Environ. Sci. Technol. 2010, 44, 1677–1682
Fungal PAH-Metabolites Resist Mineralization by Soil Microorganisms S T I N E N . S C H M I D T , †,‡,§ JAN H. CHRISTENSEN,‡ AND A N D E R S R . J O H N S E N * ,† Geological Survey of Denmark and Greenland, Department of Geochemistry, Øster Voldgade 10, DK-1350 Copenhagen K, Denmark, and University of Copenhagen, Faculty of Life Sciences, Department of Basic Sciences and Environment, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
Received November 10, 2009. Revised manuscript received January 14, 2010. Accepted January 20, 2010.
This study investigated the mineralization of water-soluble polycyclic aromatic hydrocarbon (PAH) metabolites produced by the soil fungus Cunninghamella elegans. Eleven soil fungi were screened for their ability to metabolize 14Cphenanthrene, 14C-fluoranthene, and 14C-pyrene into watersoluble compounds. Eight fungi produced water-soluble metabolites from all or some of the PAHs. The composition of the water-soluble PAH-metabolites from the most effective solubilizer C. elegans was analyzed by an ultraperformance liquid chromatograph interfaced to a quadrupole time-of-flight mass spectrometer. Thirty-eight metabolites were detected. All of 34 identified metabolites were sulfate-conjugated. The mineralization of 14C-metabolites, produced by C. elegans, was compared to mineralization of the parent 14C-PAHs in soil slurries. It was hypothesized that the increased bioavailability and metabolic activation of the metabolites would increase mineralization in soil slurries compared to mineralization of the parent PAHs. Unexpectedly, the mineralization of the 14Cmetabolites was in all cases extremely slow compared to the mineralization of the parent 14C-PAHs. Slow 14C-metabolite mineralization was not caused by metabolite toxicity, neither was cometabolic mineralization of 14C-metabolites stimulated by the presence of active PAH-degraders. High water solubility, low lipophilicity, and extremely slow mineralization of the metabolites indicate a potential problem of leaching of fungal PAHmetabolites to the groundwater.
Introduction Polycyclic aromatic hydrocarbons (PAHs) are formed during gasification and incomplete combustion of biomass and fossil fuels. They are consequently ubiquitous in the soil environment, where they accumulate in the topsoil of industrialized or urban areas (1). The microbial degradation of PAHs in soil has been extensively reviewed (e.g., 2-4). Soil bacteria may use PAHs with up to four rings as sole source of carbon and energy, * Corresponding author e-mail:
[email protected]. † Geological Survey of Denmark and Greenland. ‡ University of Copenhagen. § Present address: Dept. of Environmental Chemistry and Microbiology, National Environmental Research Institute (NERI), Aarhus University, P.O. Box 358, DK-4000 Roskilde, Denmark. 10.1021/es903415t
2010 American Chemical Society
Published on Web 02/05/2010
resulting in the complete mineralization to carbon dioxide and water. Such bacterial PAH degradation is limited by the strong sorption of PAHs, resulting in low pore-water concentration and hence low bioavailability (4). Many soil fungi can also degrade PAHs, though rarely all the way to carbon dioxide. Fungal PAH degradation proceeds either by extracellular processes mediated by lignin-degradation enzymes, or by intracellular detoxification processes. The present study focuses on the latter. During PAH-detoxification, nonligninolytic fungi produce oxidized metabolites, e.g., hydroxy-, dihydroxy-, dihydrodiol-, and quinone-derivatives (phase one), that may subsequently be conjugated with methyl-, sulfate-, xylose-, glucose-, or glucuronic acid groups (phase two) (5-8), which increases the solubility and facilitates excretion. A number of screening studies have previously demonstrated that the ability to oxidize PAHs is very common among nonligninolytic environmental isolates (9-12). For instance, 36 out of 72 strains isolated from PAH-contaminated soils oxidized pyrene (10). Previous studies of fungal PAH-metabolites have focused on the total amount of organic-solvent-extractable metabolites (e.g., 7, 8, 13, 14) that are mainly phase-one metabolites. Here, we have instead focused on the truly water-soluble phase-two metabolites that are more likely to be mobile in soil pore-water. We screened the ability of common, nonligninolytic soil fungi to bring 14C-phenanthrene, 14C- fluoranthene, and 14C-pyrene into aqueous solution. The most efficient fungus was selected for further investigation. Watersoluble PAH-metabolites produced by this fungus were tentatively identified by ultraperformance liquid chromatography interfaced to a quadrupole time-of-flight mass spectrometer (UPLC-qTOF MS). It has been hypothesized that many bacteria involved in PAH degradation in soil live on eukaryotic PAH-metabolites rather than on parent PAHs due to the increased bioavailability of the metabolites (4). In the present study, we investigated if this hypothesis is true by comparing the mineralization of water-soluble, fungal, phase-two metabolites and their parent PAHs in soil slurries. The central hypothesis was that fungi may solubilize PAHs by detoxification, and those fungal PAH-metabolites are mineralized much faster than parent PAHs in soil, due to metabolic activation, increased aqueous concentration, and hence greatly increased bioavailability of the PAH-metabolites.
