Chronic metal contamination shapes the size structure of Gammarus fossarum populations in French headwater rivers.

Assessing the effects of multigenerational exposure of aquatic animal populations to chemical contamination is essential for ecological risk assessment. However, beyond rare examples reporting the sporadic emergence of a toxicological tolerance within populations that persist in contaminated environments, conclusive results are even more limited from field studies when it comes to the alteration of life-history traits. Here, we investigated whether long-term exposure to cadmium (Cd) influences size-related life-history traits (i.e., size at puberty, median adult size, maximum size) in Gammarus fossarum, a keystone species of European stream ecosystems. We studied 13 field populations of G. fossarum (cryptic lineage B) living in headwater rivers located in natural areas scattered at a large geographical scale and exposed to contrasted bioavailable Cd contamination levels due to different local geochemical backgrounds. We achieved a detailed description of the physical and physicochemical conditions of the river reaches investigated. Land-use parameters, hydrological characteristics (flow, slope, river width, flow structure, mosaic of substrates), and physicochemical conditions (temperature, conductivity, dissolved oxygen) were measured. Metallic bioavailable contamination was assessed using a standardized active biomonitoring procedure (Gammarus caging). Based on the field demographic census of the 13 populations, our results demonstrated that chronic Cd contamination significantly influences life-history in the G. fossarum species, with a significant reduction in all size traits of populations (size at puberty, median adult size, maximum size). In addition, we confirmed Cd-tolerance in contaminated populations during exposure tests in the laboratory. Various hypotheses can be then put forward to explain the modification of size-related life-history traits: a direct toxic effect of Cd, a cost of Cd-tolerance, or an adaptive evolution of life-history exposed to toxic pressure.

Avoidance behaviour of aquatic macroinvertebrates for real-time detection of micropollutant surge in wastewater effluents.

Micropollutants are regularly detected at the outlets of wastewater treatment plants (WWTPs). Across urban and industrial WWTPs, monitoring directives only require assessment for a handful of chemicals via sampling methods that fail to capture the temporal variability in micropollutant discharge. In this study, we develop a biotest for real-time on-line monitoring of micropollutant discharge dynamics in WWTPs effluents. The selected biomonitoring device ToxMate uses videotracking of invertebrate movement, which was used to deduce avoidance behaviour of the amphipod Gammarus fossarum. Organism conditioning was set up to induce a state of minimal locomotor activity in basal conditions to maximise avoidance signal sensitivity to micropollutant spikes. We showed that with a standardised protocol, it was possible to minimise both overall movement and sensitivity to physio-chemical variations typical to WWTP effluents, as well as capture the spikes of two micropollutants upon exposure (copper and methomyl). Spikes in avoidance behaviour were consistently seen for the two chemicals, as well as a strong correlation between avoidance intensity and spiked concentration. A two-year effluent monitoring case study also illustrates how this biomonitoring method is suitable for real-time on-site monitoring, and shows a promising non-targeted approach for characterising complex micropollutant discharge variability at WWTP effluents, which today remains poorly understood.

Nongenetic inheritance of increased Cd tolerance in a field Gammarus fossarum population: Parental exposure steers offspring sensitivity.

While the phenomenon of increased population tolerance to environmental contaminants has been widely reported for aquatic animal species living in contaminated ecosystems, the question of the nature of the tolerance, i.e., genetic adaptation or phenotypic plasticity, has not yet been studied to the same degree and remains under debate when explaining the variability of toxicological sensitivity among field populations. The occurrence of both processes challenges the ecological risk assessment (ERA) of chemicals, questioning the relevance of toxicity assessment procedures based on bioassays employing organisms from naive reference populations. Nonetheless, given that genetic adaptation and phenotypic acclimation are based on different mechanisms, with different persistence periods over time when exposure is stopped, gaining insight into the mechanism of tolerance to contaminants of field populations is a major concern for ERA. In this context, this study aimed to test whether genetic adaptation underlies the tolerance to cadmium (Cd) in a field Gammarus fossarum population identified in a previous study. Breeding and exposure experiments conducted in laboratory conditions revealed that: (1) first and second cohorts of neonates produced from field-collected parents present an increased Cd tolerance, which is explained by a reduction of between-brood variability of Cd sensitivity with an absence of sensitive broods compared to two reference populations rather than by the existence of more tolerant individuals; and (2) Cd tolerance was no longer evident for broods of the third cohort produced by these breeders when maintained under Cd-free conditions in the laboratory. Hence, this study drew the striking conclusion that the observation of Cd tolerance inheritance within the field population studied does not demonstrate genetic adaptation, but it results from an effect of parental exposure inducing transgenerational plasticity, which deeply influences the variability of population sensitivity. To our knowledge, this study is only the second report of increased tolerance to contaminants in a field population explicitly attributed to the phenomenon of transgenerational acclimation.

Identification, expression, and endocrine-disruption of three ecdysone-responsive genes in the sentinel species Gammarus fossarum.

