Adaptation of NO₂⁻ Extraction Methods to Different Agricultural Soils: Fine-Tuning Based on Existing Techniques

Abstract

Soil nitrite (NO₂⁻) is an important reactive intermediate in many nitrogen transformation processes, but it is unstable under acidic conditions and may be lost as gaseous N. The canonical extraction method of soil NO₂⁻ using a potassium chloride (KCl) solution greatly underestimates its concentration. In order to reflect the soil NO₂⁻ concentration more accurately, we made minor adjustments on the basis of the existing soil NO₂⁻ extraction method, so that it can be adapted to agricultural soils with different textures and pH values (alkalic fluvo-aquic soil, acidic Mollisol, and Ultisol soils, respectively). Both extractable soil ammonium (NH₄⁺) and nitrate (NO₃⁻) were systematically investigated to refine the simultaneous extraction of soil inorganic nitrogen. The effects of different extractants (deionized water (DIW), un-buffered 2 mol L⁻¹ KCl, and pH-buffered 2 mol L⁻¹ KCl), shaking time (10 and 30 min), and storage duration of the extracts (stored at −20 °C for 1 day, and at 4 °C for 1, 3, and 6 days) on the determination of soil inorganic nitrogen were investigated. The results showed that the un-buffered KCl extractant significantly underestimated soil NO₂⁻ concentration compared to DIW. The highest recovery of NO₂⁻ was obtained by extracting with DIW at 10 min of shaking for all three soils. Compared with DIW, the concentration of NH₄⁺ and NO₃⁻ in soil extracted from the KCl solution increased significantly. Furthermore, the soil inorganic nitrogen content of extracts stored at 4 °C for one day was closer to the direct measurements of fresh samples than with the other storage methods. In conclusion, soil NO₂⁻ extraction was carried out by DIW extraction, shaking for 10 min, and filtering with a 0.45 µm filter, while soil NH₄⁺ and NO₃⁻ were extracted with a KCl solution and shaken for 30 min. The extract should be stored at 4 °C and analyzed within 24 h. We have made minor adjustments to our previously published method for soil NO₂⁻ analysis to make it more adaptable to soils with different textures and pH values. These adjustments make the method more effective in soil geochemical nitrogen cycling studies, especially in the assessment of soil nitrite (HONO) and nitric oxide (NO) emissions.

1. Introduction

Nitrite (NO₂⁻) plays a crucial role in the soil nitrogen cycle as a key intermediate in several nitrogen transformation processes (nitrification, denitrification, nitrifier denitrification, dissimilatory nitrate reduction to ammonium (DNRA), and chemo-denitrification) [1,2,3,4,5,6] and is also closely associated with gaseous nitrogen emissions as an independently existing nitrogen reservoir [7]. For example, NO₂⁻ is directly associated with NO and N₂O gas formation. When NO₂⁻ < 1 mg N kg⁻¹, it still promotes NO production [8]. NO₂⁻ is also an important precursor for N₂O formation, and more than 44% of N₂O changes are associated with NO₂⁻ [9]. Due to its extremely rapid turnover, it is considered an instantaneous product and has often been neglected in previous soil nitrogen (N) cycle studies [10,11,12]. However, NO₂⁻ accumulation occurs when some environmental factors lead to the decoupling of ammonia oxidation from nitrite oxidation [13,14]. For example, the application of urea or ammonium-based fertilizers may cause instantaneous accumulation of NO₂⁻ during nitrification [9,10,15], which is exacerbated at higher soil pH [16]. Soil NO₂⁻ accumulation may cause a number of environmental problems; for example, NO₂⁻ reacts with other substances to form gases such as nitrous acid (HONO), nitric oxide (NO), nitrogen dioxide (NO₂), and nitrous oxide (N₂O). This affects the oxidizing capacity of the atmosphere, air quality, and human health, and it can also act as a strong greenhouse gas [17,18,19]. The importance of NO₂⁻ in water and soil for gaseous N emissions has been increasingly acknowledged. Accurate and rapid determination of soil NO₂⁻ is therefore needed to achieve a deeper understanding of N transformation processes in soil.

