Biotransformation of 2,4,6-trinitrotoluene (TNT) by Enchytraeids (Enchytraeus albidus) in vivo and in vitro

2,4,6-Trinitrotoluene (TNT) is toxic to soil invertebrates, but little is known about its toxicokinetic behavior in soil. Tissue residue analysis was used to evaluate whether the presence of TNT and its reduced metabolites in soil invertebrates was due to uptake of these compounds from the soil into the organism, or due to microbial transformation of TNT associated with the organism followed by uptake. Adult white potworms (Enchytraeus albidus) were exposed to non-lethal concentrations of TNT in amended artificial soil for 21 d, or to TNT in solution for 20 h. Soil exposure studies confirmed earlier reports that TNT was transformed in enchytraeids in vivo to 2- and 4-aminodinitrotoluenes. However, enchytraeid exposure to TNT in solution led to the additional presence of 2,4-diaminonitrotoluene as well as 2- and 4- hydroxyamino-dinitrotoluenes and azoxy-compounds, suggesting that TNT can be metabolized in vivo in the absence of soil. Incubation of unexposed enchytraeid homogenates with TNT led to a protein-dependent appearance of these metabolites in vitro after gtoreq 16 h incubation. Cellular fractionation studies indicated that most of this activity resided in the 8000 x g pellet, and was completely inhibited by broad-spectrum antibiotics. These studies demonstrate that enchytraeids can transform TNT in vivo and in vitro, at least in part, by bacteria associated with the host organism. Crown Copyright Copyright 2004 Published by Elsevier Ltd. All rights reserved.NRC publication: Ye

Lethal and subchronic effects of 2,4,6-trinitrotoluene (TNT) on Enchytraeus albidus in spiked artificial soil

The effects of 2,4,6-trinitrotoluene (TNT) exposure in spiked artificial soil on the survival and reproduction rate of the white potworm Enchytraeus albidus were studied. Based on the initial concentrations, TNT in freshly spiked soil decreased enchytraeid survival (21-day LC50=422+-63 (SD)mg/kg, N=3) and fecundity (42-day EC50=111+-34, N=4). Data also indicated that TNT was 5-10 times more lethal to juveniles than adults, and lethality was less pronounced in TNT-spiked soils aged for 21 days. A time-dependent decrease in the TNT concentrations, as well as a concomitant increase in the levels of 2- and 4-aminodinitrotoluene, was observed during the 42-day toxicity test. Taken together, TNT (or one of its metabolites) is more lethal to juvenile than adult enchytraeids. This effect may explain, at least in part, the ability of TNT to decrease fecundity as determined using the enchytraeid mortality-reproduction test. (c) Biosciences Information Services.NRC publication: Ye

Ecotoxicological assessment of a high energetic and insensitive munitions compound : 2,4-Dinitroanisole (DNAN)

The high explosive nitroaromatic 2,4-dinitroanisole (DNAN) is less shock sensitive than 2,4,6-trinitrotoluene (TNT), and is proposed as a TNT replacement for melt-cast formulations. Before using DNAN in munitions and potentially leading to environmental impact, the present study examines the ecotoxicity of DNAN using selected organisms. In water, DNAN decreased green algae Pseudokirchneriella subcapitata growth (EC50=4.0mg/L), and bacteria Vibrio fischeri bioluminescence (Microtox, EC50=60.3mg/L). In soil, DNAN decreased perennial ryegrass Lolium perenne growth (EC50=7mg/kg), and is lethal to earthworms Eisenia andrei (LC50=47mg/kg). At sub-lethal concentrations, DNAN caused an avoidance response (EC50=31mg/kg) by earthworms. The presence of DNAN and 2-amino-4-nitroanisole in earthworms and plants suggested a role of these compounds in DNAN toxicity. Toxicity of DNAN was compared to TNT, tested under the same experimental conditions. These analyses showed that DNAN was equally, or even less deleterious to organism health than TNT, depending on the species and toxicity test. The present studies provide baseline toxicity data to increase the understanding of the environmental impact of DNAN, and assist science-based decision makers for improved management of potential DNAN contaminated sites. \ua9 2013.Peer reviewed: YesNRC publication: Ye

Role of soil interstitial water in the accumulation of hexahydro-1,3,5-trinitro-1,3,5-triazine in the earthworm Eisenia andrei

The uptake of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) from soil by the earthworm Eisenia andrei was examined by using the equilibrium partitioning (EqP) theory and a three-compartment model including soil (S), interstitial water (IW), and earthworms (E). The RDX concentrations were measured using U.S. Environmental Protection Agency (U.S. EPA) Method 8330A and high-performance liquid chromatography (HPLC). The S-IW studies were conducted using four natural soils with contrasting physicochemical properties that were hypothesized to affect the bioavailability of RDX. Each soil was amended with nominal RDX concentrations ranging from 1 to 10,000 mg/kg. The HPLC analysis showed that the IW extracted from soil was saturated with RDX at 80 mg/kg or greater soil concentrations. The calculated S-IW coefficient (Kp) values for RDX ranged from 0.4 to 1.8 ml/g soil, depending on the soil type, and were influenced by the organic matter content. In the IW-E studies, earthworms were exposed to nonlethal RDX concentrations in aqueous media. The uptake of RDX by the earthworms correlated well (r\ub2 = 0.99) with the dissolved RDX concentrations. For the E-S studies, earthworms were exposed to RDX-amended soils used in the S-IW studies. The bioconcentration factors (BCF; ratios of E-to-IW RDX concentrations) were relatively constant (~5) up to 80 mg/kg soil RDX concentrations, which encompass the RDX saturation limit in the interstitial water of the tested soils. At this concentration range, the RDX uptake from interstitial water was likely dominated by passive diffusion and could be used as an indicator of bioavailability. Other mechanisms may be involved at greater RDX soil concentrations.Peer reviewed: YesNRC publication: Ye

