Microplastic Retention in Secondary Sewage Sludge: Characterization and Influence of Solid Concentration
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors1.The introduction does not adequately compare the mechanisms of chemical agents on MPs removal as discussed in references [12,27]. It is recommended to supplement the analysis of differences from existing studies (e.g., "Previous studies focused on Al-based coagulants, while this study is the first to systematically compare the synergistic effects of FeCl₃ and nonionic PAM").
2.The research boundaries are unclear, and the specific contribution of "TSS influencing MPs retention" needs to be explicitly defined (e.g., is this the first study to quantify the threshold of TSS affecting floc-MPs interactions?).
3.The dosages of FeCl₃ (5%) and PAM (5 ppm) lack optimization evidence. It is suggested to cite similar studies or provide preliminary screening data.
4.In Figure 5, clarify the relationship between the red curve (retention efficiency %) and the bar chart (MPs concentration) in the figure caption.
Author Response
Comment 1. The introduction does not adequately compare the mechanisms of chemical agents on MPs removal as discussed in references [12,27]. It is recommended to supplement the analysis of differences from existing studies (e.g., "Previous studies focused on Al-based coagulants, while this study is the first to systematically compare the synergistic effects of FeCl₃ and nonionic PAM").
Response 1. More detailed information on the coagulant and flocculant values ​​used in this study is included in the introduction section to clarify it (page 2, lines 51-55, 58-60 and 62-70). The novelty of this work is now indicated at the end of the “Introduction” section (page 2, lines 83-86).
Comment 2. The research boundaries are unclear, and the specific contribution of "TSS influencing MPs retention" needs to be explicitly defined (e.g., is this the first study to quantify the threshold of TSS affecting floc-MPs interactions?).
Response 2. More detailed information about the research boundaries is now included at the end of the introduction section (page 2, lines 83-86).
Comment 3. The dosages of FeCl₃ (5%) and PAM (5 ppm) lack optimization evidence. It is suggested to cite similar studies or provide preliminary screening data.
Response 3. This was just a first approach and we are now preparing a new work that investigate the effect of the dosages. In any case, more detailed information on the coagulant and flocculant concentrations ​​used in this study has been included in the results and discussions section to clarify it (pages 15, lines 397 to 402).
Comment 4. In Figure 5, clarify the relationship between the red curve (retention efficiency %) and the bar chart (MPs concentration) in the figure caption.
Response 4. The mathematical procedure used to extract the data from the red curve (% MP retention) has been described (page 10, lines 300-302). The percentage of MP retained was calculated by subtracting the number of MPs that remained in the supernatant after settling from the number of MPs that were initially in the water-sludge mixture and dividing the result by the initial number of MPs.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsThis work analyzes microplastics contained in thickened secondary sludge from a local WWTP over a period of six months (sampling at monthly intervals), monitors the shape, chemical composition and size of microplastics in the sludge. Here I have a question, whether microplastics are also found in the wastewater itself, or in the water during sludge thickening. And how to separate them from the complex matrix?
The captured secondary sludge was used for laboratory work, in which the influence of TSS and the addition of a coagulant FeCl3 to improve the efficiency of microplastic capture was tested.
After the first reading of the article, the text seemed quite incomprehensible, but after studying the Supplementary, the published data make sense of the article. I do not know if they will also be showed in the published article.
It is unintelligible because there is a lot of numerical data (sludge flakes, microplastic particle size, composition of solutions, etc.) mainly in laboratory experiments.
On the other hand, I praise the research design and the implementation of the experiments, there is not much published data on the topic of MP retention. The experiments are conducted meaningfully, precisely, correctly, contain all the necessary data, measurements, from this point of view there is not much to criticize the authors of the article.
The work is original, highly up-to-date and the scope of the experiments only documents the given issue. The text processing, evaluation of the experiment and results are at a high professional level.
