Antioxidant Astaxanthin Co-Treatment Protects Zebrafish from Dimethomorph-Induced Cardiovascular Toxicity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper is written according to the propositions of the journal.  Topic of cardiotoxicity due to various agents is quite interesting these days, and that makes this article relevant and interesting to readers. 

The introduction is of appropriate length and presents the most important and up-to-date information regarding the topic.

The applied methods are appropriate in relation to the set research goals and in accordance with recently published works in that field. Zebra fish is a relatively good model and acceptable in the field of research

 

The results are well presented and are in line with the title of the paper and methodology applied. 

 

The English language is generally good, but some corrections are suggested below :

Lines 85-86: the word functions is in the wrong order

In Table 1, genes are presented in the form of abbreviations, but their full names are missing in
the text.

In Figure 1, there is no blue arrow as indicated in the legend.

It would be good if the image descriptions were shorter. Statistical analysis can only be described in the methodology.

Half of the references are older than 5 years. Please update with newer references.

The paper lacks authors contributions

Author Response

Comments and Suggestions for Authors

The paper is written according to the propositions of the journal.  Topic of cardiotoxicity due to various agents is quite interesting these days, and that makes this article relevant and interesting to readers. The introduction is of appropriate length and presents the most important and up-to-date information regarding the topic. The applied methods are appropriate in relation to the set research goals and in accordance with recently published works in that field. Zebra fish is a relatively good model and acceptable in the field of research. The results are well presented and are in line with the title of the paper and methodology applied. 

The English language is generally good, but some corrections are suggested below :

Lines 85-86: the word functions is in the wrong order

The authors appreciate the reviewer’s comment. The word order in the sentence have been corrected to “They offer a high-throughput, cost-effective system for assessing the impact of toxicants on biological processes, including cardiovascular and neurobehavioral functions”.

In Table 1, genes are presented in the form of abbreviations, but their full names are missing in the text.

The authors are grateful for the reviewer’s reminder. The authors have added the full names of all the mentioned genes in the manuscript which are; ventricular myosin heavy chain (vmhc), atrial myosin heavy chain (amhc), vascular endothelial growth factor Aa (vegfaa), T-box transcription factor 5 (tbx5), NK2 homeobox 5 (nkx2.5), GATA binding protein 4 (gata4), and myosin heavy chain cardiac muscle alpha (myh6).

In Figure 1, there is no blue arrow as indicated in the legend.

The authors appreciate the reviewer’s reminder. The yellow arrow mentioned should be the blue arrow, pointing to the edema found in zebrafish larvae exposed to DIM.

It would be good if the image descriptions were shorter. Statistical analysis can only be described in the methodology.

The authors appreciate the reviewer’s comment. While the authors agree that image descriptions are quite long, the authors would like to provide a complete information on the statistical test used in the study, which might differ from one figure to the other, additionally mentioning it in all the manuscript, this would provide enough information for the reader to read the manuscript without going to the other sections (such as material and methods) to obtain those informations.

Half of the references are older than 5 years. Please update with newer references.

The authors are thankful for the reviewer’s comment. The authors have updated the references to newer publications, however some older publications are kept as they are the original findings and are still used in recent publications.

The paper lacks authors contributions

The authors are grateful for the reviewer’s reminder. The authors have added author contributions and funding source as required by the publisher.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript "Antioxidant Astaxanthin Co-Treatment Protects Zebrafish from
Dimethomorph-Induced Cardiovascular Toxicity " by Chia-Chen Wu et al. studies relevant problem namely the off-target toxicity of fungicide dimethomorph (DIM). In general, the study is well conducted and reported, but some short-comings are present. Here, below, are my comments to improve the manuscript.

1) ROS is strongly suggested as toxicity mechanism of DIM. Although indirectly supported, the direct evidence of DIM affecting ROS levels in zebrafish embryo are lacking. Direct evidence on increased ROS after DIM treatment would strongly support the presented conclusions.

2) The environmental levels should be directly discussed. Now the discussion is indirect (eg. line 378-379), and would be better to write down and reference the environmental levels of DIM. 

3)It is unclear how the AST dose was selected. The partial rescue could be also due to insufficient dose of AST compared to DIM (0.6ppm AST vs 10ppm DIM). The clarification of the dose selection would strengthen the manuscript.

