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The authors thank the German Environment Agency (UBA) (projects 370861402 and 371165417) for funding, the Danish EPA (DK) and INRA for the financial support and all participating laboratories for using own funds and the good cooperation so that the project could be successfully carried out. We are also grateful to Heike Heidenreich from the IHI Zittau, Germany and Olaf Wappelhorst from chemlab GmbH, Germany for performing chemical analyses.; Mollusks are known to be uniquely sensitive to a number of reproductive toxicants including some vertebrate endocrine disrupting chemicals. However, they have widely been ignored in environmental risk assessment procedures for chemicals. This study describes the validation of the Potamopyrgus antipodarum reproduction test within the OECD Conceptual Framework for Endocrine Disrupters Testing and Assessment. The number of embryos in the brood pouch and adult mortality serve as main endpoints. The experiments are conducted as static systems in beakers filled with artificial medium, which is aerated trough glass pipettes. The test chemical is dispersed into the medium, and adult snails are subsequently introduced into the beakers. After 28 days the reproductive success is determined by opening the brood pouch and embryo counting. This study presents the results of two validation studies of the reproduction test with eleven laboratories and the chemicals tributyltin (TBT) with nominal concentrations ranging from 10 to 1000 ng TBT-Sn/L and cadmium with concentrations from 1.56 to 25 mu g/L. The test design could be implemented by all laboratories resulting in comparable effect concentrations for the endpoint number of embryos in the brood, pouch. After TBT exposure mean EC10, EC50, NOEC and LOEC were 35.6, 127, 39.2 and 75.7 ng Sn/L, respectively. Mean effect concentrations in cadmium exposed snails were, respectively, 6.53, 14.2, 6.45 and 12.6 mu g/L. The effect concentrations are in good accordance with already published data. Both validation studies show that the reproduction test with P. antipodarum is a well-suited tool to assess reproductive effects of chemicals. (C) 2017 Elsevier Ltd. All rights reserved.

Tributyltin (TBT) has been recognized as an endocrine disrupting chemical (EDC) for several decades. However, only in the last decade, was its primary endocrine mechanism of action (MeOA) elucidated—interactions with the nuclear retinoid-X receptor (RXR), peroxisome proliferator-activated receptor γ (PPARγ), and their heterodimers. This molecular initiating event (MIE) alters a range of reproductive, developmental, and metabolic pathways at the organism level. It is noteworthy that a variety of MeOAs have been proposed over the years for the observed endocrine-type effects of TBT; however, convincing data for the MIE was provided only recently and now several researchers have confirmed and refined the information on this MeOA. One of the most important lessons learned from years of research on TBT concerns apparent species sensitivity. Several aspects such as the rates of uptake and elimination, chemical potency, and metabolic capacity are all important for identifying the most sensitive species for a given chemical, including EDCs. For TBT, much of this was discovered by trial and error, hence important relationships and important sensitive taxa were not identified until several decades after its introduction to the environment. As recognized for many years, TBT-induced responses are known to occur at very low concentrations for molluscs, a fact that has more recently also been observed in fish species. This review explores the MeOA and effects of TBT in different species (aquatic molluscs and other invertebrates, fish, amphibians, birds, and mammals) according to the OECD Conceptual Framework for Endocrine Disruptor Testing and Assessment (CFEDTA). The information gathered on biological effects that are relevant for populations of aquatic animals was used to construct Species Sensitivity Distributions (SSDs) based on No Observed Effect Concentrations (NOECs) and Lowest Observed Effect Concentrations (LOECs). Fish appear at the lower end of these distributions, showing that they are as sensitive as molluscs, and for some species, even more sensitive. Concentrations in the range of 1 ng/L for water exposure (10 ng/g for whole-body burden) have been shown to elicit endocrine-type responses, whereas mortality occurs at water concentrations ten times higher. Current screening and assessment methodologies as compiled in the OECD CFEDTA are able to identify TBT as a potent endocrine disruptor with a high environmental risk for the original use pattern. If those approaches had been available when TBT was introduced to the market, it is likely that its use would have been regulated sooner, thus avoiding the detrimental effects on marine gastropod populations and communities as documented over several decades.

Ecological risk assessment of chemicals in mesocosms requires measurement of a large number of parameters at the community level. Studies on invertebrate communities usually focus on taxonomic approaches, which only provide insights into taxonomic structure changes induced by chemicals. In the present study, abundance, biomass (B), theoretical production (P), and instantaneous P/B ratio were used as endpoints to assess the effects of the commercial form of the dithiocarbamate fungicide thiram (35 µg/L and 170 µg/L nominal concentrations) and of the hydrocarbon water accommodated fraction (HWAF) of a petroleum distillate (0.01 mg/L, 0.4 mg/L, 2 mg/L, and 20 mg/L loadings) on the zooplankton community in freshwater pond mesocosms. Endpoints were measured during a 4-wk treatment period (1 pulse/wk) followed by a 5-mo posttreatment period to evaluate zooplankton population recovery. The chlorophyll a concentration in water was significantly increased after treatment with HWAF, whereas it was not affected by thiram treatment. Zooplankton abundance-based analysis showed effects on a limited number of taxa, whereas other endpoints (mainly the P/B ratio) revealed that more taxa were impacted, with recovery depending on the chemical and concentration. Exposure to HWAF mainly had a negative impact on cladocerans, which resulted in top-down effects (between cladocerans and phytoplankton). Thiram negatively affected rotifers and copepods, suggesting more direct toxic effects. The results show that the use of secondary production as an endpoint provides a more comprehensive assessment of potential direct and indirect effects of chemicals on a community, and they also support evidence of alteration in functional processes.

