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.

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.

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.

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.

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.

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.

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.

Is endotoxin and cytokine release related to a decrease in gastric intramucosal pH after hemorrhagic shock?

OBJECTIVES: (a) To investigate the relationship between gut ischemia parameters (gastric intramucosal pH [pHi], mucosal-arterial carbon dioxide difference [PCO2-gap]), and endotoxin or cytokine release during hemorrhagic shock; (b) to compare the predictive value of pHi, PCO2-gap and arterial lactate concentrations. DESIGN: Prospective study. SETTING: Surgical intensive care unit of a university hospital. PATIENTS: 20 multiple trauma patients with severe hemorrhagic shock. INTERVENTIONS: Intramucosal measurements and blood samples were obtained on admission to the emergency room and repeatedly over 48 h. MEASUREMENTS AND RESULTS: Endotoxin was measured using a chromogenic limulus amoebocyte assay. Cytokine [tumor necrosis factor-alpha (TNF alpha) and interleukin-6 (IL-6)] values were evaluated by immunoradiometric assays. Only 3 patients had positive blood cultures but endotoxins were detected at least once in all patients. Endotoxin levels were similar in survivors and non-survivors over the study period and were not related to pHi or PCO2-gap. Initially, high levels of IL-6 were observed in both nonsurvivors and survivors [median 1778 pg/ml (range 435-44,540) vs 2068 pg/ml (range 996-92,300)]. IL-6 levels progressively decreased in the survivors but not significantly. On admission, TNF alpha concentrations were similar in nonsurvivors and survivors (42 +/- 35 vs 46 +/- 27 pg/ml). From the 24th h, TNF alpha values were higher in the nonsurvivors than in the survivors (24 h: 72 +/- 38 vs 34 +/- 17 pg/ml, p < 0.05). The greatest IL-6 levels were found for a pHi < 7.20 (28.5 +/- 36.5 vs 1.8 +/- 1.3 ng/ml, p < 0.05) or a PCO2-gap > 7.5 mmHg (1 kPa) (32.5 +/- 37.5 vs 1.7 +/- 1.3 ng/ml, p < 0.01). With the same pHi threshold, no difference was found in endotoxin levels. The lactate concentrations were predictive for outcome from the 12th h (9.5 +/- 5.9 vs 3.6 +/- 2.3 mmol/l, p < 0.05). CONCLUSIONS: During severe hemorrhagic shock, endotoxin translocation from the gut was a common phenomenon that seemed independent of both pHi values and outcome. It could not explain IL-6 and TNF alpha release. In severe hemorrhagic shock, neither pHi nor PCO2-gap provides additional information to the lactate measurements.

Survival tests with Chironomus riparius exposed to spiked sediments can profit from DEBtox model.

DEBtox model is a biologically based model used to analyse aquatic toxicity data (The analysis of aquatic toxicity data, VU University Press, Amsterdam, The Netherlands, 1996, 149pp.). To date, it has not been used to analyse sediment survival tests, mainly because this would require daily counting of survivors, which is difficult for benthic organisms. In the present study, we adapted survival toxicity tests with the midge Chironomus riparius to permit survival data analysis with DEBtox. To validate the adaptation proposed, we exposed organisms to two chemicals, copper and methiocarb. We exposed larvae of second, third and fourth instar to different concentrations of the tested compounds and two different diets to assess the influence of diet and instar on DEBtox parameter estimates. Daily counting of organisms did not affect survival and did not lead to much more effort when compared to usual tests. Moreover, the analysis profited much from use of the DEBtox model. It was possible, with only survival data to estimate and validate the kinetics of the compounds, to predict survival during the recovery of exposed organisms and to assess the influence of food availability on toxicity. Food availability did not influence methiocarb toxicity, but copper was more toxic to food-limited organisms when a threshold concentration was exceeded. Comparison of parameters estimate also helped in understanding the differences in toxicity responses between instars. For the two compounds we studied, the difference was entirely explained by differences in threshold values.

Impacts of 17beta-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio).

