Mode of action evaluation for reduced reproduction in Daphnia pulex exposed to the insensitive munition, 1-methyl-3-nitro-1-nitroguanidine (MeNQ).

The US Department of Defense (DOD) is developing insensitive munitions (IMs) that are resistant to unintended detonation to protect warfighters. To enable material life-cycle analysis for the IM, 1-methyl-3-nitro-1-nitroguanidine (MeNQ), ecotoxicological impacts assessment was required. A previous investigation of MeNQ exposures in Daphnia pulex revealed concentration-responsive decreases in reproduction relative to controls (0 mg/L) across a 174, 346, 709, 1385, and 2286 mg/L exposure range. The present study used those exposures to conduct global transcriptomic expression analyses to establish hypothetical mode(s) of action underlying inhibited reproduction. The number of significantly affected transcripts and the magnitude of fold-change differences relative to controls tended to increase with increasing MeNQ concentration where hierarchical clustering analysis identified separation among the "low" (174 and 346 mg/L) and "high" (709, 1385, and 2286 mg/L) exposures. Vitellogenin is critical to Daphnia reproductive processes and MeNQ exposures significantly decreased transcriptional expression for vitellogenin-1 precursor at the lowest exposure level (174 mg/L) with benchmark dose (BMD) levels closely tracking concentrations that caused inhibited reproduction. Additionally, juvenile hormone-inducible protein, chorion peroxidase, and high choriolytic enzyme transcriptional expression were impacted by MeNQ exposure having potential implications for egg production / maturation and overall fecundity. In concert with these effects on specific genes involved in Daphnia reproductive physiology, MeNQ exposures caused significant enrichment of several canonical-pathways responsible for metabolism of cellular energy substrates where BMD levels for transcriptional expression were observed at ≤100 mg/L. These observations imply possible effects on whole-organism energy budgets that may also incur indirect costs on reproduction.

Genomic investigations of acute munitions exposures on the health and skin microbiome composition of leopard frog (Rana pipiens) tadpoles.

Natural communities of microbes inhabiting amphibian skin, the skin microbiome, are critical to supporting amphibian health and disease resistance. To enable the pro-active health assessment and management of amphibians on Army installations and beyond, we investigated the effects of acute (96h) munitions exposures to Rana pipiens (leopard frog) tadpoles and the associated skin microbiome, integrated with RNAseq-based transcriptomic responses in the tadpole host. Tadpoles were exposed to the legacy munition 2,4,6-trinitrotoluene (TNT), the new insensitive munition (IM) formulation, IMX-101, and the IM constituents nitroguinidine (NQ) and 1-methyl-3-nitroguanidine (MeNQ). The 96h LC50 values and 95% confidence intervals were 2.6 (2.4, 2.8) for ΣTNT and 68.2 (62.9, 73.9) for IMX-101, respectively. The NQ and MeNQ exposures caused no significant impacts on survival in 96h exposures even at maximum exposure levels of 3560 and 5285 mg/L, respectively. However, NQ and MeNQ, as well as TNT and IMX-101 exposures, all elicited changes in the tadpole skin microbiome profile, as evidenced by significantly increased relative proportions of the Proteobacteria with increasing exposure concentrations, and significantly decreased alpha-diversity in the NQ exposure. The potential for direct effects of munitions exposure on the skin microbiome were observed including increased abundance of munitions-tolerant phylogenetic groups, in addition to possible indirect effects on microbial flora where transcriptional responses suggestive of changes in skin mucus-layer properties, antimicrobial peptide production, and innate immune factors were observed in the tadpole host. Additional insights into the tadpole host's transcriptional response to munitions exposures indicated that TNT and IMX-101 exposures significantly enriched transcriptional expression within type-I and type-II xenobiotic metabolism pathways, where dose-responsive increases in expression were observed. Significant enrichment and increased transcriptional expression of heme and iron binding functions in the TNT exposures served as likely indicators of known mechanisms of TNT toxicity including hemolytic anemia and methemoglobinemia. The significant enrichment and dose-responsive decrease in transcriptional expression of cell cycle pathways in the IMX-101 exposures was consistent with previous observations in fish, while significant enrichment of immune-related function in response to NQ exposure were consistent with potential immune suppression at the highest NQ exposure concentration. Finally, the MeNQ exposures elicited significantly decreased transcriptional expression of keratin 16, type I, a gene likely involved in keratinization processes in amphibian skin. Overall, munitions showed the potential to alter tadpole skin microbiome composition and affect transcriptional profiles in the amphibian host, some suggestive of potential impacts on host health and immune status relevant to disease susceptibility.