Materials and Methods Chemicals and Media. Phenanthrene (>98% purity) was purchased from Merck-Schuchardt, fluoranthene (99% purity) and pyrene (98% purity) were purchased from Aldrich, 9-[14C]-phenanthrene (>97% purity), 3-[14C]-fluoranthene (>95% purity), and 4,5,9,10-[14C]-pyrene (>99% purity) were from Sigma-Aldrich, and 2,2,4,4,6,8,8-heptamethylnonane (HMN, 98% purity) was from Aldrich. Difco Malt Extract Broth (per liter: malt extract, 6.0 g; maltose, 1.8 g; dextrose, 6.0 g; yeast extract 1.2 g, pH 4.7 ( 0.2) and Bushnell-Haas minimal medium (BH-medium, per liter: magnesium sulfate, 0.2 g; calcium chloride, 0.02 g; monopotassium dihydrogen phosphate, 1.0 g; diammonium hydrogen phosphate, 1.0 g; potassium nitrate, 1.0 g; ferric chloride, 0.05 g, pH 6.5) were purchased from Becton, Dickinson and Company. All media were autoclaved at 121 °C for 30 min. Scintillation cocktail Hisafe 3 was purchased from Perkin-Elmer. Cultivation of Fungi. The 11 fungi were previously isolated from agricultural soils (15-17). Unpublished strains from VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Screening of Eleven Soil Fungi for Their Ability to Solubilize PAHsa % in solution fungus abiotic control Acremonium sp. Gr161 (16) Alternaria sp. Gr174 (16) Fusarium culmorum Gr59 Phoma eupyrena CBS 118522 (17) Trichoderma hamatum Gr56 Cunninghamella elegans JS/2 (15) Mortierella sp. CBS 118520 (17) Penicillium janczewski Gr150 Cladosporium herbarum Gr51 Cladosporium cladosporioides Gr2 Cladosporium cladosporioides Gr128 a
14
C-Phe
8.6 8.8 19.5 41.6 7.5 12.8 94.8 8.0 22.2 17.3 6.5 7.7
14
C-Fla
14
2.2 4.1 6.0 6.5 1.0 3.3 79.2 5.4 7.6 2.1 1.9 1.9
C-Pyr
3.2 6.8 7.6 14.2 1.1 54.4 6.7 6.1 2.7 1.7 2.4
Phe: phenanthrene; Fla: fluoranthene; Pyr: pyrene.
these studies were identified by Deutsche Sammlung von Mikroorganismen and Zellkulturen (Braunschweig, Germany). The fungi were stored at -80 °C as mycelia-containing agar plugs in 20% glycerol. The fungi were grown by placing agar plugs on malt extract agar plates, which were incubated for 4-16 days in the dark at 25 °C. Six agar plugs (0.4 × 0.4 cm) from each plate were transferred to 100 mL of liquid malt extract medium in sterile 250-mL conical flasks. The flasks were incubated in the dark at 20 °C and 150 rev min-1 on an orbital shaker for 2-4 days until round pellets of fungal mycelia were formed. Screening of Soil Fungi for Production of Water-Soluble PAH Metabolites. Eleven soil fungi (Table 1) were screened for their ability to solubilize phenanthrene, fluoranthene, and pyrene in BH medium. Stock solutions (100 µL) of 14Cphenanthrene, 14C-pyrene, or 14C-fluoranthene in acetone (1 mg mL-1) were added to sterile 100-mL red cap bottles (Scot Duran), and the acetone was evaporated. BH medium (10 mL) was added to give a final PAH concentrations of 10 mg L-1 corresponding to 2000 dpm mL-1. The bottles were closed with Teflon-lined screw-caps, and then sonicated for five min. Round pellets of fungal mycelia (ø ) 8-12 mm, one or two pellets per 10 mL of medium) were washed 3 times in BH medium to remove the malt extract medium. The pellets were then transferred to the red cap bottles, and incubated in the dark at 20 °C and 130 rev min-1 on an orbital shaker. Abiotic controls without fungal mycelium were also prepared. Subsamples of approximately 2 mL were collected at days 4, 7, and 11. The samples were centrifuged in glass test tubes at 1200g for 10 min to remove PAH crystals and fungal mycelia. The supernatants were collected and weighed before transfer to scintillation vials. Scintillation cocktail (10 mL) was added, and the concentration of solubilized PAH was quantified by liquid scintillation counting, followed by calculation of the percentage of solubilized PAH. Characterization of PAH-Metabolites Produced by C. elegans. Metabolites from the most efficient solubilizer, C. elegans, were tentatively identified by UPLC-qTOF MS with negative electrospray ionization. The procedure for production of metabolites was as described above for 14C-labeled PAHs, except that duplicate treatments were incubated in 250-mL red cap bottles containing 35 mL of BH medium and 10 mg L-1 of nonradioactive phenanthrene, fluoranthene, or pyrene. An additional treatment containing a mixture of the three PAHs (PAH-mix) was prepared with a concentration of each compound of 10 mg L-1 leading to a total concentration of 30 mg L-1 PAH. Parallel experiments were carried out with 14 C-labeled PAHs for the quantification of total solubilized PAHs. Abiotic controls without fungal mycelium, and biotic controls incubated with C. elegans in the absence of PAHs 1678
