Correlation between biological and physical availabilities of phenanthrene in soils and soil humin...

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Environmental Toxicology and Chemistry, Vol. 18, No. 8, pp. 1720–1727, 1999q 1999 SETAC

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

CORRELATION BETWEEN BIOLOGICAL AND PHYSICAL AVAILABILITIES OFPHENANTHRENE IN SOILS AND SOIL HUMIN IN AGING EXPERIMENTS

JASON C. WHITE,†‡ MARGARET HUNTER,† KYOUNGPHILE NAM,‡ JOSEPH J. PIGNATELLO,*† andMARTIN ALEXANDER‡

†Department of Soil and Water, Connecticut Agricultural Experiment Station, P.O. Box 1106, New Haven, Connecticut 06504, USA‡Institute for Comparative and Environmental Toxicology and Department of Soil, Crop, and Atmospheric Sciences,

Cornell University, Ithaca, New York 14853, USA

(Received 14 April 1998; Accepted 11 September 1998)

Abstract—The bioavailability of an organic compound in a soil or sediment commonly declines with the soil-chemical contacttime (aging). A series of parallel desorption and bioavailability experiments was carried out on phenanthrene previously aged upto ;100 d in Mount Pleasant silt loam (Mt. Pleasant, NY, USA) or Pahokee peat soil to determine as a function of the aging periodthe degree of correlation between the reduction in bioavailability and the rate and extent of desorption and the influence of soilorganic matter composition on availability. The mineralization of phenanthrene by two bacteria and the uptake of phenanthrene byearthworms showed expected declines with aging. Likewise, the rate of phenanthrene desorption in the absence of organismsdecreased with aging. The decline in initial rate of mineralization or desorption was nearly an order of magnitude after 50 to 60d of aging. Plots of normalized rates of mineralization or desorption practically coincided. Similarly, plots of normalized fractionmineralized or fraction desorbed during an arbitrary period gave comparable slopes. The partial removal of organic matter fromthe peat by extraction with dilute NaOH to leave the humin fraction reduced the biodegradation of phenanthrene aged for 38 and63 d as compared to the nonextracted peat, but the effect disappeared at longer incubation times. The rate of desorption fromsamples of peat previously extracted with NaOH or Na4P2O7 declined with aging and, for a given aging period, was significantlyslower than from nonextracted peat. This work shows that the reduction in bioavailability of phenanthrene over time in soil isdirectly correlated with reduction of its physical availability due to desorption limitations. In addition, this study shows that removalof extractable humic substances leads to a decline in the rate of desorption and in the bioavailability of the substrate.

Keywords—Sequestration Aging Bioavailability Desorption kinetics

INTRODUCTION

It is well known that certain chemicals that are biodegrad-able in laboratory tests simulating field conditions can persistin the environment for years after their initial release [1–3].This unexpected biological resistance has been termed se-questration [1] because it is thought to be due to a segregationof the chemical in soil particle interstices from the organism.Early studies by Steinberg et al. [4] and Scribner et al. [5]showed that pesticide residues remaining after long times inthe field were biologically less available than pesticides thatwere freshly added. Sequestration might explain the inabilityto achieve complete bioremediation of petroleum hydrocar-bons [6] and other compounds at many contaminated sites.Laboratory studies show that biodegradation of freshly addedchemicals often stops before completion as biodegradation be-comes rate limited by desorption [7,8] and that increasingsolid-chemical contact time results in progressively reducedmineralization by bacteria [9,10], reduced uptake by earth-worms [11], and reduced acute toxicity to insects [12]. More-over, soils or sediments contaminated for years or decades areless toxic or result in lower body burdens than equivalentspiked samples [13–15].

Information about the relation of sequestration to toxicityis of particular interest to regulatory agencies whose soil or

* To whom correspondence may be addressed([email protected]).

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

sediment quality criteria for contaminants often assume 100%bioavailability of the amount of chemical recovered by ex-haustive extraction of the soil or are based on models thatassume equilibrium sorption conditions [16]. Kelsey and Al-exander [11] showed that the decline in the bioavailabilitiesof atrazine, phenanthrene, and naphthalene to bacteria andearthworms in soil as a function of the aging period prior tointroduction of the organisms were not accurately reflected bythe amounts of chemical recovered by exhaustive extraction.Consequently, reliance on exhaustive recovery or equilibriummodels to establish quality criteria could significantly over-estimate the bioavailability of a toxicant as well as the riskposed by a particular contaminated site.

