Interactions of Ingested Polystyrene Microplastics with Heavy Metals (Cadmium or Silver) as Environmental Pollutants: A Comprehensive In Vivo Study Using Drosophila melanogaster

Round 1

Reviewer 1 Report

given in att

Comments for author File: Comments.pdf

Author Response

Prof. Dr. Jukka Finne

Prof. Dr. Andrés Moya

Editor-in-Chief

Biology

 

Oliver Sun

Editor

Biology

                                                                                              Antalya, 21^st September 2022

Manuscript ID: biology-1924143

Title: Interactions of Ingested Polystyrene Microplastics with Heavy Metals
(Cadmium or Silver) as Environmental Pollutants: A Comprehensive In Vivo
Study Using Drosophila melanogaster

 

Dear Prof. Dr. Jukka Finne,

 

Dear Prof. Dr. Andrés Moya,

 

Dear Oliver Sun,

 

Thank you very much for your kind letter, dated on 21^st September, with the comments of the two reviewers that consider our work of interest, as well as suggestions of the revisions to this manuscript.

We have read carefully the comments of the reviewers and we expect that our responses and revisions will satisfy their suggestions. We hope that, with the revisions made in line with the comments/questions raised by the reviewers, the current version of our paper will be acceptable for publication.

In addition, we have introduced several modifications in the new version to ensure that the manuscript conforms to the format of the Journal. With the new version, I enclosed a marked with track changes copy where the modifications are highlighted.

I look forward to hearing from you at your earliest convenience.

Sincerely yours,

Prof. Dr. Eşref Demir

RESPONSE TO THE REVIEWERS

Reviewer: 1

The paper represents a study of synergistic effects of ingested polystyrene microplastics with
cadmium and/or silver as environmental pollutants using Drosophila melanogaster
as a model. The study is valuable from the aspect of different biological impact of various
pollutants which are increasing in the environment.

RESPONSE

We thank the Reviewer for the positive feedback.

1. 
   In a comprehensive introduction the authors give review of literature data regarding
   the background the research. However, that part should be more structured, less
   broadly revue of environmental significance, but focused on the goal of their study.
   The first part goes into wide details and review on microplastic, recycling,
   biodegradation, global problems, etc. I am sure it can be halved.

RESPONSE

Thank you very much for your valuable contribution. Since microplastics and nanoplastics (MNPLs) pollution has a great importance in recent years, relevant information has been added to the introduction section so that readers can access detailed information about the effects, permanence and biological effects of MNPLs in the environment. In order to emphasize the importance of this study, it is aimed to draw attention to the transformations of MNPLs in the environment and the problems they cause.

2. At the end of introduction, but not separated even with paragraph, the aim of the
   study is given. It is a bit confusing and not fully consistent with the experimental
   flow which proceeds in Material and Methods and Results. It should clearly state
   the goals. Maybe the whole that part needs English revision to make it better, clear
   and convincing. For example..“Therefore(?).this study aimed”first sentence is
   ok,…than the methodology for one part (aim?) is given in details which is
   inappropriate at that place. Last sentence even more weakens the consistency of
   study design and goals: …”Furthermore (?), we also examined in great detail
   (?)..the whole ..”..and it is actually the big part of the study..should not be given as
   “also” etc…It is contradictory with the flow given in the results.

RESPONSE

Thank you very much for your valuable suggestion. We have revised the related sentences in line with your comments.

3. 
   Material and Methods part is well covered with technical and methodology
   information but I think that except in part 2.2. in the rest of the experiments
   (staining, indigestion etc. the sample size.. the number of larvae examined, used,
   presented in the results..is missing.

RESPONSE

We thank for this reflection. We have added the relevant information. On the other hand, carbon powder is not a stain. It is just used for better visibility of Drosophila eggs and added to Drosophila medium.

