Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a relevant and timely study investigating the impact of microplastics (MPs) on soil microbial communities across different soil types. The experimental design is generally sound, and the use of high-throughput sequencing provides valuable data. However, revisions are needed, particularly concerning the misclassification of the contaminant as microplastics when nanoplastics were used. Additional clarification is also required regarding the experimental replication design for microbiome analysis. A more in-depth mechanistic discussion linking the observed microbial shifts to potential causes is strongly advised. Several critical points must be addressed to improve the manuscript’s clarity, quality, and overall impact. 

Author Response

 

 Dear Reviewer,

Thank you for your insightful comments and suggestions, which have significantly helped us improve the manuscript. A detailed list of revisions is provided below.

General Comments:

1. The study used polystyrene latex particles with a diameter of 0.55 μm (Line 163). Particles smaller than 1 μm are classified as nanoplastics, not microplastics. This is a fundamental error in terminology and contaminant classification. Please revise the title, abstract, keywords, introduction, and discussion accordingly.

Response: fixed

2. The manuscript does not clearly articulate the novelty of the research.

Response: we have attempted to more clearly outline the contribution of our research in the Introduction and Conclusion.

3. The authors are advised to avoid strong claims and/or statements.

Response: completed

4. Avoid long sentences; split them into shorter, clearer ones.

Response: completed

5. Avoid using keywords already mentioned in the title.

Response: fixed

6. Maintain consistency in the use of 'Fig' and 'Figure'.

Response: fixed

7. Provide high-resolution images and include sub-figure numbers for easy identification. Ensure that all figures and subfigures are cited and discussed appropriately in the main text to support reader comprehension.

Response: completed. All figures have been improved in quality.

Specific Comments:

Abstract

1. Revise the abstract to better present the methods and results. Include the incubation duration.

Response: completed

2. Clarify the term “model microplastic particles.” Include properties such as size and composition.

Response: completed

3. Add limitations and future directions at the end of the abstract (one to two sentences). Introduction

Response: completed

4. While the literature review is extensive, it fails to establish a clear research gap. Rewrite the section to emphasize this.

Response: completed

5. Explicitly state the knowledge gap this study intends to address.

Response: completed

6. Enhance the introduction by referencing specific studies on the impact of microplastics on soil microbial communities.

 Response: completed

7. Lines 61–66: Add citations for “Previous research... soil ecosystem functioning.”

Response: completed

8. Lines 71–74: Add citations for “Recent findings... agricultural soils.”

Response: completed

9. Lines 84–86: The statement on “increased abundance of pathogenic microorganisms... under long-term fertilisation” feels out of place since this study doesn’t involve fertilisation. Rephrase to focus on the effects of microplastics.

Response: completed

10. Discuss how soil texture and organic matter content influence microbial responses to MPs. This is central to the article.

Response: completed

11. Line 120: The phrase “(2) to isolate... original soils” is incorrectly framed as an objective. Please revise. Materials and Methods

Response: completed

12. Split this section into sub-sections for better readability.

Response: completed

13. Include the geographical coordinates of soil sampling sites. Provide key soil properties such as texture and water-holding capacity. These are crucial for understanding soil health and function.

Response: information was added

14. Line 153–154: Clarify how the soil chemical analyses were performed.

Response: clarified

15. Line 154: Remove the statement “Laboratory methods were employed.” It is redundant.

Response: fixed

16. Acknowledge that the incubation duration was relatively short and discuss its potential impact on the results.

Response: thank you for this comment. We acknowledge that long-term incubation experiments would be valuable, and we plan to conduct such studies in future research

17. Line 174: Briefly describe the RIAM DNA extraction protocol.

Response: completed

18. Justify the chosen contamination level (0.025% by weight) concerning environmental relevance or exposure scenarios.

Response: completed

19. Rephrase the description of the experimental setup to improve clarity. Simplify the explanation of the concentration used.

