A Systems Toxicology Approach to Elucidate the Mechanisms Involved in RDX Species-Specific Sensitivity Christopher M. Warner, †,‡ Kurt A. Gust, †, * Jacob K. Stanley, † Tanwir Habib, § Mitchell S. Wilbanks, † Nata ̀ lia Garcia-Reyero, ∥ and Edward J. Perkins † † Environmental Laboratory, U.S. Army Engineer Research and Development Center, Vicksburg, Mississippi, United States ‡ Keck Graduate Institute, Claremont, California, United States § Badger Technical Services, San Antonio, Texas, United States ∥ Mississippi State University, Starkville, Mississippi, United States * S Supporting Information ABSTRACT: 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 neuro- toxicant. Relative toxicity was measured through transcrip- tional, 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. ■ INTRODUCTION New chemical testing programs, including the High Production Volume (HPV) challenge in the United States and Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) in Europe are significantly increasing the number of chemicals for which toxicity data is needed. 1 In addition, human and veterinary pharmaceuticals, nanocomposites and other engineered materials promise to increase demands for regulatory testing for ecological effects. 2 Ecological testing and screening programs must become more thorough, less costly, and more rapid to make testing of thousands of chemicals feasible. A critical and often overlooked component of transitioning test data to ecological risk assessment (ERA) are the uncertainty factors associated with toxicological effects observed in model versus nonmodel species. Current methods employed for ERA rely on arbitrary uncertainty factors (typically a factor of 10) when quantitative data is not available to compare the relative sensitivity to chemical toxicity among species. 3 This study sought to develop a robust empirical cross-species comparison for consideration in ERA and to assess if a typical 10-fold uncertainty factor of is overly conservative. To attain this goal, we analyzed the relationships among multiple levels of biological organization to provide a mechanistic description of differential sensitivity among species. We utilized the Adverse Outcome Pathway (AOP) framework to contextualize our species comparisons. 4 The AOP characterizes connections from the molecular initiating event, to impacts on metabolic pathways, to impacts within a cell, through impacts on tissues, organs and so on and ultimately to the adverse outcome at the Received: February 6, 2012 Revised: May 31, 2012 Accepted: June 14, 2012 Published: June 14, 2012 Article pubs.acs.org/est © 2012 American Chemical Society 7790 dx.doi.org/10.1021/es300495c | Environ. Sci. Technol. 2012, 46, 7790−7798