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International audience; Background This study aims to describe and test a tiered approach for assessing compliance to Environmental Quality standards (EQSs) for priority substances in biota in line with the European Water Framework Directive. This approach is based on caged gammarids and trophic magnification factors (TMFs) at the first tier, with fish analyzed at the second tier at sites predicted to exceed the EQS at the first tier. A dataset was implemented by monitoring perfluorooctane sulfonate (PFOS) in caged gammarids exposed at 15 sites in French rivers, and in fish muscle and rest-of-body from the same sites. Isotopic ratios (delta C-13 and delta(15) N) were also measured in gammarids and fish. Two scenarios were developed to compare measured PFOS concentrations in fish against predicted concentrations based on measures in caged gammarids and TMFs. Scenario (1) compared measured PFOS concentrations in fish fillets with predicted PFOS concentrations based on measured concentrations in caged gammarids and delta(15) N. Scenario (2) tested whether or not EQS exceedance was correctly predicted based on measured concentrations in caged gammarids and trophic levels (TLs) from wild fish and gammarid populations. Results delta C-13 and delta(15) N variations showed that caged gammarids used local food resources during exposure in the field. PFOS concentrations in gammarids were fairly variable through time at each site. In fish, concentrations ranged from < 1 to 250 ng g(-1)(wet weight). After adjustment to the TL at which the EQS is set, 12 sites were above the EQS for PFOS. In scenario (1), predicted concentrations were almost correct at 7 sites out of 15. Most incorrect predictions were overestimations that were slightly improved by applying a lower (neutral) TMF. In scenario (2) we tested several variants for parameters involved in the predictions. The most efficient combination yielded two wrong predictions out of 15. This result was obtained with a higher (more conservative) TMF value, mean concentrations in gammarids from several field exposures during a year, and a TL for gammarids at the median of the distribution in French rivers. Conclusion The proposed tiered approach was thus efficient. However, the number of sites was relatively limited, and the dataset was biased towards EQS exceedance. The tiered approach warrants further validation.

International audience; The effectiveness of mussel caging for active microplastics (MPs) biomonitoring was investigated for the first time by comparing abundance and characteristics (shape, size, color and type of polymers) of MPs ingested by caged depurated blue mussels with those ingested by native mussels collected at the same sites and with those found in their surrounding environment (surface water and sediments). Mussels were exposed along a pollution gradient originating from a wastewater treatment plant discharge and near an abandoned coastal landfill. After 6 weeks of deployment, the majority (93%) of clean transplanted mussels had ingested MPs with a mean number of items ranging from 0.61 to 1.67 items/g. The occurrence, abundance and properties of MPs ingested by caged mussels were similar to those found in native mussels. Among the debris items detected in caged and native mussels, fragments were the most predominant type, consistent with the MPs found in their surrounding environment. MPs sizes were very similar whether in the water, sediments and both caged and native mussels, with a dominance of items

International audience; Estuaries are important areas highly vulnerable to anthropogenic pollutions. Therefore, the assessment of estuarine water quality is a major ecological issue. In this study, we sampled juveniles of the European flounder in the “pristine” Canche estuary, and caged them in Canche and in two polluted sites of the Seine estuary, Rouen and Fosse Nord. After one month, the metal and organic pollutants in these sites were assessed, and we evaluated several phenotypic indicators (condition index, RNA/DNA ratios and genotoxicity), and extracted the proteins in fish livers for analysis using a shotgun proteomics approach.The results showed strong modifications in the fish caged in both sites of the Seine estuary, as compared to those caged in Canche. In particular, many proteins involved in phase I and phase II detoxification reactions were accumulated in the liver of fish caged in the site showing the highest pollution, Rouen. In addition, we observed a general disruption of metabolism, in particular an increase in lipid synthesis and carbohydrate degradation in Rouen, and a decrease in the abundance of proteins associated to translational activity in Fosse Nord. At both sites, several stress proteins were decreased.The proteomic impact of the encagement by itself was also evaluated, by comparing the liver proteome of fish caged in Canche to that of fish stayed in natura during the same time. The results showed proteomic signatures of exposure to stressful conditions (particularly heat stress), most probably related to the micro-habitat in which the cages were placed. In conclusion, the caging technique is of great interest for ecotoxicological assessment of estuarine waters, but should consider that the results are representative of the micro-habitat around the cages, which does not necessarily represent the overall heterogeneity of the estuarine environment.

