Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions

Summary Composting was investigated as a bioremediation technology for clean-up of sediments contaminated with explosives and propellants. Two field demonstrations were conducted, the first using 2,4,6-trinitrotoluene (TNT), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and N-methyl-N,2,4,6-tetranitroaniline (tetryl) contaminated sediment, and the second using nitrocellulose (NC) contaminated soil. Tests were conducted in thermophilic and mesophilic aerated static piles. Extractable TNT was reduced from 11840 mg/kg to 3 mg/kg, and NC from 13090 mg/kg to 16 mg/kg under thermophilic conditions. Under mesophilic conditions, TNT was reduced from 11 190 mg/kg to 50 mg/kg. The thermophilic and mesophilic half-lives were 11.9 and 21.9 days for TNT, 17.3 and 30.1 days for RDX, and 22.8 and 42.0 days for HMX, respectively. Known nitroaromatic transformation products increased in concentration over the first several weeks of the test period, but decreased to low concentrations thereafter.     

Evaluation of tissue and cellular biomarkers to assess 2,4,6-trinitrotoluene (TNT) exposure in earthworms: effects-based assessment in laboratory studies using Eisenia andrei

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at ≥1.8 µg TNT cm^-2, whereas sublethal (weight loss) and lethal effects to earthworms were detected at ≥3.5 and 7.1 µg TNT cm^-2, respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations ( ≥55 mg TNT kg^-1 soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO₄, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.     

Unusual spectroscopic and ligand binding properties of the cytochrome P450-flavodoxin fusion enzyme XplA

The Rhodococcus rhodochrous strain 11Y XplA enzyme is an unusual cytochrome P450-flavodoxin fusion enzyme that catalyzes reductive denitration of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX). We show by light scattering that XplA is a monomeric enzyme. XplA has high affinity for imidazole (K(d) = 1.6 μM), explaining previous reports of a red-shifted XplA Soret band in pure enzyme. The true Soret maximum of XplA is at 417 nm. Similarly, unusually weak XplA flavodoxin FMN binding (K(d) = 1.09 μM) necessitates its purification in the presence of the cofactor to produce hallmark flavin contributions absent in previously reported spectra. Structural and ligand-binding data reveal a constricted active site able to accommodate RDX and small inhibitory ligands (e.g. 4-phenylimidazole and morpholine) while discriminating against larger azole drugs. The crystal structure also identifies a high affinity imidazole binding site, consistent with its low K(d), and shows active site penetration by PEG, perhaps indicative of an evolutionary lipid-metabolizing function for XplA. EPR studies indicate heterogeneity in binding mode for RDX and other ligands. The substrate analog trinitrobenzene does not induce a substrate-like type I optical shift but creates a unique low spin EPR spectrum due to influence on structure around the distal water heme ligand. The substrate-free heme iron potential (-268 mV versus NHE) is positive for a low spin P450, and the elevated potential of the FMN semiquinone/hydroquinone couple (-172 mV) is also an adaptation that may reflect (along with the absence of a key Thr/Ser residue conserved in oxygen-activating P450s) the evolution of XplA as a specialized RDX reductase catalyst.     

The orientationally controlled assembly of genetically modified enzymes in an amperometric biosensor

A novel, nitroreductase (NTR) containing a sequence of six cysteine amino acids, enabling strong thiolate bonds to form on a gold electrode surface without the loss of enzyme activity, was genetically engineered. The enzyme was directly immobilised at a gold electrode without the need for pre-treatment of the surface with a self-assembled monolayer or a conducting polymer. The ensemble was used to develop an amperometric biosensor for the detection of explosives containing nitroaromatic compounds. Preliminary results demonstrate detection levels down to 100 parts per trillion, signifying tremendous promise towards an in situ sensor for the detection of explosives.     

A Peat Moss-Based Technology for Mitigating Residues of the Explosives TNT,RDX, and HMX in Soil

There is increased interest in how to balance military preparedness and environmental protection at Department of Defense (DoD) facilities. This research evaluated a peat moss-based technology to enhance the adsorption and biodegradation of explosive residues at military testing and training ranges. The evaluation was performed using 30-cm-long soil columns operated under unsaturated flow conditions. The treatment materials were placed at the soil surface, and soil contaminated with 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) was spread over the surface. Simulated rainfall initiated dissolution and leaching of the explosive compounds, which was monitored at several depths within the columns. Peat moss plus soybean oil reduced the soluble concentrations of TNT, RDX and HMX detected at 10 cm depth by 100%, 60%, and 40%, respectively, compared to the no-treatment control column. Peat moss alone reduced TNT and HMX concentrations at 10 cm depth relative to the control, but exhibited higher soluble RDX concentrations by the end of the experiment. Concentrations of HMX and RDX were also reduced at 30 cm depth by the peat moss plus soybean oil treatments relative to those observed in the control column. These preliminary results demonstrate proof-of-concept of a low cost technology for reducing the contamination of groundwater with explosives at military test and training ranges.     