4) Dose-response curve in Fig.1A looks good. It would be still useful to include confidence interval of other error measure in the LC50 value.

 

Author Response

The manuscript "Antioxidant Astaxanthin Co-Treatment Protects Zebrafish from
Dimethomorph-Induced Cardiovascular Toxicity " by Chia-Chen Wu et al. studies relevant problem namely the off-target toxicity of fungicide dimethomorph (DIM). In general, the study is well conducted and reported, but some short-comings are present. Here, below, are my comments to improve the manuscript.

1) ROS is strongly suggested as toxicity mechanism of DIM. Although indirectly supported, the direct evidence of DIM affecting ROS levels in zebrafish embryo are lacking. Direct evidence on increased ROS after DIM treatment would strongly support the presented conclusions.

The authors are grateful for the reviewer’s comment. The authors would like to apologize for the limited evidence regarding ROS levels in zebrafish embryo, the authors are unable to extract these data due to inavailability of tools to test ROS levels in authors current repertoire, which will be taken into consideration by the authors in the future study. Thus, the authors used metabolic rate through observing oxygen consumption from time to time for 50 minutes after exposure to DIM, while it is possible that the difference of oxygen consumption is caused by metabolic states, the significant increase of oxygen consumption within 50 minutes from larvae of the same clutch and relatively high number (60+) should support the increase of ROS due to DIM exposure.

Hou, C.; Metcalfe, N.B.; Salin, K. Is mitochondrial reactive oxygen species production proportional to oxygen consumption? A theoretical consideration. BioEssays 2021, 43, 2000165, doi:https://doi.org/10.1002/bies.202000165.

 

2) The environmental levels should be directly discussed. Now the discussion is indirect (eg. line 378-379), and would be better to write down and reference the environmental levels of DIM. 

The authors are grateful for the reviewer’s comment. In accordance to the reviewer’s comment, the authors have tried to find a study which stated environmental levels of DIM, however the authors found DIM concentrations to be relatively low, except during DIM spraying schedule for several plants, in which DIM concentration might go up to 0.639 mg a.i./L at 41 day post spraying as stated by a research conducted by Canada health agency. This information have been included in the manuscript at line 277-279.

Canada., C.H.; Agency., C.P.M.R. Dimethomorph and its associated end-use products: final decision.; Ottawa, Ontario: Pest Management Regulatory Agency, Health Canada, 2020.: 2020.

 

3)It is unclear how the AST dose was selected. The partial rescue could be also due to insufficient dose of AST compared to DIM (0.6ppm AST vs 10ppm DIM). The clarification of the dose selection would strengthen the manuscript.

The authors are thankful for the reviewer’s suggestion. The dose of AST was decided from a preliminary test using a small number of zebrafish larvae (5 each) exposed to AST at different dose 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 ppm. Zebrafish larvae was exposed to AST at 24 hpf for 48 hours. From this test, the authors observed DIM at ≥0.8 ppm seems to reduce zebrafish larvae activity (manual observation), thus the authors decided to use AST at the highest concentration (0.6 ppm). This decision resulted in a partial rescue as observed in the manuscript.

4) Dose-response curve in Fig.1A looks good. It would be still useful to include confidence interval of other error measure in the LC50 value.

The authors would like to extend their gratitude for reviewer’s comment. The authors have added the 95% CI value (14.37 to 15.82 ppm) to the manuscript as the reviewer’s requested.

 

Reviewer 3 Report

Comments and Suggestions for Authors

 This study by Wu et al. suggests that a toxic dose of dimethomorph, a commonly used morpholine fungicide, induces cardiac hypertrophy, reduces heart rate, significantly alters the expression of key genes (gata4 and tbx5) associated with cardiovascular development and function, and increases metabolic rate in zebrafish embryos. In addition, the antioxidant astaxanthin attenuated the heart rate reduction induced by dimethomorph. The study is interesting; however, several points require clarification.

