Sabine G. Dodard

Ecotoxicity characterization of dinitrotoluenes and some of their reduced metabolites

In the present study, the toxic effects of 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT) and a selection of their respective metabolites were examined and compared to 2,4,6-trinitrotoluene (TNT) using the 15-min Microtox (Vibrio fischen) and 96-h freshwater green alga (Selenastrum capricomutum) growth inhibition tests. All of the compounds tested were less toxic than TNT. Using the Microtox assay, 2,6-DNT was more toxic than 2,4-DNT and the order of toxicity for 2,6-DNT and its metabolites was: 2,6-DNT > or = 2A-6NT >> 2,6-DAT; whereas that for 2,4-DNT was: 4A-2NT > 2A-4NT > 2,4-DNT > 2,4-DAT. For the algal test, 2,4-DNT was more toxic than 2,6-DNT and the order of toxicity for 2,4-DNT and its metabolites was: 2,4-DNT > 2,4-DAT approximately equal to 4A-2NT = 2A-4NT. The order of toxicity for 2,6-DNT and its reduced metabolites using the algal test was very similar to the Microtox bioassay. These results demonstrate that the reduced metabolites of 2,6-DNT tested in this study were less toxic than that of the parent compound, but certain partially reduced metabolites of 2,4-DNT can be more toxic than the parent molecule. These data put into question the general hypothesis that reductive metabolism of nitro-aromatics is associated with a sequential detoxification process.

Acute and chronic toxicity of the new explosive CL-20 to the earthworm (Eisenia andrei) exposed to amended natural soils.

Monocyclic nitramine explosives such as 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are toxic to a number of ecological receptors, including earthworms. The polycyclic nitramine CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) is a powerful explosive that may replace RDX and HMX, but its toxicity is not known. In the present study, the lethal and sublethal toxicities of CL-20 to the earthworm (Eisenia andrei) are evaluated. Two natural soils, a natural sandy forest soil (designated RacFor2002) taken in the Montreal area (QC, Canada; 20% organic carbon, pH 7.2) and a Sassafras sandy loam soil (SSL) taken on the property of U.S. Army Aberdeen Proving Ground (Edgewood, MD, USA; 0.33% organic carbon, pH 5.1), were used. Results showed that CL-20 was not lethal at concentrations of 125 mg/kg or less in the RacFor2002 soil but was lethal at concentrations of 90.7 mg/kg or greater in the SSL soil. Effects on the reproduction parameters such as a decrease in the number of juveniles after 56 d of exposure were observed at the initial CL-20 concentration of 1.6 mg/kg or greater in the RacFor2002 soil, compared to 0.2 mg/kg or greater in the SSL soil. Moreover, low concentrations of CL-20 in SSL soil (approximately 0.1 mg/kg; nominal concentration) were found to reduce the fertility of earthworms. Taken together, the present results show that CL-20 is a reproductive toxicant to the earthworm, with lethal effects at higher concentrations. Its toxicity can be decreased in soils favoring CL-20 adsorption (high organic carbon content).

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 =7 mg/kg), and is lethal to earthworms Eisenia andrei (LC50 = 47 mg/kg). At sub-lethal concentrations, DNAN caused an avoidance response (EC50 = 31 mg/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.

SURVIVAL AND REPRODUCTION OF ENCHYTRAEID WORMS, OLIGOCHAETA, IN DIFFERENT SOIL TYPES AMENDED WITH ENERGETIC CYCLIC NITRAMINES

Hexanitrohexaazaisowurtzitane (CL-20), a new polycyclic polynitramine, has the same functional nitramine groups (N-NO2) as the widely used energetic chemicals hexahydro-1,3,5-trinitro-1,3,5-triazacyclohexane (royal demolition explosive [RDX]) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (high-melting explosive [HMX]). Potential impacts of CL-20 as an emerging contaminant must be assessed before its use. The effects of CL-20, RDX, or HMX on adult survival and juvenile production by potworms Enchytraeus albidus and Enchytraeus crypticus were studied in three soil types, including Sassafras sandy loam (1.2% organic matter [OM], 11% clay, pH 5.5), an agricultural soil (42% OM, 1% clay, pH 8.2), and a composite agricultural-forest soil (23% OM, 2% clay, pH 7.9) by using ISO method 16387 (International Standard Organization, Geneva, Switzerland). Results showed that CL-20 was toxic to E. crypticus with median lethal concentration values for adult survival ranging from 0.1 to 0.7 mg/kg dry mass (DM) when using the three tested soils. In addition, CL-20 adversely affected juvenile production by both species in all soils tested, with median effective concentration (EC50) values ranging from 0.08 to 0.62 mg/kg DM. Enchytraeus crypticus and E. albidus were similarly sensitive to CL-20 exposure in the composite agricultural-forest soil, which supported reproduction by both species and enabled comparisons. Correlation analysis showed weak or no relationship overall among the soil properties and reproduction toxicity endpoints. Neither RDX nor HMX affected (p > 0.05) adult survival of either species below 658 and 918 mg/kg DM, respectively, indicating that CL-20 is more toxic to enchytraeids than RDX or HMX. Examination of data shows that CL-20 should be considered as a potential reproductive toxicant to soil invertebrates, and that safeguards should be considered to minimize the potential for release of CL-20 into the environment.

