PONE-D-23-43946Biomonitoring benzophenones in guano samples of wild bats in PolandPLOS ONE

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Reviewers' comments:

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Comments to the Author

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

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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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: In the paper “Biomonitoring benzophenones in guano samples of wild bats in Poland Benzophenones in wild bats in Poland” the authors assessed the concentration levels of selected BPs commonly used in the industry including benzophenone 1 (BP-1), benzophenone 2 (BP-2), benzophenone 3 (BP-3) and benzophenone 8 (BP-8) in guano samples collected from colonies of greater mouse eared bats (Myotis myotis) using liquid chromatography with the tandem mass spectrometry (LC-MS/MS) method. This manuscript is well organized, and the drawn conclusions are coherent with the obtained results. I have enjoyed reading your paper; however, the manuscript must be revised by an English native speaker to fix several grammatical errors that I detected in the paper. I hope to provide very useful suggestions to improve the overall clarity of your study as well as the quality of your analysis. I think that my suggestions look feasible to you, and I believe you will be able to address them. Thus, please take care to do a full revision of your manuscript according to all my comments. Improvements based on my comments will be crucial for acceptance. I have some concerns and suggestions for each aspect of the manuscript. Please see below.

Abstract: I would like to suggest giving more emphasis to the results.

Line 30: To arrange the keywords in alphabetic order.

Introduction: The paper is technically sound and the claims are convincing. However I think that some references should be updated. Please, note that the hypothesis and the predictions are unclear, you need to well explain them.

Lines 98 - 99: I think that you should add these important references to support your sentence: “Bats are highly sensitive to environmental pollution and any environmental change”. I would like to suggest:

Salinas-Ramos, V. B., et al., (2021). Artificial illumination influences niche segregation in bats. Environmental Pollution, 284, 117187.

Huszarik, M., et al., (2023). Increased bat hunting at polluted streams suggests chemical exposure rather than prey shortage. Science of The Total Environment, 905, 167080.

Lines 99 - 100: I think that you should add these important references to support your sentence: “which is the reason for the continuous reduction in their population and the protection of many bat species”. I would like to suggest:

Ancillotto, L., et al., (2021). Resilient responses by bats to a severe wildfire: conservation implications. Animal Conservation, 24(3), 470-481.

Gilmour, L. R., et al., (2020). Comparing acoustic and radar deterrence methods as mitigation measures to reduce human-bat impacts and conservation conflicts. PLoS One, 15(2), e0228668.

Lines 109 - 110: I think that you should add these important references as example to support your sentence: “Feces/guano samples are practically the only matrix, which may be collected without the need for significant interference in the animal life. ”. I would like to suggest:

Del Vaglio, M. A., Nicolau, H., Bosso, L., & Russo, D. (2011). A first assessment of feeding habits in the fruit bat Rousettus aegyptiacus on Cyprus island. Hystrix, 22(2), DOI:10.4404/hystrix-22.2-4587.

Provencher, J. F., Vermaire, J. C., Avery-Gomm, S., Braune, B. M., & Mallory, M. L. (2018). Garbage in guano? Microplastic debris found in faecal precursors of seabirds known to ingest plastics. Science of the Total Environment, 644, 1477-1484.

Lines 112 – 117: Please, explain in detail you hypothesis and predictions.

Materials and methods: In general, the methods are appropriate and the study seems well conducted, although some details deserve a bit more attention i.e., especially about the methodology and the data. All the script used in this paper must be added in the supplementary materials. Please, provide also all the link to source where you downloaded the data.

Line 130: Why did you choose this bat species?

Figure 1: Please, add the scale and the north symbol.

Results: Well written! The figures and the tables are all informative and necessary, but not redundant, ensuring the correct comprehension of the manuscript.

Discussion: The paper discussed appropriately the context and the theme, although there is important literature not cited by the authors. I think that the authors should be discussing their results also comparing them with those already published on other species/genus/family. In fact your paper discusses findings in relation to some of the work in the field but ignores other important work that I think should be added in your discussion.

Reviewer #2: Dear Authors,

Congratulations on this interesting article. In general, I found the manuscript to be very well written, with substantial amount of data, with a clear structure, and the manuscript was easy to follow.

The authors analysed 40 bat guano samples from 4 sites in Poland for the content of benzophenones, using LC-MS/MS. The used LC-methodology was already published, but especially the analysis and results of the method used for these emerging pollutants will be of interest for readers of PLOS one. In my opinion the article is suitable for publication in PLOS one after Minor Revision.

