Red Mullet (Mullus barbatus) Collected from North and South Euboean Gulf, Greece: Fishing Location Effect on Nutritive Quality

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments:

The authors aimed to compare the nutritional quality of fish from five different locations in the Euboean Gulf by performing various nutritional characterization analyses. Fresh fish were collected during the same period (May 2024) from five sites in the Euboean Gulf. Overall, the manuscript is well written and organized. However, the design of the statistical comparative study is not appropriate. The main issue is that the authors used only a single pooled sample for each location (15 individual samples were pooled per location, n = 1 per location) to compare among sites. With n = 1, variability among individual samples cannot be assessed (no meaningful standard deviation), which makes a comparative statistical analysis unfeasible. If I understood correctly the reported standard deviations reflect only the analysis of the pooled sample for each site.

If the authors choose to revise the manuscript with a non-comparative approach about the 5 locals, please consider the following comments and suggestions:

• Present all quantitative results using three significant figures for the average and ensure that the standard deviations (SD) are reported with the same number of decimal places as their corresponding average. For example, in Table 3, values such as 0.01 ± 0 (L3, Co), 0.68 ± 0.04 (L1, Se), 1.27 ± 0.06 (L1, Cr), 86.36 ± 3.54 (L1, Al), 485.02 ± 14.07 (L1, K), 3324 ± 113 (L1, Mg) should be reformatted to 0.01xx ± 0.xxxx (L3, Co), 0.68x ± 0.04x (L1, Se), 1.27 ± 0.06 (L1, Cr), 86.4 ± 3.5 (L1, Al), 485 ± 14 (L1, K), 3324 ± 113 (L1, Mg). This formatting should be applied consistently throughout all tables, the text, and the abstract (e.g., report 10.81% as 10.8%).
• In Section 2.1, please specify the grade and part number of the nitric acid used and cite the source of the analytical standard used for the metal analysis.
• On page 3, line 101, include the brand and model of the electrical mill used.
• On page 7, line 246, refer to the Falcon tube by its material rather than by brand (e.g., “50 mL polypropylene (PP) centrifuge tube”). Similarly, on page 7, line 253, refer to the “Eppendorf tube” as a PP microcentrifuge tube.
• On page 9, line 353, please rewrite the first sentence. If you mean that the concentration of Ca is higher than that of Mg, include the word “concentration” in the sentence to clarify. The same suggestion applies to page 10, line 404. If these results are intended to show only relative concentrations, consider either removing them or providing a detailed discussion.
• It was not immediately clear that each experiment was conducted using a pool of 15 samples per location. I just realize because in Tables 1–5, the standard deviations appear too low for being from 15 individual samples. After re-reading lines 135–136, I noted that the samples were indeed pooled (n = 1 per site). I suggest adding a footnote in the tables stating that the results represent x analytical replicates of a pooled sample from each site.
• In Table A1, please add a column indicating the m/z ratio used for each analyte and another column showing the LOD in the sample (normalized to µg/100 g). Currently, the LOD is only provided for the analytical solution, which makes it difficult to evaluate the data.
• According to my calculations, the LOD in the sample would be considerably higher than the value for the analytical solution, meaning that many results in Table 3 might actually be below the LOD. Please verify this and report any such values as “< LOD.” Also, include a description in the text of how the LOD was calculated.
• On page 5, Section 2.4.2, clarify whether the ash content was determined solely by heating or if any acid was also added to the sample. If acid was used, please mention it; if not, you may disregard this comment.
• Discuss the limitations of the ashing method for subsequent elemental analysis, especially for analytes such as As, Se, Cd, Pb, and Te. Emphasize that the results for these elements might be underestimated due to the sample preparation method employed.

Therefore, since the work was not designed adequately, I reject the publication of this manuscript as it is. The authors would need to rewrite the manuscript, changing the focus of the work for this work to be reevaluated. But as I mentioned before, I recommend that the authors rewrite the manuscript to focus on a non-comparative description of the nutritional profiles at each site. With n = 1 per site, a comparative statistical evaluation for this type of study is not possible.

Comments for author File: Comments.pdf

Author Response

The authors aimed to compare the nutritional quality of fish from five different locations in the Euboean Gulf by performing various nutritional characterization analyses. Fresh fish were collected during the same period (May 2024) from five sites in the Euboean Gulf. Overall, the manuscript is well written and organized. However, the design of the statistical comparative study is not appropriate. The main issue is that the authors used only a single pooled sample for each location (15 individual samples were pooled per location, n = 1 per location) to compare among sites. With n = 1, variability among individual samples cannot be assessed (no meaningful standard deviation), which makes a comparative statistical analysis unfeasible. If I understood correctly the reported standard deviations reflect only the analysis of the pooled sample for each site.

We would like to thank the reviewer for his in-depth comments on our manuscript. We would also like to apologize for the inconvenience regarding the handling of the samples. The samples per region (15) were divided into three groups of five (so n=3). Necessary changes have been made to the manuscript (including Tables) to ensure the process is understood by the wider public.

If the authors choose to revise the manuscript with a non-comparative approach about the 5 locals, please consider the following comments and suggestions:

• Present all quantitative results using three significant figures for the average and ensure that the standard deviations (SD) are reported with the same number of decimal places as their corresponding average. For example, in Table 3, values such as 0.01 ± 0 (L3, Co), 0.68 ± 0.04 (L1, Se), 1.27 ± 0.06 (L1, Cr), 86.36 ± 3.54 (L1, Al), 485.02 ± 14.07 (L1, K), 3324 ± 113 (L1, Mg) should be reformatted to 0.01xx ± 0.xxxx (L3, Co), 0.68x ± 0.04x (L1, Se), 1.27 ± 0.06 (L1, Cr), 86.4 ± 3.5 (L1, Al), 485 ± 14 (L1, K), 3324 ± 113 (L1, Mg). This formatting should be applied consistently throughout all tables, the text, and the abstract (e.g., report 10.81% as 10.8%).

We revised the results in order to maintain three significant numbers in the whole manuscript, as suggested by the reviewer.

• In Section 2.1, please specify the grade and part number of the nitric acid used and cite the source of the analytical standard used for the metal analysis.

The part number of nitric acid has been added to the manuscript. The analytical standard for metal analysis was already mentioned, along with its catalogue number.

• On page 3, line 101, include the brand and model of the electrical mill used.

The details of the electric mill have been added to the manuscript.

• On page 7, line 246, refer to the Falcon tube by its material rather than by brand (e.g., “50 mL polypropylene (PP) centrifuge tube”). Similarly, on page 7, line 253, refer to the “Eppendorf tube” as a PP microcentrifuge tube.

We agree with the reviewer's recommendations, so the words have been changed accordingly.

• On page 9, line 353, please rewrite the first sentence. If you mean that the concentration of Ca is higher than that of Mg, include the word “concentration” in the sentence to clarify. The same suggestion applies to page 10, line 404. If these results are intended to show only relative concentrations, consider either removing them or providing a detailed discussion.

With the intention of maintaining the distinct classification of the various elements (i.e., trace elements, essential elements, etc.) for the general public, the manuscript was reformatted accordingly.

