Study of Heavy Metals Pollution and Vitellogenin Levels in Brown Trout (Salmo trutta trutta) Wild Fish Populations

Round 1

Reviewer 1 Report

 

Review comments on the manuscript by Merola et al 

Study of heavy metal pollution and vitellogenin levels in brown trout wild fish populations

 

The aim of the study was to describe the concentrations of a variety of heavy metals, levels of vitellogenin and the condition of trout from two natural rivers in Italy to assess their pollution status and possible sources of contamination.

 

Overall, the data presented achieves the study aims and provides an original contribution to our knowledge of the metals in fish.  The analytical techniques and QA/QC procedures, sampling design and statistical analysis are appropriate to address the aims of the study and the tables and figures relevant to presenting the results of the study.  As such, I consider that a revised paper incorporating the attached track change comments and addressing the comments detailed below could be resubmitted for assessment for publication.

 

In line 180 the authors state that Al was the most abundant metal in the samples.  However Table 4 shows that the concentrations of Zn were higher.  The authors should clarify this statement.  Also in table 4 they should include the units for the metals concentrations.

 

Given that the author’s first language is not English, I have marked as track changes on the manuscript in the attached MS Word document, numerous suggested spelling and grammatical changes to improve the quality and flow of the manuscript.  The authors should review these changes and have the manuscript reviewed before resubmission.

 

There are also a number of issues with the references.  For example, for references the journal title is spelt out in full and abbreviated in other references and scientific names italicised. The authors should check previous issues of the journal regarding presentation of references.

Comments for author File: Comments.docx

Author Response

I appreciate and thankful to the Reviewer for your reading and improvement of the manuscript carefully done. I have made the necessary changes following your corrections and highlighted the words or sentences changed. I have incorporated all the suggestions. 
About your question at line 180: the Reviewer is right about the Zn concentrations but in the manuscript, I wrote that Al reported the highest concentrations "Amongst the non-essential metals", indeed I excluded the Zn from this comparison. If the Reviewer thinks that statement is not enough I could modify the sentence.
I have also checked/modified the References as requested.

Reviewer 2 Report

Comments: Manuscript ID: applsci-1233235 Title: Study of heavy metals pollution and vitellogenin levels in brown trout  (Salmo trutta trutta) wild fish populations In this work, the authors  determined the concentration of some heavy metals (As, Cd, Co, Cr, Ni, Pb, Al, Zn and Hg) in the muscles of the wild brown trout, collected from two rivers located in Abruzzi region (Italy), and the levels of vitellogenin as indicator for endocrine disturbance. They reported the presence in all analyzed samples of Al/Zn. On the contrary, they found that the levels of Pb, Cd and Hg in all samples were lower than those established from the European Commission. The paper is interesting as the authors report a strategy for monitoring eventual environment pollution and fish health also in parks or places apparently unpolluted. Thus, I recommend it for publication but with major/minor revisions below described, hoping that the suggestions will be useful for publication.     1.    Introduction and discussion: describe better why you chose these rivers to do your research, as more pollution in last years, thus you expected pollution in fishes. Are those rivers unpolluted, I imagine? So, which is the reason why you do these campaign? for example the aim is monitoring environment pollution and fish health in places apparently unpolluted. Discuss why your research is important for scientific community. 2.    Separate results and discussion according to the instructions for authors. Instead, conclusions are optional.  3.    In order to help readers orienting, you should add a geographic map of the two rivers in the Abruzzi Park, with the localization of places for sample collection. If possible, you could add some photos, to improve the manuscript. (see: Heavy metal contamination and hepatic toxicological responses in brown trout (Salmo trutta) from the Kerguelen Islands. Ali Jaffal, Ste ́phane Betoulle, Sylvie Biagianti-Risbourg, Alexandre Terreau, Wilfried Sanchez & Se ́verine Paris-Palacios. Polar Research 2015, 34, 22784, http://dx.doi.org/10.3402/polar.v34.22784) 4.    lines 49 or/and 215: you should add some other references about effects of heavy metals in marine organisms. As an example the review about neurotoxicity of many pollutants, among which heavy metals in marine organisms: Deidda,I.;Russo,R.; Bonaventura, R.; Costa, C.; Zito, F.; Lampiasi, N. Neurotoxicity in Marine Invertebrates: An Update. Biology 2021, 10, 161. https://doi.org/ 10.3390/biology10020161. 5.    Revise english in all text.  Minor revisions: •    line 20  add: then "to" investigate •    line 20  change "associate" with "associated" •    line 87 all fishes •    line 88 Immediately, blood •    line 100: add city and country to  bioresource company •    line 109: secondly, .. •    line 110: Germany) without comma •    line 198: change "a lower level" •    line 202: arsenic change in "As" •    line 205-208: revise the sentence. it is not clear. •    line 218-222: add reference •    line 227: add something like ..., indicating that Hg can be considered not harmful in the rivers object of this study. 

