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Communication
Peer-Review Record

Differential Accumulation of Particulate Pollutants in Gills and Gastrointestinal Tracts in Sphoeroides Fish from Tropical and Subtropical Estuaries in Brazil

Diversity 2025, 17(4), 300; https://doi.org/10.3390/d17040300
by Sérgio Murilo de Souza Filho 1,2, Marco Tadeu Grassi 3,4, Mayara Padovan dos Santos 3,4, Juliano Morimoto 2,5, Marcelo Soeth 6,7 and Luís Fernando Fávaro 1,2,*
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Reviewer 4: Anonymous
Diversity 2025, 17(4), 300; https://doi.org/10.3390/d17040300
Submission received: 14 February 2025 / Revised: 18 April 2025 / Accepted: 19 April 2025 / Published: 21 April 2025
(This article belongs to the Special Issue Socioecology and Biodiversity Conservation—2nd Edition)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I'm sorry, but the manuscript cannot be accepted in this state.

 

Line 56-58. It is noted that  «Microplastics can also act as carriers for persistent organic pollutants, heavy metals, and pathogenic microorganisms, further exacerbating their ecological impact (Nelms et al., 2018; Shen et al., 2019).». However, the mentioned sources do not contain information confirming the transfer of persistent organic pollutants and heavy metals on microplastics.

 

Figure 2. “provides examples of microplastics”. There is no evidence that all of these particles are microplastics. They could be fragments of cotton threads or some other formations. They could even be hairs, not threads.

Plastic is a material that comes from synthetic or human-made organic compounds containing carbon, often made with petroleum. There is no evidence in this work that the particles observed under the optical microscope are actually plastics. Modern research methods allow us to determine, what kind of plastic each particle consists of. For this purpose, µFTIR or Raman spectroscopy can be used. Pyrolysis with gas chromatography - mass detection method is also used.

Yes, such researches are painstaking and require significant effort, when each suspicious particle is examined and the resulting spectrum is compared with the spectrum in the database. But it is necessary work.

The authors refer to the microplastics determination protocol of Karami et al. (2017). However, the specified protocol used micro-Raman spectroscopy and elemental analysis with energy-dispersive X-ray spectroscopy (EDX). You can also read there, that 59.0% of 61 isolated particles were plastic polymers. Failure to comply with this protocol gives almost doubly overestimated value for the content of plastic particles.

It is also not specified how “Contamination control” was carried out in the lab or whether it was carried out.

 

Thus, the lack of precise identification of the objects under study does not allow us to make any subsequent calculations and conclusions.

Author Response

Our responses are given in bold text.

I'm sorry, but the manuscript cannot be accepted in this state. Line 56-58. It is noted that  «Microplastics can also act as carriers for persistent organic pollutants, heavy metals, and pathogenic microorganisms, further exacerbating their ecological impact (Nelms et al., 2018; Shen et al., 2019).». However, the mentioned sources do not contain information confirming the transfer of persistent organic pollutants and heavy metals on microplastics. The references have been reviewed and replaced.

 

Figure 2. “provides examples of microplastics”. There is no evidence that all of these particles are microplastics. They could be fragments of cotton threads or some other formations. They could even be hairs, not threads. We can remove the figure; however, all precautions were taken in the laboratory to prevent cross-contamination. Even though only visual analysis under a stereomicroscope was used, it is possible to distinguish different materials.

 

Plastic is a material that comes from synthetic or human-made organic compounds containing carbon, often made with petroleum. There is no evidence in this work that the particles observed under the optical microscope are actually plastics. Modern research methods allow us to determine, what kind of plastic each particle consists of. For this purpose, µFTIR or Raman spectroscopy can be used. Pyrolysis with gas chromatography - mass detection method is also used. Yes, such researches are painstaking and require significant effort, when each suspicious particle is examined and the resulting spectrum is compared with the spectrum in the database. But it is necessary work. The authors refer to the microplastics determination protocol of Karami et al. (2017). We changed it to: “The methodology used in processing the biological material for microplastic analysis was modified e based on studies of Karami, et al. (2017) and Pegado et al., (2018).

However, the specified protocol used micro-Raman spectroscopy and elemental analysis with energy-dispersive X-ray spectroscopy (EDX). You can also read there, that 59.0% of 61 isolated particles were plastic polymers. Failure to comply with this protocol gives almost doubly overestimated value for the content of plastic particles. It is also not specified how “Contamination control” was carried out in the lab or whether it was carried out.  Thus, the lack of precise identification of the objects under study does not allow us to make any subsequent calculations and conclusions. There is still no perfect and 100% efficient methodology. Raman spectroscopy also has limitations that may prevent accurate readings for various reasons, such as very small particles degrading under the strong laser exposure or fluorescence interference due to dyes used in plastic production. Therefore, while one methodology may overestimate, another may underestimate. We have included this issue in the discussion, citing the relevant study Araujo et al., 2018.

Reviewer 2 Report

Comments and Suggestions for Authors

This paper offers a comparison of microplastic contamination in Brazilian subtropical (Babitonga Bay) and tropical (Porto Seguro) estuaries Sphoeroides fish. Considering the growing concern about microplastics in aquatic environments, the work is important and timely. The authors emphasize the impact of regional human activities on contamination levels by offering insightful analysis of the varying accumulation of microplastics in fish gills and gastrointestinal systems. The work might be improved in a few areas, nonetheless, especially in terms of methodological clarity, statistical analysis, and result discussion. Considering the noted errors and suggested improvements. I suggest a major revision.

 

  1. The background on microplastics in estuaries given in the beginning is decent. The writers can improve the explanation for emphasizing Sphoeroides fish by including more detailed details on their ecological function and the reasons for their especially sensitivity to microplastics intake. Introduction is not much sufficient.

 

  1. The cited references in introduction part are too old. It is advisable to consider recent references such as https://doi.org/10.1016/j.marpolbul.2022.113590, https://doi.org/10.1016/j.psep.2023.10.002, https://doi.org/10.1016/j.marpolbul.2022.113590 .

 

  1. Line 106~ The genus name is misspelled as Sphaeroides instead of Sphoeroides. This error occurs multiple times throughout the manuscript and should be corrected.

