Environmental fate of 2,4-dinitroanisole (DNAN) and its reduced products.

Several defense departments intend to replace 2,4,6-trinitrotoluene (TNT) in munitions formulations by the less sensitive 2,4-dinitroanisole (DNAN). To help understand environmental behavior and ecological risk associated with DNAN we investigated its key initial abiotic and biotic reaction routes and determined relevant physicochemical parameters (pKa, logKow, aqueous solubility (Sw), partition coefficient (Kd)) for the chemical and its products. Reduction of DNAN with either zero valent iron or bacteria regioselectively produced 2-amino-4-nitroanisole (2-ANAN) which, under strict anaerobic conditions, gave 2,4-diaminoanisole (DAAN). Hydrolysis under environmental conditions was insignificant whereas photolysis gave photodegradable intermediates 2-hydroxy-4-nitroanisole and 2,4-dinitrophenol. Physicochemical properties of DNAN and its amino products drastically depended on the type and position of substituent(s) on the aromatic ring. Sw followed the order (TNT

Controlled expedient disposal of excess gun propellant.

The expedient field disposal of excess gun propellants on the ground is an integral part of live-fire training in many countries. However, burning excess propellant in the field will leave significant quantities of energetic residues and heavy metals in the environment. Compounds such as dinitrotoluene and nitroglycerin and metals such as lead will leach into the soil column, eventually migrating to groundwater. Contamination of the environment will lead to high remediation costs and the possible loss of the training facility. After investigating the contamination at several propellant disposal sites, a portable propellant burn pan was developed and tested. The pan was transported to training sites where excess propellant was loaded and burned in a controlled manner. Up to 120 kg of excess single-base propellant charges have been burned during two series of tests at a consumption rate of greater than 99.9%. Less than 0.03% of the energetic material was recovered outside the burn pan. Recovered lead is largely contained within the pan. The turnover rate for burns is 15 min. The residues can be collected following cool-down for proper disposal.

Assessment of a 2,4,6-trinitrotoluene-contaminated site using Aporrectodea rosea and Eisenia andrei in mesocosms.

Polynitro-organic compounds such as 2,4,6-trinitrotoluene (TNT) can be released into the environment from production and processing facilities and military firing ranges as well as through field use and disposal practices. Based on laboratory toxicity data, TNT has lethal (at >/=260 mg TNT/kg dry soil) and sublethal effects (at >/=59 mg TNT/kg dry soil) to the earthworm. However, field studies are needed to relate exposure of organisms to explosives in mixed-contaminated soil under field conditions and to define effects-based ecotoxicologic benchmarks for TNT-contaminated soil. In the present study, the lethal and sublethal effects of a 10-day in situ exposure at a TNT-contaminated field site using mesh-bag mesocosms were assessed. In addition to the survival end point, the biomarkers of earthworm exposure and effect-including tissue residues, lysosomal neutral red retention time (NRRT), and total immune activity (TIA)-were measured. Concentrations of TNT in soil mesocosms ranged from 25 to 17,063 mg/kg. Experiments indicated a trend toward decreasing survival of caged Aporrectodea rosea and Eisenia andrei as the concentration of TNT and total nitroaromatic compounds increased. E. andrei tolerated higher concentrations of TNT (up to 4050 mg/kg dry soil) in mesocosms than did indigenous earthworms, who survived only at

Phytotoxicity of 2,4,6-trinitrotoluene (TNT) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in spiked artificial and natural forest soils.

