Toxicity of a Common Glyphosate Metabolite to the Freshwater Planarian (Girardia tigrina)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The scientific objectives and sound but the experimental approach used is defficitary as only two treatments and control were used for both chronic and acute assay. Considering the scientific dimension addressed at least 3 or 4 treaments plus control should have been used to allow the establishment of functional relationships and the calculation of EC₅₀ values at least for some of the chronic and acute endpoints. I consider that using only lower and higher environmental concentrations for the tests is unsufficient.

Author Response

Reviewer 1 said: “The scientific objectives and sound but the experimental approach used is deficient as only two treatments and control were used for both chronic and acute assay. Considering the scientific dimension addressed at least 3 or 4 treatments plus control should have been used to allow the establishment of functional relationships and the calculation of EC₅₀ values at least for some of the chronic and acute endpoints. I consider that using only lower and higher environmental concentrations for the tests is insufficient.”

 

Response: While we could have used more doses for each of the eight experiments, we did not have the physical capability to do so; instead, we focused on how the planarians might respond to contamination levels they might find in the wild. We have clarified this in the concluding paragraph of the manuscript and in the concluding paragraph of the introduction. A great follow-up paper might include finding the EC50 values.

We have attached our response to you and the other reviewers, FYI

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article contributes to the knowledge of the environmental impacts produced by glyphosate. The study was well conducted, and the manuscript is well written. I have a few suggestions to improve the text.

Part of the experiments were carried out with exposure for 7 days, called acute exposure. However, acute exposures occur within 24 hours. I suggest the experiments be renamed with the number of days of exposure instead of acute and chronic. Experiments with exposure for 7 days should be mentioned before those with 14 days throughout the manuscript.

 

L.36 and 335: Roundup is a trade name, and here I believe it is referring to any glyphosate-based herbicide, and not just this product specifically

L.106: Following [27] ?

L.106-107: Did you control the pH of the water?

L.128-129: I didn't understand the reason for measuring eyespot distance. Can this distance be affected in adult planarians or only during embryonic development?

L.144-148: Statistical analysis would be a result, not a methodology. ANOVA is not the most appropriate analysis for this data, as the deviations were very different (1.62 to 4.79). A non-parametric analysis would be more appropriate.

L.162-169: idem

L.211-217: Normality was tested, but some data show considerable variations between standard deviations. Differences between survival rates should also be compared statistically.

Author Response

Reviewer 2 said: Part of the experiments were carried out with exposure for 7 days, called acute exposure. However, acute exposures occur within 24 hours. I suggest the experiments be renamed with the number of days of exposure instead of acute and chronic. Experiments with exposure for 7 days should be mentioned before those with 14 days throughout the manuscript.

 

Response: We think this is an excellent suggestion that gets rid of jargon and makes for better reading. We made these changes.

 

Reviewer 2 said: L.36 and 335: Roundup is a trade name, and here I believe it is referring to any glyphosate-based herbicide, and not just this product specifically.

 

Response: We changed these to glyphosate-based.

 

Reviewer 2 said: L.106: Following [27] ?

 

Response: We added the citation for clarity.

 

Reviewer 2 said: L.106-107: Did you control the pH of the water?

 

Response: We did not. Because this was an ecotoxicity study rather than a toxicology study, we were more interested in the impact of the AMPA exposure as it would occur in the natural world than we were in the biochemical mechanism. We do however agree that it is an interesting question and if we pursue the EC50 studies suggested by other reviewers in the future, assessing and controlling for water pH would be on our list of activities.

 

 

Reviewer 2 said: L.144-148: Statistical analysis would be a result, not a methodology.

 

Response: We moved this information to the Results.

 

Reviewer 2 said: L.162-169: idem

 

Response: We moved this information to the Results.

 

Reviewer 2 said: ANOVA is not the most appropriate analysis for this data, as the deviations were very different (1.62 to 4.79). A non-parametric analysis would be more appropriate.