 

  1. It is not quite apparent how microplastics should be categorized. The authors declare they divided microplastics into three size ranges: ≤1 mm, 1.1-3 mm, and 3.1-5 mm. Still, the justification for choosing these particular size ranges should come from references to relevant literature.

 

  1. Although the authors used two-way ANOVA, they subsequently note that the data did not satisfy homoscedasticity or normalcy presumptions. If normalcy cannot be attained by transformation, the authors must consider using a different model such as Generalized Linear Model (GLM) or non-parametric tests like the Kruskal-Wallis test instead of ANOVA.

 

  1. How were microplastics distinguished from natural particles (e.g., algae, sediment)?

 

  1. Were chemical tests (e.g., FTIR or Raman spectroscopy) used to confirm the polymer types of the microplastics?

 

  1. The authors stipulate that particles larger than 5 mm were excluded, however, they do not provide a justification for this exclusion. Since larger particles can also pose risks, it is important to provide an explanation for this decision.

 

  1. The authors indicate that 92.11% of fish samples contained microplastics, with only one specimen from Porto Seguro showing no detectable microplastics. This appears contradictory, as a contamination rate of 92.11% suggests the presence of multiple fish devoid of microplastics. Please elucidate this inconsistency.

 

 

  1. Did the authors identify whether the microplastics came from polyethylene (PE), polypropylene (PP), or polystyrene (PS)? This is very important point. If not, it recommended to check and add analysis results with some explanation.

 

Author Response

Our responses are given in bold text below.

 

 

This paper offers a comparison of microplastic contamination in Brazilian subtropical (Babitonga Bay) and tropical (Porto Seguro) estuaries Sphoeroides fish. Considering the growing concern about microplastics in aquatic environments, the work is important and timely. The authors emphasize the impact of regional human activities on contamination levels by offering insightful analysis of the varying accumulation of microplastics in fish gills and gastrointestinal systems. The work might be improved in a few areas, nonetheless, especially in terms of methodological clarity, statistical analysis, and result discussion. Considering the noted errors and suggested improvements. I suggest a major revision. Thank you for the kind comments who helped us improve the manuscript. 

 

  • The background on microplastics in estuaries given in the beginning is decent. The writers can improve the explanation for emphasizing Sphoeroides fish by including more detailed details on their ecological function and the reasons for their especially sensitivity to microplastics intake. Introduction is not much sufficient. The details requested by the reviewer can be found in the discussion section of this study. The authors deemed it more important to include these details in the discussion in order to relate them to the results. Placing the information in the introduction would make the paper repetitive.

 

  • The cited references in introduction part are too old. It is advisable to consider recent references such as https://doi.org/10.1016/j.marpolbul.2022.113590, https://doi.org/10.1016/j.psep.2023.10.002, https://doi.org/10.1016/j.marpolbul.2022.113590Thank you very much for pointing out the references, which have been incorporated into the manuscript text (Introduction and Discussion).

 

  • Line 106~ The genus name is misspelled as Sphaeroides instead of Sphoeroides. This error occurs multiple times throughout the manuscript and should be corrected.

Apologies, this has been corrected throuhgout.

 

  • It is not quite apparent how microplastics should be categorized. The authors declare they divided microplastics into three size ranges: ≤1 mm, 1.1-3 mm, and 3.1-5 mm. Still, the justification for choosing these particular size ranges should come from references to relevant literature. Particles with a maximum size of 5mm are considered microplastics. Thus, according to Bessa et al. (2018), we established the length classes. Please refer to the text."The microplastics observed in the biological samples were measured in their largest axis, characterized and classified into: fragments (irregular shape with irregular surface), fibers (thin and elongated) and films (thin flap plane), according by Karami et (2017). They were also classified as size: ≤1 mm; 1.1-3 mm and 3.1-5 mm (Bessa et al., 2018). Plastic particles larger than 5 mm in size were not considered in the analyses.”

 

  • Although the authors used two-way ANOVA, they subsequently note that the data did not satisfy homoscedasticity or normalcy presumptions. If normalcy cannot be attained by transformation, the authors must consider using a different model such as Generalized Linear Model (GLM) or non-parametric tests like the Kruskal-Wallis test instead of ANOVA. Thank you for raising this importante point. ANOVA is proven to be a robust test in non-normally distributed data (see e.g. Blanca Mena, M. J., Alarcón Postigo, R., Arnau Gras, J., Bono Cabré, R., & Bendayan, R. (2017). Non-normal data: Is ANOVA still a valid option?. Psicothema, 2017, vol. 29, num. 4, p. 552-557.). Alternative models such as  Kurskal Wallis, suggested by the reviewer, is a better option only the data is very distinct from normality (e.g., following a Beta distribution with parameters alpha = beta = 0.5; Lantz, Björn. "The impact of sample non‐normality on ANOVA and alternative methods." British Journal of Mathematical and Statistical Psychology66, no. 2 (2013): 224-244.). This is not the case in our studyNote that our statistical significance tests are extremely strong (i.e., p <0 .001) and our effect sizes support these effects (see information across all Tables). Thus, our results are robust to whichever test we use, including non-parametric ones like Kruskal Wallis. We therefore would like to retain our original analysis.

 

  • How were microplastics distinguished from natural particles (e.g., algae, sediment)? Were chemical tests (e.g., FTIR or Raman spectroscopy) used to confirm the polymer types of the microplastics? Did the authors identify whether the microplastics came from polyethylene (PE), polypropylene (PP), or polystyrene (PS)? This is very important point. If not, it recommended to check and add analysis results with some explanation. Microplastics were detected solely using a stereomicroscope. Due to logistical issues and lack of infrastructure, we were unable to employ more refined methodologies. The expertise gained from working extensively with histological analyses facilitated the process.There is still no perfect and 100% efficient methodology. Raman spectroscopy also has limitations that may prevent accurate readings for various reasons, such as very small particles degrading under the strong laser exposure or fluorescence interference due to dyes used in plastic production. Therefore, while one methodology may overestimate, another may underestimate. We have included this issue in the discussion, citing the relevant study Araujo et al., 2018.