• It was not immediately clear that each experiment was conducted using a pool of 15 samples per location. I just realize because in Tables 1–5, the standard deviations appear too low for being from 15 individual samples. After re-reading lines 135–136, I noted that the samples were indeed pooled (n = 1 per site). I suggest adding a footnote in the tables stating that the results represent x analytical replicates of a pooled sample from each site.

Thank you for your feedback. We understand the concern regarding the statistical treatment of samples by region. We apologize for any confusion with the initial description. We want to assure you that the number of fish was n=3 per site. We divided the fish samples from each location into three groups and analyzed each group in triplicate, capturing both analytical precision and biological variability. We added a footnote in Tables 1–5 stating that the results represent three analytical replicates of three groups from each site to clarify this point.

• In Table A1, please add a column indicating the m/z ratio used for each analyte and another column showing the LOD in the sample (normalized to µg/100 g). Currently, the LOD is only provided for the analytical solution, which makes it difficult to evaluate the data. According to my calculations, the LOD in the sample would be considerably higher than the value for the analytical solution, meaning that many results in Table 3 might actually be below the LOD. Please verify this and report any such values as “< LOD.” Also, include a description in the text of how the LOD was calculated.

Thank you for your valuable feedback. In response to your comments, we have added a column in Table A1 indicating the m/z ratio used for each analyte and another column showing the LOD in the sample, normalized to µg/100 g. We have verified the LOD values in the sample and ensured that any results in Table 3 that are below the LOD are reported as “< LOD.” Additionally, we have included a detailed description in the text on how the LOD was calculated and we have added an Equation. For example, the LOD for Aluminum, initially provided as 2.42 μg/L, was recalculated considering the dilution and sample preparation steps. Given that 0.01 g of ash was diluted in 10 mL of 2% v/v nitric acid solution and the fish sample had 1% ash content, the LOD was determined to be approximately 24.2 μg/100 g.

• On page 5, Section 2.4.2, clarify whether the ash content was determined solely by heating or if any acid was also added to the sample. If acid was used, please mention it; if not, you may disregard this comment.

It is now highlighted in sections 2.4.2 and 3.2.3 that the dry-ashing method was used for elemental analysis.

• Discuss the limitations of the ashing method for subsequent elemental analysis, especially for analytes such as As, Se, Cd, Pb, and Te. Emphasize that the results for these elements might be underestimated due to the sample preparation method employed.

In section 3.2.3 we highlight the drawbacks of dry-ash method, as suggested by the reviewer.

Therefore, since the work was not designed adequately, I reject the publication of this manuscript as it is. The authors would need to rewrite the manuscript, changing the focus of the work for this work to be reevaluated. But as I mentioned before, I recommend that the authors rewrite the manuscript to focus on a non-comparative description of the nutritional profiles at each site. With n = 1 per site, a comparative statistical evaluation for this type of study is not possible.

Thank you for your feedback. We acknowledge that our initial description may not have been clear. We correctly focused on a comparative description of the nutritional profiles at each site because we worked by dividing the fish samples from each location into three groups (n=3) and analyzing each group in triplicate. This approach aims to capture both analytical precision and biological variability. Despite any possible limitations of our study design, our results provide valuable insights into the nutritional composition of red mullet fish from different locations. We highlighted these aspects in the revised manuscript and proposed recommendations for future studies to include a larger number of replicates per site, seasonal variations, and other influencing factors.

Reviewer 2 Report

Comments and Suggestions for Authors

Abstract

Page 1, lines 13-15 – What is the meaning of “a fish widely recognized”? The nutrients referred are typical of almost all fish species.

Page 1, lines 17 and 19 – I suppose it is “proximate composition”.

Page 1, line 19 – Please clarify “impact of fishing location”. I think it is “moisture” and not hydration”.

Page 1, lines 24-26 – Please improve these sentences.

Page 1, lines 29 and 30 – Please specify what is “distinctive biological characteristics”.

 

Introduction

Page 2, line 56 – I think it is caught” instead of “hunted”.

Page 2, line 62 – Please clarify “trawl deeper”.

Page 2, line 70 – Please also clarify “specific meals”.

Page 2, lines 71 and 72 – Please check the sentence “although previous... several coasts” for its improvement.

Page 2, lines 80 and 81 - This is such an ambitious objective that it certainly does not fit into this study.

 

Materials and Methods

Page 3, line 98 – I suggest replacing M. barbatus with “red mullet” in the rest of the manuscript.

Page 4, Figure 1 – The location of the different fishing sites is not clear. Just increase the size of L1-L5.

Page 5, line 148 – Please clarify “After combination... sampling location...”

Pages 5 and 6 – Why were used standard methods such as those of AOAC followed to determine moisture and protein contents? For example, colorimetric methods for determining protein content in fishery products generally give lower values.

Page 6, lines 205-210 – I suggest including the equations used to calculate these indices.

Page 6, line 229 – I apologise, but I don’t think the tocopherol content was calculated by HPLC-FLD.

Page 7, line 249 – I suggest replacing “introduced” with “added”, for instance.

Page 7, line 251 – Please indicate the centrifugal force in g.

Page 7, line 252 – I suggest “One mL”.

Page 7, line 256 – I also suggest replacing “by creating” with a more appropriate word(s).

Page 7, line 278 – I thing “of the samples” could be deleted.

Page 8, line 286 – I think is “14.35 % and not “74.36 %”.

Page 8, line 289 - What do you mean that the fat and ash content of the fillets fluctuated?

Page 8, lines 293 and 294 – Please check the sentence “The  sole variation... other site” because it is not clear.

Page 8, lines 296 and following – I think your results are not comparable to those of Polat et al. These authors obtained protein content between 17.90 % and 20.43 % whereas in the current study they were between 10.81 % and 14.35 % which are relatively low. The fat content reported by Polat et al. was in the range of 3.68 % to 5.76 % with an average value around half of the results referred in the current study. Roncarati et al. also obtained protein contents in the range of 18.88 % to 19.15 % and fat contents between 1.82 % and 7.45 %. These are the results reported in the two papers cited in this study, but other authors may also find values that do not agree with those reported in this paper. On the other hand, a simple check of the results of the proximate chemical composition can be simply to calculate the total of the four main constituents. Thus, in the case of the fish samples from L1 and L4, the total values are 102.79 per cent and 101.46 per cent, respectively. For fish from sites L2 and L5, the totals are 96.47 % and 97.53 %, respectively. In the work by Polat et al., these values varied between 99.19 % and 99.97 % and in that of Roncarati et al. the variation was between 100.36 and 101.43 %. Another thumb rule indicates a value of 80 % for the sum of fat and water content. In the current work this value was between 83.37 % (L2) and 87.59 % (L1). Therefore, the results obtained in the current study do not seem reliable.

Page 8, lines 313-315 – These sentences seem irrelevant to the discussion.

Page 9, lines 316-351 - All of these phrases are generic and very much centre on environment/fish interactions. This, although important, would be better suited to another type of work.

Page 9, line 362 – What is “water culture” in this context?

Page 10, lines 366-368 – Please clarify these sentences.

Page 10, lines 369-374 – Please check these sentences and particularly “smaller sizes of meat”.

Page 10, line 378-394 – These comments don’t seem to be related with the results obtained.

