Evaluation of paraoxonase I and hemoglobin levels in farmers and agricultural workers in relation to organophosphorus and carbamate levels in their blood and urine samples: A cross sectional study

Background: Pesticides are chemicals that have become common household products in developing countries. The purpose of pesticides is to manage agricultural work. The majority of pesticides for indoor and agricultural use are carbamate and organophosphorus. Toxicity is caused due to excess and improper use or disposal of these chemical agents. Slow exposure to pesticides causes chronic poisoning whereas rapid exposure causes acute poisoning. The paraoxonase I (PON 1) enzyme has a role in detoxifying some of the oxon derivatives which thereby inhibit acetylcholinesterase and butyrylcholinesterase. Methods: This study analyzed farmers who were exposed intermittently to organophosphorus and carbamates pesticides during farming for more than five years. Serum paraoxonase I was evaluated by colorimetry method, and hemoglobin levels were evaluated using portable Fresenius Kabi haemoglobinometer. Results: The study showed that the pesticides were found in the blood and urine samples of farmers and there was an alteration of paraoxonase I and hemoglobin levels in them due to the exposure of pesticides in large quantities over some time. The present study showed around 81% of the participants who were intermittently exposed to pesticides for more than five years were detected with pesticide toxicity. The paraoxonase I level was altered in farmers who were positive for organophosphorus and carbamate pesticides. The hemoglobin level did not show much variation among the farmers exposed to pesticides. This may be due to the lifestyle of the subjects, climatic variations and also their eating habits. Conclusions: The study suggested that there was alteration in the levels of PON1 and hemoglobin in farmers and agricultural workers with positive organophosphorus and carbamates in their blood and urine samples. As our study was done without quantifying the amount of pesticides, further studies can be done by quantifying the pesticide level and comparing it with the paraoxonase I level.


Introduction
Pesticides are synthetic chemical compounds that are used to destroy pests including insects and unwanted plants.As a result of its easy availability, it has become a commonly used agricultural product in rural areas of developing countries. 1esticide poisoning is a major challenging problem all over the world. 2 Exposure to pesticides, specially of OPs is an important toxicological issue of concern, in India.Acute pesticide poisoning is an important cause of morbidity and mortality in India. 3 and CBM pesticides are common household insecticides used commonly for agricultural purposes in developing countries. 4,5In India, OP, CBM, organochlorine, and pyrethroids are the commonly used pesticides. 6Most of these pesticides are sold directly from shops due to the lack of special rules and regulations regarding the use and sale of pesticides in countries like India. 2 Chronic and acute exposure is mainly through residue of pesticides sprayed to crops and vegetables. 7According to a recent study, the various food commodities such as cereals and vegetables contain pesticide residues. 8Hence the farmers and agricultural workers exposed to pesticides are at risk of acute or chronic poisoning.PON I, an Esterase, can detoxify pesticide derivatives in human serum. 9The primary function of the PON I enzyme is likely to be related to its antioxidant properties. 10However, it plays an important role in the pesticide toxicity pathway by helping in hydrolyzing several oxon derivatives. 11PON I activity of an individual can vary to a large extent.It is reported that PON I levels can vary by at least 13-fold and the activity up to 40-fold. 7This implies that the ability of individuals to detoxify OP would depend on their PON I activity.Pesticide poisoning inhibits the PON I enzyme which in turn inhibits acetylcholinesterase in synapse and on red blood cell (RBC) membrane and butyrylcholinesterase in plasma. 12Accumulation of cholinesterases causes central nervous system depression or seizures, and respiratory failure. 13e PON-I has a role in prevention of oxidative stress and inflammation, the level of which is affected in poisoning cases. 14The lifespan of red blood cells can be shortened through oxidative damage and toxic mechanisms resulting in anaemia.Hemoglobin is also subjected to oxidative damage resulting the formation of methemoglobin which is nonfunctioning resulting in tissue hypoxia. 14e present study shows the pesticide toxicity in farmers and agricultural workers who were exposed to pesticides for more than 5 years.Exposure was confirmed by asking the participants about their use/contact with pesticides, and toxicity was determined via the paraoxonase I and hemoglobin levels measured.

