CHARACTERIZATION OF METABOLITES IN THE BIOTRANSFORMATION OF 2,4,6-TRINITROTOLUENE WITH ANAEROBIC SLUDGE - ROLE OF TRIAMINOTOLUENE

The present study describes the biotransformation of 2,4,6-trinitrotol uene (TNT) (220 mu M) by using anaerobic sludge (10%, vol/vol) supplem ented 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 ((CO2)-C-14,). A combination of solid-phase microextraction-gas chroma tography-mass spectrometry and liquid chromatography-mass spectrometry identified two distinctive cycles in the degradation of TNT, One cycl e was responsible for the stepwise reduction of TNT to eventually prod uce triaminotoluene (TAT) in relatively high yield (160 mu M). The oth er cycle involved TAT and was responsible for the production of azo de rivatives, 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'-a zoT) at pH 7.2. These azo compounds were also detected when TAT was tr eated with the anaerobic sludge but not with an autoclaved sludge, sug gesting the biotic nature of their formation, When the anaerobic condi tions in the TAT-containing culture medium were removed by aeration an d/or acidification (pH 3), the corresponding phenolic compounds, e.g., hydroxy-diaminotoluenes and dihydroxy-aminotoluenes, mere observed at room temperature. Trihydroxy-toluene was detected only after heating TAT in water at 100 degrees C, When (CH3)-C-13-labeled TNT was used as the N source in the above microcosms, me were unable to detect C-13-l abeledp-cresol or [(CH3)-C-13]toluene, indicating the absence of denit ration or deamination in the biodegradation process. The formation and disappearance of TAT were not accompanied by mineralization, suggesti ng that TAT acted as a dead-end metabolite.