In vivo fish bioaccumulation testing is a costly, time-consuming, and ethically contentious requirement under pesticide regulatory frameworks. Fish cell lines offer validated in vitro alternatives that support the 3Rs principles of replacement, reduction, and refinement. In this study, we assessed the acute cytotoxicity and bioaccumulation of three organic pesticides (trifloxystrobin, tebuconazole, and tembotrione) using RTgill-W1 cells, derived from the gills of rainbow trout (Oncorhynchus mykiss), as an in vitro model. Non-toxic concentrations, derived from acute cytotoxicity testing per OECD TG 249, were used for bioaccumulation assessment.

Bioaccumulation factors were quantified using kinetic BCF (from uptake and elimination rate constants) and steady-state BCF approaches, and compared against in vivo measurements and QSAR estimates. Chemical analysis revealed compound-specific depletion in cell-containing exposures relative to cell-free controls, and biotransformation products were detected, confirming the biotransformation capacity of the cell line. Together, these findings demonstrate that RTgill-W1 cell assays can reliably capture bioaccumulation endpoints relevant to regulatory fish toxicity assessment, offering a practical and ethical alternative to animal testing.