TNT and 4-amino-2,6-dinitrotoluene influence on germination and early seedling development of tall fescue

Cost-effective and environmentally acceptable methods are needed to remediate munitions-contaminated soil. Some perennial grass species are tolerant of soil contaminants and may promote remediation because of their high water use and extensive fibrous root systems. The effects of 2,4,6-trinitrotoluene (TNT) and its reduction product, 4-amino-2,6-dinitrotoluene (4ADNT), on germination and early seedling development of tall fescue (Festuca arundinacea Schreb.) were determined. Tall fescue seeds were germinated in nutrient-free agar containing 0-60 mg TNT litre(-1) or 0-15 mg 4ADNT litre(-1). Germination decreased linearly as TNT concentration increased but was not significantly affected by 4ADNT at these concentrations. Concentrations less than 30 mg TNT litre(-1) or 7.5 mg 4ADNT litre(-1) had little effect on seedling growth and development. Higher TNT or 4ADNT concentrations substantially delayed seedling development, caused abnormal radicle tissue development, and reduced secondary root and shoot growth. Seedling respiration rates decreased linearly with increasing TNT concentration. Experiments indicate that tall fescue may be grown in soils that maintain soil solution concentrations of 30 mg TNT litre(-1) or less.     

Degradation of 2,4,6-trinitrotoluene by selected helophytes

Four emergent plants (helophytes, synonyms emersion macrophytes, marsh plants, etc.) Phragmites australis, Juncus glaucus, Carex gracillis and Typha latifolia were successfully used for degradation of TNT (2,4,6-trinitrotoluene) under in vitro conditions. The plants took up and transformed more than 90% of TNT from the medium within ten days of cultivation. The most efficient species was Ph. australis which took up 98% of TNT within ten days. The first stable degradation products 4-amino-2,6-dinitrotoluene (4-ADNT) and 2-amino-4,6-dinitrotoluene (2-ADNT) were identified and analysed during the cultivation period. [14C] TNT was used for the detection of TNT degradation products and their compartmentalization in plant tissues after two weeks of cultivation. Forty one percent of 14C was detected as insoluble or bound in cell structures: 34% in roots and 8% in the aerial parts. These results open the perspective of using the above-mentioned plants for the remediation of TNT contaminated waters.     

Effects of heavy metals and nitroaromatic compounds on horseradish glutathione S-transferase and peroxidase

Glutathione S-transferase (GST) and peroxidase (POX) activities have a direct relation to the effect of stress on plant metabolism. Changes in the activities of the enzymes were therefore studied. Horseradish hairy roots were treated by selected bivalent ions of heavy metals (HMs) and nitroaromatic compounds (NACs). We have shown differences in GST activity when assayed with substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB). The conjugation of DCNB catalysed by GST was inhibited in all roots treated with HMs as compared to non-treated roots, whereas NACs caused induction of the activity in dependence on the exposition time and concentration of compounds. The conjugation of CDNB by GST was not affected to the same extent. The increase of GST activity was determined in cultures treated by nickel (0.1 mM) and diaminonitrotoluenes (DANTs, 0.1 mM) for 6 h, whereas the roots treated by 2,4,6-trinitrotoluene (TNT), 4-amino-2,6-dinitrotoluene (ADNT) and dinitrotoluene (DNT, 1.0 mM) needed 27 h treatment to induce the activity. The POX activity of cultures treated by HMs was inhibited to 17-35% in comparison to non-treated cultures. The POX activity of roots treated by TNT (0.1 and 1.0 mM) for 6 and 27 h and by ADNT (0.1 and 1.0 mM) for 6 h was inhibited. A partial increase of POX activity was measured in roots treated by all NACs for 27 h. The content of oxidized glutathione (GSSG) and reduced glutathione (GSH) in the roots differed significantly. It was followed as a symptom of the stress reaction of the plant metabolism to the effect of NACs and HMs.     