Page 10, lines 396-399 – Why starting by presenting the data by Korkmaz et al.?

Page 10, line 399 and 400 – This comment don’t seem to be in line with figures shown in Table 3.

Page 11, line 417 – I think it Pagelus erythrinus.

Page 11, lines 418-406 – Please clarify these sentences.

Page 11, line 428 – Please indicate the Units of the permissible tolerable daily intake of As, Cd, and Pb.

Page 11, lines 435-437 – As shown in Table 3, it seems that bioaccumulation didn’t occur with elements. Please check.

Page 12, line 459 – I suggest “These authors”

Page 12, line 469 – Please clarify “...are organisms that consume at the base of the...”

Page 13, line 487 – I think “to each other” could be deleted.

Page 13, lines 497-501 – These sentences are generic and not relevant in this context.

Page 13, line 503 – The levels of ω-3 PUFA of L1 and L2 were significantly different as shown in Table 5. Please check.

Page 13, lines 520 and 521 – I think it is mobilization of reserve lipids for the gonad maturation.

Page 13, line 527 – I suggest deleting “amounts of”. I also suggest indicating the reference values of these health indices.

Page 14, Table 5 – Is there any explanation for low ω-3/ ω-6 ratio in the fish from L1 compared to the fish from the other locations?

Page 17, lines 602 and 603 – It is not clear how the correlation between various environmental factors, not specified in this study, and the quality of fish populations, whatever this is, was pinpointed. The next sentences are even more ambitious, but it is difficult to find any relationship with the results obtained.

 

Conclusions

Page 17, lines 624-626 – This conclusion (“The study also... environments”) is surprising given that the authors only worked with fish fillets.

Page 17, lines 629 and 630 – It would be interesting if the authors gave more information about the type of industries they are talking about.

 

Comments on the Quality of English Language

The English language could be improved.

Author Response

We would like to thank the reviewer for the comprehensive point-to-point comments for our manuscript.

Abstract

Page 1, lines 13-15 – What is the meaning of “a fish widely recognized”? The nutrients referred are typical of almost all fish species.

This sentence has been rephrased, as suggested.

Page 1, lines 17 and 19 – I suppose it is “proximate composition”.

Corresponding changes were made.

Page 1, line 19 – Please clarify “impact of fishing location”. I think it is “moisture” and not hydration”.

Substantial changes were made to enhance the readability of this section.

Page 1, lines 24-26 – Please improve these sentences.

The sentences have been rephrased.

Page 1, lines 29 and 30 – Please specify what is “distinctive biological characteristics”.

The specific terms have been specified.

Introduction

Page 2, line 56 – I think it is caught” instead of “hunted”.

The correction has been made.

Page 2, line 62 – Please clarify “trawl deeper”.

The term has been changed to provide a clearer picture of the meaning of searching for food.

Page 2, line 70 – Please also clarify “specific meals”.

The terms have been revised.

Page 2, lines 71 and 72 – Please check the sentence “although previous... several coasts” for its improvement.

The specific sentences have been revised accordingly.

Page 2, lines 80 and 81 - This is such an ambitious objective that it certainly does not fit into this study.

The specific sentence has been removed from the manuscript.

Materials and Methods

Page 3, line 98 – I suggest replacing M. barbatus with “red mullet” in the rest of the manuscript.

This change has been adopted throughout the manuscript.

Page 4, Figure 1 – The location of the different fishing sites is not clear. Just increase the size of L1-L5.

Figure 1 has been revised as suggested.

Page 5, line 148 – Please clarify “After combination... sampling location...”

The sentence has been rephrased.

Pages 5 and 6 – Why were used standard methods such as those of AOAC followed to determine moisture and protein contents? For example, colorimetric methods for determining protein content in fishery products generally give lower values.

We chose a simple gravimetric procedure for determining moisture content after freeze-drying the fillets. Bradford method was chosen as it provides automation and multiple sample analysis.

Page 6, lines 205-210 – I suggest including the equations used to calculate these indices.

The equations have been inserted in the manuscript as suggested by the reviewer.

Page 6, line 229 – I apologise, but I don’t think the tocopherol content was calculated by HPLC-FLD.

We confirm that the specific chemical compounds were identified with this procedure, as it is already cited in our previous work.

Page 7, line 249 – I suggest replacing “introduced” with “added”, for instance.

This change has been made.

Page 7, line 251 – Please indicate the centrifugal force in g.

To provide clarity between the two expressions of centrifugation throughout the manuscript, we inserted centrifugal force in parentheses where needed.

Page 7, line 252 – I suggest “One mL”.

This change has been made.

Page 7, line 256 – I also suggest replacing “by creating” with a more appropriate word(s).

The specific word has been replaced by “conducting”.

Page 7, line 278 – I thing “of the samples” could be deleted.

This phrase has been deleted.

Page 8, line 286 – I think is “14.35 % and not “74.36 %”.

We apologize for the typographical error. The corresponding correction has been made.

Page 8, line 289 - What do you mean that the fat and ash content of the fillets fluctuated?

We changed the term “fluctuated” with “differed”.

Page 8, lines 293 and 294 – Please check the sentence “The  sole variation... other site” because it is not clear.

This sentence has been rephrased in order to provide a clearer picture of the measured fats in the samples L1 and L3.

Page 8, lines 296 and following – I think your results are not comparable to those of Polat et al. These authors obtained protein content between 17.90 % and 20.43 % whereas in the current study they were between 10.81 % and 14.35 % which are relatively low. The fat content reported by Polat et al. was in the range of 3.68 % to 5.76 % with an average value around half of the results referred in the current study. Roncarati et al. also obtained protein contents in the range of 18.88 % to 19.15 % and fat contents between 1.82 % and 7.45 %. These are the results reported in the two papers cited in this study, but other authors may also find values that do not agree with those reported in this paper. On the other hand, a simple check of the results of the proximate chemical composition can be simply to calculate the total of the four main constituents. Thus, in the case of the fish samples from L1 and L4, the total values are 102.79 per cent and 101.46 per cent, respectively. For fish from sites L2 and L5, the totals are 96.47 % and 97.53 %, respectively. In the work by Polat et al., these values varied between 99.19 % and 99.97 % and in that of Roncarati et al. the variation was between 100.36 and 101.43 %. Another thumb rule indicates a value of 80 % for the sum of fat and water content. In the current work this value was between 83.37 % (L2) and 87.59 % (L1). Therefore, the results obtained in the current study do not seem reliable.

It is highlighted in the manuscript that the proximate composition of red mullets is affected by a number of factors, including seasonal variation, migratory behavior, sexual maturation, and dietary cycles. The differences observed in our study compared to Polat et al. and Roncarati et al. can be attributed to these factors. Regarding fat content, it is stated that it may vary from 0.5% to 20%. In the study by Roncarati et al., similar variation was observed in terms of fat content (1.82%-7.54%). In the study by Polat et al., differences were observed in protein content. Given that moisture levels are consistent in fish samples, the other two components (i.e., fat and proteins) may be affected by the mentioned parameters. This is a limitation of our work; however, we discussed that a future study could include seasonal variation to address these inconsistencies.

