Aminodinitrotoluenes Research Articles

A novel strategy for high-throughput sample collection, analysis and visualization of explosives’ concentrations for contaminated areas

The use of explosives has led to a widespread distribution of 2,4,6-trinitrotoluene (TNT) and its by- and degradation products in the soil on former production and testing sites. The investigation of those large contaminated sites is so far based on a few selected soil samples, due to high costs of conventional HPLC and GC analysis, although huge differences in concentrations can already be found in small areas and different collection depths. We introduce a novel high-throughput screening system for those areas, which combines a smartphone-based collection of GPS data and soil characteristics with a fast MALDI-TOF MS quantification of explosives in soil sample extracts and finally a heatmap visualization of the explosives’ spread in soil and an analysis of correlation between concentrations and soil characteristics. The analysis of a 400 m2 area presented an extensive contamination with TNT and lower concentrations of the degradation and by-products aminodinitrotoluenes (ADNT) and dinitrotoluenes (DNT) next to a former production facility for TNT. The contamination decreased in deeper soil levels and depended on the soil type. Pure humus samples showed significantly lower contaminations compared to sand and humus/sand mixtures, which is likely to be caused by an increased binding potential of the humic material. No correlation was found between the vegetation and the concentration of explosives. Since the results were obtained and visualized within several hours, the MALDI-TOF MS based comprehensive screening and heatmap analysis might be valuable for a fast and high-throughput characterization of contaminated areas.

Accumulation and depuration of trinitrotoluene and related extractable and nonextractable (bound) residues in marine fish and mussels

To determine if trinitrotoluene (TNT) forms nonextractable residues in mussels (Mytilus galloprovincialis) and fish (Cyprinodon variegatus) and to measure the relative degree of accumulation as compared to extractable TNT and its major metabolites, organisms were exposed to water fortified with 14C-TNT. After 24 h, nonextractable residues made up 75% (mussel) and 83% (fish) while TNT accounted for 2% of total radioactivity. Depuration half-lives for extractable TNT, aminodinitrotoluenes (ADNTs) and diaminonitrotoluenes (DANTs) were fast initially (<0.5 h), but slower for nonextractable residues. Nonextractable residues from organisms were identified as ADNTs and DANTs using 0.1 M HCL for solubilization followed by liquid chromatography-tandem mass spectrometry. Recovered metabolites only accounted for a small fraction of the bound residue quantified using a radiotracer likely because of low extraction or hydrolysis efficiency or alternative pathways of incorporation of radiolabel into tissue.

Identification and determination of trinitrotoluenes and their degradation products using liquid chromatography–electrospray ionization mass spectrometry

In the environs of ammunition plants and former military area, contaminations caused by explosives and their degradation products are of great environmental relevance. During the production of worldwide mostly used explosive compound – 2,4,6-trinitrotoluene its isomers were distributed into the environment. Therefore determination of 14 selected nitroaromatic compounds (trinitrotoluenes, amino dinitrotoluenes and diamino nitrotoluenes) by means of LC–MS–MS coupling utilizing electrospray ionization was developed. Therewith, these compounds were identified and quantified on the basis of specific precursor/product ion traces using the high selectivity and sensitivity of multiple reaction monitoring mode (MRM) of a triple quadrupole mass spectrometer. A new stationary phase designed especially for separation of EPA explosive mixture was used for separation of specific mixture of nitroaromatics compounds. Modification of HPLC properties enables base-line separation of all analytes and therefore improving of their MS identification and quantification. Limits of detection obtained using highly specific mass spectrometric detection MRM mode were in range 4–114 pg/μL. MS–MS qualification and quantification of explosives and their biodegradation products is feasible also in case of samples with complex matrix and high amount of co-eluting compounds.

