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The explosive compounds hexahydro-1 f 3,5-trinitro-1 , 3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT) are widespread environmental contaminants commonly found as co-pollutants on military training ranges. TNT is a toxic carcinogen which remains tightly bound to the soil, whereas RDX is highly mobile leaching into groundwater and threatening drinking water supplies. We have engineered Arabidopsis plants that are able to degrade RDX, whilst withstanding the phytotoxicity of TNT. Arabidopsis thaliana (Arabidopsis) was transformed with the bacterial RDXdegrading xplA, and associated reductase xplB, from Rhodococcus rhodochrous strain 11Y, in combination with the TNT-detoxifying nitroreductase (NR), nfsl, from Enterobacter cloacae. Plants expressing XplA, XplB and NR remove RDX from soil leachate and grow on soil contaminated with RDX and TNT at concentrations inhibitory to XplA-only expressing plants. This is the first study to demonstrate the use of transgenic plants to tackle two chemically diverse organic compounds at levels comparable with those found on contaminated training ranges, indicating that this technology is capable of remediating concentrations of RDX found in situ. In addition, plants expressing XplA and XplB have substantially less RDX available in aerial tissues for herbivory and potential bioaccumulation.

The role of terrestrial vegetation in transferring chemicals from soil and air into specific plant tissues (e.g., stems, leaves, and roots) is still not well characterized. We provide here a critical review of plant‐to‐soil bioconcentration ratio (BCR) estimates based on models and experimental data. This review includes the conceptual and theoretical formulations of the BCR, constructing and calibrating empirical and mathematical algorithms to describe this ratio and the experimental data used to quantify BCRs and calibrate the model performance. We first evaluate the theoretical basis for the BCR concept and BCR models and consider how lack of knowledge and data limit reliability and consistency of BCR estimates. We next consider alternate modeling strategies for BCR. A key focus of this evaluation is the relative contributions to overall uncertainty from model uncertainty versus variability in the experimental data used to develop and test the models. As a case study, we consider a single chemical, hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine, and focus on variability of bioconcentration measurements obtained from 81 experiments with different plant species, different plant tissues, different experimental conditions, and different methods for reporting concentrations in the soil and plant tissues. We use these observations to evaluate both the magnitude of experimental variability in plant bioconcentration and compare this to model uncertainty. Among these 81 measurements, the variation of the plant‐to‐soil BCR has a geometric standard deviation (GSD) of 3.5 and a coefficient of variation (CV; i.e., ratio of the arithmetic standard deviation to the mean) of 1.7. These variations are significant but low relative to model uncertainties, which have an estimated GSD of 10, with a corresponding CV of 14.

