2016 Progress Report: Linking Biological Scales Across Generations: An Estuarine and Marine Model for Measuring The Ecological Impact of Endocrine Disrupting Compounds

EPA Grant Number: R835799
Title: Linking Biological Scales Across Generations: An Estuarine and Marine Model for Measuring The Ecological Impact of Endocrine Disrupting Compounds
Investigators: Brander, Susanne M , Mehinto, Alvine C , Connon, Richard E , White, J. Wilson
Institution: Oregon State University , Southern California Coastal Water Research Project Authority , University of California - Davis
EPA Project Officer: Lasat, Mitch
Project Period: June 1, 2015 through May 31, 2018 (Extended to May 31, 2020)
Project Period Covered by this Report: June 1, 2016 through May 31,2017
Project Amount: $399,884
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:

We plan to determine the effects of bifenthrin and levonorgestrel, and relative positive controls ethinylestradiol and trenbolone, on Menidia beryllina across the molecular (gene expression, DNA methylation) organismal (gross immune response, gonad development), and population levels (sex ratio, population model) and over three generations (P, F1, F2). Reared offspring of the exposed parent generation (exposed from fertilization through 21 dph, then reared in clean water) will be examined for epigenetic carryover effects for the same endpoints. These in vivo responses will be linked to in vitro cellular assays. A mathematical model of fish population dynamics will scale up experimental results to predict the effects of contaminant exposure on the persistence of Menidia populations and on the persistence of other species.

Experimental Approach: We are using a combination of in vitro cell assays (ER, AR, PR activity), molecular assays including qPCR and analysis of the global DNA methylome (reduced representation bisulfite sequencing), gonad histology, a PHA (phytohaemagglutinin) immune assay, a 21-day reproductive assay (on adult P and F1 generations), and an integral projection model (population dynamics). The unexposed F1 generation will undergo the same assays, the unexposed F2 generation, reared to 21 dph, will undergo the molecular assays and a newly added evaluation of embryonic development described below.

Anticipated Results: We expect to find that emerging EDCs (bifenthrin, levonorgestrel) produce higher level effects (e.g., reduced reproductive output, altered immune response and gonadal histology) similar to those of positive controls (ethyinylestradiol, trenbolone), but that the molecular initiating events (assayed in vitro and via qPCR) will differ between those compounds. In vitro effects of the studied EDCs on endocrine-related endpoints will likely be of higher magnitude than the effects measured in vivo. We also expect that exposure of P generation fertilized eggs through 21 dph will produce carryover epigenetic effects in both the F1 and F2 generations. Modeling results will likely show emergent population-level effects of different EDC exposures on both persistence and dynamics. 

Progress Summary:

We have made progress on several fronts during year one. Prior to beginning exposures on the parental embryos intended to be the progenitors of the F1 and F2 generations, we conducted a range-finding experiment to determine optimal concentrations for levonorgestrel, bifenthrin, ethinylestradiol, and trenbolone. This was determined by evaluating the expression of a suite of endocrine-related genes. We also developed a protocol for processing fertilized silverside eggs for use in toxicity testing based on techniques used with medaka species, and adapted existing published protocols for scoring and staining developing embryos. The main experiment has begun, with the exposed (egg to 21 dph) parental generation nearing reproductive maturity. During the fish exposure, water samples were collected at three time points for chemical analysis and in vitro assays. These samples have been processed by solid phase extraction and will soon be analyzed at SCCWRP. High molecular weight DNA has been sent to the University of California, Davis Genome Center for sequencing (PacBio, Illumina) of the M. beryllina genome. 

Future Activities:

In year two, the M. beryllina genome will be used in the development of RRBS (reduced representation bisulfite sequencing) techniques for detecting the methylation of CPG islands in exposed Menidia. We anticipate that the F1 generation will be spawned in late October or early November, at which time spawning assays, assessment of immune response, and gonadal histology will be conducted on subsamples of parental adults and a 21-day exposure will be performed using F1 embryos. The F1 generation will be reproductively mature in May 2017, at which time the same assay will be conducted on adults and the F2 generation reared to 21 dph, at which time rearing will be completed. While the F1 generation is reared, we will be processing parental samples and larval F1 subsamples for the above-mentioned endpoints. In vitro cell assays and chemical analyses will be completed. Results of F0 qPCR analyses will be used to investigate the linkage between in vitro response and gene expression on specific nuclear receptors.


Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other project views: All 8 publications 8 publications in selected types All 8 journal articles
Type Citation Project Document Sources
Journal Article Brander SM, Gabler MK, Fowler NL, Connon RE, Schlenk D. Pyrethroid pesticides as endocrine disruptors: molecular mechanisms in vertebrates with a focus on fishes. Environmental Science & Technology 2016;50(17):8977-8992. R835799 (2016)
R835799 (2017)
R835799 (2018)
  • Abstract from PubMed
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  • Journal Article White JW, Cole BJ, Cherr GN, Connon RE, Brander SM. Scaling up endocrine disruption effects from individuals to populations: outcomes depend on how many males a population needs. Environmental Science & Technology 2017;51(3):1802-1810. R835799 (2015)
    R835799 (2016)
    R835799 (2017)
    R835799 (2018)
  • Abstract from PubMed
  • Full-text: ACS-Full Text HTML
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  • Abstract: ACS-Abstract
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  • Other: ACS-Full Text PDF
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  • Supplemental Keywords:

    Menidia beryllina, epigenetic, methylation, qPCR, systems biology, ecotoxicology, endocrine disrupting compounds, EDCs

    Progress and Final Reports:

    Original Abstract
  • 2015 Progress Report
  • 2017 Progress Report
  • 2018 Progress Report