Endocrine disruptors are known to alter endogenous free and esterified levels of androgenic and estrogenic steroid hormones in aquatic mollusks. The origin of steroids in these animals, however, remains controversial. In the present study, free and esterified testosterone concentrations were measured in the hermaphroditic aquatic gastropod Lymnaea stagnalis exposed to molecules known for their androgenic (testosterone and tributyltin), anti-androgenic (cyproterone-acetate), and estrogenic (chlordecone) properties, by reference to their mode of action in vertebrates. In parallel, snail oviposition and fecundity were followed over a 21-d exposure period. Testosterone exposure resulted in increased esterified testosterone levels, whereas free testosterone concentrations remained stable. In contrast, cyproterone-acetate significantly increased the free form of testosterone with no changes in the esterified form, whereas chlordecone showed a tendency to reduce (though not significantly) esterified testosterone concentrations without changing free testosterone levels. Finally, tributyltin did not alter testosterone homeostasis. The production of egg clutches and eggs was significantly reduced only in the snails exposed to the highest concentrations of chlordecone (19.6 mu g/L) and tributyltin (94.2ng Sn/L). Overall, the present study demonstrates that uptake of testosterone from the exposure medium occurs in L. stagnalis. Moreover, it shows that cyproterone-acetate and, to a lesser extent, chlordecone can alter endogenous testosterone levels in this freshwater snail. However, the relationship between hormonal changes and snail reproduction has not been established. Environ Toxicol Chem 2013;32:1740-1745. (c) 2013 SETAC

International audience; Chlordecone (CLD) is an organochlorine insecticide abundant in aquatic environment of the French West Indies. However, few studies have investigated its impact on freshwater invertebrates. Whereas CLD is suspected of inducing endocrine disruption, this work aimed to study the effects of environmentally relevant concentrations of CLD on the 20-hydroxyecdysone (20-HE) hormone concentration and on the chitobiase activity, both having key roles in the molting process of crustaceans. In addition, the bioaccumulation of CLD was measured in the muscle tissue of Macrobrachium rosenbergii to underline potential dose-response relationship. The results have shown that CLD was bioaccumulated in exposed organisms according to a trend to a dose-response relationship. Moreover, it was observed that CLD decreased the 20-HE concentration in exposed prawns when compared to control, whatever the duration of exposure, as well as it inhibited the chitobiase activity after 30 days of exposure. The present study indicates that CLD could interfere with molting process of M. rosenbergii by disturbing the 20-HE concentration and the activity of chitobiase, suggesting consequences at the long term on the shrimp development. This study also confirmed that CLD could be an endocrine disruptor in decapod crustaceans, as it was already observed in vertebrates.

Interest in stream mesocosms has recently revived for higher tier aquatic macrophyte risk assessment of plant protection products mainly because 1) the highest predicted environmental concentrations for the assessment of effects are frequently derived from stream scenarios, and 2) they allow an effect assessment using stream-typical pulse exposures. Therefore, the present stream mesocosm study used an herbicide pulse exposure and evaluated the responses of Elodea canadensis and Myriophyllum spicatum. Macrophytes were exposed for 24 h to 1 μg/L, 3 μg/L, 10 μg/L, and 30 μg/L of the herbicide iofensulfuron-sodium with a subsequent recovery period of 42 d. Biological endpoints were growth rates of the main, side, and total shoot length, the shoot number, the maximum root length, and the dry weight. The total shoot length was identified as the most sensitive endpoint; the growth rate of the total shoot length was inhibited by up to 66% and 45% in M. spicatum and E. canadensis, respectively. The lowest no observed effect concentrations (NOECs) were observed at day 7 and/or day 14 after herbicide treatment and were 1 μg/L for M. spicatum and 3 μg/L for E. canadensis. The no-observed-ecologically-adverse-effect concentrations (NOEAECs) were 10 μg/L and 30 μg/L for M. spicatum and E. canadensis, respectively. Such or similar mesocosm designs are useful to simulate typical stream exposures and estimate herbicide effects on aquatic macrophytes in stream systems. Environ Toxicol Chem 2017;36:1090-1100. © 2016 SETAC.

The immediate response and recovery of the macrobenthic communities of nonisolated and isolated freshwater outdoor 9 ml mesocosms following an acute stress caused by the addition of deltamethrin were studied over a 14-month period. To discriminate between internal and external recovery mechanisms, half of the treated ponds were covered by 1-mm mesh lids that restricted aerial recolonization. Both structural (abundance of the different taxonomic groups) and functional (litter breakdown) parameters were monitored. Insects were broadly reduced in numbers by deltamethrin addition. In general, noninsect groups were not affected or increased in abundance in deltamethrin-treated ponds, probably because of relative insensitivity to deltamethrin, reduced predation, and lower competition for food. No major change in litter breakdown rates were seen, probably because of functional redundancy among the macrobenthic community. Chironominae larvae recovered in open, treated mesocosms 62 d after deltamethrin addition and most insect groups recovered 84 d after the treatment date. However, the presence of lids significantly reduced insect recovery rate, suggesting that it largely depends on the immigration of winged forms (i.e., external recovery) from surrounding non- or less affected systems. These results indicate that the recovery time of macrobenthic communities in an affected natural pond would depend on spatial characteristics of the landscape and also the season that exposure occurs. Isolated ecosystems would display posttreatment insect recovery dynamics very different from highly connected ones, evolving toward alternate pseudoequilibrium states, possibly with lower biodiversity but with preserved functionality. Consequences for higher tier risk assessment of pesticides are discussed.

Responses of circulating hemocytes were studied in Lymnaea stagnalis exposed to 10, 30, 90, and 270 microg/L fomesafen for 24 and 504 h. Flow cytometry was used to quantify fomesafen-induced production of reactive oxygen species (ROS), phagocytic activity on Escherichia coli, and oxidative burst when hemocytes were challenged by E. coli or phorbol 12-myristate-13-acetate (PMA). Lysosomal membrane damage was assessed, using the neutral-red retention time (NRRT) assay. Exposure to fomesafen for 24 h resulted in increase in ROS levels and decreases in phagocytosis and the oxidative burst in PMA-stimulated hemocytes. After 504 h, intracellular levels of ROS returned to normal, but phagocytosis of E. coli was still inhibited and the associated oxidative burst significantly reduced. After both durations of exposure, decreases of NRRT indicated that lysosome membrane fragility increased with fomesafen concentration. Potential implications for the health and survival of the snails and consequences on populations are discussed.

