Study Site ( Figure 1 ):

The Hula Project (HP) territory ( Figure 1 ) is part of the entire Hula Valley which constitutes part of the northern section of the Syrian-African Great Rift Valley. HP is bordered between 70 - 61 MASL altitudes and 33˚06'12'' North and 35˚36'33'' latitude coordinates.

Sampling Stations ( Figure 1 ):

1) Underground (GWT) waters were sampled in the 14 boreholes.

2) The entrance of Canal Z into Lake Agmon-Hula (LAH). Canal Z conveys drained water from the Central block of Peat Soil.

3) The reconstructed Jordan entrance into Lake Agmon-Hula (LAH).

Analysis of Nitrite, Nitrate and Ammonia that were carried out on sampled water. Nitrate analysis was carried out using the Cadmium Reduction (NED) method on a Millipore-filtered sample. Nitrite analysis was carried out by 4500-NO₂-Nitrite Colorimetry method, on Millipore filtered sample [11] . The analysis of ammonia was done using the Indophenol Method, which is based on the formation of a blue color resulting from the reaction between phenol and hypochlorite in the presence of ammonia [11] .

The data was obtained from the Hula Project Monitor Program (1994-2024): Migal-Galilee Scientific Research Institute, Jewish National Fund and Israel Water Authority [12] - [18] .

Statistical evaluation included the following methods: Quadratic and Linear Regressions (w/CI 95%) and scatter plots. Quadratic and linear regressions (w/CI 95%) are used for modeling relationships between variable distribution with a parabolic best-fit curve, as most likely relevant to the analyses presented in this paper.

Principal Component Analysis (PCA) is a dimensionality reduction technique used in exploratory data analysis for a multivariate Gaussian distribution. The vectors shown and the separate Biplot presentation provide a visualization of the data preprocessing results. The data is linearly transformed into a new coordinate system such that the directions (principal components) capturing the largest variation in the data can be identified. PCA is used when variables are correlated with each other and it is desirable to reduce their number to an independent set. The first principal component can equivalently be defined as a direction that maximizes the variance of the projected data. PCA defines a new orthogonal coordinate system that optimally describes variance in a single dataset. The statistical evaluation was carried out using the software STATA 17.0—Standard Edition for Statistics and Data Science. Soil features in the Hula valley are highly variated and divided into three major groups: mineral lake sediments, Peat and transitions in between. Peat soil samples (sampling Station No. 57) were collected from the outlet, which inflows into LAH. These samples are the optimal presentation of the porewater content of the central Peat soil block. The porewaters within the eastern Peat soil block (sampling station No. 73) are optimally presented in the outlet of its drainage canal, which inflows into LAH.

Denitrification is a microbial reductive process where Nitrogen is converted to the final product of molecular N₂. The intermediate stages of this process include gaseous Nitrogen oxides. These microbial stages are carried out by different bacterial species (De-nitrifiers) and their rate of activity is not similar. Oxidation of organic matter (electron donor) is responded to by Nitrogen oxide reduction (electron acceptor) which is denitrifier respiration (source of energy). Therefore, metabolic energetic demands such as oxygen or organic matter substrate are minimal, and the process condition is anaerobic. Eventually, the preference order of Nitrogen oxide stages during the denitrification process is: NO₃ (Nitrate), NO₂ (Nitrite, Nitrogen dioxide), NO (nitric oxide), N₂O (nitrous oxide), N₂ (dinitrogen).

This is a microbiological process comprised of several stages that originated as consumption of Ammonia (NH₃) and terminated as Nitrate (NO₃) production via intermediary nitrite (NO₂). The process of complete nitrification occurs through different bacterial species. The most common bottleneck (limiting factor) of the nitrification chemo-microbial chain of events is the rate of nitrite (NO₂) production. Other limitations are also predicted under substrate (organic matter, NH₃ contents), such as a reduction of availability, which is unpredictable in the Hula Peat soil, and the development of anaerobic conditions. Moreover, the rate of NO₂ is faster than NO₃ production. Therefore, the accumulation of NO₂ might be an indication of Nitrification’s existence. The entire process of nitrification is an obligatory demand of aerobic conditions.

The high content of Ammonia (NH₃) in the Hula Peat soil originated through different processes: 1) Microbial and fungal decomposition of vegetation substances during the pre-drainage period of the Hula wetlands. 2) Denitrification during the post-drainage period. Ammonia production through direct reduction of nitrate to ammonia is also maintained by denitrifier bacteria (dissimilatory nitrate reduction to ammonium) (DNRA), where natural nitrogenic compounds are broken into ammonia. Nevertheless, most of the Ammonia content in the Peat soil originates from the ammonification of organic matter containing organic Nitrogen is likely.

