PONE-D-23-29563A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita)  using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and GenotoxicityPLOS ONE

Dear Dr. Shahzad,

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.

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Amel Mohamed El Asely

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript 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

Reviewer #2: Yes

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4. Is the manuscript 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 note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

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5. 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 manuscript entitled (A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita) using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and Genotoxicity) needs major changes before being published

General comments:

The manuscript needs language editing

The introduction section should be improved

The methodology needs more details

The aim of work not clear

Specific comments

Abstract section

-line 18, insert a full stop after (Products)

-line 19 to 21 rephrase

-the abstract should be informative, the scientific name of fish should be added, number of fish used and their average weight, number of fish groups, number of replicates, and the experimental duration.

The methodology should mention briefly in the abstract.

-Add a space between T0(0.00 µg/L).

-the full name of all abbreviations written in the abstract should be written

- (Due to higher oxidative enzymes activity antioxidant enzymes potential to overcome their stress activity reduced) this sentence should be deleted, no explanations were written in the abstract

-which level of ethyl paraben that produced the results written in the abstract

Keywords: remove the word, aquatic toxicology

Introduction section

-the introduction section lack the information about the toxic impact of paraben on fish tissue, health

Also the residues of such paraben in water bodies, how paraben gain access to the water bodies. The uses of paraben in agriculture practices

-the significance of the studied fish (rohu, Labeo rohita) should be added in the introduction section

Why the authors used this fish as toxicological model

-the aim of work not clear because the problem not clarified well in the introduction section

-add the following references in the introduction section, they will be useful

1- Ahmed, S.A., Ibrahim, R.E., Elshopakey, G.E., Khamis, T., Abdel-Ghany, H.M., Abdelwarith, A.A., Younis, E.M., Davies, S.J., Elabd, H., Elhady, M.J.F., Immunology, S., 2022. Immune-antioxidant trait, growth, splenic cytokines expression, apoptosis, and histopathological alterations of Oreochromis niloticus exposed to sub-lethal copper toxicity and fed thyme and/or basil essential oils enriched diets. 131, 1006-1018.

2- Ibrahim, R.E., El-Houseiny, W., Behairy, A., Mansour, M.F., Abd-Elhakim, Y.M., 2019. Ameliorative effects of Moringa oleifera seeds and leaves on chlorpyrifos-induced growth retardation, immune suppression, oxidative stress, and DNA damage in Oreochromis niloticus. Aquaculture 505, 225-234.

3- Mohamed, W.A., El-Houseiny, W., Ibrahim, R.E., Abd-Elhakim, Y.M., 2019. Palliative effects of zinc sulfate against the immunosuppressive, hepato-and nephrotoxic impacts of nonylphenol in Nile tilapia (Oreochromis niloticus). Aquaculture 504, 227-238.

4- Ibrahim, R.E., Elbealy, M.A., Salem, G.A., Abdelwarith, A.A., Younis, E.M., Wagih, E., Elkady, A.A., Davies, S.J. and Rahman, A.N.A., 2023. Acute mancozeb-fungicide exposure induces neuro-ethology disruption, health disorders, and immune-oxidative dysfunction in Nile tilapia (Oreochromis niloticus). Aquatic Toxicology, 261, p.106630.

5- Ibrahim, Rowida E., Heba I. Ghamry, Saed Ayidh Althobaiti, Daklallah A. Almalki, Medhat S. Shakweer, Mona A. Hassan, Tarek Khamis, Heba M. Abdel-Ghany, and Shaimaa AA Ahmed. "Moringa oleifera and Azadirachta indica Leaves enriched diets mitigate chronic oxyfluorfen toxicity induced immunosuppression through disruption of pro/anti-inflammatory gene pathways, alteration of antioxidant gene expression, and histopathological Alteration in Oreochromis niloticus." Fishes 8, no. 1 (2022): 15.

Material and method section

1-line 91, replace made with assigned

2-number of fish and weight should be mentioned

3-what is the LC50 value for ethyl parapen, why authors evaluated it for 48 hours not for 96 hours

4- What is the relation between the lC50 and the different doses of parapen used in the experiment?

5-line 109, add the dose of clove oil and reference

6-the number of samples should be written for each group in all methodology (blood samples, histological samples, ….etc)

7-the procedures of histological examinations is lack many details, so superficial

The details should be added

8- All abbreviations should be defined for the first time

9- For the statistical analysis, the normality test should be performed before the ANOVA

Results

1- All tables titles should be rephrased and rewritten

2- Table footnotes should be added and the full names of all measured parameters should be inserted in the footnote

3- Line 196, the author written (propyl paraben), the authors used ethyl paraben or propyl paraben

4- All data in the figures and tables should be carefully revised by the authors

Discussion section

This part should be revised and improved

Line 320-321 why written in italic form

Many language errors are present

Conclusion section: this section not present

Reviewer #2: 1. The abstract has not been written in a clear and concise manner. For example the paragraph starting with ethyl paraben induced oxidative .....; is not stating the findings clearly.