The aim of this study was to examine sex- and age-dependent changes in carbohydrate and lipid reserves throughout the fourth larval instar of Chironomus riparius Meigen. Individuals were sexed using genital discs and aged using phases of development defined through the observation of genital and thoracic imaginal discs. Glycogen concentration was measured in the eviscerated body (fat body, muscles, and cuticle), and the levels of glucose and trehalose were followed in the hemolymph, whereas the amounts of free glycerol and triglycerides were measured in both tissues. Compared with males, females had higher levels of body reserves, whereas the levels of sugars in the hemolymph were similar for both sexes, except for free glucose, which was slightly higher in males. The levels of energy-yielding substrates in both the eviscerated body and hemolymph varied significantly with the phase of development for both sexes, except for the concentration in free glycerol in the hemolymph. Analysis of variability indicated that selection of larvae for both the sex and phase of development significantly reduces uncertainty of biochemical measures. The results are discussed with respect to potential use of the levels of energy-yielding substrates as endpoints in toxicity tests with fourth-instar C. riparius larvae.

The influence of nonylphenol polyethoxylates (NPEO), formulated astheadjuvant Agral® 90, on the effects of the diphenyl ether herbicide fomesafen in the pond snail Lymnaea stagnalis was investigated, with particular attention to the reproductive performances and underlying energetic and hormonal processes. Separate short-term exposures to low concentrations of fomesafen and fomesafen-Agral mixture were performed in the laboratory. Outdoor experimental ponds (mesocosms) were used for long-term exposures to higher chemical concentrations. At the concentrations used in the studies, NPEO were known as nontoxic in L. stagnalis. Fomesafen was mixed with the adjuvant in the 3:7 ratio recommended for agricultural uses (nominal herbicide concentrations of 22 and 40 μg/L in laboratory and mesocosm, respectively). In mesocosms, multiple application of fomesafen, leading to maximal herbicide concentrations of 60.33 ± 2.68 μg/L in water, resulted in reduced number of egg masses and altered glycogen metabolism in contaminated snails. These changes, as well as affected steroid-like levels in fomesafen-exposed snails, support the hypothesis of impaired neuroendocrine functions. When Agral 90 was added to the herbicide, results obtained in mesocosms showed that the adjuvant softened the impact of fomesafen. In mesocosms treated with the fomesafen-Agral mixture, significantly lower herbicide levels were found in the water (30.33 ± 14.91 μg/L at the end of the contamination period). Consequently, internal exposure of the snails to fomesafen was reduced when the herbicide was mixed with the adjuvant. Mitigation of the effects of fomesafen by the adjuvant may therefore result from nonionic surfactant activity of NPEO that prevented fomesafen from reaching the snails.

The sensitivity of diatom taxonomy and trait-based endpoints to chemicals has been poorly used so far in Environmental Risk Assessment. In this study, diatom assemblages in outdoor flow-through mesocosms were exposed to thiram (35 and 170 mu g/L), and a hydrocarbon emulsion (HE; 0.01, 0.4, 2 and 20 mg/L). The effects of exposure were assessed for 12 weeks, including 9 weeks post-treatment, using taxonomic structure and diversity, bioindication indices, biological traits, functional diversity indices, indicator classes and ecological guilds. For both chemicals, diversity increased after the treatment period, and responses of ecological traits were roughly identical with an abundance increase of motile taxa tolerant to organic pollution and decrease of low profile taxa. Bioindication indices were not affected. Traits provided a complementary approach to biomass measurements and taxonomic descriptors, leading to a more comprehensive overview of ecological changes due to organic chemicals, including short- and long-term effects on biofilm structure and functioning. (C) 2014 Elsevier Ltd. All rights reserved.

The hermaphroditic gastropod Lymnaea stagnalis is proposed as a candidate species for the development of OECD guidelines for testing of the reprotoxicity of chemicals, including endocrine active substances (EASs). Up to now, only a few putative EASs have been tested for their reproductive toxicity in this species. In this study, we investigate the effects of four EASs with different affinities to the vertebrate estrogen and androgen receptors (chlordecone as an estrogen; cyproterone acetate, fenitrothion and vinclozolin as anti-androgens) on the reproduction of L stagnalis in a 21-day semi-static test. Testosterone and 17 alpha-ethinylestradiol (EE2) were used as the reference compounds. The tested EASs had no significant effect on growth and survival at the tested concentration ranges (ng to mu g/L. Classical reproduction endpoints (i.e., oviposition and fecundity) were not responsive to the tested chemicals, except for chlordecone and 17 alpha-ethinylestradiol, which hampered reproduction from 19.6 mu g/L and 17.6 mu g/L, respectively. The frequency of polyembryonic eggs, used as an additional endpoint, demonstrated the effects of all compounds except EE2. The molecular pathways, which are involved in such reproduction impairments, remain unknown. Our results suggest that egg quality is a more sensitive endpoint as compared to other reproductive endpoints commonly assessed in mollusk toxicity tests. (C) 2014 Elsevier B.V. All rights reserved.

Tributyltin (TBT) and triphenyltin (TPT) are emblematic endocrine disruptors, which have been mostly studied in gonochoric prosobranchs. Although both compounds can simultaneously occur in the environment, they have mainly been tested separately for their effects on snail reproduction. Because large discrepancies in experimental conditions occurred in these tests, the present study aimed to compare the relative toxicity of TBT and TPT under similar laboratory conditions in the range of 0 ng Sn/L to 600 ng Sn/L. Tests were performed on the simultaneous hermaphrodite Lymnaea stagnalis, a freshwater snail in which effects of TPT were unknown. Survival, shell length, and reproduction were monitored in a 21-d semistatic test. Frequency of abnormal eggs was assessed as an additional endpoint. Triphenyltin hampered survival while TBT did not. Major effects on shell solidity and reproduction were observed for both compounds, reproductive outputs being more severely hampered by TBT than by TPT. Considering the frequency of abnormal eggs allowed increasing test sensitivity, because snail responses to TBT could be detected at concentrations as low as 19 ng Sn/L. However, the putative mode of action of the 2 compounds could not be deduced from the structure of the molecules or from the response of apical endpoints. Sensitivity of L. stagnalis to TBT and TPT was compared with the sensitivity of prosobranch mollusks with different habitats and different reproductive strategies.

