Review Reports - The Ability of Nitrification Inhibitors to Decrease Denitrification Rates in an Arable Soil

Round 1

Reviewer 1 Report

Notes and comments in the attached file

Comments for author File: Comments.pdf

Author Response

Responses to the Comments of Editors and Reviewers (Manuscript ID: agronomy-2007894)

The manuscript has been revised according to the suggestions and comments the reviewers have made, and the point-by-point responses are as follows:

Reviewer 1

Comments: Line 92-96. The soil carbon availability was subsequently assessed. Briefly, the soil sample (35g) was placed in a 1.8-L glass preserving jar, which was then sealed with a septum stopper-fitted lids. After incubation at 25ºC for 7 days, the accumulation of CO₂ in the headspace atmosphere of the preserving jar was evaluated using an Infrared gas analyzer as an indicator of soil respiration. What does the concept of available carbon mean and how can it be defined through CO₂ emissions? How much is it possible to determine?

Response: Available C is just a concept, meaning available C for microorganisms to use in the soil. There are several ways to determine it, but all determinations only give indications and for comparison between treatments. CO₂ (soil respiration rate) is an indirect way to suggest how C in the soil is available to microorganisms to use. Other ways to determine availability of C include water or hot water extraction (Csol), 0.5 M K₂SO₄- extractable C (similar to KCl for mineral N) (Cext), aerobically respired C (Cresp), water - soluble phenolic acids (Cphen) and potentially available C (Cpot). Available C could have a lot of chemical forms of C (such as glucose….).

Comments: Line 166-169. The degradation rate of DMPP. How was it determined?

Response: Thank you for the suggestion. The DMPP analysis has been included in the revised manuscript (Line 144-160 in the revised manuscript). ‘DMPP extraction and determination followed the protocol developed by Benckiser et al. (2013) and Chen et al. (2019). Briefly, 5 g of soil (on an oven-dried basis) was homogenized with 5 ml of ultrapure water and 0.1 ml 1 MK₃PO₄ in a 50 ml centrifuge tube and then shaken for 2 h at 250 rpm. Afterwards, 0.1 ml 1 M CaCl₂ was added, followed by shaking for 1 h at 250 rpm. For the disproportionation of DMPP into dimethylpyrazole (DMP), 0.5 ml of 1 M NaOH was added, followed by 30 min of further shaking. In order to transfer DMP into t-butyl-methyl-ether (MTBE) phase, 7.5 ml of MTBE was added followed by 1-h-shaking at 200 rpm. Subsequently, the DMP-containing MTBE was separated from the aqueous phase by adding 6.5 g water-free Na₂SO₄ and mixing on a vortex. Samples were centrifuged for 5 min at 3000 rpm and 5 ml of supernatant (DMP-containing MTBE) was collected in glass bottles. Afterwards, 5 ml of supernatant and 1.5 ml of HPLC eluent were evaporated at room temperature for 5 min using a rotary evaporator IKA RV8 (IKA®-Werke GmbH & Co. KG, Germany), and the final homogenate (1.5 ml) was filtered through a 0.45 μm membrane filter. DMP, disproportionated from DMPP, was quantified by a Shimadzu HPLC (Shimadzu, Japan) using a 5 μm, 4.6 × 250 mm Shiseido SpolarC18 column (Shiseido, Japan)’.

Cited References

Benckiser, G., Christ, E., Herbert, T., Weiske, A., Blome, J., Hardt, M., 2013. The nitrification inhibitor 3,4-dimethylpyrazole-phosphat (DMPP) - quantification and effects on soil metabolism. Plant Soil 371, 257–266.

Chen, H., Yin, C., Fan, X., Ye, M., Peng, H., Li, T., Zhao, Y., Wakelin, S.A., Chu, G., Liang, Y., 2019. Reduction of N₂O emission by biochar and/or 3,4-dimethylpyrazole phosphate (DMPP) is closely linked to soil ammonia oxidizing bacteria and nosZI-N₂O reducer populations. Science of the Total Environment, 694 (2019), 133658.

Comments: Line 111. Denitrifying enzyme activity (DEA). What did you end up measuring? What enzymes do they refer to? Would it have been better to determine the activity of each enzyme?

