Micro and Nanoplastic Contamination and Its Effects on Freshwater Mussels Caged in an Urban Area

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

The authors present the study entitled: Evidence of micro and nanoplastics contamination and effects in freshwater mussels caged at downstream an urban area.

 

It is a classic work of measuring the effect of a toxicant (musels), by evaluating biomarkers of stress and exposure. The work is well designed and executed, but there are some flaws that should be improved before publication in JoX.

 

Line 41. check the text in bold

L98. Celsius degree symbol

L102: Please, check "was were".

L108. Please, check the verb in "the mussel were..."

L147. Please indicate the brand and origin of the filters.

L152. Check micrometer abbreviation (um)

L190, Check ¨tdata" Word

 

The bibliography and citation style do not conform to the journal's guidelines.

 

Section: Material and methods

L176. Are borosilicate tubes suitable for fluorescence measurements?

 

Please, indicate the analytical procedure used for total protein quantification, I don't think it has been described/cited.

 

Indicate the coordinates (UTM) of the area where the specimens were captured.

 

Indicate the brand and origin of the polytron used and rpm/time used.

More information on the new cytometric method used (L124)

 

Indicate the significance level (*). Indicate the meaning of Ve/Vt. Rescale the Y-axis in subfigure "C).

 

Figure 3. Rescale the Y-axis of subfigure "A". Indicate the significance level (*) in the figure caption.

 

Figure4: Rescaling the y-axes of the 3 subfigures. Indicate the significance level

 

Figure 5. It would help a lot to understand the components if there was a central axis (x (0.0-0.0) Y (0.0-0.0) that divides the vectors into 4 quadrants.

 

I would appreciate a more complete commentary on the principal component analysis (PCA), it seems that there is a direct relationship between the different variables.

 

I missed a histological study revealing the presence of microplastics at the cellular level. I understand that this is outside the scope of the paper but it would have rounded out the study nicely. Perhaps in a second phase.

The bibliography and citation style do not conform to the journal's guidelines.

Comments on the Quality of English Language

There are some editing.

Author Response

Answers to comments

All comments were considered in the revision and the changes were provided in blue color for easy tracking.

Editorial comments

The work is well designed, but it has some inaccuracies that should be improved before publication in JoX.
- In the title it is indicated "Evidence of micro and nanoplastics contamination and effects in freshwater mussels caged at downstream an urban area", however, in the conclusion section, authors reported that this study 1) shows that freshwater mussels downstream large urban areas are exposed to plastic contamination and 2) strengthen the hypothesis that in-situ plastic contamination elicit deleterious physiological changes in E. complanata. So the term evidence, I think it is not appropriate and should be "Micro and nanoplastics contamination and potentiel effects in freshwater mussels caged at downstream an urban area". OK done
- Lines 97-98: Mussels were acclimated in 60 L aquariums (30-40 mussels each) for
20 days. After, lines 228-229: Changes relative to the lake mussels caged for
the same time period in the laboratory. And in the results section, lines 239-241: Nanoplastics were below the detection limit in digestive glands in mussels collected at the reference lake and caged in the laboratory for 3 months. This needs to be clarified and clearly specified in the section. Yes, the lake mussels were placed in nets in the laboratory. This point was clarified at lines 103-104 and 215.

- Lines 109-111: Water samples were also collected near the cages at the beginning, after 1.5 month, and at end of the exposure period for chemical analysis (pH, conductivity, total suspended solids, dissolved organic carbon and ammonia concentrations). Why authors did not analyzed also in these water samples, micro and nanoplastics? It was not deemed necessary since they were grap samples and the amount of materials would have been low or below the detection limit of the method.

- Lines 133-134: the fluorescence was measured (excitation 450 nm, emission 620
nm) as described above. Above there are only references cited and no description about fluorescence? This needs to be clarified. Done see lines 128-29 and 133-134.

- Lines 132-133: For each sample, 50 μL of 100 μM DCVJ (in water) were added and the fluorescence was measured. Is DCVJ react with nanoparticles and these after were measured indirectly by fluorescence? This needs to be clarified. Yes the probe DCVJ react directly on PSNPs. See lines 128-29.

- Why authors did not analyzed all physiological parameters in caged mussels after 1 week and compared results at the end (after 3 months)? As indicated in discussion section that some results can be correlated with other contaminants present in the water and/or contaminants adsorbed on micro and nanoplastics? Comparaison of results between caged mussels and reference mussels caged in laboratory, does not allow us to prove the conclusions presented although the statistical analyzes showed significant correlations. We did not measure the baseline levels of biomarkers at the beginning since we included a laboratory control that revealed the baseline levels at the same duration of exposure. In this respect, these comparisons provide evidence of plastic contamination.

