Thermodynamic analysis of trinitrotoluene biodegradation and mineralization pathways

Biodegradation of 2,4,6-trinitrotoluene (TNT) proceeds through several different metabolic pathways. However, the reaction steps which are considered rate-controlling have not been fully determined. Glycolysis and other biological pathways contain biochemical reactions which are acutely rate-limiting due to enzyme control. These rate-limiting steps also have large negative Gibbs free energy changes. Because xenobiotic compounds such as TNT can be used by biological systems as nitrogen, carbon, and energy sources, it is likely that their degradation pathways also contain acutely rate-limiting steps. Identification of these rate-controlling reactions will enhance and better direct genetic engineering techniques to increase specific enzyme levels.This article identifies likely rate-controlling steps (or sets of steps) in reported TNT biodegradation pathways by estimating the Gibbs free energy change for each step and for the overall pathways. The biological standard Gibbs free energy change of reaction was calculated for each pathway step using a group contribution method specifically tailored for biomolecules. The method was also applied to hypothetical "pathways" constructed to mineralize TNT using several different microorganisms. Pathways steps that have large negative Gibbs free energy changes are postulated to be potentially rate-controlling. The microorganisms which utilize degradation pathways with the largest overall (from TNT to citrate) negatiave Gibbs free energy changes were also determined. Such microorganisms can extract more energy from the starting substrate and are thus assumed to have a competitive advantage over other microorganisms. Results from this modeling-based research are consistent with much experimental work available in the literature. (c) 1996 John Wiley & Sons, Inc.     

Methemoglobin formation in human erythrocytes by nitroaromatic explosives

We have examined the structure-activity relationships in methemoglobin (MetHb) formation by high explosives 2,4,6-trinitrotoluene (TNT), 2,4,6-trinitrophenyl-N-nitramine (tetryl) and 2,4,6-trinitrophenyl-N-nitraminoethylnitrate (pentryl), and a number of model nitrobenzenes. In lysed human erythrocytes the rate constants of oxyhemoglobin (OxyHb) oxidation increased with an increase in single-electron reduction potential (E(1)7) or with a decrease of the enthalpies of single-electron reduction of nitroaromatics. Tetryl and pentryl oxidized OxyHb almost 3 times faster than TNT. Although the initial rates of MetHb formation in intact erythrocytes by tetryl, pentryl, and TNT matched their order of reactivity in the oxidation of OxyHb in lysed erythrocytes, TNT was a more efficient MetHb forming agent than tetryl and pentryl during a 24-h incubation. The decreased efficiency of tetryl and pentryl was attributed to their reaction with intraerythrocyte reduced glutathione (GSH) producing 2,4,6-trinitrophenyl-Sglutathione, which acted as a less efficient OxyHb oxidizing agent.     

Detection of 2,4,6-trinitrotoluene in seawater using a reversed-displacement immunosensor

Reported in this study are the experimental design and results of an immunosensor for the detection of the explosive, 2,4,6-trinitrotoluene (TNT) in seawater using a reversed-displacement format. This reversed-displacement immunosensor methodology has successfully measured TNT in seawater by direct injection, eliminating the need for preconcentration or pretreatment of samples. A microcolumn containing an Affi-Gel resin derivatized with a 2,4,6-trinitrobenzene (TNB) moiety and a fluorophore-labeled anti-TNT antibody composed the immunoassay reactive chamber. Fluorophore-labeled anti-TNT antibody was incubated with the modified Affi-Gel resin until binding equilibrium was reached. Under a constant flow, samples containing TNT were introduced into the flow stream displacing the fluorophore-labeled TNT antibody. Limits of detection were 2.5ng/mL or part-per-billion (ppb) for TNT in saline buffer and 25ppb in seawater with an analysis time of 10 min. Two anti-TNT antibodies with differing binding affinities were compared in the reversed-displacement assay format, and a correlation between affinity and detection limits was observed. Furthermore, we have demonstrated that the reversed-displacement format can be used to screen seawater samples containing TNT, remains effective after dozens of cycles, and provides significant fluorescence response before regeneration is required.     

2,4,6-trinitrotoluene reduction by an Fe-only hydrogenase in Clostridium acetobutylicum

The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a K(m) of 152 micro M. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R(2) = 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.     

