AD-A220 610D IC FILE COPY

* Environmental TransformationProducts of Nitroaromatics andNitraminesLiterature Review and Recommendationsfor Analytical Method DevelopmentMarianne E. Walsh February 1990

DTICai S ELECT ED

hmd LU APR16 990 IVobm-"U(l B "

For conversion of Simetric units to U.S./Brifish customaryunits of measurement consult ASTM Standard E380,Metric Practice Guide, published by the AmericanSociety for Testing and Materials, 1916 Race St., Phila-delphia, Pa. 19103.

Special Report 90-2U.S. Army Corpsof EngineersCold Regions Research &Engineering Laboratory

Environmental TransformationProducts of Nitroaromatics andNitraminesLiterature Review and Recommendationsfor Analytical Method DevelopmentMarianne E. Walsh February 1990

Prepared forU.S. ARMY TOXIC AND HAZARDOUS MATERIALS AGENCYCETHA-TE-CR-89205

Approved for public release; distribution is unlimited.

PREFACE

This report was prepared by Marianne E. Walsh, Research Physical Scientist,Applied Research Branch, Experimental Engineering Division, U.S. Army ColdRegions Research and Engineering Laboratory. Funding for this project was pro-vided by the U.S. Army Toxic and Hazardous Materials Agency, Aberdeen ProvingGround, Maryland (R-90 Multi-Analytical Services), Martin H. Stutz, Project Moni-tor.

The author gratefully acknowledges Dr. Thomas F. Jenkins, Dr. Charles M.Reynolds and Daniel C. Leggett of CRREL, and Martin H. Stutz of USATHAMA, fortechnical reviews of the manuscript, and Mark Hardenberg of CRREL for editorialreview. Special thanks are given to Cynthia Whitney of the CRREL library staff foracquiring the literature necessary for this review.

The contents of this report are not to be used for advertising or promotionalpurposes. Citation of brand names does not constitute an official endorsement orapproval of the use of such commercial products.

CONTENTS

Preface ................................................................................................... iiAbbreviations............................................................................................... ivIntroduction..............................................................................................Nitroaromatics ................................ :................................................. .......

2,4,6-Trinitrotoluene (TNT).....................................................................2,4-Dinitrotoluene (2.4-DNT) ...................................................................... 6Trinitrobenzene (TNB) and l,3-dinitrobenzene (DNB) ................................. ......... 7

Nitramines ............................................................................................... 9Hexahydro- I .3.5-trinitro- I .3.5-triazine (RDX) ................................................... 8Octahydro- I ,3.5,7-tetranitro- I .3,5,7-tetrazocine (HMX) ........................................ 9Tetryl..............................................................................................10

Requirements for analytical methods .................................................................... IIConclusions.............................................................................................. 12Literature cited ............................................................................................. 12Appendix A: Analytical methods.............................................................. ....... 17Abstract.................................................................................................. 23

ILLUSTRATIONS

Figure1. Pathway for TNT ..............................................................................2. Pathway to 2-amino-4,6-dinitrobenzoic acid................................................... 43. Pathway for 2,4-DA degradation................................................................ 54. Pathway for 2,4-DNT............................................................................. 75. Pathway for RDX............................................................................... 96. Chromatograms at various stagzes of HMX biotransformation .............................. 107. Photolytic products of HMX .................................................................... 108. N-methyl picramide as a transformation product of tetryl .................................... 19. Chromatograrn of tetryl degradation product .................................................. I I

TABLE

1. Summary of degradation products of TNT reported prior to 1980 ......................... 2

Accesso or

NTIS QRA&I -

DTIC TAB 0Unannounced 0Just ifioatio

J Distribution/%4 K val-ab-ility -Codes

jAva 11 and/or

Dit Special

ABBREVIATIONS

2-A 2-amino-4,6-dinitrotoluene4-A 4-amino-2,6-dinitrotoluene2,4-DA 2,4-dianiino-6-nitrotoluene2,6-DA 2,6-diamidno-4-nitrotoluene2-OFIA 2-hydroxylamidno-4,6-dinitrotoluene4-01-A 4-hydroxylamino-2,6-dintrotolune4,4'-Az 2,2',6,6'-tetranitro-4,4'-azoxytoluene2,2'-Az 4,4',6,6'-tetranitro-2,2'-azoxytoluene2,4'-Az 2',4,6,6-tetranitro-2,4'-azoxytolueneTNB trinitrobenzeneFAD flavin adenine dinucleotideNAD nicotinamide adenine dinucleotideNADH nicotinamide adenine dinucleotide, reduced formDNT dinitrotolueneDNB 1,3-dintrobenzeneTNT 1,3,5-trinitrotolueneTHB trihydroxybenzeneH-MX octahydro-1,3,5,7-tetranitro-I,3,5,7-tetrazocineRDX hexahydro-1,3,5-trinitro-1,3,5-triazineMINX hexahydro-1 -nitroso-3,5-dinitro-1,3,5-triazineDNX hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazineTNX hexahydro-1,3,5-trinitroso-1,3,5-triazineAcH-MX octahydro-l-(N)-acetyl-3,5,7-trinitro-1 ,3,5,7-tetrazocine (SEX)AcRDX hexahydro-l-(N)-acetyl-3,5-dinitro-1,3,5-triazine (TAX)GC/MS gas chromatography /mass spectrometry

iv

Environmental Transformation Products ofNitroaromatics and Nitramines

Literature Review and Recommendations forAnalytical Method Development

MARIANNE E. WALSH

INTRODUCTION and a bay. Isbister et al. (1980) reviewed studies onmicrobial degradation pathways of TNT, 2,4-DNT

One of the Army's major environmental prob- and RDX. The conclusions of both reports werelems is the contamination of soil and water with that the TNT molecule is transformed by reduc-residues of explosives at many military facilities. tion of the nitro groups to form monoaminodini-This contaminatioii occurred during the disposal trotoluenes (2-A, 4-A) and condensation productsof wastewater from production and load-and- such as tetranitroazoxytoluene. Nitroreduction ispack operations, burning or detonation of off- accomplished by various microorganisms in-specification material, and destruction of out of cluding bacteria, yeast and fungi. No definitivedate explosives.\To monitor these sites, analytical evidence of ring cleavage was found. Transforma-methods were required to detect and quantify tion products reported in the literature prior tothese military-unique compounds in environ- 1980 are listed in Table 1.mental matrices. Uniter the auspices of U.S. Army Reports published since 1980 describe the ex-Toxic and Hazardous Materials Agency (THAMA), tensive work on composting by researchers at theCRREL has developedrethods for the deter- U.S. Army Natick Research and Developmentmination of explosives in ervironmental samples. Laboratory (Kaplan and Kaplan 1982a,b,c) and atSince some of the transformatilon products of these the Atlantic Research Corporation (Isbister et al.explosives may be of environm\ tal concern, it 1980, 1984). Information on microbiological de-may also be necessary to devel6pR analytical giadation products has also been published in themethods for some of these byproducts.The objec- Russian, Chinese and Indian literature.

tive of this study was to review the literature to The biotransformation pathway for TNT inidentify the products formed after explosives en- simulated composting systems proposed by Ka-ter the environment and to identify those com- plan and Kaplan (1982a) is shown in Figure 1. The

pounds for which certified analytical methods compost was incubated with uniformly labeledshould be developed. Also, analytical methodol- 14C-TNT for 91 days. After 24 days, TNT, 2-A andogy already developed for these compounds is 4-A were identified in the test compost, with 4-A

summarized. as the predominant amine. After 91 days, TNT, 2-A, 2,4-DA, 2,6-DA, 4,4'-Az and 2,4'-Az were ex-tracted. No significant 14 CO2 was recovered.