The presence of pesticides in the environment results in potential unwanted effects on non-target species. Freshwater organisms inhabiting water bodies adjacent to agricultural areas, such as ditches, ponds and marshes, are good models to test such effects as various pesticides may reach these habitats through several ways, including aerial drift, run-off, and drainage. Diquat is a non-selective herbicide used for crop protection or for weed control in such water bodies. In this study, we investigated the effects of diquat on a widely spread aquatic invertebrate, the holarctic freshwater snail Lymnaea stagnalis. Due to the known redox-cycling properties of diquat, we studied transcript expression and enzymatic activities relative to oxidative and general stress in the haemolymph and gonado-digestive complex (GDC). As diquat is not persistent, snails were exposed for short times (5, 24, and 48 h) to ecologically relevant concentrations (22.2, 44.4, and 222.2 mu g l(-1)) of diquat dibromide. RT-qPCR was used to quantify the transcription of genes encoding catalase (cat), a cytosolic superoxide dismutase (Cu/Zn-sod), a selenium-dependent glutathione peroxidase (gpx), a glutathione reductase (gred), the retinoid X receptor (rxr), two heat shock proteins (hsp40 and hsp70), cortactin (cor) and the two ribosomal genes r18S and r28s. Enzymatic activities of SOD, Gpx, Gred and glutathione S-transferase (GST) were investigated in the GDC using spectrophoto/fluorometric methods. Opposite trends were obtained in the haemolymph depending on the herbicide concentration. At the lowest concentration, effects were mainly observed after 24 h of exposure, with over-transcription of cor, hsp40, rxr, and sod, whereas higher concentrations down-regulated the expression of most of the studied transcripts, especially after 48 h of exposure. In the GDC, earlier responses were observed and the fold-change magnitude was generally much higher: transcription of all target genes increased significantly (or non-significantly for cat) after 5 h of exposure, and went back to control levels afterwards, suggesting the onset of an early response to oxidative stress associated to the unbalance of reactive oxygen species (ROS) in hepatocytes. Although increases obtained for Gred and SOD activities were globally consistent with their respective transcript expressions, up-regulation of transcription was not always correlated with increase of enzymatic activity, indicating that diquat might affect steps downstream of transcription. However, constitutive levels of enzymatic activities were at least maintained. In conclusion, diquat was shown to affect expression of the whole set of studied transcripts, reflecting their suitability as markers of early response to oxidative stress in L. stagnalis. (c) 2012 Elsevier B.V. All rights reserved.

Freshwater pond mesocosms were used to validate xenobiotic-metabolizing enzymes as biomarkers of contamination by atrazine and hexachlorobenzene (HCB) in a basommatophoran gastropod, Lymnaea palustris (Muller). Over long-term (21-d) exposure to 5, 25, and 125 μg/L atrazine and to 0.5, 1.25, and 5 μg/L HCB, the uptake and internal concentration of both pesticides were followed, and the activities of benzo[a]pyrene hydroxylase (BaPH) and glutathione S-transferases (GSTs) of pesticide-exposed snails were compared with those of control animals maintained in untreated mesocosms. Internally recovered HCB concentrations were much higher than internal atrazine concentrations, but the uptake of atrazine was faster than that of HCB. Although it affected the integrity of microsomal membranes, HCB had no relevant effects on BaPH and GST activities at concentrations which affected growth and fecundity, thus confirming the low inducibility of mollusc xenobiotic-metabolizing enzymes by chlorinated compounds. In contrast, atrazine markedly inhibited BaPH and both postmitochondrial and cytosolic GSTs at the same concentrations, which had no effects on growth or reproduction. Enzyme inhibition was negatively correlated with the maximal internal amount of atrazine and positively correlated with the bioconcentration factor, suggesting that effects on xenobiotic-metabolizing enzymes may affect pharmacokinetics of atrazine within the snail body. Correlation between the bioconcentration factor and enzyme inhibition may serve as a descriptor of the physiological status of animals and can also be used to indirectly estimate the pesticide concentration in the environment. Laboratory data were considered for the interpretation of results obtained in the mesocosms. In the biomarker context, BaPH and GST activities are proposed, along with other biochemical markers already identified in atrazine- and HCB-exposed L. palustris, as elements of a multiparametric approach of the ecotoxicological effects of pesticides on freshwater ecosystems.

Freshwater mesocosms were used to study the long-term sublethal effects of atrazine and hexachlorobenzene (HCB) on a basommatophoran gastropod, Lymnaea palustris (Mueller). Growth, fecundity, and biochemical parameters related to polysaccharide metabolism of pesticide-exposed snails were compared with those of control animals maintained in untreated mesocosms. HCB inhibited body growth and stimulated egg production, whereas atrazine had no relevant effect on these physiological parameters. Also, HCB stimulated the activity of polysaccharide-hydrolyzing enzymes, suggesting that changes in the metabolism of reserve polysaccharides (glycogen) may be involved in the inhibition of growth and increase of fecundity. In contrast, atrazine had no effect on the metabolism of polysaccharides. It is concluded that the effects of HCB are related to its neurotoxicity that would have affected the neurohormonal control of growth and reproduction of exposed snails. It is suggested that polysaccharide-hydrolyzing enzymes may be used as biomarkers to predict the effects of neurotoxic pesticides on freshwater snail populations.

Standard ecotoxicological tests are as simple as possible and food sources are mainly chosen for practical reasons. Since some organisms change their food preferences during the life-cycle, they might be food limited at some stage if we do not account for such a switch. As organisms tend to respond more sensitively to toxicant exposure under food limitation, the interpretation of test results may then be biased. Using a reformulation of the von Bertalanffy model to analyze growth data of the pond snail Lymnaea stagnalis, we detected food limitation in the early juvenile phase. The snails were held under conditions proposed for a standardized test protocol, which prescribes lettuce as food source. Additional experiments showed that juveniles grow considerably faster when fed with fish flakes. The model is based on Dynamic Energy Budget (DEB) theory, which allows for mechanistic interpretation of toxic effects in terms of changes in energy allocation. In a simulation study with the DEB model, we compared the effects of three hypothetical toxicants in different feeding situations. The initial food limitation when fed with lettuce always intensified the effect of the toxicants. When fed with fish flakes, the predicted effect of the toxicants was less pronounced. From this study, we conclude that (i) the proposed test conditions for L. stagnalis are not optimal, and require further investigation, (ii) fish flakes are a better food source for juvenile pond snails than lettuce, (iii) analyzing data with a mechanistic modeling approach such as DEB allows identifying deviations from constant conditions, (iv) being unaware of food limitation in the laboratory can lead to an overestimation of toxicity in ecotoxicological tests. Electronic supplementary material The online version of this article (doi:10.1007/s10646-012-0973-5) contains supplementary material, which is available to authorized users.