Biological Degradation of 2,4,6-Trinitrotoluene

Nitroaromatic compounds are xenobiotics that have found multiple applications in the synthesis of foams, pharmaceuticals, pesticides, and explosives. These compounds are toxic and recalcitrant and are degraded relatively slowly in the environment by microorganisms. 2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound. Certain strains of Pseudomonas and fungi can use TNT as a nitrogen source through the removal of nitrogen as nitrite from TNT under aerobic conditions and the further reduction of the released nitrite to ammonium, which is incorporated into carbon skeletons. Phanerochaete chrysosporium and other fungi mineralize TNT under ligninolytic conditions by converting it into reduced TNT intermediates, which are excreted to the external milieu, where they are substrates for ligninolytic enzymes. Most if not all aerobic microorganisms reduce TNT to the corresponding amino derivatives via the formation of nitroso and hydroxylamine intermediates. Condensation of the latter compounds yields highly recalcitrant azoxytetranitrotoluenes. Anaerobic microorganisms can also degrade TNT through different pathways. One pathway, found in Desulfovibrio and Clostridium, involves reduction of TNT to triaminotoluene; subsequent steps are still not known. Some Clostridium species may reduce TNT to hydroxylaminodinitrotoluenes, which are then further metabolized. Another pathway has been described in Pseudomonas sp. strain JLR11 and involves nitrite release and further reduction to ammonium, with almost 85% of the N-TNT incorporated as organic N in the cells. It was recently reported that in this strain TNT can serve as a final electron acceptor in respiratory chains and that the reduction of TNT is coupled to ATP synthesis. In this review we also discuss a number of biotechnological applications of bacteria and fungi, including slurry reactors, composting, and land farming, to remove TNT from polluted soils. These treatments have been designed to achieve mineralization or reduction of TNT and immobilization of its amino derivatives on humic material. These approaches are highly efficient in removing TNT, and increasing amounts of research into the potential usefulness of phytoremediation, rhizophytoremediation, and transgenic plants with bacterial genes for TNT removal are being done.     

Combined biological–chemical procedure for the mineralization of TNT

Contamination of ground and surface water with 2,4,6-trinitrotoluene (TNT) and its biological and chemical transformation products are a persisting problem at former TNT production sites. We have investigated the photochemical degradation of TNT and its aminodinitro-(ADNT) and diaminonitrotoluene (DANT) metabolites using OH-radical generating systems like Fenton and hydrogen peroxide irradiated with UV, in order to compare the degradation and mineralization rate of ADNT- and DANT-isomers with TNT itself. As a result, we find that the aminoderivatives were mineralized much faster than TNT. Consequently, as ADNTs and DANTs are the known dead-end products of biological TNT degradations, we have combined our photochemical procedure with a preceding biological treatment of TNT by a mixed culture from sludge of a sewage plant. This consecutive degradation procedure, however, shows a reduced mineralization rate of the ADNTa and DANTs in the biologically derived supernatant as compared to the pure substances, suggesting that during the biological TNT treatment by sludge competing substrates are released into the solution, and that a more defined biological procedure would be necessary in order to achieve an effective, ecologically and economically acceptable mineralization of TNT from aqueous systems.     

Biological degradation of 2,4,6-trinitrotoluene.

Nitroaromatic compounds are xenobiotics that have found multiple applications in the synthesis of foams, pharmaceuticals, pesticides, and explosives. These compounds are toxic and recalcitrant and are degraded relatively slowly in the environment by microorganisms. 2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound. Certain strains of Pseudomonas and fungi can use TNT as a nitrogen source through the removal of nitrogen as nitrite from TNT under aerobic conditions and the further reduction of the released nitrite to ammonium, which is incorporated into carbon skeletons. Phanerochaete chrysosporium and other fungi mineralize TNT under ligninolytic conditions by converting it into reduced TNT intermediates, which are excreted to the external milieu, where they are substrates for ligninolytic enzymes. Most if not all aerobic microorganisms reduce TNT to the corresponding amino derivatives via the formation of nitroso and hydroxylamine intermediates. Condensation of the latter compounds yields highly recalcitrant azoxytetranitrotoluenes. Anaerobic microorganisms can also degrade TNT through different pathways. One pathway, found in Desulfovibrio and Clostridium, involves reduction of TNT to triaminotoluene; subsequent steps are still not known. Some Clostridium species may reduce TNT to hydroxylaminodinitrotoluenes, which are then further metabolized. Another pathway has been described in Pseudomonas sp. strain JLR11 and involves nitrite release and further reduction to ammonium, with almost 85% of the N-TNT incorporated as organic N in the cells. It was recently reported that in this strain TNT can serve as a final electron acceptor in respiratory chains and that the reduction of TNT is coupled to ATP synthesis. In this review we also discuss a number of biotechnological applications of bacteria and fungi, including slurry reactors, composting, and land farming, to remove TNT from polluted soils. These treatments have been designed to achieve mineralization or reduction of TNT and immobilization of its amino derivatives on humic material. These approaches are highly efficient in removing TNT, and increasing amounts of research into the potential usefulness of phytoremediation, rhizophytoremediation, and transgenic plants with bacterial genes for TNT removal are being done.     