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were also prepared. Subsamples of 3 mL were collected at days 1, 3, 7, and 11. PAH crystals and fungal mycelium were removed by centrifugation as described above. Approximately 2 mL of supernatant was collected and transferred to 8-mL amber vials. Methanol (500 µL) was added, and the samples were stored at -18 °C. The unlabeled PAH-metabolites were analyzed by an UPLC-qTOF MS (Waters). A BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm, Waters) was used. The binary solvent system consisted of water/acetonitrile 95:5 (v/v) with 0.1% formic acid (mobile phase A), and water/acetonitrile 5:95 (v/v) with 0.1% formic acid (mobile phase B). Gradient elution was done by a linear gradient from 99 to 88% of mobile phase A for 7 min, a linear gradient to 65% in 28 min, and decreased to 20% in 1 min (held for 3 min). The column was re-equilibrated for 5 min (99% mobile phase A) after a linear gradient from 20% A to 99% A. A flow rate of 0.325 mL min-1 was used. The metabolites were ionized by negative electrospray ionization (ESI-) with source temperature 120 °C, desolvation temperature 400 °C, capillary voltage 1.00 kV, and cone voltages of 20 and 120 V. The UPLC-MS chromatograms of sample extracts were compared to chromatograms from abiotic and biotic controls. Chromatograms from abiotic controls were used to identify possible photo-oxidation products or other impurities. Chromatograms from biotic controls were used to identify non-PAH exudates from C. elegans that might interfere with the identification of PAH-metabolites. A positive control for the detection of the phase-one compound 1-hydroxy pyrene was included. Molecular ions were determined in chromatograms obtained with a cone voltage of 20 V while insource fragment-ions were determined in chromatograms obtained with a cone voltage of 120 V. The metabolites were tentatively identified by their masses, isotope distributions, and mass spectral fragmentation patterns. Mineralization of 14C-PAHs and 14C-Metabolites in Soil Slurries. We tested the ability of indigenous soil microorganisms to mineralize parent PAHs and aqueous PAHmetabolites. Mineralization of the compounds was investigated in a natural soil collected in a forest at a densely populated rural site north of Copenhagen (Sjælsø, Denmark), and in a roadside soil collected at a heavily trafficked crossroad in central Copenhagen. The soils (0-10 cm) were homogenized and sieved twice through a 2-mm mesh. Contents of the 16 U.S. Environmental Protection Agency priority PAHs (Σ16PAHs) were determined by GC-MS as described by Boll et al. (18). 14 C-labeled metabolites from 14C-phenanthrene, 14Cfluoranthene, 14C-pyrene, and 14C-PAH-mix were produced by C. elegans, as described in the preceding section, with an adjustment to 3000 dpm mL-1 of the 14C-PAHs and 7 days incubation. The amounts of solubilized PAHs were determined by liquid scintillation counting. Unlabeled metabolites from the PAH-mix were also produced following the same procedure. BH medium, incubated with C. elegans in the absence of PAHs, was prepared to generate BH medium containing fungal exudates. This medium was used in the mineralization studies of 14C-PAHs to allow a direct comparison to the mineralization studies with 14C-metabolites produced by C. elegans. The following four types of mineralization experiments were prepared in triplicate in 100-mL sterile red cap bottles: 14 C-Metabolites. Ten mL of 14C-metabolite solution and 150 µL of HMN were added to the red cap bottles. 14 C-PAHs. Ten mL of BH medium, incubated with C. elegans in the absence of PAHs, was added to red cap bottles. Acetone was evaporated from aliquots of the four 14C-PAH stock solutions, and the 14C-PAHs were then redissolved in HMN. Amounts of 14C-PAH HMN-solutions, equivalent to the concentration of the corresponding 14C-metabolites, were
