Dissipation, Residue, and Dietary Risk Assessment of Methoxyfenozide, Chlorantraniliprole, Indoxacarb, Lufenuron, and Chlorfenapyr in Spinach Using a Modified QuEChERS Method Combined with a Tandem Mass Spectrometry Technique
, Jinju Pan 1, Sumei Yang 2, Jifeng Han 1, Xingang Ma 1, Xiaoxue Qi 1, Lin Liang 1,* and Bojun Zuo 1,*
Round 1
Reviewer 1 Report (New Reviewer)
The document is very dense in some sections, and contents that should go in another section are mixed, which makes it difficult to read. Here are some of these comments:
1. For example, the introduction is very long, it should be synthesized
2. I suggest that the information from section 2.2 be placed in a table, as well as the ionization parameters used (First transition, collision energy, second transition)
3. In section 2.6, I suggest that all the information of detection limits, quantitation limits, recovery percentages be placed in a table.
Some titles should be rewritten in a more appropriate way for the area of ​​study, for example section 2.2 “Instrument and detection conditions”, something like “instrumental conditions” is suggested; section 3.1 “Purity optimization of the agents”, could be something like “Sample preparation and clean up”
On the other hand, I think it is convenient to include in the results section a section on
“Environmental fate of pesticides” that is complemented with a table that includes Toxicological characteristics of the pesticides detected
Author Response
Reviewer 1
The document is very dense in some sections, and contents that should go in another section are mixed, which makes it difficult to read. Here are some of these comments:
- For example, the introduction is very long, it should be synthesized
Response: Thanks for the suggestion. As suggested, we carefully checked and revised the section of Introduction. Delete some unnecessary content, strive for simplicity.
- I suggest that the information from section 2.2 be placed in a table, as well as the ionization parameters used (First transition, collision energy, second transition)
Response: Thanks for the suggestion. As suggested, a table (Table S2) was added to summarize the information of the instrument conditions.
- In section 2.6, I suggest that all the information of detection limits, quantitation limits, recovery percentages be placed in a table.
Response: Thanks for the suggestion. All the information of the method such as average recovery, spiked level, LOQ, are listed in Table 2.
Some titles should be rewritten in a more appropriate way for the area of ​​study, for example section 2.2 “Instrument and detection conditions”, something like “instrumental conditions” is suggested; section 3.1 “Purity optimization of the agents”, could be something like “Sample preparation and clean up”
Response: Thanks for the suggestion. 2.2 “Instrument and detection conditions” has been revised to “Instrumental conditions”;
2.4. “Sample treatment” has been revised to “Sample preparation by modified QuEChERS”;
3.1 “Purity optimization of the agents” has been revised to “Optimization of purification”;
On the other hand, I think it is convenient to include in the results section a section on
“Environmental fate of pesticides” that is complemented with a table that includes Toxicological characteristics of the pesticides detected
Response: Thanks for the suggestion. At present, our research has focused on the residues of five pesticides in spinach, and has not yet studied the environmental fate of pesticides in the environment. In actual production, pesticide residues will not only be found in crops, but also in the environment such as soil and water. The next stage of our work will be to study the effects of pesticide residues on the environment and non-target organisms.
In addition, only one toxicological characteristic (Acceptable daily intake, ADI) of the pesticides has been used in this study, which is listed in Table 5.
We greatly appreciate the helpful and constructive comments, which improved the quality and clarity of our manuscript. Thank you very much!
Reviewer 2 Report (New Reviewer)
The article was devoted to improving the method for analyzing five insecticides in spinach leaves, determining their residues in 8 regions of China and their degradation rate in 5 regions in field and greenhouse applications, as well as assessing their long-term dietary risk. The topic was certainly relevant given the fact that spinach belongs to dietary products and for three out of five insecticides there were no national MRL values.
The purpose of the work was clearly formulated, and the article had a standard structure and was written in an understandable language. The abstract briefly reflected the main results and conclusions of the work. In the introduction, the authors explained why it was important to study pesticide residues in crops and what previous research had been done in this area. The Objects and Methods section provided a full description of the methods used to study residues and process the resulting data. The results section was logically divided into 5 subsections, in which the authors present the data obtained. The Conclusion section needs to be improved, more on that later in the text.
1. The title does not reflect that the authors developed a method for the analysis of five pesticides in spinach leaves, in particular, selected a sorbent for SPE and other parameters, and validated the method. This is also not reflected in the keywords.
2. Tables S2 and S3 contain important information about the condition of the experiment and the values ​​of the MRL, which would be better included in the article.
