Combined Effects of Polystyrene Nanoplastics and Enrofloxacin on the Life Histories and Gut Microbiota of Daphnia magna

Round 1

Reviewer 1 Report

This paper investigates combined effects of polystyrene nanoplastics and enrofloxacin on the life histories and gut microbiota of Daphnia magna

-          Name of Daphnia Magna should be in italic. You should check through the paper if the name is written correctly.

-          2.1. Experimental animals – whole section is in italic?

-          Physical units are not written correctly. As example, XX mg C × L-1 should be written XX mg C × L^-1 or XX mg C / L. Check through the paper.

-          Please use better english. As example: „Here, no organic solvent and buffer were used to increase the solubility of enrofloxacin to avoid changing water parameters.“ should be rewritten: Stock solution of enrofloxacin was prepared without using organic solvents nor buffer solutions... Please check your English through whole paper.

-          Please explain 16L:8D; I am not biologist and presume that is biologist therm.

-          Figure 1 should be in better resolution.

-          In my opinion, Figure 1 histograms show no effect. Values are near constant in different variances.

-          All figures should be in better resolution.

Author Response

Reviewer 1

Comments and Suggestions for Authors

This paper investigates combined effects of polystyrene nanoplastics and enrofloxacin on the life histories and gut microbiota of Daphnia magna

-     Name of Daphnia magna should be in italic. You should check through the paper if the name is written correctly.

• Yes, the unformatted text was pasted into a template during the submissions of the manuscript. We improved the formatting, including the name of Daphnia magna throughout the revised text.

-     2.1. Experimental animals – whole section is in italic?

• This was also done unintentionally during the submission process. Corrected.

-     Physical units are not written correctly. As example, XX mg C × L-1 should be written XX mg C × L^-1 or XX mg C / L. Check through the paper.

• Again, these mistakes were done by the system when pasting text into a template during the submission process. Corrected throughout the revised text.

-     Please use better english. As example: „Here, no organic solvent and buffer were used to increase the solubility of enrofloxacin to avoid changing water parameters.“ should be rewritten: Stock solution of enrofloxacin was prepared without using organic solvents nor buffer solutions... Please check your English through whole paper.

• The sentence has been corrected. In accordance to the comment, the revised text has been edited by a native English speaker.

-     Please explain 16L:8D; I am not biologist and presume that is biologist term.

• In accordance with the comment, we replaced (16L:8D) with (16 light : 8 dark).

-     Figure 1 should be in better resolution.

• Again, the low resolution of the figures was due to the improper pasting of the manuscript into the template during the submission. We improved the resolution of the figures in the revised text.

-     In my opinion, Figure 1 histograms show no effect. Values are near constant in different variances.

• We fully agree with the comment. In a previous version of the Ms we used incorrect statistical analysis. As the data did not met the Gaussian distribution, we shouldn't have used a parametric test. We corrected the analysis in the revised text by using non-parametric tests (Aligned Rank Transform for Nonparametric Factorial two-way ANOVA, L 311-316). Moreover, in the graphs we used more informative standard errors instead of standard deviations. Finally, we slightly modified the format of the figures to make them more informative.
• All figures should be in better resolution.
• In accordance with the comment, we improved the resolution of the figures in the revised text.

Author Response File: Author Response.doc

Reviewer 2 Report

This is a review of the manuscript entitled “Combined effects of polystyrene nanoplastics and enrofloxacinon the life histories and gut microbiota of Daphnia magna” as submitted to Water. Below are some questions for enhancing the impact of this manuscript.

1. Ambiguity of NP concentration

The authors divided the concentration (density-particle/L) of nanoparticles as low, medium, and high, but the interval is tight and ambiguous.

2. Flaw in experimental design used for combined effect

In Figure 1, some effect was observed by NP exposure only and enrofloxacinon only, and the authors used these concentrations for combined toxicity, which is very difficult to judge synergistic or antagonistic effect.

3. Reliability in taxonomic diversity of gut microbiota experiments

Not many studies investigated changes in gut microbiota after chemical exposure in daphnia. So, this study can provide insights in this area. In other words, the methods (sections of 2.4.3 and 2.4.4.)  should be standardized, and their reproducibility is needed to provided.

4. More evidences to support the combined toxicity results

In order to interpret the experimental results, the concentration of the enrofloxacin in bulk solution is needed to be measured in the absence or presence of NPs, also in experiment investigating life history variations.

5. Need the improvement in Figure quality and use of abbreviations.

Author Response

Reviewer 2

This is a review of the manuscript entitled “Combined effects of polystyrene nanoplastics and enrofloxacinon the life histories and gut microbiota of Daphnia magna” as submitted to Water. Below are some questions for enhancing the impact of this manuscript.

1. Ambiguity of NP concentration

The authors divided the concentration (density-particle/L) of nanoparticles as low, medium, and high, but the interval is tight and ambiguous.

• The misunderstanding concerning the densities used in our study stem from incorrect formatting of the text while pasting it to the journal template. We used 3 distinctly different densities of nanoplastics: N_l = 1 × 10³, N_m = 1 × 10⁶ and N_h = 1 × 10⁹ particles × L⁻¹ rather than 103, 106 and 109 particles L^-1. The bug was corrected in the revised text (L 184).

 

2. Flaw in experimental design used for combined effect

In Figure 1, some effect was observed by NP exposure only and enrofloxacinon only, and the authors used these concentrations for combined toxicity, which is very difficult to judge synergistic or antagonistic effect.

