Biochemical and Pathophysiological Responses in Capoeta capoeta under Lethal and Sub-Lethal Exposures of Silver Nanoparticles

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Comments

Responses of author

1

Lots of descriptions in Abstract were meaningless.

Some part of the abstract is deleted and abstract part is rewritten.

2

Lots of descriptions in the Introduction section are unclear and I do not think it is creative enough.

Some unnecessary descriptions in the Introduction section are deleted and the introduction part is rewritten.

3

The data in Figure 1 was only one corresponding to each concentration and it was not scientific.

The authors agree with the reviewer, and we are removing this figure from the main manuscript file; it is now included in supplementary materials.

4

Figure 2 and 3, there were not gills lesions and liver lesions of Capoeta (Capoeta capoeta) in control group.

In the two figures authors have not observed any gills lesions and liver lesions of Capoeta (Capoeta capoeta) in control group.

5

In the Discussion section, I believe that the authors failed to use current research to discuss the results of this study.

In the Results and discussion section we have included many current research to discuss the results of this study. The newly included current research are-

Khosravi-Katuli K, Shabani A, Paknejad H, Imanpoor MR. Comparative toxicity of silver nanoparticle and ionic silver in juvenile common carp (Cyprinus carpio): accumulation, physiology and histopathology. Journal of Hazardous Materials. 2018 Oct 5;359:373-81.

Kakakhel MA, Wu F, Sajjad W, Zhang Q, Khan I, Ullah K, Wang W. Long-term exposure to high-concentration silver nanoparticles induced toxicity, fatality, bioaccumulation, and histological alteration in fish (Cyprinus carpio). Environmental Sciences Europe. 2021 Dec;33(1):1-1.

Lekamge S, Miranda AF, Abraham A, Li V, Shukla R, Bansal V, Nugegoda D. The toxicity of silver nanoparticles (AgNPs) to three freshwater invertebrates with different life strategies: Hydra vulgaris, Daphnia carinata, and Paratya australiensis. Frontiers in Environmental Science. 2018 Dec 13;6:152.

Daima HK, Selvakannan PR, Kandjani AE, Shukla R, Bhargava SK, Bansal V. Synergistic influence of polyoxometalate surface corona towards enhancing the antibacterial performance of tyrosine-capped Ag nanoparticles. Nanoscale. 2014;6(2):758-65.

Syafiuddin A, Salmiati S, Hadibarata T, Kueh AB, Salim MR, Zaini MA. Silver nanoparticles in the water environment in Malaysia: inspection, characterization, removal, modeling, and future perspective. Scientific reports. 2018 Jan 17;8(1):1-5.

Schipper CA, Dubbeldam M, Feist SW, Rietjens IM, Murk AT. Cultivation of the heart urchin Echinocardium cordatum and validation of its use in marine toxicity testing for environmental risk assessment. Journal of Experimental Marine Biology and Ecology. 2008 Sep 12;364(1):11-8.

Butler JD, Varghese L, Deb N, Thornhill B. Extending international toxicity testing guidance to middle eastern test species. Science of the Total Environment. 2020 May 10;716:136343.

6

The conclusion was not detailed just a review description.

Conclusion is rewritten.

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Comments

Responses of author

1

There are many AgNPs available on the market. What are the characteristics of the AgNPs used in the experiment? It is necessary to include: size, shape, Zeta potential, coating, etc.

Characteristics of the AgNPs have been done and included in the revised paper both in materials and methods section and Results section.

The NPs were characterized in the MilliQ water. The surface plasmon resonance anal-ysis of AgNPs was carried out in a quartz cuvette with a path length of 1 cm using a Varian Cary 50 UV-visible spectrophotometer [46]. Drop casting the NP solution on carbon-coated copper grids measured the NP size using transmission electron micros-copy at 100 kV. (JEOL 1010) [46]. The zeta potential and hydrodynamic diameter (HDD) of the nanoparticles were measured on a Zetasizer using a folded capillary cell and a glass cuvette, respectively (Dynamic light scattering; Malvern Zetasizer Nano series, NanoZS).

The UV-visible absorbance spectra of AgNPs exhibit an SPR (Surface Plasmon Reso-nance) band at 406 nm, which is typical of AgNPs, and a peak at 274 nm [46, 48]. The HDD (Hydrodynamic Diameter) was 86.6 nm while core size determined by TEM was ∼30 nm in diameter. The negative charge of AgNPs, as determined by zeta potential, is -42.4mV. [48]

2

Am I to understand that the AgNPs were added directly to the aquarium water to reach the dilutions that consider their administered doses? In this way I interpret that the administration was cutaneous, oral and branchial?.  It is necessary that this issue be drafted with greater clarity

The administration was cutaneous, oral and branchial. In section “2.3.1. Acute toxicity assay”, we have described it with greater clarity.

