Basu, N. | Search bioRxiv

bioRxiv2023

Towards establishing a 24-hour, microplate-based, transcriptomics assay for rainbow trout embryos

There is interest in the development of early-life stage (ELS) tests with fish embryo models that are high-throughput and can generate transcriptomics point of departure (tPOD) values. The objective of this study was to establish a method in rainbow trout (Oncorhynchus mykiss) hatchlings that could satisfy both of these interests. We based our pilot method on recent efforts by U.S. EPA researchers to establish a larval fathead minnow high throughput transcriptomics assay. Here, 1-2 day post hatch trout were assayed in 24-well plates in which they were exposed for 24 hours to 12 different concentrations of test chemicals, including a negative control (DMSO, culture water). Test concentrations were anchored with a chemicals LC50 data from the US EPA ECOTOX database and EnviroTox database, and from this, concentrations were spaced on a half-log basis that spanned 6-7 orders of magnitude. In pilot study 1 we tested 3,4-dichloroaniline, CuSO4 (0.34 mg/L), and ethinylestradiol. In pilot study 2 we tested 3,4-dichloroaniline (58.5 mg/L), CuSO4 (0.34 and 0.41 mg/L), ethinylestradiol (>10 {micro}g/L), permethrin (>10 {micro}g/L), malathion (0.61 mg/L), 6PPD quinone (5.6 {micro}g/L), acetaldehyde (41.2 mg/L), 4-fluoroaniline (242.7 mg/L), glyphosate ([~]150 mg/L), ethanol (>1 g/L), thiamethoxam (>300 mg/L), and allyl alcohol (>30 mg/L). In both pilot studies derived LC50 values are provided in parentheses. Repeated studies of CuSO4 yielded consistent LC50 values (0.34, 0.34, 0.41 mg/L). The correlation between LC50s from the current study for rainbow trout embryos versus those from the literature on adult rainbow trout for 7 chemicals was r2 = 0.91. Work is underway to optimize transcriptomics assays from these samples using EcoToxChips and UPXome, with the ultimate goal to be able to derive transcriptomics points of departure. Taken together these results provide a foundation towards establishing a novel testing platform for chemical and environmental risk assessment that is much quicker (24 hrs), ethical (non-protected life stages), resource efficient (e.g., microplate-based, small volumes of chemicals), and more informative (molecular clues into MOA) than traditional bioassay approaches.

pharmacology and toxicology↗

bioRxiv2022

Resource requirements for ecotoxicity testing: A comparison of traditional and new approach methods

Toxicity testing is under transformation as it aims to harness the potential of New Approach Methods (NAMs) as alternative test methods that may be less resource intensive (i.e., fewer animals, cheaper costs, quicker assays) than traditional approaches while also providing more data and information. While many stakeholders are of the opinion that this unfolding transformation holds significant promise as a more efficient and ethical way forward, few studies have compared the resources required for NAMs versus those needed for traditional animal-based toxicity tests, particularly in the field of ecotoxicology. The objective was to compare resources needed for traditional animal-based ecotoxicity tests versus alternative tests using emergent NAMs. From a bibliometric review, we estimate that traditional tests for a single chemical cost $118,000 USD, require 135 animals, and take 8 weeks. In comparison, alternative tests cost $2,600, require 20 animals (or none), and take up to 4 weeks to test 16 (to potentially hundreds of) chemicals. Based on our analysis we conclude that NAMs in ecotoxicology can be more advantageous than traditional methods in terms of resources required (i.e., monetary costs, number of animals needed, and testing times). We note, however, that the evidence underpinning these conclusions is relatively sparse. Moving forward, groups developing and applying NAMs should provide more detailed accounts of the resources required. In addition, there is also a need for carefully designed case studies that demonstrate the domain of applicability of NAMs (and make comparisons to traditional tests) to ultimately build confidence among the user community.

pharmacology and toxicology↗