Desorption and Mineralization Kinetics of Phenanthrene and

Dec 30, 1996 - The relative rates of desorption and mineralization for spiked concentrations of [14C]phenanthrene and [14C]chrysene preloaded on two p...
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Environ. Sci. Technol. 1997, 31, 126-132

Desorption and Mineralization Kinetics of Phenanthrene and Chrysene in Contaminated Soils LISA M. CARMICHAEL,† RUSSELL F. CHRISTMAN, AND FREDERIC K. PFAENDER* Department of Environmental Sciences and Engineering, CB 7400 Rosenau Hall, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7400

The relative rates of desorption and mineralization for spiked concentrations of [14C]phenanthrene and [14C]chrysene preloaded on two previously contaminated soils (foc, 0.029 and 0.0026) were investigated using static, slurry phase microcosms (Vwater/Vsoil ) 10). Desorption rates of [14C]phenanthrene and [14C]chrysene preloaded on the contaminated soils were much faster than observed mineralization rates, whereas the desorption rates of native polynuclear aromatic hydrocarbons (PAH) in the higher organic content contaminated soil were equal to or slower than mineralization rates. This suggests that the desorption of aged PAH may control their degradation and may explain the persistence of PAH even in soils containing a large and active community of PAH-degrading microorganisms. In addition, using 14C-spiked PAH in contaminated soils to measure desorption and biodegradation rates may lead to misleading interpretations of the environmental fate of soilbound polynuclear aromatic hydrocarbons.

Introduction The thermodynamic and kinetic principles governing the distribution of nonionic, hydrophobic organic compounds (HOC) between an aqueous phase and soils or sediments have been the subject of controlled laboratory studies over the last two decades (1-6). The equilibrium distribution of HOCs between aquatic and soil phases is believed to result from a partitioning process controlled by soil organic carbon (SOC) (5, 6), involving aqueous solute dissolution into the hydrophobic soil organic phase, the same as partitioning between two immiscible solvents (7). For polynuclear aromatic hydrocarbons (PAH), sorptive uptake has been correlated with the degree of aromaticity in soil humic materials at normalized SOC levels (8). The facts that nonpolar organics generally do not exhibit competitive sorption effects and that HOC partitioning in solvent and solvent-water systems yield similar exothermic heats constitute strong arguments for the relevance of the partitioning mechanisms (3, 5). When SOC is present in insufficient amounts (98% purity ; [9-14C]phenanthrene, specific activity (SA) ) 10.0 and 8.3 mCi mmol-1) and from Chemsyn (Lenexa, KS; >98% purity; [5,6,11,12-14C]chrysene; SA ) 47.57 mCi mmol-1), respectively. Solutions of these compounds were prepared in 95% ethanolwater (v/v), and working solutions were 50% ethanol-water (v/v). D-[U-14C]glucose (SA ) 270 mCi mmol-1) was purchased from Amersham (Arlington Heights, IL). Solvents and inorganic chemicals were of the highest quality available and were purchased from Mallinckrodt Specialty Chemical Company (Paris, KY) and EM Science (Gibbstown, NJ). Sodium pyrophosphate, polyvinyl pyrrolidone (MW 180 000), and NaN3 were purchased from Sigma Chemical Company (St. Louis, MO). Soil Loading and Desorption Experiments with Spiked [14C]Phenanthrene and [14C]Chrysene. Soil loading and desorption experiments were performed in microcosms

S0013-936X(96)00210-6 CCC: $14.00

 1996 American Chemical Society

TABLE 1. Properties of Soils from Contaminated Sites sample designation

source

location

[PAH]a

fOCb

CECc,d

pH

% sand

% silt

% clay

SSAe

SSC DCU

St. Louis Park, MN Cantonment, FL

surface sub-surface (3-5 ft)

269 2

0.029 0.0026

25.4 2.5

8 6.3

58 87

34 6

8 7

3.8 4.6

a Units, mg kg-1. Total PAH contamination analyzed by Triangle Laboratories (EPA SW846, Method 8310). b Fraction of organic carbon (non-PAH) in the soils, analyzed by Huffman Labs (ASTM D5373). c Cation exchange capacity. d Units, mequiv(100 × cm3)-1. e Specific surface area (m2 g-1), by N2 adsorption.

