A comparison of two methods for fractionating complex mixtures in preparation for toxicity analysis

Chemical fractionation is a widely used tool for the chemical and toxicological characterization of complex mixtures. The objective of this research was to compare two frequently employed methods for fractionating a wood preserving waste (WPW) containing polycyclic aromatic hydrocarbons (PAHs) and pentachlorophenol (PCP). The first method involved fractionation of the WPW into acid, base, and neutral fractions using a liquid-liquid acid/base/neutral (A/B/N) technique. The second method utilized alumina column chromatography to produce two fractions, A1 and A2. Gas chromatography and mass spectrometry were used to quantify the chemical components in all fractions. The alumina method recovered 473,338 mg of total PAHs (tPAHs) per kilogram crude, while the A/B/N method yielded only 193,379 mg tPAHs/kg crude. In contrast, the A/B/N method recovered 13.7 mg PCP/kg crude while the alumina method yielded only 0.5 mg PCP/kg crude. Three bioassays were used to determine the toxicity of the crude extract and fractions. The neutral and A1 fractions contained the highest levels of tPAHs and benzo[a]pyrene (BaP) but failed to induce a positive response in the Salmonella/microsome assay with concentrations containing as much as 1800 and 2500 ng BaP/plate, respectively. In the Escherichia coli prophage induction assay, the acid fraction, which contained 472 mg PCP/kg fraction, induced a positive response, as did the base fraction, which did not contain detectable PCP. Significant reduction of gap junctional intercellular communication in hepatic cells occurred with the crude extract and acid, base, and neutral fractions. Overall, the results of these bioassays suggest that PCP genotoxicity was expressed in the acid fraction, whereas the cumulative genotoxicity of genotoxic PAHs appeared to be masked in the isolates from either fractionation method. The optimal fractionation method for a mixture of chlorophenols and PAHs may involve a refined hybrid method.