Phytotreatment of TNT-Contaminated Groundwater

Phytoremediation is a viable technique for treating nitroaromatic compounds, particularly munitions. Continuous flow phyto-reactor studies were conducted at the following three influent concentrations of 2,4,6-trinitrotoluene (TNT): 1, 5, and 10?ppm. A control was also prepared with an influent TNT concentration of 5 ppm. Flow rates were systematically reduced to increase hydraulic retention times (HRT) which ranged from 12 to 76 days. Initially, the control reactor removed TNT as efficiently as the plant reactors. With time, however, the efficiency of the control became less than that of the plant reactors, suggesting that adsorption was initially the mechanism for removal. Up to 100% of the TNT was removed. Aminodinitrotoluene (ADNT) effluent concentration was higher for higher TNT influent concentrations. Increasing the retention time reduced ADNT concentration in the effluent. Supplementary batch studies confirmed that ADNT and diaminonitrotoluene (DANT) were phytodegraded. Preliminary batch studies were also conducted on the degradation of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX (Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine). These batch studies indicated that the degradation of RDX was slower than that for TNT. A study with HMX indicated that the removal rates were reasonable, but required a lag phase.     

Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.     

Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.     

Sheep prefer clean forage over forage contaminated with military explosives TNT, RDX and HMX

The common military explosives 2-methyl-1,3,5-trinitrobenzene (TNT), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) and 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX) are distributed in many military training areas, and are thus encountered by grazing animals. The aim of this study was to examine small ruminant's intake of forage contaminated with explosives. An indoor, experimental setup was used to determine if contamination of forage by these compounds affected intake by sheep. The results clearly demonstrate that contamination by any of the three explosives reduced forage intake in sheep; in order of increasing avoidance: RDX < TNT < HMX. The results are discussed in a risk assessment context.     

Leaching of contaminated leaves following uptake and phytoremediation of RDX, HMX, and TNT by poplar

The uptake and fate of 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by hybrid poplars in hydroponic systems were compared and exposed leaves were leached with water to simulate potential exposure pathways from groundwater in the field. TNT was removed from solution more quickly than nitramine explosives. Most of radioactivity remained in root tissues for 14C-TNT, but in leaves for 14C-RDX and 14C-HMX. Radiolabel recovery for TNT and HMX was over 94%, but that of RDX decreased over time, suggesting a loss of volatile products. A considerable fraction (45.5%) of radioactivity taken up by whole plants exposed to 14C-HMX was released into deionized water, mostly as parent compound after 5 d of leaching. About a quarter (24.0%) and 1.2% were leached for RDX and TNT, respectively, mostly as transformed products. Leached radioactivity from roots was insignificant in all cases (< 2%). This is the first report in which small amounts of transformation products of RDX leach from dried leaves following uptake by poplars. Such behavior for HMX was reported earlier and is reconfirmed here. All three compounds differ substantially in their fate and transport during the leaching process.     

Biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine by novel fungi isolated from unexploded ordnance contaminated marine sediment

Undersea deposition of unexploded ordnance (UXO) constitutes a potential source of contamination of marine environments by hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). Using sediment from a coastal UXO field, Oahu Island, Hawaii, we isolated four novel aerobic RDX-degrading fungi HAW-OCF1, HAW-OCF2, HAW-OCF3 and HAW-OCF5, tentatively identified as members of Rhodotorula, Bullera, Acremonium and Penicillium, respectively. The four isolates mineralized 15–34% of RDX in 58 days as determined by liberated ¹⁴CO₂. Subsequently we selected Acremonium to determine biotransformation pathway(s) of RDX in more details. When RDX (100 μM) was incubated with resting cells of Acremonium we detected methylenedinitramine (MEDINA), N₂O and HCHO. Also we detected hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) together with trace amounts of hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). Under the same conditions MNX produced N₂O and HCHO together with trace amounts of DNX and TNX, but we were unable to detect MEDINA. TNX did not degrade with Acremonium. These experimental findings suggested that RDX degraded via at least two major initial routes; one route involved direct ring cleavage to MEDINA and another involved reduction to MNX prior to ring cleavage. Nitrite was only detected in trace amounts suggesting that degradation via initial denitration did take place but not significantly. Aerobic incubation of Acremonium in sediment contaminated with RDX led to enhanced removal of the nitramine.     

