ToF-SIMS imaging shows specific lipophilic vitamin alterations in chronic reprotoxicity caused by the emerging contaminant Pravastatin in Gammarus fossarum.

Blood lipid-lowering agents, such as Pravastatin, are among the most frequently used pharmaceuticals released into the aquatic environment. Although their effects on humans are very well understood, their consequences on freshwater organisms are not well known, especially in chronic exposure conditions. Gammarus fossarum is commonly used as sentinel species in ecotoxicology because of its sensitivity to a wide range of environmental contaminants and the availability of standardized bioassays. Moreover, there is an increased interest in linking molecular changes in sentinel species, such as gammarids, to observed toxic effects. Here, we performed a reproductive toxicity assay on females exposed to different concentrations of pravastatin (30; 300; 3,000 and 30,000 ng L-1) during two successive reproductive cycles and we applied ToF-SIMS imaging to evaluate the effect of pravastatin on lipid homeostasis in gammarids. Reproductive bioassay showed that pravastatin could affect oocyte development in Gammarus fossarum inducing embryotoxicity in the second reproductive cycle. Mass spectrometry imaging highlighted the disruption in vitamin E production in the oocytes of exposed female gammarids at the second reproductive cycle, while limited alterations were observed in other lipid classes, regarding both production and tissue distribution. The results demonstrated the interest of applying spatially resolved lipidomics by mass spectrometry imaging to assess the molecular effects induced by long-term exposure to environmental pharmaceutical residues in sentinel species.

N-acetyl-ß-D-glucosaminidase measurement on the freshwater amphipod Gammarus fossarum: development, biological variability and application in an ecotoxicological approach.

Due to its role in the crustacean moulting process, N-acetyl-ß-D-glucosaminidase (NAGase) is interesting to monitor the good proceeding of the moult cycle, as well as relevant in assessing changes in the moulting process caused by stressors. The present study aimed to measure the NAGase activity to monitor the moulting process of the freshwater amphipod Gammarus fossarum. Firstly, an optimised protocol measuring the NAGase activity was made, allowing a robustness and reproducibility of measurements. Then, intrinsic variability of NAGase response was checked under two physiological factors: the gammarid moult cycle and gender. For both genders, a significative increase of activity was observed during premoult, instead of a basal activity detected during postmoult and intermoult. However, the NAGase female profile was preconised to study since it was defined with more precision. Finally, a 16-day exposure of female gammarids to different levels of treated or non-treated wastewater effluents was made. If delays of tissue development appeared on effluent exposed specimens, NAGase activity was similar between the different conditions. This apparent desynchronization between tissue and molecular activities accentuates the diagnostic of moult impairment and raises the interest to use markers at different organisational levels.

Interest of a multispecies approach in active biomonitoring: Application in the Meuse watershed.

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.

A "Population Dynamics" Perspective on the Delayed Life-History Effects of Environmental Contaminations: An Illustration with a Preliminary Study of Cadmium Transgenerational Effects over Three Generations in the Crustacean Gammarus.

We explore the delayed consequences of parental exposure to environmentally relevant cadmium concentrations on the life-history traits throughout generations of the freshwater crustacean Gammarus fossarum. We report the preliminary results obtained during a challenging one-year laboratory experiment in this environmental species and propose the use of population modeling to interpret the changes in offspring life-history traits regarding their potential demographic impacts. The main outcome of this first long-term transgenerational assay is that the exposure of spawners during a single gametogenesis cycle (3 weeks) could result in severe cascading effects on the life-history traits along three unexposed offspring generations (one year). Indeed, we observed a decrease in F1 reproductive success, an early onset of F2 offspring puberty with reduced investment in egg yolk reserves, and finally a decrease in the growth rate of F3 juveniles. However, the analysis of these major transgenerational effects by means of a Lefkovitch matrix population model revealed only weak demographic impacts. Population compensatory processes mitigating the demographic consequences of parental exposure seem to drive the modification of life-history traits in offspring generations. This exploratory study sheds light on the role of population mechanisms involved in the demographic regulation of the delayed effects of environmental toxicity in wild populations.

