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Environmental Research
journal homepage: www.elsevier.com/locate/envres
Organic diet intervention significantly reduces urinary pesticide levels in
U.S. children and adults
Carly Hyland
a
, Asa Bradman
a
, Roy Gerona
b
, Sharyle Patton
c
, Igor Zakharevich
b
,
Robert B. Gunier
a
, Kendra Klein
d,⁎
a
Center for Environmental Research and Children's Health (CERCH), School of Public Health, University of California at Berkeley, Berkeley, CA, United States
b
Clinical Toxicology and Environmental Biomonitoring Laboratory, University of California at San Francisco, San Francisco, CA, United States
c
Commonweal Institute, Bolinas, CA, United States
d
Friends of the Earth U.S., 2150 Allston Way Suite 360, Berkeley, CA 94704, United States
ARTICLE INFO
Keywords:
Pesticides
Exposure
Chlorpyrifos
Neonicotinoid pesticides
Organic diet
Biomonitoring
ABSTRACT
Background: Previous diet intervention studies indicate that an organic diet can reduce urinary pesticide me-
tabolite excretion; however, they have largely focused on organophosphate (OP) pesticides. Knowledge gaps
exist regarding the impact of an organic diet on exposure to other pesticides, including pyrethroids and neo-
nicotinoids, which are increasing in use in the United States and globally.
Objective: To investigate the impact of an organic diet intervention on levels of insecticides, herbicides, and
fungicides or their metabolites in urine collected from adults and children.
Methods: We collected urine samples from four racially and geographically diverse families in the United States
before and after an organic diet intervention (n = 16 participants and a total of 158 urine samples).
Results: We observed significant reductions in urinary levels of thirteen pesticide metabolites and parent com-
pounds representing OP, neonicotinoid, and pyrethroid insecticides and the herbicide 2,4-D following the in-
troduction of an organic diet. The greatest reductions were observed for clothianidin (−82.7%; 95% confidence
interval [95% CI]: −86.6%, −77.6%; p< 0.01), malathion dicarboxylic acid (MDA), a metabolite of ma-
lathion (−95.0%; 95% CI: −97.0%, −91.8%; p< 0.01), and 3,5,6-trichlor-2-pyridinol (TCPy), a metabolite
of chlorpyrifos (−60.7%; 95% CI: −69.6%, −49.2%; p< 0.01). Metabolites or parent compounds of the
fungicides boscalid, iprodione, and thiabendazole and the neonicotinoid insecticide imidacloprid were not de-
tected among participants in our study.
Conclusion: An organic diet was associated with significant reductions in urinary excretion of several pesticide
metabolites and parent compounds. This study adds to a growing body of literature indicating that an organic
diet may reduce exposure to a range of pesticides in children and adults. Additional research is needed to
evaluate dietary exposure to neonicotinoids, which are now the most widely used class of insecticides in the
world.
1. Introduction
Diet accounts for a significant proportion of total pesticide exposure
in the general population (Curl et al., 2015; Riederer et al., 2008).
Recent data from the US Food and Drug Administration (FDA) Pesticide
Residue Monitoring Program show that approximately 47% of domestic
food and 49% of imported foods sampled had detectable pesticide re-
sidues in the 2016 examination (United States Food and Drug
Administration, 2016). Pesticide residue monitoring data also indicate
that organically grown foods have lower pesticide residues compared
with conventionally grown foods (Forman and Silverstein, 2012; United
States Department of Agriculture, 2016).
Exposure to pesticides has been associated with various adverse
health outcomes, including decreased cognitive scores (Bouchard et al.,
2011; Engel et al., 2011; Rauh et al., 2011) and increased behavioral
and attention problems in children (Quirós-Alcalá et al., 2014), asthma
(Hernandez et al., 2011; Raanan et al., 2016), cancer (Bassil et al.,
2007), and impacts on the reproductive (Bretveld et al., 2006) and
https://doi.org/10.1016/j.envres.2019.01.024
Received 21 September 2018; Received in revised form 19 December 2018; Accepted 10 January 2019
Abbreviations: ADI, Acceptable Daily Intake; CI, Confidence Interval; RfD, Reference Dose; NHANES, National Health and Nutrition Examination Survey; OP,
Organophosphate; U.S. EPA, United States Environmental Protection Agency; U.S. FDA, United States Food and Drug Administration
⁎
Corresponding author.
E-mail address: kklein@foe.org (K. Klein).
Environmental Research xxx (xxxx) xxx–xxx
0013-9351/ © 2019 Published by Elsevier Inc.
Please cite this article as: Hyland, C., Environmental Research, https://doi.org/10.1016/j.envres.2019.01.024
endocrine systems (Mnif et al., 2011). While knowledge gaps exist re-
garding specific health effects associated with chronic low-level dietary
pesticide exposure, a recent longitudinal study of nearly 70,000 adults
found that higher frequency of organic food consumption - which is
associated with lower pesticide exposure - was protective against sev-
eral cancers (Baudry et al., 2018).