Response: The short-term assay of denitrifying enzyme activity (DEA) has been developed for measuring the enzyme activity of the denitrifier population in soils when samples are collected (Luo et al. 1989; Tiedje et al. 1989; Watkins et a., 2013). The DEA provides a ‘snap-shot’ picture of the denitrifying potentials in the soil at the time of sampling. DEA is based on the principal that if conditions are optimized for enzyme catalysed reactions, the reaction rate will be proportional to the enzyme concentration in the soil. The technique involves anaerobic incubation of soil samples and the measurement of N₂O emission in the presence of C₂H₂. To avoid any effect of growth of denitrifying organisms on the DEA, the incubation period should not exceed 5 h at 20°C. The K_m value for substrate NO₃^- concentrations was in the range 21-38 μg NO₃^--N g^-1 soil, but NO₃^--N concentrations > 100 μg N g^-l soil inhibited production of N₂O. The optimum concentrations for NO₃^--N and glucose-C were 50 and 300 μg g^-1 soil, respectively. Field-moist samples of soil retained their DEA during 5 days’ storage at either 2 or 20°C, but thereafter the DEA declined with time. The denitrification activity of air-dried soil increased relative to fresh soil after 1 week’s storage, but subsequently declined. The effects were generally consistent with a change in the availability of substrate C in the stored soil (moist or air-dry), but there also appeared to be a decline in the potential activity of the soil denitrifier populations after 5-7 days’ storage (Line 135-143 in the revised manuscript).

Cited References

Tiedje, J.M.; Simkins, S.; Groffman, P.M. Perspectives on measurement of denitrification in the field including recommended protocols for acetylene based methods. Plant Soil. 1989, 115, 261284.

Luo, J.; White, R.E.; Ball R.P.; Tillman, R.W. Measuring denitrification activity in soils under pasture: optimizing conditions for the short-term denitrification enzyme assay and effects of soil storage on denitrification activity. Soil Biol. Biochem. 1996, 28 (3), 409-417.

Watkins, N.L.; Schipper, L.A.; Sparlinga, G.P.; Thorroldb, B.; Balks, M. Multiple small monthly doses of dicyandiamide (DCD) did not reduce denitrification in Waikato dairy pasture. New Zeal. J. Agri. Res. 2013, 56 (1), 37-48.

Comments: 2.6. Statistical analysis The differences in soil biochemical parameters were analyzed by two-way ANOVA was the SPSS Statistics 16.0 (SPSS Inc., Chicago, USA). The paper does not show the results of statistical processing. There are only a few references to the level of reliability. We need a detailed multivariate statistical analysis of the results. Probably better to present them in the form of a table. All conclusions and regularities obtained during the work should be confirmed by statistical analysis.

Response: Thank you for the comments, all the data has been gone through the statistical analysis according to the standard process. It is hard to show all the data in tables, but to clarify the meaning, we have shown them in the figures and results. The statistical values have been included in the results and discussion parts (Line 166-266 in the revised manuscript).

Comments: Line 175. As enzyme activity declined under conditions of constant NO₃^--N concentration…Why make this decision?

Response: Thank you, the unrelated information has been deleted.

Comments: Line 188-189 A remarkable decline (81%) in the concentrations of inhibitors was observed on day 5 after inhibitor applications, which was predominantly due to microbial degradation (Figure 2f). How can you prove this decision?

Response: Thank you for the suggestion, DMPP degradation in the field was measured to establish how long the effects of DMPP might last, thus we measured the DMPP concentrations to know how much DMPP was lost by leaching, sorption to soil organic matter or microbial degradation. To clarify the meaning, the wording has been changed to ‘A remarkable decline (81%) in the concentrations of inhibitors was observed on day 5 after inhibitor applications, which was probably due to microbial degradation, leaching or sorption to soil organic matter (Figure 2f).’ (Line 144-160, 235-239 in the revised manuscript).

Comments: Line 217 More studies are needed to clarify the molecular mechanisms underlying the inhibitory effects of nitrification inhibitor on denitrification rates. Maybe it's worth doing this research or cite data from the literature?

Response: Thank you for the suggestion. Indeed, this study is a first step in the evaluation the ability of nitrification inhibitors to decrease denitrification rates in the field study. A long-term field study is currently being conducting to further evaluate the effects of nitrification inhibitors to limit denitrification under different sites and climate conditions and clarify the molecular mechanisms underlying the inhibitory effects of nitrification inhibitors on ammonia oxidizers and denitrifies (Line 265-266 in the revised manuscript).

Comments: Results and 4. Discussion You need to write in more detail using statistics

Response: Thank you for the comments, the statistical values have been checked and included in the results and discussion part in the manuscript (Line 166-266 in the revised manuscript).

Comments: Line 219-227.5. Conclusions. Very short and uninformative conclusions.

Response: The conclusion has been revised and made sure to present more information of the manuscript. We have summarized and analyzed the results of this study again and have concluded that ‘In this study, the denitrification rates and denitrifying enzyme activities were highly variable in different growing period, but were not affected by the application of inhibitors. Partial inhibition of nitrification process was observed, as revealed by an increase in NH₄⁺-N concentration and a decrease in NO₃^--N concentration in the inhibitor treatments compared with the urea or manure-only treatments. However, the decrease in NO₃^--N was not sufficient enough to limit NO₃^--N availability to denitrifiers, and thus, denitrification rates were found to not decrease. SMB, soil pH and microbial respiration were not affected by nitrification inhibitors regardless of whether manure or urea was applied in the soil.’ (Line 267-275 in the revised manuscript).