Typically, a 2 mol L⁻¹ KCl solution is used to extract NO₂⁻ from soil, which is also a classical method for simultaneously extracting soil ammonium (NH₄⁺) and nitrate (NO₃⁻) [20]. However, KCl causes NO₂⁻ degradation under acidic and neutral conditions, greatly underestimating the NO₂⁻ concentration in soil [16,20]. Studies have shown that only 22–40% of NO₂⁻ is recovered from soils with a pH between 4.8–5.4 [20,21]. Subsequently, Homyak et al. (2015) [20] improved the extraction of NO₂⁻ from soil and identified deionized water (DIW) as the preferred extractant as a new method for extracting soil NO₂⁻.

Although DIW has become the recommended extractant for soil NO₂⁻ analysis, the presence of suspended solids in the DIW extract affects the absorbance measurements of NO₂⁻ analyzed spectrophotometrically, thus requiring filtration through a 0.45 µm filter [20]. To maximize the recovery of soil NO₂⁻, the pH of the extraction solution with calcium hydroxide [Ca(OH)₂] [22], calcium carbonate (CaCO₃) [23], or potassium hydroxide (KOH) [24] was adjusted. Stevens and Laughlin (1995) [24] adjusted the pH of the extraction solution to 8.0 with 2 mol L⁻¹ KOH and a soil/extractant ratio of 1:1, which resulted in a significant increase in NO₂⁻ recovery. However, this method is only applicable to a substantial amount of soil (200 g) [20], and deviation from this parameter may lead to dispersion of silt and clay particles and/or the dissolution of organic matter [25], making it difficult to obtain a clear solution for analysis [20]. This adjustment process is cumbersome and increases the experimental time and effort. Due to the strong buffering effect of acidic soils, it is difficult to keep the pH of the adjusted KCl extract stable, and the pH may still decrease after oscillation, which may lead to the loss of NO₂⁻. In addition, a higher pH tends to cause volatilization losses of NH₃ [26], potentially decreasing NH₄⁺ recovery if the same extraction method is applied.

In recent years, a large number of studies have been conducted on the factors affecting the determination of soil NH₄⁺ and NO₃⁻, including the extraction method [27], the concentration of the extractant [27], the storage method [28], the shaking time [27], etc. However, the effects of these factors on soil NO₂⁻ concentration have not been systematically reported. Therefore, this study aims to verify the effectiveness and accuracy of the latest soil NO₂⁻ detection method in soils with different textures and pH values in order to provide a scientific basis for the efficient extraction of NO₂⁻, NO₃⁻ and NH₄⁺.

2. Materials and Methods

2.1. Soil Sampling

The soils used here (fluvo-aquic soil, Mollisol, and Ultisol) were previously studied by Song et al. (2023) [19] and have been described in detail. Briefly, the fluvo-aquic soil was collected from Quzhou County, in the North China Plain (36°52′ N, 115°10′ E), with a pH of 7.98, soil organic matter (SOM) of 17.59 g kg⁻¹, total nitrogen (TN) of 0.11%, and C/N of 9.23, respectively. The soil clay, silt, and sand fractions were 0.65%, 84.47%, and 11.88%, respectively. The soil net nitrification rate and the net mineralization rate were 16.92 and 19.55 mg kg⁻¹ day⁻¹, respectively. The climate is a temperate monsoon climate, with an average annual temperature and precipitation of 13.2 °C and 494 mm, respectively. The crops planted are a rotation of wheat and maize. The Mollisol was collected from Lishu County in Northeast China (43°37′ N, 124°36′ E). It has a pH of 5.51, SOM of 22.76 g kg⁻¹, TN of 0.13%, and C/N of 10.13, respectively. The soil clay, silt, and sand fractions were 3.85%, 75.62%, and 20.53%, respectively. The soil net nitrification rate and net mineralization rate were 15.36 and 21.45 mg kg⁻¹ day⁻¹, respectively. The climate is a temperate monsoon climate, with an average annual temperature and precipitation of 6.5 °C and 650 mm, respectively. The planted crop is maize. The Ultisol was collected from Jinjing City in Central South China (28°38′ N, 113°19′ E). It has a pH of 5.35, SOM of 18.45 g kg⁻¹, TN of 0.11%, and C/N of 9.72, respectively. The soil clay, silt, and sand fractions were 0.07%, 77.49%, and 22.44%, respectively. The soil net nitrification rate and net mineralization rate were 11.05 and 11.05 mg kg⁻¹ day⁻¹, respectively. The climate is a subtropical monsoon climate, with an average annual temperature and precipitation of 17.5 °C and 1330 mm, respectively. The crops planted are oilseed rape and maize. Five sampling points were randomly selected on each site, and samples were taken at a depth of 20 cm before combining them into one composite sample. After removing roots, rocks, seeds, and other debris, the soil was air-dried. After grinding, the soil was sieved through a 2 mm sieve and then kept at room temperature.

