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International audience; Preservation of biodiversity and ecosystem services is critical for sustainable development and human well-being. However, an unprecedented erosion of biodiversity is observed and the use of plant protection products (PPP) has been identified as one of its main causes. In this context, at the request of the French Ministries responsible for the Environment, for Agriculture and for Research, a panel of 46 scientific experts ran a nearly 2-year-long (2020-2022) collective scientific assessment (CSA) of international scientific knowledge relating to the impacts of PPP on biodiversity and ecosystem services. The scope of this CSA covered the terrestrial, atmospheric, freshwater, and marine environments (with the exception of groundwater) in their continuity from the site of PPP application to the ocean, in France and French overseas territories, based on international knowledge produced on or transposable to this type of context (climate, PPP used, biodiversity present, etc.). Here, we provide a brief summary of the CSA's main conclusions, which were drawn from about 4500 international publications. Our analysis finds that PPP contaminate all environmental matrices, including biota, and cause direct and indirect ecotoxicological effects that unequivocally contribute to the decline of certain biological groups and alter certain ecosystem functions and services. Levers for action to limit PPP-driven pollution and effects on environmental compartments include local measures from plot to landscape scales and regulatory improvements. However, there are still significant gaps in knowledge regarding environmental contamination by PPPs and its effect on biodiversity and ecosystem functions and services. Perspectives and research needs are proposed to address these gaps.

International audience; Growing evidence shows that endocrine disruptors (EDs), known to affect the reproductive system, may also disturb other hormone-regulated functions leading to cancers, neurodevelopmental defects, metabolic and immune diseases. To reduce exposure to EDs and limit their health effects, development of screening and mechanism-based assays to identify EDs is encouraged. Nevertheless, the crucial validation step of test methods by regulatory bodies is a time- and resource-consuming process. One of the main raisons of this long duration process is that method developers, mainly researchers, are not fully aware of the regulatory needs to validate a test. We propose an online self-assessment questionnaire (SAQ) called ReadEDTest easy to be used by all researchers. The aim of ReadEDTest is to speed up the validation process by assessing readiness criteria of in vitro and fish embryo ED test methods under development. The SAQ is divided into 7 sections and 13 sub-sections containing essential information requested by the validating bodies. The readiness of the tests can be assessed by specific score limits for each sub-section. Results are displayed via a graphical representation to help identification of the sub-sections having sufficient or insufficient information. The relevance of the proposed innovative tool was supported using two test methods already validated by the OECD and four under development test methods.

Computational analysis of bio-images by deep learning (DL) algorithms has made exceptional progress in recent years and has become much more accessible to non-specialists with the development of ready-to-use tools. The study of oogenesis mechanisms and female reproductive success has also recently benefited from the development of efficient protocols for three-dimensional (3D) imaging of ovaries. Such datasets have a great potential for generating new quantitative data but are, however, complex to analyze due to the lack of efficient workflows for 3D image analysis. Here, we have integrated two existing open-source DL tools, Noise2Void and Cellpose, into an analysis pipeline dedicated to 3D follicular content analysis, which is available on Fiji. Our pipeline was developed on larvae and adult medaka ovaries but was also successfully applied to different types of ovaries (trout, zebrafish and mouse). Image enhancement, Cellpose segmentation and post-processing of labels enabled automatic and accurate quantification of these 3D images, which exhibited irregular fluorescent staining, low autofluorescence signal or heterogeneous follicles sizes. In the future, this pipeline will be useful for extensive cellular phenotyping in fish or mammals for developmental or toxicology studies.

International audience; The prevalence of metabolic diseases, such as obesity, diabetes, metabolic syndrome and chronic liver diseases among others, has been rising for several years. Epidemiology and mechanistic (in vivo, in vitro and in silico) toxicology have recently provided compelling evidence implicating the chemical environment in the pathogenesis of these diseases. In this review, we will describe the biological processes that contribute to the development of metabolic diseases targeted by metabolic disruptors, and will propose an integrated pathophysiological vision of their effects on several organs. With regard to these pathomechanisms, we will discuss the needs, and the stakes of evolving the testing and assessment of endocrine disruptors to improve the prevention and management of metabolic diseases that have become a global epidemic since the end of last century.

International audience; Regulatory assessment of the effects of chemicals requires the availability of validated tests representing different environments and organisms. In this context, developing new tests is particularly needed for marine species from temperate environments. It is also important to evaluate effects that are generally poorly characterized and seldom included in regulatory tests. In this study, we designed an exposure protocol using European sea bass (Dicentrarchus labrax) larvae. We examined classical toxicological values (LCx) as well as behavioral responses. By comparing different hatching and breeding strategies, we defined the optimal conditions of exposure as non-agitated conditions in 24- or 48-well microplates. Our exposure protocol was then tested with 3,4-dichloroaniline (3,4-DCA), a recommended reference molecule. Based on our results, the 96 h LC50 for 3,4-DCA corresponded to 2.04 mg/L while the 168 h LC50 to 0.79 mg/L. Behavioral analyses showed no effect of 3,4-DCA at low concentration (0.25 mg/L). In conclusion, the present work established the basis for a new test which includes behavioral analysis and shows that the use of sea bass is suitable to early-life stage toxicity tests.

International audience; 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.

Several studies reported changes in spontaneous electroencephalogram alpha band activity related to radiofrequency electromagnetic fields, but findings showed both an increase and a decrease of its spectral power or no effect. Here, we studied the alpha band modulation after 900 MHz mobile phone radiofrequency exposure and localized cortical regions involved in these changes, via a magnetoencephalography (MEG) protocol with healthy volunteers in a double-blind, randomized, counterbalanced crossover design. MEG was recorded during eyes open and eyes closed resting-state before and after radiofrequency exposure. Potential confounding factors, known to affect alpha band activity, were assessed as control parameters to limit bias. Entire alpha band, lower and upper alpha sub-bands MEG power spectral densities were estimated in sensor and source space. Biochemistry assays for salivary biomarkers of stress (cortisol, chromogranin-A, alpha amylase), heart rate variability analysis and high-performance liquid chromatography for salivary caffeine concentration were realized. Results in sensor and source space showed a significant modulation of MEG alpha band activity after the radiofrequency exposure, with different involved cortical regions in relation to the eyes condition, probably because of different attention level with open or closed eyes. None of the control parameters reported a statistically significant difference between experimental sessions.

International audience; 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.

International audience; Objective: Solid composite propellants combustion, in aerospace and defense fields, can lead to complex aerosols emission containing high concentrations of alumina nanoparticles (Al2O3 NPs) and hydrogen chloride gas (HClg). Exposure to these mixtures by inhalation is thus possible but literature data toward their pulmonary toxicity are missing. To specify hazards resulting from these combustion aerosols, a pilot study was implemented.Materials and methods: Male Wistar rats were nose-only exposed to Al2O3 NPs (primary size 13 nm, 10 g/L suspension leading to 20.0–22.1 mg/m3 aerosol) and/or to HClg aerosols (5 ppm target concentration) following two exposure scenarios (single exposures (SE) or repeated exposures (RE)). Bronchoalveolar lavage fluids (BALF) content and lungs histopathology were analyzed 24 h after exposures.Results: Repeated co-exposures increased total proteins and LDH concentrations in BALF indicating alveolar–capillary barrier permeabilization and cytolysis. Early pulmonary inflammation was induced after RE to Al2O3 NPs ± HClg resulting in PMN, TNF-α, IL-1β, and GRO/KC increases in BALF. Both exposure scenarios resulted in pulmonary histopathological lesions (vascular congestions, bronchial pre-exfoliations, vascular and interalveolar septum edemas). Lung oxidative damages were observed in situ following SE.Conclusion:Observed biological effects are dependent on both aerosol content and exposure scenario. Results showed an important pro-inflammatory effect of Al2O3 NPs/HClg mixtures on the lungs of rat 24 h after exposure. This pilot study raises concerns toward potential long-term pulmonary toxicity of combustion aerosols and highlights the importance for further studies to be led in order to define dose limitations and exposure thresholds for risk management at the work place.