Questions and comments on the article:
1. It would be interesting to monitor the efficiency of capturing (removing) microplastics within each step of the WWTP technological line, have such monitoring been done? Is the efficiency of the WWTP assessed based on input and output parameters? How much microplastic was at the input and output of the WWTP?
2. How did you prepare standard solutions with different MP concentrations for laboratory experiments? We have sludge flakes, a complex matrix, microplastics (61%) in the form of fibers, how can such microplastics be separated so that you get an accurate standard value (e.g. 120-240 MP, 240-480 MP...) the text states several times that "MP obtained in the field", how did you know the MP value?
3 I quote from the text: " MP obtained in the field used to enrich suspensions were isolated from thickened sludges, .... MP were obtained by filtration through glass fiber filters, what is the porosity of these filters? In my opinion, the sludge will prevent filtration, it is not easy to filter. Especially when I am interested in the solid fraction and not the filtered water, e.g. in the case of a sludge:water sample, where 100 g of sludge is added to 500 ml of distilled water.
3. Can Fenton's reagent or hydrogen peroxide have an effect on the decomposition (decomposition) of microplastics?
4. The authors focused on only a few parameters (TSS, pH, sludge moisture) during the research, it might be good to also list the basic characteristic parameters of the WWTP, e.g. COD, BOD, TOC, insoluble and soluble substances, dry matter, dry matter of dewatered sludge, the text deals with sludge, it needs to be described.
Author Response
Comment 1. This work analyzes microplastics contained in thickened secondary sludge from a local WWTP over a period of six months (sampling at monthly intervals), monitors the shape, chemical composition and size of microplastics in the sludge. Here I have a question, whether microplastics are also found in the wastewater itself, or in the water during sludge thickening. And how to separate them from the complex matrix?
Response 1. The separation of MPs from complex matrices is typically performed through an experimental procedure involving oxidation (to remove organic matter), density separation (to remove inorganic matter), and vacuum filtration of the final solution containing MPs. The MPs are then retained on a final filter, ready for quantification, classification, and analysis. It is explained in the "materials and methods" section (page 4, lines 129 – 140).
Some reviews that include information about similar procedures are listed below:
- Sol, D., Laca, A., Laca, A., and Díaz, M. Wastewater treatment approaches to remove microplastics. Polluting Textiles: The Problem with Microfibres. 2022. https://doi.org/10.4324/9781003165385-9
- Franco, A. A., Martín-García, A. P., Egea-Corbacho, A., Arellano, J. M., Albendín, G., Rodríguez-Barroso, R., and Coello, M. D. Assessment and accumulation of microplastics in sewage sludge at wastewater treatment plants located in Cádiz, Spain. Pollut., 2023, 317, 120689. https://doi.org/10.1016/j.envpol.2022.120689
- Casella, C., Sol, D., Laca, A., and Díaz, M. Microplastics in Sewage Sludge: A review. Sci. Pollut. Res., 2023, 1-34. https://doi.org/10.1007/s11356-023-27151-6
Comment 2. The captured secondary sludge was used for laboratory work, in which the influence of TSS and the addition of a coagulant FeCl3 to improve the efficiency of microplastic capture was tested.
After the first reading of the article, the text seemed quite incomprehensible, but after studying the Supplementary, the published data make sense of the article. I do not know if they will also be showed in the published article.
It is unintelligible because there is a lot of numerical data (sludge flakes, microplastic particle size, composition of solutions, etc.) mainly in laboratory experiments.
Response 2. According to the Reviewer’s suggestion the section of results has been improved with the addition of Figures and Tables from the Supplementary Material (see Figures 7, 8 and 11 in pages 12, 13 and 16, respectively; and Table 1 in page 4).
On the other hand, I praise the research design and the implementation of the experiments, there is not much published data on the topic of MP retention. The experiments are conducted meaningfully, precisely, correctly, contain all the necessary data, measurements, from this point of view there is not much to criticize the authors of the article.
The work is original, highly up-to-date and the scope of the experiments only documents the given issue. The text processing, evaluation of the experiment and results are at a high professional level.
