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.

Assessing viral freshwater hazard using a toxicokinetic model and Dreissena polymorpha.

The detection all pathogenic enteric viruses in water is expensive, time-consuming, and limited by numerous technical difficulties. Consequently, using reliable indicators such as F-specific RNA phages (FRNAPH) can be well adapted to assess the risk of viral contamination of fecal origin in surface waters. However, the variability of results inherent to the water matrix makes it difficult to use them routinely and to interpret viral risk. Spatial and temporal variability of surface waters can lead to underestimate this risk, in particular in the case of low loading. The use of bivalve mollusks as accumulating systems appears as a promising alternative, as recently highlighted with the freshwater mussel Dreissena polymorpha, but its capacity to accumulate and depurate FRNAPH needs to be better understood and described. The purpose of this study is to characterise the kinetics of accumulation and elimination of infectious FRNAPH by D. polymorpha in laboratory conditions, formalised by a toxico-kinetic (TK) mechanistic model. Accumulation and depuration experiments were performed at a laboratory scale to determine the relationship between the concentration of infectious FRNAPH in water and the concentration accumulated by D. polymorpha. The mussels accumulated infectious FRNAPH (3-5.4 × 104 PFU/g) in a fast and concentration-dependent way in only 48 h, as already recently demonstrated. The second exposure demonstrated that the kinetics of infectious FRNAPH depuration by D. polymorpha was independent to the exposure dose, with a T90 (time required to depurate 90 % of the accumulated concentration) of approximately 6 days. These results highlight the capacities of D. polymorpha to detect and reflect the viral pollution in an integrative way and over time, which is not possible with point water sampling. Different TK models were fitted based on the concentrations measured in the digestive tissues (DT) of D. polymorpha. The model has been developed to formalise the kinetics of phage accumulation in mussels tissues through the simultaneous estimation of accumulation and depuration rates. This model showed that accumulation depended on the exposure concentration, while depuration did not. Standardized D. polymorpha could be easily transplanted to the environment to predict viral concentrations using the TK model defined in the present study to predict the level of contamination of bodies of water on the basis of the level of phages accumulated by the organisms. It will be also provide a better understanding of the dynamics of the virus in continental waters at different time and spatial scales, and thereby contribute to the protection of freshwater resources.

Biochemical basis of resistance to multiple contaminations in the native and invasive populations of Dreissena polymorpha.

The zebra mussel Dreissena polymorpha (Pallas, 1771) is an invasive species and a valuable bioindicator in the inland waters. Nevertheless, the biochemical reasons for the unique competitiveness of zebra mussels are not clear. This study aimed to compare the native and invasive populations of D. polymorpha in their ability to withstand the same multiple environmental challenges (i.e. chemical: microplastics and caffeine; physical: temperatures). The specimens from the invasive population in west Ukraine (Tn) and native population at lower streams of river Dnipro (south Ukraine, Kh) were sampled in the August of 2021 y. Molluscs from both populations were treated simultaneously with microplastics (MP, 1 mg L-1, pore size 2 µm); caffeine (Caf, 20.0 µg L-1) at 18 °C, elevated temperature (25 °C) and MP and Caf combinations at 18 °C and 25 °C for 14 days. Untreated molluscs exposed at 18 °C represented control groups. A set of the 20 markers of oxidative stress, biotransformation, detoxification and apoptosis were assayed in the total soft tissues. From the two controls, Kh-group indicated lower stressful impact. However, both populations increased caspase-3 and GST activities and lysosomal instability in most exposures, and cholinesterase and phenoloxidase activities under the heating and combine exposures, indicating the remarkable properties to respond to new challenges and synergistic effect of mixtures. Inter-population differences were related to the metallothionein, cathepsin D, cytochrome P540 and oxidative stress responses that reflect population-dependent adverse outcome pathways. The discriminant analysis separated two populations with a substantially lesser magnitude of responses in the invasive population as a sign of higher resistance.

Application of the Fpg-modified comet assay on three-spined stickleback in freshwater biomonitoring: toward a multi-biomarker approach of genotoxicity.