1. Please follow the Author’s Guidelines of IMJS. The manuscript should follow the sequence: Introduction – Results – Discussion – Materials and Methods.
2. Please define all abbreviations at their first appearance in the manuscript using the following format: the full term followed by the abbreviation in parentheses. For example: LD50, hpf, CCD, SWH, et al.
3. How was the sample size determined for each experimental protocol (acute toxicity test, cardiac toxicity assay, vascular toxicity assay, metabolic rate analysis, gene expression analysis, and rescue experiment)?
4. Line 204, Statistical analysis section: How were normality and homogeneity of variance assessed in the data?
5. Figure 2B and C, and Figure 5B and C (legends):
   Please change “sd1 and sd2 of heart chamber” to “sd1 and sd2 of heart rate variability.”
   Accordingly, replace “heart chamber” with “heart rate variability” in the figure legends.
6. Figure legends for Figures 2, 3, 4, and 5:
   What does n represent? Please clarify whether it refers to biological replicates, technical replicates, or the total number of measurements.
7. Figure 3:
   Please include the mean ± SD of the data obtained at each time point (600, 1200, 1800, 2400, and 3000 s) for each group directly in Figure 3.
8. Figure 3 legend:
   What does “total oxygen consumption” mean? Does it refer to oxygen consumption over 50 minutes? Please clarify this description.
9. Figure 5 and its legend:
   Please add the dosages of DIM and AST in both the figure and the legend. In addition, please compare stroke volume between the DIM-only group and the DIM + AST group in Figure 5D. Since AST is used as a rescue treatment for DIM toxicity, statistical analysis comparing stroke volume between DIM alone and DIM + AST should be performed in Figure 5D. However, comparing control and DIM + AST groups is not appropriate.

 

Author Response

Comments and Suggestions for Authors

This study by Wu et al. suggests that a toxic dose of dimethomorph, a commonly used morpholine fungicide, induces cardiac hypertrophy, reduces heart rate, significantly alters the expression of key genes (gata4 and tbx5) associated with cardiovascular development and function, and increases metabolic rate in zebrafish embryos. In addition, the antioxidant astaxanthin attenuated the heart rate reduction induced by dimethomorph. The study is interesting; however, several points require clarification.

1. Please follow the Author’s Guidelines of IMJS. The manuscript should follow the sequence: Introduction – Results – Discussion – Materials and Methods.

The authors are grateful for the reviewer’s reminder. The authors have adjusted the manuscript content to follow IJMS manuscript sequence, following the author’s guidelines.

2. Please define all abbreviations at their first appearance in the manuscript using the following format: the full term followed by the abbreviation in parentheses. For example: LD50, hpf, CCD, SWH, et al.

The authors are grateful for the reviewer’s suggestion. In accordance to the reviewer’s suggestion, the authors have added the full term of the abbreviations used in this study as mentioned by the reviewer, for example Lethal concentration 50 (LC₅₀), hours post-fertilization (hpf), charge-coupled device (CCD), Salvador-Warts-Hippo (SWH) signaling pathway, to name the few.

3. How was the sample size determined for each experimental protocol (acute toxicity test, cardiac toxicity assay, vascular toxicity assay, metabolic rate analysis, gene expression analysis, and rescue experiment)?

The authors would like to extend their gratitude for the question, in regard of the sample size, the authors determined it from the previous study experiences. From our experience for acute toxicity test, cardiac toxicity assay, vascular toxicity assay, and rescue experiment n = 30~, for metabolic rate analysis n = 60~, and gene expression analysis n = 3 should be enough to fulfill the minimal sample size required. In order to report this claim, we have included two new references from our previous publication by Saputra et al., and Feng et al.

Saputra, F.; Uapipatanakul, B.; Lee, J.-S.; Hung, S.-M.; Huang, J.-C.; Pang, Y.-C.; Muñoz, J.E.R.; Macabeo, A.P.G.; Chen, K.H.C.; Hsiao, C.-D. Co-Treatment of Copper Oxide Nanoparticle and Carbofuran Enhances Cardiotoxicity in Zebrafish Embryos. In International Journal of Molecular Sciences, 2021; Vol. 22, p 8259.
Feng, W.-W.; Chen, H.-C.; Audira, G.; Suryanto, M.E.; Saputra, F.; Kurnia, K.A.; Vasquez, R.D.; Casuga, F.P.; Lai, Y.-H.; Hsiao, C.-D., et al. Evaluation of Tacrolimus’ Adverse Effects on Zebrafish in Larval and Adult Stages by Using Multiple Physiological and Behavioral Endpoints. In Biology, 2024; Vol. 13, p 112.

