APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1981, p. 817-8230099-2240/81/110817-07$02.00/0

Vol. 42, No. 5

Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-TriazineN. G. McCORMICK,* J. H. CORNELL, AND A. M. KAPLAN

Environmental Protection Group, U.S. Arny Natick Laboratories, Natick, Massachusetts 01760

Received 16 April 1981/Accepted 8 July 1981

Biodegradation of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)occurs under anaerobic conditions, yielding a number of products, including:hexahydro- l-nitroso-3,5-dinitro- 1,3,5-triazine, hexahydro- 1,3-dinitroso-5-nitro-1,3,5-triazine, hexahydro-1,3,5-trinitroso-1,3,5-triazine, hydrazine, 1,1-dimethyl-hydrazine, 1,2-dimethylhydrazine, formaldehyde, and methanol. A scheme for thebiodegradation of RDX is proposed which proceeds via successive reduction ofthe nitro groups to a point where destabilization and fragmentation of the ringoccurs. The noncycic degradation products arise via subsequent reduction andrearrangement reactions of the fragments. The scheme suggests the presence ofseveral additional compounds, not yet identified. Several of the products are

mutagenic or carcinogenic or both. Anaerobic treatment of RDX wastewaters,which also contain high nitrate levels, would permit the denitrification to occur,with concurrent degradation of RDX ultimnately to a mixture of hydrazines andmethanol. The feasibility of using an aerobic mode in the further degradation ofthese products is discussed.

Among pollutants unique to the military arethose arising from the manufacture, handling,and demilitarization of munitions. Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is an explo-sive widely used for military purpose. A homo-log, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazo-cine (HMX), is a by-product of the synthesis ofRDX and is also used in RDX formulations.During the manufacture of RDX, up to 12 ,ug/ml may be discharged to the environment inprocess wastewaters (14). The question of thetoxicity of RDX to aquatic life has been ad-dressed (6, 23), and studies conducted by theOffice of The Surgeon General have recom-mended a 24-h average maximum allowable con-centration of 0.30 ,ug of RDX per ml of waste-water to protect aquatic life (23). It has not beenclearly established whether or not RDX is toxicin mammalian toxicity studies (11, 16, 17). Thesereports suggest that RDX per se may not presenta serious toxicity problem but that metabolicproducts derived from RDX may be toxic.A number of studies on the products formed

from the chemical decomposition of RDX havebeen reported. Hoffsommer et al. (13) reportedthat the alkaline hydrolysis of RDX yieldednitrate, nitrogen, ammonia, nitrous oxide, formicacid, formaldehyde, and traces of H2. The firststep in this reaction was a proton abstraction bythe base with simultaneous elimination of a ni-trite group from the adjacent ring nitrogen (4).Exposure of aqueous solutions of RDX to ultra-violet (UV) light resulted in the formation ofnitrate, nitrite, ammonia, formaldehyde, nitrous

oxide, formamide, and N-nitroso-methylenedi-amine (7). The combination of UV light withozone produced C02, cyanic acid, nitrate, am-monia, and formic acid (8).The decomposition of RDX in a biological

system has been reported. Osmon and Klaus-meier (15) found that some RDX disappearedduring soil enrichment studies, but evidence forRDX degradation by microorganisms was notobtained. The complete disappearance of RDXby mixed cultures of purple photosynthetic bac-teria was reported by Soli (22), who advancedthe hypothesis that the strongly reducing con-ditions ofthe culture during photosynthesis wereresponsible for disruption of the RDX molecule.Hoffsommer et al. (12) found no disappearanceof RDX in an aerobic activated-sludge system.Sikka et al. (19) reported the disappearance ofRDX, after a 20-day lag period, from river watersamples supplemented with river sediment. Theobservation was made that '4CO2 was evolvedfrom [14C]RDX depending on the source of thesediment.The products of the biological degradation of

RDX may pose more serious toxicological prob-lems than the RDX itself. Therefore, it is im-portant not only to assess the environmentalfate of RDX, but also to determine the nature ofthe products of biotransformation or biodegra-dation or both.

