Using Muscle Element Fingerprint Analysis (EFA) to Trace and Determine the Source of Hypophthalmichthys nobilis in the Yangtze River Basin

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Thank you very much for the recognition from the expert reviewer.

Is the research design appropriate?

Must be improved

We have improved this section in the revision.

Are the methods adequately described?

Can be improved

We have improved this section in the revision.

Are the results clearly presented?

Can be improved

We have improved this section in the revision.

Are the conclusions supported by the results?

Yes

Thank you very much for the recognition from the expert reviewer.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

This study is about the use of element fingerprint analysis to trace the origin of fish in the Yangtze River basin and I considered it very interesting. However, I discovered some discrepancies in the manuscript and it needs minor revision. But I have a question that I consider very pertinent: it seems to me that few fish were analysed (in one of the groups, only 2 individuals were analysed...) which seems to me to make the statistical treatment in general unfeasible and that somehow weakens the work. I know that the fish are large in size, but the ideal would have been to analyse more muscle of different fish, at least 5 to 10 in each group. Anyway, here are some suggestions:

Response 1:

Thank you for pointing this out. We really appreciate the reviewer for recognizing our manuscript and providing valuable suggestions for revisions. We agree with the evaluation experts that the sample size in general experimental studies is usually around 5 to 10 in each group. However, in this study, we used a smaller sample size mainly because, in practical market regulation applications, the sampling volume is often limited. The discriminant indicators identified through the analysis of a small number of samples in this study can better suit with the sampling and testing analysis of limited samples in market regulation inspections. The discriminant elements identified through analyzing these few samples enable us to trace different sources effectively and yield good differentiation results. This further confirms the sensitivity and practicality of using a small sample analysis to select discriminant elements for practical applications.

Comments 2:

Introduction: Rewrite this sentence, there is no verb... “Given the different targets of application, difficulties in sample collection, and discrimination sensivity of different methods”

Response 2:

We greatly appreciate the reviewer's comments, and we have revised this section in the revision and highlighted in red.

Comments 3:

Material and methods: Include the freeze dryer manufacturer; HCLO4 manufacturer hot plate manufacturer; What is the temperature of the hot plate to carry out digestion? Which elements are analysed in ICP-OES and which are analysed in ICP-MS?

Response 3:

In this study, the temperature for freeze-drying is -48°C. The temperature for sample pretreatment digestion in this study is 150°C. The elements of K, Na, Ca are analysed in ICP-OES and the remaining elements are analysed in ICP-MS.

Comments 4:

Results: In relation to the data in the table 2: Zn and Mn content in the samples, was the content higher than Ca? It's not usual... And the Cd content close to Ca? Are you sure the values are in the same units?

Response 4:

Thank you very much to the reviewer for pointing out the issue with the units in Table 2. The different units used for various elements in Table 2 are due to the standardized processing in this study. To facilitate analysis and ensure that the ratios of different elements to Ca are in similar orders of magnitude, different units were applied for elements with significant differences in content. Specifically, the unit for Zn/Ca and Mn/Ca is µg/mg, while the unit for Cd/Ca is ng/mg. The units for the elements to Ca have been clearly specified in the Note of Table 2 and highlighted in red.

Comments 5:

Figure 2 does not seem interesting to me given that the results are identical to those in table 2.

Response 5:

We sincerely appreciate the expert's suggestions. The purpose of including Figure 2 in this study is to provide a more intuitive and visual representation of the elements that differ in Table 2. Figure 2 can be deleted or moved to the Supplementary Materials later in the article correction, based on expert opinion that it is not convenient to represent Figure 2 in the main text.

Comments 6:

Figures：In figures 3, 5 and S1 perhaps change the symbols, as they are the same and only differ in color...

Response 6:

Thank you very much for the expert's suggestions. We have revised Figures 3, 5, and S1 to make the differences between the groups more noticeable.

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Thank you very much for the valuable revision suggestions provided by the reviewing experts. We have improved this section in the revision.

Is the research design appropriate?

Can be improved

We have improved this section in the revision.

Are the methods adequately described?

Must be improved

We have improved this section in the revision.

