Microplastics’ Impact on the Environment and the Challenging Selection of Reliable Key Biomonitors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of: Microplastics impact on water and soil: recent advances in environmental biomonitoring and the challenging selection of key biomarkers and bioindicators

Water Journal

 

Recommendation: Major revision

 

Specific comments

Title

Biomonitors or biomarkers? Use the same term all through the manuscript as well as in the title.

I suggest revising the first part of title to better reflect the content of the paper.

 

Keywords

Do not repeat the same words as those presented in the title.

 

Abstract

The abstract shows promise but needs improvement in several areas.

L19 – 21: rephrase and keep it simpler.

L21 – 23: The importance of bioindicators and biomarkers in evaluating MPs can be introduced to readers.

L29 – 31: The results should be specific to the systems reviewed, rather than being more general.

L31 – 34: It is important to address the challenges associated with selecting key biomarkers and bioindicators and explore effective solutions.

 

Introduction

L65 – 67, 70 – 73: cite with appropriate references.

L79: To address the problem of plastic pollution around the world,…change

L84: please refer to and cite - https://doi.org/10.1016/j.foodres.2023.113840; https://doi.org/10.1016/j.envpol.2023.121961

The section focuses on microplastics, which is ideal. However, the core concept of biomonitors and bioindicators has not been introduced or discussed with the reader. Distinguish what are biomarkers and bioindicators. Authors are encouraged to include a separate paragraph emphasizing their significance in the field and how their contributions further research. This is a crucial aspect that distinguishes the paper and highlights its unique value.

 

The literature search to collect and analyze papers for the review must be added.

 

Section 2

L111 – 116: cite with appropriate references

L129 – 135: This study does not focus on field-based environmental monitoring of microplastics using plants as biomonitors. The example provided pertains more to the uptake of microplastics rather than air deposition on plants, which could be further analyzed. Replace it with the study that first employed plants as biomonitors for studying microplastics.

L136 – 138: rephrase, the sentence seems incomplete.

L138 – 149: which work is discussed here, cite please.

L146: how was the extraction done?

Before discussing this, it is important to understand how the  atmosphereically deposited microplastics were sampled from the plants. What methods are there and their limiations.

L153: Loppi et al. (2021) remove year and give the reference number

L153 – 155: It is premature to acknowledge the groundbreaking discovery until the results are presented. Please rephrase

L166: among various limitations ….what are they ? specify later move on to the next subject.

L178: remove “, as”

L186: spatial distribution of the ?.. fill the blank

L201 – 202: cite the three articles here

L209 – 211: The previous section primarily discussed the time period, which is important. However, it is also crucial to consider the number of samples required to accurately represent the study area and the optimal distance between each sample within the same area. Authors are encouraged to share their insights and perspectives on these aspects as well. The topic should be incorporated in section 2.3.

Table 1: change airborne microfibres to airborne microplastics

The study by Capozzi et al., 2018 can be omitted in the table 1

 

Section 3

L363 – 386: It is more generic; it can be condensed and combined with the opening paragraph provided above.

L408: What kind of comparison is this, considering that the two organisms are from different systems?

 

The authors should be mindful of when and how to introduce exposure studies in the main text. The title of Section 3 and 4 focuses on environmental monitoring, but their subsection shifts to discussing the effects of microplastics. Authors are urged to carefully address these things.

 

I would not agree entirely the title of section 4.1 effects of microplastics on mussels as it is a mixture of both field and laboraotry studies. The authors can separate them and present individually.

 

The critical issues as described in section 2.3. is missing for these sections 3 and 4.

 

Section 5 – as a reader it stands out and does not seems to fit in with the scope of the work that authors presented.

Author Response

Answer to Reviewer 1

[R: Reviewer's request; A: Authors reply]

R: Title. Biomonitors or biomarkers? Use the same term all through the manuscript as well as in the title. I suggest revising the first part of the title to better reflect the content of the paper. 

A: The title was revised: in the context of the proposed review, considered the preliminary data on the use of the described organisms to detect microplastics pollution, no specific definitions might be given, and as the reviewer suggested, it is not correct to use different terms, but rather it results more effective to describe the organisms with a unified description. The word “biomonitors” was added to the title, preferred to biomarkers and bioindicators. 

 

 R: Keywords. Do not repeat the same words as those presented in the title. 

A: The keywords were updated, according to the reviewer’s suggestion. 

 

 Abstract 

The abstract shows promise but needs improvement in several areas. 

R: L19 – 21: rephrase and keep it simpler. 

