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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.

The aim of this study was to evaluate the effects of caging constraints on multiple fish biomarkers used during ecotoxicological studies (biometric data, immune and antioxidant systems, and energetic status). Two of these constraints were linked to caging: starvation and fish density in cages, and one in relation to the post-caging handling: a short transport. Three in situ experiments were conducted with three-spined sticklebacks (Gasterosteus aculeatus). The first experiment compared the effects of three densities (low, medium, and high). The second experiment compared effects of starvation in fish fed every two days with fish that were not fed. Finally comparisons between sticklebacks which have suffered a short car transport after caging and sticklebacks killed without preliminary transport were made. The lack of food had no effect on fish energetic reserves but negatively affected their condition index and their immune system. Transport and high density induced oxidative stress, defined as an overproduction of reactive oxygen species and a stimulation of the antioxidant system. These two constraints also harmed the leucocyte viability. In order not to have any impact on ecotoxicity biomarkers during in situ experiments, it is preferable to decrease fish density in cages, prevent transport before dissections, and feed fish when the caging lasts more than two weeks.

Organism immune defences might be weakened by pollutants, largely detected in aquatic ecosystems, leading to the facilitation for opportunistic pathogens to infect organisms. In this context, destabilization of fish non-specific immune parameters and erythrocyte DNA integrity was tested, on a model fish species, the three-spined stickleback (Gasterosteus aculeatus), after exposure to chlorpyrifos (CPF). Alone, pesticide exposure induced a genotoxic potential (chlorpyrifos at 1.75 and 0.88 µg/L) in addition to a decrease in phagocytosis capacity and a stimulation of respiratory burst. Then, to mimic pathogenic infection, fish exposure to chlorpyrifos was combined with lipopolysaccharides (LPS) stress. In this second experiment, an increase of DNA damage was observed in fish exposed to a lower concentration of chlorpyrifos and LPS. Moreover, at the higher concentration of chlorpyrifos, an early destabilization of innate immunity was observed as suggested by the absence of an increase of lysosomal presence in fish injected with LPS. This study highlighted the usefulness of stress on stress responses to better understand the impact of contaminants on the organism's health.

Human activities have led to increased levels of various pollutants including metals in aquatic ecosystems. Increase of metallic concentrations in aquatic environments represents a potential risk to exposed organisms, including fish. The aim of this study was to characterize the environmental risk to fish health linked to a polymetallic contamination from former uranium mines in France. This contamination is characterized by metals naturally present in the areas (manganese and iron), uranium, and metals (aluminum and barium) added to precipitate uranium and its decay products. Effects from mine releases in two contaminated ponds (Pontabrier for Haute-Vienne Department and Saint-Pierre for Cantal Department) were compared to those assessed at four other ponds outside the influence of mine tailings (two reference ponds/department). In this way, 360 adult three-spined sticklebacks (Gasterosteus aculeatus) were caged for 28 days in these six ponds before biomarker analyses (immune system, antioxidant system, biometry, histology, DNA integrity, etc.). Ponds receiving uranium mine tailings presented higher concentrations of uranium, manganese and aluminum, especially for the Haute-Vienne Department. This uranium contamination could explain the higher bioaccumulation of this metal in fish caged in Pontabrier and Saint-Pierre Ponds. In the same way, many fish biomarkers (antioxidant and immune systems, acetylcholinesterase activity and biometric parameters) were impacted by this environmental exposure to mine tailings. This study shows the interest of caging and the use of a multi-biomarker approach in the study of a complex metallic contamination.

International audience; In freshwater ecosystems, a large number of chemical substances are able to disturb homeostasis of fish by modulating one or more physiological functions including the immune system. The aim of this study was to assess multi-biomarker responses including immunotoxicity induced by urban and agricultural pressure in European bullheads living in a small French river basin. For this purpose, a set of biochemical, immunological, physiological and histological parameters was measured in wild bullheads from five locations characterized by various environmental pressures. Moreover, to address effects of physiological status and contamination level variation on biomarker responses, fish were sampled during three periods (April, July and October). Results revealed a clear impact of environmental pressure on fish health and particularly on immunological status. An increase of EROD activity was recorded between upstream and downstream sites. Upstream sites were also characterized by neurotoxicological effects. Fish exhibited up stream/downstream variations of immunological status but strong differences were observed according to sampling season. Conversely, regarding biochemical and immunological effects, no significant response of physiological indexes was recorded related to environmental pressures. According to these results, the European bullhead appears as a valuable model fish species to assess adverse effects in wildlife due to urban and agricultural pressures.