 

4. Results. 3.1. Toxicity of PSMPLs and heavy metals (CdCl2 and AgNO3) Although
   in Material and methods the part 2.2 is, as far as I understand, aimed to choose the
   appropriate concentration and doses of Cd, Ag and PSMPLs administered
   individually and simultaneously and to measure the effects through egg-to adult
   survival, the presentation of the obtained results is missing important part. The
   Figure 1 gives toxicity results measured as loss of viability (egg-to-adult survival),
   but only for PSMPLs, CdCl2 and AgNO3 individually. There is no presented data on
   combined effect which should be the main point of the study. The authors state that
   “according to these results, PSMPLs “could be said” (to descriptive and speculative)
   to have no statistically significant toxic effects as in D. melanogaster larvae”. And
   that “significant toxic effects were observed after exposure to CdCl2 and AgNO3 at
   doses higher than 0.5 mM “. In the whole study the combined, synergistic effects is
   given and discussed except in this part. If they do not have these data than there the toxicity part is not completed and the authors should not consider it in this paper, just
   keep shortly the methodological explanation of chosen doses. Even if there are
   combined data (somehow missing in the results) my suggestion would be to skip this
   part in the presentation form in the Results section and only explain which doses are
   chosen.

RESPONSE

Thank you very much for your valuable contribution and different point of view. Viability experiments were previously carried out in our laboratory to determine the range of doses to be used in the genotoxicity studies. The criteria to choose the final selected doses were based on two reasons: First, a reduction in the percentage of developing treated larvae is a clear indication that the compounds affected the larvae and, in addition, the number of emerging adults must be high enough to perform the genotoxicity experiments. For this reason, we did not aim to find LC₅₀ values in this study. Instead, we conducted a toxicity study to detect the genotoxic effects at doses above 50 percent viability.

5. 
   The final sentence and emphasized in Conclusion should be deleted, or reephrased
   and replaces in introduction, discussion…: “In conclusion, this study confirms that
   Drosophila is an useful organism in environmental toxicity/geno-toxicity studies for
   environmental pollutants.” Because that is not the main conclusion of this study “to
   confirm” something well known from decades of genotoxicity studies on
   Drosophila, including environmental pollutants such as heavy metals.

RESPONSE

Thank you very much for your valuable contribution and different point of view. We have deleted the relevant sentence.

 

6. Reference list is too large... 146 references is too much for research arrticle, many
   overlap and some fields are overrepresented.

RESPONSE

Thank you very much for your valuable contribution. A few references were deleted.

Author Response File: Author Response.docx

Reviewer 2 Report

Manuscript entitled “Interactions of Ingested Polystyrene Microplastics with Heavy Metals (Cadmium or Silver) as Environmental Pollutants: A Comprehensive In Vivo Study Using Drosophila melanogaster” by Demir et al. A very interesting study highlighting the toxic potential of microplastic with heavy metals.  Overall data presented in the study showing the synergistic/additive toxic effect of microplastic with heavy metals. However, I have some major queries that is

1.      How microplastics with cadmium and silver nitrate are environmentally relevant ? Any reports where combinations of microplastic with cadmium and silver nitrate are reported. If any please discuss and cite. Please discuss how exposed concentrations of cadmium and silver nitrate were selected and are these concentrations reported with microplastic.

2.      In the Result section, Authors show larval viability in sterile water is 100% and after exposure to 10 mM to PSMPLs (4, 10, and 20 µm), 72, 75, and 77% of eggs successfully reached adult stage. Which is 28, 25 and 23% decrease in viability but authors show it is statistically nonsignificant. Please recheck statistics.

3.     Is co-exposure of microplastic with Cadmium and silver nitrate to increase the toxic effects synergistically or additive? If the effect is synergistic or additive, I am still not sure whether microplastic interact with heavy metals as the title of the manuscript says.

4.     Data presented in bar graph if authors present the data in bar graph with dot plot that clearly show the number of replicates and range

Author Response

Prof. Dr. Jukka Finne

Prof. Dr. Andrés Moya

Editor-in-Chief

Biology

 

Oliver Sun

Editor

Biology

                                                                                              Antalya, 21^st September 2022

Manuscript ID: biology-1924143

Title: Interactions of Ingested Polystyrene Microplastics with Heavy Metals
(Cadmium or Silver) as Environmental Pollutants: A Comprehensive In Vivo
Study Using Drosophila melanogaster

 

Dear Prof. Dr. Jukka Finne,

 

Dear Prof. Dr. Andrés Moya,

 

Dear Oliver Sun,

 

Thank you very much for your kind letter, dated on 21^st September, with the comments of the two reviewers that consider our work of interest, as well as suggestions of the revisions to this manuscript.

