Assessment of the viral contamination of fecal origin over a wide geographical area using an active approach with Dreissena polymorpha.

Biomonitoring appears to be a key approach to assess chemical or microbiological contaminations. The freshwater mussel, Dreissena polymorpha (D. polymorpha), is a suitable tool already used to monitor chemical and, more recently, microbiological pollution. In the present study, we used this sentinel species to monitor viral contamination of fecal origin over a wide geographical distribution. An active approach was implemented based on caging of calibrated and pathogen-free organisms with the same exposure conditions, allowing spatio-temporal comparisons between different water bodies. In addition, different types of sites were selected to investigate the range of environmental concentrations that D. polymorpha are able to translate. Different viral genome targets were measured: norovirus genogroup I and II (NoV GI and GII) and F-specific RNA bacteriophages belonging to the genogroup -I and -II (FRNAPH-I and -II). Total infectious FRNAPH were also monitored. D. polymorpha was able to translate a wide range of concentrations for all the viral targets studied, meaning that this sentinel species can be used for both low and highly anthropised sites. Moreover, D. polymorpha caging proved effective in achieving gradients of viral contamination of fecal origin pressure and to highlight the contribution of tributaries to the main rivers. D. polymorpha provided spatial and temporal variations of the viral contamination. It allowed to highlight the prevalence of the FRNAPH-I and -II genogroups according to the caging site. FRNAPH-II was found to be dominant in urban areas and FRNAPH-I in rural areas. This strategy uses the caging of the sentinel species D. polymorpha on selected sites with standardised analysis methods has proven to be a promising tool for characterizing viral contamination at both large and very fine scales.

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.

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.

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.

Thirtieth Anniversary of the Discovery of Laxaphycins. Intriguing Peptides Keeping a Part of Their Mystery.

Lipopeptides are a class of compounds generally produced by microorganisms through hybrid biosynthetic pathways involving non-ribosomal peptide synthase and a polyketyl synthase. Cyanobacterial-produced laxaphycins are examples of this family of compounds that have expanded over the past three decades. These compounds benefit from technological advances helping in their synthesis and characterization, as well as in deciphering their biosynthesis. The present article attempts to summarize most of the articles that have been published on laxaphycins. The current knowledge on the ecological role of these complex sets of compounds will also be examined.

Biological Activities of Cyclic and Acyclic B-Type Laxaphycins in SH-SY5Y Human Neuroblastoma Cells.

Laxaphycins are a family of non-ribosomal lipopeptides that have been isolated from several cyanobacteria. Some of these compounds have presented cytotoxic activities, but their mechanism of action is poorly understood. In this work, the already described laxaphycins B and B3, and acyclolaxaphycins B and B3 were isolated from the marine cyanobacteria Anabaena torulosa. Moreover, two new acyclic compounds, [des-(Ala4-Hle5)] acyclolaxaphycins B and B3, were purified from the herviborous gastropod Stylocheilus striatus, with this being the first description of biotransformed laxaphycins. The structure of these new compounds was elucidated, together with the absolute configuration of acyclolaxaphycins B and B3. The bioactivities of the six peptides were determined in SH-SY5Y human neuroblastoma cells. Laxaphycins B and B3 were cytotoxic (IC50: 1.8 and 0.8 µM, respectively) through the induction of apoptosis. In comparison, acyclic laxaphycins did not show cytotoxicity but affected mitochondrial functioning, so their effect on autophagy-related protein expression was analyzed, finding that acyclic peptides affected this process by increasing AMPK phosphorylation and inhibiting mTOR. This work confirms the pro-apoptotic properties of cyclic laxaphycins B and is the first report indicating the effects on autophagy of their acyclic analogs. Moreover, gastropod-derived compounds presented ring opening and amino-acids deletion, a biotransformation that had not been previously described.

Structure and biological evaluation of new cyclic and acyclic laxaphycin-A type peptides.

Five new laxaphycins were isolated and fully characterised from the bloom forming cyanobacteria Anabaena torulosa sampled from Moorea, French Polynesia: three acyclic laxaphycin A-type peptides, acyclolaxaphycin A (1), [des-Gly11]acyclolaxaphycin A (2) and [des-(Leu10-Gly11)]acyclolaxaphycin A (3), as well as two cyclic ones, [l-Val8]laxaphycin A (4) and [d-Val9]laxaphycin A (5). The absolute configuration of the amino acids, established using advanced Marfey's analysis for compounds 2-5, highlights a conserved stereochemistry at the Cα carbons of the peptide ring that is characteristic of this family. To the best of our knowledge, this is the first report of acyclic analogues within the laxaphycin A-type peptides. Whether these linear laxaphycins with the aliphatic ß-amino acid on the N-terminal are biosynthetic precursors or compounds obtained after enzymatic hydrolysis of the macrocycle is discussed. Biological evaluation of the new compounds together with the already known laxaphycin A shows that [l-Val8]laxaphycin A, [d-Val9]laxaphycin A and [des-Gly11]acyclolaxaphycin induce cellular toxicity whereas laxaphycin A and des-[(Leu10-Gly11)]acyclolaxaphycin A do not affect the cellular viability. An analysis of cellular death shows that the active peptides do not induce apoptosis or necrosis but instead, involve the autophagy pathway.