• The most important for us was the use of environmental concentrations of both stressors, i.e. 3 enrofloxacin concentrations (0, E_l = 10 and E_h = 100 ng × L⁻¹) and 4 densities of PS-NPs (0, N_l = 1 × 10³, N_m = 1 × 10⁶ and N_h = 1 × 10⁹ particles × L⁻¹). The high concentration of enrofloxacin and the highest concentration of PS-NPs are well above the environmental concentrations of these stressors. Although the nature of the effects (synergistic or additive) in some cases is not apparent in the figures, we describe the nature of the effects in the text of the results section. In accordance with the comment, we have modified Figs. 1 and 2 so that the nature of the effects is more pronounced.

3. Reliability in taxonomic diversity of gut microbiota experiments

Not many studies investigated changes in gut microbiota after chemical exposure in daphnia. So, this study can provide insights in this area. In other words, the methods (sections of 2.4.3 and 2.4.4.)  should be standardized, and their reproducibility is needed to provided.

• In accordance with the comment, we added in several places in the sections of 2.4.3 and 2.4.4. of the revised text the explanation that the methods used in our study (and their description) are standard as used in several earlier studies (L 237 and L 270-274).

4. More evidences to support the combined toxicity results

In order to interpret the experimental results, the concentration of the enrofloxacin in bulk solution is needed to be measured in the absence or presence of NPs, also in experiment investigating life history variations.

• We fully agree with this comment. Therefore, we expanded the interpretation of the results obtained that the interactions observed in our study may stem from both the interactive effect of the stressors on the animal physiology or the absorption of the enrofloxacin at the NPs’ surface (L 621-628, and L 748-755).

5. Need the improvement in Figure quality and use of abbreviations.

• The low resolution of the figures was due to the improper pasting of the manuscript into the template during the submission. We improved the resolution of the figures in the revised text. Moreover, in accordance to the comment, we used abbreviations in the figures and added explanations of these abbreviations in the figure captions in the revised text.

Author Response File: Author Response.doc

Reviewer 3 Report

GENERAL COMMENTS

In this paper, Maszczyk et al. study the individual and combined effect of nanoplastics (direct effect) and antibiotics (indirect effect through their impact on gut microbiota) on different life history traits as well as on taxonomic and functional diversity of the cladoceran Daphnia magna. The paper provides very interesting results on a topic for which there do not exist enough evidence nowadays, so I consider this study has enough interest to deserve its publication after solving some minor issues. My main concerns have to do with clarifying confusing statements, adding some extra text to acknowledge some limitations and put the results in a broader context and increasing the quality of the figures, which must be definitively improved. For instance, in its current state, it is difficult to follow when the results refer to the effect of a single stressor or the combined effect, if these effects are negative or positive, and if they are significant or not. I think this issue can be easily solved if authors put them in a tidiest way, like a routine: first, single effect of NPs (if they are positive or negative and significant or non-significant on every response variable), then the single effect of enrofloxacin (negative, positive, significant, non-significant, etc.), and then the combined effect of both stressors (negative, positive, significant, non-significant, etc.). It would be very helpful to readers if authors structure the discussion following the same structure. I must say that I know that my comments can be difficult to follow due to the lack of line numbers through the text (I hope not). By the way, authors must use italic font to write the name of the species (Dapnia magna) everywhere through the text.

 

MINOR COMMENTS

Abstract

First sentence. The use of there is confusing. The behaviour and availability of what can influence? Nanoplastics? Pollutants? Antibiotics? Please, rewrite.

 

Penultimate sentence. A result can be significant or not significant according to the level chosen but not very or less significant. So, I would say that the negative effects were significant and all the response variables measured but they were more obvious on body size and the metabolic firgerprint while less notorious on reproductive parameters and taxonomic diversity.

 

Introduction

Paragraph 2, last sentence. Authors mention plasticizers antioxidants, flame retardants and UV stabilizers. It would be great they would expand how these substances, i.e. the mechanisms, can affect organisms.

 

Last paragraph. Related to my statements in the general comments, the aims and results of the paper are here shown in a more understandable way than in the abstract. Maybe authors can take this paragraph as an inspiration to rewrite the abstract. For instance, is clearly metioned the assessment of the single and combined effect of NPs and antibiotics. However, I consider that these paragraph spoil all the results. I find correct to anticipate the main results in the last paragraph of the introduction but in a slightly way, not so detailed. I think that something like ‘We found that both NPs and enrofloxacin affect the life history traits of Daphnia magna as well as the taxonomic diversity and metabolic firgerprint of their gut microbiota, and there is and interaction in the effect of both stressors’ is enough for the introduction section. Then, all these results can be detailed in their own section.  

 

Material and Methods

2.1. This paragraph should not be in italic font. Can authors provide any difference among clones (in size or whatever variable) or it is just to reflect their different locations?

 

2.3. Please, add the units for L and W in the first brackets.

 

2.4.1. Why authors used these concentrations of enrofloxacin? Can these levels be found in natural environments?

 

2.5. If assumptions for the parametric test were not met, why authors have not employed classical non parametric alternatives such as Sheirer Ray Hare text in the case of two or less levels for any treatment or Pairwise Mann-Withney test (function pairwise.wilcox.text in R) in the case of more than two levels for any treatment? As far as I know, Tukey is the post hoc test usually employed when the assumptions of normality and homocedasticity are met. Moreover, have they used any p-value correction such as Bonferroni to be more restrictive with the significance level? I would like to see the results applying this kind of correction and check if the differ from the current results for any response variable. Why Bray-Curtis distance instance of any other? I am not saying that this is wrong, but authors must justify their decision according to their objectives as there are many other similarity indexes and each one is more appropriated to each particular objective.

