Do Single-Component and Mixtures Selected Organic UV Filters Induce Embryotoxic Effects in Zebrafish (Danio rerio)?
Round 1
Reviewer 1 Report
The paper is interesting and the main drawback of it are the following:
The Introduction section should be split into two sections: One Introduction and the second Literature review or Background.
It is important to provide and elaborate on relevant contextual ideas and background leading to research studies on the toxicological effect of the single UV filters. I suggest adding additional literature to explain why it is important for this research study.
The authors should focus on the research framework to avoid overstating your research scope. It is necessary to provide a clearer explanation of making the case to the academic world on why the proposed model raises discussions and importance in the field.
The authors should explain in further detail the research gaps that the paper seeks to close and why the paper is needed to recognize the current gaps in the literature.
It is necessary to add more details regarding the research data collection, analysis, and interpretation of results.
The findings were not well-presented to readers to understand the focus of the research study.
I suggest that the results should be interpretive rather than just descriptive and connect the research results with relevant literature citations for validity and reliability.
Due to the lack of information about the research instrument and protocol, the reader may not be convinced of the study’s findings/results.
The Discussion is not well-presented as it does not integrate with the results of the research to provide a coherent scholarly argument.
The Discussion should connect the research results with relevant literature citations for validity and reliability.
Discussing of the findings could be improved by interpreting them in support of theories related to the research topic.
The results are mainly presented by tables and figures. I think there are too many and they are not sufficiently explained.
The conclusion section is underdeveloped. This section should contain the key focus of the study.
The conclusions are not supported by the research data, which does not indicate a clearer path for future studies on the topic.
A follow-up of restating findings/results with supporting literature reviews could make the conclusion section more effective.
Good Luck
Author Response
Dear Editors,
we send you a revised manuscript „Do single-component and mixtures selected organic UV filters induce embryotoxic effects in zebrafish (Danio rerio)?“ by Cahova et al. (Ms. No. Water-1328372). We would like to thank the reviewers for the beneficial comments. The manuscript was corrected in accordance to their comments and suggestions. Our comments are below this text. We tried to improve the quality and significance of the text as much as possible, in order to fulfil request.
I hope you will find our manuscript interesting and it is in acceptable form for publishing in your journal after our revision.
Reviewer 1:
Point 1: The Introduction section should be split into two sections: One Introduction and the second Literature review or Background.
Response 1: According to reviewer suggestion, the chapter Introduction was split into separate section – (i) Ultraviolet radiation and ultraviolet filters and (ii) Literature review.
Point 2: It is important to provide and elaborate on relevant contextual ideas and background leading to research studies on the toxicological effect of the single UV filters. I suggest adding additional literature to explain why it is important for this research study.
Response 2: Thank you for this comment. We added some references to define the background leading to research.
Point 3: The authors should focus on the research framework to avoid overstating your research scope. It is necessary to provide a clearer explanation of making the case to the academic world on why the proposed model raises discussions and importance in the field.
Response 3: According to reviewer suggestion, we specify the objectives of our study in the Introduction section.
Point 4: The authors should explain in further detail the research gaps that the paper seeks to close and why the paper is needed to recognize the current gaps in the literature.
Response 4: The importance of our research was defined.
Point 5: It is necessary to add more details regarding the research data collection, analysis, and interpretation of results.
Response 5: According to reviewer suggestion, we rewritten the chapter Material and Methods to be more clearer.
Point 6: The findings were not well-presented to readers to understand the focus of the research study.
Response 6: According to reviewer suggestion, we modified the section Results. Tables and figures have been modified to better understand the results. We also reduced their numbers. Tables and figures with non-significant results were deleted from manuscript and these results are only described in the text. Nevertheless, we think that it would be appropriate to present these non-significant results in detail, therefore we have moved them into supplementary material.
Point 7: I suggest that the results should be interpretive rather than just descriptive and connect the research results with relevant literature citations for validity and reliability. Due to the lack of information about the research instrument and protocol, the reader may not be convinced of the study’s findings/results.
Response 7: Thank you for this comment. The section of results was changed. Tables and figures with non-significant results were moved into Supplementary Materials. We hope this section will be clearer.
Point 8: The Discussion is not well-presented as it does not integrate with the results of the research to provide a coherent scholarly argument. The Discussion should connect the research results with relevant literature citations for validity and reliability. Discussing of the findings could be improved by interpreting them in support of theories related to the research topic.
Response 8: Discussion section has been modified to better connect the research results with literature.