3. Table S2 gives precipitation data, but does not indicate for which period. Given the fact that the pesticide is contained not only in the leaves, but also on their surface, it is important to know the amount of precipitation during the observation period. It is also desirable to indicate the average air temperature for the observation period.
4. Table 2 shows the initial concentrations of pesticides. As can be seen, the Guangdong region has the highest initial concentrations for four of the five compounds, while Shandong also has the lowest values ​​for four of the five compounds. How can you explain it?
5. Why was the median of the concentrations used for risk analysis, while the OECD recommendations use the mean to determine MRLs? [OECD MRL CALCULATOR: STATISTICAL WHITE PAPER. ENV/JM/MONO(2011)3].
The introduction should analyze how MRL and dietary risk are assessed in the EU, USA and other countries and compare with those used in China.
6. As can be seen from the table 2, Shandong has the highest DT50. How could you explain this, despite the fact that the minimum initial concentrations were also observed here.
7. Explain why MRL selection should be done in this order? The selection of crops whose MRL values ​​have not been established in China should be in the following order: CAC > USA > EU > Australia > Korea > Japan
9. It is not stated how much water was used to treat the crop with pesticides, only that ”the recommended water consumption for the spinach was 450 kg ha-1".
11. The authors performed a full long-term risk assessment of five pesticides using STMR data from WHO and crop intake data from the global environmental monitoring system (GEMS)/Food Consumption Cluster Diets System on multiple crops. This approach to assessing the risk of pesticide consumption in a person's complete food supply does not meet the safety criteria, since the consumption of each product separately must be safe, while the diet of one person may not be sufficiently balanced. It would be more useful to refine the data on spinach consumption in China and make an estimation with the revised data.
12. The authors state: “The difference between the two conventional methods was mainly due to the dietary amount of the consumed crops”. It can be seen from formula 1, that the high rate of spinach (Food intake) and low body weight of the Chinese lead to high NEDI values. And the conclusion that you need to clarify the consumption of spinach is absolutely correct. However, it is important to remember that spinach is used as a diet food and lowering the safety factor or other means to unreasonably increase the tolerable risk level is unacceptable. Therefore, in assessments, it is important to take into account national characteristics of consumption and body parameters for a correct assessment.
13. There is no mention at all in the introduction and conclusion that the method for the determination of five insecticides in spinach leaves has been adapted. In conclusion, it is necessary to describe in more detail what observations were obtained in the experiment. The influence of factors was not considered in the text of the article, so it is not worth mentioning this in the conclusion. It is also incorrect to speak of an acceptable risk for indoxacarb, since the calculations used WHO consumption data, which do not take into account the national specifics of China.
Author Response
Reviewer 2
The article was devoted to improving the method for analyzing five insecticides in spinach leaves, determining their residues in 8 regions of China and their degradation rate in 5 regions in field and greenhouse applications, as well as assessing their long-term dietary risk. The topic was certainly relevant given the fact that spinach belongs to dietary products and for three out of five insecticides there were no national MRL values.
The purpose of the work was clearly formulated, and the article had a standard structure and was written in an understandable language. The abstract briefly reflected the main results and conclusions of the work. In the introduction, the authors explained why it was important to study pesticide residues in crops and what previous research had been done in this area. The Objects and Methods section provided a full description of the methods used to study residues and process the resulting data. The results section was logically divided into 5 subsections, in which the authors present the data obtained. The Conclusion section needs to be improved, more on that later in the text.
Response: Thanks for the positive and valuable comments.
- The title does not reflect that the authors developed a method for the analysis of five pesticides in spinach leaves, in particular, selected a sorbent for SPE and other parameters, and validated the method. This is also not reflected in the keywords.
Response: Thanks for the question. The title has been revised to “Dissipation, residue, and dietary risk assessment of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr in spinach using modified QuEChERS method combined with tandem mass spectrometry technique”.
In addition, relevant contents have been added in the abstract, keywords and conclusion sections.
- Tables S2 and S3 contain important information about the condition of the experiment and the values ​​of the MRL, which would be better included in the article.
Response: Thanks for the suggestion. As suggested, Tables S2 and S3 (Table1 and Table 5) were added in the article.
- Table S2 gives precipitation data, but does not indicate for which period. Given the fact that the pesticide is contained not only in the leaves, but also on their surface, it is important to know the amount of precipitation during the observation period. It is also desirable to indicate the average air temperature for the observation period.
Response: Thanks for the suggestion. As suggested, average temperature and precipitation during the test were added in Table1.
