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International audience; This study investigates the effect of fish density and exposure duration on trace metal elements (TME) bioaccumulation and several biomarkers response. Juvenile flounders were caged at low, medium and high densities and exposed during 15 or 30 days in the Seine estuary. The concentrations of the TME measured in the muscle of the caged fish were all in agreement with their bioavailability percentage in the sediments. Higher concentrations of TME were found in flounders' muscle exposed for 15 days compared with those caged for 30 days. For the same exposure time, the density of fish had no effect on the accumulation of the TME in the flounders' muscle. Biomarkers responses varied according to density and duration of exposure. Special care should be taken in their interpretation. We underline that for an optimal assessment of TME pollution in the field, 15 days with low densities of fish per cage are sufficient.

International audience; A biomonitoring approach based on a single model species cannot be representative of the contaminations impacts on the ecosystem overall. As part of the Interreg DIADeM program (“Development of an integrated approach for the diagnosis of the water quality of the River Meuse”), a study was conducted to establish the proof of concept that the use of a multispecies active biomonitoring approach improves diagnostic of aquatic systems. The complementarity of the biomarker responses was tested in four model species belonging to various ecological compartments: the bryophyte Fontinalis antipyretica, the bivalve Dreissena polymorpha, the amphipod Gammarus fossarum and the fish Gasterosteus aculeatus. The species have been caged upstream and downstream from five wastewater treatment plants (WWTPs) in the Meuse watershed. After the exposure, a battery of biomarkers was measured and results were compiled in an Integrated Biomarker Response (IBR) for each species. A multispecies IBR value was then proposed to assess the quality of the receiving environment upstream the WWTPs. The effluent toxicity was variable according to the caged species and the WWTP. However, the calculated IBR were high for all species and upstream sites, suggesting that the water quality was already downgraded upstream the WWTP. This contamination of the receiving environment was confirmed by the multispecies IBR which has allowed to rank the rivers from the less to the most contaminated. This study has demonstrated the interest of the IBR in the assessment of biological impacts of a point-source contamination (WWTP effluent) but also of the receiving environment, thanks to the use of independent references. Moreover, this study has highlighted the complementarity between the different species and has emphasized the interest of this multispecies approach to consider the variability of the species exposition pathway and sensibility as well as the mechanism of contaminants toxicity in the final diagnosis.

Bisphenol A (BPA) is a chemical of major concern due to its endocrine disrupting function, high production volume, and persistence in the aquatic environment. Consequently, organisms such as fish are subject to chronic exposure to BPA. However, physiologically-based toxicokinetic (PBTK) models, which are valuable tools to improve the understanding of a chemical's fate in an organism, have never been specifically adapted to model BPA toxicokinetics (TK) in fish. In our work, an existing PBTK developed for four different fish species was modified to model BPA ADME processes (absorption, distribution, metabolization and excretion). The metabolization of BPA into BPA-monoglucuronide (BPA gluc) and BPA-monosulfate (BPA sulf) and their TK in various organs was taking into account in the model. Experiments were performed to generate BPA TK data in a model species commonly used in ecotoxicology, the stickleback. The model structure had to include two sites of metabolization to simulate BPA TK accurately in stickleback organs. Thus, the fish liver may not be the only site of the metabolization of BPA: plasma or gills could also play a role in BPA metabolization. The PBTK model predictive performance evaluated on literature data in zebrafish and rainbow trout concurs with this conclusion. Finally, a calibration mixing data from the three species was compared to the calibration on stickleback data only.

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; 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; Aquatic organisms are exposed to mixtures of chemicals that may interact. Mixtures of atrazine (ATR) and chlorpyrifos (CPF) may elicit synergic effects on the permanent inhibition of acetylcholinesterase (AChE) in certain aquatic organisms, causing severe damage. Mechanistic mathematical models of toxicokinetics and toxicodynamics (TD) may be used to better characterize and understand the interactions of these two chemicals. In this study, a previously published generic physiologically-based toxicokinetic (PBTK) model for fish was adapted to ATR and CPF. A sub-model of the kinetics of one of the main metabolites of CPF, chlorpyrifos-oxon (CPF-oxon), was included, as well as a TD model. Inhibition of two esterases, AChE and carboxylesterase, by ATR, CPF and CPF-oxon, was modeled using TD modeling of quantities of total and inactive esterases. Specific attention was given to the parameterization and calibration of the model to accurately predict the concentration and effects observed in the fish using Bayesian inference and published data from fathead minnow (Pimephales promelas), zebrafish (Dunn) rerio) and common carp (Cyprinus carpio L.). A PBTK-TD for mixtures was used to predict dose-response relationships for comparison with available adult fish data. Synergistic effects of a joint exposure to ATR and CPU could not be demonstrated in adult fish.