Long-Term Explosive Contamination in Soil: Effects on Soil Microbial Community and Bioremediation

Explosive contamination in soil is a great concern for environmental health. Following 50 years of munitions manufacturing and loading, soils from two different sites contained ≥ 6,435 mg 2,4,6-trinitrotoluene (TNT), 2,933 mg hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,135 mg octahydrol-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) kg^{? 1} soil. Extractable nitrate-N was as high as 315 and ammonium-N reached 150 mg N kg^{? 1} soil. Water leachates in the highly contaminated soils showed near saturation levels of TNT and RDX, suggesting great risk to water quality. The long-term contamination resulted in undetectable fungal populations and as low as 180 bacterial colony forming units (CFU) g^–1 soil. In the most severely contaminated soil, dehydrogenase activity was undetectable and microbial biomass carbon was very low (< 3.4 mg C _mic kg^–1 soil). The diminished biological activity was a consequence of long-term contamination because short-term (14 d) contamination of TNT at up to 5000 mg TNT kg^–1 soil did not cause a decline in the culturable bacterial population. Natural attenuation may not be a feasible remediation strategy in soils with long-term contamination by high concentrations of explosives.     

Biodegradation of Nitro-Substituted Explosives 2,4,6-Trinitrotoluene, Hexahydro-1,3,5-Trinitro-1,3,5-Triazine, and Octahydro-1,3,5,7-Tetranitro-1,3,5-Tetrazocine by a Phytosymbiotic Methylobacterium sp. Associated with Poplar Tissues (Populus deltoides × nigra DN34)

A pink-pigmented symbiotic bacterium was isolated from hybrid poplar tissues (Populus deltoides × nigra DN34). The bacterium was identified by 16S and 16S-23S intergenic spacer ribosomal DNA analysis as a Methylobacterium sp. (strain BJ001). The isolated bacterium was able to use methanol as the sole source of carbon and energy, which is a specific attribute of the genus Methylobacterium. The bacterium in pure culture was shown to degrade the toxic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX). [U-ring-¹⁴C]TNT (25 mg liter⁻¹) was fully transformed in less than 10 days. Metabolites included the reduction derivatives amino-dinitrotoluenes and diamino-nitrotoluenes. No significant release of ¹⁴CO₂ was recorded from [¹⁴C]TNT. In addition, the isolated methylotroph was shown to transform [U-¹⁴C]RDX (20 mg liter⁻¹) and [U-¹⁴C]HMX (2.5 mg liter⁻¹) in less than 40 days. After 55 days of incubation, 58.0% of initial [¹⁴C]RDX and 61.4% of initial [¹⁴C]HMX were mineralized into ¹⁴CO₂. The radioactivity remaining in solution accounted for 12.8 and 12.7% of initial [¹⁴C]RDX and [¹⁴C]HMX, respectively. Metabolites detected from RDX transformation included a mononitroso RDX derivative and a polar compound tentatively identified as methylenedinitramine. Since members of the genus Methylobacterium are distributed in a wide diversity of natural environments and are very often associated with plants, Methylobacterium sp. strain BJ001 may be involved in natural attenuation or in situ biodegradation (including phytoremediation) of explosive-contaminated sites.     

Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions

Summary Composting was investigated as a bioremediation technology for clean-up of sediments contaminated with explosives and propellants. Two field demonstrations were conducted, the first using 2,4,6-trinitrotoluene (TNT), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and N-methyl-N,2,4,6-tetranitroaniline (tetryl) contaminated sediment, and the second using nitrocellulose (NC) contaminated soil. Tests were conducted in thermophilic and mesophilic aerated static piles. Extractable TNT was reduced from 11840 mg/kg to 3 mg/kg, and NC from 13090 mg/kg to 16 mg/kg under thermophilic conditions. Under mesophilic conditions, TNT was reduced from 11 190 mg/kg to 50 mg/kg. The thermophilic and mesophilic half-lives were 11.9 and 21.9 days for TNT, 17.3 and 30.1 days for RDX, and 22.8 and 42.0 days for HMX, respectively. Known nitroaromatic transformation products increased in concentration over the first several weeks of the test period, but decreased to low concentrations thereafter.     

