Effects of Different Submerged Macrophytes on the Water and Sediment in Aquaculture Ponds with Enrofloxacin Residues
Round 1
Reviewer 1 Report
In this study the authors investigated different submerged macrophytes to restore the aquaculture ponds with enrofloxacin residues in the sediment. The manuscript fits within the scope of the journal, the subject is relevant, and the work is on a good level. Moreover, the data were submitted to statistical analysis.
Author Response
Thanks for your comments on this article. Your encouragement will support us to achieve better works.
Reviewer 2 Report
Overall, this study is interesting. The study is about using plant as treatment in the antibiotic contaminated aquaculture pond. my comment to the paper as follow:
Title: accepted
Abstract: if possible please provide method of study in brief
Introduction:
Elaborate the impacts of antibiotics specifically ENR to environment, aquatic organisms and etc
Methods:
If possible provide references in each section
Must identify the source of ENR in the sediment of aquaculture pond
Separate results and discussion
References
Must ensure 80% of references are within 5 years work
moderate
Author Response
We are very grateful to the reviewers for the suggestion and revision comments for the manuscript. All of the words and sentences modified are highlighted in the manuscript. The responses to the reviewers' comments and the necessary explanations are detailed as follows:
C1. Abstract: if possible please provide method of study in brief.
R1: The method of study was added in the Abstract in line 10-14 as “A microcosm experiment was carried out to study the interaction between three kinds of submerged macrophytes and their growing environment with antibiotic residues. Ceratophyllum demersum L.with no roots, Vallisneria spiralis L. with flourish roots and Hydrilla verticillata L with little roots were chosen to planted in the sediment added with enrofloxacin (ENR). The growth of submerged macrophytes, the changes of the overlying water and sediment characteristics, and the microbial community in the sediment were analyzed.”
C2. Introduction: Elaborate the impacts of antibiotics specifically ENR to environment, aquatic organisms and etc.
R2: According to the review’s suggestion, some contents about the impacts of antibiotics specifically ENR to environment and aquatic organisms were added in Introduction (Line 44-52).
C3. Methods: If possible provide references in each section.
R3: Reference “Nie et al. [20]” was added in the section “2.3 DNA extraction and high throughout sequencing”. Line 115-117 showed that “Detailed determination methods for water (TOC, TP, TN and NH4+-N) and sediment (TN, TP, TOC, ENR and CIP) samples were described in our previous study [14]”.
C4: Must identify the source of ENR in the sediment of aquaculture pond.
R4: We identify the source of ENR in the sediment in line 99-101 as “A 6 cm layer of sediment (about 7.5 kg of wet weight) was laid in each tank, and then 50 ml of ENR solution with a concentration of 100 mg/L was added and mixed with the sediment evenly”.
C5: Separate results and discussion.
R5: We are sorry that we followed our previous writing habits and put the “Results” and “Discussion” sections together. Due to the limited time for this revision, we didn’t separate them. But we are willing to try to write “Discussion” separately in our future works. Thanks for your tolerance.
C6: References Must ensure 80% of references are within 5 years’ work.
R6: Most of the references published 5 years ago have been replaced by newer ones. Now only 3 out of 40 references cited in the manuscript are the works 5 years ago.
Reviewer 3 Report
Please refer to the attached file for my reviewer report
Comments for author File: 
Comments.docx
There are several grammar or editing mistakes. Please check them carefully again, and it is better to polish this manuscript with someone proficient in English.
Author Response
We are very grateful to the reviewers for the suggestion and revision comments for the manuscript. All of the words and sentences modified are highlighted in the manuscript. The responses to the reviewers' comments and the necessary explanations are detailed as follows:
C1: There are several grammar or editing mistakes. Please check them carefully again, and it is better to polish this manuscript with someone proficient in English.
R1: We carefully checked the grammar and editing mistakes in the manuscript and corrected them as much as possible. We hope the revised manuscript can help readers understand this research.
C2. For readers to quickly catch your contribution, it would be better to highlight major difficulties and challenges, and your original achievements to overcome them, in a clearer way in abstract and introduction. Abstract should be informative and include the main findings.
