Virgin and Photoaged Polyethylene Microplastics Have Different Effects on Collembola and Enchytraeids
, Sónia Chelinho 3, Maria J. I. Briones 1 and Jose P. Sousa 4Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe study is quite interesting and relevant. The thoughtful design of the experiment with replications and sufficient sampling is a definite plus. Nevertheless, in this form, the work is not without shortcomings, which I have pointed out in the comments below.
Title. I suggest to change the name to a more specific one: we can use a more precise characterization of the particles “photoaged polyetelene microplastics” . the same goes for “soil mesafauna” - it is too generalized name for three species.
Keywords. I suggest reducing their number. For example, the words “pristine” and “virgin” are essentially synonyms. We could keep just one. I noticed that you use the variant “virgin” more often in the text. The word “aged” could also be removed, since “photoaged” is more appropriate here. The particles “weathered” were not studied at all in this work.
Introduction
Please check if the concentrations of microplastics in soils are correct and if the reference to the study is appropriate. I looked at the article under reference 4 and did not find the relevant information there. I am quite surprised that soils can contain 67 g/kg specifically of micro-sized plastic particles.
Lines 43-46. It is not clear why examples of plastic impact on aquatic organisms (from microalgae to fish) should be given if the article is about soil mesofauna. It would be more appropriate to present examples of works on the impact of plastics on soil organisms.
Write the full names of the species used in the experiment at the first mention, as is done later in the materials and methods.
It would be useful to write more clearly the aim of the study.
Methodology
Line 91 Specify the lamp power.
Lines 93-95. Please write in more detail the device settings when measuring the spectra. (number of scans, spectral resolution, whether the frustrated total internal reflection prefix is ​​installed). what program was used to obtain and process the spectral images.
Usually, in works of this kind, methods for preventing accidental contamination with plastic are described in detail (use of cotton robes, how and with what dishes were washed, etc.). Perhaps you were guided by some protocol.
Results
Since the main text contains only one image, I propose to move all the material from the Supplementary to the main text.
Design.
Please clarify why you shorten the word "microplastics" to "MPs" and "polyethelene microplastics" to PE MP. You can also use "PE MPs".
Please pay attention to the design of the reference numbers. When listing several references in the text, there is no need to write brackets for each individual number.
Sometimes there is no space between the number and the unit of measurement, for example, lines 154, 163, 179.
In the results, when numbering subsections, there is no (.) after the second number. It should be 3.1.
The text contains empty lines (253-254, 344-345)
The list of references is not formatted according to the format accepted in the journal.
Author Response
Dear Editor,
Thank you for the opportunity to revise our ms. We have addressed all the comments and suggestions. The changes to the initial version of the manuscript are identified by Microsoft® Word - track-changes. Below, we provide a point-by-point response and explanations on how we have addressed each comment.
In response to reviewer 1
Comment 1: The study is quite interesting and relevant. The thoughtful design of the experiment with replications and sufficient sampling is a definite plus. Nevertheless, in this form, the work is not without shortcomings, which I have pointed out in the comments below.
Answer: Thank you for the positive assessment of our ms. Below, we indicate how each comment has been addressed.
Comment 2: Title. I suggest to change the name to a more specific one: we can use a more precise characterization of the particles “photoaged polyetelene microplastics” . the same goes for “soil mesafauna” - it is too generalized name for three species.
Answer: The title has been updated to read “Virgin and Photoaged Polyethylene Microplastics have different Effects on Collembola and Enchytraeids”
Comment 3: Keywords. I suggest reducing their number. For example, the words “pristine” and “virgin” are essentially synonyms. We could keep just one. I noticed that you use the variant “virgin” more often in the text. The word “aged” could also be removed, since “photoaged” is more appropriate here. The particles “weathered” were not studied at all in this work.
Answer: Keywords have been corrected to “Polyethylene; microplastic pollution; Folsomia candida; Proisotoma minuta; Enchytraeus crypticus”
Comment 4: Please check if the concentrations of microplastics in soils are correct and if the reference to the study is appropriate. I looked at the article under reference 4 and did not find the relevant information there. I am quite surprised that soils can contain 67 g/kg specifically of micro-sized plastic particles.
Answer: The article in question sampled soils near industrial sites, and the highest concentration of MPs found was 67500 mg/kg. However, we have also included more relevant citation here (L41).
Comment 5: Lines 43-46. It is not clear why examples of plastic impact on aquatic organisms (from microalgae to fish) should be given if the article is about soil mesofauna. It would be more appropriate to present examples of works on the impact of plastics on soil organisms.
Answer: We agree. However, when the paper was initially written, there were not many references available for toxicological impacts of aged microplastics on soil organisms. We have updated this section and removed reference to aquatic organisms (L47-52).