assembled from 40-mL glass vials (Fisher Scientific, Fairlawn, NJ) with Teflon-lined caps. All glassware was acid washed, solvent washed, and autoclaved prior to use. Experiments were initiated by placing 1 g of soil in a vial along with 10 mL of autoclaved, distilled deionized water containing 0.5% (v/ v) NaN3. Sodium azide, used to inhibit microorganisms in these experiments, has been known to increase soil pH (22, 23), but it did not appear to influence the uptake of [14C]phenanthrene or [14C]chrysene in the experimental soils when examined over short time intervals (30 min; data not shown). The ratio of soil to water was selected based on data showing that a 1:10 ratio produced higher rates of microbial activity than more or less dense slurries (24). This ratio is not, however, similar to those ordinarily found in the field. [14C]PAH was pipetted into each vial containing the soil/water slurry to achieve a final concentration of approximately 0.10 mg L-1 for [14C]phenanthrene while [14C]chrysene was added at 0.08 or 0.002 mg L-1. After addition of the [14C]PAH, the vials were vortexed for 30 s and then stored in the dark at 20 °C until they were sacrificed for analysis at selected time intervals. To obtain preliminary estimates of the time required to load [14C]PAH onto the soils, soil uptake rates were determined by monitoring water phase 14C-labeled concentrations from a single spiked [14C]PAH over a 2-week period at intervals of 0.5, 1, 3, 24, 48, and 72 h, and 1 and 2 weeks. At the end of each period, triplicate vials were removed from incubation and centrifuged (Sorval RC5B refrigerated centrifuge) at 270g for 20 min at 4 °C (10). After centrifugation, the supernatant was decanted and analyzed by liquid scintillation counting (LSC) on a Packard 1900TR scintillation counter (Downers Grove, IL). The soil pellet was analyzed to estimate the fraction of [14C]PAH remaining in the soils in order estimate total [14C]PAH recovered from each microcosm. Nonsequential desorption kinetics were measured to determine the time interval over which desorption occurs. Initial loading was performed by adding 14C-labeled compounds to vials containing soil and water and then storing them for 24 h prior to water phase separation by centrifugation after which the supernatant was counted by LSC. The soil pellet was retained, and desorption was initiated by adding a new 10mL aliquot of NaN3-amended water to each vial containing the centrifuged soil pellet, which was vortexed and allowed to desorb for time intervals of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 24, and 72 h and 1 and 2 weeks. At the end of each time interval, triplicate vials were centrifuged, and the water phase was decanted for analysis by LSC. The [14C]PAH remaining on the soil pellet after desorption was also counted in order to evaluate the overall recovery of [14C]PAH from the microcosms. The fraction of [14C]PAH remaining on the soil was evaluated by transferring individual, centrifuged soil pellets from the loading and nonsequential desorption experiments to a fresh vial along with 10 mL of water and 5 mL of ethyl acetate. Ethyl acetate is less dense less than water, and thus it is easily collected from the slurry microcosms. In previous experiments, ethyl acetate has also been found to efficiently extract freshly added PAH from soils (data not shown). Vials containing soil, ethyl acetate, and water were vortexed for 30 s and centrifuged, after which 1 mL of the ethyl acetate was

