182

Environmental Toxicology and Chemistry, Vol. 18, No. 2, pp. 182–187, 1999q 1999 SETAC

Printed in the USA0730-7268/99 $9.00 1 .00

ENHANCING THE BIOAVAILABILITY OF ORGANIC COMPOUNDS SEQUESTERED INSOIL AND AQUIFER SOLIDS

JASON C. WHITE,† MARTIN ALEXANDER,*† and JOSEPH J. PIGNATELLO‡†Institute for Comparative and Environmental Toxicology and Department of Soil, Crop, and Atmospheric Sciences,

Cornell University, Ithaca, New York 14853, USA‡Connecticut Agricultural Experiment Station, P.O. Box 1106, New Haven, Connecticut 06504, USA

(Received 6 January 1998; Accepted 9 May 1998)

Abstract—A study was conducted to find ways to increase the biodegradability of compounds that have aged in soil or aquifermaterial and become less bioavailable. Slurrying enhanced the rate and extent of biodegradation by individual bacterial strains ofaged and unaged phenanthrene and di(2-ethylhexyl) phthalate in soils and aquifer solids. After bacterial degradation of agedphenanthrene in unslurried soil had largely ceased, the residual compound was metabolized if the soil was slurried and reinoculatedwith a phenanthrene-degrading bacterium. The rate and extent of biodegradation of aged phenanthrene by Pseudomonas sp. wereenhanced when anthracene or pyrene was added to the soil at the same time as the bacterium, although the organism could notmetabolize anthracene or pyrene. Moreover, anthracene or pyrene increased the amount of aged phenanthrene removed from soilby a mild extractant. The data show that the bioavailability of organic compounds that become sequestered by aging can be alteredby appropriate soil treatments.

Keywords—Aging Bioavailability Di(2-ethylhexyl) phthalate Phenanthrene Sequestration

INTRODUCTION

That some organic compounds become sequestered in soilis well established. This sequestration results in a diminutionin the rate and extent of biodegradation by microorganisms ofchlorinated hydrocarbons in the field [1], the resistance tobiodegradation in the laboratory of chemicals aged in the fieldfollowing their application for pest control [2,3], a decliningability of bacteria to metabolize compounds aged in the lab-oratory for increasingly long periods of time [4], a decline inuptake by earthworms as chemicals persist in soil [5], and theprogressively lower acute toxicity to insects of dieldrin andDDT aged in soils in the laboratory [6]. Sequestration alsoapparently leads to incomplete bioremediation of petroleumhydrocarbons [7].

The decline in toxicity and bioavailability as a result ofaging and the accompanying sequestration may represent anatural remediation process because, although the chemical isnot destroyed, the exposure of susceptible organisms to theharmful effects of the toxicant, and thus the risk from it, de-creases with time. Thus, enhancement of this natural seques-tration would be beneficial. White et al. [8] showed that morephenanthrene was sequestered in soil exposed to cycles ofwetting and drying than in soil maintained at constant mois-ture. Conversely, increasing the availability of such aged com-pounds to microorganisms would make those molecules moresusceptible to bioremediation, leading to a lower final con-centration of the toxicants of concern. However, little infor-mation exists on means to increase the bioavailability of agedcompounds.

Therefore, a study was conducted to find procedures thatmight result in an increase in the bioavailability of organicmolecules aged in soil. The compounds used for the investi-

* To whom correspondence may be addressed (jae2@cornell.edu).

gation were phenanthrene and di(2-ethylhexyl) phthalate(DEHP), and bioavailability was determined by measuringtheir biodegradability and extractability.

MATERIALS AND METHODS

Aging of chemicals

The environmental samples were Lima loam (pH 7.2, 7.8%organic matter), Mount Pleasant silt loam (pH 6.2, 8.0% or-ganic matter), Edwards muck (pH 7.2, 23.0% organic matter),Bath silt loam (pH 6.8, 8.2% organic matter), Middlebury siltloam (pH 6.9, 5.1% organic matter), and aquifer solids (pH7.7, 1.3% organic matter). The soils, all of which were fromsites in New York, USA, and aquifer material collected froma depth of 10 m in Brooktondale, New York, USA, were airdried, passed through a 4-mm sieve, and sterilized with 2.5Mrad of g-irradiation from a 60Co source.

