Prerpints.org logo
Preprint
Article

Improved Extraction Method of Soil Nitrite

Altmetrics

Downloads

97

Views

39

Comments

0

A peer-reviewed article of this preprint also exists.

Submitted:

27 December 2023

Posted:

27 December 2023

You are already at the latest version

Alerts
Abstract
Soil nitrite (NO2‒) is an important reactive intermediate in many nitrogen transformation processes, but it is unstable under acidic conditions. Canonical extraction method of soil NO2‒ with potassium chloride (KCl) solution greatly underestimates its concentration. In order to reflect the concentration more accurately, we optimized the extraction method of soil NO2‒ in this study. Moreover, soil ammonium (NH4+) and nitrate (NO3‒) were also systematically investigated to achieve efficient extraction of soil inorganic nitrogen. The results showed that un-buffered KCl significantly underestimated soil NO2‒ concentration compared to DIW. The highest recovery of NO2‒ was obtained by extracting with DIW at 10 min of oscillation for three soils. Compared with DIW, the concentration of NH4+ and NO3‒ in soil extracted from KCl solution increased significantly. Furthermore, the soil inorganic nitrogen content of extracts stored at 4°C for one day were closer to directly measurements of fresh samples than the other storage methods. Overall, the recommend analysis method for soil NO2‒ was extracted by DIW, oscillated for 10 min, and filtered by a 0.45 µm filter, and soil NH4+ and NO3‒ were extracted with KCl solution and oscillated for 30 min. The extract should be stored at 4°C and analyzed within 24 hours.
Keywords: 
Subject: Environmental and Earth Sciences  -   Soil Science

1. Introduction

Nitrite (NO2‒) is 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] and is also closely associated with gaseous nitrogen emissions as an independently existing nitrogen reservoir [6]. Due to its extremely rapid metabolism, it is considered as an instantaneous product and is often neglected in previous soil nitrogen (N) cycle studies [7,8,9]. However, NO2‒ accumulation occurs when some environmental factors lead to the decoupling of ammonia oxidation from nitrite oxidation [10,11]. For example, the application of urea or ammonium-based fertilizer may cause instantaneous accumulation of NO2‒ during nitrification [8,12,13], and higher pH values may exacerbate this process [14]. 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), affecting the oxidizing capacity of the atmosphere, air quality and human health [15,16,17]. Nowadays, the source and importance of NO2‒ in water and soil are getting more and more attention. Accurate and rapid determination of soil NO2‒ will contribute to a deeper understanding of the transformation process of nitrogen in soil.
Typically, 2 mol L‒1 KCl solution is used to extract NO2‒ from soil, which is also a classical method for extracting soil ammonium (NH4+) and nitrate (NO3‒) [18]. During the extraction of NO2‒, there are generally no biological or chemical reactions that cause changes in its concentration or isotopic composition [19]. However, KCl causes NO2‒ degradation under acidic and neutral conditions, greatly underestimating the NO2‒ concentration in soil [14,18]. Studies have shown that only 22‒40% of NO2‒ was recovered from soils with pH between 4.8‒5.4 [18,20]. Nevertheless, extraction of soil NO2‒ with 2 mol L‒1 KCl solution is still a recommended method to be used in soil analysis manuals and international standards [21,22,23].
New method has been proposed to improve the low extraction efficiency of NO2‒. The recovery of NO2‒ in acidic soil was higher when it was extracted by deionized water (DIW) with a small amount (4 g) of soil, but the suspended solids in the DIW extracts may affect the absorbance measurements during NO2‒ analysis [18]. To maximize the recovery of soil NO2‒, the pH of extracting solution with calcium hydroxide [Ca(OH)2] [24], calcium carbonate (CaCO3) [25], or potassium hydroxide (KOH) [19] was adjusted. For example, Stevens and Laughlin (1995) [19] adjusted the pH of the extracting 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 large number of soil samples (200 g), and deviation from this parameter may lead to dispersion of silt and clay particles and/or dissolution of organic matter [26], making it difficult to obtain a clear solution for analysis [18]. This adjustment process is cumbersome and increases the experimental time. In addition, higher pH tends to cause volatilization losses of NH3 [27], which potentially decreases NH4+ recovery if applied the same extraction method.
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 [28], the concentration of the extractant [28], the storage method [29], and the oscillation time [28], etc. However, the effects of these factors on soil NO2‒ concentration have not been systematically reported. Therefore, this study aims to improve the effective extraction and the accuracy of soil NO2‒ determination, and to provide a basis for realizing 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) [17] and have been described in detail. Briefly, Fluvo-aquic soil was collected from Quzhou County, North China Plain (36°52′N, 115°10′E), with pH of 7.98, TN and C/N of 0.11% and 9.23 respectively. The climate is a temperate monsoon climate, with an average annual temperature and precipitation of 13.2℃ and 494 mm, respectively. The crops planted are wheat and maize rotation. Mollisol was collected from Lishu County in Northeast China (43°37′N, 124°36′E), with pH of 5.51, TN and C/N of 0.13% and 10.13, respectively. The climate is a temperate monsoon climate, with an average annual temperature and precipitation of 6.5℃ and 650 mm, respectively. The planting crop is maize. Ultisol was collected from Jinjing City in Central South China (28°38′N, 113°19′E), with pH of 5.35, TN and C/N of 0.11% and 9.72, respectively. The climate is a subtropical monsoon climate, with an average annual temperature and precipitation of 17.5℃ and 1330 mm, respectively. The crops planted are oilseed rape and maize. Five sampling points were randomly selected for each site and mixed evenly, with a depth of 0‒20 cm. After removing roots, rocks, seeds, and other debris, the soil was air-dried, and after grinding, the soil was kept at room temperature through a 2 mm sieve for further use.

