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The toxicity and ecotoxicity of pesticide active ingredients are evaluated by a number of standardized test methods using vertebrate animals. These standard test methods are required under various regulatory programs for the registration of pesticides. Over the past two decades, additional test methods have been developed with endpoints that are responsive to endocrine activity and subsequent adverse effects. This paper examines the available test methods and their endpoints that are relevant for an assessment of endocrine disrupting properties of pesticides. Furthermore, the paper highlights how weight of evidence approaches should be applied to determine whether an adverse response in (eco)toxicity tests are caused by an endocrine mechanism of action (MoA). The large number of endpoints in the current testing paradigms for pesticides make it unlikely that endocrine activity and adversity is being overlooked. This article is protected by copyright. All rights reserved. Integr Environ Assess Manag 2023;00:0-0. © 2023 SETAC.

The European Commission intends to protect vertebrate wildlife populations by regulating plant protection product (PPP) active substances that have endocrine‐disrupting properties with a hazard‐based approach. In this paper we consider how the Commission's hazard‐based regulation and accompanying guidance can be operationalized to ensure that a technically robust process is used to distinguish between substances with adverse population‐level effects and those for which it can be demonstrated that adverse effects observed (typically in the laboratory) do not translate into adverse effects at the population level. Our approach is to use population models within the adverse outcome pathway framework to link the nonlinear relationship between adverse effects at the individual and population levels in the following way: (1) use specific protection goals for focal wildlife populations within an ecosystem services framework; (2) model the effects of changes in population‐related inputs on focal species populations with individual‐based population models to determine thresholds between negligible and nonnegligible (i.e., adverse) population‐level effects; (3) compare these thresholds with the relevant endpoints from laboratory toxicity tests to determine whether they are likely to be exceeded at hazard‐based limits or the maximum tolerated dose/concentration from the experimental studies. If the population threshold is not exceeded, then the substance should not be classified as an endocrine disruptor with population‐relevant adversity unless there are other lines of evidence within a weight‐of‐evidence approach to challenge this. We believe this approach is scientifically robust and still addresses the political and legal requirement for a hazard‐based assessment. Integr Environ Assess Manag 2019;15:278–291. © 2018 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC), Key Points In this paper we consider how the European Commission's hazard‐based Plant Protection Product Regulation can be operationalized to distinguish between substances with adverse population‐level effects and those for which observed adverse effects do not translate into adverse population‐level effects.Our approach is to use population models within the adverse outcome pathways framework to link the nonlinear relationship between adverse effects at the individual and population levels.Only if the population threshold is exceeded should the active substance be classified as an endocrine disruptor with population‐relevant adversity, unless this classification is challenged by other lines of evidence (e.g., from field studies) within a weight‐of‐evidence approach.We believe this approach is scientifically robust and addresses the political and legal requirement for a hazard‐based assessment.

For ecotoxicological risk assessment, endocrine disruptors require the establishment of an endocrine mode of action (MoA) with a plausible link to a population-relevant adverse effect. Current ecotoxicity test methods incorporate mostly apical endpoints although some also include mechanistic endpoints, subcellular-through-organ level, which can help establish an endocrine MoA. However, the link between these endpoints and adverse population-level effects is often unclear. The case studies of endocrine-active substances (EAS) (tributyltin, ethinylestradiol, perchlorate, trenbolone, propiconazole, and vinclozolin) evaluated from the Society of Environmental Toxicology and Chemistry (SETAC) Pellston Workshop® “Ecotoxicological Hazard and Risk Assessment Approaches for Endocrine-Active Substances (EHRA)” were used to evaluate the population relevance of toxicity endpoints in various taxa according to regulatory endocrine-disruptor frameworks such as the Organisation for Economic Co-operation and Development (OECD) Conceptual Framework for Testing and Assessment of Endocrine Disruptors. A wide variety of potentially endocrine-relevant endpoints were identified for mollusks, fish, amphibians, birds, and mammals, although the strength of the relationship between test endpoints and population-level effects was often uncertain. Furthermore, testing alone is insufficient for assessing potential adaptation and recovery processes in exposed populations. For this purpose, models that link effects observed in laboratory tests to the dynamics of wildlife populations appear to be necessary, and their development requires reliable and robust data. As our understanding of endocrine perturbations and key event relationships improves, adverse population-level effects will be more easily and accurately predicted. Integr Environ Assess Manag 2017;13:317–330. © 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC)