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.

Brest harbor (Bay of Brest, Brittany, France) has a severe past of anthropogenic chemical contamination, but inputs tended to decrease, indicating a reassessment of its ecotoxicological status should be carried out. Here, native and caged mussels (Mytilus spp.) were used in combination to evaluate biological effects of chronic chemical contamination in Brest harbor. Polycyclic aromatic hydrocarbon (PAH) contamination was measured in mussel tissues as a proxy of harbor and urban pollution. Biochemical biomarkers of xenobiotic biotransformation, antioxidant defenses, generation of reducing equivalents, energy metabolism and oxidative damage were studied in both gills and digestive glands of native and caged mussels. In particular, activities of glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), NADP-dependent isocitrate dehydrogenase (IDP), pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) were measured and lipid peroxidation was assessed by malondialdehyde (MDA) quantification. In addition, a condition index was calculated to assess the overall health of the mussels. Moderate PAH contamination was detected in digestive glands of both native and caged individuals from the exposed site. Modulations of biomarkers were detected in digestive glands of native harbor mussels indicating the presence of a chemical pressure. In particular, results suggested increased biotransformation (GST), antioxidant defenses (CAT), NADPH generation (IDP) and gluconeogenesis (PEPCK), which could represent a coordinated response against chemically-induced cellular stress. Lipid peroxidation assessment and condition index indicated an absence of acute stress in the same mussels suggesting metabolic changes could, at least partially, offset the negative effects of contamination. In caged mussels, only GR was found modulated compared to non-exposed mussels but significant differences in oxidative stress and energy-related biomarkers were observed compared to native harbor mussels. Overall, these results suggested mussels chronically exposed to contamination have set up metabolic adaptation, which may contribute to their survival in the moderately contaminated harbor of Brest. Whether these adaptive traits result from phenotypic plasticity or genetic adaptation needs to be further investigated.

International audience; AbstractAlthough caging of Gammarus species offers promising lines of inquiry to monitor metal bioavailability in freshwaters, the interspecies responsiveness to metal exposures is still unclear. In addition, abiotic factors inherent to transplantation can hamper the interpretation of field bioaccumulation data. To assess the relevance of using gammarids as biomonitors, we investigated the seasonal influence on metal bioaccumulation in two common species, Gammarus pulex and Gammarus fossarum. During four seasons, caged gammarids were deployed on three sites along the Seine River exhibiting a diffuse gradient of multi-metal contamination: a site upstream and two sites downstream from the Paris megacity. For each seasonal deployment, metal concentrations in animals were determined after 7d-exposure in situ (Ag, Cd, Co, Cu, Mn, Ni, Pb and Zn). Results show that the seasonal patterns of metal contaminations are similar between both Gammarus species, and closely related to the river axis' contamination gradient. Statistical analyses indicate that bioaccumulation of essential metals in both species is influenced by season, especially by water temperature. This highlights the necessity to consider this climatic factor inherent to the deployment period for a reliable interpretation of bioaccumulation data in the field. The comparison of accumulation factors suggests that these two species coming from different geochemical origins display similar abilities to internalize metals. This generic responsiveness of caged gammarids supports their use as sentinel organisms to quantify low spatiotemporal variations in metal bioavailabilities. Previous article in issue

International audience; The feeding activity and subsequent assimilation of the products resulting from food digestion allow organisms to obtain energy for growth, maintenance and reproduction. Among these biological parameters, we studied digestive enzymes (amylase, cellulase and trypsin) in Gammarus fossarum to assess the impact of contaminants on their access to energy resources. However, to enable objective assessment of a toxic effect of decreased water quality on an organisms’ digestive capacity, it is necessary to establish reference values based on its natural variability as a function of changing biotic and abiotic factors. To limit the confounding influence of biotic factors, a caging approach with calibrated male organisms from the same population was used. This study applied an in situ deployment at 23 sites of the Rhone basin rivers, complemented by a laboratory experiment assessing the influence of two abiotic factors (temperature and conductivity). The results showed a small effect of conductivity on cellulase activity and a significant effect of temperature on digestive enzyme activity but only at the lowest temperature (7 °C). The experimental conditions allowed us to define an environmental reference value for digestive enzyme activities to select sites where the quality of the water impacted the digestive capacity of the organisms. In addition to the feeding rate, this study showed the relevance of digestive enzymes as biomarkers to be used as an early warning tool to reflect organisms’ health and the chemical quality of aquatic ecosystems.

