Chronic aquatic toxicity of perfluorooctane sulfonic acid (PFOS) to Ceriodaphnia dubia, Chironomus dilutus, Danio rerio, and Hyalella azteca.

Perfluorooctane sulfonic acid (PFOS) is a ubiquitous and persistent contaminant in aquatic ecosystems. Chronic toxicity information for aquatic organisms is limited, therefore we conducted chronic PFOS toxicity tests for four model organisms commonly used for freshwater toxicology assays: Chironomus dilutus (midge), Ceriodaphnia dubia (water flea), Hyalella azteca (amphipod) and Danio rerio (zebrafish). The 16-day survival test with C. dilutus resulted in the lowest PFOS exposure concentrations to cause significant impacts, with reduced survival at 1 µg/L, a LC50 of 7.5 µg/L, and a growth EC10 of 1.5 µg/L. D. rerio was the next most sensitive species, with a 30-day LC50 of 490 µg/L and reduced growth at 260 µg/L. Effects for C. dubia and H. azteca occurred at concentrations a thousand-fold higher than for C. dilutus. H. azteca had a 42-day LC50 of 15 mg/L, an EC50 of 3.8 mg/L for reproduction (neonates per female) and an EC50 of 4.7 mg/L for growth. C. dubia was similarly tolerant of PFOS, with a 6-day LC50 of 20 mg/L for survival and an EC50 of 7 mg/L for reproduction (neonates per adult). H. azteca, C. dubia, and, to a lesser extent, D. rerio, appear tolerant of PFOS concentrations typically found in the environment. However, in agreement with previous studies, C. dilutus was particularly sensitive to PFOS exposure, with lethal and sublethal effects occurring at concentration levels present at highly contaminated sites.

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.

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.

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.

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.

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.

A weight of evidence assessment approach for adverse outcome pathways.

The adverse outcome pathway (AOP) is a framework to mechanistically link molecular initiating events to adverse biological outcomes. From a regulatory perspective, it is of crucial importance to determine the confidence for the AOP in question as well as the quality of data available in supporting this evaluation. A weight of evidence approach has been proposed for this task, but many of the existing frameworks for weight of evidence evaluation are qualitative and there is not clear guidance regarding how weight of evidence should be calculated for an AOP. In this paper we advocate the use of a subject matter expertise driven approach for weight of evidence evaluation based on criteria and metrics related to data quality and the strength of causal linkages between key events. As a demonstration, we notionally determine weight of evidence scores for two AOPs: Non-competitive ionotropic GABA receptor antagonism leading to epileptic seizures, and Antagonist-binding and stabilization of a co-repressor to the peroxisome proliferator-activated receptor α (PPARα) signaling complex ultimately causing starvation-like weight loss.

Daphnia magna's sense of competition: intra-specific interactions (ISI) alter life history strategies and increase metals toxicity.

This work investigates whether the scale-up to multi-animal exposures that is commonly applied in genomics studies provides equivalent toxicity outcomes to single-animal experiments of standard Daphnia magna toxicity assays. Specifically, we tested the null hypothesis that intraspecific interactions (ISI) among D. magna have neither effect on the life history strategies of this species, nor impact toxicological outcomes in exposure experiments with Cu and Pb. The results show that ISI significantly increased mortality of D. magna in both Cu and Pb exposure experiments, decreasing 14 day LC50 s and 95 % confidence intervals from 14.5 (10.9-148.3) to 8.4 (8.2-8.7) µg Cu/L and from 232 (156-4810) to 68 (63-73) µg Pb/L. Additionally, ISI potentiated Pb impacts on reproduction eliciting a nearly 10-fold decrease in the no-observed effect concentration (from 236 to 25 µg/L). As an indication of environmental relevance, the effects of ISI on both mortality and reproduction in Pb exposures were sustained at both high and low food rations. Furthermore, even with a single pair of Daphnia, ISI significantly increased (p < 0.05) neonate production in control conditions, demonstrating that ISI can affect life history strategy. Given these results we reject the null hypothesis and conclude that results from scale-up assays cannot be directly applied to observations from single-animal assessments in D. magna. We postulate that D. magna senses chemical signatures of conspecifics which elicits changes in life history strategies that ultimately increase susceptibility to metal toxicity.

