Drivers of the apparent nitrate isotope effect during sedimentary net nitrate reduction: diffusion limitation and nitrate regeneration

Abstract. Isotope effects associated with sedimentary nitrate (NO₃^-) reduction are often strongly suppressed at the ecosystem scale in the overlying water, resulting in low apparent isotope effects (ε_app) that are commonly attributed to physical transport limitation by diffusion. However, the extent to which environmental conditions and experimental manipulations modulate diffusive substrate transport and thereby regulate the expression of sedimentary isotope signals remains unclear. We quantified coupled nitrogen (N) and oxygen (O) isotope effects associated with net NO₃^- reduction in whole-core incubations with sediments from two Swiss lakes. We isolated the potential controls on apparent isotope effects by manipulating sediment reactivity, macrofauna-mediated transport, NO₃^- regeneration via nitrification, and the extent/presence of the oxic diffusive barrier within the sediments. Under near-natural oxic conditions and across treatments with altered organic matter content and enhanced bioturbation, the apparent isotope effects remained strongly suppressed (^15,18ε_app ≤ 3.5 ‰). In Lake Baldegg, inhibition of nitrification with N-allylthiourea increased apparent isotope effects (^15/18ε_app = 6.4/8.9 ‰), demonstrating that NO₃^- regeneration contributes to suppressing isotope effects under oxic conditions within the surface sediment layer. In Lake Sarnen, however, inhibition of nitrification alone had little effect, indicating that transport limitation across the oxic diffusive barrier remained the dominant control. In anoxic experiments, when the oxic diffusive barrier was removed completely, apparent isotope effects increased further, reaching the highest values (8.3–10.5 ‰) in both lakes, reflecting alleviation of diffusion limitation and minimization or prevention of nitrification. Consistent with the stronger expression of denitrification isotope effects under anoxic conditions, dual-isotope relationships most closely reflected the characteristic denitrification signature, with near-unity Δδ¹⁸O-NO₃^-:Δδ¹⁵N-NO₃^- coupling in Lake Baldegg (0.97) and a lower ratio still consistent with denitrification in Lake Sarnen (0.79). Together, these experiments show that apparent NO₃^- isotope effects in sediment-overlying waters are transport-filtered ecosystem-scale signals that are primarily controlled by diffusion limitation across the oxic surface sediment layer and further modulated by NO₃^- regeneration through nitrification.