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).

International audience; Within the Zelcor European project, INRA applied a newly-developed process in ionic liquids to depolymerise and functionalise lignin (increase the phenol content) to two reference lignin samples (Protobind 1000 from GreenValue and Futurol residue) and their fractions. Advantages of the process are to combine short reaction time (max 40 min) and low temperatures (max 110°C) compared to alternative catalytic processes. The process conditions were selected according to the prior optimisation performed on phenolic model compounds (monomer, dimer and milled wood isolated lignins ). Thioacidolysis, 31P NMR, and SEC were applied to the ethyl acetate soluble and insoluble reaction products so as to assess the degree of depolymerisation and demethylation. The results indicate that: i) a prior extraction of the ethyl acetate soluble compounds is beneficial to the process; ii) up to 30% solubility can be reached after the treatment of insoluble fractions; iii) demethylation occurs for all samples. The samples were then tested for antioxidant properties study to assess whether the process generates fractions with improved performances compared to the lignin samples and fractions already available. In parallel to the technical development of the process, security assessment has been undertaken through close interaction with INERIS to identify possible safety advantages of the process and to check that there is no major obstacle to future industrial development of the process.

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.

International audience; With the growing interest of a circular economy, the use of lignocellulosic residues such as lignins and humins as potential renewable feedstock for biorefining processes looks more and more promising. With humins, many challenges are still remaining for their sustainable use, starting from the need of providing reference data reflecting actual usable feedstocks of such materials. With this perspective, this paper offers a first outlook on the potential environmental fate of those materials and components, all related to furanics, a family of compounds for which toxicity is still a matter of debate. During the assessment, the conventional OECD ecotoxicity and biodegradability tests demanded by the European REACH regulation for a primary evaluation of environmental hazards were performed in combination with fish immunomarker tests to study the possible long-term effects on aquatic ecosystems. These first results are promising as humins did not exhibit any immediate ecotoxicological concerns and hence would allow considering their use in environmental-friendly applications.

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.

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.

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.

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.

The effects of two protocols (density gradient versus hypotonic lysis) used for leukocyte isolation from three major lymphoid tissue of fish (head-kidney, spleen and blood) were examined on some cell functional activities (tissue leucocytes distributions, phagocytosis, basal and burst oxidative activities) classically used to estimate the fish immune status. Experiments were conducted on roach (Rutilus rutilus), a cyprinid fish model often studied in different eco-physiological contexts (aquaculture, ecotoxicology …). All of immune endpoints were assessed either immediately after cell isolation or after a 12 h of incubation in order to observe if a post-isolation incubation may influence the leukocytes activities. Compared to the density gradient, hypotonic lysis is associated with granulocytes enrichments of cell suspensions. This is particularly true for leukocyte suspensions isolated from head kidney where granulocytes are naturally abundant. However, important variabilities in leukocyte distributions were observed in head kidney and spleen cells samples obtained by the use of hypotonic lysis for two incubation conditions used (no incubation or 12 h of incubation at 4 °C). The density gradient protocol leads to a transitory increase in basal ROS production in spleen lymphocytes and macrophages The blood leukocytes isolated by this same method exhibit high basal oxidative activities after 12 h of incubation at 4 °C and for the three leukocyte types (lymphocytes, monocytes and granulocytes). The hypotonic lysis is associated with an increase in PMA-induced ROS production especially in head kidney leukocytes. The increases in cell oxidative activities are consistent with increases in granulocyte proportions observed in leukocyte suspensions obtained by hypotonic lysis. Finally, the two protocols have no effect on leukocyte mortality and phagocytic activity. Within limits of our experimental conditions, the spleen is the organ whose leukocyte oxidative activities (stimulated or not) are only slightly influenced by the methods used for leukocyte isolation. This is also the case for the anterior kidney, but for this tissue, it is necessary to incubate the isolated cells for 12 h at 4 °C before functional analyses. Each of the two methodologies used has advantages and disadvantages. The hypotonic lysis allows to isolate a greater variety of leukocytes types whereas the density gradient used ensures a better stability of cells distributions over time. However, for the same fish species and for the same tissue, the method used to isolate leukocytes influences results and must be taken into consideration during acquired data analysis for evaluation of fish immune status.

