Stability of naked and encapsulated dsRNA in water and on plant surfaces: Key data to inform environmental safety and toxicity assessments

RNA interference (RNAi) offers a sequence-specific strategy for sustainable pest management, but the environmental instability of unprotected double-stranded RNA (dsRNA) limits its practical foliar use. Minicell-encapsulated dsRNA (ME-dsRNA) formulations enhance stability, yet their environmental fate-a key requirement for regulatory approval-remains largely unknown. This study provides the first comprehensive comparison of the persistence of ME-dsRNA versus naked dsRNA on agriculturally relevant plant surfaces and across diverse aquatic ecosystems. We found minicell encapsulation significantly enhanced dsRNA stability, increasing its half-life by more than twofold in most environments. Nonetheless, ME-dsRNA half-lives remain relatively short compared to conventional chemical pesticides. Our results showed that persistence was shaped by a complex interplay of abiotic factors, such as water hardness (Ca²⁺), and biotic factors, especially microbial activity. Notably, we demonstrated for the first time that fungal communities in water, rather than bacterial populations, were more strongly correlated with dsRNA degradation. This study elucidates these complex degradation pathways and provides the first matrix-specific kinetic parameters (DT₅₀/DT₉₀) for both naked and minicell-encapsulated dsRNAs under varying conditions. Together, these findings offer the critical exposure data required for robust environmental risk assessments, supporting the safe and science-based deployment of next-generation RNA-based biopesticides.