Short-Term Toxicity of Lanthanum to Embryonic and Yolk-Sac Stage Larvae of the Rare Minnow Gobiocypris rarus Ye & Fu, 1983

Round 1

Reviewer 1 Report

1. The whole manuscript needs English editing

2. the list of abbreviations should be added

3. The experimental design should more clear and more concise. the authors should use a graphical abstract in experimental design.

4. Please, write in detail about the detection of  the heart rate of embryos

5. The methods used should be more details about each parameter

6. The discussion section should be rewritten again, focusing in the authors' findings and its interpretation

Author Response

Comments and Suggestions for Authors

1. The whole manuscript needs English editing.

Response: Thanks for your review. The original manuscript has been revised by International Science Editing. We're sorry to cause your confusion. If the language does not meet your requirements, could you recommend a language editing company? Thank you very much.

2. the list of abbreviations should be added.

Response: Thank you for your patience review and valuable suggestions. We have added it in the revised manuscript. Please see it in the page 9.

3. The experimental design should more clear and more concise. the authors should use a graphical abstract in experimental design.

Response: Thanks a lot. We have added it in the revised manuscript. Please see 2.3.2 line 19.

4. Please, write in detail about the detection of the heart rate of embryos.

Response: We appreciate the reviewer for this important comment. We have added it in the revised manuscript. Please see 2.3.2 lines 6-8.

5. The methods used should be more details about each parameter.

Response: Thank you for your patience review and valuable suggestions. We have revised it in the revised manuscript. Please see 2.3.1 lines 4-5 and 2.3.2 lines 6-8 and 14-15.

6. The discussion section should be rewritten again, focusing in the authors' findings and its interpretation.

Response: Thank you for your patience review and valuable suggestions. We reconsidered our findings and made reasonable explanations based on previous relevant studies. Please see discussion, paragraphs 3-5.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors, I found your manuscript quite interesting and focused on a topic poorly treated that deserves to be deepened. Despite this, I have some doubts related to the value of this manuscript in the present form, that in my opinion is not ready for publication in Water Journal. The manuscript is too synthetic in my opinion in some very essential parts, such as the discussion and conclusion section, not giving resonance to your results. Moreover, the reference list is consequently really short and not well focused on the topic and the treated organisms, it needs to be improved.

I briefly resumed all my comments and suggestion in detail as follow.

Considering that the manuscript format does have not the line numbers, I have to report your period or sentences to avoid misunderstanding.

Title

Please add the references after the scientific name (Ye & Fu, 1983). This suggestion is also valid for the first species mentioned in the main text.

Abstract

The abstract section is too short in my opinion. Considering that you are within the limit of words of the journal, a few sentences more in the introductory and discussion part could be added. Please absolutely avoid the use of the terms obvious/obviously, nothing in scientific research is obvious, at least could be expected (also valid for the conclusion section).

Keywords

Please try to avoid the repetition of words already present in the title, to give more resonance to your manuscript.

Introduction

"For example, reeaearchers recently reported that the contents of total dissolved REEs (14 elements) in Taihu Lake ranged from 46.86 to 112.50 ng/L [7]. Data clearly show that REE pollution exists, and its toxicity to aquatic organisms requires further study". Do the authors provide this report as the natural standard concentration of the REEs? What is the standard concentration in nature of these elements, and when is it considerable pollution? (Fix grammatical errors in researchers)

"The early life stages of fish are more sensitive to contaminants than the juvenile and adult stages. Thus, studies of the effect of La on the early development of fish species is critical to understanding the toxicity of this element and to identifying the optimal dosage of La as a feed additive." What is, if exists, the direct relation between La content in feed and its relapse in water environments? Please better argue this topic, that you have also reported among the aim of your study.

Material and Methods

"A 100 mg/L La (III) stock solution was prepared in double distilled water at the beginning of the experiment and stored in the dark at 4°C until use in this experiment." Can the authors support this method with some references that show the stability of this stock solution in time?

"During the experimental period, the photoperiod was maintained at 12L:12D from 08:00 to 20:00" Is this a normal practice for this species in captivity?

