Dissolution of microscale energetic residues in saturated porous media: visualization and quantification at the pore-scale by spectral confocal microscopy

Microscale energetic residues (<1 mm) are produced during munitions detonation and the weathering of larger residues, and may serve as mobile and fast dissolving sources of explosive compounds, such as 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). Knowledge of the behavior of microscale energetic residues in subsurface environments is quite limited. This work employed a previously unreported property of TNT, RDX, and HMX (i.e., autofluorescence under 405 nm laser illumination) to visualize and quantify the dissolution of microscale physically attrited energetic residues in saturated porous media. The results demonstrated that within the Composition B particles, TNT dissolved preferentially over RDX/HMX and the mass ratio of RDX/HMX to TNT increased by >5.3 times initially. The focused particles dissolved in a stepwise fashion, with >72% of particle volume reduction in <36 min. Moreover, the results suggested that the particle shape factor was relatively stable and the particles retained their highly irregular shape throughout the dissolution processes. This is the first work to demonstrate application of spectral confocal microscopy for visualizing and quantifying the behavior of energetic residues at the pore-scale.     

Dissolution, sorption, and kinetics involved in systems containing explosives, water, and soil

Knowledge of explosives sorption and transformation processes is required to ensure that the proper fate and transport of such contaminants is understood at military ranges and ammunition production sites. Bioremediation of 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and related nitroaromatic compounds has met with mixed success, which is potentially due to the uncertainty of how energetic compounds are bound to different soil types. This study investigated the dissolution and sorption properties of TNT and RDX explosives associated with six different soil types. Understanding the associations that explosives have with a different soil type assists with the development of conceptual models used for the sequestration process, risk analysis guidelines, and site assessment tools. In three-way systems of crystalline explosives, soil, and water, the maximum explosive solubility was not achieved due to the sorption of the explosive onto the soil particles and observed production of transformation byproducts. Significantly different sorption effects were also observed between sterile (gamma-irradiated) and nonsterile (nonirradiated) soils with the introduction of crystalline TNT and RDX into soil-water systems.     

Use of liquid chromatography/tandem mass spectrometry to detect distinctive indicators of in situ RDX transformation in contaminated groundwater

An important element of monitored natural attenuation is the detection in groundwater of distinctive products of pollutant degradation or transformation. In this study, three distinctive products of the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) were detected in contaminated groundwater from the Iowa Army Ammunition Plant; the products were MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine), DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine), and TNX (hexahydro-1,3,5-trinitroso-1,3,5-triazine). These compounds are powerful indicators of RDX transformation for several reasons: (a) they have unique chemical features that reveal their origin as RDX daughter products, (b) they have no known commercial, industrial, or natural sources, and (c) they are well documented as anaerobic RDX metabolites in laboratory studies. The products were analyzed by LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) with selected reaction monitoring and internal standard quantification using [ring-U-15N]RDX. Validation tests showed the novel LC/MS/MS method to be of favorable sensitivity (detection limits ca. 0.1 microg/L), accuracy, and precision. The products, which were detected in all groundwater samples with RDX concentrations of > ca. 1 microg/L (25 out of 55 samples analyzed), were present at concentrations ranging from near the detection limit to 430 microg/L. MNX was the typically the most abundant of the three nitroso-substituted products; concentrations of the products seldom exceeded 4 mol % of the RDX concentration, although they ranged as high as 26 mol % (TNX). Geographic and temporal distributions of RDX, MNX, DNX, and TNX were assessed. A degradation product resulting from RDX ring cleavage, methylenedinitramine, was not detected by LC/MS/MS in any sample (detection limit ca. 0.6-4 microg/L). This extensive field characterization of MNX, DNX, and TNX distributions in groundwater by a highly selective analytical method (LC/MS/MS) is significant because very little is known about the occurrence of intrinsic RDX transformation in contaminated aquifers.     

Electrochemical reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine in aqueous solutions

Electrochemical reduction of RDX, hexahydro-1,3,5-trinitro-1,3,5-triazine, a commercial and military explosive, was examined as a possible remediation technology for treating RDX-contaminated groundwater. A cascade of divided flow-through cells was used, with reticulated vitreous carbon cathodes and IrO2/Ti dimensionally stable anodes, initially using acetonitrile/water solutions to increase the solubility of RDX. The major degradation pathway involved reduction of RDX to the corresponding mononitroso compound, followed by ring cleavage to yield formaldehyde and methylenedinitramine. The reaction intermediates underwent further reduction and/or hydrolysis, the net result being the complete transformation of RDX to small molecules. The rate of degradation increased with current density, but the current efficiency was highest at low current densities. The technique was extended successfully both to 100% aqueous solutions of RDX and to an undivided electrochemical cell.     

