Microplastic Pollution in Shoreline Sediments of the Vondo Reservoir Along the Mutshindudi River, South Africa

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study provides valuable baseline data on microplastic (MP) pollution in a subtropical reservoir system, demonstrating significant spatial and seasonal variations. While the work addresses important knowledge gaps regarding MP accumulation in dammed rivers, several methodological and contextual limitations should be addressed to strengthen the manuscript. Below are specific comments and suggestions for revision: 

1- The use of NaCl solution (density: 1.22 g/cm³) for density separation likely underestimated low-density MPs (e.g., polyethylene, polystyrene), which constitute common plastic pollutants. Explicitly acknowledge this limitation in the Methods/Discussion and cite literature supporting NaCl’s restricted efficacy for polymers with densities. 
2-Discuss how this shallow sampling could bias seasonal comparisons, particularly if wet-season flows redistribute sediments vertically.  
3- While field blanks were mentioned, procedural details (e.g., air exposure during drying, lab environment particulate counts) were insufficiently documented.Provide QA/QC specifics in Supplementary Materials and quantify blank corrections applied.  

4- Airborne MPs contribute significantly to freshwater sediments but were not assessed. Recommend atmospheric sampling in future work to address this gap.  
5- Compare global reservoir trends to contextualize pollution severity

Author Response

Dear reviewer
We would like to express our sincere gratitude for reviewing our manuscript, which have greatly improved the overall quality of the paper. In response, we have thoroughly revised the manuscript. We hope that our revisions and detailed responses (provided in bold below) adequately address all concerns and meet the requirements for publication in Water

 

Reviewer #1

 

This study provides valuable baseline data on microplastic (MP) pollution in a subtropical reservoir system, demonstrating significant spatial and seasonal variations. While the work addresses important knowledge gaps regarding MP accumulation in dammed rivers, several methodological and contextual limitations should be addressed to strengthen the manuscript. Below are specific comments and suggestions for revision: 
Response: Thank you for taking the time to thoroughly review our manuscript and for providing valuable feedback. Your comments and suggestions have been instrumental in improving the quality of our work. We have carefully addressed all the comments/suggestions raised, including methodological and contextual limitations, to strengthen the manuscript.

1- The use of NaCl solution (density: 1.22 g/cm³) for density separation likely underestimated low-density MPs (e.g., polyethylene, polystyrene), which constitute common plastic pollutants. Explicitly acknowledge this limitation in the Methods/Discussion and cite literature supporting NaCl’s restricted efficacy for polymers with densities. 

Response: We have now added limitations in the methods section and cited (Line 189-192).

The sieved sediments were then subjected to density separation using a 25 L stainless steel bucket filled with 100 ml of filtered NaCl solution (1400 g NaCl in 0.6 L water; density 1.22 g/cm³), following the method described by Quinn et al. [34] and Cutroneo et al. [35]. However, it should be noted that the use of NaCl solution (1400 g NaCl in 0.6 L water; density 1.22 g/cm³) likely led to an underestimation of low-density micro-plastics (e.g., polyethylene, polystyrene), which may not float in solutions of this den-sity [34,36].

 

Duong, T.T., Le, P.T., Nguyen, T.N.H., Hoang, T.Q., Ngo, H.M., Doan, T.O., Le, T.P.Q., Bui, H.T., Bui, M.H., Trinh, V.T. and Nguyen, T.L., 2022. Selection of a density separation solution to study microplastics in tropical riverine sediment. Environmental monitoring and assessment, 194, pp.1-17. https://doi.org/10.1007/s10661-021-09664-0

Quinn, B., Murphy, F. and Ewins, C., 2017. Validation of density separation for the rapid recovery of microplastics from sediment. Analytical Methods, 9(9), pp.1491-1498. DOI: 10.1039/C6AY02542K 

2-Discuss how this shallow sampling could bias seasonal comparisons, particularly if wet-season flows redistribute sediments vertically.  

Response: Now discussed, Line 144- 146

3- While field blanks were mentioned, procedural details (e.g., air exposure during drying, lab environment particulate counts) were insufficiently documented. Provide QA/QC specifics in Supplementary Materials and quantify blank corrections applied.  

Response: QA/QC was taken into consideration during extraction process and also during MPs identification process (see line 181-184; 205-208). We have also added a supplementary file as requested.

4- Airborne MPs contribute significantly to freshwater sediments but were not assessed. Recommend atmospheric sampling in future work to address this gap.  

Response: We have now added recommendation for future research, see line 403-410. “It is also important to acknowledge that atmospheric deposition may represent an additional and often overlooked pathway for microplastic input into freshwater systems. Airborne microplastics, transported via wind and precipitation, can be deposited directly into rivers and reservoirs. Although this source was not assessed in the present study, recent research by Mutshekwa et al. (2025) has shown that MP deposition from the atmosphere occurs across seasons in South Africa and could contribute to sedimentary MP contamination, particularly in exposed open-water systems like Vondo Reservoir. Future studies should incorporate atmospheric sampling to better quantify this pathway and its role in freshwater MP pollution.”