A SETAC Pellston Workshop® “Environmental Hazard and Risk Assessment Approaches for Endocrine-Active Substances (EHRA)” was held in February 2016 in Pensacola, Florida, USA. The primary objective of the workshop was to provide advice, based on current scientific understanding, to regulators and policy makers; the aim being to make considered, informed decisions on whether to select an ecotoxicological hazard- or a risk-based approach for regulating a given endocrine-disrupting substance (EDS) under review. The workshop additionally considered recent developments in the identification of EDS. Case studies were undertaken on 6 endocrine-active substances (EAS—not necessarily proven EDS, but substances known to interact directly with the endocrine system) that are representative of a range of perturbations of the endocrine system and considered to be data rich in relevant information at multiple biological levels of organization for 1 or more ecologically relevant taxa. The substances selected were 17α-ethinylestradiol, perchlorate, propiconazole, 17β-trenbolone, tributyltin, and vinclozolin. The 6 case studies were not comprehensive safety evaluations but provided foundations for clarifying key issues and procedures that should be considered when assessing the ecotoxicological hazards and risks of EAS and EDS. The workshop also highlighted areas of scientific uncertainty, and made specific recommendations for research and methods-development to resolve some of the identified issues. The present paper provides broad guidance for scientists in regulatory authorities, industry, and academia on issues likely to arise during the ecotoxicological hazard and risk assessment of EAS and EDS. The primary conclusion of this paper, and of the SETAC Pellston Workshop on which it is based, is that if data on environmental exposure, effects on sensitive species and life-stages, delayed effects, and effects at low concentrations are robust, initiating environmental risk assessment of EDS is scientifically sound and sufficiently reliable and protective of the environment. In the absence of such data, assessment on the basis of hazard is scientifically justified until such time as relevant new information is available. Integr Environ Assess Manag 2017;13:267–279. © 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC)

Higher-tier ecological risk assessment (ERA) in mesocosms is commonly performed in lotic or lentic experimental systems. These systems differ in their physico-chemical and hydrological properties, leading to differences in chemical fate, community characteristics and potential recovery. This raises the issue of the relevance and sensitivity of community-level endpoints in different types of mesocosms. In this study, macroinvertebrate abundance and biomass estimates were used to assess the effects of a dithiocarbamate fungicide, thiram (35 and 170 A mu g l(-1)), and a petroleum middle distillate (PMD; 0.01, 0.4, 2 and 20 mg l(-1)) in outdoor stream and pond mesocosms. Streams were continuously treated during 3 weeks followed by a 2-month long post-treatment period. Ponds were treated weekly for 4 weeks, followed by a 10-month long post-treatment period. Taxonomic structure of macroinvertebrate communities was characterized using the alpha, beta and gamma components of taxa richness, Shannon and Gini-Simpson indices. Computations were based either on abundance or biomass data. Results clearly highlighted that the effects of chemicals depended on the exposure regime (for thiram) and type of system (for the PMD). Causes of the differences between streams and ponds in the magnitude and nature of effects include differential sensitivity of taxa dwelling in lentic and lotic systems and the influence of hydrology (e.g., drift from upstream) and mesocosm connectivity on recovery dynamics. This study also showed complementarities in the use of both types of mesocosms to improve the characterization of chemical effects on communities in ERA.

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.