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. 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 (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.

Effects of TiO2 nanoparticles on ROS production and growth inhibition using freshwater green algae pre-exposed to UV irradiation.

This study investigated the possibility that titanium dioxide nanoparticles (nano-TiO2) toxicity in Pseudokirchneriella subcapitata involves reactive oxygen species (ROS) production, using the dichlorodihydrofluorescein (DCF) assay. Algae were exposed to nano-TiO2 under laboratory fluorescent lamps supplemented with UV irradiation for 3h, with or without a UV filter. Results showed that nano-TiO2 increased ROS production in UV-exposed cells, with or without a UV filter (LOEC values were 250 and 10mg/L, respectively). Sublethal effects of nano-TiO2 on UV pre-exposed algae were also examined. Toxicity studies indicated that exposure to nano-TiO2 agglomerates decreased algal growth following 3h pre-exposure to UV, with or without a UV filter (EC50s were 8.7 and 6.3mg/L, respectively). The present study suggests that the growth inhibitory effects of nano-TiO2 in algae occurred at concentrations lower than those that can elevate DCF fluorescence, and that ROS generation is not directly involved with the sublethal effects of nano-TiO2 in algae.

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% 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.

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 (K(p)) 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(2) = 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 ( approximately 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.

In vitro degradation of hexanitrohexaazaisowurtzitane (CL‐20) by cytosolic enzymes of Japanese quail and the rabbit

Hexanitrohexaazaisowurtzitane (CL-20) is a polycyclic nitramine explosive and propellant, currently being considered as a potential replacement for existing cyclic nitramine explosives. Earlier studies have provided evidence suggestive of adverse liver effects in adult Coturnix spp. exposed to CL-20, yet analysis of tissue samples (plasma, liver, brain, heart, or spleen) indicated that CL-20 was not detectable in these treated animals. The present study was conducted to identify and purify the enzymes capable of CL-20 biotransformation. Results indicate that the hepatic biotransformation of CL-20 in vitro was inhibited by ethacrynic acid (93%) and by the glutathione (GSH) analogue S-octylglutathione (80%), suggesting the involvement of glutathione-S-transferase (GST). Partially purified cytosolic alpha- and mu-type GST (requiring presence of GSH as a cofactor) from quail and rabbit liver was capable of CL-20 biotransformation. The degradation of CL-20 (0.30 +/- 0.05 and 0.40 +/- 0.02 nmol/min/mg protein for quail and rabbit, respectively) was accompanied with the formation of nitrite and consumption of GSH. Using liquid chromatography/mass spectrometry, we detected two intermediates, that is, open-ring, monodenitrated GSH-conjugated CL-20 biotransformation product with the same deprotonated molecular mass ion at 699 Da, suggesting isomeric forms of the intermediate metabolites. Identity of the conjugated metabolites was confirmed by using ring-labeled [15N]CL-20 and the nitro group-labeled [15NO2]CL-20. These data suggest that the in vitro biotransformation of CL-20 by GST under the conditions tested may be a key initial step in the in vivo degradation of CL-20 in the quail and resulted in the formation of more biologically reactive intermediates than the parent compound. These data will aid in our understanding of the biotransformation processes of CL-20 in vivo.

Inhibition of soil microbial activity by nitrogen‐based energetic materials

We investigated individual toxicities of the nitrogen-based energetic materials (EMs) 2,4-dinitrotoluene (2,4-DNT); 2-amino-4,6-dinitrotoluene (2-ADNT); 4-amino-2,6-dinitrotoluene (4-ADNT); and nitroglycerin (NG) on microbial activity in Sassafras sandy loam (SSL) soil, which has physicochemical characteristics that support very high qualitative relative bioavailability for organic chemicals. Batches of SSL soil for basal respiration (BR) and substrate-induced respiration (SIR) assays were separately amended with individual EMs or acetone carrier control. Total microbial biomass carbon (biomass C) was determined from CO2 production increases after addition of 2500 mg/kg of glucose-water slurry to the soil. Exposure concentrations of each EM in soil were determined using US Environmental Protection Agency method 8330A. Basal respiration was the most sensitive endpoint for assessing the effects of nitroaromatic EMs on microbial activity in SSL, whereas SIR and biomass C were more sensitive endpoints for assessing the effects of NG in soil. The orders of toxicity (from greatest to least) were 4-ADNT > 2,4-DNT = 2-ADNT > NG for BR; but for SIR and biomass C, the order of toxicity was NG > 2,4-DNT > 2-ADNT = 4-ADNT. No inhibition of SIR was found up to and including the greatest concentration of each ADNT tested in SSL. These ecotoxicological data will be helpful in identifying concentrations of contaminant EMs in soil that present acceptable ecological risks for biologically mediated processes in soil. Environ Toxicol Chem 2017;36:2981-2990. Published 2017 Wiley Periodicals Inc. on behalf of SETAC.This article is a US government work and, as such, is in the public domain in the United States of America.