Vol. 45, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1983, p. 1234-12410099-2240/83/041234-08$02.00/0Copyright ) 1983, American Society for Microbiology

Microbial Transformation of Nitroaromatic Compounds inSewage Effluent

LAURENCE E. HALLASt AND MARTIN ALEXANDER*Laboratory of Soil Microbiology, Department ofAgronomy, Cornell University, Ithaca, New York 14853

Received 22 September 1982/Accepted 30 December 1982

The transformation of mono- and dinitroaromatic compounds was measured insewage effluent maintained under aerobic or anaerobic conditions. Most of thenitrobenzene, 3- and 4-nitrobenzoic acids, and 3- and 4-nitrotoluenes and much ofthe 1,2- and 1,3-dinitrobenzenes disappeared both in the presence and absence ofoxygen. Under anaerobiosis, 2,6-dinitrotoluene and 3,5-dinitrobenzoic acid disap-peared slowly, but no loss was evident in 28 days in aerated sewage. Aromaticamines did not accumulate during the aerobic decomposition of the mononitrocompounds. They did appear in nonsterile, but not in sterile, sewage incubatedaerobically with the dinitro compounds and anaerobically with all the chemicals.Analysis by gas chromatography and combined gas chromatography-mass spec-trometry showed that aniline was formed from nitrobenzene, toluidine wasformed from 3- and 4-nitrotoluenes, and aminobenzoic acid was formed from 3-and 4-nitrobenzoic acids under anaerobiosis, and that nitroaniline was formedfrom 1,2- and 1,3-dinitrobenzenes, aminonitrotoluene resulted from 2,6-dinitrotol-uene, and aminonitrobenzoic acid was a product of 3,5-dinitrobenzoic acid underboth conditions. The isomeric forms of the metabolites were not established.Aniline, 4-toluidine, and 4-aminobenzoic acid added to sewage disappeared fromaerated nonsterile, but not from sterile, sewage or sewage in the absence ofoxygen. 2-Nitroaniline, 2-amino-3-nitrotoluene, and 2-amino-5-nitrobenzoic acidadded to sewage persisted for at least 60 days in aerobic or anaerobic conditions.Gas chromatographic and gas chromatographic-mass spectrometric analysesdemonstrated that acetanilide and 2-methylquinoline were formed from aniline, 4-methylformanilide and 4-methylacetanilide were formed from 4-toluidine, 2-methylbenzimidazole was a product of 2-nitroaniline, and unidentified benzimid-azoles were formed from 2-amino-3-nitrotoluene in the absence of oxygen, andthat 2-nitroacetanilide and 2-methyl-6-nitroacetanilide were formed from 2-ni-troaniline and 2-amino-3-nitrotoluene, respectively, in the presence or absence ofoxygen. It is suggested that the transformations of widely used nitroaromaticcompounds should be further studied because of the persistence and possibletoxicity of products of their metabolism.

Nitroaromatic compounds such as nitrophe-nols, nitrobenzene, nitrotoluenes, and nitroben-zoic acids are used in the manufacture of pesti-cides, dyes, explosives, and industrial solvents.The annual production of nitrobenzene aloneexceeds 500 million pounds (ca. 225 x 106 kg)(7), and it has been estimated that as much as 19million pounds of this chemical is dischargedannually into natural waters (25).

Nitroaromatic compounds are believed to beresistant to microbial attack (10), although me-tabolism of a few members of this class ofchemicals by pure cultures has been reported.Cartwright and Cain (4) noted the reduction of

t Present address: Process Development Group, MonsantoAgricultural Products Co., St. Louis, MO 63167.

nitrobenzoic acids by species of Nocardia andPseudomonas fluorescens. McCormick et al.(19) found that enzyme preparations of Veillon-ella alkalescens reduced 30 mono-, di-, andtrinitroaromatic compounds. They also foundthat Mucrosporium sp. converted 2,4-dinitrotol-uene to aminonitrotoluenes and 3-azo-oxy com-pounds (18). Recently, Corbett and Corbett (6)reported that Rhodosporidium sp. transformedchloronitrobenzene to chloroacetanilide andchlorohydroxyacetanilide. However, recent re-views cite only a few reports on the fate ofnitroaromatic compounds in natural environ-ments (1, 17, 20). Because many of these chemi-cals are toxic, a metabolic product that is struc-turally similar to the parent compound may alsobe inhibitory to some organisms; hence, if ni-