Toxicity of 2,4,6-trinitrotoluene (TNT) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) using two terrestrial plant species, lettuce (Lactuca sativa) and barley (Hordeum vugare), was assessed in artificial soil (silica) and forest soil. Lettuce emergence was significantly decreased after 5 days of exposure to TNT nominal spiked concentrations >/= 1,040 mg/kg dry soil in silica. Barley emergence was significantly reduced after 14 days of exposure at initial (t = 0) TNT concentrations >/= 55.9 +/- 4.5 mg/kg dry soil in silica and at >/= 291.9 +/- 42.8 mg/kg dry forest soil. Biomasses of shoot and roots of barley seeds were significantly reduced after 14 days of exposure at TNT initial exposure concentrations >/= 55.9 +/- 4.5 (LOEC) mg/kg dry soil in silica. Results were similar with the forest soil (LOEC = 91.4 +/- 7.9 mg TNT/kg dry soil) using the root growth parameter, but the shoot biomass was reduced only at concentrations >/= 291.9 +/- 42.8 mg TNT/kg dry soil. Plants were not affected by an HMX exposure up to 3,320 +/- 1,019 mg/kg dry soil using silica or 1,866 +/- 438 mg/kg dry soil using a forest soil. During the 14-day experiments, TNT was partially transformed in the spiked soil samples, as indicated by the presence of its amino metabolites (2-ADNT and 4-ADNT). Higher quantities of metabolites were detected in forest soils having higher initial TNT concentrations (

TNT, RDX, and HMX decrease earthworm (Eisenia andrei) life-cycle responses in a spiked natural forest soil.

Sublethal and chronic toxicities of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) on earthworm Eisenia andrei in a sandy forest soil were assessed. Various reproduction parameters of fecundity (total and hatched number of cocoons, number of juveniles, and their biomass) were significantly decreased by TNT (> or = 58.8 +/- 5.1 mg/kg dry soil), RDX (> or = 46.7 +/- 2.6 mg/kg), and HMX (> or = 15.6 +/- 4.6 mg/kg). These effects occurred at much lower concentrations than those reported earlier using artificial soil preparations. Growth of adults was significantly decreased in the TNT-spiked natural soils at 136.2 +/- 25.6 mg/kg dry soil, the highest concentration having no significant mortality. In contrast, survival and growth were not significantly reduced at relatively high measured concentrations of RDX (167.3 mg/kg) and HMX (711.0 mg/kg). Although TNT, RDX, and HMX share a common life-cycle response ( i.e., decreased juvenile counts), a number of differences related to other reproduction parameters (e.g., productivity of cocoons) was observed. These results indicate that the tested explosives do not support a common mechanism of toxicity, at least in the earthworm, probably due to differences in their physical-chemical properties as well as metabolites formed during exposure.

Detection of explosives and their degradation products in soil environments.

Polynitro organic explosives [hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and 2,4,6-trinitrotoluene (TNT)] are typical labile environmental pollutants that can biotransform with soil indigenous microorganisms, photodegrade by sunlight and migrate through subsurface soil to cause groundwater contamination. To be able to determine the type and concentration of explosives and their (bio)transformation products in different soil environments, a comprehensive analytical methodology of sample preparation, separation and detection is thus required. The present paper describes the use of supercritical carbon dioxide (SC-CO2), acetonitrile (MeCN) (US Environmental Protection Agency Method 8330) and solid-phase microextraction (SPME) for the extraction of explosives and their degradation products from various water, soil and plant tissue samples for subsequent analysis by either HPLC-UV, capillary electrophoresis (CE-UV) or GC-MS. Contaminated surface and subsurface soil and groundwater were collected from either a TNT manufacturing facility or an anti-tank firing range. Plant tissue samples were taken fromplants grown in anti-tank firing range soil in a greenhouse experiment. All tested soil and groundwater samples from the former TNT manufacturing plant were found to contain TNT and some of its amino reduced and partially denitrated products. Their concentrations as determined by SPME-GC-MS and LC-UV depended on the location of sampling at the site. In the case of plant tissues, SC-CO2 extraction followed by CE-UV analysis showed only the presence of HMX. The concentrations of HMX (<200 mg/kg) as determined by supercritical fluid extraction (SC-CO2)-CE-UV were comparable to those obtained by MeCN extraction, although the latter technique was found to be more efficient at higher concentrations (>300 mg/kg). Modifiers such as MeCN and water enhanced the SC-CO2 extractability of HMX from plant tissues.

Ecotoxicological effects of hexahydro-1,3,5-trinitro-1,3,5-triazine on soil microbial activities.

