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An acute toxicity study assessed the LC50 values for eight different amino acid ionic liquids (AAILs), featuring two cations, tetrabutylphosphonium [P4444] and tetrabutylammonium [N4444], coupled with four anions [PHE], [ASP], [SER], and [GLY]. According to the OECD 203 standard for acute fish toxicity tests with guppy fish (Poecilia reticulata, all the AAILs exhibited low toxicity levels, and were practically nontoxic and harmless. The LC50 values surpassed 100 mg/L and 1000 mg/L. This study provides valuable insights for industrial professionals in utilizing tetrabutylphosphonium-based amino acid ionic liquids [P4444] [AA] and tetrabutylammonium-based amino acid ionic liquids [N4444][AA] in chemical processes, indicating their safety in aquatic environments. These promising results highlight the potential of incorporating these AAILs into diverse chemical processes while ensuring minimal ecological impact.