C. R.

Biologies 340 (2017) 188–194

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Comptes Rendus Biologies

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Ecology/Écologie

Treated wastewater phytotoxicity assessment using

Lactuca sativa: Focus on germination and root elongation test
parameters
Évaluation de la phytotoxicité d’eaux de rejets via Lactuca sativa :
paramètres des tests de germination et d’élongation
Anne Priac, Pierre-Marie Badot, Grégorio Crini *
UMR 6249, Chrono-Environment, University of Franche-Comté/CNRS, 16, route de Gray, 25000 Besançon, France

A R T I C L E I N F O A B S T R A C T

Article history: Sensitive and simple ecotoxicological bioassays like seed germination and root elongation
Received 10 June 2014 tests are commonly used to evaluate the phytotoxicity of waste and industrial discharge
Accepted after revision 10 January 2017 waters. Although the tests are performed following national and international standards,
Available online 28 February 2017 various parameters such as the number of seeds per dish, the test duration or the type of
support used remain variable. To be able to make a correct comparison of results from
Keywords: different studies, it is crucial to know which parameter(s) could affect ecotoxicological
Bioassay diagnosis. We tested four different control waters and three seed densities. No significant
Lactuca sativa differences on either germination rate or root elongation endpoints were shown.
Sensitivity scale Nevertheless, we found that the four lettuce cultivars (Appia, batavia dorée de printemps,
Cultivar grosse blonde paresseuse, and Kinemontepas) showed significantly different responses
Discharge water
when watered with the same and different metal-loaded industrial discharge water. From
the comparison, it is clear that a differential sensitivity scale occurs among not just species
but cultivars.
C 2017 Académie des sciences. Published by Elsevier Masson SAS. This is an open access

article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/

4.0/).

R É S U M É

Mots clés : Les bio-essais écotoxicologiques simples comme les tests de germination et d’élongation
Bio-essais sont fréquemment utilisés pour évaluer la phytotoxicité de rejets industriels. Si ces tests
Lactuca sativa sont réalisés selon des standards nationaux et internationaux, de nombreux paramètres
Échelle de sensibilité (nombre de graines, durée du test, type de support. . .) varient. Afin de comparer
Variété correctement les résultats tirés de différentes études, il est indispensable de savoir
Rejet
précisément quel(s) paramètre(s) pourrai(en)t affecter le diagnostic écotoxicologique.
Nous avons testé quatre eaux de contrôle et trois abondances de graines : aucune différence
significative du taux de germination ou d’élongation des plantules n’a été mise en évidence.
Néanmoins, nous avons montré que les quatre variétés de laitue testées (Appia, batavia
dorée de printemps, grosse blonde paresseuse and Kinemontepas) présentaient des
différences de réponse écotoxicologique, après avoir été mises en contact avec le même

* Corresponding author.
E-mail address: [email protected] (G. Crini).

http://dx.doi.org/10.1016/j.crvi.2017.01.002
1631-0691/ C 2017 Académie des sciences. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://

creativecommons.org/licenses/by-nc-nd/4.0/).
 A. Priac et al. / C. R. Biologies 340 (2017) 188–194 189

rejet industriel. Il apparaı̂t clairement que les différences de sensibilité sont

interspécifiques, mais également intraspécifiques.
C 2017 Académie des sciences. Publié par Elsevier Masson SAS. Cet article est publié en

Open Access sous licence CC BY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/

4.0/).

