105

(a) Dehydratase
H-(O-CH -CH )-0 -CH =CH
~
H-(0-CH 2-CH 2 )n-0-CH2-CH20H
2 2 n 2
Polyethylene glycol
H20~
o OH

H-(0-CH 2-CH 2 l,,--0H

II
+ HC-CH 3 ... I
H -(0 -CH 2-CH2 kO - CH - C~
Acetaldehyde
Hemiacetal

(b)

r
Dehydrogenase

H-(O CH 2-CH 2 )n-0-CH2-CH20H ~ 2HH -(0-CH 2- CH2 )n-OH2-0CH =CHOH

Polyethylene glycol ~

oII OH
I
H-(0-CH2-CH2)n-OH + HC-CH 2 0H ....- H-(0-CH2-CH2)n-0-CH -CH2 0H
Glycolaldehyde

Figure 5.12 Metabolic pathways for the biodegradation of polyethylene glycols (PEGs). Both
mechanisms involve formation of an olefinic group in the terminal glycol unit which then
hydrates to introduce OH at the ether-linked carbon to yield an unstable hemiacetal. The
mechanisms differ in the route to unsaturation of the terminal glycol which involves either (a)
dehydration (non-oxidative mechanism) or (b) dehydrogenation (oxidative mechanism).

(Figure 5.12b). The terminal HO group of the PEG is unaffected by this

mechanism and its retention would lead to production of glycolaldehyde
during spontaneous hydrolysis of the hemiacetal, although there is no
direct evidence for this. 123 A Flavobacterium sp. growing under aerobic
conditions, also employed an intracellular dehydrogenase enzyme, but in
this case acting on terminally oxidized PEG-carboxylates (see below) via
an unknown mechanism to produce glyoxylate (OHC-COOH) as the
putative reaction prodUCt. 1l2 ,124
In the environment and in laboratory cultures, PEGs are frequently
oxidized by microbial dehydrogenase enzymes which convert a terminal
C-OH group to COOH via CHO to yield the so-called 'PEG carboxylates'.
In several instances it appears that oxidation of the terminal hydroxyl of
the PEG to carboxyl is a prerequisite to exo-cleavage of the glycol
unit. 113,117,125,126
Biodegradation of polypropylene glycol (PPG) has been shown to occur
and, with a single notable exception,99 appears to be mediated by a
separate system from those described for PEG. 113 ,127,128 For example,
PPG was degraded to 80% in 8 days by a Corynebacterium species which
could not utilize PEGS. 129 Further studies involving this isolate acting on
106 PRESERVATION OF SURFACTANT FORMULATIONS

commercial dipropylene glycol as a model compound, were complicated by

the existence of several positional and optical isomers arising from
variation in the position of the methyl groupS.130 Nevertheless the results
indicated that isomers containing secondary alcohols were oxidized
preferentially compared with primary alcohols, that these particular PPG-
degrading enzymes were not extracellular and that the mechanisms for
PPG- and PEG-degradation were probably similar, i.e. oxidation of the
terminal alcoholic group prior to cleavage of an exo-ether bond. The
Corynebacterium also grew on PEGIPPGIPEG block copolymers but only
those with an excess of PPG. Similarly Cain 131 reports that PEG degraders
will grow on PEG in block copolymers as long as the PEG:PPG ratio is
high (at least 3:1 and preferably 5:1 or 7:1).

5.3.6 Fatty acid alkanolamides and ethoxylated fatty amines

Fatty acid alkanolamides such as fatty acid mono ethanol amide (the
un sulphated form of the structure in shown Figure 5.8a), diethanolamide
and monoisopropanolamide, are known to undergo extensive and rapid
biodegradation in mixed environmental or sewage cultures.58.132.133
Recently, studies with closed bottle tests to assess oxygen uptake as a
measure of biodegradation, have been used to propose hydrophile/
hydrophobe separation mechanisms for primary biodegradation of both
ethoxylated fatty amines and fatty alkanolamides. 134 In the proposed
mechanisms, fatty ami des undergo amide bond hydrolysis to liberate the
free fatty acid which is then rapidly oxidized. For the ethoxylated fatty
amines, the speculative mechanism is that C-N bond cleavage yields a long
chain alkanal which is oxidized to fatty acid before further catabolism.
Although these mechanisms are plausible, the data do not preclude
contributions from, for example, m/j3-oxidation. More work with pure
cultures is needed to establish with certainty the mechanisms of bio-
degradation of these fatty amine and amide surfactants, and indeed of the
related anionic surfactants as well (sections 5.2.8 and 5.2.9).

5.4 Biodegradation of cationic surfactants

Cationic surfactants particularly the quaternary ammonium chlorides

(QAC, Figure 5.13), possess potent germicidal properties and they are
used in a variety of biocides, sanitizers and disinfectants. As a consequence,
these compounds might be expected to be extremely recalcitrant to
microbial degradation, and indeed there is plenty of evidence to show that
biodegradation of cationics can be impeded by their toxicity to the
degrading microflora (see e.g. Games et al.B 5 ). However, many environ-
mental bacteria appear to be much less susceptible to cationic surfactant
 BIODEGRADATION OF SURFACTANTS 107

Dodecyltrimethylammonium chloride Dodecyldimethylbenzylammonium chloride

Didodecyldimethylammonium chloride Dodecyl pyridinium chloride

MMM
tJ> CI-

Figure 5.13 Examples of some common quarternary ammonium chloride cationic surfactants.

toxicity than are the target pathogenic bacteria such as Streptococcus. 136
There is no doubt that, given the right conditions, these compounds are
metabolized by microorganisms but an extensive review 137 has revealed a
confusing complexity of data with, for example, reported biodegradabilities
ranging between 0 and 100%. Although such factors as the test system used
and methods of analysis will contribute to this variability, two features
stand out as major determinants of the environmental fate of cationics, viz.
adsorption and acclimation of microorganisms.

5.4.1 Adsorption
The utility of cationics as fabric softeners and hair conditioners depends
upon their adsorptive capacity to surfaces. QACs are strongly sorbed to a
wide range of materials and form complexes with anionic substances.
Because both toxicity and biodegradation rates are concentration-depend-
ent, and biodegradation and adsorption reduce solution concentrations,
the interactions between biodegradation and adsorption are likely to be
very significant for cationics.138 Depletion of cationics by adsorption may
be commensurate with losses from true biodegradation and it is important
to distinguish their relative contributions to the apparent 'primary bio-
degradation' as measured by degrees in solution concentration. 5 More-
over adsorption of cationics may itself affect biodegradation rates either
positively or negatively. For example, if solution concentrations exceed
bactericidal toxicity thresholds, adsorption may alleviate toxication by
removal of surfactant from solution. Indeed it is well established that
cationic surfactants which are recalcitrant at high concentrations (in the
order of 10 1_102 ppm) are readily degraded at lower concentrations (see
e.g. Dean-Raymond and Alexander,139 Gerike et al. 140 ). There is also a
general failure to observe significant biodegradation of QACs in low
108 PRESERVATION OF SURFACTANT FORMULATIONS

biomass systems in which detoxication by adsorption to biomass is

necessarily minimal. On the other hand, at low concentrations of QAC,
biodegradation rates increase linearly with concentration 137 so that if
biodegradation occurs predominantly in solution, then adsorption reduces
bioavailability of surfactant and lowers biodegradation rates. Evidently the
extent of adsorption of cationics and the magnitude and direction of its
effects on biodegradation need to be assessed for individual systems. This
is an important point to appreciate in the present context because in
surfactant-product formulation the nature and extent of surfaces available
for adsorption and the concentration of cationics will usually be very
different from those found in wastewater treatment systems and receiving
waters, to which previously published work applies.

5.4.2 Acclimation
Acclimation (or 'adaptation') is defined operationally as an increase in the
biodegradation rate of a chemical as a result of prior exposure of the
bacterial population to the compounds. Several factors may contribute to
this phenomenon 14 including induction of enzymes, genetic changes
(mutations) producing new metabolic capabilities, and, most commonly,
growth of that part of the microbial population competent in the
biodegradation process. Challenging unexposed microbial populations
with high concentrations of toxic QACs is likely to decimate the bacterial
population beyond the point where it can recover and accomplish
biodegradation; many authors have observed the recalcitrance of QACs at
high concentrations. 137 ,139,140 However at lower doses, a higher survival
rate of potential degraders will allow them to function, reduce the toxic
threat and increase the numbers of competent bacterial cells through
growth. Subsequent additions at the same concentration will be dealt with
even more effectively, and additions at higher concentration may also be
accommodated. In this way the dose level may be progressively raised, and
the microbial population declared to be 'acclimated' to the surfactants.
Thus for example, when dodecyltrimethylammonium chloride was dosed
at 0.1 ppm into either a continuous flow river microcosm138 or into a batch
model lake ecosystem 141 over a period of about 2 weeks, there were very
significant increases in the rate of biodegradation of surfactant. Similarly
dodecyldimethylbenzylammonium chloride disappeared in river water
tests within a few days at 5 ppm but there was little degradation at 30 ppm.
Moreover, repeated addition in increasing doses, with each dose delayed
until the preceding one had degraded, enabled high concentrations to be
degraded easily.142 The effects of acclimation can be expected to be most
profound for alkylpyridinium chloride and dialkyldimethyl- and
alkyldimethylbenzyl-ammonium chlorides which are generally more resist-
ant than the alkyl trimethyl compounds. 137
 BIODEGRADATION OF SURFACTANTS 109

5.4.3 Biodegradability and metabolic pathways

Several studies have shown that rates of primary biodegradation of QACs
depend largely on the balance between long chain alkyl and methyl groups
attached to nitrogen. 5 ,136,139,142-144 Thus QACs with a single long alkyl
chain (monoalkyltrimethylammonium chloride) undergo primary bio-
degradation faster than the dialkyldimethyl analogues. Substitution of a
methyl group with benzyl in the latter type slightly retards biodegradation,
and the alkylpyridinium chlorides in which nitrogen and three of its bonds
are actually part of an aromatic ring are slower still. The most resistant
group are the trialkylmonomethyl compounds.
The length of the alkyl chains also influences primary biodegradation
rates, which increase with increasing chain length up to about C 1{}-12 but
then decrease for higher homologues. This pattern contrasts markedly with
the anionics for which the distance principle (longer chains enabling ever
faster biodegradation, see section 5.2.9) applies. The decrease in bio-
degradability at longer chain lengths is probably attributable to the
bacteriostaticlbactericidal properties of cationics which increase with
increasing chain length. Thus as chain length is increased to around C 1{}-12
toxic effects begin to outweigh the distance principle. These observations
imply operation of an m/ft-oxidation of the long alkyl chain, thus supporting
more direct evidence obtained using an isolated Xanthomonas species. 139
This organism degraded decyltrimethylammonium chloride with the
formation of 9-carboxynonyltrimethylammonium ion (the product of 00-
oxidation of the parent surfactant) and 7-carboxyheptyltrimethylammonium
ion (the product of a first round of ft-oxidation). There was also evidence
for the formation of 5-carboxypentyl-, 3-carboxypropyl- and carboxy-
methyltrimethyl ammonium ions which correspond to further rounds of
acetyl-group removal by ft-oxidation. The presence of these shorter
compounds in small amounts is consistent with the slowing of ft-oxidation
as the site of oxidation approaches the hydrophilic group, as seen in other
surfactants.
Other metabolic pathways must also contribute because in acclimated
sewage treatment units all the carbon in the long alkyl chains and in the
methyl groups in dialkyldimethylammonium chloride were equally access-
ible to microorganisms. 145 Intermediates that were formed, although
unidentified, were short-lived and converted to CO 2 , Demethylations
clearly occur and Cain 4 has accumulated several pieces of circumstantial
evidence that support the possibility, but whether they precede or follow
m/ft-oxidation is unknown.
110 PRESERVATION OF SURFACTANT FORMULATIONS

5.5 Relevance to biodeterioration

Almost all the pathways described in the foregoing sections relate to

studies with bacteria from natural (e.g. rivers, lakes) or manmade
(e.g. wastewater treatment plants) environments. Chemical and physical
conditions in these environments are likely to be quite different from those
present in a surfactant-containing formulation in terms of, for example,
physical form and properties (viscosity, fluidity), concentrations of
surfactant and other organic and inorganic components, and the microflora
with which the sample may come into contact. So how valid is it to
extrapolate from biodegradation of surfactants in environmental situations
to biodeterioration in surfactant formulations?
First, members of the genus Pseudomonas pervade the ranks of those
known surfactant degraders which have been studied mechanistically.
Pseudomonas species are also extremely versatile and ubiquitously
distributed in water supplies, in the air and on solid surfaces. They are thus
likely contaminants in surfactant preparations, and indeed have been
specifically identified in documented cases. 146 ,147
Secondly, although high concentrations of surfactant may affect microbial
cell physiology, they are unlikely to divert metabolic pathways in
competent organisms.
Thirdly, in most pathways, the surfactant undergoes an initial biochemical
step(s) which destroys its amphipathic properties (primary biodegradation),
followed by a series of catabolic steps which further degrade the molecule
to components which are normally present in the cells and which serve as
sources of carbon and energy for growth (ultimate biodegradation).
Although these latter stages involving growth and replication of the cells
may be influenced by environmental conditions such as concentrations of
surfactant and of other nutrients (e.g. nitrogen, phosphorus), the primary
biodegradation step is less likely to be affected. Thus notwithstanding
possible differences in ultimate biodegradation, the primary steps of
biodegradation in the environment and of biodeterioration in the formula-
tion, are likely to be comparable.
Finally the organisms so far studied as biodegraders of surfactants
(which include members of the genera Arthrobacter, Achromobacter,
Alcaligenes, Bacillus, Citrobacter, Corynebacterium, Flavobacterium,
Klebsiella, Micrococcus, Mycobacterium, Nocardia, Pseudomonas,
Sphaerotilus, Streptococcus and Vibrio,4 have been isolated by enrichment
culture on relatively high concentrations of surfactants (typically around
0.1-1%). Such concentrations are necessary to provide sufficient growth
and they create a selection pressure for those organisms that cannot only
withstand the lytic effects of surfactants on cells but can also utilize them as
nutrients. These are characteristics that might be expected of microbial
 BIODEGRADATION OF SURFACTANTS 111

contaminants in surfactant formulations. Moreover, the experimental

studies so far carried out on the mechanisms of biodegradation and the
associated enzymology have required work with high culture densities of
bacterial cells which in turn can only be achieved by growth in high
concentrations of surfactants. As Cain llO has pointed out, this approach
may be criticized as having little relevance to true environmental
conditions where typical concentrations are in the ppm range (i.e.
-0.0001 %), but in the present context, it is fortuitously appropriate
because it approaches the high concentrations of surfactant usually
incorporated in formulations.

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114 PRESERVATION OF SURFACTANT FORMULATIONS

67. Willetts, A.l. and Cain, R.B. (1972) Microbial metabolism of alkylbenzene sulfonates.
Bacterial metabolism of undecylbenzene-p-sulphonate and dodecylbenzene-p-
sulphonate. Biochem. J., 129, 389-402.
68. Locher, H.H., Leisinger, T. and Cook, A.M. (1989) Degradation ofp-toluenesulphonic
acid via side chain oxidation, desulphonation and meta ring cleavage in Pseudomonas
(Comamonas) testosteroni T-2. J. Gen. Microbiol., 135, 1969-1978.
69. Locher, H.H., Leisinger, T. and Cook, A.M. (1991) 4-Sulphobenzoate 3,4-
dioxygenase. Biochem. J., 274, 833-842.
70. Thurnheer, T., Zurrer, D., Hoglinger, O. et al. (1990) Initial steps in the degradation of
benzene sulfonic acid, 4-toluene sulfonic acid, and orthanilic acid in Alcaligenes sp.
strain 0-1. Biodegradation, 1, 55-64.
71. Cook, A.M. and Leisinger, T. (1991) Desulfonation of aromatic compounds. In
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72. Hopper, D.l. (1991) Aspects of the aerobic degradation of aromatics by micro-
organisms. In Biodegradation: Natural and Synthetic Materials, Betts, W.B. (ed.),
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73. Smith, M.R. (1993) The physiology of aromatic hydrocarbon degrading bacteria. In
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74. Kertesz, M.A., Kolbener, P., Stockinger, H. et al. (1994) Desulfonation of linear
alkylbenzenesulfonate surfactants and related compounds by bacteria. Appl. Environ.
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75. Swanwick, l.D., Bruce, A.M. and Vandyke, K.G. (1968) Inhibition of sludge digestion
by synthetic detergents. Water Pollution Control, 67, 91-99.
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Water Pollution Control, 69, 675-683.
77. McEvoy, l. and Giger, W. (1986) Determination of linear alkylbenzenesulfonates in
sewage-sludge by high-resolution gas-chromatography mass-spectrometry. Environ. Sci.
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78. Kolbel, H., Kurzendorfer, P. and Zahiruddin, M. (1976) Constitution and properties of
surfactants. IV. Influence of structure on the biodegradation of anionic surfactants.
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79. Sawyer, C.N., Bogan, R.H. and Simpson, l.R. (1956) Biochemical behaviour of
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80. Ryckman, D.W. and Sawyer, C.N. (1957) Chemical structure and biological oxidiz-
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81. Ikeda, K., Yamada, H. and Tanaka, S. (1963) Bacterial degradation of taurine. J.
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82. Kondo, H., Anada, H., Ohsawa, K. et al. (1971) Formation of sulfoacetaldehyde from
taurine in bacterial extracts. J. Biochem. (Tokyo), 69, 621-623.
83. Kondo, H., Kagotani, K., Oshima, M. et al. (1973) Purification and some properties of
taurine dehydrogenase from a bacterium. J. Biochem. (Tokyo), 73, 1268-1278.
84. Kondo, H. and Ishimoto, M. (1972) Enzymatic formation of sulfite and acetate from
sulfoacetaldehyde, a degradation product of taurine. J. Biochem. (Tokyo), 72, 487-
489.
85. Kondo, H. and Ishimoto, M. (1975) Purification and properties of sulfoacetaldehyde
sulfolyase, a thiamine pyrophosphate-dependent enzyme forming sulfite and acetate.
J. Biochem. (Tokyo), 78, 317-325.
86. Kondo, H. and Ishimoto, M. (1974) Requirement for thiamine pyrophosphate and
magnesium for sulfoacetaldehyde sulfo-Iyase activity. J. Biochem. (Tokyo), 76, 229-
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87. Shimamoto, G. and Berk, R.S. (1979) Catabolism of taurine in Pseudomonas
aeruginosa. Biochim. Biophys. Acta, 569, 287-292.
88. Shimamoto, G. and Berk, R.S. (1980) Taurine catabolism. II. Biochemical and genetic
evidence for sulfoacetaldehyde sulfolyase involvement. Biochim. Biophys. Acta, 632,
121-130.
89. Toyama, S., Miyasato, K., Yasuda, M. et al. (1973) Occurrence of taurine-pyruvate
aminotransferase in bacterial extract. Agric. Bioi. Chern., 37, 2939-2941.
 BIODEGRADATION OF SURFACTANTS 115

90. Toyama, S., Misono, H. and Soda, K. (1978) Properties of taurine: a-ketoglutarate
aminotransferase in Achromobacter superficialis. Inactivation and reactivation of
enzyme. Biochim. Biophys. Acta, 523, 75-81.
91. Patterson, S.J., Scott, C.C. and Tucker, K.B.E. (1970) Nonionic detergent degrada-
tion: III. Initial mechanism of the degradation. 1. Am. Oil Chern. Soc., 47, 37-41.
92. Tobin, R.S., Onuska, F.I., Brownlee, B.G. et al. (1976) The application of an ether
cleavage technique to a study of the biodegradation of a linear alcohol ethoxylate
nonionic surfactant. Water Res., 10, 529-535.
93. Ichikawa, Y., Kitamoto, Y. and Hosoi, N. (1978) Degradation of polyethylene glycol
dodecyl ethers by a Pseudomonad isolated from activated sludge. 1. Ferment. Technol.,
56, 403-409.
94. Kravetz, L., Chung, H., Guin, K.F. etal. (1982) Ultimate biodegradation of an alcohol
ethoxylate and a nonylphenol ethoxylate under realistic conditions. Part 1. Household
and Personal Products Industries, March 1982 and April 1982, 46-72 and 62-70.
95. Vashon, R.D. and Schwab, B.S. (1982) Mineralization of linear alcohol ethoxylates and
linear alcohol ethoxy sulfates at trace concentrations in estuarine water. Environ. Sci.
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96. Steber, J. and Wierich, P. (1985) Metabolites and biodegradation pathways of fatty
alcohol ethoxylates in microbial biocenoses of sewage treatment plants. Appl. Environ.
Microbiol., 49, 530-537.
97. Watson, G.K. and Jones, N. (1979) The microbial degradation of nonionic surfactants.
Soc. Gen. Microbiol. Quart., 6, 78-79.
98. Nooi, J.R., Testa, M.C. and Willemse, S. (1970) Biodegradation mechanisms of fatty
alcohol nonionics. Experiments with some 14C-labelled stearyl alcohollEO condensates.
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99. Grant, M. and Payne, W.J. (1983) Anaerobic growth of Alcaligenes faecalis var.
denitrificans at the expense of ether glycols and non-ionic detergents. Biotechnol.
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100. Wickbold, R. (1966) Analysis for non ionic surfactants in water and wastewater. Yom
Wasser, 33, 229-241.
101. Schoberl, P. and Bock, K.J. (1980) Surfactant degradation and its metabolites. Tenside
Surfact. Deterg., 17,262-266.
102. Schoberl, P. (1981) Comparative investigations on the microbial metabolism of a
nonylphenol and an oxoalcohol ethoxylate. Tenside, 18, 64-72.
103. Osburn, Q.W. and Benedict, J.H. (1966) Polyethoxylated alkyl phenols: relationship of
structure to biodegradation mechanism. 1. Am. Oil Chern. Soc., 43, 141-146.
104. Giger, W., Brunnen, P.H. and Schaffner, C. (1984) 4-Nonylphenol in sewage sludge:
accumulation of toxic metabolites from nonionic surfactants. Science, 225, 623--{i25.
105. Ahel, M., McEvoy, J. and Giger, W. (1993) Bioaccumulation of the lipophilic
metabolites of non ionic surfactants in freshwater organisms. Environ. Pollution, 79,
243-248.
106. Rudling, L. (1972) Biodegradability of non ionic surfactants: a progress report. Report
B134 of Swedish Water and Air Pollution Research Laboratory, Stockholm.
107. Stephanou, E. and Giger, W. (1982) Persistent organic chemicals in sewage effluents. 2.
Quantitative determination of nonylphenol ethoxylates by glass capillary gas chromato-
graphy. Environ. Sci. Technol., 16, 800-805.
108. Maki, H., Masuda, N., Fujiwara, Y. et al. (1994) Degradation of alkylphenol
ethoxylates by Pseudomonas sp. strain TR01. Appl. Environ. Microbiol., 60, 2265-
2271.
109. Baggi, G., Beretta, L., Galli, E. et al. (1978) Biodegradation of alkylphenol ethoxylates.
In The Oil Industry and Microbial Ecosystems, Chester, K.W.A. and Somerville, H.J.
(eds), Heyden, London, pp. 129-136.
110. Cain, R.B. (1994) Biodegradation of detergents. Curro Opinion Biotechnol., 5, 266-274.
111. Cox, D.P. (1978) The biodegradation of polyethylene glycols. Adv. Appl. Microbiol.,
23, 173-194.
112. Kawai, F. (1987) The biochemistry of degradation of polyethers. CRC Crit. Rev.
Biotechnol., 6, 273-307.
113. Watson, G.K. and Jones, N. (1977) The biodegradation of polyethylene glycols by
sewage bacteria. Water Res., 11, 95-100.
116 PRESERVATION OF SURFACTANT FORMULATIONS

114. lenkins, L.D.L. and Cook, K.A. (1979) Microbial degradation of polyethylene glycols.
J. Appl. Bacteriol., 47, 75-85.
1l5. Wagener, S. and Schink, B. (1988) Fermentative degradation of nonionic surfactants
and polyethylene glycol by enrichment cultures and by pure cultures of homoacetogenic
and propionate-forming bacteria. Appl. Environ. Microbiol., 54, 561-565.
116. Haines, 1.R. and Alexander, M. (1975) Microbial degradation of polyethylene glycols.
Appl. Microbiol., 29, 621-{)25.
117. Obradors, N. and Aguilar, 1. (1991) Efficient biodegradation of high-molecular-weight
polyethylene glycols by pure cultures of Pseudomonas stutzeri. Appl. Environ.
Microbiol., 57, 2383--2388.
1l8. Schink, B. and Stieb, M. (1983) Fermentative degradation of polyethylene glycol by a
strictly anaerobic, Gram negative, non-spore-forming bacterium, Pelobacter venetian us
sp. nov. Appl. Environ. Microbiol., 45, 1905-1913.
119. Dwyer, D.F. and Tiedje, 1.M. (1986) Metabolism of polyethylene glycol by two
anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp. Appl. Environ.
Microbiol., 52, 852-856.
120. Strass, A. and Schink, B. (1986) Fermentation of polyethylene glycol via acetaldehyde
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121. Frings, 1., Schramm, E. and Schink, B. (1992) Enzymes involved in anaerobic
polyethylene glycol degradation by Pelobacter venetianus and Bacteroides strain PG 1.
Appl. Environ. Microbiol., 58, 2164-2167.
122. Pearce, B.A. and Heydeman, M.T. (1980) Metabolism of di(ethylene glycol) [2-(2'-
hydroxyethoxy)ethanol] and other short poly(ethylene glycol)s by Gram-negative
bacteria. J. Gen. Microbiol., 118, 21-27.
123. Thelu, 1., Medina, L. and Pelmont, 1. (1980) Oxidation of poly(oxethylene) oligomers
by an inducible enzyme from Pseudomonas P400. FEMS Microbiol. Lett., 8, 187-
190.
124. Kawai, F. and Yamanaka, H. (1986) Biodegradation of polyethylene glycol by symbiotic
mixed culture (obligate mutualism). Archiv. Microbiol., 146, 125-129.
125. Kawai, F., Kimura, T., Fukaya, T. et al. (1978) Bacterial oxidation of polyethylene
glycol. Appl. Environ. Microbiol., 35, 679-{)84.
126. Kawai, F., Kimura, T., Tani, Y. et al. (1978) Purification and characterization of
polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene
glycol. Appl. Environ. Microbiol., 40, 701-705.
127. Kawai, F., Fukaya, M., Tani, Y. et al. (1977) Identification of polyethylene glycols
(PEGs )-assimilable bacteria and culture characteristics of PEG 6000 degradation in a
mixed culture. J. Ferment. Technol., 55, 429-437.
128. Ogata, K., Kawai, F., Fukaya, M. et al. (1975) Isolation of polyethylene glycol-
assimilable bacteria. J. Ferment. Technol., 53, 557-761.
129. Kawai, F., Hanada, K., Tani, Y. et al. (1977) Bacterial degradation of water-insoluble
polymer (polypropylene glycol). J. Ferment. Technol., 55, 89-96.
130. Kawai, F., Okamoto, T. and Suzuki, T. (1985) Aerobic degradation of polypropylene
glycol by Corynebacterium sp. J. Ferment. Technol., 63, 239-244.
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Pollution, Society of Microbiology Symposium 48, Fry, 1.e., Gadd, G.M., Herbert,
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135. Games, L.M., King, J.E. and Larson, R.l. (1982) Fate and distribution of a quaternary
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cationiques. Water Res., 11,833--841.
 BIODEGRADATION OF SURFACTANTS 117

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6 Preservation of agrochemicais
D.A. KNOWLES

6.1 Introduction

Agrochemical products have been used widely for many years to increase
the yield and improve the quality of food and fibre crops all over the world.
The agrochemical industry has become a major business producing
products with a total world sales value estimated in 1993 at about US$25
billion, and it plays an important part in the economies of most countries.
The agrochemical business represents a significant opportunity for surfact-
ants and other essential formulation additives and adjuvants in spray
applications. Although the agrochemical industry has reached maturity in
North America, Western Europe and Japan, there is still considerable
scope for new more environmentally friendly formulations. Developing
areas, especially the Asia-Pacific region, will show increasing need for
agrochemical products.
Changes in the population of the world and increasing urbanisation and
industrialisation of communities are placing a great demand on the
efficient use of available land for agriculture. For example, the United
Nations have forecasted that if present trends continue, the population of
the world will increase from about 5 billion now to about 10 billion by the
year 2040, and the fastest rate of growth will be in the less developed areas,
particularly the Asia-Pacific region. 1 There will, therefore, be an
increasing need for agrochemical products as an important input in the
management of food and fibre crops to improve their yield and quality.
The ability to protect growing crops from weeds, pests and diseases has
been known since ancient times in the Old World of the Middle East, Asia
and China. However, the greatest improvements in crop protection
efficiency and productivity in terms of crop yield and quality have occurred
mainly in the West and within the last century. Simple emulsifiable oils and
soaps have been used as agricultural sprays for many years. The modern
era of weed control can be said to have started in the 1940s with the
development of the phenoxy acid herbicides such as 2,4-D acid. Since the
Second World War, particularly in the 1960s, 1970s and 1980s, many new
synthetic pesticides have been introduced to combat a very wide range of
weeds, pests and diseases. A great deal of research and development has
been carried out by all the major agrochemical companies to produce
 PRESERVATION OF AGROCHEMICALS 119

formulations which can be applied easily to crops and which will optimise
the activity of the pesticide. 2
Although the last few decades have seen remarkable development in
new agrochemical active ingredients and formulations, most companies are
now reviewing their product strategies and government regulatory author-
ities are introducing controls and legislation which are leading to the
introduction of safer and more environmentally friendly active ingredients
and formulations. There is also a need to reduce the total amount of active
ingredients applied per hectare. The increasingly high cost of the
development of new products is causing the industry to consolidate by
mergers of companies or joint ventures between companies.
The wide variety of agrochemical formulations which is available
requires a range of different formulation additives to produce safe and
usable products. Two of the most important formulation additives are
surface active agents, or surfactants, and preservatives. Surfactants have
been obtained from natural products by extraction or modification for
thousands of years. Many examples of surfactants are well known, like
soaps for cleaning, greases and tallows for waterproofing and glue, egg
white and natural gums as dispersing and emulsifying agents.
Synthetic surfactants, which have been specially synthesised in order to
obtain surface active effects, represent a relatively modern development
which may be said to have evolved from the 'sulphonated oils' of the last
century. The period between the two World Wars was a very active phase
in the development of sulphated and sulphonated anionic surfactants with
long hydrocarbon chains.
Since the Second World War, the development of surfactants entered a
more specialised phase with the introduction of amphipathic molecules for
specific applications. Non-ionic surfactants became available in which the
hydrophilic part of the molecule was based on condensed chains of
ethylene oxide. A wide range of surfactant properties can be achieved by
varying the ethylene oxide chain length. This development has led to a
better understanding of the colloid and surface chemistry principles
involved in the fundamental functional properties of wetting, dispersing,
emulsification and solubilisation in the formulation of pesticides. As a
result of all this work, it is now possible for surfactant suppliers to prepare
'tailor-made' surfactants to suit particular functions. 3-5
For nearly all formulations the most important formulation additive is
the surfactant in terms of preparation and production. The surfactant often
determines the maximum concentration of the formulation that can be
achieved, the particle or droplet size, long term stability and sometimes
even the biological activity of the formulation.
Preservatives are also an important additive to formulations to prevent
biodegradation during preparation and storage, particularly where the
formulations are aqueous based and contain carbohydrates, or where the
120 PRESERVATION OF SURFACTANT FORMULATIONS

products are exposed to the atmosphere after application, as in the case of

baits and pellets.
This chapter reviews the current state-of-the-art with conventional
agrochemical formulations and the trend towards safer and more convenient
formulations. It also describes the major surfactants used in these systems
and the reasons for their choice. The chapter also reviews the major types
of spoilage microorganisms which are encountered in agrochemical
formulations, the preservatives which are used to prevent microbial
spoilage during preparation, storage and use, and testing protocols for
quality control. Finally regulatory constraints on the use of formulation
additives are discussed.

6.2 Agrochemical formulations

Since the 1940s there has been a rapid development of synthetic organic
chemical pesticides for crop protection and pest control by all the major
international chemical companies. Farmers and growers in all the main
agricultural areas of the world rely substantially upon crop protection
chemicals to help them meet the ever increasing demand for food and
other materials such as natural fibres. The consumer continues to seek
higher quality and more variety of produce. In post-war years, the
chemical industry has endeavoured to satisfy these demands by the
continuous development and introduction of novel crop protection
chemicals into the international marketplace. Today, there is an effective
herbicide, insecticide or fungicide to combat almost every significant
problem faced by the modern farmer and grower.
This development has led to a need for a wide range of product
formulations to accommodate the variety of physical and chemical
properties of the pesticide active ingredients. For example, water-soluble
active ingredients may be marketed as aqueous solutions or powder
formulations, whereas oily liquid active ingredients are sold as hydrocarbon-
based emulsifiable concentrates. Active ingredients which have very low
solubility in either water or hydrocarbon oils, may be formulated as
suspensions, powders or water-dispersible granules. 6
In recent years, pressure from the consumer has highlighted a need for
products and formulations which are safer and more convenient to use and
which will be more effective at much lower application rates. By far the
most important method of application is by spraying, usually with water
but occasionally with oils as the principal carrier. Formulations are also
made for direct application to the soil or for treating seeds before planting,
and for protecting stored crops from various pests and diseases (fungi,
insects or rodents) which in some countries could destroy as much as 30-
40% of the harvest.
 PRESERVATION OF AGROCHEMICALS 121

Pesticidal active ingredients or toxicants encompass a broad range of

chemicals each with its unique chemical and physical properties and mode
of action. The main categories of pesticide are herbicides, insecticides,
fungicides, plant growth regulators, molluscicides and rodenticides. A
great deal of research work has been carried out into understanding the
modes of action and physiological effects of active ingredients and the
influence of formulation type on the biological performance of the
pesticide. 7 The successful use of any active ingredient depends on its
correct formulation into a preparation which can be applied for crop
protection with safety to those applying the material, to animal life and to
the environment in general.
The earliest pesticide formulations were based on simple dusts, powders,
aqueous solutions and mineral oil-in-water emulsions. In the post-war
period, particularly during the period 1970 onwards, there has been a rapid
development of more sophisticated formulations based on the availability
of more powerful surfactants and a much better understanding of the
principles of colloid and surface chemistry.
The main objectives of formulation can be summarised as follows:
To provide the user with a convenient, safe product which will not deteriorate
over a period of time, and to obtain the maximum activity inherent in the active
ingredient.
Usually it is the physicochemical properties of the active ingredient
which are the main indicators of the choice of the specific formulation type,
but other factors which need to be taken into account are:
• biological activity and mode of action
• method of application
• safety in use
• formulation costs
• market preference.
Once these parameters have been determined, proper selection can be
made of the final formulation and the use of inert ingredients including
surfactants, preservatives and other additives.
The most common formulations are still soluble concentrates for water-
soluble chemicals, emulsifiable concentrates for oil-soluble chemicals and
wettable powders and suspension concentrates for insoluble solids.
Granules and seed treatments for direct application have also been
produced for many years. In recent years the number of formulation types
has increased enormously to meet the needs of operator and environmental
safety or to improve the activity and persistence of the active ingredient by
controlled release. An international coding system was, therefore, devised
by GIFAP in 1984 (GIFAP; International Group of National Associations
of Manufacturers of Agrochemical Products, based in Brussels, Belgium).
122 PRESERVATION OF SURFACTANT FORMULATIONS

Table 6.1 Major types of pesticide formulations

Formulation GIFAP Code

Granules GR
Wettable powder WP
Solution concentrate SL
Emulsifiable concentrate EC
Suspension concentrate SC
Seed treatments DS, WS, LS, FS
OfW emulsions EW
Suspoemulsions SE
Microemulsions ME
Water-dispersible granules WG
Microcapsules CS

The major types of formulation with their international codes are shown in
Table 6.1.
The most important formulations are those which are made for dilution
into water in a spray tank. In these cases the choice of formulation
additives is very important to ensure that the product mixes and dilutes
easily. Sometimes products may be mixed together in the spray tank or
may be mixed with spray adjuvants to enhance biological activity. Products
such as granules or seed treatments are usually applied undiluted to the soil
or to the seed respectively. A few products are formulated to be diluted
and sprayed in oils, and there are many minor formulations such as baits,
pellets, smokes and aerosols for special purposes.

6.2.1 Conventional formulations

6.2.1.1 Wettable powders (WP). Wettable powder formulations of

pesticides have been known for many years and are made from solid active
ingredients which are suitable for fine grinding through a hammer or pin-
type mill or a fluid energy microniser. The powders contain dry surfactants
as powder wetting and dispersing agents and inert carriers or fillers.
Wettable powders frequently contain more than 50% active ingredient
and the upper limit is usually determined by the amount of inert material
such as silica required to prevent the active ingredient particles fusing
together during processing in the dry grinding mills. This is influenced by
the melting point of the active ingredient, but an inert filler is also needed
to prevent the formulated product from caking or aggregating during
storage.
Wettable powders have a high proportion of particles less than 5 !lm and
all the particles should pass through a 44 !lm screen. Ideally, the amount of
surfactants should be sufficient to allow the spray droplets to wet and
 PRESERVATION OF AGROCHEMICALS 123

spread over the target surface, but the particles should not be easily
washed off by rain.
Powder formulations contain a wetting agent to lower the interfacial
tension between the solid particles and water and ensure that the powder
wets and mixes with water in the spray tank easily. A dispersing agent is
also necessary to prevent the particles in the spray tank from flocculating
or aggregating together. This ensures that the particles remain suspended
in water during the spraying operation.
The types of wetting agents commonly used are:
• sodium dodecylbenzene sulphonate
• sodium lauryl sulphate
• dioctyl sulphosuccinate
• fatty alcohol ethoxylates
• nonyl phenol ethoxylates.
Examples of dispersing agents are:
• sodium lignosulphonates
• naphthalene sulphonic acid-formaldehyde condensates.
A typical wettable powder formulation is shown below:
% By weight
Active ingredient 25-80
Wetting agent 1-3
Dispersing agent 2-5
Inert filler/carrier to 100

Wettable powders can also be made from liquid pesticides by using

absorbent fillers such as diatomaceous earth or high surface area synthetic
silica. However, in this case the active ingredient concentration is usually
limited to 40%.
Many pesticides, especially herbicides and fungicides are formulated as
wettable powders. However, due to their 'low tech.' image arising from
their dustiness, which creates hazards on handling, they are now being
superseded by suspension concentrates or water-dispersible granules.

6.2.1.2 Solution concentrates (SL). The simplest of all formulations to

make is the solution concentrate, an aqueous solution of the active
ingredient which merely requires dilution in the spray tank. The number of
pesticides which can be formulated in this way is limited by solubility and
hydrolytic stability. Some solution concentrate formulations contain a
surfactant, usually to assist wetting onto the leaf surface. These wetting
agents are generally of the nonionic type, such as nonylphenol condensed
with 8-9 mols of ethylene oxide.
Solution concentrate formulations are usually very stable and, therefore,
124 PRESERVATION OF SURFACTANT FORMULATIONS

present few storage problems. Some problems do occur occasionally, such

as precipitation during dilution and corrosion of metal containers or spray
applicators. A typical solution concentrate formulation is shown below:

% By weight
Active ingredient 20--50
Wetting agent 3-10
Antifreeze 5-10
Water }
Water-miscible solvent to 100

Nonylphenol or tallow amine ethoxylates are often used as tank mix

wetters for solution concentrate formulations. Alternatively, the wetting
agent may be built into the formulation to ensure that the correct rate of
wetting agent is applied to optimise biological activity. This is often the
case, for example, with paraquat and glyph os ate formulations. A consider-
able amount of work is being carried out on new surfactant wetting agents
for glyph os ate formulations. s In some cases preservatives may be
necessary to prevent mould growth or bacterial spoilage during long term
storage.

6.2.1.3 Emulsifiable concentrates (EC). Emulsifiable concentrate

formulations have been very popular for many years and represent the
biggest volume of all pesticide formulations in terms of consumption.
Emulsifiable concentrates are made from oily active ingredients or from
low melting, waxy solid active ingredients which are soluble in non-polar
hydrocarbon solvents, such as xylene, C-9-C-10 solvents, solvent naphtha,
odourless kerosene or other proprietory hydrocarbon solvents. Surfactant
emulsifiers are added to these formulations to ensure spontaneous
emulsification with good emulsion stability properties in the spray tank.
Careful selection of a 'balanced pair' emulsifier blend is frequently
necessary to ensure emulsion dilution stability is maintained over widely
differing climatic conditions and degrees of water hardness. Emulsion
droplets of 0.1-5 !-tm are produced.
The formulation of emulsifiable concentrates has been greatly facilitated
by the commercial development over the last 20 years of nonionic
emulsifying agents in which the hydrophilic portion of the molecule
consists of a polyethylene oxide chain. The nonionic surfactant which is
commonly used is a nonyl phenol hydrophobic chain condensed with 12 or
more moles of ethylene oxide. The other component of the balanced pair is
generally an anionic surfactant such as the oil-soluble calcium salt of
dodecylbenzene sulphonic acid.
The total concentration of the emulsifier blend is usually 5-10% of the
formulation. There are no definite rules to determine the ratio of anionic
to nonionic surfactant in the mixed emulsifiers, but guidance can be
 PRESERVATION OF AGROCHEMICALS 125

obtained from the HLB system. HLB stands for hydrophile-lipophile

balance and the higher the HLB the more hydrophilic (water soluble) is the
surfactant. The HLB range 8-18 will normally provide good oil-in-water
emulsions. The optimum ratio of anionic and nonionic surfactants is
determined experimentally to give spontaneous emulsification in water,
and to give a stable emulsion with very little creaming and no coales-
cence.
Emulsifiable concentrates are limited in the number of active ingredients
for which they are suitable. Many pesticides are not soluble enough to be
supplied economically in this form. However, it may be possible to boost
the solubility of the active ingredient by the addition of a more polar
solvent without increasing the risk of crystallisation in the spray tank.
A typical emulsifiable concentrate formulation is shown below:
% By weight
Active ingredient 20-70

J
Emulsifier blend 5-10
Solvent
Cosolvent to 100

The presence of solvents and emulsifiers in emulsion concentrate

formulations can sometimes give enhanced biological efficacy compared
with other formulations. Many insecticides, e.g. organophosphorous
compounds and pyrethroids, are oil-soluble active ingredients and are
readily formulated as emulsifiable concentrates, and a few active ingredients
need to be formulated with solvents for optimum biological activity.
Health, safety and environmental pressures on the use of petroleum-
based solvents generally are influencing a move away from these solvent-
based formulations. However, it seems unlikely that solvents can be
replaced entirely for some products, and safer high flash point solvents are
being introduced along with new ideas for packaging to reduce physical
contact between the product and the operator.

6.2.1.4 Suspension concentrates (SC). Suspension concentrate techno-

logy has been widely applied to the formulation of many solid crystalline
pesticides since the late 1960s and early 1970s. Pesticide particles may be
suspended in an oil phase, but it is much more usual for suspension
concentrates to be dispersions in water. Considerable attention has been
given in recent years to the production of aqueous suspension concentrates
by fine grinding processes such as bead milling. The use of surfactants as
wetting and dispersing agents has also led to a great deal of research on the
colloidal and surface chemistry aspects of dispersion and stabilisation of
solid/liquid dispersions. 9
Suspension concentrate formulations offer many advantages such as ease
of handling and application, safety to the operator and environment and
126 PRESERVATION OF SURFAC'IANT FORMULATIONS

economy. They also enable water-soluble wetting agents to be built into

the formulation to give enhanced biological activity.
In most cases, suspension concentrates are made by dispersing the active
ingredient powder in an aqueous solution of a wetting and/or dispersing
agent, followed by a wet grinding process in a bead mill to give a particle
size distribution in the range 0.1-5 f-lm. The wetting/dispersing agent aids
the wetting of the powder into water and the breaking of aggregates,
agglomerates and single crystals into smaller particles. In addition, the
surfactant which becomes adsorbed onto the freshly formed particle
surface should prevent reaggregation of the small particles and should
ensure colloidal stability of the dispersion. Typical wetting/dispersing
agents used in suspension concentrate formulations are:
• lignosulphonates
• naphthalene sulphonic acid-formaldehyde condensates
• nonyl phenol ethoxylates
• fatty alcohol ethoxylates
• tristyrylphenol ethoxylate phosphate ester
• ethylene oxide/propylene oxide copolymers
Also in development are polymeric surfactants which show considerable
potential for the stabilisation of suspension concentrates for long term
storage. lO A typical suspension concentrate formulation is shown below:

% By weight
Active ingredient 20-50
Wetting/dispersing agent 2-5
Propylene glycol antifreeze 5-10
Anti-settling agent 0.2-2
Water to 100

The antisettling agent is added to prevent separation of particles during

long term storage. These agents are often cellulose derivatives, natural
gums or other types of polysaccharides, such as xanthum gum, and they are
generally susceptible to microbial attack. For this reason, preservatives are
sometimes added to suspension concentrate formulations to prevent
degradation of the antisettling agent so that long term stability of the
product is not impaired.
Many crystalline solid active ingredients are now available as suspension
concentrates. However, there is increasing pressure, especially in Europe
and the USA, to enforce stringent pack rinsing and disposal regulations,
which may have a serious impact on the future of suspension concentrates
and their packaging.

6.2.1.5 Seed treatments (DS, WS, LS, FS). Although most pesticide
formulations are applied by spraying onto crops or weeds, a significant
 PRESERVATION OF AGROCHEMICALS 127

amount of pesticide products are applied directly onto seeds prior to

planting into the soil. It is estimated that the market value of seed
treatment formulations in 1993 was about US$750 million representing
about 3% of the total market for agrochemical products. Approximately
50% of seed treatment formulations are applied to cereal seeds.
Products for seed treatment fall into four categories:

• powder for dry seed treatment - DS

• water slurriable powder for seed treatment - WS
• non-aqueous solution for seed treatment - LS
• flowable suspension for seed treatment - FS
Of these formulations only water slurriable powders and flowable
suspensions use appreciable amounts of surfactants. The technology for
producing flowable suspensions is similar to that for producing suspension
concentrates, and therefore preservatives are needed to prevent degrada-
tion of the antisettling thickeners. Surfactants used are similar to those
used for suspension concentrate formulations.
Because seed treatments are applied directly to the seed, there is very
little loss of active ingredient. Seed treatments are, therefore, seen as a
very efficient means of targeting pesticide to crops and are regarded as an
environmentally safe way of applying pesticides.

6.2.2 New generation formulations

Over the last few years there has been increasing pressure from
government and regulatory authorities to develop formulations which have
less impact on the environment generallyY The main issues which are
being addressed are:

• safety in manufacture and use

• convenience for the user
• ease of pack disposal or re-use
• reduction of the amount of pesticide applied
• reduction of waste and effluent of all kinds.
The likely trends over the next few years in the development of pesticide
formulations are likely to be:

• To use safer solvents or to eliminate solvents wherever possible and use

aqueous emulsions.
• To replace wettable powders by aqueous suspension concentrates or
water-dispersible granules.
• To develop multi-active ingredient formulations with built-in bio-
enhancing surfactant wetters.
128 PRESERVATION OF SURFACTANT FORMULATIONS

• To control release rate and targeting of pesticides by encapsulation

techniques.
• To develop more effective spray adjuvants to enhance biological activity
and reduce pesticide dosage.
These complex requirements are being met by technical advances in
surfactants and other formulation additives, particularly blends of surfact-
ants, more powerful dispersing agents and a better understanding of the
principles of colloid and surface chemistry and rheology.12 There is also a
need for better spray application systems to reduce pesticide waste, and a
greater understanding of the physiological effects of spray droplets on leaf
surfaces.
It is recognised that some components of formulations are less desirable
than others and that some formulation types exhibit more operator
exposure problems than others. Examples of these are aromatic solvents in
emulsifiable concentrates which are coming under scrutiny due to their
flammability, toxicity both alone and in the formulation, and package
disposal problems.
Wettable powders also cause problems with toxic dust being created
during the loading operation to a spray tank. The ideal product would
seem to be one which is solvent free, gives no operator exposure hazard,
has the maximum biological activity at the lowest dose level and produces
no pack disposal problem. A dry powder or water-dispersible granule in a
water-soluble sachet which can be added directly to a spray tank goes a
long way towards meeting these requirements, and considerable work is
being carried out on this option by all the major agrochemical companies.
However, it will never be possible to formulate all active ingredients this
way and so other options are being evaluated extensively, along with ideas
for packaging and close-coupled spray application systems. Aqueous-based
formulations will be a necessary and safe alternative to water-dispersible
granule formulations and these options include (in addition to suspension
concentrates which have been already discussed):
• suspoemulsions
• emulsions or concentrated emulsions
• micro emulsions
• microencapsulation.
Other possibilities involving specialised packaging will be gels and
effervescent tablets.

6.2.2.1 Water-dispersible granules (WG). Water-dispersible granules,

or dry flowables, as they are sometimes known, are a relatively new type of
formulation and are being developed as safer and more commercially
attractive alternatives to wettable powders and suspension concentrates.
 PRESERVATION OF AGROCHEMICALS 129

They are becoming more popular because of their convenience in

packaging and use, being free-flowing granules which should disperse
quickly when added to water in the spray tank. They therefore represent a
technological improvement on wettable powders and imitate liquids in
their handling characteristics.
Water-dispersible granules are complex formulations because they can
be formulated using various processing techniques, but in each case the
resultant product must redisperse in the spray tank to give the same
particle size distribution as the original powder or suspension from which it
is made. This requires careful choice of both the surfactant additives and
the process of granulation which is usually one of the following
techniques: 13
• pan granulation
• mixing agglomeration
• extrusion granulation
• fluid bed granulation
• spray drying.
Several factors, such as the physicochemical properties of the active
ingredient and additives, need to be considered when deciding upon which
process to use. These factors and the various processing techniques used to
make water-dispersible granules determine the main properties of the final
product in terms of granule shape and size, degree of dustiness and ease of
dispersion into water. The dispersion time in water is a very important
property, and to ensure that no problems occur in the spray tank it is
usually necessary for all the granules to disperse completely within two
minutes at varying degrees of water temperature and hardness.
Water-dispersible granules usually contain a wetting agent and a
dispersing agent in the same way as a wettable powder or a suspension
concentrate. They may also contain a water-soluble salt to act as a
disintegrant in the spray tank. The remainder of the formulation is usually
a water-soluble or a water-dispersible filler. A typical water-dispersible
granule formulation is shown below:
% By weight
Active ingredient 50-90
Wetting agent 1-5
Dispersing agent 5-20
Disintegrating agent 0-15
Soluble or insoluble filler to 100
Wetting and dispersing agents commonly used in water-dispersible
granules are often similar to those used in wettable powder and suspension
concentrate formulations. Because they are essentially dry solids there is
usually no need to add a preservative to these formulations.
130 PRESERVATION OF SURFACTANT FORMULATIONS

6.2.2.2 Suspoemulsions (SE). Mixed formulations are becoming more

popular due to their convenience, because they ensure that the farmer
applies the correct amount of each component pesticide and because they
enable problems of tank mix incompatibility to be overcome. If one active
ingredient is a solid and the other is a liquid, it is necessary to produce a
suspoemulsion formulation, which consists of three phases:
• liquid oil droplets
• solid dispersed particles
• continuous phase, usually water.
Suspoemulsions can, therefore, be considered to be mixtures of
suspension concentrates with oil-in-water (O/W) emulsions with added
surfactants to prevent flocculation and thickeners to prevent separation of
the dispersed phases. Surfactants used as dispersing agents for the solid
phase are similar to those already mentioned for suspension concentrates.
Emulsifiers for the oily liquid phase will be discussed in section 6.2.2.3 on
emulsions (EW). As these formulations are aqueous based and generally
thickened with polysaccharides, it is necessary to add a preservative to
prevent degradation of the thickener. Some problems of heteroflocculation
between the solid particles and the oil droplets can occur and extensive
storage testing of these formulations is necessary. 14

6.2.2.3 O/W Emulsions (EW). Emulsions are now receiving consider-

able attention because of the need to produce stable emulsions in

phase I Ostwald
coa escence ripening
.~ion
~
••
.~
[j
Figure 6.1 OfW emulsion stability problems.
 PRESERVATION OF AGROCHEMICALS 131

suspoemulsion (SE) formulations and also as a means of producing stable

emulsion concentrates (EW) which are water based and therefore reduce
or eliminate organic solvents.
O/W emulsions can have significant advantages over emulsifiable
concentrates (Ee) in terms of cost and safety in manufacture and use.
However, they require careful selection of surfactant emulsifiers to prevent
flocculation, creaming and coalescence problems. These effects are shown
diagrammatically in Figure 6.1. Nonionic surfactants and polymeric
surfactants are now being used to produce stable emulsions. In the case of
nonionic surfactants it is sometimes useful to combine a low and a high
HLB surfactant to give an average HLB of 11-16 for optimum emulsion
stability.
Droplet size is also a good indicator of stability and should be below
2 I-lm. The emulsions are usually thickened with polysaccharides such as
xanthan gum and, therefore, a preservative is added to the formulation.
Sometimes polymers such as polyvinyl alcohol are used as both emulsifier
and thickener.

6.2.2.4 Microemulsions (ME). Microemulsions are thermodynamically

stable transparent emulsions and are stable over a wide temperature range.
They have a very fine droplet size of less than 0.1 I-lm and consist of three
components, namely:
• oily liquid or solid dissolved in solvent
• water
• surfactant/cosurfactant.
These components form a single phase containing relatively large
'swollen micelles' in which the non-aqueous phase of the active ingredient
and solvent are dissolved or solubilised.
In the preparation of microemulsions two different types of surfactant
are needed; one water soluble and one oil soluble. The water-soluble
surfactant is usually anionic or non-ionic with a very high HLB value, and
the hydrophobic part of the molecule should match the oil. The
cosurfactant should be oil soluble and should have a very low HLB value,
such as hexanol. The total concentration of surfactants for a micro emulsion
can be between 10 and 30%, compared with about 5% for an O/W
emulsion (EW).
Microemulsions have relatively low active ingredient concentrations, but
may have enhanced biological activity.

6.2.2.5 Controlled release formulations. The application of controlled

release technology has been slow to reach commercialisation despite
interesting research and development work by the major agrochemical
132 PRESERVATION OF SURFACTANT FORMULATIONS

companies over the last 10-20 years. Controlled release formulations can
have a number of advantages over conventional formulations, as shown
below: they

• reduce mammalian toxicity

• have longer residual biological activity
• control/reduce evaporation of pesticide
• reduce phytotoxicity to crop
• improve compatibility in the spray tank
• reduce fish toxicity
• reduce ground water leaching
• reduce solvent usage in formulation
• reduce pesticide application rate.

Controlled release pesticide formulations can be divided into four main

types:

• coated pesticide granules

• matrix systems containing physically trapped pesticides
• polymer systems containing covalently bound pesticides
• polymer membrane-pesticide reservoir systems, e.g. microencapsula-
tion.

Of the four main types of controlled release formulations, only the

polymer membrane, or microencapsulation, formulations use significant
amounts of surfactants. A well-known method of microencapsulation is by
interfacial polymerisation. In this process the active ingredient, usually a
liquid or low melting waxy solid, is dissolved into an aromatic solvent, such
as C-9 and C-lO solvents used for emulsifiable concentrates. An oil-soluble
monomer such as toluene diisocyanate (TDI) is dissolved in the solvent
mixture. A fine emulsion of the oil phase in water is made by high shear
mixing with an aqueous solution of an emulsifier and a reactive amine,
such as ethylene diamine. An emulsion with droplets of 10-30 flm is
formed, and polymerisation between the isocyanate and the amine occurs
at the oil/water interface giving a polyurea membrane around each droplet.
Alternatively the interfacial polymerisation process may be carried out by
allowing the isocyanate to react with water at the interface to form an
amine in situ, which then reacts with more isocyanate to form a polyurea
membrane. 1s
The rate of release of the active ingredient can be controlled by adjusting
the droplet size, the thickness of the polymer membrane and the degree of
cross linking or porosity of the polymer. This is, therefore, an example of a
diffusion controlled process.
A typical microencapsulated suspension (CS) formulation is shown:
 PRESERVATION OF AGROCHEMICALS 133

% By weight
Active ingredient 10-30
Emulsifier 1-5
Polymer 10-15
Solvent 5-15
Anti-settling agents 1-3
Water to 100
Microcapsule suspensions need to be stabilised with surfactants and
thickeners in the same way as suspension concentrates and emulsions, and
similar additives are used, including preservatives.
A number of microencapsulated products are now on the market,
including selective herbicides to reduce volatility, insecticides to reduce
toxicity and to increase residual activity, and pheromones to maintain the
required vapour concentration over a period of 10--14 days. The benefits of
microencapsulated products over conventional formulations in terms of
bioavailability may be demonstrated graphically as shown in Figure 6.2,
where the optimum level of pesticide availability can be maintained over a
much longer period than with conventional formulations.

6.2.2.6 Built-in-wetter formulations. There is increasing pressure from

regulatory authorities and for marketing reasons to include surfactant
adjuvants in the formulation in order to optimise biological activity and to
reduce the rate of active ingredient usage. In some cases, the regulatory

/Conventional
Formulation

OJ
Ol
m Microencapsulated
Ul
o For~ulation
Cl Optimum Range
...... for Effectiveness
m
....uE
OJ
L:.
U

7 14 21
Time (days)
After Application.
Figure 6.2 Bioavailability of microencapsulated formulation compared with conventional
formulations.
134 PRESERVATION OF SURFACTANT FORMULATIONS

authorities require specific data on the formulation which includes the

biological enhancing wetter.
The potential effects of built-in-wetters to formulations are:
• better foliar wetting and spreading
• better adhesion of the droplets
• reduced droplet size of the spray
• increased drying time and water retention
• increased uptake and translocation in plant.
Non-ionic surfactants are often used as built-in-wetters to give the above
benefits. They can increase the solubility of the active ingredient in the
droplet by micellisation, thus making it easier for the active ingredient to
enter the target. Built-in-wetters are useful for hydrophilic active ingredi-
ents, such as paraquat and glyphosate, to enhance their uptake into the leaf
surface. They may also improve the physical compatibility of different
pesticide formulations in the spray tank mixture.
No universal surfactant wetter exists for all pesticides and it is necessary
to carry out stability tests and biological activity tests with a range of
different surfactant wetters to find the optimum system. However,
surfactants such as nonylphenol ethoxylates, linear fatty alcohol ethoxylates
and fatty amine ethoxylates are often used. The mechanism of action of
surfactant adjuvants in contact with the target organism is not fully
understood, but it seems that lowering the interfacial tension, reducing the
contact angle and increasing the movement of pesticide through the leaf
surface are all important processes. 16

6.3 Surfactants for agrochemicals

Surfactants are essential components for the formulation of most agro-

chemical products. They have several functions the most important of
which are:
• wetting
• dispersing
• emulsifying
• solubilising
• bioenhancement.
Surfactants are able to wet powders into water by lowering the surface
and interfacial tensions so that concentrated premixes can be made. They
also help in the particle dispersion process by adsorbing onto the freshly
formed surface and preventing reaggregation. Surfactants can emulsify oils
into water and in some cases can increase the concentration of active
ingredients by solubilisation of the material in the surfactant micelles.
Surfactants playa major role in the stabilisation of pesticide formulations
 PRESERVATION OF AGROCHEMICALS 135

to impart good shelf life stability. During the spray application process they
enable solid products to wet out and disperse into the spray tank dilution,
and liquid products to emulsify and disperse. Surfactants are also used by
themselves or as components of adjuvants for tank mixing with pesticide
products. A knowledge of the physicochemical properties of surfactants is
essential for the successful design of agrochemical formulations and
adjuvants. 17 Agrochemical formulations usually contain 1-10% of surfact-
ant or a mixture of surfactants. For spray applications surfactants are
sometimes added to the spray tank at levels of 0.01 % to 0.1 % to improve
droplet wetting and adhesion on the foliage. In recent years higher
concentrations of surfactants up to 1-2% are being used to enhance the
biological performance of the pesticide by increasing uptake into the plant
and translocation within the plant. It has been estimated that the world
consumption of surfactants for agrochemical use was about 230 000 t in
1993, representing about 3.3% of the total consumption of surfactants for
all end uses. 18
The simplest surfactant molecule comprises a lipophilic part which
prefers an oil phase, attached to a hydrophilic head group which prefers
water. A common and simple example is sodium dodecyl sulphate;
C 12H 25 S04 - Na+.
Surfactants are classified into the following types:
• anionic: negatively charged hydrophilic headgroup,
• cationic: positively charged hydrophilic headgroup,
• nonionic: uncharged hydrophilic headgroup,
• amphoteric: negatively and positively charged hydrophilic headgroup.
Some common examples of the different types of surfactants used in
agrochemical formulations are shown schematically in Figure 6.3. A wide
range of surfactants is available to enable the formulator to make the best
choice for a particular formulation. Surfactants are used primarily as
wetting agents, emulsifiers and dispersing agents but also have uses as
antifoaming agents and anticaking agents and an increasingly important
use as agents to enhance the biological activity of pesticides and herbicides
by improving capture by and penetration of the biological target. Anionic
and nonionic surfactants are much more commonly used with agrochemical
formulations than cationic and amphoteric surfactants in order to prevent
flocculation problems with other anionic formulation additives. However,
where they are used, cationic surfactants may also exhibit bactericidal
properties. Amphoteric surfactants are rarely used in agrochemical
formulations, but in some cases they can have interesting effects at
different pH values. For agrochemical formulations anionic surfactants
comprise about 50% of the total surfactant usage, whereas for spray
application adjuvants nonionic surfactants comprise about 75% of the total
surfactant usage.
136 PRESERVATION OF SURFACTANT FORMULATIONS

I
I
I
I

Hydrophobic/Lipophilic Chain !: Hydrophilic

Head Group

>-+
I

Anionic C 12 H 25 <- SO ; Na +
CH i
Catiooie C 16 H 33 CH 3 ~' Br-
CH 3: I
I

Nonionic CnH2n+1 ~ > + 0 - (CH 2CH 20) mH

n=8 or 9 I
I
I

-+---
I
I
I

C n H 2n+1 I
0- (CH 2CH 20) mH
n=12-18 :
I
I
I

i /
I

(CH 2CH 2 0) x H
C n H 2n+1 -+-N
!
~
(CH 2CH 20) y H
I
I
I

CH3~ + _
Amphoteric R ~N -CH 2 COO
CH 3----- :

Hydrophobic (Lipophilic) Hydrophilic

Part Part

Figure 6.3 Surfactant classifica Ions and examples.

6.3.1 Conventional surfactants

Wettable powder formulations usually contain a wetting agent such as
sodium lauryl sulphate or a sodium sulphosuccinate derivative:
C 12H 2S S0 4- Na+

CH2COOCnH 2n+1
I
-SOrCHCOOCnH2n+1 Na+ n = 6-8
 PRESERVATION OF AGROCHEMICALS 137

The most commonly used dispersing agent for wettable powders is

sodium lignosulphonate. Another popular dispersing agent is naphthalene
sulphonic acid formaldehyde condensate sodium salt. The structures of
these two complex polyelectrolyte anionic dispersing agents are shown in
Figures 6.4 and 6.5.
Both of these polyelectrolyte anionic dispersing agents are also useful for
the preparation of suspension concentrates. They are sometimes combined
with nonionic surfactants such as alkylphenol ethoxylates or long chain
alcohol ethoxylates with the typical structures shown:

CnHzn+lO-O(CHzCHzO)m H n = 8-9 m = 6-20

CnHZn+lO(CHzCHzO)m H n = 12-17 m = 6-20

Increasing the number of ethylene oxide units (EO) in the molecule

increases the hydrophilicity of the surfactant and reduces its lipophilic
tendencies, e.g. solubility in oils.
By changing the mass of the lipophile or hydrophile, what is known as
the hydrophilicllipophilic balance (HLB) can be changed, thereby modify-
ing the surface activity and solubility of the molecule. For example,
increasing the ethylene oxide chain length increases the cloud point of the
surfactant and can prevent flocculation problems during storage at high
temperatures.
Another common group of surfactants used in suspension concentrate
formulations is based on polypropylene oxide as the hydrophobe and
polyethylene oxide as the hydrophile. These are formed as ABA blocks
where A is the polyethylene oxide unit and B is the polypropylene oxide
unit. A large number of surfactants having a wide range of properties can
be obtained by changing the A to B ratio and the molecular weights of A
and B. The number of ethylene oxide units can range from two to a few
hundred. Polypropylene oxide chains below about 12 units are not really
hydrophobic and can range from this minimum to a few hundred units.
One of the advantages of nonionic surfactants is the way that their
properties can be modified by changing the level of ethoxylation, i.e. the
hydrophile/lipophile balance (HLB). Products in the HLB range 1-4 are
likely to be immiscible in water at room temperatures, HLB range 4-7
form unstable dispersions, HLB range 7-9 give opaque stable dispersions,
HLB range 10--13 give hazy solutions and HLB 13-20 clear solutions.
Nonionic surfactants in the HLB range 2-7 are likely to form water-in-oil
(W/O) emulsions and in the range 7-18 oil-in-water (O/W) emulsions.
Wetting, foaming and defoaming properties are also HLB dependent. A
wide range of degrees of ethoxylation is available, normally in the range 4
EO to 50 EO units. Some properties are summarised in Table 6.2 for nonyl-
phenol (NP) ethylene oxide condensates with 8-20 moles of ethylene oxide.
138 PRESERVATION OF SURFACTANT FORMULATIONS

CH,OH
I
H-C-OH CO
, I I
H-C- CH,

(F
I 1 7"
H,co=4
H-C-SO,M I I
CH, H,COH HC ~
_ I I OCH,
~ II O-CH HC 0

OCH, H-L()
'- YOCH,
7" I OH

OCH,

H,COH

O--C-H
I
MO,S-C-H
I

H.CO OCH,
o OH
I
Figure 6.4 Structure of a typical section of polymeric lignosulphonate salt. Lignosulphonates
are anionic polyelectrolytes whose molecular weight varies between 1000 and 20 000. Their
organic structure has not been completely determined, but it is known that the basic lignin
monomer unit is a substituted phenylpropane.

O-CH,-
SOJNa

o
Figure 6.5 Structure of naphthalene sulphonic acid formaldehyde condensate sodium salt.
Naphthalene sulphonate formaldehyde condensates are a mixture of low polyelectrolytes in
the approximate molecular weight range 500-2200 (corresponding to a naphthalene nucleus
content of 2-9 per molecule). Major components of the mixture are believed to have the
structure shown.
 PRESERVATION OF AGROCHEMICALS 139

Table 6.2 Properties of nonylphenol ethoxylates

Product HLB Cloud point eC) Pour point Surface tension

eC) (0.1%) (mN m-I)
Water 10% NaCI

NP8 12.3 31 <0 29

NP12 13.9 82 54 14 35
NP15 15.0 97 67 21 33
NP20 16.0 73 30 42

Table 6.3 Properties of linear alcohol ethoxylates

Degree of ethoxylation 7 11 20
(moles EO)
Physical form viscous liquid soft paste hard wax
Pour point eC) 21 27 37
Cloud point eC) 45-49 84-89 100
HLB 12.2 13.9 16.2
Surface tension 28.6 32.8 41.3
(0.1%) (mN m-I)

The properties of long chain alcohol ethoxylates vary in a similar way to

the alkylphenol derivatives as determined by the degree of ethoxylation.
Data on physical properties are shown in Table 6.3 for some linear alcohol
ethoxylates based on C-13-CI5 synthetic fatty alcohols.
A very important usage of surfactants is for the emulsifier blends which
are added to emulsifiable concentrate formulations to produce stable
emulsions in the spray tank. Careful selection of a 'balanced pair' of
emulsifiers is necessary to ensure that spontaneous emulsification takes
place and stable emulsions are formed in water with varying degrees of
hardness and temperature. Popular emulsifier pairs are mixtures of
nonionic and anionic surfactants such as nonylphenol ethoxylates and the
calcium salt of dodecyl benzene sulphonic acid, shown:

C9 H 19 V-O (CH CH 0)13 2 2

(C 12 H 25 j==\
V-S03) 2
- Ca 2+

The total concentration of the emulsifier blend is usually 5-10% of the

formulation. The ratio of non-ionic to anionic surfactant is determined by
using the HLB system as a guide and carrying out dilution and emulsion
stability tests. The preferred range of HLB for good emulsion stability is 8-
18 to avoid the problems of creaming and coalescence.

6.3.2 Recent surfactant developments

In recent years there has been considerable interest in the preparation of
'tailor made' surfactants with improved properties to suit specific functions
140 PRESERVATION OF SURFACTANT FORMULATIONS

in formulations and spray adjuvants. For instance, the trend towards

aqueous suspension and emulsion formulations has provided a need for
surfactants with better adsorption characteristics and long term stabilisation
properties. The conventional surfactants have molecular weights of about
1000-2000 and generally give incomplete coverage of particle surface area.
They may also desorb from the surface, leading to flocculation and
problems of increased viscosity on storage. The need for improved long
term stability has led to the development of polymeric surfactants with
molecular weights of 20 000 to 30 000 for the hydrophobic 'backbone',
which has multiple anchoring points for adsorption onto surfaces. These
polymeric surfactants can have more than ten times the adsorption affinity
of conventional surfactants, and are much less likely to desorb from the
surface.
Polymeric surfactants, therefore, impart better stability and allow higher
volume concentrations of particles to be suspended in water without
increasing viscosity.19 A good example of a polymeric surfactant of this
type is a graft 'comb' copolymer which has a hydrophobic backbone of
polymethyl methacrylate/methacrylic acid onto which are grafted 'teeth' of
polyethylene oxide.
Examples of other surfactant developments are the silicone-based and
the fluorinated surfactants giving very low surface tensions, which can be
as low as 15-20 mN m-1 compared with 30-40 mN m-1 for conventional
surfactants.
There is also considerable interest in using surfactants which are as
environmentally friendly as possible. The trend in the future will be
towards surfactants which are fully biodegradable and have low toxicity to
mammals and fish. Surfactants based on sugars or polysaccharides are
being developed as alternatives to the conventional alkylphenol ethoxylates
and other synthetic surfactants. Sugar ethers or alkyl polysaccharides are
nonionic surfactants and may be suitable alternatives in that they give a
similar effect to petroleum-based surfactants, but are more acceptable
from a toxicology and environmental standpoint. Preservatives may have
an increasing role to play in these developments.

6.4 Preservatives for agrochemicals

Microorganisms which cause spoilage of formulated products can enter

formulations in a number of ways, such as by using unclean plant and
equipment, contaminated raw materials and water, or by exposure to the
atmosphere. In order to grow and develop, microorganisms need a
nutrient source. Most agrochemical pesticide types do not provide a
nutrient source for microorganisms and are not susceptible to microbial
degradation. Indeed, some fungicides act as preservatives themselves to
 PRESERVATION OF AGROCHEMICALS 141

prevent the growth of fungicidal spores in formulated products. However,

because of the need to formulate agrochemicals to give products which
have good biological activity and long term storage stability, it is necessary
to add preservatives to protect the formulation additives from microbial
attack and prevent spoilage of the final product. Surfactants and
carbohydrate-based thickeners may sometimes act as nutrient sources for
microorganisms and generally require preservatives to protect them.
Dry products such as dusts, wettable powders and granules, and solvent-
based formulations such as emulsifiable concentrates do not contain
enough water to support the growth of microorganisms. These formula-
tions, therefore, are not susceptible to biodegradation and do not require
the use of preservatives.
Aqueous-based formulations such as solution concentrates, suspension
concentrates and OIW emulsions are particularly vulnerable to attack by
microorganisms and this is the area where the bulk of preservatives are
used. It is likely that the use of preservatives will increase in the future
because of the trend towards safer aqueous-based formulations and also
the use of formulation additives which are more biodegradable. The
problems that can occur if preservatives are not added to aqueous-based
formulations are summarised:
• production of gas
• production of bad odours
• discoloration
• pH change
• viscosity change
• phase separation
• sedimentation.
All the above problems need to be avoided to give products which
perform satisfactorily and have long shelf life.

