Review Reports - Estimation of Effective Cation Exchange Capacity and Exchangeable Iron in Paddy Fields After Soil Flooding

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

good efforts

Comments for author File: Comments.pdf

Author Response

Reviewer # 1:

Add statistical validation for the proposed estimation equations.

The validation of the proposed subject is in the theoretical basis used in the manuscript. It is well established that the standard exchangeable Fe and CEC evaluation method does not apply to flooded soils, as it underestimates the values. Therefore, one can not use it as a reference, and since no replication was used in the soil samples, no statistical test can be applied.

This question was already addressed in the first round of corrections in response to the former reviewers' query. Due to the complexity of the soil solution collection process, it was not possible to perform replications in the soils. The idea was that covering a greater number of different soils would be similar to having replications (i.e., the various soils) and would give an idea of how often the new method improves Fe and CEC measurements. Therefore, no deviations or error bars can be generated.

Justify or verify the constancy of Ca and Mg after flooding.

In order to justify the stability of Ca and Mg, the following was added: “Despite of what occurs with Fe, there is no formation of new exchangeable Ca and Mg after flooding, but a change in the ratio of cation in the solution/exchangeable cation. Adding the concentrations of exchangeable and soluble Ca before and after flooding yields the same total values, as does adding the concentrations of exchangeable and soluble Mg. The average sum of Ca-exchangeable + Ca-solution was 10.07 cmol_c kg^-1 before flooding and 10.09 cmol_c kg^-1 after flooding. Regarding Mg, the average Mg-exchangeable + Mg-solution was 4.09 cmol_c kg^-1 before flooding and 4.10 cmol_c kg^-1 after flooding

Include standard deviations or error bars in all figures and tables.

Response: This question was already explained in the first round of corrections from the former reviewers' query. Due to the complexity of the soil solution collection process, it was not possible to perform replications in the soils. The idea was that covering a greater number of different soils would be similar to having replications (i.e., the various soils) and would give an idea of how often the new method improves Fe and CEC measurements. Therefore, no deviations or error bars can be generated.

Correct minor typographical and grammatical errors.

Response: A general grammar review was made of the entire text.

Expand discussion on the applicability and limitations of the proposed model.

Response: The following paragraph was added: “The results generated allowed a better understanding of the main cations exchangeable fractions and in solution, involved in the nutrition of rice and iron toxicity, enabling perspectives of studying this nutritional disturbance as a way to predict its occurrence really efficiently.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

This manuscript entitled Estimation of effective cation exchange capacity and exchangeable iron in paddy fields after soil flooding presents a new methodology to estimate the exchangeable Fe²⁺ and the effective CEC of flooded soils. A total of 21 surface soil samples from rice fields were collected, flooded for 50 days and the soil solution was collected and analysed for pH, Na, K, Ca, Mg, Fe and Mn contents. While the topic is interesting, however, some aspects can be improved. Details are shown below.

Abstract present mainly the background of the study, the aims and the methodology. But the main results and findings are missing. Please rewrite this to make the abstract self-explanatory for the entire manuscript.
In the introduction, please explains the role of CEC determination in soil, and possible implications for metals accumulation or mobility in soil (please see doi 10.1080/10934529.2012.650583)
Materials and Methods. Please provide information regarding the storage and transportation of soil samples from the field to laboratory
Materials and Methods. Soil analysis. Please better explains how the sum of K, Na, Ca, Mg, Mn and Al are converted to CEC value.
Materials and Methods. Lines 130-131 “…the determination of Na and K in the extracts was performed by flame photometry, the determination of Ca, Mg and Mn by atomic absorption 131 spectrophotometry” Please provide the models and producers name of the used equipment
Please provide the analytical quality control to ensure results reliability
Conclusions section should be improved

Author Response

Reviewer # 2: Comments and Suggestions for Authors

Abstract present mainly the background of the study, the aims and the methodology. But the main results and findings are missing. Please rewrite this to make the abstract self-explanatory for the entire manuscript.

Response: the description of several results and also a few comments of Reviewer 3 were added. The conclusions presented relate satisfactorily to the main goals of the manuscript.

In the introduction, please explains the role of CEC determination in soil, and possible implications for metals accumulation or mobility in soil (please see doi 10.1080/10934529.2012.650583)

Response: One paragraph in the introduction was added concerning this issue: “The CEC is an important index of soil nutrient availability [10], acts on cation mobility throughout the soil profile and is also linked to aggregate formation and stability.

Materials and Methods. Please provide information regarding the storage and transportation of soil samples from the field to laboratory

Response: the experiment was conducted in the laboratory. Therefore, there was no need to further transport the collected samples. The solution samples were immediately acidified and the soil samples were placed in contact with the extractors soon after collecting, as is already mentioned in the text. The procedure was described already: “After collection, the samples were air-dried, sieved through a 4 mm mesh and stored in plastic bags.”

Materials and Methods. Soil analysis. Please better explain how the sum of K, Na, Ca, Mg, Mn and Al are converted to CEC value.