Aerobic Biodegradation of High Explosives, Phase I - HMX

The Pantex facility near Amarillo, Texas, has soil and groundwater contaminated with differing combinations of high explosives (HEs), including 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). This project was concerned with direct treatment of HMX in groundwater withdrawn at this plant. Several physical and chemical treatment schemes for the treatment of HMX have been successful. However, the successful biological treatment of HMX has been limited to anaerobic environments. The objective of this work was to identify microbial consortia and amendments capable of aerobically biodegrading HMX in water. Microbial consortia and amendments employed were provided as livestock manure and soil with its indigenous flora from nearby historically contaminated sites. Possible losses of HMX by nonbiological means such as adsorption and photolysis were accounted for by appropriate abiotic experiments. Loss of the parent compound was measured by high-performance liquid chromatography, using a modification of U.S. Environmental Protection Agency (EPA) Method 8330. Results varied from no degradation to a reduction of parent HMX from 6 to 1 mg/L in 5.2 days. Evidence for biodegradation was supported by the appearance of metabolites. Metabolite identification was performed at Oak Ridge National Laboratory. Five metabolites (four intermediate and one final) were identified.     

Enhanced Biodegradation and Fate of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) and Octahydro-1,3,5,7-Tetranitro-1,3,5,7-Tetrazocine (HMX) in Anaerobic Soil Slurry Bioprocess

Native soil microbial populations and unadapted municipal anaerobic sludges were compared for nitramine explosive degradation in microcosm assays under various conditions. Microbial populations from an explosive-contaminated soil were only able to mineralize 12% hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (at a concentration of 800 mg/kg slurry) or 4% octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (at a concentration of 267 mg/kg slurry). In contrast, municipal anaerobic sludges were able to mineralize them to carbon dioxide, with efficiencies of up to 65%. Reduction of RDX and HMX into their corresponding nitroso-derivatives was notably faster than their mineralization. The biodegradation of HMX was typically delayed by the presence of RDX in the microcosm, confirming RDX is used as an electron acceptor preferentially to HMX. The laboratory-scale bioslurry reactor reproduced the results of the microcosm assays, yet with much higher RDX and HMX degradation rates. A radiolabel-based mass balance in the soil slurry indicated that, besides a significant mineralization to carbon dioxide, 25% and 31% of RDX and HMX, respectively, appeared as acetonitrile-extractable metabolites, while the remaining part was incorporated into biomass and irreversibly bound to the soil matrix. About 10% of the HMX derivatives were estimated to be chemically bound to the soil matrix, while for RDX the estimation was nil.     

Biodegradation of nitro-substituted explosives 2,4,6-trinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine, and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine by a phytosymbiotic Methylobacterium sp. associated with poplar tissues (Populus deltoides x nigra DN34)

A pink-pigmented symbiotic bacterium was isolated from hybrid poplar tissues (Populus deltoides x nigra DN34). The bacterium was identified by 16S and 16S-23S intergenic spacer ribosomal DNA analysis as a Methylobacterium sp. (strain BJ001). The isolated bacterium was able to use methanol as the sole source of carbon and energy, which is a specific attribute of the genus Methylobacterium. The bacterium in pure culture was shown to degrade the toxic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX). [U-ring-(14)C]TNT (25 mg liter(-1)) was fully transformed in less than 10 days. Metabolites included the reduction derivatives amino-dinitrotoluenes and diamino-nitrotoluenes. No significant release of (14)CO(2) was recorded from [(14)C]TNT. In addition, the isolated methylotroph was shown to transform [U-(14)C]RDX (20 mg liter(-1)) and [U-(14)C]HMX (2.5 mg liter(-1)) in less than 40 days. After 55 days of incubation, 58.0% of initial [(14)C]RDX and 61.4% of initial [(14)C]HMX were mineralized into (14)CO(2). The radioactivity remaining in solution accounted for 12.8 and 12.7% of initial [(14)C]RDX and [(14)C]HMX, respectively. Metabolites detected from RDX transformation included a mononitroso RDX derivative and a polar compound tentatively identified as methylenedinitramine. Since members of the genus Methylobacterium are distributed in a wide diversity of natural environments and are very often associated with plants, Methylobacterium sp. strain BJ001 may be involved in natural attenuation or in situ biodegradation (including phytoremediation) of explosive-contaminated sites.     

