*3.5. Identification of Nitrogen Contamination*

scientifically identified.

*3.5. Identification of Nitrogen Contamination*  Based on the evaluated groundwater quality results, the NO3(N) concentrations in some parts of the study area were overstandard, and the highest concentration far exceeded the level III standard. Thus, the main source of nitrate contaminations should be Based on the evaluated groundwater quality results, the NO3(N) concentrations in some parts of the study area were overstandard, and the highest concentration far exceeded the level III standard. Thus, the main source of nitrate contaminations should be scientifically identified.

As shown in Figure 10a, the Cl–/Na+ ratios of most samples were close to 1, indicating

Contaminations coming from manure and sewage would show a higher Cl– content and a lower NO3−/Cl− ratio. The Cl− molar concentration should usually be greater than 1 mmol/L, while the NO3−/Cl– molar concentration ratio should range between 0.001 and 0.1 [44,67]. The contaminations originating from synthetic NO3 fertilizer via agricultural

ratios ranged from 0.00 to 3.69 with an average of 0.49, and the *Cv* was 1.34 (>1), indicating that there was a strong spatial variation. Combined with Figure 10b, the ratios of SO42– /Na+ were smaller than those of NO3−/Na+ overall, and the groundwater samples were located near the agricultural side, meaning that the nitrate content of groundwater was mainly affected by agricultural activities. The distribution of the points in Figure 10a was scattered and the *Cv* of NO3−/Na+ was higher than 1, meaning that agricultural activities in different zones had different impacts on groundwater. The discussed results were consistent with the field survey results. The farmland in the study area was widely distributed, and the main crops were potatoes, oats and vegetables. Fertilizers and pesticides were very likely to enter groundwater along with the water from irrigation and precipi-

tation, resulting in excessive nitrogen in groundwater.

3.5.1. Relationship between NO3– and Other Ions (Cl–, SO42−, Na+ and K+)

3.5.1. Relationship between NO<sup>3</sup> − and Other Ions (Cl−, SO<sup>4</sup> <sup>2</sup>−, Na<sup>+</sup> and K<sup>+</sup> ) *Water* **2022**, *14*, 3168 20 of 26

> As shown in Figure 10a, the Cl−/Na<sup>+</sup> ratios of most samples were close to 1, indicating that the Cl−/Na<sup>+</sup> ratios were mainly affected by salt rock dissolution [16]. The NO<sup>3</sup> <sup>−</sup>/Na<sup>+</sup> ratios ranged from 0.00 to 3.69 with an average of 0.49, and the *C<sup>v</sup>* was 1.34 (>1), indicating that there was a strong spatial variation. Combined with Figure 10b, the ratios of SO<sup>4</sup> <sup>2</sup>−/Na<sup>+</sup> were smaller than those of NO<sup>3</sup> <sup>−</sup>/Na<sup>+</sup> overall, and the groundwater samples were located near the agricultural side, meaning that the nitrate content of groundwater was mainly affected by agricultural activities. The distribution of the points in Figure 10a was scattered and the *C<sup>v</sup>* of NO<sup>3</sup> <sup>−</sup>/Na<sup>+</sup> was higher than 1, meaning that agricultural activities in different zones had different impacts on groundwater. The discussed results were consistent with the field survey results. The farmland in the study area was widely distributed, and the main crops were potatoes, oats and vegetables. Fertilizers and pesticides were very likely to enter groundwater along with the water from irrigation and precipitation, resulting in excessive nitrogen in groundwater. activities should show characteristics of low Cl− concentrations and high NO3−/Cl− ratios, the Cl− content should be less than 0.1 mmol/L, and the NO3–/Cl– molar concentration ratio should range between 0.1 and 10 [18]. As shown in Figure 10c, the Cl– contents were generally more than 1 mmol/L, and the molar concentration ratios of NO3–/Cl– ranged between 0.1 and 10, with an average value of 0.58. The points were mostly distributed in the upper–right of Figure 10c, indicating that the nitrate content in groundwater was affected by a variety of factors such as manure, sewage and fertilizers. To further explore the probability of synthetic NO3 fertilizer as a probable source of nitrate, the bivariate relationship between NO3− and K+ was examined. There will be a strong correlation between NO3− and K+ if NO3 originates from synthetic NO3 fertilizer [7]. In fact, there was a weak correlation between NO3− and K+ (Figure 10d), indicating that synthetic NO3 fertilizer is not the dominant source of NO3– in the study area.

**Figure 10.** Relationships of (NO3<sup>−</sup>/Na+) vs. (Cl−/Na+) (**a**), (SO42<sup>−</sup>/Na+) vs. (NO3<sup>−</sup>/Na+) (**b**), (NO3<sup>−</sup>/Cl<sup>−</sup>) vs. Cl<sup>−</sup> (**c**), and NO3<sup>−</sup>/K+ (**d**). **Figure 10.** Relationships of (NO<sup>3</sup> <sup>−</sup>/Na<sup>+</sup> ) vs. (Cl−/Na<sup>+</sup> ) (**a**), (SO<sup>4</sup> <sup>2</sup>−/Na<sup>+</sup> ) vs. (NO<sup>3</sup> <sup>−</sup>/Na<sup>+</sup> ) (**b**), (NO<sup>3</sup> −/Cl−) vs. Cl− (**c**), and NO<sup>3</sup> <sup>−</sup>/K<sup>+</sup> (**d**).