Response: One sentence was added in the text: “The sum of cations, when correctly expressed in charge units (cmol_c kg^-1), represents the total amount of positive charges that the soil can retain, which is the practical definition of CEC”.

Materials and Methods. Lines 130-131 “…the determination of Na and K in the extracts was performed by flame photometry, the determination of Ca, Mg and Mn by atomic absorption 131 spectrophotometry” Please provide the models and producers name of the used equipment

Response: A sentence describing both equipments was added: “ELEVELAB Scientific Equipment, model FP-6400, and GBC Scientific Equipment, model GBC-SAVANTAA)

Please provide the analytical quality control to ensure results reliability

Response: A paragraph concerning this query was added: In order to guarantee the result accuracy, the evaluations were performed adding control samples to the pool of samples evaluated. Besides, the soil analysis lab from our Department is included in the quality control program led by the South Regional Nucleus of the Brazilian Society of Soil Science.

7. Conclusions section should be improved

Response: Considering that “ this paper aims to propose an alternative for estimating exchangeable Fe²⁺and ECEC after flooding”, the conclusions are adequate to the manuscript’s main goal. Adjustments suggested by reviewer # 3 were added.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

1.Line 9–16, The innovation is not sufficiently emphasized. The abstract should explicitly state *why* existing methods fail (e.g., “traditional CEC extraction oxidizes Fe²⁺/Mn²⁺, causing underestimation”) and how the proposed model overcomes this. Rephrase to highlight the novelty: *“We introduce a pH-gradient-based model to predict ECEC and exchangeable Fe²⁺ in flooded soils, circumventing oxidation artifacts inherent in conventional methods.”*

2.Line 83–89, The assumption that ECEC post-flooding can be inferred from pH shifts lacks theoretical grounding. Cite literature establishing the linear relationship between pH-dependent charge and CEC (e.g., Barrow & Hartemink 2023). Additionally, justify why Ca²⁺/Mg²⁺ are assumed stable despite displacement into the solution (Lines 324–326), referencing cation selectivity studies (e.g., Saeki et al. 2004).

3. Line 103–114, The addition of corn straw (2 t ha⁻¹) to accelerate reduction is described, but its potential impact on nutrient dynamics (e.g., K⁺ release) is not addressed. Specify whether straw was chemically characterized (C/N ratio) and discuss how this might influence cation measurements.
Lines 200-211, The increase in Fe²⁺ concentration (avg. 0.06 → 3.19 mmol L⁻¹) is attributed solely to reduction. However, pH-driven desorption from variable-charge sites also contributes (Lines 279–281). Integrate this mechanism to explain why low-Fe soils (e.g., Soil 5) exhibit smaller pH shifts.

5.Lines 399-403, The claim of a “linear relationship between ECEC and CEC pH 7.0” is unsupported by data. Include a scatter plot (ECEC vs. CEC pH 7.0 for all 21 soils) with R² values in supplementary material. If nonlinearity exists, address its impact on Eq. 1’s accuracy.

6.Lines 414-418, Equations in the conclusion are split awkwardly (e.g., Eq. 1 spans lines 414–416). Reformat as single-line equations for readability. Also, define all variables in-text (e.g., “where pH_sol.before = pre-flooding soil pH”).

7.Figures 2–5 lack statistical annotations (e.g., error bars, significance letters). Given the ANOVA/Duncan test mention (Line 197), include these to clarify variability between soils and treatments. For Figure 5, differentiate between “analyzed” and “estimated” ECEC/Fe²⁺ to visually reinforce the study’s core correction.

Comments on the Quality of English Language

The quality of English language could be improved to more clearly express the research.

Author Response

Reviewer # 3: Comments and Suggestions for Authors

Line 9–16, The innovation is not sufficiently emphasized. The abstract should explicitly state *why* existing methods fail (e.g., “traditional CEC extraction oxidizes Fe²⁺/Mn²⁺, causing underestimation”) and how the proposed model overcomes this. Rephrase to highlight the novelty: *“We introduce a pH-gradient-based model to predict ECEC and exchangeable Fe²⁺ in flooded soils, circumventing oxidation artifacts inherent in conventional methods.”*

Response: This suggestion was partially accepted. The following sentence was added to the abstract: ”We introduce a pH-gradient-based model to predict ECEC and exchangeable Fe²⁺ in flooded soils, circumventing oxidation artifacts inherent in conventional methods.”

Regarding the first query of the reviewer (The abstract should explicitly state *why* existing methods fail), we understand that our point is made very clear in the abstract (“However, these reduced forms of Mn and Fe are oxidized and precipitated during the extraction process used by traditional methods for determining CEC. This procedure underestimates the exchangeable portion of these cations and, consequently, the CEC value of the flooded soil”) and that adding this statement again would be redundant.

Line 83–89, The assumption that ECEC post-flooding can be inferred from pH shifts lacks theoretical grounding. Cite literature establishing the linear relationship between pH-dependent charge and CEC (e.g., Barrow & Hartemink 2023). Additionally, justify why Ca²⁺/Mg²⁺ are assumed stable despite displacement into the solution (Lines 324–326), referencing cation selectivity studies (e.g., Saeki et al. 2004).

