Evaluation of Cytotoxic and Genotoxic Risk Derived from Exposure to Pesticides in Corn Producers in Tlaxcala, Mexico

Round 1

Reviewer 1 Report (New Reviewer)

Dear,

I judge this paper is an interesting piece of work.

However, I have some suggestions before publishing:

Abstract:

“Grupo Vicente Guerrero (GVG) conceives a greater importance of 17 problems related to the use of pesticides for nature, as well as for health of workers and consumers.”. Please remove it. It seems an advertisement.

“Farmers exposed to pesticides (FC) 26 are urgently recommended to implement training, education, and information programs on the 27 proper use of pesticides, safety measures and regulatory provisions by local authorities.” Please remove it. It is a general recommendation, valid for all farmers of the world…

 

Text:

L51: Please be more specific regarding the word “phosphoric”, which means “regarding phosphorous..”.

L117: Do you have the ethic´s permission to do these surveys?

L163: typo in Kolmogo-rov

L180: In the whole text use p<=0.05 instead of p<0.05.

Table 1 and in the whole text: Source: Research data, 2022.... Is it necessary?

L461: Chlorophenoxy: use chlorophenoxy with lowercase. It is valid for the entire paper.

 

 

References: Are all references necessary? Please explain it.

 

I have not checked the paper regarding plagiarism and self-citations.

 

Sincerely yours.

Author Response

Reviewer 1

Thanks for the comments and suggestions.

All the observations to the document marked in green were made.

Abstract:

"Vicente Guerrero Group (GVG) conceives a greater importance of 17 problems related to the use of pesticides for nature, as well as for the health of workers and consumers." Please delete it. It looks like an ad.

"Farmers exposed to (FC)26 pesticides are urgently recommended to implement training, education and information programs on the proper use of pesticides, safety measures and regulatory provisions by local authorities." Please delete it. It is a general recommendation, valid for all farmers in the world...

Response:

Removed at reviewer's suggestion.

Text message:

• L51: Please be more specific about the word "phosphoric", which means "with respect to phosphorous...".

Response:

The wording in the text of the work is improved.

• L117: Do you have ethics permission to do these surveys?

Response:

Yes. In section 2.3 everything related to the permission by the participants is mentioned.

• L163: typo in Kolmogo-rov

Response:

The typo is removed.

• L180: Throughout the text use p<=0.05 instead of p<0.05.

Response:

All changes suggested by the reviewer were made.

• Table 1 and throughout the text: Source: Research Data, 2022.... Is it necessary?

Response:

Not required, decision is made to remove the phrase Research Data, 2022.

• L461: Chlorophenoxy: Use lowercase chlorophenoxy. It is valid for the entire article.

Response:

It is suggested to leave in capital letters.

• References: Are all references necessary? Please explain.

Response:

Yes. The work is well supported by references. The references are recent and contribute to a better understanding of the work presented, in addition the discussion is rich and well supported by the bibliography. That is why all the references are necessary, finally in the terms of reference of the journal there is no mention of a minimum or maximum number of references at the time of uploading the work for its opinion.

• I have not reviewed the document regarding plagiarism and self-citation.

Response:

The publisher of the magazine takes care of this aspect and they let us know in the final evaluation.

Author Response File: Author Response.docx

Reviewer 2 Report (New Reviewer)

This paper describes a study on the evaluation of cytotoxic and genotoxic risk derived from exposure to pesticides in corn producers in Tlaxcala (Mexico). Two different groups of farmers were assessed in this study, one more exposed to pesticides (n = 47) and a second one using more agroecological practices (n = 43).

The buccal micronucleus cytome assay (BMCA), a non-invasive tool, was used as a biomarker of genetic damage caused by exposure to pesticides. Other variables, such as age, level of education, smoking habits, and alcohol consumption were also taken into consideration in the statistical analysis.

The results (micronuclei assay, as well as nuclear abnormalities present in cells of the epithelium of the buccal mucosa) showed that there were differences between the two groups assessed, with farmers more exposed to pesticides having a higher risk of presenting genotoxic and cytotoxic damage compared to the group of farmers with a higher incidence of agroecological practices and less use of pesticides.

The work seems to be new and to have been properly carried out, and the manuscript is generally well written and organized. The discussion is rich and well supported in the bibliography.

Given the novelty of the study, i.e., the results obtained for the two groups of farmers in this particular location, I recommend publication after minor revision (please see specific comments in the pdf file attached).

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Thanks for the comments and suggestions.