Ten- or 5-g portions of sterile soil or aquifer solids wereadded to 50-ml sterile screw-cap test tubes, and 1 3 105 dpmof [9-14C]phenanthrene (8.3 mCi/mmol, .98% pure; SigmaChemical, St. Louis, MO, USA) or [U-ring-14C]DEHP (2.7mCi/mmol, .98% pure; Sigma) and unlabeled phenanthreneor DEHP were added in 50 ml of dichloromethane to give 1.1or 11.0 mg/g of soil or aquifer solids. The tubes were mixedmanually 10 times and then shaken with a vortex mixer for 6s every 10 min in a 50-min period to allow the dichloromethaneto volatilize and to mix the hydrocarbon with the soil. Steriledeionized water was added to bring the moisture level to ap-proximately 21.1 bar. The tubes were tightly capped withTeflon-lined closures, mixed on a vortex shaker, and placed inthe dark at 21 6 28C. Additional samples were prepared inthe same way to give soils in which the compound had beenaged for different times. Each treatment was in triplicate, andstatistical significance was determined by Student’s t test.

Enhancing bioavailability of sequestered compounds Environ. Toxicol. Chem. 18, 1999 183

Bacteria

A DEHP-degrading bacterium was obtained by enrichmentculture derived from a sample of Lima loam inoculated intoan inorganic salts solution containing 0.05% (v/w) DEHP assole added carbon source. The DEHP-degrading bacterium,which was a catalase- and oxidase-positive gram-negative rod,and a phenanthrene-degrading bacterium (Pseudomonas strainR) were grown at 298C in the medium containing phenanthrene(40 mg/L) or DEHP (40 ml/L) in excess of their water solu-bilities and 0.4 g of K2HPO4, 0.2 g of KH2PO4, 0.1 g ofCaCl2·2H2O, 0.1 g of NH4NO3, 0.05 g of MgSO4, and 0.01 gof FeCl3 per liter of distilled water. After 7 to 14 d of incubationon a rotary shaker operating at 100 rpm, the phenanthrene-utilizing culture was passed through a 40-mm pore-size glassfrit to remove remaining crystals, and the filtrate was centri-fuged at 10,400 g for 10 min. The cells were resuspended in0.01 M phosphate buffer, pH 7.0. The cell density was deter-mined by plating on Trypticase-soy agar. The rates of min-eralization by the bacteria were determined from a linear re-gression of the data from the first three sampling times.

Slurrying

Phenanthrene (1.1 mg/g of soil) was aged in samples ofLima loam, aquifer solids, and Mount Pleasant silt loam for92, 100, and 110 d, respectively. The DEHP (1.1 mg/g soil)was aged in Mount Pleasant silt loam, Lima loam, and aquifersolids for 68, 94, and 112 d, respectively. One half of thesamples containing unaged or aged substrate were asepticallytransferred to sterile 250-ml Erlenmeyer flasks containing 100ml of sterile distilled water. The slurries or unshaken tubes ofsoil or aquifer solids were inoculated with 108 to 109 cells ofPseudomonas strain R or the DEHP-degrading bacterium. Theslurries were incubated on a rotary shaker operating at 100rpm. All samples were incubated at approximately 228C. The14CO2 formed from mineralization of the test compounds wastrapped in 2.0 ml of 0.5 M NaOH that was contained in a 10-ml beaker suspended from a Teflon-taped rubber stopper inthe tube or flask. The alkali was sampled periodically andadded to Liquiscint scintillation fluid (National Diagnostics,Somerville, NJ, USA) for measurement of radioactivity witha liquid scintillation counter (model LS7500, Beckman In-struments, Irvine, CA, USA).

After mineralization had largely stopped, the soil or aquifersolids were separated from the liquid phase, and the solidswere mixed vigorously for 2 min with 25 ml of n-butanol. Thesuspension was passed through Whatman number 1 filter paper.The tubes were washed with 10 ml of n-butanol, and this liquidwas also filtered. Soil remaining on the filters was extractedfor 3 h in a Soxhlet extractor with 90 ml of hexanes and 2 mlof n-butanol. The hexanes were removed by evaporation, andthe remaining n-butanol was combined with the first butanolextract. The combined extracts were passed through a 0.22-mm Teflon-syringe filter and analyzed with a liquid chromato-graph (model 1050, Hewlett-Packard, Avondale, PA, USA)fitted with a Spherosorb ODS-2 octadecyl-bonded silica col-umn (Hewlett-Packard; 5 mm, 250 3 4 mm) using acetonitrile:water (86:14, v/v) as the mobile phase at a flow rate of 0.8ml/min. Phenanthrene was detected by its absorbance at 254nm. The same method was used for analysis of anthracene andpyrene.

