The Explosive 2,4,6-trinitrotoluene Research Articles

Explosives leaking from dumped munition contaminate fish from German coastal waters: a reason for chronic effects?

BackgroundConventional munition dumped into the North Sea and the Baltic Sea close to the German coastline is corroding. A major concern is that biota, including fish, are negatively affected by toxic explosives leaking into marine environments. With the present study, we investigated fish living in close proximity to munition dumping sites for contamination and for signs of health impairments. The flat fish species common dab (Limanda limanda) was used as a model, since it lives in the vicinity of dumping sites and exhibits minor migratory activity. Since explosives are excreted via the bile, the bile fluids from dab were analysed. Further on we inspected the health status of the fish.ResultsDab caught in German coastal waters of the Baltic Sea and the North Sea were contaminated with explosives. Probably due to rapid metabolization, concentrations of the explosive 2,4,6-trinitrotoluene (TNT) were always below limit of detection, but its metabolites 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were detected in bile fluid up to 26.36 ng/ml and 95.91 ng/ml, respectively. Only few fish from the Baltic Sea were positive for the explosive HMX, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine with a maximum concentration of 0.89 ng/ml. Highest concentrations of TNT metabolites in bile fluid were detected in dab collected near the dumping site “Kolberger Heide” in the bay of Kiel (Baltic Sea). However, also dab from the North Sea were significantly contaminated with TNT metabolites.ConclusionsThe present study showed for the first time that fish living close to near shore munition dumping sites in the North Sea are contaminated with explosives. Various health indicators (body condition factors, externally visible fish diseases, parasites or liver anomalies) showed differences in health status between fish living in the North Sea and in the Baltic Sea, respectively. However, the health status of fish caught at the most contaminated site in the Baltic Sea was not worse compared to fish living in less contaminated areas. We conclude that fish living in the vicinity of dumping sites in the North Sea and the Baltic Sea can be significantly contaminated with explosives. However, obvious health impairments of the fish were not observed.

Integration of heterogeneous photocatalysis (TiO2/UV) and activated sludge system operated in air lift reactor for the treatment of industrial effluent red water

In the industrial production of the explosive 2,4,6-trinitrotoluene (TNT), purification steps are required to ensure the quality of the product, procedures that generate wastewater of a complex nature and with eco-toxicological potential, called red water, which consists of soluble sulfonates, TNT isomers, and other typical nitro aromatic compounds. The present work aimed to study the effects of integrating heterogeneous photocatalysis based on commercial TiO2, with a biological process, based on activated sludge, for red water treatment. For the photocatalytic treatment, a 72% reduction in the typical absorption of nitro aromatic compounds (the region between 195 − 275 nm), a 36% reduction in chemical oxygen demand (COD), and a 68% reduction in total phenols were obtained. In the biological treatment, there was a 60% reduction in absorbance in the typical nitro aromatics region (NA), 10% reduction in COD, and 36% reduction in total phenols (FT). The integration of photocatalytic and biological treatments showed promising results compared to the individual processes. Having 94% reduction in NA absorbance, 72% reduction in FT, and 89% reduction in COD with an association of photocatalytic pretreatment followed by biological, and reductions of 88% in NA absorbance, 62% in FT, and 87% in COD for a biological pretreatment followed by the photocatalytic process. In general, when comparing the chemical and biological processes, isolated and integrated, both types of integration showed significantly superior results. They were able to remove the main nitro aromatic constituents of the Red Water effluent.

Fast Detection of 2,4,6-Trinitrotoluene (TNT) at ppt Level by a Laser-Induced Immunofluorometric Biosensor