"The short-term toxicity assay of embryonic and sac-fry stages was carried out accord- ing to the Organisation for Economic Cooperation and Development (OECD) guideline 212" and "Daily observations of abnormal development including yolk sac edema and pericar- dial edema, and mortality were performed based on the lethal and sublethal toxicological endpoints provided by OECD guidelines and on previously published methods." Please provide references.

"buffered ricaine methanesulfonate" Ricaine?

"2.3.2. Signs of embryonic and sac-fry stage toxicity." Is not clear from the main text if the eggs/larvae were replaced in the experimental solution to continue the exposure after the controls, or were discarded.

Results

"However, none of the embryos in the groups exposed to 1.00 and 1.92 mg/L La (III) survived after 72 h." This sentence is repeated two times in the first part of 3.1.

Figure 4. Please try to improve the quality of the images.

Discussion and Conclusion

Both these sections need more care, in the present form are in my opinion too synthetic and does not help the study to have soundness for other researchers. Please try to better discuss your results in comparison with other studies related to fishes early life stage and REEs exposure.

See for example:

Malhotra, Nemi, et al. "An updated review of toxicity effect of the rare earth elements (REEs) on aquatic organisms." Animals 10.9 (2020): 1663.

Dubé, M., et al. "Gene expression changes and toxicity of selected rare earth elements in rainbow trout juveniles." Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 223 (2019): 88-95.

Hanana, H., et al. "Insights on the toxicity of selected rare earth elements in rainbow trout hepatocytes." Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 248 (2021): 109097.

Qiang, Tu, et al. "Bioaccumulation of the rare earth elements lanthanum, gadolinium and yttrium in carp (Cyprinus carpio)." Environmental pollution 85.3 (1994): 345-350.

Yang, Luping, et al. "Residual levels of rare earth elements in freshwater and marine fish and their health risk assessment from Shandong, China." Marine Pollution Bulletin 107.1 (2016): 393-397.

Best regards

The Reviewer

Author Response

Comments and Suggestions for Authors

Dear authors, I found your manuscript quite interesting and focused on a topic poorly treated that deserves to be deepened. Despite this, I have some doubts related to the value of this manuscript in the present form, that in my opinion is not ready for publication in Water Journal. The manuscript is too synthetic in my opinion in some very essential parts, such as the discussion and conclusion section, not giving resonance to your results. Moreover, the reference list is consequently really short and not well focused on the topic and the treated organisms, it needs to be improved.

I briefly resumed all my comments and suggestion in detail as follow.

Considering that the manuscript format does have not the line numbers, I have to report your period or sentences to avoid misunderstanding.

Title

Please add the references after the scientific name (Ye & Fu, 1983). This suggestion is also valid for the first species mentioned in the main text.

Response: Thanks a lot. We have revised it in the revised manuscript. Please see the title.

Abstract

The abstract section is too short in my opinion. Considering that you are within the limit of words of the journal, a few sentences more in the introductory and discussion part could be added. Please absolutely avoid the use of the terms obvious/obviously, nothing in scientific research is obvious, at least could be expected (also valid for the conclusion section).

Response: Thank you for your valuable suggestion. We have added and revised it in the revised manuscript. Please see abstract.

Keywords

Please try to avoid the repetition of words already present in the title, to give more resonance to your manuscript.

Response: Thanks. Done. Please see the part of keywords.

Introduction

"For example, reeaearchers recently reported that the contents of total dissolved REEs (14 elements) in Taihu Lake ranged from 46.86 to 112.50 ng/L [7]. Data clearly show that REE pollution exists, and its toxicity to aquatic organisms requires further study". Do the authors provide this report as the natural standard concentration of the REEs? What is the standard concentration in nature of these elements, and when is it considerable pollution? (Fix grammatical errors in researchers)

Response: Thank you for your patience review and valuable suggestions. Rare earth elements are not an essential element for humans. And the concentration of REEs in natural seawater is only 0.01 μg/L [1]. But with the widespread use of REEs, the concentration of REEs in natural water is dozens or even hundreds of times compared with the normal levels. In addition, dilation of blood capillaries, congestion of liver sinusoids, and presence of irregularly-shaped hepatocytes occurred in rare minnow after exposed to 0.04 mg/L La (III) for 21 d [2]. Thus, we use the word pollution in our manuscript.

