Determination of ammonium and nitrate in soils by digital colorimetry

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V. Garmay, K. V. Oskolok, O. V. Monogarova, M. I. Demidov This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4007169/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Sep, 2024 Read the published version in Environmental Monitoring and Assessment → Version 1 posted 12 You are reading this latest preprint version Abstract A method of digital colorimetric determination of ammonium and nitrate in soils is proposed. The method is based on corresponding photometric techniques of ammonium and nitrate determination after potassium chloride extraction from soils samples. Ammonium is determined as an indophenol dye, and nitrate is determined as an azo dye. The original techniques were modified to overcome the lower sensitivity of the digital colorimetric method. For ammonium determination, the time required for the reaction to proceed completely was studied. Along with the use of a 96-well microplate protected from ambient light by a special frame, mathematical correction of scattered radiation using black ink and taking the images by a scanner in transmission mode without any post-processing, the resulting colorimetric techniques proved to provide accuracy and sensitivity close to those of the spectrophotometric techniques, and the overall analysis speed for tens of samples was even higher. The techniques’ validity was proven by the analysis of standard samples and by the analysis of soil samples collected in several districts of the Moscow region. digital colorimetry soil nitrate ammonium Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Nitrogen (N) is a vital element for plant growth and crops productivity (Hachiya and Sakakibara 2017 ; Jose 2022 ; Saleem et al. 2022 ). It is acquired from soils mostly in the form of ammonium and nitrate (Hachiya and Sakakibara 2017 ). Natural soil N content is usually not enough to provide sufficient productivity (An et al. 2022 ). Therefore, N fertilizers are used worldwide (An et al. 2022 ; Saleem et al. 2022 ). Moreover, ammonium and nitrate should be added together, since their sole use was shown to be less beneficial or even toxic for plants, and the maximum plants growth requires a certain ammonium-to-nitrate ratio depending on the species (Hachiya and Sakakibara 2017 ; Saleem et al. 2022 ). On the other hand, a great part of N is lost due to leaching (Fayose et al. 2021 ; Gurmesa et al. 2022 ; Mustafa, Hayat and Alotaibi 2022 ). The losses may vary from 10–75% (Choosang et al. 2018 ; Kabala et al. 2017 ). This leads to ecological problems like the pollution of water systems or even greenhouse gases (N 2 O and NH 3 ) production, and also causes a decrease in the economic efficiency of agriculture (An et al. 2022 ; Choosang et al. 2018 ; Fayose et al. 2021 ; Kabala et al. 2017 ; Mustafa, Hayat and Alotaibi 2022 ). For these reasons, determination of ammonium and nitrate content in soils is of great importance. Several methods can be used for ammonium and nitrate determination, including Kjeldahl steam distillation techniques, ion chromatography, ion selective electrodes (ISE) and various spectrophotometric techniques (Choosang et al. 2018 ; Griffin et al. 2011 ). The Kjeldahl method is, however, hazardous, lengthy and labor intensive, taking several hours to analyze one sample (Śaez-Plaza et al. 2013 ). Although various automated Kjeldahl procedures are long known (Harwood and Huyser 1970 ; Lennox and Flanagan 1982 ), usually it is not considered the method of choice. Ion chromatography and ISE methods face the same problem of the severe influence of interfering ions (Choosang et al. 2018 ; Fayose et al. 2021 ; Griffin et al. 2011 ). Indeed, since ammonium is strongly adsorbed by the soil adsorption complex (Bolt, Bruggenwert and Kamphorst 1976 ), its extraction usually requires the use of concentrated salt solutions like 2M KCl (Griffin et al. 2011 ). Nitrate can be extracted from soil by water or diluted salt solutions, at least in some cases, but its low ionic strength can cause the dispersion of soil, resulting in cloudy filtrates. When both ammonium and nitrate are to be determined, 2M KCl extraction is considered an ideal extractant (Griffin et al. 2011 ). Therefore, photometric procedures are among the most common for ammonium and nitrate determination (Griffin et al. 2011 ; López Pasquali, Fernández Hernando and Durand Alegrı́a 2007 ). Ammonium is usually determined by indophenol method, and nitrate is often determined by diazotation method after being reduced to nitrite (Bolleter, Bushman and Tidwell 1961 ; López Pasquali, Fernández Hernando and Durand Alegrı́a 2007 ; Schmiedt Sattolo et al. 2016 ; Singh et al. 2019 ). These methods are accepted as regulatory state standards in some countries (GOST 26488-85; GOST 26489-85; O’Dell 1993 , Method 353.2; O’Dell 1993 , Method 350.1). The disadvantage of spectrophotometry is, however, the higher cost of equipment as compared to ISE (Choosang et al. 2018 ; Fayose et al. 2021 ; Nadporozhskaya et al. 2022 ). Another challenging problem is in-field ammonium and nitrate determination (Choosang et al. 2018 ; Fayose et al. 2021 ; Heckman 2002 ; Heckman et al. 2002 ; Kabala et al. 2017 ). Portable colorimeters have been designed for these purposes, which are, however, quite expensive and are reported to be typically used for research purposes instead of practical in-field application (Choosang et al. 2018 ). ISE are sometimes considered well-suited for in-field analysis (Fayose et al. 2021 ; Choosang et al. 2018 ; Nadporozhskaya et al. 2022 ), but its use for ammonium determination remains questionable (Cuartero et al. 2020 ). One of the promising solutions of the stated problems is a digital colorimetric method rapidly developing in the recent years (Christodouleas et al. 2015 ; Choodum et al. 2013 ; Oskolok et al. 2018 ). Since it utilizes various consumer devices like smartphones and scanners for measuring analytical signal, its cost is in fact limited by the cost of chemicals and glassware (Oskolok et al. 2021 ). Although sometimes the low speed of spectrophotometric reactions could still be a problem, its influence on the overall analysis duration becomes less pronounced when numerous samples are analyzed. Indeed, ISE can take from 30 s up to several minutes for equilibration (Cheremisinoff 2001 ; HACH 2021 ; Vernier 2023 ), resulting in hours for several tens of samples. In digital colorimetry, the analysis can be performed faster by using multiwell plates allowing measuring several tens of samples simultaneously without cleaning and replacing cuvettes (Christodouleas et al. 2015 ). Besides, it requires less sample volume, which can further decrease the overall cost of analysis (Oskolok et al. 2021 ). At last, smartphone-based digital colorimetry can be easily applied in-field, allowing one to conduct analyses with accuracy and precision close to those of spectrophotometric method at significantly lower cost (Christodouleas et al. 2015 ; Choodum et al. 2013 ; Nadporozhskaya et al. 2022 ; Manbohi and Ahmadi 2022 ; Mohamed, Ismail and Ali 2020 ). Digital colorimetry has been reported to be used for both nitrate and ammonium N determination (Nadporozhskaya et al. 2022 ; Yokota, Okada and Yamaguchi 2007 ). However, the original spectrophotometric techniques were used “as is”, despite the fact that the method is clearly less sensitive. Therefore, the aim of this work was to develop digital colorimetric techniques of ammonium and nitrate determination with accuracy and sensitivity close to those of the spectrophotometric method. Materials and methods Reagents and equipment The following reagents were used: potassium chloride, potassium iodide, sodium hydroxide, sodium pyrophosphate decahydrate, sodium salicylate, sodium nitroprusside dihydrate, ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA), sodium