Integrating Bioassays and Non-Target Screening: A Review of Effect-Directed Analysis for Unknown Toxicants

The widespread use of chemicals continues to pose significant risks to ecosystems and human health. While toxicological mechanisms of many known pollutants remain incompletely understood, the emergence of numerous unknown or poorly characterized contaminants intensifies the urgent need for robust identification strategies. Effect-directed analysis (EDA) has shown effective in detecting bioactive compounds in complex environmental matrices, yet traditional EDA approaches mainly relying on target analyses are inherently limited in uncovering new or unexpected toxicants. Integrating emerging non-target analysis (NTA) techniques with EDA offers a transformative approach, enabling comprehensive profiling of unknown compounds and improving accuracy and efficiency of environmental risk assessment. Despite this potential, the lack of standardized workflows has constrained the widespread application of NTA in EDA. The present review summarized recent developments in integrating NTA with EDA, covering key aspects from sample collection and preparation to fractionation, instrumental analysis, and toxicity confirmation, with a focus on chemical analysis for various matrices. We discuss key methodological challenges such as matrix effects and confidence levels in structural elucidation. By synthesizing current knowledge and identifying future directions, this work aims to provide actionable guidance for the identification of new or less-concerned toxicants in mixtures, ultimately advancing environmental monitoring and public health protection with integrative EDA and NTA approaches.