Response: completed

20. Specify the primers targeting the 16S rRNA gene region. Include read length (e.g., 2×250 bp or 2×300 bp). Mention any controls used for DNA extraction contamination or PCR inhibition. Clarify whether rarefaction or normalization was applied before diversity analysis.

Response: primers are cited in the text. We use water as negative control for our pcr protocol. We rarefy our data before the diversity analysis.

21. Include a sub-section on statistical analysis methods, detailing the tests used and their appropriateness.

Response: corrected

22. Justify the choice of polystyrene latex as the contaminant.

Response: completed

23. While taxonomic changes are discussed, the absence of functional implications limits interpretation. Consider using predictive tools like PICRUSt2 or Tax4Fun2.

Response: we deliberately decided not to use PICRUSt2 or Tax4Fun2 tools as their predictive power is quite restricted. Usually, we stick either with taxonomic analysis via 16S rRNA gene sequencing or go further with the full metagenome. For this experiment we opted to limit ourselves with taxonomic analysis.

24. Including soil enzyme activity analysis would provide further insight into functional changes.

Response: thank you for your comment. This is an interesting topic for future research

25. Clearly state the parameters used at each step of the DADA2 pipeline (e.g., truncation lengths, minimum overlap) to ensure transparency and reproducibility.

Response: the information has been provided. The code can be made available by request.

Results and Discussion

26. This section lacks depth. Consider rewriting it with greater focus on ecological relevance.

 Response: completed

27. Lines 186–189: The statement “Following the incubation... pH and density [42–46]” belongs in the methods section.

Response: completed

28. Figure 2 caption: Indicate that different letters represent significant differences (p<0.05) within each soil type. Specify what the error bars represent.

Response: completed

29. Figure 4 caption: Clarify that “treated” means microplastic-added samples.

Response: completed

30. Lines 265–269: The taxa shifts identified by ANCOM-BC are important. Consider presenting these in a table or supplementary material. Include the direction of change (increase or decrease) and significance level.

Response: data is presented in the format of a table and may be made available by request. The parameters of ANCOM-BC analysis were clarified in the methods section.

31. Expand the discussion on the functional roles of key taxa involved in nutrient cycling and ecosystem processes. Highlight the roles of pioneer taxa.

Response: the discussion has been expanded

32. Discuss how variables such as microplastic polymer type and experimental duration may have influenced the results.

Response: completed

33. Lines 265–267: The mention of both a family and a phylum (e.g., Myxococcota) in the same

context is unclear. Reorganize or clarify the sentence.

Response: completed

Conclusion

34. Be more specific and detailed, and emphasize the novel contributions of the study. Include the

potential implications of their findings for soil ecosystem management and microplastic pollution control.

Response: completed

35. Address study limitations and provide future research recommendations

Response: completed

Sincerely yours, 

professor Evgeny Abakumov,

corresponding author

 

 

 

 

 

 

                     

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled 'The effect of applying model microplastic particles to soil on the composition of its microbial community' studies how the microbial communities in soils in the southern taiga zone (near Saint Petersburg, Russia) react to the addition of polystyrene microplastics. The work carried out is interesting. However, the manuscript needs considerable improvement. The manuscript contains numerous issues related to wording and formatting, including consistent double spacing, missing punctuation (such as periods), inappropriate use of italics, among others. Furthermore, the authors should clarify their use of the term 'microplastics', given that they have worked with 'nanoplastics', and explain why they used plastic tubes to conduct experiments on the impact of plastics on different soil types. Additionally, the discussion of the results is vague and lacks sufficient depth. These issues undermine the scientific rigour of the manuscript and should be thoroughly addressed to improve the overall quality of the paper. Therefore, the format needs to be standardised throughout the manuscript, certain questions need to be answered and some parts of the manuscript need to be corrected to fully reflect the content of the article.

 

Line 15: There is no colon after the word 'abstract'.

Lines 18 and 129: Typographical errors: double spaces.

Line 22: Authors use the present tense in this sentence, whereas in the rest of the abstract they use the past tense, as is normal.

