Hydrogen NMR metabolomics of earthworm responses to sub-lethal polycyclic aromatic hydrocarbon exposure

1H nuclear magnetic resonance (NMR) metabolomics was used to determine the response of earthworm exposure to polycyclic aromatic hydrocarbons (PAHs) in contact and soil tests. Eisenia fetida is recommended for toxicology testing, but to date this species has not frequently been used in environmental metabolomic studies. The metabolic profile of E. fetida was characterized with the goal of using this species in metabolomic studies. Testing several individual solvents for earthworm tissue extraction indicated that D₂O buffer extracted the highest concentration of the widest variety of earthworm metabolites. Sample preparation methods were evaluated to reduce variability and achieve reproducible control groups for use in metabolomic studies. 96h depuration and intact lyophilization of earthworms before homogenization resulted in the least variation between sample extracts. This sample preparation method was used to compare E. fetida and two other earthworm species (Lumbricus rubellus and Lumbricus terrestris) and E. fetida had the most reproducible 1H NMR spectra. E. fetida was then used to identify the metabolic response after exposure to several concentrations of the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene and pyrene, individually and in mixtures. With exposure to individual PAHs in contact tests, earthworm responses were both PAH- and concentration-dependent. In earthworms exposed to PAH mixtures in contact tests, an increase in amino acids was measured. Furthermore, an increase in specific amino acids and a decrease in maltose were identified as potential indicators of sub-lethal phenanthrene exposure in soil. Lastly, the relationship between earthworm response and contaminant bioavailability in soil was tested. Contaminant bioavailability is typically assessed using indirect methods [e.g., ‘soft’ extraction techniques like hydroxypropyl cyclodextrin (HPCD) extraction]. However, it was found that the directly measured response of earthworm exposure to sub-lethal concentrations of phenanthrene in soil is related to both the total and bioavailable phenanthrene. This suggests there is potential for the use of 1H NMR metabolomics for the assessment of contaminant bioavailability. This thesis has demonstrated that E. fetida are suitable for metabolomic studies and has indicated that 1H NMR metabolomics may have potential for measuring and monitoring earthworm exposure to sub-lethal concentrations of PAHs.