International audience; The Organisation for Economic Cooperation and Development (OECD) provides several standard test methods for the environmental hazard assessment of chemicals, mainly based on primary producers, arthropods, and fish. In April 2016, two new test guidelines with two mollusc species representing different reproductive strategies were approved by OECD member countries. One test guideline describes a 28-day reproduction test with the parthenogenetic New Zealand mudsnail Potamopyrgus antipodarum. The main endpoint of the test is reproduction, reflected by the embryo number in the brood pouch per female. The development of a new OECD test guideline involves several phases including inter-laboratory validation studies to demonstrate the robustness of the proposed test design and the reproducibility of the test results. Therefore, a ring test of the reproduction test with P. antipodarum was conducted including eight laboratories with the test substances trenbolone and prochloraz and results are presented here. Most laboratories could meet test validity criteria, thus demonstrating the robustness of the proposed test protocol. Trenbolone did not have an effect on the reproduction of the snails at the tested concentration range (nominal: 10-1000 ng/L). For prochloraz, laboratories produced similar EC10 and NOEC values, showing the inter-laboratory reproducibility of results. The average EC10 and NOEC values for reproduction (with coefficient of variation) were 26.2 A mu g/L (61.7%) and 29.7 A mu g/L (32.9%), respectively. This ring test shows that the mudsnail reproduction test is a well-suited tool for use in the chronic aquatic hazard and risk assessment of chemicals.

Bacillus thuringiensis var. israelensis (Bti) is commonly used for selective control of larval populations of mosquitoes in coastal wetlands. A two year-study was implemented to investigate whether repeated treatments with Bti applied either as a liquid (VectoBac (R) 12AS) or a water-dispersible granule (VectoBac (R) WG) formulation may affect the abundance and diversity of non-target aquatic invertebrates in saltmarsh pools. Taxonomic composition of the invertebrate communities was typical of brackishwater intermittent ecosystems, with a dominance of annelids, crustaceans and nematocerans. Conditions were contrasted between the two years of the survey, both in terms of annual cumulative rainfall and rainfall distribution throughout the year. As a consequence, the hydroperiod and some other environmental characteristics associated with pool drying played a major role in the dynamics of the invertebrate community. In summer 2006, pool drying reduced the abundance of the polychaete worm Nereis diversicolor, of the amphipod crustacean Corophium volutator and of chironomid larvae. These taxa were able to recolonize rapidly the pools after flooding in September 2006. In 2007, rainfall was more regularly distributed across the year, and the pools did not get dry. Hydrozoans, Chironomini and Orthocladiinae larvae, and oligochaetes were more abundant in treated than in control pools, especially in VectoBac (R) WG-treated pools. No adverse effects of the treatments were shown on the abundance of N. diversicolor, C. volutator and midge larvae, suggesting that the availability of these food sources for birds was not negatively affected by Bti applications. It is concluded that, as currently performed in Western France coastal wetlands, land-based treatments of saltmarsh pools for larval mosquito control with Bti, used either as VectoBac (R) 12AS or VectoBac (R) WG, did not adversely impact non-target aquatic invertebrate communities. (C) 2011 Elsevier Inc. All rights reserved.

This paper presents the results from two ring-tests addressing the feasibility, robustness and reproducibility of a reproduction toxicity test with the freshwater gastropod Lymnaea stagnalis (RENILYS strain). Sixteen laboratories (from inexperienced to expert laboratories in mollusc testing) from nine countries participated in these ring-tests. Survival and reproduction were evaluated in L. stagnalis exposed to cadmium, tributyltin, prochloraz and trenbolone according to an OECD draft Test Guideline. In total, 49 datasets were analysed to assess the practicability of the proposed experimental protocol, and to estimate the between-laboratory reproducibility of toxicity endpoint values. The statistical analysis of count data (number of clutches or eggs per individual-day) leading to ECx estimation was specifically developed and automated through a free web-interface. Based on a complementary statistical analysis, the optimal test duration was established and the most sensitive and cost-effective reproduction toxicity endpoint was identified, to be used as the core endpoint. This validation process and the resulting optimized protocol were used to consolidate the OECD Test Guideline for the evaluation of reproductive effects of chemicals in L. stagnalis.

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.

