Reactivity of partially reduced arylhydroxylamine and nitrosoarene metabolites of 2,4,6-trinitrotoluene (TNT) toward biomass and humic acids

Sequential anaerobic/aerobic treatment of 2,4,6-trinitro-toluene (TNT) generally results in the incorporation of residues into biomass and natural organic matter fractions of a system. To better understand the potential contribution of hydroxylamine and nitroso moieties in these reactions, studies were conducted using model systems taking advantage of the biocatalytic-activity of Clostridium acetobutylicum that does not produce aminated TNT derivatives. To evaluate binding to biomass only, systems containing cell-free extracts of C. acetobutylicum and molecular hydrogen as a reductant were employed. At the end of treatment, mass balance studies showed that 10% of the total C-14 was associated with an insoluble protein-containing precipitate that could not be extracted with organic solvents. Model reactions were conducted between a mixture of 2,4-dihydroxylamino-6-nitrotoluene (DHA6NT) and 4-hydroxylamino-2,6-dinitrotoluene (4HADNT) and 1-thioglycerol to test the involvement of the nitroso-thiol reaction in binding to biomass. It was demonstrated that DHA6NT formed a new and relatively polar product with 1-thioglycerol only in the presence of oxygen. The oxygen requirement confirmed that the nitroso functionality was responsible for the binding reaction. The reactivity of arylhydroxylamino and nitrosoarene functionalities toward International Humic Substance Society (IHSS) peat humic acid was evaluated under anaerobic and aerobic conditions, respectively. 4HADNT showed no appreciable reactivity toward peat humic acid. Conversely, the nitrosoarene compound, nitrosobenzene, showed rapid reactivity with peat humic acid (50% removal in 48 h). When tested with two other humic acids (selected on the basis of their protein content), it became apparent that the proteinaceous fraction was responsible at least in part for the nitrosoarene's removal from solution. Furthermore, the pretreatment of the humic acids with a selective thiol derivatizing agent had a considerable effect on their ability to react with nitrosobenzene. Finally, molecular modeling tools were used to compare the electrophilic characteristics of potential nitroso intermediates forming from the oxidation of arylhydroxylamino metabolites of TNT. Molecular modeling analysis demonstrated that the more reduced TNT derivative containing nitroso groups were more likely to react with nucleophiles in humic substances than the less reduced nitroso intermediates.