2.2. Experimental Design

We first compared the effect of different extraction methods on inorganic N concentrations (DIW, un-buffered 2 mol L⁻¹ KCl, and pH-buffered 2 mol L⁻¹ KCl) to assess whether increasing the pH in the extraction solution would affect the extractable NO₂⁻. Three groups of treatments were set up: (1) only DIW; (2) un-buffered 2 mol L⁻¹ KCl; (3) pH = 7.5, 6.5, and 6.0 phosphate buffer solutions (PBSs) + 2 mol L⁻¹ KCl (1:4) were added to the fluvo-aquic soil; and pH = 8.4, 8.0, 7.5, and 7.0 phosphate buffer solutions + 2 mol L⁻¹ KCl (1:4) were added to the Mollisol and Ultisol samples, respectively. Different pH values of PBS were prepared by mixing 1/15 mol L⁻¹ KH₂PO₄ and Na₂HPO₄ in different proportions. Air-dried soils (5 g; n = 3; sieved to 2 mm) were extracted in 25 mL DIW, un-buffered 2 mol L⁻¹ KCl, or pH-buffered 2 mol L⁻¹ KCl (the soil-liquid ratio of all extractions was 1:5 (weight/volume, w/v)), followed by shaking at 200 rpm and 25 °C for 30 min, centrifugation at 9000 rpm for 15 min, and filtration, first through 9 cm medium speed filter paper (aperture 15–20 µm) and then through 0.45 µm aperture filter paper. The inorganic nitrogen content of the extracting solution was measured by a SmartChem automatic chemical analyzer 450 (AMS-Alliance, Rome, Italy). The pH of the extracting solution was measured by a pH meter (Mettler Toledo FE28, Schwerzenbach, Switzerland) and mixed at a 1:2.5 soil to water ratio (w/v).

We further investigated the effect of storing the extracts for variable time periods. Briefly, air-dried soils (5 g, n = 3) were extracted in 25 mL un-buffered 2 mol L⁻¹ KCl or DIW and mixed at a 1:5 soil-liquid ratio (w/v), followed by shaking at 200 rpm and 25 °C for 30 min, centrifugation at 9000 rpm for 15 min, and filtration, first with 9 cm medium speed filter paper (aperture 15–20 µm) and then with 0.45 µm aperture filter paper. Aliquots of the extracts were taken for direct determination of soil inorganic nitrogen concentration, while the remainder was stored at −20 °C for 1 day and at 4 °C for 1, 3, and 6 days, respectively, before analysis.

Finally, we studied the effect of shaking duration on soil NO₂⁻ recovery. NO₂⁻ standard solution was added to evaluate the recovery of NO₂⁻ in the extracts. The background value of NO₂⁻ is low in acidic soils, and in order to be able to accurately detect changes in NO₂⁻ content, the amount of NO₂⁻ added in this experiment was 3 mg N kg⁻¹ soil. Soil samples were extracted with DIW and pH-buffered 2 mol L⁻¹ KCl. The extraction method was as described above. The difference was that shaking was 10 min and 30 min, respectively, and the extract was placed at 4 °C for storage and analyzed within 24 h. All the above treatments were set up in triplicates. Soil without the addition of standard solution was used as a control. The recovery of NO₂⁻ was calculated based on the difference measured in soil with and without adding the standard solution:

$$Recovery=\frac{N_b-N_a}{N_o}\times 100\%$$

(1)

where N_b and N_a represent the NO₂⁻ content of soil with added standard solution and control soil, respectively; N_o represents the NO₂⁻ concentration in the standard solution.

