The embryos and chorions in Japanese flounder eggs exposed to OPO seawater were physiologically and histologically analyzed. In control embryos, eosinophilic granules were observed from the heart beat period to immediately after hatching, and they were concluded to be hatching enzyme granules based on a comparison with other studies. Eosinophilic granules were also observed in eggs exposed to OPO (ozone-produced oxidants) seawater at the same development stages, and delayed hatching of eggs, indicating secretion of hatching enzymes. Light and electron microscopy revealed abnormalities in the chorions of eggs exposed to OPO seawater. Furthermore, the puncture pressure of the chorion of eggs exposed to OPO seawater was significantly smaller than that of the control eggs in the heart beat period. Moreover, while part of the chorion was seen to protrude immediately before hatching and hatching occurred at this protrusion in control eggs, this phenomenon was not observed in eggs exposed to OPO seawater. Therefore, the cause of delayed hatching was likely the protruded process and the abnormalities of the chorion.