Abiotic transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine by Fe(II) bound to magnetite

RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), a nitramine explosive, is often found as a subsurface contaminant at military installations. Though biological transformations of RDX are often reported, abiotic studies in a defined medium are uncommon. The work reported here was initiated to investigate the transformation of RDX by ferrous iron (Fe(II)) associated with a mineral surface. RDX is transformed by Fe(II) in aqueous suspensions of magnetite (Fe3O4). Negligible transformation of RDX occurred when it was exposed to Fe(II) or magnetite alone. The sequential nitroso reduction products (MNX, DNX, and TNX) were observed as intermediates. NH4+, N2O, and HCHO were stable products of the transformation. Experiments with radiolabeled RDX indicate that 90% of the carbon end products remained in solution and that negligible mineralization occurred. Rates of RDX transformation measured for a range of initial Fe(II) concentrations and solution pH values indicate that greater amounts of adsorbed Fe(II) result in faster transformation rates. As pH increases, more Fe(II) adsorbs and k(obs) increases. The degradation of RDX by Fe(II)-magnetite suspensions indicates a possible remedial option that could be employed in natural and engineered environments where iron oxides are abundant and ferrous iron is present.     

Phytophotolysis of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in leaves of reed canary grass

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was degraded in reed canary grass leaves exposed to simulated sunlight to primary products nitrous oxide and 4-nitro-2,4-diazabutanal. This is the first time that 4-nitro-2,4-diazabutanal, a potentially toxic degradate, has been measured in plant tissues following phytotransformation of RDX. These compounds, along with nitrite and formaldehyde, were also detected in aqueous RDX systems exposed to the same simulated sunlight. Results showed that the initial products of RDX photodegradation in translucent plant tissues were similar to products formed from aqueous photolysis of RDX. Combustion analysis of leaves following 14C-RDX uptake and subsequent light exposure revealed the presence of tissue-bound material that could not be extracted with acetonitrile. No detectable formaldehyde was emitted from the leaves. The detection of similar RDX degradation products in both aqueous and plant-based systems suggests that RDX may be initially transformed by similar mechanisms in both systems. Direct photolysis of RDX via ultraviolet irradiation passing into the leaves is hypothesized to be responsible for the observed transformations. In addition, membrane-bound "trap chlorophyll" in the chloroplasts may shuttle electrons to RDX as an indirect photolysis transformation mechanism. Results from this study indicate that reed canary grass facilitates photochemical degradation of RDX, and this mechanism should be considered along with more established phytoremediation processes when assessing the fate of contaminants in plant tissues. Plant-mediated phototransformation of xenobiotic compounds is a process that may be termed "phytophotolysis".     

Biodegradation of RDX nitroso products MNX and TNX by cytochrome P450 XplA

Anaerobic transformation of the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) by microorganisms involves sequential reduction of N-NO(2) to the corresponding N-NO groups resulting in the initial formation of MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine). MNX is further reduced to the dinitroso (DNX) and trinitroso (TNX) derivatives. In this paper, we describe the degradation of MNX and TNX by the unusual cytochrome P450 XplA that mediates metabolism of RDX in Rhodococcus rhodochrous strain 11Y. XplA is known to degrade RDX under aerobic and anaerobic conditions, and, in the present study, was found able to degrade MNX to give similar products distribution including NO(2)(-), NO(3)(-), N(2)O, and HCHO but with varying stoichiometric ratio, that is, 2.06, 0.33, 0.33, 1.18, and 1.52, 0.15, 1.04, 2.06, respectively. In addition, the ring cleavage product 4-nitro-2,4,-diazabutanal (NDAB) and a trace amount of another intermediate with a [M-H](-) at 102 Da, identified as ONNHCH(2)NHCHO (NO-NDAB), were detected mostly under aerobic conditions. Interestingly, degradation of TNX was observed only under anaerobic conditions in the presence of RDX and/or MNX. When we incubated RDX and its nitroso derivatives with XplA, we found that successive replacement of N-NO(2) by N-NO slowed the removal rate of the chemicals with degradation rates in the order RDX > MNX > DNX, suggesting that denitration was mainly responsible for initiating cyclic nitroamines degradation by XplA. This study revealed that XplA preferentially cleaved the N-NO(2) over the N-NO linkages, but could nevertheless degrade all three nitroso derivatives, demonstrating the potential for complete RDX removal in explosives-contaminated sites.     

