High uptake of 2,4,6-trinitrotoluene by vetiver grass--potential for phytoremediation?

2,4,6-Trinitrotoluene (TNT) is a potent mutagen, and a Group C human carcinogen that has been widely used to produce munitions and explosives. Vast areas that have been previously used as ranges, munition burning, and open detonation sites are heavily contaminated with TNT. Conventional remediation activities in such sites are expensive and damaging to the ecosystem. Phytoremediation offers a cost-effective, environment-friendly solution, utilizing plants to extract TNT from contaminated soil. We investigated the potential use of vetiver grass (Vetiveria zizanioides) to effectively remove TNT from contaminated solutions. Vetiver grass plants were grown in hydroponic systems containing 40 mg TNTL(-1) for 8d. Aqueous concentrations of TNT reached the method detection limit ( approximately 1 microg L(-1)) within the 8-d period, demonstrating high affinity of vetiver for TNT, without any visible toxic effects. Results from this preliminary hydroponic study are encouraging, but in need of verification using TNT-contaminated soils.     

Effectiveness of urea in enhancing the extractability of 2,4,6-trinitrotoluene from chemically variant soils

One of the major challenges in developing an effective phytoremediation technology for 2,4,6-trinitrotoluene (TNT) contaminated soils is limited plant uptake resulting from low solubility of TNT. The effectiveness of urea as a solubilizing agent in increasing plant uptake of TNT in hydroponic systems has been documented. Our preliminary greenhouse experiments using urea were also very promising, but further characterization of the performance of urea in highly-complex soil-solution was necessary. The present study investigated the natural retention capacity of four chemically variant soils and optimized the factors influencing the effectiveness of urea in enhancing TNT solubility in the soil solutions. Results show that the extent of TNT sorption and desorption varies with the soil properties, and is mainly dependent on soil organic matter (SOM) content. Hysteretic desorption of TNT in all tested soils suggests irreversible sorption of TNT and indicates the need of using an extractant to increase the release of TNT in soil solutions. Urea significantly (p < 0.0001) enhanced TNT extraction from all soils, by increasing its solubility at the solid/liquid interface. Soil organic matter content and urea application rates showed significant effects, whereas pH did not exert any significant effect on urea catalysis of TNT extraction from soil. The optimum urea application rates (125 or 350 mg kg⁻¹) for maximizing TNT extraction were within the limits set by the agronomic fertilizer-N rates used for major agricultural crops. The data obtained from this batch study will facilitate the optimization of a chemically-catalyzed phytoremediation model for cleaning up TNT-contaminated soils.     

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.     

Effects of EDTA on solubility of cadmium, zinc, and lead and their uptake by rainbow pink and vetiver grass

Rainbow pink (Dianthus chinensis), a potential phytoextraction plant, can accumulate high concentrations of Cd from contaminated soils. Vetiver grass (Vetiver zizanioides) has strong and long root tissues and is a potential phytostabilization plant since it can tolerate and grow well in soils contaminated with multiple heavy metals. Soil was moderately artificially contaminated by cadmium (20 mg/kg), zinc (500 mg/kg), and lead (1000 mg/kg) in pot experiments. Three concentrations of Na2-EDTA solution (0, 5, and 10 mmol/kg soil) were added to the contaminated soils to study the influence of EDTA solution on phytoextraction by rainbow pink or phytostabilization by vetiver grass. The results showed that the concentrations of Cd, Zn, and Pb in a soil solution of rainbow pink significantly increased following the addition of EDTA (p < 0.05). The concentrations of Cd and Pb in the shoots of rainbow pink also significantly increased after EDTA solution was applied (p < 0.05), but the increase for Zn was insignificant. EDTA treatment significantly increased the total uptake of Pb in the shoot, over that obtained with the control treatment (p < 0.001), but it did not significantly increase the total uptake of Cd and Zn. The concentrations of Zn and Pb in the shoots of rainbow pink are significantly correlated with those in the soil solution, but no relationship exists with concentrations in vetiver grass. The toxicity of highly contaminating metals did not affect the growth of vetiver grass, which was found to grow very well in this study. Results of this study indicate that rainbow pink can be considered to be a potential phytoextraction plant for removing Cd or Zn from metal-contaminated soils, and that vetiver grass can be regarded as a potential phytostabilization plant that can be grown in a site contaminated with multiple heavy metals.     

