2015 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: July 1, 2016 through June 30,2017
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


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 carry-over 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. 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. 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 a number of fronts during year two. The main experiment continues, with the F1 generation nearing reproductive maturity. We expect to spawn the F2 generation in October 2017 and to complete the rearing phase of the work by November 2017. Chemical and in vitro analysis on water samples collected at three time points has been completed at SCCWRP and all chemical concentrations were confirmed to be in line with nominal exposure levels. Parental gonadal histology is nearly complete and F1 adults will be similarly processed this fall. The M. beryllina genome was sequenced at the UC Davis Genome Center using new Illumina 10X technology. To the best of our knowledge M. beryllina is the first fish to be sequenced using this approach, and as such some method development was required. The sequenced genome is now undergoing assembly and annotation. Results from the first generation show that early-life exposure to all EDCs except for ethinylestradiol (EE2) significantly reduced fecundity in the parental generation. Conversely, only EE2 skewed sex ratios (feminized) relative to controls. Findings from the F0 and F1 generations demonstrate that exposure to EDCs increased growth in the parental generation, and that androgenic treatment groups (Levo, TB) maintain this growth through the subsequent F1 generation. In the parental generation, differences in adult immune response are apparent between bifenthrin and levonorgestrel. A related study that was published this year demonstrates the potential for transgenerational effects of bifenthrin and EE2 alone and when combined with increased temperatures (DeCourten and Brander, 2017). Additionally, we made progress towards modeling goals. An initial version of the model was parameterized for M. beryllina, including the experimentally-determined relationship between sex ratio and spawning output. A journal publication (White, et al., 2017) showed how individual-level effects of feminizing and masculinizing EDCs (and combinations of both) in a single generation scale up to affect population dynamics. Finally, as a result of a session on epigenetics chaired at the 2016 Society of Environmental Toxicology and Chemistry meeting by PI Brander and Dr. Adam Biales of the EPA, we have also written a review paper on epigenetics in the field of Aquatic Toxicology (Brander, et al., in press)

Future Activities:

In Year 3, 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 F2 generation will be spawned in October 2017, at which time spawning assays, assessment of immune response, and gonadal histology will be conducted on subsamples of F1 adults and the F2 generation will be reared until day 21 post hatch. Once the rearing phase of the work is completed, all extracted RNA samples from the three generations will be carefully relocated and analyzed simultaneously at Oregon State University, which is the new home institution of Lead PI Brander’ and co-PI White’. Future data gathered on gonadal histology, gene expression and DNA methylation will allow us to further hone in on the mechanisms causing higher order downstream effects. Once all empirical data are collected, the model will be updated to include both within-generation exposure effects and trans-generational effects of EDC exposure. We intend to request a 1 year no-cost extension due to the move, so all project goals can be sufficiently completed.

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 DeCourten BM, Brander SM. Combined effects of increased temperature and endocrine disrupting pollutants on sex determination, survival, and development across generations. Scientific Reports 2017;7(1):9310 (9 pp.). R835799 (2015)
R835799 (2017)
R835799 (2018)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: Nature-Full Text HTML
  • Abstract: Nature-Abstract
  • Other: Nature-Full Text PDF
  • 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
  • Abstract: ACS-Abstract
  • Other: ACS-Full Text PDF
  • Supplemental Keywords:

    Menidia beryllina, epigenetic, methylation, qPCR, systems biology, ecotoxicology.

    Progress and Final Reports:

    Original Abstract
  • 2016 Progress Report
  • 2017 Progress Report
  • 2018 Progress Report