- Table 2 shows the initial concentrations of pesticides. As can be seen, the Guangdong region has the highest initial concentrations for four of the five compounds, while Shandong also has the lowest values ​​for four of the five compounds. How can you explain it?
Response: Thanks for the question. Spinach is a leafy vegetable with a high surface area, making it easy for spray application to cause a higher original deposition of the chemicals being sprayed. Therefore, the growth period, variety and planting density of spinach can lead to the difference of original deposition. Through the photos taken during the experiment, it was found that Shandong spinach has smaller leaves and lower planting density, while Guangdong is just the opposite, which may be the reason for the difference between the two places.
- Why was the median of the concentrations used for risk analysis, while the OECD recommendations use the mean to determine MRLs? [OECD MRL CALCULATOR: STATISTICAL WHITE PAPER. ENV/JM/MONO(2011)3].
Response: Thanks for the question. In 2015, China issued guidelines for the risk assessment of pesticide residues in food to scientifically assess the health risks associated with dietary pesticide residues.
(http://www.moa.gov.cn/nybgb/2015/shiqi/201712/t20171219_6103890.htm)
At present, both China and the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) use the median value for risk assessment.
The introduction should analyze how MRL and dietary risk are assessed in the EU, USA and other countries and compare with those used in China.
Response: Thanks for the suggestion. The differences of MRL and dietary risk are assessed between China and other countries and regions, which are supplemented in Part 3.4. Please see below: MRLs for pesticides are set by the regulatory authorities in each country and are the limits and standards that ensure safety in agricultural products and food. Different countries and regions have slightly different methods of developing MRL and risk assessment. For example, the OECD adds the mean to 4 times standard deviation (SD) of all residue values to obtain unrounded MRL, from which the proposed MRL are derived [41]. Then based on the proposed MRL, chronic consumer risk assessment upon exposure of pesticide was determined. A slight difference with the OECD is that both China and the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) use the median value for risk assessment.
[41] OECD (Organisation for Economic Co–operation and Development). OECD MRL Calculator: Spreadsheet for Single Data Set and Spreadsheet for Multiple Data Set.http://www.epa.gov/pesticide-tolerances/oecd-maximum-residue-limit-calculator/ (2011).
- As can be seen from the table 2, Shandong has the highest DT50. How could you explain this, despite the fact that the minimum initial concentrations were also observed here.
Response: Thanks for the question. Spinach is a kind of plant that likes to grow at low temperature, and high temperature will inhibit its growth. As shown in Table 1, the average temperature in Shandong (greenhouse) was the highest during the experiment, which may inhibit the growth of spinach. As a result, the dilution effect of spinach growth slows down and the half-life in spinach is prolonged.
- Explain why MRL selection should be done in this order? The selection of crops whose MRL values ​​have not been established in China should be in the following order: CAC > USA > EU > Australia > Korea > Japan.
Response: Thanks for the question. The order set by the pesticide management departments in China.
- It is not stated how much water was used to treat the crop with pesticides, only that ” the recommended water consumption for the spinach was 450 kg ha-1".
Response: Thanks for the question. We have revised the sentence in the text to make it clearer as follows: The pesticides were sprayed once during the larvae stage of S. exigua, and the spray volume 450 L per hectare was applied by electric knapsack sprayer.
- The authors performed a full long-term risk assessment of five pesticides using STMR data from WHO and crop intake data from the global environmental monitoring system (GEMS)/Food Consumption Cluster Diets System on multiple crops. This approach to assessing the risk of pesticide consumption in a person's complete food supply does not meet the safety criteria, since the consumption of each product separately must be safe, while the diet of one person may not be sufficiently balanced. It would be more useful to refine the data on spinach consumption in China and make an estimation with the revised data.
Response: Thanks for the question. The World Health Organization (WHO) has developed the global environmental monitoring system (GEMS) / Food Consumption Cluster Diets, which provides an overview of the food consumption patterns worldwide covering through 17 dietary patterns.
The dietary data of this system considered the dietary structure of different regions and different populations, representing the dietary structure of ordinary people in the region.
At present, JMPR's dietary safety evaluation of pesticides are all based on the data of this system. The data of 5 pesticides in our study are all from the JMPR evaluation report (international risk assessment).
It is well known that dietary intake varies among different regions and populations in China, making it difficult to obtain accurate dietary data in different regions. The dietary data of spinach (dark vegetables) in this study comes from the nutrition and health status of residents released by the Chinese government (China risk assessment).
