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Pesticide Residue ExposureEnvironmental Profiles Science & Technology is published
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Silicone Wristband Passive Samplers Yield Highly
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Individualized Pesticide Residue Exposure Profiles
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Raf Aerts,*,†,‡,ǁ Laure Joly,§ Philippe Szternfeld,§ Khariklia Tsilikas,§ Koen De Cremer,⊥ Philippe
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Castelain,£ Jean-Marie Aerts,ǁ Jos Van Orshoven,‡ Ben Somers,‡ Marijke Hendrickx,∇ Mirjana
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Andjelkovic,§ and An Van Nieuwenhuyse,†,∆
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†
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Contaminants,
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Service Toxicology, and ∇Scientific Service Mycology and Aerobiology, Scientific Institute of
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Public Health (WIV-ISP), Brussels, Belgium
Scientific Service Health and Environment,
§
Scientific Service Chemical Residues and
⊥
Operational Directorate Food, Medicines and Consumer Safety, £Scientific
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‡
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ǁ
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Engineering, Department of Biosystems (BIOSYST), and ∆Environment and Health, Department
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of Public Health and Primary Care, University of Leuven (KU Leuven), Leuven, Belgium
Division Forest, Nature and Landscape, Department Earth and Environmental Sciences,
Measure, Model & Manage Bioresponses (M3-BIORES), Division Animal and Human Health
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ABSTRACT
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Monitoring human exposure to pesticides and pesticide residues (PRs) remains crucial for
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informing public health policies, despite strict regulation of plant protection product and biocide
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use.
We used 72 low-cost silicone wristbands as non-invasive passive samplers to assess
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cumulative 5-day exposure of 30 individuals to polar PRs. Ethyl acetate extraction and LC-
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MS/MS analysis were used for the identification of PRs. Thirty-one PRs were detected of which
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15 PRs (48%) were detected only in worn wristbands, not in environmental controls. The PRs
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included 16 fungicides (52%), 8 insecticides (26%), 2 herbicides (6%), 3 pesticide derivatives
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(10%), 1 insect repellent (3%) and 1 pesticide synergist (3%). Five detected pesticides were not
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approved for plant protection use in the EU. Smoking and dietary habits that favor vegetable
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consumption were associated to higher numbers and higher cumulative concentrations of PRs in
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wristbands. Wristbands featured unique PR combinations. Our results suggest both environment
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and diet contributed to PR exposure in our study group. Silicone wristbands could serve as
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sensitive passive samplers to screen population-wide cumulative dietary and environmental
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exposure to authorized, unauthorized and banned pesticides.
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INTRODUCTION
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Pesticides are widely used in agriculture, industry and in the home environment as plant
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protection products and as biocidal active substances to control weeds, fungi and pests. Strict
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international policies and regulations with respect to market approval and maximum residue
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limits have been implemented and enforced by authorities to minimize risk of pesticide use.
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However, whether or not exposure to pesticide residues (PRs) in the environment, food and
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water forms a potential threat for human health remains debated.1 The chronic and acute
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exposure, including periconceptional, pre- and perinatal exposure, to pesticidal and biocidal
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active substances and their residues, are increasingly associated to a number of adverse health
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outcomes, such as Parkinson’s disease,2 prostate and liver cancer,3,4 and reduced sperm quality in
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adults;5 cough,6 respiratory problems,7 leukemia,8,9 brain tumors,10 and neurobehavioral
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disorders in children;11 and neurodevelopmental disorders,12,13 low birth weight,14 and congenital
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disorders in infants.15,16 Many of these epidemiological exposure studies, however, have
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limitations.17 Actual exposures are hard to measure as the surrogate metrics of exposure, such as
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reported days or frequency of pesticide use, provide insufficiently precise information about
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effective exposure. Whereas there is no “gold standard” for exposure,18 there is a need for
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improving exposure estimation to discriminate surrogate from actual exposures.