酶法催化降解2,4,6-三硝基甲苯的研究进展

2,4,6-三硝基甲苯(TNT)作为一种广泛使用的含能材料,发挥巨大作用的同时也给环境带来了严重的污染,对人类健康构成一定威胁。目前国内外的TNT处置主要有物理、化学、生物及酶法等方法,其中酶法作为一种新兴的方法,显示了良好的应用潜力,受到研究者的广泛关注,。比较了各类处理方法的优缺点,重点介绍近年来涉及TNT降解的酶学研究进展,并对酶法在TNT废水处理和土壤修复中的应用前景进行展望。     

Promising Strategies for the Mineralisation of 2,4,6-trinitrotoluene

2,4,6-trinitrotoluene (TNT) is known to be one of the most common military explosives. In spite of its established toxicity and mutagenicity for many organisms, soils and groundwater are still being frequently contaminated at manufacturing, disposal and TNT destruction sites. The inability of natural aquatic and soil biota to use TNT as growth substrate has been recognized as the primary limitation in the application of bioremediation processes to contaminated environments. However, promising degradation pathways have been recently discovered which may lead to the mineralisation of TNT. Significant advances have been made in studying the mechanism of TNT denitration, which can be considered as the major reaction and the driving force towards beneficial biodegradation. The possibilities to favour TNT denitration are discussed based on current knowledge of the enzymology and genetics of denitration in nitroaromatic degrading organisms. The literature survey demonstrates that the only enzymes characterized so far for their denitrase activity towards TNT belong to the class I flavin-dependent β/α barrel oxidoreductases, also known as the “Old Yellow Enzyme” family. In addition, this review provides an overview of strategies and future directions towards a rational search for new catabolic activities, including metagenomic library screening, plus new possibilities to improve the activity of known catabolic enzymes acting on TNT, such as DNA shuffling.     

Enhanced transformation of tnt by tobacco plants expressing a bacterial nitroreductase

The manufacture, disposal, and detonation of explosives have resulted in the pollution of large tracts of land and groundwater. Historically, 2,4,6-trinitrotoluene (TNT) is the most widely used military explosive and is toxic to biological systems and recalcitrant to degradation. To examine the feasibility of enhancing the ability of plants to detoxify the explosive TNT, we created transgenic tobacco (Nicotiana tabacum) constitutively expressing the nsfI nitroreductase gene from Enterobacter cloacae. The product of TNT reduction by the nitroreductase was found to be 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT). Characterization of the transgenic lines in sterile, aqueous conditions amended with TNT demonstrated that these plants were able to remove all of the TNT from the medium at an initial concentration of 0.5 mM (113 mg L(-1)) TNT. In contrast, growth was suppressed in wild-type plants at 0.1 mM (23 mg L(-1)). Following uptake, transgenic seedlings transformed TNT predominantly to 4-HADNT and its high levels appeared to correlate with enhanced tolerance and transformation of TNT. Transformation products of TNT were subsequently conjugated to plant macromolecules to a greater degree in transgenic tobacco, indicating enhanced detoxification compared to the wild type.     

2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum

The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a K_m of 152 μM. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R² = 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.     