The proximate composition was calculated as the average value of 15 samples, the sum of which could be beyond 100%. This discrepancy can be attributed to the inherent variability in biological samples and the potential for measurement errors. While triplicate analyzes provide an estimate of the analytical precision, our study design also includes biological replicates to account for variability in composition and nutritional value. By dividing the fish samples from each location into three groups (n=3) and analyzing each group in triplicate, we aimed to capture both analytical precision and biological variability. Despite this limitation, our results are consistent with the ranges reported in the literature, taking into account the factors affecting proximate composition. Future studies will aim to minimize these discrepancies by considering seasonal variations and other influencing factors.

Page 8, lines 313-315 – These sentences seem irrelevant to the discussion.

These sentences have been deleted.

Page 9, lines 316-351 - All of these phrases are generic and very much centre on environment/fish interactions. This, although important, would be better suited to another type of work.

We have reduced the length of these sentences and potentially unnecessary information, while keeping important details in the manuscript, aligning with the reviewer's comments.

Page 9, line 362 – What is “water culture” in this context?

The term “culture” has been deleted.

Page 10, lines 366-368 – Please clarify these sentences.

It is now highlighted that increased fat consumption could lead to decreased metal absorption from fish, so the manuscript has been revised accordingly.

Page 10, lines 369-374 – Please check these sentences and particularly “smaller sizes of meat”.

We agree with the reviewer’s comments. To that end, these sentences have been substantially revised to enhance the readability of the manuscript. The study highlighted that some metals were found in higher amounts in fish samples with smaller length and weight.

Page 10, line 378-394 – These comments don’t seem to be related with the results obtained.

It was observed that fish samples with bigger size (weight and length) had lower metal/element content in their fillets. A possible mechanism was mentioned in the manuscript. Since this discussion should focus on metals/elements, any discussion involving lipids has been removed.

Page 10, lines 396-399 – Why starting by presenting the data by Korkmaz et al.?

We have added appropriate text at the beginning of the section to introduce the comparison with other works.

Page 10, line 399 and 400 – This comment don’t seem to be in line with figures shown in Table 3.

This typographical error has been corrected.

Page 11, line 417 – I think it Pagelus erythrinus.

P.erythrinus has been changed to its full name (Pagellus erythrinus).

Page 11, lines 418-406 – Please clarify these sentences.

The advantages and drawbacks of the dry-ashing method are discussed in the manuscript. These sentences have been rephrased and relocated in subsection 3.2.3.

Page 11, line 428 – Please indicate the Units of the permissible tolerable daily intake of As, Cd, and Pb.

It was already mentioned in the manuscript that these values indicated μg/kg of body weight/day, which is now made clearer. In addition, tolerable daily intake (TDI) is referred to as “μg” for each metal to a 70-kg adult, which is now highlighted. Finally, these values occur for average and non-high consumers.

Page 11, lines 435-437 – As shown in Table 3, it seems that bioaccumulation didn’t occur with elements. Please check.

We agree with the reviewer’s comment as we hypothesized that this discovery could probably be due to the metal’s concentration rather than bioaccumulation.

Page 12, line 459 – I suggest “These authors”

This change has been applied to the manuscript.

Page 12, line 469 – Please clarify “...are organisms that consume at the base of the...”

We rephrased the sentence to clarify its meaning.

Page 13, line 487 – I think “to each other” could be deleted.

This phrase has been removed from the manuscript.

Page 13, lines 497-501 – These sentences are generic and not relevant in this context.

These sentences have been removed from the manuscript.

Page 13, line 503 – The levels of ω-3 PUFA of L1 and L2 were significantly different as shown in Table 5. Please check.

We thank the reviewer for the correction. We meant that no difference was observed between L1 and L2 in terms of PUFA concentration, so ω-3 has been removed and the typographical error has been corrected.

Page 13, lines 520 and 521 – I think it is mobilization of reserve lipids for the gonad maturation.

The sentence has been rephrased accordingly.

Page 13, line 527 – I suggest deleting “amounts of”. I also suggest indicating the reference values of these health indices.

This phrase has been deleted. Given the absence of reference values for the specific indices, it is stated that the lowest possible values are the most beneficial for human health. Some indices from food have also been inserted to provide a clearer picture of our measured values.

Page 14, Table 5 – Is there any explanation for low ω-3/ ω-6 ratio in the fish from L1 compared to the fish from the other locations?

It is now discussed in the manuscript that this outcome was linked to the significantly elevated levels of ω-6 fatty acids, potentially resulting from their diet in comparison to other fish samples.

Page 17, lines 602 and 603 – It is not clear how the correlation between various environmental factors, not specified in this study, and the quality of fish populations, whatever this is, was pinpointed. The next sentences are even more ambitious, but it is difficult to find any relationship with the results obtained.

Theses sentences have been thoroughly revised to be aligned with the findings of the work.

Conclusions

Page 17, lines 624-626 – This conclusion (“The study also... environments”) is surprising given that the authors only worked with fish fillets.

The term “tissue” has been changed to “fillets”.

Page 17, lines 629 and 630 – It would be interesting if the authors gave more information about the type of industries they are talking about.

Several types of industries are now referred to in the manuscript, as suggested by the reviewer.

Comments on the Quality of English Language

The English language could be improved.

English language correction has been done throughout the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript focuses on a widely employed and consumed fish species, red mullet. The novelty of the manuscript can be the comparative study among five different locations in the Greek coast.

I think the way it has been presented ought to be notably performed before a subsequent revision is done. The authors ought to clarify several important concerns.

I would mention the following:

 

Abstract

Lines 13-15: This information is general. Additionally, the positive aspects mentioned are common to most marine species.

Line 24: L1 ? L3? No explanation has been provided before.

Line 27: The size was apparently studied.

This section ought to be written again in order to present clearly what was really done.

 

Keywords

Eliminate “fish” and “seafood”,

 

Introduction

The effect of the size is not presented in last paragraph.

 

Material and methods

Apparently, 15 individuals were pooled together. A single replicate was carried out for each location ?

Lines 198-204: Provide more concrete information on GLC analysis. How was the quantification carried out ? Was there any internal standard used ?

Line 258: The fact that analyses are carried out in triplicate will give the error of the analytical tool, but not of the composition or nutritional value. This is by far my most important concern with this manuscript.

In the fillets study, have both muscles, light and dark, being studied altogether, or has only the light muscle been studied ?

 

Results

Tables 2-5 do not provide the number of replicates. How was then calculated the standard deviation ? The discussion of results is based on such average values and standard deviations.

Author Response

The manuscript focuses on a widely employed and consumed fish species, red mullet. The novelty of the manuscript can be the comparative study among five different locations in the Greek coast.

I think the way it has been presented ought to be notably performed before a subsequent revision is done. The authors ought to clarify several important concerns.

We would like to thank the reviewer for the insightful comments.

I would mention the following:

Abstract

Lines 13-15: This information is general. Additionally, the positive aspects mentioned are common to most marine species.

The sentences have been rephrased in order to provide a clearer explanation for the significance of the study on the specific fish species.

Line 24: L1 ? L3? No explanation has been provided before.

The term “location” is now mentioned in the Abstract section.

Line 27: The size was apparently studied.

This section ought to be written again in order to present clearly what was really done.

