Olivier Geffard

Physiological and behavioural responses of Gammarus pulex (Crustacea: Amphipoda) exposed to cadmium.

The aim of this study was to investigate the effects of cadmium on physiological and behavioural responses in Gammarus pulex. In a first experiment, cadmium LC50s for different times were evaluated in 264 h experiment under continuous mode of exposure (LC50(96 h)=82.1 microgL(-1), LC50(120 h)=37.1 microgL(-1), LC50(168 h)=21.6 microgL(-1), LC50(264 h)=10.5 microgL(-1)). In a second experiment, the physiological and behavioural responses of the amphipod exposed to cadmium (0, 7.5 and 15 microgL(-1)) were investigated under laboratory conditions. The mortality and the whole body cadmium concentration of organisms exposed to cadmium were significantly higher than in controls. Concerning physiological responses, cadmium exposure exerted a significant decrease on osmolality and haemolymph Ca(2+) concentration, but not on haemolymph Na(+) and Cl(-) concentrations, whereas the Na(+)/K(+)-ATPase activity was significantly increased. Behavioural responses, such as feeding rate, locomotor and ventilatory activities, were significantly reduced in Cd exposed organisms. Mechanism of cadmium action and consequent energetic reallocation in favour of maintenance functions (i.e., osmoregulation) are discussed. The results of this study indicate that osmolality and locomotor activity in G. pulex could be effective ecophysiological/behavioural markers to monitor freshwater ecosystem and to assess the health of organisms.

A comparison between oyster (Crassostrea gigas) and sea urchin (Paracentrotus lividus) larval bioassays for toxicological studies

Abstract The respective sensitivity of oyster ( Crassostrea gigas ) and sea urchin ( Paracentrotus lividus ) embryos and larvae to salinity, and to various micropollutants (four heavy metals, three pesticides) and to two polluted sediments were compared with a simplified bioassay method. C. gigas embryos and larvae were more sensitive to copper; the sensitivity of both species to tributyltin (TBT) was practically the same; P. lividus was more sensitive to lead and mercury. The only pesticide found to be toxic was a herbicide, Dinoterbe, to which oyster larvae were more sensitive than sea urchin plutei. Of the two sediments tested, the first one had effects on P . lividus embryogenesis only; C. gigas embryos and larvae were more affected by the second sediment which was originated from a harbour and was polluted by heavy-metals. The choice of species for biomonitoring may be based on biological considerations, such as the availability of mature adults for obtaining gametes, or on the salinity of the studied area; the oyster bioassay seems to be more suitable for coastal and estuarine brackish waters, because of the broader salinity tolerance of estuarine bivalve larvae as compared to sea urchin larvae.

Acetylcholinesterase activity in Gammarus fossarum (Crustacea Amphipoda): linking AChE inhibition and behavioural alteration.

Relations between whole-body acetylcholinesterase (AChE) inhibition and changes in feeding and locomotor behaviours were investigated in adult male Gammarus fossarum during short-term exposure (96h) to the organophosphorous pesticide chlorpyrifos (CPE) and the carbamate pesticide methomyl (MT). AChE activity was measured after 24, 48 and 96h of exposure. The feeding rate was assessed after 48 and 96h of exposure and locomotor activity was measured at the end of the experiment. A concentration-dependent decrease of AChE activity and behavioural parameters was observed for both CPE and MT. However, these two compounds presented dissimilar modes of action since MT-induced effects appeared rapidly during the first 48h of the experiment and remained constant until the end of experiment, contrary to CPE-induced effects, which occurred gradually during the last 48h. For CPE, significant mortality was observed from 50% AChE inhibition, contrary to MT for which no mortality was observed despite 66% inhibition. These results suggest that, for CPE, the observed mortality was not directly related to AChE inhibition but that an additional toxic mode of action occurred. On the contrary, the feeding rate and locomotion impairment were directly correlated to levels of AChE inhibition for both chemicals, except for the lowest concentrations of MT for which an induction of the behavioural parameters was observed. Although CPE and MT have different modes of action (acting as indirect and direct inhibitors), the relations between AChE activity and inhibition of behavioural parameters were relatively close for these two compounds. This study provides a basis to interpret the biomarker AChE at the upper biological organisation level, on both the feeding rate and locomotor behaviour, which are known to be relevant ecological responses.

Effects of metals on feeding rate and digestive enzymes in Gammarus fossarum: An in situ experiment

The feeding activity and afterward the assimilation of the products resulting of the food digestion, allow organisms to obtain energy useful for growth, maintenance and reproduction. These biological parameters may be studied to assess the impact of contaminants on the energy metabolism of organisms, which could induce potential effects at an individual level. The studied species was an amphipod Gammarus fossarum, which has a high ecological relevance since it is widespread in European streams and plays a major role in the breakdown of leaf litter. Thus some G. fossarum were transplanted in four sites of a river characterized by metal contamination (Amous River, France). The following parameters were studied: digestive enzymes activities (esterase, beta-glucosidase, beta-galactosidase, amylase and endoglucanase), feeding rate, metal bioaccumulation and survival. Results showed a strong relationship between digestive enzymes activities, feeding rate and metal contents.

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.

Genotoxicity assessment in the amphipod Gammarus fossarum by use of the alkaline Comet assay.

Many xenobiotics and newly developed substances released in the aquatic environment have been found genotoxic for living organisms. There is interest in developing biomarkers of genotoxicity in different phyla and the need to increase our understanding of the impact of genotoxic insult on invertebrates, particularly on crustaceans. Freshwater invertebrates and particularly amphipods are highly relevant species ecologically. However, genotoxic responses of such species are rarely studied, whereas understanding these responses is becoming an urgent concern. The aim of this study was to develop and optimize the Comet assay in the freshwater invertebrate Gammarus fossarum by use of different cell-types: haemocytes, oocytes and spermatozoa. In a first step, the Comet assay was performed on these three cell types after exposure to the model genotoxicant methyl methanesulfonate (MMS) in vitro and in vivo. Results showed a clear dose-response relationship for all tissues, a low variability and a high sensitivity of the response, demonstrating the effectiveness of the Comet assay to detect genotoxic insult in amphipods. In a second step, to explore the potential of this technique for use in ecotoxicological studies with amphipods, these organisms were exposed to five known or suspected genotoxic compounds. The results demonstrated the possibility to use the freshwater amphipod G. fossarum in environmental genotoxicity studies with the Comet assay.

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.

Subcellular compartmentalization of cadmium, nickel, and lead in Gammarus fossarum: Comparison of methods

Knowledge of the subcellular behavior of toxicants, mainly metals, in living organisms is fundamental for understanding their toxicological consequences at individual and trans-generation levels and throughout the food web. However, different partitioning methodologies are available in the literature, particularly concerning the cytosolic fraction. Different cytosolic compounds, metallothionein-like proteins (MTLPs), and non-metallothionein-like proteins (non-MTLPs) could be separated by size exclusion chromatography or by heat treatment and centrifugation. Our results show changes of metal partitioning in the cytosolic fraction according to the methodologies used. After heat treatment, the majority of cytosolic cadmium (Cd) was found with MTLPs compounds and the opposite results were observed with size exclusion chromatography. Metal partitioning could also play an important role in the metal trophic transfer. Therefore, metal partitioning in Gammarus fossarum, an ecologically relevant species and good metal bioaccumulator, defining the trophically available metal fraction, is also discussed.