Ethics statement
Ethical approval was obtained from Institutional Ethics Committee (Kasturba Hospital, Manipal, which is a part of Kasturba Medical College, Manipal; No: IEC143/2017 dated 15.02.2017) before starting the study.The detailed plan of the study was explained to the 100 farmers and agricultural workers through the Participant Information Sheet (PIS) and on willingness to participate in the study, written informed consent in a printed form was obtained from the study participants.After agreeing to participate in the study, the participants voluntarily signed the form.The consent form was pre-validated and approved from the ethics committee.The consent was obtained for both participation and for academic publication purposes.

Study design
This cross-sectional prospective observational study was conducted between October 2017 to April 2018 in the poison information center, part of the Kasturba tertiary care teaching hospital in South India.Both male and female agricultural workers were included in the study belonging to the age group 25-75 years.100 farmers and agricultural workers (50 males and 50 females) of the native district (Udupi district of Karnataka) were included in the study and the blood and urine samples were collected from them in the hospital by the postgraduate medical students (interns) and faculty

REVISED Amendments from Version 1
The title of the article is modified along with few changes in the manuscript as suggested by the reviewers.One new table ( Any further responses from the reviewers can be found at the end of the article members.These farmers and agricultural workers were part of the health checkup camp arranged for them in the native district.A total of 240 agricultural workers were part of health checkup camp.Out of them based on inclusion criteria i.e. those with minimum 5 years of work in the field and more than 25 years of age were included in the study.Those farmers and agricultural workers were identified based on their identity card and initial information collected.The effects of chronic toxicity can be seen in individuals in several months to years after the exposure. 15Hence, only the farmers and agricultural workers who were involved in agriculture for a period of at least 5 years (and who confirmed their exposure to pesticides) were included in the study.
During the study, the antecubital fossa was cleaned with antiseptic solution and through venipuncture by puncturing medial cutaneous vein or cubital vein or brachial vein, 2 ml of the blood was collected.The urine was collected in the labelled plastic container of 20 ml capacity.

Estimation of hemoglobin level
Haemoglobin levels of each participant was evaluated using portable Fresenius Kabi haemoglobinometer.It is essentially a photometer which allows measurement of color intensity of solutions.The disposable micro cuvette acts as reaction vessel in which measurements are made.The reagents necessary for both release of Hb from erythrocytes and conversion of Hb to a stable-colored product are present in dried form on the walls of the cuvette.A small sample (typically 10 μL) of venous blood to the micro cuvette is introduced and micro cuvette is introduced into the instrument.The instrument is factory precalibrated using HiCN standard, and absorbance of the test solution is automatically converted to ctHb.The result is displayed in less than a minute.The displayed results were recorded.