Systems toxicology identifies mechanistic impacts of 2-amino-4,6-dinitrotoluene (2A-DNT) exposure in Northern Bobwhite.

BACKGROUND: A systems toxicology investigation comparing and integrating transcriptomic and proteomic results was conducted to develop holistic effects characterizations for the wildlife bird model, Northern bobwhite (Colinus virginianus) dosed with the explosives degradation product 2-amino-4,6-dinitrotoluene (2A-DNT). A subchronic 60 d toxicology bioassay was leveraged where both sexes were dosed via daily gavage with 0, 3, 14, or 30 mg/kg-d 2A-DNT. Effects on global transcript expression were investigated in liver and kidney tissue using custom microarrays for C. virginianus in both sexes at all doses, while effects on proteome expression were investigated in liver for both sexes and kidney in males, at 30 mg/kg-d. RESULTS: As expected, transcript expression was not directly indicative of protein expression in response to 2A-DNT. However, a high degree of correspondence was observed among gene and protein expression when investigating higher-order functional responses including statistically enriched gene networks and canonical pathways, especially when connected to toxicological outcomes of 2A-DNT exposure. Analysis of networks statistically enriched for both transcripts and proteins demonstrated common responses including inhibition of programmed cell death and arrest of cell cycle in liver tissues at 2A-DNT doses that caused liver necrosis and death in females. Additionally, both transcript and protein expression in liver tissue was indicative of induced phase I and II xenobiotic metabolism potentially as a mechanism to detoxify and excrete 2A-DNT. Nuclear signaling assays, transcript expression and protein expression each implicated peroxisome proliferator-activated receptor (PPAR) nuclear signaling as a primary molecular target in the 2A-DNT exposure with significant downstream enrichment of PPAR-regulated pathways including lipid metabolic pathways and gluconeogenesis suggesting impaired bioenergetic potential. CONCLUSION: Although the differential expression of transcripts and proteins was largely unique, the consensus of functional pathways and gene networks enriched among transcriptomic and proteomic datasets provided the identification of many critical metabolic functions underlying 2A-DNT toxicity as well as impaired PPAR signaling, a key molecular initiating event known to be affected in di- and trinitrotoluene exposures.

Relating suborganismal processes to ecotoxicological and population level endpoints using a bioenergetic model.

Ecological effects of environmental stressors are commonly evaluated using organismal or suborganismal data, such as standardized toxicity tests that characterize responses of individuals (e.g., mortality and reproduction) and a rapidly growing body of "omics" data. A key challenge for environmental risk assessment is relating such information to population dynamics. One approach uses dynamic energy budget (DEB) models that relate growth and reproduction of individuals to underlying flows of energy and elemental matter. We hypothesize that suborganismal information identifies DEB parameters that are most likely impacted by a particular stressor and that the DEB model can then project suborganismal effects on life history and population endpoints. We formulate and parameterize a model of growth and reproduction for the water flea Daphnia magna. Our model resembles previous generic bioenergetic models, but has explicit representation of discrete molts, an important feature of Daphnia life history. We test its ability to predict six endpoints commonly used in chronic toxicity studies in specified food environments. With just one adjustable parameter, the model successfully predicts growth and reproduction of individuals from a wide array of experiments performed in multiple laboratories using different clones of D. magna raised on different food sources. Fecundity is the most sensitive endpoint, and there is broad correlation between the sensitivities of fecundity and long-run growth rate, as is desirable for the default metric used in chronic toxicity tests. Under some assumptions, we can combine our DEB model with the Euler-Lotka equation to estimate longrun population growth rates at different food levels. A review of Daphnia gene-expression experiments on the effects of contaminant exposure reveals several connections to model parameters, in particular a general trend of increased transcript expression of genes involved in energy assimilation and utilization at concentrations affecting growth and reproduction. The sensitivity of fecundity to many model parameters was consistent with frequent generalized observations of decreased expression of genes involved in reproductive physiology, but interpretation of these observations requires further mechanistic modeling. We thus propose an approach based on generic DEB models incorporating few essential species-specific features for rapid extrapolation of ecotoxicogenomic assays for Daphnia-based population risk assessment.

Coral-zooxanthellae meta-transcriptomics reveals integrated response to pollutant stress.