R2: According your suggestion, some content was added in the Abstract in line 7-14 as “Submerged macrophyes are widely used to restore aquaculture ponds in recent years. But whether the residual antibiotics in ponds will affect the remediation effect of submerged macrophyes, and the effect of different submerged macrophyes on the water and sediment in aquaculture ponds with antibiotic residues are unclear. A microcosm experiment was carried out to study the interaction between three kinds of submerged macrophytes and their growing environment with antibiotic residues. Ceratophyllum demersum L.with no roots, Vallisneria spiralis L. with flourish roots and Hydrilla verticillata L with little roots were chosen to planted in the sediment added with enrofloxacin (ENR).”
C3. Keywords should include “sediment”.
R3: “Sediment” has been added as one of the Keywords in line 25.
C4. The authors can update their literature in the introduction with newer ones (2018-2023).
R4: Thanks for your valuable suggestion. Most of the references published 5 years ago have been replaced by newer ones. Now only 3 out of 40 references cited in the manuscript are the works 5 years ago.
C5: From the summary of the introduction of the manuscript, the idea of the article is good, which is worth affirming. However, the data of the article is too single, and the main line is not clear enough, especially the descriptive data in the article is too many, which makes the article look more like a survey report. I'm not sure whether the author's discussion is not deep enough to affect the readability of the article.
R5: Thanks for your comments on this manuscript again. Some content in the manuscript where the writing was indeed unclear and the discussion was insufficient. We have carefully revised the description of some results and added some discussions which are highlighted. We hope the manuscript can be better now.
C6: Moreover, the manuscript could be substantially improved by relying and citing more on recent literatures about contemporary real-life case studies on sustainability of water and/or sediment such as the followings:
R6: Thanks for your suggestion. According to the sections of “Introduction” and “Discussion”, we have cited more references published in recent 5 years to improve the quality of this manuscript.
C7: Please check the legends on the figures and/or tables for completeness, such as if values are means, SD or SE, n = replicates, and also the details and explanations of any statistical labels. Please check for some typo/grammar (few places).
R7: Thanks for your friendly suggestion. We carefully checked the legends, statistical labels and grammar mistakes in the manuscript and corrected them as much as possible. We hope the revised manuscript can be better now.
C8: In the conclusion section, the limitations of this study, suggested improvements of this work and future directions should be highlighted. Mention about limitations of your study if you have any in conclusion section.
R8: According to your suggestion, related content as “However, due to the limitations of the water depth in this study, the effect of submerged macrophytes on the removal of ENR was not clear. Further studies are needed to explore the ENR removal efficiencies by different submerged macrophytes and related mechanisms such as the effect of released oxygen and exudates from the roots on the removal of antibiotics” was added into the “Conclusion” section.
C9: There are too many descriptive words in the results and discussion part. It is obvious that the author's discussion is not deep enough. It is suggested that further in-depth discussion should be conducted rather than just describing the survey results. Obviously, the interpretation of data should not only stay on the surface but should dig deeper conclusions from a broader perspective.
R9: Thanks for your valuable suggestion. According to the section of “Results and Discussion”, we have made every effort to modify and improve it by citing and comparing with more references published in recent 5 years. We hope the quality of the revised manuscript can be better.
Author Response File: Author Response.docx
Reviewer 4 Report
The paper has novel findings however the following issues must be addressed carefully:
Generally, the manuscript is well-written with updated references. However, I suggest searching again and citing related published papers in high-quality journals (regarding three used plants only).
Some experimental images must be added (Design of tanks, plants in different stages, their root, and more details.
The study location should be shown on the map.
Why authors don't have any imaging analysis from the roots of used plants?
In discussion, authors can add more comparisons with similar studies to show the importance of their findings.
Add some quantitative results to the conclusion section.
Author Response
We are very grateful to the editors and reviewers for the suggestion and revision comments for the manuscript. All of the words and sentences modified are highlighted in the manuscript. The responses to the reviewers' comments and the necessary explanations are detailed as follows:
C1: Generally, the manuscript is well-written with updated references. However, I suggest searching again and citing related published papers in high-quality journals (regarding three used plants only).
R1: Thanks for your suggestion. We have searched again and cited more references with the same submerged macrophytes (such as reference [24], [26] and [36]) published in recent 5 years to improve the quality of this manuscript.
C2: Some experimental images must be added (Design of tanks, plants in different stages, their root, and more details).
