In-Gel Assay to Evaluate Antioxidant Enzyme Response to Silver Nitrate and Silver Nanoparticles in Marine Bivalve Tissues

Round 1

Reviewer 1 Report

The manuscript “In-gel assay to evaluate antioxidant enzyme response to silver nitrate and silver nanoparticles in marine bivalve tissues” focuses on a very important issue - marine pollution. Metals are known to be harmful to the environment, including sea creatures. Today, silver nitrate is actively used in the marine industry, particularly when used for checking for chloride contaminants on steel products. In this regard, the need to monitor biomarkers of oxidative stress is extremely important. For these purposes, the authors very successfully chose the sea mussel as a bioindicators of marine pollution, namely, the tissues of the digestive gland and gills of marine bivalves.

The authors give a clear justification for the choice of in-gel electrophoretic techniques to measure the activity of the antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase. Careful preliminary analysis of this method allowed them to improve the test protocol. Developed design of the protocol of electrophoretic approach give a better definition of the CAT band in the gel without loss of linearity and with a significant increase in sensitivity, detect the specific glutathione-dependent peroxidase activity in case of GPX.

The article can be published with minor modifications.

Minor remarks:

Line 42 OECD - Full name is required.

Line 45-46 present in over two hundred consumer medical products [?] - It would be great if you add a reference here.

Line 179 When needed, - Please note that information has been lost.

Line 196 Please clarify where are Panels A-D in Fig.1

Comments for author File: Comments.docx

Author Response

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Reviewer 2 Report

The authors describe the activity of enzymes in molluscs after treatment with silver nanoparticles and silver nitrate. This work cannot be accepted for publication in its current form. It is suggested to make the following modifications and consider some suggestions.

1.- The use of experimental animals must be approved by a bioethics committee and this must be named in the manuscript.

2.- You must indicate what the acronym OECD means,

3.- The quality of all the images must be improved.

4.- The foot of each figure must be reviewed in detail, since in some it describes panels that are not highlighted in the figure.

5.- Regarding the treatment applied to the mussels, three concentrations of silver nitrate or silver nanoparticles have been considered. What is the reason for this choice of concentrations? Is there any support? The toxic activity of the nanoparticles and silver nitrate on the mussels must be considered through the determination of LDH, and based on this, select the concentrations to be used for the treatments.

6.- It should be taken into account that the determined activity will be due to the accumulation of nanoparticles and silver nitrate, so the final concentration will be different. Does the silver content bioaccumulate? What are the final concentrations of each treatment?

Author Response

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Reviewer 3 Report

Authors present a method for detecting the effect of silver particles on antioxidant activity in a species of mussels.

The manuscript is poorly written, and it needs a deep modification before be publised in any journal.

As general comments:

• Details of equipments (country, brands) must be provided. There is a lack of information at this point as well as no information concerning chemical reactives used.
• Figures should be extensively edited. Most of them are not clear or there is lack of information. As an example: figure 1, which are the differences among the wells? why some samples have activity and others no?
• Calibration curves need more data points, and they should include the error bars in order to certify that the calibration was succesful. Data fitting to cuadratic equations should be adequately justified. Maybe it is a logarithmic behaviour?
• Please increase the quality of figure 5
• HSP70 increase is something expected... I cannot see the novelty at this point.

Some minor comments:

• Line96: spelling
• Line101: what 1:6 w/v means? this is a strange unit... please clarify this to the readers
• Line129: SOD activity? It is catalase assay... it is not clear
• Line 179: sentence not completed...
• Line231: out or our?
• Lines231 to 234: these are materials and methods. Please, move to the appropiate section

Author Response

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Round 2

Reviewer 2 Report

the manuscript is accepted in its present form

Reviewer 3 Report

The manuscript has been improved considerably, and it could be considered for publication