2.2. Experimental Design

We first compared inorganic N concentrations extracted by different extraction methods (DIW, un-buffered 2 mol L‒1 KCl, or pH-buffered 2 mol L‒1 KCl) to assess whether increasing the pH of the extracting solution would affect soil NO2‒ concentrations. Three groups of treatments were set up in the experiment, as follows: (1) Only DIW; (2) un-buffered 2 mol L‒1 KCl; (3) pH = 7.5, 6.5, and 6.0 phosphate buffer solution (PBS) + 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 solution + 2 mol L‒1 KCl (1:4) were added to the Mollisol, and Ultisol, respectively. Different pH of PBS were prepared by mixing 1/15 mol L‒1 KH2PO4 and Na2HPO4 in different proportions. The soil-liquid ratio of all the above treatments was 1:5 (weight/volume, w/v), followed by shaking at 200 rpm and 25°C for 30 min, centrifuged at 9000 rpm for 15 min, and filtration first through 9cm medium speed filter paper (aperture 15-20µm) and then by 0.45µm aperture filter. The pH and inorganic nitrogen content of the extracting solution were simultaneously measured by a pH meter (Mettler Toledo FE28, Switzerland) and a SmartChem automatic chemical analyzer 450 (AMS-Alliance, Italy), respectively.
We further investigated the effect of different storage methods and duration on soil inorganic nitrogen concentration. Air-dried soils (5 g, n = 3) were extracted in 25 mL un-buffered 2 mol L‒1 KCl or DIW (filtered through 0.45 µm). The extraction methods were as described above. The difference is that one set of extracting solution was taken for direct determination of soil inorganic nitrogen concentration, while the others were stored at −20°C for 1 day, and at 4°C for 1, 3, and 6 days, respectively, before determination.
Finally, we studied the effect of oscillation duration on soil NO2‒ recovery. NO2‒ standard solution was added to evaluate the recovery of NO2‒ in the extracts (3 mg N kg‒1 soil, extracted with DIW and pH-buffered 2 mol L‒1 KCl). The extraction method was as described above. The difference is that the oscillation was 10 min and 30 min, respectively, and the extraction solution was placed at 4°C for storage and analyzed within 24 h. Three replicates were set for all the above treatments. Soil without adding standard solution was used as a control. The recovery of NO2‒ was calculated based on the difference measured in soil with and without adding standard solution:
R e c o v e r y = N b − N a N o × 100 %
where Nb and Na represent the NO2‒ concentration extracted from the added standard solution and control soil, respectively; No 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, USA) was used for one-way ANOVA or two-factor ANOVA to determine the differences in soil inorganic nitrogen concentrations between treatments (t and LSD test, P<0.05). When necessary, data were transformed to meet the assumption of normality.