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TRANSFORMATION OF NITROAROMATICS IN SEWAGE 1235

troaromatics are modified by microorganisms innatural waters, soil, or sewage, it is important toidentify the products that are generated.The present investigation was designed to

determine whether nitroaromatic compoundsare metabolized in sewage effluent and to identi-fy products that might accumulate.

MATERIALS AND METHODSPrimary effluent of raw municipal sewage from the

Ithaca, N.Y. sewage treatment plant was amendedwith 10 gu.g of the test compounds per ml. When sterilesewage was used, it was first autoclaved for 20 min,cooled, and amended with 100 pLg of HgCl2 per ml toprevent subsequent contamination during sampling.The sewage samples, which had pH values of 7.3 to8.5, were incubated in the dark at 29°C in 250-mlflasks. For sewage maintained aerobically, 125-mlsamples were incubated on a rotary shaker operatingat 100 rpm. For sewage incubated under anaerobicconditions, side-arm flasks completely filled with sew-age were placed in an anaerobic incubator (NationalAppliance Co., Portland, Oreg.). Before and after eachsampling, the side-arm flasks were evacuated andflushed with N2 through a rubber stopper fitted withglass tubing packed with cotton. Samples were ob-tained through serum stoppers placed in the side-arm.Fresh sewage (5%, vol/vol) was added every 7 days toall samples of nonsterile sewage to provide additionalnutrients. Samples were taken at 0, 2, and 7 days andat weekly intervals thereafter. The sample suspensionswere centrifuged for 10 min at 4,100 x g, and thesupernatant fluid was stored at -10°C. The sampleswere again centrifuged after they were thawed, andanalyses were performed on the supernatant fluid.The disappearance of the test compounds, which

was measured with a double-beam spectrophotometer(model DB-G; Beckman Instruments, Fullerton, Cal-if.), was determined by dividing the area of the UVpeak in nonsterile samples by the area in sterilecontrols analyzed at the same time. Thus, disappear-ance of the compounds resulted from microbial activi-ty and not evaporation or another abiotic mechanism.The wavelengths scanned were: 250 to 300 nm fornitrobenzene and nitrobenzoic acids; 260 to 300 nm for3-nitrotoluene; 270 to 320 nm for 4-nitrotoluene; 220 to280 nm for 1,2-dinitrobenzene and 2-amino-5-nitroben-zoic acid; 220 to 260 nm for 3,5-dinitrobenzoic acid,1,3-dinitrobenzene, and 2,6-dinitrotoluene; 220 to 300nm for aniline; 260 to 320 nm for 4-toluidine and 2-nitroaniline; 270 to 340 nm for 4-aminobenzoic acid;and 270 to 310 nm for 2-amino-3-nitroaniline.The formation of aromatic amines was detected by a

modification of the procedure of Villanueva (26). A1.5-ml sample was adjusted to pH 2.0 with 0.375 ml of1 N HCI, and the liquid was mixed with 0.187 ml of afreshly prepared aqueous solution of 0.1% NaNO3.After 5 min, 0.187 ml of a 0.5% ammonium sulfamatesolution was added. The liquid was mixed, and 0.187ml of a 0.1% N-(1-naphthyl)ethylenediamine dihy-drochloride solution was added 3 min later. The colorwas allowed to develop for 1 h, and the absorbancywas then measured at 520 nm with a Bausch and Lomb(Rochester, N.Y.) Spectronic 20 spectrophotometer.The aromatic amine corresponding to the nitro com-

pound added as the test chemical was used as astandard for quantification.To identify the products of metabolism, the pH of

