Ecotoxicological Effects of Silver Nanoparticles (Ag-NPs) on Parturition Time, Survival Rate, Reproductive Success and Blood Parameters of Adult Common Molly (Poecilia sphenops) and Their Larvae

Round 1

Reviewer 1 Report

General Comments

Straightforward examination of effects of silver nanoparticles on health of a fish.  Results suggest that while LC50 is fairly high, negative effects are observed at much lower concentrations.  There are some issues with providing details of methods and presentation of results that warrant revision.

Specific Comments

Introduction

The whole manuscript needs to be carefully proofed for proper use of English.

Do the authors know the relationship between the quantity of silver nanoparticles and actual water silver concentration?

Methods

Can the authors provide more detail about the salt concentrations of the water used in these studies?

It is not clear how many fish were put in each tank for the acute toxicity tests.

In these static water type tests where a fixed amount of nanoparticles was added at the beginning I wonder if the dissolved silver concentration was constant throughout the duration of the test period.  Did the authors sample the water and test it for silver concentration?

Given that silver is known to negatively impact ion regulation in fish it would have been worthwhile to measure plasma salt levels.

Results

Results from Tables 1 and 2 are presented again in Fig. 1.  I suggest presenting the data in one or the other, but not both.

I presumed that triplicate aquaria for each concentration were set up so that the authors were able to get some measure of variability in order to statistically analyze their results.  Instead they seem to lump the findings together so that there is no measure of variability.

I don’t see the purpose of Table 3 or Table 4.  Table 4 seems like it could be covered briefly in the discussion.

Discussion

The authors calculate LC50 at 96 h of around 25 mg/L, but the reproductive and blood data suggest that much lower concentrations were having profound negative impacts on the fish.  Some discussion about this seems in order.

Author Response

Reviewer #1 comments:

• Comments and Suggestions for Authors
• General Comments
• Straightforward examination of effects of silver nanoparticles on health of a fish. Results suggest that while LC50 is fairly high, negative effects are observed at much lower concentrations. There are some issues with providing details of methods and presentation of results that warrant revision.

Authors

• The authors thank you for your attention. The manuscript was sent to proofreading. Also, new details for methods and presentation are added to the main text.

Rev 1

• Specific Comments
• Introduction
• The whole manuscript needs to be carefully proofed for proper use of English.

Authors

• Done, the English writing in the manuscript was proofread

• Do the authors know the relationship between the quantity of silver nanoparticles and actual water silver concentration?
• Authors didn't check actual concentration of silver nanoparticles in the test tanks; because, the method and parameters of present study were set and modified according to laboratory facilities and references. According to product specification, silver actual concentration was 1000 mg.l^-1 in stock solution (Nanocid L-2000, Nanonasb Pars Co., Tehran, Iran) and author just measured and report commercial solution of Ag-NPs (lines 97-99).

• Methods
• Can the authors provide more detail about the salt concentrations of the water used in these studies?
• Yes we can, the used water in present study was freshwater and concentration of salt (NaCl) was less than 1 mg.L^-1 during the test (lines 94-95).

• It is not clear how many fish were put in each tank for the acute toxicity tests.
• Done, there were 7 fish per each tank (line 105).

• In these static water type tests where a fixed amount of nanoparticles was added at the beginning I wonder if the dissolved silver concentration was constant throughout the duration of the test period. Did the authors sample the water and test it for silver concentration?
• Done, according to laboratory facilities and references concentration of commercial solution of Ag-NPs was fixed by daily water replacing (lines 115-116).

• Given that silver is known to negatively impact ion regulation in fish it would have been worthwhile to measure plasma salt levels.
• Thank you for your comment; this should be included in our future study.

• Results
• Results from Tables 1 and 2 are presented again in Fig. 1. I suggest presenting the data in one or the other, but not both.
• Thank you for your attention and suggest; the figure 1 just showed mortality rate of fish 96 h after exposed to Ag-NPs commercial solution (Nanocid L-2000, Nanonasb Pars Co., Tehran, Iran) and compared it between two groups. Also, it suggested a trend line for it. (lines 168-171)

• I presumed that triplicate aquaria for each concentration were set up so that the authors were able to get some measure of variability in order to statistically analyze their results. Instead they seem to lump the findings together so that there is no measure of variability.

• Results of previous studies showed toxicity effect of pollutants clearly depends on environmental conditions, species, size, gender and weight of organism. Hence, authors according to these references and laboratory facility set 3 replications for the each treatment, because it was a static test in laboratory conditions. Also, replications helped the results were comparable and had average and variance. (lines 60-63)

• I don’t see the purpose of Table 3 or Table 4. Table 4 seems like it could be covered briefly in the discussion.
• Table 3 showed lethal concentration of Ag-NPs 24, 48, 72 and 98 h after exposed. These concentrations can be useful for management of aquatic environment (lines 177-181). Table 4 moved to discussion and changed its number (lines 241-242)

• Discussion
• The authors calculate LC50 at 96 h of around 25 mg/L, but the reproductive and blood data suggest that much lower concentrations were having profound negative impacts on the fish. Some discussion about this seems in order
• Toxicity of Ag-NPs may dependent on the environmental or test conditions, species, age, size and diet of the organism [21, 23]. Also, other reason of this diference can be solvent of used commercial suspension in present study (Ag-NPs, Nanocid L-2000, Nanonasb Pars Co., Tehran, Iran) that according product specification it was distilled water. (lines 226-231)
• New text in discussion was added (282-289).