MATERIALS AND METHODS

Cultures and media. Biodegradation studies werecarried out in nutrient broth (Difco Laboratories, De-

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818 McCORMICK, CORNELL, AND KAPLAN

troit, Mich.). For aerobic studies the media were in-oculated with activated sludge obtained from theMarlboro Easterly Municipal Sewage TreatmentPlant, Marlboro, Mass. The volume of liquid did notexceed 10% of the total volume of the flask; the flaskswere incubated at 30°C on a reciprocating shaker (150strokes/min). For anaerobic studies the vessels werefilled to approximately 95% of their capacity, inocu-lated with anaerobic sewage sludge (obtained from theNut Island Sewage Treatment Plant, Boston, Mass.),and incubated as stationary cultures at 37°C. Theinocula were prepared by diluting the sludge with twovolumes of distilled water and filtering through glasswool. A 2% (vol/vol) inoculum was used.Due to the relative insolubilities of the compounds

used as substrates, weighed amounts were added toempty flasks and dissolved in a small amount of ace-tone. The acetone was evaporated by a stream of N2,leaving a thin film of material on the inside surface ofthe flask. The culture medium was deoxygenated byboiling, poured into the flask containing the deposit ofmaterial, and stirred vigorously until solution wasattained. The medium was allowed to cool to 35 to40°C before inoculation. Samples were removed fromthe culture vessels after various periods of incubationand centrifuged, and the supernatant solutions werefiltered through 0.22-,um membrane filters. The result-ing culture medium filtrates (CMF) were used for allanalytical procedures.

Liquid chromatography. Samples of CMF (10 to20 p1) were injected without further treatment into aWaters model 6000A liquid chromatograph equippedwith a ,tBondapak C-18 column and a model 450variable wavelength detector (Waters Associates, Inc.,Milford, Mass.). The solvent system used to monitorthe disappearance ofRDX was 20% methanol in water;solvent flow was 2.5 to 3.0 ml/min, UV detector at 230nm. For determination of formaldehyde, the solventwas 60% methanol in water, UV detector at 340 nm.Vacuum distillation. Samples of CMF (100 ml)

were placed in a 500-ml boiling flask fitted with anadaptor to which an empty flask was attached. TheCMF was frozen in liquid N2, a vacuum (0.01 mmHg,1.3 Pa) was established, and the system was closed off.The empty flask was placed in liquid N2, and thesample-containing flask was allowed to warm. After 2h the system was opened to atmospheric pressure, andthe colorless material which had collected in the sec-ond flask was assayed for radioactivity and subjectedto analysis by gas chromatography-mass spectrometry(GC/MS) for the presence of methanol.Determination of formaldehyde. CMF (125 ml)

was adjusted to pH 2 with H2SO4 and distilled. Thedistillate was collected with the delivery tube sub-merged below the surface of a small amount of waterto retain volatile components. To 100 ml of distillatewas added 1 ml of 10% 5,5-dimethyl-1,3-cyclohexane-dione (methone) in 95% ethanol. The mixture washeated to boiling for 5 min, cooled, and stored at 4°Cfor several days to allow crystallization to occur. Crys-tals were collected, washed with a small amount of ice-cold water, and dried (mp 192°C; literature 1890C[24]). The colorimetric determination of formaldehydewas carried out on 0.1-. to 1.0-ml portions of distillateby the chromotropic acid method as described by

APPL. ENVIRON. MICROBIOL.

Grant (9). The absorbance was determined at 570 nmwith a Coleman Junior spectrophotometer. A high-performance liquid chromatographic (HPLC) methodwas developed for determining formaldehyde directlyin aqueous systems, based on a method described byBeasley et al. (1) for the determination of formalde-hyde in air. The method involved the addition of 1.0ml of a filtered 0.1% solution of 2,4-dinitrophenylhy-drazine in 2 N HCl to 1.0 ml of CMF contained in asmall test tube. The contents were mixed and allowedto react for 10 min, and 10 to 20,ul was injected directlyinto the HPLC. Authentic 2,4-dinitrophenylhydrazoneof formaldehyde was synthesized (18) as a referencematerial (mp 166°C; literature 166°C).