thiosulfate (Titrisol® concentrated standard), sodium hypochlorite water solution, potassium sodium tartrate tetrahydrate, copper (II) sulfate pentahydrate, hydrazine sulfate, 1-naphthylamine, sulfanilamide, hydrochloric acid 37% water solution, phosphoric acid 85% water solution. All the chemicals were purchased at Sigma Aldrich and were reagent grade. To determine the level of scattered radiation, liquid black ink was used (JSC “GAMMA”, Russian Federation). To prepare soil suspensions, a LOIP LS-120 (JSC "Laboratory Equipment and Instruments", Russian Federation) laboratory shaker was used. Photometric analysis was performed using a UV-mini 1240 spectrophotometer (Shimadzu, Japan). Images for digital colorimetric analysis were taken using an EPSON Perfection 1670 office flatbed photoscanner (Japan) in transmission mode. For this purpose, the solutions were placed into a 96-well transparent polystyrene microplate (Greiner AG, Austria) with a flat bottom (well volume 350 µL). A special frame was put into the scanner that protected the microplate from ambient light. The images were saved as 48-bit *.tiff files, with no scanner post-processing being used. For processing the images, the free IrfanView application was applied (IrfanView 2024 ). Extraction of ammonium and nitrate from soil samples Soil extracts were prepared as described in (GOST 26488-85; GOST 26489-85). 16,00 ± 0,01 g of soil were put into a conical flask, then 40,0 ml of 2M potassium chloride solution were added. The suspension was shaken for 1 h and filtered through a grade 2 filter paper (Whatman ®). At the first stage, standard samples of soddy-podzolic medium loamy soils, alkaline and saline soils, podzolized medium loamy chernozemic soils and carbonate deep chernozemic soils were analyzed by the digital colorimetric method to estimate its accuracy. Then, the method was applied along with the standard spectrophotometric procedures for the analysis of soil samples to compare their metrological characteristics. About 250 samples were analyzed during 2022–2023 years. The samples were collected in the Moscow region. Nitrate determination Since only a few mL of a colored solution are required for the analysis, the volumes of the analyzed solutions and of the solution of the reagents were reduced 10 times compared to the original procedure (GOST 26488-85). 0.500 mL of the analyzed solution was added to a 5.0 mL test-tube and mixed with 1.00 mL of sodium pyrophosphate solution and 1.00 mL of hydrazine sulfate solution. Sodium pyrophosphate solution was prepared by dissolving 5.00 ± 0.01 g of sodium pyrophosphate and 8.00 ± 0.01 g of sodium hydroxide in 1.00 L of deionized water. To prepare hydrazine sulfate solution, 5.50 ± 0.01 g of hydrazine sulfate were dissolved in several mL of deionized water in a 1.00 L flask, then 6.00 mL of 0.01 M copper sulfate solution were added, and the solution was diluted to the mark with deionized water. 10 minutes after adding sodium pyrophosphate solution, 2.50 mL of a coloring solution was added. The coloring solution was prepared by dissolving 5.00 ± 0.01 g of sulfanilamide, 1.00 ± 0.01 g of 1-naphthylamine, 1.00 ± 0.01 g of EDTA and 100 mL of phosphoric acid in 1.00 L of deionized water, followed by diluting of the obtained solution 5 times with deionized water. The color develops in 15 minutes after adding the coloring solution and is stable for 1.5 h. The overall scheme of the analysis is following: In order to adapt the original procedure to the digital colorimetric method, we also considered using the concentrated coloring solution without dilution. The aliquot of the analyzed solution was also variated. See Section 3 for details. For digital colorimetric analysis, 320 µL of each sample were poured into a 96-well plate. One well was filled with 2M KCl solution as a blank sample. Ammonium determination Since only a few mL of a colored solution are required for the analysis, the volumes of the analyzed solutions and of the solution of the reagents were reduced 10 times compared to the original procedure (GOST 26489-85). 0.200 mL of the analyzed solution were added to a 5.0 mL test-tube and mixed with 4.00 mL of a coloring solution and 0.200 mL of 0.125% sodium hypochlorite solution. To prepare the coloring solution, 14.17 ± 0.01 g of sodium salicylate, 5.60 ± 0.01 g of potassium sodium tartrate tetrahydrate and 6.67 ± 0.01 g of sodium hydroxide were dissolved in 180 mL of water and boiled for 20 minutes to remove ammonia. After cooling the solution, 0.1000 ± 0.0002 g of sodium nitroprusside and 5.00 ± 0.01 g of EDTA were added, and the solution was diluted to 0.2500 L by deionized water. For analysis, the solution should be diluted 10 times with deionized water. The overall scheme can be presented as follows: 0.125% sodium hypochlorite solution was prepared by dilution of the original solution with deionized water. Concentration of the original solution was established by iodometric titration as described in (GOST 26489-85) and occurred to be 1.7%. In order to adapt the original procedure to the digital colorimetric method, we also considered varying the aliquot of the analyzed solution. See Section 3 for details. For digital colorimetric analysis, 320 µL of each sample were poured into a 96-well plate. One well was filled with 2M KCl solution as a blank sample. Results and discussion Nitrate determination The first question about nitrate determination was whether the original spectrophotometric procedure was suitable for digital colorimetric analysis. Therefore, calibration curves were constructed using the scanner according to the procedure described in the first paragraph of Section 2.3. Eight nitrate solutions were prepared with N concentration equal to 0; 1.0; 2.0; 4.0; 6.0; 8.0; 10.0 and 12.0 mg·L –1 , corresponding to nitrate N content in soil varying from 2.5 to 30 ppm. Five points were selected for each well of the 96-well plate and averaged. Absorbance A i for the i th color channel was calculated using a formula $${A}_{i}=\text{lg}\left(\frac{{L}_{blank,i}-{L}_{scatter,i}}{{L}_{sample,i}-{L}_{scatter,i}}\right),$$ where L blank, i is the lightness of the i th color channel for a blank sample, L scatter, i is the scattered radiation level for the i th color channel (the lightness of the i th color channel of the well filled with black ink), and L sample, i is the lightness of the i th color channel for a well filled with a sample. The most significant variations were observed for the green channel of the RGB-space. The curve is given in Fig. 1 a. Although it is linear, the absorbances do not exceed 0.14. Moreover, the lowest concentration with a non-zero absorbance is 4.0 mg·L –1 , which is 4 times higher than the limit of quantitation of the standard method (GOST 26488-85). Assuming that this can be overcome by increasing nitrate concentration, we tried to construct calibration curves adding 1.00 and 1.50 mL of calibration solutions instead of 500 µL used earlier (Fig. 1 , b, c). In these cases, absorbances were statistically higher than zero for 2.0 mg·L –1 for 1.00 mL aliquot and for 1.0 mg·L –1 for 1.50 mL aliquot. However, the curves’ linearity occurred to be violated, beginning with 10.0 mg·L –1 solution, although the corresponding absorbances did not exceed 0.27. This effect was more pronounced for 1.50-mL aliquot of the nitrate solution. Curves constructed using a spectrophotometer demonstrated a similar behavior, reaching a plateau at high concentrations (Fig. 2 ). This indicates that, most probably, the coloring solution concentration is not high enough, and exceeding diazo intermediates recombine with water. Therefore, we decided to use the concentrated coloring solution without dilution. The aliquot was 1.50 mL. The corresponding calibration curve is given in Fig. 3 . It is linear up to 12.0 mg·L –1 , which agrees with the standard method (GOST 26488-85). Therefore, we applied these aliquot