Metabolism of 4-fluoroaniline and 4-fluorobiphenyl in the earthworm Eisenia veneta characterized by high-resolution NMR spectroscopy with directly coupled HPLC-NMR and HPLC-MS

Abstract

1. Little is known about metabolism of xenobiotics by earthworms, despite their importance in soil ecotoxicity testing. Normal earthworms and earthworms treated with antibiotics to ensure inhibition of gut microflora were exposed to two model xenobiotic compounds, 4-fluoroaniline and 4-fluorobiphenyl, to determine which metabolites were produced, and whether the pattern of metabolism was affected by the presence of microbial transformation ability. 2. 19 F-NMR spectroscopy detected the number and relative proportions of metabolites and directly coupled HPLC- 1 H-NMR spectroscopy and HPLC-MS then identified the metabolites. 3. Despite uptake, no metabolism of 4-fluorobiphenyl was observed at any stage, which appears to be a consequence of the lack of oxidative Phase I metabolic activity of the earthworms towards this substrate. In contrast, 4-fluoroaniline exhibited dose-dependent metabolism. At high doses (leading to mortality within 24 h) one predominant metabolite was observed, which was identified as the N - β -glucoside conjugate. At lower dose levels, the predominant metabolite was the γ-glutamyl conjugate, although the glucoside and another as yet unidentified metabolite were also detected. 4. The inhibition of gut microflora did not have any influence on metabolism. The study represents the first evidence for glucoside and glutamyl conjugation as a pathway for xenobiotic metabolism in earthworms.