Effect of California’s 2020 Chlorpyrifos Ban on Urinary Biomarkers of Pesticide Exposure in Agricultural Communities

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study attempted to examine the impact of California’s chlorpyrifos ban on urinary pesticide concentrations among urban and agricultural communities in the Central Valley, however there are serious methodological flaws in the design of the study that do not support the authors’ conclusions. First, the authors conducted pre-ban data collection in the non-spray season and post-ban urine collection in the spray-season, confounding the impacts of the ban. Use of OPs was significantly higher in the post-ban urine sampling phase due to natural seasonal fluctuations in pesticide use. Second, the focus on differences in DM metabolites is confusing, given that chlorpyrifos is not a DM OP and other OPs increased in the post-ban sampling phase because the data collection was conducted during the spray season. Third, the authors collected a single urine sample during the pre- and post-ban phases; these metabolites have extraordinarily short half-lives, and the authors simply cannot make sweeping generalizations about the impact of this policy on urinary concentrations before and after the ban, particularly given other methodological flaws with this work. Fourth, the authors only recruited four participants from urban communities, yet describe this work as looking at the impact of the ban in both urban and agricultural communities. The authors’ conclusions that the only explanation for their findings is the persistence of chlorpyrifos in the environment is not supported from this research.  This study has major methodological flaws and the conclusions are not supported.

Author Response

Please see the attached Author Response letter (pdf).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a well written study about the presence of chlorpyrifos after California's 2020 ban. The manuscript was well-written and the aim is well-defined. The study is relevant and the study plann was rigorous to answered the question raised. The references are quite new and the figures and tables helped in making the results easier to understand. I have some specific comments that are well described in my analysis. Overall, the manuscript could be accepted for publication after a minor revision.

Some points to be improved:

• Which was the method used to quantify the OPs and metabolites?

• Discussion at section 4.1 do not properly discuss the data, but brings an overview of the work, which I suggest to be suppressed and to gocus the discussion section on data discussion.

Author Response

Please see the attached Author Response letter (pdf).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript investigates the effect of California’s 2020 chlorpyrifos ban on urinary biomarkers of pesticide exposure in the Central Valley of California, USA, including Fresno and Tulare Counties. The topic is relevant to the journal Environments. However, the validity of the study design needs further justification. Moreover, the readability could be improved by some clarification and editorial amendments. More specific comments are listed in the Comments to Author section.

Comments on the Quality of English Language

Careful proofreading could be needed to correct some errors and awkward sentences/word choices. Some examples are listed below.  

L144, “Magzamen-Reynolds-Schaeffer lab”, perhaps “Magzamen-Reynolds-Schaeffer Lab”

L201, “mean (standard deviation (SD)),” perhaps “mean and standard deviation (SD)” could be more appropriate.

L242, “Among the study sample for biomarker analysis, there was a wide range of ages”, perhaps “Among the study participants for biomarker analysis, there was a wide range of ages”.

L261, “Table 2 display “, should read “Table 2 displays”.

L361, “260 thousand kg”, perhaps “260,000 kg” is more appropriate.

The manuscript could be more concise, e.g., “Supplemental Table 1” could be replaced with “Table S1”. Some phrases could omitted, e.g., “n(%)” (L201), “(Figure 1)” (L301), “with significant post-ban increased average concentrations compared to pre-ban (estimate 1.51, 95% CI 0.98, 2.04)” (L304, Fig 1 caption). “4.1. Overview”, this is section could be placed in the Introduction section and consolidated with statements there.

The authors may want to avoid the use of first person, i.e., “we”.

Kindly check the journal’s format requirement of the Reference section. Based on the citation format used in the manuscript, the numbers may not be needed.

Author Response

Please see the attached Author Response letter (pdf).

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Organophosphate insecticides are among the most commonly used pesticides. However, with their widespread use, more and more information is emerging about their chronic toxicity to both humans and the natural environment. This leads to a ban on the use of these substances. In the introduction, the authors should, in addition to presenting the local situation, present the general picture. For example, is chlorpyrifos also banned in other countries, are its residues tested in American food? Does the ban also apply to the ban on use on pets?

