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.

Concomitant investigation of crustacean amphipods lipidome and metabolome during the molting cycle by Zeno SWATH data-independent acquisition coupled with electron activated dissociation and machine learning.

BACKGROUND: DIA (Data-Independent Acquisition) is a powerful technique in Liquid Chromatography coupled with high-resolution tandem Mass Spectrometry (LC-MS/MS) initially developed for proteomics studies and recently emerging in metabolomics and lipidomics. It provides a comprehensive and unbiased coverage of molecules with improved reproducibility and quantitative accuracy compared to Data-Dependent Acquisition (DDA). Combined with the Zeno trap and Electron-Activated Dissociation (EAD), DIA enhances data quality and structural elucidation compared to conventional fragmentation under CID. These tools were applied to study the lipidome and metabolome of the freshwater amphipod Gammarus fossarum, successfully discriminating stages and highlighting significant biological features. Despite being underused, DIA, along with the Zeno trap and EAD, holds great potential for advancing research in the omics field. RESULTS: DIA combined with the Zeno trap enhances detection reproducibility compared to conventional DDA, improving fragmentation spectra quality and putative identifications. LC coupled with Zeno-SWATH-DIA methods were used to characterize molecular changes in reproductive cycle of female gammarids. Multivariate data analysis including Principal Component Analysis and Partial Least Square Discriminant Analysis successfully identified significant features. EAD fragmentation helped to identify unknown features and to confirm their molecular structure using fragmentation spectra database annotation or machine learning. EAD database matching accurately annotated five glycerophospholipids, including the position of double bonds on fatty acid chain moieties. SIRIUS database predicted structures of unknown features based on experimental fragmentation spectra to compensate for database incompleteness. SIGNIFICANCE: Reproducible detection of features and confident identification of putative compounds are pivotal stages within analytical pipelines. The DIA approach combined with Zeno pulsing enhances detection sensitivity and targeted fragmentation with EAD in positive polarity provides orthogonal fragmentation information. In our study, Zeno-DIA and EAD thereby facilitated a comprehensive and insightful exploration of pertinent biological molecules associated with the reproductive cycle of gammarids. The developed methodology holds great promises for identifying informative biomarkers on the health status of an environmental sentinel species.

Advancing environmental monitoring across the water continuum combining biomarker analysis in multiple sentinel species: A case study in the Seine-Normandie Basin (France).

Nowadays, biomarkers are recognized as valuable tools to complement chemical and ecological assessments in biomonitoring programs. They provide insights into the effects of contaminant exposures on individuals and establish connections between environmental pressure and biological response at higher levels. In the last decade, strong improvements in the design of experimental protocols and the result interpretation facilitated the use of biomarker across wide geographical areas, including aquatic continua. Notably, the statistical establishment of reference values and thresholds enabled the discrimination of contamination effects in environmental conditions, allowed interspecies comparisons, and eliminated the need of a reference site. The aim of this work was to study freshwater-estuarine-coastal water continua by applying biomarker measurements in multi-species caged organisms. During two campaigns, eight sentinel species, encompassing fish, mollusks, and crustaceans, were deployed to cover 25 sites from rivers to the sea. As much as possible, a common methodology was employed for biomarker measurements (DNA damage and phagocytosis efficiency) and data interpretation based on guidelines established using reference values and induction/inhibition thresholds (establishment of three effect levels). The methodology was successfully implemented and allowed us to assess the environmental quality. Employing multiple species per site enhances confidence in observed trends. The results highlight the feasibility of integrating biomarker-based environmental monitoring programs across a continuum scale. Biomarker results align with Water Framework Directive indicators in cases of poor site quality. Additionally, when discrepancies arise between chemical and ecological statuses, biomarker findings offer a comprehensive perspective to elucidate the disparities. Presented as a pilot project, this work contributes to gain insights into current biomonitoring needs, providing new questions and perspectives.