1. Introduction wheat Triticum aestivum. Previous studies [20,24] com-

pared some of these species and recommended L. sativa as
Surface treatment (ST) is a very important industrial a bioindicator to determine the toxicity of soil and water
sector in Europe and in France, Franche-Comté is especially samples.
concerned. ST industry supplies a great variety of products Haugland and Brandsaeter [25] previously studied the
for various industrial and domestic sectors including the effects of various procedures and conditions on bioassay
motor, building, electronic, military and also clothing sensitivity in allelopathic studies. They pointed out that
industries. However, their activities are energy- and water- the lack of real standardized bioassays makes comparison
consuming as well as highly chemically polluting. Indeed, between studies very difficult. It is nowadays in fact the
ST is well known to be one of the largest consumers of proceedings that are not standardized: even when
chemicals (toxic metals known to be harmful to humans phytotoxicological bioassays using lettuce are performed
and to the environment in particular) and to generate large in accordance with national or international standards,
amounts of toxic waste water with a complex composition multiple parameters remain variable (Table 2). Di Salva-
[1]. The main pollutants are metal ions such as Cr(III), tore [10], studying synthetic solutions containing metal
Cr(VI), Zn, Sn, Cu, Ni, Ag and Fe, organic substances such as ions, found that lettuce GR and RE were not affected by
chloroform and polycyclic aromatic hydrocarbons, and substrate, agar agar vs. filter paper. However, there is no
other non-metallic pollutants such as cyanide, boron, and literature comparing the parameters used of industrial
fluoride. Despite the efforts made to clean up their effluent, as that of the ST industry.
polycontaminated effluents, most commonly by physico- The present study is based on the assessment that
chemical treatment [2], industry and scientists are proceeding parameters could affect the ecotoxicity diag-
confronted with a great challenge: to remove the entire nosis. Indeed, we tested three of the most variable
load of organic and inorganic pollutants to assess their parameters, using GR, root and total lengths as end points:
ecotoxicological effects and hence move towards zero control water quality, number of seeds per germination
pollution discharge [3,4]. While pollutant mixtures present dish and lettuce cultivar.
in discharge water after treatment are relatively easy to
characterize chemically, assessing their impact on the
2. Material and methods
environment is usually difficult [2]: over the past few
decades, ecotoxicological methods have been developed to
2.1. Industrial discharge waters
complete chemical analysis [5].
Bioassays are widely carried out following national and
During this study, discharge waters were once collected
international recommendations. Some very different
in three different surface treatment companies (denoted
organisms are commonly used in ecotoxicological bio-
Co1, Co2 and Co3) in the Franche-Comté region. Effluent
monitoring: primary producers (algae, i.e.
samples were collected at the outlet of the decontamina-
Pseudokirchneriella subcapitata [6]), primary consumers
tion station of each company. The main activities of each
(aquatic invertebrates, i.e. Daphnia magna, Gammarus pulex
company and the related major environmental concerns
[7]) or secondary consumers (aquatic vertebrates, i.e.
are reported in Table 3. The table also shows the
Gambusia holbrooki [8]). Less frequently used in compari-
son with faunal tests [9], toxicity studies using higher
plants have however increased in recent years Table 1
[11,12]. Ratsch and Johndro [13] first concluded that the Major advantages of phytotoxicity assays using vascular plant seeds.

inhibition of root elongation (RE) is a valid and sensitive Advantages of phytotoxicity tests involving seeds
indicator of environmental toxicity. Several articles (seed germination rate, root elongation, etc.)a
[10,14–20] have since shown that phytotoxicity tests like Simple and very reproducible method
seed germination rate (GR) and RE tests present many Applicable in situ and in vitro
advantages as summarized in Table 1. These bioassays are No requirement for major equipment
Minimal maintenance costs
simple, inexpensive and only require a relatively small
Seeds are self-sufficient (no adjuvants/nutrients needed in the
amount of sample. Moreover, the seeds remain usable for a test water)
long time. The most common plant species recommended Only small sample size required (e.g. water, effluent, soil, sediment)
by, among others, the US Environmental Protection Agency No seasonality
[21], the US Food and Drug Administration [22], and the Seeds can be easily purchased in bulk
Seeds remain viable a long time
Organization for Economic Cooperation and Development Rapid germination
[23] are cucumber Cucumis sativus, lettuce Lactuca sativa L., a
Based on multiple references including [10,14,15,17].
radish Raphanus spp L., red clover Trifolium pratense L., and
190 A. Priac et al. / C. R. Biologies 340 (2017) 188–194

Table 2
Non-exhaustive list of parameters that remain variable in seed germination bioassaysa.