NITROAROMATICS However, with time, a progressively greater per-centage of 14C-labeled material appeared to bind

2,4,6-Trinitrotoluene (TNT) to the organic matter fractions of the compost. TheThe microbial transformation of 2,4,6-trinitro- authors (Kaplan and Kaplan 1982b) caution that

toluene (TNT) has been studied extensively. Two the "mere disappearance of a compound underliterature reviews concerning the environmental composting conditions is not sufficient evidencefate of TNT were published in 1980. Spanggord et that the resulting environmental hazard has beenal. (1980a) compiled the literature on the transport effectively eliminated." They (Kaplan and Kaplanand transformation products of TNT and ten other 1983) found similar binding reactions betweenmunition wastewater constituents in two rivers TNT and its metabolites and humic material in

.1_

Table 1. Summary of degradation products of TNT reported prior to

1980.

Media End products

Jerger and Chynoweth Anaerobic 4-OHA(1966) digesters

Enzinger (1970) Broth cultures Probably 4-A and 4,4'-Az

Klausmeier et al. Fungi, yeasts, Reduction products(1973) actinomycetes,

gram-positive

Won et al. (1974) Enrichment Azoxytoluenes, 2-A, 4-cultures with OHA, DA'sPseudomonas-likeorganisms

Traxler et al. (1974) Gram-negative Some CO 2bacteria, mineralsalt basic media

McCormick et al. Aerobic and Arylamines(1976) anaerobic cond.

(enzymes fromVeillonellaalkalescens)

Jerger et al. (1976) Broth cultures 4-OHA, 2-OHA, 4-A, 2-A,4,4'-Az, 2,4-DA

Parrish (1977) Cultures of fungi 4-A, 4-OHA, 4,4'-Az

Amerkhanova and Ps. denitrificans 2-A and 4-ANaumova (1978) cultured on basic

synthetic media

Carpenter et al. (1978) Activated sludge 4-A, 2-A, 2,4-DA, 2,6-DA,polyamide macromolecules

Hoffsommer et al. Activated sludge Amines and complex(1978) with supplemental polar products

nutrients

Osmon and Andrews Soil amended 4-A

(1978) with nutrients

Pereira tu al. (1979) Groundwater 2-A, 4-A

Burlinson (1980) Aqueous systems: 1,3,5-TNB, 2,4,6-Sunlight trinitrobenzaldehyde,

trace 2-A and 4-A, 3,5-dinitroaniline

Dark Known biotransformation products

soil. Hoffsommer et al. (1978) also reported the amino compounds accounted for 12% of the con-presence, in decreasing order, of 4-A, 2-A, 2,4-DA tinuously fed TNT, and the relative amounts on aand 2,6-DA when 10-50 mg/L of aqueous TNT weight basis were 4-A:2-A:2,4-DA:2,6-DA, 1.0/was pumped into an aerated oxidation ditch facil- 0.12/0.10/0.013. Hoffsommer et al. (1978) also didity containing bacterial floc from activated sludge bioconversion experiments using uniformly la-microorganisms and supplemental nutrients. The beled TNT. After 3 days, 34% of the total 14C

2

00, N NO,

O CH 3 H 3-c 2_2'-Azz

NO2 NO2

2-A 2,6-DA2-0OHA CH 3 CH 3 CH,

0 2 N '* NHOH 0O2 N 0 NH 2 H2 NO NH2

NO 2 NO2 NO 2

ON NO2 2N0N 2

N O2

aN 0D C0 2,4'-Az

NO2 . .. r NO2 N O2

TNT__

CH2 CH3 CH 3

02 N N0 2 0N NO2 _ _ 02 N NH,

4-OHA NHOH NH 2 NH 2

4-A 2.4-DA

0

O2 NO N=N-6 NO2 4,4'-Az

HC CH,NO 2 NO2

Figure 1. Pathway for TNT (after Kaplan and Kaplan 1982a).

activity was accounted for by the amines (26% in mus, and that composting may be used to immo-the supernate and 8% in the floc). After 30 days, the bilize high concentrations of explosives in sedi-activity associated with the amines had decreased ments and soils. No evidence of ring cleavage wasto only 3% (less than 0.28% in the supernate and found by Kaplan and Kaplan or Isbister et al.2.8% in the floc). The largest portion of the 14C To study the degradation of TNT, RDX, HMXactivity (40-64%) was associated with the floc. The and 2,4-DNT in soil, Greene et al. (1985) appliedauthors postulated that this material was corn- simulated pink water to a series of soil columnsposed of water-insoluble polymeric-likesubstances that varied in carbon supplementation and pinkhaving a high molecular weight. water flow rate. The experimental columns were

Isbisteretal. (1984) used uniformlyring-labeled inoculated with organisms from an activated14C-explosives in a laboratory-scale composting sludge, from an anaerobic sludge digest and fromstudy. The reduction of TNT levels in the compost garden soil. One column was not inoculated andcorrelated with a significant increase in the corn- was sterilized with 0.5% mercuric chloride. Thepost-bound 1

4C activity. TNT reduction products columns were irrigated continuously for 110 days,(2-A, 4-A, 2,6-DA or 2,2'-Az) were not identified in and the influent and leachate were sampled weekly.the 3-week composts and only minor amounts For all columns, including the sterile column, thewere determined in the 6-week composts. The amount of TNT recovered was low (0-24%), indi-authors conclude that TNT is transformed into cating that the TNT remained in the soil column orinsoluble macromolecules that behave like hu- was transformed. Analysis of the leachate from

3

TNT -A4-A

O 2N N0 2 0irbo 02N N H2 0 2 N NO 2

NO2 NO, NH 24hiTN B 2, 4. 6- Tr inijtr obenz y I

AlcoholNO02 C-OH

Op' N 2N '

N 2

NO02

tvl 0hi/

2-Hydroxy-2-Am -4,.3-Dinitrobenzoic 4,6- Dint robenzoic

Acid Acid 3.5-DinitrophenolNH 2 COOH COOH OH

0NON NH 2 ON O OH

02P Q N02 A2) H 2 0 - ?N6N-

3,5 - Dritroono line NO 2 NO,1 0 Diozo 1110110

S2) ReductionCOO H

NO2

2,4 - DOntrobenzoic Acid

Figure 2. Pathzviay to 2-ainoii-4,6-diniitrolbtoic acid (after Spanggord 1 983a).

the columns with fast flow (100 mL/day), with groups. Equal amounts of 2-A and 4-A wereand without a carbon supplement (2.0 gIL glu- formed. They did not detect ring cleavage.cose), revealed the presence of 4-A. Also, 2-A was Spanggord et al. (1 980b) also reported TNTdetected in the leachate from the column with fast photoproducts. Less than 20% of the TNT de-flow and carbon supplementation. graded could be accotunted for by single aromatic

Spanggord et al. (1980b), while studying the ring compounds such as 1,3,5-TNB, 4,6-dini-environmental fate of TNT in selected water bod- troanthranil, 2,4,6-trinitrobenzaldeliyde, 2,4,6-ies, identified the monoaminodinitrotoluenes (2- trinitrobenzonitrile and 2,4,6-trinitrobenzoic acid.A and 4-A), diaminonitrotoluenes (2,4-DA and Another 20% of the TNT is transformed to ni-2,6-DA), and tetranitroazoxytoluene (4,4'-Az) troazoxy derivatives formed by the coupling ofderivatives of TNT as metabolites. In addition, an nitroso and hydroxylamine products. Whileintermediate metabolite was found that contained Spanggord and co-workers identified most of thea thio-methyl group, indicating that a sulfur above products when more than 50% of the avail-compound may play a role in the reduction of nitro able TNT was photolyzed, only one product was

4

detected when UV exposure was so short that only of TNT by several yeasts and fungi. Most of the10% of the TNT was degraded. This unidentified TNT was reduced to 2-A and 4-A, with 2-A pre-compound formed a pink solution when collected vailing. Naumova et al. (1983) found the amountfrom an HPLC column in 30/70 acetonitrile:water of 2-A was 2-3 times greater than the 4-A whenand reverted thermally to TNT. Properties of this TNT was transformed byPseudomonasdenitrificauiscompound, such as its water solubility, NMR and Escherichia coli. Nitroreduction was stimu-spectrum and low volatility, implied a TNT anion- lated by FAD, Mg and Mn, and occurred at a pHtype structure. range of 5.5-7.8. Reduction was dependent on

