Suspect List-Guided High-Throughput Screening (SLIG-HTS) Identifies Environmental Substances Associated with Advanced Embryonic Developmental Timing

Retardation of embryonic development by environmental contaminants has been extensively documented, whereas coordinated advancement in developmental timing has received far less attention despite its risks to organisms as well. To address this gap, we developed a Suspect List-Guided High-Throughput Screening strategy that integrates literature-informed suspect list construction with developmental trend-guided, stage-resolved end points evaluation to identify environmental substances associated with advanced embryonic developmental timing across a broad chemical space. By retrospectively screening over 9,300 toxicity records from public repositories, we curated a suspect list of 46 substances linked to affect developmental timing-related end points. Experimental validation in zebrafish confirmed that 27 compounds (58.7%) significantly affected at least one stage-resolved morphological end point, including epiboly progression, somite formation, and/or yolk sac dynamics. Notable, the sulfonamide antibiotics sulfadiazine (SDZ) and sulfamethoxazole (SMZ) exhibited pronounced effects at environmentally relevant concentrations (from ng/L to low μg/L). Exploratory transcriptomic analyses further revealed coordinated pathway-level responses related to neuromuscular and cardiovascular development, supporting a shift in overall developmental timing rather than overt toxicity. Furthermore, eight additional sulfonamides produced comparable effects, suggesting a potential class-wide phenomenon. Together, our integrated approach identified a set of environmental substances associated with advanced embryonic developmental timing at environmentally relevant levels and highlighted their potential ecological relevance.