Response: In the Introduction, a sentence was added regarding the linearity of the relationship: “The linear relationship between pH-dependent charge and CEC was established by Barrow & Hartemink [10]”

In order to justify the stability of Ca and Mg, the following was added: “Despite of what occurs with Fe, there is no formation of new exchangeable Ca and Mg after flooding, but a change in the ratio of cation in the solution/exchangeable cation. Adding the concentrations of exchangeable and soluble Ca before and after flooding, result in the same total values, and the same occurs for Mg . The average sum of Ca-exchangeable + Ca-solution was 10.07 cmol_c kg^-1 before flooding and 10.09 cmol_c kg^-1 after flooding. Regarding Mg, the average Mg-exchangeable + Mg-solution was 4.09 cmol_c kg^-1 before flooding and 4.10 cmol_c kg^-1 after flooding.

Line 103–114, The addition of corn straw (2 t ha⁻¹) to accelerate reduction is described, but its potential impact on nutrient dynamics (e.g., K⁺ release) is not addressed. Specify whether straw was chemically characterized (C/N ratio) and discuss how this might influence cation measurements.

Response: The following statements were added: “the effect of corn straw must be restrained to redox reaction stimuli, since the nutrient contents present in the straw are not sufficient to significantly alter exchangeable contents. Considering K for example, if the corn straw does present 3% of K, this amount would correspond to a 0.08 cmo_c kg K, which is a very diminute value when compared to total exchangeable cations in the soils studied.

4. Lines 200-211, The increase in Fe²⁺ concentration (avg. 0.06 → 3.19 mmol L⁻¹) is attributed solely to reduction. However, pH-driven desorption from variable-charge sites also contributes (Lines 279–281). Integrate this mechanism to explain why low-Fe soils (e.g., Soil 5) exhibit smaller pH shifts.

Response: The increase in Fe in the solution occurs due to the reduction process. The addition of corn straw provided sufficient carbon for the redox process in soils with lower organic matter (soil 5). Therefore, the soil 5 presented the lowest soil pH values after flooding, since this soil presented the lowest Fe contents going through reduction (Fe extracted with ammonium oxalate at pH 6.0 was 2.7 mmol kg^-1). Since the iron reduction was more minor, there was less consumption of H⁺ comparatively to the other soils, making the pH stable at lower values. The redox reactions consume H+ ions^, so the pH increases with flooding.

5. Lines 399-403, The claim of a “linear relationship between ECEC and CEC pH 7.0” is unsupported by data. Include a scatter plot (ECEC vs. CEC pH 7.0 for all 21 soils) with R² values in the supplementary material. If nonlinearity exists, address its impact on Eq. 1’s accuracy.

Response: a scatter plot ECEC vs. CEC pH 7.0 for all 21 soils was included in the supplementary material. The graph presented a linear tendency with and R² = 0.988

6. Lines 414-418, Equations in the conclusion are split awkwardly (e.g., Eq. 1 spans lines 414–416). Reformat as single-line equations for readability. Also, define all variables in-text (e.g., “where pH_sol.before = pre-flooding soil pH”).

Response: The corrections were performed directly in the abstract.

7. Figures 2–5 lack statistical annotations (e.g., error bars, significance letters). Given the ANOVA/Duncan test mention (Line 197), include these to clarify variability between soils and treatments. For Figure 5, differentiate between “analyzed” and “estimated” ECEC/Fe²⁺ to visually reinforce the study’s core correction.

Corrections on Figure 5, as suggested by reviewer #3.

Comments on the Quality of English Language

The quality of English language could be improved to more clearly express the research.

Response: The English grammar was reviewed throughout the text.

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript was improved and can be accepted for publication.

Author Response

Your consideration of our manuscript for publication is greatly appreciated.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Flood is the second threat after drought to agricultural production. It is necessary to determine the concentration of soil cations to understand the change process of CEC and other ions under flooding conditions. However, traditional methods may underestimate CEC, improving determine method would give us provide more accurate results to know the real situation of flooding soil. The word of this manuscript is very important and interesting. In my opinion, the objective of this manuscript is comparing the difference between the results of traditional and improving determine method of soil ions under flooding conditions. However, I did not find the contents about the objective in the Results Section.

Text: the expression of ions is incorrect, such as Fe2+, which should be Fe^2+, and same problem exists for other ions. Effective CEC is suggested to express as ECEC.

Methods: Table should be followed after text.

Results: all study results should be described with significant analysis of F test and Duncan test, including tables.

Discussion: discussion on the topic is necessary and needs to be supplemented.

Conclusions: try to display the mechanism of main findings in the study rather repeat some results.

Figure 1: the boundary is not clear, especially the study region of this MS.

Figure 7 and 10: improve the resolutions of the images.

Table 2: the positions of pH, Mn and Fe are suggested to adjust to the center of the columns.