Spanggord etal. (1983a) investigated the persis- NAD and NADH.tence of TNT and RDX in waste disposal lagoons Selivanovskaya et al. (1986) studied the degra-at the Louisiana Army Ammunition plant. TNT dation of 2,4-DA, which was produced by micro-was transformed by both photochemical and micro- bial catabolism of TNT. They proposed the follow-biological processes. The major biotransforma- ing pathway: 2,4-DA -- phloroglucinol carboxyliction products were identified as 2-A and 4-A, acid -- phloroglucinol (1,3,5-THB) -- pyrogallolwhile a major stable phototransformation product (1,2,3-THB) - ring cleavage (Fig. 3).was 2-amino-4,6-dinitrobenzoic acid (Fig. 2). Sleivanovskaya et al. (1987) found that aerobic

Kearney et al. (1983) examined whether ultra- biodegradation of TNT by Pseudonionasfluorsceiisviolet-ozonation (LV-O) of aqueous 14C-TNT was expedited by the addition of clay. The bacte-(uniformly labeled) facilitated subsequent TNTmetabolism by soil microorganisms. The productsof UV-O3 were 1,3,5-TNB, 2,4,6-trinitrobenzalde-hyde, 3,5-dinitrophenol, 3,5-dinitrocatechol, 3,5-dinitrohydroquinone and oxalic acid. There was 02 N NH2

substantial binding of TNT and its bioconversion 2,4-D Aproducts to soil. No evidence of ring cleavage wasfound. While some metabolic 14CO evolution NH202 Hwas measured, it was attributed to the methylcarbon of TNT.

Kanekar and Godbole (1984) isolated microor-ganisms from soil samples collected along the COO H

banks of the Mula River, India, opposite the waste- HO OH

water discharge point of a TNT manufacturing Pharogliucidolfacility. The most numerous genera present wasPseudomonas, and among these a new species was OHidentified, Pseudomonas trinitrotoluenophila. In asynthetic medium having TNT as the sole source +of carbon and nitrogen, this microorganism gave OH. / OH

100% TNT degradation after 30 days of incuba- Phioroglucinoltion. The authors detected CO2 as one of the P9products of microbial degradation and claim thatcleavage of the benzene ring may have occurred, OH

although it is possible that all the carbon wasprovided by the methyl group, not the benzenering, of TNT. No attempts to identify other OH

degradation products were reported and 14C- OH

labeled TNT was not used. PyrogollolThe microbial degradation of TNT has been

studied at Kazan State Univer,,ity, USSR, by OH

Naumova, Selivanovskaya and co-workers. Whilethe papers describing this work are published inRussian, the abstracts are available in English. The Ring CI e ova efollowing is a summary of the information gleanedfrom the abstracts. Figure 3. Pathway for 2,4-DA degradation (after Seli-

Naumova et al. (1982) studied the metabolism vanovskaya et al. 1986).

5

rial cells, TNT and TNT degradation products predominating. In some samples, the sum of thesorbed onto the clay particles, amounts of 2-A and 4-A have actually exceeded

Naumova et al. (1988) compared the rates of the amount of TNT detected. Therefore, our expe-TNT transformation by Psetudononas fluorescens rience leads us to believe that significant amountsunder conditions of oxygen and nitrate respira- of these intermediates may be detected in soilstion. While TNT was reduced to 2-A and 4-A under contaminated by TNT.both aerobic and anaerobic conditions, nitroreduc-tion was somewhat faster for nitrate respiration. 2,4-Dinitrotoluene (2,4-DNT)The subsequent reduction of these monoamino McCormick et al. (1978) identified the productscompounds to 2,4-DA was much morelikely under of biotransformation of 2,4-DNT by Mucrosporhmnitrate respiration conditions. In contrast, degra- sp. as 2-amino-4-nitrotoluene, 4-amino-2-nitrotolu-dation of 2,4-DA was faster under conditions of ene, 2,2'-dinitro-4,4'-azoxytoluene, 4,4'-dinitro-oxygen respiration. As a result, 2-A and 4-A pre- 2,2'-azoxytoluene and 4-acetamide-2-nitrotoluene.dominated under aerobic conditions and 2,4-DA The pathway proposed for this biotransformationpredominated under anaerobic conditions. When is shown in Figure 4. Although 2,4-diaminotolu-

bacteria were grown on a medium with 2,4-DA as ene was not detected, previous work with anaero-the sole source of nitrogen, phloroglucinol (1,3,5- bic bacteria demonstrated that both nitro groupsTHB) and pvrogallol (i,2,3-THB) were identified can be enzymatically reduced to amino groupsin the culture. (McCormick et al. 1976), and that the 4-nitro group

Two reports, published in Chinese with ab- was reduced first.stracts in English, describe TNT decomposition Experiments at the Stanford Research Instituteproducts in aqueous media. Xiong et al. (1986) were conducted on the biodegradation of 14C-2,4-treated TNT wastewater in an oxidative pond DNTbymixedmicrobialpopulations.Asreportedcontaining both algae and fungi. Biodegradation by Isbister et al. (1980), 60% of the 14C-label wasproducts included 4-A and 2-A. Lu et al. (1987) evolved as 14CO, within 48 hours (how the DNTused UV to irradiateaqueousTNT solution (260-300 was labeled is not specified), and total degrada-mg/L) at 50'C in the presence of ozone. After 3.5 tion occurred within I week. Some amino deriva-hours, - 99%/( of the TNT was degraded anc 2,3- tives and the condensation products were identi-dinitrophenylamine, 1,3-dinitrobenzene and 1,3,5- fied. The same microbial pop ulation did not yieldTNB were formed. Since the total organic carbon WCO 2 in experiments with A C-2,6-DNT.concentration decreased to less than 95 mg/L after Spanggord et al. (1980b) found similar results.70 hours of irradiation, the authors suggest that Microorganisms from natural water bodies thatTNT ring cleavage may have occurred with CO, could use 2,4-DNT as a sole source of carbonevolution. biotransformed more than 90% of the 2,4-DNT in

The aminodinitrotoluenes have also been iden- 6 days. In studies with ring-labeled 14C-2,4-DNT,tified as metabolic products of TNT in plants 59%ofthe1 4Cwasevolvedas 14 COafter7daysof(Palazzo and Leggett 1986) and mammals (Yinon incubation. They identified one metabolite as 4-and Hwang 1986). Also, Palazzo and Leggett (1986) amino-2-nitrotoluene; nitroazoxytoluenes werefound that 2-A and 4-A were chemically bound to also tentatively identified. These metabolites didthe plant material, not accumulate. The isomer 2,6-DNT was not

In summary, TNT is readily biotransformed in degraded.the environment to amino, diamino and azoxy Liu et al. (1984) identified intermediates in thecompounds. Metabolites have been reported to po- biotransformation of 2,4-DNT. They observed nolymerize and become tightly bound to organic degradation of 2,4-DNT after 14 days of incuba-materials. Some reports have been made of com- tion in aerobic fermenters. However, in anaerobicplete degradation of TNT to form CO 2, but the fermenters with methanol as the solvent carrier,conditions under which this transformation took 2,4-DNT was biotransformed and the metabolicplace were very specialized and not likely to be a products 2-nitroso-4-nitrotoluene, 2-amino-4-ni-major pathway in the environmental fate of TNT trotoluene and 4-amino-2-nitrotoluene accumu-under natural conditions. Therefore, further ana- lated and then disappeared. The nitroso compoundlytical method development for the amino and was only detected between 48 and 72 hours of in-diamino compounds is recommended. In the analy- cubation, while the two amino compounds lastedses of field-contaminated soils by CRREL's labora- almost the whole 14 days. In a subsequent experi-tory, 2-A and 4-A are often identified with 2-A ment using a higher concentration of 2,4-DNT,

