α-, β-, and γ-Hexabromocyclododecanes (HBCDs) were subjected to in vitro biotransformation experiments with rat and trout liver S9 fractions for different incubation times (10, 30, and 60 min) at 2 concentration levels (1 and 10 μM). The metabolic degradation of target HBCDs followed first order kinetics. Whereas β-HBCD undergoes rapid biotransformation (t0.5 = 6.4 and 38.1 min in rat and trout, respectively), α-HBCD appears the most resistant to metabolic degradation (t0.5 = 17.1 and 134.9 min). The biotransformation rate in trout was slower than in rat. Investigation of HBCD degradation profiles revealed the presence of at least 3 pentabromocyclododecene (PBCD) and 2 tetrabromocyclododecadiene (TBCD) isomers indicating reductive debromination as a metabolic pathway for HBCDs. Both mono- and di- hydroxyl metabolites were identified for parent HBCDs, while only mono hydroxyl metabolites were detected for PBCDs and TBCDs. Interestingly, δ-HBCD was detected only in trout S9 fraction assays indicating metabolic interconversion of test HBCD diastereomers during biotransformation in trout. Finally, enantioselective analysis showed significant enrichment of the (-)-α-HBCD enantiomer (EF = 0.321 and 0.419 after 60 min incubation in rat and trout, respectively). The greater enrichment of (-)-α-HBCD in rat than in trout underlines the species-specific differences in HBCD metabolism and the need for caution when extending similar results from animal studies to humans.

Prediction of xenobiotic fate in fish is important for the regulatory assessment of chemicals under current legislation.

1. In vitro screens are sought as informative, alternatives to the use of animals in vivo and to improve upon the current use of fish liver 9000 g supernatants (S9) in environmental risk assessment. 2. The rates of ethoxyresorufin-O-deethylation (relative to S9 protein) measured under different conditions of culture of rainbow trout hepatocytes were significantly higher than those detected in S9, in the order of freshly isolated hepatocytes > 10-day spheroid cultures > primary hepatocytes in culture > S9. The percentage of conjugated metabolites was also similar between freshly isolated and spheroid cultured hepatocytes (9.9 and 13.5%). 3. The rate of oxidation was enhanced (1.7 fold) when S9 was supplemented with cofactors for phase II conjugation but this was only approximately one tenth of the rate in freshly isolated hepatocytes (7.1 pmol/min/mg S9 protein equivalent). 4. Hepatocytes also hydroxylated ibuprofen, producing two metabolites, in contrast to only one (identified as the 1-hydroxy derivative) using hepatic S9 fractions. 5. Since the bioaccumulation potential of chemicals is often based on un-supplemented S9 in incubations ≥ 1 h when activity declines, it is recommended that predictability would be greatly improved through the use of hepatocyte spheroids, due to their maintenance of activity and longevity.