Enzymatic and microbial transformation assays for the evaluation of the environmental fate of diclofenac and its metabolites

Diclofenac has been of environmental concern due to the potential harmful effects on non-target organisms at environmentally relevant concentrations. In this study, we evaluated the transformation kinetics of diclofenac and its two major metabolites in two laboratory-scale experiments: the transformation of diclofenac in the presence of rat liver S9 fraction with co-factors, and the transformation of diclofenac, 4′-hydroxy-diclofenac and diclofenac β-O-acyl glucuronide in the inoculum used for the OECD 301C ready-biodegradability test. 4′-Hydroxy-diclofenac was identified as the major phase I metabolite and diclofenac β-O-acyl glucuronide was identified as the major phase II metabolite in the S9 assay. Transformation of diclofenac in the microbial degradation test did not occur significantly for 28 d, whereas 4′-hydroxy-diclofenac degraded slowly, indicating that the biological removal of diclofenac is not likely to occur in conventional STPs unless sorptive removal is significant. However, diclofenac β-O-acyl glucuronide deconjugated to form equimolar diclofenac within 7 d, in the microbial degradation test. The mixture of diclofenac and its two metabolites, formed after incubating diclofenac in S9 medium for 2 h, was spiked in the inoculum to link both assays. The concentrations of diclofenac and its metabolites, measured over time, agreed well with predicted values, using rate parameters obtained from independent experiments. The results show that phase II metabolites generated in mammals may deconjugate easily in conventional STPs to form a parent compound and that these processes should be considered during the environmental monitoring and risk assessment of diclofenac.