Preparation of Anti-dinitrotoluene Polyclonal Antibody and Effect of the Hapten Spacer Length in Coating Antigen on Immunoassay Sensitivity

Environmental pollution of soil, river water, and underground water by explosive–related compounds is a serious problem in the areas of buried landmines and/or landmine factories (Hilmi et al., 1999). These compounds are suspected to have human toxicity, mutagenicity, and carcinogenicity (Kennel et al., 2000; Sayama et al., 1998; Tchounwou et al., 2001; Banerjee et al., 1999). Therefore, there is a pressing need to determine the concentrations and distributions of these compounds in the environment. The main component of landmines is 2,4,6–trinitrotoluene (TNT). TNT released into the environment is decomposed and converted to dinitrotoluenes (DNTs) and aminodinitrotoluenes (ADNTs), among other products, by microorganisms, light, and heat. These compounds have been measured by using HPLC or other instrumental analytical devices (Borch and Gerlach, 2004; Ahmad and Roberts; 1995). These instrumental analyses, however, require tedious pre–treatments such as extraction from samples (Halasz et al., 2002). On the other hand, detection methods using an antigen–antibody reaction, such as enzyme–linked immunosorbent assay (ELISA) and fluorescent and chemiluminescent immunosensors, are more useful and are able to detect ppb levels of explosive compounds (Goldman et al., 2003; Green et al., 2002; Wilson et al., 2003). We have been engaged in development of an antibody with high avidity to explosive–related compounds. In recent years, we have focused on the development of highly sensitive surface plasmon resonance (SPR) sensing methods for the detection of TNT using an antigen– antibody reaction (Sakai et al., 2003; Shankaran et al., 2004; Shankaran et al., 2005a; Shankaran et al., 2005b; Matsumoto et al., 2005; Shankaran et al., 2006). According to the reports of Jenkins et al. (2000, 2001), the concentrations of 2,4–DNT and other decomposed nitroaromatic compounds around and over buried landmines are higher than the concentration of TNT. In the present study, a polyclonal antibody against 2,4–DNT was raised in a rabbit, and the antibody was applied to the detection of 2,4–DNT using ELISA. The aim was to prepare anti–DNPh–KLH antibody from a rabbit and to clarify the characteristics for the detection of 2,4–DNT based on the principle of indirect competitive ELISA. In the course of the detection of free 2,4–DNT, we recognized that the combination of the structure of the coating antigen–protein, free 2,4–DNT, and the anti– DNPh–KLH antibody are an important factor in sensitivity using an indirect competitive immunoassay. Therefore, the effect of the structures of the coating antigen–protein conjugates on the sensitivity of the detection of free 2,4–DNT was investigated in detail. Preparation of Anti–dinitrotoluene Polyclonal Antibody and Effect of the Hapten Spacer Length in Coating Antigen on Immunoassay Sensitivity

Toxicity and fate of two munitions constituents in spiked sediment exposures with the marine amphipod Eohaustorius estuarius

The lethal toxicity of the explosive compounds 14C-labeled 2,4,6-trinitrotoluene (TNT) and nonradiolabeled hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to the estuarine amphipod Eohaustorius estuarius was investigated in 10-d spiked sediment exposures. The 10-d median lethal concentration (LC50) was determined using the sum molar initial concentration of TNT, aminodinitrotoluenes (ADNTs), and diaminonitrotoluenes (DANTs), as determined by high-performance liquid chromatography (HPLC), and collectively referred to as HPLC-TNT*. Despite expectations of higher toxicity in sandy sediment (Yaquina Bay [YB], OR, USA) compared to relatively fine-grained sediment (San Diego Bay [SDB], CA, USA), LC50 values were similar: 159 and 125 micromol/kg, for YB and SDB sediments, respectively. When expressed as the sum of TNT and all its degradation products (14C-TNT*), LC50s were approximately two times the corresponding LC50s determined by HPLC. The HPLC-TNT* fraction likely corresponds to the most bioavailable and toxic transformation products. The concentrations of 14C-TNT* in tissues were substantially higher than those for HPLC-TNT*, suggesting that compounds other than TNT and its major aminated transformation products were prevalent. Critical body residues were similar for exposures to SDB (11.7 micromol/kg) and YB sediments (39.4 micromol/kg), despite marked differences in the nature of compounds available for uptake in the exposure media. The critical body residues for E. estuarius are lower than those reported for other aquatic invertebrates (83-172 micromol/kg). Unlike observations for TNT, RDX was only loosely associated with SDB sediment, with near complete recovery of the parent compound by chemical analysis. Exposure to RDX did not result in significant mortality even at the highest measured sediment concentration of 10,800 micromol/kg dry weight, nor tissue concentrations as high as 96 micromol/kg wet weight. The lack of RDX lethal effects in this study is consistent with results reported for other invertebrate species.

