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Due to the potential hazard of diclofenac on aquatic organisms and the lack of higher-tier ecotoxicological studies, a long-term freshwater mesocosm experiment was set up to study the effects of this substance on primary producers and consumers at environmentally realistic nominal concentrations 0.1, 1 and 10 µg/L (average effective concentrations 0.041, 0.44 and 3.82 µg/L). During the six-month exposure period, the biovolume of two macrophyte species (Nasturtium officinale and Callitriche platycarpa) significantly decreased at the highest treatment level. Subsequently, a decrease in dissolved oxygen levels was observed. High mortality rates, effects on immunity, and high genotoxicity were found for encaged zebra mussels (Dreissena polymorpha) in all treatments. In the highest treatment level, one month after the beginning of the exposure, mortality of adult fish (Gasterosteus aculeatus) caused effects on the final population structure. Total abundance of fish and the percentage of juveniles decreased whereas the percentage of adults increased. This led to an overall shift in the length frequency distribution of the F1 generation compared to the control. Consequently, indirect effects on the community structure of zooplankton and macroinvertebrates were observed in the highest treatment level. The No Observed Effect Concentration (NOEC) value at the individual level was

The use of a multi-biomarker approach with three-spined sticklebacks (Gasterosteus aculeatus) through an active biomonitoring strategy appears to be a promising tool in water quality assessment. The present work proposes to assess the efficiency of these tools in the discrimination of some sites in a large scale on the Meuse basin in Europe. The study was part of an EU program which aims to assess water quality in the Meuse across the French-Belgian border. Sticklebacks were caged 21 days upstream and downstream from the wastewater treatment plants (WWTPs) of Namur (Belgium), Charleville-Mezieres (France), Bouillon (Belgium) and Avesnes-sur-Helpe (France). First, the state of a variety of physiological functions was assessed using a battery of biomarkers that represented innate immunity (leucocyte mortality and distribution, phagocytosis activity, respiratory burst), antioxidant system (GPx, CAT, SOD and total GSH content), oxidative damages to the membrane lipids (TBARS), biotransformation enzymes (EROD, GST), synaptic transmission (AChE) and reproduction system (spiggin and vitellogenin concentration). The impacts of the effluents were first analysed for each biomarker using a mixed model ANOVA followed by post-hoc analyses. Secondly, the global river contamination was assessed using a principal component analysis (PCA) followed by a hierarchical agglomerative clustering (HAC). The results highlighted a small number of effects of WWTP effluents on the physiological parameters in caged sticklebacks. Despite a significant effect of the “localisation” factor (upstream/downstream) in the mixed ANOVA for several biomarkers, post-hoc analyses revealed few differences between upstream and downstream of the WWTPs. Only a significant decrease of innate immune responses was observed downstream from the WWTPs of Avesnes-sur-Helpe and Namur. Other biomarker responses were not impacted by WWTP effluents. However, the multivariate analyses (PCA and HAC) of the biomarker responses helped to clearly discriminate the different study sites from the reference but also amongst themselves. Thus, a reduction of general condition (condition index and HSI) was observed in all groups of caged sticklebacks, associated with a weaker AChE activity in comparison with the reference population. A strong oxidative stress was highlighted in fish caged in the Meuse river at Charleville-Mezieres whereas sticklebacks caged in the Meuse river at Namur exhibited weaker innate immune responses than others. Conversely, sticklebacks caged in the Helpe-Majeure river at Avesnes-sur-Helpe exhibited higher immune responses. Furthermore, weak defence capacities were recorded in fish caged in the Semois river at Bouillon. This experiment was the first to propose an active biomonitoring approach using three-spined stickleback to assess such varied environments. Low mortality and encouraging results in site discrimination support the use of this tool to assess the quality of a large number of water bodies.

International audience; The development of predictive, sensitive and reliable biomarkers is of crucial importance for aquatic biomonitoring to assess the effects of chemical substances on aquatic organisms, especially when it comes to combined effects with other stressors (e.g. temperature). The first purpose of the present study was to evaluate the single and combined effects of 90 days of exposure to an environmental cadmium concentration (0.5 μg. L−1) and two water temperatures (16 and 21 °C) on different parameters. These parameters are involved in (i) the antioxidant system (superoxide dismutase activity –SOD– and total glutathione levels –GSH–), (ii) the energy metabolism, i.e. energy reserves (glycogen, lipids, proteins) and digestive enzymes (trypsin, amylase, intestinal alkaline phosphatase–IAP–), and (iii) biometric parameters (weight, length, Fulton’s condition factor, and the gonadosomatic index –GSI–) of threespine stickleback (Gasterosteus aculeatus). The second purpose was to determine the interest of the three digestive enzymes as biomarkers in comparison with the other parameters. The higher temperature (21 °C) impacted the anti-oxidant and energy reserve parameters. In liver, GSH levels increased on day 60, while SOD decreased on days 15 and 90, with a significant decrease of protein and lipid energy reserves on day 90. In muscle, the higher temperature decreased SOD activity only on day 90. G. aculeatus biometric parameters were also impacted by the higher temperature, which limited stickleback growth after 90 days of exposure. In female sticklebacks, the GSI peaked on day 60 and decreased sharply on day 90, while the highest values were reached at day 90 in the control groups, suggesting impaired reproduction in sticklebacks raised at 21 °C. These results suggest that 21 °C is an upper-limit temperature for long-term physiological processes in sticklebacks. In contrast, very low-concentration cadmium exposure had no effect on classical biomarkers (energy reserves, antioxidant parameters, biometric parameters). However, digestive enzymes showed an interesting sensitivity to cadmium, which was emphasized by high temperature. The activity of the three digestive enzymes decreased significantly on day 90 when sticklebacks were exposed to cadmium alone, while the decrease was stronger and was recorded earlier (from day 15) when they were exposed to the cadmium-temperature combination. Compared to conventional measurements, digestive enzymes responded rapidly. This could be an important advantage for them to be used as early warning tools to reflect the health status of organisms, particularly for trypsin and IAP activities.

