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Effects of salinity and temperature on the expression of enzymatic biomarkers in Eurytemora affinis (Calanoida, Copepoda)
Comparative Biochemistry and Physiology, Part A 147 (2007) 841 – 849 www.elsevier.com/locate/cbpa
Effects of salinity and temperature on the expression of enzymatic biomarkers in Eurytemora affinis (Calanoida, Copepoda) K. Cailleaud a,b,c , G. Maillet c , H. Budzinski a , S. Souissi b , J. Forget-Leray c,⁎ a b
Université Bordeaux 1, CNRS, LPTC-UMR 5472 (Laboratory of Physico- and Toxico-Chemistry), 351 crs de la Libération, 33405 Talence, France Ecosystèmes Littoraux et Côtiers (UMR 8013 CNRS), Université des Sciences et Technologies de Lille, 28 avenue Foch, 62930 Wimereux, France c Laboratoire d'Ecotoxicologie-Milieux Aquatiques (UPRES EA3222), GDR IMOPHYS, Faculté des Sciences et Techniques du Havre, 25 rue Philippe Lebon, 76058 Le Havre, France Received 21 July 2006; received in revised form 24 September 2006; accepted 26 September 2006 Available online 30 September 2006
Abstract In order to establish effective enzymatic biomarkers that could provide in situ early warning of contaminant exposure in estuarine ecosystems, the potential effects of the principal abiotic factors (temperature and salinity) were investigated on common biomarkers, the acetylcholinesterase (AChE) and the glutathione S-transferase (GST) in Eurytemora affinis. Short term salinity stress effects simulated during an experimental tide indicated that enzymatic activities of this species are characterized by maximum expression related to an optimal salinity range (between 5 and 15 psu). Moreover, longer time exposure to various salinity tanks confirmed the effects of this factor on both AChE and GST activities. Therefore, optimal AChE activity was measured at 10 psu, while optimal GST activity was measured at 5 psu. Furthermore, significant effects of temperature were also recorded, particularly for AChE expression (slight effects were measured on GST expression) with an optimal condition at 11 °C. These experiments indicated a more pronounced effect of salinity over temperature especially on the AChE expression and confirmed the need to standardize sampling procedures in relation with environmental parameters for biomonitoring studies based on enzymatic analyses. © 2006 Elsevier Inc. All rights reserved. Keywords: AChE; GST; Physicochemical parameters; Estuary; Invertebrate
1. Introduction In order to assess the water quality of various aquatic ecosystems and to estimate the stress encountered by the different populations that inhabitate these environments, the concentrations of a number of organic contaminants (PAHs, PCBs…) as well as trace metals (Hg, Cd, Pb, Cu, Zn) have been regularly monitored in many contaminated locations in the world. Nevertheless, chemical analyses alone are not sufficient to describe the effects of pollutants at contaminated areas. During the past years, many studies focusing on the biological effects of these contaminants have been conducted on several aquatic species, especially on fish and mollusks. Indeed, several contaminant effect indicators have been developed (Van der
⁎ Corresponding author. Tel.: +33 2 32744379; fax: +33 2 32744314. E-mail address: [email protected] (J. Forget-Leray). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.09.012
Oost et al., 2003). Thus, a number of promising and sensitive early warning signals, or biomarkers, that reflect adverse biological responses consecutive to anthropogenic contaminants and environmental toxin exposure and stress, have been identified (McCarthy and Shugart, 1990). Therefore, a wide range of biomarkers and particularly enzymatic activities, that provide information on uptake, biotransformation and detoxification patterns (Livingstone, 1993; Snyder, 2000), has been developed, tested and proposed for application in monitoring and assessing deleterious effects of chemical stress on organisms (Handy et al., 2003; Lam and Gray, 2003). Besides, the use of a battery of biomarkers in combination with chemical analyses is recommended (Cajaraville et al., 2000). Among the most reliable biomarkers, the acetylcholinesterase enzymatic activity (AChE) is one of the most frequently used in Ecotoxicology. AChE is responsible for the breakdown of acetylcholine in cholinergic synapses, preventing continuous nerve firings, which is vital for normal cellular neurotransmitter
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functioning (Payne et al., 1996). As an indicator of exposure to contaminants, the inhibition of this biomarker is commonly used to detect environmental pollution caused by neurotoxic compounds and is particularly specific of organophosphate and carbamate contaminations (Galgani and Bocquené, 1990; Forget et al., 2003). These compounds, that bind in reversible or irreversible manner to the catalytic site of the enzyme, cause the free unbound acetylcholine to accumulate at cholinergic receptor sites and thus result in the overstimulation of muscarinic and nicotinic cholinergic receptors in the central and peripheral nervous system. In addition to anticholinesterase insecticides, other contaminants such as PAHs (Kang and Fang, 1997), surfactants (Guilhermino et al., 2000), and pharmaceuticals (Nunes et al., 2006) have been shown to cause AChE inhibition. Other enzymatic biomarkers related to the detoxification potential of the studied organisms are also of great interest. Among these enzymatic activities, GSTs are a group of widely distributed enzymes that catalyze the conjugation of reduced glutathione (GSH) with compounds having reactive electrophilic groups (especially xenobiotics), generating less toxic and more hydrophilic molecules (Olsen et al., 2001). This metabolic pathway allows the protection of nucleophilic groups in macromolecules such as proteins and nucleic acids. GSTs were determined as effect biomarkers induced by different natural and synthetic compounds such as organochlorine pesticides, PAHs and PCBs (Lee et al., 1988; Fitzpatrick et al., 1997; Hoareau et al., 2001). Although these biomarkers reflect adverse biological responses to anthropogenic contamination, they could also be related to natural environmental temporal and spatial variations. Thus, several studies have investigated the seasonal variations on some biochemical biomarkers, linking them to seasonal abiotic parameter variations such as temperature, salinity, turbidity and food availability (Sheehan and Power, 1999; Lau et al., 2004; Leiniö and Lehtonen, 2005). Estuaries in particular present marked physicochemical small scale changes (mainly salinity, turbidity and current flow) associated with tidal cycles (Chapman and Wang, 2001) and large scale seasonal variations (essentially temperature and food bioavailability). Therefore, ecotoxicological in situ studies in estuaries constitute a challenge to integrate and distinguish biomarker responses to natural abiotic factor variations from contaminant-induced effects. To manage pollution monitoring in such ecosystems, the principal issues are to select an appropriate sentinel species and suitable biomarkers. E. affinis is a common estuarine copepod species, widely distributed in world estuaries, particularly in the Seine River Estuary where it represents between 90 and 100% of the zooplankton biomass (Mouny and Dauvin, 2002). This euryhaline and eurythermal species is distributed all over the estuary, with a well known biology characterized by a short life cycle (Katona, 1970); it is abundant all year round and is easily sampled; this copepod is omnivorous, playing a key role in the estuarine food web, and could therefore ensure transfers and bioaccumulation of contaminants from dissolved water and organic material to upper organisms in the food chain. Besides,
this species could be easily kept in laboratory. For these reasons E. affinis is an ideal candidate for ecotoxicological studies. The principal objectives of this work were to investigate experimentally the potential effects of the main physicochemical variations (salinity and temperature) that occurred in naturally fluctuating ecosystems such as estuaries, which could induce variable responses on biochemical biomarkers and bias pollution-caused alterations of these biological indicators. This study is the first step in the development of an in situ estuarine bioassay using E. affinis as sentinel species. Indeed, it could allow to minimize biomarker response variability in field studies related to physicochemical variations by normalizing sampling procedures focusing on salinity and temperature. 2. Materials and methods 2.1. Sample collection Several sampling were conducted in spring 2005. The copepods E. affinis were collected using subsurface tows of WP2 plankton net (200 μm mesh size) at ebb tide, in Tancarville Station in the oligohaline part of the Seine Estuary (Fig. 1). Immediately after sampling, copepods were sorted using 500 μm sieves in order to eliminate predators (especially Mysidacea and Gammaridae), then transferred into containers using filtered estuarine water and brought back to the laboratory for further precise sorting. Then, the copepods were sorted quickly (b 3 h) using several times the sequence: Dilution–Decantation–Photo attraction. To dilute the natural samples, mineral clean water (adjusted to the sampling site salinity) was used. The objectives were to separate the copepods attracted by the light on the water surface, from the underlying particles in order to make sure of the composition of the matrix for the following experiments. 2.2. Experimentation After sorting by photo-attraction, copepods were kept in the laboratory for an acclimatization period of one day in glass beakers, with a mixture of 300 ml of filtered estuarine water and 1500 ml of mineral water adjusted to the salinity of the sampling site at controlled temperature (11 °C) in a climatic chamber (LMS Cooled Incubator, Bioblock Scientific, Illkirch, France). The copepods were fed one time during the acclimatization period with Isochrysis galbana, and then kept starving during the experiments in order to limit feeding movements which could enhance the AChE activity. 2.2.1. Experimental tide The Seine Estuary is characterized by two tidal cycles a day. Each one lasts approximately 12 h and is composed of an ebb tide during which freshwater influence increases with a salinity scale ranging from 25 psu to 0 psu, and a flow tide dominated by marine influence with a salinity reaching back 25 psu. Salinity variations during the tide depend on the location in the estuary and are maximum in the euryhaline part of the estuary. In the laboratory, such small scale salinity variations were preliminarily tested during one simulated flow tide. Therefore, different
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Fig. 1. Map of the sampling location.
