Review Reports - Selecting the Most Sustainable Phosphorus Adsorbent for Lake Restoration: Effects on the Photosynthetic Activity of <i>Chlorella</i> sp.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript ID: sustainability-3075866

Title: Effects of novel phosphorus adsorbents used for combating eutrophication on the photosynthetic activity of Chlorella sp.

This paper covers an investigation on the effects of emerging phosphorus adsorbents on the growth and photosynthetic parameters of a Chlorella strain. The manuscript is well written and well structured; however, some revisions must be considered prior to potential acceptance by the journal Sustainability.

Abstract: The abstract is presented in a poor manner. It would be interesting to specify which photosynthetic parameters were evaluated. Furthermore, it is mentioned that the initial hypothesis was rejected, but it has not yet been presented previously. Additionally, the last sentence should provide a conclusion about the main finding of the study.

Line 34-36: This phrase is written in a confuse way.

Line 73: Remove the double space in this phrase.

Line 104: What do you mean by 'good sensibility'? You can justify the use of Chlorella by the fact that they are cosmopolitan and easy to cultivate in the laboratory. However, Chlorella strains generally exhibit high lethal concentration 50% values for various chemical pollutants.

Line 124 – Table 1: The references must be grouped into a new column “Refs.”.

Line 264: Why was cell concentration measured only at the end of the experiment?

Line 303 – Figure 3: As no significant difference was detected between the treatments in graphs A and B, you do not need to insert identical letters in all the bars.

Line 307-308: This information is unnecessary here.

Line 328 – Discussion: In general, discussion section is too long and there are a number of unnecessary phrases, such as: “Next we aim on comparing the effects of P adsorbents on algal concentration based […]”. The authors must consider rewrite the discussion, removing 'catchphrases' and focusing on justifying the results found.

Comments on the Quality of English Language

Overall, english language is fine, only minor revisions are necessary.

Author Response

Reviewer #1: This paper covers an investigation on the effects of emerging phosphorus adsorbents on the growth and photosynthetic parameters of a Chlorella strain. The manuscript is well written and well structured; however, some revisions must be considered prior to potential acceptance by the journal Sustainability.

We appreciate the reviewer's comment.

Comments 1: Abstract: The abstract is presented in a poor manner. It would be interesting to specify which photosynthetic parameters were evaluated. Furthermore, it is mentioned that the initial hypothesis was rejected, but it has not yet been presented previously. Additionally, the last sentence should provide a conclusion about the main finding of the study.

Response 1: According to the referee, we have re-written the abstract (lines 11 to 27). In relation to the referee´s comment about including a last sentence with a conclusion, we do not actually understand his comment since abstract already concludes with the consequences of the addition of P adsorbents in a real scenario.

Comments 2: Line 34-36: This phrase is written in a confuse way.

Response 2: We agree with this comment and accordingly, we have modified the sentence in introduction, page 2, lines 43-45.

Comments 3: Line 73: Remove the double space in this phrase.

Response 3: Thank you for pointing this out. We have, accordingly, removed the additional space in page 3, line 84.

Comments 4: Line 104: What do you mean by 'good sensibility'? You can justify the use of Chlorella by the fact that they are cosmopolitan and easy to cultivate in the laboratory. However, Chlorella strains generally exhibit high lethal concentration 50% values for various chemical pollutants.

Response 4: We have considered the referee´s suggestion and accordingly, we have rewritten the sentence in page 3, lines 114-116.

Comments 5: Line 124 – Table 1: The references must be grouped into a new column “Refs.”.

Response 5: As the referee suggested us, we have added a new column in table 1, page 4, section 2.2.

Comments 6: Line 264: Why was cell concentration measured only at the end of the experiment?

Response 6: Cell concentration was measured only at the end of the experiment with the aim to identify if there exist, after the maximum contact time, significant differences among control and treatments when adding P adsorbents.

Comments 7: Line 303 – Figure 3: As no significant difference was detected between the treatments in graphs A and B, you do not need to insert identical letters in all the bars.

Response 7: We thank the referee´s comment and we completely agree. We have removed the letters in Figure 3 (page 12, section 3.3.) and in Figure 1 (page 10, section 3.2.).

Comments 8: Line 307-308: This information is unnecessary here.

Response 8: As the referee suggested us, we have removed this information in the manuscript (page 12, lines 344-345).

Comments 9: Line 328 – Discussion: In general, discussion section is too long and there are a number of unnecessary phrases, such as: “Next we aim on comparing the effects of P adsorbents on algal concentration based […]”. The authors must consider rewrite the discussion, removing 'catchphrases' and focusing on justifying the results found.

