Stable isotopes reveal organic nitrogen pollution and cycling from point and non-point sources in a heavily cultivated (agricultural) Mediterranean river basin

The Pinios River Basin (PRB) is the most intensively cultivated area in Greece, which hosts numerous industries and other anthropogenic activities. The analysis of water samples collected monthly for ∼1 ½ years in eight monitoring sites in the PRB revealed nitrate pollution of organic origin extending from upstream to downstream and occurring throughout the year, masking the signal from the application of synthetic fertilizers. Nitrate concentrations reached up to 3.6 mg/l as NO₃^--N, without exceeding the drinking water threshold of ∼11.0 mg/l (as NO₃^--N). However, the water quality status was "poor" or "bad" in ∼50 % of the samples based on a local index, which considers the potential impact of nitrate on aquatic biological communities. The δ¹⁵Ν-ΝΟ₃^- and δ¹⁸O-NO₃^- values ranged from +4.4 ‰ to +20.3 ‰ and from -0.5 ‰ to +14.4 ‰, respectively. The application of a Bayesian model showed that the proportional contribution of organic pollution from industries, animal breeding facilities and manure fertilizers exceeded 70 % in most river sites with an overall uncertainty of ∼0.3 (UI90 index). The δ¹⁸O-NO₃^- and its relationship with δ¹⁸O-H₂O revealed N-cycling and mixing processes, which were difficult to identify apart from the uptake of nutrients by phytoplankton during the growing season and metabolic activities. The strong correlation of δ¹⁵Ν-ΝΟ₃^- values with a Land Use Index (LUI) and a Point Source Index (PSI) highlighted not only the role of non-point nitrate sources but also of point sources of nitrate pollution on water quality degradation, which are usually overlooked. The nitrification of organic wastes is the dominant nitrate source in most rivers in Europe. The systematic monitoring of rivers for nitrate isotopes will help improve the understanding of N-cycling and the impact of these pollutants on ecosystems and better inform policies for protection measures so to achieve good ecological status.