Molecular Evidence of the Promotion of Refractory Dissolved Organic Matter through Anthropogenic DOM Inputs to the Fen River, China

Dissolved organic matter (DOM) represents a critical nexus in the global carbon cycle, with rivers acting as pivotal reservoirs and transporters of terrestrially derived carbon. Anthropogenic nitrogen (N) and sulfur (S) inputs complicate DOM dynamics, altering its bioavailability and persistence, yet the molecular drivers of refractory DOM (RDOM) formation in human-altered river systems remain unresolved. Here, we integrate Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), 13C stable isotope analysis (delta 13CDOC) analysis, and advanced optical characterization to unravel DOM sources and RDOM transformation pathways in the anthropogenically stressed Fen River basin. Longitudinal analyses revealed increasing dissolved organic carbon (DOC) concentrations (2.18-10.37 mg/L), RDOC concentrations (0.07-7.43 mg/L) and S-containing DOM contributions (64.9 +/- 4.1% to 69.6 +/- 10.1%) along the river continuum, tightly coupled with sewage-derived inputs. S-enriched CHOS compounds emerged as robust molecular markers of RDOM production, dominated by aliphatic structures, hydrophobic units (HUs), and humic acids (HAs). This study deciphers the molecular architecture of DOM persistence in human-impacted rivers, identifying CHOS as both tracers and enablers of RDOM formation. By linking sewage inputs to S-mediated DOM stabilization, these findings provide actionable insights for mitigating legacy organic pollution in freshwater ecosystems under intensifying anthropogenic and climatic pressure.