added to the red cap bottles. Additional HMN was added to give a total of 150 µL. 14 C-Metabolites and Unlabeled PAHs. Ten mL of 14Cmetabolite solution, produced from the 14C-PAH-mix, was added to red cap bottles. Unlabeled PAH-mix in HMNsolution, equivalent to the concentration of the 14Cmetabolites, was added to the red cap bottles. Additional HMN was added to give a total of 150 µL. 14 C-PAHs and Unlabeled Metabolites. Ten mL of unlabeled metabolite solution, produced from the PAH-mix, was added to red cap bottles. 14C-PAH-mix in HMN, equivalent to the concentration of the unlabeled metabolites, was added to the red cap bottles. Additional HMN was added to give a total of 150 µL. The flasks were placed on an orbital shaker (80 rev min-1) in the dark at 15 °C to allow the PAHs and metabolites to phase-partition between the aqueous and the HMN phases. After 20 h, soil (1.0 g) was added followed by incubation in the dark at 15 °C and 80 rev min-1 on an orbital shaker. 14CO2 was captured in glass test tubes containing 2 mL of NaOH (1 M) placed inside the bottles. The NaOH solution was changed regularly during the 96-day incubation period. The amount of 14CO2 in the NaOH was quantified by liquid scintillation counting as described above. Phase Distribution of PAH-Metabolites. We estimated the distribution ratio between HMN and BH-medium for metabolites produced by C. elegans. The 14C-metabolites were produced from the 14C-PAH-mix as described above. BH medium with 14C-metabolites was sterilized by adding NaN3 (100 µg mL-1, final concentration) to avoid microbial growth. Two mL of BH-medium with metabolites and 2 mL of HMN were added to a 10-mL glass test tube in duplicate. The glass tubes were closed with Teflon-lined screw-caps, and vortexed. The glass tubes were then placed horizontally on an orbital shaker, and incubated in the dark at 15 °C and 80 rev min-1 for 72 h. Phase separation was allowed for 2 h before sampling 1 mL of the HMN. The remaining HMN was removed, and 1 mL of BH medium was sampled. The amounts of metabolites in the two phases were quantified by liquid scintillation counting.
Results and Discussion Solubilization of PAHs. Eleven soil fungi (Table 1) were screened for their ability to bring phenanthrene, fluoranthene, and pyrene into solution. These PAHs were selected as model substrates because they are found in high concentrations in soil polluted by pyrogenic PAHs (1). The fungi were scored positive when the aqueous concentration of radiolabel was at least 50% higher than that of the abiotic controls, indicating metabolization of the PAHs. Alternaria sp. Gr174, Fusarium culmorum Gr59, Penicillium janczewskii Gr150, and Cunninghamella elegans JS/2 solubilized all three PAHs. Cladosporium herbarum Gr51 only solubilized phenanthrene, while Acremonium sp. Gr161 and Mortierella sp. CBS 118520 solubilized only fluoranthene and pyrene. Trichoderma hamatum Gr56 solubilized phenanthrene and fluoranthene. Phoma eupyrena CBS118522, Cladosporium cladosporioides Gr2 and Gr128 did not solubilize any of the three PAHs. Most of the eight fungi showed continuous increases in solubilized phenanthrene, fluoranthene, and pyrene during the 11-day period (data not shown). However, for C. elegans the amounts of PAHs in solution did not increase after day 7. Scintillation counting of solubilized metabolites was not supported by UPLC-MS analyses, so there is a theoretical risk that small amounts of parent PAHs may have been brought into solution by complexation to soluble polymers or surface active compounds excreted by the fungi.