Author Response

Response to reviewer #1:

- Flood is the second threat after drought to agricultural production. It is necessary to determine the concentration of soil cations to understand the change process of CEC and other ions under flooding conditions. However, traditional methods may underestimate CEC, improving determine method would give us provide more accurate results to know the real situation of flooding soil. The word of this manuscript is very important and interesting. In my opinion, the objective of this manuscript is comparing the difference between the results of traditional and improving determine method of soil ions under flooding conditions. However, I did not find the contents about the objective in the Results Section.

Response: We agree regarding the importance of the present work, since it is focused on filling a gap on the evaluation of exchangeable Fe and a ECEC in soils subjected to flooding. However, we should explain that the goal of the present work is not to make a simple comparison between traditional and improved method, as commented by reviewer 1. This comparison is not needed because it is already known that traditional methods underestimate exchangeable Fe and CEC values, since the soil sample gets in contact with oxygen and the Fe that was in the reduced form (Fe²⁺) and, therefore, soluble, assumes an insoluble oxidized form (Fe³⁺). Thus, the objective of the present work is to propose an alternative to correct the error in the exchangeable Fe and CEC measurements resulting from Fe oxidation. The working hypothesis and the objective were described in the last paragraph of the introduction. Since this was clear in the introduction section, we found that it was unnecessary to repeat it in the results section.

- Text: the expression of ions is incorrect, such as Fe2+, which should be Fe^2+, and same problem exists for other ions. Effective CEC is suggested to express as ECEC.

Response: The term was corrected throughout the text. The cations had their valences shown as superscript and Effective CEC was changed to ECEC throughout the text.

- Methods: Table should be followed after text.

Response: We are not sure about this request. Table 1 is the only table in the item Material and Methods and it was placed right below the paragraph where it was cited.

- Results: all study results should be described with significant analysis of F test and Duncan test, including tables.

Response: it is not possible to run F and Duncan tests, since the complexity and involved in the process of soil solution collection, it was not possible to perform replications in the soils. The idea was that cover a higher number of different soils would work similar to having replications (i.e., the different soils) and would give and idea of how often the new method causes an improvement in the Fe and CEC measurements.

- Discussion: discussion on the topic is necessary and needs to be supplemented.

Response: We tried to separate results and discussion. However, given the nature of the results in the present article, the new text became very tedious and repetitive. Therefore, if the journal guidelines allow, we would prefer to keep these sections united as Results and Discussion.

Conclusions: try to display the mechanism of main findings in the study rather repeat some results.

Response: the section conclusions was rewritten.

= Figure 1: the boundary is not clear, especially the study region of this MS.

Response: the figure was refined and there was a significant improvement in its definition and the size of the font used.

- Figure 7 and 10: improve the resolutions of the images.

Response: There is no figure 7 and 10 in the manuscript. However, the resolution of all figures was improved.

- Table 2: the positions of pH, Mn and Fe are suggested to adjust to the center of the columns.

Response: Table 2 was transformed in a figure according to the suggestions of one of the other reviewers.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

MS: Estimation of effective cation exchange capacity and exchangeable iron in paddy fields after soil flooding

I have carefully read the entire manuscript and provide the following comments for your consideration:

-It is crucial to have a native English speaker review and edit the language throughout the MS.

-I am not entirely clear on the necessity of conducting this study. In my view, the determination of CEC and exchangeable Fe is not particularly difficult, and the relationship between pH and CEC has already been extensively investigated in previous studies.

-As the authors stated, iron toxicity is one of the most important abiotic stresses limiting rice yields. However, this threat is mostly confined to strongly acidic soils, while other types of soils are not significantly affected. This raises the question of whether the focus on Fe²⁺ is necessary, and whether the authors should have been more selective in choosing their study soils.

-In my opinion, instead of investing time and effort in measuring the concentrations of Ca, Mg, Fe, and Mn, it would have been more direct and informative to simply measure Fe²⁺ itself.

-Figure 1 needs improvement. Currently, the text in the figure is too small, and the image resolution is too low, making it difficult to discern the information the authors intend to present.

-Table 2 appears distorted. The alignment of pH, Mn, and Fe data within the table needs to be corrected.

-The "Results and Discussion" section must be divided into smaller, clearly titled subsections. As it stands, it is difficult to follow the authors' line of reasoning and the flow of information.

-More importantly, the manuscript lacks a critical discussion of the findings. A dedicated Discussion section must be added to interpret the results more thoroughly.

-The Conclusion section is currently inadequate. The authors need to revise it to succinctly summarize the main findings of the study, covering the majority of the data presented.

-References. It would be best to consult the specific guidelines provided by the journal for instructions on formatting and referencing.

Author Response

Response to reviewer # 2

- -It is crucial to have a native English speaker review and edit the language throughout the MS.

The english was revised again

- -I am not entirely clear on the necessity of conducting this study. In my view, the determination of CEC and exchangeable Fe is not particularly difficult, and the relationship between pH and CEC has already been extensively investigated in previous studies.