Assessment of the impact of chemical pollution on endangered migratory fish in two major rivers of France, including spawning grounds.

Water pollution is a one of the most contributors to aquatic biodiversity decline. Consequently, ecological risk assessment methods have been developed to investigate the effects of existing stresses on the environment, including the toxic effects of chemicals. One of the existing approaches to quantify toxic risks is called "Potentially Affected Fraction of species" (PAF), which estimates the potential loss of species within a group of species studied. In this study, the PAF method was applied to the Garonne catchment (southwest France) due to the limited information available on the involvement of water pollution in the decline of diadromous fish populations. This approach was used to quantify the potential toxic risk associated with chemical contamination of water for fish species. The objectives were to quantify this risk (1) in the Garonne and Dordogne rivers and (2) in the spawning grounds of two endangered anadromous fish species: the allis shad and the European sturgeon during the development period of their early life stages. Environmental pollution data was provided for 21 sites within the Garonne catchment between 2007 and 2022, and toxicity data was obtained specifically from freshwater toxicity tests on fish species. Then, for each site and each year, the potential toxic risk for a single substance (ssPAF) and for a mixture of substances (msPAF) was calculated and classified as high (>5 %), moderate (>1 % and < 5 %) or low (<1 %). Potential toxic risks were mostly moderate and mainly associated with: metals > other industrial pollutants and hygiene and care products > agrochemicals. In summary, this study highlights the probable involvement of water contamination on the decline, fate and restoration of diadromous fish populations in the Garonne catchment, focusing notably on the toxic effects on early life stages, a previously understudied topic.

Electron-activated dissociation (EAD) for the complementary annotation of metabolites and lipids through data-dependent acquisition analysis and feature-based molecular networking, applied to the sentinel amphipod Gammarus fossarum.

The past decades have marked the rise of metabolomics and lipidomics as the -omics sciences which reflect the most phenotypes in living systems. Mass spectrometry-based approaches are acknowledged for both quantification and identification of molecular signatures, the latter relying primarily on fragmentation spectra interpretation. However, the high structural diversity of biological small molecules poses a considerable challenge in compound annotation. Feature-based molecular networking (FBMN) combined with database searches currently sets the gold standard for annotation of large datasets. Nevertheless, FBMN is usually based on collision-induced dissociation (CID) data, which may lead to unsatisfying information. The use of alternative fragmentation methods, such as electron-activated dissociation (EAD), is undergoing a re-evaluation for the annotation of small molecules, as it gives access to additional fragmentation routes. In this study, we apply the performances of data-dependent acquisition mass spectrometry (DDA-MS) under CID and EAD fragmentation along with FBMN construction, to perform extensive compound annotation in the crude extracts of the freshwater sentinel organism Gammarus fossarum. We discuss the analytical aspects of the use of the two fragmentation modes, perform a general comparison of the information delivered, and compare the CID and EAD fragmentation pathways for specific classes of compounds, including previously unstudied species. In addition, we discuss the potential use of FBMN constructed with EAD fragmentation spectra to improve lipid annotation, compared to the classic CID-based networks. Our approach has enabled higher confidence annotations and finer structure characterization of 823 features, including both metabolites and lipids detected in G. fossarum extracts.

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.

Organ-oriented proteogenomics functional atlas of three aquatic invertebrate sentinel species.

Proteogenomic methodologies have enabled the identification of protein sequences in wild species without annotated genomes, shedding light on molecular mechanisms affected by pollution. However, proteomic resources for sentinel species are limited, and organ-level investigations are necessary to expand our understanding of their molecular biology. This study presents proteomic resources obtained from proteogenomic analyses of key organs (hepatopancreas, gills, hemolymph) from three established aquatic sentinel invertebrate species of interest in ecotoxicological/ecological research and environmental monitoring: Gammarus fossarum, Dreissena polymorpha, and Palaemon serratus. Proteogenomic analyses identified thousands of proteins for each species, with over 90% of them being annotated to putative function. Functional analysis validated the relevance of the proteomic atlases by revealing similarities in functional annotation of catalogues of proteins across analogous organs in the three species, while deep contrasts between functional profiles are delimited across different organs in the same organism. These organ-level proteomic atlases are crucial for future research on these sentinel animals, aiding in the evaluation of aquatic environmental risks and providing a valuable resource for ecotoxicological studies.