the supernatant fluid from the spent sewage wasadjusted to 12.0 with NaOH, and the solution wasextracted three times with ether. The aqueous phasewas then acidified with HCl to pH 2.0 and reextractedthree times with ether. The acidic and basic extractswere dried over anhydrous Na2SO4 and evaporated to1.0 ml. Each extract (0.2 ml) was then derivatized in awater bath for 1 h at 90°C with 0.05 ml of N-methyl-N-t-butyldimethylsilyltrifluoroacetamide (Regis Chemi-cal Co., Morton Grove, Ill.) or left underivatized. Theextracts of sewage amended with nitrobenzoic acidswere derivatized in a water bath for 5 min at 90°C with0.05 ml of boron trichloride in 15% methanol (Supelco,Bellefonte, Pa.).The samples were then analyzed with a Perkin

Elmer (Norwalk, Conn.) gas chromatograph, model3920B. For the detection of ether-soluble metabolites,each sample was analyzed on separate columnspacked with Tenax-GC, 10%o SP-2100 on 100/i20Supelcoport, and 3% Silar IOC on 100/120 Gas-Chrom Q. The Tenax column was operated at 200°Cfor 8 min and then programmed to 300°C at 4°C/min.The SP-2100 column was operated at 100°C for 4 minand programmed to 300°C at 8°C/min. The Silar col-umn was operated at 225°C. The temperature of theinjection port was maintained 10°C higher than themaximum temperature of the column oven, and thetemperature of the manifold was 300'C. The flow rateof the carrier gas, N2, was 20 ml/min. Individual peakswere further characterized by gas chromatography-mass spectrometry with a Finnigan (Sunnyvale, Calif.)9500 gas chromatograph connected to a Finnigan 3300quadrupole mass spectrometer and a Systems Indus-tries 150 Data System. For electron impact analysis,the mass spectrometer was operated at 70 eV. TheProbability Based Matching Computer Package (Cor-nell University, Ithaca, N.Y.) was used to interpretthe mass spectra.

Nitrobenzene, 2,6-dinitrotoluene, 3,5-dinitroben-zoic acid, 2- and 4-aminobenzoic acids, 2-amino-3-nitrotoluene, and 2-amino-5-nitrobenzoic acid wereobtained from Aldrich Chemical Co., Milwaukee, Wis.Aniline, 2- and 4-nitrotoluenes, 2- and 4-nitrobenzoicacids, 2- and 4-aminotoluenes, and 2- and 3-nitroani-lines were obtained from Eastman Kodak Co., Roch-ester, N.Y. The 1,2- and 1,3-dinitrobenzenes wereobtained from K & K Laboratories, Plainview, N.Y.All chemicals and analytical reagents were of thehighest purity available and were not purified further.Stock solutions (1%) of the compounds were stored at-10°C in the dark.

RESULTSThe disappearance of mononitro compounds

from sewage effluents under aerobic and anaero-bic conditions is shown in Fig. 1. Sterile controlswere run concurrently with experimental sew-age samples to assess possible changes fromabiotic mechanisms. Most of the UV absorbancefrom these compounds had disappeared in theabsence or presence of 02 in 14 days. Theapparently large amounts of 3-nitrotoluene re-

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1236 HALLAS AND ALEXANDER

wO 50z

E 25co

-J

z 100

LA775

8DAYS

FIG. 1. Decrease in absorbance of nitrobenzene(NB), 3-nitrobenzoic acid (3-NBA), 4-nitrobenzoicacid (4-NBA), 3-nitrotoluene (3-NT), and 4-nitrotolu-ene (4-NT) in sewage incubated in the presence orabsence of oxygen.

maining in the presence of air, and those ofnitrobenzene remaining in its absence, resultednot from appreciable quantities of residual sub-strate but rather from the formation of productsabsorbing UV light at similar wavelengths; thiswas confirmed when samples were extractedand analyzed by gas chromatography and massspectrometry.

