## 2 Materials and Methods  
### Study Area  
The Po River catchment, the largest hydrographic system in Italy, covers about one-quarter of the national territory, and it is a strategic area for the national economy, accounting for \(\sim\)40% of the Italian gross domestic product. The Po basin is one of the most densely populated and agriculturally exploited areas in Europe; thus, eutrophication and NO\({}_{3}\)\({}^{-}\) pollution of surface and groundwater are the most critical consequences in terms of water quality deterioration [24; 25; 26]. The Po di Volcano-Sacca di Goro watershed (925 km\({}^{2}\), Figure 1) is located in the terminal part of the Po basin, and it is a recently reclaimed deltaic territory included within the administrative borders of the Ferrara Province (Emilia-Romagna Region, Northeastern Italy). The basin covers an area extending from the city of Ferrara to the Adriatic Sea, and it is limited to the north by the Po River main course and to the south by the Po di Volcano, an ancient branch of the Po River Delta, which is now an artificial canal that carries water to the Sacca di Goro Lagoon [27; 28]. The climate is warm temperate (type Cfa, according to Koppen-Geiger classification) [29], with a mean wet deposition of \(\sim\)600 mm-year\({}^{-1}\) (late spring and autumn peaks), elevated humidity, low wind speed, minimum monthly mean air temperature of \(\sim\)1 \({}^{\circ}\)C in January, and maximum monthly mean air temperature of \(\sim\)25 \({}^{\circ}\)C in July [30]. From the late 1960s when the land reclamation was completed, the large availability of water for irrigation purposes and the completely flat topography of this territory favored the establishment of farming activity becoming increasingly mechanized, scarcely amended with livestock manure and almost exclusively subsidized with synthetic fertilizers, mostly in the form of urea and ammonium nitrate [31]. From the mid-1980s, the Sacca di Goro, a shallow eutrophic lagoon where the main economic activity is clam farming, suffered from summer anoxic crises and dystrophic outbreaks as a consequence of macroalgal blooms [30]. Under the European Nitrates Directive (91/676/EEC) and the European Water Framework Directive (2000/60/EC), the whole Po di Volcano-Sacca di Goro watershed was designated as a nitrate-vulnerable zone from agricultural sources. Restrictions with regard to the timing and quantity of fertilizer application were put into practice in 2006 by the Water Protection Plan of Emilia-Romagna Region. Agriculture is the dominant land use with \(\sim\)80% of the watershed area classified as utilized agricultural land (UAA). Wheat is the main crop cultivated in the Po di Volcano-Sacca di Goro watershed (29% of UAA), followed by maize (23%), industrial and horticultural crops (22%), rice (9%), fruit trees (8%), and feed crops (7%) (National Statistics Institution, Sixth Agricultural Census 2010; [[http://dati-censimentoagricoltura.istat.it](http://dati-censimentoagricoltura.istat.it)]([http://dati-censimentoagricoltura.istat.it](http://dati-censimentoagricoltura.istat.it))). Artificial surfaces cover \(<\)10% of the watershed area (Emilia-Romagna land-use/land-cover database, 2008), and the urban planning scheme is spread with a relatively low population density concentrated in the city of Ferrara and other small towns. Since this basin lies in a depressed area partially below the sea level, the system is automatically controlled by pumping stations and gates managed by the land reclamation authority, and the hydrological regime is artificially regulated by a dense network of canals serving for both drainage and irrigation. Irrigation water (40-50 \(\times\) 10\({}^{6}\) m\({}^{3}\)-month\({}^{-1}\) from April to September) is derived from the Po River through a series of mechanically controlled gates, and it feeds the surface water system formed by some west-east-oriented main canals and several smaller secondary canals, with an overall linear extension of 1780 km.