Zebrafish (Danio rerio) were exposed for 3 weeks to low concentrations of estradiol including environmentally relevant concentrations (5, 25 and 100 ng/l), encompassing either their embryo-larvae (from fertilization to 21 day post-fertilization (dpf)), juvenile (from 21 to 42 dpf) or adult life stages (>200 dpf) with a view to investigating the most sensitive life stage of the zebrafish to 17beta-estradiol (E2). At all sampling points, whole-body vitellogenin concentrations and gonadal development were analyzed in order to investigate the effects of estrogen exposure on these endpoint in the zebrafish. In the adult stage, additional endpoints were measured including secondary sexual characteristics (manifestation of the uro-genital papillae (UGP) in males), gonadal growth (the gonado-somatic index (GSI)) and sex ratio. For all the different life stage exposures, reproductive performance of the F0 generation was assessed (egg production) and survival and development of the F1 embryo-larvae. Exposure to low concentrations of E2 resulted in vitellogenin induction whatever the life stage exposed but these effects were reversible after depuration. The effective concentration for vitellogenin induction in zebrafish early life stages was 100 ng E2/l, and in adult male zebrafish the effective concentration for vitellogenin induction (between 5 and 25 ng/l) was lower than for the early life stage fish. Exposure to E2 prior to (from fertilization to 21 dpf) and during the time of sex differentiation (from 21 to 42 dpf) also caused disruptions in the process of sexual differentiation (resulting in formation of a retrogonadal cavity in presumptive male, germ cell development and leading to a significant change of the sex ratio towards the female sex at the dose of 100 ng E2/l for the fish exposure as embryo-larvae) and altered patterns of egg production in the subsequent adults. Exposure of adult fish to E2 resulted in a modification of the secondary sexual characteristic in males at 25 and 100 ng E2/l as well as a dose-dependent inhibition of egg production. The findings from this study show that the nature and intensity of the reproductive effects of E2 are dependent of the time and concentration of exposures of zebrafish to E2, some of these effects being permanent (effect on the sexual differentiation) while others being reversible (effect on the Vtg induction). This study demonstrated that early life stages of zebrafish are sensitive to low concentrations of E2 and provides relevant data that could be used for the adaptation of existing fish early life stage test for the in vivo testing of estrogenic compounds. The data presented raise further concerns about the effects of steroid estrogens in the environment on fish reproductive health.

The measurement of cholinesterase activities as a biomarker in chub (Leuciscus cephalus): the fish length should not be ignored.

Biomarkers are early warning systems of the exposure of aquatic organisms to pollutants. Among them, the measurement of the cholinesterase (ChE) activities in fish muscle is a biomarker of the exposure to organophosphosphates and carbamates pesticides. As such it has been used in numerous field studies both in marine and continental waters. Cyprinids (chub, Leuciscus cephalus) were sampled in river sites (France) in relatively clean and polluted areas. We performed the statistical analysis of the ChE activities and we generally observed a statistical relationship between ChE activities and fish length, the larger fish having the lower ChE activities. We concluded that the great majority of the significant differences in ChE activities between sites could be due in fact to differences in fish length between field samples. We stress the importance of taking into account the fish length whenever differences in ChE levels between field sites must be interpreted.

Effects of advanced oxidation processes (AOPs) on the toxicity of a mixture of pharmaceuticals.

The possibility of applying main AOP techniques, namely ozonation, H2O2/UV photolysis and TiO2 photocatalysis to provide a significant reduction of toxicity of pharmaceutical mixtures has been evaluated. For the preparation of the mixture six pharmaceuticals were chosen among those found at highest concentrations in Sewage Treatment Plant effluents, namely carbamazepine, clofibric acid, diclofenac, sulfamethoxazole, ofloxacin and propranolol. The blue-green alga Synechococcus leopoliensis and the rotifer Brachyonus calyciflorus were utilised to assess the toxicity of the mixtures after AOP treatments. All the toxicity tests were performed using chronic standardized bioassays. The best results were obtained with ozonation. With this type of treatment a complete removal of mixture toxicity on S. leopolensis was obtained even after the shortest time of application (1 min). The ozonation treatment leads also to removal of all the pharmaceutical mixture toxicity on B. calyciflorus, by applying the oxidizing agent for at least for 2 minutes.

