Reducing the reliance on early-life stage testing with relevance to euryhaline fishes: Development and implementation of in-vitro assays predictive of early life stage toxicity and population-level effects in Menidia beryllina

EPA Grant Number: R839503
Title: Reducing the reliance on early-life stage testing with relevance to euryhaline fishes: Development and implementation of in-vitro assays predictive of early life stage toxicity and population-level effects in Menidia beryllina
Investigators: Brander, Susanne M , Armbrust, Kevin , Chappell, Patrick , White, Wilson
Institution: Oregon State University , Louisiana State University , Oregon State University , Oregon State University
EPA Project Officer: Lasat, Mitch
Project Period: August 1, 2019 through July 31, 2022
Project Amount: $849,988
RFA: Advancing Actionable Alternatives to Vertebrate Animal Testing for Chemical Safety Assessment (2018) RFA Text |  Recipients Lists
Research Category: Safer Chemicals

Description:

Although the Tox21 directive has been in place for over a decade, toxicity assessment of aquatic pollutants, particularly for estuarine and marine ecosystems, still requires large numbers of live fishes. A handful of in vitro assays have been developed for euryhaline species, however, many are not in EPA-approved models. Further complicating euryhaline in vitro model development is the documented alteration in uptake and bioavailability of many compounds at higher salinities, which is difficult to account for with cell lines.

Objective:

As such, we describe three major objectives: 1. Develop in vitro assays complementary to assays such as the fish early life stage (FELS) and larval growth and survival (LGS), thus reducing reliance on in vivo testing, 2. Quantify internal exposure concentrations of model developmental toxicants across a salinity gradient during in vivo testing, to generate dosimetry for mimicking exposures in vitro, and 3. Use a combination of genomic tools and demographic modeling in a "middle-out" approach that links the phenotypic anchors (e.g. deformities) measured in vivo to biomarker candidates and population-level outcomes.

Approach:

Objectives will be met via a highly interdisciplinary approach tapping expertise across cell and molecular biology, aquatic toxicology, analytical chemistry, and quantitative ecology. Firstly, cell lines will be developed from both embryonic and differentiated tissues (cardiomyocytes, osteoblasts, hepatocytes). Concurrently, early life stage testing in M. beryllina will be conducted using a suite of pesticides established to cause tissue specific effects (e.g. cardiotoxicity, skeletal deformities, hepatoxicity), across a concentration (5 levels) and salinity gradient (0, 10, 20, 30 PSU). Internal concentrations will be measured in embryos and larvae, and used for cell line exposures. Both RNA seq and targeted qPCR, with a focus on common genes underlying cardiotoxicity, hepatoxicity, and osteotoxicity, will be conducted in embryos, fish larvae, and cell lines. We will thus generate plausible candidates for molecular initiating events (MIEs) that trigger observed phenotypic or population-level alterations.

Expected Results:

We hypothesize that MIE candidates linked to adverse phenotypes and population-level changes (e.g. growth, rate of hatch or survival) will be identified through this combination of in vitro and in vivo approaches used to probe the transcriptome and that as a result the M. beryllina cell lines created could then be implemented as a predictive assay for organismal and population-level effects, and used as a first tier, high throughput approach to decrease the number of live animals needed for testing of pollutant impacts in marine and estuarine ecosystems. Ultimately this would both reduce the cost and increase the accuracy of risk assessment, which is increasingly important given the number of new chemicals that arrive on the market each year and the limited resources available for toxicity testing.

Supplemental Keywords:

pesticides, molecular level, toxicology, inland silverside, estuarine, salinity, ecological effects, exposure, modeling, environmental chemistry, bioavailability