Comments: Figure 1 and 2. Poor quality of drawings, it is very difficult to make out the variants of the experience.

Response: To clarify the meaning, the figures have been revised according to the suggestions.

Comments: A very small list of references for such a complex topic of a scientific article.

Response: Thank you for the comments, all the references have been checked and more new ones have been included in the manuscript (Line 289-357 in the revised manuscript).

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

Detailed notes on the manuscript are as follows:

1) The introduction to the manuscript is insufficient. They should be expanded and additional literature items included.

2) In the introduction (final part), the purpose of the work should be specified; … Monitored the dynamics ..

3) 2.6. Statistical analysis - The 5% confidence level; the notation should be p <0.05

4) 2.6. Statistical analysis - What kind of ANOVA was used - parametric or nonparametric? If parametric ANOVA, are the conditions for its application met (normality of the distribution, homogeneity of variance, etc.)

5) 2.6. Statistical analysis - Pearson's correlation?

6) Figure 1 Denitrification rate ... - in my opinion, the data in the figure should not be combined (after all, it is not known what is happening between 2 points; the authors did not forecast the data)

7) Figure 2 Soil microbial biomass ... - as above

8) 5. Conclusions - the title should be changed to "Summary"

9) Additional literature should be included. There is a wealth of literature on soil nitrification / denitrification. Take advantage of the works published after 2017.

Author Response

Responses to the Comments of Editors and Reviewers (Manuscript ID: agronomy-2007894)

The manuscript has been revised according to the suggestions and comments the reviewers have made, and the point-by-point responses are as follows:

Reviewer 2

Comments: The introduction to the manuscript is insufficient. They should be expanded and additional literature items included.

Response: Thank you for the valuable comments. We have considered your comments carefully and revised our manuscript according to the suggestions (Line 28-83 in the revised manuscript). Nitrification inhibitors were originally intended to improve N retention in soil by blocking the microbial oxidation of ammonium to nitrate. However, the inhibitors also have the potential to alter other components of the nitrogen cycle such as denitrification. If widespread use of nitrification inhibitor is to be encouraged to reduce NO₃^- leaching and/or N₂O emissions, it is important to know what other aspects of the N cycle are affected by this compound. Denitrification is dependent on anaerobic conditions in the soil, available carbon and a sufficient supply of nitrate to act as electron acceptor. We were interested to test whether DMPP would decrease denitrification (and hence N₂O emissions) by limiting the nitrate supply from arable soils, where N fertilizer and cattle manure are the major sources of N. To our knowledge, animal manure applications would input large amounts of metabolizable C, mineral N, and water into soil simultaneously, which may favor both the nitrification and denitrification processes. Previous researches have mainly focused on changes in the soil N mineralization process that may influence the dynamics of soil inorganic N and the soil N cycle, lacking comparison with denitrification rates from other N fertilizers. Moreover, observations of denitrification process under various fertilizers with DMPP application during the whole maize growing period was lack. Hence, a field experiment was performed to determine whether the DMPP can affect denitrification rates by limiting NO₃^- availability. Therefore, the specific objectives of this study were to (1) clarify the characteristics of denitrification rates affected by nitrification inhibitor from different N fertilizers types applied to soils; (2) explore the influencing factors of the dynamics in annual denitrification rates (e.g. DMPP concentrations, soil properties). This will provide insightful information for our understanding of the achievement of inhibitors on the mitigation of N losses in arable soil under field conditions.

Comments: In the introduction (final part), the purpose of the work should be specified; … Monitored the dynamics .

Response: Thank you for the suggestion, the specific objectives of this study were to (1) clarify the characteristics of denitrification rates affected by nitrification inhibitor from different N fertilizers types applied to soils; (2) explore the influencing factors of the dynamics in annual denitrification rates (e.g. DMPP concentrations, soil properties). The related information has been included in the revised manuscript (Line 68-83 in the revised manuscript).

Comments: 2.6. Statistical analysis - The 5% confidence level; the notation should be p <0.05

Response: The wording has been changed to ‘The differences in soil biochemical parameters were analyzed by two-way ANOVA was the SPSS Statistics 16.0 (SPSS Inc., Chicago, USA). The 5% confidence level (p < 0.05) was considered statistically different (Line 161-165 in the revised manuscript).

Comments: 2.6. Statistical analysis - What kind of ANOVA was used - parametric or nonparametric? If parametric ANOVA, are the conditions for its application met (normality of the distribution, homogeneity of variance, etc.)

Response: Thank you for your helpful suggestion, yes, we did the statistical analysis according to the standard process, all the conditions for its application met (Line 161-165 in the revised manuscript).

Comments: 2.6. Statistical analysis - Pearson's correlation?

