Development of a new methodology for the determination of PET microplastics in sediment, based on microwave-assisted acid digestion

Marco Perez; Sonnia Parra; Cristofher Ferrada; Manuel Bravo; Pablo A. Perez; Waldo Quiroz

doi:10.1371/journal.pone.0314520

Peer Review History

Original SubmissionJuly 8, 2024
6 Oct 2024 Decision Letter - Amitava Mukherjee, Editor PONE-D-24-22018Development of a new methodology for the determination of PET microplastics in sediment, based on microwave-assisted acid digestion.PLOS ONE Dear Dr. Quiroz, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Nov 20 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below. Reviewer #1: No: The overall flow of the manuscript must be improved. ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The overall methodology focuses on the recovery of PET particles. Kindly check the comments which have been included as an attachment. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Review comments.pdf https://doi.org/10.1371/journal.pone.0314520.r001
Revision 1
24 Oct 2024 Author Response Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 5. Is the article presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below. Reviewer #1: No: The overall flow of the manuscript must be improved. R: Several grammatical errors were corrected, and the overall flow of the text was improved by incorporating connecting paragraphs and repositioning entire sections. We believe this new structure better follows the logical sequence of the validation process: first reproduction of staining Nile Red method, second the sample treatment validation; third, the color identification process; and finally, the application of the method to real samples. ________________________________________ 6. Review Comments to the Author The authors have formulated a way to recover PET particles from the sample by subjecting the samples to differential conditions, including different temperature and excitation wavelength. These conditions have successfully eliminated other interferences at each consequent stages and resulted in the detection of PET MPs. I would like to suggest the following: General comments: Applies to the entire manuscript. 1. Check the manuscript for grammatical and spelling errors. For example: In keywords “Red Nyle” is not correct. R: Red Nyle was corrected to Nye Red or NR in all manuscript 2. For all the statistical representation, mention the software used for the preparation of graphs. Additionally, include a statistical analysis section for this in the methodology head. R: The "Statistical Analysis" section included details on the software used, the type of statistical tests, and the confidence level applied for each data analysis. 3. I suggest it would be appropriate if a pictorial representation or a flowchart is included for methodology, including the effect of each step (such as elimination of chitin fluorescence after digestion) R: Fig. 2. Flowchart of the method was included according to reviewer advice. Representative pictures and the explicit purpose of each analytical step were included. This figure replace Figure 1 on this new version of the manuscript 4. The font consistency is lacking. For example: “Result and Discussions” and “Conclusions” are in different forms; also check font in Table 1. R: Font and size font was checked on all manuscript including head titles, table and figure labels “Vancouver font” was used in all manuscript and the size of the font and formatting were corrected according to Plos One “Manuscript body formatting guidelines”, Hence, I recommend the authors to rewrite the manuscript considering all the above stated suggestions and thus improving the overall flow of the manuscript. Specific comments: Major comments- 1. The addition of the possible real-life applications of this methodology would be beneficial for the readers. Additionally, the methodology referred to in reference number 9 has successfully recovered five polymer types. I suggest the authors to clearly include a reason for their focus only on PET. R: At the end of the discussion, an explanatory paragraph was added to clarify why this study focused on PET. Briefly, there are two main reasons. First, PET is one of the most frequently detected plastics in municipal waste. Locally, a published study has shown that many floating plastic containers have been found in Chilean rivers, including the Loa River, which is part of our study. Finally, our results indicate that this type of polymer was the most frequently observed in these samples. 3 references were included to support our choice. Finaly a monitoring was performed along sediments of Loa River in Chile. 4 sampling locations were included in this new version and results are presented in Figure 12. 2. It would be effective if the authors stated the reasons for each and every step as it will provide great understanding for the readers. R: This suggestion aligns with that made by the other reviewer. We recognize the importance of clarifying the purpose of each step in the methodology. For this reason, we have removed Figure 1 and replaced it with a pictorial flow diagram that includes the description and purpose of each methodological step. This is now Figure 2 in the revised manuscript. 