3. Results

3.1. DIW Extraction Can Achieve Higher Soil NO2‒ Content

The pH of the un-buffered KCl extracts was 7.68, 4.16, and 4.65 for the Fluvo-aquic soil, Mollisol, and Ultisol, respectively, which was significantly lower than the pH of the DIW extracts (Figure 1). After adding phosphate buffer solution, pH of the extracts were significantly decreased in Fluvo-aquic soil and increased in Mollisol and Ultisol. The pH was 7.14, 6.17, and 5.96 for the pH-buffered KCl extracts in Fluvo-aquic soil, while that were maintained at 6.0‒7.5 in Mollisol and Ultisol.
The variation of inorganic nitrogen in different treatments are shown in Figure 2. The NO2‒ content was significantly higher in 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). Extraction with DIW can achieve higher NO2‒ content than un-buffered KCl solution in all three soils. The extracted NO2‒ concentrations were mostly increased after adding of phosphate buffer solution with different pH, but which were 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 un-buffered KCl, respectively. Soil NO3‒ concentrations were significantly higher in un-buffered KCl than DIW extracts of Fluvo-aquic soils, while the opposite was true for Mollisol and Ultisol.

3.2. Storage Increased NO2‒ and NH4+ Content

Storage methods significantly affected the concentration of inorganic nitrogen in the three soils (Figure 3). Soil NO2‒ content was closer to directly measured content of fresh samples when stored at 4°C than −20°C, regardless of extraction with DIW or un-buffered KCl. However, there was a tendency of increase with longer storage time (P < 0.05). Compared with storage 1 d at 4°C, storage 6 d at 4°C increased soil NO2‒ content by 3.4 (Fluvo-aquic soil), 1.6 (Mollisol), and 1.8 folds (Ultisol), respectively. Soil NO3‒ concentration extracted by un-buffered KCl decreased with the storage time (4℃), while NH4+ was the opposite. Soil NH4+ concentration increased by 51% (Fluvo-aquic soil), 59% (Mollisol), and 20% (Ultisol) after stored for 6 d compared to 1 d at 4°C. We also found no significant difference between stored for 1 d at 4°C and directly measured for DIW extracted NO2‒ and un-buffered KCl extracted NH4+ concentrations (P > 0.05).

3.3. Effect of Oscillation Time on NO2‒ Recovery

Oscillation time had a significant effect on NO2‒ recovery in acidic soils (Mollisol and Ultisol) rather than alkaline soil (Fluvo-aquic soil) (Figure 4). When oscillating 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, in the two extraction methods (DIW and pH-buffered KCl). No significant difference was found between each treatment (P > 0.05). With the increase of oscillation time, the recovery of NO2‒ in acidic soil decreased, especially in the DIW treatment. The NO2‒ recovery was also affected by the pH of extraction solution in acidic soils. After oscillation 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 two-way ANOVA showed that there was a significant interaction effect between pH and oscillation time on the recovery of NO2‒ in acidic soils, but not in alkaline soil (P > 0.05). In summary, extraction with DIW and oscillating for 10 min could be suitable for both acidic and alkaline soils.

