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1 Glsgow Cledonin University Urinry biomrker concentrtions of cptn, chlormequt, chlorpyrifos nd cypermethrin in UK dults nd children living ner griculturl lnd Gle, Kren S.; McClmn, Lur; Jones, Kte; Cocker, John; Teedon, Pul; Cherrie, John W.; vn Tongeren, Mrtie Published in: Journl of Exposure Science nd Environmentl Epidemiology DOI: /jes Publiction dte: 2015 Document Version Publisher's PDF, lso known s Version of record Link to publiction in ReserchOnline@GCU Cittion for published version (APA): Gle, K. S., McClmn, L., Jones, K., Cocker, J., Teedon, P., Cherrie, J. W., & vn Tongeren, M. (2015). Urinry biomrker concentrtions of cptn, chlormequt, chlorpyrifos nd cypermethrin in UK dults nd children living ner griculturl lnd. Journl of Exposure Science nd Environmentl Epidemiology, 25, /jes Generl rights Copyright nd morl rights for the publictions mde ccessible in the public portl re retined by the uthors nd/or other copyright owners nd it is condition of ccessing publictions tht users recognise nd bide by the legl requirements ssocited with these rights. Users my downlod nd print one copy of ny publiction from the public portl for the purpose of privte study or reserch. You my not further distribute the mteril or use it for ny profit-mking ctivity or commercil gin You my freely distribute the URL identifying the publiction in the ReserchOnline@GCU portl Tke down policy If you believe tht this document breches copyright plese contct us t: repository@gcu.c.uk providing detils, nd we will remove ccess to the work immeditely nd investigte your clim. Downlod dte: 29. Jn. 2019

2 OPEN Journl of Exposure Science nd Environmentl Epidemiology (2015) 25, Nture Americ, Inc. All rights reserved /15 ORIGINAL ARTICLE Urinry biomrker concentrtions of cptn, chlormequt, chlorpyrifos nd cypermethrin in UK dults nd children living ner griculturl lnd Kren S. Gle 1, Lur McClmn 1, Kte Jones 2, John Cocker 2, Pul Teedon 3, John W. Cherrie 1,4 nd Mrtie vn Tongeren 1 There is limited informtion on the exposure to pesticides experienced by UK residents living ner griculturl lnd. This study imed to investigte their pesticide exposure in reltion to spry events. Frmers treting crops with cptn, chlormequt, chlorpyrifos or cypermethrin provided spry event informtion. Adults nd children residing 100 m from spryed fields provided first-morning void urine smples during nd outwith the spry seson. Selected smples (1 2 dys fter spry event nd t other times (bckground smples)) were nlysed nd cretinine djusted. Generlised Liner Mixed Models were used to investigte if urinry biomrkers of these pesticides were elevted fter spry events. The finl dt set for sttisticl nlysis contined 1518 urine smples from 140 prticipnts, consisting of 523 spry event nd 995 bckground smples which were nlysed for pesticide urinry biomrkers. For cptn nd cypermethrin, the proportion of vlues below the limit of detection ws greter thn 80%, with no difference between spry event nd bckground smples. For chlormequt nd chlorpyrifos, the geometric men urinry biomrker concentrtions following spry events were 15.4 μg/g cretinine nd 2.5 μg/g cretinine, respectively, compred with 16.5 μg/g cretinine nd 3.0 μg/g cretinine for bckground smples within the sprying seson. Outwith the sprying seson, concentrtions for chlorpyrifos were the sme s those within sprying seson bckgrounds, but for chlormequt, lower concentrtions were observed outwith the sprying seson (12.3 μg/g cretinine). Overll, we observed no evidence indictive of dditionl urinry pesticide biomrker excretion s result of spry events, suggesting tht sources other thn locl sprying re responsible for the reltively low urinry pesticide biomrkers detected in the study popultion. Journl of Exposure Science nd Environmentl Epidemiology (2015) 25, ; doi: /jes ; published online 16 September 2015 Keywords: exposure; pesticides; residents INTRODUCTION The use of pesticides cn give rise to public concern. The Royl Commission on Environmentl Pollution (RCEP) published report on bystnder nd resident exposure to pesticides nd recognised tht the epidemiologicl literture showing ssocitions between chronic ftigue syndrome nd multiple chemicl sensitivity nd pesticide exposure ws plusible but equivocl. 