Synthesis of [14CJRDX. The synthesis of [14C]-RDX was based on the method described by Schiesslerand Ross (U.S. patent 2,434,230, 1948). The reactionwas conducted in the screw-capped vial (60 by 17 mm)in which [14C]paraformaldehyde (1 mCi, 2.3 mg) wasreceived (New England Nuclear Corp., Boston, Mass.).The cap was fitted with a Teflon liner, and 23.4 mg offinely powdered paraformaldehyde was added to thevial to bring the total paraformaldehyde to 0.85 mmol.Finely powdered NH4NO3 (68.5 mg, 0.85 mmol) wasintroduced into the vial, together with a 10-mm Tef-lon-clad miniature stirring bar. Acetic anhydride (0.3ml, 3.2 mmol) was added, followed by 50 pl of borontrifluoride etherate. The vial was tightly closed andheated for 7 h at 65 to 70°C with stirring. At the endof the reaction time the vial was opened, and thestirrer was removed and washed with several drops ofacetic acid. The product was precipitated by the ad-dition of 0.3 ml of water and brought into solution byboiling the reaction mixture. The product was allowedto crystallize at room temperature for several hours.The supernatant solution was removed by decanta-tion, and the crystals were washed twice with theirvolume of water. The product was dried under acurrent of air at 70°C and finally at 0.1 (13.3 Pa)mmHg at 25°C. The yield of product was 28.9 mg(45.9% of theoretical). The infrared spectrum was iden-tical to that of a twice recrystallized authentic sampleof RDX obtained from Holston Army AmmunitionPlant, Holston, Tenn. Both samples exhibited thesame Rf on silica gel thin-layer cochromatography(TLC). A trace of HMX was eliminated by recrystal-lization from acetone.

Synthesis of TNX. Hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) was prepared by the method ofBrockman et al. (3). The crude product was recrystal-lized twice from 95% ethanol and then from benzeneto give 4.8 g of yellow needles (mp 105 to 107°C;literature 105 to 107°C). The recrystallized productgave a single spot when subjected to TLC analysis onEastman silica gel plates with fluorescent indicator.The developing solvent was benzene-ethanol (95:5),and the material was visualized by fluorescencequenching under UV light. The infrared spectrum wasconsistent with the structure, exhibiting bands at 1,449cm-l and 1,495 cm-' (N-NO). The mass spectrum ofthe product had a peak at m/z 174, corresponding tothe molecular ion, and a major fragment ion at m/z100.Synthesis of MNX and DNX. Hexahydro-l-ni-

troso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-

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BIODEGRADATION OF RDX 819

1,3-dinitroso-5-nitro-1,3,5-triazine (DNX) were syn-thesized from TNX by the method described by Si-macek (20). The synthesis yielded a mixture of MNX,DNX, and unreacted TNX. MNX and DNX wereisolated as a single slow-moving band by preparativeTLC and further purified by HPLC. MNX was sub-jected to analysis by GC/MS and exhibited a molec-ular ion at m/z 206 and a major fragment ion at m/z132. GC/MS analysis ofDNX showed a molecular ionat m/z 190 and fragment ion at m/z 116.

Isolation of hydrazine and its dimethyl deriv-atives. CMF was made alkaline with NaOH and dis-tilled in a rotary evaporator at 500C under 15 mmHg(2.0 kPa). The distillate was collected in dilute HCL.The colorless distillate was evaporated to dryness inthe same manner to yield residue A. For the isolationof salicylazine, residue A was dissolved in a smallvolume of water and neutralized with NaOH. Theresulting solution was shaken with 25 ml of 4% sali-cylaldehyde in benzene. The benzene layer was sepa-rated and evaporated to dryness at room temperatureunder a stream of N2. The residue was stored oversilica gel in a vacuum desiccator for several days toremove excess salicylaldehyde. The residue was sub-jected to TLC analysis with benzene as solvent. Areference sample of salicylazine, prepared by themethod of Blout and Gofstein (2), was cochromato-graphed with the unknown. On visualization with UVlight the TLC exhibited two principal spots, the lowerofwhich had the same Rfas salicylazine. The materialswas analyzed by GC/MS, and two principal peakswere observed. The larger had the same retention timeas salicylazine (molecular weight, 240), exhibiting amolecular ion at m/z 240. Its spectrum was identicalto that of the reference standard. A small peak withmolecular ion at m/z 239 was not futher identified.For the isolation of the dimethylhydrazines, residue Awas refluxed with 10 ml of methanol and cooled toroom temperature. The supernatant was removed toa small vial and evaporated to dryness in a blockheated at 700C. The methanol extraction of residue Awas repeated twice more with 3 ml each of methanol.The residue from the final evaporation was treatedwith 250 pl of acetic anhydride. The vial was sealedwith a Teflon-lined cap and heated at 650C for 10 min.Standards were concurrently prepared with 3 pl eachof methylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine treated in the same manner. Anadditional standard was prepared containing 3 ul of1,1-dimethylhydrazine and 10 mg of ammonium chlo-ride in addition to the acetylating mixture. The sam-ples were analyzed with a Bendix model 2500 gaschromatograph with a 6-ft (183 cm) Pyrex column(0.25-in [0.64 cm] diameter) packed with Tenax GC.The carrier gas was N2 at 40 ml/min; a flame ionizationdetector was used at 2500C, and the injection port wasmaintained at the same temperature. With the oventemperature at 2300C, the derivatized extracts fromthe CMF gave two small peaks. The peak at 4.6 mincorresponded to acetyl-1,2-dimethylhydrazine; theother peak at 3.5 min was not identified. No peakscorresponding to acetyl-1,1-dimethylhydrazine (1.6min) or acetylmethylhydrazine (4.3 min) were ob-served. However, when the oven temperature wasreduced to 1900C a larger peak was observed at 4.1

