Untargeted analysis of nanoLC-HRMS data by ANOVA-PCA to highlight metabolites in Gammarus fossarum after in vivo exposure to pharmaceuticals.

In Western Europe, river water quality can be assessed using sentinel species such as the amphipod Gammarus fossarum. In this work of environmental metabolomics, the objective was to develop suitable chemometrics methods, using a limited number of individuals, to assess the modification of the metabolism of G. fossarum exposed to two human pharmaceuticals. Males and females gammarids were exposed to a mixture of the anxiolytic oxazepam and the antiepileptic carbamazepine (1000 ng L-1) for 14 days under laboratory conditions according to a full factorial design 2² (repeated 5 times). They were analyzed at the single individual scale using a method including a µQuEChERS type extraction followed by a nanoliquid chromatography analysis coupled to high-resolution mass spectrometry. The molecular fingerprints obtained were investigated using XCMS. Several corrections of experimental drifts (by using lock mass and Quality Control samples) were tested prior to using APCA + method for the exploitation of the unbalanced designed data. Signal reproducibility was greatly improved by the lock mass normalisation. From the experimental design, a significant effect of both experimental factors "exposure to the mixture" and "gammarid gender" on the signals measured were highlighted by APCA+. Finally, the results obtained made it possible to identify variables responsible for each of the factor effects.

Immune responses in the aquatic gastropod Lymnaea stagnalis under short-term exposure to pharmaceuticals of concern for immune systems: Diclofenac, cyclophosphamide and cyclosporine A.

This is a pioneering study in the ecotoxicological assessment of immunotoxic effects of the three selected drugs of concern to a freshwater gastropod species. Lymnaea stagnalis was exposed in the laboratory for 3 days to three drugs used for immune systems: diclofenac (nonsteroidal anti-inflammatory drug), cyclophosphamide (anti-cancer immunosuppressive drug) or cyclosporine A (anti-xenograft immunosuppressive drug). Exposure ranges included environmental realistic (1-10µgL-1) and therapeutic concentrations (100-1000µgL-1). At the end of exposure times, the immune parameters of individual snails were measured: hemocyte density and viability, hemocyte phagocytosis capacity and hemocyte-related oxidative activities (basal and NADPH-oxidase stimulated with zymosan particles). Diclofenac and cyclosporine A induced immune responses, although the effects were not strong. No immunosuppression was observed. Such subtle immunomodulations bring further interrogations regarding their long-term immunotoxicity and possible resulting tradeoffs with life-history traits. On the other hand, the prodrug cyclophosphamide did not induce significant immune responses. Since metabolism pathways differ greatly between vertebrates and invertebrates, this study also suggests that relevant vertebrate metabolites should be included in the immunotoxicity assessment of pharmaceuticals in non-target invertebrate species. Finally, the possible interactive effects of these pharmaceuticals sharing similar modes of action or effects features should also be explored.

Caged mudsnail Potamopyrgus antipodarum (Gray) as an integrated field biomonitoring tool: exposure assessment and reprotoxic effects of water column contamination.

This study highlights the usefulness of gastropods for water quality monitoring. Gastropods were caged upstream and downstream of an effluent discharge. Exposure was assessed by measurement of organic contaminants in water. Contamination of the Potamopyrgus antipodarum mudsnail was also measured using innovative techniques at the end of the 42 days of exposure. Biological effects were measured at the individual level (growth, reproduction) and subindividual level (energy reserves, vitellin-like proteins, steroid levels, expression of genes involved in estrogen signaling pathways), thus providing a better understanding of reprotoxic effects. The effluent was mainly contaminated by pharmaceutical compounds, as was the mudsnail. The highest concentrations were measured for oxazepam and were higher than 2 mg/kg downstream of the effluent discharge. Alkylphenols, bisphenol A, and vertebrate-like sex-steroid hormones were also bioaccumulated by the mudsnail downstream of the effluent. The combined use of water and snail contamination provided a complete exposure assessment. Exposure was further linked to biological effects. The mudsnail was shown to be a better adapted species for in situ exposures than Valvata piscinalis. Reproduction was sharply decreased after 6 weeks of exposure in the mudsnail. Feeding issues were excluded, confirming the toxic origin. These effects were related to estrogen signaling pathways using genomic analysis. Genes coding for proteins involved in nongenomic signaling pathways were inhibited, and those of genomic pathway repressors were induced. These results suggest that the chemical contamination due to the effluent discharge altered steroid control of reproduction and blocked the transition between oocyte and unshelled embryo, resulting in a drastic decrease of embryo production, while survival was not affected.

