Effect of Chronic Exposure to Pesticide Methomyl on Antioxidant Defense System in Testis of Tilapia (Oreochromis niloticus) and Its Recovery Pattern

Round 1

Reviewer 1 Report

This manuscript looks interesting, the investigation is well set up, rigorous and complete.

The figures are well made and easy to understand. The bibliography is extensive and updated

The work is an important contribution to evaluate the state of the art regarding the study of Methomyl toxicity in farmed fish and establish/confirm the NOAEL value.

I have no revisions to propose.

Just a suggestion: considering that the aim of this work is also to propose a NOAEL value, given the WHO definition: Greatest concentration or amount of a substance, found by experiment or observation, which causes no detectable adverse alteration of morphology, functional capacity, growth, development or life span of the target organism under defined conditions of exposure. Alterations of morphology, functional capacity, growth, development or life span of the target may be detected which are judged not to be adverse.

Therefore it is better to specify in the conclusions that the data of this work, together with those of your recent works on damages tissue to the gonads (*), done on the same fish, allow to propose a  NOAEL value.

In fact, the works are integrated and one completes the other in order to establish a value of NOAEL.

(*):Meng, S.L.; Liu, T.; Chen, X.; Qiu, L.P.; Hu, G.D.; Song, C.; Fan, L.M.; Zheng, Y.; Chen, J.Z.; Xu, P. Effect of Chronic Exposure to Methomyl on Tissue Damage and Apoptosis in Testis of Tilapia (Oreochromis niloticus) and Recovery Pattern. B Environ Contam Tox. 2019, 102, 371-376.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

This is a straightforward experimental paper, with good methodology and clear results on the metabolic effects of methomyl. I particularly like the determination of actual concentrations using up-to-date analytical methods, which is often neglected in this type of papers. The data contribute to a better understanding of the environmental effects of methomyl.

The paper can be accepted provided the following issues can be improved.

1. The paper ends to be corrected for English style throughout.
2. Please remove all abbreviations (GPx, CAT, etc.) from the Abstract. Spell out the names of the enzymes. Please also spell out the enzymes again in line 107 (beginning of the Methods).
3. The Abstracts is full of percentages. Please summarize the data in words, avoid excessive listing of percentages.
4. It is unclear how many replicates were used in the calculation of error terms in the significance tests. Please specify in the legend of Fig. 1 what are the error bars in the graphs (standard errors or standard deviations?). Also specify over how many replicates the mean and the error were calculated. The Methods section says there were three replicates (three aquaria with the same exposure). It also says that there were 30 fish per aquarium. Is the error bar taken over 90 replicates (which is incorrect, as 30 of them came from the same aquarium), or over 18 replicates (6 fish sampled per aquarium) of over 3 replicates after pooling the data for each aquarium (which is correct, since the experimental unit is the aquarium).
5. The acute toxicity of methomyl to Nile tilapia has been documented in an earlier paper. It is important to discuss here how the NOAEL for effects on oxidative enzyme activity is related to the toxicity on the level of the whole fish. Please report LC50 or EC50 values and discuss how far are these above the NOAEL.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript described the effects of the pesticide Methomyl on the antioxidant functionality of testis in Tilapia. Methomyl is a very common water-soluble pesticide used in agriculture, found at various concentrations in water bodies and food, and therefore of big interest for its toxicity for human and wildlife health.

The study analyzed chronic exposure to Methomyl (4 different concentrations), compared to a control group (no exposure) for 30 days. The expression of several antioxidant enzymes was evaluated, as well as the recovery trend (i.e. expression levels compared to the control group) 18 days post-exposure.

The results show that the lowest tested concentration has basically no effect on the antioxidant system of testis in Tilapia (i.e. NOAEL). The second and third highest tested concentrations reported significant increase (or decrease, whether the metabolic pathway was enhanced of blocked) on the expression of the enzymes, in a dose-dependent trend, but returned to basal level (i.e. no permanent alteration or oxidative damage) after the recovery time in pesticide-free water. On the other hand, the highest concentration showed a downward trend on the effect on these enzymes, and no significant recovery occurred.

The study is conceptually designed in a correct way, with replicates and control tanks to assess the potential variability in such outcomes. However, there are no specifications of the techniques employed to assess the functionality of these enzyme, other than referring to past literature. More details (even "briefly") should be included.

