Comparative toxicity of plant protection products, their active substances and mixtures in zebrafish embryos and HepaRG cells

The intensive use of plant protection products (PPPs) is essential in modern agriculture to ensure food security. PPPs are complex formulations of active substances (ASs) and co-formulants, yet regulatory risk assessment primarily focuses on AS toxicity, often overlooking combined effects. This study assessed the toxicity of three commercial PPPs, their AS-mixtures, and a co-formulant using the in vitro HepaRG liver cell assay and the in vivo zebrafish embryo toxicity test (ZFET). Product 1 contained the AS Benzovindiflupyr (Benzo), and Product 2 contained Benzo and Prothioconazole (Pro). Product 3 contained Pro and Tebuconazole (Teb) and the co-formulant N,N-Dimethyldecanamide (DDA), which was singled out for further investigation. AS were tested in different concentration ranges from 0.03 to 105 µmol/L in the ZFET and from 12.8 to 454.8 µmol/L in HepaRG cells. AS-mixtures were tested from 0.06 to 50 µmol AS/L (ZFET) and from 7.7 to 312.0 µmol AS/L (HepaRG). PPPs were tested from 0.02 to 29.8 µmol AS/L (ZFET) and from 2.9 to 312.0 µmol AS/L (HepaRG). PPPs were either more or as toxic as their respective AS-mixtures. In the ZFET, Products 1 and 2 showed similar toxicity to their AS-mixtures, while Product 3 showed a fourfold increase. In HepaRG cells, Product 1 was four times and Product 3 three times more toxic than their mixtures. Concentration addition models underestimated observed effects, particularly for Product 3, where co-formulants increased internal AS concentrations in zebrafish embryos. These findings underscore the need for whole mixture-based risk assessment for selected PPPs and support using integrated in vitro/in vivo approaches. Our study highlights the need for holistic PPP evaluation to improve safety assessments and regulatory strategies.