A zinc-induced perivitelline fluid phenotype as a complementary early-life endpoint in rare minnow embryos

Early-life metal toxicity assessments typically rely on apical endpoints such as mortality, hatching, and malformation, potentially overlooking early localized responses at the embryo-environment interface. Here, we characterized a zinc (Zn)-induced perivitelline fluid (PVF) membrane-like sloughing phenotype in rare minnow (Gobiocypris rarus) embryos. The phenotype was operationally defined as a distinguishable membrane-like structure within the perivitelline space and progressed through formation, compression, and sloughing stages. Independent scoring of 190 embryo images showed 92.1% inter-observer agreement (Cohen's κ = 0.836), supporting the reproducibility of the microscopy-based classification. Zn exposure produced clear concentration-response relationships, with EC50 values of 0.318 mg/L (95% CI: 0.288-0.348) at 12 hpf and 0.258 mg/L (95% CI: 0.233-0.281) at 24 hpf. Similar PVF abnormalities were induced by Cu²⁺, Mn²⁺, Fe²⁺, and Cd²⁺, whereas Cr(VI) did not produce a comparable phenotype under the tested conditions. DEPC and NEM pretreatment reduced phenotype incidence, while transfer of embryos with preformed PVF structures to EDTA resulted in progressive dispersal, reaching 73.1% complete dispersal after 120 min compared with 2.6% in dilution water. Whole-embryo RNA-seq revealed stage-dependent Zn-responsive transcriptional changes, with significant enrichment of the ECM-receptor interaction pathway at the blastula stage. RT-qPCR, qualitative immunofluorescence, and Sirius Red staining further indicated ECM-related transcriptional and spatial changes associated with phenotype development. The PVF phenotype occurred at Zn concentrations and developmental stages at which significant mortality was not yet evident. In addition, compression score was not significantly associated with spontaneous tail-coiling activity, and no broad changes in Na, K, Ca, or Fe contents were detected within the observation window. Collectively, the Zn-induced PVF phenotype represents a reproducible, concentration-responsive early sublethal morphological readout that may complement conventional embryo toxicity endpoints. Its relationship with subsequent developmental outcomes remains to be established.