Accumulation of Insensitive Munition Compounds in the Earthworm Eisenia andrei from Amended Soil: Methodological Considerations for Determination of Bioaccumulation Factors.

The present study investigates the bioaccumulation of the insensitive munition compounds 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO), developed for future weapons systems to replace current munitions containing sensitive explosives. The earthworm Eisenia andrei was exposed to sublethal concentrations of DNAN or NTO amended in Sassafras sandy loam. Chemical analysis indicated that 2- and 4-amino-nitroanisole (2-ANAN and 4-ANAN, respectively) were formed in DNAN-amended soils. The SumDNAN (sum of DNAN, 2-ANAN, and 4-ANAN concentrations) in soil decreased by 40% during the 14-d exposure period. The SumDNAN in the earthworm body residue increased until day 3 and decreased thereafter. Between days 3 and 14, there was a 73% decrease in tissue uptake that was greater than the 23% decrease in the soil concentration, suggesting that the bioavailable fraction may have decreased over time. By day 14, the DNAN concentration accounted for only 45% of the SumDNAN soil concentration, indicating substantial DNAN transformation in the presence of earthworms. The highest bioaccumulation factor (BAF; the tissue-to-soil concentration ratio) was 6.2 ± 1.0 kg/kg (dry wt) on day 3 and decreased to 3.8 ± 0.8 kg/kg by day 14. Kinetic studies indicated a BAF of 2.3 kg/kg, based on the earthworm DNAN uptake rate of 2.0 ± 0.24 kg/kg/d, compared with the SumDNAN elimination rate of 0.87 d-1 (half-life = 0.79 d). The compound DNAN has a similar potential to bioaccumulate from soil compared with trinitrotoluene. The NTO concentration in amended soil decreased by 57% from the initial concentration (837 mg NTO/kg dry soil) during 14 d, likely due to the formation of unknown transformation products. The bioaccumulation of NTO was negligible (BAF ≤ 0.018 kg/kg dry wt). Environ Toxicol Chem 2021;40:1713-1725. © 2021 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Environmental fate of 2,4-dinitroanisole (DNAN) and its reduced products.

Several defense departments intend to replace 2,4,6-trinitrotoluene (TNT) in munitions formulations by the less sensitive 2,4-dinitroanisole (DNAN). To help understand environmental behavior and ecological risk associated with DNAN we investigated its key initial abiotic and biotic reaction routes and determined relevant physicochemical parameters (pKa, logKow, aqueous solubility (Sw), partition coefficient (Kd)) for the chemical and its products. Reduction of DNAN with either zero valent iron or bacteria regioselectively produced 2-amino-4-nitroanisole (2-ANAN) which, under strict anaerobic conditions, gave 2,4-diaminoanisole (DAAN). Hydrolysis under environmental conditions was insignificant whereas photolysis gave photodegradable intermediates 2-hydroxy-4-nitroanisole and 2,4-dinitrophenol. Physicochemical properties of DNAN and its amino products drastically depended on the type and position of substituent(s) on the aromatic ring. Sw followed the order (TNT

Microaerophilic degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by three Rhodococcus strains.

AIM: The goal of this study was to compare the degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by three Rhodococcus strains under anaerobic, microaerophilic (<0.04 mg l(-1) dissolved oxygen) and aerobic (dissolved oxygen (DO) maintained at 8 mg l(-1)) conditions. METHODS AND RESULTS: Three Rhodococcus strains were incubated with no, low and ambient concentrations of oxygen in minimal media with succinate as the carbon source and RDX as the sole nitrogen source. RDX and RDX metabolite concentrations were measured over time. Under microaerophilic conditions, the bacteria degraded RDX, albeit about 60-fold slower than under fully aerobic conditions. Only the breakdown product, 4-nitro-2,4-diazabutanal (NDAB) accumulated to measurable concentrations under microaerophilic conditions. RDX degraded quickly under both aerated and static aerobic conditions (DO allowed to drop below 1 mg l(-1)) with the accumulation of both NDAB and methylenedinitramine (MEDINA). No RDX degradation was observed under strict anaerobic conditions. CONCLUSIONS: The Rhodococcus strains did not degrade RDX under strict anaerobic conditions, while slow degradation was observed under microaerophilic conditions. The RDX metabolite NDAB was detected under both microaerophilic and aerobic conditions, while MEDINA was detected only under aerobic conditions. IMPACT AND SIGNIFICANCE OF THE STUDY: This work confirmed the production of MEDINA under aerobic conditions, which has not been previously associated with aerobic RDX degradation by these organisms. More importantly, it demonstrated that aerobic rhodococci are able to degrade RDX under a broader range of oxygen concentrations than previously reported.

