Determination of Median Lethal Concentration (LC50) and Nitrite Accumulation in the Blood and Tissue of Blood Cockle (Tegillarca granosa, Linnaeus 1758)

Round 1

Reviewer 1 Report

Determination of Median Lethal Concentration (LC50) and Nitrite Accumulation in Blood and Tissue of Blood Cockle (Tegillarca granosa, Linnaeus 1758)

This study aims to evaluate the nitrite toxicity on Tellargica granosa by the determination of the median lethal concentration (LC50), the accumulation of NO_2- in blood and tissues and the relationship of NO_2- accumulation with mortality percentage. Cockles were exposed to different concentrations of nitrite for 72 hr and NO_2- accumulation was determined by a nitrite/nitrate colorimetric assay kit. Findings suggest NO_2- accumulation in blood and tissues and a percentage of mortality increase proportional with exposure concentrations.

The manuscript is written in poor English, many parts are redundant and there are too much repetitions which make reading complicated. Many sentences are too long and sometimes the grammar is not correct such that understanding is in part not possible (e.g. chapter 4.2 and 4.3). Large part of the text, especially in in the result and discussion part should be rewritten. There are inconsistencies in the use of capitalization in the main text, capitalization and italics in the literature. The figure references are not always corresponding to the correct figures. The Figures 6 and 7 are repetitive, showing the same result in slightly different ways.

The Material and Methods section should be more comprehensive. Details regarding the experimental design (maybe it could be better to represent it graphically?) and the procedure of the Nitrate/Nitrite Colorimetric assay should be added.

The conclusion section is too short. The authors should give more detailed conclusion. What is the implications for this study? What is your further study in the future?

However, I must admit that this is a potentially interesting study and its findings can be useful to the scientific community and the aquaculture farmers and fishers.

Therefore I suggest to reconsider the manuscript after a major revision is carried out.  These corrections must attend the points previously expressed.

Author Response

We sincerely and gratefully appreciate your in-depth reviews, they are completely valuable and supportive to our manuscript.

We again appreciate your great cooperation and consideration in helping improve the manuscript. Thank you once again.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors present a study focused on evaluating the toxicity of nitrite to blood cockle (Tegillarca granosa) and linking toxicity to accumulation within the blood and tissue of exposed organisms by collecting test organisms from the field, depurating the organisms, and then using them within toxicity tests. Based on the results of the study, the authors conclude that the toxicity associated with nitrite exposure is due to accumulation of nitrite within the blood and tissue of exposed organisms that causes methaemoglobinaemia, leading to death. Considering the economic demand for the blood cockle, presence of nitrite in waterways where these species live is a concern that should be addressed.

Overall, the basic attributes of the study seem experimentally appropriate and relevant to the objectives of the study. The implementation of the results, however, relative to environmental contamination and the means by which toxicity occurs in the species expands outside of the data collected by the authors. Similarly, there are some concerns that exist in reference to the experimental design, outlined below, and significant grammatical errors made throughout the manuscript that confound interpretation of the information presented. Therefore, it is recommended that the manuscript undergo major revisions prior to being accepted for publication. Specific comments on the manuscript are outlined below.

Specific Comments

Line 35: Revise “nitrite, thus may result” to “nitrite, and may result”

Line 38: “Penaeus setiferus” should be italicized.

Line 39: “…while the study…” should be “…while a study…”

Lines 50-56: This paragraph seems largely irrelevant considering the study is focused on blood cockles, not fish. Is the uptake mechanism in blood cockles the same as in fish? If so, the information presented here should be related to blood cockles more clearly.

Line 80: It appears that sampling only occurred on one day out of the year of 2018. While this may have been sufficient to provide the necessary animals for the experiment, it is not likely that the water quality measurements obtained were sufficient to indicate the environmental concentrations of nitrite within this environment. If one of the objectives of the study is to compare the toxicity of nitrite to blood cockles to environmental concentrations, there should be much more data available on the environmental concentrations of nitrite within the habitat than a measurement from a single day. Therefore, it is recommended that any comparisons of the LC50 obtained from the study to environmental concentrations measured from the site be removed from the manuscript, as there is not enough data to support this comparison.

