## 4 Discussion
### The Characterization of Nitrogen-Saturated Plots Using Physiological Properties  
Physiological results obtained from the nitrogen-saturated plots within this study showed a reduction in net ammonium (NH4+-N) concentrations, whilst net nitrate (NO3-N) levels increased considerably. This outcome is indicative of the processes governing the nitrogen cycle and subsequent nitrogen saturation [28]. More specifically, once the required nitrogen uptake of the plant and microbial communities within the grassland were exceeded, the majority of excess ammonium (NH4+-N) was converted into nitrate (NO3-N) through the process of nitrification [16]. Thereafter, soils within the saturated plots began to acidify as pH levels decreased due to the abundance of NO3- cations [30]. The negatively charged NO3- cations combined with the high annual rainfall within the site (694 mm) attracted and facilitated the leaching of positively charged calcium and magnesium anions from the saturated plots [13]. This resulted in a highly acidic physiochemical environment with reduced and unbalanced soil nutrient profiles. These results are congruent with the early indicators and associated effects of early nitrogen saturation documented by [PERSON] et al. [16]. Moreover, [PERSON] et al. [28] achieved comparable results in their study of the impact of nitrogen deposition on the interactions between plants and microbes within Chinese grasslands. Their study demonstrated that at high nitrogen deposition, NO3-N concentrations were significantly elevated, whilst both NH4+-N and pH values were considerably lower. In addition, at extreme nitrogen deposition, they documented a decrease in both shoot and root biomass as well as species richness within the grassland [28]. Likewise, [PERSON] et al. [13], in their assessment of the effects of nitrogen fertilization on grassland soil within northern China, obtained similar outcomes. They stressed that fertilizer treatments in excess of 80 kg N ha-1 (which was exceeded within Ukulina) will facilitate the accumulation of mobile NO3- within the soil, with possible NO3- and mineral leaching likely to occur soon after. Lastly, they found that rainfall above 655 mm per annum was significantly correlated to the rate of nitrification within Mongolian grasslands [13]. Finally, [PERSON] et al. [18] found that nitrogen deposition at an average rate of 50 kg N ha-1 can cause a non-linear relationship with soil respiration; however, this varied with grass type in addition to the N gradients applied in the study. Furthermore, the high net foliar nitrogen concentrations obtained for saturated plots, as well as a similar biomass yield between saturated and non-saturated plots demonstrated by this study, indicate that the ecosystem had not yet reached the final stage of nitrogen saturation. Moreover, this outcome is congruent with the indicators and related ecosystem impacts associated with the effects of early onset nitrogen saturation that was documented by [PERSON] et al. [16]. This is further consolidated by the high nitrogen enrichment factor obtained for nitrogen-saturated grassland plots (Figure 4f), which correspond to findings by [PERSON] and [PERSON] [82]. [PERSON] and [PERSON] [82] highlighted that the natural abundance of foliar nitrogen would increase for systems approaching nitrogen saturation. Hence, this study maintains that the saturated grassland plots identified were within the initial stages of nitrogen saturation. From a spectral perspective, however, the increased availability of foliar nitrogen within saturated plots would intensify chlorophyll concentrations [83]; this will in turn facilitate greater visible leaf reflectance within the Red-Edge and Near-Infrared parts of the spectrum and thereby allow for a greater discernment of nitrogen-saturated plots [39; 83]. Nonetheless, we caution that if further nitrogen deposition occurs within this ecosystem, the negative consequences associated with the final stage of nitrogen saturation will likely ensue.