Accumulation of 14C-trinitrotoluene and related nonextractable (bound) residues in Eisenia fetida

To determine if trinitrotoluene (TNT) forms nonextractable residues in earthworms and to measure the relative degree of accumulation as compared to TNT and its deaminated metabolites, Eisenia fetida was exposed to ¹⁴C-TNT using dermal contact to filter paper or exposure to soil. Nonextractable residues made up 32-68% of total body burden depending on exposure media and depuration time. Parent TNT accounted for less than 3% of radioactivity, while ADNTs accounted for 7-38%. Elimination half-lives were 61-120 h for TNT, ADNTs, and DANTs, which was significantly lower than the half-lives found for nonextractable residues, 201-240 h. However, over 80% of the nonextractable residue was solubilized using weak acid (pH 2). Based on our findings that TNT accumulation occurs primarily as nonextractable residues, which have a longer half-life, and that nonextractable residues can be solubilized, we propose that nonextractable residues could be used as a selective biomarker for assessing TNT contamination.     

Comparison of TNT removal from seawater by three marine macroalgae

Axenic plantlets derived from three species of marine macroalgae, the temperate green alga Acrosiphonia coalita, the temperate red alga Porphyra yezoensis, and the tropical red alga Portieria hornemannii, all possessed a similar metabolic route to remove the explosive compound 2,4,6-trinitrotolune (TNT) from seawater. At a biomass density of 1.2 g l(-1) and initial TNT concentrations of 10 mg l(-1) or less, TNT removal from seawater was 100% within 72 h for P. hornemannii and P. yezoensis. Specific rate constants for TNT uptake were 0.016-0.018 l g(-1)FWh(-1) for A. coalita filaments, 0.047-0.062 l g(-1)FW h(-1) for P. yezoensis blades, and 0.037-0.049 l g(-1)FW h(-1) for P. hornemannii microplantlets. Only trace amounts of TNT were found within the biomass. All species reduced TNT to 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dintrotoluene, but these products never accounted for more than 20% of the initial TNT.     

Toxicity and bioaccumulation of reduced TNT metabolites in the earthworm Eisenia andrei exposed to amended forest soil

Soils contaminated with 2,4,6-trinitrotoluene (TNT) and TNT primary reduction products have been found to be toxic to certain soil invertebrates, such as earthworms. The mechanism of toxicity of TNT and of its by-products is still not known. To ascertain if one of the TNT reduction products underlies TNT toxicity, we tested the toxicity and bioaccumulation of TNT reduction products. 2-Amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-diamino-6-nitrotoluene (2,4-DANT) and 2,6-diamino-4-nitrotoluene (2,6-DANT) were tested separately in adult earthworms (Eisenia andrei) following a 14-d exposure to amended sandy loam forest soil. TNT, 4-ADNT, and 2-ADNT were lethal to earthworms (14-d LC(50) were: 580, 531 and 1088 micromol kg(-1), or 132, 105 and 215 mgkg(-1) dry soil, respectively) and gave the following order of toxicity: 4-ADNT>TNT>2-ADNT. Exposure to 2,4-DANT and to 2,6-DANT caused no mortality at 600 micromol kg(-1) or 100 mgkg(-1) dry soil. We found that all four TNT reduction products accumulated in earthworm tissues and 2-ADNT reached the highest levels at 3.0+/-0.3 micromol g(-1) tissue. The 14-d bioaccumulation factors were 5.1, 6.4, 5.1 and 3.2 for 2-ADNT, 4-ADNT, 2,4-DANT and 2,6-DANT, respectively. Results also suggest that some TNT metabolites are at least as toxic as TNT and should be considered when evaluating the overall toxicity of TNT-contaminated soil to earthworms.     