We have modified the grammatical errors in the revised manuscript. Please see Introduction, paragraph 1, line 13.

Reference:

[1] Elderfield, H., Greaves, M.J. The rare earth elements in seawater. Nature. 1982, 296, 214-219.

[2] Dong, Hua, Jianwei, Wang, Denghang, & Yu, et al. Lanthanum exerts acute toxicity and histopathological changes in gill and liver tissue of rare minnow (gobiocypris rarus). Ecotoxicology (London, England). 2017, 26:1207–1215.

"The early life stages of fish are more sensitive to contaminants than the juvenile and adult stages. Thus, studies of the effect of La on the early development of fish species is critical to understanding the toxicity of this element and to identifying the optimal dosage of La as a feed additive." What is, if exists, the direct relation between La content in feed and its relapse in water environments? Please better argue this topic, that you have also reported among the aim of your study.

Response: We appreciate the reviewer for this important comment. The original intention of us is that the significance of this study is to provide a theoretical basis for further research on rare earth elements as feed additives. And we have revised it in the revised manuscript. Please see Introduction, paragraph 2, line 12-14.

Material and Methods

"A 100 mg/L La (III) stock solution was prepared in double distilled water at the beginning of the experiment and stored in the dark at 4°C until use in this experiment." Can the authors support this method with some references that show the stability of this stock solution in time?

Response: Rare earth elements are metals. They are relatively stable. Liu et al. found that the nanogram/gram levels of rare earth elements in wheat flour was stable. In addition, this manipulation also appeared in Trifuoggi et al.’s study of the toxic effects of some rare earth elements on the early life stages of sea urchin Paracentrotus lividus.

Reference:

[1] Liu H.S., Wang X.Y., Wang N.F. Determination of ng/g rare earth elements in human hair and wheat flour by ICP-MS. PTCA (PART B:CHEMICAL ANALYSIS). 2001, 37(8):1.

[2] Trifuoggi M., Pagano, M., Guida, G., Palumbo, M., Siciliano, A., & Gravina, A., et al. Comparative toxicity of seven rare earth elements in sea urchin early life stages. Environmental Science & Pollution Research. 2017, 24:20802-20810.

"During the experimental period, the photoperiod was maintained at 12L:12D from 08:00 to 20:00" Is this a normal practice for this species in captivity?

Response: Thanks a lot. Feeding and management standard for rare minnow has been established.  The standard stipulates that the light/dark ration of rare minnow is 12 h:12 h. To make it easier to turn off the lights, we choose 8:00 to 20:00. This standard has been preliminarily completed and are prepared for local standard in Hubei province. The first author Liangxia Su is honored to participate in the formulation of this standard.

"The short-term toxicity assay of embryonic and sac-fry stages was carried out accord- ing to the Organisation for Economic Cooperation and Development (OECD) guideline 212" and "Daily observations of abnormal development including yolk sac edema and pericar- dial edema, and mortality were performed based on the lethal and sublethal toxicological endpoints provided by OECD guidelines and on previously published methods." Please provide references.

Response: Thanks a lot. Done. Please see 2.3.1 line 3 and 2.3.2 line 3.

"buffered ricaine methanesulfonate" Ricaine?

Response: We are sorry for our careless mistake in the statement. This is a clerical error. We have modified it in the revised manuscript. Please see part of 2.3.2, lines 8-9.

"2.3.2. Signs of embryonic and sac-fry stage toxicity." Is not clear from the main text if the eggs/larvae were replaced in the experimental solution to continue the exposure after the controls, or were discarded.

Response: We are sorry for confusing you. During the experimental period, the observed embryos/larvae were returned to the cylindrical glass containers for further observation. We have added it in the revised manuscript. Please see part of  2.3.2, lines 12-14.