Lines 21-23: 'The alpha diversity parameters of the microbial community are also decreasing, as is the number of operational taxonomic units and biodiversity indices (Shannon and Simpson)': In what type of soil does the microbial community decrease? The authors should clarify this point.

Lines 23-24: How should be interpreted the authors' assertion that the dynamics of alpha diversity are less pronounced in podzolic soil?

Lines 101-102 and lines 257-261: Phyla should not be written in italics. Authors should standardise the format throughout the manuscript. Furthermore, line 101 refers to the Firmicutes phylum, which is currently known as the Bacillota phylum. The authors should correct this.

Figure 1 is low resolution and the images of the different soil types need to be enlarged. As they are currently sized, the details of the different soil types cannot be seen.

Lines 163-164: 'Polystyrene latex with a particle diameter of 0.55 μm…': They are not microplastics. This study is working with nanoplastics. The authors should clarify and correct this point. The same plastic cannot be both microplastic and nanoplastic, as the authors claim referring to the reference 25. The reviewer suggests that the authors use the correct term: 'nanoplastics', which is used in a wide variety of articles, such as:

• Thompson, R. C., Courtene-Jones, W., Boucher, J., Pahl, S., Raubenheimer, K., & Koelmans, A. A. (2024). Twenty years of microplastic pollution research—what have we learned? Science, 386(6720), eadl2746.
• Thacharodi, A., Meenatchi, R., Hassan, S., Hussain, N., Bhat, M. A., Arockiaraj, J., ... & Pugazhendhi, A. (2024). Microplastics in the environment: a critical overview on its fate, toxicity, implications, management, and bioremediation strategies. Journal of Environmental Management, 349, 119433.
• Tirkey, A., & Upadhyay, L. S. B. (2021). Microplastics: An overview on separation, identification and characterization of microplastics. Marine pollution bulletin, 170, 112604.

Line 165: 'The experiment was conducted in 50 ml plastic tubes. ' The use of these plastic tubes may have influenced the results. Moreover, the authors should specify the type of plastic from which the tubes are made. Furthermore, it is essential that they justify why they chose to use these plastic supports in an experiment aimed at evaluating the influence of plastic presence in different soils. Although it is true that using the same plastic tubes across all experiments helps minimize error, it must be made clear that the selection and use of these tubes itself constitutes a source of experimental error. The authors are urged to clarify this issue.

Line 183: Missing a final period.

Line 188: Remove the period before the bracket.

Line 208: Would the idea be that the soil is resistant? Or would it be that the microbiota present in the soil is more resilient to the changes caused by the presence of plastic particles?

Figure 3: The resolution of this figure needs improving. The figure legend should provide descriptions of the sample codes appearing in the box plot graphs.

Figure 6: In the legend, specify the sample codes and parameters, and note that CCA stands for 'canonical correspondence analysis'.

Lines 281-284: Which references support this evidence? The discussion is vague and should be improved by the authors through a more in-depth discussion of the results and integration of additional references that corroborate or refute the findings of this study.

Author Response

Dear Reviewer,

Thank you for your insightful comments and suggestions, which have significantly helped us improve the manuscript. A detailed list of revisions is provided below.

Line 15: There is no colon after the word 'abstract'

Response: fixed

Lines 18 and 129: Typographical errors: double spaces.

Response: fixed

Line 22: Authors use the present tense in this sentence, whereas in the rest of the abstract they use the past tense, as is normal.

Response: fixed

Lines 21-23: 'The alpha diversity parameters of the microbial community are also decreasing, as is the number of operational taxonomic units and biodiversity indices (Shannon and Simpson)': In what type of soil does the microbial community decrease? The authors should clarify this point.

Response: it was found that Sandy Podzols were more resistant to the addition of nanoplastics  than loamy Retisols and Fluvisols.

Lines 23-24: How should be interpreted the authors' assertion that the dynamics of alpha diversity are less pronounced in podzolic soil?

Response: as these are natural, undisturbed soils, they may exhibit greater resistance to contamination. We have added this clarification to the discussion section.

