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Environmental Toxicology and Chemistry, Vol. 18, No. 3, pp. 420–425, 1999q 1999 SETAC

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

SOLID-PHASE GENOTOXICITY ASSAY FOR ORGANIC COMPOUNDS IN SOIL

RENEE R. ALEXANDER, NAMHYUN CHUNG, and MARTIN ALEXANDER*Department of Soil, Crop, and Atmospheric Sciences and Institute of Comparative and Environmental Toxicology,

Cornell University, Ithaca, New York 14853, USA

(Received 28 January 1998; Accepted 1 June 1998)

Abstract—A genotoxicity assay was developed for samples from environments in which toxic organic compounds are largelysorbed. The assay entails measurement of the rate of mutation of a strain of Pseudomonas putida to rifampicin resistance. Theratio of induced to spontaneous mutants was a function of the concentration of a test mutagen in soil. In studies of the utility ofthe assay in samples amended with 2-aminofluorene as a test mutagen, the ratio of induced to spontaneous mutants declined withtime. The decline paralleled the disappearance of extractable 2-aminofluorene from the soil. The ratio of induced to spontaneousmutants also fell in four other soils with dissimilar properties. We suggest that this solid-phase assay is more appropriate for theestimation of genotoxicants sorbed in soil than assays involving extractants or suspensions of soil or sediment samples.

Keywords—Bioassays Genotoxicants Pseudomonas putida 2-Aminofluorene

INTRODUCTION

Many of the toxic compounds present in soil are extensivelysorbed to particulate matter, and often the rate of desorptionis so slow that little of the toxicant appears in solution. Con-siderable evidence exists that the sorbed form is biologicallyavailable, often with little of the compound being detected insolution. For example, although 2,3,7,8-tetrachlorodibenzo-p-dioxin has not moved appreciably with leaching water despiteits presence for many years in field sites, it is toxic after in-gestion [1,2] or pulmonary exposure [3]. Similarly, despite itsextensive sorption, benzo[a]pyrene is available for dermalsorption [4] and by ingestion [5]. In addition, certain bacteriautilize organic molecules sorbed on surfaces of particles, acapacity not possessed by other microorganisms that use onlythe substrate in solution [6,7].

Bioassays that make use of the aqueous phase present to-gether with the particulate matter of soils or sediments do notgive an appropriate assessment of the detrimental effect of thecompound that remains sorbed. An aqueous suspension con-taining soil or sediment may provide some contact of organismwith solid, but the relevance of the data obtained by such assaysmay be misleading because of the lack of information on thefrequency or duration of the organism–particle contact. A num-ber of microbiological assays have been used for assessing thetoxicity of sediments and soils [8–10], but these are not ac-tually solid-phase assays because of the use of suspensions orjust the aqueous phase without prolonged or known frequencyof contact of the test microorganism with the sediment or soilparticles.

Therefore, a study was initiated to devise a solid-phasemicrobiological assay. Because such solid-phase assays havebeen widely used to measure the acute toxicity of pesticidesto the microorganisms responsible for nitrification and organicmatter decomposition [11] and no procedure exists for as-sessing the chronic effects of carcinogens that are stronglysorbed to soil, the investigation was designed to devise a solid-

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

phase genotoxicity assay. Some of the methods described foraqueous-phase tests [12] served as models.

MATERIALS AND METHODS

Materials

Antibiotics, methyl methanesulfonate, dimethyl sulfoxide,and 2-aminofluorene (2AF) were purchased from SigmaChemical (St. Louis, MO, USA). S9 rat liver homogenate wasobtained from Microbiological Associates (Rockville, MD,USA). Biology GN microplates were purchased from Biolog(Hayward, CA, USA). The soils used were Catalina silt loam(pH 4.8, 0.41% [w/v] organic C) from Puerto Rico, Amor siltyclay loam (pH 8.4, 0.62% organic C) from North Dakota, andAngola silty clay loam (pH 6.1, 4.64% organic C), Langfordsilt loam (pH 6.6, 4.73% organic C), and Lima loam (pH 7.2,7.16% organic C) from New York.

Counts of the parent culture were performed on Luria-Ber-tani (LB) agar [13], and mutants were grown on the samemedium, to which 50 mg of rifampicin and 100 mg of cyclo-heximide per milliliter were added. ‘‘Top agar’’ tubes (13 3100 mm) contained 2.5 ml of molten agar (0.6% [w/v] agarand 0.5% NaCl [w/v]) at 458C [14]. The antibiotics were ster-ilized by passage through 0.22-mm sterile nylon membranefilters and added to molten agar before the plates were poured.Dilutions of cell suspensions were made in an aqueous 0.9%NaCl (w/v) solution.

