Multi-species Aquatic Toxicity Assessment of 1-Methyl-3-Nitroguanidine (MeNQ).

The Army is replacing traditional munitions with insensitive munitions (IM) resistant to accidental detonation. The aquatic toxicity of 1-methyl-3-nitroguanidine (MeNQ), which is being assessed for potential use in IM formulations, remains largely untested. The present study fills a number of critical data gaps for MeNQ aquatic toxicity by evaluating effects across two vertebrate and five invertebrate species. Specifically, responses in larval Pimephales promelas, Rana pipiens tadpoles, Chironomus dilutus, Lumbriculus variegatus, Hydra littoralis, Hyalella azteca, and Daphnia pulex were assessed in MeNQ exposures across various acute, subchronic, and chronic bioassays. Overall, survival was unaffected in most of the MeNQ exposures where significant lethal effects were only observed in D. pulex, H. littoralis, and C. dilutus and only at concentrations ≥ 2186 mg/L. Significant sublethal effects on growth were observed for C. dilutus at 903 mg/L and H. azteca at 1098 mg/L in 10-d assays. Significantly decreased reproduction was observed at 2775 mg/L for H. azteca in a chronic 35-d assay and at 174 mg/L for D. pulex in the 11-d three-brood assay representing a sublethal effect one order of magnitude more sensitive than the effective lethal concentration for D. pulex (2987 mg/L). Degradation of MeNQ in ultraviolet light (UV) greatly increased toxicity to D. pulex. Specifically, exposure to a MeNQ solution that was completely UV-degraded prior to D. pulex exposures resulted in an 11-d LC50 of 6.1 mg/L and a 50% reduction in reproduction at 3.125 mg/L, based on the original MeNQ parent-compound concentrations.

Comparative Toxicological Evaluation of UV-Degraded versus Parent-Insensitive Munition Compound 1-Methyl-3-Nitroguanidine in Fathead Minnow.

The US Army is replacing traditional munitions with insensitive munitions resistant to accidental detonation. Although the parent insensitive munition compound nitroguanidine (NQ) is generally not acutely toxic at concentrations >1000 mg/L in aquatic exposures, products formed by intensive ultraviolet (UV) degradation resulted in multiple-order of magnitude increases in toxicity. A methylated congener of NQ, 1-methyl-3-nitroguanidine (MeNQ), is also being assessed for potential use in insensitive munition explosive formulations; therefore, the present study investigated the hazard of parent versus UV-degraded MeNQ using fathead minnows (Pimephales promelas). Although up to 716 mg/L parent MeNQ caused no significant mortality or effects on growth in larval P. promelas fish in 7-d exposures, a similar concentration of MeNQ subjected to UV treatment resulted in 85% mortality. The UV treatment degraded only 3.3% of the MeNQ (5800 mg/L stock, UV-treated for 6 h), indicating that MeNQ degradation products have potentially high toxicity. The parent MeNQ exposure caused significantly decreased transcriptional expression of genes within the significantly enriched insulin metabolic pathway, suggesting antagonism of bioenergetics pathways, which complements observed, although nonsignificant, decreases in body weight. Significant differential transcriptional expression in the UV-degraded MeNQ treatments resulted in significant enrichment of pathways and functions related to the cell cycle, as well as erythrocyte function involved in O2 /CO2 exchange. These functions represent potential mechanistic sources of increased toxicity observed in the UV-degraded MeNQ exposures, which are distinct from previously observed mechanisms underlying increased toxicity of UV-degraded NQ in fish. Environ Toxicol Chem 2020;39:612-622. © 2019 SETAC.

Molecular Evaluation of Impacted Reproductive Physiology in Fathead Minnow Testes Provides Mechanistic Insights into Insensitive Munitions Toxicology.

