In groundwater-dependent ecosystems (GDEs) a significant contribution of nitrogen contamination of the surface waters arises from groundwater input by shallow and deep flow systems. In general, nitrogen surface-groundwater exchanges and processes at the hyporheic scale are difficult to monitor. In order to have a better understanding about these interactions, it is proposed to use stable isotopes (15N-NO3, 18O-NO3, 15N-NH4+) as environmental tracers to identify the sources and fate of the nitrogen compounds in groundwater in the Sagittario River basin, central Italy. The detrimental effect of the groundwater ammonium on the meiofaunal organisms is also evaluated in the hyporheic zone, where N-NH4+ concentrations reach a maximum value of 0.6 mg L−1. The main source of the nitrogen contamination originates from agricultural and urban activities at the catchment scale. The nitrogen compounds reach the gaining stretches of the hyporheic zone along the streambed as nitrate associated to a shallow flow system and ammonium related to a deep flow system. δ15N data demonstrate that sewage and fertilizers including manure are the main sources of NO3 and NH4+, the latter being the major pollutant in the study area. δ18O-NO3 data are consistent with the nitrification of ammonium to nitrate, along with the contribution from inorganic fertilizers. Furthermore, δ15N-NO3 and δ18O-NO3 data reveal that denitrification does not play any role on nitrate attenuation in the shallow aquifer and near the discharge areas to the river. The meiofaunal communities, represented by the Crustacea Copepoda selected as the target group, were shown to be sensitive to the high concentration of ammonium found at several hyporheic sites. The statistical analyses indicated a significant reduction of species abundance in the sites polluted by N-NH4+ concentrations > 0.0653 mg L−1.

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