Project description:Production, usage and disposal of the munitions constituent (MC) cyclotrimethylenetrinitramine (RDX) has led to environmental releases on military facilities. The chemical attributes of RDX are conducive for leaching to surface water which may put aquatic organisms at risk of exposure. Because RDX has been observed to cause aberrant neuromuscular effects across a wide range of animal phyla, we assessed the effects of RDX on central nervous system (CNS) function in the representative aquatic ecotoxicological model species, fathead minnow (Pimephales promelas). A brain-tissue based cDNA library enriched for transcripts differentially expressed in response to RDX exposure was developed for fathead minnow and was transitioned to custom cDNA-based microarrays. All 4,128 cDNAs were sequenced, quality filtered and assembled yielding 3,018 unique sequences and 945 significant blastx matches (E ≤ 10-5). Bioassays were conducted exposing fathead minnows to RDX at 0.625, 1.25, 2.5, 5, 10 mg/L or an acetone-spike control for 10d. Overt toxicity of RDX in fathead minnow occurred only at the highest exposure concentration resulting in 50% mortality. Conversely, Bayesian analysis of microarray data indicated significant changes in transcript expression in fathead minnow brain tissue at RDX concentrations as low as 0.625 mg/L. In total, 154 microarray targets representing 44 unique transcript identities were differentially expressed in RDX exposures, the majority of which were validated by RT-qPCR. Investigation of molecular pathways, gene ontology and individual gene functions indicated that RDX exposures affected metabolic processes involved in: oxygen transport, neurological function, calcium binding / signaling, energy metabolism, cell cycle / cell proliferation, oxidative stress and ubiquitination. In total, our study indicated that RDX exposure affected molecular processes critical to CNS function in fathead minnow. 10 Day RDX Exposure, Brain Tissue Investigation: Sub-adult fathead minnows were exposed to RDX in a 10d dose-series experiment (0.625, 1.25, 2.5, 5.0, or 10 mg/L RDX) which included an acetone-spike control (1% acetone). Each experimental treatment included 8 replicate fish (48 total fish) and endpoints included mortality, total weight and neurotoxicogenomics. The 1.25mg/L dose was not included in the microarray experiment. Please see attached PDF file for detailed 'Balanced, Interwoven Loop Design'.

Project description:Paper Abstract. Extrapolating toxicological sensitivity information across species can require the use of highly conservative safety factors as prescribed by ecological risk assessment protocols. The objective of our study was to use multipule factors to understand the dynamics of stress responses by comparing ecotoxicological, developmental, and genomic data to quantitatively determine the relative sensitivity of two common model aquatic vertebrates to assess the potential for developing refined safety factors among species. Larval (3-d old) zebrafish (Danio rerio) and fathead minnows (Pimephales promelas) were concurrently exposed in 96-h static exposures to the widely-used military energetic compound cyclotrimethylenetrinitramine (RDX). Endpoints assessed included mortality, incidence of vertebral deformities, swimming behavior, and differential gene (transcript) expression. Analytically verified treatment concentrations ranged from 0.9 to 27.7 mg/L RDX. While RDX concentrations prepared for exposures included 0, 0.9, 1.8, 3.5, 7.5, 15.0, and 30.0 mg/L, analytically verified treatment concentrations were 0, 0.88, 1.75, 3.46, 6.96, 13.79, and 27.7 mg/L, respectively. Changes in prepared RDX concentrations and analytically verified treatment concentrations are a result of chemical breakdown, a well known characteristic of RDX Our results indicated a higher sensitivity to RDX in P. promelas than in D. rerio in both mortality (LOECs 13.79 and 27.7 mg/L, respectively) and vertebral deformity (LOECs 13.79 and >13.79, respectively) endpoints. Swimming behavior was assessed in 10 min behavioral trials using Noldus EthoVision® digital tracking software. Swimming behavior trials indicated non-significant trends of decreased velocity, distance moved, and increased meander (change in direction per cm traveled) for P. promelas. No such trends were apparent in D. rerio swimming behavior. A comprehensive genomic analysis is in progress to determine the genes, gene ontology elements, metabolic pathways and metabolic networks affected in common among species in the RDX exposure. Taken as a whole, the ecotoxicological, developmental and genomic results will provide valuable insights into the similarity in response among these fish species to RDX. Three-day-old fathead minnow (FHM, Pimephales promelas) or Zebrafish (ZF, Danio rerio) fry were exposed to either control water or one of six concentrations of RDX (0.9, 1.9, 3.8, 7.5, 15, or 27.7 mg/L) in 96-h exposures. However, microarray analysis was only performed on FHM exposed to 0, 0.9, 1.9, 7.5, or 15 mg/L RDX and ZF exposed to 0, 0.9, 1.9, 3.8, 7.5, or 15 mg/L RDX due to insufficient tissue for microarray analysis due to animal death at the other RDX concentrations. The experimental design was based on the U.S. EPA (2002) acute testing method for P. promelas, test method 2000.0 and included four replicates per treatment with ten fish per replicate. Whole fish (10 fish per replicate) were investigated for differential expression in response to RDX exposure.

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Pimephales promelas

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