BACKGROUND: Corals represent symbiotic meta-organisms that require harmonization among the coral animal, photosynthetic zooxanthellae and associated microbes to survive environmental stresses. We investigated integrated-responses among coral and zooxanthellae in the scleractinian coral Acropora formosa in response to an emerging marine pollutant, the munitions constituent, 1,3,5-trinitro-1,3,5 triazine (RDX; 5 day exposures to 0 (control), 0.5, 0.9, 1.8, 3.7, and 7.2 mg/L, measured in seawater). RESULTS: RDX accumulated readily in coral soft tissues with bioconcentration factors ranging from 1.1 to 1.5. Next-generation sequencing of a normalized meta-transcriptomic library developed for the eukaryotic components of the A. formosa coral holobiont was leveraged to conduct microarray-based global transcript expression analysis of integrated coral/zooxanthellae responses to the RDX exposure. Total differentially expressed transcripts (DET) increased with increasing RDX exposure concentrations as did the proportion of zooxanthellae DET relative to the coral animal. Transcriptional responses in the coral demonstrated higher sensitivity to RDX compared to zooxanthellae where increased expression of gene transcripts coding xenobiotic detoxification mechanisms (i.e. cytochrome P450 and UDP glucuronosyltransferase 2 family) were initiated at the lowest exposure concentration. Increased expression of these detoxification mechanisms was sustained at higher RDX concentrations as well as production of a physical barrier to exposure through a 40% increase in mucocyte density at the maximum RDX exposure. At and above the 1.8 mg/L exposure concentration, DET coding for genes involved in central energy metabolism, including photosynthesis, glycolysis and electron-transport functions, were decreased in zooxanthellae although preliminary data indicated that zooxanthellae densities were not affected. In contrast, significantly increased transcript expression for genes involved in cellular energy production including glycolysis and electron-transport pathways was observed in the coral animal. CONCLUSIONS: Transcriptional network analysis for central energy metabolism demonstrated highly correlated responses to RDX among the coral animal and zooxanthellae indicative of potential compensatory responses to lost photosynthetic potential within the holobiont. These observations underscore the potential for complex integrated responses to RDX exposure among species comprising the coral holobiont and highlight the need to understand holobiont-species interactions to accurately assess pollutant impacts.

Survival, Growth, and Reproduction Responses in a Three-Generation Exposure of the Zebrafish (Danio rerio) to Perfluorooctane Sulfonate.

A prior multigenerational perfluorooctane sulfonic acid (PFOS) exposure investigation in zebrafish reported adverse effects at 0.734 µg/L, among the lowest aquatic effect levels for PFOS reported to date. The present three-generation PFOS exposure quantified survival, growth, reproduction, and vitellogenin (VTG; egg yolk protein) responses in zebrafish, incorporating experimental design and procedural improvements relative to the earlier study. Exposures targeting 0.1, 0.6, 3.2, 20, and 100 µg/L in parental (P) and first filial (F1) generations lasted for 180 days post fertilization (dpf) and the second filial generation (F2) through 16 dpf. Survival decreased significantly in P and F2 generation exposures, but not in F1, at the highest PFOS treatment (100 µg/L nominal, 94-205 µg/L, measured). Significant adverse effects on body weight and length were infrequent, of low magnitude, and occurred predominantly at the highest exposure treatment. Finally, PFOS had no significant effects on P or F1 egg production and survival or whole-body VTG levels in P or F1 male fish. Overall, the predominance and magnitude of adverse PFOS effects at <1 µg/L reported in prior research were largely nonrepeatable in the present study. In contrast, the present study indicated a threshold for ecologically relevant adverse effects in zebrafish at 117 µg/L (SE 8 µg/L, n = 10) for survival and 47 µg/L (SE 11 µg/L, n = 19) for all statistically significant negative effects observed. Environ Toxicol Chem 2024;43:115-131. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Interspecific effects of 4A-DNT (4-amino-2,6-dinitrotoluene) and RDX (1,3,5-trinitro-1,3,5-triazine) in Japanese quail, Northern bobwhite, and Zebra finch.

