Bioassay-Directed Fractionation and such contaminated site is in St.

Louis Park,
Minnesota, the former site of the Reilly Tar
Chemical Identification of Mutagens and Chemical Corporation's coal tar
in Bioremediated Soils distillation and wood preserving plant.
From 1917 to 1972, dumping from this
plant contaminated about 80 acres of soil
Lance R. Brooks,1 Thomas J. Hughes,1 Larry D. Claxton,1 and the underlying groundwater with
Barry Austern,2 Richard Brenner,2 and Fran Kremer2 creosote wood-preserving waste (3). In
1978, the Minnesota Department of
1Environmental Carcinogenesis Division, National Health and Health discovered significant concentra-
Environmental Effects Research Laboratory, U.S. Environmental tions of polycyclic aromatic hydrocarbons
Protection Agency, Research Triangle Park, North Carolina; 2National (PAHs) in six municipal drinking water
Risk Management Research Laboratory, U.S. Environmental Protection wells near the Reilly Tar Site plant (3).
Agency, Cincinnati, Ohio Currently, St. Louis Park is pumping and
treating the contaminated groundwater
Soil from a Superfund site (Reilly Tar Site, St. Louis Park, Minnesota) contaminated with plume leaching from the creosote-conta-
polycyclic aromatic hydrocarbons (PAHs) from creosote was treated with several bioremediation minated soil and has placed a soil cover
technologies including bioslurry (BS), biopile (BP), compost (CMP), and land treatment (LT). These with grass over the contaminated soil.
treatment technologies are being evaluated in pilot scale laboratory systems by the U.S. To reduce the length of time required
Environmental Protection Agency's National Risk Management Research Laboratory in Cincinnati, for pump and treat operations, the U.S.
Ohio. To evaluate the genotoxicity and identify the mutagens in the soil before and after the Environmental Protection Agency (U.S.
various treatments, fractionated extracts of five soils were bioassayed for mutagenic activity with EPA) National Risk Management Research
a microsuspension modification of the Salmonella histidine reversion assay. Soils were extracted Laboratory (Cincinnati, Ohio) is evaluating
by sonication using dichloromethane (DCM). The five extracts were fractionated in triplicate (two various bioremediation technologies for
for bioassay and one for chemical analysis) by reverse-phase high-performance liquid their efficiency in removing PAHs and
chromatography (HPLC) using hexane/DCM/methanol, and the fractions for bioassay were reducing toxicity of soil collected from St.
solvent-exchanged into dimethyl sulfoxide by nitrogen evaporation. Forty HPLC fractions for each Louis Park. The bioremediation technolo-
sample were bioassayed in strain YG1041 with and without exogenous liver metabolic activation. gies that are being examined in pilot-scale
As shown in a companion paper, the mutagenicity of two treatments (BS and BP) was laboratory studies are bioslurry (BS), biopile
significantly greater than the mutagenicity of the untreated soil. Mutagenic fractions (>500 (BP), compost (CMP), and land treatment
revertants) were analyzed by gas chromatography/mass spectrometry (GC/MS). PAH analysis of (LT). These technologies are presented and
the soils indicated that all treatments were effective in reducing the total PAH concentration
(48-74%). Qualitative GC/MS analysis of the mutagenic fractions from the BS and BP treatments described by Hughes et al. (4). The goal of
indicated that they contained azaarenes, which are mutagens. The CMP and LT processes were these bioremediation technologies is to
the most effective and least toxic bioremediation procedures based on mutagenic potency and reduce the PAH concentrations and toxicity
chemical analysis. This research demonstrated that the combination of bioassays and chemical of contaminated soils. The PAH concentra-
analysis provided a more accurate determination of toxicity in these complex environmental tions were chemically monitored through-
mixtures. Environ Health Perspect 1 06(Suppl 6):1435-1440 (1998). http.//ehpnetl.niehs.nih.gov/ out this study. The mutagenic activity of the
docs/l998/Suppl-6/1435-1440brooks/abstract.html soil extracts was measured in the untreated
soil (UTS) and in the four treatment soils at
Key words: fractionation, bioremediation, polycyclic aromatic hydrocarbons, Salmonella, end of the study. Mutagenic activity was
mutagenicity
measured using the Salmonella mutagenicity
assay developed by Maron and Ames (5).
The bioassay indicated higher mutagenicity
There are over 700 wood-preserving treatment of wood products, excess creosote in the BS (163.3x 106 revertants [rev]/kg
facilities documented in the United States is released from the treated materials, and dry soil) and BP (3.0 x 106 rev/kg dry soil)
(1,2). Many designated Superfund sites are the leaching of creosote wastes from treated treatments than in the UTS (0.008x 106
a result of wood treatment activities involv- materials contaminating the soil and rev/kg dry soil) (4). A significant increase in
ing creosote (1,2). During the pressure groundwater has been common (1,2). One mutagenicity in the CMP and LT extracts