6.4.1 Spoilage microorganisms

Once present in a formulation, a viable population of microorganisms may
grow very rapidly. A few organisms will normally become dominant and
this is determined by the nature of the formulation components, and other
parameters such as pH, temperature and oxygen and water concentration.
Microorganisms tend to grow best at around neutral pH and from 15 to
40°C, although significant growth can occur between pH 4 to 9 and
temperatures of 7 to 60°C. Examples of the wide range of microorganisms
typically associated with spoilage of aqueous-based formulations are shown
in Table 6.4.
The presence of a suitable carbon source is necessary to support the
growth of microorganisms. Many microorganisms secrete enzymes which
142 PRESERVATION OF SURFACTANT FORMULATIONS

Table 6.4 Microorganisms in formulations

Bacteria Pseudomonas aeruginosa

Pseudomonas putida
Escherichia coli
Enterobacter cloacae
Proteus vulgaris
Serratia marcescens
Rhodopseudomonas capsulata
Staphylococcus aureus
Streptococcus lactis
Streptococcus faecalis
Bacillus subtilis
Fungi Aspergillus niger
Penicillium nota tum
Aureobasidium pullulans
Yeast Saccharomyces cerevisiae
Rhodotorula rubra
Endomycopsis albicans

break down polymeric molecules into smaller fragments to act as a source

of carbon. These enzymes may persist after the organism has been killed,
making it important that microorganisms are controlled throughout the
manufacturing process. Good plant hygiene is essential to minimise the
likelihood of microbial contamination of agrochemical products. However,
in order to avoid problems occurring during long term storage, it is
necessary to add a suitable preservative.

6.4.2 Types of preservatives

Preservatives are usually required for aqueous-based formulations and
baits or pellets used as molluscicides and rodenticides. The general
requirement for preservatives is that they should have the widest possible
spectrum of antimicrobial activity and should be effective at very low
concentrations.
For many years formaldehyde, used as Formalin (a 40% solution in
water and ethanol) has been added to formulations as a general
bactericide. However, in recent years its use has been very much reduced
due to the potential mammalian carcinogenicity of formaldehyde. Cationic
surfactants, especially quaternary salts such as cetyl trim ethyl ammonium
bromide or cetyl pyridinium chloride, are also well known as antimicrobial
agents. However, many agrochemical formulations contain anionic surfact-
ants or anionic polysaccharide thickeners which would be destabilised by
cationic surfactants.
Other preservatives which are sometimes used for aqueous-based
formulations are:
 PRESERVATION OF AGROCHEMICALS 143

• propionic acid and its sodium salt

• sorbic acid and its sodium or potassium salts
• benzoic acid and its sodium salt
• p-hydroxy benzoic acid sodium salt
• methyl p-hydroxy benzoate.
These preservatives are regarded as quite safe to use and some are used
as food product preservatives. 2o However, problems have sometimes
occurred due to deactivation of the bactericide during storage of the
formulated product. This effect is thought to be due to the presence of
non ionic surfactants which can solubilise the preservative inside the
surfactant micelle, thus reducing the availability of the bactericide. 21
In recent years some new preservatives have been developed which are
active against a wide range of microorganisms, effective over a wide range
of pH values and not deactivated by surfactants in the formulations. One of
the most popular of these newer bactericides is 1,2-benzisothiazalin-3-one
which is sold under the trade name 'Proxel' by Zeneca Biocides (formerly
leI Biocides). 1,2-Benzisothiazolin-3-one is compatible with most anionic
and nonionic surfactants and also with polymeric and clay-type antisettling
thickeners used in aqueous suspension and emulsion formulations. It is
available as a suspension in water or as a solution in propylene glycol, and
is normally added to formulations at about 0.03 to 0.06%. 1,2-Benziso-
thiazolin-3-one has very good long term stability.
Another isothiazolin-3-one in common use is Kathon, a mixture of 5-
chloro-2-methylisothiazolin-3-one and 2-methylisothiazolin-3-one. This is
available from Rohm and Haas.
Other newly developed bactericides which are sometimes recommended
are:
• 6-Acetoxy-2,4-dimethyl-m-dioxane,
sold as 'Giv-Gard DXN' by Givaudan,
use rate about 0.03 to 0.1 %.
This is an active aldehyde release agent. A number of formaldehyde
release agents are also sometimes used (local regulations permitting).
They are:
• Hexahydro 1,3,5,-tris(2-hydroxyethyl)-sym-triazine,
sold as 'Glokill 77' by Rhone-Poulenc,
use rate about 0.04 to 0.2%.
• Sodium hydroxymethyl glycinate,
sold as 'Integra 44' by ISP,
use rate about 0.05 to 0.2%.
• N-(Hydroxymethyl)-N-(1,3-dihydroxymethyl)-2,5-dioxo-4-
imidazolidinyl)-N-(hydroxymethyl) urea,
sold as 'Integra 22' by ISP,
use rate about 0.05 to 0.3%.
144 PRESERVATION OF SURFACTANT FORMULATIONS

In the formulation of aqueous suspension concentrates and emulsions it

is sometimes necessary to make a stock solution of the polysaccharide
thickener, and this is often done in the case of xanthum gum. These
thickeners are stable in the dry state but when made up as 2% stock
solutions the use of a preservative is necessary to prevent the growth of
microorganisms which eventually lead to spoilage and a reduction in
viscosity. This applies to stock solutions which may be held for longer than
24 h before use.
It is difficult to make general recommendations on the use of
preservatives since many factors affect their efficiency. The choice depends
on the composition of the product being made, on the type of micro-
organisms likely to be present and on the required period of stability. Each
candidate preservative must be tested under conditions of manufacture and
storage in order to determine the most suitable type. In some cases the use
of more than one type may be necessary.
Solid agrochemical formulations such as molluscicide baits and rodent-
icide pellets are a special case, where the product may be attacked by soil-
borne or air-borne microorganisms during prolonged exposure after
application. In these cases preservatives are sometimes used to inhibit
mould growth. However, the presence of a preservative can sometimes
lead to 'bait shyness' which prevents the target pest from eating the bait.
Some of the active ingredients used in biocides are also used in
agrochemical products such as rodenticides, avicides, insecticides and
acaricides and repellents.

6.4.3 Testing and regulatory protocols

Agrochemical formulations are normally SUbjected to a series of storage
tests at different temperatures to check chemical and physical stability and
predict shelf life stability under normal conditions of use. A shelf life of at
least two years at ambient temperature in a sales pack is usually required.
Accelerated storage tests are also carried out at 54°C for two weeks, and
sometimes at intermediate temperatures such as 35 or 40°C. Chemical and
physical stability data are usually required in order to register the product
for sale in many countries.
If spoilage of a formulation by microorganisms is thought to be a
problem, it may be possible to identify the type of bacteria or fungi which is
causing the problem by incubating a sample of the diluted formulation on
agar plates. Various preservatives can then be tested to determine the
minimum concentration of biocide which will prevent growth after
incubation for 48 h for bacteria and 120 h for fungi.
At present the regulation of agrochemicals for sale in various countries is
controlled by the relevant government authority in each country, who may
request companies to provide sufficient data on the active ingredients and
 PRESERVATION OF AGROCHEMICALS 145

the formulated products to show that they are safe to use, and unlikely to
have a deleterious effect on the environment. In some countries, data on
product shelf life and biological efficacy may also be requested.
In the USA pesticides are regulated by The Environmental Protection
Agency (EPA) under two statutes; Federal Insecticide, Fungicide and
Rodenticide Act (FIFRA) and Federal Food, Drug and Cosmetics Act
(FFDCA).
Recognising that, while pesticidally inert, some formulation additives
may present a toxicological or environmental risk, the EPA is categorising
inerts into four lists:

List 1. Inerts of known toxicological concern.

List 2. Inerts of potential toxicity requiring further testing.
List 3. Inerts with insufficient data.
List 4. Inerts known to be of minimal concern and generally recognised as
safe (GRAS).

Wherever possible only those inerts which are included in List 4 should
be used, and inerts included in List 1 should be avoided.
Until recently, individual countries in the European Union (EU) were
responsible for registering products in their own countries using their own
procedures and test data. However, the member states of the EU have now
agreed on a system to harmonise registration procedures and testing
throughout the EU following the issue of Directive 911414IEEC in 1991,
which regulates the placing on the market of pesticide products. Under
Directive 9114141EEC, the EU will develop a positive list (Annex I) of
active ingredients, for a ten-year period, which may be used in any member
state, and the individual countries will be responsible for approving the
sale of formulated products of the active ingredients. Data generated for
registration in one member state will be allowed for registration of the
same formulation in other member states. Other Annexes will define the
testing protocols for toxicology, biological efficacy, product chemistry and
physical properties (Annex II for active ingredients and Annex III for
formulations. )
It is proposed that the scope of Directive 911414IEEC should be
extended to include inert materials added to the formulation and adjuvants
added to the spray tank. It seems likely that inert formulation additives
such as surfactants and preservatives will continue to be considered only as
part of the pesticide-formulated product for the purposes of product safety
and biological efficacy. The EU has recently published a proposal
concerning the placing of biocidal products on the market (Official Journal
ofthe European Communities, Volume 36, No. C239/3, 1993).22 However,
the proposal states 'Plant protection products and other biocides that are
already covered by Community requirements are excluded from scope.'
146 PRESERVATION OF SURFACTANT FORMULATIONS

References

1. Sugavanam, B. (1990) UNIDO's activities on pesticides. In Recent Developments in the

Field of Pesticides and their Application to Pest Control, Holly, K., Copping, L.S. and
Brooks, G.T. (eds), UNIDO, Vienna, pp. 262-71.
2. Green, M.B., Hartley, G.S. and West, T.F. (1987) Chemicals for Crop Improvement and
Pest Management, Pergamon Press, Oxford.
3. Karsa, D.R. (ed.) (1987) Industrial Application of Surfactants I, Royal Society of
Chemistry, Cambridge.
4. Karsa, D.R. (ed.) (1990) Industrial Application of Surfactants II, Royal Society of
Chemistry, Cambridge.
5. Karsa, D.R. (ed.) (1992) Industrial Application of Surfactants III, Royal Society of
Chemistry, Cambridge.
6. Valkenburg, W. van (ed.) (1973) Pesticide Formulations, Marcel Dekker, New York.
7. Chow, P.N.P., Hinshalwood, A.M. and Simundsson, E. (eds) (1989) Adjuvants and
Agrochemicals, Vols. 1 and 2, CRC Press, Boca Raton, Florida.
8. Foy, C.L. (ed.) (1992) Adjuvants for Agrichemicals, CRC Press, Boca Raton, Florida.
9. Tadros, T.F. (ed.) (1987) Solid/Liquid Dispersions, Academic Press, London.
10. Heath, D., Knott, R.D., Knowles, D.A. and Tadros, T.F (1984) Stabilisation of aqueous
pesticidal suspensions by graft copolymers. In Advances in Pesticide Formulation
Technology, ACS Series 254, Scher, H.B. (ed.), American Chemical Society, Washington
D.C., pp. 11-28.
11. Holden, W.T.C. (1992) Future formulation trends - the likely impact of regulatory and
legislative pressures, Brighton Crop Protection Conference, Vol. 1, BCPC, Brighton, pp.
313-20.
12. Seaman, D. (1990) Trends in the formulation of pesticides - an overview. J. Pesticide
Sci., 29, 437-49.
13. Capes, C.E. (1980) Particle Size Enlargement, Vol. 1, Elsevier, Amsterdam.
14. Tadros, T.F. (1988). In Proceedings of 2nd World Surfactants Congress, Section D,
ASPA, Paris, pp. 271-83.
15. Scher, H.B. (1983) Human welfare and the environment. In IUPAC Pesticide Chemistry,
Miyamote, J. and Kearney, P.c. (eds), Pergamon Press, Oxford, pp. 295-300.
16. Holloway, P.J. and Stock, D. (1990). Factors affecting the activation of foliar uptake of
agrochemicals by surfactants. In Industrial Applications of Surfactants II, Karsa, D.R.
(ed.), Royal Society of Chemistry, Cambridge, pp. 303-37.
17. Porter, M.R. (1991) Handbook of Surfactants, Blackie, Glasgow.
18. Reports on Agricultural Surfactants and Related Materials, Vols. I and II, (1994), Hewin
International, Amsterdam.
19. Knowles, D.A. (1994) Trends in the use of surfactants in agricultural formulations. In
Proceedings of 8th IUPAC International Congress of Pesticide Chemistry, American
Chemical Society, Washington D.C. (to be published).
20. Smith, J. (ed.) (1991) Food Preservatives, Blackie, Glasgow.
21. Lehmann, R.H. (1988) Synergisms in disinfectant formulations. In Industrial Biocides,
Payne, K.R. (ed.), published for SCI by John Wiley, London, pp. 82-87.
22. EU (1993). Official J. European Communities, 36, No. C239/3.
7 Preservation of personal care products
D.K. BRANNAN

7.1 Introduction

7.1.1 History of personal care products and preservation concerns

The Sumerians, Babylonians, Hebrews and Egyptians used cosmetics for
ceremonial, medicinal and ornamental purposes as early as 4000 Be. Their
cosmetics were mainly face and body paints, skin oils, ointments packaged
in pots or jars, and pigments contained in sticks (crayons) or pencils. Their
focus was on the eyes. Not only could cosmetics highlight and emphasize
these expressions of the soul, but they had a practical side as well by
protecting the eyes from flies and the Sun's glare. Lashes, lids and
eyebrows were painted black with kohl. The lower lid was edged with a
green paste made from malachite. Malachite green is an excellent
antibacterial dye and so this may have been the first instance of a preserved
cosmetic. 1
But cosmetic history goes back even farther to a more primitive time.
Men and women seem to have a basic primal need to change their
appearance using cosmetics. Archaeologists claim humans have used
cosmetics since the Paleolithic period nearly 50 000 years ago exploiting
pigments blended with fats to decorate their bodies. The most commonly
used pigments were made from white lead, chalk or gypsum for white
coloration. For black coloration, they used charcoal or manganese ores. Or
they used kohl, a paste made from soot and antimony, or galena, a lead
ore. For red, orange and yellow they used ocher from crushed and
powdered iron ores such as the hydrous iron oxide limonite for yellow or
the red iron oxide crystal hematite for red. Heating the ocher powders
produced the reddish brown pigment known as burnt ocher.1
Life was tougher in the Paleolithic period; nature was mysterious and
frightening. Magic, wizardry and the supernatural pervaded the society.
The hallmark of being human seems to be the ability to anticipate how
one's actions today will affect our and others' futures. Ritual helped reduce
the anxiety of anticipating the future. It gave them the ability to believe
they had control over their fate. During these rituals, primitive man
apparently used the pigments for body painting. Perhaps the painting
transformed the participants into entities no longer associated with self and
148 PRESERVATION OF SURFACTANT FORMULATIONS

gave them a sense of defying fate. The Judea-Christian creation story of

Adam and Eve mimics this idea of hiding one's true self. Once they defied
the rule God gave them (eating forbidden fruit from the tree of knowledge
of good and evil), they covered themselves to hide who they really were.
Hiding who we really are has ever since been ingrained in our psyche.
Cosmetics are the tools we use to accomplish this. Cosmetics are the cures
for the disease of being someone whom we do not want to be.
Pigments remained in popular use by Neanderthals, a variety of Homo
sapiens. They painted their dead with them before burial to return the pink
blush of life into the body. When Cro-Magnons appeared, they continued
to use the mixtures of pigments and fat. But when Egyptian culture
flourished, the simple pigments were replaced with elegant cosmetics and
hair dyes. They used henna to dye everything from hair and fingernails to
palms and soles. Scents and essences from almond, thyme, oregano,
frankincense and myrrh, saffron and rosewater were compounded into
perfumes, creams and lotions. The upper classes even used rouges,
whitening powders, bath oils and lipsticks. The Greeks used imported
perfumes, depilatories, rouges and kohl eye paint. Greek women even
dyed their hair or lightened it with colored pigments. The Romans used
perfumes, henna, rouge, face paint, hair dye and bleaches as status and
wealth symbols to differentiate between upper and lower classes. 1 ,2
The refined cosmetics were now luxuries. The lower classes could not
afford them. No longer was the use of cosmetics needed for rituals to ease
the anxiety of the future. Now, the upper class defied their common fate
with the commoners (death) by claiming they were special and separate
from them. How better to announce this separation from the lower classes
than by a simple changing of one's appearance with cosmetics? Modifying
our appearance is integral to our human nature and cosmetics have been
with us throughout the development of culture.
In medieval Europe, face paint was fashionable but restricted to noble
males, high born ladies and courtesans. The medieval ideal of feminine
beauty was skin as white as the lily and cheeks as red as the rose. While the
rich had special cosmetics to achieve this, the commoners used wheat-flour
powder and beet-juice rouge. In 18th century Europe, the beauty mark
(made from a velvet patch) was invented to cover smallpox blemishes.
What started out as simple dots, the patches became large and shaped as
birds, stars, flowers or as symbols of the wearer's occupation or avocation. 1
Microbial contamination of cosmetics, however, did not become much
of an issue until about 50 years ago. 2 The first microbial contamination was
mold spoilage which affected cosmetics once large scale manufacture
began. Parabens were used to provide adequate protection. As World War
II ended, we entered an antibiotic euphoria and bacterial phobia.
Everywhere there were new antibacterial skin creams, toothpastes,
mouthwashes, deodorants, deodorant soaps and shampoos. Microbiologists
 PRESERVATION OF PERSONAL CARE PRODUCTS 149

frenetically screened the latest biocide. There were as many different

preservative challenge test protocols as there were different companies.
Since most of these tests were for the use of the companies rather than
contributions to science, little of the work was published. This meant that
none of the test protocols ever underwent the scientific process of peer
review.
As we entered the space age and became moonstruck, a more scientific
paradigm began emerging. Both industry and medicine became aware of
bacterial resistance particularly among the Gram-negative organisms.
Contamination by Escherichia, Klebsiella, Enterobacter, Serratia and
Pseudomonas spp. became a major issue in non-sterile drugs, cosmetics,
disinfectants and hand cream dispensers.3--6 During 1966, 1967 and early
1968 the US Food and Drug Administration (FDA) recalled 25 microbially
contaminated cosmetics. 7 In two US surveys conducted in 1969, anywhere
from 20--24% of the cosmetics tested were contaminated with micro-
organisms; 3.6-6.4% of the cosmetics were contaminated with Pseudomonas
Spp.8,9 The Cosmetic, Toiletry and Fragrance Association (CTFA) quickly
established a form of peer review known as the Microbiology and Quality
Assurance committees to develop technical guidelines for the industry.
More effective and more responsible preservation practices began as a
result of this collaboration between microbiologists from the various
companies. The 1970s saw a flurry of literature contributions. Not all the
contributions were in prestigious or even recognized scientific journals but
at least cosmetic microbiology was out of the closet. The CTFA issued
technical guidelines covering all aspects of good manufacturing and
microbiological practices. This time (1972-1975) when surveys of cosmetics
were conducted by FDA and again by Wolven, only 3.6--5% of
marketplace cosmetics showed contamination. 1O ,11
But we still were not out of the woods. By mid-1970s, eye cosmetics
came under special scrutiny.12,13 There were several cases of blindness
occurring due to Pseudomonas-contaminated mascaras. 14 The finding was
that most products reached the consumer in good microbiological
condition, but they could not withstand the insult of organisms added to
the product during use. 15- 20 This was the main issue to be addressed over
the next few years. As a result, the FDA gave a contract to Ahearn and
Wilson at Georgia State University in 1975 to develop eye area challenge
tests. 21 Coming out of this contract was the 'direct contact membrane'
method for preservative efficacy testing of mascaras22 which was further
tested in another FDA contract given to the University of California at San
Francisco in 1977. The CTFA became involved quickly by setting up an
Eye Area Task Force. This group arranged a collaborative study
comparing the direct contact membrane method for evaluating preservative
efficacy with the CTFA direct inoculation procedure. Both tests were
proved to be equally satisfactory for measuring preservative activity.23
150 PRESERVATION OF SURFACTANT FORMULATIONS

The FDA has continued its campaign to develop preservative challenge

methods that predict the risk of consumer contamination. Since cosmetics
are viewed by FDA as providing a zero health benefit, they must present
zero risk. In 1985, the FDA gave a contract to Schering-Plough and the
University of North Carolina to develop preservative challenge testing
methods for creams and lotions that were predictive of consumer
contamination. The FDA has still not published the data from this study.
In 1990, the CTFA published the results of a survey to determine if
companies tried to correlate their challenge test data with consumer use
data. 24 Nearly all companies claimed they had correlation programs in
place. 25 None presented their claim support data, however. There are only
two challenge test methods that have published validation data proving
their ability to predict the in-use potential for consumer contamina-
tion. 2 6-29 Both tests are modifications of the CTFA test.
In 1990, the FDA established a joint program with CTFA and the
Association of Official Analytical Chemists (AOAC) to develop standard
preservative challenge tests. This represents the first major step in
developing a statistically based protocol for preservative efficacy testing.
Rather than rely on a compendial method, the CTFNAOACIFDA
collaborative intends to be a study that infuses reproducibility and
reliability into the method. The collaborative test will be conducted using
the same inoculum (pools of similar organisms), the same four types of
products and with the same materials at 17 different laboratories. The four
products to be evaluated are preserved and unpreserved versions of a
shampoo, a conditioner, a water-in-oil emulsion and an oil-in-water
emulsion. There will be three replicates of each product to be subjected to
the challenge organisms. The testing phase should have been done by the
end of summer 1994 and a first draft available for review in mid-1995. The
major negative of the test method developed by this collaborative is that
the goal of having a challenge test correlate with the risk of consumer
contamination will continue to be absent. Despite the tests' stated purpose
'To demonstrate the ability of products to withstand microbial insult which
may occur during intended use', there will be no consumer-use studies
conducted prospectively to validate this modified CTFA method.
The United States Pharmacopeia (USP) is also looking at ways to refine
their preservative effectiveness test. The final revision should be available
by 1995. Some of the more significant changes under consideration by the
panel will be to eliminate the phenol coefficient test (replacing it with a five
subcultures away from the original source rule) and eliminating the
requirement for a 56 day evaluation (demoting that requirement to a
suggestion by moving it to an information chapter). Finally, the 150%
variability rule between sampling times will be replaced with a 0.5 log
varibility.
Perhaps the final goal of all these revisions should one day be to combine
 PRESERVATION OF PERSONAL CARE PRODUCTS 151

all the preservative efficacy tests from all the various nations and
organizations into one coherent method that is scientifically and statistically
sound and that is truly a validated predictor of consumer contamination
potential.

7.1.2 A look at the future

The world is continuing to become a global economy. The breaking down
of the Berlin Wall, the establishment of the European Commerce
Commission, the North American Free Trade Agreement and moves
trying to re-establish most favored nation trade agreements with China will
continue to help us along the way to a global economy. In light of this
movement, microbiologists must be concerned wtih developing tests,
manufacturing practices, labeling requirements and other details that are
uniform throughout all the nations and compendial sources rather than
maintain a parochial attitude. This blending of the methods will be the
biggest demand we face in the future.
The best way to accomplish globally valid methods is to stop developing
compendial methods based on a panel of industry experts who eventually
come to consensus on a protocol. Instead, we should rely on scientifically
sound statistically valid procedures that go through rigorous peer review
and testing by a variety of laboratories. The industry is now alert to the fact
that it is responsible for its products during manufacturing and consumer
use. Good industrial microbiologists anticipate microbial resistance and
adaptability. A high awareness of asepsis, hygiene and sanitation now
mitigate our once overdependence on antimicrobials. To continue on this
road of continuous improvement, we must develop new globally valid
approaches and methods if we are to persist in winning microbiological
battles.

7.2 Importance of preservation

7.2.1 Types of personal care products needing preservation

Cosmetics include skin care and hair care products. Skin care products can
be as ordinary as deodorants, antiperspirants (which are also drugs) and
hand and body lotions. Hair care products can be as ordinary as shampoos
and conditioners. But cosmetics also include a plethora of other products:
make-up foundations, eye shadows, eye liners, mascaras, lipsticks, rouges,
powders, perfumes, mousses, styling gels, finishing sprays, night creams,
eye creams, sunscreens (which are also drugs), nail treatment oils, nail
polish, cuticle removers, nail polish binder and nail fortifiers, nail polish
remover, depilatories, hair spray, permed hair treatments and hair
152 PRESERVATION OF SURFACTANT FORMULATIONS

coloring agents. Of these products, the ones that most need preservatives
are those with the most water; shampoos, conditioners, emulsions
including make-up creams and water-based mascaras and gels. Products
with low water activity (Aw ~ 0.90) usually need little more than anti-
fungals such as methyl or ethyl parabens to provide adequate preserva-
tion. Table 7.1 provides the water activity limits for microorganisms. 30 ,31
Most of the products needing biocide preservatives are emulsions. The
common characteristic is the presence of some type of surfactant and a
water activity above 0.95. Whenever water is present, so is life. Regardless
of temperature, pH or any other environmental factor, if water is in a

Table 7.1 Water activity and the potential for adaptation31

Water activity pH Microbial Problem Examples of

adaptation organisms cosmetic
potential capable of products
growth

0.98-1.00 Any pH High Most Gram- Shampoos and

negatives emulsion products
0.95--D.97 Above 5.5 Moderate Most Gram- Liquid make-
negatives ups and eye area
products
Below 5.5 Low Some Gram- Some hair
negatives conditioners
0.92--D.95 Above 5.5 Low Few Gram- Some pressed
negatives powders
Below 5.5 Very low Spoilage
bacteria
0.9O--D.92 Any pH None Gram-positive Some rouges
Lactobacilli (non-water based)
0.8O--D.90 Any pH Potential for Staphylococcus Lipsticks (non-
Staphylococcus, Molds water based)
mold and yeast Yeasts
adaptation
0.7O--D.80 Any pH Some potential Molds Some talcs
for yeast and Yeasts
mold
adaptation
0.65--D.70 Any pH Very little Osmotolerant
potential for yeasts
0.6O--D.70 Any pH microbial Osmotolerant Some anti-
adaptation molds perspirants
Below 0.60 Any pH None (all None
microbes)
 PRESERVATION OF PERSONAL CARE PRODUCTS 153

liquid form, a microorganism will live there. The goal then is to add
biocides in the hope of limiting the organisms that adapt to the aqueous
environment while the customer uses the product. To think that one can
develop the fail-safe biocide or process that will prevent contamination by
microorganisms is the ultimate delusion. In the words of Winogradsky,
'Microbes are everywhere, the environment only selects.' They will
eventually grow in even the most hostile of environments provided water is
available in liquid form.

7.2.2 Surfactants used

Some of the most common surfactants used in the manufacture of
cosmetics include ammonium lauryl and lauryl ether sulfates, sodium
lauryl and lauryl ether sulfates, triethanolamine lauryl sulfates, ammonium
xylene sulfonate, mono- and di-alkyl quaternary compounds and diethanol-
amides. The general categories of surfactants used are a-olefin sulfonates,
betaines, lauryl sulfates, silicone-based surfactants, amide-based surfact-
ants, quaternary compounds, ethoxylated surfactants, ethers and alcohols.
Nearly all surfactants require preservatives with a few exceptions where
the pH extremes are below 3.5 or above 10. The most common biocide
preservative for surfactants is formaldehyde at about 100 to 200 ppm. This
is added as a processing aid and therefore does not have to be included on
the cosmetic label. Other preservatives used are dimethyloldimethyl
(DMDM) hydantoin and the isothiazolinones. The formulator should
always be very careful not to use the same preservative in the surfactant as
he or she does in finished product. Adaptation and contamination occur
much easier in the raw surfactant. A carryover of those adapted organisms
into a product using the same preservative results in contamination and
thus recalls due to the contaminated finished product.

7.2.3 Consequences of not adding a preservative

The person who believes that a thorough microbiological testing or
monitoring program will ensure product quality simily does not understand
that biological systems are constantly evolving. Bacterial evolution is so
rapid that the agents which kill today can become bacterial food within less
than a year after exposure to the microbial world. Bacteria adapt rapidly.
Preservative failure should be expected and anticipated. As a result, at
least two suitable preservative systems should be identified for each new
product early in the development process. One way to slow the adaptation
process is to protect cosmetics from contaminants by strict sanitation rules.
Items to enforce rigidly are cleanliness of process water, raw materials,
manufacturing and personnel hygiene. Nevertheless, one still needs to be
ready for the surprises that the microbial world has in store.
154 PRESERVATION OF SURFACTANT FORMULATIONS

Microbial contamination presents a substantial risk to product quality,

regulatory compliance and consumer health. In terms of affecting product
quality, contamination will produce adverse changes in product odor, color
and viscosity. 32 The microorganisms metabolize and digest the product
ingredients to produce their waste products in the process. They do this by
producing a wide variety of hydrolytic enzymes. These changes happen
rapidly if the invading microbes have an environment conducive for high
reproduction. They happen slowly if the microbes reproduce more slowly.
In fact, microbial contamination is usually not all that obvious. At first,
there is no immediate evidence of growth in the product. Months can go by
before the insidious creatures affect the product.
By far, the most critical issue associated with microbial contamination of
cosmetics is the risk to consumer health. Fortunately, health-related
incidences involving contaminated cosmetics are very rare. These in-
cidences include infection from a hand lotion,33 eye infections from use
of eye area cosmetics 14 ,34 and the death of one immunocompromised
individual. 35
Microbiological safety of a cosmetic is also a regulatory responsibility
administered by the US Food and Drug Administration. The Food, Drug
and Cosmetic Act,36 gives FDA the authority to protect the public against
adulterated cosmetics. Adulterated products contain injurious or patho-
genic microorganisms or their metabolites. 37-40 Adulteration can be real or
even just perceived. By the wording of the law, even if conditions are such
that contamination 'may have occurred' this perception gives the FDA the
right to claim adulteration. Determination of adulteration will result in
product recalls. It may even lead to seizure of the cosmetic resulting in
significant economic consequences for the manufacturer.
Cosmetics do not need to be sterile, but they must be adequately
preserved to withstand consumer use and manufacturing processes.
Manufacturing contamination can be reasonably controlled, but consumer
use and abuse cannot be controlled. Consumers repeatedly challenge the
cosmetic with microorganisms in saliva, dirty hands and in tap water. The
bathroom, where most cosmetics and toiletry articles are kept, is a perfect
incubator providing heat and humidity ideal for stimulating microbial
growth. 41 ,42 With all these potential insults and our litigious society, it only
makes sense to be sure that cosmetic products can withstand consumer use
and resist becoming contaminated.

7.2.4 Microorganisms encountered

Cosmetics can be contaminated with spoilage microorganisms and a variety
of organisms found in the household environment. 43 Table 7.2 lists the
types of organisms that contaminate shampoos and skin lotions. 44 The
contaminants include bacteria, yeasts and molds. Most are spoilage
 PRESERVATION OF PERSONAL CARE PRODUCTS 155

Table 7.2 Types and percentages of microorganisms contaminating cosmetics after

use. 44

Organisms Isolated Isolated

from from skin
shampoo lotion

Citrobacter freundii 18 0
Enterobacter s~p." 37 9
Klebsiella spp. 9 9
Pseudomonas spp. c 9 21
Serratia spp. d 18 4
GNRe (non-fermentative) 0 4
GNR (fermentative) 9 0
CDC serotype rVC2 0 4
Bacillus spp. 0 4
Staphylococcus epidermidis 0 4
Propionibacterium sp. 0 4
Sarcina sp. 0 4
Diphtheroid 0 4
Yeasts and molds 0 29

"E. aerogenes, E. agglomerans and E. cloacae. bK. pneumoniae and K. oxytoca. cpo putida, P.
fluorescens, P. paucimobilis, P. aeruginosa and P. maltophilia. dS. liquefaciens, S. odorifera
and S. rubidaea. eGNR, Gram-negative rod.

organisms, however, even non-pathogenic spoilage microbes in a cosmetic

may cause disease under appropriate conditions. A product contaminated
with spoilage organisms may invade and create disease if it is applied to a
break in the skin. 45 Use of a shampoo that irritates the eye will
compromise it and allow microbial contaminants to infect the eye. With
more and more immunocompromised individuals in the population from
the pandemic of AIDS, the problem becomes critical from a product
liability standpoint.
The biggest contamination concern, however, are pathogens that
present a frank health risk. An historic example of the organisms involved
here are the pseudomonads. 46 Cosmetics intended for eye area use are
particularly susceptible to providing health risks. The cornea, when
compromised, is highly vulnerable to infection. There have been several
instances of mascara contamination from Pseudomonas Spp.14,31 These
cause infection as well as product discoloration and odor formation. Other
typical contaminants of cosmetics include Enterobacter spp., Klebsiella
spp., Serratia spp. and a variety of Pseudomonas spp. (see Table 7.2).

7.2.5 Considerations for preservation

In order to understand fully how to preserve a product, the microbiologist
must consider all the variables that contribute to a product being well
preserved against microbiological contamination. Adding a biocide to a
156 PRESERVATION OF SURFACTANT FORMULATIONS

product is not all there is to preservation. Even disinfectants with some of

the most powerful biocides have been contaminated47--49 and recalled.
Minimally, one should use the biocide that gives the product the best
ability to withstand the microbial challenges to which it will be subjected
during manufacturing and consumer use. However, a good microbiologist
goes beyond this minimal approach. Microbiologists must be holistic
practitioners of preservation if they expect to formulate a cosmetic product
with appropriate antimicrobial hostility. He or she will also ensure that the
biocide is safe, stable, compatible with the product and its container,
inexpensive, readily available, approved by appropriate regulatory
agencies, have a positive consumer perception and is environmentally
friendly.
In addition, preservation includes having good manufacturing practices
that insist on only the cleanest of manufacturing conditions. Those
cleanliness standards should even include the surfactant supplier being
under good manufacturing practices (GMP) and strict sanitation practices.
Raw material quality, container and cap design, expected shelf life and
exposure conditions, and even how the consumer will use and misuse the
product should be considered. Consideration of all these elements is
critical to maintain the microbiological quality of a product.
The entire product matrix should be considered as the preservative. A
biocide may be adversely affected by the other ingredients in the product.
For example, some of the suspended solids in a formulation such as
carbonates and silicates, talc and metal oxides, cellulose and starch react
by absorbing out preservatives. Minor pH changes can inactivate some
preservatives especially weak acids (e.g. sorbic acid) which rely on
hydrogen ions that disassociate to disrupt chemiosmotic membrane
balance. 50-52 Minor shifts in ionic strength or changing the buffering
system in a product can also alter a bacterium's susceptibility to a biocide
or affect how a preservative partitions between the water matrix and the
microbial cell. 53 .54 Preservatives are usually water soluble and so exist in
the water phase of emulsions. Some parabens, however, present unique
formulation challenges because they have an affinity for the oil phase. This
presents a particular problem for water-in-oil emulsions since the microbes
live in the water phase only. 55 Even the surfactant system used can affect
biocide performance. 52 .56--58 In fact, nonionic surfactants are used to
neutralize some preservatives (e.g. parabens)59-61 however they tend to
enhance quats. 62 Finally, high levels of protein (often used in conditioners
and lotions) will also reduce the antimicrobial activity of many preserva-
tives 63-65 and the presence of hydrophilic polymers will affect others. 66
In considering choice of container, the microbiologist should also
consider compatibility with the preservative. 67 ,68 Most preservative manu-
facturers do not include a statement of container compatibilities. Many of
the negative container effects are due to long term storage. The
 PRESERVATION OF PERSONAL CARE PRODUCTS 157

preservative may either be absorbed into the container material in the case
of lipid-soluble preservatives or inactivated because of complexation of the
preservative with the dyes used in the plastic or lost because of the
volatility of the preservative (e.g. phenoxyethanol). In one instance, 50%
of the parabens used to preserve a mascara were absorbed into the
polyethylene container after storage and could only be extracted from the
container in 50% methanol (personal communication, S. Richards). The
microbiologist should test long term stability of the preserved product in
the container expected for marketing. Sometimes, these tests are done
under accelerated conditions (high temperatures) making use of the
assumption that the inactivation will follow Arrhenius kinetics. Chemical
analyses for the preservative should be included as well as conducting
preservative efficacy tests. In this way a failure in the preservative efficacy
test can be traced to a breakdown or loss of the preservative. If the
preservative is still present, then some other factor is acting to make it
unavailable to kill the organisms such as partitioning out of the water
phase.
When considering containers, one should not overlook the impact that
dispensing closures have in preventing microbial contamination especially
during consumer use. Flip caps and pump tops provide more protection for
shampoos and skin lotions than screw caps.44 Some closures can even
inactivate the preservative. 69 Preservation is a function of formula and
even container, not just the biocide. The wise microbiologist inserts herself
or himself into the product development team early on to help decide upon
the formula, the package and even specific instructions for use on the
label.

7.3 Preservative efficacy testing

7.3.1 General considerations of preservative efficacy testing (PET)

Test protocols for determining preservation efficacy in cosmetics
vary.32,67,70,71 The protocol used in Europe is one found in the European
Pharmacopeia. Although Britain has her own pharmacopeia, it is likely she
will adopt the European Pharmacopeia. The three protocols that are fairly
widely used in the United States include the protocols from the CTFA, the
American Society for Testing and Materials (ASTM) and the USP. The
AOAC is also attempting to define a protocol in collaboration with CTFA.
The reason for such a plethora of methods is the types of products for
which they are designed. Each trade organization must address a variety of
different products. The CTFA focuses on cosmetics, the ASTM on
commercial products such as cutting oils and paints and USP focuses on an
assortment of drugs.
158 PRESERVATION OF SURFACTANT FORMULATIONS

Many of the specific differences among the procedures are a result of the
unique concerns of the organization developing the test. Some of the
differences are due to lack of agreement on what the purpose of the
preservative efficacy test is. Sitting on both the CTFA and the USP
committees responsible for developing such tests has reminded me of the
maxim 'one does not want to know how laws and sausage are made.' At
other times, the cooperation and concern shown by the participants to
develop an equitable method was impressive. The logic and arguments that
go into establishing these protocols are primarily based on consensus of
what is being done by the various participants on the panel developing the
protocol. Certainly, these efforts represent 'state-of-the-art', but they do
not necessarily represent rigidly controlled protocols subjected to multiple
laboratory-controlled experiments and statistical analysis. To develop
these kinds of tests, a company must employ a microbiologist who provides
the scientific expertise needed to develop a validated 'in-house protocol'
that is specific for the company.
The primary purpose of a PET might be to have a test that predicts
consumer contamination. Two other purposes of a PET might be that it
demonstrate the presence of a biocide or that it predict the change of
manufacturing contamination. Regarding these latter two purposes, the
first can be met by chemical analysis, the second can be met by controlling
manufacturing insults to the product through good manufacturing practices
and sanitary processing. However, no controls exist to provide protection
from the consumer except what can be designed into the product itself and
into its container. Several publications have already shown that a modified
version of the CTFA preservative efficacy test is a valid predictive model of
the risk of consumer contamination. 2 6-29
The FDA has tried several times to develop a PET that predicts
consumer contamination as described in detail above. If the FDA had such
a standard test, then products failing it would be subject to recall. The
FDA is now collaborating with CTFA and AOAC to develop a
standardized laboratory preservative efficacy test complete with multi-lab
comparisons and statistical analysis. However, the purpose of having the
test correlate with the risk of consumer contamination will continue to be
absent. Despite the AOAC/CTFAlFDA collaborative's protocol stating
that its purpose is 'to demonstrate the ability of products to withstand
microbial insult which may occur during intended use,' it does not have this
ability to predict consumer contamination since no correlative consumer
testing is planned using the same products. The omission of this goal was
not entirely unplanned. If the test is developed with this purpose in mind,
then it might be used to enforce a recall on those products that fail it.
Regardless of which philosophy one adopts to define the purpose of a PET,
at a minimum the goal should be to develop a database to rank the
antimicrobial hostility of the company's products.
 PRESERVATION OF PERSONAL CARE PRODUCTS 159

Although preservative efficacy (or challenge) tests are deceptively

simple in concept, they are complex to perform. All the protocols involve
introducing microorganisms into the cosmetic, sometimes referred to as
'challenging the product.' All the protocols have several other similar
steps: product preparation, inoculum selection, inoculation, incubation,
estimating the surviving microorganisms and interpreting the data. The
ideal PET does not exist. Nevertheless, the cosmetic industry has been
fairly successful without a standard PET. The rarity of contamination
incidents speaks well for an industry that properly uses the right methods,
the right manufacturing and the right packaging controls to prevent
microbial contamination.

7.3.2 Common issues shared by the PET methods

7.3.2.1 Product sample. The product sample used in a PET should

replicate formulation, packaging, manufacturing conditions and other
factors significant to preservation. All raw materials should be of the same
quality and from the same source when testing preservation options. As
the product becomes refined, minor changes will be made to adjust
viscosity, color, perfume or pH. These minor changes can alter the
antimicrobial capacity of the product. They require the product to be
reassessed for preservative efficacy since even small composition changes
in individual ingredients can adversely affect the hostility of a product.
Even the quality of the process water affects a product's antimicrobial
activity. Changing raw material suppliers whose standards and processes
are different should prompt a re-evaluation of the product's hostility since
even the presence of trace chemicals in the new supplier's materials can be
the cause of preservative inactivation.
Even going from lab to pilot plant to plant (scale-up), may cause
preservative inactivation. As a result, going from small batch to continuous
large volume manufacturing requires that the product be tested for its
preservative efficacy. Scale-up provides the opportunity for inadequate
mixing times, inappropriate temperature and pH changes in localized areas
of the mixers and slow cooling times that may inactivate the preservative.
These variables need to be identified and controlled. The final nationally
delivered product produced and packaged for retail sale should, once
again, be evaluated for its preservative efficacy.

7.3.2.2 Microbial inoculum. An appropriate microbiological challenge

of the product is the most critical factor in determining the validity of a
preservative efficacy test. All the tests currently specify a set of inoculum
microorganisms. 32 ,67,70,71 Some of the methods list specific strains from
American Type Culture Collection (ATCC). Both the CTFA and USP
160 PRESERVATION OF SURFACTANT FORMULATIONS

tests also allow the manufacturer to include any other organisms that can
be likely contaminants. Most cosmetic manufacturers include the recom-
mended organisms from the compendial tests. However, they also use their
own selection of resistant strains recovered from consumer-used product
samples, raw materials or manufacturing sites. These microorganisms are
sometimes considered more resistant to preservatives and other hostile
conditions. This kind of parochial thinking, however, should be re-
evaluated if the company has not maintained a rigorous program of
preserving the originally isolated culture.
Most companies use Gram-negative bacteria such as Enterobacter,
Serratia, Pseudomonas, Klebsiella, Gram-positive bacteria such as
Staphylococcus aureus and fungi such as Candida, Aspergillus, Fusarium,
Penicillium and Saccharomyces. The isolates are chosen based on their
resistance to preservatives or their adaptation potential and their potential
as opportunistic pathogens. Resistance to antimicrobials includes resist-
ance to even what might be referred to as whole cell poisons such as
chlorine. 72 ,73 However, rather than a genetic mode of resistance develop-
ment (e.g. plasmid acquisition or mutation) the development of chlorine
resistance is more likely to be a result of population shifts in the amount of
production of capsule or even physical community developments within
biofilms where certain organisms exist as protector guilds for other
organisms. S. aureus (ATCC 6538) is used to challenge frequently used
cosmetics since it is an opportunistic pathogen that inhabits the skin.1 1 P.
aeruginosa is a non-fermentative Gram-negative rod used in all three
methods. The ASTM and USP use ATCC strains 9027 while CTFA uses
strains 15442 and 13388. Pseudomonas spp. are ubiquitous and show high
resistance to many preservatives as well as being potentially pathogenic.
Both CTFA and USP use Escherichia coli (ATCC 8739), a fermentative
Gram-negative rod used as an indicator of fecal contamination in water.
Within the same family of organisms (the enterobacteriaceae) are
Enterobacter, Klebsiella and Proteus. These may be adequate replacements
of E. coli. For example, ASTM uses Enterobacter aerogenes (ATCC
13048) as the inoculum. ASTM, CTFA and USP all use Candida albicans
for the yeast inoculum. It is an opportunistic pathogen. ASTM and USP
use A TCC strain 10231; CTFA makes no specific recommendation on strain.
Filamentous fungi may also contaminate cosmetics. All three methods use
Aspergillus niger (ASTM and USP use ATCC strains 16404; CTFA uses
ATCC 9642). CTFA also suggests Penicillium luteum ATCC 9644 be used
to represent fungal potential for contamination.
Finally, CTFA recommends using microorganisms that are indigenous to
the eye, clinically significant isolates and product isolates for challenging
eye cosmetics. They also include Gram-positive spore formers such as
Bacillus subtilis. However, when using B. subtilis for a challenge inoculum,
most of the cells should be in the vegetative form rather than the spore
 PRESERVATION OF PERSONAL CARE PRODUCTS 161

form when the challenge is done. The practice of using spore-forming

bacteria as a challenge is questionable at best. If the challenge inoculum
consists of spores and a product is developed to be sporicidal, the product
will probably be unsafe for use.
Most of the tests make recommendations regarding the organisms to
use. However, they disagree on which organisms to use, whether the
challenge should be pure or mixed, the inoculum concentration and how to
maintain the organisms. These and other issues related to inoculum will be
discussed next.

7.3.2.2.1 Use of ATCC cultures as the inoculum. The ATCC cultures

used mayor may not have the specific biocide resistance that the
manufacturer needs for its products. The ATCC is a repository for
researchers to deposit cultures. As a result, it depends on the depositor to
correctly characterize the originally deposited strain and its origin. The
ATCC cannot be expected to verify the claims of all depositors. It can,
however, be expected to maintain the resistance of the culture by ensuring
that subcultures sold are never more than two to three transfers removed
from the original strain deposited.
Using as an example some of the preservative strains deposited in 1940,
the ATCC does a remarkable job. The strains were deposited as
lyophilized vials that were kept at -70°C. In the 1960s, when ATCC began
using liquid nitrogen, the original strain was cracked open, grown up and a
population was lyophilized and frozen in liquid nitrogen as the main stock.
The lyophilized vials serve as the working vials from which cultures are
distributed to investigators today. The cultures received today, therefore,
are never more than three transfers away from the original culture
deposited. The ATCC also uses a popUlation biology approach for
preserving cultures. After verifying that the culture is pure, they put up an
entire popUlation derived from the original culture rather than a single
cloned representative from the population. This population approach,
which is not the typical clinical microbiology approach, enhances the
likelihood of receiving the originally deposited strain. Thus, the manu-
facturer with its own preservative-resistant strains should rely on submitting
cultures to ATCC for maintenance.
An alternative approach in lesser culture houses would be the following
nightmare. If more culture requests are made than the original set of vials
preserved, then the last remaining vial is subcultured. From this vial an
isolated colony (obtained by streaking for isolation) is selected to grow up
and harvest. This is preserved in another set of lyophilized vials. This
process represents a departure from the originally deposited culture
because the progeny of only a single individual was selected to represent
the original population. If the requests are very high, several subcultures
using this process may occur. If enough subcultures occur, the risk of losing
162 PRESERVATION OF SURFACTANT FORMULATIONS

the original resistance is high due to loss of resistance factors from

the artificial selection process known as 'cloning from an isolation
streak.'

7.3.2.2.2 Maintaining preservative resistance of the inoculum. Main-

taining culture strains in their original biocide-resistant state repre-
sents quite a challenge for the cosmetic microbiologist. Routine sub-
culturing on non-selective growth media will eventually cause the loss of
this resistance since the selective pressure to keep the resistance is no
longer present. Since most resistance genes are plasmid located, the
organism may lose the plasmid unless selective pressure is kept on the
population to maintain the resistance. The routine practice of pure culture
techniques in microbiology is to isolate a clone for subsequent transfer to a
new tube of medium. This approach is invalid when trying to maintain a
population of resistant organisms. The likelihood of selecting out the
organisms without the resistant factor is high when using traditional 'streak
for isolation' pure culture concepts. Instead, one should rely on assessing
the purity of the population by its homogenous appearance on a lawned
agar plate. Preferably this should be done on a medium that has the
preservative in an active state (not neutralized) incorporated into the agar.
Subcultures should be done from a heavy inoculation off this lawn. The
concept here is that a population of organisms is being employed most of
which will carry the resistance genes. A sampling of the entire population is
needed to ensure that the organisms within the population that carry the
resistance genes are capable of producing plenty of progeny that will
maintain the resistance within the population. In addition, the organism
should be lyophilized or kept in liquid nitrogen as a back-up.
The working cultures should never be subcultured more than a set
number of passages away from the original strain. The USP is planning to
recommend a five passage limit. This recommendation is not based on any
particularly scientific rationale, it just seems reasonable and is pragmatic.
In fact, the five passage limit was an arbitrary number that originated in the
Bureau of Biologics (FDA) for virus passages for vaccines.
In order to maintain a culture collection of resistant organisms a 'house
organism' is isolated from a contaminated product. The organism is grown
and checked to see if it looks pure (the colonies are all identical). Then the
population is grown up as a lawn spread on an agar plate incorporating the
preservative to which the organism is resistant. The entire lawn is
harvested for freezing or lyophilization. The culture should be frozen in at
least twenty vials in liquid nitrogen and another twenty vials put up for
lyophilization. When the organism is scheduled for use in inoculation, one
of the lyophilized vials is used by subculturing the entire vial and using
plates or slants as the working stock (this is the first subculture from the
original). If a PET is conducted each week, then four more weekly
 PRESERVATION OF PERSONAL CARE PRODUCTS 163

transfers take place before a new lyophilized vial must be cracked open.
With this schedule, a culture collection can last two years on the
lyophilized vials. At that time, a new liquid nitrogen vial is cracked open
and another twenty lyophilized vials set up. The liquid nitrogen vials will
be used up over the next forty years. By that time, the company will
probably be using new kinds of preservatives anyway and the resistant
strains to today's preservatives will be obsolete! Alternatively, another
culture collection can be set up for another forty years and still only be one
subculture away from the original strain.
Contaminated product may also be used directly as the inoculum. The
advantage of this approach is that the organism can be maintained in a
biocide-resistant state because the population contaminating it is con-
tinuously selected by subculturing it into fresh product. However, direct
use of the contaminated product can create the problem of carrying over
the growth product into the test product. If the two products are not
compatible (e.g. a shampoo with anionic surfactants and a conditioner with
quaternary surfactants), then confounding results may be obtained.

7.3.2.2.3 Growth and harvesting of the inoculum. The method of

harvesting the culture and preparing it may also influence the organism's
viability. Challenge inocula may be grown in broth or on solid media. 74,75
When grown on broth, the challenge is often adjusted to a standard
concentration (measured with optical density) with sterile saline or water
added directly to the broth. When grown on solid media, the culture is
washed off the slant or plate with sterile saline or water and adjusted to the
standard optical density. The ASTM and USP methods mention harvesting
techniques generally. Both of these methods use solid agar medium to
grow the inoculum. The ASTM uses sterile water while the USP uses
sterile saline when washing the organisms from the transferred stock
culture. The organisms are then diluted to 1 X 108 colony forming units
(cfu) per ml. The CTFA does not specify any recommendations for
harvesting. None of the methods discusses properly buffering the harvesting
solution to the correct pH or osmolarity. The ideal approach would be to
resuspend the cells in a solution of the same pH and osmolarity as the
culture medium.
Greater resistance to preserved product has been described for broth-
grown cultures compared to cultures grown on solid media. 74 ,75 However,
this result may have been due to the carry-over of broth into the product
acting as a neutralizing agent of the preservative in the product rather than
any intrinsic resistance gained by the bacteria by growing in broth.
Another area needing further research is investigating the importance of
the growth phase of the challenge inoculum. The growth phase affects the
physiological state of the organisms used as the inoculum. For example,
Holm-Hansen found that A TP per cell is decreased as cells reach
164 PRESERVATION OF SURFACTANT FORMULATIONS

stationary.76 This physiological change and other potential changes may

affect an organism's resistance to preservatives.
Finally, considerable guidance is needed in the preparation of the fungal
inoculum for any PET. One of the most difficult steps in the challenge
methods is to obtain standardized fungal spores. In the USP test, the fungi
are harvested in sterile saline supplemented with 0.05% polysorbate 80
to help disperse the spores. No one has assessed the effect this non-
ionic detergent has on inactivating preservatives when spores harvested
by this method are used as inoculum. The spore count is adjusted to
1 X 108 cfu ml-1 by standard methods. The suspensions should be used
immediately. The biggest problem is the ability to harvest a high enough
concentration of spores. ASTM recommends harvesting fungal spores by
putting distilled water onto the surface of the plate and rubbing the lawn of
growth on the plate with a sterile inoculating loop or sterile glass hockey
stick to dislodge the spores. The liquid is poured off and filtered with
sterile non-absorbent cotton. This removes the hyphae to give pure spores.
One may also refer to several US patents on how to harvest fungal spores
(3 294 647; 3 300 390; 3 357 895; 3 616 246; 4 046 593; 4280000). The
other area of concern is how to keep the spores from germinating. Within
2 h, some of the fungal spores will germinate if at room temperature.
Refrigeration of the fungal preparation will slow this germination down.
Light also has an effect on germination.

7.3.2.2.4 Concentration of inoculum and rechallenge. The bacterial

concentration of the inoculum should be about 1 X 108 cfu ml-1 . If 20 g of
product are inoculated with 0.2 ml, then the final recommended con-
centration of 1 X 106 colony forming units per gram (cfu g-l) product is
obtained. All of the tests recommend this level of organisms in the
product. However, USP will allow the inoculum to drop to 1 X 105 cfu g-l
product. Any 1:100 ratio of inoculum to product should be adequate; the
ASTM recommends using 100 g of product rather than 20 g. The goal is to
keep the dilution of product by the inoculum to a minimum; a good rule is
to not dilute the product over 1% with the inoculum. In like manner, fungi
and yeast are introduced into the product. However, their concentration
ranges from 1 X 104 cfu g-l product in the CTFA method to 1 X 105 or 1 X
106 cfu g-l product in the USP and ASTM methods. Assuming a 1:100
ratio of inoculum to product, the concentration of the inoculum must be
1 X 106 cfu ml-1 (CTFA) or 1 X 107 to 1 X 108 cfu ml-1 (USP and ASTM).
The assumption that these counts represent fungal spores may not be valid
since hyphae can also give rise to fungal colonies.
How to standardize the concentration of the inoculum is left up to the
microbiologist in the USP and CTFA methods. Only the ASTM method
provides specific recommendations regarding spectrophotometer, spectro-
photometer tubes and wavelength of light used. The transmittance is
 PRESERVATION OF PERSONAL CARE PRODUCTS 165

measured at 425 nm to yield 1.0 X 108 cfu ml- I (generally about 30--40%
transmittance). ASTM suggests use of a hemocytometer count to adjust
the spore level to 1.0 X 107 cfu ml- I . However, any reference to standard
microbiological methods will provide the specific detail for determining
microbial concentrations. 77 A qualified microbiologist should have no
trouble developing a valid method.
Rechallenge is the addition of fresh inoculum to a product that has
already killed off the first challenge after an appropriate time. CTFA
provides for a rechallenge if desired but does not require it; ASTM
recommends a rechallenge if the product is expected to be used repeatedly
by the consumer. Some studies suggest this practice does not provide any
more information than single challenges. 78 The manufacturer, however,
may be able to make a case for multiple challenges. For example, mascaras
are commonly subjected to repeat insults by the consumer. If this is the
case, the microbiologist should select a challenge level that is reasonable
and likely from consumer use (1 X 102 cfu gm- I ) rather than the high levels
recommended in the compendial methods. These levels could be deter-
mined by allowing people to use unpreserved products and then immedi-
ately analyze the level of organisms introduced into the product after that
use.

7.3.2.2.5 Pure versus mixed challenge inoculum. There is a consider-

able debate regarding whether pure or mixed challenges should be used.
Part of the debate is confusion over semantics. The term mixed challenge
does not refer to use of impure cultures. Instead, it is the use of several
pure cultures which have been mixed together after they have been grown
up and harvested. Use of this mixed inoculum may be more representative
of actual conditions of contamination; microorganisms do not exist as pure
cultures in nature but as interacting populations within communities of
microorganisms. Some feel that the mixed cultures provide greater stress
to the preservative system. 79 This view is intrinsically logical if one assumes
that cometabolism or synergism occurs within a community biology
dynamic. In fact, cometabolism, vitamin and cofactor synthesis help
stimulate mixed interactions within communities of microorganisms. 80 ,81
However, no published information exists to support the hypothesis that
these interactions take place during a preservative efficacy test of a
cosmetic. The idea is supported by observations by Henriette et al. 82 who
described a mixed bacterial community that developed in disinfectants and
antibiotics. None of the individual species was resistant to the anti-
microbials. Only the community showed resistance.
It is the theory that mixed populations may be more robust, however,
that forms one of the objections to mixed challenges. The opponents of
mixed challenges claim that it introduces the variable of microbial
population dynamics into the challenge test. Therefore a failure may occur
166 PRESERVATION OF SURFACTANT FORMULATIONS

since the mixed challenge was more capable of contaminating the product.
Alternatively, there is the theory that the mixed community may be less
stringent than pure challenges because one organism may produce
metabolic factors which are antagonistic to other microorganisms in the
challenge. 83 Another idea is that the organisms will compete with each
other for limiting substrates and growth factors such as iron. 84 Resolution
of the issue will take more research, possibly more than is practical in an
industrial microbiology laboratory.
Some methods permit the use of mixed challenges composed of similar
groups of microorganisms. For example, a group of Gram-positive bacteria
and yeasts or a group of Gram-negative bacteria is permitted for the
challenge in the CTFA and ASTM methods. This permission is not based
on scientific concerns but is purely pragmatic. It is far less work to
challenge one container with multiple microorganisms than it is to
challenge multiple containers with each of the microorganisms.

7.3.2.3 Product concentrations. Most published preservative methods

test full strength (100%) product. 24 However, Brannan et al. 26 •27 and
Cooke et al. 85 added diluted product to the challenge test as well. The
dilutions stress the product and also provide a means of ranking the
product. For example, products that can be diluted and continue killing the
inoculum may be classified as well preserved whereas products that kill the
inoculum only when the product is at full strength are classified as
marginally preserved. In addition, this approach is another way of
mimicking expected use patterns. For example, products that are diluted
during normal use such as shampoos should be able to continue killing
microbial challenges. One may even make a case for the product being
diluted due to manufacturing error. The product should, it is argued,
remain hostile with water added since microorganisms can adapt easier to
low levels of biocide in the diluted product. Once acclimated, they are
better able to adapt and grow in full strength product.
Microbial adaptation to biocides is certainly a possibility.86 However,
several papers claiming to have demonstrated this phenomenon are really
either a case of neutralization of the biocide (by carry-over of the growth
medium) or a case of saturating the biocide with more organisms than
available biocide to the point of inactivating it. 87 .88 A mistaken idea is to
think of chemical biocides as enzymes. In reality, one mole of biocide
reacts with 'one mole' of microorganisms to give one mole of spent biocide
and 'one mole' of dead organism. Certainly, biochemical mechanisms, true
adaptation, for resistance to biocides exist in the case of formaldehyde and
parabens. 89 ,90 However, resistance to all biocides by similar biochemical
mechanisms must not be assumed for all microorganisms. In some cases,
the resistance claimed may be a result of physical factors such as capsule
formation or existing within a clump of organisms or within a biofilm. The
 PRESERVATION OF PERSONAL CARE PRODUCTS 167

most naive idea is that the resistance mechanisms against biocides are
similar to those mechanisms found in antibiotic resistance. Whereas
antibiotic resistance can be described based on molecular activity at a
single site, the resistance to biocides cannot. Biocides attack at multiple
sites.

7.3.2.4 Plating methods and neutralization. One of the easiest ways of

conducting a challenge test is to place the product in large 50 ml plastic
throwaway pill vials with snap on lids. Most of these are made with the
same polypropylene or polyethylene plastics from which the majority of
cosmetic packages are made. One can then easily use sterile tongue
depressors to stir the product before sampling. In the ASTM method, the
product samples are put in lidded glass jars. The other two methods specify
that the test be conducted in the product container or at least a container
made of similar material. The product (20 g for USP and CTFA; 100 g for
ASTM) is put into the containers. After inoculating, the inoculum should
be thoroughly mixed into the product. The inoculated product is then
incubated for at least 28 days at 20-25°C. These storage conditions mimic
shelf conditions but they also eliminate the potential that some preservatives
are actually more biocidal at elevated temperatures. 91
Aliquots of inoculated product are taken (after thorough mixing) at
sp~cific times. Sampling of viscous materials should be done with a syringe.
Usually a 1 ml sample is pulled and diluted into 9 ml of the proper
neutralization broth in order to inactivate the preservative. Both physical
dilution and chemical inactivation operate to neutralize the preservative.
The number of surviving microorganisms is then determined by plating the
diluted product onto suitable media. This estimate of the numbers of
survivors is typically done by plating 1 ml of diluted sample into melted
agar for pour plates or by spread plating 0.1 ml of the diluted sample onto
prepoured plates of agar (a special gel-like microbial growth medium).
After the organisms grow for a few days, they appear as visible colonies.
The colonies are then counted as colony forming units (cfu) and multiplied
by any dilution factors to estimate the numbers of organisms in the original
sample.
The problems with this traditional approach are the assumptions that we
microbiologists never question. Does one organism give rise to one
colony? Are the organisms evenly distributed as single cells or do they exist
as clumps? Are they all equally exposed to the product? Do they all grow
well in the medium provided?
It may be that these assumptions are false. The paradigm of organisms
existing as non-uniformly distributed clumps that later break up into
individual cells helps explain the anomalous results which are occasionally
obtained in preservative efficacy testing. The following scenario is
sometimes seen. An initial kill occurs at 7 days (seen as a decrease in cfu)
168 PRESERVATION OF SURFACTANT FORMULATIONS

but is followed by an increase in cfu at 14 days, followed by another

decrease at 21 days.
Using the paradigm of microbes existing in clumps, this occurrence is
now explainable (Figure 7.1). The initial kill at 7 days may have been due
to killing of the cells in smaller clumps where the entire clump of cells is
killed but the larger clumps have a few cells within them that remain alive
because they were protected. Here, our model is that cfu are really derived
from clumps rather than individual cells. The surviving cells in clumps then
disperse to result in single cells that give a higher cfu at 14 days but now are
more susceptible to the biocide allowing a reduction to follow at 21 days.
The paradigm of organisms existing as attached cells to each other and to
any other surface will also help explain 'grow-back' without resorting to
claims of microbial adaptation, injured cells or the fanciful term of 'the
phoenix phenomenon,.92 Without this model and continuing with the false

.. .' --. .t

. .' ". e
f1I
II if ~
.,.
"
II

!!f1 ~
~
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• ~
,
(a) (b)

a
~IJt 'G •
i'
,a ~

-
I) Q

-'"It a
,
f9)

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Sl
(c) (d)

Figure 7.1 The paradigm of bacteria existing as clumps helps explain anomalous results in
PET testing. Microorganisms exist in clumps in Poisson distribution. Each clump gives rise to
a colony rather than each individual doing so. After 7 days, the CFU/ml is reduced due to the
death of organisms existing outside of the protection from living within a clump. A few of the
organisms at the periphery of the clump are killed but, since it is clumps that produce a single
colony, the clump shows as an individual colony and the fact that some of the organisms in the
clump died is not detected upon plating. After 14 days, the clumps break up to provide more
CFU. Now the individuals are no longer within the protection of the clump and are more
susceptible to exposure to the biocide. Therefore, at day 21, the total CFU is decreased.
(a) At time T = 0 days, population = 18 cfu ml-\ (b) T = 7 days, population = 10 cfu ml- 1 ;
(c) T = 14 days, population = 21 cfu ml- 1 ; (d) T = 21 days, population = 6 cfu ml- 1 .
 PRESERVATION OF PERSONAL CARE PRODUCTS 169

assumption of organisms existing as individuals rather than attached to

each other, we will continue to have what appear to be anomalous results.
Certainly there are cases where adaptation occurs, antibiotics for example
and some food preservatives. However, where adaptation is claimed for
preservatives that have multiple modes of action, the clump paradigm is
more likely.
Traditionally, only those dilutions giving countable plate growth of 30-
300 cfu/plate are included. This dogma can be accepted or rejected
depending on one's expertise. A well-trained technician can count plates
accurately with considerably more than 300 cfu/plate. Additionally, if
dilutions are made meticulously, it is possible to rely on numbers less than
the 30 cfu/plate value. Use of good judgement is a better guideline than the
antiquated, clinically based, dogma of 30-300 cfu/plate.
Either pour plates or spread plates can be used to determine how many
cfu gm-1 survive. In pour plates, the diluted product (about 1 ml) is
vortexed into a test tube of about 15 ml of melted agar at 46 ± 2°C. The
agar is then poured into a petri dish. With spread plates, the diluted
product (about 0.1 ml) is spread onto prepoured solidified agar plates using
a bent glass rod. Spread plating allows easy processing of samples. The main
advantage is that it avoids exposing microorganisms to heated media.
However, pour plating allows for more exposure to neutralizing agents in
the agar. Some published information finds that the two methods give
similar results. 93 ,94
Appropriate use of neutralizers is often overlooked when conducting
preservative efficacy tests. Dilution of the product allows for biocide
neutralization. Chemical neutralization can also be used. Filtration is
another approach but is limited to those products that can be filtered.
Singer paraphrases work done by the CTFA Microbiology Committee
where they describe some of the chemical neutralizers useful for
preservatives. 95 Sutton's work also describes a number of neutralization
methods for preservatives. 9 6--98 Russell describes several neutralizers for
disinfectants. 99 Both CTFA and ASTM suggest using neutralizers but
neither provides extensive guidance regarding which ones are effective.
ASTM does, however, offer a method to determine if a neutralizer is non-
toxic and effective. First the organism's maximum tolerated concentration
(MTC) of neutralizer is determined. This is essentially a comparison of
growth medium to the medium containing various increasing levels of the
neutralizer. The MTC is the largest amount of neutralizer put into the
medium that did not decrease the microbial count as compared to the
normal growth medium which serves as the control. Once the MTC is
determined, then the maximum amount of biocide that the neutralizer can
inactivate is determined. This is done by adding various concentrations of
biocide to the neutralizer at its MTC. With this approach, one is using
microorganisms as biological indicators of a neutralizer-biocide reaction
170 PRESERVATION OF SURFACTANT FORMULATIONS

that results in inactivated biocide. The colony counts of the neutralizer-

biocide combinations (square-root transforms) should not differ signific-
antly (p = 0.05) from the counts in the growth medium/neutralizer controls
using at-test.
In addition, ASTM offers a retroactive check for neutralization of the
preservative. If a plate shows no growth, it is streaked using a nutrient
broth culture at about 102 cfu ml- 1 . Lack of growth after incubation
suggests the biocide was not neutralized. A positive growth result,
however, does not prove that neutralization actually occurred. Retroactive
procedures like these tell us only that neutralization had finally occurred by
the time the plate was streaked with the test organisms. We have no idea if
the product was neutralized at the time it was originally plated. Therefore,
positive growth using a retroactive check is not proof that biocide
neutralization took place at the time of sampling. This kind of retroactive
check on neutralization is invalid.
Neutralizers can be used in the diluent or the plating medium, or both.
The goal of a neutralizer is to inactivate the biocide before the biocide
inactivates the microorganism and thus allow uninhibited microbial
growth. Failure immediately to inactivate the biocide upon sampling
causes overestimation of the biocide's killing potential. This failure is
actually a measure of the kill that continues even in the recovery of plating
medium because active biocide is carried over into the medium. 1oo Of
course the neutralizer itself must not be toxic to the organism. For
example, sodium bisulfite is a good neutralizer for formaldehyde and
glutaraldehyde but it inhibits the growth of some bacteria and the
germination of spores. In order to determine neutralizer toxicity the
number of survivors after exposure to the neutralizer are compared to
those surviving after exposure to the growth medium. Lecithin, polysorbate
80 and sodium thiosulfate are neutralizers for quaternary ammonium
compounds, phenolics and halogens, respectively. Studies by Sutton and
Brannan suggest that the type of neutralizer used for each biocide and
microorganism combination is unique. 101,102 However, a fairly effective all
purpose (universal) neutralizing medium is the Dey-Engley. It contains
sodium thioglycollate, sodium thiosulfate, sodium bisulfite, lecithin and
polysorbate 80.100,103 It is available in broth or agar preparation. Some
preservatives only require dilution to be inactivated (e.g. some alcohols,
organic acids and aldehydes).

7.3.2.5 Recovery of inoculum after apparent kill. All of the recognized

tests require long incubation periods (28-56 days). These long periods are
supposed to account for the phenomenon of adaptation. After a lag phase
the microbes 'grow back' to high enough levels to be detected again. The
mechanism(s) for this regrowth are not well understood. Perhaps it is due
to survival and adaptation. Perhaps it is due to in situ recovery of injured
 PRESERVATION OF PERSONAL CARE PRODUCTS 171

organisms. 104 It may be due to container-associated organisms that slough

off into the prodUCt. lOS •I06 It may even be due to inadequate mixing and
inconsistent plating methods, since bacteria display Poisson distribution in
the sample. Bacteria rarely, if ever, are evenly distributed in a sample
despite the vigorous shaking to which it is subjected. Thus the adapting
microorganisms may be located in a portion of the product where they are
never sampled. Unless the entire test volume is cultured at each sampling
interval complete kill of the inoculum cannot be certain. Late sampling
intervals must be completed even when total and rapid kill of the
challenge inoculum seems to have occurred.
Finally, the question of bacteria living not as isolated cells but in clumps
(perhaps in communities) should not be ignored as previously addressed.
We need to throw off the dogma that bacteria exist as single evenly
dispersed entities that can be isolated from one another. We even need to
look at microbial contamination from a community of several species
viewpoint rather than a solitary species population standpoint. Even
though historically, most contamination incidences have appeared to be
due to individual species, it may be more likely that what is observed is just
a transient successional event set up by previous contaminants. The cause
and effect relationships of pure cultures that cause disease (Koch's
postulates) apply when studying isolated populations that are selected by a
host's defenses. However, the pure culture, clinical microbiology paradigms
do not apply to environmental microbiology problems including cosmetic
contamination. We should be studying multi-species communities as
contaminants rather than isolated species in pure cultures.

7.3.2.6 Enrichment. Enrichments of the product can be performed to

detect low levels of potentially recoverable organisms. I07 The sensitivity or
detection limit of typical dilution and plate count methods is usually from
10 to 20 cfu g-I of product. In enrichment, at least 10 g of product is put
into 1 I of broth and incubated. Any turbidity (or color change if one uses
either a pH or redox indicator) indicates at least one organism was present
in the 10 g sample. This approach makes the detection limit 1 cfu per 10 g of
product (theoretically 0.1 cfu gm- I). Thus the sensitivity is increased by
100 times compared to traditional plate count methods. It is most useful in
determining if, after the 28 day period, low levels of inoculum still exist
that may still be capable of adapting and growing later on.

7.3.2.7 Interpretation of data. All the compendial tests prescribe how to

interpret the data. This interpretation is not related to any field data on
how the product should behave in the hands of the consumer. Instead, it is
based on anecdotal evidence and opinion regarding how long a product
should take to reduce the numbers of the challenge inocula.
The best way of interpreting the data is to compare how the test product
 172 PRESERVATION OF SURFACTANT FORMULATIONS

performs compared to known well-preserved and poorly preserved control

products. The well-preserved products should be defined as those that do
not become contaminated during consumer use and the poorly preserved
products should be those that do become contaminated (on a statistical
basis using a chi-square analysis with p = 0.05). This allows a test of the
inoculum to be sure it is capable of contaminating a product that is poorly
preserved but not so robust as to grow in well-preserved product.
Some microbiologists use the PET to predict the likelihood of
manufacturing contamination. They develop their preservatives to with-
stand manufacturing abuse from a dirty environment or contaminated raw
materials. This is the wrong approach for designing a PET. The
microbiologist should spend more time focused on bringing the plant into
sanitary control than developing preservatives to hide sloppy manufacture.
The performance criteria that characterize an acceptably preserved
product vary according to the compendial method used. The data are in the
form of cfu g-l product. For most PETs, the data points are the plate
counts from samples taken at 0, 7, 14, 21 and 28 days after inoculation.
USP does not include the zero day count. CTF A also includes a day one
count and, for eye cosmetics, a three day count. In the CTFA and ASTM
methods, the vegetative bacterial counts should be reduced to 0.1% of
their original numbers within seven days of inoculation. This is a 99.9% (or
3 log) reduction (from 106 to 103 cfu g-l). The counts should remain at 103
or continue to decrease for the rest of the test. The USP (for category I-A
products) allows 14 days for a 3 log reduction of bacteria to occur and then
the final 28 day count should be no more than 150% of the 14 day count.
The yeast and fungal counts must be reduced by 90.0% (a 1 log reduction)
after seven days for the CTFA and after 28 days for the ASTM. The USP
(for category I-A products) expects that by 14 and 28 days the level of
yeasts and fungi will be 'not more than 150% of the initial inoculum.'
Other USP product categories (IB-D and II) require less stringent
microbial reductions. The 150% variability rule is being reconsidered for a
change to 'not more than a 0.5 log increase over the initial inoculum.' The
USP does not give details how to treat the seven day data at this time.