In Situ Reproductive Bioassay with Caged Gammarus fossarum (Crustacea): Part 2-Evaluating the Relevance of Using a Molt Cycle Temperature-Dependent Model as a Reference to Assess Toxicity in Freshwater Monitoring.

Active biomonitoring approaches are now recognized as relevant for monitoring water contamination and toxicity. Nevertheless, due to the confounding influence of variable and uncontrolled environmental conditions such as temperature, biological markers measured on transplanted individuals to assess water quality are difficult to interpret. The purpose of the present study is to propose a methodology for adapting a laboratory test of chronic sublethal toxicity based on the molting cycle of Gammarus fossarum to in situ assays. To this end, we 1) adapted the molt cycle temperature-dependent model developed in Part 1 (Chaumot et al. 2020, this issue) to the fluctuating temperatures measured in the field; 2) assessed the predictive power of our approach as a "reference value" from gammarids caged in 9 nonimpacted sites at different seasons; and 3) tested the relevance of our tool to interpret in situ reproductive bioassays from 5 upstream/downstream studies and a large-scale deployment in 12 sites. Our approach based on modeling the progress of gammarid molting cycle as a function of temperature appeared to be a relevant and robust tool for interpreting in situ observations in different environmental contexts in time and space. By avoiding using a "reference" or upstream situation as a baseline from which water quality could be assessed, this approach provides a real added value to water quality diagnosis in biomonitoring programs. Environ Toxicol Chem 2020;39:678-691. © 2019 SETAC.

In Situ Reproductive Bioassay with Caged Gammarus fossarum (Crustacea): Part 1-Gauging the Confounding Influence of Temperature and Water Hardness.

Monitoring the adverse effects of environmental contaminants on the reproduction of invertebrate species in the field remains a challenge in aquatic ecotoxicology. To meet the need for reliable tools for in situ toxicity assessment, we present the first part of a methodological study of the in situ implementation of a reproductive bioassay in Gammarus previously developed for screening the toxicity of chemical compounds during laboratory exposure. To ensure the correct interpretation of the modulation of reproductive markers (molting, fecundity, follicle growth, and embryonic development) in uncontrolled environmental conditions, we experimentally assessed and statistically modeled the variability in the female reproductive cycle during laboratory exposure under several temperature and water hardness conditions. Whereas water hardness did not influence the reproductive cycle, the significant accelerating effect of temperature on the dynamics of molting and marsupial development was finely modeled, by detailing the influence of temperature on the probability of transition between all molt and embryonic stages along the female cycle. In addition, no effect of temperature or water hardness was detected on the number of oocytes and embryos carried by females. Furthermore, the finding that the relative durations of the first 4 molt and embryonic stages are constant whatever the temperature makes it possible to predict the molting dynamics in fluctuating temperature conditions. Because this could allow us to take into account the confounding influence of temperature on the measurement of reproductive markers, the implications of these findings for an optimal in situ implementation of the reproductive bioassay with G. fossarum are discussed. The relevance of this modeling approach during in situ implementation is tested in a companion study. Environ Toxicol Chem 2020;39:667-677. © 2019 SETAC.

A Bayesian framework for estimating parameters of a generic toxicokinetic model for the bioaccumulation of organic chemicals by benthic invertebrates: Proof of concept with PCB153 and two freshwater species.

