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Environmental Toxicology and Chemistry, Vol. 18, No. 8, pp. 1728–1732, 1999q 1999 SETAC

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

INCREASE IN BIOAVAILABILITY OF AGED PHENANTHRENE IN SOILS BYCOMPETITIVE DISPLACEMENT WITH PYRENE

JASON C. WHITE,†‡ MARGARET HUNTER,† JOSEPH J. PIGNATELLO,*† and MARTIN ALEXANDER‡†Department of Soil and Water, Connecticut Agricultural Experiment Station, P.O. Box 1106, New Haven, Connecticut 06504, USA

‡Institute of Comparative and Environmental Toxicology and Department of Soil, Crop, and Atmospheric Sciences,Cornell University, Ithaca, New York 14583, USA

(Received 14 April 1998; Accepted 11 September 1998)

Abstract—Competitive sorption to natural solids among mixtures of organic compounds has been documented in the literature.This study was conducted to determine co-solute competitive effects on the biological and physical availability of polycyclicaromatic hydrocarbons in soils after long contact periods (aging). Sterile suspensions of Mount Pleasant silt loam (Mt. Pleasant,NY, USA) and Pahokee peat soils were spiked with phenanthrene and allowed to age for 3 or 123 d before inoculation with aphenanthrene-degrading bacterium in the presence or absence of the nonbiodegradable co-solute pyrene. As expected, mineralizationdecreased with aging in the samples not amended with pyrene. However, addition of pyrene just prior to inoculation at 123 dsignificantly mitigated this decrease; that is, the extent of mineralization was greater in the 123-d pyrene-amended samples thanin the 123-d nonamended samples. Parallel experiments on sterile soils showed that pyrene increased the physical availability ofphenanthrene by competitive displacement of phenanthrene from sorption sites. First, the addition of pyrene increased recovery of123-d-aged phenanthrene by mild solvent extraction. Second, addition of pyrene (at three concentrations) dramatically reduced theapparent distribution coefficient ( ) of several concentrations of 60-, 95-, and 111-d-aged phenanthrene. At the lowest phenanthreneappKd

and highest pyrene concentrations, reductions in the of phenanthrene in the peat soil reached 83%. The competitive displacementappKdeffect observed in this study adds further support to the dual mode model of sorption to soil organic matter. The displacement ofan aged contaminant by a nonaged co-solute might also prove useful in the development of novel remediation strategies.

Keywords—Competition Aging Bioavailability Sorption Displacement

INTRODUCTION

The long-term contact of hydrophobic compounds with soiloften leads to progressive immobilization or sequestration ofthe compounds with time because of physical sorption pro-cesses. This has important consequences for the natural atten-uation [1,2], toxicity [3,4], and bioremediation of such com-pounds [5]. Several recent studies have shown that sorptionof hydophobic compounds in soils and soil organic matter(SOM) is subject to co-solute competitive effects [6–10]. Wesought to determine whether competitive effects could influ-ence the bioavailability of contaminants, especially after longperiods of contact (aging) in soil.

In multisolute systems a mutual suppressive effect on sorp-tion can occur when the co-solutes compete for a limited num-ber of specific sorption sites. SOM is the dominant sorbent ofhydrophobic compounds in soils, yet the classical solid-phasedissolution, or partition model, for SOM predicts an absenceof competition [11,12]. To explain competition and other non-ideal behaviors, Xing and Pignatello proposed a dual modemodel [13,14] that presumes that concurrent partition and ad-sorption-like mechanisms can take place in SOM. The latteris subject to competitive effects. In this polymer-based model,SOM is viewed as having a gradation of physical domainsfrom highly rubbery to highly glassy in character. The less

* To whom correspondence may be addressed(jpignat@caes.state.ct.us).

Presented at the 218th Meeting of the American Chemical Society,Las Vegas, NV, USA, September 7–11, 1997.

flexible, less solvated glassy domain is believed to contain aninternal, quasi-permanent nanopore structure that provides spe-cific sorption sites in which competition can occur. Weber andcoworkers [15–19] have proposed a similar model in whichSOM consists of hard and soft domains; this model predictsnonlinear sorption and competitive effects in relation to bind-ing within the rigid or crystalline regions of organic matterthat form during diagenesis of the soil. Although some reportsof competition involve polar or ionizable compounds that cansorb through functional group interactions [6,7], competitionhas also been observed between apolar compounds, such aspolycyclic aromatic hydrocarbons (PAHs) [8], alkyl-substi-tuted benzenes [9], and halogenated aliphatic hydrocarbons[10,15,16], that cannot interact specifically.

