Nitrogen Fertilization Alleviates Microplastic Effects on Soil Protist Communities and Rape (Brassica napus L.) Growth
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe research presented in the manuscript "Nitrogen fertilization alleviates microplastic effect on soil protist communities and rape (Brassica napus L.) Growth" is important for the current context of plastic accumulation in soil. The exploration of combined effect between microplastic and nitrogen addition is beneficial for the higher understanding of changes in protist communities and, thus, the potential changes in soil biological activity and stability.
The abstract is clear and point specifically the most important trends observed by the authors.
The Introduction section state the research problem, the importance of the study for the field, and the hypotheses associated with the aim.
The Materials and methods section present all the necessary information to replicate the experiment, the design used by the authors and the data extraction and analysis.
Results section
For figure 1 I suggest adding in the caption the Legend of treatment abbreviations. This will ensure the standalone status of the figure and an easier reading process.
Same suggestion for table 1, figure 2, 3.
Figure 3. The text "* and ns indicate significant and nonsignificant differences, respectively." I do not think it belongs in this figure caption. Please remove it or add the for * significance for all the PCoA`s.
Add the legend of treatments in figure 4 and for SWC in figure 5.
Overall, the Results section is well organized, with each graph and table well explored.
The Discussion section links the findings of the study with international references and provide a good discussion basis for other researches in this field.
Conclusion section. I suggest the removal of the first and second sentences. They just repeat the aim of this work. The same suggestion for the last two sentences (lines 398-403), which are perspectives and belong better at the end of the Discussion section.
The manuscript is interesting and deserve to be improved.
Author Response
Dear Reviewer,
We sincerely appreciate the time you have taken to review our manuscript and the thoughtful suggestions you provided. Below, we address your comments point-by-point, outlining the revisions we have made. For your convenience, all modifications in the manuscript are highlighted in red.
Comments 1: The abstract is clear and point specifically the most important trends observed by the authors.
Response 1: Thank you for your positive feedback on the abstract.
Comments 2: The Introduction section state the research problem, the importance of the study for the field, and the hypotheses associated with the aim.
Response 2: We appreciate your positive evaluation of the Introduction.
Comments 3: The Materials and methods section present all the necessary information to replicate the experiment, the design used by the authors and the data extraction and analysis.
Response 3: Thank you for your careful review.
Comments 4: For figure 1 I suggest adding in the caption the Legend of treatment abbreviations. This will ensure the standalone status of the figure and an easier reading process. （Same suggestion for table 1, figure 2, 3.）
Response 4: Thank you for this valuable suggestion. We have amended the captions for Figures 1, 2, 3, and 4 to include the necessary legends for treatment abbreviations, thereby improving the clarity and standalone status of these figures.
Comments 5: Figure 3. The text "* and ns indicate significant and nonsignificant differences, respectively." I do not think it belongs in this figure caption. Please remove it or add the for * significance for all the PCoA`s. （Add the legend of treatments in figure 4 and for SWC in figure 5.）
Response 5: Thank you for your detailed feedback. We have revised Figure 3 accordingly to ensure that all significance markers are clearly explained, and we have added the legends for treatments in Figure 4 and for SWC in Figure 5 to enhance clarity.
Comments 6: Overall, the Results section is well organized, with each graph and table well explored.
Response 6: We appreciate your positive evaluation of the Results section.
Comments 7: The Discussion section links the findings of the study with international references and provide a good discussion basis for other researches in this field.
Response 7: Thank you for your encouraging feedback on the Discussion section.
Comments 8: Conclusion section. I suggest the removal of the first and second sentences. They just repeat the aim of this work. The same suggestion for the last two sentences (lines 398-403), which are perspectives and belong better at the end of the Discussion section.
Response 8: Thank you for your valuable suggestion. In response, we have removed the redundant sentences from the Conclusion section, and the perspective content has been relocated to the end of the Discussion section.
We are grateful for your expert insights, which have significantly enhanced both the scientific content and the overall presentation of our manuscript. Thank you once again for your constructive feedback.