Although hexahydro-1,3,5-trinitro-1,3,5-triazine (also called RDX or hexogen) is a potentially toxic explosive compound that persists in soil, its ecotoxicological effects on soil organisms have rarely been assessed. In this study, two uncontaminated garden soils were spiked with 10 to 12,500 mg RDX/kg dry soil. Soil microbial activities, i.e., potential nitrification, nitrogen fixation, dehydrogenase, basal respiration, and substrate-induced respiration were chosen as bioindicators and were determined after 1-, 4-, and 12-weeks of exposure. Experimental results indicate that RDX showed significant inhibition (up to 36% of control) on indigenous soil microbial communities over the period of this study. All five bioindicators responded similarly to the RDX challenge. The length of exposure also affected the microbial toxicity of RDX, with 12-week exposure exerting more significant effects than the shorter exposure periods, suggesting that soil microorganisms might become more vulnerable to RDX when exposure is extended. The estimated lowest observable adverse effect concentration of RDX was 1,235 mg/kg. No biodegradation products of RDX were detected at all three sampling times. Compared with 2,4,6-trinitrotoluene (TNT), RDX is less toxic to microbes, probably because of its resistance to biodegradation under aerobic conditions, which precludes metabolic activation of nitro groups.

Biotransformation routes of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine by municipal anaerobic sludge.

Recently we demonstrated that hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a trimer of methylene nitramine (CH2=N-NO2) undergoes spontaneous decomposition following an initial microbial attack using a mixed microbial culture at pH 7 in the presence of glucose as carbon source. The present study describes whether the second cyclic nitramine octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), a more strained tetramer of CH2=N-NO2, degrades similarly using sludge of the same source. Part of HMX biotransformed to give products that are tentatively identified as the nitroso derivatives octahydro-1-nitroso-3,5,7-trinitro-1,3,5,7-tetrazocine (mNs-HMX) and octahydro-1,3-dinitroso-5,7-dinitro-1,3,5,7-tetrazocine and its isomer octahydro-1,5-dinitroso-3,7-dinitro-1,3,5,7-tetrazocine (dNs-HMX). Another fraction of HMX biotransformed, apparently via ring cleavage, to produce products that are tentatively identified as methylenedinitramine (O2NNHCH2-NHNO2) and bis(hydroxymethyl)nitramine ((HOCH2)2NNO2). None of the above intermediates accumulated indefinitely; they disappeared to predominantly form nitrous oxide (N2O) and formaldehyde (HCHO). Formaldehyde biotransformed further to eventually produce carbon dioxide (14CO2). Nitrous oxide persisted in HMX microcosms containing glucose but denitrified rapidly to nitrogen in the absence of glucose. The presence of nitrous oxide was accompanied by the presence of appreciable amounts of hydrogen sulfide, a known inhibitor of denitrification.

The fate of the cyclic nitramine explosive RDX in natural soil.

The sorption-desorption behavior and long-term fate of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was examined in sterilized and nonsterilized topsoil. Results of this study indicate that although RDX is not extensively sorbed by the topsoil (Ks(d) of 0.83 L/kg), sorption is nearly irreversible. Furthermore, there was no difference in the sorption behavior for sterile and nonsterile topsoil. However, over the longterm, RDX completely disappeared within 5 weeks in nonsterile topsoil, and hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) metabolites formed in the aqueous phase. Over the same period, recovery of RDX from sterile topsoil was high (55-99%), and the nitroso metabolites were not detected. Only traces of RDX were mineralized to CO2 and N2O by the indigenous microorganisms in nonsterile topsoil. Of the RDX that was mineralized to N2O, one N originated from the ring and the other from the nitro group substituent, as determined using N15 ring-labeled RDX. However, N2O from RDX represented only 3% of the total N2O that formed from the process of nitrification/denitrification.

Explosive biodegradation in soil slurry batch reactors amended with exogenous microorganisms.

