Developmental‐Stage‐Dependent Gape Limitation in the Toxic Effect of Polystyrene Microbeads on the Water Flea, <scp> <i>Daphnia magna</i> </scp>

Complex mechanisms by which microplastics exert toxicity in natural environments are poorly understood, and their ecotoxicological assessment remains challenging due to their heterogeneous nature and physical properties. Methods for standard toxicity tests, originally developed for soluble chemicals, often fail to account for microplastic-specific behaviors such as sedimentation and variable ingestion by organisms. In this study, we used a rotator-based, semi-static exposure system to evaluate developmental-stage-specific toxicity of polystyrene microbeads of two sizes (3 μm and 30 μm) on Daphnia magna. Three exposure designs were employed: continuous exposure to single bead sizes, exposure to size mixtures, and sequential exposure aligned with developmental stages. These results demonstrated that 3-μm beads exerted stronger toxic effects in early life stages, whereas 30-μm beads had greater impacts in later stages, likely due to gape limitation and ontogenetic changes in ingestion capacity. Mixed-size exposure revealed potential additive or synergistic effects, particularly for body length. The rotator system ensured homogeneous particle suspensions and reproducible data, overcoming limitations of conventional static systems. These findings highlight the need to consider particle size heterogeneity, organismal developmental stage, and exposure method when assessing microplastic toxicity. Furthermore, our findings also suggest that chronic exposure to mixed particle sizes may better represent environmental situations and reveal stronger biological impacts than single-size exposures. By integrating particle size with organismal traits and realistic exposure dynamics, this study provides insight into the multifactorial nature of microplastic toxicity and supports the development of more ecologically relevant assessment methods.