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Introduction: In the field of biomedicine, marine algae have gained significant attention due to the abundance of bioactive compounds they contain. However, studies on the chronic toxicity of algae are limited. Aim: To evaluate the in-vitro, in-vivo acute, and subchronic toxicity of Sargassum tenerrimum (S. tenerrimum) in an adult zebrafish model. Materials and Methods: The current preclinical toxicological interventional study was conducted at the Department of Pharmacology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, Tamil Nadu, India, in September 2022. The algae S. tenerrimum was extracted with methanol using Soxhlet extraction. In-vitro toxicity of S. tenerrimum was performed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay on the SH-SY5Y cell line. Acute toxicity was carried out for 96 hours according to Organisation for Economic Cooperation and Development (OECD) 203 guidelines, and subchronic toxicity was carried out for 14 days on the zebrafish model using the immersion method. Additionally, histopathological changes were observed after the acute toxicity study, and the Lethal Concentration 50 (LC50) value was analysed. Results: The MTT assay of the methanolic extract of S. tenerrimum revealed an LC50 value of 140.014 µg/mL. An acute toxicity study conducted on zebrafish for 96 hours showed an LC50 value of 504.669 mg/L. Subchronic toxicity was done for 14 days, and on the 14th day of exposure, the LC50 value was found to be 404.196 mg/L. Histopathological changes were observed at higher concentrations (800 mg/L) of algal extract. Conclusion: The present study revealed that the methanolic extract of S. tenerrimum showed a toxic effect at higher concentrations on the zebrafish model. However, at lower concentrations, S. tenerrimum was deemed safe for further exploration of pharmacological activities in a zebrafish model. [ABSTRACT FROM AUTHOR]