Disruption of oogenesis and molting by methoprene and glyphosate in Gammarus fossarum: involvement of retinoic acid?

In the last decade, the freshwater amphipod Gammarus fossarum proved to be a promising sentinel species in active biomonitoring programs to assess the effects of environmental contamination on non-target organisms. Given that the highly conserved retinoid (RETs) metabolism supports many biological functions and is perturbed by xenobiotics and used as biomarker for vertebrates, we explored the RETs functions in the crustacean model Gammarus fossarum. More specifically, we studied the implication of all -trans retinoic acid (atRA) in the reproduction (embryo, oocyte, and juvenile production) and development (success and delay of molting) by exposing G. fossarum females to atRA and citral (CIT), a known inhibitor of RA synthesis. In parallel, we exposed gammarids to methoprene (MET) and glyphosate (GLY), two pesticides suspected to interfere with atRA metabolism and signaling and frequently found in water systems. After 14 days of exposure, atRA, CIT, and MET reduced the number of oocytes, whereas only MET caused a reduced number of embryos. After 44 days, MET and GLY showed a tendency to decrease juvenile production. The duration of the molting cycle increased following the exposures to atRA and MET, while the treatment with CIT caused a typical endocrine disruptive inverted U-shaped curve. The exposure to GLY led to increased duration of the molting cycle at the lowest concentrations and lowered molting success at the highest concentration tested. This study highlights for the first time the implication of RA in the oogenesis and molting of G. fossarum and suggests that it may be a potential mediator of MET-induced effects on these processes. This study adds to the comprehension of the reproductive and developmental control in G. fossarum and opens new research avenues to study the effects of xenobiotics on the RET system in this sentinel species. Ultimately, our study will drive the development of RET-based biomarkers for non-target aquatic invertebrates exposed to xenobiotics.

Dynamic Multiple Reaction Monitoring of amphipod Gammarus fossarum caeca expands molecular information for understanding the impact of contaminants.

Mass spectrometry in multiple reaction monitoring (MRM) mode is a powerful technique that can provide highly selective, multiplexed, and reproducible quantification of peptides derived from proteins. Ideal for the application of molecular biomarkers in biomonitoring surveys, MRM tools have been recently developed to quantify sets of pre-selected biomarkers in freshwater sentinel species. Still limited to the validation and application phase of biomarkers, dynamic MRM (dMRM) acquisition mode has increased the multiplexing capacity of mass spectrometers, expanding opportunities to explore proteome modulations in sentinel species. This study evaluated the feasibility to propose dMRM tools for investigating sentinel species proteomes at the organ level and demonstrated its potential for screening contaminant effects and discovering new protein biomarkers. As a proof of concept, a dMRM assay was developed to comprehensively capture the functional proteome of the caeca of Gammarus fossarum, a freshwater crustacean, commonly used as a sentinel species in environmental biomonitoring. The assay was then used to assess the effects of sub-lethal concentrations of cadmium, silver, and zinc on gammarid caeca. Results showed dose-response and specific metal effects on caecal proteomes, with a slight effect of zinc compared to the two non-essential metals. Functional analyses indicated that cadmium affected proteins involved in carbohydrate metabolism, digestive and immune processes, while silver affected proteins related to oxidative stress response, chaperonin complexes and fatty acid metabolism. Based on these metal-specific signatures, several proteins modulated in a dose-dependent manner were proposed as candidate biomarkers for tracking the level of these metals in freshwater ecosystems. Overall, this study highlights the potential of dMRM to decipher the specific modulations of proteome expression induced by contaminant exposure and pinpoints specific response signatures, offering new perspectives for the de novo identification and development of biomarkers in sentinel species.