and coloring solution concentration values for the following measurements. To test the validity of the resulting colorimetric technique, several standard soil samples were analyzed. Some results are given in Table 1 . Relative errors fall well within the nominal accuracy of the method, and the relative standard deviation values are also satisfactory. At the next step, the colorimetric technique was applied for various soil samples analysis along with the standard spectrophotometric method (GOST 26488-85). The corresponding scatter diagram is given in Fig. 4 . The normalized root mean square deviation (NRMSD) between the two datasets was about 4%, and the mean absolute percentage error (MAPE) was about 8%, which is well within the limits specified by the standard method (GOST 26488-85). Since nitrate is readily extracted from soil in several minutes (Griffin et al. 2011 ), the developed technique is possibly applicable for in-field nitrate determination, although it requires following investigation. Table 1 The results of nitrate N determination in some soil standards (P = 0.95; n = 3) Sample Nitrate N content, ppm Relative error, % Certified Measured Soddy-podzolic heavy loamy soil SADPP-07/3 26.0 25.1 ± 2.0 3.5 Soddy-podzolic medium loamy soil SADPP-10/5 3.01 3.13 ± 0.73 4.0 Carbonate deep chernozem SACHkP-08/1 13.1 12.5 ± 1.2 4.6 Podzolized medium loamy chernozem SACHopP-04/1 91.0 85.2 ± 1.5 6.4 Ammonium determination In case of ammonium determination, we decided to take into account the results of the previous section and, therefore, we started with constructing six calibrations, the aliquot of standard ammonium solutions being varied from standard to double and the concentration of the hypochlorite solution varied from standard (0.125%) to double (0.25%) and original (1.7%). The standard solutions contained 0; 2.0; 4.0; 8.0; 12.0; 16.0; 20.0; 24.0 mg·L –1 of ammonium N, corresponding to ammonium N content in soil varying from 5 to 60 ppm. A double aliquot of the ammonium solutions led to increased calibration slopes (Fig. 5 ). When a standard hypochlorite aliquot was applied, the lowest points (2 and 4 mg·L –1 ) absorbances were negligible. Increased hypochlorite concentration led to increased absorbance and calibration slopes. However, for the original solution, absorbances were slightly lower than for 0.25% solution, and the lowest points in this case were also negligible. Therefore, we applied 0.25% solution for further measurements. To test the validity of the resulting colorimetric technique, several standard soil samples were analyzed. Some results are given in Table 2 . Relative errors fall well within the nominal ac-curacy of the method, and the relative standard deviation values are also satisfactory. The next stage was an optimization of the reaction time. For this purpose, the images of the standard solutions after adding all the necessary reagents were taken each 10 minutes beginning with the 5th minute of reaction. It was found that the maximum absorption is reached by the 25th minute and then remains constant for at least an hour independently of NH 4 + concentration. At the next step, the colorimetric technique was applied for various soil samples analysis along with the standard spectrophotometric method (GOST 26489-85). The corresponding scatter diagram is given in Fig. 6 . The normalized root mean square deviation between the two datasets was about 4%, and the mean absolute percentage error was about 9%, which is well within the limits specified by the standard method (GOST 26489-85). Table 2 The results of ammonium N determination in some soil standards (P = 0.95; n = 3) Sample Ammonium N content, ppm Relative error, % Certified Measured Soddy-podzolic heavy loamy soil SADPP-07/3 5.60 5.23 ± 0.78 6.6 Saline soil SAZP-2011 11.7 11.0 ± 1.1 6.0 Carbonate deep chernozem SACHkP-08/1 7.53 7.2 ± 1.1 4.4 Podzolized medium loamy chernozem SACHopP-04/1 10.7 10.6 ± 1.1 0.9 As for in-field ammonium determination, the limiting stage will obviously be its extraction from soil, requiring at least an hour of shaking its suspension. On the other hand, ISE-based techniques will face the same problem. Therefore, in-field ammonium determination requires further research. Conclusions The standard methods (GOST 26488-85; GOST 26489-85) of nitrate and ammonium N determination were successfully adapted for the colorimetric method. Using a scanner instead of a spectrophotometer allows to decrease the cost of the analysis and to measure several tens of samples at once due to the use of a multiwell plate. Taking the images by a scanner in transmission mode and taking into account scattered radiation makes it possible to achieve an accuracy close to that of the spectrophotometric method. Nitrate in-field determination is potentially available by the developed technique, since NO 3 – ions are readily extracted from soil in several minutes (Griffin et al. 2011 ). On the other hand, in-field ammonium determination in soils require following investigation to find optimal extraction conditions, if they even exist. Declarations Data availability All relevant data are available upon request from the authors. Competing interests The authors declare no conflict of interest. Funding This research was funded by M.V. Lomonosov Moscow State University (No. AAAA-A21-121011590089-1 “Development of highly informative and high-tech methods of chemical analysis for the protection of ecosystems, the creation of new materials and advanced production technologies, the transition to environmentally friendly and resource-saving energy, the development of nature-like technologies, high-tech healthcare and rational use of natural resources”). Author Contribution K.V. Oskolok and O.V. Monogarova developed a modified digital colorimetric method, including consideration of scattered light. A.V. Garmay and M.I. Demidov modified the reaction conditions to provide the required sensitivity and dynamic range and performed most of the experiments. A.V. Garmay wrote the main manuscript text and prepared figures and tables. All authors reviewed the manuscript. References An, T., Wang, F., Ren, L., Ma, Sh., Li, Sh., Liu, L., and Wang, J. (2022). 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Environmental Monitoring and Assessment , 192, 657. https://doi.org/10.1007/s10661-020-08615-5 Mustafa, Gh., Hayat, N., and Alotaibi, B. A. (2022). How and why to prevent over fertilization to get sustainable crop production. In T. Aftab, and Kh. R. Ha-keem (editors), Sustainable Plant Nutrition. Molecular Interventions and Advancements for Crop Improvement , (Chapter 15, pp. 339-354). Academic Press. https://doi.org/10.1016/B978-0-443-18675-2.00019-5 Nadporozhskaya, M., Kovsh, N., Paolesse, R., and Lvova, L. (2022). Recent Advances in Chemical Sensors for Soil Analysis: A Review. Chemosensors , 10(1), 35. https://doi.org/10.3390/chemosensors10010035 O’Dell, J. W. (Ed.). (1993). Method 353.2. Determination of Nitrate+Nitrite Nitrogen by automated colorimetry. Revision 2.0. Cincinnati, Ohio: Environmental Monitoring Systems Laboratory, Office of Research and Development, U.S. Environmental Protection Agency. O’Dell, J. W. (Ed.). (1993). Method 350.1. Determination of Ammonia Nitrogen by automated colorimetry. Revision 2.0. Cincinnati, Ohio: Environmental Monitoring Systems Laboratory, Office of Research and Development, U.S. Environmental Protection Agency. Oskolok, K. V., Shults, E. V., Monogarova, O. V., and Chaplenko, A. A. (2018). Optical molecular analysis using office flatbed photo scanner: New approaches and solutions. Talanta , V. 178, 377-383. https://doi.org/10.1016/j.talanta.2017.09.049 Oskolok, K. V., Monogarova, O. V., Garmay, A. V., and Pastukhova, A. A. (2021). Simultaneous Determination of Two Components of Nickel Silver by Digital Colorimetry. Moscow University Chemistry Bulletin , 76, 33-37. https://doi.org/10.3103/S0027131421010119 