Response: Yes, to clarify the meaning, the wording has been modified to ‘Pearson correlation analysis was employed to analyze the relationships between soil properties and denitrification rates (161-165 in the revised manuscript).

Comments: Figure 1 Denitrification rate ... - in my opinion, the data in the figure should not be combined (after all, it is not known what is happening between 2 points; the authors did not forecast the data)

Response: Thank you for the comments, we did not combine the data in the figures, hourly denitrification rates were presented at each sampling time in the figure 1. The field measurements of in situ denitrification rates were made using the static soil core incubation system. Briefly, the intact soil cores consisting of PVC pipe with uniformly distributed holes were isolated and transferred into a glass preserving jar sealed with septum stopper-fitted lids. Then, 120 ml acetylene (10% v/v of headspace) was placed in the jars, mixed thoroughly, and kept in a temperature-controlled room. Gases (22 ml) were collected from the jars at 30 min, 3, 6 and 24 h following acetylene addition, and then injected into a vacutainer until further use. All samples were analyzed using a gas chromatography system (Philips, USA) equipped with an electron capture detector at 350ºC. The accumulation of N₂O in the presence of acetylene represents the total production of N₂O and N₂ from denitrification. Hourly denitrification rates were calculated from headspace concentration at each sampling time, and corrected for the solubility of N₂O in the soil water using the temperature-dependent Bunsen absorption coefficients (Line 122-134 in the revised manuscript).

Comments: Figure 2 Soil microbial biomass ... - as above

Response: Thank you for the comments, we did not combine the data in the figure 2, soil microbial biomass were analyzed and presented at each sampling time in the figure 2.

Comments: Conclusions - the title should be changed to "Summary"

Response: The wording has been modified accordingly (Line 267 in the revised manuscript).

Comments: Additional literature should be included. There is a wealth of literature on soil nitrification / denitrification. Take advantage of the works published after 2017.

Response: We have considered your comments carefully and revised our manuscript thoroughly according to the suggestions. The new additional literatures have been included in the revised manuscript (Line 289-357 in the revised manuscript).

Author Response File: Author Response.docx

Reviewer 3 Report

Refers to the manuscript: agronomy-2007894: The ability of nitrification inhibitors to decrease denitrification rates in an arable soil.

The manuscript presented for review concerns research on the influence of nitrification inhibitors on the rate of denitrification in arable soils. The issues discussed in it are not new, and the influence of nitrification inhibitors on soil nitrogen losses is widely known, which has been published in many reputable journals. Therefore, in the present state, there are not enough new findings to justify the publication of this manuscript. In my humble opinion, I do not recommend it for publication in its current form.

Comments:

1. The abstract should be more precise, detailing the purpose, concept and methodology of the research. The abstract of a good journal article always ends with an outline of the benefits of the results obtained and recommendations as a solution to the presented problem. There is no such information in the presented abstract.

2. Keywords should not repeat phrases contained in the title of the manuscript: „Nitrification inhibitors; Denitrification rate; Arable soil”. Please change it.

3. The authors reviewed very old literature. But what innovations will be introduced to the literature in this article? Therefore, the current state of knowledge should be clearly and detailed in the introduction. A hypothesis must be made. How is this work different from available literature? The last paragraph of the introductory part always emphasizes the innovative aspects of the experiments carried out with a clear purpose and significance of the results obtained. The introduction lacks goals, which is unacceptable. The manuscript introduction should be expanded and redrafted to provide a more comprehensive approach.

4. Chapter Materials and Methods: point 2.1 and 2.2 - information on the properties of the tested soil and the fertilizers and additives used, should be collected in the Table.

5. Chapter Results:

· Please provide detailed statistical verification of the obtained results.

· The presented figures are hardly legible. Maybe they should be made in color?

· Point 3.3: please correct “losees” to “losses”.

6. The conclusions need to be significantly improved. In my humble opinion, the conclusions must be convincing statements about what was found to be innovative, strongly supporting the results and discussions. They should highlight the main results of the work. As it stands, the conclusions presented are obvious and expected.

7. Please check and correct any grammatical errors in the entire manuscript carefully.

The authors supported the conducted research with very old references (1984-2008). After 2008, have there been any studies on the topic presented? Or maybe everything is already explained in this topic? In my opinion, the presented manuscript requires very major corrections.

Author Response

Responses to the Comments of Editors and Reviewers (Manuscript ID: agronomy-2007894)

The manuscript has been revised according to the suggestions and comments the reviewers have made, and the point-by-point responses are as follows:

Reviewer 3

General Comments: The manuscript presented for review concerns research on the influence of nitrification inhibitors on the rate of denitrification in arable soils. The issues discussed in it are not new, and the influence of nitrification inhibitors on soil nitrogen losses is widely known, which has been published in many reputable journals. Therefore, in the present state, there are not enough new findings to justify the publication of this manuscript. In my humble opinion, I do not recommend it for publication in its current form.