To date, studies examining competition in multisolute sys-tems have used relatively short equilibration times. However,at field sites (where mixtures are common) contaminants havebeen in contact with the soil ordinarily for long periods andcan be extensively sequestered. This study seeks to examinethe competitive displacement of aged phenanthrene by pyrene.Phenanthrene is a three-ring PAH, and pyrene is a four-ringanalog of phenanthrene. We hypothesized that these com-pounds would compete for some sites in SOM. This study wasprompted by the findings of White et al. [20], who observedenhancement of phenanthrene biodegradation on addition ofpyrene and speculated that the enhancement could be due tocompetitive displacement. White et al. [20] showed that Pseu-domonas strain R used in that study and in the present one isunable to metabolize pyrene, making pyrene useful in exper-

Competitive displacement of phenanthrene Environ. Toxicol. Chem. 18, 1999 1729

iments involving competition for sites in soil. This study wasconducted in conjunction with the one reported in anotherpaper [21] that showed a direct correlation between bioavail-ability and the rate of desorption of phenanthrene from soilparticles as a function of the aging period.

MATERIALS AND METHODS

Materials

A description of the soils, Mount Pleasant silt loam (5.1%organic carbon), Pahokee peat soil (43.9% organic carbon),the sterilization technique, and the sources of the chemicalsare given in a previous paper [21]. The bacterium used wasPseudomonas strain R, isolated from soil by Rebecca Ef-roymson [22]. In all experiments, the aqueous phase was aninorganic salts solution [21]. For physical availability studies,the salts solution was amended with 200 mg/L of HgCl2 tomaintain sterility. A concern that sterility was lost because ofprecipitatation of mercury with phosphate [21] turned out tobe unfounded, as high recoveries of radioactivity (98.8 67.3%) were obtained after all aging periods. In the adjoiningpaper [21], we confirmed by gas chrematography(GC) that all(101%) of the radioactivity recovered from samples of peataged up to 100 d was attributable to phenanthrene.

Biodegradation

Phenanthrene was applied to each soil sample uniformlyby desorption from a coated flask [21]. The ratio of soil towater was 1:10, and the phenanthrene concentration was 2mg/g (1 3 105 dpm/g). The samples were placed in glass vialsor bottles with Teflont-lined closures (VWR, Canlab, Missis-sauga, ON, Canada) and stored at 218C for 3 or 123 d of aging.After aging, subsamples of peat (11.2 g wet, 5.0 g dry) or siltloam (9.2g wet, 5.0 g dry) were transferred to 250-ml Erlen-meyert flasks containing 50 ml of the sterile salts solution.Half the subsamples were amended with 250 mg of pyrene in50 ml of methanol(less than 1% of pyrene’s water solubilityafter sorption) and half with 50 ml methanol to serve as con-trols. Pseudomonas strain R was added (6 3 108 cells perflask), and the evolution of 14CO2 was determined as describedbefore [20]. After mineralization had stopped, the slurries wererigorously extracted to quantify undegraded parent phenan-threne by high-performance liquid chromatography (HPLC)[20].

Extractability

Samples of silt loam and peat containing phenanthrene thathad been prepared and aged in the same manner as those inthe biodegradation experiments were transferred to 50-ml Tef-lon centrifuge tubes. Half the 123-d-aged samples received250 mg of pyrene in 50 ml of methanol and half received just50 ml of methanol. The samples were then subjected to se-quential extractions. Each tube received 25 ml of an ethanol:water (45:55, v/v) solution, and the tubes were incubated ona rotary shaker operating at 75 rpm at 308C for 24 h. Thetubes were then centrifuged at 48C for 10 min at 12,000 g,and a 1-ml portion of the supernatant was sampled for deter-mination of radioactivity by liquid scintillation counting. Thesoils were further extracted by shaking with 25 ml of n-butanolfor 24 h at 308C. Finally, 25 ml of hexanes was added, andthe soils were shaken for 72 h at 308C.