Integrative biomarker response - Threshold (IBR-T): Refinement of IBRv2 to consider the reference and threshold values of biomarkers.

The Integrated Biomarker Response (IBR) is one of the most used index in biomonitoring, especially the IBRv2 integrating a reference condition. However, some limitations remain for its routine and large-scale use. The IBRv2 is proportional to the total number of biomarkers, is dependent on the nature of biomarkers and considers all biomarkers modulations, even small and biologically non-significant. In addition, IBRv2 relies on reference values but the references are often different between each study, making it difficult to compare results between studies and/or campaigns. To overcome these limitations, the present work proposed a new index called IBR-T ("Integrated Biomarker Response - Threshold") which considers the threshold values of biomarkers by limiting the calculation of the IBR value to biomarkers with significant modulations. The IBRv2 and the IBR-T were calculated and compared on four datasets from active biomonitoring campaigns using Dreissena polymorpha, a bivalve widely used in freshwater biomonitoring studies. The comparison between indices has demonstrated that the IBR-T presents a better correlation (0.907 < r2 < 0.998) with the percentage of biomarkers significantly modulated than the IBRv2 (0.002 < r2 < 0.759). The IBRv2 could not be equal to 0 (0.915 < intercept <1.694) because the value was dependent on the total number of biomarkers, whereas the IBR-T reached 0 when no biomarker was significantly modulated, which appears more biologically relevant. The final ranking of sites was different between the two index and the IBR-T ranking tends to be more ecologically relevant that the IBRv2 ranking. This IBR-T have shown an undeniable interest for biomonitoring and could be used by environmental managers to simplify the interpretation of large datasets, directly interpret the contamination status of the site, use it to decision-making, and finally to easily communicate the results of biomonitoring studies to the general public.

The retinoid metabolism of Gammarus fossarum is disrupted by exogenous all-trans retinoic acid, citral, and methoprene but not by the technical formulation of glyphosate.

Over the last decade, fluctuations of retinoids (RETs), also known as vitamin A and derivatives, have proved to be useful biomarkers to assess the environmental chemical pressure on a wide variety of non-target vertebrates. This use of RET-based biomarkers is of particular interest in the non-target sentinel species Gammarus fossarum in which RETs were shown to influence crucial physiological functions. To study and probe this metabolism in this crustacean model, a UHPLC-MS/MS method was developed to 1) identify and 2) monitor several endogenous RETs in unexposed females throughout their reproductive cycle. Then, females were exposed in controlled conditions to exogenous all-trans retinoic acid (atRA) and citral (CIT), a RA synthesis inhibitor, to simulate an excess or deficiency in RA. Perturbation of vitamin A metabolism by pesticides was further studied in response to methoprene (MET), a juvenile hormone analog as well as glyphosate (GLY). The developed method allowed, for the first time in this model, the identification of RA metabolites (all-trans 4-oxo and 13-cis 4-oxo RA), RA isomers (all-trans and 13-cis RA) as well as retinaldehyde (RALD) isomers (all-trans, 11-cis, and 13-cis RALD) and showed two distinct phases in the reproductive cycle. Retinoic acid successfully increased the tissular concentration of both RA isomers and CIT proved to be efficient at perturbating the conversion from RALD to RA. Methoprene perturbed the ratios between RA isomers whereas GLY had no observed effects on the RET system of G. fossarum females. We were able to discriminate different dynamics of RET perturbations by morphogens (atRA or CIT) or MET which highlights the plausible mediation of RETs in MET-induced disorders. Ultimately, our study shows that RETs are influenced by exposure to MET and strengthen their potential to assess aquatic ecosystem chemical status.