International audience; The relevance of a biomarker for biomonitoring programs was influenced both by the knowledge on biomarker natural inter-individual and site variabilities and by the sensitivity of the biomarker towards environmental perturbations. To minimize data misinterpretation, robustness reference values for biomarkers were important in biomonitoring programs. Specific three-spined stickleback, Gasterosteus aculeatus, immune reference ranges for field studies had been determined based on laboratory data and one reference station (Contentieuse river at Houdancourt). In this study, data obtained in one uncontaminated and three contaminated sites were compared to these reference ranges as a validation step before considering them for larger scale biomonitoring programs. When the field reference range were compared to data from the uncontaminated station (Béronelle), only few deviations were shown. In this way, data coming from uncontaminated station (Béronelle) was integrated in the field reference ranges to improve the evaluation of site variability. The new field reference ranges provided better discrimination of sites and spanned a larger range of fish lengths than the initial reference ranges. Furthermore, the results suggest lysosomal presence during several months and phagocytosis capacity in autumn may be the most relevant immunomarkers towards identifying contaminated sites. In the future, combining this reference value approach with active biomonitoring could facilitate the obtention of data in multiple stream conditions.

Pharmaceutical substances are ubiquitous in the aquatic environment and their concentration levels typically range from ng/L up to several μg/L. Furthermore, as those compounds are designed to be highly biologically active, assessing their impacts on non-target organisms is important. Here, we conducted a mesocosm experiment testing a mixture of five pharmaceuticals (diclofenac, carbamazepine, irbesartan, acetaminophen and naproxen) on fish, three-spined stickleback (Gasterosteus aculeatus). The mixture concentration levels were chosen on the basis of the contamination of the Meuse river in Belgium which had been measured previously during a monitoring campaign undertaken in 2015 and 2016. Three nominal mixture concentration levels were tested: the lowest concentration level mixture was composed by environmentally-relevant concentrations that approximate average realistic values for each pharmaceuticals (Mx1); the two other levels were 10 and 100 times these concentrations. Although no impact on stickleback prey was observed, the mixture significantly impaired the survival of female fish introduced in the mesocosms at the highest treatment level without causing other major differences on fish population structure. Impacts on condition factors of adults and juveniles were also observed at both individual and population levels. Using a modelling approach with an individual-based model coupled to a bioenergetic model (DEB-IBM), we concluded that chronic exposure to environmentally-relevant concentrations of five pharmaceuticals often detected in the rivers did not appear to strongly affect the three-spined stickleback populations. Mechanisms of population regulation may have counteracted the mixture impacts in the mesocosms.

International audience; Knowledge about combined effects of chemicals and temperature on reproductive capacity of fish are rare in literature, especially when it comes to the effects of chronic low-dose chemical exposure combined to the thermal stress. The aim of the study was to evaluate the single and combined effects of temperature (16, 18, 21 °C) and an environmentally relevant concentration of waterborne cadmium (1 µg L−1, nominal concentration) on the reproductive outputs of threespine stickleback (Gasterosteus aculeatus), and their consequences on offspring survival parameters. The high temperature (21 °C) was the only factor that affected parental parameters (gonadosomatic index “GSI”, and vitellogenin “VTG” particularly). On females, 21 °C had a stimulating effect on gonadal development evaluated by an early increase, followed by a sharp decrease of GSI, probably indicating gonadal atresia. Promoting effect of temperature was corroborated by an early production of VTG. In vitro fertilization assays showed interesting results, particularly cadmium effects. As it was supposed, high temperature had a negative impact on offspring parameters (significant decrease in survival and an increase of unhatched embryos). Parental exposure to the very low concentration of cadmium had also negative consequences on mortality rate (significant increase) and hatching rate (significant decrease). Our results indicate that in a global warming context, high temperature and its combination with contaminant may impact reproductive capacity of G. aculeatus, by decreasing parental investment (low eggs and/or sperm quality).

Pharmaceuticals are emerging organic contaminants ubiquitously present in the environment due to incessant input into the aquatic compartment mainly resulting from incomplete removal in wastewater treatment plants. One of the major preoccupations concerning pharmaceuticals released into surface waters is their potential for bioaccumulation in biota, possibly leading to deleterious effects on ecosystems especially as they could affect a broad variety of organisms living in or depending on the aquatic environment. Thus, the development of accurate and sensitive methods is necessary to detect these compounds in aquatic ecosystems. Considering this need, this study deals with the analytical development of a methodology to quantify traces of diclofenac together with some of its biotic and abiotic transformation products in whole-body tissue of three-spined stickleback. A simple and reliable extraction method based on a modified QuEChERS extraction is implemented on 200 mg of fish. The detection and quantification of the ten target compounds are performed using liquid chromatography-tandem mass spectrometry. The whole process was successfully validated regarding linearity, recovery, repeatability, and reproducibility. The method limits of detection and quantification do not exceed 1 ng/g. To reproduce environmental conditions, we measured the concentration of DCF and its transformation products in three-spined sticklebacks after a 6-month exposure in mesocosms at several levels of DCF ranging from 0.05 to 4.1 μg/L. The phase I metabolite 4'-hydroxydiclofenac was detected in fish samples exposed at the highest DCF concentration. Graphical abstract Analysis of diclofenac and some of its transformation products in the three-spined stickleback, Gasterosteus aculeatus, by QuEChERS extraction followed by LC-MS/MS.