 

Results

Figure 1. If the figure includes the test results, I consider that is more informative to show the error standard barrs (as in Figure 2) or confidence interval barrs instead of SD barrs.

 

Figure 4. Please, provide the stress value. This result does not seem very logical according to the clustering of treatments. Could this mess be clarified adding a third dimension?

 

3.3. Low taxonomic diversity of bacteria living in the digestive tract of Daphnia… compared to what? In the third sentence, there is one ‘the’ (with increasing the concentrations of enrofloxacin) that should be removed.

 

Figure 5. The consistent increase in Firmicutes is very interesting. I think authors should expand the explanations and implications of this increase in the discussion section.

 

Figure 6. Please, provide the stress value. Again, the clustering of treatments does not seem to follow any logical order. Could this mess be clarified adding a third dimension?

 

Discussion and Conclusions

I think that the last paragraph of the discussion are the real conclusions (author should go deeper on these ideas and expand them a little) and the conclussions paragraph should be placed as the last paragraph of the discussion. Also, given the information provided in section 4.1.1, where authors remark the scarcity of studies on this topic and the contradictory results found by these studies, I miss an extra sentence in the conclusions where authors emphasize the urgent need for further studies to accumulate more scientific evidence which help to disentangle these questions.

Author Response

Reviewer 3

GENERAL COMMENTS

In this paper, Maszczyk et al. study the individual and combined effect of nanoplastics (direct effect) and antibiotics (indirect effect through their impact on gut microbiota) on different life history traits as well as on taxonomic and functional diversity of the cladoceran Daphnia magna. The paper provides very interesting results on a topic for which there do not exist enough evidence nowadays, so I consider this study has enough interest to deserve its publication after solving some minor issues. My main concerns have to do with clarifying confusing statements, adding some extra text to acknowledge some limitations and put the results in a broader context and increasing the quality of the figures, which must be definitively improved. For instance, in its current state, it is difficult to follow when the results refer to the effect of a single stressor or the combined effect, if these effects are negative or positive, and if they are significant or not. I think this issue can be easily solved if authors put them in a tidiest way, like a routine: first, single effect of NPs (if they are positive or negative and significant or non-significant on every response variable), then the single effect of enrofloxacin (negative, positive, significant, non-significant, etc.), and then the combined effect of both stressors (negative, positive, significant, non-significant, etc.). It would be very helpful to readers if authors structure the discussion following the same structure. I must say that I know that my comments can be difficult to follow due to the lack of line numbers through the text (I hope not). By the way, authors must use italic font to write the name of the species (Dapnia magna) everywhere through the text.

• In accordance with the general comment, we have rewritten the results section by presenting first the single effect of NPs, then the single effect of enrofloxacin, and then the combined effect of both stressors. We also structured the Discussion section following the same structure, and added an extra text in several places to acknowledge some limitations and put the results in a broader context (e.g. L 570-576, 582-589, 622-627, 691-700, 719-730, 748-755, 763-768). Finally, we also increased the quality of the figures and improved text formatting of the revised manuscript, including Latin species name formatting.

MINOR COMMENTS

Abstract

First sentence. The use of there is confusing. The behaviour and availability of what can influence? Nanoplastics? Pollutants? Antibiotics? Please, rewrite.

• In accordance with the comment, we have rewritten the first sentence of the abstract in the revised text by stating that: “The effect of nanoplastics (NPs) has been shown to interact with the effect of pollutants, including antibiotics.”

Penultimate sentence. A result can be significant or not significant according to the level chosen but not very or less significant. So, I would say that the negative effects were significant and all the response variables measured but they were more obvious on body size and the metabolic fingerprint while less notorious on reproductive parameters and taxonomic diversity.

• In accordance with the comment, we have rewritten the penultimate sentence. In the revised text we stated that “Our results supported the hypothesis as each of the stressors on its own influenced most of the measured parameters and as there was an interaction in the effect of both stressors on all of the measured parameters.”

Introduction

Paragraph 2, last sentence. Authors mention plasticizers antioxidants, flame retardants and UV stabilizers. It would be great they would expand how these substances, i.e. the mechanisms, can affect organisms.

• In accordance with the comment, we expanded the text with the explanation how the common groups of additives may affect the organisms by stating that: “Among the most common additives are plasticisers, which may affected life history and morphology in Daphnia magna (Schrank et al., 2019), flame retardants that may cause induced significant sublethal chronic toxicity to D. magna (Song et al., 2021), antioxidant that may reduce hatching rates, increase malformation rates and decrease the length of calcified vertebrae (Zhao et al., 2020), and UV stabilisers that recently have been revealed causing potential immune dysfunction (Li et al., 2019).”

Schrank, I., Trotter, B., Dummert, J., Scholz-Böttcher, B.M., Löder, M.G., Laforsch, C. Effects of microplastic particles and leaching additive on the life history and morphology of Daphnia magna. Environment. Pollut. 2019, 255, 113233.

Song, J., Na, J., An, D., Jung, J. Role of benzophenone-3 additive in chronic toxicity of polyethylene microplastic fragments to Daphnia magna. Sci. Tot. Environm. 2021, 800, 149638.

Zhao, H.J., Xu, J.K., Yan, Z.H., Ren, H.Q., Zhang, Y. Microplastics enhance the developmental toxicity of synthetic phenolic antioxidants by disturbing the thyroid function and metabolism in developing zebrafish. Environ. Internat., 2020, 140, 105750.