4. Discussion

4.1. Soil NO2‒ Concentration

Compared with DIW, extracting with 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 (pKa=3.3, 25°C), which subsequently decomposes to gaseous nitrogen products such as HONO, NO, or NO2, and can explain the loss of NO2‒ in acidic environments [17,18,30]. These results were confirmed by lower NO2‒ concentrations (35%‒59%) in the un-buffered KCl extract than that of DIW (Figure 2a‒c). In addition, chemical fixation of NO2‒ by insoluble soil organic matter [20,31], reaction with metal oxides [32], or rapid conversion to NO3‒ [25,31] 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 [24], CaCO3 [25], or KOH [19]. Indeed, the pH of the pH-buffered KCl extracts increased significantly (Figure 1b‒c), and the NO2‒ concentration also increased significantly (Figure 2b‒c). The extracted NO2‒ by pH 8.4 buffer solution was even comparable with that of DIW in Mollisol and Ultisol. It was found that the recovery of NO2‒ was about 69% when the extracts pH was 4.8‒6.0, and as high as 95% when the pH was adjusted to 7.8 [32]. In conclusion, extracted by un-buffered KCl could greatly underestimate soil NO2‒ concentrations.
Although DIW is capable of extracting higher concentrations of NO2‒, it produces a brown extract that interferes with colorimetric analysis (Figure 5). Homyak et al. (2015) [18] found that the concentration of NO2‒ in DIW extract measured by colorimetric analysis was 35% higher than that by ion chromatography. However, conventional filters did not remove suspended particles, and the color reagent flocculates with the suspended solids, resulting in higher absorbance readings and overestimation of the NO2‒ concentration. The measured results were consistent after filtration by 0.45 µm filter [18].
Oscillation time was also a key factor affecting soil NO2‒ recovery. Acidic soil NO2‒ recovery decreased significantly with increasing oscillation time (Figure 4). It was shown that NO2‒ recovery at 10 min of oscillation was 86%, which was three times higher than that at 30 min of oscillation [19]. Our study showed that the NO2‒ recovery of acidic Mollisol and Ultisol extracted by DIW was greater than 90% at 10 min of oscillation. Higher NO2‒ recovery were obtained by keeping the extraction process at a higher pH when oscillating for 30 min.
During the analysis process, the extracts may not be determined immediately and stored for a period of time because of various reasons. However, few studies have considered the effect of the storage duration on soil NO2‒ concentration. We found that NO2‒ concentration of samples stored at 4°C was closer to the directly measured value of fresh sample, rather than that of −20°C (Figure 3a‒c). The reason may be due to NO2‒ degradation caused by freezing and thawing. In addition, the NO2‒ concentration gradually increased with the increase of storage time (4°C). Therefore, it is recommend 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 NH4+ concentration in the three soils, and NO3‒ concentration in the Fluvo-aquic soil (Figure 2d‒f). Tu et al. (2021) [28] also found that measured concentrations of NH4+ and NO3‒ in KCl extracts were 1.6‒2.6 and 1.1‒1.8 folds higher than those of DIW, respectively. Lim et al. (2018) [14] 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 [14], 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 "freely" exists in soil solution.
Although most of the literature suggests that the oscillation time for extracting NH4+ and NO3‒ is 60 min [28,33], some studies believe that the concentration of NO3‒ does not change significantly after 15‒30 min of oscillation [34,35]. NH4+ and NO3‒ concentrations were relatively stable at the late stage of oscillation (30‒60 min), which indicate that 30 min of oscillation is long enough to extract soil NH4+ and NO3‒ [35]. For soils with very high mineral nitrogen content, the oscillation time may be extended to 60 min. In this study, NH4+ and NO3‒ were extracted with an oscillation time of 30 min.
Storage conditions significantly affected soil NH4+ and NO3‒ concentrations. We found that soil NH4+ or NO3‒ concentrations were close to the directly measured values of fresh samples when un-buffered KCl or DIW extracts were stored at 4°C, which were also higher than that at −20°C (Figure 3d‒i). NH4+ and NO3‒ concentrations 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 freezing or thawing process. However, a gradual increase in NH4+ concentration and decrease in NO3‒ concentration with soil sample extracts were stored at 4°C (Figure 3d‒i), which were also confirmed by the results from Li et al. (2012) [33]. Thus, we recommend the extracted solution could be stored at 4°C and analyzed within 24 h.

