2,4-Dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO) are two insensitive munitions compounds (IMCs) being used to replace conventional munitions, TNT and RDX, respectively. Relatively little is known on how these compounds will perform in the environment with respect to their biotransformation and retention in soil systems. The objectives of this study were to evaluate the interaction of abiotic and biotic factors contributing to the attenuation of IMCs in the soil leading to the formation of environmentally safe end-points.
A series of soil or soil mineral microcosm experiments were conducted in which IMC biotransformation products or IMC sorption were monitored with liquid chromatography (including quadrupole time of flight mass spectrometry detection), toxicity assays and 14C fractionation studies. Staggered toxicity tests were used in which microbial and zebrafish embryo toxicity was monitored as a function of biotransformation time and product composition. To evaluate mineralization of IMC, common N ions were measured with an ion chromatograph and CO2 and N2 were measured with a gas chromatograph.
DNAN was initially converted to 2-methoxy-4-nitroaniline (MENA) and 2,4-diaminoanisole (DAAN) in soil especially under reducing conditions provided with electron donating substrate in a reaction involving both abiotic and biotic mechanisms. The rate of reduction was correlated strongly to the soil organic carbon (OC). The aromatic amines reacted chemically with nitroso-intermediates of DNAN reduction to form azo-linked dimers and subsequently both the aromatic amines and dimers were subjected to secondary metabolism causing O-demethylation and N-methylation and N-acetylation. The aromatic amines also reacted with soil organic matter (NOM) to become covalently and irreversibly bound with the insoluble humus fraction (humin). The extent of 14C-DNAN incorporation into humin could be accurately predicted by the mass ratio of OC and initial DNAN (mg:mg) regardless of the source of the soil organic matter. Comparing incubation conditions indicates that the most rapid incorporation of 14C-DNAN into humin occurs under completely anaerobic conditions. The most likely mechanism of anaerobic incorporation is a nucleophilic substitution reaction between quinone moieties in humus and the aromatic amine daughter products.
The biotransformation intermediates of DNAN were observed to be toxic to some microbial systems such as acetoclastic methanogenesis, nitrification and the marine bioluminescent marine bacterium, Aliivibrio fischeri (Microtox assay). The toxicity was often either in the same range or moderately lower than the parent DNAN depending on the test system and the specific intermediate. However, only an N-acetylated DAAN metabolite was distinctly less toxic than DNAN by many orders of magnitude. Staggered bioassays revealed methanogen inhibition increased sharply early as reactive intermediates formed during nitro-group reduction but the inhibition reversed when dimers were formed. Aliivibrio fischeri tolerated the early intermediates but progressively became more inhibited as dimer levels accumulated over longer biotransformation time periods.
To evaluate ecotoxicity, zebrafish embryo were utilized as the model system. Intermediates of DNAN biotransformation caused detectable developmental and behavioral toxicity in zebrafish embryos. Most concerning was the high level of developmental toxicity caused by a surrogate azo-dimer intermediate and 4-methoxy-5-nitroaniline (iMENA) (both at 6.4 μM) as well as evidence of locomotor toxicity caused by DAAN at higher concentrations. In staggered assays significant increase in acute mortality was observed at the onset of dimer formation. The toxicity tests clearly indicated that intermediates of DNAN biotransformation are often still quite toxic, thus the goal of remediation should be to achieve environmentally safe end points such as irreversible covalent incorporation into humin (“bound residue”) or complete biodegradation to mineralized products (“mineralization”).
NTO was also readily reduced to its corresponding heterocyclic amine, 3-amino-1,2,4 triazol- 5-one (ATO) under anaerobic conditions in soil via a microbially catalyzed reaction requiring an electron donor. Under aerobic conditions in aqueous soil suspensions, NTO was not biotransformed, whereas under anaerobic conditions, ATO was not degraded further. Full biodegradation could be achieved by properly sequencing redox reactions. NTO must first be reduced to ATO to subsequently be able to mineralize it under aerobic conditions. NTO degradation was achieved in a continuously fed aerobic biotrickle reactor. The reduction of NTO in an aerobic bulk environment of the reactor was plausible due to the presence of putative anaerobic microniches in the reactor’s biofilm. ATO was fully biodegraded as the sole C and N source by a sustainable enrichment culture (EC) developed from soil inoculum. The EC mineralizes C in ATO completely to CO2 and the N in ATO by approximately 50% to NH3 and the other 50% to N2. The EC requires O2, indicating at least one O2- dependent step. Clone library studies indicated a possible role of Hydrogenophaga previously implicated in oxidative 4- aminobenzenesulfonate degradation and Hyphomicrobium, known for C1 metabolism.
IMC adsorption studies were carried out with the clay, montmorillonite and the iron oxides, goethite and ferrihydrite. DNAN and its daughter product, MENA were adsorbed strongly by the layer silicate clay; whereas NTO and to a lesser extent ATO was adsorbed by the iron oxides. K+ enhanced DNAN adsorption to the clay due to its lower hydration shell compared to other major cations, enhancing the innersphere complexes.
Common occurring soil minerals can cause transformation reactions. Birnessite (MnO2) and ferrihydrite. During reductive (bio)transformation of DNAN and NTO, the progressive replacement of electron withdrawing nitro-groups by amino groups, increases the oxidative susceptibility of the IMC molecules and they were rapidly oxidized by birnessite. In contrast, the parent compounds, DNAN and ATO were completely resistant to oxidation. ATO was extensively oxidized to safe end products consisting of CO2, NH3 and urea during the reaction with birnessite. Lastly, the mixed valent iron containing mineral, green rust, reduced DNAN in the time scale of a few days and NTO in the time scale of minutes to the corresponding amine daughter products.
The most important benefit of the project is the recognition that IMCs can be converted by a sequence of reduction and oxidation (or substitution) reactions to environmentally safe end points. The sequence is required since our study shows that primary daughter products are still quite toxic and yet significantly more prone to oxidation. The sequence enables extensive degradation of NTO by forming ATO which in turn is extensively mineralized by aerobic bacteria or birnessite to CO2, NH3, N2 and urea. The sequence also enables extensive incorporation of DNAN into NOM by first reducing it to aromatic amines that later undergo nucleophilic substitution reactions with quinone moieties in NOM creating irreversible covalent bonds. The mass ratio of OC to DNAN was identified as a parameter predicting humus-bound residue formation, with the implication of NOM addition enhancing the remediation of amines.
Other benefits include the discovery that iron oxides in soil can significantly adsorb NTO. Adsorption of DNAN in clay soils can be greatly increased by adding K+ to soil. The project also demonstrates the importance of both soil minerals and bacteria in the transformation of IMCs. And lastly, an enrichment culture was developed that reliably mineralizes ATO as sole C and N-source to benign products, CO2, NH3 and N2, with potential for bioaugmentation applications.