Questions and comments on the article:
Comment 3. It would be interesting to monitor the efficiency of capturing (removing) microplastics within each step of the WWTP technological line, have such monitoring been done? Is the efficiency of the WWTP assessed based on input and output parameters? How much microplastic was at the input and output of the WWTP?
Response 3. This study was focused on the presence and behaviour of MPs contained in the secondary sludge, which has been less studied than the presence of MPs in the water line (nobody has previously studied the effect of solid concentrations). Indeed, several works have been already published investigating the removal efficiency of microplastics within each step of the municipal wastewater treatment. MP concentrations in the influent (raw wastewater) and effluent (treated wastewater) are considered to calculate overall MP removal efficiencies, but also partial efficiencies for each treatment can been found in previous works. In particular, we have recently published several works on this subject:
- Sol, D., Laca, A., Laca, A., & Díaz, M. (2021). Microplastics in wastewater and drinking water treatment plants: occurrence and removal of microfibres. Applied Sciences, 11(21), 10109. https://doi.org/10.3390/app112110109
- Menéndez-Manjón, A., Martínez-Díez, R., Sol, D., Laca, A., Laca, A., Rancaño, A & Díaz, M. Long-term occurrence and fate of microplastics in WWTPs: a case study in southwest europe (2022) Applied Sciences-Basel, 12:4, 2133. https://doi.org/10.3390/app12042133
- Sol, D., Solís-Balbín, C., Laca, A., Laca, A., & Díaz, M. (2023). A standard analytical approach and establishing criteria for microplastic concentrations in wastewater, drinking water and tap water. Science of the Total Environment, 899, 165356. https://doi.org/10.1016/j.scitotenv.2023.165356
Comment 4. How did you prepare standard solutions with different MP concentrations for laboratory experiments? We have sludge flakes, a complex matrix, microplastics (61%) in the form of fibers, how can such microplastics be separated so that you get an accurate standard value (e.g. 120-240 MP, 240-480 MP...) the text states several times that "MP obtained in the field", how did you know the MP value?
Response 4. MP concentrations were measured in the initial sludge samples. Then, mixtures water-sludge were prepared and enriched with MPs isolated from other previous sludge. MPs were isolated using methods developed by other authors, which included oxidation, density separation, filtering and quantification by counting in the microscopy. Some changes have been made on "Materials and methods" section to clarify it. As we know the initial MP concentration in the sludge and the amount of MPs added, we know the initial MP concentrations in the mixtures (we tried that they were similar in all the experiments). Below you can see some works that used similar procedures to quantify MPs in sludges.
- Franco, A. A., Martín-García, A. P., Egea-Corbacho, A., Arellano, J. M., Albendín, G., Rodríguez-Barroso, R., and Coello, M. D. Assessment and accumulation of microplastics in sewage sludge at wastewater treatment plants located in Cádiz, Spain. Pollut., 2023, 317, 120689. https://doi.org/10.1016/j.envpol.2022.120689
- Casella, C., Sol, D., Laca, A., and Díaz, M. Microplastics in Sewage Sludge: A review. Sci. Pollut. Res., 2023, 1-34. https://doi.org/10.1007/s11356-023-27151-6
Comment 5. I quote from the text: " MP obtained in the field used to enrich suspensions were isolated from thickened sludges, .... MP were obtained by filtration through glass fiber filters, what is the porosity of these filters? In my opinion, the sludge will prevent filtration, it is not easy to filter. Especially when I am interested in the solid fraction and not the filtered water, e.g. in the case of a sludge:water sample, where 100 g of sludge is added to 500 ml of distilled water.
Response 5. Sludge samples are not filtered directly, as explained in material and methods, the sludge was subjected to a pre-treatment including double oxidation, first for 24 hours with H2O2 and then for another 24 hours at room temperature with Fenton's reagent. Lastly, ZnCl2 was used for density separation. Subsequently, the solution that contain MPs was filtered through filters made of glass microfiber (pore size 0.7 μm, Whatman) (Page 4, line 129-140).