Aquatic species are exposed to a wide spectrum of substances, which can compromise their genomic integrity by inducing DNA damage or oxidative stress. Genotoxicity biomarkers as DNA strand breaks and chromosomal damages developed on sentinel species have already proved to be relevant in aquatic biomonitoring. However, these biomarkers do not reflect DNA oxidative lesions, i.e., the 8-oxodG, recognized as pre-mutagenic lesion if not or mis-repaired in human biomonitoring. The relevance to include the measure of these lesions by using the Fpg-modified comet assay on erythrocytes of the three-spined stickleback was investigated. An optimization step of the Fpg-modified comet assay considering enzyme buffer impact, Fpg concentration, and incubation time has been performed. Then, this measure was integrated in a battery of genotoxicity and cytotoxicity biomarkers (considering DNA strand breaks, DNA content variation, and cell apoptosis/necrosis and density) and applied in a freshwater monitoring program on six stations of the Artois Picardie watershed (3-week caging of control fish). These biomarkers allowed to discriminate the stations regarding the genotoxic potential of water bodies and specifically by the measure of oxidative DNA lesions, which seem to be a promising tool in environmental genotoxicity risk assessment.

Insights on the Organ-Dependent, Molecular Sexual Dimorphism in the Zebra Mussel, Dreissena polymorpha, Revealed by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Metabolomics.

The zebra mussel, Dreissena polymorpha, is extensively used as a sentinel species for biosurveys of environmental contaminants in freshwater ecosystems and for ecotoxicological studies. However, its metabolome remains poorly understood, particularly in light of the potential molecular sexual dimorphism between its different tissues. From an ecotoxicological point of view, inter-sex and inter-organ differences in the metabolome suggest variability in responsiveness, which can influence the analysis and interpretation of data, particularly in the case where males and females would be analyzed indifferently. This study aimed to assess the extent to which the molecular fingerprints of functionally diverse tissues like the digestive glands, gonads, gills, and mantle of D. polymorpha can reveal tissue-specific molecular sexual dimorphism. We employed a non-targeted metabolomic approach using liquid chromatography high-resolution mass spectrometry and revealed a significant sexual molecular dimorphism in the gonads, and to a lesser extent in the digestive glands, of D. polymorpha. Our results highlight the critical need to consider inter-sex differences in the metabolome of D. polymorpha to avoid confounding factors, particularly when investigating environmental effects on molecular regulation in the gonads, and to a lesser extent in the digestive glands.

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.

Toward better monitoring of human noroviruses and F-specific RNA bacteriophages in aquatic environments using bivalve mollusks and passive samplers: A case study.

Monitoring pathogenic enteric viruses in continental and marine water bodies is essential to control the viral contamination of human populations. Human Noroviruses (NoV) are the main enteric viruses present in surface waters and foodstuff. In a context of global change, it is currently a challenge to improve the management of viral pollutions in aquatic environments and thereby limit the contamination of vulnerable water bodies or foodstuffs. The aim of this study is to evaluate the potential of specific accumulation systems for improving the detection of NoV in water bodies, compared to direct water analyses. Passive samplers (Zetapor filters) and three species of bivalve molluscan shellfish (BMS) (Dreissena polymorpha, Mytilus edulis and Crassostreas gigas) were used as accumulation systems to determine their performance in monitoring continental and marine waters for viruses. F-specific RNA bacteriophages (FRNAPH) were also analyzed since they are described as indicators of NoV hazard in many studies. During a one-year study in a specific area frequently affected by fecal pollution, twelve campaigns of exposure of passive samplers and BMS in continental and coastal waters were conducted. Using suitable methods, NoV (genome) and FRNAPH (infectious and genome) were detected in these accumulation systems and in water at the same time points to determine the frequency of detection but also to gain a better understanding of viral pollution in this area. The reliability of FRNAPH as a NoV indicator was also investigated. Our results clearly showed that BMS were significantly better than passive samplers and direct water analyses for monitoring NoV and FRNAPH contamination in water bodies. A dilution of viral pollution between the continental and the coastal area was observed and can be explained by the distance from the source of the pollution. Viral pollution is clearly greater during the winter period, and stakeholders should take this into consideration in their attempts to limit the contamination of food and water. A significant correlation was once again shown between NoV and FRNAPH genomes in BMS, confirming the reliability of FRNAPH as a NoV indicator. Moreover, a strong correlation was observed between NoV genomes and infectious FRNAPH, suggesting recent viral pollution since infectious particles had not been inactivated at sufficient levels in the environment. More generally, this study shows the value of using BMS as an active method for improving knowledge on the behavior of viral contamination in water bodies, the ranking of the contamination sources, and the vulnerability of downstream water bodies.

