Matrix-driven hydrolysis of an antimicrobial peptide reveals limitations of standardized aquatic degradation tests

Hydrolysis is commonly assessed using buffered ultrapure water to predict the environmental persistence of organic contaminants, yet the relevance of this approach for complex aquatic systems remains uncertain. Here, we investigate the hydrolytic degradation of Pep16, a newly developed antimicrobial peptide, in buffered ultrapure water following Organisation for Economic Co-operation and Development guideline No. 111 and in sterilized surface waters from the Seine River. While Pep16 was stable under all buffered conditions and is thus classified as non-hydrolysable according to guideline criteria, it degraded measurably in surface waters, with half-lives ranging from 346 to 2,665 hr depending on sampling time and sterilization method. Additional experiments reveal that Pep16 undergoes hydrolysis mediated by microbial extracellular enzymes as well as chemically catalyzed abiotic hydrolysis, mediated by aqueous matrix components such as organic matter. Degradation was also enhanced in the presence of Cu2+, whereas nitrate, nitrite, sulfate, and Fe2+ exerted no consistent effects. Moreover, additional undetermined factors catalyzing Pep16 hydrolysis remain to be investigated. Several transformation products were identified using high-resolution mass spectrometry and were consistent with peptide backbone hydrolysis, with limited evidence for potential oxidative degradation. Overall, this study highlights the complementarity of buffer-based hydrolysis assessments and hydrolysis experiments conducted in river water. These approaches allow us to understand both pure abiotic, pH-dependent hydrolysis, matrix-driven hydrolysis, and potential co-occurring biotic hydrolysis, which is crucial for accurately evaluating the environmental fate of peptide-based contaminants in surface waters.