Recent studies demonstrated that degradation of the military explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by species of Rhodococcus, Gordonia, and Williamsia is mediated by a novel cytochrome P450 with a fused flavodoxin reductase domain (XplA) in conjunction with a flavodoxin reductase (XplB). Pulse field gel analysis was used to localize xplA to extrachromosomal elements in a Rhodococcus sp. and distantly related Microbacterium sp. strain MA1. Comparison of Rhodococcus rhodochrous 11Y and Microbacterium plasmid sequences in the vicinity of xplB and xplA showed near identity (6,710 of 6,721 bp). Sequencing of the associated 52.2-kb region of the Microbacterium plasmid pMA1 revealed flanking insertion sequence elements and additional genes implicated in RDX uptake and degradation.
For decades, the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) has been used for military and industrial applications. Residues of RDX pollute soils in large areas globally and the persistence and high soil mobility of these residues can lead to leaching into groundwater. Dendroremediation, i.e. the long-term use of trees to clean up polluted soils, is gaining acceptance as a green and sustainable strategy. Although the coniferous tree species Norway spruce and Scots pine cover large areas of military land in Central Europe, the potential of any coniferous tree for dendroremediation of RDX is still unknown. In this study, uptake experiments with a 14C-labelled RDX solution (30 mg L−1) revealed that RDX was predominantly retained in the roots of 6-year-old coniferous trees. Only 23 % (pine) to 34 % (spruce) of RDX equivalents (RDXeq) taken up by the roots were translocated to aboveground tree compartments. This finding contrasts with the high aerial accumulation of RDXeq (up to 95 %) in the mass balances of all other plant species. Belowground retention of RDXeq is relatively stable in fine root fractions, since water leaching from tissue homogenates was less than 5 %. However, remobilisation from milled coarse roots and tree stubs reached up to 53 %. Leaching from homogenised aerial tree material was found to reach 64 % for needles, 58 % for stems and twigs and 40 % for spring sprouts. Leaching of RDX by precipitation increases the risk for undesired re-entry into the soil. However, it also opens the opportunity for microbial mineralisation in the litter layer or in the rhizosphere of coniferous forests and offers a chance for repeated uptake of RDX by the tree roots.
Polymer-bonded explosives (PBX) fulfil the need for insensitive munitions. However, the environmental impacts of PBX are unclear, even though it is likely that PBX residues from low-order detonations and unexploded ordnance are deposited on military training ranges. The release of high explosives from the polymer matrix into the environment has not been studied in detail, although as polymers degrade slowly in the environment we anticipate high explosives to be released into the environment. In this study, PBXN-109 (nominally 64% RDX) samples were exposed to variable UK climatic conditions reproduced in the laboratory to determine the effects of temperature, UV irradiation and rainfall on the release of RDX from the polymer binder. The most extreme conditions for spring, summer and winter in the UK were artificially reproduced. We found that up to 0.03% of RDX was consistently released from PBXN-109. The rate of RDX release was highest in samples exposed to the summer simulation, which had the lowest rainfall, but the highest temperatures and longest UV exposure. This was confirmed by additional experiments simulating an extreme summer month with consistently high temperatures and long periods of sunlight. These results probably reflect the combination of polymer swelling and degradation when samples are exposed to higher temperatures and prolonged UV irradiation.
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitroamine explosive that is a major component in many military high-explosive formulations. In this study, two aerobic bacteria that are capable of using RDX as the sole source of carbon and nitrogen to support their growth were isolated from surface soil. These bacterial strains were identified by their fatty acid profiles and 16S ribosomal gene sequences as Williamsia sp. KTR4 and Gordonia sp. KTR9. The physiology of each strain was characterized with respect to the rates of RDX degradation and [U-14C]RDX mineralization when RDX was supplied as a sole carbon and nitrogen source in the presence and absence of competing carbon and nitrogen sources. Strains KTR4 and KTR9 degraded 180 μM RDX within 72 h when RDX served as the only added carbon and nitrogen source while growing to total protein concentrations of 18.6 and 16.5 μg/ml, respectively. Mineralization of [U-14C]RDX to 14CO2 was 30% by strain KTR4 and 27% by KTR9 when RDX was the only added source of carbon and nitrogen. The addition of (NH4)2SO4 greatly inhibited KTR9's degradation of RDX but had little effect on that of KTR4. These are the first two pure bacterial cultures isolated that are able to use RDX as a sole carbon and nitrogen source. These two genera possess different physiologies with respect to RDX mineralization, and each can serve as a useful microbiological model for the study of RDX biodegradation with regard to physiology, biochemistry, and genetics.