Modulation of haemocyte motility by chemical and biological stresses in Mytilus edulis and Dreissena polymorpha.

Mussels are constantly exposed to various pollutants in the environment, which can impair their immune defences against microbes and thus threaten their survival. In this study, we expand the insight into a key parameter of immune response in two mussel species by exploring the impact of exposure to pollutants or bacteria or simultaneous chemical and biological exposure on haemocyte motility. Basal haemocyte velocity in primary culture was high and increasing over time in Mytilus edulis (mean cell speed of 2.32 µm/min ± 1.57) whereas Dreissena polymorpha showed a constant and rather low cell motility with time (mean cell speed of 0.59 µm/min ± 0.1). In the presence of bacteria, the motility of haemocytes was instantly enhanced and slowed down after 90 min for M. edulis. In contrast, in vitro exposure of haemocytes to chemicals, either Bisphenol A, oestradiol, copper, or caffeine, induced an inhibition of cell motility in both mussel species. Finally, the cellular activation observed during bacterial challenges was inhibited by simultaneous exposure to bacteria and pollutants. Overall, our results indicate that chemical contaminants can alter haemocyte migration in mussels which can weaken their response to pathogens and therefore increase their susceptibility to infectious diseases.

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.

Metal Contamination and Biomarkers in Cerastoderma glaucum: A Multi-level Approach.

In this study, we focused on evaluating the responses of the cockle, Cerastoderma glaucum to in situ exposures to metals at three sites in the Gulf of Gabes in the coastal zone of Tunisia differing in levels of metal contamination. Firstly, we examined the general physiological state of the organisms. Secondly, we evaluated the bioaccumulation of several metals (Cd, Cu, Zn, Ni) in the cockles. Thirdly, we focused on evaluating histologically changes in gametogenesis and sexual maturity of the organisms. Finally, we determined the expression of seven key genes encoding enzymes or proteins involved in responses to different types of environmental stressors. Results showed a decrease in the general physiological status of the cockles, including a reduced condition index, sex ratios skewed to females (70% and 80% females in the intermediate and the contaminated site, respectively) and greater mortalities in tests under anoxic conditions (i.e., stress on stress test) in cockles collected from the most contaminated site (LT50 = 2.88 days) compared to the cockles from the intermediate site (LT50 = 5 days) and the less contaminated site (LT50 = 6 days). Results for metal bioaccumulation showed that the levels of Cd, Cu, Zn and Ni in cockles were consistent with the contaminant gradient, with the highest levels in cockles from the most contaminated site (1.04; 4.92; 52.76 and 13.81 µg/g dw, respectively), followed by those from the intermediate site (0.34; 2.94; 36.94; 17.40 µg/g dw, respectively) and then the less contaminated site (0.065; 1.27; 21.62 and 5.40 µg/g dw, respectively). Results from the gametogenesis and maturity index showed few differences in the reproductive cycle of cockles collected from the three study sites. There were different patterns of gene expression that were divided into three groups in terms of responses: (1) expression of genes involved in metal detoxification, ATP Binding Cassette Subfamily B Member 1 (ABCB1) and metallothionein MT) and genes for superoxide dismutases (i.e., Mn SOD and CuZn SOD), which did not show any difference in their levels of expression; (2) heat shock protein 70 (HSP70) gene expression, which decreased in cockles according to the pollution gradient, and (3) expression of catalase (CAT) and cytochrome oxidase subunit 1 (COI) genes was threefold and 1000-fold higher in cockles from intermediate and most contaminated sites compared to the less contaminated site. Therefore, changes in overall physiological condition, sex ratios and expression of HSP70, CAT and COI genes may be appropriate biomarkers for in situ studies of the impacts of metals in cockles. However, these biomarkers should be coupled to proteomics studies.

Comparative immune responses of blue mussel and zebra mussel haemocytes to simultaneous chemical and bacterial exposure.