- The authors state: “The difference between the two conventional methods was mainly due to the dietary amount of the consumed crops”. It can be seen from formula 1, that the high rate of spinach (Food intake) and low body weight of the Chinese lead to high NEDI values. And the conclusion that you need to clarify the consumption of spinach is absolutely correct. However, it is important to remember that spinach is used as a diet food and lowering the safety factor or other means to unreasonably increase the tolerable risk level is unacceptable. Therefore, in assessments, it is important to take into account national characteristics of consumption and body parameters for a correct assessment.
Response: Thanks for the question. At present, the dietary data used in China are based on crop classification, that is, the consumption of certain agricultural products is not used alone, and only the total consumption of crop categories is used. Therefore, this assessment method maximizes the risk of dietary intake.
For example, the spinach diet in this assessment is 0.0915kg (China risk assessment), which is actually the total intake of dark vegetables, and the actual spinach intake is far lower than this value.
Currently, the overestimated risk assessment method adopted by China involves treating the consumption risk with caution and increasing the guarantee coefficient for the health and safety of the population.
In the revised draft, we deleted the statements that could cause ambiguity in the text.
- There is no mention at all in the introduction and conclusion that the method for the determination of five insecticides in spinach leaves has been adapted. In conclusion, it is necessary to describe in more detail what observations were obtained in the experiment. The influence of factors was not considered in the text of the article, so it is not worth mentioning this in the conclusion. It is also incorrect to speak of an acceptable risk for indoxacarb, since the calculations used WHO consumption data, which do not take into account the national specifics of China.
Response: Thanks for the questions. As suggested, we carefully checked and revised the sections of introduction and conclusion.
Please see below: “Introduction: …To evaluate the safety of the five pesticides in spinach, we explored and validated a sensitive and effective method for 6 compounds in spinach based on the QuEChERS method….
Conclusion: In this study, an analytical method for determination of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, chlorfenapyr, and tralopyril in spinach was developed upon the basis of QuEChERS with the LC-MS/MS (GC-MS/MS) technique. The method was satisfactory considering linearity, selectivity, accuracy, and precision. Based on the method, the dissipation and terminal residues of 5 pesticides in spinach under field condition was investigated.
The residue dissipation of methoxyfenozide, chlorantraniliprole, indoxacarb, chlorfenapyr, and lufenuron in spinach showed a rapid degradation with a half-life range of 1.1-4.4 d under the two field experiment conditions and conformed to the first-order kinetic equation. The terminal residues of indoxacarb were below the MRL set by China. The long-term dietary risk assessment suggested that the risk of dietary intake for methoxyfenozide, chlorantraniliprole, chlorfenapyr, and lufenuron was acceptable, and spinach obtained under the good agricultural practices conditions will not pose a threat to humans through their dietary intake. Finally, this work will be useful to establish the MRLs of methoxyfenozide, chlorantraniliprole, chlorfenapyr, and lufenuron in spinach and to provide guidance on their safe use.
We greatly appreciate the helpful and constructive comments, which improved the quality and clarity of our manuscript. Thank you very much!
Round 2
Reviewer 1 Report (New Reviewer)
The authors took into account the recommendations
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
I don't understand why the authors claim that the WHO estimate of daily intake is more accurate than China's. Just because China's is more cautious doesn't mean it wrong. Also, I would imagine there is cultural variation in spinach intake, which could result in people in some places being much more likely to receive too much indoxacarb residue in their diet. It sounds like caution should be taken.
p. 2 Not sure what is meant by "single agent". Does that mean not in formulation with other ingredients? Or just not other active ingredients? Are they available in combination with something else? Same paragraph, I don't think "unexplainably" is the word you mean here.
On page 6 you state that "these agents exhibit shorter half-lives in vegetables" but it isn't clear to me...shorter than what?
p. 7. The end of the first paragraph makes important points about variation in diet.
p. 8 top: "would hinder reasonable pesticide usage later"... non sure what the later refers to.
The title of Table S4 doesn't make sense. Maybe it should be Crops for which the five compounds are registered in China.
Author Response
We thank the reviewer very much for his helpful comments and suggestions, which greatly improved the quality of our manuscript.
Responses to Reviewer:
1.I don't understand why the authors claim that the WHO estimate of daily intake is more accurate than China's. Just because China's is more cautious doesn't mean it wrong. Also, I would imagine there is cultural variation in spinach intake, which could result in people in some places being much more likely to receive too much indoxacarb residue in their diet. It sounds like caution should be taken.
Thanks for the insight. We have deleted the statements that could cause ambiguity in the text and only compared the assessment methods adopted by China and WHO.