Toxicokinetic (TK) models are relevant and widely used to predict chemical concentrations in biological organisms. The importance of dietary uptake for aquatic invertebrates has been increasingly assessed in recent years. However, the model parameters are estimated on limited specific laboratory data sets that are bounded by several uncertainties. The aim of this study was to implement a Bayesian framework for simultaneously estimating the parameters of a generic TK model for benthic invertebrate species from all data collected. We illustrate our approach on the bioaccumulation of PCB153 by two species with different life traits and therefore exposure routes: Chironomus riparius larvae exposed to spiked sediment for 7 days and Gammarus fossarum exposed to spiked sediment and/or leaves for 7 days and then transferred to a clean media for 7 more days. The TK models assuming first-order kinetics were fitted to the data using Bayesian inference. The median model predictions and their 95% credibility intervals showed that the model fit the data well. From a methodological point of view, this paper illustrates that simultaneously estimating all model parameters from all available data by Bayesian inference, while considering the correlation between parameters and different types of data, is a real added value for TK modeling. Moreover, we demonstrated the ability of a generic TK model considering uptake and elimination routes as modules to add according to the availability of the data measured. From an ecotoxicological point of view, we show differences in PCB153 bioaccumulation between chironomids and gammarids, explained by the different life traits of these two organisms.

Natural variability and modulation by environmental stressors of global genomic cytosine methylation levels in a freshwater crustacean, Gammarus fossarum.

To improve the assessment of aquatic organism responses to environmental stressors, there is an interest in studying epigenetic marks in addition to other validated biomarkers. Indeed, the epigenetic marks may be influenced by the surrounding environment. Non-model invertebrates such as gammarids are sentinel organisms representative of the diversity of natural stream communities. Despite their ecologically relevance, the epigenetic responses have been to date poorly documented in these species. The present study explores the measurement of the global cytosine methylation level in the genome of the freshwater crustacean Gammarus fossarum. In a first step, natural variability of global cytosine methylation level (basal level) was assessed by studying the effect of sex, age and sampling site of organisms. Results showed a significant effect of age and sampling site. In a second step, effects of water temperature and food starvation were studied. For both factors, a hypermethylation was observed after 1 month of exposure. In a third step, gammarids were exposed to a range of environmentally relevant cadmium concentrations (0.05-5 µg/L) in order to assess the effect of a chemical stress. Whatever the cadmium concentration used, a significant hypomethylation was observed after 14 days followed by a trend for hypermethylation after 1 month of exposure. These results are the first ones dealing with the 5C-methylation status in gammarids. The results constitute potential markers of environmental stresses in relevant sentinel species widely used in ecotoxicological studies.

Application of a multidisciplinary and integrative weight-of-evidence approach to a 1-year monitoring survey of the Seine River.

Quality assessment of environments under high anthropogenic pressures such as the Seine Basin, subjected to complex and chronic inputs, can only be based on combined chemical and biological analyses. The present study integrates and summarizes a multidisciplinary dataset acquired throughout a 1-year monitoring survey conducted at three workshop sites along the Seine River (PIREN-Seine program), upstream and downstream of the Paris conurbation, during four seasonal campaigns using a weight-of-evidence approach. Sediment and water column chemical analyses, bioaccumulation levels and biomarker responses in caged gammarids, and laboratory (eco)toxicity bioassays were integrated into four lines of evidence (LOEs). Results from each LOE clearly reflected an anthropogenic gradient, with contamination levels and biological effects increasing from upstream to downstream of Paris, in good agreement with the variations in the structure and composition of bacterial communities from the water column. Based on annual average data, the global hazard was summarized as "moderate" at the upstream station and as "major" at the two downstream ones. Seasonal variability was also highlighted; the winter campaign was least impacted. The model was notably improved using previously established reference and threshold values from national-scale studies. It undoubtedly represents a powerful practical tool to facilitate the decision-making processes of environment managers within the framework of an environmental risk assessment strategy.

Osmoregulatory responses to cadmium in reference and historically metal contaminated Gammarus fossarum (Crustacea, Amphipoda) populations.

