Metabolomics reveals energetic impairments in Daphnia magna exposed to diazinon, malathion and bisphenol-A.

(1)H nuclear magnetic resonance (NMR)-based metabolomics was used to study the response of Daphnia magna to increasing sub-lethal concentrations of either an organophosphate (diazinon or malathion) or bisphenol-A (BPA). Principal component analysis (PCA) of (1)H NMR spectra were used to screen metabolome changes after 48h of contaminant exposure. The PCA scores plots showed that diazinon exposures resulted in aberrant metabolomic profiles at all exposure concentrations tested (0.009-0.135 μg/L), while for malathion the second lowest (0.08μg/L) and two highest exposure concentrations (0.32μg/L and 0.47μg/L) caused significant shifts from the control. Individual metabolite changes for both organophosphates indicated that the response to increasing exposure was non-linear and described perturbations in the metabolome that were characteristic of the severity of exposure. For example, intermediate concentrations of diazinon (0.045μg/L and 0.09μg/L) and malathion (0.08μg/L) elicited a decrease in amino acids such as leucine, valine, arginine, glycine, lysine, glutamate, glutamine, phenylalanine and tyrosine, with concurrent increases in glucose and lactate, suggesting a mobilization of energy resources to combat stress. At the highest exposure concentrations for both organophosphates there was evidence of a cessation in metabolic activity, where the same amino acids increased and glucose and lactate decreased, suggesting a slowdown in protein synthesis and depletion of energy stocks. This demonstrated a similar response in the metabolome between two organophosphates but also that intermediate and severe stress levels could be differentiated by changes in the metabolome. For BPA exposures, the PCA scores plot showed a significant change in metabolome at 0.1mg/L, 1.4mg/L and 2.1mg/L of exposure. Individual metabolite changes from 0.7 to 2.1mg/L of BPA exposure showed increases in amino acids such as alanine, valine, isoleucine, leucine, arginine, phenylalanine and tyrosine. These metabolite changes were correlated with decreases in glucose and lactate. This pattern of response was also seen in the highest organophosphate exposures and suggested a generalized stress response that could be related to altered energy dynamics in D. magna. Through studying increasing exposure responses, we have demonstrated the ability of metabolomics to identify discrete differences between intermediate and severe stress, and also to characterize how systemic stress is manifested in the metabolome.