Similar observations could be made on the statistical analysis. On this regard, overall data should include SD, while highest points don’t need that. But this brings to another problem, which is the way the results are showed: it’s reasonable to present the variation in expression of enzymes between the tested group and the control group as percentage, but instead of showing the highest induction/highest inhibition rate for each enzyme (which is dependent on the individual specific response), data should show the overall value (+SD) of each group tested at each time. From what it is presented in the manuscript, only “highest” data are shown, and not overall value.

Also, given that the data have been collected, it would useful to add the results from the testing occurred throughout the experiment, to assess whether these upward or downward trends on antioxidant functionality are consistent during the 30 days exposure, or not. These data could be added as "Supplementary data".

Finally, the paper is written in fairly poor English. There are several typos, and entire sections (e.g. Line 141-173) should be rewritten to increase clarity. For example, when discussing how a mechanism takes place (e.g. explaining how a metabolic pathway works) and how the results obtained in the experiment compare with previous studies, in general the results from the previous should be mentioned first, not last.

The use of a format where Results and Discussion are merged definitely didn’t help a clear presentation of the data. I’m not sure whether this is the format required by the journal but, if not, I strongly recommend the division of that section in two separate sections.

Overall, the data presented in the paper are worth it the publication, but a deep, major revision of the data presentation and of the English writing are required.

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The manuscript has significantly improved since the first versions. The English is now smooth and with very few typos. The addition of the Discussion section also helped in keeping the flow of the writing.

There are a few things that need to be fixed (see below in details), but those are minor changes.

Once those changes are addressed, the manuscript could be considered acceptable for publications.

L22: Stop the sentence after “significantly” and start the new sentence with: “On the contrary, …”

L47: change “antioxidants” with “antioxidant enzymes”

L54: what does “biochemical materials” means? I believe these words were lost in translation. I would remove “Among biochemical material, “.

L57: “endpoints”??? Maybe you mean “parameters”? Please clarify and correct.

L60: change “materials” with "substances”

L147-152: I really don’t see the utility of reporting only the highest induction/inhibition amongst all the different concentrations, once you reported the trend. Fig 1 is already quite compelling. I would remove all citations of values (which would have also needed SD, if cited) like:

“, and the 147 highest induction levels were 159.35% and 137.69%, respectively”

“and the highest inhibition level was 63.95%”, “reaching values of 150 161.23%, 149.85%, 138.75% and 120.59%, respectively”

“reaching values of 68.12%, 71.22%, 76.55% and 74.11%, respectively.”

189-192: as I mentioned in the previous review, a standard way to present data is by:

• Presenting the hypothesis
• Presenting previous work that support/negate the hypothesis
• Presenting your data and show how they fit in that hypothesis

This pattern is not followed in the next few sentences. In this case, move “A similar decreasing trend in GSH content accompanied by a rise in the GST level was reported by other researchers [7,23].” just before “In our experiment, a significant increase in GST was accompanied by a decrease of GSH in testis of fish exposed to 2 and 20 μg L−1 methomyl (Figure 1c).  

L194-199: same thing as above: move “Previous researchers have found decreasing levels of GSH accompanied by increases in GSSG and GPx in Nile tilapia exposed to methomyl [11], in rainbow trout (Oncorhynchus mykiss) exposed to carbamazepine [22], and in Nile tilapia exposed to domoic acid [25].” just before “In the present study, the steady rise in GSSG 195 and GPx was accompanied by a decrease in GSH in testis of the tilapia exposed to methomyl concentrations 2, 20, and 200 μg L −1 (Figure 1g).”

L209: stop the sentence at “methomyl”, and start the new sentence with “A similar trend…”

L230-231” change the sentence: “This phenomenon might be explained 231 by the following sequence:: with “It could be speculated that, …”

L266-271: just as mentioned earlier, remove the highest values, as they don’t provide significant values here and make the writing flow very cumbersome. Basically eliminate:

“with the highest induction levels of 159.35% 267 and 137.69%, respectively”

“with the highest inhibition value of 63.95%”

“with the highest induction rates of 161.23%, 149.85%, 138.75% and 120.59% 271 and highest inhibition values of 68.12%, 71.22%, 76.55% and 74.11%, respectively.”

Author Response

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Author Response File: Author Response.docx