Chitobiase is involved in exoskeleton degradation and recycling during the moulting process in arthropods. In aquatic species, the moulting fluid is released into the aqueous environment, and chitobiase activity present therein can be used to follow the dynamics of arthropod populations. Here, chitobiase activity was used for monitoring the impact of mosquito candidate larvicides on Daphnia pulex and Daphnia magna under laboratory conditions. Both species were exposed to spinosad (2, 4, 8 mu g L-1) and diflubenzuron (0.2, 0.4, 0.8 mu g L-1) for 14 days. Bacillus thuringiensis var. israelensis (Bti: 0.25, 0.5, 1 mu L L-1) was used as the reference larvicide. Chitobiase activity, adult survival, individual growth and fecundity, expressed as the number of neonates produced, were measured every 2 days. Average Exposure Concentrations of spinosad were ten-fold lower than the nominal concentrations, whereas only a slight deviation was observed for diflubenzuron. In contrast to Bti, spinosad and diflubenzuron significantly affected both species in terms of adult survival, and production of neonates. As compared to D. pulex, D. magna was more severely affected by diflubenzuron, at low and medium concentrations, with reduced adult growth and much lower chitobiase activity. Chitobiase activity was positively correlated with the individual body length, number of neonates produced between two consecutive observation dates, and number of females and neonates. In addition, the significant positive correlations between chitobiase activity measured on the last sampling date before the first emission of neonates and the cumulative number of neonates produced during the whole observation period strongly support the potential of the activity of this chitinolytic enzyme as a proxy for assessing the dynamics of arthropod populations exposed to larvicides used for mosquito control. (C) 2010 Elsevier Inc. All rights reserved.

In vitro testosterone hydroxylation was compared between susceptible and insecticide-resistant Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) larvae. The susceptible larvae were topically applied with a non-lethal dose of lindane (γ-HCH) and the effects on microsomal P 450 were studied within the 48 h following treatment. In vitro testosterone hydroxylation by phenobarbital-induced rat liver microsomes was used as a biological standard to investigate the functional role of P 450 isozymes in S. littoralis . Depending on either the strain or the treatment, various amound of identical hydroxylated derivatives of testosterone were formed. Lindane increased cytochrome P 450 content in susceptible S. littoralis but isoforms specific for testosterone hydroxylation were unaffected or even inhibited. In contrast, most of testosterone hydroxylase activities were enhanced in the resistant strain, uggesting either that not only xenobiotic-specific cytochromes P 450 were overproduced, or that overproduced cytochrome P 450 forms were not only specific for xenobiotics.

Activity of the newly developed nitroguanidine insecticide imidacloprid against fourth-instar larvae of insecticide-susceptible strains of the tobacco budworm, Heliothis virescens F., and the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) was determined from topical application and oral ingestion tests. The activity of imidacloprid was compared to that of cyfluthrin. The LD50 at 48 h of topically applied imidacloprid was estimated as 7.7 and 36.7 μg larva−1 for H. virescens and S. littoralis, respectively. In oral tests, the LC50 at 48 h of imidacloprid incorporated into artificial diet was estimated at 821.0 and 17.7 μg (g food)−1 for H. virescens and S. littoradis larvae, respectively. Imidacloprid showed lower contact and oral toxicities to both species than cyfluthrin, but the difference was much less important in ingestion tests. Mortality caused by both insecticides occurred more slowly in dietary exposures than in topical applications, but imidacloprid acted much more rapidly than cyfluthrin in ingestion tests. When incorporated into the diet, imidacloprid did not produce feeding deterrence, in contrast to cyfluthrin which exhibited a slight repulsive/antifeedant effect. Though cyfluthrin was the more active compound against both H. virescens and S. littoralis larvae, imidacloprid acted much more rapidly in ingestion bioassays. However, the level of activity of the compound is not high enough to provide efficient control of cotton insect pest populations in the field.

International audience; The ecotoxicological impact of nitrate-induced photodegradation products of diuron and chlorotoluron was studied through monospecific biotests conducted in conjunction with experiments in outdoor aquatic mesocosms. Organisms representing three trophic levels were used: two heterotrophic microorganisms, the luminescent bacterium Vibrio fischeri and the ciliated protozoa Tetrahymena pyriformis, and one metazoa, the gastropod Lymnaea stagnalis. Among the variety of the phenylurea photoproducts, the N-formylated ones appeared clearly more toxic than the parent compounds towards the microorganisms, whereas the nitroderivatives showed a similar toxicity. Using photodegraded solutions of diuron, toxicity was maintained or even increased during disappearance of the initial herbicide, demonstrating that some of the photoproducts may have an impact additively or in synergy. Enzymatic biomarker assays performed on Lymnaea stagnalis exposed under monospecific conditions showed significant effects, due to the combination of nitrate with the pesticide and its photoproducts. A positive impact on snail fecundity was observed with chlorotoluron both under monospecific laboratory and integrated mesocosm conditions. Oviposition stimulation took place when first- and second-generation photoproducts were predominant.