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. Received: 14 February 2000 / Received revision: 9 June 2000 / Accepted: 13 June 2000     

A Peat Moss-Based Technology for Mitigating Residues of the Explosives TNT,RDX, and HMX in Soil

There is increased interest in how to balance military preparedness and environmental protection at Department of Defense (DoD) facilities. This research evaluated a peat moss-based technology to enhance the adsorption and biodegradation of explosive residues at military testing and training ranges. The evaluation was performed using 30-cm-long soil columns operated under unsaturated flow conditions. The treatment materials were placed at the soil surface, and soil contaminated with 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) was spread over the surface. Simulated rainfall initiated dissolution and leaching of the explosive compounds, which was monitored at several depths within the columns. Peat moss plus soybean oil reduced the soluble concentrations of TNT, RDX and HMX detected at 10 cm depth by 100%, 60%, and 40%, respectively, compared to the no-treatment control column. Peat moss alone reduced TNT and HMX concentrations at 10 cm depth relative to the control, but exhibited higher soluble RDX concentrations by the end of the experiment. Concentrations of HMX and RDX were also reduced at 30 cm depth by the peat moss plus soybean oil treatments relative to those observed in the control column. These preliminary results demonstrate proof-of-concept of a low cost technology for reducing the contamination of groundwater with explosives at military test and training ranges.     

Metabolism of Explosive Compounds by Sulfate-Reducing Bacteria

The metabolism of various explosive compounds—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-tetraazocine (HMX)—by a sulfate-reducing bacterial consortium, Desulfovibrio spp., was studied. The results indicated that the Desulfovibrio spp. used all of the explosive compounds studied as their sole source of nitrogen for growth. The concentrations of TNB, RDX, and HMX in the culture media dropped to below the detection limit (<0.5 ppm) within 18 days of incubation. We also observed the production of ammonia from the nitro groups of the explosive compounds in the culture media. This ammonia served as a nitrogen source for the bacterial growth, and the concentration of ammonia later dropped to <0.5 mg/L. The sulfate-reducing bacteria may be useful in the anaerobic treatment of explosives-contaminated soil. Received: 23 January 1998 / Accepted: 5 March 1998     

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.     

Isolation of three hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading species of the family Enterobacteriaceae from nitramine explosive-contaminated soil.

Three species of the family Enterobacteriaceae that biochemically reduced hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) were isolated from nitramine explosive-contaminated soil. Two isolates, identified as Morganella morganii and Providencia rettgeri, completely transformed both RDX and the nitroso-RDX reduction intermediates. The third isolate, identified as Citrobacter freundii, partially transformed RDX and generated high concentrations of nitroso-RDX intermediates. All three isolates produced 14CO2 from labeled RDX under O2-depleted culture conditions. While all three isolates transformed HMX, only M. morganii transformed HMX in the presence of RDX.     