Solubilization of nitrotoluenes in micellar nonionic surfactant solutions

A key factor in selecting surfactants to enhance chemical or biological transformation or physical removal of an organic pollutant from contaminated soil is knowledge of the pollutant's solubility behavior in the surfactant solution. This study investigated the influence of nonionic surfactant structure on the solubility of 4-nitrotoluene (NT), 2,3-dinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, and 2,4,6-trinitrotoluene (TNT) at room temperature. For a series of alkyl phenol ethoxylates (Tergitol NP-8 to NP-40), decreasing the ethoxylate chain length increased the solubility of these nitrotoluenes by a factor of two or less in 10 g l(-1) surfactant solutions, but did not significantly change their molar solubilization ratios (MSR, e.g. 0.02 for TNT) or their micelle-water partition coefficients (K(m), e.g. 3.4 for TNT). For Tergitol NP-8 solutions ranging from 1.0 to 12.4 g l(-1), no enhancement in NT solubility was found, suggesting that the cloud point was reached. The MSRs for Tween 80 were higher than those of Tween 20 and the MSRs of Brij-58 were higher than those for Brij-35. When comparing solutes, NT had the highest solubility and MSR (0.28-0.41), while TNT had the lowest solubility and MSR (0.02-0.03). A linear relationship between K(m) values and octanol-water partition coefficients based on Triton X-100 predicted the logK(m) values within 0.5 of their measured values. A linear solvation free energy correlation for K(m) suggested the importance of solute volume and effective hydrogen bond basicity in the partitioning process while implying that the nitrotoluenes are solubilized in a polar portion of the micelle.     

Environmental behavior of explosives in groundwater from the Milan Army Ammunition Plant in aquatic and wetland plant treatments. Uptake and fate of TNT and RDX in plants

Uptake and fate of TNT and RDX by three aquatic and four wetland plants were studied using hydroponic, batch, incubations in explosives-contaminated groundwater amended with [U-14C]-TNT or [U-14C]-RDX in the laboratory. Substrates in which the plants were rooted were also tested. Plants and substrates were collected from a small-scale wetland constructed for explosives removal, and groundwater originated from a local aquifer at the Milan Army Ammunition Plant. This study demonstrated rapid uptake of [U-14C]-TNT derived 14C, concentration at the uptake sites and limited transport in all plants. Per unit of mass, uptake was higher in submersed than in emergent species. Biotransformation of TNT had occurred in all plant treatments after 7-day incubation in 1.6 to 3.4 mg TNT L-i, with labeled amino-dinitrotoluenes (ADNTs), three unidentified compounds unique for plants, and mostly polar products as results. Biotransformation occurred also in the substrates, yielding labeled ADNT, one unidentified compound unique for substrates, and polar products. TNT was not recovered by HPLC in plants and substrates after incubation. Uptake of [U-14C]-RDX derived 14C in plants was slower than that of TNT, transport was substantial, and concentration occurred at sites where new plant material was synthesized. As for TNT, uptake per unit of mass was higher in submersed than in emergent species. Biotransformation of RDX had occurred in all plant treatments after 13-day incubation in 1.5 mg RDX L-1, with one unidentified compound unique for plants, and mostly polar products as results. Biotransformation had occurred also in the substrates, but to a far lower extent than in plants. Substrates and plants had one unidentified 14C-RDX metabolite in common. HPLC analysis confirmed the presence of RDX in most plants and in three out of four substrates at the end of the incubation period.     

Chemically catalyzed uptake of 2,4,6-trinitrotoluene by Vetiveria zizanioides

The efficiency of vetiver grass (Vetiveria zizanioides) in removing 2,4,6-trinitrotoluene (TNT) from aqueous media was explored in the presence of a common agrochemical, urea, used as a chaotropic agent. Chaotropic agents disrupt water structure, increasing solubilization of hydrophobic compounds (TNT), thus, enhancing plant TNT uptake. The primary objectives of this study were to: (i) characterize TNT absorption by vetiver in hydroponic media, and (ii) determine the effect of urea on chemically catalyzing TNT uptake by vetiver grass in hydroponic media. Results showed that vetiver exhibited a high TNT uptake capacity (1.026 mgg(-1)), but kinetics were slow. Uptake was considerably enhanced in the presence of urea, which significantly (p<0.001) increased the 2nd-order reaction rate constant over that of the untreated (no urea) control. Three major TNT metabolites were detected in the roots, but not in the shoot, namely 1,3,5-trinitrobenzene, 4-amino 2,6-dinitrotoluene, and 2-amino 4,6-dinitrotoluene, indicating TNT degradation by vetiver grass.     

