This paper examines processes in plants for the formation of fate products of TNT beyond its aminated reduction products, 2-amino-4,6-dinitrotoluene and 4-amino-2,6dinitrotoluene. TNT metabolites were isolated and characterized in combination with temporal analyses of product profiles and 14 C distribution, in microbe-free, axenic root cultures of Catharanthus roseus. Four unique TNTderived compounds were isolated. Using evidence from 1 H NMR, mass spectroscopy, HPLC, acid hydrolysis, and enzymatic hydrolysis with β-glucuronidase and β-glucosidase, they were established as conjugates formed by reactions of the amine groups of 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene. From the mass spectral evidence, at least a six-carbon unit from the plant intracellular millieu was involved in conjugate formation. Mass balance analysis indicated that, by 75 h after TNT amendment of the initial TNT radiolabel, extractable conjugates comprised 22%, bound residues comprised another 29%, 2-amino-4,6dinitrotoluene was 4%, and the rest remained unidentified. Isolates from TNT-amended roots versus monoaminodinitrotoluene-amended roots were not identical, suggesting numerous possible outcomes for the plant-based conjugation of 2-amino-4,6-dinitrotoluene or 4-amino-2,6-dinitrotoluene. This study is the first direct evidence for the involvement of the primary reduction products of TNTs2-amino-4,6dinitrotoluene and 4-amino-2,6-dinitrotoluenesin conjugation processes in plant detoxification of TNT.

TNT transformation processes in sediment-free, “natural”, aquatic phytoremediation systems of Myriophyllum aquaticum were investigated with specific interest in oxidation products. Extraction procedures combining liquid−liquid extractions and solid-phase extractions were developed for the isolation of the mostly acidic, oxidized TNT metabolites. Six compounds unique from the reduction products of TNT were isolated and characterized by UV−vis, 1H, and 13C NMR spectroscopy, by mass spectroscopy, and by chemical synthesis where feasible. These compounds include 2-amino-4,6-dinitrobenzoic acid, 2,4-dinitro-6-hydroxy-benzyl alcohol, 2-N-acetoxyamino-4,6-dinitrobenzaldehyde, 2,4-dinitro-6-hydroxytoluene, and two binuclear metabolites unique from the customary azoxytetranitrotoluenes. The monoaryl compounds show clear evidence of oxidative transformations, methyl oxidation and/or aromatic hydroxylation. It is possible that oxidative transformation(s) preceded nitro reduction since studies on exposure of M. aquaticum to either 2-amino-4,6-dinitrotoluene or 4-amino-2,6-dinitrotoluene did not yield any of the oxidation products identified here. The accumulation of oxidation products was significant:  2-amino-4,6-dinitrobenzoic acid, 4.4%; 2,4-dinitro-6-hydroxy-benzyl alcohol, 8.1%; 2-N-acetoxyamino-4,6-dinitrobenzaldehyde, 7.8%; and, 2,4-dinitro-6-hydroxytoluene, 15.6%. The binuclear metabolites accounted for an estimated 5.6%. This study is the first direct evidence for oxidative transformations in aquatic phytoremediation systems.

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