All the observations to the document marked in yellow were made.

Author Response File: Author Response.docx

Reviewer 3 Report (New Reviewer)

The manuscript is interesting, well written, easy to read and completely understandable. In the work, the authors consider an important problem that needs to be published in the journal. I have no comments on the manuscript. I think that the manuscript should be published in a journal Applied Sciences.

Author Response

Reviewer 3

Thank you for the comments on the work presented.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

 

The manuscript titled ‘Genotoxicity due to Pesticide Exposure in Maize Producers of the Vicente Guerrero Community, Tlaxcala-Mexico’ attempts to measure the cytotoxic and genotoxic effects of pesticides on maize producers in Tlaxcala-Mexico. The manuscript will need some changes before it can be considered for publication.  

 

1.       There are several small paragraphs in the manuscript which can be consolidated in longer paragraphs based on the themes.

2.       A summary figure to highlight the proposed pesticide effects will be useful.

3.       Did authors measure any mRNA or protein biomarkers for cytotoxic and genotoxic effects?

 

Author Response

Thanks for the comments and suggestions.

All the observations to the document marked in yellow were made.

1. There are several small paragraphs in the manuscript that can be consolidated into longer paragraphs depending on the topics.

The manuscript was consolidated at the reviewer's suggestion.

2. A summary figure will be helpful to highlight the proposed effects of the pesticide.

Table 1 is attached, where the effects on health exerted by the different pesticides are highlighted.

3. Did the authors measure any mRNA or protein biomarkers for cytotoxic and genotoxic effects?

The work did not contemplate measuring any mRNA or protein biomarker for cytotoxic and genotoxic effects. The MN assay used in this research is an alternative to the conventional chromosomal aberration test, in which the alterations present in mitotic metaphases are analyzed and it allows the detection of chromosomal aberrations that respond to alterations of a structural type (clastogenic effect) or numerical alterations ( aneugenic effect) of the agent under study.

In addition, due to the covid-19 pandemic, access to laboratories was very limited, just as the participation of peasant corn producers was very limited. However, their proposal to use a biomarker for other studies could be considered.

Author Response File: Author Response.docx

Reviewer 2 Report

The results presented in this research show the need for protection measures for 425 farmers exposed to higher use of pesticides (PC). They consider as urgent to implement training,  education, and information programs on the proper use of pesticides, as well as the provision of protective clothing and supervision by local authorities. 

Although the article is well written and relevant to the farmers in the area (Vicente Guerrero Community), I consider the article is not novel or original enough to publish it in ijerph. This article is useful in order to implement training and education programs in the farmers of this community but the international relevant of this article is limited. It has already been demonstrated in different communities around the world the toxicity of these pesticides and it has also been shown that they are still widely used. The measures and programs derived from this article all only of local interest.

Author Response

Thanks for the comments and suggestions.

However, I say the following:

The MN assay used in this research is an alternative to the conventional chromosomal aberration test, in which the alterations present in mitotic metaphases are analyzed and it allows the detection of chromosomal aberrations that respond to alterations of a structural type (clastogenic effect) or numerical alterations ( aneugenic effect) of the agent under study.
In addition, the MN assay manages to: a) reduce the difficulty in performing the count, b) improve the sensitivity of the assay, since MN are only counted in cells that have completed a nuclear division, c) increase the power statistics of the studies by analyzing thousands of cells instead of hundreds of them, d) reduce the cost of the test. Therefore, the MN assay is a practical, universally validated and technologically accessible assay useful for evaluating genetic instability induced by genotoxic agents.

In addition to this point, and due to the covid-19 pandemic, access to laboratories was very limited, as was the participation of the agricultural sectors, which is why this study is adapted to these disadvantages that we all experience and As you rightly mention, it is useful to implement training and education programs for farmers in this community. However, these results should be disclosed not only locally or regionally. We believe that it has the academic quality to make these results known internationally, and to continue adding to the results that show the toxicity of these pesticides in different communities around the world.

Finally, in the International Journal of Environmental Research and Public Health IJERPH allows us to publicize this type of results, in addition, it should be noted that I have found a great variety of publications referring to genotoxicity of regional and local importance, as is the case of our study (I mention some examples), that is why I consider it novel and original enough to be published.