Results

"However, none of the embryos in the groups exposed to 1.00 and 1.92 mg/L La (III) survived after 72 h." This sentence is repeated two times in the first part of 3.1.

Response: We are sorry for our careless mistake in the statement. And we have modified it in the revised manuscript. Please see part of 3.1.

Figure 4. Please try to improve the quality of the images.

Response: Thank you for your patience review and valuable suggestions. Due to the low resolution of the camera instrument used in this experiment, the resolution of our picture is not high.  But the experiment has ended, we are sorry that we can't make reshoots.  In future experiments, we will pay attention to this problem and correct it.

Discussion and Conclusion

Both these sections need more care, in the present form are in my opinion too synthetic and does not help the study to have soundness for other researchers. Please try to better discuss your results in comparison with other studies related to fishes early life stage and REEs exposure.

See for example:

Malhotra, Nemi, et al. "An updated review of toxicity effect of the rare earth elements (REEs) on aquatic organisms." Animals 10.9 (2020): 1663.

Dubé, M., et al. "Gene expression changes and toxicity of selected rare earth elements in rainbow trout juveniles." Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 223 (2019): 88-95.

Hanana, H., et al. "Insights on the toxicity of selected rare earth elements in rainbow trout hepatocytes." Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 248 (2021): 109097.

Qiang, Tu, et al. "Bioaccumulation of the rare earth elements lanthanum, gadolinium and yttrium in carp (Cyprinus carpio)." Environmental pollution 85.3 (1994): 345-350.

Yang, Luping, et al. "Residual levels of rare earth elements in freshwater and marine fish and their health risk assessment from Shandong, China." Marine Pollution Bulletin 107.1 (2016): 393-397.

Response: We appreciate the reviewer for this important comment. We have carefully read these literatures you recommended, and revised the part of discussion. Due to the lack of research on the toxic effects of lanthanum on the early life stage of fish species, we have to analyze our results from the studies on the toxic effects of lanthanum on adults and the mechanisms of toxic effect of other hazardous chemicals on the early life stage of fish species. Please see Discussion, paragraphs 3-5.

Author Response File: Author Response.pdf

Reviewer 3 Report

This MS investigates the effect of various concentrations of La on embryonic development in the rare minnow. It is well-written and deserves to be published. Since it’s a toxicology paper, it should use the relevant terms when discussing exposure and response, e.g., calculate LC50 for La; discuss acute exposure (at the 2 highest concentrations of La) versus chronic effects at lower doses. E.g., Intro paragraph 2, line 3-4: what concs of La were used in these studies (acute vs chronic). 

Can you please comment on the concs of La you chose for your experiment? They are all quite high relative to the values found in the environment in China that you reported in the intro. Eg., your lowest tested value was 0.06 mg/L, while the maximum value found in aquatic environments in the mining area was 0.12 mg/L. I’m wondering why more values weren’t tested between 0 and 0.06 mg/L (e.g., 0.01 and 0.02 mg/L), given the ug/L values found in the environment. This would have helped nail down a more accurate La LC50 value.

In many of the figures, not all 6 concentrations of La are included in the results (0-1 mg/L). E.g., in fig. 2, there is no 1 mg/L; in fig. 5, the top 3 values are missing. State why these data are missing, e.g., perhaps because higher concentrations elicited acute effects.

Specific comments below should also be addressed in the revised version.

Intro line 7: …Province mining area… presumably this is in the aquatic environment there? Specify where (rivers, lakes etc.).

Intro last line paragraph 1: give some context for the levels of REEs in the environment. What are the China, EU, US regulatory levels for these elements? How many fold above these levels are those observed? Also 2.3.2: what are the OECD lethal (acute) and sublethal (chronic?) endpoints?

Intro paragraph 2, line 5: need a reference for the cyanobacterial study.

Intro last para page 1: Give background on the use of this fish in aquaculture. Is it consumed directly by people or is it used to feed other farmed fish?

Intro 2nd last paragraph: 

• “Toxic effects”: were the 2 previous studies using chronic or acute levels of La? 