Previous toxicological investigations of the insensitive munition (IM), 3-nitro-1,2,4-triazol-5-one (NTO), demonstrated histopathological and physiological impacts in mammalian testes. The implications of these findings for fish was unknown, therefore we investigated the effects of chronic (21 day) exposures to NTO and an NTO-containing IM formulation called IMX-101 (composed of 2,4-dinitroanisole (DNAN), nitroguanidine (NQ), and NTO) in adult male fathead minnows to assess if impacts on testes were conserved. The NTO exposure caused no significant mortality through the maximum exposure concentration (720 mg/L, measured), however NTO elicited testicular impacts causing significant asynchrony in spermatogenesis and necrosis in secondary spermatocytes at the two highest exposure concentrations (383 mg/L and 720 mg/L) and testicular degeneration at the highest exposure. Microarray-based transcriptomics analysis identified significant enrichment of steroid metabolism pathways and mTORC-signal control of spermatogonia differentiation in NTO exposures each having logical connections to observed asynchronous spermatogenesis. Additionally, NTO impaired transcriptional expression for genes supporting sperm structural and flagellar development including sperm-associated antigen 6 (Spag6). These functional transcriptomic responses are hypothesized contributors to impacted reproductive physiology in NTO exposures that ultimately lead to reductions in spermatozoa. In contrast to NTO, the IMX-101 formulation elicited significant mortality at the two highest exposure concentrations of 25.2 and 50.9 mg/L (DNAN nominal + NTO measured + NQ measured). Unlike NTO and NQ, the DNAN component of the IMX-101 formulation underwent significant transformation in the 21d exposure. From previous investigations, neither NTO nor NQ caused mortality in fish at >1000 mg/L suggesting that mortality in the present study arose from DNAN / DNAN-attributable transformation products. The 12.6 mg/L IMX-101 exposure caused significant sublethal impacts on testes including sperm necrosis, interstitial fibrosis, and Sertoli-like cell hyperplasia. Transcriptional profiles for IMX-101 indicated significant enrichment on multiple signaling pathways supporting spermatogenesis, mitosis / meiosis, and flagellar structure, all logically connected to observed sperm necrosis. Additionally, pronounced transcriptional increases within the PPARα-RXRα pathway, a known DNAN target, has been hypothesized to correspond to Sertoli cell hyperplasia, presumably as a compensatory response to fulfill the nurse-function of Sertoli cells during spermatogenesis. Overall, the transcriptional results indicated unique molecular responses for NTO and IMX-101. Regarding chemical hazard, NTO impacted testes and impaired spermatogenesis, but at high exposure concentrations (≥ 192 mg/L), whereas the IMX-101 formulation, elicited mortality and impacts on reproductive physiology likely caused by DNAN and its transformation products present at concentrations well below the NTO-component concentration within the IMX-101 mixture formulation.

Subchronic, chronic, lethal and sublethal toxicity of insensitive munitions mixture formulations relative to individual constituents in Hyalella azteca.

Insensitive munitions (IMs) are replacing conventional munitions, improving safety from unintended detonation. IMs are deployed in mixture formulations but little is known about their mixture toxicology. We characterized mixture effects of the IM formulations IMX-101 (mixture of 2,4-dinitroanisole [DNAN], 3-nitro-1,2,4-triazol-5-one [NTO], and nitroguanidine [NQ]) and IMX-104 (DNAN, NTO, and hexahydro-1,3,5-trinitro-1,3,5-triazine [RDX]) in subchronic (10 d) and chronic (35 d) water-only tests in Hyalella azteca assessing impacts on survival, growth and reproduction. In 10-d single chemical exposures, DNAN was the most potent constituent, eliciting an LC50 of 16.0 mg/L; the LC50s for NTO and NQ were 891 and 565 mg/L, respectively. RDX did not elicit significant mortality up to 29.5 mg/L, a concentration near its solubility limit. Based on toxic-units (TUs), the toxicity of IMX-101 was driven by the effective concentration of DNAN; however, the presence of NTO, RDX, or both elicited interactive effects causing an approximately 2-fold decrease in lethality for IMX-104. Growth reduction was observed in 10-d exposures to DNAN, IMX-101 and IMX-104, but not for NQ, NTO, or RDX. Longer exposure duration (35 d) to IMX-101, IMX-104, and DNAN resulted in 3-6 times higher sensitivity for lethality and resulted in the most sensitive endpoint for DNAN, RDX, and IMX-101 exposures, decreased reproduction. Slight, but statistically significant, antagonistic responses among IMX-101 constituents were observed for survival and reproduction at 35d. Overall, the results support response-additive summation as a sufficient method to provide conservative hazard assessments of subchronic, chronic, and sublethal IMX-101 and IMX-104 mixture impacts in H. azteca.

Transcriptomics provides mechanistic indicators of mixture toxicology for IMX-101 and IMX-104 formulations in fathead minnows (Pimephales promelas).