In order to better understand the variable sensitivities of crustaceans to metals, we investigated the impact of cadmium exposure in 3 populations of Gammarus fossarum from different rivers of France. The first population lives in a Cd-contaminated river from a geochemical background, while the others inhabit Cd-free sites. Osmoregulation, a relevant biomarker to evaluate crustacean health following metal contamination, was used as a proxy to evaluate the intra- and inter-populationnal sensitivities to Cd. Specimens from each population were experimentally exposed to 9 µg Cd2+/L Cd for 7 days and hemolymph osmolality (HO) was then individually measured. In exposed populations, high inter-individual variations in HO values were noted, resulting in their separation into non-impacted and slightly or highly Cd-impacted (with lower HO) animals. In gills of impacted organisms, deep histopathological alterations and protein overexpression of Na+/K+-ATPase and V-H+-ATPase were observed through histology and immunolocalization, while non-impacted animals showed profiles comparable to controls. Moreover, the osmoregulatory processes in the population living in the Cd-contaminated site were impacted by acute Cd exposure in the laboratory as much as for one of the two populations originating from Cd-free sites. The observed changes did not reveal any obvious adaptive osmoregulatory phenomena at the population scale, but they may be due to differences in fitness between individuals and between populations in relation to the features of their respective environments, unrelated with the presence of the metal.

Use of Gammarus fossarum (Amphipoda) embryo for toxicity testing: A case study with cadmium.

The effects of environmental contaminants on arthropod embryo stages have been poorly investigated in ecotoxicology. Moreover, many of these tests used hatching success as the sole metric, although it is possible to detect many more subtle effects. After a detailed description of embryogenesis in Gammarus fossarum, the present study reports on the sublethal effects of cadmium (Cd) exposure during embryonic development in G. fossarum. Embryos were first directly exposed in multiwell plates throughout the entire embryonic cycle (23 d) to increasing Cd concentrations (0, 1.5, and 3.0 µg/L; 120 embryos/concentration). Then, to assess the representativeness of the gammarid embryo assay performed in multiwell plates, embryos were exposed to similar Cd concentrations through the maternal open brood pouch. Next, to pinpoint sensitive periods of development, embryos were directly exposed to 3.0 µg/L of Cd for shorter periods of time: during gastrulation, organogenesis, and hatching. After hatching, the following parameters were measured in the newborn individuals: 1) body mass; 2) activity of the enzyme phenoloxidase, a key enzyme of the arthropod immune system; and 3) locomotor activity. Phenoloxidase activity was strongly inhibited in newborn individuals of embryos exposed (either in multiwell plates or in the maternal brood pouch) to 3.0 µg/L Cd throughout embryonic development. Furthermore, strong detrimental locomotor effects were observed in newborn individuals of embryos directly exposed to 3.0 µg/L. Exposures for shorter periods of time were not sufficient to induce such effects; no sensitive period could be determined. By bringing new insights into a critical time window of exposure, the gammarid embryo assay could provide a novel and interesting addition to existing bioassays in gammarids. Environ Toxicol Chem 2017;36:2436-2443. © 2017 SETAC.

Validation of a two-generational reproduction test in Daphnia magna: An interlaboratory exercise.

Effects observed within one generation disregard potential detrimental effects that may appear across generations. Previously we have developed a two generation Daphnia magna reproduction test using the OECD TG 211 protocol with a few amendments, including initiating the second generation with third brood neonates produced from first generation individuals. Here we showed the results of an inter-laboratory calibration exercise among 12 partners that aimed to test the robustness and consistency of a two generation Daphnia magna reproduction test. Pyperonyl butoxide (PBO) was used as a test compound. Following experiments, PBO residues were determined by TQD-LC/MS/MS. Chemical analysis denoted minor deviations of measured PBO concentrations in freshly prepared and old test solutions and between real and nominal concentrations in all labs. Other test conditions (water, food, D. magna clone, type of test vessel) varied across partners as allowed in the OECD test guidelines. Cumulative fecundity and intrinsic population growth rates (r) were used to estimate "No observed effect concentrations "NOEC using the solvent control as the control treatment. EC10 and EC-50 values were obtained regression analyses. Eleven of the twelve labs succeeded in meeting the OECD criteria of producing >60 offspring per female in control treatments during 21days in each of the two consecutive generations. Analysis of variance partitioning of cumulative fecundity indicated a relatively good performance of most labs with most of the variance accounted for by PBO (56.4%) and PBO by interlaboratory interactions (20.2%), with multigenerational effects within and across PBO concentrations explaining about 6% of the variance. EC50 values for reproduction and population growth rates were on average 16.6 and 20.8% lower among second generation individuals, respectively. In summary these results suggest that the proposed assay is reproducible but cumulative toxicity in the second generation cannot reliably be detected with this assay.