Pesticide use patterns have shifted in recent decades. While the use
of organophosphate (OP) pesticides has declined in the U.S. since the
passage of the Food Quality Protection Act in 1996 (United States
Environmental Protection Agency, 2017), they are one of the most
widely used classes of insecticides in U.S. agriculture (California
Department of Pesticide Regulation, 2016; Curl et al., 2015), and diet is
the primary source of OP exposure in the general population (Curl
et al., 2015). As OP pesticide use has declined, use of pyrethroids and
neonicotinoids has increased over the past two decades (Burns and
Pastoor, 2018). Neonicotinoids are now the most widely used class of
insecticides worldwide (Lu et al., 2018) and residues have been found
in food (Chang et al., 2018). Because they are systemic (i.e., readily
taken up into the tissue of plants), these residues cannot be washed off
food (Abreu-Villaca and Levin, 2017). Biomonitoring data also indicate
widespread pyrethroid exposure in the general U.S. population
(Saillenfait et al., 2015), with 3-phenoxybenzoic acid (3PBA), a non-
specific metabolite of multiple pyrethroids, being detected in over 70%
of samples from the 1999–2002 National Health and Nutrition Ex-
amination Survey (NHANES) assessment (Barr et al., 2010). Ad-
ditionally, the latest U.S. Environmental Protection Agency (EPA) re-
port on pesticide use indicates that the herbicide 2,4-D was the fifth
most commonly used active pesticide ingredient in the U.S in 2012
(United States Environmental Protection Agency, 2017).
Previous observational and intervention studies have reported de-
creased urinary pesticide concentrations among people who consume
organic food compared with those who follow a conventional diet
(Bradman et al., 2015; Curl et al., 2015, 2003; Fenske et al., 2002; Göen
et al., 2016; Lu et al., 2008, 2006a; Oates and Cohen, 2011; Oates et al.,
2014). However, these studies have primarily focused on exposure to
OPs and, to a lesser extent, pyrethroids. In this paper, we address ex-
isting data gaps by investigating whether an organic diet intervention
reduced urinary levels of OPs, pyrethroids, neonicotinoids, fungicides,
and one herbicide among adults and children from four geographically
and racially diverse families in the United States.
2. Methods
2.1. Study participants
Four racially diverse families of three to five members were re-
cruited from four locations: Oakland, CA, Minneapolis, MN, Baltimore,
MD, and Atlanta, GA. Families in these cities were originally contacted
via a brief recruitment email that explained the purpose of the study
and study procedures. If interested, families contacted study staffand a
phone script was read to screen for eligibility in the study, including: 1)
willingness to alter their diet for six days, 2) no more than six family
members and with two to three children between the ages of three to
eighteen living at home, 3) all children toilet-trained and able to have
their breakfast, lunch, and dinner prepared at home during the organic
phase of the study, 4) English speaking, 5) no pregnant family mem-
bers, 6) no family members with severe food allergies, and, 7) the fa-
mily did not typically consume an organic diet. Families participated in
the study between February and May 2017. The Western Institutional
Review Board reviewed and approved all study procedures. Written
informed consent was obtained from parents before data collection
began.
2.2. Data collection
Each family participated in the study over twelve consecutive days.
Prior to beginning data collection, participants were contacted via an
online video call and provided with instructions on how to collect urine
samples and complete food diaries. Food diaries included information
about the type of food (produce, grain, dairy, meat, etc.) and portion
size. A questionnaire was administered by phone to one adult in each
family to collect information about potential pesticide exposure, in-
cluding pesticide use and storage in and around the home, proximity of
the home to locations known to use pesticides, such as golf courses, and
potential occupational exposure to pesticides.
2.3. Dietary intervention
During days one through five, study participants were asked to
follow their normal conventional diet (conventional phase). During
days six through eleven, all family members were provided with cer-
tified organic food while at home, work, school, or daycare, including
all beverages other than water, all food categories, and oils, condi-
ments, and spices (organic phase). After the participants collected the
first morning void urine sample on day twelve, they could choose to eat
either organic or conventional food. Organic food was provided to fa-
milies in two ways: 1) Participants were asked to compile a list of all
groceries they would need for six days, and research assistants pur-
chased organic foods from this list and delivered the groceries to the
participants’homes for their use, and, 2) dinners were prepared with all
organic foods by a licensed chef or caterer and delivered to study
participants by the research assistants. All organic foods for the six-day
period were provided free of charge to the families.
2.4. Urine collection
Prior to the start of the study, participants were given urine col-
lection instructions and urine collection kits were mailed to their
homes. First morning void urine samples were collected into specimen
cups and immediately stored in sealed plastic bags in the family's
freezer each day during the study period. Research assistants picked up
the frozen urine samples for each phase of the study and shipped them
overnight on dry ice to the laboratory.
For one family, collection of urine samples from the conventional
phase was repeated after the organic phase (with washout time) due to
an error in maintaining the samples frozen in the laboratory.
2.5. Laboratory analysis of urine samples
Urine samples were analyzed for eighteen pesticide analytes, in-
cluding nine specific OP, neonicotinoid, pyrethroid, fungicide, and
herbicide analytes, three non-specific pyrethroid analytes, and six non-
specific OP dialkylphosphate (DAPS) analytes (Table 1). In choosing
pesticides for analysis, we assessed amount of use in U.S. agriculture
(United States Environmental Protection Agency, 2017) and frequency
of detection as food residues (United States Food and Drug
Administration, 2016).
