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<p>Large agricultural streams receive excessive inputs of nitrogen. However, quantifying the role of these streams in nitrogen processing remains limited because continuous direct measurements of the interacting and highly time-varying nitrogen processing pathways in larger streams and rivers are very complex. Therefore, we employed a monitoring-driven modelling approach with high-frequency in situ data and the river water quality model Water Quality Analysis Simulation Program (WASP) 7.5.2 in the 27.4 <span class="inline-formula">km</span> reach of the sixth-order agricultural stream called Lower Bode (central Germany) for a 5-year period (2014–2018). Paired high-frequency sensor data (15 <span class="inline-formula">min</span> interval) of discharge, nitrate, dissolved oxygen, and chlorophyll <span class="inline-formula"><i>a</i></span> at upstream and downstream stations were used as model boundaries and for setting model constraints. The WASP model simulated 15 <span class="inline-formula">min</span> intervals of discharge, nitrate, and dissolved oxygen with Nash–Sutcliffe efficiency values higher than 0.9 for calibration and validation, enabling the calculation of gross and net dissolved inorganic nitrogen uptake and pathway rates on a daily, seasonal, and multiannual scale. Results showed daily net uptake rate of dissolved inorganic nitrogen ranged from <span class="inline-formula">−</span>17.4 to 553.9 <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M6" display="inline" overflow="scroll" dspmath="mathml"><mrow class="unit"><mi mathvariant="normal">mg</mi><mspace linebreak="nobreak" width="0.125em"/><mi mathvariant="normal">N</mi><mspace linebreak="nobreak" width="0.125em"/><msup><mi mathvariant="normal">m</mi><mrow><mo>-</mo><mn mathvariant="normal">2</mn></mrow></msup><mspace linebreak="nobreak" width="0.125em"/><msup><mi mathvariant="normal">d</mi><mrow><mo>-</mo><mn mathvariant="normal">1</mn></mrow></msup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="65pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="f34846d5bf23921be3e11f7b532accbe"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="hess-26-5817-2022-ie00001.svg" width="65pt" height="15pt" src="hess-26-5817-2022-ie00001.png"/></svg:svg></span></span>. The highest daily net uptake could reach almost 30 % of the total input loading, which occurred at extreme low flow in summer 2018. The growing season (spring and summer) accounted for 91 % of the average net annual uptake of dissolved inorganic nitrogen in the measured period. In spring, both the <span class="inline-formula">DIN</span> gross and net uptake were dominated by the phytoplankton uptake pathway. In summer, benthic algae assimilation dominated the gross uptake of dissolved inorganic nitrogen. Conversely, the reach became a net source of dissolved inorganic nitrogen with negative daily net uptake values in autumn and winter, mainly because the release from benthic algae surpassed uptake processes. Over the 5 years, average gross and net uptake rates of dissolved inorganic nitrogen were 124.1 and 56.8 <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M8" display="inline" overflow="scroll" dspmath="mathml"><mrow class="unit"><mi mathvariant="normal">mg</mi><mspace linebreak="nobreak" width="0.125em"/><mi mathvariant="normal">N</mi><mspace width="0.125em" linebreak="nobreak"/><msup><mi mathvariant="normal">m</mi><mrow><mo>-</mo><mn mathvariant="normal">2</mn></mrow></msup><mspace width="0.125em" linebreak="nobreak"/><msup><mi mathvariant="normal">d</mi><mrow><mo>-</mo><mn mathvariant="normal">1</mn></mrow></msup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="65pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="6358d316a327c7ab9128e04cc5660cb0"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="hess-26-5817-2022-ie00002.svg" width="65pt" height="15pt" src="hess-26-5817-2022-ie00002.png"/></svg:svg></span></span>, which accounted for only 2.7 % and 1.2 % of the total loadings in the Lower Bode, respectively. The 5-year average gross <span class="inline-formula">DIN</span> uptake decreased from assimilation by benthic algae through assimilation by phytoplankton to denitrification. Our study highlights the value of combining river water quality modelling with high-frequency data to obtain a reliable budget of instream dissolved inorganic nitrogen processing which facilitates our ability to manage nitrogen in aquatic systems. This study provides a methodology that can be applied to any large stream to quantify nitrogen processing pathway dynamics and complete our understanding of nitrogen cycling.</p>