Purification and characterization of NAD(P)H-dependent nitroreductase I from Klebsiella sp. C1 and enzymatic transformation of 2,4,6-trinitrotoluene

Three NAD(P)H-dependent nitroreductases that can transform 2,4,6-trinitrotoluene (TNT) by two reduction pathways were detected in Klebsiella sp. C1. Among these enzymes, the protein with the highest reduction activity of TNT (nitroreductase I) was purified to homogeneity using ion-exchange, hydrophobic interaction, and size exclusion chromatographies. Nitroreductase I has a molecular mass of 27 kDa as determined by SDS-PAGE, and exhibits a broad pH optimum between 5.5 and 6.5, with a temperature optimum of 30-40 degrees C. Flavin mononucleotide is most likely the natural flavin cofactor of this enzyme. The N-terminal amino acid sequence of this enzyme does not show a high degree of sequence similarity with nitroreductases from other enteric bacteria. This enzyme catalyzed the two-electron reduction of several nitroaromatic compounds with very high specific activities of NADPH oxidation. In the enzymatic transformation of TNT, 2-amino-4,6-dinitrotoluene and 2,2',6,6'-tetranitro-4,4'-azoxytoluene were detected as transformation products. Although this bacterium utilizes the direct ring reduction and subsequent denitration pathway together with a nitro group reduction pathway, metabolites in direct ring reduction of TNT could not easily be detected. Unlike other nitroreductases, nitroreductase I was able to transform hydroxylaminodinitrotoluenes (HADNT) into aminodinitrotoluenes (ADNT), and could reduce ortho isomers (2-HADNT and 2-ADNT) more easily than their para isomers (4-HADNT and 4-ADNT). Only the nitro group in the ortho position of 2,4-DNT was reduced to produce 2-hydroxylamino-4-nitrotoluene by nitroreductase I; the nitro group in the para position was not reduced.

Biological reduction of TNT as part of a combined biological-chemical procedure for mineralization.

The explosive 2,4,6-trinitrotoluene (TNT), one of the most abundant and persistent contaminants at former armament factories and military sites, was cometabolically reduced by sludge (mixed culture) from a sewage plant in order to facilitate mineralization in a subsequent photochemical treatment. Under aerobic conditions, the main reduction products were aminodinitrotoluenes (ADNTs). A greater amount of the nitroaromatics (ca. 30%) was adsorbed by the sludge as was shown by a complete balance of the process using 14C-TNT. Under anaerobic conditions, TNT was further converted into ADNTs and diaminonitrotoluenes (DANTs) while only negligible adsorption to the sludge occured.

Models of 2,4,6-trinitrotoluene (TNT) initial conversion by yeasts.