Effect of 2,4-dinitrotoluene on the anaerobic bacterial community in marine sediment.

AIMS: To study the impact of added 2,4-dinitrotoluene (DNT) on the anaerobic bacterial community in marine sediment collected from an unexploded ordnance dumping site in Halifax Harbour. METHODS AND RESULTS: Marine sediment was spiked with 2,4-DNT and incubated under anaerobic conditions in the presence and absence of lactate. Indigenous bacteria in the sediment removed 2,4-DNT with subsequent formation of its mono- and diamino-derivatives under both conditions. PCR-DGGE and nucleotide sequencing were used to monitor the change in the bacterial population in sediment caused by the presence of 2,4-DNT. The results showed that denaturing gradient gel electrophoresis banding patterns of sediment microcosms treated with 2,4-DNT were different from controls that did not receive 2,4-DNT. Bacteroidetes, Firmicutes and delta-Proteobacteria were present in sediment incubated in the absence of 2,4-DNT. However, several gamma-Proteobacteria became dominant in sediment in the presence of 2,4-DNT, two of which were 99% similar to Shewanella canadensis and Shewanella sediminis. In the presence of both 2,4-DNT and lactate, two additional delta-Proteobacteria were enriched, one closely related (98% similarity) to Desulfofrigus fragile and the other affiliated (96% similarity) to Desulfovibrio sp. In contrast, none of the above four Proteobacteria were enriched in sediment incubated with lactate alone. CONCLUSIONS: Presence of 2,4-DNT led to a significant change in bacterial population of marine sediment with the enrichment of several gamma- and delta-Proteobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provided the first evidence on the impact of the pollutant 2,4-DNT on the indigenous bacterial community in marine sediment, and provided an insight into the composition of bacterial community that degrade 2,4-DNT.

Maple sap as a rich medium to grow probiotic lactobacilli and to produce lactic acid.

AIMS: To demonstrate the feasibility of growing lactobacilli and producing lactic acid using maple sap as a sugar source and to show the importance of oligosaccharides in the processes. METHODS AND RESULTS: Two maple sap samples (Cetta and Pinnacle) and purified sucrose were used as carbon sources in the preparation of three culture media. Compared with the sucrose-based medium, both maple sap-based media produced increased viable counts in two strains out of five by a factor of four to seven. Maple sap-based media also enhanced lactic acid production in three strains. Cetta sap was found to be more efficient than Pinnacle sap in stimulating lactic acid production and, was also found to be richer in various oligosaccharides. The amendment of the Pinnacle-based medium with trisaccharides significantly stimulated Lactobacillus acidophilus AC-10 to grow and produce lactic acid. CONCLUSIONS: Maple sap, particularly if rich in oligosaccharides, represents a good carbon source for the growth of lactobacilli and the production of lactic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a proof-of-concept, using maple sap as a substrate for lactic acid production and for the development of a nondairy probiotic drink.

Production of polyhydroxyalkanoates from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2.

A new bacterial strain, isolated from groundwater contaminated with explosives, was characterized as a pink-pigmented facultative methylotroph, affiliated to the genus Methylobacterium. The bacterial isolate designated as strain GW2 was found capable of producing the homopolymer poly-3-hydroxybutyrate (PHB) from various carbon sources such as methanol, ethanol, and succinate. Methanol acted as the best substrate for the production of PHB reaching 40 % w/w dry biomass. PHB accumulation was observed to be a growth-associated process, so that there was no need for two-step fermentation. Optimal growth occurred at 0.5 % (v/v) methanol concentration, and growth was strongly inhibited at alpha concentration above 2 % (v/v). Methylobacterium sp. strain GW2 was also able to accumulate the copolyester poly-3-hydroxybutyrate-poly-3-hydroxyvalerate (PHB/HV) when valeric acid was supplied as an auxiliary carbon source to methanol. After 66 h, a copolymer content of 30 % (w/w) was achieved with a PHB to PHV ratio of 1:2. Biopolymers produced by strain GW2 had an average molecular weight ranging from 229,350 to 233,050 Da for homopolymer PHB and from 362,430 to 411,300 Da for the copolymer PHB/HV.

Effect of iron(III), humic acids and anthraquinone-2,6-disulfonate on biodegradation of cyclic nitramines by Clostridium sp. EDB2.