Quantification of the nine pesticide-specific analytes and three non-
specific pyrethroid metabolites was performed using liquid-chromato-
graphy-tandem mass spectrometry (LC-MS/MS) on an Agilent LC 1260-
AB Sciex 5500 system. The urine specimens (1 mL) were prepared for
LC-MS/MS analysis by solid phase extraction (SPE) using Waters Oasis
WAX cartridges (10 mg, 30 µm, 1 cc). All urine samples were decon-
jugated prior to LC-MS/MS analysis by addition of 450 U H. pomatia
glucuronidase (Sigma-Aldrich, St Louis, MO) and incubated at 37 °C for
two hours with constant shaking. The compounds were ionized in the
negative mode using electrospray ionization (ESI) and monitored by
multiple reaction monitoring. Each compound was monitored using
two transitions (See Supplementary material S1) along with 2,4-D-d3
and Cloth-d3 as internal standards. Each batch of samples was injected
in duplicates and run alongside calibration standards that were run at
the beginning, between the duplicate sample injections, and after the
C. Hyland et al. Environmental Research xxx (xxxx) xxx–xxx
2
sample set. Additionally, low and high spiked quality control (QC)
samples were run for each analysis batch. A batch run was accepted if
both QC samples were within 20% of their target values and had
coefficients of variation (CV) ≤20%.
The biomarkers for the nine pesticide-specific analytes and three
non-specific pyrethroid metabolites were selected based on those that
have been previously measured and reported by other studies for the
more common analytes (e.g., TCPy, MDA). For novel analytes (e.g.,
clothianidin, imidacloprid), we conducted a literature search of meta-
bolic studies to identify targets. Because the measurements were con-
ducted in urine, we prioritized metabolites over the parent compound
when reference standards of the metabolites were available. The parent
compound was used as a biomarker when metabolite reference stan-
dards were not available or cost-prohibitive to custom synthesize.
To quantify the six non-specific OP pesticide DAP metabolites (DEP,
DETP, DEDTP, DMP, DMTP and DMDTP), 0.1 mL of the urine speci-
mens were enriched with internal standards (DEP-
13
C
4,
DETP-
13
C
4,
DEDTP-
13
C
4,
DMP-d
6
, DMTP-d
6
, DMDTP-d
6
). After adding 1 mL of
acetonitrile and 200 mg of potassium carbonate, the samples were de-
rivatized with 15 µL of pentafluorobenzyl bromide (PFBBr) at 70 °C for
2 h. The derivatized products were extracted with 7 mL of a mixture
dichloromethane: hexane (8:92), mixed 15 min and centrifuged 5 min
at 1500 rpm. The solvent was then evaporated to dryness, taken up in
500 µL of dichloromethane: hexane (20:80) and analyzed for pesticide
metabolites on an Agilent 6890 Network gas chromatograph (GC)
(Agilent Technologies; Mississauga, Ontario, Canada) coupled to a
Waters Quattro Micro GC mass spectrometer in tandem (MS/MS)
(Waters; Milford, MA). The GC was fitted with an Agilent 30 m HP-5MS
column (0.25 mm i.d., 0.25 µm film thickness) to the MS/MS. The in-
ternal reference materials used to control the quality of the analyses
were the non-certified reference material ClinChek (Urine Level 1;
RECIPE Chemicals; Munich, Germany) and 3 in-house reference mate-
rials (Low, Medium, High QC) prepared by the Centre de Toxicologie du
Québec (CTQ), Institut national de santé publique du Québec (INSPQ).
The overall quality and accuracy of the analytical method was mon-
itored by the participation to the interlaboratory program as the
German External Quality Assessment Scheme (G-EQUAS; Erlangen,
Germany).
2.6. Data analysis
Urine samples from days one and seven were considered washout
days and were excluded from data analysis. We included ten urine
samples from each of the participants in the analyses, including five
samples each from the conventional diet and organic diet phases. For
one participant missing DETP data from the fourth day of the organic
Table 1
Summary of parent compounds and measured metabolites in urine.
Chemical class and precursor compounds Analyte measured (abbreviation) LOD (ng/mL) Overall DF
(%)
a
Conventional DF
(%)
b
Organic DF
(%)
c
Organophosphate Insecticides
Chlorpyrifos 3,5,6-trichloro-2-pyridinol (TCPy) 0.2 93.05 97.5 82.28
Malathion Malathion dicarboxylic acid (MDA) 0.02 78.48 87.50 43.04
Azinphos-methyl, chlorpyrifos-methyl, dichlorvos,
dicrotophos, dimethoate, fenitrothion, fenthion,
isazofos-methyl, malathion, methidathion, methyl
parathion, naled, oxydemeton-methyl, phosmet,
pirimiphos-methyl, temephos, tetrachlorvinphos,
trichlorfon
Total dimethylphosphates (total DMs =
DMP + DMTP + DMDTP)
DMP: 0.2
DMTP: 0.4
DMDTP: 0.07
94.34
d
100.00
d
88.61
d
Chlorethoxyphos, chlorpyrifos, coumaphos, diazinon,
disulfoton, ethion, parathion, phorate, sulfotepp,
terbufos
Total diethylphosphates (total DEs = DEP
+ DETP + DEDTP)
DEP: 0.25
DETP: 0.09
DEDTP: 0.06
100.00
e
100.00
e
100.00
e
Totals Total dialkylphosphates (total DMs + total
DEs)
–94.34
f
100.00
f
88.61
f
Pyrethroid Insecticides
Allethrin, cyhalothrin, cypermethrin deltamethrin,
fenpropathrin, permethrin, trialomethrin
3-Phenoxybenzoic acid (3-PBA) 0.02 100.00 100.00 100.00
Cyfluthrin 4-fluoro-3-phenoxybenzoic acid (F-PBA) 0.01 86.39 91.25 77.22
cis-Cypermethrin, cis-cyfluthrin, cis-permethrin cis-2,2-(Dichloro)-2-
dimethylvinylcyclopropane carboxylic acid
(cis-DCCA; cDCCA)
0.1 95.25 93.75 96.20
trans-Cypermethrin, trans-cyfluthrin, trans-permethrin trans-2,2-(Dichloro)-2-
dimethylvinylcyclopropane carboxylic acid
(trans-DCCA; tDCCA)
0.05 98.73 96.25 100.00
Neonicotinoid Insecticides
Imidacloprid 5-Hydroxy-Imidacloprid (5OH-Imd) 0.1 ND ND ND
Clothianidin Clothianidin 0.05 49.37 85.00 13.92
Fungicides
Thiabendazole 5-Hydroxy-Thiabendazole (5OH-TBZ) 0.2 ND ND ND
Boscalid Boscalid 0.02 ND ND ND
Iprodione Iprodione 0.05 ND ND ND
Herbicides
2,4-Dichlorophenoxyacetic acid 2,4-Dichlorophenoxyacetic acid (2,4-D) 0.01 100.00 100.00 100.00
Abbreviations: DF, detection frequency; LOD, limit of detection; ND, not detected.