We have read carefully the comments of the reviewers and we expect that our responses and revisions will satisfy their suggestions. We hope that, with the revisions made in line with the comments/questions raised by the reviewers, the current version of our paper will be acceptable for publication.

In addition, we have introduced several modifications in the new version to ensure that the manuscript conforms to the format of the Journal. With the new version, I enclosed a marked with track changes copy where the modifications are highlighted.

I look forward to hearing from you at your earliest convenience.

Sincerely yours,

Prof. Dr. Eşref Demir

RESPONSE TO THE REVIEWERS

Reviewer: 2

Manuscript entitled “Interactions of Ingested Polystyrene Microplastics with Heavy Metals (Cadmium or Silver) as Environmental Pollutants: A Comprehensive In Vivo Study Using Drosophila melanogaster” by Demir et al. A very interesting study highlighting the toxic potential of microplastic with heavy metals.  Overall data presented in the study showing the synergistic/additive toxic effect of microplastic with heavy metals. However, I have some major queries that is

RESPONSE

We thank the Reviewer for the positive feedback.

1. How microplastics with cadmium and silver nitrate are environmentally relevant ? Any reports where combinations of microplastic with cadmium and silver nitrate are reported. If any please discuss and cite. Please discuss how exposed concentrations of cadmium and silver nitrate were selected and are these concentrations reported with microplastic.

RESPONSE

Thank you very much for your valuable contribution and different point of view.

Silver (Ag) content in the surface soils varies between <0.01 and 5 mg/kg (Sterckeman et al., 2002; Murray Rogers, and Kaufman, 2004). In addition, general concentration of cadmium (Cd) ranges from 0.1 to 1 mg/kg in soil (Alloway, 2013).

Microplastic (MPL) concentrations in rivers and inland waters now exceed alarming levels— samples from the river Elbe in Europe were shown to contain 3.35 x 10⁶ microplastic particles in sediments and 5.57 MPLs per cubic meter (Scherer et al. 2020).

The concentrations of PSMPLs were based on our previous toxicity studies into particulate matter on Drosophila (0.01-10 mM) (Zhang et al. 2020; Demir 2021; Matthews et al. 2021). On the other hand, the concentrations of CdCl₂ and AgNO₃ were determined based on the data from previous toxicity and genotoxicity research with CdCl₂ (0.01-0.2 mM) and AgNO₃ (0.05, 0.2 and 1 mM) on Drosophila (Alaraby et al. 2022b; Zhang et al. 2020; Demir et al. 2011).

The studies in the literature and the amounts found in the environment were taken into consideration for choosing the tested concentrations.

2. In the Result section, Authors show larval viability in sterile water is 100% and after exposure to 10 mM to PSMPLs (4, 10, and 20 µm), 72, 75, and 77% of eggs successfully reached adult stage. Which is 28, 25 and 23% decrease in viability but authors show it is statistically nonsignificant. Please recheck statistics.

RESPONSE

Thank you very much for your valuable contribution. For PSMPLs, our results showed that a partial decrease in viability was detected as dose-dependent. These reductions did not show statistically significance.

3. Is co-exposure of microplastic with Cadmium and silver nitrate to increase the toxic effects synergistically or additive? If the effect is synergistic or additive, I am still not sure whether microplastic interact with heavy metals as the title of the manuscript says.

RESPONSE

We thank for this reflection. The results obtained in co-exposure of microplastic with Cadmium and silver nitrate are higher than in single exposures. In this context, it is considered that there is an interaction between microplastics and metals.

4. Data presented in bar graph if authors present the data in bar graph with dot plot that clearly show the number of replicates and range

RESPONSE

Thank you very much for your valuable contribution and different point of view. We have revised the related graph in line with your comments.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors responded in 48h hours to serious comments I made which is impressive but obviously not enough.

1. Introduction is still not structured and is maybe appropriate for review paper, but for research article should be more straightforward.

2. RESULTS, Figure 1. 

The Figure 1 gives toxicity results measured as loss of viability (egg-to-adult survival), but only for PSMPLs, CdCl2 and AgNO3 individually. There is no presented data on combined effect which should be the main point of the study. In the whole study the combined, synergistic effects is given and discussed except in this part. If they do not have these data than there the toxicity part is not completed and the authors should not consider it in this paper, just keep shortly the methodological explanation of chosen doses. Even if there are combined data (somehow missing in the results) my suggestion would be to skip this part in the presentation form in the Results section and only explain which doses are chosen. and describe the effect on viability. Without Figures.

