Revision of Test Method for Acute Biological Toxicity — Semi-static Method Using Zebrafish Embryos

This method applies to the determination of acute toxicity to zebrafish embryos of effluent, wastewater, and environmental agents. Testing laboratories shall conduct semi-static tests using fertilized zebrafish embryos, replace test solutions every 24 hours, observe embryo development and mortality, and calculate the 96-hour median lethal concentration (LC50) or acute toxicity unit. The method specifies requirements for sample collection, light-protected refrigerated or frozen storage, pre-test preparation, embryo source and health conditions, test vessels and water-quality control, and negative and positive controls. Where a cosolvent is used for poorly soluble environmental agents, a solvent control test shall be included.