A: The sentence was rephrased, as requested: “The accumulation of MPs in various trophic levels mostly depends on weathering phenomena, their reduced dimensions and improved bioavailability: this ultimately causes their ingestion by organisms living in different niches.” 

 

R: L21 – 23: The importance of bioindicators and biomarkers in evaluating MPs can be introduced to readers. 

A: The abstract was revised to better focus on the aim of the review. 

 

R: L29 – 31: The results should be specific to the systems reviewed, rather than being more general. 

A: Done. 

 

R: L31 – 34: It is important to address the challenges associated with selecting key biomarkers and bioindicators and explore effective solutions. 

A: Done. 

 

Introduction 

L65 – 67, 70 – 73: cite with appropriate references. 

A: The appropriate references were cited. Authors thank the reviewer for the comment. 

 

 

R: L79: To address the problem of plastic pollution around the world,…change 

A: Authors are not able to find the referred paragraph and/or text, neither in the indicated line, or in the whole manuscript. 

 

R: L84: please refer to and cite: 

https://doi.org/10.1016/j.foodres.2023.113840; https://doi.org/10.1016/j.envpol.2023.121961 

A: The references were added to the section, as suggested by the reviewer, providing more information to the retrieval of microplastics in the environment, in particular in food. 

 

The section focuses on microplastics, which is ideal. However, the core concept of biomonitors and bioindicators has not been introduced or discussed with the reader. Distinguish what are biomarkers and bioindicators. Authors are encouraged to include a separate paragraph emphasizing their significance in the field and how their contributions further research. This is a crucial aspect that distinguishes the paper and highlights its unique value. 

A: Authors thank the reviewer for the suggestion and the opportunity to reflect on the focus of the review and agreed on the fact that it is not needed to differentiate the concepts, but rather generally indicating the term “biomonitors” for the species described. Considering the preliminary status of the research on microplastics, our main attention was focused on the selection of the species that might be identified as valuable biomonitors, that, in the future perspectives, might be considered bioindicators, or whose specific biomarkers might be identified. Some additional comments were added to the introduction to reinforce the concept and the aims of the review. 

 

 R: The literature search to collect and analyze papers for the review must be added. 

A: Using Scholar and Scopus search engines, the following keywords were searched: plants; biomonitor; bioaccumulation; mosses; lichens; microplastics; anthropogenic microfibers 

A: The literature search was integrated, as required. 

 

Section 2 

R: L111 – 116: cite with appropriate references 

A: The following articles were cited in the document, as requested: 

- Bates, J. W. (1992). Mineral nutrient acquisition and retention by bryophytes. Journal of Bryology, 17(2), 223-240.  

- Berg, T., Røyset, O., & Steinnes, E. (1995). Moss (Hylocomium splendens) used as biomonitor of atmospheric trace element deposition: estimation of uptake efficiencies. Atmospheric environment, 29(3), 353-360. 

- Nieboer, E., Richardson, D. H. S., & Tomassini, F. D. (1978). Mineral uptake and release by lichens: an overview. Bryologist, 226-246. 

- Branquinho, C. (2001). Lichens. In Metals in the Environment (pp. 137-178). CRC Press. 

 

R: L129 – 135: This study does not focus on field-based environmental monitoring of microplastics using plants as biomonitors. The example provided pertains more to the uptake of microplastics rather than air deposition on plants, which could be further analyzed. Replace it with the study that first employed plants as biomonitors for studying microplastics. 

A: L129-138: Although the study by Capozzi et al 2018 did not deal with the air "matrix", it is the first evidence that mosses have the ability to retain nanoplastics and, since mosses are known as biomonitors of air pollutants, it was cited in the text to underline their possible role as biomonitors of airborne MFs. Therefore, we would keep the citation; the same reference was used indeed, for the same reason by Roblin & Aherne (2020), in their first work based on biomonitoring of airborne atmospheric microfibers. 

 

R: L136 – 138: rephrase, the sentence seems incomplete. 

A: We rephrased: “proved the feasibility of using the naturally growing Hylocomium splendidens (Hedw.) Schimp. for the biomonitoring of airborne anthropogenic MFs.” 

 

R: L138 – 149: which work is discussed here, cite please. 

A: The work was already cited:  Roblin & Aherne (2020). Please refer to the manuscript, where the references were updated 

 

R: L146: how was the extraction done? 

A: The extraction was done using a wet peroxide oxidation (WPO), we added this into the text. 