Abiotic transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by green rusts

The rate and extent of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) transformation was measured in the presence of carbonate and sulfate green rust suspended in solutions containing common groundwater anions. Formaldehyde (HCHO), nitrous oxide gas (N2O(g)), and ammonium (NH4+) were the major end products, accounting for about 70% of the carbon mass balance and about half of the nitrogen mass balance. Results from experiments with both 14C-RDX and LC-MS analysis indicate that the remaining carbon products are soluble and most likely small (< 50 Da). The transient appearance of 1,3-dinitro-5-nitroso-1,3,5-triazacyclohexane (MNX), 1,3-dinitroso-5-nitro-1,3,5-triazacyclohexane (DNX), and 1,3,5-trinitroso-1,3,5-triazacyclohexane (TNX) indicate that some nitro-group reduction occurred. The kinetics of RDX transformation was rapid with a half-life of less than an hour in a pH 7.0 KBr solution. Little difference in rates of RDX transformation or product distribution was observed between carbonate and sulfate green rust, and an apparent reaction order of 1.0 was measured with respect to Fe(II) in both green rusts. Phosphate anions completely inhibited RDX reduction, and carbonate and sulfate anions resulted in slower kinetics, and in some cases, an initial lag period, compared to bromide and chloride. Our results suggest that green rusts may contribute to abiotic natural attenuation of RDX in Fe-rich subsurface environments, but that it will be important to consider groundwater composition when assessing rates of attenuation.     

Dissolution of composition B detonation residuals

Composition B (Comp B) detonation residuals pose environmental concern to the U.S. Army because hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a constituent, has contaminated groundwater near training ranges. To mimic their dissolution on surface soils, we dripped water at 0.51 ml/h onto individual Comp B particles (0.1-2.0 mg) collected from the detonation of 81-mm mortars. Analyses of the effluent indicate thatthe RDX and 2,4,6-trinitrotoluene (TNT) in Comp B do not dissolve independently. Rather, the relatively slow dissolution of RDX controls dissolution of the particle as a whole by limiting the exposed area of TNT. Two dissolution models, a published steady-flow model and a drop-impingement model developed here, provide good agreementwith the data using RDX parameters for time scaling. They predict dissolution times of 6-600 rainfall days for 0.01-100 mg Comp B particles exposed to 0.55 cm/h rainfall rate. These models should bracket the flow regimes for dissolution of detonation residuals on soils, but they require additional data to validate them across the range of particle sizes and rainfall rates of interest.     

Degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) using zerovalent iron nanoparticles

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a common contaminant of soil and water at military facilities. The present study describes degradation of RDX with zerovalent iron nanoparticles (ZVINs) in water in the presence or absence of a stabilizer additive such as carboxymethyl cellulose (CMC) or poly(acrylic acid) (PAA). The rates of RDX degradation in solution followed this order CMC-ZVINs > PAA-ZVINs > ZVINs with k1 values of 0.816 +/- 0.067, 0.082 +/- 0.002, and 0.019 +/- 0.002 min(-1), respectively. The disappearance of RDX was accompanied by the formation of formaldehyde, nitrogen, nitrite, ammonium, nitrous oxide, and hydrazine by the intermediary formation of methylenedinitramine (MEDINA), MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine), DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine), TNX (hexahydro-1,3,5-trinitroso-1,3,5-triazine). When either of the reduced RDX products (MNX or TNX) was treated with ZVINs we observed nitrite (from MNX only), NO (from TNX only), N2O, NH4+, NH2NH2 and HCHO. In the case of TNX we observed a new key product that we tentatively identified as 1,3-dinitroso-5-hydro-1,3,5-triazacyclo-hexane. However, we were unable to detect the equivalent denitrohydrogenated product of RDX and MNX degradation. Finally, during MNX degradation we detected a new intermediate identified as N-nitroso-methylenenitramine (ONNHCH2NHNO2), the equivalentof methylenedinitramine formed upon denitration of RDX. Experimental evidence gathered thus far suggested that ZVINs degraded RDX and MNX via initial denitration and sequential reduction to the corresponding nitroso derivatives prior to completed decomposition but degraded TNX exclusively via initial cleavage of the N-NO bond(s).     