3.5.2. Denitrification and Nitrification As shown in Figure 11a, the relationship between NO3– and δ15N(NO3) was not negative, indicating that denitrification in groundwater was not obvious. Previous studies [45,46] have shown that the enrichment coefficient (εN/εO) should range between 1.3 –2.1 if intensive denitrification occurs in groundwater, in other words, the slope of δ15N(NO3) and δ18O(NO3) should range from 0.48 to 0.77. As shown in Figure 11c, the slope of δ15N(NO3) and δ18O(NO3) was −0.09, far lower than 0.48 and 0.77, respectively. It was indicated that no obvious denitrification occurred during the transforming process of nitrate in the study area. Under an oxidative environment, NH4+ is transformed into NO3– by Nitrobacter, which is called nitrification. According to the experimental research of Kendall [68], one O atom of NO3 comes from the atmosphere, and the other two O atoms come from H2O in environment, i.e., δ18O(NO3) = 2/3δ18O(H2O) + 1/3δ18O(O2). The δ18O(H2O) average of Contaminations coming from manure and sewage would show a higher Cl− content and a lower NO<sup>3</sup> −/Cl− ratio. The Cl− molar concentration should usually be greater than 1 mmol/L, while the NO<sup>3</sup> −/Cl− molar concentration ratio should range between 0.001 and 0.1 [44,67]. The contaminations originating from synthetic NO<sup>3</sup> fertilizer via agricultural activities should show characteristics of low Cl− concentrations and high NO<sup>3</sup> −/Cl− ratios, the Cl− content should be less than 0.1 mmol/L, and the NO<sup>3</sup> −/Cl− molar concentration ratio should range between 0.1 and 10 [18]. As shown in Figure 10c, the Cl− contents were generally more than 1 mmol/L, and the molar concentration ratios of NO<sup>3</sup> −/Cl− ranged between 0.1 and 10, with an average value of 0.58. The points were mostly distributed in the upper–right of Figure 10c, indicating that the nitrate content in groundwater was affected by a variety of factors such as manure, sewage and fertilizers. To further explore the probability of synthetic NO<sup>3</sup> fertilizer as a probable source of nitrate, the bivariate relationship between NO<sup>3</sup> <sup>−</sup> and K<sup>+</sup> was examined. There will be a strong correlation

groundwater in the study area was −9.07‰, and the δ18O(O2) value was +23.5‰[69]. Based

between NO<sup>3</sup> <sup>−</sup> and K<sup>+</sup> if NO<sup>3</sup> originates from synthetic NO<sup>3</sup> fertilizer [7]. In fact, there was a weak correlation between NO<sup>3</sup> <sup>−</sup> and K<sup>+</sup> (Figure 10d), indicating that synthetic NO<sup>3</sup> fertilizer is not the dominant source of NO<sup>3</sup> − in the study area. the δ15N(NO3) values among multiple sources. To overcome this problem, the 15N(NO3) –18O(NO3) dual isotopes technique has been proposed [8] (Figure 11c). The δ15N(NO3) values ranged from +0.29 to +14.39‰, and the δ18O(NO3) values ranged from −6.47 to +1.24‰. 50% of the groundwater samples were in

As shown in Figure 11a and Figure 11b, the distribution of samples was an irregular plane, indicating that NO3 contamination was a mixture of point sources and nonpoint sources. The δ15N(NO3) values were within the overlap range of multiple sources (Figure 11a), such as precipitation, soil organic nitrogen, manure and sewage, while the δ18O(NO3) values were in the range of soil organic nitrogen (Figure 11b). The results based on single isotope to identify the source of nitrogen were not accurate enough due to the overlaps of

δ18O(NO3) in the study area ranged from −6.47 to +1.24‰, and the average was −2.40‰. There was a difference between the actual values and theoretical value, the main possible reasons were: (1) the real conditions were different from the laboratory cultures; (2) the δ18O(O2) value was cited from previous study not from actual measurement. According to the previous research, the δ18O(NO3) value produced by microbial nitrification ranged from −10 to +10‰ in general [47,68,70]. As shown in Figure 11b, the δ18O(NO3) values all fell within the above range and indicated that nitrification was the main process of nitro-

### 3.5.2. Denitrification and Nitrification the range of manure and sewage, 41.67% of the groundwater samples were in the overlap

gen transformation.