The present work aimed to assess the efficiency of a constructed wetland as tertiary treatment on urban wastewater with a multibiomarker approach using caged three-spined sticklebacks (Gasterosteus aculeatus L.). Fish were caged on three sites: at the entrance of the Constructed Wetland (CW), directly inside the wastewater effluent, at the exit of the CW, and in a weakly impacted site considered as a reference. After 21 days of caging, sticklebacks state of health was assessed using several biomarkers representing some biological functions such as innate immune and antioxidant systems, biotransformation enzymes, reproduction parameters and synaptic transmission. A strong inhibition of the innate immune system, an induction of EROD activity and an alteration of the hepatosomatic index were observed in fish caged at the entrance of the CW compared to those caged in the reference site. In addition, wastewater effluent induced a decrease of antioxidant system without induced oxidative damage on cell membranes. No improvement of these biomarkers was observed for antioxidant parameters at the exit of the CW. However, in fish caged at the exit of the CW, the EROD induction observed at the entrance was reduced and the innate immune system presented the same level compared to fish caged in the reference site, underlying the beneficial effect of the CW for these parameters. Integrated Biomarker response (IBR) was equal to 25.2 at the entrance of the CW and 17.4 at the exit of the CW which highlighted the global positive effect of the CW on water quality based on fish biomarker measurement.

Caging is an active biomonitoring strategy that employs a sentinel species, sometimes a species naturally absent from the studied site, in the surveillance of water bodies to verify whether biota may be at risk. The main advantage of caging is the possibility to standardize several biotic and abiotic parameters. However, little knowledge is available about the effects of confinement on physiology and metabolism of caged organisms. The aim of this study is to characterize confinement and food access restriction effects, induced via caging experiments using a multi-biomarker approach (biometric data, immunity, antioxidant, metabolic detoxication, and digestive enzymes). The study has been undertaken using the same experiment conducted in ecosystem conditions using three-spined stickleback (Gasterosteus aculeatus) during two different periods: one in April, corresponding to breeding season, and the other in October, outside breeding season. Fifteen fish were maintained for 21 days in different conditions (caged or uncaged and with or without food supply). The main result was that confinement stress had little impact on the biological markers of sticklebacks. However, the stressors seemed to increase the negative effects of food restriction on these biomarkers, when sticklebacks needed more energy, that is, during their breeding period. Outside breeding period, most investigated biomarkers were not impacted by caging. This study showed a way to specify the conditions of application and interpretation of biomarkers during active monitoring to ensure an effective, reliable diagnosis of water body quality.

One of the goals of environmental risk assessment is to protect the whole ecosystem from adverse effects resulting from exposure to chemicals. Many research efforts have aimed to improve the quantification of dose-response relationships through the integration of toxicokinetics. For this purpose, physiologically-based toxicokinetic (PBTK) models have been developed to estimate internal doses from external doses in a time-dependent manner. In this study, a generic PBTK model was developed and adapted for rainbow trout (Onchorhynchus mykiss), zebrafish (Danio rerio), fathead minnow (Pimephales promelas), and three-spined stickleback (Gasterosteus aculeatus). New mechanistic approaches were proposed for including the effects of growth and temperature in the model. Physiological parameters and their inter-individual variability were estimated based on the results of extensive literature searches or specific experimental data. The PBTK model was implemented for nine environmental contaminants (with log k(ow) from - 0.9 to 6.8) to predict whole-body concentrations and concentrations in various fish's organs. Sensitivity analyses were performed for a lipophilic and a hydrophilic compound to identify which parameters have most impact on the model's outputs. Model predictions were compared with experimental data according to dataset-specific exposure scenarios and were accurate: 50% of predictions were within a 3-fold factor for six out of nine chemicals and 75% of predictions were within a 3-fold factor for three of the most lipophilic compounds studied. Our model can be used to assess the influence of physiological and environmental factors on the toxicokinetics of chemicals and provide guidance for assessing the effect of those critical factors in environmental risk assessment.

The aim of this study was to evaluate the effects of caging constraints on multiple fish biomarkers used during ecotoxicological studies (biometric data, immune and antioxidant systems, and energetic status). Two of these constraints were linked to caging: starvation and fish density in cages, and one in relation to the post-caging handling: a short transport. Three in situ experiments were conducted with three-spined sticklebacks (Gasterosteus aculeatus). The first experiment compared the effects of three densities (low, medium, and high). The second experiment compared effects of starvation in fish fed every two days with fish that were not fed. Finally comparisons between sticklebacks which have suffered a short car transport after caging and sticklebacks killed without preliminary transport were made. The lack of food had no effect on fish energetic reserves but negatively affected their condition index and their immune system. Transport and high density induced oxidative stress, defined as an overproduction of reactive oxygen species and a stimulation of the antioxidant system. These two constraints also harmed the leucocyte viability. In order not to have any impact on ecotoxicity biomarkers during in situ experiments, it is preferable to decrease fish density in cages, prevent transport before dissections, and feed fish when the caging lasts more than two weeks.

The Champagne region (France) is characterized by various chemical environmental pressures which could interfere with the immune status of natural populations of European bullhead, Cottus sp. Some adult fish were caught by electrofishing in spring, summer and autumn to determined immune effect of urban (Muizon), intensive agricultural (Bouy; Prunay) or viticultural (Serzy; Prunay) influences. The major results demonstrated an increase of cellular mortality and a decrease of phagocytosis activity in the stations impacted by agri-viticultural chemicals. These immunomodulations followed the temporal variability due to different treatments (agricultural impacts on spring; viticultural effects on autumn). At the present time, not enough data was provided to confirm the impact of agri-viticultural chemicals on fish immune system without interaction with other environmental factors. For example, in summer, the immunomarkers seems to be not only correlated with water contamination but also with other environmental factors (pathogens, physical field degradation, nutrients, temperature …). Nevertheless, immune parameters give a global view of organism and ecosystem health explaining growing interest for these biomarkers in environmental risk assessment.