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Possible routes of entry of pesticides and their derivatives are transdermal absorption, inhalation
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and oral ingestion.19 Occupational exposure and the handling of items that have been treated with
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pesticides (e.g. cut flowers,20,21 plant cultivation infrastructure and materials22) increase the
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probability of uptake and subsequent acute or chronic adverse health effects15,23. Such exposure
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can be minimized by using personal protective equipment,22,24,25 by implementing engineering
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controls during handling,25 by using proper ventilation,26 and by reducing or avoiding pesticide
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use.1,27 Environmental exposure to contaminated water, soil or air from runoff, dust and spray
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drift generated by domestic or agricultural pesticide applications is also an important pathway of
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exposure to PRs.1,16,28 At the level of the general population, residential pesticide use,8,10,29
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contact with contaminated household surfaces,30 and notably diet1,29,31-35 have been identified as
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important exposure pathways to PRs. Short-term and cumulative dietary exposure to a number of
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PRs have been shown to be a public health concern,36,37 in particular for children.34 PR screening
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in food and feed and monitoring of human exposure to PRs therefore play vital roles in
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safeguarding public health.38
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Human biomarker monitoring, for instance urinary biomarker measurements using liquid
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chromatography (LC) and mass spectrometry (MS),29,38,39 is the principal method to monitor
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human exposure to PRs. Urine, sweat, saliva, breast milk, nails and hair are suitable non-
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invasive human matrices for pesticide exposure monitoring.19,40-42
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include waste water sampling for population-wide exposure assessment43 and (personal) passive
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sampling devices (PSDs). Recently polyethylene, polyurethane foam and silicone matrices have
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been proposed as passive samplers for environmental44,45 and individual assessment of exposure
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to persistent organic pollutants, pesticides, brominated diphenyl ethers, organophosphate flame-
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retardants and other potentially harmful substances.46-53
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In this cross-sectional, non-interventional study, we tested the suitability of low-cost
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commercially available silicone wristbands as personal passive sampling devices to assess
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personal exposure to LC-amenable pesticide residues. We hypothesized that PR spectra detected
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in the wristbands would reflect the participants’ exposure to pesticide residues, which is
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determined by their pesticide use, outdoor and indoor environmental exposure and personal food
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choices.
Less intrusive methods
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MATERIALS AND METHODS Study Design.
Study participants were recruited from the Department of Earth and
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Environmental Sciences of the University of Leuven, Leuven, Belgium (n = 30; Table S1) in
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September 2016. The protocol for a cross-sectional, non-interventional study was reviewed and
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approved by the Social and Societal Ethics Committee of the University of Leuven (SMEC
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protocol number G-2016 09 636).
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information, anonymity and the possibility of withdrawing from the study at any time without
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the need for justification. All participants were adults and gave written prior informed consent.
Participants were assured confidentiality of personal
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Participants agreed to wear a silicone wristband for 5 days, to place a second wristband near their
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residence, and to complete questionnaires.
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Wristband Cleaning. Adult-size (200 mm L × 12 mm W × 2 mm T; weight 5.33 g, SD =
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0.10 g) silicone wristbands with embossed serial numbers were purchased (pdc Healthcare,
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Nivelles, Belgium; wristbands.com).
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extractions in an overhead shaker (Reax 2®, Heidolph Instruments GmbH & Co. KG,
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Schwabach, Germany) to remove potentially present analytes of interest and compounds that
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could cause chromatographic interference. The first extraction of 30 minutes with 1:1 ethyl
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acetate/hexane (v/v) was followed by a second extraction of 30 minutes with 1:1 ethyl
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acetate/methanol (v/v). After the extractions, the wristbands were dried under a nitrogen stream,
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and stored in a dark place in labeled, neutral glass vials of 60 ml with screw closure. This
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cleaning method was adapted from the procedure described by O’Connell et al.46 to minimize the
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consumption of organic solvents.
The wristbands were cleaned using two successive
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Wristband Exposure. Participants wore the wristbands continuously for a 5-day period
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(starting on the Monday morning of a work week) during all daily activities. Stationary silicone
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wristbands were placed by the participants on a location outside their residence (protected from
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rain) and served as controls of the residential outdoor environment. Participants recorded start
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and stop time of the wristband exposure in a log book. Additional stationary control bands were
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placed in the main building of the department where the participants were recruited (n = 4,
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occupational environment controls) and in Heverleebos-Meerdaalwoud, a high-value mixed
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deciduous forest 3 km south of Leuven (n = 8, clean air environment controls). The purpose of
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the stationary control bands was to identify airborne PRs. Six cleaned wristbands were kept in
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their vials in the laboratory at room temperature to serve as blanks. At the end of the 5-day
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period, participants placed the control and worn wristbands in their original vials and kept the
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vials refrigerated at 4−6°C.
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84.3−88.2 hours. In the laboratory, returned vials were stored at −20°C awaiting chemical
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analysis.50
The wristband exposure time (95% confidence interval) was
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Questionnaires. Participants provided three types of background information needed to
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interpret the results: daily outdoor exposure information, demographic data and personal data.
