Leonard, J A, et al. Metabolomic, behavioral, and reproductive effects of the synthetic estrogen 17 alpha-ethinylestradiol on the unionid mussel Lampsilis fasciola. Aquat Toxicol, 2014. 150(103-16).
The endocrine disrupting effects of estrogenic compounds in surface waters on fish, such as feminization of males and altered sex ratios, may also occur in aquatic invertebrates. However, the underlying mechanisms of action and toxicity, especially in native freshwater mussels (Order Unionoida), remain undefined. This study evaluated the effects of a 12-day exposure of 17alpha-ethinylestradiol (EE2), a synthetic estrogen in oral contraceptives commonly found in surface waters, on the behavior, condition, metabolism, and reproductive status of Lampsilis fasciola. Adult mussels of both sexes were exposed to a control and two concentrations of EE2 (0ng/L, 5ng/L considered to be environmentally relevant, and 1000ng/L designed to provide a positive metabolic response), and samples of gill tissue were taken on days 4 and 12; gills were used because of the variety of critical processes they mediate, such as feeding, ion exchange, and siphoning. Observations of mussel behavior (mantle display, siphoning, and foot movement) were made daily, and condition of conglutinates (packets of eggs and/or glochidia) released by females was examined. No significant effects of EE2 on glochidia mortality, conglutinate condition, female marsupial gill condition, or mussel foot extension were observed. However, exposure to both concentrations of EE2 significantly reduced male siphoning and mantle display behavior of females. Metabolomics analyses identified 207 known biochemicals in mussel gill tissue and showed that environmentally relevant EE2 concentrations led to decreases in glycogen metabolism end products, glucose, and several essential fatty acids in females after 12 days, indicating reductions in energy reserves that could otherwise be used for growth or reproduction. Moreover, males and females showed significant alterations in metabolites involved in signal transduction, immune response, and neuromodulation. Most of these changes were apparent at 1000ng/L EE2, but similar metabolites and pathways were also affected at 5ng/L EE2. Components of the extracellular matrix of gill tissue were also altered. These results demonstrate the utility of metabolomics when used in conjunction with traditional physiological and behavioral toxicity test endpoints and establish the usefulness of this approach in determining possible underlying toxicological mechanisms of EE2 in exposed freshwater mussels.