Response: The determination of exchangeable Fe will be essential for the development of a methodology to predict the risk of iron toxicity (see comments made in response to reviewer 1). However, during the process of extracting cations to evaluate the CEC, Fe is oxidized and precipitates, causing an underestimation of its value. A mathematical procedure is necessary to correct this underestimation, which is what is being proposed in the work.

Response: As justified in the response to comments of Reviewer 1, this work corresponds to the first stage of a study that aims at a methodology for predicting iron toxicity, so it was necessary to use soils that presented different reactions to Fe toxicity, from unaffected soils to soils proven to be affected by toxicity.

-In my opinion, instead of investing time and effort in measuring the concentrations of Ca, Mg, Fe, and Mn, it would have been more direct and informative to simply measure Fe²⁺ itself.

Response: As explained in the comments of reviewer 1, the occurrence of Fe toxicity is more related to the proportion of exchangeable Fe in the soil CEC than to the absolute content of exchangeable Fe. Attempts to relate Fe toxicity to absolute contents of exchangeable Fe were inefficient in predicting the occurrence of toxicity.

-Figure 1 needs improvement. Currently, the text in the figure is too small, and the image resolution is too low, making it difficult to discern the information the authors intend to present.

Response: The figure was improved.

-Table 2 appears distorted. The alignment of pH, Mn, and Fe data within the table needs to be corrected.

Response: The tables were transformed in figures, according to suggestions of reviewer #3.

-The "Results and Discussion" section must be divided into smaller, clearly titled subsections. As it stands, it is difficult to follow the authors' line of reasoning and the flow of information. More importantly, the manuscript lacks a critical discussion of the findings. A dedicated Discussion section must be added to interpret the results more thoroughly.

Response: Results and discussion were divided in subtitles.

-The Conclusion section is currently inadequate. The authors need to revise it to succinctly summarize the main findings of the study, covering the majority of the data presented.

Response: done

-References. It would be best to consult the specific guidelines provided by the journal for instructions on formatting and referencing.

Response: done

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This experimental work addresses an soil chemistry and mineralogy subject but the analysis looks like it is written without addressing soil chemistry literature and norms. There is nowhere to be found herein the theoretical link between pH changes and Fe and Mn reduction, or the cause of reduction of exchangeable acidity (H+Al) with pH increase, or really what is the difference between effective CEC and CEC(pH7) or its cause.

There are copious tables of soluble and exchangeable cations and CECs, flooded and freshly-flooded. Nobody will read these tables. This type of data belongs in a Data Repository like Open Science Framework or Dryad, if MDPI does not make its own repository for supplemental files available. In this modern era, tables in the manuscript should be limited to analysis of the deposited data, descriptive things like means, ranges and standard deviations.

The authors' effort should go to use the wide ranging data to draw illustrative figures that present their data analysis, the relationship between CECeff freshly flooded vs CECeff, with&w/o Fe+Mn vs CECpH7. Maybe relationship between Fe and Mn in solution and pH or delta-pH. If they measured bicarbonate in solution, then perhaps they could say something about the limitation that siderite (FeCO3) formation played on limiting soluble Fe2+. They can go further with the data they have and determine the apparent exchange coefficients for Fe/Ca, Fe/Mg, Mn/Ca, and Mn/Mg based on their data on soluble and exchangeable forms of these cations. If they measured Eh in their soil solutions, that would be great to analyze too.

A pdf file is presented with some of the minor edits to be considered by authors.

It would be meaningful if they could provide some information about the mineralogy and organic carbon content of these soils, also.

Any evidence in the literature that these trends they put forward as a model hold true for flooded rice soils elsewhere in the world? That would go a long way to establishing Originality/Novelty, Significance of Content, and Interest to Readers.

Comments for author File: Comments.pdf

Author Response

Response to reviewer # 3:

-This experimental work addresses an soil chemistry and mineralogy subject but the analysis looks like it is written without addressing soil chemistry literature and norms. There is nowhere to be found herein the theoretical link between pH changes and Fe and Mn reduction, or the cause of reduction of exchangeable acidity (H+Al) with pH increase, or really what is the difference between effective CEC and CEC (pH7) or its cause.

Response: The issues raised by the reviewer are related to basic concepts of soil chemistry that are common knowledge and occur in all soils and are not specific to soils with iron toxicity. Therefore, we believe that it is not necessary to detail these reactions in the text of the paper beyond what has been covered. However, if the reviewer believes that there is a need to delve deeper into these topics, we may do so in a new round of corrections.

- There are copious tables of soluble and exchangeable cations and CECs, flooded and freshlyflooded. Nobody will read these tables. This type of data belongs in a Data Repository like Open Science Framework or Dryad, if MDPI does not make its own repository for supplemental files available. In this modern era, tables in the manuscript should be limited to analysis of the deposited data, descriptive things like means, ranges and standard deviations. The authors' effort should go to use the wide ranging data to draw illustrative figures that present their data analysis, the relationship between CECeff freshly flooded vs CECeff, with&w/o Fe+Mn vs CECpH7. Maybe relationship between Fe and Mn in solution and pH or delta-pH

Response: the larger tables were transformed into illustrative figures, keeping only table 7, which seems to us to be the most appropriate way of presenting the data contained therein.- If they measured bicarbonate in solution, then perhaps they could say something about the limitation that siderite (FeCO3) formation played on limiting soluble Fe2+.

