Grantee Research Project Results

2024 Progress Report: 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 , Chappell, Patrick , Armbrust, Kevin , White, Wilson
Institution: Oregon State University , Louisiana State University
EPA Project Officer: Chung, Serena
Project Period: August 1, 2019 through April 23, 2025
Project Period Covered by this Report: August 1, 2023 through July 31,2024
Project Amount: $849,988
RFA: Advancing Actionable Alternatives to Vertebrate Animal Testing for Chemical Safety Assessment (2018) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability

Objective:

Over the past decade great strides have been made in minimizing the use of vertebrates for toxicity testing focused on human health outcomes. However, although numerous approaches and models that facilitate the reduction of vertebrates in human toxicity testing currently exist, far fewer alternatives are available for use in ecotoxicological applications, particularly for marine and estuarine ecosystems. For example, the current marine and estuarine species approved for EPA Whole Effluent Toxicity (WET) testing are Cyprinodon variegatus (Sheepshead minnow) and Menidia beryllina (Inland silverside). Neither species has appropriate in vitro alternatives available to limit the use of live animals for regulatory and research purposes. Assessing ecotoxicological effects in estuarine systems is critical, given the importance and value of those ecosystems (and their vulnerability to pollution. Further complicating the assessment of toxicity in estuarine systems is the altered behavior of chemicals in seawater, which influences water solubility and concomitant bioavailability and thus raises the potential for differential responses to chemicals dependent on salinity, not to mention the likelihood of differences in osmoregulatory strategies influencing the uptake and metabolism of chemicals between fishes inhabiting fresh and saline waters. For example, fish in saline waters drink constantly to remain isotonic with their surroundings, while freshwater fish urinate frequently to achieve the same goal. Regardless of these challenges, a marine model that uses cell lines to reduce reliance on live fish for the purposes of identifying, prioritizing, and evaluating potential toxicants is greatly needed. Additionally, the development of dosimetry that accurately reflects bioavailability across a salinity gradient would refine this testing, allowing cell lines to be used at a minimum as a first-tier approach to limit the number of toxicants that necessitating in vivo testing.

Progress Summary:

Following the publication of acute exposures conducted by Hutton et al. (2021), in 2022-2023 bioconcentration and preliminary sublethal exposures were completed at 5 and 25 ppt using Inland Silverside eggs by now graduated Ph.D. student Scott St. Romain (LSU). During this process capelin eggs were developed as a reliable surrogate for Silverside eggs, so enough biomass was available for chemical extractions. Exposures for six biocides (excluding paraquat) were run at these two contrasting salinities to determine if there were differences in chemical partitioning dependent on salinity. We found that five of the six biocides bioconcentrate at significantly higher concentrations at 25 PSU, when compared to levels detected in the same eggs at 5 PSU. Related work completed and published in 2023 and 2024 on behavioral toxicity has resulted in protocols that can be applied to the remainder of our EPA-funded research.

For in-vivo bioconcentration exposures we have confirmed via the work described above that the LC₁₀ (lethal concentration for 10% of the population) can be detected in Silverside and surrogate capelin eggs. Originally LC₂₅ values derived from Hutton et al. (2021) were being used to inform future sub-lethal exposures in larval fish and cell lines, but high toxicity was observed with triademefon in fish, especially at higher salinities (25 ppt), caused our research team to revisit the concentrations planned for use. Exposures based on LC₁₀ concentrations are now completed by new Masters student Kate Berreman (OSU). In accordance with our original hypothesis, it appears that behavioral toxicity is enhanced at higher salinities across the majority of chemicals. In addition to traditional sublethal endpoints (e.g. growth) and behavioral toxicity, RNA sequencing on exposed larvae is now completed and in the process of being analyzed. We expect to submit this work for publication in 2025.

In 2022-2023, cardiomyocytes and hepatocytes isolated from silversides were successfully immortalized using lentiviral infection of dispersed tissues with plasmids encoding large T antigen in 2022, but generating a sufficient number of cells without contamination was a major challenge. We have modified protocols to focus on generating an embryonic cell line to ensure earlier elimination of pathogens and equal exposure of all individuals. We are in the process of testing a new sterilization protocol that will reduce contamination in cell lines to allow for sufficient cell division and immortalization. Once we can achieve adherence with an adequate number of cells, exposures that match those completed in larval fish will be run using the embryonic cell line.

Future Activities:

We have completed sublethal exposures led by Masters student Kate Berreman (OSU) and supported by graduated Ph.D. student Sara Hutton (2023), on the bioconcentration of biocides at 5 and 25 PSU described above. This will be submitted in for publication by spring 2025 and will include both behavioral toxicity and gene expression (RNA seq). Berreman has also developed a new approach for cell lines using embryos rather than dissected tissues from adult fish, Should immortalization and adherence be successful, we will conduct in vitro exposures to all biocides. We have requested a final NCE through 2025 to ensure Berreman has enough time to complete her work and that modeling approaches can be completed once sublethal and cell line data are in hand.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Publications Views
Other project views:	All 9 publications	9 publications in selected types	All 8 journal articles

Publications
Type	Citation	Project	Document Sources
Journal Article	Hutton SJ, Brander SM. Epigenetics in Aquatic Toxicology. Epigenetics in Aquaculture 2023 Aug 2:301-23.	R839503 (2024)	Abstract: Wiley - Abstract HTML Exit

Supplemental Keywords:

Menidia beryllina, in vivo, in vitro, salinity, biocide, cell line

Relevant Websites:

Brander Lab Exit

Progress and Final Reports:

Original Abstract

The 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.