2-l tanks filled with 300 ml of filtered estuarine water and 1500 ml of mineral water were adjusted at the following selected salinities, 0, 5, 10, 15, 20 and 25 psu with Sera PREMIUM salt (Heinsberg, France) and kept at a controlled temperature of 11 °C. Then, the copepods which were acclimatized during one day to the laboratory conditions were transferred into the first beaker containing water at 0 psu and kept 1 h in this tank. After that, the copepods were filtered and transferred to the second beaker at 5 psu and kept in this tank during one hour and so on until the last beaker with a water salinity of 25 psu. Pools of approximately 200 mg (wet weight) of copepods were sampled after 1 h in each tank at the different salinities for AChE and GST activity analyses (two replicate samples for each sampling time and each exposure condition). 2.2.2. Effects of salinity The E. affinis population is not homogeneously distributed in the Seine Estuary. Therefore, all copepods are not submitted to the same salinity range according to their location in the different zones of the estuary (oligohaline, mesohaline and euryhaline). Thus, copepods distributed in zones under maximal marine or freshwater influences (oligohaline and euryhaline zones) are submitted to slighter salinity variations than copepods from the mesohaline zone. Besides, these copepods are submitted to a short scale salinity variation but during a longer time. In order to test the effects of salinity on AChE and GST enzymatic activities, different water tanks filled with 300 ml of filtered estuarine water and 1500 ml of mineral water were adjusted at the following selected salinities, 0, 5, 10, 15, 20 and 25 psu and kept at 11 °C in a climatic chamber. After the acclimatization period of one day in the laboratory, the initial pool of copepods was divided into 6 homogeneous groups and each group was transferred to one of the tanks previously prepared, containing the different salinity waters. At the beginning of the experiment and after 1, 2, 4, 18 and 72 h, pools of approximately 200 mg of copepods were sampled in each tank at the different salinities for AChE and GST activity analyses (two replicate samples for each sampling time and each exposure condition for the AChE activity).
2.2.3. Effects of temperature The copepod E. affinis is present in the Seine Estuary during all the year. Therefore, the successive copepod populations are submitted to variable temperatures depending on the season (from 4 °C in winter to 25 °C in summer periods). To study the effects of temperature on AChE and GST enzymatic activities, different water tanks filled with 300 ml of filtered estuarine water and 1500 ml of mineral water were adjusted at 15 psu and kept at the following selected temperatures representative of the annual temperature range in the Seine Estuary: 4, 11, 18 and 25 °C in 4 climatic chambers. After the acclimatization period of one day in the laboratory, the initial pool of copepods was divided into 4 homogeneous groups and each group was transferred to one of the tanks previously prepared, kept at the different temperatures. At the beginning of the experiment and after 1, 2, 4, 18 h, pools of approximately 200 mg of copepods were sampled in each tank at the different temperatures for AChE and GST activity analyses (two replicate samples for each sampling time and each exposure condition). In comparison to salinity experiments, there were not sufficient amounts of copepods after 72 h to measure both AChE and GST activities for all the exposure conditions. 2.3. Biomarker assays A pool of approximately 200 mg of whole-body E. affinis was used to determine both AChE and GST activities. A pool of copepods was homogenized on ice, in ice-cold 0.02 M pH 7 phosphate buffer+/0.1% Triton X100 (1/4, volume/weight) using an Ultra-Turax homogenizer. The homogenates were then centrifuged two successive times at 10,000 ×g for 5 min at 4 °C, with an intermediate shaking. Measurements of AChE activity were performed using the colorimetric method of Ellman et al. (1961), modified by Bocquené and Galgani (1998) at 412 nm, with acetylthiocholine iodide (AcSCh) as substrate and dithiobis-nitrobenzoate as reagent at a controlled temperature of 20 °C. GST activity was evaluated as the conjugation between glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm (Habig et al., 1974). The extinction coefficients used to
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determine GST and AChE specific activities were respectively 9.6 × 10− 3 M− 1cm− 1 at 340 nm and 12,600 M− 1cm− 1 at 412 nm. AChE and GST activities are expressed respectively as μmol min−1 mg−1 protein and as mol min− 1 mg− 1 protein. Proteins for standardisation of the above biomarkers were determined using Bradford's (1976) method modified for use with a micro-plate reader (Bocquené and Galgani, 1998). All assays were performed in quadruplicate. All chemicals were purchased from Sigma-Aldrich (St Quentin Fallavier, France). 2.4. Statistical analyses All biomarker results are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used, after testing the variable normality (Kolmogorov– Smirnoff test) and the variance homogeneity (Levene test), to compare both AChE and GST activities between the different exposure conditions, with salinity, time and temperature as factors. These analyses were followed by Tukey's multiple comparison tests to discriminate significantly different treatments. The significance p-level used was set to 0.05. When normal distribution and homogeneity of the variances were not verified (for experimental tide, and effect of temperature), non parametric Kruskal–Wallis ANOVA by ranks was performed both on AChE and GST data followed by the non parametric Mann–Whitney U test to identify significant differences between groups ( p b 0.05). All data were statistically analyzed by the STATISTICA Software v6.0 (Statsoft. Inc., 2002). 3. Results 3.1. Experimental tide Data obtained during the experimental tide related to AChE and GST activities are reported in Table 1. Average AChE and GST activities range respectively from 765 to 1023 μmol min− 1 mg− 1 protein and from 622 ± 82 to 842 ± 42 mol min− 1 mg− 1 protein. Besides, the Kruskal–Wallis test indicates a significant effect of salinity variations during the experimental tide both on AChE and GST activities (respectively, H = 59.76, p b 0.00001, df = 6; and H = 22.97, p b 0.0008, df = 6). Therefore, during the experimental tide, AChE levels increase from 0 psu to 10 psu exposures then decrease for all the following salinity exposures until 25 psu. Thus, the highest AChE level is measured for 10 psu salinity while the lowest one is measured for 25 psu. Moreover, AChE level for 10 psu is significantly higher than AChE levels for all other salinities ( p b 0.05), while AChE levels for 20 and 25 psu salinity are significantly lower than all other AChE levels ( p b 0.005). In the same way, during the experimental tide, GST levels increase from 0 to 5 psu exposures and then decrease for all the following salinity exposures until 25 psu. Moreover, the highest GST level is measured for a salinity exposure of 5 psu and is significantly higher than GST levels for all other salinity exposures ( p b 0.05) except for 0 and 10 psu. Furthermore, the significantly lowest GST levels are measured for salinity exposures of 20 and 25 psu ( p b 0.05).
