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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 48 h 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 (0.08 μg/L) and two highest exposure concentrations (0.32 μg/L and 0.47 μg/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 (0.045 μg/L and 0.09 μg/L) and malathion (0.08 μg/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 0.1 mg/L, 1.4 mg/L and 2.1 mg/L of exposure. Individual metabolite changes from 0.7 to 2.1 mg/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. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) is the primary platform used in high-throughput environmental metabolomics studies because its non-selectivity is well suited for non-targeted approaches. However, standard NMR probes may limit the use of NMR-based metabolomics for tiny organisms because of the sample volumes required for routine metabolic profiling. Because of this, keystone ecological species, such as the water flea Daphnia magna, are not commonly studied because of the analytical challenges associated with NMR-based approaches. Here, the use of a 1.7-mm NMR microprobe in analyzing tissue extracts from D. magna is tested. Three different extraction procedures (D2O-based buffer, Bligh and Dyer, and acetonitrile : methanol : water) were compared in terms of the yields and breadth of polar metabolites. The D2O buffer extraction yielded the most metabolites and resulted in the best reproducibility. Varying amounts of D. magna dry mass were extracted to optimize metabolite isolation from D. magna tissues. A ratio of 1-1.5-mg dry mass to 40 µl of extraction solvent provided excellent signal-to-noise and spectral resolution using 1H NMR. The metabolite profile of a single daphnid was also investigated (approximately 0.2 mg). However, the signal-to-noise of the 1H NMR was considerably lower, and while feasible for select applications would likely not be appropriate for high-throughput NMR-based metabolomics. Two-dimensional NMR experiments on D. magna extracts were also performed using the 1.7-mm NMR probe to confirm 1H NMR metabolite assignments. This study provides an NMR-based analytical framework for future metabolomics studies that use D. magna in ecological and ecotoxicity studies. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Highlights: [•] Daphnia magna responses to sub-lethal metal exposure using NMR-based metabolomics. [•] Copper and lithium had similar changes indicating a similar mode of action. [•] The changes suggest altered production of neurotransmitters and impaired energy metabolism. [•] Arsenic exposure shows changes in the amino acids lysine and alanine. [•] 1H NMR metabolomics provides insight into metal contamination on aquatic species. [Copyright &y& Elsevier]