5- Compare global reservoir trends to contextualize pollution severity

Response: Thank you for the suggestion. We have now expanded the comparison of our findings with additional studies, particularly those focused on microplastic pollution in African freshwater systems, to better contextualize and validate our results.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear. Authors

The submitted manuscript (water-3680154) investigates the distribution of microplastics in sediment at the Vondo Reservoir on the Mutshindudi River. The topic is of interest, and the study has potential; however, substantial revisions are required to improve the clarity, rigor, and overall quality of the manuscript. Therefore, I recommend a major revision.

 

1. There are major comments.

1.1. Justification for the study area

Lines 97–101 present the objective of the study. However, the rationale for selecting this particular site is insufficient. I suggest incorporating Lines 140–143 directly after the objective to highlight the importance and relevance of the Vondo Reservoir for microplastic pollution research.

1.2. Originality of the manuscript

In the Introduction, the manuscript mentions that this is the first study investigating microplastic pollution in the Vondo Reservoir. To enhance the originality, the authors should compare their study with previous research on microplastics in other African freshwater systems. This also should be highlighted in the Abstract section.

1.3. Identification of microplastics

Lines 178–191: The authors should provide detailed information about the FT-IR analytical conditions, including the detection technique (e.g., ATR), wavenumber range, resolution, number of particles analyzed per sample, and the identification rate. Additionally, the authors should clearly describe the criteria used to identify polymers, such as by comparing the obtained spectra with those from reference materials or literature sources.

1.4. Calculation of microplastic content

Lines 184–186: The manuscript states that 50–60% of randomly selected particles were analyzed by FT-IR. However, the method used to extrapolate the total microplastic content (particles/kg) from these subsamples needs to be clarified.

1.5. Size distribution of microplastics

Information on the size of microplastics is essential for understanding their behavior and transport in aquatic systems. Did the authors measure or categorize the size of the particles?

1.6. Limitations of density separation

While sodium chloride is commonly used for density separation, it may not recover denser polymers such as PET or PVC. Since they were detected, the authors should discuss the potential underestimation of denser microplastics in the Discussion section.

1.7. Equations (1) and (2)

Lines 203–204: The Pollution Load Index (PLI) is typically calculated using the geometric mean of contamination factors and not by taking the square root of a single factor. The current representation appears to be incorrect and should be revised.

1.8. Figure 2.

Figure 2 shows overall MP characteristics (color, shape, polymer type), but the text states that there are seasonal and spatial differences. The authors should expand on these variations and discuss differences among seasons and sampling sectors.

1.9. n-MDS

Lines 293–296 mention n-MDS analysis, but no results or interpretations are provided. This section should either be elaborated upon or removed.

1.10. Correlation with water quality variables

Lines 144-148, 220-236, 292-297, Table 1, and Figure 4.

The authors measured water quality parameters (e.g., temperature, pH, conductivity, and TDS), but the MPs were found in sediment, not water. Unless relevant pollution indicators (e.g., DO, BOD, eutrophication factors) are provided, drawing correlations between water parameters and sediment MPs is not appropriate. I recommend removing or reconsidering this section.

1.11. Comparison with other studies

In the Discussion, the authors should compare their findings with other studies on microplastic pollution, especially in African freshwater systems. This would help to contextualize and validate the results.

 

2. Minor Comments

2.1. Figure 1

The caption is missing for "Site 7." Please revise.

2.2. Unit

Ensure consistent formatting for all units throughout the manuscript (e.g., g/cm³, m², particles/kg, mg/L). Use superscripts and spacing consistently.

2.3.2.3. Typos

Line 339: “... water run-off directly into the reservoir., recreational parks, improper disposal of ...” → Remove the comma after "reservoir."

Line 362: “... various colours, shapes and colours ...” → Repetition of “colours” should be corrected.

 

 

I hope these comments will be helpful in revising the manuscript. I look forward to seeing the improved version.

Sincerely,
The Reviewer

 

Comments for author File: Comments.pdf

Author Response

Dear reviewer
We would like to express our sincere gratitude for reviewing our manuscript, which have greatly improved the overall quality of the paper. In response, we have thoroughly revised the manuscript. We hope that our revisions and detailed responses (provided in bold below) adequately address all concerns and meet the requirements for publication in Water

 

Review #2

The submitted manuscript (water-3680154) investigates the distribution of microplastics in sediment at the Vondo Reservoir on the Mutshindudi River. The topic is of interest, and the study has potential; however, substantial revisions are required to improve the clarity, rigor, and overall quality of the manuscript. Therefore, I recommend a major revision.
Response: Thank you for your constructive comments and for recognizing the potential of our study. We appreciate your detailed feedback, which has been invaluable in improving the clarity, rigor, and overall quality of the manuscript. We have carefully considered and addressed all your comments and suggestions in the revised version.