This paper is based on a presentation at the Conference on Current Issues on Chemical Mixtures held 11-13 August 1997 in Fort Collins, Colorado. Manuscript received
at EHP 17 February 1998; accepted 30 June 1998.
The authors thank L. Stephens, A. Warren, P. Matthews, H. Carlsen, and B. Eischen for their valuable assistance. The Reilly Tar Site samples were prepared by the fol-
lowing people at the U.S. Environmental Protection Agency (U.S. EPA) in Cincinnati, Ohio: untreated soil (P. McCauley), bioslurry (J. Glaser), biopiles and compost (C.
Potter), and land treatment (G. Sayles). The authors thank them for supplying soil samples and the chemical analysis (percent reduction in PAH) for this study. The
authors thank J. Ryan and E. George for their critical technical review of the manuscript.
Address correspondence to L.R. Brooks, Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental
Protection Agency, MD-68, Research Triangle Park, NC 27711. Telephone: (919) 541-1365. Fax: (919) 541-3966. E-mail: [email protected]
Abbreviations used: BP, biopile; BS, bioslurry; CMP, compost; DCM, dichloromethane; DMSO, dimethyl sulfoxide; GC/MS, gas chromatography/mass spectrometry;
HPLC, high-performance liquid chromatography; LT, land treatment extract; rev, revertants; +S9, with aroclor-induced rat liver; -S9, without arochlor-induced rat liver;
PAH, polycyclic aromatic hydrocarbon; U.S. EPA, U.S. Environmental Protection Agency; UTS, untreated creosote-contaminated soil.

Environmental Health Perspectives * Vol 106, Supplement 6 * December 998 1435

 BROOKS ET AL.