N-15 NMR study of the immobilization of 2,4- and 2,6-dinitrotoluene in aerobic compost

Large-scale aerobic windrow composting has been used to bioremediate washout lagoon soils contaminated with the explosives TNT (2,4,6-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) at several sites within the United States. We previously used 15N NMR to investigate the reduction and binding of T15NT in aerobic bench-scale reactors simulating the conditions of windrow composting. These studies have been extended to 2,4-dinitrotoluene (2,4DNT) and 2,6-dinitrotoluene (2,6DNT), which, as impurities in TNT, are usually presentwherever soils have been contaminated with TNT. Liquid-state 15N NMR analyses of laboratory reactions between 4-methyl-3-nitroaniline-15N, the major monoamine reduction product of 2,4DNT, and the Elliot soil humic acid, both in the presence and absence of horseradish peroxidase, indicated that the amine underwent covalent binding with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and non-heterocyclic condensation products. Liquid-state 15N NMR analyses of the methanol extracts of 20 day aerobic bench-scale composts of 2,4-di-15N-nitrotoluene and 2,6-di-15N-nitrotoluene revealed the presence of nitrite and monoamine, but not diamine, reduction products, indicating the occurrence of both dioxygenase enzyme and reductive degradation pathways. Solid-state CP/MAS 15N NMR analyses of the whole composts, however, suggested that reduction to monoamines followed by covalent binding of the amines to organic matter was the predominant pathway.     

Role of organically complexed iron(II) species in the reductive transformation of RDX in anoxic environments

Organically complexed iron species can play a significant role in many subsurface redox processes, including reactions that contribute to the transformation and degradation of soil and aquatic contaminants. Experimental results demonstrate that complexation of Fe(II) by catechol- and thiol-containing organic ligands leads to formation of highly reactive species that reduce RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and related N-heterocyclic nitramine explosive compounds to formaldehyde and inorganic nitrogen byproducts. Under comparable conditions, relative reaction rates follow HMX < RDX < MNX < DNX < TNX. Observed rates of RDX reduction are heavily dependent on the identity of the Fe(II)-complexing ligands and the prevailing solution conditions (e.g., pH, Fe(II) and ligand concentrations). In general, reaction rates increase with increasing pH and organic ligand concentration when the concentration of Fe(II) is fixed. In solutions containing Fe(II) and tiron, a model catechol, observed pseudo-first-order rate constants (k(obs)) for RDX reduction are linearly correlated with the concentration of the 1:2 Fe(II)-tiron complex (FeL2(6-)), and kinetic trends are well described by -d[RDX]/dt= k(FeL2)6-[FeL2(6-)][RDX], where k(FeL2)6- = 7.31(+/-2.52) x 10(2) M(-1) s(-1). The reaction products and net stoichiometry (1 mol of RDX reduced for every 2 mol of Fe(II) oxidized) support a mechanism where RDX ring cleavage and decomposition is initiated by sequential 1-electron transfers from two Fe(II)-organic complexes.     

Reduction of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine by zerovalent iron: product distribution

RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) are cyclic nitramines ((CH2NNO2)n; n = 3 or 4, respectively) widely used as energetic chemicals. Their extensive use led to wide environmental contamination. In contrast to RDX, HMX tends to accumulate in soils due to its unique recalcitrance. In the present study, we investigated the potential of zerovalent iron (ZVI) to transform HMX under anoxic conditions. HMX underwent a rapid transformation when added in well-mixed anoxic ZVI-H2O batch systems to ultimately produce formaldehyde (HCHO), ammonium (NH4+), hydrazine (NH2NH2), and nitrous oxide (N2O). Time course experiments showed that the mechanism of HMX transformation occurred through at least two initial reactions. One reaction involved the sequential reduction of N-NO2 groups to the five nitroso products (1NO-HMX, cis-2NO-HMX, trans-2NO-HMX, 3NO-HMX, and 4NO-HMX). Another implied ring cleavage from either HMX or 1NO-HMX as demonstrated by the observation of methylenedinitramine (NH(NO2)CH2NH(NO2)) and another intermediate that was tentatively identified as (NH(NO2)CH2N(NO)CH2NH-(NO2)) or its isomer (NH(NO)CH2N(NO2)CH2NH(NO2)). This is the first study that demonstrates transformation of HMX by ZVI to significant amounts of NH2NH2 and HCHO. Both toxic products seemed to persist under reductive conditions, thereby suggesting that the ultimate fate of these chemicals, particularly hydrazine, should be understood prior to using zerovalent iron to remediate cyclic nitramines.     

