Urinary Glyphosate, 2,4-D and DEET Biomarkers in Relation to Neurobehavioral Performance in Ecuadorian Adolescents in the ESPINA Cohort

Background: Herbicides are the most used class of pesticides worldwide, and insect repellents are widely used globally. Yet, there is a dearth of studies characterizing the associations between these chemical groups and human neurobehavior. Experimental studies suggest that glyphosate and 2,4-dichlorophenoxyacetic acid (2,4-D) herbicides can affect neurobehavior and the cholinergic and glutamatergic pathways in the brain. We aim to assess whether herbicides and insect repellents are associated with neurobehavioral performance in adolescents. Methods: We assessed 519 participants (11–17 years of age) living in agricultural communities in Ecuador. We quantified urinary concentrations of glyphosate, 2,4-D, and two N,N-diethyl-meta-toluamide (DEET) insect repellent metabolites [3-(diethylcarbamoyl)benzoic acid (DCBA) and 3-(ethylcarbamoyl)benzoic acid (ECBA)] using isotope-dilution mass spectrometry. We assessed neurobehavioral performance using 9 subtests across 5 domains (attention/inhibitory control, memory/learning, language, visuospatial processing, and social perception). We characterized the associations using generalized estimating equations and multiple imputation for metabolites below detection limits. Models were adjusted for demographic and anthropometric characteristics, urinary creatinine, and sexual maturation. Mediation by salivary cortisol, dehydroepiandrosterone, 17β-estradiol, and testosterone was assessed using structural equation modeling. Results: The mean of each neurobehavioral domain score was between 7.0 and 8.7 [standard deviation (SD) range: 2.0–2.3]. Glyphosate was detected in 98.3% of participants, 2,4-D in 66.2%, DCBA in 63.3%, and ECBA in 33.4%. 2,4-D was negatively associated with all neurobehavioral domains, but statistically significant associations were observed with attention/inhibition [score difference per 50% higher metabolite concentration (β)=−0.19 95% confidence interval (CI): −0.31, −0.07], language [β=−0.12 (95% CI: −0.23, −0.01)], and memory/learning [β=−0.11 (95% CI: −0.22, 0.01)]. Glyphosate had a statistically significant negative association only with social perception [β=−0.08 (95% CI: −0.14, −0.01)]. DEET metabolites were not associated with neurobehavioral performance. Mediation by gender and adrenal hormones was not observed. Conclusion: This study describes worse neurobehavioral performance associated with herbicide exposures in adolescents, particularly with 2,4-D. Replication of these findings among other pediatric and adult populations is needed. https://doi.org/10.1289/EHP11383


Table of Contents
Table S1.Change in estimate of adjusted associations of pesticide metabolite concentrations or summary scores with the Attention & Inhibitory Control domain, after adding whether the participant's mother worked in agriculture while pregnant, or whether the participant's mother lived with an agricultural worker while pregnant.Participants completed the Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study July to October 2016 examination that took place in Pedro Moncayo, Ecuador (n=519).
Table S2.Change in estimate of adjusted associations of pesticide metabolite concentrations or summary scores with the Language domain, after adding whether the participant's mother worked in agriculture while pregnant, or whether the participant's mother lived with an agricultural worker while pregnant.Participants completed the Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study July to October 2016 examination that took place in Pedro Moncayo, Ecuador (n=519).
Table S3.Change in estimate of adjusted associations of pesticide metabolite concentrations or summary scores with the Memory & Learning domain, after adding whether the participant's mother worked in agriculture while pregnant, or whether the participant's mother lived with an agricultural worker while pregnant.Participants completed the Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study July to October 2016 examination that took place in Pedro Moncayo, Ecuador (n=519).
Table S4.Change in estimate of adjusted associations of pesticide metabolite concentrations or summary scores with the Visuospatial Processing domain, after adding whether the participant's mother worked in agriculture while pregnant, or whether the participant's mother lived with an agricultural worker while pregnant.Participants completed the Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study July to October 2016 examination that took place in Pedro Moncayo, Ecuador (n=519).