Pesticide use leads to complex exposure and response patterns in non-target aquatic species, so that the analysis of data from standard toxicity tests may result in unrealistic risk forecasts. Developing models that are able to capture such complexity from toxicity test data is thus a crucial issue for pesticide risk assessment. In this study, freshwater snails from two genetically differentiated populations of Lymnaea stagnalis were exposed to repeated acute applications of environmentally realistic concentrations of the herbicide diquat, from the embryo to the adult stage. Hatching rate, embryonic development duration, juvenile mortality, feeding rate and age at first spawning were investigated during both exposure and recovery periods. Effects of diquat on mortality were analysed using a threshold hazard model accounting for time-varying herbicide concentrations. All endpoints were significantly impaired at diquat environmental concentrations in both populations. Snail evolutionary history had no significant impact on their sensitivity and responsiveness to diquat, whereas food acted as a modulating factor of toxicant-induced mortality. The time course of effects was adequately described by the model, which thus appears suitable to analyse long-term effects of complex exposure patterns based upon full life cycle experiment data. Obtained model outputs (e.g. no-effect concentrations) could be directly used for chemical risk assessment.

[Departement_IRSTEA]Eaux [TR1_IRSTEA]BELCA [Departement_IRSTEA]Eaux [TR1_IRSTEA]BELCA; Long-term effects of endocrine disruptors (EDs) on aquatic invertebrates remain difficult to assess, mainly due to the lack of appropriate sensitive toxicity test methods and relevant data analysis procedures. This study aimed at identifying windows of sensitivity to EDs along the life-cycle of the freshwater snail Lymnaea stagnalis, a candidate species for the development of forthcoming test guidelines. Juveniles, sub-adults, young adults and adults were exposed for 21 days to the fungicide vinclozolin (VZ). Survival, growth, onset of reproduction, fertility and fecundity were monitored weekly. Data were analyzed using standard statistical analysis procedures and mixed-effect models. No deleterious effect on survival and growth occurred in snails exposed to VZ at environmentally relevant concentrations. A significant impairment of the male function occurred in young adults, leading to infertility at concentrations exceeding 0.025 μg/L. Furthermore, fecundity was impaired in adults exposed to concentrations exceeding 25 μg/L. Biological responses depended on VZ concentration, exposure duration and on their interaction, leading to complex response patterns. The use of a standard statistical approach to analyze those data led to underestimation of VZ effects on reproduction, whereas effects could reliably be analyzed by mixed-effect models. L. stagnalis may be among the most sensitive invertebrate species to VZ, a 21-day reproduction test allowing the detection of deleterious effects at environmentally relevant concentrations of the fungicide. These results thus reinforce the relevance of L. stagnalis as a good candidate species for the development of guidelines devoted to the risk assessment of EDs.

The fate of gamma-HCH (hexachlorocyclohexane) was studied in a susceptible strain of the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.). A non-lethal dose (0·2 μg larva−1) was topically applied to fourth-instar larvae, and the pharmacokinetics of the insecticide were studied for a period of 48 h following treatment. Penetration showed biphasic kinetics, with a rapid increase of the internal gamma-HCH concentration during the first 6 h after the application, followed by a plateau up to 48 h. The rate of metabolic degradation was highest between 6 and 24 h. During this period, the rapid decrease in the amount of gamma-HCH in the larvae was associated with an accelerated excretion of metabolites. A compartmental model with curve-fitting was used to quantify the rates of uptake through the cuticle (penetration) and of loss from the tissues (degradation and elimination). Chromatographic analysis of the aqueous and organic fractions extracted from the larvae and their faeces provided direct evidence that our susceptible strain of S. littoralis was able to metabolise a nonlethal dose of gamma-HCH.

The influence of tank-mix adjuvants on pesticide toxicity remains largely unknown. Agral 90, a nonylphenol polyethoxylated tank-mix adjuvant, has been used with diquat (bipyridylium herbicide) and fomesafen (diphenyl-ether herbicide) in aquatic indoor microcosms in order to compare the toxicity of the single compounds and of binary herbicide-adjuvant mixtures to Lemna minor. Twenty-four microcosms were used and treatments were performed with substances alone or with herbicide-adjuvant binary mixtures, at two concentrations levels (44.4 and 222.2 microg/L for the herbicides, and 100 and 500 microg/L for Agral 90). Toxicity was assessed weekly for 1 month through growth measurements, as inferred from the relative frond number (RFN) and relative frond area (RFA). Concentrations of diquat and fomesafen in water and sediments were measured weekly. The herbicides showed very different behaviour in microcosms, with a rapid disappearance of diquat from the aqueous phase whereas fomesafen levels remained almost constant over time. Diquat strongly inhibited the growth of L. minor whereas fomesafen had no effect on plant growth. Presence of the adjuvant only slightly reduced the effect of the lowest concentration of diquat, probably as a result of dispersion of the herbicide at the water surface. It is concluded that tank-mix adjuvant designed to improve herbicide efficiency in the terrestrial environment did not have any effect on aquatic plants when applied to the aquatic environment.

Effects of the bipyridylium herbicide diquat and tank-mix adjuvant Agral ® 90 were investigated on various life history traits of the freshwater pulmonate snail Lymnaea stagnalis . Trait expression was measured in simple laboratory bioassays on small size groups of snails, and under more complex, indoor microcosm conditions, on larger groups of snails. Microcosms were provided with sediment, plants, and fish, thus allowing a more complex level of intra and inter-specific interactions to develop. Treatments were performed with substances alone or in mixture, at concentrations ranging from 4.4 to 222.2 μg l −1 for diquat, and from 10 to 500 μg l −1 for Agral 90, under a fixed ratio design. Adult growth was negatively affected by diquat and its mixture with Agral 90 both at the highest concentrations (222.2 and 500 μg l −1 , respectively). Fecundity expressed differently in bioassays and microcosms, but no effect of the chemicals could be observed on this trait. Progeny development was impaired by 222.2 μg l −1 diquat and its mixture with 500 μg l −1 Agral 90, as reflected by longer development time and reduced hatching rate of clutches laid by the exposed animals, as compared to the controls. Hatching data suggested that diquat bioavailability was lower in microcosms than under bioassay conditions. Consistently, chemical analysis showed that diquat disappeared more rapidly from the water in microcosms than in bioassays. Moreover, the differential expression of several life history traits under bioassays and microcosms conditions was probably also influenced by the level of intraspecific interaction, which differed among the systems. When significant, the effect of diquat was attenuated by the presence of Agral 90, indicating antagonistic interaction between the two substances. Such a deviation from additivity was partly validated statistically.