1. There are major comments.

1.1. Justification for the study area

Lines 97–101 present the objective of the study. However, the rationale for selecting this particular site is insufficient. I suggest incorporating Lines 140–143 directly after the objective to highlight the importance and relevance of the Vondo Reservoir for microplastic pollution research.
Response: We have now incorporated line 140-143 after our objective, see line 97

1.2. Originality of the manuscript

In the Introduction, the manuscript mentions that this is the first study investigating microplastic pollution in the Vondo Reservoir. To enhance the originality, the authors should compare their study with previous research on microplastics in other African freshwater systems.

This also should be highlighted in the Abstract section.
Response: We have incorporated comparisons of our findings with other studies conducted both within Africa and globally in the Discussion section. Additionally, we have highlighted these in the Abstract.

1.3. Identification of microplastics

Lines 178–191: The authors should provide detailed information about the FT-IR analytical conditions, including the detection technique (e.g., ATR), wavenumber range, resolution, number of particles analyzed per sample, and the identification rate. Additionally, the authors should clearly describe the criteria used to identify polymers, such as by comparing the obtained spectra with those from reference materials or literature sources.
Response: We have now added such information
“For polymer identification, 50% - 60% of the total MP particles found (smaller than 5 mm) were randomly selected and analysed using a vibrational Platinum-ATR Fourier-transform infrared spectroscopy (FT-IR) (Bruker Alpha model, Germany) technique with a spectral region of 400–3200 cm−1, resolution of 8 cm−1, and a rate of 16 scans per analysis.” Line 204-206

“The MP particle spectra were compared to polymer databases, including the Hummel Polymer Sample Library, HR Polymer Additives and Plasticizers, HR Hummel Polymers and Additives, and Synthetic Fibres by Microscope, with a matching degree of >75% between sample and standard spectra considered acceptable.” Line 211-215

1.4. Calculation of microplastic content

Lines 184–186: The manuscript states that 50–60% of randomly selected particles were analyzed by FT-IR. However, the method used to extrapolate the total microplastic content (particles/kg) from these subsamples needs to be clarified.
Response: Now addressed, see line 179-180.

1.5. Size distribution of microplastics

Information on the size of microplastics is essential for understanding their behavior and transport in aquatic systems. Did the authors measure or categorize the size of the particles?
Response: Thank you for your observation. Unfortunately, we did not measure the size of the microplastic particles in this study. However, we acknowledge its importance and will incorporate particle size measurements in our future research.

1.6. Limitations of density separation

While sodium chloride is commonly used for density separation, it may not recover denser polymers such as PET or PVC. Since they were detected, the authors should discuss the potential underestimation of denser microplastics in the Discussion section.
Response: Now discussed, see line 380-384.

1.7. Equations (1) and (2)

Lines 203–204: The Pollution Load Index (PLI) is typically calculated using the geometric mean of contamination factors and not by taking the square root of a single factor. The current representation appears to be incorrect and should be revised.
Response: PLI now removed from the manuscript following another review suggesting. Thank you feedback regarding the error we made.

1.8. Figure 2.

Figure 2 shows overall MP characteristics (color, shape, polymer type), but the text states that there are seasonal and spatial differences. The authors should expand on these variations and discuss differences among seasons and sampling sectors.
Response: Thank you for the valuable comment. In this study, colors, shapes, and polymer types were combined to describe the overall MP composition, while only the MP data were grouped according to sites and seasons. We recognize that a more detailed fingerprinting approach would strengthen the analysis, and we will incorporate this in future research. Thank you again

1.9. n-MDS

Lines 293–296 mention n-MDS analysis, but no results or interpretations are provided. This section should either be elaborated upon or removed.
Response: Removed. Thank You for noticing our mistake.

1.10. Correlation with water quality variables Lines 144-148, 220-236, 292-297, Table 1, and Figure 4.

The authors measured water quality parameters (e.g., temperature, pH, conductivity, and TDS), but the MPs were found in sediment, not water. Unless relevant pollution indicators (e.g., DO, BOD, eutrophication factors) are provided, drawing correlations between water parameters and sediment MPs is not appropriate. I recommend removing or reconsidering this section.
Response: Thank you for this valuable comment. While we agree that microplastics (MPs) were measured in sediment, we respectfully argue that water quality parameters such as temperature, pH, conductivity, and TDS are still relevant, as they can influence the transport, distribution, and deposition of MPs in sediment. For example, variations in pH and conductivity can affect the aggregation behavior of particles, while temperature influences water movement and biological activity, both of which play roles in sedimentation processes.