compared to the UTS was not detected (4). mm, Alltech, Deerfield, Illinois). The fractionated. The HPLC fractions were
In this study the mutagenic BS and BP extracts were eluted at 7 ml/min using a evaporated to dryness and then dissolved
extracts, along with the UTS extracts, were Waters 600E HPLC (Millipore) equipped into 100 pl DCM. The fractions were
fractionated by high-performance liquid with a 717 autosampler, a M996 photodi- then analyzed using a Hewlett-Packard
chromatography (HPLC) and the result- odearray detector, and an Isco Foxy Jr. 5890 gas chromatograph with a 5973 mass
ing mutagenic fractions were analyzed by (Isco, Lincoln, Nebraska) fraction collector. spectrometer interfaced to a dedicated data
gas chromatography/mass spectrometry The gradient initially was 99% hexane system. The gas chromatograph oven con-
(GC/MS) to identify the mutagens (HPLC grade, Burdick & Jackson) and 1% tained an HPB-5ms column (30 mx 0.25
present in the BS and BP. DCM, which was held for 5 min. This was mm, 0.25-pm film thickness) (Hewlett
followed by a linear gradient to 100% Packard), which was at 40°C initially and
Materials and Methods DCM in 20 min and then held for 1 min. then increased to 300°C at a rate of
Soil Exaction This was followed by a linear gradient to 5°C/min. GC/MS data were acquired
100% methanol (HPLC grade, Burdick & from 35 to 500 atomic mass units. The
Five soil samples from the end of the study Jackson) in 5 min and then held for 10 min. results interpretation was done on the
(UTS, BS, BP, CMP, and LT) were placed Fractions were collected at the rate of 1/min basis of data collected in a computer
individually in glass jars, as described by during the 40-min run in 8-ml vials con- library containing 275,821 mass spectrom-
Hughes et al. (4). Twice the volume of taining 5 pl dimethyl sulfoxide (DMSO) for etry spectra (9). All identifications are
dichloromethane (DCM) (GC grade, bioassay. The vials contained no DMSO tentative, as no authentic standards were
Burdick & Jackson, Muskegon, Michigan) when collected for chemical analysis. The used for comparison.
was added to each jar. Each sample was resulting fractions were then concentrated
sonicated for 15 min and the extract was by nitrogen evaporation. Results
decanted into a glass collection vessel. This Polycyclic aromatic hydrocarbon analysis
procedure was repeated twice more. Each Mutagenicity Assay of the soils before and after each treat-
organic extract was then dried of water Whole extracts of the five soils (UTS, BS, ment demonstrated observable decreases
with sodium sulfate and filtered through a BP, CMP, LT) were bioassayed in the plate (from 48 to 74%) in the concentrations of
0.45-pm Teflon filter (Millipore, Marl- incorporation assay using strains YG 1041 priority pollutant PAHs, as seen in Table
borough, Massachusetts). Sample volume and YG 1042 with and without exogenous 1. The greatest reduction (59 to 92%) was
was reduced by rotoevaporation and nor- mammalian metabolic activation (S9) (4). in the two- and three-ring PAHs for all
malized to 25 ml. The concentration of The HPLC fractions were bioassayed in a treatments. The next greatest reduction
each extract was then determined by gravi- microsuspension modification of the (40 to 75%) was in the four-ring PAHs.
metric analysis (milligrams of organics per Salmonella mutagenicity assay (4,7) using The percent reduction (-10 to 20%) in
