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Previous studies of the biodegradation of nonpolar nitroaromatic compounds have suggested that microorganisms can reduce the nitro groups but cannot cleave the aromatic ring. We report here the initial steps in a pathway for complete biodegradation of 2,4-dinitrotoluene (DNT) by a Pseudomonas sp. isolated from a four-member consortium enriched with DNT. The Pseudomonas sp. degraded DNT as the sole source of carbon and energy under aerobic conditions with stoichiometric release of nitrite. During induction of the enzymes required for growth on DNT, 4-methyl-5-nitrocatechol (MNC) accumulated transiently in the culture fluid when cells grown on acetate were transferred to medium containing DNT as the sole carbon and energy source. Conversion of DNT to MNC in the presence of 1802 revealed the simultaneous incorporation of two atoms of molecular oxygen, which demonstrated that the reaction was catalyzed by a dioxygenase. Fully induced cells degraded MNC rapidly with stoichiometric release of nitrite. The results indicate an initial dioxygenase attack at the 4,5 position of DNT with the concomitant release of nitrite. Subsequent reactions lead to complete biodegradation and removal of the second nitro group as nitrite.

The short-term oral toxicity of 2,4,6-trinitrotoluene (alpha-TNT) was determined in dogs, rats, and mice. Single-dose oral LD50s for alpha-TNT in corn oil were 1320 and 794 mg/kg in male and female rats, respectively, and 660 mg/kg in both male and female mice. For multiple-dose studies, dogs were dosed daily for up to 13 wk with alpha-TNT at 0, 0.2, 2.0, or 20 mg/kg by capsule; rats received 0, 0.002, 0.01, 0.05, or 0.25% and mice received 0, 0.001, 0.005, 0.025, or 0.125% alpha-TNT in their diets over the same period. All species receiving the highest doses exhibited anemia, with reduced erythrocytes, hemoglobin, and hematocrit. Alterations were observed in organ weights, including enlarged spleens (accompanied by hemosiderosis) and livers, and depressed body weight and/or body weight gain (temporary in dogs and mice). Alterations in clinical chemistry values included elevated cholesterol and depressed serum glutamicpyruvic transaminase activity in dogs and rats; no effect on serum glutamic-oxaloacetic transaminase activity was observed. Some effects, such as SGPT depression in rats, appeared after 13 wk, suggesting a cumulative toxicity. Reduced testes size was observed in rats at the highest dose regardless of length of exposure. Most of the toxic effects were reversible, but testicular atrophy was not in rats allowed a 4-wk recovery period after treatment. Signs of anemia were present at intermediate dose levels. "No observable effects" levels for alpha-TNT were: dogs, 0.20; rats, 1.42; and mice, 7.76 mg/kg . d.

TNT transformation processes in sediment-free, “natural”, aquatic phytoremediation systems of Myriophyllum aquaticum were investigated with specific interest in oxidation products. Extraction procedures combining liquid−liquid extractions and solid-phase extractions were developed for the isolation of the mostly acidic, oxidized TNT metabolites. Six compounds unique from the reduction products of TNT were isolated and characterized by UV−vis, 1H, and 13C NMR spectroscopy, by mass spectroscopy, and by chemical synthesis where feasible. These compounds include 2-amino-4,6-dinitrobenzoic acid, 2,4-dinitro-6-hydroxy-benzyl alcohol, 2-N-acetoxyamino-4,6-dinitrobenzaldehyde, 2,4-dinitro-6-hydroxytoluene, and two binuclear metabolites unique from the customary azoxytetranitrotoluenes. The monoaryl compounds show clear evidence of oxidative transformations, methyl oxidation and/or aromatic hydroxylation. It is possible that oxidative transformation(s) preceded nitro reduction since studies on exposure of M. aquaticum to either 2-amino-4,6-dinitrotoluene or 4-amino-2,6-dinitrotoluene did not yield any of the oxidation products identified here. The accumulation of oxidation products was significant:  2-amino-4,6-dinitrobenzoic acid, 4.4%; 2,4-dinitro-6-hydroxy-benzyl alcohol, 8.1%; 2-N-acetoxyamino-4,6-dinitrobenzaldehyde, 7.8%; and, 2,4-dinitro-6-hydroxytoluene, 15.6%. The binuclear metabolites accounted for an estimated 5.6%. This study is the first direct evidence for oxidative transformations in aquatic phytoremediation systems.