Response 9: We have considered the referee´s comment and accordingly we have removed some sentences of the discussion section (page 14, lines 384-385; page 17, lines 475-476). However, and based on referees´ (referees 2 and 3) comments, we have had to expand some aspects of the discussion so in the end the discussion is still but necessarily long.

Response to Comments on the Quality of English Language

Point 1: Overall, English language is fine, only minor revisions are necessary.

Response 1: We thank the referee´s comment.

Reviewer 2 Report

Comments and Suggestions for Authors

The study that formed the basis of the manuscript "Effects of novel phosphorus adsorbents used for combating eutrophication on the photosynthetic activity of Chlorella sp." addresses a very important topic of preserving and maintaining the delicate balance of ecosystems. The authors did not mention enough that anthropogenic impact is the main source of water P pollution. I think that more should be said about it in the introduction. Although the study is very relevant, it has a number of very serious shortcomings.

Firstly, it is still not clear whether the inhibition of photosynthesis is due to the introduction of drugs or to the fact that the amount of P in the cultivation medium decreased and starvation started? No methods were used to estimate the amount of phosphorus in the medium after application and during the experiment. This makes all photosynthesis measurement results unrelated to anything!!!! I believe this is critical for this study and without this data all conclusions of the authors are speculative.

Secondly, the authors chose only one object for toxicity assessment, which is not acceptable, as in classical toxicology at least two objects from different toxonomic groups should be subjected to the study. Chlorella is very sensitive to change, but it is also quite stable and plastic. It is necessary to take several more reference species, preferably cyonobacteria and diatoms, so that the study can be truly comprehensive.

Thirdly, the authors consider chemical and physical methods of controlling excessive P in water bodies. But they say nothing about biological methods of control. For example, it is known that Synechocystis https://doi.org/10.1007/s00709-019-01374-2 is able to accumulate significant doses of phosphorus from the environment in just a few minutes, leading to a decrease in its free forms in it. This aspect should also be discussed in the article.

There are also a number of minor remarks: - the introduction of statistics in a separate table makes the data analysis unusual, and detached from real values, it seems to me at least strange; - the authors give the composition of preparations, particle sizes only according to literature data, but storage conditions, transportation, etc. can make significant adjustments in the composition of preparations, and it would be good to study them immediately before the experiment.

In conclusion, I would like to say that the research topic is very topical, one can even say acute. However, much more research should be done by the authors in order to make clear and precise statements.

Author Response

Reviewer #2: The study that formed the basis of the manuscript "Effects of novel phosphorus adsorbents used for combating eutrophication on the photosynthetic activity of Chlorella sp." addresses a very important topic of preserving and maintaining the delicate balance of ecosystems. The authors did not mention enough that anthropogenic impact is the main source of water P pollution. I think that more should be said about it in the introduction. Although the study is very relevant, it has a number of very serious shortcomings.

We have considered the referee´s suggestion and accordingly, we have included some sentences and references about the anthropogenic impact on eutrophication in the introduction section (pages 1 and 2, lines 40-42).

Comments 1: Firstly, it is still not clear whether the inhibition of photosynthesis is due to the introduction of drugs or to the fact that the amount of P in the cultivation medium decreased and starvation started? No methods were used to estimate the amount of phosphorus in the medium after application and during the experiment. This makes all photosynthesis measurement results unrelated to anything!!!! I believe this is critical for this study and without this data all conclusions of the authors are speculative.

Response 1: We do not understand the reviewer's comment since the phosphorus concentration has been measured at the end of the experiment. Please, have a look on: Material and Methods (page 5, line 163); Results (page 7, lines 249-254 and page 8, Table 3) and Discussion (page 16, lines 432-439) sections.

Comments 2: Secondly, the authors chose only one object for toxicity assessment, which is not acceptable, as in classical toxicology at least two objects from different toxonomic groups should be subjected to the study. Chlorella is very sensitive to change, but it is also quite stable and plastic. It is necessary to take several more reference species, preferably cyonobacteria and diatoms, so th at the study can be truly comprehensive.