ISI Document Delivery No.: CF3II Times Cited: 0 Cited Reference Count: 52 Cited References: Szoecs Eduard, 2012, SCIENCE OF THE TOTAL ENVIRONMENT, V437, P121 Szoecs, Eduard Van den Brink, Paul J. Lagadic, Laurent Caquet, Thierry Roucaute, Marc Auber, Arnaud Bayona, Yannick Liess, Matthias Ebke, Peter Ippolito, Alessio ter Braak, Cajo J. F. Brock, Theo C. M. Schafer, Ralf B. Interface Recherche-Expertise pour l'Evaluation du Risque en Ecotoxicologie; Ministry of Ecology, Sustainable Development and Energy; TOTAL S.A. [10, 11]; Region Bretagne; INRA; Dutch Ministry of Economic Affairs; TOTAL S.A.; [BO-20-002-001] The authors gratefully acknowledge the contributions from scientists and technicians from the INRA Experimental Unit of Aquatic Ecology and Ecotoxicology and Ecotoxicology and Quality and Aquatic Research Group during mesocosm experiments. These experiments were conducted in programmes funded by the Interface Recherche-Expertise pour l'Evaluation du Risque en Ecotoxicologie coordinated by the Structure Scientifique Mixte INRA-Direction Generale de l'Alimentation (Study no. 8), by the Ministry of Ecology, Sustainable Development and Energy through its "Pesticides" research programme (Study Nos. 3, 4), and by TOTAL S.A. (Study Nos. 10, 11). A. Auber and Y. Bayona PhDs were funded through a grant from The Region Bretagne and INRA and by TOTAL S.A., respectively. The studies conducted at Wageningen UR (Study nos. 1, 7 and 9) were financially supported by the Dutch Ministry of Economic Affairs, as well as the contribution of Paul Van den Brink and Theo Brock to this paper (Project BO-20-002-001). The data sets of these studies are available upon request. Springer Dordrecht Environmental Sciences & Ecology; Toxicology; International audience; Mesocosm experiments that study the ecological impact of chemicals are often analysed using the multivariate method 'Principal Response Curves' (PRCs). Recently, the extension of generalised linear models (GLMs) to multivariate data was introduced as a tool to analyse community data in ecology. Moreover, data aggregation techniques that can be analysed with univariate statistics have been proposed. The aim of this study was to compare their performance. We compiled macroinvertebrate abundance datasets of mesocosm experiments designed for studying the effect of various organic chemicals, mainly pesticides, and re-analysed them. GLMs for multivariate data and selected aggregated endpoints were compared to PRCs regarding their performance and potential to identify affected taxa. In addition, we analysed the inter-replicate variability encountered in the studies. Mesocosm experiments characterised by a higher taxa richness of the community and/or lower taxonomic resolution showed a greater inter-replicate variability, whereas variability decreased the more zero counts were encountered in the samples. GLMs for multivariate data performed equally well as PRCs regarding the community response. However, compared to first axis PRCs, GLMs provided a better indication of individual taxa responding to treatments, as separate models are fitted to each taxon. Data aggregation methods performed considerably poorer compared to PRCs. Multivariate community data, which are generated during mesocosm experiments, should be analysed using multivariate methods to reveal treatment-related community-level responses. GLMs for multivariate data are an alternative to the widely used PRCs.