The project to ban the use of chlorpyrifos in California created a unique opportunity to conduct epidemiological studies on the presence of this insecticide and its metabolites in human bodies. The research plan presented by the authors of the publication is exemplary, containing both demographic data and properly planned chemical analyses. The only drawback that had a negative impact on the study results was the low population size. Such a small number of the studied population, with such a low level of metabolites in urine, could not allow for drawing statistically significant conclusions. And the authors already knew about this from the pre-ban study. So why did research continue?

In the paper, the authors state that some of the indicators are general indicators. It should be stated which groups of compounds their presence may indicate.

Have any previous epidemiological studies been conducted to determine the levels of chlorpyrifos metabolites in humans, e.g. comparative studies in exposed and unexposed individuals? 

Author Response

Please see the attached Author Response letter (pdf).

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I still feel the conclusions from this work are too strong and not supported by the data and study design. I do not think the authors can categorically conclude that the findings "suggest persistence of chlorpyrifos in the environment" and think they should frame these findings as a potential explanation, not a definitive conclusion. 

Author Response

Please see the attached pdf document with our point-by-point response letter.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript investigates the effect of California’s 2020 chlorpyrifos ban on urinary biomarkers of pesticide exposure in the Central Valley of California, USA, including Fresno and Tulare Counties. The topic is relevant to the journal Environments. It appears that the revision is limited to “Comments on the Quality of English Language”. In the response file, the authors stated that “We have addressed each of your comments and concerns, shown below and in the track changes manuscript.” In the revised manuscript, some passages were yellow highlighted in the introduction and discussion sections. However, the reviewer could not find any track changes in the revised manuscript or response to any of the reviser’s comments and suggestions regarding the “Major Concern” or “Clarification Issues”.  Some yellow highlighted phrases are the same as in the original manuscript (e.g., L318) and changes in some yellow highlighted sections are citation format only (e.g., L322-326). As pointed out last time, the validity of the study design needs further justification, and the readability could be improved by some clarification. My previous comments and suggestions are listed below. Kindly note that the line numbers below refer to the original manuscript.

Major Concern

The validity of the study design needs further justification. As the authors pointed out, “Given growing concerns of the widespread use of OPs, particularly chlorpyrifos, and its established associations with adverse health effects among infants, children, and adults, California took an unprecedented step for a statewide ban of this pesticide’s sale in February 2020 and agricultural applications by the end of the 2020 calendar year. This unique environmental policy to ban a pesticide from use statewide offered an opportunistic observational study to investigate changes in chlorpyrifos and other OP exposures among residents living in areas with likely high-exposure, particularly those in agricultural communities, plus an urban region. California applied approximately 16,135 metric tons of chlorpyrifos over the previous 27 years, leading to a potentially drastic reduction in exposure among residents following the ban.” (L312). However, “there were notably higher OP applications in the study region of Fresno and Tulare Counties comparing the pre-ban campaign (December 1-30, 2020) with 697 kg vs. the post-ban campaign (February 19 to April 22, 2022) with 3,659 kg (California Department of Pesticide Regulation, 2022).” (L362).

The significantly increased use of OPs after the ban casts doubt on the validity of the study design. The question is, could one assume that the ban on one type of OPs could lead to a reduction of exposure to OPs, regardless of the total use of OPs. It appears that the observed increases in some bio-maker levels are attributed to “persistence of chlorpyrifos in indoor and environmental sources”, and “likelihood of other routes of exposure, such as ingestion as mentioned above, lack of photodegradation indoors, which can reduce ambient levels of pesticides, and lack of wind to disperse pesticide residue”, with little consideration of the increased OP application post-ban. More evidence is needed to support that the “persistence” and other exposure pathways are the major causes of increased bio-marker levels when the AP use has increased.   

The follow up question is, in case the major driver of the observed higher level of some bio-markers post-ban is the higher application amount of APs post-ban due “to seasonal variation in OP application as the post-ban campaign was conducted as OP application began to increase for the growing season”, could the study design “assess the impacts of this policy ban among residents in the Central Valley of California by measuring pre- and post-ban personal biomarkers of exposure to OPs”.   