 

Response: You are quite right! We missed a number of these. We therefore went through the data and redid all of the normality tests and replaced the standard ANOVAs with Kruskal-Wallis ANOVAS where appropriate. We also clarified the paragraph describing the stat we used and when we used them.

 

Reviewer 2 said: L.211-217: Normality was tested, but some data show considerable variations between standard deviations.

 

Response: You are quite right. See above.

 

Reviewer 2 said Differences between survival rates should also be compared statistically.

 

Response: We added chi-square tests to rectify this.

We have attached a file with responses to all the reviewers, FYI

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

SUSTAINABILITY-2716512

Toxicity of AMPA to the freshwater planarian (Girardia tigrina)

This study investigates the toxicity effects of AMPA, a metabolite of the herbicide glyphosate, on the freshwater planarian G. tigrina. The researchers conducted chronic (14 day) and acute (7 day) toxicity experiments exposing planarians to environmentally relevant, low, and high doses of AMPA. They declared that 1. Chronic high dose AMPA exposure significantly reduced tail regeneration rates and modestly impacted mortality during regeneration; 2. Acute exposure had no measurable effect on regeneration, survival, or behavior; 3. Results suggest chronic environmental AMPA exposures could negatively impact planarian populations and freshwater ecosystems. However, some methodologies were incomplete, and the logical flow must also be supplemented. Some concerns need to be addressed or clarified before the manuscript can be considered for publication in the journal.

Major comments:

1.         What statistical tests were used to analyze the data on regeneration rates and mortality? Were any corrections applied for multiple comparisons? Please clarify.

2.         How were the AMPA exposure concentrations selected? What is the evidence that these reflect realistic environmental levels? Were all experiments replicated? How many true replicates were performed per treatment?

3.         The title of this article should be more specific. The authors used a very broad title, yet their research only looked at some common markers (regeneration, survival, or behavior).

4.         In addition, I strongly suggest the authors present some of the data in their paper in table form (Table 1, 2, 3...), rather than just listing their exposure diagrams. Diagrams cannot really be called data or figures.

5.         The English language needs to check carefully in the revision stage because of there are some careless mistakes in the text.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Reviewer 3 said: What statistical tests were used to analyze the data on regeneration rates and mortality? Were any corrections applied for multiple comparisons? Please clarify.

 

Response: We clarified statistical methods in the Materials and Methods section and in the Results. We used a chi-square analysis for the survivorship data, and we used a Kruskal-Wallis ANOVA when the data was non-normally distributed and a one-way ANOVA when the data was normally distributed.

 

Reviewer 3 said: How were the AMPA exposure concentrations selected? What is the evidence that these reflect realistic environmental levels?

 

Response:  The third paragraph of the Introduction reviews the several papers that measure AMPA concentrations in the wild, and we selected the high and low concentrations from those publications to reflect what the USGS and others have detected over decades of research. We also added values detected in European waters.

 

Reviewer 3 said: Were all experiments replicated? How many true replicates were performed per treatment?

 

Response: Each of our experiments have 20-25 replicates per treatment. Because each planaria was individually housed once experimentation began, we believe these to be true replicates. Additionally, the planaria used in the 7-day experiments were not the same individuals used in the 14-day experiments, adding a second level of independence.

 

Reviewer 3 said: The title of this article should be more specific. The authors used a very broad title, yet their research only looked at some common markers (regeneration, survival, or behavior).

 

Response: We changed the title to remove the abbreviation AMPA; we feel the phrase aminomethylphosphonic acid is too long for the title. Similarly, we feel that spelling out all the parameters that we test in the title makes it long and unwieldly. Studies show that papers with longer titles get fewer citations (Paiva et al., 2012). Furthermore, the variables of interest that could potentially be spelled out in the title occur both in the Abstract and the in the Keywords.

 

Reviewer 3 said: In addition, I strongly suggest the authors present some of the data in their paper in table form (Table 1, 2, 3...), rather than just listing their exposure diagrams. Diagrams cannot really be called data or figures.