 

  • The authors stipulate that particles larger than 5 mm were excluded, however, they do not provide a justification for this exclusion. Since larger particles can also pose risks, it is important to provide an explanation for this decision. The explanation is that microplastics are characterized by a maximum size of 5mm. Since the study focuses on microplastics, we preferred to maintain the focus of the analysis on particles of these dimensions.

 

  • The authors indicate that 92.11% of fish samples contained microplastics, with only one specimen from Porto Seguro showing no detectable microplastics. This appears contradictory, as a contamination rate of 92.11% suggests the presence of multiple fish devoid of microplastics. Please elucidate this inconsistency. A total of 39 specimens were obtained, resulting in 78 biological samples. One uncontaminated specimen (2 uncontaminated samples) left 76 remaining samples. Out of these 76 samples (100%), only 70 were contaminated, representing 92.1% of the total biological samples analyzed.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript deals with the contamination of two estuarine ecosystems by particulate pollutants. The topic is interesting, in the context of the worldwide contamination with those pollutants and their deleterious effects.

Here I am willing to raise a few concerns of mine regarding the quality of the manuscript.

In the abstract: please use consistent significant figures to present the obtained results.

In order to increase confidence in the results obtained, please detail the experimental part.

The authors mentioned that the methodology for particulate pollutants analysis followed protocols from Karami et al. (2017) and Pegado et al. (2018). In order to analyse the microplastics in fish, Karami et al. (2017) used Raman spectroscopy and FESEM-EDX and Pegado et al. (2018) used the ATR-FTIR analysis. The authors declared that they used only a stereomicroscope, this is a visual identification. The authors should add the type, producer, country of origin and the magnification of the microscope. Please mention what you used to take pictures? Also, please mention the precision of the measurement in order to categorize by size the particulate pollutants.

The authors should add the concentration of formaldehyde, the producer and country of origin of ethanol and formalin.

Page 5: The equations should be numbered.

The comments in the caption of the tables 2 and 3 would be more appropriate to be located in the text, not in the captions.

In the tables, please use a period, and not a comma to separate the decimal places.

Page 10, lines 262-270: there are some phrases that are repeated. Please correct that.

Line 281: please finish the sentence.

Lines 279-281: there are some phrases that are repeated in lines 298-299. Please correct that.

For the 5th affiliation, I recommend the English name of the institution. For the affiliations nos. 5, 6, 7, I recommend adding the name of the country.

Author Response


The manuscript addresses the contamination of two estuarine ecosystems by particulate pollutants. The topic is relevant, considering the global contamination by these pollutants and their harmful effects. Here, I would like to raise some concerns regarding the quality of the manuscript.

Response: Thank you for the appraisal of our work and for the time to make useful comments that improved the readability of the paper.

Abstract: Use consistent significant figures to present the results.
Response: Done as requested by the reviewer.

To increase confidence in the results, please provide more details on the experimental section.
Response: Done as requested by the reviewer..

The authors mentioned that the methodology for analyzing particulate pollutants followed the protocols of Karami et al. (2017) and Pegado et al. (2018). Karami et al. (2017) used Raman spectroscopy and FESEM-EDX, and Pegado et al. (2018) used ATR-FTIR analysis.
Response: As explained in lines 151 to 156, the methodology used in our study was based on and modified from the protocols of Karami et al. (2017) and Pegado et al. (2018) to fit the nature of our data.

The authors stated that they used only a stereomicroscope, which is a visual identification method. Please add the model, manufacturer, and country of origin.
Response: Done as requested by the reviewer..

Please indicate what was used to take the photographs.
Response: Done as requested by the reviewer..

The authors should add the concentration of formaldehyde, and the manufacturer and country of origin for both ethanol and formalin.
Response: Done as requested by the reviewer..

Page 5: Equations should be numbered.
Response: Done as requested by the reviewer..

The comments in the captions of Tables 2 and 3 would be more appropriate if included in the main text instead of the figure captions.
Response: Done as requested by the reviewer..

In the tables, use a period instead of a comma to separate decimal places.
Response: Done as requested by the reviewer..

Page 10, lines 262–270: Some sentences are repeated. Please correct.
Response: We have now reworded the section for conciseness.

Line 281: Please complete the sentence.
Response: This sentence has now been deleted following comments from the reviewer.

Lines 279–281: Some sentences are repeated in lines 298–299. Please correct.
Response: See response to comment above.

For the 5th affiliation, we recommend using the English name of the institution. For affiliations 5, 6, and 7, we recommend adding the country name.
Response: Done as requested by the reviewer.

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

I have read and reviewed the study “Differential Accumulation of Microplastics/Particulate pollutants in Gills and Gastrointestinal Tracts in Sphoeroides Fish from Tropical and Subtropical Estuaries in Brazil”. Overall, it is very interesting study. Here are my specific suggestions:

  1. I would rather suggest to keep the “microplastic” instead on “particulate pollutants” if you really want to refer to small plastic particles, not metal/wood/glass. I really think it’s not appropriate to use the term “particulate pollutants” if you refer to plastic
  2. Why “bleaching” in the keywords? I would suggest to change it also you state “freshwater” but at my knowledge Porto Seguro Estuary even Babitonga Bay Estuary is not an entirely and 100% defined as freshwater, Please revise
  3. Line 91 – please insert citations at the end of the sentence
  4. Line 116-131 and 151-156 – please rewrite the entire sentence and make it shorter, keep the unnecessary explanatory things for Discussion section
  5. M&M - Please state the month/season and year of sampling the fish
  6. I think you can combine the number of microplastics found and FO in the same table, one per each fish species analyzed, it could give a more organized appearance and will be easier to follow
  7. I would suggest a smaller font in the table, I think also in the template the standard is 9
  8. Line 250-254 – if you can make a figure, or a graphic or something to visualize the data you mentioned is summary will be great and will be clearer
  9. Please respect the author guidelines in terms of referencing system “In the text, reference numbers should be placed in square brackets [ ], and placed before the punctuation; for example [1], [1–3] or [1,3].”
  10. Add the author contribution section
  11. I would like to see a small update in the bibliography of the manuscript, at least 5 articles from 2024/2025. There are already a lot of publications on the presence of microplastics in the intestinal tract from different key points of the world, or on specific concentrations of microplastics in the environment that have been recently identified.