This section has been revised taking into account the hypotheses and obtained results. Indeed, the size of each fish sample was examined as variations in metals/elements were observed, as stated in the Abstract.

Keywords

Eliminate “fish” and “seafood”,

These keywords have been removed from the manuscript.

Introduction

The effect of the size is not presented in last paragraph.

The impact of size is now referred to in the last paragraph.

Material and methods

Apparently, 15 individuals were pooled together. A single replicate was carried out for each location ?

Each location had 15 fish, and from each location, we selected 5 fish samples. The samples were divided into three groups, and each group was analyzed in triplicate (n=3). This approach ensured that we captured variability within each location and provided reliable data for our analysis.

Lines 198-204: Provide more concrete information on GLC analysis. How was the quantification carried out ? Was there any internal standard used ?

We have included all the necessary information on the analysis by gas chromatography with flame ionization detection (GC–FID). The identification was achieved by comparing the individual peaks to FAME Mix C8–C24 reference standards. The percentage of each component was calculated based on the average of three GC–FID analyzes.

Line 258: The fact that analyses are carried out in triplicate will give the error of the analytical tool, but not of the composition or nutritional value. This is by far my most important concern with this manuscript.

We understand the concern. While triplicate analyzes provide an estimate of the analytical precision, our study design also includes biological replicates to account for variability in composition and nutritional value. By dividing the fish samples from each location into three groups (n=3) and analyzing each group in triplicate, we aimed to capture both analytical precision and biological variability.

In the fillets study, have both muscles, light and dark, being studied altogether, or has only the light muscle been studied ?

Both light and dark muscles were analyzed together in this study to provide a comprehensive assessment of the nutritional composition of the fish fillets. This approach ensures that our results accurately reflect the overall composition of the fish, without separating the muscle types.

Results

Tables 2-5 do not provide the number of replicates. How was then calculated the standard deviation ? The discussion of results is based on such average values and standard deviations.

We apologize for the oversight. Each value in Tables 2-5 represents the mean ± standard deviation (SD) of three independent replicates (n=3) for each location. The standard deviation was calculated from the triplicate analyzes of each group within each location. This ensures that the reported averages and standard deviations accurately reflect the variability in our data.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Comments:

• I believe there might be some missing information or a misunderstanding, as the results tables show a very low SD, which is characteristic of replicates from the same sample or even instrumental replicates. The SD presented in Tables 2, 3, 4, and 5 is relatively low compared to what would be expected for individually distinct samples. As noted in previously cited studies (29, 31, 35, 36, etc), the standard deviation between individual samples from the same location can reach the same order of magnitude as the average for certain analytes, with the relative standard deviation even exceeding 100% in some cases, particularly for non-essential analytes. However, in your study, I see that most results have a relative standard deviation below 7%, even for values reported as below the LOD, which is characteristic of replicates from the same pool of sample.

1) Could you please confirm if those SD presented in tables 2 to 5 are coming from 3 independent pools or from 1 pool in triplicate?

2) I saw that your reference #53 presents low SD like you but they present a big range between parentheses in table 4 that doesn’t represent the SD. Therefore, I did not understand how the SD came from, maybe you are in the same situation than this paper, using the same approach. So, if your SD presented in tables 2 to 5 it is like that, where is the range found in this triplicate? and if the case what means the SD?

In case if n = 3 independent sample pool, consider the comments below:

• In line 31 and line 88 replace the word “heavy-metal” to “arsenic”, since As is the only one that you were able to see.
• In line 206 include the word “in analytical solution”, such as: LOD values in analytical solution were measured according to the following Equation (1).
• Please check whether Figure 3, Figure 4 and table 6 are still applicable or if it needs to be updated after removing results below the LOD and incorporating the entire discussion above.
• Please check whether all the results Figure 3, Figure 4 and table 6 are still applicable or if it needs to be updated after removing results below the LOD and incorporating the entire discussion above.
• In the line 477 you mention As concentration as high as 12.6 ug/100 g wet fillet. However, this value is relatively low considering other works (https://doi.org/10.3390/w13020224 ; DOI: 4194/1303-2712-v17_6_07) evaluating the same fish. So, I recommend not emphasize the result as elevated As. And as I mentioned before, I still think that maybe these arsenic values are underestimated because the sample preparation is not suitable for this type of volatile analyte in this matrix.
• Add the LOD in the sample to Table A2 and update the values below the LOD in Tables 4 and 5 (normalized as ug/100g wet fillet).
• In Table A1: There is no natural abundance of ⁶⁴Cu, ⁶⁵Zn, or ⁵⁹Ni. Please review all m/z values, as it appears you cited the ponderated natural abundance mass of the elements from the periodic table rather than the actual isotope used in ICP-MS. Please verify this for all the analytes.
• In Table A1: Another point that call me my attention is that the LOD in analytical solution is in the middle of the calibration range. The calibration range is correct? Please verify this.
• Are ⁵⁶Fe and ⁴⁰Ca being measured in No-Gas mode by ICP-MS? Please verify this setting.

Comments for author File: Comments.pdf

Author Response

We would like to thank the reviewer for the comprehensive comments regarding our manuscript.

Comments:

• I believe there might be some missing information or a misunderstanding, as the results tables show a very low SD, which is characteristic of replicates from the same sample or even instrumental replicates. The SD presented in Tables 2, 3, 4, and 5 is relatively low compared to what would be expected for individually distinct samples. As noted in previously cited studies (29, 31, 35, 36, etc), the standard deviation between individual samples from the same location can reach the same order of magnitude as the average for certain analytes, with the relative standard deviation even exceeding 100% in some cases, particularly for non-essential analytes. However, in your study, I see that most results have a relative standard deviation below 7%, even for values reported as below the LOD, which is characteristic of replicates from the same pool of sample.

Thank you for pointing this out. In our study, we tried to ensure that fish in each pool had about the same age, weight, and length. For this reason, most results have a relative standard deviation below 7%, even for values reported as below the LOD, which might indicate replicates from the same sample pool but calculated from three independent pools.

• Could you please confirm if those SD presented in tables 2 to 5 are coming from 3 independent pools or from 1 pool in triplicate?

We can confirm that the SD presented in Tables 2 to 5 are calculated from three independent pools. We acknowledge that this information may not have been clear in the text, but we have updated the methodology section to clarify this.

• I saw that your reference #53 presents low SD like you but they present a big range between parentheses in table 4 that doesn’t represent the SD. Therefore, I did not understand how the SD came from, maybe you are in the same situation than this paper, using the same approach. So, if your SD presented in tables 2 to 5 it is like that, where is the range found in this triplicate? and if the case what means the SD?

Thank you for your observation. Reference #53 does present a low SD similar to ours, but they also provide a large range between parentheses in Table 4, which doesn't represent the SD. We did not use the same approach as this paper. The SD values in Tables 2 to 5 were calculated based on three independent sample pools. The SD in our context represents the variability among the independent sample pools.

 

In case if n = 3 independent sample pool, consider the comments below:

Given that the SD values are from three independent sample pools (n=3), we appreciate the following comments and have addressed them accordingly.

• In line 31 and line 88 replace the word “heavy-metal” to “arsenic”, since As is the only one that you were able to see.