R2: According to your suggestion, “Figure 1. Schematic of the experimental set-up” has been added in section “2.1 Experimental mesocosms” to provide readers with a clearer understanding of the experimental design in this study.
C3: The study location should be shown on the map.
R3: This study is not a field study, but a microcosm experiment conducted indoors. The supplementary introduction of the study location is added in line 88 as follows “The experiment was carried out in a transparent glass greenhouse located in the downtown area of Shanghai, China”. If you still think this paper need a map, we can consider supplementing it again.
C4: Why authors don't have any imaging analysis from the roots of used plants?
R4: We are sorry that we really don’t have any imaging analysis of the roots. We have added “Figure 1. Schematic of the experimental set-up” into section “2.1 Experimental mesocosms” to provide readers with a clearer understanding of the experimental design in this study.
C5: In discussion, authors can add more comparisons with similar studies to show the importance of their findings.
R5: Thanks for your valuable suggestion. We have added more comparisons with similar studies, such as reference [24-27], [31] and [36]. We hope the revised manuscript can be better than before.
C6: Add some quantitative results to the conclusion section.
R6: Some quantitative results were added to the conclusion section in line 323-326 as follows “The TOC, TP, NH4+-N and TN concentrations in the overlying water were decreased by V. spiralis with 25.0%, 71.7%, 38.1% and 24.8%, respectively. The planting of V. spiralis also increased the richness and diversity of the microorganisms in the sediment with 21.7% for the number of OUTs, 17.8% for Chao1 index and 19.3% for ACE index”.
Author Response File: Author Response.docx
Reviewer 5 Report
The study aimed to the effect of different submerged macrophytes growth on the enrofloxacin residus in the sediment in aquaculture pomds. There showed good removal effect of V. spiralis for the water quality, such as TP. And the conclusion showed that V. spiralis was more suitable for the remediation of the aquaculture ponds with enrofloxacin residue. However, I could not find any evidence for this conclusion. Although this manuscript is presenting an interesting method, I think it still needs some major revisions. There are many deficiencies in the article.
1. Abstract: There are many results on TOC, N and P, even microorganisms in the sediments. Few results were showen on the impact of submerged macrophytes on enrofloxacin removal. Obviously, this deviates from the study objectives.
2. The experimental environmental parameters were lack, like temperature, light etc.. The experiment lasted for over three months. How to replenish water during the experiment? How to detect enrofloxacin in sediment?
3. What was the growth status of submerged macrophytes during the experiment? I think there shoule be added some figures.
4. Will enrofloxacin be released into overlying water? The enrofloxacin characteristics in the water effected the submerged macrophytes should be added.
5. For the total removal efficiencies of enrofloxacin, the removal ability of Control was the highest for the degradation of enrofloxacin. The should be indicated that the role of submerged macrophytes was limited. How to correspond to the objectives of the study?
6. I think that the results of this paper are not sufficient to support the restore of enrofloxacin in pond sediment by submerged macrophytes. The title of this paper should be reconsidered.
Further improvement is needed.
Author Response
We are very grateful to the editors and reviewers for the suggestion and revision comments for the manuscript. All of the words and sentences modified are highlighted in the manuscript. The responses to the reviewers' comments and the necessary explanations are detailed as follows:
C1: The conclusion showed that V. spiralis was more suitable for the remediation of the aquaculture ponds with enrofloxacin residue. However, I could not find any evidence for this conclusion.
R1: Among the three kinds of submerged macrophytes, V. spiralis had the highest growth rate, N and P accumulation and P removal ability in the aquaculture ponds with ENR residues. It also increased the richness and diversity of the microorganisms in the sediment with 21.7% for OUTs number, 17.8% for Chao1 index and 19.3% for ACE index. This was also the highest among the three kinds of submerged macrophytes. So we concluded that V. spiralis was more suitable for the remediation of the aquaculture ponds with enrofloxacin residue.
C2: Abstract: There are many results on TOC, N and P, even microorganisms in the sediments. Few results were showen on the impact of submerged macrophytes on enrofloxacin removal. Obviously, this deviates from the study objectives.