 

All in all, the study is very interesting and certainly a lot of many hours of hard work. Please note that correct use of terminology is essential for acceptance for publication. Please harmonize throughout the manuscript and update the bibliography!

Author Response

  1. Terminology – “microplastic” vs. “particulate pollutants”
    Response: The replacement of the term “microplastic” with “particulate pollutants” was previously and forcefully suggested by previous reviewers and the handling editor. We adopted this new terminology and changed the entire structure of the manuscript to accommodate this. We therefore kindly ask that the new terminology is maintained.

 

  1. Keywords – “bleaching” and “freshwater”
    Response: Apologies, the keyword "bleaching" was removed, and we revised the terminology regarding estuarine salinity, acknowledging that neither Porto Seguro nor Babitonga Bay can be classified as purely freshwater systems.

 

  1. Line 91 – Add references

Response: Done. The relevant citations were now inserted at the end of the sentence.

 

  1. Lines 116–131 – Rewrite for clarity and conciseness
    Response: The paragraph was now revised for conciseness.

 

  1. Lines 151–156 – Rewrite for clarity and conciseness
    Response: This section was written according to suggestions from another reviewer, who requested a clear explanation regarding the adaptation of the methodology. We chose to keep this part as written to avoid conflict between reviewer recommendations.

 

  1. Materials & Methods – Add collection dates/seasons
    Response: Done. The month and year of fish sampling were added for both estuaries.

 

  1. Tables – Suggest combining microplastic counts and FO% by species
    Response: The current table structure follows the flow of the manuscript and was previously adjusted according to another reviewer’s suggestion. Thus, we opted to maintain the layout.

 

  1. Table formatting – Suggest using a smaller font size (e.g., size 9)
    Response: The tables were formatted according to the journal’s submission template, which used the current font size. Therefore, we have chosen to maintain it.

 

  1. Lines 250–254 – Consider visual representation (graph/figure)
    Response: Our manuscript has many tables and figures. Given that the paragraph that the reviewer mentions provides only summary statistics, and it is concise, we prefer to maintain this information in the main text.

 

  1. Reference formatting – Follow journal style ([1], [1–3], etc.)
    Response: Thank you for pointing this out. We have now reviewed the journal guidelines and formatted all in-text citations accordingly. This will also be done in collaboration with the journal when the paper is formally accepted.

 

  1. Author Contributions section

Response: Thank you for the observation. We were not initially aware of this requirement. The Author Contributions section has now been added following the journal’s format.

 

  1. Update bibliography with recent literature (2024/2025)
    Response: The change from microplastics to particulate pollutants was based on the editor's recommendation, which required restructuring the manuscript and updating the literature accordingly. We conducted a new literature search to support the updated terminology and incorporated relevant and recent references throughout the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have corrected the references and significantly improved the introduction.

 

However, as I wrote for the first time, the authors were unable to improve the study in general.

In new lines 242 - 245, the authors indicate that they were unable to determine the chemical nature of the pollutants. It means that the authors cannot guarantee that these pollutants are microplastics and are not other substances. This fact completely negates all subsequent reasoning and conclusions, as I wrote in my first review.

Undoubtedly, each method of determination, such as Raman spectroscopy, or any other one, has its disadvantages. However, these disadvantages are not as fatal as using only one method of optical microscopy. The authors could have used methods such as Pyrolysis Gas Chromatography Mass Spectrometry, Scanning Electron Microscopy, micro Fourier transform infrared spectroscopy and etc.

Researchers use several methods to confirm the nature of the origin of pollutants. This is especially important for studying of biological samples. For example, the authors prove their material using the following methods: Pyrolysis Gas Chromatography Mass Spectrometry + Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) + ATR-FTIR ( doi.org/10.1038/s41591-024-03453-1)

The authors claim that they followed the methodology (lines 124–125) of Karami et al. (2017) and Pegado et al. (2018). This is not the case. Pegado et al. (2018) used ATR-FTIR to identify particles. Karami et al. (2017) used micro-Raman spectroscopy and elemental analysis with energy-dispersive X-ray spectroscopy (EDX).

 

All modern researchers use instrumental methods for chemical identification of microplastic particles. The authors can verify this by studying the articles from their own introduction. For example: doi.org/10.1016/j.envpol.2018.01.083 used μ-FT-IR. doi.org/10.1016/j.marpolbul.2018.06.035 used ATR-FTIR. doi.org/10.1016/j.marpolbul.2015.11.043 used FT-IR.

Using those methods for identification is not something exotic. It is normal for modern researches. This must be done at least for some of the samples. Then it is possible to determine the percentage of the studied pollution particles visible in an optical microscope are actually microplastics.

 

Comments on the Quality of English Language

-

Author Response

Reviewer 1 raised an important, and the entirety of their comment relates to the chemical characterisation of the particles we found. As such, we paste their full comments below, and provide a response thereafter:

he authors have corrected the references and significantly improved the introduction.

However, as I wrote for the first time, the authors were unable to improve the study in general.

In new lines 242 - 245, the authors indicate that they were unable to determine the chemical nature of the pollutants. It means that the authors cannot guarantee that these pollutants are microplastics and are not other substances. This fact completely negates all subsequent reasoning and conclusions, as I wrote in my first review.

Undoubtedly, each method of determination, such as Raman spectroscopy, or any other one, has its disadvantages. However, these disadvantages are not as fatal as using only one method of optical microscopy. The authors could have used methods such as Pyrolysis Gas Chromatography Mass Spectrometry, Scanning Electron Microscopy, micro Fourier transform infrared spectroscopy and etc.

Researchers use several methods to confirm the nature of the origin of pollutants. This is especially important for studying of biological samples. For example, the authors prove their material using the following methods: Pyrolysis Gas Chromatography Mass Spectrometry + Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) + ATR-FTIR ( doi.org/10.1038/s41591-024-03453-1)

The authors claim that they followed the methodology (lines 124–125) of Karami et al. (2017) and Pegado et al. (2018). This is not the case. Pegado et al. (2018) used ATR-FTIR to identify particles. Karami et al. (2017) used micro-Raman spectroscopy and elemental analysis with energy-dispersive X-ray spectroscopy (EDX).