Significant amounts of the UV absorbancy ofdinitro compounds persisted for 28 days (Fig. 2).Small amounts of 2,6-dinitrotoluene and 3,5-dinitrobenzoic acid but appreciable amounts of1,2- and 1,3-dinitrobenzenes disappeared underaerobic conditions. Under anaerobiosis, all ofthe absorbancy associated with 1,2-dinitroben-zene disappeared in 14 days, but significantamounts of the absorbancy associated with 1,3-dinitrobenzene, 2,6-dinitrotoluene, and 3,5-dini-trobenzoic acid remained after 28 days. Verylittle absorption of UV light at 200 to 350 nm wasdetected in sewage incubated aerobically withthe mononitro compounds. However, in sewageincubated anaerobically with the mononitrocompounds or aerobically and anaerobicallywith dinitro compounds, products were formedwith UV spectra different from those of theadded substrates.

Accordingly, samples of the amended sewage

were analyzed for the presence of aromaticamines. None was detected in sewage incubatedwith the mononitro compounds in the presenceOf 02. In contrast, large amounts were formedfrom the mononitro compounds under anaerobicconditions (Fig. 3). The apparently high yield ofthe amine relative to the amount of added pre-cursor results from the fact that the UV absor-bancy method is only semiquantitative. Signifi-cant amounts of aromatic amines were alsoformed from 1,2- and 1,3-dinitrobenzenes, 2,6-dinitrotoluene, and 3,5-dinitrobenzoic acid, bothanaerobically and aerobically (Fig. 4). Aromaticamines were not detected in sterile sewage incu-bated for the same period of time with the mono-or dinitro compounds.Samples of nonsterile and sterile sewage that

had been incubated with the nitro compoundswere extracted with ether, and the extracts wereanalyzed by gas chromatography using threecolumn packings and by combined gas chroma-tography-mass spectrometry. Only the test com-pounds were found in the sterile controls. Aro-matic amines were found in sewage incubatedanaerobically with the mononitro compounds,and aminonitroaromatic compounds were de-tected in sewage incubated aerobically or anaer-obically (Table 1). The identifications werebased on cochromatography of the unknowns

40z0-m° 20

-J

F-80z

oU.0* 60r

FIG. 2. Decrease in absorbance of dinitroaromaticcompounds in sewage incubated in the presence orabsence of oxygen.

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TRANSFORMATION OF NITROAROMATICS IN SEWAGE 1237

10

,-- 8

z20

0

4 8 12DAYS

FIG. 3. Formation of aromatic amines in sewageincubated in the absence of oxygen with five nitroaro-matic compounds (10 ,ug/ml).

with authentic standards and comparisons of themass spectra of the unknowns with publishedspectra for the authentic chemicals (23). Thespecific isomeric forms of the products were notestablished, although gas chromatographic anal-yses of the products were performed with theisomers corresponding to the nitro compoundused as substrate. The retention times given inTable 1 are those from the Tenax-GC column for

all chemicals except for the mono- and dinitro-benzoic acids, in which cases the values arefrom the Silar lOC column. These analyses con-firmed that some of the substrate remained insewage amended with dinitro compounds.Aromatic amines and aminonitro compounds

at the concentration of 10 ,ug/ml were added tofresh sewage, and their disappearance with timewas measured spectrophotometrically. Nochange was evident in 53 days in the UV absor-bancy of 4-toluidine and 4-aminobenzoic acidunder anaerobiosis in sewage amended withthese chemicals. Little decline was evident inthe absorbancy of aniline in this period of time(Fig. 5). In contrast, the UV absorbancy of thesethree chemicals disappeared under aerobic con-ditions. Moreover, a significant amount of theabsorbancy of 2-amino-3-nitrotoluene and 2-amino-5-nitrobenzoic acid remained under aero-bic as well as anaerobic conditions, and onlyunder aerobiosis and after 28 days was theabsorbancy of 2-nitroaniline appreciably re-duced. These results confirm the resistance ofthese compounds to microbial attack.To determine whether the amines were toxic,

each was added at concentrations of 1, 10, and50 ,ug/ml to 100-ml portions of a liquid mediumcontaining 60 mg of beef extract, 100 mg ofpeptone, and 10 ml of fresh sewage. Turbiditywas observed in each instance within 24 h.Sewage that had been amended with 10 jig of

the amines or aminonitroaromatic compoundsper ml and incubated for 60 days was extractedwith ether and analyzed by gas chromatographyand combined gas chromatography-mass spec-trometry. The original substrates, but no prod-ucts, were detected in sterile sewage amendedwith these substances. In contrast, neither thesubstrates nor organic products were found insewage incubated aerobically with the mononi-tro compounds. The original substrates, but no

