Environ. Sci. Technol. 2002, 36, 633-638
Insights into the Formation and Degradation Mechanisms of Methylenedinitramine during the Incubation of RDX with Anaerobic Sludge ANNAMARIA HALASZ,† JIM SPAIN,‡ LOUISE PAQUET,† CHANTALE BEAULIEU,† AND J A L A L H A W A R I * ,† Biotechnology Research Institute, National Research Council of Canada, Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2, and U.S. Air Force Laboratory, 139 Barnes Drive, Tyndall Air Force Base, Florida 32403
In an earlier study, we reported that hexahydro-1,3,5trinitro-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 N2O 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.
Introduction Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro1,3,5,7-tetranitro-1,3,5,7-tetrazine (HMX) are highly energetic * Corresponding author telephone: (514)496-6267; fax: (514)4966265; e-mail:
[email protected]. † National Research Council of Canada. ‡ U.S. Air Force Laboratory. 10.1021/es011071g CCC: $22.00 Published on Web 01/12/2002
2002 American Chemical Society
chemicals and are used in various propellants and explosives. Several reports describe the thermal decomposition of RDX and HMX with the subsequent formation of the end products N2, N2O, NO2, HCHO, CO, and CO2 (1, 2). RDX and HMX are toxic (3-5), and their use has resulted in soil and groundwater contamination. Recently, several studies demonstrated that biodegradation of RDX and HMX is possible under both aerobic and anaerobic conditions, but little information is available on their initial and intermediate products and their biodegradation mechanisms (6-12). Understanding the intermediate products and the underlying mechanisms of their formation is essential to allow prediction and enhancement of the complete biodegradation of the two explosives. McCormick et al. (6) studied the biodegradation of RDX in anaerobic sludge and proposed a pathway based on the sequential reduction to hexahydro-1-nitroso-3,5-dinitro1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). Recently, we reported that in addition to the above nitroso products both RDX and HMX can undergo direct ring cleavage in domestic sludge to eventually produce nitrous oxide (N2O) and formaldehyde (HCHO). The latter biodegraded further to carbon dioxide (11, 12). An intermediate of RDX incubation with anaerobic sludge was tentatively identified as methylenedinitramine, O2NNHCH2NHNO2, based on LC/MS (ES-) analysis. Earlier studies suggested the presence of methylenedinitramine as an intermediate, often as a biphosphate salt, during the thermal and photochemical degradation of RDX (13, 14). Because of the significant role of methylenedinitramine in the biodegradation of RDX and HMX, we recently obtained this chemical and used it as a reference standard to confirm the identity of the suspected RDX intermediate. Also, we studied biodegradation of RDX in the presence and absence of D2O in an attempt to gain insight about the route(s) leading to the formation of methylenedinitramine. The decomposition of methylenedinitramine in water in the presence and absence of sludge was also investigated to help understand its role on the complete degradation of RDX to N2O and CO2.
Experimental Section Materials. Commercial grade RDX (with a purity >99%) was provided by Defense Research Establishment Valcartier, Quebec, Canada. Mono- and trinitroso-RDX were synthesized according to the methods described by Brockman et al. (15). Deuterium oxide (99.9%) and [14C]-HCHO (53 mCi/mmol) were purchased from Aldrich, Canada. We obtained a sample of methylenedinitramine from the Rare Chemical Department of Aldrich, Canada. All other chemicals were reagent grades. Anaerobic sludge was obtained from the biological waste treatment section of a nutrient factory (Sensient Flavors Canada) located at Cornwall, ON, Canada. The sludge was always obtained fresh and stored at 4 °C when not in use. The viability of the sludge was measured using a glucose activity test. On average, the biomass concentration of the sludge was 3.6 g/L dry weight with -20 mV reduction potential (Eh) before incubation. BBL dry anaerobic indicator (VWR, Canlab, ON, Canada) was frequently used to detect air leaks and to ensure anaerobic conditions. Biotransformation and Mineralization Study. Biodegradation experiments were prepared in serum bottles (100 mL) containing anaerobic sludge (5 mL) and a 45-mL mineral salt medium containing NaH2PO4 (0.15 g/L), K2HPO4 (0.45 g/L), Na2SO4 (0.24 g/L), and glucose (2.1 g/L) following the procedure described earlier (11). The microcosms were supplemented with either RDX (200 mg/L) taken from VOL. 36, NO. 4, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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acetone stock solution (40 g/L) or methylenedinitramine (20 mg/L) taken from water stock solution (200 mg/L). In the case of acetone, the organic solvent was removed by evaporation prior to the addition of the aqueous medium. We added RDX in excess of its water solubility (ca. 60 mg/L) to generate sufficient amounts of metabolites for detection. To account for insoluble or suspended portion of RDX (water solubility is ca. 60 mg/L; 5), all the content of the microcosm was extracted in acetonitrile. In experiments with deuterated water, we added sludge (1.5 mL) and diluted it with the proper volume of D2O to produce different D2O/H2O solutions (90, 50, and 0% v/v). The