Qualitative estimation of pesticides
A total of 2 ml of each extracted blood and urine sample were processed qualitatively by performing thin-layer chromatography (TLC) by running against organophosphorus (Cypermethrin, Thimet and Chlorpyrifos) and carbamate (Anuferon) standards.
To one portion of the sample, two portions of the diethyl ether was added and vortexed.The upper layer was collected and considered as the treated sample.The extraction process was repeated two to three times.
The thin layer silica sheets were cut according to the number of samples and the size of the chamber.Linea were cut 1 cm away from the top and bottom cut ends of TLC sheets.The line below was for the sample application and the line above was considered as the solvent front.Simultaneously the glass chamber with the solvent system consisting of cyclohexane: acetone:chloroform [70:25:5] in it, was kept for saturation (at least for 10-15 min).
Spotting the standards and treated sample was done using the capillary tubes.The capillary tube was placed vertically on the TLC sheet repeatedly so that the solution was applied in several portions with intermediate drying (blow with cold or hot air).The organophosphorus and carbamate standards were spotted 5-6 times to increase the concentration and the treated samples are spotted 16-18 times for the same reason at the base line drawn.Then the TLC plates were kept in the saturated TLC chamber carefully without disturbing the solvent and allowed the solvent to run till the solvent front.
The solvent ascends through the layer by capillary action and causes the substances to separate.Once the solvent reaches the solvent front (near the top line) the TLC plates are taken out and kept for drying.The dried TLC plates are carefully placed in the visualizing chamber (iodine chamber) for 5-10 min till brown spots are visible.
The plates were then taken out and the spots were marked immediately.Localization of a substance was done with parallel runs with reference substances.The value used for evaluation was R f value (retention factor).R f values of samples were calculated, and comparison was done with the R f value of the standards.R f value was defined as follows: R f = Distance from starting point to middle of spot/Distance from starting point to solvent front.
The farmers and agricultural workers were categorized depending on the presence or absence of pesticides in their blood or urine samples into following 4 groups; carbamate toxicity, organophosphorus toxicity, both carbamate and organophosphorus toxicity and no pesticide toxicity.
Estimation of paraoxanase I level 250 μl of diluted serum was added in 2 ml of tris HCl buffer containing 0.5 ml of 1 mmol of CaCl 2 , 0.5 ml of 2.5% methanol, and 0.5 ml of p-nitrophenyl acetate.The absorbance of the solution is measured at 405 nm.The intensity of the color developed is directly proportional to the amount of Paraoxanase present in the serum.
The data obtained were tabulated and analyzed using Statistical Package for Social Sciences (SPSS) version 21.Data was expressed as Mean AE SD.The significance value was set to p > 0.05.Inter comparison of data between the groups was assessed by ANOVA (One Way Analysis of Variance).
Results 100 samples were collected from 100 farmers and agricultural workers belonging to the age group of 18 to 75 years involving both males and females.All the samples were divided into four groups based on the presence of the type of pesticide as; Group 1: CBM which was detected in 60 farmers; Group 2: OP positive in 13 farmers; Group 3: Absence of pesticide which was seen in 19 participants and Group 4: Both OP and CBM, positive in 8 farmers, as mentioned in Table 1 and Underlying data. 16e four groups were analyzed statistically but did not show any significant relation with a p-value (p < 0.005).PON I level was comparatively low in participants positive for CBM, OP, and both CBM and OP positive when compared with participants who were detected negative for pesticide (Table 2).
Hemoglobin level was considerably low in participants containing both CBM and OP positive cases.
When the levels of haemoglobin and PON I, were compared between the groups, the statistically significant results were not shown (Table 3).F value was 1.856 for intergroup comparison for hemoglobin and 1.780 for PON I which were statistically unsignificant with p value.We had outliers in the study.As a result, the data of one participant was not included in the analysis of intergroup comparison as shown in Table 3.We are confining our study to only adjusted estimates since we have no regression analysis included in our study.
When the inter group comparison i.e.OP positive cases (group 2 and 4) done, the PON I level was raised which was statistically significant as shown in Table 4.