removed and analyzed by LSC to obtain the fraction of 14Clabeled compound that was solvent extractable from the soil. After removal of the remaining ethyl acetate, the extracted slurry was agitated, and 1 mL was subjected to LSC to estimate 14C-labeled compounds remaining in the soil. Total recoveries of [14C]phenanthrene and [14C]chrysene in the microcosms ranged from 75 to 105% (data not shown). The loss of 14C-labeled compounds to glass surfaces was evaluated in the vials used in the loading and desorption experiments by removing the soil pellet and then rinsing the vial with 1 mL of water to remove any remaining soil particles. The empty vials were vortexed with 5 mL of ethyl acetate for 30 s prior to analysis of the ethyl acetate by LSC. Loss of [14C]PAH to the walls of the vials was also examined after the addition of similar concentrations of [14C]PAH to individual vials containing only sterile, distilled, deionized water. In these experiments, five different vials were sacrificed daily for 1 week when the water was analyzed by LSC. The empty vials were then extracted with 5 mL of ethyl acetate that was analyzed by LSC. To estimate apparent partition coefficients for [14C]PAH, nonequilibrium sorption and desorption isotherms were obtained at 20 °C. Microcosms were spiked with a range of initial concentrations for [14C]-phenanthrene (862, 441, 85, 52, and 34 µg L-1) and [14C]chrysene (2, 1.7, and 1.2 µg L-1) and then incubated using the times derived from the single spiked value kinetic experiments (loading, 1 day; desorption, 1 h). After incubation, the microcosms were centrifuged and the water phase was analyzed by LSC. In order to determine the fraction of [14C]PAH that can desorb from the soils over time, the sequential desorption of spiked [14C]PAH was examined in five vials for each soil and [14C]PAH combination. The vials were loaded with [14C]PAH using the procedure described for the nonsequential desorption experiments. After soil loading, the vials were centrifuged to separate the water phase for analysis by LSC, and the soil pellet was mixed with 10 mL of fresh NaN3amended water. This procedure was repeated daily for 85 days, after which time the [14C]PAH remaining in the soil pellet was extracted and subjected to LSC as described previously. Desorption Kinetics of Native [12C]PAH in SSC. Desorption of native [12C]PAHs was monitored with the aid of a 10-fold larger reactor to provide the amount necessary for analytical measurements. Ten-gram samples of SSC soil were placed in 250-mL glass bottles along with 100 mL of NaN3/ water solution. At 1, 3, 7, 14, and 28 days, triplicate bottles were removed, and the water phase was decanted and delivered to Triangle Laboratories of Ohio (Dublin, OH) for analysis of the 16 PAH priority pollutants present in the water phase (EPA Method 8310). Additionally, the PAH content of unmanipulated SSC soil was measured by Triangle Laboratories using similar techniques following hot toluene:methanol (10:1) Soxhlet extraction of the soil for 18 h. Biodegradation Rate Experiments. Mineralization rates of [14C]phenanthrene and [14C]chrysene were assessed in vials containing either 1 g of soil and 10 mL of water or 9 mL of water and 1 mL of a suspension of microorganisms washed from the soil [approximately 106 cells (g of soil)-1]. Triplicate vials with the intact soil or the washed culture were then

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FIGURE 1. Water phase concentrations (CW) from the sorption (% of added [14C]PAH in the water phase) and desorption (% of soil bound [14C]PAH in the water phase) of 0.10 mg L-1 [14C]phenanthrene (water solubility: 1.29 mg L-1) and 0.08 mg L-1 (solid line) or 0.002 mg L-1 (dashed line) [14C]chrysene (water solubility: 0.002 mg L-1) in two soils, 2.9% organic content (b) and 0.26% organic content (O) over time (days). (A) Sorption of [14C]phenanthrene; (B) sorption of [14C]chrysene; (C) desorption of [14C]phenanthrene; (D) desorption of [14C]chrysene. Mean (n ) 3) and standard deviation. spiked with a single concentration of either [14C]PAH added below solubility limits; six concentrations for phenanthrene (0.65, 0.38, 0.24, 0.17, 0.06, and 0.04 mg L-1) and three for chrysene (2.8, 1.7, and 1.6 µg L-1). The vials were closed with Teflon-lined caps with a center well (Kontes,Vineland, NJ) containing a fluted piece of Whatman No. 1 chromatography paper (Maidstone, England) saturated with 200 µL of 2 N KOH to collect 14CO2 (25, 26) and were incubated for 10 h at 20 °C. After incubation, the vials were acidified to pH 2 with 20% (v/v) H3PO4 and placed on a rotary shaker at 50 rpm for at least 16 h to liberate and trap the evolved 14CO2. The filter paper was then subjected to LSC to estimate the amount of [14C]phenanthreneor[14C]chrysenethatwasmineralized.[14C]CO2 recovery efficiencies were estimated with triplicate [14C]NaHCO3 amended control vials that were processed and analyzed in a manner identical to the microcosms containing [14C]PAH. Recovery efficiencies of [14C]NaHCO3 were used to correct mineralization recoveries in the sample vials up to 100%. Mineralization was also monitored in vials that were manipulated in the same manner as the sample vials but

