Imidacloprid Transformation Products Exhibit Stronger Developmental Neurotoxicity than the Parent Compound in Zebrafish ( <i>Danio rerio</i> ) via the Glutamate Signaling Pathway

Imidacloprid (IMI) and its transformation products (TPs; DNIMI, IMIOL, DNIMIOL, and 5OHIMI) are ubiquitous in aquatic ecosystems, yet their ecotoxicological impacts remain poorly characterized. Here, we investigated the developmental neurotoxicity of IMI and its TPs. Zebrafish were exposed to IMI, DNIMI, IMIOL, DNIMIOL, and 5OHIMI (0.005-500 μg/L) for 120 h postfertilization (hpf). DNIMI and IMIOL showed greater developmental and behavioral toxicity than IMI and other TPs and were therefore selected (0.05-500 μg/L) for further toxicokinetic and recovery investigations. Although IMI, IMIOL, and DNIMI accumulated to comparable levels by 72 hpf, DNIMI and IMIOL were cleared substantially more slowly than IMI, which resulted in higher internal residues after the depuration period. Consistent with their prolonged internal exposure, zebrafish exposed to DNIMI and IMIOL exhibited sustained hypoactivity postdepuration, accompanied by significantly reduced glutamate (GLU) levels and alternated transcription of GLU-related genes. The partial least-squares discriminant analysis (PLS-DA) identified GLU as a pivotal role (variable importance projection scores ≥1, <i>p</i> < 0.05). DNIMI and IMIOL downregulated genes involved in GLU synthesis and metabolism, concurrently decreasing glutamate decarboxylase and glutaminase levels while increasing glutamine synthetase levels. Exogenous GLU coexposure alleviated the locomotor activity, neurotransmitter alternations, and transcriptional perturbations, confirming disrupted GLU signaling contributes to the neurotoxicity of DNIMI and IMIOL. Overall, IMI-TPs induced stronger developmental neurotoxicity than IMI in zebrafish via GLU pathway disruption, highlighting their enhanced neurotoxic risks in aquatic environments.