The heterocyclic polynitramine hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a highly energetic compound found as a soil contaminant at some defense installations. Although RDX is not lethal to soil invertebrates at concentrations up to 10,000 mg/kg, it decreases earthworm cocoon formation and juvenile production at environmentally relevant concentrations found at contaminated sites. Very little is known about the uptake of RDX in earthworms and the potential risks for food-chain transfer of RDX in the environment. Toxicokinetic studies were conducted to quantify the bioaccumulation factors (BAFs) using adult earthworms (Eisenia andrei) exposed for up to 14 d to sublethal concentrations of nonlabeled RDX or [14C]RDX in a Sassafras sandy loam soil. High-performance liquid chromatography of acetonitrile extracts of tissue and soil samples indicated that nonlabeled RDX can be accumulated by the earthworm in a concentration- and time-dependent manner. The BAF, expressed as the earthworm tissue to soil concentration ratio, decreased from 6.7 to 0.1 when the nominal soil RDX concentrations were increased from 1 to 10,000 mg/kg. Tissue concentrations were comparable in earthworms exposed to nonlabeled RDX or [14C]RDX. The RDX bioaccumulation also was estimated using the kinetically derived model (BAF(K)), based on the ratio of the uptake to elimination rate constants. The established BAF(K) of 3.6 for [14C]RDX uptake was consistent with the results for nonlabeled RDX. Radioactivity also was present in the tissue residues of [14C]RDX-exposed earthworms following acetonitrile extraction, suggesting the formation of nonextractable [14C]RDX metabolites associated with tissue macromolecules. These findings demonstrated a net accumulation of RDX in the earthworm and the potential for food-chain transfer of RDX to higher-trophic-level receptors.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been widely used as an explosive in munition formulations, resulting in contamination of wildlife habitat on military installations. To estimate health effects for reptilian species, acute, subacute, and subchronic oral toxicity studies were conducted using the Western fence lizard (Sceloporus occidentalis). Estimated oral median lethal doses were 72 (95% confidence interval [CI], 49-106) mg/kg body weight (slope, 3.754) for males and 88 (95% CI, 65-119) mg/kg (slope, 4.525) for females. Toxicity from RDX suggested the neurological system as the critical target tissue. A 14-d subacute study followed with males dosed orally with RDX (corn oil) at 0, 10, 20, 25, 30, 45, and 60 mg/kg/d. Signs of toxicity frequently included a characteristic body posture. A significant dose-survival relationship was seen over the range of doses, with a significant decrease in survival at 20 mg/kg/d. Males in the 60-d subchronic study were dosed at 0, 1, 2.5, 5, 8, and 11 mg/ kg/d, and signs of toxicity included lethargy, cachexia, and anorexia. Survival was decreased at 8 and 11 mg/kg/d. Reduced growth rate and food consumption occurred at 5 mg/kg/d. Brain tissue was assayed for RDX when seizures were observed at a residue concentration of at least 18 microgram/g. No abnormalities were observed in the hematologic indices, whereas plasma proteins were reduced. Hepatic enlargement and decreased testes mass occurred at 8 and 11 mg/kg/d. Plasma testosterone concentrations, sperm counts, and motility measures were variable for all treatment levels. Based on survival, growth rate, food intake, and testes to brain weight ratios, these data suggest a lowest-observed-adverse effect level of 5 mg/kg/d and a no-observed-adverse effect level of 2.5 mg/ kg/d.

Hexanitrohexaazaisowurtzitane (CL‐20) and hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX), both energetic compounds, share some degree of structural similarity. A noninvasive electrophysiological technique was employed to assess the impacts of acute sublethal exposures on impulse conduction in medial (MGF) and lateral (LGF) giant nerve fiber pathways of the earthworm Eisenia fetida and to evaluate the reversibility of neurotoxic effects. Earthworms were exposed to either 0.02 to 2.15 μg/cm2 of CL‐20 or 0.04 to 5.35 μg/cm2 of RDX, for 1 to 14 d, on moistened filter paper. Conduction velocities of MGF and LGF were recorded on a digital oscilloscope before and after exposure. Results indicate that at exposure levels as low as 0.02 μg/cm2 of CL‐20 or 0.21 μg/cm2 of RDX, worms exhibited physiological impacts such as retardation, stiffness, and body shrink. Both MGF and LGF conduction velocities were negatively correlated with increasing doses of CL‐20 or RDX. However, such neurotoxic effects were alleviated or even eliminated within a few days after exposed worms were transferred to an uncontaminated environment, indicating that the neurotoxicity is reversible even after 6‐d exposure. The CL‐20 is more potent than RDX, which is consistent with previous studies on lethality, growth, and reproduction endpoints in soil oligochaetes.

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, ami‐nodinitrotoluenes (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 μmol/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 μmol/kg) and YB sediments (39.4 μmol/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 μmol/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 μmol/kg dry weight, nor tissue concentrations as high as 96 μmol/kg wet weight. The lack of RDX lethal effects in this study is consistent with results reported for other invertebrate species.

