Environmental and Molecular Mutagenesis. DOI 10.1002/em 406 Lemieux et al. MATERIALS AND METHODS Chemicals All chemicals used for soil extraction and fractionation were analytical grade and obtained from EMD Chemicals (Gibbstown, NJ) unless other- wise speciﬁed. Methyl methanesulfonate, 2-nitroﬂuorene, and 2-aminoan- thracene were obtained from Moltox (Boone, NC). The methodology employed to quantify PAHs in soil extracts was validated using a stand- ard reference material (SRM 2260, National Institute of Standards and Technology, Gaithersburg, MD). Soil, Bioslurry Treatment, and Sampling A 150-kg sample of PAH-contaminated soil was obtained from a for- mer gasworks site at Husarviken in Stockholm, Sweden [Lundstedt et al., 2003]. The gasworks factory on the site was in use from 1893 to 1972. At the time of collection, the site was composed of sandy soil, ash, and demolition debris, all of which is heterogeneously contaminated with coal tar, heavy metals, and cyanide. The earlier work by Lundstedt et al. provides a thorough description of the bioslurry treatment [Lundstedt et al., 2003]. Brieﬂy, soil from the Husarviken gasworks site was sieved, mixed with water, and the result- ing slurry treated in a bioreactor containing a microbial culture designed for the degradation of PAHs in soil (Deutsche Montan Technologie, Essen, Germany), nutrients (Bio-D; Medina Agriculture Products, Hondo, TX), and oxygen (6 mg/l), and was continuously mixed for 29 days. The pH was adjusted to neutral with concentrated sulfuric acid and thus var- ied from 6.0 to 8.5 throughout the duration of the treatment. One liter aliquots were collected at the beginning of treatment and after 3, 7, 24, and 29 days of treatment. The samples were stored for several weeks at 220 8C until analysis. Sample Pretreatment, Extraction, and Fractionation The pretreatment, extraction, and fractionation of the bioslurry sam- ples was carried out as described in Lundstedt et al [2003]. Brieﬂy, pres- surized liquid extraction was used to extract organic compounds from dried samples of remediated soil obtained on days 0, 3, 7, 24, and 29 of the bioremediation treatment. The extracts were applied to 15-mm (inner diameter) open silica gel columns (10% w/w deactivated with water) that were sequentially eluted with 5 ml hexane, 15 ml hexane:dichloro- methane (3:1 v/v), and 30 ml dichloromethane. These fractions were reconstituted in 500 ll DMSO (analytical grade, Sigma Aldrich Canada, Oakville, ON, Canada) and stored at 48C until mutagenicity testing with the Salmonella reverse mutation assay. The ﬁrst fraction contains ali- phatic hydrocarbons. The second fraction includes nonpolar neutral com- pounds such as PAHs, alkyl-PAHs, and S- and O-heterocyclics. The third fraction contains semipolar aromatics including N-heterocyclics and oxy-PAHs. Extensive validation of the extraction and fractionation pro- cedures has been carried out [Lundstedt et al., 2000, 2003, 2006], and analyses of certiﬁed reference material showed that recoveries of all PAHs tested except phenanthrene and anthracene were greater than 85%. Acceptable reproducibility has been demonstrated for several PAHs, alkyl-PAHs, oxy-PAHs, and N- and S- heterocyclics (relative standard deviation < 13%) using a standard reference material and a contami- nated gasworks soil. Salmonella Reverse Mutation Assay The Salmonella reverse mutation assay was used to assess the muta- genic activity of the same samples examined in the aforementioned Lundstedt et al. [2003] study. Mutagenic activity of the nonpolar neutral and semipolar aromatic fractions of each bioslurry sample (i.e., day 0, 3, 7, 24, and 29) was evaluated using the standard plate incorporation ver- sion of the Salmonella reverse mutation assay [Mortelmans and Zeiger, 2000]. The assay was performed according to Mortelmans and Zeiger [2000] with one exception; 0.2 mM histidine and 0.4 mM biotin were added directly to the minimal agar plates rather than the top agar. The ﬁrst fraction (i.e., aliphatics) was not evaluated for mutagenicity. Earlier analyses on a limited number of soil extracts failed to detect mutagenic activity in this fraction (data not shown). Fractions 2 and 3 (i.e., nonpo- lar neutral and semipolar aromatic) were tested with and without the addition of a post-mitochondrial supernatant (S9) fraction from Aroclor 1254 induced rat liver (male Sprague-Dawley rats, protein content between 35.7 and 43.5 mg/ml; Moltox, Boone, NC) on Salmonella strains TA98, TA100, YG1041, and YG1042. Strains TA98 and YG1041 detect frameshift mutations, whereas strains TA100 and YG1042 detect base pair substitution mutations. YG1041 and YG1042 contain the multi- copy pYG233 plasmid, which contains genes encoding O-acetyltransfer- ase (OAT) and the Salmonella classic nitroreductase (Cnr) [Hagiwara et al., 1993]. As such, they are highly sensitive to nitroarenes and aro- matic amines. Each fraction of each bioslurry sample was tested at ﬁve concentra- tions (1, 3, 9, 27, and 50 mg equivalent dry soil/plate for the nonpolar neutral fractions and 1, 3, 9, 27, and 81 mg equivalent dry soil/plate for the semipolar aromatic fractions). Each concentration was tested in tripli- cate, and revertant colonies on each plate were counted using an auto- mated colony counter (Protocol RGB, Model # 9000, Synoptics Ltd, UK). Positive and negative controls were included in each experiment. When metabolic activation was not used, 2-nitroﬂuorene was used as a positive control for TA98 (2.5 lg per plate), YG1041 (0.5 lg per plate), and YG1042 (0.5 lg per plate). Methylmethane sulfonate (0.4 lg per plate) was used as a positive control for TA100 without metabolic acti- vation. 2-Aminoanthracene (1 lg per plate) was used as a positive con- trol for all strains with metabolic activation. Negative control samples contained only top agar and bacteria (and S9 where necessary), and did not include DMSO or sample. Previous work in our laboratory has noted identical responses for negative controls that contain DMSO and those that do not (data not shown). All positive and negative controls were tested in triplicate. Data Analysis All statistical analyses were carried out using SAS v. 8.02 for Win- dowsTM (Statistical Analysis Institute, Cary, NC). A sample was classi- ﬁed as mutagenic if it induced a twofold increase in the number of revertants compared to the negative control, and a reproducible concen- tration-response function was generated in one or more strains. For each mutagenic soil sample, a mutagenic potency (revertants per mg equiva- lent of dry soil) was calculated using ordinary least squares linear regres- sion on the linear portion of the concentration-response function. All raw data (e.g., plate counts) are readily available from the corresponding author. RESULTS AND DISCUSSION Transformation of PAHs During Bioremediation This study tracked changes in the mutagenicity of a PAH-contaminated gasworks soil throughout the course of a pilot-scale bioremediation. Nevertheless, a brief discus- sion of the changes in the chemical composition of the soil that occurred during the bioremediation is essential for interpretation of the bioassay results. The results pre- sented below constitute an abbreviated summary of the chemical changes noted in the aforementioned Lundstedt et al. [2003] study. For a more thorough description and