milliliter DCM). strain YG1041. Hughes et al. (4) showed the five- and six-ring PAHs indicated no
that YG1041 was the most sensitive strain real change in these concentrations.
Polycycic Aromatic for detecting mutagenicity of the whole Higher-ring PAHs take longer to be
Hydrocbon Analysis extracts from the Reilly Tar Site. The degraded (10) because of the induction of
A 40-ml volatile organic analysis vial served extracts and fractions were bioassayed with complex biodegradation enzymes in
as the extraction vessel. Four grams of the arochlor-induced rat liver (+S9) and without microorganisms, low solubility, and sorp-
soil was mixed with 10 g sodium sulfate and arochlor-induced rat liver (-S9) metabolic tion to the soil. The largest average reduc-
1000 pg surrogate (2-fluorobiphenyl) (two activation according to the method of tion in total PAHs for the four extracts
to six replicates of each treatment were used DeMarini (8). Briefly, 100 pl of 10-fold tested in the mutagenicity assay was in the
for analysis). After mixing the soil and concentrated cells in buffer (from 16-hr cul- CMP (74%), followed by the BS (62%),
sodium sulfate, 20 ml of a DCM/acetone tures) and 100 pl of 0.015 M phosphate LT (62%), and BP treatments (48%).
extraction solvent mixture was added to the buffer or 100 pl S9 mix (6%), pH 7.2, were After the bioassay of each fraction was
vial. The vial was then placed on a recipro- added to each fraction that had been completed, the resulting mutagenicity
cating shaker for 18 hr. After the vial was solvent-exchanged into 5 pl DMSO. profile of the HPLC fractions constituted a
centrifuged at 1500 rpm for 30 min, a 1-ml Contents of the tubes were mixed, incu- mutagram (11). Mutagrams from all four
aliquot was removed for GC analysis. U.S. bated at 370C for 90 min, then poured onto bioremediation treatments, UTS, and a
EPA method 8100 (6) was used to quantify minimal medium plates. 2-Nitrofluorene solvent (DCM) method blank, bioassayed
19 PAHs in the sample extracts. A Hewlett- (0.3 pg/plate) served as the positive control in the Salmonella mutagenicity assay in
Packard 5890A gas chromatograph for YG1041 (-S9) and yielded a mean of YG1041 +S9 and -S9, are shown in
(Hewlett-Packard, Wilmington, Delaware), 204 rev. 2-Aminoanthracene was the posi- Figures 1 and 2. The average mutagenicity
a Supelco SPB-5 column (Supelco, tive control for YG1041 (+S9) with 0.25 pg of the solvent blank fractions was 138 ± 55
Bellefonte, Pennsylvania) (30 mx0.53 mm, yielding 738 rev. Solvent (DMSO) control rev +S9 and 131 ±41 rev -S9. A treatment
0.50-pm film thickness), and a flame values for YG1041 were 109 (-S9) and 106 fraction was not considered mutagenic
ionization detector were used for this analysis. (+S9). A laboratory blank (DCM) was also unless it was . 500 rev (greater than three
tested as a control for the HPLC system. times the solvent blank) per fraction. The
i1gb-Performance Liquid BS treatment had the most mutagenic frac-
Chromatography Fractionation Gas Chromatography/lass tions (both revertants per fraction and the
For fractionation, 5 mg of each of the five Spectrometry Analysis number of fractions) both +S9 and -S9.
soil extracts was injected onto a silica For qualitative GC/MS analysis, a total of The BP treatment showed less mutagenicity
column (Econsil Silica, 10 pm, 250 x 10 25 mg for each extract (UTS, BS, BP) was (both revertants per fraction and the