AIMS: To determine the biodegradation of cyclic nitramines by an anaerobic marine bacterium, Clostridium sp. EDB2, in the presence of Fe(III), humic acids (HA) and anthraquinone-2,6-disulfonate (AQDS). METHODS AND RESULTS: An obligate anaerobic bacterium, Clostridium sp. EDB2, degraded RDX and HMX, and produced similar product distribution including nitrite, methylenedinitramine, nitrous oxide, ammonium, formaldehyde, formic acid and carbon dioxide. Carbon (C) and nitrogen (N) mass balance for RDX products were 87% and 82%, respectively, and for HMX were 88% and 74%, respectively. Bacterial growth and biodegradation of RDX and HMX were stimulated in the presence of Fe(III), HA and AQDS suggesting that strain EDB2 utilized Fe(III), HA and AQDS as redox mediators to transfer electrons to cyclic nitramines. CONCLUSIONS: Strain EDB2 demonstrated a multidimensional approach to degrade RDX and HMX: first, direct degradation of the chemicals; second, indirect degradation by reducing Fe(III) to produce reactive-Fe(II); third, indirect degradation by reducing HA and AQDS which act as electron shuttles to transfer electrons to the cyclic nitramines. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study could be helpful in determining the fate of cyclic nitramine energetic chemicals in the environments rich in Fe(III) and HA.

Effects of dietary administration of CL-20 on Japanese quail Coturnix coturnix japonica.

Hexanitrohexaazaisowurtzitane, or CL-20, is an emerging highly energetic compound currently under consideration for military applications. With the anticipated wide use of CL-20, there is the potential for soil and groundwater contamination resulting in adverse toxicologic effects on environmental receptors. Presently, there is a lack of data describing the toxic effects of CL-20 on avian species. The present study describes the effect of CL-20 on Japanese quail (Coturnix coturnix japonica) modified from standard toxicity test guidelines. First, a 14-day subacute assay was adopted using repeated gavage doses (0, 307, 964, 2439, 3475, or 5304 mg CL-20/kg body weight (BW)/d for 5 days followed by no CL-20 exposure (vehicle only) for 10 days. Second, a subchronic feeding assay (0, 11, 114, or 1085 mg CL-20/kg feed) was done for 42 days. During both studies, no overt toxicity was observed in the CL-20-treated birds. During the first 5 days of the subacute study, CL-20-exposed birds showed a dose-dependent decrease in BW gain, whereas increased liver weight, plasma sodium, and creatinine levels were observed in birds receiving the highest dose tested. For the subchronic study, embryo weights were significantly decreased in a dose-dependent manner. Embryos from CL-20-exposed birds were observed to have multiple cranial and facial deformities, beak curvatures, possible mid-brain enlargement, and classic one-sided development with micro-opthalamia (nonstatistical comparisons with control embryos). A trend toward decreased number of eggs laid per female bird was also observed. We conclude that CL-20 (or its degradation products) elicits few effects in adults but may affect avian development, although these preliminary findings should be confirmed.

Assessment of a 2,4,6-trinitrotoluene-contaminated site using Aporrectodea rosea and Eisenia andrei in mesocosms.

Polynitro-organic compounds such as 2,4,6-trinitrotoluene (TNT) can be released into the environment from production and processing facilities and military firing ranges as well as through field use and disposal practices. Based on laboratory toxicity data, TNT has lethal (at >/=260 mg TNT/kg dry soil) and sublethal effects (at >/=59 mg TNT/kg dry soil) to the earthworm. However, field studies are needed to relate exposure of organisms to explosives in mixed-contaminated soil under field conditions and to define effects-based ecotoxicologic benchmarks for TNT-contaminated soil. In the present study, the lethal and sublethal effects of a 10-day in situ exposure at a TNT-contaminated field site using mesh-bag mesocosms were assessed. In addition to the survival end point, the biomarkers of earthworm exposure and effect-including tissue residues, lysosomal neutral red retention time (NRRT), and total immune activity (TIA)-were measured. Concentrations of TNT in soil mesocosms ranged from 25 to 17,063 mg/kg. Experiments indicated a trend toward decreasing survival of caged Aporrectodea rosea and Eisenia andrei as the concentration of TNT and total nitroaromatic compounds increased. E. andrei tolerated higher concentrations of TNT (up to 4050 mg/kg dry soil) in mesocosms than did indigenous earthworms, who survived only at

Biosorption of heavy metals by red algae (Palmaria palmata).

