Virgin Polystyrene Microparticles Exposure Leads to Changes in Gills DNA and Physical Condition in the Mediterranean Mussel Mytilus Galloprovincialis

Round 1

Reviewer 1 Report

Dear Authors,

you can find my comments and suggestions in the attached report.

 

 

Best regards

The Reviewer

Comments for author File: Comments.pdf

Author Response

RESPONSE TO REVIEWER 1

 

Title:

Title is descriptive of the report, but it should be better related to the gills tissue. Indeed, the Authors evaluated the effects on DNA only for this tissue in their study.

• OK, the title has been changed.

 

Simple Summary and Abstract:

These sections should describe the study in an essential way, but in the parts related to the conclusions supported by the results obtained, in this form there are no firm points but only assumptions (This may indicate, Results could be interpreted as). Please be sure to give the right resonance to your results.

• OK, the simple summary has been improved according to your recommendations.

 

In addition, the conservation aspects mentioned in the title are not mentioned here. I invite the authors to consider whether to leave them in the title.

• Thank you for the comment. This has been eliminated from the title and added as a keyword instead (Marine conservation).

 

 

Keywords:

Its better to avoid the repetition of words already reported in the title among keywords. Please take care of this aspect.  

• OK, three of the keywords that appeared in the title have been changed. Now, they are: Condition Index; Future trends; Marine biota; Microplastics; Marine conservation.

 

 

Introduction:

Introduction section provide a good overview of the topic, but needs some improvement, especially in the first part, regarding references related to this pollution field that nowadays are really varied and abundant, and this is not evident from the use made by the Authors. For example, to enrich your statements use these related references:

 

Line 37, mention several animal species and groups:

• Thank you very much for this suggestion and the references, they have been added when describing ingestion of microplastics by different species: “Due to their ubiquity and small size, microplastics are bioavailable for a great number of marine organisms [8], and therefore reports of ingestion by marine animals are numerous [9], as well as their physical and ecotoxicological implications [e.g 10, 11, 12, 13]. Animals reporting ingestion of microplastics goes from planktonic species [14, 15], corals [16], and cnidarians [17] to fishes [18] and top predators [19]. However, filter-feeders and pelagic feeders exhibit the highest rates of microplastic consumption [20].” à Lines 85 to 91

 

 

Albanon M., Panarello, G., Di Paola, D., D’Angelo, G., Granata, A., Savoca, S., Capillo, G.

The mauve stinger Pelagia noctiluca (Cnidaria, Scyphozoa) plastics

contamination, the Strait of Messina case. (2021) International Journal of

Environmental Studies.

DOI: 10.1080/00207233.2021.1893489

 

Savoca, S., Matanovic, K., D'Angelo, G., Vetri, V., Anselmo, S., Bottari, T.,… Gjurcevic, E.

Ingestion of plastic and non-plastic microfibers by farmed gilthead sea bream

(Sparus aurata) and common carp (Cyprinus carpio) at different life stages.

(2021) Science of the Total Environment, 782, art. no. 146851.

DOI: 10.1016/j.scitotenv.2021.146851

 

Line 39, extend also to deep marine environments: 

D’iglio, C., Savoca, S., Rinelli, P., Spanò, N., Capillo, G.

Diet of the deep-sea shark galeus melastomus rafinesque, 1810, in the

mediterranean sea: What we know and what we should know.

(2021) Sustainability (Switzerland), 13 (7), art. no. 3962.

DOI: 10.3390/su13073962

• OK, thanks for that, deep environments have been added and the reference has been included. à Line 44

 

 

Line 46, support this sentence related to filter feeders:

Albano, M., Panarello, G., Di Paola, D., Capparucci, F., Crupi, R., Gugliandolo,

E., Spanò, N., Capillo, G., Savoca, S.

The influence of polystyrene microspheres abundance on development and feeding

behavior of Artemia salina (Linnaeus, 1758. (2021) Applied Sciences (Switzerland), 11 (8), art. no. 3352.

DOI: 10.3390/app11083352

• OK, the reference has been added in order to support the sentence. à Line 57.

 

 

The lines between 54 and 59 seems to be a repetition of the previous ones, and don’t give any additional information useful for the readers. Please consider merging this period with the previous one.

• Ok, done. Thank you for the advice. The entire paragraph has been changed, as it makes more sense to talk about it when talking about the ubiquity of microplastics and where can we find them (including biota). à Lines 44 to 50.

 

 

Materials and Methods:

Sincerely, I have several doubts about the experimental design adopted in this study. Are the Authors able to support their choice of exposure mode, with references in which a similar trend of 2h per day was used? I do not know what kind of exposure this can be considered whether acute or chronic. In fact, it does not represent any of the two in my opinion, as it has been given the opportunity to animals to purify daily, just because of the filtration speed that distinguishes the mussels, I would have opted for an exposure of classic type. This in my opinion could lead to a sublethal effect only, as reported. Please better explain and support this crucial question.  