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were not inoculated with microorganisms to account for abiotic transformations of [14C]PAH. The washed microorganism suspensions were obtained by placing 100 g of soil (SSC or DCU) in a sterile 500-mL flask along with 250 mL of sterile water containing 0.5% sodium pyrophosphate (v/v) and 0.05% polyvinyl pyrrolidone, neutralized to pH 7. The flask was placed on a rotary shaker for 1 h at 100 rpm (25). After agitation, the soil was allowed to settle for several minutes prior to centrifugation at 120g for 15 min to remove large soil particles. The soil pellet was discarded, and the water phase was decanted and centrifuged again at 3020g for 20 min. The resulting pellet of microorganisms was washed by resuspending the pellet in a sterile 0.25% modified M9 buffer/water solution and centrifuging as mentioned previously. The M9 buffer consisted of 3 g of Na2PO4, 1.5 g of KH2PO4, 2.5 g of NH4Cl, and 0.12 of g MgSO4/L of distilled deionized water, pH 7.3. After washing, the microorganisms were resuspended in the buffer solution so that the final concentration of microorganisms equaled approximately 106 cells mL-1, which was verified by acridine

FIGURE 2. Isotherms of [14C]phenanthrene (PHEN) and [14C]chrysene (CHRY) sorption (0) and desorption (9) in SSC and DCU. All plots were fit with linear isotherms. Mean (n ) 3) and standard deviation.

TABLE 2. Sorption and Desorption Rates of Phenanthrene (PHEN) and Chrysene (CHRY) in Contaminated Soils sorption of [14C]PAH (µg L-1 h-1)

desorption of [14C]PAH (µg L-1 h-1)

desorption of [12C]PAH (µg L-1 h-1)

soil

PAH

rapid (30 min)a

slow (1 day)a

log CS/CWb

rapid (30 min)a

log CS/CWb

rapid (1 day)a

log CS/CWc

SSC

PHEN CHRY PHEN CHRY

74.2 55.8 100.0 40.0

0.034 0.27 1.94 0.80

4.15 (0.994) 4.26 (0.895) 4.20 (0.997) 4.99 (0.986)

2.63 2.04 13.78 3.91

4.32 (0.998) 4.54 (0.851) 4.46 (0.995) 5.24 (0.937)

0.001 0.009 NTd NT

6.83 7.41 NT NT

DCU

a Interval over which each rate was calculated. not tested.

b

Log CS/CW values and r 2 from isotherms. c Log CS/CW value obtained from desorption data.

orange direct counts (27). The nonspecific metabolic activity of the washed culture and the intact soils was compared by monitoring the mineralization of [U-14C]glucose over similar time intervals. Calculations. The distribution of a solute between aqueous and solid phases is described by the distribution coefficient, Kd:

Kd ) (CS)(CW)-1 ) [mmol of PAH kg-1] [mmol of PAH L-1]-1 (1) where CS and CW refer to the equilibrium concentration of solute per liter of water phase and kilogram of soil phase, respectively. Due to the fact that the soil loading procedures used in these experiments did not always achieve equilibrium, we will simply refer to the solute distribution by the ratio CS/CW.

Results Loss of [14C]Phenanthrene or [14C]Chrysene to Glass Walls. In microcosms containing only distilled deionized water, up to 10% of the [14C]chrysene and 2% of the [14C]phenanthrene was extracted from the glass walls of the vials after 24 h of solution contact. In the presence of soil, however, these values decreased to 2.5% and 1.0%, respectively, and were subsequently ignored in determinations of liquid and solid phase concentrations. Soil Loading and Desorption Experiments with Spiked [14C]Phenanthrene and [14C]Chrysene. It is evident from

d

NT,

Figure 1, panels A and B, that equilibrium was not achieved between the aqueous and soil phases during the 2 week period following addition of the [14C]PAH. The loading curves for [14C]PAH were similar in shape and achieved over 90% uptake in 24 h when [14C]phenanthrene was added below water solubility and [14C]chrysene was added above water solubility. Examination of the loading curve for [14C]chrysene loaded below solubility limits (Figure 1B) suggests that transport limitation may have been a factor in the higher loadings of [14C]chrysene. In contrast, desorption equilibrium was achieved in less than 30 min for each [14C]PAH and soil combination (Figure 1, panels C and D). The transport limitation noted above for the higher loadings of [14C]chrysene was not evident in the desorption data (data not shown). Loading and desorption isotherms, shown in Figure 2, demonstrate hysteresis effects for each [14C]PAH and soil combination in that the sorptive and desorptive behavior for these systems do not lead to the same (Cs)(Cw)-1 values (Table 2). The degree of hysteresis was greater for the higher molecular weight PAH on the same soil and greater for both [14C]phenanthrene and [14C]chrysene on the higher organic soil. Despite the fact that the reactor vials were spiked with amounts of 14C-labeled compounds approaching aqueous solubility ([14C]phenanthrene) or in excess of aqueous solubility ([14C]chrysene), the equilibrium water concentrations were relatively low. Therefore, Figure 2 does not represent the full range of possible values of solute saturation in the water phase, and it is likely that the degree of hysteresis would be greater at larger equilibrium water concentrations. The