Reductive (pre)treatment with elemental iron is a potentiallyuseful method for degrading nitramine explosives in water and soil. In the present study, we examined the kinetics, products, and mechanisms of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) and octahydro‐1,3,5,7‐tetranitro‐1,3,5,7‐tetrazocine (HMX) degradation with elemental iron. Both RDX and HMX were transformed with iron to formaldehyde, NH +4, N2O, and soluble products. The yields of formaldehyde were relatively constant (71% ± 5%), whereas the yields of NH +4 and N2O varied, depending on the nitramine and the mechanism. The reactions most likely were controlled by a surface process rather than by external mass transfer. Methylenedinitramine (MDNA) was an intermediate of both RDX and HMX and was transformed quantitatively to formaldehyde with iron. However, product distributions and kinetic modeling results suggest that MDNA represented a minor reaction path and accounted for only 30% of the RDX reacted and 14% of the formaldehyde produced. Additional experiments showed that RDX reduction with elemental iron could be mediated by graphite and Fe2+ sorbed to magnetite, as demonstrated previously for nitroaromatics and nitrate esters. Methylenedinitramine was degraded primarily through reduction in the presence of elemental iron, because its hydrolysis was slow compared to its reactions with elemental iron and surface‐bound Fe2+. Our results show that in a cast iron‐water system, RDX may be transformed via multiple mechanisms involving different reaction paths and reaction sites.

Contamination with hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (Royal Demolition Explosive [RDX]) has been identified at areas of explosive manufacturing, processing, storage, and usage. Thus, the potential exists for exposure to N‐nitroso compounds, hexahydro‐1‐nitroso‐3,5‐dinitro‐1,3,5‐triazine, hexahydro‐1,3‐dinitroso‐5‐nitro‐1,3,5‐triazine, and hexahydro‐1,3,5‐trinitroso‐1,3,5‐triazine (TNX), formed via anaerobic transformation of RDX. Following exposure, reproductive toxicity of TNX was evaluated in three consecutive litters of deer mice (Peromyscus maniculatus). Hexahydro‐1,3,5‐trinitroso‐1,3,5‐triazine was administered ad libitum via drinking water at four doses: 0 (control), 1, 10, and 100 μg/L. Endpoints investigated included reproductive success, offspring survival, offspring weight gain, offspring organ weights, and liver TNX residues. Data from the present study indicate that TNX bioaccumulates in the liver and is associated with postpartum mortality, dose‐dependent decrease in body weight from birth to weaning, and decrease in kidney weight of deer mice offspring.

Concerns have been raised over potential bioavailability and biotransfer of energetic materials such as hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX). The present study assessed plant‐incorporated [14C]RDX and plant‐derived [14C]RDX‐metabolite ingestion by the prairie vole (Microtus ochrogaster). The animals were fed labeled chow (maximum, ≤ 10 g/d) for 5 or 7 d, followed by a 6‐ or 4‐d chase period. More than 95% of all label presented was recovered in the summed excreta, with 74% of this in the fecal nonabsorbed bulk. Greater than 20% of the presented [14C]RDX and plant‐derived [14C]RDX metabolites were absorbed by the animals' digestive tracts. These materials were either metabolized to 14CO2 (8–10% of the total label) or removed as nitrogenous waste through the kidneys (10–14%). Both 14C‐urine and 14CO2 excretion continued after the feces cleared, indicating ongoing metabolism of the labeled material. Approximately 4% was retained within the tissues at death, with the highest total activity in the liver and the highest specific activity in the testes. Other labeled tissues included the lung, heart, brain, spleen, skeletal muscle, bone, and pancreas. All these tissues containing [14C]RDX‐derived materials are available to subsequent predators, indicating a potential for transfer to a higher trophic level.

Several H2-producing fermentative anaerobic bacteria including Clostridium, Klebsiella and Fusobacteria degraded octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (36 micromolar) to formaldehyde (HCHO) and nitrous oxide (N2O) with rates ranging from 5 to 190 nmol h-1 g [dry weight] of cells-1. Among these strains, C. bifermentans strain HAW-1 grew and transformed HMX rapidly with the detection of the two key intermediates the mononitroso product and methylenedinitramine. Its cellular extract alone did not seem to degrade HMX appreciably, but degraded much faster in the presence of H2, NADH or NADPH. The disappearance of HMX was concurrent with the release of nitrite without the formation of the nitroso derivative(s). Results suggest that two types of enzymes were involved in HMX metabolism: one for denitration and the second for reduction to the nitroso derivative(s).