a
Overall analyte DF (%) is the ratio of the total number of urine samples with analyte concentration > LOD during the entire study to the total number of urine
samples analyzed in the study (158) multiplied by 100.
b
Conventional DF (%) is the ratio of the total number urine samples > LOD to the number of samples analyzed during the convectional phase (80).
c
Organic DF (%) is the ratio of the total number of urine samples > LOD to the number of samples analyzed during the organic phase (78).
d
Total DM counted as detect if either DMP, DMTP, or DMTP > LOD.
e
Total DE counted as detect if either DEP, DETP, or DEDTP > LOD.
f
Total DAPs counted as detect if both total DM and total DE counted as detect.
C. Hyland et al. Environmental Research xxx (xxxx) xxx–xxx
3
diet phase, we imputed the average concentration from their four other
organic diet samples. One participant was missing a urine sample from
the eighth day of the study (second day of the organic phase) and one
participant was missing urine samples from the eighth, tenth, and
twelfth days of the study (second, fourth, and sixth days of the organic
diet phase). For these two participants, we included the urine sample
collected on day seven of the study, which was the first day of the or-
ganic phase, and considered a washout day for other participants. These
criteria resulted in a total of 158 urine samples included in the analysis.
For the six non-specific OP metabolites, we converted concentra-
tions to their molar equivalents and summed them to produce total DM
(DMP + DMTP + DMDTP), DE (DEP + DETP + DEDTP), and DAP
(DMs + DEs) concentrations for each sample. All analyte concentra-
tions were log-transformed for statistical analyses, and we computed
descriptive statistics for the fourteen analytes detected: specific OPs
(MDA, TCPy), six non-specific OPs (summarized as total DMs, total DEs,
and total DAPs), pyrethroids (3-PBA, FPBA, cDDCA, tDDCA), neonico-
tinoid (clothianidin), and herbicide (2,4-D). In order to account for the
correlation among repeat urine samples collected from the same in-
dividual, we used linear mixed-effects models to estimate the percent
change in urinary analyte concentrations from the conventional to or-
ganic phase using the formula % Change = [exp(β)-1] * 100, where βis
the regression coefficient for organic diet from the mixed effects
models.
We also conducted sensitivity analyses to confirm our results: 1) we
repeated the statistical analyses with creatinine-adjusted analyte con-
centrations; and 2) we omitted outliers based on model standardized
residuals > 3 or < -3 (Pardoe, 2018).
3. Results
3.1. Demographic characteristics
Seven adults ages 36–52 years and nine children ages 4–15 years
participated in this study. The mean ( ± SD) age for adults and children
in our study was 42.3 ± 6.1 years and 8.3 ± 4.1 years, respectively.
Nine of the participants were Caucasian, four were Hispanic/Latino,
and three were African American. All participants lived above the U.S.
federal poverty threshold.
3.2. Household pesticide use during study
None of the families reported using pesticides inside their home
within three months of beginning the study or during the study period.
One family reported hiring a pest control company to treat termites
with imidacloprid (Premise 75, Bayer, Whippany, NJ, USA) on the ex-
terior of their foundation and at entry points one week prior to be-
ginning the study.
3.3. Urinary measurements
Detection frequencies (DFs) of the three non-specific pyrethroid
analytes ranged from 91–100% to 77–100% during the conventional
and organic diet phases, respectively (Table 1). DFs of the five specific
Table 2
Urinary analyte concentrations (ng/mL) and percent change from conventional to organic diet.