There is a paucity of large-scale field investigations on the effects of organic toxicants on stream macro invertebrate community structure and ecosystem functions. We investigated a total of 29 streams in two study areas of France and Finland for pesticide exposure, invertebrates and leaf-litter breakdown. To link pesticide exposure and community composition we applied the trait-based Species At Risk (SPEAR) indicator system. In the French region, pesticide stress was associated with a decrease in the relative abundance and number of sensitive species in the communities. The presence of undisturbed upstream reaches partly compensated the effects of pesticide contamination. Functional effects of pesticides were identified by a 2.5-fold reduction of the leaf-litter breakdown rate that was closely correlated with the structural changes in the contaminated streams. No effects of pesticides were observed in Finnish streams since contamination with pesticides was very low. In a follow-up analysis, the SPEAR approach successfully discriminated between reference and contaminated sites across different biogeographical regions, also including results of a previous field study in North Germany. Furthermore, change of the community structure was detectable at a concentration range as low as 1/100 to 1/1000 the acute 48 h-LC50 of Daphnia magna. Our findings demonstrate that pesticides may influence the structure and function of lotic ecosystems and that the SPEAR approach can be used as a powerful tool in biomonitoring over large spatial scales. (c) 2007 Elsevier B.V. All rights reserved.

International audience; The influence of relative isolation on the ecological recovery of freshwater outdoor mesocosm communities after an acute toxic stress was assessed in a 14-month-long study. A single concentration of deltamethrin was applied to 8 out of 16 outdoor 9-m3 mesocosms to create a rapid decrease of the abundance of arthropods. To discriminate between external and internal recovery mechanisms, four treated and four untreated (control) mesocosms were covered with 1-mm mesh screen lids. The dynamics of planktonic communities were monitored in the four types of ponds. The abundance of many phytoplankton taxa increased after deltamethrin addition, but the magnitude of most increases was relatively small, probably due to low nutrient availability and the survival of rotifers. The greatest impact on zooplankton was seen in Daphniidae and, to a lesser extent, calanoid copepods. Recovery (defined as when statistical analysis failed to detect a difference in the abundance between the deltamethrin-treated ponds and corresponding control ponds for two consecutive sampling dates) of Daphniidae was observed in the water column 105 and 77 d after deltamethrin addition in open and covered mesocosms, respectively, and 42 d for both open and covered ponds at the surface of the sediments. Rotifers did not proliferate, probably because of the survival of predators (e.g., cyclopoid copepods). These results confirm that the recovery of planktonic communities after exposure to a strong temporary chemical stress mostly depends upon internal mechanisms (except for larvae of the insect Chaoborus sp.) and that recovery dynamics are controlled by biotic factors, such as the presence of dormant forms and selective survival of predators.

International audience; As compared to other groups of aquatic gastropods, documented examples of endocrine disruption in pulmonates are rather limited. This is quite surprising because the endocrine control of physiological functions has been extensively studied in these animals. In the model-species Lymnaea stagnalis, the neurohormonal regulation of reproduction has been thoroughly investigated, and the primary structure of several peptides and receptors involved in endocrine processes has been established. However, the use of this knowledge has been fairly limited in the context of ecotoxicology, to investigate the effects of endocrine-disrupting chemicals. The present review summarizes the main and more recent findings on the neuroendocrine control of reproduction in aquatic pulmonate snails (Basommatophora). It then comprehensively describes selected in vivo laboratory and semi-field studies which provide evidence for possible endocrine disrupting effects of estrogenic and androgenic test compounds [e.g., ethynylestradiol, methyltestosterone (MT)], and of environmental contaminants [e.g., cadmium (Cd), tributyltin (TBT), and nonylphenol (NP), pesticides]. Finally, challenging perspectives for future research are discussed.

This study examined the response of 7-ethoxyresorufine-O-deethylase (EROD), glutathioneS-transferase (GST), glutathione peroxidase (GPx), glutathione content (GSH), level of thiobarbituric acid reactive compounds (TBARS) and circulating vitellogenin, in three-spined sticklebacks after 21 days of exposure to Diquat herbicide, commercial nonylphenol polyethoxylate (NPEO) adjuvant and mixture between Diquat and adjuvant. The results showed that adjuvant exerted more important oxidative effects than Diquat and that mixture effects were unlike to single additivity. This study argues for ecotoxicological risk assessment of adjuvants and mixtures of adjuvants and pesticides.

When spiked in sediments, copper is known to reduce growth of Chironomus riparius larvae and the production of eggs by adult females. The aim of this work was to better understand the origin of these phenomena by studying the effects of copper using developmental and energetic biomarkers, such as changes in larval weight and age and changes in the levels of sugars and lipids. Four-day-old C. riparius larvae were exposed to nominal concentrations of copper of 0, 6.5, 12.5, 25 and 50 mg/kg of dry sediment (silica) in 0.6 l beakers. They were fed ad libitum and exposures were stopped at 7 and 9 days after the beginning of the tests. The larvae were weighed, sexed and aged. For each sex, the larvae belonging to the phases the most frequently found in the beakers were selected for dissection and measurement of energy reserves. The increase in the concentration of copper resulted in an increasing delay in larval growth in both sexes. Desynchronized development was observed, as shown by the increase in the number of individuals that remained in the third instar or early phases of the fourth instar, as well as by a reduction in age of males. Concerning energy reserves, the levels of sugars (glycogen, trehalose and glucose) in the dissected larvae remained almost constant among levels of exposure. In contrast, at the highest copper concentration (50 mg/kg), triglyceride levels suffered a slight reduction whereas the level of free glycerol significantly increased. It is concluded that selection of C. riparius larvae for both sex and age improves the relevance of some energy-yielding substrates as indicators of adverse physiological effects of copper.