Śaez-Plaza, P., Navas, M. J., Wybraniec, S., Michałowski, T., and Asuero, A. G. (2013). An Overview of the Kjeldahl Method of Nitrogen Determination. Part II. Sample Preparation, Working Scale, Instrumental Finish, and Quality Control. Critical Reviews in Analytical Chemistry , 43, 224-272.https://10.1080/10408347.2012.751787 Saleem, S., Ul Mushtaq, N., Rasool, A., Shah, W.H., Tahir, I., and Ul Rehman, R. (2022). Plant nutrition and soil fertility: physiological and molecular avenues for crop improvement. In T. Aftab, and Kh. R. Hakeem (Editors), Sustainable Plant Nutrition. Molecular Interventions and Advancements for Crop Improvement (Chapter 2, pp. 23-49). Elsevier. https://doi.org/10.1016/B978-0-443-18675-2.00009-2 Schmiedt Sattolo, Th. M., Otto, R., Mariano, E., and Kamogawa, M. Ya. (2016). Adaptation and Validation of Colorimetric Methods in Determining Ammonium and Nitrate on Tropical Soils. Communications in Soil Science and Plant Analysis , 47(22), 2547-2557. https://doi.org/10.1080/00103624.2016.1243710 Singh, P., Singh, M., Beg, Y. R., and Nishad, G. R. (2019). A review on spectroscopic methods for determination of nitrite and nitrate in environmental samples. Talanta , 191, 364-381. https://doi.org/10.1016/j.talanta.2018.08.028 Vernier (2023). Vernier NH4-BTA Ammonium Ion-Selective Electrode User manual. Yokota, M., Okada, T., and Yamaguchi, I. (2007). An optical sensor for analysis of soil nutrients by using LED light sources. Measurement Science and Technology , 18, 2197-2201. https://doi.org/10.1088/0957-0233/18/7/052 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 18 Sep, 2024 Read the published version in Environmental Monitoring and Assessment → Version 1 posted Editorial decision: Revision requested 09 May, 2024 Reviews received at journal 24 Apr, 2024 Reviews received at journal 23 Apr, 2024 Reviewers agreed at journal 02 Apr, 2024 Reviews received at journal 02 Apr, 2024 Reviewers agreed at journal 02 Apr, 2024 Reviewers agreed at journal 02 Apr, 2024 Reviewers agreed at journal 01 Apr, 2024 Reviewers invited by journal 01 Apr, 2024 Editor assigned by journal 27 Mar, 2024 Submission checks completed at journal 27 Mar, 2024 First submitted to journal 02 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4007169","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285521183,"identity":"2893a00b-c825-47e0-8287-bbc58c85a551","order_by":0,"name":"A. V. Garmay","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYHACAyR2BYhIIEnLGZK1MLYRoYV/dvO2Dx/bGOz6Z59O/Mw777C8OXvyMQnGPYdxapG4c6x45sw2huQZ53I3S/NuO2y4s+dZsgHDM9xaGG7kGDPzbvufzHCGdwNIC+OGGzmGDxgO4NYiD9LydxtDsvwZ3s2/eecctt9wI//DAXxaDEBaGLcx2Bmc4d0mzdtwOBFoCyNeWwxvpBUz9v5jSDAEarGccyw9ecOZZ8YGCQfScWqRu5G8meHHGQZ7OaDDbrypsbbdcDz5mcSHA9a4vQ8FiQ1AgomHoRnCTSCogYHBHkQw/mCoI0LtKBgFo2AUjDQAAEv9XNn14XPvAAAAAElFTkSuQmCC","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":true,"prefix":"","firstName":"A.","middleName":"V.","lastName":"Garmay","suffix":""},{"id":285521184,"identity":"fda0bfa4-db20-4217-b98c-340d80efccb2","order_by":1,"name":"K. V. Oskolok","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"K.","middleName":"V.","lastName":"Oskolok","suffix":""},{"id":285521185,"identity":"305beef8-138f-43a7-9a32-67c4775f04ac","order_by":2,"name":"O. V. Monogarova","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"O.","middleName":"V.","lastName":"Monogarova","suffix":""},{"id":285521186,"identity":"a85d7ef4-0506-431a-8d53-f8492dd8ab1f","order_by":3,"name":"M. I. Demidov","email":"","orcid":"","institution":"Lomonosov Moscow State University","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"I.","lastName":"Demidov","suffix":""}],"badges":[],"createdAt":"2024-03-03 00:29:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4007169/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4007169/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10661-024-13068-1","type":"published","date":"2024-09-18T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53890251,"identity":"81f2a954-e024-4fc0-a665-99c985d52235","added_by":"auto","created_at":"2024-04-01 20:56:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":76373,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curves “G-channel absorbance– nitrate N content, ppm” with standard solutions aliquots: (a) 0.50 mL; (b) 1.00 mL; (c) 1.50 mL\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4007169/v1/bbb9a5487422e250b7f5892a.png"},{"id":53890240,"identity":"a4f92dd8-d1d6-498e-a110-56a34b52be7c","added_by":"auto","created_at":"2024-04-01 20:56:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90446,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4007169/v1/d2e204c9779123e94234649a.png"},{"id":53890262,"identity":"8808f220-45c2-4b35-9fdd-cb9eb2696e4f","added_by":"auto","created_at":"2024-04-01 20:56:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60391,"visible":true,"origin":"","legend":"\u003cp\u003eCalibration curves “G-channel absorbance– nitrate N content, ppm” with standard solutions aliquot 1.50 mL, constructed using the concentrated coloring solution\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4007169/v1/367263781d046d0fb9c1998c.png"},{"id":53890252,"identity":"4eb76e33-a827-48b5-a2c4-44794338b280","added_by":"auto","created_at":"2024-04-01 20:56:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94732,"visible":true,"origin":"","legend":"\u003cp\u003eScatter diagram “nitrate N by spectrophotometry – nitrate N by digital colorimetry”. NRMSD ~ 4%, MAPE ~ 8%\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4007169/v1/f55bdf9a2723c64426f58643.png"},{"id":53890253,"identity":"ea45df66-0631-4102-a616-21b94c4b3530","added_by":"auto","created_at":"2024-04-01 20:56:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90117,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4007169/v1/248ed13c20629bd288d7411c.png"},{"id":53890260,"identity":"c5ec7c66-eca4-4006-86df-7fcbb72650a6","added_by":"auto","created_at":"2024-04-01 20:56:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":95034,"visible":true,"origin":"","legend":"\u003cp\u003eScatter diagram “ammonium N by spectrophotometry – ammonium N by digital colorimetry”. NRMSD ~ 4%, MAPE ~ 9%\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4007169/v1/b284194e4f15786830cf1b89.png"},{"id":65432682,"identity":"028f44f6-459d-494d-9b13-a961b4f687ad","added_by":"auto","created_at":"2024-09-27 12:06:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":897114,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4007169/v1/11a7efd2-6731-4325-a3c9-564392d7f501.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Determination of ammonium and nitrate in soils by digital colorimetry","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNitrogen (N) is a vital element for plant growth and crops productivity (Hachiya and Sakakibara \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Jose \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Saleem et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is acquired from soils mostly in the form of ammonium and nitrate (Hachiya and Sakakibara \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Natural soil N content is usually not enough to provide sufficient productivity (An et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, N fertilizers are used worldwide (An et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Saleem et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, ammonium and nitrate should be added together, since their sole use was shown to be less beneficial or even toxic for plants, and the maximum plants growth requires a certain ammonium-to-nitrate ratio depending on the species (Hachiya and Sakakibara \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Saleem et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). On the other hand, a great part of N is lost due to leaching (Fayose et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gurmesa et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mustafa, Hayat and Alotaibi \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The losses may vary from 10\u0026ndash;75% (Choosang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kabala et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This leads to ecological problems like the pollution of water systems or even greenhouse gases (N\u003csub\u003e2\u003c/sub\u003eO and NH\u003csub\u003e3\u003c/sub\u003e) production, and also causes a decrease in the economic efficiency of agriculture (An et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Choosang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fayose et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kabala et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mustafa, Hayat and Alotaibi \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For these reasons, determination of ammonium and nitrate content in soils is of great importance.