Grantee Research Project Results
2016 Progress Report: System Toxicological Approaches to Define Flame Retardant Adverse Outcome Pathways
EPA Grant Number: R835796Title: System Toxicological Approaches to Define Flame Retardant Adverse Outcome Pathways
Investigators: Tanguay, Robyn L. , Du, Jane La , Sullivan, Chris , Reif, David , Simonich, Mike
Current Investigators: Tanguay, Robyn L. , Reif, David , Simonich, Mike , Sullivan, Chris , Du, Jane La
Institution: Oregon State University , North Carolina State University at Raleigh
EPA Project Officer: Aja, Hayley
Project Period: June 1, 2015 through May 31, 2018 (Extended to May 31, 2019)
Project Period Covered by this Report: June 1, 2015 through May 31,2016
Project Amount: $798,661
RFA: Systems-Based Research for Evaluating Ecological Impacts of Manufactured Chemicals (2014) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
The objectives of this research are:
- Expose embryonic zebrafish to a comprehensive list of flame retardant chemicals (FRCs) and observe their morphology and behavior for signs of toxicity.
- Grow exposed zebrafish to adulthood and measure their physiology and behavior for signs of persistent toxicity.
- Bin FRC outcomes across the levels of biological organization (i.e., chemical structure, similarity of gene expression profiles, early and adult life stage adverse outcomes), and thereby define adverse outcome pathways (AOP) for mechanistic FRC hazard prediction.
- Share our data with EPA and the broader research community.
The following were the focus of this year’s efforts:
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Complete the evaluation of US EPA-NCCT 42 flame retardants library
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Complete statistical analysis to integrate behavioral and morphological endpoints
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Optimize adult behavioral assays
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Select FRCs and begin developmental exposures and grow outs to assess adverse behavioral effects
Progress Summary:
We have made considerable progress and the highlights are summarized below
(1) Complete the evaluation of US EPA-NCCT 42 flame retardants library
The U.S. EPA National Center of Computational Toxicology ToxCast chemical library consists of 42 flame retardants that were nominated by various sources. As a collaborative effort, EPA-NCCT provided this subset of the ToxCast library to evaluate their developmental toxicity in our high-throughput zebrafish toxicity model. Embryos were exposed from 6-120 hours post fertilization (hpf) to 11 concentrations of Flame Retardant Chemicals (FRCs) (0.1-80 µM). At 24 and 120 hpf, 22 morphological endpoints were evaluated. Additionally, exposed embryos underwent two behavioral assays.
(2) Complete statistical analysis to integrate behavioral and morphological endpoints
For each chemical, the lowest effect level (LEL) was computed for the morphology, 24 h behavior and 5 d behavioral endpoints. The FRCs fell into four clusters, which can be described as not bioactivity (not a hit in any of the three assays, those that induced both morphological and hyper active behavioral response, or hypo activity, and those that caused neurobehavioral effects. These important findings position us to identify representative FRCs for which to conduct whole-genome transcriptomic and developmentally exposed grow out studies.
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(3) Optimize adult behavioral assays
We identified that new strategies were needed to examine potentially persistent effects from developmental exposures to flame retardant chemicals. We addressed this by designing and building multi-use behavioral units to evaluate conditioned place preference, anxiety and social behaviors. Prior to applying these assays to a select set of FRCs, they were evaluated with known neurotoxicants, and the infrastructure to analyze the data was established. The experimental design consists of developmental exposure to a chemical from 6 to 120 hpf, followed by rearing to adulthood (90 days). At approximately 3 months, the zebrafish undergo 4 behavioral assays: (1) schooling behavior, (2) predator avoidance response, (3) startle response, and (4) conditioned place preference learning in a shuttlebox. Assays 1-3 are conducted in the same instrument platform. The shuttlebox assay is a separate platform that the Tanguay lab optimized and deployed prior to the start of this grant. For each assay, individual fish performances are tracked, and compared to the mean response of the control fish. While the neurotoxicant exposed fish showed this same trend, the zebrafish of the lowest concentration group showed significantly less avoidance of the area adjacent the predator image than the controls or the higher concentration group. We have identified the most informative time window and statistical analysis method for all 3 of the assays conducted on this instrument platform. We are now positioned to apply this basic design workflow to the subset of FRCs we will identify from the developmental toxicity screen.
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(4) Select FRCs and begin developmental exposures and grow outs to assess adverse behavioral effects
Based on the four bins identified from the FRC screen, we have exposed embryos to 10 FRCs: TBBPA (Tetrabromobisphenol A), TBBPA-DBPE (Tetrabromobisphenol A bis(2,3-dibromopropyl ether), TDCPP (Tris(1,3-dichloro-2-propyl)phosphate, TBEP (Tris(2-butoxyethyl) phosphate, TCPP (Tris(1,3-dichloro-2-propyl)phosphate, IPP-1 (Tris(1,3-dichloro-2-propyl)phosphate, TPP (Triphenyl phosphate), BPBP (tert-Butylphenyl diphenyl phosphate), TBPH (Bis(2-ethylhexyl) tetrabromophthalate), TCEP (Tris(2-chloroethyl) phosphate). Half are coming of age to be assessed in our optimized assay this upcoming year.
Future Activities:
Future activities include the following:
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Complete the validation studies to calculate EC80 for the selected FRCs
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Evaluate the new library of FRCs in our high-throughput developmental zebrafish assay
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Author two manuscripts: (1) The results from 42 FRC EPA-NCCT chemical library and (2) adult behavior responses based on the developmental response bin for the FRCs
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Conduct genome-wide transcriptomics on the FRCs
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 32 publications | 7 publications in selected types | All 7 journal articles |
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Type | Citation | ||
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Chen J, Tanguay RL, Simonich M, Nie S, Zhao Y, Li L, Bai C, Dong Q, Huang C, Lin K. TBBPA chronic exposure produces sex-specific neurobehavioral and social interaction changes in adult zebrafish. Neurotoxicology and Teratology 2016;56:9-15. |
R835796 (2015) R835796 (2016) |
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Chen J, Tanguay RL, Xiao Y, Haggard DE, Ge X, Jia Y, Zheng Y, Dong Q, Huang C, Lin K. TBBPA exposure during a sensitive developmental window produces neurobehavioral changes in larval zebrafish. Environmental Pollution 2016;216:53-63. |
R835796 (2015) R835796 (2016) |
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Noyes PD, Haggard DE, Gonnerman GD, Tanguay RL. Advanced morphological-behavioral test platform reveals neurodevelopmental defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants.Toxicological Sciences 2015;145(1):177-195. |
R835796 (2015) R835796 (2016) |
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Supplemental Keywords:
Dose-response, teratogen, animal, stressor, toxics, chemical mixtures, adverse outcome pathwaysProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.