4. Line 204, Statistical analysis section: How were normality and homogeneity of variance assessed in the data?

The authors are grateful for the reviewer’s question. Data normality were assessed through normality test in the GraphPad Prism. The test includes Anderson-Darling test, D’Agostino & Pearson test, Shapiro-Wilk test, and Kolmogorov-Smirnov test. From the results on these tests, we decided the type of ANOVA test compatible for the data. In accordance to the reviewer’s question, the information have been added to the manuscript.

5. Figure 2B and C, and Figure 5B and C (legends): Please change “sd1 and sd2 of heart chamber” to “sd1 and sd2 of heart rate variability.” Accordingly, replace “heart chamber” with “heart rate variability” in the figure legends.

The author would like to thank the reviewer for the comment. The authors have changed the term used in the figures as requested by the reviewer. The authors hope that by changing this, it provides better clarity to the reviewer and the readers.

6. Figure legends for Figures 2, 3, 4, and 5: What does n represent? Please clarify whether it refers to biological replicates, technical replicates, or the total number of measurements.

The authors are grateful for the reviewer’s comment. n represents total sample size used in the test. While we have mentioned the information regarding our sample size in the material and methods part of the manuscript, as the reviewer’s mentioned this might not be enough to inform the reader, thus we have mentioned it again in the earliest part of the manuscript that mention the n, which is Figure 2.

7. Figure 3: Please include the mean ± SD of the data obtained at each time point (600, 1200, 1800, 2400, and 3000 s) for each group directly in Figure 3.

The authors are grateful for the reviewer’s comment. The authors would like to clarify the reviewer regarding the data in this figure. The data in this figure is measured at every second, thus it is not limited to 600, 1200, 1800, 2400, and 3000s. The current data shown in the manuscript is the mean of the data, while the mean ± SD of the aforementioned figure is attached below. However, the authors think that by putting this data, it will be difficult to observe the values, as the data overlapped with each other. Thus the authors have decided to keep the current figure. However, if the reviewer still preferred to use the mean ± SD data, the authors are open to replace it with the figure below.

8. Figure 3 legend: What does “total oxygen consumption” mean? Does it refer to oxygen consumption over 50 minutes? Please clarify this description.

The authors are grateful for the reviewer’s question. Yes, total oxygen consumption mean the total oxygen consumed during the 50 minutes of metabolic rate assessment. We have added the information in the figure legend as the reviewer’s request to add more clarity on this matter.

9. Figure 5 and its legend:
Please add the dosages of DIM and AST in both the figure and the legend. In addition, please compare stroke volume between the DIM-only group and the DIM + AST group in Figure 5D. Since AST is used as a rescue treatment for DIM toxicity, statistical analysis comparing stroke volume between DIM alone and DIM + AST should be performed in Figure 5D. However, comparing control and DIM + AST groups is not appropriate.

The authors are grateful for the reviewer’s comment. As the reviewer’s suggested, the authors have added the dosage of DIM and AST to both the figures and legend. Secondly, the authors would like to clarify that the comparison was conducted to compare all possible pairwise combinations (control and DIM, control and DIM + AST, and DIM and DIM + AST). The authors choose these combinations to show the degree of rescue provided by AST, if the AST is very effective in relieving DIM effect, the authors expect that the results statistical significance between control group and DIM + AST treatment group will be non significant, which can be observed in the stroke volume in this study.

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript investigates the cardiovascular toxicity of dimethomorph (DIM) in zebrafish larvae and explores the potential protective effects of astaxanthin. DIM exposure leads to cardiovascular toxicity, including decreased heart rate, increased stroke volume, cardiac edema, and altered heart rate variability. In addition, the authors assessed metabolic rate by measuring oxygen consumption and found that DIM exposure significantly increased metabolic activity in zebrafish larvae. Gene expression analysis further revealed changes in several cardiovascular development–related genes, including gata4, myh6, tbx5, and vegfaa. By the rescue experiment using the antioxidant astaxanthin and observed partial recovery of cardiac function. However, several issues need to be addressed.

 

1. The abstract states that DIM exposure results in a “substantial enlargement in heart size.” However, the Results section does not present any direct measurement or quantitative analysis of heart size to support this statement

 

2. Cardiac edema was observed at 96 hours post-exposure to DIM exposure at 10 ppm, whereas the cardiac and vascular performance assays were performed at 72 hours of exposure. If overt pericardial edema becomes apparent only after 96 hours of exposure, how should the functional changes measured at the earlier time point be interpreted?