Comment 5: Write the full names of the species used in the experiment at the first mention, as is done later in the materials and methods.
Answer: Done. The full names of the species used in this study have been written in the introduction.
Comment 6: It would be useful to write more clearly the aim of the study.
Answer: The aim of the study has been edited to improve clarity (L61-68)
Comment 7: Line 91 Specify the lamp power.
Answer:. The specification of the lamp power has been added (L97):
Comment 8: Lines 93-95. Please write in more detail the device settings when measuring the spectra. (number of scans, spectral resolution, whether the frustrated total internal reflection prefix is ​​installed). what program was used to obtain and process the spectral images.
Answer: More details regarding the FTIR specification has been provided (L102-110).
Comment 9: Usually, in works of this kind, methods for preventing accidental contamination with plastic are described in detail (use of cotton robes, how and with what dishes were washed, etc.). Perhaps you were guided by some protocol.
Answer: More information on contamination control has been provided (L120-121):
Comment 10: Since the main text contains only one image, I propose to move all the material from the Supplementary to the main text.
Answer: As requested, we have moved the figure and table of the supplementary material to the main text paper.
Comment 11: Please clarify why you shorten the word "microplastics" to "MPs" and "polyethelene microplastics" to PE MP. You can also use "PE MPs".
Answer: Yes, this was a typo. PE MP has been replaced with PE MPs for consistency.
Comment 12: Please pay attention to the design of the reference numbers. When listing several references in the text, there is no need to write brackets for each individual number.
Answer: The reference numbers have been corrected. Thank you.
Comment 13: Sometimes there is no space between the number and the unit of measurement, for example, lines 154, 163, 179.
Answer: All necessary spacing issues have been addressed.
Comment 14: In the results, when numbering subsections, there is no (.) after the second number. It should be 3.1.
Answer: This error has been rectified.
Comment 14: The text contains empty lines (253-254, 344-345)
Answer: This formatting issue has been addressed.
Comment 15: The list of references is not formatted according to the format accepted in the journal.
Answer: The bibliography has now been updated to follow the Journal formatting guidelines.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript is generally well-structured; however, several issues need to be addressed to improve clarity and consistency:
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Citation format: When multiple references are listed, they should be grouped within a single square bracket. If the references are consecutive, a dash should be used to indicate the range (e.g., [9–12]). Please ensure consistency throughout the manuscript.
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Line 61: A closing bracket is missing—please revise accordingly.
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Line 72 (or relevant section): The sentence "Particle size was informed by ingestion capabilities of our test species" is unclear. Consider rephrasing it to more clearly explain whether the particle size was selected based on known ingestion limits of the test species, and if so, please provide a reference.
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pH measurement: It is not specified whether the pH was measured in Hâ‚‚O, CaClâ‚‚, or KCl.
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Line 147 and elsewhere: "Twenty" should not be capitalized unless it begins a sentence. Please carefully review the manuscript for similar inconsistencies with capital letters used unnecessarily in common nouns.
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Data analysis: It is not clear whether the normality of the data was tested prior to conducting statistical analyses. Please clarify the statistical workflow and justify the methods used depending on the data distribution.
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Lines 268–270: The sentence is difficult to follow and should be rephrased for clarity. Consider breaking it into two shorter sentences or restructuring for better readability.
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Figures: Since there is only one figure in the manuscript, I recommend separating the survival and reproduction data into two distinct figures. This would enhance clarity and allow for more detailed visual interpretation of the results.
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Supplementary material: The FTIR-ATR spectra are important to support your findings. I suggest incorporating them into the main manuscript rather than leaving them as supplementary material, unless space or formatting constraints prevent it.
Author Response
Dear Editor,
Thank you for the opportunity to revise our ms. We have addressed all the comments and suggestions. The changes to the initial version of the manuscript are identified by Microsoft® Word - track-changes. Below, we provide a point-by-point response and explanations on how we have addressed each comment.
In response to Reviewer 2,
Comment 1: The manuscript is generally well-structured; however, several issues need to be addressed to improve clarity and consistency:
Answer: Thank you for your positive comment. Below, each of your comments have been addressed.
Comment 2: Citation format: When multiple references are listed, they should be grouped within a single square bracket. If the references are consecutive, a dash should be used to indicate the range (e.g., [9–12]). Please ensure consistency throughout the manuscript.
Answer: The citations have been edited accordingly.
Comment 3: Line 61: A closing bracket is missing—please revise accordingly.
Answer: This typo has been addressed.
Comment 4: Line 72 (or relevant section): The sentence "Particle size was informed by ingestion capabilities of our test species" is unclear. Consider rephrasing it to more clearly explain whether the particle size was selected based on known ingestion limits of the test species, and if so, please provide a reference.