- p. 2 Not sure what is meant by "single agent". Does that mean not in formulation with other ingredients? Or just not other active ingredients? Are they available in combination with something else? Same paragraph, I don't think "unexplainably" is the word you mean here.
Thank you for the questions. We have revised the sentence in the text to make it clearer. It reads, “Since these pesticides have not been registered for spinach, it is essential to investigate their residual levels in spinach and assess their dietary intake risks.”
- On page 6 you state that "these agents exhibit shorter half-lives in vegetables" but it isn't clear to me...shorter than what?
Thanks for this. We have revised the sentence in the text to make it clearer. The revised sentence reads, “Previous studies have shown that these agents have short half-lives in vegetables such as asparagus….”
- p. 7. The end of the first paragraph makes important points about variation in diet.
Thanks. Spinach intake indeed differs in different countries and regions due to the differences in their dietary habits. Therefore, the MRLs of spinach pesticides formulated by different countries and regions are slightly different.
- p. 8 top: "would hinder reasonable pesticide usage later"... non sure what the later refers to.
Thanks for highlighting this. We have revised the sentence to “However, when used on crops with high residue levels, the pesticide intake increases several times; thus, the estimated higher risk levels would hinder reasonable pesticide usage and increase the assessment cost.”
- The title of Table S4 doesn't make sense. Maybe it should be Crops for which the five compounds are registered in China.
Thanks. The title has been revised to “Crops registered for the five compounds in China.”
Reviewer 2 Report
This paper describes a dissipation, residue and dietary risk assessment of five pesticides residues in samples of spinach in China. The study was correctly conducted and well written but I consider that the methodology and results of risk assessment presented were too scarce. The Table 5 and 6 are not contributing to the discussion of data obtained for the five pesticides studied in spinach.
However, the most significant drawback is related to the lack of novelty/relevance for a global audience. The methodology used in this study is rutinary and the data provided is commonly required for the authorization of pesticides at national level.
Some comments are included to improve the manuscript:
· The title should be clear and more specific
· Why the QUECHERs method was not included in the study?
· The dosages used to control S. exigua are recommend by the national authorities or pesticides companies?
· Include some references in the sample treatment section
· What is GCB?
· Why chlorfenapyr assessment? (methodology section) five pesticides were evaluated in this study.
Author Response
We thank the reviewer very much for his helpful comments and suggestions, which greatly improved the quality of our manuscript.
Responses to Reviewer:
This paper describes a dissipation, residue and dietary risk assessment of five pesticides residues in samples of spinach in China. The study was correctly conducted and well written but I consider that the methodology and results of risk assessment presented were too scarce. The Table 5 and 6 are not contributing to the discussion of data obtained for the five pesticides studied in spinach. However, the most significant drawback is related to the lack of novelty/relevance for a global audience. The methodology used in this study is rutinary and the data provided is commonly required for the authorization of pesticides at national level.
Thanks very much for the valuable comments.
Some comments are included to improve the manuscript:
1.The title should be clear and more specific
Thanks for the important points. The title has been revised to “Dissipation, residue, and dietary risk assessment of methoxyfenozide, chlorantraniliprole, indoxacarb, lufenuron, and chlorfenapyr in spinach.”
2.Why the QUECHERs method was not included in the study?
Thanks for the question. The proposed extraction and clean-up procedures for analyzing the six compounds were based on the modified QuEChERS method.
- The dosages used to control S. Exigua are recommend by the national authorities or pesticides companies?
Thanks for the question. The dosages used to control S. Exigua are recommended by the companies manufacturing the pesticides.
- Include some references in the sample treatment section
Thanks for the suggestion. The reference containing the method used in the sample treatment section has been added to the first sentence of the section, reading, “The proposed extraction and clean-up procedures for the six compounds were based on modified QuEChERS method.”
5.What is GCB?
Thanks for pointing this out. We have provided the full name of the adsorbent in Section 2.1 as graphitized carbon black (GCB). We have also defined PSA denotes primary secondary amine.
- Why chlorfenapyr assessment? (methodology section) five pesticides were evaluated in this study.
Thanks for the question. It was discovered that chlorfenapyr metabolite (tralopyril) could also potentially harm environmental organisms. The definition of the dietary risk assessment of chlorfenapyr residues was the sum of chlorfenapyr and its metabolite tralopyril. This has been presented in formula form, which is easier to understand. Since methoxyfenozide and other agents were evaluated as the parent compounds, there was no need for detailed explanation.
Round 2
Reviewer 2 Report