Higher-tier ecological risk assessment of chemicals often relies upon studies in dynamic and/or static mesocosms. Physico-chemical and hydrological properties of each type of mesocosm result in specific chemicals fate, community functioning, and potential recovery. In the present study, macroinvertebrate abundance- and biomass-weighted biological and ecological trait matrices were used to assess the effects of a dithiocarbamate fungicide, thiram (35 and 170 A mu g l(-1)), and of a petroleum middle distillate (0.01, 0.4, 2 and 20 mg l(-1)) in outdoor stream and pond mesocosms. Trait sensitivity was characterized using functional diversity indices and trait modality distributions to assess the influence of the type of experimental systems and the ability of traits to disentangle chemical-induced effects from temporal and stochastic variations. In addition, leaf litter breakdown was used as an integrative functional endpoint. Regardless to the substance, treatments had a direct effect on the functional structure of benthic macroinvertebrate communities in streams but not in ponds, suggesting that global functional responses to chemicals are system-specific. Although both substances had an effect in streams, differences were noticed in the nature of the affected traits suggesting that chemical mode of action plays a role in functional alterations. This was illustrated by the link between negative effects of chemical exposure on detritivorous taxa and reduced litter breakdown rate in streams. Therefore, characterisation of macroinvertebrate biological traits associated with the measurement of a functional process such as litter breakdown may provide a comprehensive understanding of the effects occurring in mesocosms exposed to organic chemicals.

International audience; To optimize their efficacy, some insecticides used for mosquito control are introduced into aquatic ecosystems where mosquito larvae develop (marshes, ponds, sanitation devices) and cannot escape from the treated water. However, this raises the question of possible effects of mosquito larvicides on nontarget aquatic species. Bacillus thuringiensis var. israelensis (Bti), which is well-known for its selectivity for Nematocera dipterans, is widely used for mosquito control all over the world. Spinosad, a mixture of spinosyns A and D known as fermentation products of a soil actinomycete (Saccharopolyspora spinosa), is a biological neurotoxic insecticide with a broader action spectrum. It is a candidate larvicide for mosquito control, but some studies showed that it may be toxic to beneficial or non-target species, including non-biting midges. The present study was therefore undertaken to assess the impact of Bti and spinosad on natural populations of Polypedilum nubifer (Skuse) and Tanytarsus curticornis Kieffer (Diptera: Chironomidae) in field enclosures implemented in Mediterranean coastal wetlands. Unlike Bti, spinosad had a strong lethal effect on Polypedilum nubifer and seems to affect Tanytarsus curticornis at presumed recommended rates for field application. Differences in the sensitivity of these two species to spinosad confirm that population dynamics need to be known for a proper assessment of the risk encountered by chironomids in wetlands where larvicide-based mosquito control occurs. (C) 2015 Elsevier Inc. All rights reserved.

Micro- and mesocosms are frequently required in regulatory procedures of aquatic risk assessment for pesticides. However, many questions are still a matter of debate with regard to the use of these systems for environmental risk assessment, especially considering the inter-system variability of the measured parameters and its consequences on experimental design and data analysis. In this paper, variability of physico-chemical and biological parameters measured during two long-term experiments (8 to 9 months) in uncontaminated outdoor freshwater lentic mesocosms (8 m3) is analysed. Consequences on the design of ecotoxicity tests in mesocosms and on data analysis are also addressed. Water temperature, pH, dissolved oxygen concentration and concentration of suspended solids exhibited a very low variability whereas nutrient concentrations displayed elevated levels of variability. Among biological parameters, those measured at the individual level were less variable than those measured at the community level. Functional descriptors frequently exhibited a lower inter-mesocosm variability than structural descriptors. Aggregation of data proved to significantly reduce inter-mesocosm variability. The results indicate that univariate statistical methods may be used for physico-chemical or species-level (e.g. biometric parameters) data which exhibit a moderate inter-mesocosm variability. The use of multivariate techniques is suggested for other levels of investigation. Nevertheless, variability is not sufficient to identify useful parameters. The sensitivity towards chemicals and ecological relevance of descriptors within the experimental context must also be considered.

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.