• unknown, not unknow 

• 0 to 3 C: this seems like a narrow temp tolerance range. Should this be 0 to 30 C, given that the fish were cultured at 23 C for this study?

2.3.1: 

• what is the previously determined LC50 value for La?

• Monitored using a dissecting microscope?

2.3.2

• Give concs of MS222 and sodium bicarbonate used for anaesthesia

2.4 and throughout section 3: Use the terms “% hatch” and “% survival” and not “hatching/survival rate”. A rate is a change in something versus time; what you are showing is a count = % of control at a given time.

3.1: “At 148 h, all larvae…”: this should be “At 120 h…”

Fig 4: for visual observations reported, what concentration of La did you use? What was exposure length?

4. Para 2 line 5: these experiments are not “short-term exposure to La”; the eggs are exposed to La for their entire developmental period, from fertilization through hatching. A toxicology term like “chronic exposure” is more accurate here.

4 paragraph 4: the link between high La levels and inhibited “hatching enzyme” activity is speculative and needs to be better supported by the literature. E.g., name a key “hatching enzyme” and give evidence from the literature that its activity and effect on hatch rate is inhibited by the La concentrations used in your study.

4 para 5: again need to use appropriate toxicology terms: instead of “killed”, say “was acutely toxic to”. Also 5: not “obvious lethal effects” but “was acutely toxic to”. What is 10^-5 M La expressed as mg/L as in your study? This makes comparison of your results to the Oral et al. urchin study easier.

5 Conclusions: Based on your results, state what the maximum conc of La should be in feed for farmed rare minnows.

Author Response

Reviewer 3

Comments and Suggestions for Authors

This MS investigates the effect of various concentrations of La on embryonic development in the rare minnow. It is well-written and deserves to be published. Since it’s a toxicology paper, it should use the relevant terms when discussing exposure and response, e.g., calculate LC50 for La; discuss acute exposure (at the 2 highest concentrations of La) versus chronic effects at lower doses. E.g., Intro paragraph 2, line 3-4: what concs of La were used in these studies (acute vs chronic). 

 Response: Thank you for your review and important suggestions. As suggested by the reviewer, we have analyzed and discussed these questions, and then revised our manuscript. We hope the revised manuscript will meet the acceptance requirements of reviewer and Water.

Can you please comment on the concs of La you chose for your experiment? They are all quite high relative to the values found in the environment in China that you reported in the intro. Eg., your lowest tested value was 0.06 mg/L, while the maximum value found in aquatic environments in the mining area was 0.12 mg/L. I’m wondering why more values weren’t tested between 0 and 0.06 mg/L (e.g., 0.01 and 0.02 mg/L), given the ug/L values found in the environment. This would have helped nail down a more accurate La LC50 value.

 Response: Thank you for your valuable suggestions. When our team previously studied the genotoxicity of lanthanum on the adult rare minnow, we found that the 0.4 mg/L lanthanum exposed group had little effect on it, but 0.08 mg/L lanthanum exposed group had a significantly effect on it. Therefore, the minimum concentration used in our experiments was 0.06 mg/L. This is a small omission in our experimental design, we will correct it in subsequent experiments.

In many of the figures, not all 6 concentrations of La are included in the results (0-1 mg/L). E.g., in fig. 2, there is no 1 mg/L; in fig. 5, the top 3 values are missing. State why these data are missing, e.g., perhaps because higher concentrations elicited acute effects.

Response: Thank you for your patience review and valuable suggestions. For figure 2, we found that the development of embryos in the 1 mg/L La (III) exposure group was about 6 h later than that in the control group. Thus, there was no result about spontaneous movement frequency of rare minnow embryos at 38 h of exposure in the 1 mg/L La (III) exposure group. For figure 5, the results in this section were about body length and body weight of rare minnow larvae at the end of the experiment.  Data were not available because all larvae in these three exposed groups died at the end of the experiment. Because the survival of rare minnow was detailed in Table 1, I did not describe them in the following results.

Specific comments below should also be addressed in the revised version.

Intro line 7: …Province mining area… presumably this is in the aquatic environment there? Specify where (rivers, lakes etc.).