Line 89: Units for salinity are missing.

Line 93: It is not clear what 90% concentration means in the context of the oxygen levels that were monitored in the artificial sea water. Clarification should be provided.

Line 115: The authors state that a 24 h preliminary range finding test was used to determine the nitrite concentrations to use in the toxicity test. The definitive toxicity test, however, was 72 h in length. Why was a 24 h test used to approximate the range of concentrations for a 72 h toxicity test? Range finding tests should be the same length as definitive tests. This may explain why the toxicity data obtained did not reach 100% mortality, even in the highest concentration, which can skew interpretation of the 72 h LC50. The authors need to provide justification for this decision.

Line 115-117: The authors report that the water in the test chambers was renewed twice a day during the 72 h toxicity test, but later in the results (Figure 4), report that the final nitrite concentrations dropped to the levels of the control in all treatments. Why is the nitrite concentration declining in the test chambers if the water was being renewed during the test? How does this impact the results of the study?

Line 127: The authors state that blood and tissue samples were taken from blood cockles that survived the 72 h exposure to nitrite within the treatment beakers. There were differing levels of survival at each treatment level, however. Therefore, the replication of blood and tissue samples would be different at each nitrite concentration. How did the authors address this issue? How many blood and tissue samples were analyzed at each concentration? More information on the replication that was utilized in this analysis is needed.

Line 133: Are these replicates technical replicates (3 replicates of the same blood/tissue samples) or experimental replicates (blood/tissue samples were taken from three cockles at each nitrite concentration)?

Line 146-148: Clarification on why the nitrite concentrations dropped despite the renewals occurring during the test needs to be included.

Line 150: The authors do not present any data that would support conclusions drawn related to the effects of nitrite exposure duration on blood cockles as only one exposure duration was tested in the current study.

Line 151: The LC50 reported by the authors is based on the nominal nitrite concentrations used in the toxicity test, but it is clear from the results presented that the nitrite concentrations changed drastically during the 72 h exposure. Therefore, the use of nominal concentrations in the determination of the LC50 using the Probit analysis seems inappropriate. It would be better to use a time weighted average of the nitrite concentrations in the Probit analysis, as this would more accurately represent the nitrite concentration to which the cockles were actually exposed. This could be done by finding the difference in the initial and final nitrite concentration and dividing it by the length of the exposure. Using the time weighted concentrations in the Probit analysis would likely change the reported LC50 and more accurately represent the concentrations to which the organisms were exposed.

Line 151: Confidence intervals should be reported with the LC50 estimate.

Lines 154-156: Are the statistical differences reported for the tissue and blood concentrations reported as comparisons made between tissue concentrations and blood concentrations at each nitrite concentration or comparisons between blood concentrations at each nitrite concentration and tissue concentrations at each nitrite concentration? More clarification on the exact comparisons that are being made is needed.

Line 161-162: The LC50 is already reported earlier in this paragraph so this sentence is redundant.

Lines 301-304: This conclusion may change if the LC50 estimate is made using the adjusted nitrite concentrations. Also, comparisons of the LC50 to the field concentrations seems inappropriate as it appears the nitrite concentration was only measured on one day out of the year. This is hardly representative of the actual nitrite concentrations in the water, such that this comparison is largely unfounded.

Line 309: The inclusion of the human habit of consuming cockles as a reason for sampling the entire tissue as opposed to particular organs is not in line with any of the objectives for the study presented earlier. More relevant justification for this decision is needed.

Lines 312-313: The comparisons of nitrite concentrations in the blood and tissues reported in the current study to previous studies is confusing. It is not made clear if these previous studies were evaluating the same species of cockle or different species. Also, would it not be expected that the concentrations in the whole tissue would be higher than that in the specific tissues as you are sampling more mass of tissue when looking at the entire organism compared to specific organs isolated from individuals? More justification and clarification for this comparison is needed.

