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The estrogenicity of polylactic-co-glycolic acid (PLGA) nanoparticles (NPs) with average size diameters of 100, 200, and 350 nm was investigated using the Japanese medaka (Oryzias latipes) bioassay. The estrogenicity of PLGA NPs was evaluated using a mixed-mode assay that included a zebrafish embryo (Danio rerio) bioassay as an initial screening step, followed by a medaka embryo bioassay. We used the zebrafish bioassay because this assay provides better sensitivity to detect the estrogenicity of NPs with low toxicity compared with the medaka bioassay. In the zebrafish bioassay, the NPs were added to the water at concentrations of 1, 10, and 100 microg/L and were incubated for 72 hr. In the medaka bioassay, the NPs were injected into the pericardial sac of one-day-old embryos at a concentration of 1 mg/L. 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