Sources and migration similarly determine nitrate concentrations: Integrating isotopic, landscape, and biological approaches.

Rapid land use change has significantly increased nitrate (NO) loading to rivers, leading to eutrophication, and posing water security problems. Determining the sources of NO to waters and the underlying influential factors is critical for effectively reducing pollution and better managing water resources. Here, we identified the sources and influencing mechanisms of NO in a mixed land-use watershed by integrating stable isotopes (δN-NO and δO-NO), molecular biology, water chemistry, and landscape metrics measurements. Weak transformation processes of NO were identified in the river, as evinced by water chemistry, isotopes, species compositions, and predicted microbial genes related to nitrogen metabolism. NO concentrations were primarily influenced by exogenous inputs (i.e., from soil nitrogen (NS), nitrogen fertilizer (NF), and manure & sewage (MS)). The proportions of NO sources seasonally varied. In the wet season, the source contributions followed the order of NS (38.6 %) > NF (31.4 %) > atmospheric deposition (ND, 16.2 %) > MS (13.8 %). In the dry season, the contributions were in the order of MS (39.2 %) > NS (29.2 %) > NF (29 %) > ND (2.6 %). Farmland and construction land were the original factors influencing the spatial distribution of NO in the wet and dry seasons, respectively, while slope, basin relief (HD), hypsometric integral (HI), and COHESION, HD were the primary indicators associated with NO transport in the wet and dry seasons, respectively. Additionally, spatial scale differences were observed for the effects of landscape structure on NO concentrations, with the greatest effect at the 1000-m buffer zone scale in the wet season and at the sub-basin scale in the dry season. This study overcomes the limitation of isotopes in identifying nitrate sources by combining multiple approaches and provides new research perspectives for the determination of nitrate sources and migration in other watersheds.