Organic solvents impair life-traits and biomarkers in the New Zealand mudsnail Potamopyrgus antipodarum (Gray) at concentrations below OECD recommendations.

Potamopyrgus antipodarum is a gastropod mollusk proposed for use in the development of reproduction tests within the Organization for Economic Cooperation and Development (OECD). Numerous chemicals, including endocrine disrupters, are relatively water-insoluble, and water-miscible solvents are currently used for testing them. OECD recommends a maximum concentration of 100 µll(-1). As several studies highlighted effects of lower concentrations of solvents, this study assessed the effects of 20 µll(-1) acetone, ethanol, methanol and dimethylsulfoxide (DMSO) on juvenile and adult snails during 42 days. Ethanol decreased juvenile growth, while acetone increased the rate of embryonic development. All solvents increased estradiol-like levels in adult snails. DMSO only increased mRNA expression of vitellogenin-like gene, while acetone, ethanol and methanol decreased mRNA expression of three nuclear receptor (estrogen receptor-like, ecdysone-induced protein and chicken ovalbumin upstream promoter transcription factor) genes as well as of genes encoding proteins involved in genomic (prohibitin-2) and non-genomic (striatin) pathways of estrogens activity in vertebrates. This study highlights the confounding effects of low concentrations of solvents and recommends avoiding their use. Where solvent use is inevitable, their concentrations and type should be investigated for suitability for the measured endpoints prior to use in chemical testing strategies.

Effects of short-term exposure to environmentally relevant concentrations of different pharmaceutical mixtures on the immune response of the pond snail Lymnaea stagnalis.

Pharmaceuticals are pollutants of potential concern in the aquatic environment where they are commonly introduced as complex mixtures via municipal effluents. Many reports underline the effects of pharmaceuticals on immune system of non target species. Four drug mixtures were tested, and regrouped pharmaceuticals by main therapeutic use: psychiatric (venlafaxine, carbamazepine, diazepam), antibiotic (ciprofloxacine, erythromycin, novobiocin, oxytetracycline, sulfamethoxazole, trimethoprim), hypolipemic (atorvastatin, gemfibrozil, benzafibrate) and antihypertensive (atenolol, furosemide, hydrochlorothiazide, lisinopril). Their effects were then compared with a treated municipal effluent known for its contamination, and its effects on the immune response of Lymnaea stagnalis. Adult L. stagnalis were exposed for 3 days to an environmentally relevant concentration of the four mixtures individually and as a global mixture. Effects on immunocompetence (hemocyte viability and count, ROS and thiol levels, phagocytosis) and gene expression were related to the immune response and oxidative stress: catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), Selenium-dependent glutathione peroxidase (SeGPx), two isoforms of the nitric oxide synthetase gene (NOS1 and NOS2), molluscan defensive molecule (MDM), Toll-like receptor 4 (TLR4), allograft inflammatory factor-1 (AIF) and heat-shock protein 70 (HSP70). Immunocompetence was differently affected by the therapeutic class mixtures compared to the global mixture, which increased hemocyte count, ROS levels and phagocytosis, and decreased intracellular thiol levels. TLR4 gene expression was the most strongly increased, especially by psychiatric mixture (19-fold), while AIF-1, GR and CAT genes were downregulated. A decision tree analysis revealed that the immunotoxic responses caused by the municipal effluent were comparable to those obtained with the global pharmaceutical mixture, and the latter shared similarity with the antibiotic mixture. This suggests that pharmaceutical mixtures in municipal effluents represent a risk for gastropods at the immunocompetence levels and the antibiotic group could represent a model therapeutic class for municipal effluent toxicity studies in L. stagnalis.

Immunotoxicity of surface waters contaminated by municipal effluents to the snail Lymnaea stagnalis.