3.1.1. Effects of salinity AChE activity variations in E. affinis after exposure to different salinities are presented in Fig. 2. Average AChE activities range respectively from 583 to 728, 546 to 962, 528 to 1156, 565 to 849, 583 to 813 and 444 to 738 μmol min− 1 mg− 1 protein for 0 to 25 psu. Besides, significant effects of salinity over the time of exposure on AChE activity are recorded (ANOVA; p b 0.00025, F = 4.92, degree of freedom: 5). High AChE activities are measured during all the experiments in copepods exposed to 10 psu water, particularly after 18 and 72 h of exposure when increases are significant ( p b 0.00005). Therefore, after 72 h of exposure, AChE level is enhanced by 98% in copepods exposed to 10 psu water. Besides, AChE activity increases significantly in copepods exposed to 15 and 20 psu between 4 and 18 h of exposure ( p b 0.00005) after which the activity remains constant at 20 psu or decreases significantly at 15 psu ( p b 0.00005). AChE activities measured in copepods kept at 5 and 25 psu show a higher variability and no significant increase during the experiments. Finally, copepods exposed to 0 psu present slightly increasing AChE levels between 1 and 2 h of exposure in comparison to the other experiments, then decreasing levels until 18 h; no copepod has survived at the end of the experiments after 72 h. Moreover, after one hour of exposure, the highest AChE levels are measured for copepods exposed to 5 psu water. Then, after 2 and 4 h of exposure, slight differences are measured in AChE levels between the different experimental exposures. After 18 and 72 h of exposure, the highest AChE levels are measured for copepods exposed to 10 psu water ( p b 0.00005), and these AChE levels are the highest ones measured during all the experiments. GST activity variations in E. affinis after exposure to different salinities are presented in Fig. 3. Average GST activities range respectively from 700 to 125, 1138 to 60, 700 to 108, 700 to 122, 700 to 175 and 700 to 132 mol min− 1 mg− 1 protein for 0 to 25 psu. Besides, significant effects of salinity over the time of exposure on GST activity are recorded (ANOVA; p b 0.0003, F = 5.13, degree of freedom: 5). Therefore, a linear significant Table 1 Variations of AChE and GST activities (means values ± SEM) in E. affinis during the experimental tide Exposure time
T0 In situ 1H 2H 3H 4H 5H 6H
Treatment salinity (PSU)
2.5 0 5 10 15 20 25
Enzymatic activities AChE
GST
μmol min− 1 mg− 1 protein
mol min− 1 mg− 1 protein
818 ± 64b, c, d, e 894 ± 73a, d, f, g 945 ± 52a, d, f, g 1023 ± 79a, b, c, e, f, 906 ± 44a, d, f, g 810 ± 54b, c, d, e 765 ± 57b, c, d, e
622 ± 8c, d 718 ± 121 842 ± 42a, e, 822 ± 58a, e, 725 ± 45c, d 685 ± 66c, d 690 ± 123c
g
f, g f
Means (n = 8: i.e. 2 experimental replicates and 4 enzymatic measures/pooled samples) and standard deviations are mentioned. Significant differences between salinities (p b 0.05) are indicated using letters (a, b, c, d, e, f and g which refer respectively to 2.5, 0, 5, 10, 15, 20 and 25 psu).
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Fig. 2. AChE activity variations in the copepod E. affinis after exposure to different salinities (from 0 to 25 psu). Means (n = 8: i.e. 2 experimental replicates and 4 enzymatic measures/pooled samples) and standard deviations are mentioned. Significant differences between sampling times ( p b 0.05) are indicated using letters (a, b, c, d, e and f which refer respectively to 0, 1, 2, 4, 18 and 72 h of exposure).
GST decrease is recorded during all the experiment in copepods exposed to 15 psu ( p b 0.05). After 72 h of exposure to 15 psu water, GST level decreases to reach 17% of the GST level of the beginning of the experiment. In the same way, non linear decreasing GST levels are measured at 20 psu. Besides,
copepods exposed to 0, 10 and 25 psu waters present the same GST variation pattern: significant decreases during the first hour of the experiments ( p b 0.0005), then non significant GST variation until the 18th hour of experiment and finally significant decreases until the 72nd hour of the experiment ( p b 0.05).
Fig. 3. GST activity variations in the copepod E. affinis after exposure to different salinities (from 0 to 25 psu). Means (n = 4 i.e. 4 enzymatic measures) and standard deviations are mentioned. Significant differences between sampling times ( p b 0.05) are indicated using letters (a, b, c, d, e and f which refer respectively to 0, 1, 2, 4, 18 and 72 h of exposure).
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Fig. 4. Effects of temperature on AChE activity expression during 18h of experimental exposure. Means (n = 8: i.e. 2 experimental replicates and 4 enzymatic measures/pooled samples) and standard deviations are mentioned. Significant differences between sampling times (p b 0.05) are indicated using letters (a, b, c, d and e which refer respectively to 0, 1, 2, 4 and 18 h of exposure).
Moreover, GST activities measured in copepods kept at 5 psu show a higher variability compared to that of copepods exposed to other salinities. Indeed, a significant increasing GST level ( p b 0.0005) followed by a significant decrease ( p b 0.0005) are measured at the beginning of the experiment. Then no significant variation is recorded between 2 and 18 h of exposure and finally the GST activity significantly decreases again ( p b 0.0005). 3.1.2. Effects of temperature Average AChE activity of E. affinis exposed to an 11 °C water temperature increases during all the experiment from 716 to 875 μmol min− 1 mg− 1 protein (Fig. 4). The total increase of AChE activity between the beginning and the end of the experiment represents 22%. Furthermore, after two hours of exposure and until the end of the experiment AChE levels in
copepods exposed to this temperature condition are significantly higher than AChE level at the beginning of the experiments ( p b 0.05). In addition, after 1, 4 and 18 h of experiment, AChE levels in copepods exposed to an 11 °C water temperature are significantly higher than AChE levels measured in copepods exposed to the other water temperature conditions at the same time of the experiment. For all other temperature conditions, AChE levels are more variable. Moreover, for 4, 18 and 25 °C conditions, AChE levels at the beginning and at the end of the experiment are not statistically significant. In relation to a high mortality after 4 h of exposure, there was no sufficient quantity of copepods to measure both AChE and GST activities at the end of the experiment, after 18 h of exposure to an 18 °C water temperature. The lowest AChE activities are measured in copepods exposed to the highest water temperature (25 °C)
Fig. 5. Effects of temperature on GST activity expression during 18 h of experimental exposure. Means (n = 8: i.e. 2 experimental replicates and 4 enzymatic measures/ pooled samples) and standard deviations are mentioned. Significant differences between sampling times ( p b 0.05) are indicated using letters (a, b, c, d and e which refer respectively to 0, 1, 2, 4 and 18 h of exposure).
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during all the experiment. Besides, the higher the water temperature is, the lower the AChE level is. Furthermore, Kruskal–Wallis tests indicate significant influence of both temperature (H = 67.6, p b 0.00001, df = 3) and time (H = 26.13, p b 0.00001, df = 4) factors on AChE activity variations. No significant GST activity variations are recorded after exposure at 4, 18 and 25 °C between the beginning and the end of the experiment (Fig. 5). Besides, GST activity is quite constant all over the experiment at 18 and 25 °C, while this enzymatic activity is more variable at 4 °C with significant increase during the first hours of the experiment (p b 0.0005) and then significant decrease at the end of the experiment to reach back the initial level. In opposition, slight (17%) but significant increasing levels (p b 0.0) are recorded after exposure at 11 °C between the beginning and the end of the experiment. Furthermore, Kruskal– Wallis tests confirm significant effects of the temperature factor (H = 26.1, p b 0.00001, df = 3) at each sampling time. Significant effects of the time of exposure factor (H = 15.19, p b 0.0043, df = 4) are also measured at 4 and 11 °C temperature conditions. 4. Discussion In order to use biochemical biomarkers for assessing pollution effects in variable ecosystems like estuaries, the natural variability of these biomarkers has to be studied. However, few experimental or in situ studies have reported the effects of temperature and salinity factors on enzymatic biomarker activities. Besides, reported data concern mainly vertebrate and especially marine fish species while effects on estuarine invertebrates have been poorly investigated. Nevertheless, effects of both temperature and salinity have been reported for different species including blue mussels (Galgani and Bocquené, 2000; Pfeifer et al., 2005), Nereis diversicolor (Scaps and Borot, 2000) and brown shrimps (Menezes et al., 2006). The data obtained in this study indicate an increase of AChE levels under controlled conditions (Fig. 2) which suggests the presence of and/or multiple inhibiting factors in the field (contaminants as well as natural parameters like temperature or salinity). Besides, decreasing GST levels are recorded (Fig. 3) during the experiment in the laboratory which implies the occurrence of inducing factors in the field which could be the same as those responsible for the inhibition of the AChE activity in the case of an environmental contamination. Similar results have been reported in the shrimp Crangon crangon after maintaining the field collected animals in the laboratory (Menezes et al., 2006). Furthermore, the results of the effects of an experimental tide on both AChE and GST activity expression indicate significant variations even at small time scale. Besides, these results indicate an optimal physiological salinity range for E. affinis between 5 and 10 psu related to the enzyme, beyond which the enzymatic activity expression decreased. Moreover, these results show that this species is better adapted to low salinity zones as the highest biomarker levels are measured for the lowest salinity and because the highest salinity induces the maximal inhibition. To our knowledge, no work has studied up to now the effects of tidally related salinity variations on AChE and GST activities. Therefore, no comparison can be made with other estuarine species.