Discussion
Pesticides poisoning is an important toxicological issue all over the world.In rural areas of developing countries like India, pesticidal poisoning causes a greater number of deaths than rest of other diseases or infections. 17Lack of regulatory control to the use and distribution of pesticides is one of the major reasons for these poisoning cases. 18Organophosphorus and carbamate toxicity are the most common cases seen in agriculturally rich areas. 19nagement of pesticide poisoning cases is a greater challenge in developing countries.Though there are specific management protocols available, 20,21 research is still being performed around the world in view of their early diagnosis and more effective management.A number of biomarkers and bedside diagnostic tools are being developed for easy detection of pesticide exposure. 22The levels of biomarkers of pesticide exposures in human invasive samples such as blood and non-invasive samples such as urine have been studied. 3,23,24N I is a high-density lipoprotein-associated enzyme that plays a critical role in the metabolism and detoxification of Organophosphorus pesticides as shown in many studies. 8,10In the present study, the PON I level is comparatively less in Organophosphorus poisoning than in the other poisoning groups.6][27] A low level of PON I enzyme will increase the susceptibility to pesticides causing respiratory and neurological problems. 28The higher levels of PON I activity in the patients may have substantially better detoxifying capacity against OP compounds as shown in research done by Hettagowdanahally V Rahul et al. 29 However, previous researchers have shown that the organophosphorus compounds directly inactivate the PON I affecting neither their synthesis nor clearance of the enzyme. 1 However, PON level is a risk factor, not the effect.Its level and polymorphism is affecting the capacity of detoxication and so the level of OP in blood may be higher as they are hydrolyzed and eliminated at lower rate.Therefore, it should be considered that the level of PON I in the individual is affecting the level of OPs remaining in blood and the possibility of toxic effects.
When the difference in the levels of paraoxonase and hemoglobin was considered, the lower values were observed for OP and CBM positive workers, although it was not statically significant.Cholinesterase inhibition due to pesticide poisoning leads to cellular dysfunction leading to oxidative damage of the RBC membrane.This leads to a lower level of hemoglobin levels in pesticide poisoning cases.In these cases, there is no statistical significance in hemoglobin levels which may be due to environmental factors or due to the food habits of the patients.
In the present study, around 85% of the participants were evaluated positively to various pesticides like OP and CBM.This concludes that there is no proper management for the use and disposal of pesticides.The studies have shown that PON I levels to a larger extent and detoxification of pesticides majorly Organophosphorus would depend on Paraoxanase I. 7 In those people with low level of PON I have lower capacity of detoxication, and consequently, higher level may be detected in their blood.

Limitations
The study had no statistical significance since it had certain limitations.The quantification of organophosphorus and carbamates was not done in our study.Only the samples were analysed qualitatively for presence of organophosphorus and carbamates.Hence the considerable proportionate of lowering of PON I level probably was not statistically significant.

Conclusions
Our study suggests that around 85% of the participants who were exposed to pesticides for more than 5 years were detected with presence of OP and CBM.The PON I level was low in participants who were positive for presence of pesticide than in the participants who were normal.Further studies can be done by quantifying the pesticide level and comparing it with the PON I level.
Early identification followed by effective management in the initial stages increases the rate of survival among pesticide toxic patients.

Data availability
Underlying data Figshare: Evaluation of paraoxonase I and hemoglobin levels in farmers and agricultural workers in relation to pesticide levels in their blood and urine samples: A cross sectional study.https://doi.org/10.6084/m9.figshare.22595113.v3. 16is project contains the following underlying data: -FARMERS LIST -ALL.xlsx.
Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).
This article brings forth all the required details and has analysed in depth the relation of Paraoxonase I and hemoglobin levels in relation to OPC and Carbamate exposure.
The structured abstract helps in the understanding of the relevant details of the study.The introduction and methodology is described in detail.The results have been tabulated and significance levels have been discussed.The discussion is elaborate.
This article has brought forth the importance of evaluation of paraoxonase I and hemoglobin levels in conjunction with organophosphorus and carbamate levels which provides a comprehensive approach to assessing the health and safety of farmers and agricultural workers exposed to pesticides, offering valuable insights for both individual health management and broader public health considerations.
I consider this is a remarkable study .