5. Conclusions

The soil NO2‒ extraction method was optimized in order to more accurately reflect the true concentration of NO2‒ in soil. Un-buffered KCl significantly underestimated soil NO2‒ concentration compared to DIW. Further studies showed that the oscillation time and preservation method significantly affected the NO2‒ concentration. The highest NO2‒ recoveries for the three soil NO2‒ extracted by DIW were obtained at 10 min of oscillation. Soil inorganic nitrogen content in extracts stored at 4°C for one day was closer to direct measurements of fresh samples than other preservation methods. Overall, the optimal conditions for determination of soil NO2‒ was using DIW as the extractant, oscillating for 10 min, and passing through 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 efficient extraction of soil NO2‒, NO3‒, and NH4+, respectively, and further optimization is needed if we want to achieve their efficient extraction at the same time.

Author Contributions

Yaqi Song: Data analysis and visualization, writing the manuscript. Dianming Wu: Conception, funding acquisition, methodology, review and editing. Peter Dörsch: Review and editing. Lanting Yue: Measurement. Lingling Deng: Measurement. Chengsong Liao: Supervision. Zhimin Sha: Supervision, editing. Wenxu Dong: Funding acquisition, supervision. Yuanchun Yu: Funding acquisition, supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2023YFC3707401), National Natural Science Foundation of China (42077083), State Key Laboratory of Atmospheric Boundary Layer Physics and Atmospheric Chemistry (LAPC-KF-2022-09), the Fundamental Research Funds for the Central Universities (2017–2023), Shanghai “Science and Technology Innovation Action Plan” Project (22002400300) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