The biosorption of heavy metals from aqueous solutions was investigated, using a cheap and abundant dry biomass of red algae P. palmata. The Freundlich, Langmuir and Brunauer Emmer and Teller (BET) models were used to describe the uptake of lead (pb2+), copper (Cu2+), nickel (Ni2+), cadmium (Cd 2+) and zinc (Zn2+) on P. palmata. The good fits of the Langmuir and BET models to the experimental data reflected that the sorption on P. palmata was a multi-layer sorption, in which a Langmuir equation could be applied to each layer. The highest maximum sorption capacity q(max), derived from the Langmuir model was 15.17 mg g(-1) for lead and 6.65 mg g(-1) for copper (dry weight metal/dry weight biosorbent) at a pH of 5.5-6. The affinity of metals for P. palmata was found to decrease in the order: Pb2+ > Cd2+ > Cu2+ > Ni2+. The factors influencing copper and lead uptake were found to be contact time, pH, initial concentration and temperature. Biosorption of copper and lead was a rapid process, with 70% and 100% of the respective uptakes occurring within the first 10 minutes.

Metabolism of hexahydro-1,3,5-trinitro-1,3,5-triazine through initial reduction to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine followed by denitration in Clostridium bifermentans HAW-1.

A fast hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-degrading [28.1 micromol h(-1) g (dry weight) cells(-1); biomass, 0.16 g (dry weight) cells(-1)] and strictly anaerobic bacterial strain, HAW-1, was isolated and identified as Clostridium bifermentans using a 16S-rRNA-based method. Based on initial rates, strain HAW-1 transformed RDX to 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) with yields of 56, 7.3 and 0.2%, respectively. Complete removal of RDX and its nitroso metabolites produced (%, of total C or N) methanol (MeOH, 23%), formaldehyde (HCHO, 7.4%), carbon dioxide (CO2, 3.0%) and nitrous oxide (N2O, 29.5%) as end products. Under the same conditions, strain HAW-1 transformed MNX separately at a rate of 16.9 micromol h(-1) g (dry weight) cells(-1) and produced DNX (25%) and TNX (0.4%) as transient products. Final MNX transformation products were (%, of total C or N) MeOH (21%), HCHO (2.9%), and N2O (17%). Likewise strain HAW-1 degraded TNX at a rate of 7.5 micromol h(-1) g (dry weight) cells(-1 )to MeOH and HCHO. Furthermore, removal of both RDX and MNX produced nitrite (NO2-) as a transient product, but the nitrite release rate from MNX was quicker than from RDX. Thus, the predominant pathway for RDX degradation is based on initial reduction to MNX followed by denitration and decomposition. The continued sequential reduction to DNX and TNX is only a minor route.

Phytotoxicity of 2,4,6-trinitrotoluene (TNT) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in spiked artificial and natural forest soils.

Toxicity of 2,4,6-trinitrotoluene (TNT) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) using two terrestrial plant species, lettuce (Lactuca sativa) and barley (Hordeum vugare), was assessed in artificial soil (silica) and forest soil. Lettuce emergence was significantly decreased after 5 days of exposure to TNT nominal spiked concentrations >/= 1,040 mg/kg dry soil in silica. Barley emergence was significantly reduced after 14 days of exposure at initial (t = 0) TNT concentrations >/= 55.9 +/- 4.5 mg/kg dry soil in silica and at >/= 291.9 +/- 42.8 mg/kg dry forest soil. Biomasses of shoot and roots of barley seeds were significantly reduced after 14 days of exposure at TNT initial exposure concentrations >/= 55.9 +/- 4.5 (LOEC) mg/kg dry soil in silica. Results were similar with the forest soil (LOEC = 91.4 +/- 7.9 mg TNT/kg dry soil) using the root growth parameter, but the shoot biomass was reduced only at concentrations >/= 291.9 +/- 42.8 mg TNT/kg dry soil. Plants were not affected by an HMX exposure up to 3,320 +/- 1,019 mg/kg dry soil using silica or 1,866 +/- 438 mg/kg dry soil using a forest soil. During the 14-day experiments, TNT was partially transformed in the spiked soil samples, as indicated by the presence of its amino metabolites (2-ADNT and 4-ADNT). Higher quantities of metabolites were detected in forest soils having higher initial TNT concentrations (

TNT, RDX, and HMX decrease earthworm (Eisenia andrei) life-cycle responses in a spiked natural forest soil.