Is the work clearly and accurately presented and does it cite the current literature?
Yes

Eugenio Vilanova
Universidad Miguel Hernandez de Elche, Elche, Valencian Community, Spain In this paper, blood and urine of farmers working 5 years potentially exposed to pesticides were qualitatively detected organophosphorus (OP) and carbamates (CBM) and measured paraoxonase 1 (PON1) and hemoglobin in serum.
I consider this is an interesting study that does warrant to be published.It is not easy to get direct information about pesticide exposure in humans, therefore any available information is very convenient to be reported, including in studies in which like this, the reported information is very limited.
However, there are some concerns about the lack of some information, and the interpretation of the observations.Therefore, I suggest authors to consider my comment in order to improve the manuscript.
Sorry, I apologise that I have done a long series of comments but it reflects my interest in the topic and my willingness to help for improving the manuscript.

(0). ONE GENERAL CONCERN
The study is studying 100 people, all them farmers with potential exposure to pesticides.The study is lacking of stabilizing a control group with a clear absence of potential exposure.I understand that author is comparing several groups, and may be considered that the equivalent to a control is the group with negative detection by the qualitative procedures.However, the negative detection by such weak procedure does not means absence of exposure.As I consider that any observed data in humans are convenient to be reported, I still consider of interest its publication beside of this deficiency.In any case authors had to include a short comment about this limitation in the Discussion section.
(1).-SOME MINOR GENERAL ISSUES: (1.1).-As the wording organophosphorus and carbamates are used many times along the manuscript (and more if you consider my next suggestion), I recommend define an abbreviation first time mentioned and use the abbreviation along the manuscript.I suggest to use the most usual abbreviation of OP and CbM or CBM.
(1.2).-There are several inconsistencies.Note that the word "organophosphorus" is a common name, therefore, it should be named in lower case always (except when initiating a paragraph, or using an abbreviation).
(1.3).-You are not studying "pesticides" in general, but only OPs and CB, then please delete most of the expression of "pesticides" along the manuscript for the specific kind of evaluated compounds (OP, CB, or OP and CB).
(1.4).-The number of decimal digits (four) in Table 2 for the values of PON and hemoglobin levels has no meaning as the Standard error are in the level of decimals or units, therefore I suggest to show only a maximum of 2 decimal digits which mean up to 4 total significant digits.The same for the PON1 values in the complementary raw data in the Excel table. (