The illegal use of explosives by terrorists and other criminals is an increasing issue in public spaces, such as airports, railway stations, highways, sports venues, theaters, and other large buildings. Security in these environments can be achieved by different means, including the installation of scanners and other analytical devices to detect ultra-small traces of explosives in a very short time-frame to be able to take action as early as possible to prevent the detonation of such devices. Unfortunately, an ideal explosive detection system still does not exist, which means that a compromise is needed in practice. Most detection devices lack the extreme analytical sensitivity, which is nevertheless necessary due to the low vapor pressure of nearly all explosives. In addition, the rate of false positives needs to be virtually zero, which is also very difficult to achieve. Here we present an immunosensor system based on kinetic competition, which is known to be very fast and may even overcome affinity limitation, which impairs the performance of many traditional competitive assays. This immunosensor consists of a monolithic glass column with a vast excess of immobilized hapten, which traps the fluorescently labeled antibody as long as no explosive is present. In the case of the explosive 2,4,6-trinitrotoluene (TNT), some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and chip-based mixing-devices and flow-cells. The system achieved limits of detection of 1 pM (1 ppt) of the fluorescent label and around 100 pM (20 ppt) of TNT. The total assay time is less than 8 min. A cross-reactivity test with 5000 pM solutions showed no signal by pentaerythritol tetranitrate (PETN), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). This immunosensor belongs to the most sensitive and fastest detectors for TNT with no significant cross-reactivity by non-related compounds. The consumption of the labeled antibody is surprisingly low: 1 mg of the reagent would be sufficient for more than one year of continuous biosensor operation.

Degradation of trinitrotoluene by transgenic nitroreductase in Arabidopsis plants

The explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. TNT is toxic to many organisms, it is known to be a potential human carcinogen, and is persistent in the environment. This study presents a system of phytoremediation by Arabidopsis plants developed on the basis of overexpression of NAD(P)H-flavin nitroreductase (NFSB) from the Sulfurimonas denitrificans DSM1251. The resulting transgenic Arabidopsis plants demonstrated significantly enhanced TNT tolerance and a strikingly higher capacity to remove TNT from their media. The highest specific rate constant of TNT disappearance rate was 1.219 and 2.297 mL/g fresh weight/h for wild type and transgenic plants, respectively. Meanwhile, the nitroreductase activity in transgenic plant was higher than wild type plant. All this indicates that transgenic plants show significantly enhanced tolerances to TNT; transgenic plants also exhibit strikingly higher capabilities of removing TNT from their media and high efficiencies of transformation.

A High Aspect Ratio Bifurcated 128-Microchannel Microfluidic Device for Environmental Monitoring of Explosives.

Design and evolution of explosives monitoring and detection platforms to address the challenges of trace level chemical identification have led investigations into the use of intricately designed microfluidic devices. Microfluidic devices are unique tools that possess distinct characteristics that, when designed properly and configured with optical and fluidic components, can produce detection platforms with unmatched performance levels. Herein, we report the design, fabrication and integration of a bifurcated high aspect ratio microfluidic device containing 128 microchannels (40 mm × 40 μm × 250 μm; L × W × H) for explosives detection at trace levels. Aspect ratios measuring >6:1 support improved receptor-target molecule interactions, higher throughput and extremely low limits of detection (LOD). In addition to superior assay sensitivity, the bifurcated microfluidic device provides greater durability and versatility for substrate modification. Using the explosive 2,4,6-trinitrotoluene (TNT) as the model compound in a fluorescence-based displacement immunoassay, we report LODs for TNT at 10 parts-per-trillion (pptr) using a neutravidin-coated biotinylated anti-TNT microfluidic device. Solution to wall interactions were also simulated in COMSOL Multiphysics to understand fluid flow characteristics. Reynolds numbers were calculated to be 0.27–2.45 with a maximum pressure of 1.2 × 10−2 psi.

Understanding the hydrogen transfer mechanism for the biodegradation of 2,4,6-trinitrotoluene catalyzed by pentaerythritol tetranitrate reductase: molecular dynamics simulations.

The explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic pollutant. Biodegradation is inevitably one of the most cost-effective and enviromentally friendly means of removing TNT pollution. However, the aromatic derivatives from the reduction of nitro groups by several classic enzymes are still toxic. Besides the reduction of nitro groups, pentaerythritol tetranitrate reductase (PETNR) offers a potential route to ring fission and complete degradation of TNT through the pathway of the Meisenheimer complex. This work is devoted to deeply understand the essence of the Meisenheimer pathway and mainly focus on the crucial hydrogen-transfer reaction by means of molecular dynamics (MD) simulations. We obtain three valuable findings. Firstly, the parallel π-π stacking between TNT and the flavin mononucleotide (FMN) cofactor is a precondition. The key residue controlling this conformation is His181. Although His184 does not interact with TNT, the mutation from His184 to Asn184 would abolish the π-π structure. Secondly, the data of the empirical valence bond (EVB) show that the Meisenheimer pathway is predominant because its activation barrier is 6.7 kcal mol-1 far less than that of nitro reduction (26.6 kcal mol-1). Finally, based on the results of thermodynamic integration (TI), the type of transferred hydrogen is also ensured, that is, the H anion (H-) for the Meisenheimer complex and the H radical (H˙) for nitro reduction. Our findings provide an exhaustive understanding for the first hydrogen transfer reaction that has a decisive effect on two competing pathways, and help in searching for and designing new enzymes that can effectively degrade TNT.