*Water* **2022**, *14*, 3168 21 of 26

3.5.3. δ15. N(NO3) – δ18O(NO3) Dual Isotope Technique

As shown in Figure 11a, the relationship between NO<sup>3</sup> − and δ <sup>15</sup>N(NO3) was not negative, indicating that denitrification in groundwater was not obvious. Previous studies [45,46] have shown that the enrichment coefficient (εN/εO) should range between 1.3–2.1 if intensive denitrification occurs in groundwater, in other words, the slope of δ <sup>15</sup>N(NO3) and δ <sup>18</sup>O(NO3) should range from 0.48 to 0.77. As shown in Figure 11c, the slope of δ <sup>15</sup>N(NO3) and δ <sup>18</sup>O(NO3) was <sup>−</sup>0.09, far lower than 0.48 and 0.77, respectively. It was indicated that no obvious denitrification occurred during the transforming process of nitrate in the study area. area of soil organic nitrogen and manure and sewage, and 8.33% of the samples were located below the range of manure and sewage. High concentration of nitrate in groundwater indicated that soil organic nitrogen was not the main source of nitrate contamination. The mixed contamination of manure, sewage and NH4 fertilizers increased the nitrate contents in groundwater, and the contribution of manure and sewage was greater than that of NH4 fertilizers. The δ15N(NO3) and δ18O(NO3) data further affirmed the hydrochemical interpretation that precipitation, industrial activities and synthetic NO3 were unlikely to be the main sources of NO3 in the study area.

**Figure 11.** Relationships of NO<sup>3</sup> − vs. δ 15N(NO<sup>3</sup> ) (**a**), NO<sup>3</sup> − vs. δ <sup>18</sup>O(NO<sup>3</sup> ) (**b**) and δ 15N(NO<sup>3</sup> ) vs. δ <sup>18</sup>O(NO<sup>3</sup> ) (**c**).

Under an oxidative environment, NH<sup>4</sup> + is transformed into NO<sup>3</sup> − by Nitrobacter, which is called nitrification. According to the experimental research of Kendall [68], one O atom of NO<sup>3</sup> comes from the atmosphere, and the other two O atoms come from H2O in environment, i.e., δ <sup>18</sup>O(NO3) = 2/3δ <sup>18</sup>O(H2O) + 1/3δ <sup>18</sup>O(O2). The δ <sup>18</sup>O(H2O) average of groundwater in the study area was −9.07‰, and the δ <sup>18</sup>O(O2) value was +23.5‰ [69]. Based on this, the δ <sup>18</sup>O(NO3) theoretical value was calculated as 1.79‰. The actual values of δ <sup>18</sup>O(NO3) in the study area ranged from <sup>−</sup>6.47 to +1.24‰, and the average was <sup>−</sup>2.40‰. There was a difference between the actual values and theoretical value, the main possible reasons were: (1) the real conditions were different from the laboratory cultures; (2) the δ <sup>18</sup>O(O2) value was cited from previous study not from actual measurement. According to the previous research, the δ <sup>18</sup>O(NO3) value produced by microbial nitrification ranged from −10 to +10‰ in general [47,68,70]. As shown in Figure 11b, the δ <sup>18</sup>O(NO3) values all fell within the above range and indicated that nitrification was the main process of nitrogen transformation.

### 3.5.3. δ15. N(NO3) – δ <sup>18</sup>O(NO3) Dual Isotope Technique

As shown in Figure 11a,b, the distribution of samples was an irregular plane, indicating that NO<sup>3</sup> contamination was a mixture of point sources and nonpoint sources. The δ <sup>15</sup>N(NO3) values were within the overlap range of multiple sources (Figure 11a), such as precipitation, soil organic nitrogen, manure and sewage, while the δ <sup>18</sup>O(NO3) values were in the range of soil organic nitrogen (Figure 11b). The results based on single isotope to identify the source of nitrogen were not accurate enough due to the overlaps of the δ <sup>15</sup>N(NO3) values among multiple sources.

To overcome this problem, the <sup>15</sup>N(NO3) –18O(NO3) dual isotopes technique has been proposed [8] (Figure 11c). The δ <sup>15</sup>N(NO3) values ranged from +0.29 to +14.39‰, and the δ <sup>18</sup>O(NO3) values ranged from <sup>−</sup>6.47 to +1.24‰. 50% of the groundwater samples were in the range of manure and sewage, 41.67% of the groundwater samples were in the overlap area of soil organic nitrogen and manure and sewage, and 8.33% of the samples were located below the range of manure and sewage. High concentration of nitrate in groundwater indicated that soil organic nitrogen was not the main source of nitrate contamination. The mixed contamination of manure, sewage and NH<sup>4</sup> fertilizers increased the nitrate contents in groundwater, and the contribution of manure and sewage was greater than that of NH<sup>4</sup> fertilizers. The δ <sup>15</sup>N(NO3) and δ <sup>18</sup>O(NO3) data further affirmed the hydrochemical interpretation that precipitation, industrial activities and synthetic NO<sup>3</sup> were unlikely to be the main sources of NO<sup>3</sup> in the study area.