Role of cellular compartmentalization in the trophic transfer of mercury species in a freshwater plant-crustacean food chain.

Mercury (Hg) represents an important risk for human health through the food webs contamination. Macrophytes bioaccumulate Hg and play a role in Hg transfer to food webs in shallow aquatic ecosystems. Nevertheless, the compartmentalization of Hg within macrophytes, notably major accumulation in the cell wall and its impact on trophic transfer to primary consumers are overlooked. The present work focusses on the trophic transfer of inorganic Hg (IHg) and monomethyl-Hg (MMHg) from the intracellular and cell wall compartments of the macrophyte Elodea nuttallii - considered a good candidate for phytoremediation - to the crustacean Gammarus fossarum. The results demonstrated that Hg accumulated in both compartments was trophically bioavailable to gammarids. Besides IHg from both compartments were similarly transferred to G. fossarum, while for MMHg, uptake rates were ∼2.5-fold higher in G. fossarum fed with the cell wall vs the intracellular compartment. During the depuration phase, Hg concentrations in G. fossarum varied insignificantly suggesting that both IHg and MMHg were strongly bound to biological ligands in the crustacean. Our data imply that cell walls have to be considered as an important source of Hg to consumers in freshwater food webs when developing procedures for enhancing aquatic environment protection during phytoremediation programs.

Mothers and not genes determine inherited differences in cadmium sensitivities within unexposed populations of the freshwater crustacean Gammarus fossarum.

Deciphering evolutionary processes occurring within contaminated populations is important for the ecological risk assessment of toxic chemicals. Whereas increased tolerance to contaminants is well documented in aquatic animal populations, whether such phenotypic changes occur through genetic adaptation is still debated. In that sense, several studies with the freshwater crustacean Gammarus concluded in a weak potential for genetic adaptation to cadmium (Cd), while others reported inheritable increased tolerance in Cd-contaminated populations. Using quantitative genetics and selection experiments, this study sought to further assess the potential of Gammarus populations to genetically adapt to Cd. By combining the control of the reproductive cycle of this species in the laboratory and protocols of individual Cd exposure, we conducted half-sib analyses to establish the genetic and environmental sources of variance in Cd sensitivity of neonates. Prior to experiments, computations allowed optimizing the experimental design in order to increase the power to detect additive genetic variance. The main findings are the existence of strong between-brood variability along with weak heritability of Cd sensitivity within Gammarus populations. This study also revealed a significant maternal effect on individual Cd sensitivity. This sheds new light on the importance of maternal influence in microevolutionary processes occurring in contaminated environments.

Linking feeding inhibition with reproductive impairment in Gammarus confirms the ecological relevance of feeding assays in environmental monitoring.

The in situ feeding bioassay in Gammarus fossarum is recognized as a reliable tool for monitoring the toxicity of freshwater contamination. However, whether recorded feeding inhibitions can potentially provoke population-level adverse outcomes remains an open question. In the present study, the authors present an experimental study in G. fossarum, which contributes to the quantitative description of the links between feeding inhibitions and impacts on female reproductive performance. The authors studied the impacts of food deprivation on reproductive endpoints (i.e., fecundity, fertility, molt cycle) during 2 successive molting cycles. Among the main results, the authors found that food deprivation triggered a slowdown of the molting process and a reduction in fertility but no alteration to embryonic development. These reproductive impairments appeared for feeding inhibition values usually recorded in monitoring programs of environmental pollution. Using a population model translating Gammarus life-history, the authors predicted that the observed reproductive alterations predict a strong degradation of population dynamics. The present study underlines the importance of feeding inhibition in population-level risk assessment and discusses how establishing upscaling schemes based on quantitative mechanistic links between impacts at different levels of biological organization can be applied in environmental monitoring to propose an ecotoxicological assessment of water quality, which would be sensitive, specific, and ecologically relevant.