Chemical methods and phytoremediation of soil contaminated with heavy metals

The effects of chemical amendments (calcium carbonate (CC), steel sludge (SS) and furnace slag (FS)) on the growth and uptake of cadmium (Cd) by wetland rice, Chinese cabbage and wheat grown in a red soil contaminated with Cd were investigated using a pot experiment. The phytoremediation of heavy metal contaminated soil with vetiver grass was also studied in a field plot experiment. Results showed that treatments with CC, SS and FS decreased Cd uptake by wetland rice, Chinese cabbage and wheat by 23-95% compared with the unamended control. Among the three amendments, FS was the most efficient at suppressing Cd uptake by the plants, probably due to its higher content of available silicon (Si). The concentrations of zinc (Zn), lead (Pb) and Cd in the shoots of vetiver grass were 42-67%, 500-1200% and 120-260% higher in contaminated plots than in control, respectively. Cadmium accumulation by vetiver shoots was 218 g Cd/ha at a soil Cd concentration of 0.33 mg Cd/kg. It is suggested that heavy metal-contaminated soil could be remediated with a combination of chemical treatments and plants.     

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.     

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.     

Ecological rehabilitation and phytoremediation with four grasses in oil shale mined land

Vetiver grass (Vetiveria zizanioides), bahia grass (Paspalum notatum), St. Augustine grass (Stenotaphrum secundatum), and bana grass (Pennisetum glaucumxP. purpureum) were selected to rehabilitate the degraded ecosystem of an oil shale mined land of Maoming Petro-Chemical Company located in Southwest of Guangdong Province, China. Among them, vetiver had the highest survival rate, up to 99%, followed by bahia and St. Augustine, 96% and 91%, respectively, whereas bana had the lowest survival rate of 62%. The coverage and biomass of vetiver were also the highest after 6-month planting. Fertilizer application significantly increased biomass and tiller number of the four grasses, of which St. Augustine was promoted most, up to 70% for biomass, while vetiver was promoted least, only 27% for biomass. Two heavy metals, lead (Pb) and cadmium (Cd) tested in this trial had different concentrations in the oil shale residue, and also had different contents and distributions in the four grass species. Concentrations of Pb and Cd in the four grasses presented a disparity of only 1.6-3.8 times, but their uptake amounts to the two metals were apart up to 27.5-35.5 times, which was chiefly due to the significantly different biomasses among them. Fertilizer application could abate the ability of the four species to accumulate heavy metals, namely concentration of heavy metals in plants decreased as fertilizer was applied. The total amount of metals accumulated by each plant under the condition of fertilization did not decrease due to an increase of biomass. In summary, vetiver may be the best species used for vegetation rehabilitation in oil shale disposal piles.     

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.     

Analysis of phytochelatin complexes in the lead tolerant vetiver grass [Vetiveria zizanioides (L.)] using liquid chromatography and mass spectrometry

Ethylenediamene tetraacetic acid (EDTA) has been used to mobilize soil lead (Pb) and enhance plant uptake for phytoremediation. Chelant bound Pb is considered less toxic compared to free Pb ions and hence might induce less stress on plants. Characterization of possible Pb complexes with phytochelatins (PC_n, metal-binding peptides) and EDTA in plant tissues will enhance our understanding of Pb tolerance mechanisms. In a previous study, we showed that vetiver grass (Vetiveria zizanioides L.) can accumulate up to 19,800 and 3350 mg Pb kg⁻¹ dry weight in root and shoot tissues, respectively; in a hydroponics set-up. Following the basic incubation study, a greenhouse experiment was conducted to elucidate the efficiency of vetiver grass (with or without EDTA) in remediating Pb-contaminated soils from actual residential sites where Pb-based paints were used. The levels of total thiols, PC_n, and catalase (an antioxidant enzyme) were measured in vetiver root and shoot following chelant-assisted phytostabilization. In the presence of 15 mM kg ⁻¹ EDTA, vetiver accumulated 4460 and 480 mg Pb kg⁻¹ dry root and shoot tissue, respectively; that are 15- and 24-fold higher compared to those in untreated controls. Despite higher Pb concentrations in the plant tissues, the amount of total thiols and catalase activity in EDTA treated vetiver tissues was comparable to chelant unamended controls, indicating lowered Pb toxicity by chelation with EDTA. The identification of glutathione (referred as PC₁) (m/z 308.2), along with chelated complexes like Pb-EDTA (m/z 498.8) and PC₁-Pb-EDTA (m/z 805.3) in vetiver root tissue using electrospray tandem mass spectrometry (ES-MS) highlights the possible role of such species towards Pb tolerance in vetiver grass.     

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.     

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.     