Taking advantage of a large transcriptomic dataset recently obtained in the sentinel crustacean amphipod Gammarus fossarum, we developed an approach based on sequence similarity and phylogenetic reconstruction to identify key players involved in the endocrine regulation of G. fossarum. Our work identified three genes of interest: the nuclear receptors RXR and E75, and the regulator broad-complex (BR). Their involvement in the regulation of molting and reproduction, along with their sensitivity to chemical contamination were experimentally assessed by studying gene expression during the female reproductive cycle, and after laboratory exposure to model endocrine disrupting compounds (EDCs): pyriproxyfen, tebufenozide and piperonyl butoxide. RXR expression suggested a role of this gene in ecdysis and post-molting processes. E75 presented two expression peaks that suggested a role in vitellogenesis, and molting. BR expression showed no variation during molting/reproductive cycle. After exposure to the three EDCs, a strong inhibition of the inter-molt E75 peak was observed with tebufenozide, and an induction of RXR after exposure to pyriproxyfen and piperonyl butoxide. These results confirm the implication of RXR and E75 in hormonal regulation of female reproductive cycles in G. fossarum and their sensitivity towards EDCs opens the possibility of using them as specific endocrine disruption biomarkers.

Assessing the relevance of a multiplexed methodology for proteomic biomarker measurement in the invertebrate species Gammarus fossarum: A physiological and ecotoxicological study.

Recently, a protein sequence database was built specifically for the sentinel non-model species Gammarus fossarum using a proteogenomics approach. A quantitative multiplexed targeted proteomics assay (using Selected Reaction Monitoring mass spectrometry) was then developed for a fast and simultaneous quantification of dozens of biomarker peptides specific of this freshwater sentinel crustacean species. In order to assess the relevance of this breakthrough methodology in ecotoxicology, the response patterns of a panel of 26 peptides reporting for 20 proteins from the Gammarus fossarum proteome with putative key functional roles (homeostasis, osmoregulation, nutrition, reproduction, molting,…) were recorded through male and female reproductive cycles and after exposure to environmental concentrations of cadmium and lead in laboratory-controlled conditions. Based on these results, we validated the implication of annotated vtg-like peptides in the oogenesis process, and the implication of Na+/K+ ATPase proteins in the molt cycle of organisms. Upon metal (cadmium and lead) contamination, peptides belonging to proteins annotated as involved in antioxidant and detoxification functions, immunity and molting were significantly down-regulated. Overall, this multiplex assay allowed gaining relevant insights upon disruption of different main functions in the sentinel species Gammarus fossarum. This breakthrough methodology in ecotoxicology offers a valid and high throughput alternative to currently used protocols, paving the way for future practical applications of proteogenomics-derived protein biomarkers in chemical risk assessment and environmental monitoring.

Environmental relevance of laboratory-derived kinetic models to predict trace metal bioaccumulation in gammarids: Field experimentation at a large spatial scale (France).

Kinetic models have become established tools for describing trace metal bioaccumulation in aquatic organisms and offer a promising approach for linking water contamination to trace metal bioaccumulation in biota. Nevertheless, models are based on laboratory-derived kinetic parameters, and the question of their relevance to predict trace metal bioaccumulation in the field is poorly addressed. In the present study, we propose to assess the capacity of kinetic models to predict trace metal bioaccumulation in gammarids in the field at a wide spatial scale. The field validation consisted of measuring dissolved Cd, Cu, Ni and Pb concentrations in the water column at 141 sites in France, running the models with laboratory-derived kinetic parameters, and comparing model predictions and measurements of trace metal concentrations in gammarids caged for 7 days to the same sites. We observed that gammarids poorly accumulated Cu showing the limited relevance of that species to monitor Cu contamination. Therefore, Cu was not considered for model predictions. In contrast, gammarids significantly accumulated Pb, Cd, and Ni over a wide range of exposure concentrations. These results highlight the relevance of using gammarids for active biomonitoring to detect spatial trends of bioavailable Pb, Cd, and Ni contamination in freshwaters. The best agreements between model predictions and field measurements were observed for Cd with 71% of good estimations (i.e. field measurements were predicted within a factor of two), which highlighted the potential for kinetic models to link Cd contamination to bioaccumulation in the field. The poorest agreements were observed for Ni and Pb (39% and 48% of good estimations, respectively). However, models developed for Ni, Pb, and to a lesser extent for Cd, globally underestimated bioaccumulation in caged gammarids. These results showed that the link between trace metal concentration in water and in biota remains complex, and underlined the limits of these models, in their present form, to assess trace metal bioavailability in the field. We suggest that to improve model predictions, kinetic models need to be complemented, particularly by further assessing the influence of abiotic factors on trace metal uptake, and the relative contribution of the trophic route in the contamination of gammarids.

Evolution of cadmium tolerance and associated costs in a Gammarus fossarum population inhabiting a low-level contaminated stream.