Nitrogen Fixation (NF) is a microbial process in which atmospheric dinitrogen (N₂) is converted into Ammonia (NH₃), catalyzed by the enzyme Nitrogenase. Several terrestrial Nitrogen-fixing bacteria are symbionts of vascular plants that occupy root system tissues. Alfalfa (Medicago sativa) of the legume Fabaceae family is well-known to host species. Alfalfa was intensively cultivated on the Hula Peat soil land but later was drastically reduced, resulting in frequent rodent (Microtus socialis) outbreak infestations. Nevertheless, a relation between Gypsum-sourced sulfate distribution and nitrogenase rate of activity was documented. Information about terrestrial nitrogen fixation is very scarce.

Nutrient migrations from the Hula Valley into Lake Kinneret through the Jordan River are a long-term subject of research. Shortly after the completion of the Hula drainage, the major concern about potential Kinneret water quality deterioration was directed to nitrates. The reason for that was the newly developed oxidative conditions within the drained Peat soil land. Nonetheless, optional opportunities for the increase of ammonium concentration in the peat soil were not highly considered. Lesser scientific attention was attributed towards the creation of anaerobic conditions accompanied by denitrification, and/or ammonification enhancement. Nitrogen fixation did not attract research during earlier stages, but it was enhanced in relation to the expanded cultivation of Alfalfa. Nitrogen processes became an intensive research issue during agricultural development and the implementation of the reclamation project (Hula Project, HP, 2006) [1] [2] . Public awareness after drainage was directed to Nitrate migration whilst during the 1970s and early 1980s, organic nitrogen accompanied by Ammonia, Phosphorus and intestinal microbiota became major awareness factors. As a result, potential sources were managed: raw sewage was removed and fishponds were restricted from 1700 to 350 ha.

The fate of nitrogen within the Hula Valley ecosystem during the post-drainage period is indicated as a key factor in the management design. The development of geochemical transformations of Nitrogen forms is defined as simultaneously multidirectional. Thought-chained environmental conditions determine the potential outcome direction. In water that contains a high proportion of organic nitrogen, the ammonification process will predominate. The process of organic matter oxidation, mostly nitrification, dominated the nitrogen transformations after drainage. The produced nitrate is partly consumed and mostly migrated [5] . The most important inorganic forms of nitrogen in wetlands (or drained wetlands, as Hula Valley) are Ammonia (NH₃), Ammonium (NH₄), Nitrite (NO₂), Nitrate (NO₃), Nitrous Oxide (N₂)) and dissolved or gas elemental Nitrogen (N₂). The Ammonia/Ammonium proportion depends on temperature and pH. Denitrification depletes and nitrification enriches atmospheric nitrogen. Nonetheless, denitrification accompanied the heterotrophic metabolism of bacteria under conditions of anoxia [5] .

The chained sequence of rain capacity, headwater discharges, irrigation and water availability control soil moisture and consequently nitrogen migration. Another sequenced chain direction of aerobic/anaerobic reflecting oxygen availability determines nitrification or denitrification control [4] [5] [7] [8] . Moreover, the impact of the drainage, enhanced significantly the ecosystem’s internal interactions and external consequences as well whilst during pre-drainage, the ecosystem was more stable [6] [9] [10] [20] [21] . The drainage resulted in the loading enhancement of nutrients in the Jordan River. The oxidative degradation of the peat was followed by aeolian and riverine transport of nutrients into Lake Kinneret. During the 1980s, the decline in the aeolian transport of dusty peat to LK was recorded [22] [23] . The fishpond restriction and sewage removal carried out during 1970-1980, in the Hula Valley resulted in the decline of the annual mean of organic-N in the Jordan (Huri, sampling station) from 1.3 to 0.4 ppm [22] [23] .

