Adverse outcome pathway networks of microplastic ecotoxicity to aquatic organisms: A critical review

Numerous studies have documented the ecotoxicity of microplastics in aquatic organisms, yet the mechanistic pathways linking early molecular disturbances to higher-level biological outcomes remain poorly understood. In this paper, we conducted a comprehensive multi-level ecotoxicological review integrated with automated text-mining using AOP-helpFinder 3.0. This approach retrieved confidence scores for stressor-event and event-event relationships, enabling systematic identification of candidate molecular initiating events (MIEs), key events (KEs), and adverse outcomes (AOs). A putative adverse outcome pathway (AOP) network was then constructed, anchored on susceptible tissues (gill, gut, liver/gonad, and brain) and incorporating downstream cascades of interlinked KEs. From this network, a linear AOP was selected and qualitatively evaluated using Bradford Hill criteria to assess causal linkages. The weight-of-evidence assessment revealed strong support for early and midstream key event relationships, whereas downstream relationships remained weakly substantiated, highlighting critical knowledge gaps. Importantly, this review emphasizes the need to adapt the classical definition of the MIE for non-chemical particulate stressors such as microplastics. We propose that the initial mechanistic interaction occurring at epithelial surfaces represents the putative MIE for microplastic toxicity in aquatic organisms. Overall, this review applies the AOP framework as a qualitative tool to organize mechanistic evidence of microplastic toxicity. By capturing the cascading effects of microplastic exposure across biological levels, the proposed AOP network provides a structured basis for predictive ecotoxicological assessment and supports the development of regulatory risk management strategies for microplastic pollution in aquatic ecosystems.