Examples:

• Hutter, H.-P.; Khan, A.W.; Lemmerer, K.; Wallner, P.; Kundi, M.; Moshammer, H. Cytotoxic and Genotoxic Effects of Pesticide Exposure in Male Coffee Farmworkers of the Jarabacoa Region, Dominican Republic. Int. J. Environ. Res. Public Health 2018, 15, 1641. https://doi.org/10.3390/ijerph15081641
• Hutter, H.-P.; Poteser, M.; Lemmerer, K.; Wallner, P.; Shahraki Sanavi, S.; Kundi, M.; Moshammer, H.; Weitensfelder, L. Indicators of Genotoxicity in Farmers and Laborers of Ecological and Conventional Banana Plantations in Ecuador. Int. J. Environ. Res. Public Health 2020, 17, 1435. https://doi.org/10.3390/ijerph17041435
• Valencia-Quintana, R.; López-Durán, R.M.; Milić, M.; Bonassi, S.; Ochoa-Ocaña, M.A.; Uriostegui-Acosta, M.O.; Pérez-Flores, G.A.; Gómez-Olivares, J.L.; Sánchez-Alarcón, J. Assessment of Cytogenetic Damage and Cholinesterases’ Activity in Workers Occupationally Exposed to Pesticides in Zamora-Jacona, Michoacan, Mexico. Int. J. Environ. Res. Public Health 2021, 18, 6269. https://doi.org/10.3390/ijerph18126269

Author Response File: Author Response.docx

Reviewer 3 Report

 

This study performed buccal micronucleus cytome assay (BMCA) among farmers in Espanita in Mexico. Of these, 43 worked in organic farming while 47 were exposed to a range of pesticides on a more regular basis. Those exposed to pesticides displayed a higher rate of cytological signs of cytotoxicity and genotoxicity. So far, the study is straightforward and plausible. BMCA is in general a valid test for recent cellular damage. Usually, buccal cells should be viewed under the microscope both under visible light and under UV-light. Under visible light alone some structures (e.g. dirt) cannot be discerned with certainty from nuclear material. But only nuclear material will display fluorescence under UV-light.

The authors only examined cells under visible light. Therefore, they likely found also false positive pathologies. This is not a big issue as this false positive rate likely affected both groups of farmers in a similar manner and thus would not pose a confounder. But the authors at least should mention this as a shortcoming in their discussion. Indeed, false positives would even add statistical noise to their analysis and thus would even increase the likelihood that a true effect were missed.

Generally, I am not fully convinced by the statistical methods applied. The dependent variables of the study are count data (number of cells with certain pathologies, number of micronuclei). Count data most likely show a Poisson distribution. To examine the effects of exposure together with the effect of possible confounders (age, smoking, alcohol, education) and moderator variables (personal protection), a Poisson regression analysis would be the natural way to do. Instead, the authors performed linear regression analysis or comparison of the mean, stratified analyses (e.g. per smoking category or per age group), and also Spearman correlation tests. The results are rather confusing or – in the case of the stratified analysis – often not very informative. For example, they show that among light smokers (1-5 cigarettes per day), there is no significant effect of exposure. But this is likely only due to low power of that analysis as only 4 and 2 farmers were included in this stratum.

Besides these two main issues (mentioning lack of fluorescence microscopy and using an improved statistical analysis strategy), I only have some minor concerns.

I did get confused by geographical facts. I (as likely also other readers) am not very familiar with the Mexican geography. Why do the authors mention (line 54) the states Sinaloa, Colima, Sonora, Chiapas, Veracruz, Michoacán, Tabasco, Campeche, Yucatán, Puebla, and Oaxaca? As far as I understand, Espanita lies in none of these. Since I do not know if the mentioned states represent less or more than 70% of the arable land of Mexico used for the cultivation of maize, I cannot even say if 70% of all pesticides used is much or not. The description of the study area (line 85 onwards) also causes more confusion than it provides enlightenment. If the authors believe that this information is important, maybe a map would help? Why was Vicente Guerrero chosen in the first place? I would accept the fact that it was chosen because of personal contacts of the researchers. But it might be that the research area is special in some way. For example, it might provide the unique opportunity to examine exposed and not exposed farmers that are very similar in most (other, e.g. socio-economic) characteristics. For me, it is less important to learn the names of the neighboring communities. And since the study was performed in Vicente Guerrero, which is only one part of the municipality Españita, the size of that municipality is maybe also not as relevant as the size of the locality. That the authors call Vicente Guerrero a municipality, not a locality, in the next paragraph (line 98) even adds to my confusion.

From the results section I understand that the 47 + 43 farmers were also asked about life-style, socio-economic and work related issues. But in the methods section we only learn that a “questionnaire on exposure to pesticides was applied”.