Soil preparation

Air-dried samples of soil were passed through a 2-mm sieveand mixed with CaCO3 to a concentration of 2% (w/w). Limewas not added to Lima loam because of its high pH. Five-gram samples of soil were placed in 50-ml glass tubes, and1.5 ml of distilled H2O was added. Because the test bacteriumdid not grow in soil sterilized by g irradiation, the soils weresterilized by autoclaving twice for 2-h periods with a 1-d in-terval between the heat treatments. Sterility was confirmed byadding some of the sterilized soil to LB agar plates and ob-serving no growth after 48 h of incubation at 308C.

Solid-phase genotoxicity assay Environ. Toxicol. Chem. 18, 1999 421

After sterilization, the soils were allowed to air dry for 72h at 308C. A solution of 2AF dissolved in dimethyl sulfoxideor the solvent alone then was added to the tubes. The soil wasmixed twice with a Vortex mixer for 30 s, and the soil wasallowed to stand for 72 h at 308C. The soil then was moistenedto 80% of field capacity with M-9 inorganic salts solution [13]containing glucose to give a final concentration of 1% (w/v)[15] and 0.1 ml of a 16- to18-h culture containing approx. 104

cells in saline. The soils were incubated at 308C for 48 h topermit growth of the test organism and to allow for mutantexpression.

Strain selection

To obtain antibiotic-resistant mutants, 10-g samples of non-sterile Lima loam were added to petri dishes, and 0.1 g ofglucose and sufficient M-9 inorganic salts solution were addedto moisten the soil, which was then pressed down to providea smooth surface. The plates were incubated at 308C for 72h, and 48 of the bacterial colonies that developed on the surfaceof the soil were picked. The isolates were cultivated in liquidLB medium and streaked on LB agar plates, and single colonieswere selected. These isolates were tested for sensitivity to 50mg of rifampicin, 15 mg of tetracycline, 50 mg of kanamycin,25 mg of streptomycin, 40 mg of ampicillin, and 30 mg ofchloramphenicol per milliliter. A strain more sensitive to tox-icants was obtained by incubating a fully grown culture ofPseudomonas putida A11 with 0.4% (v/v) methyl methane-sulfonate at 378C for 1 h. The cells were collected by centri-fugation, washed with cold 0.9% (w/v) NaCl solution, andstreaked on LB agar.

A strain of P. putida more sensitive to ultraviolet (UV)light (A11r) was obtained by transferring a suspension of fullygrown cells to a petri dish and exposing the cells for 8 s to agermicidal lamp (15 W) at a distance of 33 cm. Then, 0.1 mlof the irradiated suspension was spread on LB agar. The plateswere incubated for 24 h at 308C. A number of colonies weretransferred to tubes containing liquid LB medium. The cultureswere grown, and portions were streaked on LB agar plates.One-half of each plate was covered and irradiated as describedabove.

In a preincubation test to increase mutation rates, 0.1 mlof a 14- to 18-h culture was incubated for 20 min at 378C ina solution containing 0.9 ml 0.2 M phosphate buffer, pH 7.4,and either 5.0 mg 2AF in 0.1 ml of dimethyl sulfoxide or 2.5mg sodium azide in 0.1 ml of water. The cells were then platedon LB agar containing rifampicin.

To test the effect of commercial arochlor-induced S9 ratliver homogenate on mutation rates, a suspension containing10% (v/v) of S9 liver homogenate was prepared [14], and 0.5ml was added to molten top agar tubes, as was 50 mg of 2AFand 0.1 ml of a culture grown for 14 to 18 h. After mixing,the contents of the tubes were poured onto LB agar containing50 mg rifampicin/ml. Agar without 2AF and agar with mutagenbut no S9 liver homogenate were also prepared [14,16].

Enumeration of bacteria

To count cells in soil, 15 ml of a 0.9% NaCl (w/v) solutionwas added to the soil, the tube was mixed thoroughly, serialdilutions were made from this slurry, and 0.1-ml portions werespread on duplicate LB agar plates with a spreader. The plateswere incubated at 308C for 18 to 24 h and counted.

