Characterization of Metabolites in the Biotransformation of 2,4,6-Trinitrotoluene with Anaerobic Sludge: Role of Triaminotoluene

Abstract

The present study describes the biotransformation of 2,4,6-trinitrotoluene (TNT) (220 μM) by using anaerobic sludge (10%, vol/vol) supplemented with molasses (3.3 g/liter). Despite the disappearance of TNT in less than 15 h, roughly 0.1% of TNT was attributed to mineralization (14CO2). A combination of solid-phase microextraction–gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry identified two distinctive cycles in the degradation of TNT. One cycle was responsible for the stepwise reduction of TNT to eventually produce triaminotoluene (TAT) in relatively high yield (160 μM). The other cycle involved TAT and was responsible for the production of azo derivatives, e.g., 2,2′,4,4′-tetraamino-6,6′-azotoluene (2,2′,4,4′-TA-6,6′-azoT) and 2,2′,6,6′-tetraamino-4,4′-azotoluene (2,2′,6,6′-TA-4,4′-azoT) at pH 7.2. These azo compounds were also detected when TAT was treated with the anaerobic sludge but not with an autoclaved sludge, suggesting the biotic nature of their formation. When the anaerobic conditions in the TAT-containing culture medium were removed by aeration and/or acidification (pH 3), the corresponding phenolic compounds, e.g., hydroxy-diaminotoluenes and dihydroxy-aminotoluenes, were observed at room temperature. Trihydroxytoluene was detected only after heating TAT in water at 100°C. When 13CH3-labeled TNT was used as the N source in the above microcosms, we were unable to detect13C-labeled p-cresol or [13CH3]toluene, indicating the absence of denitration or deamination in the biodegradation process. The formation and disappearance of TAT were not accompanied by mineralization, suggesting that TAT acted as a dead-end metabolite.

Enhancing the attenuation of explosives in surface soils at military facilities: Combined sorption and biodegradation

“…Stimulation of explosives biodegradation in different environments with molasses has been reported previously by several investigators [14,15,[30][31][32][33][34], so its effectiveness in these studies was not surprising. Potato starch and corn starch also have been observed to stimulate significant RDX biodegradation in anaerobic soil-water slurries under anaerobic conditions [35].…”

Spread, Behavior, and Ecosystem Consequences of Conventional Munitions Compounds in Coastal Marine Waters

“…This also implies a greater immobilization of the ADNT forms compared with DANT. TAT is not stable in solution, undergoing hydrolysis to azo-(under anoxic conditions) and hydroxy-(under oxic conditions) intermediate forms, and eventually polymerizing and precipitating (Hawari et al, 1998). Unknown products formed under anoxic conditions appear to be derived from a TAT precursor (Elovitz and Weber, 1999), consistent with formation of TAT hydrolysis azo-products (Hawari et al, 1998).…”

“…There is extensive evidence for regioselective reduction of TNT nitro moieties, with the para form dominating over the ortho form to a variable degree depending on the relative importance of abiotic and biotic pathways (Barrows et al, 1997). In one study conducted with sludge incubation, concentrations of 4-ADNT and 2,4-DANT reached two-and ten-fold higher, respectively, than the 2-ADNT and 2,6-DANT forms (Hawari et al, 1998). Similar preferential formation of 4-ADNT has been shown in other soil and plant systems (Harvey et al, 1990).…”

Soil properties affect the toxicities of 2,4,6‐trinitrotoluene (TNT) and hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) to the enchytraeid worm Enchytraeus crypticus

“…These transformation products can be either more or less bioavailable and more or less toxic than the parent compound [2,26,37,[45][46][47][48][49][50][51]. The aminonitrotoluene intermediates can be formed by soil bacteria under either aerobic or anaerobic conditions [2,47,52]; they are the most commonly detected and influential TNT transformation products found in weathered and aged natural soils. Ongoing studies with aminonitrotoluene intermediates of TNT transformation will provide additional information regarding the toxicity of these compounds.…”