 

Furthermore, several studies (e.g., Mbedzi et al., 2023, to name few) have explored correlations between water quality parameters and MP abundance in sediments. We believe that including this information adds environmental context to our findings, though we acknowledge that more direct pollution indicators (e.g., DO, BOD) would provide additional insight and will be considered in future studies.

 

1.11. Comparison with other studies

In the Discussion, the authors should compare their findings with other studies on microplastic pollution, especially in African freshwater systems. This would help to contextualize and validate the results.

Response: Thank you for the suggestion. We have now expanded the comparison of our findings with additional studies, particularly those focused on microplastic pollution in African freshwater systems, to better contextualize and validate our results.

2. Minor Comments

2.1. Figure 1

The caption is missing for "Site 7." Please revise.

Response: Thank you for noticing the error, new figure with site 7 now added

2.2. Unit

Ensure consistent formatting for all units throughout the manuscript (e.g., g/cm³, m², particles/kg, mg/L). Use superscripts and spacing consistently.
Response: Now corrected throughout the manuscript.

2.3.2.3. Typos

Line 339: “... water run-off directly into the reservoir., recreational parks, improper disposal of ...” → Remove the comma after "reservoir."
Response: Now corrected,  Line 332.

Line 362: “... various colours, shapes and colours ...” → Repetition of “colours” should be corrected.

Response: Now corrected,  Line 359.

I hope these comments will be helpful in revising the manuscript. I look forward to seeing the improved version. Sincerely, The Reviewer
Response: Thank for your feedback.

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Summary of manuscript

Seasonal sediment surveys were conducted at nine sites situated upstream and downstream of a reservoir, with two sampling campaigns completed in 2023 and three replicate samples collected at each location. The hypotheses were (1) that the reservoir functions as a sink for microplastics (MPs) and (2) that MP densities would peak during the dry season when reduced flows facilitate sediment accumulation. The resulting data are reported to support both hypotheses. However, these findings are based on an inappropriate statistical model and the number of sampling events is likely too small to adequately assess seasonality.

Introduction

The introduction is complete with sufficient recent sources, however there should be more support for the author’s second hypothesis on Line 105. Some logical connection between the MP description in the earlier sections of the Introduction and dry season conditions.

Methods

The methods are adequately described, but are two samples sufficient for determining differences in seasons? How do the authors separate potential sampling variability versus temporal variability? This is very weakly supported and should be de-emphasized in the text. If the authors had monthly samples this would be a result that is far stronger.

Why use the pollution load index? Why not use the MP data itself?

ANOVA assumptions are not met by this data. The residuals here cannot be normally distributed as they must be truncated at zero (e.g., there cannot be a negative concentration of MPs). Zero-truncated regressions with an appropriate distribution, a generalized linear mixed model for count data, or some other statistical method should be used.

The color, MP shape, and polymer type data seem underutilized. A fingerprinting methodology (possibly through a combination of principal component analysis and grouping analysis) could strengthen the author’s conceptual model of the reservoirs trapping MP and source attribution.

Results

The results section starts with water quality comparisons by season, this isn’t the most important finding so I would recommend the authors move this below the MP results. Additionally, Table 2 supports my comments about the variability in the data not sufficiently captured by two sampling events, the dry season downstream numbers are less than the wet season, counter to what is in the abstract.

I’m still not sure why indices are required in addition to the MP results.

Does Figure 4 need to be in the manuscript? Isn’t it sufficient to write in the text that no significant relationships were observed?

Discussion/Conclusion

No major comments within this section other to say that I still feel the seasonal comparison lacks the sample numbers to be an accurate test of MP seasonality. And that the statistics used are not appropriate and this text should be updated once those new methods are used.

 

Minor Comments

Line 30, define MP pollution indices for readers before using the term.

Line 32, why would white fibers indicate diverse sources?

Line 116, is the unit for surface area correct?

Line 240-250, the units are not superscripted.

Author Response

Dear reviewer
We would like to express our sincere gratitude for reviewing our manuscript, which have greatly improved the overall quality of the paper. In response, we have thoroughly revised the manuscript. We hope that our revisions and detailed responses (provided in bold below) adequately address all concerns and meet the requirements for publication in Water

 

Reviewer #3

Seasonal sediment surveys were conducted at nine sites situated upstream and downstream of a reservoir, with two sampling campaigns completed in 2023 and three replicate samples collected at each location. The hypotheses were (1) that the reservoir functions as a sink for microplastics (MPs) and (2) that MP densities would peak during the dry season when reduced flows facilitate sediment accumulation. The resulting data are reported to support both hypotheses. However, these findings are based on an inappropriate statistical model and the number of sampling events is likely too small to adequately assess seasonality.