3. Line 130- It would be good if the authors explained the use of only 254nm wavelength filter and not the 365nm wavelength as mentioned in the reference paper. R: The results of the Nile red-stained microplastics exposed to a 365 nm excitation wavelength were included, along with a discussion demonstrating that selective fluorescence was not achieved at this wavelength. This is now Figure 4 in the revised version of the manuscript. Also state the efficiency of this method over already existing methods. Additionally, clarification of the possibility to recover other particles using different excitation wavelengths would be an added advantage. This information would be helpful since many researchers aim to isolate all the potential plastic constituents from the environmental samples. Again, I suggest the authors to clearly include a reason for their focus only on PET. For this addition of microscopic images depicting the particles in bright field, the selected fluorescence wavelength (254nm) and in other wavelengths, superimposed on each other would substantiate the present study. R: We greatly appreciate this reviewer’s comment, as it highlights the broader potential of the method we presented in the initial version of the manuscript. Indeed, this method has the potential to be extended to the identification and quantification of PE and PP. The reason we focused solely on PET is because this method was designed to be applied to the Loa River, which is primarily contaminated by PET plastic bottles, as described in a previous study that has been added to the introduction. However, the identification of PE and PP still faces a selectivity challenge that we have not yet fully resolved. As shown in Figure 3, both PE and PP fluorescence colors are yellow with similar tones. Nevertheless, we agree with the reviewer that it is important to make readers aware of the real potential of this method. Therefore, we have taken two actions: First, we used RGB color recognition through the artificial intelligence platform ChatGPT-4.0 to demonstrate two key points. Firstly, the method selectively detects PET. Secondly, while AI-based color recognition is possible for PE and PP, selectivity remains a challenge that must be addressed to fully unlock the method's potential. For this reason, we have included Figure 9 to demonstrate the power of RGB color recognition for the three plastics (PET, PE, and PP) and Figure 10 to show the method’s selectivity for detecting PET, as well as the limitations regarding selectivity for PE and PP Minor comments- 1. Considering that the density of PET is less than that of CaCl2, causing it to float, and that LDPE, PP, and PS are recovered in both solutions due to their lower density, I have a question regarding the use of NaCl (1.20 g/cm3). Since PET would settle in NaCl at the above density, I would like to suggest the authors to consider recovering the PET in a denser NaCl solution. If not, it would be good if explanation was provided for why this approach was not considered for the recovery of PET in NaCl. R: We acknowledge that presenting the flotation step results after the direct digestion method may give the impression that this step was not sufficiently optimized. However, the purpose of reproducing the flotation step is not to incorporate it into our procedure, but rather to demonstrate that it is unnecessary and that good recovery results can still be achieved by omitting this step and directly applying microwave digestion to the sample. To avoid further confusion, the flotation method results were presented first and discussed in this context, prior to introducing the recovery results from the direct digestion method. 2. Mention the purpose of the rapid cooling of the samples in line 126 and also provide the temperature. R: By apply this cycle of 50°C for staining and then 0°C for cooling have shown us that the particles exhibit more intense and longer-lasting fluorescence. We include the temperature and a proper discussion about this process. 3. Line 146: Briefly describe the calcination process and why it is used here? R: Considering the heterogeneity in the distribution of microplastics in the environment, spike and recovery studies on real samples cannot be conducted using naturally contaminated samples because “native microplastics” cannot be subtracted mathematically. For this reason a calcination procedure was applied to a real sediment sample, to eliminate native MPs. A proper explanation was included at the end of “Trueness determination” method description according to reviewer advice. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0314520.r002
12 Nov 2024 Decision Letter - Amitava Mukherjee, Editor Development of a new methodology for the determination of PET microplastics in sediment, based on microwave-assisted acid digestion. PONE-D-24-22018R1 Dear Dr. Quiroz, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Amitava Mukherjee, ME, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes ****** 4. If the manuscript contains new data, have the authors made this data fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the article presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Most of the explanations are appropriate and the article has been refined. This article is noow helpful to thee scientific community. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No ******** https://doi.org/10.1371/journal.pone.0314520.r003
Formally Accepted
27 Nov 2024 Acceptance Letter - Amitava Mukherjee, Editor PONE-D-24-22018R1 PLOS ONE Dear Dr. Quiroz, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Dr. Amitava Mukherjee Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0314520.r004

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