The authors may want to clarify the study hypotheses and factors being considered to explain the differences in the pre- and post-ban biomarker levels, and justify that the study design could indeed “assess the impacts of this policy ban among residents in the Central Valley of California by measuring pre- and post-ban personal biomarkers of exposure to OPs”.  

Clarification Issues

The title, “Effect of California's 2020 chlorpyrifos ban on urinary biomarkers of pesticide exposure in agricultural communities”. As stated in the main body, some participants resided in “an urban community”. 

For international readers, more specific information about the study area would be helpful, i.e., its coordinates, population, and country.

Kindly clarify the meaning of “Blank Urine samples” with the values of other parameters, e.g., the pH value of the blanks. 

L195, kindly explain the reason of “98 valid TCPy concentrations” but “96 valid urinary metabolite concentrations for all other biomarkers” and whether “98” samples, “96” samples, or both were used in sequential analysis.  

L236, “There were no meaningful differences”, the statement is a bit arbitrary, perhaps the ranges of percent difference between the two could be more informative.

L266, “Contrary to our hypotheses”, kindly provide the hypotheses.

L368, “No differences were reported in chlorpyrifos applications for the two study periods with virtually zero kg of chlorpyrifos reported for campaign 1 (1.6 kg) vs. campaign 2 (0.0 kg) (California Department of Pesticide Regulation, 2022).” Kindly clarify the reason of “1.6 kg” being considered as “zero kg” or rephrase.

L375, “the increase post-ban of TCPy supports the likely persistence of chlorpyrifos in indoor and environmental sources.”  Kindly rephrase and explain how long the TCPy levels are expected to increase post-ban or to research its maximum.     

L400-402, a lack of photodegradation or weak dispersion indoors may not be “other routes of exposure”.

L401, “lack of photodegradation indoors, which can reduce ambient levels of pesticides”, not sure a lack of photodegradation would reduce levels of pesticides. Also, “ambient” refers to outdoor environment. Did you mean “a lack of photodegradation indoors, which hinders the reduction of levels of pesticides”.

L402, “lack of wind to disperse pesticide residue”, maybe “a lack of strong air movement indoors to disperse pesticide residue”. It is uncommon to encounter outdoor environments where   wind is absent all the time.

Equations, all equations should be numbers, and all variables in each equation explained.

Some abbreviations are defined in the main body, some are not. It would be better to be consistent. Either way, all abbreviations in the Abstract should be defined.

Comments on the Quality of English Language

Could be improved. 

Author Response

Please see the attached pdf point-by-point response letter.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Responses to comments and corrections in the manuscript are adequate.

Author Response

Please see the attached pdf point-by-point response letter.

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the author's responses. 

Author Response

Please see attached Response Letter.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors should be applauded for their efforts to revise the manuscript again. All comments were responded to this time. The charity and the scientific soundness of the manuscript have improved. The following comments are the same as in the Original and R1. Line numbers refer to R2.  

1) Equations, all equations should be numbered (at the far right of the line) and all variables in each equation should be explained.

2) L425, “Furthermore, the evidence of chlorpyrifos exposure, despite virtually zero agricultural applications during the study period, highlights the likelihood of other routes of exposure, such as ingestion of chlorpyrifos residue on food as mentioned above, as well as the potential lack of photodegradation indoors and lack of strong air movement indoors to disperse pesticide residue.” As pointed out previously, “the potential lack of photodegradation indoors and lack of strong air movement indoors to disperse pesticide residue” itself may not be “other routes of exposure”. If “indoor exposure” is referred to, kindly clarify.  

Additional comment

L400 and L484, “second time period”, perhaps “second time period (post-ban)” would be more clear.

Comments on the Quality of English Language

Overall, very good. There are a few awkward phrases, e.g., L486, “potential other sources”, kindly rephrase.  

Author Response

Please see attached Response letter.

Author Response File: Author Response.pdf