Reproduction stage specific dysregulation of Daphnia magna metabolites as an early indicator of reproductive endocrine disruption

Abstract

Rapid biomolecular observation in model indicator organisms has been considered as a potential predictor of water pollution from chronic and trace toxicants. This study evaluated the use of Daphnia magna metabolomic measurements as indicators for exposure to reproductive endocrine disruptors by using the model juvenile hormone analogue fenoxycarb. Because D. magna reproduction controls metabolic regulation, the reproduction stage was also carefully considered in metabolic observations and data analysis to examine differences. Comparisons of metabolite abundance regulation between 1 and 12 days of fenoxycarb exposure were performed to investigate the predictability of the sub-chronic (12 days) adverse impacts on reproduction and metabolic regulation based on acute (1 day) metabolic observations. ANOVA-simultaneous component analysis (ASCA) detected reversed patterns in direction of time-course metabolite abundance regulation with fenoxycarb exposure. For example, decreases in the abundances of leucine, asparagine, methionine, and isoleucine which then changed to increases were observed with time during fenoxycarb exposures. The reversed regulation pattern was observed at the last reproduction stage (stage 3), exclusively. Pearson correlation analysis showed that correlations of pairwise metabolites were disrupted with fenoxycarb exposure. Similar to ASCA, data normalization based on the reproduction stage improved the detectability of significant correlations. The disruption on ambient metabolite regulation patterns and pairwise metabolite correlations was consistently observed with both 1 and 12 days of fenoxycarb exposures for sets of select metabolites. The observed regulatory disruptions to these specific metabolites suggest altered oogenesis as the affected metabolites and the specific reproduction stage are related to successful oogenesis. This study demonstrates that D. magna metabolic dysregulation is a predictor of water contamination by endocrine disrupting compounds. The high predictability of sub-chronic (12 days) endocrine disruption was confirmed based on acute (1 day) metabolic observations. Furthermore, integration of the reproduction cycle information in D. magna metabolomics was validated by observing a reproduction stage specific dysregulation in metabolite abundance regulation, which was not observable from the broader data analysis. Consequently, this study confirms the potential for establishing a quantitative relationship between water quality and indicator species metabolic observations. Additionally, it was found that constraining variables relevant to toxicity mechanisms of interest, such as the reproduction stage, is a key consideration for extraction of ecologically meaningful information in environmental metabolomics.