The Use of Aquatic Macrophytes as a Nature-Based Solution to Prevent Ciprofloxacin Deleterious Effects on Microalgae

Round 1

Reviewer 1 Report

Manuscript number water-2380971

Title: The use of aquatic macrophytes as a nature-based solution to prevent ciprofloxacin deleterious effects to non-target organisms

 

The paper was submitted to section: Water and Climate Change

Special Issue:

 Environmental Feedbacks between Global Changes, Contaminants and Aquatic Plants

 

The paper presents the results of experimental studies focus on water plant responses to the different concentrations of antimicrobial ciprofloxacin. The research was conducted in terms of the phytoremediation potential of selected aquatic plant species.

 

The proposed subject is interesting and worth publishing. I have some comments which should be addressed before publication of the manuscript

 

I would consider changing the title. The term macrophytes should not be used. Macrophytes means vascular plants and multicelular algae, like charophytes which superficially resembling some higher plants. Although Salvinia molesta and Egeria densa belong to macrophytes, the third Desmodesmus subspicatus, represents phytoplankton.

 

2.2.1. Phytoremediation using  Aquatic Macrophytes

How long did the experiment last?

How the plant biomass (10 g plants L⁻¹) for the experiment was estimated?

Whether the loss of water due to evaporation has been replenished?

Please specify how many Cipro concentrations were tested

What was the initial biomass/cells numer of Desmodesmus subspicatus?

 

Table 2 is unreadable, it should be formatted

Comments for author File: Comments.docx

Author Response

We would like to thank the reviewer for all the valuable comments. They were very fruitful in improving the quality of our manuscript.

Comments

Title: The use of aquatic macrophytes as a nature-based solution to prevent ciprofloxacin deleterious effects to non-target organisms

 

The paper was submitted to section: Water and Climate Change

Special Issue:

 Environmental Feedbacks between Global Changes, Contaminants and Aquatic Plants

The paper presents the results of experimental studies focus on water plant responses to the different concentrations of antimicrobial ciprofloxacin. The research was conducted in terms of the phytoremediation potential of selected aquatic plant species.

The proposed subject is interesting and worth publishing. I have some comments which should be addressed before publication of the manuscript

I would consider changing the title. The term macrophytes should not be used. Macrophytes means vascular plants and multicelular algae, like charophytes which superficially resembling some higher plants. Although Salvinia molesta and Egeria densa belong to macrophytes, the third Desmodesmus subspicatus, represents phytoplankton.

We thank the reviewer for the comment. In order to clearly convey the aim of the paper, we have changed the title to “The use of aquatic macrophytes as a nature-based solution to prevent ciprofloxacin deleterious effects to microalgae”

2.2.1. Phytoremediation using Aquatic Macrophytes

How long did the experiment last?

Thank you for the comment. We included the experiment last in the text.

How the plant biomass (10 g plants L⁻¹) for the experiment was estimated?

Thank you for the comment. We included the description in the text

Whether the loss of water due to evaporation has been replenished?

Thank you for the comment. All Erlenmeyer flasks were stoppered with cotton wool to avoid both evaporation and contamination. This information was added to the text.

Please specify how many Cipro concentrations were tested

Thank you for the comment. The Cipro concentrations were added

What was the initial biomass/cells numer of Desmodesmus subspicatus?

Thank you for the comment. The initial, and not the final (as previously stated in the text) cell number were 1 x 10⁶ cells mL⁻¹. We corrected the text.

Table 2 is unreadable, it should be formatted

Thank you for the comments. The table was fixed.

 

 

Reviewer 2 Report

I have reviewed the manuscript titled " The Use of Aquatic Macrophytes as a Nature-Based Solution to Prevent Ciprofloxacin Deleterious Effects to Non-Target Organisms" and I would like to recommend its publication in the journal. The paper is well-written and the methodology is well-described. The authors have provided a comprehensive analysis of the data. The contribution of this paper to the literature is significant. While the study is well-written and the methodology is clearly described, I am concerned about the generalizability of the results.