The changes have been applied to the manuscript as suggested.

• In line 206 include the word “in analytical solution”, such as: LOD values in analytical solution were measured according to the following Equation (1).

This term has been inserted into the manuscript.

• Please check whether Figure 3, Figure 4 and table 6 are still applicable or if it needs to be updated after removing results below the LOD and incorporating the entire discussion above.

Thank you for your suggestion. We have revised Figures 3 and 4 and Table 6 after removing the results below the LOD. This ensures that all the data presented is accurate and consistent with the updated information.

• Please check whether all the results Figure 3, Figure 4 and table 6 are still applicable or if it needs to be updated after removing results below the LOD and incorporating the entire discussion above.

The same comment as above.

• In the line 477 you mention As concentration as high as 12.6 ug/100 g wet fillet. However, this value is relatively low considering other works (https://doi.org/10.3390/w13020224 ; DOI: 4194/1303-2712-v17_6_07) evaluating the same fish. So, I recommend not emphasize the result as elevated As. And as I mentioned before, I still think that maybe these arsenic values are underestimated because the sample preparation is not suitable for this type of volatile analyte in this matrix.

It is already mentioned that the concentration of arsenic in this fish sample (L1) was found to be higher compared to the other fish samples and it is stressed that it may not be related to the size of the fish, as expected. To avoid confusion, the term 'elevated' has been removed. In addition, in the same section it was also highlighted as a disadvantage of the dry-ash method that some metals are probably determined at a lower concentration than the actual concentration.

• Add the LOD in the sample to Table A2 and update the values below the LOD in Tables 4 and 5 (normalized as ug/100g wet fillet).

Thank you for your suggestion. We have added the LOD (limit of detection) values in the sample to Table A2, normalizing them as µg/100 g wet fillet. This ensures that all data is accurately reported and provides a clearer understanding of the detection limits for each analyte. However, in Table 4, the measurements were reported in mg/100 g wet fillet, and in Table 5, the results are expressed as a percentage of total fatty acids.

• In Table A1: There is no natural abundance of ⁶⁴Cu, ⁶⁵Zn, or ⁵⁹Ni. Please review all m/z values, as it appears you cited the ponderated natural abundance mass of the elements from the periodic table rather than the actual isotope used in ICP-MS. Please verify this for all the analytes.

We appreciate your attention to detail. We have reviewed all m/z values in Table A1 to ensure that the correct isotopes used in ICP-MS are cited, rather than the natural abundance masses from the periodic table. Some necessary corrections have been made to accurately reflect the isotopes measured in our study.

• In Table A1: Another point that call me my attention is that the LOD in analytical solution is in the middle of the calibration range. The calibration range is correct? Please verify this.

It is possible for the LOD (limit of detection) to fall within the calibration range. Ideally, the calibration range should encompass the LOD to ensure accurate quantification of the analytes. However, it is essential to verify that the calibration range is appropriately set and that it accurately reflects the concentration ranges relevant to the analysis.

• Are ⁵⁶Fe and ⁴⁰Ca being measured in No-Gas mode by ICP-MS? Please verify this setting.

All the minerals were measured in Helium [He] mode by ICP-MS. This mode helps to reduce polyatomic interferences and ensures more accurate measurements of the analytes in the fish samples.

Reviewer 2 Report

Comments and Suggestions for Authors

Abstract

Page 1, lines 27 and 28 – Total PUFA values of samples from L1 and L3 were not significantly different from those obtained in the samples from the other locations as mentioned in line 548 and shown in Table 5. Then, I suggest revise the sentence in these two lines. I also suggest “...was only detected in fish samples from these two locations”.

 

Introduction

Page 2, lines 74 and 75 – The sentence “Dietary preferences... meals” is a kind of tautology and I think “meals” is not appropriate.

Page 2, lines 76-88 – In line 77 is said that "the primary scope of this research was...", but in line 86 "the aim was to determine how the fish size..." In line 84 it is added that determinations of antioxidants were also made. This does not seem to be the best way to present the objective of the study, which should be simple and clear.

 

Materials and methods

Page 5, line 154 – It is “Proximate composition”.

Page 6, lines 180-182 – I have no doubt that the Bradford method is widely used for the determination of soluble protein content. However, the methods more adequate to determine the protein content in materials like fish are the Kjeldahl method or that based on the Dumas method. On the other, I don’t think necessary indicating that the reference [12] has more than 200 000 citations because the number of citations doesn’t mean that this method, which very good, could be successfully used in all matrices for the determination of protein content.

Page 7, line 246 - In the commentary to the first version of this manuscript, I wanted to say that “The tocopherol content was determined by HPLC-FLD”.

Page 8, lines 273 and 274 – Please consider this alternative “...quantification was done using an ascorbic acid standard curve”.

 

Results and Discussion

Page 9, lines 313 and 314 – I suggest revising this sentence because the fat content of samples from L1, L2 and L3 was not significantly different.

Page 9, lines 315 and next - Considering, for example, fish of the same species, there may be or may not be significant differences in the proximate composition of the various fish. The proximate composition of a single fish varies throughout its life and also among the fishes in a school, even if they have similar lengths or weights. However, in the case of a single fish, the sum of the percentages of the four main constituents (moisture, protein, fat and ash) at each point in its life is very close to 100%. In the case of a school, even if it is made up of very different fishes, if you were to make a pool of all these fishes, you would get a proximate average composition in which the percentage of each constituent would be between the maximum and minimum of that constituent recorded in that group of fish and the sum of the percentages of these constituents should be very close to 100 %. This is therefore a simple criterion for testing our results. When this is not the case, it means that there may have been an error in the determinations, in the preservation of the samples or in the type of method used, for example. This is my opinion on your comments and why I consider the results obtained in this study on the approximate composition of red mullet to be unreliable.

Page 9, lines 321 and 322 - The authors Koubaa et al. cite an FAO study with the approximate composition of many species, which is why the range of variation in fat content is so wide that it includes the composition of all species. So, the right thing to do is obviously to compare the values of the same or very similar species.

Page 10, lines 364-367 – Please consider this alternative: “Mollusks may bioaccumulate metals in greater quantities from their diets [31] and have been regarded as promising bioindicator species because of this capacity [31–33]”.

Page 10, lines 375-376 – Please revise this sentence for clarification.

Page 11, lines 386 – I suggest “moisture” instead of “humidity”.

Page 11, lines 387 and 388 – Please clarify “and tissue accumulation”.

Page 11, lines 392 and 393 – What I read in the paper by Morais et al [40] is that diets rich in fat, particularly saturated, may contribute to reduce the bone mass leading to an increase of bone fractures.

Page 11, lines 395 and 396 – Morais et al. mention hyperlipidemic diets in general and not necessarily fish. I think that the aspects mentioned by these authors are important in human nutrition, but irrelevant in the current work.

Page 11, lines 396-398 – The authors Duarte et al. [39] concluded “that fish oil added to tilapia feed nutritionally enriches the fish fillets and it is a good option for commercial production”. Concerning minerals, these authors say that fish is an important source of minerals and also reported the mineral content of feeds used in the trials with tilapia.

Page 11, lines 398-402 – The results by Reshetnyak [41] concern aspects of fish physiology and do not appear to be relevant to the discussion of this study.