The purpose of this study was to assess the toxicological effects of two munition compounds, 4-amino-2,6-dinitrotoluene (4A-DNT) and 1,3,5-trinitro-1,3,5-triazine (RDX), on three different bird species: two common toxicological model species-the Northern Bobwhite (Colinus virginianus) and the Japanese Quail (Coturnix japonica), and a representative passerine-the Zebra Finch (Taeniopygia guttata). Bobwhite were exposed to 4A-DNT at 0, 8, 15, 30, 60, or 150 mg/kg body weight (bw) d by oral gavage for seven days; because the high dose of 4A-DNT was lethal to bobwhite, the maximum dose was changed to 100 mg/kg bw d for Japanese quail and finches to ensure tissue could be used for future toxicogenomic work. RDX was similarly administered at 0, 0.5, 1.5, 3, 6, or 12 mg/kg bw d. Blood was drawn prior to euthanasia for blood cellularity and chemistry analyses. Finches were clearly least affected by 4A-DNT as evidenced by a lack of observable effects. Bobwhite appeared to be the most sensitive species to 4A-DNT as observed through changes in blood cellularity and plasma chemistry effects. Bobwhite appeared to be more sensitive to RDX than Japanese Quail due to increased effects on measures of plasma chemistries. Finches exhibited the greatest sensitivity to RDX through increased mortality and seizure activity. This study suggests that sensitivity among species is chemical-specific and provides data that could be used to refine current avian sensitivity models used in ecological risk assessments.

Cellular and molecular responses to ethyl-parathion in undifferentiated SH-SY5Y cells provide neurotoxicity pathway indicators for organophosphorus impacts.

High-fidelity nonanimal screening methods are needed that can rapidly and accurately characterize organophosphorus compound (OP)-induced neurotoxicity. Herein, the efficacy of human neuroblastoma cell line (SH-SY5Y) to provide molecular and cellular responses characteristic of the OP neurotoxicity pathway was investigated in response to the OP-model compound, ethyl-parathion. Undifferentiated SH-SY5Y cells were exposed to ethyl-parathion for 30 min at 0 (control), 0.5, 2.5, 5, 10, and 25 µg/ml. Dose-responsive reductions in cell viability were observed with significant reductions at ≥10 µg/ml. From these results, ethyl-parathion exposures of 0 (control), 5, and 10 µg/ml were selected to examine bioindicators underlying the OP neurotoxicity pathway including: reactive oxygen species (ROS), cell membrane peroxidation, mitochondrial membrane potential (MMP), and apoptosis. Ethyl-parathion elicited highly significant increases in ROS relative to controls (p < .01) at both exposure concentrations, confirmed using N-acetyl cysteine (NAC) as a ROS quencher which alleviated ROS increases. A response characteristic of increased ROS exposure, cell membrane-lipid peroxidation, significantly increased (p < .05) at the highest ethyl-parathion exposure (10 µg/ml). As a likely consequence of membrane-lipid peroxidation, ethyl-parathion-induced reductions in MMP were observed with significant effects at 10 µg/ml, reducing MMP by 58.2%. As a culmination of these cellular-damage indicators, apoptosis progression was investigated by phosphatidylserine translocation where ethyl-parathion-induced dose-responsive, highly significant (p < .01) increases at both 5 and 10 µg/ml. Overall, the mechanistic responses observed in undifferentiated SH-SY5Y cells corresponded with in vivo mammalian results demonstrating potential for this nonanimal model to provide accurate OP neurotoxicology screening.

A systems toxicology approach to elucidate the mechanisms involved in RDX species-specific sensitivity.

Interspecies uncertainty factors in ecological risk assessment provide conservative estimates of risk where limited or no toxicity data is available. We quantitatively examined the validity of interspecies uncertainty factors by comparing the responses of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) to the energetic compound 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), a known neurotoxicant. Relative toxicity was measured through transcriptional, morphological, and behavioral end points in zebrafish and fathead minnow fry exposed for 96 h to RDX concentrations ranging from 0.9 to 27.7 mg/L. Spinal deformities and lethality occurred at 1.8 and 3.5 mg/L RDX respectively for fathead minnow and at 13.8 and 27.7 mg/L for zebrafish, indicating that zebrafish have an 8-fold greater tolerance for RDX than fathead minnow fry. The number and magnitude of differentially expressed transcripts increased with increasing RDX concentration for both species. Differentially expressed genes were enriched in functions related to neurological disease, oxidative-stress, acute-phase response, vitamin/mineral metabolism and skeletal/muscular disorders. Decreased expression of collagen-coding transcripts were associated with spinal deformity and likely involved in sensitivity to RDX. Our work provides a mechanistic explanation for species-specific sensitivity to RDX where zebrafish responded at lower concentrations with greater numbers of functions related to RDX tolerance than fathead minnow. While the 10-fold interspecies uncertainty factor does provide a reasonable cross-species estimate of toxicity in the present study, the observation that the responses between ZF and FHM are markedly different does initiate a call for concern regarding establishment of broad ecotoxicological conclusions based on model species such as zebrafish.

