N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is used as a ubiquitous rubber antioxidant worldwide and has been shown to be potentially toxic to aquatic organisms. In this study, zebrafish embryos were exposed to 6PPD for five days starting at two hours post-fertilization at concentrations of 0, 0.0022, 0.022, and 0.22 mg/L to investigate its effects on embryonic development, the growth hormone/insulin-like growth factor (GH/IGF) axis, and the hypothalamic-pituitary-thyroid (HPT) axis. The results showed that the 96 h LC50 of 6PPD was 2.2 mg/L. 6PPD exposure decreased hatchability, lowered autonomous movement, reduced body length in zebrafish embryos and caused deformities. The hormones levels and the expression of genes related to GH/IGF and HPT axis were altered after exposure to 6PPD in zebrafish larvae. These results indicated that the GH/IGF and HPT axis was disturbed. Moreover, treatment of 6PPD produced oxidative stress in zebrafish embryos. Overall, the present study thus demonstrated that exposure to 0.22 mg/L 6PPD caused developmental toxicity and disrupted the GH/IGF and HPT axis of zebrafish, which could be responsible for developmental impairment and growth inhibition.

Copper (Cu²⁺) and zinc (Zn²⁺) are two kinds of heavy metals essential to living organisms. Cu²⁺ and Zn²⁺ at excessive concentrations can cause adverse effects on animals, but little is known about the thyroid-disrupting effects of these metals in fish, especially in the early developmental transition stage from embryos to larvae. Wild-type zebrafish embryos were used to expose to Cu²⁺ (0, 1.5, 15, and 150 μg/L) and Zn²⁺ (0, 20, 200, and 2000 μg/L) for 120 h. Thyroid hormone contents and transcriptional changes of the genes connected with the hypothalamic-pituitary-thyroid (HPT) axis were measured. Results showed that zebrafish embryos/larvae malformation rates were significantly increased in the Cu²⁺ and Zn²⁺ groups. Remarkably elevated thyroxine (T4) concentrations and reduced triiodothyronine (T3) concentrations were observed in Cu²⁺ and Zn²⁺ exposure fish. And the expression patterns of genes connected with the HPT axis were changed after Cu²⁺ and Zn²⁺ treatment. Based on principal component analysis (PCA) results, Zn²⁺ caused significant effects on the thyroid endocrine system at 200 μg/L, while Cu²⁺ resulted in thyroid disruption as low as 1.5 μg/L. In short, our study demonstrated that exposure to Cu²⁺ and Zn²⁺ induced developmental toxicity and thyroid disruption to zebrafish embryos/larvae.