6

CH 3 0 CH,

'<_

NO 2 N02

CH 3, CH 3

N O N H O H

N H -

0(D I -_ - C1NO, 2 CH 3 0 NO2 H

N 2 NO2 O2/"- NON -. NH 2

_ = 'N -, 2-A-4-NT

'\ ]CH, X yNO02 CH"O2N_' l H

2,4-DNT / CHI 3 2,4'-Az 3 OAT

N0 2 NO2 ---- N2

CH3

NONHOH N> NO 2 N H 2 C H,

4FNO-2-NT 4-HA 24D a M oc4--2-NT N 0talN

N

N- 4,4'-A z NHC0H

r9 N024-Ac-2-NT

CH I

Figutre 4. Pathwuay for 2,4-DNT (after McCormick e~t al. 7978).

they tentatively identi fied another intermediate as to those reported for TNT have also been found,4-nitroso-2-nitrotoluene. Couch et al. (1987) found namely the amino and azoxy derivatives. Pre-that 2-amino-4-nitrotoluene and 2-nitroso-4-nitro- sumably, these compounds would also be formedtoluene were more mutagenic then 2,4-DNT itself in soils. The development of analytical methodswhen tested using a quantitative reversion assay for the amino compounds is recommended, espe-with Salmonella typhiniuriuin TA98. cially in light of their mutagenicity.

Mori et al. (1984) found that the isomers of DNTwere reduced via hydroxylaminonitrotoluenes to Trinitrobenzene (TNB) andmonoaminonitrotoluenes by Escherichia coli incu- 1,3-dinitrobenzene (DNB)bated aerobically for 28 hours. In an experiment Like TNT and DNT, the principal mode ofwith 2,4-DNT, the first metabolites detected were microbial transformation of the nitroaromatic4-hydroxylamino-2-nitrotoluene and 2-hydroxyl- compounds TNB and DNB is reduction of the nitroamino-4-nitrotoluene; peak concentrations were groups to form amino groups. McCormick et al.reached at 8 hours. As the concentration of the hy- (1976) studied the reduction pathway of severaldroxylaminonitrotoluenes declined, there was a nitroaromatic compounds including TNT, DNT,simultaneous increase in the concentrations of 2- TNB and DNB in the presence of enzymes fromamino-4-nitrotoluene and 4-amino-2-nitrotoluene. Veillonella alkalescens. The pathway proceededOther metabolic products such as the nitroso inter- through a nitroso intermediate to hydroxylaminomediates and diaminotoluene were not found. compounds. Further reduction yielded amino

In summary, the fate of 2,4-DNT in water bod- compounds.ies and soils has not been systematically defined, Marino (1986) studied the reduction of the iso-but the biotransformation of 2,4-DNT by cell cul- mers of DNB using a variety of laboratory strainstures has been studied. Unlike TNT, 2,4-DNT may of plant, animal and microbial cells. Metabolitesbe completely metabolized to CO2 by mixed mi- were confirmed by GC/MS to be the correspond-crobial populations. Reduction products similar ing isomers of nitroaniline. Intermediates were

7

identified as the nitroso and hydroxylarnine de- pathway for RDX proposed by McCormick et al.rivatives. (1981) is presented in Figure5. The products include

Evidence suggests thatTNB is persistent in soils hexahydro-l-nitroso-3,5-dinitro-1,3,5-triazine(Jerger 1976, Keirn et al. 1981) and in groundwater, (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-while DNB is not. Incubation of DNB in water triazine (DNX), hexahydro-1,3,5-trinitroso-1,3,5-samples taken from the Tennessee River down- triazine(TNX),hydrazine, 1,1-dimethylhydrazine,stream from the Volunteer Army Ammunition 1,2-dimethylhydrazine, formaldehyde andPlant resulted in the microbially mediated conver- methanol. Upon subsequent exposure to aerobicsion of the compound to CO. Mineralization of conditions, the methanol may be metabolized toDNB did not occur in sterile controls or in water CO 2. The authors caution that the biodegradationobtained from a local (Frederick, Maryland ) source, products of RDX may pose more seriousindicating that the capability to degrade DNB by toxicological problems than RDX itself. Spaldingmicroorganisms will vary with the water source. and Fulton (1988) found RDX to be persistent inIn contrast, incubation of TNB in Tennessee River groundwater near Grand Island, Nebraska; nonewater did not result in biodegradation of the com- of theRDX degradation products mentioned abovepound. A slow decline in TNB concentration was were found, even though some anaerobic condi-observed, and the transformation product 3,5,- tions may have existed in some deep water wells.dinitroaniline was identified (Mitchell and Dennis Isbister et al. (1984) reported aerobic degrada-1982, Mitchell et al. 1982). These results are consis- tion in laboratory- and greenhouse-scale com-tent with our experience at CRREL, where hun- posting experiments using uniformly ring-labeleddreds of groundwater samples from present and 4C-RDX. After 6 weeks of composting, the solventformer Army sites have been analyzed. TNB is extractable RDX decreased to 21.6%. An averageroutinely observed in those samples containing of 54.5% of the 14C was evolved as CO 2, indicatingTNT. In some samples, the level of TNB exceeded cleavage of the RDX ring structure. The remain-that of TNT, 2-A and 4-A, while the levels of DNB ing 14C in the solids was attributed to adsorbedare usually close to the level of detection. RDX or breakdown products incorporated into

the cell material. The leachates from the compostswere nonmutagenic when tested using the Ames

NITRAMINES Assay.Aerobic degradation of 14C-RDX during com-

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) posting was also reported by Doyle et al. (1986).The biodegradation of RDX has been studied For hay-horsefeed and sewage sludge composts,

under aerobic and anaerobic conditions. Several 46.5 and 37.2% of the ' 4C added as 14C-RDXinvestigators have observed that RDX does not was recovered as 14CO2. Most of the remainingdegrade under aerobic conditic . For example, label (29.7 and 54.6%) was in residues that couldHoffsommer et al. (1978) found no bioconversion not be extracted from the compost with acetoni-of RDX by aerobic-activated sludge micro- trile using a 30-minute multiple extraction proce-organisms. Spanggord et al. (1980b) reported that dure. More RDX may have been extracted withRDX was persistent in aerobic water bodies. How- a longer extraction time. Our studies at CRRELever, RDX degradation was significant under with field-contaminated soils have shown that atanaerobic conditions when extra organic nutrients least 18 hours of extraction in a sonic bath iswere supplied. Soli (1973) reported a reduction in required to achieve equilibrium.RDX concentration from 20 to 0.6 mg/L after 5 In the study conducted by Greene et al. (1985),days in an anaerobic medium containing purple in which simulated pink water was applied to soilphotosynthetic bacteria. Soli hypothesized that columns, the mononitroso (MNX) and dinitrosoRDX was not metabolized; rather it was reduced (DNX) derivatives of RDX were identified in theduring the photosynthetic process. McCormick et leachate of the column with fast flow (100 mL/al. (1981, 1984a) monitored the degradation of day) and carbon supplement (2.0 g/L glucose).RDX in nutrient broth cultures incubated MNX was also identified in the leachates from theanaerobically. As the concentration of RDX columns with slow flow (40 mL/day) with anddeclined, there was an increase and sequential without carbon supplement.decrease of the mono-, di-, and trinitroso analogs RDX is susceptible to photodegradation. Theof RDX formed by reduction of the nitro groups to photoproducts obtained when aqueous solutionsnitroso groups. The anaerobic biodegradation of RDX were exposed to UV were reported by

8

2 3 4

0 N,\ ' NO 0 2OZ"N0 )N\ NNG O N - N 0 ON, N ,NO

N N N

N 0 NO NO NO

5 6 7 100N ~ONNNANiOHO2N\,N/N 0 O' N'N, N NHOH ONN ONN ,,.N NHOH

- N N N NHkN L.N,) K.N) - , H

9 C NHOH NO NO

N // H2 c\ /CH 3 NCH 302 N 8 NO, 10 N NN \N 02N NHOH I *I1

02ON 10 / N

NHOH I H N N N 0 H 2 cNo

H2 H HIHO

22

12 13 H3C ,CH 3 /CH 3HCHO - ON \ /NO 2 N N

N N 1 116 OHH H N 0 N\0

N 12 14

HCHO + H2 N-NO2 19 23NH 2 , H 3 C CH 3 CH 3

N/ HN /

NH 2 NHH3C

12 15

HCHO H2 N-NH 2

ICH30H

Figure 5. Pathway for RDX (after McCormick et al. 1981).