International audience; Clotrimazole is a pharmaceutical fungicide known to inhibit several cytochrome P450 enzyme activities, including several steroidogenic enzymes. This study aimed to assess short-term in vivo effects of clotrimazole exposure on blood 11-ketotestosterone (11-KT) levels and on the transcriptional activity of genes in pituitary and testis tissue that are functionally relevant for androgen production with the view to further characterize the mode of action of clotrimazole on the hypothalamus-pituitary-gonad axis in zebrafish, a model vertebrate in toxicology. Adult male zebrafish were exposed to measured concentrations in water of 71, 159 and 258 micro g/L of clotrimazole for 7 days. Expression of pituitary gonadotropins ß subunit (lhb, fshb), testicular gonadotropins receptors (lhcgr, fshr) and testicular steroidogenesisrelated genes (e.g., star, cyp17a1, cyp11c1) were assessed. Blood concentrations of 11-KT were measured. Short-term exposure to clotrimazole induced a concentration-dependent increase of star, cyp17a1, and cyp11c1 gene expression and Cyp17a1 and Cy11c1 protein synthesis in Leydig cells, but androgen levels in blood remained unchanged. fshb, but not lhb mRNA levels in the pituitary tended to increase in clotrimazole-exposed zebrafish. Testicular expression of the Fsh receptor gene was significantly upregulated following exposure, when expression of this receptor was significantly correlated to the expression of steroidogenesis-related genes. Moreover, the Fsh-regulated insulin-like growth factor 3 (igf3) gene, a fish-specific Igf peptide expressed in Sertoli cells, was induced in testes. By using a network of genes functioning in pituitary and testis tissue, our study demonstrated that clotrimazole induced a cascade of molecular and cellular events which are in agreement with a role for Fsh in stimulating Leydig cell steroidogenesis to compensate the inhibitory action of clotrimazole on 11-KT synthesis and in inducing the expression of Fsh-regulated igf3 in Sertoli cells.

To characterize environmental risks linked to former uranium mines in the Limousin region of France, a study was conducted on fish health effects from uranium releases. Two private ponds were compared in this study, one with uranium contamination and one background site, upstream of the mining zone. Roach, Rutilus rutilus, were caged for 28 days in both ponds. Physico-chemical parameters of water and sediments and bioaccumulation of metals in several organs were determined. After 14 and 28 days of caging, immune, oxidative stress, biotransformation, neurotoxicity and physiological parameters were measured. Iron and aluminium were quantified in the water of both sites; however, barium and manganese were only present in the water of the uranium contaminated site. Uranium was present in both sites but at very different concentrations. The sediments from the uranium contaminated site contained high levels of radioactive elements coming from the disintegration chain of uranium. Results of biological parameters indicated stimulation of immune parameters and of oxidative stress and a decrease of AChE in fish caged in the uranium contaminated pond compared to the uranium-free pond. Overall, the results determined roach health status in the context of pollution from poly-metallic mining. The data strengthen our knowledge of the environmental risk assessment associated with radioactive substances in the environment.

International audience; Clotrimazole is an azole fungicide used as a human pharmaceutical that is known to inhibit cytochrome P450 (CYP) enzymatic activities, including several steroidogenic CYP. In a previous report, we showed that a 7-day exposure to clotrimazole induced the expression of genes related to steroidogenesis in the testes as a compensatory response, involving the activation of the Fsh/Fshr pathway. In this context, the aim of the present study was to assess the effect of an in vivo 21-day chronic exposure to clotrimazole (30-197 µg/L) on zebrafish testis function, i.e., spermatogenesis and androgen release. The experimental design combined (1) gene transcript levels measurements along the brain-pituitary-gonad axis, (2) 11-ketotestosterone (11-KT) quantification in the blood, and (3) histology of the testes, including morphometric analysis. The chronic exposure led to an induction of steroidogenesis-related genes and fshr in the testes as well as fshß in the pituitary. Moreover, increases of the gonadosomatic index and of the volume proportion of interstitial Leydig cells were observed in clotrimazole-exposed fish. In accordance with these histological observations, the circulating concentration of 11-KT had increased. Morphometric analysis of the testes did not show an effect of clotrimazole on meiotic (spermatocytes) or postmeiotic (spermatids and spermatozoa) stages, but we observed an increase in the number of type A spermatogonia, in agreement with an increase in mRNA levels of piwil1, a specific molecular marker of type A spermatogonia. Our study demonstrated that clotrimazole is able to affect testicular physiology and raised further concern about the impact of clotrimazole on reproduction.