 

All modern researchers use instrumental methods for chemical identification of microplastic particles. The authors can verify this by studying the articles from their own introduction. For example: doi.org/10.1016/j.envpol.2018.01.083 used μ-FT-IR. doi.org/10.1016/j.marpolbul.2018.06.035 used ATR-FTIR. doi.org/10.1016/j.marpolbul.2015.11.043 used FT-IR.

Using those methods for identification is not something exotic. It is normal for modern researches. This must be done at least for some of the samples. Then it is possible to determine the percentage of the studied pollution particles visible in an optical microscope are actually microplastics.

Reply: Thank you very much for this comment. We agree that our manuscript cannot categorically state that the pollutants are microplastics, nor their chemical composition. As a result – and following the editors' suggestions – we have changed the title and terminology of manuscript. Instead of "microplastic" we now present the analysis of "particulate pollutants". 


Particulate pollutants (from which microplastics is but one type) more adequately characterise the subject of our study, in light of the reviewers' comments.  We have changed the terminology throughout the manuscript. We have revised the introduction and discussion accordingly, giving a more proper background of the definition for particulate pollutants (lines 50- 65) and better justify our approach in the methods (lines 143 - 147 ) and discussion (lines 249-267). 

We appreciate that many laboratories are well-equipped to run chemical characterisation of particulate pollutants, especially in places like North America, Europe, and Australasia (e.g., China, Australia, New Zealand). Unfortunately, this is not the case in Latin America, where the research has taken place and where all of the analyses were conducted. We agree that a full characterisation would strengthen our original claims relating the pollutants to microplastic, but we feel that our revised use of the terminology, focusing on particulate pollutants, properly addresses the concern of the reviewer and acknowledges the significance of our work. We hope the reviewer understands our position. 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have properly responded to my queries. However, I could not find the FTIR analysis in the main manuscript (please refer to comment #7). If the authors have conducted the FTIR analysis, it is recommended that they include the corresponding discussion in the main draft. Once this point is addressed, this manuscript can be accepted for publication in Diversity.

Author Response

The authors have properly responded to my queries. However, I could not find the FTIR analysis in the main manuscript (please refer to comment #7). If the authors have conducted the FTIR analysis, it is recommended that they include the corresponding discussion in the main draft. Once this point is addressed, this manuscript can be accepted for publication in Diversity.

REPLY: Thank you for recommending our manuscript for publication. We would like to apologise for our reply regarding FTIR: this was a mistake on our side. We meant that one of the papers we used to adapt the methodology (Pegado et al 2018) used FTIR. We did not have access to this technique for this study. 

Reviewer 4 Report

Comments and Suggestions for Authors

Even if we use different terminology (microplastic vs. particulate pollutants) this does not mean that we have to find articles strictly with "particulate pollutants" you can also use "microplastic" just mention other terminology in the text. A simple search on Google Scholar shows 4,820 results for the year 2025 alone. Please add at least a few articles from the years 2024/2025 to be "up to date", especially since the samples were only taken in 2020.

Author Response

Even if we use different terminology (microplastic vs. particulate pollutants) this does not mean that we have to find articles strictly with "particulate pollutants" you can also use "microplastic" just mention other terminology in the text. A simple search on Google Scholar shows 4,820 results for the year 2025 alone. Please add at least a few articles from the years 2024/2025 to be "up to date", especially since the samples were only taken in 2020.

Reponse: Thank you. We understand that the main issue is in the Introduction and the lack of recent references, particularly those involving microplastic which is also a particulate pollutant. The reviewer is right that we could incorporate some of the more recent literature on microplastics. We have now done so, by citing 12 new papers from 2025 that addresses contamination by microplastics and its consequences worldwide. 

Here is how the revised Introduction sections read: 

Line 63-69:

"Several studies have investigated particulate pollutants related to plastic, such as microplastic, in Brazilian estuaries (Ribeiro et al., 2025; Menezes et al., 2025; Krelling et al., 2025; Parra et al., 2025; Pegado et al., 2018; Possato et al., 2011; Vendel et al., 2017), as well as in other countries, such as Canada (Kabir et al., 2025), Mexico (Sanchez-Paz et al., 2025), Portugal (Bessa et al., 2018), China (Wang et al., 2025; Li et al., 2018), and India (Kalangutkar et al., 2025; Goswami et al., 2020)."


Lines 71 to 80

"For instance, in Malaysia, an average of ~2.5 particles of microplastic per liter of water was found in the Klang River estuary, originating from fishing gear and synthetic clothes (Zaki et al., 2021a; Zaki et al., 2021b). This has implications to biodiversity of the estuary which also accumulated microplastics in their bodies, for example gastropods (Zaki et al.,2021b). Similar results were found in bivalves in an urbanized estuary in Brazil (Santos-Sao Vicente), for which bioavailability of microplastics were linked to industrial activities and negatively correlated with population density, and bioaccumulated in decreasing concentrations from clams, mussels and oysters, respectively (Ribeiro et al., 2025).

Particulate plastic pollutants can act as vectors, facilitating the entry of secondary pollutants into the environment (Sinha et al., 2025; Akdogan & Guven, 2019). "


And the new reference list:

