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).     

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.     

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.     

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.     

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 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.     

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.     

Biodegradation of RDX and MNX with Rhodococcus sp. strain DN22: new insights into the degradation pathway

Previously we demonstrated that Rhodococcus sp. strain DN22 can degrade RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) aerobically via initial denitration. The present study describes the role of oxygen and water in the key denitration step leading to RDX decomposition using (18)O(2) and H(2)(18)O labeling experiments. We also investigated degradation of MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine) with DN22 under similar conditions. DN22 degraded RDX and MNX giving NO(2)(-), NO(3)(-), NDAB (4-nitro-diazabutanal), NH(3), N(2)O, and HCHO with NO(2)(-)/NO(3)(-) molar ratio reaching 17 and ca. 2, respectively. In the presence of (18)O(2), DN22 degraded RDX and produced NO(2)(-) with m/z at 46 Da that subsequently oxidized to NO(3)(-) containing one (18)O atom, but in the presence of H(2)(18)O we detected NO(3)(-) without (18)O. A control containing NO(2)(-), DN22, and (18)O(2) gave NO(3)(-) with one (18)O, confirming biotic oxidation of NO(2)(-) to NO(3)(-). Treatment of MNX with DN22 and (18)O(2) produced NO(3)(-) with two mass ions, one (66 Da) incorporating two (18)O atoms and another (64 Da) incorporating only one (18)O atom and we attributed their formation to bio-oxidation of the initially formed NO and NO(2)(-), respectively. In the presence of H(2)(18)O we detected NO(2)(-) with two different masses, one representing NO(2)(-) (46 Da) and another representing NO(2)(-) (48 Da) with the inclusion of one (18)O atom suggesting auto-oxidation of NO to NO(2)(-). Results indicated that denitration of either RDX or MNX and denitrosation of MNX by DN22 did not involve direct participation of either oxygen or water, but both played major roles in subsequent secondary chemical and biochemical reactions of NO and NO(2)(-).     

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.     

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.     

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".     

Transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by permanganate

The chemical oxidant permanganate (MnO(4)(-)) has been shown to effectively transform hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) at both the laboratory and field scales. We treated RDX with MnO(4)(-) with the objective of quantifying the effects of pH and temperature on destruction kinetics and determining reaction rates. A nitrogen mass balance and the distribution of reaction products were used to provide insight into reaction mechanisms. Kinetic experiments (at pH ～ 7, 25 °C) verified that RDX-MnO(4)(-) reaction was first-order with respect to MnO(4)(-) and initial RDX concentration (second-order rate: 4.2 × 10(-5) M(-1) s(-1)). Batch experiments showed that choice of quenching agents (MnSO(4), MnCO(3), and H(2)O(2)) influenced sample pH and product distribution. When MnCO(3) was used as a quenching agent, the pH of the RDX-MnO(4)(-) solution was relatively unchanged and N(2)O and NO(3)(-) constituted 94% of the N-containing products after 80% of the RDX was transformed. On the basis of the preponderance of N(2)O produced under neutral pH (molar ratio N(2)O/NO(3) ～ 5:1), no strong pH effect on RDX-MnO(4)(-) reaction rates, a lower activation energy than the hydrolysis pathway, and previous literature on MnO(4)(-) oxidation of amines, we propose that RDX-MnO(4)(-) reaction involves direct oxidation of the methylene group (hydride abstraction), followed by hydrolysis of the resulting imides, and decarboxylation of the resulting carboxylic acids to form N(2)O, CO(2), and H(2)O.     

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.     

Abiotic degradation of hexahydro-l,3,5-trinitro-1,3,5-triazine in the presence of hydrogen sulfide and black carbon.

We report that hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was rapidly destroyed by sulfides in the presence of black carbon, forming nitrite and formaldehyde, rather than toxic nitrosated reduction products. Although traditionally viewed as inactive sorbents, black carbons have been noted to participate in the destruction of certain contaminants, such as azo dyes, via quinonoid groups. However, in our experiments sulfide modification of quinones did not seem to be involved. Although at least 1.2 mM sulfides were needed for the reaction to proceed, abiotic natural attenuation of RDX in marine sediments may occur, because these concentrations are found in certain marine sediments, together with black carbon. In the absence of natural black carbons, synthetic black carbons, such as activated carbon, may be added to sediments. As compared with other in situ techniques, such as bioremediation and zero-valent iron cutoff trenches, which often generate nitrosated byproducts, this in situ, abiotic technique may be an attractive alternative.     