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The copepod E. affinis shows strong osmoregulation abilities to maintain the population distribution in all the Seine Estuary (Roddie et al., 1984; Kimmel and Bradley, 2001). However, deviation from the optimal salinity range previously described can be stressful and the cost of osmotic regulation, at the molecular level, could directly affect biological molecules such as enzymes like AChE and GST activities. These observations make sense especially in longer time exposure as shown in Figs. 2 and 3. Thus, significant effects of salinity are recorded after the experiment. Therefore, increasing AChE levels are observed during the experiments and the most pronounced ones are measured for the exposition to 10 psu water salinity. These results confirm an optimal salinity around 10 psu as previously mentioned since the maximum increase in AChE activity is measured in copepods exposed to 10 psu water. Moreover, differences observed with the other exposition conditions could be explained by a decrease in energetic cost allocated to AChE activity and increasing energy devoted to osmotic regulation. Moreover, Gyllenberg and Lundqvist (1979) and Gaudy et al. (2000) specified that increasing respiration rates have been observed respectively in Eurytemora hirundoides and in Acartia tonsa and Acartia clausi when salinity conditions diverge from the optimal salinity zone of these species and that this increase of respiration is related to the need of supplementary energy for osmoregulation. Furthermore, Scaps and Borot (2000) have reported similar effects of salinity on Nereis diversicolor with however, adaptation for higher salinity zone. In addition, decreasing GST levels (Fig. 3) are measured during the experiment which could result from the combination of the transfer into non contaminated water with the effect of salinity stress. The most linear decrease is reported for 15 psu water exposition. Besides, for salinity different from the optimal one, more variable GST activities are measured (decreasing levels alternate with increasing levels). These results suggest that salinity stress could interfere with oxidative stress responses in E. affinis. However, as no similar study is available, no comparison could be made with other estuarine species. Nevertheless, we could hypothesize that salinity stress could modify antioxidative biomarker activities with however slighter effect than for AChE activity. To date, the effects of temperature on biochemical biomarkers have been essentially studied in vertebrate species and especially fish (Hazel, 1969; Hogan, 1970). However, some recent works have been performed with invertebrate species (Scaps and Borot, 2000; Pfeifer et al., 2005; Menezes et al., 2006). Thus, it is well established that the increase of water temperature induces significantly the expression of the AChE activity. Furthermore, temperature can directly affect the activity of enzymes by changing their physical structure and thereby changing their catalytic efficiency or binding capacity (Hochachka and Somero, 1984). Our experiments show that the maximum AChE levels are obtained at the 11 °C water condition (Fig. 4). Besides, significant increases of AChE activity at this temperature are recorded during the experiment. For the other water temperature conditions, rapid heat-shock responses resulting in a decrease of AChE levels, are observed after one hour of exposure. Then, AChE activity increases again for all
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temperature conditions. According to these results, the hypothesis of a short adaptation time to water temperature transitions could be made. Furthermore, Gyllenberg and Lundqvist (1979), Pagano and Gaudy (1986), and Gaudy et al. (2000) have mentioned, in different estuarine copepod species, metabolisms reduced to a minimum to maintain the physiological activity at low temperatures and to save energy at high temperatures. These observations are consistent with the results obtained in this study. Concerning GST activity, slighter significant effects of temperature are observed compared to those on the AChE activity. The highest differences are measured for the first two hours of the experiment for the exposure condition of 4 °C. Similar higher anti-oxidative activities are detected in in situ studies on Perna viridis (Lau et al., 2004). The authors hypothesized that these increases could be due to concentrating effects related to the general decrease in protein level during cold seasons or to stress induced by a chemical contamination of the sampling site. In the same way, GST activity variations related to seasonal variations in temperature have been reported in the blue mussel (Power and Sheehan, 1996). In opposition, Lushchak and Bagnyukova (2006) have reported no effect of temperature increase in the GST activity of goldfish. However, as no similar experimental study has been previously performed, no comparison could be made. To conclude, the results of this study constitute the first step before using biochemical markers such as AChE and GST activities to assess contaminant exposure of the copepod E. affinis in estuarine ecosystems. Indeed, attention should be paid to the effects of abiotic environmental parameters and particularly salinity and temperature variations for in situ biomonitoring programs based on enzymatic activity studies. Moreover, a minimum reduction or activation of 30% of respectively AChE and GST activities are advisable to conclude on contaminant exposure. Furthermore, E. affnis is a fundamental species in the estuarine food web and presents a wide world distribution. Besides, enzymatic activities of this species are sensitive to chemical exposition and changes in water temperature do not appear to be an important source of enzymatic activity alteration (especially for GST activity). In opposition, salinity variations significantly modulate these enzyme activities, which require for biomonitoring projects to repeat sampling in the same salinity conditions during all the study in order to avoid fluctuations of enzymatic activities related to environmental parameters. According to these observations, E. affinis represents a promising sentinel species in estuarine ecosystems. Acknowledgements This work was financially supported by the multidisciplinary Seine Aval scientific program by the Regi on Haute-Normandie and by the PNETOX scientific program. References Bocquené, G., Galgani, F., 1998. Biological effect contaminants: cholinesterase inhibition by organophosphate and carbamate compounds. ICES Tech. Mar. Environ. Sci. 22, 1–12.
Bradford, M., 1976. A rapid and sensitive assay of protein utilizing the principle of dye binding. Anal. Biochem. 772, 248–264. Cajaraville, M.P., Bebianno, M.J., Blasco, J., Porte, C., Sarasquete, C., Viarengo, A., 2000. The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach. Sci. Total Environ. 247, 295–311. Chapman, P.M., Wang, F., 2001. Assessing sediment contamination in estuaries. Environ. Toxicol. Chem. 20, 3–22. Ellman, G., Courtney, K., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Fitzpatrick, P.J., O'Halloran, J., Sheehan, D., Walsh, A.R., 1997. Assessment of a glutathione-S-transferase and related proteins in the gill and digestive gland of Mytilus edulis (L.) as potential organic pollution biomarkers. Biomarkers 2, 51–56. Forget, J., Beliaeff, B., Bocquené, G., 2003. Acetylcholinesterase activity in copepods (Tigriopus brevicornis) from the Vilaine River estuary, France, as a biomarker of neurotoxic contaminants. Aquat. Toxicol. 62, 195–204. Galgani, F., Bocquené, G., 1990. In vitro inhibition of acetylcholinesterase from four marine species by organophosphates and carbamates. Bull. Environ. Contam. Toxicol. 45, 243–249. Galgani, F., Bocquené, G., 2000. Molecular biomarkers of exposure of marine organisms to organophosphorus pesticides and carbamates. In: Lagadic, L. (Ed.), Use of Biomarkers for Environmental Quality Assessment. Elsevier Science Publisher, pp. 113–137. Gaudy, R., Cervetto, G., Pagano, M., 2000. Comparison of the metabolism of Acartia clausi and A. tonsa: influence of temperature and salinity. J. Exp. Mar. Biol. Ecol. 247, 51–65. Guilhermino, L., Lacerda, M.N., Nogueira, A.J.A., Soares, A.M.V., 2000. In vitro and in vivo inhibition of Daphnia magna acetylcholinesterase by surfactant agents: possible implications for contamination biomonitoring. Sci. Total Environ. 247, 137–141. Gyllenberg, G., Lundqvist, G., 1979. The effects of temperature and salinity on the oxygen consumption of Eurytemora hirundoides (Crustacea, Copepoda). Ann. Zool. Fenn. 16, 205–208. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione-S-Transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Handy, R., Galloway, T.S., Depledge, M.H., 2003. A proposal for the use of biomarkers for the assessment of chronic pollution and in regulatory toxicology. Ecotoxicology 12, 331–343. Hazel, J., 1969. The effect of thermal acclimation upon brain acetylcholinesterase activity of Carassius auratus and Fundulus heteroclitus. Life Sci. 8, 775–784. Hoareau, P., Gnassia-Barelli, M., Roméo, M., Girard, J.P., 2001. Differential induction of GST in the clam Ruditapes decussatus. Eur. J. Biochem. 269, 4359–4366. Hochachka, P.W., Somero, G.M., 1984. Biochemical Adaptation. Princeton University Press, New York. Hogan, J.W., 1970. Water temperature as a source of variation in specific activity of brain acetylcholinesterase of bluegills. Bull. Environ. Contam. Toxicol. 5, 347–353. Kang, J.J., Fang, H.W., 1997. Polycyclic aromatic hydrocarbons inhibit the activity of acetylcholinesterase purified from electric eel. Biochem. Biophys. Res. Commun. 238, 367–369. Katona, S.K., 1970. The developmental stages of Eurytemora affinis (Poppe, 1880) (Copepoda, Calanoida) raised in laboratory cultures, including a comparison with the larvae of Eurytemora Americana Williams, 1906, and Eurytemora herdmani Thompson and Scott, 1897. Crustaceana 21, 5–20. Kimmel, D.G., Bradley, B.P., 2001. Specific protein responses in the calanoid copepod Eurytemora affinis (Poppe, 1880) to salinity and temperature variation. J. Exp. Mar. Biol. Ecol. 266, 135–149. Lam, P.K.S., Gray, J., 2003. The use of biomarkers in environmental monitoring programmes. Mar. Pollut. Bull. 46, 182–186. Lau, P.S., Wong, H.L., Garrigues, Ph., 2004. Seasonal variation in antioxidative responses and anticholinesterase activity in Perna viridis in eastern oceanic and western estuarine waters of Hong Kong. Cont. Shelf Res. 24, 1969–1987.