TABLE 1. Products detected in sewage incubated in the presence or absence of oxygen with mono- anddinitroaromatic compounds

Condition ProductAdded chemical of Retention

incubation' time (s) Identty

Nitrobenzene An 498 Aniline3-Nitrotoluene An 624 Toluidine4-Nitrotoluene An 636 Toluidine3-Nitrobenzoic acid An 432 Aminobenzoic acid4-Nitrobenzoic acid An 444 Aminobenzoic acid

1,2-Dinitrobenzene Aer, an 1,152 Nitroaniline1,3-Dinitrobenzene Aer, an 1,110 Nitroaniline2,6-Dinitrotoluene Aer, an 1,200 Aminonitrotoluene3,5-Dinitrobenzoic acid Aer, an 540 Aminonitrobenzoic

acid

a Aer, Aerobic; an, anaerobic.

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1238 HALLAS AND ALEXANDER

7 14 21 28DAYS

FIG. 4. Formation of aromatic amines in sewageamended with dinitroaromatic compounds and incu-bated in the presence or absence of oxygen.

products, were found in sewage supplementedwith 4-aminobenzoic acid and incubated anaero-bically and in sewage amended with 2-amino-5-nitrobenzoic acid and incubated aerobically oranaerobically. Most of the aniline and 4-tolu-idine was recovered from sewage incubated an-aerobically, but none remained in samples incu-bated aerobically. Most of the 2-nitroaniline and2-amino-3-nitrotoluene was also recovered fromsewage that was incubated both aerobically andanaerobically. The relative amounts of thechemicals present after 60 days were establishedby comparing recoveries in the incubated sew-age with those in sewage supplemented withknown concentrations of the compounds.A variety of products were found in sewage

amended with aniline, 4-toluidine, 2-nitroani-line, or 2-amino-3-nitrotoluene. These productswere detected by gas chromatography and iden-tified by mass spectrometry (Table 2). The etherextracts of amended sewage were chromato-graphed on columns containing three packingmaterials, but the retention times presented arethose from the 10% SP-2100 column. The identi-fications of acetanilide, 4-methylacetanilide, 2-nitroacetanilide, 2-methyl-6-nitroacetanilide,and 4-methylformanilide were confirmed bycomparisons of their mass spectral fragmenta-tion patterns with those of the authentic chemi-

cals (23). Particularly noteworthy were the find-ings of 2-methylquinoline (quinaldine) and 2-methylbenzimidazole in sewage amended withaniline and 2-nitroaniline, respectively. This isthe first report of the microbial formation ofthese two products. These products were identi-fied by mass spectrometry with the aid of theProbability Based Matching Computer Package.The mass spectra are given in Fig. 6. Resultsfrom the computer system also suggested that 2-amino-3-nitrotoluene was converted in sewageto metabolites having fragmentation patternssimilar to benzimidazoles, but the spectra ofthese unknown benzimidazoles did not coincidewith published spectra (23).

DISCUSSIONMicroorganisms in sewage effluent can appar-

ently metabolize a variety of mono- and dinitrocompounds under aerobic and anaerobic condi-tions. The UV absorbancy of the mononitrocompounds disappeared in 7 days in the pres-ence of oxygen, suggesting ring cleavage andpossible mineralization. Under anaerobiosis,however, these molecules were transformed toaromatic amines. The confirmed or likely prod-ucts formed are shown in Fig. 7. Each chemicalwas shown to be reduced to a monoamino

80

60

UJX 40z

m 20

Co

Xi 100

Z 80U-

0

* 60

401-

201-

10 20 30DAYS

40 50

FIG. 5. Decrease in absorbance of aromatic aminesand aminonitroaromatic compounds in sewage incu-bated in the presence or absence of oxygen.