Reviewer 2 Report

Water-1524240

Ecotoxicological effects of silver nanoparticles (Ag-NPs) on parturition time, survival rate, reproductive success and blood parameters of adult common molly, Poecilia sphenops and their larvae

Vali S, Majidiyan N, Yalsuyi AM, Vajargah MF, Prokic MD, Faggio C

In this article, the authors described toxic effects of Ag-nanoparticles on survival rate, reproductive system, and blood biochemical properties in a teleost fish, common molly, Poecilia sphenops. The toxic effects showed obvious exposure concentration-dependency, and the physiological significance of changes in sub-lethal effect, such as blood parameters are discussed. The experiment was well done and the text was well-written and the contents mentioned in the text give new insight in the relevant topic.

I have only minor comments as follows:

P.3 l.95~ How many fish are taken in an aquarium? Is it seven? And the authors used mature fish in the acute toxicity test. Are they male or female? Is there any difference in mortality between male and female fish?

P.5. Legend of Figure 1, the author describe “mortality rate”, but it is not rate, but number. It is better to show mortality rate, because number of dead fish are described in Table 1.  

P.5. Regarding to footnote of Table 3, the authors describe “All concentrations are nominal concentrations”, it is better to change the sentence to “All toxicity values are calculated based on nominal concentrations”.

P7. L.208. Remove az after (Oryzias latipes).

P.7 In first paragraph in the discussion section, the authors discuss effect of Ag-NPs on white blood cells. Are there any literatures referring adverse effect of Ag-NPs on immune system in other fish or organisms, and molecular mechanism how Ag-NPs decrease white blood cells?

P.9 Please check ref no. 17.18. mistypo?

This article discussed only hazard of Ag-NPs, and from ecotoxicological point of view, it is weak point.

Please discuss the ecological risk also. Is it possible that the concentrations around the toxicity values occur in natural environment?

Author Response

Reviewer #2 comments:

Ecotoxicological effects of silver nanoparticles (Ag-NPs) on parturition time, survival rate, reproductive success and blood parameters of adult common molly, Poecilia sphenops and their larvae

Vali S, Majidiyan N, Yalsuyi AM, Vajargah MF, Prokic MD, Faggio C

In this article, the authors described toxic effects of Ag-nanoparticles on survival rate, reproductive system, and blood biochemical properties in a teleost fish, common molly, Poecilia sphenops. The toxic effects showed obvious exposure concentration-dependency, and the physiological significance of changes in sub-lethal effect, such as blood parameters are discussed. The experiment was well done and the text was well-written and the contents mentioned in the text give new insight in the relevant topic

Authors

• The authors thank you for your time and valuable comments that improved our paper.

• I have only minor comments as follows:

• 3 l.95 How many fish are taken in an aquarium? Is it seven? And the authors used mature fish in the acute toxicity test. Are they male or female? Is there any difference in mortality between male and female fish?
• There were 7 fishes in each aquarium and all of them were female; because previous studies clearly showed size, weight and gender of organisms can directly effect on toxicity of pollutants. The differences lethal concentration between male and female organisms can be tittle of future study (lines 60-63); also, Male fishes just were used for fertilize the female fishes (lines 89-90).

• 5. Legend of Figure 1, the author describe “mortality rate”, but it is not rate, but number. It is better to show mortality rate, because number of dead fish are described in Table 1.
• Done, it was changed according to reviewer suggest (lines 168-171)

• 5. Regarding to footnote of Table 3, the authors describe “All concentrations are nominal concentrations”, it is better to change the sentence to “All toxicity values are calculated based on nominal concentrations”.
• Done, it was replaced (lines 180-181)

• L.208. Remove az after (Oryzias latipes).
• Done, it was removed. (line 224)

• 7 In first paragraph in the discussion section, the authors discuss effect of Ag-NPs on white blood cells. Are there any literatures referring adverse effect of Ag-NPs on immune system in other fish or organisms, and molecular mechanism how Ag-NPs decrease white blood cells?
• Done, new information added to the text (line 236-240)

• 9 Please check ref no. 17.18. mistypo?
• Done, it was improved (lines 352-354)

• This article discussed only hazard of Ag-NPs, and from ecotoxicological point of view, it is weak point. Please discuss the ecological risk also. Is it possible that the concentrations around the toxicity values occur in natural environment?
• Previous evaluations estimated concentrations of total silver in water to be in the range of 0.09 to 320 μg.l-1. However, due to the uncertainty of these estimates and the widespread use of nanoparticles in various industries, concerns about its further spread in aquatic environments are increasing; because as mentioned, aquatic environments are last destination of pollutants. (lines 282-289)