Cosolute addition

To assess the possible effect of a second compound on thebiodegradation of an aged compound, phenanthrene was addedto the soil, and sterile deionized water was added to bring themoisture level to 21.1 bar. Edwards muck (10 g) received 11mg of phenanthrene and 4.0 ml of water. Five- and 10-g sam-ples of Mount Pleasant silt loam received 26 or 11 mg ofphenanthrene and 1.1 or 2.3 ml of water, respectively. Thephenanthrene was aged for 259 d in the muck and for 38 or74 d in the 5- or 10-g samples of silt loam, respectively. Afteraging, the samples were aseptically transferred to sterile 250-ml Erlenmeyer flasks that were covered loosely with aluminumfoil and stored at 308C to allow the soil to dry. Phenanthreneis not lost under these conditions [9]. After 6 to 10 d, by whichtime the soil had reached constant weight, the samples wereaseptically transferred to sterile 50-ml test tubes. Replicatesamples of muck received 200 mg of anthracene, replicate 5.0-g samples of silt loam received 100 mg of anthracene, andreplicate 10-g samples of silt loam were amended with 300mg of pyrene. The compounds were added in 30 ml of di-chloromethane. The remaining replicates of each soil received30 ml of the solvent alone. The samples were shaken manuallyand with a vortex mixer as described above to mix the chem-icals with the soils. The samples were brought to a moisturelevel of 21.1 bar and inoculated with Pseudomonas strain R,and mineralization was measured.

To assess the effect of anthracene and pyrene on the ex-tractability of an aged compound, 11.0 mg of phenanthrenewas added to 10 g of Mount Pleasant silt loam or Bath siltloam, and water was added to bring the moisture level to 21.1bar. The phenanthrene was aged for 192 d in Bath silt loamand for 69 or 121 d in Mount Pleasant silt loam. Anthraceneor pyrene was added, and the samples then were extracted withethanol:water (45:55, v/v) for 24 h, and the quantity of phe-nanthrene in the extracts was determined. The soils then weresequentially extracted with n-butanol followed by hexanes toprovide a mass balance of phenanthrene.

Surfactants

To measure the influence of surfactants Novel 1412-56 andAlfonic 810-60 (both from Vista Chemical Company, Austin,TX, USA) on the biodegradability and extractability of agedcompounds, 1.1 mg of phenanthrene per gram of soil was addedto Edwards muck and Mount Pleasant silt loam, and 10 mg ofDEHP per gram of soil was added to Middlebury silt loam.The soils were brought to a moisture level of 21.1 bar. Thechemicals were aged for 496, 124, and 58 d in the muck,Middlebury silt loam, and Mount Pleasant silt loam, respec-tively. The soils were then aseptically transferred to sterile250-ml Erlenmeyer flasks, and the flasks were covered looselywith aluminum foil and stored at 308C. After 7 to 10 d, bywhich time the soils had dried to a constant weight, one halfof the samples were brought to 21.1 bar with 4.0 (muck) or2.3 ml (silt loams) of sterile water amended with 10 mg ofeither Alfonic 810-60 or Novel 1412-56 per g of soil. Theremaining samples received sterile water. The muck and Mid-dlebury silt loam were inoculated with 108 to 109 cells ofPseudomonas strain R or the DEHP-degrading bacterium, andmineralization was measured. The samples of Mount Pleasantsilt loam were extracted with 45:55 (v/v) ethanol:water for 24 h.