Sincerely,
Dongyan Liu
Sichuan Normal University
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsSoil and Microplastic Material Preparation
Line 77: Please specify the amount of soil collected.
Line 83: The meaning of “(<5 mm)” is unclear; please clarify.
Experimental Design
A more detailed description of the experimental design is required, including information on the number of replicates, number of pots, number of plants, amount of soil used, irrigation protocol, and environmental conditions.
Line 90: The nitrogen application rate is defined as 180 kg N·hm⁻², which is a weight-per-area unit. In pot experiments, expressing nitrogen addition in this way is not the most appropriate approach. Since pots have a controlled volume and soil mass, nitrogen addition should be reported as either:
Concentration per soil mass (e.g., mg N per kg of soil), or
Absolute nitrogen amount per pot (e.g., mg or g N per pot).
Line 95: It is recommended to use the phrase "2 cm in height" for improved clarity.
Sample Collection and Soil Physicochemical Property Determination Methods
Line 102: Please specify the drying time used during sample processing.
High-Throughput Sequencing and Bioinformatics Analysis
Line 110: Please specify the name of the DNA extraction kit used.
Line 117: To ensure accuracy in diversity calculations and multivariate statistical analyses, it is strongly recommended to rarefy the sequences before proceeding with subsequent steps.
Statistical Analysis
Line 121: Parametric statistical methods rely on assumptions about the underlying data distribution. For these methods to be valid and yield reliable results, the assumptions of normality, homoscedasticity, and independence must be met. Since the experimental design is not clearly described, it is not possible to determine whether the authors established an appropriate sample size or tested the data to confirm the suitability of parametric statistics.
Line 128: It is preferable to state “Generalized Linear Mixed Model using the glmm.hp package in R” rather than “glmm.hp” alone for clarity and accuracy.
Results
Changes in Soil Protist Diversity and Community Composition
Lines 178-197: The manuscript presents diversity indices (Chao1 and Shannon) and community composition analyses without addressing rarefaction. While rarefaction is not strictly required for diversity indices, it is essential to ensure that differences in sequencing depth do not introduce bias.
Chao1 estimates species richness and is sensitive to sequencing effort. If sequencing depths differ significantly between treatments, rarefaction is necessary to avoid biases. The Shannon index accounts for both richness and evenness, making it less sensitive to sequencing depth than Chao1. However, if sequencing depth varies greatly, rarefaction may still be beneficial.
Community composition analyses, including Principal Coordinate Analysis (PCoA) and relative abundance comparisons, can be influenced by variations in sequencing depth. If rarefaction was not performed, the authors should justify this decision (e.g., sequencing depth was consistent across samples). If rarefaction was performed, it should be explicitly mentioned to confirm that diversity and composition analyses were not biased.
Changes in the Functional Group of the Soil Protist Community
Line 206: There are inconsistencies between the percentage values reported for PCoA and those presented in Figures 3a and 3b. These discrepancies should be verified and corrected.
Figure 3
Line 232: Since Bray-Curtis is not a distance method, it is more appropriate to refer to it as "Bray-Curtis dissimilarity" rather than "Bray-Curtis distance method."
Discussion and Conclusions
Since there are methodological and statistical issues in the manuscript, the Discussion and Conclusion sections must be revised to ensure consistency with the data and analyses presented.
Author Response
Dear Reviewer,
Thank you for your detailed review and the valuable suggestions for improving our manuscript. We have carefully addressed each of your comments, and all modifications in the revised manuscript are clearly marked in red for your convenience.
Comments 1: Line 77: Please specify the amount of soil collected.
Response 1: Thank you for pointing this out. Approximately 100 kg of soil was collected via drilling and brought to the laboratory for pot experiments. We have added this information on line 87.
Comments 2: The meaning of “(<5 mm)” is unclear; please clarify.
Response 2: Thank you for your detailed feedback. The term “<5 mm” originally referred to microplastics, defined as plastic particles or fragments less than 5 mm in diameter or length. However, since the microplastics used in our experiment actually range from 25–150 μm, we have removed this reference from the text.