Are the results clearly presented?

Can be improved

We have improved this section in the revision.

Are the conclusions supported by the results?

Must be improved

We have improved this section in the revision.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

The contents and the topic of the paper are interesting and with practical applications both by FBO and for official controls.

Response 1:

Thanks to the reviewer for recognizing our paper. We will further enhance the overall quality of the paper based on the reviewer's suggestions to maximize its application value.

Comments 2:

Paragraph 2.1. The authors must explain wich was the statistical rationale behind the sample numbers employed in the trial for tuning the conditons and segregating the elements to use as a marker, just 2 samples for WH and 5 samples respectively for AQ and TH.

Response 2:

Thanks for the reviewer’s suggestions. Regarding the issue of the small sample size in this study, it is common in general experimental research to select sample sizes of 5-10 or more. However, in this study, we chose a smaller sample size primarily due to practical considerations in market regulation, where the sample quantity is often limited. The goal of this study is to identify the most distinct elements as discriminant indicators using a smaller sample size. This approach aims to establish a more sensitive and practical basis for discrimination based on elemental differences, meeting the demands of market regulation where both limited sampling and high sensitivity in practical applications are required.

Comments 3:

In the same paragraph it must be indicated how the fishes were suppressed.

Response 3:

In this study, in order to obtain samples of different sizes, the gill nets with different mesh size (from 3cm to 21cm) were set up for sampling and the samples were collected after the gill nets were placed underwater for 12h.

Comments 4:

Still in the paragraph 2.1. Considering that the focus of the study was the analysis of elements it seems critical, by a procedural viewpoint, to use metallic devices to prepare the samples (such as stainless steel surgical knives, tissue grinder??) that could release own elements that contaminated the samples. The authors have to explain how this criticity was managed.

Response 4:

In this study, the dissection tools and sample processing devices, such as scissors and grinders, used during the dissection and grinding of samples were thoroughly cleaned six times with Milli-Q water before and after use. This was to ensure that there was no cross-contamination between different samples and to avoid any impact of dissection tools and processing devices on the different sample groups. Additionally, the dissection and processing devices used for different sample groups were the same, ensuring that any mechanical background influence were consistent across groups, which will not affect the final discriminative analysis results.

Comments 5:

In addition, it must be explained from which section or sections of the dorsal muscles were taken the analytical samples

Response 5:

In this study, due to the large size of the samples, pieces of muscle were taken from the anterior, middle, and posterior regions of the fish's back to form a composite sample. This sampling method ensures that the obtained sample adequately represents the elemental composition of the fish overall muscles.

Comments 6:

Paragraph 2.2. Which was the reason to use two different instruments to measure the elements?

Response 6:

Given that the content of different elements in fish muscle tissue varies, selecting different instruments can ensure precise detection of elements at various concentrations.

Comments 7:

Paragraph 3.1. Explain why the calcium was used to do a sort of normalization of the elements concentration.

Response 7:

In this study, among the four constant elements (K, Ca, Na, Mg), K has the highest content, while Na and Mg have similar and relatively low contents. Ca has a moderate content, and there are no significant differences in its content among different groups. This makes it suitable for use as a standardized reference element in this research. After standardization by dividing different elements by calcium, we can reduce the differences between different samples and better present the elemental fingerprint characteristics for different individuals.

Comments 8:

Why it was employed Non-parametric tests and then One way ANOVA to study significant differences among the three sampling stations?

Response 8:

In the data processing of this study, nonlinear analysis was first applied to initially determine whether there are differences among groups. Under the premise of differences exist, One-way ANOVA was then used to compare the differences between the groups. The data processing and analysis approach in this study shows a progressive relationship, while also allowing for a comparison of the results obtained from the two analysis methods to see if they are consistent. This comparison of the differences between the analysis methods lays the basis for further in-depth analysis.

Comments 9:

In the paragraph 3.3. The authors write "There was no significant difference of these four discriminative elements ratios between AQ and PC-AQ. It can be seen that the WH samples can be distinguished by Pb element ratios from these four groups, and the TH samples can be distinguished by Al element ratios, while the composition of the four discriminative elements between AQ and PC-AQ is relatively consistent, which is expected to identify and trace the source of samples from different sources by the composition differences of these four discriminative elements ratios". It not clear why the PC samples were attributed to AQ station by the boxplot of Figure 4.