Batch soil adsorption and column transport studies of 2,4-dinitroanisole (DNAN) in soils

The explosive 2,4,6-trinitrotoluene (TNT) is currently a main ingredient in munitions; however the compound has failed to meet the new sensitivity requirements. The replacement compound being tested is 2,4-dinitroanisole (DNAN). DNAN is less sensitive to shock, high temperatures, and has good detonation characteristics. However, DNAN is more soluble than TNT, which can influence transport and fate behavior and thus bioavailability and human exposure potential. The objective of this study was to investigate the environmental fate and transport of DNAN in soil, with specific focus on sorption processes. Batch and column experiments were conducted using soils collected from military installations located across the United States. The soils were characterized for pH, electrical conductivity, specific surface area, cation exchange capacity, and organic carbon content. In the batch rate studies, change in DNAN concentration with time was evaluated using the first order equation, while adsorption isotherms were fitted using linear and Freundlich equations. Solution mass-loss rate coefficients ranged between 0.0002h−1 and 0.0068h−1. DNAN was strongly adsorbed by soils with linear adsorption coefficients ranging between 0.6 and 6.3Lg−1, and Freundlich coefficients between 1.3 and 34mg1−nLnkg−1. Both linear and Freundlich adsorption coefficients were positively correlated with the amount of organic carbon and cation exchange capacity of the soil, indicating that similar to TNT, organic matter and clay minerals may influence adsorption of DNAN. The results of the miscible-displacement column experiments confirmed the impact of sorption on retardation of DNAN during transport. It was also shown that under flow conditions DNAN transforms readily with formation of amino transformation products, 2-ANAN and 4-ANAN. The magnitudes of retardation and transformation observed in this study result in significant attenuation potential for DNAN, which would be anticipated to contribute to a reduced risk for contamination of ground water from soil residues.

Expression of a Drosophila glutathione transferase in Arabidopsis confers the ability to detoxify the environmental pollutant, and explosive, 2,4,6-trinitrotoluene.

The explosive 2,4,6-trinitrotoluene (TNT) is a significant, global environmental pollutant that is both toxic and recalcitrant to degradation. Given the sheer scale and inaccessible nature of contaminated areas, phytoremediation may be a viable clean-up approach. Here, we have characterized a Drosophila melanogaster glutathione transferase (DmGSTE6) which has activity towards TNT. Recombinantly expressed, purified DmGSTE6 produces predominantly 2-glutathionyl-4,6-dinitrotoluene, and has a 2.5-fold higher Maximal Velocity (Vmax ), and five-fold lower Michaelis Constant (Km ) than previously characterized TNT-active Arabidopsis thaliana (Arabidopsis) GSTs. Expression of DmGSTE6 in Arabidopsis conferred enhanced resistance to TNT, and increased the ability to remove TNT from contaminated soil relative to wild-type plants. Arabidopsis lines overexpressing TNT-active GSTs AtGST-U24 and AtGST-U25 were compromised in biomass production when grown in the absence of TNT. This yield drag was not observed in the DmGSTE6-expressing Arabidopsis lines. We hypothesize that increased levels of endogenous TNT-active GSTs catalyse excessive glutathionylation of endogenous substrates, depleting glutathione pools, an activity that DmGST may lack. In conclusion, DmGSTE6 has activity towards TNT, producing a compound with potential for further biodegradation. Selecting or manipulating plants to confer DmGSTE6-like activity could contribute towards development of phytoremediation strategies to clean up TNT from polluted military sites.

Monodehydroascorbate reductase mediates TNT toxicity in plants.

The explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. Due to the scale of affected areas, one of the most cost-effective and environmentally friendly means of removing explosives pollution could be the use of plants. However, mechanisms of TNT phytotoxicity have been elusive. Here, we reveal that phytotoxicity is caused by reduction of TNT in the mitochondria, forming a nitro radical that reacts with atmospheric oxygen, generating reactive superoxide. The reaction is catalyzed by monodehydroascorbate reductase 6 (MDHAR6), with Arabidopsis deficient in MDHAR6 displaying enhanced TNT tolerance. This discovery will contribute toward the remediation of contaminated sites. Moreover, in an environment of increasing herbicide resistance, with a shortage in new herbicide classes, our findings reveal MDHAR6 as a valuable plant-specific target.

Draft Genome Sequence of Pseudomonas putida JLR11, a Facultative Anaerobic 2,4,6-Trinitrotoluene Biotransforming Bacterium

We report the draft genome sequence of Pseudomonas putida JLR11, a facultative anaerobic bacterium that has been studied in detail for its capacity to use the explosive 2,4,6-trinitrotoluene (TNT) as a nitrogen source. The sequence confirms the mechanisms used by this versatile strain to reduce and assimilate nitrogen from TNT.

Detection of Explosives in a Dynamic Marine Environment Using a Moored TNT Immunosensor

A field demonstration and longevity assessment for long-term monitoring of the explosive 2,4,6-trinitrotoluene (TNT) in a marine environment using an anti-TNT microfluidic immunosensor is described. The TNT immunosensor is comprised of a microfluidic device with 39 parallel microchannels (2.5 cm × 250 μm × 500 μm, L × W × D) fabricated in poly(methylmethacrylate) (PMMA), then chemically functionalized with antibodies possessing a high affinity for TNT. Synthesized fluorescence reporter complexes used in a displacement-based assay format were used for TNT identification. For field deployment the TNT immunosensor was configured onto a submersible moored steel frame along with frame controller, pumps and TNT plume generator and deployed pier side for intermittent plume sampling of TNT (1h increments). Under varying current and tidal conditions trace levels of TNT in natural seawater were detected over an extended period (>18 h). Overnight operation and data recording was monitored via a web interface.

Microfabricated Gas Chromatograph for Rapid, Trace-Level Determinations of Gas-Phase Explosive Marker Compounds

A prototype microfabricated gas chromatograph (μGC) adapted specifically for the rapid determination of selected gas-phase marker compounds of the explosive 2,4,6-trinitrotoluene (TNT) at sub-parts-per-billion (<ppb) concentrations in complex mixtures is described. Si-microfabricated focuser, separation column, and sensor array components are integrated with a high-volume sampler of conventional construction to reduce analysis time and the limit of detection (LOD). The primary markers selected as target analytes were 2,4-dinitrotoluene (2,4-DNT, a persistent impurity of TNT) and 2,3-dimethyl-2,3-dinitrobutane (DMNB, a taggant), along with 2,6-dinitrotoluene (2,6-DNT, a less-prominent TNT impurity), which was also included in numerous tests. Selective preconcentration, on-column focusing, temperature-programmed chromatographic separation, and sensor array detection/recognition facilitated determinations of the primary markers in the presence of 20 (or more) interferences within ∼2 min under laptop control. Estimated LODs of 2.2, 0.48, and 0.86 ng were achieved for DMNB, 2,6-DNT, and 2,4-DNT, respectively, which correspond to 0.30, 0.067, and 0.12 ppb in each 1-L air sample collected.

Screen-printed electrode-based electrochemical detector coupled with in-situ ionic-liquid-assisted dispersive liquid–liquid microextraction for determination of 2,4,6-trinitrotoluene

A novel method is reported, whereby screen-printed electrodes (SPELs) are combined with dispersive liquid-liquid microextraction. In-situ ionic liquid (IL) formation was used as an extractant phase in the microextraction technique and proved to be a simple, fast and inexpensive analytical method. This approach uses miniaturized systems both in sample preparation and in the detection stage, helping to develop environmentally friendly analytical methods and portable devices to enable rapid and onsite measurement. The microextraction method is based on a simple metathesis reaction, in which a water-immiscible IL (1-hexyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide, [Hmim][NTf2]) is formed from a water-miscible IL (1-hexyl-3-methylimidazolium chloride, [Hmim][Cl]) and an ion-exchange reagent (lithium bis[(trifluoromethyl)sulfonyl]imide, LiNTf2) in sample solutions. The explosive 2,4,6-trinitrotoluene (TNT) was used as a model analyte to develop the method. The electrochemical behavior of TNT in [Hmim][NTf2] has been studied in SPELs. The extraction method was first optimized by use of a two-step multivariate optimization strategy, using Plackett-Burman and central composite designs. The method was then evaluated under optimum conditions and a good level of linearity was obtained, with a correlation coefficient of 0.9990. Limits of detection and quantification were 7 μg L(-1) and 9 μg L(-1), respectively. The repeatability of the proposed method was evaluated at two different spiking levels (20 and 50 μg L(-1)), and coefficients of variation of 7 % and 5 % (n = 5) were obtained. Tap water and industrial wastewater were selected as real-world water samples to assess the applicability of the method.