1436 Environmental Health Perspectives * Vol 106, Supplement 6 * December 1998

 BIOASSAY-DIRECTED FRACTIONATION OF BIOREMEDIATED SOILS

Table 1. Polycyclic aromatic hydrocarbon concentration of the four treatment soils.a and BP treatments. In both the BS and the
Average initial Average final
BP treatments, fraction 16 contained
concentration, concentration, Percent acenaphthene and fractions 30 and 31
Soil treatment mg/kg soil mg/kg soil reduction contained acenaphthylene, anthracene,
Bioslurry benzo[a]anthracene, and chrysene. Fraction
Two- and three-ring PAHsb 443 ± 62 114 ± 75 74 16 of the UTS contained acenaphthene, and
Four-ring PAHsc 874±219 217±81 75 fraction 31 contained acenaphthylene and
Five- and six-ring PAHsd 388 ± 90 309± 71 20 benzo[a]anthracene. The other compounds
Total PAHs 1705±361 640±161 62 seen in the remaining BS and BP fractions
Biopile were mainly N-heterocyclics (italicized in
Two- and three-ring PAHs 1490 ± 183 458±159 69 Table 2) such as acenaphthopyridine,
Four-ring PAHs 1094±85 651 ±253 40
Five- and six-ring PAHs 408 ±75 450 ±49 [1i0ie acridine, 1-azapyrene, benzo(c)carbazole,
Total PAHs 2992 ±326 1559 ±726 48 benzoquinoline, benzothiazolylphenol,
Compost carbazoles, diaminotriazole, diphenylpyra-
Two- and three-ring PAHs 2628± 57 217 ± 23 92 zole, indenopyridine, methylacridine,
Four-ring PAHs 1245±35 468 ± 9 62 methylazaphenanthrene, methylbenzacri-
Five- and six-ring PAHs 444±40 457 ±3 [3Ve dine, and methylisothiazole. Some fractions
Total PAHs 4317 ±132 1142 ± 11 74
Land treatment also contained S- and 0- heterocyclics such
Two- and three-ring PAHs 1232 ± 111 295±102 76 as benzothiazolylphenol, benzocoumarin,
Four-ring PAHs 1126 ±73 361 ± 243 68 benzofuranol, fluoroscein, methylisothiazole,
Five- and six-ring PAHs 438 ± 116 399±159 68 and phenanthrofuran (Table 2).
Total PAHs 2796 ± 292 1055± 719 62
Untreated soil Discussion
Two- and three-ring PAHs 1580 ±75 NA NA
Four-ring PAHs 880±44 NA NA It has been estimated that creosote
Five- and six-ring PAHs 325±134 NA NA consists of 85% PAHs, 10% phenolics,
Total PAHs 2785±230 NA NA and 5% other N-, S-, and O-heterocyclics
aAs determined by U.S. EPA method 8100 (6). bTwo- and three-ring PAHs include naphthalene, 2-methyinaphtha- (1). In this study, the bioremediation
lene, acenaphthylene, acenaphthene, dibenzofuran, fluorene, phenathrene, and anthracene. cFour-ring PAHs treatments (BP, BS, CMP, and LT) were
include fluoranthene, pyrene, benzo[alanthracene, and chrysene. dFive- and six-ring PAHs include benzo[b]fluoran- successful in reducing the priority pollu-
thene, benzo[k]fluoranthene, benzo[e]pyrene, benzo[alpyrene, indeno[123-cd]pyrene, dibenzo[a,h]anthracene, and tant PAHs by 48% or more (Table 1).
dibenzo[g,h,l]perylene. 'Denotes an increase in concentration. However, when the soil extracts from the
bioremediation treatments and the UTS
(extracted at the same time as the treat-
number of fractions) than the BS treatment GC/MS identification of compounds in ment soils) were tested in the Salmonella
but was more mutagenic than the other the mutagenic fractions from the BS and BP mutagenicity assay in strain YG1041, two
treatments (CMP, LT) and the UTS. The HPLC fractionations is summarized in treatments (BS and BP) had increased
mutagenicity of the CMP and LT frac- Table 2. The HPLC fractionations from the mutagenic activity, both with and without
tions was not qualitatively different from UTS also were analyzed by GC/MS for S9 addition, when compared to UTS (4).
that in the UTS fractions both in our comparison to the BS and BP fractions and All four bioremediation treatments and
study and in Hughes et al. (4). Therefore, are included in Table 2. The priority pollu- the UTS were fractionated by HPLC and
extracts from these two treatments were tant PAHs (listed in Table 1) only were tested in a microsuspension modification
not chemically analyzed. detected in three fractions for both the BS of the Salmonella mutagenicity assay using

