Individual and Combined Toxic Effects of Nano-ZnO and Polyethylene Microplastics on Mosquito Fish (Gambusia holbrooki)

Round 1

Reviewer 1 Report

Nano-ZnO and microplastics have been a topic of worldwide concern because of their high concentration and potential ecological risk in the environment. In this work, the authors investigated Individual and combined toxic effects of Nano-ZnO and polyethylene microplastics on mosquito fish. This is well designed and of great practical significance, which provide useful information on understanding combined toxic mechanism of nanoplastics and Nano-ZnO in fish. The manuscript is well written and structured, the research methods are timely and innovative, and the results are analyzed in depth. However, some details should be revised to fulfill the goals of this study. Thus, I recommend accepting this manuscript after a few minor amendments listed below.

1.      The abstract section should briefly introduce the research background and significance, clarify the research methods, introduce the main research results, and finally, give the corresponding conclusions. The abstract of this article looks ok, but some descriptions are redundant, and I hope to make a comprehensive modification.

2.      The material and method section needs to be re-integrated and checked to avoid repetition and confusion. All experimental protocols should be better explained.

3.      Line 222: H₂O₂. Please check it throughout the manuscript.

4.      Line 230: More relevant reference should be added, e.g. doi.org/10.1016/j.chemosphere.2021.132941; doi.org/10.1016/j.envpol.2022.120029

5.      Define and explain all acronyms and abbreviations on first mention in the text.

On first mention of a species in the text give both the common (trivial) and formal name, and make sure that the presentation is correct and consistent.

Make sure that symbols, sub- and super-scripts, upper- and lower-case are presented correctly, and that there is correct and consistent use of italics, brackets and punctuation etc.

Author Response

Authors are thankful of the reviewers for their insightful comments

All the revisions are made in the manuscript with the track changes mode.

Comments from Reviewer 1:

Response: Authors have incorporated the suggestion made by reviewer.

1. The abstract section should briefly introduce the research background and significance, clarify the research methods, introduce the main research results, and finally, give the corresponding conclusions. The abstract of this article looks ok, but some descriptions are redundant, and I hope to make a comprehensive modification.

• Response: Made the revisions as per the suggestions while considering the limitations of 200 words only.

2. The material and method section need to be re-integrated and checked to avoid repetition and confusion. All experimental protocols should be better explained.

• Response: Modified and reframed as per the suggestion.

3. Line 222: H₂O₂. Please check it throughout the manuscript.

• Response: Correction made

4. Line 230: More relevant reference should be added, e.g. org/10.1016/j.chemosphere.2021.132941; doi.org/10.1016/j.envpol.2022.120029

• Response: Incorporated the suggestions.

5. Define and explain all acronyms and abbreviations on first mention in the text.

• Response: Modified and reframed as per the suggestion.

6. On first mention of a species in the text give both the common (trivial) and formal name, and make sure that the presentation is correct and consistent.
   • Response: Made the changes as per the suggestion.

7. Make sure that symbols, sub- and super-scripts, upper- and lower-case are presented correctly, and that there is correct and consistent use of italics, brackets, and punctuation etc.

• Response: Modified and checked

Reviewer 2 Report

Overall, the abstract provides a clear summary of the research conducted, but there are some areas that could be improved for clarity and precision.

• Line 12: Specify the type of aquatic system being studied (e.g., freshwater, marine).
• Line 13: Clarify what is meant by "interaction" of microplastic and zinc oxide nanoparticles. Are they being studied in combination or separately?
• Line 14: Provide a brief background on why Gambusia was chosen as the model organism.
• Line 17: Specify what the abbreviations CAT, SOD, GPx, and GR stand for.
• Line 18: Mention the direction of the alteration in antioxidant system activity (e.g., increased or decreased).
• Line 21: Clarify whether the reduction in total antioxidant levels is significant compared to the control group or within the treated groups only.
• Line 22-23: Provide more details on the accumulation study, such as the duration of exposure and the method of analysis.
• Line 24: Clarify whether the PE-MPs are causing the ZnO-NPs-induced toxicity or if they are simply facilitating it.