Noncontact optical detection of explosive particles via photodissociation followed by laser-induced fluorescence

High-sensitivity (ng/cm²) optical detection of the explosive 2,4,6-trinitrotoluene (TNT) is demonstrated using photodissociation followed by laser-induced fluorescence (PD-LIF). Detection occurs rapidly, within 6 laser pulses (~7 ns each) at a range of 15 cm. Dropcasting is used to create calibrated samples covering a wide range of TNT concentrations; and a correspondence between fractional area covered by TNT and PD-LIF signal strength is observed. Dropcast data are compared to that of an actual fingerprint. These results demonstrate that PD-LIF could be a viable means of rapidly and remotely scanning surfaces for trace explosive residues.

A Nanosensor for TNT Detection Based on Molecularly Imprinted Polymers and Surface Enhanced Raman Scattering

We report on a new sensor strategy that integrates molecularly imprinted polymers (MIPs) with surface enhanced Raman scattering (SERS). The sensor was developed to detect the explosive, 2,4,6-trinitrotoluene (TNT). Micron thick films of sol gel-derived xerogels were deposited on a SERS-active surface as the sensing layer. Xerogels were molecularly imprinted for TNT using non-covalent interactions with the polymer matrix. Binding of the TNT within the polymer matrix results in unique SERS bands, which allow for detection and identification of the molecule in the MIP. This MIP-SERS sensor exhibits an apparent dissociation constant of (2.3 ± 0.3) × 10−5 M for TNT and a 3 μM detection limit. The response to TNT is reversible and the sensor is stable for at least 6 months. Key challenges, including developing a MIP formulation that is stable and integrated with the SERS substrate, and ensuring the MIP does not mask the spectral features of the target analyte through SERS polymer background, were successfully met. The results also suggest the MIP-SERS protocol can be extended to other target analytes of interest.

Attenuation of TNT in seawater microcosms

The ability of two differing marine sediments (one clayey, the other sandy) to attenuate the explosive 2,4,6-trinitrotoluene (TNT), dissolved in intertidal seawater from the eastern English coast of the North Sea, was examined using aerobic microcosms. Analysis of the seawater from the microcosms revealed an initial sharp decline in TNT concentration with clayey sediment in both sterilized (to prevent microbial activity) and unsterilized microcosms. This effect did not occur to such a marked extent in similar sterile and non-sterile sandy sediment microcosms and was attributed mainly to sorption of TNT to the fine clay particles of the clayey sediment. As time progressed, the attenuation of TNT in microcosms containing either type of sediment was found to be less in those that had been sterilized compared with those where microbial action proceeded unhindered. Feeding the microcosms, (i.e. supplying extra carbon sources for the microbial communities), appeared to have a small, but perceptible, enhancing effect upon TNT dissipation. The attenuation of TNT was also measured in large microcosms containing 2.5 L of seawater and no sediment. Analysis of the seawater revealed a gradual decline in TNT concentration in non-sterile and fed microcosms compared to their sterile counterpart. Overall, this laboratory study showed that the attenuation of TNT is slow (half-life in seawater ca.1900 days; half-life sand sediment <700 days; half life in clay sediment 130 days) under conditions commonly encountered in coastal waters of the North Sea.

Bioaccumulation of TNT and DDT in Sheepshead Minnows, Cyprinodon variegatus L., Following Feeding of Contaminated Invertebrates

The aim of this study was to determine the potential for dietary uptake by trophic transfer using the explosive 2,4,6-trinitrotoluene (TNT) and the substantially more hydrophobic dichlorodiphenyltrichloroethane (DDT) utilizing the amphipods Leptocheirus plumulosus as prey and the fish Cyprinodon variegatus as predator. Bioaccumulation did not change significantly over time for TNT but apparent steady-state was not reached for DDT at exposure termination after 7 days of dietary exposure. The bioaccumulation factor was 0.09 mg/mg for TNT and 0.34 mg/mg for DDT, confirming the low potential of TNT to bioaccumulate in fish.