)
7.3.3 Other PET methods

7.3.3.1 D-value methods. Long term tests are intended to mimic

incubation times that reasonably parallel the life of the product while in use
by the consumer. Within individual companies, however, rapid tests are
sometimes used for quick impressions of which preservatives to use in a
product. One such method is the D-value method. Aside from one author,
no one else claims D-value methods are valid for final testing of nationally
distributed product. 108 In fact, there is evidence that D-value methods are
 PRESERVATION OF PERSONAL CARE PRODUCTS 173

inappropriate for at least some consumer products 109 since D-values rely
on first order rate kinetics and biocide kills are not described by first order
rate kinetics.
The D-value method uses short sample times and estimates the final
response at 28 days by linear regression. The claim is that each organism
has a characteristic rate of death. By multiplying the death rate by the log
of the inoculum challenge, the method is supposed to predict the time it
takes to inactivate the entire challenge. This method is an adaptation from
food microbiology's heat or radiation destruction D-values. These two
methods of microbial kill do indeed follow first order rate kinetics and
therefore the D-values determined for them are quite valid. The major
weakness with applying the D-value technique to preservatives and other
chemical means of disinfection is that these follow second order rate
kinetics where the microorganism must first react with the disinfectant to
result in the product: dead microbe and spent disinfectant. When
disinfecting with heat or radiation, the reaction does not depend on the
microbe and the agent interacting first before the 'reaction' product of
dead microbe can take place. The only case where a second order reaction
can approach pseudo-first order rate kinetics is when the second reactant is
present in such large excess that it is virtually in constant concentration. It
is rarely the case however that preserved products have an excess of
preservative in such great abundance that the biocide remains in constant
concentration. The second criticism of the D-value technique is that it
extrapolates beyond the measured data by falsely assuming a linear
relationship between biocide exposure time and the number of surviving
microorganisms.
In defense of the rapid D-value methods, it allows at most a preliminary
screening of preservatives despite its invalidity. This approach assumes
that appropriate reproducible controls are in place such that the various D-
values can be ranked amongst a wide variety of products and then the data
can be correlated to full scale PET results on the same products. However,
the microbiologist should never rely on D-values as the sole method of
testing for preservative efficacy.

7.3.3.2 Capacity tests. A capacity test determines how many bacterial

challenges are needed before the product begins growing micro-
organisms. 110 After each challenge the products are sampled and challenged
again until the product either receives 15 challenges without showing
growth (a well-preserved product) or until three consecutive positive
results occur (a lesser preserved product). The goal is for the product to
reduce the number of viable organisms in the inoculated formulation by
3 logs (99.9%) in 48 h. With each subsequent challenge, this ability
diminishes as a result of dilution, neutralization and reaction with the
inoculum. The value of such a test may be for multi-use products.
174 PRESERVATION OF SURFACTANT FORMULATIONS

However, to obtain a more predictive test of consumer contamination

potential, the challenge inocula should probably be lowered to levels likely
to be encountered during use. Once done, the capacity test may be a more
quantitative and valid way of understanding a product's ability to handle
contamination by use.

7.4 Preservatives available for use

7.4.1 Mode of action of preservatives

The modes of action of antibiotics are known at the molecular level
because of their highly specific biochemical reactions. With preservatives
and biocides, the modes of action are far more generalized with points of
attack at numerous sites. With few exceptions, nearly all biocides work by
denaturing proteins throughout the cell or by affecting membrane
permeability so that either transport or energy generation is blocked.
Biocides such as formaldehyde, formaldehyde releasers, isothiazolinones,
bromine and chlorine compounds act by protein denaturation. The
parabens and weak acids like sorbic, benzoic and dehydroacetic acid act by
disrupting control of membrane electrical potential and its permeability to
block energy generation and loss of transport, respectively. Of course, any
compound that denatures proteins will also denature membrane proteins
to inhibit transport of essential materials via permeases.
With some biocides, it is almost as simplistic to ask what is the mode of
action of heat in killing microorganisms. The effect of chlorine, for
example, is to oxidize every reduced site that exists in a cell and, as a
result, denaturation of proteins takes place. Chlorine is a virtual poison.
Thus, very few cases of true chlorine resistance occur. Instead, the
resistance seen is really a popUlation or community effect of cells existing
within the protection of a biofilm or surrounded by an incredible capsule
that excludes the chlorine.
One compound that is not technically a biocide but rather an adjuvant to
most biocides is ethylenediaminetetraacetic acid (EDTA). This chelating
agent and others remove magnesium divalent cations from the cell wall and
cell membranes which are needed for stability. Once destabilized, they
permit easier access of biocides into the cell. One illustrative event
personally encountered was a formaldehyde-resistant Enterobacter spp. in
ammonium lauryl sulfate. A simple capsule stain indicated that this
organism had an incredible capsule surrounding the cell that was nearly
five times as thick as the organism's width. The capsule was scavenging out
the formaldehyde to allow the organism to grow. A simple addition of
0.1 % EDTA (disodium salt) permitted the formaldehyde once again to be
effective in this surfactant apparently because the EDTA removed the
 PRESERVATION OF PERSONAL CARE PRODUCTS 175

capsule. Had the organism been a Pseudomonas spp. with a formaldehyde

dehydrogenase we would not have been so fortunate with simple EDT A
addition.
When bacterial cells are exposed to parabens, nutrient transport is
inhibited. 111 Thus the parabens apparently inhibit nutrient uptake by
shutting down permeases, disrupting porin channels, or by disrupting the
membrane pH gradient or electrical charge potential across the membrane
to prevent substrate transport and A TP generation. This inhibition is
apparently reversible and is consistent with other observations that the
mode of action of parabens is by disruption of the membrane electrical
potential. 112
Organic acids probably work in the same fashion 112 ,113 however, they
may even be enzyme inhibitors as wellY4,115 Typically, they are only
biocidal at pH values below their pKa . In this protonated form, they pass
through the membrane and the hydrogen ion dissociates from the weak
acid to decrease the cytoplasmic pH. As a result, both substrate transport
and oxidative phosphorylation are uncoupled from the electron transport
system. This effectively starves the cell of needed substrate and energy
derived from ATP kinase driven by hydrogen ions.
Phenolics also disrupt the proton motive force of the cell membrane 116 ,117
but they have the ability non-specifically to denature cytoplasm, cell walls
and cell membranes. 118 The more lipophilic phenolics have the greater
antibacterial capacity, perhaps because of a greater ability to partition out
of the water phase and into the lipid membrane. 119 ,120 Alcohols likewise
disrupt the membrane causing permeability loss, 121 but they also appear to
inhibit enzymes. 122
Perhaps one of the most widely used of the newer preservatives are the
isothiazolinones. These are usually compounded into a single product
composed of chloromethylisothiazolinone and methylisothiazolinone, but
they can also include benzyl-type compounds as well. 123 ,124 The iso-
thiazolinones inhibit glucose oxidation and active transport without
significantly affecting membrane integrity.125 In fact, these compounds
denature thiol-containing proteins and enzymes (e.g. ATPase and
glyceraldehyde-3-phosphate dehydrogenase). The isothiazolinones
inactivate proteins and enzymes that contain thiol groups (e.g.
asparaginase).125 Thus, the isothiazolinones attack proteins with thiol-
containing amino acids. Initially, the isothiazolinone forms a disulfide link
with the thiol group on the amino acid. Occasionally, the chloromethyl-
isothiazolinone may facilitate linkage with another thiol group to establish
a new disulfide linkage and release the biocide as a mercaptoacrylamide.
This mercaptoacrylamide can tautomerize to a thioacyl chloride which may
react again by denaturing nucleic acids. 126
Formaldehyde also denatures protein but by alkylating amino and
sulfhydril groups; it can also alkylate the nitrogens of purine rings to
176 PRESERVATION OF SURFACTANT FORMULATIONS

denature DNA.127 Most of the formaldehyde donors (e.g. Glydant®,

Germall®, Dowicil®, Nuosept®, etc.) act in this basic manner since
these compounds release formaldehyde into the microbial cell. Differences
seen between the formaldehyde donors may exist as a result of when or
what triggers the compound to release formaldehyde. For example, a
compound with a long hydrophilic chain connected to the formaldehyde
donating region (e.g. Nuosept®) may release formaldehyde only when the
long chain enters into the LPS section of the membrane.
Brominated compounds such as bromonitropropanediol and bromo-
nitrodioxane act by oxidation of thiol groupS128-131 or by causing thiols to
convert to disulfides 132 ,133 where the thiol group first becomes brominated
and then reacts with another thiol group to yield a disulfide and free
bromide. As a result, enzymes involved in respiratory activity (e.g.
dehydrogenases) and nucleic acid synthesis are inhibited, cell membrane
integrity is compromised, and the cell wall may even be affected. 133 ,134

7.4.2 Selection of preservative

The microbiologist uses a variety of chemical preservatives to prevent
contamination by pathogens or spoilage microorganisms. The ideal
preservative would be broad spectrum, safe and completely free of any
sensitization issues, completely water soluble, completely stable to all
extremes of pH and temperature, completely compatible with all ingredi-
ents and packages, impart no color or odor to the product and be
inexpensive. This ideal does not exist. Instead, a preservative must be
selected based on empirical testing. The only approach bordering on a
theoretical basis for choosing a preservative is a qualified microbiologist's
intuition finely honed by experience. There are published lists of available
preservatives135 and certainly no dearth of advertisements in trade
magazines. 136 These are good places for getting ideas of what might work
in a formulation, but only the novice relies entirely on these lists or places
much faith in the advertising claims when choosing a preservative. Every
formulation must be considered unique. Factors such as the physical and
chemical nature of the product, how it is to be used, the container type and
the shelf life must be considered when choosing the preservative. Often the
selection of a preservative must be a compromise between efficacy,
stability and safety.

7.4.3 Safety considerations of preservatives

One must always balance the risk of microbial contamination with the risk
that a biocide may give to a product. For example, many eye area products
were permitted to contain phenyl mercuric acetate because the risk of
infection to the eye was greater than the risk of exposure to the compound.
 PRESERVATION OF PERSONAL CARE PRODUCTS 177

The key consideration is to judge whether the product will be safe for the
consumer under normal use and foreseeable misuse conditions. This
section will not make a microbiologist into a good toxicologist any more
than reading this chapter would make a toxicologist a good microbiologist.
Instead, it will allow both to have a basic understanding of the other's
responsibility to allow a positive and productive interaction.
One of the first considerations of a preservative is its acute toxicity. In
cosmetics, the route of exposure is dermal but often oral routes of acute
toxicity are also needed in cases of accidental ingestion. Occular irritation,
subchronic and chronic toxicity tests are performed via the expected
consumer exposure route to determine at what level the preservative can
exhibit any irritant, toxic or carcinogenic properties. These levels will then
be compared with typical use exposure levels to determine safety ratios.
Perhaps more important than these tests are the skin responses to
biocides. Basic irritant responses can be a result of corrosion, acute
irritation, cumulative irritation or photoirritation. In cosmetic use of a
preservative, most testing involves placing the biocide onto the skin using
an occlusive patch dosed with the compound. After a time, the patch is
removed and the skin graded as to how irritated it became.
Skin sensitization is another key concern when using biocides. Nearly all
biocides used today will elicit sensitization in some people. Sensitization
testing is performed in much the same way as irritant patch testing except
much lower concentrations are used. Even in-use sensitization studies can
be used as well.
Another concern for biocides is mutagenicity testing to determine if the
biocide has the potential to mutate somatic or germ cells. In addition to
this testing, embryological (or developmental) toxicity testing is done to
determine if the biocide may be a teratogen capable of causing birth
defects.
In all these tests, the results must be compared to the ordinary use and
foreseeable misuse exposure levels to give a reasonable risk assessment.
Often the test data are expressed as no observed effect level (NOEL - the
highest dose without causing a toxic effect). Consumer exposure levels of
one-tenth the NOEL (a lOx safety factor) based on human testing may be
considered safe to use under some circumstances. This safety factor may
increase up to 1000 times depending on whether the data used are animal
data or where insufficient chronic exposure data are lacking. The definition
of reasonable risk must include considerations based on the benefits of
using the biocide, the ability to use less risky biocides for the same use, the
economic benefits of using the biocide (can the biocide help prevent costly
recalls due to contamination?), even how the biocide may affect quality of
life, environment and public opinion about the company.
178 PRESERVATION OF SURFACTANT FORMULATIONS

7.5 Conclusion

The personal care product (cosmetic) microbiologist must balance a variety

of factors to provide safe, unspoiled quality products. 137 In addition to
knowing preservatives, he or she must understand microbial physiology,
pathogenic microbiology and microbial ecology. In addition to micro-
biology, he or she must understand organic and physical chemistry,
toxicology, engineering, manufacturing and processing, sanitation, and
regulatory/environmental law. The cosmetic microbiologist must use all
this education and knowledge within the context of the business needs of
the company and be able to balance risk/benefit to the consumer using the
product. The microbiologist that best fits this description is the one who
has a good solid liberal arts undergraduate education followed by post
graduate training in microbiology (preferably in microbial physiology and
ecology). He or she must be a critical thinker, a philosopher dedicated to
lifelong learning, and a true renaissance man or woman interested in a
broad range of subjects pertaining to the field. Finally and most
importantly, this person must have the highest of ethical standards. They
must consider themselves as part of the cadre of health care providers in
the world dedicated to serving humankind via the mission of providing
them with microbially safe and efficacious products.

Acknowledgements

I would like to thank the many colleagues who have helped me prepare this
chapter and, in particular, those who allowed me to base parts of this
chapter on drafts they have prepared for other publications. Their insight
has been very valuable. I thank Drs. Phil Geis, Bill Apel, Scott Sutton and
Tom Sox especially. I also thank Ms. Jan Curry, Mr. Stephen Richards and
Mr. Rich Hennessy.

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103. Engley, F.B. and Dey, B.P. (1970) A universal neutralizing medium for antimicrobial
chemicals. In Proceedings of the 56th Mid-Year Meeting of the Chemical Specialties
Manufacturers Association, Chemical Manufacturers Association, New York, pp. 100-
106.
104. Russell, A.D. (1991) Injured bacteria: occurrence and possible significance. Lett. Appl.
Microbial., 12, 1.
 PRESERVATION OF PERSONAL CARE PRODUCTS 183

105. Van Loosdrecht, M.C.M., Lyklema, J., Norde, W. and Zehnder, A.J.B. (1990)
Influence of interfaces on microbial activity. Microbiol. Rev., 54, 75-87.
106. Delaquis, P.J., Caldwell, D.E., Lawrence, J.R. and McCurdy, A.R. (1989) Detachment
of Pseudomonas fluorescens from biofilms on glass surfaces in response to nutrient
stress. Micro. Ecol., 18, 1999.
107. Curry, J. (1987) Thoughts on preservation testing of water-based products. Cosmet.
Toiletries, 102, 93-95.
108. Orth, D.S. (1979) Linear regression method for rapid determination of cosmetic
preservative efficacy. f. Soc. Cosmet. Chern., 30, 321-332.
109. Sutton, S.V.W., Franco, R.J., Mowrey-McKee, M.F., Busschaert, S.C., Hamberger, J.
and Proud, D.W. (1991) D-value determinations are an inappropriate measure of
disinfecting activity of common contact lens disinfecting solutions. Appl. Environ.
Microbiol., 57, 2021-26.
110. Barnes, M. and Denton, G.W. (1969) Capacity tests for the evaluation of preservatives
in formulations. Soap, Perfum. and Cosmet., 42, 729-733.
111. Eklund, T. (1980) Inhibition of growth and uptake processes in bacteria by some
chemical food preservatives. f. Appl. Bacteriol., 48, 423-432.
112. Eklund, T. (1985). The effect of sorbic acid and esters of p-hydroxybenzoic acid on the
proton motive force in Escherichia coli membrane vesicles. f. Gen. Microbiol., 131, 73-
76.
113. Freese, E., Sheu, C.W. and Galliers, E. (1973) Function of lipophilic acids as
antimicrobial food additives. Nature, 24, 321-325.
114. Martoadiprawito, W., and Whitaker, J.R. (1963) Potassium sorbate inhibition of yeast
alcohol dehydrogenase. Biochim. Biophys. Acta, 77, 536-544.
115. Troller, J.A. (1965) Catalase inhibition as a possible mechanism of the fungistatic action
of sorbic acid. Can. f. Microbiol., 11,611-617.
116. Prindle, P.F. (1983). Phenolic compounds. In Disinfection, Sterilization, and Preservation,
3rd edn, Block, S.S. (ed.), Lea and Febiger, Philadelphia, pp. 197-224.
117. Hugo, W.B. (1978) Membrane-active antimicrobial compounds - a reappraisal of their
mode of action in light of the chemi-osmotic theory. Internat. f. Pharm., 1, 127-131.
118. Hugo, W.B. (1983) Mode of action of non-antibiotic antibacterial agents. In
Pharmaceutical Microbiology, 3rd edn, Hugo, W.B. and Russell, A.D. (eds). Blackwell
Scientific, London, pp. 258-64.
119. Fogg, A.H. and Lodge, R.M. (1945) The mode of antibacterial action of phenols in
relation to drug-fastness. Trans. Faraday Soc., 41, 359-65.
120. Richardson, E.M. and Reid, E.E. (1940) Alpha, gamma-di-p-hydroxyphenyl-alkanes.
f. Am. Chem. Soc., 62, 413-415.
121. Silver, S. and Wendt, L. (1976) Mechanism of action of phenethyl alcohol: breakdown
of the cellular permeability barrier. f. Bacteriol., 93, 560.
122. Gilbert, P., Beveridge, E.G. and Crone, B.P. (1977) The lethal action of 2-
phenoxyethanol and its analogues upon Escherichia coli NCTC 5933. Microbios, 19,
125-14l.
123. Law, A.B., Moss, J.N. and Lashen, E.S. (1984) Kathon CG®, a new single-
component, broad spectrum preservative system for cosmetics and toiletries. In
Cosmetic and Drug Preservation; Principles and Practice, Kabara, J.J. (ed.), Marcel
Dekker, New York, pp. 129-141.
124. Collier, P.J., Ramsey, A., Austin, P. and Gilbert, P. (1990) Growth inhibitory and
biocidal activity of some isothiazolone biocides. f. Appl. Bacteriol., 69, 569-577.
125. Fuller, S.J. (1985) The mode of action of 1,2-benzisothiazolin-3-one on Staphylococcus
aureus. Lett. Appl. Microbial., 1, 13.
126. Collier, P.J., Ramsey, A., Waigh, R.D., Douglas, K.T., Austin, P. and Gilbert P.
(1990). Chemical reactivity of some isothiazolone biocides. f. Appl. Bacterial., 69, 578.
127. Grossman, L., Levine, S.S. and Allison, W.S. (1961) The reaction of formaldehyde with
nucleotides and T2 bacteriophage DNA. f. Malec. Bioi., 3, 41-60.
128. Shepherd, J.A., Buerby, M.R., Pemberton, D., Gilbert, P. and Waigh, R.D. (1987)
The reactions of Bronopol® (2-bromo-2-nitropropan-l,3-diol) with thiols. f. Pharm.
Pharmacol., 39, 116P Supplement.
129. Zsolnai, T. (1961) Versuche zur entdeckung neuer fungistatika II. Nitroverbindungen.
Biachem. Pharmacal., 5, 287-304.
184 PRESERVATION OF SURFACTANT FORMULATIONS

130. Croshaw, B., Groves, M.l. and Lessel. (1964) Some properties of Bronopol®, a new
antimicrobial agent active against Pseudomonas aeruginosa. J. Pharm. Pharmacol.
Suppl., 16, t27T-130T.
131. Croshaw, B. and Holland, V.R. (1984) Chemical preservatives - use of Bronopol® as a
cosmetic preservative. In Cosmetic and Drug Preservation; Principles and Practice,
Kabara, 1.1. (ed.), Marcel Dekker, New York, pp. 31--62.
132. Ghannoum, M., Thompson, M., Bowman, W. and AI-Khalil, S. (1986) Mode of action
of the antimicrobial compound 5-bromo-5-nitro-1,3-dioxane (Bronidox®), Folia Micro-
bioI., 31, 19.
133. Stretton, R.I. and Manson, T.W. (1973) Some aspects of the mode of action of the
antibacterial compound Bronopol® (2-bromo-2-nitropropan-1,3-diol). J. Appl.
Bacteriol., 36, 61-76.
134. Naito, R., Itoh, T., Hasegawa, H., Arimura, H., Fujita, Y., Hasegawa, K., Inaba, T.,
Kagitani, Y., Komeda, S., Matsumoto, T., Okamoto, H., Okano, K., Oguro, Y. and
Ogushi, T. (1974) Bronopol® as a substitute for thimersal. Develop. Bioi. Stand., 24,
39-48.
135. Wallhausser, K.H. (1984) Antimicrobial preservatives used by the cosmetic industry. In
Cosmetic and Drug Preservation; Principles and Practice, Kabara, 1.1. (ed.), Marcel
Dekker, New York, pp. 605-745.
136. One trade magazine that routinely publishes what they call a 'Cosmetic Preservative
Encyclopedia' is Cosmetics and Toiletries published by Allured Publishing Corp. IL. The
last of these was published in their October 1993 issue.
137. Brannan, D.K. (1993) Cosmetic Microbiology. In Encyclopedia of Microbiology,
Lederberg, J. (ed.), Academic Press, San Diego, pp. 593--603.
8 Preservation of paint formulations
R.D. YORE and l.A. PIERCE

8.1 Introduction

Cost effective use of modern biocides in paint formulations requires an

understanding of the susceptibility of the formulation, consequences of
degradation, laboratory optimization and proper plant hygiene. Poor
hygiene, the use of spoiled raw materials and ineffective preservative
selection can lead to intermittent spoilage and produce long term
contamination problems. Understanding the relationship between these
factors is essential. This chapter is intended to provide the formulation
chemist or microbiologist with the background necessary to produce high
quality, cost effective product in light of the possibility of microbial
contamination.

8.2 The chemistry and manufacture of paint

The terminology used to describe paint formulations is quite varied

depending on the company, nationality and industry involved. Powder
finishes are dried mixtures of pigment and binder that are electrodeposited
on the substrate. Bake-on, UV curables, epoxy systems (both one and two
component) and industrial finishes are normally solvent-borne systems.
With reductions in solvents being mandated under environmental pressures
most major manufacturers are developing water-borne systems. Solvent-
borne alkyd paints are rapidly being replaced by water-borne latex
systems. Durability and application characteristics of the water-borne
systems are beginning to match critical characteristics of solvent-borne
alkyds in many areas. The latex area itself is divided between exterior and
interior use.
Dispersants, wetting agents and coalescing agents are functional
surfactants in paints. Paint, reduced to its simplest form, consists of
pigment, a binder and a solvent. Pigments are any material that imparts a
final color. Pigments may be either mineral or organic. Mineral pigments
include various iron oxides, calcium carbonate and titanium dioxide. The
binders or film formers cross link after application to form a solid surface
trapping the pigments in place. In latex paint these are usually acrylic and
186 PRESERVATION OF SURFACTANT FORMULATIONS

vinyl acrylics though polyvinyl acetates are also used. Solvents are
designed to aid in application and delivery of the system to the intended
target. Prior to the introduction of water-borne systems hydrocarbon
solvents were used in paint manufacturing. These solvents also acted as
preservatives. In-can spoilage of solvent-based paints is quite rare. This
chapter will be limited to spoilage of water-borne latex paints. Water-
borne latices require the use of a co-solvent and contain up to 38% ethylene
and propylene glycol. The exception to this is low volatile organic carbon
(VOC) paints which contain minimal volatile organic solvents. The binder
and solvent together constitute the vehicle for the pigment. Numerous
modifications to the basic formulations are possible. White washes are
formulations consisting of pigment, calcium carbonate and water. Varnishes
are binders and solvent without pigment. Solid materials added to the
formulation that do not impart color are called extenders or thickeners. 1
Cellulosic extenders are inexpensive materials and are used in place of
expensive pigments. They also provide high viscosity to hold ingredients in
a homogeneous blend and provide rheological properties important during
application. Clays and associative thickeners are also used. 2
Paint manufacturing consists of two steps: dispersion or grind and let
down. During the manufacturing of mineral pigments, large blocks of
material are reduced in physical size to produce uniform small particles. In
shipping and storage the primary particles absorb moisture and air on to
their surfaces and then clump together to form agglomerates or secondary
particles and aggregates or tertiary particles. Agglomerates are primary
particles in edge-to-edge contact. Aggregates are primary particles in face-
to-face contact. Agglomerates are stabilized by weak electrostatic charges
whereas aggregates are stabilized by strong charges. In the dispersion step
of paint manufacture the agglomerates are disrupted to produce a
homogeneous slurry of primary pigment particles. Aggregates are difficult
to disperse without imparting large kinetic energies that may destroy the
crystal structure of the primary particles. The dispersion process relies both
on chemical dispersants and high shear forces. Dispersants are added to
electrically charge the pigment particles and thus allow uniform distribu-
tion. 3 Tamol 731 is commonly used. Tamol 731 is a sodium salt of a
polymeric carboxylic acid. Wetting agents are used to allow uniform
contact of the aqueous phase over the surface of the solid particles.
Commonly used wetting agents are Igepals from Rhone Poulenc and
Tritons from Rohm and Haas. 4 Igepals are nonionic surfactants of
nonylphenoxypoly( ethyloxy)ethanol. Tritons are polyoxyethylene ethers.
Dispersants and wetting agents reduce the kinetic energy input required to
produce a uniform product. Once the pigment and mineral fillers have
been incorporated the binders and extenders can be added. These are
added at low speed and low shear. Additional dispersants are added at this
time.
 PRESERVATION OF PAINTS 187

Paints must be shelf stable emulsions and dispersions. Excess settling

will alter the application rheology and coverage of the product. Viscosity
stability during storage is thus of vital importance. Rheology is maintained
by incorporating thixotropic agents and the correct selection of dispersants.
Paints rely on more than one pigment component. Organic pigments have
very large hydrophobic surface areas compared to the smaller hydrophilic
surfaces of mineral pigments. The stability of the emulsion requires that all
of the particles of varying surface tensions and charges be maintained in
dispersion at relatively constant concentrations, otherwise selected particles
may either flocculate or settle and the performance of the paint will be
diminished. Combinations of dispersants and wetting agents are required
to achieve adequate stability.
Properties of the paint formulation during application are critical to user
acceptance. The binder resins must blend smoothly with one another
during drying; this is referred to as coalescing. Coalescing agents are added
during the let down. During the initial drying process water evaporation
leaves large voids in the film. The coalescing agent allows the film to
maintain fluidity and thus form a uniform film of the binder and pigment
phases. Coalescing agents are high boiling point solvents that are miscible
with the liquid phase. The coalescing agents thus act as surfactants by
coupling the binder and liquid phase while wet and the binder and pigment
during the drying period. Texanol and butyl carbitol are commonly used.
Butyl carbitol is diethylene glycol mono butyl ether.
Reduced solvent latex paints are being introduced by a number of major
companies in the USA and Europe. These include both architectural paints
and water-borne latices to replace solvent-borne marine maintenance
coatings. In the United States VOCs are measured by the imprecise US
Environmental Protection Agency Method 24 where solvent contents are
inferred by drying loss rather than quantitatively measured. This rather
poor method has caused the definition of Zero VOC to be questioned
scientifically and hotly debated among raw material suppliers and paint
producers. Simultaneously, both the total volatile content and the types of
volatiles released are being scrutinized for additional legislation. 5 Present
labeling requirements in the United States reveal little useful information
on the actual solvent content of paints. 6 It is generally agreed that solvent
levels will be reduced in future latex paints. How this trend will affect their
microbial susceptibility remains to be seen.

8.3 Consequences of microbial spoilage

The preservation and spoilage of paint concerns two quite different

physical states of the material. The wet state is an aqueous state prior to
use and desiccation associated with final application. The spoilage, or more
188 PRESERVATION OF SURFACTANT FORMULATIONS

properly the defacement, of the dried paint is the result of surface growth
on the applied and cured film itself. The organisms involved and the
substrates utilized in these two different types of contamination are consider-
ably different. In the wet state water is the dominant solvent with lesser
amounts of surfactants and binders also being present. In the wet state
spoilage is normally a result of bacterial growth with yeast as an occasional
factor. The microorganisms utilize either the surfactants or the cellulosic
thickeners during spoilage of the wet state. The defacement of dried paint
films is apparently the result of a complex community of microorganisms
including bacteria, fungi and algae. This succession of organisms has only
recently begun to be investigated. 7
Exterior paints are subjected to continuous UV damage, changes in
temperature and humidity and nearly every microorganism that can
possibly survive in the soil. Interior paints are applied, used and survive in
the much more hospitable environment of the indoor human habitat. As
such, the extremes of environmental conditions are lacking. Formulations
of components utilized to survive in these two environments are quite
different. These changes are significant in that they affect not only the
performance of the dried film but also the resistance of wet paint
to microbial degradation. The defacement of exterior paints is the result of
either fungal or algal growth. Therefore, exterior latex paints incorporate a
fungicide. The fungicide is designed to leach slowly from the film and
inhibit fungal growth. Commonly used fungicides are presented in Table
8.l.
Algal growth is a recognized problem in masonry paints. Both algae and
fungal growth may appear as black stains. The actual amount of algal
defacement on house paint in unknown but it is now under extensive
investigation. 8 The defacement of dried films does not involve the
degradation of surfactants since these have been volatilized during the
process. Defacement processes are beyond the scope of this work and are
not addressed further. The investigations and studies of fungal defacement

Table 8.1 Commercial paint film fungicides used in the USA

Chemical name of active CAS number Trade name Producer

agent of
active agent

Diiodomethyl-p-tolylsulfone 20018--09-1 Amical® Angus

3-iodo-2-propynyl butyl 55406-5H Troysan® Polyphase Troy Chemical, Inc.

carbamate

Tetrachloroisophthalonitrile 1897-45-6 Nuocide® 960 Hills

2-n-octyl-4-isothiazoline-3-one 26530--20--1 Skane® Rohm and Haas

 PRESERVATION OF PAINTS 189

and control are numerous. Several articles are cited for further
reference. 9 - 13
To prevent UV damage to latex film many exterior formulations use zinc
oxide. The presence of a fungicide with or without zinc oxide in exterior
formulations makes these paints naturally resistant to most bacterial attack
in the wet state. Neither of these are true antibacterial agents in the wet
state but they do raise the bioresistance level of the paint. Since it is
bacteria that are responsible for wet state spoilage the exterior paints of
most US companies are able to survive with approximately half of the
preservative required in interior paints. This rule holds true independent of
the preservative type utilized. Spoilage patterns of interior paints are also
subject to formulation vagaries. Gloss, semi-gloss and egg shell formula-
tions contain more binder than pigments. Formulations where the binder
content exceeds that of the pigment binder are called under critical. Flat
formulations contain more pigment than binder. Where the pigment
exceeds the binder content the paints are referred to as overcritical paints.
Quality factors, component types and variation of the ratio of components
allow the production of first, second and third lines of paints. This
characterization refers not only to cost but also to the tangible factors such
as scrubability. The demands on the surfactant in these two conditions are
different. Flat formulations are cheaper to produce since they are based
on more inexpensive components. Not surprisingly the microbial suscept-
ibility of gloss and egg shell paints is quite different from that of flat paints.
This appears to be the rule whether acrylic or PVA-based paints are
examined.
Spoilage of paint in the wet state is noticeable to the untrained observer
as an off odor, off color, bulged container, broken emulsion and loss of
viscosity. The symptoms observed depend on the type of species of
microorganism involved. Bacteria are responsible for the vast majority of
in-can spoilage. In measuring contamination levels the standard unit of
measure is colony forming units per gram (cfu g-l). In most cases a single
colony of bacteria arises from one bacterial cell. Some bacteria clump or
form chains, such as Staphylococcus and Bacillus, respectively. In these
cases bacterial levels may not be equivalent to colony forming units. For
other than philosophical reasons the distinction between the two terms is
meaningless. Paint is normally sterile. Vegetative growth in the can is
indicative of some spoilage. Bacterial levels below 105 cfu ml-1 will have
little long term impact on the paint. As levels rise above 105 the rate of
deterioration becomes a relationship between level and time. At popula-
tions of 106 it may take weeks or months before any detectable change is
noticed. Off odors are noticed as the spoilage proceeds and bacterial levels
rise past 106 . With populations of 108 the complete and irreversible
destruction of the product may occur in several days. Growth rates and
associated spoilage rates are also temperature dependent. In pragmatic
190 PRESERVATION OF SURFACTANT FORMULATIONS

terms this results in three or four times faster spoilage rates during hot
summer months than the cold winter months. Once the emulsion has been
broken the paint is no longer salvageable. Interventions with appropriate
antimicrobials prior to breaking the emulsion are possible and used
commonly by manufacturers. The production of organic acids may occur in
both bacterial and yeast spoilage. If sufficient acid production occurs the
pH of the paint will be compromised. If the formulation has a pH-sensitive
rheology it may be necessary to make corrections after biocidal treatment.
A comprehensive review of biodeterioration of paint is mentioned for
further reference. 14
Can bulging and copious out-gassing are normally associated with yeast
spoilage due to the genus Candida. Candida spp. are able to degrade a
number of hydrocarbon compounds under the anaerobic conditions that
exist inside of a sealed paint can. The out-gassing observed with Candida
spoilage is a result of the liberation of carbon dioxide during the
fermentation process. The gas pressure developed in the sealed cans may be
several atmospheres and the resulting explosion at can failure is dramatic.
The controlling factor in pressure buildup is the integrity of the container,
not biological since few metabolic systems are inhibited by even hyperbaric
pressures. Candida induced spoilage is associated with a sweet, apple-like
aroma of rising bread.
The loss of viscosity in-can is a substantially different spoilage problem.
Viscosity loss is almost always the result of degradation of the cellulosic
extenders. Cellulose is a biologically intractable polymer of glucose linked
through B1,4 bonds. Few organisms are able to degrade this particular
linkage, but among those that can are the soil fungi Trichoderma and
members of bacterial genera Bacillus. 15 The actual degradation of cellulose
is the result of secretion of the extracellular enzyme cellulase. Cellulases
are stable enzymes that are able to depolymerize considerable quantities
of cellulose with no biological cofactors or viable cells present. Spoilage
due to cellulose degradation is rare in US paints but stories of this problem
have been elevated to mythical levels. Industry lore is abundant with
stories of viscosity loss during the late 1970s through the mid 1980s. Most
of these stories are not substantiated by detailed microbiological reports.
Lack of documentation has contributed to the lore. Furthermore, non-
microbiologists are often baffled by cellulase degradation problems since
viable cells need not be present.
Cellulase spoilage incidents appear to occur in similar patterns. Paints
are produced and held without an adequate in-can preservative, allowing
the development of spoilage microorganisms. As the spoilage is noticed the
bulk storage tanks are treated with a biocide to kill all viable cells and thus
recover the lot. This approach is not appropriate for cellulose degradation
since enzyme degradation is the culprit and biocides are designed to kill
viable cells. Biocides may interfere with specific enzymes but these are
 PRESERVATION OF PAINTS 191

endogenous enzyme systems vital to cellular function not exo-enzymes

liberated from the cells which are of little consequence to the continued
viability of the cell. The inappropriate use of biocides to recover cellulase-
damaged paints is problematic. After 'recovery' the paint technologist may
believe that spoilage incident is under control since standard practice is to
measure spoilage by the number of viable microorganisms present. But in
this case recovered batches will still contain active cellulase but no viable
bacteria. The recovered material is then either packaged or blended off
with new paints. In the latter cases the dilution approach allows one lot of
cellulase-containing paint to comingle with a large number of otherwise
good lots. In either system the cellulase continues to function and the
viscosity of the paint approaches that of the solvent, water. Cellulase-
degraded paints are seldom recoverable for this reason. In typical incidents
of this type, production plant personnel try repeated conventional
approaches to kill the offending bacteria over a period of several months.
It is the hours of fruitless efforts and usually total loss of all material
produced during these incidents that allow the lore of cellulase.
Effective control of cellulase spoilage can only be regained by complete
cleaning of the entire production facility or by switching to sterically
hindered thickeners. The potential for viscosity loss remains so significant
in manufacturers' minds than any possible indication results in a rapid and
significant knee-jerk reaction. Present manufacturing facilities and formula-
tions now in commercial production are not particularly susceptible to
cellulose degradation. Concerns about cellulose degradation are misplaced
since preservatives used for in-can protection also provide activity against
both surfactant and cellulose-degrading species. As long as adequate
preservative levels and types are maintained in cellulose slurries incidents
of viscosity loss are unlikely to occur. Since this pattern of spoilage is
dependent on cellulose degradation the surfactant systems are not
involved. Further discussion on cellulose degradation and viscosity loss is
beyond the scope of this chapter. 16
The commercial implications of paint spoilage are of great interest to
manufacturers. Both professional and do-it-yourself users demand that
paint be of acceptable quality in the price range they are using. Off-
specification paint is returned to the point of purchase and replaced by the
sales agent. Profit margins in paint manufacture can only be maintained by
steady and high plant outputs. Spoilage incidents require both adminis-
trative and production personnel to modify their work patterns until the
situation is resolved. Contaminated materials must either be treated with
additional antimicrobial agent or disposed of. The material must be held
until then. If the material is recovered it is usually held in bulk tanks. Thus,
even small spoilage incidents result in reduced plant production, reduced
profit-oriented labor and increased biocide use. Under present environ-
mental legislation the disposal of non-recoverable paint is very expensive.
192 PRESERVATION OF SURFACTANT FORMULATIONS

In major incidents companies may institute a product recall. Recalls are

the nightmare of both production and marketing departments. Personnel,
shipping both the bad and replacement paint, lost production and disposal
costs are enormous in recalls. The loss of prestige associated with spoilage
and recalls is viewed as devastating to market managers. Loss of
permanent sales to established customers due to brand switching is likely if
the situation is poorly handled by the paint manufacturer. For these
reasons most paint companies are risk aversive where there is a possibility
of spoilage.

8.4 Requirements for microbial growth

The general requirements to support microbial growth include utilizable

nutrients, favorable environmental conditions including temperature, pH
and a reliable water supply. Oxygen is not required by most bacteria, yeast
and some fungi. Nutritional requirements for microorganisms range from
simple inorganic compounds to quite complex biopolymers. With very few
exceptions, microorganisms isolated from industrial products have simple
nutritional requirements and do not have obligate requirements for growth
factors such as vitamins. Yet these species are able to attack many
surfactants and some complex polymers such as cellulose and gums.
Nutrients include both carbon in appropriate structural form and all other
elements. Nitrogen in the correct form is second in importance to carbon.
Members of the genus Pseudomonas are frequent causes of industrial
spoilage. This genus is able to utilize over 2000 separate carbon compounds
as primary sources of energy and carbon skeletons. Appropriate tempera-
tures for bacterial growth range from 5°C to over 50°C. The pH range of
most microorganisms is about 3.5 to over 9.5. Water is critical for growth
of both bacteria and fungi. Products with very high solids and very low
water activity (a w < 0.9) experience few bacterial problems but remain
quite susceptible to fungal attack. Spackling and pre-mixed wall board
compounds are two examples. Bacteria typically reproduce by doubling
once every hour in industrial products. An unpreserved storage tank or
paint allowed to sit overnight could have over 108 bacteria per ml by the
next morning using a very small inoculation from previous batches or the
water. Aqueous-based products contain a variety of organic compounds,
are formulated in the 5.0 to 9.0 pH range, are produced, stored and used in
temperatures comfortable to life processes and by their nature supply
water. With this perspective, the necessity for incorporation of preservatives
into paint formulations should not be surprising.
 PRESERVATION OF PAINTS 193

8.5 Typical flora associated with paint spoilage

Microorganisms are ubiquitous in the environment. Several thousand

bacterial genera have been identified and millions of strains potentially
exist. However only a handful of types of bacteria and fungi are commonly
isolated throughout the world in paint formulations. Microorganisms
associated with paint spoilage in practice do not present a significant health
hazard to exposed personnel. The species involved are weakly pathogenic
at best and lack the general infectivity of opportunistic pathogens.

8.5.1 Bacteria
Pseudomonas spp. account for about 75% of the isolates from paint. The
most common species is Ps. aeruginosa with Ps. cepacia and Ps. fluorescens
also isolated. Ps. putida, Ps. putrifaciens and Ps. vesiculoans are rare.
Alcaligenes spp. are the second most common group isolated. E. coli and
Proteus spp. are also occasionally isolated. Bacteria that account for 5% or
less of spoilage include Citrobacter freundii, Morganella morganii, Bacillus
spp. and Corynebacterium spp. Yeasts are isolated from less than 5% of
spoiled paints. Candida albicans is the most frequent yeast isolated.
Rhodotorula spp. and Torulopsis spp. are occasionally isolated. I7

8.5.1.1 Pseudomonads. Pseudomonads produce a wide variety of

enzymes and thus have the ability to metabolize many different compounds
as sources of carbon and energy. Compounds such as aromatics, amines
and organics in the form of thickeners, surfactants, pH regulators,
antifoams and dispersants are prime candidates in a paint formulation.
This includes organic acids, sugars and polymers made from those sugars,
proteins, alcohols and surfactants and dispersants. Pseudomonads are
found in soil, water and every wet material including most water. Swabs of
facilities after rinsing with potable water should produce pseudomonads.
These are usually introduced from the potable water supply. Nearly every
moist environment will harbor some members of this genus. It should not
be surprising that this hardy group is responsible for most industrial
spoilage.

8.5.1.2 Bacillus. Members of the genus Bacillus are Gram-positive

spore-forming bacteria mainly associated with soil. They are occasional
primary spoilers of wet paint. They are readily controlled by all in-can
preservatives now in use. Misunderstandings among paint chemists exist
concerning the spoilage threat from Bacillus spores. Spores, regardless of
their origin, are dormant and lack any detectable metabolic activity.
Spores do not in themselves lead to spoilage. It is only after the spores
194 PRESERVATION OF SURFACTANT FORMULATIONS

germinate and lead to vegetative cells that spoilage is possible. Once

germination has begun the in-can preservative is able to kill the resulting
vegetative cell. Confusion occurs during microbiological examinations of
paints containing spores. As stated above if the paint is preserved
adequately outgrowth is not possible. During standard testing protocols
the paint is diluted by the addition of water and nutrients in the biological
growth medium employed. The resulting dilution decreases the in-can
preservative level to below that required to kill germinating spores. Thus
inadequately trained laboratory personnel may assume that spoilage due to
spores has occurred or is imminent. In reality the paint is quite stable and
under no threat of spoilage. Spoilage due to spores will only occur where
inadequate or unstable in-can preservatives are employed. Due to both the
possibility of confusion and lost laboratory time investigating spore
occurrences, spores should be eliminated as soon as possible. This is easier
said than done. Spores are able to survive several minutes exposure to
boiling water, repeated freeze thaw cycles and attack from most chemicals
including in-can preservatives. Elimination programs must include a
thorough cleaning of contaminated equipment and known sporicidal
compounds such as hypochlorous acid (bleach) and glutaraldehyde.

8.5.2 Yeasts

8.5.2.1 Candida. The genus Candida contains both industrially signific-

ant and pathogenic species. Most human infections caused by Candida spp.
are caused by personal microbial flora. 18 Candida spp. are notable for their
ability to transform hydrocarbon compounds under the anaerobic condi-
tions that exist inside of a sealed paint can. 19 Can bulging and copious out-
gassing are normally associated with yeast of the genus Candida. The out-
gassing is a result of the liberation of carbon dioxide during the
fermentation process. This is the same metabolic pathway employed by
Saccharomyces cereviseae in the production of bread, beer and wine. The
sweet, apple-like aroma from Candida growth is due to the accumulation
of acetaldehyde during fermentation. This is the same aroma usually
associated with rising bread and green beer. This aroma can be used as a
presumptive indicator of yeast spoilage since no industrial spoilage
bacteria will accumulate acetaldehyde in such large amounts. Yeasts are
more fragile than bacteria in that they have a narrower and lower optimal
pH and temperature growth range.

8.5.3 Fungi
Fungi are not usually a primary cause of in-can spoilage of paint and are
almost never isolated from spoiled paint. The pH and lack of oxygen in a
 PRESERVATION OF PAINTS 195

sealed can are formidable barriers to fungi. This is in spite of the fact that
fungi are ubiquitous soil inhabitants. Examination of mineral pigments,
both dry and slurries, and cellulosic fillers may reveal the presence of
fungal spores.z° In nature fungi must compete against the rapid growth of
bacteria and yeast. Their competitive advantage lies in the diversity of
complex organic substrates they are able to utilize. In the ecological
succession of microorganisms bacteria and yeasts are primary colonizers.
They deplete the relatively simple organic substrates such as sugars first.
Proteins and simple polymers are degraded next. Complex polymers with
multiple branch points such as cellulose and lignin are beyond the
metabolic range of most bacteria and yeasts. After the initial colonization
of the niche only the complex substrates are left intact. Without sufficient
substrate the primary colonizers decrease in both number and diversity.
This allows secondary colonizers, including the fungi, the opportunity to
degrade the remaining substrates. This explains why fungi are not isolated
until after considerable degradation of the system. In reviewing the records
of approximately 4500 evaluations of paint in the Zeneca Biocides
Laboratories we have only isolated fungi from two paint lines, both of which
were from Latin America.

8.6 The problem with solvent-free systems: spoilage

Prior to the introduction of water-borne systems hydrocarbon solvents

were very extensively used in paint manufacturing. These solvents acted as
delivery agents and inadvertently as preservatives. The functional
membranes of living cells are bilayer micelles of C lO to CIS fatty acids
bound to glycerol backbones. The solvents disrupted the cellular
membranes of all the microorganisms introduced during manufacturing or
application. Preservatives were only required to prevent attack on applied
and dried product.
The reduction and elimination of solvents exposes the paint formulation
chemist to the problem of wet state microbial spoilage. Both raw materials
and water itself are sources of microorganisms. Good quality drinking
water may contain up to 103 non-pathogenic bacteria per ml. 21 Improperly
.zz
stored rinse water may contain over 107 cfu ml- 1 Air blowing through a
manufacturing plant may have several hundred fungal spores per cubic
meter. Raw materials may have 1 to 10 million bacteria per ml. In all of
these cases raw materials would be viewed as good and appropriate for use
in production if microbiological monitoring did not occur. Paint chemists
and production personnel with years of experience in solvent-borne
production report that the transition to water-borne systems is difficult if
inadequate microbiological precautions are in place prior to production.
196 PRESERVATION OF SURFACTANT FORMULATIONS

8.7 The chemistry of industrial preservatives

8.7.1 Biocide terminology

Two different functional types of biocides are used in paints today.
Fungicides are designed to inhibit the surface defacement of dried films by
fungi. Fungicides are also called mildewcides. Bacteriocides are designed
to preserve the wet paint until application. Bacteriocides are designed to
function in the can, not in the dried film. The terms in-can preservative,
preservative, biocide, bicide, microbiocide and microbicide are synonym-
ous with bacteriocide.

8.7.2 Mercurials
In the last several decades the paint industry has had experience of a wide
range of products and compounds for antimicrobial preservation. Mercury
was first introduced in the USA in organometallic form as phenylmercuric
acetate (PMA) in the late 1930s23 and was used extensively as both an in-
can antibacterial and film fungicide for water-based paints. The biological
activity of mercury is non-specific and is highly effective against both
bacteria and fungi. Many paint producers utilized the broad spectrum of
mercury as both an in-can aqueous and dry film single biocide. 24
The current regulatory status of mercury depends on the environmental
control agency. Following much review and many hearings with respect to
the safety of using mercurials, the United States Environmental Protection
Agency (USEPA) determined that use of these compounds presented an
unreasonable health risk. The USEPA cancelled all registrations for use of
mercury-based compounds in interior line paints in 1990.25 Mercurials
have also been banned in Japan, the United Kingdom and Germany and
use is declining in Spain, yet mercurials are still used in Asia Pacific
countries, Latin America and Greece.

8.7.3 Desirable properties of biocides

Factors that are of concern in selecting a biocide are persistence,
mammalian toxicology (especially if volatile), cost effectiveness, spectrum
of activity, ease of use, thermal stability, and its effects on color rheology
and pH. Biocides must provide persistent protection during the manu-
facturing, storage, shipment and end use. They must not only be able to
kill spoilage organisms introduced from raw materials but be persistent
enough to kill organisms introduced during several use periods of final
application. In the case of consumer paints these use patterns may be
separated by weeks or months. This type of use pattern requires that the
 PRESERVATION OF PAINTS 197

biocide be stable in formulated paint for at least one year and preferably
two. Mammalian toxicology is important not only to the end users but also
to production personnel. All biocides will cause adverse immune reactions
in humans after repeated uncontrolled exposure. The human toxicology of
the biocides in use varies widely (see chapter 12).

B.7.4 Biocides presently available

B.7.4.1 Formaldehyde release preservatives. Due to the extreme safety

and regulatory pressure placed on mercurials, many organic synthetic
antimicrobials were researched and developed. A number of the commer-
cially available biocides today are formaldehyde condensates which rely on
the release of free formaldehyde (CH 2 0) from the hydrolysis of a parental
nitrogen-based structure. Again, with free formaldehyde as the active
mechanism for antimicrobial activity, considerations were raised around
the safety of products based on these chemistries. Formaldehyde is now
listed as a known human carcinogen and the US Occupational Safety and
Health Administration (OSHA) has placed a limit on the concentration of
free formaldehyde which is allowable in both the work environment air and
in the liquid product in which it is being used. The fate, therefore, for
continued and expanded use of formaldehyde-based antimicrobials in
industrial products is questionable. Table 8.2 lists commercially available
preservatives based on the chemistry. 26
The exact mechanism of action of biocides in this class is poorly
documented. In all cases the compounds can be shown to decompose and
release significant amounts of formaldehyde into the solution. The release
of free formaldehyde can be demonstrated with both the neat product and
in industrial matrices. Disputes as to the precise chemistry of these
compounds are ongoing. 27-29

Table 8.2 Putative formaldehyde release biocides used in paints in the United States

Chemical name of active agent CAS number Trade name Producer

of
active agent

2((Hydroxymethyl) amino) ethanol 34375-28-5 Troysan® 174 Troy Chemical

2((Hydroxymethyl) amino)- 52299-20-4 Troysan® 186 Troy Chemical
2-methyl propanol Troysan® 192
5-Hydroxymethyl-1-aza-3,7- 56709-13-8 Nuosept® 95 Huls
dioxybicycio(3,3,0) octane
[[(1-Methyl-2-(5-methyl-3-oxazolidinyl)- 97553-90-7 Nuosept® 145 Huls
ethoxy) methoxy]methoxy]methanol
1-(3-Chloraliyl)-3,5,7- 4080-31-3 Dowicil® 75 Dow Chemical
triza-1-azoniaadamantane
198 PRESERVATION OF SURFACTANT FORMULATIONS

8.7.4.2 Isothiazolinones. Isothiazolinone biocides have been researched

and developed to target defined metabolic functions in the bacterial cell.
They are effective generally at low use levels «1000 ppm), have a broad
spectrum of antibacterial and antifungal activity and generally have a more
acceptable portfolio with respect to toxicology.
Table 8.3 lists isothiazolinone paint preservatives. Proxel GXL is the
most commonly utilized paint biocide in the United States today. Kathon
LX1.5 is used in limited quantities. Promexal is the latest isothiazolinone
to be commercialized. It is presently available in Europe. Commercial
availability in the United States is pending EPA approval.
The chemistry and stability of isothiazolinones are quite varied. The
5-chloro-2-methyl-4-isothiazolin-3-one component in Kathon is the most
biologically active of the isothiazolinones. It is used in most industrial
systems at 25 ppm or less. This is compared to a typical use level of 100 to
150 ppm of 1,2-benzisothiazolin-3-one in Proxel. The higher antimicrobial
activity of the Kathon system is also reflected in its higher mammalian
toxicity. The stability of the actives in Proxel and Kathon are on the
opposite ends of the spectrum. Whereas Proxel is stable in an environment
with high amine levels and an alkaline pH, Kathon is stable at acid pHs and
in more oxidizing environments. Kathon is rapidly inactivated in the
presence of primary and secondary amines. The pH of most US acrylic and
vinyl acrylic paints is 8.5 or above. This coupled with amines used as
corrosion inhibitors and dispersants produces a very stable environment
for Proxel. Research in the Zeneca Biocides laboratory has been able to
detect, both chemically and biologically, more than 90% of the Proxel
added after one year storage in typical paint. The stability of Kathon in
paint is limited and poorly documented in published literature. The 2-
methyl-4,5-trimethylene-4-isothiazolin-3-one found in Promexal displays
intermediate properties between Kathon and Proxel formulations.
Promexal active use levels are near that of Kathon but display chemical
stability closer to Proxel. Because of paint manufacturers' desire to

Table 8.3 Isothiazolinone paint preservatives

Chemical name of active agent CAS number Trade name Producer

of
active agent

1,2-Benzisothiazolin-3-one 2634-33-5 Proxel® GXL Zeneca

Proxel® BD
2-Methyl-4,5-trimethylene-4- 082633-79-2 Promexal® X50 Zeneca
isothiazolin-3-one
5-Chloro-2-methyl-4- 26172-55-4 Kathon® LX1.5 Rohm and Haas
isothiazolin-3-one Kathon® LX
2682-20-4
2-Methyl-4-isothiazolin-3-one
 PRESERVATION OF PAINTS 199

maintain long shelf lives of finished product they should conduct stability
studies of all actives prior to incorporation into new product lines. This
should be in addition to biological challenge tests.

B.7.5 The future of biocides

As toxicological and ecological pressures surrounding the use of biocides
increase, producers will be faced with developing 'greener' antimicrobials.
These are likely to be lower in volatile organics (VOCs). There will need to
be a careful balancing act of locating molecules that are effective at lower
concentrations and are more toxicologically sound. This may be accom-
plished by researching and developing active agents which target more
specific biological pathways in microorganisms, thus decreasing risk factors
to higher organisms, induding humans.

8.8 Methods to establish the correct biocide level for preservation

B.B.1 In-can preservation

Proper preservation of paint formulations requires that the biocide
candidate be evaluated for resistance to bacterial insult. This is primarily
accomplished by 'optimizing' the usage level using standard microbiological
protocols. Evaluations should be carried out using multiple 'challenges'.
This method was developed to best simulate varying plant production
conditions and insult microbes. Preservatives passing this test will be able
to provide the most long term protection of the formulation during plant
hygiene breakdown and in field applications. In doing this, the actual paint
formulation is acquired and used to serve as a substrate for bacterial
growth. It is challenged repeatedly with 'typical' industrial contaminants.
A number of protocols have been suggested. 3 O-33 Described below is the
protocol presently employed by the Zeneca Biocides western hemisphere
laboratory .

B.B.1.1 Challenge testing. The paint used should be relatively free from
established bacterial populations. A mixture of several predominant
spoilage bacteria are employed for this type of evaluation. These
organisms may be acquired from actual contaminated product or stock
strains from a bacterial culture collection center such as the American
Type Culture Collection (ATCC). Careful maintenance is essential not
only to maintain purity but also the desired biochemical properties of the
strains used. Wild isolates that have undergone more than five passes, or
about 25 generations, in the laboratory are essentially converted to
laboratory strains. If wild isolates are employed they must be maintained
200 PRESERVATION OF SURFACTANT FORMULATIONS

as frozen stocks. Improper care of the culture collection will result in

erratic results. This potential variability will eliminate the possibility of
duplicating results at remote times. For this reason ZENECA has adopted
the use of A TCC strains. These strains are periodically replaced with fresh
vials from the A TCe. This approach also has the added advantage of
reducing the maintenance requirements.
Typical organisms used in challenge tests include:
• Pseudomonas aeruginosa
• Pseudomonas stutzeri
• Pseudomonas putida
• Pseudomonas cepaci
• Pseudomonas aureofaciens
• Aeromonas hydrophilia
• Enterobacter cloacae
• Serratia marcescens
• Escherichia coli
• Acinetobacter calcoaceticus
Prior to testing microorganisms used should be grown as stock
laboratory cultures. Culturing at varying pH levels on appropriate media,
such as Tryptic Soy Agar, assists the development of viable controls in the
challenge test. Organisms are then chosen from a stock culture pH which
most closely matches the pH of the test sample. This will reduce pH shock
on the organism when introduced to the industrial matrix. Lawns of
bacteria are harvested from a solid medium using a small amount of sterile
dilution water. The resulting bacterial suspension is then used to inoculate
separate samples of the unpreserved product. The American Society for
Testing and Materials (ASTM) protocol and a number of biocide suppliers
inoculate paints directly with these bacterial suspensions. Zeneca has
found that this does not always reflect real world spoilage patterns. To
simulate more closely the manufacturing plant it is advisable to pre-adapt
the bacteria to the paint. This is essentially a pre-challenge step where
individual species of bacteria are repeatedly inoculated into the paint until
a stable population is established. This is specifically designed to simulate a
plant condition where a transitory bacterium has established an adapted
population in one section of the plant. This might be in a manifold or dead-
end T-fitting. The viability of these inoculations can be monitored after
24 and 48 h by evaluating the degree of persistence of the organisms on
agar plates. When the majority of the organisms are found to persist at
107 cfu ml-1 , the bacterial adaptation process is completed and the various
bacteria are pooled to form the combined inoculum.
In the challenge test the paint sample is then divided into aliquots and
the desired preservatives are added at various levels. A biocide-free
control is included as a control. If growth is not established in the control
 PRESERVATION OF PAINTS 201

the results of the test are not valid in themselves. All samples are
subsequently challenged at least three times with the adapted bacterial
inoculum. The efficacy of the added preservative in controlling contamina-
tion is monitored 24, 48 and 72 h following each inoculation. During the
interpretation of challenge test results the time between inoculation and
measuring viable bacterial counts is very important. Formaldehyde
condensates have more rapid kill rates than isothiazolinones. Isothiazol-
inones are biostatic on contact but may take 72 h for a complete kill at use
levels. For consistency of results all challenge test comparisons must
recognize this issue. The concentration of preservative affording protection
(no viable growth) during the third challenge at 72 h is considered to be the
effective dosage.
Results from a typical challenge test are shown in Table 8.4. Samples on
day 1 and 2 after challenge are qualitative streak plates. Growth is scored
on a 0 to 5 scale. The day 3 samples are standard plate counts of viable
bacteria and are expressed as cfu ml-I. Quantitative assessment of the
effective treatment level is defined as the lowest level that provides
consistent biological control in all challenges. In this example the 750 ppm
provides the desired effect.
Interpolating between data points should be avoided in testing of this
type. Although the 'lowest' effect level will lie between 500 and 750 ppm
its exact end point is imprecisely defined. This coupled with inconsistent
dose levels of the biocide during manufacture and varying biological load
from raw materials makes interpolation very risky. Efforts to reduce
overall biocide use would be better spent in improving plant hygiene and
reducing biocide used in recovering spoiled products.

8.9 Implementing the laboratory report in practice

The trend in biocides today is to use more environmentally friendly

biocides at the lowest levels practical. Because of the time and effort
management spends on biocide decisions plant personnel frequently
perceive biocides as exotic and dangerous materials. Indeed, few trained
scientists outside of biocide suppliers understand the biochemistry of
product spoilage and biocide function. This combination of general
ignorance lends itself to misunderstandings. The implementation of
adequate biocide usage patterns requires extensive training of personnel
including management, purchasing and production. Biocides by their
nature are reactive with living systems, including human beings. Changes
in biocides must be accompanied by adequate training for all potentially
exposed plant personnel. This training should include types of exposure
hazard (inhalation or direct contact) and training on personal protection
devices and clothing. Procedures for spill clean up and proper disposal of
 l growth
concentrations to determin e biocide level required to prevent microbia
Table 8.4 Challenge test of laborato ry strains against various biocide
Challenge test on latex wall paint

2nd Challenge 3rd Challenge

1st Challeng e Concent ration Product (cfu g-l)
Product (cfu g-l) Concentration Product (cfu g-l)
Concent ration of biocide (ppm) Days post inoculation
Days post inoculation of biocide (ppm) Days post inoculation
of biocide (ppm)
2 3 1 2 3
2 3 1
5 3.6 X 106 0 5 5 2.2 X lOS
0 5 5 3.4 X 108 0 5
5 3.4 X 106 250 5 5 1.9 X 108
250 5 5 3.7 X 106 250 5
4 1.7 X l(f 500 5 5 1.2 X lOS
500 5 3 <10 500 5
0 <10 750 2 2 <10
750 3 0 <10 750 1
0 <10 1000 0 0 <10
1000 0 0 <10 1000 0
0 <10 1250 0 0 <10
1250 1 0 <10 1250 0
1 <10 1500 0 0 <10
1500 1 0 <10 1500 0
0 <10 2000 0 0 <10
2000 0 0 <10 2000 0
6-15 cfu;
l growth. 0 = no growth; 1 = 1-5 colony forming unit (cfu); 2 =
Streak plates are ranked from 0-5 according to the degree of microbia d as colony forming units per gram (cfu g-l).
are expresse
3 = 16-30 cfu; 4 = 31-45 cfu; 5 = >45 cfu. Serial dilution counts
 PRESERVATION OF PAINTS 203

empty containers should be in place prior to use and not asked after
incidents and spills have already occurred.
The time period in switching to an unfamiliar biocide is the most likely
period when spoilage may occur. It is best to start new biocides at high
dose levels and lower the use level with experience. When problems are
encountered immediate action should be taken. Dosages used to recover
spoiled material should be at least twice that used for normal preservation.
Creeping the dose up in periods of sporadic spoilage will only serve to
select for resistant strains. Failure to instruct plant personnel on the
effective limitation of biocides is a major source of spoilage during initial
conversions. Biocides vary in their mode and rate of action. Heavy metals
are persistent but kill over a period of several days. Some formaldehyde
donors are volatile in open systems but kill within 24 h of contact. It would
be unwise to use a formaldehyde donor to preserve an open tank of warm
latex for several weeks. Hypochlorous acid (household bleach) has been
used for several years by a number of latex and paint companies to recover
spoilage bulk tanks. This does provide a rapid and inexpensive recovery
but may create more long term problems than it solves. Hypochlorite is
reactive with most non-metallic biocides. The hypochlorite used not only
kills the microorganism but it also leaves the material with no long term
preservation and a hostile chemical environment to other biocides.
Additional biocide should be added after the use of bleach but only after
allowing sufficient time for all hypochlorite to dissipate. Glutaraldehyde is
similarly affected by amines in alkaline conditions.
Biocide rotation programs are not required when adequate levels of
biocide are maintained in raw material and finished products. Repetitive
underdosing of any biocide may lead to the development of adapted
bacterial popUlations. Eliminating these types of populations frequently
requires thorough cleaning and sanitization of the entire plant. The best
analogy to this situation is prescription antibiotics for humans. If the
prescription is not entirely used the infection may reoccur. Upon the
reoccurrence the infection is frequently more severe and more intractable
to antibiotic treatment.

8.10 Plant sanitation and hygiene audits

The most critical aspect of preventive maintenance from a microbiological

aspect is routine housekeeping coupled with microbiological plant audits.
Fundamental practices such as cleaning up spills, keeping lids closed on
storage tanks and sanitization complement the overall effectiveness of any
preservative in use. The implementation of a sanitization program
throughout the plant will reduce the contamination levels and should also
reduce the incidence of finished product spoilage. By reducing the
204 PRESERVATION OF SURFACTANT FORMULATIONS

bioburden in the raw materials and production equipment the biocide will
be more effective in preserving the quality of the finished product. A
number of articles have been written on the role of sanitation and hygiene
in spoilage control. 34-36 These articles tend to be brief and assume some
level of comprehensive understanding of microbial physiology and ecology.
Technologists desiring to understand and establish a hygiene program
would be rewarded by investigating a number of articles written for the
food industry?7 A full consideration of this topic is given in chapter 3.
However, consideration of how it may be specifically applied to the paint
industry is outlined below.

8.10.1 Cleaning equipment

The first step in implementing a hygiene plan must be a thorough cleaning.
The objective is to remove all of the accumulated material and debris. Hot
water with detergents or caustic cleaning solutions are usually effective.
Operator experience in cleaning should be considered when selecting the
cleaning method. All of the built-up debris must be removed and bare
metal or plastic should be exposed prior to sanitization or the effort will be
wasted. The organics in the debris serve to deactivate sanitizers, they also
serve as breeding sites for bacteria and fungi and restrict the access of
sanitizers to the resident flora thus defeating the purpose of sanitization.
High pressure hot water washers are ideal for industrial environments. The
hot water used in cleaning by itself will not kill bacteria or fungi but may
help the cleaning agents injure the cells and allow the sanitizers used later
to work more effectively. Water over 160°F (71°C) is desirable.
After all of the debris is removed the equipment is sanitized. A solution
of 500 ppm free chlorine in good quality water works well. A direct contact
time of 10 min using 75°F (24°C) water will kill all industrially important
bacteria and fungi. The 500 ppm level of free chlorine is specified because
residual organics will stoichiometrically deactivate chlorine. Glutar-
aldehyde is also a very effective sanitizer but the smell may lead to worker
complaints. The equipment should then be rinsed with drinking quality
water. This is especially true with chlorine as chloride ions will cause
pitting on both mild and stainless steel. Rinse waters containing residual
sanitizer should be evaluated for compatibilty before being used in the
manufacture of new products. Residual sanitizers may lead to inactivation
of biocides, discoloration of the product or modification of the emulsion
stability.
Once the equipment has been treated it should be stored so no further
contamination occurs. This is not always easy when beginning a system
wide hygiene plan. All tanks should have tight fitting covers to exclude
contamination from dust, dripping water, birds, mice and foreign objects
like hard hats. Covers should not be made of wood or soft steel. Wood
 PRESERVATION OF PAINTS 205

retains moisture and provides large areas not treatable during surface
application of the sanitizer. Soft steel will pock mark with time. Both wood
and pock marked steel provide large areas for bacterial colonization that
are impossible to decontaminate effectively. Water should not be allowed
to stand in the plant. This includes soak tanks and troughs. Flexible
transfer hoses should be cleaned, sanitized and rinsed frequently. These
hoses should never be allowed to lay horizontally full of material or water.
Hoses should be hung up to drain after each use. The areas that result in
the most contamination are always the filtering and pumping units where
different batches and products intermingle. These areas are open to air-
borne and water-borne contamination.
As stated, housekeeping and plant hygiene must be a routine and
regularly scheduled practice to be effective, 'once a year or so' is not
adequate. If skins are not allowed to accumulate, then the time required
for cleaning is minimized. Once the equipment is rinsed it is back in service
again. If the time involved in a hygiene program is compared to the
expense of product rework, or worse yet a product return, then plant
hygiene is seen as a very cost effective quality control method.

8.10.2 Treating wash/rinse water

The wash/rinse water that is recycled through the plumbing system is
frequently a major source of contamination in industrial plants. The
residual chlorine level of potable water is instantaneously depleted by
contact with organic material. This leaves the wash water with no bacterial
protection and very large quantities of organic material, which provides an
ideal situation for bacterial growth. The most important issue to keep in
mind is the residence time of the water prior to re-use. If the water stands
less than 8 h no treatment may be required. If wash/rinse water is allowed
to stand over the weekend a program to check for bacterial growth should
be started. A key factor in selecting a biocide for wash water is the interval
between addition of biocide and the use of the water. Preventing growth
may prove more effective than killing established populations. Over
weekends growth may be prevented by the addition of adequate levels of
biocide at the end of the last Friday shift.

8.10.3 Hygiene audits

Routine inspections or 'audits' are useful to determine if these practices are
in place and to check for the presence of spoilage in a variety of locations
throughout the production facilities. Plant hygiene audits can be performed
internally by trained personnel or by external vendors who provide this
type of service. Some biocide companies even provide training for in-house
personnel on effective auditing programs. It is recommended by the
206 PRESERVATION OF SURFACTANT FORMULATIONS

authors that monthly internal inspections be performed and a database be

generated to compile a historical perspective for a particular plant. This
program should also be coupled with an external audit every year. External
auditing will keep plant personnel informed as to changes in methodology.
External audits frequently are able to locate areas overlooked by plant
personnel due to every day familiarity.
Listed below are five of the most critical areas to monitor with respect to
the potential for plant hygiene failure. Also listed are five general
recommendations for building a fundamental program of plant hygiene.
Table 8.5 illustrates the locations and results from a typical industrial plant
hygiene audit.
Critical plant inspection locations are:
1. Bulk input manifolds and raw material tanks
2. Water system and holding tanks
3. Wash water and holding tanks
4. Rework material
5. Product filters (basket, stacked, shakers)
General hygiene recommendations are:
1. Treat wash water daily
2. Add biocide early in manufacturing process
3. Make monthly hygiene audits
4. Seek outside audit annually
5. After database is established look for patterns in (a) transient
populations and (b) resident flora.
Samples indicated by S in Table 8.5 were swabs of solid surfaces. These
are qualitative samples. Growth is detected by streaking the swab onto an
agar plate and reading the plate following incubation. Ratings are given for
the degree of growth present according to the following scale:
0= no growth
1 = 1-5 cfu (colony forming units); lightly contaminated
2 = 6-15 cfu; lightly contaminated
3 = 16-30 cfu; moderately contaminated
4 = 31--45 cfu; heavily contaminated
5 = >45 cfu; severely contaminated.
All other samples were liquids. Samples are checked for the presence of
viable microorganisms using a serial dilution plate counting technique on
standard laboratory media. Zeneca Biocides uses tryptone glucose extract
agar (TGEA) because this particular medium supports a very large number
of organisms and is able to provide rapid recovery from weak cells. The
correct biological unit is cfu g-l. Results were generated after incubation
for 48 h at 30°C. Counts between 103 and 105 cfu g-l are considered to be
 PRESERVATION OF PAINTS 207

Table 8.5. An industrial paint company plant hygiene audit evaluation results

Sample no. Location Growth detected

Raw materials
1 PV A latex - rail car <10
2 PVA latex - storage tank <10
3 Acrylic latex - rail car <10
4 Ti0 2 - rail car <10
5 Ti0 2 - storage tank <10
6 Clay slurry - storage tank <10
7 CaC0 3 - rail car 3.7 X 104
8 CaC0 3 - storage tank 7.3 X 104
9 Water supply 3.1 X 103

Production line
10 Tank 14 1.4X103
11 Let down tank no. 26 <10
12 Water entering grind tanks 2.0 X 103
13 (S) Slurry hose to grind tanks 4
14 (S) Let down tank no. 17 0
15 (S) Let down tank no. 15 5
16 (S) Let down tank no. 14 5
17 (S) Transfer hose at finishing tank 5
18 (S) Let down tank no. 13 5
19 (S) Let down tank no. 12 0
20 (S) Transfer hose to let down tanks 5
21 (S) Let down tank no. 11 3
22 (S) Let down tank no. 10 0
23 (S) Grind tank no. 9A 0
24 (S) PV A line into tank no. 9A 0
25 (S) PYA line into tank no. lOA 0
26 (S) Tank 27 water inlet 5

Finished products
27 Paint retain - enamel 1.8 X 105
28 paint retain - gloss 1.72 X lOS
29 Paint retain - satin <10
30 Paint retain - eggshell <10
31 Paint retain - flat <10
32 Paint retain - flat <10

S = Swab sample

substantial. Counts of 106 cfu g-l or greater indicate that the stability of
the emulsion is in danger.

8.11 When spoilage occurs

Sanitization of all tanks, mills, plumbing and transfer vessels is required to

regain control of highly contaminated facilities. After the equipment has
208 PRESERVATION OF SURFACTANT FORMULATIONS

been cleaned and sanitized with hypochlorous acid, glutaraldehyde or

quaternary ammonium compounds it must be rinsed with high quality
water. Rinsing with potable water after sanitization is essential. Cleaning
solutions contain surfactants that may affect the viscosity of products if
they are not removed from the system prior to restarting production.
Sanitizers may interfere with biocide action in the finished product or may
lead to color changes.

8.11.1 Handling contaminated product

The best judge in handling and treating contaminated product is past
experience with similar situations. The nature and the scope of the
problem should be determined. Was adequate biocide added during
manufacture? Is the problem unique or a repetition of historical problems?
Repeated occurrences call for plant hygiene audits designed to locate point
sources of inoculation and a thorough examination of all manufacturing
procedures. Single occurrences, while still serious, do not necessarily
require the same level of intervention and treatment as a recurrent
problem.