Biomonitoring at the scale of the aquatic continuum and based on biomarkers, requires various representative species and a knowledge of their sensitivity to contaminants. Mussel immunomarkers are established tools for evaluating immunotoxic stress, but little is known about the consequences of an immune activation by local microorganisms on their response to pollution. This study aims to compare the sensitivity of cellular immunomarkers in two mussel species from different environments, the marine mussel Mytilus edulis (blue mussel) and the freshwater mussel Dreissena polymorpha (zebra mussel), to chemical stressors combined with bacterial challenge. Haemocytes were exposed ex vivo to the contaminants (bisphenol A, caffeine, copper chloride, oestradiol, ionomycin) for 4 h. The chemical exposures were coupled with simultaneous bacterial challenges (Vibrio splendidus and Pseudomonas fluorescens) to trigger activation of the immune response. Cellular mortality, phagocytosis efficiency and phagocytosis avidity were then measured by flow cytometry. The two mussel species had different basal levels since D. polymorpha showed higher cell mortality than M. edulis (23.9 ± 11% and 5.5 ± 3% dead cells respectively), and lower phagocytosis efficiency (52.6 ± 12% and 62.2 ± 9%), but similar phagocytosis avidity (17.4 ± 5 and 13.4 ± 4 internalised beads). Both bacterial strains led to an increase in cellular mortality (+8.4% dead cells in D. polymorpha, +4.9% in M. edulis), as well an activation of phagocytosis (+9.2% of efficient cells in D. polymorpha, +6.2% efficient cells and +3 internalised beads per cell in M. edulis). All chemicals triggered an increase in haemocyte mortality and/or phagocytotic modulations, except for bisphenol A. The two species differed in the amplitude of their response. The addition of a bacterial challenge significantly altered cell responses to chemicals with synergetic and antagonistic variations compared to a single exposure, depending on the compound used and the mussel species. This work highlights the species-specific sensitivity of mussel immunomarkers to contaminants, with or without bacterial challenge, and the necessity of considering the presence of in natura non-pathogenic microorganisms for future in situ applications of immunomarkers.

Time and dose-dependent impairment of liver metabolism in Gasterosteus aculeatus following exposure to diclofenac (DCF) highlighted by LC-HRMS untargeted metabolomics.

Anthropogenic chemicals as emerging contaminants, such as pharmaceuticals, increased worldwide in the environment. This study aimed to apply metabolomics-based approaches on the fish model species three-spined stickleback (Gasterosteus aculeatus) exposed to diclofenac (DCF) to identify toxicity pathways and potential biomarkers. For this purpose, males and females were exposed to a continuous flow of diclofenac solution in laboratory for 21 days, followed by 3 days of depuration, to nominal concentrations of 1 (low) and 100 µg/L (high) of DCF. A methodology based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) was employed. Uni- and multivariate statistical analyses were combined to evaluate the modulations of the liver metabolome of G. aculeatus after exposure to DCF. The metabolomics data revealed variations both as a function of time and of the DCF concentration. We observed 2487 altered metabolites, with 1460 and 1027 specific to males and females, respectively. Some of them were significantly impaired by the experimental conditions. However, we showed that several metabolites were impacted by other factors as they were already modulated in the control individuals. The results indicated that the energy metabolism was up-modulated in females and down-modulated in males, with the presence of DCF. The antioxidant system was impacted in males, suggesting oxidative stress in the metabolism, while the immunity system was down-regulated in females following exposure. Moreover, our results revealed 1 and 4 metabolites as potential metabolic biomarkers in male and female sticklebacks, respectively. Among them, the glutaryl-carnitine and the adipoyl-carnitine were putatively identified in females, known to be implicated in the energy metabolism. These 5 metabolites showed to be promising biomarkers since they were early modulated during exposure to the stress and showed a notable trend through time.

Evaluation of the accumulation of the iodinated contrast agents diatrizoic acid and iohexol in Dreissena polymorpha mollusks.

Mollusks are very sensitive to aquatic environmental alterations and then, are important bio-indicators for monitoring the contamination of water bodies. Iodinated X-ray contrast media (ICMs) are ubiquitously present in the aquatic environment, primarily due to their high consumption for diagnosis purposes, high injection levels, low biodegradability, and low removal rates by wastewater treatment plants. Although these compounds are assumed to be of low toxicity, aquatic organisms are continuously exposed to these agents, which may result in adverse effects as ICMs can act as iodine source and disrupt the endocrine system. Thus, the evaluation of their environmental risk, especially on aquatic fauna is of great interest. To this end, we first compared the accumulation behavior, based on iodine analysis, of two ICM exhibiting different osmolality, diatrizoic acid and iohexol in Dreissena polymorpha bivalves exposed under laboratory conditions at concentrations of 0, 100, and 1000 µg/L during 4 and 7 days. This study was the first to provide information on iodine concentration in whole soft tissues and several organs in control zebra mussels. Moreover, it showed, after exposure, an increase of iodine content mainly in the digestive glands, followed by gills and gonads, highlighting that ICMs actually enter the organisms. Thus, bioaccumulation of ICMs studies were then performed, by liquid chromatography coupled to tandem mass spectrometry, on entire mollusks and digestive glands of organisms exposed at 0, 10, 100, and 1000 µg/L of both ICMs during 21 days, followed by 4 days of depuration. These first data on ICMs concentrations in zebra mussels, showed a clear accumulation of ICMs in mussels as a function of relative exposure level, as well as a rapid depuration. Osmolality did not seem to have a significant impact on the accumulation level, but a slight difference was observed on the accumulation pattern between both ICMs.

