Protocols for measuring in vitro and in vivo biotransformation rates of hydrophobic substances in fish

The effective management of commercial chemicals is important for achieving sustainable development goals while reducing risks to human and ecological health. National and international practices for the management of chemicals involve identifying substances with bioaccumulative properties which can lead to elevated chemical concentrations in organisms and associated toxic effects. Current metrics for identifying bioaccumulative properties of chemicals are the fish-water bioconcentration factor (BCF) and the octanol-water partition coefficient (KOW). The currently recommended method for measuring the BCF is expensive, difficult, time consuming, and requires the use of many animals. The objective of this research is to develop and test alternative methods for assessing the bioaccumulation of substances. Methods include a dietary in-vivo bioaccumulation test, in-vitro biotransformation tests and in-vitro to in-vivo extrapolation. The research involved the development and testing of a dietary bioaccumulation test for determining the BCF as well as biotransformation rates in the intestines and the body of the fish that involved the use of non-metabolizable reference chemicals. The results show that gastro-intestinal biotransformation plays a dominant role in the bioaccumulation of a large number of the tested hydrophobic organic chemicals when fish are exposed via the diet; while somatic biotransformation (including hepatic biotransformation) plays a dominant role in the bioaccumulation of tested chemicals in fish exposed via the water. The results demonstrate that the BCF can be measured in a dietary bioaccumulation tests and that biotransformation pathways and rates differ between aqueous and dietary tests. The research also involved the development and testing of an in-vitro fish liver S9 biotransformation testing method. The results show that biotransformation rates using fish liver fractions are highly dependent on the concentration of the test chemical in the test. As a result, the recommended 1 µM initial substrate concentration may underestimate the in vitro biotransformation rate constant and, therefore, an overestimation of the whole fish BCF. To avoid challenges presented by concentration dependence, multiple solvent delivery based depletion experiments at a range of initial concentrations are recommended for determining the maximum depletion rate constant. Meanwhile, a single sorbent phase dosing experiment may also provide reasonable approximations of maximum depletion rates of very hydrophobic substances. Lastly, the research involved extrapolating in vitro maximum depletion rate constants to somatic biotransformation rate constants and comparing the results with those measured from in vivo dietary tests. The results show a good agreement with empirical measurements from various in vivo experiments for the majority of test chemicals. However, a significant underestimation of the in vitro-extrapolated somatic biotransformation rate constant for 9-methylanthracene may suggest that the fish liver S9 in vitro system may not contain all of the enzymes and/or co-factors to biotransform the chemical compared to the whole fish. Overall, the results demonstrate potential for fish liver S9 extracts to assess in vivo biotransformation potential in the fish body. Both in vivo and in vitro research indicate that extrahepatic may not be considered using standardized in vivo (BCF) and liver S9 in vitro testing. For gastro-intestinal biotransformation to be considered, streamlined in vivo dietary bioaccumulation tests are recommended. Meanwhile, in vitro S9 protocols may be best supplemented with in vitro gastro-intestinal biotransformation tests in future research, especially when extrapolating to endpoints such as BMF and BAF where the diet is a significant route of exposure.