Human activities are responsible of the introduction of a large number of chemical contaminants in the environment. In order to assess the ecological status of water bodies, it becomes necessary to assess the impact of chemical contamination in the aquatic environment. The use of genotoxicity biomarkers allows the evaluation of the impact of chemical contaminants on the DNA integrity as predictors of effects at the population level. The main objective of this work was to develop a sensitive biomarker of early genotoxic effects of chemical contamination in Dreissena polymorpha caged in urban area. We used the comet assay, to measure DNA strand breaks and alkali-labile sites. We have coupled this assay with a specific endonuclease of oxidative DNA damage (hOGG1) to enhance the significance of the comet-hOGG1 assay, by the oxidative DNA damage detection, such as 8-oxodG, involved in cellular aging and carcinogenesis. The DNA oxidation formation has been demonstrated by the comet assay applied to mussel gills cells exposed to BaP and Cd. The oxidative DNA damage induced by BaP exposure were faster repaired than in mussels exposed to Cd. Then, the intrinsec variability of some genotoxicity biomarkers such as the level of DNA strand breaks, the micronuclei frequency and the number of DNA adducts, was assessed in order to eliminate this parameter in future biomonitoring studies. Results obtained in field studies showed the oxidative DNA damage induction in caged zebra mussels in urban sites of the Seine river basin. In addition, these studies showed that the mussels transplantation did not induce change either in the biological status of organisms or changes in the DNA strand break levels and micronuclei frequency. Finally, the comparison of the expression of genotoxicity biomarkers between spring and autumn showed that these two seasons are favourable to biomonitoring studies by the transplantation of Dreissena polymorpha., Les activités anthropiques sont responsables de l'introduction d'un grand nombre de substances chimiques dans l'environnement. Dans le cadre de la Directive Cadre Européenne sur l'eau, il est nécessaire d'évaluer l'impact de la contamination chimique sur les milieux aquatiques afin d'établir un diagnostic de l'état écologique des masses d'eau. Les biomarqueurs de génotoxicité permettent l'évaluation de l'impact des contaminants chimiques sur l'intégrité structurelle de l'ADN et comme indicateurs prédictifs d'effets au niveau populationnel. L'objectif général de cette thèse est de développer un biomarqueur de génotoxicité sensible et précoce des effets de la contamination chimique urbaine sur Dreissena polymorpha. Dans ces travaux, nous avons utilisé le test comète, permettant de mesurer les cassures de brins d'ADN et les sites alcali-labiles. Nous avons couplé ce test à une endonucléase spécifique de la détection des lésions oxydatives (hOGG1) afin d'améliorer sa significativité, par la recherche de lésions oxydatives, telle que la 8-oxodG, impliquée dans le vieillissement cellulaire et la cancérogénèse. La formation d'oxydation de l'ADN a été mise en évidence par le test comet-hOGG1 appliqué à des cellules de branchies de moules exposées au BaP et au Cd. La réparation des lésions oxydatives induites par le BaP s'est avérée plus rapide que celles induites par le Cd. D'autre part, la variabilité naturelle de certains biomarqueurs de génotoxicité tels que le taux de cassures à l'ADN, la fréquence des micronoyaux et le nombre d'adduits à l'ADN, a été étudiée dans le but d'éliminer ce biais dans de futures études de biomonitoring. Les résultats des études in situ ont permis de mettre en évidence une induction de lésions oxydatives sur les moules transplantées sur les sites urbains du bassin de la Seine. De plus, ces travaux montrent que la technique de transplantation des moules n'induit pas de modification du statut biologique des organismes, ni de variation de l'expression du taux de cassures à l'ADN et de la fréquence de micronoyaux. Enfin, la comparaison de l'expression des biomarqueurs de génotoxicité entre le printemps et l'automne montre que ces deux saisons sont favorables aux études de biomonitoring par la transplantation de dreissènes.

