In this study, we assessed the chronic effects of the two antimicrobial substances triclocarban (TCC) and triclosan (TCS) on reproduction of a mollusk species by using the reproduction test with the New Zealand mudsnail Potamopyrgus antipodarum. Snails coming from a laboratory culture were exposed for 28 days to nominal concentrations ranging from 0.1 up to 10 µg/L for both chemicals (measured 0.082-8.85 µg TCC/L; 0.068-6.26 µg TCS/L). At the end of the experiment, snails were dissected and embryos in the brood pouch were counted to assess the individualized reproductive success of adult snails. Exposure to TCC resulted in an inverted u-shaped concentration-response relationship, with a stimulation of reproduction at low concentrations followed by an inhibition at higher concentrations. The no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were 0.082 and 0.287 µg/L, respectively. TCS caused significantly increased embryo numbers at all tested concentrations, except in the group of 0.170 µg/L. Therefore, the NOEC for TCS was 0.170 µg/L and the LOEC was 0.660 µg/L. These results indicate that TCC and TCS may cause reproductive effects at environmentally relevant concentrations indicating a potential risk for aquatic organisms in the environment.

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 μg/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 EC, EC, 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 μg/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.

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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 EC and NOEC values, showing the inter-laboratory reproducibility of results. The average EC and NOEC values for reproduction (with coefficient of variation) were 26.2 µg/L (61.7%) and 29.7 µg/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.