The present study explores the feasibility of biotreatment of 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro- 1,3,5-triazine (RDX)-contaminated soils in slurry batch reactors. Radiorespirometric assays showed that anaerobic sludge was able to mineralize 59% RDX to CO2 although significant mineralization of TNT was not observed in all cases. TNT and RDX at concentrations higher than 50 and 100 mg/L respectively were inhibitory to methanogenesis (used as a bioindicator), however, methanogenesis recovered after TNT was transformed into less toxic triaminotoluene. Bioslurry batch reactors containing 40% of contaminated soil (2000 mg RDX and 1000 mg TNT/kg dry soil) were operated under various conditions. Both TNT and RDX were persistent to soil indigenous microbes. Degradation of both TNT and RDX was enhanced by the municipal sludge amendment, although degradation of RDX was only achieved under anaerobic conditions.

Transformation of 2,4,6-trinitrotoluene (TNT) by immobilized Phanerochaete chrysosporium under fed-batch and continuous TNT feeding conditions.

The cometabolic transformation of 2,4,6-trinitrotoluene (TNT) by an immobilized Phanerochaete chrysosporium culture was investigated under different TNT and/or glycerol feeding conditions in a 5-L reactor. In the fed-batch feeding mode, as a result of four spiking events at an average feeding rate of 20 mg TNT L(-1) d(-1) and 250 mg glycerol L(-1) d(-1), the initial TNT transformation rate and the glycerol uptake rate of the 7-day-old immobilized cell culture were 2.41 mg L(-1) h(-1) and 16.6 mg L(-1) h(-1), respectively. Thereafter, the TNT fed into the reactor depicted a negative effect on the cell physiology of P. chrysosporium, i.e., both rates decreased constantly. At 32 mg TNT L(-1) d(-1) feeding rate, also in the presence of glycerol (200 mg L(-1) d(-1)), this effect on the fungal cell metabolism was even more significant. When TNT was fed alone at 3.7 mg L(-1) d(-1), it showed an initial 0.75 mg L(-1) h(-1) rate of TNT transformation, i.e., one-third the initial level observed in the presence of glycerol. In contrast, in the continuous feeding mode (dilution rate, D = 0.11 d(-1)), at 5.5 mg TNT L(-1) d(-1) and 220 mg glycerol L(-1) d(-1), the immobilized cell culture exhibited a constant TNT transformation rate for cultivation periods of 50 and 61 days, under uncontrolled and controlled pH conditions, respectively. Thereafter, during the latter experiment, 100% TNT biotransformation was achieved at 1,100 mg L(-1) d(-1) glycerol feeding rate. Immobilized cells (115-day-old), sampled from a continuous TNT feeding experiment, mineralized [(14)C]-TNT to a level of 15.3% following a 41-day incubation period in a microcosm.

Chronic toxicity of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in soil determined using the earthworm (Eisenia andrei) reproduction test.

The sublethal and chronic effects of the environmental contaminant and explosive octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in artificial soil were assessed using the earthworm (Eisenia andrei). Based on various reproduction parameters (total and hatched number of cocoons, number of juveniles and their biomass), fecundity was reduced at the different concentrations of HMX tested (from 280.0 +/- 12.3 to 2502.9 +/- 230.0 mg kg-1 dry soil) in spiked artificial soil (LOEC: 280.0 +/- 12.3 mg kg-1 dry soil). The growth of adult E. andrei was also reduced at the different concentrations tested, though no mortality occurred, even at the highest tested concentrations. The number of juveniles produced was correlated with the number of total and hatched cocoons, and the biomass of juveniles was correlated with the number of cocoons. Pooled results of these and earlier studies on explosives (TNT, RDX) using the E. andrei reproduction test confirm that effects of HMX on cocoon production are indicative of some reproductive consequences (number of juvenile and their biomass), whereas adult growth, in general, does not correlate strongly with change in reproduction capacity.

Microbial degradation of explosives: biotransformation versus mineralization.