·         Overall, the introduction provides a good overview of the problem and the background information related to plastic pollution, microplastics, and their impact on aquatic ecosystems. However, some changes can be made to improve the clarity and flow of the text.

• Line 30: "usually break down" can be changed to "can break down" for more accuracy.
• Line 33: "raw materials" can be changed to "ingredients" to be more precise.
• Line 36: "invertebrates, fish, birds, and mammals" can be changed to "a variety of aquatic organisms" for clarity.
• Line 41: "could affect" can be changed to "have been shown to affect" for more clarity.
• Line 45: "the intestine’s microflora" can be changed to "intestinal microbiota" for accuracy.
• Line 49: "potential" can be changed to "properties" to be more specific.
• Line 51: "is found to be toxic for aquatic animals" can be changed to "has been shown to be toxic to aquatic animals" for accuracy.
• Line 58: "invertebrates and fish" can be changed to "various aquatic organisms" for clarity.
• correct spelling of "peroxide hydrogen" to "hydrogen peroxide"
• Change the spelling of "aquaria" to "aquariums"
• Line 158: It might be helpful to provide a brief introduction or context for the FTIR assay. For example, you could mention why this assay was used or what it measures.
• Line 159: "FTIR graphs" could be more specific. Are these spectra or something else? It would be good to clarify.
• Line 160: Consider providing a bit more information about what the standard graph is, as it might not be immediately clear to the reader.
• In line 191, change "µgL-1" to "µg L-1".
•  In line 194, add a comma after "combined".
•  In line 174, add a comma after "200 µg L-1 PE-MPs".
•  In line 179, change "following" to "after".
• It may be helpful to specify that the units for SOD activity are missing, as they are provided for GPx and CAT activities.
• In the sentence "No significant change … group (Figure 4)", it may be clearer to state whether the lack of significance was determined by statistical analysis.
• In the sentence "Although …… reference group", it may be clearer to state whether the lack of significance was determined by statistical analysis.
• In the sentence "Results showed ….. in all exposure groups", it may be helpful to specify the unit for MDA contents (e.g., µmol/L).
• In the sentence "There was a significant …… and in combined", it may be clearer to specify whether the decrease was compared to the control group or to the baseline value before exposure.
• It may be helpful to provide a brief explanation of what is meant by "synergistic impact" and "development of oxidative stress" in this context, for readers who may not be familiar with the terminology.

Overall, the discussion provides a comprehensive analysis of the findings of the study. However, there are a few areas where the text could be improved:

·         In line 202, it would be helpful to provide some context or background information to support the statement about the importance of understanding the effects of microplastics and nanoparticles on biota.

·         In line 207, it would be helpful to specify the type of fish used in the study.

·         In line 213, it would be helpful to provide some additional information about the significance of enhanced Zn bioaccumulation.

·         In line 218, it would be helpful to explain what ROS are and why their production is a concern.

·         In lines 220-221, it would be helpful to clarify what GR is.

·         Instead of saying "non-significant modifications," it would be clearer to say "no significant differences."

·         It would be helpful to state the direction of the change (i.e., increase or decrease) in SOD, CAT, GPx, and GR activity in response to exposure to nanoparticles and PE-MPs.

·         It would be better to rephrase "Similar elevated values of SOD" to something like "Similarly, increased levels of SOD activity have also been reported."

·         230-231. Instead of using "in contrast" to introduce a contradictory study, it would be better to use a more neutral transition phrase like "On the other hand" or "In a different study."

·         232-233. It would be clearer to state explicitly that the recorded significantly higher levels of MDA in the tissue indicate damage caused by reactive oxygen species.

·         235-236. It would be helpful to explain what HSP expression and catalase activity are and how they relate to the response to ZnO-NPs.

·         237-238. Instead of "an accordant increase," it would be better to say "a similar increase" to avoid repeating the word "increase" twice in the same sentence. Additionally, it would be helpful to explain what LPO is and how it relates to the study.