International audience ; Repeated use of the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on military land has resulted in significant soil and groundwater pollution. Rates of degradation of RDX in the environment are low, and accumulated RDX, which the U.S. Environmental Protection Agency has determined is a possible human carcinogen, is now threatening drinking water supplies. RDX-degrading microorganisms have been isolated from RDX-contaminated land; however, despite the presence of these species in contaminated soils, RDX pollution persists. To further understand this problem, we studied RDX-degrading species belonging to four different genera (Rhodococcus, Microbacterium, Gordonia, and Williamsia) isolated from geographically distinct locations and established that the xplA and xplB (xplAB) genes, which encode a cytochrome P450 and a flavodoxin redox partner, respectively, are nearly identical in all these species. Together, the xplAB system catalyzes the reductive denitration of RDX and subsequent ring cleavage under aerobic and anaerobic conditions. In addition to xplAB, the Rhodococcus species studied here share a 14-kb region flanking xplAB; thus, it appears likely that the RDX-metabolizing ability was transferred as a genomic island within a transposable element. The conservation and transfer of xplAB-flanking genes suggest a role in RDX metabolism. We therefore independently knocked out genes within this cluster in the RDX-degrading species Rhodococcus rhodochrous 11Y. Analysis of the resulting mutants revealed that XplA is essential for RDX degradation and that XplB is not the sole contributor of reducing equivalents to XplA. While XplA expression is induced under nitrogen-limiting conditions and further enhanced by the presence of RDX, MarR is not regulated by RDX.
International audience ; Repeated use of the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on military land has resulted in significant soil and groundwater pollution. Rates of degradation of RDX in the environment are low, and accumulated RDX, which the U.S. Environmental Protection Agency has determined is a possible human carcinogen, is now threatening drinking water supplies. RDX-degrading microorganisms have been isolated from RDX-contaminated land; however, despite the presence of these species in contaminated soils, RDX pollution persists. To further understand this problem, we studied RDX-degrading species belonging to four different genera (Rhodococcus, Microbacterium, Gordonia, and Williamsia) isolated from geographically distinct locations and established that the xplA and xplB (xplAB) genes, which encode a cytochrome P450 and a flavodoxin redox partner, respectively, are nearly identical in all these species. Together, the xplAB system catalyzes the reductive denitration of RDX and subsequent ring cleavage under aerobic and anaerobic conditions. In addition to xplAB, the Rhodococcus species studied here share a 14-kb region flanking xplAB; thus, it appears likely that the RDX-metabolizing ability was transferred as a genomic island within a transposable element. The conservation and transfer of xplAB-flanking genes suggest a role in RDX metabolism. We therefore independently knocked out genes within this cluster in the RDX-degrading species Rhodococcus rhodochrous 11Y. Analysis of the resulting mutants revealed that XplA is essential for RDX degradation and that XplB is not the sole contributor of reducing equivalents to XplA. While XplA expression is induced under nitrogen-limiting conditions and further enhanced by the presence of RDX, MarR is not regulated by RDX.