The uptake or release of a chemical by soil often showsslow or very slow stages that represent an appreciable fractionof the total quantity present. This fraction might also resistextraction by less-than-exhaustive techniques. Rate-limitedsorption and its ramifications for solute transport have beenreviewed [2,17–19]. Slow sorption phenomena are usually ex-plained by hindered molecular diffusion within soil organicmatter or in the fixed intraparticle pore network of soil min-erals. Soil organic matter plays a dual role in the sorption ofhydrophobic compounds by providing expanded (soft, rub-bery) regions in which linear, rapidly reversible partitioningcan occur and condensed (hard, glassy) regions in which non-linear, slowly reversible sorption can occur to a heterogeneoussuite of sites [2,20–23]. The pore structure of minerals canprovide steric resistance to molecular diffusion of contami-

Biological and physical availabilities of phenanthrene Environ. Toxicol. Chem. 18, 1999 1721

nants. In addition, a fraction of soil organic matter (and thusof chemical sorbed to it) can be trapped in the pore networkaway from equilibrium with bulk fluid phases [18].

Movement of molecules into inaccessible regions of theorganic and/or mineral matrices is thought to limit the amountof substrate available for degradation or uptake by organisms[1,2,19,24]. Investigators have shown a reduction in biodeg-radation of phenanthrene [25,26] and 4-nitrophenol [25] whenthese compounds were sorbed to a range of porous and non-porous model solids. There have been several attempts to mod-el coupled sorption-biodegradation processes [27–29]. Mostof these studies pertain to substrate disappearance on simul-taneous addition of substrate and active organisms and do notinclude an aging period. However, to date no study has at-tempted to directly correlate the biological and physical avail-abilities of compounds as they become sequestered in naturalsolids over time, and that is the purpose of this work. Wecompared the biological availability of phenanthrene as mea-sured by its mineralization by two bacterial isolates and itsuptake by earthworms with the physical availability of phen-anthrene as determined by its desorption as a function of theaging period. The soils included a peat soil with high organicmatter content and a silt loam with a moderate organic mattercontent. In addition, we compared the effect on biological andphysical availability of phenanthrene of removal of the base-extractable organic matter fraction of the peat, which increasesthe ratio of glassy to rubbery organic matter. In a second paper[30], we examine the competitive displacement of aged phen-anthrene by freshly added pyrene using biological and physicalassays.

MATERIALS AND METHODS

Materials

The soils were a Mount Pleasant silt loam (Mt. Pleasant,NY, USA; pH 6.2, 5.1% organic carbon) and Pahokee peat(pH 6.4, 43.9% organic carbon), a reference material from theInternational Humic Substances Society (University of Min-nesota, St. Paul, MN, USA). The silt loam was air-dried andpassed through a 4-mm sieve. The peat was received dry andpassed through a 0.5-mm sieve. Both soils were sterilized with2.5 Mrad of g radiation from a 60Co source (Ward Laboratories,Cornell University, Ithaca, NY, USA). [9-14C]Phenanthrenewas purchased from Sigma Chemical (St. Louis, MO, USA);the stated radiolabel purity (.98%) was confirmed by gaschromatography (GC) and scintillation analysis (101%). Un-labeled phenanthrene was also from Sigma. The bacteria usedin the bioavailability experiments were either Pseudomonasstrain R or isolate P5-2 isolated from soil by Rebecca Ef-roymson [31] and Wei-chih Tang [32] of Cornell University,respectively. The aqueous phase for all experiments was aninorganic salts solution commonly used to grow bacteria thatcontained 0.1 g CaCl2·2H2O, 0.1 g NH4NO3, 0.049 g MgSO4,0.2 g KH2PO4, 0.8 g K2HPO4, and 0.01 g FeCl3 per liter ofdistilled water. The (unadjusted) pH of the salts solution was7.1. In sorption and desorption studies, the salts solution wasamended with 200 mg/L of HgCl2 to maintain sterility. Aftercompletion of the study we realized that Hg21 precipitated outas mercuric phosphates because a large stoichiometric excessof phosphate ion was present and mercuric phosphate has alow solubility product. Whether sterility was achieved underthis condition is debatable; however, the high recoveries ofradioactivity (95–100%) after all aging periods indicate thatthe possible loss of sterility did not affect the results. Because

most of the mercury was precipitated, the possible effect ofHg21 complexation with soil organic matter on phenanthrenesorption is assumed negligible. (In any case, Xing et al. [20]showed that the Freundlich parameters of metolachlor in a soilwere identical for NaN3 or HgCl2 as chemosterilants.)