To detect rifampicin-resistant (rfpr) mutants, 0.1-ml por-tions of dilutions of soil suspensions were added to tubes with

2.5 ml of top agar held at 458C. The tubes were then mixed,and the contents were applied to the surface of rifampicin-containing LB agar plates and spread by tilting and rotatingthe plates to evenly distribute the warm agar. Four replicatesof each soil sample and four antibiotic-containing plates wereprepared. The plates were incubated right side up for 48 h at308C.

The effect of the mutagen is expressed as a mutagenicityratio, which is the ratio of induced mutants per 109 parentalcells to the number of spontaneous mutants per 109 parentalcells.

Sample size

The number of replicates needed to compare soil sampleswith and without the mutagen was determined by applying atwo-sample test at a power of 0.80 at p 5 0.05 [17]. Thecalculations were made using data that gave the highest mu-tagenicity ratios. The data presented were obtained after 48 hof growth in samples containing 0 and 100 mg of 2AF pergram of soil.

From the number of mutants formed in soil samples freshlytreated with 100 mg 2AF/g soil, the sample size (n) requiredto detect a reduction of 50% or more in mutation frequencywas calculated to be 3.5 [17]. Therefore, four soil replicateswere analyzed, and four plate counts of each replicate weremade.

Mutagen concentration and growth period

The effect of concentration was assessed using four rep-licates of 2AF concentrations from 0 to 200 mg/g. The solutionswere thoroughly mixed with 5.0-g samples of dry, sterile soilusing a Vortex mixer, and then 50 mg of glucose [15], 1.8 mlof M-9 solution, and approx. 104 cells were added. The sampleswere incubated for 48 h at 308C.

To determine the growth period for optimum mutant ex-pression, approx. 104 cells of P. putida A11rUV, 1.8 ml of M-9 solution, and 50 mg of glucose were added to samples ofsterile Lima loam. Four replicate soil samples containing no2AF (to count spontaneous mutants) and four containing 100mg 2AF/g were prepared for each sampling time.

Effect of soil treatment

Cycles of wetting and drying were imposed on soil con-taining 0 and 100 mg 2AF/g using the procedure of White etal. [18]. Sterile 5-g samples of dry Lima loam in 250-ml Er-lenmeyer flasks were amended with 1.8 ml of sterile distilledwater, and the flasks were loosely capped and stored until thesoils were air dry as determined by weight loss, at which time1.8 ml of water was added. Four wetting–drying cycles wereimposed on the soil. The water additions were made at 0, 10,18, and 28 d. At the end of each cycle, four samples of soilcontaining 0 mg 2AF/g and four containing 100 mg 2AF/gwere amended with glucose, inorganic nutrients, and P. putidaA11rUV cells as described previously. The flasks were incu-bated at 308C for 48 h, and the numbers of parents and mutantswere determined.

Effect of residence time

To study the effect of residence time of 2AF in the soils,five sets of four replicate tubes were prepared. Each contained5 g of dry, sterile soil and either 50 ml of dimethyl sulfoxideor 0.5 mg of 2AF dissolved in 50 ml of dimethyl sulfoxide.Sufficient salts solution was added to bring the soils to 80%

422 Environ. Toxicol. Chem. 18, 1999 R.R. Alexander et al.

Fig. 1. Effect of concentration of 2-aminofluorene on number of ri-fampicin-resistant mutants of Pseudomonas putida A11rUV appear-ing in Lima loam.

of their field capacities. The tubes were tightly capped, coveredwith parafilm, and stored in the dark at 21 6 28C for 0, 15,30, 60, and 90 d, at which time glucose and an inoculum wereadded. The soils were incubated at 308C for 48 h (Lima, Lang-ford, and Amor soils) or 72 h (Angola and Catalina soils), andtotal cell counts and the number of spontaneous and inducedmutants were determined.

Analysis of 2AF

To extract the mutagen from soil, 2AF was freshly addedto Lima loam to give a concentration of 100 mg/g. The com-pound was quantitatively recovered by shaking 5 g of soil at478C for 16 h in 25 ml of methanol. The slurries were thencentrifuged for 10 min at 12,000g, and the soil was extractedtwice for 18 h in a Soxhlet apparatus with 120 ml of methanol.Portions of the extracts were passed through 0.22-mm Teflontsyringe filters (Micron Separations, Westboro, MA, USA). Thesamples were analyzed by high-performance liquid chroma-tography (Hewlett-Packard Series 1050, Hewlett-Packard,Avondale, PA, USA) using a Spherisorb ODS-2 octadecyl-bonded silica column. The solvent system was acetonitrile:water (70:30) at a flow rate of 1.0 ml/min. Absorbance wasdetermined at 284 nm.

