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

Abstract

ABSTRACTThere 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 chemical’s 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 µg/L), permethrin (>10 µg/L), malathion (0.61 mg/L), 6PPD quinone (5.6 µg/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 CuSO4yielded 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.