International audience; Estuarine areas represent complex and highly changing environments at the interface between freshwater and marine aquatic ecosystems. Therefore, the aquatic organisms living in estuaries have to face highly variable environmental conditions. The aim of this work was to study the influence of environmental changes from either natural or anthropogenic origins on the physiological responses of Mytilus edulis. Mussels were collected in the Vilaine estuary during early summer because this season represents a critical period of active reproduction in mussels and of increased anthropogenic inputs from agricultural and boating activities into the estuary. The physiological status of the mussel M. edulis was evaluated through measurements of a suite of biomarkers related to: oxidative stress (catalase, malondialdehyde), detoxication (benzopyrene hydroxylase, carboxylesterase), neurotoxicity (acetylcholinesterase), reproductive cycle (vitelline, condition index, maturation stages), immunotoxicity (hemocyte concentration, granulocyte percentage, phagocytosis, reactive oxygen species production, oxidative burst), and general physiological stress (lysosomal stability). A selection of relevant organic contaminant (pesticides, (polycyclic aromatic hydrocarbons, polychlorobiphenyls) was measured as well as environmental parameters (water temperature, salinity, total suspended solids, turbidity, chlorophyll a, pheopigments) and mussel phycotoxin contamination. Two locations differently exposed to the plume of the Vilaine River were compared. Both temporal and inter-site variations of these biomarkers were studied. Our results show that reproduction cycle and environmental parameters such as temperature, organic ontaminants, and algal blooms could strongly influence the biomarker responses. These observations highlight the necessity to conduct integrated environmental approaches in order to better understand the causes of biomarker variations.

Global change, in particular climate change, will affect agriculture worldwide in many ways: increased drought or flooding amplitude and frequency, variable temperature increases, loss of natural depuration of waters, soil erosion, loss of soil carbon content, invasion by alien species, increased pest events, changes in plant phenology, increased sensitivity of crops to stress and diseases etc. (Fisher et al. 2005; Howden et al. 2007). These anticipated or even already occurring stresses raise concerns about the sustainability of production and the ability of agriculture to feed human populations. All these changes could lead to an increased use of pesticides (Kattwinkel et al. 2011). Moreover, demographic pressure continues to rise, in particular in tropical and sub-tropical regions, where greater threats to agriculture and food sustainability are anticipated by the Intergovernmental Panel on Climate Change (IPCC) (Easterling et al. 2007). These trends will certainly lead to mounting conflicts involving water uses (irrigation versus drinking water production or freshwater ecosystem maintenance, sanitation etc.) and food production. This appeals to an “ecologically intensive agriculture” (Griffon 2006), i.e. a sustainable agriculture providing ecosystem services more efficiently than today and causing fewer adverse impacts on the environment and water resources. With EU Directive 2009/128/EC (EC 2009a) enforcement, requesting Member States to adopt action plans aiming to reduce risks and impacts related to pesticide uses, there will be a focus in the public and political debates in Europe on achieving a more sustainable use of pesticides. This should consequently lead to a reduction of the risks or impacts of pesticides on the environment. In Europe, there is currently a strong focus on source (including dose) reduction. This approach may nevertheless be too restrictive if the goal is to reduce the agriculture footprint while maintaining or increasing yield. Depending on the chemical properties of pesticides as well as environmental factors, decreasing the amounts of pesticides applied to crops will not automatically produce a decrease in the risk to non-target species or water supply. How could society meet the challenge of the forthcoming climate change? What adaptations should be envisaged for agriculture/pesticide risk management (RM)? These changes will probably have a profound effect on agricultural systems (crop selection, farming practices etc.) and to a lesser extent influence the fate and effects of chemicals (Schiedek et al. 2007). These questions have been addressed by two European research networks, namely Euraqua (the European Network of Freshwater Research Organisations, http://www.euraqua.org/) and PEER (Partnership for European Environmental Research, http://www.peer.eu/), which organised a workshop aiming to identify research needs and strategies induced by these questions in October 2011 in Montpellier, France. The workshop's specific goals were to (1) discuss the pesticide risk assessment (RA) approach, its limitations (e.g. spatial scale and multi-stress situations), the connections between different policies (pesticide regulation and Water Framework Directive), the use of models, (2) review integrated practices and innovative technologies which could or are intended to reduce pesticides' environmental impacts and (3) contribute to the future research and development agenda. This review summarises the workshop discussions.

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.

