Altération de la physiologie des poissons exposés à des hydrocarbures aromatiques polycycliques (HAP) : comportement et reproduction

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POPs) which are found in complex mixtures in the environment including aquatic ecosystems. They adsorb on particles, accumulate in sediment and in the first link of the food web. The aim of this study was to measure long term effects of PAHs contamination on a vertebrate model : the zebrafish. In a first study, embryos were exposed on natural sediment spiked with 3 individual PAH (phenanthrene, pyrene and benzo[a]pyrene) during the first four days of their development. In a second study, to evaluate the consequences of long-term chronic exposure to PAHs, zebrafish were exposed, from their first meal (5 days post fertilisation) until they became reproducing adults, to diets spiked with three PAHs fractions at environmentally relevant concentrations (0.3X, 1X and 3X; with the 1X concentration being in the range of 4.6-6.7 μg.g-1 for total quantified PAHs). The fractions used were representative of PAHs of pyrolytic (PY) origin or of two different oils of differing compositions (a heavy fuel (HO) and a light crude oil (LO)). Long term effects on growth, survival, reproduction and behavior were evaluated at different ages. Effects were observed at molecule, tissue, organ and individual scales. In general, for contaminated fish, growth was reduced, larval survival decreased in HO, and reproduction was strongly impaired at hormonal, tissue and phenotypic levels, and behavioral responses were lethargic or hyperactive depending on fractions and stages. Sediment exposure still revealed visible effects when fish were 6 month old. These results hereby showed significant late effects, especially in behavioral responses after an early exposure, including in larvae issued from contaminated fish. In spiked diets contaminated fish, we observed a gradation of effects. Combining all functions, we established of hierarchy in toxicity in the studied fractions: PY < LO ≈ HO. Finally, our results gathered at a multiscale from molecule, to tissue and individuals, indicate that PAH mixtures of different compositions, representative of situations encountered in the wild, can promote lethal and sublethal effects which are likely to be detrimental for fish survival and recruitment into future generations.Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants organiques persistants qui sont présents en mélange complexe dans l’environnement et notamment dans les milieux aquatiques. Ils s’adsorbent sur les particules en suspension, s’accumulent dans les sédiments et dans les premiers maillons de la chaîne trophique. L’objectif de cette étude était de mesurer les effets à long terme d’une contamination par des HAP chez un modèle de vertébrés : le poisson zèbre. Dans un premier temps, des embryons ont été exposés pendant les 4 premiers jours de leur développement sur du sédiment naturel enrobé de 3 HAP individuel (phénanthrène, pyrène et benzo[a]pyrène) puis transférés et élevés en eau propre. Dans un deuxième temps, des poissons ont été exposés par voie alimentaire tout au long du cycle de vie et dès le premier repas à 3 fractions de HAP à doses environnementales enrobées sur les aliments : une fraction pyrolytique (PY) extraite d’un sédiment échantillonné dans la Seine et deux fractions pétrogéniques (un fuel lourd (HO) provenant de l’Erika et un pétrole brut type Arabian light (LO)). Les effets à long terme sur la croissance, la survie, la reproduction et le comportement ont été évalués à différents âges. Ces effets ont pu être observés à plusieurs échelles, i.e. de la molécule, aux tissus et organes puis à l’échelle de l’individu. Globalement, chez les poissons contaminés, la croissance est ralentie, la survie larvaire diminuée chez les HO, la fonction reproduction est altérée fortement à la fois au niveau hormonal, tissulaire et phénotypique ainsi que les réponses comportementales léthargiques ou hyper-réactives selon les cas. L’exposition par voie sédimentaire a révélé des effets visibles lorsque les individus atteignaient 6 mois soulignant l’existence d’effets tardifs, notamment sur les réponses comportementales, d’une exposition très précoce y compris chez la descendance. Quant à l’exposition par voie alimentaire, elle a montré une gradation des effets. Toutes fonctions confondues, les résultats obtenus permettent d'établir une hiérarchie dans la toxicité des fractions étudiées : PY < LO ≈ HO. L'approche multi-échelle utilisée dans ce travail a permis, en partant d'informations acquises au niveau moléculaire, tissulaire et individuel, d'aboutir à une compréhension intégrée des conséquences qu'une exposition à des HAP peut avoir sur l'aptitude à la survie des poissons dans leur milieu naturel ainsi qu'à leur capacité à participer au renouvellement des générations futures

Long term physiological disruptions in fish after exposure to polycyclic aromatic hydrocarbons (PAHs) : behavior and reproduction

Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants organiques persistants qui sont présents en mélange complexe dans l’environnement et notamment dans les milieux aquatiques. Ils s’adsorbent sur les particules en suspension, s’accumulent dans les sédiments et dans les premiers maillons de la chaîne trophique. L’objectif de cette étude était de mesurer les effets à long terme d’une contamination par des HAP chez un modèle de vertébrés : le poisson zèbre. Dans un premier temps, des embryons ont été exposés pendant les 4 premiers jours de leur développement sur du sédiment naturel enrobé de 3 HAP individuel (phénanthrène, pyrène et benzo[a]pyrène) puis transférés et élevés en eau propre. Dans un deuxième temps, des poissons ont été exposés par voie alimentaire tout au long du cycle de vie et dès le premier repas à 3 fractions de HAP à doses environnementales enrobées sur les aliments : une fraction pyrolytique (PY) extraite d’un sédiment échantillonné dans la Seine et deux fractions pétrogéniques (un fuel lourd (HO) provenant de l’Erika et un pétrole brut type Arabian light (LO)). Les effets à long terme sur la croissance, la survie, la reproduction et le comportement ont été évalués à différents âges. Ces effets ont pu être observés à plusieurs échelles, i.e. de la molécule, aux tissus et organes puis à l’échelle de l’individu. Globalement, chez les poissons contaminés, la croissance est ralentie, la survie larvaire diminuée chez les HO, la fonction reproduction est altérée fortement à la fois au niveau hormonal, tissulaire et phénotypique ainsi que les réponses comportementales léthargiques ou hyper-réactives selon les cas. L’exposition par voie sédimentaire a révélé des effets visibles lorsque les individus atteignaient 6 mois soulignant l’existence d’effets tardifs, notamment sur les réponses comportementales, d’une exposition très précoce y compris chez la descendance. Quant à l’exposition par voie alimentaire, elle a montré une gradation des effets. Toutes fonctions confondues, les résultats obtenus permettent d'établir une hiérarchie dans la toxicité des fractions étudiées : PY < LO ≈ HO. L'approche multi-échelle utilisée dans ce travail a permis, en partant d'informations acquises au niveau moléculaire, tissulaire et individuel, d'aboutir à une compréhension intégrée des conséquences qu'une exposition à des HAP peut avoir sur l'aptitude à la survie des poissons dans leur milieu naturel ainsi qu'à leur capacité à participer au renouvellement des générations futures.Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POPs) which are found in complex mixtures in the environment including aquatic ecosystems. They adsorb on particles, accumulate in sediment and in the first link of the food web. The aim of this study was to measure long term effects of PAHs contamination on a vertebrate model : the zebrafish. In a first study, embryos were exposed on natural sediment spiked with 3 individual PAH (phenanthrene, pyrene and benzo[a]pyrene) during the first four days of their development. In a second study, to evaluate the consequences of long-term chronic exposure to PAHs, zebrafish were exposed, from their first meal (5 days post fertilisation) until they became reproducing adults, to diets spiked with three PAHs fractions at environmentally relevant concentrations (0.3X, 1X and 3X; with the 1X concentration being in the range of 4.6-6.7 μg.g-1 for total quantified PAHs). The fractions used were representative of PAHs of pyrolytic (PY) origin or of two different oils of differing compositions (a heavy fuel (HO) and a light crude oil (LO)). Long term effects on growth, survival, reproduction and behavior were evaluated at different ages. Effects were observed at molecule, tissue, organ and individual scales. In general, for contaminated fish, growth was reduced, larval survival decreased in HO, and reproduction was strongly impaired at hormonal, tissue and phenotypic levels, and behavioral responses were lethargic or hyperactive depending on fractions and stages. Sediment exposure still revealed visible effects when fish were 6 month old. These results hereby showed significant late effects, especially in behavioral responses after an early exposure, including in larvae issued from contaminated fish. In spiked diets contaminated fish, we observed a gradation of effects. Combining all functions, we established of hierarchy in toxicity in the studied fractions: PY < LO ≈ HO. Finally, our results gathered at a multiscale from molecule, to tissue and individuals, indicate that PAH mixtures of different compositions, representative of situations encountered in the wild, can promote lethal and sublethal effects which are likely to be detrimental for fish survival and recruitment into future generations

Changes in Brain Monoamines Underlie Behavioural Disruptions after Zebrafish Diet Exposure to Polycyclic Aromatic Hydrocarbons Environmental Mixtures