  1. Akdogan, Z., Guven, B. Microplastics in the environment: A critical review of current understanding and identification of future research needs. Environmental pollution 254, p. 113011. 2019. https://doi.org/10.1016/j.envpol.2019.113011
  2. Anderson, J. C., Park, B. J., & Palace, V. P. (2016). Microplastics in aquatic environments: Implications for Canadian ecosystems. Environmental Pollution, 218, 269–280. https://doi.org/10.1016/j.envpol.2016.06.074
  3. Andrady, A. L. (2011). Microplastics in the marine environment. Marine Pollution Bulletin, 62(8), 1596–1605. https://doi.org/10.1016/j.marpolbul.2011.05.030
  4. Araujo, C.F., Nolasco, M.M., Ribeiro, A.M.P., Ribeiro-Claro, P.J.A. (2018) Identification of microplastics using Raman spectroscopy: Latest developments and future prospects. Water Research, v. 142, p. 426–440. https://doi.org/10.1016/j.watres.2018.05.060
  5. Arias, A. H., Ronda, A. C., & Oliva, A. L. (2019). Evidence of microplastic ingestion by fish from the Bahía Blanca Estuary in Argentina, South America. Bulletin of Environmental Contamination and Toxicology, 102(5), 750–756. https://doi.org/10.1007/s00128-019-02604-2
  6. Batel, A., Borchert, F., Reinwald, H., Erdinger, L., & Braunbeck, T. (2018). Microplastic accumulation patterns and transfer of benzo[a]pyrene to adult zebrafish (Danio rerio) gills and zebrafish embryos. Environmental Pollution, 235, 918–930. https://doi.org/10.1016/j.envpol.2018.01.028
  7. Bessa, F., Barría, P., Neto, J. M., Frias, J. P. G. L., Otero, V., Sobral, P., & Marques, J. C. (2018). Occurrence of microplastics in commercial fish from a natural estuarine environment. Marine Pollution Bulletin, 128, 575–584. https://doi.org/10.1016/j.marpolbul.2018.01.044
  8. Browne, M. A., Crump, P., Niven, S. J., Teuten, E. L., Tonkin, A., Galloway, T., & Thompson, R. C. (2011). Accumulations of microplastic on shorelines worldwide: Sources and sinks. Environmental Science & Technology, 45(21), 9175–9179. https://doi.org/10.1021/es201811s
  9. Carpenter, E. J., Smith, K. L., Jr. Plastics on the Sargasso Sea surface. Science, 175, p. 1240–1241. 1972. https://www.science.org/doi/10.1126/science.175.4027.1240
  10. Cole, M., Lindeque, P., Halsband, C., & Galloway, T. S. (2011). Microplastics as contaminants in the marine environment: A review. Marine Pollution Bulletin, 62(12), 2588–2597. https://doi.org/10.1016/j.marpolbul.2011.09.025
  11. Cozar, A., Echevarría, F., Gonzalez-Gordillo, J. I., Irigoien, X., Ubeda, B., Hernandez-León, S., Palma, A. T., Navarro, S., Garcia-de-Lomas, J., Ruiz, A., Fernandez-de-Puelles, M. L., & Duarte, C. M. (2014). Plastic debris in the open ocean. Proceedings of the National Academy of Sciences of the United States of America, 111(28), 10239–10244. https://doi.org/10.1073/pnas.1314705111
  12. Cverenkárová, K., Valachovicová, M., Mackul’ak, T., Žemlicka, L., Bírošová, L. Microplastics in the food chain. Life, 11, n. 12, p. 1349, 2021. doi: 10.3390/life11121349
  13. Davison, P., & Asch, R. G. (2011). Plastic ingestion by mesopelagic fishes in the North Pacific Subtropical Gyre. Marine Ecology Progress Series, 432, 173–180. https://doi.org/10.3354/meps09142
  14. Einoder, L. D., MacLeod, C. K., & Coughanowr, C. (2018). Metal and isotope analysis of bird feathers in a contaminated estuary reveals bioaccumulation, biomagnification, and potential toxic effects. Archives of Environmental Contamination and Toxicology, 75(1), 96–110. https://doi.org/10.1007/s00244-018-0532-z
  15. Figueiredo, J. L., & Menezes, N. A. (2000). Manual de peixes marinhos do sudeste do Brasil. IV. Teleostei (5). Museu de Zoologia - USP.
  16. Filgueras, A. S., Silva, T. S., & Corrêa, I. C. S. (2023). Impacts of sea level rise and land use on coastal wetlands: Methodology applied to Baía da Babitonga (SC). Sociedade & Natureza, 36, e69403. https://doi.org/10.14393/SN-v36-2024-69403
  17. Foekema, E. M., de Gruijter, C., Mergia, M. T., van Franeker, J. A., Murk, A. T. J., & Koelmans, A. A. (2013). Plastic in North Sea fish. Environmental Science & Technology, 47(15), 8818–8824. https://doi.org/10.1021/es400931b
  18. Goswami, P., Vinithkumar, N. V., & Dharani, G. (2020). First evidence of microplastics bioaccumulation by marine organisms in the Port Blair Bay, Andaman Islands. Marine Pollution Bulletin, 155, 111163. https://doi.org/10.1016/j.marpolbul.2020.111163
  19. Haward, M. (2018). Plastic pollution of the world’s seas and oceans as a contemporary challenge in ocean governance. Nature Communications, 9(1), 667. https://doi.org/10.1038/s41467-018-03104-3
  20. IBGE - Brazilian Institute of Geography and Statistics. (2010). Demographic census 2010.
  21. IBGE - Brazilian Institute of Geography and Statistics. (2022). Demographic census 2022.
  22. (2006). Joinville - City in data. Research and Planning Institute for the Sustainable Development of Joinville. Prefeitura Municipal de Joinville.
  23. Jabeen, K., Su, L., Li, J., Yang, D., Tong, C., Mu, J., & Shi, H. (2017). Microplastics and mesoplastics in fish from coastal and fresh waters of China. Environmental Pollution, 221, 141–149. https://doi.org/10.1016/j.envpol.2016.11.055
  24. Kabir, A.E., Michon, E., Mingelbier, M., Robert, D., Soubaneh, Y.D., Xie, H. and Lu, Z., 2025. Microplastics in the benthic fish from the Canadian St. Lawrence River and Estuary: Occurrence, spatial distribution and ecological risk assessment. Marine Pollution Bulletin, 212, p.117509. https://doi.org/10.1016/j.marpolbul.2024.117509