Deciphering evolutionary processes occurring within long-term contaminated wild populations is essential for the ecological risk assessment of persistent chemical contaminations. Using field populations of Gammarus, a commonly-used genus in aquatic ecotoxicology, the present study sought to gain insights into the extent to which long-term exposure to metals in the field could effectively lead to shifts in toxicological sensitivities. For this, we identified a Gammarus population inhabiting a stream contaminated by cadmium (Cd). We compared the Cd-exposure and Cd-sensitivity of this population to those of five reference populations. Active biomonitoring determined in different years and seasons that significant levels of Cd were bioavailable in the contaminated site. Laboratory sensitivity tests under common garden conditions established that this long-term field exposure led to the development of a moderate Cd tolerance, which was maintained after a 3-week acclimatization in the laboratory, and transmitted to offspring produced under clean conditions. The potential physiological costs of tolerance were assessed by means of feeding rate measurements (in the laboratory and in situ). They revealed that, unlike for reference populations, the feeding activity of organisms from the tolerant population was greatly decreased when they were maintained under laboratory conditions, potentially indicating a high population vulnerability to environmental perturbations. Because dissolved Cd concentrations in water from the contaminated site were low (averaging 0.045 µg L(-1)) and below the current European environmental quality standard for Cd for inland surface waters (fixed at 0.08 µg L(-1) in soft water environments), this case study sheds light onto the extent to which current environmental quality standards are protective against potential adverse outcomes of adaptive and micro-evolutionary processes occurring in contaminated environments.

Effect of water quality and confounding factors on digestive enzyme activities in Gammarus fossarum.

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.

Natural variability and response interpretation of fecundity, vertebrate-like sex-steroid levels and energy status in the New Zealand mudsnail Potamopyrgus antipodarum (Gray).

Potamopyrgus antipodarum is a promising test organism that is often used in ecotoxicology, both in laboratory and field exposures. As no data are available on the physiological variation range of its life-traits and the biomarkers it uses, we studied the variation of fecundity, steroid levels and energy reserves over the course of a year in a field population. The reproductive cycle was described and showed seasonal activity during summer and autumn. Steroid levels (17ß-estradiol and testosterone) varied significantly during the year and were correlated with the reproductive cycle, which suggested a potential role for sex-steroids in P. antipodarum reproduction. Energy status also showed seasonal variations. Triglycerides (TG) seemed to be the main energy lipid, whereas cholesterol appeared to be mostly used as a structural lipid. Proteins were also involved in the reproductive cycle, but only when TG were not sufficient to support the reproductive strain, similar to cholesterol. Glycogen seemed to be used as an early reserve. Threshold values under which no reproduction occurred were defined in starved snails. We proposed a range of variation in the measured parameters, allowing for a better understanding and interpretation of their levels during laboratory or in situ exposures. The data suggest that the variability of fecundity in snails has not been fully appreciated in literature.

Influence of biotic and abiotic factors on metallothionein level in Gammarus pulex.

Detection and assessment of the impact of pollution on biological resources imply increasing research on early-warning markers such as metallothioneins in metal exposure. Metallothioneins are cytosolic, low molecular weight proteins, involved principally in essential metal homeostasis and non-essential metal detoxication. Metallothionein synthesis could be influenced by abiotic (season) or biotic (reproduction process) factors directly or indirectly by its effect on metal bioaccumulation (i.e., sex, weight). In a view of using metallothioneins as metal-exposure biomarkers in Gammarus pulex, this study attempts to define the effect of several factors (sex, weight/size and season) on the level of this protein. Metallothionein levels recorded in individuals over a large range of weights indicate a negative correlation between them. Inversely in our conditions, no difference was observed between male and female organisms. During field study, metallothionein level changes were observed with the highest levels in autumn and winter periods. The highest metallothionein levels were observed after the reproduction period, perhaps linked with the metabolic needs of biologically available essential metal such as zinc.

Importance of metallothioneins in the cadmium detoxification process in Daphnia magna.

Good knowledge of the relationship between toxic metals and biological systems, particularly the sub-cellular fraction, could be a suitable early indicator of toxic effects. These effects and the sub-cellular behaviour of cadmium were studied with a widely used species in freshwater toxicity bioassays, Daphnia magna. In spite of this very commonplace usage in ecotoxicological studies, very few data are available on its toxicant metabolism and in particular metal homeostasis. Combining multi-tools analysis, a soluble protein was found: it is heat-stable, rich in sulfhydryl groups (differential pulse polarography), characterised by a molecular mass of approximately 6.5 kDa, with a G-75 chromatographic profile corresponding to the rabbit metallothioneins monomer, with few if any aromatic-containing amino acids, it binds metals (e.g. Cd, Cu), and its concentration increases with Cd exposure. This evidence led us to hypothesise that metallothioneins (MTs) are present in D. magna. Up to 75% of the Cd body burden with Cd exposure is bound to the MTs fraction. The increase in the Cd concentration in the surrounding medium and concomitantly in daphnids induces sub-cellular reorganisation of essential metals such as Cu and Zn. The rate of metals in the soluble cellular fraction and associated with MTs increases with the Cd body burden. Monitoring sub-cellular distribution of metals after exposure in the natural environment could be very useful for ecotoxicological assessment.