Response: Thank you for your thorough review of our manuscript and for providing insightful feedback that has greatly contributed to improving our work. We have carefully addressed all the corrections and suggestions you provided.

 

Introduction

The introduction is complete with sufficient recent sources, however there should be more support for the author’s second hypothesis on Line 105. Some logical connection between the MP description in the earlier sections of the Introduction and dry season conditions.

Response: We have now added logical connection as requested, see line 108-114.

Methods

The methods are adequately described, but are two samples sufficient for determining differences in seasons? How do the authors separate potential sampling variability versus temporal variability? This is very weakly supported and should be de-emphasized in the text. If the authors had monthly samples this would be a result that is far stronger.

Response: Thank you for your thoughtful review and valuable feedback on our manuscript. We greatly appreciate your time and expertise in evaluating our work. We agree with your comment regarding the limitations of using only two sampling events to assess seasonal differences in microplastic (MP) contamination. To address this, we have revised the manuscript to de-emphasize the seasonal findings and included a statement acknowledging that the limited number of sampling timepoints may not fully capture seasonal trends and could reflect sampling variability rather than true temporal changes.

 

Why use the pollution load index? Why not use the MP data itself?
Response: Following another reviewer suggestion, we have decided to remove the pollution indices calculations from the manuscripts.

ANOVA assumptions are not met by this data. The residuals here cannot be normally distributed as they must be truncated at zero (e.g., there cannot be a negative concentration of MPs). Zero-truncated regressions with an appropriate distribution, a generalized linear mixed model for count data, or some other statistical method should be used.

Response: Thank you for the comment. We acknowledge the limitations associated with using ANOVA when assumptions of normality and homoscedasticity are not met. However, ANOVA was applied only to the environmental variables (e.g., temperature, pH, conductivity), which in our dataset were approximately normally distributed and met the required assumptions based on diagnostic checks. For microplastic (MP) density data, which are zero-truncated and non-normally distributed, we now used non-parametric Kruskal–Wallis tests to assess seasonal and sites differences.

The color, MP shape, and polymer type data seem underutilized. A fingerprinting methodology (possibly through a combination of principal component analysis and grouping analysis) could strengthen the author’s conceptual model of the reservoirs trapping MP and source attribution.

Response: Thank you for the valuable comment. In this study, colors, shapes, and polymer types were combined to describe the overall MP composition, while only the MP data were grouped according to sites and seasons. We recognize that a more detailed fingerprinting approach would strengthen the analysis, and we will incorporate this in future research. Thank you again

Results

The results section starts with water quality comparisons by season, this isn’t the most important finding so I would recommend the authors move this below the MP results. Additionally, Table 2 supports my comments about the variability in the data not sufficiently captured by two sampling events, the dry season downstream numbers are less than the wet season, counter to what is in the abstract.
Response: Thank you for the comment. We have moved the water quality section to the end of the Results as suggested. We agree with your observation and have revised the abstract accordingly. Furthermore, an explanation for the observed seasonal variability is now provided in Lines 343–352.

 

I’m still not sure why indices are required in addition to the MP results.
Response: Thank you for the comment. We have decided to remove the indices from the manuscript as suggested.

Does Figure 4 need to be in the manuscript? Isn’t it sufficient to write in the text that no significant relationships were observed?
Response: Agree, we have now removed figure 4

Discussion/Conclusion

No major comments within this section other to say that I still feel the seasonal comparison lacks the sample numbers to be an accurate test of MP seasonality. And that the statistics used are not appropriate and this text should be updated once those new methods are used.

Response: Thank you very much for your comments and suggestions. We have addressed them accordingly and updated the discussion section. However, the Kruskal–Wallis analysis produced similar results—showing differences between sites and no significant differences between seasons—therefore only minimal revisions were necessary in the discussion.

 

Minor Comments

Line 30, define MP pollution indices for readers before using the term.
Response: We have removed pollution indices in our study as suggested earlier and by another reviewer

Line 32, why would white fibers indicate diverse sources?

Response: Agree, we have now rephrased our statement , Line 32.

Line 116, is the unit for surface area correct?

Response: Thank you for noticing, it was incorrect. We have now corrected it “km2”

Line 240-250, the units are not superscripted.
Response: We noticed that in the originally submitted document, the units appeared superscripted due to the journal's formatting. We have now corrected this issue.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed all comments. No other comments 

Author Response

Comments and Suggestions for Authors

The authors addressed all comments. No other comments 

Response: Thank you

Reviewer 2 Report

Comments and Suggestions for Authors

Dear. Authors

This is a 2^nd round review report for the manuscript (water-3680154). Thank you for your responses to the comments. However, the reviewer found it difficult to follow the changes because the line numbers the authors referred to in the response letter did not match those in the revised manuscript. It would be helpful if you could include the actual revised text in your future response letters to make the revisions clear to verify.