Response: Thank you very much for giving us the opportunity to revise our manuscript. We very much appreciated the insightful and constructive comments you provided on our manuscript. We have carefully revised the whole manuscript accordingly. Nitrification inhibitors were originally intended to improve N retention in soil by blocking the microbial oxidation of ammonium to nitrate. However, the inhibitors also have the potential to alter other components of the nitrogen cycle such as denitrification. If widespread use of nitrification inhibitor is to be encouraged to reduce NO₃^- leaching and/or N₂O emissions, it is important to know what other aspects of the N cycle are affected by this compound. Denitrification is dependent on anaerobic conditions in the soil, available carbon and a sufficient supply of nitrate to act as electron acceptor. We were interested to test whether DMPP would decrease denitrification (and hence N₂O emissions) by limiting the nitrate supply from arable soils, where N fertilizer and cattle manure are the major sources of N. To our knowledge, animal manure applications would input large amounts of metabolizable C, mineral N, and water into soil simultaneously, which may favor both the nitrification and denitrification processes. Previous researches have mainly focused on the effects of nitrification inhibitors (e.g., DCD, nitripyrin) on changes in the soil N mineralization process that may influence the dynamics of soil inorganic N and the soil N cycle, lacking comparison with denitrification rates from other N fertilizers and nitrification inhibitor (DMPP). Moreover, observations of denitrification process under various fertilizers with DMPP application during the whole maize growing period was lack. Hence, a field experiment was performed to determine whether the DMPP can affect denitrification rates by limiting NO₃^- availability. Therefore, the specific objectives of this study were to (1) clarify the characteristics of denitrification rates affected by nitrification inhibitor from different N fertilizers types applied to soils; (2) explore the influencing factors of the dynamics in annual denitrification rates (e.g. DMPP concentrations, soil properties). This will provide insightful information for our understanding of the achievement of inhibitors on the mitigation of N losses in arable soil under field conditions.

Comments: The abstract should be more precise, detailing the purpose, concept and methodology of the research. The abstract of a good journal article always ends with an outline of the benefits of the results obtained and recommendations as a solution to the presented problem. There is no such information in the presented abstract.

Response: Thank you for the suggestion, we have summarized and analyzed the results of this study again and improved the abstract section in the revision. ‘This study investigated the dynamics in annual denitrification rates affected by nitrification inhibitors from a maize field for the first time. Our observations showed that denitrification and denitrification enzyme activity were highly variable and no significant effect of DMPP in decreasing denitrification was detected. Our results concluded that the formation of NO₃^--N and nitrification rates could be markedly reduced by DMPP, while NO₃^--N availability did not affect denitrification rates. Our results may ultimately help to clarify the characteristics of denitrification rates affected by nitrification inhibitor from different N fertilizers types applied to soils and explore the influencing factors of the dynamics in annual denitrification rates denitrification rates. However, more field studies on evaluating the effectiveness of nitrification inhibitors in reducing denitrification under different sites and climate conditions and molecular mechanisms driving denitrification rates changes need to be explored in the future’ (Line 8-27 in the revised manuscript).

Comments: Keywords should not repeat phrases contained in the title of the manuscript: „Nitrification inhibitors; Denitrification rate; Arable soil”. Please change it.

Response: Thank you for the suggestions, the keywords have been changed to ‘denitrification process, inhibitors, N transformation, nitrification rates’ (Line 29-30 in the revised manuscript).

Comments: The authors reviewed very old literature. But what innovations will be introduced to the literature in this article? Therefore, the current state of knowledge should be clearly and detailed in the introduction. A hypothesis must be made. How is this work different from available literature? The last paragraph of the introductory part always emphasizes the innovative aspects of the experiments carried out with a clear purpose and significance of the results obtained. The introduction lacks goals, which is unacceptable. The manuscript introduction should be expanded and redrafted to provide a more comprehensive approach.

Response: Thank you for the suggestion. Nitrification inhibitors were originally intended to improve N retention in soil by blocking the microbial oxidation of ammonium to nitrate. However, the inhibitors also have the potential to alter other components of the nitrogen cycle such as denitrification. We were interested to test whether DMPP would decrease denitrification (and hence N₂O emissions) by limiting the nitrate supply from arable soils, where N fertilizer and cattle manure are the major sources of N. To our knowledge, animal manure applications would input large amounts of metabolizable C, mineral N, and water into soil simultaneously, which may favor both the nitrification and denitrification processes. Previous researches have mainly focused on the effects of nitrification inhibitors (e.g., DCD, nitripyrin) on changes in the soil N mineralization process that may influence the dynamics of soil inorganic N and the soil N cycle, lacking comparison with denitrification rates from other N fertilizers and nitrification inhibitor (DMPP). Moreover, observations of denitrification process under various fertilizers with DMPP application during the whole maize growing period was lack. Hence, a field experiment was performed to determine whether the DMPP can affect denitrification rates by limiting NO₃^- availability. Therefore, the specific objectives of this study were to (1) clarify the characteristics of denitrification rates affected by nitrification inhibitor from different N fertilizers types applied to soils; (2) explore the influencing factors of the dynamics in annual denitrification rates denitrification rates (e.g. DMPP concentrations, soil properties). This will provide insightful information for our understanding of the achievement of inhibitors on the mitigation of N losses in arable soil under field conditions. We have revised the introduction section in lines 31-83 in the revision.

