Journal of Environmental Quality 31:150-154 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
TECHNICAL REPORT
Ecological Risk Assessment
Genotoxicity is Unrelated to Total Concentration of Priority Carcinogenic Polycyclic Aromatic Hydrocarbons in Soils Undergoing Biological Treatment
Renee R. Alexander,
Jixin Tang and
Martin Alexander*
Department of Crop and Soil Sciences and Institute for Comparative and Environmental Toxicology, Cornell Univ., Ithaca, NY 14853
* Corresponding author (ma59{at}cornell.edu)
Received for publication May 21, 2001.
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ABSTRACT
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A solid-phase microbiological assay was used to determine the changes in genotoxicity associated with sequestration or biodegradation of carcinogenic compounds in contaminated soils. The concentration of six carcinogenic polycyclic aromatic hydrocarbons (PAHs) did not change in 59 d in sterile soil, but the genotoxicity declined markedly. In a soil undergoing bioremediation in the field for 147 d or biodegradation in the laboratory for 180 d, the concentrations either changed little or declined at different rates, but the genotoxicity increased followed by a decline. The genotoxicity of a second soil declined as a result of biological treatment. The data show that genotoxicity of contaminated soils may be unrelated to the concentration of carcinogenic PAHs because of aging or new mutagens formed during biological treatment.
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INTRODUCTION
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MANY STUDIES involving chemical analysis have shown that a variety of compounds are destroyed during bioremediation of soils. Fewer investigations have demonstrated that the toxicity of polluted soils diminishes as a result of bioremediation, and those studies commonly involve only the assay of acute toxicants. For example, bioremediation of oil-contaminated soils resulted in a progressive diminution and ultimate disappearance of acute toxicity to earthworms (Salanitro et al., 1997), and biological treatment had a similar beneficial effect on earthworms in a soil contaminated with polycyclic aromatic hydrocarbons (PAHs) (Hund and Traunspurger, 1994). Bioremediation also reduces or eliminates acute toxicity to growing plants or seed germination (Hund and Traunspurger, 1994; Salanitro et al., 1997; Wang and Bartha, 1990).
However, a major purpose of remediation of contaminated soils is to destroy chronic toxicants, particularly carcinogens. Prominent among the carcinogenic compounds that contaminate soils are some PAHs, and their biological activity can be measured by use of a test for genotoxicity (Alexander and Alexander, 1999; Alexander et al., 1999). The genotoxicity assays that have been conducted sometimes are performed with aqueous extracts or aqueous suspensions of soils (Kwan, 1995; Kwan and Dutka, 1995; Marwood et al., 1998; Randerath et al., 1999), but the carcinogenic PAHs have very low solubility in water (Mackay and Shiu, 1977) and also are extensively sorbed to particulate matter in natural environments (Karickhoff et al., 1979). In some cases, organic solvents are used to extract contaminated soils (Malachova, 1999), but organic solvents may extract the nonbioavailable fraction of PAHs in soil as well as the fraction that is available for genotoxicity (Alexander and Alexander, 1999, 2000).
We have previously described a solid-phase bioassay of genotoxicity of soils (Alexander et al., 1999). Differing from tests with the aqueous phase, which fail to show the influence of compounds that are sorbed, and also tests with suspensions of soil, which do not give information on duration or frequency of contact of organism with soil particles, the solid-phase assay allows for prolonged contact of the test organism with the contaminated soil containing sorbed toxicants. The purpose of the present study was to use that genotoxicity assay to evaluate changes that occur as a result of the bioremediation in the field of a soil contaminated with waste from a manufactured-gas plant (MGP), the bench-scale biodegradation of a soil naturally contaminated with MGP wastes, and a second soil that was deliberately contaminated. Sites containing PAHs from MGPs are common because of the widespread, early use of coal to manufacture gaseous fuels that were used for heating and lighting (Hayes et al., 1996).
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MATERIALS AND METHODS
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Mutations in Laboratory-Contaminated Soil
Coal tar oil designated 97C0977:CTI Standard Oil from a gas-manufacturing plant was obtained from ThermoRetec Corp. (Ithaca, NY). The coal tar was an oily liquid, and its major PAH constituents were alkylnaphthalenes (36 g kg-1), naphthalene (18 g kg-1), alkylphenanthrenes and alkylanthracenes (17 g kg-1), phenanthrene(12 g kg-1), and acenaphthene (9.2 g kg-1). Hexane was added to a homogenized sample to a concentration of 10% (w/v), and the suspension was mixed gently on a magnetic stirrer for ca. 16 h to permit the PAHs to go into solution. The resulting coal-tar extract was decanted and sterilized by passage through a 0.22-µm sterile nylon membrane filter. Five-gram portions of moist (30% water, w/w) Lima loam (pH 7.2, 7.16% organic C) in 50-mL screw-cap tubes fitted with Teflon liners were sterilized by autoclaving twice for 2-h periods with a 1-d period between the heat treatments, the soils were allowed to dry in air, and 0.5 mL of coal-tar extract was added to each tube. The soil was vigorously shaken for 1 min on a vortex mixer, and the hexane was allowed to evaporate for 72 h. No colonies appeared when portions of the soil were incubated for 72 h on Luria Bertani (LB) agar (Sambrook et al., 1989).