Overall, the revisions for 1^st round have been generally well modified. However, the reviewer believes that some parts still require further revision, so the reviewer has decided to classify this as a major revision.

 

1. 1. The term "density" is uncommon to describe microplastics present in the sediment matrix. The reviewer recommends using either "abundance" or "content" consistently throughout the main text instead of "density".
2. The statement in Lines 256–258, which suggests that low-density microplastics may have been underestimated in this study, is inappropriate. This contradicts the explanation provided in Lines 851–855. A relatively low-density solution was used for density separation in this study. Microplastics with densities higher than that of the solution might be underestimated, not the low-density ones.

Lines 256-258: However, it should be noted that the use of NaCl solution (1400 g 256NaCl in 0.6 L water; density 1.22 g/cm³) likely led to an underestimation of low-density microplastics (e.g., polyethylene, polystyrene), which may not float in solutions of this density [34,36].

3. Although the manuscript states that the correlation between the distribution of microplastics and water quality variables was examined using Pearson correlation analysis (Lines 615-617), the statistical method is not described in the Materials and Methods section under Statistical Analysis (Lines 308-316). Furthermore, even if the analysis showed no statistically significant correlations, the lack of significance itself should still be reported with corresponding R² values. A results table presenting the R² and associated significance levels would enhance the clarity of the results.
4. The data in a disaggregated format is essential to support any correlation analysis and to ensure logical consistency between the stated objectives and the presented results. Therefore, the results of the water variables in Table 1 should be organized by both seasons and sites.
5. Line 321-334. The Kruskal–Wallis test is a non-parametric statistical method that is appropriate for comparing medians across independent samples. It is a valid choice for analyzing differences in microplastic content among the three sampling point groups (upstream, reservoir, downstream), where the samples can reasonably be considered independent. However, for comparing microplastic contents between two different seasons at the same sampling points, the samples are paired, not independent. Therefore, the Kruskal–Wallis test is not appropriate for this type of analysis, as it does not account for the paired nature of the data. Additionally, since the study compares microplastic content between two seasons, not three or more seasons, the Wilcoxon signed-rank test would be more suitable for assessing median differences in paired samples. This choice of test may influence the statistical significance of the results, and revisiting the analysis with the appropriate method is recommended.
6. Lines 610-614. For the same reason, since the samples are collected from the same sampling points across different seasons, they should be treated as paired (dependent) samples, not independent. Therefore, if the data follow a normal distribution, the paired t-test is more appropriate than ANOVA to compare mean values between two seasonal datasets at the same sampling points.
7. 7. Lines 102-160. The final paragraph of the Introduction section is rather long, which reduces its readability. The reviewer recommends splitting it into two or more paragraphs based on the key points to improve clarity and enhance the logical flow of the text.

 

I hope these comments will help revise the manuscript.

Sincerely,
The Reviewer

 

Comments for author File: Comments.pdf

Author Response

Review  #2

Thank you for the comments/suggestions to improve our manuscript

Dear. Authors

This is a 2^nd round review report for the manuscript (water-3680154). Thank you for your responses to the comments. However, the reviewer found it difficult to follow the changes because the line numbers the authors referred to in the response letter did not match those in the revised manuscript. It would be helpful if you could include the actual revised text in your future response letters to make the revisions clear to verify.

 

Response: We did also upload a clean version of the manuscript. We truly apologise if it did not reach you

 

Overall, the revisions for 1^st round have been generally well modified. However, the reviewer believes that some parts still require further revision, so the reviewer has decided to classify this as a major revision.
Response: Thank you

 

1. 1. The term "density" is uncommon to describe microplastics present in the sediment matrix. The reviewer recommends using either "abundance" or "content" consistently throughout the main text instead of "density"
   Response: We appreciate the reviewer’s suggestion regarding the use of terminology. However, we have intentionally used the term "density" to describe microplastic concentrations in sediment, as our values were standardized and reported in particles per kilogram (particles/kg) of dry sediment. This usage aligns with numerous published studies in the field, where “density” is commonly employed to reflect microplastic counts relative to sediment mass. Given the consistency of this terminology in the existing literature and the methodological basis of our calculations, we respectfully maintain the use of "density" throughout the manuscript. See examples below
   