Identification of oxidized TNT metabolites in soil samples of a former ammunition plant

Water extracts of soil samples of the former ammunition plant “Tanne” near Clausthal-Zellerfeld, Lower Saxony, Germany, were investigated for highly polar oxidized 2,4,6-trinitrotoluene (TNT) metabolites. 0.4 to 9.0 mg/kg dry soil 2,4,6-trinitrobenzoic acid (TNBA) and 5.8 to 544 mg/kg dry soil 2-amino-4,6-dinitrobenzoic acid (2-ADNBA) were found. In addition to the oxidized metabolites, TNT, 4- and 2-aminodinitrotoluene (4- and 2-ADNT), and 2,4-dinitrotoluene (2,4-DNT) were extractable with water. Most interestingly, in one sample, 2-ADNBA represented the main contaminant. The origin of the oxidized nitroaromatics is unknown at this time. They might be generated chemically or photochemically. Furthermore, a biological synthesis seems possible.     

Fractionation and extractability of sulfur, iron and trace elements in sulfidic sediments

This study describes iron and sulfur fractionation, and the related extractability of selected trace elements (As, Cd, Cr, Cu, Ni, Pb and Zn) in estuarine sediments. The sediments were sulfidic, with moderately high concentrations of pore-water sulfide (200-600 micromol l(-1)) and acid-volatile sulfide (AVS; 9.9-129 micromol g(-1)). Pyrite-S concentrations increased with depth, with 63-251 micromol g(-1) at site W1 and 312-669 micromol g(-1) at site W2. The degree of sulfidisation was generally high (>80%), indicating that Fe may be limiting pyrite accumulation. The ratios of AVS to pyrite-S increased with sediment depth, as expected for the pyritisation of solid-phase AVS. Cadmium, Pb and Zn extractability in 1M HCl indicated that these elements are not significantly sequestered during pyritisation, whereas sequestration may be important for As, Cu and possibly Ni. Extractability trends for Cr suggest that diagenesis in sulfidic sediments may enhance Cr reactivity. Overall, replacement of AVS by pyrite during diagenesis may enhance the reactivity of Cd, Cr, Pb and Zn, whereas As, Cu and possibly Ni may be rendered less reactive.     

Dendroremediation of trinitrotoluene (TNT) Part 2: Fate of radio-labelled TNT in trees

BACKGROUND, AIM AND SCOPE: Problems of long-term existence of the environmental contaminant 2,4,6-trinitrotoluene (TNT) and necessities for the use of trees ('dendroremediation') in sustainable phytoremediation strategies for TNT are described in the first part of this paper. Aims of the second part are estimation of [14C]-TNT uptake, localisation of TNT-derived radioactivity in mature tree tissues, and the determination of the degree of TNT-degradation during dendroremediation processes. METHODS: Four-year-old trees of hybrid willow (Salix spec., clone EW-20) and of Norway spruce (Picea abies) were cultivated in sand or ammunition plant soil (AP-soil) in wick supplied growth vessels. Trees were exposed to a single pulse application with water solved [U-14C]-TNT reaching a calculated initial concentration of 5.2 mg TNT per kg dry soil. Two months after application overall radioactivity and extractability of 14C were determined in sand/soil, roots, stem-wood, stem-bark, branches, leaves, needles, and Picea May sprouts. Root extracts were analysed by radio TLC. RESULTS: 60 days after [14C]-TNT application, recovered 14C is accumulated in roots (70% for sand variants, 34% for AP-soil variant). 15-28% of 14C remained in sand and 61% in AP-soil. 3.3 to 14.4% of 14C were located in aboveground tree portions. Above-ground distribution of 14C differed considerably between the angiosperm Salix and the gymnosperm Picea. In Salix, nearly half of above-ground-14C was detected in bark-free wood, whereas in Picea older needles contained most of the above-ground-14C (54-69%). TNT was readily transformed in tree tissue. Approximately 80% of 14C was non-extractably bound in roots, stems, wood, and leaves or needles. Only quantitatively less important stem-bark of Salix and Picea and May shoots of Picea showed higher extraction yields (up to 56%). DISCUSSION: Pulse application of [14C]-TNT provided evidence for the first time that after TNT-exposure, in tree root extracts, no TNT and none of the known metabolites, mono-amino-dinitrotoluenes (ADNT), diaminonitrotoluenes (DANT), trinitrobenzene (TNB) and no dinitrotoluenes (DNTs) were present. Extractable portions of 14C were small and contained at least three unknown metabolites (or groups) for Salix. In Picea, four extractable metabolites (or groups) were detected, where only one metabolite (or group) seemed to be identical for Salix and Picea. All unknown extractables were of a very polar nature. CONCLUSIONS: Results of complete TNT-transformation in trees explain some of our previous findings with 'cold analytics', where no TNT and no ADNT-metabolites could be found in tissues of TNT-exposed Salix and Populus clones. It is concluded that 'cold' tissue analysis of tree organs is not suited for quantitative success control of phytoremediation in situ. RECOMMENDATIONS AND OUTLOOK: Both short rotation Salicaceae trees and conifer forests possess a dendroremediation potential for TNT polluted soils. The degradation capacity and the large biomass of adult forest trees with their woody compartments of roots and stems may be utilized for detoxification of soil xenobiotics.     