Within the US military, new insensitive munitions (IMs) are rapidly replacing conventional munitions improving safety from unintended detonation. Toxicity data for IM chemicals are expanding rapidly, however IM constituents are typically deployed in mixture formulations, and very little is known about their mixture toxicology. In the present study we sought to characterize the mixture effects and toxicology of the two predominant IM formulations IMX-101 and IMX-104 in acute (48 h) larval fathead minnow (Pimephales promelas) exposures. IMX-101 consists of a mixture of 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), and nitroguanidine (NQ) while IMX-104 is composed of DNAN, NTO, and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). DNAN was the most potent constituent in IMX-101 eliciting an LC50 of 36.1 mg/L, whereas NTO and NQ did not elicit significant mortality in exposures up to 1040 and 2640 mg/L, respectively. Toxic unit calculations indicated that IMX-101 elicited toxicity representative of the component concentration of DNAN within the mixture. Toxicogenomic responses for the individual constituents of IMX-101 indicated unique transcriptional expression and functional responses characteristic of: oxidative stress, impaired energy metabolism, tissue damage and inflammatory responses in DNAN exposures; impaired steroid biosynthesis and developmental cell-signaling in NQ exposures; and altered mitogen-activated protein kinase signaling in NTO exposures. Transcriptional responses to the IMX-101 mixture were driven by the effects of DNAN where expression and functional responses were nearly identical comparing DNAN alone versus the fractional equivalent of DNAN within IMX-101. Given that each individual constituent of the IMX-101 mixture elicited unique functional responses, and NTO and NQ did not interact with DNAN within the IMX-101 mixture exposure, the overall toxicity and toxicogenomic responses within acute exposures to the IMX-101 formulation are indicative of "independent" mixture toxicology. Alternatively, in the IMX-104 exposure both DNAN and RDX were each present at concentrations sufficient to elicit lethality (RDX LC50 = 28.9 mg/L). Toxic-unit calculations for IMX-104 mixture formulation exposures indicated slight synergistic toxicity (ΣTU LC50 = 0.82, 95% confidence interval = 0.73-0.90). Unique functional responses relative to DNAN were observed in the IMX-104 exposure including responses characteristic of RDX exposure. Based on previous transcriptomics responses to acute RDX exposures in fathead minnow larvae, we hypothesize that the potentially synergistic responses within the IMX-104 mixture are related to interactive effects of each DNAN and RDX on oxidative stress mitigation pathways.

The increased toxicity of UV-degraded nitroguanidine and IMX-101 to zebrafish larvae: Evidence implicating oxidative stress.

Insensitive munitions (IMs) improve soldier safety by decreasing sympathetic detonation during training and use in theatre. IMs are being increasingly deployed, although the environmental effects of IM constituents such as nitroguanidine (NQ) and IM mixture formulations such as IMX-101 remain largely unknown. In the present study, we investigated the acute (96h) toxicity of NQ and IMX-101 to zebrafish larvae (21d post-fertilization), both in the parent materials and after the materials had been irradiated with environmentally-relevant levels of ultraviolet (UV) light. The UV-treatment increased the toxicity of NQ by 17-fold (LC50 decreased from 1323mg/L to 77.2mg/L). Similarly, UV-treatment increased the toxicity of IMX-101 by nearly two fold (LC50 decreased from 131.3 to 67.6mg/L). To gain insight into the cause(s) of the observed UV-enhanced toxicity of the IMs, comparative molecular responses to parent and UV-treated IMs were assessed using microarray-based global transcript expression assays. Both gene set enrichment analysis (GSEA) and differential transcript expression analysis coupled with pathway and annotation cluster enrichment were conducted to provide functional interpretations of expression results and hypothetical modes of toxicity. The parent NQ exposure caused significant enrichment of functions related to immune responses and proteasome-mediated protein metabolism occurring primarily at low, sublethal exposure levels (5.5 and 45.6mg/L). Enriched functions in the IMX-101 exposure were indicative of increased xenobiotic metabolism, oxidative stress mitigation, protein degradation, and anti-inflammatory responses, each of which displayed predominantly positive concentration-response relationships. UV-treated NQ had a fundamentally different transcriptomic expression profile relative to parent NQ causing positive concentration-response relationships for genes involved in oxidative-stress mitigation pathways and inhibited expression of multiple cadherins that facilitate zebrafish neurological and retinal development. Transcriptomic profiles were similar between UV-treated versus parent IMX-101 exposures. However, more significant and diverse enrichment as well as greater magnitudes of differential expression for oxidative stress responses were observed in UV-treated IMX-101 exposures. Further, transcriptomics indicated potential for cytokine signaling suppression providing potential connections between oxidative stress and anti-inflammatory responses. Given the overall results, we hypothesize that the increased toxicity of UV-irradiated NQ and the IMX-101 mixture result from breakdown products with elevated potential to elicit oxidative stress.

Toxicity of the conventional energetics TNT and RDX relative to new insensitive munitions constituents DNAN and NTO in Rana pipiens tadpoles.

An initiative within the US military is targeting the replacement of traditional munitions constituents with insensitive munitions to reduce risk of accidental detonation. The purpose of the present study was to comparatively assess toxicity of the traditional munitions constituents 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) with the new insensitive munitions constituents 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO). The following exposure durations were performed with Rana pipiens (leopard frog) tadpoles: TNT and DNAN, 96 h and 28 d; RDX, 10 d and 28 d; NTO, 28 d. The 96-h 50% lethal concentration (LC50) values and 95% confidence intervals for TNT and DNAN were 4.4 mg/L (4.2 mg/L, 4. 7 mg/L) and 24.3 mg/L (21.3 mg/L, 27.6 mg/L), respectively. No significant impacts on survival were observed in the 10-d exposure to RDX up to 25.3 mg/L. Effects on tadpole swimming distance were observed with a lowest-observed-effect concentration (LOEC) of 5.9 mg/L RDX. In the 28-d exposures, the LOECs for survival for TNT, DNAN, and NTO were 0.003 mg/L, 2.4 mg/L, and 5.0 mg/L, respectively. No significant mortality was observed in the RDX chronic 28-d exposure up to the highest treatment level tested of 28.0 mg/L. Neither tadpole developmental stage nor growth was significantly affected in any of the 28-d exposures. Rana pipiens were very sensitive to chronic TNT exposure, with an LOEC 3 orders of magnitude lower than those for insensitive munitions constituents DNAN and NTO.