Genetic and ecological factors may interact in their effects on fitness. Such interactions are thus to be expected between inbreeding and exposure of a population to a toxicant. The magnitude of inbreeding depression is thought to increase in stressful environments. To test this hypothesis, we investigated the combined effects of environmental conditions and inbreeding on fitness in the self-fertile snail Lymnaea stagnalis, using a stress gradient (0–2) applied to a 100 isolated and paired lineages: laboratory control (0), outdoor microcosm control (1) and pesticide exposure under outdoor microcosm conditions (2). Outdoor stress conditions were maintained for 28 days prior to measurements of fitness traits (fecundity, hatching success, and size at hatching) under laboratory conditions, so that delayed environmental effects could be estimated. Under laboratory control conditions, we found significant initial family level heterogeneity for most measured traits, including physiological performances as assessed through energetic biomarkers. Whatever the environmental conditions, inbreeding depression was very low for progeny performances. Negative values of self-fertilization depression (SFD) were obtained. Unexpectedly, SFD showed a negative relationship with the assumed stress intensity, reflecting a higher sensitivity under pairing than under selfing, mostly due to parental fecundity. This suggests that stressful conditions may favour selfing. Stress intensity increased the distribution limits of both depression indices, suggesting that changes in fitness are less predictable in a population under stress. Implications of such findings for environmental risk assessment of pesticides are discussed

Sudden exposure of an aquatic system to an insecticide can have significant effects on populations other than susceptible organisms. Although this is intuitively obvious, little is actually known about how such exposure might affect bacterial communities and their relative metabolic activity in ecosystems. Here, we assessed small sub-unit (ssu)-RNA levels in open and shaded 9 m3 aquatic mesocosms (16 units – 2 × 2 factorial design in quadruplicate) to examine the effects of sudden addition of deltamethrin to the units. When deltamethrin was added, a cascade of bacterial then phytoplankton "blooms" occurred over time. The bacterial bloom, which most likely included organisms from the plastid/cyanobacterial phylogenetic guild, was almost immediate (within hours), whereas the phytoplankton (algal) bloom lagged by about 4 days. This sequential response can be explained by an apparent sudden release of nutrients consequent to arthropod death that triggered a series of responses in the microbial loop. Interestingly, bacterial blooms were noted in both open and shaded mesocosms, whereas the algal bloom was only seen in open units, suggesting that both deltamethrin addition (and presumptive nutrient release) and an adequate light supply was required for the phytoplankton response. Overall, this work shows that microbial activities as reflected by ssu-rRNA levels can respond dramatically via apparently indirect effects following insecticide application.

Immunotoxicological effects of environmentally relevant concentrations (10, 23, 50, 100 microg/l) of atrazine were studied in Lymnaea stagnalis. Individual hemolymph sampling was performed at 0, 24, 48, 72, 96, 168, 336, 504 and 672 h during exposure. Every atrazine concentration induced a significant increase in the mean number of circulating hemocytes, without any concentration-response relation. A peak (1.6-fold increase) of hemocyte density was observed after 96 h of exposure. After 504 h, the number of hemocytes remained higher only in the snails exposed to the two highest concentrations. Granulocytes contributed most to the increase in hemocyte density in herbicide-exposed snails. Both short- (24 and 96 h) and long-term (504 h) exposures resulted in significant inhibition of hemocyte phagocytic activity upon E. coli. Over the long-term, phagocytosis recovered for the two lowest concentrations. After 504 h of exposure, every herbicide level resulted in a significant reduction of reactive oxygen species production in E. coli-stimulated hemocytes, which was not observed for short-term exposures.

Many studies have reported perturbations of mollusc reproduction following exposure to low concentrations (ng/L range) of endocrine disrupting chemicals (EDCs). However, the mechanisms of action of these molecules on molluscs are still poorly understood. Investigation of the modifications of protein expression in organisms exposed to chemicals using proteomic methods can provide a broader and more comprehensive understanding of adverse impacts of pollution on organisms than conventional biochemical biomarkers (e.g., heat-shock proteins, metallothioneins, GST, EROD). In this study we have investigated the impacts of four chemicals, which exhibit different endocrine disrupting properties in vertebrates, on the proteome of the hermaphroditic freshwater pulmonate gastropod Lymnaea stagnalis after 21 days of exposure. Testosterone, tributyltin, chlordecone and cyproterone acetate were chosen as tested compounds as they can induce adverse effects on the reproduction of this snail. The 2D-DIGE method was used to identify proteins whose expression was affected by these compounds. In addition to modifying the expression of proteins involved in the structure and function of the cytoskeleton, chemicals had impacts on the expression of proteins involved in the reproduction of L. stagnalis. Exposure to 19.2 mu g/L of chlordecone increased the abundance of ovipostatin, a peptide transmitted during mating through seminal fluid, which reduces oviposition in this species. The expression of yolk ferritin, the vitellogenin equivalent in L. stagnalis, was reduced after exposure to 94.2 ng Sn/L of tributyltin. The identification of yolk ferritin and the modification of its expression in snails exposed to chemicals were refined using western blot analysis. Our results showed that the tested compounds influenced the abundance of yolk ferritin in the reproductive organs. Alteration in proteins involved in reproductive pathways (e.g., ovipostatin and yolk ferritin) could constitute relevant evidence of interaction of EDCs with reproductive pathways that are under the control of the endocrine system of L. stagnalis.

Cytosolic GST activity was followed after a single topical application of a non-lethal dose of lindane to fourth instar larvae of a susceptible strain of Spodoptera littoralis (Boisd.). The induction reached its peak 8 h after treatment for 1-chloro-2,4-dinitrobenzene (CDNB) conjugating GSTs (1.5-fold), and 12 h after the treatment for 1,2-dichloro-4-nitrobenzene (DCNB) comjugating enzymes (1.3-fold). CDNB conjugating GST activity in an insecticide-resistant strain of S. littoralis was twice that measured in the non-induced suceptible larvae. No significant difference was observed with DCNB. GSTs of both larvae were purified by affinity chromatography using a glutathione-agarose column, which resulted in a 30-fold prufication and a yield of 50–70%. Purified GSTs were characterized using CDNB as the substrate. Apparent K m and V max values calculated for CDNB and reduced glutathione (GSH) were significantly different between the resistant strain and the susceptible one, whether induced by lindane or not. The lindane treatment modified only K m values for CDNB and GSH in susceptible larvae. Monodimensional SDS-PAGE of GSTs purified from both strains revealed a large band of 27 kDa apparent molecular weight.