• Line 240: "Besides regular antioxidant assay" is a bit vague. It might be better to specify what type of antioxidant assay was used.
• Line 245: "Bessemer along with his coworkers" should be rephrased as "study found that..".
• Line 249: "Synergism analysis of interaction" could be rephrased as "Analysis of the synergistic interaction".

Author Response

Authors are thankful of the reviewers for their insightful comments

All the revisions are made in the manuscript with the track changes mode.

Comments from Reviewer 2

1. Line 12: Specify the type of aquatic system being studied (e.g., freshwater, marine).
   • Response: Incorporated as per the suggestion.

2. Line 13: Clarify what is meant by "interaction" of microplastic and zinc oxide nanoparticles. Are they being studied in combination or separately?
   • Response: In this study, we assayed toxicity effects of microplastics and ZnO-nanoparticles, alone and in combination, on Gambusia.

3. Line 14: Provide a brief background on why Gambusia was chosen as the model organism.
   • Response: Modification has been made considering the limitations of 200 words and has been detailed in introduction section.
   • The mosquitofish (Gambusia holbrooki) is a viviparous fish (Cyprinodontiformes, Poeciliidae, Gambusia Sp.) that live in freshwater habitats. This specie often feeds from mosquito larvae. Therefore, the mosquitofish is usually used to biological control the larvae of vector insects.

4. Line 17: Specify what the abbreviations CAT, SOD, GPx, and GR stand for.
   • Response: Modified as per the suggestion and restraining to limitation of 200 words.

1. Line 18: Mention the direction of the alteration in antioxidant system activity (e.g., increased or decreased).
• Response: With due respect, it is already written as ‘elevated’ activity.

1. Line 21: Clarify whether the reduction in total antioxidant levels is significant compared to the control group or within the treated groups only.
• Response: Completed the result.

1. Line 22-23: Provide more details on the accumulation study, such as the duration of exposure and the method of analysis
• Response: After 14 days, the oxidative biomarkers and ZnO concentrations were assayed by spectrophotometric methods and inductively coupled plasma-optical emission spectrometry (ICP-OES), respectively. The microplastic and ZnO were detected by Fourier Transform Infrared Spectroscopy (FTIR).

8. Line 24: Clarify whether the PE-MPs are causing the ZnO-NPs-induced toxicity or if they are simply facilitating it.
   • Response: After comparative analysis on the endpoints studied such as antioxidant enzyme activity, TAN and MDA levels in the individual and combination experiment groups. Further, followed by the synergism analysis has inferred to the positive association of the combination in the toxicity.
   • MPs have been shown to be effective carriers of heavy metals in the environment. Studies have shown that MPs can absorb heavy metals from the surrounding environment and increase their bioavailability.

9. Line 30: "usually break down" can be changed to "can break down" for more accuracy.
   • Response: incorporated the suggestion.

1. Line 33: "raw materials" can be changed to "ingredients" to be more precise.

• Response: incorporated the suggestion.

11. Line 36: "invertebrates, fish, birds, and mammals" can be changed to "a variety of aquatic organisms" for clarity.
    • Response: incorporated the suggestion.

12. Line 41: "could affect" can be changed to "have been shown to affect" for more clarity.
    • Response: incorporated the suggestion.

13. Line 45: "the intestine’s microflora" can be changed to "intestinal microbiota" for accuracy.
    • Response: incorporated the suggestion.

14. Line 49: "potential" can be changed to "properties" to be more specific.
    • Response: Changed as per the suggestion.

1. Line 51: "is found to be toxic for aquatic animals" can be changed to "has been shown to be toxic to aquatic animals" for accuracy.

• Response: incorporated the suggestion.

1. Line 58: "invertebrates and fish" can be changed to "various aquatic organisms" for clarity.

• Response: incorporated the suggestion.

1. correct spelling of "peroxide hydrogen" to "hydrogen peroxide"

• Response: Made the correction.

1. Change the spelling of "aquaria" to "aquariums"

• Response: incorporated the suggestion.

19. Line 158: It might be helpful to provide a brief introduction or context for the FTIR assay. For example, you could mention why this assay was used or what it measures.