_AIO5NTOEZI ACID

2-NITROANILINE2-AMINO-3-NITROTOLUENE

- 4-AMINOBENZOIC ACID

* 2-ITRANNILINEAN ROCLUIDINE AEROBIC

- aN~~~~~~~~~E

\2-AMINO-3-\NITROTOLUENE

2-NITROANIIE

ANAEROBIC

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TRANSFORMATION OF NITROAROMATICS IN SEWAGE 1239

TABLE 2. Products detected in sewage incubated in the presence or absence of oxygen with aminoaromaticand aminonitroaromatic compounds

ProductAdded chemical Condition ofincubationa Retention Identity

time (s)

Aniline An 192 AcetanilideAn 216 2-Methylquinoline

4-Toluidine An 282 4-MethylformanilideAn 306 4-Methylacetanilide

2-Nitroaniline Aer, an 372 2-NitroacetanilideAn 420 2-Methylbenzimidazole

2-Amino-3-nitrotoluene Aer, an 450 2-Methyl-6-nitroacetanilide

An 492, 510 Benzimidazolesa Aer, Aerobic; an, anaerobic.

product, R in reaction 1 referring to CH3 orCOOH in the 3 or 4 position, and R in reaction 3indicating an H or CH3. In not every instancewas the structure of the isomer confirmed. Sev-eral of the monoamino products were then acet-ylated to yield the acetanilide and, in one in-stance, the corresponding formanilide. Inreaction 2, R represents CH3 in the 4 position. Inone instance each, a quinoline and a benzimid-azole were identified as a product of the trans-formation.Axenic cultures of microorganisms have been

found to reduce nitro compounds. The sub-strates include a number of mono- (4, 6), di- (18,19), and trinitroaromatics (19). The intestinalmicroflora of several species of mammals hasbeen found to reduce 2,4-dinitrotoluene, withthe reaction sequence involving the formation ofthe aminonitro compound before the productionof diaminotoluene (13).The persistence of the reduced products in the

present study suggests that the supply of nutri-ents needed for further metabolism of theamines is depleted, that inhibitors accumulate insewage, that the aromatic amines themselves aretoxic, or that they are inherently resistant todecomposition. Nutrient depletion was probablynot significant because fresh sewage was usedand added weekly. Because the amines persistedeven when added to fresh sewage, their longev-ity in sewage amended with the original nitrocompounds is probably not a result of the accu-mulation of toxins. Moreover, the fact thatgrowth was observed in diluted nutrient brothcontaining 100 ,ug of the amines per ml andinoculated with sewage suggests that somemicroorganisms can grow in the presence ofthese compounds, although the chemicals maybe toxic to the very microorganisms capable ofmetabolizing them.

Horowitz et al. (15) reported that severalaromatic amines are highly resistant to microbialattack under anaerobic conditions. Yet, pub-lished reports suggest that aromatic amines aremetabolized in pure culture, albeit slowly. Forexample, Corbett and Corbett (6) found thatRhodosporidium graminis formed 4-chloroacet-anilide and 4-chloro-2-hydroxyacetanilide from

CH3

6'|; ; : i 143

2-METHYLOUINOLINEW IItILslI s5iII Z7 11 1,.15z | | T128

50 63 76 10Z 38 Ii 89

JiIINTrCH3 132

2-METHYLBENZIMIDAZOLE

6339 5210

Ii~.liii 77 901030 50 70 90 1 10

MASS NUMBER (m/z)130 150

FIG. 6. Mass spectra of unknowns identified as 2-methylquinoline and 2-methylbenzimidazole.