Glover and Hoffsommer (1979) to be nitrate, ni- tified in soil column studies (Greene et al. 1985),trite, ammonia, formaldehyde, nitrous oxide, form- analytical methodology is tentatively recom-amide and N-nitroso-methylenediamine. The mended for these compounds.products of UV-O 3 produced CO2' cyanic acid,nitrate, ammonia, and formic acid. Spanggord et Octahydro-1,3,5,7-tetranitro-1,3,5,7-al. (1980b) identified formaldehyde, nitrite and tetrazocine (HMX)nitrate as photoproducts of RDX in water bodies. Only two reports were found that identified

In summary, RDX has been reported to be resis- biodegradation products of HMX. Both Spanggordtant to biodegradation under aerobic conditions. et al. (1983b) and McCormick et al. (1984b) studiedHowever, under anaerobic conditions with sup- the degradation of HMX under anaerobic condi-plemental organic nutrients, RDX degrades sub- tions and detected the nitroso derivatives of HMX.stantially. Byproducts include methanol, formal- Both studies described a pattern of peaks thatdehyde, hydrazine and the nitroso-reduction appeared in chromatograms at various stages ofproducts of RDX. RDX has also been reported to HMX biotransformation (Fig. 6). As the peak areadegrade in aerobic composts with evolution of for HMX decreased, a second peak with an earlierCO2 . We may surmise that RDX may anaerobi- elution time appeared, followed by a third peakcally transform in soils, groundwater and stag- with a still earlier elution time. GC/MS analysisnant surface water, but the rate of biotransforma- indicated that these peaks were attributable to thetion is unknown. Since the reduction mechanism mono, di- and in some cases trinitroso derivativesproceeds through the nitroso derivatives (MNX, of HMX. The same pattern was observed for RDX,DNX and TNX) and nitroso derivatives were iden- AcHMX and AcRDX (McCormick et al. 1984b), but

9

quential reduction to form nitroso derivatives.× The situation is similar to that of RDX; the poten-

tial for this transformation exists, but the relativeamounts of reduction products are unknown.

00 oTetrylNo reports were found that identified biode-

gradation products of tetryl. However, thermal

Day and photolytic products have been detected. Tamiriand Zitrin (1986), while conducting analyses forexplosives by GC/MS, identified N-meth-

C j ylpicramide as a thermal degradation product ofo_ tetryl (Fig. 8). Kayser et al. (1984) reported picrate

ion, N-methylpicramide, methylnitramine, nitrate- and nitrite as photolytic products of tetryl in aque-

ous solution, and estimated the environmental0 hydrolysis half-life of tetryl to be 302 ± 76 days at

20 0C and pH 6.8. Farey and Wilson (1975) also2 identified N-methylpicramide in tetryl samples

00

D N0 2

NO 2

-00

0, N-N N-NO?

N00 NO2

NO0

Figure 6. Chromatograms at Nvarious stages ofHMXbiotrans- o >N-N NH 2

formation (after McCormick .N 19

1983b). NNo,

h I/Radical Scavenger

the reaction rates for the eight-membered-ringedHMX and AcHMX were slower than for six-mem-bered-ringed RDX and AcRDX. McCormick et al. NO

(1984b) also detected methanol, which, they hy- N H2 0pothesized, resulted from the reduction of formal- 0 2 N-N N+ NO2 P HNO 2 -HN03

dehyde, even though they did not detect formal- LI

dehyde.IIn their soil column experiments, Greene et al.

(1985) detected no transformation productsof HMXin the column leachates. This result is consistent /

with the report of McCormick et al. (1984b) in that N-NH/C H2 N CH 2 +N 2 0.H 2 0the eight-membered ring of HMX is less suscep-tible to biotransformation than the six-membered H

ring of RDX. 40

Spanggordetal.(1983b)alsoidentifiedthepho- HOCH 2- NH CH 2 0H N CHO NH, NHC

tolytic products of HMX. The stable end productswere nitrite, nitrate and formaldehyde (Fig. 7). Figure 7. Photolytic products of HMX (after

In the environment, HMX may undergo se- Spanggord 1983b).

10

H

0HA2

CH3 NO 2 CH 3 N0 2 CH3 H

0 2 N , . NO2 H O 0N 'ON0 2 O 2 NN + HN--I - --I,- I-HN 03

N02 NO 2 NO,

TETRYL N-METHYLPICRAMIDE

Figure 8. N-etiyl picramide as a transformation product of tetrlrI (after Tamiri andZitrin 1986).

heated under various conditions. These condi-tions included heating tetryl alone for 4 weeks at80'C, alone for 4.5 days at 120'C and with leadazide for 4 weeks at 80'C. Other thermal degrada-tion products found were picramide, picric acid,p-nitroaniline and trinitroanisole. Yasuda (1970) 45 °C

reported that production grade tetryl contains -oU

impurities such as N-methyl-N,2,4-trinitroaniline,picryl chloride, picric acid, picramide or N,2,4,6- C.tetranitroaniline and N-methyl-2,4,6-trinitroanil-ine. When tetryl samples were heated at 120'C for48 hours, the major decomposition products werepicric acid and 2,4,6-trinitroanisole. Smaller con-centrations were also found of picramide or N,2,4,6-tetranitroaniline, N-methyl-2,4,6-trinitroaniline,1,3,5-trinitrobenzene and N-methyl-N,2,4-trini-troaniline, or all five.

While developing an analytical method fornitroaromatics and nitramines in our laboratory,we found that tetryl thermally degraded while 1_.csoils were extracted for 18 hours in a sonic bath at45°C (Bauer et al. 1989). When chromatograms ofsoil extracts were examined (Fig. 9), we found apeak at 10.5 minutes for those samples in whichtetryl had degraded. The information presentedabove suggests this product is probably N-methylpicramide.

REQUIREMENTS FOR ANALYTICALMETHODS

Chromatography has been used to detect and o5 10 15

quantify the transformation products of ni- Retention Time (mm)

troaromatics and nitramines. Appendix A con-tains some examples of parameters used for analy-sis and some sample chromatograms. Figure 9. Chromatogram of tetryl degradation product.

11

When monitoring requirements are developed field-contaminated soil should be investigated.for these compounds, analytical methods will need Analytical methodology is already established,to be certified at the appropriate contaminant levels, but a source for standards is required.This certification procedure will require that ana- Other compounds whose presence in the envi-lytical standards be made available. The stability ronment should be investigated include 2-amino-of standards, both pure and in solution, must be 4-nitrotoluene, 4-amino-2-nitrotoluene, 3,5-dini-clarified. Since most of the transformation prod- troaniline, 3-nitroaniline, 2-amino-4,6-dinitroben-ucts are transitory in the environment, sample zoic acid, DNX and TNX.handling methodology is of great importance and In conclusion, the immediate need is a source ofmust be developed so that a true measure of ana- standards so that environmental contaminationlytes present at the time of sampling may be ob- may be validated. Next, certification of the analyti-tained. Standard practice for nitroaromatics and cal methods is required for those compoundsnitramines in soil and water is to store samples at deemed a true environmental concern. Established4°C for up to 1 week. Additional precautions may methods already exist for groups of compoundsbe needed when transition products are of interest (primarily the source compound and reduction(i.e., sterilization, protection from light, shorter products). Since it is unlikely that a single analyti-storage times). As the standards and methods cal protocol will be adequate for all the byproductsbecome available, in all likelihood, reports of trans- that may be of interest, the most effective approachformation products in environmental samples will would be to emphasize the development of twoincrease as more analysts gain the capability for separation schemes, one for nitroaromatic bypro-detection and identification of these compounds. ducts and one for nitramine byproducts.

