The in vivo ECOD_Sed assay: an optimised method for measuring cytochrome P450 activity in sediment-dwelling organisms

Cytochrome P450 (CYP450) enzymes are central to xenobiotic biotransformation, yet traditional in vivo water-based ethoxycoumarin-O-deethylase (ECOD) assays are not an efficient method for deposit-feeding species. We developed and validated ECOD_Sed, a sediment-integrated in vivo assay, by incorporating sediment through both pre-exposure and assay phases. ECOD_Sed reflects environmentally relevant exposure pathways for species exposed to sediment-associated contaminants during ingestion. We evaluated three assay formats representing different pre-exposure scenarios prior to assessing ECOD in vivo, namely ECOD_Sed14 (14-day pre-exposure), ECOD_Sed7 (7-day pre-exposure), and Quick ECOD_Sed (no pre-exposure). We found that CYP inducers, such as polychlorinated biphenyls (PCBs) and the polycyclic aromatic hydrocarbon fluoranthene (FLUO), had the strongest induction of ECOD activity after 14 days in ECOD_Sed14 (∼55%, PCB), and after 7 days in ECOD_Sed7 (∼129%; FLUO). In both ECOD_Sed7 and ECOD_Sed14, the antidepressant sertraline (SER; CYP inhibitor) produced persistently lower activity (∼30% on average) than controls. In the Quick assay, sertraline significantly inhibited activity (∼50%), while fluoranthene showed no deviation from controls. Overall, the hierarchy of ECOD activity followed: fluoranthene > PCBs > control > sertraline. Our results demonstrate that ECOD_Sed reliably captures chemical-specific metabolic dynamics across multiple exposure durations in vivo. Together, the three ECOD_Sed assays establish a tiered framework that allows users to match assay duration and complexity. The Quick assay offers rapid screening of chemical modulators, species responses, substrate suitability; ECOD_Sed7 captures early phase induction dynamics; ECOD_Sed14 reveals sustained metabolic responses under long term exposure. ECOD_Sed advances CYP450 mediated activity assessment in sediment dwelling organisms, enabling rapid evaluations and deeper mechanistic insight into contaminant effects.