Li, Z., Li, W., Zha, J., Chen, H., Martyniuk, C.J., Liang, X. Transcriptome analysis reveals benzotriazole ultraviolet stabilizers regulate networks related to inflammation in juvenile zebrafish (Danio rerio) brain. Environmental Toxicol., 2019, 34, 112-122.

Last paragraph. Related to my statements in the general comments, the aims and results of the paper are here shown in a more understandable way than in the abstract. Maybe authors can take this paragraph as an inspiration to rewrite the abstract. For instance, is clearly mentioned the assessment of the single and combined effect of NPs and antibiotics. However, I consider that these paragraph spoil all the results. I find correct to anticipate the main results in the last paragraph of the introduction but in a slightly way, not so detailed. I think that something like ‘We found that both NPs and enrofloxacin affect the life history traits of Daphnia magna as well as the taxonomic diversity and metabolic fingerprint of their gut microbiota, and there is and interaction in the effect of both stressors’ is enough for the introduction section. Then, all these results can be detailed in their own section.  

• We followed the recommendation and have rewritten the result section of the abstract.

Material and Methods

2.1. This paragraph should not be in italic font.

• Many formatting errors were made when pasting text into a template during submission procedure. We improved the formatting in the revised text, including the italic font in this paragraph.

Can authors provide any difference among clones (in size or whatever variable) or it is just to reflect their different locations?

Each replicate of the experiment was performed using a different clone of D. magna in order to assess the species- rather than clone-specific effects. This is a standard procedure in experiments using Daphnia as a model. In accordance with the comment, we added this explanation in the revised text (L 137-140). In the same section, we also added the mean body size at first reproduction of each of the clones.

2.3. Please, add the units for L and W in the first brackets.

• Corrected (L 166)

2.4.1. Why authors used these concentrations of enrofloxacin? Can these levels be found in natural environments?

• In accordance with the comment, we added the statement in the revised text that: “The concentrations used in our study are within the environmental concentration range (Fair et al., 2014, Zainab et al., 2020, Zahou et al., 2018, Kovalakova et al., 2020, Duan et al., 2022).” (L 184-185).

Fair, R.J., Tor, Y. Antibiotics and bacterial resistance in the 21st century. Perspectiv. Medicin. Chem. 2014, 6, PMC-S14459.

Zainab, S.M., Junaid, M., Xu, N., Malik, R.N. Antibiotics and antibiotic resistant genes (ARGs) in groundwater: A global review on dissemination, sources, interactions, environmental and human health risks. Water Res. 2020, 187, 116455.

Zhou, L., Limbu, S. M., Shen, M., Zhai, W., Qiao, F., He, A., Zhang, M. Environmental concentrations of antibiotics impair zebrafish gut health. Environment. Pollut. 2018, 235, 245-254.

Kovalakova, P., Cizmas, L., McDonald, T. J., Marsalek, B., Feng, M., Sharma, V.K. Occurrence and toxicity of antibiotics in the aquatic environment: A review. Chemosphere 2020, 251, 126351.

Duan, W., Cui, H., Jia, X., Huang, X. Occurrence and ecotoxicity of sulfonamides in the aquatic environment: A review. Sci. Tot. Environ., 2022, 153178.

2.5. If assumptions for the parametric test were not met, why authors have not employed classical non parametric alternatives such as Sheirer Ray Hare text in the case of two or less levels for any treatment or Pairwise Mann-Withney test (function pairwise.wilcox.text in R) in the case of more than two levels for any treatment? As far as I know, Tukey is the post hoc test usually employed when the assumptions of normality and homocedasticity are met. Moreover, have they used any p-value correction such as Bonferroni to be more restrictive with the significance level? I would like to see the results applying this kind of correction and check if the differ from the current results for any response variable. Why Bray-Curtis distance instance of any other? I am not saying that this is wrong, but authors must justify their decision according to their objectives as there are many other similarity indexes and each one is more appropriated to each particular objective.

• In the previous version of the manuscript, we winsorized the data in order to obtain the normality of their distribution, and then we performed a standard parametric two-way ANOVAs with Tukey post-hoc tests. However, we agree with the comment that using a non-parametric test is a more appropriate solution. Therefore, in the revised manuscript we reanalysed the data by using a non-parametric test, the recently recommended Aligned Rank Transform (ATR) ANOVA (e.g. Wobbrock et al. 2011, Elkin et al. 2021, Kay et al. 2021), which offers higher statistical power compared to classical non-parametric tests. After the main analysis, we used the Bonferroni-Holm correction for p-values for multiple comparisons. (L 311-321). In accordance with the comment, we also added the explanation of why we used Bray-Curtis and not another similar statistical tool (L 322-327).

Kay, M., Elkin, L. A., Higgins, J. J., and Wobbrock, J. O. (2021). ARTool: Aligned Rank Transform for Nonparametric Factorial ANOVAs. R package version 0.11.1, https://github.com/mjskay/ARTool. DOI: 10.5281/zenodo.594511.

Wobbrock, J. O., Findlater, L., Gergle, D., and Higgins, J. J. (2011). The Aligned Rank Transform for Nonparametric Factorial Analyses Using Only ANOVA Procedures. Proceedings of the ACM Conference on Human Factors in Computing Systems (CHI 2011). Vancouver, British Columbia (May 7-12, 2011). New York: ACM Press, pp. 143-146. https://depts.washington.edu/acelab/proj/art/. DOI: 10.1145/1978942.1978963.