Table 2 reports active ingredients and chemical substance group besides the commercial names of the pesticides. Since this is an English language paper, active ingredients and chemical substance group should be written in English, not in Spanish!

I do not fully understand table 4. But maybe it only repeats the results also presented in figure 1? But how can a “Range” sometimes from negative to positive (KL) and sometimes from positive to negative values (KR)? How is it possible that a range that includes zero (KR) can still be highly significant? Besides, I believe the U-statistic is used to calculate the p-value and it is not a result relevant by itself.

Author Response

Thanks for your comments

All comments were made on the document

1. This study performed buccal micronucleus cytome assay (BMCA) among farmers in Espanita in Mexico. Of these, 43 worked in organic farming while 47 were exposed to a range of pesticides on a more regular basis. Those exposed to pesticides displayed a higher rate of cytological signs of cytotoxicity and genotoxicity. So far, the study is straightforward and plausible. BMCA is in general a valid test for recent cellular damage. Usually, buccal cells should be viewed under the microscope both under visible light and under UV-light. Under visible light alone some structures (e.g. dirt) cannot be discerned with certainty from nuclear material. But only nuclear material will display fluorescence under UV-light.

Response:
The year 1999 was crucial for the MN assay, since the technique was validated worldwide and considered as an effective biomarker of DNA damage, using the visual means of visible light optical microscopy as the standard, and to date it has been continues to use with favorable.
Currently, the application of fluorescence in situ hybridization (FISH) techniques allows the identification of the origin of MN, allowing the precise determination of the clastogenic and/or aneugenic effect of any genotoxic agent. However, in the use of fluorescence, the following disadvantages must be considered: a) acridine orange dye and phosphates are more expensive than other dyes, b) using this dye decreases the analysis time even by 50%, taken into account that an expert will take an average of one hour to analyze a sample with other dyes, of course, depending on the skill of the technician and the quality of the sample, c) the handling of the dye requires extreme precautionary measures, since it is a highly genotoxic reagent because it is a base analog, so it tends to be mutagenic when introduced into the DNA and causing a shift in the reading frame, d) the fluorescence equipment requires a mercury lamp, which For safety, it must be replaced every 100-120 hours of use, which increases costs.
The validation created an international human micronucleus program (HUMN: HUman MicroNucleus Project), designed by Michael Fenech and Stefano Bonassi in order to collect basal MN frequencies obtained in different laboratories and populations around the world [http://ehs.sph .berkeley.edu/holland/humn/].
In conclusion, the MN assay is an alternative to the conventional chromosomal aberration test, in which the alterations present in mitotic metaphases are analyzed and it allows the detection of chromosomal aberrations that respond to structural alterations (clastogenic effect) or numerical alterations (aneugenic effect). ) of the agent under study.
In addition, with the MN assay it is possible to: a) reduce the difficulty in performing the count, b) improve the sensitivity of the assay, since MN are only counted in cells that have completed a nuclear division, c) increase the statistical power of the studies by analyzing thousands of cells instead of hundreds of them, d) reducing the cost of the assay.
The MN assay is therefore a practical, universally validated and technologically accessible assay useful for evaluating genetic instability induced by genotoxic agents.

Finally, I annex publications referring to the micronucleus technique under visible light:

• Sommer, S.; Buraczewska, I.; Kruszewski, M. Micronucleus assay: The State of art, and future directions. Int J Mol Sci. 2020, 21(4): 1534. https://doi: 10.3390/ijms21041534.
• Rincón, G., Sánchez, C. Short assaydesign for micronucleus detection in human lymphocytes. Biomed Res Int. 2021, 11;2021:2322257. https://doi: 10.1155/2021/2322257.
• Shen, X.; Chen, Y.; Li, C.; Yang, F.; Wen, Z.; Zheng, J.; Zhou, Z. Rapid and automatic detection of micronucleiin binucleated lymphocytes image. Sci Rep. 2022, 10; 12(1): 3913. https://doi: 10.1038/s41598-022-07936-4.
• Heaven, C.J.; Wanstall, H.C.; Henthorn, N.T.; Warmenhoven, J.W.; Ingram, S.P.; Chadwick, A.L.; Santina, E.; Honeychurch, J.; Schmidt, C.K.; Kirkby, K.J.; Kirkby, N.F.; Burnet, N.G.; Merchant, M.J. The suitability of micronucleias markers of relative biological Mutagenesis. 2022, 2;37(1):3-12. https://doi: 10.1093/mutage/geac001.
• Arnaoutoglou, C.; Keivanidou, A.; Dragoutsos, G.; Tentas, I.; Meditskou, S.; Zarogoulidis, P.; Matthaios, D.; Sardeli, C.; Ioannidis, A.; Perdikouri, E.I.; Giannopoulos, A. Factors affecting the nuclei in newborn and children. Int J Environ Res Public Health. 2022, 1;19(7):4226. https://doi: 10.3390/ijerph19074226