Page 11, line 408 – As above, I suggest “moisture” instead of “humidity”.

Page 11, line 414 and 415 –Please consider this alternative sentence: “Smaller red mullet often inhabit shallower waters [43], ...”

Page 11, lines 426 and 427 – The meaning of the sentence “Most of the... studies” is not clear. Please clarify.

Page 11, lines 427-430 and page 12, lines 431-443 - I suggest that you first present and comment on your results, emphasising certain aspects, such as: Mn content <LOD at three sites; Se content < LOD in samples from all sites; Mo content <LOD at three sites, etc. Present the permissible tolerable daily intake and comment on your results taking these values into account. Include your sentence “The concentrations of essential trace minerals were comparable...” and compare your results with those obtained by other authors, including the results of Kormaz et al.

Page 12, lines 450 and 451 – I suggest this alternative sentence: “The Pb levels in red mullet samples collected in all sites were <LOD.

Page 12, lines 476-480 – Please consider this alternative: “It is to highlight that larger fish from L1 region did not present elevated...expectations”. Please clarify the sentence that begins ‘This discovery’. Also, it would seem better to start this sentence with “These results...”

Page 14, lines 511-514 – Please consider this alternative: “The presence of carotenoids in the red mullet samples, as in other fish species, is due to the ingestion of crustaceans and molluscs because they are unable to synthesise these biomolecules”.

Page 15, line 575 – I suggest replacing “These results” instead of “This discovery”.

Page 15, line 582 – It is “Fernandes”.

Page 15, lines 566-568 – Please take into consideration this alternative: “The observed change in the overall lipid composition might suggest the mobilization of reserve lipids for the gonad’s maturation throughout the spring season”.

Page 16, line 652 – Why “fish health”?

Comments on the Quality of English Language

The quality of English language need to be improved.

Author Response

We would like to thank the reviewer for the insightful comments in order to improve the readability of our manuscript.

Abstract

Page 1, lines 27 and 28 – Total PUFA values of samples from L1 and L3 were not significantly different from those obtained in the samples from the other locations as mentioned in line 548 and shown in Table 5. Then, I suggest revise the sentence in these two lines. I also suggest “...was only detected in fish samples from these two locations”.

The changes have been applied to the manuscript as suggested by the reviewer.

Introduction

Page 2, lines 74 and 75 – The sentence “Dietary preferences... meals” is a kind of tautology and I think “meals” is not appropriate.

This term has been replaced by “preys”.

Page 2, lines 76-88 – In line 77 is said that "the primary scope of this research was...", but in line 86 "the aim was to determine how the fish size..." In line 84 it is added that determinations of antioxidants were also made. This does not seem to be the best way to present the objective of the study, which should be simple and clear.

The whole paragraph has been revised correspondingly to the reviewer’s suggestions.

Materials and methods

Page 5, line 154 – It is “Proximate composition”.

The term “analysis” has been changed to “composition”.

Page 6, lines 180-182 – I have no doubt that the Bradford method is widely used for the determination of soluble protein content. However, the methods more adequate to determine the protein content in materials like fish are the Kjeldahl method or that based on the Dumas method. On the other, I don’t think necessary indicating that the reference [12] has more than 200 000 citations because the number of citations doesn’t mean that this method, which very good, could be successfully used in all matrices for the determination of protein content.

We have removed the citations from Bradford method and we highlighted those other methods (i.e., Kjeldahl) that could be more effective in our study as a limitation to our study.

Page 7, line 246 - In the commentary to the first version of this manuscript, I wanted to say that “The tocopherol content was determined by HPLC-FLD”.

This phrase has been corrected, as suggested by the reviewer.

Page 8, lines 273 and 274 – Please consider this alternative “...quantification was done using an ascorbic acid standard curve”.

The sentence has been revised accordingly.

Results and Discussion

Page 9, lines 313 and 314 – I suggest revising this sentence because the fat content of samples from L1, L2 and L3 was not significantly different.

The sentence has been revised accordingly.

Page 9, lines 315 and next - Considering, for example, fish of the same species, there may be or may not be significant differences in the proximate composition of the various fish. The proximate composition of a single fish varies throughout its life and also among the fishes in a school, even if they have similar lengths or weights. However, in the case of a single fish, the sum of the percentages of the four main constituents (moisture, protein, fat and ash) at each point in its life is very close to 100%. In the case of a school, even if it is made up of very different fishes, if you were to make a pool of all these fishes, you would get a proximate average composition in which the percentage of each constituent would be between the maximum and minimum of that constituent recorded in that group of fish and the sum of the percentages of these constituents should be very close to 100 %. This is therefore a simple criterion for testing our results. When this is not the case, it means that there may have been an error in the determinations, in the preservation of the samples or in the type of method used, for example. This is my opinion on your comments and why I consider the results obtained in this study on the approximate composition of red mullet to be unreliable.

Thank you for your insightful comments. We agree that the proximate composition of fish can vary throughout their life and among individuals in a school. In our study, we aimed to minimize variability by selecting fish with similar age, weight, and length for each pool. This approach was taken to ensure more consistent results, and we acknowledge that this might not fully capture the natural variability in the proximate composition. We understand that the sum of the percentages of the four main constituents (moisture, protein, fat, and ash) should be close to 100% in both individual fish and pooled samples. We applied this criterion to validate our results, and our findings showed that the sum of these percentages was indeed close to 100%. Nevertheless, we acknowledge your concerns regarding potential errors in the determinations, preservation of samples, or the methods used. Your feedback is invaluable, and we will incorporate it into our revised manuscript to address these issues comprehensively.

Page 9, lines 321 and 322 - The authors Koubaa et al. cite an FAO study with the approximate composition of many species, which is why the range of variation in fat content is so wide that it includes the composition of all species. So, the right thing to do is obviously to compare the values of the same or very similar species.

To enhance the readability of this study, we opted to remove the specific study and use another cited study to compare the fat content of red mullet, aligning with the reviewer’s recommendation.

Page 10, lines 364-367 – Please consider this alternative: “Mollusks may bioaccumulate metals in greater quantities from their diets [31] and have been regarded as promising bioindicator species because of this capacity [31–33]”.

These changes have been made as recommended.

Page 10, lines 375-376 – Please revise this sentence for clarification.

This sentence has been revised and enhanced in order to assure its understanding.

Page 11, lines 386 – I suggest “moisture” instead of “humidity”.

This change has been applied to manuscript.

Page 11, lines 387 and 388 – Please clarify “and tissue accumulation”.

This term has been removed in order to avoid confusion.

Page 11, lines 392 and 393 – What I read in the paper by Morais et al [40] is that diets rich in fat, particularly saturated, may contribute to reduce the bone mass leading to an increase of bone fractures.

Page 11, lines 395 and 396 – Morais et al. mention hyperlipidemic diets in general and not necessarily fish. I think that the aspects mentioned by these authors are important in human nutrition, but irrelevant in the current work.

Page 11, lines 396-398 – The authors Duarte et al. [39] concluded “that fish oil added to tilapia feed nutritionally enriches the fish fillets and it is a good option for commercial production”. Concerning minerals, these authors say that fish is an important source of minerals and also reported the mineral content of feeds used in the trials with tilapia.

