Hydroxyl radical bursts triggered by long-term nitrate accumulation accelerate deep soil organic carbon...

Soils below 1 m depth store approximately 50% of global terrestrial soil organic carbon (SOC), yet the mechanisms by which long-term nitrate (NO_3^-) accumulation influences deep SOC mineralization remain poorly understood. Here, we combined depth-resolved field measurements from a 26-year fertilization experiment with short-term microcosm incubations to examine whether long-term NO_3^- accumulation promotes hydroxyl radical (•OH) production and deep SOC mineralization. The study was conducted in a calcareous fluvo-aquic soil under a winter wheat-summer maize rotation in the North China Plain, China, with a semi-arid to semi-humid temperate monsoon climate. We compared an unfertilized control with an excessive nitrogen input treatment (N600, 600 kg N ha^-1 yr^-1 applied as urea) across a 0-10 m profile. We quantified NO_3^-, redox-related, biochemical, and carbon turnover indicators, including nicotinamide adenine dinucleotide (NADH), hydrogen peroxide, ferrous iron, •OH, phenolic compounds, hydrolytic enzyme activities, and CO₂ emissions, along the soil profile. Compared with the unfertilized control, long-term excessive N fertilization significantly increased NO₃^- concentrations, raised NADH concentrations by 5-25% and enhanced •OH production by 8-16% in deep soils at 4-10 m depth, increased β-glucosidase, β-xylosidase, and chitinase activities by 2-10 fold, 1.5-5 fold, and 2-8 fold, respectively, and stimulated CO₂ emissions by 30-60%. NADH was positively correlated with •OH, whereas •OH was negatively correlated with phenolic compounds and positively correlated with hydrolytic enzyme activity. In short-term incubations, quenching •OH with tertiary butanol reduced cumulative CO₂ emissions by 5-25% and increased phenolic content by 40-65%. Conversely, exogenous NO₃^- addition to control soils recapitulated field-observed •OH production and cumulative CO₂ release. Together, these field patterns and incubation responses support a mechanism whereby long-term NO₃^- accumulation enhances •OH production, weakens phenolic constraints on enzyme activity, and accelerates deep SOC mineralization. These findings highlight a previously overlooked mechanism by which chronic NO₃^- leaching may increase the vulnerability of deep carbon in agricultural soils.