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.

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.

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.

To reduce health and environmental risks of chemicals and to minimize the environmental footprint of human activities, green chemistry searches for alternative, environment-friendly reaction media and at the same time strives to increase reaction rates and milder reaction conditions. A growing area of research is devoted to ionic liquids (ILs) often termed as “designer” solvents due to the extreme tunability of sought functional properties. As the aftermath of earlier misleading messages reporting ILs generically as intrinsically “safe and risk free” whatever ILs considered, their increase in potential or emerging applications in key domains (like bio-refining, energy storage, nano-material processing, pollution remediation…) dictates a more scientifically-sound approach of actual risk profiles of ILs, including both physico-chemical, health and environmental risks that might be triggered by inadequate selection of use of ionic liquids. In this context, we proposed a advanced approach serving comprehensive evaluation of environmental hazard by combined use of OECD standardized tests (Daphnia magna acute immobilization test ; algal growth inhibition test) and fish ex vivo leucocyte mortality analysis. This innovative approach integrating multicriteria assessment has been tested to on a first set of seven imidazolium-based ([BMIM][Cl], [EMIM][Cl], [EMIM][DCA], [EMIM][MeSO3], [EMIM][EtOSO3], [EMIM][BF4], [HMIM][Cl]) and four phosphonium-based ionic liquids ([P6,6,6,14][Cl], [P6,6,6,14][DCA], [P6,6,6,14][(iC8)2PO2], [Pi4,i4,i4,1][TOS]) and is expected to be further used to examine other ILs (biobased, ILs, pyrrolidinium, …) in soon future. Results of ecotoxicity tests obtained so far have revealed a clear potential to rank clearly the families of ILs according to the studied risk and even to identify influencing structural factors within a same family of ILs and compounds within the same family. Complementarity of these tests and ecotoxic properties of IL will be discussed specifically in terms of ecological footprint reduction strategies in chemical engineering processes for successful IL utilization in the future.

During the XXth century, the industrial chemistry development play an important role in the economic sector, based on the petroleum process. At the same time, the green chemistry and the biorefineries have been developed and based on the agricultural and forest biomass transformation in a variety of biobased and bioenergy process. To understand the environmental impact of these industries on the biologic environment, this project presents an approach between the laboratory and the ground research. The aims of this work correspond to the characterization on the aquatic environmental impact around an industrial plant by different investigation at the upstream and downstream industrial localization such as: - The identification molecule potentially product ; - The development and the establishment of analytical method with the determination of the detection and quantification limit by GC, GC/MS, HPLC... ; - The target molecules metabolism analysis in order to understand the mechanism action ; - The characterization effect on fish and bivalves indigenous ; - The impact assessment on the microbial diversity sediments. In a first time, by the chemistry approach, we have developped some different gas chromatography methods for the characterization of the pertinent bio-refinerie compounds. These first results give the limit of detection (LOD) and quantification (LOQ) for more 40 chemistry compounds. To complete the chemical investigation of industrial effluent, some ecotoxicological tests were made including standardized tests on representative organisms of the aquatic compartment (green algae growth inhibition test – OECD test guideline 201; Daphnia magna, acute immobilization test – OECD test guideline 202) and an chronic assay of 21 days using adult sticklebacks, a well described model fish species used to assess impact of environmental compounds.

International audience; The health risks of nanoparticles remain a serious concern given their prevalence from industrial and domestic use. The primary route of titanium dioxide nanoparticle exposure is inhalation. The extent to which nanoparticles contribute to cellular toxicity is known to associate induction of oxidative stress. To investigate this problem further, the effect of titanium dioxide nanoparticles was examined on cell lines representative of alveolo-capillary barrier. The present study showed that all nanoparticle-exposed cell lines displayed ROS generation. Macrophage-like THP-1 and HPMEC-ST1.6R microvascular cells were sensitive to endogenous redox changes and underwent apoptosis, but not alveolar epithelial A549 cells. Genotoxic potential of titanium dioxide nanoparticles was investigated using the activation of γH2AX, activation of DNA repair proteins and cell cycle arrest. In the sensitive cell lines, DNA damage was persistent and activation of DNA repair pathways was observed. Moreover, western blot analysis showed that specific pathways associated with cellular stress response were activated concomitantly with DNA repair or apoptosis. Nanoparticles-induced oxidative stress is finally signal transducer for further physiological effects including genotoxicity and cytotoxicity. Within activated pathways, HSP27 and SAPK/JNK proteins appeared as potential biomarkers of intracellular stress and of sensitivity to endogenous redox changes, respectively, enabling to predict cell behavior.