Table S6 .
Pearson correlation (r [p-value]) matrix of urinary pesticide biomarker concentrations of participants of the Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study July to October 2016 examination that took place in Pedro Moncayo, Ecuador (n=519).

Table S7 .
Curvilinear results of generalized estimating equations (GEE) of associations between pesticide exposure and the five NEPSY-II domains for participants of the Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study July to October 2016 examination that took place in Pedro Moncayo, Ecuador (n=519).

Table S8 .
Interaction terms between gender and herbicide or DEET metabolite concentrations or summary scores with neurobehavioral domain scores for participants of the Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study July to October 2016 examination that took place in Pedro Moncayo, Ecuador (n=519).

Table S9 .
Mediation analyses of testosterone on the relationship between each pesticide metabolite or summary score and cognitive domains using structural equation modeling in participants of the July to October 2016 Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study examination (N=519) based in Pedro Moncayo, Ecuador.Statistically significant associations reflect the presence of mediation.Directed acyclic graph for the generalized estimated equation models that will assess whether herbicide or DEET biomarker concentration is associated with NEPSY-II domain scores for participants of the Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study July to October 2016 examination in Pedro Moncayo, Ecuador.

Table S1 .
Change in estimate of adjusted associations of pesticide metabolite concentrations or summary scores with the Attention & Inhibitory Control domain, after adding whether the participant's mother worked in agriculture while pregnant, or whether the participant's mother lived with an agricultural worker while pregnant.Participants completed the Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study July to October 2016 examination that took place in Pedro Moncayo, Ecuador (n=519).

Difference in Attention & Inhibitory Control domain scores per 50% higher biomarker concentration, β (95% CI) Original Estimate a Mother Worked in Agriculture While Pregnant b Mother Lived with Agricultural Worker While Pregnant c Both Prenatal Exposures d n β 95% CI β 95% CI % Change in Estimate β 95% CI % Change in Estimate β 95% CI % Change in Estimate Herbicide Summary Score 508
a Models adjusts for retest learning effect, age, gender, race, creatinine, z-BMI-for-age, monthly salary, and average parental education.

Language domain scores per 50% higher biomarker concentration, β 95% CI) Original Estimate a Mother Worked in Agriculture While Pregnant b Mother Lived with Agricultural Worker While Pregnant c Both Prenatal Exposures d
Models adjusts for retest learning effect, age, gender, race, creatinine, z-BMI-for-age, monthly salary, and average parental education.

& Learning domain scores per 50% higher biomarker concentration, β (95% CI) Original Estimate a Mother Worked in Agriculture While Pregnant b Mother Lived with Agricultural Worker While Pregnant c Both Prenatal Exposures d
Models adjusts for retest learning effect, age, gender, race, creatinine, z-BMI-for-age, monthly salary, and average parental education. a

Processing domain scores per 50% higher biomarker concentration, β (95% CI) Original Estimate a Mother Worked in Agriculture While Pregnant b Mother Lived with Agricultural Worker While Pregnant c Both Prenatal Exposures d
Models adjusts for retest learning effect, age, gender, race, creatinine, z-BMI-for-age, monthly salary, and average parental education.

Perception domain scores per 50% higher biomarker concentration, β (95% CI) Original Estimate a Mother Worked in Agriculture While Pregnant b Mother Lived with Agricultural Worker While Pregnant c Both Prenatal Exposures d
Models adjusts for retest learning effect, age, gender, race, creatinine, z-BMI-for-age, monthly salary, and average parental education.

Table S6 .
Pearson correlation (r [p-value]) matrix of urinary pesticide biomarker concentrations of participants of the Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study July to October 2016 examination that took place in Pedro Moncayo, Ecuador (n=519).

Table S7 .
Curvilinear results of generalized estimating equations (GEE) of associations between pesticide exposure and the five NEPSY-II domains for participants of the Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) study July to October 2016 examination that took place in Pedro Moncayo, Ecuador (n=519).