The original models of the initial steps of 2,4,6-trinitrotoluene (TNT) conversion by yeasts are presented. Saccharomyces sp. ZS-A1 reduced nitro groups of TNT producing isomeric monohydroxylaminodinitrotoluenes (HADNT) as the key initial metabolites (molar ratio HADNT/TNT was up to 0.81), whereas aminodinitrotoluenes (ADNT) and the hydride-Meisenheimer complex of TNT (H-TNT) were the minor products. Conversely, Candida sp. AN-L13 transformed TNT almost quantitatively into H-TNT, thus realizing the alternative attack, consisting of the TNT aromatic ring reduction. The third type of conversion, revealed in Candida sp. AN-L14, is the combination of both above mechanisms and produces an equimolar mix of HADNT and H-TNT. In the toxicity tests with Paramecium caudatum, the supernatant of Saccharomyces sp. ZS-A1, which converts TNT into HADNT, was most toxic while the supernatant of Candida sp. AN-L13 (TNT --> H-TNT) was least toxic. The microorganisms converting TNT quantitatively to the reactive metabolites can be useful for their immobilization through the detoxifying interaction with the soil components such as humic compounds.

Models of 2,4,6-trinitrotoluene (TNT) initial conversion by yeasts

The original models of the initial steps of 2,4,6-trinitrotoluene (TNT) conversion by yeasts are presented. Saccharomyces sp. ZS-A1 reduced nitro groups of TNT producing isomeric monohydroxylaminodinitrotoluenes (HADNT) as the key initial metabolites (molar ratio HADNT/TNT was up to 0.81), whereas aminodinitrotoluenes (ADNT) and the hydride–Meisenheimer complex of TNT (H-TNT) were the minor products. Conversely, Candida sp. AN-L13 transformed TNT almost quantitatively into H-TNT, thus realizing the alternative attack, consisting of the TNT aromatic ring reduction. The third type of conversion, revealed in Candida sp. AN-L14, is the combination of both above mechanisms and produces an equimolar mix of HADNT and H-TNT. In the toxicity tests with Paramecium caudatum, the supernatant of Saccharomyces sp. ZS-A1, which converts TNT into HADNT, was most toxic while the supernatant of Candida sp. AN-L13 (TNT→H-TNT) was least toxic. The microorganisms converting TNT quantitatively to the reactive metabolites can be useful for their immobilization through the detoxifying interaction with the soil components such as humic compounds.

Evaluation of continuous flow‐through phytoreactors for the treatment of TNT‐contaminated water

AbstractThe proof of concept for created wetlands to treat groundwater or industrial water contaminated with trinitrotoluene (TNT) was established using bench‐scale, continuous flow reactors with aquatic plants. Contaminant loadings from 0.0132 to 2.488 g/m3/day were tested on phytoreactors using parrotfeather (Myriophyllum aquaticum) as the plant. Reactor removal efficiency and elimination capacity were linearly related to the load of TNT. An algal reactor was also tested at a loading of 0.9671 g/m3/day and removed 93% of the TNT. Some removal was also found in control reactors that contained no plants and minimized algal growth by shielding them from light. However, the removal efficiencies in the controls were much lower. Transformation products, aminodinitrotoluenes (ADNT) and diaminonitrotoluenes (DANT), were detected in the algae and phytoreactors, but not in the controls, indicating that the TNT was being transformed. However, ADNT persisted in the effluent, representing a reactor‐design issue that must be further investigated. ADNT concentrations in the plant material increased with contaminant loading. TNT was only found in plant tissue at the highest loading of 2.488 g/m3/day. Higher concentrations of ADNT were found in the root of the plant, with approximately equal concentrations in the stem and leaf portions. Ratios of 4‐aminodinitrotoluene (4ADNT) versus 2‐aminodinitrotoluene (2ADNT) were higher in the water phase versus what was found in plant material. Because microbial processes tend to favor the formation of 4ADNT, the higher ratio suggests that the removal of TNT is actually a combination of microbial and phyto processes.

