## 4 Discussion
The outcome of the extensive monitoring campaign of the shallow aquifer, performed at the scale of the entire watershed, showed that no severe contamination by NO3- is present, with the exception of a few hot spots where concentrations peak at 4-6 mg N-L-1 on some transient occasions, in correspondence to sandy permeable soils, belonging to the coastal paleo-dunes and to the Po River paleo-channels. The very low NO3- concentrations support the evidence that no actual downward vertical N migration takes place and that effective microbial processes, faster compared to groundwater fluxes, buffer the excess NO3-, preventing its accumulation in the shallow aquifer except in paleochannels. Nitrogen reactive forms were generally scarce, not only in waters, as here reported, but also in surficial soils, where only NO3- was found in the amount of 10-35 kg-ha-1-year-1, as demonstrated by repeated analytical campaigns [31; 56]. Multiple findingsreported in the present study exclude the relevance of accumulation in groundwater and in soils. About 37% of the N input is not accounted for in the considered N outputs from agricultural soils of the Po di Volcano-Sacca di Goro basin. Thus, the fate of this missing N is driven by soil denitrification (i.e., the average unaccounted term of 72 kg N:ha\({}^{-1}\)-year\({}^{-1}\) in the soil N budget), which is likely the leading process responsible for N excess attenuation. Denitrification occurs when three conditions are simultaneously fulfilled, i.e., hypoxia/anoxia, availability of NO\({}_{3}\)\({}^{-}\), and availability of an electron donor or an energy source for the heterotrophic denitrifying bacteria, mostly labile organic carbon [57; 58]. In temperate regions, agricultural soils remain generally wet from the late autumn to the early spring and they are occasionally completely saturated after heavy rainfall events, in cases where the water table is proximal to the ground surface as in the lowland deltaic area of the Po river system. By affecting redox status, soil water content is considered a key driver for N removal via denitrification and the chance for creating anoxic microsites is higher in soils with low porosity (clayey and loamy soil) and greater water retention than in more permeable coarsely textured soils [17; 59]. Thus, denitrification may be boosted in the soils of the studied basin, characterized by the fine texture, low hydraulic conductivity, flat topography, and shallow water table [22; 34]. This is consistent with the outcomes of previous laboratory experiments demonstrating high NO\({}_{3}\)\({}^{-}\) consumption and correspondent N\({}_{2}\) production in soil mesocosms under saturated conditions [21]. Moreover, pyrite oxidation could contribute to remove excess NO\({}_{3}\)\({}^{-}\) in reducing environments lacking organic carbon substrates [60]. In conclusion, the present study confirms the importance of denitrification, representing more than one-third of the total N inputs, as a crucial N sink in heavily fertilized fine-texture soils, prone to waterlogged conditions as a consequence of rainfall. The outcomes of black-box studies highlighting efficient N retention in agricultural watersheds support the need for spatially and temporally resolved measurements of denitrification to accurately take into account this process in explaining the fate of missing N. More work is required to perform denitrification estimates at spatial and temporal scales relevant to N pollution, suitable to be included in catchment modeling and management, especially in vulnerable areas to NO\({}_{3}\)\({}^{-}\). The following are available online at [[http://www.mdpi.com/2076-3417/10/6/2136/s1](http://www.mdpi.com/2076-3417/10/6/2136/s1)]([http://www.mdpi.com/2076-3417/10/6/2136/s1](http://www.mdpi.com/2076-3417/10/6/2136/s1)), Table S1: Livestock categories, live weights, and N excretion rates; Table S2: N fertilization, yield, and N uptake coefficient of the harvested portions of the main crops cultivated in the Po di Volcano-Sacca di Goro watershed. Conceptualization, G.C. and E.S.