2).-A MAIN CONCERN about interpretations:
In abstract and in several points of the manuscript, it is mentioned that paraoxonase (PON) is altered in worker with positive detection of OP and carbamates.It seems that authors suggest that the change of PON1 is due to the exposure to pesticides, but the interpretation might be just the reverse as I am commenting as follows: Paraoxonase is well known that is able to hydrolyze organophosphorus compounds (OPs) (biodegradation and detoxification) but not inhibited by them.Authors could consider the interpretation that just those people with low level of paraoxonase have lower capacity of detoxication, and consequently, higher level may be detected in their blood.
Actually, for example, in the reference 1, have in the title the same mistake with a inconsistency, as in that paper in the last sentence in abstract, the author concluded with the reverse line of causeeffect relationship: that the level and polymorphism of PON1 is a risk factor affecting to the susceptibility to the exposure to OPs.In your Discussion, I agree with your comment in which you confirm this interpretation.However, at the end of 3rd paragraph you mention this reference (1); I consider that that paper of Sozmen, Mackness et al (2002) did not concluded that OP are directly affecting PON1, although they mentioned this possibility.No confirmation of inhibition of PON1 by OPs have been demonstrated.They actually observed a reduction of level, that could be interpreted by a reduction of liver synthesis.
So, I strongly suggest author to change the rational interpretation, changing the direction of the correlation PON<> (OP LEVEL AND TOXICITY).PON level is a cause (risk factor), not the effect.PON1 level and polymorphism, is affecting the capacity of detoxication and so the level of OP in blood may be higher as they are hydrolyzed and eliminated at lower rate.
Therefore, it is not true that OP exposure is strongly altering PON (what is mainly genetically conditioned), while it should be considered that the level of PON1 in the individual is affecting the level of OPs remaining in blood and the possibility of toxic effects.
This comment does not invalidate the value of the paper and that in my opinion the recorded data is of interest to be published.My concern is with the interpretation.
(3).-OTHER MINOR ISSUES (3.1).-The difference in the levels of paraoxonase and hemoglobin, seems not be statistically significant, therefore author should be more prudent describing the differences in the level of the different groups and indicated in results and abstract expressions as "lower values were observed for OP positive workers, although no statically significant.
(3.2).-Under my point of view the relationship with PON1 level has actual sense in relationship with OPs.Consequently, it should be of interest comparing all positive OP (group 2+4) with all negative OP (group 1 + 3) cases.
(3.3).-For hemoglobin, I have more difficult to give an interpretation of why OP positives (group 2 and 4) seems to show lower level .The suggested interpretation of an effects in the cells producing hemoglobin seems a reasonable interesting speculation, although there is not published data supporting it, but perhaps it worth to be investigated in future.
(4).-ABSTRACT (4.1).-Abstract is a very important section of the paper as may reader only read it, and it should reflect clearly and objectively the content of the manuscript.I consider that it needs to be improved significantly.
(4.2).-In Method in abstract should be mentioned that OP and CB pesticides were detected by a qualitative method with thin layer chromatography, and assigned a positive or negative to OP or carbamates.
(4.3).-In method in abstract, please indicate the kind of epidemiological study (cross-sectional, longitudinal, other).In title you say "cross sectional".However, in the abstract you are mentioning 5 years.Have you been studying these workers continually or periodically during this time, or on the contrary, in a specific time but to workers that had been working during the las 5 years?(4.4).-Taking into consideration my main concern, please modify abstract to show more objective and precise description of the observations.Some examples as follows: The sentence: "The study showed that the pesticides were found in the blood and urine samples of farmers and there was an alteration of paraoxonase I and hemoglobin levels in them due to the exposure of pesticides in large quantities over some time." For something like: "Farmers were classified as a function of positive/negative detection of OP and CB and the level of PON1 and hemoglobin were compared in the different groups." The sentence: "The present study showed around 81% of the participants who were intermittently exposed to pesticides for more than five years were detected with pesticide toxicity." For: "The 81% of the participants showed positive detection for OPs, CB or both" (4.5).-You also indicated in abstract that 81% workers "were detected with pesticide toxicity".What you want to mean with "toxicity"?Are you wanting to say that all they suffer illness or disease and some symptoms and signs of health alteration compatible with toxicity OP/carbamate?In that case, please be more precise and specific, indicating the specific sign of toxicity.I understand that you are not evaluating clinically toxicological effects, therefore delete the wording "toxicity" in all related comments along the manuscript.I understand that you are using as criteria the positive detection with the qualitative methods you have used.
(4.6) Also, you say that "alteration in the levels of PON1 and hemoglobin in farmers and agricultural workers with positive organophosphorus and carbamates in their blood", however later you say that "done without quantifying the amount of pesticides" Therefore it is no clear how you could consider "positive in OP and CBM in blood?For this reason is needed my suggestion ( -please, consider my previous comments before for abstract, also in those place in the section of Methods, Results and Discussion, related with the same topic.-I disagree and suggest delete or modify your first sentence in Discussion, in which you say: "Pesticides poisoning is the major global issue all over the world".This an absurd exaggeration, compare with many other severe issues in the world (cancer, tobacco, COVID, wars, hunger).Therefore, I suggest to change by something like as: "Pesticide poisoning is an important toxicological issue".
The reference (16) says "a major", while you say "the major".This reference is not good evidence, because it is just a statement with no information about the studies that demonstrate the figures they show.Therefore may be better to delete as it is not needed for the purpose of you paper.
(6.2).-Related with the previous comment, I also strongly disagree and I suggest deleting the sentence in the introduction that say "According to World Health Organization (WHO) in 1990, all together around three million pesticide poisoning cases occur annually around the world (WHO  1990)".This and similar sentences are written in many publications but if you think that it is true, please show an appropriate reference of the original objective study demonstrating such statement.Please do not give an indirect reference of reference of reference.It is also common to state that 300.000 fatalities due to OPs intoxication is occurring every year.There are some reports (not official from WHO but supported by WHO) in which, based of some few data were suggesting such kind of figures.These statements are coming from reports in 1990, from data coming from 1970s, without appropriate sufficient data obtained from regions that are not representing the world.It has been assumed that data from a few situations with special incidence due to special social circumstances, can be extrapolated to all the world, and that today is still occurring the same situation.In those statements, 3 million of cases are also suggested from which 2 million by suicides and the other 1 M by occupational and other unintentional intoxication.
In India it was officially recorded about 400,000 suicides during 11 years (1995-2015) by pesticides (around 35,000 `per year) with about 20% fatalities, 90 % by OPs and with a strong tendency to decrease after 2014.If in India with not very good situation with 1.4 thousand million inhabitants, assuming all the world with 8 thousand million people in the same bad situation than India, the extrapolation would be about 300,000 cases (very far from the 2 million in those reports).Assuming about 20% fatalities as described in India, they would be not more than 60.000.Indeed, this extrapolated number would be a very high exaggeration, because the tendency since 2010s is a clear decrease, OPs is strongly decreasing their use in all the world.For example, in my country (40 million population) zero lethal cases by pesticides have been registered during last 5 years.
Can you suggest an original objective study demonstrating that 3 million pesticide acute intoxication?Probably not, therefore it may be better to delete it to avoid something not sure and discussable, and what is not needed for the purpose of your paper.Probably a more simple and neutral statement as "Exposure to pesticides and specially of OPs is an important toxicological issue of concern, in India" This problem is still a big problem in some countries, and may be better to give reference about the specific situation in your country with objective data.If you have not such appropriate reference, better not to give not validated data.You do not need for the purpose of your paper that I still consider of interest its publication.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?Partly