Alternative pathways of the initial transformation of 2,4,6-trinitrotoluene by yeasts

A new model for the initial transformation of 2,4,6-trinitrotoluene (TNT) by facultatively anaerobic and aerobic yeasts is presented. The model is based on the data that Saccharomyces sp. ZS-A1 was able to reduce the nitrogroups of TNT with the formation of 2- and 4-hydroxyaminodinitrotoluenes (2-HADNT and 4-HADNT) as the major early TNT metabolites (the molar HADNT/TNT ratio reached 0.81), whereas aminodinitrotoluenes (ADNTs) and the hydride-Meisenheimer complex of TNT (H-TNT) were the minor products. Candida sp. AN-L13 almost completely transformed TNT into H-TNT through the reduction of the aromatic ring. Candida sp. AN-L14 transformed TNT through a combination of the two mechanisms described. Aeration stimulated the production of HADNT from TNT, whereas yeast incubation under stationary conditions promoted the formation of HADNT. The transformation of TNT into HADNT led to a tenfold increase in the acute toxicity of the TNT preparation with respect to Paramecium caudatum, whereas the increase in the toxicity was about twofold in the case of the alternative attack at the aromatic ring.

Oxidation of Aminodinitrotoluenes with Ozone: Products and Pathways

An investigation of the products from the reaction of ozone with aminodinitrotoluenes (ADNTs) provides information about the oxidation pathway. Studies conducted at low conversions of 2- and 4-ADNT show 2:1 ozone/ADNT stoichiometries, prompt formation of glyoxylic and pyruvic acids, and NO2- and NO3- (NOx) ions. Reaction schemes to account for these results involve a 1,3-dipolar cycloaddition of ozone to selected double bonds of the aromatic ring, leading to ring cleavage. 15N-Labeling experiments indicate that the amino function is not involved in the initial ozone oxidation and eventually is incorporated into pyruvamide (2-ADNT) and oxamic acid (4-ADNT) before being oxidized to nitrate.

Tolerance to nitrogenous explosives and metabolism of TNT by cell suspensions of Datura innoxia

Cell suspension cultures of Datura innoxia were incubated in the presence of the nitro-substituted explosives 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5-triazine (RDX), and 1,3,5,7-tetranitro-1,3,5,7-tetraazocyclooctane (HMX). Cellular tolerance levels and TNT biotransformation kinetics were examined. Tolerance to TNT varied as cell suspensions aged. Concentrations of RDX or HMX in excess of reported solubility limits produced no observable changes in cell viability. GC/MS analysis of TNT-treated cell media and cell lysates revealed rapid removal of TNT. Within 12 h, less than 1% of the initial TNT remained in the growth medium. Aminodinitrotoluenes (ADNTs), known metabolites of TNT, accumulated transiently in cell lysates, and to a lesser extent in cell media. ADNT concentrations started to decrease after 3 h. After 12 h, less than 5% of the initial TNT could be detected as ADNT. Total ADNTs never exceeded 26% of initial TNT, suggesting that additional biotransformation steps also occurred. No other nitroaromatics were detected. A pseudo-first order rate constant for TNT clearance was calculated, k=0.40 h−1. D. innoxia cell suspension cultures demonstrated virtually complete clearance of TNT and of subsequent ADNT metabolites in less than 12 h. This rapid metabolism of nitroaromatics by the Datura cell suspension system indicates the utility of this system for further molecular and biochemical studies.

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 −1, 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.

Kinetics of the Alkaline Hydrolysis of 2,4,6-Trinitrotoluene in Aqueous Solution and Highly Contaminated Soils

During the two World Wars, large amounts of TNT were released into the environment. Until today, high concentrations of TNT can be found in the soil of former ammunition plants. To obtain basic data for a novel treatment process for highly contaminated soils, the homogeneous aqueous hydrolysis of TNT in the pH range from 10 to 12 and the alkaline treatment of two contaminated soils at pH 11 and pH 12 were investigated. The experimental data were described for their respective pH values using a pseudo-first-order model. In the homogeneous experiments, 95−97% of the TNT was hydrolyzed. During alkaline hydrolysis, up to two nitrogoups per TNT molecule were released, indicating the irreversible destruction of TNT. Except for the formation of small traces of amino dinitrotoluenes and trinitrobenzenes, no nitroaromatic benzenes or toluenes were detected during GC analysis. For the less contaminated soil, ELBP2, with an initial TNT concentration of 116 mg/kg, a destruction of 99% was achieved. The highly contami...