Author Response

Prof. Dr. Jukka Finne

Prof. Dr. Andrés Moya

Editor-in-Chief

Biology

 

Oliver Sun

Editor

Biology

                                                                                              Antalya, 26^th September 2022

Manuscript ID: biology-1924143

Title: Interactions of Ingested Polystyrene Microplastics with Heavy Metals
(Cadmium or Silver) as Environmental Pollutants: A Comprehensive In Vivo
Study Using Drosophila melanogaster

 

Dear Prof. Dr. Jukka Finne,

 

Dear Prof. Dr. Andrés Moya,

 

Dear Oliver Sun,

 

Thank you very much for your kind letter, dated on 26^th September, with the comments of the two reviewers that consider our work of interest, as well as suggestions of the revisions to this manuscript.

We have read carefully the comments of the reviewers and we expect that our responses and revisions will satisfy their suggestions. We hope that, with the revisions made in line with the comments/questions raised by the reviewers, the current version of our paper will be acceptable for publication.

In addition, we have introduced several modifications in the new version to ensure that the manuscript conforms to the format of the Journal. With the new version, I enclosed a marked with track changes copy where the modifications are highlighted.

I look forward to hearing from you at your earliest convenience.

Sincerely yours,

Prof. Dr. Eşref Demir

 

RESPONSE TO THE REVIEWERS

Reviewer: 1

The authors responded in 48h hours to serious comments I made which is impressive but obviously not enough.

RESPONSE

We thank the Reviewer for the positive feedback.

 

1. Introduction is still not structured and is maybe appropriate for review paper, but for research article should be more straightforward.

RESPONSE

Thank you very much for your valuable suggestion. We have revised the Introduction in line with your comments.

 

2. RESULTS, Figure 1. 

The Figure 1 gives toxicity results measured as loss of viability (egg-to-adult survival), but only for PSMPLs, CdCl2 and AgNO3 individually. There is no presented data on combined effect which should be the main point of the study. In the whole study the combined, synergistic effects is given and discussed except in this part. If they do not have these data than there the toxicity part is not completed and the authors should not consider it in this paper, just keep shortly the methodological explanation of chosen doses. Even if there are combined data (somehow missing in the results) my suggestion would be to skip this part in the presentation form in the Results section and only explain which doses are chosen. and describe the effect on viability. Without Figures.

 

RESPONSE

We thank for this reflection. We have introduced the suggestions in the manuscript. The Figure 1 was deleted. We only explained which doses are chosen and described the effect on viability.

Reviewer 2 Report

Authors satisfactory answered the queries raised by reviewer. Manuscript recommended for publication. 

Author Response

Prof. Dr. Jukka Finne

Prof. Dr. Andrés Moya

Editor-in-Chief

Biology

 

Oliver Sun

Editor

Biology

                                                                                              Antalya, 26^th September 2022

Manuscript ID: biology-1924143

Title: Interactions of Ingested Polystyrene Microplastics with Heavy Metals
(Cadmium or Silver) as Environmental Pollutants: A Comprehensive In Vivo
Study Using Drosophila melanogaster

 

Dear Prof. Dr. Jukka Finne,

 

Dear Prof. Dr. Andrés Moya,

 

Dear Oliver Sun,

 

Thank you very much for your kind letter, dated on 26^th September, with the comments of the two reviewers that consider our work of interest, as well as suggestions of the revisions to this manuscript.

We have read carefully the comments of the reviewers and we expect that our responses and revisions will satisfy their suggestions. We hope that, with the revisions made in line with the comments/questions raised by the reviewers, the current version of our paper will be acceptable for publication.

In addition, we have introduced several modifications in the new version to ensure that the manuscript conforms to the format of the Journal. With the new version, I enclosed a marked with track changes copy where the modifications are highlighted.

I look forward to hearing from you at your earliest convenience.

Sincerely yours,

Prof. Dr. Eşref Demir

 

RESPONSE TO THE REVIEWERS

 

Reviewer: 2

Authors satisfactory answered the queries raised by reviewer. Manuscript recommended for publication. 

RESPONSE

We thank the Reviewer for the positive feedback. Thank you very much for your valuable contribution and different point of view.