Cells were grown at 308C in 100 ml of inorganic saltssolution containing either 40 mg (Pseudomonas strain R) or1 g (strain P5-2) of phenanthrene. After 3 to 7 d, the turbidcultures were centrifuged, and the cells were washed and thenresuspended in the inorganic salts solution prior to their ad-dition to the soils.

Removal of organic matter

Humic acid was removed from the Pahokee peat with 0.1M NaOH or 0.1 M Na4P2O7 as described by Schnitzer [33].Sodium hydroxide is the classic extractant of humic materialsfrom soil. It works by deprotonating acidic functional groups,causing the macromolecules to unfold and dissolve. Sodiumpyrophosphate was suggested later as a more efficient extract-ant; although solutions of it also are alkaline (pH ; 9) andcapable of deprotonating most acidic functional groups, thepyrophosphate ion acts additionally as a chelating agent fordi- and trivalent metal ions complexed to the humic macro-molecules, further promoting dissolution. The extracted ma-terials in both cases are ordinarily defined as humic acids. Theremaining insoluble organic matter is known as humin, al-though differences can exist in the nature of this residue leftby the two extractants.

Briefly, 100 g of peat was mixed with 1 L of extractant ina 2-L flask, and the headspace was displaced with N2. Themixture was placed on a rotary shaker at 120 rpm and 218C.After 48 h, the mixture was transferred to 250-ml centrifugebottles and centrifuged at 28,000 g for 30 min. The extractedsoil was washed with distilled water several times. The pH ofnonextracted, NaOH-extracted, and Na4P2O7-extracted soils(1:1 soil:water ratio) was approx. 6.4, 7.4, and 7.3, respec-tively. The extracted peat samples were oven-dried at 508Cuntil the excess water was removed and then air-dried. Thepercentage organic carbon of NaOH-extracted and Na4P2O7-extracted samples was 41.7% and 33.5%, respectively. Allsamples for mineralization experiments were then sterilizedwith g radiation.

Aging of chemicals

Phenanthrene was applied to each soil sample by the meth-od of Karickhoff et al. [34]. The method involved coating thewalls of a flask with [14C]-phenanthrene dissolved in dichlo-romethane, evaporating the dichloromethane, and then con-tacting the flask with an aqueous soil slurry (soil to deionizedwater ratio of 1:10) for 3 to 4 d. Preliminary experiments withglass beads showed that phenanthrene is phase transferred fromthe glass surface to the soil within this time. The soil phen-anthrene concentration was 2 mg/g, and the radioactivityranged from (1.0–1.2) 3 105 dpm/g of soil for bioavailabilityexperiments and (2.5–3.0) 3 106 dpm/g of soil for sorption/desorption experiments. The samples were placed in Teflont-lined screw cap test tubes or vials and stored at 218C forprescribed times.

Biodegradation

After a given aging period, samples of peat (11.2 g) or siltloam (9.2 g) in triplicate were transferred to 150- or 250-mlErlenmeyert flasks containing 50 ml of sterile salts solution.

1722 Environ. Toxicol. Chem. 18, 1999 J.C. White et al.

Fig. 1. Effect of 3, 14, or 30 d of aging on the mineralization ofphenanthrene by Pseudomonas strain R in Mount Pleasant silt loamand Pahokee peat soils. Error bars represent one standard deviationof triplicates. In all figures, error bars not visible are obscured by thesymbol.

Pseudomonas strain R or isolate P5-2 were inoculated into thesample (about 108 cells per sample), and the samples wereincubated at 218C on a rotary shaker at operating at 100 rpm.Within a set of experiments, all samples received an identicalnumber of cells. The evolved 14CO2 was trapped in 1.5 ml of0.5 M NaOH contained in a vial suspended from a Teflon-taped rubber stopper, and the amount of radioactivity was de-termined by liquid scintillation counting in Liquiscint scintil-lation cocktail (National Diagnostics, Somerville, NJ, USA).