• Response: FTIR stands for Fourier Transform Infrared Spectroscopy, which is a technique used to analyze the chemical composition of materials by measuring how they interact with infrared light. It works by passing infrared light through a sample and measuring the amount of light that is absorbed at different frequencies. This absorption pattern produces a unique spectral fingerprint that can be used to identify and quantify the presence of various functional groups and chemical bonds in the sample. FTIR is commonly used in fields such as chemistry, biochemistry, pharmaceuticals, and materials science for analyzing and characterizing a wide range of samples, including solids, liquids, and gases.
1. Line 159: "FTIR graphs" could be more specific. Are these spectra or something else? It would be good to clarify
• FTIR (Fourier Transform Infrared Spectroscopy) is a common analytical technique for identifying and characterizing polyethylene plastics. The most prominent peaks in the FTIR spectrum of polyethylene are typically associated with vibrations of the CH₂ groups in the polymer backbone. Polyethylene has several characteristic peaks in its FTIR spectrum, including a strong peak at approximately 2916 cm^-1 corresponding to CH₂ stretching vibrations and a weaker peak at approximately 1460 cm^-1 corresponding to CH₂ bending vibrations. Other peaks may also be present depending on the specific type of polyethylene and any additives present. Also, the low-density polyethylene (LDPE) typically peaks at around 720 cm^-1, which is associated with rocking vibrations of the CH₂ groups. On the other hand, high-density polyethylene (HDPE) shows a peak at around 1375 cm^-1, which is attributed to CH₃ deformation vibrations.

21. Line 160: Consider providing a bit more information about what the standard graph is, as it might not be immediately clear to the reader.

• Response: In FTIR spectroscopy, a standard graph can be used to quantify the amount of a particular chemical component, such as an additive or impurity, in a polyethylene sample.

1. In line 191, change "µgL-1" to "µg L-1".
• Response: Made the correction.

1.  In line 194, add a comma after "combined".
• Response: With due respect we found adding a comma is not appropriate.

1.  In line 174, add a comma after "200 µg L-1 PE-MPs".
• Response: made the incorporation

1.  In line 179, change "following" to "after".
• Response: modified as per the suggestion.

1. It may be helpful to specify that the units for SOD activity are missing, as they are provided for GPx and CAT activities.
• Response: made the incorporation

1. In the sentence "No significant change … group (Figure 4)", it may be clearer to state whether the lack of significance was determined by statistical analysis.
• Response: Made the incorporation and modified as per the suggestion
2. In the sentence "Although …… reference group", it may be clearer to state whether the lack of significance was determined by statistical analysis.
• Response: Modified as per the suggestion.

29. In the sentence "Results showed ….. in all exposure groups", it may be helpful to specify the unit for MDA contents (e.g., µmol/L).
    • Response: Improvised as per the suggestion.

1. In the sentence "There was a significant …… and in combined", it may be clearer to specify whether the decrease was compared to the control group or to the baseline value before exposure.
• Response: Suggested the incorporation.

1. It may be helpful to provide a brief explanation of what is meant by "synergistic impact" and "development of oxidative stress" in this context, for readers who may not be familiar with the terminology.

• Response: Synergistic impact refers to the combined effect of two or more things that is greater than the sum of their individual effects.
• A suppressive effect on biochemical parameters refers to the ability of a substance or intervention to reduce the levels or activity of specific biomolecules in the body.
• Oxidative stress is a biological process that occurs when cellular and molecular components of the body are exposed to high levels of reactive oxygen species (ROS) and free radicals.

1. In line 202, it would be helpful to provide some context or background information to support the statement about the importance of understanding the effects of microplastics and nanoparticles on biota.