The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a reactive molecule that biotransforms readily under both aerobic and anaerobic conditions to give aminodinitrotoluenes. The resulting amines biotransform to give several other products, including azo, azoxy, acetyl and phenolic derivatives, leaving the aromatic ring intact. Although some Meisenheimer complexes, initiated by hydride ion attack on the ring, can be formed during TNT biodegradation, little or no mineralization is encountered during bacterial treatment. Also, although the ligninolytic physiological phase and manganese peroxidase system of fungi can cause some TNT mineralization in liquid cultures, little to no mineralization is observed in soil. Therefore, despite more than two decades of intensive research to biodegrade TNT, no biomineralization-based technologies have been successful to date. The non-aromatic cyclic nitramine explosives hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) lack the electronic stability enjoyed by TNT or its transformed products. Predictably, a successful enzymatic change on one of the N-NO2 or C-H bonds of the cyclic nitramine would lead to a ring cleavage because the inner C-N bonds in RDX become very weak (<2 kcal/mol). Recently this hypothesis was tested and proved feasible, when RDX produced high amounts of carbon dioxide and nitrous oxide following its treatment with either municipal anaerobic sludge or the fungus Phanaerocheate chrysosporium. Research aimed at the discovery of new microorganisms and enzymes capable of mineralizing energetic chemicals and/or enhancing irreversible binding (immobilization) of their products to soil is presently receiving considerable attention from the scientific community.

An in situ respirometric technique to measure pollution-induced microbial community tolerance in soils contaminated with 2,4, 6-trinitrotoluene.

Long-term exposure to 2,4,6-trinitrotoluene (TNT) can induce changes in the structure and activities of soil microbial communities. Such changes may be associated with an elevated microbial tolerance. An in situ respirometry technique based on the analysis of the substrate-induced respiration response to freshly added TNT was used to examine soil microbial tolerance to TNT at the community level. The specific growth rate derived by fitting an exponential equation to respiration data was taken as the measurement endpoint. Microbial tolerance was evaluated using a tolerance index defined as the ratio of the specific growth rate at a spiking dose of 2000 microg TNT/g soil to that of the control with no spiked TNT. Three soils with long-term exposure histories (TNT level in soil: 1.5, 32, and 620 microg TNT/g, respectively) exhibited significantly higher microbial community tolerance to TNT than two uncontaminated control soils. A soil containing 29,000 microg TNT/g exhibited the highest tolerance. Findings from this study support the hypothesis that pollution-induced community tolerance can be used as a means of identifying those compounds that have exerted selective pressure on the community.

Characterization of metabolites during biodegradation of hexahydro-1, 3,5-trinitro-1,3,5-triazine (RDX) with municipal anaerobic sludge.

The biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in liquid cultures with municipal anaerobic sludge showed that at least two degradation routes were involved in the disappearance of the cyclic nitramine. In one route, RDX was reduced to give the familiar nitroso derivatives hexahydro-1-nitroso-3,5-dinitro-1,3, 5-triazine (MNX) and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX). In the second route, two novel metabolites, methylenedinitramine [(O(2)NNH)(2)CH(2)] and bis(hydroxymethyl)nitramine [(HOCH(2))(2)NNO(2)], formed and were presumed to be ring cleavage products produced by enzymatic hydrolysis of the inner C---N bonds of RDX. None of the above metabolites accumulated in the system, and they disappeared to produce nitrous oxide (N(2)O) as a nitrogen-containing end product and formaldehyde (HCHO), methanol (MeOH), and formic acid (HCOOH) that in turn disappeared to produce CH(4) and CO(2) as carbon-containing end products.

Tween 80 enhanced TNT mineralization by Phanerochaete chrysosporium.

The effect of a nonionic surfactant (Tween 80) on 2,4,6-trinitrotoluene (TNT) mineralization by the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767, was investigated in a liquid culture at 20, 50, and 100 mg TNT.L-1. The presence of 1% (w/v) Tween 80, at 20 mg.L-1 TNT, added to a 4-d-old culture, allowed the highest TNT mineralization level, that is 29.3% after 24 d, which is two times more than the control culture, without Tween 80 (13.9%). The mineralization of TNT resumed upon additional Tween 80 supplementation, consequently, 39.0% of the TNT was respired on day 68. Orbital agitation of the fungal culture was found detrimental to TNT mineralization, with or without Tween 80 in the culture medium. The surfactant also stimulated the growth of P. chrysosporium without any notable effect on either the glycerol consumption rate or the extracellular LiP and MnP activity levels. Respirometric assays highlighted some differences between the oxygen uptake rate of the fungal culture supplemented with or without Tween 80.

