Grantee Research Project Results
2023 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: R839503Title: 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: Callan, Richard
Project Period: August 1, 2019 through July 31, 2022 (Extended to July 31, 2024)
Project Period Covered by this Report: August 1, 2022 through July 31,2023
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. This work was just accepted for publication in the journal Environmental Toxicology and Chemistry. Related work completed and published in 2023 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 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. We have successfully lowered the planned sublethal concentrations to the LC10 for each chemical being our highest sublethal concentration. These exposures are now nearly completed by new Masters student Kate Berreman (OSU), and we expect to submit this work for publication in 2024.
Cardiomyocytes and hepatocytes isolated from silversides were recently successfully immortalized using lentiviral infection of dispersed tissues with plasmids encoding large T antigen in 2022. We are continuing to fine-tune approaches to reducing contamination, which had involved exposing adult Silversides to Baytril (enroflaxocin) via their dry food for 18 days prior to humane sacrifice and dissection of tissues for cell line generation. However, we discovered that it may be more productive to expose embryos aqueously to ensure earlier elimination of pathogens and equal exposure of all individuals. This combined with recently developed visualization approaches to assess cell health should ensure success. Once we can achieve adherence with an adequate number of cells, exposures that match those described above for larval fish will be run using cardiomyocytes and hepatocytes.
Future Activities:
We are in the process of completing sublethal exposures led by Masters student Kate Berreman (OSU) and supported by recently graduated Ph.D. student Sara Hutton who now works in environmental consulting, on the bioconcentration of biocides at 5 and 25 PSU described above. This will be submitted in for publication in summer / fall 2024. Berreman will also be validating immortalized cell lines (cardiomyocytes, hepatocytes) via qPCR, and conducting in vitro exposures to all biocides. Due to a no-cost extension granted in 2022 we had planned to finish work in 2024, but may request a second NCE to 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 : 2 Displayed | Download in RIS Format
Other project views: | All 7 publications | 7 publications in selected types | All 7 journal articles |
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Hutton SJ, Siddiqui S, Pedersen EI, Markgraf CY, Segarra A, Hladik ML, Connon RE, Brander SM. Comparative behavioral ecotoxicology of Inland Silverside larvae exposed to pyrethroids across a salinity gradient. Science of The Total Environment 2023;857:159398. |
R839503 (2023) |
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St. Romain SJ, Basirico LM, Hutton S, Brander S, Armbrust KL. Influence of Salinity on the Partitioning Behavior of Six Commonly Used Pesticides in Fish Eggs. Environmental Toxicology and Chemistry 2024;43(2):299-306. |
R839503 (2023) |
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Supplemental Keywords:
Menidia beryllina, in vivo, in vitro, salinity, biocide, cell lineRelevant Websites:
Progress and Final Reports:
Original AbstractThe 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.