Biotransformation routes of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine by municipal anaerobic sludge

Recently we demonstrated that hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a trimer of methylene nitramine (CH2=N-NO2) undergoes spontaneous decomposition following an initial microbial attack using a mixed microbial culture at pH 7 in the presence of glucose as carbon source. The present study describes whether the second cyclic nitramine octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), a more strained tetramer of CH2=N-NO2, degrades similarly using sludge of the same source. Part of HMX biotransformed to give products that are tentatively identified as the nitroso derivatives octahydro-1-nitroso-3,5,7-trinitro-1,3,5,7-tetrazocine (mNs-HMX) and octahydro-1,3-dinitroso-5,7-dinitro-1,3,5,7-tetrazocine and its isomer octahydro-1,5-dinitroso-3,7-dinitro-1,3,5,7-tetrazocine (dNs-HMX). Another fraction of HMX biotransformed, apparently via ring cleavage, to produce products that are tentatively identified as methylenedinitramine (O2NNHCH2-NHNO2) and bis(hydroxymethyl)nitramine ((HOCH2)2NNO2). None of the above intermediates accumulated indefinitely; they disappeared to predominantly form nitrous oxide (N2O) and formaldehyde (HCHO). Formaldehyde biotransformed further to eventually produce carbon dioxide (14CO2). Nitrous oxide persisted in HMX microcosms containing glucose but denitrified rapidly to nitrogen in the absence of glucose. The presence of nitrous oxide was accompanied by the presence of appreciable amounts of hydrogen sulfide, a known inhibitor of denitrification.     

Hexahydro-1,3,5-trinitro-1,3,5-triazine transformation by biologically reduced ferrihydrite: evolution of Fe mineralogy, surface area, and reaction rates

Microbial respiration of Fe(III) oxides has been shown to produce reduced Fe phases that are capable of transforming a variety of oxidized contaminants. Little data, however, are available on how these Fe phases evolve over time and how this evolution may affect their ability to reduce contaminants. Here,the evolution and reactivity of biologically reduced ferrihydrite were monitored over a period of 14 months. Solids were collected from a culture of Geobacter metallireducens (GS-15) thatwas incubated with ferrihydrite (as the electron acceptor) for 0, 7, 10, 20, 75, and 400 days. Mineralogical composition and surface area of the biologically reduced solids were characterized using M?ssbauer spectroscopy, X-ray diffraction, and BET with N2 adsorption. By day 10, ferrihydrite began to transform, and a nanoparticle magnetite/maghemite phase, as well as two ferrous phases, was observed. One of the ferrous phases was identified as siderite, whereas the other could not be positively identified. Likely candidates, however, include Fe(OH)2(s) or an adsorbed Fe(II) species. Over the next few months, ferrihydrite was completely reduced and evolved into a mixture containing about 70% magnetite/maghemite, 19% siderite, and 11% of the second Fe(II) phase. The effect of incubation time on the reactivity of the biologically reduced solids was evaluated by measuring the kinetics of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) transformation. The only products observed were the three reduced nitroso products. Rate coefficients (k) for RDX transformation were dramatically influenced by incubation time with half-lives of about 1 month observed in the presence of solids incubated for 10 and 20 days, 3 months with solids incubated for 75 days, and negligible removal with solids incubated for 400 days. The loss of reactivity was not directly correlated to any one mineralogical variable but may be due to particle size or surface chemistry changes in the reactive Fe phase or to cell die-off and the accumulation of cell lysis products after consumption of the electron acceptor. The dramatic effect of incubation time on the rate of RDX removal highlights a potential limitation of studying complex systems, as we have here, in batch reactors and suggests that incubation time is an important variable to consider when measuring and comparing rates of contaminant reduction.     

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.     

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.     

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.