184 Environ. Toxicol. Chem. 18, 1999 J.C. White et al.

Table 1. Effect of slurrying on the mineralization of unaged and agedphenanthrene in two soils and aquifer solids

Environmentalsample

Period ofaging (d) Treatment

Mineraliza-tion rate

(%/d)

Extent ofminerali-

zation(%)a

Mount Pleasantsilt loam

0

110

UnslurriedSlurriedUnslurriedSlurried

3.08 Bb

8.03 D1.45 A3.20 C

22.8 B45.6 D8.2 A

32.9 CLima loam 0

92

UnslurriedSlurriedUnslurriedSlurried

1.91 B18.6 D0.89 A

14.3 C

28.5 B52.9 D9.4 A

37.1 CAquifer solids 0

100

UnslurriedSlurriedUnslurriedSlurried

5.18 A19.7 C5.80 A

14.0 B

21.2 A35.2 B21.6 A34.1 B

a Extent determined after 28, 20, and 36 d in Lima loam, aquifer solids,and Mount Pleasant silt loam, respectively.

b Values followed by the same letter within each type of environmentalsample and each column are not significantly different (p , 0.05).

Table 2. Effect of slurrying and reinoculation on the biodegradationof unaged and aged phenanthrene in Mount Pleasant silt loam

Period ofaging (d) Treatment Reinoculation

Phenanthreneremaining

(mg)

0 UnslurriedSlurried

NoNo

1.9 Ba

0.6 A41 Unslurried

UnslurriedSlurriedUnslurried/slurriedb

Unslurried/slurried

NoYesNoNoYes

4.1 E3.1 D2.3 C2.2 C1.4 A

a Values followed by the same letter are not significantly different (p, 0.05).

b Soil incubated 27 d without slurrying, followed by slurrying for 25 d.

Table 3. Effect of slurrying on the mineralization of unaged and ageddi(2-ethylhexyl)phthalate in two soils and aquifer solids

Environmentalsample

Periodof aging

(d) Treatment

Mineraliza-tion rate(%/d)a

Extent ofmineraliza-

tion (%)

Mount Pleasantsilt loam

0

68

UnslurriedSlurriedUnslurriedSlurried

0.12 Ab

9.73 C0.07 A3.37 B

2.7 A54.5 D

3.2 A35.3 B

Lima loam 0

94

UnslurriedSlurriedUnslurriedSlurried

0.29 A15.8 C

0.44 A6.51 B

10.8 B53.8 D

7.8 A41.4 C

Aquifer solids 0

112

UnslurriedSlurriedUnslurriedSlurried

8.20 A15.8 B

7.09 A18.2 C

22.5 B34.7 C19.1 A37.2 Cc

a Extent determined after 24, 18, and 14 d in Mount Pleasant silt loam,Lima loam, and aquifer solids, respectively.

b Values followed by the same letter within each type of environmentalsample are not significantly different (p , 0.05).

c Significantly less than corresponding unaged sample at p , 0.10.

RESULTS

Slurrying

Phenanthrene was aged in samples of Lima loam, aquifersolids, and Mount Pleasant silt loam, and then the mineral-ization of the unaged and aged compound in samples that wereunslurried or slurried was determined. The rates and extentsof mineralization of aged phenanthrene were significantly lessthan the unaged compound in both unslurried and slurried soil,but 100 d of aging had little or no effect on the rate or extentof mineralization in samples of aquifer solids (Table 1). Re-gardless of aging, the rate and extent of mineralization weremarkedly greater in slurried than in nonslurried soil and aquifersolids.

After mineralization had nearly stopped in the silt loam (36d), the soil was subjected to sequential n-butanol and Soxhletextractions, and the extracts were combined and analyzed byhigh-performance liquid chromatography (HPLC) for residualphenanthrene. After biodegradation of unaged phenanthrenein slurried and unslurried soil, 2.5 and 5.3 mg of the compoundremained. The values are significantly different (p , 0.01).Following biodegradation of aged phenanthrene in slurried andunslurried soil, 4.0 and 8.3 mg, respectively, of the compoundwere still present. These values are significantly different (p, 0.01). Aging also significantly reduced (p , 0.05) the bio-availability of the compound in both unslurried and slurriedsoil.