1.       Based on MS content, it’s better to alter title to fit the content and clearly represent the paper topic.

2.       Some of keywords have been already stated in the title of the manuscript. The author should revise the keywords to provide complimentary information of the title.

3.       Abstract: Define acronyms when they first appear; thereafter directly use them. Please delete acronyms that are only used once.

4.       The novelty of the work has to be highlighted in the abstract.

5.       Keywords should reflect the key points of the work. Be corrected.

6.       The literature review is comprehensive, but it needs to be streamlined to focus on the specific gaps in knowledge that this paper addresses. The following references are helpful for the modification of the introduction. Please consider them.

https://doi.org/10.1016/j.chemosphere.2023.138580

https://doi.org/10.1021/acs.iecr.2c03833

https://dx.doi.org/10.22034/ecc.2021.268819.1122

https://xs2.zidianzhan.net/scholar?oi=bibs&hl=zh-CN&cluster=7479047946711920426

7.       Please minimize the use of technical terms and acronyms and explained when necessary.

8.       The discussion section would be better to more explicit in linking the findings to the literature and the research question.

9.       The results section would be better to concise and focused on the main findings.

10.    The methodology section would be revised to provide more detailed explanations of the models and methods used.

11.   In order for the audience and readers to benefit from the superiority of the present work, the comparison of the results of the present work with previous works should be reported in the form of an individual table.

12.   It needs to modify conclusion with more data.

13.   The format of symbol and unit should be double-checked.

 Minor editing of English language required

Author Response

We would like to thank the reviewer for all the valuable comments. They were very fruitful in improving the quality of our manuscript.

Comments

I have reviewed the manuscript titled " The Use of Aquatic Macrophytes as a Nature-Based Solution to Prevent Ciprofloxacin Deleterious Effects to Non-Target Organisms" and I would like to recommend its publication in the journal. The paper is well-written and the methodology is well-described. The authors have provided a comprehensive analysis of the data. The contribution of this paper to the literature is significant. While the study is well-written and the methodology is clearly described, I am concerned about the generalizability of the results.

Based on MS content, it’s better to alter title to fit the content and clearly represent the paper topic.

Thank you for the comments. We have changed the title to “The use of aquatic macrophytes as a nature-based solution to prevent ciprofloxacin deleterious effects to microalgae”.

Some of keywords have been already stated in the title of the manuscript. The author should revise the keywords to provide complimentary information of the title.

Thank you for your comments. We have revised the keywords.

Abstract: Define acronyms when they first appear; thereafter directly use them. Please delete acronyms that are only used once.

Thank you for your comments. We have revised the acronyms and their corresponding definitions.

The novelty of the work has to be highlighted in the abstract.

Thank you for your comments. We have made changes to the text.

Keywords should reflect the key points of the work. Be corrected.

Thank you for your comments. We have made changes to the text.

The literature review is comprehensive, but it needs to be streamlined to focus on the specific gaps in knowledge that this paper addresses. The following references are helpful for the modification of the introduction. Please consider them.

https://doi.org/10.1016/j.chemosphere.2023.138580

https://doi.org/10.1021/acs.iecr.2c03833

https://dx.doi.org/10.22034/ecc.2021.268819.1122

https://xs2.zidianzhan.net/scholar?oi=bibs&hl=zh-CN&cluster=7479047946711920426

Thank you for your comments. We have noticed that none of the references suggested by the reviewer are related to our manuscript or its subject matter. We suspect that there may have been a mistake in their selection, and therefore, we did not use these references in our article.

Please minimize the use of technical terms and acronyms and explained when necessary.

Thank you for your comments. We have made changes to the text.

The discussion section would be better to more explicit in linking the findings to the literature and the research question.

Thank you for your comments. We have made changes to the text. After including the table suggested by Reviewer 2, we agree that the discussion has improved.

The results section would be better to concise and focused on the main findings.

Thank you for your comments. However, we presented the major results that involved a general explanation of the physiological effects caused before and after the phytoremediation treatment.

The methodology section would be revised to provide more detailed explanations of the models and methods used.

