The assessment of the environmental footprint of an organization or product is based on methods published by the European Union Joint Research Centre, which take 16 impact areas into account. Among the listed categories are human and freshwater ecosystem toxicities. Standard protocols utilize just chemical parameters as input data, hindering the determination of the full impact of complex mixes, such as pollutants released into the environment. Biological assays enable us to overcome this gap: in the present work, assays were employed to determine both baseline and specific toxicity to aquatic species (green algae, luminescent bacteria, and crustacean cladocera) as well as specific toxicity (mutagenicity and carcinogenicity). Ecological footprint was estimated with regard to the impact categories “freshwater toxicity” and “human cancer toxicity” following the standard methodology. In parallel, the impact on the above categories was estimated using the results of biological assays as input. Standard and bioassay-based results are not always congruent, and conventional methods generally underestimate the effects. Likewise, the choice of reference substance (metals or organics) influences the quantification of impact. Appropriate batteries of biological assays could therefore be utilized to complement LCA (Life Cycle Assessment) techniques in order to make them more sensitive when considering toxicity in mid-term impact categories.

Per- and polyfluoroalkyl substances (PFAS) represent a diverse group of chemicals widely used in industrial and consumer products due to their unique physicochemical properties. This review critically examines the ecotoxicological profiles of six key PFAS—PFOA, PFOS, PFBA, PFBS, GenX, and ADONA—focusing on their effects on standard aquatic model organisms (Daphnia magna, Raphidocelis subcapitata, and Aliivibrio fischeri). The data highlight that legacy compounds such as PFOA and PFOS generally show greater toxicity, particularly with reproductive and growth endpoints in D. magna and growth inhibition in R. subcapitata, at lower concentrations compared to their short-chain analogues (PFBA, PFBS). GenX and ADONA, introduced as replacements for long-chain PFAS, have been the subject of far fewer studies. Available data, mainly from regulatory sources, suggest lower acute and chronic toxicity for these alternatives under standard test conditions. However, the lack of comprehensive data—especially regarding sub-lethal, chronic, and mixture effects—precludes firm conclusions about their environmental safety. The findings underscore the need for further ecotoxicological research on emerging PFAS and call for integrating bioassays with chemical analyses to better assess risks associated with PFAS mixtures in aquatic ecosystems.