\u003c/p\u003e \u003cp\u003eSeveral methods can be used for ammonium and nitrate determination, including Kjeldahl steam distillation techniques, ion chromatography, ion selective electrodes (ISE) and various spectrophotometric techniques (Choosang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Griffin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The Kjeldahl method is, however, hazardous, lengthy and labor intensive, taking several hours to analyze one sample (Śaez-Plaza et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although various automated Kjeldahl procedures are long known (Harwood and Huyser \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Lennox and Flanagan \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1982\u003c/span\u003e), usually it is not considered the method of choice. Ion chromatography and ISE methods face the same problem of the severe influence of interfering ions (Choosang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fayose et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Griffin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Indeed, since ammonium is strongly adsorbed by the soil adsorption complex (Bolt, Bruggenwert and Kamphorst \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1976\u003c/span\u003e), its extraction usually requires the use of concentrated salt solutions like 2M KCl (Griffin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Nitrate can be extracted from soil by water or diluted salt solutions, at least in some cases, but its low ionic strength can cause the dispersion of soil, resulting in cloudy filtrates. When both ammonium and nitrate are to be determined, 2M KCl extraction is considered an ideal extractant (Griffin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Therefore, photometric procedures are among the most common for ammonium and nitrate determination (Griffin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; L\u0026oacute;pez Pasquali, Fern\u0026aacute;ndez Hernando and Durand Alegrı́a \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Ammonium is usually determined by indophenol method, and nitrate is often determined by diazotation method after being reduced to nitrite (Bolleter, Bushman and Tidwell \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1961\u003c/span\u003e; L\u0026oacute;pez Pasquali, Fern\u0026aacute;ndez Hernando and Durand Alegrı́a \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Schmiedt Sattolo et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These methods are accepted as regulatory state standards in some countries (GOST 26488-85; GOST 26489-85; O\u0026rsquo;Dell \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Method 353.2; O\u0026rsquo;Dell \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Method 350.1).\u003c/p\u003e \u003cp\u003eThe disadvantage of spectrophotometry is, however, the higher cost of equipment as compared to ISE (Choosang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fayose et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nadporozhskaya et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Another challenging problem is in-field ammonium and nitrate determination (Choosang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fayose et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Heckman \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Heckman et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Kabala et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Portable colorimeters have been designed for these purposes, which are, however, quite expensive and are reported to be typically used for research purposes instead of practical in-field application (Choosang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). ISE are sometimes considered well-suited for in-field analysis (Fayose et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Choosang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nadporozhskaya et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but its use for ammonium determination remains questionable (Cuartero et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne of the promising solutions of the stated problems is a digital colorimetric method rapidly developing in the recent years (Christodouleas et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Choodum et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Oskolok et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Since it utilizes various consumer devices like smartphones and scanners for measuring analytical signal, its cost is in fact limited by the cost of chemicals and glassware (Oskolok et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although sometimes the low speed of spectrophotometric reactions could still be a problem, its influence on the overall analysis duration becomes less pronounced when numerous samples are analyzed. Indeed, ISE can take from 30 s up to several minutes for equilibration (Cheremisinoff \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; HACH \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Vernier \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), resulting in hours for several tens of samples. In digital colorimetry, the analysis can be performed faster by using multiwell plates allowing measuring several tens of samples simultaneously without cleaning and replacing cuvettes (Christodouleas et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Besides, it requires less sample volume, which can further decrease the overall cost of analysis (Oskolok et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). At last, smartphone-based digital colorimetry can be easily applied in-field, allowing one to conduct analyses with accuracy and precision close to those of spectrophotometric method at significantly lower cost (Christodouleas et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Choodum et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nadporozhskaya et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Manbohi and Ahmadi \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mohamed, Ismail and Ali \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDigital colorimetry has been reported to be used for both nitrate and ammonium N determination (Nadporozhskaya et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yokota, Okada and Yamaguchi \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, the original spectrophotometric techniques were used \u0026ldquo;as is\u0026rdquo;, despite the fact that the method is clearly less sensitive. Therefore, the aim of this work was to develop digital colorimetric techniques of ammonium and nitrate determination with accuracy and sensitivity close to those of the spectrophotometric method.