International audience; Innate immunomarkers reflect both environmental contamination and fish health status, providing useful information in environmental risk assessment studies. Nevertheless, the lack of knowledge about the effect of confounding factors can lead to data misinterpretation and false diagnoses. The aim of this study was to evaluate the impact of three confounding factors (season, sex and body size) on three-spined stickleback innate immunomarkers in laboratory conditions. Results shown strong seasonal variations in stickleback innate immunomarkers, with higher immune capacities in late winter-early spring and a disturbance during the spawning period in late spring-summer. Sex and body size had a season dependant effect on almost all tested immunomarkers. Reference ranges were established in laboratory-controlled conditions (i.e. laboratory reference ranges) and compared with data obtained from in vivo chemical expositions. The predictive power of the statistical model depended on the immunomarker, but the control data of the in vivo experiments, realized in same laboratory conditions, were globally well include in the laboratory reference ranges. Moreover, some statistical effects of the in vivo exposures were correlated with an augmentation of values outside the reference ranges, indicating a possible harmful effect for the organisms. As confounding factors influence is a major limit to integrate immunomarkers in biomonitoring programs, modelling their influence on studied parameter may help to better evaluated environmental contaminations.

To characterize environmental risks linked to former uranium mines in the Limousin region of France, a study was conducted on fish health effects from uranium releases. Two private ponds were compared in this study, one with uranium contamination and one background site, upstream of the mining zone. Roach, Rutilus rutilus, were caged for 28 days in both ponds. Physico-chemical parameters of water and sediments and bioaccumulation of metals in several organs were determined. After 14 and 28 days of caging, immune, oxidative stress, biotransformation, neurotoxicity and physiological parameters were measured. Iron and aluminium were quantified in the water of both sites however, barium and manganese were only present in the water of the uranium contaminated site. Uranium was present in both sites but at very different concentrations. The sediments from the uranium contaminated site contained high levels of radioactive elements coming from the disintegration chain of uranium. Results of biological parameters indicated stimulation of immune parameters and of oxidative stress and a decrease of AChE in fish caged in the uranium contaminated pond compared to the uranium-free pond. Overall, the results determined roach health status in the context of pollution from poly-metallic mining. The data strengthen our knowledge of the environmental risk assessment associated with radioactive substances in the environment.

Organism immune defences might be weakened by pollutants, largely detected in aquatic ecosystems, leading to the facilitation for opportunistic pathogens to infect organisms. In this context, destabilization of fish non-specific immune parameters and erythrocyte DNA integrity was tested, on a model fish species, the three-spined stickleback (Gasterosteus aculeatus), after exposure to chlorpyrifos (CPF). Alone, pesticide exposure induced a genotoxic potential (chlorpyrifos at 1.75 and 0.88 µg/L) in addition to a decrease in phagocytosis capacity and a stimulation of respiratory burst. Then, to mimic pathogenic infection, fish exposure to chlorpyrifos was combined with lipopolysaccharides (LPS) stress. In this second experiment, an increase of DNA damage was observed in fish exposed to a lower concentration of chlorpyrifos and LPS. Moreover, at the higher concentration of chlorpyrifos, an early destabilization of innate immunity was observed as suggested by the absence of an increase of lysosomal presence in fish injected with LPS. This study highlighted the usefulness of stress on stress responses to better understand the impact of contaminants on the organism's health.

Due to their sensitivity to environmental contamination and their link with fish health status, innate immunomarkers are of great interest for environmental risk assessment studies. Nevertheless, the lack of knowledge about the effect of confounding factors can lead to data misinterpretation and false diagnostics. So, the determination of reference values was of huge interest for the integration of biomarkers in biomonitoring programs. Laboratory immunomarker reference ranges (including cellular mortality, leucocyte distribution, phagocytosis activity, respiratory burst and lysosomal presence) that consider three confounding factors (season, sex and body size) were previously developed in three-spined stickleback, Gasterosteus aculeatus, from our husbandry. Usefulness of these reference ranges in biomonitoring programs depends on how they can be transposed to various experimental levels, such as mesocosm (outdoor artificial pond) and field conditions. Immunomarkers were therefore measured every 2 months over 1 year in one mesocosm and in one site assumed to uncontaminated (Houdancourt, field). Differences between immunomarker seasonal variations in mesocosm and field fish on one side and laboratory fish on the other side were quantified: in some cases, seasonal trends were not significant or did not differ between mesocosm and laboratory conditions, but overall, models developed based on data obtained in laboratory conditions were poorly predictive of data obtained in mesocosm or field conditions. To propose valuable field reference ranges, mesocosm and field data were integrated in innate immunomarker modelling in order to strengthen the knowledge on the effect of confounding factors. As in laboratory conditions, sex was overall a confounding factor only for necrotic cell percentage and granulocyte-macrophage distribution and size was a confounding factor only for cellular mortality, leucocyte distribution and phagocytosis activity. Confounding factors explained a large proportion of immunomarker variability in particular for phagocytosis activity and lysosomal presence. Further research is needed to test the field models in a biomonitoring program to compare the sensitivity of immunomarkers to the confounding factors identified in this study and the sensitivity to various levels of pollution.