The immunotoxic effects of surface waters contaminated by a municipal effluent dispersion plume were examined in the snail Lymnaea stagnalis. Snails were exposed to surface waters where changes in hemocyte counts, viability, levels of reactive oxygen species (ROS), reduced thiols and phagocytic activity were tracked following exposure periods of 3h and 3 and 7d. Changes in mRNA expression of some genes in the hemocytes were also assessed after 7d of exposure, as follows: genes coding for catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSR), selenium-dependent glutathione peroxidase (SeGPX), two isoforms of the nitric oxide synthetase (NOS1 and NOS2), molluscan defensive molecule (MDM), toll-like receptor 4 (TLR4), allograft inflammatory factor-1 (AIF), and heat-shock protein 70 (HSP70). At the sites closest to the discharge point, exposure led to impaired hemocyte viability and intracellular thiol levels and also an increase of hemocyte count, ROS levels and phagocytosis. Phagocytosis and ROS levels in hemocytes were correlated with heterotrophic bacterial counts in snails. We found four genes with increased mRNA expression as a response to exposure of municipal wastewaters: TLR4 (6-fold), HSP70 (2-fold), SeGPx (4-fold) and CAT (2-fold). Immunocompetence responses were analyzed by canonical analysis to seek out relationships with mRNA expression of the genes involved in stress, pattern recognition, cellular and humoral responses. The data revealed that genes involved in oxidative stress were strongly involved with immunocompetence and that the resulting immune responses were influenced both by the bacterial and pollutant loadings of the effluent.

Natural variability and response interpretation of fecundity, vertebrate-like sex-steroid levels and energy status in the New Zealand mudsnail Potamopyrgus antipodarum (Gray).

Potamopyrgus antipodarum is a promising test organism that is often used in ecotoxicology, both in laboratory and field exposures. As no data are available on the physiological variation range of its life-traits and the biomarkers it uses, we studied the variation of fecundity, steroid levels and energy reserves over the course of a year in a field population. The reproductive cycle was described and showed seasonal activity during summer and autumn. Steroid levels (17ß-estradiol and testosterone) varied significantly during the year and were correlated with the reproductive cycle, which suggested a potential role for sex-steroids in P. antipodarum reproduction. Energy status also showed seasonal variations. Triglycerides (TG) seemed to be the main energy lipid, whereas cholesterol appeared to be mostly used as a structural lipid. Proteins were also involved in the reproductive cycle, but only when TG were not sufficient to support the reproductive strain, similar to cholesterol. Glycogen seemed to be used as an early reserve. Threshold values under which no reproduction occurred were defined in starved snails. We proposed a range of variation in the measured parameters, allowing for a better understanding and interpretation of their levels during laboratory or in situ exposures. The data suggest that the variability of fecundity in snails has not been fully appreciated in literature.

Is exposure temperature a confounding factor for the assessment of reproductive parameters of New Zealand mudsnails Potamopyrgus antipodarum (Gray)?

Potamopyrgus antipodarum is a promising test organism often used in ecotoxicology testing, both in laboratory and in field exposure experiments. It has been recommended for use in the development of an OECD reproduction test. However, exposure temperature is important to take into account when assessing reproduction and related biomarkers, because it can act as a confounding factor inducing variability in physiological values. The effect of three environmentally realistic exposure temperatures (8, 16 and 24°C) was examined with respect to the number of neonates born, the number of embryos in the brood pouch and the duration of embryonic development. We also measured additional markers likely to be related to the modulation of reproductive performance, such as vertebrate-like sex steroid, energy status and vitellin-like proteins. Exposure temperature had a significant effect on reproduction in P. antipodarum, on both the duration of embryonic development and the quantity of embryos and neonates. The consequences of these observations must not be neglected when using this species in laboratory and field experiments. This study determined suitable temperatures for field experiments and a mean duration for embryonic development independent of temperature. In addition to steroid levels, energy status and Vn-like protein levels were only slightly modified by exposure temperature between 8 and 24°C. Thus, they can be easily implemented and their variations related to anthropogenic factors during field exposure of mudsnails.

Comprehensive biological effects of a complex field poly-metallic pollution gradient on the New Zealand mudsnail Potamopyrgus antipodarum (Gray).