Response: We are sorry for confusing you. That specific location is Wojiang river. We have added it in the revised manuscript. Please see Introduction, paragraph1, line 8.

Intro last line paragraph 1: give some context for the levels of REEs in the environment. What are the China, EU, US regulatory levels for these elements? How many fold above these levels are those observed? Also 2.3.2: what are the OECD lethal (acute) and sublethal (chronic?) endpoints?

Response: Thanks. Normally, the concentration of rare earth elements in natural seawater is only 0.01 μg/L. But, due to the wide use of rare-earth elements in China for aquacultural purpose and many other applications, the content of REEs increased dozens or even hundreds of times. The description of this part has been slightly revised. The OECD lethal endpoint is death, so we can calculate the LC₅₀. The sublethal endpoint is malformation, mortality, and growth of fish species.

Intro paragraph 2, line 5: need a reference for the cyanobacterial study.

 Response: Thanks a lot. Done. Please see introduction, paragraph 2, line 6.

Intro last para page 1: Give background on the use of this fish in aquaculture. Is it consumed directly by people or is it used to feed other farmed fish?

 Response: Thanks a lot. Rare minnow is a model animal. Currently, it is the only native Chinese fish species recommended by the National Standard of China (GB/T29763-2013) for use in biological tests of aquatic environment. This is described in paragraph 3. In addition, it is a small animal. And it has o edible value.

Intro 2nd last paragraph: 

• “Toxic effects”: were the 2 previous studies using chronic or acute levels of La? 
• Response: Thanks a lot. The previous studies have looked at chronic toxic effects in adult fish. We have revised it in the revised manuscript. Please see Introduction, paragraph 3, line 5.
•  
• unknown, not unknow 
• Response: Thanks. Done. Please see Introduction, paragraph 3, line 7.
•  
• 0 to 3 C: this seems like a narrow temp tolerance range. Should this be 0 to 30 C, given that the fish were cultured at 23 C for this study?
• Response: We are sorry for our careless mistake in the statement. And we have modified it in the revised manuscript. Please see Introduction, paragraph 3, line 2.
•  

2.3.1: 

• what is the previously determined LC50 value for La?
• Response: Thanks. The 96h LC₅₀ for adult rare minnow is 1.92 mg/L. We have added it in the revised manuscript. Please see 2.3.1, lines 4-5.
•  
• Monitored using a dissecting microscope?
• Response: Yes. Because the egg diameter of the embryo and the total length of the new hatched larvae are 1.3-1.6 mm and 4-5 mm, respectively, the embryos and larvae can only be observed through a dissecting microscope. The reason for not choosing a microscope is that the dissecting microscope is convenient for observation and does not cause damage to the embryo or larvae.
•  

2.3.2

• Give concs of MS222 and sodium bicarbonate used for anaesthesia
• Response: Thanks a lot. We have added it in the revised manuscript. Please see 2.3.2, lines 8-9.
•  

2.4 and throughout section 3: Use the terms “% hatch” and “% survival” and not “hatching/survival rate”. A rate is a change in something versus time; what you are showing is a count = % of control at a given time.

Response: Thanks. We are sorry for not understanding this very well. In fish species, there is a technical term for hatching rate and survival rate. To avoid confusing you, we have revised it to hatchability in the revised manuscript. Please see the revised manuscript.

3.1: “At 148 h, all larvae…”: this should be “At 120 h…”

Response: Thanks a lot. Done. Please see 3.1, line 4.

Fig 4: for visual observations reported, what concentration of La did you use? What was exposure length?

Response: Thank you for your patience review. Deformities in embryos and larvae were observed throughout the experimental period. Each exposed group will cause deformity to embryos and larvae. There is no difference between high and low concentrations of exposed solution in the form of deformity. In addition, the same deformed form will be produced in different exposure times. The results in this part suggest that lanthanum exposure can cause primarily egg condensation, melanin depletion, embryo autolysis, and eyespot disappearance in embryos and yolk sac edema, pericardium edema, deflated swim bladder, weak pigmentation, spine curvature, and body shortening in larvae. Thus, we did not write the exposure concentration and time in the paper.