The ecotoxicity of artificially alterated cerium dioxide nanoparticles (nano-CeO2) suspensions was determined using the freshwater microalgae growth inhibition test. The agglomeration or aggregation state of the alterated suspensions was followed because it represents one of the obvious modifications when nanoparticles reached the environment. In addition, its influence on the ecotoxicity of nanoparticles is currently not well-addressed. Our results showed that the suspensions were stable within the first 24 h and then agglomerate up to 10 μm after 3 and 30 days. The inhibitory effect on the growth of exposed algae was however similar whatever the tested suspension. This supports the fact that the agglomeration state of nano-CeO2, in our conditions, has few influences on the ecotoxicity toward these organisms. The EC50 values were 5.6; 4.1 and 6.2 mg L(-1), after exposure to non aged, 3 and 30 days aged suspensions respectively. The interaction between algal cells and nano-CeO2 was also addressed.

Human activities have led to increased levels of various pollutants including metals in aquatic ecosystems. Increase of metallic concentrations in aquatic environments represents a potential risk to exposed organisms, including fish. The aim of this study was to characterize the environmental risk to fish health linked to a polymetallic contamination from former uranium mines in France. This contamination is characterized by metals naturally present in the areas (manganese and iron), uranium, and metals (aluminum and barium) added to precipitate uranium and its decay products. Effects from mine releases in two contaminated ponds (Pontabrier for Haute-Vienne Department and Saint-Pierre for Cantal Department) were compared to those assessed at four other ponds outside the influence of mine tailings (two reference ponds/department). In this way, 360 adult three-spined sticklebacks (Gasterosteus aculeatus) were caged for 28 days in these six ponds before biomarker analyses (immune system, antioxidant system, biometry, histology, DNA integrity, etc.). Ponds receiving uranium mine tailings presented higher concentrations of uranium, manganese and aluminum, especially for the Haute-Vienne Department. This uranium contamination could explain the higher bioaccumulation of this metal in fish caged in Pontabrier and Saint-Pierre Ponds. In the same way, many fish biomarkers (antioxidant and immune systems, acetylcholinesterase activity and biometric parameters) were impacted by this environmental exposure to mine tailings. This study shows the interest of caging and the use of a multi-biomarker approach in the study of a complex metallic contamination.

International audience; In freshwater ecosystems, a large number of chemical substances are able to disturb homeostasis of fish by modulating one or more physiological functions including the immune system. The aim of this study was to assess multi-biomarker responses including immunotoxicity induced by urban and agricultural pressure in European bullheads living in a small French river basin. For this purpose, a set of biochemical, immunological, physiological and histological parameters was measured in wild bullheads from five locations characterized by various environmental pressures. Moreover, to address effects of physiological status and contamination level variation on biomarker responses, fish were sampled during three periods (April, July and October). Results revealed a clear impact of environmental pressure on fish health and particularly on immunological status. An increase of EROD activity was recorded between upstream and downstream sites. Upstream sites were also characterized by neurotoxicological effects. Fish exhibited up stream/downstream variations of immunological status but strong differences were observed according to sampling season. Conversely, regarding biochemical and immunological effects, no significant response of physiological indexes was recorded related to environmental pressures. According to these results, the European bullhead appears as a valuable model fish species to assess adverse effects in wildlife due to urban and agricultural pressures.

International audience; The in vitro effects of polycyclic aromatic hydrocarbons (PAHs) on two plasmatic immune parameters, lysozyme concentration and haemolytic alternative complement activity, of the European sea bass, Dicentrarchus labrax, were tested using field (10(-7) and 10(-9) mg mL(-1)) and high concentrations (10(-3) and 10(-5) mg mL(-1)) observed during oil spills. Peripheral blood from 105 fish was collected, centrifuged at 1200 g, for 10 min, at 4 degrees C and three plasma pools, each of 35 fish, were constituted. Two oils (heavy fuel oil and light cycle oil) and 16 pure PAHs, selected on the basis of the American Environmental Protection Agency list (US EPA), were tested in vitro on the two humoral immune parameters. Only three pure PAHs (anthracene, chrysene and dibenz[a,h]anthracene) modulated lysozyme concentration. Acenaphthene, acenaphthylene, anthracene, benzo[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, pyrene and light cycle oil modified the haemolytic alternative complement activity after 4h of incubation. This study investigates the direct effects of several PAHs on fish humoral immune functions and describes the haemolytic complement activity of fish as suitable biomarkers of oil pollution.

This work aimed at assessing the influence of different exposure systems to perform the commonly used OECD 201 freshwater algal growth inhibition test in the context of nanoparticles hazard assessment. Two distinct TiO2 nanoparticles were considered and three different exposure systems were investigated: Erlenmeyers flasks and 24-well microplates (both using an orbital shake system), and an alternative system using cylindrical vials and magnetic stirring. All three systems are in accordance with the OECD 201 test guideline recommendations. We concluded that the exposure systems applied to achieve the test can substantially affect the ecotoxicological results and the subsequent calculated ECx. The selected systems influenced both the interaction between algal cells and TiO2 nanoparticles as well as the growth inhibition recorded. Disparities in ecotoxicity relative to the TiO2 nanoparticles tested were also observed and are finally discussed.

The prophenoloxidase (ProPO) system is the origin of melanin production and is considered to be an innate defence mechanism in invertebrates. In different bivalve species, phenoloxidase (PO) is present in the haemolymph as an inactive form of ProPO. The present study focuses on the Pacific adult oyster, Crassostrea gigas, an economically important bivalve species along French coasts. The results indicate that many factors may inhibit the PO-like activity. These include: phenylthiourea (PTU), sodium diethylthiocarbamate (DETC), beta-mercaptoethanol and tropolone, which repressed the spontaneous PO activity. The activation of PO-like activity in C. gigas acellular fraction by lipopolysaccharide (LPS) involved participation of other factors, including at least one serine protease. PO was present as proPO in the acellular fraction of haemolymph and haemocytes of C. gigas and could be activated by an exogenous protease (trypsin-N-tosyl-L-phenylalanine chloromethyl ketone) when used at 1 g L-1. Treatment of acellular fractions with other modulators/activators namely LPS (1 g L-1), zymosan (0.6 g L-1) or laminarin (0.6 g L-1) also increased PO-like activity but to a less important way. The study demonstrated the evidence of a true phenoloxidase activity in Pacific oyster, C. gigas (Thunberg). The activation of a proPO system by non-self molecules suggests the role played by PO in vivo in the internal defence mechanisms. Understanding the activation of the ProPO system could enable the evaluation of the health of oyster stocks. (c) 2006 Published by Elsevier Ltd.