The fate of the cyclic nitramine explosive RDX in natural soil

The sorption-desorption behavior and long-term fate of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was examined in sterilized and nonsterilized topsoil. Results of this study indicate that although RDX is not extensively sorbed by the topsoil (Ks(d) of 0.83 L/kg), sorption is nearly irreversible. Furthermore, there was no difference in the sorption behavior for sterile and nonsterile topsoil. However, over the longterm, RDX completely disappeared within 5 weeks in nonsterile topsoil, and hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) metabolites formed in the aqueous phase. Over the same period, recovery of RDX from sterile topsoil was high (55-99%), and the nitroso metabolites were not detected. Only traces of RDX were mineralized to CO2 and N2O by the indigenous microorganisms in nonsterile topsoil. Of the RDX that was mineralized to N2O, one N originated from the ring and the other from the nitro group substituent, as determined using N15 ring-labeled RDX. However, N2O from RDX represented only 3% of the total N2O that formed from the process of nitrification/denitrification.     

Alkaline hydrolysis of the cyclic nitramine explosives RDX,HMX, and CL-20: new insights into degradation pathways obtained by the observation of novel intermediates

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX, I) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) hydrolyze at pH > 10 to form end products including NO2-, HCHO, HCOOH, NH3, and N2O, but little information is available on intermediates, apart from the tentatively identified pentahydro-3,5-dinitro-1,3,5-triazacyclohex-1-ene (II). Despite suggestions that RDX and HMX contaminated groundwater could be economically treated via alkaline hydrolysis, the optimization of such a process requires more detailed knowledge of intermediates and degradation pathways. In this study, we hydrolyzed the monocyclic nitramines RDX, MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine), and HMX in aqueous solution (pH 10-12.3) and found that nitramine removal was accompanied by formation of 1 molar equiv of nitrite and the accumulation of the key ring cleavage product 4-nitro-2,4-diazabutanal (4-NDAB, O2NNHCH2NHCHO). Most of the remaining C and N content of RDX, MNX, and HMX was found in HCHO, N2O, HCOOH, and NH3. Consequently, we selected RDX as a model compound and hydrolyzed it in aqueous acetonitrile solutions (pH 12.3) in the presence and absence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) to explore other early intermediates in more detail. We observed a transient LC-MS peak with a [M-H] at 192 Da that was tentatively identified as 4,6-dinitro-2,4,6-triaza-hexanal (O2NNHCH2NNO2CH2NHCHO, III) considered as the hydrolyzed product of II. In addition, we detected another novel intermediate with a [M-H] at 148 Da that was tentatively identified as a hydrolyzed product of III, namely, 5-hydroxy-4-nitro-2,4-diaza-pentanal (HOCH2NNO2CH2NHCHO, IV). Both III and IV can act as precursors to 4-NDAB. In the case of the polycyclic nitramine 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), denitration (two NO2-) also led to the formation of HCOOH, NH3, and N2O, but neither HCHO nor 4-NDAB were detected. The results provide strong evidence that initial denitration of cyclic nitramines in water is sufficient to cause ring cleavage followed by spontaneous decomposition to form the final products.     

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-tiazine catalyzed by a NAD(P)H: nitrate oxidoreductase from Aspergillus niger