Evaluation of pollutant effects in environment is one of the major issues of the European Water Framework Directive 2000. Regulations have particularly the objective to reach to a good chemical and ecological status of water bodies. In this context, the aim of our study was to develop a multibiomarker approach on juvenile marine fish in order to evaluate the biological effects of chemical pollution in coastal areas. Molecular detoxification parameters (EROD, GST) and an antioxidant enzyme (CAT) were used as early warning tools of toxicity allowing the prevention of irreversible damages. In parallel, different physiological biomarkers (somatic and recent growth, RNA:DNA ratio, morphometric and lipidic indices) were analysed as reflecting damages on juveniles health. Sensitivity and relevance of molecular and physiological biomarkers were tested experimentally on juvenile : i) sea bass exposed to acute petroleum pollution, ii) sea bass and turbot submitted to a mix of contaminants in environmental concentrations during controlled and semi-controlled (caging) conditions. Our results show the ability of EROD, and in lower degree the GST, to detect short exposure (2 and 4 days) of organisms to petroleum and to reflect their deleterious effects on fish health. This relationship between molecular and physiological biomarkers was more difficultly established under multiple pollutions. Growth and condition indices were found to be more sensitive to the different levels of chemical contamination analysed (metal, PAHs). Their analyses allowed us to evaluate the weakened condition of organisms caged in the harbour area during 38 days. This caging experiment was relevant especially for juvenile sea bass in which no physiological stress was detected in the reference station. Deleterious effects of chemical contaminant on turbot juvenile health were also observed in controlled condition after 21 days exposure to the same harbour sediments and to an estuarine sediment. In complement to these experiments, a field study was realized on juvenile flounders sampled in some estuaries along the French and Belgium coast. A decrease of morphometric and lipidic indices were found in juvenile flounders from the three anthropogenic estuaries showing the highest metal bioconcentrations compared to the reference estuary. Results from these different studies showed the potentiality of growth and condition indices to reflect biological effects of chemical contaminants on juvenile marine fish. However, their use could be limited by their lower specificity to pollutant than parameters involved in detoxification. These works show therefore the need to use biomarkers at different level of biological organization in biomonitoring programs.; L’évaluation de l’impact des polluants dans l’environnement est une des préoccupations majeures qui s’inscrit dans la Directive Européenne Cadre Eau 2000. Les réglementations préconisées ont notamment pour objectif de parvenir au bon état chimique et écologique des masses d’eau. Dans ce contexte, notre étude a consisté à développer une approche multibiomarqueur sur des juvéniles de poisson afin d’évaluer les effets biologiques de la pollution chimique en milieu littoral. Des paramètres moléculaires de détoxification (EROD, GST) et une enzyme antioxydante (CAT) ont été utilisés en tant que «système d’alarme » susceptibles de détecter une perturbation avant l’apparition de signes pathologiques irréversibles. En parallèle, différents biomarqueurs physiologiques (croissance somatique et récente, rapport ARN/ADN, indices morphométrique et lipidique) ont été analysés en considérant que ceux-ci pourraient révéler les dommages induit par les polluants sur l’état de santé des juvéniles. La sensibilité et la pertinence des biomarqueurs moléculaires et physiologiques ont été testés expérimentalement sur des juvéniles : i) de bar exposés à une pollution aigüe de pétrole, ii) de bar et de turbot soumis à des mélanges de contaminants en concentrations environnementales en conditions contrôlée et semi-contrôlée (« caging »). Nos résultats montrent la capacité de l’EROD, et à un degré moindre de la GST, à détecter une exposition courte (2 et 4 jours) des organismes au pétrole et à refléter ses effets délétères sur leur état de santé. Cette relation entre biomarqueurs moléculaires et physiologiques a par contre été plus difficilement établie dans un contexte de pollution multiple. Les indices de croissance et de condition utilisés se sont avérés plus sensibles aux différents niveaux de contamination analysés (métaux et HAPs). Leur utilisation a permis d’évaluer la condition affaiblie des organismes mis en cage en milieu portuaire pendant 38 jours. Cette expérience de « caging » s’est révélée concluante, notamment pour les juvéniles de bar, sur lesquels aucun stress physiologique de la mise en cage n’a été détecté dans la station de référence. Les effets délétères des contaminants chimiques sur l’état de santé des juvéniles de turbot ont également été observés en condition contrôlée après exposition de 21 jours aux mêmes sédiments portuaires et à un sédiment estuarien. En complément de ces expériences, une étude de terrain a été réalisée sur des juvéniles de flet prélevés dans des estuaires le long de la côte française et belge. Une diminution des indices morphométrique et lipidique des juvéniles de flet, issu des trois estuaires anthropisés, a été observée en relation avec des bioconcentrations en métaux plus élevées que l’estuaire de référence. Les résultats issus de ces différentes études montrent la potentialité des indices de croissance et de condition à révéler les effets biologiques des contaminants chimiques sur les juvéniles de poissons marins. Cependant, leur spécificité vis à vis des polluants étant plus faible que les paramètres de détoxification, leur utilisation peut être limitée. Ces travaux montrent ainsi le besoin d’utiliser des biomarqueurs à différents niveaux d’organisation biologique dans les programmes de biosurveillance.

