Measurement of kinetic parameters for biotransformation of polycyclic aromatic hydrocarbons by trout liver S9 fractions: Implications for bioaccumulation assessment.

substrate depletion methods developed by the pharmaceutical industry are being used with increasing frequency to support chemical bioaccumulation assessments for fish. However, the application of these methods to high log chemicals poses special challenges. Biotransformation of three polycyclic aromatic hydrocarbons (PAHs) was measured using trout liver S9 fractions. Measured activity declined with incubation time and was reduced by acetone (used as a spiking solvent) at concentrations greater than 0.5%. Addition of alamethicin, a pore-forming peptide used to support UDP-glucuronosyltransferase activity, also reduced activity in a concentration-dependent manner. The substrate concentration dependence of activity was evaluated to estimate and values for each compound. Derived kinetic constants suggested that all three PAHs are transformed by the same reaction pathway and indicated an inverse correlation between and chemical log . Binding effects on activity were evaluated by measuring unbound chemical concentrations across a range of S9 protein levels. Reaction rates were proportional to the unbound concentration except when these concentrations approached saturating levels, providing a direct demonstration of the free chemical hypothesis. These findings suggest that previous work with high log compounds was conducted at inappropriately high substrate concentrations resulting in underestimation of true activity. Preliminary calculations also indicate that PAH metabolism in fish may approach saturation during standardized testing efforts, potentially resulting in concentration-dependent accumulation and/or steady-state levels of accumulation greater than those which occur in a natural setting.