Major remarks:

• L23/abstract: Using the median makes more sense as already used in Table 2

• L26-28: Rephrase the sentence. The results show that BPs can be determined by sampling guano and LC-MS/MS analysis and not to be an alternative to urine or blood samples. Blood samples were not compared to guano samples

• L32-117: I would suggest providing the structures of the BPs

• L32-117: Which other approaches are available for the analysis of BPs?

• L32-117: It should already be mentioned in the Introduction that the determined levels are compartment dependent as mentioned in the discussion

• L134: Rounding the population to hundreds is better, the exact number of citizens is irrelevant; e.g. 11,300

• L136-143: Good job!

• L176: Which commercial guano was used/ from which origin. I would assume there would be differences from the composition of the guano from insectivores, frugivores and nectarivores bats. The composition could have an influence on the sample preparation.

• L178: Why was 0.01 ng/g used as calibration point when the MDL is 0.04 ng/g. That makes no sense for me. Data should be reanalysed.

• L181-185: Good job!

• L196: Thank you for providing the single data in the supplementary material

• L271-277: I would assume that the used commercial guano is from a big frugivores bat colony (Madagascar?) and the colony is not next to a town/village/city. I a therefore not surprised that the commercial guano is not highly contaminated. It should be mentioned that Myotis myotis is an insectivore, feeding on various arthropods which could have an influence on the detected compounds.

• L278-290: Good point!

• L294-305: It is also an advantage that whole bat populations can be analysed by “one” sample. To my experience the difference of the pollutant load between individuals and the whole colony is not that great.

• L 320-335: I would assume there are differences in the distribution of the BPs in different compartments, and data must be available for other mammals (e.g. mice, humans). The faeces contain mainly fat-soluble substances whereas urine contains more hydrophilic substances

• L348-352: Good point!

• Figure 2: Should be revised. It is a bit confusing.

Minor remarks:

• L23: the concentration is already a level

• L32-117: The article of Schanzer et al could be useful (Chemosphere 2022, 135342)

• L121: disperse

• L122: What means high purity, please provide %

• L127: -18 space °C; no zero symbol

• L312/ L314: 47,400 and 2400

Reviewer #3: Studies on chemical pollutants in terrestrial environments are extremely important, especially emerging ones for which there is little or no information in the literature. Bats are mammals with unique characteristics, and therefore, they perform fundamental ecosystem services for the balance of the environment. Precisely performing these services and their proximity to agricultural and industrial areas, they become contaminated with the most diverse chemical pollutants.

Taking into account the difficulty in obtaining non-invasive samples to the individual, alternative matrices, such as the use of guano, are welcome.

The manuscript is easy to read and understand. I consider the article suitable for publication in “PLOS ONE”. However, some modifications must be taken into consideration.

Main concerns:

*Add information about benzophenone can increase significantly under favorable temperature and time conditions, probably due to the additional degradation of Octocrylene.

*L92: Add information about on the effects of benzophenone on laboratory rats.

*L131: Georeference the map.

*L134: Add information about recreational or tourist water areas. The use of UV-filter in these places is higher and can influence contamination.

*L220: Rephrase the figures, especially 2 and 3.

*The study needs to discuss more about the species’ characteristics, such as food guild, foraging areas and behavior, since the bioaccumulation rate can be affected by these topics. Furthermore, it is important to clarify some intraspecific information on the species based on the chosen matrix in the study.

Minor concerns:

*L62: Add information about carcinogenic potential.

*L87: Add current data on benzophenone-3 in zebrafish.

*L88: Add article "Comparative acute toxicity of benzophenone derivatives and bisphenol analogues in the Asian clam Corbicula fluminea."

*L88: Add article about freshwater planarian Dugesia japonica

*L144: As this is an unprecedented work, all details are important for future replications. Therefore, I suggest better developing how the samples were collected and stored in a way that prevents their accidental extrinsic pollution with BPs.

*L252: “Okereke and Abdel-Rhaman 1994; Okereke et al. 1994”

*L280: “Okereke”

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Monteiro-Alves P.S

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Submitted filename: Reviewer Attachments PONE-D-23-43946. PSMA.docx

Biomonitoring of benzophenones in guano samples of wild bats in Poland

PONE-D-23-43946R1

Dear Dr. Rytel,

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.

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Kind regards,

Lyi Mingyang, Ph.D.

Academic Editor

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Additional Editor Comments (optional):

Dear Authors,

Thank you very much for sending us this new and improved version of the manuscript.

I have personally read this R1 version and found it suitable for publication in PLOSONE.

Best Regards,

LM

Reviewers' comments: