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

## TL;DR

This study compares the toxicity of plant protection products and their ingredients in zebrafish embryos and liver cells, showing that whole products can be more toxic than their individual components.

## Contribution

The study demonstrates that whole PPP formulations can be more toxic than their active substance mixtures, emphasizing the need for holistic risk assessment.

## Key findings

- Plant protection products were more or equally toxic compared to their active substance mixtures in zebrafish embryos and HepaRG cells.
- Co-formulants in PPPs increased internal concentrations of active substances in zebrafish embryos, leading to higher toxicity than predicted by concentration addition models.
- Integrated in vitro and in vivo approaches are needed for accurate safety assessments of plant protection products.

## Abstract

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.

The online version contains supplementary material available at 10.1007/s00204-025-04290-y.