Lines 101-102 and lines 257-261: Phyla should not be written in italics. Authors should standardise the format throughout the manuscript. Furthermore, line 101 refers to the Firmicutes phylum, which is currently known as the Bacillota phylum. The authors should correct this.

Response: fixed

Figure 1 is low resolution and the images of the different soil types need to be enlarged. As they are currently sized, the details of the different soil types cannot be seen.

Response: fixed

Lines 163-164: 'Polystyrene latex with a particle diameter of 0.55 μm…': They are not microplastics. This study is working with nanoplastics. The authors should clarify and correct this point. The same plastic cannot be both microplastic and nanoplastic, as the authors claim referring to the reference 25. The reviewer suggests that the authors use the correct term: 'nanoplastics', which is used in a wide variety of articles, such as:

• Thompson, R. C., Courtene-Jones, W., Boucher, J., Pahl, S., Raubenheimer, K., & Koelmans, A. A. (2024). Twenty years of microplastic pollution research—what have we learned? Science, 386(6720), eadl2746.
• Thacharodi, A., Meenatchi, R., Hassan, S., Hussain, N., Bhat, M. A., Arockiaraj, J., ... & Pugazhendhi, A. (2024). Microplastics in the environment: a critical overview on its fate, toxicity, implications, management, and bioremediation strategies. Journal of Environmental Management, 349, 119433.

Sincerely yours,

professor Evgeny Abakumov,

coreesponding author

Reviewer 3 Report

Comments and Suggestions for Authors

I suggest to review the English, there are some points difficult to understand beacause of the poor quality of the language.

Other suggestions are about a better explication, sometimes it is hard to understand the point of view from the authors.

line 43= do you thin plastic production is recent. It is 70 years plastic is produced. I suggest you to highlight in the last 20-30 decade the production has increased

line 57= why did you write in compost there is plastic? Could you explain please?

line 70= what do you mean with beahaviour is changing due to the presence of MP? Explain better, please

line 107-108= Could you explain how MPs shape can affect enzymatic activity, please?

line 156= what is the dimension of the soil after sieving?

line 156= what do you mean with morphology of soil, maybe characteristichs?

line 163= with modern marine clay do you mean from  Pleistocene?

line 178-179= where did you get the control soil? I didn't understand. Later you wrote you add plastic. I suggest to explain better

line 184= I think Pravia stands for Pavia

line 204= did you recycle tubes and caps? DId you wash with distilled water or bidistilled?

line 209= maybe three replicates for each samples

line 271=what does pronouced in Podzoil soil. crobiologica studies mean? There are many mistakes in English, pay attention please

line 342= composition is not changing, the type of microbiome is changing. Speaking about composition I may think to the chemical or mineralogical one, too. I suggest you to rewrite this sentence, please

line 343= So it is not possible to say "composition" is changing

line 350= is M2_NP or M2_MP? In caption and figure are different

line 352-353= where is the principal sentence? what you wrote has no meaning

line 353-355= this is already knonw

359-361= I don't agree, maybe these soils are only not  or less anthropogenic disturbed!

Comments on the Quality of English Language

I suggest to improve the quality of English language

Author Response

 

Dear reviewer!

We appreciate the reviewer's insightful comments, which have allowed us to improve our manuscript.

A detailed list of revisions based on their suggestions is provided below.

 

I suggest to review the English, there are some points difficult to understand because of the poor quality of the language.

Response: we have made an effort to improve the English language throughout the manuscript

 

Other suggestions are about a better explication, sometimes it is hard to understand the point of view from the authors.

line 43= do you thin plastic production is recent. It is 70 years plastic is produced. I suggest you to highlight in the last 20-30 decade the production has increased

Response: the introduction has been revised to address the comment

line 57= why did you write in compost there is plastic? Could you explain please?

Response: a description of the pathways by which microplastics enter compost and subsequently agricultural soils has been added."

line 70= what do you mean with beahaviour is changing due to the presence of MP? Explain better, please

Response: the phrasing has been corrected

line 107-108= Could you explain how MPs shape can affect enzymatic activity, please?