K. Cailleaud et al. / Comparative Biochemistry and Physiology, Part A 147 (2007) 841–849 Lee, R.F., Keeran, W.S., Pickwell, G.V., 1988. Marine invertebrate glutathioneS-transferase: purification, characterization and induction. Mar. Environ. Res. 24, 97–100. Leiniö, S., Lehtonen, K.K., 2005. Seasonal variability in biomarkers in the bivalves Mytilus edulis and Macoma balthica from the northern Baltic Sea. Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol. 140, 408–421. Livingstone, D.R., 1993. Biotechnology and pollution monitoring: use of molecular biomarkers in the aquatic environment. J. Chem. Technol. Biotechnol. 57, 195–211. Lushchak, V.I., Bagnyukova, T.V., 2006. Temperature increase results in oxidative stress in goldfish tissues. 2. Antioxidant and associated enzymes. Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol. 143, 36–41. McCarthy, J.F., Shugart, L.R., 1990. Biomarkers of Environmental Contamination. Lewis Publishers, Boca Raton, FL. Menezes, S., Soares, A.M.V.M., Guilhermino, L., Peck, M., 2006. Biomarker responses of the estuarine brown shrimp Crangon crangon L. to non-toxic stressors: temperature, salinity and handling stress effects. J. Exp. Mar. Biol. Ecol. 335, 114–122. Mouny, P., Dauvin, J.C., 2002. Environmental control of mesozooplankton community structure in the Seine estuary (English Channel). Oceanol. Acta 25, 13–22. Nunes, B., Carvalho, F., Guilhermino, L., 2006. Effects of widely used pharmaceuticals and a detergent on oxidative stress biomarkers of the crustacean Artemia parthenogenetica. Chemosphere 62, 581–594. Olsen, T., Ellerbeck, L., Fisher, T., Callaghan, A., Crane, M., 2001. Variability in acetylcholinesterase and glutathione S-transferase activities in Chironomus riparius Meigen deployed in situ at uncontaminated field sites. Environ. Toxicol. Chem. 20, 1725–1732.
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Pagano, M., Gaudy, R., 1986. Biologie d'un copépode des mares temporaires du littoral méditerranéen français, Eurytemora velox. II. Respiration et excrétion. Mar. Biol. 90, 551–564. Payne, J.F., Mathieu, A., Melvin, W., Fancey, L.L., 1996. Acetylcholinesterase, an old biomarker with a new future? Field trials in association with two urban rivers and a paper mill in Newfoundland. Mar. Pollut. Bull. 32, 225–231. Pfeifer, S., Schiedek, D., Dippner, J.W., 2005. Effect of temperature and salinity on acetylcholinesterase activity, a common pollution biomarker, in Mytilus sp. from the south-western Baltic Sea. J. Exp. Mar. Biol. Ecol. 320, 93–103. Power, A., Sheehan, D., 1996. Seasonal variation in the anti-oxidant defence systems of gill and digestive gland of the Blue Mussel Mytilus edulis. Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol. 114, 99–103. Roddie, B.D., Leakey, R.J.G., Berry, A.J., 1984. Salinity–temperature tolerance and osmoregulation in Eurytemora affinis (Poppe) (Copepoda: Calanoida) in relation to its distribution in the zooplankton of the upper reaches of the forth estuary. J. Exp. Mar. Biol. Ecol. 79, 191–211. Scaps, P., Borot, O., 2000. Acetylcholinesterase activity of the polychaete Nereis diversicolor: effects of temperature and salinity. Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol. 125, 377–383. Sheehan, D., Power, A., 1999. Effects of seasonality on xenobiotic and antioxidant defense mechanisms of bivalve mollusks. Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol. 123, 193–199. Snyder, M.J., 2000. Cytochrome P450 enzymes in aquatic invertebrates: recent advances and future directions. Aquat. Toxicol. 48, 529–547. Van der Oost, R., Beyer, J., Vermeulen, N.P.E., 2003. Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ. Toxicol. Pharmacol. 13, 57–149.
Effects of salinity and temperature on the expression of enzymatic biomarkers in Eurytemora affinis (Calanoida, Copepoda) K. Cailleaud a,b,c , G. Maillet c , H. Budzinski a , S. Souissi b , J. Forget-Leray c,⁎ a b
Université Bordeaux 1, CNRS, LPTC-UMR 5472 (Laboratory of Physico- and Toxico-Chemistry), 351 crs de la Libération, 33405 Talence, France Ecosystèmes Littoraux et Côtiers (UMR 8013 CNRS), Université des Sciences et Technologies de Lille, 28 avenue Foch, 62930 Wimereux, France c Laboratoire d'Ecotoxicologie-Milieux Aquatiques (UPRES EA3222), GDR IMOPHYS, Faculté des Sciences et Techniques du Havre, 25 rue Philippe Lebon, 76058 Le Havre, France Received 21 July 2006; received in revised form 24 September 2006; accepted 26 September 2006 Available online 30 September 2006
Abstract In order to establish effective enzymatic biomarkers that could provide in situ early warning of contaminant exposure in estuarine ecosystems, the potential effects of the principal abiotic factors (temperature and salinity) were investigated on common biomarkers, the acetylcholinesterase (AChE) and the glutathione S-transferase (GST) in Eurytemora affinis. Short term salinity stress effects simulated during an experimental tide indicated that enzymatic activities of this species are characterized by maximum expression related to an optimal salinity range (between 5 and 15 psu). Moreover, longer time exposure to various salinity tanks confirmed the effects of this factor on both AChE and GST activities. Therefore, optimal AChE activity was measured at 10 psu, while optimal GST activity was measured at 5 psu. Furthermore, significant effects of temperature were also recorded, particularly for AChE expression (slight effects were measured on GST expression) with an optimal condition at 11 °C. These experiments indicated a more pronounced effect of salinity over temperature especially on the AChE expression and confirmed the need to standardize sampling procedures in relation with environmental parameters for biomonitoring studies based on enzymatic analyses. © 2006 Elsevier Inc. All rights reserved. Keywords: AChE; GST; Physicochemical parameters; Estuary; Invertebrate
1. Introduction In order to assess the water quality of various aquatic ecosystems and to estimate the stress encountered by the different populations that inhabitate these environments, the concentrations of a number of organic contaminants (PAHs, PCBs…) as well as trace metals (Hg, Cd, Pb, Cu, Zn) have been regularly monitored in many contaminated locations in the world. Nevertheless, chemical analyses alone are not sufficient to describe the effects of pollutants at contaminated areas. During the past years, many studies focusing on the biological effects of these contaminants have been conducted on several aquatic species, especially on fish and mollusks. Indeed, several contaminant effect indicators have been developed (Van der
⁎ Corresponding author. Tel.: +33 2 32744379; fax: +33 2 32744314. E-mail address: [email protected] (J. Forget-Leray). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.09.012
Oost et al., 2003). Thus, a number of promising and sensitive early warning signals, or biomarkers, that reflect adverse biological responses consecutive to anthropogenic contaminants and environmental toxin exposure and stress, have been identified (McCarthy and Shugart, 1990). Therefore, a wide range of biomarkers and particularly enzymatic activities, that provide information on uptake, biotransformation and detoxification patterns (Livingstone, 1993; Snyder, 2000), has been developed, tested and proposed for application in monitoring and assessing deleterious effects of chemical stress on organisms (Handy et al., 2003; Lam and Gray, 2003). Besides, the use of a battery of biomarkers in combination with chemical analyses is recommended (Cajaraville et al., 2000). Among the most reliable biomarkers, the acetylcholinesterase enzymatic activity (AChE) is one of the most frequently used in Ecotoxicology. AChE is responsible for the breakdown of acetylcholine in cholinergic synapses, preventing continuous nerve firings, which is vital for normal cellular neurotransmitter