min, corresponding to acetamide-dimethylhydrazone.This compound is formed from 1,1-dimethylhydrazineunder derivatization conditions when an excess ofammonium chloride is present, as was the case inCMF. The identities of acetamide-dimethylhydrazone(m/z 101) and acetyl-1,2-dimethylhydrazine (m/z 102)were confirmed by GC/MS.

Distribution of radioactivity. CMF was adjustedto pH 3 with HCI and sparged with helium for 2 h.The gas passed sequentially through a series of trapswhich contained 0.1 M Na2SO3, 0.1 N HCI, water, and0.1 N NaOH, respectively. The sparging gas continuedthrough a copper oxide-packed column heated to7000C and, finally, through a second 0.1 N NaOH trap.After purging the acidified mixture for 2 h, the mixturewas adjusted to pH 11, and the sparging was continuedfor an additional 2 h. The resulting purged aqueousphase was neutralized and subjected to continuousether extraction for 24 h. The aqueous phase wasadjusted to pH 3 and similarly extracted with ether.Finally, the aqueous phase from the pH 3 ether ex-traction was adjusted to pH 11 and again extractedwith ether for 24 h. Samples (usually 1.0 ml) wereplaced in scintillation vials to which 10 ml of Aquasol-2 (New Enkland Nuclear) was added. Measurementsfor radioactivity were carried out in a model 3255Packard Tri-Carb liquid scintillation spectrometer.

RESULTSRDX disappearance. RDX at concentra-

tions of 50 or 100 fig/ml disappeared rapidlyfrom nutrient broth cultures inoculated withanaerobic sewage sludge andmincubated anaero-bically (Fig. 1). RDX disappearance was essen-tially complete after 4 days. Concentrations ofRDX remained unchanged when cultures wereinoculated with aerobic activated sewage sludgeand incubated aerobically. No RDX disappearedin uninoculated controls.Intermediate formation. HPLC analysis of

anaerobic reaction mixtures revealed the pres-ence of intermediates formed during the disap-pearance of RDX (Fig. 1). The fact that thereaction proceeded only under anaerobic condi-tions suggested that these intermediates mightbe reduced forms of RDX. Chloroform extrac-tion of an 8-day incubation mixture which ini-tially contained 50 mg of RDX per ml yieldedyellow crystals having the same melting point,infrared spectrum, and GC/MS as authenticTNX. Extraction of 2- to 3-day-old cultures (ap-preciable quantities ofMNX and DNX present)with ethyl acetate and subsequent 500-fold con-centration allowed MNX and DNX to be sepa-rated in sufficient quantity by HPLC to establishtheir identity by GC/MS. As with the synthe-sized reference compounds, a molecular ion atm/z 206 and a major fragment with m/z 132were detected from the compound correspond-ing to curve B (Fig. 1), and a molecular ion atm/z 190 and fragment ion at m/z 116 were

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820 McCORMICK, CORNELL, AND KAPLAN

E0)3%-

C0-a

h.O-

C

C0u

D

%% 0

N. ...

o 1 2 3 4 5 6 7 " 18Days

FIG. 1. Disappearance of RDX and production ofintermediates during anaerobic incubation. A, RDX;B, MNX; C, DNX; D, TNX; E, RDX incubated underaerobic conditions.