Plant processes important for the transformation and degradation of explosives contaminants

Environmental contamination by explosives is a worldwide problem. Of the 20 energetic compounds, 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are the most powerful and commonly used. Nitroamines are toxic and considered as possible carcinogens. The toxicity and persistence of nitroamines requires that their fate in the environment be understood and that contaminated soil and groundwater be remediated. This study, written as a minireview, provides further insights for plant processes important for the transformation and degradation of explosives. Plants metabolize TNT and the distribution of the transformation products, conjugates, and bound residues appears to be consistent with the green liver model concept. Metabolism of TNT in plants occurs by reduction as well as by oxidation. Reduction probably plays an important role in the tolerance of plants towards TNT, and, therefore a high nitroreductase capacity may serve as a biochemical criterion for the selection of plant species to remediate TNT. Because the activities and the inducibilities of the oxidative enzymes are far lower than of nitroreductase, reducing processes may predominate. However, oxidation may initiate the route to conjugation and sequestration leading ultimately to detoxification of TNT, and, therefore, particularly the oxidative pathway deserves more study. It is possible that plants metabolize RDX also according to the green liver concept. In the case of plant metabolism of HMX, a conclusion regarding compliance with the green liver concept was not reached due to the limited number of available data.     

Biodegradation of the nitramine explosives hexahydro-1,3,5-trinitro-1,3,5-triazine and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine in cold marine sediment under anaerobic and oligotrophic conditions

The in situ degradation of the two 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), was evaluated using a mixture of RDX and HMX, incubated anaerobically at 10 degrees C with marine sediment from a previous military dumping site of unexploded ordnance (UXO) in Halifax Harbor, Nova Scotia, Canada. The RDX concentration (14.7 mg.L-1) in the aqueous phase was reduced by half in 4 days, while reduction of HMX concentration (1.2 mg.L-1) by half required 50 days. Supplementation with the carbon sources glucose, acetate, or citrate did not affect the removal rate of RDX but improved removal of HMX. Optimal mineralization of RDX and HMX was obtained in the presence of glucose. Using universally labeled (UL)-[14C]RDX, we obtained a carbon mass balance distributed as follows: CO2, 48%-58%; water soluble products, 27%-31%; acetonitrile extractable products, 2.0%-3.4%; and products covalently bound to the sediments and biomass, 8.9% (in the presence of glucose). The disappearance of RDX was accompanied by the formation of the mononitroso derivative hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and formaldehyde (HCHO) that subsequently disappeared. In the case of HMX, mineralization reached only 13%-27% after 115 days of incubation in the presence or absence of the carbon sources. The disappearance of HMX was also accompanied by the formation of the mononitroso derivative. The total population of psychrotrophic anaerobes that grew at 10 degrees C was 2.6 x 10(3) colony-forming units.(g sediment dry mass)-1, and some psychrotrophic sediment isolates were capable of degrading RDX under conditions similar to those used for sediments. Based on the distribution of products, we suggest that the sediment microorganisms degrade RDX and HMX via an initial reduction to the corresponding mononitroso derivative, followed by denitration and ring cleavage.     

Metabolite production during transformation of 2,4,6-trinitrotoluene (TNT) by a mixed culture acclimated and maintained on crude oil-containing media

Metabolites formed during 2,4,6-trinitrotoluene (TNT) removal by a mixed bacterial culture (acclimated and maintained on crude oil-containing medium and capable of high rates of TNT removal) were characterized. In resting cell experiments in the absence of glucose, 46.2 mg/l TNT were removed in 171 h (87.5% removal), with a combined total formation of 7.7 mg/l amino-4,6-dinitrotoluene (ADNT) and 0.3 mg/l 4,4-azoxytetranitrotoluene and 2,4-azoxytetranitrotoluene, leaving 70% of the initial TNT unaccounted for. In the presence of glucose, resting cells removed 45.4 mg/l TNT in 49 h (95.5% removal), with 9.1 mg/l ADNT and 2.4 mg/l azoxy compounds being produced, leaving 70.3% of the TNT unaccounted for. Growing cells (glucose present) were capable of removing 44.2 mg/l TNT within 21 h (97.9% removal), with the concomitant formation of 1.8 mg/l ADNTs and 2.2 mg/l azoxy compounds. Denitrated TNT in the form of 2,6-dinitrotoluene was also produced in growing cells with a maximum amount of 1.31 mg/l after 28 h, followed by a slight decrease with time, leaving 88.5% of the initial TNT unaccounted for after 171 h. Radiolabeled ¹⁴C-TNT studies revealed 4.14% mineralization after an incubation period of 163 days with growing cells.