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and equipment\u003c/h2\u003e \u003cp\u003eThe following reagents were used: potassium chloride, potassium iodide, sodium hydroxide, sodium pyrophosphate decahydrate, sodium salicylate, sodium nitroprusside dihydrate, ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA), sodium thiosulfate (Titrisol\u0026reg; concentrated standard), sodium hypochlorite water solution, potassium sodium tartrate tetrahydrate, copper (II) sulfate pentahydrate, hydrazine sulfate, 1-naphthylamine, sulfanilamide, hydrochloric acid 37% water solution, phosphoric acid 85% water solution. All the chemicals were purchased at Sigma Aldrich and were reagent grade. To determine the level of scattered radiation, liquid black ink was used (JSC \u0026ldquo;GAMMA\u0026rdquo;, Russian Federation).\u003c/p\u003e \u003cp\u003eTo prepare soil suspensions, a LOIP LS-120 (JSC \"Laboratory Equipment and Instruments\", Russian Federation) laboratory shaker was used.\u003c/p\u003e \u003cp\u003ePhotometric analysis was performed using a UV-mini 1240 spectrophotometer (Shimadzu, Japan). Images for digital colorimetric analysis were taken using an EPSON Perfection 1670 office flatbed photoscanner (Japan) in transmission mode. For this purpose, the solutions were placed into a 96-well transparent polystyrene microplate (Greiner AG, Austria) with a flat bottom (well volume 350 \u0026micro;L). A special frame was put into the scanner that protected the microplate from ambient light. The images were saved as 48-bit *.tiff files, with no scanner post-processing being used. For processing the images, the free \u003cem\u003eIrfanView\u003c/em\u003e application was applied (IrfanView \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExtraction of ammonium and nitrate from soil samples\u003c/h2\u003e \u003cp\u003eSoil extracts were prepared as described in (GOST 26488-85; GOST 26489-85). 16,00\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01 g of soil were put into a conical flask, then 40,0 ml of 2M potassium chloride solution were added. The suspension was shaken for 1 h and filtered through a grade 2 filter paper (Whatman \u0026reg;). At the first stage, standard samples of soddy-podzolic medium loamy soils, alkaline and saline soils, podzolized medium loamy chernozemic soils and carbonate deep chernozemic soils were analyzed by the digital colorimetric method to estimate its accuracy. Then, the method was applied along with the standard spectrophotometric procedures for the analysis of soil samples to compare their metrological characteristics. About 250 samples were analyzed during 2022\u0026ndash;2023 years. The samples were collected in the Moscow region.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNitrate determination\u003c/h2\u003e \u003cp\u003eSince only a few mL of a colored solution are required for the analysis, the volumes of the analyzed solutions and of the solution of the reagents were reduced 10 times compared to the original procedure (GOST 26488-85). 0.500 mL of the analyzed solution was added to a 5.0 mL test-tube and mixed with 1.00 mL of sodium pyrophosphate solution and 1.00 mL of hydrazine sulfate solution. Sodium pyrophosphate solution was prepared by dissolving 5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g of sodium pyrophosphate and 8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g of sodium hydroxide in 1.00 L of deionized water. To prepare hydrazine sulfate solution, 5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g of hydrazine sulfate were dissolved in several mL of deionized water in a 1.00 L flask, then 6.00 mL of 0.01 M copper sulfate solution were added, and the solution was diluted to the mark with deionized water. 10 minutes after adding sodium pyrophosphate solution, 2.50 mL of a coloring solution was added. The coloring solution was prepared by dissolving 5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g of sulfanilamide, 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g of 1-naphthylamine, 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g of EDTA and 100 mL of phosphoric acid in 1.00 L of deionized water, followed by diluting of the obtained solution 5 times with deionized water. The color develops in 15 minutes after adding the coloring solution and is stable for 1.5 h. The overall scheme of the analysis is following:\u003c/p\u003e \u003cp\u003eIn order to adapt the original procedure to the digital colorimetric method, we also considered using the concentrated coloring solution without dilution. The aliquot of the analyzed solution was also variated. See Section 3 for details.\u003c/p\u003e \u003cp\u003eFor digital colorimetric analysis, 320 \u0026micro;L of each sample were poured into a 96-well plate. One well was filled with 2M KCl solution as a blank sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAmmonium determination\u003c/h2\u003e \u003cp\u003eSince only a few mL of a colored solution are required for the analysis, the volumes of the analyzed solutions and of the solution of the reagents were reduced 10 times compared to the original procedure (GOST 26489-85). 0.200 mL of the analyzed solution were added to a 5.0 mL test-tube and mixed with 4.00 mL of a coloring solution and 0.200 mL of 0.125% sodium hypochlorite solution. To prepare the coloring solution, 14.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g of sodium salicylate, 5.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g of potassium sodium tartrate tetrahydrate and 6.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g of sodium hydroxide were dissolved in 180 mL of water and boiled for 20 minutes to remove ammonia. After cooling the solution, 0.1000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0002 g of sodium nitroprusside and 5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g of EDTA were added, and the solution was diluted to 0.2500 L by deionized water. For analysis, the solution should be diluted 10 times with deionized water. The overall scheme can be presented as follows:\u003c/p\u003e \u003cp\u003e0.125% sodium hypochlorite solution was prepared by dilution of the original solution with deionized water. Concentration of the original solution was established by iodometric titration as described in (GOST 26489-85) and occurred to be 1.7%.\u003c/p\u003e \u003cp\u003eIn order to adapt the original procedure to the digital colorimetric method, we also considered varying the aliquot of the analyzed solution. See Section 3 for details.\u003c/p\u003e \u003cp\u003eFor digital colorimetric analysis, 320 \u0026micro;L of each sample were poured into a 96-well plate. One well was filled with 2M KCl solution as a blank sample.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNitrate determination\u003c/h2\u003e \u003cp\u003eThe first question about nitrate determination was whether the original spectrophotometric procedure was suitable for digital colorimetric analysis. Therefore, calibration curves were constructed using the scanner according to the procedure described in the first paragraph of Section 2.3. Eight nitrate solutions were prepared with N concentration equal to 0; 1.0; 2.0; 4.0; 6.0; 8.0; 10.0 and 12.0 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, corresponding to nitrate N content in soil varying from 2.5 to 30 ppm. Five points were selected for each well of the 96-well plate and averaged. Absorbance \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e for the \u003cem\u003ei\u003c/em\u003e\u003csup\u003eth\u003c/sup\u003e color channel was calculated using a formula\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$${A}_{i}=\\text{lg}\\left(\\frac{{L}_{blank,i}-{L}_{scatter,i}}{{L}_{sample,i}-{L}_{scatter,i}}\\right),$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eL\u003c/em\u003e\u003csub\u003eblank, \u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the lightness of the \u003cem\u003ei\u003c/em\u003e\u003csup\u003eth\u003c/sup\u003e color channel for a blank sample, \u003cem\u003eL\u003c/em\u003e\u003csub\u003escatter, \u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the scattered radiation level for the \u003cem\u003ei\u003c/em\u003e\u003csup\u003eth\u003c/sup\u003e color channel (the lightness of the \u003cem\u003ei\u003c/em\u003e\u003csup\u003eth\u003c/sup\u003e color channel of the well filled with black ink), and \u003cem\u003eL\u003c/em\u003e\u003csub\u003esample, \u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the lightness of the \u003cem\u003ei\u003c/em\u003e\u003csup\u003eth\u003c/sup\u003e color channel for a well filled with a sample.