Acute, subacute, and subchronic exposure to 2A-DNT (2-amino-4,6-dinitrotoluene) in the northern bobwhite (Colinus virginianus)

2-Amino-4,6-dinitrotoluene (2A-DNT) is a metabolite of the explosive 2,4,6-trinitrotoluene (TNT) which is present in the soil at numerous U.S. Army installations as the result of TNT manufacture or training activities. Although many avian species are known to inhabit areas where 2A-DNT has been found in the environment, no published studies of the effects of 2A-DNT exposure in birds are available. In this study, we conducted an evaluation of the oral toxicity of 2A-DNT in a representative ground foraging species of management concern, the northern bobwhite (Colinus virginianus). Subacute (14 days) and subchronic (60 days) oral gavage exposure studies were conducted following determination of the median acute lethal dose (LD50 = 1167 mg/kg). In the subacute study, survival occurred at 50 mg/kg/day. This helped to determine dose groups for the subchronic study: 0, 0.5, 3, 14, and 30 mg 2A-DNT/kg body weight-d in corn oil. The lowest observed adverse effects level (LOAEL) was determined to be 14 mg/kg/day based on mortality, and the no observed adverse effects level (NOAEL) was determined to be 3 mg/kg/day based on lack of effects at this exposure level.

Fluorescence-based Sensing of 2,4,6-Trinitrotoluene (TNT) Using a Multi-channeled Poly(methyl methacrylate) (PMMA) Microimmunosensor

Fluorescence immunoassays employing monoclonal antibodies directed against the explosive 2,4,6-trinitrotoluene (TNT) were conducted in a multi-channel microimmunosensor. The multi-channel microimmunosensor was prepared in poly (methyl methacrylate) (PMMA) via hot embossing from a brass molding tool. The multi-channeled microfluidic device was sol-gel coated to generate a siloxane surface that provided a scaffold for antibody immobilization. AlexaFluor-cadaverine-trinitrobenzene (AlexaFluor-Cad-TNB) was used as the reporter molecule in a displacement immunoassay. The limit of detection was 1–10 ng/mL (ppb) with a linear dynamic range that covered three orders of magnitude. In addition, antibody crossreactivity was investigated using hexahydro-1,3,5-triazine (RDX), HMX, 2,4-dinitrotoluene (DNT), 4-nitrotoluene (4-NT) and 2-amino-4,6-DNT.

Detoxification of the explosive 2,4,6‐trinitrotoluene in Arabidopsis: discovery of bifunctional O‐ and C‐glucosyltransferases

Plants, as predominantly sessile organisms, have evolved complex detoxification pathways to deal with a diverse range of toxic chemicals. The elasticity of this stress response system additionally enables them to tackle relatively recently produced, novel, synthetic pollutants. One such compound is the explosive 2,4,6-trinitrotoluene (TNT). Large areas of soil and groundwater are contaminated with TNT, which is both highly toxic and recalcitrant to degradation, and persists in the environment for decades. Although TNT is phytotoxic, plants are able to tolerate low levels of the compound. To identify the genes involved in this detoxification process, we used microarray analysis and then subsequently characterized seven uridine diphosphate (UDP) glycosyltransferases (UGTs) from Arabidopsis thaliana (Arabidopsis). Six of the recombinantly expressed UGTs conjugated the TNT-transformation products 2- and 4-hydroxylaminodinitrotoulene, exhibiting individual bias for either the 2- or the 4-isomer. For both 2- and 4-hydroxylaminodinitrotoulene substrates, two monoglucose conjugate products, confirmed by HPLC-MS-MS, were observed. Further analysis indicated that these were conjugated by either an O- or C-glucosidic bond. The other major compounds in TNT metabolism, aminodinitrotoluenes, were also conjugated by the UGTs, but to a lesser extent. These conjugates were also identified in extracts and media from Arabidopsis plants grown in liquid culture containing TNT. Overexpression of two of these UGTs, 743B4 and 73C1, in Arabidopsis resulted in increases in conjugate production, and enhanced root growth in 74B4 overexpression seedlings. Our results show that UGTs play an integral role in the biochemical mechanism of TNT detoxification by plants.