Detection of 2,4,6-Trinitrotoluene-Utilizing Anaerobic Bacteria by 15N and 13C Incorporation

2,4,6-Trinitrotoluene (¹⁵N or ¹³C labeled) was added to Norfolk Harbor sediments to test whether anaerobic bacteria use TNT for growth. Stable-isotope probing (SIP)-terminal restriction fragment length polymorphism (TRFLP) detected peaks in the [¹⁵N]TNT cultures (60, 163, and 168 bp). The 60-bp peak was also present in the [¹³C]TNT cultures and was related to Lysobacter taiwanensis.It has been estimated that there are over 1 million cubic yards of material contaminated with 2,4,6-trinitrotoluene (TNT) in the United States at concentrations as high as 600,000 to 700,00 mg/kg of material (9). Marine and estuarine sediments have also been impacted through the manufacturing, use, and/or disposal of TNT. Microbial biodegradation of these pollutants in situ is preferable due to the large volume of contaminated soils/sediments. However, it is unclear whether in situ bacteria can utilize TNT as a nitrogen or carbon source. Under aerobic conditions, TNT appears to be largely unavailable to bacteria but can be used by a variety of fungi as a carbon and nitrogen source (7). Under anaerobic conditions, only a few bacterial strains (Clostridium and Desulfovibrio strains and Pseudomonas sp. strain JLR11) have been reported to utilize TNT as a sole nitrogen source (6, 7). It is widely believed that nitroaromatic compounds cannot serve as growth substrates under anaerobic conditions in situ (11), and coamendment strategies are suggested for stimulating TNT transformation to 2,4,6-triaminotoluene (TAT) (1, 7, 18). Given these difficulties, there is no direct evidence that TNT can be biodegraded in situ and there is little proof that anaerobic bacteria can utilize TNT as a sole carbon or nitrogen source in organic-rich sediments. This study tested whether bacteria in Norfolk Harbor sediment are able to incorporate nitrogen (N) or carbon (C) from TNT into biomass under sulfidogenic conditions using stable-isotope probing (SIP). The findings indicate that bacteria assimilate ¹⁵N and ¹³C from TNT into their genomes during anaerobic incubations (2 to 35 days). Interestingly, one small-subunit (SSU) gene, related to Lysobacter taiwanensis, was observed in both the ¹⁵N and the ¹³C incubations.     

Escherichia coli has multiple enzymes that attack TNT and release nitrogen for growth

There has been a growing interest in the degradation of 2,4,6-trinitrotoluene (TNT) over the last decade, ever since its removal from polluted sites was declared an international environmental priority. Certain aerobic and anaerobic microorganisms are capable of using TNT as an N source, although very few studies have proven the mineralization of this compound. An unexpected observation in our laboratory led us to discover that certain Escherichia coli bench laboratory strains have multiple enzymes that attack TNT. One of the NemA products is responsible for the release of nitrite from the nitroaromatic ring: among the metabolites observed in vitro include Meisenheimer dihydride complexes of TNT from which 2-hydroxylamino-6-nitrotoluene is slowly formed during their rearomatization under concomitant release of nitrite. Furthermore, NemA, together with NfsA and NfsB reduce the nitro groups on the aromatic ring to the corresponding hydroxylamino derivatives, which probably results in the release of ammonium ions which can, in turn be used as a nitrogen source by E. coli for growth.     

Assimilation of nitrogen from nitrite and trinitrotoluene in Pseudomonas putida JLR11

Pseudomonas putida JLR11 releases nitrogen from the 2,4,6-trinitrotoluene (TNT) ring as nitrite or ammonium. These processes can occur simultaneously, as shown by the observation that a nasB mutant impaired in the reduction of nitrite to ammonium grew at a slower rate than the parental strain. Nitrogen from TNT is assimilated via the glutamine syntethase-glutamate synthase (GS-GOGAT) pathway, as evidenced by the inability of GOGAT mutants to use TNT. This pathway is also used to assimilate ammonium from reduced nitrate and nitrite. Three mutants that had insertions in ntrC, nasT, and cnmA, which encode regulatory proteins, failed to grow on nitrite but grew on TNT, although slower than the wild type.     

