Munitions Constituents Research Articles

Neurotoxicogenomic Investigations to Assess Mechanisms of Action of the Munitions Constituents RDX and 2,6-DNT in Northern Bobwhite (Colinus virginianus)

Munitions constituents (MCs) including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT), and TNT derivatives are recognized to elicit aberrant neuromuscular responses in many species. The onset of seizures resulting in death was observed in the avian model Northern bobwhite after oral dosing with RDX beginning at 8 mg/kg/day in subacute (14 days) exposures, whereas affective doses of the TNT derivative, 2,6-dinitrotoluene (2,6-DNT), caused gastrointestinal impacts, lethargy, and emaciation in subacute and subchronic (60 days) exposures. To assess and contrast the potential neurotoxicogenomic effects of these MCs, a Northern bobwhite microarray was developed consisting of 4119 complementary DNA (cDNA) features enriched for differentially-expressed brain transcripts from exposures to RDX and 2,6-DNT. RDX affected hundreds of genes in brain tissue, whereas 2,6-DNT affected few (<or= 17), indicating that 2,6-DNT exposure had relatively little impact on the brain in comparison to RDX. Birds exhibiting RDX-induced seizures accumulated over 20x more RDX in brain tissues in comparison to non-seizing birds even within a common dose. In parallel, expression patterns were unrelated among seizing and non-seizing birds exposed to equivalent RDX doses. In birds experiencing seizures, genes related to neuronal electrophysiology and signal transduction were significantly affected. Comparative toxicology revealed strong similarity in acute exposure effects between RDX and the organochlorine insecticide dichlorodiphenyltrichloroethane (DDT) regarding both molecular mechanisms and putative mode of action. In a manner similar to DDT, we hypothesize that RDX elicits seizures by inhibition of neuronal cell repolarization postaction potential leading to heightened neuronal excitability and seizures facilitated by multiple molecular mechanisms.

Biomarker Discovery and Transcriptomic Responses in Daphnia magna exposed to Munitions Constituents

Ecotoxicogenomic approaches are emerging as alternative methods in environmental monitoring because they allow insight into pollutant modes of action and help assess the causal agents and potential toxicity beyond the traditional end points of death, growth, and reproduction. Gene expression analysis has shown particular promise for identifying gene expression biomarkers of chemical exposure that can be further used to monitor specific chemical exposures in the environment. We focused on the development of gene expression markers to detect and discriminate between chemical exposures. Using a custom cDNA microarray for Daphnia magna, we identified distinct expression fingerprints in response to exposure at sublethal concentrations of Cu, Zn, Pb, and munitions constituents. Using the results obtained from microarray analysis, we chose a suite of potential biomarkers for each of the specific exposures. The selected potential biomarkers were tested in independent chemical exposures for specificity using quantitative reverse transcription polymerase chain reaction. Six genes were confirmed as differentially regulated bythe selected chemical exposures. Furthermore, each exposure was identified by response of a unique combination (suite) of individual gene expression biomarkers. These results demonstrate the potential for discovery and validation of novel biomarkers of chemical exposures using gene expression analysis, which could have broad applicability in environmental monitoring.

The effects of grinding methods on metals concentrations in soil

Multi-increment sampling (MIS) has been most extensively used for munitions constituents at environmental sites where a high degree of contaminant heterogeneity exits. A revised method (USEPA Method 8330B) for explosives that uses MIS was announced in the fall of 2006, but similar guidance has not been reported for testing metals in soils. Questions have been raised as how to prepare representative analytical samples for metals determination from field composites. Three different grinding procedures were used in this study for three soil types to determine if grinding (relative to homogenizing soil without grinding) increases metal concentrations and decreases variability. The performance of these procedures was demonstrated via the analysis of replicates ( n = 16 for two soil types) using statistical evaluations that included calculations of various descriptive statistics (e.g., medians, means and standard deviations), Kruskal–Wallis (KW) tests for the medians and two tests for the variances (Bartlett's and Levene's test). There was a slight increase in concentrations for several of the metals in the clay loam soil after grinding, although the increases were a small percentage of the concentrations measured. The standard deviations (and variances) for replicate digestions and analyses generally decreased, although anomalies were observed. The grinding methods increased precision overall, however, the data indicated that the roller mill grinding was not as effective as the other grinding methods tested.