2. The authors only examined cells under visible light. Therefore, they likely found also false positive pathologies. This is not a big issue as this false positive rate likely affected both groups of farmers in a similar manner and thus would not pose a confounder. But the authors at least should mention this as a shortcoming in their discussion. Indeed, false positives would even add statistical noise to their analysis and thus would even increase the likelihood that a true effect were missed.

Response:

The methodology explains that the participants in both groups rinsed their mouths twice with tap water precisely to avoid false positives with food remains.

3. Generally, I am not fully convinced by the statistical methods applied. The dependent variables of the study are count data (number of cells with certain pathologies, number of micronuclei). Count data most likely show a Poisson distribution. To examine the effects of exposure together with the effect of possible confounders (age, smoking, alcohol, education) and moderator variables (personal protection), a Poisson regression analysis would be the natural way to do. Instead, the authors performed linear regression analysis or comparison of the mean, stratified analyses (e.g. per smoking category or per age group), and also Spearman correlation tests. The results are rather confusing or – in the case of the stratified analysis – often not very informative. For example, they show that among light smokers (1-5 cigarettes per day), there is no significant effect of exposure. But this is likely only due to low power of that analysis as only 4 and 2 farmers were included in this stratum.

Response:

Thanks to notice the error of the presentation of the results.

We made an error in Table 3 in the “Personal protective equipment” section by showing the variables without categories, which created confusion for the validity of the test.

The error is corrected and the changes are shown in table 3 in green, in this way the statistical analysis between the two groups according to the Mann-Whitney U test (p ≤0.05) is significant, as shown in Table 4. In addition, we consider performing a Power analysis of a study (1-β) and the effect of the sample size of the study to give greater statistical validity between the comparison of the groups studied.

With respect to Spearman's correlation, it is worth mentioning that only the total number of micronuclei found was correlated by group with each variable that did not present a normal distribution (Age, Education level, Smoking habits, Alcohol consumption, Personal protection), obtaining only a statistical difference in the Personal protection variable for both groups.

4. I did get confused by geographical facts. I (as likely also other readers) am not very familiar with the Mexican geography. Why do the authors mention (line 54) the states Sinaloa, Colima, Sonora, Chiapas, Veracruz, Michoacán, Tabasco, Campeche, Yucatán, Puebla, and Oaxaca? As far as I understand, Espanita lies in none of these.

Response:

A map is added to better indicate spatial location.
Españita belongs to the state of Tlaxcala and is not found in the states with the highest use of pesticides, however, there are farmers who use this type of product; since it is widely used in Mexico in the production of corn.
In the municipality of Españita, in the town of Vicente Guerrero, there are corn-producing farmers who have been applying agroecological practices and less use of pesticides for 30 years, which is why this town was selected to carry out the study.

The error of calling the Vicente Guerrero locality marked in blue a municipality has been corrected.
The questionnaire is complemented with sociodemographic characteristics of the study groups in the methodology section marked in blue.

5. Table 2 reports active ingredients and chemical substance group besides the commercial names of the pesticides. Since this is an English language paper, active ingredients and chemical substance group should be written in English, not in Spanish!

Response:

Table 2 is corrected

6. I do not fully understand table 4. But maybe it only repeats the results also presented in figure 1? But how can a “Range” sometimes from negative to positive (KL) and sometimes from positive to negative values (KR)? How is it possible that a range that includes zero (KR) can still be highly significant? Besides, I believe the U-statistic is used to calculate the p-value and it is not a result relevant by itself.

Response:

Table 4 is improved

Author Response File: Author Response.docx

Reviewer 4 Report

The study is of interest, comparing cell damage in agricultural workers with different degrees of pesticide exposure. However, it has deficiencies in the descriptions of the statistical analysis, in addition to other minor deficiencies that are easy to improve.

Please see the attached file

Comments for author File: Comments.pdf

Author Response

Thanks for the comments and suggestions.

All the observations to the document marked in green were made.

2.2 I suggest improvement with sample size and participant selection

Thanks for this remark, however it is not possible to extend the sample selection, the paper it is mentioned that the sample is sufficient to detect sample effect sizes of about 2/3 standard deviation. This gives us the reliability of the sample in the selection of participants.