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Partly Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Toxicology of organophosporus compounds I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Shankar Bakkannavar
Dear Reviewer, Thanks a lot for detailed review and letting us know the points to be added/modified in our manuscript.Accordingly we have modified the manuscript pointwise as per your suggestions which are attached below for your perusal.
The study is lacking of stabilizing a control group with a clear absence of potential exposure.I understand that author is comparing several groups, and may be considered that the equivalent to a control is the group with negative detection by the qualitative procedures. 1.
In our study the control group was comprised of participants who were non farmers.The estimation of samples did not reveal the presence of OP and CBM in these samples.I suggest to use the most usual abbreviation of OP and CbM or CBM. 1.

Changed
You are not studying "pesticides" in general, but only OPs and CB, then please delete most of the expression of "pesticides" along the manuscript for the specific kind of evaluated compounds (OP, CB, or OP and CB) 1.
Changes done.However at few places we have kept pesticides as we are introducing the topic and wherever necessary we have removed pesticides and and change to organophosphorus and carbamates.Table 2 for the values of PON and hemoglobin levels has no meaning as the Standard error are in the level of decimals or units, therefore I suggest to show only a maximum of 2 decimal digits which mean up to 4 total significant digits.The same for the PON1 values in the complementary raw data in the Excel table. 1.
Changes done and only 2 decimal points included.ON<> (OP LEVEL AND TOXICITY).PON level is a cause (risk factor), not the effect.PON1 level and polymorphism, is affecting the capacity of detoxication and so the level of OP in blood may be higher as they are hydrolyzed and eliminated at lower rate.

1.
Your statement if highly appreciated and we have added the same thing in the discussion part of our manuscript.Therefore, it is not true that OP exposure is strongly altering PON (what is mainly genetically conditioned), while it should be considered that the level of PON1 in the individual is affecting the level of OPs remaining in blood and the possibility of toxic effects.