R2: We have explained it in the section “Conclusion” as “Due to the limitations of the water depth in this study, the effect of submerged macrophytes on the removal of ENR was not clear. Further studies are needed to explore the ENR removal efficiencies by different submerged macrophytes and related mechanisms such as the effect of released oxygen and exudates from the roots on the removal of antibiotics”. After careful consideration, we also accepted your suggestion and revised the title of the manuscript.
C3: The experimental environmental parameters were lack, like temperature, light etc. The experiment lasted for over three months. How to replenish water during the experiment? How to detect enrofloxacin in sediment?
R3: We added the method of water replenishment and water temperature as follows “Then the tanks were filled with tap water and which was replenished weekly to compensate for the evaporated water. During the experimental period, the water temperature was between 19.2 and 33.9 oC with an average value of 26.8 oC” in line 104-106. This experiment was carried out in a transparent glass greenhouse under natural light. So we didn't measure the light intensity. The detection method of enrofloxacin in sediment was cited one of our previous papers (Zhang et al., 2019), in which ENR and CIP concentrations of the sediment were measured using solid phase extraction (SPE)-high performance liquid chromatography (HPLC, Agilent 1100, USA).
C4: What was the growth status of submerged macrophytes during the experiment? I think there shoule be added some figures.
R4: According to your suggestion, “Figure 1. Schematic of the experimental set-up” has been added in section “2.1 Experimental mesocosms” to provide readers with a clearer understanding of the experimental design in this study.
C5: Will enrofloxacin be released into overlying water? The enrofloxacin characteristics in the water effected the submerged macrophytes should be added.
R5: Thanks for your valuable suggestion. We have tried to measure the ENR concentrations in the overlying water. But 2L sample was needed for one measurement. And we may need to measure multiple times. The total capacity of the tank was about 80L. We concerned the amount of water samples needed would be too large to affect the experiment. So we gave up measuring the concentration of ENR in the overlying water. On the other hand, the concentration of ENR in the overlying water was low, the photodegradation by natural light may make it difficult for us to detect it.
C6: For the total removal efficiencies of enrofloxacin, the removal ability of Control was the highest for the degradation of enrofloxacin. The should be indicated that the role of submerged macrophytes was limited. How to correspond to the objectives of the study?
R6: We have explained it in line 231-236 as “Lin et al. [29] reported that natural irradiation plays a major role in the degradation of ENR and CIP in the water and sediment, with biodegradation playing just a minor role. The depth of the water in this study was no more than 60 cm, which may facilitate the photodegradation of ENR and CIP in the sediment of Control. The covering of submerged macrophytes hindered the photodegradation of ENR and CIP in the sediment of the three planted treatments”.
C7: I think that the results of this paper are not sufficient to support the restore of enrofloxacin in pond sediment by submerged macrophytes. The title of this paper should be reconsidered.
R7: Thanks for your suggestion. We revised the title of the manuscript to “Effects of different submerged macrophytes on the water and sediment in aquaculture ponds with enrofloxacin residues”. We hope this title would be better
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
The authors has taken the serious effort to revise the manuscript. Now the paper has improved significantly.
Author Response
Thanks for your comments on the revised manuscript.
Reviewer 4 Report
All my comments have been addressed. only one issue needs to be considered:
Figure 1: it should be real experimental images not schematic.
Author Response
Thanks for your suggestion. Now we provide the real experimental image in the revised manuscript. But we are not sure whether its better than the schematic. We are very willing to follow your and the editor's suggestions and choose one to include in the manuscript.
Author Response File: Author Response.docx
Reviewer 5 Report
The revised manuscript has been improved followed the commnets of reviewer. There was less results referes to the ENR residus affected by different submerged macrophytes. The obvious conclusions should be presented in the abstract. There is no experimental set-up without ENR in sediment?
Author Response
We have added the content "The effects of the three submerged macrophytes on the removal of ENR from sediment was not reflected due to the limitation of water depth" in red into the Abstract.
In one of our previous studies (Zhang et al, 2019) which was cited in this paper, we compared the effects of different ENR concentrations in the sediment on the plant growth, water quality and microbial community. In that paper, the Control with no ENR added into the sediment was set up. But in this paper ,We just wanted to compare different plant species on the aquaculture ponds with ENR. So the Control was set up with ENR but no plants in this paper.
Author Response File: Author Response.docx