Due to their ability to explore whole genome response to drugs and stressors, omics-based approaches are widely used in toxicology and ecotoxicology, and identified as powerful tools for future ecological risk assessment and environmental monitoring programs. Understanding the long-term effects of contaminants may indeed benefit from the coupling of genomics and eco-evolutionary hypotheses. Next-generation sequencing provides a new way to investigate pollutants impact, by targeting early responses, screening chemicals, and directly quantifying gene expression, even in organisms without reference genome. Lymnaea stagnalis is a freshwater mollusk in which access to genomic resources is critical for many scientific issues, especially in ecotoxicology. We used 454-pyrosequencing to obtain new transcriptomic resources in L. stagnalis and to preliminarily explore gene expression response to a redox-cycling pesticide, diquat. We obtained 151,967 and 128,945 high-quality reads from control and diquat-exposed individuals, respectively. Sequence assembly provided 141,999 contigs, of which 124,387 were singletons. BlastX search revealed significant match for 34.6 % of the contigs (21.2 % protein hits). KEGG annotation showed a predominance of hits with genes involved in energy metabolism and circulatory system, and revealed more than 400 putative genes involved in oxidative stress, cellular/molecular stress and signaling pathways, apoptosis, and metabolism of xenobiotics. Results also suggest that diquat may have a great diversity of molecular effects. Moreover, new genetic markers (putative SNPs) were discovered. We also created a Ensembl-like web-tool for data-mining ( http://genotoul-contigbrowser.toulouse.inra.fr:9095/Lymnaea_stagnalis/index.html ). This resource is expected to be relevant for any genomic approach aimed at understanding the molecular basis of physiological and evolutionary responses to environmental stress in L. stagnalis.

Although a number of biomarkers of pollutant exposure have been identified in invertebrate species, direct linkage with changes at population and/or community levels are poorly documented and, despite the ecological importance of invertebrates, there is no conclusive evidence that individual measurements of biochemical parameters may allow the effects of pollutants on populations and communities to be predicted. Among the various biochemical parameters used as biomarkers in invertebrates exposed to pollutants in the field, only those for which changes at population or community level can be suggested are discussed in the present review. At population and community levels, the development of resistance to pesticides and changes in behaviour, reproduction and development are analysed as putative consequences of biochemical and physiological alterations. Limits to the use of biochemical parameters as biomarkers of invertebrate exposure to pollutants are discussed. Future research trends and experimental approaches to the validation of invertebrate biomarkers in environmental pollution assessment are suggested.

Intestinal pH, determined in situ from the colours of indicator dyes ingested with sucrose, ranged between 6.5-5.5 in the foregut and midgut, and 5.5-4.5 in the hindgut. An identical optimal pH value of 5.5 was determined for sucrase, maltase and amylase. Under physiological conditions of pH (6.0), the kinetics of α-glycosidase activity showed that maltase was by far the most active, followed by sucrase and then amylase. Substrate specificities and preliminary characterization of the major digestive α-glycosidases of B. affinis suggest a well-fitted adaptation to the seasonal variations of diet composition.

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.

Because exposure to toxicants not only results in mortality but also in multiple sublethal effects, the use of life-table data appears particularly suitable to assess global effects on exposed populations. The present study uses a life table response approach to assess population-level effects of two insecticides used against mosquito larvae, spinosad (8 mu g/l) and Bacillus thuringiensis var. israelensis (Bti, 0.5 mu l/l), on two non target species, Daphnia pulex and Daphnia magna (Crustacea: Cladocera), under laboratory versus field microcosms conditions. Population growth rates were inferred from life table data and Leslie matrices under a model with resource limitation (ceiling). These were further used to estimate population risks of extinction under each tested condition, using stochastic simulations. In laboratory conditions, analyses performed for each species confirmed the significant negative effect of spinosad on survival, mean time at death, and fecundity as compared to controls and Bti-treated groups; for both species, population growth rate lambda was lower under exposure to spinosad. In field microcosms, 2 days after larvicide application, differences in population growth rates were observed between spinosad exposure conditions, and control and Bti exposure conditions. Simulations performed on spinosad-exposed organisms led to population extinction (minimum abundance = 0, extinction risk = 1), and this was extremely rapid (time to quasi-extinction = 4.1 one-week long steps, i.e. one month). Finally, D. magna was shown to be more sensitive than D. pulex to spinosad in the laboratory, and the effects were also detectable through field population demographic simulations.