RESULTS

Strain selection

Rates of mutation to antibiotic resistance were chosen asselective markers because nutritional requirements, which arecommonly used in microbiological genotoxicity assays, mightbe met by soil constituents. Bacillus subtilis, Escherichia coli,Azotobacter vinelandii, Rhizobium meliloti, and Agrobacter-ium tumefaciens either failed to grow appreciably in soil orproduced few if any mutants. Therefore, bacteria growing wellin soil and capable of generating mutants resistant to one ormore antibiotics were sought. Three isolates were obtainedthat were sensitive to one or more of the antibiotics and showeda mutagenic response. The one that was selected (strain A11)yielded the greatest number of mutants resistant to rifampicin.That bacterium was identified by the Biolog GN microplatesystem [19] as P. putida (similarity ratio index of 0.801).Pseudomonas putida A11 produces smooth colonies on LBagar and is sensitive to rifampicin, tetracycline, and kana-mycin, but is resistant to streptomycin, ampicillin, and chlor-amphenicol.

The penetration of large molecules through the cell wall,which results in enhanced mutation rates, is characteristic ofthe rough-colony type of Salmonella typhimurium (rfamarker), which is widely used for studying genotoxicity[16,20,21]. A rough-colony type was isolated and designatedP. putida A11r. On lawns of cells growing on LB agar con-taining a filter paper disk impregnated with a 0.1% (w/v) so-lution of crystal violet, which diffuses into the agar [14,16],the diameters of the clear zones of inhibition surrounding theapplication sites were 8 and 14 mm for the parent and mutantstrains, respectively, indicating greater permeability of the mu-tant to the large molecule of crystal violet and presumably toother large molecules.

Salmonella strains that have the uvrB mutation are sensitiveto UV light and more sensitive to mutagenic compounds[14,16,20]. Many of the bacteria obtained after exposure toUV light in this study grew on the irradiated side of an agarplate, but the strain selected and designated P. putida A11rUV

showed sensitivity to UV light by growing only sparsely inthe exposed area. This culture has been deposited with theAmerican Type Culture Collection and is designated ATCC700478.

The applicability of two other techniques [14,16] to im-prove mutation rates was evaluated. The preincubation pro-cedure did not result in higher frequencies of mutation to ri-fampicin resistance. The second approach involved the use ofcommercial aroclor-induced S9 rat-liver homogenate, whichusually is effective in testing the mutagenesis of hydrophobiccompounds. This approach resulted in an increase in the fre-quency of mutation of 40%, which is much lower than the six-to eightfold increases obtained with S. typhimurium [14,16].

Effect of concentration and time

The effect of concentration of 2AF on rates of mutation ofP. putida A11rUV to rifampicin resistance was measured. Thenumber of mutants was approximately the same with 0 and25 mg mutagen/g soil (Fig. 1). At higher concentrations, therelationship between number of rfpr mutants and 2AF con-centration was essentially linear up to 100 mg/g. The numberof mutants was lower at a concentration of 200 mg/g than at100 mg/g.

The growth period that permits optimum mutant expressionwas also determined. The parent grew at essentially the samerate and reached the same final density in the presence orabsence of the mutagen (Fig. 2). The numbers of spontaneousrifampicin-resistant mutants, which are reflected by the col-onies appearing when soil not amended with 2AF was platedon agar containing the antibiotic, increased with time andreached a maximum value of approx. 800 per 5 g before de-clining. The numbers of induced mutants, which are reflectedby the colonies derived from cells in 2AF-amended soil, alsorose with time, but their densities at days 1, 2, and 3 werehigher than the numbers of spontaneous mutants. The muta-genicity ratios continued to increase after 48 h, but these valuesmay reflect mere proliferation of existing mutants and not newmutations. The 2-d incubation period was used in subsequentstudies.

Solid-phase genotoxicity assay Environ. Toxicol. Chem. 18, 1999 423

Fig. 2. Effect of incubation time on growth of Pseudomonas putidaA11rUV in Lima loam and mutant induction in the presence of 0 and100 mg of 2-aminofluorene (2AF) per gram of soil. The values fortotal number of cells at 0 and 1 d were the same. Values in closedsymbols are from soil with no 2AF, and values with open symbolsare from soil with 2AF.

