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

Soils below 1 m depth store approximately 50% of global terrestrial soil organic carbon (SOC), yet the mechanisms by which long-term nitrate (NO3-) 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 NO3- 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 NO3-, 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.