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FIGURE 3. Sequential removal of [14C]phenanthrene in DCU, after 24-h intervals. Panel A presents data from water (0) and estimated soil phase (O) concentrations over an 85-day extraction period. Panel B shows the change in apparent Kd over time. The Kd calculated from sorption isotherms is indicated with an arrow. Mean (n ) 3) and standard deviation. concentrations of [14C]PAH used in the isotherm experiments are appropriate, however, for studies of microbial metabolism that we wished to conduct. Furthermore, the degree of hysteresis is a marked function of contact time between the PAH and the soil as is evident in the water and solid phase concentrations resulting from 85 daily sequential desorptions of single spiked concentration of [14C]phenanthrene or [14C]chrysene. For each soil and [14C]PAH combination, water phase concentrations declined to less than 0.1 ng mL-1 over 85 days while the residual concentrations of [14C] PAH in the soil remained relatively high (100-300 ng g-1), as illustrated by the sequential desorption of [14C]phenanthrene in DCU in Figure 3. It is important to note that in these experiments, the concentration of [14C]PAH in the soil could not be measured each day but was estimated based on the amount of [14C]PAH that had been previously removed from the vial in the water phase. As Pignatello (13) observed in similar experiments with tetrachloroethylene on fine sandy loam soil, this behavior leads to significantly increased apparent Kd values as compared to the Kd value obtained from the sorption or loading isotherm. This effect was qualitatively similar for [14C]chrysene in SSC and DCU and for both [14C]phenanthrene and [14C]chrysene on the higher organic soil (SSC) (data not shown). The overall pattern of sorption kinetics for [14C] phenanthrene and [14C] chrysene consisted of a relatively rapid phase followed by a much slower phase (Table 2), in agreement with the literature (28, 16). The rapid-phase sorption values are minimum estimates, however, due to the fact that our first data point was at 0.5 h. Rates for the slow-phase sorption for [14C]phenanthrene and [14C]chrysene are similar as might be expected from adjacent members of the homologous PAH series. Desorption rates for both labeled compounds are uniformly higher than the slow phase of sorption rates in both soils (Table 2). With the exception of the rapid-phase sorption for [14C]chrysene, sorption and desorption equilibria were more rapid in the lower organic soil (DCU).

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FIGURE 4. PAH water phase concentration: (A) % of soil bound PAH present in the water and (B) PAH present in the water (ppb), from the desorption of the preexisting PAH contamination (CS) from the SSC soils. The soil contamination profile is presented in panel C. The 16 PAHs are 1, naphthalene; 2, acenaphthalene; 3, acenaphthene; 4, fluorene; 5, phenanthrene; 6, anthracene; 7, fluoranthene; 8, pyrene; 9, benz[a]anthracene; 10, chrysene; 11, benzo[k]fluoranthene; 12, benzo[b]fluoranthene; 13, benzo[a]pyrene; 14, dibenz[a,h]anthracene; 15, benzo[g,h,i]perylene; and 16, ideno[1,2,3-cd]pyrene. Desorption Kinetics of Native [12C]PAH in SSC. These experiments were conducted with a soil from a closed wood treating facility that was exposed to multiple doses of PAH since the late 1800’s. Addition of PAHs to this soil was stopped during the 1950’s and since that time the PAH in the soil have been subject to sorption-desorption and microbial metabolism. The magnitude of these effects on the current profile of PAH in the soil, however, is unknown as is the initial PAH profile in the soil. Figure 4 describes the desorption of 16 native PAH in soil SSC. Comparison of the data in Figure 4A with that in Figure 1, panels C and D, shows that the desorption rates for the native [12C]PAH in soil SSC were markedly slower than the desorption rates for [14C]phenanthrene and [14C]chrysene (Table 2). The maximum extent of desorption for native compounds varied over time but was

TABLE 3. Desorption and Mineralization Rates for Phenanthrene (PHEN) and Chrysene (CHRY) soil

PAH

desorption of [14C]PAH (µg L-1 h-1)