Point 9: The results are mainly presented by tables and figures. I think there are too many and they are not sufficiently explained.
Response 9: As we mentioned in our previous response (repsonse 6), the number of tables and figures was reduced and some of them were moved to Supplementary Materials.
Point 10: The conclusion section is underdeveloped. This section should contain the key focus of the study. The conclusions are not supported by the research data, which does not indicate a clearer path for future studies on the topic. A follow-up of restating findings/results with supporting literature reviews could make the conclusion section more effective.
Response 10: The conclusion section has been rewritten to keep the key focus of the study and was supported by the research data.
Reviewer 2 Report
- Lines 45-50. Add some references at the end of all these sentences, please.
- UVs as an acronym of UV filters may be misleading, better to use UVFs?
- Lines 65-66. Please, add a reference.
- Lines 66-67. Which matrices? May you clarify?
- Lines 81-82. This sentence is a bit awkward and I do not follow it. May you rephrase?
- Lines 90-91 and elsewhere in the text. I do not understand what are the “non-target organisms”. I guess that they are all the living organisms with the exception of humans, in this context?
- Line 95. Carassius auratus is not the carp, but the goldfish.
- Line 135. I would delete “naturally”.
- Lines 147-150. Aims and predictions needs to be rewritten, as nothing is obvious in science.
- Line 171. “Checked eggs” should be clarified. Checked by who? For what?
- Lines 218-228. This part is definitely to be rewritten. Which statistical analysed did you perform? How? Did you test for the applicability of these statistics to your context? Please rewrite this part with relevant references.
- Results are clear, but figures are too much. Please create some tables including some of this graphs and keep only those really useful to the MS.
Author Response
Dear Editors,
we send you a revised manuscript „Do single-component and mixtures selected organic UV filters induce embryotoxic effects in zebrafish (Danio rerio)?“ by Cahova et al. (Ms. No. Water-1328372). We would like to thank the reviewers for the beneficial comments. The manuscript was corrected in accordance to their comments and suggestions. Our comments are below this text. We tried to improve the quality and significance of the text as much as possible, in order to fulfil request.
I hope you will find our manuscript interesting and it is in acceptable form for publishing in your journal after our revision.
Reviewer 2:
Point 1: Lines 45-50. Add some references at the end of all these sentences, please.
Response 1: The reference was added.
Point 2: UVs as an acronym of UV filters may be misleading, better to use UVFs?
Response 2: With all the respect, abbreviation UVs is common used in the scientific literature and therefore we prefer to use it in our manuscript.
Point 3: Lines 65-66. Please, add a reference.
Response 3: References were added.
Point 4: Lines 66-67. Which matrices? May you clarify?
Response 4: We have clarified the matrices in our manuscript.
Point 5: Lines 81-82. This sentence is a bit awkward and I do not follow it. May you rephrase?
Response 5: These sentences were changed for better understanding.
Point 6: Lines 90-91 and elsewhere in the text. I do not understand what are the “non-target organisms”. I guess that they are all the living organisms with the exception of humans, in this context?
Response 6: The non-target organisms were defined in manuscript.
Point 7: Line 95. Carassius auratus is not the carp, but the goldfish.
Response 7: The Latin name for common carp was changed to Cyprinus carpio.
Point 8: Line 135. I would delete “naturally”.
Response 8: According to reviewer suggestion, the word “naturally” was deleted.
Point 9: Lines 147-150. Aims and predictions needs to be rewritten, as nothing is obvious in science.
Response 9: Thank you for this benefical comment. According to reviewer suggestion, the section with aims of our study was rephrased to be more clearer.
Point 10: Line 171. “Checked eggs” should be clarified. Checked by who? For what?
Response 10: The “Checked eggs” was replace for “the selected eggs”. We hope it is better for understanding.
Point 11: Lines 218-228. This part is definitely to be rewritten. Which statistical analysed did you perform? How? Did you test for the applicability of these statistics to your context? Please rewrite this part with relevant references.
Response 11: The section with results of statistical analysis was completely rewritten.
Point 12: Results are clear, but figures are too much. Please create some tables including some of this graphs and keep only those really useful to the MS.
Response 12: Thank you for your reminder, tables and figures with non-statistical significant changes were moved to Supplementary Materials (i.e. table 1, 2, 4 and figure 4, 6).
Round 2
Reviewer 1 Report
GOOD LUCK!