References

  1. Butterbach-Bahl K, Dannenmann M. Denitrification and associated soil N2O emissions due to agricultural activities in a changing climate. Environmental Sustainability. 2011;3:389-395. [CrossRef]
  2. Li Y, Ju X, Wu D. Transient nitrite accumulation explains the variation of N2O emissions to N fertilization in upland agricultural soils. Soil Biology and Biochemistry. 2023;177:108917. [CrossRef]
  3. Kool DM, Dolfing J, Wrage N, Van Groenigen JW. Nitrifier denitrification as a distinct and significant source of nitrous oxide from soil. Soil Biology and Biochemistry. 2011;43:174-178. [CrossRef]
  4. Pandey A, Suter H, He JZ, Hu HW, Chen DL. Dissimilatory nitrate reduction to ammonium dominates nitrate reduction in long-term low nitrogen fertilized rice paddies. Soil Biology and Biochemistry. 2019;131:149-156. [CrossRef]
  5. Wang M, Hu R, Ruser R, Schmidt C, Kappler A. Role of Chemodenitrification for N2O Emissions from Nitrate Reduction in Rice Paddy Soils. Acs Earth and Space Chemistry. 2020;4:122-132. [CrossRef]
  6. Müller C, Laughlin RJ, Spott O, Rütting T. Quantification of N2O emission pathways via a 15N tracing model. Soil Biology and Biochemistry. 2014;72:44-54. [CrossRef]
  7. Serna M, Bañuls J, Quiñones A, Primo-Millo E, Legaz F. Evaluation of 3,4-dimethylpyrazole phosphate as a nitrification inhibitor in a Citrus-cultivated soil. Biology and Fertility of Soils. 2000;32:41-46. [CrossRef]
  8. Yang L, Zhang X, Ju X, Wu D. Oxygen-depletion by rapid ammonia oxidation regulates kinetics of N2O, NO and N2 production in an ammonium fertilised agricultural soil. Soil Biology and Biochemistry. 2021;163:108460. [CrossRef]
  9. Zhu G, Song X, Ju X, Zhang J, Müller C, Sylvester-Bradley R, Thorman RE, Bingham I, Rees RM. Gross N transformation rates and related N2O emissions in Chinese and UK agricultural soils. Science of the Total Environment. 2019;666:176-186. [CrossRef]
  10. Giguere AT, Taylor AE, Suwa Y, Myrold DD, Bottomley PJ. Uncoupling of ammonia oxidation from nitrite oxidation: Impact upon nitrous oxide production in non-cropped Oregon soils. Soil Biology and Biochemistry. 2017;104:30-38. [CrossRef]
  11. Venterea R, Coulter J, Clough T. Nitrite accumulation and nitrogen gas production increase with decreasing temperature in urea-amended soils: Experiments and modeling. Soil Biology and Biochemistry. 2020;142:10772. [CrossRef]
  12. Maharjan B, Venterea RT. Nitrite intensity explains N management effects on N2O emissions in maize. Soil Biology and Biochemistry. 2013;66:229-238. [CrossRef]
  13. Ma L, Shan J, Yan X. Nitrite behavior accounts for the nitrous oxide peaks following fertilization in a fluvo-aquic soil. Biology and Fertility of Soils. 2015;51:563-572. [CrossRef]
  14. Lim NYN, Frostegård Å, Bakken LR. Nitrite kinetics during anoxia: The role of abiotic reactions versus microbial reduction. Soil Biology and Biochemistry. 2018;119:203-209. [CrossRef]
  15. Medinets S, Skiba U, Rennenberg H, Butterbach-Bahl K. A review of soil NO transformation: Associated processes and possible physiological significance on organisms. Soil Biology and Biochemistry. 2015;80:92-117. [CrossRef]
  16. Oswald R, Behrendt T, Ermel M, Wu D, Su H, Cheng Y, Breuninger C, Moravek A, Mougin E, Delon C, Loubet B, Pommerening-Roser A, Sorgel M, Poschl U, Hoffmann T, Andreae MO, Meixner FX, Trebs I. HONO emissions from soil bacteria as a major source of atmospheric reactive nitrogen. Science. 2013;341:1233-1235. [CrossRef]
  17. Song Y, Wu D, Ju X, Dörsch P, Wang M, Wang R, Song X, Deng L, Wang R, Gao Z, Haider H, Hou L, Liu M, Yu Y. Nitrite stimulates HONO and NOx but not N2O emissions in Chinese agricultural soils during nitrification. Science of the Total Environment. 2023;902:166451. [CrossRef]
