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A 2‐tier standardized protocol was designed to test the toxicity of microplastics to planktonic organisms. This approach uses sea urchin (Paracentrotus lividus) and copepod (Acartia clausi) larvae because they are common biological models in marine research, and standard methods for toxicity testing with regulatory applications are available. In Tier I, leachates obtained at a 100 to 1 liquid to solid ratio are tested, and toxic units are calculated using a probit dose–response model to quantify the toxicity of the plastics. In Tier II, which is conducted only if significant toxicity (> 1 toxic unit) is found in Tier I, particles less than 20 μm in size are tested at concentrations between 0.1 and 10 mg L−1, and a toxicity threshold suitable for ranking materials according to their toxicity is obtained from the 10% effect concentration (EC10) values. Results point to chemical additives as being responsible for the toxicity found in certain plastic materials. This process is suitable for both a priori identification of the hazard posed by plastic objects in the aquatic environment, and a posteriori assessment of environmental risk caused by microplastic pollution. The method also provides a quantitative procedure appropriate for ranking plastic materials according to their toxicity to aquatic organisms. Environ Toxicol Chem 2019;38:630–637. © 2018 SETAC Standard protocol for plastics' toxicity testing including micronization of plastic objects, Tier I leachate test and calculation of toxic units (toxic units), and Tier II microparticle test and calculation of the toxicity threshold. EC10 = 10% effect concentration. [ABSTRACT FROM AUTHOR]