Response: currently, there is a lack of sufficient research that reliably examines the similarities or differences in the impact of different types, sizes, and shapes (fibers, fragments, spheres) of micro- and nanoplastics on various soil processes. However, a difference is presumed, and a precise description of all parameters of the selected micro/nanoplastic is essential to improve the reproducibility of the experiment

line 156= what is the dimension of the soil after sieving?

Response: the soil was sieved through a 1 mm sieve

line 156= what do you mean with morphology of soil, maybe characteristichs?

Response: by morphology, we meant the differing genesis of the studied soils, which we briefly described in subsection 2.1.

line 163= with modern marine clay do you mean from  Pleistocene?

Response: holocene lake sediments with a clay texture

line 178-179= where did you get the control soil? I didn't understand. Later you wrote you add plastic. I suggest to explain better

Response: the experimental design has been further clarified (lines 157-163)

line 184= I think Pravia stands for Pavia

Response: corrected

line 204= did you recycle tubes and caps? DId you wash with distilled water or bidistilled?

Response: the experimental design has been further clarified.

line 209= maybe three replicates for each samples

Response: the experimental design has been further clarified (lines 157-163).

line 271=what does pronouced in Podzoil soil. crobiologica studies mean? There are many mistakes in English, pay attention please

Response: corrected

line 342= composition is not changing, the type of microbiome is changing. Speaking about composition I may think to the chemical or mineralogical one, too. I suggest you to rewrite this sentence, please

Response: the sentence has been corrected

line 343= So it is not possible to say "composition" is changing

Response: the sentence has been corrected

line 350= is M2_NP or M2_MP? In caption and figure are different

Response: corrected

line 352-353= where is the principal sentence? what you wrote has no meaning

Response: duplicate information removed

line 353-355= this is already knonw

Response: duplicate information removed

359-361= I don't agree, maybe these soils are only not or less anthropogenic disturbed!

Response: we suggest that, among other things, it is anthropogenic impact and associated changes that may to some extent determine the response of the soil microbiome to the introduction of nanoplastics

English spelling and grammar has been revised in current version of the manuscript.

 

Sincerely yours,

Evgeny Abakumov,

Corresponding author.

Professor, Saint-Petersburg State University, Russia

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

1. Lines 31–36: Move the section starting with “Soil microorganisms… and other parameters)” to the beginning of the abstract for better context.

2. Abbreviate nanoplastics as NPs after first mention.

3. Line 90: “Selective enrichment of certain microbial taxa, such as Acidobacteriota,” is incomplete — please revise to complete the sentence.

4. Recheck citations: reference 22 (line 111) appears after reference 16 (line 101); reorder them chronologically.

5. Line 191: Change the section title “2.1. Microplastic preparation and application” to “2.2. Microplastic preparation and application.”

6. Line 228: Briefly describe the RIAM protocol.

7. Line 266: Remove the unnecessary statement “One of the… (OTUs)[47]”.

8. Lines 272–273: “In this regard, it is frequently employed in mi” is incomplete — please revise.

9. Lines 321 and 345: Correctly format the citations: “[10.1016/j.scitotenv.2023.167972]” and “[10.3390/ijms25010593, 10.1016/j.chemosphere.2024.142933]”.

10. Provide details on the raw sequence database and make it publicly accessible.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

We thank you for the time and effort you have dedicated to improving our manuscript.

Below, we address each comment:

1. Lines 31–36: Move the section starting with “Soil microorganisms… and other parameters)” to the beginning of the abstract for better context.

Response: We have restructured the abstract as suggested, placing the mentioned section at the beginning to provide clearer context.

1. Abbreviate nanoplastics as NPs after first mention.

Response: The term "nanoplastics" is now abbreviated as "NPs" after its first full mention, as recommended.

1. Line 90: “Selective enrichment of certain microbial taxa, such as Acidobacteriota,” is incomplete — please revise to complete the sentence.

Response: The phrase has been removed.

Recheck citations: reference 22 (line 111) appears after reference 16 (line 101); reorder them chronologically.

