Dependence of genotoxicity of benzo[a]pyrene suspensions in Mutatox test on dissolved concentration and S9 addition

The Mutatox test is a novel genotoxicity test measuring the ability of a test chemical to restore the luminescent state in dark mutants of Vibrio fischeri. Chemicals can be tested with or without rat hepatic S9 enzymes for metabolic activity, so that promutagenic agents can be detected as well. In the Microbics Mutatox Manual (1993, Microbics Corp., Carlsbad, CA), benzo[a]pyrene is recommended as positive control for S9 medium at a nominal concentration of 10 mg/liter with 2% DMSO as carrier solvent. The concentration of benzo[a]pyrene dissolved in this test suspension without addition of S9 was determined by GC-MS. It was significantly lower than the nominal concentration and did not reveal a genotoxic effect in the Mutatox test with S9. The analytical concentration of benzo[a]pyrene in the supernatant of the test suspension in S9 medium was significantly higher and induced the luminescence of bacteria. Therefore, S9 microsomes seem, apart from metabolizing benzo[a]pyrene to its genotoxic epoxide, to increase transfer of benzo[a]pyrene into V. fischeri in such a way that the genotoxic effect is produced. This suggests that sparingly soluble compounds might have different ecotoxicological effects as suspensions than as solutions. Therefore, the possible uptake of suspended particles by organisms without being completely dissolved before should be considered in ecotoxicological tests.