   Rodrigues, C., Rodríguez, Y., Frias, J., Carriço, R., Sobral, P., Antunes, J., Duncan, E.M. and Pham, C.K., 2024. Microplastics in beach sediments of the Azores archipelago, NE Atlantic. Marine Pollution Bulletin, 201, p.116243.
   Nkosi, M.S., Cuthbert, R.N., Wu, N., Shikwambana, P. and Dalu, T., 2023. Microplastic abundance, distribution, and diversity in water and sediments along a subtropical river system. Environmental Science and Pollution Research, 30(39), pp.91440-91452.
   Gabriel, A.D. and Bacosa, H.P., 2024. Microplastic abundance and distribution in the sediment of Cagayan de Oro River, Philippines. Soil and Sediment Contamination: An International Journal, 33(8), pp.1252-1268.
   Themba, N.N., Dondofema, F., Cuthbert, R.N., Munyai, L.F. and Dalu, T., 2024. Abundance and distribution of microplastics in benthic sediments and Cladocera taxa in a subtropical Austral reservoir. Integrated Environmental Assessment and Management, 20(6), pp.2256-2270.
   Dalu, T., Ngomane, N., Dondofema, F. and Cuthbert, R.N., 2023. Water or sediment? Assessing seasonal microplastic accumulation from wastewater treatment works. H2Open Journal, 6(2), pp.88-104.
   Mutshekwa, T., Munyai, L.F., Mugwedi, L., Cuthbert, R.N., Dondofema, F. and Dalu, T., 2023. Seasonal occurrence of microplastics in sediment of two South African recreational reservoirs. Water Biology and Security, 2(3), p.100185.

 

2. The statement in Lines 256–258, which suggests that low-density microplastics may have been underestimated in this study, is inappropriate. This contradicts the explanation provided in Lines 851–855. A relatively low-density solution was used for density separation in this study. Microplastics with densities higher than that of the solution might be underestimated, not the low-density ones.
   
   

Lines 256-258: However, it should be noted that the use of NaCl solution (1400 g 256NaCl in 0.6 L water; density 1.22 g/cm³) likely led to an underestimation of low-density microplastics (e.g., polyethylene, polystyrene), which may not float in solutions of this density [34,36].
Response: We have removed the statement, Thank you. Line 189

3. Although the manuscript states that the correlation between the distribution of microplastics and water quality variables was examined using Pearson correlation analysis (Lines 615-617), the statistical method is not described in the Materials and Methods section under Statistical Analysis (Lines 308-316). Furthermore, even if the analysis showed no statistically significant correlations, the lack of significance itself should still be reported with corresponding R² values. A results table presenting the R² and associated significance levels would enhance the clarity of the results.
   Response: We did describe (see line 225-229). We chose not to include a table of correlation results as no statistically significant relationships were detected, and including non-significant R² values could be misleading or overinterpreted, especially in an exploratory context with limited explanatory power. In such cases, highlighting non-significant trends without biological or ecological relevance may detract from the central findings of the study.
4. The data in a disaggregated format is essential to support any correlation analysis and to ensure logical consistency between the stated objectives and the presented results. Therefore, the results of the water variables in Table 1 should be organized by both seasons and sites.
   Response: We appreciate the reviewer’s suggestion. However, we chose to present the water quality data in Table 1 as overall averages across seasons to emphasize the statistically significant differences between seasons, which aligns with our stated objectives. Detailed values for both seasons and individual sites are already presented in the main text, ensuring transparency and accessibility of the disaggregated data. We felt that including all values in the table would result in redundancy and reduce clarity.
5. Line 321-334. The Kruskal–Wallis test is a non-parametric statistical method that is appropriate for comparing medians across independent samples. It is a valid choice for analyzing differences in microplastic content among the three sampling point groups (upstream, reservoir, downstream), where the samples can reasonably be considered independent. However, for comparing microplastic contents between two different seasons at the same sampling points, the samples are paired, not independent. Therefore, the Kruskal–Wallis test is not appropriate for this type of analysis, as it does not account for the paired nature of the data. Additionally, since the study compares microplastic content between two seasons, not three or more seasons, the Wilcoxon signed-rank test would be more suitable for assessing median differences in paired samples. This choice of test may influence the statistical significance of the results, and revisiting the analysis with the appropriate method is recommended.

Response: We thank the reviewer for the thoughtful comment. We acknowledge that the Wilcoxon signed-rank test is suitable for paired data; however, in our study, we treated seasonal samples from the same sites as independent due to logistical and temporal constraints—the samples were collected during distinct seasonal events and not as true pairs from the exact same sampling event or conditions. Additionally, this approach aligns with several published studies on seasonal comparisons of microplastics in freshwater systems, which also used Kruskal–Wallis tests when comparing distributions across seasons, especially in cases where data are not normally distributed and sample sizes are unbalanced. Given the non-parametric nature of our data and the goal to identify general differences between seasons across multiple sites, we found the Kruskal–Wallis test appropriate and robust for our analysis. We respectfully maintain this statistical approach for consistency with related literature and the structure of our dataset. Others reviewers are happy with our statistical approach.

Graca, B., Rychter, A., Bełdowska, M. and Wojdasiewicz, A., 2024. Seasonality of mercury and its fractions in microplastics biofilms-comparison to natural biofilms, suspended particulate matter and bottom sediment. Science of The Total Environment, 948, p.174814.