After mineralization had stopped, some of the samples weresubjected to a two-step extraction procedure to quantify theamount of undegraded phenanthrene [10]. Briefly, the solidswere mixed vigorously with n-butanol for 2 min, and the mix-ture was then passed through Whatman No. 1 filter paper. Thesoil remaining on the filter was extracted for 3 h in a Soxhlettextractor with 90 ml of hexanes and 2 ml of n-butanol. Thehexanes were removed with a rotary evaporator, and the bu-tanol extracts were combined. The combined extracts werereduced to less than 5 ml and passed through a 0.2-mm syringefilter (Millex-FG13, Millipore, Bedford, MA, USA) in prepa-ration for liquid chromatographic (LC) analysis [10].

Earthworm uptake

Triplicate samples of silt loam (18.4 g, g irradiated) con-taining phenanthrene aged for 3, 14, or 30 d were placed in50-ml test tubes, and three earthworms (Eisenia foetia) wereadded. After an 8-d exposure, the worms were removed andstored live for 24 h on filter paper to ensure complete elimi-nation of soil from the gut. The worms were then frozen withliquid nitrogen, ground with a mortar and pestle, and Soxhlet-extracted with 90 ml of hexanes. The extracts were analyzedby LC for phenanthrene using the same method as that forsoil analysis.

Desorption

Samples of peat (0.15–0.25 g) in 20 ml of solution wereprepared in duplicate except for the 3- and 11-d samples oforiginal peat, which were single replicates. The desorption ofphenanthrene was carried out in the presence of a polymericsorbent (Tenax, 20/35 mesh, Supelco, Bellefonte, PA, USA)as a third-phase sink for phenanthrene [18,24]. After a givenaging period, samples were transferred to 30-ml separatoryfunnels with Teflon stopcocks (VWR, Canlab, Mississauga,ON, Canada). Then 0.1 g of Tenax was added, and the funnelswere sealed with Teflon-lined septa in 22-mm crimp-cap clo-sures. The funnels were mixed end-over-end on a Glas-Colrugged rotator (Glas-Col, Terre Haute, IN, USA) at a rate ofabout 30 rpm. Periodically, the slurry was transferred to a newseparatory funnel containing new Tenax [24]. The remainingused Tenax was rinsed into a glass vial and extracted withhexane or acetonitrile, the latter more effectively removing theTenax from the funnel.

We confirmed the findings of others [35] that sorption ofphenanthrene to Tenax is rapid (within hours) and similar inmagnitude on a mass basis to soil organic matter. The Tenaxbehaves essentially as an infinite sink because the soil-waterdistribution of phenanthrene is always far out of equilibriumand because the Tenax is replaced numerous times in a de-sorption experiment (every time a point is taken). However,to verify that Tenax is acting as an infinite sink, a study wasperformed to determine the effect of two different amounts ofTenax (0.1 and 1.0 g) on the release of phenanthrene fromsoil. The mass of phenanthrene trapped at short desorption

times (,24 h) was somewhat greater using 1.0 g Tenax, butthe mass trapped after 4 d of desorption was independent ofthe amount of Tenax used. The difference observed at shorttimes is likely due to the much greater difficulty of separatingsoil from Tenax when the larger amount was used.

At the end of desorption, soils were extracted with aceto-nitrile at 708C for 4 h. The average recovery was 97.8 6 2.5%14C. Moreover, a spot check by GC of seven randomly selectedextracts (aged 3–100 d) indicated that all (101 6 14%) of therecovered radioactivity was attributable to phenanthrene. Thisis important because it verifies the biotic and abiotic integrityof phenanthrene during the sorption/desorption cycle and en-sures that radioactivity is a valid surrogate for phenanthreneitself in these experiments.