SSC

PHEN CHRY PHEN CHRY

2.6 2.0 13.8 3.9

DCU

mineralization ratea (µg L-1 h-1)

desorption of [12C]PAH (µg L-1 h-1)

soil

washed culture

0.001 0.009 NTb NT

0.002 0.002 0.02 0.04

0.002 0.01 0.05 0.02

a Mineralization rate values for microorganisms in soils and microorganisms washed from the soils were calculated using the mineralization rate applied to the concentrations in column 5. b NT ) not tested.

approximately 0.30% of the soil-bound native material (Figure 4A) whereas the maximum extent of desorption for 14C-labeled compounds was on the order of 2.5-12% of soil-bound material (Figure 1, panels C and D). In addition, comparison of Figure 4 shows variation in the desorption rates for the individual native compounds and no correlation with present native compound abundance in the soil. Biodegradation Rate Experiments. Rates of mineralization for [14C]phenanthrene and [14C]chrysene in SSC and DCU were compared with the rates of desorption of freshly added 14C-labeled compounds and native 12C-labeled compounds (SSC only). Rates of mineralization of 12C-labeled materials were not measured. Mineralization was used as a metabolic end point because it has been found to be the primary biotic fate of phenanthrene and chrysene in these soils (29). Although both [14C]phenanthrene and [14C]chrysene were tested over a range of concentrations in both soils, mineralization did not demonstrate saturation kinetics with either the microbial communities in the intact soils or microorganisms washed from the soils (data not shown). The metabolic activity of the washed culture of microorganisms, used to estimate the effect of sorptive interactions on the rate of mineralization, was found to be similar to that of the unmanipulated soil community when the mineralization of a nonsorbing and readily degradable substrate (D-[U-14C]glucose) was monitored over 1 day (data not shown). Desorption rates of [14C]phenanthrene and [14C]chrysene were much faster than the rate of mineralization for the same soil and 14C-labeled compound combination using both the unmanipulated soil and the washed culture of microorganisms (Table 3). Comparison, however, of the [14C] PAH data with native 12C-labeled data indicates that desorption rates of [12C]PAH were equal to or slightly slower than mineralization rates and that mineralization of 12C-labeled compounds may be controlled by the rate at which they desorb (Table 3).

Discussion Isotherm nonsingularity has been observed in many HOC sorption studies (10, 11, 30, 31). Hysteresis effects are also evident in this work (Figures 2 and 3), and this behavior may be inherent in PAH-soil interactions. Kd values will, therefore, be dependent upon whether equilibrium is approached from the sorptive or desorptive directions. Hysteresis effects have been attributed to a variety of factors: removal of HOC by microorganisms (32); failure to reach equilibrium in both the sorptive and desorptive directions; artifacts due to methods; the sorption of HOC to dissolved organic carbon (DOC) or colloids that may artificially overestimate water phase concentrations of HOC (33, 34); or in some cases that a portion of the HOC is resistant to desorption (e.g., ref 10). A complete discussion of these issues is found in Pignatello (13). In this work, only the first reason can be excluded because the experiments were performed in a metabolically inhibited environment. The importance of the remaining mechanisms, however, cannot be distinguished. Sorption equilibrium was not reached in the sorptive direction after 1 day, and therefore, we have not used the sorption isotherm data in Figure 2 to compute Kd values and refer only to the ratio CS /CW as noted