International audience; Pharmaceuticals are emerging pollutants of concern for aquatic ecosystems where they are occurring in complex mixtures. In the present study, the chronic toxicity of an environmentally relevant pharmaceutical mixture on juvenile rainbow trout (Oncorhynchus mykiss) was investigated. Five pharmaceuticals (paracetamol, carbamazepine, diclofenac, naproxen and irbesartan) were selected based on their detection frequency and concentration levels in the Meuse river (Belgium). Fish were exposed for 42 days to three different concentrations of the mixture, the median one detected in the Meuse river, 10-times and 100-times this concentration. Effects on the nervous, immune, antioxidant, and detoxification systems were evaluated throughout the exposure period and their response standardized using the Integrated Biomarker Response (IBRv2) index. IBRv2 scores increased over time in the fish exposed to the highest concentration. After 42 days, fish exposed to the highest concentration displayed significantly higher levels in lysozyme activity (p < 0.01). The mixture also caused significant changes in brain serotonin turnover (p < 0.05). In short, our results indicate that the subchronic waterborne exposure to a pharmaceutical mixture commonly occurring in freshwater ecosystems may affect the neuroendocrine and immune systems of juvenile rainbow trout.

Contamination of the marine environment by antidepressants may affect neurophysiological processes in nontarget organisms, such as the common cuttlefish, Sepia officinalis. The present study tested whether environmentally realistic concentrations of antidepressants, that is, fluoxetine alone (5 ng L-1 ) or cumulated with venlafaxine (2.5 or 5 ng L-1 ), affect camouflage in newly hatched cuttlefish. The results show that antidepressants improved uniform body patterns, whereas disruptive body patterns were not affected. Environ Toxicol Chem 2021;40:2571-2577. © 2021 SETAC.

International audience; Before their placing on the market, the safety of plant protection products (PPP) towards both human and animal health, and the environment has to be assessed using experimental and modelling approaches. Models are crucial tools for PPP risk assessment and some even help to avoid animal testing. This review investigated the use of modelling approaches in the ecotoxicology section of PPP active substance assessment reports prepared by the authorities and opened to consultation from 2011 to 2021 in the European Union. Seven categories of models (Structure-Activity, ToxicoKinetic, ToxicoKinetic-ToxicoDynamic, Species Sensitivity Distribution, population, community, and mixture) were searched for into the reports of 317 active substances. At least one model category was found for 44 % of the investigated active substances. The most detected models were Species Sensitivity Distribution, Structure-Activity and ToxicoKinetic for 27, 21 and 15 % of the active substances, respectively. The use of modelling was of particular importance for conventional active substances such as sulfonylurea or carbamates contrary to microorganisms and plant derived substances. This review also highlighted a strong imbalance in model usage among the biological groups considered in the European Regulation (EC) No 1107/2009. For example, models were more often used for aquatic than for terrestrial organisms (e.g., birds, mammals). Finally, a gap between the set of models used in reports and those existing in the literature was observed highlighting the need for the implementation of more sophisticated models into PPP regulation.

International audience; Cyanobacteria are a potential threat to aquatic ecosystems and human health because of their ability to produce cyanotoxins, such as microcystins (MCs). MCs are regularly monitored in fresh waters, but rarely in estuarine and marine waters despite the possibility of their downstream export. Over a period of two years, we monthly analyzed intracellular (in phytoplankton) and extracellular (dissolved in water) MCs at five stations along a river continuum from a freshwater reservoir with ongoing cyanobacterial blooms to the coast of Brittany, France. MCs were quantified using two integrative samplers placed at each site: solid phase adsorption toxin tracking (SPATT) samplers for collecting extracellular MCs and caged mussels (Anodonta anatina and Mytilus edulis) filter-feeding on MC-producing cyanobacteria. The MC transfer was demonstrated each year during five months at estuarine sites and sporadically at the marine outlet. SPATT samplers integrated extracellular MCs, notably at low environmental concentrations (0.2 µg/L) and with the same variant profile as in water. The mussel A. anatina highlighted the presence of MCs including at intracellular concentrations below 1 µg/L. M. edulis more efficiently revealed the MC transfer at estuarine sites than water samplings. Bivalves showed the same MC variant profile as phytoplankton samples, but with differential accumulation capacities between the variants and the two species. Using SPATT or bivalves can give a more accurate assessment of the contamination level of a freshwater-marine continuum, in which the MC transfer can be episodic. MC content in M. edulis represents a potent threat to human health if considering updated French guideline values, and particularly the total (free and protein-bound) MC content, highlighting the necessity to include cyanotoxins in the monitoring of seafood originating from estuarine areas.

Phthalate esters, mainly di-ethylhexylphthalate (DEHP), represent a class of chemicals primarily used as plasticizers for polyvinyl chloride in a wide range of domestic and industrial applications. These phthalate esters are low-toxicity environmental contaminants. To address these drawbacks, POLYSORB® ID 37, a blend of diesters obtained from esterification of isosorbide with plant-based fatty acids, was developed. The company can now offer PVC manufacturers a new product which competes with phthalates and other such chemicals. The market for plasticizers is very important, and ROQUETTE intends to provide a more sustainable and safer product. Isosorbide diester is bio-based (made from glucose and vegetable fatty acids). This plasticizer is registered in REACH regulation for high volumes (>1000 T/year). Risk assessment was obtained by conducting a wide range of biodegradability and toxicological protocols, using rodent models, according to established guidelines. Overall, all of the toxicological and biodegradability studies demonstrated that POLYSORB® ID 37 is nontoxic to mammalian life and is readily biodegradable.

International audience; Synthetic glucocorticoids (GCs) are a group of steroids that are massively used as anti-inflammatory drugs to treat a broad spectrum of deseases such as asthma or arthritis. They may enter surface waters via urban, hospital and/or pharmaceutical industry effluents and were already detected in surface waters at concentrations ranging from < 1 to tens of ng/L. In organisms, GCs are designed to act through the glucocorticoid receptor (GR) and some studies have reported adverse effects on aquatic vertebrates, such as fish. Overall, there is still a need for knowledge on the sources and driver compounds for GR activity in different contexts. In the frame of a French national survey of EDCs in surface waters using in vitro bioassays, we previously identified a hotspot river site with high GR activity. To further identify the source(s) and the nature of this contamination, an investigative monitoring based on bioassay (MDA-Kb2) and chemical analyses was implemented in the current study. A contamination mapping was established on the basis of bioassay data, along a 40 km streatch upstream and downstream of the contaminated site allowing identification of the discharge of the contamination in the river and of its highly probable industrial origin in the sewerage network, although the contribution of waste waters from a hospital could not be excluded. By using a suspect screening approach based on HR-MS, fluticasone propionate as well as few other GCs were identified in the samples. Mass balance analysis based on quantitave chemical analysis confirmed fluticasone propionate main role in the measured GR activity. The high mobility and persistence of this contamination points out possible high environmental concern in terms of its fate and hazard for aquatic organisms.