Repeated use of the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on military land has resulted in significant soil and groundwater pollution. Rates of degradation of RDX in the environment are low, and accumulated RDX, which the U.S. Environmental Protection Agency has determined is a possible human carcinogen, is now threatening drinking water supplies. RDX-degrading microorganisms have been isolated from RDX-contaminated land; however, despite the presence of these species in contaminated soils, RDX pollution persists. To further understand this problem, we studied RDX-degrading species belonging to four different genera (Rhodococcus, Microbacterium, Gordonia, and Williamsia) isolated from geographically distinct locations and established that the xplA and xplB (xplAB) genes, which encode a cytochrome P450 and a flavodoxin redox partner, respectively, are nearly identical in all these species. Together, the xplAB system catalyzes the reductive denitration of RDX and subsequent ring cleavage under aerobic and anaerobic conditions. In addition to xplAB, the Rhodococcus species studied here share a 14-kb region flanking xplAB; thus, it appears likely that the RDX-metabolizing ability was transferred as a genomic island within a transposable element. The conservation and transfer of xplAB-flanking genes suggest a role in RDX metabolism. We therefore independently knocked out genes within this cluster in the RDX-degrading species Rhodococcus rhodochrous 11Y. Analysis of the resulting mutants revealed that XplA is essential for RDX degradation and that XplB is not the sole contributor of reducing equivalents to XplA. While XplA expression is induced under nitrogen-limiting conditions and further enhanced by the presence of RDX, MarR is not regulated by RDX.
AbstractBiodegradation has been applied to remediate explosives contaminants, and bacteria have a high potential for the degradation of explosives, such as hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) and 2,4,6‐trinitrotoluene (TNT). The present study aims to screen and characterize explosive biodegradable Actinomycetes from water, wastewater, and sludge. Actinomycetes isolates were recovered from 80 environmental samples from diverse environmental resources in explosive contaminated areas of Iran and identified to the genus and species levels using conventional and molecular methods. The growth rate in the presence of pollutants and chromatography was used to determine their biodegradation capability. Twenty‐nine isolates (36.25%) of Actinomycetes were characterized from the cultured samples that belonged to 6 genus and 24 validated species. The most prevalent Actinomycetes isolated were genus Mycobacterium with 11 isolates (37.94%), genus Rhodococcus with seven isolates (24.13%), genus Nocardia with four isolates (13.8%), and genus Streptomyces with three isolates (10.33%). Moreover, our results showed that these isolates could degrade and consume 50–80% of RDX and TNT as their sole carbon and energy source. In conclusion, we showed that Actinomycetes from explosive‐contaminated areas of Iran could degrade TNT and RDX. Hence, seeking and screening untapped ecosystems that possess unexplored Actinomycetes will increase the chances of discovering the resident microorganism that has been capable of degrading TNT and RDX for application in the bioremediation process. The results of this study can be useful in using intact bacteria in nature to eliminate environmental pollution, which is one of the major environmental problems in the world.
Contamination of soils with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX, Research Department Explosive) as a result of military applications is a large-area problem globally. Since coniferous trees dominate the vegetation of large areas of military land in Central Europe, particularly in Germany, the long-term fate of 14C-RDX in the conifers Scots pine and Dwarf Alberta spruce was studied. Acetic acid was the most effective solvent for the removal of extractable RDX residues from homogenates of RDX-laden tree material (85%, 80–90% and 64–80% for roots, wood and needles, respectively). On average, only a fifth of RDX-derived 14C was bound in non-extractable residues (NER). Within the main cell wall compartments, lignin was the dominant binding site for NER (needles: 32–62%; roots: 38–42%). Hemicellulose (needles: 11–18%; roots: 6–11%) and cellulose (needles: 12–24%; roots: 1–2%) were less involved in binding and a considerable proportion of NER (needles: 15–24%; roots: 59–51%) was indigestible. After three-year incubation in rot chambers, mineralisation of tree-associated 14C-RDX to 14CO2 clearly dominated the mass balance in both tree species with 48–83%. 13–33% of 14C-RDX-derived radioactivity remained in an unleachable form and the remobilisation by water leaching was negligible (< 2%).
Insensitive High-Explosive (IHE) typically comprises up to five constituents including 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), which are mixed in various ratios to achieve desired performance and increase insensitivity. Insensitive munitions, which are designed to detonate on command and not accidentally, are currently in use in military operations and training areas around the world. However, there is minimal literature available on the physiochemical behavior of these materials in the environment, therefore the actual consequence of residues being deposited post-detonation is still an unexplored area of research. Three 155 mm artillery shells filled with an IHE mixture of 53 % NTO, 32 % DNAN, and 15 % RDX were detonated in an inert sand arena to collect and quantify residues. Post detonation, approximately 0.02 % NTO, and 0.07 % DNAN were deposited in the environment which may rapidly accumulate dependent on the number of rounds fired. This is of concern due to the toxicity of DNAN and its degradation products, and the potential for increased acidity of soil and discoloration of watercourses from NTO contamination.
