Life stage-specific endocrine disruption by Tris(2-ethylhexyl) trimellitate (TOTM) in zebrafish: Thyroid-neurobehavioral effects and metabolic-reproductive alterations

Tris(2-ethylhexyl) trimellitate (TOTM) is a widely used plasticizer alternative to bis(2-ethylhexyl) phthalate in medical and consumer products, yet its toxicological profile remains poorly characterized. Given the increasing detection of TOTM in environmental matrices, concerns regarding its potential biological activity are rising. This study evaluated the endocrine-disrupting potential of TOTM across zebrafish life stages, integrating apical endpoints with transcriptomic profiling to investigate potential mechanisms. In 7 days post-fertilization (dpf) larvae, TOTM exposure produced a hyperthyroid-like hormone profile, with elevated whole-body thyroid hormones accompanied by altered transcription of thyroid axis–related genes, including some responses in the lowest exposure group (<0.047 mg/L). This hormonal imbalance was accompanied by significant hypoactivity in the light–dark transition assay, with reductions in total distance moved, mean velocity, and acceleration observed during the second light cycle. Transcriptomic analysis at 5.00 mg/L revealed that these neurobehavioral alterations were potentially linked to disrupted phototransduction and circadian rhythm pathways. In contrast, adult male zebrafish exposed to TOTM exhibited distinct metabolic and reproductive toxicity. While thyroid hormones showed no significant changes, reduced hepatic total cholesterol and sex steroid imbalance were observed at < 0.047 and 0.38 mg/L. These changes were supported by transcriptional alterations in steroidogenic and lipid metabolism genes. Collectively, the current findings demonstrate that TOTM exposure disrupts endocrine function, with different susceptibility by life stage in zebrafish: primarily targeting the thyroid-neurobehavioral axis in developing larvae while disrupting lipid and sex steroid homeostasis in adults. These results highlight the complex endocrine-disruption profile of TOTM and underscore the need for subsequent observational studies in human populations.