Nitrogen transformations are operated by bacteria: nitrifiers, denitrifiers, ammonifiers, and nitrogen fixers, which are at different stages by different bacterial species. Protein-contained substances disintegrate through the ammonification process, where ammonium is the final product. Ammonia is oxidized by Nitrosomonas to nitrite, which is converted by Nitrobacter to nitrate. The backward process is operated by denitrifiers with the end product of molecular dinitro (N₂). The maintenance of the terrestrial denitrification process exists under anaerobic conditions by bacteria hosted in the roots of Papilionaceae (Fabaceae) plants. Nitrate (NO₃) is the end product of nitrification whilst Nitrite (NO₂) might be an intermediate stage of both nitrification and denitrification [24] . Moreover, the rate of aquatic and terrestrial denitrification is similarly dependent on anaerobic conditions and nitrate availability. The higher is the nitrate available, the higher the power of the denitrification process. Aquatic nitrogen oxidation is progressed by free oxygen consumption and when it is used up, the oxygen demand for nitrate reduction is consumed and finally when this is out, the executive source of oxygen is reduced sulfate producing sulfide [24] . As of today, such full-chained processes producing very low concentrations of sulfide have rarely been documented in bottom sediments of LAH [17] - [19] [25] .

The hypsometrical distribution of nutrients was defined for three depth levels [26] : the shallowest level of surface water, the intermediate level of the underground water table (GWT) and the deepest level of Lignite waters. Organic Nitrogen, Sulfate and Nitrates are mostly Hula Valley-originated nutrients whilst most of the Phosphorus contributed externally to Lake Kinneret which originates outside the Hula Valley. An underground north-south hydrological gradient and nutrient migration were indicated. Hypsometrical downward migrated nutrients probably accumulate within the “Lignite” level [26] . Information about the underground water dynamics in the Hula Valley was previously documented [27] . A vertical plastic sheet (4.5 m deep) was placed 2.8 km across the valley, dividing the underground space into northern-organic and southern mineral soil blocks. The groundwater table (GWT) in the northern part of the valley is shallower than in the south, and seasonal fluctuation amplitudes are smaller in the north. A higher level of ammonium was documented in the southern underground waters but not for Nitrate (NO₃) concentrations. Nutrient concentrations in the Southern underground waters were higher than those in the North partly because of mediated nutrient water migration. This migration occurred underneath and/or aside from the plastic barrier, driven by the hydraulic gradient as water moved from north to south [27] .

The information about the temporal, seasonal and hypsometrical distribution of Nitrogen compounds in the Hula Valley prevailed from routine long-term (1994-2024) Hula Project monitor program in the sampling stations of Lake Agmon Hula (LAH) outlet (No. 49), drainage canal Z (No. 57), reconstructed Jordan (No. 48), Hula East which conveys drained water from the eastern block of Peat soil No.73), and Ground Water Table (GWT) pumped from 14 boreholes ( Figure 1 ). The evaluation of the role of nitrogenic microbial-chemical process impact is the result of the interpretation of nitrogen species distribution, climate conditions, and the location of the sampling station.

The potential impact of N cycling in the Hula Peat soil and seasonal variations in N isotope composition have been documented in Lake Kinneret [28] . It is a useful methodology providing a biogeochemical tool for studying N cycling in lakes and ecological changes in N source and cycling in response to watershed land use changes and climate change [27] [28] . The interaction between deposited nitrogen and salinization in the tree (Torreya grandis) tissues that were studied indicated that an increase in H₂O₂ in the leaves was induced by the elevation of salinity and moderate nitrogen enhancement can mitigate salt damage [29] . Those terrestrial and aquatic case studies of nitrogen conversion are likely relevant to the analysis of nitrogen dynamics in the Hula Valley.

Before the Hula drainage, the ecosystem maintained rather stable anaerobic conditions within the wetlands-swampy ecosystem [3] . The ecological Instability is induced mostly by precipitations and headwater river discharges and, to a lesser extent, by anthropogenic intervention such as fishery, livestock grazing and moderate commercialized removal of reed (Typha domingensis), including intentioned controlled fire. None of these ecological factors caused fundamental habitat changes. Therefore, under the existing anaerobic conditions, the majority of the ecosystem’s nitrogen load was in the form of ammonia and organic nitrogen. After Hula drainage, the diversity of geochemical conditions was enriched and external (atmospheric and terrestrial) impact became more effective and intensive. This resulted in the occurrence of nitrogenic processes such as nitrification, denitrification, and later also nitrogen fixation through Alfalfa cultivation. The drainage expedited the elimination of factors of ecological stability in the newly created ecosystem, and the impact of climate change intensified. The positive relation between rain capacity and soil moisture and the depth of GWT-induced nitrogen dynamics fluctuations within the various habitats in the valley. Different habitats include Jordan waters, LAH, Pure Peat and other soil types such as transition-Peat and mineral soil [1] [2] [30] [31] . The occurrence of denitrification in wet Pure Peat soil became more frequent whilst the potential for nitrification in transition and mineral soil is higher.