Table 2. Mutagenicity as a function of residence time of 2-aminofluorene in soil

Time(d)

Mutagenicity ratioa

Lima Amor Catalina Angola Langford

0 4.79 A 3.43 A 2.11 A 4.23 A 2.83 A15 1.99 BC 1.61 B 1.18 B 1.72 B 1.81 AB30 2.65 B 2.08 B BDLb 1.09 B 1.13 B60 1.18 C 1.08 B NDc BDL 1.10 B90 1.22 C 1.14 B ND ND 1.32 B

a Values followed by the same capital letter are not significantly dif-ferent (p 5 0.05).

b BDL 5 below detection limit.c ND 5 not done.

Table 3. Effect of residence time in Lima loam on recovery of 2-aminofluorene by sequential extractionsa

Residencetime (d)

% Extracted

Shaking at478C

FirstSoxhlet

extraction

SecondSoxhlet

extractionTotal

recovered

0 76.0 6 2.8 25.1 6 3.4 NDb 101.1 Ac

5 49.0 6 1.4 34.3 6 8.3 6.3 6 1.3 89.6 B14 10.1 6 0.4 19.4 6 0.3 12.4 6 0.3 41.9 C33 22.1 6 5.1 13.1 6 4.0 BDLd 35.2 C63 10.7 6 3.3 10.3 6 2.0 BDL 20.9 D

a Values are the mean 6 SD of duplicate or triplicate samples.b ND 5 not done.c Values followed by the same capital letter are not significantly dif-ferent (p 5 0.05).

d BDL 5 below detection limit.

Table 1. Growth and 2-aminofluorene–induced mutations ofPseudomonas putida A11rUV in various soils

Soil

Doublingtime(h)

In stationary phase

Cells(3 106/g

soil)Mutagenicity

ratioa

Lima loam 1.4 720 4.71 6 0.58Langford silt loam 1.4 1,040 2.61 6 0.79Amor silty clay loam 1.8 160 3.25 6 0.95Angola silty clay loam 2.1 680 4.38 6 0.25Catalina silty clay 3.8 200 2.10 6 0.10

a Values are the mean 6 SD of duplicate sets of soil samples.

Effect of soil treatment

The wetting and drying of soil affects the availability oforganic compounds to microorganisms [18], so cycles of wet-ting and drying were imposed on the soil. The mutagenicityratio was initially 2.65, and the ratios after 1, 2, and 3 cyclesof wetting and drying were 2.89, 1.23, and 1.52, respectively.After the fourth cycle, the number of mutants in the 2AF-treated soils was below that in the soils without mutagen.Therefore, this treatment decreased bioavailability and muta-genicity of the test compound.

Five soils were selected to study the effect of soil type onthe changes in bioavailability of the test mutagen with time.The growth and mutation rates of the bacterium in the presenceof 0 and 100 mg 2AF/g of each soil were measured. Pseu-domonas putida A11rUV grew fastest in Lima loam, Langfordsilty loam, and Amor silty clay loam and reached the stationaryphase after 48 h, but 72 h was required to reach the stationaryphase in Angola silty loam and Catalina silty clay (Table 1).The numbers of parental cells were the same in these soils inthe presence and absence of 2AF (data not shown), and themutagenicity ratios for these samples increased with incuba-tion time and reached their maximum values at the stationary

phase. The data suggest that the mutagenicity ratios, whichvaried among the soils, are independent of cell density at thestationary phase or the doubling time.

A study of the influence of residence time on frequency ofmutant formation showed that fewer mutants were obtained infive soils at 15 d than at 0 d, and the mutagenicity remainedlow thereafter (Table 2). The small increase in ratio between15 and 30 d in two soils was not statistically significant. InCatalina silty clay and Angola silty loam, mutagenicity wasno longer detectable after 30 and 60 d, respectively.

Recovery of mutagen

Tests were conducted to determine whether 2AF could beextracted from the soil after varying periods of persistence.As shown in Table 3, 2AF freshly added to Lima loam wascompletely recovered after one Soxhlet extraction. However,the longer the compound remained in soil, the lower the re-covery.