Response 2: We agree with the referee that a toxicological assessment based on different taxonomic groups would be very valuable, however it was not the aim of the present study. This research is a complementary study about the toxicity assessment that this research group has been performing with different P adsorbents used in lake restoration strategies during the last 15 years. Our previous studies have been mainly focused on carrying ecotoxicological tests by using magnetic particles with two different approaches: single-species phytoplankton (Álvarez-Manzaneda and de Vicente, 2017; Álvarez-Manzaneda et al., 2019a), zooplankton (Álvarez-Manzaneda et al., 2017; Álvarez-Manzaneda and de Vicente, 2017; Álvarez-Manzaneda et al., 2019a), and benthic organisms (Álvarez-Manzaneda et al., 2017) and experiments on the phytoplankton and zooplankton communities (Álvarez-Manzaneda et al., 2019b; del Arco et al., 2021). However, there is still a lack in the comprehensive toxicity assessment of the most frequently used P adsorbents. Actually, as far as we know, Álvarez-Manzaneda et al. (2019) is the only paper assessing the effects of different P adsorbents on the algal growth of Raphidocelis subcapitata.

As a result, the aim of this study was to assess the possible ecotoxicological effects of three phosphorus adsorbents on two different parameters of the algae Chlorella sp. with the objective of being able to compare these results with the previous works carried out.

In addition, we completely disagree with the referee when he/she states “the authors chose only one object for toxicity assessment, which is not acceptable, as in classical toxicology at least two objects from different taxonomic groups should be subjected to the study” as lots of toxicity assessments with one species have been performed and published in high quality specialized journals (e.g. Ma et al., 2002; Lin et al., 2007; Lürling and Tolman, 2010; Geiger et al., 2016; Xiao et al., 2016) by proving the importance of these experiments on the ecotoxicological assessment of different compounds.

Comments 3: Thirdly, the authors consider chemical and physical methods of controlling excessive P in water bodies. But they say nothing about biological methods of control. For example, it is known that Synechocystis https://doi.org/10.1007/s00709-019-01374-2 is able to accumulate significant doses of phosphorus from the environment in just a few minutes, leading to a decrease in its free forms in it. This aspect should also be discussed in the article.

Response 3. Thank you very much for your comment. In previous papers (see Álvarez-Manzaneda et al., 2021) we discussed the role of a helophytic species: Phragmites australis in the removal of phosphorus since this species was present in the study site. However, since the adsorbents that were assessed in this experiment are not biological methods for controlling eutrophication, we mainly focused on those that have similar mechanisms in phosphorus adsorption. Additionally, since we have shortened the discussion, as it has been required by referee 1, we consider that the addition of more information not directly related to our research would result in a much longer discussion.

Comments 4: - the introduction of statistics in a separate table makes the data analysis unusual, and detached from real values, it seems to me at least strange

Response 4: We appreciate the referee´s suggestion but we disagree with him/her. We have decided to present the main statistical results in a table (table 4) as all details for a better comparison among adsorbents is obtained. Moreover, the report of statistical results in tables has been proposed by several statistical experts (e. g. Morgan et al., 2016; Ross and Willson, 2018).

Comments 5: the authors give the composition of preparations, particle sizes only according to literature data, but storage conditions, transportation, etc. can make significant adjustments in the composition of preparations, and it would be good to study them immediately before the experiment.

Response 5: We thank the referee´s comment but we disagree as all P adsorbents used in this experiment are commercially obtained, solid and have been fully characterized by other researchers as we have reported in the manuscript. All the adsorbents were transported and stored in sealed containers protected from environmental extremes as recommended by the manufacturer. About the preparations that the reviewer mention, the liquid solutions were prepared by using different concentrations for every adsorbent (see page 4, section Materials and Methods, lines 143-148) only and just before performing this experiment.

By contrast, we would agree with the referee that a complete characterization of the adsorbents would be necessary if we had used particles synthesized by the researchers themselves, as we have previously done: de Vicente et al. (2010) and Funes et al. (2017).

Comments 6: In conclusion, I would like to say that the research topic is very topical, one can even say acute. However, much more research should be done by the authors in order to make clear and precise statements.

Response 6: We appreciate the reviewer's comments, which surely help to improve the work, although there are some comments, as we have already mentioned above, with which we completely disagree.

Reviewer 3 Report

Comments and Suggestions for Authors

The study reported the toxicity assay of several P adsorbents towards model organism Chlorella sp. The topic is interesting and the study is clear and logic, and is useful for the practical application of these P adsorbents.

Comments for further improvement:

Major concern:

-acute toxicity may not be the only concern considering the long-term safety assay of these chemicals. In introduction and discussion part, other side effects or potential hazards of often seen P adsorbents should be discussed. For example, the HQ chemical has obviously smaller size (around micrometer level) comparing with CHF12 & PHOSLOCK (millimeter level). How about the long-term magnification effect of HQ? durable in eco-system? similar with micro-plastic? These questions cannot be answered via toxicity assay towards producer model in this study. However should be discussed in depth.

Minor concern:

-abstract. Quantitative data is lacked in abstract to present the key results.