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.

Different as night and day: Behavioural and life history responses to varied photoperiods in Daphnia magna.

Nearly all animal species have utilized photoperiod to cue seasonal behaviours and life history traits. We investigated photoperiod responses in keystone species, Daphnia magna, to identify molecular processes underlying ecologically important behaviours and traits using functional transcriptomic analyses. Daphnia magna were photoperiod-entrained immediately posthatch to a standard control photoperiod of 16 light/ 8 dark hours (16L:8D) relative to shorter (4L:20D, 8L:16D, 12L:12L) and longer (20L:4D) day length photoperiods. Short-day photoperiods induced significantly increased light-avoidance behaviours relative to controls. Correspondingly, significant differential transcript expression for genes involved in glutamate signalling was observed, a critical signalling pathway in arthropod light-avoidance behaviour. Additionally, period circadian protein and proteins coding F-box/LRR-repeat domains were differentially expressed which are recognized to establish circadian rhythms in arthropods. Indicators of metabolic rate increased in short-day photoperiods which corresponded with broadscale changes in transcriptional expression across system-level energy metabolism pathways. The most striking observations included significantly decreased neonate production at the shortest day length photoperiod (4L:20D) and significantly increased male production across short-day and equinox photoperiods (4L:20D, 8L:16D and 12L:12D). Transcriptional expression consistent with putative mechanisms of male production was observed including photoperiod-dependent expression of transformer-2 sex-determining protein and small nuclear ribonucleoprotein particles (snRNPs) which control splice variant expression for genes like transformer. Finally, increased transcriptional expression of glutamate has also been shown to induce male production in Daphnia pulex via photoperiod-sensitive mechanisms. Overall, photoperiod entrainment affected molecular pathways that underpin critical behavioural and life history traits in D. magna providing fundamental insights into biological responses to this primary environmental cue.

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.

Multiple environmental stressors induce complex transcriptomic responses indicative of phenotypic outcomes in Western fence lizard.

BACKGROUND: The health and resilience of species in natural environments is increasingly challenged by complex anthropogenic stressor combinations including climate change, habitat encroachment, and chemical contamination. To better understand impacts of these stressors we examined the individual- and combined-stressor impacts of malaria infection, food limitation, and 2,4,6-trinitrotoluene (TNT) exposures on gene expression in livers of Western fence lizards (WFL, Sceloporus occidentalis) using custom WFL transcriptome-based microarrays. RESULTS: Computational analysis including annotation enrichment and correlation analysis identified putative functional mechanisms linking transcript expression and toxicological phenotypes. TNT exposure increased transcript expression for genes involved in erythropoiesis, potentially in response to TNT-induced anemia and/or methemoglobinemia and caused dose-specific effects on genes involved in lipid and overall energy metabolism consistent with a hormesis response of growth stimulation at low doses and adverse decreases in lizard growth at high doses. Functional enrichment results were indicative of inhibited potential for lipid mobilization and catabolism in TNT exposures which corresponded with increased inguinal fat weights and was suggestive of a decreased overall energy budget. Malaria infection elicited enriched expression of multiple immune-related functions likely corresponding to increased white blood cell (WBC) counts. Food limitation alone enriched functions related to cellular energy production and decreased expression of immune responses consistent with a decrease in WBC levels. CONCLUSIONS: Despite these findings, the lizards demonstrated immune resilience to malaria infection under food limitation with transcriptional results indicating a fully competent immune response to malaria, even under bio-energetic constraints. Interestingly, both TNT and malaria individually increased transcriptional expression of immune-related genes and increased overall WBC concentrations in blood; responses that were retained in the TNT x malaria combined exposure. The results demonstrate complex and sometimes unexpected responses to multiple stressors where the lizards displayed remarkable resiliency to the stressor combinations investigated.