International audience; Aquatic organisms are exposed to mixtures of chemicals that may interact. Mixtures of atrazine (ATR) and chlorpyrifos (CPF) may elicit synergic effects on the permanent inhibition of acetylcholinesterase (AChE) in certain aquatic organisms, causing severe damage. Mechanistic mathematical models of toxicokinetics and toxicodynamics (TD) may be used to better characterize and understand the interactions of these two chemicals. In this study, a previously published generic physiologically-based toxicokinetic (PBTK) model for fish was adapted to ATR and CPF. A sub-model of the kinetics of one of the main metabolites of CPF, chlorpyrifos-oxon (CPF-oxon), was included, as well as a TD model. Inhibition of two esterases, AChE and carboxylesterase, by ATR, CPF and CPF-oxon, was modeled using TD modeling of quantities of total and inactive esterases. Specific attention was given to the parameterization and calibration of the model to accurately predict the concentration and effects observed in the fish using Bayesian inference and published data from fathead minnow (Pimephales promelas), zebrafish (Danio rerio) and common carp (Cyprinus carpio L.). A PBTK-TD for mixtures was used to predict dose-response relationships for comparison with available adult fish data. Synergistic effects of a joint exposure to ATR and CPF could not be demonstrated in adult fish.

International audience; In order to improve the diagnosis of environmental contamination of freshwater, multi-biomarkers approaches have been developed to better reflect the physiological state of an organism and potentially detect exposures to xenobiotics. Genotoxic biomarkers seem to be quite relevant due to the multitude of substances interacting with the DNA molecule which can affect its integrity in somatic and germ cells. Among the existing tools used to highlight the genotoxicity on fish erythrocytes, the alkaline comet assay (SCGE) is the most widely used method to detect primary DNA strand breaks and flow cytometry (FCM) is becoming a fast and convenient tool to notice chromosomal damages for aquatic biomonitoring. The present work proposes to evaluate the relevance to include two genotoxic biomarkers (primary DNA strand breaks and chromosomal damages) in the existing battery of multi-biomarkers developed on three-spined stickleback (Gasterosteus aculeatus). After 21 days of exposure, biological samples were recovered from sticklebacks caged in six sites in Northern France to constitute the battery. The inclusion of genotoxicity biomarkers seems to bring a little improvement to the discriminating power of sites as obtained by a Principal Component Analysis (PCA) explaining 30.8% of the variance, although they provide a complementary information on the toxicity of sites. The “Rhonelle” river was the most affected by primary DNA strand breaks measured in fish but this parameter was not well represented on axis, in opposition to fish caged in “Deule” and “Sensée Bouchain” sites showing lower levels of chromosomal damages, in accordance with the first component axis of the PCA.

International audience; Physiologically-based toxicokinetic models (PBTK) are useful tools to improve understanding of the fate of a chemical inside an organism. Recently, Grech et al. (2019) proposed a generic PBTK model for four different fish, including zebrafish (Danio rerio), threespine stickleback (Gasterosteus aculeatus), rainbow trout (Oncorhynchus mykiss) and fathead minnow (Pimephales promelas). Meanwhile, bisphenol A (BPA) is considered as very high concern substance due to its endocrine disruptor function, its high production volume and its persistence in the aquatic environment. In this work, the PBTK will be adapted to accurately model the ADME processes (absorption, distribution, metabolization and excretion) of BPA. A particular effort will be carried out to predict internal concentration of its two main metabolites, BPA-monoglucuronide (BPA-g) and BPA-monosulfate (BPA-s), in various organs. In vitro data from zebrafish, rat (Sprague-Dawley) and mouse (CD-1) hepatocytes will be used to calculate Michaelis-Menten parameters related to metabolism. Moreover, TK data in zebrafish and rainbow trout for BPA, BPA-G and BPA-S in different organs will be retrieved from literature to calibrate the model. The objective will be to accurately predict TK data measured in vivo in external datasets. Further development will later add TD sub-models to predict the dynamic of biomarkers measured in organs.

International audience; Introduction The aquatic systems are the ultimate repository of a large part of the compounds due to human activities. In response to these chemical stresses which aquatic environment may suffer, most studies have focused on the use of biomarkers as early responses induced by these pressures. In recent years, several studies showed that metabolomics can be used to compare and determine the causal link between exposure and effects for predicting toxic effects of contaminants in aquatic organisms. Especially, untargeted approaches based on high resolution mass spectrometry has gained more and more popularity due to its benefit of analysing the whole metabolome in a global view. This approach is a promising tool to provide biomarkers of effect and information on exposure ways in organisms in view of aquatic biomonitoring and environmental risk assessment. The aims of this study were : - To develop an exhaustive untargeted approach able to cover a huge range of metabolites within three-spined sticklebacks (Gasterosteus aculeatus) - To identify impacted metabolites after a sub-chronic exposure of this species to diclofenac. Materials and methods A solid liquid extraction followed by liquid chromatography (RSLC U3000 from Thermo Scientific®) coupled to QtoF (Maxis Plus from Bruker Daltonics®) were used. The data were processed using Metaboscape 4.0 (Bruker Daltonics®). The extraction process was performed on 25 or 50 mg of whole fish, liver, gills or brain with 2.5 mL of MeOH/H2O/heptane. Two complementary analytical methods were then combined : a reverse phase separation on HSS T3 C18 column for positive ionization mode, and an HILIC separation on Nucleodur column for negative ionization mode. This development was done to identify finer and then more relevant biomarkers by a comparative strategy between control and exposed individuals to diclofenac. Results and discussion Two LC-HRMS methods have been developed based on the responses of analytical standards representative of different metabolite groups (amino acids, sugars...). The extraction protocol has been optimized in all matrices, taking into account the number of features (m/z, tr) observed in LC-HRMS. The study shows the influence of the nature of the solvents during the extraction stage as well as the choice of chromatographic conditions. These methods were applied to adult fish exposed to diclofenac, a non-steroidal anti-inflammatory drug ubiquitous in the environment. Individuals were exposed for 21 days at 1 and 100 µg/L. The results of the untargeted metabolomic approach will be presented during the speech. Conclusions This study provides a complete reliable analytical method for the characterization of metabolites in three-spined sticklebacks. Thus, it enables to identify biomarkers through a comparative strategy, for biomonitoring of water bodies quality.

River water that receives treated wastewater can be contaminated by pathogens including enteric viruses due to fecal pollution, which may represent an important public health hazard. There is a great diversity of enteric viruses and fecal bacteriophages, especially F-specific RNA bacteriophages (FRNAPHs), are commonly proposed as indicators of viral pollution due to a variety of characteristics such as their structural similarities to the main enteric viruses, their high concentrations in raw wastewater and their environmental survival rate, which is better than other cultivable enteric viruses. However, evaluating the viral contamination of water on the basis of FRNAPH concentration levels continues to present a challenge. This is because the quality of detection is strongly dependent on the quantity of viral particles, high spatio-temporal variabilities and the physico-chemical conditions of the water during sampling. To overcome these limitations, the present study aims to evaluate whether the bivalve mollusk Dreissena polymorpha (zebra mussel) could be considered a suitable experimental model for assessing the viral contamination of rivers. In order to determine this, the capacity of D. polymorpha to accumulate FRNAPHs and assimilate them into their soft tissue was studied. This provided a proof of concept for the use of D. polymorpha to evaluate the viral contamination of surface water. Two experiments were conducted: (1) an in situ experiment to confirm that zebra mussels naturally accumulated FRNAPHs and (2) a laboratory experiment to determine the accumulation and depuration kinetics of FRNAPHs in D. polymorpha tissue. The study highlights the capacity of the mussels to accumulate infectious FRNAPHs both on a laboratory scale under controlled conditions as well as in situ at different sites that are representative of different bodies of water. An analysis of the mussels’ soft tissue showed that they were capable of reflecting the water’s contamination level very quickly (within less than 24 h). Moreover, the soft tissue retained the viral load much longer than the water due to a low depuration rate. The analysis of FRNAPH concentrations in mussels exposed in situ suggested that there were differences in contamination levels between sites. These preliminary results underline the potential utility of zebra mussels in assessing viral contamination by measuring the accumulation of FRNAPHs in their tissue. This may ultimately enable stakeholders to use zebra mussels as a means of monitoring viral pollution in surface water.