Analyte Conventional Organic Percent change (95% CI) p-Value Median (95% CI) NHANES
a
n Median (IQR) Max n Median (IQR) Max
All (n = 16)
2,4-D 80 0.65 (0.36, 0.95) 2.43 78 0.40 (0.20, 0.78) 1.45 −36.9 (−47.0, −25.0) 0.01 0.28 (0.25, 0.32)
3PBA 80 2.70 (0.93, 4.83) 47.74 78 1.11 (0.67, 1.77) 13.09 −42.7 (−57.7, −22.2) < 0.01 0.40 (0.35, 0.46)
cDDCA 80 1.98 (0.70, 4.48) 39.64 78 0.68 (0.43, 1.20) 15.58 −53.2 (−65.7, −36.1) < 0.01 < LOD
d
Clothianidin 80 0.24 (0.10, 0.52) 6.60 78 < LOD 1.06 −82.7 (−86.6, −77.6) < 0.01 –
FPBA 80 0.04 (0.02, 0.07) 7.11 78 0.02 (< LOD, 0.03) 0.13 −57.2 (−66.3, −45.6) < 0.01 < LOD
e
MDA 80 1.03 (0.20, 2.63) 1.04 78 < LOD (< LOD, 0.09) 3.54 −95.0 (−97.0, −91.8) < 0.01 < LOD
d
TCPy 80 2.78 (1.91, 4.85) 19.48 78 1.34 (0.64, 2.45) 7.57 −60.7 (−69.6, −49.2) < 0.01 –
tDCCA 80 2.54 (0.90, 5.58) 47.20 78 1.23 (0.58, 2.31) 20.03 −46.1 (−60.9, −25.6) < 0.01 < LOD
f
Adults
b
(n = 7)
2,4-D 35 0.56 (0.29, 0.86) 2.43 32 0.33 (0.15, 0.81) 1.45 −41.9 (−55.4, −24.4) < 0.01 0.27 (0.23, 0.31
g
3PBA 35 3.71 (1.69, 5.57) 47.74 32 0.71 (0.44, 1.47) 3.79 −71.2 (−82.0, −54.0) < 0.01 0.39 (0.33, 0.47)
g
cDDCA 35 3.44 (1.36, 8.33) 39.64 32 0.71 (0.41, 1.59) 7.16 −71.7 (−83.1, −52.6) < 0.01 < LOD
d,g
Clothianidin 35 0.38 (0.24, 0.73) 5.57 32 < LOD (< LOD, < LOD) 1.06 −88.0 (−92.0, −82.7) < 0.01 –
FPBA 35 0.03 (0.02, 0.06 0.19 32 < LOD (< LOD, 0.02) 0.06 −55.3 (−67.0, −39.0) < 0.01 < LOD
e,g
MDA 35 1.01 (0.14, 2.99) 11.41 32 < LOD (< LOD, 0.10) 3.54 −94.1 (−97.3, −87.1) < 0.01 < LOD
d,g
TCPy 35 2.55 (1.79, 3.92) 12.09 32 0.63 (< LOD, 1.25) 7.57 −77.7 (−85.8, −65.1) < 0.01 –
tDCCA 35 3.35 (1.87, 6.96) 47.20 32 0.97 (0.34, 2.11) 6.14 −71.2 (−82.9, −51.5) < 0.01 < LOD
f,g
Children
c
(n = 9)
2,4-D 45 0.67 (0.38, 0.96) 2.27 46 0.43 (0.25, 0.78) 1.44 −32.4 (−46.2, −15.1) < 0.01 0.35 (0.29, 0.44)
h
3PBA 45 1.64 (0.68, 3.89) 19.25 46 1.37 (0.99, 1.91) 13.09 −4.4 (−33.9, 38.1) 0.81 0.48 (0.35, 0.70)
h
cDDCA 45 1.27 (0.35, 3.02) 17.89 46 0.67 (0.44, 1.10) 15.58 −31.7 (−52.9, −1.0) 0.04 < LOD
d,h
Clothianidin 45 0.17 (0.07, 0.39) 6.60 46 < LOD 0.69 −77.2 (−84.0, −67.3) < 0.01 –
FPBA 45 0.05 (0.03, 0.08) 7.11 46 0.02 (< LOD, 0.03) 0.13 −59.0 (−71.4, −41.2) < 0.01 < LOD
e,h
MDA 45 1.04 (0.37, 2.63) 35.80 46 < LOD (< LOD, 0.08) 0.83 −95.5 (−977, −91.4) < 0.01 < LOD
d,h
TCPy 45 2.99 (1.94, 5.32) 19.48 46 1.77 (1.19, 3.03) 7.25 −41.9 (−56.2, −22.9) < 0.01 –
tDCCA 45 2.35 (0.72, 5.53) 22.41 46 1.45 (0.78, 2.31) 20.03 −14.1 (−41.3, 25.6) 0.43 < LOD
f,h
Abbreviations: LOD, limit of detection; CI, confidence interval; IQR, interquartile range; NHANES, National Health and Nutrition Examination Survey.
a
Median and 95% confidence interval of non-creatinine adjusted analyte levels in ug/L from 2009 to 2010 NHANES.
b
Adults in study 36–52 years old.
c
Children in study 4–15 years old.
d
LOD = 0.5 μg/L.
e
LOD = 0.1 μg/L.
f
LOD = 0.6 μg/L.
g
Adults from NHANES 20–59 years old.
h
Children from NHANES 6–11 years old.
C. Hyland et al. Environmental Research xxx (xxxx) xxx–xxx
4
analytes (e.g. MDA, TCPy, clothianidin, FPBA, and 2,4-D) ranged from
85–100% to 14–100% during the conventional and organic diet phases,
respectively (Table 1). Parent compounds or metabolites of the in-
secticide imidacloprid and fungicides thiabendazole, boscalid, and
iprodione were not detected in urine samples (Table 1).