Zebrafish were exposed through diet to two environmentally relevant polycyclic aromatic hydrocarbons (PAHs) mixtures of contrasted compositions, one of pyrolytic (PY) origin and one from light crude oil (LO). Monoamine concentrations were quantified in the brains of the fish after six month of exposure. A significant decrease in noradrenaline (NA) was observed in fish exposed to both mixtures, while a decrease in serotonin (5HT) and dopamine (DA) was observed only in LO-exposed fish. A decrease in metabolites of 5HT and DA was observed in fish exposed to both mixtures. Several behavioural disruptions were observed that depended on mixtures, and parallels were made with changes in monoamine concentrations. Indeed, we observed an increase in anxiety in fish exposed to both mixtures, which could be related to the decrease in 5HT and/or NA, while disruptions of daily activity rhythms were observed in LO fish, which could be related to the decrease in DA. Taken together, these results showed that (i) chronic exposures to PAHs mixtures disrupted brain monoamine contents, which could underlie behavioural disruptions, and that (ii) the biological responses depended on mixture compositions

Maturation of behaviour in the fathead minnow

International audienceFathead minnow (FM, Pimephales promelas) are a species of small fish native to North America. Their small size, fast development, and ability to breed in the lab make them an ideal species to use in research, especially in toxicology. Behaviour in general is poorly studied in FM. The aim of this study was to characterize the normal behaviour of fathead minnow at 3 different stages of development in a light-dark box and in a social behaviour test. Fish larvae showed a preference for the light area, and then an increase in dark preference was seen as the fish aged. FM preferred to be with conspecifics at each age, but this preference was much stronger at the adult stage. The time of first entry into the conspecific area was reduced with increasing age of the fish. The time spent in the conspecific area increased between the juvenile and adult stage, and adults stayed more in this area when they entered it. Maturation of behavior in FM was demonstrated in our study. The FM is another good model fish to assess behavioral effects of chemicals, and this study helps to define the appropriate ages for behavioral studies with FM

Exposure to microplastic: ingestion/egestion rates and behavioural impact on <i> Solea solea <i/>

International audienceMicroplastic (MP) occurrence is ubiquitous in fresh and marine waters and species living inEstuary are particularly exposed to this kind of pollution. In this context, the Seine River isinteresting for investigating the impacts of MP on fish, because of intense and numerous humanactivities through its watershed. The goal of this study is to evaluate the fate and impact of MPingestion using experimental exposure of the common sole, Solea solea, a representative species ofthe Seine Estuary. Larvae of fish (from 7 days post hatch (dph) to 42 dph) were exposed during 2hours to MP particles before (7 dph), during (21 and 22 dph) and after metamorphosis (41 and 42dph). Three types of industrial MP were chosen with different properties (middle size floating (27-35μg mL-1, DC: 50 ng mL-1). Post-exposurem, d=0.99), middle size sinking (27-32μg mL-1, DC: 50 ng mL-1). Post-exposurem, d=1.20) and large size sinking (63- 75μg mL-1, DC: 50 ng mL-1). Post-exposurem, d=1.20).In addition, at the last stage, fish were also exposed to natural plastic particles collected in the SeineRiver and ground into a fraction with a size less than 100 μg mL-1, DC: 50 ng mL-1). Post-exposurem (PVC, PE, PET, PS and PP testedseparately). After exposure, fish which had ingested MP were counted (Figure 1) and isolated tomonitor egestion. Solea solea ingested all types of plastics in variable (1 to more than 100)quantities and egested them on average less than 24 hours after exposure at all stages. A behaviouralchallenge, was performed on MP contaminated larvae. Behavioral responses of fish did not seem tobe affected by MP exposures before metamorphosis compared to controls but distance moveddecreased during metamorphosis for fish that had ingested MP particles. This could be explained bythe fact that before metamorphosis fish can still use yolk energy reserve and are thus less affected byMP than later on when external feeding becomes mandatory

Exposition chronique au diclofénac de Lymnaea stagnalis, un gastéropode d'eau douce : Impact sur l'éclosion des embryons et sur la croissance des juvéniles

National audienceLe diclofénac est un anti-inflammatoire non stéroïdien largement utilisé en France, avec une vente de médicaments remboursés représentant 30 tonnes de principe actif en 2016. Cette molécule étant faiblement dégradée au niveau des stations d'épuration par les traitements classiques, elle fait partie des polluants émergents des milieux aquatiques. En effet, le diclofénac est retrouvé à des concentrations allant du ng/L au µg/L dans les eaux de surface, ce qui pourrait impacter la faune aquatique. Des études montrant un effet de ce médicament sur les organismes vivant en eau douce ont déjà été réalisées, cependant il reste nécessaire d'obtenir plus d'informations sur les conséquences à long terme du diclofénac aux concentrations environnementales. L’objectif de cette étude est de mieux caractériser les effets du diclofénac à long terme et à concentration environnementale sur Lymnaea stagnalis, un mollusque gastéropode d’eau douce. Le taux d’éclosion des pontes et la taille et le poids des juvéniles ont été mesurés

