Arnaud Chaumot

Acetylcholinesterase activity in Gammarus fossarum (Crustacea Amphipoda) Intrinsic variability, reference levels, and a reliable tool for field surveys

The appropriate use of an enzyme activity as a biomarker requires good knowledge of its basal level and its natural variability related to intrinsic biotic and environmental abiotic factors. In view of using whole-body acetylcholinesterase (AChE) activity in Gammarus fossarum as a reliable biomarker of exposure to anti-cholinesterase agents in aquatic ecosystems, (i) the effects of the main biotic (sex, reproductive status, and weight) and abiotic (water temperature) factors on the basal activity level of this enzyme were measured in the laboratory and (ii) the spatio-temporal variability of basal enzyme activity was followed in wild populations over a 1-year period. The results show no direct effect of sex. However, significant differences in AChE activity were observed between females depending on gonadal and embryonic development. A strong negative correlation between the AChE activity levels and organism body weight was observed. Indeed, AChE activity decreases drastically during the early life stages and tends to stabilise in larger individuals. These reports led us to select a standard organism (male; weight range, 15-20mg) to minimise inter-individual variability. No effect of temperature on basal AChE activity was observed in the laboratory for the tested range (6-24 degrees C). Similarly, no spatio-temporal change relative to season or the physico-chemical characteristics of the water (such as conductivity and temperature) was recorded during the field survey. On the basis of field-collected data, we defined the standard organism having a reference activity level with minimal and maximal threshold values. Finally, the value of AChE activity normalisation by protein contents is discussed.

Ovarian cycle and embryonic development in Gammarus fossarum: Application for reproductive toxicity assessment

Among freshwater invertebrates, Gammarus fossarum is an important test organism and is currently used in ecotoxicology for acute and chronic assays; nevertheless, reproductive toxicity test methods are not yet available for these species. In the present study, the reproductive cycle in Gammarus fossarum was characterized in order to propose a reproductive toxicity test encompassing molting, follicle growth, and embryonic development that will provide a better understanding of the mode of action of chemicals disrupting these hormone-regulated processes. A detailed description of the reproductive cycle in Gammarus fossarum was obtained. As in some amphipods, molt and reproductive cycles of G. fossarum females occur concurrently, lasting 30 d at 12°C. Each molt stage is characterized by a specific marsupial embryonic development stage and the size of developing follicles visible on the ovarian membrane. Based on these results, a 21-d reproductive toxicity test is proposed for this species. This new bioassay was applied to identify the specific impact of different stressors: cadmium, methomyl, nonylphenol, and a starvation diet. Good reproducibility was obtained for different endpoints under control conditions and throughout the experiments. Preliminary robust reference values or benchmarks were proposed for these endpoints. Cadmium was found to specially inhibit secondary vitellogenesis. Nonylphenol had a specific concentration-dependent effect on embryonic development, with an increase in the percent abnormality from a concentration of 0.05 µg/L. A restricted food diet led to a significant delay in the molt cycle, which in turn induced inhibition of secondary vitellogenesis.

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.

Towards a renewed research agenda in ecotoxicology

New concerns about biodiversity, ecosystem services and human health triggered several new regulations increasing the need for sound ecotoxicological risk assessment. The PEER network aims to share its view on the research issues that this challenges. PEER scientists call for an improved biologically relevant exposure assessment. They promote comprehensive effect assessment at several biological levels. Biological traits should be used for Environmental risk assessment (ERA) as promising tools to better understand relationships between structure and functioning of ecosystems. The use of modern high throughput methods could also enhance the amount of data for a better risk assessment. Improved models coping with multiple stressors or biological levels are necessary to answer for a more scientifically based risk assessment. Those methods must be embedded within life cycle analysis or economical models for efficient regulations. Joint research programmes involving humanities with ecological sciences should be developed for a sound risk management.

Caged Gammarus fossarum (Crustacea) as a robust tool for the characterization of bioavailable contamination levels in continental waters: towards the determination of threshold values.

We investigated the suitability of an active biomonitoring approach, using the ecologically relevant species Gammarus fossarum, to assess trends of bioavailable contamination in continental waters. Gammarids were translocated into cages at 27 sites, in the Rhône-Alpes region (France) during early autumn 2009. Study sites were chosen to represent different physico-chemical characteristics and various anthropic pressures. Biotic factors such as sex, weight and food availability were controlled in order to provide robust and comparable results. After one week of exposure, concentrations of 11 metals/metalloids (Cd, Pb, Hg, Ni, Zn, Cr, Co, Cu, As, Se and Ag) and 38 hydrophobic organic substances including polycyclic aromatic hydrocarbons (PAHs), polychlorobiphenyles (PCBs), pentabromodiphenylethers (PBDEs) and organochlorine pesticides, were measured in gammarids. All metals except Ag, and 33 organic substances among 38 were quantified in G. fossarum, showing that this species is relevant for chemical biomonitoring. The control of biotic factors allowed a robust and direct inter-site comparison of the bioavailable contamination levels. Overall, our results show the interest and robustness of the proposed methodological approach for assessing trends of bioavailable contamination, notably for metals and hydrophobic organic contaminants, in continental waters. Furthermore, we built threshold values of bioavailable contamination in gammarids, above which measured concentrations are expected to reveal a bioavailable contamination at the sampling site. Two ways to define such values were investigated, a statistical approach and a model fit. Threshold values were determined for almost all the substances investigated in this study and similar values were generally derived from the two approaches. Then, levels of contaminants measured in G. fossarum at the 27 study sites were compared to the threshold values obtained using the model fit. These threshold values could serve as a basis for further implementation of quality grids to rank sites according to the extent of the bioavailable contamination, with regard to the applied methodology.