 

Response: Figures 1 and 2 are meant to clarify our methods, which are potentially confusing, given the multiple pathways to exposure and timing of bisection. The graphics are not meant to convey data. While we agree that the use of graphics in a materials and methods section is slightly unconventional, we think they are best left where they are because they convey methodology rather than results.

 

Reviewer 3 said: The English language needs to check carefully in the revision stage because of there are some careless mistakes in the text.

 

Response: We wholly agree and apologize. After making the major revisions and before resubmitting, we had a second reader edit the manuscript.

 

We have attached a file with responses to all our reviewers FYI

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Abstract

The abstract is not detail enough, some of the manuscript contents were not captured here. Kindly revisit.

Introduction

The introduction was well written, but I have issues with how the introduction was structured.  Why the study? How do AMPA come about, and from what I understand, it is a metabolite of glyphosate, this must be captured from inception.  What are the effects of AMPA on freshwater systems? How do they find their way into the systems? Then narrow it down to how you came about choosing planaria for this study. Tell us briefly about the biology and ecology of planaria. Again, most of your claims were centred on studies conducted in USA and the America. I would advise you widen your search and tell us about similar studies conducted elsewhere. Remember, your findings will be a world reference, hence there should be element of studies conducted elsewhere, to increase the wide acceptance of this study.

Materials and methods

The methodology was well thought out and reported, but I wonder why you were reporting results of means, standard deviations, graphs, etc. in the methodology. This must be corrected. Move all results interpretation to results section. Then, all associated tests and analyses must be moved to Data analysis section. See attached document for detailed comments.

Results

 See attached document for comments.

Discussion

 See attached document for comments.

Comments for author File: Comments.pdf

Author Response

Reviewer 4, Thanks so much for the detailed and thoughtful critique. We found all of your suggestions very helpful.

 

Reviewer 4 said: The abstract is not detail enough, some of the manuscript contents were not captured here. Kindly revisit.

 

Response: We revised the abstract to both reflect the restructured Introduction and to add more details regarding the experiments themselves.

 

Reviewer 4 said: The introduction was well written, but I have issues with how the introduction was structured.  Why the study? How do AMPA come about, and from what I understand, it is a metabolite of glyphosate, this must be captured from inception.  What are the effects of AMPA on freshwater systems? How do they find their way into the systems? Then narrow it down to how you came about choosing planaria for this study. Tell us briefly about the biology and ecology of planaria. Again, most of your claims were centred on studies conducted in USA and the America. I would advise you widen your search and tell us about similar studies conducted elsewhere. Remember, your findings will be a world reference, hence there should be element of studies conducted elsewhere, to increase the wide acceptance of this study.

 

Response: We restructured the Introduction according to these comments and the comments in the edited manuscript. By changing the title, we also made the Introduction more powerful. We added other countries. We also updated the information about the ecosystem services delivered by freshwater invertebrates, and brought in information about AMPA contamination on a global level. We thank the reviewer for such helpful and thoughtful ways to make the Intro more relevant.

 

Reviewer 4 said: The methodology was well thought out and reported, but I wonder why you were reporting results of means, standard deviations, graphs, etc. in the methodology. This must be corrected. Move all results interpretation to results section. Then, all associated tests and analyses must be moved to Data analysis section. See attached document for detailed comments.

 

Response: We have made all but one of the suggested changes, leading to a very different Materials and Method Section and a very different results section. We think the suggested changes lead to a much stronger Results section.

 

The one change we didn’t make involved Figure 1 and 2. We created these figures because the timing of exposures and bisections was complicated and hard to describe. We understand that putting figures into Materials and Methods is slightly unconventional, but we strongly believe they belong there. The contain no data. Optionally, we could remove them, especially if the reviewers think that our written explanation of our experimental design is clear. For now, we’ve left them where they are.

 

Reviewer 4 said: Results. See attached document for comments.

 

Response: We have made all changes.

 

Reviewer 4 said: Discussion. See attached document for comments.

 

Response: We have made all changes.

 FYI, we have attached a file with the responses to all reviewers' comments below.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors answered my questions.