Bioaccumulation, Biotransformation, and Synergistic Effects of Binary Fungicide Mixtures in <i>Hyalella azteca</i> and <i>Gammarus pulex</i>:How Different/Similar are the Two Species?

Aquatic organisms are consistently exposed to a mixture of micropollutants that can bioaccumulate, undergo biotransformation, and may exert mixture effects. However, little is known on the underlying mechanisms and species-specificity. Herein we investigated bioaccumulation, biotransformation and synergistic effects of azole (i.e., prochloraz) and strobilurin (i.e., azoxystrobin) fungicides in the two aquatic invertebrate species, <i>Hyalella azteca</i> and <i>Gammarus pulex</i>. Bioaccumulation of azoxystrobin was similar, whereas bioaccumulation of prochloraz was slightly different in the two species but was still significantly below the REACH criteria for bioaccumulative substances. Similar biotransformation patterns were observed in both species, and only a few unique biotransformation reactions were detected in <i>H. azteca</i> such as malonyl-glucose and taurine conjugation. Toxicokinetic modeling additionally indicated that biotransformation is a more important elimination pathway in <i>H. azteca</i>. In mixtures, no-observed-adverse-effect levels of prochloraz decreased the LC₅₀s of azoxystrobin in both species which correlated well with increased internal azoxystrobin concentrations. This synergistic effect is partly due to the inhibition of cytochrome P450 monooxygenases by prochloraz which subsequently triggered the reduced biotransformation of azoxystrobin (lower by five folds in <i>H. azteca</i>). The largely similar responses in both species suggest that the easier-to-cultivate <i>H. azteca</i> is a promising representative of invertebrates for toxicity testing

Multigenerational responses in the Lymnaea stagnalis freshwater gastropod exposed to diclofenac at environmental concentrations

International audienceOver the last decade, there has been increased concern about the occurrence of diclofenac (DCF) in aquatic ecosystems. Living organisms could be exposed to this “pseudo-persistent” pharmaceutical for more than one generation. In this multigenerational study, we assessed the DCF impact at environmentally relevant concentrations on the life history and behavioral parameters of two offspring generations (F1 and F2) of the Lymnaea stagnalis freshwater gastropod. Snail growth was affected by DCF in the F1 generation, with increased shell sizes of juveniles exposed to 0.1 μg L 1 concentration and a decreased shell size at 2 and 10 μg L 1. DCF also lowered food intake, enhanced locomotion activity and reduced the number of eggs/egg mass in the F1 generation. For the F2 generation, shorter time to hatch, faster growth, increased food intake and production of more egg masses/snail were induced by DCF exposure at 10 μg L 1. Over time, DCF exposure led to maximization of L. stagnalis reproductive function. These results show that multigenerational studies are crucial to reveal adaptive responses to chronic contaminant exposure, which are not observable after short-term exposure

Systematic Screening of Behavioral Responses in Two Zebrafish Strains

Wild-type (WT) zebrafish are commonly used in behavioral tests, but the term WT is not a precise description, and corresponds to many different strains (e.g., AB, TU, WIK, and others). Previous studies compared the physiological, behavioral, or metabolic characteristics of different zebrafish strains (indigenous WT populations versus laboratory WT strains). AB and TU are widely used, but at least one study has demonstrated behavioral differences between them. To choose the most appropriate strain for our experiments, we systematically screened behavioral responses of AB and TU fish in several assays. We analyzed the locomotion activity and responses to a light/dark challenge in adults and larvae, and exploratory behavior and color conditioning in adults. Differences were observed for all tests, the strains displaying particular behavior depending on the tests. As larvae, TU displayed a wider activity range than AB larvae at the onset of locomotor behavior; as adults, TU were more reactive to sudden light transitions and recovered the swimming activity faster in T-maze or homebase release in novel tank tests, whereas AB fish had more contrasted circadian rhythms and performed better in color learning. Strain-specific behavior should be considered when designing experiments using behavior

Long term effects of an early exposure and continuous exposure to pyrolitic PAHs on zebrafish behavioral responses.

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