DISCUSSION

The procedure described here represents an inexpensive andreasonably rapid method for assessing the genotoxicity of com-pounds present in soil. Of particular importance is the fact thatit is a solid-phase assay. Many toxicants, especially those thatare hydrophobic or cationic, are sorbed to soil, and use ofbioassays that rely on analysis of only the aqueous phase mayincorrectly assess toxicity because more of a compound thatis sorbed may be bioavailable than is reflected by the amountthat appears in water brought into contact with the solids. Thisis amply demonstrated by toxicity [1,2] and bioavailability

424 Environ. Toxicol. Chem. 18, 1999 R.R. Alexander et al.

[3,4] tests with mammals, uptake by ingestion by an amphipod[5], and use of organic compounds for growth of bacteria [6,7].Undoubtedly, the compound must be desorbed from soil par-ticles to become available to a living organism, but that de-sorption may require some biological intervention, as by sur-factant production or release by the organism of a metabolitethat effects the desorption. A simple aqueous solution placedin contact with the particulate matter may not reflect the bi-ological processes that cause the desorption. Shaking the soilin water likewise may not be appropriate for a solid-phaseassay because of the unknown frequency and intimacy of con-tact of organism with the particle surface. On the other hand,the use of organic solvents may result in an overestimate oftoxicity because more of the inhibitor may be mobilized thanis actually available in the sorbed form. Solid-phase assaysusing microorganisms are not new, and they were widely usedeven more than three decades ago for assaying acute effectsof pesticides in soil [11], but the present report extends thatold approach by showing how it can be applied for a geno-toxicity assay. Different test bacteria may give somewhat dif-ferent results if they have dissimilar capabilities of gainingaccess to sorbed compounds [6].

The polycyclic aromatic hydrocarbon 2AF was chosen asa test mutagen for this study because it is often used in theAmes test with strains of Salmonella [14]. Methyl methane-sulfonate, a potent mutagen for Pseudomonas [22,23], wasused to obtain a rough-colony isolate and a strain that wasalso sensitive to UV light. These characteristics are similar tothe rfa and uvrB traits of the Salmonella tester strains [20,21].Because of these alterations, the cells of P. putida A11rUVpresumably are more permeable and more sensitive to foreigncompounds than are the cells of the original culture, therebyincreasing the potential mutagenic effect of genotoxic chem-icals. Pseudomonas putida is known to have P-450 oxidativecapabilities [23], which may account for the small increase inmutation frequency obtained when 2AF was treated with S9liver homogenate. This treatment is widely used in other mi-crobiological genotoxicity assays to solubilize hydrophobiccompounds, including 2AF [12,14,16].

Several cycles of wetting and drying soil reduced theamount of 2AF available for mutagenesis. This loss of bio-availability after wetting and drying has been observed pre-viously in tests of biodegradability of phenanthrene [21]. Aloss of bioavailability for mutagenesis occurred in several dis-similar soils, but this loss was not apparently related to theorganic matter content of the soils. In several of the soils, theloss in genotoxicity was sufficiently marked that a mutage-nicity ratio could not be determined. Because of the differencesin growth rates and maximum cell densities of P. putidaA11rUV in these soils, it may be necessary to determinegrowth rates and extents as well as conditions for mutagenicityin soils to be tested by this assay.

The decline in bioavailability of 2AF for mutagenesis wasaccompanied by a decline in the percentage of the compoundthat was recovered by extraction. It is known that the ease ofextraction of organic compounds decreases as the moleculespersist, as has been shown for several polycyclic aromatichydrocarbons in soil [24] and sediment [25]. On the other hand,the decrease in extractable 2AF may result from its covalentbinding to the organic fraction of the soil inasmuch as aromaticamines readily form covalent complexes with humic substanc-es [26]. Regardless of the explanation, however, the simulta-

neous loss of both mutagenic activity and extractability isnoteworthy.

The solid-phase assay proposed herein represents a realisticmethod for evaluating the carcinogenic activity of sorbed mol-ecules. Additional evaluations of the assay are required, nev-ertheless, including determinations with other compounds, dis-similar soils, and complex mixtures of pollutants. Ultimately,the assay may be useful for monitoring soils for mutagens andcarcinogens and may allow for assessing factors affecting thebioavailability of these compounds.

Acknowledgement—This research was supported by research grantES05950 from the National Institute of Environmental Health Sci-ences, research grant F49620-95-1-0336 from the U.S. Air Force Of-fice of Scientific Research, and a grant-in-aid from duPont deNemours.

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