8.11.2 Treating contaminated product

Treatment of contaminated paint requires the addition of a supplemental
dose of biocide. Depending on preservative type, this may require holding
the product at least 72 h after addition to allow the biocide time to kill the
bacteria and verify microbiologically sound product. Quick acting dis-
infectants such as bleach, glutaraldehyde and some formaldehyde donor
preservatives may also be used as these will produce a rapid rate of kill, but
it is advised to check for formulation compatibility especially with bleach
and glutaraldehyde in polymeric formulations. These compounds may alter
color, viscosity or even break the emulsion. All potential candidates for
such uses should be validated in all product lines as part of the in-house
microbiological procedures. These efforts will save time when it becomes
necessary to recover spoiled product as timing in eliminating established
bacterial populations is critical to the integrity of the paint. In any case,
when a quick acting biocide such as bleach is used, final dosing with a
persistent preservative is required to provide subsequent protection. Quick
acting biocides are usually depleted in providing antimicrobial action.
If possible, it is recommended that contaminated material be examined
by a microbiology laboratory to determine an effective concentration of
biocide to eliminate spoilage in the product. This is important in
determining both the correct biocide and the effective level that will
eliminate and prevent subsequent spoilage. Use of an ineffective biocide or
sublethal concentrations will allow a multitude of future problems to
 PRESERVATION OF PAINTS 209

develop. This is especially true if the questionable product will be

eventually released for sale. Timing in laboratory examination is critical. If
the spoiled product is allowed to sit for extended periods of time the change
in formulation caused by bacterial growth will become irreversible.
Samples should be sent by overnight express. 'Recovery' testing is one
microbiological protocol employed to determine effective biocide levels.
Contaminated material is simply divided into aliquots, a spectrum of
concentrations is added and the material is monitored for 24 to 72 h for
growth. The level of preservative which kills all viable cells is considered to
be the effective level. The working concentration determined by this
method is usually 100 to 200% greater than normally required to prevent
contamination. Therefore, usage recommendations provided by this
method are usually adequate to prevent further spoilage of the system.
Any final product that proves untreatable should be disposed of by
established procedures in accordance with local, state and federal
regulations. Such product should never be reworked back into the
production stream. Reworking would serve to inoculate otherwise good
batches with untreatable bacteria. With adequate preservative usage and
preventive programs this should never be required.

8.12 Implementing a HACCP program

Hazard analysis and critical control point (HACCP) is an entire approach

at manufacturing control designed to identify all areas where contamination
may occur and then develop a control system to address each area. It grew
out of a concern that NASA had for the health of its astronauts in orbit. If
an astronaut were to become ill from contaminated food the completion of
the mission and safe return from orbit of the crew would become
uncertain. NASA worked closely with the food industry to develop the
overall approach to food safety. The lessons learned from HACCP are
easily transferable to industrial non-food manufacturing. Plant quality
audit personnel responsible for establishing a hygiene system will find large
numbers of well written guides on the subject. 38 Much of the above
discussion is intended to introduce HACCP into this review?9

8.13 Summary

Industrial formulations such as paints contain constituents which are prime

candidates for degradation by a multitude of microorganisms. Bacterial
species will cause unprotected formulations to suffer from pH swings,
viscosity loss, emulsion destabilization, off odors and off colors and gas
production. Fungal organisms will cause cracking, change adhesion
210 PRESERVATION OF SURFACTANT FORMULATIONS

properties and deface the final paint after application. These effects can
wreak havoc on production and marketing departments when lost time,
plant contamination and product recalls develop. Ultimate losses are
measured by a company's reputation suffering. Protection of industrial
days of the use of mercury and its broad spectrum, more 'forgiving'
qualities have passed. The use of formalin or formaldehyde releasing
qualities have passed. The use of formalin or formaldehyde released
preservatives is also becoming less acceptable. Today, due to ever
changing environmental and regulatory pressures, manufacturers are faced
with using more selective biocides which target very defined biochemical
processes in microorganisms. Because of their specificity, preservatives are
becoming 'greener' with more acceptable human toxicology profiles. As
regulatory pressures increase both the number of biocides and the
companies capable of developing new products will decrease. Producers of
aqueous products are faced with efforts to reduce usage levels of these
greener products for both toxicological and financial reasons. Under these
conditions plant hygiene has become a major concern. Manufacturers are
now faced with implementing fundamental plant hygiene practices to
reduce the bioload on the preservative system in place. Frequent
sanitization of production and storage facilities is required to optimize
protection. In summary, protection of industrial formulations from
microbial attack will remain a necessity for years to come.

References

1. Turner, G.P.A. (1988) Introduction to Paint Chemistry and Principles of Paint

Technology, 3rd edn., Chapman & Hall, London.
2. Blake, M.D. (1987) Thickeners for waterborne coatings. In Handbook of Coatings
Additives, Calbo, L.J. (ed.), Marcel Dekker, New York.
3. Vash, R. (1987) Wetting and dispersing. In Handbook of Coatings Additives, Calbo, L.J.
(ed.) Marcel Dekker, New York.
4. Flick, E.W. (1988) Water-Based Trade Formulations, Noyes Publications, Park Ridge,
NJ.
5. Tilton, H. (1992) Shades of Health, Chemical Marketing Reporter, Oct 1992, 3-18.
6. House Paints and Stains (1993) Consumer Reports, 58: 610-615.
7. O'Neill, T.B. (1986) Succession and interrelationships of microorganisms on painted
surface. J. Coat. Technol., 58, 51-56.
8. Smith, R.E. (1993) Zeneca Biocides, personal communication.
9. Simpson, D.K. and Kappock, P. (1994) The role of fungicides and algaecides in
architectural paint. Paint & Coatings Ind., October.
10. Gillat, J. and Wood, B. (1990) Prevention of organic growth on coatings. Polymers, Paint
Colour J., 180,540--541.
11. Vannoy, W.G. (1948) Mildew inhibitors for house paints. Fed. Paint Varnish Clubs, 277,
163-175.
12. Goll, M., Snyder, H.D. and Birnbaum, H.A. (1952) New developments in the diagnosis
of paint mildew. Official Digest Fed. Paint Varnish Prod. Clubs, 326, 149-155.
13. Harnden, R.C. (1945) Mildew control in the paint industry. Paint Oil Chem. Rev., 108,
32-34.
 PRESERVATION OF PAINTS 211

14. Bravery, A.F. (1988) Biodeterioration of paint; a state-of-the-art comment. Bio-

deterioration 7, Selected papers presented 7th Internat. Biodeteriation Symposium, 7,
466-485.
15. Alexander, M. (1977) Introduction to Soil Microbiology, 2nd edn., John Wiley and Sons,
New York.
16. Springle, W.R. (1988) Liquefaction of cellulosic paint thickeners. Part 2: Quantitative
aspects of enzymatic degradation. J. Oil Colour Chem. Assoc., 71(4),109-113.
17. Zeneca Biocides, unpublished research data.
18. Boyd, R. F. and Hoerl, B.G. (1986) Basic Medical Microbiology, 3rd edn., Little, Brown
and Company, Boston.
19. Schwartz, R.D. and Leathen, W.W. (1976) Petroleum microbiology. In Industrial
Microbiology, Miller, B.M. and Litsky, W. (eds), McGraw-Hili, New York, pp. 384-413.
20. Carter, G. and Huddart, G. (1974) The 'in-can' preservation of aqueous paints. Pigment
Resin Technol., 3-10.
21. American Water Works Association (1990) Water Quality and Treatment - A Handbook
of Community Water Supplies, 4th edn., McGraw-Hili, New York.
22. Siegert, W. (1990) Production hygiene in the manufacture of water-based coating
materials. Polymers Paint Colour J., 180, 584.
23. Block, S.S. (1991) Disinfection, Sterilization, and Preservation, 4th edn., Lea & Febiger,
Malvern, PA.
24. Matta, B.L. and Breindel, K. (1991) Mercurial versus Non-Mercurial Biocides, 2°
Congressa Internacional de Tintas, pp. 528-540.
25. Block, S.S. (1991) Disinfection, Sterilization, and Preservation, 4th edn., Lea & Febiger,
Malvern PA.
26. Rossmoore, H.W. (1991) Nitrogen compounds. In Disinfection, Sterilization, and
Preservation, 4th edn., Block, S.S. (ed.) Lea and Febiger, Philadelphia, pp. 290--321.
27. Scott, C.R. and Wolf, P.A. (1962) The antibacterial activity of a series of quaternaries
prepared from hexamethylenetetramine and halohydrocarbons. Appl. Microbiol, 10,
211-215.
28. Rossmoore, H.W. and Sondossi, M. (1988) Applications and mode of action of
formaldehyde condensate biocides. Adv. Appl. Microbiol., 33, 223-277.
29. The Dow Chemical Company (1981) Dowicil 75 Preservative 'Formaldehyde Release' in
Aquous Systems, Form no. 192-833-981.
30. Jakubowski, J .A., Simpson, S.L. and Gyuris, J. (1982) Microbiological spoilage of latex
emulsions: causes and prevention. J. Coat. Technol., 54, 39-44.
31. Ross, R.T. and Hollis, C.G. (1976) Microbiological deterioration of pulpwood, paper
and paint. In Industrial Microbiology, Miller, B.M. and Litsky, W. (eds), McGraw-Hill,
New York, pp. 309-354.
32. Carter, G. and Huddart, G. (1974) The 'in-can' preservation of aqueous paints. Pigment
Resin Technol., 3-10.
33. ASTM (1992) Standard method for resistance of emulsion paints in the container to
attack by microorganisms (#D2574-86). Annual Book of ASTM Standards, Vol 6.01.
34. Carman, J.R. and Goll, M. (1970) The role of industrial hygiene in the manufacture
of latex paint. Develop. In dust. Microbiol., 29, 327-329.
35. Siegert, W. (1990) Production hygiene in the manufacture of water-based coating
materials. Polymers Paint Colour J., 180, 584.
36. Jakubowski, J.A., Simpson, S.L. and Gyuris, J. (1982) Microbiological spoilage of latex
emulsions: causes and prevention. J. Coat. Technol., 54, 39-44.
37. Marriott, N.G. (1989) Principles of Food Sanitation, 2nd edn. Van Nostrand Reinhold,
New York.
38. Sperber, W.H. (1991) The modern HACCP system. Food Technol., 45, 116, 118, 120.
39. Pierson, M.D. and Corlett, D.A. Jr. (1992) HACCP: Principles and Applications, Van
Nostrand Reinhold, New York.
9 Preservation of aqueous-based synthetic polymer
emulsions and adhesive formulations
M.A. CRESSWELL and K. HOLLAND

9.1 General introduction

The need for protection from microorganisms has been recognised for
many centuries, but only since the late 18th and 19th centuries have orderly
programmes been mounted to control this problem. Much of the early
effort was concentrated in agriculture where success was mandatory for
economic, social or even physical survival. The great majority of this
pioneering work was directed toward preventing fungal damage on natural
substances: wood, paper, other cellulosics, seeds and crops.
The development of water-based and surfactant-based systems in the
past 50 years raised the additional challenge of bacterial attack and the
need for antimicrobial agents with broader spectrum effectiveness. 1-15 The
concern of the user was expanded from end-use protection of the finished
product where fungi were long recognised as causative organisms, to
preservation during processing, storage and sampling where wider varieties
of microorganisms playa major role. Biological problems, never simple,
became more complex, and the search for preservatives that control the
myriad of organisms that flourish in many of the new formulations became
more difficult.
The last 30-35 years have been characterised by a growing awareness,
triggered in no small part by regulatory pressures, that to be accepted, and
thereby achieve economical success, a preservative must provide the user
with security beyond the old basics: efficacy, compatibility and cost
effectiveness. These, although necessary, form only a few of the broad
requisites for 'modern' antimicrobial agents. The other requirement is that
of minimal hazard in use as well as a low toxicological, environmental and
ecological impact. Trends in development of new antimicrobial
compounds, their uses, advantages and disadvantages will be considered
and discussed in this chapter.
Synthetic polymer emulsions play an important role in everyday
products from household domestic goods to many industrial uses. Though
polymer emulsions may not be apparent as the emulsion system they are
still very much in abundance in our everyday life and items we use. Some
examples where polymer emulsions are to be found include: home 'do-it-
 PRESERVATION OF AQUEOUS EMULSIONS 213

yourself' products (emulsions paints, wallpaper pastes, glues, grouts and

cements), domestic products (food packaging, cartons etc.), synthetic
fabricated goods (non-woven textile materials, waddings, insulation), as
well as vast numbers of industrial uses. Some of these uses and applications
will be highlighted in more detail later in the chapter.
Synthetic polymer emulsions or latices or resins are fine dispersions or
suspensions of synthetic polymer particles (0.1 /lm to 5--6/lm) in an
aqueous stabilisation medium. The emulsion systems considered in this
chapter are all of the vinyl type whereby polymerisation reactions of alkene
monomers form very large, high molecular weight polymers. The process
of polymerisation can be either of two types: addition polymerisation or
condensation polymerisation. The main components of the aqueous
stabilisation medium, apart from water, are colloids such as polyvinyl
alcohol, cellulosic materials or starch materials and/or surfactant, being
anionic, cationic or even nonionic depending on the polymer type. A
common feature, however, with most types of these polymer emulsions is
that they are susceptible to spoilage by microorganisms.
Adhesives, like polymer emulsions, play an important part in day-to-day
life, from the simple glues on the back of postage stamps which are
remoistened to allow their positioning on letters, to the glues used for
bonding labels on bottles and the high performance adhesives designed to
withstand severe stresses in automotive manufacture. Adhesives are used
both in the domestic environment as well as in industrial situations in many
operations.
An adhesive can be defined by many definitions that range from a 'sticky
substance' to a 'material uniting other materials either temporarily or
permanently', but each retains the central theme that an adhesive is
capable of binding or adhering to a surface and can act simultaneously on
two surfaces to hold them together. Adhesives provide an alternative to
the common mechanical methods of combining materials together, e.g.
screwing, nailing, riveting and fusing as well as having many advantages
such as stress distribution, versatility and simplicity.
Numerous theories abound on how adhesives work and on the
mechanisms of adhesion and these are covered in some of the classical
texts, though the common mechanisms are by short range intermolecular
interactions, physical interlocking or chemical bonding.
Formulations of adhesives vary immensely and a variety of raw materials
are employed in their manufacture. A knowledge of these raw materials
and their effects is necessary to understand the details of an adhesive
formulation. Surfactants are used as raw materials in their own right to
provide certain properties but are also frequently present in many of the
other raw materials themselves.
Given the care taken in designing an adhesive to ensure its effectiveness
in an application, the significance of this chapter is to show that
214 PRESERVATION OF SURFACTANT FORMULATIONS

contamination and growth of microorganisms in the adhesive can reduce its

effectiveness. Several types of microorganism can be encountered in spoilt
polymer and adhesive systems and their harmful effects can be judged by
the deleterious results they may have on the end-use properties critical to
their performance. Often the harmful effects are similar for different
microorganisms and the need to identify the specific nature of the
microorganism may only be important if it falls outside the spectrum of
activity of the selected preservative. This chapter will consider ways of
preventing microbiological spoilage in both polymer emulsions and
adhesives by the judicious choice of preservative and control of the supply
chain.
The prevention of microbiological contamination by the incorporation of
a preservative into the formulation requires a careful choice of biocide-
active agent which is governed by the raw materials in the formulation, by
the chemical environment and by legislative restrictions relevant to the end
use of the manufactured product. Prevention in the supply chain requires
an emphasis on the importance of manufacturing plant hygiene, especially
during formulation processing combined with the use of sampling and
testing protocols in order to confirm the efficacy of the chosen preservative
system. This involves closely monitoring each step in the chain from receipt
of the raw materials through to the final use of the product and often even
beyond this stage if the dry film is also susceptible to microbial attack.

9.2 Aqueous-based synthetic polymer emulsions

9.2.1 Applications and uses of polymer emulsions

Polymer emulsions are widely used as raw materials in an extensive range
of industries. 16 Some of the uses of these emulsions are listed in Table 9.1.

9.2.2 Range and chemical types of polymer emulsions

There is a wide range of different types of synthetic polymer emulsions,
incorporating different monomers in a variety of combinations. 16 Examples
of emulsion types are listed here:
• Polyvinyl vinyl acetate (PVA) homopolymers
• Vinyl acetate/ethylene (VAlE) copolymers
• Vinyl acetate/vinyl chloride/ethylene (V AIENC) terpolymers
• Acrylic-containing (Ac) polymers
• Styrene/acrylic (Sty/Ac) polymers
• Vinyl acetate/vinyl versatate (VANeoVA) copolymers
If gaseous monomers such as ethylene or vinyl chloride are used, the
production processes involve polymerisation at high pressures and the
 PRESERVATION OF AQUEOUS EMULSIONS 215

Table 9.1 Some uses of polymer emulsions

Industry Examples of applications and uses

Adbesives Packaging and converting, metal and plastic foil

laminating, building, wood bonding, ceramic tiles fixing,
bookbinding, cold seal, automotive assembly
Emulsion paints Emulsion paint, from full gloss to matt for exterior or
purely interior use - the emulsion acts as the binding
medium, contributing durability and resistance to water,
scrubbing etc.
Textured coatings Exterior and interior paints - the emulsion has the same
function as in normal emulsion paints above.
Industrial finishes Wood and metal including anticorrosive finishes - special
emulsions ensure requisite hardness, solvent resistance
etc. in air drying of stoving finishes.
Printing inks Alkali-soluble emulsion polymers provide a reliable,
consistent varnish base, particularly for flexographic inks.
Building auxiliaries and Emulsions with stability to high concentrations of ions, and
cement additives with polymer components resisting the weakening action
of water, oils, fats, etc. are widely used for general
purpose bonding, and for upgrading the adhesion and
resilience of renderings and floor toppings.
Carpets Polymer emulsions increase the tuft anchorage in woven
carpets, without distracting from easy lay; in tufted carpet
emulsions act as anchor coats for backing compounds,
and in reinforcing and primary binders in backing.
Non-woven fabrics Emulsions provide the full range of cost performance
properties meeting the various requirements of
disposable and permanent non-woven fabrics, e.g.
J-cloths, disposable nappies/diapers, feminine hygiene
products.
Textile finishings, printing The versatility of emulsions covers the wide range of fabric
and flocking finishing requirements, as well as the specific binding
requirements for flocking printing, pigment binding etc.
Glass fibre sizes and binders Special emulsions bind glass strands and chopped strand
mat for easy handling during lay-up.
Paper and board coating Emulsion binders for mineral coated stock enable good
colour and printing properties.
Wallpaper Emulsions are used in all front coating operations, as well
as duplexing and pre-pasting adhesives.
Soil and mineral stabilisation Surface binding of loose soil and mineral deposits by
emulsions is widely undertaken to improve crop yields
and reduce dust and similar environmental problems.
Biological applications Special emulsions with entrapped fungicides/mildewicides
are used to coat seeds to prevent their decay prior to
germination. Also, emulsions are used as cryo-preserving
aids in the storage of harvested microbial cells.
216 PRESERVATION OF SURFACTANT FORMULATIONS

polymers formed are commonly referred to as 'pressure polymers', e.g.

copolymers of V AlE or terpolymers of V AlENC or V AlE/2-EHA. Other
non-gaseous monomers may be polymerised together to form polymers in
low pressure systems and these polymers are commonly referred to as
'conventional polymers' or 'atmospheric polymers', e.g. VA homo-
polymers, Ac polymers of methylmethacrylate. Furthermore, the use of
other functional monomeric units to give the polymer specific application
properties, such as cross linking in cured textile fabric applications (e.g. N-
methylol acrylamide, acrylamide and many others), are often employed
and these polymers are commonly referred to as 'speciality polymers'.
Common to all polymerisation reactions the processes are usually carried
out in a batch-wise system, but continuous processes can also be employed.
The majority of polymer emulsions have a pH range between 3.5 to 7.5.
In most of the emulsions mentioned in this chapter the pH is acidic,
between 4 to 6.5, but some emulsions, however, have an alkaline pH, e.g.
acrylic types of polymers with pH of greater than 8. It is often therefore the
pH range, and the fact that polymer emulsions are aqueous-based
products, that determine the degree of susceptibility of the emulsion to the
various classes of microorganisms. Acidic pH systems tend to favour the
growth of yeasts and moulds, whereas neutral to alkaline pH systems tend
to favour the growth of bacteria. Knowing this also helps when selecting
and choosing the right biocide to preserve a particular polymer emulsion
type.

9.2.3 The chemical nature of polymer emulsions

Apart from water, three main components are required to manufacture a
polymer emulsion 16
• surfactant and/or colloid
• monomer
• initiator
These will now be considered separately.

9.2.3.1 Surfactants. Surfactants, by definition, are substances which

lower the surface tension of liquids in which they are dissolved or the
interfacial tension between two or more mutually immiscible phases. It has
long been recognised that the most effective surface tension depressants
contain highly water-attracting (hydrophilic) and highly water-repellent
(hydrophobic) groups, joined together in the same molecule.
The most important hydrophilic (polar) groups found in surfactants are,
for anionics, ionised sulphonate, sulphate, phosphate and carboxyl groups;
for nonionics, polyoxyethylene chains and polyols; and for cationics,
ionised tertiary or quaternary ammonium groups. The hydrophobic
 PRESERVATION OF AQUEOUS EMULSIONS 217

portion of the surfactant molecule usually consists of hydrocarbon radicals

with more than eight carbon atoms, i.e. alkyl-aryl or long chain alkyl.
Since the hydrophobic hydrocarbon groups have considerable affinity for
oils and fats they have also been termed lipophilic or fat loving. Long
polyoxypropylene chains, though less hydrophobic than hydrocarbons,
nevertheless possess sufficient repellency to confer strong surface activity
to compounds, wherein they are linked to hydrophilic groupings, such as
polyoxyethylene chains. Thus a very versatile range of surfactants consists
of block copolymers of ethylene oxide and propylene oxide in which the
ratios and the molecular weights of the two different types of polyether
chains are varied over a wide range. Table 9.2 gives some examples
illustrating the chemical make-up found in typical commercial surfactants.
The behaviour of amphiphilic compounds in either aqueous or non-
aqueous media is determined by the relative effectiveness of their
hydrophilic groups and hydrophobic groups. In aqueous media the
hydrophilic groups are strongly associated with the water molecules whilst
the hydrophobic groups are repelled and tend to concentrate at the air-
water interface. In the presence of both aqueous and oily phases the
affinities of both groups can be satisfied by concentration of the surfactant
molecules at the oil-water interface. The surfactant molecules will be
orientated in such a way that the hydrophobic groups are associated with
the non-aqueous phase while the hydrophilic groups are firmly anchored in
the water layer.
If the interfacial area is small, it can only accommodate a small number
of molecules. When, as usual, many more surfactant molecules than this
are present, the majority cannot escape from the bulk liquid to the
interface and the affinities of the hydrophilic and lipophilic groups must be
satisfied by other means if thermodynamic stability is to be achieved. This
again occurs by a process of orientation; in an aqueous medium the
hydrophobic groups turn towards and associate with one another, forming
in effect their own oil phase, surrounded by the hydrophilic groups turned
outwards and anchored in the water. This type of internal association and
orientation has been termed micelle formation. Micelles are usually
spherical in shape. The 'escape mechanism' of micelle formation only

Table 9.2 Some typical commercial surfactants

Anionics Nonionics

Stearate soaps Polyethoxylated alkanol

Dodecyl sulphate Polyethoxylated nonyl phenol
Dodecylbenzene sulphonate Polyethoxylated polypropylene glycol
Dioctyl sulphosuccinate (pluronic)
Nonyl phenol polyether sulphate Glyceryl monolaurate
Dodecyl polyether phosphate
218 PRESERVATION OF SURFACTANT FORMULATIONS

becomes operative above a certain minimum surfactant concentration.

This concentration has been termed the critical micelle concentration
(CMC). CMCs vary from about 5 X 10-2 mol 1-1 for the most hydrophilic to
about 5 X 10-4 mol 1-1 for the most hydrophobic types of surfactant. They
are influenced by electrolytes, especially in the case of ionic surfactants,
and also by other polar/non-polar chemical compounds such as alcohols,
ami des and, of course, other surfactants.
The most important consequence of micelle formation in the process of
emulsion polymerisation is the solubilisation of organic compounds in
aqueous media. Since the association of lipophilic groups inside a micelle
leads to a formation of centres of attraction for organic compounds it is
possible to dissolve appreciably higher proportions of sparingly water-
soluble monomers in micellar solutions than in water alone. Monomers
which are essentially non-polar in character may be expected to dissolve
only inside the hydrocarbon portion of the micelle. Compounds with polar
groups can at least partially be accommodated in the water phase or at the
micelle surface so that the requirements for the micelle dimensions are less
critical.

9.2.3.1.1 Surfactants in emulsion polymerisation: the mechanism. The

role played by surfactants in emulsion polymerisation l7 •18 can best
be demonstrated in a simple recipe involving only water, surfactant,
monomer(s) and polymerisation initiator. When a sparingly water-soluble
monomer is stirred into an aqueous surfactant solution it will be broken
into droplets of varying size and give a more or less stable emulsion
depending on the choice and quantity of surfactant present. If the
surfactant concentration exceeds the CMC, some of the monomer will be
solubilised in micelles. On the addition of a polymerisation initiator, and
this almost invariably means a substance capable of producing free
radicals, polymerisation will begin as soon as the initiator has been
activated either thermally or chemically to yield free radicals. 19
The process of emulsion polymerisation begins when the free radicals
derived from the, usually water-soluble, polymerisation initiator enter the
monomer-saturated micelles where they find a sufficient number of
solubilised molecules to start a rapid chain reaction. 19 Each polymer
radical first exhausts the monomer contained in the micelle and then
captures additional supplies from 50 or more other micelles before the
chain reaction is terminated. Some of the depleted micelles then break up
and the released emulsifier molecules are adsorbed at the surface of the
newly formed primary polymer particles. 18 The remainder are replenished
by diffusion from the emulsified monomer droplets which act essentially as
reservoirs.
The formation of fresh polymer particles continues until all the
emulsifier originally contained in the micelles is adsorbed at the larger
 PRESERVATION OF AQUEOUS EMULSIONS 219

polymer/water interface and the surfactant concentration has dropped

below the CMC. 18 A new and different situation is thus created for the
polymerisation which would come to a halt if it could only proceed inside
surfactant micelles. The growing polymer particle is, however, still very
similar in general characteristics to a true (spherical) micelle. The chief
difference is the composition and size of its lipophilic centre. On the
outside there are still the layers of orientated surfactant molecules with
their hydrophilic groups pointed towards the external water phase while
the lipophilic groups are now associated, not with each other, but with the
dispersed internal polymer phase. The conditions for monomer solubilisa-
tion are in fact greatly improved. Quite apart from the fact that the
polymer particles have a far greater affinity for monomer molecules than
the micelles ever had, there is now a considerable interfacial area available
where interface solubilisation, akin to intramicellar solubilisation, can take
place. It is this interface solubility of the monomers which is chiefly
concerned in the second phase polymerisation, i.e. after the disappearance
of the micelles. The loci of polymerisation have then shifted from the
micelles to the polymer particles to which all the emulsified monomer is
gradually transferred.
Now consider the case of polymerisation of monomers with an
appreciable water solubility and their copolymerisation with less water
soluble comonomers. Monomers such as vinyl acetate or methyl acrylate
have sufficient water solubility to permit their rapid polymerisation in the
water phase even in the absence or after the disappearance of surfactant
micelles. New polymer particles can be formed as long as the monomer
concentration in the water phase remains high enough. In most cases, there
is strong monomer/polymer affinity so that more and more monomer will
be extracted from the water phase. As polymer concentrations increase,
polymerisation in the water phase will finally cease and with it the
formation of new particles. Henceforth, conversion will proceed at the
surface of the polymer/monomer particles in much the same way as in the
case of water-insoluble monomers after the disappearance of the surfactant
micelle.

9.2.3.1.2 Stabilisation of polymer particles. At the end ofthe emulsion

polymerisation polymer particles will exist in the dispersed water phase
with surfactant molecules adsorbed on the particle surface. It is usually
considered that it is the hydrophobic portion of the surfactant that is
adsorbed onto the surface while the hydrophilic portion goes into the water
phase. The role of the surfactant is now to keep the system stable by
preventing coalescence of the polymer particles in the dispersion. If
particle coalescence is not prevented the settling of the coagulated particles
can take place giving a non-redispersible sediment.
For anionic surfactants stabilisation of the polymer particles is achieved
220 PRESERVATION OF SURFACTANT FORMULATIONS

by the negative charge on the adsorbed surfactant molecules repelling

neighbouring particles which are similarly charged. Stabilisation by this
means is termed 'electrostatic stabilisation'. In effect, an energy barrier to
particle coalescence is set up by charge-charge repulsion between
particles. This energy barrier can be reduced by the addition of electrolyte,
e.g. salts.
Nonionic surfactants by definition carry no charge and with these
surfactants stabilisation is mainly by volume reaction. Again the hydro-
phobic portion is adsorbed on the polymer surface while the hydrophilic
portion, usually long ethylene oxide chains, extends into the water phase.
Because of the volume occupied by these ethylene oxide chains the
polymer particles cannot easily approach each other, i.e. there is an energy
barrier to coalescence due to the spatial presence of the adsorbed
surfactant. Stabilisation by this means is termed 'steric stabilisation'. The
energy barrier to coalescence can be reduced by reducing the proportion of
the ethylene oxide chains, e.g. either by salt addition or by heating the
latex.
It should be noted that instead of stabilisation by surfactants the
stabilisation of polymer particles may also be achieved by long chain water-
soluble polymers. These are usually termed 'colloids' and typical examples
are polyvinyl alcohol,z0.21 hydroxyethyl cellulose and starch. Stabilisation
is again by steric means. The main functions of colloids are:
• to give increased viscosity to the latex and to influence the structure and
rheological properties, and
• to influence the application properties of the latex, e.g. give increased
bond strength in adhesives.
Polyvinyl alcohol is supplied as a white free-flowing powder. Although
the term 'polyvinyl alcohol' is used it is generally a copolymer of vinyl
alcohol and vinyl acetate. 20,21 The degree of hydrolysis represents the
percentage number of moles of vinyl alcohol to the total number of moles
present. Typical degrees of hydrolysis encountered in emulsion poly-
merisation are 80-100%. Quite different emulsion polymer properties and
application results will be obtained using different hydrolysis grades of
polyvinyl alcohol. The viscosity effects produced by polyvinyl alcohol will
also depend very much on the molecular weight (MW) , since higher
molecular weight species produce higher viscosity latices. 22
Polyvinyl alcohol is rarely used in paint systems as irreversible structures
may be obtained with the gelling agent. For paints the structure may be
achieved by using colloidal agents, such as hydroxyethyl cellulose.

9.2.3.2 Monomers. The polymers existing in emulsion form are

produced by addition polymerisation of one or more unsaturated
monomers, the commonest of which are listed in Table 9.3.
 PRESERVATION OF AQUEOUS EMULSIONS 221

Table 9.3 Monomers

Monomer Monomer name Approx.

MW

HOOC.CH:CHz Acrylic acid 72

N:C.CH.CH z Acrylonitrile 53
C4 H 9 0.OC.CH:CH z Butyl acrylate 128
C4 H s O.OC.CH:CH2 Ethyl acrylate 100
CHz:CH2 Ethylene 28
CH30.0C.C(CH3):CH2 Methyl methacrylate 100
HOCH2·NH.OC.CH:CH2 N-methylol acrylamide 102
C6 H s ·CH:CH2 Styrene 120
CH3·COO.CH:CHz Vinyl acetate 86
CI.CH:CH 2 Vinyl chloride 62.5
CHz--CH.OH:O-C9 H 19 Vinyl versa tate

If a single monomer is reacted, the polymer obtained is termed a

homopolymer. Many of the above monomers, however, can also react with
each other, forming copolymers; varying the proportions of these
comonomers affords an excellent means of controlling polymer properties.
Not all the monomers listed in Table 9.3 react together satisfactorily:
thus vinyl acetate and styrene, ethylene and styrene, are examples of
unfavourable pairs. Moreover, monomers without terminal unsaturation,
e.g. the fumarates, and the related maleates, do not generally form
homopolymers although readily forming copolymers with, for example,
vinyl acetate.
The properties of the polymers are governed mainly by the monomer(s)
on which they are based. All are thermoplastic, that is to say they soften
progressively as their temperature is increased, but regain their original
condition when cooled. Except when special reactive monomers are
included, there is no cross-linking reaction, as occurs in thermosetting
polymers, for example melamine-formaldehyde or urea-formaldehyde
systems.
The hardness and softness of the polymer at a given temperature is
predominantly determined by the monomer(s) employed. In polymers of
identical make-up, differences in molecular weight are found to have a
secondary effect, with higher molecular weight tending to reduce thermo-
plasticity slightly and lead to greater toughness and better resistance to
solvents and water. Examples of monomers yielding hard generally brittle
homo polymers requiring external plasticisers include: vinyl acetate, vinyl
chloride, styrene, methyl methacrylate and acrylonitrile. Examples of
monomers yielding soft internally plasticised homopolymers include: butyl
acrylate, 2-ethylhexyl acrylate and the vinyl esters. Ethylene also acts as a
plasticising comonomer with vinyl acetate and vinyl chloride but by a
different mechanism.
All the polymers are fully saturated, i.e. they contain no residual double
222 PRESERVATION OF SURFACTANT FORMULATIONS

bonding. In consequence they are not prone to ageing effects of

embrittlement and discoloration.
All monomers impart certain characteristic properties to polymers of
which they form a part, but some have a particularly marked effect and can
bring about significant changes in polymer behaviour even when present as
only a few percent of the total monomer mix. As examples, acrylic,
methacrylic acids and other unsaturated acids are used to give alkali-
soluble copolymers in conjunction with methacrylic acid and acrylic esters.
N-methyl acrylamide and other polyfunctional monomers produce
reactive copolymers capable of secondary polymerisation or cross linking
by etherification or ionic bonding, usually after the watery portion of the
emulsion has dried off.

9.2.3.3 Initiators. Initiators produce free radicals which act on the

monomer to form the polymer in aqueous conditions. Initiation may be
thermal using a single chemical type or a redox system may be used. 19
Redox systems are suitable for polymerisation at low temperatures. Some
common initiator systems are listed in Table 9.4.
These redox chemicals may also be added at the end of the polymerisation
process, as a finishing off stage (FOS), in order to get maximum
polymerisation and to reduce residual unreacted monomer.
Other additives may be found in a polymer emulsion system which
contribute to its total composition. Examples of these additives are:
• Buffering agents - for pH control, e.g. sodium bicarbonate
• Antifoams - for preventing foaming
• Salts, e.g. sodium sulphate
• Metal catalysts, e.g. ferric chloride
• Preservatives - for antimicrobial protection from spoilage.

9.2.4 Manufacture of synthetic polymer emulsions

Emulsion polymerisation is carried out in a reactor where temperatures
may reach 90°C during the process. Thermally initiated emulsion processes
are manufactured at temperatures between 70--90°C. In redox initiated

Table 9.4 Initiators for emulsion polymerisation

Oxidising agents Reducing agents

Potassium persulphate Sodium formaldehyde sulphoxylate

Sodium persulphate Sodium metabisulphite
Ammonium persulphate Sodium thiosulphite
t-Butyl hydroperoxide Ascorbic acid
Cumene hydroperoxide
Hydrogen peroxide
 PRESERVATION OF AQUEOUS EMULSIONS 223

processes temperatures of between 40-50°C are usually the norm. It is

worthy of note that in the authors' experience microbial contamination has
not been found in emulsion samples taken directly from the reactor after
polymerisation. In many instances, the temperatures and chemical
environment during which polymerisation is carried out are sufficient to
destroy many microbial contaminants arising from raw materials.
From the reactor the emulsion is pumped through a network of
pipework to the blenders, degassing tanks, an open filtration system and
finally to the bulk storage systems. During this time the emulsion is cooling
and possibly could become contaminated by microorganisms. This is the
reason for adding a chemical biocide in order to preserve the emulsion
against spoilage. Biocides, though required in very small amounts, are
often expensive and complex chemicals that are very labile to the hostile
chemical environment of the polymerisation process and therefore their
addition must be carefully controlled and monitored. 23 ,24 The biocide is
therefore added into the blender at a time determined when the
temperature is low enough «50°C) to prevent thermal degradation and
when the free radicals have been consumed in the process. 24 This time is
determined by a simple test to monitor the redox state of the emulsion. 25 ,26
Under these conditions the biocide has the best chance of survival to allow
it to perform its function as a long term preservative against microbial
spoilage of the emulsion. The chemical stability of the biocide and the
accuracy of dosing of the biocide into the emulsion is determined by high
performance liquid chromatography. 27 This is a quality control test method
carried out to confirm if the emulsion is adequately preserved and is a
standard specification along with other specifications of solids content, pH,
viscosity, particle size distribution, residual monomer levels and so on.
Another reason for checking the level of the biocide is for regulatory
purposes where different emulsions depending on their end use have limits
on their preservative type and content. Examples of these regulations
include: FDA and BGA regulations for direct and indirect food contact in
food packaging applications for adhesives; BGA regulations for non-
woven applications where binders are used in sensitive areas, e.g. feminine
hygiene products, medical wipes and disposable nappies/diapers; and last
but by no means least, areas where only food grade biocides can be
incorporated, e.g. the adhesive used in cigarette manufacture for the seams
in the paper and in the wax coatings for certain cheeses.

9.3 Adhesive formulations

9.3.1 Applications and uses of adhesives

To demonstrate the key properties required for an adhesive, consider the
choice of an adhesive in two cases, a broken china mug to be repaired and a
224 PRESERVATION OF SURFACTANT FORMULATIONS

wooden wine rack to be assembled. For the wine rack, the likely adhesive
choice is a standard wood glue, commonly one based on aqueous synthetic
polymer emulsions; a small 'blob' of glue would be placed in each of the
joints, the joint made and the final assembly left to dry for 24 h before
subjecting it to stress, i.e. filling it with bottles. For the china mug, a
cyanoacrylate adhesive is chosen; its use requires the cleaning of both
surfaces to be bonded, followed by the application of a small amount of
adhesive to one or both surfaces and then holding these close together for a
short time while the adhesive dries.
The choice of these two adhesives is not by accident since they are
effective in different ways. The wood glue, which contains a polymer
dispersed in water, acts by using water as a carrier to facilitate penetration
of the wood fibres by the polymer. Then, slowly, the water dries leaving
only polymer which holds the joint together since it is intrinsically
entangled with the wood fibres. Important adhesive characteristics are: the
viscosity, in that the adhesive must be thin enough to penetrate the wood
but not so thin that it penetrates one side of the joint completely before
assembly or that it drips readily; the solids content, whereby water is
necessary as a carrier but too much water slows the drying and may distort
the joint; the set speed which is different for different emulsion types; and
the bond strength which is a requirement determined by the stress which
the adhesive receives when the wine rack is full.
This wood adhesive may perform well in this application, but it would
not be suitable for bonding a mug designed to hold liquids and so be
impervious to water; no mechanical adhesion could be obtained by
transport of the polymer into the china. Without transport, other
mechanisms of adhesion would be ineffective with this adhesive/substrate
combination since the low intermolecular forces would be insufficient for
the demanding end use (e.g. hot tea and cleaning in dishwashers) and again
chemical adhesion is low since there are no chemical groups which can
react between the polymer and the china surface.
The cyanoacrylate, on the other hand, acts in a totally different way with
no carrier involved though the adhesives are usually of low inherent
viscosity. Although little penetration is seen, a good bond can be obtained
since a chemical polymerisation occurs after application forming strong
bonds between the adhesive and itself and between the adhesive and the
surface of the mug. The final bond is much stronger than with the
woodworking emulsions due to the strength of this reacted polymer. So
why not use this to make the wine rack? One reason is the higher cost in
use, another is the irreversibility if mistakes are made aligning the joints.
These two adhesives comprise two of the four main types of adhesive,
namely aqueous-based (i.e. solution and dispersion) and reactive adhesives.
The third adhesive type consists of hot melt types and the fourth is based
on solvents.
 PRESERVATION OF AQUEOUS EMULSIONS 225

9.3.2 Range and chemical types of adhesive

9.3.2.1 Hot melt adhesives. Hot melt adhesives are formulated to give a
material which is solid at room temperature but which softens on heating.
At application temperatures, commonly in the region of 150 to 170°C, the
hot melt is liquid enough to act as its own carrier to allow the polymer to
penetrate into the substrate. These adhesives are generally faster setting
than aqueous-based adhesives since less energy change is required to cool
them to form a reasonable bond than is required to drive off water to set
aqueous-based adhesives. Their disadvantage often lies in their heat
sensitivity, since a bond subjected to high temperatures may fall apart.
These adhesives will not be mentioned further in this chapter since
preservatives are rarely required for hot melt adhesives because the
fundamental requirements for microbial growth, nutrients, moisture and
oxygen are not present in either the adhesive or the adhesive film in the
finished assembly. In addition, the high application temperatures would
kill any microorganisms that may be present.

9.3.2.2 Solvent-based and reactive adhesives. Solvent-based adhesives use

solvent as a carrier to transport dissolved polymer into the substrates. Their
history and efficacy is well documented but increasingly environmental
pressures are causing these 'older' formulations to be replaced by aqueous-
based materials. In some cases these environmental pressures may be mis-
placed since the risks of environmental damage are simply redirected from
the air (solvent evaporation) to the ground (waste water). Key differences
from aqueous-based adhesives are that they are often of a lower molecular
weight, higher tack, lower solids content and a faster set speed. Again,
these types of adhesive are much less prone to microbial attack than the
aqueous-based adhesives.
In addition to the cyanoacrylates mentioned above, other types of
reactive adhesives include the anaerobics and epoxies which can be cured
by heat or by two part reactions with amines or polyurethanes which cure
by reaction of isocyanate groups with polyols and silicones. Preservation is,
as above, often not an issue with these adhesives.

9.3.2.3 Aqueous-based adhesives. The various types of aqueous-based

adhesives are the most conducive to microbial growth. They can be
classified in many ways with the simplest classification dependent on their
solubility. The solution type of adhesives are based on starch, dextrine,
casein or polyvinyl alcohol and the disperson type adhesives are based on
synthetic polymer emulsions ranging from vinyl acetate systems to rubber
latex or polyurethane emulsions.

9.3.2.3.1 Solution adhesives. Starch, a biopolymer, from various

226 PRESERVATION OF SURFACTANT FORMULATIONS

sources including potato, maize and tapioca is one of the oldest raw
materials 28 for adhesive manufacture and in its various guises or modifica-
tions is still commonly used as an adhesive. 29 Starch is a natural polymer of
repeating glucose units which are linked by a-glycosidic bonds to give
linear (amylose) or branched (amylopectin) chains and the relative amount
of each of these chains determines in part the starch properties. In the
highly branched amylopectin chains the starch polymers are packed less
densely than in the straight chain amylose starch polymers and have a
greater stability, greater water solubility and lower strength.
Starch itself is processed to provide discrete granules of 5--40 ~m in size,
but to be useful as an adhesive these particles have to be dissolved by
heating a slurry of the starch with water to a 'gel point' where the granule
will be swollen and, if subjected to strong agitation, can rupture. Native
starches can be treated in this way but give only a low solids content, a
high viscosity and form an unstable paste, and as such it is generally the
modified starches that are used in the manufacture of starch pastes. The
simplest modifications that are employed involve acid conversion (when
the starch slurry is treated with a mineral acid) to give a fluidity starch or
chemical oxidation (when the starch slurry is treated with oxidising
agents), but again the adhesives produced from these after gelatinisation
are fairly low stability pastes and they are used for simple applications like
paper bag manufacture.
More importantly, chemical modifications of starch are employed to
widen the scope of these adhesives using the chemical reactivity of the
highly reactive hydroxyl groups on the glucose ring. Wet phase modifica-
tions are performed by adding reagents to the aqueous starch slurry under
controlled reaction conditions. Cross-linking starches with, for example,
epichlorohydrin, esterifying with acetic anhydride or etherifying with
propylene oxide, all give starches with greater viscosity stability than native
or fluidity starches and this increases the range of adhesive end uses. For
example, wallpaper pastes are often made from chemically modified
starches.
An alternative to cooking starches to give a final adhesive is to drum dry
them after partial gelatinisation during the conversion process, which gives
rise to cold water-soluble starches which may then be prepared in situ
without the need for further cooking. Dry conversion of the starches is
achieved by roasting with an acid catalyst to give dextrines. Two classes are
produced but many variations are possible due to the complexity of the
various starches and the various parameters of the process which can be
independently controlled. White dextrines are less converted than the
yellow dextrines and have slower set speeds, lower tack and lower viscosity
stability. Conversely, yellow dextrines can be used at very high solids
content and are often treated with borax to give high tack products used in
paper converting, for example tube winding.
 PRESERVATION OF AQUEOUS EMULSIONS 227

Jelly gums are made by cooking starch in strong alkali to give thick,
tacky adhesives for bottle labelling applications.
Other natural sources of solution or semi-colloidal adhesives are the
caseins which use milk-derived products to give pale, high tack, high
viscosity and high adhesion products. These properties and the strongly
temperature-dependent viscosity profile of caseins also makes them
suitable for this bottle labelling application.
Like starch, cellulose is also a biopolymer and has a similar molecular
origin, again it is based on glucose units but with the units linked by f3-1,4-
(D )-glycosidic bonds as opposed to the a-glycosidic bonds in starch
polymers. Cellulose polymers are also used as adhesives in simple
applications such as tissue and towel lamination.
Other biopolymers, namely polyhydroxybutyrate produced from micro-
bial metabolic transformations are increasingly being used in the field of
adhesive applications. A commercial process for the production of
polyhydroxybutyrate by microbial transformations with the microorganisms
has been patented by ZENECA BioProducts in Billingham, Cleveland,
UK and called Biopol. Biopolymers, being natural products, are becoming
more interesting to industry due to an increased environmental awareness
and needs for recydability of packing materials.
Natural rubbers also provide a source of adhesive raw materials. Natural
rubber latex adhesives may be difficult to handle due to the high pH (often
ammonia-based) required for stability but show great advantages in the
ability of latex to bond well to itself but little to other substrates. This
makes it an ideal choice for self-seal applications such as in envelope
manufacture.
Solution adhesives do not all come from natural sources. Polyvinyl
alcohol 2o ,21 is used to make adhesives used in applications such as multi-ply
board lamination, as well as its use as a formulation tool for emulsion-
based adhesives. The polyvinyl alcohol is prepared by hydrolysis of
polyvinyl acetate and it is supplied as a dry powder which must be dissolved
prior to use.

9.3.2.3.2 Dispersion adhesives. Applications for solution adhesives

from starch or other sources are often limited by poor adhesive properties
such as a low adhesion to certain materials, for example plastics, a
relatively poor viscosity stability often coupled with a low solids level and a
relatively slow drying speed (since a great proportion of the water must be
lost to allow film forming). Synthetic polymer emulsions were developed to
overcome these shortcomings and a description of these and their chemical
constitution and manufacture have already been discussed.
The great advantages of using synthetic emulsion polymers in adhesives
lies in their ability to set quickly due to the hydrophobic nature of the
polymer, their relatively high molecular weight and their facility for
228 PRESERVATION OF SURFACTANT FORMULATIONS

manufacture at high solids level (40-50%) at a stable, low viscosity. The

thermoplastic polymers also have a good adhesion onto plastics.
Several types of polymer are used. The polyvinyl acetate homopolymer
(PVA) from vinyl acetate is a fairly hard polymer due to restriction of
rotation in the molecular chain caused by the proximity of bulky acetate
groups and the use of homopolymer emulsions usually requires formulation
to soften or swell the polymer particles by the addition of plasticisers. An
alternative route to a softer emulsion comes from the use of copolymer
emulsions from the copolymerisation of vinyl acetate and ethylene
monomers. The relatively low steric volume of the ethylene links allows
greater freedom for chain movement giving a polymer which is less hard
when dry and the use of softening agents can be avoided. This effect can be
measured by looking at the glass transition temperature of these two
polymers when dry, that of the copolymer is around 30°C lower. The
drawback of using these vinyl acetate/ethylene (VAlE) copolymers is their
higher cost which arises from the capital investment required for their
manufacture but this is often offset by the advantages of their higher
adhesion. In general, PVA is suitable for use in bonding involving paper,
cardboard or wood and VAlE is also suitable for these materials and for
more difficult substrates, e.g. treated paper or plastics.
Other copolymers commonly used are between vinyl acetate and dibutyl
maleate or between vinyl acetate and vinyl acrylate. The latter gives
acrylic-type emulsions which are characterised by very low glass transition
temperatures and are seen to be permanently tacky or pressure sensitive.
Adhesives based on these are commonly used for self-adhesive labels.
Terpolymer emulsions are also used, e.g. vinyl acetate, ethylene and vinyl
acrylate.
More recently polyurethane emulsions have gained importance and
these show the advantages of high adhesion and high heat resistance
(especially if cross linked in situ with isocyanates) though they have
restricted compatibility with some other emulsions and can be both shear
and pH sensitive. The manufacturing process is very different from those
above since the polymerisation is essentially done in a non-aqueous organic
phase which is then dispersed in water and the residual organic phase
stripped off by vacuum.

9.3.3 The key properties and tests on adhesives

Some of the requirements for adhesives used in the home have been
considered. When industrial applications are involved the requirements
become considerably more varied and demanding. Adhesives may be
applied by spray systems allowing the deposit of several kilos of adhesive
each minute or by systems of rollers, this requires not just a specific
viscosity but also a specific rheology (shear dependence of viscosity). The
 PRESERVATION OF AQUEOUS EMULSIONS 229

end-use requirements may also be stringent since although the adhesive

may be a small part of an assembly and contribute only a small part to its
cost, the consequences of adhesive failure could be severe with the whole
assembly being rendered useless.
To summarise, some of the key characteristics required of aqueous-
based adhesives are described below.

9.3.3.1 Viscosity and rheology. This determines the ability to penetrate

into surfaces and the suitability for different types of application
equipment. Viscosity is generally measured on a Brookfield viscometer
and rheology on various types of rheometer. Pseudoplastic adhesives have
lower viscosities as shear is increased, dilatant adhesives have higher
viscosities as shear increases and thixotropic adhesives show a time-
dependent behaviour.

9.3.3.2 Solids. This is a measure of the amount of material left after

drying, i.e. after water loss.

9.3.3.3 Set speed. This quantifies how quickly a given adhesive forms a
workable bond under given conditions. The bond must not develop before
the assembled pieces are in their final position but must set quickly enough
to withstand the stresses in any subsequent manufacturing operation.

9.3.3.4 Bond strength. The final bond strength of an assembly is

determined by the weakest of three types of interaction, the adhesion of
the adhesive onto the substrate, the internal strength of the adhesive and
the internal strength of the substrate itself. The finished assembly may be
subjected to many stresses in its lifetime and the bond strength must be
high enough to withstand these both initially and after ageing.

9.3.3.5 pH. Specific pH ranges may be required, for example to

prevent attack of the adhesive on aluminium machine parts.

9.3.3.6 Adhesion. Adhesives may be designed for specific adhesion to

difficult substrates. The adhesive strength can be quantified by measuring
the force required to remove adhesive strips from a substrate on a
tensiometer.

9.3.3.7 Wet tack. This qualifies the amount of grab/hold an adhesive has
before drying. It is important in holding assemblies together temporarily,
especially in high speed operations.

9.3.3.8 Redispersibility. The properties of redispersibility are often

important in allowing an adhesive to run cleanly on a machine, since
230 PRESERVATION OF SURFACTANT FORMULATIONS

buildup of dried adhesive can cause non-uniform adhesive coverage. This

property is also becoming increasingly important in the search for
recyclable packaging materials since in paper packaging, it may be the
adhesive which is the most difficult part of the package to recycle.

9.3.3.9 Odour. A low odour can be important, especially in adhesives

which are used for food packaging but also for the comfort of operators in
large scale industrial processes.

9.3.3.10 Stability. Adhesives are required to remain stable and suitable

for use, often for one year from their date of manufacture. This places
requirements on their ability to maintain all of the above properties over
time and not to settle out of phase. In order to do this, the adhesives must
be capable of resisting extremes of storage temperature and, of course,
attack from microorganisms.

9.3.4 Chemical nature and formulation of aqueous-based adhesives and

their raw materials
An aqueous adhesive is designed to have specific values for the properties
described in section 9.3.3, with the actual values determined by the
adhesive end use and its method of application. Each adhesive is
formulated with various raw materials to produce these properties and
these raw materials contribute to the susceptibility of the product to
microbial attack.
In addition to an emulsion or a blend of emulsions, a product may
contain the following components.

9.3.4.1 Plasticiser. Plasticisers such as dibutyl phthalate, triacetin or

diethylene glycol dibenzoate are added at up to 15%. They usually come in
the form of non-volatile organic liquids and act by swelling emulsion
particles, increasing the viscosity of the wet adhesive and the softness of
the dry film. They are particularly important in the formulation of PYA
homopolymers, without them these polymers are usually too brittle.
Copolymers can be formulated without plasticiser.

9.3.4.2 Humectant. Up to 5% glycerine, urea, sugar or glycols can be

added to emulsion adhesives. These hygroscopic materials reduce the
speed at which the adhesive dries due to their affinity for water. They also
control surface drying or skinning on the emulsion surface and they can be
used to make the dry film very susceptible to picking up moisture, hence
their use in adhesives for high speed machines.

9.3.4.3 Polyvinyl alcohol. Polyvinyl alcohols are water-soluble polymers

that are added to emulsions to increase open time, tack and redispersibility
 PRESERVATION OF AQUEOUS EMULSIONS 231

and to control the viscosity. They are supplied as dry powders and need to
be cooked in water to dissolve, this can be done in situ before adding the
emulsions or as a separate premix. Both partially and fully hydrolysed
polymers are used.

9.3.4.4 Tackifiers. Boric acid is often added to emulsions used in fast

packaging applications since it cross links any polyvinyl alcohol present and
significantly augments the tack. Tackifying resins from rosin or hydrocarbon
sources can also be added for this purpose.

9.3.4.5 Thickeners. Small quantities of materials such as acrylics,

cellulosics or polyurethanes are added to give a required viscosity or
rheology or to control solids by allowing various dilution factors for a
constant end viscosity.

9.3.4.6 Fillers. Calcium carbonates, calcium sulphates, clay or starch

can be added at up to 50% to emulsion products. As well as enabling cost
control in the application due to their gap filling properties, fillers help to
give hard films and can affect rheology. Titanium dioxide can be used to
control adhesive colour. Processing of filled emulsions requires strong
mechanical agitation to ensure uniform dispersion ..

9.3.4.7 Defoamers. Foam control agents or surfactants are added as

antifoam to prevent foam formation or as a specific defoamer to destroy
foam both during adhesive manufacture and during the final adhesive use.
A foamed adhesive will give a poor, non-homogeneous film.

9.3.4.8 Surfactants. Wetting agents such as surfactants are also added to

modify the surface tension of the final adhesive and then control its ability
to wet out on certain surfaces. This is particularly important in the realm of
pressure-sensitive adhesives and when bonding to plastics.

9.3.4.9 Preservatives. These are used to prevent microbial attack.

9.4 Microbial spoilage of polymer emulsions and adhesives

Having considered the uses, applications and chemistry of different types

of polymer emulsions and adhesives it is clearly indicated that their
aqueous-based formulations provide, in the water phase, all the essential
ingredients required for microorganisms to reproduce and flourish. 30-33
Although the limited amount of unreacted residual monomer present will
give some antimicrobial protection this is becoming less important as levels
are reduced due to increasing legislation and awareness of environmental
232 PRESERVATION OF SURFACTANT FORMULATIONS

pollution. The trend is towards manufacturing and supplying emulsions

free of organic emissions by employing methods to 'scrub out' these
components so as to gain market share for more 'environmentally friendly'
products. Conversely, certain monomers will actually aid in the spoilage of
the emulsion itself. This statement, however, contradicts a widely held
belief in the polymer industry that a residual level of monomer will act as a
microbicide. 33 It is very much a question of levels. Microbial attack of vinyl
chloride has been shown to occur34 and still further it is known that vinyl
acetate breaks down to give acetaldehyde and acetate 35 and both these
metabolities are microbial nutrients at the levels found in typical polymer
emulsions, where free vinyl acetate levels are of the order of 0.1 %. At
higher levels, as may have been found some years ago, acetaldehyde would
have been at preservative concentrations. Free acrylate monomers may be
available to some types of bacteria especially Pseudomonads. The precise
mechanism by which they are utilised is not clearly demonstrated but
probably occurs by hydrolysis of the monomer to acrylate and the alcohol.
In the past, the residual monomer was regarded as an essential component
of the emulsion system to afford some degree of antimicrobial protection,
although this is only adequate for normal storage of emulsions in closed
containers. For prolonged storage, or in vessels that are not well closed,
the protective action from the residual monomer will slowly evaporate, so
additional preservative will need to be added. Moreover, the protective
effects from the residual monomer are not sufficient to withstand the
additional populations of microorganisms likely to be introduced with
pigments, fillers etc., or withstand heightened microbial activity in the
presence of additional colloids.
The stabilising colloids are also a good source of microbial nutrient.
Some surfactants contain long chain fatty acids and are in consequence
fairly easily degraded. Substituted celluloses which are commonly used as
stabilisers may be attacked by microorganisms, the amount of nutrient
available from this source depending on the type and degree of
substitution. Cell uloses may also provide nutrients from their catalytic
chemical breakdown throughout the reaction. The degradation of
cellulosic materials by a variety of microorganisms has been reported
abundantly in the scientific literature over the past 100 years as reported in
two recent reviews on the subject. 36 ,37
The polymer component itself is virtually unaffected by microorganisms,
but the degradative effect on the water phase can lead to spoilage of the
emulsion manifested by the production of a number of undesirable effects.

9.4.1 Consequences and effects of microbial spoilage

If microbial spoilage does occur, the consequences can be severe since it
can cause deterioration in both the polymer emulsion and adhesive
 PRESERVATION OF AQUEOUS EMULSIONS 233

properties and potentially render the formulated product unsuitable for its
intended purpose. The effects of microbiological spoilage of these
aqueous-based synthetic products may often go unnoticed until it is too late
to take remedial action. As such, if substrates are contaminated and the
microorganisms and their byproducts, e.g. extracellular enzymes, are not
inactivated, or at least their growth and activity inhibited heavily, they are
able to decompose the product's ingredients, a consequence which
manufacturers or consumers will realise when one or more of the following
unacceptable symptoms are noticed. 3O-32

9.4.1.1 Effects on products.

1. Viscosity loss of the emulsion and of formulated products by breakdown
of colloids and surfactant-stabilising systems of emulsions by microbial
attack or by extracellular enzymes. Since the viscosity and rheology are
fundamental to the product's applicability characteristics, a contamin-
ated product can be unusable. Another consequence of viscosity
instability is that phasing may be induced leading to a non-
homogeneous product with thinner liquid on the top and thicker
material on the bottom. This phenomenon is often termed 'syneresis'
and is the result of settling and destabilisation of the product after
effects to its rheological and viscocity properties.
2. An unpleasant odour or foul smells, e.g. sulphurous 'bad egg' odours
produced by the sulphate-reducing bacteria which under anaerobic
conditions can utilise oxygen from sulphates leading to hydrogen
sulphide production. This type of odour production is often combined
with a noticeable blackening in the colour of the product. In itself this
odour can be unpleasant for the user but potentially more serious is the
possibility that the odour will reappear if a dried adhesive film is
remoistened. A contaminated adhesive used for food packaging could
lead to product rejection by the final consumer. Other unpleasant
rancid or musty odours are derived from various microbial by-products
such as butyric acid and 1,6-dimethyl-3-methoxypyrazine.
3. Colour changes and discoloration as a result of the formation of
insoluble sulphides such as iron sulphide produced by sulphate bacteria.
Other microorganisms, particularly the pink yeasts such Rhodotorula
rubra and Sporobolomyces roseus, and pigmented mould fungi can
produce other discolorations. This is often visualised in the appearance
of an oily yellow film on the product's surface.
4. Gas formation, often accompanied by unpleasant odours, produced by
fermentative bacteria from the breakdown of colloidal thickeners, e.g.
cellulose to glucose which is then fermented to yield acid and carbon
dioxide. Such gas production is not usually noticed during production
but can cause distortion and even splitting or 'lid popping' of containers.
234 PRESERVATION OF SURFACTANT FORMULATIONS

5. Surface growth of the aerobic mould microfungi on the surface of

products leading to surface disfiguration. This is particularly true where
moulds grow on the surface giving a 'green carpet' on the surface of a
product. In these cases, testing can reveal that only the surface is
contaminated and the bulk of the product below the surface is free from
microorganisms.
6. pH changes, e.g. lowering of pH due to metabolic activity of microbial
cells by fermentative metabolic processes breaking down colloidal
components of the product. This acid production can lead to product
destabilisation as well as possible corrosion to storage vessels and
containers.
7. Changes in properties affect the use of the formulated product due to
microbial contamination. The resulting destabilisation and viscosity loss
of the product as well as attack on the polymer particles can lead to a
reduction in the average molecular weight of the polymer. In this
context the bond strength of a contaminated adhesive would be
expected to be less than that of a corresponding non-contaminated
adhesive. In practice, this is not often seen since if the deterioration has
progressed to this stage, the contamination is likely to be detected
before the adhesive is applied.
8. Enzyme production, e.g. cellulases and amylases produced by bacteria
and mould fungi may lead to viscosity loss in cellulosic and starch based
products. Cellulase enzymes are effective at concentrations as low as
10-5 enzyme units per ml in breaking down long chain cellulosic
molecules into shorter oligomers, thereby destroying their viscosity
regulating ability and will thus cause a dramatic viscosity change of
formulated products. 38
9. Corrosion from metabolic bypro ducts and acid production.

Once a polymer emulsion or adhesive has been used, contamination can

still take place especially in high moisture environments. To prevent this,
dry film preservatives can be incorporated into the formulated product
prior to application, e.g. ceramic tile adhesives and in water-based
emulsion interior paints and exterior masonry paints. 5 ,13,39-41 However,
dry film preservatives are not generally necessary in most aqueous-based
adhesives and polymer emulsions.
In general, for end uses where a polymer emulsion or adhesive will be in
contact with an environment conducive to the propagation of microbial
infestation, a contaminated product cannot be ignored as a possible source
of further contamination. All the effects detailed above resulting from
microbial spoilage of polymer emulsions and adhesives must be fully
investigated in terms of product quality complaints because they can lead
to significant economic loss to the manufacturer if products reach the
consumer in a deteriorated condition.
 PRESERVATION OF AQUEOUS EMULSIONS 235

9.4.1.2 Effects on the environment.

• Appearance of sour and putrid odours.
• Health risks.
These effects are less apparent to the manufacturer than to the end user
of the polymer emulsion, but they must not be overlooked. Both the foul
odours, particularly from hydrogen sulphide, and the actual presence of
microbial spores can have a serious effect on man. Hydrogen sulphide
levels in contaminated products have never been shown to reach toxic
concentrations but even very small amounts can render a product
unsaleable. In both these examples there are strict guidelines on
occupational exposure. In the case of microbial spores, overexposure can
lead to respiratory disorders and asthmatic symptoms.

9.4.1.3 Effect on production plants.

• Blocking of pipes,
• Foam formation,
• Filtration problems,
• Corrosion of plant vessels and machinery.
These effects are more physical and are often associated with the result
of a microbiological spoilage outbreak in a manufacturing plant. They can
range from simple situations that are relatively easy to eradicate with the
use of sanitation measures combined with the use of effective biocides, to
more complex situations if spoilage is allowed to go unchecked for
prolonged periods. Then significant economic loss to the company is
realised, ranging from that caused by long periods of downtime whilst the
problem is resolved to the need for major capital investments to replace
pipework and tanks or even corroded metalwork in the plant.

9.4.1.4 Effects on production. The effect on product quality can

severely damage a manufacturer's reputation in the market to the extent
that they may lose the business in part or totally, or even suffer high
financial claims for the end-user's losses which may be settled in or out of
court. All this can lead to interrupted work in production and additional
time which must be put in to rectify the quality incident.

9.4.2 Types of spoilage microorganism encountered

The majority of bacteria, yeasts and mould microfungi commonly living in
the environment can be found in samples of polymer emulsions, as well as
in formulated products thereof. Microorganisms are extremely versatile in
their modes of nutrition, many organisms have very simple requirements
needing only an organic carbon source and inorganic source of nitrogen,
236 PRESERVATION OF SURFACTANT FORMULATIONS

phosphorus and sulphur. Some individual genera are capable of utilising a

wide variety of compounds, e.g. Pseudomonas spp. which can grow in
many hundreds or organic compounds ranging from simple compounds
like acetate to complex organic chemicals used as disinfectants in hospitals,
e.g. phenols and organohalogen compounds often used as ingredients in the
formulation of many biocides.
Various chemical, physical and environmental factors will determine the
types of microorganism that can be isolated from different synthetic
polymer emulsion and adhesive systems. For example, Evertsen in 198831
noted that products with a pH of less than 4-5 were rarely susceptible to
microbial attack although slight contamination was occasionally found in
acidic plasticised vinyl acetate homopolymers. Evertsen also found that
alkaline acrylic and styrene acrylic types of polymers were particularly
susceptible to bacteria. In contrast to these findings the authors (un-
published data) and Elsom in 198830 found that acidic polymers, including

Table 9.5 Types of microorganism isolated from polymer emulsions

Microorganism type Genera and/or species

Fungi and moulds Geotrichum candidum

Aspergillus niger
Aspergillus terreus
Penicillium ochrochloron
Fusarium solani
Trichoderma viride
Cladosporum herbarum
Alternaria alternata
Yeasts Saccharomyces cerevisiae
Candida albicans
Rhodotorula rubra
Pichia spp.
Torulopsis spp.
Bacteria Pseudomonas aeruginosa
Pseudomonas cepaciae
Pseudomonas fluorescens
Pseudomonas paucilomobilis
Pseudomonas putida
Pseudomonas stutzeri
Aeromonas hydrophila
Escherichia coli
Alcaligenes faecalis
Arthrobacter spp.
Achromobacter spp.
Citrobacter fruendii
Bacillus subtilis
Ochrobactrum anthropi
Micrococcus luteus
Xanthomonas maltophilia
Proteus vulgaris
Anaerobic bacteria Delsulphovibrio desulphuricans
 PRESERVATION OF AQUEOUS EMULSIONS 237

homo polymers of vinyl acetate and copolymers of vinyl acetate and

ethylene that are stabilised with polyvinyl alcohol and/or starch or cellulose
colloidal systems, in the pH range 3.5-6.5, were particularly susceptible to
yeast and mould fungi. It is postulated that the colloid system has an
influence on the susceptibility of the product and in the case of the
copolymers of vinyl acetate and ethylene it may be that ethylene has a
growth-enhancing effect. 42 Table 9.5 lists some of the major contaminating
microorganisms that have been encountered, isolated and identified by the
authors from spoilt samples of polymer emulsions as well as formulated
products.

9.4.2.1 Moulds and microfungi. In the authors' experience the principal

organism isolated is Geotrichum candidum. This organism is very
important in the biodeterioration of polymer emulsions and has been
isolated in many cases of spoiled product. The reason for its prevalence is
due to its ability to spread rapidly and disperse throughout the substrate
matrix by the production of arthrospores or fragmenting hyphae. This
arthrospore-forming process of reproduction has often led some micro-
biologists to misclassify Geotrichum as a yeast since the colony charac-
teristics on agar plates make it appear like a powdery yeast-like species.
Geotrichum candidum is a known degrader of polyvinyl alcohol and
cellulose and starch-based colloids and its degradative powers have also
been reported in the polyvinyl acetate system. 43- 51
Other moulds and fungal organisms have also been isolated but these are
more likely to be general spoilage organisms growing on components of the
system. In 1983 Jakubowski et al. 8 found many mould contaminants
comprising most of the well-known biodeteriogenic species such as
Aspergillus, Penicillium, Fusarium, Geotrichum, Scopulariopsis, etc. These
mould species are more often found on the surface of the emulsion in
storage vessels at the air/surface interface. This is seen as a surface layer of
mould growth, or small coloured spots, and arises when non-volatile
preservatives are employed in the emulsion giving no surface protection.
As such condensate from the lid of the containers will drop onto the
emulsion's surface and being unpreserved will thus become rapidly
contaminated from spores in the environment. Moulds and fungi,
however, are unlikely to have a major role in the polymer degradation
itself. However, their control is essential in order to maintain a well-
preserved product of extended shelf life.

9.4.2.2 Yeasts. Various types of yeast are regularly isolated from

polymer emulsions. The yeast types found and isolated are often
fermentative and distributed throughout the emulsion. The common types
of yeast include the genera of Saccharomyces, Rhodotorula and Torula and
these have been noted by the authors as well as being reported by
238 PRESERVATION OF SURFACTANT FORMULATIONS

Jakubowski et al. 8.52 who has also isolated Sporobolomyces on a number of

occasions.

9.4.2.3 Bacteria. Bacteria may be found in specific types of polymer

emulsions often of high pH, e.g. acrylic and styrene acrylic types reported
by Evertsen?l After isolating Gram-negative Pseudomonads from every
contaminated low viscosity paint examined, Winters and Guidetti53 and
Oppermann and Goll 54 concluded that this group of bacteria is involved
in the great majority of instances of loss of viscosity of emulsion/dispersion
paints. This conclusion was supported by Carter55 who stated that the
Gram-negative, non-sporing microorganisms such as those belonging to
the genera Pseudomonas, Proteus, Enterobacter, Escherichia and Flavo-
bacterium are most frequently associated with degradation, whilst Gram-
positive microorganisms are regarded as secondary invaders. However,
Buono et al. 56 studied the cellulolytic activity of several strains of Gram-
negative and Gram-positive bacteria and found the Gram-positive micro-
organisms (Bacillus spp.) to be more active than the Gram-negative
microorganisms. Furthermore, in more recent work (unpublished) the
authors have isolated other less common species as well as those previously
found by other workers on a number of occasions (see Table 9.5).
In the authors' experience the most common spoilage bacteria isolated
from polymer emulsions are non-lactose fermenting Gram-negative rods.
The most predominant genera are the pseudomonads, which are organisms
with very powerful biodeteriogenic capacities. Other genera are also
isolated from time to time in specific emulsion types, including occasionally
Gram-positive organisms.
It is probable that, as strains of both Gram-negative and Gram-positive
bacteria are capable of polymer degradation, the type of microorganism
responsible for the degradation of particular polymers will be dependent
upon the polymer constituents and upon the accessibility of each
microorganism to the polymer.43---45 In either case, it is imperative that the
polymer manufacturer uses a biocide which is active against both Gram-
negative and Gram-positive bacteria.

9.4.2.4 Anaerobic sulphate-reducing bacteria. Anaerobic sulphate-

reducing bacteria (SRBs) have also been found in polymer emulsion
systems by the authors and many other workers including Elsom30 and
Gillatt. 57 These microorganisms tend to occur in systems that have been
previously contaminated with numerous aerobic species and are generally
regarded as the last organisms to arise in microbial ecological succession.
SRBs tend to occur at the bottom of bulk storage vessels/tanks and cause
blackening of the emulsion and are responsible for foul sulphurous odours
due to the terminal electron acceptor in their respiratory chain being
hydrogen sulphide.
 PRESERVATION OF AQUEOUS EMULSIONS 239

Table 9.6 Sources of microbial contamination

Source Control

Raw materials (including process water)

Environment
Manufacturer can control
Manufacturing equipment
Operators
Packaging materials
In use Manufacturer cannot control

Oppermann and Goll54 also investigated the incidence of anaerobic

microorganisms and in their study of seventy contaminated samples found
a wide variety of species of which Bacteroides, Clostridium, Desulphovibrio
and Bifidobacterium were the most common.

9.4.3 Sources of microbial contamination

As stated previously polymer emulsions when first produced are sterile. It
can therefore be assumed that spoilage microorganisms must gain access
subsequent to the completion of the polymerisation reaction. When a
major outbreak of contamination does occur, initial responses tend to
centre around methods of wiping out the immediate problem. Identification
of the source of contamination is considered as an afterthought, if at all.
The sources of contamination are limited and generally within the
control of the manufacturer (Table 9.6). Realistic action taken to identify
these sources, quantify the potential risk from each source and reduce or
eliminate the degree of contamination at that source can save a great deal
of time and expense later.