Reviewer 2 Report
Authors cobsidered my previous comments and improved the ms accordingly
, which could now be accepted for publication.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The manuscript animals-1259069 entitled “Do single-component and mixtures selected organic UV filters induce embryotoxic effects in zebrafish (Danio rerio)?” analyzed the toxicity of UV filters (single substance and their mixtures) in zebrafish embryos and larvae during 96 h of exposure. The MS is current and relevant; however, some points should be better clarified.
Line 97, 103, 107, .... Scientific names of species are italicized (confirm throughout the text).
Introduction. Describe the Fish Embryo Acute Toxicity Test (FET) and zebrafish embryos as a model system to assess traditional and emerging pollutants.
Line 146-148. Add a hypothesis.
Line 185. “dose-dependent” = “concentration-dependent”
Line 196. “hours post-fertilization (hpf)” or “hours of exposure”?
Delete Figure 1.
Change the order of results. First the isolated pollutants and then the mixture
Table 1. “The results of cumulative mortality (%) of zebrafish embryos exposed to various mixtures of UV filters” = “Cumulative mortality (%) of zebrafish embryos exposed to various mixtures of UV filters during 96 h”.
Results. Add solvent control group.
Line 238. Add results (toxicity test with BP-3)
Figure 3-5. “The results of the hatching rate” = “Hatching rate”.
Figures. Check the position of legends.
Was the experiment conducted in triplicate? Add the mean ± standard deviation of the replicates (results – Figures/Tables).
Figure 10. Add bar scale. Add a representative figure of zebrafish larvae from all experimental groups.
For future studies, I recommend analyzing the frequency of spontaneous contractions (neurotoxicity), heart rate (cardiotoxicity), oxidative stress, and morphometric parameters.
Introduction and discussion. Update the literature on the effects of UV filters on fish.
Author Response
reviewer #1:
Point 1: Line 97, 103, 107, .... Scientific names of species are italicized (confirm throughout the text).
Response 1: Manuscript was checked and italic font was used for Latin names of fish.
Point 2: Introduction. Describe the Fish Embryo Acute Toxicity Test (FET) and zebrafish embryos as a model system to assess traditional and emerging pollutants.
Response 2: The description of FET test was mentioned in chapter Materials and Methods.
Point 3: Line 146-148. Add a hypothesis.
Response 3: The hypothesis was added.
Point 4: Line 185. “dose-dependent” = “concentration-dependent”
Response 4: According to reviewer suggestion, phrase „concentration-dependent“ was used instead of „dose-dependent“.
Point 5: Line 196. “hours post-fertilization (hpf)” or “hours of exposure”?
Response 5: Thank you for this comment, but we prefer to use phrase „hours post-fertilization (hpf). This phrase is commonly used in many scientific studies focusing on embryonic toxicity test as well as in OECD methods.
Point 6: Delete Figure 1.
Response 6: In our opinion, this figure is sufficiently illustrative and we prefer to keep it in our manuscript.
Point 7: First the isolated pollutants and then the mixture
Response 7: According reviewer suggestion, the order of tables with cummulative mortalites was changed. At first, we present results of isolated pollutants (table 1 and 2) and last one is table of mixture (table 3).
Point 8: Table 1. “The results of cumulative mortality (%) of zebrafish embryos exposed to various mixtures of UV filters” = “Cumulative mortality (%) of zebrafish embryos exposed to various mixtures of UV filters during 96 h”.
Response 8: According reviewer suggestion, we changed titles of tables.
Point 9: Results. Add solvent control group.
Response 9: Results of control group with solvent were added in the manuscript.
Point 10: Line 238. Add results (toxicity test with BP-3)
Response 10: No mortality of zebrafish embryos was observed in the toxicity test with BP-3. For this reason, the table with zero values is not given in our manuscript.
Point 11: Figure 3-5. “The results of the hatching rate” = “Hatching rate”.
Response 11: According reviewer suggestion, we changed titles of figures.
Point 12: Figures. Check the position of legends.
Response 12: All figures have been revised to make them clearer to readers.
Point 13: Was the experiment conducted in triplicate? Add the mean ± standard deviation of the replicates (results – Figures/Tables). Add the mean ± standard deviation of the replicates (results – Figures/Tables).
Response 13: The experiment was conducted in duplicates (it is mentioned in Material and method). Twenty four eggs were used for each concentration. In the embryonic toxicity test, results of hatching rate, mortality and malformation are commonly expressed in %. Use of absolute values (number of eggs) and the associated expression as an average ± SD is not commonly used. We prefere to express our results in %, because if other author want to compare their results with our data it will be more easier.