  18. Homyak PM, Vasquez KT, Sickman JO, Parker DR, Schimel JP. Improving Nitrite Analysis in Soils: Drawbacks of the Conventional 2 M KCl Extraction. Soil Science Society of America Journal. 2015;79:1237-1242. [CrossRef]
  19. Stevens RJ, Laughlin RJ. Nitrite Transformations during Soil Extraction with Potassium Chloride. Soil Science Society of America Journal. 1995;59:933-938. [CrossRef]
  20. Islam A, Chen D, White RE, Weatherley AJ. Chemical decomposition and fixation of nitrite in acidic pasture soils and implications for measurement of nitrification. Soil Biology and Biochemistry. 2008;40:262-265. [CrossRef]
  21. Maynard DG, Kalra YP, Crumbaugh JA. Nitrate and exchangeable ammonium nitrogen; M.R. Carter and E.G. Gregorich: Soil sampling and methods of analysis. 2nd ed. CRC Press, Boca Raton, FL, 2007; 71–80.
  22. Pansu M, Gautheyrou J. Handbook of soil analysis: Mineralogical, organic and inorganic methods; Springer, New York; 2006.
  23. Soil quality Determination of nitrate, nitrite and ammonium in field-moist soils by extraction with potassium chloride solution-Part 2: Automated method with segmented flow analysis: ISO 14256-2: 2005. https://www.iso.org/obp/ui/#iso:std:iso:14256:-2:ed-1:v1:en.
  24. Reuss JO, Smith RL. Chemical Reactions of Nitrites in Acid Soils. Soil Science Society of America Journal. 1965;29:267-270. [CrossRef]
  25. Nelson DW, Bremner JM. Factors affecting chemical transformations of nitrite in soils. Soil Biology and Biochemistry. 1969;1:229-239. [CrossRef]
  26. Fujitake N, Kusumoto A, Tsukamoto M, Kawahigashi M, Suzuki T, Otsuka H. Properties of soil humic substances in fractions obtained by sequential extraction with pyrophosphate solutions at different pHs. Soil Science and Plant Nutrition;44:253-260. [CrossRef]
  27. Ju X, Zhang C. Nitrogen cycling and environmental impacts in upland agricultural soils in North China: A review. Journal of Integrative Agriculture. 2017;16:2848-2862. [CrossRef]
  28. Tu Q, Dou F, Salem HM. Optimizing Conditions for the Measurement of Soil Inorganic Nitrogen with a Micro-Plate Reader. Global Journal of Agricultural Innovation, Research & Development. 2021;8:22-31. [CrossRef]
  29. Ma BL, Ying J, Balchin D. Impact of Sample Preservation Methods on the Extraction of Inorganic Nitrogen by Potassium Chloride. Journal of Plant Nutrition. 2005;28:785-796. [CrossRef]
  30. Venterea RT, Burger M, Spokas KA. Nitrogen Oxide and Methane Emissions under Varying Tillage and Fertilizer Management. Journal of Environmental Quality. 2005;34:1467-1477. [CrossRef]
  31. Isobe K, Koba K, Suwa Y, Ikutani J, Kuroiwa M, Fang Y, Yohb M, Mo J, Otsukaa S, Senoo S. Nitrite transformations in an N-saturated forest soil. Soil Biology and Biochemistry. 2012;52:61-63. [CrossRef]
  32. Nelson DW, Bremner JM. Gaseous products of nitrite decomposition in soils. Soil Biology and Biochemistry. 1970;2:203-215. [CrossRef]
  33. Li K, Zhao Y, Yuan X, Zhhao H, Wang Z, Li S, Malhi S. Comparison of Factors Affecting Soil Nitrate Nitrogen and Ammonium Nitrogen Extraction. Communications in Soil Science and Plant Analysis. 2012;43:571-588. [CrossRef]
  34. Yang L, Zhou G, Qiu Z, Wang Z, Yu B. Study on the Methods of Sample Collection and Keeping in Forest Soil NO3--N Determination. Forest Research (in Chinese). 2005;18:209-213. [CrossRef]
  35. Zhu J, Wang C, Paerhati, Xiao Y, Cao X, Hua Z. Effect of Oscillation and Extraction Time on Measurement of Content of NO3--N and NH4+-N in Soil Held by Different Saving Methods. Xinjiang Agricultural Sciences (in Chinese). 2014;51:761-767.
Preprints 94612 g001
Figure 1. pH variation of soil extracts from different treatments. DIW, deionized water; KCl, 2 mol L‒1 KCl; PBS 8.4, 8.0, 7.5, 7.0, 6.5, and 6.0 denote phosphate buffer solution with the pH of 8.4, 8.0, 7.5, 7.0, 6.5, and 6.0, respectively. FS: Fluvo-aquic soil; MS: Mollisol; US: Ultisol. Different lowercase letters mean significant difference at P < 0.05.