Comment 6. Can Fenton's reagent or hydrogen peroxide have an effect on the decomposition (decomposition) of microplastics?
Response 6. This aspect has been studied in previous works. Large amounts of organic matter are present in the sewage sludge samples, thus it's important to strike a balance between eliminating organic matter and harming or degrading MPs. This equilibrium can be reached using H2O2 and Fenton, resulting in a high extraction capacity of MPs and an organic matter (OM) clearance rate greater than 80%. MPs may suffer damage or deterioration if H2O2 and Fenton are used at high temperatures or for very long times. For instance, it is known that using H2O2 at 60–70°C can destroy polyamide (nylon, PA-6,6) and degrade polystyrene (PS). Conversely, using H2O2 at room temperature does not harm polymers unless exposure is prolonged for 7 days, in which case polypropylene (PP) and polyethylene terephthalate (PET) may be harmed. The breakdown of ethylene-vinyl acetate (EVA), polylactic acid (PLA), polymethyl methacrylate (PMMA), PET, and PA can be impacted by the heat released during the oxidation of the organic matter by Fenton.
In the present study, the samples were pre-treated at room temperature for relatively short time periods (48 h) (Page 4, lines 134–144). The majority of the organic matter was removed during the first treatment with H2O2 (24 hours), a weaker oxidizing agent than Fenton. Afterwards, the Fenton reaction was conducted at room temperature to guarantee complete OM removal (24 hours). Since there was very little OM in this instance, the reaction is less exothermic, meaning that the sample was not heated. It has been reported that to prevent MP degradation in sewage sludge samples, reasonably short (< 7 days) periods and keeping the temperature reaction below 50ºC are crucial.
This information and more details about the pre-treatment of sewage sludge samples for MP measurement can be found for instance in the next works:
- Sol, D., Solís-Balbín, C., Laca, A., Laca, A., & Díaz, M. (2023). A standard analytical approach and establishing criteria for microplastic concentrations in wastewater, drinking water and tap water. Science of The Total Environment, 899, 165356. https://doi.org/10.1016/j.scitotenv.2023.165356
- Cavazzoli, S., Ferrentino, R., Scopetani, C., Monperrus, M., & Andreottola, G. (2023). Analysis of micro-and nanoplastics in wastewater treatment plants: key steps and environmental risk considerations. Environmental Monitoring and Assessment, 195(12), 1483. https://doi.org/10.1007/s10661-023-12030-x
- Munno, K., Helm, P. A., Jackson, D. A., Rochman, C., & Sims, A. (2018). Impacts of temperature and selected chemical digestion methods on microplastic particles.Environmental toxicology and chemistry, 37(1), 91-98. https://doi.org/10.1002/etc.3935
Comment 7. The authors focused on only a few parameters (TSS, pH, sludge moisture) during the research, it might be good to also list the basic characteristic parameters of the WWTP, i.e., COD, BOD, TOC, insoluble and soluble substances, dry matter, dry matter of dewatered sludge, the text deals with sludge, it needs to be described.
Response 7. Several parameters such as total organic carbon (TOC), dry matter (DM), and chemical oxygen demand (tCOD) values are now included in Table 1 (page 4). In addition, information about these parameters is included in lines 113-123 (pages 3-4) from Materials and methods section.
Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for Authors1. Please explain the abbreviation WWTP
2. All used and listed devices should include: the device model, and manufacturer (City, Country). Please complete this.
3. Please briefly describe the procedure described in [28] - Line 98
4. There is no information about where the reagents used were obtained. Please complete the manufacturer (City, Country) for each reagent used.
5. Please provide the magnifications used during microscopic analysis.
6. Please explain the abbreviation TSS.
7. Please provide the number of scans performed for FTIR analysis and in what wavenumber range.
8. Please describe "the Quality assurance and Quality control" procedure. Simply indicating sources 30-31 is not sufficient.