\u003c/p\u003e \u003cp\u003eThe most significant variations were observed for the green channel of the RGB-space. The curve is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. Although it is linear, the absorbances do not exceed 0.14. Moreover, the lowest concentration with a non-zero absorbance is 4.0 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, which is 4 times higher than the limit of quantitation of the standard method (GOST 26488-85). Assuming that this can be overcome by increasing nitrate concentration, we tried to construct calibration curves adding 1.00 and 1.50 mL of calibration solutions instead of 500 \u0026micro;L used earlier (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, b, c). In these cases, absorbances were statistically higher than zero for 2.0 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e for 1.00 mL aliquot and for 1.0 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e for 1.50 mL aliquot. However, the curves\u0026rsquo; linearity occurred to be violated, beginning with 10.0 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e solution, although the corresponding absorbances did not exceed 0.27. This effect was more pronounced for 1.50-mL aliquot of the nitrate solution. Curves constructed using a spectrophotometer demonstrated a similar behavior, reaching a plateau at high concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This indicates that, most probably, the coloring solution concentration is not high enough, and exceeding diazo intermediates recombine with water.\u003c/p\u003e \u003cp\u003eTherefore, we decided to use the concentrated coloring solution without dilution. The aliquot was 1.50 mL. The corresponding calibration curve is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It is linear up to 12.0 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, which agrees with the standard method (GOST 26488-85). Therefore, we applied these aliquot and coloring solution concentration values for the following measurements.\u003c/p\u003e \u003cp\u003eTo test the validity of the resulting colorimetric technique, several standard soil samples were analyzed. Some results are given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Relative errors fall well within the nominal accuracy of the method, and the relative standard deviation values are also satisfactory.\u003c/p\u003e \u003cp\u003eAt the next step, the colorimetric technique was applied for various soil samples analysis along with the standard spectrophotometric method (GOST 26488-85). The corresponding scatter diagram is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The normalized root mean square deviation (NRMSD) between the two datasets was about 4%, and the mean absolute percentage error (MAPE) was about 8%, which is well within the limits specified by the standard method (GOST 26488-85).\u003c/p\u003e \u003cp\u003eSince nitrate is readily extracted from soil in several minutes (Griffin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), the developed technique is possibly applicable for in-field nitrate determination, although it requires following investigation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe results of nitrate N determination in some soil standards (P\u0026thinsp;=\u0026thinsp;0.95; n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eNitrate N content, ppm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRelative error, %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCertified\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMeasured\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoddy-podzolic heavy loamy soil\u003c/p\u003e \u003cp\u003eSADPP-07/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoddy-podzolic medium loamy soil\u003c/p\u003e \u003cp\u003eSADPP-10/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbonate deep chernozem\u003c/p\u003e \u003cp\u003eSACHkP-08/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePodzolized medium loamy chernozem\u003c/p\u003e \u003cp\u003eSACHopP-04/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e91.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e85.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAmmonium determination\u003c/h2\u003e \u003cp\u003eIn case of ammonium determination, we decided to take into account the results of the previous section and, therefore, we started with constructing six calibrations, the aliquot of standard ammonium solutions being varied from standard to double and the concentration of the hypochlorite solution varied from standard (0.125%) to double (0.25%) and original (1.7%). The standard solutions contained 0; 2.0; 4.0; 8.0; 12.0; 16.0; 20.0; 24.0 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e of ammonium N, corresponding to ammonium N content in soil varying from 5 to 60 ppm. A double aliquot of the ammonium solutions led to increased calibration slopes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). When a standard hypochlorite aliquot was applied, the lowest points (2 and 4 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) absorbances were negligible. Increased hypochlorite concentration led to increased absorbance and calibration slopes. However, for the original solution, absorbances were slightly lower than for 0.25% solution, and the lowest points in this case were also negligible. Therefore, we applied 0.25% solution for further measurements.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test the validity of the resulting colorimetric technique, several standard soil samples were analyzed. Some results are given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Relative errors fall well within the nominal ac-curacy of the method, and the relative standard deviation values are also satisfactory.\u003c/p\u003e \u003cp\u003eThe next stage was an optimization of the reaction time. For this purpose, the images of the standard solutions after adding all the necessary reagents were taken each 10 minutes beginning with the 5th minute of reaction. It was found that the maximum absorption is reached by the 25th minute and then remains constant for at least an hour independently of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration.\u003c/p\u003e \u003cp\u003eAt the next step, the colorimetric technique was applied for various soil samples analysis along with the standard spectrophotometric method (GOST 26489-85). The corresponding scatter diagram is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The normalized root mean square deviation between the two datasets was about 4%, and the mean absolute percentage error was about 9%, which is well within the limits specified by the standard method (GOST 26489-85).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe results of ammonium N determination in some soil standards (P\u0026thinsp;=\u0026thinsp;0.95; n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAmmonium N content, ppm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRelative error, %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCertified\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMeasured\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoddy-podzolic heavy loamy soil\u003c/p\u003e \u003cp\u003eSADPP-07/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSaline soil\u003c/p\u003e \u003cp\u003eSAZP-2011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbonate deep chernozem\u003c/p\u003e \u003cp\u003eSACHkP-08/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePodzolized medium loamy chernozem\u003c/p\u003e \u003cp\u003eSACHopP-04/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e10.