Origin of p-cresol in the anaerobic degradation of trinitrotoluene.

p-Cresol was repeatedly detected as a trace metabolite in anaerobic slurry reactors treating 2,4,6-trinitrotoluene (TNT)-contaminated soils. This study shows that p-cresol was not a metabolite of the anaerobic degradation of TNT, by using a combination of analytical techniques and 13C-labelled TNT. Instead, p-cresol, an intermediate in the degradation pathway of some amino acids, was shown to be inhibited by TNT and its metabolites. The range and persistence of inhibition to p-cresol microbial degradation decreased with the level of amino-substitution of the derivatives. This explains why p-cresol accumulated within the TNT-treating anaerobic bioslurry, as it could not be further biodegraded in the presence of TNT.

Acute toxicity of 2,4,6-trinitrotoluene in earthworm (Eisenia andrei).

2,4,6-Trinitrotoluene (TNT) is an worldwide recalcitrant environmental contaminant and is toxic to a number of organisms including humans. This study examines the acute effects (lethal and biomass changes) of TNT on the oligochaetes species Eisenia andrei, using the 3-day filter paper, and the 7- and 14-day direct contact spiked soil (OECD artificial and forest soil) toxicity tests. Studies using the filter paper test indicated that the lethality of TNT could be detected in the range 1.5 to 14.2 microg/cm(2), with significant biomass (body weight) changes occurring at the lowest concentration. Acute effects (lethality) could not be measured when earthworms were placed on filter paper containing a saturated aqueous solution of TNT. This may indicate that with these exposure conditions, TNT may have been adsorbed to the filter paper, and that this matrix should be saturated with TNT before becoming available to the earthworms. Spiked soil toxicity tests indicated that the E. andrei lethality by TNT was >1.5 times higher when earthworms were exposed to TNT-spiked forest soil (LOEC:260 mg/kg; LC(50) 14 days 222.4 mg/kg) than to spiked OECD artificial soil (LOEC:420 mg/kg; LC(50) 14 days: 364.9 mg/kg). The sublethal effect on biomass change at the selected TNT concentrations in soil was not significant compared to controls. Results indicate that the bioanalytical methods described in this article could be used as TNT toxicity assessment tools. This soil quality test method gives valuable information for the screening of soil toxicity.

Cytotoxic and genotoxic effects of energetic compounds on bacterial and mammalian cells in vitro.

The mutagenicity and toxicity of energetic compounds such as 2,4, 6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), hexahydro-1,3, 5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3, 5,7-tetrazocine (HMX), and of amino/nitro derivatives of toluene were investigated in vitro. Mutagenicity was evaluated with the Salmonella fluctuation test (FT) and the V79 Chinese hamster lung cell mutagenicity assay. Cytotoxicity was evaluated using V79 and TK6 human lymphoblastic cells. For the TK6 and V79 assays, TNB and 2, 4,6-triaminotoluene were more toxic than TNT, whereas RDX and HMX were without effect at their maximal aqueous solubility limits. The primary TNT metabolites (2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2, 6-diamino-4-nitrotoluene) were generally less cytotoxic than the parent compound. The FT results indicated that TNB, TNT and all the tested primary TNT metabolites were mutagenic. Except for the cases of 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene in the TA98 strain, addition of rat liver S9 resulted in either no effect, or decreased activity. None of the tested compounds were mutagenic for the V79 mammalian cells with or without S9 metabolic activation. Thus, the FT assay was more sensitive to the genotoxic effects of energetic compounds than was the V79 test, suggesting that the FT might be a better screening tool for the presence of these explosives. The lack of mutagenicity of pure substances for V79 cells under the conditions used in this study does not preclude that genotoxicity could actually exist in other mammalian cells. In view of earlier reports and this study, mutagenicity testing of environmental samples should be considered as part of the hazard assessment of sites contaminated by TNT and related products.