The Lot River is known to be contaminated by metals, mainly cadmium and zinc, due to a former Zn ore treatment plant in the watershed of the Riou-Mort, a tributary of the Lot River. Many studies have been performed to characterize contamination, but few have assessed its consequences on the biological responses of organisms along the gradient. We exposed adult and juvenile New Zealand freshwater mudsnails Potamopyrgus antipodarum at several sites along the gradient of metal contamination for 28 days. Biological responses were monitored at different levels: individual (survival, growth and fecundity), tissue and biochemical (energy status and vertebrate-like sex steroid levels) to better understand the toxicity mechanisms involved. Accumulation of Cd and Zn was high during exposure. Most of the biological effects observed could be linked to this contamination and were concentration-dependent. Histological lesions of the digestive gland were observed, with hypertrophy of calcium cells and vacuolization of digestive cells. Such effects are likely to explain the decrease of energy status (triglycerides and proteins), juvenile growth and adult fecundity observed at the most polluted site. However the magnitude of the fall in fecundity cannot be attributed only to these tissular effects, indicating another mode of action of Cd or possible confounding factors. Steroid accumulation in snails indicated only organic pollution. Histopathological effects proved the most sensitive endpoint to metal (Cd and Zn) contamination.

Development, validation and comparison of LC-MS/MS and RIA methods for quantification of vertebrates-like sex-steroids in prosobranch molluscs.

The role of vertebrate-like sex-steroids (testosterone, T, progesterone, P, and 17beta-estradiol, E2) in molluscs is still debated, but they could represent potential biomarkers of endocrine disruption. A radioimmunoassay (RIA) and a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods have been developed and compared to measure their levels in a gastropod snail Potamopyrgus antipodarum. Both methods showed a good reproducibility despite the complex matrix and the very low levels of vertebrate-like sex-steroids. Only T and P were detected using the LC-MS/MS method, while the RIA method reached lower detection limits and enabled the detection of all three steroids. Results indicated that T and P were mainly present as unconjugated forms. Both methods were compared in the analysis of snails exposed to waste water treatment plant effluents and led to the same conclusions concerning the modulation of steroids levels. Moreover, they both were in agreement concerning T measurements. On the other hand, LC-MS/MS appeared to be more suitable when analyzing P levels due to a low sensitivity of the RIA method. As E2 was not measured using the LC-MS/MS method because of a higher detection limit compared to the other steroids, the results obtained with the RIA method should be interpreted with caution. LC-MS/MS remains the gold standard for sex-steroid determinations, however a relevant and alternative method based on RIA was developed, requiring fewer organisms. RIA seems a promising method as a screening tool for experimental use, allowing comparison of sex-steroid levels in the mudsnail both in laboratory and in field experiments.

Sensitivity of New Zealand mudsnail Potamopyrgus antipodarum (Gray) to a specific aromatase inhibitor.

The freshwater prosobranch Potamopyrgus antipodarum (Molluska, Hydrobiidea, Smith 1889) has been proposed as a suitable species to assess the impact of endocrine disrupting compounds (EDC) in aquatic ecosystems. Steroid hormone biosynthesis pathway is potentially an important target for EDC, and vertebrate-like sex steroids seem to play a functional role in the control of mollusk reproduction. To assess the response and the sensitivity of P. antipodarum to disrupters of the steroid hormone biosynthesis pathway, we have experienced the action of a specific vertebrate aromatase inhibitor, fadrozole, acting on 17beta-estradiol synthesis in two separate 28 and 42d exposures. Fadrozole had effects consistent with the expected mechanism of action. A decrease of the reproduction parameters (such as on the number of neonates and number of embryos in the brood pouch) in a dose-dependant manner was observed. The steroids levels were also impaired with the ratio 17beta-estradiol/testosterone decreased by half in exposed snails. This shift of the steroids balance was accompanied by some alteration in the gonads histology and immunohistochemistry in fadrozole-exposed snails. This study highlights the value role of P. antipodarum as a test species for assessing EDC effects in aquatic wildlife.

Effects of fluoxetine on the reproduction of two prosobranch mollusks: Potamopyrgus antipodarum and Valvata piscinalis.