 

3. If the authors intend to demonstrate cardiomegaly, should consider providing additional quantitative evidence, such as direct measurement of heart size, histological analysis of cardiac structure, such as determining the maximal thickness of the Tnnt⁺ compact muscle in hearts.

 

4. The qRT-PCR data appear to show relatively large standard deviations (Fig. 4 E F). Could you add the number of biological replicates and present the individual data points in the graphs?

 

5. The authors suggest that astaxanthin may act as a protective agent against DIM-induced cardiotoxicity. The discussion should more thoroughly address the underlying mechanisms and biological feasibility of this protective effect, supported by relevant literature.

 

6. “Due to the observed increased metabolic rate and oxygen consumption, which may lead to elevated ROS production, a rescue experiment was conducted using the strong antioxidant Astaxanthin (AST)”. However, no ROS assay (e.g., DCFH-DA staining) was performed.

 

 

 

Author Response

This manuscript investigates the cardiovascular toxicity of dimethomorph (DIM) in zebrafish larvae and explores the potential protective effects of astaxanthin. DIM exposure leads to cardiovascular toxicity, including decreased heart rate, increased stroke volume, cardiac edema, and altered heart rate variability. In addition, the authors assessed metabolic rate by measuring oxygen consumption and found that DIM exposure significantly increased metabolic activity in zebrafish larvae. Gene expression analysis further revealed changes in several cardiovascular development–related genes, including gata4, myh6, tbx5, and vegfaa. By the rescue experiment using the antioxidant astaxanthin and observed partial recovery of cardiac function. However, several issues need to be addressed.

1. The abstract states that DIM exposure results in a “substantial enlargement in heart size.” However, the Results section does not present any direct measurement or quantitative analysis of heart size to support this statement

The authors are grateful for the reviewer’s suggestion. In accordance to the reviewer’s suggestion, the authors have measured the heart size of zebrafish used in the study. From the measurement, the authors observed a significant increase in zebrafish heart volume at 10 ppm DIM. The manuscript have been revised to mention this finding.

2. Cardiac edema was observed at 96 hours post-exposure to DIM exposure at 10 ppm, whereas the cardiac and vascular performance assays were performed at 72 hours of exposure. If overt pericardial edema becomes apparent only after 96 hours of exposure, how should the functional changes measured at the earlier time point be interpreted?

The authors would like to apologize for the lack of information. While we observed and presented the data of cardiac edema at 96 hpf from acute toxicity test. We actually observed the edema again at 72 hpf during the cardiac and vascular performance assays, however we did not photograph any image to present in this time point, thus to avoid misunderstanding, we revised the manuscript to not mention the edema at 72 hpf (cardiac performance assay).

3. If the authors intend to demonstrate cardiomegaly, should consider providing additional quantitative evidence, such as direct measurement of heart size, histological analysis of cardiac structure, such as determining the maximal thickness of the Tnnt⁺ compact muscle in hearts.

The authors are thankful for the reviewer’s comment. As the reviewer have suggested, the authors have included heart size measurement data in the manuscript to provide a direct evidence of cardiomegaly. From the result, DIM exposure at 10 ppm induced significantly larger heart size compared to control, while DIM exposure at lower concentrations did not show any significant alteration.

4. The qRT-PCR data appear to show relatively large standard deviations (Fig. 4 E F). Could you add the number of biological replicates and present the individual data points in the graphs?

The authors are grateful for the reviewer’s input. However, currently we have limited budget thus it is not possible to add biological replicates for the qRT-PCR, however to reviewer’s request, we have added the individual data points in the graphs.

5. The authors suggest that astaxanthin may act as a protective agent against DIM-induced cardiotoxicity. The discussion should more thoroughly address the underlying mechanisms and biological feasibility of this protective effect, supported by relevant literature.