Response: bicarbonate in solution was not measured

- They can go further with the data they have and determine the apparent exchange coefficients for Fe/Ca, Fe/Mg, Mn/Ca, and Mn/Mg based on their data on soluble and exchangeable forms of these cations.

Response: the authors are not sure what the reviewer is asking for

- If they measured Eh in their soil solutions, that would be great to analyze too.

Response: Eh was not measured

- A pdf file is presented with some of the minor edits to be considered by authors.

Response: The minor changes were presented in the PDF file. The major corrections are presented here.

- It would be meaningful if they could provide some information about the mineralogy and organic carbon content of these soils, also.

Response: soil organic carbon contents have been added in Figure 3, but we do not have assessments of soil mineralogy.

- Any evidence in the literature that these trends they put forward as a model hold true for flooded rice soils elsewhere in the world? That would go a long way to establishing Originality/Novelty, Significance of Content, and Interest to Readers.

Response: Considering that the manifestation of iron toxicity in Southern Brazil presents characteristics similar to those observed in other rice-producing regions, it is believed that what is being proposed also has applicability to other rice-producing regions around the world. A statement regarding this was added to the text.

In the text of the pdf file, the minor changes were added directly to the doc file. Regarding the major changes, some comments are as follows:

- Line 109 (PDF): rate is per area. How much per pot?:

Response: it was added to the text, the amount of soil per pot.

- Line 127 to 134 (PDF): Single extraction with 1 M KCl and CaOAc or multiple?

How was Al determined? AAS? this is important because apparently the difference btwn these CEC

Response: Changes were made in the text to make it clear.

- Above, line 128, different units were used for the same KCl.

Response: Changes were made in the text

- Line 157: wrong word. Perhaps "subtracted"?

Response: Changes were made in the text

- Line 184: What is the origin of this model? Is there a literature source that is being tested, i.e., it is an a priori model, or is it one that is suggested by the results, post hoc?

Response: The model was developped as a theoretical possibility and was validated by the manuscript results.

- Here Mn2+ whereas elsewhere, Mn2+. This reviewer prefers the superscripted version here and for all ions but defers to authors' and publisher's choice.

Response: the superscripted version was adopted in the Whole text.

- Incidentally, the analytical methods used cannot distinguish between Mn2+ and Mn4+ or Fe2+ and Fe3+. There is therefore another logical step taken that must be disclosed: Only Mn2+ and Fe2+ are sufficiently soluble at these pH values, 5 to 7, to be significant. Of course, any organic complexes with Mn and Fe could also increase their solubility

Response: Other Mn e Fe soluble forms can appear, especially in acidic pHs (as pH5). However, almost all soluble Mn and Fe do occurr in the reduced forms ( Mn²⁺ and Fe²⁺). Nevertheless, it is not possible to make a distinction with the methods used. Therefore, one has opted to present the, as Mn²⁺ and Fe²⁺

- Table 6: where is comparable data for exchangeable Mn?

Response: There is no need to present Mn2+ data, as it is less sensitive than Fe2+ to small variations in O2 and pH concentrations in the samples during the analytical procedure used. Therefore, the Mn2+ values evaluated are considered valid.

- Table 7: Average. By why just averages, how about ranges, and standard deviations?

Response: This table with the averages was included to prove that the Fe values evaluated by the extraction method were actually underestimated and that the proposed correction of the values can correct the underestimation satisfactorily. On the other hand, it proves that there was no error in the evaluation of exchangeable Mn, therefore not requiring corrections. We do not believe that there is a need to include ranges or standard deviations.

- Table 7: what is the meaning of the subscripted and underlined a for all of the soil solution cations except for Ca, which has a C? Not clear what means were tested for significant differences.

Response: There was some formatting or automatic correction error in Word. The superscripts were corrected. They were all actually capital A's. Below the table, the statistical test was identified and what the letters inside the table refer to.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This study examined the effective cation exchange capacity (CEC) under three conditions: before flooding, after flooding, and at pH 7. The author observed that the post-flood effective CEC did not change as significantly as expected, particularly when compared to the effective CEC at pH 7. Due to this discrepancy, the author questioned the reliability of the two iron (Fe) determination methods used and proposed a new calibration method for Fe under flooding conditions. While the topic is intriguing and the negative results are clearly presented, I doubt that an MDPI journal would accept a manuscript that fails to prove their hypothesis. Nevertheless, I believe the author should have the opportunity to thoroughly revise the paper, addressing the following comments:

If the author cannot validate the reliability of their proposed framework for estimating flood-induced Fe concentrations, I recommend avoiding the claim of having a "new method" for Fe estimation under flooding conditions.
The title, abstract, final paragraph of the introduction, results, and conclusion require substantial revision. Given the findings, the study’s focus could be reframed to investigate the factors influencing effective CEC changes during flooding—such as pH variability or potential inconsistencies in Fe concentration measurements. From my view, this is the easiest way that I could find to avoid additional lab work.
I doubt the methods of making pH=7 will also affect effective CEC results.
While I initially considered recommending rejection, I would prefer to give the author an opportunity to revise. However, given the extent of the required revisions, I do not believe the author can reasonably complete them within two weeks. I recommend contacting the editorial office to request an extension of the revision period.