Integration of Genotoxic Biomarkers in Environmental Biomonitoring Analysis Using a Multi-Biomarker Approach in Three-Spined Stickleback (Gasterosteus aculeatus Linnaeus, 1758).

Water is impacted by a variety of increasing pressures, such as contaminants, including genotoxic pollutants. The proposed multi-biomarker approach at a sub-individual level gives a complementary indicator to the chemical and ecological parameters of the Water Framework Directive (WFD, 2000/60/EC). By integrating biomarkers of genotoxicity and erythrocyte necrosis in the sentinel fish species the three-spined stickleback (Gasterosteus aculeatus) through active biomonitoring of six stations of the Artois-Picardie watershed, north France, our work aimed to improve the already existing biomarker approach. Even if fish in all stations had high levels of DNA strand breaks, the multivariate analysis (PCA), followed by hierarchical agglomerative clustering (HAC), improved discrimination among stations by detecting an increase of nuclear DNA content variation (Etaing, St Rémy du Nord, Artres and Biache-St-Vaast) and erythrocyte necrosis (Etaing, St Rémy du Nord). The present work highlighted that the integration of these biomarkers of genotoxicity in a multi-biomarker approach is appropriate to expand physiological parameters which allow the targeting of new potential effects of contaminants.

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.

First evidence of SARS-CoV-2 genome detection in zebra mussel (Dreissena polymorpha).

The uses of bivalve molluscs in environmental biomonitoring have recently gained momentum due to their ability to indicate and concentrate human pathogenic microorganisms. In the context of the health crisis caused by the COVID-19 epidemic, the objective of this study was to determine if the SARS-CoV-2 ribonucleic acid genome can be detected in zebra mussels (Dreissena polymorpha) exposed to raw and treated urban wastewaters from two separate plants to support its interest as bioindicator of the SARS-CoV-2 genome contamination in water. The zebra mussels were exposed to treated wastewater through caging at the outlet of two plants located in France, as well as to raw wastewater in controlled conditions. Within their digestive tissues, our results showed that SARS-CoV-2 genome was detected in zebra mussels, whether in raw and treated wastewaters. Moreover, the detection of the SARS-CoV-2 genome in such bivalve molluscans appeared even with low concentrations in raw wastewaters. This is the first detection of the SARS-CoV-2 genome in the tissues of a sentinel species exposed to raw and treated urban wastewaters. Despite the need for development for quantitative approaches, these results support the importance of such invertebrate organisms, especially zebra mussel, for the active surveillance of pathogenic microorganisms and their indicators in environmental waters.

Blue mussel (Mytilus edulis)-A bioindicator of marine water contamination by protozoa: Laboratory and in situ approaches.

AIMS: The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii are identified as public health priorities and are present in a wide variety of environments including the marine ecosystem. The objective of this study was to demonstrate that the marine bivalve blue mussel (Mytilus edulis) can be used as a tool to monitor the contamination of marine waters by the three protozoa over time. METHODS AND RESULTS: In order to achieve a proof of concept, mussels were exposed to three concentrations of G. duodenalis cysts and Cryptosporidium parvum/T. gondii oocysts for 21 days, followed by 21 days of depuration in clear water. Then, natural contamination by these protozoa was sought for in wild marine blue mussels along the northwest coast of France to validate their relevance as bioindicators in the field. Our results highlighted that: (a) blue mussels bioaccumulated the parasites for 21 days, according to the conditions of exposure, and parasites could still be detected during the depuration period (until 21 days); (b) the percentage of protozoa-positive M. edulis varied under the degree of protozoan contamination in water; (c) mussel samples from eight out of nine in situ sites were positive for at least one of the protozoa. CONCLUSIONS: The blue mussel M. edulis can bioaccumulate protozoan parasites over long time periods, according to the degree of contamination of waters they are inhabiting, and can highlight recent but also past contaminations (at least 21 days). SIGNIFICANCE AND IMPACT OF THE STUDY: Mytilus edulis is a relevant bioaccumulators of protozoan (oo)cysts in laboratory and field conditions, hence its potential use for monitoring parasite contamination in marine waters.

