In vivo measurement of xenobiotic detoxification in annelids: ECOD activity in a terrestrial, a freshwater, and an estuarine worm

Cytochrome monooxygenases (CYPs) play a pivotal role in xenobiotic detoxification through chemical biotransformation. In vitro methods for measuring these CYPs commonly rely on samples obtained from homogenised tissues. Such preparations, however, often lead to enzyme degradation and auto-inhibition, rendering them unsuitable for some species. Methods to quantify CYP activity in vivo have been proposed as an alternative to the use of homogenised tissues. However, such methods remain limited, especially for soil- and sediment-dwelling species. This study aimed to adapt and validate an in vivo 7-ethoxycoumarin-O-deethyllase (ECOD) assay previously used for aquatic invertebrates to measure CYP-dependent enzyme activity in three annelid species: Enchytraeus crypticus (terrestrial oligochaete), Capitella teleta (estuarine sediment-dwelling polychaete), and Tubifex tubifex (freshwater sediment-dwelling oligochaete) exposed in water. The successful development and use of the assay demonstrated ECOD activity in E. crypticus and C. teleta, with activity in C. teleta up to 17 times higher than in E. crypticus per milligram wet weight. In contrast, no measurable ECOD activity was observed in T. tubifex, likely reflecting assay sensitivity limitations for this species. CYP inhibition studies with azole fungicides (propiconazole, imazalil) confirmed that the assay is able to detect concentration-dependent CYP inhibition in E. crypticus, highlighting the potential of the method for toxicological assessments of CYP activity and its inhibition or induction. To our knowledge, this study is the first to develop and apply an in vivo assay to measure CYP activity in a terrestrial and annelid species.