Multiple environmental stressors elicit complex interactive effects in the western fence lizard (Sceloporus occidentalis).

Evaluation of multiple-stressor effects stemming from habitat degradation, climate change, and exposure to chemical contaminants is crucial for addressing challenges to ecological and environmental health. To assess the effects of multiple stressors in an understudied taxon, the western fence lizard (Sceloporus occidentalis) was used to characterize the individual and combined effects of food limitation, exposure to the munitions constituent 2,4,6-trinitrotoluene (TNT), and Plasmodium mexicanum (lizard malaria) infection. Three experimental assays were conducted including: Experiment I--TNT × Food Limitation, Experiment II--Food Limitation × Malaria Infection, and Experiment III--TNT × Malaria Infection. All experiments had a 30 day duration, the malaria treatment included infected and non infected control lizards, food limitation treatments included an ad libitum control and at least one reduced food ration and TNT exposures consisting of daily oral doses of corn oil control or a corn oil-TNT suspension at 5, 10, 20, 40 mg/kg/day. The individual stressors caused a variety of effects including: reduced feeding, reduced testes mass, anemia, increased white blood cell (WBC) concentrations and increased mass of liver, kidney and spleen in TNT exposures; reduced cholesterol, WBC concentrations and whole body, testes and inguinal fat weights given food limitation; and increased WBC concentrations and spleen weights as well as decreased cholesterol and testes mass in malaria infected lizards. Additive and interactive effects were found among certain stressor combinations including elimination of TNT-induced hormesis for growth under food limitation. Ultimately, our study indicates the potential for effects modulation when environmental stressors are combined.

Effect of UV-light exposure duration, light source, and aging on nitroguanidine (NQ) degradation product profile and toxicity.

Previous studies have reported increased aquatic toxicity of UV-degraded nitroguanidine (NQ), but many details underlying the dynamics of NQ degradation and toxicity remain unknown. These data gaps represent critical barriers to assessing the environmental relevance of laboratory-generated UV-degradation results and extrapolation to environmental risk. In the present study, the toxicity of NQ increased with increasing proportional degradation of the parent compound. Specifically, while the LC50 of undegraded NQ was 1485 mg/L, the toxicity at the lowest degradation level examined (7% parent compound degraded) increased by nearly two-orders of magnitude (LC50 = 17.3 mg/L) and increased by nearly three-orders of magnitude (LC50 = 6.23 mg/L) in the highest percent NQ degradation (90%) treatment. Similar LC50 values between immediately tested and aged (8-13 days) NQ degradation products suggested the degradation product(s) causing the toxicity were stable, although concentrations of nitrite and nitrate increased after aging. Finally, experiments where NQ was degraded in natural sunlight confirmed increased toxicity in environmentally relevant D. pulex exposures; however, the two-order of magnitude increase in toxicity (LC50 = 21.3 mg/L) at 53% degradation was less than NQ degraded by a laboratory photoreactor by a similar percentage (46% degraded). Identification of principal toxic agents in the UV-degraded NQ product mixture remains a critical data gap. Mass balance calculations were generated for our experimental results and literature values revealing difficulty in accounting for all NQ degradation products. Products with suspected high potency in D. pulex were identified which require further testing including: nitrosoguanidine, nitrosourea, and hydroxylamine. SYNOPSIS: The toxicity of NQ increased with increasing UV-degradation where toxicity-inducing degradation products were stable over 1-2 weeks; increased toxicity was validated from natural-sunlight degradation of NQ, however toxicity was lower than UV-photoreactor degraded NQ; and the identity of specific toxic UV-degradation products remains elusive where carefully-designed mass-balance experiments and toxicity testing are needed to provide definitive identification.