Effects of Chemical Compounds on the Activity of the N-acetyl-ß-D-Glucosaminidase of the Marine Prawn, Palaemon serratus: Screening In Vitro.

N-acetyl-ß-D-glucosaminidase (NAGase) is important for crustaceans because the enzyme activity is necessary for the molting process. The present study aimed to assess the sensitivity of Palaemon serratus NAGase activity to a set of compounds of diverse chemical families in the context of in vitro exposures. Compounds representing different chemical families were selected according to their abundance, impact in the environment, and relevance as disruptors of the molting process. In a first step, four solvents (dimethylsulfoxide [DMSO], methanol, acetone, and ethanol) were tested to determine their suitability to dissolve hydrophobic compounds without affecting NAGase activity. Exclusively, ethanol had no effect on enzyme activity and on the integrity of the proteins present in the enzyme extract. The 18 other compounds were tested and four of these compounds, pentoxifylline, fenoxycarb, dithiocarbamate, and RH5849, showed a specific alteration on the activity of NAGase, without affecting the protein content. However, cadmium, zinc, and glyphosate showed a nonspecific alteration, affecting both the enzyme activity and the proteins, whereas ibuprofen exclusively altered the protein content. Finally, 10 of the 22 tested compounds (including DMSO, acetone, and methanol) showed a direct alteration of NAGase activity. Environ Toxicol Chem 2023;42:846-858. © 2023 SETAC.

Influence of the exposure concentration of dissolved cadmium on its organotropism, toxicokinetic and fate in Gammarus fossarum.

Information on the relationship between the exposure concentrations of metals and their biodistribution among organs remained scarce in invertebrates. The objective of this study was to investigate the effects of Cd concentration on the organotropism, toxico-kinetic and fate of this metal in different organs of gammarids exposed to dissolved radioisotope 109Cd. Gammarids male were exposed for 7 days to three environmental Cd concentrations (i.e. 4, 52 and 350 ng.L-1) before being placed in depuration conditions (i.e. uncontaminated water). At several sampling times, Cd concentrations were determined by 109Cd γ-counting in water, caeca, cephalon, gills, intestine and remaining tissues. Bioconcentration Factors (BCF) and Cd relative proportions in organs were calculated to assess the exposure concentration effect on the bioaccumulation capacities. The dependance of the organ-specific kinetic parameters to Cd water concentrations were estimated by fitting nested one-compartment toxico-kinetic (TK) models to both the accumulation and depuration data, by Bayesian inference. Then, for each Cd concentrations, the metal exchanges among organs over time were formalized by a multi-compartments TK model fitted to all organ data simultaneously. Our results highlighted that, at the end of the exposure phase, BCF and Cd relative proportions, in each organ, were not significantly modulated by water concentrations. Kinetically, Cd accumulation rates in all organs (except intestines) were depended on the exposure concentration, but not the elimination rates. The in vivo management of Cd (i.e. metal exchanges among organs) remained qualitatively unchanged according to exposure concentration. All these results also highlighted key role of that organs in the management of Cd: bioconcentration by caeca, storage by gills and main entry pathway by intestine. This study shows the interest of implementing TK approaches to test the effect of environmental factors on bioaccumulation, inter-organ exchanges and fate of contaminants in invertebrate body to enhance the understanding of the toxicity risk.

Identifying the impact of toxicity on stream macroinvertebrate communities in a multi-stressor context based on national ecological and ecotoxicological monitoring databases.