-in HQ group, pH is increased by HQ addition, what’s the reason? If negative effects of higher pH was not found to Chlorella sp, how about other algae? Discuss them more in detail.

Author Response

Reviewer #3: The study reported the toxicity assay of several P adsorbents towards model organism Chlorella sp. The topic is interesting, and the study is clear and logic, and is useful for the practical application of these P adsorbents.

We appreciate the reviewer's opinion about our manuscript.

Comments 1: Acute toxicity may not be the only concern considering the long-term safety assay of these chemicals. In introduction and discussion part, other side effects or potential hazards of often seen P adsorbents should be discussed. For example, the HQ chemical has obviously smaller size (around micrometer level) comparing with CHF12 & PHOSLOCK (millimeter level). How about the long-term magnification effect of HQ? durable in eco-system? similar with micro-plastic? These questions cannot be answered via toxicity assay towards producer model in this study. However, should be discussed in depth.

Response 1: We completely agree with the referee about the key role of particle size in determining ecotoxicological effects. In the resubmitted version, and according to the referee´s suggestion, we have included more information for remarking the importance of the different particle sizes the material and methods section (page 15, lines 419-427). In relation to the long-term magnification of HQ, no long-term effects are expected to occur as these particles are removed from the medium.

Comments 2: abstract. Quantitative data is lacked in abstract to present the key results.

Response 2: We thank the referee´s suggestion and accordingly, we have rewritten the abstract by using track changes (lines 11 to 27).

Comments 3: in HQ group, pH is increased by HQ addition, what’s the reason? If negative effects of higher pH was not found to Chlorella sp, how about other algae? Discuss them more in detail.

Response 3: We have considered the referee suggestion, and a new paragraph has now been included in Discussion (page 16, lines 460-467).

Reviewer 4 Report

Comments and Suggestions for Authors

The work focuses on comparing and selecting the best adsorbent for the practical task of capturing phosphorus in an aqueous medium and studying the effect of adsorbents on the photosynthetic activity of Chlorella sp. The authors tested their concept of the eco-effect of magnetic and non-magnetic particles in the composition of the studied adsorbents on the growth and photosynthetic activity of Chlorella sp. and presented further prospects for the application of the results obtained and the directions of their future research. After correcting minor remarks, the manuscript can be considered for acceptance to the Sustainability.

Abstract, Lines 24-27. The authors talk about their intentions, not about what has been done in this particular work. It is unlikely that this part is appropriate for an abstract.
The authors mention the economic inefficiency of the adsorbent used (Lines 59-67). At the same time, they themselves suggest using another adsorbent containing carbonyl iron (Lines 85-97). Carbonyl Fe is a material containing high purity iron used in appropriate technological processes, and therefore cannot be cheap. The authors should mention and explain the economic feasibility of using carbonyl Fe in their study.
Lines 82-84. It would be nice to provide a ref to the work here. If the work of the authors of 2010 is meant, then the term "recently" is hardly appropriate.
The "Materials and Methods" section. In addition to the above equations (1-4), it may be interesting for readers to give chemical equations describing the processes of action of adsorbents.
For all figures, add and provide signatures along the axes, for example, in Figure 1 add "Concentration" on the Y axis, and in Figure 2 indicate, that the measurements were made in hours on the X axis, and so on.
The Discussion section. The authors cite to a large number of works, but mainly (in terms of the volume of the text) the previous works of the authors themselves are discussed. At the same time, the citation is in the third person, for example Lines 495-497: "... All in all, our results are more consistent with those observed for Raphidocelis subcapitata by Álvarez-Manzaneda et al [38] who found EC50 of 1.50 g HQ L^-1... ". Perhaps it will be clearer for future readers if the authors simply mention that this is their past recent work. This remark is entirely at the discretion of the authors themselves.
You should consider splitting large paragraphs into several, as they are somewhat difficult to understanding. For example, one paragraph occupies the entire page 15. The other occupies almost the entire page 17, respectively.
List of references. There is an extensive bibliography of 82 references, but novel works (for the last 5 years) this is a total of 13 refs, while 5 of them are the work of the authors themselves, which is a little confusing in terms of the relevance of this manuscript by the authors. It should be noted that the refs are perfectly designed, maybe only except for refs 49 and 71, which may need to be redesigned.

Author Response

Reviewer #4: The work focuses on comparing and selecting the best adsorbent for the practical task of capturing phosphorus in an aqueous medium and studying the effect of adsorbents on the photosynthetic activity of Chlorella sp. The authors tested their concept of the eco-effect of magnetic and non-magnetic particles in the composition of the studied adsorbents on the growth and photosynthetic activity of Chlorella sp. and presented further prospects for the application of the results obtained and the directions of their future research. After correcting minor remarks, the manuscript can be considered for acceptance to the Sustainability.