To characterize environmental risks linked to former uranium mines in the Limousin region of France, a study was conducted on fish health effects from uranium releases. Two private ponds were compared in this study, one with uranium contamination and one background site, upstream of the mining zone. Roach, Rutilus rutilus, were caged for 28 days in both ponds. Physico-chemical parameters of water and sediments and bioaccumulation of metals in several organs were determined. After 14 and 28 days of caging, immune, oxidative stress, biotransformation, neurotoxicity and physiological parameters were measured. Iron and aluminium were quantified in the water of both sites; however, barium and manganese were only present in the water of the uranium contaminated site. Uranium was present in both sites but at very different concentrations. The sediments from the uranium contaminated site contained high levels of radioactive elements coming from the disintegration chain of uranium. Results of biological parameters indicated stimulation of immune parameters and of oxidative stress and a decrease of AChE in fish caged in the uranium contaminated pond compared to the uranium-free pond. Overall, the results determined roach health status in the context of pollution from poly-metallic mining. The data strengthen our knowledge of the environmental risk assessment associated with radioactive substances in the environment.

This study aimed to determine the link between reproductive status and immune activity in Cottus sp. Immune capacities were more correlated with environmental factors than reproduction. Autumn was the best season for environmental immunomonitoring. European bullhead is a relevant fish species to assess adverse effects of environmental stress on wild fish. Nevertheless, their complex reproductive cycle is very different between sites and could interfere with many physiological processes. Thus, prior to use biomarker to statute on environmental quality of rivers, we wanted to characterize reproductive profile (spawn number, GSI, gonad development). The major results demonstrated that the two types of reproductive cycle shown were strongly correlated to water temperature variation. In a second time, even if innate immunomarkers are highly relevant on biomonitoring program, hormonal variation seems to impact severely their responses. Thus, the link between reproductive status and immune activity (leucocyte distribution, cellular mortality, respiratory burst, phagocytosis activity) must also be study. Nonetheless, in the present work, immune capacities seems to be more correlated with season and environmental factors than reproduction.

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on eleven organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity iii) immobilization in the crustacean Daphnia magna, iii) fish embryo toxicity, iv) impaired frog embryo development and v) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations., JRC.H.1-Water Resources

Today, the list of endocrine disrupting compounds (EDCs) in freshwater and marine environments that mimic or block endogenous hormones is expanding at an alarming rate. As immune and reproductive systems may interact in a bidirectional way, some authors proposed the immune capacities as attractive markers to evaluate the hormonal potential of environmental samples. Thus, the present work proposed to gain more knowledge on direct biological effects of natural and EDCs on female fish splenic leucocyte non-specific immune activities by using ex vivo assays. After determining the optimal required conditions to analyze splenic immune responses, seven different EDCs were tested ex vivo at 0.01, 1 and 100 nM over 12 h on the leucocyte functions of female three-spined stickleback, Gasterosteus aculeatus. In summary, we found that natural hormones acted as immunostimulants, whilst EDCs were immunosuppressive.

This work focuses on the effects of two commercial formulations of dispersants on juvenile turbot after 48 h of contamination and 15 days of recovery. Oxidative stress, gill, and immune functions were assessed in seven conditions: exposition to the water-soluble fraction of an oil, mechanical dispersion, two dispersants alone, two types of chemical dispersion and a control group. In the contaminated groups, nominal concentrations of oil and dispersants were 66 and 3.3 mg L−1, respectively. Dispersants alone had weak effects; the soluble fraction induced leucopenia and gill alteration. Chemical and mechanical dispersion induced similar effects. After contamination, a principal component analysis showed two distinct areas: the first one included the control and dispersants groups, the second one dispersion of the oil. After the 15-day recovery period, it was not possible to differentiate the groups. This study shows that, in the experimental conditions tested, the dispersion, either chemical or mechanical, enhances the consequences of exposure to crude oil without long-lasting consequences.

International audience; The use of a biochemical multi-biomarker approach proved insufficient to obtain clear information about ecosystem health. The fish immune system is considered as an attractive non-specific marker for environmental biomonitoring which has direct implications in individual fitness and population growth. Thus, the present work proposes the use of fish immunomarkers together with more common biochemical biomarkers in sampling conditions optimized to reduce biomarker variability and increase parameter robustness. European bullheads (Cottus sp.) from 11 stations in the Artois-Picardie watershed (France) were sampled. In the multiple discriminant analysis, the sites were highly correlated with apoptosis, respiratory burst, GST and EROD activities. Moreover, the use together of biochemical and immune markers increased the percentage of fish correctly classed at each site and enhanced site separation. This study argues in favor of the utilization of apoptosis, necrosis and respiratory burst for the determination of environmental risk assessment in addition to the set of biochemical biomarkers commonly used in fish.