• Response: Zinc oxide (ZnO) nanoparticles have unique physical and chemical properties that make them useful in a variety of applications, including electronics, optical devices, cosmetics, and sunscreens. Although, ZnO-NPs are using in drug delivery and cancer therapy, there are also concerns about the potential toxicity of ZnO-NPs. Studies have suggested that they may cause oxidative stress, inflammation, and DNA damage in cells.
• Microplastics can indeed act as carriers for heavy metals. Heavy metals are often attached to the surface of microplastics, which can then be transported by water and wind currents. This is a concern because microplastics are becoming increasingly prevalent in the environment, and their ability to transport heavy metals means that these toxic substances can potentially enter the food chain when ingested by aquatic organisms.
• Additionally, the small size of microplastics means that they can be ingested by a wide range of organisms, from zooplankton to larger marine mammals. Once ingested, these microplastics can release the heavy metals they carry into the digestive tract, where they can be absorbed by the organism's body tissues. This can lead to a range of negative health effects, including cellular damage and organ dysfunction.
• Overall, the presence of microplastics in the environment is a growing concern, and their role as carriers of heavy metals only adds to this problem. It is important to take steps to reduce the amount of plastic waste that enters the environment and to properly dispose of any plastic materials to prevent them from contributing to the accumulation of microplastics in marine, ocean and freshwater ecosystems.

33. In line 207, it would be helpful to specify the type of fish used in the study.
    • Response: Made the incorporation.

34. In line 213, it would be helpful to provide some additional information about the significance of enhanced Zn bioaccumulation.
    • Response: Explained as per the suggestion.

35. In line 218, it would be helpful to explain what ROS are and why their production is a concern.
    • Response: Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen, including superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (OH−). The excessive accumulation of ROS can cause damage to cellular components such as proteins, lipids, and DNA, leading to oxidative stress and contributing to the development of various diseases such as cancer, neurodegenerative disorders, and cardiovascular diseases.

36. In lines 220-221, it would be helpful to clarify what GR is.
    • Response: Clarified as per the suggestion.

37. Instead of saying "non-significant modifications," it would be clearer to say "no significant differences."
    • Response: Improvised as per the suggestion.

38. It would be helpful to state the direction of the change (i.e., increase or decrease) in SOD, CAT, GPx, and GR activity in response to exposure to nanoparticles and PE-MPs.
    • Response: Revised as per the suggestion.

39. It would be better to rephrase "Similar elevated values of SOD" to something like "Similarly, increased levels of SOD activity have also been reported."
    • Response: Improvised as per the suggestion.

40. 230-231. Instead of using "in contrast" to introduce a contradictory study, it would be better to use a more neutral transition phrase like "On the other hand" or "In a different study."
    • Response: Modifies as per the requirement.

41. 232-233. It would be clearer to state explicitly that the recorded significantly higher levels of MDA in the tissue indicate damage caused by reactive oxygen species.
    • Response: Revised as per the suggestion

42. 235-236. It would be helpful to explain what HSP expression and catalase activity are and how they relate to the response to ZnO-NPs.
    • Response: Although we did not measure HSP expression in this study, high levels of HSP expression have been associated with improved cell survival and resistance to stress-induced damage.
    • Catalase is important for preventing the accumulation of toxic levels of hydrogen peroxide within cells, which can damage cell membranes, proteins, and nucleic acids.

43. 237-238. Instead of "an accordant increase," it would be better to say "a similar increase" to avoid repeating the word "increase" twice in the same sentence. Additionally, it would be helpful to explain what LPO is and how it relates to the study.

• Response: Malondialdehyde (MDA) is a reactive organic compound that is formed as a byproduct of lipid peroxidation. In this process, reactive oxygen species (ROS) attack polyunsaturated fatty acids in cell membranes, forming lipid hydroperoxides. These hydroperoxides can then break down into various products, including MDA.

44. Line 240: "Besides regular antioxidant assay" is a bit vague. It might be better to specify what type of antioxidant assay was used.

• Response: Specified as per the suggestion.

45. Line 245: "Bessemer along with his coworkers" should be rephrased as "study found that..".
    • Response: Revised accordingly.

46. Line 249: "Synergism analysis of interaction" could be rephrased as "Analysis of the synergistic interaction".

• Response: Revised as per the suggestion.

Round 2

Reviewer 2 Report

recommend to accept.