Selenium toxicity to oviparous vertebrates is often attributed to selenomethionine (SeMet), which can maternally transfer to developing embryos. The mechanism of SeMet toxicity is unclear. Furthermore, salinity of fresh waterways is increasing due to climate change and anthropogenic disturbance. Hypersalinity can potentiate SeMet toxicity to Japanese medaka (Oryzias latipes). The current study aimed to characterize the molecular mechanisms of SeMet and hypersalinity at sensitive developmental stages. Developmental toxicity of seawater was compared to desalination brine (DSB) and artificial water based on the San Joaquin River (SJR), CA. DSB toxicity was equal to seawater, while SJR water was the most toxic to embryos and larvae. Flavin-containing monooxygenases (FMOs) initiate SeMet toxicity and are induced by hypersalinity. However, developmental expression and regulation of FMOs in fish are unknown. Five putative medaka FMOs were identified with differential developmental mRNA expression patterns: two FMOs increased during mid-organogenesis; two FMOs decreased beginning at early organogenesis; and one FMO remained constant. Promoter analysis indicated regulation by developmental factors and the UPR. Treatments with UPR-inducer tunicamycin increased expression of two FMOs. In contrast, dithiothreitol inhibited the UPR and three FMOs, suggesting that FMOs are differentially regulated by the UPR. The developmental stage sensitivity of medaka embryos to SeMet was investigated in freshwater and SJR water. Stages 9-25 were most sensitive to SeMet; and hypersalinity potentiated SeMet toxicity during the onset of liver organogenesis, osmoregulation, and chondrogenesis. The mechanisms behind the potentiation of SeMet toxicity by hypersalinity indicated no involvement of oxidative stress or apoptosis; however, results suggested a role for the unfolded protein response (UPR) when animals were treated with 50μM SeMet for 12hrs. Mechanisms of SeMet-induced spinal deformities (5μM and 2.5μM for 24hrs) were further elucidated using imaging methods and showed increased oxidative stress and apoptosis in tails of embryos with spinal malformations. Gene expression analysis demonstrated a UPR activation pattern unique from UPR positive controls. Furthermore, these effects prematurely repressed chondrogenesis and induced osteogenesis. Overall, results will be useful for the risk assessment of hypersalinity and Se under hypersaline conditions; and inform studies on developmental mechanisms of toxicity