14C-labeled

in Table 2. In addition, the presence of DOC and colloidal materials was not accounted for in our experimental measurements of water phase [14C]PAH, both of which can increase the apparent water solubility of the HOC (34, 35). Thus, the data for soil SSC may have been more affected by DOC and colloidal materials because it has a larger fOC and fraction of silt and clay than DCU. No suggestion of “non labile” material was evident in the [14C]PAH isotherms on either soil, as the desorption curves in Figure 2 return to zero CS at zero CW. Material for these isotherms had been sorbed for only 1 day. The sequential desorption experiments using the same 14C-labeled compounds and soils, however, showed that over an 85-day period of residence on the soil, between 15% and 30% of the spiked [14C]phenanthrene and 15-40% of the spiked [14C]chrysene had become nonlabile. A much larger fraction of the native [12C]PAH in soil SSC appears to be nonlabile, as less than 0.5 wt % of total soil-bound PAH was removed over a 1-month desorption interval (Figure 4A). This is similar to results found with other HOC and environmental medias (12, 36, 37). Limited desorption of native [12C]PAH in soil SSC can be explained by restricted diffusion of [12C]PAH through the soil or by the irreversible binding of [12C]PAH to components of SSC soil. The results from these experiments do not allow us to determine which phenomenon is impacting the desorption of native PAH. If the desorption of the native PAH is limited by diffusion, however, these limitations must be extreme because the native PAHs in SSC have had several decades to desorb from the soil. Desorption of [12C]PAH in SSC also continued over the 28-day test period (Figure 4B) with no apparent correlation between native PAH size and desorption time. Although it would have been helpful to study the behavior of spiked [14C]PAH that were aged to the same extent as the native contamination, it was not possible to re-create the conditions to which the native PAH were subjected, as they are unknown. It is attractive to assume that the sorption-desorption of spiked PAH in soils SSC and DCU is explained by a general partition mechanism with diffusion limitations. During the sorptive phase, 24 h of uptake accounts for a mass transfer of 90% of starting solute concentration and occupation of the more accessible sorption sites. Longer time intervals are required, however, for the remaining material to penetrate the less accessible pores of the soil, and thus sorptive equilibrium may only be achieved after extended time intervals (17). Desorption may be more rapid since the material loaded onto the soil was from the rapid sorptive phase (24 h) and much less mass transfer (less than 1% of adsorbed solute) would be needed. Our principal objective was to compare the rates of desorption of freshly added and aged PAH to the rates of mineralization for these compounds. Many investigators have compared rates of desorption and mineralization indirectly either by relying on the behavior of spiked HOC (9), by measuring microbial metabolism with a laboratory culture of HOC degrading microorganisms (19, 31), or by comparing the extent of mineralization for freshly added verses aged contamination (12, 20). Our work, on the other hand, is a

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direct comparison between desorption rates of both spiked PAH and PAH aged under field conditions to rates of mineralization by indigenous microorganisms. Similar experiments were performed by Rijnaarts et al. (38) looking at the fate of the chlorinated pesticide R-hexachlorocyclohexane using a different experimental system and metabolic end point. The data in Table 3 shows, under the test conditions used in these experiments, that the rate of mineralization of spiked 14 C-labeled compounds is significantly slower than the rate of desorption for these compounds. Consistent with the findings of others (9, 30) who used similar methods (spiked compounds), it would appear that the rate of mineralization controls environmental fate, since desorption appears to be more rapid than degradation. Importantly, the data in Table 3 also suggest that the rates of mineralization of [14C]PAH are equal to or slightly more rapid than the rates of desorption for native [12C]PAH. This suggests that desorption would control the fate of aged [12C]PAH. Rijnaarts et al. (38) showed that the metabolism of native material was faster than rates of desorption, based on measurements of chloride release in a soil reactor. These results and ours suggest that the microbial community may have adapted to be able to use native materials as rapidly as they become available. Limited desorption of [12C]PAH explains the persistence of PAH in our soils, both of which contain active communities of PAH degrading microorganisms (29). The persistence of PAH in soil may not be solely based on their suitability as substrates for microorganisms, since readily degradable PAH, such as naphthalene and phenanthrene, persist in the soils along with the less degradable higher molecular weight PAH (eg., benzo[a]pyrene) (39). The data in Table 3 suggest that native/spiked differences are larger than differences observed in mineralization experiments in the presence or absence of soil (Table 3). The similarity between the rates of desorption of native PAH and mineralization supports the idea that the soil microorganisms may have adapted to the concentrations of PAH to which they had been previously exposed. These biodegradation results also support the previous finding that freshly added PAH do not behave in the same manner as aged PAH (eg., refs 12, 19, and 20).

Acknowledgments This work was funded by a National Institute of Environmental Health Sciences Superfund Center grant (P42ES05948) to the University of North Carolina at Chapel Hill. We would like to thank J. Pedit and M. D. Aitken for a critical review of an earlier version of this manuscript and F. Kramer and M. Fite of the U.S. EPA for help in obtaining contaminated soil samples. Chemsyn, vendor for [14C]PAH, is associated with the Cancer Research Program of the National Cancer Institute, Division of Cancer Cause and Prevention, Bethesda, MD.

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Received for review March 5, 1996. Revised manuscript received August 12, 1996. Accepted September 3, 1996.X ES9602105 X

Abstract published in Advance ACS Abstracts, November 1, 1996.