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functioning (Payne et al., 1996). As an indicator of exposure to contaminants, the inhibition of this biomarker is commonly used to detect environmental pollution caused by neurotoxic compounds and is particularly specific of organophosphate and carbamate contaminations (Galgani and Bocquené, 1990; Forget et al., 2003). These compounds, that bind in reversible or irreversible manner to the catalytic site of the enzyme, cause the free unbound acetylcholine to accumulate at cholinergic receptor sites and thus result in the overstimulation of muscarinic and nicotinic cholinergic receptors in the central and peripheral nervous system. In addition to anticholinesterase insecticides, other contaminants such as PAHs (Kang and Fang, 1997), surfactants (Guilhermino et al., 2000), and pharmaceuticals (Nunes et al., 2006) have been shown to cause AChE inhibition. Other enzymatic biomarkers related to the detoxification potential of the studied organisms are also of great interest. Among these enzymatic activities, GSTs are a group of widely distributed enzymes that catalyze the conjugation of reduced glutathione (GSH) with compounds having reactive electrophilic groups (especially xenobiotics), generating less toxic and more hydrophilic molecules (Olsen et al., 2001). This metabolic pathway allows the protection of nucleophilic groups in macromolecules such as proteins and nucleic acids. GSTs were determined as effect biomarkers induced by different natural and synthetic compounds such as organochlorine pesticides, PAHs and PCBs (Lee et al., 1988; Fitzpatrick et al., 1997; Hoareau et al., 2001). Although these biomarkers reflect adverse biological responses to anthropogenic contamination, they could also be related to natural environmental temporal and spatial variations. Thus, several studies have investigated the seasonal variations on some biochemical biomarkers, linking them to seasonal abiotic parameter variations such as temperature, salinity, turbidity and food availability (Sheehan and Power, 1999; Lau et al., 2004; Leiniö and Lehtonen, 2005). Estuaries in particular present marked physicochemical small scale changes (mainly salinity, turbidity and current flow) associated with tidal cycles (Chapman and Wang, 2001) and large scale seasonal variations (essentially temperature and food bioavailability). Therefore, ecotoxicological in situ studies in estuaries constitute a challenge to integrate and distinguish biomarker responses to natural abiotic factor variations from contaminant-induced effects. To manage pollution monitoring in such ecosystems, the principal issues are to select an appropriate sentinel species and suitable biomarkers. E. affinis is a common estuarine copepod species, widely distributed in world estuaries, particularly in the Seine River Estuary where it represents between 90 and 100% of the zooplankton biomass (Mouny and Dauvin, 2002). This euryhaline and eurythermal species is distributed all over the estuary, with a well known biology characterized by a short life cycle (Katona, 1970); it is abundant all year round and is easily sampled; this copepod is omnivorous, playing a key role in the estuarine food web, and could therefore ensure transfers and bioaccumulation of contaminants from dissolved water and organic material to upper organisms in the food chain. Besides,
this species could be easily kept in laboratory. For these reasons E. affinis is an ideal candidate for ecotoxicological studies. The principal objectives of this work were to investigate experimentally the potential effects of the main physicochemical variations (salinity and temperature) that occurred in naturally fluctuating ecosystems such as estuaries, which could induce variable responses on biochemical biomarkers and bias pollution-caused alterations of these biological indicators. This study is the first step in the development of an in situ estuarine bioassay using E. affinis as sentinel species. Indeed, it could allow to minimize biomarker response variability in field studies related to physicochemical variations by normalizing sampling procedures focusing on salinity and temperature. 2. Materials and methods 2.1. Sample collection Several sampling were conducted in spring 2005. The copepods E. affinis were collected using subsurface tows of WP2 plankton net (200 μm mesh size) at ebb tide, in Tancarville Station in the oligohaline part of the Seine Estuary (Fig. 1). Immediately after sampling, copepods were sorted using 500 μm sieves in order to eliminate predators (especially Mysidacea and Gammaridae), then transferred into containers using filtered estuarine water and brought back to the laboratory for further precise sorting. Then, the copepods were sorted quickly (b 3 h) using several times the sequence: Dilution–Decantation–Photo attraction. To dilute the natural samples, mineral clean water (adjusted to the sampling site salinity) was used. The objectives were to separate the copepods attracted by the light on the water surface, from the underlying particles in order to make sure of the composition of the matrix for the following experiments. 2.2. Experimentation After sorting by photo-attraction, copepods were kept in the laboratory for an acclimatization period of one day in glass beakers, with a mixture of 300 ml of filtered estuarine water and 1500 ml of mineral water adjusted to the salinity of the sampling site at controlled temperature (11 °C) in a climatic chamber (LMS Cooled Incubator, Bioblock Scientific, Illkirch, France). The copepods were fed one time during the acclimatization period with Isochrysis galbana, and then kept starving during the experiments in order to limit feeding movements which could enhance the AChE activity. 2.2.1. Experimental tide The Seine Estuary is characterized by two tidal cycles a day. Each one lasts approximately 12 h and is composed of an ebb tide during which freshwater influence increases with a salinity scale ranging from 25 psu to 0 psu, and a flow tide dominated by marine influence with a salinity reaching back 25 psu. Salinity variations during the tide depend on the location in the estuary and are maximum in the euryhaline part of the estuary. In the laboratory, such small scale salinity variations were preliminarily tested during one simulated flow tide. Therefore, different
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Fig. 1. Map of the sampling location.
2-l tanks filled with 300 ml of filtered estuarine water and 1500 ml of mineral water were adjusted at the following selected salinities, 0, 5, 10, 15, 20 and 25 psu with Sera PREMIUM salt (Heinsberg, France) and kept at a controlled temperature of 11 °C. Then, the copepods which were acclimatized during one day to the laboratory conditions were transferred into the first beaker containing water at 0 psu and kept 1 h in this tank. After that, the copepods were filtered and transferred to the second beaker at 5 psu and kept in this tank during one hour and so on until the last beaker with a water salinity of 25 psu. Pools of approximately 200 mg (wet weight) of copepods were sampled after 1 h in each tank at the different salinities for AChE and GST activity analyses (two replicate samples for each sampling time and each exposure condition). 2.2.2. Effects of salinity The E. affinis population is not homogeneously distributed in the Seine Estuary. Therefore, all copepods are not submitted to the same salinity range according to their location in the different zones of the estuary (oligohaline, mesohaline and euryhaline). Thus, copepods distributed in zones under maximal marine or freshwater influences (oligohaline and euryhaline zones) are submitted to slighter salinity variations than copepods from the mesohaline zone. Besides, these copepods are submitted to a short scale salinity variation but during a longer time. In order to test the effects of salinity on AChE and GST enzymatic activities, different water tanks filled with 300 ml of filtered estuarine water and 1500 ml of mineral water were adjusted at the following selected salinities, 0, 5, 10, 15, 20 and 25 psu and kept at 11 °C in a climatic chamber. After the acclimatization period of one day in the laboratory, the initial pool of copepods was divided into 6 homogeneous groups and each group was transferred to one of the tanks previously prepared, containing the different salinity waters. At the beginning of the experiment and after 1, 2, 4, 18 and 72 h, pools of approximately 200 mg of copepods were sampled in each tank at the different salinities for AChE and GST activity analyses (two replicate samples for each sampling time and each exposure condition for the AChE activity).