Due to the potential hazard of diclofenac on aquatic organisms and the lack of higher-tier ecotoxicological studies, a long-term freshwater mesocosm experiment was set up to study the effects of this substance on primary producers and consumers at environmentally realistic nominal concentrations 0.1, 1 and 10 µg/L (average effective concentrations 0.041, 0.44 and 3.82 µg/L). During the six-month exposure period, the biovolume of two macrophyte species (Nasturtium officinale and Callitriche platycarpa) significantly decreased at the highest treatment level. Subsequently, a decrease in dissolved oxygen levels was observed. High mortality rates, effects on immunity, and high genotoxicity were found for encaged zebra mussels (Dreissena polymorpha) in all treatments. In the highest treatment level, one month after the beginning of the exposure, mortality of adult fish (Gasterosteus aculeatus) caused effects on the final population structure. Total abundance of fish and the percentage of juveniles decreased whereas the percentage of adults increased. This led to an overall shift in the length frequency distribution of the F1 generation compared to the control. Consequently, indirect effects on the community structure of zooplankton and macroinvertebrates were observed in the highest treatment level. The No Observed Effect Concentration (NOEC) value at the individual level was

For the determination of acute toxicity of chemicals in zebrafish (Danio rerio) embryos, the OECD test guideline 236, relative to the Fish Embryo Toxicity Test (FET), stipulates a dose-response analysis of four lethal core endpoints and a quantitative characterization of abnormalities including their time-dependency. Routinely, the data are analysed at the different observation times separately. However, observations at a given time strongly depend on the previous effects and should be analysed jointly with them. To solve this problem, we developed multistate models for occurrence of developmental malformations and live events in zebrafish embryos exposed to eight concentrations of valproic acid (VPA) the first five days of life. Observations were recorded daily per embryo. We statistically infer on model structure and parameters using a numerical Bayesian framework. Hatching probability rate changed with time and we compared five forms of its time-dependence; a constant rate, a piecewise constant rate with a fixed hatching time at 48 hours post fertilization, a piecewise constant rate with a variable hatching time, as well as a Hill and Gaussian form. A piecewise constant function of time adequately described the hatching data. The other transition rates were conditioned on the embryo body concentration of VPA, obtained using a physiologically-based pharmaco­kinetic model. VPA impacted mostly the malformation probability rate in hatched and non-hatched embryos. Malformation reversion probability rates were lowered by VPA. Direct mortality was low at the concentrations tested, but increased linearly with internal concentration. The model makes full use of data and gives a finer grain analysis of the teratogenic effects of VPA in zebrafish than the OECD-prescribed approach. We discuss the use of the model for obtaining toxicological reference values suitable for inter-species extrapolation. A general result is that complex multistate models can be efficiently evaluated numerically.

The use of a multi-biomarker approach with three-spined sticklebacks (Gasterosteus aculeatus) through an active biomonitoring strategy appears to be a promising tool in water quality assessment. The present work proposes to assess the efficiency of these tools in the discrimination of some sites in a large scale on the Meuse basin in Europe. The study was part of an EU program which aims to assess water quality in the Meuse across the French-Belgian border. Sticklebacks were caged 21 days upstream and downstream from the wastewater treatment plants (WWTPs) of Namur (Belgium), Charleville-Mezieres (France), Bouillon (Belgium) and Avesnes-sur-Helpe (France). First, the state of a variety of physiological functions was assessed using a battery of biomarkers that represented innate immunity (leucocyte mortality and distribution, phagocytosis activity, respiratory burst), antioxidant system (GPx, CAT, SOD and total GSH content), oxidative damages to the membrane lipids (TBARS), biotransformation enzymes (EROD, GST), synaptic transmission (AChE) and reproduction system (spiggin and vitellogenin concentration). The impacts of the effluents were first analysed for each biomarker using a mixed model ANOVA followed by post-hoc analyses. Secondly, the global river contamination was assessed using a principal component analysis (PCA) followed by a hierarchical agglomerative clustering (HAC). The results highlighted a small number of effects of WWTP effluents on the physiological parameters in caged sticklebacks. Despite a significant effect of the “localisation” factor (upstream/downstream) in the mixed ANOVA for several biomarkers, post-hoc analyses revealed few differences between upstream and downstream of the WWTPs. Only a significant decrease of innate immune responses was observed downstream from the WWTPs of Avesnes-sur-Helpe and Namur. Other biomarker responses were not impacted by WWTP effluents. However, the multivariate analyses (PCA and HAC) of the biomarker responses helped to clearly discriminate the different study sites from the reference but also amongst themselves. Thus, a reduction of general condition (condition index and HSI) was observed in all groups of caged sticklebacks, associated with a weaker AChE activity in comparison with the reference population. A strong oxidative stress was highlighted in fish caged in the Meuse river at Charleville-Mezieres whereas sticklebacks caged in the Meuse river at Namur exhibited weaker innate immune responses than others. Conversely, sticklebacks caged in the Helpe-Majeure river at Avesnes-sur-Helpe exhibited higher immune responses. Furthermore, weak defence capacities were recorded in fish caged in the Semois river at Bouillon. This experiment was the first to propose an active biomonitoring approach using three-spined stickleback to assess such varied environments. Low mortality and encouraging results in site discrimination support the use of this tool to assess the quality of a large number of water bodies.

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.

International audience; Antidepressants in coastal waters may affect ontogeny of predatory behaviour in cuttlefish, which may, as a result, affect growth of newly-hatched cuttlefish. We investigated the effects of two of the most prescribed antidepressants, fluoxetine (FLX) and venlafaxine (VEN) in environmentally realistic concentrations on the predatory behaviour of hatchlings of Sepia officinalis. Newly-hatched cuttlefish were exposed from 1 hour (i.e., day 1) to 5 days after hatching to either FLX alone (5 ng·L-1) combined with VEN (2.5 ng·L-1 or 5 ng·L-1 each) to simulate an environmentally realistic exposure scenario. Their predatory behaviour was analysed through several parameters: prey detection, feeding motivation and success in catching the prey. All parameters improved in control animals over the first five days. The combination of FLX and VEN at 5 ng·L-1 each altered the predatory behaviour of the hatchlings by increasing the latency before attacking the prey, i.e., reducing feeding motivation, as well as by reducing the number of successful attacks. The changes in predatory behaviour tended to reduce food intake and affected growth significantly at 28 days post-hatching. Exposures to either FLX at 5 ng·L-1 or FLX and VEN in mixture at 2.5 ng·L-1 each tended to produce similar effects, even though they were not statistically significant. It is likely that the antidepressants affect maturation of the predatory behaviour and/or learning processes associated with the development of this behaviour. The slightest delay in maturation processes may have detrimental consequences for growth and population fitness.

International audience; Contexte Pour se conformer au cadre réglementaire, des évaluations d’impact sanitaire sont réalisées pour les installations susceptibles de rejeter des substances radioactives et chimiques, en vue d’obtenir l’autorisation de les exploiter. Lors d’exposition à des niveaux faibles, de plus en plus d’experts sont tentés d’utiliser une approche graduelle ou proportionnée pour comparer le risque radiologique au risque chimique.Méthode Les méthodes de calcul de risques sanitaires chimiques et radiologiques seront présentées pour des effets sans seuil, essentiellement des effets cancérogènes. Les différentes étapes constitutives de chacune des deux approches seront détaillées de manière séquentielle : origine des données d’incidence des cancers, méthodes de raisonnement utilisées et processus de calcul.Résultats Concernant le risque chimique, la méthode s’appuie sur la détermination de valeurs toxicologiques de référence pour des effets sans seuil, les excès de risques unitaires, pour chacune des voies d’exposition de chaque substance concernée puis d’une sommation des risques pour l’ensemble des substances du mélange. Concernant le risque radiologique, le concept de détriment radiologique utilisé par la Commission Internationale de Protection Radiologique permet de quantifier l’ensemble des effets stochastiques nocifs de l’exposition aux rayonnements ionisants à faible dose et faible débit de dose. Il est déterminé à partir des coefficients de risques nominaux par type de cancer (organe/tissu), en tenant compte de la gravité de la maladie en termes de létalité, de qualité de vie et d’années de vie perdues.Conclusion En conclusion, il s’agit d’une analyse détaillée des deux approches mettant en exergue les différents éléments pour une meilleure compréhension des démarches et de leur interprétation. Les deux approches de calcul du risque chimique et radiologique seront mises en parallèle afin de déterminer à quel niveau elles sont comparables.

