Nitrate accumulation and transformation processes within a karst conduit system: Insights from hydrochemistry, stable isotopes, metagenomics, and tracer tests.

Nitrate (NO3-) pollution in karst groundwater threatens drinking water security. However, limited subsurface accessibility makes tracing its migration and transformation within karst conduits challenging. This study integrated hydrochemistry, multiple isotopes, functional genes, and tracer tests to elucidate NO3- sources and transformation in the Lanfeng watershed, a representative karst conduit system in southwestern China. Nitrification dominated the watershed nitrogen cycle, with soil organic nitrogen (SON) as the primary NO3- source, contributing 69.4% and 74.0% to surface and groundwater, respectively. High dissolved oxygen (DO) and low dissolved organic carbon (DOC) conditions in the conduit favored nitrification. Along the flow path, NO3- concentrations increased from 1.56 mg L-1 (inlet) to 14.23 mg L-1 (outlet), accompanied by increases in the NO3-/Cl- molar ratio (0.14→1.55) and nitrification functional gene abundances (24.74→97.28 CPM), supporting nitrification as an important nitrogen transformation process within the conduit. However, lateral recharge or water mixing cannot be completely excluded as contributors to the observed NO3- enrichment. A conditional apparent NO3- concentration change index, based on spatial concentration differences normalized by tracer-derived peak-time differences, ranged from 0.20 to 0.89 mg L-1 d-1. This study demonstrates that karst conduits are not passive transport pathways, but rather "reactors" with an intrinsic capacity for NO3- production. These findings highlight the need to consider endogenous NO3- production within subsurface flow paths, alongside external anthropogenic nitrogen inputs, in NO3- risk assessment and mitigation, providing broader insights into NO3- pollution management in karst aquifers worldwide.