decreases in toxicological activity. Some studies have also shown that the genotoxicity of PAH-contaminated soils decreases during bioremediation [Aprill et al., 1990; Alexander et al., 2002], and as a result of soil aging [Alexander and Alexander, 1999]. However, others have shown substantial increases in genotoxicity following remediation [Hughes et al., 1998], or initial increases in mutagenicity followed by decreases during extended peri- ods of remediation [Barbee et al., 1992, 1996]. The latter results as well as other published works that examined temporal changes in the mutagenic activity of contami- nated soils undergoing remediation [e.g., Brown et al., 1986; Aprill et al., 1990; Donnelly et al., 1990] suggest that longer periods of remediation may be required to sig- niﬁcantly reduce the mutagenic hazard of a contaminated soil. Thus, it seems clear that no single trend will be ap- plicable to all contaminated sites and all bioremediation methods. Interestingly, for the nonpolar fractions, some strains detected peaks of mutagenic activity at several time- points during the treatment. For example, TA98 detected maximal mutagenic activity without S9 on Day 3, and maximal S9-activated mutagenic activity on Day 24. The somewhat cyclical nature of the mutagenic activity may suggest the formation, and subsequent degradation, of mu- tagenic compounds; however, the identities of these muta- gens are unknown. Semipolar Aromatic Fractions The semipolar aromatic fractions of the bioslurry sam- ples generally exhibited higher mutagenic activity than their corresponding nonpolar neutral fractions, suggesting that the compounds present in these fractions (i.e., oxy- PAHs, nitro-PAHs, aromatic amines) are more abundant or more potent than those in the nonpolar fractions. Moreover, the addition of S9 almost always resulted in an increase in mutagenicity (Fig. 1). This result was not anticipated, since this fraction would be expected to con- tain oxy-PAHs and nitro-PAHs that are mainly direct- acting mutagens that induce frameshift mutations [Choudhury, 1982; Eide et al., 2002] and whose activity is frequently decreased with the addition of S9 [Choud- hury, 1982; Rosenkranz and Mermelstein, 1983]. The elevated level of S9-mediated activity observed in this study was particularly noteworthy for the metabolically enhanced Salmonella strains (i.e., YG1041 and YG1042), and therefore may be due to the presence of oxygenated aromatic amines in the soil, rather than oxy-PAHs or nitroarenes. Aromatic amines require oxidation of the amino group to a hydroxylamine by CYP enzymes, whereas oxy-PAHs and nitroarenes are generally direct- acting mutagens that are active without S9. Aromatic amines, including N-heterocyclic compounds, were detected in the soil by Lundstedt et al. [2003] and some Environmental and Molecular Mutagenesis. DOI 10.1002/em Mutagenicity of Soil During Bioremediation 409 of these compounds are known to be potent mutagens [Tokiwa and Ohnishi, 1986; Aeschbacher and Turesky, 1991]. Alternatively, this increased activity could be due to the presence of oxygenated derivatives of S-heterocy- clic compounds, which would be expected to elute in this fraction, and that are in fact mutagenic. Kumar et al. [2004] demonstrated that the sulfoxide metabolite of benzo[b]naphtha[2,1-d]thiophene is mutagenic in TA100 with and without metabolic activation [Kumar et al., 2004], and a recent study by Swartz et al. demon- strates that some dihydrodiol and sulfone derivatives of benzo[c]phenanthrene, phenanthro[3,4-b]thiophene, and phenanthro[4,3-b]thiophene are mutagenic in TA98, TA100, and YG1041 [Swartz et al., in press]. Like the nonpolar fractions, the semipolar aromatic fractions also exhibited far greater activity in the frame- shift strains (i.e., TA98 and YG1041), as compared to the base-pair substitution strains (i.e., TA100 and YG1042), with no activity observed in TA100 at all. This observa- tion was surprising since strong base-pair activity has pre- viously been observed for PACs, including some oxy- PAHs [Moller et al., 1985] expected to be present in these bioslurry fractions. The mutagenic potencies of the semipolar aromatic fractions also displayed considerable temporal variability, with peaks in activity observed at various points during the treatment. Although the treatment was terminated at 29 days, the results suggest a cyclic pattern of formation and degradation of mutagenic substances similar to that noted for the nonpolar neutral fractions. Moreover, the pattern of responses across strains and S9 conditions sug- gests the formation and degradation of speciﬁc types of substances at different points throughout the treatment. For example, without S9, the activity of the semipolar fraction in YG1042 drops signiﬁcantly between days 24 and 29, while the activity in YG1041 increases. This sug- gests the degradation of some compounds that are frame- shift mutagens, and the formation of other compounds that induce base-pair substitutions. Other signiﬁcant changes include a peak in activity in the absence of meta- bolic activation for YG1042 and TA98 on Day 24; this peak was not apparent with YG1041 (Fig. 1). In contrast, YG1041 direct-acting activity displayed a distinctly cyclic pattern with the maximum value observed at the end of the treatment (i.e., Day 29). The formation of a variety of PAH transformation products via biological processes dur- ing remediation has been documented. For example, two studies have noted the formation of hydroxylated, oxygen- ated, and methoxy derivatives during microbial and fungal degradation of PAHs [Wunder et al., 1997; Andersson et al., 2003]. In some instances the transformation prod- ucts may be more persistent, bioavailable, or mutagenic than the parent compounds. The mutagenicity of the bioslurry fractions cannot be fully explained by the corresponding chemical proﬁles