DFT M06-2X investigation of alkaline hydrolysis of nitroaromatic compounds

The nitroaromatic compounds 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT) and 2,4-dinitroanisole (DNAN) are potential environmental contaminants and their transformations under a variety of environmental conditions are consequently of great interest. One possible method to safely degrade these nitrocompounds is alkaline hydrolysis. A mechanism of the initial stages of this reaction was investigated computationally. Simulations of UV-VIS and NMR spectra for this mechanism were also produced. The results obtained were compared to available experimental data on the alkaline hydrolysis of TNT and suggest that the formation of Meisenheimer complexes and an anion of TNT are potential first-step intermediates in the reaction path. As the reaction proceeds, computational results indicate that polynegative complexes dominate the degradation pathway, followed by cycles of carbon chain opening and breaking. A second possible pathway was identified that leads to polymeric products through Janovsky complex formation. Results from this study indicate that the order of increasing resistance to alkaline hydrolysis is TNT, DNT and DNAN.     

Phytotoxicity of nitroaromatic energetic compounds freshly amended or weathered and aged in sandy loam soil

The toxicities of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), 2,4-dinitrotoluene (2,4-DNT), and 2,6-dinitrotoluene (2,6-DNT) to terrestrial plants alfalfa (Medicago sativa L.), Japanese millet (Echinochloa crusgalli L.), and perennial ryegrass (Lolium perenne L.) were determined in Sassafras sandy loam soil using seedling emergence, fresh shoot, and dry mass measurement endpoints. A 13-week weathering and aging of energetic materials in soils, which included wetting and drying cycles, and exposure to sunlight of individual soil treatments, was incorporated into the study design to better reflect the soil exposure conditions in the field than toxicity determinations in freshly amended soils. Definitive toxicity tests showed that dinitrotoluenes were more phytotoxic for all plant species in freshly amended treatments based on EC20 values for dry shoot ranging from 3 to 24mgkg(-1) compared with values for TNB or TNT ranging from 43 to 62mgkg(-1). Weathering and aging of energetic materials (EMs) in soil significantly decreased the toxicity of TNT, TNB or 2,6-DNT to Japanese millet or ryegrass based on seedling emergence, but significantly increased the toxicity of all four EMs to all three plant species based on shoot growth. Exposure of the three plant species to relatively low concentrations of the four compounds initially stimulated plant growth before the onset of inhibition at greater concentrations (hormesis).     