9.4.3.1 Aerial contaminants and the environment. This is often over-

looked because microorganisms cannot be seen with the naked eye. It is
accepted that the human race lives in equilibrium with a wide spectrum of
microorganisms in the environment. A dusty atmosphere from building
operations, animal pollution from farms, stables etc., proximity to
orchards, gardens, etc. leading to yeast and mould contamination, are
typical hazards for any factory making aqueous-based products that are
vulnerable to microbiological spoilage.
Polymer emulsion and adhesive manufacturing plants tend to be open to
the atmosphere, in many locations, due to the nature of the chemicals
used. The open filtration and sieving processes are often the stage where
most microbial contamination can occur. For example, DiehllO,ll reported
that air may contain 500-2000 microorganisms per m 3 which may contain
spores of some of the polymer emulsion spoilage species.
240 PRESERVATION OF SURFACTANT FORMULATIONS

9.4.3.2 Water. The process water is unlikely to be a primary source of

microbial contamination, as any microorganisms present tend to be
destroyed in the reactor, however, some workers have reported that
certain spore-forming microorganisms and some yeasts can survive the
polymerisation process and these have been isolated from fresh polymer
emulsions soon after production. It is therefore necessary to ensure that
process water is treated before its use since water may come from a number
of sources including wells, boreholes or even rivers. Chlorination alone
may not be totally effective to treat the water, especially if organic matter
is present, when residual contamination may be left. As suggested by
Duddridge58 even supposedly good quality town tap water will contain
bacteria, for example Pseudomonas species at up to 1000 cells per ml. In
order to improve the process water quality, an ultraviolet treatment system
for the water has been installed in one of the Vinamul polymer emulsion
production plants. This process is checked on a monthly basis to ensure it is
functioning properly to produce sterile water.
Assuming adequate control measures are taken to ensure the supply
water is sterile the most likely source of microorganisms from water would
be from untreated residual wash water if it is left in mixing vessels and
hoses, etc. which could lead to contamination.

9.4.3.3 Post-added raw materials. Any new materials added at the end
of the manufacturing process can be contaminated with microorganisms,
e.g. addition of thickening agents such as polyvinyl alcohol. Nichols 12
suggested that raw materials were probably the single biggest potential
contamination source. Malcolm6 arbitrarily classed raw materials into
three groups; non-aqueous synthetics, aqueous synthetics and natural.
Some of the powdered raw materials, especially those originating from
natural sources, such as extenders/fillers can often be contaminated with
dormant spores of bacteria and fungi which can germinate once an aqueous
environment is provided.
Liquid raw materials such as defoamers, surfactants, starch solutions
(Liquid Natural Polymer, LNP) and hydroxyethyl cellulose solutions may
themselves be susceptible to microbial attack and, unless carefully
manufactured and protected with biocides, can also introduce contamina-
tion into the product.

9.4.3.4 Manufacturingprocedures. Increased attention to quality control,

and the introduction of ISO 9000 quality systems, has led to more detailed
examination of the raw materials used. However, even if raw materials are
free from infection before use, the method of manufacture itself may result
in contamination of the end product. The addition of biocide to a product
as soon as practicably possible after the polymerisation process is therefore
recommended. Ideally, it should be added to the product as soon as it
 PRESERVATION OF AQUEOUS EMULSIONS 241

leaves the reactor on pump out to the blender. Incorporation of the biocide
can be made with the dilution water required to bring the product into its
solids content specification. It must also be noted that if thickening
solutions of colloids are employed post-manufacture these also should be
protected with a biocide.
A number of other areas in the manufacturing procedures are also
important sources of microbial contamination and these should be noted.
These are detailed further below.

9.4.3.5 Pipelines. It is not uncommon that excessively long and compli-

cated runs of transfer pipelines with sharp bends and deadspots are present
in manufacturing plants which contain accumulated product, often diluted
with wash water. Microbiological contamination of the dilute emulsion can
occur rapidly in such pipelines which can then be a source of inoculum for
fresh product that is pumped through the system. Furthermore, flexible
hoses used for transfer of product can, if improperly cleaned and stored,
also become contaminated, especially if diluted product, resulting from
ineffective cleaning, accumulates.

9.4.3.6 Mixing vessels and bulk storage tanks. Dirty vessels and bulk
storage tanks, at either the manufacturer or the customer, may be an
important source of microbial contaminants.
Open hatches for the passage of air during the filling and emptying of
storage tanks will also allow aerial inoculation of the product with
contaminating microorganisms from the environment. This can easily be
resolved by fitting an air filter suitable to permit the passage of air to
prevent a vacuum from building up and filter out particulate dusts and
microorganisms.
Condensation will wash contaminating microorganisms onto the surface
of the bulk phase of the product where, especially if the vessel remains
unstirred for some time, dilution of the surface layer occurs along with
some degree of syneresis of the product and hence dilution of the biocide
allowing profuse microbial growth to occur. When mixing recommences, a
high microbial loading will enter the bulk phase and may 'overwhelm' a
biocide.
Empty vessels and storage tanks should be cleaned down at regular
intervals but the residual wash water, if left in them, is effectively diluted
product and will rapidly become contaminated, even in just a few hours in
a warm environment. If the residual wash water is left in the vessel or tank
it will provide a heavy source of contamination for the next batch
produced. The use of an effective biocide or disinfectant can be employed
to prevent this situation from arising.
In addition to regular cleaning and sterilising of vessels and storage tanks
other measures can be taken to prevent problems in such vessels. These
242 PRESERVATION OF SURFACTANT FORMULATIONS

can include fitting a stirrer to ensure the product is well mixed at the
surface and in the bulk, or if this option is too expensive a simple device
fitted to spray a dilute biocide or disinfectant onto the surface of the
product at regular intervals can be employed. Either of these options will
minimise microbial contamination in vessels and storage vessels.

9.4.3.7 Road tankers. Road tankers are often not cleaned properly
between loads and therefore may be an important source of microbial
contaminants. Strict guidelines should be rigorously adhered to to ensure
tankers are properly cleaned between loads and as with storage tanks the
residual wash water should be treated with a biocide.

9.4.3.8 Product storage containers: drums, mausers and buckets. If a

sterile emulsion is filled into a sterile, impervious container, microbial
growth will not occur.
First the container itself may not be biologically clean. If stored in
unsuitable conditions, microorganisms may be present before the product
is filled into it. Plastic containers will often attract microbial spores and
dusts electrostatically and these can lead to contamination of the product
when it is filled into the container. When the container is filled and the lid
is applied, condensation may irrigate the lid and form a pool of diluted,
underprotected product on the product's surface in which contaminants
can grow. It is therefore important that biocide is used that has broad
spectrum activity against bacteria, yeast and the mould fungi as well as
some degree of headspace protection and is stable in the product for the
duration of the product's life.

9.4.3.8.1 Case study: a polymer emulsion spoilage scenario. A typical

scenario for microbial contamination of polymer emulsions exists due to
the fact that an emulsion will spend most of its time in a storage system,
whether it is a bulk tank, road tanker, drums, etc., and it is here that the
most likely sources of infection will arise. When a storage tank has been in
use for some time with repeated filling and emptying then polymer
inevitably builds up on the walls of the tank. Water condensing on the
polymer film will wash out any preservative leaving it open to microbial
contamination, which enters with air that is drawn into the tank on
emptying. In the absence of preservative the microorganisms may
multiply. The rate of formation of the polymer film and its subsequent
infection is influenced by the type of emulsion, the type of storage system,
the nature of the tank lining and the ambient temperature. It is, however, a
fairly slow process taking months rather than weeks to occur. Once an
infection becomes established in the polymer film then microorganisms can
potentially be washed continually onto the surface of the contents of the
 PRESERVATION OF AQUEOUS EMULSIONS 243

tank by condensation. In an unstirred tank, this may give rise to a thin

layer of infected water on the surface of the emulsion. On emptying the
tank it may be hosed down, leaving a pool of diluted microorganisms at the
bottom of the tank. As those organisms washed off the polymer film on the
tank wall are those which are capable of growth on the constituents of the
emulsion then rapid growth should occur in this diluted emulsion. On
receipt of the next delivery polymer emulsion is introduced into the tank
with its infected material. Thus, a high level of contamination can occur.
Another means by which microorganisms may be introduced is by way of
a 'wet breather' system which is fitted to some storage tanks to prevent the
contents from skinning over. There are a number of arrangements whereby
air entering the tank is first drawn over wet surfaces. This type of system
will also act as a filter removing dust from the atmosphere. The dust
filtered out may provide nutrients and the wet breather may become
heavily fouled with microorganisms. Air that is drawn through a fouled wet
breather may contain large numbers of droplets containing significant
numbers of microorganisms.

9.5 Prevention and control of microbial spoilage in polymer emulsions and

adhesives

In the previous sections of this chapter the emphasis was on the

susceptibility of synthetic polymer emulsions and formulated adhesives,
the ease at which they can be spoiled by microorganisms and some of the
effects of contamination both in the emulsion and the formulated products.
Therefore to prevent microbial contamination of aqueous-based products
and so retain their beneficial properties, it is essential that biodeterioration
is prevented. This is effected in two ways: by controlling the number of
microorganisms which may come into contact with the product and by the
use of an adequate level of a suitable preservative. It is also very important
to ensure that the preservative is stable in the product for the time frame
over which protection is required. These criteria equally apply to the end
user of the emulsion system, e.g. the paint and adhesive manufacturer for
without their cooperation the useful life of the emulsion will be greatly
reduced.
Preservatives are designed to protect clean products from occasional
microbial contamination. At typical use levels, preservatives are not able
to withstand continual repeated challenges of high levels of microorganisms.
In such situations, the preservative can be consumed and the product will
no longer be protected from subsequent microbial contamination.
Unlike formaldehyde, most other preservatives have very low vapour
pressures which result in little or no preservative present in the storage
tank headspaces. As a result, microorganisms introduced into the
244 PRESERVATION OF SURFACTANT FORMULATIONS

headspace of a storage tank can often proliferate in the condensation on

the walls and ceiling of the tank and on the agitator shaft. When new
material is added to the tank, all of the preservative can be consumed in
eradicating these microorganisms.
In addition, the greater the range of microorganisms exposed to a
preservative system, the greater the likelihood of encountering micro-
organisms able to survive in the presence of the preservative. Thus,
continued poor storage and handling conditions can lead to development
of a microbial population which is not controlled by the preservative
system, particularly when an inadequate level of preservative is being used.
In situations where large microbial populations are exposed to inadequate
levels of preservative, some microorganisms may respond to the preserv-
ative and alter their susceptibility. Development of a resistant, or more
precisely tolerant, microbial population is a common problem for some
preservatives such as formaldehyde. However, due to the complex
chemical nature of preservative systems true resistance is an uncommon
event.
Microbial contamination in storage tanks can be greatly reduced or
eliminated if the following procedures are implemented in conjunction
with the use of adequate levels of a suitable preservative.
1. Supply a microbe-free atmosphere to the tank headspace. After
thoroughly cleaning and sanitising the storage tanks, options such as the
following can be initiated:
(a) Use nitrogen or air purified with ultraviolet radiation or submicron
filters, or use clean compressed air.
(b) Keep a small positive pressure (a couple of inches of water) of the
clean atmosphere in the tank, or keep a low continuous flow
through the tank. The advantage of a low continuous flow is a
significant reduction in condensation. If using a low continuous
flow, ensure that the flow sweeps the entire headspace and that no
other air enters the tank.
2. Monitor the microbial quality of raw materials and products, particularly
water.
The microbiological quality of water supplies varies considerably.
Bore holes and wells usually contain relatively high levels of a range of
organisms including Pseudomonas spp. Thus it is important to maintain
water quality by the adequate use of chlorine, or other biocides or even
by physical agents such as ultraviolet sterilisation systems and more
recently by the use of ozone. Deionised water which is stored, generally
needs to be chlorinated to prevent microbial growth. Maintaining a
residual chlorine level of 2 ppm in water storage tanks is an excellent
means of controlling microbial growth.
Other heavily contaminated raw materials should also be treated
 PRESERVATION OF AQUEOUS EMULSIONS 245

prior to introducing them into the manufacturing environment, especi-

ally those materials that are aqueous-based products, e.g. surfactants,
defoamers, emulsifiers, thickeners and colloids etc.
Typical microbiological tests for quality control can range from agar-
plate techniques using specific microbiological media or dipslides to
rapid methods such as impedance measurements. The newer rapid
methods allow for screening of products in less than 24 h. Systems such
as the Bactometer have been used for a number of years in the food 59
industry and are increasingly employed in the detergents,60 personal
products, cosmetics60 and toiletries 61 and pharmaceutical industries. 62 ,63
Vinamul currently use such a procedure and results have shown that
impedance methodology is equally applicable to the emulsion and
related industries, e.g. paints, adhesives, etc. 52 ,64,65
3. Ensure that microorganisms are not introduced into the storage tanks
when new shipments, batches, etc., are pumped into the tanks:
(a) Flush and sanitise all the lines and hoses frequently and do not
allow product to stagnate in pipes.
(b) When possible, eliminate bends in piping and hoses, valves, lips,
ridges and lines that do not drain «5% slope). These all provide
excellent environments for bacteria to gain a foothold and establish
biofilms. Biofilms are protective layers produced by bacteria and
are difficult to remove.
(c) Ensure that ambient air is not inadvertently pumped into storage
tanks when new additions are made to tanks.
(d) Ensure microorganisms are not introduced into storage tanks by
level indicator air sources. If level indicators bubble ambient air
through the tank, install a submicron filter in the air line or switch
to purified compressed air.

9.5.1 Manufacturing plant hygiene, cleaning and sanitisation methods

In order to prevent contamination problems, a suitable sanitisation
programme is extremely important. 66-68 All good emulsion producers and
end users will have such a regime in place. Typically, this would involve a
cleaning and sanitisation programme to clean regularly the storage tanks,
pipework etc. with high pressure water jetting apparatus to remove
polymer residues on surfaces combined with caustic washing and possibly
sterilisation with dilute sodium hypochlorite (e.g. 1000 ppm available
chlorine). Such a regime carried out three to four times a year at the
emulsion producer and the end-users' plants will be adequate to reduce the
risk of microbial contamination. Additionally, this sanitisation programme
should be carried out in the event of an infection prior to refilling with
fresh product. To prevent skinning at the emulsions surface as well as
surface contamination at the air interface the most appropriate design of
246 PRESERVATION OF SURFACTANT FORMULATIONS

bulk tank is one with a stirrer. This greatly improves storage times and
quality of the product.
The following procedure is commonly used for cleaning and sanitisation
of storage tanks.

9.5.1.1. Cleaning. Drain the lines and flush well with water. If possible,
combine with mechanical cleaning, such as water pressure surges, or
putting a 'pig' through the line, to aid in the removal of any biofilm that
may have formed.

9.5.1.2 Sanitisation.

9.5.1.2.1 Halogenation. Fill the tank with 0.02-0.20% sodium or

calcium hypochlorite, or similar bromine-release compounds which pose
less of a threat than the organochlorine effluents after hypochlorite
treatment. Allow the solution to stand in the tank for 24 h. Flush all lines
well with 0.02% of the sanitising solution. Rinse the tank and lines well with
microbially clean water.
Alternatively, tanks can be thoroughly swabbed with a 5% hypochlorite
solution, allowed to stand for 24 h, then rinsed well with microbially clean
water. This method reduces the volume of waste water.

9.5.1.2.2 Steam. Stainless steel tanks and lines can be steam cleaned.
When using steam, ensure all parts of the system being cleaned reach 75-
80a C for 15-30 min. One way to monitor the steam sanitisation process is
to place temperature stickers on equipment throughout the system.
Stickers that do not change colour indicate areas which are not sufficiently
sanitised.

9.5.2 Preservation of polymer emulsions and adhesives with antimicrobial

agents
It is of course also important that an emulsion and adhesive is adequately
preserved and to this end a preservative is added during the manufacturing
process. 69 ,70 The type and level of the preservative system used may vary
with the type of emulsion, because as previously stated due to differences in
available nutrients, pH and chemistry of the emulsion matrix some are
more difficult to preserve than others. Generally, the emulsion manu-
facturers aim to protect and preserve their products against microbial
spoilage under reasonably good storage conditions for up to 12 months. It
is not the aim to add sufficient preservative to provide protection for any
finished product arising from it. New impending European legislation, the
Biocidal Products Directive due in 1997,71 will ensure that the amount of
 PRESERVATION OF AQUEOUS EMULSIONS 247

preservative used is adequate for protection of the product it is contained

within without being in excessive quantities. This will be determined by
appropriate efficacy tests for the preservative in that product. If a product
was to be supplied with a higher level of a preservative, it would be
regarded by the Directive as a biocidal product itself. This would mean it
would have to be listed with toxicity data, environmental fate and
biodegradability data as well as efficacy data in the annexe of the
Directive. 71
In order to choose the right preservative from a wide range of biocides
available that are based on many different active ingredient chemicals and
stabilisation systems a wide variety of factors must be considered. Some of
these factors are listed here.
• Broad spectrum antimicrobial activity
• Stable over a wide pH range
• Stable at elevated temperatures (to 50°C)
• Stable to various chemical agents
• Aqueous-based solutions or dispersions
• Compatible with a wide range of polymer types
• Low environmental impact
• Low toxicity, e.g. skin irritancy
• Ease of handling and safe to factory workers
• Relevant regulatory approvals, e.g. FDA, BGA, EPA, EEC
• Cost effective
Though cost effectiveness is a factor, less emphasis is placed on this
attribute now than 5-10 years ago. Toxicity has taken on an increasing
importance in recent years as have the environmental implications of the
waste disposal of the emulsion. Related to this is the issue of safety of
handling and the use of diluted solutions is preferred. Also dilute solutions
are easier to pump and meter into products. Solid biocides pose handling
difficulties. A dispersion of a solid biocide is easier to handle if a solution
cannot be prepared. 72- 74 In the case of compatibility with manufacturing
processes some preservatives appear to give good results in the laboratory
but when used on the plant scale have failed to perform. Careful
consideration of compatibility of preservatives with the emulsion in
question must be given and appropriate tests carried out to verify it. It is
also necessary to look for colouration effects, viscosity changes, gelling,
grit formation, etc. Some biocides fail in the plant due to the presence of
excess chemical species that are antagonistic to most preservatives, e.g.
redox chemicals. The stability of the preservative to these sorts of chemical
must therefore be understood. 24 Lastly, suitable efficacy tests must be
carried out in order to ensure that the biocide is adequately biostatic or
biocidal for its intended end use.
248 PRESERVATION OF SURFACTANT FORMULATIONS

9.5.3 Chemistry of active ingredients as antimicrobial agents

Some of the different active ingredient chemicals that have been employed
as preservatives over the years are summarised here. 74
• Heavy metals, e.g. Hg, As, Sn
• Phenolic compounds, e.g. halogenated cresols
• Aldehydes, e.g. formaldehyde, glutaraldehyde
• Oxoazolines, e.g. Amical
• 1,2-Dibromo-2,4-cyanobutanes, e.g. Tektamer
• Thiocyanates, e.g. Tolcide
• Triazines, e.g. Dazomet
• N,S-heterocycles: various substituted isothiazolinones and benziso-
thiazolinones, e.g. Kathon, Proxel and Promexal
One of the major groups of biocides used for aqueous-based product
preservation are the substituted isothiazolinones. These classes of anti-
microbial chemicals have been known for more than 15 years. Both the
1,2-benzisothiazolin-3-one ('Proxel' types) and the mixtures of the two
substituted isothiazolinones, 5-chloro-2-methyl-4-isothiazolin-3-one and 2-
methyl-4-isothiazolin-3-one ('Kathon' and 'Acticide' types) have been
employed for the longest as aqueous-based product preservatives. Both
have advantages and disadvantages, depending upon the system, and these
have to be determined when evaluating and selecting the right biocide for
the system. Two other substitutions are the 2-n-octyl-isothiazolin-3-one
and the 4,5-dichloro-2-n-octyl-isothiazolin-3-one, which have primarily
been employed as dry-film preservatives in surface coatings.
By their chemical nature, biocides are toxic to the microbial cell by
targeting the microbial cell wall cytoplasmic membrane, cytoplasm and
various organelles or combinations of these cellular constituents. The
effect of biocides on several cell functions and their relative importance
may vary depending on the value of that function to the species of
microorganism challenged. Altered sensitivity towards biocides may reflect
change in accessibility of targets within cells and the way it affects that
target. These changes in sensitivity may be brought about by changes in the
physiology of the organism or the environment (such as the formulation or
process).

9.5.4 Physical and chemical factors influencing the choice of preservative

strategies
The choice of preservative must be taken knowing the nature of the raw
materials in the polymer emulsion and adhesive formulation and the
chemical environment they create. Some of the factors which must be
considered are:
 PRESERVATION OF AQUEOUS EMULSIONS 249

9.5.4.1 Chemical constituents. The choice and level of preservative

required is partly determined by the raw materials in a formulation since
they may be contaminated when received or particularly prone to spoilage.
A guideline to susceptibility can be written to predict the extent of
protection required:
- Most susceptible. Fillers such as china clay and calcium carbonate
(strong differences can be seen due to the origins of
the filler); natural materials such as starch;
dextrines.
Susceptible. Cellulose derivatives; polyvinyl alcohol solutions.
Less susceptible. Emulsion bases, these are generally attacked
through their stabilising system rather than inherent
attack on the polymer. With these we can produce
an approximate susceptibility series starting with
the most susceptible.
• PvOH-stabilised PYA
• PvOH-stabilised VAlE
• Cellulosic-stabilised VAlE
• Rubber emulsions (SBR, natural and poly-
urethane)
• Surfactant-stabilised V AlE or acrylic
• Styrene acrylate
• Acrylamide, N-methyl acrylamide, N-butyl
methyl acrylamide
- Least susceptible. Concentrated or solid surfactants; defoamers.
With emulsion bases, the residual monomer levels are important since
these free monomers can themselves have an antimicrobial effect. The
current trend of using low VOC (volatile organic component) emulsions
for adhesive manufacture has to be run hand-in-hand with preservative
testing. This is particularly important in Scandinavian countries where the
move to reduce VOC levels is being accompanied by moves to restrict
allowed preservative levels. In some cases, the techniques used in emulsion
polymerisation to reduce the VOC level can destroy the preservative. 75 ,76

9.5.4.2 pH. Preservatives are active over specific pH ranges and may
prove completely ineffectual or decompose outside of these. Generally
polymer emulsions and adhesives are prone to yeast and mould growth at
pH of 3-7 and to bacteria at pH 6--8. Over pH 9 and under pH 2, adhesives
are less prone to attack. Some of the acid-catalysed cross-linking adhesives
used at very low pH as woodworking adhesives may not require
preservative addition at all. Standard V AlE and PV A emulsions are
generally in the pH range 4--6 so a wide range of preservatives can be used.
For alkali-converted starches and casein adhesives where the pH is over 8.5
250 PRESERVATION OF SURFACTANT FORMULATIONS

the chlorinated isothiazolinones (e.g. 'Kathon') would be ineffective and

the benzisothiazolinones (e.g. 'Proxel') or the dibromocyanobutanes (e.g.
'Tektamer'), or the cationic biguanide hydrochlorides (e.g. 'Vantocil') are
used instead.
Care with pH levels must also be taken when adding pre-dissolved
preservatives. Sodium benzoate and the sodium salts of p-(4)-hydroxy-
benzoic esters all give alkaline pre-mixes which may cause coagulation if
added directly to acidic emulsions. Drift of pH with time should also be
monitored for these.

9.5.4.3 Solids. Dilution may also play an important role in preservation.

Very low solids products have shown a particular sensitivity to attack and
this can be exacerbated if regulatory restrictions fix allowable levels based
on the dry component of the adhesive and not the wet state.

9.5.4.4 Climate. Polymer emulsions and adhesives destined for export

to warmer countries can be particularly susceptible to growth due to
condensation of dilute films onto the product's surface. These low solids
films are readily attacked by bacteria.

9.5.4.5 Compatibility. Compatibility problems can be observed in

addition to simply acid/base interactions and should be verified before
finalising an adhesive formulation. This can be seen with polyurethane
emulsions where addition of some biocides can cause coagulation or with
caseins where formaldehyde or formaldehyde-release preservatives can
cause gelatinisation.

9.5.4.6 Processing. Processes involving 'cook-ups' require the preserv-

ative to be heat stable or to be post-added after cooling. Formaldehyde-
release preservatives are generally unstable over 50°C.

9.5.4.7 Redox state. Excess oxidising agents occasionally used in adhes-

ives, e.g. hypochlorite, can have deleterious effects on certain preservatives.
The same is true for reducing agents such as sodium metabisulphite which
is used to maintain a light colour in starch-based adhesives as well as being
used as initiators and end-stripping chemicals in polymer emulsions.

9.5.4.8 Nucleophiles. Heterocyclic preservatives can be decomposed by

nucleophilic attack rendering them inactive. Primary, secondary and even
tertiary amines can deactivate some isothiazolinone preservatives (see
chapter 2). Sulphur nucleophiles like mercaptans show the same effect.

9.5.4.9 Physical form. For aqueous-based products, preservatives in

liquid form are easier to incorporate than powders. Liquids can also be
 PRESERVATION OF AQUEOUS EMULSIONS 251

dispensed using automatic metering systems to reduce the amount of

operator exposure to the preservatives during manufacture.

9.5.4.10 Solubility. Given the importance of preservation during pro-

cessing and storage for polymer emulsions and adhesives, the preservatives
used should be soluble in the aqueous phase.

9.5.4.11 Versatility. A broad spectrum of antimicrobial activity is

required.

9.5.4.12 Cost. In some cases the preservative can contribute a relatively

high proportion of the raw material cost of a formulation. The high cost of
some preservatives can be offset by their potency which may allow them to
be used in low doses.

9.5.5 Test methodology for the selection of biocides and analytical

methods for their quantification
In order to make recommendations for the use of a chosen or selected
biocide for the preservation of polymer emulsions and formulated products
the first criterion that must be satisfied is that of its microbiological
efficacy, i.e. how effective and good it is at giving prolonged protection to
the product formulation in terms of preventing microbial infection. In
order to test for the microbiological efficacy of biocides in product
formulations two specific testing procedures should be employed.

9.5.5.1 Microbiological challenge testing for biocide efficacy. The micro-

biological effectiveness of a biocide is tested by repeated sequential
challenges with a specific acclimatised mixed microbial inoculum. 51 •64 ,65,77
At present there are no standard methods in existence for determining
the biocidal activity of products intended for the preservation of polymer
emulsions and adhesives. However, there are a number of international
standards such as ISO, CEN, ASTM, BSI, DIN, AFNor, as well as many
industry associations and groups. However, it is more often the case at
present that in-house methods are preferred. These in-house methods can
be adapted to manufacturer's own products and use spoilage micro-
organisms that are highly specific to the industry in question. There is of
course a disadvantage with such testing in that it is not very reproducible
and as such comparisons are often not easy if at all possible.
The International Biodeterioration Research Group (IBRG) was origin-
ally established under the auspices of the Organisation for Economic
Cooperation and Development (OECD). It is an international group of
scientists from industry, academic institutes, national testing institutes and
organisations and government and privately funded test laboratories. One
252 PRESERVATION OF SURFACTANT FORMULATIONS

aim of the IBRG is to develop standard test methods to determine the

antimicrobial activity of microbicides in a range of products that can be
acceptable to the EU for the Biocidal Products Directive requirements on
efficacy testing of active substances. 71 One IBRG project is currently to
develop a standardised method for testing biocides for use in polymer
emulsions78 that can equally be applied to adhesives and related formula-
tions with some minor adaptations.
The group is currently developing a standardised method for biocides for
polymer emulsions that will give statistically valid results when comparing
different biocides in different polymer formulations. Areas of interest
include the microorganisms in the challenge inoculum, how to adapt them
to growth in the product formulation, inoculum density and whether a
specific diluent should be employed, pH of the substrate and whether
ageing of the polymer is important. Some workers go further by
questioning whether it would be possible to develop a chemically defined
medium that is representative of a typical polymer matrix (a very complex
medium) which could be used as a model system. In this way, biocide
efficacy for such products can be evaluated under standard laboratory
parameters thus generating results that are statistically viable without the
need to work in the complex matrix itself. The ultimate aim of the IBRG is
to have a suitable test method available more quickly, in time for the EU
Directive, which both biocide producers and users alike can use to test
active ingredients and thus generate meaningful comparisons of relative
effectiveness and efficacy.
Once a satisfactory biocide has been found which gives the best
microbiological protection in the product formulation, the next stage in the
assessment of the antimicrobial compound is to ensure its stability in the
specific polymer emulsion and/or adhesive system.

9.5.5.2 Biocide active ingredient compatibility with the formulation.

Some relatively simple tests can be performed in order to check the
biocide's chemical compatibility in the polymer or adhesive matrix to
ensure there are no undesirable side reactions such as colour changes,
odours, gelation, precipitation and sedimentation. These tests essentially
involve dosing the biocide into the product at various levels, below and
above the recommended dose, and then storing the samples for prolonged
periods of time at low and elevated temperatures and observing any side
effects. Other factors such as pH compatibility, viscosity loss and chemical
interactions are also evaluated.

9.5.5.3 Biocide active ingredient chemical stability in the product formula-

tion. Testing the chemical stability of a biocide in a polymer matrix or
adhesive formulation can be achieved by the use of quantitative analytical
methods to monitor the level of active ingredient. This will demonstrate if
 PRESERVATION OF AQUEOUS EMULSIONS 253

the preservative is stable over the product's lifetime, as well as discovering

if the formulation is antagonistic to the biocide. 79 This form of procedure is
necessary and critical and must be adopted to assess the potential of a
biocide for preservation of polymer systems where it is known that there
may be antagonistic components to the active ingredients present, e.g.
redox chemical-containing systems.
Thus, it is important that analytical procedures for the active ingredient
of the biocide are available. This will also be a requirement for the EU
Biocidal Products Directive positive list of active substances. Furthermore,
methods are increasingly required in order to carry out quality control
(QC) checks such as:
• QC checking of dosing accuracy at recommended set level,
• QC checking of biocide stability in the polymer emulsion or adhesive
batch in question.
• QC checking of biocide active ingredient content with suppliers'
certificate of analysis,
• Marketing purposes where biocide type and level need to be stated on
safety data sheets where there are regulatory limits for their inclusion.

9.5.6 Regulatory issues governing the choice and selection of antimicrobial

agents and biocides
The amount and level of preservative which can be used in a synthetic
polymer emulsion or an adhesive formulation can be subjected to
regulatory control. Various legislation exists and is applied according to
requirements laid down by the specific nature of the application and the
country where this is to be used. The form of the law is often a positive list
of acceptable substances. In addition, for some applications many adhesive
customers have their own guidelines on acceptable preservatives and these
may be more stringent than local regulations.
We can consider some of the legislation relevant to emulsion polymers
and adhesives.

9.5.6.1 American Food and Drug Administration (FDA) legislation. This

governs all the ingredients which are permitted in adhesives for certain end
uses and although designed for the USA it is also widely used and
recognised in Europe and elsewhere. Three levels are important for
adhesive manufacture with different degrees of severity or applicability.
GRAS status (generally recognised as safe) is given to ingredients,
including preservatives which can be used as food additives. These
preservatives are used for example in the preservation of fruit juices and
they include some of the less potent preservatives such as sodium benzoate
and potassium sorbate. These can also be used in adhesives but show low
inhibitory powers in many cases.
254 PRESERVATION OF SURFACTANT FORMULATIONS

The two main areas which concern adhesive manufacturers in the FDA
are the direct and the indirect food contact regulations.
If there is a possibility of contact between the dry adhesive film and the
food then Title 21 Part 176 must be consulted since Title 21 Part 176
(Indirect Food Additives: Paper and Paperboard Components) contains
two sections relevant to coatings which can be extended to adhesives.
These sections differ according to the type of food which may be in contact
with the coated board. Section 176.170 names components allowed for
contact with aqueous and fatty food and Section 176.180 names those
allowed for contact with dry food. Materials with GRAS status automati-
cally conform.
Title 21 Part 175 Section 105 (Indirect Food Additives: Adhesives and
Components of Coatings) names the ingredients which are allowed for
adhesives where the adhesive is separated from the food by a functional
barrier. This regulation is relevant to packaging adhesives for food, for
example a 'cornflakes' packet or box as long as the dry adhesive film can
not touch the food, i.e. the 'cornflakes'. Again, materials with GRAS
status are automatically included here.

9.5.6.2 German BGA legislation. In Germany, substances which are

used in food are subject to restrictions decreed in the BGA legislation.
This is published by Carl Heymanns Verlag KG under the title 'Kunstoffe
im Lebensmittelverkehr' and contains various chapters detailing allowed
components for various materials. Restrictions on the ingredients allowed
for use in adhesives are governed by chapter XIV. As well as polymers,
surfactants and other raw materials, the acceptable preservatives are listed.
Some subparts of this legislation restrict the use of preservatives even
further. In the tobacco industry, the DTV regulations (Verordnung iiber
Tabakerzeugnisse Tabakverordnung) retain essentially only food grade
preservatives as acceptable for use in adhesives for tipping and seaming
applications. These restrictions are echoed in the tobacco legislation of
other European countries.
Generally, in Europe, conformance to the BGA or the indirect FDA
statements are sufficient for adhesives for many applications and these are
often used as substitute guidelines where there is no directly relevant
legislation, as in the tissue and towel market where, although food contact
is not involved, adhesives are often required to conform to the same
standards.
Though not always governed directly by legislation, the choice of
preservatives for a system is equally guided by a desire for systems showing
low impact on the environment and also a low toxicity or tendency to cause
irritation or sensitisation to both workers handling the preservatives during
adhesive manufacture and during end-use processing of the adhesive.
 PRESERVATION OF AQUEOUS EMULSIONS 255

9.5.6.3 EEC biocidal products legislation. During the time when the EC
Council for European Member States prepared the Directive 911414IEEC
concerning the placing on the market of plant protection products,?1 it was
felt that another Directive should be drafted dealing specifically with other
dangerous substances and preparations in the field of non-agricultural
pesticides. After taking advice from experts the EC decided that such
products should be grouped together under the heading of biologically
active products for use in non-agricultural applications. It was then found
that the nature and the applications of these products made them very
different from pesticides, and so the term 'biocides' was selected. These
biocidal products can be used both on microscopic organisms, such as
viruses, bacteria, moulds and yeasts, and on larger organisms, such as
insects, small rodents (rats, mice, etc.), molluscs, algae and even some
birds. The Directive thus became known as 'The Council Directive
concerning the placing of biocidal products on the market'. 71
The organisms detailed above can harm human and animal health,
damage natural or manufactured products and attack materials, such as
plastics, masonry, wood and paint. They can also cause pollution in
sanitary and air-conditioning systems, and can weaken, if not undermine
dykes.
A summary of the types of biocidal products in this current EU Directive
legislative process includes the following wide range of biologically active
substances: rodenticides, molluscicides, insecticides, acaricides, fumigants,
disinfectants for swimming pools and food applications, antimicrobial
active ingredients such as the general biocides, sanitary biocides, air-
conditioning biocides; wood preservatives; textile preservatives; artwork
preservatives; masonry preservatives; consumer-product preservative; and
other specialist biocides; and antifouling paints and other products where a
biological functionality is claimed.
In essence this Directive will have many implications in the adhesive and
polymer emulsions and related industries in that it will govern and regulate
what can be used, at what level and how it is employed. The Directive will
authorise the actual placing of active substances on the market that have
proven technical efficacy in specific applications at the prescribed dosage
without being harmful to human and animal health or having other harmful
effects, directly or indirectly (e.g. through drinking water, food or feed).
The Directive will also require the substances that are authorised to have
substantial toxicological and environmental fate dossiers.
Once a product has been given authorisation it will be filed with the
following information: the applicant (name and address), chemical
identity, physical and chemical properties, methods of detection and
identification, effectiveness against target organisms, toxicological profile
for man and animals including metabolism, ecotoxicological profile
including environmental fate and behaviour, measures necessary to protect
256 PRESERVATION OF SURFACTANT FORMULATIONS

man, animals and the environment and classification and labelling. Each of
these categories is subdivided into many other classifications to substantiate
the filing of appropriate data, but will not be discussed further in this
chapter.
In conclusion, this Directive will limit and control the use of antimicrobial
preservatives which can be used in both adhesives and synthetic polymer
emulsions and related products to the benefit of human and animal health
and as such will assist in the choice of the right product for a specific
application. With standard test methods also available for efficacy testing
the best preservative systems for adhesives and polymers should be able to
be selected more easily from a list of acceptable active substances. No
known harmful and toxic ingredients will be included in the Directives'
annexes, for example mercury-based materials.

9.6 Preservation strategies and related issuesaffecting polymer emulsions

and adhesives for the next millennium

The following points summarise the requirements or attributes for biocides

that will be employed for the preservation of polymer emulsions and
adhesives in the future. With increasing public awareness of environmental
issues and waste disposal, toxicological issues, regulatory approval
requirements and the advent of the Biocidal Products Directive biocide
manufacturers will have to become more environmentally aware and
supply of biocides will be very much market driven.
The following issues are some of those that will increasingly need to
be addressed by reputable biocide manufacturers:
1. Lower environmental impact: no VOCs, no solvent, no halogen,
2. Lower toxicological effects: Daphnia magna test for 'EcoLabel'.
3. Low skin sensitisation: local lymph node assay,
4. Biodegradable below use concentrations: waste effluent treatment,
5. Ease of handling and safe to factory workers,
6. Chemically stable and compatible with polymer system,
7. Regulatory approvals: EPA, EU, FDA, BGA,
8. Use in low cost-effective doses.

9.6.1 Safety, health and environmental issues

9.6.1.1 Ease and safety of application. The biocide user, as part of his
own manufacturing process, must incorporate the biocide into the product
to be protected. The ease with which the biocide can be added and
incorporated into that product is an important factor. 72,73
Liquid formulations, either solutions or dispersions with low viscosity,
are the easiest to handle as they can be stored in holding tanks and then
 PRESERVATION OF AQUEOUS EMULSIONS 257

automatically pumped and metered into the final product. Mixing, to

ensure uniform distribution of the preservative throughout the whole batch
of the product, is also easier with a liquid. Solids, on the other hand, are
more difficult to use. Free-flowing powders and granules are relatively easy
to add, but still require manual rather than pumped addition. Precise
dosing is also difficult, requiring either the weighing of a solid, or the
addition of a whole number of sacks or drums of preservative. Addition of
wet or sticky solids is labour intensive and messy.
Safety in use is an important consideration for any biocide user. Because
biocides are toxic to the microbial cell, there is potential to be toxic to
other cells, including mammalian cells. There is therefore a fine balance
between efficacious concentrations of the active ingredient of the biocides
in products and processes, and safety of those concentrations to humans
and the environment. 80 In the concentrated forms in which biocides are
available on the market, the toxic potential of the product is extenuated
and eye and skin irritation and sensitisation do occur. 80

9.6.1.2 Toxicological and environmental features. The toxicological and

environmental features of biocides are becoming increasingly important.
This encompasses not only the active ingredients, but also the additives
used in formulation, and containers and packaging used in transport.

9.7 Concluding remarks

In conclusion to this chapter, the following questions can be posed, which

can only be answered with time and continued testing of new molecules
and novel antimicrobial systems in many different formulations.
1. What will be the active ingredients for future antimicrobial agents and
biocide systems?
2. Do these active ingredients meet the needs of the industry?
3. Are these active ingredients cost effective?
4. Are these active ingredients safe from an environmental and
toxicological point of view?
5. What does the future hold for the biocide industry and preservation of
aqueous- and surfactant-based formulated products and are there any
other developments yet to be discovered?
It must be recognised that the biocide is the only raw material added to
polymer emulsions and adhesive formulations with the specific intent to
kill living organisms and therefore it will have some degree of toxicity. The
future biocide is likely to exhibit a fine balance between the high toxicity to
micoorganisms with low toxicity toward higher organisms.
Given the required properties for a biocidal preservative development
258 PRESERVATION OF SURFACTANT FORMULATIONS

we are unlikely to see the emergence of a truly universal system. Modern

trends have shown that the way forward is in the production and
formulation of a number of active agents that together may fulfil the
requirements of the ideal biocide. It is possible that the ideal biocide does
exist. Perhaps it is just waiting to be discovered!

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27. Vinamul Microbiology Test Method, VMTM/07, Analysis of Preservative Active
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28. Rosenbaum, J.L. (ed.) (1945) Industrial Cold Adhesives, C. Griffin, London.
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31. Evertsen, P. (1988) The biodeterioration of polymer emulsions/dispersions. Paper
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33. Cheesman, G.C.N. and Conquer, L. (1979) Some aspects of microbiology of synthetic
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34. Nelson, Y.M. and Jewell, W.J. (1993) Vinyl chloride biodegradation with methano-
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35. Nieder, M., Sunarko, B. and Meyer, O. (1990) Degradation of vinyl acetate by soil,
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36. Doelle, H.W. (1984) Microbial cultures in the utilisation of cellulosic materials. Biotech.
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37. Enari, T.M. (1983) Microbial cellulases: In Microbial Enzymes and Biotechnology (ed.
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38. Gillat, J.W. (1992) Bacterial and fungal spoilage of water-borne formulations during
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39. Paulus, W. (1992) Microbicides for material protection. Farg ah Lack Scandinavia, 9,
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40. Paulus, W. (ed.) (1992) Microbicides for Material Protection, Chapman and Hall,
London.
41. Miller, G.A. and Lovegrove, T. (1980) 3-(2H)-Isothiazolinone: A new class of
antifouling toxicant. J. Coat. Technol., 52 (661), 69-72.
42. Cresswell, M.A. (unpublished) Vinamul Technical Report on the Ethylene-enhancing
effect on Microbial Colonisation of Polymer Emulsions.
43. Hesketh, A.J., Cresswell, M.A. and Gowland, P.C. (1995) Microbial biodegradation of
polyvinyl acetate (PVA) emulsions. Biodeterioration & Biodegradation, 9 (eds. Bouscher,
A. and Edyvean, R.G.J.), Inst. Chern. Engrs. In press.
44. Hesketh, A.J., Cresswell, M.A., Gowland, P. and Gowland, P.c. (1995) Detection of
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Environ. Polymer Degradation. In press.
45. Hesketh, A.J., Cresswell, M.A., Gowland, P. and Gowland, P.c. (1994) Approaches to
studying the biodegradation in emulsion polymers. Paper presented at Int. Biodet. and
Biodegr. Research Group Meeting in Braunschweig, Germany. Paper number IBRGI
P94/13. The International Biodeterioration and Biodegradation Research Group
Secretariat, Building Research Establishment, Watford, UK.
260 PRESERVATION OF SURFACTANT FORMULATIONS

46. Heyn, c., Petersen, K. and Krumbein, W.E. (1995) Investigations on the microbial
degradation of synthetic polymers used in the conservation and restoration of art objects.
Biodeterioration & Biodegradation, 9 (eds. Bouscher, A. and Edyvean, R.G.I.), Int.
Chern. Engrs. In press.
47. Kay, M.J., McCabe, R.W. and Morton, L.H.G. (1993) Chemical and physical
degradation occurring in polyester polyurethane during biodegradation. Int. Bio-
deterioration & Biodegradation, 31, 209-225.
48. Trejo, G. (1988) Fungal degradation of polyvinyl acetate. Ecotox. & Environ. Safety, 16,
25-35.
49. Griffin, G.J.L. and Mivetchi, H. Biodegradation of ethylene/vinyl acetate copolymers.
Proc. Biodet. Soc., Sept., 8 pages.
50. Pahren, H.R. and Bloodgood, D.E. (1961) Biological oxidation of vinyl compounds. J.
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51. Smith, L.H. and Cresswell, M.A. (1994) Biodegradation of textile nonwoven binders:
Theories and applications for using the Bactometer. Vinamul Internal report, TP1426,
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52. Jakubowski, J.A., Gyuris, J. and Simpson, S.L. (1982) Microbiological spoilage of latex
emulsions: Causes and prevention. J. Coat. Technol., 54 (595), 39-44.
53. Winters, H. and Guidetti, G. (1974) Growth of a typical paint bacterial isolate in aqueous
emulsion paint. J. Coat. Technol., 46 (594), 69-72.
54. Oppermann, R.A. and Goll, M. (1984) Presence and effects of anaerobic bacteria in
water based paints. I. J. Coat. Technol., 56 (712), 51-56.
55. Carter, G. (1982) The preservation of latex emulsions. Proceedings Plastics and Rubber
Institute Conference on Emulsion Polymers, paper 12, 1-9.
56. Buono, F., Stewart, W.J. and Friefeld, M. (1973) Evaluation of latex preservatives. J.
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57. Gillatt, J.W. (1990) The biodeterioration of polymer emulsions and its prevention with
biocides. Internat. Biodeterioration Biodegr., 26, 205-216.
58. Duddridge, J.E. (1987) Problems associated with microbiologically contaminated mains
water. Notes from Preservation Towards 1990 and Beyond, Ladbroke Hotel, Warwick,
organised by Rohm and Haas 11112 June.
59. Holah, J.T., Higgs, C., Robinson, S., Worthington, D. and Spence ley, H. (1990) A
conductance-based surface disinfection test for food hygiene. Letts. Appl. Microbiol., 11,
255-259.
60. Kahn, P. and Firstenberg-Eden, R. (1984) A new cosmetic sterility test. Soaps/Cosmetics/
Chemical Specialities, May, 4 pages.
61. Whittingham, L. (1988) Use of the Bactometer for Cosmetics and Toiletries. Vitek Internal
Paper, 4 pages.
62. Booth, P.E. (1987) Evaluation of the Bactometer for microbial bioburden testing of
challenged fire products. In Bactomatic's User's Seminar. Proc. 2nd Annual Advanced
Impedance Workshop, Las Vegas, Nevada, 15-16 June. Princeton, New Jersey:
Bactomatic Inc.
63. Connolly, P., Bloomfield, S.F. and Denyer, S.P. (1994) The use of impedance for
preservative efficacy testing of pharmaceuticals and cosmetics. J. Appl. Bacteriol., 7, 68-
74.
64. Provan, F. and Cresswell, M.A (1994) Bactometer-Impedance method for the rapid
detection of microbial activity. Development and standardisation of procedures for
polymer emulsion testings. Vinamul Internal report, TP1421, Unpublished.
65. Jacques, P.A. and McLaurin, M.C. (1993) An efficient impedimetric procedure to
demonstrate bacterial contamination in water-based coatings and their raw materials. J.
Coatings Technol., 8, 1-5.
66. Rigarlsford, J. (1987) Plant hygiene and disinfection. Notes from Preservation Towards
1990 and Beyond, Ladbroke Hotel, Warwick, organised by Rohm and Haas 11112 June.
67. Siegert, W. (1994) Production of microbiologically faultless cosmetics. Euro-Cosmetics,
7,45-48.
68. Siegert, W. (1993) Production hygiene in the manufacture of water-based coating
materials. Farbe & Lack, Scandinavia, 9.
 PRESERVATION OF AQUEOUS EMULSIONS 261

69. Wood, W.B. (1982) Prevention of microbial spoilage of latex paint. f. Waterborne Coat.,
111,2.
70. Springle, W.R. (1990) Guide to Preservatives for Water-Based Coatings. Paint Research
Association report.
71. EU (1993) Proposal for a Council Directive concerning the placing of biocidal on the
market. 0.1. of the E. c., C, (239/3).
72. Payne" J. (1992) Formulation of industrial biocides. Spec. Chem., 485-489.
73. Godfrey, D. (1993) Blending for the future. Cosmetics and Toiletries Manufacture
Worldwide, 4/5, 149-151.
74. Gillat, J.W. (1993) The ideal biocide for the protection water-based formulations. Spec.
Chem., 18(6), 336-342.
75. Conquer, L. (1991) Environmental needs shape biocides future. Perf. Chems., 617, 31-
34.
76. Conquer, L. (1993) Volatile organic compounds (VOC) and the biocide industry.
EuroCoat, 9, 592-597.
77. DePasquale, D., Kahn, P., and Firstenberg-Eden, R. (1985) Impedimetric determination
of antimicrobial preservative efficacy testing. An automated challenge test. Presented at
the Annual Meeting of the American Society for Microbiology, Las Vegas, Nevada.
78. Gillatt, J.W. (1994) Development of a method for evaluating biocidal components in
aqueous polymer emulsions - I: Establishment of a recommended mixed microbial
inoculum. Paper presented at International Biodeterioration and Biodegradation Research
Group Meeting in Helsinki, Finland. Paper number IBRGIP94/13. The International
Biodeterioration and Biodegradation Research Group Secretariat, Building Research
Establishment, Watford, UK.
79. Parkin, M. (1992) Evaluating biocides using analytical methods. Spec. Chem., 390-392.
80. DeGroot, A.C. (1991) Choosing preservatives: Dermatoallergenic considerations.
Cosmet. Toiletries, 106 (2), 37-38.

Further reading
Corbett, R. and Wood, P. (1994) Novel biocide design using an inorganic composite. Spec.
Chem., 223-228.
Barman, B.N. and Preston, H.G. (1992) The effect of pH on the degradation of
isothiazolinone biocides. Tribiology Int., 25(4), 281-287.
Krzeminski, S.F., Brackett, C.F. and Fisher, J.D. (1975) Fate of microbial 3-isothiazolinone
compounds in the environment: Modes and rates of dissipation. Agricult. and Food Chern. ,
23(6), 1060-1064.
Krzeminski, S.F., Brackett, C.F. and Fisher, J.D. (1975) Fate of microbial3-isothiazolinone
compounds in the environment. Products of degradation. Agricult. and Food Chem., 23(6),
1064-1074.
Hopton, J.W. and Hill, E.C. (eds) (1987) Industrial Microbiological Testing, Society for
Applied Bacteriology Technical Series No. 23, Blackwell Scientific Publications, London.
Board, R.G., Allwood, M.C. and Banks, J.G. (eds) (1986) Preservatives in the Food,
Pharmaceutical and Environmental Industries, Society for Applied Bacteriology Technical
Series No. 22, Blackwell Scientific Publications, London.
Collins, C.H., Allwood, M.C., Bloomfield, S.F. and Skinner, F.A. (eds) (1981) Disinfectants:
Their Use and Evaluation of Effectiveness, Society for Applied Bacteriology Technical
Series No. 17, Blackwell Scientific Publications, London.
Gould, G.W. and Correy, J.E.L. (eds) (1980) Microbial Survival in Extreme Environments,
Society for Applied Bacteriology Technical Series No. 15, Blackwell Scientific Publica-
tions, London.
Board, R.G. and Lovelock, D.W. (eds) (1975) Some Methods for Microbiological Assay,
Society for Applied Bacteriology Technical Series No.8, Blackwell Scientific Publications,
London.
Board, R.G. and Lovelock, D.W. (eds) (1973) Sampling - Microbiological Monitoring of
Environments, Society for Applied Bacteriology Technical Series No.7, Blackwell
Scientific Publications, London.
10 Preservation of inorganic systems
P.M. PRICHARD and J. MARTIN

10.1 Introduction

Microbiological contamination is prevalent in inorganic mineral processing

where the mineral is either processed or sold as an aqueous suspension.
Severe contamination can seriously affect processing and product quality.
Because processing inorganic minerals generally requires suspension of the
mineral in aqueous media, microbiological growth must be controlled.
Mineral refining requires discrete, individual particles. Refining includes
such aqueous processes as degritting, dispersion, blunging, classification,
flotation, selective flocculation, delamination, screening, chemical modi-
fication and magnetic separation.
Aqueous suspensions of inorganic minerals provide a suitable environ-
ment for the growth of microorganisms. Temperature and pH conditions
during processing/shipping are often physiologically ideal for micro-
biological growth. Aerobic organisms are supported by oxygen that is
introduced through mixing, pumping, flotation, etc. Processing also
requires the addition of any number of chemicals, many of which can serve
as nutrients and, quite often, sources of microbiological contamination.
Areas that are not circulated tend to become anaerobic, supporting the
growth of anaerobic microorganisms.
The manufacturing process utilizes apparatus which are rarely cleaned,
much less sterilized. Therefore, the primary source of microbiological
contamination may be the processing equipment.
The advantages of processing minerals as aqueous suspensions (slurry)
are many. Particle size separation can be achieved with centrifuges,
hydrocyclones and settling tanks. Slurry is passed through electromagnets
for the removal of paramagnetic materials. Chemical processing, such as
reductive bleaching with sodium hydro sulfite , oxidation of organic
substances by ozonation, and removal of impurities by flotation, requires
aqueous media. The removal of residue is not only more efficient and
effective in aqueous media, but much smaller residue fractions can also be
removed. Product uniformity can be easily obtained through mixing of
dispersed, aqueous suspensions. Sampling is much more representative.
Transportation and blending (blending may be needed at the beginning or
end of the process) are greatly facilitated. The environmental issue of dust
is eliminated.
 PRESERVATION OF INORGANIC SYSTEMS 263

There are several advantages of shipping slurry to customers. Unloading

and storage are easier; the effect of moisture on dry discharge is
eliminated. The cost of drying is eliminated. The homogeneous suspension
of dried minerals in water (makedown) is very energy and chemical
intensive. The customer typically does not have a make down facility.
There are also distinct disadvantages in shipping slurry to customers.
There is the cost of transporting water; therefore, it is necessary to achieve
the highest solids possible. This is possible only with the use of costly
dispersants. Mixing (using energy and apparatus) is required. There can be
settling, gelation, corrosion and microbiological growth, all of which can
diminish the quality of the slurry. Dispersants and suspension aids are used
to keep the mineral particles in suspension; all of these greatly enhance
microbiological growth.
It is important to use water of good microbiological quality for
processing. A strong oxidant, such as chlorine, is normally used to treat
water; however, the amount of residual oxidant is usually grossly
inadequate to control the levels of microorganisms that will be encountered
during processing. The control of microbiological growth in aqueous
suspensions of inorganic minerals during processing and in final product is
typically accomplished with the use of biocides.

10.1.1 Kaolin and calcium carbonate

Kaolin is a naturally occurring mineral which contains, and is processed
with, large amounts of water. The economic kaolin deposits in Georgia
were formed by the erosion of feldspar and their subsequent transportation
by water to the coast of Georgia during the Cretaceous period. Because of
their sedimentary formation, the kaolin deposits contain varying amounts
and types of both nutrients and microorganisms. Both kaolin and water are
potential sources of microorganisms.
Calcium carbonate used for ground calcium carbonate (GCC) is in the
form of marble. It is a naturally occurring, metamorphic mineral which is
essentially devoid of nutrients, water and microorganisms. However, it is
also processed with large amounts of water and is therefore subject to
microbiological contamination from water, processing apparatus and
added chemicals.

10.1.2 Titanium dioxide

Ti0 2 is used as a white pigment. It is typically produced from ore that
contains ilmenite (FeTi0 3) and ferric oxide (Fe203). The ore is digested
with an aqueous sulfuric acid solution to produce an aqueous solution of
titanyl sulfate and ferrous sulfate. Iron sulfate is removed. Water is added
to hydrolyse the titanyl sulfate to H 2Ti03, which is insoluble and
264 PRESERVATION OF SURFACTANT FORMULATIONS

precipitates, and H 2S04 , The precipitate is heated to drive off water,

leaving Ti0 2 . The use of water for processing and the introduction of
sulfate makes the control of aerobic and anaerobic organisms necessary.

10.1.3 Processing kaolin and calcium carbonate

10.1.3.1 Kaolin. Kaolin is typically processed by 'blunging' (mixing) the

crude clay with water containing first, dispersants such as sodium
polyacrylate or sodium hexametaphosphate and second pH modifiers
such as ammonium hydroxide, sodium carbonate or sodium hydroxide.
Blunging is followed by degritting, where sand is removed by gravity
sedimentation, classification (particle size selection), electromagnetic
separation, reductive bleaching with sodium hydrosulfite and water/salts
removal by filtration. Blunging, degritting, classification and electro-
magnetic separation are done at neutral pH. Reductive bleaching and
filtration take place at very low pH, inhibiting the growth of bacteria.
Following filtration, the suspension is made pH neutral again with the
addition of sodium hydroxide, ammonium hydroxide or sodium carbonate.
Additional dispersants are also added.
The filter product is pumped to holding tanks, after which solids are
increased by various methods. It may be pumped to spray driers for drying,
or mixing facilities where spray dried clay is added to increase solids
content, or evaporators where water is removed under decreased pressure
and increased temperature. The spray drying process kills the majority of
bacteria; however, spores survive and become active again when the spray
dried material is resuspended in water. The evaporative process also kills
the majority of bacteria, but some survive and must be controlled with
biocides. Nutrients remain plentiful after the spray drying and evaporative
processes.
In addition to these processing steps, some clays are processed using
flotation techniques to remove impurities and improve brightness. The
flotation process uses additional chemicals which can serve as nutrients/
contaminants; also, temperature, pH and oxygen are generally optimal for
microbiological growth. Air is bubbled through the suspension to 'force'
the less dense impurity complexes to the top where they are removed as
froth.
Settling of coarser products during transport is prevented by the addition
of suspension aids such as xanthan gum, alginate and carboxymethyl
cellulose. Unfortunately, these carbon-based complex materials are
excellent nutrients and frequently microbiological contaminants. The use of
nutrient-type suspension aids makes preservation more difficult.
Some kaolin deposits are grey in color because they contain organics;
these deposits do not respond to the usual processing. Oxidation is
required to remove the organics. This is generally accomplished by either
 PRESERVATION OF INORGANIC SYSTEMS 265

ozonation or calcination. Obviously, the use of ozone or calcination for

processing has a positive effect on microbiological control. Microorganisms
are killed and food sources are oxidized. However, ozone is very short
term and microbiological control is absent on further contamination.
Calcination takes place at extremely high temperatures (800-1000°C)
which oxidize all organic material including microorganisms. When the
calcined clay is made into aqueous suspension and transported, a
suspension agent is typically used to prevent settling. The suspension
agents provide sufficient nutrients to support rapid microbiological growth
and some suspension aids may be contaminated with microorganisms.
Some suspension aids, such as Kelzan, are produced using microbiological
processes. Once again, by suspending an otherwise sterile mineral in water
and adding chemicals which serve as nutrients, the dilemma of controlling
microbiological growth is created.

10.1.3.2 Calcium carbonate. Ground calcium carbonate is typically

processed by 'autogenous' grinding which generates biocidal temperatures.
Like kaolin, processing includes centrifugation, screening and sometimes
flotation. Coarser products include the use of 'settling bowls' at ambient
temperature which, in the absence of biocide, contribute to the level of
microbiological contamination. Like kaolin, settling of coarser final
products is prevented by the addition of suspension aids. On the other
hand, the finer products are 'sand ground' near the end of the process.
Therefore, biocidal temperatures are achieved at the beginning and end of
the process for the finer products. Once the slurry cools, however,
microbial activity will increase in the absence of biocide.
Precipitated calcium carbonate (PCC) is produced in two steps: first
'slaking' (addition of water to calcium oxide to form calcium hydroxide)
and second addition of carbon dioxide to the calcium hydroxide to form
calcium carbonate. Calcium oxide is produced from calcium carbonate at
very high temperatures and the 'slaking' reaction is highly exothermic.
Therefore, the starting material, calcium oxide, and the slaking reaction
are not conducive to microbiological contamination or growth. The
carbonation reaction also takes place at biocidal temperatures.
Generally, PCC is used immediately after production. Extended storage
of PCC suspensions will require biocide treatment. The use of flue gas as a
source of carbon dioxide can introduce unwanted sulfur compounds which,
as nutrients for anaerobic microorganisms, can cause odor and discoloration
if the suspension is allowed to become anaerobic.

10.1.4 Uses

10.1.4.1 Kaolin. Kaolin is sold as bulk and slurry material. It has many
industrial applications, primarily in the paper industry. It is also used in
266 PRESERVATION OF SURFACTANT FORMULATIONS

rubber, catalysts, fiberglass, insulation, paint and ceramics. In the paper

industry kaolin is used as a filler, i.e. it is added to the fiber furnish prior to
sheet formation to improve light scatter (opacity), paper smoothness and
brightness. It is also used as a coating pigment to improve the gloss and
printing properties of paper.

10.1.4.2 Calcium carbonate. Ground calcium carbonate is sold as both a

bulk and slurry material. It is a filler in paper, rubber, paint and plastics.
GCC is also used in paper coating. Precipitated calcium carbonate is sold
as a slurry material and is generally used as a filler in paper manufacturing.

10.1.4.3 Titanium dioxide. Ti0 2 is an excellent pigment with excellent

light scattering properties due to its very high refractive index. Ti0 2 is used
in paper and print manufacturing. Its short supply and high price have
forced the paper manufacturer to look at other pigments such as kaolin for
use as Ti0 2 extenders and replacements.

10.2 Contamination sources

Microbiological contamination includes both aerobic and anaerobic micro-

organisms; it is excessive and ubiquitous. Contamination is found in the
crude mineral, water, air, blungers, pipes, centrifuges, pumps, flotation
cells, magnets, storage tanks and other processing apparatus. Pockets of
anaerobic microorganisms will be present where oxygen is depleted, such
as tank, blunger and flotation cell bottoms. A primary source of
contamination is plant apparatus that has not been cleaned.

10.2.1 Types of organisms found in kaolin aqueous manufacturing

processes

10.2.1.1 Kaolin. As expected, microbiological surveys have shown

many types of microorganisms to be prevalent in our kaolin manufacturing
facility. These include Alcaligenes faecalis, Pseudomonas diminuta, Erwinia
carnegieana, Yersinia, Arthrobacter protophormiae, Micrococcus luteus,
Acinetobacter baumannii, Aeromonas hydrophila, Serratia marcescens,
Arthrobacter globiformis, Pseudomonas syringae, Bacillus mycoides,
Xanthobacter fiavus, Ochrobactrum anthropi, Acinetobacter haemolyticus,
Pasturella multocida, Phyllobacterium rubiacearum and Pseudomonas
aeruginosa.
 PRESERVATION OF INORGANIC SYSTEMS 267

10.3 Consequences of microbiological contamination

If microorganisms are not controlled, the consequences of severe contam-

ination can range from slightly detrimental to catastrophic for processing,
final product and the paper making/coating process itself. In the absence of
biocide, aerobic microorganisms, such as Pseudomonas aeruginosa, prolif-
erate rapidly in kaolin and carbonate slurry and deplete oxygen to the
point where anaerobic bacteria such as the sulfate-reducing bacteria
Desulfovibrio desulfuricans can grow.

10.3.1 Kaolin
During kaolin processing, several sulfur-containing compounds are used,
including sulfuric acid, sodium hydrosulfite and alum. The sulfate-reducing
bacteria use sulfur instead of oxygen as a terminal electron acceptor and
form hydrogen sulfide. The H 2 S reacts with iron hydroxide that has been
formed by the reaction of sodium hydrosulfite with iron oxide to form the
black compound, iron sulfide. Hence, kaolin slurry that has become
anaerobic has the typical rotten egg odor (H2 S) and the level of iron sulfide
is sufficient to turn the clay grey. Reclamation of kaolin slurry that has
become anaerobic and chemically altered by the reaction of H 2 S with iron
to form FeS must be accomplished with a strong oxidant, such as H 2 0 2 .
The quality of kaolin slurry that has become anaerobic can not be
recovered with the addition of biocides alone.
Calcination of kaolin removes sulfur. Even though calcined kaolin slurry
will not become discolored due to the lack of sulfur, it will become highly
contaminated with aerobic microorganisms and become anaerobic if
microbiological growth is not controlled.

10.3.2 Calcium carbonate

Ground calcium carbonate contains very little sulfur. If aerobes are not
controlled, the slurry will still become anaerobic and if sulfate-reducing
bacteria are present, enough sulfur is present to detect the odor of H 2S.
Iron content is minimal and significant discoloration is not observed.
Precipitated calcium carbonate also contains very little sulfur because
calcium carbonate has been heated at high temperatures (800--1000°C) to
drive off the CO 2 • However, the use of flue gas that contains sulfur will
introduce sufficient sulfur that, if the PCC suspension is allowed to become
anaerobic in the presence of sulfate-reducing bacteria, H 2 S will be formed.
Although the level of iron is minimal, the FeS formed is obvious because of
its color contrast to the very high whiteness of PCC.
268 PRESERVATION OF SURFACTANT FORMULATIONS

10.3.3 General
In addition to foul odor and discoloration, viscosity and pH can also be
adversely affected. The excretion of organic acids by the bacteria lowers
the pH. Viscosity is adversely affected by both a lowering of pH and
microbiological degradation of the dispersant, for example sodium
polyacrylic acid. Degradation of dispersant can lead to settling and
unloading difficulties.

10.3.4 Paper
The introduction of slurry that is highly contaminated with microorganisms
into the customer's storage tank can lead to very serious problems with
fillers and coatings. These include paper breaks, holes, fish eyes, poor
runnability, excessive microbiological contamination in food grade paper,
loss of strength, loss of brightness and loss of gloss. High aerobic
contamination will ultimately contaminate the customer's system and, if
left unchecked, will result in an anaerobic condition. All machine additives
must be microbiologically acceptable for optimum performance. The use
of the proper biocides for both head box and coating color sterilization!
preservation will be discussed later.

10.4 Sampling

The collection of a representative slurry sample is, of course, very

important when testing a slurry sample for any quality parameter. In order
for the sample to be truly representative, the suspension must be as
homogeneous as possible and the sample either taken from the suspension
itself or from an uncontaminated valve. Homogeneity requires good
mixing.
Compounding the difficulty of collecting a representative slurry sample
is the problem of obtaining a sample that will be representative of the
microbiological contamination. Not only is homogeneity required, contam-
ination must be avoided. Although most quality parameters are static,
microbiological quality is dynamic. If one contaminates a sample for
microbiological testing with only a minimal amount of microbiological
contaminant, the data can be completely erroneous.
Prerequisites for microbiological sampling are clean, sterile containers
and careful handling of the sample to avoid contamination. Perhaps the
most difficult part is obtaining the sample itself. Assuming homogeneity,
the best sample is taken with a sterile container introduced directly into the
top of the suspension. However, this can be very time consuming and the
sterile container holding apparatus must be washed after each sample is
 PRESERVATION OF INORGANIC SYSTEMS 269

taken. The use of strong oxidants to wash/decontaminate the apparatus

risks introducing oxidants into the next area of sampling which may alter
the microbiological population.
Samples taken from side valves on static tanks or other containers
containing mineral suspensions cannot be assumed to be representative.
The valves themselves can also create erroneous results. These valves plug
easily, contain contaminated material and the flow from them is typically
insufficient to 'clean out' the valve. The dead space will tend to become
contaminated and contaminate the clay at the entrance of the valve which,
during mixing, can contaminate the entire tank.
Samples taken from large bottom valves are also suspect because they
are typically below the level of the mixing paddles, are not aerated and
become contaminated with anaerobic microorganisms. A suspension that
is not mixed and contains insufficient biocide to control microbiological
growth will become anaerobic. This phenomenon is observed wherever
'dead spaces' occur.
If time permits, the sample should be taken from the suspension itself
without any intermediate pipes, valves, etc. to contaminate the sample. If
this is not possible, then obtaining the sample from a moving stream is
acceptable. A stainless steel valve on the outlet side of the pump in a
recirculating/unloading system provides sufficient quantity of material and
stream velocity to clear the valve of most residual material. The valve
should be thoroughly flushed before taking the sample.

10.5 Equipment design

Typically a system has to be dealt with that was not designed to facilitate
microbiological control: flat-bottom tanks that do not drain properly, lack
of recirculation system, poor mixing, inadequate sampling valves, tank
composition, inability to drain slurry from pipes/loading lines, etc. No
mineral suspension processing/storage facility should be built without the
appropriate design for cleaning, recirculation, mixing, sampling and
chemical addition.

10.6 Exponential growth

Once the work force understands the deleterious effects of excess

microbiological contamination, perhaps one of the most difficult facts
about microbial growth to communicate to them is the rate at which
microorganisms multiply. As was discussed earlier, unlike other quality
parameters which tend to be static, microbiological growth is extraordinarily
dynamic. The growth rate is exponential with a doubling time every 20 min
under optimum conditions.
270 PRESERVATION OF SURFACTANT FORMULATIONS

The following method is effective in conveying this growth phenomenon

to operators. Assuming that bacteria can double every 20 min, and starting
with only one bacterium per ml, operators are asked to calculate the
number of bacteria that will accumulate in 1 ml of slurry during an eight-
hour work shift. Since there are 24 twenty-minute increments in 8 h, they
are asked to enter '1' into their calculator and multiply by two 24 times in
succession. When the operators see that the answer is greater than 10
million, they begin to realize just how rapidly bacteria can grow and how
important it is to prevent microbiological growth. Of course, the
suspension typically contains> 1 bacterium per ml prior to adding biocide,
which of course exacerbates the problem. What may have seemed like
minor contamination can become catastrophic in a matter of hours.

10.7 Microbiological test methods

The level of microbiological contamination must be monitored continuously

to ensure control is effective. The primary goal in mineral suspension
preservation is to control the aerobic microorganisms, thus preventing the
suspension from becoming anaerobic. Techniques are used to measure and
monitor the levels of aerobic organisms while biocides are used to prevent
their growth.
The two techniques typically used in the mineral industry are standard
plates and 'dip slides'. Although the dip slide method is generally
acceptable, the standard plate method is more accurate, especially for low
levels of contamination. However, at moderate to high contamination
levels, the two methods are reasonably correlatable. In a quality control
(QC) environment where there are large numbers of tests and time is of the
essence, the dip slide method is preferred.
To carry out effectively a meaningful microbiological test requires an
uncontaminated, representative sample and a test duration of 72 h. It is
extremely important to collect samples in a clean sterile container. Also,
the samples should not be contaminated by residual material in valves,
hoses, etc.; these should be kept clean and flushed thoroughly before
catching the sample.
The test procedure requires a 24 h incubation at the optimal growing
temperature (this provides sufficient time for the microorganisms to react
with the biocide), dilution and plating of the sample and further incubation
for 48 h to observe microbiological growth.
This time requirement is certainly a limiting factor in most production
situations. 'Just-in-time shipments', scheduling, and rail space typically
require test results to be provided in a matter of hours, certainly less than
24 h. By the time the microbiological test results have been obtained, the
slurry shipment is usually on its way to the customer. A microbiological
 PRESERVATION OF INORGANIC SYSTEMS 271

test of slurry shipments, therefore, becomes more of a secondary check

than test for shipment approval. The quantitation of residual biocide
(which has been shown to be effective and stable in a system) is a more
expeditious test for shipment approval. This will be discussed later.

10.7.1 Standard plate count procedure

The slurry sample is serially diluted and an aliquot of each dilution is
placed in or on suitable culture media. Each colony is assumed to have
grown from one viable unit, which may be one organism or a group of
many. This standard method is described in most microbiology text books.
One method is the use of tryptose glucose extract (TGE) agar. Dilutions
are made with sterile Butterfield buffer.

10.7.2 Dip slide method

Dip slides are available from several manufacturers and are of various
types, depending on the medium and microbiological population to be
examined. Each side of the paddle will be coated with a specific medium
containing nutrients optimum for the microbiological species to be
examined. The 'paddle' may have different media on each side such that
two types of microorganisms, such as bacteria and fungi, can be examined
simultaneously.

10.7.2.1 Equipment needed.

1. 1 ml Sterile syringes or sterile pipettes - syringes are preferred to
eliminate the hazard of mouth pipetting.
2. Tubes containing 9 ml of sterile buffer solution - (Butterfield-
monopotassium phosphate).
3. Dip slides with Tryp--Soy agar impregnated with TIC - (Triphenyl-
tetrazolium chloride) on one side and Sabouraud (SAB) dextrose and
malt extract agar on the other. TIC, colorless at neutral pH, is changed
to the red-colored formozan by the acids excreted by most species of
bacteria such that bacterial colonies appear as red dots.
The SAB dextrose side is adjusted slightly acidic to favor the growth
of fungi/yeast/mold but suppress bacterial growth. This side is also
impregnated with chloramphenicol, an antibiotic which inhibits the
growth of prokaryotic (anucleated) cells but not eukaryotic (nucleated)
cells such as fungi. Thus, bacterial growth is inhibited on the 'fungus'
side which allows one to differentiate between bacteria and fungus/
yeast/mold contamination. Numerous types of dip slides are available
from several companies.
4. Incubator typically set at 35°C.
272 PRESERVATION OF SURFACTANT FORMULATIONS

10.7.2.2 Procedure. (Be very careful not to contaminate mineral

suspension and test equipment):
1. Obtain 1 ml of slurry using a syringe or pipette. Ensure that there are no
bubbles present.
2. Introduce the 1 ml of slurry into the buffer tube with 9 ml Butterfield
buffer to give a 1 to 10 dilution. Dilution is required because of the
opaqueness of the mineral suspension and to dilute out any biocide
present. Undiluted slurry will prevent observation of the bacterial!
fungal colonies. Of course the dilution factor must be taken into
consideration when the assessment of colonies is made.
3. Shake the slurry sample-buffer mixture until homogenous (10-20 s).
4. Remove the cap from the dip slide container (being very careful not to
contaminate the dip slide) and pour the diluted sample into the
container, replace dip slide, tighten and gently agitate the container
such that the agar surface comes into contact with the slurry for a
minimum of 5 s. Do not agitate too vigorously as the agar can be
dislocated from the paddle. Remove the dip slide and pour excess slurry
from the container over the dip slide; let drain.
5. Replace the dip slide into its container and label accordingly. incubate
for 24 h at 35°C.
6. After 24 h, count the colonies (red dots) present. If there are no
colonies, incubate for a further 24 h and count. Sometimes the bacteria
grow slowly and, quite often, the bacteria colonies are present as very
tiny dots but have not excreted sufficient acid to tum the TIC red.
7. Bacteria will appear on the non-chloramphenicol-treated side of the dip
slide as colonies that look like red or clear dots. Fungi appear as fluffy
colonies and yeast appears as 'dry' colonies on the chloramphenicol-
treated side of the dip slide.
8. Pictorial guides are provided by the dip slide manufacturer for an
estimate of bacterial!fungal population. The guides are typically
photographs of dip slides that have been exposed to solutions of
bacteria whose populations have been determined by the standard plate
method.