The Interreg DIADeM program (development of an integrated approach for the DIAgnosis of the water quality of the river Meuse) was created in order to develop new tools to evaluate the quality of water bodies in the Meuse waterway across the FrenchBelgian border. The project aimed to bring together the various crossborder stakeholders concerned with the protection of aquatic environments (academics, operators, users) while informing citizens of the risks caused by the degradation of water bodies. The use and the relevance of the multibiomarker approach in different model species (fish, crustacean, mussel) to assess the water quality was an important part of the program. Among the different model species used, the threespined stickleback has gained interest for biomonitoring for several years, due to its ubiquity and its tolerance to pollution. For many years, a new active strategy based on the importation of caged sticklebacks in a study site have been developed. Within the scope of the DIADeM program, the caging strategy was used with stickleback as model species to discriminate some sites in a large scale on the Meuse basin. In that way, sticklebacks were caged for 21 days upstream and downstream the wastewater treatment plants (WWTPs) of Namur (Belgiun), CharlevilleMézière (France), Bouillon (Belgium) and Avesnes-sur-Helpe (France). A battery of biomarkers was assessed to represent different physiological functions, such as innate immunity (leucocyte mortality and distribution, phagocytosis activity, respiratory burst, antioxidant system (GPx, CAT, SOD and GSH content) and oxidative damages (TBARS), biotransformation enzymes (EROD, GST), synaptic transmission (AChE) and reproduction system (spiggin and vitellogenin concentration). Physiological responses were compared between each site but also with biomarkers levels measured in sticklebacks under control conditions. Study highlighted a decrease of innate immune system downstream the WWTPs, especially through an increase of leucocyte mortality and a decrease in phagocytosis activity. A slight induction of EROD activity was also noticed downstream the WWTP of Bouillon and Namur. Moreover, the use of active biomonitoring clearly discriminated the different study sites from the reference but also amongst themselves, demonstrating the efficiency of used tools to assess the quality of water and characterize types of physiological impacts on fish.

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (Test Guideline 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 µg/L). In addition to “classical” endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that the highest PCZ concentration significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 µg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. These experiments showed that the physiological responses observed in control and PCZ-exposed cyp19a1aeGFP transgenic zebrafish (E2, VTG, reproduction) are similar to those of wild-type zebrafish, both qualitatively and quantitatively. Besides, the cyp19a1a-eGFP model reliably informs on the time- and concentrationdependent effects of PCZ on ovarian aromatase through in vivo GFP fluorescence of the ovaries, thereby providing novel mechanistic information without increasing the number of animals needed. Overall, the cyp19a1a-eGFP transgenic zebrafish line proved to be a relevant model to study the effects of EDCs in fish, which might also be wisely used to refine mechanism-based assays such as OECD TG 229.

International audience; Regulatory assays available from the OECD for the testing of chemicals, such as endocrine disrupting chemicals (EDC) testing (TG229, TG230), can be performed with zebrafish without any recommendation regarding the strain that should be used. In addition to endpoints defined in the TG, transgenic fish could inform on mechanisms underlying EDC effects. By avoiding interference with pigmentation during imaging, use of casper background may improve tests sensitivity. In the present study, we compared 4 zebrafish lines (wild type AB, casper, cyp19a1a-eGFP and cyp19a1a-eGFP-casper) to determine whether transgene insertion and mutations could modify their basal physiology and their response to pollutant, thereby impeding their use for EDC testing. We thus compared the behavior (anxiety, sociability) and reproductive parameters (circulating vitellogenin (VTG) concentrations, female fecundity, egg viability) of control fish of the 4 lines and evaluated their responses to a model pollutant, e.g. clotrimazole. Sociability was evaluated by monitoring the time spent near congeners in a shuttle box while anxiety was evaluated using the novel tank diving test. No difference was observed between lines for either sociability or anxiety level. Concerning reproduction, no significant difference in the number of eggs laid per female, in the viability of eggs or in the female circulating VTG concentrations was noted between the 4 lines studied. Clotrimazole exposure (28 days; 250 µg/L) had similar effects in the 4 zebrafish lines despite a slightly higher sensitivity in wild type AB. Clotrimazole led to a masculinization of one part of the females, an inhibition of VTG, an inhibition of aromatase A expression (GFP measurement and qPCR) and an induction of 11-ketotestosterone. In summary, no critical difference in the behavior, reproductive parameters and response to pollutant was found between the 4 zebrafish lines, indicating that the transgene insertion and the mutations did not modify basal physiology of the fish. Therefore, their use in OECD tests for the study of EDC appears to be relevant. Further work will be conducted on strains sensitivity by evaluating dose-response effects of pollutants.

Within the Water Framework Directive Common Implementation Strategy (WFD CIS) context, the derivation of Environmental Quality Standards (EQS) is framed by recommendations given in the European Technical Guidance Document for Deriving EQS (TGD EQS, E.C., 2018). This guidance recommends the use of data that are deemed relevant and reliable. Those are most often generated using standardised test methods, but not confined to standard studies and species. The concern raised by endocrine disruption should be tackled in diverse regulatory frameworks, among which the WFD. In this context, endocrine disruption (ED) is quoted several times as an issue in the TGD EQS but recommendations as this regards are not prescriptive. Our study attempts to assess how far can the ECHA/EFSA Guidance for the identification of endocrine disruptors (ECHA/EFSA, 2018) and its weight of evidence approach may be used within the context of the derivation of EQS for tebuconazole.The work consisted in (i) gathering ED toxicity data for aquatic wildlife and mammals not included in the initial dataset underlying the current EQS, (ii) assembling them in a database as lines of evidence of toxicity adressing endocrine activity in order to assess as rigorously as possible the ED potential of tebuconazole and finally (iii) analysing these data to evaluate how far they allowed to improve reliability of the current EQS for tebuconazole.The collection of data allowed to assemble more than 200 lines of evidence consisting in vivo mammals data (52%), in vivo wildlife data (34%) and in vitro mechanistics data (14%).The assessment did not allow to conclude on the ED potential of tebuconazole for wildlife strictly applying the guidance, whereas this ED potential was demonstrated for mammals for which at least two modes of action analysis enabled to conclude on the link between endocrine activity and adversity.Wildlife toxicity assessment would need more in-depth analysis, inter alia to collate and analyse invertebrates toxicity data in order to assess other taxa than fish and amphibians which are already well-represented in the dataset.This analysis allowed implementation of the weight of evidence approach of non-standard toxicity test within the assessment in order to strenghten relevancy and reliability of its EQS. It gives a good opportunity to evaluate how ECHA/EFSA guidance may be used in another regulatory context for which it is meant and allows highlighting the pros and cons of this exercise.

Corrigendum: Aquatic Toxicology, Volume 247, June 2022, Pages 106166 https://doi.org/10.1016/j.aquatox.2022.106166 WOS:000799284400001; International audience; Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 μg/L). In addition to “classical” endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 μg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 μg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals.