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.

Proteogenomics of Gammarus fossarum to document the reproductive system of amphipods

Because of their ecological importance, amphipod crustacea are employed worldwide as test species in environmental risk assessment. Although proteomics allows new insights into the molecular mechanisms related to the stress response, such investigations are rare for these organisms because of the lack of comprehensive protein sequence databases. Here, we propose a proteogenomic approach for identifying specific proteins of the freshwater amphipod Gammarus fossarum, a keystone species in European freshwater ecosystems. After deep RNA sequencing, we created a comprehensive ORF database. We identified and annotated the most relevant proteins detected through a shotgun tandem mass spectrometry analysis carried out on the proteomes from three major tissues involved in the organisms reproductive function: the male and female reproductive systems, and the cephalon, where different neuroendocrine glands are present. The 1,873 mass-spectrometry-certified proteins represent the largest crustacean proteomic resource to date, with 218 proteins being lineage specific. Comparative proteomics between the male and female reproductive systems indicated key proteins with strong sexual dimorphism. Protein expression profiles during spermatogenesis at seven different stages highlighted the major gammarid proteins involved in the different facets of reproduction.

Proteomic Investigation of Male Gammarus fossarum, a Freshwater Crustacean, in Response to Endocrine Disruptors

While the decrease in human sperm count in response to pollutants is a worldwide concern, little attention is being devoted to its causes and occurrence in the biodiversity of the animal kingdom. Arthropoda is the most species-rich phyla, inhabiting all aquatic and terrestrial ecosystems. During evolution, key molecular players of the arthropod endocrine system have diverged from the vertebrate counterparts. Consequently, arthropods may have different sensitivities toward endocrine disrupting chemicals (EDCs). Here alteration of sperm quality in a crustacean, Gammarus fossarum, a popular organism in freshwater risk assessment, was investigated after laboratory exposure to various concentrations of three different xenobiotics: cadmium, methoxyfenozide, and pyriproxyfen. The integrity of the reproductive process was assessed by means of sperm-quality markers. For each substance, semiquantitative/relative proteomics based on spectral counting procedure was carried out on male gonads to observe the biological impact. The changes in a total of 871 proteins were monitored in response to toxic pressure. A drastic effect was observed on spermatozoon production, with a dose-response relationship. While exposure to EDCs leads to strong modulations of male-specific proteins in testis, no induction of female-specific proteins was noted. Also, a significant portion of orphans proved to be sensitive to toxic stress.

Effects of chronic dietary and waterborne cadmium exposures on the contamination level and reproduction of daphnia magna

Regulatory assessments of metal toxicity on freshwater organisms assume that toxic effects are caused by dissolved metals. In aquatic systems, organisms are exposed to both dissolved and particulate-bound metals. In this study, the chronic toxicity of dietary cadmium (Cd) on the reproduction and Cd body burden of Daphnia magna was investigated. Daphnids (<24 h) were successively exposed to dissolved Cd (8 h) and then to uncontaminated or contaminated algae (16 h) for 21 d. The results show a higher Cd burden in daphnids because of the addition of contaminated food and reveal that Cd uptake by D. magna from water and food was additive for the lowest Cd concentrations tested. Similar Cd distributions (cytosolic and insoluble fractions) were observed in the two groups of organisms, showing similar potential toxicity of Cd accumulated from the two exposure routes. Dietary Cd induces deleterious effects on D. magna reproduction. On the basis of Cd body burden of daphnids, the results support the claim that waterborne and dietary Cd exposures were additive in causing toxicity for Cd concentrations lower than 25 microg/L. At the highest Cd concentrations, the importance of dietary Cd on the daphnid contamination level decreases and confounding factors such as feeding rate reduction seem to appear, which induce an effect on neonate reproduction. In this study, we illustrate the need to take the dietary pathway into account in regulatory assessments and to establish effective concentrations with particulate-bound metals.

Next-Generation Proteomics: Toward Customized Biomarkers for Environmental Biomonitoring

Because of their ecological representativeness, invertebrates are commonly employed as test organisms in ecotoxicological assessment; however, to date, biomarkers employed for these species were the result of a direct transposition from vertebrates, despite deep evolutionary divergence. To gain efficiency in the diagnostics of ecosystem health, specific biomarkers must be developed. In this sense, next-generation proteomics enables the specific identification of proteins involved in key physiological functions or defense mechanisms, which are responsive to ecotoxicological challenges. However, the analytical investment required restricts use in biomarker discovery. Routine biomarker validation and assays rely on more conventional mass spectrometers. Here, we describe how proteomics remains a challenge for ecotoxicological test organisms because of the lack of appropriate protein sequences databases, thus restricting the analysis on conserved and ubiquitous proteins. These limits and some strategies used to overcome them are discussed. These new tools, such as proteogenomics and targeted proteomics, should result in new biomarkers specific to relevant environmental organisms and applicable to routine ecotoxicological assessment.