The soil in half the tubes was moistened to 80% of field capacity with sterile M-9 salts solution (Sambrook et al., 1989), and the tubes were tightly capped and stored in the dark at 21 ± 2°C to allow components of the oil to age. The remaining tubes received ca. 0.1 g of unremediated loam from an MGP-contaminated field site to serve as a microbial inoculum, and M-9 salts solution was added to bring the soil to 80% of field capacity. The contents of the tubes were mixed, and the tubes were loosely capped and incubated at 30°C to allow for biodegradation. The soils were mixed with a spatula once or twice per week to increase their aeration, and M-9 salts solution was added as needed to maintain the moisture level.
At each sampling time, the soils in six tubes for tests of aging and six for tests of biodegradation were allowed to dry in air. Three of the dry soil samples from each treatment were sterilized by 
-irradiation (2.5 Mrad) from a 60Co source and, after 3 wk, tested for genotoxicity. The remaining six samples of soil were analyzed to determine the PAH concentrations, three replicates being used for each analysis.
Mutations in Field-Contaminated Soils
Samples of a loam (pH 7.5, 1.45% organic C) from a land-treatment site originally contaminated with residue from a manufactured gas plant were obtained from ThermoRetec Corp. Portions of soil that had been bioremediated in the field for 0, 58, 101, and 147 d were air dried, passed through a 2-mm sieve, and analyzed for their content of PAHs and the capacity to induce mutations. The 0-d sample represents the contaminated soil before the initiation of bioremediation.
For tests of changes associated with biodegradation in the laboratory, samples of untreated loam (0-d sample) were dried and passed through a 2-mm sieve. Portions (20 g dry weight) were placed in screw-cap jars (7-cm diam x 9-cm depth), ca. 0.5 g of the unremediated contaminated loam from the land-treatment site was added as a microbial inoculum, and M-9 salts solution was added to bring the soil to 80% of field capacity. Two jars were prepared for each sampling time, one to provide soil for chemical analysis and one for genotoxicity testing. The soils were thoroughly mixed and incubated at 30°C with caps slightly loosened to permit air circulation. Twice per week, the soils were mixed and M-9 salts solution was added as needed to maintain the moisture level.
At each sampling time, the soil in one jar of the loam treated in the laboratory was allowed to dry for ca. 72 h, and three 5-g portions were transferred to Whatman cellulose thimbles, extracted, and analyzed for PAH content. Three 5-g portions also were transferred to 50-mL screw-cap tubes fitted with Teflon liners, and the soils were sterilized by -irradiation and tested for genotoxicity.
Chemical Analysis
Three 5-g samples of soil were extracted in a Soxhlet apparatus with 100 mL of a mixture of hexanes and acetone (9:1). The extracts were concentrated 10-fold in a Buchi rotary evaporator (Buchler Instruments, Fort Lee, NJ), and the concentrated solutions were analyzed with a gas chromatograph (Model HP5890A; Hewlett-Packard, Avondale, PA) with a flame-ionization detector and fitted with a 30-m silica capillary column (Hewlett-Packard HP-5; diam 0.32 mm; film thickness, 0.25 µm). The column temperature was maintained at 35°C for 5 min, and it was raised at 6°C min-1 to 310°C and then held at 310°C for 20 min. At the time of each analysis, a standard mixture containing 20 µg mL-1 of PAHs was also analyzed. Unless otherwise stated, values represent the means and standard deviations of triplicate samples.
