Effects of nanoplastics and 17α-ethinylestradiol exposure on skeletal development in zebrafish (Danio rerio) larvae

Nanoplastics (NPs) accumulate in aquatic environments, cross biological barriers, and may hinder or harm early skeletal development in aquatic organisms. Estrogenic endocrine disruptors such as 17α-ethinylestradiol (EE2) can impair craniofacial ossification in fish. However, the effects of NPs on skeletal development in these organisms are not well characterized. Here, we compared the impact of 50 nm polystyrene NPs and EE2 on craniofacial skeletal development and skeletal formation-related gene expression in zebrafish (Danio rerio) larvae, using commercially available fluorescent polystyrene nanoparticles (nominal diameter: 50 nm, aqueous suspension). Embryos were exposed to NPs (5, 50, or 200 μg/mL) or EE2 (10 or 1000 ng/L) between 3 h post-fertilization (hpf) and 10 days post-fertilization (dpf). Operculum area and craniofacial skeletal length were significantly lower in all NPs- and EE2-exposed groups than in the controls. Even low NPs concentrations reduced early-forming skeletal bones. In contrast, it was mainly higher NPs concentrations that inhibited late-forming bones. Principal component analysis (PCA) and hierarchical clustering revealed overlapping skeletal length reduction patterns in the NPs- and EE2-exposed groups. Low NPs exposure downregulated the osteoblast differentiation-related genes runx2 and sp7 whereas higher NPs concentrations repressed the mineralization-related genes alpl, enpp1, and bglap. Similar gene expression patterns were observed for the EE2-exposed groups. The osteoclast-related gene ctsk was downregulated and, therefore, bone resorption was suppressed in the groups exposed to high concentrations of NPs and the EE2-exposed groups. Relative sensitivity to NPs and EE2 type and dosage differed between estrogen receptor subtypes esr1 and esr2a. The findings suggest that NPs exposure may induce alterations in skeletal developmental similar to those observed following EE2 exposure. They also indicate that early-life NPs exposure in zebrafish could influence their skeletal formation.