(1) Background: Mercury is a threat for the aquatic environment. Nonetheless, the entrance of Hg into food webs is not fully understood. Macrophytes are both central for Hg entry in food webs and are seen as good candidates for biomonitoring and bioremediation; (2) Methods: We review the knowledge gained on the uptake and effects of inorganic Hg (IHg) and methyl-Hg (MMHg) in the macrophyte Elodea nuttallii found in temperate freshwaters; (3) Results: E. nuttallii bioaccumulates IHg and MMHg, but IHg shows a higher affinity to cell walls. At the individual level, IHg reduced chlorophyll, while MMHg increased anthocyanin. Transcriptomics and metabolomics in shoots revealed that MMHg regulated a higher number of genes than IHg. Proteomics and metabolomics in cytosol revealed that IHg had more effect than MMHg; (4) Conclusions: MMHg and IHg show different cellular toxicity pathways. MMHg’s main impact appears on the non-soluble compartment, while IHg’s main impact happens on the soluble compartment. This is congruent with the higher affinity of IHg with dissolved OM (DOM) or cell walls. E. nuttallii is promising for biomonitoring, as its uptake and molecular responses reflect exposure to IHg and MMHg. More generally, multi-omics approaches identify cellular toxicity pathways and the early impact of sublethal pollution.

BackgroundData on the effects of extremely low frequency electromagnetic fields (ELF-EMF) on pregnancy outcomes are inconclusive.ObjectiveTo study the relation between maternal cumulative exposure to ELF-EMF during pregnancy and the risk of prematurity or small for gestational age (SGA) in a pooled analysis of two French birth cohorts.MethodsElfe and Epipage2 are both population-based birth cohorts initiated in 2011 and included 18 329 and 8400 births, respectively. Health data and household, mother and child characteristics were obtained from medical records and questionnaires at maternity and during follow-up. A job exposure matrix was used to assess cumulative exposure to ELF-EMF during three periods: (1) until 15 weeks of gestation, (2) until 28 weeks of gestation and (3) until 32 weeks of gestation. Analyses were restricted to single live births in mainland France and to mothers with documented jobs (N=19 894). Adjusted logistic regression models were used.ResultsAccording to the period studied, 3.2%–4% of mothers were classified as highly exposed. Results were heterogeneous. Increased risks of prematurity were found among low exposed mothers for the three periods, and no association was observed among the most exposed (OR1=0.92 (95% CI 0.74 to 1.15); OR2=0.98 (95% CI 0.80 to 1.21); OR3=1.14 (95% CI 0.92 to 1.41)). For SGA, no association was observed with the exception of increased risk among the low exposed mothers in period 2 and the most exposed in period 3 (OR=1.25 (95% CI 1.02 to 1.53)).ConclusionSome heterogeneous associations between ELF-EMF exposure and prematurity and SGA were observed. However, due to heterogeneity (ie, their independence regarding the level of exposure), associations cannot be definitely explained by ELF-EMF exposure.

BackgroundBreast cancer is the most frequent cancer in women in industrialized countries. Lifestyle and environmental factors, particularly endocrine-disrupting pollutants, have been suggested to play a role in breast cancer risk. Current epidemiological studies, although not fully consistent, suggest a positive association of breast cancer risk with exposure to several International Agency for Research on Cancer Group 1 air-pollutant carcinogens, such as particulate matter, polychlorinated biphenyls (PCB), dioxins, Benzo[a]pyrene (BaP), and cadmium. However, epidemiological studies remain scarce and inconsistent. It has been proposed that the menopausal status could modify the relationship between pollutants and breast cancer and that the association varies with hormone receptor status. ObjectiveThe XENAIR project will investigate the association of breast cancer risk (overall and by hormone receptor status) with chronic exposure to selected air pollutants, including particulate matter, nitrogen dioxide (NO2), ozone (O3), BaP, dioxins, PCB-153, and cadmium. MethodsOur research is based on a case-control study nested within the French national E3N cohort of 5222 invasive breast cancer cases identified during follow-up from 1990 to 2011, and 5222 matched controls. A questionnaire was sent to all participants to collect their lifetime residential addresses and information on indoor pollution. We will assess these exposures using complementary models of land-use regression, atmospheric dispersion, and regional chemistry-transport (CHIMERE) models, via a Geographic Information System. Associations with breast cancer risk will be modeled using conditional logistic regression models. We will also study the impact of exposure on DNA methylation and interactions with genetic polymorphisms. Appropriate statistical methods, including Bayesian modeling, principal component analysis, and cluster analysis, will be used to assess the impact of multipollutant exposure. The fraction of breast cancer cases attributable to air pollution will be estimated. ResultsThe XENAIR project will contribute to current knowledge on the health effects of air pollution and identify and understand environmental modifiable risk factors related to breast cancer risk. ConclusionsThe results will provide relevant evidence to governments and policy-makers to improve effective public health prevention strategies on air pollution. The XENAIR dataset can be used in future efforts to study the effects of exposure to air pollution associated with other chronic conditions. International Registered Report Identifier (IRRID)DERR1-10.2196/15167

High-power microwaves are used to inhibit electronics of threatening military or civilian vehicles. This work aims to assess health hazards of high-power microwaves and helps define hazard threshold levels of modulated radiofrequency exposures such as those emitted by the first generations of mobile phones.Rats were exposed to the highest possible field levels, under single acute or repetitive exposures for eight weeks. Intense microwave electric fields at 1 MV/m of nanoseconds duration were applied from two sources at different carrier frequencies of 10 and 3.7 GHz. The repetition rate was 100 pps, and the duration of train pulses lasted from 10 s to twice 8 min. The effects were studied on the central nervous system, by labelling brain inflammation marker GFAP and by performing different behavioural tests: rotarod, T-maze, beam-walking, open-field, and avoidance test. Long-time survival was measured in animals repeatedly exposed, and anatomopathological analysis was performed on animals sacrificed at two years of life or at death if earlier. One group was sham exposed.Few effects were observed on behaviour. With acute exposure, an avoidance reflex was shown at very high, thermal level (22 W/kg); GFAP was increased some days after exposure. Most importantly, with repeated exposures, survival time was 4-month shorter in the exposed group, with eleven animals exhibiting a large sub-cutaneous tumour, compared to two in the sham group. A residual X-ray exposure was also present in the beam (0.8 Gy), which is not a bias for the observed result.High power microwaves below thermal level in average, can increase cancer incidence and decrease survival time in rats, without clear effects on behaviour. The parameters of this effect need to be explored further, and a more precise dosimetry to be performed.

Pharmaceutical substances are ubiquitous in the aquatic environment and their concentration levels typically range from ng/L up to several μg/L. Furthermore, as those compounds are designed to be highly biologically active, assessing their impacts on non-target organisms is important. Here, we conducted a mesocosm experiment testing a mixture of five pharmaceuticals (diclofenac, carbamazepine, irbesartan, acetaminophen and naproxen) on fish, three-spined stickleback (Gasterosteus aculeatus). The mixture concentration levels were chosen on the basis of the contamination of the Meuse river in Belgium which had been measured previously during a monitoring campaign undertaken in 2015 and 2016. Three nominal mixture concentration levels were tested: the lowest concentration level mixture was composed by environmentally-relevant concentrations that approximate average realistic values for each pharmaceuticals (Mx1); the two other levels were 10 and 100 times these concentrations. Although no impact on stickleback prey was observed, the mixture significantly impaired the survival of female fish introduced in the mesocosms at the highest treatment level without causing other major differences on fish population structure. Impacts on condition factors of adults and juveniles were also observed at both individual and population levels. Using a modelling approach with an individual-based model coupled to a bioenergetic model (DEB-IBM), we concluded that chronic exposure to environmentally-relevant concentrations of five pharmaceuticals often detected in the rivers did not appear to strongly affect the three-spined stickleback populations. Mechanisms of population regulation may have counteracted the mixture impacts in the mesocosms.