Initial denitration of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 produces CO 2 and the dead-end product 4-nitro-2,4-diazabutanal (NDAB), OHCNHCH 2 NHNO 2 , in high yield. Here we describe experiments to determine the biodegradability of NDAB in liquid culture and soils containing Phanerochaete chrysosporium. A soil sample taken from an ammunition plant contained RDX (342 mol kg ؊1 ), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 3,057 mol kg ؊1 ), MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine; 155 mol kg ؊1 ), and traces of NDAB (3.8 mol kg ؊1 ). The detection of the last in real soil provided the first experimental evidence for the occurrence of natural attenuation that involved ring cleavage of RDX. When we incubated the soil with strain DN22, both RDX and MNX (but not HMX) degraded and produced NDAB (388 ؎ 22 mol kg ؊1 ) in 5 days. Subsequent incubation of the soil with the fungus led to the removal of NDAB, with the liberation of nitrous oxide (N 2 O). In cultures with the fungus alone NDAB degraded to give a stoichiometric amount of N 2 O. To determine C stoichiometry, we first generated [ 14 C]NDAB in situ by incubating [14 C]RDX with strain DN22, followed by incubation with the fungus. The production of 14 CO 2 increased from 30 (DN22 only) to 76% (fungus). Experiments with pure enzymes revealed that manganese-dependent peroxidase rather than lignin peroxidase was responsible for NDAB degradation. The detection of NDAB in contaminated soil and its effective mineralization by the fungus P. chrysosporium may constitute the basis for the development of bioremediation technologies.The cyclic nitramine hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (Fig. 1) is a widely used energetic compound which has caused severe soil and groundwater contamination (13,24). RDX is toxic (27, 33), and substantial research has been done to evaluate its fate in the environment and to develop efficient bioremediation processes (19). The degradation of RDX under anaerobic conditions was extensively studied, and the studies revealed the involvement of an important microbial diversity (14,17,22,23,(35)(36)(37)(38). Aerobic degradation studies using RDX as a nitrogen source were more recently reported and have led to the isolation of three Rhodococcus strains, namely, Rhodococcus sp. strain DN22 (9), Rhodococcus sp. strain A (20), and Rhodococcus rhodochrous strain 11Y (29), and a strain of Stenotrophomonas maltophilia (7). Coleman et al. (9) reported the release of nitrite (NO 2 Ϫ ) during RDX degradation with strain DN22, whereas Fournier et al. (12) reported the formation of several other products, including nitrous oxide (N 2 O), ammonia (NH 3 ), formaldehyde (HCHO), and a dead-end product (C 2 H 5 N 3 O 3 ) that was tentatively identified as either 4-nitro-2,4-diazabutanal (NDAB; O 2 NNHCH 2 NHCHO) or 2-nitro-3-amino-2-azapropanal (NH 2 CH 2 NNO 2 CHO). Subsequent work by Bhushan et al. (6) rigorously identified the metabolite as NDAB using a reference standard and nuclear magn...

The mutagenicity of 36 polynitroaromatic compounds was investigated with five strains of Salmonella typhimurium. Isomeric trinitrotoluenes (TNT), with the exception of 2,4,6-TNT and 2,3,4-TNT, exhibit mutagenicity independently of nitroreductase enzymes, but isomeric aminodinitrotoluenes (ADNT) and isomeric dinitrotoluenes (DNT) need nitroreductase to induce mutation. Within groups of isomeric TNTs, DNTs, and ADNTs, mutagenic response was enhanced by a para orientation of nitro groups. The mutagenic response of isomeric DNTs was found to correlate with the compound's ability to undergo charge-transfer complexation with reductive enzymes, whereas further complexation (such as a Janovsky complex) appears to be required for inducing mutation in dinitrobenzenes. These results indicate that polynitroaromatic compounds in TNT wastewaters possess a significant potential for biologic activity.

The environmental fate of nitroguanidine (NQ) in surface waters is dominated by photolysis with surface half‐lives at 40°N ranging from 0.6 d in summer to 2.3 d in winter. The environmental quantum yield is 0.01. The NQ is initially photolyzed to nitrite and hydroxyguanidine; nitrite is photochemically converted to nitrate and hydroxyguanidine undergoes sensitized photolysis to unknown products. The photooxidation of nitrite is assisted by organic material in a process not involving H2O2 or singlet oxygen. Nitroguanidine biotransforms cometabolically; in the absence of extra organic nutrients the second‐order rate constant was (3.8 ± 0.9) × 10−10 ml cell−1 h−1. Half‐life estimates for aerobic, aquatic biotransformation range from 1 to 100 d. Cyanamide appears to be an end product of NQ use and no intermediate biotransformation products were observed. Nitroguanidine is expected to move readily through soils (soil sorption coefficient Kp < 0.1); however, anaerobic biotransformation occurs readily in soil, with an estimated half‐life of 4 d. Other fate parameters measured at 25°C are a water solubility of 2,600 ± 100 ppm, octanol/water partition coefficient of 0.148 ± 0.001 (dimensionless), Henry's constant of < 7 × 10−6 (dimension‐less), base hydrolysis constant of (3 ± 1) × 10−4 M−1 S−1, neutral hydrolysis constant ≤ 2 × 10−8 s−1, and biouptake constants of 110 g dry cells/g water for Anabena flos‐aquae and 150 g dry cells/g water for Selenastrum capricornutum.

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