Comments 3: A more detailed description of the experimental design is required, including information on the number of replicates, number of pots, number of plants, amount of soil used, irrigation protocol, and environmental conditions.
Response 3: We appreciate your suggestions and have expanded the experimental design description to include the requested details in lines 104–113.
Comments 4: Line 90: The nitrogen application rate is defined as 180 kg N·hm⁻², which is a weight-per-area unit. In pot experiments, expressing nitrogen addition in this way is not the most appropriate approach. Since pots have a controlled volume and soil mass, nitrogen addition should be reported as either:
Response 4: Thank you for your valuable suggestion. We have revised the nitrogen application rate description in lines 100–101 to better reflect the controlled volume and soil mass in pot experiments.
Comments 5: It is recommended to use the phrase "2 cm in height" for improved clarity.
Response 5: Thank you for the suggestion. We have updated the description to “2 cm in height” on line 109.
Comments 6: Line 102: Please specify the drying time used during sample processing.
Response 6: We appreciate your review. The drying time information has been added in lines 117–119.
Comments 7: Please specify the name of the DNA extraction kit used.
Response 7: Thank you for your suggestion. The DNA extraction kit we used is a self-made kit developed in our laboratory. We have included the appropriate reference in lines 128–129.
Comments 8: To ensure accuracy in diversity calculations and multivariate statistical analyses, it is strongly recommended to rarefy the sequences before proceeding with subsequent steps.
Response 8: We acknowledge your suggestion and have performed rarefaction of the sequences to a minimum of 56,478 reads, as noted on line 136.
Comments 9: Parametric statistical methods rely on assumptions about the underlying data distribution. For these methods to be valid and yield reliable results, the assumptions of normality, homoscedasticity, and independence must be met. Since the experimental design is not clearly described, it is not possible to determine whether the authors established an appropriate sample size or tested the data to confirm the suitability of parametric statistics.
Response 9: We thank you for your thorough analysis. Given that plant and soil samples were measured in four replicates, we performed statistical comparisons of each physicochemical parameter using a non-parametric Kruskal-Wallis test (p < 0.05) combined with Dunn's multiple comparisons post-hoc test, which is now detailed in lines 141–144.
Comments 10: Lines 178-197: The manuscript presents diversity indices (Chao1 and Shannon) and community composition analyses without addressing rarefaction. While rarefaction is not strictly required for diversity indices, it is essential to ensure that differences in sequencing depth do not introduce bias.
Response 10: We have added the rarefaction analysis at the minimum sequence depth to ensure that differences in sequencing depth do not introduce bias.
Comments 11: Chao1 estimates species richness and is sensitive to sequencing effort. If sequencing depths differ significantly between treatments, rarefaction is necessary to avoid biases. The Shannon index accounts for both richness and evenness, making it less sensitive to sequencing depth than Chao1. However, if sequencing depth varies greatly, rarefaction may still be beneficial.
Response 11: As suggested, we have performed rarefaction prior to analyzing both alpha and beta diversity indices.
Comments 12: Line 232: Since Bray-Curtis is not a distance method, it is more appropriate to refer to it as "Bray-Curtis dissimilarity" rather than "Bray-Curtis distance method."
Response 12: Thank you for your valuable feedback. We have revised this section to refer to it as "Bray-Curtis dissimilarity" rather than "Bray-Curtis distance method."
Comments 13: Since there are methodological and statistical issues in the manuscript, the Discussion and Conclusion sections must be revised to ensure consistency with the data and analyses presented.
Response 13: We appreciate your comprehensive review. We have incorporated additional statistical information (lines 141–144).
We sincerely appreciate the constructive feedback, which has greatly enhanced the quality and clarity of our manuscript.
Sincerely,
Dongyan Liu
Sichuan Normal University
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsAbstract;
You must mention what mean TC before mentioned (line 14), CK LINE 19;.
Introduction;
I suggest developing more.