Response 9:

We sincerely appreciate the valuable comments provided by the reviewer. We have made a clearer statement in this section and highlighted it in red in the text. The revised portion is as follows: "As shown in Figure 4, it can be concluded through Pb/Ca analysis that there is a significant difference between PC and WH group, while through Al/Ca analysis that there is a significant difference between PC and TH group. However, for the PC and AQ groups, not only is there no difference in Pb and Al between these two groups, but there is also no difference in Na and Cr between these two groups. It can be seen that the distribution of these four elements to Ca is similar between PC and AQ groups, and it can be preliminarily inferred that the PC and AQ groups have similar sources."

Comments 10: Discussion. The first half of the discussion chapter it is not true discussion, and it seems more appropiate to move it in the Introduction chapter.

Response 10: We greatly appreciate the valuable comments from the reviewer, and we have made adjustments to the relevant content in the introduction and discussion. Some content discussed in this study is also presented similarly in the introduction, but the manner of presentation and the purpose of expression differ between the two sections. In the introduction, we primarily explain why muscle tissue was chosen for analysis and why element fingerprint analysis was selected as the method, mainly based on literature collection and analysis. In the discussion, we focus on analyzing experimental results to validate the rationale and effectiveness of choosing muscle tissue and element fingerprint analysis for this study. The introduction leans more towards literature and theoretical analysis, while the discussion is more oriented towards results and validation analysis.

Comments 11: In a speculative perspective I wonder whether pollutant heavy metals, such as Pb and Hg, and others like All that clearly contaminate the river by anthropogenic activities can be considered as reliable markers insted of other element whose origin is due to the natural background. It can expect that future variations of the industrial activities could deeply change the water concentration of these elements making the EFA model developed in this study to trace and determine the source of Hypophthalmichthys nobilis in the Yangtze River Basin less reliable. The authors should discuss this criticity.

Response 11: We sincerely appreciate the suggestions and guidance from the reviewer. In this study, we also considered similar issues, so we analyzed the distinguishing elements selected from different sites and their possible sources. The selected distinguishing elements are somewhat correlated with the pollutants in the corresponding water bodies. However, considering the distribution of typical elements in the water environment of different sections of the Yangtze River, this distribution represents a long-term accumulation effect that is relatively stable and will not show significant changes in the long-term background values due to variations in local pollution sources in the short term. For fish that appear in the market, which have a relatively short time from birth to market entry, the accumulation of elements in their muscle is primarily influenced by the elemental distribution patterns in their broader living environment. Given the stability of the long-term accumulated background values formed in different sections of the Yangtze River, using the discriminant elements selected from different sites in this study for source tracing remains effective and has practical value.

4. Response to Comments on the Quality of English Language

Point 1: The english language seems sufficiently good and comprehensible

Response 1: We sincerely appreciate the reviewers' recognition of our English expression.

Comments 1:

Thank you for following my suggestions. After your clarification, I understand that you were only able to use a few fish specimens in this study.

I just think you should put figure 2 in the supplementary files. Everything else seems OK to me.

Response 1:

Thank you very much for the recognition of our Response.

We have moved Table 2 to the supplementary files as Figure S1 according to your valuable suggestions.

Thank you again to the reviewer for your recognition of our article.

Comments 1:

Due to missing of the lines numbering it is difficult to explain duly, but it seems there is a repetition phrase in the chapter 4 (discussion).

Response 1:

Thank you for pointing this out. We really appreciate the reviewer for recognizing our previous Response and providing valuable suggestions again. We have revised and streamlined the repetitive content in the Discussion.

Comments 2:

Occasionally, especially close or into the revised text (red color) the english language can be improved.

Response 2:

Thank you very much for your valuable suggestions on the Quality of English Language. We have revised and improved the English expression, especially about the revised text (red color).

Thank you again to the reviewer for your recognition of our article.