10.7.3 Assessment of bacterial populations using dip slides

10.7.3.1 Counts of <1 K organisms per ml of slurry. Slurry is considered

within tolerable limits and does not require biocide treatment. Recheck
every 24 h if slurry is to remain stored in tank for an extended period
(longer than 1 week).

10.7.3.2 Counts of>lK but <lOOK organisms per ml of slurry. Slurry is

considered contaminated with aerobic microorganisms but sufficient
 PRESERVATION OF INORGANIC SYSTEMS 273

oxygen remains to inhibit the growth of anaerobic microorganisms.

However, slurry must be treated with biocide immediately. Retest after
24 h and retreat with biocide as required.

10.7.3.3 Counts of >100K organisms per ml of slurry. Slurry is con-

sidered seriously contaminated and in danger of becoming anaerobic.
Slurry must be treated with biocide immediately. Test for anaerobic
bacteria and treat accordingly. Retest after 24 h and retreat with biocide as
required. Once the contaminated slurry has been treated and utilized, the
holding tank must be washed with water and sterilized before new slurry is
introduced.

10.8 Anaerobic contamination

Depletion of oxygen by aerobic microorganisms in the slurry allows

anaerobic microorganisms to flourish. In the case of anaerobic mineral
suspensions containing sulfur, iron and sulfate-reducing bacteria, hydrogen
sulfide and iron sulfide are formed.
The FDA approved biocides will not destroy the iron sulfide. If the
suspension has become anaerobic, fouled with H 2 S and discolored by iron
sulfide, a strong oxidant such as hydrogen peroxide must be used to oxidize
the iron sulfide. It should be noted that the absence of aerobic
microorganisms does not necessarily preclude the existence of contamina-
tion. The suspension may have already gone through the aerobic phase,
become anaerobic, and only anaerobic bacteria are viable. Obviously,
discoloration and/or odor indicates that an anaerobic microbiological test
for sulfate-reducing bacteria should be done.

10.8.1 Anaerobic microbial analysis

The following 'stab test' method is used to assess a population of anaerobic
sulfate-reducing bacteria in a kaolin or calcium carbonate slurry sample.

10.8.1.1 Equipment needed.

1. One ml sterile syringe or sterile pipette - syringes are preferred to
eliminate the hazard of mouth pipetting.
2. One tube containing 9 ml sterile buffer solution - (monopotassium
phosphate).
3. One tube containing 8 ml of Peptone Iron Agar Butt.
4. One inoculation wire - (6 inch chromed 'A' wire).
5. Bunsen burner or incinerator.
6. Incubator.
274 PRESERVATION OF SURFACTANT FORMULATIONS

10.8.1.2 Procedure.
1. Using the sterile syringe or pipette, obtain 1 ml of slurry. Ensure there
are no bubbles present.
2. Place the 1 ml of slurry into the buffer tube with 9 ml of buffer
solution.
3. Shake the mixture until homogeneous (10-20 s).
4. Sterilize the inoculation wire by passing it through the bunsen burner
flame or into the incinerator until the wire turns red.
5. Allow the wire to cool for 5 s.
6. Insert wire into slurry and buffer mixture.
7. Remove cap from Peptone Iron Agar Butt.
8. Remove the wire from the buffer tube and immediately insert the wire
into the agar tube, being careful not to touch the sides of the tube with
the wire when inserting or extracting it. Make sure wire penetrates
agar to the bottom of the tube. Allow the wire to remain for 5 sand
remove.
9. Recap the Peptone Agar Butt.
to. Store agar tube in incubator at 35°C for 48 h.
11. Inspect the tube after the 24 and 48 h incubation period. If anaerobic
sulfate-reducing bacteria are present, agar will turn black where the
inoculation wire penetrated. The black color is due to iron sulfide
formed from iron and hydrogen sulfide produced by the sulfate-
reducing bacteria.
Note that this method is basically a qualitative method.

10.9 Microbiological control

The control of microbiological growth can be accomplished physically with

frequent housekeeping (cleaning/sanitation). It can also be accomplished
with radiation (UV, X-ray, gamma radiation) and chemicals such as
antibiotics, biocides and strong oxidants (chlorine, ozone, chlorine dioxide
and hydrogen peroxide).

10.9.1 Cleaning/housekeeping
Microbiological control cannot be properly maintained through the use of
biocides alone. Equally important is the routine cleaning and sterilizing of
the processing system itself. This will greatly enhance microbiological
control by reducing any recontamination of the slurry as it flows through
the system.
Typically, frequent cleaning and sterilization of plant equipment are
 PRESERVATION OF INORGANIC SYSTEMS 275

very difficult because of both the continuous use and inaccessibility of

equipment. However, it must be recognized that this facet of a micro-
biological control program is very important. The higher the contamination
in the equipment the greater the risk of spoilage and the greater the biocide
demand.
Equipment should be cleaned on a regular basis to eliminate the
contamination that will accumulate. This includes biofilm which is very
difficult to remove and not only provides a continuous source of
contamination but also contributes to corrosion.
Equipment (tanks, valves, hoses, couplings, etc.) is usually cleaned with
sodium hypochlorite solution. Sodium hypochlorite must be thoroughly
removed by rinsing with good quality water because of its detrimental
effect on biocides and viscosity. Equipment can also be cleaned with strong
acids, strong bases, detergents, solvents or dispersants.

10.9.2 Radiation
The use of ultraviolet radiation for sterilization is not practical because of
the inability of UV radiation to penetrate a high solids slurry and the large
quantity of material. The use of X-ray or gamma radiation is also not
practical to sterilize such large amounts of material and, of course, safety
becomes a primary consideration. Radiation techniques, like strong
oxidants, have no residual effect. Any further contamination after the
radiation treatment will result in uncontrolled microbiological activity.

10.9.3 Oxidants
Hydrogen peroxide, ozone, chlorine dioxide and chlorine (chlorine gas,
sodium hypochlorite) are used for sterilization. However, although strong
oxidants provide good sterilization, they do not provide preservation.
Strong oxidants can also destroy other important processing chemicals,
such as dispersants, suspension aids and biocides, as well as oxygenate the
system to support aerobic microorganisms once the biocide has been
depleted. Where iron has been converted to the reduced, soluble form for
extraction purposes, peroxide will oxidize it to the insoluble iron oxide.

10.10 Biocides

Chemicals are used in conjunction with housekeeping for microbiological

control. Chemicals have been developed which are toxic to microorganisms
and provide not only quick kill, but intermediate to long term micro-
biological control. These chemicals are termed biocides. Biocides that
provide long term control are called biostats.
The primary function of biocides used in kaolin and carbonate slurry
276 PRESERVATION OF SURFACTANT FORMULATIONS

preservation is to control the growth of aerobic microorganisms such that

sufficient oxygen is present in the slurry to inhibit the growth of anaerobic
microorganisms, especially the sulfate-reducing bacteria. Obviously, the
user of the kaolinlcarbonateltitanium dioxide product is concerned about
both aerobic and anaerobic microbiological contamination in his system.
So the key is to keep the aerobes in check so that the anaerobes cannot
grow. By doing this, deterioration in the quality of the slurry is prevented
and the customers' pigments, storage tanks, paper machine, etc. are not
contaminated.
The mechanism of action of biocides varies with the specific product.
Where available, these will be discussed later.

10.10.1 FDA approval

Biocides that are used for the preservation of pigment slurry are generally
those FDA approved for food contact. They meet the following approvals:
CFR 176.170, CFR 176.180 and CFR 176.230. Other non-FDA approved
biocides used to preserve mineral suspensions are methylene bisthiocyanate
(MBT) , 2-dibromonitrilopropionamide (DPNPA) and 2-bromo-2-nitro-
propane-1,3-diol (BNDP or Bronopol).

10.10.1.1 CFR 176.170. (Components of paper and paperboard in

contact with aqueous and fatty foods). These biocides are those that may
be safely used as components of the uncoated or coated food-contact
surface of paper and paperboard intended for use in producing, manu-
facturing, packaging, processing, preparing, treating, packing, transporting
or holding aqueous and fatty foods.

10.10.1.2 CFR 176.180. (Components of paper and paperboard in

contact with dry food). These biocides are substances that may be safely
used as components of the uncoated or coated food-contact surface of
paper and paperboard intended for use in processing, manufacturing,
packaging, transporting or holding dry food of the type identified in CFR
176.170.

10.10.1.3 CFR 176.230. (Special section for thione biocides). The

general requirement of this section is that the thione be volatilized by heat
in the drying and finishing of the paper, coated paper or paperboard.

10.10.2 Approval outside the United States

It must be ensured that biocides used in mineral suspensions that are
exported comply with regulations of the country of destination. Generally,
US FDA approval is sufficient. However, this is not always the case.
 PRESERVATION OF INORGANIC SYSTEMS 277

Typically, Europe will adhere to the German BGA guidelines. Scandinavian

countries may have different regulations. Japan will generally accept US
FDA approved biocides.
On the other hand, imported mineral suspensions would have to meet
US FDA guidelines. Biocides such as Bronopol are accepted for mineral
suspensions used in food grade packaging in Europe but not in the US.
Another biocide issue is that the biocide must also meet the regulations of
the country in which the paper product is used.

10.10.3 Types of biocide

FDA approved biocides are discussed below. Other biocides, depending
on the end use, which can be considered for pigment slurry preservation
but are not discussed here are dodecylguanidine hydrochloride (DGH), 2-
bromo-2-nitropropane-1,3-diol (Bronopol) and 2-bromo-4' -hydroxy aceto-
phenone (Bus an 90).

10.10.3.1 Tetrahydro-3,5-dimethyl-2H-1,3,5-thiadiazine-2-thione. This is

generally referred to as thione or dazomet. Thione is the biocide generally
used in the kaolin and carbonate industry for slurry preservation. Thione
hydrolyzes.to several compounds including formaldehyde, methylamine,
carbon disulfide, methylisothiocyanate, carbon monoxide, carbon dioxide,
nitrogen oxides, sulfur oxides, disodium oxide and hydrogen sulfide.
The primary biocidal compound is methylisothiocyanate (MITC); the
small amount of formaldehyde present contributes very little to the
biocidal activity. Thione does not polymerize and is effective over a broad
pH range. It is incompatible with strong acids, mercurials or other
compounds that are not compatible with sulfur compounds, oxidizing
agents, copper, carbon steel and epoxy drum liners. Color will vary from
pale yellow to amber to green.
Thione is manufactured in the USA by Vinings Chemicals and Buckman
and sold in three formulations: dry, 10% NaOH and aqueous dispersion.
Dry thione is relatively stable but on contact with water rapidly breaks
down to methylaminomethyldithiocarbamate which subsequently yields
MITC and hydrogen sulfide. In acid solution, the dry product hydrolyses to
form carbon disulfide.
Thione is the most cost effective FDA approved biocide. Unlike other
FDA approved biocides, the maximum allowable amount that can be
added is not restricted except as is stated in CFR 176.230 (b); 'The quantity
used shall not exceed the least amount reasonably required to accomplish
the intended technical effect and shall not be intended to nor, in fact,
accomplish any physical or technical effect in the food itself.' The primary
requirement is that the preservative is volatilized by heat in the drying and
finishing of paper, coated paper and paperboard.
278 PRESERVATION OF SURFACTANT FORMULATIONS

Thione has the following disadvantages. Some of the volatile breakdown

products are malodorous, irritating and lachrimatory. Thione also emits
small amounts of formaldehyde. Thione and its breakdown products
are incompatible with several of the other FDA approved biocides:
glutaraldehyde, Kathon and Tektamer. It is also incompatible with
hydrogen peroxide which is commonly used to sterilize/recover pigment
slurry.
Thione is most effective when it is added to a system containing low
microbiological contamination. Thione is not as effective when added to a
slurry that is in a high contamination/nutrient state.

10.10.3.2 1,2-Benzisothiazolin-3-one. This is generally referred to as

BIT or its trade name, Proxel. Proxel is manufactured by Zeneca and used
throughout the kaolin industry as a preservative. A very effective biocide,
one of its greatest advantages is its compatibility with all the FDA
approved biocides. It is incompatible with strong oxidizing agents and
corrodes mild steel, aluminum, copper and other metals. It is a brown
liquid with a slight odor and a pH of 12.
BIT does not polymerize and is effective over a pH range of 4 to 12. It is
odorless and does not react with ammonia and amines. It has relatively low
toxicity and can be used up to 475 ppm active ingredient (BIT) per wet ton.
It is a good biostat, has a high percentage of active ingredient and does not
emit formaldehyde.
BIT's disadvantages include cost, especially when used with a commodity-
type material, and mild skin sensitization. It is also inactivated by
reduction to mercaptobenzamide and BIT diamide by reducing agents such
as those typically found in kaolin slurry (sodium hydrosulfite and sodium
bisulfite) and hydrogen sulfide. It is also inactivated by strong oxidants
such as hydrogen peroxide and sodium hypochlorite.
Proxel GXL (19% BIT in dipropylene glycol) becomes very viscous in
cold weather. This should be taken into consideration when designing the
chemical addition system.

10.10.3.3 Glutaraldehyde(1,5-pentanedial}. This is generally referred to

by its trade name, Ucarcide. Glutaraldehyde is manufactured by Union
Carbide. It is a broad spectrum biocide due to its ability to cross link
primary amines, such as those present in lysine residues in a microbial cell
wall. This fixative action ultimately can prevent a microorganism from
carrying on its metabolic functions by limiting the cell permeability. It is
effective against aerobic and anaerobic organisms.
Glutaraldehyde is typically sold as a 50% active material in water. It is a
colorless liquid with a strong odor and a pH of 3.7 to 4.5 and is corrosive to
steel, galvanized iron, aluminum, tin and zinc.
Glutaraldehyde reacts with and is inactivated by ammonia, ammonium
 PRESERVATION OF INORGANIC SYSTEMS 279

salts, primary amines and protein. It does not react with secondary,
tertiary, quaternary or protonated amines. pH has a dramatic effect on the
efficacy of glutaraldehyde. As the pH is increased, the amino groups are
deprotonated and reactivity is rapid. However, above pH 9 glutaraldehyde
will be inactivated by polymerization. As the pH is decreased, the amino
groups become protonated and do not react with glutaraldehyde. The
inactivation of glutaraldehyde with ammonia, primary amines and protein
can be offset by lowering the pH.
Glutaraldehyde is stable in kaolin slurry to the presence of low
concentrations of hydrogen peroxide (up to lIb active H 202/ton). It is an
excellent, rapidly acting, long term biocide in kaolin slurry at pH 7 when
ammonia and primary amines are absent. The use of ammonia, primary
amines or proteins excludes the use of glutaraldehyde.
Glutaraldehyde has not received widespread acceptance in the kaolin
industry because of inconsistency. Particularly, it may be converted to the
inactive valerolactone by a component(s) in kaolin and/or process water.
This inactivation reaction is easily eliminated by pretreatment with
hydrogen peroxide.
Although glutaraldehyde is an excellent, rapidly acting biocide in
calcium carbonate slurry, it is not biostatic. The high pH of carbon-
ate slurry results in inactivation of glutaraldehyde by polymerization.
Glutaraldehyde must be used in combination with a stable biocide in
carbonate slurry for preservation.

10.10.3.4 5-Chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-iso-

thiazolin-3-one. This is generally known by its trade name Kathon.
Kathon is manufactured by Rohm and Haas. It is a combination of
chlorinated methyl and methyl isothiazolin-3-ones at a ratio of 3.3:1. It is a
broad spectrum biocide, controlling Gram-negative and Gram-positive
bacteria as well as fungi and is typically sold as a water-based 1.5% active
material at pH 3-5 with a copper stabilizer. It is pale yellow to green in
color with a mild aromatic odor and is corrosive to mild steel, carbon steel,
copper and 304 stainless. It does not polymerize.
Kathon reacts with and is inactivated by reducing agents, such as
hydrosulfite, sulfite and bisulfite typically found in kaolin slurry. It is
also inactivated by primary, secondary and tertiary amines, ammonia,
mercaptans, sulfides, strong oxidants, and high pH typically found in
carbonate slurry. These reactions are exacerbated by high temperature.
Kathon is a cost effective, quick killing biocide at low active concentra-
tions. However, its low percent active ingredient and its maximum usage
rate result in low levels of active ingredient in the slurry. Thus, the
presence of minimal amounts of reducing agents or nucleophiles will result
in its inactivation. It is an excellent preservative in systems where it can be
stabilized.
280 PRESERVATION OF SURFACTANT FORMULATIONS

Kathon stability in kaolin slurry can be attained by ensuring the

elimination of reducing agents, primarily sulfites resulting from the use of
sodium hydrosulfite for iron reduction, and the use of sodium hydroxide or
sodium carbonate instead of ammonia.
Reducing agents can be removed by oxidation with hydrogen peroxide.
However, because of isothiazolin's inactivation by strong oxidants such as
hydrogen peroxide, hydrogen peroxide and isothiazolin have to be used
sequentially. Hydrogen peroxide should be eliminated before the addition
of isothiazolin.
The chlorinated methyl isothiazolin is subject to nucleophilic attack by
amines. This makes this molecule unstable in systems where ammonium
hydroxide/amines are used. It also makes it unstable in the presence of
thione biocide and its hydrolysis products. As with all biocides, Kathon
should be handled with extreme caution. Kathon, in addition to its
corrosivity, is also a strong skin sensitizer.

10.10.3.5 1,2-Dibromo-2,4-dicyanobutane. This is generally known by

its trade name Tektamer or DBDCB. DBDCB is a broad spectrum
biocide controlling bacteria, mold, yeast and sulfate reducers. It is effective
at a pH range of 2-8.5 but is incompatible with sulfites, amines and strong
nucleophilic agents. It is compatible with materials such as PVC,
polyethylene, polypropylene, Tygon, Teflon and glass. It is corrosive to
mild steel.

10.10.3.6 Biocide blends. Several FDA approved biocide blends are

available. Also, one can add biocides that are compatible with one
another, as long as they are added separately. These combinations can
consist of fast acting biocides and long term, but slower acting, biostats,
such as a mixture of glutaraldehyde and Proxel. This allows the immediate
killing of highly contaminated suspensions plus long term preservation by
the bios tat. Once control is established, the slower acting biostats, such as
Proxel, effectively maintain control.

10.10.3.7 Alternating biocideslresistance. Although biocides tend to be

fairly non-specific, some biocides can be more effective in controlling
certain microorganisms than others. Microbiological competition for
nutrients, oxygen, etc. is reduced by ridding the aqueous suspension of
certain bacterialyeast/fungi/mold. This allows the 'more resistant' micro-
organisms to flourish. The limited number of FDA approved biocides for
indirect food contact coatings as well as biocide-biocide/biocide-process
chemical incompatibility make it difficult to alternate biocides in most
cases.
 PRESERVATION OF INORGANIC SYSTEMS 281

10.11 Biocide performance testing

Although biocide brochures provide excellent information concerning the

performance of biocides, one must determine biocide performance for a
mineral suspension 'in house'. Cost and compatibility issues must be
considered.
Initially, tests are done in the laboratory with appropriate slurry samples
and a wide range of biocide concentrations. A minimum of 30-day control
is required. Most FDA approved biocides have maximum addition levels.
If a particular biocide is effective, one must then reinoculate ('reinsult')
the suspension with additional bacteria to ensure that the biocide is
sufficiently stable to control further contamination. Strong oxidants like
hydrogen peroxide are short lived and, on reinoculation, a lack of control
may be observed. Once satisfactory laboratory results are obtained, the
biocide must be tested in the actual process.

10.12 Biocide addition systems

Biocides can be added manually or automatically. The manual addition of

biocides is not recommended because of the danger inherent to these types
of chemicals. If manual addition is necessary appropriate protective
clothing must be worn such as chemical aprons and goggles. Ventilation
must be sufficient. The addition of biocide should be controlled by mass
flow meters to ensure accurate dosing regardless of slurry flow.
The effect of biocide pH on mineral slurries must be taken into
consideration. Very acidic (such as Kathon or glutaraldehyde) or alkaline
biocide solutions (such as thione, which is typically formulated in 10%
sodium hydroxide) can flocculate a suspension. Kaolin particles, which
have an overall negative charge at neutral pH, are kept separated or
deflocced by maintaining the surface negative charge. The overall negative
charge is also increased by using anionic dispersants. If the negative
charges are neutralized, the Van der Waals attraction forces cause
flocculation.
Biocides should be added at the earliest possible point in the process to
control microbiological growth. Temperature, pH and process chemical
compatibility must be taken into consideration when determining the point
of addition. Of course, temperature will exacerbate chemical reactions.
Biocides are often added to the make down water prior to addition of
pigment.
282 PRESERVATION OF SURFACTANT FORMULATIONS

10.13 Residual biocide testing

As discussed earlier, microbiological tests are very time consuming and

sterile representative sampling is crucial. A positive test may mean that the
sample was contaminated. Testing for residual biocide is one way to
expedite the shipment approval process. Of course, the biocide must have
been shown to be effective and stable in the system. Several analytical
methods are available for the determination of residual biocide in slurry.
Also available are bacteria-impregnated strips to determine if there is
biocide present.
The importance of a minimum amount of the right biocide to maintain
long term microbiological quality as well as the high cost of biocides makes
accurate biocide quantitation in final product necessary. The addition of an
accurate dose of biocide does not always ensure that sufficient biocide is
available. The residual biocide level depends on its stability to pH,
temperature, bacteria and chemicals. Minimal biocide levels for acceptable,
long term control must be determined for the mineral suspension being
preserved.

10.13.1 Thione
The primary biocidal product in thione biocide is methylisothiocyanate
(MITC). This compound can be detected by GC or HPLC. Formaldehyde
is another byproduct which can be detected by HPLC.

10.13.2 Glutaraldehyde
Glutaraldehyde levels may be quantified with the Union Carbide 'Ucarcide
antimicrobials field test kit'. Glutaraldehyde may also be quantified by gas
chromatographic (GC) or high performance liquid chromatographic
(HPLC) methods. Rapid tests for glutaraldehyde are also commercially
available. If inconsistent control is observed, it may be due to valerolactone
formation; this compound can also be quantified with HPLC or Gc.

10.13.3 Benzisothiazolin-3-one
Benzisothiazoline (Proxel) levels in slurry may be quantified by high
performance liquid chromatographic methods. This method is available
from Zeneca. If inconsistency of control is observed, it may be due to the
formation of mercaptobenzamide and/or BIT diamide; these compounds
can also be quantified with HPLC.
 PRESERVATION OF INORGANIC SYSTEMS 283

10.13.4 Kathan
Kathon levels in slurry may be quantified by HPLC. This method is
available from Rohm and Haas or Vinings. A field test using UV
spectroscopy is also available from Rohm and Haas and Vinings.

10.14 Working with the external customer

A close working relationship with the external customer is required to have

a successful microbiological control program. The extended process
requires the supplier to work with the customer so the fitness for use
quality of the slurry is maintained.
Slurry transported in rail cars or trucks will typically be at optimum
temperatures for microbiological growth. Shipment samples stored at
room temperature will not always indicate what actually happens to the
slurry en route. Representative samples should be taken on arrival for both
microbiological and biocide analysis.
Several questions have to be asked. What biocides is the customer using?
Is the customer making food grade paper? What process chemicals is the
customer using? Are they compatible with the biocides in the slurry? Are
the process chemicals in the slurry compatible with the biocides the
customer is using? Will the customer blend one supplier's slurry with
another supplier's slurry? How long will the slurry be stored? Is there a
housekeeping program at the customer's facility? How clean is the
unloading system? Does the customer drain/clean apparatus used to
unload slurry? Is there a sampling/testing program at the customer site? Is
there concern about formaldehyde? Is there concern about odor? Safety
must always be thoroughly discussed and closely monitored. The shipment
of slurry to customers requires that not only must the slurry arrive with
minimal microbiological growth, it must contain residual, compatible
biocide.

10.15 Slurry made from dry material and slurry storage

Quite often, dry kaolin and/or carbonate are made into suspensions at an
intermediate site or at the customer site. Chlorinated water and clean
equipment should be used during slurry make down to prevent initial
contamination. Biocide should always be added during slurry make down
to control subsequent microbiological growth in the slurry. If the slurry is
to be stored for an extended period (longer than one week), it should be
tested regularly for microbiological growth.
11 Preservation of metalworking fluids
P.S.K. LEE

11.1 Introduction to metalworking fluids

Metalworking fluids are extensively used in a wide range of machining

operations such as turning, milling, tapping, drilling, grinding, broaching,
rolling, drawing and stamping. 1 ,2 The primary functions of metalworking
fluids during these metalworking operations are to provide cooling effects
on the tool and workpiece by dissipating the generated heat and to
lubricate the work area by reducing the amount of frictional heat
generated. Other secondary functions of metalworking fluids include waste
removal, rust protection or corrosion prevention, surface part finish and
tool protection during metalworking operations.

11.2 Consequences of metalworking fluids failure

Metalworking fluid plant operations are usually related to a central fluid

system serving several metalworking machines or individual sumps on
metalworking machines. Metalworking system sizes can therefore vary
from 25 gallons to several thousand gallons of metalworking fluids use.
These metalworking fluid operation systems are frequently subjected to
microbial contamination which is detrimental to the working life of the
metalworking fluids and is a major cause of metalworking fluids failure.3--6
The growth of microorganisms in metalworking fluids is usually kept in
check by the addition of chemical preservatives. However, unchecked
microbial growth in preservative-free metalworking fluids may result in
modifications to the metalworking fluids as previously determined by
chemical analytical methods. 7- 9 Uncontrolled microbial growth in metal-
working fluids will result in offensive odor development, decrease in pH
and increase in corrosion rates due to production of organic acids or
harmful microbial metabolites, darkening of metalworking fluids color that
may cause surface finish blemishes, selective depletion of balanced
metalworking fluid components which become utilized as microbial
nutrient sources and which may cause changes in emulsion stability, and
the visible presence of slimy microbial biomass that will clog up fluid lines,
filters and screens in metalworking fluid systems. All these microbial
 PRESERVATION OF METALWORKING FLUIDS 285

activities will eventually result in the deterioration or failure of metal-

working fluids.
The deterioration or failure of metalworking fluids will in turn result in
the loss of plant worker productivity, the need to rework finished parts, the
premature disposal and increase in dumping of metalworking fluids into
the system to replenish the rapidly spoilt metalworking fluids, increase in
plant operation downtime and decrease in metalworking tool life.
Most of these factors will add up to higher cost needed to operate the
metalworking plant system and may not be economically beneficial. There
also exists the possibility of unchecked growth of opportunistic microbial
pathogens in metalworking fluids that may represent an increase in health
risk to the metalworking plant workers. 10- 17

11.3 Ranges and types of metalworking fluid

There are four basic metalworking fluid types used in metal cutting and
metal forming operations. 1 8--22 Soluble, semi-synthetic and synthetic-based
metalworking fluids are three out of the four metalworking fluids that are
diluted with water by end users. 23 ,24 These three types of metalworking
fluids are often called water-based or water-miscible metalworking fluids.
Water-based metalworking fluids are usually sold as water-free con-
centrates which can be mixed with water by end users to 1: 10 or 1:40
dilutions depending on the fluid types and the metalworking applications.
Straight or neat oils are not water-based metalworking fluids and are used
as they are in the non-diluted form by end users.

11.3.1 Straight or neat oils

These are solely oils of petroleum (naphthenic or paraffinic mineral oils),
animal, vegetable or marine in origin. These base oils may be used alone or
in combination with or without additives. Additives are usually added to
improve lubricity and prevent welding between metal surfaces via extreme
pressure properties of the additives. These metalworking fluids offer
excellent lubricating properties but have only minimal heat transfer or
cooling effect during metalworking operations. Typical compositions of
straight or neat oils and their applications are given below.

11.3.1.1 Base mineral oil blends. Base mineral oils are blended with a
fatty oil or polar additive, e.g. lard, castor or rapeseed oil. This blended
mineral oil is typically used in metalworking operations involving light duty
machining of cast iron, aluminum, brass and magnesium. Other applica-
tions include fine honing and lapping metalworking operations.
286 PRESERVATION OF SURFACTANT FORMULATIONS

11.3.1.2 Sulfured fatty mineral oils and sulfurized fatty mineral oils.
Sulfur is added to the base mineral oils to provide extreme pressure
lubricity. Sulfured fatty mineral oil consists of dissolving 1 to 1.5% sulfur in
hot mineral base oil. The sulfur in this particular blend is loosely bound
and is very reactive at low temperature metalworking operations, hence
the term 'active oil'. The sulfide-forming reaction occurs at low temperature
with metals like copper which can cause staining to the metal worked on.
Sulfurized fatty mineral oil, however, consists of sulfur added to the fatty
oil. This blended combination results in a stronger chemical bond between
the fatty oil and sulfur. The resulting blend will prevent the sulfur from
being active and has less tendency to stain non-ferrous metals like copper.

11.3.1.3 Chlorinated mineral oils. This mineral oil has chlorine dissolved
in base oils as an additive to provide an extreme pressure lubricity
property. This blend of mineral oil is typically used in metal machining
operations such as turning, drilling and tapping including ferrous and non-
ferrous forms of grinding.

11.3.1.4 Sulfochlorinated mineral or fatty oils. This oil consists of a

combination of both chlorine and sulfur additives which can either be
blended as a sulfochlorinated mineral or sulfochlorinated fatty oil. This
allows both mineral and fatty oils to be available for use. Typical
metalworking applications of this blend of mineral oil are in thread
grinding, gear cutting and automatic screw machining of steel.

11.3.2 Soluble oils

These are essentially mineral oils with or without fatty acids that contain an
emulsifier such as petroleum sulfonates, i.e. oil-based emulsifiable
concentrates. These metalworking fluids are milky white dispersions of oil-
in-water with the oil particle size ranging between 2 to 10 !lm. Soluble oils
are used mainly in low duty machining operations. The three general
compositions of soluble metalworking fluid are shown in Table 11.1.24

11.3.3 Semi-synthetic metalworking fluids

These are fluids that contain mineral base oil at a lower percentage of 5 to
40% than that of emulsion or soluble oils at 40 to 65% mineral base oil
content. Semi-synthetic metalworking fluids usually impart better lubrica-
tion than true synthetic metalworking fluids while still retaining their heat
dissipating character. 25 Semi-synthetic metalwork fluids usually form
translucent or transparent emulsions with oil particles size equal to 0.1 !lm.
This metalworking fluid type is used in high speed machining and some fine
grinding metalworking operations. Water hardness has also been reported
 PRESERVATION OF METALWORKING FLUIDS 287

Table 11.1 General composition of three soluble metalworking fluids

Soluble or emulsifiable oils Functional composition

Simple mineral base oil

emulsifier
coupling agent
preservative

Fatty mineral base oil

emulsifier
coupling agent
preservative
fatty acids or soaps

Extreme pressure (EP) mineral base oil

emulsifier
coupling agent
fatty acids or soaps
preservative
EP additives (chlorine, sulfur or
phosphorus)

Table 11.2 General composition of a semi-synthetic metalworking fluid

Functional components Composition by weight (%)

Mineral base oil 15.0

Emulsifier/secondary or coemulsifier 20.0
Corrosion inhibitors (multi-package) 6.0
Coupling agent 1.0
Preservative 2.0
Water 56.0

Total 100.0

to affect the lubricity of a semi-synthetic metalworking fluid,z6 A general

composition of a semi-synthetic metalworking fluid is given in Table 11.2.
It was noted that an optional extreme pressure or EP package may also
be included into the semi-synthetic formulation.

11.3.4 Synthetic metalworking fluids

These are water-based solutions free of mineral oils which rely on glycols
and esters for lubricity.27.28 Water-soluble additives such as corrosion
inhibitors, detergents and defoamers can be present in these metalworking
fluids. These metalworking fluids are typically transparent in appearance
and are used mainly in high heat metalworking operations like grinding
where cooling is of importance. These metalworking fluids have high
detergency properties but lack the lubricating properties for machining
288 PRESERVATION OF SURFACTANT FORMULATIONS

Table 11.3 General composition of a synthetic metalworking fluid

Functional components Composition by weight (%)

Water 72.0
Corrosion inhibitor 10.0
Corrosion inhibitor pH buffer 4.0
Boundary lubricant 4.0
Extreme pressure lubricant 4.0
Synthetic lubricant 4.0
Preservative 2.0

Total 100.0

operations. A general composition of a synthetic metalworking fluid is as

shown in Table 11.3 minus mineral base oils.
It not only helps for metalworking fluids end users to know about the
various types and applications of these metalworking fluids, but the proper
care, handling and disposal of these metalworking fluids are important
toO. 29 ,30

11.4 Major surfactant types used in metalworking fluids

Surfactants are primarily used in metalworking fluids as functional

components of the emulsifier system of mainly water-based metalworking
fluidsY-33 Secondary functions of surfactants in metalworking fluids
include corrosion inhibiting and lubricating properties. Major types of
surfactant used in the various ranges of metalworking fluid types are
described here. 34-36

11.4.1 Anionics

11.4.1.1 Petroleum sulfonates. Petroleum sulfonates can be derived

from either a natural or synthetic source. There are three types of oil-
soluble sodium petroleum sulfonates based on molecular weight differ-
ences, i.e. low (420-440), medium (450-470), and high (490-510). These
surfactants are typically used as primary emulsifiers and secondary
corrosion inhibitors in water-based metalworking fluids, especially in
soluble and semi-synthetic metalworking fluid types. Petroleum sulfonates
like the calcium petroleum sulfonates can also provide secondary oil
soluble corrosion inhibitory functions by utilizing a film formation
mechanism on metal surfaces. These are inexpensive surfactants but are
typically dark in color and may contain unsulfonated hydrocarbons.
 PRESERVATION OF METALWORKING FLUIDS 289

11.4.1.2 Metal carboxylate soaps. These are the sodium and potassium
soaps derived from long chain fatty acids that include mixed tallow,
coconut oil, tall oil or palm oil fatty acids and purified lauric fatty acids.
These surfactants are effective in stabilizing oil-to-water metalworking
fluid emulsions. In addition, these surfactants are stable in high pH environ-
ment of pH 10 or higher, relatively inexpensive and are used as emulsifying
components in emulsifier packages for water-based metalworking fluids.
Amine carboxylates such as fatty acid soaps of triethanolamine or
morpholine are used in a milder alkaline environment.

11.4.1.3 Alkylsulfamido and arylsulfamido carboxylic acids. These

surfactants are soluble in water as salts and are used as emulsifiers or
corrosion inhibitors in metalworking fluids systems involving water-based
metalworking fluid types like the semi-synthetics and synthetics.

11.4.1.4 Phosphate esters. These surfactants are complex phosphoryl-

ated molecules derived from phosphated ethoxylated phenols and long
chain alcohols and may vary from free acid form to amine or alkali metals.
The aromatic phosphate esters provide corrosion inhibitory, strong EP,
good wetting and lubricating properties for synthetic metalworking fluids.
The aliphatic phosphate esters also provide corrosion inhibitory, strong
EP, good lubricating, plus emulsifying properties for water-based metal-
working fluid formulations.

11.4.1.5 Sulfated triglyceride oils. These surfactants are derived from

the sulfation of hydroxy group and/or the fatty acid portion of the
triglyceride. Examples are sulfated castor oil, sulfated soya oil, sulfated
oleic acid and sulfonated tall oil fatty acid. Used primarily as lubricants and
emulsifiers in metalworking fluids.

11.4.2 Nonionics

11.4.2.1 Alkanolamides. The 2:1 amides tend to be more water soluble

and are used to a greater extent in metalworking fluids than the 1:1 amides.
The 2:1 amides are prepared from the reaction of 2 mol of alkanolamine
(diethanolamine, monoethanolamine or monisopropanolamine) with
1 mol of free fatty acid. They contain an excess amine content of 25 to 30%
with a lesser amide content of 60 to 70% than the 1:1 amides, and are
usually neutralized with fatty acids to make amine soaps which can
function as coemulsifiers with the amide component. An example of an
alkanolamide used in metalworking fluids is the 2:1 diethanolamine
(DEA) tall oil fatty acid alkanolamide used as the emulsifier, lubricant and
corrosion inhibitor in soluble, synthetic and semi-synthetic metalworking
290 PRESERVATION OF SURFACTANT FORMULATIONS

fluids. The 2:1 DEA lard oil soaped alkanolamide is another alkanolamide
used as a secondary emulsifier with good lubricating properties in
water-based metalworking fluid types. Other alkanolamines like
2-amino-2-methyl-propanol (AMP) have been considered as secondary
ethanolamine replacements in light of potential health concerns about
n-nitrosodiethanolamine contamination in metalworking fluids. 37

11.4.2.2 Alcohol, alkyl phenol, natural oil ethoxylates. Ethoxylated

alcohol may be coconut oil-derived, tallow-derived or synthetic linear
chain alcohol. These surfactants are used as secondary or coemulsifiers in
metalworking fluids, e.g. ethoxylated C 12-C I5 primary alcohols with a
typical HLB (hydrophilic-lipophilic balance) value equal to 7.S. Ethoxyl-
ated fatty alcohols are also used as mineral oil emulsifiers in soluble and
semi-synthetic metalworking fluids. The ethoxylated alkyl phenol molecule
contains the hydrophilic ethoxylate chains and the hydrophobic alkyl
phenol portions. These surfactants are used as secondary or co emulsifiers
in soluble or emulsifiable oils. Examples of these surfactants are the octyl
(C8 Hn) phenol ethoxylates and the nonyl (C9 H I9 ) phenol ethoxylates.
Ethoxylated natural oil derivatives such as the ethoxylated castor oil are
the emulsifiers for fats and soaps used in the compounding of water-soluble
metalworking fluids.

11.4.2.3 EO/PO block copolymers/polyglycols. EO/PO (ethylene

oxide/propylene oxide) block copolymers offer a wider range of products
by varying the length of both the hydrophobic (polyoxypropylene) and
hydrophilic (polyoxyethylene) portions of the molecule for specific
applications. An example is an EO/PO block copolymer with HLB values
between 7 and 12, which is used as the emulsifying and lubricating
component in water-based metalworking fluids. Molecular transposition of
the previously mentioned EO/PO block copolymer structure will result in
an example of a reverse EO/PO block copolymer with HLB values
between 2 and 7, used to lubricate oil-soluble metalworking fluids.
Tetrafunctional EO/PO block copolymers are derived from the addition of
ethylene oxide and propylene oxide to ethylenediamine. These EO/PO
block copolymers provides lubricating properties for water-based metal-
working fluids. EO/PO esters can also be prepared from their corresponding
methyl esters and EO/PO glycols for use as emulsifiers and lubricants in
semi-synthetic fluids. The more hydrophobic EO/PO esters have defoam-
ing properties and combine with alkanolamides for additional corrosion
protection properties. 38
Polyglycols are used primarily as the lubricant component in water-
based metalworking fluids. An example is an unmodified random
polyalkylene glycol which contributes inverse solubility and hydrodynamic
lubricity properties to the metalworking fluids. This surfactant is typically
 PRESERVATION OF METALWORKING FLUIDS 291

used in combination with fatty acids or phosphate esters in metalworking

fluids to provide good lubrication in ferrous and non-ferrous metalwork-
ing operations. Another example is a modified triethanolamine acid
polyalkylene glycol mixture which is used as the sole lubricant base for
water-based metalworking fluids. This surfactant is used to provide
extreme pressure lubrication for heavy duty machining operations.

11.4.2.4 Long chain carboxylic acid esters. These surfactants are the
sorbitan esters which are typically sorbitan fatty acid esters such as sorbitan
monooleate or the ethoxylated derivative of polyoxyethylene sorbitan(5)-
monooleate, and the glycerol esters such as glycerol monooleate. These
two surfactant groups are primarily used as coemulsifiers in soluble
metalworking fluids. The polyfunctional alcohol or polyol esters are used
for compounding synthetic metalworking fluids and are the boundary
lubricant component of the fluids. Polyethylene glycol or PEG esters are a
wide range of surfactants with different HLB values for selective
applications. They are used primarily as the emulsifiers in synthetic
metalworking fluids and in some cases as the lubricant component of the
fluids.

11.4.3 Cationics and amphoterics

These surfactant types are normally not used in metalworking fluid
formulations. Factors that prevent the utilization of these surfactants to a
greater extent than either the anionic or non ionic surfactants are mainly
cost and incompatibility with typical anionics found in most metalworking
fluid formulations.
Cationic surfactants used in metalworking fluids are the imidazolines
surfactants. Examples of these surfactants are the oleyl, coco and soya
imidazolines derived from oleic acids, coco and soya fatty acids, respect-
ively. Imidazolines are primarily used as oil-soluble emulsifiers and
corrosion inhibitors in straight or neat oil metalworking fluid formulations.
Amphoteric surfactants used in metalworking fluid are the amphoteric
carboxylate salts like the tall amphopropionate derived from tall oil fatty
acid imidazolines. The amphoteric surfactants are usually sodium chloride
free and primarily used as oil emulsifiers in soluble oils.

11.4.4 Bioresistant surfactants

The surfactant components in metalworking fluid formulations are
susceptible to microbial degradation during in-use metalworking fluid
operations. 5,38 Degradation of metalworking fluid surfactants or emulsifiers
by microbial action has been reported for petroleum sulfonates,9 as well as
for oil-soluble nonionic ethoxylated alcohols or water-dispersable nonionic
292 PRESERVATION OF SURFACTANT FORMULATIONS

ethoxylated esters? Metalworking fluid formulators have started to

investigate surfactants that are more resistant to biodegradation,39,40 but
are constrained by biodegradability requirements for proper waste disposal
of spent metalworking fluids.

11.5 Types of microorganism in metalworking fluids

The primary source of microbial contamination of a fresh coolant is the

residual biomass left behind from the previous charge. Although cleaning
the system between charges is a good practice, it may not necessarily
remove all the microbial biomass in the system. 3,4,41 Other sources of
microbial contamination are from the metalworking fluid concentrate
itself, the water used to dilute the metalworking fluid concentrates for use,
dirt entering the plant system from the air, plant workers' clothing or from
the workers themselves through poor personal hygiene, general poor plant
hygiene, metal stocks and hydraulic oilleakage. 4,25,42,43 In order for the
microorganisms to survive in the metalworking fluid system, nutrient
requirements for microorganisms are often obtained from the various
components present in the metalworking fluids formulation itself. 38,44--46
These nutrients can be organic in nature such as mineral oil base stocks,
fatty acids soaps, fatty oils, synthetic esters, phosphate esters, glycols,
amines or inorganic mineral salts. Three common components of metal-
working fluids such as naphthenic petroleum-based mineral oil, petroleum
sulfonates and fatty acids (linoleic and oleic acids) were evaluated as
sources of carbon for microbial growth. The study indicated that the fatty
acids component of metalworking fluids was the primary source of carbon
for bacterial growth. 47
Other primary factors that contribute to the growth and proliferation of
microorganisms in the metalworking fluid system are microbial growth
conditions of pH between 9.2 and 9.5, optimal bulk temperatures of
metalworking fluids in the system for microbial growth and oxygen
availability for certain microorganisms. Secondary factors that influence
microbial growth in metalworking fluid systems include the effects of iron
fines,48 oil-to-water rati0 49 and water hardness. 50
There are usually three types of microorganisms associated with
metalworking fluids deterioration. They are the aerobic bacteria, anaerobic
bacteria and fungi (mold and yeast). The presence of biofilm has also been
reported in water-based metalworking fluids which is different from the
typical planktonic or free-floating microorganisms associated with an
aqueous system. 51 The biofilm present in a metalworking system is made up
of bacteria and/or fungi species and may account for residual biomass that
recontaminates fresh additions of metalworking fluid into the operating
system. Biofilm presence in the metalworking fluid system may also be
 PRESERVATION OF METALWORKING FLUIDS 293

responsible for the regrowth of the microbial population following

preservative treatment for microbial control of the system. 52

11.5.1 Aerobic bacteria

These microorganisms include the commonly found Pseudomonas species
which are the prevalent spoilage agents of cutting fluids. 4,38,53--57 Other
aerobic bacteria found in metalworking fluids are the Enterobacter species,
Klebsiella species, Escherichia species and Proteus species. 4 The Acineto-
bacter bacterial species have also been indicated as a primary bacterial
colonizer in metalworking fluids. Although not the dominant species, its
presence may maintain the other bacterial species in contaminated
metalworking fluids. 47 These microorganisms are highly oxidative and
adapt very well to a wide variety of metalworking fluids. Their growth and
proliferation lead to emulsion destabilization, loss of metalworking fluids
lubricating properties and increase in corrosive properties of metalworking
fluids used in the plant system. A microbial ecological study of a
metalworking fluid system has indicated that the levels of fungi and
anaerobic or sulfate-reducing bacteria are quite dependent on the initial
levels of aerobic bacteria. 58

11.5.2 Anaerobic bacteria

These microorganisms are essentially sulfate reducers such as Desulfovibrio
desulfuricans which reduce sulfate to sulfide. 59 Anaerobic conditions can
exist in stagnant areas within the metalworking fluid system for anaerobic
bacterial growth. The mechanism of anaerobic growth stems from an
interrelationship with the aerobic bacteria,6o,61 where the aerobic bacteria
lower the redox potential of the metalworking fluids and synthesize
nutrients for themselves. The presence of iron swarf or metal fines may
contribute to the increase in sulfate reducing bacteria growth. 48,62
Anaerobic bacteria which generate sulfide by the reduction of sulfur in
petroleum sulfonates, organic polysulfides and sulfurized oiVfats instead of
sulfate, are better known as sulfide-generating bacteria. 45 Growth of
sulfate-reducing and/or sulfate-generating bacteria usually causes metal-
working fluids to blacken or grey, offensive odor to form and corrosion to
metalworking tools and the workplace.

11.5.3 Fungi
These microorganisms grow well in synthetic metalworking fluids and have
been implicated in the spoilage of metalworking fluids. 44 ,63---65 Most
commonly found fungi in metalworking fluids include the Fusarium
species, Cephalosporium species and Candida species. Fungi can grow
294 PRESERVATION OF SURFACTANT FORMULATIONS

well when the preservatives added to metalworking fluids are more

active against bacteria and would then clear the way for the growth of
fungi. 51 ,66--68 Fungi found in metalworking fluid systems are mainly slime
formers and can physically plug system pipelines and thereby disrupt the
flow of metalworking fluids. Fungi can be found free living in the water
phase and at the same time be found metabolizing the ingredients that
migrate from the oily phase of metalworking fluids. Due to susceptibility
of metalworking fluids to fungal contamination and uncontrolled fungal
growth, metalworking fluids are commonly treated with preservatives
active against fungal contamination.

11.6 Types of preservative used in metalworking fluids

Metalworking preservatives are used to prevent the biodeterioration of the

metalworking fluid functions and extend the life of the metalworking fluids
used in metalworking operations. 4,69,7o Metalworking fluid preservatives
are incorporated as a functional component of metalworking fluid
concentrates by metalworking fluid formulators, or added by metalwork-
ing fluids end users as regular tankside additions to the diluted metalworking
fluids.
Factors to keep in mind when selecting a preservative for a specific
metalworking fluid application are toxicology profile, environmental
friendliness, biocidal effectiveness, chemical compatibility and physical
stability.
A favorable and extensive toxicity profile is a must. Preservative users
should be informed of potential health hazards associated with the
preservative for safe handling, storage and use of the product. 41 ,70,71
To prevent contamination of the environment, safe disposal procedures
must be obtained from the preservative manufacturer. This information
should be made available to the preservative users and in compliance with
all relevant disposal laws and regulations to prevent contamination of the
environment.
Preservative biocidal effectiveness should be broad spectrum against a
wide variety of bacteria and fungi commonly found in biodeteriorated or
spoilt metalworking fluids at recommended end-use dosages. The biocidal
effectiveness of preservatives used in a metalworking fluid system may be
impacted by the presence of biofilm52 ,72 and microbial resistance to the
preservative treatments. 73-75
The preservative should also be chemically compatible with the other
components of the metalworking fluid concentrates,16 or with other
constituents or byproducts present in the in-use metalwork fluid system,
such as filtering agents,77 metals78 and hydraulic fluids. 79
Physical stability of the preservative should include the pH stability in an
 PRESERVATION OF METALWORKING FLUIDS 295

alkaline environment of pH 8.5 to 9.5,80 thermal stability,81 corrosivity

effects on worked metals82 and oil-in-water solubility where the relative
volume of oil-to-water influences the equilibrium concentration of the
preservative in the aqueous phase during end-use diluted metalworking
applications. 49
The Federal Insecticide, Fungicide and Rodenticide Act (FIFRA)
requires the United States Environment Protection Agency to regulate
legally the pesticide registration of a stated application use of a pre-
servative in the United States including preservatives used in metalworking
fluids applications. 83 Registration of metalworking fluid preservative use
may also apply to other countries where pesticide registration is also
required by law. 84. Although there are a number of metalworking fluid
preservatives presently registered for use in metalworking fluid applica-
tions, the bulk of the metalworking fluid control is accomplished through a
limited number of preservative manufacturers. The various chemical types
used as metalworking fluid preservatives include the formaldehyde
releasers, e.g. triazines and hexamines,66,67,85,86 phenolic derivatives,87
thiazoles,88-90 alkane derivatives, 91,92 morpholines,93 glutaraldehyde94 ,95
and others. 96
A sampling of metalworking fluid preservatives registered for use in the
United States is described below based on manufacturer, trade name,
active ingredient/s and applications.

11.6.1 ANGUS Chemical Company

11.6.1.1 Bioban® CS-1J35 (EPA Reg. No.48301-8). Contains 4,4-

dimethyloxazolidine as 74% active ingredient (AI) and 3,4,4-trimethyl-
oxazolidine as 2.5% AI. This water-based preservative is used in soluble
oils, semi-synthetics and synthetic metalworking fluid concentrates and
tankside additions. Recommended end-use level in final diluted metal-
working fluid is 0.10% w/w of product.

11.6.1.2 Bioban® N-95 (EPA Reg. No. 1100-82-4830l). Contains 5-

hydroxymethoxymethylaza-3,7-dioxabicyclo(3.3.0)octane as 24.5% AI, 5-
hydroxymethyl-l-aza-3,7-dioxabicyclo(3.3.0)octane as 7.7% AI and 5-
hydroxypoly[methyleneoxy(74%C-2, 21 %C-3, 4%C-4, 1%C-5)]methyl-l-
aza-3,7-dioxabiocyclo(3.3.0)octane as 7.8% AI. Recommended end-use
level in final diluted metalworking fluid is 0.1 to 0.3% w/w of the product.

11.6.1.3 Bioban® P-1487 (EPA Reg. No.48301-7). Contains 4-(2-nitro-

butyl)morpholine as 70% AI and 4,4'-(2-ethyl-2-nitrotrimethylene)-
dimorpholine as 20% AI. This water-based preservative is used in soluble
oils, semi-synthetics and synthetic metalworking fluid concentrates and
296 PRESERVATION OF SURFACTANT FORMULATIONS

tankside additions. Recommended end-use level in final diluted metal-

working fluids is an initial dose of 0.1 % active ingredient followed by a
maintenance dose of 0.01 % w/w active ingredient on a weekly basis.

11.6.1.4 Tris Nitro® (EPA Reg. No.48301-11). Contains tris(hydroxy-

methyl)nitromethane as 50% AI. Used mainly with metalworking fluids as
tankside additions. Recommended end-use level in final diluted metal-
working fluids is 0.05% w/w of the product.

11.6.2 Buckman Laboratories, Inc.

11.6.2.1 Busan® 30WB (EPA Reg.No.1448-55). Contains 2-(thiocyano-

methylthio )benzothiazole as 30% AI. This is a water-based preservative
used in water-diluted soluble oils, semi-synthetic and synthetic metal-
working fluids especially as tankside additions. Recommended end-use
level in final diluted metalworking fluid is 0.01 % w/w of product.

11.6.2.2 Busan® 77 (EPA Reg. No. 1448-242). Contains poly[oxyethyl-

ene(dimethyliminio )ethylene( dimethylimino )ethylene dichloride] as 60%
AI. This preservative is used in water-based synthetic and oil-based
metalworking fluids with emulsified nonionic surfactants. However, it is
not recommended for use in straight oils or metalworking fluids that
contain anionic surfactants in their formulation. Recommended end-use
level in final diluted metalworking fluids is 0.01 to 0.1 % w/w of product.

11.6.2.3 Busan® 1030 (EPA Reg. No. 1448-55). Contains 2-(thiocyano-

methylthio )benzothiazole as 30% AI. This is a solvent-based preservative
used in soluble oils, semi-synthetic and synthetic metalworking fluid
concentrates. Recommended end-use level in final diluted metalworking
fluids is 0.005 to 0.01 % w/w of product.

11.6.2.4 Busan® 1060 (EPA Reg. No. 1448-243). Contains hexahydro-

1,3,5-tris(2-hydroxyethyl)-s-triazine as 78.5% AI. This preservative is
stable and water soluble. Used in soluble oils, semi-synthetic and
synthetic metalworking fluid concentrates and tankside additions.
Recommended end-use level in final diluted metalworking fluid is 0.10 to
0.15% w/w of product.

11.6.3 The Dow Chemical Company

11.6.3.1 Dowicide® A (EPA Reg. No.464--78). Contains sodium salt of

O-phenylphenol as 97% AI. This water-soluble preservative is used in
soluble oil, semi-synthetic and synthetic metalworking fluid concentrates
 PRESERVATION OF METALWORKING FLUIDS 297

as well as tankside additions. Recommended end-use level in final diluted

metalworking fluid is O.OOS to 0.2% w/w of product.

11.6.3.2 Dowicide® 1 (EPA Reg. No.464-70). Contains O-phenyl-

phenol as 99% AI. This is mainly an oil-soluble preservative used in
soluble oils, semi-synthetic and synthetic metalworking fluid concentrates
as well as tankside additions. Recommended end-use level in final diluted
metalworking fluid is O.OS to O.lS% w/w of product.

11.6.3.3 Dowicil® 75 (EPA Reg.No.46~03). Contains 1-(3-chloro-

allyl)-3,S,7-tria-1-azoniaadamantane chloride as 67.S% AI combined with
an inert sodium bicarbonate stabilizer. Used in water-diluted soluble oils,
semi-synthetic and synthetic metalworking fluids especially as tankside
additions. Recommended end-use level in final diluted metalworking fluid
is O.OlS to 0.2S% w/w of product.

11.6.4 Olin Corporation

11.6.4.1 Triadine® 3 (EPA Reg. No. 1258-1071). Contains hexahydro-

1,3,S-tris(2-hydroxyethyl)-s-triazine as 78.S% AI. This preservative is
stable and water soluble. Used in soluble oil, semi-synthetic and synthetic
metalworking fluid concentrates and tankside additions. Recommended
end-use level in final diluted metalworking fluid is 0.10 to O.lS% w/w of
product.

11.6.4.2 Triadine® 10 (EPA Reg. No. 1258-990). Preservative combina-

tion of hexahydro-1,3,S-tris(2-hydroxyethyl)-s-triazine as 63.6% AI and
sodium 2-pyridinethiol-1-oxide as 6.4% AI. This preservative is used in
soluble oil, semi-synthetic and synthetic metalworking fluid concentrates
and tanks ide additions. Maximum end-use level in final diluted metal-
working fluid is 0.2% w/w of product. Typical end-use level in diluted
metalworking fluid is 0.07% w/w in synthetic fluids or 0.1 % w/w in oil-
containing fluids.

11.6.4.3 Triadine® 20 (EPA Reg. No. 1258-1205). Preservative com-

bination of hexahydro-1,3 ,S-tris(2-hydroxyethyl)-s-triazine as 71.4% AI
and sodium 2-pyridinethiol-1-oxide as 3.64% AI. This preservative is used
in soluble oil, semi-synthetic and synthetic metalworking fluid concentrates
and tankside additions. Recommended end-use level in final diluted
metalworking fluid is O.lS to 0.20% w/w of product.

11.6.4.4 Sodium Omadine® 10% aqueous solution (EPA Reg.No. 1258-

1213). Contains sodium 2-pyridinethiol-1-oxide as 10% AI. This pre-
servative is used in soluble oil, semi-synthetic and synthetic metalworking
298 PRESERVATION OF SURFACTANT FORMULATIONS

fluid concentrates and tankside additions. Its lower use-concentration

allows for easier tankside additions of smaller sumps. Maximum end-use
level in final diluted metalworking fluid is 0.5% w/w of product.

11.6.4.5 Sodium Omadine® 40% aqueous solution (EPA Reg. No. 1258-
843). Contains sodium 2-pyridinethiol-1-oxide as 40% AI. This pre-
servative is used in soluble oil, semi-synthetic and synthetic metalworking
fluid concentrated and tankside additions. Maximum end-use level in final
diluted metalworking fluid is 0.125% w/w of product. Recommended end-
use level in final diluted metalworking fluid is 0.016% to 0.025% w/w of
product.

11.6.5 Rohm and Haas Company

11.6.5.1 Kathon® 886MW (EPA Reg.No.707-129). Contains 5-chloro-

2-methyl-4-isothiazolin-3-one as 8.6% AI and 2-methyl-4-isothiazolin-3-
one as 2.6% AI. Used in soluble oil, semi-synthetic and synthetic
metalworking fluid concentrates and diluted tankside additions. Recom-
mended end-use level in final diluted metalworking fluids is an initial dose
of 10 to 21.8 ppm active ingredient followed by a maintenance dose of
5 ppm active ingredient every 4 weeks or 5-21.8 ppm active ingredient
every 8-12 weeks.

11.6.5.2 Kathon® 893MW (EPA Reg.No.707-195). Contains 2-n-octyl-

4-isothiazolin-3-one as 45% AI. Used in soluble oil, semi-synthetic and
synthetic metalworking fluid concentrates and diluted tankside additions.
Recommended end-use level in final diluted metalworking fluid is an initial
dose of 25-75 ppm active ingredient followed by a maintenance dose of 5-
30 ppm active ingredient every four weeks.

11.6.5.3 Kathon® MWC (EPA Reg.No.707-220). Contains 5-chloro-2-

methyl-4-isothiazolin-3-one as 2.69% AI and 2-methyl-4-isothiazolin-3-one
as 0.8% AI. Used in soluble oil, semi-synthetic and synthetic metalworking
fluid concentrates and diluted tankside additions. Recommended end-use
level in final diluted metalworking fluids is an initial dose of 4.9-20.5 ppm
active ingredient followed by a maintenance dose 4.9-20.5 ppm active
ingredient every 1 to 3 weeks.

11.6.6 R. T. Vanderbilt Company, Inc.

11.6.6.1 Vancide® TH (EPA Reg. No. 1965-55). Contains hexahydro-

1,3,5-triethyl-s-triazine as 95% AI. This preservative is stable as well as
water soluble and oil soluble. Used in soluble oil, semi-synthetic and
 PRESERVATION OF METALWORKING FLUIDS 299

synthetic metalworking fluid concentrates and diluted tankside additions.

Recommended end-use level in final diluted metalworking fluid is 0.04 to
0.15% of product.

11.6.6.2 Vancide® 51 (EPA Reg.No.1965--8). Preservative combination

of sodium dimethyldithiocarbamate as 27.6% AI and sodium 2-mercapto-
benzothiazole as 2.4% AI. This preservative is used in water-diluted
soluble oils, semi-synthetic and synthetic metalworking fluids especially
tankside additions. Recommended end-use level in final diluted metal-
working fluid is 0.5 to 0.1 % w/w of the product.

11.6.7 Stepan Company

11.6.7.1 Onyxide® 200 (EPA Reg. No. 1839-99). Contains hexahydro-

1,3,5-tris(2-hydroxyethyl)-s-triazine as 78.5% AI. This preservative is
stable and water soluble. Used in soluble oil, semi-synthetic and synthetic
metalworking fluid concentrates and tankside additions. Recommended
end-use level in final diluted metalworking fluid is 0.10 to 0.15% w/wof
product.

11.6.8 Union Carbide Corporation

11.6.8.1 UCONE)(® Antimicrobial 345 (EPA Reg. No. 10352-28).

Aqueous solution of glutaraldehyde as 45% AI. Used in water-diluted
soluble oil, semi-synthetic and synthetic metalworking fluids especially
tanks ide additions. Recommended end-use level in final diluted metal-
working fluid is an initial dose of 220 to 670 ppm followed by maintenance
dose of 90 to 440 ppm (daily to weekly) of the product.

11.6.9 US Professional Laboratories (L & F Products Group)

11.6.9.1 Grotan® (EPA Reg. No. 365-76). Contains hexahydro-1,3,5-

tris(2-hydroxethyl)-s-triazine as 78.5% AI. This preservative is stable and
water soluble. Used in soluble oil, semi-synthetic and synthetic metalwork-
ing fluid concentrates and tankside additions. Recommended end-use level
in final diluted metalworking fluid is 0.10 to 0.15% of product.

11.6.10 ZENECA Biocides

11.6.10.1 Proxel® MW200 Preservative (EPA Reg. No. 10182-30). Con-

tains a solution of 1,2-benzoisothiazolin-3-one as 17% AI. This preserv-
ative is stable and water soluble. Used in soluble oil, semi-synthetic and
300 PRESERVATION OF SURFACTANT FORMULATIONS

synthetic metalworking fluid concentrates and tankside additions. Recom-

mended end-use level of the product in the final diluted metalworking fluid
is 0.025 to 0.100% w/w for soluble oils, 0.050 to 0.125% w/w for semi-
synthetics and 0.075 to 0.150% w/w for synthetics.

11.6.10.2 Proxe[® MW300 Preservative (EPA Reg.No.10182-l). Contains

an aqueous solution of 1,2-benzoisothiazolin-3-one as 30% AI. This
preservative is stable and water soluble. Used in soluble oil, semi-synthetic
and synthetic metalworking fluid concentrates and tankside additions.
Recommended end-use level of the product in the final diluted metal-
working fluid is 0.02 to 0.07% w/w for soluble oils, 0.03 to 0.08% w/w for
semi-synthetics and 0.05 to 0.10% w/w for synthetics.

11.6.11 Potentiation of preservatives

Potentiation of the biocidal effectiveness of metalworking preservatives
was reported with the use of corrosion inhibitors such as alkanol-
amines,97-100 metal chelating agents such as ethylenediaminetetraacetic
acid (EDTA),101-103 copper citrate lO4-106 and even in combination with
other preservatives. 89
However, there is no universally potent preservative; each user has to
evaluate and select the ideal preservative in terms of whether it works or
not in a formulated concentrate or in-use diluted metalworking fluid
operation, with the help of standard testing methods.

11.7 Testing protocols used in metalworking fluid preservative selection

Many test protocols have been developed to evaluate and select various
types of preservative for use in metalworking fluid systems. 107- 109 There
are usually two testing phases involved in the selection of the right
preservative for a particular metalworking fluid plant system. The first
phase involves preliminary laboratory screening of the test preservative for
efficacy to control microbial growth in a laboratory simulated metalwork-
ing plant system. The second phase is the confirmatory field testing of the
selected preservative for efficacy to control microbial growth in an actual
metalworking plant operation. In some instances, both phases can be done
in parallel to each other in order to select the right preservative for
microbial growth control for a metalworking fluid system.
To facilitate the standardization of preservative testing and selection, the
microbial inoculum used for the testing has to be predetermined. The
microbial inoculum selection can either be obtained by isolating the
primary microorganisms indigenous to the contaminated metalworking
fluid system of interest, 110,111 or be a defined microbial inoculum
 PRESERVATION OF METALWORKING FLUIDS 301

developed specifically for metalworking fluid preservative testing. 112-114

The defined microbial inoculum can survive and grow in a large number of
metalworking fluids and usually consists of three bacterial species of
Pseudomonas aeruginosa (dominant metalworking fluid biodeteriorant),
Proteus mirabilis (produces strong fecal odor), Klebsiella pneumoniae
(slime former), including two fungal species of Fusarium and Cephalo-
sporium. The microbial count of the bacterial inoculum is approximately
109 colony forming units or cfu ml -1 for each bacterial species and
approximately 105 cfu ml-1 for each fungal species. Other factors to be
considered in determining whether to adopt a particular test method are
the general acceptance of that particular test method by metalworking fluid
end users, the ability to simulate metalworking plant operation use,
reliability and statistical reproducibility, ease of conduct, and possibility of
completion within a reasonable amount of time.

11.7.1 Laboratory testing of metalworking fluid preservatives

A number of laboratory tests for evaluating and selecting metalworking
fluid preservatives have been previously covered. 109 These include the
recirculating test methods that have aerobic/anaerobic cycles, repeated
microbial inoculations of the test systems, aeration and circulation of
metalworking fluids through iron chips,108,115-117 and the aerated bottle
test methods,4,112 upon which the current American Society for Testing
and Material (ASTM) testing protocols for preservative selection for use
with water-based metalworking fluids were based. Steps were originally
initiated by ASTM to develop these standard test protocols for anti-
microbial agents which included testing of metalworking fluid preserv-
atives. 118 This resulted in the development of the two-week bioresistance
test (ASTM# D 396-92) and the six-week bioresistance test (ASTM# E
686-91) standard methods for testing.
The two-week bioresistance test is a standard test method for evaluating
the microbial resistance of water-based metalworking fluids. 119 The level
of bioresistance of the water-based metalworking fluids is evaluated by
microbiologically challenging the fluids with an inoculum prepared from
actual biodeteriorated metalworking fluid obtained from the end-user's
site. This method allows the end users to determine whether a particular
metalworking fluid is susceptible or not to microbial spoilage, and if so, to
select a preservative for microbial control.
The six-week bioresistance test is a standard test method for evaluating
the selecting preservatives for use in water-based metalworking fluids. 120
The efficacy of any antibacterial and antifungal preservatives at use
concentrations are evaluated in controlling the microorganisms responsible
for the biodeterioration of metalworking fluids. This test method can also
be used to evaluate the efficacy of preservatives which are directly
302 PRESERVATION OF SURFACTANT FORMULATIONS

formulated into the metalworking fluid concentrates. The test runs for six
weeks with multiple microbial challenge either isolated from the spoilt
metalworking fluid or a defined inoculum. The metalworking fluids are
then evaluated for physical changes and pH changes, including bacterial
and fungal microbial counts. The metalworking fluids either pass or fail the
preservative selection test based on these criteria.

11.7.2 Field testing of metalworking preservatives

Field testing allows for metalworking fluid users to assess the actual
interactions of prior microbial growth and its bypro ducts with the actual
preservative used in a metalworking plant operation. 121 Efforts were
attempted in the past to correlate laboratory testing results with actual field
evaluation testing of the selected preservative of choice for microbial
control in the plant system being evaluated.122-I24 The field test confirms
the efficacy of the selected preservative from the laboratory screen to
protect the metalworking fluid system against uncontrolled microbial
contamination and eventual spoilage of the metalworking fluids. Once a
preservative has been selected to treat successfully a particular metal-
working fluid system, it is important for the end users to know when to add
more preservative for continuous control of microbial contamination. This
can be accomplished by routine monitoring and record keeping of
microbial levels in the metalworking system using specific microbial assays
for determining microbial growth and proliferation levels in metalworking
fluids.

11.7.3 Monitoring for microbial contamination in metalworking fluid

systems
Early microbial assays for monitoring the microbial growth and prolifera-
tion levels of metalworking fluid systems included the use of the Warburg
apparatus, 'red-spot' test, methylene blue reduction test and the 'corn
meal' test methods. 107 The Warburg apparatus was based on the
measurement of oxygen consumption of the actively growing micro-
organisms in metalworking fluids?8 The red-spot test is based on the
ability of actively growing microorganisms to reduce a tetrazolium salt to
form a red colored formazan. 125 The methylene blue test is also based on
dye reduction chemistry where methylene blue is reduced by the actively
growing microorganisms in the metalworking fluids to a colorless leuco
base. 115 ,126 The corn meal test measures the microbiological stability of
metalworking fluids for at least 21 days without going 'sour'. This prepared
metalworking fluid mixture would be evaluated at periodic intervals for
slime formation, stability and odor production. l27 These early microbial
assays did not provide enough information concerning the identity or types
 PRESERVATION OF METALWORKING FLUIDS 303

of spoilage microorganisms present in the contaminated metalworking

system and the microbial counts obtained as the 'actual' number of
microorganisms were not accurate or consistent, and were marginally
quantitative. 107
More practical microbial assays were developed to determine the level of
microbial load in the metalworking fluid system. These were based on the
enumeration of microorganisms by viability testing, microbial metabolic
activity measurements and microbial cell components measurements. 128
Viability assay is based on the enumeration of microbial numbers
present in the metalworking fluid system. This can be done through
standard plate counts or by five tube dilution-to-extinction microbial
assays.4,110 The 'dip stick' or 'dip slide' method is a simplified plate count
method developed for the on-site estimation of specific microorganisms
found in the metalworking fluid system. 108 ,129,130 The microbial assay
consists of a plastic paddle or filter paper coated with an appropriate
microbial recovery medium and/or indicator, dipped into the metalworking
fluid sample, returned to its container and incubated appropriately. The
microorganisms left behind on the dip slide can grow and produce visible
colonies. Instead of counting microbial numbers in cfu ml-1 , the observer
compares the results obtained to a standard colonies density chart (ranging
in order of magnitude from 103 to 107 cfu ml- 1 for total aerobic bacterial
numbers). The results obtained are then measured according to the
corresponding order of magnitude of colonies density in cfu ml-1.
An increase of more than two orders of magnitude, i.e. from 104 to
106 cfu ml- 1 , indicates that microbial growth is out of control and the
metalworking fluid system requires appropriate additions of preservative
for control. This method can provide specific microbial isolates for
inoculum preparation and is relatively easy to do but limited to time
requirements of 24 h or one week for results to develop and for safe
disposal considerations. These microbial assays are available to detect total
aerobic bacteria and fungi or combinations of both bacteria/fungi. The
detection of sulfate-reducing bacteria utilizes specially prepared media
tubes. This involves a reaction by the sulfate-reducing bacteria with iron
present in the tube to form a visible black iron sulfide precipitate. l3l
Metabolic activity assay measures the overall metabolic activity of the
microorganisms present in the metalworking fluid system. A modified
biochemical oxygen demand (BOD) assay specifically measures the
metabolic rate at which the microbial population is consuming oxygen
within the metalworking fluid system. 128 Loss of more than half of the
dissolved oxygen in a test sample left standing for four hours indicates
significant microbial contamination. The microbial assay is relatively rapid
and measures the overall impact of the microbial population in the system
but it requires more technical skill and purchase of expensive equipment.
Microbial cell component assays involve the selective measurement of
304 PRESERVATION OF SURFACTANT FORMULATIONS

specific constituents present in microbial cells growing in the metalworking

fluid system. Specific microbial assays were developed for measuring cell
components such as adenosine triphosphate (ATP) and catalase. The ATP
microbial assay utilizes indirect measurement of A TP concentration with
the luciferase enzyme and luciferin to form A TP-Iuciferin-Iuciferase
complex that is extremely reactive and emits a photon of light. Detection
of this emitted light can be measured using a photometer and the value
obtained is proportional to the amount of A TP per unit of microbial
biomass present in the metalworking system. 132 The A TP assay has not
been successful due to the quenching effect of the enzyme reaction by
components of the metalworking fluids. The catalase microbial assay
estimates microbial loads by indirectly measuring the concentration of
catalase enzyme present in most bacteria and all fungi found in
metalworking fluids. Good correlation between standard plate counts and
catalase activity of microbial load were determined for all the three water-
based metalworking fluid types.133 Pressure detection used for measure-
ment of microbial load was also confirmed to be proportional to oxygen
evolution and microbial catalase concentration in the metalworking
system. 134 This assay requires some operator training to yield reproducible
results, purchase of expensive equipment and generation of individual
standard calibration for each metalworking system, but the rapid results
allow for real-time monitoring and control of microbial contamination in
the system.
Another microbial assay included the development of the significance
tests or sig-tests that address the Eh, pH, oxygen and nutrient gradients
found in metalworking fluid sumps using a gel system to determine the
overall contribution of the metalworking fluid microbial community to
spoilage. 45,135
Other potential microbial assays may be developed, such as using
molecular DNA or rRNA probes, to quantify specific microbial populations
like Pseudomonas aeruginosa, commonly associated with the biodeteriora-
tion of metalworking fluids.
There is no single best method for measuring and monitoring microbial
load in a metalworking fluid system. Each metalworking system will
establish its own characteristic operating profile. The best approach is to
select a microbial assay that will provide the most economical and efficient
routine microbial monitoring process for that particular metalworking fluid
system.
Monitoring microbial loads using the different microbial assays is just
one parameter for monitoring microbial contamination in metalworking
systems. Other parameters that also impact the overall control of microbial
contamination in metalworking systems include gross observations, system
operating parameters, physical analysis and chemical analysis of the in-use
metalworking fluids. 128 ,132,136,137
 PRESERVATION OF METALWORKING FLUIDS 305

11.8 Summary

If constructive efforts are made to monitor and control microbial

contamination in metalworking fluid systems, with proper know-how and
use of the selected preservative, marked improvement in the overall plant
management of metalworking fluid systems will occur, with significant
reduction in metalworking plant operation costs.

