Molecular-level responses highlight physiological stress in muscle and liver tissue of apparently healthy European sardine specimen

This article is remarkably well written and argued . The title is appropriate, and the abstract presents well the objectives and main finding of the article. The background and questions are clearly and synthetically presented, and the methods are precisely and clearly outlined. The exploration and interpretation of proteomic data and their relation to trace metal contamination data is thorough and well argued, making the approach convincing, which is not always the case in literature. It however lacks an information concerning the functional enrichment execution, that make for now impossible to judge about the relevance of the functional enrichment (see below, comment on L273).

The discussion of the data is enriched by a comparison with the available bibliography through an additional figure and table in supplementary materials, which provides transparency to the discussion.

I only have some recommendations and points of discussion on the manuscript :

> L114-115 : Summarizing liver function in terms of detoxification is a bit simplistic, and all liver functions should be listed.

> L119 : Please define “LFQ”

> L273 – “Functional enrichment analysis and pathway network” section: The background used to perform the functional enrichment on clusters have to be specified. Indeed, using a generic background (not organ specific) could lead to incorrect/biased pathway enrichment and lead to biased biological interpretation. (for example, discussed in Wijesooriya K, Jadaan SA, Perera KL, Kaur T, Ziemann M (2022) Urgent need for consistent standards in functional enrichment analysis. PLoS Comput Biol 18(3): e1009935. https://doi.org/10.1371/journal.pcbi.1009935)

According to the answer to this question, there may be some adjustments to perform to data analysis that may influence the content of the article.

> L280 : Can some arguments can be provided to justify the choice ton consider the top 10 enriched biological processes ?

> Results section “Physiological response to inorganic contamination”: It would be important to provide, for each tissue, the total number of protein detected (not formally provided for muscle) and the proportion of this background included in significantly correlated modules.

> Figure 5: Term names are illegible in the PDF deposited as a preprint. It would be appreciable to find a solution to insert this figure with a greater resolution.

> L489: I'm not convinced that the number of overexpressed pathways can be used to compare the intensity of organ response. It's more a reflection of the diversity or heterogeneity of the response. The absolute number of proteins included in clusters correlated with mixtures, or the part of detected proteins that are included in clusters correlated with mixtures in each tissue seems to be a better metric to compare the intensity of organ response – and should be considered by the authors.

>L513 - “Red muscle proteome response to mixture 1 was less marked than that of the liver proteome”: It is unclear on what criteria the categorization of “less marked” is based, and it should be specified.

>L548-549: Given that mTOR activity is dependent on several post-translational modifications, considering only its abundance as a biomarker of metals contamination may not fully respond the objectives - Authors should consider the characterization of mTOR post-translational modifications in addition to its abundance. (see for example Yin et al, Int. J. Mol. Sci. 2021, 22(4), 1784; https://doi.org/10.3390/ijms22041784)

my comments

Title and abstract

Does the title clearly reflect the content of the article? Yes, 
Does the abstract present the main findings of the study?  Yes

Introduction Are the research questions/hypotheses/predictions clearly presented? Yes, 
Does the introduction build on relevant research in the field? Not at all. There are several points that you can explain better. Just in the last two years a new campaign on Mediterranean see has been condcted and more data are now available on metals and legacy pesticides. Pelase check and add also for comparisons.You have also to consider the existence of legacy and emergent POP's and the cocktail is made also by them, not only by trace elements. Please discuss in this section also the "problem" connnected with the realism of the use of biomarkers which is quite controversial. I would consider also the role of Temperature in time, during the last years as a probable factor causing the decreaase of number of fish (less or diverse preys as it is hppening in freshwater environments). You should explain better this aspect, since of course (specify it better in the introduction) contamination of trace elements can't be the only cause (other fish? other equilibrium conditions?)

Materials and methods Are the methods and analyses sufficiently detailed to allow replication by other researchers? not at all:  please explain how you measured wet weight of fish - pleas esplain the method by Fold since it is quite old and not easy to find - explain why you did not take into account methyl mercury

I'm not an expert of proteomi, so I've nothing to say about.

Are the methods and statistical analyses appropriate and well described?  Yes 

Results In the case of negative results, is there a statistical power analysis (or an adequate Bayesian analysis or equivalence testing)? I don’t know
Are the results described and interpreted correctly?  Yes, - why did you look for the relationshiè between lenght and size and you corrected the calues? It is not clear to me

Discussion Have the authors appropriately emphasized the strengths and limitations of their study/theory/methods/argument? Yes, even if you did not consider the importance of temperature variations. I wouldn't compare concentrations in seas which are so different and concentrations are to low and methods probably too different to be comparable.

please consider the size of the preys, thanks

Beauvieux et al. provided data on the contamination of the European sardine and the relationship between the level of pollution with potential physiological responses in the Mediterranean sea. Considering that this basin has been longed reported as one of the most polluted sea at wordwide level, it is surely important to provides new updated data on marine ecotoxicology.

Despite its potential, this manuscript would surely benefict from an exhaustive bibliographic search as it is keepen very superficial throughtout its introduction and discussion as well. For instance:

line 44-45 need a reference;

line 55-57: the authors mentioned multiple TE stating they can cause several adverse effects, however the cited work is only one and it is reffered to only copper. The authors need to cite proper references. 

line 457-460: the levels of TE are potentially higher in the Med compared to other basins is likely linked to multiple sources of contamination, both natural and human's. It is much more complicated than just stating "higher levels because the Mediterranean throphic webs show enhanced abilities to better accumulate TE pollutants". 

Other observations:

- authors need to be consistent throughout the manuscript. They need to use the acronym the first time the written about TE and then use either the acronym or the full name. However, in the discussion they still refer to Hg (mercury) etc. (Line 476).

- References need to be uniformed in style (e.g. something is "et al.," other time "et al.")

Line 154: No need for "Trace-element", the authors could simply say "Trace element"

Line 159 and 170 etc.: be careful in writing correctly (in style) the chemical formula as well as the unit

Line 208: Latin name goes italics, moreover, if authors decide to use the common name or the Latin name is ok, but they need to be consistent.