Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca, Gastropoda).

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 608µgCdL(-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 56days were reproducible between laboratories (68

Development and validation of an OECD reproductive toxicity test guideline with the mudsnail Potamopyrgus antipodarum (Mollusca, Gastropoda).

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 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 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.

Endocrine-disrupting effect of the ultraviolet filter benzophenone-3 in zebrafish, Danio rerio.

The chemical ultraviolet (UV) filter benzophenone-3 (BP-3) is suspected to be an endocrine disruptor based on results from in vitro and in vivo testing. However, studies including endpoints of endocrine adversity are lacking. The present study investigated the potential endocrine-disrupting effects of BP-3 in zebrafish (Danio rerio) in the Fish Sexual Development Test (Organisation for Economic Co-operation and Development TG 234) and a 12-d adult male zebrafish study. In TG 234, exposure from 0 d to 60 d posthatch caused a monotone dose-dependent skewing of the phenotypic sex ratio toward fewer males and more female zebrafish (no observed effect concentration [NOEC]: 191 µg/L, lowest observed effect concentration [LOEC]: 388 µg/L). Besides, gonad maturation was affected in both female fish (NOEC 191 µg/L, LOEC 388 µg/L) and male fish (NOEC 388 µg/L, LOEC 470 µg/L). Exposure to BP-3 did not affect the vitellogenin concentration in TG 234. After 12 d exposure of adult male zebrafish, a slight yet significant increase in the vitellogenin concentration was observed at 268 µg/L but not at 63 µg/L and 437 µg/L BP-3. Skewing of the sex ratio is a marker of an endocrine-mediated mechanism as well as a marker of adversity, and therefore the conclusion of the present study is that BP-3 is an endocrine-disrupting chemical in accordance with the World Health Organization's definition.

Evaluation of yolk protein levels as estrogenic biomarker in bivalves; comparison of the alkali-labile phosphate method (ALP) and a species-specific immunoassay (ELISA).

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 17ß-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.

Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT).

Results are presented from a validation (with 5 laboratories) of the Fish Sexual Development Test (FSDT) developed to detect endocrine disrupters (EDs) and included in the OECD (Organisation for Economic Co-operation and Development) working program. The aromatase-inhibiting fungicide prochloraz was tested in zebrafish (Danio rerio) and fathead minnow (Pimephales promelas). The fish were exposed during sexual differentiation and development from 0 to 60 days post hatch (dph). After exposure, the vitellogenin (VTG) concentrations were quantified in head/tail homogenate and the sex ratio was determined (defined as female, male, intersex or undifferentiated). NOEC/LOEC and EC(x) designs were compared to optimize the test approach. Results show that both species are highly sensitive to prochloraz during sexual development. They respond by skewing of the sex ratio towards male phenotype and by a VTG decline in females. The NOEC/LOEC approach is preferred because sex ratio is difficult to analyze with a regression model. The mean NOEC/LOEC for prochloraz on the sex ratio was 43.3/134 µg/L and 101/293 µg/L for zebrafish and fathead minnow, respectively. The mean NOEC/LOEC on the decline in female VTG concentration was 65/110 µg/L and ~30/68 µg/L respectively. In conclusion, zebrafish and fathead minnow are suitable species in the FSDT and their sexual differentiation is equally labile to EDs.