Author Response

I appreciate and thankful to the Reviewer for the careful reading of the manuscript.

I have made the necessary changes in the manuscript and highlighted the changes done.

I have incorporated all the suggestions and have added the map.

 

Answers to the questions of the reviewers:

 

1. We added in the manuscript some sentences regarding the reasons we have chosen these two rivers, as suggested by the Reviewer (lines 79-819.
2. About this point, we prefer do not separate results from discussion because it would be a repetition to add a new subheading (for biometric data, VTG, and heavy metals) in the discussion. In Applied Science authors guidelines is reported that the discussion section can be combined with Results, then if possible, we prefer to follow this way.
3. Done, we added a map showing the sampling rivers.
4. Done we added the suggested reference.
5. The English editing of the manuscript has been made by a professional editing service also.

Minor revisions: we corrected the mistakes, added the references, and followed the Reviewer's suggestions.

 

Reviewer 3 Report

Please see the attached file.

Comments for author File: Comments.pdf

Author Response

I appreciate and thankful to the Reviewer for the careful reading of the manuscript.

I have made the necessary changes in the manuscript and highlighted the changes done.

1. This research fits into a broad study including the environmental monitoring program in another part of the Abruzzi region. The Reviewer is right about the control, but we used as control trouts coming from another river, and data were reported in a published article (Zezza, D. ; Bisegna, A. ; Angelozzi, G. ; Merola, C. ; Conte, A. ; Amorena, M. ; Perugini, M. Impact of endocrine disruptors on vitellogenin concentrations in wild brown trout (Salmo trutta trutta). Bull. Environ. Contam. Toxicol. 2020, 105, 218–223.)
2. The Reviewer is right about the SD values, but this is not an inaccuracy in the analysis process. The analyses have been made by Chemistry Department, of Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta (CReAA). The CReAA center was designed as National Reference Laboratory for heavy metals and nitrogenous compounds in feed from 2006, and they use validated methods to analyze metal. The main problem is due to the variability in the trout populations, where we had several outliers for weight and length and VTG values (for example we had a fish of 1210 g and 48cm in the group showing a mean and SD of 258.44±362.04). It is normal that the SD becomes very high, but when we performed the statistical analyses we decided to include all outliers because these were representative of the natural population, and we were interested to have data on trouts population. The aim of this study was not to have homogenous groups of fish and we did not sample only trouts in a length/weight specific range, but if the Reviewer suggests excluding these values we can perform a new statistical analysis reducing the total number of samples. 
3. The Reviewer is right. The correlation between vitellogenin levels and heavy metals concentration was not significant, we added a sentence in the text (lines 168-170) to discuss this point. 
4. We thank the reviewer for the stimulating suggestion, it could be useful to do comparative analyses in the future. The present research fits into a local biomonitoring plan program carried out from the National Park of Gran Sasso e Monti della Laga office to confirm the wild fish health status living in a protected area, apparently unpolluted. About vitellogenin levels in male trouts, there was no significant difference between the two rivers (lines 159-161). 
5. Done. 

 

Round 2

Reviewer 2 Report

the manuscript has been sufficiently improved, and is acceptable for publication