Comparative toxicogenomics of three insensitive munitions constituents 2,4-dinitroanisole, nitroguanidine and nitrotriazolone in the soil nematode Caenorhabditis elegans.

BACKGROUND: Ecotoxicological studies on the insensitive munitions formulation IMX-101 and its components 2,4-dinitroanisole (DNAN), nitroguanidine (NQ) and nitrotriazolone (NTO) in various organisms showed that DNAN was the main contributor to the overall toxicity of IMX-101 and suggested that the three compounds acted independently. These results motivated this toxicogenomics study to discern toxicological mechanisms for these compounds at the molecular level. METHODS: Here we used the soil nematode Caenorhabditis elegans, a well-characterized genomics model, as the test organism and a species-specific, transcriptome-wide 44 K-oligo probe microarray for gene expression analysis. In addition to the control treatment, C. elegans were exposed for 24 h to 6 concentrations of DNAN (1.95-62.5 ppm) or NQ (83-2667 ppm) or 5 concentrations of NTO (187-3000 ppm) with ten replicates per treatment. The nematodes were transferred to a clean environment after exposure. Reproduction endpoints (egg and larvae counts) were measured at three time points (i.e., 24-, 48- and 72-h). Gene expression profiling was performed immediately after 24-h exposure to each chemical at the lowest, medium and highest concentrations plus the control with four replicates per treatment. RESULTS: Statistical analyses indicated that chemical treatment did not significantly affect nematode reproduction but did induce 2175, 378, and 118 differentially expressed genes (DEGs) in NQ-, DNAN-, and NTO-treated nematodes, respectively. Bioinformatic analysis indicated that the three compounds shared both DEGs and DEG-mapped Reactome pathways. Gene set enrichment analysis further demonstrated that DNAN and NTO significantly altered 12 and 6 KEGG pathways, separately, with three pathways in common. NTO mainly affected carbohydrate, amino acid and xenobiotics metabolism while DNAN disrupted protein processing, ABC transporters and several signal transduction pathways. NQ-induced DEGs were mapped to a wide variety of metabolism, cell cycle, immune system and extracellular matrix organization pathways. CONCLUSION: Despite the absence of significant effects on apical reproduction endpoints, DNAN, NTO and NQ caused significant alterations in gene expression and pathways at 1.95 ppm, 187 ppm and 83 ppm, respectively. This study provided supporting evidence that the three chemicals may exert independent toxicity by acting on distinct molecular targets and pathways.

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.

Predicting chemical bioavailability using microarray gene expression data and regression modeling: A tale of three explosive compounds.

BACKGROUND: Chemical bioavailability is an important dose metric in environmental risk assessment. Although many approaches have been used to evaluate bioavailability, not a single approach is free from limitations. Previously, we developed a new genomics-based approach that integrated microarray technology and regression modeling for predicting bioavailability (tissue residue) of explosives compounds in exposed earthworms. In the present study, we further compared 18 different regression models and performed variable selection simultaneously with parameter estimation. RESULTS: This refined approach was applied to both previously collected and newly acquired earthworm microarray gene expression datasets for three explosive compounds. Our results demonstrate that a prediction accuracy of R(2) = 0.71-0.82 was achievable at a relatively low model complexity with as few as 3-10 predictor genes per model. These results are much more encouraging than our previous ones. CONCLUSION: This study has demonstrated that our approach is promising for bioavailability measurement, which warrants further studies of mixed contamination scenarios in field settings.

Inter- and intraspecies chemical sensitivity: a case study using 2,4-dinitroanisole.