Determination of Genotoxicity
The mutagenicity of the sterilized soils was measured by adding to each tube 50 mg of glucose, sufficient M-9 salts solution to bring the soil to 80% of field capacity, and 0.1 mL of a 0.9% NaCl solution containing ca. 104 cells of a 16- to 18-h culture of Pseudomonas putida A11rUV. Full details of the assay have been published (Alexander et al., 1999). The soils were incubated at 30°C for 48 h, a period that permitted the population to reach 109 to 1010 g-1 of soil and allowed for mutant expression (Alexander et al., 1999). To each tube, 15 mL of 0.9% NaCl solution was added, the tube was vigorously shaken on a Vortex mixer, and 0.1 mL of this suspension was added to tubes containing 2.5 mL of "top agar" (0.6% agar and 0.5% NaCl) held at 45°C (Maron and Ames, 1983). The molten agar was gently mixed, and the contents of the tubes were layered on plates containing LB agar supplemented with 50 µg of rifampicin mL-1. The plates were incubated for 48 h at 30°C, and the number of rifampicin-resistant (Rfpr) colonies was counted. The numbers of spontaneous mutants were determined in the same manner using uncontaminated soil, and those values were subtracted from the total numbers to give the abundance of mutants induced per g of test soil. Four replicate plates were inoculated for each sample tested. The total number of cells in each tube of soil was determined by serial dilutions in 0.9% NaCl solution and plating duplicate samples of the dilutions on LB agar. The plates were incubated for 24 h at 30°C, and the numbers of colonies were counted. The counts were made after the soils were incubated for 48 h, at which time the population size had reached its maximum value. The number of mutants was also counted at 48 h, and therefore the mutation rate was not affected by population size (Alexander et al., 1999).
The statistical significance of differences in numbers of mutants was determined by analysis of variance.
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RESULTS
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Mutations in Sterile Lima Loam with Aged Polycyclic Aromatic Hydrocarbons
Analyses were conducted of the concentrations of benzanthracene (BA), benzo(k)fluoranthene (BkF), benzo(b)fluoranthene (BbF), benzo(a)pyrene (BaP), indeno(1,2,3-c,d) pyrene (IP), and dibenzo(a,h)anthracene (diBA) in moist, sterile Lima loam contaminated with the extract of coal tar. The concentrations of these carcinogenic PAHs detected at 0 d differed markedly, with the level of BaP being more than 20 times higher than that of diBA (Table 1). Determinations made at 14, 29, and 59 d showed no significant decline in the concentrations of each of the test compounds. Thus, none of the compounds was subject to abiotic degradation.
Determinations were also made of the changes in genoxicity of the sterile soil in which the PAHs had aged. The number of induced mutants was 835, 840, 770, and 390 g-1 at 0, 14, 30, and 59 d, respectively. The last value was significantly less (P = 0.05) than the preceding three values. Thus, the decline in genotoxicity is not related to the concentration of the six carcinogens, and the decline in genotoxicity without a change in concentration shows that the compounds become less bioavailable as a result of aging.
Mutations and Chemical Changes During Bioremediation
Bioremediation in the laboratory of the tar-contaminated Lima loam resulted in a decline in the concentration of each of the carcinogenic PAHs (Table 2). More than half of the BA and BkF had disappeared during the 59-d period of treatment, but destruction of the other four compounds was also evident. The apparent increases in BbF after 59 d and IP after 29 d are anomalous. The numbers of induced mutants also were determined during the period of bioremediation. The data show that their abundance did not change during the first 14 d but then declined by about half.
In the field-contaminated loam that was being bioremediated in the field, approximately half of the BA and BaP had disappeared during a 147-d treatment period (Table 3). The percentage of BbF that disappeared in this period was smaller, and little or no loss of the other three compounds was evident. However, the number of induced mutants was significantly greater at 58 d than at 0 d, despite the decline or lack of change in concentration of the test compounds. With longer treatment times, the numbers of mutants fell, and their abundance after 147 d was significantly below the values at 0 d.
Other samples of soil from the field-contaminated loam were brought to the laboratory and there subjected to bioremediation. Because the samples were taken from different sites in the field, where the distribution of contaminants was not uniform, the initial PAH concentrations were different from those given in Table 3. Under laboratory conditions, marked declines in levels of each of the test compounds were apparent (Table 4). Large changes were evident in the concentrations of BA, BkF, BaP, and diBA, the percentage decline after 180 d in each case being greater than 50%. The fall in level was also marked with BbF and IP. The changes in abundance of induced mutants, however, did not parallel the changes in concentrations of these PAHs. For the first 30 d, the numbers did not change significantly, but in contrast with the values from the chemical analyses, the numbers of mutants rose thereafter and were greater at 61 and 122 d than in the first month. Nevertheless, by 180 d, their numbers had again fallen to values much below those at 61 and 122 d. Although the value (340 g-1) at 180 d was not significantly different from the values at 0, 15, and 30 d at P = 0.05, it was significantly different at P = 0.10.