We appreciate the reviewer's comment.

Comments 1: Abstract, Lines 24-27. The authors talk about their intentions, not about what has been done in this particular work. It is unlikely that this part is appropriate for an abstract.

Response 1: We thank the referee´s comment and we completely agree. The comparison of the effects of the studied phosphorus adsorbents in a real application scenery was performed in this study (in discussion, page 18, lines 517-524). For this reason, we modified the abstract (page 1, line 24) with the aim of clarifying what was done in this study.

Comments 2: The authors mention the economic inefficiency of the adsorbent used (Lines 59-67). At the same time, they themselves suggest using another adsorbent containing carbonyl iron (Lines 85-97). Carbonyl Fe is a material containing high purity iron used in appropriate technological processes, and therefore cannot be cheap. The authors should mention and explain the economic feasibility of using carbonyl Fe in their study.

Response 2: We appreciate the referee´s comment. In page 2, lines 78-80 we explained that carbonyl iron particles (HQ) can be recovered from the medium after their application and reused up to 4 times by achieving a high phosphorus removal efficiency as we showed in previous papers (Álvarez-Manzaneda et al., 2021 [37]). This makes HQ not only more cost-effective than other phosphorus adsorbents but also for reusing the recovered phosphorus as a fertilizer (Álvarez-Manzaneda et al., 2021 [31]).

Comments 3: Lines 82-84. It would be nice to provide a ref to the work here. If the work of the authors of 2010 is meant, then the term "recently" is hardly appropriate.

Response 3: We have considered the referee´s comment and accordingly we have

removed the term “recently” of the introduction section (page 2, line 83) and we added some references (page 3, line 85).

Comments 4: The "Materials and Methods" section. In addition to the above equations (1-4), it may be interesting for readers to give chemical equations describing the processes of action of adsorbents.

Response 4: We thank the referee´s comment. However, since in this paper we focused on the toxicological assessment of the adsorbents, we consider that the description of the processes of action of these adsorbents would fall out the scope of this study.

Comments 5: For all figures, add and provide signatures along the axes, for example, in Figure 1 add "Concentration" on the Y axis, and in Figure 2 indicate, that the measurements were made in hours on the X axis, and so on.

Response 5: Thank you very much for your comment. In the resubmitted version, and according to the referee´s suggestion, we have included more information to the axes of all the figures.

Comments 6: The Discussion section. The authors cite to a large number of works, but mainly (in terms of the volume of the text) the previous works of the authors themselves are discussed. At the same time, the citation is in the third person, for example Lines 495-497: "... All in all, our results are more consistent with those observed for Raphidocelis subcapitata by Álvarez-Manzaneda et al [38] who found EC50 of 1.50 g HQ L-1... ". Perhaps it will be clearer for future readers if the authors simply mention that this is their past recent work. This remark is entirely at the discretion of the authors themselves.

Response 6: We really appreciate the referee’s suggestion. We will take this comment in consideration for further publications.

Comments 7: You should consider splitting large paragraphs into several, as they are somewhat difficult to understanding. For example, one paragraph occupies the entire page 15. The other occupies almost the entire page 17, respectively.

Response 7: We thank the referee’s suggestion. We split the paragraphs in page 15, lines 421-422 and in page 17, lines 497-498.

Comments 8: List of references. There is an extensive bibliography of 82 references, but novel works (for the last 5 years) this is a total of 13 refs, while 5 of them are the work of the authors themselves, which is a little confusing in terms of the relevance of this manuscript by the authors. It should be noted that the refs are perfectly designed, maybe only except for refs 49 and 71, which may need to be redesigned.

Response 8: We thank the referee´s comment and we completely agree. The lack of studies about the toxicological effects of these adsorbents makes a challenge for the discussion of our results. We modified the references 49 and 71 accordingly with the referee’s suggestion.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for their detailed answer and explanation of their opinion. Unfortunately, apart from the proposal in the introduction and the automatic cancellation of my first question, the rest of the authors categorically disagree with my opinion, so I do not see any possibility of further work on this manuscript.

Author Response

Comments 1: I thank the authors for their detailed answer and explanation of their opinion. Unfortunately, apart from the proposal in the introduction and the automatic cancellation of my first question, the rest of the authors categorically disagree with my opinion, so I do not see any possibility of further work on this manuscript.
Response 1: We appreciate the reviewer's comment.