Parameter Examples

Cultivar Regina; Buttercrunch; Trocadero; Divina; Iceberg; non-specified

Support Agar agar; filter paper; germination paper; non-specified
Seed pre-treatment Yes; no; non-specified–When yes: 10 or 30% hypochlorite solution
Temperature [in 8C] 20; 24; 28; room temperature; non-specified
pH 5.5 to 8.2; non-specified
Dish Glass; polyethylene; non-specified
Number of seeds 10; 20; 50; non-specified
Amount of sample 4 mL; 9 mL; non-specified
Duration 72 to 192 h
Control water Distilled; deionized; milliQ; non-specified
a
Based on multiple references including [1,5,10,13,19,20,26–30].

concentration threshold in discharge for key pollutants. 2.3. Control and toxicity test
Table 4 shows the characteristics of the samples studied
here, taken from three different surface treatment Germination rates for samples were evaluated using the
companies (Co1S1, Co2S1 and Co3S1). The effluents are French normalized method ISO 17126 [31]. Tests were
average samples, characteristic of daily activity. Each conducted using 100  15 mm disposable plastic Petri
treated water sample was tested following the same dishes and two layers of filter paper. Thirty plump
concentration range: 25, 50, 75 and 100%. All dilutions undamaged seeds of almost identical size were laid on
were prepared in Reverse Osmosis Water (ROW). the filter paper in each dish, which contained 4 mL of
industrial discharge water (pH  8.4). Each condition was
2.2. Lettuce seeds tested in triplicate. All dishes were kept in the dark, at
24  1 8C, for seven days of exposure. As recommended by
Four lettuce (L. sativa L.) cultivars were germinated: the normalized method [31], a control test with distilled
Appia (A), batavia dorée de printemps (B), grosse blonde water was performed in triplicate for every condition tested.
paresseuse (GBP) and Kinemontepas (K). They were chosen Multiple parameters were tested as described in Table
among the 1500 or so commercially available cultivars. The 5. After seven days, germinated seeds were counted (GR)
seeds (Caillard, Avignon, France) were all kept under using equation (1) (where GSS is the number of Germinated
laboratory conditions, in the dark and shielded from large Seeds in the Sample and GSC the number of Germinated
modifications of temperature and moisture [1]. Seeds in the Control) and plantlet growth measured (root and
total lengths; RL and TL; the total length refers to the root and
hypocotyl of the plantlets).
Table 3
Main environmental issues encountered by the two surface treatment
GSS
companies and the regulatory values (in mgL1) for different pollutants GR ¼ (1)
contained in the water discharges (French law of 5th September 2006). GSC

Company and main Contaminant(s) Threshold emission As recommended by the normalized method [31], GR
activity of major concern value (mgL1) under 90% is unacceptable for control conditions. Control
Co 1 Zn 3.5 water pH did not skew germination test results as long as it
Treatment by electrolysis Ni 3.5 remained between 5.5 and 9.5.
Co2 Fe 5
Plating with precious Ni 2
metals
2.4. Statistical analysis
Co3 Al 5
Surface treatment All homoscedasticities were tested using a Bartlett test
of aluminum as prerequisite for parametric test. The GRs were compared
using the Chi2 test and lengths (root and total) using

Table 4 Table 5
Physicochemical characteristics of three discharge waters (Co1S1, Co2S1, Parameters assessed here.
Co3S1) from the three industrial sites investigated in this study. Quality of water Distilled water DW (pH 7.3)
Mineral water Evian1, E (pH 7.2)
Parameter/Metal Co1S1 Co2S1 Co3S1
Reverse osmosis water ROW (pH 6)
pH 8.5 8.4 6.9 Ultra pure water UPW (6.05)
Conductivity – 1730 3280 Number of seeds per Petri dish 15
Fe 1.97 5.18 0.117 20
Cr 0.13 0.079 0.12 30
Zn 2.67 0.15 0.05 Cultivar of lettuce var. Appia (A)
Ni 0.6 0.96 0.49 Lactuca sativa var. batavia dorée de printemps (B)
var. grosse blonde paresseuse (GBP)
All concentrations are expressed in mgL1, except the conductivity in var. Kinemontepas (K)
mScm1.
 A. Priac et al. / C. R. Biologies 340 (2017) 188–194 191

Table 6
Comparison between germination rate (GR) and root length RL means ( SD; n = 3) of L. sativa var Batavia dorée de printemps (B) watered with four different
control waters (E, UPW, ROW, DW).