Mashamba, R., Cuthbert, R.N., Dondofema, F., Munyai, L.F., Wu, N. and Dalu, T., 2024. Spatiotemporal variation in macroplastic abundances along a subtropical Austral river system. Environmental Monitoring and Assessment, 196(3), p.273.

Dalu, T., Themba, S.T., Dondofema, F., Wu, N. and Munyai, L.F., 2024. Assessing microplastic abundances in freshwater fishes in a subtropical African reservoir. Discover Sustainability, 5(1), p.360.

 

6. Lines 610-614. For the same reason, since the samples are collected from the same sampling points across different seasons, they should be treated as paired (dependent) samples, not independent. Therefore, if the data follow a normal distribution, the paired t-test is more appropriate than ANOVA to compare mean values between two seasonal datasets at the same sampling points.
   Response: See response above, thank you
7. 7. Lines 102-160. The final paragraph of the Introduction section is rather long, which reduces its readability. The reviewer recommends splitting it into two or more paragraphs based on the key points to improve clarity and enhance the logical flow of the text.
   
   We have now split into two paragraphs

 

I hope these comments will help revise the manuscript.

Sincerely,
The Reviewer

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have adequately addressed my comments. 

Author Response

Comments and Suggestions for Authors

The authors have adequately addressed my comments. 

Response: Thank You

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Dear. Authors

Thank you for the authors' second response to the manuscript (water-3680154). The reviewer finds the reply to my comments appropriate and largely acceptable. However, the reviewer remains unconvinced by the justification provided for the use of the Kruskal–Wallis test.

When comparing results from two different seasons at the same sampling points, the observations inherently reflect all environmental changes that occurred between seasons, such as water temperature and TDS, as reported in the submitted manuscript. Therefore, seasonal comparisons of microplastic median values should be based on paired data from each site, since site-specific characteristics remain constant except for temporal variation, unless the sampling points themselves were not the same.

The discussion on whether the data are parametric or non-parametric is not central to the current issue concerning the appropriateness of the Kruskal–Wallis test. The Wilcoxon signed-rank test, as suggested by the reviewer, is also a non-parametric test. Hence, the concern lies in the data structure (paired vs. independent), not distributional assumptions.

While the reviewer respects the perspectives of other reviewers and decided “accept after minor revisions,” the reviewer maintains that the statistical method used in the current manuscript is not the most appropriate for comparing medians in this dataset.

If the authors choose to retain the Kruskal–Wallis test for consistency with previous studies, the reviewer recommends that the authors explicitly cite the supporting previous studies and clearly acknowledge in the manuscript that this method is not statistically optimal for paired data. Such clarification is important to prevent misinterpretation by readers and to uphold analytical transparency.

 

I hope these comments are helpful to the authors in the proper selection of statistical analysis skills in their further studies.

 

Sincerely,
The Reviewer

Comments for author File: Comments.pdf

Author Response

Dear. Authors

Thank you for the authors' second response to the manuscript (water-3680154). The reviewer finds the reply to my comments appropriate and largely acceptable. However, the reviewer remains unconvinced by the justification provided for the use of the Kruskal–Wallis test.

When comparing results from two different seasons at the same sampling points, the observations inherently reflect all environmental changes that occurred between seasons, such as water temperature and TDS, as reported in the submitted manuscript. Therefore, seasonal comparisons of microplastic median values should be based on paired data from each site, since site-specific characteristics remain constant except for temporal variation, unless the sampling points themselves were not the same.

The discussion on whether the data are parametric or non-parametric is not central to the current issue concerning the appropriateness of the Kruskal–Wallis test. The Wilcoxon signed-rank test, as suggested by the reviewer, is also a non-parametric test. Hence, the concern lies in the data structure (paired vs. independent), not distributional assumptions.

While the reviewer respects the perspectives of other reviewers and decided “accept after minor revisions,” the reviewer maintains that the statistical method used in the current manuscript is not the most appropriate for comparing medians in this dataset.

If the authors choose to retain the Kruskal–Wallis test for consistency with previous studies, the reviewer recommends that the authors explicitly cite the supporting previous studies and clearly acknowledge in the manuscript that this method is not statistically optimal for paired data. Such clarification is important to prevent misinterpretation by readers and to uphold analytical transparency.

 

I hope these comments are helpful to the authors in the proper selection of statistical analysis skills in their further studies.

 

Sincerely,
The Reviewer


Response: Thank you for the helpful comment. We confirm that sampling was conducted at the exact same points across seasons, and we agree that a paired analysis is more appropriate. Accordingly, we have replaced the Kruskal–Wallis test with the Wilcoxon signed-rank test, and the results have been updated in the manuscript. We appreciate your guidance in improving the statistical robustness of our study.