As a first step in identifying those transforma-tion products that may be present in munitions-contaminated soils, previously collected samplescould be examined. CRREL has a large inventory LITERATURE CITEDof contaminated soils from Army sites from overten states, and could use the samples to develop Amerkhanova, N.N. and R.P. Naumova (1978) 2,the necessary methodology. 4, 6-trinitrotoluene as a source of nutrition for

bacteria. Mikrobiologia, 47: 393-395.Bauer, C.F., T.F. Jenkins, S. Koza, P.W. Schuma-

CONCLUSIONS cher, P. Miyares and M.E. Walsh (1989) Develop-ment of an analytical method for the determina-

Nitroaromatics and nitramines are susceptible tion of explosive residues in soil, Part II--Col-to environmental transformation. Since some of laborative test results. USA Cold Regions Researchthe byproducts of this transformation may be per- and Engineering Laboratory, CRREL Report 89-9.sistent, and therefore of environmental concern, Burlinson, N.E. (1980) Fate of TNT in an aquaticdevelopment of analytical standards and method- environment: photodegradation vs. biotransfor-ology is required to establish the prevalence of mation. Final report. Silver Spring, Maryland:these compounds. Much research has been con- Naval Surface Weapons Center, NSWC/TR-79-ducted on the environmental transformation of 445.TNT. Since it is well established that 2-A, 4-A, 2,4- Couch, D.B., D.J. Abernethy and P.F. Allen (1987)DA and 2,6-DA are frequently present in TNT The effect of biotransformation of 2, 4-dinitrotolu-contaminated matrices, certification of analytical ence on its mutagenic potential. Mutagenesis, 2(6):methodology is highly recommended for these 415-418.compounds. In addition, the presence of the cou- Carpenter, D.F., N.G. McCormick, J.H. Cornellpling products 2,2'-Az, 2,4'-Az and 4,4'-Az should and A.M. Kaplan (1978) Microbial transformationbe validated. Since protocols for the liquid chro- of ' 4C-labeled 2,4, 6-trinitrotoluene in an activatedmatographic analysis of TNT reduction products sludgesystem.AppliedEnvironmentalBiology,35(5):have been published, the availability of standards 949-954.is the limiting factor for the validation. Doyle, R.C., J.D. Isbister, G.L. Anspach and J.F.

The environmental fate of RDX is less defined Ketchens (1986) Composting explosives/organ-than that of TNT. However, the nitroso derivative, ics contaminated soils. Alexandria, Virginia: At-MNX, has been detected in laboratory studies with lantic Research Corporation. ADA169994.soil columns. The presence of this compound in Enzinger, R.M. (1970) Special study of the effect of

12

alpha-TNT on miicrobiological ~vt indd thc Kaplan, L).L. and A.M. Kaplan (1982a) Thermo-determination of biodegrad abil it ofaclpha-I NI philio. biotransformation of 2, 4, 6-trinitrotoluene

US Evionintl \giene A,,en, AD72, 1,'. uder ,iliu.lated Colnposting conditions. AppliedFarey, M.G. and S.E. Wilson (11Q75) QuanlltitatiVe! dlil Eiroiunneltal Microbiologt,, 44(3): 757-760.determination of tetryl and its degraidation prod- Kaplan, D.L. and A.M. Kaplan (1982b) Compost-ucts by high-pressure liqUid chuoinaitograpliv ink, industrial %xastes-biochemical consideration.Journal of Chroinmatoapliu,, 114. 2b I --2o,-. B)iOC m ic/', 23(3): 42-44.Glover, D.J. and j.C. Hoffsornmer (1 979) I 'lioto ii Kaplan, D.L. and A.M. Kaplan 01982c0 Separationsis of RDX in aqueous solution with and w,,ithiout of mlixture.-of 2_4 (-trinitrotOlUene reduction prod-ozone. Silver Spring, N/iarviand: Mival Suirfacc ucts With liqluid chromatography. Analiytica Clii,,-Weapons Center, NSkVC//WUI.-I R-78-1,':. iraAud 136: 425-428.ADA080195. Kaplan, D.L. and A.M. Kaplan (1983) ReactivityGreene, B., D.L. Kaplan, and A.M. Kaplan (19:; ol TNT & TNT-Microbial reduction productsDegradation of pink water compouinds in soil-- w.ith soil components. USA Natick Research andTNT, RDX, HM/X. USA Natick IResearch an1d Development Laboratory, TR-83/041.Development Center, NATICK/TR-63/04(h. ADA Kayser, E.G., N.E. Burlinson and D.H. Rosenblatt157954. (1984) Kinetics of hydrolysis and products ofHoffsommer, J.C., L.A. Kaplan, D.J. G lover, D.A. hyd 'crol 's"is and photolysis of tetryl. Silver Spring,Kubose, C. Dickinson, H. Goya, E.G. Kayser, C.L. 11 Ma r lanrd: Na val Sur 1face Weapons Center, Tech-Groves and M.E. Sitzma nn01970 tBik d et;ra JabilI nical Report 84-78. ADA153144.ity of TNT: A three-year pilot studyv. Silver Spring, Kearney, P.C., Q. Zeng and J.M. Ruth (1983)Maryland: Naval Surface WeIta pon'. (en toi, Odaiepret eatmnent accelerates TNT metabo-NSWC/WOL-TR-77-13o. lismn ini soils. c-liciiispli're, 12(11 /12): 1583-1597.Isbister, J.D., R.C. Doyle and 1.1. K itchenms 'I 1) Keirn, I. A., C. I. Stratton, J.J. Mousaj.D. Donds,Engineering and dev'elopnient suppor t otgeiwral J.L.. IWinegardner, H.S. Prentice, W.D. Adamsdecontamination techniques. Task 6. adaptcd/' anid I.). Powell (1981) Environmental survey ofmutant biological treatment. Phase I - literati, 'Alabamal' Armyv Ammunition Plant. Gainsville,review. Alexandria, Virginia: Atlantir1 KReearch FloIRIa Lnvir-Onilental Science and Engineering,Corporation. Inlc. A1713(159906 I.Isbister,J.D., G.L. Anspach,.K Kitchien anidR.(C. Klatisneier, R.E., J.L. Osmon and D.R. WallsDoyle (1984) Comnposting for decontamination oi ( 1973) The effect of trinitrotoluene on micro-soils con taininrg explosm i es. M icrwbi, a (/oI: N''0 , Olga n ismZls. D~'/u'uut nIzntulMrloo,7: 47-73. I5: 309-3 17.Jenkins, T.F. and P.11. Miyares (ini pivp) Sti Liu, D.-S., K. Thomson and A.C. Anderson (1984)out extraction for low level deternii tion of ii - Identification of nitroso compounds from biotrans-troaromatics andinitrai nes in ground o'a ,,tt ;J_;\ A urinal ion of 2, 4,-dinitrotoluene. Applied and Envi-Cold Regions Research and Enginee~ring Laboia- uuuulmicital Mioluiotoy, 47(6): 1295-1298.tory, CRREL Special Report. L~u Miaoqlin, Wang Xiangming, GongYuhua, LinJerger, D.E. and D.P. Chynoweth (l9houu 'Ilie Wenjie and He Fcnzhu (1987) Photodegradationmicrobiological degradation of 2,4, 6i-trinitrotolti of alpha-Il NT in a(queouIssoLu-tion. Haanjing K.ut('ene under anaerobic conditions. Alu Irulaui ui (Olunese), 8(2): 1F-20 (abstract in English).Meetings of the, Ainerican Socit Of NI icr L~il .s Ma rino, V. A. (1986) Biotranlsformi-ation ofnitroaro-Q63. mat16C il Ccell cUItwres. Ph.D. Dissertation, Univer-Jerger, D.E., P.B. Simon, R.L. Weitzel and [[K. ,ity' of Colorado (un1puiblished).Schenk (1976) Aqulatic field suirvey at Iowva, MlcCormiick, N.G., F.E. Feeherry and H.S.Radford, and Joliet Arjny Amimunitioun l'al. Levinson (I Q 70) Microbial transformation of 2,4,6-Vol. 111. Microbiological investigation, Iowa and tri ni trotolumene and other nitroaromaticJoliet Army Ammunition Plants. Anni Arbor, compounds. Applied andi Enviironmnental Micro-Michigan: Environmental Control Technology l'ioloxy, 31(6): 949-958.Corp. (Prepared for USA Medical 1ResvCrchl and McCormick, N.G., J.H. Cornell and A.M. KaplanDevelopment Command.) A DA036778. (19~78) Identification of biotransformation prod-Kanekar, P. and S.H. Godbole (1984) Microioial uicts froin 2,4-dinitrotolUene. Applied and Environl-degradation of trini trotohilene (TNTl). idji i jour inciuhul Mwrohiolo..y, 35(5): 945-948.nal of Environmiental Health, 26(2): 89-I 11. McCormick, N.G., J.H. Cornell and A.M. Kaplan