The abiotic controls showed virtually constant aqueous concentrations during the 11-day period (data not shown). At day 7, the solubilized 14C-PAHs in the abiotic controls corresponded to 0.83 mg L-1 of phenanthrene, 0.22 mg L-1 of fluoranthene, and 0.32 mg L-1 of pyrene (Table 1). For pyrene, this is above the aqueous solubility, probably due to minor radio-chemical impurity for instance by photooxidation of a small fraction of the 14C-pyrene. To our knowledge, this is the first screening of soil fungi for the metabolic ability to bring PAHs into aqueous solution. Several researchers have carried out somewhat comparable screenings (9-11), but the PAH metabolites were in these studies extracted with the organic solvent ethyl acetate. Considerably different results are obtained when analyzing the aqueous phase because the relatively hydrophobic phaseone metabolites sorp to the hyphae in the aqueous environment, and because the hydrophilic phase-two metabolites have low solubility in ethyl acetate. Identification of PAH-Metabolites. C. elegans was by far the most efficient solubilizer and was therefore chosen as model-organism. A total of 38 water-soluble metabolites were found, 23 from phenanthrene, 11 from fluoranthene, and 4 from pyrene (Figure 1, Table 2). No new metabolites appeared after day 7, which was also the day when the 14C-metabolites reached the maximum aqueous concentrations (phenanthrene 80%, fluoranthene 74%, pyrene 68%, PAH-mix 68%). Parent PAHs cannot be detected by the applied electrospray ionization method, so traces of parent PAHs may be present in the metabolite solution. All of 34 tentatively identified metabolites were hydroxylated and conjugated with sulfate (OSO3-). The presence of sulfate in the metabolite structures was confirmed by characteristic losses of 80 Da (SO3-) in the mass spectral fragmentation patterns (Table 2, fragment I). This is in line with a previous study of C. elegans where sulfateconjugated phenanthrene-metabolites were reported (19). Sulfate-conjugated PAH-metabolites have also been reported for Aspergillus terreus, Penicillium glabrum, Aspergillus niger, and Syncephalastrum racemosum (7, 8, 19, 20). The metabolites were mono-, di-, or trihydroxylated (Table 2). The number of hydroxy-groups in the metabolite structure was identified by the m/z of the molecular ion (with the sulfate-conjugate) and the m/z of mass spectral fragment ions. Characteristic losses of 18 Da (H2O) in the mass spectral fragmentation patterns showed that ten metabolites were dihydrodiols (Table 2, fragment II). Due to small differences in polarity, metabolite isomers were expected to have only slightly different retention times. Hydroxy phenanthrene sulfates (m/z 289), however, eluted in two different parts of the chromatogram (Figure 1), suggesting that some hydroxy phenanthrene sulfates may have had additional, nonidentified groups. Except for the positive controls with 1-hydroxypyrene, we did not detect any oxidized, nonconjugated metabolites, presumably due to sorption of these relatively nonpolar compounds to the fungal mycelia. The phase-one metabolites hydroxy-, dihydroxy-, and trans-dihydrodiol phenanthrenes (13, 19, 21, 22), hydroxy- and trans-dihydrodiol fluoranthenes (23, 24), and hydroxy-, dihydroxy-, and quinone pyrenes (8) have been reported for C. elegans in studies where organic solvent extraction was used. C. elegans has also been reported to produce glucosideconjugated phase-two metabolites from phenanthrene (19, 20, 25), fluoranthene (23, 24), and pyrene (6), but none of these were seen in our study. Phase Distribution. It is difficult to determine the bioavailability of pure PAHs in shaken aqueous systems because the bioavailability is influenced by factors such as the shape and size of the PAH crystals and sorption to VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Extracted ion chromatograms of PAH metabolites produced by C. elegans. The metabolites are labeled with the m/z of the molecular ion.
TABLE 2. Tentatively Identified PAH-Metabolites Produced by C. elegans m/za parent PAH
phenanthrene
fluoranthene
pyrene a
Mass/charge.
b
PAH-metabolite
MH
fragment I
phenanthrene sulfate hydroxy phenanthrene sulfate dihydroxy phenanthrene sulfate dihydrodiol phenanthrene sulfate hydroxy dihydrodiol phenanthrene sulfate unidentified phenanthrene-metabolite A unidentified phenanthrene-metabolite B fluoranthene sulfate hydroxy fluoranthene sulfate dihydroxy fluoranthene sulfate dihydrodiol fluoranthene sulfate pyrene sulfate hydroxy pyrene sulfate unidentified pyrene-metabolite
273 289 305 307 323 291 532 297 313 329 331 297 313 353
193 209 225 227 243
9
217 233 249 251 217 233
fragment II
209 225
233
isomersb 2 8 3 4 3 2 1 2 4 2 3 1 2 1
Number of structural isomers.