Sorption

The effect of co-solute addition on the sorption of seques-tered phenanthrene in Pahokee peat was studied using bottleswith crimp-cap closures containing a soil-to-water ratio of 0.1g soil to 160 ml solution. Replicates of peat samples wereamended with phenanthrene at a rate of 0.005 and 0.17 mg/Lof solution (6.0 3 106 dpm/g) added in methanol as carrier.The carrier volume never exceeded 0.1% of the total volumeof solution. The uptake rate of phenanthrene was measured inone set of samples during 99 d of aging by periodically cen-trifuging the suspension (750 g, IEC Model UV centrifuge,Needham Heights, MA, USA) and sampling the aqueousphase. At 95 d, half the samples were amended with pyreneat a rate of 2 mg/L. After an additional 4 d of incubation, theaqueous phase was sampled. A second set of samples was agedfor 111 d. At 111 d, some of the replicates were amended withpyrene at the rate of 1 or 10 mg/L. After an additional 5 d ofaging, the aqueous phase was sampled. Liquid samples wereassayed for radioactivity by scintillation counting.

Sorption experiments for the silt loam were conducted sim-ilarly. In this case, aging was carried out in flame-sealed am-poules containing 0.1 g soil and 5 ml solution—a larger soil-to-solution ratio than in the case of peat because of the weakersorption of phenanthrene. Replicates were amended with phen-anthrene at 0.005, 0.17, and 0.39 mg/L of solution, and theampoules were placed on a rotary shaker operating at 75 rpm.After 53 d of aging, the ampoules were cracked open and theslurries transferred to 7-ml glass vials with Teflon-lined screwcaps. An additional 1 ml of solution was used to rinse eachof the ampoules, leaving a total aqueous volume of 6 ml ineach vial. After an additional 7 d, half the vials were amendedwith pyrene at the rate of 10 mg/L of solution. The vials wereallowed to incubate for 5 d, and thereafter the amount of[14C]phenanthrene in solution was determined by scintillationcounting.

RESULTS

Biodegradation

The mineralization of phenanthrene (2 mg/g) in MountPleasant silt loam or Pahokee peat soil was measured after anaging period of 3 or 123 d. In both soils, the rate and extentof mineralization declined significantly with increasing age ofthe sample (Fig. 1). However, the addition of pyrene (50 mg/g)in large excess over phenanthrene at the time of inoculationsignificantly increased the extent of mineralization of the 123-d-aged phenanthrene as compared to 123-d-aged phenanthrenein samples not amended with pyrene. The percentage of initialphenanthrene recovered by vigorous solvent extraction aftermineralization had stopped increased with aging but at thesame aging time decreased in the presence of pyrene. Therecovered phenanthrene from the silt loam was 6.8% (3-d aged,without pyrene), 13.6% (123-d aged, with pyrene), and 20.5%(123-d aged, without pyrene). The recovered phenanthrenefrom the peat was 9.3% (3-d aged, without pyrene), 16.0%(123-d aged, with pyrene), and 31.0% (123-d aged, withoutpyrene).

Extractability

Phenanthrene was extracted from the soils by solvents ofsequentially higher solvating power with respect to nonpolarcompounds (45:55 ethanol:water, n-butanol, and hexane). Thefirst extraction was carried out by shaking samples for 24 h

1730 Environ. Toxicol. Chem. 18, 1999 J.C. White et al.

Fig. 1. Mineralization of 3- or 123-d-aged phenanthrene (2 mg/g) inMount Pleasant silt loam or Pahokee peat soil amended or not amend-ed with pyrene (50 mg/g) prior to inoculation.

Fig. 2. (a) The effect of pyrene (initially 2 mg/L) added at 95 d onthe of phenanthrene (initially 0.0052 mg/L) in Pahokee peat soil.appKd

(b) The effect of pyrene (initially 2 mg/L) added at 95 d on theof phenanthrene (initially 0.17 mg/L) in Pahokee peat soil.appKd

Table 1. Effect of aging and pyrene addition on the cumulativerecovery of phenanthrene from Mount Pleasant silt loam and Pahokee

peat by solvent extractants of increasing strength

Agingtime (d)

Addedpyrene(mg/g)

