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Endorheic basins located in semiarid or arid regions constitute one of the most vulnerable and exposed environments to NO3- pollution. The Petrola basin (Central Spain) is an outstanding example of one of these endorheic system. Several constraints have been observed that impede correct identification of the denitrification pathway and its degree at the field scale. To better understand the key factors controlling NO3- attenuation, a five-stage column experiment was performed using organic and pyrite rich sediments from the Utrillas Facies (Lower Cretaceous) as a possible electron donor source to promote denitrification. A chemical and multi-isotopic characterization (delta N-15(NO3), delta O-18(NO3), delta C-13(DIC), delta S-34(SO4), delta O-18(SO4)) of the outflow of the column experiment showed that NO3- attenuation was driven by organic matter. The amount of organic C consumed during denitrification was 2% of the total organic C present in the sediment. Both the degree and rate of denitrification were related to flow rate variations. Lower flow rates favored bacterial growth at the beginning of the experiment producing an increase of complete denitrification rate (CDR). When NO3- reduction was greater than 15%, CDR remained constant (around 30 mu mol L-1 d(-1)) under different flow rates. In the last stage, flow rate had no effect on output NO3- concentration because the organic C supply was limited and had become the main kinetic factor in denitrification. Isotopic fractionations (epsilon of N-15-NO3- and O-18-NO3- between reaction products and remaining reactants were calculated using representative column samples. The results for the two attenuation stages observed were -11.6 parts per thousand and -15.7 parts per thousand for epsilon N, and -12.1 parts per thousand and -13.8 parts per thousand for epsilon O. Most of the field samples showed a percentage of attenuation ranging from 0% to 60% of NO3- removal. (c) 2012 Elsevier Ltd. All rights reserved.