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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. The effects of size and surface charge were assessed by varying the amount of cetyltrimethyl ammonium bromide (CTAB) in the NP dispersion and by decreasing or increasing the electrostatic repulsion between the NPs using poly-D-lysine (PDL) or polyethylene glycol (PEG). The estrogenicity of PLGA NPs was not significantly affected by particle size, surface charge, or shape. We used the zebrafish bioassay to evaluate the effects of these properties on the estrogenicity of PLGA NPs. The estrogenicity of PLGA NPs was not influenced by their surface properties. A mixed-mode bioassay was established to evaluate the estrogenicity of PLGA NPs. This bioassay is a simple and sensitive approach that may be applicable to the assessment of the estrogenicity of other NPs.Ensuring quality in the operating room. A multidisciplinary evaluation. The continuing emphasis on quality assurance has mandated a multidisciplinary approach to the evaluation of the operating room. 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