Analyses for 10 other PAHs were also performed to verify that biodegradation was proceeding in the soils. Samples were analyzed at 0, 14, 29, and 59 d for Lima loam, at 0, 58, 101, and 147 d for field-contaminated loam remediated in the field, and at 0, 15, 30, 61, 122, and 180 d for field-contaminated soil incubated in the laboratory. However, only the initial and final values are presented. Nine of the 10 compounds had disappeared to a greater or lesser extent in all four soils, but the benzo(g,h,l)perylene was not evidently degraded in two soils (Table 5). Analyses at the intermediate times showed that most of the acenaphthalene, fluorene, phenanthrene, and pyrene had disappeared from all soils in the final few weeks, that naphthalene disappeared quickly only if the initial concentration was high, and that benzo(g,h,l)perylene was refractory to biodegradation. The disappearance of the lower-molecular-weight PAHs was more extensive than that of the higher-molecular-weight, genotoxic molecules. Thus, ignoring benzo(g,h,l)perylene, only a total of 1.9, 18.8, and 11.1% of these compounds remained in contaminated Lima loam and the laboratory- and field-treated field soil, respectively, after the bioremediation.
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DISCUSSION
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The decline in genotoxicity observed in the three soils with time of treatment is not surprising. Part of the decline is probably a result of the microbial metabolism of the carcinogenic PAHs as well as other genotoxic compounds in the soil. The data indeed show that the concentration of most or all of the six carcinogenic PAHs fell during the course of treatment of soil in the field and in the laboratory. A diminution in acute toxicity has been observed in a number of tests (Hund and Traunspurger, 1994; Salanitro et al., 1997; Wang and Bartha, 1990). Malachova (1999) reported a reduced genotoxicity resulting from biodegradation, but the significance of the data is questionable because of the use of solvent extracts rather than intact soil. Part of the reduced genotoxicity may result from sequestration of the PAHs in soil, at least in soil to which the coal tar was added in the laboratory. We have previously shown that the aging of BaP and 9,10-dimethyl-1,2-dibenzanthracene in soil is associated with a decline in genotoxicity (Alexander and Alexander, 1999). On the other hand, Bordelon et al. (2000) observed no decline in genotoxicity of coal tar in artificially amended soil when measured by analysis of DNA adducts in liver and lung cells of rats fed the contaminated soil. The absence of detectable aging in their study is not surprising because it is likely that appreciable percentages of the genotoxic compounds did not migrate from the coal tar (added at a rate of 5% by weight) into the soil matrix in the 9-month period allowed for aging.
Particularly noteworthy is the statistically significant increase in genotoxicity in the field-contaminated soil during bioremediation in the field as well as biodegradation in the laboratory. These findings suggest that microorganisms are generating new genotoxic substances, either from PAHs or from other constituents of the coal tar. Additional sampling times would have defined more clearly the timing and duration of the increases, but the sampling protocol during the field bioremediation was established by the contractors of the clean-up of the site. Neverthless, the fact that analogous changes occurred during the laboratory biodegradation in the same contaminated soil lends credence to the field data. Unknown soil properties appear to influence the occurrence of the increase in genotoxicity with time because inoculation of Lima loam with a small amount of unremediated field-contaminated soil did not lead to a rise in genotoxicity.
The finding of increases into toxicity are not without precedent. Thus, a rise in genotoxicity has also been reported in solvent extracts of soils amended with diesel fuel and subjected to biodegradation in the laboratory (Randerath et al., 1999). Acutely toxic products have also been reported to be formed during the course of biodegradation (Wang and Bartha, 1994). Microbial activation, in which nontoxic compounds or compounds with little toxicity are converted to potent toxicants, has been reported for many compounds, and some of the products are known carcinogens (Alexander, 1999). With time, however, the genotoxicity in the soils tested here fell to levels appreciably below the initial values.
In evaluating the carcinogenic risks posed by exposure to PAHs, the U.S. Environmental Protection Agency (1993) proposed a scale that measures the potency of a compound relative to the effects attributed to BaP. This model has been used to assess the potential harm resulting from exposure to PAHs at hazardous waste sites (Linz and Nakles, 1997). The following "relative potency estimates" are based on mouse skin carcinogenesis: BaP, 1.0; diBA, 1.11; BbF, 0.167; BA, 0.145; IP, 0.055; BkF, 0.02; and chrysene, 0.0044. The coal-tar-contaminated soils used contained all these PAHs, and the concentrations of six of the seven hydrocarbons were measured at regular intervals. Chrysene was omitted because of its low potency.
The data show that the genotoxicity of the soils is not directly related to the concentration of the six priority carcinogenic PAHs. Neither the increase in genotoxicity nor the diminished bioavailability would have been anticipated by monitoring the changes in the levels of these compounds. Therefore, a more appropriate means than analysis for total concentration of a few genotoxic compounds in contaminated soils needs to be found, a method that takes into account the potential formation of other toxic products and also the bioavailability of the range of compounds with biological activity.
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ACKNOWLEDGMENTS
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This research was supported by Grant ES05950 from the National Institutes of Health with funding provided by the U.S. Environmental Protection Agency and funds provided by the Gas Technology Institute.
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