In this paper we consider the transport of a chemical species in a two phase system with exchange of matter at the interface (so-called active dispersion). The boundary conditions at the interface between the two phases existing within the pore space are either of thermodynamic equilibrium or of reactive type. Saturation changes with the exchange of matter at the interface. The medium itself is characterized by different levels of heterogeneity starting from the pore-scale. In this work, problems related to pore-scale to Darcy-scale upscaling and then to Darcy-scale to Large-scale upscaling are considered. The first upscaling [1] leads to different problems depending on the boundary conditions and various assumptions, in particular the role of the total mass exchange rate, non-standard terms, etc. The role of geometry, reaction rate on dispersion and tortuosity is investigated for simple 1D/2D/3D unit cells. The second upscaling is only considered for the case of a local non-equilibrium Darcyscale model in the case of small variations of density and viscosity with concentration. It leads to a complex coupled problem between Darcy-scale deviations and large-scale averaged values. In particular, the solution exhibits memory and history effects. An algorithm is proposed to solve for the case of small Damköhler numbers (the Diffusive Damköler for low Péclet numbers or the convective Damköhler for high Péclet numbers). The resulting large-scale model is of the non-equilibrium type. The algorithm allows for the calculation of large-scale effective properties such as large-scale relative permeability, large-scale mass exchange coeffcients as a function of the large-scale phase saturation. References [1] J. Guo, M. Quintard and F. Laouafa, Dispersion in Porous Media with Heterogeneous Nonlinear Reactions, Transport in Porous Media, 109, pp. 541-570, 2015.

International audience; The development of predictive, sensitive and reliable biomarkers is of crucial importance for aquatic biomonitoring to assess the effects of chemical substances on aquatic organisms, especially when it comes to combined effects with other stressors (e.g. temperature). The first purpose of the present study was to evaluate the single and combined effects of 90 days of exposure to an environmental cadmium concentration (0.5 μg. L−1) and two water temperatures (16 and 21 °C) on different parameters. These parameters are involved in (i) the antioxidant system (superoxide dismutase activity –SOD– and total glutathione levels –GSH–), (ii) the energy metabolism, i.e. energy reserves (glycogen, lipids, proteins) and digestive enzymes (trypsin, amylase, intestinal alkaline phosphatase–IAP–), and (iii) biometric parameters (weight, length, Fulton’s condition factor, and the gonadosomatic index –GSI–) of threespine stickleback (Gasterosteus aculeatus). The second purpose was to determine the interest of the three digestive enzymes as biomarkers in comparison with the other parameters. The higher temperature (21 °C) impacted the anti-oxidant and energy reserve parameters. In liver, GSH levels increased on day 60, while SOD decreased on days 15 and 90, with a significant decrease of protein and lipid energy reserves on day 90. In muscle, the higher temperature decreased SOD activity only on day 90. G. aculeatus biometric parameters were also impacted by the higher temperature, which limited stickleback growth after 90 days of exposure. In female sticklebacks, the GSI peaked on day 60 and decreased sharply on day 90, while the highest values were reached at day 90 in the control groups, suggesting impaired reproduction in sticklebacks raised at 21 °C. These results suggest that 21 °C is an upper-limit temperature for long-term physiological processes in sticklebacks. In contrast, very low-concentration cadmium exposure had no effect on classical biomarkers (energy reserves, antioxidant parameters, biometric parameters). However, digestive enzymes showed an interesting sensitivity to cadmium, which was emphasized by high temperature. The activity of the three digestive enzymes decreased significantly on day 90 when sticklebacks were exposed to cadmium alone, while the decrease was stronger and was recorded earlier (from day 15) when they were exposed to the cadmium-temperature combination. Compared to conventional measurements, digestive enzymes responded rapidly. This could be an important advantage for them to be used as early warning tools to reflect the health status of organisms, particularly for trypsin and IAP activities.

International audience; The relevance of a biomarker for biomonitoring programs was influenced both by the knowledge on biomarker natural inter-individual and site variabilities and by the sensitivity of the biomarker towards environmental perturbations. To minimize data misinterpretation, robustness reference values for biomarkers were important in biomonitoring programs. Specific three-spined stickleback, Gasterosteus aculeatus, immune reference ranges for field studies had been determined based on laboratory data and one reference station (Contentieuse river at Houdancourt). In this study, data obtained in one uncontaminated and three contaminated sites were compared to these reference ranges as a validation step before considering them for larger scale biomonitoring programs. When the field reference range were compared to data from the uncontaminated station (Béronelle), only few deviations were shown. In this way, data coming from uncontaminated station (Béronelle) was integrated in the field reference ranges to improve the evaluation of site variability. The new field reference ranges provided better discrimination of sites and spanned a larger range of fish lengths than the initial reference ranges. Furthermore, the results suggest lysosomal presence during several months and phagocytosis capacity in autumn may be the most relevant immunomarkers towards identifying contaminated sites. In the future, combining this reference value approach with active biomonitoring could facilitate the obtention of data in multiple stream conditions.

International audience; Aquatic organisms are exposed to mixtures of chemicals that may interact. Mixtures of atrazine (ATR) and chlorpyrifos (CPF) may elicit synergic effects on the permanent inhibition of acetylcholinesterase (AChE) in certain aquatic organisms, causing severe damage. Mechanistic mathematical models of toxicokinetics and toxicodynamics (TD) may be used to better characterize and understand the interactions of these two chemicals. In this study, a previously published generic physiologically-based toxicokinetic (PBTK) model for fish was adapted to ATR and CPF. A sub-model of the kinetics of one of the main metabolites of CPF, chlorpyrifos-oxon (CPF-oxon), was included, as well as a TD model. Inhibition of two esterases, AChE and carboxylesterase, by ATR, CPF and CPF-oxon, was modeled using TD modeling of quantities of total and inactive esterases. Specific attention was given to the parameterization and calibration of the model to accurately predict the concentration and effects observed in the fish using Bayesian inference and published data from fathead minnow (Pimephales promelas), zebrafish (Dunn) rerio) and common carp (Cyprinus carpio L.). A PBTK-TD for mixtures was used to predict dose-response relationships for comparison with available adult fish data. Synergistic effects of a joint exposure to ATR and CPU could not be demonstrated in adult fish.

International audience; Acer 35 EC is a widely used insecticide (a binary mixture of lambda-cyhalothrin and acetamiprid) in pest control in many West African countries, particularly in the cotton culture in north Benin. The aim of this study was to investigate the chronic effects of Acer 35 EC on Nile tilapia Oreochromis niloticus juveniles using a multi-biomarker approach under laboratory conditions. For this purpose, fish were exposed to sublethal concentrations of Acer 35 EC (0, 1 and 10% of LC50- 96 h value). After 28 and 56 days of exposure, several biomarkers were measured in males and females including enzymatic activities related to detoxification and oxidative stress, neurotoxicity and immune responses, sex steroid hormones (testosterone, 17β-estradiol and 11-keto-testosterone) and histological alterations of liver, kidney and gonads. An Integrated Biomarker Response (IBR) was then calculated. The results showed a reduction of cholinesterase activity in muscles, and intercellular superoxide anion production in both sexes. Female steroidogenesis and gametogenesis were affected, especially testosterone levels and oocyte growth. More alterations were observed in liver after exposure to Acer 35 EC. In both sexes, IBR values were higher after 56 days than after 28 days of exposure. In conclusion, based on a large set of biomarkers and IBR values, the chronic exposure to low doses of insecticide Acer 35 EC seems to impair different physiological functions in Nile tilapia juveniles on a time-dependent manner, with a stronger impact on females than on males.