Comments: Chapter Materials and Methods: point 2.1 and 2.2 - information on the properties of the tested soil and the fertilizers and additives used, should be collected in the Table.

Response: Thank you for the comments, the information has been presented in Table 1 as suggested.

Comments: Chapter Results: Please provide detailed statistical verification of the obtained results. The presented figures are hardly legible. Maybe they should be made in color? Point 3.3: please correct “losees” to “losses”.

Response: The p values have been shown in result section, as the following 6 places. (1) The increased DEA in the urea or manure-amended soils was most obvious on day 5 after the fertilizer application and decreased with time (p < 0.05, Figure 1 b) (Line 177 in the revised manuscript). (2) DEA was higher in manure-amended soils than other treatments (p < 0.05) (Line 178 in the revised manuscript). (3) The manure-amended soils had an increase in SMB compared with the urea-amended soils on day 5 after manure application (p < 0.05) (Line 186 in the revised manuscript). (4) The manure-amended soils demonstrated an obvious reduction in C availability over the sampling period (p < 0.05) (Line 190 in the revised manuscript). (5) After application of NPK and manure soils, the concentrations of NH₄⁺-N and NO₃^--N were first increased and then declined. In the urea and manure treatments, the addition of inhibitors could lead to higher soil NH₄⁺-N concentrations compared to the soil without inhibitors (Figure 2d, p < 0.05) (Line 200 in the revised manuscript). (6) The manure-amended soils with inhibitors also had a lower NO₃^--N content compared to those without inhibitors between 45 and 70 days after fertilization (p < 0.05) (Line 203 in the revised manuscript). The figures are revised according to the suggestion (Figure 1 and 2). The wording has been changed to ‘losses’ (Line 213 in the revised manuscript).

Comments: The conclusions need to be significantly improved. In my humble opinion, the conclusions must be convincing statements about what was found to be innovative, strongly supporting the results and discussions. They should highlight the main results of the work. As it stands, the conclusions presented are obvious and expected.

Response: The conclusion has been revised and made sure to present more information of the manuscript (Line 267-276 in the revised manuscript). We have summarized and analyzed the results of this study again and have concluded that ‘In this study, the denitrification rates and denitrifying enzyme activities were highly variable in different growing period, but were not affected by the application of inhibitors. Partial inhibition of nitrification process was observed, as revealed by an increase in NH₄⁺-N concentration and a decrease in NO₃^--N concentration in the inhibitor treatments compared with the urea or manure-only treatments. However, the decrease in NO₃^--N was not sufficient enough to limit NO₃^--N availability to denitrifiers, and thus, denitrification rates were found to not decrease. SMB, soil pH and microbial respiration were not affected by nitrification inhibitors regardless of whether manure or urea was applied in the soil.’

Comments: Please check and correct any grammatical errors in the entire manuscript carefully. The authors supported the conducted research with very old references (1984-2008). After 2008, have there been any studies on the topic presented? Or maybe everything is already explained in this topic? In my opinion, the presented manuscript requires very major corrections.

Response: Thank you for the suggestion, the language in the revised manuscript has been polished up thoroughly by Elsevier language Editing Services. The new additional literatures have been included in the revised manuscript (Line 289-357 in the revised manuscript). Previous researches have mainly focused on the effects of nitrification inhibitors (e.g., DCD, nitripyrin) on changes in the soil N mineralization process that may influence the dynamics of soil inorganic N and the soil N cycle, lacking comparison with denitrification rates from other N fertilizers and nitrification inhibitor (DMPP). Moreover, observations of denitrification process under various fertilizers with DMPP application during the whole maize growing period was lack. Hence, a field experiment was performed to determine whether the DMPP can affect denitrification rates by limiting NO₃^- availability. (Line 31-83 in the revised manuscript).

Author Response File: Author Response.docx

Reviewer 4 Report

Material and methods need to explain DMPP degradation. The authors have a figure showing the time course DMPP degradation.
Why do they use Corn in the field? There is no data on growth or productivity associated with the treatments used. They do sampling from 5 to 160 days.
They perform Correlation analysis during the data analysis (data not shown, Why?).
In the discussion section, "In this study, multiple-stepwise regression analysis revealed........" where are they? Do the authors confuse correlation with regression?
In the discussion section, "More studies are needed to clarify the molecular mechanisms underlying the inhibitory effects of nitrification inhibitor on denitrification rates". The authors need more data before undertaking molecular studies.