Thank you for your comments. We have made changes to the text.

In order for the audience and readers to benefit from the superiority of the present work, the comparison of the results of the present work with previous works should be reported in the form of an individual table.

Thank you for your comments. We have included the table.

It needs to modify conclusion with more data.

Thank you for your comments. We have made changes to the text.

The format of symbol and unit should be double-checked.

Thank you for your comments. We have made changes to the text.

Reviewer 3 Report

Dear Authors,

I found the work interesting but there are some errors and issues that are not clear. There are some formal typographical errors (the way in which units are expressed) and some doubts about the study that I would like to be clarified.

 

 

Line 167. Why were the samples filtered through a 45 μm filter? Did you mean 0.45 μm? Please check the abbreviation for hydrochloric acid (HCl).

L180. Please check the degree symbol, as it is not the correct form.

Please add the country in all instruments and software used in the study (L148). Additionally, add the trademark and model of the optical microscope (L191).

In Section 2.2.2, according to the OECD 201 Technical Guideline for Freshwater Alga and Cyanobacteria, Growth Inhibition Test, the initial biomass concentration for Desmodesmus subspicatus should be 2-5 x 10³ cells/mL. However, in your assays, the initial biomass concentration is 1 million cells/mL, which is three orders of magnitude higher than the value established by OECD 201. This high initial biomass can affect the growth rate as the culture may not be in the exponential growth phase. Did you follow a standardized guideline such as ISO or OECD? Additionally, please provide information on the type of illumination and light intensity (lux) used during the assay.

The procedure followed for the biochemical parameters is not well cited in the text. Citation #55 does not describe any biochemical procedure. Please cite the actual authors for each parameter. For example, CAT (Aebi, 1984), MDA (Hodges et al., 1999), total protein (Bradford, 1976), and H2O2 (Velikova et al., 2000).

In Section 3.1, you mentioned that no Cipro was observed in water samples of the control treatments (0 μg/L). Please indicate the detection limit.

Table 1: In the first column, please put the Latin name on a separate line to avoid cutting the name.

 

Author Response

Reviewer 3

 

Dear Authors,

 

I found the work interesting but there are some errors and issues that are not clear. There are some formal typographical errors (the way in which units are expressed) and some doubts about the study that I would like to be clarified.

 

Line 167. Why were the samples filtered through a 45 μm filter? Did you mean 0.45 μm? Please check the abbreviation for hydrochloric acid (HCl).

Thank you for your comments. We apologize for the error, the correct is 0.45 μm. We have made changes to the text.

 

L180. Please check the degree symbol, as it is not the correct form.

Thank you for your comments. We have made changes to the text.

 

Please add the country in all instruments and software used in the study (L148).

Thank you for your comments. We have made changes to the text.

 

Additionally, add the trademark and model of the optical microscope (L191).

Thank you for your comments. We have included this information in the text.

 

 

In Section 2.2.2, according to the OECD 201 Technical Guideline for Freshwater Alga and Cyanobacteria, Growth Inhibition Test, the initial biomass concentration for Desmodesmus subspicatus should be 2-5 x 10³ cells/mL. However, in your assays, the initial biomass concentration is 1 million cells/mL, which is three orders of magnitude higher than the value established by OECD 201. This high initial biomass can affect the growth rate as the culture may not be in the exponential growth phase. Did you follow a standardized guideline such as ISO or OECD?

 

We appreciate the concern of the reviewer regarding the initial biomass concentration we used in our experiment for Desmodesmus subspicatus, which was higher than the range recommended by the OECD 201 Technical Guideline for Freshwater Alga and Cyanobacteria, Growth Inhibition Test.

While we understand the importance of adhering to established guidelines, we would like to present our rationale for using a higher initial biomass concentration of 1 million cells/mL and explain why it does not significantly impact the validity of our results.