4. Para 2 line 5: these experiments are not “short-term exposure to La”; the eggs are exposed to La for their entire developmental period, from fertilization through hatching. A toxicology term like “chronic exposure” is more accurate here.

 Response: Thanks. Done. Please see Discussion, paragraph 2, line 5.

4 paragraph 4: the link between high La levels and inhibited “hatching enzyme” activity is speculative and needs to be better supported by the literature. E.g., name a key “hatching enzyme” and give evidence from the literature that its activity and effect on hatch rate is inhibited by the La concentrations used in your study.

Response: Thank you for your patience review and valuable suggestions. Fish hatching is completed by breakage of the egg membrane under the action of chorionic enzymes hatching enzymes and twisting of the embryo. The hatching enzymes means chorionic enzymes. We have revised it in the manuscript. But we are sorry that we have not found relevant literature on the relationship between enzyme activity and lanthanum exposure on hatchability. Thus, we will consider this direction in the further research.

4 para 5: again need to use appropriate toxicology terms: instead of “killed”, say “was acutely toxic to”. Also 5: not “obvious lethal effects” but “was acutely toxic to”. What is 10^-5 M La expressed as mg/L as in your study? This makes comparison of your results to the Oral et al. urchin study easier.

Response: Thanks a lot. Done. Please see discussion, paragraph 5, line 1 and 3, and conclusion, lines 1-2.

5 Conclusions: Based on your results, state what the maximum conc of La should be in feed for farmed rare minnows.

Response: We are sorry for confusing in the Introduction.  The mainly purpose of this study is to explore the toxic effect of lanthanum on the early developmental stages of rare minnow. In addition, if there are researchers who want to explore lanthanum as an additive for rare minnow feed, this study can provide a theoretical basis for their research. We have revised our description in the revised manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Dear Authors, 

thank you for seriously revising your manuscript based on my previous suggestions.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

1. the 'Background' section should be rewritten to make it more compact and scientific in its approach. It is better not to use abbreviations in this section.
2. How the authors renewed the exposure solutions daily in order to maintain the test 83 concentrations
3. What is the type of food provided?
4. How the authors examined the data for homogeneity
5. How do the authors interpret the survival rate at conc 13
6. The introduction section is too short. Authors are suggested to update this section with recent information
7. Please list used in the biochemical assays in 'Chemicals' section
8. I suggest that the author’s used graph for the experimental design

Reviewer 2 Report

Although the type of study described in this manuscript probably falls within the general scope of the journal, an environmental science journal with a greater focus on aquatic toxicology or ecotoxicology may be a more appropriate outlet for the work.

There are some shortcomings with the study and there are weaknesses in the way the authors have presented their material. These include problems with the structure of the manuscript, and with the way in which the authors have presented their data.

There are problems with the description of the design, sampling protocols and analysis given in both the Abstract and M & Ms; the description is incomplete, the presentation is sometimes cumbersome and imprecise, and some of the information given is confusing.

There are shortcomings with the technical presentation, and several corrections and improvements are required.

Make sure that all works cited in the text are used correctly, that the presentation is consistent and that correct information is given in the reference list.

Define and explain all acronyms and abbreviations on first mention in the text, make sure that the presentation is correct and be consistent in the way that they are used.

On first mention of a species in the text give both the common (trivial) and formal names, and make sure that the presentation is correct and consistent; for example, compare the presentation given on lines 38-39, where the authority is included for one fish species but not the other.

Make sure that symbols, sub- and super-scripts, upper- and lower-case are presented correctly, and that there is correct and consistent use of italics, brackets, and punctuation etc.

The authors do not have complete command of English and the language is often cumbersome or imprecise. There are problems with choices of wording and phraseology, and there are many sentences in which there are syntax errors. The manuscript requires quite substantial linguistic changes and correction.

The title provides a reasonable indication of the nature of the work described in the manuscript.