detected from the compound corresponding tocurve C. The material corresponding to curve Dwas identified as TNX by TLC, HPLC, and GC/MS, yielding a molecular ion at m/z 174 and amajor fragment ion at m/z 100.Distribution of radioactivity from [14C]-

RDX. A reaction mixture containing 50 mg ofRDX per ml, including 2 MCi of [14C]RDX, wasincubated anaerobically for 7 days. No 14C-la-beled gas was evolved during incubation. Theincubated mixture was sparged with helium andextracted with ether as described. The resultsare shown in Table 1. No more than 1.5% of thetotal 14C added to the system was found involatile (spargeable) material. Even after threecontinuous ether extractions, approximately 42%of the 14C remained in the aqueous phase. Mostof this remaining radioactivity disappeared uponevaporation of the sample to dryness at 45 to50°C under a stream of N2, regardless of pH.Radioactivity from 14C-labeled RDX was foundalmost exclisively in the soluble fraction fromthe earliest measurement (24 h) to the 21st day,with only 2% associated with the pellet or re-tained by membrane filters.Formaldehyde. The presence of HCHO in

distillates was demonstrated by the formation ofthe dimethone derivative and by the character-

istic and specific color reaction obtained withchromotropic acid. The amount of HCHO pro-duced from RDX increased to a maximum after1 to 2 days of incubation (Fig. 2), after which theconcentration declined to a negligible value.Methanol. The total "C found in the distil-

late could not be reconciled with the specificactivity expected if all of the "C in the distillatewas present as HCHO. However, initial attemptsto identify and quantitate the other '4C-contain-ing material led to the loss of most of the radio-activity. Since the entire reaction was the resultof an anaerobic process, there was the possibilitythat the polar, volatile, low-molecular-weight,carbon-containing compound might be metha-nol. Radioactive CMF from reaction mixtureswhich had been incubated 2 to 3 weeks weresubjected to vacuum distillation. Radioactivematerial collected in a liquid N2 trap was ana-lyzed by GC/MS. The presence of up to 300 ygof MeOH per ml (9 mM) in the distillate wasconfirmed. Kinetic data on the formation ofMeOH are not yet available.

TABLE 1. Distribution of radioactivity afteranaerobic incubation with ['4C]RDXTreatment Radioactivity (%)

None.. 100.0Volatiles (from traps) .1..1.5aEther extraction, pH 7' ..... 23.7Ether extraction, pH 3b 25.4Ether extraction, pH llb 7.8Aqueous phase .. 41.6

a Total radioactivity recovered from purging vola-tiles under acid and alkaline conditions.

b Continuous ether extraction for 24 h.

0 1 2 3 4 5 6 7Days

FIG. 2. Production offormaldehyde during the an-aerobic degradation ofRDX.

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0---I .-.

`111.E50

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BIODEGRADATION OF RDX 821

Hydrazines. The presence of hydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazinewas confirmed by GC/MS analysis of the hydra-zine derivatives. The bases were isolated as theirhydrochloride salts and were then treated withsalicylaldehyde or with acetic anhydride to yieldeither the disalicyl derivative of hydrazine (sal-icylazine) or the acetyl derivatives of the substi-tuted hydrazines. Infrared and GC/MS analysisand comparison with authentic salicylazine con-firmed the presence of hydrazine in the reactionmixture. Treatment of the salts of the bases withacetic anhydride yielded acetyl derivatives ofthe dimethylhydrazines. Comparisons with au-thentic compounds confirmed the presence ofboth 1,2-dimethylhydrazine and 1,1-dimethyl-hydrazine.

DISCUSSIONThe biodegradation ofRDX occurs only under

anaerobic conditions. Concurrent with the dis-appearance of RDX is the sequential buildupand disappearance of the mono-, di-, and trini-troso analogs of RDX (Fig. 1). The fact thatHCHO formation reaches a maximum in a shorttime (Fig. 2), whereas that of the nitroso deriv-atives lags behind, tends to rule out the possi-bility thatHCHO is produced solely by reactionssubsequent to the formation of the trinitrosoderivative and suggests that formaldehyde isproduced early in the reaction sequence fromprecursors of trinitroso-RDX. Uninoculated

O2N,N,NO2

NO,

OgN N"-N,NOtN)NO2

O2^N~N.NO2KNHONHOH

8

NHOH9

controls containing RDX orTNX and incubatedfor the same periods of time as the inoculatedflasks produced no HCHO. From the accumu-lated data we propose a pathway for the biodeg-radation of RDX as illustrated in Fig. 3.

