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ObjectivesThis study focused on the assessment of acute toxicity caused by Osthol, a major component of environment-friendly biological pesticides, by using two aquatic organisms.MethodsThe assessment of acute toxicity caused by Osthol was conducted in Daphnia magna and by examining the morphological abnormalities in Danio rerio embryos.ResultsThe median effective concentration value of Osthol in D. magna 48 hours after inoculation was 19.3 μM. The median lethal concentration of D. rerio embryo at 96 hours was 30.6 μM. No observed effect concentration and predicted no effect concentration values of Osthol in D. magna and D. rerio were calculated as 5.4 and 0.19 μM, respectively. There was an increase in the morphological abnormalities in D. rerio embryo due to Osthol over time. Coagulation, delayed hatching, yolk sac edema, pericardial edema, and pigmentation were observed in embryos at 24–48 hours. Symptoms of scoliosis and head edema occurred after 72 hours. In addition, bent tails, ocular defects, and symptoms of collapse were observed in fertilized embryo tissue within 96 hours. Ocular defects and pigmentation were the additional symptoms observed in this study.ConclusionsBecause Osthol showed considerable toxicity levels continuous toxicity evaluation in agro-ecosystems is necessary when bio-pesticides containing Osthol are used.