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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 10 , EC 50 , 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. [ABSTRACT FROM AUTHOR]

The OECD test guideline development program has been extended in 2011 to establish a partial life-cycle protocol for assessing the reproductive toxicity of chemicals to several mollusk species, including the great pond snail Lymnaea stagnalis . In this paper, we summarize the standard draft protocol for a reproduction test with this species, and present inter-comparison results obtained in a 56-day prevalidation ring-test using this protocol. Seven European laboratories performed semi-static tests with cultured snails of the strain Renilys® exposed to nominal concentrations of cadmium chloride (from 53 to 608 μg Cd L −1 ). Cd concentrations in test solutions were analytically determined to confirm accuracy in the metal exposure concentrations in all laboratories. Physico-chemical and biological validity criteria (namely dissolved oxygen content >60% ASV, water temperature 20 ± 1 °C, control snail survival >80% and control snail fecundity >8 egg-masses per snail over the test period) were met in all laboratories which consistently demonstrated the reproductive toxicity of Cd in snails using the proposed draft protocol. Effect concentrations for fecundity after 56 days were reproducible between laboratories (68 < EC 50–56d < 124 μg L −1 ) and were consistent with literature data. EC 50–56d and EC 10–56d values were comprised within a factor of 1.8 and 3.6, respectively, which is in the range of acceptable variation defined for reference chemicals in OECD test guidelines for invertebrates. The inter-laboratory reproducibility coefficient of variation (CV) for the Cd LC 50–56d values was 8.19%. The inter-laboratory comparison of fecundity within the controls gave a CV of 29.12%, while exposure to Cd gave a CV of 25.49% based on the EC 50–56d values. The OECD has acknowledged the success of this prevalidation exercise and a validation ring-test involving 14 laboratories in Europe, North- and South-America is currently being implemented using four chemicals (Cd, prochloraz, trenbolone and tributyltin). [ABSTRACT FROM AUTHOR]

Altered concentration of the vertebrate yolk protein precursor vitellogenin is a recognized biomarker for endocrine disruption in fish, and within recent years yolk protein alteration has also been associated with endocrine disruption in bivalves. Species-specific, direct and sensitive methods for quantification of vitellogenin in fish have been available for years whereas bivalve yolk protein levels have been estimated indirectly by alkali-labile phosphate (ALP) liberated from high molecular weight proteins because the sequence and biochemical structure of most bivalve yolk proteins are unknown. By applying a species-specific enzyme-linked immunosorbent assay (ELISA) for accurate determination of yolk protein level the impact of 17β-estradiol (57, 164 and 512 ng/L) on the freshwater bivalve Unio tumidus was investigated and compared with ALP estimations. Seven weeks of exposure during the pre-spawning and spawning period had no consistent effect on yolk protein concentration in hemolymph, and ALP levels in hemolymph also remained unchanged in both males and females. Further, basal male and female ALP levels were indistinguishable whereas the ELISA demonstrated that yolk protein levels of females exceeded male levels at the time of sampling, although male basal levels were high compared to fish. Altogether it is shown that individual ALP levels do not reflect yolk protein levels and hence hemolymph ALP levels cannot serve as biomarker for estrogenic exposure during the pre-spawning and spawning period in U. tumidus . The necessity of sensitive and validated biomarkers for reliable interpretation of data and the utility of ALP and yolk protein levels as biomarkers in bivalves are discussed. [ABSTRACT FROM AUTHOR]