Chemicals in the aquatic environment can be harmful to organisms and ecosystems. Knowledge on effect concentrations as well as on mechanisms and modes of interaction with biological molecules and signaling pathways is necessary to perform chemical risk assessment and identify toxic compounds. To this end, we developed criteria and a pipeline for harvesting and summarizing effect concentrations from the US ECOTOX database for the three aquatic species groups algae, crustaceans, and fish and researched the modes of action of more than 3,300 environmentally relevant chemicals in literature and databases. We provide a curated dataset ready to be used for risk assessment based on monitoring data and the first comprehensive collection and categorization of modes of action of environmental chemicals. Authorities, regulators, and scientists can use this data for the grouping of chemicals, the establishment of meaningful assessment groups, and the development of in vitro and in silico approaches for chemical testing and assessment.