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1240 HALLAS AND ALEXANDER

NO2 NH2

NO2 1,NH2

2 _

NHCOCH3

0+NHCOCH3

0+NO2 NH2 NHCOCH3ON02 N02 ON02

NO2

ON02

9 H3

NHCHO

R

NI=CH3N

NO2

WNH2

NO2 NH

HOOC$ 02HOOhNO2FIG. 7. Products of the metabolism of mono- and

dinitroaromatic compounds in sewage. The majormass fragments are designated by numbers.

degradation has been reported for structurallysimilar molecules. Thus, quinoline is degradedin soil (21), the compound probably being me-tabolized under aerobic conditions through kyn-urenic acid (3). Imidazoles are thought to bemetabolized by way of formiminoglycine andglycine (3).The formation and accumulation of some of

these products from widely used nitro com-pounds are causes for potential concern or atleast further study. This is particularly truebecause aminonitro compounds such as 4-ami-no-2-nitrophenol and 5-nitro-o-toluidine are re-ported to be carcinogenic (14). As little as 10 ,ugof 2-methylquinoline per ml is teratogenic whentested on Xenopus laevis (8), and methylbenzim-idazoles may also be acutely toxic (9). Hence,attention should be given to establishing whichcompounds are formed and persist in naturalecosystems receiving the nitro compounds.

ACKNOWLEDGMENTS

We thank T. Wachs and J. E. Houser for assistance withmass spectrometry.

This research was supported by Public Health Servicetraining grant ES-07052 from the Division of EnvironmentalHealth Sciences, National Institutes of Health.

4-chloroaniline. Furthermore, although severalchlorinated anilines persist in soil (24), smallamounts of azobenzene derivatives are knownto be formed (2). In addition, Cerniglia et al. (5)reported that two cyanobacteria strains trans-formed aniline to formanilide and acetanilide,reactions similar to those noted in this study.

This is the first report of the microbial forma-tion of 2-methylquinoline and of the biosynthe-sis of benzimidazoles from nitroaniline and 2-amino-3-nitrotoluene. Benzimidazoles havebeen reported to be formed in soil from dini-troaniline herbicides such as Dinitramine (16),Oryzalin (12), and Pendimethalin (22). Golab(11) found that nine benzimidazoles were pro-duced from Trifluralin in flooded soil, and hesuggested that acetanilides and hydroxyacetani-lides were intermediates in the formation of thebenzimidazoles. Similarly, the 2-methylbenzim-idazole noted in this study could be formed from2-nitroaniline with 2-nitroacetanilide and then 2-hydroxylaminoacetanilide as intermediates. It isalso possible that 2-methylbenzimidazole isformed by a high temperature-mediated ringclosure of the microbiologically formed 2-amino-acetanilide in a thermal reaction occurring dur-ing gas chromatographic analysis. The 2-methyl-quinoline could be formed by 2-hydroxylation ofthe acetanilide generated from aniline and then acondensation of a C2 compound with 2-hydroxy-acetanilide to yield the nitrogen heterocycle.The accumulation of 2-methylquinoline and 2-methylbenzimidazole is noteworthy because

LITERATURE CITED

1. Alexander, M. 1981. Biodegradation of chemicals of envi-ronmental concern. Science 211:132-138.

2. Bartha, R., and D. Pramer. 1967. Pesticide transformationto aniline and azo compounds in soil. Science 156:1617-1618.

3. CaUely, A. G. 1978. The microbial degradation of hetero-cyclic compounds. Prog. Ind. Microbiol. 14:205-281.

4. Cartwright, N. J., and R. B. Cain. 1959. Bacterial degra-dation of the nitrobenzoic acids. 2. Reduction of the nitrogroup. Biochem. J. 73:305-314.

5. Cerniglia, C. E., J. P. Freeman, and C. Van Bateen. 1981.Biotransformation and toxicity of aniline and aniline de-rivatives in cyanobacteria. Arch. Microbiol. 130:272-275.

6. Corbett, M. D., and B. R. Corbett. 1981. Metabolism of 4-chloronitrobenzene by the yeast Rhodosporidium sp.Appl. Environ. Microbiol. 41:942-949.

7. Dorigan, J., and J. Hushon. 1976. Air pollution assess-ment of nitrobenzene. Technical report MITRE-7228. TheMitre Corp., McLean, Va.