Elkin, L. A., Kay, M, Higgins, J. J., and Wobbrock, J. O. (2021). An Aligned Rank Transform Procedure for Multifactor Contrast Tests. Proceedings of the ACM Symposium on User Interface Software and Technology (UIST 2021). Virtual Event (October 10-14, 2021). New York: ACM Press, pp. 754-768. DOI: 10.1145/3472749.3474784.

Results

Figure 1. If the figure includes the test results, I consider that is more informative to show the error standard bars (as in Figure 2) or confidence interval bars instead of SD bars.

• In accordance with the comment, we added error standard bars instead of standard deviation bars in Fig. 1.

Figure 4. Please, provide the stress value. This result does not seem very logical according to the clustering of treatments. Could this mess be clarified adding a third dimension?

• Thank you for drawing our attention to the lack of stress values. The stress value was lost from the chart by mistake while editing it. We added the stress value (stress = 0.045) in Figure 4. In the literature, stress values equal to or below 0.1 are considered fair, while values equal to or below 0.05 indicate good fit. The stress values in our analyses suggest that the grouping is statistically significant and allowed for further interpretation of results. The three-dimensional analysis only slightly reduces the stress value to about 0.024. The relationship between the variants remained as in the case of two-dimensional analyses.

3.3. Low taxonomic diversity of bacteria living in the digestive tract of Daphnia… compared to what? In the third sentence, there is one ‘the’ (with increasing the concentrations of enrofloxacin) that should be removed.

• We agree that the comparison needs a point of reference. Our intention was to compare the Daphnia microbiome of our experiment with data describing the typical diversity of bacteria living in lake waters and bacteria from Daphnia digestive tracts described in other studies. We have added information about this and appropriate references in the text of the article (page 11 section 3.3). We removed ‘the’ as suggested.

Figure 5. The consistent increase in Firmicutes is very interesting. I think authors should expand the explanations and implications of this increase in the Discussion section.

• In accordance to the comment, we expand the explanations and implications of the Firmicutes’ increase in several places in the Discussion section of the revised text by stating that: “On the one hand, the relative increase of Firmicutes may have a positive effect on Daphnia, as it increases the diversity of the microbial composition, which is often equated with the improvement of the host's health [101]. It has been disclosed that Firmicutes produce short-chain fatty acids, which could be used for de novo synthesis of lipids or glucose and an additional energy source for the host [23, 102, 103]. Therefore, from the perspective of host energy input, Firmicutes in the gut may play a positive role. In other studies, it has been displayed that an increased proportion of Firmicutes to other bacteria phyla is an indicator of metabolic disorders in animals [104, 105], which may explain the reduction in body size and reproductive potential of Daphnia by each of the stressors on its own in our study.” (L 691-700) and that “However, our study contradicts the results of He et al. [23], who found a decrease in the relative abundance of Firmicutes in males exposed to (3.45 mg × g⁻¹) dietary polystyrene NPs.” (L 712-714). We also refer to other studies where the selective effect of antibiotics on the microbiota and the potential importance of the increase in Firmicutes share for Daphnia was found stating that: “In other study Motiei et al. [69] showed that G + bacteria, mostly Actinobacteria and Firmicutes, were better equipped to withstand ciprofloxacin (similar in mechanism of action to enrofloxacin) exposure as their relative abundance increased with antibiotic concentration.” (L 719-730).

Figure 6. Please, provide the stress value. Again, the clustering of treatments does not seem to follow any logical order. Could this mess be clarified adding a third dimension?

• Similar to Figure 4, we added the stress value to the drawing. As in the case of the metabolic analysis, the level of the stress value (0.055) allowed for a reliable interpretation of the NMDS taxonomic clustering results.
  

Discussion and Conclusions

I think that the last paragraph of the discussion are the real conclusions (author should go deeper on these ideas and expand them a little) and the conclusions paragraph should be placed as the last paragraph of the discussion. Also, given the information provided in section 4.1.1, where authors remark the scarcity of studies on this topic and the contradictory results found by these studies, I miss an extra sentence in the conclusions where authors emphasize the urgent need for further studies to accumulate more scientific evidence which help to disentangle these questions.

• In accordance with the comment, we switched earlier conclusions with the last paragraph of the Discussion section and expanded the new paragraph on the conclusions of the scarcity of studies on the topic concerning the combined effect of the stressors on the life history parameters of freshwater organisms, as well as the metabolomic and taxonomic diversity of their intestinal microbial community and the contradictory results found by these studies.

Reviewer 4 Report

See attached file for full comments on each section of the manuscript.

Comments for author File: Comments.pdf

Author Response

Reviewer 4

Combined effects of polystyrene nanoplastics and enrofloxacin on the life histories and gut microbiota of Daphnia magna

Full text:

Overall, the construction of the paper made it difficult to read. There were many incomplete and run-on sentences that were hard to follow, and a small number of grammatical errors. More citations are necessary in the introduction and discussion. There were several instances in both sections where the authors wrote “several earlier studies” or “many earlier studies”, but then only cited a single study. Additionally, there are repetitive sentences throughout the text, some which are exact duplicates. Therefore, I believe extensive English changes are necessary for this manuscript. Daphnia magna and D. magna should be italicized throughout the text.