RESULTS

Effect of aging on biodegradation

The mineralization of phenanthrene aged in Mount Pleasantsilt loam or Pahokee peat for 3, 14, or 30 d by Pseudomonasstrain R was measured. In each soil, both the initial rate andthe extent of mineralization declined with aging (Fig. 1). Fur-thermore, the percentage of initial phenanthrene recovered bysolvent extraction after mineralization had stopped decreasedwith aging: that is, the percentage of phenanthrene aged for3, 14, or 30 d that was recovered after biodegradation in thesilt loam was 6.9, 28.4, and 35.8%, respectively (significantlydifferent at p , 0.05), and in the peat was 9.3, 18.8, and 35.5%,respectively (significantly different at p , 0.05).

Similarly, the mineralization by strain P5-2 of phenanthreneaged for 3, 38, 63, or 103 d in samples of Pahokee peat wasdetermined. Both the rate and the extent of mineralization ofphenanthrene declined significantly (p , 0.05) with aging (Fig.2). Thus, the effect of aging on biodegradation occurs for bothbacteria.


Fig. 2. Mineralization of aged phenanthrene in Pahokee peat soil bysoil isolate P5-2. Error bars represent one standard deviation of trip-licates.

Fig. 3. (A) Desorption of phenanthrene from Mount Pleasant silt loamafter 3 or 49 d of aging. (B) Desorption of phenanthrene from Pahokeepeat soil after 3, 11, 35, 60, or 100 d of aging. Error bars representrange of duplicates.

Effect of aging on uptake by earthworms

The effect of aging on earthworm uptake in Mount Pleasantsilt loam was assessed. The percentages of phenanthrene agedfor 3, 14, or 30 d that was taken up by earthworms during an8-d exposure were 6.1, 4.3, and 3.8%, respectively. The per-centage uptake of phenanthrene was decreased after 14 d ofaging (significant at p , 0.05), but no significant differencein uptake was observed between samples aged for 14 or 30 d.An earthworm assay could not be done with the peat becausethe worms would not burrow into the soil or would return tothe surface if deliberately buried.

Effect of aging on desorption

The course of phenanthrene desorption was followed underconditions of continuous phenanthrene removal from the aque-ous phase, and the results are shown in Figure 3A and B. Thetwo soils behaved similarly. The initial rate of desorption de-creased as the aging period increased. At long desorption timesthe rates generally dropped to very low values well beforedesorption was complete. The transition from rapid to veryslow desorption became more gradual with increasing aging.The results show that a fraction of the phenanthrene was pro-duced in a strongly resistant state. The strongly resistant frac-tion increased with aging but was appreciable even for theshortest aging period (3 d). In the peat soil the strongly resistantfraction remaining after 63 d of continuous desorption was 20,26, 38, 55, and 58% of initially sorbed phenanthrene for 3,11, 35, and 100 d of aging, respectively. Likewise, in the siltloam the strongly resistant fraction after 30 d of desorptionwas 11 and 35% for 3 and 48 d of aging, respectively.

Correlation between biological and physical availability

The correlation between microbial bioavailability and phys-ical availability as a function of aging time is shown in Figures4A and B and 5A and B. In Figure 4A and B, bioavailabilityis taken to be the fraction of 14C mineralized within an arbitrary

period of 21 d of incubation. Likewise, physical availabilityis taken to be the fraction desorbed to an infinite sink within21 d. Because the fraction of total 14C utilized by cells that isconverted to 14CO2 is unknown, the bioavailability data havebeen normalized by setting the fraction mineralized at theshortest aging period (3 d) equal to the fraction desorbed dur-ing that same period. Figure 5A and B shows the normalizedinitial rate of biodegradation or desorption as a function ofaging time. In this case, initial rate was determined on thebasis of the first ;20% of added phenanthrene mineralized ordesorbed. The initial rates were then normalized to the initialrate for the 3-d aging period of the silt loam soil (set equal to1.0).


Fig. 4. The fraction of phenanthrene desorbed to an infinite sink in21 d or mineralized by bacterial degraders within 21 d as a functionof the aging period. Error bars represent propagated error.

Fig. 5. The normalized initial rate of biodegradation or desorption toan infinite sink as a function of the aging period.

The combined results of Figures 4 and 5 show that theeffect of aging on biodegradation is quantitatively similar tothe effect of aging on desorption and that these changes aregreater in the silt loam than in the peat.