Experimental Context: In our study, we aimed to investigate the effects of ciprofloxacin on Desmodesmus subspicatus. By using a higher initial biomass concentration, we were able to establish a more robust experimental setup, ensuring a sufficient cell population to withstand potential adverse effects and maintain a viable culture throughout the entire experiment. We measured biochemical biomarkers as well as assessed net photosynthesis and respiration. Consequently, when a lower initial biomass concentration was used, we were unable to evaluate all the necessary parameters. Considering the growth kinetics exhibited by the strain, our experiment was conducted during the exponential growth phase of the microalgae (as shown by the growth curve below)

 

(see the attached file)

 

Previous Research and Comparability: We conducted an extensive literature review and found several studies that successfully used higher initial biomass concentrations in similar experiments with Desmodesmus subspicatus or other algal species. These studies provided valuable insights and established a precedent for the use of elevated initial biomass concentrations, suggesting that it may not be a critical factor affecting the overall outcomes and interpretation of the results.

 

For example:

https://doi.org/10.1016/j.chemosphere.2022.135796

https://doi.org/10.1016/j.envpol.2023.121672

https://doi.org/10.1016/j.scitotenv.2023.164309

https://doi.org/10.1590/0001-3765202020190050

https://doi.org/10.1016/j.aquatox.2019.02.021

https://doi.org/10.1007/s11356-021-12950-6

https://doi.org/10.1016/j.scitotenv.2021.151008

https://doi.org/10.1016/j.jhazmat.2023.131215

 

Considering these factors, we firmly believe that the use of a higher initial biomass concentration of 1 million cells/mL in our experiment did not compromise the validity or reliability of our findings. We are confident that our study contributes valuable insights to the field, and the results remain meaningful and relevant to the scientific community.

If the reviewer has any further questions or concerns, we are more than willing to provide additional clarification or discuss this matter further.

 

Additionally, please provide information on the type of illumination and light intensity (lux) used during the assay.

Thank you for your comments. Information was added.

 

The procedure followed for the biochemical parameters is not well cited in the text. Citation #55 does not describe any biochemical procedure. Please cite the actual authors for each parameter. For example, CAT (Aebi, 1984), MDA (Hodges et al., 1999), total protein (Bradford, 1976), and H2O2 (Velikova et al., 2000).

Thank you for your comments. We have made changes to the text.

 

In Section 3.1, you mentioned that no Cipro was observed in water samples of the control treatments (0 μg/L). Please indicate the detection limit.

Thank you for your comments. The detection limit of quantification is present in lines 154-155. “Recovery rates were 94.4% and the limit of detection and limit of quantification were 0.3 and 1.0 µg.Cipro.L⁻¹, respectively.”

 

Table 1: In the first column, please put the Latin name on a separate line to avoid cutting the name.

Thank you for your comments. We have made changes to the text.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The presentation of this part of work is not fine to fit as a formal manuscript for peer-review, and the authors did not consider the reviewers comments. They failed to report the significance and novelty of this research. The quality of this work is too week and has not reach the standard of the water Journal. 

In this manuscript “The Use of Aquatic Macrophytes as a Nature-Based Solution to Prevent Ciprofloxacin Deleterious Effects to Non-Target Organisms” were involved. Although the topic seems good, however, the authors have not added good technical values to the field and there are several errors in this work. In the introduction, the authors did not make clear why these materials need to be designed with the knowledge of the current progress of relative researches. Nevertheless, the presentation of this part of work is not fine to fit as a formal manuscript for peer-review, and the authors did not consider the reviewers comments. They failed to report the significance and novelty of this research. The quality of this work is too week and has not reach the standard of the water Journal. Therefore, it is not recommended for publication in its present form, and I would like to recommend again as major revision.

 

 

Author Response

Reviewer 3

 

Dear Authors,

 

I found the work interesting but there are some errors and issues that are not clear. There are some formal typographical errors (the way in which units are expressed) and some doubts about the study that I would like to be clarified.

 

Line 167. Why were the samples filtered through a 45 μm filter? Did you mean 0.45 μm? Please check the abbreviation for hydrochloric acid (HCl).

Thank you for your comments. We apologize for the error, the correct is 0.45 μm. We have made changes to the text.

 

L180. Please check the degree symbol, as it is not the correct form.