Minor comments:

L67-L71: These are about Fe toxicity. I do not think it should belong to a new paragraph. Please merge these sentences to the third paragraph.

L72-L75: These two sentences have no logic to connect context. I recommend deleting them.

L75: Should start a new paragraph after “However…”.

L84: Should start a new paragraph from “the hypothesis of the work is…”.

Table 2: pH, Mn, and Fe are not in the right position in the first row. The unit “mmol/L” is not in the right position in the third row.

Author Response

Response to Reviewer #4:

Reviewer 4 questions the validity of the article, suggesting, among other things, a change in the focus of the study. We consider it important and convenient to maintain the current focus of the article, based on the following arguments: - Iron toxicity in irrigated rice is a nutritional disorder in which high absolute iron levels are not a determining factor for the occurrence of symptoms. The occurrence of toxicity is more related to the proportion of iron levels in relation to the levels of other cations present in the soil solution and exchangeable phase. With flooding, low-soluble Fe3+ is reduced to soluble Fe2+. Thus, this study represents the first stage of a study in which the ultimate objective is to develop a methodology for predicting iron toxicity based on the saturation of the soil CEC with Fe2+. In this first stage of the study, it is crucial to determine the soil CEC under flooding conditions, where exchangeable Fe2+ can represent a significant portion of the CEC. However, in traditional methods of determining CEC during the process of agitation and extraction of exchangeable nutrients, the soil sample comes into contact with oxygen and the Iron that was in the reduced form of Fe2+ oxidizes and precipitates as Fe3+, ceasing to be exchangeable. These methods, therefore, underestimate the concentration of exchangeable Fe during flooding. The same does not happen with Mn2+ because it is less susceptible than Fe2+ to small variations in O2 and pH concentrations in the samples during the analytical procedure used. However, the concentrations of the other exchangeable elements (Na, Ca, Mg and K) are not affected by the oxidation-reduction condition of the soil, so the evaluation of their exchangeable concentrations is valid by the usual methods. Therefore, if we can estimate exchangeable Fe by the procedure proposed in the article, the other cations can be evaluated by the traditional methods of determining the CEC. In addition, to date, the article has more than 50 downloads, even in pre-print, which is an indicator of the interest aroused by the article in the scientific community, since it seeks to fill a gap in scientific knowledge related to the topic of iron toxicity in rice.

- Minor comments:

L67-L71: These are about Fe toxicity. I do not think it should belong to a new paragraph. Please merge these sentences to the third paragraph.

Response: done

L72-L75: These two sentences have no logic to connect context. I recommend deleting them

Response: done

L75: Should start a new paragraph after “However…”

Response: done

L84: Should start a new paragraph from “the hypothesis of the work is…”

Response: done

Table 2: pH, Mn, and Fe are not in the right position in the first row. The unit “mmol/L” is not in the right position in the third row.

Response: This table was transformed in figure, according to the suggestion made by reviewer 3.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Significant analysis results of the results of before and after flooding should be presented in relative figures.

Discussions still are requested to supplement combined with previous research results after the line 292.

Author Response

Significant analysis results of the results of before and after flooding should be presented in relative figures.
Response: A reply to this query was already produced in the first round of corrections. Since the process of soil solution collection was complex, it was not possible to perform replications in the soils. The idea was that covering a higher number of different soils would work similar to having replications (i.e., the broad range of different soils would randomly deviate from a mean value, giving a good estimate of the method). Therefore, this procedure would give and idea of how often the new method causes an improvement in the Fe and CEC measurements.

Discussions still are requested to supplement combined with previous research results after the line 292.
Response: A new section for discussion was introduced, as suggested by reviewer 2. In this process, new elements were created in the discussion

Reviewer 2 Report

Comments and Suggestions for Authors While I appreciate the authors’ efforts to revise the manuscript, I must point out that further improvements are still necessary to meet the journal’s publication standards. First, although the overall English language has been improved, there remain several lengthy and complex sentences that would benefit from careful editing by a native English speaker to enhance clarity and readability. Second, the resolution and legibility of some figures should be further improved to ensure that all text, labels, and data points are easily discernible. Finally, the “Results and Discussion” section remains overly long and somewhat unfocused. It is strongly recommended that the authors separate the discussion elements from the results presentation and create a dedicated “Discussion” section. This would help to better highlight the key findings, provide a more critical interpretation of the results, and improve the logical flow of the manuscript.