Evaluation of real-time qPCR-based methods to detect the DNA of the three protozoan parasites Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii in the tissue and hemolymph of blue mussels (M. edulis).

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4-400 parasites/g, DNA-RR: 19-80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10-1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.

An optimized LC-HRMS untargeted metabolomics workflow for multi-matrices investigations in the three-spined stickleback.

Environmental metabolomics has become a growing research field to understand biological and biochemical perturbations of organisms in response to various abiotic or biotic stresses. It focuses on the comprehensive and systematic analysis of a biologic system's metabolome. This allows the recognition of biochemical pathways impacted by a stressor, and the identification of some metabolites as biomarkers of potential perturbations occurring in a body. In this work, we describe the development and optimization of a complete reliable methodology based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) for untargeted metabolomics studies within a fish model species, the three-spined stickleback (Gasterosteus aculeatus). We evaluated the differences and also the complementarities between four different matrices (brain, gills, liver and whole fish) to obtain metabolome information. To this end, we optimized and compared sample preparation and the analytical method, since the type and number of metabolites detected in any matrix are closely related to these latter. For the sample preparation, a solid-liquid extraction was performed on a low quantity of whole fish, liver, brain, or gills tissues using combinations of methanol/water/heptane. Based on the numbers of features observed in LC-HRMS and on the responses of analytical standards representative of different metabolites groups (amino acids, sugars…), we discuss the influence of the nature, volume, and ratio of extraction solvents, the sample weight, and the reconstitution solvent. Moreover, the analytical conditions (LC columns, pH and additive of mobile phases and ionization modes) were also optimized so as to ensure the maximum metabolome coverages. Thus, two complementary chromatographic procedures were combined in order to cover a broader range of metabolites: a reversed phase separation (RPLC) on a C18 column followed by detection with positive ionization mode (ESI+) and a hydrophilic interaction chromatography (HILIC) on a zwitterionic column followed by detection with negative ionization mode (ESI-). This work provides information on brain, gills, liver, vs the whole body contribution to the stickleback metabolome. These information would help to guide ecotoxicological and biomonitoring studies.

Toxic effects of a mixture of five pharmaceutical drugs assessed using Fontinalis antipyretica Hedw.

The potential health risks associated with the pharmaceuticals released into the environment through effluents from sewage treatment plants have become a major cause for concern. Owing to the lack of effective indicators, monitoring the concentration of these pollutants in the aquatic environment is challenging. The aim of this study was to assess the toxicity of a mixture of five pharmaceutical drugs (paracetamol, carbamazepine, diclofenac, irbesartan, and naproxen) using the aquatic moss Fontinalis antipyretica as a bioindicator and bioaccumulator. We examined the effects of the drug mixture on the cellular antioxidant system, chlorophyll content, and morphological traits of F. antipyretica. The plant was exposed for 5 months to three concentrations of the mixture, including the environmental concentration (MX1), and 10- (MX10) and 100-times (MX100) the environmental concentration. The results showed that only carbamazepine and irbesartan were accumulated by the species. The bioconcentration level increased with exposure time, with the maximum uptake at the 4th month of exposure. The increase in bioaccumulation with exposure time was more evident in plants exposed to MX100. Analysis of the activity of antioxidant enzymes showed that superoxide dismutase (SOD, EC 1.15.1.1.) and catalase (EC 1.11.1.6.) were highly sensitive to the drug mixture. The activity of the enzymes was significantly higher in plants exposed to MX100; however, the activity of guaiacol peroxidase (GPX, EC 1.11.1.7.) was not significantly affected. Plants exposed to MX10 and MX100 had significantly lower total chlorophyll content and chlorophyll a/b ratio compared with those of plants in the control group; however, photosynthetic activity was restored after 5 months of exposure. The morphological characteristics of F. antipyretica were less sensitive to the treatment conditions.