Enhancing the Potential for in situ Bioremediation of RDX Contaminated Soil from a Former Military Demolition Range

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a toxic, mobile groundwater contaminant common to military sites. Biodegradation of RDX is an alternative, cost effective and environmentally friendly remediation approach. The effects of carbon amendments (waste glycerol and cheese whey) used alone or with a potential electron shuttle (ammonium lignosulfonate) on RDX biodegradation were assessed. These substrates are readily available waste materials that can be used as nutrients to promote oxygen consumption, creating a more reducing environment. Nutrient amended batch assays were conducted using RDX spiked contaminated demolition range soil under anaerobic conditions. The amendments that improved RDX mineralization the most were subsequently tested in a scaled up repacked soil column study to verify if this strategy could be effectively implemented on-site. Microcosm results indicated that RDX mineralization by indigenous anaerobic microorganisms was enhanced the most by the low carbon amendment concentration. The use of ammonium lignosulfonate was not effective, exhibiting an inhibitory effect on RDX biodegradation that was stronger at higher concentrations. The soil column study showed that the low concentration of waste was the most promising treatment scenario. These results offer good prospects for the use of waste glycerol for in situ treatment of soils contaminated with energetic-materials, such as RDX.     

Right on target: using plants and microbes to remediate explosives

Abstract

While the immediate effect of explosives in armed conflicts is frequently in the public eye, until recently, the insidious, longer-term corollaries of these toxic compounds in the environment have gone largely unnoticed. Now, increased public awareness and concern are factors behind calls for more effective remediation solutions to these global pollutants. Scientists have been working on bioremediation projects in this area for several decades, characterizing genes, biochemical detoxification pathways, and field-applicable plant species. This review covers the progress made in understanding the fundamental biochemistry behind the detoxification of explosives, including new shock-insensitive explosive compounds; how field-relevant plant species have been characterized and genetically engineered; and the major roles that endophytic and rhizospheric microorganisms play in the detoxification of organic pollutants such as explosives.     

Modeling Fate of RDX at Demolition Area 2 of the Massachusetts Military Reservation

The movement of explosive RDX residue from soil to groundwater at Demolition Area 2 of the Massachusetts Military Reservation was studied with mathematical models to assess its utility and limitations and to determine requisite model improvements and data needs. The Munitions Residue Characterization and Fate model, which is based on the MEPAS source term model for soil modified for solid phase residue with dissolution, the MEPAS vadose zone model, and the MEPAS aquifer model were used in this study. All three models were applied within ARAMS? to facilitate model-to-model connectivity for computing movement of RDX from soil to vadose zone, and from vadose zone to groundwater. Model parameters and a hypothetical RDX residue loading rate were adjusted to match model results to measured RDX concentrations in surface soil and in groundwater. Through an iterative process, a loading rate of 1 kg/yr for RDX residues applied for 10 years starting in 1978 was found to fit measured conditions 20 years later. Model results were sensitive to the C4-RDX dissolution flux rate and the aqueous RDX degradation rate. Results indicate that dissolution of C4 and degradation of RDX in soil and groundwater could be quite slow, and such processes warrant further study. Mechanistic models such as those presented will be useful for estimating fate of constituent residue in soil and transport to receiving waters for evaluating range residue carrying capacity and compliance issues.     

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.     

Bioremediation of tetryl-contaminated soil using sequencing batch soil slurry reactor

A laboratory study was conducted to determine whether tetryl (2,4,6-trinitrophenlymethylnitramine) contaminated soil could be bioremediated using a sequencing batch soil slurry reactor (SBR) operated under anoxic–aerobic sequence. The results indicated that tetryl was co-metabolically converted to aniline under anoxic conditions with molasses as the growth substrate. The gas chromatographic/mass spectrometric analysis of the soil slurry showed various metabolites, identified as trinitrobenzeneamine, dintrobenzenediamine, nitroaniline and aniline. Aniline was not metabolized further under anoxic conditions. When the soil slurry reactor was operated under aerobic conditions, the aniline concentration was reduced to below the detection limit (0.05 ppm). This metabolic conversion of tetryl is probably of value in the treatment of tetryl-contaminated soil and ground water, such as those found at the Joliet army ammunition plant site in Illinois and the Iowa army ammunition plant site in Burlington, Iowa.