Periodic Reporting for period 1 - ECOPFAS (Ecotoxicogical assessment of banned and novel PFAS as individuals or in mixture)
Período documentado: 2023-10-01 hasta 2025-09-30
Per- and polyfluoroalkyl substances (PFASs) are a large group of synthetic organofluorine compounds widely used for their exceptional physicochemical properties. Among them, legacy PFAS such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been extensively used and are now found globally in the environment due to their persistence, bioaccumulation, and described toxicity. To replace these regulated substances, industry is developing new organofluorine compounds, such as perfluoroalkyl ether carboxylic acids (PFECAs) — e.g. hexafluoropropylene oxide trimer acid (HFPO-TA) and perfluoro-3,6,9-trioxadecanoic acid (PFO3DA) — and perfluoroalkyl ether sulfonic acids (PFESAs), including NBP1 and NBP2. These alternatives were designed with modified that were presumed to improve degradability and reduce environmental persistence. However, emerging evidence shows that many of these new compounds are ubiquitously detected in human serum and aquatic systems with sometimes concentrations comparable to or higher than legacy PFAS.
Despite their increasing global occurrence, the ecotoxicological properties of these new PFAS remain insufficiently characterised. Underlying a critical need to understand their effects on aquatic organisms of both individual compounds and their mixtures. The ECOPFAS project addresses this gap by aiming to elucidate the ecotoxicological impacts of banned and emerging PFAS through an integrative approach combining apical toxicity testing with mechanistic (omics) analyses. The project specifically seeks to (i) characterise dose-dependent effects of individual PFAS, (ii) evaluate mixture toxicity and potential synergistic interactions, and (iii) develop mechanistic insights to support improved environmental risk assessment frameworks.
Despite their increasing global occurrence, the ecotoxicological properties of these new PFAS remain insufficiently characterised. Underlying a critical need to understand their effects on aquatic organisms of both individual compounds and their mixtures. The ECOPFAS project addresses this gap by aiming to elucidate the ecotoxicological impacts of banned and emerging PFAS through an integrative approach combining apical toxicity testing with mechanistic (omics) analyses. The project specifically seeks to (i) characterise dose-dependent effects of individual PFAS, (ii) evaluate mixture toxicity and potential synergistic interactions, and (iii) develop mechanistic insights to support improved environmental risk assessment frameworks.
Extensive zebrafish embryo-larval exposure studies following the OECD TG 236 “Fish Embryo Toxicity Test” were conducted using legacy PFAS (PFOA, PFOS) and novel alternatives (HFPO-TA, PFO3DA, NBP1, NBP2), individually and in mixtures. Several endpoints were measured including survival, hatching, developmental malformations, cardiac activity and behavioural activities. Results have demonstrated that the six tested PFASs have different dose-dependent toxicity with NBP1 exhibiting the highest toxicity, followed by PFOS-K and PFO3DA. NBP2, HFPO-TA, and PFOA having the lower toxicity. Explaining why the LC50 values for PFOA, NBP2, and HFPO-TA were not determined. The results have also demonstrated dose-dependent developmental disturbances different for each PFASs and the mixture. In addition, the mixture was inducing deformities at lower concentrations emphasizing the importance of performing mixture-based testing.
Extensive Daphnia magna exposure studies were also performed following both the OECD TG 202 “Daphnia sp., Acute Immobilisation Test” and 211 “Daphnia magna Reproduction Test”. The D. magna acute immobilisation test has also demonstrated different dose-dependent toxicity after 48 h exposure for each PFASs with from most toxic NBP-1, HFPO-TA, PFOS-K, NBP-2 and PFO3DA and PFOA having the lower toxicity. Those results guided the selection of the mixture concentrations to perform the reproduction test with the PFAS mixture that significantly affected growth, reproduction, swimming activities, and survival in D. magna at lower doses.
In addition, the mechanism of how PFAS chemicals disturb the normal functioning of organisms was analysed at the gene level.
In zebrafish, the results showed that PFAS affect key biological processes such as fat metabolism, hormone production, and the ability of cells to protect themselves from stress. Both the legacy PFASs and the newer alternatives interfered with similar molecular pathways showing that these alternatives may not be safer than the legacy ones.
In adult D. magna, PFAS exposure activated the organism’s detoxification and stress-response systems, while reducing its ability to produce energy and build essential proteins. Furthermore, in juvenile D. magna, the effects were even more pronounced. Genes involved in growth, molting, and the development of the outer shell were strongly repressed, while stress and detoxification genes were activated. This shows that young organisms are more sensitive than adults, especially during key developmental stages.
Extensive Daphnia magna exposure studies were also performed following both the OECD TG 202 “Daphnia sp., Acute Immobilisation Test” and 211 “Daphnia magna Reproduction Test”. The D. magna acute immobilisation test has also demonstrated different dose-dependent toxicity after 48 h exposure for each PFASs with from most toxic NBP-1, HFPO-TA, PFOS-K, NBP-2 and PFO3DA and PFOA having the lower toxicity. Those results guided the selection of the mixture concentrations to perform the reproduction test with the PFAS mixture that significantly affected growth, reproduction, swimming activities, and survival in D. magna at lower doses.
In addition, the mechanism of how PFAS chemicals disturb the normal functioning of organisms was analysed at the gene level.
In zebrafish, the results showed that PFAS affect key biological processes such as fat metabolism, hormone production, and the ability of cells to protect themselves from stress. Both the legacy PFASs and the newer alternatives interfered with similar molecular pathways showing that these alternatives may not be safer than the legacy ones.
In adult D. magna, PFAS exposure activated the organism’s detoxification and stress-response systems, while reducing its ability to produce energy and build essential proteins. Furthermore, in juvenile D. magna, the effects were even more pronounced. Genes involved in growth, molting, and the development of the outer shell were strongly repressed, while stress and detoxification genes were activated. This shows that young organisms are more sensitive than adults, especially during key developmental stages.
The ECOPFAS project has generated substantial advances in understanding the risks posed by both banned and new alternatives, contributing directly to the scientific state of the art in ecotoxicology and risk assessment. Across the models tested, the results show that the new PFAS alternatives are not benign substitutes and may produce toxic effects comparable or even exceeding the legacy PFASs. Importantly, mixture exposures produced effects that cannot be predicted from single-chemical assessments, highlighting a major gap in current regulatory practices. ECOPFAS broadened the classical OECD endpoints by integrating more sensitive biomarkers such as cardiac rhythm, brood timing, behavioural responses, and maternal transfer. These refinements strengthen predictive toxicology and may help shape future testing guidelines. In parallel, the use of gene expression has produced a new set of tools, concepts, and molecular datasets that go beyond what is currently available in PFAS toxicology. One major innovation is the comparative omics framework between zebrafish embryos and both juvenile and adult D. magna. This multi-species, multi-life-stage approach enables the identification of cross-species molecular signatures of PFAS toxicity. The framework is particularly valuable because it directly links molecular responses—genes, pathways, and biomarkers—to higher-level apical outcomes such as growth, reproduction, and developmental abnormalities. It also provides the first mechanistic comparison of legacy PFAS (PFOA, PFOS) and emerging alternatives (HFPO-TA, PFO3DA, NBP1, NBP2). Another key outcome is the generation of PFAS mixture-specific transcriptomic fingerprints. These datasets reveal clear signs that mixture doesn’t lead additive effects during combined exposures and identify molecular endpoints that predict mixture toxicity more accurately than assessments of individual compounds. Together, these innovations provide a new foundation for mixture risk assessment and offer highly relevant insights for future regulatory decision-making.