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To characterize the intensity of outdoor exposure, participants recorded on a daily basis
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information on their whereabouts and travels. For each travel the participants recorded start and
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end point addresses, start and end time, and type of transportation used. This information was
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later used to calculate total time spent moving outdoors, and by using Google Maps routes, total
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distance traveled (km). Participants completed a short questionnaire to provide demographic
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data and information on their living environment, smoking habits and occupational or domestic
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exposure to pesticides. After the experiment, participants provided information on pet ownership
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and treatment, the number of showers taken during the 5-day period, and general dietary choices
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(organic vs. conventional and vegetarian vs. conventional).
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Wristband Extraction. For detection of pesticide residues, a tandem mass spectrometry
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(MS/MS) protocol was developed, simplifying the protocol from O’Connell et al.46 To extract
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pesticides, 40 ml of ethyl acetate was added to the vial containing the wristband. Vial, wristband
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and extraction solvent were shaken mechanically during 30 min by an overhead shaker (Reax
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2®, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). This procedure was
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repeated two times with the same volume and time. The ethyl acetate phase (120 ml) was
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evaporated with a sample concentrator at 40°C (Genevac Ltd, Ipswich, UK) and under a nitrogen
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stream until the volume was reduced to 200 µl. To prepare the final extract, the raw extract was
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spiked with 200 µl internal standard solution (Oxfendazole, a broad spectrum veterinary
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anthelmintic compound, at 100 ng/ml), then diluted with 400 µl mobile phase A (water/methanol
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(90/10 v/v) containing 5 mM ammonium acetate) and adjusted to 1 ml with methanol. An aliquot
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(500 µl) was transferred in a filtration vial prior to injection into the UHPLC-MS/MS system.
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Wristband Chemical Analysis. Based on production, maximum residue levels in food
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and frequency of detection, LC-MS/MS systems have a wider scope and better sensitivity to
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detect pesticide residues than GC-MS systems.54,55 Therefore, the screening was focused on LC-
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amenable residues of polar plant protection products and biocides. UHPLC-MS/MS analyses
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were performed on an UPLCTM system (Waters, Milford, MA) coupled to a Quattro PremierTM
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mass spectrometer (Waters, Milford, MA) following the protocol of Hanot et al.56 Five microliter
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of sample was injected onto a reverse-phase ACQUITY™ UPLC BEH C18 column (1.7 µm,
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2.1×100 mm) (Waters, Milford, MA). The mobile phases were composed of water/methanol
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(90/10 v/v) (phase A) and methanol/water (90/10 v/v) (phase B) both containing 5 mM
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ammonium acetate. The chromatographic separation was performed at a flow rate of 0.45 ml
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min-1 with an elution gradient starting with 99.9% of mobile phase A and linearly evolving until
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0.1% in 10 min and maintained during two minutes. The column was re-equilibrated for the next
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injection during 3 min with 99.9% of mobile phase A. Three UHPLC runs were necessary to
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acquire two transitions for each of 200 pesticides. For the two first runs the sample was ionized
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with an electrospray probe in the positive mode; for the third run the negative mode was used.
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Selected quantities of 200 LC-amenable pesticide residues were added just before the vial
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filtration. The linearity range of the method was 0.4−100 ng g-1 wristband. In all cases, good
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linearity was achieved with correlation coefficients > 0.99. The limit of quantification (LOQ)
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was 0.2 ng g-1 wristband.
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Quality Control. To ensure reliability of results, a blank and a spiked sample were
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analysed in each of 5 batches. For the spiked sample (analytical quality control sample), a multi-
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residue solution was spiked on wristband, which was allowed to dry for 1 h, and then analysed.
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Multi-residue solutions containing 200 LC-amenable pesticide residues were prepared in
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methanol according to the European quality criteria for pesticide residue analysis in food and
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feed.57 Wristbands were spiked by spraying small droplets directly on the wristbands following
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the procedure of Toumi et al.20 The spike was calculated to reach an average concentration of
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about 2 ng g-1 wristband for each LC-amenable pesticide residue (equivalent to a concentration
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of 10 ng of pesticide residue on a wristband of 5 g). No pesticides were detected in the blank
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samples with a concentration above LOD, proving an absence of pesticides in the cleaned
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wristbands themselves, except for the insect repellent N,N-diethyl-meta-toluamide (DEET). The
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concentration of DEET was similar in all the blank samples (0.5 ng g-1 wristband). This
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background value was subtracted from the detected DEET values for all samples. Detections
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below LOQ were assigned the value of ½ LOQ (0.1 ng g-1) to enable statistical analyses.
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Statistical Analyses.