Heterotrophic diazotrophs are potentially important agents in freshwater ecosystems and terrestrial cultivations of special plant species. The contribution of freshwater heterotrophic diazotrophs as free-living or aggregate-associated cells to total N₂ fixation in the Jordan River was recently documented [32] . Heterotrophic diazotrophs accounted for 25% to 56% of the total diazotrophs and were commonly found as free-living cells or attached to aggregates in the Jordan River.

The major load of nitrogen in the Hula Valley is accumulated in the central block of the Peat soil ( Table 1 ). Nevertheless, internal interactions between ecological parameters within the LAH ecosystem are not different from those known in shallow lakes [20] [33] : The oxidation of the accumulated organic matter, as well as nitrification and denitrification, are dependent on dissolved oxygen (DO) content which is diffused from the atmosphere and by submerged vegetation photosynthesis. Nevertheless, a significant impact on the entire ecosystem is due to regional rain capacity dictating headwater discharges, soil moisture and accumulated organics in LAH. Heavier discharge enhances organic matter accumulation in the LAH whilst reducing DO content, which encourages denitrification. Although the supply of Jordan water (Station 48) mediated ammonia exhibits seasonal and temporal changes, these fluctuations are relatively minor ( Figure 2 ). It is therefore likely that the impact of Peat soil on Jordan water nitrogen content is minor. Unlike low fluctuated ammonia in the Jordan waters, its concentration in LAH highly fluctuates: significant summer decline ( Figure 3 ) resulted from intensive oxidation of organic matter due to the enhancement of photosynthetic Oxygen produced by the biomass increase of submerged vegetation. Nevertheless, following the peak in vegetation biomass, plant tissues rapidly degrade, consuming excess oxygen and leading to partial anoxia. This is accompanied by denitrification and a subsequent decline in nitrate levels ( Figure 8 ; Station 57). Most of the ammonia in the Hula Valley accumulates in the Peat soil of the central block, as confirmed by its content in drained water ( Figure 4 ; Station 57). Evaluation of the long-term record of nitrogen migration in the Hula Valley justifies the conclusion about its correlation with soil moisture. During summer months (seasonal) or periods of low rainfall, reduced soil moisture and diminished drain capacity typically occur, followed by a decline in ammonia concentrations in Canal Z ( Figure 4 , Station 57). The impact of two factors is involved in the case of ammonia accumulation in the Peat Soil: The climate effect of rain-river discharges and soil moisture enhancement as an intermediate stage within the nitrification and denitrification process. The similar seasonal fluctuation patterns observed in NO₃ and NH₄ concentrations ( Figure 5 and Figure 8 ) in the drained waters of central and eastern Peat soil blocks further confirm that both factors are interconnected. The nitrate stock that accumulated in the central block of the Peat soil is very high and temporal climate change is therefore confounded ( Figure 8 ) and not significant. The summer decline of nitrate in LAH ( Figure 7 ) is due to both denitrification [8] [9] [20] , and reduction of lake water mass, water body shrinkage, and inflows decline of mediated nutrients input. The summer decline of Nitrate concentration in the eastern Peat soil block drained waters is likely a result of soil moisture decline as well as reduced freshwater seepage and nitrate migration ( Figure 9 ). Evaluation of ammonia and nitrate concentrations in drained waters throughout the entire valley (Stations 48, 49, 67, 73) confirms pattern uniformity of concentration changes ( Figure 10 ). The response of nutrient migration patterns to climate changes confirms similarities. Moreover, the correlation between rain capacity and the depth of GWT confirms the impact of climate change on nitrogen form concentration dynamics in drained waters. During spring-summer-fall months, soil moisture declines and GWT deepens, whereas they rise during the rainy season ( Figure 11 , Figure 12 ).

The record of NO₂ samples analysis covers a shorter period of 1994-2006 ( Figure 13 ). Since NO₂ is an intermediate stage in both nitrification and denitrification processes, relying solely on chemical analysis at a specific sampling point is insufficient. Additional environmental information is necessary to support and contextualize the findings. As previously indicated, nitrate migration within the Peat soil is dependent on soil moisture and therefore nitrite changes reflect nitrification and soil moisture fluctuations.

Conclusively, the sources, migration, and dynamic properties of nitrogen in the Hula Valley are crucial for the present and future hydrological and agricultural management design, especially under global climate changes and regional water scarcity. Beneficial properties include agricultural efficient production accompanied by Kinneret water quality protection. The control of factors of limitation, such as hydrology and nutrient resources, are located in neighboring countries, which are outside of this paper’s scope.