Acute toxicity of AgNPs to fish was assessed by exposing them to four concentrations of AgNPs plus a blank control (0 ml/L).

3

In my view, it is necessary in the design of experiments such as the one presented, at least to put a positive control,  as a source of metallic silver, especially for the type of exposure they used, We do not know what happens with the AgNPs once diluted in the aquarium, if the silver ions are released and it is not the complete AgNPs but the dissociated silver ions that induce toxicity. 

We thank the reviewer for the valuable comment.

While we agree that to put a positive control,  as a source of metallic silver study would have been good, we however submit that it was not part of our study design, neither was it an omission.  The two standard references employed for this study i.e., Lekamge S, Miranda AF, Abraham A, Li V, Shukla R, Bansal V, Nugegoda D. The toxicity of silver nanoparticles (AgNPs) to three freshwater invertebrates with different life strategies: Hydra vulgaris, Daphnia carinata, and Paratya australiensis. Frontiers in Environmental Science. 2018 Dec 13;6:152. And

Khosravi-Katuli K, Shabani A, Paknejad H, Imanpoor MR. Comparative toxicity of silver nanoparticle and ionic silver in juvenile common carp (Cyprinus carpio): accumulation, physiology and histopathology. Journal of Hazardous Materials. 2018 Oct 5;359:373-81.

did not emphasize to put a positive control, as a source of metallic silver as fundamental to bioassays.

We may however consider the positive control, as a source of metallic silver studies for a future experiment. Thank you

4

Its discussion should be improved, especially in view of the fact that there is a problem with the dosage and stability of the AgNPs it uses which does not include characterization, it is very difficult to know to what extent the changes in toxicity they report are due exclusively to the AgNPs.

The characterization of AgNPs has been done and added to the Results and discussion part (Line number 185-188).

We also added the reason for choosing such concentrations of AgNPs and the reason for selecting the duration of experiment (Line number 190-195).

Overall, the Results and discussion part is improved now.

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Comments

Responses of author

1

It is necessary to add a brief conclusion to abstract.

Brief conclusion to abstract is added. The gradients of toxic responses across exposure concentrations and portrayals of disrupted fish health with increasing silver nanoparticle exposure time indicate a reduced physiological ability for surviving in the wild.

2

line112: Why did you choose these concentration “0, 5, 10, 15, and 20 ml/L”? what is the basis for selecting these concentrations? Why did you choose these time points “24-, 48-, 72-, and 96-hours”?

Syafiuddin, Salmiati [46] recorded AgNP values in rivers and sewage treatment plants ranging from 0.13 to 10.16 mg/L and 0.13 to 20.02 mg/L, respectively in Malaysia. Thus, we selected these concentrations of AgNPs for toxicity testing on fish Hematological and biochemical endpoints.

Based on the standard protocols of 96-hours toxicity tests we chose the durations of experiments [47-49].

3

the author explored the acute toxicity of AgNPs. However, AgNPs exist in the environment. What might be the effects of long-term exposure at low dose of AgNPs?

Kakakhel et al. tested freshwater fish Cyprinus carpio for toxicity, mortality, bioaccu-mulation, and histological changes after exposing them to silver nanoparticles for longer term [42]. The findings indicate bioaccumulation of silver nanoparticles in many organs of fish. The liver had the highest bioaccumulation of silver nanoparticles, followed by the intestine, gills, and muscles [42]. Furthermore, the study revealed that silver nano-particle bioaccumulation resulted in histopathological changes, including damaged gill tissue and intestinal structure [42].

4

Add some descriptions about animal welfare in the method.

Throughout the experimental period, all ethical concerns were met as per the regulation provided by the Institutional Biosafety Committee- Gorgan University of Agricultural Science and Natural Resources.

5

line 181, line 251, and line 278: The full name of the abbreviation should be unified. Such as "fig 1" should be written as “Figure1”.

Corrected

6

Scale bar should be added in the Figures.

Scale bar has been added in figure 2 and figure 3.

7

Punctuation marks in illustrations shall be unified throughout the text, such as “Table 2,” should be written as “Table 2.” In line 195, and “Figure 2,” should be written as “Figure 2.”

In line 269. The first letter of the sentence in the figure legends should be capitalized, such as line 270 and line 296. The author should examine them carefully.

Corrected.