3.4. Effect of diet on urinary pesticide levels
Following the organic diet intervention, we observed significant
reductions in urinary levels of all analytes detected in the study, except
for DEP (percent change from conventional to organic phase: −16.7%
(95% confidence interval [95% CI]: −37.0%, 10.0%; p = 0.20)). The
largest changes were observed for MDA, clothianidin, and TCPy, which
decreased by −95.0% (95% CI: −97.0%, −91.8%; p < 0.01),
−82.7% (95% CI: −86.6%, −77.6%; p < 0.01), and −60.7% (95%
CI: −69.6%, −49.2%; p < 0.01), respectively (Table 2 and Fig. 1).
During the organic diet phase, urinary levels of total DE, total DM, and
total DAP metabolites decreased by −26.0% (95% CI: −43.7%,
−2.6%; p< 0.01), −83.9% (95% CI: −88.0%, −78.4%; p< 0.01),
and −69.5% (95% CI: −76.6%, −60.2%; p< 0.01), respectively
(Table 3 and Fig. 2). Fig. 3 presents the mean and 95th CI for urinary
levels of clothianidin, MDA, TCPy, total DE, total DM, and total DAPs
among all participants, adults, and children.
We also observed significant reductions in levels of 2,4-D, 3PBA,
FBPA, cDDCA, and tDCCA among all participants following the organic
diet intervention (Table 2). Although statistically significant reductions
in 3PBA and tDCCA were observed among all participants and adults,
these metabolites did not decrease significantly among children in our
study (Table 2).
3.5. Sensitivity analysis
Results from linear mixed-effects models using creatinine-adjusted
analytes did not differ appreciably from models not adjusted for crea-
tinine (See Supplementary material Tables S2 and S3). Overall, the
results also did not differ appreciably when outliers based on model
residuals were excluded from analyses (See Supplementary material
Tables S4 and S5). However, among all participants, the percent change
in total urinary DE metabolites from the conventional to organic diet
phase changed from −26% to −23% and the p-value increased
from < 0.01 to 0.05 after excluding one outlier with the highest ur-
inary DE concentration. The percent change in total urinary DE meta-
bolites from the conventional to organic diet phase among adults
changed from negative to positive after outliers were excluded. While
the percent change in total DE exposure was not statistically significant
among adults either with or without the outliers, it appears that urinary
total DE levels from two samples from one of the participants during the
conventional phase may have influenced the overall effect estimate for
adults.
4. Discussion
We detected fourteen pesticides and pesticide metabolites in all of
Fig. 1. Percent decrease in urinary metabolite concentration from conventional
to organic diet phase from mixed effects models (n = 16 participants; n=158
urine samples).
Table 3
Urinary DAP analyte concentrations (nmol/L) during conventional and organic diet.
Metabolite Conventional Organic Percent change (95% CI) p-Value
n Median (IQR) Max n Median (IQR) Max
All (n = 16)
DEs 78 31.40 (14.87, 60.21) 819.54 77 24.79 (12.24, 42.08) 223.98 −26.0 (−43.7, −2.6) < 0.01
DMs 78 101.42 (48.47, 257.98) 1730.72 77 18.34 (9.78, 35.95) 172.50 −83.9 (−88.0, −78.4) < 0.01
DAPs 78 138.39 (70.93, 315.56) 2039.93 77 45.46 (25.50, 77.43) 310.61 −69.5 (−76.6, −60.2) < 0.01
Adults (n = 7)
DEs 34 20.96 (14.11, 36.01) 819.54 32 24.62 (11.71, 34.42) 142.28 −10.7 (−42.5, 38.5) 0.61
DMs 34 71.77 (50.45, 135.44) 472.59 32 10.27 (3.88, 19.71) 172.50 −87.6 (−91.8, −81.3) < 0.01
DAPs 34 93.34 (67.30, 167.10) 927.13 32 39.22 (20.52, 59.07) 230.77 −68.5 (−78.7, −53.4) < 0.01
Children (n = 9)
DEs 44 44.01 (22.23, 78.74) 309.21 45 26.00 (12.62, 51.58) 223.98 −35.2 (−54.4, −7.9) 0.02
DMs 44 127.09 (48.07, 315.02) 1730.72 45 28.30 (15.57, 40.49) 86.63 −80.5 (−87.0, −70.6) < 0.01
DAPs 44 212.98 (85.25, 415.09) 2039.93 45 57.64, 32.03, 110.84) 310.61 −69.8 (−79.2, −56.2) < 0.01
Abbreviations: LOD, limit of detection; CI, confidence interval; IQR, interquartile range.
DMs = DMP + DMTP + DMDTP; DEs = DEP + DETP + DEDTP; DAPs = DMs + DEs.
Fig. 2. Percent decrease in urinary DAP metabolite concentrations from con-
ventional to organic diet phase from mixed effects models (n = 16 participants;
n=158 urine samples).
C. Hyland et al. Environmental Research xxx (xxxx) xxx–xxx
5
the study participants' urine. These chemicals represent potential ex-
posure to over 40 different pesticides including OP, neonicotinoid, and
pyrethroid insecticides as well as the herbicide 2,4-D (Table 1). Fol-
lowing an organic diet intervention, urinary levels of all but one of
these exposure biomarkers (DEP) decreased significantly.
