Integrated isotopic and molecular insights reveal agricultural legacy phosphorus driving Nanyihu Lake eutrophication.

Phosphorus (P)-driven eutrophication remains a major ecological threat to aquatic ecosystems, particularly in intensively farmed regions. However, the relative contributions of agricultural legacy P and internal sediment-derived P remain poorly constrained due to complex transformation pathways and the lack of integrated source-tracing approaches. In this study, we combined chemical fractionation, phosphate oxygen isotope (δ18OP) tracing, and high-resolution molecular characterization to identify dominant P sources and their bioavailability in Nanyihu Lake, China. Results from lake and estuarine sediments, as well as adjacent agricultural soils, indicate that labile (LP) and moderately labile phosphorus (MLP) are the main bioavailable fractions. Bayesian mixing models indicate that agricultural sources attributed 46 % of total P inputs, including agricultural soils (25 %) and feces (21 %), exceeding contributions from internal sediment release (38 %) and wastewater treatment plants (16 %). In situ profiling using diffusive gradients in thin films (DGT) and Peeper methods reveals that iron (oxy)hydroxide reduction facilitates sedimentary P release, with soluble reactive phosphorus (SRP) fluxes reaching 6.32 ± 0.11 mg m-2d-1, underscoring its substantial role in annual internal P loading. Furthermore, Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) indicates that lipid- and protein-like organic P compounds in sediments can be readily mineralized, continuously replenishing bioavailable P pools. These findings demonstrate that agricultural activities not only drive external P loading but also promote accumulation of reactivatable P in sediment, sustaining internal P cycling and long-term eutrophication. Effective watershed management requires integrating control of external inputs and sediment legacy P across the landscape.