• We have carefully revised the manuscript in accordance with the comments received from the Reviewer. All modified sentences in the revised Ms are marked in blue. We have addressed all the points raised in the review and provided an itemised list of Reviewer comments (in black) and our responses (in blue). Mainly, we (1) corrected several incomplete and run-on sentences and improved formatting throughout the text, (2) added 14 new citations to the Introduction and Discussion sections, (3) clarified the description of our experimental design by rewriting several parts of the text of the Methods section, (4) reanalysed the data by using non-parametric test, removed all statistical tables from the main text and added the p-values at the ends of statements of significance, (5) made several corrections concerning the figures clarity and the correctness of the presented data, and (6) modified several parts of the Discussion section to diversify the interpretation of the results. Finally, the revised text has been edited by a native English speaker.

Abstract:

They talk about the results as either “very significant” or “less significant”. I believe that this is misleading and should either be significantly different, or not significantly different.

• We have rewritten the description of the results in the abstract by stating that: “Our results supported the hypothesis as each of the stressors on its own significantly influenced most of the measured parameters and as there was a significant interaction in the effect of both stressors on all of the measured parameters.”

Introduction:

Some sentences state facts that require citation.

• In accordance with the comment, we have added additional citations in the Introduction section of the revised text (e.g. Line 45, 57, 59, 71, 72, 74, 75, 101, 122).

Materials and Methods:

What was the purpose of using 3 different clones for the replicates? Because the clones come

from different bodies of water, are there natural differences in their populations and gut

microbiome that would exclude them from being “replicates” of one another?

• Each replicate of the experiment was performed using a different clone of D. magna originating from different ponds in order to assess the species- rather than clone-specific effects. This is a standard procedure in experiments using Daphnia as a model. In accordance with the comment, we added this explanation in the revised text (L 137-140). In the same section, we also added the mean body size at first reproduction of each of the clones.

They state that high temperature and low food conditions were used to “increase the Daphnia

filtration rate”. Could these conditions also be considered added stressors themselves, and

therefore potentially inflate observed impacts of the toxins. What were the effects under

“normal” laboratory conditions?

• The temperature used in our study was high (23 °C), but very close to the optimal temperature range of Daphnia magna, which was set between 16 and 22 °C (Bruijning et al., 2018). In accordance with the comment, we added this information in the revised text (L 187-188). Moreover, the temperature of 23^oC is common in ponds inhabited by D. magna during summer. The food concentration used in our study (0.6 mg C_org × L^-1) was also only slightly limiting (the incipient limiting level for D. magna is approximately between 0.6 and 0.8 mg C_org × L^-1), which enabled the production of a high number of eggs (up to 11) during the first reproduction. Additionally, it is worth adding that the conditions were the same for all of the treatments, and therefore potential stressful conditions due to slightly suboptimal temperature and food conditions were the same in all of the treatments. In accordance with the comment, we added in the Discussion section of the revised text the explanation on how suboptimal temperature and food conditions may have impacted the results obtained in our study by stating that: “…since we used in our study temperature and food conditions very close to optimal (23 ^°C and 0.6 mg C_org × L^-1), it is rather unlikely that the stress caused by suboptimal experimental conditions had any impact on the observed effects of NPs and enrofloxacin. On the other hand, it can be expected that the use of lower temperature and higher food concentration could reduce the Daphnia filtration rate and consequently could reduce the observed effects of NPs and enrofloxacin, although they would not change the direction of the effects.” (L 570-576)

Bruijning, M., ten Berge, A.C., Jongejans, E. Population‐level responses to temperature, density and clonal differences in Daphnia magna as revealed by integral projection modelling. Funct. Ecol., 2018, 32, 2407-2422.

Two conflicting statements are given about the way that media changes were conducted:

- “we removed Daphnia from each container using a strainer with a plankton net and

placed it temporarily in 250-ml glass containers with the respective media”

- “We removed individuals from each container using a strainer with a plankton net and

placed temporarily in 250-ml plastic containers with milliQ water”

I am not sure which of these is correct, but it would be improper to place the Daphnia in milliQ water while the exposure media was changed.

• We have rewritten this part of the Methods section (2.4.1. Experimental protocol) to make it clearer that we used “250-ml glass containers with the respective media” during media exchange and when the Daphnia were photographed, and that we used “sterile 100-ml plastic containers with milliQ water to remove non-symbiotic bacteria from their guts” only at the end of the experiments after Daphnia were photographed. (L 211-218).

Section 2.4.2 states that:

“Additionally, in the first replicate of the experiments… Then we used two randomly selected individual groups from each variant...” From this I conclude that two random groups were selected from only the first experimental replicate to assess gut microbiota. If this is correct, than you do not have true replicates from the microbiota portion of this research. This would need to be repeated for true replication.

I do not believe it is appropriate to Winsorize this type of data. Winsorization modifies a data set, and the authors do not provide a reason for choosing to do this instead of simply using nonparametric methods of analysis.

• In accordance with the comment, we corrected the incorrect statement “in the first replicate of the experiments…” by replacing it with “At the end of each of the replicates of the experiments…”.

We agree with the comment that using a non-parametric test is a more appropriate solution to analyse our data. Therefore, in the revised manuscript we reanalysed the data by using a non-parametric test, the recently recommended Aligned Rank Transform (ATR) ANOVA (e.g. Wobbrock et al. 2011, Elkin et al. 2021, Kay et al. 2021), which offers higher statistical power, compared to classical non-parametric tests. After the main analysis, we used the Bonferroni-Holm correction for p-values for multiple comparisons. (L 311-321).

Kay, M., Elkin, L. A., Higgins, J. J., and Wobbrock, J. O. (2021). ARTool: Aligned Rank Transform for Nonparametric Factorial ANOVAs. R package version 0.11.1, https://github.com/mjskay/ARTool. DOI: 10.5281/zenodo.594511.