Author Response

Responses to the Comments of Editors and Reviewers (Manuscript ID: agronomy-2007894)

The manuscript has been revised according to the suggestions and comments the reviewers have made, and the point-by-point responses are as follows:

Reviewer 4

Comments: Material and methods need to explain DMPP degradation. The authors have a figure showing the time course DMPP degradation.

Response: Thank you for the comments, the material and methods to analysis DMPP concentration have been included in the revised manuscript. DMPP extraction and determination followed the protocol developed by Benckiser et al. (2013). Briefly, 5 g of soil (on an oven-dried basis) was homogenized with 5 ml of ultrapure water and 0.1 ml 1 MK₃PO₄ in a 50 ml centrifuge tube and then shaken for 2 h at 250 rpm. Afterwards, 0.1 ml 1 M CaCl₂ was added, followed by shaking for 1 h at 250 rpm. For the disproportionation of DMPP into dimethylpyrazole (DMP), 0.5 ml of 1 M NaOH was added, followed by 30 min of further shaking. In order to transfer DMP into t-butyl-methyl-ether (MTBE) phase, 7.5 ml of MTBE was added followed by 1-h-shaking at 200 rpm. Subsequently, the DMP-containing MTBE was separated from the aqueous phase by adding 6.5 g water-free Na₂SO₄ and mixing on a vortex. Samples were centrifuged for 5 min at 3000 rpm and 5 ml of supernatant (DMP-containing MTBE) was collected in glass bottles. Afterwards, 5 ml of supernatant and 1.5 ml of HPLC eluent were evaporated at room temperature for 5 min using a rotary evaporator IKA RV8 (IKA®-Werke GmbH & Co. KG, Germany), and the final homogenate (1.5 ml) was filtered through a 0.45 μm membrane filter.’ (Line 144-160 in the revised manuscript).

Comments: Why do they use Corn in the field? There is no data on growth or productivity associated with the treatments used. They do sampling from 5 to 160 days.

Response: Thank you for the comments. Maize is one of the major cereal crops in Northeast China, its yield accounting for 62% of local grain products and 26% of national maize production. Maize cultivation also has been identified as one of the main leading source of anthropogenic N₂O emission, since excessive inputs of chemical or organic fertilizers, thus we did the study in maize field. As the reviewer emphasized, gaseous loses, plant growth, yield and soil properties may also have been influenced by inhibitors. We did do these analyses in this study, but for this manuscript, we mainly focused on the ability of nitrification inhibitors to decrease denitrification rates, thus we chose the related parameters to present. Following such valuable suggestions, we are going to perform long-term field studies to further evaluate the effects of nitrification inhibitors to limit denitrification under different sites and climate conditions and clarify the molecular mechanisms underlying the inhibitory effects of nitrification inhibitors on ammonia oxidizers and denitrifies (Line 26-27, 265-266 in the revised manuscript).

Comments: They perform Correlation analysis during the data analysis (data not shown, Why?). In the discussion section, "In this study, multiple-stepwise regression analysis revealed........" where are they? Do the authors confuse correlation with regression?

Response: In this study, Pearson correlation analysis was employed to analyze the relationships between soil properties and denitrification rates. The results revealed that the effect of inhibitors on denitrification rates was not markedly associated with DEA, soil NH₄⁺-N, NO₃^--N soil pH and carbon availability. Following the valuable suggestions, the data has been included in the revised manuscript (Line 208-211 in the revised manuscript, Table 2).

Table 2. Pearson correlation analysis of denitrification rates and soil properties

Variable factors	NPK		NPK+DMPP			Manure		Manure + DMPP
Variable factors	R²	P		R²	P	R²	P	R²	P
DEA	0.121	0.532		0.247	0.064	0.178	0.074	0.674	0.421
NH₄⁺-N	0.378	0.126		0.498	0.079	0.452	0.145	0.546	0.178
NO₃^--N	0.236	0.214		0.216	0.142	0.312	0.126	0.201	0.097
pH	0.145	0.145		0.347	0.078	0.147	0.245	0.394	0.076
Carbon availability	0.189	0.321		0.421	0.231	0.325	0.365	0.414	0.069

Comments: In the discussion section, "More studies are needed to clarify the molecular mechanisms underlying the inhibitory effects of nitrification inhibitor on denitrification rates". The authors need more data before undertaking molecular studies.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have corrected all the comments. I am satisfied with the answers.

Author Response

Responses to the Comments of Editors and Reviewers (Manuscript ID: agronomy-2007894)

The manuscript has been revised according to the suggestions and comments the reviewers have made, and the point-by-point responses are as follows:

Reviewer 1

Comments: The authors have corrected all the comments. I am satisfied with the answers.