In this scheme RDX is reduced sequentiallyto the nitroso derivatives, 2 (MNX), 3 (DNX),and 4 (TNX), each of which may undergo fur-ther reduction of a nitroso group to form thehypothetical compounds 5 (1-hydroxylamino-3,5-dinitro-1,3,5-triazine), 6 (1-hydroxylamino-3-nitroso-5-nitro-1,3,5-triazine), and 7 (1-hydroxy-lamino-3,5-dinitroso-1,3,5-triazine). We postu-late that the molecule becomes unstable whenany one of the nitro groups is reduced beyondthe nitroso level. At this point hydrolytic cleav-age, followed by rearrangement and further re-ductions of the fragments, gives rise to the endproducts observed. Cleavage of 5 via one routeyields products 8 (N-hydroxymethyl-methyl-enedinitramine) and 9 (N-hydroxymethylene-hydrazone), and cleavage of 6 via another routeyields 10 (N-hydroxylamino-N'-nitromethyl-enediamine) and 11 (dimethylnitrosamine).Compound 7 undergoes cleavage via eitherroute.Figure 4 shows the postulated reactions of the

fragments arising from the initial cleavage reac-tion. Cleavage of 8 releases 12 (HCHO) and 13(methylenedinitramine), which decomposes toyield HCHO and 14 (nitramide), which in turnis reduced to 15 (hydrazine). Compound 9 rear-

LINI)NO31~

OtKN N""N HOH

NO6

OIN'N"-N AHOHH H

10

HJXNCH,

11

ONI.NNNOK,N)_ kN|J

NO

4

ONeNHOH

I

ON.'NAHOHH H

10O

FIG. 3. Proposed pathway for the anaerobic biodegradation of RDX. Compounds: 1, RDX; 2, MNX; 3,DNX; 4, TNX; 5, 1-hydroxylamino-3,5-dinitro-1,3,5-triazine; 6, 1-hydroxylamino-3-nitroso-5-nitro-1,3,5-tria-zine; 7, 1-hydroxylamino-3,5-dinitroso-1,3,5-triazine; 8, N-hydroxymethylmethylenedinitramine; 11, dimeth-ylnitrosamine radical.

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822 McCORMICK, CORNELL, AND KAPLAN

HCF

OPN%./WNO,2 12

CHH

HO'C28

N-tCH2

NHOH \9 HN,CH2PH

NH2

16

FIG. 4. Proposed pathwaytion (continued). Compounds.enedinitramine; 14, nitramid,droxymethylhydrazine; 17, m

ranges and is reduced to 1drazine), which yields HiUnder the strongly reducidehyde is reduced to metbAs Fig. 3 shows, the ot]

(via compounds 6 and 7)methylenediamine, 10 andino-N'-nitroso-methylenecthe pathway taken by 13.-other product of this cle

d O N"NN'NO2 fold concentration was achieved. Since as muchH H as 300 ,ug of methanol per ml was detected in one

distillate sample, the original sample contained15 jig of methanol per ml, which is consistentwith the above. Thus, even in the absence of

HCHO + H2N-NO quantitative kinetic information on the forma-tion of methanol or the hydrazines, an apprecia-12 14 ble portion of the carbon can be accounted for.Since some of the carbon must be found in themethyl groups of the dimethylhydrazines, it is

I likely that we will be able to account for theHCHO + H2N-NH? majority of the carbon.

12 15 Several facts emerge pertaining to the nature12 15of the final products remaining in solution: (i) no

I spargeable, volatile '4C-containing compoundsCH3OH were detected; (ii) 98% of the radioactivity re-

17mained in the supernatant after the disappear-17

RDX biodegradaance of RDX, formaldehyde, and the nitroso

2for RDX biodegrada- derivatives of RDX; (iii) '4C was ether extract-

; 12,HCHO; 13,methyl- able from acidic, alkaline, or neutral solutions;e; 15 hydrazine; 16, hy- and (iv) '4C disappeared when either acidic, al-

kaline, or neutral solutions were evaporated todryness. These findings strongly suggested the

L6 (hydroxymethylhy- presence of carbon-containing, low-molecular-CHO and hydrazine. weight, polar, neutral compounds. Methanol andng conditions formal- formaldehyde were subsequently identified aslanol. two compounds with these properties. The pro-her cleavage reaction posed pathway includes several additional com-yields derivatives of pounds that fit this description, namely, dimeth-

I 10a (N-hydroxylam- ylnitrosamine and 1,2-dimethyldiazine-1-oxideliamine), which follow (azoxymethane) (compounds 18 and 22, respec-ks shown in Fig. 5, the tively); however, we have not detected these,avage, compound 11 compounds in reaction mixtures.