1.
PON level is a risk factor, not the effect.Its level and polymorphism is affecting the capacity of detoxication and so the level of OP in blood may be higher as they are hydrolysed and eliminated at lower rate.Therefore, it should be considered that the level of PON1 in the individual is affecting the level of OPs remaining in blood and the possibility of toxic effects.
The difference in the levels of paraoxonase and hemoglobin, seems not be statistically significant, therefore author should be more prudent describing the differences in the level of the different groups and indicated in results and abstract expressions as "lower values were observed for OP positive workers, although no 1.
statically significant.When the difference in the levels of paraoxonase and hemoglobin was considered, the lower values were observed for OP and CBM positive workers, although it was not statically significant.
Under my point of view the relationship with PON1 level has actual sense in relationship with OPs.Consequently, it should be of interest comparing all positive OP (group 2+4) with all negative OP (group 1 + 3) cases. 1.
Values were compared between groups 2 & 4. Abstract is a very important section of the paper as may reader only read it, and it should reflect clearly and objectively the content of the manuscript.I consider that it needs to be improved significantly.

1.
It is improved by changing the statements as we have a word limit to structure the abstract.
In Method in abstract should be mentioned that OP and CB pesticides were detected by a qualitative method with thin layer chromatography and assigned a positive or negative to OP or carbamates. 1.
Included the suggested point in the manuscript.
In method in abstract, please indicate the kind of epidemiological study (crosssectional, longitudinal, other).In title you say "cross sectional".However, in the abstract you are mentioning 5 years.Have you been studying these workers 1.
continually or periodically during this time, or on the contrary, in a specific time but to workers that had been working during the las 5 years?Cross sectional study was included.
"The study showed that the pesticides were found in the blood and urine samples of farmers and there was an alteration of paraoxonase I and hemoglobin levels in them due to the exposure of pesticides in large quantities over some time." 1.
Changes done in the manuscript.Farmers were classified as a function of positive/negative detection of OP and CB and the level of PON1 and hemoglobin were compared in the different groups. 1.
The present study showed around 81% of the participants who were intermittently exposed to pesticides for more than five years were detected with pesticide toxicity. 1.

Changes done
The 81% of the participants showed positive detection for OPs, CB or both 1.

Changes done
delete the wording "toxicity" in all related comments along the manuscript.1.
Toxicity word deleted wherever it was appropriate.
in their blood", however later you say that "done without quantifying the amount of pesticides" Therefore it is no clear how you could consider "positive in OP and CBM in blood? 1.
Positive or negative was decided by qualitative method (TLC)."Pesticides poisoning is the major global issue all over the world".This an absurd exaggeration, compare with many other severe issues in the world (cancer, tobacco, COVID, wars, hunger).Therefore, I suggest to change by something like as: "Pesticide poisoning is an important toxicological issue". 1.

This Statement is considered and added
The reference (16) says "a major", while you say "the major".1.
Reference 16 is deleted as suggested.
"Exposure to pesticides and specially of OPs is an important toxicological issue of concern, in India" 1.
Statement was considered in the manuscript.
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Table 2 .
Mean AE SD between hemoglobin and paraoxanase I levels.

Table 1 .
Number of participants in carbamate, organophosphorus, combination of both, and no pesticide groupings.

Table 3 .
F (test value) and Sig (significance value) of haemoglobin and paraoxanase I level.

Table 4 .
F (test value) and Sig (significance value) of paraoxanase I level in OP positive groups.

the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Reviewer Report 02 January 2024 https://doi.org/10.5256/f1000research.156722.r215720©2024Vilanova E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Eugenio VilanovaUniversidad Miguel Hernandez de Elche, Elche, Valencian Community, Spain Author have considered my comment and suggestions.Therefore I suggest to APPROVE the manuscript.

Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results?
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Version 1
https://doi.org/10.5256/f1000research.144558.r195014© 2023 Vilanova E. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.