Wobbrock, J. O., Findlater, L., Gergle, D., and Higgins, J. J. (2011). The Aligned Rank Transform for Nonparametric Factorial Analyses Using Only ANOVA Procedures. Proceedings of the ACM Conference on Human Factors in Computing Systems (CHI 2011). Vancouver, British Columbia (May 7-12, 2011). New York: ACM Press, pp. 143-146. https://depts.washington.edu/acelab/proj/art/. DOI: 10.1145/1978942.1978963.

Elkin, L. A., Kay, M, Higgins, J. J., and Wobbrock, J. O. (2021). An Aligned Rank Transform Procedure for Multifactor Contrast Tests. Proceedings of the ACM Symposium on User Interface Software and Technology (UIST 2021). Virtual Event (October 10-14, 2021). New York: ACM Press, pp. 754-768. DOI: 10.1145/3472749.3474784.

Results:

P-values should be given at the ends of statements of significance instead of forcing the reader to reference the supplementary material each time. It is very difficult to go back and forth. ANOVA tables are not necessary in the text if the p-values are given in the text.

• In accordance with the comment, in the revised text, we removed all statistical tables from the main text and added the p-values at the ends of statements of significance for the results of life history parameters and metabolic rate, for main effects (ART ANOVA) and for planned contrast for interactions. We left the tables with the detailed statistical analysis in the supplementary materials.

The bar graphs are very busy and difficult to decipher. They need a better way to designate the important significant differences for the reader. Additionally, The amount of significant

differences they identify seem high in relation to the figures they present. Specifically, the top few graphs in Figure 1. The figures all appear very blurry. How is “clutch volume” telling you something different than “eggs per clutch” and “egg volume.” It seems that if you have the later two, than the former can simply be inferred.

• In accordance with the comment, we made several corrections concerning the figures’ clarity and the correctness of the presented data in the revised manuscript. First, we improved the resolution of the figures. Second, we modified the formatting of Figure 1 and added the standard error bars instead of standard deviations. Third, we reanalysed the data of life history traits in different treatments (Fig. 1) by using more appropriate statistics (a non-parametric Aligned Rank Transform ANOVA with the Bonferroni-Holm correction for multiple comparisons). Finally, we removed from the Fig. 1 the graph showing the clutch volume in different treatments. We left the description of the results for the clutch volume and their interpretation in the text of the manuscript as all of the measurements concerning the fecundity (eggs per clutch, egg volume, clutch volume) have different meaning and are important from the ecological point of view: (1) eggs per clutch shows how many offspring will be produced, (2) egg volume shows how many resources is invested in producing an individual, and (3) clutch volume is the common currency of the total reproductive investment.

Section 3.3 gives no statistical values or indicates that “significant” changes were identified. If no significant differences were found, then that should be stated.

• The taxonomic analyses were done after rarefaction curves analyses and data were rarefied to a sampling-depth equal to the lowest frequency among samples - 23500 reads. The frequency analyses at the Phylum and Family levels were done using the entire dataset. The percentages of taxons at the Phylum level presented in Fig. 5 in Chapter 3.3. were also calculated for the full dataset and reflect the complete analyses of illumine sequencing data from experimental variants. The technique of analysing this kind of data that we use are commonly accepted in the literature, and we think that the differences we have found and described are informative. Additionally, the analyses of stress in NMDS analyses of taxonomic results at the Family level (we added stress values in Fig. 6 in the revised manuscript) inform that the grouping of variants is statistically significant.

Discussion:

They do not discuss the warmer/low food conditions that they used and how these may have

impacted the results of their study in comparison to others.

• In accordance with the comment, we added in the Discussion section of the revised text the explanation on how suboptimal temperature and food conditions may have

impacted the results of the results obtained in our study by stating that: “On the one hand, since we used in our study temperature and food conditions very close to optimal (23 ^°C and 0.6 mg C_org × L^-1), it is rather unlikely that the stress caused by suboptimal experimental conditions had any impact on the observed effects of NPs and enrofloxacin. On the other hand, it can be expected that the use of lower temperature and higher food concentration could reduce the Daphnia filtration rate and consequently could reduce the observed effects, although they would not change the direction of the effects.” (L 570-576)

They are only exposing the daphnia until they are 5 days old, and though they note that clutch sizes and volumes are lower in certain treatments, they should discuss if exposure to the stressors is simply delaying start of reproduction.

• In accordance with the comment, we added in the results section the information that: “The number of ovigerous females in relation to the females without eggs was the greatest in the control (84%), moderate in the presence of NPs on its own (81-83%) and in the presence of enrofloxacin on its own (79-80%), and was the lowest in N_mE_l (70%) and N_hE_h (75%) treatments. (L 364-367) Also in accordance with the comment, we added the short interpretation of the results in the context of the effect of both stressors on the age of the first reproduction by stating that: “Although we did not assess the age of the first reproduction of Daphnia in the experiments, the greatest number of ovigerous females (in relation to the females without eggs) during the fifth day of the experiment in the control, moderate in the presence of each of the stressors on its own, and the lowest in the treatments with the combined stressors, suggest that each of the stressors delayed the start of reproduction and that there was an interaction in the effect of both stressors.” (L 583-589)

If they say “several earlier studies” or “many earlier studies” then more than one study should be provided in citation. Some sections are repetitive. They repeat multiple times they their hypotheses were confirmed. Much of the discussion feels like a restating of the results.