The authors are thankful for the reviewer’s suggestion. The authors have included additional discussion as the reviewer’s requested. In short, we have found reference that highlighted DIM dose-dependent toxicity on zebrafish which are very apparent in zebrafish heart area. Meanwhile, AST have been shown to be especially effective in scavenging and inhibiting ROS, as well as free radicals. This means that while DIM increase ROS levels, the addition of AST might regulate ROS levels to a more acceptable state. However, from our result we found that AST was only able to partially alleviate DIM toxicity, therefore there might be another toxic properties other than increasing ROS, which was thyroid hormone dysregulation found in previous study.

Wei, Y.; Meng, Y.; Jia, K.; Lu, W.; Huang, Y.; Lu, H. Dimethomorph induces heart and vascular developmental defects by disrupting thyroid hormone in zebrafish embryos. Ecotoxicology and Environmental Safety 2025, 289, 117413, doi:https://doi.org/10.1016/j.ecoenv.2024.117413.

Wei, Y.; Meng, Y.; Huang, Y.; Liu, Z.; Zhong, K.; Ma, J.; Zhang, W.; Li, Y.; Lu, H. Development toxicity and cardiotoxicity in zebrafish from exposure to iprodione. Chemosphere 2021, 263, 127860.

 

6. “Due to the observed increased metabolic rate and oxygen consumption, which may lead to elevated ROS production, a rescue experiment was conducted using the strong antioxidant Astaxanthin (AST)”. However, no ROS assay (e.g., DCFH-DA staining) was performed.

The authors are grateful for reviewer’s input. In the current state, the authors have limited resources, therefore we are unable to perform the ROS assay as the reviewer have mentioned. However, we found a study that stated a proportional increase of ROS production with oxygen consumption, although it might be varies depending on mitochondrial respiratory rate. By providing an identical condition during the metabolic rate measurement and used zebrafish larvae from the same clutch per replication, we tried to keep then variation within a replication low, thus we expect the variation to only be caused from DIM treatment. In conclusion, we used total oxygen consumption to indirectly show ROS production due to DIM exposure in zebrafish larvae.

Hou, C.; Metcalfe, N.B.; Salin, K. Is mitochondrial reactive oxygen species production proportional to oxygen consumption? A theoretical consideration. BioEssays 2021, 43, 2000165, doi:https://doi.org/10.1002/bies.202000165.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I thank authors for the revised manuscript. My concerns are satisfactorily addressed and I have not further comments.

Author Response

I thank authors for the revised manuscript. My concerns are satisfactorily addressed and I have not further comments.

Thank you for your progessional comments

Reviewer 3 Report

Comments and Suggestions for Authors

Some parts of the authors’ responses to the reviewer’s comments are unclear. Therefore, the following issues should be addressed:

1) Figure Legends (Figures 2, 3, 4, and 5):

1. Please specify which normality test was used in each figure legend. It is unclear whether the authors applied multiple tests, such as the Anderson–Darling test, D’Agostino–Pearson test, Shapiro–Wilk test, or Kolmogorov–Smirnov test.

In addition, please describe the method used to assess normality (for example, whether an F-test or other statistical procedures were performed) in each figure legend.

1. Please provide detailed information on the number of biological replicates, independent experimental repeats, and the total number of measurements in each figure legend.

For example: each group consisted of 15 fish (biological replicates) for cardiovascular performance measurements, the experiment was conducted in duplicate (independent experimental repeats), resulting in a total of 30 measurements.

 

2) Figure 3:

Please include the mean ± standard deviation (SD) values at each time point (600, 1200, 1800, 2400, and 3000 seconds) for each group directly in Figure 3.

 

Author Response

Comments and Suggestions for Authors

Some parts of the authors’ responses to the reviewer’s comments are unclear. Therefore, the following issues should be addressed:

1) Figure Legends (Figures 2, 3, 4, and 5):

Please specify which normality test was used in each figure legend. It is unclear whether the authors applied multiple tests, such as the Anderson–Darling test, D’Agostino–Pearson test, Shapiro–Wilk test, or Kolmogorov–Smirnov test. In addition, please describe the method used to assess normality (for example, whether an F-test or other statistical procedures were performed) in each figure legend.

The authors appreciate the reviewer’s input. In accordance to the reviewer’s input, the authors had included the normality test used in the figure legend. The authors used Anderson-Darling test, D’Agostino & Pearson test, Shapiro-Wilk test, and Kolmogorov-Smirnov test as normality test (multiple test). As the data passed normality test (α = 0.05), the authors decided to use one-way ANOVA/two-way ANOVA depending on the data type as F-test to observe statistical difference, additionally to compare statistical difference between treatment group, Dunnet’s multiple comparison test (compare against select group (control)) and Tukey’s multiple comparison test (compare all possible pairwise combination) was used depending on the necessity. This information has been included in the figure legend as requested by the reviewer.

