Evaluation of nitrate sources and the percent contribution of bacterial denitrification in hyporheic zone using isotope fractionation technique and multi-linear regression analysis.

Denitrification has documented as a promising pathway to permanently remove nitrate from a system. Numerous studies have used the isotope fractionation technique (IFT) to evaluate the denitrification rate in the constructed wetlands (CWs), but the potential of IFT method to quantify the denitrification rate in hyporheic zone (saturated sediments beneath a stream) is still challenging. Thus, more studies are required to investigate that if measurements of the natural abundance of δN-NO and δO-NO (IFT) can be employed to calculate the fate of nitrate in hyporheic zone. Therefore, in this study, the possibility of the IFT to quantify the hyporheic-denitrification rate was investigated. Then, the results were verified by the combined application of the pre-established net Sediment N flux and multi-linear regression analysis (p < 0.01). Finally, the groundwater bacterial groups (Fecal coliform (FC) and Escherichia coli (EC)), and the mass balance isotope mixing model were used to investigate the dominant sources of hyporheic-nitrate. The IFT reveals that denitrification contributes 74.1% and 29.1% of the hyporheic-nitrate removal during dry and wet seasons, respectively. The multi-linear regression analysis, considering at 99% confidence interval (R = 92.1%; n = 44; p < 0.01), slightly overestimates the rate and the percent contribution of denitrification in the dry season (475.15 ± 101.18 μmol/m2d; 80.7%) and underestimates it during the wet season (205.072 ± 35.39 μmol/m2d; 24.01%). The analysis of EC and FC demonstrates that manure (41.9 ± 4.2%) and sewage (54.1 ± 8.9%) are the dominant contributors of the hyporheic-nitrate load. In addition, the results achieved by the analysis of the fecal bacterial indicators (EC and FC) were confirmed by NO/Cl vs Cl diagram. This study provides an alternative-initiative framework to accurately quantify the spatio-seasonal variations in the hyporheic-nitrate sources and hyporheic-denitrification rate that enables decision-makers to apply appropriate and targeted strategies to regulate nitrate load in river-aquifer systems.