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.

Developpes et largement utilises dans les programmes de recherche, les biomarqueurs sont des outils ecotoxicologiques permettant d’evaluer l’exposition des organismes aquatiques et les effets associes. Ils sont toutefois peu utilises pour la surveillance reglementaire de la qualite des milieux aquatiques. Cet article met en lumiere les potentialites des biomarqueurs dans le cadre du controle d’enquete ou de l’investigation environnementale. Deux etudes de cas initiees suite a des observations de pecheurs et portant respectivement sur les effets d’un rejet industriel et sur la diminution des captures de poissons sont analysees. Dans ce contexte, les biomarqueurs mesures chez le poisson permettent de determiner les mecanismes d’action des polluants susceptibles d’etre a l’origine des effets observes. Des travaux complementaires restent necessaires pour etablir de maniere formelle des relations entre les effets precoces et les effets individuels et populationnels, permettant ainsi d’utiliser les biomarqueurs comme des signaux d’alerte.

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.

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.

Ionic liquids (ILs), often termed as "designer" solvents due to the extreme tunability of sought functional properties, are promoted by many stakeholders as green alternatives to the conventional volatile organic solvents. Nevertheless, this qualification was stated without sufficient caution versus versatility of ILs chemical structures which could induce significant variations in hazardous properties. Moreover, this class of compounds which contained organic cations and most inorganic anions may often present a high water solubility and poor biodegradability. These specific properties could influence the environmental behavior of these ILs and implied IL potential environmental and health effects. With regards to the exponential increase of applications, publications and patent for ten years, concerning ILs without halogen atoms, we may anticipate that some ILs could attain multi ton industrial production in the near future. In this context, according to the EC regulation 1907/2006 regarding REACH, each manufacturer or importer of a substance in quantity of minimum one ton by year should submit a registration application to the European Chemicals Agency (ECHA). In this context, a global view of some physico-chemical, toxicological and ecotoxicological data available in the literature, in accordance with the information requested by REACH regulation, is proposed here. In fact, it seems to be important to identify the relationship between LI structures and their (eco-)toxicological properties to help manufacturer for design of the more safe IL structures.

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.

International audience

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.

Ionic liquids belong to a sub-category of salts being liquids below 100°C. They are currently raising fast increasing interest as multipurpose chemicals for applications in a variety of key industrial sectors. One great expectation lies with their potential use in the emerging bio-based economy (e.g. where they may be used as reactants, solvents or can be used as catalysts or for the production of advanced catalysts). Erroneously considered for a while as green chemicals per se, while referring to potentially millions of Ionic Liquids, more consideration must be paid to their true and uncommon properties. Indeed, to support their sustainable use, more proactive consideration of their potential hazards is highly desirable, beyond conventional rating of chemical hazards as demanded by the CLP or REACh regulations. We recently showed that actual properties of ionic liquids justify a dedicated approach for phys-chem hazard assessment. In order to reveal genuine hazards, they must be evaluated in the context of their intended use, for an appropriate risk-based approach. As a matter of facts, pool fire burning mode of ionic liquids considerably differs from conventional diffusion flames from hydrocarbon liquid fires, as illustrated at figure 1. This presentation focuses on such a topic, based on a recent thesis work performed in collaboration by INERIS, UTC/ESCOM, and two well known ionic liquids providers BASF and CYTEC, with further assistance of URCA, UDRI (USA), and U-Lyon1. Examples provided to illustrate our views cover hazards of phys-chem nature as well as those addressing the potential impact on the environment, from data obtained and processed essentially on imidazolium and phosphonium based ionic liquids. This work constitutes partial contribution to the PIVERT (http://www.institut-pivert.com/sas-p-i-v-e-r-t/) and the ECORBIO on-going projects.

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.