Environmental Fate and Risk Assessment in Soil of RNAi-based biopesticide dsRNA V-ATPase subunit A against small hive beetle Aethina tumida

Abstract

Pest control is essential for agricultural production. Many conventional pesticides present a risk for human/animal health and ecological biodiversity because of their environmental persistence and broad-spectrum action. Thus, the development of novel biopesticides which are less persistent and more selective in their action, and thus more eco-friendly, represent a promising avenue for more sustainable food production. However, European Union regulation like (EC) 1107/2009 requires to fully assess the risks of all new active substances. Double stranded RNA (dsRNA) is a biomolecule that triggers the mechanism of RNA interference (RNAi), which is a protein synthesis disruptor mechanism in eukaryotic cells. dsRNA has considerable potential as a tool for selective insect pest control. Analytical protocols for its extraction, purification and quantification from soil are essential for the tests required to parameterize environmental fate assessments. This study systematically developed a procedure for recovering and quantifying dsRNA from loamy sand soil using RT-qPCR. During adsorption experiments following the OECD 106 Guidelines for Testing of Chemicals, it was found that pre-equilibrating the soil with CaCl2 solution (0.01M) resulted in precipitation of dsRNA (CaCl2 solution + dsRNA), because CaCl2 might neutralize the charges on the phosphate backbone of dsRNA. This enhanced adsorption was compared to soil equilibration with distilled water. Thus, soil solution composition and ionic strength are important considerations when following OECD guidelines in assessing the sorption of a new generation of biopesticides consisting of nucleic acids. In adsorption tests, the dsRNA reached equilibrium within 1h in loamy sand soil with a sorption coefficient (Kd) 0.55 L kg -1 . Next, biodegradation tests in soil were carried out following the OECD 307 guidelines. The experimental data were fitted with three different biodegradation models (Single First-Order Rate Model, Double FirstOrder in Parallel, First Order Multi-Compartment). The results showed that dsRNA has a short half-life (DT50) of 10h. This informed an environmental risk assessment in soil for dsRNA within European legislation EU 1107/2009 which is necessary for the registration of dsRNA as biopesticide. Furthermore, a new screening test procedure was proposed to identify sensitive species for biopesticide toxicity by using a bioinformatics tool (BLAST searching in the NCBI database) to find in the genome of non-target organisms regions of local similarity with the dsRNA sequence. Overall, it was concluded that even though the dsRNA had a low risk profile due to its ready biodegradability, considerable uncertainty remains around potentially high application rates and potential interference of the smaller siRNAs generated from the dsRNA with mRNA of non-target organisms.