Dissolved organic matter (DOM) exerts a crucial role in biogeochemical processes and ascertaining water quality in reservoirs, where it is vulnerable to the dynamic impacts of surface water inflows. However, understanding how DOM quantity and biochemical features responds to hydrodynamic forces in tropical reservoirs remains limited. To enhance our understanding of the vertical profiles of DOM characteristics under varying hydrodynamic forces (strong, moderate, and weak regions) in the Chitian Reservoir (18°43′–18°42′ N, 109°68′–109°70′ E), in December 2023, we investigated the concentrations and biochemical characteristics of water column DOM samples using multispectral techniques, a parallel factor model, and two-dimensional correlation analysis. Our results indicated that DOM concentrations (4.34 ± 0.36 mg/L) are the highest in the reservoir center, whereas total nitrogen (0.52 ± 0.04 mg/L), total phosphorus (0.02 ± 0.03 mg/L), and nitrate nitrogen (1.01 ± 0.07 mg/L) present their highest values in the inlet region. As hydrodynamic force decreases, microbial activity increases, whereas DOM’s humification degree and molecular weight decline. DOM in the Chitian Reservoir comprises humic-like components, including three terrestrial sources (accounting for 85.38%~87.03%) and one microbial source, with dominant characteristics of allochthonous origin. The relative abundance of microbial components decreased from 14.62% to 12.97% with the increasing hydrodynamic force and increased with depth. DOM functional groups in the strong hydrodynamic force region and the reservoir’s upper layer show high consistency and uniformity. Phenolic O–H is the most reactive functional group concerning changes in water depth across all hydrodynamic areas, followed by polysaccharide C–O, owing to its high photoactivity. In contrast, aromatic C–H demonstrates the weakest reactivity. DOM’s spectral features are closely linked to nutrient form concentrations (N and P).
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