Fluoxetine is a widely used antidepressant, frequently found in aquatic ecosystems. We investigated its effects on two freshwater prosobranch gastropods: Valvata piscinalis (European valve snail) and Potamopyrgus antipodarum (New Zealand mudsnail), which have different reproductive modes. The fecundity of V. piscinalis (cumulate number of eggs at day 42) was not affected with an NOEC of 100 mvg/L nominal concentration (69 microg/L measured concentration). The mudsnail P. antipodarum responded in a biphasic dose-effect curve at low concentrations. The cumulate number of neonates at day 42 had an LOEC of 100 microg/L (69 microg/L) and an NOEC of 33.3 microg/L (13 microg/L), whereas the embryos in the brood pouch at day 42 only showed an LOEC of 3.7 microg/L (1 microg/L). We also observed histological effects in P. antipodarum (gonadal thickness). Among the sexual steroids we measured only testosterone which varied, independent of reproduction. Moreover the use of two closely related species highlights the interspecific variability.

Fluoxetine effects assessment on the life cycle of aquatic invertebrates.

Fluoxetine is a serotonin re-uptake inhibitor, generally used as an antidepressant. It is suspected to provoke substantial effects in the aquatic environment. This study reports the effects of fluoxetine on the life cycle of four invertebrate species, Daphnia magna, Hyalella azteca and the snail Potamopyrgus antipodarum exposed to fluoxetine spiked-water and the midge Chironomus riparius exposed to fluoxetine-spiked sediments. For D. magna, a multi-generational study was performed with exposition of newborns from exposed organisms. Effects of fluoxetine could be found at low measured concentrations (around 10microgl(-1)), especially for parthenogenetic reproduction of D. magna and P. antipodarum. For daphnids, newborns length was impacted by fluoxetine and the second generation of exposed individuals showed much more pronounced effects than the first one, with a NOEC of 8.9microgl(-1). For P. antipodarum, significant decrease of reproduction was found for concentrations around 10microgl(-1). In contrast, we found no effect on the reproduction of H. azteca but a significant effect on growth, which resulted in a NOEC of 33microgl(-1), expressed in nominal concentration. No effect on C. riparius could be found for measured concentrations up to 59.5mgkg(-1). General mechanistic energy-based models showed poor relevance for data analysis, which suggests that fluoxetine targets specific mechanisms of reproduction.

Physiological and behavioural responses of Gammarus pulex (Crustacea: Amphipoda) exposed to cadmium.

The aim of this study was to investigate the effects of cadmium on physiological and behavioural responses in Gammarus pulex. In a first experiment, cadmium LC50s for different times were evaluated in 264 h experiment under continuous mode of exposure (LC50(96 h)=82.1 microgL(-1), LC50(120 h)=37.1 microgL(-1), LC50(168 h)=21.6 microgL(-1), LC50(264 h)=10.5 microgL(-1)). In a second experiment, the physiological and behavioural responses of the amphipod exposed to cadmium (0, 7.5 and 15 microgL(-1)) were investigated under laboratory conditions. The mortality and the whole body cadmium concentration of organisms exposed to cadmium were significantly higher than in controls. Concerning physiological responses, cadmium exposure exerted a significant decrease on osmolality and haemolymph Ca(2+) concentration, but not on haemolymph Na(+) and Cl(-) concentrations, whereas the Na(+)/K(+)-ATPase activity was significantly increased. Behavioural responses, such as feeding rate, locomotor and ventilatory activities, were significantly reduced in Cd exposed organisms. Mechanism of cadmium action and consequent energetic reallocation in favour of maintenance functions (i.e., osmoregulation) are discussed. The results of this study indicate that osmolality and locomotor activity in G. pulex could be effective ecophysiological/behavioural markers to monitor freshwater ecosystem and to assess the health of organisms.

Physiological and behavioural responses of Gammarus pulex exposed to acid stress.