The present study was undertaken to assess the impact of a candidate mosquito larvicide, spinosad (8, 17 and 33 μg L−1) on a field population of Daphnia magna under natural variations of water temperature and salinity, using Bti (0.16 and 0.50 μL L−1) as the reference larvicide. Microcosms (125 L) were placed in a shallow temporary marsh where D. magna was naturally present. The peak of salinity observed during the 21-day observation period may have been partly responsible for the decrease of daphnid population density in all the microcosms. It is also probably responsible for the absence of recovery in the microcosms treated with spinosad which caused a sharp decrease of D. magna abundance within the first two days following treatment whereas Bti had no effect. These results suggest that it may be difficult for a field population of daphnids to cope simultaneously with natural (water salinity and temperature) and anthropogenic (larvicides) stressors. Significant interaction between salinity and spinosad exposure impairs the recovery of a natural population of Daphnia magna

[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 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.

Spinosad, a candidate biological larvicide for mosquito control, was evaluated for its effects on a field population of Daphnia pulex, using Bacillus thuringiensis serovar israelensis (Bti) as a reference larvicide. Microcosms (125L enclosures) were placed in a shallow temporary oligohaline marsh where D. pulex was present. Three concentrations of spinosad (8, 17 and 33 microg L(-1)) and two concentrations of Bti (0.16 and 0.50 microL L(-1)) were applied (5 replicates per concentration, including the controls). Effects of larvicides on D. pulex were evaluated after 2, 4, 7, 14 and 21d of exposure, through measurements of abundance and individual size. Dissipation of spinosad from the water phase was rapid. Four days after treatment, residue concentration represented 11.8%, 3.9% and 12.7% of the initial exposure level for the nominal concentrations of 8, 17 and 33 microg L(-1), respectively. Spinosyns A and D dissipated at similar rates. Analysis of abundance and size structure of the D. pulex population showed an impact of spinosad. Both survival and size structure were affected. However, at the lowest concentration (8 microg L(-1)), population recovered after the first week. In microcosms treated with Bti, the abundance of D. pulex was not affected but the size structure of the population changed after 21d. As compared to laboratory tests, the use of in situ microcosms improved the environmental risk assessment of larvicides, taking into account the influence of environmental factors (e.g., temperature, light, salinity) and intrinsic capacity of recovery of D. pulex under field conditions.

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.

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

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.

Outdoor artificial ponds (mesocosms) of 12 m 3 were designed for long-term ecotoxicological studies. Sediment, macrophytes ( Typha angustifolia and Elodea canadensis ), and free and caged freshwater snails [ Lymnaea palustris (Muller)] and wood lice ( Asellus aquaticus L.) were collected in nearby natural ecosystems and introduced in the mesocosms. Sixty goldfish ( Carassius auratus L.) were caged in each pond. Introduced species developed and reproduced in every mesocosm. Animal species (mainly insects and amphibians) spontaneously colonized the ponds, developed, and reproduced. The resulting communities qualitatively resemble those living in natural lentic systems in the surrounding area. Homogenity in physical and chemical conditions and in abundance of phytoplanktonic, periphytic, and macroinvertebrate communities between the different mesocosms was assessed during the stabilization period (8 months). Except for periphyton biomass, no divergent evolution was observed between the ponds. Mesocosm water was slightly eutrophic, alkaline (mean pH: 8.47 ± 0.09), and moderately hard and mineralized. The homogenous and realistic environmental conditions and high ecological representativity of the outdoor experimental ponds were suitable for extensive ecotoxicological studies. Considerations on the choice and origin of introduced species and on possible interactive effects of the transfer of organisms from natural environments, maintainance conditions, and pollutant exposure are discussed.