Key challenges and developments in wildlife ecological risk assessment: Problem formulation.

Problem formulation (PF) is a critical initial step in planning risk assessments for chemical exposures to wildlife, used either explicitly or implicitly in various jurisdictions to include registration of new pesticides, evaluation of new and existing chemicals released to the environment, and characterization of impact when chemical releases have occurred. Despite improvements in our understanding of the environment, ecology, and biological sciences, few risk assessments have used this information to enhance their value and predictive capabilities. In addition to advances in organism-level mechanisms and methods, there have been substantive developments that focus on population- and systems-level processes. Although most of the advances have been recognized as being state-of-the-science for two decades or more, there is scant evidence that they have been incorporated into wildlife risk assessment or risk assessment in general. In this article, we identify opportunities to consider elevating the relevance of wildlife risk assessments by focusing on elements of the PF stage of risk assessment, especially in the construction of conceptual models and selection of assessment endpoints that target population- and system-level endpoints. Doing so will remain consistent with four established steps of existing guidance: (1) establish clear protection goals early in the process; (2) consider how data collection using new methods will affect decisions, given all possibilities, and develop a decision plan a priori; (3) engage all relevant stakeholders in creating a robust, holistic conceptual model that incorporates plausible stressors that could affect the targets defined in the protection goals; and (4) embrace the need for iteration throughout the PF steps (recognizing that multiple passes may be required before agreeing on a feasible plan for the rest of the risk assessment). Integr Environ Assess Manag 2022;00:1-16. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Conserved toxic responses across divergent phylogenetic lineages: a meta-analysis of the neurotoxic effects of RDX among multiple species using toxicogenomics.

At military training sites, a variety of pollutants such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), may contaminate the area originating from used munitions. Studies investigating the mechanism of toxicity of RDX have shown that it affects the central nervous system causing seizures in humans and animals. Environmental pollutants such as RDX have the potential to affect many different species, therefore it is important to establish how phylogenetically distant species may respond to these types of emerging pollutants. In this paper, we have used a transcriptional network approach to compare and contrast the neurotoxic effects of RDX among five phylogenetically disparate species: rat (Sprague-Dawley), Northern bobwhite quail (Colinus virginianus), fathead minnow (Pimephales promelas), earthworm (Eisenia fetida), and coral (Acropora formosa). Pathway enrichment analysis indicated a conservation of RDX impacts on pathways related to neuronal function in rat, Northern bobwhite quail, fathead minnows and earthworm, but not in coral. As evolutionary distance increased common responses decreased with impacts on energy and metabolism dominating effects in coral. A neurotransmission related transcriptional network based on whole rat brain responses to RDX exposure was used to identify functionally related modules of genes, components of which were conserved across species depending upon evolutionary distance. Overall, the meta-analysis using genomic data of the effects of RDX on several species suggested a common and conserved mode of action of the chemical throughout phylogenetically remote organisms.

Perfluorooctanesulfonic Acid-Induced Toxicity on Zebrafish Embryos in the Presence or Absence of the Chorion.

Perfluorooctanesulfonic acid (PFOS) is a perfluorinated compound used in many industrial and consumer products. It has been linked to a broad range of adverse effects in several species, including zebrafish (Danio rerio). The zebrafish embryo is a widely used vertebrate model to elucidate potential adverse effects of chemicals because it is amenable to medium and high throughput. However, there is limited research on the full extent of the impact the chorion has on those effects. Results from the present study indicate that the presence of the chorion affected the timing and incidence of mortality as well as morphometric endpoints such as spinal curvature and swim bladder inflation in zebrafish embryos exposed to PFOS. Furthermore, removal of the chorion prior to exposure resulted in a lower threshold of sensitivity to PFOS for effects on transcriptional expression within the peroxisome proliferator-activated receptor (PPAR) nuclear signaling pathway. Perturbation of PPAR pathway gene expression can result in disruption of metabolic signaling and regulation, which can adversely affect development, energy availability, and survival. It can be concluded that removal of the chorion has significant effects on the timing and incidence of impacts associated with PFOS exposure, and more research is warranted to fully elucidate the protective role of the chorion and the critical timing of these events. Environ Toxicol Chem 2021;40:780-791. Published 2020. This article is a US Government work and is in the public domain in the USA. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.