3000
* Bioslurry (59S) 0 Land treatment (-S9) * Bioslurry (-S9) 0 Land treatment (-S9)
* Biopiles (-S9) A Blank (-S9) * Biopiles (-S9) A Blank (-S9)
oI Compost (-S91 vUntreated 1-S91 2500
D Compost (-S9) v Untreated (-S9)

,, 2000

50 0 c 1500
aC
c, cc

1000

0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40
Fractions Fractions
Figure 1. Mutagram of high-performance liquid chromatography fractions tested in Figure 2. Mutagram of high-performance liquid chromatography fractions tested in
the Salmonella mutagenicity assay without metabolic activation (-S9). the Salmonella mutagenicity assay with metabolic activation (+S9).

Environmental Health Perspectives * Vol 106, Supplement 6 * December 1998 1437

 BROOKS ET AL.

Table 2. Results of the gas chromatography/mass spectrometry analysis of the mutagenic high-performance liquid chromatography fractions.a
Fraction
no. Untreated soil Bioslurry Biopile
14 Not analyzed Methylated and oligomethylated Methylated and oligomethylated
carbazoles, naphazulenone, carbazoles, benzonitrile,
indenoanthracenone phenylquinoline
15 9H-carbazole, 9H-carbazole, acenaphthalene, 9H-carbazole
benzo[c]carbazole methylbenzacridine
16 Acenaphthene, Acenaphthene, Acenaphthene,
benzo[c]carbazole, benzo[c]carbazole, benzo[c]carbazole,
anthracenecarbonitrile anthracenecarbonitrile, anthracenecarbonitrile,
benzanthracenone, benzanthracenone,
fluorenecarbonitrile, fluorenecarbonitrile,
1-azapyrene phenethrol,
indenoisoquinoline,
hydroxy-thienyl-quinoline
18 Benzoquinoline, antracenedione, Tetramethylphenol, fluoroscein, Anthracenecarbonitrile,
cyclopenta(deflphenanthrenone, 9,1 0-anthraquinone, aromatic ketones
fluorenecarbonitrile indacenedione, benzanthracenone
20 9H-fluoren-9-one, anthracenone Benzoquinoline, Aromatic ketones
cyclopentaphenanthrenone,
hydroxypyrene, anthracene dione,
cyanopyrene, benzothiazolylphenol
23 Dihydrocyclobuta[b]naphthalene Benzanthracenone, diphenylpyrazole Benzanthracenone
24 Benzanthracenone, 2-Hexanol, methylcyclopentanone, 2-Hexanol, benzanthracenone,
dihydrocyclobuta[b]naphthalene, tetrachloroethane, tetrahydrophenanthrenone,
naphthopyrandione 3-methylpentanone, diaminotriazole styrylquinoline,
therahydrobenzanthracene,
phenanthrofuran
25 No identified compounds Dihydrocyclobuta[b]naphthalene, Phenanthrofuran,
phenanthrofuran aromatic ketones
27 No identified compounds No identified compounds Phenthrofuran, benzofuranol,
benzocoumarin
30 Not analyzed Acenaphthylene, anthracene, Acenaphthylene anthracene,
dihydroindenone, pyrene, chrysene, dihydroindenone, methylindene,
hexanols, heptanols, hexones, heptanones, aliphatic ketones and alcohols
methylisothiazole, acenaphtylenedione,
anthracendiamine, benzoanphthothiophene
31 Acenaphthylene, Acenaphthylene, Acenaphthylene,
benzo[a]anthracene, benzo[a]anthracene, anthracene, benzo[alanthracene,
benzo[c]acridine, hexanediol, chrysene, hexanediol, benzo[clacridine,
dihydroindenone, dihydroindenone, anthracene, chrysene
acenaphthopyridine, acenaphthopyridine,
methylindanone, 2-pentanone, indenopyridine,
demethylfurylpuridine, acridine, methylacridine,
naphthopyran, indenquinoline, methylazaphenanthrene,
phenylcarbazole naphthopyrandione,
anthracenecarbonitrile, palmitic acid
aCompounds in italics are azaarenes.

strain YG1041, which yielded mutagrams analyzed by GC/MS, only 3 fractions structure). Several of these azaarenes
(Figures 1 and 2). Again, the two treat- contained any of the priority pollutant (acridine, 1-azapyrene, diaminotriazole, and
ments (BS and BP) had increased muta- PAHs (Table 1). The mutagenicity (+S9) methylazaphenanthrene) are mutagenic -S9
genicity as compared to the UTS, seen in fractions 30 and 31 of the BS and (Table 3).
especially in 11 of 40 fractions of the BS BP treatments is likely due to PAHs that Nitroarenes are another class of
(+S9, -S9) and in 6 fractions of the BP are positive in the Salmonella mutagenic- chemicals that were of concern in these
(+S9, -S9) (Figures 1 and 2). ity assay (Table 3). The mutagenicity in samples. Many nitroarenes (i.e., a nitro
To determine the cause of the increased other fractions, therefore, must be attrib- group attached to a PAH) are direct-acting
mutagenicity in these two treatments, the uted to compounds other than the 19 mutagens (i.e., they do not need S9 to be
mutagenic HPLC fractions, along with priority PAHs. Many of the other com- mutagenic). For a review of their muta-
corresponding fractions from the UTS, were pounds identified by GC/MS in the frac- genicity see Rosenkranz and Mermelstein
analyzed by GC/MS. Of the 11 mutagenic tions are azaarenes (i.e., PAHs where a (17). Nitroarenes are metabolized by the
fractions of the BS and BP treatments nitrogen replaces a carbon in the ring bacterial enzymes nitroreductase and