2.2.3. Effects of temperature The copepod E. affinis is present in the Seine Estuary during all the year. Therefore, the successive copepod populations are submitted to variable temperatures depending on the season (from 4 °C in winter to 25 °C in summer periods). To study the effects of temperature on AChE and GST enzymatic activities, different water tanks filled with 300 ml of filtered estuarine water and 1500 ml of mineral water were adjusted at 15 psu and kept at the following selected temperatures representative of the annual temperature range in the Seine Estuary: 4, 11, 18 and 25 °C in 4 climatic chambers. After the acclimatization period of one day in the laboratory, the initial pool of copepods was divided into 4 homogeneous groups and each group was transferred to one of the tanks previously prepared, kept at the different temperatures. At the beginning of the experiment and after 1, 2, 4, 18 h, pools of approximately 200 mg of copepods were sampled in each tank at the different temperatures for AChE and GST activity analyses (two replicate samples for each sampling time and each exposure condition). In comparison to salinity experiments, there were not sufficient amounts of copepods after 72 h to measure both AChE and GST activities for all the exposure conditions. 2.3. Biomarker assays A pool of approximately 200 mg of whole-body E. affinis was used to determine both AChE and GST activities. A pool of copepods was homogenized on ice, in ice-cold 0.02 M pH 7 phosphate buffer+/0.1% Triton X100 (1/4, volume/weight) using an Ultra-Turax homogenizer. The homogenates were then centrifuged two successive times at 10,000 ×g for 5 min at 4 °C, with an intermediate shaking. Measurements of AChE activity were performed using the colorimetric method of Ellman et al. (1961), modified by Bocquené and Galgani (1998) at 412 nm, with acetylthiocholine iodide (AcSCh) as substrate and dithiobis-nitrobenzoate as reagent at a controlled temperature of 20 °C. GST activity was evaluated as the conjugation between glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm (Habig et al., 1974). The extinction coefficients used to
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determine GST and AChE specific activities were respectively 9.6 × 10− 3 M− 1cm− 1 at 340 nm and 12,600 M− 1cm− 1 at 412 nm. AChE and GST activities are expressed respectively as μmol min−1 mg−1 protein and as mol min− 1 mg− 1 protein. Proteins for standardisation of the above biomarkers were determined using Bradford's (1976) method modified for use with a micro-plate reader (Bocquené and Galgani, 1998). All assays were performed in quadruplicate. All chemicals were purchased from Sigma-Aldrich (St Quentin Fallavier, France). 2.4. Statistical analyses All biomarker results are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used, after testing the variable normality (Kolmogorov– Smirnoff test) and the variance homogeneity (Levene test), to compare both AChE and GST activities between the different exposure conditions, with salinity, time and temperature as factors. These analyses were followed by Tukey's multiple comparison tests to discriminate significantly different treatments. The significance p-level used was set to 0.05. When normal distribution and homogeneity of the variances were not verified (for experimental tide, and effect of temperature), non parametric Kruskal–Wallis ANOVA by ranks was performed both on AChE and GST data followed by the non parametric Mann–Whitney U test to identify significant differences between groups ( p b 0.05). All data were statistically analyzed by the STATISTICA Software v6.0 (Statsoft. Inc., 2002). 3. Results 3.1. Experimental tide Data obtained during the experimental tide related to AChE and GST activities are reported in Table 1. Average AChE and GST activities range respectively from 765 to 1023 μmol min− 1 mg− 1 protein and from 622 ± 82 to 842 ± 42 mol min− 1 mg− 1 protein. Besides, the Kruskal–Wallis test indicates a significant effect of salinity variations during the experimental tide both on AChE and GST activities (respectively, H = 59.76, p b 0.00001, df = 6; and H = 22.97, p b 0.0008, df = 6). Therefore, during the experimental tide, AChE levels increase from 0 psu to 10 psu exposures then decrease for all the following salinity exposures until 25 psu. Thus, the highest AChE level is measured for 10 psu salinity while the lowest one is measured for 25 psu. Moreover, AChE level for 10 psu is significantly higher than AChE levels for all other salinities ( p b 0.05), while AChE levels for 20 and 25 psu salinity are significantly lower than all other AChE levels ( p b 0.005). In the same way, during the experimental tide, GST levels increase from 0 to 5 psu exposures and then decrease for all the following salinity exposures until 25 psu. Moreover, the highest GST level is measured for a salinity exposure of 5 psu and is significantly higher than GST levels for all other salinity exposures ( p b 0.05) except for 0 and 10 psu. Furthermore, the significantly lowest GST levels are measured for salinity exposures of 20 and 25 psu ( p b 0.05).
3.1.1. Effects of salinity AChE activity variations in E. affinis after exposure to different salinities are presented in Fig. 2. Average AChE activities range respectively from 583 to 728, 546 to 962, 528 to 1156, 565 to 849, 583 to 813 and 444 to 738 μmol min− 1 mg− 1 protein for 0 to 25 psu. Besides, significant effects of salinity over the time of exposure on AChE activity are recorded (ANOVA; p b 0.00025, F = 4.92, degree of freedom: 5). High AChE activities are measured during all the experiments in copepods exposed to 10 psu water, particularly after 18 and 72 h of exposure when increases are significant ( p b 0.00005). Therefore, after 72 h of exposure, AChE level is enhanced by 98% in copepods exposed to 10 psu water. Besides, AChE activity increases significantly in copepods exposed to 15 and 20 psu between 4 and 18 h of exposure ( p b 0.00005) after which the activity remains constant at 20 psu or decreases significantly at 15 psu ( p b 0.00005). AChE activities measured in copepods kept at 5 and 25 psu show a higher variability and no significant increase during the experiments. Finally, copepods exposed to 0 psu present slightly increasing AChE levels between 1 and 2 h of exposure in comparison to the other experiments, then decreasing levels until 18 h; no copepod has survived at the end of the experiments after 72 h. Moreover, after one hour of exposure, the highest AChE levels are measured for copepods exposed to 5 psu water. Then, after 2 and 4 h of exposure, slight differences are measured in AChE levels between the different experimental exposures. After 18 and 72 h of exposure, the highest AChE levels are measured for copepods exposed to 10 psu water ( p b 0.00005), and these AChE levels are the highest ones measured during all the experiments. GST activity variations in E. affinis after exposure to different salinities are presented in Fig. 3. Average GST activities range respectively from 700 to 125, 1138 to 60, 700 to 108, 700 to 122, 700 to 175 and 700 to 132 mol min− 1 mg− 1 protein for 0 to 25 psu. Besides, significant effects of salinity over the time of exposure on GST activity are recorded (ANOVA; p b 0.0003, F = 5.13, degree of freedom: 5). Therefore, a linear significant Table 1 Variations of AChE and GST activities (means values ± SEM) in E. affinis during the experimental tide Exposure time
T0 In situ 1H 2H 3H 4H 5H 6H
Treatment salinity (PSU)
2.5 0 5 10 15 20 25
Enzymatic activities AChE
GST
μmol min− 1 mg− 1 protein
mol min− 1 mg− 1 protein
818 ± 64b, c, d, e 894 ± 73a, d, f, g 945 ± 52a, d, f, g 1023 ± 79a, b, c, e, f, 906 ± 44a, d, f, g 810 ± 54b, c, d, e 765 ± 57b, c, d, e
622 ± 8c, d 718 ± 121 842 ± 42a, e, 822 ± 58a, e, 725 ± 45c, d 685 ± 66c, d 690 ± 123c
g
f, g f
Means (n = 8: i.e. 2 experimental replicates and 4 enzymatic measures/pooled samples) and standard deviations are mentioned. Significant differences between salinities (p b 0.05) are indicated using letters (a, b, c, d, e, f and g which refer respectively to 2.5, 0, 5, 10, 15, 20 and 25 psu).
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Fig. 2. AChE activity variations in the copepod E. affinis after exposure to different salinities (from 0 to 25 psu). Means (n = 8: i.e. 2 experimental replicates and 4 enzymatic measures/pooled samples) and standard deviations are mentioned. Significant differences between sampling times ( p b 0.05) are indicated using letters (a, b, c, d, e and f which refer respectively to 0, 1, 2, 4, 18 and 72 h of exposure).
GST decrease is recorded during all the experiment in copepods exposed to 15 psu ( p b 0.05). After 72 h of exposure to 15 psu water, GST level decreases to reach 17% of the GST level of the beginning of the experiment. In the same way, non linear decreasing GST levels are measured at 20 psu. Besides,
copepods exposed to 0, 10 and 25 psu waters present the same GST variation pattern: significant decreases during the first hour of the experiments ( p b 0.0005), then non significant GST variation until the 18th hour of experiment and finally significant decreases until the 72nd hour of the experiment ( p b 0.05).
Fig. 3. GST activity variations in the copepod E. affinis after exposure to different salinities (from 0 to 25 psu). Means (n = 4 i.e. 4 enzymatic measures) and standard deviations are mentioned. Significant differences between sampling times ( p b 0.05) are indicated using letters (a, b, c, d, e and f which refer respectively to 0, 1, 2, 4, 18 and 72 h of exposure).
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Fig. 4. Effects of temperature on AChE activity expression during 18h of experimental exposure. Means (n = 8: i.e. 2 experimental replicates and 4 enzymatic measures/pooled samples) and standard deviations are mentioned. Significant differences between sampling times (p b 0.05) are indicated using letters (a, b, c, d and e which refer respectively to 0, 1, 2, 4 and 18 h of exposure).
Moreover, GST activities measured in copepods kept at 5 psu show a higher variability compared to that of copepods exposed to other salinities. Indeed, a significant increasing GST level ( p b 0.0005) followed by a significant decrease ( p b 0.0005) are measured at the beginning of the experiment. Then no significant variation is recorded between 2 and 18 h of exposure and finally the GST activity significantly decreases again ( p b 0.0005). 3.1.2. Effects of temperature Average AChE activity of E. affinis exposed to an 11 °C water temperature increases during all the experiment from 716 to 875 μmol min− 1 mg− 1 protein (Fig. 4). The total increase of AChE activity between the beginning and the end of the experiment represents 22%. Furthermore, after two hours of exposure and until the end of the experiment AChE levels in
copepods exposed to this temperature condition are significantly higher than AChE level at the beginning of the experiments ( p b 0.05). In addition, after 1, 4 and 18 h of experiment, AChE levels in copepods exposed to an 11 °C water temperature are significantly higher than AChE levels measured in copepods exposed to the other water temperature conditions at the same time of the experiment. For all other temperature conditions, AChE levels are more variable. Moreover, for 4, 18 and 25 °C conditions, AChE levels at the beginning and at the end of the experiment are not statistically significant. In relation to a high mortality after 4 h of exposure, there was no sufficient quantity of copepods to measure both AChE and GST activities at the end of the experiment, after 18 h of exposure to an 18 °C water temperature. The lowest AChE activities are measured in copepods exposed to the highest water temperature (25 °C)
Fig. 5. Effects of temperature on GST activity expression during 18 h of experimental exposure. Means (n = 8: i.e. 2 experimental replicates and 4 enzymatic measures/ pooled samples) and standard deviations are mentioned. Significant differences between sampling times ( p b 0.05) are indicated using letters (a, b, c, d and e which refer respectively to 0, 1, 2, 4 and 18 h of exposure).