References

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2. Nachtman, E.S. and Kalpakjian, S. (1985) Lubricants and Lubrication in Metalworking
Operations, Marcel Dekker, New York.
3. Hill, E.C. (1967) The significance and control of microorganisms in rolling mill oils and
emulsions. Metals Mater., 1,294-297.
4. Bennett, E.O. (1974) The deterioration of metal cutting fluids. Progr. Indust.
Microbiol., 13, 121-149.
5. Rossmoore, H.W. (1974) Microbiological causes of cutting fluid deterioration. Soc.
Manu/act. Eng. Tech. Paper, #MR74-169.
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11. Rossmoore, H.W. and Williams, B.W. (1967) Survival of coagUlase-positive staphylo-
cocci in soluble cutting oils, Health Lab. Sci., 4, 160-165.
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oil emulsions a health hazard to workers and to the public? Tribology Internat., 9,
271-281.
13. Bennett, E.O. (1983) Water based cutting fluids and human health. Tribology Internat.,
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306 PRESERVATION OF SURFACTANT FORMULATIONS

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 PRESERVATION OF METALWORKING FLUIDS 307

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61. Isenberg, D.L. and Bennett, E.O. (1959) Bacterial deterioration of emulsion oils II.
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63. DeMare, J., Rossmoore, H.W. and Smith, T.H.F. (1972) Comparative study of triazine
biocides. Develop. Indust. Microbiol., 13,341-347.
64. Rossmoore, H.W. and Holtzman, G.H. (1974) Growth of fungi in cutting fluids.
Develop. Indust. Microbiol., 15, 273-280.
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enumeration of cutting fluid fungi. Develop. Indust. Microbiol., 16, 475-482.
66. Bennett, E.O. (1973) Formaldehyde preservatives for cutting fluids. Internat. Bio-
deterioration Bull., 9, 95-100.
67. Rossmoore, H.W., DeMare, J. and Smith, T.H.F. (1972) Anti- and pro-microbial
activity of hexahydro-l,3,5-tris-(2-hydroxyethyl)-s-triazine in cutting fluid emulsion. In
Biodeterioration of Materials Vol. 2, Walters, A.H. and Hueck-Van Der Plas, E.H.
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68. Rossmoore, H.W., Holtzman, G.H. and Kondek, L. (1976) Microbiology with a cutting
edge. In Proceedings of the Third International Biodeterioration Symposium, Sharpley,
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69. Hill, E.C., Gibbon, O. and Davies, P. (1976) Biocides for use in oil emulsions.
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preservatives. Lubrication Eng., 25, 313-320.
72. Keevil, C.W., MacKerness, C.W. and Colbourne, J.S. (1990) Biocide treatment of
biofilms. Internat. Biodeterioration, 26, 169-179.
73. Eagon, R.G. and Barnes, c.P. (1986) The mechanism of microbial resistance to
hexahydro-l,3,5,-triethyl-s-triazine. 1. Indus!. Microbiol., 1, 113-118.
308 PRESERVATION OF SURFACTANT FORMULATIONS

74. Sondossi, M., Rossmoore, H.W. and Wireman, J.W. (1986) The effect of fifteen
biocides on formaldehyde-resistant strain of Pseudomonas aeruginosa. J. Indust.
Microbiol., 1, 87-96.
75. Sondossi, M., Rossmoore, H.W. and Williams, R. (1989) Relative formaldehyde
resistance among bacterial survivors of biocide-treated metalworking fluid. Internat.
Biodeterioration, 25, 423-437.
76. Onyekwelu, LV., Bennett, E.O. and Gannon, J.E. (1981) The effective life of
preservatives in cutting fluid concentr)ltes. Tribology Internat., 14, 7-9.
77. Onyekwelu, LV. and Bennett, E.O. (1979) The effects of filtering agents upon the
activity of preservatives in cutting fluids. Internat. Biodeterioration Bull., 15, 88--95.
78. Bennett, E.O. (1982) The effects of metals upon the inhibitory activities of cutting fluid
preservatives. Internat. Biodeterioration Bull., 7-12.
79. Bennett, E.O., Oppong, D. and Lee, P. (1988) The effects of hydraulic fluids on cutting
fluid rancidity control. Soc. Manufact. Eng. Tech. Paper, #MR88--182.
80. Barman, B.N. and Preston, H.G. (1992) The effects of pH on degradation of
isothiazolone biocides. Tribology Internat., 25, 281-287.
81. Barman, B.N. (1994) Influence of temperature on the degradation of isothiazolone
biocides in aqueous media and in metalworking fluid concentrate. Lubrication Eng., 50,
351-355.
82. Heenan, D.F., Burrell, R.E., Hajscar, E.E., Lauzon, D.C. and Dowdle, W.R. (1991)
Isothiazolone microbiocide-mediated steel corrosion and its control in aluminum hot
rolling emulsions. Lubrication Eng., 47, 545-548.
83. Engler, R. (1980) Regulations pertaining to the use of industrial biocides. Develop.
Indust. Microbiol., 22, 117-122.
84. HalJeux, P. (1990) Regulatory demands for biocides today. Internat. Biodeterioration,
26,251-258.
85. Barnes, C.P. and Eagon, R.G. (1986) The mechanism of action of hexahydro-l,3,5-
triethyl-s-triazine. J. Indust. Microbiol., 1, 105-112.
86. Rossmoore, H.W. and Sondossi, M. (1988) Applications and mode of action of
formaldehyde condensate biocides. Adv. Appl. Microbiol., 33, 223--277.
87. Wheeler, H.O. and Bennett, E.O. (1956) Bacterial inhibitors for cutting oil. Appl.
Microbiol., 4, 122-126.
88. Singer, M. (1976) Laboratory procedures for assessing the potential of antimicrobial
agents as industrial biocides. Process Biochem., 11, 3()"'35.
89. Rossmoore, H.W. (1979) Heterocyclic compounds as industrial biocides. Develop.
Microbiol., 20, 41-71.
90. Collier, P.J., Ramsey, A.J., Austin, P. and Gilbert, P. (1990) Growth inhibitory and
biocidal activity of some isothiazolone biocides. J. Appl. Bacteriol., 69, 569-577.
91. Bennett, E.O. and Futch, H.N. (1960) Nitroparafin inhibitors for cutting fluids.
Lubrication Eng., 16, 228--230.
92. Bennett, E.O., Gannon, J.E. and Bennett, D.L. (1983) Inhibitory properties of 1,3-
propanediols in cutting fluids. Tribology Internat., 16, 199-202.
93. Bennett, E.O., Onyekweiu, LV., and Gannon, J.E. (1979) Corrosion inhibitors as
preservatives for metalworking fluids-morpholine compounds. Mater. Perform., 18,
40-45.
94. Leder, J. (1987) Glutaraldehyde: A new microbiocide for metalworking©. Lubrication
Eng., 43, 666--671.
95. Leder, J. and Russo, M.R. (1989) Biocide usage in metalworking fluids: The effect of
treatment patterns on efficacy. Lubrication Eng., 45, 217-220.
96. Rossmoore, H.W. (1983) Nitrogen compounds. In Disinfection, Sterilization and
Preservation, 3rd edn, Block, S.S. (ed.), Lea & Febiger, Philadelphia, pp. 271-307.
97. Bennett, E.O. (1978) The antimicrobial properties of diethylene triamines in metal-
working fluids. Internat. Biodeterioration Bull., 14, 21-29.
98. Bennett, E.O., Onyekwelu, LV., Bennett, D.L. and Gannon, J.E. (1980) Inhibitory
activities of triazole compounds in metalworking fluids. Lubrication Eng., 36, 215-218.
99. Oppong, D. and Bennett, E.O. (1989) Vse of a mixture of alcoholamines to potentiate
the antimicrobial activities of certain cutting fluid preservatives. Tribology Internat., 22,
343--346.
 PRESERVATION OF METALWORKING FLUIDS 309

100. Sandin, M., Mattsby-Baltzer, I. and Edebo, L. (1991) Control of microbial growth in
water-based metal-working fluids. Internat. Biodeterioration, 27, 61-74.
101. Izzat, LN. and Bennett, E.O. (1978) The potentiation of the antimicrobial activities of
cutting fluid preservatives by EDTA. Lubrication Eng., 35, 153-159.
102. Izzat, LN., Bennett, E.O., Gannon, J.E. and Onyekwelu, LV. (1981) Effects of EDTA
on the antimicrobial properties of mixtures of cutting fluids preservatives. Tribology
Intern at. ,14, 171-175.
103. Bennett, E.O., Gannon, J.E. and Bennett, D.L. (1982) Effects of EDTA on the
antimicrobial properties of mixtures of cutting fluids preservatives. Part II. Tribology
Internat., 15, 187-189.
104. Piet, L. and Rossmoore, H.W. (1985) A further study of the use of monocopper(lI)
citrate as an antimicrobial agent in metalworking fluid©. Lubrication Eng., 41,
103-105.
105. Riha, V.F., Sondossi, M. and Rossmoore, H.W. (1990) The potentiation of industrial
biocide activity with Cu2 +. II. Synergistic effects with 5-chloro-2-methyl-4-isothiazolin-
3-one. Internat. Biodeterioration, 26, 303-313.
106. Sondossi, M., Riha, V.F. and Rossmoore, H.W. (1990) The potentiation of industrial
biocide activity with Cu2 +. I. Synergistic effects with formaldehyde. Internat. Bio-
deterioration, 26, 51-61.
107. Bennett, E.O. (1974) The biologial testing of cutting fluids. Lubrication Eng., 30,
128-135.
108. Hill, E.C. (1976) Evaluation of biocides for use with petroleum products. Process
Biochem., 11,36-38,41.
109. Shennan, J.L. (1983) Selection and evaluation of biocides for aqueous metal-working
fluids. Tribology Internat., 16, 317-330.
110. Rossmoore, H.W. (1977) Evaluation techniques for biodeterioration of water-miscible
metalworking fluids. In Biodeterioration Investigation Techniques, Walters, A.H. (ed.),
Applied Science, London, pp. 227-242.
111. Morton, L.H.G. (1987) Detection of significant spoilage microorganisms in soluble oil-
in-water metal-working fluids. In Industrial Microbiological Testing, Hopton, J.W. and
Hill, E.C. (eds.), Blackwell Scientific, Palo Alto, California, pp. 221-225.
112. Rossmoore, H.W., Seszny, P. and Rossmoore, L.A. (1977) Evaluation of source
bacterial inoculum in development of a cutting fluid test procedure. Lubrication Eng. ,
33, 372-377.
113. Rossmoore, H.W. and Rossmoore, L.A. (1980) The identification of a defined
microbial inoculum for the evaluation of biocides in water-based metalworking fluids.
Lubrication Eng., 36, 6-20.
114. Rossmoore, H.W. (1982) The role of the inoculum in the microbiological evaluation of
water-based metalworking fluids. In International Yearbook of Tribology, Bartz, W.J.
(ed.), Expert Verlag, West Germany, pp. 791-797.
115. Pivnick, H. and Fabian, F.W. (1953) Methods for testing the germicidal value of
chemical compounds for disinfecting soluble oil emulsions. Appl. Microbiol., 1,
204-207.
116. Himmelfarb, P. and Scott, A. (1968) Simple circulating tank test for evaluation of
germicides in cutting fluid emulsions. Appl. Microbiol., 16, 1437-1438.
117. Shennan, J.L. and Chater, K.W.A. (1984) The philosophy and compatibility of biocide
additions. In Monitoring and Maintenance of Aqueous Metal-Working Fluids, Chater,
K.W.A. and Hill, E.C. (eds), John Wiley, New York, pp. 113-129.
118. Sharpell, F.H. (1979) Development of test protocols for antimicrobial agents by the
ASTM. Develop. Indust. Microbiol., 20, 73-80.
119. ASTM (1993) Standard test method for evaluating the bioresistance of water-dilutable
metalworking fluids. In Standards on Materials and Environmental Microbiology, 2nd
edn, Philadelphia, pp. 642-644.
120. ASTM (1993) Standard test method for evaluation of antimicrobial agents in aqueous
metalworking fluids. In Standards on Materials and Environmental Microbiology, 2nd
edn, Philadelphia, pp. 277-279.
121. Rossmoore, H.W. and Rossmoore, L.A. (1991) Effect of microbial growth products on
biocide activity in metalworking fluids. Intern at. Biodeterioration, 27, 145-156.
310 PRESERVATION OF SURFACTANT FORMULATIONS

122. Brandeberry, L.J. and Myers, H.V., Jr. (1960) Test procedures for compounds used as
preservatives in industrial coolants. Lubrication Eng., 16, 161-164.
123. Rossmoore, H.W. and Williams, B.W. (1971) An evaluation of a laboratory and plant
procedure for preservation of cutting fluids. Internat. Biodeterioration Bull., 7, 55--60.
124. Rogers, M.R., Kaplan, A.M. and Beaumont, E. (1975) A laboratory in-plant analysis of
a test procedure for biocides in metalworking fluids. Lubrication Eng., 31, 301-310.
125. Hill, E.C., Davies, 1., Pritchard, J.A.V. and Byron, D. (1967) The estimation of
microorganisms in petroleum products. J. Inst. Petroleum, 53, 275-279.
126. Rossmoore, H.W. (1971) Methylene blue reduction for rapid inplant detection of
coolant breakdown. Internat., Biodeterioration Bull., 7, 147-154.
127. Yanis, R.J. and Wolfe, G.F. (1960) Test procedures for the evaluation of cutting fluids.
Lubrication Eng., 16, 164-170.
128. Passman, F.J. (1989) Monitoring microbial contamination in metalworking fluds. Soc.
Manufact. Eng. Tech. Paper, #MR89-454.
129. Genner, C. (1976) Evaluation of the dip-slide technique for the mcirobiological testing
of industrial fluids. Process Biochem., 11, 39-48.
130. Genner, C. and Hill, E.C. (1981) Evaluation of the 'dip-slide' technique for cutting oils.
Tribology Internat., 14, 11-13.
131. Rossmoore, L.A., Wireman, J.W. and Rossmoore, H.W. (1986) Rapid field method for
the detection and enumeration of sulfate reducers. In Biodeterioration 6 - Proceedings
of the Sixth International Biodeterioration Symposium, Sheila, B. and Houghton, D.R.
(eds), C.A.B. Intnl. Mycological Inst., The Biodeterioration Society, United Kingdom,
pp. 413-419.
132. McCoy, J.S. (1978) A practical approach to central system control. Lubrication Eng.,
34, 180-186.
133. Gannon, J.E. and Bennett, E.O. (1981) A rapid technique for determining microbial
loads in metalworking fluids. Tribology Internat., 14, 3--6.
134. Passman, F.J. (1984) A catalase test for quickly estimating microbial loads in
metalworking fluids. Soc. Manufact. Eng. Tech. Paper, #MR84-916.
135. Hill, E.C. (1987) Spatial distribution of spoilage microbes in fluids using the gel system.
In Industrial Microbiological Testing, Hopton, J.W. and Hill, E.C. (eds), Blackwell
Scientific, Palo Alto, California, pp. 195-199.
136. Holdum, R.S. (1977) Microbial spoilage of engineering materials: Part 6 - Improving
monitoring and control. Tribology Internat., 10, 273-280.
137. Yust, P.R. and Becket, G.J.P. (1980) Microbiology theory and practice in metalwoking
fludis. Indust. Lubrication Tribology, NovemberfDecember, 220-225.
12 Toxicology of preservatives
R.A. RaDFORD

12.1 Introduction

Preservatives are, by their very nature, biologically active molecules.

Basically, they need to be aggressive to do a good job. Therefore, most
biocides, particularly those used for product preservation, are subject
to some form of regulation specifying scope and concentration for use.
In today's tightly regulated environment, the development of a new pre-
servative is a very expensive and time-consuming operation. The test
requirements for regulatory approval of a new chemical vary depending on
where it is made and marketed. In addition, many countries have positive
lists for preservatives, particularly when they are used in cosmetics or
foods. To obtain a positive listing, a package of toxicological studies needs
to be presented to and approved by the relevant regulatory authority.
Exposure to a preservative may be by ingestion, inhalation or by skin
absorption. Skin irritation and/or skin sensitization may result from direct
contact of the preservative with the skin. All new preservatives should
therefore be regarded as potential health hazards until the required
toxicological evaluation has been completed and a risk assessment
performed for the proposed use. In general, the manufacturer will need to
supply data from a package of acute studies including oral, dermal and
possibly inhalation, skin and eye irritation, sensitization and genotoxicity.
A recommendation for an acceptable air-borne concentration in the
working environment, termed an occupational exposure limit (OEL) in the
UK, a threshold limit value (TLV) in the US or a maximum Arbeitskonzen-
tration (MAK) in Germany, should be proposed. The particular application
and extent of human exposure will indicate which subacute or chronic
studies should be performed to provide an adequate safety support
package.
In view of the intrinsic aggressiveness of preservatives towards biological
material, it should come as no surprise that they often give positive results
in toxicological studies especially in vitro mutagenicity assays and stringent
skin sensitization studies such as the Magnusson and Kligman guinea pig
maximization test. This is now better appreciated and any assessment of
their safety should be based on the fullest possible understanding of their
toxicity together with a risk assessment for the particular use.
Detergent formulations generally provide ideal media for the propagation
312 PRESERVATION OF SURFACTANT FORMULATIONS

of microorganisms. The Gram-negative type such as Pseudomonas are the

most common although Gram-positive organisms, moulds and yeasts may
also be found. These organisms not only spoil the product but can pose a
serious health hazard to the consumer. Therefore, an effective preservative
should be used to control the microorganisms and prevent spoilage of the
product. For products needing an extended shelf life, regulations require
the use of a preservative which is both effective and safe.
The use of nonionic surfactants in both personal care and detergent
formulations have increased the problems of product preservation as it has
been shown that the efficiency of certain preservatives is reduced when the
ratio of the surfactant to the preservative exceeds a certain critical value.
This ratio varies depending on the surfactant, the preservative and the test
organism. 1 The mechanism of inactivation of preservatives by nonionic
surfactants has been studied by many investigators including Higuchi and
coworkers 2 ,3 who showed the complexing of phenolic preservatives with
polyethylene glycols and Dyer4 and Evans5 who attributed inactivation to a
partitioning of the preservative between the micelles of the surfactant and
the aqueous phase. Anionic and cationic surfactants can also interact with
preservatives and this has been reviewed by Coates. 6
Many other factors, including the pH and partitioning in oil-in-water
systems can also affect the activity of any particular preservative. The
overall assessment of preservative performance can, therefore, only be
made on the finished product.

12.2 Preservatives in general use

In considering a new product formulation, at least four points need to be

addressed~ The first of these is the spoilage potential of the product.
Within this context, it is necessay to take into account the presence of
chemical substrates for microbial growth, physical conditions, i.e. pH,
water activity, etc., microbial challenge during manufacture, storage
conditions, pattern of use and storage time and temperature. Normal in-
use conditions, expected shelf working life and the probability of consumer
abuse should also be considered.
The second point to consider is whether product preservation is possible
without the use of a biocide. In the case of preservative-free formulations,
the physical or chemical properties should be inimical to growth and the
exclusion of microorganisms should be easy to achieve during manufacture.
It would also be beneficial if one or more of the ingredients has
bacteriostatic or bacteriocidal action.
Thirdly, it has to be considered which biocides are appropriate.
Chemical compatibility with the product has to be taken into account along
with suitability to meet the expected microbial challenge, legislative
 TOXICOLOGY 313

restrictions, costs and safety approval implications. Last but not least, the
requirements of occupational health and environmental measures must be
taken into account.
The preservatives listed below have found application generally in
surfactant-containing formulations for cosmetic and detergent products.
Other product types, for example, pharmaceuticals, paints and adhesives
can also be preserved with these chemicals. Some compounds are also
active against algae and have been used as slimicides, for example,
quaternary ammonium salts or the isothiazolinones.

12.2.1 Organic acids

Included within this group of compounds are acetic, lactic, benzoic, sorbic
and propionic acids. These preservatives generally have slight to moderate
activity against both Gram-positive and Gram-negative bacteria. Benzoic
and sorbic acids also have strong activity against yeasts whilst sorbic and
propionic acids have a strong activity against moulds. Evidence has been
produced that many organic acids act as uncoupling agents by preventing
the uptake of substrates which depend on a protonmotive force for their
entry into the cell. 7 The major incompatibility of organic acids is a high pH
formulation.

12.2.2 Phenolics
This class of biocides includes ortho-phenyl phenol, hexachlorophene,
chloroxylenol, chlorhexidene, phenoxyethanol, triclosan and dichlorophen.
These compounds generally have a moderate to strong activity against
Gram-positive and/or Gram-negative bacteria. Ortho-phenyl phenol and
dichlorophen also have good activity against both yeasts and moulds.
Various possible modes of action have been suggested for the different
phenolic biocides. 8 Phenoxyethanol is thought to inhibit active transport
by uncoupling oxidation from phosphorylation and protein coagulation.
Chlorhexidene and hexachlorophene are suggested to act by general
coagulation and disruption of the permeability barrier of the cell. Phenol
itself causes generalized membrane damage by possible lysis of the
cytoplasmic membrane which results in a leakage of amino acids, purines,
pyrimidines, potassium ions and pentoses.
Ortho-phenyl phenol is poorly soluble in surfactants, chloroxylenol is
incompatible with both cationic and nonionic surfactants and chlorhexidene
is incompatible with anionics and gums. Phenoxyethanol is also incompat-
ible with nonionics. Some of these phenolics, especially the chlorinated
ones, do not have an acceptable toxicological or environmental profile for
use in high volume products.
314 PRESERVATION OF SURFACTANT FORMULATIONS

12.2.3 Quaternary ammonium salts

Benzalkonium chloride, benzethonium chloride and cetrimide are
members of this group of materials which have their strongest biocidal
activity against Gram-positive bacteria. The mode of action of quaternary
ammonium salts has not yet been completely elucidated but certain
accepted and well-defined processes have been reported. 9 •10 Firstly, the
preservative is adsorbed onto the bacterial cell surface. Diffusion then
takes place through the cell wall and binding to, and disruption of, the
cytoplasmic membrane follows. Next there is leakage of potassium ions
and cytoplasmic constituents and finally, precipitation of the cell contents
and cell death.
Quaternary ammonium compounds are useful at pH ranges above 5 but
are incompatible with anionics, proteins and salts.

12.2.4 Mercury salts

Two examples of this group are thiomersal and phenyl mercuric acetate
which both have broad spectrum biocidal activity and are also very
effective against both yeasts and moulds. Mercury salts can combine with
the -SH groups of enzymes. 11 This can occur with enzymes associated with
the membrane or with enzymes and proteins found in the cytoplasm. Low
concentrations of some mercuric salts can lyse growing cultures of
Escherichia coli.
The effective in-use concentration is very low, typically below 0.05%
w/w and the useful pH range is less than 8. Major incompatibilities are
nitrogen- and sulphur-containing organics, plastics and halides. Mercury
salts are not considered appropriate for consumer products from both
toxicological and environmental standpoints.

12.2.5 Ureas
Imidazolidinyl-urea and diazolidinyl-urea are both preservatives in this
class of compounds. The former has strong activity against Gram-positive
bacteria and yeasts whilst the latter has strong activity against both Gram-
positive and Gram-negative bacteria and moderate activity against both
yeasts and moulds. It is presumed that this class of preservatives owes its
antimicrobial activity to the release of formaldehyde. The useful pH range
for these urea compounds is 3-9.

12.2.6 Isothiazolinones
The biocidal materials in this group are benzyl, methyl or chloro derivatives.
They are broad spectrum preservatives active against both Gram-positive
 TOXICOLOGY 315

and Gram-negative bacteria. They are also very effective against yeasts
and moulds with a wide useful pH range from 1-9. The mechanism of
antibacterial action of 1,2-benzisothiazolin-3-one, the active ingredient in
the Proxel range of preservatives from Zeneca, has been shown to involve
reaction with the thiol groups associated with the organisms. Interaction at
the cytoplasmic membrane surface is indicated. Proxel inhibits the
oxidation of carbohydrate substrates which are transported across the
cytoplasmic membrane by thiol-dependent enzymes. It inhibits the
oxidation of glycerol which enters the cell by diffusion and also inhibits the
utilization of the electron transport chain by bacteria, possibly by action on
the dehydrogenase enzymes. 12 The mode of action of other isothiazolinone
preservatives is still under investigation but a reasonable assumption would
be that initially, a process of generalized damage to the cell membrane is
involved, followed by interaction with proteins and enzymes probably via
disulphide linkages. Major incompatibilities of this group of biocides are
amines, sulphites and mercaptans.

12.2.7 Hydantoins (formaldehyde donors)

Dimethyloldimethyl hydantoin and monomethyloldimethyl hydantoin are
most effective against Gram-positive bacteria. Activity against yeasts and
moulds is slight and the useful pH range is around 4-9. It is assumed that
the biocidal action of these chemicals is due to the release of formaldehyde.
Thus, the use of these preservatives may be restricted in certain countries
in which adverse pUblicity about formaldehyde has led to a retail ban.

12.2.8 Miscellaneous organics

Other notable examples of compounds with biocidal actIvIty are, the
methyl, propyl and butyl esters of parabens, 2-bromo-2-nitropropane-
1,3-diol (Bronopol), formaldehyde, quaternium 15 (Dowicil 200), oxa-
zolooxazole (Nuosept 95 or Nuosept C), 4,4-dimethyl-l,3-oxazolidine
(Oxaban A), l-azo-3,7-diox-5-ethylbicyclo(3.3.0)octane (Oxaban E),
glutaraldehyde, N-( trichloromethylthio )cyclohex-4-ene-l ,2-dicarboximide
(Captan), glyceryl monolaurate and zinc pyrithione.
The strongest activity of the parabens esters is against Gram-positive
bacteria and moulds. The mechanism of action of parabens esters is
concentration dependent. 13 Low concentrations inhibit transport whilst
higher concentrations affect membrane integrity. This has the effect of
leakage of intracellular constituents and selective inhibition of ~pH across
the cell membrane. A major drawback of the parabens esters is their
incompatibility with nonionics and cationics.
Both Bronopol and formaldehyde are effective preservatives with strong
activity against both Gram-positive and Gram-negative bacteria with
316 PRESERVATION OF SURFACTANT FORMULATIONS

slightly less activity against yeasts and moulds. Bronopol acts by interfering
with cytoplasmic membrane enzymes oxidizing thiol groups to di-
sulphides. 11 Very low concentrations of formaldehyde cause lysis of the
cell wall. In addition, formaldehyde interacts with amino groups in the
cytoplasm. 11 Bronopol and formaldehyde are useful across a wide range of
pH around 3/4--10. The former is incompatible with -SH compounds and
aluminium and the latter is incompatible with proteins.
Dowicil 200 and Nuosept C are both strongly active against both Gram-
positive and Gram-negative bacteria, moderately effective against moulds
but only slightly active against yeasts. The mode of action of Dowicil200, a
hexamine derivative quatemized with cis-1,3-dichloropropene, has been
studied by Rossmoore and Sondossi.14 Hexamine breaks down by acid
hydrolysis to release formaldehyde so the activity of Dowicil 200 is almost
certainly due to this mechanism. The activity of Nuosept C is also assumed
to be due to the release of formaldehyde. The useful pH range for both
these preservatives is 4--9/10 but Nuosept C is incompatible with bisulphite
at high or low pH.
Oxabans A and E and glutaraldehyde are effective against all bacteria,
yeasts and moulds, the latter particularly so at pH levels of 7.5-8.5.
Oxabans A and E are only useful at a pH of greater than 6 as below this
they break down. The mode of action of glutaraldehyde is complex due to
its diverse chemical reactivity. Its activity is, therefore, probably due to
more than one process. The following effects on bacteria have been
observed: crosslinking of the peptidoglycan in Gram-positive bacteria;
interaction with protein in the cell walls of Gram-negative bacteria;
reaction with spore cortex constituents; and increased resistance to lysis of
protoplasts and spheroplasts. 15 Glutaraldehyde is inactivated by ammonia
and primary amines at neutral to basic pH values.
Capt an and glyceryl monolaurate are both moderately effective against
bacteria, yeasts and moulds although glyceryl monolaurate is less effective
against Gram-negative bacteria. Captan is an important agricultural
fungicide. Its mode of action is centred on the reaction with cellular thiol
groups. 16-18
Zinc pyrithione is strongly active against Gram-positive bacteria, yeasts
and moulds and moderately effective against Gram-negative bacteria. It is
useful across a pH range of 4.5-9.5 but is incompatible with EDTA and
some nonionics.
The toxicology of the materials discussed above obviously varies widely
as there is such a range of chemical types. It is therefore proposed to limit
the discussion on the various toxicological aspects to a selection of the most
effective all-round preservatives, namely, the isothiazolinones (Proxel and
Kathon), Bronopol, formaldehyde, Oxabans A and E and glutaraldehyde.
 TOXICOLOGY 317

12.3 Acute toxicity

12.3.1 Formaldehyde
Formaldehyde is a normal metabolite in mammals and, in small quantities,
is rapidly metabolized. 19 The major route of biotransformation appears to
be oxidation to formic acid, followed by further oxidation to carbon
dioxide and water. 20,21 Most of the formaldehyde absorbed by ingestion or
inhalation is rapidly oxidized in the blood and excreted via the kidneys.
The half-life of formaldehyde in blood is considered to be 1.S min or less in
rats, guinea pigs, rabbits and cats. 22 At concentrations considerably
greater than those likely to be experienced by the general public from its
use as a preservative, formaldehyde is severely irritating to the oral mucosa
and cases of fatal ingestion of formaldehyde are known. Industrial
exposure represents a potential worst case situation, and therefore
maximum exposure limits are set by government bodies to ensure any
inconvenience to the work force is minimized. The absolute concentrations
of formaldehyde permitted industrially vary slightly from country to
country. Examples of permissible exposure limits for formaldehyde are:
• UK - 2 ppm (MEL - TWA and STEL?3
• USA - 0.3 ppm (TLVc?4
• Germany - O.S ppm (MAK?5
where MEL = maximum exposure limit, TWA = 8 h time weighted
average, STEL = 10 min short term exposure limit TLV(c) = threshold
limit, value (ceiling) and MAK = maximum Arbeitskonzentration.
Acute ingestion of formaldehyde by humans has resulted in loss of
consciousness, vascular collapse, pneumonia, haemorrhagic nephritis and
abortion. Formaldehyde has occasionally injured the larynx and trachea,
but damage to the gastrointestinal tract has occurred primarily in the
stomach and lower oesophagus. 26

12.3.2 Isothiazolinones
The two best known preservatives in this class are benzisothiazolinone (the
Proxel range of biocides from Zeneca) and a 3:1 mixture of S-chloro-2-
methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (the Kathon
range of biocides manufactured by Rohm and Haas). Neither of these
products is acutely toxic by ingestion, inhalation or skin absorption. In
tests on animals conducted many years ago, oral LDSOs in the rat (the dose
required to killSO% of the animals) were 1100 mg kg-1 for Proxel GXL27
which is 20% active to >2000 mg kg-1 for Proxel XL227 which is 9.S%
active. For Kathon CG (1.S% active),zs the rat oral LDSO was established
318 PRESERVATION OF SURFACTANT FORMULATIONS

at 3350 mg kg-1 (males) and 2630 mg kg- 1 (females). The dermal LD50 in
the rabbit was >5000 mg kg-1 for Kathon CG. 28 The LC50, four-hour
exposure, to the rat (the air-borne concentration required to kill 50% of
the animals) was about > 13.7 mg 1-1 of air for Kathon CG. 28

12.3.3 2-Bromo-2-nitropropane-1,3-diol
The trade name of this compound is Bronopol manufactured by the Boots
Company pIc. Following acute administration, Bronopol was classified as
moderately toxic to rats and mice by the oral route using the classification
system of Hodge and Sterner29 intraperitoneal, subcutaneous and percuta-
neous acute toxicity data were consistent with this assessment. 30 The oral
LD50s to rats were 307 mg kg-1 (males) and 342 mg kg-1 (females) whilst
the corresponding data for mice showed 374 mg kg-1 (males) and
327 mg kg-1 (females).31

12.3.4 4,4-Dimethyl-1,3-oxazolidine and 1-azo-3,7-diox-5-ethylbicyclo

(3.3.0) octane
These preservatives are produced by Angus Chemicals and are known
commercially as Oxaban A and Oxaban E, respectively. The acute oral
LD50 of Oxban A to male rats was established at about 950 mg kg-1 32
indicating that this chemical would be classified in the EC as harmful. The
acute dermal LD50 of Oxaban A to rabbits was estimated to be around
1400 mg kg -4 with the skin response being moderate to severe?2 In an
acute inhalation study, the LC50 to rats was found to be about 11.7 mg 1-1
for Oxaban A.32 On the other hand, Oxaban E is less acutely toxic to rats
than Oxaban A, particularly by the oral route where the LD50s were found
to be 5250 mg kg- 1 and 3680 mg kg-1 for males and females, respectively.33
The acute dermal LD50 of Oxaban E in rabbits was found to be
1948 mg kg-1 and the LC50, following a four-hour mist inhalation study in
rats was determined to be 3.1 mg 1-1?3

12.3.5 Glutaraldehyde
Data indicate that glutaraldehyde vapour may produce complaints of
'irritation' of the eyes and respiratory tract in man at around 0.3 ppm in
air. 34 Therefore, in the UK24 a STEL has been set at 0.2 ppm and, likewise
in the USA 25 and Germany26 a ceiling TLV-eight-hour TWA and a
MAK, respectively, have been set at 0.2 ppm to control these effects.
Animal studies35 indicate that aqueous solutions of glutaraldehyde
(Ucarcide) are of moderate acute toxicity. LD50s in the rat ranged from
410-733 mg kg-1 body weight for a concentration range of 25-50% (w/w).
However, further studies with dilute solutions of Ucarcide show a very low
 TOXICOLOGY 319

order of acute oral toxicity with an LD50 of 17.5 ml kg- J in the rat. Studies
in rabbits have given dermal LD50s ranging from 897 mg kg-1 to
3045 mg kg- 1 body weight for 50% to 25% aqueous solutions. The former
would be classified as harmful by the EC. Concentrations of 5% or less
were shown to have a very low order of acute dermal toxicity. Acute
inhalation studies in rats show that atmospheres generated from saturated
vapour from 25%, 45% or 50% could produce irritant effects but there
were no effects to indicate they were harmful.

12.4 Skin and eye irritation

Most biocides in a concentrated form are corrosive or irritant to skin and!

or eyes.

12.4.1 Formaldehyde
On contact with the skin formaldehyde in solution may cause primary
irritation. 36 ,37 Although its irritancy potential is generally recognized, few
quantitative data exist for formaldehyde. The only relative irritancy data
are from an open epicutaneous test using guinea pigs where marked
irritation occurred at 30% and 10% of formaldehyde. Some irritation was
also produced at 3%, 0.1 % and 0.03%, although there were no effects at
1% nor at 0.3% formaldehyde?8 No comment was made on this apparent
discrepancy; possibly it is an artefact of this type of test.
Human data indicate that formaldehyde is a severe irritant to the eyes
and upper respiratory tract. Formaldehyde, at a concentration of 4 ppm,
can produce lachrymation but the irritancy threshold may be much lower.
At 10 ppm, formaldehyde causes severe eye irritation in man?9

12.4.2 Isothiazolinones
Single applications of 1,2-benzisothiazolin-3-one (BIT) in aqueous solution
to rat skin were irritant or strongly irritant at concentrations above 8%,
whereas a single application of an aqueous solution containing 1.65% BIT
was not irritant. 4o Dilute aqueous solutions of BIT «0.07%) were not
irritant to the rabbit eye but more concentrated solutions (say >0.5%)
were irritant or very irritant with the possibility of causing serious damage
to eyes. 40
Studies carried out according to OECD guidelines have indicated that
Kathon CG (1.5% active ingredient (a.i.» is irritant to rabbit eyes but not
to rabbit skin.41 Other animal studies, however, using the Draize eye
irritation test showed Kathon 886 (14% a.i. as supplied), ranging in
concentration from 1.1% to 14% a.i., and Kathon CG at 1.5% a.i. were
320 PRESERVATION OF SURFACTANT FORMULATIONS

corrosive when tested as supplied. Kathon CG was also found to be

severely irritating to rabbit skin using occlusive patches on both intact and
abraded test sites. 42

12.4.3 Bronopol
In rabbit eye tests, Bronopol was not irritating at a concentration of 0.5%
in normal saline applied once a day on four successive days. Following
single applications, 5% Bronopol in polyethylene glycol 400 was irritant to
rabbit eyes but a concentration of 2% was not.31 Single applications of
0.5% Bronopol in 2.5% aqueous methyl cellulose were not irritant when
applied to intact rabbit skin for six hours under occlusion but 2% Bronopol
was irritant using the same test conditions. Since 5% Bronopol in
polyetheylene glycol was not irritant under these test conditions, the
irritancy potential of Bronopol would appear to be vehicle dependent. This
was confirmed in human studies where Bronopol was not irritant to skin
under occlusive conditions at 0.5% in petrolatum or 0.1% in aqueous
buffer at pH 5.5 but concentrations of 1% in petrolatum or 0.25% in
aqueous buffer were slightly irritant. 31 These data indicate that irritation
reactions due to Bronopol are unlikely to occur in man even if an undiluted
product containing 0.02% Bronopol were in contact with the skin or
entered the eye.

12.4.4 Oxabans A and E

Due to its alkaline nature, Oxaban A can cause severe burns to eyes,
although at a concentration of 5000 ppm in water, eye irritation potential is
extremely low. 32 Using the US Department of Transport test method,
Oxaban A is not corrosive to skin. However, the undiluted chemical is still
severely irritant to skin. Animal studies also indicate that Oxaban E is
severely irritating to skin and eyes but not corrosive. 33

12.4.5 Glutaraldehyde
Undiluted glutaraldehyde caused severe irritation and corrosion in rabbit
eye tests. 43 ,44 Whilst a 5% solution of glutaraldehyde also caused corneal
damage, 0.1 % and 0.5% did not cause inflammation or corneal damage to
rabbit eyes. 45 In an accidental exposure, a 2% aqueous solution of
glutaraldehyde has caused eye irritation in man. 46 In a four-hour skin
covered patch test in rabbits, a 25% aqueous solution of glutaraldehyde
caused severe inflammation and corrosion. 45 Whilst these investigators
identified 1% as a 'threshold' for inflammation, other workers saw no local
effects in rabbits at concentrations up to 7% and only mild inflammation
and some crusting at 25% following a 24-hour covered patch test. 47 In
 TOXICOLOGY 321

man, nine out of 13 patients with contact dermatitis had irritant reactions
to 1% glutaraldehyde in petrolatum following 48-hour covered patch tests,
whilst at 0.1 % no irritation was experienced by 884 patients. 48 Exposure to
glutaraldehyde vapour at 2-11 ppm for ~ h was irritating to rats eyes,45
as was exposure to 3 ppm for unknown daily periods for 90 days.49

12.5 Skin and respiratory sensitization

Many biocides can cause allergic contact dermatitis or respiratory

sensitization.

12.5.1 Formaldehyde
Formaldehyde is a frequent cause of sensitization, as assessed in patients
attending dermatology clinics. In a series of patch tests carried out on
preservatives and fragrance materials used in consumer products, the
incidence of positive patch test reactions ranged from 2.1 % to 4.7%.50-54
There have also been numerous reports of occupational contact dermatitis
to formaldehyde. 55 ,56 In addition, some cases of formaldehyde-associated
occupational asthma have been reported. 57- 59 However, the number of
cases is small and the evidence for an immune response is not clear. 60 This
response of the respiratory tract to formaldehyde would appear to be
considerably less prevalent than the cutaneous response to the substance.
Formaldehyde is a sensitizer in predictive guinea pig tests, although the
observed responses may differ between laboratories. 61 In human predictive
tests, repeated applications of a 5% or 10% aqueous solution of
formaldehyde followed by a challenge with a 1% aqueous formaldehyde
solution led to 8% of the 154 subjects tested becoming sensitized. 62 Jordan
et al. 63 determined a minimum eliciting level of formaldehyde by patch
testing and observed that four out of nine formaldehyde-sensitive subjects
reacted to 30 ppm formaldehyde after 120 continuous hours of testing.
These tests indicate that levels of formaldehyde below 30 ppm should be
tolerated by sensitive subjects when used repeatedly on normal skin. This
conclusion is supported by the fact that contact dermatitis from formalde-
hyde in cosmetics is rare. 55

12.5.2 Isothiazolinones
Cases of allergic contact dermatitis were reported in man following
exposure to concentrations of Proxel greater than the recommended use
level. 64-66
Both human and animal studies have shown Kathon CG to have strong
skin sensitization potential. 67 ,68 Much work was carried out to establish a
322 PRESERVATION OF SURFACTANT FORMULATIONS

use level for Kathon CG which would not pose any significant risk of
allergic contact dermatitis to the consumer. The level determined was
15 ppm in 'rinse-off' products such as shampoos and shower gels and
7.5 ppm in 'leave-on' products such as skin creams and lotions. 42

12.5.3 Bronopol
Overall, Bronopol has a low, but variable, sensitization in the Magnusson
and Kligman guinea pig maximization test and is, at most, a weak sensitizer
by this method. There are some inconsistencies in published literature
concerning the skin sensitization potential of Bronopol in man. This has
probably arisen from the use of irritant concentrations of Bronopol for
patch tests. Initial studies indicated that 2.5% Bronopol in petrolatum was
not irritant and that Bronopol was a potential sensitizer. 69 However, later
studies70 showed that Bronopol was irritant at concentrations above 1%
and a test in 93 subjects using the Draize procedure, with an induction
concentration of 5% and a non-irritant challenge concentration of 0.25%,
showed no evidence of skin sensitization. A test using a single application
of 0.25% Bronopol in petrolatum in 149 patients attending a dermatitis
clinic also found no evidence of skin sensitization. 31 A study on cosmetic
intolerance in over 5000 Belgian patients being tested for contact
dermatitis found only 0.1 % of the sample gave a positive reaction to
Bronopol in a patch test. 71 The divergence in the reported incidence of
reactions to Bronopol may result from the inclusion in some studies of
subjects who have been occupationally exposed to high levels of Bronopol.
These data indicate a very low sensitization potential for Bronopol; it is
unlikely to induce allergic contact dermatitis in man at normal preservative
use concentrations.

12.5.4 Oxabans A and E

In the Magnusson and Kligman guinea pig maximization test, Oxaban A
was found to be a strong sensitizer at a challenge of 1% following
intradermal and topical inductions of 0.1 % and 2.5% a.i., respectively. In
human skin irritation and sensitization studies, a 3% solution of Oxaban A
in water was found to be irritant; 60% of the 101 test panel reacting by the
end of the second week. The concentration of the test material was
reduced to 0.3% for the third week and applied to a fresh test site.
Following two weeks rest, a further fresh test site was challenged with the
0.3% solution of Oxaban A. Two of the panellists reacted, so that two
weeks later these two were rechallenged with a concentration of 0.1 %
Oxaban A. There was no reaction to this concentration. 32 The conclusion
may be drawn that whilst Oxaban A has been shown to possess a fairly
strong sensitization potential in animal studies, the risk of consumers
 TOXICOLOGY 323

exhibiting allergic contact dermatitis from contact with products containing

Oxaban A at the normal preservative use concentration of 0.1 % is very
low. 32
In human studies, one out of 201 subjects showed sensitization to 3%
Oxaban E although some 12% of the panellists showed varying degrees of
skin irritation during the induction period. 33 However, Oxaban E was
shown to be a strong sensitizer in the Magnusson and Kligman guinea pig
maximization test at a challenge concentration of 2.5% following
intradermal and topical application inductions of 2.5% and 5%, respect-
ively. At a second challenge following a week's rest, a weak response was
observed at a concentration of 0.1 %, but there was no response at 0.01 %.
Strong cross reactions to formaldehyde were also demonstrated.

12.5.5 Glutaraldehyde
Glutaraldehyde is a well known skin contact allergen in animals and man.
The lowest induction or challenge concentration producing skin sensitiza-
tion in man was shown to be 0.5%. Concentrations of 0.2% and below
have shown no potential for allergic contact dermatitis in man. 69 •72 ,73
Glutaraldehyde is also on the prescribed list of agents known to cause
occupational asthma. 74

12.6 Subacute and cbronic toxicity including carcinogenicity

12.6.1 Formaldehyde
In a chronic inhalation study, rats and mice were exposed to 0, 2.0, 5.6 or
14.3 ppm formaldehyde in air for six hours per day five days per week for
two years. 75 Squamous cell carcinoma of the nasal passages was induced
in some rats (44% at 14.3 ppm and 0.85% at 5.6 ppm). Polyploid
adenomas were found in all exposed rats. Nasal lesions also occurred in
mice, but at a much lower incidence of only 0.93% at the top dose. This
difference in response may be related to a species difference in physio-
logical responses to respiratory irritants. This study leads to the conclusion
that formaldehyde does have carcinogenic potential in rats and mice.
The issue is to determine the relevance of these animal data to man. Many
epidemiological studies have examined the effects of industrial exposure to
formaldehyde. One such study has examined the mortality of men
employed in the British chemical and plastics industry from 1920 to 1989. 76
There were slight increases in lung cancer, respiratory disease and stomach
cancer, but no correlation was found with the estimated cumulative dose or
the time since the first exposure. It was concluded that these findings do
not provide a clear conclusion on the potential human carcinogenicity of
formaldehyde. A previous study examined the mortality in the UK of
324 PRESERVATION OF SURFACTANT FORMULATIONS

pathologists and medical laboratory technicians occupationally exposed to

formalin 77 and found no health problems related to formaldehyde
exposure.
A case-control study of cancer of the nose and paranasal sinuses was
conducted in France. 78 Out of 207 cases and 409 controls, no significant
association was found between exposure to formaldehyde and squamous
cell carcinomas of the sinonasal cavities. However, the study did suggest
that exposure to both formaldehyde and wood dust may increase the risk of
nasal adenocarcinoma, when compared with the risk due to wood dust
alone. Another earlier case control study of cancer mortality among Du
Pont workers exposed to formaldehyde 79 showed no differences between
the company's formaldehyde-exposed workers and non-exposed co-
workers.
A recent meta-analysis of the epidemiologic evidence of an association
between formaldehyde and respiratory cancer80 did not indicate an excess
risk or an exposure-response gradient for lung cancer. An exposure-
response gradient was seen for both sinonasal and nasopharyngeal cancers
which suggested that at least substantial levels (which induce chronic
irritation) of occupational exposure to formaldehyde are associated with a
risk of these cancers. This information supports the contention that a
threshold exists for formaldehyde carcinogenicity.
A review suggests that man is less susceptible to formaldehyde than rats
and that the risk of carcinogenic effects from even occupational exposures
is very low. 81 It is concluded that there is no risk presented to man from use
of formaldehyde as a preservative.

12.6.2 Kathon CG
In a three-month drinking water study in rats, no treatment-related effects
were observed with Kathon CG except for mild gastric irritation. 82 A
dermal carcinogenicity study, in which Kathon CG was applied to the skin
of mice three times a week for 30 months, showed no treatment-related
tumours or other adverse effects apart from local tissue irritation. 82

12.6.3 Bronopol
Repeated oral administration of Bronopol to rats 31 established the
following no-effect levels:
1. 20 mg kg-1 body weight per day by gavage for 90 days.
2. 100 mg kg-1 body weight per day in the diet for 12 weeks.
3. 10 mg kg-1 body weight per day in the drinking water for two years.
Oral administration of Bronopol to dogs at 20 mg kg-1 body weight per
day for 90 days produced no significant toxic reaction, although some
 TOXICOLOGY 325

vomiting was noted. 31 These data indicate that Bronopol is moderately

toxic following oral administration to rats and dogs. Bronopol was not
carcinogenic when applied dermally to mice at doses of up to 1.5 mg per
day for 80 weeks. 31 When Bronopol was administered to rats in the
drinking water at doses up to 160 mg kg-1 body weight per day for two
years, there was no effect on the incidence of tumours over the controls. 31

12.6.4 Oxaban A
In a 90-day dermal study in rats with Oxaban A, no deaths occurred and no
treatment-related effects were observed in serum chemistry, haematology,
urine or organ pathology. The top dose of 195 mg kg-1 body weight,
representing a level of Oxaban A equal to 100 times normal preservative
use concentration, produced moderate to severe skin irritation after the
third week. At actual Oxaban A use concentrations, equivalent to
1.95 mg kg-1 body weight per day, no adverse effects were observed. 32

12.6.5 Glutaraldehyde
In an ll-week rat drinking water study with glutaraldehyde at concentra-
tions up to 0.5% there were no clinical signs of toxicity during the live
phase of the study. Subsequent histopathology revealed no morphological
abnormality in central or peripheral nervous tissue. 83 Rats exposed to up
to 10 ppm glutaraldehyde vapour for an unknown period daily for 90 days,
showed no overt neuromuscular abnormalities. 49 A six-week dermal study
in rabbits using 5 mg kg-1 body weight per day of 2% aqueous glutar-
aldehyde in unoccluded applications, produced no observable adverse
effects other than skin irritation. 84 Carcinogenicity studies in rats and mice
by the inhalation and dermal routes were in progress in 1988 as part of the
National Toxicology Program in the USA.

12.7 Genetic toxicology

Genotoxicity studies are conducted to provide information on the

capability of a chemical to mutate, or change, genes or cause disruption of
chromosomes. If it can be demonstrated that a chemical can cause gene
mutations, then it may have the capability to cause cancer or changes to the
heritable gene line of man.

12.7.1 Formaldehyde
Studies by various investigators have concluded that formaldehyde is not
mutagenic in the Ames test. 85-88 However, other studies have indicated
326 PRESERVATION OF SURFACTANT FORMULATIONS

that formaldehyde is mutagenic in this test but that the mutagenicity is

masked by its high toxicity at low doses. 89-93 In addition, formaldehyde
was shown to be mutagenic in male larvae of Drosophila melanogaster,
when added to the feed medium whereas female larvae and adults were
unaffected. Injection of formaldehyde into male flies was also mutagenic
but to a lesser degree. Formaldehyde vapour was not mutagenic to
Drosophila melanogaster. 94 ,95 Other studies have shown that formalde-
hyde is mutagenic in prokaryotic and in lower eukaryotic systems, in
Drosophila and in mammalian cells in vitro. 94-98 The results of muta-
genicity tests in mammals in vivo are, however, negative or equivoca1. 96 ,99
It is considered that the mutagenic activity of formaldehyde is comparatively
weak, except under very specific conditions in Drosophila. In addition, it is
possible that it may be detoxified by metabolism before it causes damage to
cellular DNA.

12.7.2 Isothiazolinones
The genotoxicity of Kathon CG has been assessed in many different assays,
both in vitro and in vivo; viz. Ames, in a variety of bacterial strains, mouse
micronucleus, in vitro and in vivo cytogenetics, Drosophila meianogaster,
unscheduled DNA synthesis, cell transformation, mouse lymphoma and
DNA binding. 42 Positive results were obtained in the Ames test 1OO-103 and
the mouse lymphoma assay.103 The mutagenicity data have been reviewed
by a variety of experts and, taking into account negative results from a
valid dermal carcinogenicity study (see previous section) the conclusion
has been drawn that Kathon CG does not represent a genotoxic hazard for
man.42

12.7.3 Bronopol
Limited reported mutagenicity testing has shown that Bronopol was not
mutagenic in the Ames test (in vitro) nor in the mouse dominant lethal
assay (in vivo) when administered at the maximum tolerated dose. 31

12.7.4 Oxabans A and E

The mutagenic potentials of Oxaban A and E have been assessed in a
variety of in vitro and in vivo assays. There are some positive mutagenicity
data in vitro for Oxaban A, but in vivo assays have been negative. Oxaban
E does not appear to have mutagenic potential either in vitro or in vivo. 33
Oxaban A and E do not, therefore, pose a genotoxic hazard to man.
 TOXICOLOGY 327

12.7.5 Glutaraldehyde
The genotoxicity of glutaraldehyde has been investigated in in vitro tests
which have given both positive and negative results and in vivo where only
negative results have been shown. Glutaraldehyde has demonstrated a
positive response in Ames tests,91,93.104-109 and also in other tests using the
same bacterium Salmonella typhimuriumYO.lll Both positive108 and
negative 112,1l3 results have been obtained in mutagenicity assays using the
bacterium Escherichia coli. Glutaraldehyde also gave a positive result in a
Bacillus subtilis rec assay which measures DNA damage indirectly. 106
Negative results have been achieved in Drosophila melanogaster
assays.1l4-117 In in vitro tests using mammalian cells, glutaraldehyde
induced mutations in mouse cells 117 .118 and caused DNA damage in
hamster cells. 1l9 Equivocal results were obtained in other assays for
mutagenicity, chromosome damage and sister chromatid exchange.107.120
Glutaraldehyde was also negative in an unscheduled DNA test using rat
liver cells 107 and a cell transformation assay using hamster cells. 121
Glutaraldehyde showed no evidence of mutagenicity in two in vivo tests,
viz. a dominant lethal assay using female mice 122 and an unscheduled DNA
synthesis assay using male rats. 123 Evaluation of these latter data indicates
that glutaraldehyde is not an in vivo genotoxin, and therefore is probably
not a risk to man from its use as a preservative.

12.8 Reproductive and developmental effects

12.8.1 Formaldehyde
The teratogenic potential of formaldehyde has been examined in many
studies, but none has demonstrated a teratogenic effect. 124

12.8.2 Isothiazolinones
In a rabbit study, Kathon CG was not teratogenic but was embryotoxic and
foetotoxic at high doses. 42 Kathon CG was not teratogenic to rats at doses
up to 15 mg kg-1 body weight per day.42 In addition, in a 1S-week rat
drinking water study, no adverse effects were observed on fertility,
reproduction, foetal survival or health or maternal health up to a dose of
20 mg kg- 1 body weight per day.42

12.8.3 Bronopol
Bronopol produced no significant embryotoxic or teratogenic effects when
administered by oral intubation to rats throughout pregnancy at doses up
328 PRESERVATION OF SURFACTANT FORMULATIONS

to 100 mg kg-1 body weight per day.31 Bronopol had no effect on

parturition, litter size, post-natal survival or development of the young
when given orally to rat dams from day 15 of gestation and throughout
lactation at doses up to 40 mg kg-1 body weight per day. There was also no
effect on reproductive performance or fertility in both male and female rats
up to this dose level. 31 Bronopol had no embryotoxic or teratogenic effects
when administered orally to rabbits from days 8-16 of pregnancy at doses
up to 10 mg kg-1 body weight per day.31

12.8.4 Glutaraldehyde
Glutraldehyde produced no evidence of foetotoxicity or teratogenic effects
in a rat study up to a dose of 50 mg kg-1 body weight per day.19 In mice,
glutaraldehyde had no effects on maternal survival and weight gain, the
number of live foetuses, foetal weight or foetal malformation up to
24 mg kg-1 body weight per day. Maternal survival and weight gain were
decreased at 40 mg kg-1 body weight per day and above, and at 50 mg kg-1
body weight per day a slight increase in the incidence of foetal mal-
formations was observed. At 100 mg kg-1 body weight per day, glutaralde-
hyde produced reduced foetal weight and an increase in the number of
stunted foetuses and in the incidence of foetal malformations. 125 At
15 mg kg-1 body weight per day, glutaraldehyde had no effects on rabbits
when administered by stomach tube for 13 days during pregnancy. Even at
40 mg kg-1 body weight per day there were no foetal malformations but
there were indications of maternal toxicity as evidenced by early foetal
deaths and reduced foetal weights. 126

12.9 Environmental considerations

Formaldehyde is a naturally occurring substance which is a normal

metabolite in mammals. It is reported to be present in all fruit and
vegetables in the diet at a concentration of 4 mg kg-\ 3-7.5 mg of
formaldehyde are formed when the 50-150 mg of caffeine in a cup of
coffee are metabolized in the human body. 127 Formaldehyde occurs in the
atmosphere by the photochemical decomposition of organic products (e.g.
methane). The natural background concentration offormaldehyde in clean
maritime air is 0.0001 ppm and in clean continental air 0.001 ppm.
Formaldehyde in solution does not pose a hazard to the environment as it
has a low octanol:water partition coefficient, which means that it will
remain in the aqueous phase not significantly adsorbing or bioaccumulating
and, at the levels likely to be released to the sewage treatment works, it can
biodegrade both aerobically and anaerobically.
Proxel is also biodegradable and has a low potential for bioaccumulation.
 TOXICOLOGY 329

Also, it has a relatively low toxicity to birds and mammals. Proxel is,
however, acutely toxic to aquatic organisms and to sewage treatment
operations at concentrations similar to those at which surfactants are
toxic. 4o The concentrations of Proxel in the environment likely to arise
from its use as a preservative in surfactants are extremely low (a few ppb
and below) and are several (>3) orders of magnitude lower than
concentrations at which adverse effects might arise.
The environmental fate of Kathon CG has been extensively investi-
gated. 128 ,129 Studies have shown that although the active ingredients of
Kathon CG are toxic to aquatic species and wildlife, they are bio-
degradable into simple, non-toxic products which are rapidly dissipated
from soil, natural waters and sewage treatment systems. It is known that
the biodegradation of Kathon CG does not lead to a chlorinated organic
species; the 5-chloro constituent breaks down and releases the chloride
ion. Therefore, Kathon CG does not persist in the environment. However,
from a factory handling standpoint, Kathon CG-containing wastes must
not be discharged into public waters. Such wastes should either be
deactivated or adequately diluted before discharge into any public water or
sewage treatment system. 41 Deactivation can be effected by the addition of
a slightly acidic sodium metabisulphite or sodium bisulphite solution.
Bronopol does not biodegrade at concentrations as low as 3 mg 1-1.
Hydrolysis at environmental pH values is unlikely to reduce the Bronopol
level during sewage treatment, but photolysis could completely degrade it
in seven days. The degradation products formed, whilst potentially more
toxic than the parent material, will not persist in the environment. It is
expected that Bronopol will tend to remain in the aqueous phase during
sewage treatment and subsequent discharge to rivers. The bacterial
minimum inhibitory concentrations of Bronopol (12.5-50 ppm) are several
orders of magnitude higher than the levels likely to be present in sewage so
that adverse effects on the microbiological processes of sewage treatment
are unlikely. The concentrations of Bronopollikely to occur in rivers are
orders of magnitude below the levels which produce toxic effects in
daphnia (48-hour EC50 = 1.4 mg 1-1), fish (96-hour LC50 = 20 mg 1-1) or
algae (EC50 = 0.2 mg 1-1). The potential for bioconcentration of Bronopol
in aquatic organisms should be low.

12.10 Conclusion

It is important in the preparation of cosmetic, toiletry or speciality

chemical products that there is adequate protection from microbiogical
contamination. This contamination can arise from the consumer, the
environment during the shelf life of the product, the manufacturing plant
or the raw materials, including water. Good preservatives, such as those
330 PRESERVATION OF SURFACTANT FORMULATIONS

which have been discussed in this chapter, can provide peace of mind to the
manufacturer that the product will be effectively protected from micro-
biological attack. This in turn provides the consumer with a good quality
product free from any microbiological contamination which can lead to
serious adverse health effects. However, it must be remembered that
preservatives are biologically active molecules, they have to be to kill or
prevent the growth of the organisms'responsible for microbiological attack.
The assessment of the risk of potential adverse health effects on man from
the use of these preservatives is a complex process in which the known
animal and human toxicity data available have to be assessed against the
likely human exposure. Because preservatives are so biologically active,
their suppliers should ensure that there is an adequate data base from
which a risk assessment can be made. In addition, suppliers should adopt
the good practice of providing recommended use levels which provide
effective preservation but which do not represent unacceptable risks to
either man or the environment.
Generally, preservatives are used at such low levels that any irritation or
skin sensitization potential is irrelevant at the recommended use con-
centration. This being said, there are still certain applications, such as in
leave-on cosmetics in which some preservatives close to their recommended
use levels may cause either irritation or, in particular, sensitization. There
are many published papers which testify to this effect. Some manufacturers
have worked extremely hard to make sure that their preservatives are
adequately tested to provide the information which they need to set
suitable limits for use and which will ensure that the risk of adverse health
effects during normal use of the product are minimized. Indeed, some
manufacturers even monitor their customers' products in the marketplace
to ensure that their preservatives are being used at the recommended
levels.
One important and often forgotten aspect of preservative management is
to communicate effectively with the consumer. Formaldehyde is a good
example of a technically excellent, cost-effective preservative which has
now been banned in several countries because the risk assessment process
was not effectively managed and communicated to the general public. Bad
public relations has led to these marketing restrictions for formaldehyde
despite regulatory approval for the use of this preservative in many other
countries. Extensive marketing experience, together with critical evaluation
of many epidemiological studies on workers exposed industrially to
concentrations of formaldehyde which are considerably higher than those
which would occur following the use of products preserved with formalde-
hyde, show that formaldehyde in its use as a preservative is safe.
Formaldehyde is a very effective preservative for personal products. It is
effective at very low concentrations and is functional with most ingredients
across a wide pH range. It is generally only incorporated in products which
 TOXICOLOGY 331

are diluted during use and subsequently rinsed from the skin. Thus
consumer exposure, and the risk in terms of skin irritation and sensitization,
from these sources is only minimal.

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bronchial fibroblasts. Science, 228, 89-91.
99. Fontignie-Houbrechts, N. (1981) Genetic effects of formaldehyde in the mouse. Mutat.
Res., 88, 109-114.
100. Wright, C. et al. (1983). Mutagenic activity of Kathon, an industrial biocide and
cosmetics preservative containing 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-
4-isothiazolin-3-one. Mutat. Res., 119, 35-43.
101. Ashby, J. et af. (1985). Chloracetamide-N-metholol: an example of an in vitro and in
vivo cIastogen which is non-mutagenic to Salmonella. Mutat. Res., 156, (1-2), 19-32.
 TOXICOLOGY 335

102. Monte, W.C. et al. (1983) Mutagenicity of two nonformaldehyde-forming antimicrobial

agents. Food Chern. Toxicol., 21, (5), 695-696.
103. Scribner, H.E. et al. (1983) The genetic toxicology of Kathon biocide, a mixture of 5-
chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one. Mutat. Res.,
118, (3), 129-152.
104. Marnett, L.J. et al. (1985) Naturally occurring carbonyl compounds are mutagens in
Salmonella tester strain TA104. Mutat. Res., 148, (1-2), 25-34.
105. NTP (1989) Review o/Current DHHS, DOE and EPA Research Related to Toxicology,
Fiscal year 1989, National Toxicology Program, NTP-89-168. US Department of Health
and Human Services, Research Triangle Park, North Carolina.
106. Sakagami, Y. et al. (1988) DNA repair test of disinfectants by liquid rec-assay. Mutat.
Res., 193, (1),21-30.
107. Slesinski, R.S. et al. (1983) Mutagenicity evaluation of glutaraldehyde in a battery of in
vitro bacterial and mammalian test systems. Food Chern. Toxicol., 21, (5), 621-629.
108. Wilcox, P. et al. (1990) Comparison of Salmonella typhimurium TA102 with Escherichia
coli WP2 tester strains. Mutagenesis,S, (3), 285-291.
109. Zeiger, E. (1980) Unpublished data (cited in Wade et al. (1982) FDA By-lines No.1, p.
7).
110. Ruiz-Rubio, M. et al. (1985) Oxidative mutagens specific for A.T. base pairs induce
forward mutations to L-arabinose resistance in Salmonella typhimurium. Mutat. Res.
147, 153-163.
111. Sakagami, Y. et al. (1988) The evaluation of genotoxic activities of disinfectants and
their metabolites by umu test. Mutat. Res., 209, (3/4), 155-160.
112. Hemminki, K. et al. (1980) Comparison of alkylation rates and mutagenicity of directly
acting industrial and laboratory chemicals. Arch. Toxicol., 46, 277-285.
113. von der Hude, W. et al. (1988) Evaluation of the SOS chromotest. Mutat. Res., 203, (2),
81-94.
114. NTP (1982) National Toxicology Program Technical Bulletin, No.8, p. 11.
115. NTP (1985) Fiscal Year 1985 Annual Plan, National Toxicology Program, NTP-85-055,
US Department of Health and Human Services, Research Triangle Park, North
Carolina.
116. Yoon, J.S. et al. (1985) Chemical mutagenesis testing in Drosophila. IV. Results of 45
coded compounds tested for the National Toxicology Program. Environ. Mutagen., 7,
(3), 349-367.
117. Zimmering, S. et al. (1989) Chemical mutagenesis testing in Drosophila. VII. Results of
22 coded compounds tested in larval feeding experiments. Environ. Molec. Mutagen.,
14, (4), 245-251.
118. McGregor, D.B. et al. (1988). Responses of the L5178 tK+/tK-mouse lymphoma cell
forward mutation assay. II: 18 coded chemicals. Environ. Malec. Mutagen. 11, (1),91-
118.
119. Anon (1970) Symp. Fund Cancer Res., 23, 346 (cited in NIOSH, 1987. Registry of Toxic
Effects of Chemical Substances, 1987-1988 edition, DHHS (NIOSH) Pub. No. 87-114.
National Institute for Occupational Safety and Health, Cincinnati, Ohio, 1987).
120. Galloway, S.M. et al. (1985) Development of a standard protocol for in vitro cytogenetic
testing with Chinese hamster ovary cells: comparison of results for 22 compounds in two
laboratories. Environ. Mutagen., 7,1-51.
121. Tsutsui, T. et al. (1987) Assessment of the carcinogenic hazard of 27 substances used in
dental practices. Jap. J. Pharmacol., 43, 132P.
122. Tamada, M. et al. (1978) Mutagenicity of glutaraldehyde in mice. Bokin Bobai, 6, (2),
62-68 (Japan). (Chern. Abstr. 91, 135063X).
123. Mirsalis, J.C. et al. (1989) Measurement of unscheduled DNA synthesis and S-phase
synthesis in rodent hepatocytes following in vivo treatment: testing of 24 compounds.
Environ. Molec. Mutagen., 14, (3), 155-164.
124. Staples, R.E. (1983) Teratogenicity of formaldehyde. In Formaldehyde Toxicity,
Gibson, J.E. (ed.), Hemisphere New York, Chap. 6, pp. 51-59.
125. Marks, T.A. et at. (1980) Influence of formaldehyde and Sonacide (potentiated acid
glutaraldehyde) on embryo and fetal development in mice. Teratology, 22, (1),51-58.
336 PRESERVATION OF SURFACTANT FORMULATIONS

126. Anon. (1990) (Preliminary results of a gavage study of prenatal toxicity of glutar-
aldehyde in rabbits). Pestic. Toxic. Chern. News. 18, (33), 28.
127. Schmahl, D. (1985) Was the fuss about formaldehyde necessary? No risk of cancer to
the ordinary citizen. Rhein-Neckar-Zeitung, 22 February, 1985.
128. Krzeminski, S.F., et al. (1975) Fate of microbicidal 3-isothiazolone compounds in the
environment: modes and rates of dissipation. Agric. Food Chern., 23, 1060--1068.
129. Krzeminski, S.F., et al. (1975) Fate of microbicidal 3-isothiazolone compounds in the
environment: products of degradation. Agric. Food Chern., 23, 1068-1075.
13 The safe use of preservatives
D.N. MUNRO

The safe use of preservatives represents a specific example of a more

general need. That is, to ensure that in harnessing chemical and other
technologies for the benefit of man, the potential risks to health, to the
environment, and to plant and equipment, are all adequately assessed and
managed.
This chapter will focus on the avoidance of adverse occupational health
effects. This encompasses both the prevention of any such effects and
avoidance of significant interference with the wellbeing of those involved
in the manufacture or use of preservatives. It should not however be
forgotten that there will be a need, and often specific legal duty, to assess
and control adequately risks of physical and environmental damage.
Predominant amongst the ill health effects associated with the use of
preservatives are skin effects such as irritant and allergic dermatitis 1 ,2
which may occasionally also include photo allergenic effects. 3 There may
also be the potential for serious respiratory disease, 'biocide lung', which
comprises pneumonia and chronic pulmonary fibrosis 4 arising from
exposure to certain biocides.
Cases of ill health have been seen in manufacture of preservatives, but
they occur more commonly at the user stage. Many applications have been
implicated, including the textile finishing industry,S paint manufacturing
and use,6,7 metal working,S agriculture,9 the pottery industry,lO and health
care and food industry workers. 11 It is not always necessary to have contact
with the preservative itself for such problems to arise. There are reports for
example of symptoms arising from accidental exposure to formulations
containing only small quantities of preservative,12 and from exposure to
glues and pastes containing biocides.13 Exposure may not in fact be
immediately obvious, as in the unfortunate case of the deep sea diver who
developed generalised allergic dermatitis following the disinfection of his
wet suit with a 'preservative-containing' cleaning solution. 14
How then are we to ensure that ill health effects do not arise? A first
principle to establish is that there is nothing which has no inherent
toxicological hazard. This is hardly a novel observation, but one which is
frequently overlooked. Clearly even substances vital to life such as salt and
sugar will have a significant toxic effect if the dose is sufficient, although
they are usually regarded as being 'safe'. Paracelsus, the 'founding father'
of toxicology summarised this concept over four hundred years ago!
338 PRESERVATION OF SURFACTANT FORMULATIONS

AIle Dinge sind Gift

und nicht ohn Gift;
allein die Dosis macht,
dass ein Ding kein Gift ist.
Paracelsus (1493-1541).
Loosely translated this states that 'All things are poisonous, it is the dose
which separates the medicine from the poison'. This insight established for
the first time the concept of a 'dose response curve' and more importantly
that of a 'safe dose'.
This leads us to the question of what it is that we regard as 'safe'. The
title of this chapter is 'Safe use of preservatives', yet there can be nosuch
thing as absolute safety. Beware all who demand or promise absolute
safety; they will either be naive or corrupt!
The concept of safety is better regarded in terms of risk. When we talk
about managing safety it is actually risk that we are concerned to manage;
to reduce risk to an acceptable or accepted level. That these two levels are
not necessarily the same becomes immediately obvious when one considers
everyday risk. Consider the risk of premature death associated with
cigarette smoking or motorbike riding in comparison with the requirements
imposed on the nuclear and aviation industries!
The first stage in managing risk is to identify the hazard with which we
are dealing. This, in the case of a preservative, or any other chemical, will
require us to identify the inherent toxicological hazard. For substances in
commercial use this will be facilitated by regulatory requirements for
toxicological testing and product registration. For example, in the USA the
Environmental Protection Agency (EPA) initiates and administers require-
ments, derived primarily from the Toxic Substances Control Act, for the
registration and use of such substances. In Europe, various European
directives and member state regulations, largely based on the Dangerous
Substances Directive (7th Amendment), cover the issues such as the
notification of new substances and the marketing, labelling and transport
of hazardous materials. Similar specific regulatory provisions apply in
countries such as Canada, Australia, Japan and elsewhere.
The consequence for the user is that the preservative will be supplied
with information relating to its hazardous properties. This information
will be in the form of a manufacturer's safety data sheet, labels and possibly
supporting technical information. This documentation will include data
derived from standardised toxicological testing packages specified by the
regulatory authorities. It will often also be supplied in a standard format
and be interpreted as standard phrases and symbols. These data are the key
that unlocks the door leading to risk assessment and ultimately to 'safe
working'.
Although the hazard data is key, it will not in fact be the hazard that
necessarily predominates in the level of risk that arises in use. It must be
 SAFE USE OF PRESERVATIVES 339

risk = f X hazard

function f is largely a measure of exposure

task
intensity
f is dependent upon duration
frequency etc.

risk = exposure X hazard

Figure 13.1 Relationship between hazard and risk.

recognised that whilst the toxicity of a material is an 'inherent property' of

the substance, the risk will also be influenced by many other factors.
Amongst these we must include all those factors which can contribute to
the actual or potential exposure of workers to the material in question.
This concept is illustrated in Figure 13.1.
If an effective strategy to minimise 'risk' is to be defined then clearly this
can be usefully divided into two key elements. First we can seek to
minimise the hazard. Thereafter we must concentrate on managing those
risks which might arise.
Considering then the first element, the hazard of the material, how
should this be approached? Clearly it would be advantageous to select the
least hazardous preservative practicable. In practice however the choice of
a particular agent is going to be based on a range of criteria. Regulatory
status, availability, technical suitability, cost and other factors will all
obviously be crucial and usually predominant influences in the selection of
a particular agent. It is important however to evaluate the hazard profile of
short-listed candidate preservatives before a final selection is confirmed.
The nature of the hazard is likely to have consequences throughout the
product lifecycle, from manufacture through to use and ultimate disposal,
and thus it merits serious consideration.
The starting point for such consideration will usually be the manu-
facturer's safety data sheet and possibly supporting technical literature.
Detailed specific guidance on 'interpreting biocide health and safety data
sheets' has been published. 15 A guide to the key elements of a typical
safety data sheet format and its interpretation and implications is given in
Table 13.1.
The second stage in the strategy is to manage those risks which might
arise as a consequence of use of the chosen agent. This process will
essentially seek to eliminate or at least minimise actual and potential
exposures to a level below those at which we can expect to see evidence of
any ill health effects or significant nuisance.
The risk management process will be dependent upon the context. For a
new workplace or process there are opportunities to influence effectively
340 PRESERVATION OF SURFACfANT FORMULATIONS

Table 13.1 'Typical' hazard data sheet format

1. Identification What is the name?

Are there alternatives?