Please provide detailed information on the number of biological replicates, independent experimental repeats, and the total number of measurements in each figure legend. For example: each group consisted of 15 fish (biological replicates) for cardiovascular performance measurements, the experiment was conducted in duplicate (independent experimental repeats), resulting in a total of 30 measurements.

The authors are thankful for the reviewer’s suggestion. The authors have included the information regarding biological replicates, experimental repeat, and total sample size in the manuscript according to reviewer’s suggestion. However, the authors would like to mention that the final n number might vary due to mortality during the exposure, which we have also mentioned in the figure legends.

2) Figure 3:

Please include the mean ± standard deviation (SD) values at each time point (600, 1200, 1800, 2400, and 3000 seconds) for each group directly in Figure 3.

The authors are grateful for the reviewer’s response. Following this confirmation, the authors have updated the time chronology figure in the manuscript with Mean ± standard deviation (SD).

 

Reviewer 4 Report

Comments and Suggestions for Authors

I recommend acceptance of this manuscript.

Author Response

Comments and Suggestions for Authors

I recommend acceptance of this manuscript.

Thank you for your professional comment

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

The revised manuscript has been improved. However, the following comments still need to be addressed:

1. Figure 2 legend:
   Please revise
   “As the data passed normality test (α = 0.05), one-way ANOVA with Dunnett’s multiple comparison post-hoc test was used to compare the variance between treatment group to control group” to “As the data passed the normality test (α = 0.05), one-way analysis of variance followed by Dunnett’s multiple comparison test was used to compare the means among groups.” Additionally, please change “Mean ± Standard Deviation (SD)” to “mean ± standard deviation”.

1. Figure 3 legend:
   Please change “two-way ANOVA” to “two-way analysis of variance (ANOVA)” and “Mean” to “mean”.
2. Figure 4 legend:
   Please change “ANOVA” to “analysis of variance” and “Mean” to “mean”.
3. Figure 5 legend:
   Please revise “one-way ANOVA was used to check the variance with Tukey’s multiple comparison test as post-hoc test to compare all possible pairwise combinations.”
   to “one-way analysis of variance was performed to compare the means among groups, followed by Tukey’s multiple comparison test for pairwise comparisons.” Additionally, please change “Mean” to “mean”.

Author Response

Reviewer 3

Comments and Suggestions for Authors

The revised manuscript has been improved. However, the following comments still need to be addressed:

2. Figure 2 legend:
Please revise “As the data passed normality test (α = 0.05), one-way ANOVA with Dunnett’s multiple comparison post-hoc test was used to compare the variance between treatment group to control group” to “As the data passed the normality test (α = 0.05), one-way analysis of variance followed by Dunnett’s multiple comparison test was used to compare the means among groups.” Additionally, please change “Mean ± Standard Deviation (SD)” to “mean ± standard deviation”.

The authors are grateful for the reviewer’s for the reviewer’s suggestion. The figure legends have been adjusted as suggested by the reviewer.

2. Figure 3 legend:
Please change “two-way ANOVA” to “two-way analysis of variance (ANOVA)” and “Mean” to “mean”.

The authors are grateful for the reviewer’s for the reviewer’s suggestion. The figure legends have been adjusted as suggested by the reviewer.

3. Figure 4 legend:
Please change “ANOVA” to “analysis of variance” and “Mean” to “mean”.

The authors are grateful for the reviewer’s for the reviewer’s suggestion. The figure legends have been adjusted as suggested by the reviewer.

4. Figure 5 legend:
Please revise “one-way ANOVA was used to check the variance with Tukey’s multiple comparison test as post-hoc test to compare all possible pairwise combinations.” to “one-way analysis of variance was performed to compare the means among groups, followed by Tukey’s multiple comparison test for pairwise comparisons.” Additionally, please change “Mean” to “mean”.

The authors are grateful for the reviewer’s for the reviewer’s suggestion. The figure legends have been adjusted as suggested by the reviewer.