Degradation of the Insensitive Munitions Compound 3-Nitro-1,2,4-triazol-5-one (NTO) via Sequential Reducing-Oxidizing Conditions

Insensitive Munitions Compounds (IMCs) have been used by the U.S. Army as replacements for conventional explosives. The detonation of these compounds requires higher activation energy, avoiding the occurrence of accidental explosions. The nitro heterocyclic compound 3-nitro-1,2,4-triazol-5-one (NTO) is one of the main ingredients of some insensitive formulations, such as IMX-101 and IMX-104. The use of IMCs is expected to increase, and since NTO is very soluble in water and does not adsorb onto soil particles, it is very mobile in the environment. Therefore, concerns about NTO toxicity and fate once it reaches the soil and water bodies have been raised. In saturated soils, microbes are known to reduce the nitro group of NTO to an amine, yielding its corresponding heterocyclic amine, 3-amino-1,2,4 triazol-5-one (ATO). However, NTO does not undergo any microbial transformation under aerobic conditions. In this study, we aimed to achieve full degradation of NTO by properly sequencing redox reactions. The transformation of NTO in soil was investigated in batch bioassays containing soil as the microbial inoculum. Under anaerobic conditions and using pyruvate as electron donor, NTO was reduced to ATO, and after switching the conditions to aerobic, ATO was completely removed. Complete NTO and ATO removal were also achieved in an aerated continuous flow reactor using soil as the inoculum. An excess of pyruvate was added to the medium to promote oxygen consumption through the microbial biofilm allowing for simultaneous NTO reduction and ATO oxidation. In addition, enrichment cultures (EC) were developed to identify the microorganisms responsible for NTO reduction and ATO oxidation. ATO was fully biodegraded aerobically as the sole carbon and nitrogen source by a sustainable EC developed from soil inoculum, being mineralized to CO2, NH3 and N2. We also investigated the oxidation of NTO and 2,4-dinitroanisole (DNAN), another ingredient of insensitive munitions formulations, and their respective reduced daughter products, ATO and 2-methoxy-5-nitroaniline (MENA), by potassium permanganate (KMnO4), a common oxidant used for in situ chemical oxidation (ISCO). The results demonstrated that lower concentrations of KMnO4 and shorter timescales are needed to oxidize MENA and ATO than to oxidize DNAN and NTO. In another experiment, NTO was anaerobically reduced to ATO by soil microorganisms and then spiked with KMnO4, showing that the reductive biological pretreatment enhances the effectiveness of the oxidant. These results indicate that prior microbial reduction of NTO and DNAN can enhance the effectiveness of ISCO, making it a feasible option for remediating sites contaminated with IMCs. Lastly, NTO and ATO toxicity was assessed towards various organisms, including microorganisms (i.e., methanogenic archaea, aerobic heterotrophs, and Aliivibrio fischeri (Microtox assay)), the microcrustacean Daphnia magna, and zebrafish embryos (Danio rerio). The results indicate that the reductive biotransformation of NTO could enhance or lower its toxicity according to the target organism. Overall, this study indicates that NTO can be converted to environmentally safe endpoints through a combination of reducing and oxidizing conditions