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) can be efficiently mineralized with anaerobic domestic sludge, but the initial enzymatic processes involved in its transformation are unknown. To test the hypothesis that the initial reaction involves reduction of nitro group(s), we designed experiments to test the ability of a nitrate reductase (EC 1.6.6.2) to catalyze the initial reaction leading to ring cleavage and subsequent decomposition. A nitrate reductase from Aspergillus niger catalyzed the biotransformation of RDX most effectively at pH 7.0 and 30 degrees C under anaerobic conditions using NADPH as electron donor. LC/MS (ES-) chromatograms showed the formation of hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and methylenedinitramine as key initial products of RDX, but neither the dinitroso neither (DNX) nor trinitroso (TNX) derivatives were observed. None of the above detected products persisted, and their disappearance was accompanied by the accumulation of nitrous oxide (N20), formaldehyde (HCHO), and ammonium ion (NH4+). Stoichiometric studies showed that three NADPH molecules were consumed, and one molecule of methylenedinitramine was produced per RDX molecule. The carbon and nitrogen mass balances were 96.14% and 82.10%, respectively. The stoichiometries and mass balance measurements supported a mechanism involving initial transformation of RDX to MNX via a two-electron reduction mechanism. Subsequent reduction of MNX followed by rapid ring cleavage gave methylenedinitramine which in turn decomposed in water to produce quantitatively N2O and HCHO. The results clearly indicate that an initial reduction of a nitro group by nitrate reductase is sufficient for the decomposition of RDX.     

Photodegradation of RDX in aqueous solution: a mechanistic probe for biodegradation with Rhodococcus sp

Recently we demonstrated that Rhodococcus sp. strain DN22 degraded hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (1) aerobically via initial denitration followed by ring cleavage. Using UL 14C-[RDX] and ring labeled 15N-[RDX] approximately 30% of the energetic chemical mineralized (one C atom) and 64% converted to a dead end product that was tentatively identified as 4-nitro-2,4-diaza-butanal (OHCHNCH2NHNO2). To have further insight into the role of initial denitration on RDX decomposition, we photolyzed the energetic chemical at 350 nm and pH 5.5 and monitored the reaction using a combination of analytical techniques. GC/ MS-PCI showed a product with a [M+H] at 176 Da matching a molecular formula of C3H5N5O4 that was tentatively identified as the initially denitrated RDX product pentahydro-3,5-dinitro-1,3,5-triazacyclohex-1-ene (II). LC/MS (ES-) showed that the removal of RDX was accompanied by the formation of two other key products, each showing the same [M-H] at 192 Da matching a molecular formula of C3H7N5O5. The two products were tentatively identified as the carbinol (III) of the enamine (II) and its ring cleavage product O2NNHCH2NNO2CH2NHCHO (IV). Interestingly, the removal of III and IV was accompanied by the formation and accumulation of OHCHNCH2NHNO2 that we detected with strain DN22. At the end of the experiment, which lasted 16 h, we detected the following products HCHO, HCOOH, NH2CHO, N2O, NO2-, and NO3-. Most were also detected during RDX incubation with strain DN22. Finally, we were unable to detect any of RDX nitroso products during both photolysis and incubation with the aerobic bacteria, emphasizing that initial denitration in both cases was responsible for ring cleavage and subsequent decomposition in water.     

Metabolism and mineralization of hexahydro-1,3,5-trinitro-1,3,5-triazine inside poplar tissues (Populus deltoides x nigra DN-34)

Poplar tissue cultures and leaf crude extracts (Populus deltoides x nigra DN-34) were exposed to [U-14C]hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and incubated under light and in the dark. Poplar tissue cultures were able to partially reduce RDX to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), regardless of the presence or absence of light. However, further transformation of RDX, MNX, and DNX required exposure to light and resulted in the formation of formaldehyde (CH2O), methanol (CH3OH), and carbon dioxide (CO2). Similarly, transformation of RDX by poplar leaf crude extracts required exposure to light. Neither reduction of RDX to MNX and DNX nor mineralization into CO2 were recorded in crude extracts, even when exposed to light, suggesting that both processes were light-independent and required intact plant cells. Control experiments without plant material showed that RDX was partially transformed abiotically, by the sole action of light, but to a lesser extent than in the presence of plant crude extracts, suggesting the intervention of plant subcellular structures through a light-mediated mechanism. Poplar tissue cultures were also shown to mineralize 14CH2O and 14CH3OH, regardless of the presence or absence of light. These results suggest that transformation of [U-14C]RDX by plant tissue cultures may occur through a three-step process, involving (i) a light-independent reduction of RDX to MNX and DNX by intact plant cells; (ii) a plant/light-mediated breakdown of the heterocyclic ring of RDX, MNX, or DNX into C1-labeled metabolites (CH2O and CH3OH); and (iii) a further light-independent mineralization of C1-labeled metabolites by intact plant cells. This is the first time that a significant mineralization of RDX into CO2 by light-exposed plant tissue cultures is reported.