1 438 Environmental Health Perspectives * Vol 106, Supplement 6 * December 1998

 BIOASSAY-DIRECTED FRACTIONATION OF BIOREMEDIATED SOILS

Table 3. Identified compounds tested in the Salmonella mutagenicity assay.ab

Fraction Bioslurry Biopile
no. Compound Result Reference Compound Result Reference
14 No identified mutagens in the literature No identified mutagens in the literature
15 9H-carbazole Negative (12) 9H-carbazole Negative (12)
16 Acenaphthene Negative (13) Acenaphthene Negative (13)
Benzanthrone Positive, -S9 (12) Benzanthrone Positive, -S9 (12)
1-Azapyrene Positive, -S9, +S9 (12,14)
18 Fluorescein Negative (12,15) No identified mutagens in the literature
20 Anthracene dione Positive, -S9, +S9 (12) No identified mutagens in the literature
23 No identified mutagens in the literature No identified mutagens in the literature
24 Diaminotriazole Positive, -S9, +S9 (12) No identified mutagens in the literature
25 No identified mutagens in the literature No identified mutagens in the literature
27 No identified mutagens in the literature No identified mutagens in the literature
30 Pyrene Negative (12,15) Acenaphthylene Positive, +S9 (12)
Chrysene Positive, +S9 (12,16) Anthracene Positive, +S9 (13,15)
Hexone Negative (13)
31 Acenaphthylene Positive, +S9 (12) Acenaphthylene Positive, +S9 (12)
Benzo[alanthracene Positive, +S9 (15) Benzo[alanthracene Positive, +S9 (15)
Anthracene Positive, +S9 (13,15) Anthracene Positive, +S9 (13,15)
Chrysene Positive, +S9 (12,16) Chrysene Positive, +S9 (12,16)
Acridine Positive, -S9 (12)
Methylazaphenanthrene Positive, -S9, +S9 (12,16)
aResults are shown only for the mutagenic fractions. bCompounds in italics are azaarenes.

acetyltransferase. Salmonella strains strains YG1041 and YG1042 (7). The facilities contain azaarenes, oxygenated
YG1 041 and YG 1042 were developed from detection limits of the GC/MS were not PAH derivatives, and nitroarenes (21-23).
the standard Salmonella tester strains TA98 low enough to detect any nitroarenes that Azaarenes were also detected in BS frac-
and TAIOO (5), respectively, and they con- may have been present in the BS. Examples tions 20 and 24 (Table 2) and may have
tain elevated levels (50 x) of both nitrore- of the azaarenes found in these fractions been increased in the BS by the addition of
ductase and acetyltransferase activities (7).were acenaphthopyridine, acridine, 1-aza- the activated sludge. For example, acridine,
If higher mutagenic activity is detected in pyrene, benzo[c]carbazole, benzoquinoline, 1-azapyrene, diaminotriazole, and methy-
these strains than in TA98 and TA100, benzothiazolylphenol, carbazoles, diamino- lazaphenanthrene were uniquely identified
especially without S9 addition, the presence triazole, diphenylpyrazole, indenopyridine, in the BS. All have been cited as direct-
of nitroarenes could account for the muta- methylacridine, methylazaphenanthrene, acting mutagens (Table 3). The mutagenic
genicity in the test sample. This condition methylbenzacridine, and methylisothiazole. fractions for the BS in Figure 2 may be due
was true for the BS and BP extracts in this A large number of azaarenes have been to these compounds and the higher
study. The BS had mutagenic slopes (rever- reported in various creosotes (18) and in number of PAHs in the BS. In addition,
tants per microgram; calculated from the creosote-contaminated soil (19). The acridine, fluorescein (dyes), and tetra-
linear regression of the data) of 38.6 (-S9) azaarenes are of concern because of their chloroethane (an industrial product) were
and 31.4 (+S9), and the BP had mutagenic mutagenic activity and because the nitrogen identified in BS fractions 31, 18, and 24,
slopes of 3.0 (-S9) and 5.0 (+S9) (4). In atom in the ring system causes these com- respectively. The presence of these three
addition, when TA98NR (which lacks pounds to be weakly polar and considerably compounds may be evidence that the acti-
nitroreductase and has limited ability more water soluble than related PAHs. vated sludge added to the BS contained
to metabolize nitroarenes into active Azaarenes have been reported in ground- industrial chemicals.
mutagens) was used, the mutagenic activity water near creosote-contaminated sites Another possible reason the BS was
of the BS extract was reduced by 50% when including the Reilly Tar Site (1,2,10). more mutagenic than the other treatments
mutagenic results were compared to TA98, One reason the BS treatment was much was that the BS was the only treatment
which contains normal Salmonella nitrore- more mutagenic than the other treatments that was constantly aerated and mixed.
ductase (4). Therefore it was hypothesized could be that it was amended with 1% The BS had air bubbled to the bottom of
from these data that the mutagenic activity activated sludge (primary solid waste aer- the reaction vessels at the rate of 1.5
in the BS extract may be due to nitroarenes. ated and stirred to encourage bacterial ml/min and was stirred at 500 rpm (4).
This hypothesis, however, was not upheld growth) from a municipal wastewater- BS had 1% activated sludge added and BP
by chemical analysis. The mutagenic HPLC treatment facility that processed both had 1% cow manure added in an effort to
fractions in the BS and BP extracts were industrial and municipal waste (4). The increase the diversity of the bacterial pop-
analyzed by GC/MS and nitroarenes were sludge was added in an effort to increase ulation. The constant aeration in the BS
not detected; however, azaarenes were the diversity of the bacterial population in caused maximum mixing of all compo-
detected (Table 2). Nitroarenes can be the BS. Municipal wastewater and sludge nents, including the azaarenes. The BP
detected as mutagens at nanogram levels in from municipal wastewater-treatment contained azaarenes but mixing did not