  25. Kalangutkar, N., Mhapsekar, S. and Rajagopal, P., 2025. Comparative assessment of microplastic pollution in Terekhol and Sal estuaries, Goa, India. Environmental Earth Sciences, 84(1), p.48. https://doi.org/10.1007/s12665-024-12042-x
  26. Kane, I. A., Clare, M. A., Miramontes, E., Wogelius, R., Rothwell, J. J., Garreau, P., & Poh, F. (2020). Seafloor microplastic hotspots controlled by deep-sea circulation. Science, 368(6495), 1140–1145. https://doi.org/10.1126/science.aba5899
  27. Karami, A., Golieskardi, A., Ho, B. Y., Larat, V., & Salamatinia, B. (2017). Microplastics in eviscerated flesh and excised organs of dried fish. Scientific Reports, 7(1), 5473. https://doi.org/10.1038/s41598-017-05828-6
  28. Krelling, A.P., Possatto, F.E., Maia, G.R.A., Araki, K., Olivieri, G.B.R. and Nogueira, H.P., 2025. First Evidence of High Microplastic Concentrations in Estuarine Litter Windrows of a World Heritage Site: The Paranaguá Estuarine Complex, Brazil. Water, Air, & Soil Pollution, 236(5), p.266. https://doi.org/10.1007/s11270-025-07899-y
  29. Lana, P. C., & Bernardino, A. F. (Eds.). (2018). Brazilian estuaries: A benthic perspective. Springer.
  30. Law, K. L., & Thompson, R. C. (2014). Microplastics in the seas. Science, 345(6193), 144–145. https://doi.org/10.1126/science.1254065
  31. Lebreton, L. C. M., van der Zwet, J., Damsteeg, J., Slat, B., Andrady, A., & Reisser, J. (2017). River plastic emissions to the world’s oceans. Nature Communications, 8(1), 15611. https://doi.org/10.1038/ncomms15611
  32. Li, H. X., Ma, L. S., Lin, L., Ni, Z. X., Xu, X. R., Shi, H. H., Yan, Y., Zheng, G. M., & Rittschof, D. (2018). Microplastics in oysters Saccostrea cucullataalong the Pearl River Estuary, China. Environmental Pollution, 236, 619–625. https://doi.org/10.1016/j.envpol.2018.01.083
  33. Lusher, A. L., O’Donnell, C., Officer, R., & O’Connor, I. (2016). Microplastic interactions with North Atlantic mesopelagic fish. ICES Journal of Marine Science, 73(4), 1214–1225. https://doi.org/10.1093/icesjms/fsv241
  34. Marinsek, G.P. Gusso-Choueri, P.K., Choueri, R.B., Abessa, D.M.S., Gonçalves, A.R.N., Bortolotto, L.B., Mari, R.B. Integrated analysis of fish intestine biomarkers: Complementary tools for pollution assessment, Marine Pollution Bulletin, v.178, 113590. 2022. https://doi.org/10.1016/j.marpolbul.2022.113590
  35. Mazzer, A. M., & Gonçalves, M. L. (2011). Aspectos geomorfológicos da Baía da Babitonga, Santa Catarina, Brazil: Caracterização morfométrica. Revista Brasileira de Geomorfologia, 12(3), 115–120. https://doi.org/10.20502/rbg.v12i0.264
  36. Menezes, K.M., da Costa, M.B., Schuab, J.M., Dalbó, G.Z., Alves, M.M., Ocarisd, E.R.Y. and Rodrigues, F.L., 2025. Microplastic contamination in the mangroves of Piraquê-Açu and Piraquê-Mirim rivers, Aracruz (Brazil): An analysis in sediment, water, and biota. Marine Pollution Bulletin, 214, p.117696. https://doi.org/10.1016/j.marpolbul.2025.117696
  37. Paitach, P. L., Simões-Lopes, P. C., & Cremer, M. J. (2017). Tidal and seasonal influences in dolphin habitat use in a southern Brazilian estuary. Scientia Marina, 81(1), 49–56. https://doi.org/10.3989/scimar.04495.25A
  38. Parra, D.F., Gimiliani, G.T., Dos Santos, J.L., Wetter, N.U., Schepis, W.R., Bereczki, A. and Cotrim, M.E.B., 2025. Microplastics in Santos São Vicente estuarine–Hotspot in sediments caused by low energy hydrodynamic events in strongly populated areas. Marine Pollution Bulletin, 210, p.117286. https://doi.org/10.1016/j.marpolbul.2024.117286
  39. Pegado, T. S. S., Schmid, K., Winemiller, K. O., Chelazzi, D., Cincinelli, A., Dei, L., & Giarrizzo, T. (2018). First evidence of microplastic ingestion by fishes from the Amazon River estuary. Marine Pollution Bulletin, 133, 814–821. https://doi.org/10.1016/j.marpolbul.2018.06.035
  40. Possatto, F. E., Barletta, M., Costa, M. F., Ivar do Sul, J. A., & Dantas, D. V. (2011). Plastic debris ingestion by marine catfish: An unexpected fisheries impact. Marine Pollution Bulletin, 62(5), 1098–1102. https://doi.org/10.1016/j.marpolbul.2011.01.036
  41. Ribeiro, V.V., Casado-Coy, N., Rangel, D.F., Sanz-Lazaro, C. and Castro, Í.B., (2025). Microplastic in bivalves of an urbanized Brazilian estuary: Human modification, population density and vegetation influence. Journal of Hazardous Materials, 482, p.136546. https://doi.org/10.1016/j.jhazmat.2024.136546
  42. Ribeiro de Sena, S., de Souza Veiga, R., & De Amorim Silva, V. (2023). Análise de áreas de risco à degradação ambiental no município de Porto Seguro, Bahia, Brasil. Brazilian Journal of Physical Geography, 16(6), 3059–3072. https://doi.org/10.26848/rbgf.v16.6.p3059-3072
  43. Romeo, T., Battaglia, P., Pedá, C., Consoli, P., Andaloro, F., & Fossi, M. C. (2015). First evidence of presence of plastic debris in stomach of large pelagic fish in the Mediterranean Sea. Marine Pollution Bulletin, 95(1), 358–361. https://doi.org/10.1016/j.marpolbul.2015.04.048
  44. Rummel, C. D., Löder, M. G. J., Fricke, N. F., Lang, T., Griebeler, E. M., Janke, M., & Gerdts, G. (2016). Plastic ingestion by pelagic and demersal fish from the North Sea and Baltic Sea. Marine Pollution Bulletin, 102(1), 134–141. https://doi.org/10.1016/j.marpolbul.2015.11.043
  45. Rummel, C. D., Jahnke, A., Gorokhova, E., Kühnel, D., Schmitt-Jansen, M. Impacts of biofilm formation on the fate and potential effects of microplastic in the aquatic environment. Environmental science & technology letters, v. 4, p. 258–267, 2017. https://doi.org/10.1021/acs.estlett.7b00164
