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The Rio Grande, a semi-arid river in the American Southwest, is a major source of surface water for agriculture and municipal purposes in New Mexico and western Texas. In addition to increasing salinity, considerable increases in nitrate (NO3-) concentrations have been observed in the semi-arid portion of the Rio Grande. It is possible that elevated water salinity inhibits denitrification on irrigated fields and, thus fails to mediate the excess nutrient load from anthropogenic activities. Therefore, the two major goals of this study were to i) characterize and quantify major NO3- sources, and ii) assess whether elevated water salinity affects microbial denitrification in the watershed. In fall 2014 and summer 2015, the Rio Grande surface water, irrigation drains, precipitation (urban runoff), and municipal waste effluents were sampled between Elephant Butte, New Mexico and Tornillo, Texas (similar to 260 km distance) for chemical and stable isotope analyses. The highest NO3- concentrations, up to similar to 70-140 mg/L, were observed in waste effluents and agricultural drains irrigated with the reclaimed city water. Conversely, NO3- concentrations in the river and agricultural drains were significantly lower (<1-10 mg/L) in the areas farther away from urban centers. Two major NO3- sources were identified using isotope tracers: fertilizers, with low delta N-15 and high delta O-18 (average + 0.6 and + 18.3 parts per thousand, respectively), and waste water effluents from cities, with high delta N-15 and low delta O-18 (average + 10.5 and -5.1 parts per thousand, respectively). According to nitrogen isotope mass balance constraint, the contribution of waste effluent-derived NO3- was the smallest in upstream locations, between Elephant Butte and Las Cruces, and accounted for up to 0-25% (+/- 10%) compared to the fertilizer-derived NO3-. Further downstream near big urban centers, the effluent contributions increased and accounted for up to 70-100% between Las Cruces and El Paso. The highest effluent-derived NO3- contributions of 90-100% were measured in the agricultural district located below El Paso where the reclaimed city water is commonly used for irrigation. Elevated salinity does not appear to limit microbial denitrification. Locally, the strongest isotopic evidence of microbial denitrification was observed in a couple of water samples showing elevated salinity (EC 2.9-4.2 mS/cm). The results of this study suggest that urban centers are important NO3- contributors into the aquatic system of the semi-arid Rio Grande watershed, and that microbial processes (e.g., denitrification) do not appear to significantly reduce NO3- loads from anthropogenic sources. (C) 2017 Elsevier Ltd. All rights reserved.