2.3. Statistical Analyses

All test data were preliminarily sorted and in-depth calculated by Microsoft Excel 2019. SPSS Statistics 26.0 (IBM, Armonk, NY, USA) was used for a one-way ANOVA or a two-factor ANOVA to determine the differences in extractable soil inorganic nitrogen between treatments (t and LSD test, p < 0.05). When necessary, data were transformed to meet the assumption of normality.

3. Results

3.1. Higher Soil NO₂⁻ Contents with DIW Extraction

The pH values of the un-buffered KCl extracts were 7.68, 4.16, and 4.65 for the fluvo-aquic soil, Mollisol, and Ultisol, respectively. These values were significantly lower than the pH of the DIW extracts (Figure 1). Adding a phosphate buffer solution decreased the pH of the extracts significantly in the fluvo-aquic soil, while it increased the pH in the Mollisol and Ultisol samples. The pH values were 7.14, 6.17, and 5.96 for the pH-buffered KCl extracts in thefluvo-aquic soil, while they were maintained at pH 6.0–7.5 in Mollisol and Ultisol.

The variation of inorganic nitrogen in different treatments is shown in Figure 2. The NO₂⁻ content was significantly higher in the fluvo-aquic soil (1.18–2.06 mg N kg⁻¹) than in Mollisol (0.02–0.03 mg N kg⁻¹) and Ultisol (0.02–0.08 mg N kg⁻¹) (p < 0.05, Figure S1). Extraction with DIW resulted in a higher NO₂⁻ content as compared with the un-buffered KCl solution in all three soils. Extractable NO₂⁻ increased when using a phosphate buffer solution with different pH but was still lower than that in the DIW extractions. However, soil NH₄⁺ concentration was reduced by 25% (fluvo-aquic soil), 35% (Mollisol), and 60% (Ultisol) when extracted by DIW compared with the un-buffered KCl treatment, respectively. The soil NO₃⁻ concentrations were significantly higher in the un-buffered KCl treatment than in the DIW extracts of fluvo-aquic soils, while the opposite was true for Mollisol and Ultisol. It was further found that the NH₄⁺ and NO₃⁻ content of Ultisol was significantly higher than that of the other two soils in the un-buffered KCl treatment (p < 0.05, Figure S1b).

3.2. Storage Increased NO₂⁻ and NH₄⁺ Content

Storage significantly affected the concentration of inorganic nitrogen in the extracts (Figure 3). When calculating the soil NO₂⁻ content, it was closer to the directly measured content of fresh samples when stored at 4 °C than at −20 °C, regardless of extraction with DIW or un-buffered KCl treatment. There was a tendency for DIW extracts to increase with longer storage time at 4 °C (p < 0.05). Compared to storage for 1 d, storage for 6 d increased the soil NO₂⁻ content by 3.4 (fluvo-aquic soil), 1.6 (Mollisol), and 1.8 folds (Ultisol), respectively. The soil NO₃⁻ concentration extracted by un-buffered KCl treatment decreased with the storage time (4 °C), while the opposite was the case for NH₄⁺. The soil NH₄⁺ concentration increased by 51% (fluvo-aquic soil), 59% (Mollisol), and 20% (Ultisol) after storing the extracts for 6 d compared to 1 d. We found no significant difference between storing for 1 d at 4 °C and directly measuring DIW-extracted NO₂⁻ and un-buffered KCl-extracted NH₄⁺ concentrations (p > 0.05). Further studies revealed that the inorganic nitrogen concentration differed significantly among the three soils (p > 0.05, Figure S2).

3.3. Effect of Shaking Time on NO₂⁻ Recovery

Shaking time had a significant effect on NO₂⁻ recovery in acidic soils (Mollisol and Ultisol) but not in the alkaline soil (fluvo-aquic soil) (Figure 4). When shaking for 10 min, the NO₂⁻ recovery rates of the three soils were 85–87% (fluvo-aquic soil), 92–95% (Mollisol), and 91.39–91.72% (Ultisol), respectively, for both extraction methods (DIW and pH-buffered KCl). No significant difference was found among the treatments (p > 0.05). With the increase of shaking time, the recovery of NO₂⁻ in acidic soil decreased, especially in the DIW treatment. The NO₂⁻ recovery in acidic soils was affected by the pH of the extractant. After shaking for 30 min, the NO₂⁻ recovery rates of the pH-buffered treatments were 88% (Mollisol) and 90% (Ultisol), respectively, which were significantly higher than those of the DIW treatments (p < 0.05). The results of a two-way ANOVA showed that there was a significant interaction effect between pH and shaking time on the recovery of NO₂⁻ in the acidic soils, but not in the alkaline soil (p > 0.05). In summary, extraction with DIW and shaking for 10 min could be suitable for both acidic and alkaline soils.

4. Discussion

4.1. Soil NO₂⁻ Concentration

Compared with DIW, extracting with an un-buffered KCl solution significantly reduced the pH of extracts in Mollisol and Ultisol (Figure 1b,c), which may be due to the release of protons by K⁺ adsorption, an increase in ionic strength, and the hydrolysis of extractable aluminum. In acidic environments, NO₂⁻ protonates into HNO₂ (pK_a = 3.3, 25 °C), which subsequently decomposes to gaseous nitrogen products such as HONO, NO, or NO₂, a process that can explain the loss of NO₂⁻ in acidic environments [19,20,29]. These results were confirmed by lower NO₂⁻ concentrations (35–59%) in the un-buffered KCl extract compared to those extracted by DIW (Figure 2a–c). In addition, the chemical fixation of NO₂⁻ by insoluble soil organic matter [21,30], the reaction with metal oxides [31], or the rapid conversion to NO₃⁻ [23,30] may also be responsible for the low NO₂⁻ concentration in the un-buffered KCl extract. To maximize the recovery of soil NO₂⁻, the pH of the extracting solution was adjusted with Ca(OH)₂ [22], CaCO₃ [23], or KOH [24]. Indeed, the pH of the pH-buffered KCl extracts increased significantly (Figure 1b,c), and the NO₂⁻ contents also increased significantly (Figure 2b,c). The extracted NO₂⁻ by the pH 8.4 buffer solution was even comparable to that of DIW in Mollisol and Ultisol. It was found that the recovery of NO₂⁻ was about 69% when the extract’s pH was between 4.8 and 6.0, and it increased to as much as 95% when the pH was adjusted to 7.8 [31]. In conclusion, unbuffered KCl solutions considerably reduced NO₂⁻ concentrations, in agreement with previous studies [16,20]. Although more NO₂⁻ can be extracted with DIW, it produces a brown extract that interferes with colorimetric analysis [20]. Homyak et al. (2015) [20] obtained more accurate measurements of NO₂⁻ concentration by filtering through a 0.45 micron filter [20].

Shaking time was also a key factor for soil NO₂⁻ recovery. NO₂⁻ recovery from acidic soil decreased significantly with an increase in shaking time (Figure 4). Stevens and Laughlin (1995) [24] showed that the NO₂⁻ recovery after 10 min of shaking was 86%, which was three times higher than that after 30 min of shaking. Our study showed that the NO₂⁻ recovery from acidic Mollisol and Ultisol extracted by DIW was greater than 90% at 10 min of shaking. The NO₂⁻ recovery was higher when extracting for 30 min at a higher pH.

During the analysis process, the extracts may not be determined immediately and may be stored for a period of time because of logistical reasons. However, few studies have considered the effect of the storage duration on soil NO₂⁻ concentration. We found that the NO₂⁻ concentration of samples stored at 4 °C was closer to the directly measured values of fresh samples, whereas freezing to −20 °C resulted in severe losses of NO₂⁻ (Figure 3a–c). The reason may be due to NO₂⁻ degradation caused by freezing and rapid NO₂⁻ turnover during thawing [32]. There were also significant differences in NO₂⁻ concentration among different soils (p < 0.05, Figure S2). In addition, the NO₂⁻ concentration gradually increased with the increase in storage time (4 °C), likely due to nitrification in the non-sterile extract [32]. Therefore, it is recommended that NO₂⁻ should be measured as soon as possible after extraction to obtain more accurate value.

