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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.

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Diet quality can have a strong impact on organismal fitness although diet quality is infrequently considered as a factor in toxicity tests. The purpose of this study was to assess how diets differing in nutritional content affect sensitivity to Cd as measured by several sublethal responses related directly to bioenergetics. We evaluated feeding rate, growth rate, behavior and macronutrient content in the pond snail Lymnaea stagnalis exposed to Cd and fed two different diets. Hatchlings were fed either lettuce or high-caloric pellets, and exposed to 5, 10, and 20 µg/L Cd for 12 days. Length and weight were measured at the beginning and end of the test. The amount of food eaten and behavior were determined every two days. Total lipids, proteins and carbohydrates were biochemically measured at test end. For the second part of the study, snails were fed either lettuce or pellets for 2 weeks and then exposed to high challenge concentrations of Cd. Growth coefficients based on length and weight were significantly higher for snails fed pellets. In addition, snails exposed to Cd had significantly smaller growth coefficients than those in the control for both diets. Total carbohydrates and lipids were higher for snails fed pellets while the protein content was not significantly affected by Cd or diets. Even though snails fed pellets grew significantly faster, contrary to expectations they were significantly more sensitive to Cd compared to those fed lettuce. This study provides evidence that a bioenergetics-based approach can been used to better understand how diet can affect the ecotoxicity of chemical stressors to freshwater gastropods.

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