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during all the experiment. Besides, the higher the water temperature is, the lower the AChE level is. Furthermore, Kruskal–Wallis tests indicate significant influence of both temperature (H = 67.6, p b 0.00001, df = 3) and time (H = 26.13, p b 0.00001, df = 4) factors on AChE activity variations. No significant GST activity variations are recorded after exposure at 4, 18 and 25 °C between the beginning and the end of the experiment (Fig. 5). Besides, GST activity is quite constant all over the experiment at 18 and 25 °C, while this enzymatic activity is more variable at 4 °C with significant increase during the first hours of the experiment (p b 0.0005) and then significant decrease at the end of the experiment to reach back the initial level. In opposition, slight (17%) but significant increasing levels (p b 0.0) are recorded after exposure at 11 °C between the beginning and the end of the experiment. Furthermore, Kruskal– Wallis tests confirm significant effects of the temperature factor (H = 26.1, p b 0.00001, df = 3) at each sampling time. Significant effects of the time of exposure factor (H = 15.19, p b 0.0043, df = 4) are also measured at 4 and 11 °C temperature conditions. 4. Discussion In order to use biochemical biomarkers for assessing pollution effects in variable ecosystems like estuaries, the natural variability of these biomarkers has to be studied. However, few experimental or in situ studies have reported the effects of temperature and salinity factors on enzymatic biomarker activities. Besides, reported data concern mainly vertebrate and especially marine fish species while effects on estuarine invertebrates have been poorly investigated. Nevertheless, effects of both temperature and salinity have been reported for different species including blue mussels (Galgani and Bocquené, 2000; Pfeifer et al., 2005), Nereis diversicolor (Scaps and Borot, 2000) and brown shrimps (Menezes et al., 2006). The data obtained in this study indicate an increase of AChE levels under controlled conditions (Fig. 2) which suggests the presence of and/or multiple inhibiting factors in the field (contaminants as well as natural parameters like temperature or salinity). Besides, decreasing GST levels are recorded (Fig. 3) during the experiment in the laboratory which implies the occurrence of inducing factors in the field which could be the same as those responsible for the inhibition of the AChE activity in the case of an environmental contamination. Similar results have been reported in the shrimp Crangon crangon after maintaining the field collected animals in the laboratory (Menezes et al., 2006). Furthermore, the results of the effects of an experimental tide on both AChE and GST activity expression indicate significant variations even at small time scale. Besides, these results indicate an optimal physiological salinity range for E. affinis between 5 and 10 psu related to the enzyme, beyond which the enzymatic activity expression decreased. Moreover, these results show that this species is better adapted to low salinity zones as the highest biomarker levels are measured for the lowest salinity and because the highest salinity induces the maximal inhibition. To our knowledge, no work has studied up to now the effects of tidally related salinity variations on AChE and GST activities. Therefore, no comparison can be made with other estuarine species.
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The copepod E. affinis shows strong osmoregulation abilities to maintain the population distribution in all the Seine Estuary (Roddie et al., 1984; Kimmel and Bradley, 2001). However, deviation from the optimal salinity range previously described can be stressful and the cost of osmotic regulation, at the molecular level, could directly affect biological molecules such as enzymes like AChE and GST activities. These observations make sense especially in longer time exposure as shown in Figs. 2 and 3. Thus, significant effects of salinity are recorded after the experiment. Therefore, increasing AChE levels are observed during the experiments and the most pronounced ones are measured for the exposition to 10 psu water salinity. These results confirm an optimal salinity around 10 psu as previously mentioned since the maximum increase in AChE activity is measured in copepods exposed to 10 psu water. Moreover, differences observed with the other exposition conditions could be explained by a decrease in energetic cost allocated to AChE activity and increasing energy devoted to osmotic regulation. Moreover, Gyllenberg and Lundqvist (1979) and Gaudy et al. (2000) specified that increasing respiration rates have been observed respectively in Eurytemora hirundoides and in Acartia tonsa and Acartia clausi when salinity conditions diverge from the optimal salinity zone of these species and that this increase of respiration is related to the need of supplementary energy for osmoregulation. Furthermore, Scaps and Borot (2000) have reported similar effects of salinity on Nereis diversicolor with however, adaptation for higher salinity zone. In addition, decreasing GST levels (Fig. 3) are measured during the experiment which could result from the combination of the transfer into non contaminated water with the effect of salinity stress. The most linear decrease is reported for 15 psu water exposition. Besides, for salinity different from the optimal one, more variable GST activities are measured (decreasing levels alternate with increasing levels). These results suggest that salinity stress could interfere with oxidative stress responses in E. affinis. However, as no similar study is available, no comparison could be made with other estuarine species. Nevertheless, we could hypothesize that salinity stress could modify antioxidative biomarker activities with however slighter effect than for AChE activity. To date, the effects of temperature on biochemical biomarkers have been essentially studied in vertebrate species and especially fish (Hazel, 1969; Hogan, 1970). However, some recent works have been performed with invertebrate species (Scaps and Borot, 2000; Pfeifer et al., 2005; Menezes et al., 2006). Thus, it is well established that the increase of water temperature induces significantly the expression of the AChE activity. Furthermore, temperature can directly affect the activity of enzymes by changing their physical structure and thereby changing their catalytic efficiency or binding capacity (Hochachka and Somero, 1984). Our experiments show that the maximum AChE levels are obtained at the 11 °C water condition (Fig. 4). Besides, significant increases of AChE activity at this temperature are recorded during the experiment. For the other water temperature conditions, rapid heat-shock responses resulting in a decrease of AChE levels, are observed after one hour of exposure. Then, AChE activity increases again for all
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temperature conditions. According to these results, the hypothesis of a short adaptation time to water temperature transitions could be made. Furthermore, Gyllenberg and Lundqvist (1979), Pagano and Gaudy (1986), and Gaudy et al. (2000) have mentioned, in different estuarine copepod species, metabolisms reduced to a minimum to maintain the physiological activity at low temperatures and to save energy at high temperatures. These observations are consistent with the results obtained in this study. Concerning GST activity, slighter significant effects of temperature are observed compared to those on the AChE activity. The highest differences are measured for the first two hours of the experiment for the exposure condition of 4 °C. Similar higher anti-oxidative activities are detected in in situ studies on Perna viridis (Lau et al., 2004). The authors hypothesized that these increases could be due to concentrating effects related to the general decrease in protein level during cold seasons or to stress induced by a chemical contamination of the sampling site. In the same way, GST activity variations related to seasonal variations in temperature have been reported in the blue mussel (Power and Sheehan, 1996). In opposition, Lushchak and Bagnyukova (2006) have reported no effect of temperature increase in the GST activity of goldfish. However, as no similar experimental study has been previously performed, no comparison could be made. To conclude, the results of this study constitute the first step before using biochemical markers such as AChE and GST activities to assess contaminant exposure of the copepod E. affinis in estuarine ecosystems. Indeed, attention should be paid to the effects of abiotic environmental parameters and particularly salinity and temperature variations for in situ biomonitoring programs based on enzymatic activity studies. Moreover, a minimum reduction or activation of 30% of respectively AChE and GST activities are advisable to conclude on contaminant exposure. Furthermore, E. affnis is a fundamental species in the estuarine food web and presents a wide world distribution. Besides, enzymatic activities of this species are sensitive to chemical exposition and changes in water temperature do not appear to be an important source of enzymatic activity alteration (especially for GST activity). In opposition, salinity variations significantly modulate these enzyme activities, which require for biomonitoring projects to repeat sampling in the same salinity conditions during all the study in order to avoid fluctuations of enzymatic activities related to environmental parameters. According to these observations, E. affinis represents a promising sentinel species in estuarine ecosystems. Acknowledgements This work was financially supported by the multidisciplinary Seine Aval scientific program by the Regi on Haute-Normandie and by the PNETOX scientific program. References Bocquené, G., Galgani, F., 1998. Biological effect contaminants: cholinesterase inhibition by organophosphate and carbamate compounds. ICES Tech. Mar. Environ. Sci. 22, 1–12.
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