The reactivity of partially reduced metabolites of 2,4,6-trinitrotoluene (TNT), namely arylhydroxylamines and nitrosoarenes, was evaluated with a simple biological system and with components of soil natural organic matter (NOM). This study was carried out to address the long-standing problem of irreversible binding to soil NOM and biomass, commonly observed during the reductive transformation of polynitroaromatic contamination. The study focused on partially reduced metabolites rather than the completely reduced arylamine metabolites that have already been extensively investigated for their role in binding to soil NOM. In the simple bioreduction system of Clostridium acetobutylicum cell-free extract/molecular hydrogen (electron donor), 10% of the initial 14C was found bound to solid proteinaceous material following sequential anaerobic/aerobic treatment. A review of the nitroso and hydroxylamino functional group chemistry revealed that the nitroso-thiol reaction was most likely responsible for the reaction with proteins. The introduction of a model thiol, 1-thioglycerol, into an anaerobic mixture of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT) and 2,4-dihydroxylamino-6-nitrotoluene (DHA6NT) resulted in the formation of a new product, only when the reaction mixture was exposed to air. The results from the model reaction confirmed that thiols could act as competing nucleophiles for nitroso compounds, which are readily formed from hydroxylamino compounds upon exposure to air. The reactivity of arylhydroxylamines and nitrosoarenes with standard humic acids was investigated using 4HADNT and nitrosobenzene as model compounds, respectively. Contrary to results reported by others, 4HADNT was found to be nonreactive towards humic acid at humic acid concentrations in excess of dissolved organic matter concentrations found in nature. Conversely, nitrosobenzene reacted rapidly with humic acids, with the extent of reaction being highest for humic acids that had a high protein content. Humic acids that were pretreated with a thiol derivatizing agent showed diminished capacity for reaction with nitrosobenzene. Since nitroso intermediates from TNT reduction are difficult to synthesize and are rarely observed in nature due to their high instability, their electrophilic characteristics were evaluated using a molecular modeling approach. Molecular models of potential TNT nitroso intermediates were compared with those of the strongly electrophilic nitrosobenzene. The comparison revealed that 2-nitroso-4-hydroxylamino-6-nitrotoluene was more likely to react similarly to nitrosobenzene than 4-nitroso-2,6-dinitrotoluene
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