Reviewer 1

The authors present the study entitled: Evidence of micro and nanoplastics contamination and effects in freshwater mussels caged at downstream an urban area.

 

It is a classic work of measuring the effect of a toxicant (mussels), by evaluating biomarkers of stress and exposure. The work is well designed and executed, but there are some flaws that should be improved before publication in JoX.

 

Line 41. check the text in bold. ? I do not see the bold face

L98. Celsius degree symbol. Changed to ⁰

L102: Please, check "was were". Corrected

L108. Please, check the verb in "the mussel were..." Changed

L147. Please indicate the brand and origin of the filters. Done

L152. Check micrometer abbreviation (um). Done

L190, Check ¨tdata" Word. Checked

 

The bibliography and citation style do not conform to the journal's guidelines. The format of the references was changed following the journal’s guideline.

 

Section: Material and methods

L176. Are borosilicate tubes suitable for fluorescence measurements? Yes, this was included.

 

Please, indicate the analytical procedure used for total protein quantification, I don't think it has been described/cited. OK included at lines 134-136.

 

Indicate the coordinates (UTM) of the area where the specimens were captured. We did not include this since the lakes are under the jurisdiction of Québec fisheries and resources under a permit. The lakes were pristine i.e. far from roads and devoid of housings.

Indicate the brand and origin of the polytron used and rpm/time used. See lines 112-13.

More information on the new cytometric method used (L124). This was added in the revision.

 

Indicate the significance level (*). Indicate the meaning of Ve/Vtàlines 129-31. Rescale the Y-axis in subfigure "C). Not able to rescale it by the software.

 

Figure 3. Rescale the Y-axis of subfigure "A". Indicate the significance level (*) in the figure caption. Done

 

Figure4: Rescaling the y-axes of the 3 subfigures. Indicate the significance level Done.

 

Figure 5. It would help a lot to understand the components if there was a central axis (x (0.0-0.0) Y (0.0-0.0) that divides the vectors into 4 quadrants. Done

 

I would appreciate a more complete commentary on the principal component analysis (PCA), it seems that there is a direct relationship between the different variables. Lines 281-88.

 

I missed a histological study revealing the presence of microplastics at the cellular level. I understand that this is outside the scope of the paper but it would have rounded out the study nicely. Perhaps in a second phase. Indeed !

The bibliography and citation style do not conform to the journal's guidelines.

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript "Evidence of micro and nanoplastics contamination and effects in freshwater mussels caged at downstream an urban area" shed light on the current situation regarding plastic pollution.

I found article written well using current literature to justify the outcomes of study.

The methodology used is up to the mark, results are well described and justified.

·     Authors have evaluated stress biomarkers and correlated them with the exposure of toxicants using well designed methodology and planning. The number of animals in each group is sufficient looking into the current trends for using animals in experiments.

·       The pollution research has great significance in current situation, present study adds little more about the ways for evaluation the assessment of pollution from one place to distant one. Thus contributes sufficiently to the field.

·       Though the several researchers are contributing significantly and publishing or sharing the research, present one adds valuable inputs to it.

·       The specific improvements/editorial changes may be required in reference section; authors are advised to follow journal guidelines for the same.

Therefore I have no hesitation to recommend it for its publication, except any editorial changes.

Thanks

Author Response

All modifcations are highlighted in blue in the revised manuscript.

Reviewer 2

Manuscript "Evidence of micro and nanoplastics contamination and effects in freshwater mussels caged at downstream an urban area" shed light on the current situation regarding plastic pollution.

I found article written well using current literature to justify the outcomes of study.

The methodology used is up to the mark, results are well described and justified.

•    Authors have evaluated stress biomarkers and correlated them with the exposure of toxicants using well designed methodology and planning. The number of animals in each group is sufficient looking into the current trends for using animals in experiments.
• The pollution research has great significance in current situation, present study adds little more about the ways for evaluation the assessment of pollution from one place to distant one. Thus contributes sufficiently to the field.
• Though the several researchers are contributing significantly and publishing or sharing the research, present one adds valuable inputs to it.
• The specific improvements/editorial changes may be required in reference section; authors are advised to follow journal guidelines for the same. Done

Therefore I have no hesitation to recommend it for its publication, except any editorial changes.

Thanks

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,
Thank you for your improved manuscript and for the inclusion of my suggestions in it. I believe that the work is correct and could be published as is.