4.1. Organophosphates
Previous studies have indicated that dietary ingestion is the primary
route of OP pesticide exposure in the general U.S. population (Curl
et al., 2015; Lu et al., 2008, 2006a; Smith-Spangler et al., 2012; United
States Environmental Protection Agency, 2006), and the results from
our study and other investigations have provided consistent evidence
that an organic diet may reduce exposure to OP pesticides (Bradman
et al., 2015; Göen et al., 2016; Lu et al., 2006a; Oates et al., 2014). For
example, in a dietary intervention study assessing OP pesticide ex-
posure among 23 elementary school-aged children, Lu et al. (2006a)
observed that median levels of MDA and TCPy immediately fell below
the limit of detection following the organic diet intervention and re-
mained nondetectable until a conventional diet was reintroduced. In a
crossover study among thirteen adults in Australia, Oates et al. (2014)
found that, compared with the conventional diet phase, mean total DAP
and DM concentrations were 89% and 96% lower, respectively, during
a one-week organic diet phase. Bradman et al. (2015) conducted an
organic diet intervention with 40 children living in urban and rural
agricultural locations and found that, compared to the two conven-
tional diet phases, an organic diet was associated with a −39.9% (95%
CI: −58.6, −12.6%; p< 0.01) and −13.3% (95% CI: −28.9, 5.8;
p= 0.16) decrease in mean urinary total DAP and MDA levels, re-
spectively. Most recently, Göen et al. (2016) conducted a pilot organic
diet intervention study with two adults in Switzerland and found that
levels of DMP, DMTP, DEP, DETP, and TCPy were generally lower
during the organic phase.
In our study, levels of all OP metabolites except for DEP decreased
significantly following the organic diet intervention. Among the most
significant decreases during the organic diet phase were MDA, a me-
tabolite of the insecticide malathion, and TCPy, a metabolite of the
insecticide chlorpyrifos. Chlorpyrifos is a neurotoxic pesticide that was
effectively banned for residential use in 2001 due to concerns about its
health impacts on children (United States Environmental Protection
Agency, 2000; Williams et al., 2008). It has been estimated that
chlorpyrifos residues on food crops exceed Federal Food, Drug, and
Cosmetic Act (FFDCA) safety standards (United States Environmental
Protection Agency), and the most recent U.S. EPA data on pesticide
sales and usage indicate that chlorpyrifos was the most widely used OP
in U.S. agriculture from 2007 to 2012 (United States Environmental
Protection Agency, 2017). In 2018 a court ordered the U.S. EPA to ban
the insecticide form agricultural use (Dennis, 2018); if implemented,
chlorpyrifos food residues would likely decrease.
4.2. Pyrethroids
The role of diet in pyrethroid exposure is not clearly understood
because pyrethroids are commonly used in agriculture, as well as for
residential and structural pest control purposes (Lu et al., 2006b). Göen
et al. (2016) found that levels of cDCCA, tDCCA, and 3-PBA decreased
significantly following the introduction of an organic diet, however the
study only included two adults, limiting inferences in the broader po-
pulation. In contrast to these findings, Bradman et al. (2015) found that
3-PBA levels were not associated with an organic diet. We observed
significant decreases in 3-PBA and three other pyrethroid metabolites,
FPBA, cDDCA, and tDDCA, among all participants following the organic
diet intervention. However, levels of 3-PBA and tDCCA did not decrease
significantly among children in our study, suggesting potential ex-
posure from other sources.
Notably, we observed significantly higher levels of urinary pyre-
throid metabolites than those reported in data from the 2009–2010
NHANES assessment, even during the organic diet phase. Although
participants did not report household pyrethroid use during the study,
residential use has been identified as the biggest risk factor for
Fig. 3. Estimated mean and 95% CIs for select urinary analytes during conventional and organic diet phase among all participants, adults, and children.
C. Hyland et al. Environmental Research xxx (xxxx) xxx–xxx
6
pyrethroid exposure (Lu et al., 2006b) and it is possible that the high
analyte concentrations observed in our populations were due to ex-
posure at school or work, or from residential use that was not reported.
However, while pyrethroids are commonly used around homes, schools,
and other buildings (Williams et al., 2008), the significant decreases in
pyrethroid metabolites among participants in our study indicate that
exposure may be at least partially attributable to diet. Our findings are
consistent with a study that found that dietary intake, seasonal differ-
ences, and household pesticide use all contribute to children's pyre-
throid exposures (Lu et al., 2009). Additional research is needed to
elucidate the relative contributions of the various sources of pyrethroid
exposure among both children and adults.