In this study, we investigated the effects of depleted uranium (DU), the byproduct of nuclear enrichment of uranium, on several parameters related to defence system in the zebrafish, Danio rerio, using flow cytometry. Several immune cellular parameters were followed on kidney leucocytes: cell proportion, cell mortality, phagocytosis activity and associated oxidative burst and lysosomal membrane integrity (LMI). Effects of DU were tested ex vivo after 17 h of contact between DU and freshly isolated leucocytes from 0 to 500 A[micro]g DU/L. Moreover, adult zebrafish were exposed in vivo during 3 days at 20 and 250 A[micro]g DU/L. Oxidative burst results showed that DU increased reactive oxygen species (ROS) basal level and therefore reduced ROS stimulation index in both ex vivo and in vivo experiments. ROS PMA-stimulated level was also increased at 250 A[micro]g DU/L in vivo only. Furthermore, a decrease of LMI was detected after in vivo experiments. Cell mortality was also decreased at 20 A[micro]g DU/L in ex vivo experiment. However, phagocytosis activity was not modified in both ex vivo and in vivo experiments. A reduction of immune-related parameters was demonstrated in zebrafish exposed to DU. DU could therefore decrease the ability of fish to stimulate its own immune system which could, in turn, enhance the susceptibility of fish to infection. These results encourage the development and the use of innate immune analysis by flow cytometry in order to understand the effects of DU and more generally radionuclides on fish immune system and response to infectious diseases.

International audience; Uranium is ubiquitous in natural waters at concentrations ranging from a few ng/L to low micro g/L depending on the composition of surrounding rocks and up to 1 mg/L at the vicinity of uraniferous sites.1 Depleted uranium (DU) is a byproduct of enriched uranium and is used in military, aviation, medical and research applications. Freshwater ecosystems may constitute the final receptor areas of DU to which they may be chronically exposed both to its chemical toxicity.2 Among all physiological processes possibly disturbed by pollutants, the immune system is likely to be one of the more sensitive physiological systems.3 Fish innate immune responses, which are the first line of immune system defence of these organisms may be suppressed by xenobiotics and seem to represent relevant immunotoxic endpoints.4 Keller et al.5 reported effects of cadmium, copper and zinc on immune parameters including total white blood cells, macrophage activity, and oxidative burst in several fish species. Mercury and selenite were also proved to be toxic to lysozyme activity and lymphocyte proliferation in blue gourami.6 Phagocytosis and oxidative burst were also reduced in the presence of cadmium or mercury in rainbow trout.7 However, data on effects of DU on fish immune system are scarce. The objective of this work was to test the potential impacts of DU on zebrafish immune system using several endpoints measured by flow cytometry: cell population, cell mortality, reactive oxygen species (ROS) production, lysosome integrity and phagocytosis activity. Two separate experiments were conducted on zebrafish in order to assess DU effects in vivo on adults and ex vivo on leucocytes.

International audience; The neutral red retention assay has been proposed to determine the lysosomal membrane stability in immune cells. Nevertheless, this assay implies many examinations under a microscope at short time intervals and therefore the analysis of few samples. The present study proposes two more rapid, efficient, and sensitive sample analyses using flow cytometry method. Lysosomal presence and lysosomal membrane integrity (LMI) were evaluated on the three-spined stickleback, Gasterosteus aculeatus (L.), a well-described model fish species for aquatic ecotoxicology studies. After development of the two biomarkers, they were validated by ex vivo contamination with endosulfan and copper and by in situ sampling. These immunomarkers were clearly modulated by pollutants and their variations seemed to be correlated with leucocyte mortality. Thus, from a practical point of view, lysosomal presence and LMI may provide novel and efficient means of evaluating immune capacities and indicating the toxic effects of environmental pollution.

International audience; The aim of this study was to identify a signal that could be used as an androgen exposure indicator in the European bullhead (Cottus sp.). For this purpose, the ultra-structure of the kidney was characterized to identify normal structure of this organ, and histological changes previously described in the kidney of breeding male bullheads were quantified using the kidney epithelium height (KEH) assay previously developed and validated for the stickleback. In the next step, the effect of trenbolone acetate (TbA), a model androgen, was assessed to identify potential androgenic regulation of bullhead kidney hypertrophy. Measurement of KEH performed on adult non-breeding male and female bullheads exposed for 14 and 21 days to 0, 1.26 and 6.50 mu g/L showed that kidney hypertrophy is induced in a dose-dependent manner, confirming the hypothesis that the European bullhead possesses a potential biomarker of androgen exposure. Combined with the wide distribution of the European bullhead in European countries and the potential of this fish species for environmental toxicology studies in field and laboratory conditions, the hypothesis of a potential biomarker of androgen exposure offers interesting perspectives for the use of the bullhead as a relevant sentinel fish species in monitoring studies. Inducibility was observed with high exposure concentrations of TbA. Further studies are needed to identify molecular signals that could be more sensitive than KEH

International audience; Hydrocarbons are major contaminants that may affect biota at various trophic levels in estuaries and coastal ecosystems. The effects of accidental pollution by light cycle oil (LCO), a refined product of heavy fuel oil, on bioaccumulation, depuration processes and immune-related parameters in the European sea bass, Dicentrarchus labrax, were investigated in the laboratory after 7 days of exposure and a 2-week recovery period. Exposure of fish to the soluble fraction of LCO (1600 ng L(-1)) for 7 days led to the bioaccumulation of some polycyclic aromatic hydrocarbons (PAHs) in muscles: naphthalene, acenaphthene, fluorene, phenanthrene and anthracene. After 7 days of recovery period, half-elimination of naphthalene was reported in fish muscles due to facilitated diffusive loss by the epithelium and a faster elimination rate proven by the presence of a high level of naphthalene biliary metabolites. The other bioaccumulated molecules displayed a slower deputation rate due to their elimination by the formation of hydrophobic metabolites excreted through bile or urine. Three days after the beginning of the recovery period, each contaminated fish showed severe external lesions (tissue necrosis, suppurative exudates, haemorrhagic area). The hypothesis of a possible link with inflammatory phenomenon was supported by (i) an inversion of the leucocyte sub-population percentage, (ii) a significant up-expression in the spleen of the tumour necrosis factor alpha gene, (iii) a significant increase in ACH(50). Moreover, the lack of C3 gene regulation in the spleen suggested a non-renewal of this component. The reduction of phagocytic activity and lysozyme concentration reflected immune suppression. Finally, LCO toxicity in this fish was clearly demonstrated to be related to inflammatory reaction and immune depletion.