International audience; The aim of this study was to propose a tool for freshwater environmental genotoxicity assessment using Gammarus fossarum, a high ecologically relevant species. In a first part, gammarids were caged upstream and downstream wastewater treatment plant effluent output. The sensitivity of genotoxic responses of haemocytes, oocytes and spermatozoa was compared using the Comet assay. Spermatozoa appeared to be the most sensitive, suitable and relevant cell type for genotoxicity risk assessment. In a second part, a watershed-scale study was conducted over 2 years to evaluate the applicability of our caging procedure. The genotoxic impact of a contamination was followed, taking into account seasonal variability. DNA damage in spermatozoa exhibited low basal level and low variability in control upstream sites, providing a reliable discrimination of polluted sites. Finally, DNA damage in caged G. fossarum has been proved to be a sensitive and reproducible tool for freshwater genotoxicity assessment. (C) 2011 Elsevier Ltd. All rights reserved.

International audience; In situ bioassays are recognized as relevant tools for stressor and effect diagnosis in aquatic environments by allowing realistic exposure scenarios. However, most in situ bioassays are based on individual-level response assessment and do not enable to predict and/or assess the risk toward higher biological levels, such as population. Today, one of the challenges is to develop methods to extrapolate from effects measured in individual to population-level effects. This study aimed to enhance ecological relevance of this extrapolation step by using caging methods. The first step was to establish in situ relationships between life history traits (potentially used as markers of contaminant effects) and population dynamics of Gammarus fossarum (recognised as an ecologically relevant species). For this two methodologies were employed for a native population living in an uncontaminated stream: individual caging & demographic census. The second step was to assess the biological water quality of Ardières river, based on individual ecotoxicological marker measurements in transplanted organisms and then the projection of observed effects on the demography of the native population used as reference.This approach (coupling in situ bio-tests and population modeling) allows : (1) to enhance ecological relevance of diagnostic assessment, (2) to propose integrated ecotoxicological indicators.

International audience

International audience; Seasonal variations of six mussel (Mytilus galloprovincialis) biomarkers at two sites in the Mediterranean Sea were compared with physiological indices (condition, growth and gonad maturation), environmental parameters (temperature, salinity and turbidity), and chemical contamination levels. The basal levels of acetylcholinesterase (AChE), DNA adducts, benzo[a]pyrene hydroxylase (BPH), heat-shock proteins (HSP70), metallothioneins (MT) and P-glycoprotein (P-gp)-mediated multixenobiotic resistance (MXR) were estimated as early warning signals in caged mussels sampled at Carteau (native site) and La Fourcade (transplantation site) over a 2-year period. The Carteau and La Fourcade mussels have specific chemical contamination profiles but a similar range of values. For example, both are highly contaminated by heavy metals (201 and 258.4 mg kg−1 dw, respectively) and considered as moderately impacted for polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). However, contamination levels at Carteau are twice as high for PAHs (101.5 mg g−1 dw) and PCBs (90.2 mg g−1 dw) than La Fourcade. The seasonal contamination trend at Carteau showed six-fold higher levels of pyrolytic pollutants in winter. Although few tissue lesions were detected in individuals studied at either site, greater parasitic infestation was observed at Carteau. The results of findings from the two Mediterranean pilot studies support the adaptability of transplanted mussels to be used as biomarkers and to establish physiological endpoints for chemical contaminant exposure.