All of the keywords are in the title so alternatives should be found: toxicity test, early life stages, rare-earth elements, ecotoxicology, cyprinid, larval malformations.

The Abstract gives insufficient information about the study design and sampling, and there are too few details about the findings. What was the treatment replication, how many eggs and larvae were exposed to each La dose within the test chambers, and what was the size of each chamber? What was the sampling regime; how often were the developing eggs and larvae observed, how many samples were taken, and how many eggs or larvae were taken from each test chamber to form a sample? What observations were made and registered, which metrics were calculated, and how were data analyzed to make treatment comparisons?

The presentation of the findings is cumbersome and disjointed. In addition, there is much that is imprecise because very little quantitative (numerical) information is given.

The Introduction contains some background information, but the presentation has a cumbersome and disjointed structure. There are problems with both sentence structure and choices of wording and phraseology, and there are abrupt transitions that give a text that lacks continuity and cohesion.

In addition, there are some problems with the use of sources. For example, the impression is given that some sources are overviews of a topic when they are not (e.g. lines 27, 29). There are several cases of dubious or incorrect citation of sources (e.g. line 29). The publication information given for several of the sources in the reference list is incomplete.

There are problems with the use of singular and plural, meaning that it is often difficult to see whether the authors are making general statements about REE or are referring directly to La, its occurrence in the environment and its potential negative effects.

Considerable re-structuring and re-writing of the Introduction will be required to correct the mistakes and improve the presentation.

The amount of information given about the M & Ms is variable, with some protocols described in sufficient detail, but insufficient information given about others. There are some problems with sentence structure and wording in the M & Ms that introduce same lack of clarity to the presentation. At times the presentation is so cumbersome and confusing that it is not possible to see exactly how sampling and observations were carried out.

On lines 63-67, the authors should probably write ‘Fertilized eggs were examined at 8hpf using a dissecting microscope (Olympus B41, Germany) and 630 eggs that appeared to be developing normally (i.e. without yolk coagulation or signs of deformation) were selected for study. The eggs were transferred to 600 mL (12 cm diameter) glass containers (Huaou Industry, Yanchang, China), with 30 eggs being put into each container’.

How were the different test concentrations prepared from the stock solution of La, and were the La concentrations in each container analyzed to check that they were as specified?

Were the developing eggs/larvae in each container examined every 24h for 168h, dead embryos (or larvae) recorded and removed, and numbers of newly-hatched yolk-sac larvae also recorded (section 2.3.1)?

How does the information given in section 2.3.2 relate to that given in section 2.3.1?

Were the 6 embryos from each container that were used for observation, placed back in their rearing container following the recording of movement and heart rate? For how long was each embryo observed to make the measurements? In section 2.3.1 it is stated that observations were made at 24h intervals, but on line 98 there is mention of sampling at 38h; this is confusing.

There is no information given as to exactly how the metrics were calculated; provide complete information, including the equations used for making the calculations.  

Given the problems with the study design and the presentation, including those relating to incomplete and confusing information about the M & Ms, it would be premature to provide detailed comments about the Results and Discussion sections of the manuscript.

There is some confusion in the way the authors have presented the results. For example, the survival rate data are given in table 1, but the table has no sampling time 148 h (line 136). Figure 1 gives hatching rate data, but the label on the Y axis is Survival rate.

Reviewer 3 Report

In the present study the authors have investigated the effects of an exposure to La3+ on eggs and larvae of rare minnow following a guideline 212. In this study they have revealed effects of the La3+ on the different parameters that were investigated. However, the present manuscript needs to be improved before being considered for publication.

1/ My first major comment is about the purpose of this study. In the introduction the authors are stating that one of the higher concentrations of La is about 8.26 microg/L, however in the present work the lower used exposure concentration is 60 microg/L. This around 8-time what might be measured. Why not working with concentrations in range with what is measured in the natural ecosystems?

2/ I would strongly suggest improving the description of the results, and mainly on the description of the stage when the observations are done. Does the exposure time refer to the very beginning when the eggs are exposed or after hatching? This is really confusing.

3/As last major concern the quality of the English as to be significantly improved.