ENSAFE: ENvironmental SAFEty of Biotechnological Plant Protection Products

The ENSAFE project is exploring innovative bio-based alternatives, such as RNA interference (RNAi) technology and bioactive peptides, which offer effective pest and disease control with minimal environmental impact. However, the EU currently lacks a dedicated regulatory framework to assess these innovations. ENSAFE aims to bridge this gap by developing a science-based environmental risk assessment framework, ensuring the safe and sustainable use of bio-based plant protection products.  

The project aims to develop tools to understand biological effect and environmental fate of siRNA- and peptide-based Plant Protection Products (PPPs) as a basis to assess their risk and address how they can be regulated in a way that ensures environmental safety of the products. Biological effects will be addressed using both in silico models (WP1, hypothesis i) and biological tests on a wide range of aquatic and terrestrial non-target species and model ecosystems (WP2, hypothesis ii and iii). Fate will be addressed through new methodologies to reliably measure siRNA and peptide molecules in environmental matrices and parametrize and test existing fate models in model ecosystems (PW3, hypothesis iv). Regulatory needs and approaches used outside EU will be combined with the project knowledge to provide guidance towards a more efficient regulatory approach designed for siRNA- and peptide-based PPPs (WP4).

The above figure illustrates how Plant Protection Products (PPPs) based on dsRNA and peptides are different from the chemical PPPs on which the EU-legislation is based. Chemicals (purple) diffuse through cell membranes, are broken down fully or partly by detoxification enzymes (P450s), esterases, transferases) and/or bind to their molecular protein target (red cross on protein), which is usually an enzyme, transport channel or a signal receptor. Double stranded RNA (dsRNA shown in red), on the other hand, are large molecules and are either taken up through membrane channels or through endocytosis. When inside the cell, they are cut to smaller pieces creating small interfering RNA, siRNA. These can either be degraded by cellular RNAses, or in some species be multiplied by RNA-polymerases, or they are recognized by the RISK enzyme complex, which pairs them to matching messenger RNAs which are destroyed, thereby silencing the translation of that specific protein (red cross before mRNA translation). The peptides (green) are large as dsRNA and uptake therefore believed to be mainly through endocytosis, though other processes cannot be ruled out. Peptides are degraded by cellular proteases, or they bind to protein targets as chemicals do, thereby potentially disturbing similar biochemical pathways as do chemical PPPs. Both siRNA and peptides may thicker the immune system of cells, as both resemble invading foreign infections. The toxocokinetic processes are hypothesized to play a large role for variations in species sensitivity. The processes we will focus on in ENSAFE are given with broken arrows and question marks.

ENSAFE will develop a comprehensive scientific foundation and practical tools to evaluate the environmental safety of siRNA- and peptide-based PPPs. The project is structured around the following hypotheses:  

1. The vulnerability of species to siRNA-based PPPs can be predicted by identifying conserved nucleotide sequences from genomic databases and analyzing receptor conservation for peptide-based PPPs.  
2. siRNA and specific peptides may elicit generic immunotoxin effects, as organisms may respond to siRNAs in a manner similar to their responses to viral infections and antigens.  
3. The toxicity of siRNA- and peptide-based PPPs is influenced by their formulation and the processes of Adsorption, Distribution, Metabolism, and Excretion (ADME). Factors such as surface contact (e.g., cuticles, gut membranes, respiratory surfaces) and physiological conditions (e.g., extreme pH, enzymatic activity) affect molecular stability and toxicity.  
4. The dissipation half-lives of siRNA products, which determine their persistence and fate in the environment, are more influenced by formulation technology than by nucleotide sequence. In contrast, the persistence of peptide-based PPPs depends on their amino acid composition and resulting stability.