In situ bioassays are used to measure the harmful effects induced by mixtures of toxic chemicals in watercourses. In France, national-scale biomonitoring data are available including invertebrate surveys and in-field chemical toxicity measures with caged gammarids to assess environmental toxicity of mixtures of chemicals. The main objective of our study is to present a proof-of-concept approach identifying possible links between in-field chemical toxicity, stressors and the ecological status. We used two active biomonitoring databases comprising lethal toxicity (222 in situ measures of gammarid mortality) and sublethal toxicity (101 in situ measures of feeding inhibition). We measured the ecological status of each active biomonitoring site using the I2M2 metric (macroinvertebrate-based multimetric index), accounted for known stressors of nutrients and organic matter, hydromorphology and chemical toxicity. We observed a negative relationship between stressors (hydromorphology, nutrients and organic matter, and chemical toxicity) and the good ecological status. This relationship was aggravated in watercourses where toxicity indicators were degraded. We validated this hypothesis for instance with nutrients and organic matter like nitrates or hydromorphological conditions like percentage of vegetation on banks. Future international assesments concerning the role of in-field toxic pollution on the ecological status in a multi-stressor context are now possible via the current methodology.

Development of a multi-omics extraction method for ecotoxicology: investigation of the reproductive cycle of Gammarus fossarum.

Omics study exemplified by proteomics, lipidomics or metabolomics, provides the opportunity to get insight of the molecular modifications occurring in living organisms in response to contaminants or in different physiological conditions. However, individual omics discloses only a single layer of information leading to a partial image of the biological complexity. Multiplication of samples preparation and processing can generate analytical variations resulting from several extractions and instrumental runs. To get all the -omics information at the proteins, metabolites and lipids level coming from a unique sample, a specific sample preparation must be optimized. In this study, we streamlined a biphasic extraction procedure based on a MTBE/Methanol mixture to provide the simultaneous extraction of polar (proteins, metabolites) and apolar compounds (lipids) for multi-omics analyses from a unique biological sample by a liquid chromatography (LC)/mass spectrometry (MS)/MS-based targeted approach. We applied the methodology for the study of female amphipod Gammarus fossarum during the reproductive cycle. Multivariate data analyses including Partial Least Squares Discriminant Analysis and multiple factor analysis were applied for the integration of the multi-omics data sets and highlighted molecular signatures, specific to the different stages.

Multiple reaction monitoring mass spectrometry for the discovery of environmentally modulated proteins in an aquatic invertebrate sentinel species, Gammarus fossarum.

Multiple reaction monitoring (MRM) mass spectrometry is emerging as a relevant tool for measuring customized molecular markers in freshwater sentinel species. While this technique is typically used for the validation of protein molecular markers preselected from shotgun experiments, recent gains of MRM multiplexing capacity offer new possibilities to conduct large-scale screening of animal proteomes. By combining the strength of active biomonitoring strategies and MRM technologies, this study aims to propose a new strategy for the discovery of candidate proteins that respond to environmental variability. For this purpose, 249 peptides derived from 147 proteins were monitored by MRM in 273 male gammarids caged in 56 environmental sites, representative of the diversity of French water bodies. A methodology is here proposed to identify a set of customized housekeeping peptides (HKPs) used to correct analytical batch effects and allow proper comparison of peptide levels in gammarids. A comparative analysis performed on HKPs-normalized data resulted in the identification of peptides highly modulated in the environment and derived from proteins likely involved in the environmental stress response. Overall, this study proposes a breakthrough approach to screen and identify potential proteins responding to relevant environmental conditions in sentinel species.

Metal bioavailable contamination engages richness decline, species turnover but unchanged functional diversity of stream macroinvertebrates at the scale of a French region.

Freshwater ecosystems are the main source of water for sustaining life on earth, and the biodiversity they support is the main source of valuable goods and services for human populations. Despite growing recognition of the impairment of freshwater ecosystems by micropollutant contamination, different conceptual and methodological considerations can newly be addressed to improve our understanding of the ecological impact into these ecosystems. Here, we originally combined in situ ecotoxicology and community ecology concepts to unveil the mechanisms structuring macroinvertebrate communities along a regional contamination gradient. The novelty of our study lies in the use of an innovative biomonitoring approach (measurement of metal contents in caged crustaceans) allowing to quantify and compare on a regional scale the levels of bioavailable metal contamination to which stream communities are exposed. We were hence able to identify 23 streams presenting a significant gradient of bioavailable metal contamination within the same catchment area in the South West of France, from which we also obtained data on the composition of resident macroinvertebrate communities. Analyses of structural and functional integrity of communities revealed an unexpected decoupling between taxonomic and functional diversity of communities in response to bioavailable metal contamination. We show that despite the negative impact of bioavailable metal contamination exposure on taxonomic diversity (with an average species loss of 17% in contaminated streams), functional diversity is maintained through a process of non-random species replacement by functional redundant species at the regional scale. Such unanticipated findings call for a deeper characterization of metal-tolerant communities' ability to cope with environmental variability in multi-stressed ecosystems.