Point 14: Figure 10. Add bar scale. Add a representative figure of zebrafish larvae from all experimental groups.
Response 14: The bar scale was added in all figures of zebrafish. It is impossible to add representative figures of zebrafish from all experimental groups, because we tested five individual substances and their mixtures in many concentrations. We add only some representative figures with frequently occurring malformations such as pericard edema or total body deformation.
Point 15: For future studies, I recommend analyzing the frequency of spontaneous contractions (neurotoxicity), heart rate (cardiotoxicity), oxidative stress, and morphometric parameters.
Response 15: Thank you for this benefical comment and suggestion for improving our future research. We are planning some new experiments in this area, so we will definitely consider including the proposed marker in our experimental studies.
Point 16: Introduction and discussion. Update the literature on the effects of UV filters on fish.
Response 16: Updating of the literature has been done. We also renumbered all references. We found one reference (Zhou et al. 2019) which was mentioned only in references (not in text). We add this reference in the text.
Reviewer 2 Report
The study is sound and interesting, however, there are few important objections I have to state. First, if one considers the freshwater concentrations of UV filters used in this study (PBSA; EHMC; OC; 4-MBC; BP-3) it is obvious that testing concentrations above 100 ug/L (and especially above 250 and 500 ug/L) is environmentally unrealistic and therefore of no particular interest to the scientific community. Furthermore, I do not see the results of control groups with solvents. Also, there is a huge variation in observed hatching of control animals at 72 hpf (20-80%!) making this parameter unreliable and all the conclusions drawn from it irrelevant.
How do authors explain that sometimes the highest concentration is exerting lower effect (Table 1.: OC+4-MBC; Table 2: 4-MBC)
Minor points:
All the Latin names of species should be written in italic and some species (f. ex. Carassius auratus) should be indicated as fish (carp) but only when mentioned for the first time in the text.
Figures are of bad quality considering that legends for concentrations are missing (Figs 3 and 6).
Author Response
Point 1: The study is sound and interesting, however, there are few important objections I have to state. First, if one considers the freshwater concentrations of UV filters used in this study (PBSA; EHMC; OC; 4-MBC; BP-3) it is obvious that testing concentrations above 100 ug/L (and especially above 250 and 500 ug/L) is environmentally unrealistic and therefore of no particular interest to the scientific community. Furthermore, I do not see the results of control groups with solvents. Also, there is a huge variation in observed hatching of control animals at 72 hpf (20-80%!) making this parameter unreliable and all the conclusions drawn from it irrelevant.
Response 1: Thank you for your comments. The lowest concentrations represent the environmentaly relevant ones, higher concentration should reflect concentration-dependent effect.
The results of control group with solvents were added in manuscript. Due to a non-significant difference in all monitored indices between the control group and the control groups with a solvent, for next statistical evaluation only the control group was used.
Hatching rate in the control group was unique for every tested UVs and as such was statistically evaluated.
Point 2: How do authors explain that sometimes the highest concentration is exerting lower effect (Table 1.: OC+4-MBC; Table 2: 4-MBC)
Response 2: Your comment is absolutely correct, we need to replicate our results to find a link between UVs - how they can react with each other and whether they can be more toxic to non-target organisms in the mixture. According to our results, it seems that some UVs could have an additive or antagonistic effects when they are exposed to zebrafish embryo together.
Point 3: All the Latin names of species should be written in italic and some species (f. ex. Carassius auratus) should be indicated as fish (carp) but only when mentioned for the first time in the text.
Response 3: It was corrected.
Point 4: Figures are of bad quality considering that legends for concentrations are missing (Figs 3 and 6).
Response 4: All figures have been corrected.
Round 2
Reviewer 2 Report
The response of the authors to my comments regarding the extreme variations in observed hatching of control animals at 72 hpf (20-80%) is not satisfactory. As I stated in the first review – such extreme variations are making this parameter unreliable and all the conclusions drawn from it irrelevant. Especially since the hatching rate is, apart from mortality, the most important end-point measured in this study.
Also, other responses like: „higher concentration should reflect concentration-dependent effect“ are not acceptable for me as they are not providing reasonable answer. Concentration dependent effect can be sought with lower concentrations also. What are we to gain with the concentrations that are several hundred or thousand times higher than those measured in the environment?
Furthermore, there are again mistakes in figures (Fig. 3)