Ecological modeling for the extrapolation of ecotoxicological effects measured during in situ assays in Gammarus.

Evaluating the effects of chemical contamination on populations and ecological communities still constitutes a challenging necessity in environmental management. However, the toxic effects of contaminants are commonly measured by means of organism-level responses. Linking such effects measures with ecological models is a promising way to determine population-level impacts. In this way, population models are currently increasingly used in predictive risk assessment procedures, but their use in environmental diagnostic framework remains limited due to their lack of ecological realism. The present study with the crustacean Gammarus fossarum, a sentinel species in freshwater monitoring, combines a dual field and laboratory experimental approach with a population modeling framework. In this way, we developed an ecologically relevant periodic matrix population model for Gammarus. This model allowed us to capture the population dynamics in the field, and to understand the particular pattern of demographic sensitivities induced by Gammarus life-history phenology. The model we developed provided a robust population-level assessment of in situ-based effects measures recorded during a biomonitoring program on a French watershed impacted by past mining activities. Thus, our study illustrates the potential of population modeling when seeking to decipher the role of environmental toxic contamination in ecological perturbations.

Life-history phenology strongly influences population vulnerability to toxicants: a case study with the mudsnail Potamopyrgus antipodarum.

One of the main objectives of ecological risk assessment is to evaluate the effects of toxicants on ecologically relevant biological systems such as populations or communities. However, the effects of toxicants are commonly measured on selected subindividual or individual endpoints due to their specificity against chemical stressors. Introducing these effects into population models is a promising way to predict impacts on populations. The models currently employed are very simplistic, and their environmental relevance needs to be improved to establish the ecological relevance of hazard assessment. The present study with the gastropod Potamopyrgus antipodarum combines a field experimental approach with a modeling framework. It clarifies the role played by seasonal variability of life-history traits in the population's vulnerability to the alteration of individual performance, potentially due to toxic stress. The present study comprised 3 steps: 1) characterization of the seasonal variability in life-history traits of a local population over 1 yr by using in situ experiments on caged snails, coupled with a demographic follow-up; 2) development of a periodic matrix population model that visualizes the monthly variability of population dynamics; and 3) simulation of the demographic consequences of an alteration in life-history traits (i.e., fertility, juvenile, and adult survival). The results revealed that demographic impacts strongly depend on the season when alterations of individual performance occur. Model analysis showed that this seasonal variability in population vulnerability is strongly related to the phenology of the population. The authors emphasize that improving the realism of population models is a major objective for ecological risk assessment, and that taking into account species phenology in modeling approaches should be a priority.

Vitellogenin-like protein measurement in caged Gammarus fossarum males as a biomarker of endocrine disruptor exposure: inconclusive experience.

A vitellogenin (Vg) mass spectrometry-based assay was recently developed to actively biomonitor and assess the exposure of the amphipod Gammarus fossarum to endocrine-disrupting chemicals in freshwater hydrosystems. This paper focuses on the appropriate use of this biomarker, which requires good knowledge of its basal level in males and its natural variability related to intrinsic biotic and environmental abiotic factors. To obtain the lowest biomarker variability, we first studied some of these confounding factors. We observed that the spermatogenesis stage did not have an impact on the Vg level, allowing flexibility in the choice of transplanted gammarids. In the second part of the study, males were transplanted in two clean stations for 21 days, with results indicating a spatial and temporal variability of Vg levels. These Vg changes could not be correlated to environmental factors (e.g., temperature, pH and hardness of waters). Vg induction was then assessed in 21 stations having various levels of contamination. Inductions were observed for only two of the impacted stations studied. Under reference and contaminated conditions, a high interindividual variability of Vg levels was observed in caged organisms, severely limiting the sensitivity of the biomarker and its ability to detect a significant endocrine-disruptor effect. This may be explained by unidentified environmental factors that should later be determined to improved the use of Vg as a biomarker in male G. fossarum. Moreover, as discussed in this paper, recent advancements regarding the pleiotropic functions of the Vg gene in some species may complicate the application of this biomarker in males of invertebrate species.