Cumulative % recovery underextraction conditions

45:55ethanol : water Butanol Hexanes

Silt loam3

123123

00

50

24.0 Aa

18.4 B31.9 C

103 A88.3 B90.9 B

103 A101 A103 A

Peat3

123123

00

50

0.69 A0.61 A0.75 A

49.4 A32.9 B39.9 C

106 A100 A104 A

a Within soil types and for the same extractant, values (average oftriplicate samples) in a column followed by the same letter are notsignificantly different (p , 0.05).

with the ethanol-water extractant containing or not containingpyrene. Calculations showed that during this extraction almostall the pyrene was sorbed and thus was predisposed to com-petitively displace phenanthrene if that were possible. Usingpublished values of pyrene solubility in ethanol-water mixtures[23] and a linear free energy relationship between the organic-matter-based distribution coefficient Kom (L/kg) and water sol-ubility (mole/L) for PAHs [24], Kom is estimated to be about1,000 L/kg for the ethanol-water mixture used, correspondingto a soil-water distribution coefficient (Kd) of 80 L/kg for thesilt loam and 930 L/kg for the peat. Thus, the fraction of totaladded pyrene sorbed to the soil in these experiments exceeded93% for the silt loam and 99% for the peat.

The stepwise cumulative recoveries obtained for the se-quential extractions are given in Table 1. The effects of com-petition are most clearly manifested at an intermediate recov-ery of phenanthrene. Such recovery was obtained in the first

extraction (ethanol-water) for the silt loam and after the secondextraction (n-butanol) for the peat. In those cases, the recoveryof phenanthrene was significantly decreased after 123 d ofaging compared to 3 d of aging (p , 0.05); moreover, therecovery of 123-d-aged phenanthrene was significantly greaterwhen pyrene was added to the ethanol-water than when it wasabsent (p , 0.05). The same trends were observable at lowerrecovery (peat in ethanol-water) or higher recovery (silt loamin n-butanol), but the differences were not statistically signif-icant. The final extraction with hexane resulted in quantitativeoverall recovery in all cases.

Sorption

The uptake of phenanthrene by the peat was followed dur-ing a 99-d period. During the first 95 d, the sorption of phen-anthrene added initially at a rate of 0.005 or 0.17 mg/L in-creased with time; this is shown in Figure 2a and b by anincrease in the apparent soil-water distribution coefficient

Competitive displacement of phenanthrene Environ. Toxicol. Chem. 18, 1999 1731

Table 2. Effect of unaged pyrene on the apparent distribution coefficient of aged phenanthrene in Mount Pleasant silt loam and Pahokee peatsoils

SoilAging time

(d)Added phenanthrene

(mg/L)Added pyrene

(mg/L)Kapp

d

(mol·Kg21/mol·L21)aPercentage reduction inK relative to controlapp

d

Silt loam 60

60

60

0.00550.00550.170.170.390.39

010

010

010

2,530844

1,600707

1,220640

—66.6—

55.8—

47.5Peat 95

111

95

111

0.00520.00520.00520.00520.170.170.170.170.17

020

100201

10

96,60024,60099,70016,70034,40018,80035,50020,00011,400

—74.5—

83.3—

45.3—

43.767.9

a Within groups of equal phenanthrene concentration and aging time, values are significantly different at p , 0.05 or better.

( ), the single-point ratio of sorbed to aqueous-phase con-appKd

centration of phenanthrene. At 95 d, some of the samplesreceived pyrene added at the rate of 2 mg/L of solution. Theintroduction of pyrene caused a dramatic reduction in appKd

during the following 4 d, whereas no change occurred in thesamples receiving only the methanol carrier. The reductionsin were 74.6% for the 0.005-mg/L samples and 45.2% forappKd

the 0.17-mg/L samples. It should be noted that the values ofare not corrected for possible sorption to colloids or dis-appKd

solved organic matter in the aqueous phase; however, all sam-ples were treated identically, and it is unlikely that pyreneaffected the colloid or dissolved organic matter concentrations.