A reevaluation of the Hofmann rearrangement in electron deficient systems: preparation of 2-(15N)-amino-4,6-dinitrotoluene and 4-(15N)-amino-2,6-dinitrotoluene

The Hofmann rearrangement is an excellent synthetic method to introduce amino functions into aromatic molecules; however, it becomes less efficient in electron-deficient systems because of competitive hydrolytic reactions. This report describes the preparation of the aminodinitrotoluenes (ADNTs) 2-(15N)-amino-4,6-dinitrotoluene and 4-(15N)-amino-2,6-dinitrotoluene by developing conditions using HPLC as a reaction monitoring technique. Both ADNTs were prepared in >70% yield and >95% isotropic purity by using organic solvents and controlling pH during the formation of N-chlorobenzamides and then performing the rearrangement. © 1998 John Wiley & Sons, Ltd.

Pseudomonas aeruginosa strain MA01 aerobically metabolizes the aminodinitrotoluenes produced by 2,4,6-trinitrotoluene nitro group reduction.

Many microbes reduce the nitro substituents of 2,4,6-trinitrotoluene (TNT), producing aminodinitrotoluenes (ADNTs). These compounds are recalcitrant to further breakdown and are acutely toxic. In a search for organisms capable of metabolizing ADNTs, a bacterial strain was isolated for the ability to use 2-aminobenzoate (anthranilate) as sole C-source. This isolate, Pseudomonas aeruginosa MA01, metabolized TNT by first reducing one nitro group to form either 2-amino-4,6-dinitrotoluene (2ADNT) or 4-amino-2,6-dinitrotoluene (4ADNT). However, strain MA01 was distinct from other TNT-reducing organisms in that it transformed these compounds into highly polar metabolites through an O2-dependent process. Strain MA01 was able to cometabolize TNT, 2ADNT, and 4ADNT in the presence of a variety of carbon and energy sources. During aerobic cometabolism with succinate, 45% of uniformly ring-labeled [14C]TNT was transformed to highly polar compounds. Aerobic cometabolism of purified [14C]2ADNT and [14C]4ADNT with succinate as C-source produced similar amounts of these polar metabolites. During O2-limited cometabolism with succinate as C-source and nitrate as electron acceptor, less than 8% of the [14C]TNT was transformed to polar metabolites. Purified 2,6-diamino-4-nitrotoluene was not metabolized, and while 2,4-diamino-6-nitrotoluene was acetylated, the product (N-acetyl-2,4-diamino-6-nitrotoluene) was not further metabolized. Therefore, strain MA01 metabolized TNT by oxidation of the ADNTs and not by reduction the remaining nitro groups on the ADNTs.

Mutagenicity in Salmonella typhimurium and structure-activity relationships of wastewater components emanating from the manufacture of trinitrotoluene.

The mutagenicity of 36 polynitroaromatic compounds was investigated with five strains of Salmonella typhimurium. Isomeric trinitrotoluenes (TNT), with the exception of 2,4,6-TNT and 2,3,4-TNT, exhibit mutagenicity independently of nitroreductase enzymes, but isomeric aminodinitrotoluenes (ADNT) and isomeric dinitrotoluenes (DNT) need nitroreductase to induce mutation. Within groups of isomeric TNTs, DNTs, and ADNTs, mutagenic response was enhanced by a para orientation of nitro groups. The mutagenic response of isomeric DNTs was found to correlate with the compound's ability to undergo charge-transfer complexation with reductive enzymes, whereas further complexation (such as a Janovsky complex) appears to be required for inducing mutation in dinitrobenzenes. These results indicate that polynitroaromatic compounds in TNT wastewaters possess a significant potential for biologic activity.