1D

(1981) Biodegradation of hexahydro-1,3,5-trinitro- Environmental Microbiology, 34(2): 232-233.1,3,5-triazine. Applied and Environmnental Pereira, W.E., D.L. Short, D.B. Manigold and P.K.Microbiology, 42(5): 817-823. Roscio (1979) Isolation and characterization ofMcCormick, N.G., J.H. Cornell and A.M. Kaplan TNT and its metabolites in groundwater by gas(1984a) The anaerobic biotransformation of RDX, chromatography-mass spectrometer computerHMX, and the acetylated derivatives. USA Natick techniques. Bulletin of Environmental Contamina-Researcnh and Development Laboratory, TR-85/ tion and Toxicology, 21: 554-562.007. Selivanovskaya, S.Yu., D.Z. Akhmetova and R.P.McCormick, N.G., J.H. Cornell and A.M. Kaplan Naumova (1986) Terminal steps in preparatory(1984b) The fate of hexahydro-1,3,5-trinitro-1,3,5- metabolism of2,4,6-trinitrotoluene in Pseudoinonastriazine (RDX) and related compounds in an- fluorescens. Mikrobiologia (Russian), 55(6): 1040-aerobic denitrifying continuous culture systems 1041 (abstract in English).using simulated wastewater. USA Natick Research Selivanovskaya, S.Yu., T.A. Gorkunova and R.P.and Development Laboratory, TR 85/008. Naumova (1987) Effects of clay on aerobic bacte-ADA149462. rial degradation of trinitrotoluene. Khimiia IMitchell, W. R. and W.H. Dennis (1982) Biode- Teckhnologiia Vody (Russian), 9(1): 23-25 (abstractgradation of 1,3-dinitrobenzene. Journal of Envi- in English).ronental Science and Health, A17(6): 837-853. Soli, G. (1973) Microbial degradation of cycloniteMitchell, W.R., W.H. Dennis and E.P. Burrows (RDX). China Lake, California: Naval Weapons(1982) Microbial interactions with several muni- Center. AD762751.tions compounds: 1,3-dinitrobenzene, 1,3,5-trini- Spalding, R.F. and J.W. Fulton (1988) Groundwa-trobenzene, and 3,5-dinitroaniline. USA Medical ter munition residues and nitrate near Grand Is-Bioengineering Research and Development Labo- land, Nebraska, U.S.A. Journal of Contaminantratory, Technical Report 8201. ADA116651. Hydrology, 2:139-153.Mori, M., T. Miyahara, Y. Hasegawa, Y. Kudo Spanggord, R.J., T. Mill, T. Chou, W. Mabey, J.and H. Kozuka (1984) Metabolism of dinitrotolu- Smith and S. Lee (1980a) Environmental fate stud-ene isomers by Escherichia coli isolated from hu- ies on certain munition wastewater constituents-man intestine. Clienical and Pharmaceutical Bul- Literature review. Menlo Park, California: SRIletin, 32(10): 4070-4075. International. ADA082372.Naumova, R.P., T.O. Belousova and R.M. Gi- Spanggord, R.J., T. Mill, T. Chou, W. Mabey, J.lyazova (1982) Conversion of 2,4,6-trinitrotoluene Smith and S. Lee (1980b) Environmental fate stud-under the influence of microorganism. Prikladnaia ies on certain munition wastewater constituents-Biokhimiia I Mikrobiologiia (Russian), 18(1): 85-90 Lab studies. Menlo Park, California: SRI Interna-(abstract in English). tional. ADA09Q256.Naumova, R.P., N.N. Amerkhanova and L.M. Spanggord, R.J., W. Mabey, T. Mill, T.W. Chou,Zolotukhine (1983) Nitroreduction as a key J.H. Smith, S. Lee and D. Roberts (1983a) Envi-stage in microbial destruction of aromatic nitro ronmental fate studies on certain munition waste-compounds. Prikladnaia Biokhimiia I Mikrobiolo- water constituents. Phase IV-Lagoon modelgiia (Russian), 19(4): 507-512 (abstract in English). studies. Menlo Park, California: SRI International.Naumova, R.P., S. Selivanovskaya, S. Yu and I.E. ADA138550.Cherepnera (1988) Transformation of 2,4,6-trini- Spanggord, R.J., W.R. Mabey, T.W. Chou, S. Leetrotoluene in Pseudomnonasfluorescens undercondi- and P.L. Alferness (1983b) Environmental fatetions of oxygen and nitrate respiration. Prikladnaia studies of HMX. Phase 2-Detailed studies. MenloBiokii diia I Mikrobiologiia (Russian), 24(4): 493-498 Park, California: SRI International. ADA145122.(abstract in English). Tamiri, T. and S. Zitrin (1986) Capillary columnOsmon, J.L. and C.C. Andrews (1978) The biode- gas chromatography/mass spectrometry of ex-gradation of TNT in enhanced soil and compost plosives. Journal of Energetic Materials, 4: 215-237.systems. Crane, Indiana: Naval Weapons Support Traxler, R.W., E. Wood and J.M. Delaney (1974)Center, ARLCD-TR-77032. ADA054375. Bacterial degradation of alpha-TNT. DevelopmentsPalazzo, A.J. and D.C. Leggett (1986) Effect and in Industrial Microbiology, 16: 71-76.disposition of TNT in a terrestrial plant. Journal of Walsh, M.E. and T.F. Jenkins (in press) HighEnvironmental Quality, 15: 49-52. performance liquid chromatographic separationParrish, F.W. (1977) Fungal transformation of 2,4- of TNT and its principal reduction products.dinitrotoluene and 2,4,6-trinitrotoluene. Applied Analytica Chimica Acta.

14

Won, W.D., R.J. Heckly, D.G. Glover and J.C. of tetryl and related compounds by two-dimen-Hoffsommer(1974) Metabolic disposition of2,4,6- sional thin-layer chromatography. Journal oftrinitrotoluene. Applied Microbiology, 27(3): 513- Chromatography, 50: 453-457.516. Yinon, J. and D.-G. Hwang (1986) Metabolic stud-Xiong Chaucai, Hu Jinghui and Xu Cheng (1986) ies of explosives. IV. Determination of 2,4,6-trini-Decomposition products of TNT in oxidation trotoluene and its metabolites in blood of rabbitsponds. Huanjing Huaxue (Chinese), 5(4): 40-42 by high-performance liquid chromatography-(abstract in English). mass spectrometry. Journal of Chromatography, 375:Yasuda, S.K. (1970) Separation and identification 154-157.