Sublethal and chronic toxicities of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) on earthworm Eisenia andrei in a sandy forest soil were assessed. Various reproduction parameters of fecundity (total and hatched number of cocoons, number of juveniles, and their biomass) were significantly decreased by TNT (> or = 58.8 +/- 5.1 mg/kg dry soil), RDX (> or = 46.7 +/- 2.6 mg/kg), and HMX (> or = 15.6 +/- 4.6 mg/kg). These effects occurred at much lower concentrations than those reported earlier using artificial soil preparations. Growth of adults was significantly decreased in the TNT-spiked natural soils at 136.2 +/- 25.6 mg/kg dry soil, the highest concentration having no significant mortality. In contrast, survival and growth were not significantly reduced at relatively high measured concentrations of RDX (167.3 mg/kg) and HMX (711.0 mg/kg). Although TNT, RDX, and HMX share a common life-cycle response ( i.e., decreased juvenile counts), a number of differences related to other reproduction parameters (e.g., productivity of cocoons) was observed. These results indicate that the tested explosives do not support a common mechanism of toxicity, at least in the earthworm, probably due to differences in their physical-chemical properties as well as metabolites formed during exposure.

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.

Biotransformation and partial mineralization of the explosive 2,4,6-trinitrotoluene (TNT) by rhizobia.

Three strains, T10, B5, and M8, each belonging to a different species of the family Rhizobiaceae and isolated from atrazine-contaminated soils, were tested for their ability to transform 2,4,6-trinitrotoluene (TNT) (50 microg x mL(-1)) in liquid cultures using glucose as the C-source. All three strains were able to transform TNT to hydroxylaminodinitrotoluenes (2-HADNT, 4-HADNT), aminodinitrotoluenes (2-ADNT, 4-ADNT), and diaminonitrotoluene (2,4-DANT). The transformation was significantly faster in the presence of glutamate. Furthermore, the major metabolites that accumulated in cultures were 2-ADNT with glucose, and 4-ADNT with glutamate plus glucose. Rhizobium trifolii T10 was also tested for its ability to transform high levels of TNT (approximately 350 microg x mL(-1)) in a soil slurry. Almost 60% of the TNT was transformed within 2 days in bioaugmented soil slurries, and up to 90% when cultures were supplemented with glucose and glutamate. However, mineralization was minimal in all cases, less than 2% in 78 days. This is the first report on the degradation of TNT by rhizobial strains, and our findings suggest that rhizobia have the potential to play an important role in the safe decontamination of soils and sites contaminated with TNT if bioaugmentation with rhizobia is shown to have no ecotoxicological consequence.

Ecotoxicological effects of hexahydro-1,3,5-trinitro-1,3,5-triazine on soil microbial activities.

Although hexahydro-1,3,5-trinitro-1,3,5-triazine (also called RDX or hexogen) is a potentially toxic explosive compound that persists in soil, its ecotoxicological effects on soil organisms have rarely been assessed. In this study, two uncontaminated garden soils were spiked with 10 to 12,500 mg RDX/kg dry soil. Soil microbial activities, i.e., potential nitrification, nitrogen fixation, dehydrogenase, basal respiration, and substrate-induced respiration were chosen as bioindicators and were determined after 1-, 4-, and 12-weeks of exposure. Experimental results indicate that RDX showed significant inhibition (up to 36% of control) on indigenous soil microbial communities over the period of this study. All five bioindicators responded similarly to the RDX challenge. The length of exposure also affected the microbial toxicity of RDX, with 12-week exposure exerting more significant effects than the shorter exposure periods, suggesting that soil microorganisms might become more vulnerable to RDX when exposure is extended. The estimated lowest observable adverse effect concentration of RDX was 1,235 mg/kg. No biodegradation products of RDX were detected at all three sampling times. Compared with 2,4,6-trinitrotoluene (TNT), RDX is less toxic to microbes, probably because of its resistance to biodegradation under aerobic conditions, which precludes metabolic activation of nitro groups.

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.

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.

Explosive biodegradation in soil slurry batch reactors amended with exogenous microorganisms.

The present study explores the feasibility of biotreatment of 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro- 1,3,5-triazine (RDX)-contaminated soils in slurry batch reactors. Radiorespirometric assays showed that anaerobic sludge was able to mineralize 59% RDX to CO2 although significant mineralization of TNT was not observed in all cases. TNT and RDX at concentrations higher than 50 and 100 mg/L respectively were inhibitory to methanogenesis (used as a bioindicator), however, methanogenesis recovered after TNT was transformed into less toxic triaminotoluene. Bioslurry batch reactors containing 40% of contaminated soil (2000 mg RDX and 1000 mg TNT/kg dry soil) were operated under various conditions. Both TNT and RDX were persistent to soil indigenous microbes. Degradation of both TNT and RDX was enhanced by the municipal sludge amendment, although degradation of RDX was only achieved under anaerobic conditions.