RS-VSP: A New Approach to Support Military Range Sustainability

The Navy, through Range Sustainability and Environmental Program Assessment (RSEPA), is taking the initiative to identify potential off-range transport of munitions constituents. To accomplish this initiative, the Quality Assurance Project Plan (QAPP) for sampling and testing at operational ranges was developed. A key goal of environmental sampling plan design is to specify representative sampling requirements necessary to determine the presence and distribution of environmental constituents. Range Sustainability-Visual Sample Plan (RS-VSP) is a software tool that was developed to achieve the environmental sampling goal of representative sampling for RSEPA QAPP implementation so that data collected provide the data user with the required information for knowledgeable decision making. The VSP-RSM supports a sampling approach designed to bound a “source area” suspected of containing concentrations of munitions constituents above the relevant screening levels. RS-VSP incorporates an innovative approach to selecting data quality objectives, estimate sampling and testing uncertainty, and determining the number and location of samples. Preliminary assessments of off-range migration of explosives particles by windblown transport mechanisms, and the potential environmental impacts to surface water, aquifer recharge zones, and groundwater are planned under RSEPA. Besides windblown transport, infiltration of dissolved explosives residues into groundwater is another important migration pathway being investigated for range sustainability by other DoD agencies.

Toxicity and fate of two munitions constituents in spiked sediment exposures with the marine amphipod Eohaustorius estuarius

The lethal toxicity of the explosive compounds 14C-labeled 2,4,6-trinitrotoluene (TNT) and nonradiolabeled hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to the estuarine amphipod Eohaustorius estuarius was investigated in 10-d spiked sediment exposures. The 10-d median lethal concentration (LC50) was determined using the sum molar initial concentration of TNT, aminodinitrotoluenes (ADNTs), and diaminonitrotoluenes (DANTs), as determined by high-performance liquid chromatography (HPLC), and collectively referred to as HPLC-TNT*. Despite expectations of higher toxicity in sandy sediment (Yaquina Bay [YB], OR, USA) compared to relatively fine-grained sediment (San Diego Bay [SDB], CA, USA), LC50 values were similar: 159 and 125 micromol/kg, for YB and SDB sediments, respectively. When expressed as the sum of TNT and all its degradation products (14C-TNT*), LC50s were approximately two times the corresponding LC50s determined by HPLC. The HPLC-TNT* fraction likely corresponds to the most bioavailable and toxic transformation products. The concentrations of 14C-TNT* in tissues were substantially higher than those for HPLC-TNT*, suggesting that compounds other than TNT and its major aminated transformation products were prevalent. Critical body residues were similar for exposures to SDB (11.7 micromol/kg) and YB sediments (39.4 micromol/kg), despite marked differences in the nature of compounds available for uptake in the exposure media. The critical body residues for E. estuarius are lower than those reported for other aquatic invertebrates (83-172 micromol/kg). Unlike observations for TNT, RDX was only loosely associated with SDB sediment, with near complete recovery of the parent compound by chemical analysis. Exposure to RDX did not result in significant mortality even at the highest measured sediment concentration of 10,800 micromol/kg dry weight, nor tissue concentrations as high as 96 micromol/kg wet weight. The lack of RDX lethal effects in this study is consistent with results reported for other invertebrate species.

Acquisition of “Green” products and munitions—Automated assessments

Executive Orders 13101 and 13148 established the federal priorities to improve the use of environmentally preferable (”green”) products, including munitions, and prevent or reduce pollution at its source. Quantitative criteria for “green” chemicals and products have not been previously defined. Acquisition personnel, manufacturers, and regulatory agencies have heretofore been restricted to qualitative and generic, intuitive considerations (e.g., “less harmful to human health and the environment”). We have overcome this deficiency by compiling and/or integrating more than 75 million data elements for over 210,000 chemicals and 350,000 chemical and munition products over the past 22 years and synthesizing these data into quantitative “green” chemical and product ratings with our “Green” Products Compliance Analytical System (GP-CAS) and our “Green” Munitions Analytical Compliance System (G-MACS). G-MACS also uses the Munitions Items Disposition Action System (MIDAS) munitions constituent database from the U.S. Army Defense Ammunition Center as the “core” reference resource. GP-CAS and G-MACS are both based upon 47 chemical “green” criteria, each normalized on a scale of 0 percent (worst “green”) to 100 percent (best “green”). These criteria encompass ecological (airwater-soil), health (acute-chronic), and safety (fire-reactive) hazards. In addi- tion, these systems both identify which of 57 state, federal, or international regulatory lists include any chemical constituent of a product. The only difference between GP-CAS and G-MACS is the product constituent reference database source—chemicals or munition products. “Green” analyses can be completed in 5–30 seconds, depending upon the complexity of the product or munition. Hierarchical “green” assessments are performed at the chemical, part, or product levels, and “green” ratings are calculated for the holistic “greenness” of the entire product, the separate ecological/health/safety scores, each of their subcategory scores, and the individual 47 endpoints for each chemical. These assessments, therefore, not only provide a quantitative “green” rating for a chemical/munition product within a few seconds, but also quantitatively identify the type of compromising hazard(s) and the contributing hazardous chemical constituent(s) within the product. These Web-based systems can be utilized for any type of product analyses in any industry, facility, or location, since analyses are chemical-based, not product-specific. GP-CAS and G-MACS fulfill the objectives of Executives Orders 13101 and 13148 to accurately identify environmentally preferable products and prevent pollution at the source. © 2005 Wiley Periodicals, Inc.