Materials and methods
Table 1. Soil physicochemical characteristics
You must mention each individual parameter at the top of the table (TC (g/kg), SWC.....
Author Response
Dear Reviewer,
Thank you very much for your thoughtful review and constructive comments regarding our manuscript. In response to your suggestions, we have revised the manuscript accordingly, with all modifications clearly highlighted in red.
Comments 1: You must mention what mean TC before mentioned (line 14), CK LINE 19.
Response 1: Thank you for your careful review. We have provided detailed explanations for “TC” and “CK” at lines 14 and 19, respectively, to ensure clarity for our readers.
Comments 2: I suggest developing more（Introduction）.
Response 2: We appreciate your valuable suggestion. We agree that the introduction could benefit from a more in-depth discussion on the relationship between native organisms and soil nutrients, in addition to the research progress on microplastics. Accordingly, we have expanded the introduction on lines 54–62.
Comments 3: You must mention each individual parameter at the top of the table (TC (g/kg), SWC.....
Response 3: Thank you for this professional advice. We have revised the table to include the full names and units of each parameter.
We trust that these revisions address your concerns and enhance the overall clarity and quality of our manuscript. Thank you once again for your invaluable feedback.
Sincerely,
Dongyan Liu
Sichuan Normal University
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThank you for your thorough review of the article. However, I must request a new review considering the next point:
Comments 8: To ensure accuracy in diversity calculations and multivariate statistical analyses, it is strongly recommended to rarefy the sequences before proceeding with subsequent steps.
Response 8: We acknowledge your suggestion and have performed rarefaction of the sequences to a minimum of 56,478 reads, as noted on line 136.
Thank you for your response. However, while you mention that rarefaction was performed, the manuscript does not reflect this update. Since rarefaction can influence diversity indices and PCoA results, it is important to clarify whether this adjustment affected your findings. Please ensure that this methodological step is explicitly stated in the text and that any potential impact on the results, discussion and conclusions is addressed.
Author Response
Dear Reviewer,
Thank you very much for your detailed and constructive feedback, which has significantly improved our manuscript. We have carefully considered your comments, and the corresponding modifications in the revised manuscript have been marked clearly in red for your convenience.
Comments 8: To ensure accuracy in diversity calculations and multivariate statistical analyses, it is strongly recommended to rarefy the sequences before proceeding with subsequent steps.
Response 8: We acknowledge your suggestion and have performed rarefaction of the sequences to a minimum of 56,478 reads, as noted on line 136.
Thank you for your response. However, while you mention that rarefaction was performed, the manuscript does not reflect this update. Since rarefaction can influence diversity indices and PCoA results, it is important to clarify whether this adjustment affected your findings. Please ensure that this methodological step is explicitly stated in the text and that any potential impact on the results, discussion and conclusions is addressed.
Response: We sincerely appreciate your valuable suggestion. Initially, we mistakenly stated that the rarefaction had been completed; however, upon carefully reviewing all data-processing scripts, we realized that the original analyses were inadvertently conducted using non-rarefied ASV tables. We thank you for pointing out this oversight, which allowed us the opportunity to correct our data processing method. In response, the original ASV data has now been rarefied to a minimum sequencing depth. We subsequently reanalyzed all diversity indices and PCoA plots with the rarefied dataset. The reanalysis results demonstrate more robust and statistically convincing trends. Specifically, Figure 2 (a, b, c) has been updated, and the corresponding statistical results in the main text were adjusted accordingly (line 228). Additionally, we updated Figure 3 (a, b, c) at line 265, and revised descriptions of these results have been incorporated at lines 236-247 of the manuscript. For further transparency, we have attached Figures 2 and 3 as supplementary materials with this response to verify our revised methodological approach and enhanced results.
Once again, thank you for your constructive feedback, which has greatly improved the accuracy and clarity of our manuscript.
Figure2. Unrarefied (left) and rarefied (right) ASV data
Figure3. Unrarefied (left) and rarefied (right) ASV data
Sincerely,
Dongyan Liu
Sichuan Normal University
Author Response File: 
Author Response.docx