International audience; The global dynamic spread of chemical contamination through the aquatic environment calls for the development of biomarkers of interest. Reproduction is a key element to be considered because it is related to the sustainability of species. Spermatogenesis is a complex process that leads to the formation of mature germ cells, whose steps and impairments need to be finely described in ecotoxicological analyses. The physiological process has been commonly described by histological analyses of gonads in different taxa. In the present paper, we describe the development of a novel technique to characterize spermatogenesis based on the analysis of the DNA content of germ cells by flow cytometry, using a DNA-intercalating agent. This new biomarker, referred to as an index of sexual maturity, proved relevant to describe the seasonal reproductive cycle of the zebra mussel, Dreissena polymorpha (Pallas, 1771), used as a sentinel species in the biomonitoring of continental waters and sensitive to highlight the reprotoxicity of carbamazepine (an anti-epileptic pharmaceutical) tested under ecosystemic conditions (mesocosms).

Contamination of aquatic ecosystems is considered as one of the main threats to global freshwater biodiversity. Within the European Water Framework Directive (EU-WFD) a particular attention is dedicated to assess ecological risks of surface water contamination and mitigation of chemical pressures on aquatic ecosystems. In this work, we evaluated ecological risks of surface water contamination for fish populations in four EU-WFD rivers through an integrative approach investigating three Lines of Evidence (chemical contamination, biomarker responses as early warning signals of contamination impacting individuals and ecological analyses as an indicator of fish community disturbances). This work illustrates through 4 case studies the complementary role of biomarkers, chemical and ecological analyses which, used in combination, provide fundamental information to understand impacts of chemical pressures that can affect fish population dynamics. We discuss the limitations of this approach and future improvements needed within the EU-WFD to assess ecological risk of river contamination for fish populations.

International audience

Erratum in : Correction: Liquid biopsies for omics-based analysis in sentinel mussels. [PLoS One. 2019]; International audience; Liquid biopsy of plasma is a simple and non-invasive technology that holds great promise in biomedical research. It is based on the analysis of nucleic acid-based biomarkers with predictive potential. In the present work, we have combined this concept with the FTA technology for sentinel mussels. We found that hemocytes collected from liquid biopsies can be readily fixed on FTA cards and used for long-term transcriptome analysis. We also showed that liquid biopsy is easily adaptable for metagenomic analysis of bacterial profiles of mussels. We finally provide evidence that liquid biopsies contained circulating cell-free DNA (ccfDNA) which can be used as an easily accessible genomic reservoir. Sampling of FTA-fixed circulating nucleic acids is stable at room temperature and does not necessitate a cold-chain protection. It showed comparable performance to frozen samples and is ideally adapted for sampling in remote areas, most notably in polar regions threatened by anthropogenic activities. From an ethical point of view, this minimally-invasive and non-lethal approach further reduces incidental mortality associated with conventional tissue sampling. This liquid biopsy-based approach should thus facilitate biobanking activities and development of omics-based biomarkers in mussels to assess the quality of aquatic ecosystems.

Developing population models is of great interest as these models enable to extrapolate toxicity observed at the molecular or individual levels to the population level, and thus improve environmental risk assessment of chemical substances. For this purpose, accounting for natural variations of environmental conditions and prey dynamics along with the chemical stress is needed. In this study, an individual-based model (IBM) coupled to a Dynamic Energy Budget (DEB) model was developed in order to predict three-spined stickleback population dynamics in semi-controlled stream experiments (in mesocosms). Datasets obtained in mesocosms offer the opportunity to develop and evaluate the model predictions. The most sensitive parameters of the DEB-IBM were identified by sensitivity analyses and were calibrated based on data from two independent mesocosm experiments. The predictive capacities of our model were subsequently evaluated using three independent mesocosm datasets under different environmental scenarios. Finally, our model was applied to a theoretical case of toxic effects to show an example of application of the model in a regulatory context. While the most uncertain population processes (in particular, competition for food in mesocosms) in our three-spined stickleback DEB-IBM could be modelled more accurately, our model can already serve to assess the impacts of toxicants at the population level by improving the analyses of mesocosm experiments, in decreasing the uncertainty of the experimental results. Therefore, in a second step, it could be used to predict the consequences on viability of a population exposed to a contaminant under various environmental and exposure scenarios.

Bisphenol A, a well-known endocrine-disrupting chemical, is ubiquitously present in an aquatic environment. Its impacts at populational level on three-spined sticklebacks have been studied in a long-term mesocosm experiment at low-dose BPA, however the causes explaining the observed effects have remained unclear. Here, we used an individual-based model coupled to a Dynamic Energy Budget model for (i) assessing the potential of modelling to predict impacts on population viability from organism ecotoxicological endpoints and (ii) provide insights on the mechanisms of BPA toxicity in the artificial streams. To do that, both direct and indirect effects of BPA on three-spined sticklebacks were incorporated in the model. BPA impacts on the physiological processes of fish have been identified based on literature data and effects on stickleback preys have been t into account using sampling data from exposed mesocosms. Simulations were made with the model integrating dose-responses only calibrated on literature data to assess the predictive capacity of the model. Then, simulations were made with a re-calibration of the toxic parameters on the populational data to better understand the observed BPA impacts found in mesocosms. Results showed that direct BPA effects on fish (impacts on gonad formation, growth, male reproductive behavior, eggs and larvae survival) mostly explained the three-spined stickleback population structure in mesocosms, but indirect effects were not negligible. Hence, this study showed the potential of modelling in risk assessment to predict the impacts on the fish population viability from behavioural and physiological effects measured on organism in controlled conditions.

Endocrine disruptors (EDs) are a major concern because of their effects on the main physiological functions, particularly on the reproductive function.In vertebrates, reproduction is under the control of a neuroendocrine axis called hypothalamic-pituitary-gonadal (HPG) axis. It includes various actors atthe brain (gonadotropin releasing hormones (GnRH) and kisspeptins), pituitary (gonadotropic hormones) and gonadal (sex steroids) levels. Theirdévelopment and functioning are finely regulated. Among the regulators, steroids play an important role by performing feedback loops. EDs mimickingsteroids are therefore potentially able to impact these regulations. The objective of this work was to evaluate the effects of xenoestrogens on theimplementation of cerebral actors involved in the HPG axis in the European sea bass (Dicentrarchus labrax). We first described the ontogeny of thekisspeptin, GnRH and gonadotropin systems during larval development. We have highlighted a differential regulation of these actors suggesting a roleduring this developmental process. In addition, we show that 17α-ethinylestradiol (EE2) is able to disrupt the ontogeny of GnRH2 and GnRH3 systemsduring the early stages of larval development.These data provide a basis for evaluation of the EDs effects on the ontogeny of neuroendocrine systems.; Les perturbateurs endocriniens (PE) sont une préoccupation majeure de par leurs effets sur les grandes fonctions physiologiques des organismes etparticulièrement sur la fonction de reproduction. Chez les vertébrés, la reproduction est sous le contrôle d’un axe neuroendocrinien nommé axegonadotrope. Celui-ci comprend différents acteurs cérébraux (gonadolibérines et kisspeptines), hypophysaires (gonadotropines) et gonadiques(stéroïdes sexuels) dont la mise en place et le fonctionnement sont régulés finement. Parmi les mécanismes de régulation, les stéroïdes vont jouer unrôle important en effectuant des boucles de rétrocontrôle. Les PE mimant ces stéroïdes sont donc potentiellement capables d’impacter ces régulations.L’objectif de ce travail était d’évaluer les effets d’un xénooestrogène sur les mises en place des acteurs cérébraux impliqués dans l’axe gonadotrope chezle bar européen (Dicentrarchus labrax). Nous avons d’abord décrit l’ontogénèse des systèmes à kisspeptine, à gonadolibérine et à gonadotropine aucours du développement larvaire. Nous avons mis en évidence une régulation différentielle de ces acteurs suggérant un rôle durant ce processusdéveloppemental. De plus, nous montrons que le 17α-éthinylestradiol (EE2) est capable de perturber l'ontogenèse des systèmes à GnRH2 et à GnRH3au cours des stades précoces du développement larvaire.Ces données constituent une base permettant l’évaluation des effets des PE sur l’ontogénèse des systèmes neuroendocriniens.