2. Product description Solid or liquid?

What does it look like?
Appearance/odour?

3. Summary Is the material hazardous?

Exposure standard?

4. Physicochemical data Liquid/solid?

Boiling point?
Flash point?
Other properties?

5. Stability and reactivity Avoid contact with other materials?

6. Storage How to store?

Special requirements?

7. Handling Special handling precautions?

8. Personal protection Is PPE generally needed?

What about my specific use?

9. Spillage/accidental release What do I need to do?

10. Waste disposal Any special advice?

Any specific requirements?

11. First aid measures What procedures?

Antidotes etc?

12. Fire and explosion Will it burn/explode?

How do I deal with it?

13. Health hazard What if I breathe it?

It gets on skin/eyes?
Is swallowed?
Long term effects?

14. Environmental information Effects?

15. Regulatory information Labelling?

Rules for transport

PPE = personal protective equipment.

the level of risk right from the initial conceptual level. The later the points
of influence in implementing change to process, to equipment or to
procedures are effected the more difficult, more expensive and less
effective they will be. The major stages where input on the control of risk
can be effected are illustrated in Table 13.2.
 SAFE USE OF PRESERVATIVES 341

Table 13.2 Strategic opportunities to control risk

Conceptual Best use of expertise.

Strategic Expense largely time only.
Process design Opportunity for step changes and continuous
improvement (CI).

Project specification Expensive in time, materials and cash. Can

Project implementation achieve sustained improvements.
Workplace modification

Audit of control Involves local expertise.

Workplace monitoring Ongoing requirement.
Mainly preventive but can be used as a tool
for CI.

Worker exposure monitoring Expensive in time, expertise and cash.

Health surveillance Ongoing requirement.
Essentially quantification of level of failures.

Costs

TIME
(Stage of project)

Figure 13.2 Cost and effectiveness of risk control measures.

The relative costs and effectiveness of intervention at various stages are

highlighted in Figure 13.2.
The more common scenario unfortunately is that an existing application
or facility exists, and it is within these constraints that it is required to
establish and maintain safe working. We will consider in some detail how
the strategy outlined above can be applied in these circumstances.
The initial stage should be to carry out an appropriate risk assessment.
This assessment will not necessarily invoke sophisticated analysis or
quantitative techniques. The objective of the risk assessment is to
342 PRESERVATION OF SURFACTANT FORMULATIONS

determine whether or not it is probable that there will be exposure

sufficient to trigger an adverse effect.
The initial stage of such a risk assessment will be the identification of the
hazard. What is the potential to induce adverse effects? The answer can be
based on an understanding of the toxic and physicochemical properties of
the substance gleaned from the safety data sheet or other sources.
The next stage involves an assessment of exposure potential. The routes
and patterns of all potential exposures should be established. The nature,
duration, frequency and magnitude of exposures should be estimated
based on knowledge of the specific operation. It is important that all work
patterns are considered, including abnormal situations like plant start up
and shut down, routine and 'breakdown' maintenance, and factors such as
shift and seasonal work variations.
A judgement then needs to be made as to whether or not exposure is
adequately controlled. Very often the answer will be obvious. Either the
exposure will be insignificant, or, alternatively so gross that it is clear that
measures should be taken to mitigate the exposure. In other cases there
may be established exposure standards such as threshold limit values
(TLV) published by the American Conference of Governmental Industrial
Hygienists (ACGIH) ,16 permitted exposure limits (PEL) published by the
National Institute for Occupational Safety and Health (NIOSH) or other
mandatory national standards such as the occupational exposure standards
(OES) and maximum exposure limits (MEL) which apply in Great
Britain. 17 In the absence of such limits for the substance in question it may
be necessary to refer to industry practice, advice from trade associations or
from the supplier.
Alternatively it may be possible to classify the substance into a particular
toxic hazard category, and then assume some form of generic exposure
limit towards which control of exposure should be targeted. An example of
one such approach is illustrated in Figure 13.3. Such approaches have been
widely debated,18.19 have been adopted by a number of leading chemical
manufacturers 20 and have been published in various industry guidance
documents. 21 The prime objective is to enable the relative hazards of
substances to be readily compared and to suggest generic exposure limits
against which the requirements for, and the effectiveness of, control
measures can be determined.
In order to sensibly estimate exposure potential, and subsequently
possibly facilitate its elimination or minimisation, it is first important to
understand the nature of this exposure. An individual's total occupational
exposure to a substance in use represents the sum of the individual's task
exposures, incident-related exposures and background exposures. These
contributory exposures may be defined as follows.
Task-related exposures arise from planned process tasks, where the
nature of the task may lead to a release to atmosphere or to direct contact
 SAFE USE OF PRESERVATIVES 343

COSHH TOXICITY CLASSIFICATION GRID

Clas Properties D.E.S, Range Risk Phrases

R20 Harmful by inhalation
R21 Harmful by skin contact
VetyToxic < 0,1 mgJtvf R26,27,28 R22 Harmful if swallowed
H1 Respiratory Sens~iser < 0,1 ppm R42 R23 Toxic by inhalation
Potent Carcinogen (Human) R45 R24 Toxic by skin contact
R25 Toxic ff swallowed
R26 Very toxic by inhalation
Toxic > 0,1 ppm R23,24,25 R27 Very toxic byskin
Corrosive < 10 ppm R34,35,41 R28 Very toxic ff swallowed
H2 R34 Causes bums
3
Animal Carcinogen < 1 mg/M R45 R35 Causes severe bums
Skin Sens~iser R43 R36 Irritating to eyes
'Unknown' Toxicity R37 Irr~ating to respiratory system
Harmful > 10 ppm R20,21 ,22 R38 Irritating to skin
Skin/Eye Irritants < 500 ppm R36,37,38 R39 Danger of very serious
M 3 irreversible effects
Low Potency Carcinogene < 10 mglM R40 R40 Risk of irreversible effects
(E,C, Cat 3,) R41 Risk of serious eye damage
R42 May cause sens~isation by
Non Hazardous inhalation
Non Irritant R43 May cause skin sens~isation
L Non Genotoxic
> 500 ppm R45 May cause cancer
Non Sens~ising

Figure 13.3 Illustration of a scheme to classify substances by toxicological hazard. Redrawn

from Zeneca Specialties 'Control of Hazardous Substances'.

with the substance. Such tasks typically include filling or emptying vessels
or packages, maintenance work etc.
Incident-related exposures result from unplanned, although possibly
foreseen, events such as spillages or a planned task going wrong. Minor
frequent events may contribute to personal exposure either directly, e.g.
someone exposed locally to a leak, or by increasing the background
concentration. Infrequent events may cause acute problems but are
unlikely to contribute significantly to routine exposures.
Background exposure is that exposure which would be experienced by
someone being present in the workplace, but not directly carrying out any
operations involving the specific substance. Background exposures will
arise from fugitive emissions, such as continual leaks from equipment,
vents and drains and so forth and additionally from emissions from tasks
and incidents.
Moving on to the practical application of these understandings it is usual
to subdivide a workplace into a succession of smaller elements in order to
facilitate the risk assessment process. This involves splitting operational
activities down into a series of 'unit operations' such as charging materials,
sampling, packing off and so forth. The exposure potential arising from
each of these elements of the work can then easily be assessed taking into
account factors such as the methods employed, the scale and intensity of
344 PRESERVATION OF SURFACTANT FORMULATIONS

OCCUPATIONAL HYGIENE GUIDANCE-GENERIC CONTROL MEASURES

INHALATION Section INH Sheet 01

CHARGING TO PROCESS PLANT ITEMS

VOLATILE UOOIDS
I
I
•
PACKAGE TYPE I
I DRUM I IBC I BULK I

.
IHAZARD CLASSIFICATION I
I t
LCharging System I
1
.
IHAZARD CLASSIFICATION I
I Open I Sealed I
I H I M I L I I H I M I L I
---.J L-
! I

REQUIRES
I
• REQUIRES
! I I
•
REQUIRES REQUIRES
Suction lance Suction lance High integrity, dry Well ventilated area
with local exhaust with local exhausl break couplings
ventilation ventilation Self-draining CONSIDER
Ventilated booth transfer lines High integrity, dry
Expelled air cleaning CONSIDER Vent gas control break couplings
Ventilated booth CONSIDER Self-draimng
CONSIDER ~Pelled air cleaning transfer lines
Segregation Segregation Vent gas control
Inlegral package & Integral package &
system detoxing system deloxing

Figure 13.4 Example of generic guidance on exposure controls. Redrawn from Zeneca
Specialties 'Control of Hazardous Substances'.

operations, and the physical properties of the material being handled. The
level of risk arising from the exposure potential can then be matched to an
appropriate control regime. It is possible to develop 'generic guidance' in
this area (an illustrative example is given in Figure 13.4).
In practice it will be necessary to consider each task element in tum and
then apply any necessary control, taking into account the hierarchy of
available occupational hygiene control measures. This hierarchy represents
both 'best practice' and, in certain cases, specific legal requirements. 22 It is
based on the simple premise that control at source, resulting in a safe
workplace, will be more effective and easier to sustain, than a strategy
which relies upon an individual routinely to use personal protective
equipment (PPE), or to follow rigorous procedures in order to prevent
excessive exposure.
The hierarchy of occupational hygiene controls is as follows:
• elimination
• substitution
• total enclosure
• segregation
 SAFE USE OF PRESERVATIVES 345

• partial enclosure (with LEV)

• local exhaust ventilation (LEV)
• general ventilation (dilution)
• personal protective equipment
• procedural controls
• provision of amenities
• training.

Considering the hierarchy of available controls the first option is to

substitute a toxic hazard with a material which is either non-toxic or, at
least, is of lesser toxicity. As referred to earlier, other technical
considerations may determine the agent of choice. They may not however
preclude changes in formulation, e.g. aqueous rather than solvent-based
formulations, changes in physical form, e.g. pellets, granules or flakes
rather than powders or changes in particle size such that adequate control
may be more readily achieved.
Where use of a 'non-hazardous' material is impracticable, consideration
should be given to enclosing the process totally, preferably under slight
negative pressure, so as totally to separate the operator from the process.
Consideration must however also be given to ease of maintenance and the
potential exposures that might arise from such activities including any
cleaning.
Where complete enclosure is not practicable then incomplete enclosure,
supplemented by effective local exhaust ventilation, can often achieve
similar standards of containment. It will of course be necessary to ensure
that the local exhaust ventilation is properly designed, installed and
regularly tested in order to achieve, demonstrate and sustain acceptable
standards of control. Fortunately authoritative guidance on these topics is
readily available. 2 3-25 Examples of this approach range from fume
cupboards, through various types of flow booths (e.g. horizontal laminar
flow booths and downdraught booths), to the use of 'through pan
draughting' on chemical reactors.
If, due to the nature of the operation, even partial enclosure is
impracticable, local exhaust ventilation can still be used to good effect. It
should be noted however that effectiveness will be critically dependent on
the position of the exhaust inlet in comparison with the point(s) of emission
of the substance being controlled. Effectiveness will also be dependent
upon the volume and velocity of the exhaust air supplied. The velocity, at
the furthest point of capture, will need to be sufficient to capture the
pollutant, and to convey it in the desired direction, despite any
interference from thermal effects or other induced draughts. It should be
noted as a rule of thumb that the velocity of air will have fallen to one tenth
of the duct velocity at a distance 'one duct diameter' away from the orifice.
If that seems hard to believe then blowout the flame from a match at a
346 PRESERVATION OF SURFACTANT FORMULATIONS

distance of approximately six inches. Now try to suck out the flame at the
same distance!
Good general ventilation, even where supplemented by positive forced
ventilation, should not normally be relied upon for control of exposure.
Although there will be dilution of the contaminant this may be inadequate
in the case of an operator close to the source of exposure. It will also lead
to general contamination of the workplace, permit contamination of others
who would not otherwise be affected, and may well be expensive in terms
of volumes of air extracted and input.
Where an effective standard of control cannot otherwise be achieved it
may be necessary to rely upon personal protective equipment (PPE). This
should generally be seen as a temporary measure whilst the standard of
control is improved. It must be recognised however that there will be some
situations, e.g. certain maintenance activities, where PPE may be the only
viable alternative. In these situations, as in all others where there is
reliance on PPE as a prime means of protection, consideration needs to
be given to a range of consequent issues. These will include selection
of appropriate equipment, issues such as nominal protection factors,
permeation and degradation rates, useability for the tasks in question,
regulatory compliance etc. There will be a need to provide for suitable
maintenance, cleaning, storage, repair and replacements. Employees must
be suitably trained, validated and supervised in the use of PPE. These
considerations are only indicative of the issues to be addressed and are not
exhaustive. They do however begin to illustrate that, whilst PPE is never
the-preferred solution, neither is it likely to be a cheap option in the long
term.
Whatever the control mechanisms implemented they will need to be
supported by effective training and implementation of appropriate
procedures. The effectiveness of the whole should never be assumed. The
effectiveness must be demonstrated in some way and potential enhance-
ments identified and incorporated into improvement plans. An illustrative
example is given in Table 13.3.
Monitoring of the effectiveness of control may involve checks on
performance of the equipment, monitoring of exposures to contamination,
health surveillance of workers, or a mixture of all three techniques. In the
absence of any specific overriding legal requirement the emphasis should
be on the former technique. Ideally there will be some easily measured
parameter, face velocity, duct air velocity, pressure drop etc., that equates
to previously established effective control. Such data are usually required
for design purposes, but if not already documented can readily be obtained
at commissioning or later. This affords the opportunity to monitor the
success of control on an ongoing basis, and to detect and remedy any trend to
reduced effectiveness before it becomes otherwise obvious.
A second alternative is to implement a comprehensive monitoring
 SAFE USE OF PRESERVATIVES 347

Table 13.3 Task assessment record

Risk assessment
Task Transfer of biocide (50 kg) from 200 I drum to a mixer tank.
Method Pumped via flexible hose. Weight measured 'by difference'
from drum on weighscale.
Material hazard Causes burns. Harmful if swallowed. A potential skin
sensitiser. Non-volatile viscous liquid.
Risk Primarily arises from skin contact.
Protection needed to avoid burns will be adequate to prevent
skin sensitisation arising.
Control Must avoid skin and eye contact and the inhalation of any
mists or aerosols which might be generated. (Vapour will not
be a problem - unless the material is heated)
Conclusion Current controls are unacceptable.

Improvement/action plan
Immediate (1) Transfer material by vacuum (applied to the mixer tank)
rather than by pump - eliminates risk of mists, minimises
the exposure from maintenance/cleaning.
(2) Provide drip can + detox solution in order to collect/
neutralise drainings from the transfer line and prevent any
contamination of the area.
(3) Introduce a PPE regime (gloves, face shield and apron or
disposable overall) and check adequacy of washing and
showering facilities.
(4) Formalise written procedure in consultation with the
operatives. Include all foreseeable circumstances.
(5) Train and validate, supervise and audit.
Medium term As above +
(1) Seek alternative less viscous/hazardous biocide.
(2) Modify charging facility - e.g. 'drt-break' couplings,
bundedlsumped area, easy clean finishes etc.
Longer term As above +
(1) Automated enclosed dosing system.
(2) Integral detox of equipment and packages.

programme to establish workplace and personal exposures. Thus we

quantify the dose delivered to the workers, but probably will not detect any
loss of control until after the event! The need for a comprehensive
monitoring programme is referred to here as, unfortunately, 'spot samples'
are unlikely to have any real significance. Atmospheric concentrations will
vary rapidly with time, distance and circumstance. Plots of data tend to
follow a distribution not unlike that of a log normal population. 26 Thus a
single or a few isolated samples are unlikely to have statistical significance
and hence will defy scientifically valid and defensible interpretation.
Monitoring does of course have its role to play, but it is enough to state
here that it may as often be abused as used! References are given to
introductory and other texts for those who would wish to investigate
further. 27 ,28
A third alternative is to rely upon some form of health surveillance.
348 PRESERVATION OF SURFACTANT FORMULATIONS

Again, although health surveillance has many useful roles to play,22 it is

not ideally placed as a measure of effective workplace control. A whole
chain of events will need to be in place before health surveillance identifies
a problem. First, we will need to have a failure of control, secondly this will
need to have led to exposure, at a concentration, time, duration etc.
leading to symptoms. Thirdly this must be coincident with health
surveillance, and finally the link between condition and exposure must be
made before any queries about control will be raised for investigation.
Clearly this is a somewhat insensitive and unreliable feedback loop!
In summary then the key to safe working with preservatives is as follows.
To ensure that all hazards are identified. From the hazard data, taking into
account all the factors which might contribute to exposure, assess the risks
which may arise. Where this risk is not acceptable it should be controlled
by direct and effective means to a level representing an insignificant risk.
Thus matching and sustaining control measures appropriate to the level
of risk is the answer. This may not require sophisticated techniques but
must encompass all actual and foreseeable work activities and circum-
stances.
I referred earlier to Paracelsus who expounded the truism that all
substances are hazardous to a greater or lesser extent. I would also suggest
a corollary - that there are none so hazardous that they cannot be handled
safely! Certainly preservatives have the demonstrated capacity to cause
harm if they are not adequately controlled. They are not however, by any
means the most aggressively toxic materials handled by industry. III health
and discomfort must never be accepted as an inevitable consequence of
their use. The application of the approach outlined above can go a
long way towards preventing ill health effects being realised in those
manufacturing and using preservatives.

References

1. Goh, C.L. (1985) Irritant dermatitis from tri-N-butyl tin oxide in paint. Contact
Dermatitis, 12 (3), 161-163.
2. O'Driscoll, J.B. and Beck, M.H. (1988) Occupational allergic contact dermatitis from
Kathon WT. Contact Dermatitis, 19 (1), 63.
3. Norris, P.G., Hawk, L.J.M. and White, LR. (1987) Photoallergic contact dermatitis from
Fentichlor. Contact Dermatitis, 18 (5), 318-320.
4. Lings, S. (1982) Pesticide lung: a pilot investigation of fruit growers and farmers during
the spraying season. Br. J. Indust. Med., 39 (4), 370-376.
5. Kawai, K., Nakagawa, M., Sasaki, Y. and Kawai, K. (1993) Contact dermatitis from
Kathon 930. Contact Dermatitis, 28 (2), 117-118.
6. Sanz-Gallen, P., Planas, J., et al. (1992) Allergic contact dermatitis due to 1,2-
benzisothiazolin-3-one in paint manufacture. Contact Dermatitis, 27 (4) 271-271.
7. Oleage, J.M., Aguirre, A. et al. (1992) Allergic contact dermatitis from Kathon 893.
Contact Dermatitis, 27 (5), 345-346.
8. de-Boer, E.M., van-Ketel, W.G. and Bruynzeel, D.P. (1989) Dermatoses in metal
workers. Contact Dermatitis, 20 (4),280-286.
 SAFE USE OF PRESERVATIVES 349

9. Anon. (1983) Precautions to be taken when cooking narcissus bulbs in formalin.

Veiligheid, 59 (10), 503.
10. Roberts, D.L., Messenger, A.G. and Summerly, R. (1981) Occupational dermatitis due
to 1,2-benzisothiazalin-3-one in the pottery industry. Contact Dermatitis, 7 (3), 145-147.
11. Wilkinson, S.M., Schouten, P. et al. (1991) Allergic dermatitis from Tego 51. Contact
Dermatitis, 24 (1), 74, 75.
12. Clark, E.G. (1987) Risk of isothiazolines. f. Soc. Occupational Med., 37 (1), 30-31.
13. Mathias, c.G.T. (1983) Contact dermatitis to a new biocide (Tektamer 38) used in a
paste glue formulation. Contact Dermatitis, 9 (5), 418.
14. Munro, C.S., Shields, T.G. and Lawrence, C.M. (1989) Contact allergy to Tego 103G in
a deep sea diver. Contact Dermatitis, 21 (4),278-279.
15. Health and Safety Commission Paper and Board Advisory Committee (1990) Interpreting
Biocide Health and Safety Data Sheets.
16. ACGIH (1993) Threshold Limit Values for Chemical Substances and Physical Agents and
Biological Exposure Limits, 1993-1994.
17. Health and Safety Executive London (1994) EH40194 Occupational Exposure Limits
1994.
18. Gardner, R.J. and Oldershaw, P.J. (1991) Development of pragmatic exposure control
concentrations based on regulatory risk phrases. Ann. Occupational Hygiene, 35, 57-59.
19. Money, C.D. and Munro, D.N. (1992) An integrated system to assist in ensuring
satisfactory occupational hygiene performance within a diverse business. Proceedings of
the First International Occupational Hygiene Association Meeting, Brussels 1992.
20. Zeneca Specialties (1990) Control of Substances Hazardous to Health - Assessment
Training Manual.
21. Chemical Industries Association London (1993) Safe Handling of Colourants (2) -
Hazard classification and the selection of occupational hygiene strategies.
22. HMSO London (1989) Control of Substances Hazardous to Health Regulations 1988.
23. Health and Safety Executive London (1993) HS(G)37 An Introduction to Local Exhaust
Ventilation, HSE Books.
24. ACGIH (1992) Industrial Ventilation - A Manual of Recommended Practice, 21st edn,
ACGIH Ann Arbor, Michigan, USA: Edward Brothers.
25. Health and Safety Executive London (1990) HS(G)54 The Maintenance, Examination
and Testing of Local Exhaust Ventilation, HSE Books.
26. Rappaport, S.M. et al. (1981) Sampling in the assessment of continuous exposure to
acutely toxic chemicals. Part 1 Strategy. f. Am. Indust. Hygiene Assoc., 42, 831-888.
27. Health and Safety Executive London (1989) EH42 Monitoring Strategies for Toxic
Substances.
28. Corn, M. (1987) Strategies of air sampling. In Recent Advances in Occupational Health,
McDonald (ed.), Churchill Livingstone, London, pp. 199-202.
14 Regulation of preservatives in the USA
M.E. BURT

14.1 Introduction

Depending on their use, antimicrobials or preservatives are regulated in

the United States by the US Environmental Protection Agency (EPA) and
the US Food and Drug Administration (FDA). The use of preservatives
is regulated by the EPA under the Federal Insecticide, Fungicide and
Rodenticide Act, as amended (FIFRA). This law regulates the use of all
pesticides, induding antimicrobials, in the USA. FIFRA was first enacted
in 1947 and has been amended several times, most recently in 1988. The
use of preservatives in indirect food additive situations is regulated by the
FDA, operating under the Federal Food, Drug and Cosmetic Act
(FFDCA). The FFDCA was first enacted in 1938 and the Food Additive
Amendment was passed in 1958. The purpose of this chapter is to provide
an overview of the regulation of preservatives in the USA by the EPA,
FDA, and other relative schemes.

14.2 Industrial preservatives

The use of antimicrobials or preservatives in industrial use patterns and as

preservatives in agrochemicals is regulated by the EPA under FIFRA. The
EPA is charged with the promulgation of regulations, policy and
procedures so that the use of antimicrobials can be registered or accepted
for use. Prior to registering the use of any antimicrobial, the EPA requires
that certain data requirements be fulfilled. These data requirements
address the areas of product chemistry, environmental fate, toxicology,
and wildlife and aquatic organism testing. Data requirements are specified
in EPA's pesticide regulations 1 and are reviewed herein.
The applicant for registration is required to provide complete information
on the composition of the antimicrobial active ingredient, induding
certified upper and lower limits of concentration for both the technical
product and end-use formulations, CAS registry number, and chemical
identity. The applicant must also provide a description of the manufacturing
or formulation process, a discussion on the formation of impurities, the
results of chemical analysis and an analytical method, suitable for use by
 REGULATION IN THE USA 351

regulatory authorities to analyse samples collected in the marketplace.

Additionally, certain physical/chemical properties data are required for
both the technical and end-use formulations.
Environmental fate data requirements for most industrial antimicrobial
uses are fulfilled with the submission of a hydrolysis study. Wildlife and
aquatic organism data requirements are as follows: avian oral LD50
(mallard duck or bobwhite quail); avian dietary LC50 (mallard duck and
bobwhite quail); rainbow trout and bluegill LC50; Daphnia LC50.
The registration of all pesticides must be supported by a complete acute
toxicity data base for both the technical grade of the active ingredient and
the end-use formulations. These requirements include: acute oral toxicity-
rat; acute dermal toxicity; primary eye irritation; primary dermal irritation;
and dermal sensitization. The results of these studies are used to determine
appropriate label warning and precautionary statements as specified in the
regulations. 1 Also, inhalation toxicity testing will be required if the end-use
pattern specifies spray or aerosol application methods. The EPA toxicity
categories and related precautionary statements are reviewed in Table
14.1.
352 PRESERVATION OF SURFACTANT FORMULATIONS

Table 14.1 Continued

Toxicity Precautionary statements by toxicity category

category
Oral, inhalation or dermal toxicity Skin and eye local effects

Fatal (poisonous) if swallowed Corrosive, causes eye and skin

(inhaled or absorbed through damage (or skin irritation). Do
skin). Do not breathe vapor (dust not get in eyes, on skin, or on
or spray mist). Do not get in clothing. Wear goggles or face
eyes, on skin, or on clothing. shield and rubber gloves when
Front panel statement of practical handling. Harmful or fatal if
treatment required. swallowed. Appropriate first aid
statement required.

II May be fatal if swallowed (inhaled Causes eye (and skin) irritation.

or absorbed through the skin). Do not get in eyes, on skin, or on
Do not breathe vapors (dust or clothing. Harmful if swallowed.
spray mist). Do not get in eyes, Appropriate first aid statement
on skin, or on clothing. required.
Appropriate first aid statements
required.

III Harmful if swallowed (inhaled or Avoid contact with skin, eyes or

absorbed through the skin). clothing. In case of contact
A void breathing vapors (dust or immediately flush eyes or skin
spray mist). Avoid contact with with plenty of water. Get medical
skin (eyes or clothing). attention if irritation persists.
Appropriate first aid statement
required.

IV No precautionary statements No precautionary statements

required. required.

The requirement for sub chronic and chronic toxicity testing varies,
depending on whether or not the proposed use is of low, medium or high
exposure (1987 EPA antimicrobial data call-in). For the most part,
industrial uses of antimicrobials are considered to be low exposure uses
and a minimum set of sub chronic data are required. These studies include a
90-day study (oral or dermal, as appropriate) and a teratology study in one
species. Also, a genotoxicity test battery to assess the active ingredient's
potential for gene mutation, structural chromosomal aberration or other
genotoxicity effects are required for all antimicrobials. EPA will require
exposure assessment information. This information would confirm the low
potential for human exposure to industrial use antimicrobials following the
labeled use directions, thus obviating the requirement for chronic toxicity
testing.
Data submitted to EPA in support of product registrations are subject to
exclusive use by the original registrant for a period of 10 years after initial
registration, as provided by FIFRA. Also, FIFRA entitles the original data
submitter to data ownership protection for a period of 15 years after
 REGULATION IN THE USA 353

submission to EPA. During this time, the original data submitter can claim
compensation for the use of data by any other party citing that data at EPA
in order to gain product registration.
Although the use of antimicrobials for agrochemical preservatives is
registered by the EPA, the EPA must also be petitioned for an exemption
from the requirement to provide a tolerance for residues of the
antimicrobial when used as an inert ingredient in pesticide formulations
applied to growing crops (or raw agricultural commodities after ha~est, or
to animals). The amount of preservative in agrochemicals is at a low level
and residues in or on crops would usually not be of concern to the Agency.
Generally, data requirements to support a petition for the exemption from
the requirement of a tolerance are similar to those for antimicrobial
registration.
In addition to obtaining EPA registration, each state or US territory,
with few exceptions, requires that antimicrobials be registered in the state
prior to marketing. Registration requirements vary from state to state but
most require that an application for registration be supported by a copy of
the product label, proof of EPA registration (copy of EPA stamped
accepted label), a material safety data sheet and the appropriate
registration fee. Only the state of California requires that applications for
registration be supported by a complete set of product safety data and
efficacy data. Products are then registered by the California Environmental
Protection Agency after a complete and thorough data review.
If an antimicrobial is to be registered by EPA as a preservative in
adhesives used for food packaging or in components of paper and
paperboard (i.e. paper coating compositions), appropriate indirect food
contact clearances must first be obtained from the FDA. The clearances
are granted upon the submission and review of a petition containing
appropriate chemistry, product safety and efficacy data.

14.3 Indirect food additives

The FDA's chemistry requirements for indirect food additives are

reviewed. 2 In addition to basic chemical identity information, the FDA
recommendations address the requirement for food migration data. Food
migration studies are designed to estimate the amount of test substance
(i.e. preservative) that would migrate from food packaging material into
food. The results of the food migration study are used in conjunction with
the product safety data to estimate human exposure. An acceptable daily
intake (ADI) of the test substance is derived from produc~ safety data and
an estimated daily intake (EDI) is derived from the food migration study.
Simply put, if the EDI does not exceed the ADI, the FDA would approve
the proposed use and publish the regulation. 3
354 PRESERVATION OF SURFACTANT FORMULATIONS

The FDA's requirements for product safety data for direct food additives
or color additives are outlined in the Agency's 'Redbook'. 4 However, the
Redbook states that these guidelines do not specifically apply to indirect
food additives since they are not intentionally added to food but are
articles or components of articles used for food packaging. Any presence of
the indirect food additive in food items would then be caused by the
migration or extraction from the food contact surface into food. FDA
indicates that product safety data recommendations for indirect food
additives differ from those of direct additives and recommends that the
Agency be contacted for additional information.
Briefly, the FDA's requirement for product safety or toxicity testing for
indirect food additives vary, depending on dietary exposure potential
(determined by migration studies). For 'virtually nil exposure' (less than
0.05 ppm), only an acute oral rodent study would be required. Toxicity
tests for indirect additives with insignificant exposure (greater than
0.05 ppm but less than 1.0 ppm) would include subchronic studies and
genotoxicity studies. Petitions for indirect food additives with significant
dietary exposure (greater than 1.0 ppm) would be supported by a complete
chronic data base, including rodent lifetime feeding studies and possibly a
metabolism study.
Preservatives permitted by FDA for use in adhesives and as paper
and paperboard components in contact with food items are listed in
Tables 14.2, 14.3 and 14.4. In some cases, the regulations 6 provide cross
references between specific areas (e.g. preservatives permitted for use as
components of paper and paperboard in contact with fatty or aqueous food
may be safely used as components of paper and paperboard in contact with
dry food).

14.4 Cosmetic preservatives

In the United States, cosmetics have been self regulated since 1938. A
voluntary registration program was initiated by the Cosmetics, Toiletries
and Fragrance Association (CTFA) with the cooperation and support of
the US FDA in the early 1970s. Only finished cosmetics are registered.
Cosmetic ingredients, per se, are no longer included in the voluntary
registration program. However, it is important for ingredients to be listed
in the CTFA Ingredient Dictionary5 as part of the self-regulation process.
There are three levels in the FDA voluntary cosmetic registration
program. They are: registration of the manufacturing facility; registration
of the cosmetic product; and submission of cosmetic experience reports. 6
Cosmetic registration includes complete disclosure of all ingredients,
including preservatives. The experience reports deal only with verified
medical complaints and not with product performance complaints.
 REGULATION IN THE USA 355

Table 14.2 Preservatives permitted for use as components of adhesives intended for use in
packaging, transporting, or holding food. Title 21 CFR Part 175.105. 6

n-Alkyl (C 12 , C 14 , or CIS) dimethyl (ethylbenzyl) ammonium cycloehxylsulfamate

Ammonium benzoate
1,2-Benzisothiazolin-3-one
p-Benzoxyphenol
Benzyl bromoacetate
p-Benzoxyphenol
Bis(tri-n-butylin) oxide
Bis( trichloromethyl)sulfone
2-Bromo-2-nitro-l,3-propanediol
Chlorinated pyridine mixture with active ingredients conslstmg of 2,3,5,6-tetrachloro-4-
(methylsulfonyl) pyridine, 2,3,5,6-tetrachloro-4-(methylsulfinyl) pyridine and
pentachloropyridine
1-(3-Chloroallyl)-3,5, 7 -triaza-I-azoniaadamantane chloride
4-Chloro-3,5-dimethylphenol (p-chloro-m-xylenol)
4-Chloro-3-methylphenol
5-Chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one mixture at a ratio
of 3 parts to I part, manufactured from methyl-3-mercaptopropionate. The mixture may
contain magnesium nitrate at a concentration equivalent to the isothiazolone active
ingredients (weight/weight).
Coconut fatty acid amine salt of tetrachlorophenol
Copper 8-quinolate
1,2-Dibromo-2,4-dicyanobutane
2,6-Di-t-butyl-4-methylphenol
4-(Diiodomethylsulfonyl) toluene
3,5-Dimethyl-l ,3,5 ,2H-tetrahydrothiadiazine-2-thione
Ethyl hydroxyethylcellulose
Glutaraldehyde
5-Hydroxymethoxymethyl-l-aza-3, 7-dioxabicyclo[3.3.0]octane, 5-hydroxymethyl-I-aza-3,7-
dioxabicyclo[3.3.0]octane, and 5-hydroxypoly-[ methyleneoxy ]methyl-l-aza-3, 7-
dioxybicyclo[3.3.0]octane mixture
4-4' -Isopropylidenediphenol, polybutylated mixture
Magnesium fluoride
2-Mercaptobenzothiazole and dimethyl dithiocarbamic acid mixture, sodium salt
2-Mercaptobenzothiazole, sodium or zinc salt
Pentaerythritol monostearate
Phenol
o-Phenylphenol
Potassium pentachlorophenate
Quaternary ammonium chloride (hexadecyl, octadecyl derivative)
Salicylic acid
Sodium de hydroacetate
Sodium fluoride
Sodium pentachlorophenate
Sodium !-\-phenylphenate
Sodium salicylate
Sodium salt of I-hydroxy 2(1H)-pyridine thione
Thymol
Tri-t-butyl-p-phenyl phenol
Tributylin chloride complex of ethylene oxide condensate of dehydroabietylamine
Tri-n-butyltin acetate
Tri-n-bytyItin neodecanoate
356 PRESERVATION OF SURFACTANT FORMULATIONS

Table 14.3 Preservatives permitted for use as components of paper and paperboard in contact
with aqueous and fatty foods. Title 21 CFR Part 176.170. 6

Part 176.170 (a)(5)

1,2-Benzisothiazolin-3-one
n-Dodecylguanidine acetate
n- Dodecylguanidine hydrochloride
Glutaraldehyde
1,3,5-Triethylhexahydro-1,3 ,5-triazine
Part 176.170 (b )(2)
1-(3-Chloroal\yl)-3,5,7-triaza-1-azoniaadamantane chloride
5-Chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one mixture at a ratio
of 3 parts to 1 part, manufactured from methyl-3-mercaptopropionate. The mixture may
contain magnesium nitrate at a concentration equivalent to the isothiazolone active
ingredients (weight/weight).
1 ,2-Dibromo-2 ,4-dicyanobutane
Dihydroxy dichlorophenyl methane
Formaldehyde
Sodium pentachlorophenate
Sodium o-phenylphenate

Table 14.4 Preservatives permitted for use as components of paper and paperboard in contact
with dry food. Title 21 CFR Part 176.180. 6

Barium metaborate
1,2-Benzisothiazolin-3-one
Bis(trichloromethyl) sulfone
Borax
Boric acid
5-Hydroxymethoxymethyl-1-aza-3, 7-dioxabicyclo[3.3.0Joctane, 5-hydroxymethyl-1-aza-3,7-
dioxabicyclo[3.3.0Joctane, and 5-hydroxypoly-(methyleneoxy)methyl-1-aza-3,7-
dioxabicyclo[3.3.0Joctane mixture

When a cosmetic manufacturer files a cosmetic registration form, FDA

reviews the ingredient statement in order to determine if any new
ingredients are listed. A new ingredient would be one not previously listed
and not included in the CTFA Cosmetic Ingredient Dictionary. FDA will
contact the manufacturer if there are illegal or deleted products listed on
the cosmetic ingredient statement. Preservatives labeled only for use in
cosmetics or personal care products are not subject to EPA registration as
pesticides.

14.5 Summary

Preservatives are regulated in the USA by the US EPA, the US FDA, the
various states and are self regulated in the case of cosmetic preservatives.
Depending on the use pattern, preservatives can be regulated by more than
one Federal agency.
 REGULATION IN THE USA 357

References

1. Title 40 Code of Federal Regulations (updated annually) Protection of Environment, Parts

150 to 189. Office of the Federal Register, National Archives and Records Administration,
Washington, DC.
2. Recommendations for Chemistry Data for Indirect Food Additive Petitions 1.2 (1993)
Chemistry Review Branch, Office of Premarket Approval, Center for Food Safety and
Applied Nutrition, US Food and Drug Administration, Washington, DC, March.
3. Title 21 Code of Federal Regulations (updated annually) Food and Drugs, Parts 170 to
199, Office of the Federal Register, National Archives and Records Administration,
Washington, DC.
4. Toxicological Principles for the Safety Assessment of Direct Additives and Color Additives
Used in Food (1993) Draft, Center for Food Safety and Applied Nutrition, US Food and
Drug Administration, Washington, DC.
5. Nikitakis, J.M., McEwen, G.N. and Wenninger, J.A. (eds) (1991) CTFA International
Cosmetic Ingredient Dictionary, The Cosmetic, Toiletry and Fragrance Association,
Washington, DC.
6. Title 21 Code of Federal Regulations (updated annually), Food and Drugs, Parts 600 to
799, Office of the Federal Register, National Archives and Records Administration,
Washington, DC.
15 European preservative legislation
G. LLOYD

15.1 Introduction

Legislation governing the use of preservatives within Europe is highly

complex, involving a number of sub-legislative areas. For example, before
consideration of the regulatory requirements (Table 15.1) for preservatives
can be made, all chemicals used in the European Community must appear
on either the European Inventory of Existing Commercial Substances
(EINECS) or the European List of New Chemical Substances (ELINCS).
There are also specific applications where an alternative approval must
first be sought before entry into the marketplace, for instance in the
manufacture of food contact paper and the exploration and production of
oil.
The approval of industrial preservatives in Europe can best be described
as fragmented. There are countries where it is mandatory to gain full
approval for all preservative applications, others require the approval for
use in a limited number of applications, and there are countries where no
approval is required at all (Appendix 1).
Approval is normally granted for the end use of formulated product,
however before an approval can be granted the authority will require
access to a data file covering all active ingredients used in the formulation.
The registration of a formulated, or end use product, requires a much
smaller data package and is restricted to information on the product to be
submitted; physical/chemical data and a series of acutes. The application
must also include a letter of access from the owner of the data on each and
every active used in the formulation, as these data are necessary for the
calculation of the overall risk assessment. 1
Again consideration to the fees charged, and language to be used must
be taken. In addition to the overall registration position in Europe being
fragmented, we also have other government departments involved in the
granting of an approval for the use of preservatives.

15.2 Preservatives in the oil industry

The registration of preservatives for use in the oil industry (see Appendix
2), in the main, only applies to those member states with an oil production
 EUROPEAN PRESERVATIVE LEGISLATION 359

Table 15.1 Regulatory compliance in Europe - typical data requirements (active ingredients)

Data Typical registration a Proposed Biocidal Products

Directive b
Paint - Metalworking Paper Paint - Metalworking Paper
incan fluids incan fluids
General information
Name of substance X X X X X X
EINECS/ELINCS/CAS No X X X X X X
Synonyms X X X X X X
Purity X X X X X X
Impurities X X X X X X
Molecular weight X X X X X X
Structural formula X X X X X X
Type of substance X X X X X X
Who is submitting the data X X X X X X
set?
Quantity producedl X X X X X X
imported
Produced during last X X X X X X
12 months?
Classification and labelling X X X X X X
Use pattern X X X X X X
Amount of additives X X X
Method of analysis X X X X X X
Spectral data X X X X X X
Means of personal X X X X X X
protection
Side effects X X X
Safety precautions X X X X X X
Methods to decontaminate X X X X
Incineration conditions X X X
Other remarks X X X X X
Efficacy X X X X X X

Physico-chemical data
Physical state X X X X X X
Particle size X X X X X
Melting point X X X X X X
Boiling point X X X X X X
Thermal stability X X X X X X
Vapour pressure X X X X X X
Octanol/water partition X X X X X X
coefficient
Water solubility X X X X X X
Solvent solubility X X X
Fat solubility X X X X X X
Flammability X X X X X X
Crystallisation X X X X X X
Flash point X X X X X X
Autoflammability X X X X X X
Explosivity X X X X X X
Surface tension X X X X X X
Oxidising properties X X X
Other data and remarks X X X X X X
360 PRESERVATION OF SURFACTANT FORMULATIONS

Table 15.1 continued

Data Typical registration a Proposed Biocidal Products

Directiveb
Paint - Metalworking Paper Paint - Metalworking Paper
incan fluids incan fluids

Toxicity
Acute oral X X X X X X
Acute dermal (X) (x) (x) X X X
Acute inhalation X X X X X X
Skin irritation X X X X X X
Eye irritation X X X X X X
Sensitisation X X X X X X
Mutagenicity - Ames X X X X X X
Mutagenicity - (2) X X X X X X
Mutagenicity - (3) X X X X X
Reproductive toxicity screen X X X
28 day toxicity X X X X X X
90 day sub acute a (x) X X (x) (x) (x)
90 day sub acuteb X X (X)
Neurotoxicity X X X
Cholinesterse inhibition X X X
Teratogenicity X X X X
One generation reproduction X X
Two generation reproduction X (X)
Toxicokinetics X X X X X
Carcinogenicity screen (X) (X) (X)
Carcinogenicity (X) (X) (X) X X
Other relevant information X X X X X
Experience with human X X X X X X
exposure
Toxicological mechanism X X X
Worker health records X X X
Epidemiology X X X X X X

Ecotoxicology
Acute toxicity to birds X X
Subacute toxicity to birds X X
Acute toxicity to fish X X X X X X
Fish reproduction X X
Prolonged toxicity to fish X X X
Bioaccumulation if X X X X X X
Pow >1000
Acute toxicity to Daphnia X X X X X X
Prolonged toxicity to X X
Daphnia
Toxicity to algae X X X X X X
Toxicity to bacteria X X X X
Toxicity to terrestrial X X
organisms
Toxicity to soil organisms X
(e.g. worms)
Toxicity to higher plants X X
Toxicity to non target X X X X
organisms
Other remarks X X X X X
 EUROPEAN PRESERVATIVE LEGISLATION 361

Table 15.1 continued

Data Typical registration a Proposed Biocidal Products

Directive b
Paint - Metalworking Paper Paint - Metalworking Paper
incan fluids incan fluids

Environmental fate
Biodegradation X X X X X X
BOD/COD X X X
Activated sludge respiration X X X
inhibition
Degradation/transformation, X X X X
water/soil
Transport and distribution X
betweeen environmental
compartments
Adsorption/desorption X X X X
soil screen
Distribution in the X X X
environment
Mobility X X
Photodegradation X X X
Full soil adsorption/ X X
desorption study
Hydrolysis X X X X X
Other remarks X X X X X X

Parentheses indicate conditional requirement. aData requirements from more than one
industry. bData requirement is a proposal at this stage.

industry, although countries which border the North Sea do have

legitimate concerns with regard to pollution control, and therefore take an
active role in the activities of PARCOM (the Paris and Oslo Commission).
This independent organisation, based in London, was set to advise
Ministers on the best way to control, minimise or eliminate pollution in the
North Sea.
At the present time, even with the increasing activities of PARCOM,
there is little harmonisation between the various regulatory schemes
operating in the offshore oil industry. 2 An alliance between the main
contracting parties of PARCOM and industry has resulted in the
development of a risk assessment programme known as CHARM
(Chemical Hazard Assessment and Risk Management). 3 Once fully
adopted it will form the basis of the approval schemes for all offshore
chemicals of which preservatives are an important sector.
CHARM calls for a significant amount of marine toxicity and environ-
mental fate data, plus information on the potential for the product to cause
taint in fish. In the UK, approval is granted under the Offshore Chemical
Notification Scheme (OCNS) administered by the Department of Trade
362 PRESERVATION OF SURFACTANT FORMULATIONS

and Industry, with technical assistance from the Ministry of Agriculture

Fisheries and Foods. The scheme, introduced in 1979, has undergone a
number of changes with the more recent ones being influenced by the
activities of PARCOM.
In The Netherlands approval should be sought from the State Supervisor
of Mines, whilst in Norway contact must be made with the State Pollution
Control Authority. Both organisations will require conformation to the
requirements of PARCOM including an assessment carried out using the
CHARM model.

15.3 Preservatives in the paper industry

Regulatory conformance for preservatives used in the manufacture of

paper is required by countries such as Sweden, the Netherlands,4 Finland5
and Denmark. There are national authorities set up to administer the
approval schemes for chemicals, including preservatives, used in the
manufacture of food contact paper, although approval from the FDA in
the USA and German BGA (Bundesgesundheitamtes) are the most
influential in the industry.
Approval by the BGA requires a considerable amount of information,
similar but not quite as demanding as that for the registration of
preservative in The Netherlands. There are however requirements specific
to the paper industry such as proof of efficacy against slime-formers,
migration data and field trial data.
Inevitably we have the influence of the EU in this area.
A number of directives have been published and adopted under a 'frame
directive' established to control the use of material which might come into
contact with foodstuffs. Within this frame directive is a proposal to
produce a Community Directive that will regulate preservatives used in the
manufacture of food contact paper. It is anticipated that this directive will
replace all national legislation, e.g. the BGA.

15.4 The proposed Biocidal Products Directive (93/239/03)6

Following on from the introduction of the Crop Protection Directive 91/

414/EEC,7 this proposal, submitted by the commission in 1993, is designed
to harmonise the regulatory requirements for biocides throughout the
European market. The proposed scope is very wide covering preservatives
in all applications, and as the directive is intended to harmonise the
regulatory procedures throughout Europe it is essential that it takes into
consideration the systems currently in place now.
Data required to register a preservative, fall under the heading of the
 EUROPEAN PRESERVATIVE LEGISLATION 363

common principles. These stipulate the data required for each application
area, with a core data set of acutes, subacutes, wildlife and environmental
data, supplemented by additional data of relevance to the particular
application.
Like the established programmes the registration of end use products
has a much smaller data requirement the cost of which is likely to be in the
order of £60 000-£100 000 (sterling).
It may be possible to reduce the cost of a registration by data sharing.
Data used in the registration process can be protected for up to 15 years;
no member state will be allowed to use a company's data in support of a
second company's registration without the permission of the original
registrant.
The assessment of all active ingredients will be the responsibility of the
European Commission with member states carrying out the assessment
and approval of end use products. Once a member state has completed its
assessment and granted an approval, all other member states will be
required to grant approval for the preservative in their country under the
principles of mutual acceptance, unless there are very good reasons not to.
After adoption of the directive by the commission and the member states
there will be a 10 year transitional period, during which all active
ingredients and formulated products will be put through a health and
environmental risk assessment. Any product classified as very toxic, a
category 1 or 2 carcinogen, a mutagen or is toxic for reproduction
(category 1 or 2) will not be authorised for sale to the general public.
At the present time the Biocidal Products Directive is still a proposal, it
still has some way to go before it becomes law in the member states.
To conclude, legislation concerning preservatives currently lies with a
variety of government departments within the boundaries of Europe.
Although there are some countries with long standing registration
procedures the introduction of the biocidal products directive will bring
regulatory harmonisation to the industry.

References

1. Product Development of Data Requirements and Common Principles for the Evaluation
and Risk Assessment of Biocidal Products. Ministry of Environment National Agency of
Environment Protection. April 1994.
2. Harmonised Offshore Chemical Notification Scheme. 6 September 1994.
3. CHARM Wizard (V2.1), Chemical Hazard Assessment and Risk Management of Offshore
Exploration & Production Chemicals. 21 December 1994.
4. Guidelines for Submitting Applications for the Licensing of Pesticides. Commissie
Toelating, Bestrijdingsmiddelen, Wageningen, The Netherlands.
5. Applications for the Advance Approval of Wood Preservative and Slimicides. 13 April
1994. National Board of Waters and the Environment, Chemical Control Unit, PO Box
250, Helsinki, Finland.
364 PRESERVATION OF SURFACTANT FORMULATIONS

6. Proposal for a Council Directive concerning the placing of biocidal products on the
market. 93/C/239/03. Official Journal of the European Community. 3.9.93.
7. Council Directive 911414IEEC (concerning the placing of plant protection products on the
market). Official Journal of the European Commission. 15.7.91.

Appendix 1 - Countries with registration procedures for preservatives

All applications
The Netherlands
Belgium
Limited applications
UK
Denmark
Spain
Sweden
Norway
Finland
Food contact paper (e.g. BGA)
All member states of the EU
Oilfield applications (Majors)
Denmark
The Netherlands
Norway
UK
 EUROPEAN PRESERVATIVE LEGISLATION 365

Appendix 2 - Typical data requirements for preservatives used in the

offshore oil drilling and production industry

General information
Tradename
Supplier
Use/volumes
Composition
Is it on annex A?
Label
Physical/chemical
Ecotoxicology
Bioacummulation
Biodegradability
Taint potential
Marine LCso values:
Fish (optional)
Algae
Crustacea
Sediment Reworker
Bioconcentration factor
Animal toxicology
Acute LDso mammal
Index

acceptable daily intake 353 Alcaligenes faecalis 266

6-acetoxy-2,4-dimethyl-m-dioxane 143 alcohol 175
Achromobacter 110, 236 Alternaria alternata 236
Acinetobacter baumannii 266 American Conference of Government
Acinetobacter calcoaceticus 200 Hygienists 342
Acinetobacter haemolyticus 266 American Society of Testing and Material
acrylamide 249 see ASTM
acrylic acid 221 Amical 22, 188
acrylic latex 185 ammonium persulphate 222
acrylic polymers 214 amphoterics 41, 135
acrylonitrile 221 anaerobic contamination 275
Actinobacter 291 Angus 15,295-296,318
acute toxicity 317-319 anionic surfactants 74, 75, 86--100, 135,
adenosine triphosphate (ATP) assay 304 153, 288-289
adhesives 212-257 ester sulphonates 75
adhesion 229 phosphates 75, 87-88, 289
aqueous-based 225 sulphates 73,75,86--87,89, 153,289
bond strength 229 sui phonates 73, 75, 92-100, 153, 288
dispersion adhesives 227-228 anionic surfactants, biodegradation of
hot melt 225 84-99
odour 230 alkane sulphonates 92-93
pH 229 alkyl ethoxy sulphates 89-92
properties of 228-231 alkyl sulphates 86--87
redispersibility 229 dialkyl sulphosuccinates 88-89
set speed 229 fatty acid alkanolamide sulphonates
solids 229 97-98
solution adhesives 225-227 sulphonated esters and ami des of fatty
solvent-based 225 acids 98, 99
stability 230 anti-settling agent 126
uses 223 AOAC ISO, 157, 158
viscosity 229 aqueous emulsions, factors affecting
wet tack 229 preservation choice 248-251
adhesives, components of 230--231 chemical constituents 249
defoamers 231 climate 250
fillers 231 compatibility 250
humectant 230 cost 251
plasticiser 230 nucleophiles 250
polyvinyl alcohol 230--231 pH 249
preservatives 231 physical form 251-257
surfactants 231 processing 250
tackifiers 231 redox state 250
thickeners 231 solids 250
Aeromonas hydrophila 200,237,266 solubility 251
AFNOR 251 versatility 251
agglomerates 187 aqueous suspension concentrates 127
aggregates 186 Arthrobacter 110, 236
agrochemical formulations 120--134 Arthrobacter globiformis 266
air 45-50, 244 Arthrobacter protophormiae 266
Alcaligenes 110, 266 ascorbic acid 222
368 INDEX

Aspergillus 142, 160, 237 cationic surfactants 74, 75, 135, 291
Aspergillus niger 142, 160, 236 betaines 75, 153
Aspergillus terreus 236 quaternaries 74, 75, 135 (see also
ASTM 157,160,163, 164, 166,200,251, quaternary amines)
301 cationic surfactants, biodegradation of
ATCC 160, 161, 199,200 106-109
Aureobasidium pullulans 142 CCP 45
CDC serotype IVC2 155
Bacillus 110, 142, 155, 160, 189, 190, 192, cellulose 190, 191, 234
266 cement additives 215
Bacillus mycoides 266 CEN 251
Bacillus subtilis 142, 160, 236 Cephalosporium 301
Bacteroides 238 challenge testing 199, 251
barium metaborate 28 Chemical Hazard Assessment and Risk
1,2-benzisothiazolin-3-one 6, 8, 11-13, Management (CHARM) 361,362
143,175,198,248,249,278,282,317,319 chlorine gas 275
benzoic acid 143, 174 chlorine release agents 41, 42, 174, 203,
BGA 223,254,256,277,362 204,208
Bifidobacterium 238 5-chloro-2-methyl-isothiazolin-3-one
biguanides 41 7-13, 175, 198,248,249,279-281,317
Bioban CS1135 295 Citrobacter 110, 155, 192, 236
Bioban N-95 295 Citrobacter freundii 155, 192, 236
Bioban P-1487 295 Cladosporium 236, 293
Biocidal Products Directive 145,251, Cladosporium herbarium 236
252, 254-256, 359-361, 362-363 clay 186, 248
biofilm 244 cleaning 37--40, 204-205, 245, 274-275
biological oxygen demand (BOD) assay monitoring 44
303 procedures 43
Boots 15, 318 techniques 42
British Pharmacopeia 157 Clostridium 238
2-bromo-2-nitropropane-1,3-diol see coalescence 62, 63
Bronopol conditioners 151, 152
Bronopol 15-18, 176, 276-277, 315, 318, containers 156, 157
320, 322, 324-325, 326, 328 contamination
Buckman Laboratories 296 air-borne 45-50
building auxiliaries 215 personnel route 50-51
built-in-wetter agrochemical formulations Control of Hazardous Substances 343
133 controlled release agrochemical
Busan 30WB 296 formulations 131
Busan 77 296 corrosion 4, 234, 284
Busan 1030 296 Corynebacterium 110, 192
Busan 1060 296 COSHH toxicity classification grid 343
butyl acrylate 221 cosmetics preservative efficacy testing
t-butyl hydroperoxide 222 157-174
N-butyl methyl acrylamide 249 A TCC cultures 161
concentration and rechallenge 164
calcium carbonate 183, 263-267 growth and harvesting 163-164
processing 265 inoculum recovery 170-172
uses 266 maintaining preservative resistance 162
Calgon Corporation 18 microbial inoculum 159
California Environmental Protection neutralisation 167, 169
Agency 351 plating methods 167
Candida 142, 160, 190, 194, 293 product concentration 166-167
Candida albicans 142, 160, 192, 238 product sample 159
capacity tests 173 pure versus mixed inoculum 165-166
Captan 316 creaming 61, 62
carpets 215 critical control point see CCP
catalase assay 304 critical micelle concentration 59, 60, 63
 INDEX 369

CTFA 149, 150, 157, 158, 159, 160, 163 Enterobacter 142,149, 154, 160, 175,238,
164, 166, 354--356 ' 293
cumene hydroperoxide 222 Enterobacter cloacae 142, 160, 200
Enterobacteriaceae 160
Environmental Protection Agency see
Dangerous Substances Directive 338
EPA
death 154
environmental toxicology 328-329
Densil P 13
EPA 145,256,338,350,351,352 353
deodorants 151 356 ' ,
Desulfovibrio 238
EPA toxicity category definitions
Desulfovibrio desulfuricans 236, 266, 293
351-352
detergency 67, 68
Erwinia carnegieana 266
dialk~l sulphosuccinates 88, 123,217
Escherichia 142, 149, 160, 238, 293
1,2-dlbromo-2 ,4-dicyanobutane 18-19
Escherichia coli 142, 160,200, 236
247,249,280 '
estimated daily intake 355
dibromonitrilopropionamide 280
ethyl acrylate 221
d!iodomethyl-p-tolylsulphone 22, 188
ethylene 221
dlmethyl-2H-l,3,5-tetrahydrothidiazine-2-
ethylene oxide 73
thione 277
European Directive 911414/EEC 145
DIN 251
European Inventory of Existing
dioctyl sulphosuccinate 123 217
Commercial Substances see EINECS
di(phenylmercury)dodecyl su'ccinate 28
European List of New Chemical
dip slides 271-273, 303
Substances see ELINCS
diphtheroid 155
European Pharmacopeia 157
discoloration 4, 141, 154,232
Euxkyl K400 18
disinfectants 40-42
exponential growth 269-270
concentration 42
extended anionic surfactants 74, 75
contact time 42
ether carboxylate 75
selection of 40-41
ether phosphate 75
disinfection 37-40, 40-42, 241, 245
ether sulphates 75
dispersions 65-66, 68
eye infections 154, 155
monitoring 44
eye shadow 151
procedures 43
dithio-2,2' -bis(benzmethylamide) 13
fatty alcohol ethoxylates 123
dithiocarbamates 21
FDA 149, 150, 154, 158, 162, 223,
(DMDM) hydantoin 153 253-254,256,276,280,281,350 353 354
dodecyl polyether phosphate 217 356,362 ' , ,
dodecyl sulphate 217
Federal Food, Drug and Cosmetic Act
dodecylbenzene sulphonate 217
see FFDCA
Dow 197, 296-297 Federal Insecticide, Fungicide and
Dowicide A 296-297 Rodenticide Act see FIFRA
Dowicide 1 297 FFDCA 145,350
Dowicil 176, 197, 297
FIFRA 145, 350, 352
Dowicil75 197,297
Flavobacterium 110, 238
D-values 172
flocculation 61, 62
flotation 262, 264
EDTA 174 foams 63,64
EINECS 358, 359 Food and Drug Administration see FDA
ELINCS 358, 359 formaldehyde 23-27, 153, 174, 175, 176,
emulsifiable concentrate 122, 124, 125, 197, 243, 247, 315, 317, 328
130 formaldehyde release agents 23-27, 143,
emulsification 60-61 153, 174, 176, 197,203,247,314,315
emulsion polymerisation 218 formulating 77-81
emulsions 60, 61, 63 chemical compatibility 80
breakdown 61-63 pH stability 80
oil-in-water 61 physical and chemical compatibility 80
water-in-oil 61 temperature of use 80
encapsulation of agrochemicals 128, 132 viscosi ty 80
370 INDEX

fungicides 21-23, 123, 188 indirect food additives 353

Fusarium 160,236,237,293,301 adhesives 355
Fusarium solani 236 paper and paperboard, aqueous or fatty
food 355
gas buildup 4, 141,232 paper and paperboard, dry food 356
genetic toxicology 325-327 initiators, polymer emulsions 222
Geotrichum candidum 230 insecticides 125
Germall 176 Integra 22 143
Gibbs-Marangoni effect 63 Integra 44 143
GIFAB 121 interfacial tension 54, 56, 58
Giv-Gard 143 inversion 62
glass fibre 215 iodophor 41, 42
Glokill 143 3-iodopropargyl-N-butyl carbamate 21,
glutaraldehyde 24,27,203,208,247, 188
278-279,282,318-319,320-321,323,325, isolation of spoilage microorganisms 110
327,328 isothiazolinones 6-13, 153, 174, 248
glyceryl monolaurate 217 ISP 143
Glydant 176
Gram-negative bacteria 152, 155, 160, kaolin 263-267
166, 238, 312 processing 264-265, 267
Gram-positive bacteria 160,166,235,312 uses 266
granulation processes 129 Kathon 8, 143, 198, 248, 249, 278, 282,
GRAS 253, 254 298,317
Grotan 299 Kathon CG 319,320,321,324,326,327,
ground calcium carbonate 265, 267 329
Kathon 886MW 298, 319
HACCP 45, 209 Kathon 893MW 298
hair care products 151 Kathon MWC 298
halogen-releasing agents 41, 174, 246 Klebsiella 110, 149, 155, 160, 293
hazard data sheet 340-341 Klebsiella pneumoniae 301
hazard, relationship to risk 339
herbicides 123 laboratory reports 201
HLB scale 77, 78 laboratory testing 199, 251, 300-304
Huls 197 Lactobacilli 152
hydrogen peroxide 41,222,275
hydrophile-lipophile balance 77,78,79, MAK 311,317
125, 130, 137, 139 maximum Arbeitskonzentration see MAK
hydrophilising technologies 73, 74 maximum exposure limit see MEL
ethoxylation 73 MBT 20
sulphation 73 MEL 317
sulphonation 73 2-mercaptopyridine N-oxide 13
p-hydroxy benzoic acid 143 2-mercaptopyridine N-oxide, sodium salt
2-(hydroxymethyl) amino ethanol 197 13-15, 297-298
2-(hydroxymethyl) amino-2-methyl 2-mercaptopyridine N-oxide, zinc complex
propanol 197 13-15, 315
hygiene audits 205-207 mercury salts 28, 196,247,314
hygienic design 34-37, 245, 269 metalworking fluids, preservative selection
aerial contamination 238 300-305
manufacturing procedures 240 field testing 302
mixing vessels 241 laboratory testing 301-302
pipelines 240 monitoring 302-304
product storage containers 242 metalworking fluids, types 285-288
raw materials 240 base mineral oil blend 285
road tankers 241 chlorinated mineral oils 286
hypochlorous acid 208 semi-synthetic 284-287
soluble oils 286
IBRG 251,252 straight oil 285-286
Igepal's 186 sulfochlorinated mineral oils 286
 INDEX 371

sulfured fatty mineral oils 286 no observed effect level see NOEL
synthetic 287-288 Nuosept 95 197
N-methyl acrylamide 249 Nuosept 145 197
methyl p-hydroxy benzoate 143
2-methyl-isothiazolin-3-one 7-13, 175, occupational exposure limit see OEL
198, 248, 279 occupation exposure standards 342
methyl methacrylate 221 occupational hygiene 342-345
methylene bisthiocynanate 20, 276 Ochrobactrum anthropi 236, 266
N-methylol acrylamide 221 2-octylisothiazolin-3-one 7, 10, 11, 188,
2-methyl-4,5-trimethyleneisothiazolin- 248
3-one 7, 10 odour 4, 154, 232, 284
micellisation 58-60 OECD 251
microbial growth, effect of 234 OEL 311
oxidation-reduction potential 2 Offshore Chemical Notification Scheme
pH 1, 141, 152, 192, 249, 284 (OCNS) 361
presence of antimicrobial components oil industry 358-362
3 oleochemicals 69-71
suitable carbon source 3, 141, 192 condensation with alcohols or pol yo Is
temperature 1, 141, 189, 192 71
water 2, 192 condensation with amines 71
water activity 2, 152 reduction with hydrogen 71
Micrococcus 110, 236, 266 reductive animation 71, 72
Micrococcus luteus 236, 266 Olin Corporation 15, 297
microemulsions 66,67, 131 Omadine see 2-mercaptopyridine N-oxide
monomer, antimicrobial effect of 232 Onyxide 200 299
monomers, polymer emulsions 219-222 organic acids 28, 143, 156, 175, 313
Morganella morganii 193 organoiodine compounds 21-23
moulds 152, 155, 164, 195,200 OSHA 197
mutagenicity 177 Ostwald ripening 62
Mycobacterium 110 OIW emulsions 130
Oxaban A 315-316, 318, 320, 322-323,
naphthalene sulphonic acid-formaldehyde 325, 326-327
condensates 123, 126, 138 Oxaban E 315-316, 318, 320, 322-323,
National Institute for Occupational Safety 325, 326-327
and Health 342 oxoazolines 247
Nocardia 110 10,1O'-oxybisphenoxyarsine 28
NOEL 177 ozone 275
non-ionic surfactants 75, 100-106, 135, ozonation 262
153, 289-291
amine oxides 75, 106 paint chemistry 185-186
ethanolamines 75, 106, 289-290 paint manufacture 186-187
ethoxylates 73,74,75,76,85, 100-106, paint, solvent-borne 186
137, 139, 153,290 paint, water-based 186
ethylene oxide/propylene oxide paper, coating 215, 268
copolymers 126, 290-291 parabens 156, 157, 174, 175,315
long chain carboxylic acid esters 291 Paris and Oslo Commission (P ARCOM)
non-ionic surfactants, biodegradation of 361, 362
100-106 particle size separation 262, 264
alkyl phenol ethoxylates 103-104 particulate dispersion 68
branched alcohol ethoxylates 101-103 Pasteurella multocida 266
ethoxylated fatty acids 106 Penicillium 142, 160, 237
fatty acid alkanolamides 106 Penicillium luteum 160
linear alcohol ethoxylates 100-101 Penicillium notatum 142
polyglycols 104-106 Penicillium ochrochloron 236
non-woven fabrics 215 peracetic acid 41
nonyl phenol ethoxylates 123, 126, 137, permitted exposure limits 342
139 peroxy acids 41
nonyl phenol polyethcr sulphate 217 personal protective equipment 346
372 INDEX

petrochemicals 72 Rhodotorula species 142, 193,236, 237

phase separation 4, 41, 189 Rhone-Poulenc 143
phenols 27,28, 175, 313 rinse water 205
2-phenoxyethanol 157 risk, relationship to hazard 339
phenyl mercuric acetate 28, 176, 196 risk assessment 342-348
Phyllobacterium rubiacearum 266 risk control, cost versus effectiveness 401
Pichia 236 risk phase 343
polyethoxylated alkanol 217 Rohm & Haas 8, 143, 186, 198,279,298,
polyethoxylated nonyl phenol 217 317
polyethoxylated polypropylene glycol roll-up mechanism 67
217 R.T. Vanderbilt Company Inc. 298-299
polymer emulsions 212-257 rubber emulsions 249
polymer manufacture 222-223
polymeric surfactants 140 Saccharomyces 142, 160, 194, 236
polyurethane 249 Saccharomyces cerevisiae 142, 194, 236
polyvinyl acetate (PVA) homopolymers sampling 267
186, 214, 248 SBR 249
polyvinyl alcohol 248 Schuelke & ~ayer 18
potassium persulphate 222 Scopulariopsis 237
precipitated calcium carbonate 265, 267 sedimentation 61, 62
printing inks 215 seed treatments 122
processing equipment 4, 235 Serratia 142, 149, 154, 160
production areas 31,32,37 Serratia marcescens 142, 200
Promexal 7, 10, 249 shampoo 151, 152
Propionibacterium 155 short term exposure limit see STEL
propionic acid 143 Skane 7, 10,11, 188
propylene oxide 74 skin and eye irritation 177,319-321,337
Proteus 142, 160, 293, 301 skin and respiratory sensitisation 177,
Proteus mirabilis 301 321-323, 337
Proteus vulgaris 142, 236 soap 69,71,73,75,83
Proxel 6, 11-13, 143, 248, 249, 278, sodium dodecylbenzene sulphonate 123
299-300, 315, 317, 328-329 sodium formaldehyde sulphoxylate 222
Proxel GXL 317 sodium hydroxymethyl glycinate 143
Proxel ~VV200 297-300 sodium lauryl sulphate 123
Proxel ~VV300 300 sodium lignosulphonate 123, 126, 136,
Proxel XL2 317 138
Pseudomonas species 110, 142, 149, 155, sodium hexametaphosphate 262
160,192,235,238,244,266,267,301,312 sodium hydrosulphite 262, 264
Pseudomonas aeruginosa 142, 193,236, sodium metabisulphite 222
266, 267, 301, 304 sodium persulphate 222
Pseudomonas aureofaciens 200 sodium polyacrylate 264
Pseudomonas cepacia 193,200,237 sodium thiosulphite 222
Pseudomonas diminuta 264 solubilisation 66
Pseudomonas fluorescens 193, 237 solution concentrates 122, 123, 124
Pseudomonas putida 193, 200, 237 sorbic acid 143, 174
Pseudomonas putrefaciens 193 spray adjuvants 128
Pseudomonas stutzeri 200, 236 stabilisation of polymer particles 219
Pseudomonas syringae 266 Staphylococcus 142, 152, 155, 160
Pseudomonas vesiculams 193 Staphylococcus aureus 142, 160
pyrithione see 2-mercaptopyridine N-oxide Staphylococcus epidermidis 155
starch 248
quaternaryamines 41,42, 203, 314 stearate soaps 217
STEL 317
radiation 273 Stepan Company 299
reproductive and developmental Streptococcus 110, 142
toxicology 327-328 Streptococcus faecalis 142
Rhodopseudomonas capsulata 142 styrene 221
Rhodotourula rubra 142, 236 styrene acrylate 249
 INDEX 373

styrene/acrylic polymers 214 Ucarcide 278

subacute and chronic toxicity 323-325 Uconex 299
sulphate reducing bacteria 238, 293 Union Carbide 278,299
surfactant molecular interactions 54-55 US Professional Laboratories 299
surfactants as bacterial nutrients 84, tOO USP 150, 157, 159, 160, 164
suspoemulsion formulations 130, 131
synergy, biocide 298 Vancide TH 298-299
synergy, surfactant 79 Vancide 51 299
Vantocil 249
Tamol731 186 Vibrio ItO
TCMTB 20 vinyl acetate 221
Tektamer 18, 249 vinyl acetate/ethylene (V AlE) copolymers
teratogens 177 214, 216, 248
textile finishings 215 vinyl acetate/vinyl chloride/ethylene
thiocyanates 2~21, 247 terpolymer 214
2-( thiocyanomethylthio )-benzothiazole vinyl acetate/vinyl versatate (ValVe Va)
20 copolymers 214, 216
thinning 4, 141, 154, 189 vinyl acrylic latex 186
threshold limit value see TLV vinyl chloride 221
time weighted average see TWA vinyl versatate 221
titanium dioxide 185, 263, 266 VOC 186, 187,249
TLV 311,317,342 volatile organic carbon see VOC
Torula 237
Torulopis 193, 236, 238 wall paper 15
Toxic Control Substances Directive 338 wash water 205, 206
Triadine 3 297 water 3~32, 205, 206, 239, 244
Triadine to 297 microbiological quality 30, 244
Triadine 20 297 pest control 31-32
tributyl tin oxide 28 water-dispersible granules 127, 128, 129
Trichoderma 190 129
Trichoderma viride 236 wettable powders 122
Tris Nitro 296 wetting 64-65
tristyryl phenol ethoxylate phosphate ester
126 Xanthobacter flavus 264
Tritons 186 Xanthomonas maltophilia 236
Troy Chemical Inc 21,188,197
Troysan 174 197 yeast 152, 153, 190, 194
Troysan 186 197
Troysan polyphase 21, 188 Zeneca 6, to, 13, 143, 198, 199, 278,
TWA 315 282, 299-300, 315, 317