To determine whether slurrying and reinoculation enhancedbiodegradation after mineralization had already stopped, slur-ried and unslurried samples of Mount Pleasant silt loam con-taining phenanthrene (2 mg/g of soil aged for 0 and 41 d) wereinoculated with Pseudomonas strain R. After 27 d, at whichtime mineralization had stopped, two thirds of the unslurriedsamples containing aged phenanthrene were slurried and onehalf of those slurries were reinoculated. The remaining samplesof unslurried soil were also reinoculated. After an additional25 d, at which time mineralization had largely stopped, thesamples were extracted and analyzed by HPLC. Less unagedand aged phenanthrene remained following biodegradation inslurried than in nonslurried soil (Table 2). If the unslurriedsoil, which still contained 3.3 mg of phenanthrene, was slurried

for an additional 25 d, the amount of the compound remainingfell to 2.2 mg without reinoculation and 1.4 mg with reinoc-ulation.

Di(2-ethylhexyl) phthalate was aged in samples from thesame three environmental sources, and mineralization of theunaged and aged phthalate in samples that were unslurried orslurried was determined. Slurrying DEHP-containing soil oraquifer solids greatly enhanced both the rates and extents ofmineralization of unaged and aged DEHP (Table 3). Agingcaused a statistically significant reduction in the extent of min-eralization in the unslurried and slurried environmental sam-ples, except for the unslurried silt loam and the slurried aquifersolids. Aging also decreased the rates of mineralization in thesoils, although the effect was not always statistically signifi-cant, and the effects of aging were not apparent on the ratesof biodegradation in the aquifer solids.

Cosolute addition

To determine whether Pseudomonas strain R could metab-olize pyrene or anthracene, 100 mg of each compound wasseparately added to sterile culture tubes containing 10 ml ofinorganic salts solution, 10 mg of phenanthrene, and 108 to109 cells of Pseudomonas strain R. After 14 d of incubation,the solution was extracted with n-butanol and analyzed byHPLC for undegraded anthracene or pyrene. The recoveries

Enhancing bioavailability of sequestered compounds Environ. Toxicol. Chem. 18, 1999 185

Table 4. Effect of anthracene and pyrene on the biodegradation ofaged phenanthrene in soil

Soil

Periodof aging

(d)

Compet-ing

solutea

Mineralization

Rate(%/d)

Extent(%)b

Phenan-threne

remaining(mg)

Edwards muck 0259259

NoneNoneAnth

6.39 Cc

1.32 A1.78 B

25.5 C8.3 A

11.2 B

2.0 A3.3 C3.0 B

Mount Pleasantsilt loam

03838

NoneNoneAnth

8.49 C3.77 A4.53 B

36.0 C21.5 A26.3 B

1.8 A6.4 C4.7 B

Mount Pleasantsilt loam

07474

NoneNonePyr

6.17 C1.97 A2.43 B

31.2 C17.3 A22.5 B

2.1 A4.9 C2.6 B

a Anth 5 anthracene; Pyr 5 pyrene. The soils received 200 (muck)or 100 mg (silt loam) of anthracene or 300 mg of pyrene.

b Determined after 49, 22, and 44 d in the muck and the two samplesof silt loam, respectively.

c Values in the same column of soil samples followed by the sameletter are not significantly different (p , 0.05).

Table 5. Effect of anthracene or pyrene on the extraction of unagedand aged phenanthrene from soil

SoilPeriod ofaging (d)

Displacingcompounda

Recovery (%)

Ethanol:waterb Total

Mount Pleasantsilt loam

0121121121

NoneNoneAnth, 50 mgAnth, 1,000 mg

21.0 B16.6 A18.9 Ac

29.2 C

116.296.596.3

103.1Bath silt loam 0

0192192

NoneAnth, 500 mgNoneAnth, 500 mg

34.1 C37.5 Cc

22.1 A30.4 B

109.0113.6100.5102.0

Mount Pleasantsilt loam

06969

NoneNonePyr, 500 mg

25.9 B22.1 A35.3 C

104.4100.8

98.2

a Anth 5 anthracene; Pyr 5 pyrene.b Values within each soil followed by the same letter are not signif-

icantly different (p , 0.05).c Significantly greater than corresponding sample with no anthracene

at p , 0.10.