 

R: Before discussing this, it is important to understand how the  atmosphereically deposited microplastics were sampled from the plants. What methods are there and their limiations. 

A: This is precisely the point, before this article (Roblin & Aherne (2020) there are no bibliographic references that suggest different extraction methods or protocols to extract microplastics accumulated by plants. The research on the sampling of airborne MPs is at its beginning. 

 

R: L153: Loppi et al. (2021) remove year and give the reference number 

A: Done 

 

R: L153 – 155: It is premature to acknowledge the groundbreaking discovery until the results are presented. Please rephrase 

A: We rephrased: “The authors showed that the thalli of native F. caperata collected near the landfill accumulated a greater number of MPs compared to those collected from more distant sites, indicating the lichen ability to highlight a gradient in the MFs fallout. 

 

R: L166: among various limitations ….what are they ? specify later move on to the next subject. 

A: Passive biomonitoring techniques would require extensive treatment in a separate chapter, and it is not the focus of this review. We rephrased to make the concept less vague. “The non-homogeneous distribution of the target species in the study area limit the use of passive biomonitoring (i.e., based on the harvesting of native material).” 

 

R: L178: remove “, as” 

A: Done. 

 

R: L186: spatial distribution of the ?.. fill the blank 

A: Done. 

 

R: L201 – 202: cite the three articles here 

A: Done. 

 

R: L209 – 211: The previous section primarily discussed the time period, which is important. However, it is also crucial to consider the number of samples required to accurately represent the study area and the optimal distance between each sample within the same area. Authors are encouraged to share their insights and perspectives on these aspects as well. The topic should be incorporated in section 2.3. 

A: The minimum number of sampling points and replicates, but also the repetition over time of the monitoring campaign, are critical points for the use of moss and lichen bags and this regardless of the target pollutant. According to some authors, the minimum number of sampling sites must be calculated based on the equations available for calculating adequate sample sizes (e.g. Elzinga et al., 2001; ISPRA, Manuals and Guidelines 189/2019 ISBN 978-88-448-0966-9). To this purpose, it is always useful to collect preliminary information such as data variability in a study area. As for the number of replicas for each site, a minimum number of 3 bags per sampling site placed at the centre of an area with a radius of 50 m is considered by many authors to be adequate (e.g. Elzinga et al., 2001; ISPRA, Manuals and Guidelines 189/2019 ISBN 978-88-448-0966-9). 

 

R: Table 1: change airborne microfibres to airborne microplastics 

A: Done. 

 

R: The study by Capozzi et al., 2018 can be omitted in the table 1 

 A: Authors prefer to keep it, see the above response. 

 

Section 3 

R: L363 – 386: It is more generic; it can be condensed and combined with the opening paragraph provided above. 

A: The authors thank the reviewer for the suggestion. We have reduced this part and combined it with the previous paragraph.  

 

R: L408: What kind of comparison is this, considering that the two organisms are from different systems? 

A: The comparison reported is relative to a study of Panebianco et al. (reference [52]) on some edible land snails for which the authors demonstrated MPs bioaccumulation. In their conclusions, they compared the results with that of other mollusks much consumed by humans as the mussels also if are marine species. From this comparison, Panebianco et al. have evidenced that land snail as MPs bioaccumulators is less dangerous for human health because they are consumed little by humans. 

 

 The authors should be mindful of when and how to introduce exposure studies in the main text. The title of Section 3 and 4 focuses on environmental monitoring, but their subsection shifts to discussing the effects of microplastics. Authors are urged to carefully address these things. 

 I would not agree entirely the title of section 4.1 effects of microplastics on mussels as it is a mixture of both field and laboraotry studies. The authors can separate them and present individually. 

The critical issues as described in section 2.3. is missing for these sections 3 and 4. 

A: Thank you for this comment. We have modified the text of paragraph 3 adding the critical issues to paragraph 3.2: 3.2 Critical issues in biomonitoring of MPs by land snails. Regarding section 4.1 as suggested by the reviewer, we have split the text into two subsections 1) environmental exposure 2) laboratory exposure.  

 

 R: Section 5 – as a reader it stands out and does not seems to fit in with the scope of the work that authors presented. 