Insensitive munitions offer increased safety because of their "insensitivity" to unintended detonation relative to historically used formulations such as 2,4,6-trinitrotoluene (TNT). Dinitroanisole (DNAN) is an insensitive munition constituent, and its solubility and stability warrant investigations of potential toxicological hazard related to manufacturing discharges and training ranges. Although ecotoxicology data are available for other insensitive munition constituents, few data are available for DNAN. In the present study, acute and chronic exposures of a fish (Pimephales promelas) and 2 cladocerans (Ceriodaphnia dubia, Daphnia pulex) were conducted. The 50% lethal concentration (LC50) values of DNAN ranged from 14.2 mg/L to 42.0 mg/L, depending on species. In chronic exposures, fish survival (LC50 = 10.0 mg/L) was more sensitive than cladoceran survival (LC50 = 13.7 to >24.2 mg/L). However, cladoceran reproduction was equally or more sensitive to DNAN (50% inhibition values 2.7-10.6 mg/L, depending on species) than fish endpoints. Daphnia pulex was the most sensitive species, with only slight differences between the 3 populations tested. Although the aquatic toxicity of DNAN was lower than previously reported in the literature for TNT, future research is needed to determine the potential synergistic toxicity of all the constituents in insensitive munition mixtures and the implications of photo-oxidation.

SeqAssist: a novel toolkit for preliminary analysis of next-generation sequencing data.

BACKGROUND: While next-generation sequencing (NGS) technologies are rapidly advancing, an area that lags behind is the development of efficient and user-friendly tools for preliminary analysis of massive NGS data. As an effort to fill this gap to keep up with the fast pace of technological advancement and to accelerate data-to-results turnaround, we developed a novel software package named SeqAssist ("Sequencing Assistant" or SA). RESULTS: SeqAssist takes NGS-generated FASTQ files as the input, employs the BWA-MEM aligner for sequence alignment, and aims to provide a quick overview and basic statistics of NGS data. It consists of three separate workflows: (1) the SA_RunStats workflow generates basic statistics about an NGS dataset, including numbers of raw, cleaned, redundant and unique reads, redundancy rate, and a list of unique sequences with length and read count; (2) the SA_Run2Ref workflow estimates the breadth, depth and evenness of genome-wide coverage of the NGS dataset at a nucleotide resolution; and (3) the SA_Run2Run workflow compares two NGS datasets to determine the redundancy (overlapping rate) between the two NGS runs. Statistics produced by SeqAssist or derived from SeqAssist output files are designed to inform the user: whether, what percentage, how many times and how evenly a genomic locus (i.e., gene, scaffold, chromosome or genome) is covered by sequencing reads, how redundant the sequencing reads are in a single run or between two runs. These statistics can guide the user in evaluating the quality of a DNA library prepared for RNA-Seq or genome (re-)sequencing and in deciding the number of sequencing runs required for the library. We have tested SeqAssist using a synthetic dataset and demonstrated its main features using multiple NGS datasets generated from genome re-sequencing experiments. CONCLUSIONS: SeqAssist is a useful and informative tool that can serve as a valuable "assistant" to a broad range of investigators who conduct genome re-sequencing, RNA-Seq, or de novo genome sequencing and assembly experiments.

Building quantitative prediction models for tissue residue of two explosives compounds in earthworms from microarray gene expression data.

Soil contamination near munitions plants and testing grounds is a serious environmental concern that can result in the formation of tissue chemical residue in exposed animals. Quantitative prediction of tissue residue still represents a challenging task despite long-term interest and pursuit, as tissue residue formation is the result of many dynamic processes including uptake, transformation, and assimilation. The availability of high-dimensional microarray gene expression data presents a new opportunity for computational predictive modeling of tissue residue from changes in expression profile. Here we analyzed a 240-sample data set with measurements of transcriptomic-wide gene expression and tissue residue of two chemicals, 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), in the earthworm Eisenia fetida. We applied two different computational approaches, LASSO (Least Absolute Shrinkage and Selection Operator) and RF (Random Forest), to identify predictor genes and built predictive models. Each approach was tested alone and in combination with a prior variable selection procedure that involved the Wilcoxon rank-sum test and HOPACH (Hierarchical Ordered Partitioning And Collapsing Hybrid). Model evaluation results suggest that LASSO was the best performer of minimum complexity on the TNT data set, whereas the combined Wilcoxon-HOPACH-RF approach achieved the highest prediction accuracy on the RDX data set. Our models separately identified two small sets of ca. 30 predictor genes for RDX and TNT. We have demonstrated that both LASSO and RF are powerful tools for quantitative prediction of tissue residue. They also leave more unknown than explained, however, allowing room for improvement with other computational methods and extension to mixture contamination scenarios.