4.3. Neonicotinoids
Neonicotinoids have become one of the most widely used class of
insecticides in the world (Lu et al., 2018), and it is estimated that more
than four million pounds of neonicotinoids are applied to cropland
annually in the United Sates (Cimino et al., 2017). The 2012 FDA Total
Diet study indicated that neonicotinoids were among the most com-
monly reported pesticide residues in infant and toddler foods (Cimino
et al., 2017), however little is known about the health effects of chronic
low-dose exposure to these insecticides (Lu et al., 2018). We tested two
neonicotinoid compounds: the parent compound clothianidin and the
imidacloprid metabolite 5-hydroxy-imidacloprid (5OH-Imd). The most
recent Pesticide Use Reporting (PUR) data indicate that in 2016, imi-
dacloprid use on crops in California was nearly 26 times higher than
clothianidin use (California Environmental Protection Agency
Department of Pesticide Regulation, 2018). Yet, clothianidin was de-
tected in all study participants during the conventional diet phase,
while 5OH-Imd was below the limit of detection (LOD) for all study
participants (0.1 ng/mL). Furthermore, imidacloprid is frequently
found as a residue on food (Chang et al., 2018), suggesting that 5OH-
Imd may not be the best biomarker to assess dietary imidacloprid ex-
posure. Previous studies have quantified imidacloprid (Ueyama et al.,
2014) and 6-chloronicotinic acid (6CN), a non-specific metabolite of
imidacloprid and other neonicotinoids (Göen et al., 2016; Nomura
et al., 2013), in urine with detection frequencies > 50%. Future studies
should consider measuring the parent compound imidacloprid or me-
tabolites such as 6CN, which may be more reliable indicators of dietary
imidacloprid exposure.
Despite the widespread use of neonicotinoids and high detection
rates of residues on food samples, this is only the second organic diet
intervention study to include neonicotinoids, with the first taking place
in Switzerland and enrolling only two participants (Göen et al., 2016).
To date, the Centers for Disease Control and Prevention (CDC) has not
included neonicotinoids in NHANES testing and there are significant
data gaps regarding levels of exposure among children and adults in the
United States, underscoring the importance of additional research.
Future biomonitoring studies should continue to evaluate exposure to
imidacloprid and other common neonicotinoids.
4.4. 2,4-D
Similar to Bradman et al. (2015), we found that levels of the her-
bicide 2,4-D decreased significantly among children, as well as adults,
following the introduction of an organic diet. Several studies have de-
tected 2,4-D in food samples and diet has been identified as an im-
portant pathway for 2,4-D exposure among children (Wilson et al.,
2003), supporting our finding that the lower urinary levels observed
during the organic phase in our study were attributable to lower dietary
exposure.
4.5. Food choice comparison
Consistent with findings from Bradman et al. (2015) and Oates et al.
(2014), fruit and grain consumption increased slightly following the
introduction of the organic diet (See Supplementary information Table
S6). Given that fruit and grain are potential sources of dietary pesticide
exposure, these minor changes in food choices between the diet phases
would not have confounded our results.
4.6. Limitations
This study has several limitations. First, the study has a relatively
small sample size of sixteen participants, although 158 urine samples
were analyzed. Sampling for the families took place at different times of
the year between February and May 2017. Differences in seasonal
pesticide use could potentially result in different exposures. However,
given the global food supply, we do not expect that seasonal or regional
differences in agricultural pesticide use had a significant impact on
dietary exposures, and our participants lived in urban or suburban
areas. Another limitation of our study is that none of the participants
were able to identify whether they or their children were exposed to
pesticides outside of the home. Additionally, previous studies have
noted higher concentrations of OP metabolite among vegetarians
(Berman et al., 2016; Van Audenhaege et al., 2009). However, all
participants in our study reported consuming meat during both diet
phases, and we do not believe that following a vegetarian diet was a
predictor of pesticide exposure in this investigation. Notably, urinary
concentrations from non-specific pyrethroid and OP insecticides (e.g.
3PBA, cDCCA, tDCCA and DAPs) may reflect exposure not only to a
range of parent pesticide compounds, but also to preformed metabolites
from diet and the environment (Chen et al., 2012; Lu et al., 2005; Zhang
et al., 2008). It is possible that the decreases observed in urinary con-
centrations of these non-specific pyrethroid and OP insecticide meta-
bolites were at least partially attributable to lower exposure to pre-
formed metabolites on food during the organic diet phase, which may
be less toxic than the parent compounds. Finally, our laboratory
methods did not allow measurement of spinosad, the single pesticide
approved for organic production with a U.S. EPA food tolerance.
5. Conclusion
We observed significant reductions in urinary levels of thirteen
pesticide metabolites and parent compounds representing exposure to
OP, neonicotinoid, and pyrethroid insecticides and the herbicide 2,4-D
following the introduction of an organic diet. The largest changes were
observed for clothianidin and metabolites of malathion and chlorpyr-
ifos. Our study builds on prior research by assessing dietary exposure to
pesticides that to date have not been examined in diet intervention
studies among both children and adults. Additional research is needed
to better understand dietary exposure to neonicotinoids, now the most
widely used class of insecticides worldwide.
Acknowledgements
We gratefully acknowledge the families that participated in this
study. We thank Dr. Rosemary Castorina, who provided technical gui-
dance in this manuscript and Georgina del Valle, who helped in dif-
ferent stages of this project.
Funding
This work was funded by Friends of the Earth U.S, United States.
Author statements
Asa Bradman, Ph.D. is a volunteer member of the Board of Trustees
for The Organic Center, a non-profit organization addressing scientific
issues about organic food and agriculture and is also a member of the
USDA National Organic Standards Board. Bradman also advises organic
C. Hyland et al. Environmental Research xxx (xxxx) xxx–xxx
7
and conventional food producers on issues related to pesticides.
Kendra Klein, Ph.D. is Senior StaffScientist at Friends of the Earth
U.S.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.envres.2019.01.024.
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