Abstract Explosive compounds, including known toxicants 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), are loaded to soils during military training. Their fate in soils is ultimately controlled by soil mineralogical and biogeochemical processes. We detonated pure mineral phases with Composition B, a mixture of TNT and RDX, and investigated the fate of detonation residues in aqueous slurries constructed from the detonated minerals. The pure minerals included Ottawa sand (quartz and calcite), microcline feldspar, phlogopite mica, muscovite mica, vermiculite clay, beidellite (a representative of the smectite clay group), and nontronite clay. Energy-dispersive X-ray spectrometry, X-ray diffraction, and gas adsorption surface area measurements were made of the pristine and detonated minerals. Batch slurries of detonated minerals and deionized water were sampled for 141 days and TNT, RDX, and TNT transformation products were measured from the aqueous samples and from the mineral substrates at day 141. Detonated samples generally exhibited lower gas adsorption surface areas than pristine ones, likely from residue coating, shock-induced compaction, sintering, and/or partial fusion. TNT and RDX exhibited analyte loss in almost all batch solutions over time but loss was greater in vermiculite, beidellite, and phlogopite than in muscovite and quartz. This suggests common phyllosilicate mineral substrates could be used on military training ranges to minimize off-site migration of explosive residues. We present a conceptual model to represent the physical and chemical processes that occurred in our aqueous batches over time.
Explosives compounds, known toxins, are loaded to soils on military training ranges predominantly during explosives detonation events that likely fracture soil particles. This study was conducted to investigate the fate of explosives compounds in aqueous slurries containing fractured and pristine soil particles. Three soils were crushed with a piston to emulate detonation-induced fracturing. X-ray diffraction, energy-dispersive X-ray spectrometry, gas adsorption surface area measurements, and scanning electron microscopy were used to quantify and image pristine and fractured soil particles. Aqueous batches were prepared by spiking soils with solutions containing 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro 1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and 2,4-dinitrotoluene (2,4-DNT). Samples were collected over 92 d and the concentrations of the spiked explosives compounds and TNT transformation products 2-amino-4,6-dinitrotoluene (2ADNT) and 4-amino-2,6-dinitrotoluene (4ADNT) were measured. Our results suggest soil mineralogical and geochemical compositions were not changed during piston-induced fracturing but morphological differences were evident with fractured soils exhibiting more angular surfaces, more fine grained particles, and some microfracturing that is not visible in the pristine samples. TNT, 2,4-DNT, RDX, and HMX exhibited greater analyte loss over time in batch solutions containing fractured soil particles compared to their pristine counterparts. 2ADNT and 4ADNT exhibited greater concentrations in slurries containing pristine soils than in slurries containing fractured soils. Explosives compound transformation is greater in the presence of fractured soil particles than in the presence of pristine soil particles. Our results imply fractured soil particles promote explosive compound transformation and/or explosives compounds have a greater affinity for adsorption to fractured soil particle surfaces.
<p class="p1">The effect of replacing hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in HTPB-binder on the performance, sensitivity, thermal, and mechanical properties of the sheet explosive formulation has been studied. The maximum loading of HMX was achieved up to 78 per cent in HTPB-binder system. The velocity of detonation (VOD) of HMX-based sheet explosive was observed about 7300 m/s which is marginally higher than existing RDX-based sheet explosive formulation (RDX/HTPB-binder, 80/20). The VOD trends were verified by theoretical calculation by BKW code using FORTRAN executable program. The thermal decomposition kinetics of sheet explosive formulations was investigated by differential scanning calorimetry. The activation energy for sheet explosive formulation HMX/HTPB-binder (78/22) was calculated using Kissinger kinetic method and found to be 170.08 kJ/mol, infer that sheet explosive formulation is thermally stable.</p>
