Xenoestrogen Status of Wuling Farm to Surrounding Water Bodies: An Application of Biochemical Parameters Using Onychostoma barbatulum
Round 1
Reviewer 1 Report
Subject: Xenoestrogen status of Wuling Farm to surrounding water bod- 1 ies: an application of biochemical parameters using Ony- 2 chostoma barbatulum (Pellegrin, 1908).
The paper brings a research that aims to evaluate the was to apply biochemical parameters to Onychostoma barbatulum to understand whether Wuling Farm activities have caused organic pollution from xenoestrogens to flow into surrounding streams and thereby affected area aquatic ecosystems. This paper have high ecological importance. But despite this is well written for the most part, this I found that the theoretical explanation and the description of some important points were not satisfactory. Therefore, I recommend that an important review is necessary. Thus suggest that the manuscript not be accepted for puplication in this journal.
I explain my concerns in more detail below.
1. How can the authors prove that the localities are really contaminated by the xenobiotic studied? I suggest that the authors add chemical analyzes and a detailed characterization of the water quality of each location. If this is not possible, the authors can also carry out chemical analyzes in order to quantify 17β-estradiol in the liver and/or fillet of fish. Thus, the authors show the readers that the studied areas are actually being contaminated with the studied xenobiotic. Another concern: the fact that there are no farms close to the area considered as a reference by the authors that no contamination by pollutants. Therefore, it is extremely important to characterize the areas and chemical quantification, proving that the reference point is free or at lower levels of contamination than the other analyzed areas.
3. I suggest that the authors clearly explain how and why they chose the 10 ng/L concentration to study the effects of exposure to 17β-estradiol em cativeiro.
4.In the methodology, the number of animals kept in captivity per aquarium/box must be indicated. If all animals were kept in a single aquarium/box, this could lead to an estimation error.
4. In biochemical analyses, how long were the samples stored at -20ºC? The tissue homogenizeds stored for more than 30 days in a -20° freezer undergoes changes in enzymatic activity.
5. Line 161: Exchanging “induced” by “increased” the VIT concentration and “increased” Mon and GST enzymatic activity.
6. The presentation of results in boxplot format is only suitable if the authors have used statistical tests for non-parametric data. However, at according to section 2.6 of the paper, they only used ANOVA test, that means they only had data for analyzi with normal and parametric distribution. Another point, why present fig2 as a percentile and the other figures with mean +- standard deviation? There is probably a typo in the caption or in the statistical analyses.
7. The calculation to estradiol equivalent concentration (EEQ) should be presented in the material and methods section.
8. The discussion can be more exploraded and data most discussion.
Author Response
- Reviewer suggested add chemical analyzes in order to quantify 17β-estradiol in the liver and/or fillet of fish. However, it is not easy to directly measure 17β-estradiol in fish liver. Therefore, this study used VTG in fish liver as a biomarker for assess their exposure to 17β-estradiol.
- Reviewer suggested add the description about why chose the 10 ng/L concentration to study the effects of exposure to 17β-estradiol. As suggested, we have added more description in the “Materials and Methods”.
- About “The calculation to estradiol equivalent concentration (EEQ)”, we have added description in “material and methods” section, as reviewer suggested
- Reviewer suggested add the information about biochemical analyses and exposure test. As suggested, we have added more description in the “Materials and Methods”.
- The reviewer confuses about the boxplot format and used ANOVA test in this study. In our study, our collected data in each group was in the form of normal distribution, so ANOVA was used to analyze the differences among the groups. About the figure of boxplot format, we presented within-group differences for each treatment group.
- About “Discussion”, we have rewritten the description of model more clearly as reviewer suggested.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Dear Authors,
the study presents the assessment of xenoestrogen presence in water bodies and their impact on biochemical parameters of Onychostoma barbatulum - as an assessment of the pollution of reservoirs with these substances. The study showed that farming activities, including endocrine disrupting compounds, have not significantly affected these aquatic organisms. However, pollution sources are still releasing pollutants containing xenoestrogens into the Yusheng River and Ikawan River and this kind of contamination should be monitored. Overall, the manuscript is well written and structured, therefore I recommend its publication, but after major revision. I think the work may still be improved - results, discussion and conclusions sections should be expanded. Changes and suggestions would be found in the attached PDF-file, in the specific comments for all sections of the paper.
Kind regards
Reviewer
Comments for author File: Comments.pdf
Author Response
About “Discussion”, we have rewritten the description of model more clearly as reviewer suggested.
All comments marked in original manuscript by the reviewer(s) were corrected accordingly. We have attempted to edit the manuscript in Microsoft Word with the tracking feature on to show our corrections.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The manuscript is ready for publication.