E UPW ROW DW P value

GR (%) 100 99  1.6 98  3.1 94  1.6 0.067

RL (mm) 14.28  4.47 14.64  5.99 17.80  9.11 14.44  5.79 0.147
TL (mm) 63.14  4.20 61.33  6.26 65.21  6.18 67.73  1.16 0.441

Table 7
Comparison between germination rate GR and root length RL means ( SD; n = 3) of 4 lettuce cultivars watered with ROW.

A B K GBP P value

GR (%) 95  4.1 96  2.9 93  3.9 96  3.4 0.357

RL (mm) 16.20  5.40 25.70  9.49 28.07  8.16 24.49  9.91 0.08
TL (mm) 57.49  10.56 71.50  14.55 81.05  12.86 74.27  15.02 0.02

Kruskal–Wallis tests, with a significance threshold of 3.2. Number of seeds per dish
p < 0.05. All statistical analyses were performed with R
(2.15.1) (R Development Core Team, 2013, www.r-project. Three seed densities (15, 20 and 30 seeds per dish;
org). ROW) were tested, using the same lettuce cultivar (var. B).
Dose-dependent curves and EC50 values were calculat- The results for the three bioassay endpoints are detailed in
ed with Hill’s model using the macro Excel Regtox free Table 8. It can be seen that there was no significant
version EV 7.0.6. difference between GR (100%; 95%; 98%) and both root
Germination Index (GI) [32–35] were used to assess the (17.5; 18.5; 17.8 mm) and total (62; 71; 65.4 mm) length of
response variability between lettuce cultivars. Calcula- the plantlet grown in dishes containing 15, 20, and
tions of these indexes were performed using the equations 30 seeds, respectively.
(2) where RLS is the Root Length of the Sample, RLC the Weidenhamer et al. [36] reported that the number of
Root Length of the Control. seeds relative to the solution volume used in a seed
germination bioassay was a factor in the results obtained
RLSGSS as the amount of ferulic acid available to each seed
GI ¼ (2)
RLCGSC influenced germination, rather than chemical concentra-
tion of the tested solution. It seems that there is not such a
3. Results and discussion consensus about the effect of seed number on length: some
report a correlation (e.g., [37]) and some do not (e.g., [38]).
3.1. Control water Our results show that for a 4 mL sample, the number of
seeds (15 to 30 seeds) did not affect either germination or
Four kinds of water (distilled, mineral, reverse osmosis, elongation. For practical reasons, the number of seeds per
and ultra pure) were used as controls. Table 6 reports the dish was fixed at 20.
relative data of GR and RL of seeds watered with the
different control waters. The assay was performed on the 3.3. Seed cultivar
same cultivar of lettuce L. sativa. The results showed that
neither GR nor root length showed significant differences. Bioassays were conducted using a sample of raw
For practical reasons, the control water chosen was ROW. discharge water, taken from three different surface
We synchronically tested the potential ecotoxicological treatment companies (Co1S1, Co2S1 and Co3S1). The
differences between the four cultivars watered with ROW. characteristics of the samples are reported in Table 4. Fig. 1
Results (Table 7) showed no statistical GR or RL differences shows the four different dose–response curves of the four
between the different cultivars. Total lengths appeared to lettuce cultivars watered with Co1S1. It can be seen that
be different, especially Appia’s total length from the three when watered with the same effluent sample, the four
others. Differential cultivar total length was attributed cultivars did not show the same ecotoxicological response.
intrinsic natural differences as far as root lengths were not This was confirmed by the results described in Table 9,
significantly different. which presents the GR and GI values for every diluted

Table 8
Number of seeds per Petri dish versus germination rate GR (%), root and total lengths RL and TL (mm) of L. sativa var B (ROW; n = 3 replicates).