The Price of Preparing for War

Located a few miles from Anchorage, Alaska’s Eagle River Flats is a coastal saltwater marsh teeming with fish, wildlife—and unexploded mortar and artillery shells. The marsh lies on the Department of Defense’s (DOD) 62,000-acre training facility at Fort Richardson, headquarters to the Army’s Alaskan command and control units. Since World War II, Eagle River Flats has been Fort Richardson’s primary “ordnance impact zone,” where soldiers stationed at the fort come to train with live munitions. Environmental assessments undertaken at the Flats by the Army have revealed high levels of contaminants including heavy metals, explosive compounds, and white phosphorus, a toxic agent used to generate smoke cover on the battlefield. It was this contamination with white phosphorus, which can damage bones and major internal organs, that in 1994 landed Eagle River Flats on the Superfund National Priorities List, a U.S. Environmental Protection Agency (EPA) compilation of the nation’s most polluted properties. Since then, the Army has been conducting an EPA-approved effort to clean up the white phosphorus. But in April 2002 the DOD was sued by a citizens’ coalition urging the Army to address remaining contamination problems at the Flats. Among the plaintiffs were the indigenous Chickaloon Indians, who claimed the Army’s use of live munitions was polluting traditional hunting and fishing grounds. The suit also charged that unexploded mortar rounds and artillery shells in the area were leaching toxic chemicals that were migrating to nearby Cook Inlet. The plaintiffs’ attorney, Scott Allen of the San Francisco, California–based law firm Cox and Moyer, says the suit requested that the Army remove some 10,000 unexploded mortar rounds and artillery shells from the area (the number estimated in the Army’s 1998 proposed Superfund cleanup plan), remediate toxic contamination, and abstain from using the range for bombing exercises until a Clean Water Act permit had been obtained for munitions discharges. When confronted with the lawsuit, the DOD took its case to Congress. There, it argued that the laws on the books were not intended to be applied to operational military ranges in this way, citing long-standing past state and federal regulatory interpretation and practice. The DOD further argued that suits like those brought at Eagle River Flats, if successful, could set a legal precedent whereby environmental litigants could halt military training and thus undermine troop readiness on the battlefield. Before the 2002 lawsuit even arose, the DOD had proposed new legislation called the Readiness and Range Preservation Initiative (RRPI) to prevent just such lawsuits attempting to use hazardous waste laws to limit training. The RRPI calls for exemptions from a number of environmental laws on more than 8,000 operational DOD training ranges, a land area equal to roughly 24 million acres. Under this proposed new legislation, munitions would not be subject to hazardous waste permitting or cleanup requirements as long as they remain on operational ranges.

Comparison of environmental fate and transport process descriptors of explosives in saline and freshwater systems

Environmental process descriptors are necessary to evaluate the fate and transport of munitions constituents that have been introduced into the environment. An extensive database exists for freshwater environments; however, explosives fate and transport parameters such as dissolution rates, transformation rates, and adsorption of explosives have not been evaluated under both freshwater and saline conditions to determine the applicability of the freshwater data to saline environments. The objective of this study was to determine if freshwater fate and transport processes were similar to those determined under saline water conditions. We evaluated TNT, RDX, and HMX dissolution rates, transformation rates, and adsorption under freshwater and saline conditions in batch tests. Results showed a generally close agreement. Therefore, the existing freshwater database for explosives fate and transport process descriptors can be used in marine environments.