Structures and Activities of Tiahuramides A-C, Cyclic Depsipeptides from a Tahitian Collection of the Marine Cyanobacterium Lyngbya majuscula.

The structures of three new cyclic depsipeptides, tiahuramides A (1), B (2), and C (3), from a French Polynesian collection of the marine cyanobacterium Lyngbya majuscula are described. The planar structures of these compounds were established by a combination of mass spectrometry and 1D and 2D NMR experiments. Absolute configurations of natural and nonproteinogenic amino acids were determined through a combination of acid hydrolysis, derivitization with Marfey's reagent, and HPLC. The absolute configuration of hydroxy acids was confirmed by Mosher's method. The antibacterial activities of tiahuramides against three marine bacteria were evaluated. Compound 3 was the most active compound of the series, with an MIC of 6.7 µM on one of the three tested bacteria. The three peptides inhibit the first cell division of sea urchin fertilized eggs with IC50 values in the range from 3.9 to 11 µM. Tiahuramide B (2), the most potent compound, causes cellular alteration characteristics of apoptotic cells, blebbing, DNA condensation, and fragmentation, already at the first egg cleavage. The cytotoxic activity of compounds 1-3 was tested in SH-SY5Y human neuroblastoma cells. Compounds 2 and 3 showed an IC50 of 14 and 6.0 µM, respectively, whereas compound 1 displayed no toxicity in this cell line at 100 µM. To determine the type of cell death induced by tiahuramide C (3), SH-SY5Y cells were costained with annexin V-FITC and propidium iodide and analyzed by flow cytometry. The double staining indicated that the cytotoxicity of compound 3 in this cell line is produced by necrosis.

Bioaccumulation of Toxoplasma and Cryptosporidium by the freshwater crustacean Gammarus fossarum: Involvement in biomonitoring surveys and trophic transfer.

The protozoa Toxoplasma gondii and Cryptosporidium parvum are public health priorities because their oocysts can persist in recreational, surface, drinking, river, and sea water sources for a long time. To evaluate the capacity of the freshwater crustacean Gammarus fossarum to accumulate T. gondii and C. parvum oocysts, gammarids were exposed to 200, 2000 or 20,000 oocysts per gammarid and per day for 21 days followed by 5 days of depuration. C. parvum DNA was detected by qPCR in G. fossarum in only one out of four pools for the highest concentration and after 14 days of exposure, and T. gondii DNA was detected after 7 days of exposure to the two highest concentrations. Our results document the capacity of G. fossarum to accumulate T. gondii in its tissues proportionally to the ambient concentration; the maximum number of oocysts was detected in gammarid tissues after exposure to 20,000 oocysts per day. Mean values of 3.26 (±3), 21.71 (±15.18), and 17.41 (±10.89) oocysts were detected in gammarids after 7, 14, and 21 days, respectively, and after 5 days of depuration, T. gondii oocysts were still present in gammarid tissues. These results show for the first time that a freshwater crustacean can bioaccumulate T. gondii oocysts, suggesting that G. fossarum is a potential effective bioindicator of protozoan contamination in biomonitoring studies. Moreover, due to its key position in freshwater food webs, G. fossarum could also play a role in the trophic transfer of protozoa.

Isolation and Synthesis of Laxaphycin B-Type Peptides: A Case Study and Clues to Their Biosynthesis.

The laxaphyci's B family constitutes a group of five related cyclic lipopeptides isolated from diverse cyanobacteria from all around the world. This group shares a typical structure of 12 amino acids from the l and d series, some of them hydroxylated at the beta position, and all containing a rare beta-amino decanoic acid. Nevertheless, they can be differentiated due to slight variations in the composition of their amino acids, but the configuration of their alpha carbon remains conserved. Here, we provide the synthesis and characterization of new laxaphycin B-type peptides. In doing so we discuss how the synthesis of laxaphycin B and analogues was developed. We also isolate minor acyclic laxaphycins B, which are considered clues to their biosynthesis.

Benthic cyanobacterial metabolites interact to reduce coral larval survival and settlement.