Table 6. Effect of surfactants on the mineralization of agedphenanthrene and di(2-ethylhexyl) phthalate (DEHP)

Test compound

Periodof aging

(d) Surfactant

Mineralization

Rate (%/d)a Extent (%)a

Phenanthrene 0496496496

NoneNoneAlfonicNovel

4.02 Cb

0.25 A1.34 B0.65 A

22.2 C3.3 A9.5 B6.1 Ac

DEHP 0124124

NoneNoneAlfonic

1.81 B1.39 A2.14 B

25.0 A23.2 Ad

28.7 B

a Determined after 58 d for phenanthrene and 115 d for DEHP.b Values followed by the same letter are not significantly different (p

, 0.05).c Significantly different at p , 0.10 from aged phenanthrene in soil

not amended with surfactant.d Significantly less at p , 0.10 than in soil with unaged DEHP.

of anthracene and pyrene were 96.4 and 97.0% of the chemicalthat was added to the culture tubes, respectively; thus, thebacterium could not use these two hydrocarbons.

Measurements were made of the maximum rates and extentsof mineralization of unaged and aged phenanthrene in Edwardsmuck and Mount Pleasant silt loam and of aged phenanthrenein soil amended with anthracene or pyrene. Aging significantlyreduced (p , 0.01) the rates and extents of mineralization inall samples of soil (Table 4). Moreover, the addition of an-thracene or pyrene at the time of inoculation significantly in-creased the rates and extents of mineralization of aged phe-nanthrene (p , 0.05). After mineralization had slowed, thesoils were extracted first with n-butanol and then subjected toSoxhlet extraction with hexanes, and the combined extractswere analyzed by HPLC. The data show that more phenan-threne remained after biodegradation in Edwards muck andMount Pleasant silt loam if the compound had been aged thanif freshly added and that the presence of anthracene or pyrenesignificantly increased the bioavailability of aged phenan-threne to bacteria.

An assessment was made of the effect of anthracene andpyrene on the extractability of unaged and aged phenanthrenewith a mild extractant, 45:55 (v/v) ethanol:water. Aging re-duced the amount of phenanthrene extracted with ethanol:wa-ter (Table 5). However, the presence of anthracene at 50, 500,or 1,000 mg or pyrene at 500 mg added just prior to the ex-tractant significantly increased the amount of aged phenan-threne extracted from the soils. On the other hand, all of thephenanthrene was recovered by sequential extractions with n-butanol followed by hexanes.

Surfactants

Tests were conducted of the effect of two nonionic surfac-tants on the mineralization of phenanthrene aged for 496 d inEdwards muck and DEHP aged for 124 d in Middlebury siltloam. The rate and extent of mineralization of phenanthreneaged for 496 d and DEHP aged for 124 d were significantlyless than for the freshly added compounds (Table 6). Alfonic810-60 or Novel 1412-56 added at the time of inoculationincreased the extent of mineralization of phenanthrene orDEHP aged in muck or silt loam as compared to unamendedsoil, respectively. The samples of muck were subjected to n-

butanol and Soxhlet extraction with hexanes and analyzed byHPLC for undegraded phenanthrene. The recoveries from soilof phenanthrene that was unaged, aged, or aged and amendedwith Alfonic 810-60 or Novel 1412-56 were 2.9, 7.1, 3.6, and4.6 mg, respectively. Thus, less aged than unaged phenanthrenewas destroyed, and Alfonic 810-60 significantly (p , 0.05)increased the biodegradation of aged phenanthrene. The effectof Novel 1412-56 on the biodegradation of aged phenanthrenewas not statistically significant (p , 0.05). Neither surfactantaffected phenanthrene or DEHP degradation in inorganic saltssolution (data not shown).

The effect of surfactants on the recovery of phenanthreneaged for 58 d in Mount Pleasant silt loam by ethanol:water(45:55, v/v) was tested. The recoveries from soil of unagedphenanthrene, aged phenanthrene, and aged phenanthrene thatwas treated with Alfonic 810-60 or Novel 1412-56 were 27.9,19.6, 21.4, and 22.8%, respectively. These results show thatthe recovery of phenanthrene was significantly reduced by 58d of aging and that both surfactants significantly enhanced theextractability of aged phenanthrene (p , 0.05).