incubation mixtures were each supplemented with RDX as described above so that the final concentration stayed at 200 mg/L. Total volume of the incubation mixture was kept at 15 mL to keep the final concentration of sludge constant at 10% v/v throughout the study. We omitted glucose from the experiments with deuterium to allow monitoring of the formation of methylenedinitramine without the interference of volatile fatty acid metabolites. The headspace in each microcosm was flushed with either nitrogen or argon gas to maintain anaerobic conditions and then sealed with butyl rubber septa and aluminum crimp seals. Two control microcosms were prepared in D2O, both in the absence of sludge; one was supplemented with RDX, and the other was supplemented with methylenedinitramine. The stability of methylenedinitramine and its spontaneous decomposition in water and in the sludge was determined by dissolving a predetermined weight (20 mg/L) of the amine in H2O in one case and in the sludge in the second case, followed by monitoring its concentration and the concentration of its products (HCHO and N2O) as discussed below. Analytical Methods. RDX and its metabolites including the nitroso derivatives (MNX, DNX and TNX) and methylenedinitramine were measured by using the procedure described by Hawari et al. (11). A Micromass Platform benchtop single-quadrupole mass detector connected to a Hewlett-Packard 1100 series HPLC system equipped with a photodiode array detector was used. Briefly, samples (1 mL) from the above-treated culture medium were filtered through a 0.45-mm pore-size Millex-HV filter before analysis. A Synergi Polar-RP column (15 cm × 4.6 mm, particle size 4 mm) was used for separation with a solvent system consisting of a gradient of methanol/water in HCOOH (200 mM) at a flow rate of 0.75 mL/min at 25 °C. An initial MeOH-H2O composition of 10% v/v was maintained for 10 min, which then changed to 90% v/v over 10 min and was kept at that composition for 5 min. The composition of the eluent was changed to its original value (10% v/v) over a period of 10 min. Detection of RDX, its nitroso derivatives, and methylenedinitramine was done with a photodiode array detector at 230 nm. For mass analysis, ionization was done in a negative electrospray ionization mode ES(-), producing mainly the deprotonated mass ions [M - H]. In deuteriumlabeled experiments, especially biodegradation of RDX in D2O, we also searched for [M - D] mass ions. The electrospray probe tip potential was set at 3.5 kV with a skimmer voltage of 30 V at an ion source temperature of 150 °C. The mass range was scanned from 40 to 400 Da with a cycle time of 1.6 s, and the resolution was set to 1 Da (width at half-height). The analysis of formaldehyde (HCHO) was determined as described by Summers (16) with slight modification. HCHO was first derivatized with 2,4-pentanedione in the presence of ammonium acetate and glacial acetic acid at pH 6 at 40 °C for 1 h. Then the derivative was analyzed by HPLC using a Supelcosil LC-8 column (25 cm × 4.6 mm, 5 mm) maintained at 40 °C. An acetonitrile gradient of 15-27% was performed at a flow rate of 1.5 mL/ min for 6 min. The derivative was detected and quantified by excitation at 430 nm and emission at 520 nm (detection limit 25 ppb). N2O 634
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and N2 were determined using an SRI 8610 GC (INSUS Systems Inc.) connected to a Supelco Porapack Q column (2 m) and coupled with a electron capture detector (ECD) (330 °C). The gaseous products from the headspace of the microcosms (prepared under a blanket of argon) of the culture medium were sampled using a gastight syringe for subsequent injection inside the GC using helium as a carrier gas (21 mL/min) at 60 °C.
Results and Discussion Cleavage of RDX with Sludge and the Production of Methylenedinitramine. Previously, we reported the tentative identification of methylenedinitramine as a ring cleavage product from the incubation of both RDX (11) and HMX (12) with domestic anaerobic sludge. In the present study, we obtained a standard sample of methylenedinitramine and discovered that the major initial product obtained during the anaerobic biodegradation of both cyclic nitramine explosives RDX and HMX is identical to the standard as determined by its deprotonated molecular mass ions [M H] and chromatographic retention time (rt). Figure 1A is a typical LC/MS (ES-) spectrum of the methylenedinitramine metabolite obtained after incubating RDX in the anaerobic sludge for 20 h at pH 7.0, whereas Figure 1B is the LC/MS (ES-) spectrum of a standard of methylenedinitramine. Both the product from RDX biodegradation (Figure 1A) and the standard (Figure 1 B) showed their [M - 1] and rt at 135 Da and 4.3 min, respectively. The present results confirmed our hypothesis that methylenedinitramine is an important ring cleavage intermediate of the explosive RDX under reducing conditions. We found that methylenedinitramine itself is unstable in water and decomposed spontaneously to give N2O and HCHO in the presence and absence of sludge (Figure 2a,b). The main difference between the decomposition of methylenedinitramine in the absence and presence of sludge is the conversion of HCHO to CO2 in the latter case. Interestingly, we found that methylenedinitramine (7.5 µmol) decomposed much faster in deionized water alone (>90%) than in the incubation mixture with sludge (