surfaces. By adding the PAHs dissolved in an organic phase in the mineralization experiments, the distribution ratio between the aqueous medium and the organic phase could be used as a proxi of the bioavailability, this is because the aqueous equilibrium concentrations are determined by the distribution ratios. Phase distribution ratios are often reported for octanol/ water systems (log[Kow]), but octanol would be a bad choice in our mineralization experiments as it can be degraded by many soil microorganisms. Instead we used heptamethylnonane (HMN) which is considered biologically inert due to the heavy branching of the molecule. The distribution ratio between HMN and BH medium, KHMN/BH medium, was 0.013 for the 14C-metabolite mix in both duplicates. Thus, the average initial concentration of 14Cmetabolites in the aqueous phase in the mineralization experiment was 77 times higher than the concentration in the HMN-phase. 1680
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Since the metabolites and PAHs were added in equal amounts in the mineralization experiment discussed below, and both in the presence of 150 µL of HMN, the initial, relative differences in bioavailability could be estimated from the change in distribution ratio. Log(Kow) for the parent PAHs ranges from 4.5 to 4.8 (26, 27). Assuming that Kow can be used as a rough estimate of the KHMN/BH medium, the fungus decreased the distribution ratio of the PAHs, and consequently increased the bioavailability, by 6 orders of magnitude due to oxidation and subsequent sulfate-conjugation. We therefore expected the metabolites to be mineralized much faster than the parent PAHs by soil microorganisms. Mineralization of 14C-PAHs and 14C-Metabolites in Soil Slurries. Mineralization of the compounds was investigated in two soils; one containing background levels of PAHs (Σ16PAHs ) 0.53 ( 0.20 mg kg-1) and the other affected by PAHs from a heavily trafficked road (Σ16PAHs ) 1.23 ( 0.29 mg kg-1). The mineralizations in soil slurries are presented
FIGURE 2. Mineralization of 14C-PAHs and 14C-metabolites in soil slurries (n ) 3). Phe: phenanthrene; Fla: fluoranthene; Pyr: pyrene: PAH mix: mixture of phenanthrene, fluoranthene, and pyrene. as the cumulative percent of 14CO2 produced from 14C-PAHs and 14C-metabolites during a 96-day period (Figure 2). The parent PAHs were mineralized fast and reproducibly by microorganisms in both the natural soil and the roadside soil, and both as pure compounds and as a mixture. HMN was added in the mineralization experiment as a means of providing standardized conditions under which the bioavailability and mineralization of both PAHs and metabolites could be directly compared. The increased bioavailability of the metabolites, however, did not result in the expected increase in mineralization compared to the parent PAHs. On the contrary, the mineralization was in all cases extremely slow, and only 6-8% of phenanthrenemetabolites, and 2-3% of metabolites from pyrene, fluoranthene, and the PAH-mix was mineralized during the 96 days (Figure 2).
Metabolite toxicity was not the reason for the slow mineralization since mineralization of 14C-PAHs was the same with and without unlabeled metabolites (Figure 3A). We increased the potential for cometabolic mineralization of the metabolites by adding parent PAHs. Metabolite mineralization was, however, the same with and without unlabeled parent PAHs, demonstrating that metabolite mineralization could not be cometabolically stimulated (Figure 3B). The results are in line with a previous study (28) where the phase-one, phenanthrene metabolites 9-hydroxy phenanthrene and 9,10-phenanthraquinone were not mineralized to any detectable extent in garden soil slurries and activated sludge. The anthracene metabolites 9-anthrone and 9,10anthraquinone, on the other hand, were easily mineralized. The high water solubility, low lipophilicity, and extremely slow mineralization of PAH-conjugates indicate that the VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Effects of metabolites on the mineralization of 14 C-PAH (A), and effects of PAH on the mineralization of 14 C-metabolites (B) in roadside soil slurries (n ) 3). conjugates may leach from PAH-contaminated soil to groundwater aquifers. Whether this is a problem will depend on the toxicity and carcinogenicity of the conjugates. Our results also indicate that the often suggested use of fungi for bioremediation of PAH polluted soil may have negative side effects.
Acknowledgments This work was funded by the Villum Kann Rasmussen Foundation via the Center for Environmental and Agricultural Microbiology (A.R.J. and S.N.S.), by the COWI Foundation (J.H.C.), and by the Lundbeck Foundation (J.H.C.).
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