(dimethylnitrosamine radical), either undergoessequential reduction to 19 (1,1-dimethylhydra-zine) via 18 (dimethylnitrosamine) or rearrangesvia 20 (hypothetical intermediate) to yield 21(dimethyldiazene-1-oxide radical), which is re-duced to 23 (1,2-dimethylhydrazine) via 22 (di-methyldiazine-l-oxide). This scheme allows forthe formation of all of the observed products.

Figure 2 represents a steady-state concentra-tion, reflecting the formation of HCHO and itsconcomitant reduction to methanol, presumablyas soon as it is formed; thus the time of maxi-mum net generation of HCHO may not be trulyrepresented. If all the carbon in a solution of 50jig of RDX per ml (225 ,uM) were converted toHCHO, a concentration of 676 yM HCHO (20.3ug/ml) would result. Complete reduction tomethanol would yield a methanol concentrationof 21.6 ,ug/ml. The maximum steady-state con-centration of HCHO indicated in Fig. 2 was 5,ug/ml; thus, even under steady-state conditionsat least 25% of the carbon is accounted for.During the vacuum distillation of 100 ml ofCMF, approximately 5 ml of distillate was col-lected. If 100% efficiency were attained for thequantitative distillation of methanol, then a 20-

HtC&N,,,CH3 H2K NCH3 CH3I K0

N %.N4J _ N11 20 21

H,C,,WCH3

18

I~H3C22%#O

22

HCXN,.CH3 HNtCHS

NH2 H3C.**HNH19 23

FIG. 5. Proposed pathway for RDX biodegrada-tion (continued). Compounds: 18, dimethylnitrosa-mine; 19, 1,1-dimethylhydrazine; 20, hypothetical in-termediate; 21, dimethyldiazene-l-oxide radical; 22,dimethyldiazene-l-oxide; 23, 1,2-dimethylhydrazine.

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BIODEGRADATION OF RDX 823

The biological treatment of RDX-containingwastes must include an anaerobic mode since noreaction occurs aerobically. Since munitionswastes generally contain high levels of nitrate,RDX wastes can be treated in an anaerobicdenitrification mode. The result of such an op-eration would be a mixture of reduced com-pounds, including the hydrazines and methanol.Upon subsequent exposure to an aerobic stage,methanol would be oxidized to C02. Insufficientinformation is available on the biological fate ofhydrazine and the dimethyl hydrazines. Both1,1- and 1,2-dimethylhydrazine and their imme-diate precursors, dimethylnitrosamine and azox-ymethane, as well as hydrazine, are known mu-tagens or carcinogens or both (5, 10, 21), andtherefore it is most important to determine thebiodegradability ofthese compounds. Such stud-ies are currently in progress.

ACKNOWLEDGMENTSWe thank Carmine Di Pietro for performing the mass

spectral analyses and acknowledge the assistance of DianaFoster, Michael Whalen, Deborah Rollins, Linda Faulk, andBarry Jacobs in certain phases of this work. We expressappreciation to John Hoffsommer for his helpful discussions.

LITERATURE CITED1. Beasley, R. K., C. E. Hoffman, M. L. Rueppel, and J.

W. Worley. 1980. Sampling offormaldehyde in air withcoated solid sorbent and determination by high per-formance liquid chromatography. Anal. Chem. 52:1110-1114.

2. Blout, E. R., and R. M. Gofstein. 1945. The absorptionspectra of certain aldazines. J. Am. Chem. Soc. 67:13-17.

3. Brockman, F. J., D. C. Downing, and G. F. Wright.1949. Nitrolysis of hexamethylenetetramine. III. Prep-aration of pure cyclonite. Can. J. Res. 27:469-474.

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