Page 11, lines 398-402 – The results by Reshetnyak [41] concern aspects of fish physiology and do not appear to be relevant to the discussion of this study.

Discussion including studies from Morais, Duarte, and Reshetnyak et al. was removed, aligning with the reviewer’s suggestions.

Page 11, line 408 – As above, I suggest “moisture” instead of “humidity”.

This typographical error has been corrected throughout the manuscript.

Page 11, line 414 and 415 –Please consider this alternative sentence: “Smaller red mullet often inhabit shallower waters [43], ...”

This sentence has been revised.

Page 11, lines 426 and 427 – The meaning of the sentence “Most of the... studies” is not clear. Please clarify.

This section has been substantially revised.

Page 11, lines 427-430 and page 12, lines 431-443 - I suggest that you first present and comment on your results, emphasising certain aspects, such as: Mn content <LOD at three sites; Se content < LOD in samples from all sites; Mo content <LOD at three sites, etc. Present the permissible tolerable daily intake and comment on your results taking these values into account. Include your sentence “The concentrations of essential trace minerals were comparable...” and compare your results with those obtained by other authors, including the results of Kormaz et al.

This section has been revised accordingly, including tolerable upper intake level of Mn, Mo, and Se.

Page 12, lines 450 and 451 – I suggest this alternative sentence: “The Pb levels in red mullet samples collected in all sites were <LOD.

This sentence has been correspondingly revised.

Page 12, lines 476-480 – Please consider this alternative: “It is to highlight that larger fish from L1 region did not present elevated...expectations”. Please clarify the sentence that begins ‘This discovery’. Also, it would seem better to start this sentence with “These results...”

The specific sentences have been rephrased as suggested.

Page 14, lines 511-514 – Please consider this alternative: “The presence of carotenoids in the red mullet samples, as in other fish species, is due to the ingestion of crustaceans and molluscs because they are unable to synthesise these biomolecules”.

The sentence has been revised accordingly.

Page 15, line 575 – I suggest replacing “These results” instead of “This discovery”.

This term has been changed correspondingly.

Page 15, line 582 – It is “Fernandes”.

This typographical error has been corrected, as the word ”n” is now inserted in “Fernandes”.

Page 15, lines 566-568 – Please take into consideration this alternative: “The observed change in the overall lipid composition might suggest the mobilization of reserve lipids for the gonad’s maturation throughout the spring season”.

The reviewer’s suggestions have been applied and the sentence has been revised.

Page 16, line 652 – Why “fish health”?

The term has been removed.

Comments on the Quality of English Language

The quality of English language need to be improved.

Revision in English language have been conducted, as suggested by the reviewer.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has been performed. However, some of the aspects previously mentioned to be revised are still not clearly expressed.

 

Abstract

Line 15: Abundance of fatty acids ? Is it ω3 fatty acids what the authors mean ?

Line 19: Instead of “L”, it would be preferable to indicate the location names as L1-L-5.

Lines 33-34: fatty acid content ? Are the authors meaning ω3 fatty acid contents ?

 

Material and methods

Lines 146-147: The sampling procedure is still not clear. Five fish samples divided into three groups (n=3) ? How many individuals in each group ?

No additional information regarding the GLC analysis has been provided. In spite of the fact that two references are mentioned (numbers 14 and 15), the authors ought to clarify the method employed for FAME preparation, chromatographic separation of FAMEs, and quantification of FAMEs.

Author Response

The manuscript has been performed. However, some of the aspects previously mentioned to be revised are still not clearly expressed.

We thank the reviewer for the pertinent comments on our manuscript.

Abstract

Line 15: Abundance of fatty acids ? Is it ω3 fatty acids what the authors mean ?

Since we were mentioning other health-promoting chemical compounds, we added the term “unsaturated” before “fatty acids”.

Line 19: Instead of “L”, it would be preferable to indicate the location names as L1-L-5.

This change has been applied to the term “L”.

Lines 33-34: fatty acid content ? Are the authors meaning ω3 fatty acid contents ?

We refer to the differences found in total fatty acids.

Material and methods

Lines 146-147: The sampling procedure is still not clear. Five fish samples divided into three groups (n=3) ? How many individuals in each group ?

We apologize for the lack of clarity in the sampling procedure. Fifteen fish samples were divided into three groups, with each group consisting of a pooled sample from five individuals, making the total number of samples (n=3). We have revised the manuscript to clearly state the number of individuals in each group for better understanding.

No additional information regarding the GLC analysis has been provided. In spite of the fact that two references are mentioned (numbers 14 and 15), the authors ought to clarify the method employed for FAME preparation, chromatographic separation of FAMEs, and quantification of FAMEs.

We acknowledge the need for more detailed information regarding the GC-FID analysis for fatty acid composition. We have included this detailed information in the revised manuscript to ensure clarity and transparency in our methodology.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

After all doubts have been clarified and the work revised, I believe that it can be published in the Fishes.

Author Response

After all doubts have been clarified and the work revised, I believe that it can be published in the Fishes.

We would like to thank the reviewer for their comprehensive review and valuable suggestions, which have significantly enhanced the readability of our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Abstract

Page 1, lines 26 and 27 – Please take into consideration this alternative sentence: “The level of total PUFAs was above 30 % in all the samples and no significant differences were observed between them.”

 

Introduction

Page 2, line 80 – The fatty acid profile is missing.

 

Results and Discussion

Page 9, line 327 – Please take into account that fat content of L5 is not significantly different from L2.

Page 11, lines 427 and 428 – Please consider this alternative: “The concentration of most of the essential trace metals was similar to that reported in other studies.”

Comments on the Quality of English Language

The English language was improved.

Author Response

We would like to thank the reviewer for their insightful comments and cooperation.

Abstract

Page 1, lines 26 and 27 – Please take into consideration this alternative sentence: “The level of total PUFAs was above 30 % in all the samples and no significant differences were observed between them.”

Thank you for your suggestion. We have revised the sentence in the abstract.

 

Introduction

Page 2, line 80 – The fatty acid profile is missing.

Thank you for pointing this out. We have now included the fatty acid profile in the introduction section.

 

Results and Discussion

Page 9, line 327 – Please take into account that fat content of L5 is not significantly different from L2.

Thank you for your observation. We have noted that the fat content of L5 is not significantly different from L2.

Page 11, lines 427 and 428 – Please consider this alternative: “The concentration of most of the essential trace metals was similar to that reported in other studies.”

Thank you for your suggestion. We have revised the sentence.

 

Comments on the Quality of English Language

The English language was improved.

Thank you for your feedback.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has been performed. However, one aspect does not seem to be correct.

Please, revise the mentioned methodology for the FAME preparation, i.e., the presence of n-hexane and the content on methanol.

Author Response

The manuscript has been performed. However, one aspect does not seem to be correct.

Please, revise the mentioned methodology for the FAME preparation, i.e., the presence of n-hexane and the content on methanol.

We would like to thank the reviewer for their insightful comments and cooperation. We have clarified that n-hexane is used as a dissolving agent and 2 N methanolic potassium hydroxide solution is used as a methylation agent. The FAME Mix C8-C24 required only dissolution and not methylation.