• In accordance with the comment, we added more citations in each sentence in which we state “several earlier studies” or “many earlier studies” (e.g. L 602, 704). Moreover, we reduced several references to our hypothesis leaving only the main references at the beginning of the 4 paragraphs of Discussion section. Finally, we also diversified the discussion by adding additional interpretations to it (e.g. L 570-576, 582-589, 625-628, 633-636, 691-700, 712-714, 748-755, 763-768).

There is very little attempt to explain the results they found other than stating that they agree with existing literature. Furthermore, I believe they need to explain why the impacts they found were not dose dependent. What are the implications if significant differences were only seen in medium or low doses, for example. What trends do we draw here?

• If we understand correctly, the Reviewer’s comment concerns the issue of the literature context of the changes in the taxonomic structure of the Daphnia microbiota. In the Discussion section, we tried to relate the results we obtained to the results found by other researchers. Further research would be required to elucidate the complex interactions between nanoplastics and antibiotics. In accordance with the comment, we suggest in the last paragraph in the Discussion section of the revised text (Chapter 4.1.3) that our observations “may result from the system’s complexity, where NPs constitute the sorption surface for dissolved organic compounds and antibiotics, causing both their local concentration to increase and a change in the conformation of sorbed molecules that can change their physical properties. Thus, it can cause both inactivation of the antibiotic and a difference in the ability of the adsorbed organic compounds. The formation of biofilms on aggregated plastic particles may also change the dominant bacteria’s taxonomic structure”. Moreover, to clarify the role of antibiotics, we added a short information (Discussion section of the revised text, Chapter 4.1.3) about the possible enrofloxacin level selective pressure on Firmicutes, which in other studies (Motiei et al. 2020) have been suggested to be “better equipped to withstand ciprofloxacin (similar in mechanism of action to enrofloxacin) exposure”. Finally, we also expanded the interpretation of our results stating that “the interactive effects observed in our study may stem not only from the interactive effect of the stressors on the animal physiology but also from absorption of the enrofloxacin at the NPs surface.” (L 625-628).

 

Motiei, A., Brindefalk, B., Ogonowski, M., El-Shehawy, R., Pastuszek, P., Ek, K., Gorokhova, E. Disparate effects of antibiotic-induced microbiome change and enhanced fitness in Daphnia magna. PloS One, 2020, 15, e0214833.

Additionally, they claim taxonomic diversity was impacted, but this is not in agreement with the results section (3.3) which stated no significant differences.

• In section 3.3, we made the statement that “The presence of each of the stressors, especially high concentration of NPs increased Firmicutes participation.” We also emphasise that the result of the NMDS analysis showed significant changes in the taxonomic structure of the Daphnia microbiota, especially in the presence of high concentrations of stressors, which may result from the variability within Phylum Firmicutes. The only non-significant correlation we found was between taxonomic and metabolic diversity, which may suggest a high redundancy of Daphnia guts bacterial community.

Finally, they do not tell the reader if the impacts observed in this study actually negatively affect the daphnia. There needs to be a better explanation of the connection between the changes observed and their negative impacts.

• In accordance with the comment, we added a discussion of the results in the context of the negative effects of both stressors and interaction in their effect in numerous places in the revised text (e.g. L 582-583, 601-604, 606-607, 621-628, 633-636, 659-662, 710-712, 734-739, 745-747).

Author Response File: Author Response.doc

Round 2

Reviewer 2 Report

The authors revised the manuscript by reflecting previous comments. Still, as entitled, the combined effect is the major part, however, it is insufficient to explain the combined effects and to discuss.

Also, the figures are too complicated, it is not easy to know the significance.

Author Response

Comments and Suggestions for Authors

 

Reviewer 2

 

We would like to thank the Reviewer for further important comments. All corrections in the revised manuscript are marked in blue.

 

The authors revised the manuscript by reflecting previous comments. Still, as entitled, the combined effect is the major part, however, it is insufficient to explain the combined effects and to discuss.

• In accordance with the comment, we rewritten the significant part of the Results section describing the results more clearly and eliminating some inaccuracies. Moreover, we also improved the visualization of our results: (1) by adding a new set of figures (Fig. 2 and 4) presenting the combined data from all levels of NPs density (N_l = 10³, Nm = 10⁶, and N_h = 10⁹ particles L⁻¹) and from all levels of enrofloxacin concentration (E_l = 10 and E_h = 100 ng L⁻¹), for which the interactive effect of both factors is more transparent, (2) by modifying already existing figures (Fig. 5 and 7 in the revised text) by adding an additional panel presenting the combined data from all levels of NPs density and from all levels of enrofloxacin concentration, and (3) by modifying Fig. 1-4 in the revised text by marking the effect of NPs and enrofloxacin in different colors. Finally, we also rewritten several parts of the Discussion section to interpret our results more clearly and precisely.

 

Also, the figures are too complicated, it is not easy to know the significance.

• In accordance with the comment, we improved the visualization of our results: (1) by adding a new set of figures (Fig. 2 and 4) presenting the combined data from all levels of NPs density and from all levels of enrofloxacin concentration, for which the interactive effect of both factors is more transparent, (2) by modifying already existing figures (Fig. 5 and 7 in the revised text) by adding an additional panel presenting the combined data from all concentrations of both factors, and (3) by modifying Fig. 1-4 in the revised text by marking the effect of NPs and enrofloxacin in different colors.

 

Author Response File: Author Response.doc

Reviewer 4 Report

My comments have been addressed satisfactorily by the authors and I have no further changes to suggest.

Author Response

Reviewer 4

 

In accordance with the remark, we made numerous linguistic correction in the revised text – all corrections are marked in blue.