Author Response

While I appreciate the authors’ efforts to revise the manuscript, I must point out that further improvements are still necessary to meet the journal’s publication standards. First, although the overall English language has been improved, there remain several lengthy and complex sentences that would benefit from careful editing by a native English speaker to enhance clarity and readability. Second, the resolution and legibility of some figures should be further improved to ensure that all text, labels, and data points are easily discernible. Finally, the “Results and Discussion” section remains overly long and somewhat unfocused. It is strongly recommended that the authors separate the discussion elements from the results presentation and create a dedicated “Discussion” section. This would help to better highlight the key findings, provide a more critical interpretation of the results, and improve the logical flow of the manuscript.

Response: At first, the whole text (including the long sentences) was revised by a native speaker. Also, Figure resolution was improved in order to obtain a better visualization of the presented elements. At last, the section Results and Discussion was split in separate sections.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is much improved upon revision. A number of small edits are provided with the attached pdf and a couple of significant items.

The link between change in pH and redox is still not articulated. Ref 20 should make this clear.

Nonstandard units of exchangeable cations are used, cmolc dm-3 instead of cmolc kg-1. Soil is measured in weight not volume for almost all purposes.

Consider reordering the soil IDs, from lowest CEC to highest, to make the figures 4 and 5 more pleasing.

Taking CEC at pH 7 as the flooded ECEC and estimating Fe2+ as the difference is an interesting approach to describing the discrepancy. Don't know how you'd prove it. Maybe give this some thought.

Removing the multiple data tables was a good thing to do. Now the data must be placed in a data repository and a link provided in the manuscript itself before publication.

Comments for author File: Comments.pdf

Author Response

The manuscript is much improved upon revision. A number of small edits are provided with the attached pdf and a couple of significant items.

Response: one tried to follow all the suggestions in the pdf document. Among the required changes, two columns were added on table 2 in order to facilitate the comparison of obtained results.

The link between change in pH and redox is still not articulated. Ref 20 should make this clear.

Response: This point was made clear in the text.

Nonstandard units of exchangeable cations are used, cmolc dm-3 instead of cmolc kg-1. Soil is measured in weight not volume for almost all purposes.

Response: this change was performed throughout the text.

Consider reordering the soil IDs, from lowest CEC to highest, to make the figures 4 and 5 more pleasing.

Response: changes were made in Figures 4 and 5 as requested.

Taking CEC at pH 7 as the flooded ECEC and estimating Fe2+ as the difference is an interesting approach to describing the discrepancy. Don't know how you'd prove it. Maybe give this some thought.

Response: please see reply to reviewer 4 below.

Removing the multiple data tables was a good thing to do. Now the data must be placed in a data repository and a link provided in the manuscript itself before publication.

Response: ok. We agree. A repository will be created.

Reviewer 4 Report

Comments and Suggestions for Authors

The author did not address my major concerns. I summarize it again as follows:

The author claimed "the hypothesis of the work is that, based on a sample collected before flooding, it is possible to estimate the ECEC after flooding through a linear relationship between the variation of pH before and after flooding with the variation of the ECEC and CEC pH7.0.". However, end up with "it was not possible to test the hypothesis of the study experimentally". I have not seen any paper that denies one of the hypotheses that can be published in this journal.
Even if the first hypothesis can be denied, the so-called new exchangeable Fe content after flooding might be pH-related rather than independent. However, the author only used pH 7.0 as a base value.

Since these are fundamental problems and there has been no major improvement after the revision, I recommend rejection.

Author Response

The author did not address my major concerns. I summarize it again as follows:

The author claimed "the hypothesis of the work is that, based on a sample collected before flooding, it is possible to estimate the ECEC after flooding through a linear relationship between the variation of pH before and after flooding with the variation of the ECEC and CEC pH7.0.". However, end up with "it was not possible to test the hypothesis of the study experimentally". I have not seen any paper that denies one of the hypotheses that can be published in this journal.
Even if the first hypothesis can be denied, the so-called new exchangeable Fe content after flooding might be pH-related rather than independent. However, the author only used pH 7.0 as a base value.

Since these are fundamental problems and there has been no major improvement after the revision, I recommend rejection.

Response: One agrees partially with reviewer 4 regarding the difficulty in testing the hypothesis in the present study. One disagrees regarding the rejection of the work. One can consider that the present work has assumptions that are justified along the display of results and throughout the discussion, taking into account the chemistry of flooded soils. One understands that during the discussion of collected data, one raises enough justifications that support the estimates of exchangeable Fe and ECEC of flooded soils are very close to the real values, being an important alternative, since there is no better alternative in the literature than the one here proposed. One highlights that upon validation of this alternative of estimating the exchangeable Fe and ECEC, one expects to open the way for additional methods that can predict iron toxicity in rice, which represents a current gap in the understanding of flooded soil chemistry.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The MS has been greatly improved, especially with the addition of the discussion section. However, the results of the text should not include any discussion. How can the correctness of the method proposed in the paper be verified?

Author Response

1) We have added a final paragraph in the discussion that should clarify the effectiveness of this method.