Benthic cyanobacterial mats (BCMs) are becoming increasingly abundant on coral reefs worldwide. High growth rates and prolific toxin production give them the potential to cause widespread coral recruitment failure through allelopathic effects, but few studies have made the link between their toxicity for coral larvae and in situ toxin concentrations. Here we investigated the allelopathic effects of the benthic cyanobacterium Anabaena sp.1 on larvae of the coral Pocillopora acuta. This cyanobacterium produces several non-ribosomal cyclic lipopeptides of the laxaphycin family with cytotoxic properties. Therefore, we measured the concentration of laxaphycins A and B in Anabaena mats and in the water column and tested their effects on coral larvae. We found that Anabaena crude extract reduces both larval survivorship and settlement and that laxaphycin B reduces settlement. When larvae were exposed to both laxaphycins, there was a reduction in both larval survival and settlement. In the natural reef environment, laxaphycin A and B concentrations increased with increasing proximity to Anabaena mats, with concentrations being consistently above LC50 and EC50 thresholds within a 1 cm distance of the mats. This study demonstrates that laxaphycins reduce the survival and inhibit the settlement of coral larvae at concentrations found near Anabaena mats in situ. It further shows a combined effect between two cyanobacterial metabolites. As BCMs become more common, more of their secondary metabolites might be released in the water column. Their occurrence will lead to a reduction in coral recruitment rates, contributing to the continuing decline of coral reefs and shift in community structure.

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.

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.

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.

Bioaccumulation and molecular effects of carbamazepine and methylmercury co-exposure in males of Dreissena polymorpha.

Dreissena polymorpha is a bivalve promising for biomonitoring in freshwater ecosystems thanks to its abundance and high filtration activity allowing rapid uptake of toxicants and identification of their negative effects. Nonetheless, we still lack knowledge on its molecular responses to stress under realistic scenario, e.g. multi-contamination. Carbamazepine (CBZ) and Hg are ubiquitous pollutants sharing molecular toxicity pathways, e.g. oxidative stress. A previous study in zebra mussels showed their co-exposure to cause more alterations than single exposures, but molecular toxicity pathways remained unidentified. D. polymorpha was exposed 24 h (T24) and 72 h (T72) to CBZ (6.1 ± 0.1 µg L-1), MeHg (430 ± 10 ng L-1) and the co-exposure (6.1 ± 0.1 µg L-1CBZ and 500 ± 10 ng L-1 MeHg) at concentrations representative of polluted areas (~10× EQS). RedOx system at the gene and enzyme level, the proteome and the metabolome were compared. The co-exposure resulted in 108 differential abundant proteins (DAPs), as well as 9 and 10 modulated metabolites at T24 and T72, respectively. The co-exposure specifically modulated DAPs and metabolites involved in neurotransmission, e.g. dopaminergic synapse and GABA. CBZ specifically modulated 46 DAPs involved in calcium signaling pathways and 7 amino acids at T24. MeHg specifically modulated 55 DAPs involved in the cytoskeleton remodeling and hypoxia-induced factor 1 pathway, without altering the metabolome. Single and co-exposures commonly modulated proteins and metabolites involved in energy and amino acid metabolisms, response to stress and development. Concomitantly, lipid peroxidation and antioxidant activities were unchanged, supporting that D. polymorpha tolerated experimental conditions. The co-exposure was confirmed to cause more alterations than single exposures. This was attributed to the combined toxicity of CBZ and MeHg. Altogether, this study underlined the necessity to better characterize molecular toxicity pathways of multi-contamination that are not predictable on responses to single exposures, to better anticipate adverse effects in biota and improve risk assessment.

High-dose erythropoietin has no effect on short- or long-term graft function following deceased donor kidney transplantation.

We evaluated short- and long-term effects of high-dose recombinant human erythropoietin (rHuEPO) in kidney transplantation in a prospective double-blind, placebo-controlled study. Patients with chronic kidney disease following receipt of a deceased donor kidney allograft were randomized to 3 doses of 40,000 units rHuEPO or placebo. The primary study end point was kidney function 6 weeks after transplantation with secondary end points of incidence of delayed graft function and kidney function 12 months after transplantation. Six weeks or 12 months after transplantation, the difference between estimated glomerular filtration rates was not significant comparing 44 patients who received rHuEPO to 44 patients receiving placebo. There was no significant difference regarding the incidence of delayed graft function (10 of 44 with rHuEPO compared with 14 of 44 on placebo). Protocol biopsies at 6 weeks and 6 months post transplant showed no significant differences in all assessed histological indices. The number and severity of adverse events were comparable between groups, as was patient and graft survival after 12 months. Thus, treatment with high-dose rHuEPO after kidney transplantation, although well tolerated, had no effect on long-term graft function or histology.

Integrative multi-biomarker approach on caged rainbow trout: A biomonitoring tool for wastewater treatment plant effluents toxicity assessment.