8. Dumont, J. N., T. W. Schultz, and R. D. Jones. 1979.Toxicity and teratogenicity of aromatic amines to Xeno-pus laevis. Bull. Environ. Contain. Toxicol. 22:159-166.

9. Evans, C. H., and E. G. Brown. 1974. Observations on theinhibition of the growth of Saccharomyces cerevisiae bybenzimidazole. Biochem. Soc. London Trans. 2:422-424.

10. Fewson, C. A. 1981. Biodegradation of aromatics withindustrial relevance, p. 141-179. In T. Leisinger, A. M.Cook, R. Hutter, and J. Nuesch (ed.), Microbial degrada-tion of xenobiotics and recalcitrant compounds. Academ-ic Press, London.

11. Golab, T., W. A. Althaus, and H. L. Wooten. 1979. Fateof [(4C]Trifluralin in soil. J. Agric. Food Chem. 27:163-179.

12. Golab, T., C. E. Bishop, A. L. Donoho, J. A. Manthey,and L. L. Zornes. 1975. Behavior of 14C oryzalin in soiland plants. Pestic. Biochem. Physiol. 5:196-204.

13. Guest, D., S. R. Schnell, D. E. Rickert, and J. G. Dent.1982. Metabolism of 2,4-dinitrotoluene by intestinalmicroorganisms from rat, mouse, and man. Toxicol.Appl. Pharmacol. 64:160-168.

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14. Hehnes, C. T., V. A. Fung, B. Lewin, K. E. McCaleb, S.Malko, and A. M. Pawlovlch. 1982. A study of aromaticn:ro compounds for the selection of candidates for car-

cinogen bioassay. J. Environ. Sci. Health Part A Environ.Sci. Eng. 17 75-128.

15. Horowitz, A., D. R. Shelton, C. P. Cornell, and J. M.Tiedje. 1982. Anaerobic degradation of aromatic com-

pounds in sediment and digested sludge. Dev. Ind. Micro-biol. 23:435-444.

16. Kearney, P. C., J. R. Plimmer, W. B. Wheeler, and A.Kontson. 1976. Persistence and metabolism of dinitroani-line herbicides in soils. Pestic. Biochem. Physiol. 6:229-238.

17. Kobayashi, H., and B. E. Rittmann. 1982. Microbial re-

moval of hazardous organic compounds. Environ. Sci.Technol. 16:170A-183A.

18. McCormick, N. G., J. H. Cornell, and A. M. Kaplan.1978. Identification of biotransformation products from2,4-dinitrotoluene. Appl. Environ. Microbiol. 35:945-948.

19. McCormick, N. G., F. E. Feeherry, and H. S. Levinson.1976. Microbial transformation of 2,4,6-trinitrotolueneand other nitroaromatic compounds. Appl. Environ. Mi-crobiol. 31:949-958.

20. Patterson, J. W., and P. S. Kodukala. 1981. Biodegrada-tion of hazardous organic pollutants. Chem. Eng. Prog.77:48-55.

21. Robblns, W. J. 1916. The cause of the disappearance ofcumarin, vanillin, pyridine and quinoline in the soil.Science 44:894-895.

22. Smith, R. H., J. E. Olver, and W. R. Lusby. 1979. Degra-dation of pendimethalin and its N-nitroso and N-nitroderivatives in anaerobic soil. Chemosphere 8:855-861.

23. Stenhagen, E., S. Abrahamson, and F. W. McLafferty.1974. Registry of mass spectral data, vol. 1. John Wileyand Sons, New York.

24. Thompson, F. R., and C. T. Corke. 1969. Persistence andeffects of some chlorinated anilines on nitrification in soil.Can. J. Microbiol. 15:791-7%.

25. United States Environmental Protection Agency. 1978. Ni-trobenzene. Initial report of the TSCA Interagency Test-ing Committee to the administrator. EPA 560-10-78/001.U.S. Environmental Protection Agency, Washington,D.C.

26. Villanueva, J. R. 1961. Organic nitro compounds reducedby Nocardia. V. Microbiol. Esp. 14:157-162.

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