Development of a Lymnaea stagnalis embryo bioassay for chemicals hazard assessment

The validation of high-throughput toxicity tests with invertebrate species is a key priority to improve hazard assessment of new chemicals and increase the available test guidelines with organisms from a representative set of taxa. This work aimed to contribute to the validation of an embryo test with the freshwater gastropod Lymnaea stagnalis, which has been identified by Organization for Economic Co-operation and Development (OECD) as a potential invertebrate test model, and provide the basis for such an endeavor. Recently, a L. stagnalis reproductive test was standardized by the OECD. However, to encompass the entire life cycle, it is crucial to addresses embryogenic development - a phase highly susceptible to various anthropogenic chemicals, which is covered in the proposed methodology. The approach used in the present study is in line with the OECD guidelines and other published studies, namely the Detailed Review Paper (DRP) on Mollusks life-cycle toxicity testing. Here, the assay quality criteria such as basal mortality and abnormality rates, development, growth and hatching rates, the appropriated testing media, and the optimal assay duration were investigated. Cadmium was chosen as the positive test substance, due to the available data and the verified model sensitivity to this compound, namely in the OECD reproductive test validation process. The obtained data demonstrate that L. stagnalis embryogenesis using the developed methodology is highly sensitive to cadmium. High concentration-response correlation was observed using this reference compound, the EC10 and EC50 for growth are 13.57 and 21.84 μg/L, respectively, after 168 h of exposure. The development EC's 10 and 50 were 15.75 and 38.66 μg/L, respectively, after 240 h. This demonstrates the model sensitivity to this compound when compared with other embryo test models, as well as the model sensitivity during the embryogenesis, if compared with the adult stage. Further, given the determined sensitivity parameters, and incubation times, the test can be performed at 240 h as over 95 % of the control embryos were hatched and no further significant changes in the exposure groups were determined. Overall, the findings of the present study demonstrate that the embryo test with L. stagnalis has potential to high-throughput testing and the model has a high sensitivity to cadmium during this life cycle period. The background data provide by this study will be essential to foster the future standardization of this assay.