Vitellogenin-like proteins in the freshwater amphipod Gammarus fossarum (Koch, 1835): functional characterization throughout reproductive process, potential for use as an indicator of oocyte quality and endocrine disruption biomarker in males.

This work focused on the validation of biological specificity of the quantitative LC-MS/MS assay by checking the natural variability of Vg levels during the reproductive cycle in Gammarus fossarum (i.e., including oogenesis and embryogenesis). Laboratory tests were performed for 21 days under controlled conditions to assess Vg changes in male and female gammarids after exposure to chemical stress. Females were exposed to two crustacean hormones, 20-hydroxyecdysone (0.01, 1 and 100 µg L⁻¹) and methyl-farnesoate (0.01, 1 and 100 µg L⁻¹). No effect was recorded for 20-hydroxyecdysone, whereas in females exposed to methyl-farnesoate a deleterious impact on Vg production was observed. Males were exposed to crustacean hormones 20-hydroxyecdysone (0.01, 1 and 100 µg L⁻¹) and methyl-farnesoate (0.01, 1 and 100 µg L⁻¹), the insecticide methoxyfenozide (0.001, 0.1 and 10 µg L⁻¹), the fungicide propiconazole (0.001, 0.1, 10 and 1000 µg L⁻¹), and the pharmaceutical products benzophenone, carbamazepine, cyproterone, and R-propranolol (0.001, 0.1, 10 and 1000 µg L⁻¹). Induction of Vg synthesis was recorded in males exposed to cyproterone, methoxyfenozide, methyl-farnesoate, and propiconazole. Finally, we validated the function of the ILIPGVGK peptide used to track vitellogenin in G. fossarum across reproductive processes (vitellogenesis and embryogenesis), and results confirmed the energy reserve role of Vg during embryo development. We show that oocyte surface measurement is directly related to Vg levels in the oocyte, constituting a reliable indicator of egg quality in G. fossarum. Consequently, it could be used as a reliable tool for biomonitoring programs. We recorded induction of Vg in male G. fossarum; however, the possible use of this tool as a specific biomarker of exposure to endocrine disruption should be confirmed in further studies.

In situ feeding assay with Gammarus fossarum (Crustacea): Modelling the influence of confounding factors to improve water quality biomonitoring.

In situ feeding assays implemented with transplanted crustacean gammarids have been claimed as promising tools for the diagnostic assessment of water quality. Nevertheless the implementation of such methodologies in biomonitoring programs is still limited. This is explained by the necessity to improve the reliability of these bioassays. The present study illustrates how modelling the influence of confounding factors could allow to improve the interpretation of in situ feeding assay with Gammarus fossarum. We proceeded in four steps: (i) we quantified the influence of body size, temperature and conductivity on feeding rate in laboratory conditions; (ii) based on these laboratory findings, we computed a feeding inhibition index, which proved to be robust to environmental conditions and allowed us to define a reference statistical distribution of feeding activity values through the data compilation of 24 in situ assays among diverse reference stations at different seasons; (iii) we tested the sensitivity of the feeding assay using this statistical framework by performing 41 in situ deployments in contaminated stations presenting a large range of contaminant profiles; and (iv) we illustrated in two site-specific studies how the proposed methodology improved the diagnosis of water quality by preventing false-positive and false-negative cases mainly induced by temperature confounding influence. Interestingly, the implementation of the developed protocol could permit to assess water quality without following an upstream/downstream procedure and to compare assays performed at different seasons as part of large-scale biomonitoring programs.