International audience; The prophenoloxidase (proPO) system catalysing the melanin production is considered as implicated in the innate immune system in invertebrates. The phenoloxidase (PO)-like activity was detected in the cuttlefish embryo sampled at the end of the organogenesis and few hours before hatching. Various modulators of the PO activity were used to assess the triggering of the proPO activating system. The results demonstrated the evidence of a true phenoloxidase activity in the cuttlefish embryo. However, SDS and LPS granted contrasting effects on the PO-like activity between the developmental stages suggesting a progressive maturation of the proPO system from the embryonic to the juvenile stages. In eggs exposed to dissolved trace metals all along the embryonic development, Ag (1.2 µg L-1) inhibited the PO-like activity in the cuttlefish embryo except at hatching time, suggesting the synthesis of a new ”juvenile” form of the phenoloxidase enzyme. In similar conditions as for Ag, Cu (230 µg L-1) stimulated and then inhibited the PO-like activity according to a progressive metal accumulation within the egg and suggesting the occurrence of a threshold, above which the toxicity of the essential metal reduced the PO activity.

International audience; The effects of a soluble fraction of light cycle oil (LCO) on haemocyte parameters, phenoloxidase (PO) activity and mRNA expression of immune-related genes, in the Pacific oyster, Crassostrea gigas, were tested after seven days of exposure and two weeks of recovery period. Five polycyclic aromatic hydrocarbons (PAHs) out of ten detected in tank water had bioaccumulated at the end of the contamination period. The concentration of PAHs in oyster tissues decreased during the recovery period and 14 days after the exposure, 69% of bioaccumulated PAHs were detected in contaminated oysters. The exposure induced severe oyster mortality (21%), external and internal green colouration of the shell and a significant decrease of PO activity. The mRNA expression of several genes was altered. As a conclusion, a modulation of immune-related parameters was demonstrated using three different approaches, namely cellular (flow cytometry), biochemical (spectrophotometry) and genomics (gene transcription) in oysters after contact with soluble fraction of LCO.

International audience; The European sea bass, Dicentrarchus labrax, and the Pacific oyster, Crassostrea gigas, were exposed to a soluble fraction of heavy fuel oil for 5 and 9 days, respectively. The organisms were then transferred to non-contaminated seawater for 1 month. The bioaccumulation and elimination of PAHs in contaminated tissues were dissimilar between species. In fish, acenaphthene and naphthalene were detected and naphthalene was still detectable 30 days after the beginning of the recovery period. In oysters, on the other hand, pyrene and phenanthrene were bioaccumulated and 14 days after exposure no more PAHs were detected. Concerning innate immune parameters, the increase of haemolytic activity of the alternative complement pathway in fish and the reduction of phenoloxidase activity in oysters endured, respectively, 1 and 2 weeks in contaminated organisms. This indicates that these two enzymatic cascades could be quite useful for monitoring pollution by oil.

The presence of phenoloxidase (PO) activity was detected in different developmental stages of the Pacific oyster, Crassostrea gigas. A significant reduction in PO activity was observed from the 6 h embryo stage to the day 11 larvae by spectrophotometry. A progressive increase was also observed from the day 13 larvae right through to the juvenile stage. The microscopy studies with ‘6 h embryo’ and adult samples confirmed the presence of PO activity. Various modulators of PO activity were used to study the triggering of pro-phenoloxidase (proPO) activating system of C. gigas but also to confirm the exact nature of the monitored activity. The enzyme activation mechanisms appear to differ with the developmental stage: bacterial lipopolysaccharides constitute an early elicitor of the proPO–PO system, whereas a purified trypsin triggers proPO–PO system in C. gigas spat. Phenoloxidase activity was totally suppressed by PO-specific inhibitors such as β-2-mercaptoethanol, sodium diethyldithiocarbonate and tropolone. This study demonstrated the selective response of PO-like activity by different elicitors and suggested that proPO–PO activating system, which is supposed to play an important function in non-self recognition and host immune reactions in oyster, is expressed early in the Pacific oyster, C. gigas.

The in vitro effects of polycyclic aromatic hydrocarbons (PAHs) on haemocyte and haemolymphatic parameters of the Pacific oyster, Crassostrea gigas, were tested using field concentrations (10(-7) and 10(-9) mg mL(-1)) observed in the Marennes-Oleron Basin (Charente-Maritime, France) and high concentrations (10(-3) and 10(-5) mg mL(-1)) observed during oil spills. As a first step, the effects of pollutants, after a 24 h contamination period, were monitored on individual and pooled haemolymph samples and similar results were observed for both sample types. In a second step, haemolymphs from 45 oysters were withdrawn and pooled. Eighteen pollutants were tested in vitro after a 4 and 24 h contamination period and 10 of them showed significant modulations of the five haemocyte parameters tested. Seven pollutants (fluorene, pyrene, anthracene, phenanthrene, chrysene, indeno[1,2,3-c,d]pyrene and heavy fuel oil (HFO)) induced a decrease in haemocyte mortality. Fluorene, pyrene and HFO significantly decreased phagocytosis activity. Percentage of non-specific esterase positive cells, phenoloxidase activity and lysosome presence were increased by naphthalene, benzo[b]fluoranthene and dibenz[a,h]anthracene, respectively. Modulation of immune parameters in the Pacific oyster by PAHs suggested that PAH pollution may be related to enhanced susceptibility to diseases. (C) 2008 Elsevier Ltd. All rights reserved.