2. There is reference to genotoxicity in the abstract. However, it is not clear which marker for genotoxicity was used to arrive at this conclusion. If this was based on the COMET Assay, then indicate that "based on the comet assay, ....

3. Under methods: it is not clear how the dosages of 2000, 4000, and 6000 were chosen. Is this based on actual estimated concentrations of paraben in contaminated waterways? How much paraben is fish exposed to in water? Do they bio-accumulate in fish tissues? What steps were taken to ensure that prior exposure to paraben and related chemicals did not occur in regard to fish used for the study? This needs to come out clearly.

4. Quite significantly, the manuscript has recurring grammar issues, and poor prose/sentence construction. For example, the sentence that reads..." all experiments were formed.... The word "formed" is quite out of place. This is repeated throughout (line 75). Experiments are "conducted or performed" etc... Do not use don't..., rather use do not etc...

5. Did the study investigate levels of glutathione or glutathione reductase? In the results, the sub-title is GSH, which is used to abbreviate glutathione and not glutathione reductase. In the discussion and figures, references have been made in regard to antioxidants including glutathione (GSH). Where does glutathione reductase come in line 140-144?

6. Hematology data has been presented in a table format. This is not correct. Dose response studies are best presented using line graphs to easily decipher the trend. This observation applies to antioxidant analysis (GSH, TBARS, ROS, SOD, CAT etc..)

7. The data on antioxidant assay is quite confusing for liver/kidney. ROS is going up, consistent with TBARS; but antioxidant enzymes that are meant to be up-regulated in response to exposure to chemical toxicants such as paraben are actually being down-regulated. This is a paradox. Note that there is a dose-dependent rise in ROS. Antioxidants are expected to rise in concert to the rise in ROS. For this study, antioxidants are going down as the dose of paraben goes up. However, many studies have demonstrated depletion of GSH levels in response to overwhelming chemical-induced oxidative stress. This may explain the decrease in GSH. Not sure about the SOD, CAT etc..,. This scenario may be possible when there is overwhelming oxidative stress, depleting all the antioxidants. Clarify this phenomenon. Notably, liver function markers are all up-regulated, signaling severe paraben-driven liver injury.

8. Data for hematology shows evidence for paraben-driven significant up-regulation of platelets. This elevation will have serious implications for blood-related disorders like DVT and patients who may be on blood thinning medications. This aspect needs to be discussed more broadly in the discussion section to highlight implications in various circumstances. Infact, RBCs and HGB will be expected to be down-regulated due to a possible paraben-driven impediment to hematopoiesis.

9. It is not clear why paraben will result in elevation of HGB, MCV, MCHC and platelets.

10. Notably, at low dosages, the effect of paraben on blood indices was low, validating the premise of this study. This, probably needs to come out well in the discussion.

11. It is very important that the grammar issues are corrected, and the manuscript is written carefully in a concise and logical manner using appropriate english.

12. There is need for a more concise title tied to the major findings of the study.

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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

Reviewer #2: Yes: Alfred Orina Isaac

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Attachments

Attachment

Submitted filename: reviewer comments to PONE-D-23-29563.docx

PONE-D-23-29563R1A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita)  using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and GenotoxicityPLOS ONE

Dear Dr. Shahzad,

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 Jan 22 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Amel Mohamed El Asely

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript 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

Reviewer #2: Yes

**********

5. Is the manuscript 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 note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

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 authors made all comments and the revised manuscript now accepted for publication

no further correction were needed

Reviewer #2: I gave detailed review proposals in my first analysis of this work. The authors have made reasonable effort to correct the figure for dose response studies as i guided. However, the grammar issues have not been addressed across the board starting with the abstract, through to the discussion section. A lot of work needs to be done here.

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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

Reviewer #2: 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.

PONE-D-23-29563R2A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita)  using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and GenotoxicityPLOS ONE

Dear Dr. Shahzad,

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 Apr 04 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Amel Mohamed El Asely

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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[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.

PONE-D-23-29563R3A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita)  using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and GenotoxicityPLOS ONE

Dear Dr. Shahzad,

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. I have observed that your response to the reviewers' comments was not addressed carefully, and the quality of English in your manuscript still needs improvement. Therefore, we recommend that you adhere to the instructions by providing detailed responses to the reviewers in a separate file and enhancing English in your manuscript. The attached file contains the comments from the reviewers. Please carefully consider every point and respond to them individually, addressing each one separately.

Please submit your revised manuscript by May 17 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Amel Mohamed El Asely

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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A study on assessing the toxic effects of ethyl paraben on rohu (Labeo rohita)  using different biomarkers; Hemato-biochemical assays, Histology, Oxidant and Antioxidant activity and Genotoxicity

PONE-D-23-29563R4

Dear Dr. Shahzad,

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Kind regards,

Amel Mohamed El Asely

Academic Editor

PLOS ONE

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