Organ-specific accumulation of cadmium and zinc in Gammarus fossarum exposed to environmentally relevant metal concentrations.

One of the best approaches for improving the assessment of metal toxicity in aquatic organisms is to study their organotropism (i.e., the distribution of metals among organs) through a dynamical approach (i.e., via kinetic experiments of metal bioaccumulation), to identify the tissues/organs that play a key role in metal regulation (e.g., storage or excretion). This study aims at comparing the organ-specific metal accumulation of a non-essential (Cd) and an essential metal (Zn), at their environmentally relevant exposure concentrations, in the gammarid Gammarus fossarum. Gammarids were exposed for 7 days to 109Cd- or 65Zn-radiolabeled water at a concentration of 52.1 and 416 ng.L-1 (stable equivalent), respectively, and then placed in clean water for 21 days. At different time intervals, the target organs (i.e., caeca, cephalons, intestines, gills, and remaining tissues) were collected and 109Cd or 65Zn contents were quantified by gamma-spectrometry. A one-compartment toxicokinetic (TK) model was fitted by Bayesian inference to each organ/metal dataset in order to establish TK parameters. Our results indicate: i) a contrasting distribution pattern of concentrations at the end of the accumulation phase (7th day): gills > caeca ≈ intestines > cephalons > remaining tissues for Cd and intestines > caeca > gills > cephalons > remaining tissues for Zn; ii) a slower elimination of Cd than of Zn by all organs, especially in the gills in which the Cd concentration remained constant during the 21-day depuration phase, whereas Zn concentrations decreased sharply in all organs after 24 h in the depuration phase; iii) a major role of intestines in the uptake of waterborne Cd and Zn at environmentally relevant concentrations.

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.

Quantification of multi-scale links of anthropogenic pressures with PAH and PCB bioavailable contamination in French freshwaters.

Aquatic ecosystems are exposed to multiple environmental pressures including chemical contamination. Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs) known as preoccupying substances for the environment. Active biomonitoring (ABM) is a surveillance method for polluted aquatic ecosystems measuring bioavailable contamination. In this work, the aim was to quantify the total links between environmental pressures and bioavailable contamination (for PAHs and PCBs) at the French national scale. Based on 245 sites experimented by ABM from 2017 to 2019, environmental pressures (anthropogenic pressures and environmental parameters) were defined (point source landfill density, point source urban density, point source industry density, point source road density, nonpoint source industry density, nonpoint source road density, nonpoint source urban density, nutrients and organic matter, slope, dams, straightness, coarse sediment, summer precipitation, hydrographic network density and watershed size) and characterized by one or a combination of measures called stressor indicators. The links between environmental pressures and bioavailable POPs contamination (ABM measure) at a large spatial scale were defined and quantified via structural equation modeling. Point source urban density, nutrients and organic matter, summer precipitation, straightness and point source industry density are correlated positively with PAH bioavailable contamination. In contrast, nonpoint source urban density, nonpoint source industry density, nonpoint source road density and watershed size are positively correlated with PCB bioavailable contamination. The dominant pressures linked to PAHs and PCBs were different, respectively local and large-scale pressures were linked to PAH bioavailable contamination, and only large-scale pressures were linked to PCB bioavailable contamination.