Subcellular distribution of Cd in the aquatic oligochaete Tubifex tubifex, implications for trophic availability and toxicity

We studied the compartmentalization of cadmium and zinc in the oligochaete Tubifex tubifex. The subcellular distribution was followed over time and levels of metallothionein-like proteins were measured. The impact of the speciation on the trophic transfer was studied by calculating the assimilation efficiencies of metals from Tubificidae fed to carp. It was found that carp were able to assimilate 9.8% of the cadmium. The expected assimilated amount of cadmium, based on the subcellular fractions which are thought to be trophically available, is however 72%. The zinc assimilation results suggest that the debris fraction is at least partially available to predators. Differential centrifugation techniques provide information about the tissue compartmentalization in aquatic organisms but it is not straightforward to directly link internal speciation in prey items to the actual assimilation in the predator. The possible impact that the compartmentalization of cadmium in T. tubifex will have on the toxicity to the organism is also discussed.     

Coupling of 2,4,6-trinitrotoluene (TNT) metabolites onto humic monomers by a new laccase from Trametes modesta

During degradation of trinitrotoluene (TNT) by Trametes modesta, addition of humic monomers prevented the accumulation of all major stable TNT metabolites (aminodinitrotoluenes [AMDNT]) by at least 92% in the presence of 200 mM ferulic acid and guaiacol. Acute toxicity tests with individual TNT metabolites and in T. modesta cultures supplemented with 200 microM TNT demonstrated that the TNT biodegradation process lead to less toxic metabolites. Toxicity decreased in the order TNT>4-HADNT (4-hydroxylaminodinitrotoluene)>2-HADNT>2,6-DNT (2,6-dinitrotoluene)>2',2',6,6-azoxytetranitrotoluene>4-AMDNT>2-AMDNT>2,4-diamninonitrotoluene (2,4-DAMNT) while 2,4-DNT and 2,6-DAMNT were the least toxic. Ferulic acid is the best candidate for immobilization TNT biodegradation metabolites since it prevented the accumulation of AMDNTs in cultures during TNT biodegradation and its products were less toxic. All humic monomers were very effective in immobilizing 2-HADNT [100%], 4-HADNT [100%] and 2,2,6,6-azoxytetranitrotoluene [100%]. Two distinct laccase isoenzymes (LTM1 and LTM2) potentially involved in immobilization of TNT degradation products were purified to electrophoretic homogeneity. LTM1 and LTM2 have molecular weights of 77.6 and 52.5 kDa, are 18% and 24% glycosylated, have pI values of 3.6 and 4.2, respectively. Both enzymes oxidized all the typical laccase substrates tested. LTM1 showed highest kinetic constants (K(m)=0.03 microM; K(cat)=8.8 4x 10(7)s(-1)) with syringaldazine as substrate.     

Enhanced bioavailability of sorbed 2,4,6-trinitrotoluene (TNT) by a bacterial consortium

Large quantities of trinitrotoluene (TNT) have been associated with past and present military activities worldwide. Because this contaminant is highly toxic and strongly sorbs to soil particles, bacteria that are able to transform it have had very little success, if any. This study was conducted to evaluate the bioavailability of 14C-labeled TNT in soil for microbial mineralization. Sorption-desorption experiments indicated that a Kendaia loam soil effectively adsorbs this explosive compound, with approximately 30-45% of the added TNT remaining sorbed to the soil after a total of 10 washings. A bacterial consortium isolated from explosive-contaminated sites was prepared in liquid medium and then tested in a TNT-spiked Kendaia loam soil. The concentration of TNT in the soil that was inoculated with the bacterial consortium was reduced by more than 30% of the initial concentration compared to the soil that did not contain the bacterial consortium within a period of 20 weeks. Nearly half of the TNT was mineralized as determined by the percentage of 14CO2 produced. Only one member of the consortium (i.e., Enterobacter sp.) significantly mineralized 25% of TNT although the extent of mineralization was significantly enhanced to 35% in the presence of the other two members of the consortium. The data suggest that some of the strongly adsorbed TNT may be accessible for metabolism if conditions for the right combination of microorganisms with specialized capabilities are optimized. The remaining sorbed fraction of substrate is presumably sequestered and thus unavailable to the microorganisms.     