Hydride-mediated reduction of 2,4,6-trinitrotoluene by yeasts as the way to its deep degradation

Broad screening of microorganisms from natural and anthropogenic ecological niches has revealed strains Candida sp. AN-L15 and Geotrichum sp. AN-Z4 which transform 2,4,6-trinitrotoluene (TNT) via alternative pathways (with the domination of hydride ion-mediated reduction of the aromatic ring) and produce relatively high amounts of nitrites. According to the spectrophotometry data, the hydride attack of TNT by Candida sp. AN-L15 and Geotrichum sp. AN-Z4 grown at pH 5.0-8.0 leads to the mono- and dihydride complexes of TNT (H(-)-TNT and 2H(-)-TNT, respectively) and to protonated forms of the latter. Analysis by HPLC, GC-mass spectrometry, and ion chromatography revealed the products of deep conversion of TNT. The growth of the yeast strains in a weakly acidic medium with TNT (440 microM) is accompanied by formation of 2,4-dinitrotoluene (2,4-DNT, up to 18.2 microM). Together with accumulation of nitrites (up to 76.0 microM, depending on pH of the medium), these findings demonstrate the capacity of both strains for TNT denitration. Formation of 2,4-DNT reflects the realization of one of the possible mechanisms of TNT ortho-nitro group elimination and switching over to the pathways of metabolism of dinitrotoluenes, which are much more easily biodegradable than TNT. Simultaneously with the dominating TNT hydride attack, the mechanism of 4- and 6-electron reduction of the nitro group also functions in Candida sp. AN-L15 and Geotrichum sp. AN-Z4. Realization of the studied mechanisms of TNT transformation under growth of Candida sp. AN-L15 on n-alkane is important for bioremediation in the cases of combined pollution by oil products and explosives.     

Biodegradation of nitro-explosives

Environmental contamination by nitro compounds is associated principally with the explosives industry. However, global production and use of explosives is unavoidable. The presently widely used nitro-explosives are TNT (Trinitrotoluene), RDX (Royal Demolition Explosive) and HMX (High Melting Explosive). Nevertheless, the problems of these nitro-explosives are almost parallel due to their similarities of production processes, abundance of nitro-explosives and resembling chemical structures. The nitro-explosives per se as well as their environmental transformation products are toxic, showing symptoms as methaemoglobinaemia, kidney trouble, jaundice etc. Hence their removal/degradation from soil/water is essential. Aerobic and anaerobic degradation of TNT and RDX have been reported, while for HMX anaerobic or anoxic degradation have been described in many studies. A multisystem involvement using plants in remediation is gaining importance. Thus the information about degradation of nitro-explosives is available in jigsaw pieces which needs to be arranged and lacunae filled to get concrete degradative schemes so that environmental pollution from nitro-explosives can be dealt with more successfully at a macroscale. An overview of the reports on nitro-explosives degradation, future outlook and studies done by us are presented in this review.     

Enhanced transformation of TNT by Arabidopsis plants expressing an old yellow enzyme

2,4,6-Trinitrotoluene (TNT) is released in nature from manufacturing or demilitarization facilities, as well as after the firing or detonation of munitions or leakage from explosive remnants of war. Environmental contamination by TNT is associated with human health risks, necessitating the development of cost-effective remediation techniques. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. In this study, we present a system for TNT phytoremediation by overexpressing the old yellow enzyme (OYE3) gene from Saccharomyces cerevisiae. The resulting transgenic Arabidopsis plants demonstrated significantly enhanced TNT tolerances and a strikingly higher capacity to remove TNT from their media. The current work indicates that S. cerevisiae OYE3 overexpression in Arabidopsis is an efficient method for the phytoremoval and degradation of TNT. Our findings have the potential to provide a suitable remediation strategy for sites contaminated by TNT.     

Transformations of TNT and related aminotoluenes in groundwater aquifer slurries under different electron-accepting conditions