Environmental Health Perspectives m Vol 106, Supplement 6 * December 1998 1 439

 BROOKS ET AL.

occur. CMP had 1% cow manure added, was most successful not only in reducing mutagenicity assay. Amendments should
but the treatment vessel was not aerated, the total PAH concentration but also the be evaluated for toxicity before addition to
and was mixed once a day by rolling the toxic potential of this soil. The PAH analy- a bioremediation process to ensure that
vessel for 30 min. LT had nothing added sis demonstrated that each treatment was they are nontoxic. Complex environmental
and was tilled once per week. Each of successful in reducing the total PAH mixtures such as these soil extracts can
these processes, therefore, had different concentration. However, the mutagenic contain hundreds of chemicals. Bioassay-
levels of anaerobic and aerobic metabolismactivity demonstrated that some treatments directed fractionation is an efficient
occurring. The differences in aeration, (BP and BS) actually increased the toxic method to identify signature mutagenic
mixing, and addition of sludge/manure potential of these soils. Also, chemical chemicals in such mixtures. The combina-
between the mutagenic treatments (BS, analysis along with the bioassay identified tion of bioassays and chemical analysis pro-
BP) and the other nonmutagenic treat- several problems. The initial PAH concen- vided a more complete and accurate
ments (CMP, LT) may account for the trations (Table 1) varied among the treat- evaluation of the four bioremediation
mutagenicity. Weak mutagenic activity ments and were due to the different sieving treatments in this pilot study.
was also detected in all five extracts in sizes (1/4 to 1 in) used for each of them. In DISCLAIMER: This manuscript has been
TA102 (4). This strain detects mutagenic the future, uniform particle sizes should be reviewed by the National Health and
aldehydes and ketones, which were used to more accurately compare the treat- Environmental Effects Research Laboratory,
detected in the BS and BP (Table 2). ments. Also, the increased mutagenicity in U.S. EPA, and approved for publication.
the treatments (BP and BS) could not be Approval does not signify that the contents
Conclusion directly associated with the amendments necessarily reflect the views and policies of
The pilot scale laboratory experiments of (i.e., activated sludge) added to these treat- the agency, and the mention of trade names
the creosote-contaminated field soil were ments because they were not analyzed or commercial products does not constitute
conducted to determine which treatment directly in the chemical analysis and the endorsement or recommendation for use.

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1440 Environmental Health Perspectives . Vol 106, Supplement 6 * December 1998