The soil humin fraction

Samples of peat were extracted with NaOH or Na4P2O7 toremove the humic acid fraction of the organic matter, leavingthe base-insoluble humin behind. To test whether extractionresulted in conditions at the solid phase that were toxic tocells, [14C]glucose (2.01 mg/g, 1 3 105 dpm) and soil isolateP5-2 were added to samples of extracted or nonextracted peat.The mineralization of glucose was rapid and extensive in allsamples (Fig. 6). Although the rate of mineralization in theNaOH-extracted peat was slightly reduced during the 2- to 8-dperiod after inoculation as compared to nonextracted peat, theextent of mineralization after 20 d (the minimum time usedto measure phenanthrene degradation) was only slightly af-fected by treatment.

The extents of mineralization of phenanthrene aged for 3,38, 63, or 103 d in extracted or nonextracted peat are presented

in Table 1. Extraction had no effect on the mineralization ofnonaged phenanthrene, and this can be taken as a further in-dication of the absence of a toxic effect of the soil due to theextraction process. In all materials, the extent of mineralizationdeclined with aging. At 38 and 63 d of aging, mineralizationin NaOH-extracted peat was significantly less than minerali-zation in nonextracted or Na4P2O7-extracted peat, but the dif-ference disappears after 103 d of aging. Interestingly, the ex-tent of mineralization of 103-d-aged phenanthrene is greaterin Na4P2O7-extracted peat than in nonextracted peat.

Both the rate and the extent of desorption of phenanthrenefrom peat extracted with NaOH or Na4P2O7 decreased withincreasing aging time (Fig. 7A and B) in a similar manner tothe nonextracted peat described in the section ‘‘Effect of Agingon Desorption’’. The fraction of phenanthrene that stronglyresisted desorption after 60 d of continuous desorption fromthe NaOH-extracted soil was 36, 49, 69, and 72% of initiallysorbed phenanthrene for 3, 35, 60, and 100 d of aging, re-spectively; likewise, the same fraction in the Na4P2O7-extractedsoil was 37, 52, 53, and 69% for the same aging times, re-spectively. Comparison of Figure 7A and B with Figure 3Breveals that for a given aging period the initial rate of de-


Fig. 6. Mineralization of glucose added to extracted or nonextractedPahokee peat soil by isolate P5-2. Error bars represent one standarddeviation of triplicates.

Fig. 7. (A) Desorption of phenanthrene from nonextracted or NaOH-extracted peat soil after 3 or 3, 35, 60, and 100 d of aging, respectively.(B). Desorption of phenanthrene from nonextracted or Na4P2O7-treatedpeat soil after 3 or 3, 35, 60, and 100 d of aging, respectively. Errorbars represent range of duplicates.

Table 1. Extents of mineralization of aged phenanthrene by soil isolateP5-2 in Pahokee peat humin

Treatment

% Mineralizeda

3 db 38 d 63 d 103 d

No treatmentNaOHNa4P2O7

47.1 Aa42.8 Aa44.1 Aa

26.2 Ba17.4 Bb31.3 Ba

26.7 Ba16.4 Bb23.0 Ca

16.1 Ca13.3 Babc

25.4 Cbc

a Values followed by different uppercase letters within rows or low-ercase letters within columns are significantly different (p , 0.05).

b Days of aging.c These values are significantly different at p , 0.10.

sorption from the extracted peat is considerably lower thanfrom the nonextracted peat. For comparison purposes, the 3-d-aged desorption curve of the nonextracted peat first shown inFigure 3B is reproduced in Figure 7A and B. Similarly, at agiven aging period the strongly resistant fraction is consid-erably greater for the extracted peat than for the nonextractedpeat. However, pairwise differences between the peats extract-ed with NaOH and Na4P2O7 were not obvious.

DISCUSSION

Parallel experiments have shown that aging of phenanthrenedecreases the rate and extent of desorption, reduces the rateand extent of mineralization by two bacteria, and reduces up-take by earthworms. The decline in bioavailability to micro-organisms is noticeable after only short periods of aging. After14 d of aging, twice as much phenanthrene in the peat andfour times as much phenanthrene in the silt loam remainedundegraded compared to the corresponding 3-d-aged soils.This agrees with Hatzinger and Alexander [9], who observeddecreases in the mineralization of phenanthrene after as littleas 13 d of aging. The mineralization of phenanthrene in thepeat soil continued to decline after aging periods of up to 103d. Similarly, Hatzinger and Alexander [9] noted that the bio-

degradation of phenanthrene continued to decrease after agingfor as long as 315 d. The availability of phenanthrene foruptake by earthworms was significantly reduced on aging.These findings agree with those of Kelsey and Alexander [11]and White et al. [10], who observed reduced assimilation byearthworms of phenanthrene, naphthalene, and atrazine withincreasing residence time of the compounds in soils.