Number of seeds GR (%) RL (mm) TL (mm) P value

15 100 17.5  8.5 62  12.7

20 95  4 18.5  5.75 71  8.75 > 0.05
30 98  3 17.8  9.1 65.4  16.8
192 A. Priac et al. / C. R. Biologies 340 (2017) 188–194

Table 9
Germination rate, EC50 and germination index values for the four lettuce cultivars watered with the four effluent samples.

Sample Lettuce cultivar Treatment (tested effluent concentration)

25% 50% 75% 100% EC50 Sensitivity Scale

Germination rate Co1S1 B 98  2.4 92  2.4 97  2.4 45  4.1 99.75 B > K > GBP > A
GR [%] K 70  14.7 58  11.8 42  11.8 20  8.2 59.15
A 47  2.4 7  2.4 2  2.4 2  2.4 25.11
GBP 83  6.2 35  8.2 18  6.2 0 42.93
Co2S1 B 73  2.4 93  2.4 87  6.2 77  14.3 n.a. BKGBP > A
K 95 92  5 92  2.5 87  2.5 n.a.
A 82  8.5 52  14.3 33  6.2 28  14.3 61.17
GBP 97  4.7 97  6.2 97  4.7 68  6.2 n.a.
Co3S1 B 98  2.4 90  4.1 88  2.4 85  4.1 n.a BK > GBP > A
K 97  4.7 100 98  2.4 92  2.4 n.a
A 93  2.4 82  6.2 67  6.2 35  4.1 90.01
GBP 100 98  2.4 88  2.4 72  4.7 n.a
Germination index Co1S1 B 1.5 1.45 1.42 0.4 B > >K > GBP > A
GI K 0.96 0.67 0.33 0.13
A 1.12 0.13 0.03 0.05
GBP 0.89 0.51 0.23 0
Co2S1 B 1.21 1.16 1.42 1.18 B > GBPK >A
K 0.73 0.98 1.01 0.98
A 1.23 0.74 0.59 0.32
GBP 1.21 1.16 1.13 0.85
Co3S1 B 1.01 0.77 0.7 0.53 BGBP > K > >A
K 1.04 0.88 0.7 0.56
A 0.49 0.42 0.32 0.23
GBP 1.32 0.92 0.68 0.49

sample assessed on the four lettuce cultivars, and the EC50 considering all the tested samples and EC50s (Table 4). After
values for every sample. GR decreased with all the four raw comparing GR and EC50 values, var A was found to be the
samples. The intensity of the decrease depends on the most sensitive cultivar, vars K and GBP medium, and var B the
sample (e.g., var B’s GR varied from 45  4.1 to 93  2.4%). most resistant to the three effluents tested. This was
Indeed, Charles et al. [1] showed that lettuce ecotoxicological confirmed by comparing the GR/EC50 sensitive scale with
response variability can be linked with the chemical the GIs sensitive scale.
composition of the samples, which varies on a daily basis Although GR (lethal endpoint) is the most commonly
(Table 4). Differences between GRs were significant: for used endpoint, it is not the most sensitive [39]: root length
example, values for undiluted Co1S1 were 2  2.4%, (sublethal endpoint) has often proved to be a more
45  4.1%, 0% and 20  8.2% for var A, var B, var GBP and sensitive parameter, but not as easy to measure as
var K, respectively (Fig. 1). The same observation was made germination. This is the reason why GI, first defined to

Fig. 1. Lettuce germination rate versus concentration of Co1S1 raw discharge waters for the four lettuce varieties (batavia B, Kinemontepas K, Appia A and
grosse blonde parresseuse GBP).
 A. Priac et al. / C. R. Biologies 340 (2017) 188–194 193

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