Ecotoxicity characterization of dinitrotoluenes and some of their reduced metabolites

In the present study, the toxic effects of 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT) and a selection of their respective metabolites were examined and compared to 2,4,6-trinitrotoluene (TNT) using the 15-min Microtox (Vibrio fischen) and 96-h freshwater green alga (Selenastrum capricomutum) growth inhibition tests. All of the compounds tested were less toxic than TNT. Using the Microtox assay, 2,6-DNT was more toxic than 2,4-DNT and the order of toxicity for 2,6-DNT and its metabolites was: 2,6-DNT > or = 2A-6NT > 2,6-DAT; whereas that for 2,4-DNT was: 4A-2NT > 2A-4NT > 2,4-DNT > 2,4-DAT. For the algal test, 2,4-DNT was more toxic than 2,6-DNT and the order of toxicity for 2,4-DNT and its metabolites was: 2,4-DNT > 2,4-DAT approximately equal to 4A-2NT = 2A-4NT. The order of toxicity for 2,6-DNT and its reduced metabolites using the algal test was very similar to the Microtox bioassay. These results demonstrate that the reduced metabolites of 2,6-DNT tested in this study were less toxic than that of the parent compound, but certain partially reduced metabolites of 2,4-DNT can be more toxic than the parent molecule. These data put into question the general hypothesis that reductive metabolism of nitro-aromatics is associated with a sequential detoxification process.     

Phytotoxicity and phytoremediation of 2,6-dinitrotoluene using a model plant, Arabidopsis thaliana

Biochemical and genetic studies of xenobiotic metabolism in the model plant Arabidopsis have significant potential in providing information for phytoremediation. This paper presents the toxicity of 2,6-dinitrotoluene (2,6-DNT) to Arabidopsis under axenic conditions, the fate and transformation of 2,6-DNT after uptake by the plant, and the effect of a putative glutathione S-transferase (GST), which is highly induced by 2,4,6-trinitrotoluene (TNT) in the previous study, on the detoxification of 2,6-DNT. 2,6-DNT had toxic effects on the growth of Arabidopsis based on whole seedling as well as root growth assays. Using [U- 14C]2,6-DNT, the recovery was over 87% and less than 2% accounted for the mineralization of 2,6-DNT in axenic liquid cultures during the 14d of exposure. About half (48.3%) of the intracellular radioactivity was located in the root tissues in non-sterile hydroponic cultures. 2-Amino-6-nitrotoluene (2A6NT) and two unknown metabolites were produced as transformation products of 2,6-DNT in the liquid media. The metabolites were further characterized by proton NMR spectra and the UV-chromatograms when the plant was fed with either 2,6-DNT or 2A6NT. In addition, polar unknown metabolites were detected at short retention times from radiochromatograms of plant tissue extracts. The GST gene of the wild-type of Arabidopsis in response to 2,6-DNT was induced by 4.7-fold. However, the uptake rates and the tolerance at different concentrations of 2,6-DNT and TNT were not significantly different between the wild-type and the gst mutant indicating that induction of the GST gene is not related to the detoxification of 2,6-DNT.     

Fate of RDX and TNT in agronomic plants

Phytoremediation is of great interest to remediate soil contaminated with hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT). The ability of 4 agronomic plants (maize, soybean, wheat and rice) to take up these explosives and their fate in plants were investigated. Plants were grown for 42 days on soil contaminated with [(14)C]RDX or [(14)C]TNT. Then, each part was analyzed for its radioactivity content and the percentage of bound and soluble residues was determined following extractions. Extracts were analyzed by radio-HPLC. More than 80% of uptaken RDX was translocated to aerial tissues, up to 64.5 mgg(-1) of RDX. By contrast, TNT was little translocated to leaves since less than 25% of uptaken TNT was accumulated in aerial parts. Concentrations of TNT residues were 20 times lower than for RDX uptake. TNT was highly metabolized to bound residues (more than 50% of radioactivity) whereas RDX was mainly found in its parent form in aerial parts.