• Thank you for your much for this point, I totally agree with you. It does not represent any of the exposures to which usually these organisms are studied (nor chronic or acute). However, because mussels live in the intertidal, we have chosen a daily acute exposure, to simulate the conditions they can find in their habitats. Following your advice, this has been explained carefully in material and methods section:

 

“Paul-Pont et al. [41] recommend to consider realistic ecosystem scenarios when designing experiments to assess the effects of exposure to microplastics on marine organisms. à Lines 150 to 152

 

In realistic conditions, intertidal mussels living in fluctuating environments, are rarely exposed constantly to the same concentration of microplastics. Microplastics coming from the ocean or from adjacent rivers are carried by tidal movements and washed by waves, thus exposure is irregular and often recurrent. Thus, we have opted for an experiment of acute exposure (a short time), intermittent acute exposure.” à 158 to 162

 

and in the end of the discussion:

 

“Regarding the experimental design, the type of exposure to which mussels have been exposed does not fit with the typical models of exposure of most of the studies (acute or chronical), since mussels were exposed for only two hours to microplastics exposure every day. It is known that mussels have a rapidly ingestion and egestion rates of microplastics when exposed to acute exposure, showing inflammatory responses due to the cleaning and recycling processes occurring during digestion [69]. On the other hand, it is known that mussels have the ability to acclimate to a chronical long-term exposure [45]. Therefore, mussels living in the intertidal would not fit with neither of these models, and by exposing mussels to acute and daily exposition, mussels are forced to a daily depuration process, without having the opportunity to acclimate, simulating environmental conditions of the intertidal. This type of exposure will be consistent with the recommendation of Paul-Pont et al. [41] which recommend considering realistic scenarios when designing experiments to assess the effects of exposure on marine organisms. Microplastics coming from the ocean or from adjacent rivers are carried by tidal movements and washed by waves, thus exposure is irregular and often recurrent. In addition, this type of exposure, especially the higher doses, can also represent punctual microplastics spillages in the region, as it is known that during periods of heavy storms, processes of sewer overflow and drop in the efficiency of wastewater treatment plants can occur [70]. For all this reasons we have opted for an experiment of acute exposure (a short time) repeated over time.” Lines 380 to 397

 

Moreover, the Authors have conducted histological analyses or fresh visualizations that confirmed an accumulation of microplastics in the gill tissue, related to the different exposure? This is another crucial point that should be clarified and linked to the DNA analysis carried out, which are certainly of a higher level but actually not well supported without the evidence of microplastics ingestion.

• Microplastic presence has been reported in the ethanol where gills were stored: the precipitate of the Eppendorf tubes were taken and analysed directly under the microscope to determine the presence of microplastics in gills. This has been added to materials and methods:

“After leaving them to settle, the precipitate found in the bottom of the tubes were taken and placed on a glass slide and visualized under the microscope for examination of microplastics in the gills.” Lines 257 to 259

 

and also in the discussion:

“In fact, in our study microspheres were detected in gills, in the precipitate of the tubes where gills were stored, which concur with the aforementioned studies.” Lines 330 to 332

 

 

Results:

Please double check with accuracy the text layout, the figures quality and tables contents, because there are some blunders in this section.

• OK, done, figures and tables contents and layouts have been doble checked.

 

 

Discussion and conclusions:

Please take care of the comments related to experimental design and methods that are essential to address for well evaluate the entire manuscript. Indeed, discussion and conclusion section are in the present form well written and related to the obtained results, but I want to review them carefully after the Authors comment on the suggestions on materials and methods.  

• The discussion has been changed in order to fit with the previous comments, following your advices.

 

Thank you for all your comments, we think that the article has been improved a lot thanks to them.

 

 

Reviewer 2 Report

Although I was initially skeptical about the content of the article, I must admit that I liked it. The experiments were conducted as well as the discussion of the data. I list some typos and personal considerations:

Line 94: “did not found” -> maybe better “had not found”

Line 104: “technic” -> “technique”

Line 108-110: add “then” (..... by the Comet assay – THEN a test of general DNA degradation...)

Line 164: I think you don’t need to specify this “...and this was beyond the scope of the present investigation.”

Tab.1/2: please format them in a more clean/stylish way such as without vertical lines.

Figure 4: maybe a boxplot would me more appropiate.

Figure 4/5: please try to format the graphs in the same way

Author Response

RESPONSE TO REVIEWER 2

Thank you very much for your comments and improvements, we have changed all of them:

• Line 94: “did not found” -> maybe better “had not found”

OK, done. à

• Line 104: “technic” -> “technique”.

OK, done. We have changed it throughout the manuscript.

• Line 108-110: add “then” (... by the Comet assay – THEN a test of general DNA degradation...)

OK, done à Line 124

• Line 164: I think you don’t need to specify this “...and this was beyond the scope of the present investigation.”.

OK, done. This affirmation has been erased.  

• 1/2: please format them in a more clean/stylish way such as without vertical lines.

OK, done

• Figure 4: maybe a boxplot would me more appropriate.

OK, both figure 3 and 4 have been changes for a boxplot

• Figure 4/5: please try to format the graphs in the same way.

OK, done.

Reviewer 3 Report

This is an interesting study, especially in the sense it is using realistic microplastic concentrations. 

There is one error I would like to be corrected, in line 63, citing Fernández and Albentosa you said that in Mytilus mussels 85% of the microplastics were excreted after 6 days of depuration, which is not right. Only metabolic waste is excreted, in this case it is an elimination, as it is stated in the paper cited.

Other than that It is a publishable paper, after some language corrections.

 

Author Response

RESPONSE TO REVIEWER 3

 

There is one error I would like to be corrected, in line 63, citing Fernández and Albentosa you said that in Mytilus mussels 85% of the microplastics were excreted after 6 days of depuration, which is not right. Only metabolic waste is excreted, in this case it is an elimination, as it is stated in the paper cited.

Other than that It is a publishable paper, after some language corrections.

Dear reviewer,

Thank you so much for your comments and improvements, we have corrected the error you referred to (Line 74), and double checked the English grammar.

Round 2

Reviewer 1 Report

Dear Authors,

thank you for seriously following my suggestion on your manuscript, which now seems to me improved.

 

Good job.

The Reviewer