A: Authors retain that Section 5, and in particular the exploration of how microplastics pollution impact microbial communities poses a notable strength to the manuscript itself, particularly concerning biofilm formation and AMR. This section represents the real challenge on which several scientists working on microplastics is focusing, thus representing, in authors’ opinion, the innovative insights in the field of microplastics monitoring. In a One Health perspective, although the identification and definition of biomonitors is essential to assess the chemical risk for humans and animals, the metagenomic study of aquatic and terrestrial environments’ microbiota might constitute the most efficient mean to evaluate the microbiological risks connected to the presence of the pathogens on microplastics, aided by the extreme persistence of MPs in the above mentioned habitats. In the context of the manuscript, at microorganisms' level, the outcomes coming from the microplastics’ biofilm composition might constitute a biomarker of the potential hazards related to the persistence of plastic pollutants in the described habitats. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors in their article presented an overview of selected issues related to the effects of exposure to microplastics of selected representatives of plants and animals, as well as a certain microbiological aspect. The individual information is very interesting, but does not give the impression that it makes a coherent whole. I am not inclined to recommend total rejection of the article, but I recommend quite a significant rethinking of its content. The main suggestions are as follows:

First of all - the title does not correspond to the content of the review. The first part of the title is very extensive and this makes it unrealistic to fulfill its assumptions. Impact on water? In other words, on what? Physicochemical parameters of water were not what the authors had in mind. Even if they had meant aquatic ecosystems and included this in the title, the content is limited to two aspects: toxicity towards only one group of aquatic animals: mussels and transfer of antimicrobial resistance in the aquatic environment. In my opinion, this does not exhaust the announced "impact on water".

Similarly, impact on soil: the issue of soil, in my opinion, has not been fully exhausted either. Epigeic animals, such as land snails, are not soil animals and in my opinion are not an appropriate model for analyzing the impact of microplastics on soil. The second fragment in which the Authors refer to soil is the one where the weathering of plastic is mentioned. However, this is still not "impact on soil". Instead, the Authors devote a lot of space to microplastics as a component of airborne particles, associating them rather with monitoring using plants, which has no direct connection with soil and water.

In turn, the issue of biofilm and other genetic issues related to bacteria is an extremely interesting and innovative issue in itself, which is worth developing, in my opinion, as a completely separate review.

In the form in which the manuscript was submitted for review, it is rather a compilation of separate issues - independently very interesting and valuable, but not clearly connected by a common main idea.

To make it easier to organize the content, I would suggest trying to put the aim at the end of the introductory part. This would make it easier to keep a coherent narrative ending with a conclusion that closely corresponds to the aim. In its current form, the conclusion is more of a summary of the most important content of the article.

 

Comments on the Quality of English Language

Finally, I would suggest a linguistic revision: the English is generally understandable, but some sentences are very long and complex.

Author Response

Answer to Reviewer 2

[R: Reviewer's request; A: Authors reply]

The authors in their article presented an overview of selected issues related to the effects of exposure to microplastics of selected representatives of plants and animals, as well as a certain microbiological aspect. The individual information is very interesting, but does not give the impression that it makes a coherent whole. I am not inclined to recommend total rejection of the article, but I recommend quite a significant rethinking of its content. The main suggestions are as follows: 

First of all - the title does not correspond to the content of the review. The first part of the title is very extensive and this makes it unrealistic to fulfill its assumptions. Impact on water? In other words, on what? Physicochemical parameters of water were not what the authors had in mind. Even if they had meant aquatic ecosystems and included this in the title, the content is limited to two aspects: toxicity towards only one group of aquatic animals: mussels and transfer of antimicrobial resistance in the aquatic environment. In my opinion, this does not exhaust the announced "impact on water". 

Similarly, impact on soil: the issue of soil, in my opinion, has not been fully exhausted either. Epigeic animals, such as land snails, are not soil animals and in my opinion are not an appropriate model for analyzing the impact of microplastics on soil. The second fragment in which the Authors refer to soil is the one where the weathering of plastic is mentioned. However, this is still not "impact on soil". Instead, the Authors devote a lot of space to microplastics as a component of airborne particles, associating them rather with monitoring using plants, which has no direct connection with soil and water. 

In turn, the issue of biofilm and other genetic issues related to bacteria is an extremely interesting and innovative issue in itself, which is worth developing, in my opinion, as a completely separate review. 

In the form in which the manuscript was submitted for review, it is rather a compilation of separate issues - independently very interesting and valuable, but not clearly connected by a common main idea. 

To make it easier to organize the content, I would suggest trying to put the aim at the end of the introductory part. This would make it easier to keep a coherent narrative ending with a conclusion that closely corresponds to the aim. In its current form, the conclusion is more of a summary of the most important content of the article. 

A: Authors thank the reviewer for the useful and essential comments and suggestions. The entire document was entirely revised, to make the review more comprehensible and to create a “path” and define a common aim among the different contributions. 