Preliminary ecotoxicological characterization of a new energetic substance, CL-20

A new energetic substance hexanitrohexaazaisowurtzitane (or CL-20) was tested for its toxicities to various ecological receptors. CL-20 (epsilon-polymorph) was amended to soil or deionized water to construct concentration gradients. Results of Microtox (15-min contact) and 96-h algae growth inhibition tests indicate that CL-20 showed no adverse effects on the bioluminescence of marine bacteria Vibrio fischeri and the cell density of freshwater green algae Selenastrum capricornutum respectively, up to its water solubility (ca. 3.6 mg 1-1). CL-20 and its possible biotransformation products did not inhibit seed germination and early seedling (16-19 d) growth of alfalfa (Medicago sativa) and perennial ryegrass (Lolium perenne) up to 10 000 mg kg-1 in a Sassafras sandy loam soil (SSL). Indigenous soil microorganisms in SSL and a garden soil were exposed to CL-20 for one or two weeks before dehydrogenase activity (DHA) or potential nitrification activity (PNA) were assayed. Results indicate that up to 10 000 mg kg-1 soil of CL-20 had no statistically significant effects on microbial communities measured as DHA or on the ammonium oxidizing bacteria determined as PNA in both soils. Data indicates that CL-20 was not acutely toxic to the species or microbial communities tested and that further studies are required to address the potential long-term environmental impact of CL-20 and its possible degradation products. Crown Copyright Copyright 2004 Published by Elsevier Ltd. All rights reserved.NRC publication: Ye

Accumulation of hexahydro-1,3,5-trinitro-1,3,5-triazine by the earthworm Eisenia andrei in a sandy loam soil

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. Highperformance 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 (BAFK), based on the ratio of the uptake to elimination rate constants. The established BAFK 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.Peer reviewed: YesNRC publication: Ye

Phytotoxicity and uptake of nitroglycerin in a natural sandy loam soil

Nitroglycerin (NG) is widely used for the production of explosives and solid propellants, and is a soil contaminant of concern at some military training ranges. NG phytotoxicity data reported in the literature cannot be applied directly to development of ecotoxicological benchmarks for plant exposures in soil because they were determined in studies using hydroponic media, cell cultures, and transgenic plants. Toxicities of NG in the present studies were evaluated for alfalfa (Medicago sativa), barnyard grass (Echinochloa crusgalli), and ryegrass (Lolium perenne) exposed to NG in Sassafras sandy loam soil. Uptake and degradation of NG were also evaluated in ryegrass. The median effective concentration values for shoot growth ranged from 40 to 231 mg kg- 1 in studies with NG freshly amended in soil, and from 23 to 185 mg kg- 1 in studies with NG weathered-and-aged in soil. Weathering-and-aging NG in soil did not significantly affect the toxicity based on 95 percent confidence intervals for either seedling emergence or plant growth endpoints. Uptake studies revealed that NG was not accumulated in ryegrass but was transformed into dinitroglycerin in the soil and roots, and was subsequently translocated into the ryegrass shoots. The highest bioconcentration factors for dinitroglycerin of 685 and 40 were determined for roots and shoots, respectively. Results of these studies will improve our understanding of toxicity and bioconcentration of NG in terrestrial plants and will contribute to ecological risk assessment of NG-contaminated sites.Peer reviewed: YesNRC publication: Ye

Phytotoxicity of nitroaromatic energetic compounds freshly amended or weathered and aged in sandy loam soil

The toxicities of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), 2,4-dinitrotoluene (2,4-DNT), and 2,6-dinitrotoluene (2,6-DNT) to terrestrial plants alfalfa (Medicago sativa L.), Japanese millet (Echinochloa crusgalli L.), and perennial ryegrass (Lolium perenne L.) were determined in Sassafras sandy loam soil using seedling emergence, fresh shoot, and dry mass measurement endpoints. A 13-week weathering and aging of energetic materials in soils, which included wetting and drying cycles, and exposure to sunlight of individual soil treatments, was incorporated into the study design to better reflect the soil exposure conditions in the field than toxicity determinations in freshly amended soils. Definitive toxicity tests showed that dinitrotoluenes were more phytotoxic for all plant species in freshly amended treatments based on EC20 values for dry shoot ranging from 3 to 24mgkg(-1) compared with values for TNB or TNT ranging from 43 to 62mgkg(-1). Weathering and aging of energetic materials (EMs) in soil significantly decreased the toxicity of TNT, TNB or 2,6-DNT to Japanese millet or ryegrass based on seedling emergence, but significantly increased the toxicity of all four EMs to all three plant species based on shoot growth. Exposure of the three plant species to relatively low concentrations of the four compounds initially stimulated plant growth before the onset of inhibition at greater concentrations (hormesis).NRC publication: Ye