Response: The citations have been reordered chronologically to maintain proper sequencing.

1. Line 191: Change the section title “2.1. Microplastic preparation and application” to “2.2. Microplastic preparation and application.”

Response: The section numbering has been corrected to "2.2." as indicated.

1. Line 228: Briefly describe the RIAM protocol.

Response: We have added a concise description of the RIAM (Relative Impact Assessment Method) protocol

1. Line 266: Remove the unnecessary statement “One of the… (OTUs)[47]”.

Response: The redundant statement has been removed.

1. Lines 272–273: “In this regard, it is frequently employed in mi” is incomplete — please revise.

Response: The phrase has been removed.

1. Lines 321 and 345: Correctly format the citations: “[10.1016/j.scitotenv.2023.167972]” and “[10.3390/ijms25010593, 10.1016/j.chemosphere.2024.142933]”.

Response: The citations have been reformatted to journal-specific standards (e.g., author-year or numbered style, as required).

1. Provide details on the raw sequence database and make it publicly accessible.

Response: The raw sequencing data have been uploaded

Reviewer 2 Report

Comments and Suggestions for Authors

The cover letter does not address all the points raised in the reviewer’s report. In fact, issues that compromise the experimental design, as well as certain aspects of the discussion, remain unresolved. A significant number of the reviewer’s suggestions remain unaddressed by the authors, with no explanation provided in the cover letter. Among these, the ambiguous use of the terms microplastics and nanoplastics persists: although in multiple parts of the text microplastics has been replaced with nanoplastics, in other sections they are used interchangeably, including the term microplastics in the manuscript title. The figures still have inadequate resolution, and the discussion has not been expanded as recommended by the reviewer, leaving it vague and superficial. Given these, the reviewer is unable to recommend the publication of this manuscript at this stage.

Author Response

Dear Reviewer,

We sincerely appreciate your thorough evaluation and the time you have dedicated to improving our manuscript. We have made every effort to address all remaining concerns in this revised version.

The Discussion section has been significantly expanded to provide a deeper interpretation of the results and has been revised to better establish connections between our findings and broader implications.

The article title has been updated to ensure proper use of the term "nanoplastic" ("The effect of applying model nanoplastic particles to soil on the composition of its microbial community").

All figures have been saved in high resolution.

We apologize for the incompleteness of our previous response and hope these revisions now meet your expectations. Should any additional improvements be needed, we remain fully committed to addressing them.

Reviewer 3 Report

Comments and Suggestions for Authors

line 67-68= by whom? I think the reference is missing

line 107= I suggest you to describe better when you wrote more manageable molecules. IF they are going to be break down are they becaming nano-plastic particles?

line 107= I suggest you to describe better when you wrote more manageable molecules. IF they are going to be break down are they becaming nano-plastic particles?

line 167= it is better to write soil profile instead of soil morphology

line 176-177= lithology of the Entic Podzol (M3) is missing

line 210= I asked to explain this sentence also in the previous review. What do you mean same tubes and caps are the same for all of the experiments. Data are altered

Author Response

Dear Reviewer,

we thank you for your time and effort in reviewing our manuscript.

All revisions made in accordance with your comments are provided below.

 

line 67-68= by whom? I think the reference is missing

Response: the reference has been added

line 107= I suggest you to describe better when you wrote more manageable molecules. IF they are going to be break down are they becaming nano-plastic particles?

Response: degradation is possible, both into smaller microplastics and gradually into nanoplastics.

line 167= it is better to write soil profile instead of soil morphology

Response: corrected

line 176-177= lithology of the Entic Podzol (M3) is missing

Response: corrected

line 210= I asked to explain this sentence also in the previous review. What do you mean same tubes and caps are the same for all of the experiments. Data are altered

Response: clarified (to minimize variability in results, all experiments utilized tubes and caps made of identical material)

 

Sincerely Yours,

Professor

Evgeny Abakumov,

Dept. Applied Ecology,

Saint-Petersburg State University.