Figure 1. pH variation of soil extracts from different treatments. DIW, deionized water; KCl, 2 mol L‒1 KCl; PBS 8.4, 8.0, 7.5, 7.0, 6.5, and 6.0 denote phosphate buffer solution with the pH of 8.4, 8.0, 7.5, 7.0, 6.5, and 6.0, respectively. FS: Fluvo-aquic soil; MS: Mollisol; US: Ultisol. Different lowercase letters mean significant difference at P < 0.05.
Preprints 94612 g001
Preprints 94612 g002
Figure 2. Variation of inorganic nitrogen concentration in different treated soils. DIW, deionized water; KCl, 2 mol L‒1 KCl; PBS 8.4, 8.0, 7.5, 7.0, 6.5, and 6.0 denote phosphate buffer solution with pH of 8.4, 8.0, 7.5, 7.0, 6.5, and 6.0, respectively; FS: Fluvo-aquic soil; MS: Mollisol; US: Ultisol. Different lowercase letters mean significant difference at P < 0.05.
Figure 2. Variation of inorganic nitrogen concentration in different treated soils. DIW, deionized water; KCl, 2 mol L‒1 KCl; PBS 8.4, 8.0, 7.5, 7.0, 6.5, and 6.0 denote phosphate buffer solution with pH of 8.4, 8.0, 7.5, 7.0, 6.5, and 6.0, respectively; FS: Fluvo-aquic soil; MS: Mollisol; US: Ultisol. Different lowercase letters mean significant difference at P < 0.05.
Preprints 94612 g002
Preprints 94612 g003
Figure 3. Changes in inorganic nitrogen concentration in extracts from different storage methods. DM, directly measured; −20°C (1d), stored at −20°C for 1 day; 4°C (1d), (3d), and (6d) mean stored at 4°C for 1, 3, and 6 days, respectively; FS: Fluvo-aquic soil; MS: Mollisol; US: Ultisol. Different lowercase letters mean significant difference at P < 0.05.
Figure 3. Changes in inorganic nitrogen concentration in extracts from different storage methods. DM, directly measured; −20°C (1d), stored at −20°C for 1 day; 4°C (1d), (3d), and (6d) mean stored at 4°C for 1, 3, and 6 days, respectively; FS: Fluvo-aquic soil; MS: Mollisol; US: Ultisol. Different lowercase letters mean significant difference at P < 0.05.
Preprints 94612 g003
Preprints 94612 g004
Figure 4. Soil NO2‒ recovery rate with different oscillation time. DIW, deionized water; 2 M KCl + PBS, 2 mol L‒1 KCl and PBS of different pH: pH 7.5 of PBS is added to the Fluvo-aquic soil, and pH 8.4 of PBS is added to Mollisol and Ultisol; FS: Fluvo-aquic soil; MS: Mollisol; US: Ultisol. Different lowercase letters mean significant difference between the DIW and KCl + PBS treatments at P < 0.05. Different uppercase letters mean significant difference between different oscillation times at P < 0.05.
Figure 4. Soil NO2‒ recovery rate with different oscillation time. DIW, deionized water; 2 M KCl + PBS, 2 mol L‒1 KCl and PBS of different pH: pH 7.5 of PBS is added to the Fluvo-aquic soil, and pH 8.4 of PBS is added to Mollisol and Ultisol; FS: Fluvo-aquic soil; MS: Mollisol; US: Ultisol. Different lowercase letters mean significant difference between the DIW and KCl + PBS treatments at P < 0.05. Different uppercase letters mean significant difference between different oscillation times at P < 0.05.
Preprints 94612 g004
Preprints 94612 g005
Figure 5. Soil NO2‒ was extracted with deionized water to produce brown extract, which was filtered by a 0.45 µm filter to obtain clear extract.
Figure 5. Soil NO2‒ was extracted with deionized water to produce brown extract, which was filtered by a 0.45 µm filter to obtain clear extract.
Preprints 94612 g005
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Copyright: This open access article is published under a Creative Commons CC BY 4.0 license, which permit the free download, distribution, and reuse, provided that the author and preprint are cited in any reuse.
Prerpints.org logo

Preprints.org is a free preprint server supported by MDPI in Basel, Switzerland.

facebook logotwitter logolinkedin logo
MDPI Initiatives
Important Links
Subscribe

Disclaimer

Terms of Use

Privacy Policy

© 2024 MDPI (Basel, Switzerland) unless otherwise stated