Physiological and behavioural responses of the acid-sensitive amphipod Gammarus pulex exposed to a wide range of acid conditions (pH 4.1, 5.1, 6.0) under laboratory conditions were investigated. An exposure of 38 h to acid conditions caused significant decreases in survival rate, osmolality, haemolymph Na+ concentration, ventilatory and locomotor activity compared to organisms exposed to a circumneutral medium (pH 7.9). We highlighted the interest of using individual response distribution, since drastic effects can be detected in organisms exposed to pH 6.0, in particular for osmolality: The response can be divided into two groups, unimpacted and impacted organisms. Moreover this representation permitted to evaluate the health level of individual organisms through the determination of threshold values. Significant correlations between mean pH and mean physiological/behavioural responses were observed. The relationships between individual responses permitted not only to compare endpoints, but also to show that affected organisms were impacted by ionoregulation failure, hypoventilation and low locomotor activity. The energetic reallocation in favour of maintenance functions, such as osmoregulation, is discussed. The results of this study indicate that the values of haemolymph Na+ concentration, osmolality and locomotor activity in G. pulex could be effective ecophysiological/behavioural markers to monitor freshwater ecosystems and to assess the health of organisms or populations.

Measurement of dynamic mobilization of trace metals in sediments using DGT and comparison with bioaccumulation in Chironomus riparius: first results of an experimental study.

Sediments in aquatic ecosystems are often contaminated as a result of anthropogenic activities. Sediments and benthic organisms have been used to monitor trace metals contamination. However, due to the high variability of contaminant bioavailability, the attempt to link metal concentration in sediments and contamination of the organisms or ecotoxicological effect often lead to disappointing results. The technique of diffusive gradients in thin films (DGT) has been proposed as a relevant tool to study metal bioavailability, for example for accumulation in plants. In the present study, laboratory microcosm experiments were conducted with six contaminated sediments to compare metal accumulation in DGT and bioaccumulation in a chironomid (Chironomus riparius) for Cu, Cd and Pb . Metal accumulation in DGT was measured over time then modelled to determine two parameters of the dynamic response of the metals to DGT deployment: the size of the particulate labile pool and the kinetic of the solid-dissolved phase exchange. The mobility of metals was found metal and sediment dependent. A significant relationship between metal accumulated in DGT and bioaccumulated in chironomids was found for Cu and Pb. However, total metals in sediments were the best predictors of bioaccumulation. Nevertheless, the knowledge of the metals dynamic enhanced our ability to explain the different biological uptake observed in sediments of similar total metal concentrations.

Do differences between metal body residues reflect the differences between effects for Chironomus riparius exposed to different sediments?

Sediment characteristics are well known to interfere with toxicity, mainly through differences in terms of bioaccumulation. Here, with chironomids exposed to zinc in an artificial and a field sediment, we investigated the differences of zinc accumulation and of effects on the life cycle, at individual and population level. We used biology and energy-based modeling to analyze the data at all the levels of biological organization. This permits a reliable estimation of thresholds values for tissue residues. Differences in zinc tissue residues accounted for most of the differences between the results for both sediments (a factor of 11 for differences from 20 to 100 depending on the parameter which is considered). Taking into account accumulation and background variability, the differences relative to thresholds could be accounted for. However, it appeared that, once the threshold was passed, effects were much more pronounced for organisms exposed to artificial sediment compared to field sediment. We concluded that some sediment characteristics can enhance toxicity, in addition to their influence on the compound accumulation, even if the latter was the major source of differences in our study.

Non destructive in vivo measurement of ethoxyresorufin biotransformation by zebrafish prolarva: development and application.

Biotransformation enzymatic activities, such as the cytochrome P450 one, have been used as biomarkers for pollution assessment for a long time. Nevertheless, such biochemical tools are destructive processes, because they are performed on fish liver or total larvae homogenates. Moreover, the adaptation of this bioassay to some fish larvae, like Danio rerio ones, is ineffective because it needs a lot of organisms. We thus developed an original, nondestructive method to detect the induction of a biotransformation activity in the prolarva of the fish, Danio rerio, exposed to chemicals. This methodology is based on the assessment of EROD activity, by measurement in the culture medium of the fluorescence increase due to the excretion of resorufin by fish during an ethoxyresorufin exposure. After exposure of fish embryos to known inducers (BaP and beta-naphtoflavone), the prolarvae were exposed to the substrate (ethoxyresorufin), and the kinetic of the fluorescence increase was measured. A dose-effect relationship was observed, with a three to five fold increase of EROD basal activity. This methodology also allowed us to compare between EROD activity induction by different environmental samples. The proposed methodology thus allows to perform a simple, sensitive, and reproducible microbiotest for the detection of sublethal concentrations of AhR chemical inducers in environmental samples.