Environmental context. Phenanthrene is a persistent soil contaminant, whose toxic mode of action in earthworms has not been fully examined. We adopt a metabolomics approach, using 1 H nuclear magnetic resonance (NMR) spectroscopy, to measure the response of earthworms to sub-lethal phenanthrene exposure. The results indicate that NMR-based metabolomics may be used to monitor responses to sub-lethal levels of contaminants and to delineate their toxic mode of action.
Abstract.1 H NMR-based metabolomics was used to examine the response of the earthworm Eisenia fetida to sublethal phenanthrene exposure. E. fetida were exposed via contact tests to six sub-lethal (below the measured LC 50 of 1.6 mg cm
À2) concentrations of phenanthrene (0.8-0.025 mg cm À2 ) for 48 h. Multivariate statistical analysis of the 1 H NMR spectra of earthworm tissue extracts revealed a two-phased mode of action (MOA). At exposures below 1/16th of the LC 50 , the MOA was characterised by a linear correlation between the metabolic response and exposure concentration. At exposures !1/16th of the LC 50 , the metabolic response to phenanthrene appeared to plateau, indicating a distinct change in the MOA. Further data analysis suggested that alanine, lysine, arginine, isoleucine, maltose, ATP and betaine may be potential indicators for sub-lethal phenanthrene exposure. Metabolite variation was also found to be proportional to the exposure concentration suggesting that NMR-based earthworm metabolomics is capable of elucidating concentrationdependent relationships in addition to elucidating the MOA of sub-lethal contaminant-exposure.