The decline in physical availability of phenanthrene wasalso noticeable after only short periods of aging. After only 3d of aging, 15 to 20% of the sorbed phenanthrene remainsstrongly resistant to desorption after long periods of continuousdesorption in the presence of Tenax as an infinite third-phase


sink. The strongly resistant fraction continues to increase withaging; after 100 d of aging about 60% of phenanthrene in thepeat remained undesorbed after 2 months of continuous de-sorption. The decline in physical availability of phenanthrenewith aging is also manifested in its reduced extractability withorganic solvents under mild extraction conditions [9,30,36].

Figures 4 and 5 show the strong correlation between thedecline of physical and biological availability of phenanthrenewith aging time with respect to both the magnitude and therate of decline in these parameters. This correlation holds re-gardless of the soil type and regardless of the bacterial strainused. This correlation clearly shows that bioavailability withrespect to microorganisms is primarily dependent on the sorp-tion process and that bioavailability and desorption are simi-larly dependent on contact time of the chemical with the soil.The effect of aging can be quite severe; in both soils the initialrate of mineralization or desorption of phenanthrene drops byabout one order of magnitude after only 50 to 60 d. Biologicaland physical availability is generally more limited in the peatthan in the silt loam soil, most likely because of the higherorganic matter content of the peat soil, as phenanthrene haslittle affinity for mineral surfaces.

Removal of the humic acid fraction caused a decrease inthe initial rate of desorption and an increase in the stronglyresistant fraction remaining at long desorption times. On theother hand, the effect of humic acid removal on bioavailabilitywas less clear. No significant effect of extraction on bioavail-ability was seen after 3 d of aging. With increased aging,removal of humic acid by NaOH decreased the extent of min-eralization compared to the unextracted soil, whereas removalby Na4P2O7 had no significant effect. Alkaline extraction ofhumic acid from soil organic matter produces a denser, higher-molecular-weight material (humin) [2,37]. Sorption of hydro-phobic compounds to humin is greater, less linear, and subjectto stronger co-solute competitive effects than the unalteredsoil organic matter [21]. We have proposed that sorption tosoil organic matter occurs by dual mechanisms, one involvingsolid-phase dissolution (partitioning) in rubbery regions of soilorganic matter and the other by a site-specific, internal void-filling mechanism taking place in condensed glassy regions ofsoil organic matter [21,38]. Huang and Weber [23] have pro-posed a similar model in which organic matter is viewed as aheterogeneous matrix of soft and hard regions of organic mat-ter having fundamentally different sorption behaviors. As sug-gested by carbon dioxide adsorption isotherms, humin appearsto be enriched in subnanometer-size voids that might providespecific sorption sites for organic compounds [21,39]. Male-kani et al. [40] showed that selective removal of organic matterfrom a soil by alkaline extraction resulted in increased surfacearea and decreased average pore size. The nanovoids in soilorganic matter represent potential sites for steric entrapmentof sorbing molecules. To the extent that this is true, the en-hanced concentration of such sites in the humin fraction shouldlead to increased sequestration of organic chemicals comparedto the original soil. Although the desorption experiments sus-tain this prediction, further work is needed to clarify the am-biguities of the bioavailability experiments.

The findings presented in this paper are significant in thatthey represent the first direct correlation between the reducedbioavailability of a compound and parallel decreases in itsdesorption with increasing residence time. In addition, thisstudy shows that biological and physical availability are re-duced in the humin fraction. Future research should focus on

the development of mathematical models to predict and elab-orate on the link between reduced desorption and correspond-ing reductions in bioavailability using different contaminantsystems as well as on methods to overcome slow desorptionand enable successful bioremediation.

Acknowledgement—We thank Antonio Quinones Rivera for technicalassistance. Partial support for this research was provided by TrainingGrant 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 EnergyJoint Program on Bioremediation R825959-01-0.

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