The frequencies of pesticide residue detection were compared
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between worn and stationary wristbands using chi-square tests for independent samples. The
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cumulative concentration of PRs (log∑PR) was calculated as the log10-transformed sum of the
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concentrations of all PRs (ng g-1) detected in each wristband (log∑PR = log10[∑PRi+1]; the +1
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component of the transformation was included to transform total concentrations < 1 ng g-1 to
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positive values), with and without DEET. Differences in the average number of PRs detected
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and differences in log∑PR between worn and stationary wristbands were assessed using Kruskal-
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Wallis one-way analysis of variance. Generalized linear models were used to estimate the
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effects of environmental and demographic variables on the number of PR detected and log∑PR
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(without DEET). For the number of PRs, a Poisson log-linear model was used, with sex,
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smoking habit and food choices as factors and age, BMI, number of showers and total distance
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traveled as covariates. Estimated marginal means and 95% confidence intervals were obtained
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and pairwise comparisons were made for the significant (p < 0.05) factors. A linear model with
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the same factors and covariates was used for log∑PR. To identify groups with similar PR
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profiles, individual PR profiles (based on log10[PR+1]-transformed values) were clustered into k
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= 2–6 groups using the Sørensen distance measurement. The most informative number of groups
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was determined using Indicator Species Analysis58 Nonmetric multidimensional scaling (NMS)
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based on the Sørensen distance measure was then used to place individual participants and PR
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profile groups in a 2-dimensional ordination space. Clustering and ordination were conducted
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using PC-ORD 5.0 for Windows (MjM Software, Gleneden Beach, OR). All other statistical
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analyses were performed in IBM SPSS Statistics 20 (IBM Corporation, Armonk, NY).
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RESULTS AND DISCUSSION
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All participants (n = 30, 100%) completed the exposure experiment, returned the worn
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wristbands and controls, and completed the questionnaires. The study population comprised 16
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males (53%) and 14 females (47%). At the time of the survey, participants were on average 32.6
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years old (range 24−72 years) and had a normal weight (average BMI 22.8, range 18.5−28.3).
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Participants spent on average 8.8 hours in traffic during the 5-day period (range 2.8−17.7 hours)
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and covered on average a distance of 251 km (range 31.5−711.3 km) (Table S1).
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occupational or accidental exposure to pesticides was reported. Four participants reported to
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have used pet care products (spot on flea treatment and flea collars) that contained a number of
No
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antiparasitic agents that included fipronil, methoprene, selamectine, imidacloprid and
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flumethrine (methoprene, selamectine and flumethrine were not included in the LC-MS/MS
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screening).
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PRs in Wristbands. In total, 31 LC-amenable pesticide residues were detected in the 72
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deployed wristbands. These PRs included 16 fungicides (52%), 8 insecticides (26%), 2
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herbicides (6%), 1 insect repellent (3%), 3 pesticide derivatives (10%) and 1 pesticide synergist
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(3%) (Table 1). Five substances which are not approved for plant protection use in the EU were
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detected: fipronil (and its desulfinyl photoproduct and sulfone metabolite),59,60 carbendazim,
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bendiocarb, tolylfluanid (via the metabolite DMST) and ethirimol (Table 1). Carbendazim and
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ethirimol are also degradation products of thiophanate-methyl and bupirimate, respectively, and
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those compounds are approved for plant protection use in the EU. Five fungicides and 4
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insecticides had the dual use of plant protection product and biocidal active substance (BAS)
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(Table 1). The insect repellent N,N-diethyl-meta-toluamide (DEET) was detected in 93% of all
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worn bands and in 81% of the stationary controls. The high prevalence of DEET in the controls,
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even in controls that were placed in forest with high ecological value, indicates that DEET is a
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pervasive contaminant in the environment, not only in water,61,62 but also in aerosols.49,63 Apart
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from DEET, the PRs with the highest prevalence were the pesticide synergist piperonyl butoxide
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(PBO; 63% of the worn bands, 9.5% of the controls), the broad spectrum fungicide azoxystrobin
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(47% of the worn bands, 2% of the controls) and the fungicide and parasiticide thiabendazole
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(TBZ) (40% of the worn bands, 2% of the controls). PBO is added to pesticide formulations to
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increase their effectiveness, in particular pyrethrin and pyrethroid insecticides (not included in
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our screening), and has been detected in personal and indoor residential air6 and in PSDs.49
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Prenatal exposure to PBO has been associated to negative health outcomes in children.6,12 The
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fungicides azoxystrobin and TBZ are widely used as plant protection products and as biocidal
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active substances to enhance preservation of film, fiber, leather, rubber, polymerised materials,
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wood and construction material. Environmental exposure, via aerosols, dust or direct contact
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with treated materials and/or food items (e.g. TBZ on orange, lemon and other citrus peels), may
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therefore be partially responsible for the detection of these compounds.
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Nine PRs had a significantly higher prevalence in worn wristbands than in controls (all p