The complex mixtures of contaminants released in wastewater treatment plant (WWTP) effluents are a major source of pollution for aquatic ecosystems. The present work aimed to assess the environmental risk posed by WWTP effluents by applying a multi-biomarker approach on caged rainbow trout (Oncorhynchus mykiss) juveniles. Fish were caged upstream and downstream of a WWTP for 21 days. To evaluate fish health, biomarkers representing immune, reproductive, nervous, detoxification, and antioxidant functions were assayed. Biomarker responses were then synthesized using an Integrated Biomarker Response (IBR) index. The IBR highlighted similar response patterns for the upstream and downstream sites. Caged juvenile females showed increased activities of innate immune parameters (lysozyme and complement), histological lesions and reduced glycogen content in the hepatic tissue, and higher muscle cholinergic metabolism. However, the intensity of the observed effects was more severe downstream of the WWTP. The present results suggest that the constitutive pollution level of the Meuse River measured upstream from the studied WWTP can have deleterious effects on fish health condition, which are exacerbated by the exposure to WWTP effluents. Our results infer that the application of IBR index is a promising tool to apply with active biomonitoring approaches as it provides comprehensive information about the biological effects caused by point source pollution such as WWTP, but also by the constitutive pollutions levels encountered in the receiving environment.

Metabolic, cellular and defense responses to single and co-exposure to carbamazepine and methylmercury in Dreissena polymorpha.

Carbamazepine (CBZ) and Hg are widespread and persistent micropollutants in aquatic environments. Both pollutants are known to trigger similar toxicity mechanisms, e.g. reactive oxygen species (ROS) production. Here, their effects were assessed in the zebra mussel Dreissena polymorpha, frequently used as a freshwater model in ecotoxicology and biomonitoring. Single and co-exposures to CBZ (3.9 µg L-1) and MeHg (280 ng L-1) were performed for 1 and 7 days. Metabolomics analyses evidenced that the co-exposure was the most disturbing after 7 days, reducing the amount of 25 metabolites involved in protein synthesis, energy metabolism, antioxidant response and osmoregulation, and significantly altering cells and organelles' structure supporting a reduction of functions of gills and digestive glands. CBZ alone after 7 days decreased the amount of α-aminobutyric acid and had a moderate effect on the structure of mitochondria in digestive glands. MeHg alone had no effect on mussels' metabolome, but caused a significant alteration of cells and organelles' structure in gills and digestive glands. Single exposures and the co-exposure increased antioxidant responses vs control in gills and digestive glands, without resulting in lipid peroxidation, suggesting an increased ROS production caused by both pollutants. Data globally supported that a higher number of hyperactive cells compensated cellular alterations in the digestive gland of mussels exposed to CBZ or MeHg alone, while CBZ + MeHg co-exposure overwhelmed this compensation after 7 days. Those effects were unpredictable based on cellular responses to CBZ and MeHg alone, highlighting the need to consider molecular toxicity pathways for a better anticipation of effects of pollutants in biota in complex environmental conditions.

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.

Effect of reproduction cycle stage on energy metabolism responses in a sentinel species (Dreissena polymorpha) exposed to cadmium: What consequences for biomonitoring?

Metal trace elements such as cadmium (Cd) are commonly present in ecosystems and could lead to impairment of mitochondrial functions and energy imbalance in aquatic organisms including molluscs. Combined exposure to increasing temperatures and Cd could enhance such an impact on animals. Seasonal fluctuations, such as temperature, and the corresponding reproduction cycle can affect biomarker responses. However, the reproduction cycle stage is rarely taken into account in ecotoxicological studies. Thus, this work aimed at understanding energy metabolism responses in a sentinel species, Dreissena polymorpha. Mussels were collected during the rest and the reproduction periods and were exposed to 10 µg.L-1 of cadmium (Cd) at two temperatures (in situ temperature and in situ temperature + 5°C) during 7 days. Energy metabolism was monitored by measuring reserves and energy nucleotides charge and by assessing aerobic and anaerobic metabolism markers, and upstream regulation pathways. Markers related to OXPHOS activity revealed seasonal variations under laboratory conditions. Conversely, adenylate nucleotides, glycogen, lipid and transcript levels of AMP-activated protein kinase, citrate synthase, ATP synthase and cytochrome b encoding genes remained steady after the acclimation period. No evident effect of Cd on energy metabolism markers was noticed for both exposures although the transcript level of succinate dehydrogenase and citrate synthase encoding genes decreased with Cd during the rest period. Cellular stress, revealed by lipid peroxidation and catalase mRNA levels, only occurred in Cd and warming co-exposed mussels during the reproduction period. These results suggest that contaminant impact might differ according to the reproduction cycle stage. The effect of confounding factors on biomarker variations should be further investigated to have a deeper knowledge of metabolism responses under laboratory conditions.