Biotransformation of 2,4,6-trinitrotoluene (TNT) by enchytraeids (Enchytraeus albidus) in vivo and in vitro

2,4,6-Trinitrotoluene (TNT) is toxic to soil invertebrates, but little is known about its toxicokinetic behavior in soil. Tissue residue analysis was used to evaluate whether the presence of TNT and its reduced metabolites in soil invertebrates was due to uptake of these compounds from the soil into the organism, or due to microbial transformation of TNT associated with the organism followed by uptake. Adult white potworms (Enchytraeus albidus) were exposed to non-lethal concentrations of TNT in amended artificial soil for 21 d, or to TNT in solution for 20 h. Soil exposure studies confirmed earlier reports that TNT was transformed in enchytraeids in vivo to 2- and 4-aminodinitrotoluenes. However, enchytraeid exposure to TNT in solution led to the additional presence of 2,4-diaminonitrotoluene as well as 2- and 4- hydroxyamino-dinitrotoluenes and azoxy-compounds, suggesting that TNT can be metabolized in vivo in the absence of soil. Incubation of unexposed enchytraeid homogenates with TNT led to a protein-dependent appearance of these metabolites in vitro after > or =16 h incubation. Cellular fractionation studies indicated that most of this activity resided in the 8000 x g pellet, and was completely inhibited by broad-spectrum antibiotics. These studies demonstrate that enchytraeids can transform TNT in vivo and in vitro, at least in part, by bacteria associated with the host organism.     

Environmental behavior of explosives in groundwater from the Milan Army Ammunition Plant in aquatic and wetland plant treatments. Removal, mass balances and fate in groundwater of TNT and RDX.

Phytoremediation of 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in groundwater using constructed wetlands is a potentially economical remediation alternative. To evaluate Explosives removal and fate was evaluated using hydroponic batch incubations of plant and substrate treatments with explosives-contaminated groundwater amended with [U-14C]-TNT or [U-14C]-RDX. 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. The study surveyed three aquatic, four wetland plant species and two substrates in independent incubations of 7 days with TNT and 13 days with RDX. Parent compounds and transformation products were followed using 14C and chemical (HPLC) analyses. Mass balance of water, plants, substrates and air was determined. It was demonstrated that TNT disappeared completely from groundwater incubated with plants, although growth of most plants except parrot-feather was low in groundwater amended to contain 1.6 to 3.4 mg TNT L-1. Highest specific removal rates were found in submersed plants in water star-grass and in all emergent plants except wool-grass. TNT declined less with substrates, and least in controls without plants. Radiolabel was present in all plants after incubation. Mineralization to 14CO2 was very low, and evolution into 14C-volatile organics negligible. RDX disappeared less rapidly than TNT from groundwater. Growth of submersed plants was normal, but that of emergent plants reduced in groundwater amended to contain 1.5 mg RDX L-1. Highest specific RDX removal rates were found in submersed plants in elodea, and in emergent plants in reed canary grass. RDX failed to disappear with substrates. Mineralization to 14CO2 was low, but relatively higher than in the TNT experiment. Evolution into 14C-volatile organics was negligible. Important considerations for using certain aquatic and wetland plants in constructed wetlands aimed at removing explosives from water are: (1) plant persistence at the explosives level to which it is exposed, (2) specific plant-mass based explosives removal rates, (3) plant productivity, and (4) fate of parent compounds and transformation products in water, plants, and sediments.     

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.     

Treatment of trinitrotoluene by crude plant extracts

Crude plant extract solutions (spinach and parrotfeather) were prepared and spiked with 2,4,6-trinitrotoluene (TNT) (20 mgl(-1)). 90-h TNT removal by these solutions was compared to controls. Spinach and parrotfeather extract solutions removed 99% and 50% of the initial TNT, respectively; TNT was not eliminated in the controls or in extract solutions where removal activity was deactivated by boiling. A first-order removal constant of 0.052 h(-1) was estimated for spinach extract solutions treating 20 mgl(-1) TNT concentrations, which compared favorably to intact plant removal. Concentration variation was described by Michaelis-Menton kinetics. Detectable TNT degradation products represented only a fraction of the total TNT transformed, and the transformation favored the formation of 4-aminodinitrotoluene. The results indicated that crude plant extracts transform TNT, without the presence of the live plant.     

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.     

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.     

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.     

Microtox toxicity test: detoxification of TNT and RDX contaminated solutions by poplar tissue cultures

Poplar (Populus deltoidesxnigra DN34) tissue cultures removed 2,4,6-trinitrotoluene (TNT) from an aqueous solution in five days, reducing the toxicity of the solution from highly toxic Microtox EC value to that of the control. 1,3,5-Trinitro-1,3,5-triazacyclohexane (RDX) was taken up by the plant tissue cultures more slowly, but toxicity reduction of the solution was evident. The measurement of toxicity reduction of aqueous solutions containing TNT and RDX was performed using a novel methodology developed for use with the Microtox testing system. Radiolabeled TNT and RDX were used to confirm removal of explosives from hydroponic solutions containing plant tissue cultures and to verify that toxicity did not change in solutions where no plant cultures were present (positive controls). High Performance Liquid Chromatography (HPLC) and Liquid Scintillation Counter (LSC) measurements confirmed removal of TNT and RDX from solutions containing poplar plant tissue cultures and constancy of the plant-free controls. In addition, metabolites were identified in remediated solutions by HPLC, confirming the mechanism by which plants can remediate groundwater, surface water, and soil solutions.