The biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in liquid cultures with municipal anaerobic sludge showed that at least two degradation routes were involved in the disappearance of the cyclic nitramine. In one route, RDX was reduced to give the familiar nitroso derivatives hexahydro-1-nitroso-3,5-dinitro-1,3, 5-triazine (MNX) and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX). In the second route, two novel metabolites, methylenedinitramine [(O(2)NNH)(2)CH(2)] and bis(hydroxymethyl)nitramine [(HOCH(2))(2)NNO(2)], formed and were presumed to be ring cleavage products produced by enzymatic hydrolysis of the inner C---N bonds of RDX. None of the above metabolites accumulated in the system, and they disappeared to produce nitrous oxide (N(2)O) as a nitrogen-containing end product and formaldehyde (HCHO), methanol (MeOH), and formic acid (HCOOH) that in turn disappeared to produce CH(4) and CO(2) as carbon-containing end products.
In an earlier study, we reported that hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) biodegraded with domestic anaerobic sludge to produce a key RDX ring cleavage intermediate that was tentatively identified as methylenedinitramine (O2NNHCH2NHNO2) using LC/MS with negative electrospray ionization (ES-). Recently, we obtained a standard material of methylenedinitramine and thus were able to confirm its formation as the key initial RDX intermediate. In water alone or in the presence of sludge, methylenedinitramine decomposed to N20 and HCHO. Only in the presence of sludge HCHO converted further to carbon dioxide. To test our hypothesis that water was involved in the formation of methylenedinitramine during incubation of RDX with sludge, we allowed the energetic compound to biodegrade in several D2O/H2O solutions (90, 50, and 0% v/v). We observed three distinctive deprotonated or dedeuterated mass ions at 135, 136, and 137 Da that were attributed to the formation of nondeuterated (H-methylenedinitramine), monodeuterated (D1-methylenedinitramine), and dideuterated methylenedinitramine (D2-methylenedinitramine), respectively. Two controls were prepared in D2O both in the absence of sludge; the first contained methylenedinitramine, and the second contained RDX. Neither control produced any deuterated methylenedinitramine, thus excluding the occurrence of any abiotic D/H exchange between D2O and either methylenedinitramine or RDX. The results supported the occurrence of an initial enzymatic reaction on RDX, yet they did not provide compelling evidence on whether methylenedinitramine was an initial RDX enzymatic hydrolysis product or simply formed via the spontaneous hydrolysis of an anonymous initial RDX enzymatic product.
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