Bioaccumulation kinetics of the conventional energetics TNT and RDX relative to insensitive munitions constituents DNAN and NTO in Rana pipiens tadpoles.

The manufacturing of explosives and their loading, assembling, and packing into munitions for use in testing on training sites or battlefields has resulted in contamination of terrestrial and aquatic sites that may pose risk to populations of sensitive species. The bioaccumulative potential of the conventional explosives 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and of the insensitive munitions (i.e., less shock sensitive) compound 2,4-dinitroanisole (DNAN) were assessed using the Northern leopard frog, Rana pipiens. Trinitrotoluene entering the organism was readily biotransformed to aminodinitrotoluenes, whereas no transformation products were measured for RDX or DNAN. Uptake clearance rates were relatively slow and similar among compounds (1.32-2.19 L kg(-1) h(-1) ). Upon transfer to uncontaminated water, elimination rate was very fast, resulting in the prediction of fast time to approach steady state (5 h or less) and short elimination half-lives (1.2 h or less). A preliminary bioconcentration factor of 0.25 L kg(-1) was determined for the insensitive munitions compound 3-nitro-1,2,4-trizole-5-one (NTO) indicating negligible bioaccumulative potential. Because of the rapid elimination rate for explosives, tadpoles inhabiting contaminated areas are expected to experience harmful effects only if under constant exposure conditions given that body burdens can rapidly depurate preventing tissue concentrations from persisting at levels that may cause detrimental biological effects.

The good, the bad, and the toxic: approaching hormesis in Daphnia magna exposed to an energetic compound.

A hormetic response is characterized by an opposite effect in small and large doses of chemical exposure, often resulting in seemingly beneficial effects at low doses. Here, we examined the potential mechanisms underlying the hormetic response of Daphnia magna to the energetic trinitrotoluene (TNT). Daphnia magna were exposed to TNT for 21 days, and a significant increase in adult length and number of neonates was identified at low concentrations (0.002-0.22 mg/L TNT), while toxic effects were identified at high concentrations (0.97 mg/L TNT and above). Microarray analysis of D. magna exposed to 0.004, 0.12, and 1.85 mg/L TNT identified effects on lipid metabolism as a potential mechanism underlying hormetic effects. Lipidomic analysis of exposed D. magna supported the hypothesis that TNT exposure affected lipid and fatty acid metabolism, showing that hormetic effects could be related to changes in polyunsaturated fatty acids known to be involved in Daphnia growth and reproduction. Our results show that Daphnia exposed to low levels of TNT presented hormetic growth and reproduction enhancement, while higher TNT concentrations had an opposite effect. Our results also show how a systems approach can help elucidate potential mechanisms of action and adverse outcomes.

Genomic investigation of year-long and multigenerational exposures of fathead minnow to the munitions compound RDX.

We assessed the impacts of exposure to an environmentally representative concentration (0.83 mg/L) of the explosive cyclotrimethylenetrinitramine (RDX) on fathead minnows (Pimephales promelas) in one-year and multigenerational bioassays. In the one-year bioassay, impacts were assessed by statistical comparisons of females from breeding groups reared in control or RDX-exposure conditions. The RDX had no significant effect on gonadosomatic index or condition factor assayed at 1 d and at one, three, six, nine, and 12 months. The liver-somatic index was significantly increased versus controls only at the 12-month timepoint. RDX had no significant effect on live-prey capture rates, egg production, or fertilization. RDX caused minimal differential-transcript expression with no consistent discernable effect on gene-functional categories for either brain or liver tissues in the one-year exposure. In the multigenerational assay, the effects of acute (96 h) exposure to RDX were compared in fish reared to the F(2) generation in either control or RDX-exposure conditions. Enrichment of gene functions including neuroexcitatory glutamate metabolism, sensory signaling, and neurological development were observed comparing control-reared and RDX-reared fish. Our results indicated that exposure to RDX at a concentration representing the highest levels observed in the environment (0.83 mg/L) had limited impacts on genomic, individual, and population-level endpoints in fathead minnows in a one-year exposure. However, multigenerational exposures altered transcript expression related to neural development and function. Environ.