9. There are no error bars in Figure 3. Is this the average result of several measurements or just one measurement?
10. Referring to supplementary figures and tables is problematic for the reader. It would be best if the figures and tables to which the authors refer were placed in the work. Please then standardize the numbering of Figures and Tables throughout the document.
11. The authors write that they detected 14 different polymers - please show sample FTIR spectra. A verbal description alone is insufficient.
12. Please expand the conclusions with a summary of each study. Please show what practical significance the obtained results have and what future perspectives they give in the interdisciplinary area.
The English could be improved to more clearly express the research.
Author Response
Following Reviewer´s suggestion, the English of the entire manuscript has been revised and corrected by a native speaker.
Comments and Suggestions for Authors
Comment 1. Please explain the abbreviation WWTP
Response 1. It is now indicated in the abstract and introduction sections (page 1, lines 12 and 27).
Comment 2. All used and listed devices should include: the device model, and manufacturer (City, Country). Please complete this.
Response 2. The manuscript provides now detailed information on the brands and technical specifications of all equipment, devices, and reagents used in this study. For example, please see lines 113, 122, 125, 133, 135, 139, 142, etc.
Comment 3. Please briefly describe the procedure described in [28] - Line 98
Response 3. The experimental procedure described in reference [28] has been included (page 3, lines 116-119).
Comment 4. There is no information about where the reagents used were obtained. Please complete the manufacturer (City, Country) for each reagent used.
Response 4. According to the Reviewer’s suggestion, a detail information about reagents has been included (page 4, lines 129-140; and page 5, lines 157 to 165).
Comment 5. Please provide the magnifications used during microscopic analysis.
Response 5. According to the Reviewer’s suggestion, a detail information about zoom of stereomicroscope analysis has been included (page 5, line 174).
Comment 6. Please explain the abbreviation TSS.
Response 6. It is now indicated in the abstract and introduction sections (page 1, line 17 and page 2, line 79).
Comment 7. Please provide the number of scans performed for FTIR analysis and in what wavenumber range.
Response 7. According to the Reviewer’s suggestion, a detail information about FTIR analysis has been included (Pages 5 and 6, lines 185 to 188).
Comment 8. Please describe "the Quality assurance and Quality control" procedure. Simply indicating sources 30-31 is not sufficient.
Response 8. According to the Reviewer’s suggestion, a detail information about QA/QC process is now included (Page 6, lines 190 to 205).
Comment 9. There are no error bars in Figure 3. Is this the average result of several measurements or just one measurement?
Response 9. Percentages were calculated on the basis of the total number of flocs of each size counted in two samples (by microscopy). That is why error bars aren´t indicated.
Comment 10. Referring to supplementary figures and tables is problematic for the reader. It would be best if the figures and tables to which the authors refer were placed in the work. Please then standardize the numbering of Figures and Tables throughout the document.
Response 10. According to the Reviewer’s suggestion, some Figures and Tables from Supplementary Materials have been included in the manuscript to facilitate the understanding of the article (see Figures 7, 8 and 11 in pages 12, 13 and 16, respectively; and Table 1 in page 4).
Comment 11. The authors write that they detected 14 different polymers - please show sample FTIR spectra. A verbal description alone is insufficient.
Response 11. The "Supporting Information" file now includes Figure S3, displaying the FTIR spectra for all 14 MP types analyzed in this study.
Comment 12. Please expand the conclusions with a summary of each study. Please show what practical significance the obtained results have and what future perspectives they give in the interdisciplinary area.
Response 12. According to the Reviewer’s suggestion, the conclusion section has been improved (page 17, lines 454-472).
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsThis article could be accept in present form.
Comments on the Quality of English LanguageThe English could be improved to more clearly express the research.
Author Response
Comment 1. The English could be improved.
Response 1. Thank you for your comment. We certainly made some typos when transcribing the latest English revision. The entire text has been again revised and corrected by a native speaker (in blue). Attached you can find a Revisor´s certificate.
Author Response File: 
Author Response.pdf