As it is noticeable from the Abstract, we attempted to better define the rationale that brought us to describe specific species or biological models: 

“The identification and selection of appropriate biomonitors for MPs pollution risk assessment should focus on the identification of easy-to-implement assays, rapidly interpretable results (e.g. based on the MPs bioaccumulation capabilities in their tissues), and standardizable methodologies. The present review analyzed some emerging bioindicators exploited for MPs evaluation, selected and examined according to the potential use as specific biological indicators for diverse environments.” 

The same approach was applied in the Introduction and Conclusions sections, passing through the specific sections, where further paragraphs focused on the detection of critical issues, advantages, limitations and challenges connected to the potential employment of the described species as biomonitors to assess the risk connected to MPs pollution in the various environments. We hope that the text is more fluid and that the scope of the work is more clearly articulated. 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The MS aims to review ways in which microplastics affect various environments, particularly antimicrobial resistance. The MS well summarizes the interactions between microplastics and microbial communities and provides valuable insights into their environmental impact. In general, the text is well-organized covering a wide array of topics related to microplastics. A notable strength is its exploration of how MPs impact microbial communities, particularly concerning biofilm formation and AMR. This is an important aspect that adds depth to the review MS.

 

However, there are several issues to be addressed before the paper could better serve its purpose and enhance its overall quality:

- The paper would benefit from a more critical look at the limitations and gaps in the existing research. Authors should address potential issues such as biases in sampling, inconsistencies in detection methods, and how these factors might influence the study results.

- The discussion on using plants as indicators for microplastics is limited and lacks clarity. Although the potential for plant-based biomonitoring is mentioned, there isn’t enough detail on the current challenges and limitations. A deeper analysis of existing research and methodologies would strengthen this section.

- The section on terrestrial ecosystems and the soil plastisphere should be improved using a more detailed exploration of soil variability. Although soil heterogeneity is briefly mentioned, there are no details on how different soil types might affect the behavior and effects of microplastics. A more thorough examination of this aspect would enhance the understanding of microplastic interactions in various soil conditions. - the sections covering aquatic environments, including rivers and seas, but it lack a critical review of the methodologies used in the studies discussed. A more detailed critique of approaches such as metagenomic and metatranscriptomic sequencing would provide better insight into the reliability and applicability of these findings.

- Although the study summarizes individual studies well but does not fully connect these findings to present a unified picture of how MPs impact environmental health. The MS would benefit from a more cohesive discussion that ties together the findings from different studies. This will offer clearer implications for future research and policy.

- Most of the reference studies are from Italy and many self-citations were included. I suggest reviewing a broader range of studies from different regions to provide a more comprehensive global perspective.

- Some sections and sentences (like the detailed description of mosses) seem less relevant to the main topic and only make the MS longer. I suggest focusing on the key issues and removing less pertinent details would improve clarity and relevance.

- In general, the review lacks a distinct original contribution. I suggest highlighting new insights or emerging trends to add originality and distinguish this review from others already published in the field.

Author Response

Answer to Reviewer 3 

[R: Reviewer's request; A: Authors' reply]

The MS aims to review ways in which microplastics affect various environments, particularly antimicrobial resistance. The MS well summarizes the interactions between microplastics and microbial communities and provides valuable insights into their environmental impact. In general, the text is well-organized covering a wide array of topics related to microplastics. A notable strength is its exploration of how MPs impact microbial communities, particularly concerning biofilm formation and AMR. This is an important aspect that adds depth to the review MS. 

  

However, there are several issues to be addressed before the paper could better serve its purpose and enhance its overall quality:  

 

R: - The paper would benefit from a more critical look at the limitations and gaps in the existing research. Authors should address potential issues such as biases in sampling, inconsistencies in detection methods, and how these factors might influence the study results. 

 A: As further indicated, each section was integrated with further discussion and the presentation of critical issues. In some cases, a specific paragraph was added. Additionally, the conclusions were integrated with critical comments on the difficulties in implementing the proposed methods and the possible solutions. 

 

R: - The discussion on using plants as indicators for microplastics is limited and lacks clarity. Although the potential for plant-based biomonitoring is mentioned, there isn’t enough detail on the current challenges and limitations. A deeper analysis of existing research and methodologies would strengthen this section. 

A: The use of plants as biomonitors of anthropogenic microplastic is in its beginnings. In section 2 the aims are to highlight the state of the art and suggest future developments starting from the few articles published so far. Following your suggestion, we have implemented the section 2 and added new references that have been published after the first draft of this review. 

