1H NMR-based Metabolomics for Elucidating the Mode of Action of Ccontaminants in the Earthworm Eisenia Fetida after Sub-lethal Exposure

There is a growing need to develop rapid and cost-effective ecotoxicological tools for risk assessment because traditional methods examine endpoints such as mortality, which do not provide any insight into the mode of action (MOA) of the chemical. Research presented within this thesis illustrates the potential of 1H NMR-based metabolomics as a rapid and routine ecotoxicological tool that can elucidate a chemical’s MOA and also aid in the identification of metabolites of exposure. Metabolomics involves measuring the fluctuations in the endogenous metabolites of an organism within a cell, tissue, bio-fluid or whole organism in response to an external stressor. We focused on the model polycyclic aromatic hydrocarbon (PAH) phenanthrene, and the perfluoroalkyl acids (PFAAs) perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), due to their recalcitrant nature and widespread prevalence in soil environments. 1H NMR-based metabolomics analysis of the exposure of Eisenia fetida earthworms to sub-lethal phenanthrene exposure via filter paper contact tests revealed a concentration-dependent two-phased MOA: a linear correlation between the metabolic response and exposure concentration at low concentrations followed by a plateau in the responses at high concentrations. Alanine, glutamate, maltose, cholesterol and phosphatidylcholine emerged as potential indicators of phenanthrene exposure. An increased energy demand and an interruption in the conversion of succinate to fumarate in the Krebs cycle were observed due to phenanthrene exposure. Sub-lethal PFOA and PFOS exposure to E. fetida via contact tests for two days revealed heightened responses with higher PFOA and PFOS concentrations. Leucine, arginine, glutamate, maltose, and ATP were identified as potential indicators of PFOA or PFOS exposure. E. fetida responses were then investigated after exposure for two, seven and fourteen days to an artificial soil that was spiked with sub-lethal PFOS concentrations. An exposure time-dependent operation of two separate MOAs were identified. Both the contact tests and artificial soil exposure studies identified an elevation in fatty acid oxidation, a disruption in energy metabolism and biological membrane structure, and also an interruption of ATP synthesis following PFOA and PFOS exposure. This thesis illustrates the promise of NMR-based metabolomics as a routine tool for ecotoxicological assessment of contaminated sites.Ph

Analysis of Sub-Lethal Toxicity of Perfluorooctane Sulfonate (PFOS) to Daphnia magna Using 1H Nuclear Magnetic Resonance-Based Metabolomics

1H nuclear magnetic resonance (NMR)-based metabolomics was used to characterize the response of Daphnia magna after sub-lethal exposure to perfluorooctane sulfonate (PFOS), a commonly found environmental pollutant in freshwater ecosystems. Principal component analysis (PCA) scores plots showed significant separation in the exposed samples relative to the controls. Partial least squares (PLS) regression analysis revealed a strong linear correlation between the overall metabolic response and PFOS exposure concentration. More detailed analysis showed that the toxic mode of action is metabolite-specific with some metabolites exhibiting a non-monotonic response with higher PFOS exposure concentrations. Our study indicates that PFOS exposure disrupts various energy metabolism pathways and also enhances protein degradation. Overall, we identified several metabolites that are sensitive to PFOS exposure and may be used as bioindicators of D. magna health. In addition, this study also highlights the important utility of environmental metabolomic methods when attempting to elucidate acute and sub-lethal pollutant stressors on keystone organisms such as D. magna

1H NMR-Based Metabolomic Analysis of Sub-Lethal Perfluorooctane Sulfonate Exposure to the Earthworm, Eisenia fetida, in Soil

1H NMR-based metabolomics was used to measure the response of Eisenia fetida earthworms after exposure to sub-lethal concentrations of perfluorooctane sulfonate (PFOS) in soil. Earthworms were exposed to a range of PFOS concentrations (five, 10, 25, 50, 100 or 150 mg/kg) for two, seven and fourteen days. Earthworm tissues were extracted and analyzed by 1H NMR. Multivariate statistical analysis of the metabolic response of E. fetida to PFOS exposure identified time-dependent responses that were comprised of two separate modes of action: a non-polar narcosis type mechanism after two days of exposure and increased fatty acid oxidation after seven and fourteen days of exposure. Univariate statistical analysis revealed that 2-hexyl-5-ethyl-3-furansulfonate (HEFS), betaine, leucine, arginine, glutamate, maltose and ATP are potential indicators of PFOS exposure, as the concentrations of these metabolites fluctuated significantly. Overall, NMR-based metabolomic analysis suggests elevated fatty acid oxidation, disruption in energy metabolism and biological membrane structure and a possible interruption of ATP synthesis. These conclusions obtained from analysis of the metabolic profile in response to sub-lethal PFOS exposure indicates that NMR-based metabolomics is an excellent discovery tool when the mode of action (MOA) of contaminants is not clearly defined