Data for the previous experiment and for several others inwhich the uptake was not monitored over time are given inTable 2. Pyrene was added at up to 10-mg/L of solution; judg-ing from the soil-to-water ratio and the published values ofKom, this resulted in aqueous-phase pyrene concentrations atleast 70% below its water solubility. The generally de-appKd

creased with increasing phenanthrene concentration, all otherconditions being constant. This is due to the nonlinearity ofsorption for phenanthrene in these soils such that sorptionaffinity decreases with increased loading. For both soils at allaging times and at all concentrations tested, the addition ofpyrene dramatically increased the amount of aged phenan-threne in solution, thereby reducing the single-point . InappKd

general, the competitive effect increased with increasing py-rene concentration and with decreasing phenanthrene concen-tration. The maximum observed reductions in were 67%appKd

for the silt loam and 83% for the peat.

DISCUSSION

This study shows that addition of nondegradable pyrenecan significantly enhance the biodegradability of aged phen-anthrene. As indicated by the sorption and extractability ex-periments, the important finding of the present study is thatthis enhancement is almost certainly a result of competitivedisplacement of phenanthrene from sorption sites in the soil.To our knowledge, this phenomenon has not been previouslyreported and thus represents a novel link between the physicaland biological availabilities of substrates in soils. In addition,the results here provide additional support for the dual modemodel of sorption of hydrophobic compounds to soil organicmatter [14,25].

The effect of pyrene on the apparent distribution coefficient( ) of aged phenanthrene in both soils was dramatic, rangingappKd

from 44 to 83% reduction with respect to the appropriate con-trol without pyrene. The greatest reductions occurred whenconcentrations of phenanthrene were minimized and those ofpyrene were maximized. The findings of competitive effectsare in line with those of Pignatello [10], who demonstratedcompetition between pairs of halogenated hydrocarbons insoils, including the competitive displacement into solution ofa previously sorbed compound; of Abdul and Gibson [8] andStuart et al. [9], who observed competitive sorption of PAHsto soil and aquifer solids; of McGinley et al. [16], who ob-served competition between tetrachloroethylene and trichlo-robenzene; of Xing et al. [25], who observed competition be-tween atrazine and several of its analogs in soils and modelsorbents; and of Xing and Pignatello [26], who described com-petitive effects between 1,2-dichlorobenzene or 2,4-dichloro-phenol and several aromatic acids naturally occurring in soils.

According to the dual mode hypothesis, the sites wherecompetition takes place are nanometer-size voids located with-in the glassy domain. De Jonge and Mittelmeijer-Hazeleger[27] and Xing and Pignatello [14] studied the adsorption ofCO2 to SOM and concluded that organic matter has a poly-merlike structure with 95 to 99% of its surface area in poreswith size restrictions below 1 nm. Presumably, compoundshaving overlapping affinities for a subset of these voids willexhibit competitive effects. Schlautman and Morgan [28] pro-posed that the binding of PAHs to humic substances wasstrongly influenced by the steric characteristics of the moleculethat controlled its ability to fit into the hydrophobic cavitiesof the organic structure. Xing et al. [25] showed that com-petitive effects are absent in rubbery polymers and greatlyreduced in humic acid particles, which have less nanoporositythan the parent SOM from which the humic acid was extracted.

It is hypothesized that, with time, phenanthrene moleculesdiffuse into glassy organic matter. When an excess of pyreneis added to the soil, these molecules also diffuse into thesedomains and displace phenanthrene from a finite number ofsites located there. Further studies are necessary to determinethe structural properties of the competitor that influence itsactivity, the degree of competition in relation to the structureof the soil and the soil organic matter, and exactly how thecompetitive process changes with aging time.

1732 Environ. Toxicol. Chem. 18, 1999 J.C. White et al.

The findings presented in this paper are important becausethe existence of such marked competitive effects also stronglysupports the dual mode model for sorption of hydrophobicchemicals to SOM. Also, given the likelihood of finding thecontaminants at hazardous waste sites in a sequestered stateand given the drastic reduction in apparent distribution coef-ficients ( ) observed here, the concept of competitive dis-appKd

placement by innocuous co-solutes is worthy of further studyfor potential use in remediation technologies. Such studies arecurrently under way.

Acknowledgement—Support for this research was provided by Train-ing Grant ES07052-19 from the National Institute of EnvironmentalHealth Sciences, by the U.S. Department of Agriculture National Re-search Initiative 97-35102-4201, and by the U.S. Environmental Pro-tection Agency/National Science Foundation/Department of Energy/Office of Naval Research Joint Program on Bioremediation R 825959-01-0.

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