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs for in-field ammonium determination, the limiting stage will obviously be its extraction from soil, requiring at least an hour of shaking its suspension. On the other hand, ISE-based techniques will face the same problem. Therefore, in-field ammonium determination requires further research.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe standard methods (GOST 26488-85; GOST 26489-85) of nitrate and ammonium N determination were successfully adapted for the colorimetric method. Using a scanner instead of a spectrophotometer allows to decrease the cost of the analysis and to measure several tens of samples at once due to the use of a multiwell plate. Taking the images by a scanner in transmission mode and taking into account scattered radiation makes it possible to achieve an accuracy close to that of the spectrophotometric method. Nitrate in-field determination is potentially available by the developed technique, since NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e ions are readily extracted from soil in several minutes (Griffin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). On the other hand, in-field ammonium determination in soils require following investigation to find optimal extraction conditions, if they even exist.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll relevant data are available upon request from the authors.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by M.V. Lomonosov Moscow State University (No. AAAA-A21-121011590089-1 \u0026ldquo;Development of highly informative and high-tech methods of chemical analysis for the protection of ecosystems, the creation of new materials and advanced production technologies, the transition to environmentally friendly and resource-saving energy, the development of nature-like technologies, high-tech healthcare and rational use of natural resources\u0026rdquo;).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eK.V. Oskolok and O.V. Monogarova developed a modified digital colorimetric method, including consideration of scattered light. A.V. Garmay and M.I. Demidov modified the reaction conditions to provide the required sensitivity and dynamic range and performed most of the experiments. A.V. Garmay wrote the main manuscript text and prepared figures and tables. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAn, T., Wang, F., Ren, L., Ma, Sh., Li, Sh., Liu, L., and Wang, J. (2022). Ratio of nitrate to ammonium mainly drives soil bacterial dynamics involved in nitrate reduction processes. \u003cem\u003eApplied Soil Ecology\u003c/em\u003e, 169, 104164. https://doi.org/10.1016/j.apsoil.2021.104164\u003c/li\u003e\n\u003cli\u003eBolleter, W. T., Bushman, C. J., and Tidwell, P. W. (1961). Spectrophotometric Determination of Ammonia as Indophenol. \u003cem\u003eAnalytical Chemistry\u003c/em\u003e 33(4), 592-594. https://doi.org/10.1021/ac60172a034\u003c/li\u003e\n\u003cli\u003eBolt, G. H., Bruggenwert, M. G. M., and Kamphorst, A. (1976). Adsorption of Cations by Soil. In G. H. Bolt, M. G. M. Bruggenwert (Editors), \u003cem\u003eDevelopments in Soil Science\u003c/em\u003e (Vol. 5A, pp. 54-90). 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An optical sensor for analysis of soil nutrients by using LED light sources. \u003cem\u003eMeasurement Science and Technology\u003c/em\u003e, 18, 2197-2201. https://doi.org/10.1088/0957-0233/18/7/052\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-monitoring-and-assessment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"emas","sideBox":"Learn more about [Environmental Monitoring and Assessment](http://link.springer.com/journal/10661)","snPcode":"10661","submissionUrl":"https://submission.nature.com/new-submission/10661/3","title":"Environmental Monitoring and Assessment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"digital colorimetry, soil, nitrate, ammonium","lastPublishedDoi":"10.21203/rs.3.rs-4007169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4007169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA method of digital colorimetric determination of ammonium and nitrate in soils is proposed. The method is based on corresponding photometric techniques of ammonium and nitrate determination after potassium chloride extraction from soils samples. Ammonium is determined as an indophenol dye, and nitrate is determined as an azo dye. The original techniques were modified to overcome the lower sensitivity of the digital colorimetric method. For ammonium determination, the time required for the reaction to proceed completely was studied. Along with the use of a 96-well microplate protected from ambient light by a special frame, mathematical correction of scattered radiation using black ink and taking the images by a scanner in transmission mode without any post-processing, the resulting colorimetric techniques proved to provide accuracy and sensitivity close to those of the spectrophotometric techniques, and the overall analysis speed for tens of samples was even higher. The techniques\u0026rsquo; validity was proven by the analysis of standard samples and by the analysis of soil samples collected in several districts of the Moscow region.\u003c/p\u003e","manuscriptTitle":"Determination of ammonium and nitrate in soils by digital colorimetry","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-01 20:56:09","doi":"10.21203/rs.3.rs-4007169/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-09T17:02:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-24T18:43:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-23T20:25:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36d9b573-3797-4831-8506-da6252d9b628","date":"2024-04-02T15:43:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-02T07:09:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"766c5395-1362-45ba-bb2d-c195921c9327","date":"2024-04-02T05:48:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c29cf619-3210-4ed3-b232-5a1dc6544b48","date":"2024-04-02T05:01:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86a989e8-17f8-4dfc-8ec4-ca7b32417e97","date":"2024-04-02T03:12:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-01T18:33:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-27T18:28:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-27T18:28:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Monitoring and Assessment","date":"2024-03-03T00:19:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-monitoring-and-assessment","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"emas","sideBox":"Learn more about [Environmental Monitoring and Assessment](http://link.springer.com/journal/10661)","snPcode":"10661","submissionUrl":"https://submission.nature.com/new-submission/10661/3","title":"Environmental Monitoring and Assessment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6e2f7de0-6b26-4f99-a72e-9e5f91891fac","owner":[],"postedDate":"April 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-27T10:46:56+00:00","versionOfRecord":{"articleIdentity":"rs-4007169","link":"https://doi.org/10.1007/s10661-024-13068-1","journal":{"identity":"environmental-monitoring-and-assessment","isVorOnly":false,"title":"Environmental Monitoring and Assessment"},"publishedOn":"2024-09-18 15:57:15","publishedOnDateReadable":"September 18th, 2024"},"versionCreatedAt":"2024-04-01 20:56:09","video":"","vorDoi":"10.1007/s10661-024-13068-1","vorDoiUrl":"https://doi.org/10.1007/s10661-024-13068-1","workflowStages":[]},"version":"v1","identity":"rs-4007169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4007169","identity":"rs-4007169","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}