186 Environ. Toxicol. Chem. 18, 1999 J.C. White et al.

DISCUSSION

The data show that the bioavailability of phenanthrene andDEHP sequestered in soil and, in some instances, aquifer solidscan be altered by a number of simple techniques such as slur-rying and the addition of surfactants or competing solutes.Slurrying soil or aquifer solids containing unaged or agedphenanthrene and DEHP dramatically enhanced the rate andextent of biodegradation. Slurrying also increased the biodeg-radation of freshly added phenanthrene by nearly 30%. Thesefindings agree with our previous results that more desorption-resistant phenanthrene was metabolized in slurried than in un-slurried soil [10]. Similarly, Rittman and Johnson [11] ob-served that the rates of degradation of lubricating oil were 10times faster in slurry reactors containing soil than in plots ofsoil, and Mueller et al. [12] noted more rapid and extensivedegradation of pentachlorophenol and 42 creosote constituentsin slurried than in unslurried soil. When unslurried soil inwhich the mineralization of phenanthrene had slowed was ei-ther reinoculated or slurried and reinoculated, more aged sub-strate was degraded than if the samples were not reinoculated.This suggests that, although not having to compete for nutri-ents with other organisms, the activity of the bacterial popu-lation had declined appreciably even when available substrateremained. The reason for the stimulatory effect of slurryingis uncertain, but the agitation improves dispersion of the sub-strate, bacteria, and nutrients, and it increases aeration anddisrupts aggregates.

The reason that the nondegradable hydrocarbon, namelypyrene or anthracene, increased the biodegradability and ex-tractability of sequestered phenanthrene is uncertain, but ev-idence exists to support the hypothesis of competitive dis-placement. Stuart et al. [13] and Abdul and Gibson [14] ob-served the competitive sorption of polycyclic aromatic hydro-carbons in soil. Pignatello and coworkers showed that pairsof halogenated hydrocarbons [15], chlorinated benzenes [16],and triazine herbicides [17] sorb competitively to soils, andthat addition of a competing solute could displace a fractionof a previously sorbed compound (1,2-dibromoethane) backinto solution [15]. Similarly, McGinley et al. [18] observedthe competitive sorption of tetrachloroethylene, 1,4-dichlo-robenzene, and 1,2,4-trichlorobenzene in soil.

Competitive sorption is related to physical and chemicalcharacteristics of soil organic matter. Young and Weber [19]described the sorption of organic chemicals to soil as a processthat occurs in a combination of amorphous, flexible, and con-densed microcrystalline regions of organic matter, and theystated that soil that has been subjected to diagenetic alterationswill be increasingly dominated by condensed and microcrys-talline organic matter. Diagenesis has been described as thethermal maturation of amorphous sedimentary organic matterinto increasingly aromatic and cross-linked structures [20], andcompetitive sorption may be extensive in such organic matter.McGinley et al. [21] observed competitive effects in a dia-genetically old soil but not in an immature soil rich in amor-phous organic matter.

Xing et al. [17] and Xing and Pignatello [16] hypothesizedthat the properties of organic matter vary in a continuum fromrubbery (loose, flexible) to glassy (condensed, rigid), and thatcompetitive effects between cosolutes increase with the glassycharacter. They observed competitive sorption between pairsof organic compounds in glassy polymers. Glassy polymerscontain nanometer-size voids in which site-specific sorption

can take place. Such voids are fleeting or nonexistent in rub-bery polymers. With time, phenanthrene molecules presum-ably diffuse into these regions of condensed, glassy organicmatter where sorption is site-specific. When pyrene or an-thracene molecules are introduced in abundance, they alsodiffuse into these regions of organic matter and competitivelydisplace phenanthrene from these finite number of sorptionsites within the soil.

The nonionic surfactant Alfonic 810-60 but not Novel1412-56 significantly enhanced the biodegradation of agedphenanthrene and DEHP in soil. Aronstein and Alexander [22]showed that the addition of nonionic surfactants enhanced thedegradation of phenanthrene and biphenyl sorbed to soil andaquifer solids. It has been suggested that surfactants enhancebiodegradation by solubilizing or emulsifying the sorbed com-pound [23] or that surfactants above the critical micelle con-centration enhance intrasorbent diffusion, thereby increasingavailability [24].

These observations show that the biodegradability of se-questered organic chemicals can be increased by a number ofsimple methods. In view of the enormous cost entailed in usingcurrent technologies for remediation of the large number ofcontaminated sites, the utility of the methods described hereto promote the bioremediation of aged compounds in contam-inated sites is worthy of further investigation.

Acknowledgement—This research was supported by funds providedby the National Institute of Environmental Health Sciences, traininggrant ES07052, and the Air Force Office of Scientific Research, grantF49620-95-1-0336.

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