1H NMR-based metabolomic analysis of polar and non-polar earthworm metabolites after sub-lethal exposure to phenanthrene

Abstract

1H nuclear magnetic resonance (1H NMR)-based metabolomics was utilized to elucidate the earthworm sub-lethal toxicity after exposure to the persistent environmental contaminant phenanthrene. Earthworms were exposed to 0.05, 0.2 and 0.4 mg/cm2 of phenanthrene [which correspond to 1/32nd to 1/4th of the 48-h LC50 (concentration that causes 50 % mortality), respectively] via contact tests over 1, 2 and 3 days of dermal contact. 1H NMR-based metabolomic analysis of the polar and non-polar fractions of the earthworm tissue extracts revealed heightened Eisenia fetida toxic responses with both longer exposure times and higher phenanthrene concentrations. Principal component analysis (PCA) of the polar fraction showed significant separation between control and exposed earthworms along PC1 for all phenanthrene concentrations on each day. The PCA of the non-polar fraction showed significant separation between the controls and exposed earthworms for only the first day of exposure. These results suggested that alanine, glutamate, maltose, and fatty acids were potential indicators of phenanthrene exposure. Interruption in energy production due to a deactivation of the succinate dehydrogenase enzyme in the Krebs cycle was also postulated in exposed earthworms. Cross-validated partial least squares-regression models showed that the polar metabolic profile of E. fetida was weakly but significantly correlated to phenanthrene exposure concentrations after day 1 and day 2 of exposure. Overall, this study indicates that with longer exposures, contact time becomes more important than concentration in discriminating between control and exposed earthworms. This study also shows that NMR-based metabolomics has promise as a powerful ecotoxicological tool for elucidating the mode of toxicity of contaminants.