Lines 315-316: There is no evidence obtained in the current study to support the claim that nitrite exposure is occurring through the gills. It is clear that the cockles are being exposed to nitrite considering the increase in blood and tissue concentrations with increasing exposure concentrations, but the mechanism of uptake is not clearly defined by the experimental results.

Line 342-344: The conclusion drawn here relating to the potential risk of blood cockles exposed to nitrite in the field is not well supported by the data within the manuscript. As only one datapoint defining the nitrite concentration in the field location exists, there is no real comparison that can be made between the LC50 and the field data. Therefore, it would be better to focus on comparing the LC50 to the regulatory standard for nitrite within seawater, and discussing the results of the study in that context. The authors should better focus their discussion around the results that they have obtained from their study (nitrite LC50 for cockles, tissue and blood concentrations, potential mode of action for nitrite toxicity) and remove any speculation surrounding impacts on populations of cockles within the field. If the discussion is refined to center more on these points rather than trying to compare the data to field concentrations, the manuscript will be much cleaner and more impactful.

Author Response

We sincerely and gratefully appreciate your in-depth reviews, they are completely valuable and supportive to our manuscript. 

We again appreciate your great cooperation and consideration in helping improve the manuscript. 

Sincerely,

Ferdaus Mohamat Yusuff (Ph.D)

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript by Hashim and colleagues has the purpose to determine nitrite toxicity on blood cockle (Tegillarca granosa), calculate the median lethal concentration (LC50) and evaluate the accumulation level of nitrite in T. granosa. Moreover, also the relationship between nitrite accumulation and mortality percentage was examined.

Overall, the results of this manuscript may be interesting but the paper lacks clarity and there are numerous significant errors in the manuscript that do not allow for its publication.

I found too many hard periods that may lead the reader to make an arbitrary sense of sentences. In some parts of the entire Ms the authors use more colloquial than scientific language and this is particularly evident in the introduction, result, and discussion paragraphs. Authors are invited to rewrite the Ms in order to emphasize data related to their results and may an effort to make the entire Ms clearer and more concise.

Despite some interesting results contained, the Ms fails to reach a high level of scientific quality and cannot be published in Water.

Below I summarize the general comments of the Ms. Specific comments are annotated in the attached .pdf.

 

• First, the English language should be completely revised by a native speaker. The entire work presents serious linguistic errors that make it difficult to read and clearly understand the actual meaning; in my opinion, this could lead the readers to an arbitrary interpretation of some data.
• In the introduction, the authors should clearly explain (also mentioning the relevant NEW literature) how nitrite is released into the environment and how it reaches the aquatic habitat.
• Surely the materials and methods section needs essential improvements. The experimental set up is not clear. It is never explicated within the manuscript on which tissues the analysis of bioaccumulation were performed (on the whole organism?).
• The results section is completely confusing.
• For what concerns the discussion section, in my opinion, the authors can make an effort to make it more clear and concise. There is general disorganization in the explanation of the topic.

 

Formal points:

Please see attached .pdf

Comments for author File: Comments.pdf

Author Response

Dear reviewer,
Thank you for taking my manuscript into consideration, your comments are very constructive, and the requested modifications will certainly support our work.

Again thank you  for taking the time to review our manuscript.

Sincerely,

Ferdaus Mohamat Yusuff (Ph.D)

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I think the authors have satisfactorily addressed all the concerns I had in my previous review. The text is now better organised and more readable. The english has been improved. As a result, I now recommend the current form can be accepted for publication.

Reviewer 2 Report

The authors have done significant revisions to the manuscript based on the comments provided, and the overall manuscript has been much improved. It is my opinion that the manuscript is now ready for publication.

Reviewer 3 Report

The authors have modified the MS in accordance with the required fields