Answer: Sorry about this. This sentence has been edited to improve clarity (L96-97)
Comment 5: pH measurement: It is not specified whether the pH was measured in Hâ‚‚O, CaClâ‚‚, or KCl.
Answer: pH was measured in KCL and this information has been added in the Materials and Methods section (L132) and in the heading of Table 1.
Comment 6: Line 147 and elsewhere: "Twenty" should not be capitalized unless it begins a sentence. Please carefully review the manuscript for similar inconsistencies with capital letters used unnecessarily in common nouns.
Answer: Thank you for pointing this out. The text has been carefully revised to check for inconsistencies..
Comment 7: Data analysis: It is not clear whether the normality of the data was tested prior to conducting statistical analyses. Please clarify the statistical workflow and justify the methods used depending on the data distribution.
Answer: Yes, the data was checked prior the statistical analysis to ensure that the met the normality and homoscedasticity criteria and this information has been included in the Materials and Methods section (L189-191).
Comment 8: Lines 268–270: The sentence is difficult to follow and should be rephrased for clarity. Consider breaking it into two shorter sentences or restructuring for better readability.
Answer: Sorry about this. The sentence has been edited to improve clarity (L310-313).
Comment 9: Figures: Since there is only one figure in the manuscript, I recommend separating the survival and reproduction data into two distinct figures. This would enhance clarity and allow for more detailed visual interpretation of the results.
Answer: Thank you for this suggestion. We have re-drawn the graphs accordingly.
Comment 10: Supplementary material: The FTIR-ATR spectra are important to support your findings. I suggest incorporating them into the main manuscript rather than leaving them as supplementary material, unless space or formatting constraints prevent it.
Answer: As suggested the FTIR-ATR spectra has been moved to the main text.
Reviewer 3 Report
Comments and Suggestions for AuthorsLine 18: I recommend to give a diapason of varied concentration of Pampas 0.2-2000 mg/kg instead of specifying all the studied concentrations of polyethylene microplastics.
Line20: “F. candida” should be italicized. Additionally, all living organisms in the List of References also should be in italic.
Line 46: If several references are mentioned in one place in the text, they should be combined in common square brackets, rather than presenting them individually in this way, such as in Line 46 “[9], [10], [11], [12]” or Line 51“[13], [15], [16], [17], [18]” and etc..
Lines 170 and 176: The phrase “FTIR spectra” appears here, however there is no information about conditions of using this analytical method in the section of Methods.
Line 177: The phrase “Carbonyl index” appears here, however there is no explanation of this parameter in the section of Methods.
Section 3.1: The investigation of the chemical changes in the samples of PE MP is very short, and without explanation of conditions of obtaining such results (FTIR spectra) it is not possible to accept them.
Section 3.2: It is not necessary to divide this section of the text into small subsections. All of them (3.2.1, 3.2.2, 3.2.3) I recommend to combine in one joint part.
Supplementary materials: the title of Supplementary materials (“Microplastics’ Effect on the Survival and Reproduction of Soil Mesofauna”) differs from the title of the main article (“Aged Microplastics’ Effect on Soil Mesofauna”).
Resume: the article contains a very small number of actual results, despite the fact that, unfortunately, it is not clear how the results presented in Supplementary materials were obtained (there is no description in the Methods), and why they were taken outside the main text. Based on the amounts of the References in the article and investigations conducted in this scientific direction, the originallity of the performed results is not high.
Author Response
Dear Editor,
Thank you for the opportunity to revise our ms. We have addressed all the comments and suggestions. The changes to the initial version of the manuscript are identified by Microsoft® Word - track-changes. Below, we provide a point-by-point response and explanations on how we have addressed each comment.
Comment 1: Line 18:
I recommend to give a diapason of varied concentration of Pampas 0.2-2000 mg/kg instead of specifying all the studied concentrations of polyethylene microplastics.
Answer: These specifications have been updated per your suggestion (L20).
Comment 2: Line20: “F. candida” should be italicized. Additionally, all living organisms in the List of References also should be in italic.
Answer: All taxonomic names have been italicized.
Comment 3: Line 46: If several references are mentioned in one place in the text, they should be combined in common square brackets, rather than presenting them individually in this way, such as in Line 46 “[9], [10], [11], [12]” or Line 51“[13], [15], [16], [17], [18]” and etc.
Answer:. The citations have been corrected.
Comment 4: Lines 170 and 176: The phrase “FTIR spectra” appears here, however there is no information about conditions of using this analytical method in the section of Methods.
Answer: Thank you for this comment. A description of this methodology is now given in L103-111):
Comment 5: Line 177: The phrase “Carbonyl index” appears here, however there is no explanation of this parameter in the section of Methods.
Answer: This omission has been rectified (L113-116):
Comment 6: Section 3.1: The investigation of the chemical changes in the samples of PE MP is very short, and without explanation of conditions of obtaining such results (FTIR spectra) it is not possible to accept them.