[Clonidine premedication and isoflurane anesthesia to reduce bleeding in otologic surgery]. / Clonidine en prémédication et anesthésie à l'isoflurane pour la réduction du saignement en chirurgie otologique.

Seventy-seven ASA 1 patients scheduled for ear surgery were premedicated orally, 90 min before anaesthesia. They were randomly assigned to two groups, according to the drug used: hydroxyzine alone (group T, n = 39) or combined with clonidine (4.9 +/- 0.3 micrograms.kg-1) (group C, n = 38). Anaesthesia was induced with midazolam (0.3 mg.kg-1) and alfentanil (30 micrograms.kg-1). Ventilation was controlled with a 50/50 v/v mixture of oxygen and nitrous oxide (FETCO2 = 4 to 4.5%), and anaesthesia was maintained with repeated injections of alfentanil (15 micrograms.kg-1 at the start of surgery, and then every 15 min) and with isoflurane (mean end-expiratory concentration 0.6 +/- 0.3 vol %). Surgical bleeding was assessed every ten minutes on a numerical scale with four values. A bloodless surgical field was obtained by adjusting the isoflurane concentration up to 2 MAC, and by using a trinitrine infusion as required. Cardiovascular monitoring included an electrocardioscope and automatic blood pressure measurements. Before induction of anaesthesia, the blood pressure was lower in group C (84.7 +/- 11.2 vs. 95.9 +/- 106 mmHg) (p less than 0.001); the difference in heart rate was not significant (65 +/- 15 vs. 70.6 +/- 14 b.min-1). Moderate stable intraoperative hypotension was obtained in both groups. However, mean arterial blood pressure (C:65.8 +/- 7.8 mmHg; T: 73 +/- 9.4 mmHg) and heart rate (C: 53.4 +/- 6.8 b.min-1; T: 60.4 +/- 8 b.min-1) were significantly lower in the patients premedicated with clonidine (p less than 0.001). There were more periods of sinus bradycardia (heart rate less than or equal to 50 b.min-1), mostly seen before the beginning of surgery, in group C patients (p less than 0.01); atropine was also required more often (when the heart rate was less than or equal to 40 b.min-1) in this group of patients (NS). The comparative assessment of surgical field quality was in favour of group C (no troublesome bleeding) as opposed to the control group (16% troublesome bleeding); there were also more bloodless surgical fields in the former group (73.7% vs. 48.7% in group T, p less than 0.05). This study therefore demonstrated that clonidine premedication before anaesthesia with isoflurane was helpful in decreasing bleeding during ear surgery.

[Calculation of the blood volume to be removed for intentional normovolemic hemodilution]. / Règle à calcul du volume sanguin à prélever pour réaliser une hémodilution normovolémique intentionnelle.

Intentional haemodilution is more and more frequently used. Although it is a simple technique, it can only be carried out with a maximum of safety if the operator has an idea of the amount of blood that needs to be removed to obtain the required haematocrit value. Several methods have been suggested, using more or less complex calculations, or nomogrammes. A new simple rule is presented here which gives an estimation of the amount of blood to be removed in three steps: 1) the theoretical total blood volume (VS), according to weight, height and sex of the patient, 2) the ratio (R) between the initial and required final haematocrit, and 3) the volume of blood to be removed (Vp) according to VS and R. A mono-compartment model with identical inflow and outflow was used. A multicentre trial with 229 haemodilutions showed that the volume removed (mean = 1,325 ml; SD = 642 ml) was 15% less than the calculated volume (mean = 1,526 ml; SD = 561 ml). No one factor was found to explain this difference. The haematocrit values obtained by microcentrifuge were compared with those obtained by a laboratory automatic counter. The mean of the differences was 0.002 (SD = 0.029). Because of the possible errors involved in estimating the total blood volume and the haematocrit before dilution, it is essential that the haematocrit be checked at least once during the haemodilution.

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.

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.

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.

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.

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.