The transport and fate of pollutants is often governed by both their tendency to sorb as well as their susceptibility to biodegradation. We have evaluated these parameters for 2,4,6-trinitrotoluene (TNT) and several biodegradation products. Slurries of aquifer sediment and groundwater depleted TNT at rates of 27, 7.7 and 5.9 μM day⁻¹ under methanogenic, sulfate-reducing and nitrate-reducing conditions, respectively. Abiotic losses of TNT were determined in autoclaved controls. Abiotic TNT loss and subsequent transformation of the products was also observed. These transformations were especially important during the first step in the reduction of TNT. Subsequent abiotic reactions could account for all of the transformations observed in bottles which were initially nitrate-reducing. Other controls removed TNT reduction products at much slower rates than slurries containing live organisms. 2-Amino-4,6-dinitrotoluene was produced in all slurries but disappeared in methanogenic and in sulfate-reducing slurries within several weeks. This compound was converted to 2,4-diamino-6-nitrotoluene in all slurries with subsequent removal of the latter from methanogenic and sulfate-reducing slurries, while it persisted in autoclaved controls and in the nitrate-reducing slurries. Aquifer slurries incubated with either 2,4- or 2,6-diaminotoluene showed losses of these compounds relative to autoclaved controls under nitrate-reducing conditions but not under sulfate-reducing or methanogenic conditions. These latter compounds are important as reduced intermediates in the biodegradation of dinitrotoluenes and as industrial chemicals. In experiments to examine sorption, exposure to landfill sediment resulted in losses of approximately 15% of diaminotoluene isomers and 25% of aminodinitrotoluene isomers from initial solution concentrations within 24 h. Isotherms confirmed that the diaminotoluenes were least strongly sorbed and the amino-dinitrotoluenes most strongly sorbed to this sediment, while TNT sorption capacity was intermediate. In our studies, 2,4,6-triaminotoluene sorption capacity was indeterminate due to its chemical instability. Coupled with biodegradation information, isotherms help describe the likelihood of contaminant removal, persistence, and movement at impacted sites. Received 11 March 1996/ Accepted in revised form 24 July 1996     

Differences in the biotransformation of 2,4,6-trinitrotoluene (TNT) between wild and axenically grown isolates of Myriophyllum aquaticum

The aim of this study was to demonstrate the potential for aquatic plants and their associated microbes to bioremediate wetland sites contaminated with 2,4,6-trinitrotoluene (TNT). The transformation of TNT was studied using both wild and axenically grown isolates of Myriophyllum aquaticum (parrot feather). Differences in TNT transformation rates and nitroaromatic metabolites were observed between different plants. The wild isolates, containing a consortium of associated microorganisms, transformed TNT into 2-amino-4,6-dinitrotoluene (2-A-DNT) and 4-amino-2,6-dinitrotoluene (4-A-DNT) via 2- and 4-hydroxylamino-dinitrotoluene, which were detected as intermediates. The wild M. aquaticum also converted the metabolites, 2-A-DNT and 4-A-DNT, into low levels of 2,4-diaminotoluene (2,4-DAT). The axenically grown plants, containing no cultureable microorganisms, also transformed TNT into 2-A-DNT and 4-A-DNT, but at a much lower rate than that observed for the wild isolates. Unlike the wild plants, axenically grown M. aquaticum could not transform either 2-A-DNT or 4-A-DNT into 2,4-DAT over the incubation period. The differences in the performance between these plants could indicate that plant-associated microorganisms assisted in the overall transformation of TNT. For each plant, unidentifiable metabolites were observed and the soluble monoamino-derivatives present in the wild and axenic medium accounted for 14 and 7% of the initial TNT concentration, respectively. Thus, the majority of nitroaromatic derivatives remained associated with the plant tissues. Furthermore, only 7 and 3% of the initial TNT concentration were extracted as monoamino-derivatives from the tissues of the wild and axenically grown plants, respectively.     

Use of a Salmonella microsuspension bioassay to detect the mutagenicity of munitions compounds at low concentrations

Past production and handling of munitions has resulted in soil contamination at various military facilities. Depending on the concentrations present, these soils pose both a reactivity and toxicity hazard and the potential for groundwater contamination. Many munitions-related chemicals have been examined for mutagenicity in the Ames test, but because the metabolites may be present in low environmental concentrations, a more sensitive method is needed to elucidate the associated mutagenicity. RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), TNT (2,4,6-trinitrotoluene), tetryl (N-methyl-N-2,4,6-tetranitroaniline), TNB (1,3,5-trinitrobenzene) and metabolites were examined for mutagenicity in a microsuspension modification of the Salmonella histidine reversion assay with and without metabolic activation. TNB and tetryl were positive in TA98 (32.5, 5.2revertants/nmole) and TA100 (7.4, 9.5revertants/nmole) without metabolic activation and were more potent than TNT (TA98, 0.3revertants/nmole; TA100, 2.4revertants/nmole). With the exception of the tetranitroazoxytoluene derivatives, TNT metabolites were less mutagenic than TNT. RDX and two metabolites were negative in both strains, however, hexahydro-1,3,5-trinitroso-1,3,5-triazine was positive in TA100 with and without S9. Microsuspension bioassay results tend to correlate well with published Ames test data, however, there are discrepancies among the published data sets and the microsuspension assay results.