Response: Thank you for the encouraging comments and the valuable suggestions.

Reviewer 2 Report

Dear Authors,

Thank you for considering my comments. In my opinion, the introduction should be even more elaborate. There is still a lack of the latest literature on the topic.

Author Response

Responses to the Comments of Editors and Reviewers (Manuscript ID: agronomy-2007894)

The manuscript has been revised according to the suggestions and comments the reviewers have made, and the point-by-point responses are as follows:

Reviewer 2

Comments: Thank you for considering my comments. In my opinion, the introduction should be even more elaborate. There is still a lack of the latest literature on the topic.

Response: Thank you for the valuable comments. Following the suggestions, we have revised our manuscript and added new additional literatures according to the suggestions (Line 40-91, 299-304, 329-339 in the revised manuscript).

Reviewer 3 Report

1. In the second version of the manuscript presented for review: agronomy-2007894-peer-review-v2, the sentence:

„This study investigated the dynamics in annual denitrification rates affected by nitrification inhibitors from a maize field for the first time. Our observations showed that denitrification and denitrification enzyme activity were highly variable and no significant effect of DMPP in decreasing denitrification was detected. Our results concluded that the formation of NO3--N and nitrification rates could be markedly reduced by DMPP, while NO3--N availability did not affect denitrification rates.”

it has not been included in the abstract. Line 25: please remove the duplicate words (“denitrification rates”).

2. In response to the improvement in the introduction, the authors informed me that they had made changes to lines 31-83. In the presented second version of the manuscript, slight changes are visible only on lines 66-83. Only the supplementation of the introduction with the objectives of the presented research speaks in favor of the authors.

3. The conclusions section has been replaced with a summary. The authors did not improve this chapter by adding a very short sentence (lines 268-269).

The only arguments in favor of the authors are the addition of a few new literature items.

Author Response

Responses to the Comments of Editors and Reviewers (Manuscript ID: agronomy-2007894)

The manuscript has been revised according to the suggestions and comments the reviewers have made, and the point-by-point responses are as follows:

Reviewer 3

Comments: In the second version of the manuscript presented for review: agronomy-2007894-peer-review-v2, the sentence ‘This study investigated the dynamics in annual denitrification rates affected by nitrification inhibitors from a maize field for the first time. Our observations showed that denitrification and denitrification enzyme activity were highly variable and no significant effect of DMPP in decreasing denitrification was detected. Our results concluded that the formation of NO3--N and nitrification rates could be markedly reduced by DMPP, while NO3--N availability did not affect denitrification rates.”it has not been included in the abstract. Line 25: please remove the duplicate words (“denitrification rates”).

Response: Thank you for the valuable suggestions. The wording has been included in the abstract (Line 13, 15-16, 21-22 in the revised manuscript). The duplicate wording ‘denitrification rates’ has been deleted.

Comments: In response to the improvement in the introduction, the authors informed me that they had made changes to lines 31-83. In the presented second version of the manuscript, slight changes are visible only on lines 66-83. Only the supplementation of the introduction with the objectives of the presented research speaks in favor of the authors.

Response: Thank you for the valuable comments. Following the suggestions, we have further revised our manuscript and added new additional literatures according to the suggestions (Line 40-76, 299-304, 329-339 in the revised manuscript).

Comments: The conclusions section has been replaced with a summary. The authors did not improve this chapter by adding a very short sentence (lines 268-269). The only arguments in favor of the authors are the addition of a few new literature items.

Response: The conclusion has been revised and made sure to present more information of the manuscript. ‘The results showed that the denitrification rates and denitrifying enzyme activities were highly variable in different growing period, but were not affected by the application of inhibitors. Partial inhibition of nitrification process was observed, as revealed by an increase in NH₄⁺-N concentration and a decrease in NO₃^--N concentration in the inhibitor treatments compared with the urea or manure-only treatments. However, the decrease in NO₃^--N was not sufficient enough to limit NO₃^--N availability to denitrifiers, and thus, denitrification rates were found to not decrease. SMB, soil pH and microbial respiration were not affected by nitrification inhibitors regardless of whether manure or urea was applied in the soil. Our results concluded that the formation of NO₃^--N and nitrification rates could be markedly reduced by DMPP, while NO₃^--N availability did not affect denitrification rates. Furthermore, to confirm the findings of this study, field studies under different sites to explore more mechanisms driving changes over longer time periods are needed (Line 265-277 in the revised manuscript).

Reviewer 4 Report

No comments on the authors modifications.

Author Response

Responses to the Comments of Editors and Reviewers (Manuscript ID: agronomy-2007894)

The manuscript has been revised according to the suggestions and comments the reviewers have made, and the point-by-point responses are as follows:

Reviewer 4

Comments: No comments on the authors modifications.

Response: Thank you for the previous valuable suggestions.