PNNL

Abstract

Agrichemicals such as herbicides, fungicides, insecticides, and biocides are widely used in agriculture, yet some are associated with adverse health effects in humans and the environment. While many of these chemicals have been extensively studied in vitro and are included in the EPA’s ToxCast program, comprehensive in vivo comparisons using RNA sequencing across structurally diverse agrichemicals within a single screening platform are lacking. In this study, we examined 69 structurally diverse agrichemicals by statically exposing early life stage zebrafish from 6 to 120 hours post fertilization (hpf) at concentrations ranging from 0.25–100 µM. Morphological outcomes were assessed at 120 hpf across 10 endpoints, including yolk sac edema, craniofacial malformations, and axis abnormalities. Of these, 45 chemicals produced robust concentration-response relationships, enabling the calculation of effective concentrations (EC) at which 13–100% of animals showed morphological effects or mortality; these were selected for transcriptomic profiling. For transcriptomic analysis, zebrafish were statically exposed to EC13-100 concentrations (confirmed for each agrichemical) and sampled at 48 prior to the onset of adverse morphological effects observed at 120 hpf. Differential gene expression (DEG) analysis identified between 0 and 4,538 DEGs per chemical, with no clear correlation to morphological severity. Both DEG and gene coexpression network analyses revealed chemical-specific expression patterns that converge on shared biological pathways, including neurodevelopment and cytoskeletal organization. Key regulatory genes such as mylpfa and krt4 were identified within coexpression modules, suggesting their potential role in conserved toxicity mechanisms. Semantic similarity analysis of enriched gene ontology (GO) terms, when compared to existing datasets, highlighted gaps in the annotation of neurodevelopmental processes, indicating that some in vivo effects may not be fully captured by current curated resources. These findings provide new insights into the modes of action of diverse agrichemicals and establish a framework for linking chemical structure to biological function in vivo. This chemical network represents a foundational step toward a deeper mechanistic understanding of agrichemical toxicity and supports future regulatory assessments.