2015 Progress Report: System Toxicological Approaches to Define Flame Retardant Adverse Outcome Pathways

EPA Grant Number: R835796
Title: System Toxicological Approaches to Define Flame Retardant Adverse Outcome Pathways
Investigators: Tanguay, Robert L. , Du, Jane La , Reif, David , Simonich, Mike , Sullivan, Chris
Institution: Oregon State University , North Carolina State University at Raleigh
EPA Project Officer: Lasat, Mitch
Project Period: June 1, 2015 through May 31, 2018 (Extended to May 31, 2019)
Project Period Covered by this Report: July 1, 2014 through June 30,2015
Project Amount: $798,661
RFA: Systems-Based Research for Evaluating Ecological Impacts of Manufactured Chemicals (2014) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Ecosystems , Safer Chemicals

Objective:

The project's objectives are to:

  • 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:

1)    Complete the evaluation of US EPA-NCCT 42 flame retardants library.

2)    Complete statistical analysis to integrate behavioral and morphological endpoints.

3)    Optimize adult behavioral assays.

4)    Select FRCs and begin developmental exposures and grow outs to assess adverse behavioral effects.

Progress Summary:

1) Complete the evaluation of US EPA-NCCT 42 flame retardants library

The U.S. EPA National Center of Computational Toxicology (NCCT) 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 FRC (0.1 – 80 µM). At 24 and 120 hpf, 22 morphological endpoints were evaluated. Additionally, exposed embryos underwent 2 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, 24h behavior and 5d behavioral endpoints (Figure 1). Figure 1 summarizes our finding for the 42 FRCs. The dot-heatmap illustrates higher toxicity (bigger circles), hyper- (yellow), and hypo- (blue) activity. The FRCs fell into 4 clusters, which can be described as not bioactivity (not a hit in any of the 3 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. 

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. Figure 2 illustrates the predator avoidance assay in which a zebrafish tank faces a monitor displays a moving predator fish. Prior to display of the predator image, control fish show no spatial bias regarding the area adjacent the monitor, but once the predator is displayed, the control fish spatial distribution significantly shifts away from the monitor window. 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. 

4) Select FRCs and begin developmental exposures and grow outs to assess adverse behavioral effects.

Based on the 4 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:

Our future activities are to:

  • Complete the validation studies to calculate EC80 for the selected FRCs.
  • Evaluate the new library of FRCs in our high-throughput developmental zebrafish assay.
  • 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.
  • Conduct genome-wide transcriptomics on the FRCs.


Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 30 publications 7 publications in selected types All 7 journal articles
Type Citation Project Document Sources
Journal Article 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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  • Journal Article 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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  • Journal Article 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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  • Progress and Final Reports:

    Original Abstract
  • 2016 Progress Report
  • 2017 Progress Report