15

APPENDIX A: ANALYTICAL METHODS

Comipounds Parameters

TNT and reduction products Method: RP-HPLC (Fig. Al)in compost and soil Column: Waters VtBondapak C18 (30 cm x 3.9 mm)extracts Mobile phase: Methanol-Water, gradient

40 -- 78% MeOH, 2.5 mL/minDetection: UV 230 nmReference: Kaplan and Kaplan (1982c)

TNT and reduction products Method: RP-HPLC (Fig. A2)in water Column: Waters VtBondapak C18 (30 cm x 3.9 mm)

Mobile phase: Methanol-Water, gradient0 -- 70% MeOH, 2.0 mL/min

Detection: UV 254 nmReference: Spanggord et al. (1980b)

TNT, 2-A, 4-A, 2,4-DA Method: RP-HPLC (Fig.A3)2,6-DA, RDX Column: Supelco C18 (3.3 cm x 4.6 mm) in line with

Supelco CN (25 cm x 4.6 mm)Mobile phase: Water-Methanol-THF 60.5:25:14.5

1.5 mL/minDetection: UV 254 nmReference: Walsh and Jenkins (in prep.)

2-A in soil Method: RP-HPLCColumn: Supelco C18 (25 cm x 4.6 mm)

Mobile phase: Methanol-Water 1:1, 1.5 mL/minDetection: UV 254 nmReference: Bauer et al. (1989)

2-A in water Method: RP-HPLCColumn: Supelco C3 (3.3 cm x 4.6 mm)

Mobile phase: Water-Methanol-THF 70.7:27.8:1.52 mL/min

Detection: UV 254 nmReference: Jenkins and Miyares (in prep)

HMX, RDX, 2-A, 4-A, TNT, Method: RP-HPLC (Fig. A4)TNB, Dinitroaniline Column: Waters C18 Radial-Pak Ain water Mobile phase: Methanol-Acetonitrile-Water

Gradient, 30 - 80%Detection: UV 254 nmReference: Spanggord et al. (1983a)

2-amino-4,6-dinitrobenzoic Method: RP-HPLC (Fig. AS)acid, HMX, RDX, TNT, 2-A, 4-A Column: Altech C18 (25 cm x 4.6 mm)in water Mobile phase: Gradient, 40 - 50% B in A

A = 0.1M phosphate buffer (pH 7)with 0.05M TBAP; B = 0.0005M TBAPin methanol, 1.8 mL/min

17

Detection: UV 254 nmReference: Spanggord et al. (1983a)

TNT reduction products Method: GC-ECDin compost extracts Column: 2 mm i.d., 1.8-m glass column

1.5% PS2250/1.95% SP2401 on100 / 120 Supelcoport

Carrier: Nitrogen, 28 mL/minTemp: Port (210'C), Oven (180*C), Det (300*C)

Reference: Isbister et al. (1984)

TNT azoxy derivatives Method: RP-HPLCin compost extracts Column: C18 (10 cm x 7.5 mm x 10 pm)

Mobile phase: 70:30 Acetonitrile-Water2 mL/min

Detection: UV 232 nmReference: Isbister et al. (1984)

DNT reduction products Method: GC (Fig. A6)in culture broths Column: Chromosorb W(AW-DMCS), 3% OV-1

(2 mm x 180 cm)Oven temp: 130 -- 210 0C (4°C/min)

Detection: FID or ECDReference: Liu et al. (1984)

Nitroso derivatives of RDX Method: RP-HPLCin culture medium Column: Waters pBondapak C18 (30 cm x 3.9 mm)

Mobile phase: 20:80 Methanol-Water, 2.5 mL/minDetection: UV 230 nmReference: McCormick et al. (1984a)

Nitroso derivatives of HMX Method: RP-HPLC (Fig. A7)in culture medium Column: Waters pBondapak C18 (30 cm x 3.9 mm)

Mobile phase: 20:80 Acetonitrile-Water2.5 mL/min

Detection: UV 230 nmReference: McCormick et al. (1984a)

Hydrolysis products Method: Ion-pairing RP-HPLC (Fig. A8)of tetryl Column: Hewlett Packard C8 (25 cm x 4.6 mm x

10p m)Mobile phase: 50:50 Methanol-Water, 0.005 M

tetrabutyl ammoniumphosphate, pH 7.5 (Pic A reagent)

Detection: UV 254 nmReference: Kayser et al. (1984)

18

N N 0

01

I In2CU zFr

1. C N

LO IT 44

4 C\! ;II

o 44 t 4 to

iL N

IIO

ID C

f LI

Figure Al. Separation of TNT rdcinpout(aerKaplan andKaplan 1982c).

Figure A2. Separation of TNT reductionproducts (after Spanggord et al. 1980b).

II1--.

{V,

ix

, ! J _______ c l 1FigureMA. Separation of TNT4 8 12 16 20 reduction products.

Time (rein)

19

c' CO

0 n Figure A4. Chromatogram of lagoon waterfrom Louisiana Army Ammunition Plant(after Span ggord et al. 1 983a).

JCO

z

C

NO z -

Figure A5. Chromatograin of lagoon waterfrom Louisiana Army Ammunition Plantshowing resolution of 2-amino-4,6-dini-trobenzoic acid from other wastewater-components (after Span ggord et al. 1 983a).

00

N

x T-

202

Peak Peak Retention Time (s)2 1 No No

I 2-NO-4-NT 1 (158)2 2,4-DNT 2 (250)

34-A-2-NT 3 (285)

4 2-A-4-NT 4 (344)5 5 4-NO-2-NT 5 (1 33)

3

0

I Day 2 7

Lo 45 3

4L

Temp

Figure A6. Gas chromatograms of extracts from an anaerobic fermenter contain-ing 2,4-DNT (after Liu et al. 1984).

X

X

I0Z

0 "

x0

- IE

o01

0 0 U

.2 0-- z

•z

rivatives of HMX (after McCormick Figure A8. Separation of tetryl hydrolysis productset al. 1984a). (after Kayser et al. 1984).

£ IA 6 I 21

~2.

Form ApprovedREPORT DOCUMENTATION PAGE OMB No. 0704-0188Pubic reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering andmaintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information.including suggestion for reducing this burden, to Washington Headquarters Services. Directorate for Information Operations and Reports. 1215 Jefferson Davis Highway. Suite 1204. Arlington.VA 22202-4302. and to the Office of Management and Budget, Paperwork Reduction Project (0704-0188). Washington. DC 20503.

1. AGENCY USE ONLY (Leave blank) 2. REPORT DATE 3. REPORT TYPE AND DATES COVERED

February 19904. TITLE AND SUBTITLE 5. FUNDING NUMBERS

Environmental Transformation Products of Nitroaromatics and Nitramines: LiteratureReview and Recommendations For Analytical Development

6. AUTHORS

Walsh. Marianne E.

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION

REPORT NUMBER

U.S. Army Cold Regions Research and Engineering Laboratory72 Lyme Road Special Report 90-2Hanover, New Hampshire 03755-1290

9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORING/MONITORINGAGENCY REPORT NUMBER

U.S. Army Toxic and Hazardous Materials Agency CETHA-TE-CR-89205Aberdeen Proving Ground, Maryland 21010

11. SUPPLEMNTARY NOTES

12a. DISTRIBUTION/AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE

Approved for public release; distribution is unlimited.

13. ABSTRACT (Maximum 200 words)

The literature describing the environmental transformation of organic explosives and related compounds isreviewed in an attempt to identify those byproducts for which certified analytical methods should be developed.Among those compounds identified are TNT reduction products (aminodinitrotoluenes and diaminonitrotolu-enes) and coupling products (azoxytoluenes). The development of methods is also recommended for the aminoderivatives of DNT, TNB and DNB, as well as the nitroso derivatives of HMX and RDX.

14. SUBJECT TERMS 15. NUMBER OF PAGES29

Analytical methods Explosives' Nitroaromatics 16. PRICE CODEEnvironmental transformation Nitramines TNT " -

17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACTOF REPORT OF THIS PAGE OF ABSTRACT

UNCLASSIFIED UNCLASSIFIED UNCLASSIFIED ULNSN 7540-01-280-5500 Standard Form 298 (Rev. 2-89)

Prescuibed byr ANSI Sid. ZW-IS2M8-102