Thank you for your comments. We have made changes to the text.

 

Please add the country in all instruments and software used in the study (L148).

Thank you for your comments. We have made changes to the text.

 

Additionally, add the trademark and model of the optical microscope (L191).

Thank you for your comments. We have included this information in the text.

 

 

In Section 2.2.2, according to the OECD 201 Technical Guideline for Freshwater Alga and Cyanobacteria, Growth Inhibition Test, the initial biomass concentration for Desmodesmus subspicatus should be 2-5 x 10³ cells/mL. However, in your assays, the initial biomass concentration is 1 million cells/mL, which is three orders of magnitude higher than the value established by OECD 201. This high initial biomass can affect the growth rate as the culture may not be in the exponential growth phase. Did you follow a standardized guideline such as ISO or OECD?

 

We appreciate the concern of the reviewer regarding the initial biomass concentration we used in our experiment for Desmodesmus subspicatus, which was higher than the range recommended by the OECD 201 Technical Guideline for Freshwater Alga and Cyanobacteria, Growth Inhibition Test.

While we understand the importance of adhering to established guidelines, we would like to present our rationale for using a higher initial biomass concentration of 1 million cells/mL and explain why it does not significantly impact the validity of our results.

Experimental Context: In our study, we aimed to investigate the effects of ciprofloxacin on Desmodesmus subspicatus. By using a higher initial biomass concentration, we were able to establish a more robust experimental setup, ensuring a sufficient cell population to withstand potential adverse effects and maintain a viable culture throughout the entire experiment. We measured biochemical biomarkers as well as assessed net photosynthesis and respiration. Consequently, when a lower initial biomass concentration was used, we were unable to evaluate all the necessary parameters. Considering the growth kinetics exhibited by the strain, our experiment was conducted during the exponential growth phase of the microalgae (as shown by the growth curve below)

 

 

Previous Research and Comparability: We conducted an extensive literature review and found several studies that successfully used higher initial biomass concentrations in similar experiments with Desmodesmus subspicatus or other algal species. These studies provided valuable insights and established a precedent for the use of elevated initial biomass concentrations, suggesting that it may not be a critical factor affecting the overall outcomes and interpretation of the results.

 

For example:

https://doi.org/10.1016/j.chemosphere.2022.135796

https://doi.org/10.1016/j.envpol.2023.121672

https://doi.org/10.1016/j.scitotenv.2023.164309

https://doi.org/10.1590/0001-3765202020190050

https://doi.org/10.1016/j.aquatox.2019.02.021

https://doi.org/10.1007/s11356-021-12950-6

https://doi.org/10.1016/j.scitotenv.2021.151008

https://doi.org/10.1016/j.jhazmat.2023.131215

 

Considering these factors, we firmly believe that the use of a higher initial biomass concentration of 1 million cells/mL in our experiment did not compromise the validity or reliability of our findings. We are confident that our study contributes valuable insights to the field, and the results remain meaningful and relevant to the scientific community.

If the reviewer has any further questions or concerns, we are more than willing to provide additional clarification or discuss this matter further.

 

Additionally, please provide information on the type of illumination and light intensity (lux) used during the assay.

Thank you for your comments. Information was added.

 

The procedure followed for the biochemical parameters is not well cited in the text. Citation #55 does not describe any biochemical procedure. Please cite the actual authors for each parameter. For example, CAT (Aebi, 1984), MDA (Hodges et al., 1999), total protein (Bradford, 1976), and H2O2 (Velikova et al., 2000).

Thank you for your comments. We have made changes to the text.

 

In Section 3.1, you mentioned that no Cipro was observed in water samples of the control treatments (0 μg/L). Please indicate the detection limit.

Thank you for your comments. The detection limit of quantification is present in lines 154-155. “Recovery rates were 94.4% and the limit of detection and limit of quantification were 0.3 and 1.0 µg.Cipro.L⁻¹, respectively.”

 

Table 1: In the first column, please put the Latin name on a separate line to avoid cutting the name.

Thank you for your comments. We have made changes to the text.