4.2. Soil NH₄⁺ and NO₃⁻ Concentrations

Compared with un-buffered KCl, extraction with DIW significantly underestimated the NH₄⁺ concentration in the three soils and the NO₃⁻ concentration in the fluvo-aquic soil (Figure 2d–f). Tu et al. (2021) [27] also found that the measured concentrations of NH₄⁺ and NO₃⁻ in KCl extracts were 1.6–2.6 and 1.1–1.8 folds higher than those in DIW, respectively. Lim et al. (2018) [16] showed that DIW-extracted NO₃⁻ concentrations were only 50–60% of those extracted by 2 mol L⁻¹ KCl. Due to the significant ion exchange capacity of soil [16], soil colloids are usually negatively charged and can adsorb NH₄⁺, while high concentrations of K⁺ can displace NH₄⁺ adsorbed on soil colloids. However, the negatively charged NO₃⁻ ions will not be adsorbed by the soil and will “freely” exist in soil solution.

Although most of the literature suggests that the shaking time for extracting NH₄⁺ and NO₃⁻ should be 60 min [27,33], some studies believe that the concentration of NO₃⁻ does not change significantly after 15–30 min of shaking [34,35]. The NH₄⁺ and NO₃⁻ concentrations were relatively stable at the late stage of shaking (30–60 min), indicating that 30 min of shaking is long enough to quantitatively extract soil NH₄⁺ and NO₃⁻ [35]. For soils with very high mineral nitrogen content, the shaking time may be extended to 60 min. In this study, NH₄⁺ and NO₃⁻ were extracted with a shaking time of 30 min.

The storage conditions of the extracts significantly affected the soil NH₄⁺ and NO₃⁻ contents. We found that the soil NH₄⁺ or NO₃⁻ contents were close to the directly measured values of fresh samples when the un-buffered KCl or DIW extracts were stored at 4 °C. These values were also higher than those at −20 °C (Figure 3d–i). The NH₄⁺ and NO₃⁻ contents in extracts stored at −20 °C were only 50–60% and 60–80% of those at 4 °C, respectively. This may be due to the degradation of NH₄⁺ and NO₃⁻ due to the freezing or thawing process. A gradual increase in NH₄⁺ and decrease in NO₃⁻ in soil extracts stored at 4 °C (Figure 3d–i) was also reported by Li et al. (2012) [33]. Thus, we recommend that the extracted solution be stored at 4 °C and analyzed within 24 h. In addition, we observed significant differences between the NH₄⁺ and NO₃⁻ concentrations in Ultisol in the above treatments and other soils (p < 0.05, Figure S2), a phenomenon which suggests that the nature of the soil has an influence on the results of the study.

5. Conclusions

We have fine-tuned our published soil NO₂⁻ extraction methods to make them applicable to agricultural soils of different textures and pHs. Un-buffered KCl significantly underestimated soil NO₂⁻ concentration compared to DIW. Further studies showed that the shaking time and the storage method significantly affected the NO₂⁻ concentration. The highest NO₂⁻ recoveries for the three soil NO₂⁻ extracted by DIW were obtained at 10 min of shaking. The soil inorganic nitrogen content in extracts stored at 4 °C for one day was closer to the direct measurements of fresh samples than with other storage methods. Overall, the optimal conditions for determination of soil NO₂⁻ involved using DIW as the extractant, shaking for 10 min, and passing the solution through a 0.45 µm filter. Soil NH₄⁺ and NO₃⁻ were extracted using conventional KCl solution. Both extracts should be measured immediately. Otherwise, they should be stored at 4 °C and analyzed within 24 h. Our study achieved the efficient extraction of soil NO₂⁻, NO₃⁻, and NH₄⁺, respectively. Further optimization is needed if we want to achieve their efficient extraction at the same time.