Integrated supercritical fluid extraction, bioassay and chemical screening methods for analyzing vapor-phase compounds of an environmental complex mixture: Diesel exhaust

The integration of bioassay and chemical analyses for vapor-phase compounds in environmental complex mixtures is a challenging process due to the volatility and the limited amounts of compounds collected and subsequently assayed. The advantage of an integrated approach is that the bioassay could aid in determining the most biologically active fractions for further chemical analyses. Previously, we reported on the use of supercritical fluid extraction (SFE) in conjunction with a Salmonella microsuspension assay to measure mutagenic activity in model vapor-phase mutagens. The procedure was effective in minimizing losses of the vapor-phase samples. In the present study, we performed the bioassay and chemical characterization of the vapor-phase mutagens using the exhaust from a medium heavy-duty diesel truck as a model complex mixture. Compounds present in undiluted diesel exhaust were trapped using a sampling train consisting of a Teflon filter, polyurethane foam (PUF) and XAD-4 (XAD), in series. Compounds in the PUF and XAD samples were extracted by SFE using supercritical carbon dioxide, and fractions were collected based on increasing pressures. For the bioassay, the Salmonella microsuspension assay was used throughout with tester strains TA98 and TAIOO, with and without the addition of metabolic enzymes (± S9). The two most mutagenic fractions from PUF were further fractionated using high performance liquid chromatography (HPLC). Chemical analyses of the HPLC fractions were by gas chromatography with a mass selective detector (GC/MS). Compounds present in these PUF SFE fractions included substituted naphthalenes, biphenyls, fluorenes, dibenzothiophenes and phenanthrenes or anthracenes. Compounds present in the XAD SFE fractions included substituted benzenes and naphthalenes, alkyl naphthalenes, and indenes. The vapor-phase mutagens present in diesel exhaust appeared to be more mutagenic in tester strain TA100 (with S9) compared to TA98, suggesting that the mutagenic compounds are different than those responsible for mutagenic activity in particulate matter where TA98 mutagenicity dominates. Specific mutagenic activities of the total vapor-phase component in TA 100 (with or without S9) were very similar to those of the total particulate component, suggesting that the vapor-phase of diesel exhaust may be an important source of mutagenic compounds in the environment.