  46. Sánchez-Paz, A., Encinas-García, T., Ávila-Félix, L. and Mendoza-Cano, F., 2025. Microplastics in surface waters surrounding a touristic beach at the Gulf of California, Mexico. International Journal of Environmental Health Research, pp.1-12. https://doi.org/10.1080/09603123.2025.2483356
  47. Santos, T. A., Bonfim, T. M., Silva, F. S., Silva, A. G., Bandeira, M. L. S. F., Jesus, R. M., & Nascimento, L. D. (2018). Determination of metals in an estuarine system: Laguncularia racemosaas a potential indicator of contamination. The Brazilian Journal of Environmental Sciences, 49, 51–65. https://doi.org/10.5327/Z2176-947820180352
  48. Silva, A. C. R. S., Bernardes, M. E. C., Assireu, A. T., Siegle, E., Sousa, P. H. G. O., & Brown, D. (2018). Hydrodynamics of a tropical estuary: Buranhém River, Porto Seguro, Brazil. Brazilian Journal of Water Resources, 23(5), 1–9. https://doi.org/10.1590/2318-0331.0318170141
  49. Sinha, P., Saini, V., Varshney, N., Pandey, R.K. and Jha, H.C., 2025. The Infiltration of Microplastics in Human Systems: Gastrointestinal Accumulation and Pathogenic Impacts. Heliyon. (11)4 e42606. https://doi.org/ 10.1016/j.heliyon.2025.e42606.
  50. Sheraz, M., Kim, J., Kim, J. Nano/microplastics in indoor air: A critical review of synthesis routes for toxicity testing and preventative measure strategies. Process Safety and Environmental Protection, v. 180, p. 274-304. 2023. https://doi.org/10.1016/j.psep.2023.10.002.
  51. Sun, X., Li, Q., Shi, Y., Zhao, Y., Zheng, S., Liang, J., Liu, T., & Tian, Z. (2019). Characteristics and retention of microplastics in the digestive tracts of fish from the Yellow Sea. Environmental Pollution, 249, 878–885. https://doi.org/10.1016/j.envpol.2019.01.110
  52. Tanaka, K., & Takada, H. (2016). Microplastic fragments and microbeads in digestive tracts of planktivorous fish from urban coastal waters. Scientific Reports, 6(1), 34351. https://doi.org/10.1038/srep34351
  53. Trevizani, T. H., Domit, C., Santos, M. C. O., & Figueira, R. C. L. (2023). Bioaccumulation of heavy metals in estuaries in the southwest Atlantic Ocean. Environmental Science and Pollution Research, 30(11), 26703–26717. https://doi.org/10.1007/s11356-022-23974-x
  54. van Sebille, E., Wilcox, C., Lebreton, L., Maximenko, N., Hardesty, B. D., van Franeker, J. A., Eriksen, M., Siegel, D., Galgani, F., & Law, K. L. (2015). A global inventory of small floating plastic debris. Environmental Research Letters, 10(12), 124006. https://doi.org/10.1088/1748-9326/10/12/124006
  55. Vargas, J.G.M., Silva, V.B., Oliveira, L.K., Molina, E.F. (2022). Microplásticos: uso na indústria cosmética e impactos no ambiente aquático. Química Nova, 45(6), 705–711. https://doi.org/10.21577/0100-4042.20170870
  56. Vasconcelos, R. P., Reis-Santos, P., Costa, M. J., & Cabral, H. N. (2011). Connectivity between estuaries and marine environment: Integrating metrics to assess estuarine nursery function. Ecological Indicators, 11(5), 1123–1133. https://doi.org/10.1016/j.ecolind.2010.12.012
  57. Vendel, A. L., Bessa, F., Alves, V. E. N., Amorim, A. L. A., Patrício, J., & Palma, A. R. T. (2017). Widespread microplastic ingestion by fish assemblages in tropical estuaries subjected to anthropogenic pressures. Marine Pollution Bulletin, 117(1–2), 448–455. https://doi.org/10.1016/j.marpolbul.2017.01.081
  58. VishnuRadhan, R., Thresyamma, D. D.,  Eldho, T. I., Bhagat, J.(2021). Atmospheric plastics- a potential airborne fomite with an emerging climate signature. The Journal of Climate Change and Health, 3,100037. https://doi.org/10.1016/j.joclim.2021.100037.
  59. Wang, J., Peng, J., Tian, Z., Gao, Y., Zhan, Z., Chen, Q., Cai, L. Microplastics in the surface sediments from the Beijiang River littoral zone: Composition, abundance, surface textures and interaction with heavy metals. Chemosphere, v. 171, p. 248–258. 2017. https://doi.org/10.1016/j.chemosphere.2016.12.074
  60. Wang, W., Guo, S., Li, B., Xu, D. and Gao, B., 2025. Microplastics in Pearl River Estuary and Yangtze River Estuary, China: Occurrence, fragmentation and pollution risk. Journal of Hazardous Materials Advances, 17, p.100529. https://doi.org/10.1016/j.hazadv.2024.100529
  61. Wang, L. C., Lin, J. C., Dong, C., Chen, C., Liu, T. The sorption of persistent organic pollutants in microplastics from the coastal environment. Journal of hazardous materials, v. 420, p. 126658. 2021. https://doi.org/10.1016/j.jhazmat.2021.126658
  62. Zaki, M. R. M., Ying, P. X., Zainuddin, A. H., Razak, M. R., & Aris, A. Z. (2021a). Occurrence, abundance, and distribution of microplastics pollution: an evidence in surface tropical water of Klang River estuary, Malaysia. Environmental Geochemistry and Health, 1-16. https://doi.org/10.1007/s10653-021-00872-8
  63. Zaki, M.R.M., Zaid, S.H.M., Zainuddin, A.H. and Aris, A.Z., (2021b). Microplastic pollution in tropical estuary gastropods: Abundance, distribution and potential sources of Klang River estuary, Malaysia. Marine pollution bulletin, 162, p.111866. https://doi.org/10.1016/j.marpolbul.2020.111866

Round 3

Reviewer 4 Report

Comments and Suggestions for Authors

Please use the correct referencing system with [ ] and numbers according to the authors guidelines 

Author Response

Please use the correct referencing system with [ ] and numbers according to the authors guidelines 

Response: Done.

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