 

R: - The section on terrestrial ecosystems and the soil plastisphere should be improved using a more detailed exploration of soil variability. Although soil heterogeneity is briefly mentioned, there are no details on how different soil types might affect the behavior and effects of microplastics. A more thorough examination of this aspect would enhance the understanding of microplastic interactions in various soil conditions.  

A: The manuscript was integrated in the terrestrial ecosystems section. The modifications and the integrated citations are available in Section 5. 

 

R: - the sections covering aquatic environments, including rivers and seas, but it lack a critical review of the methodologies used in the studies discussed. A more detailed critique of approaches such as metagenomic and metatranscriptomic sequencing would provide better insight into the reliability and applicability of these findings. 

A: The manuscript was integrated in the terrestrial ecosystems section. The modifications and the integrated citations are available in Section 5. 

 

R: - Although the study summarizes individual studies well but does not fully connect these findings to present a unified picture of how MPs impact environmental health. The MS would benefit from a more cohesive discussion that ties together the findings from different studies. This will offer clearer implications for future research and policy. 

A: The manuscript was thouroughly revised and integrated to present the contents in a more unified and comprehensive way.  and Introduction and Conclusions were revised aiming at merging the unique scope of the review. 

 

R: - Most of the reference studies are from Italy and many self-citations were included. I suggest reviewing a broader range of studies from different regions to provide a more comprehensive global perspective. 

A: As for plants, only 15 works have been published so far, 3 of which belong from some of the co-authors of this review. As for mussels, 6 papers belong from some of the co-authors of this review, while 2 manuscripts on land snails have been published by the authors of this review. Over the 143 total citations, 11 (less than 10%) are self-citations. Self-citations are connected to the fact that authors mostly work in the field on which the present manuscript is focused, and this is one of the reasons why they decided to start joining forces, collaborate in the future on the topic, just starting collaborating from this review. 

 

R: - Some sections and sentences (like the detailed description of mosses) seem less relevant to the main topic and only make the MS longer. I suggest focusing on the key issues and removing less pertinent details would improve clarity and relevance. 

A: The description of the structure of the thalli and of the structure of the leaves of higher plants is functional to explain the possible differences in their capacity to accumulate and retain microplastics, therefore we believe it is correct to keep them in the text. 

 

R: - In general, the review lacks a distinct original contribution. I suggest highlighting new insights or emerging trends to add originality and distinguish this review from others already published in the field. 

A:  Each section was integrated with further discussion and the presentation of critical issues, Authors retain that Section 5, and in particular the exploration of how microplastics pollution impact microbial communities poses a notable strength to the manuscript itself, particularly concerning biofilm formation and AMR. This section represents the real challenge on which several scientists working on microplastics are focusing, thus representing, in authors’ opinion, the innovative insights in the field of microplastics monitoring. In a One Health perspective, although the identification and definition of biomonitors is essential to assess the chemical risk for humans and animals, the metagenomic study of aquatic and terrestrial environments’ microbiota might constitute the most efficient mean to evaluate the microbiological risks connected to the presence of the pathogens on microplastics, aided by the extreme persistence of MPs in the above mentioned habitats. In the context of the manuscript, at microorganisms' level, the outcomes coming from the microplastics’ biofilm composition might constitute a biomarker of the potential hazards related to the persistence of plastic pollutants in the described habitats. 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

There are no additional comments to add. The manuscript has been greatly improved and is suggested for acceptance and publication. 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors revised the MS by addressing the feedback provided. More critical discussions have now been added to discuss possible problems such as sampling biases and inconsistent detection techniques. The revised MS's conclusions address the difficulties in putting the suggested strategies into practice and potential fixes. The part on employing plants as microplastic indicators has been also improved. New updated references as well as more information about the present difficulties and constraints have also been provided to give a more accurate perspective of the current state of the art in this field. The discussion on terrestrial ecosystems and the soil plastisphere, has also been expanded offering a more detailed look at soil variability. The revised MS now delves deeper into how different soil types might affect the behaviour and effects of microplastics, enhancing our understanding of these interactions in various soil conditions. In the sections on aquatic environments, the authors have incorporated the suggestions by providing a more detailed critique of methodologies such as metagenomic and metatranscriptomic sequencing making the review more comprehensive. Both the introduction and conclusions have been reworked to tie the content better together, providing clearer implications for future research and policy.

 

The MS can be accepted for publication in this form.