Answer: As clarified in comments 4 and 5, we have added more details regarding this methodology and edited the text to provide a better explanation of these results (L205-208).
Comment 7: Section 3.2: It is not necessary to divide this section of the text into small subsections. All of them (3.2.1, 3.2.2, 3.2.3) I recommend to combine in one joint part.
Answer: We have edited the results section to combine the text into the same section.
Comment 8: Supplementary materials: the title of Supplementary materials (“Microplastics’ Effect on the Survival and Reproduction of Soil Mesofauna”) differs from the title of the main article (“Aged Microplastics’ Effect on Soil Mesofauna”).
Answer: Thank you for pointing this out, the title of the supplementary materials has been edited to match the title of the ms.
Comment 9: Resume: the article contains a very small number of actual results, despite the fact that, unfortunately, it is not clear how the results presented in Supplementary materials were obtained (there is no description in the Methods), and why they were taken outside the main text. Based on the amounts of the References in the article and investigations conducted in this scientific direction, the originality of the performed results is not high.
Answer: We strongly feel that the novelty of our study lies in the integrative comparison of PE MPs effects across three ecologically relevant soil invertebrate species, along with the assessment of both virgin and photoaged particles under broad concentrations (from environmentally realistic to potentially stressful ones). This approach provides valuable insights into species-specific sensitivities, which are critical for improving ecological risk assessment frameworks for terrestrial microplastic pollution — an area still underexplored compared to aquatic systems.
Furthermore, our study focused on standardized survival and reproduction endpoints (ISO guidelines), which are internationally recognized as robust indicators of population-level impacts and are widely applied in regulatory ecotoxicology. The observation of limited effects across endpoints and species (even at higher concentrations) suggest that these particles might exhibit low toxicity under the studied conditions, but we cannot rule out cumulative (long term) effects. These findings contribute with valuable data to inform species sensitivity distribution for soil’s microplastic contamination. Moreover, previous literature [1,2] showed that indirect microplastic effects can occur even in the absence of demonstrable interaction or ingestion, via soil-mediated exposure pathways or indirect physiological stress. We have clarified these points in the revised Discussion (L402-407) to better articulate the scientific contribution and rationale.
References
- Kim, S.W.; Liang, Y.; Zhao, T.; Rillig, M.C. Indirect Effects of Microplastic-Contaminated Soils on Adjacent Soil Layers: Vertical Changes in Soil Physical Structure and Water Flow. Front Environ Sci 2021, 9, doi:10.3389/fenvs.2021.681934.
- de Souza Machado, A.A.; Lau, C.W.; Till, J.; Kloas, W.; Lehmann, A.; Becker, R.; Rillig, M.C. Impacts of Microplastics on the Soil Biophysical Environment. Environ Sci Technol 2018, 52, 9656–9665, doi:10.1021/acs.est.8b02212.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have significantly revised the text of the article. I have no more comments.
Author Response
Dear reviewer 1,
Thank you very much for your contribution and acceptance!
All the best,
Elise
Reviewer 2 Report
Comments and Suggestions for AuthorsI do not have any additional comments to the manuscript.
Author Response
Dear reviewer 2,
Thank you very much for your contribution and acceptance!
All the best,
Elise
Reviewer 3 Report
Comments and Suggestions for AuthorsI have minor commnets to the current form of the articel:
- the numbrs of the pages should be added to the References ##1 and 72 in the List with references;
- the sources of information about all characteristics appeared in the part of Supplementary material (Table S1) are not clear. The situation should be clarified in Line 119 of the main text. Did the authors make all these estimations of the soil parameters by themselves? If yes, the methods used for this purpose should be mentioned in the section of Methods because that is a part of this work. If this information was taken by the authors from some other sources of information and all these estimations were conducted by other investigators, so all references should be added to the main text and near the Table S1. This is imposrtant and should be correctly performed in the article.
Author Response
Dear Reviewer 3,
Thank you for the follow up critique.
Comment 1: the numbrs of the pages should be added to the References ##1 and 72 in the List with references
Answer 1: Thank you for pointing this out. Page numbers have been added to references 1 and 72.
Comment 2: the sources of information about all characteristics appeared in the part of Supplementary material (Table S1) are not clear. The situation should be clarified in Line 119 of the main text. Did the authors make all these estimations of the soil parameters by themselves? If yes, the methods used for this purpose should be mentioned in the section of Methods because that is a part of this work. If this information was taken by the authors from some other sources of information and all these estimations were conducted by other investigators, so all references should be added to the main text and near the Table S1. This is imposrtant and should be correctly performed in the article.
Answer 2: Soil analysis was conducted by another laboratory and properly credited now on line 119.