Study area: it would be nice to have a map of the area.

Added map as suggested by reviewer

Table 1: Add a column to the right with the statistical significance of the comparison of variables (p value).

I consider that it is not necessary, the table shows frequencies and percentages and could cause confusion for many readers when putting the significance with the percentage, that is why we decided to keep table 1 without changes.

The main topic in the manuscript is statistical analysis.

Thanks for noticing the error.

We made an error in Table 3 in the “Personal protective equipment” section by showing the variables without categories, which created confusion for the validity of the test.

The error is corrected and the changes are shown in table 3, in this way the statistical analysis between the two groups according to the Mann-Whitney U test (p ≤0.05) is significant, as shown in table 4. In addition, we consider performing a Power analysis of a study (1-β) and the effect of the sample size of the study to give greater statistical validity between the comparison of the groups studied.

With respect to Spearman's correlation, it is worth mentioning that only the total number of micronuclei found was correlated by group with each variable that did not present a normal distribution (Age, Education level, Smoking habits, Alcohol consumption, Personal protection), obtaining only a statistical difference in the Personal protection variable for both groups.

The topic of age: I suggest revising and improving the explanation of age.

In the paragraph referring to age, the indicated criteria are taken into account, please verify it.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

the paper has been improved substantially. Still, some minor features should still be tackled:

Table 1: improve lay-out, for example by adding lines between the cells. It is difficult to see where the "health effects" text of one line ends and the next begins.

Line 95ff: what is the meaning of the fact that "70% of the total pesticides used ... are applied in the following states"? The states listed do not even include Tlaxcala. And is 70% much? That might depend on the population size or size of the agricultural alnd of these states. Most likely the statement is meaningless in relation to the study at hand.

Line 143: what is the meaning of "large" and "small" producers? Has the size of the field (?) an impact on pesticide use? Has the number of producers an impact on the sample size?

Figure 1: still not familiar with the geography of Mexico, I find it difficult to link the 4 maps. That each map has the same size (0-8, albeit at different units), does not help. Maybe add some lines linking the maps together!

Line 212 and later: what is the meaning of "PC" and "PA"? Why did you chose these abbreviations. They do not come to mind easy in English language. So the reader has always to check again what they stand for. And you even do not use these abbrevations consistently. You also use the abbreviations "FC" and "FA" instead (e.g. figure 2).

Line 352: "The present study presented a lower proportion of cells... in the exposed group" I do not understand this. If you mean that KL, BN, and CB are lower in the exposed compared to the unexposed group, this statement is simply not correct!

 

Author Response

Thanks for the comments and suggestions.

All the observations to the document marked in yellow were made.

Comments by reviewer

1. Line 95ff: what is the meaning of the fact that "70% of the total pesticides used ... are applied in the following states"? The states listed do not even include Tlaxcala. And is 70% much? That might depend on the population size or size of the agricultural alnd of these states. Most likely the statement is meaningless in relation to the study at hand.

Respuesta

The text was improved according to what was suggested by the reviewer

2. Line 143: what is the meaning of "large" and "small" producers? Has the size of the field (?) an impact on pesticide use? Has the number of producers an impact on the sample size?

Respuesta

The text has been improved and this part is better explained, according to what was suggested by the reviewer

3. Figure 1: still not familiar with the geography of Mexico, I find it difficult to link the 4 maps. That each map has the same size (0-8, albeit at different units), does not help. Maybe add some lines linking the maps together!

Respuesta

Made at the request of the reviewer

4. Line 212 and later: what is the meaning of "PC" and "PA"? Why did you choose these abbreviations. They do not come to mind easy in English language. So the reader has always to check again what they stand for. And you even do not use these abbrevations consistently. You also use the abbreviations "FC" and "FA" instead (e.g. figure 2).

Respuesta

The term for the document is homogenized and the abbreviation is changed at the suggestion of the reviewer

5. Line 352: "The present study presented a lower proportion of cells... in the exposed group" I do not understand this. If you mean that KL, BN, and CB are lower in the exposed compared to the unexposed group, this statement is simply not correct!

Respuesta

The observation is correct, the text is modified.

Author Response File: Author Response.docx

Reviewer 4 Report

The quality of the manuscript has evidently improved.

The authors made almost all the suggested changes.

 

Author Response

Thanks for the comments and suggestions.

All comments on the document were made.

Author Response File: Author Response.docx