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Pesticide Residue ExposureEnvironmental Profiles Science & Technology is published

Raf Aerts, Laure Philippe by Joly, the American Chemical Society. 1155 Sixteenth Szternfeld, Khariklia Tsilikas, Street N.W., Washington, DC 20036 Koen Deaccess Cremer, Philippe Subscriber provided by READING Published by American UNIV Chemical Society. Copyright © American Chemical Society.

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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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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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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