1 The RCEP report nd the responses to it hve ensured tht the issue hs remined in the public eye. 2 4 In the UK, Government Ministers must pprove ll pesticides before they cn be mrketed or used, including those used in griculture, horticulture, forestry, food storge nd the home or grden, to ensure tht they do not present helth risks to the generl public, consumers, people who pply them or work in or ner res tht hve been treted with pesticides, the environment nd nimls. The Chemicls Regultion Directorte of the UK Helth nd Sfety Executive cts s the Regultor for pesticide products nd uthorises their sle, supply, use nd storge in the UK nd represents the UK in the Europen Union process for the registrtion of new ctive substnces nd for the renewl (review) of ctive substnces lredy pproved. The helth regultory risk ssessment (RRA) underpinning the pprovl of pesticides involves the comprison of estimted potentil humn exposures with toxicologicl reference vlues levels, for exmple, cceptble opertor exposure level nd cceptble dily intke (ADI), t nd below which there is considered to be high confidence tht there will be no dverse helth effects. The RRA is therefore generlly considered to be conservtive estimte of exposure. There re no specific studies on the exposures of people living ner griculturl lnd to pesticides in the UK. There re number of reported studies from elsewhere in the world which hve explored residents exposures. For exmple, Koures et l. 5 found no difference in urinry dilkylphosphtes (generic biomrkers of orgnophosphte pesticides) between urbn nd rurl residents in Thessly, Greece, whilst pesticide spryers hd significntly incresed levels. A Cndin study 6 showed significnt difference in urbn nd rurl residents for exposure to pyrethroids, wheres in Jpn 7 no differences were seen between rurl nd suburbn residents. Rurl residents in Nicrgu showed significntly lower cetylcholinesterse ctivity which were ssocited with eril pesticide sprying from plnes. 8 Crop mps were used by Wrd et l. 9 to predict tht incresing crege of corn nd soyben fields within 750 m of homes in Iow, USA, ws ssocited with significntly elevted odds of detecting 1 Centre for Humn Exposure Science, Institute of Occuptionl Medicine (IOM), Edinburgh, UK; 2 Helth nd Sfety Lbortory, Buxton, UK; 3 School of Engineering nd the Built Environment, Glsgow Cledonin University, Glsgow, UK nd 4 School of Life Sciences, Heriot Wtt University, Edinburgh, UK. Correspondence: Dr. K Gle, Centre for Humn Exposure Science, Institute of Occuptionl Medicine (IOM), Reserch Avenue North, Riccrton, Edinburgh EH14 4AP, UK. Tel.: +44(0) Fx: +44(0) E-mil: kren.gle@iom-world.org Received 1 Mrch 2015; revised 19 June 2015; ccepted 29 July 2015; published online 16 September 2015

3 624 Tble 1. Pesticides of interest. Pesticide Clss Function Relevnt crops pproved for use Cptn Phthlimide Fungicide Apple, per Chlormequt Chlorocholine Growth regultor Cerels Chlorpyrifos Orgnophosphte Insecticide Apple, cerels, vegetbles including potto Cypermethrin Pyrethroid Insecticide Apple, vrious rble crops including potto Penconzole b Trizole Fungicide Apple, blckcurrent, hops Approvl in b Results pertining to penconzole re presented elsewhere. griculturl herbicides inside homes compred with homes with no crops within 750 m. These studies present n uncler picture of pesticide exposure in rurl residents nd re difficult to trnslte to the UK sitution. This mnuscript reports on study ( Biologicl monitoring of pesticide exposure in residents ) funded by the UK Government Deprtment of Environment, Food nd Rurl Affirs (DEFRA), which imed to ssess exposure to pesticides for dults nd children living within 100 m from the edge of griculturl lnd nd to investigte whether exposures were elevted following spry events. METHODS The study received ethicl pprovl from the South Est Scotlnd Reserch Ethics Committee 3 (study number 10/S1103/63). Our study protocol provides detils of the methodology employed nd is briefly summrised below. 10 To obtin n estimte of the number of subjects required for the study, rnge of conservtive power clcultions were crried out priori for number of pesticides identified s being most likely to be pplied during the spry sesons in the trget res. 10 Recruitment of Study Prticipnts Smple nd dt collection took plce in three loctions in the UK: Est Lothin, Kent nd Norfolk. Dt in Est Lothin nd Kent were collected in 2011 nd 2012, while in Norfolk dt collection took plce in 2012 only. Est Lothin nd Norfolk re mjor rble crop growing res, while most of the orchrds in the UK re locted in Kent. Recruitment of, nd liison with, frmers nd residents prticipting in the study were crried out by community reserchers who hd locl knowledge of the study res nd communities. 11 Frmers were identified through publicly vilble resources nd contcted vi letter explining the ims nd objectives of the study. This ws then followed by telephone contct. The pesticides of interest, the crops on which they were likely to be pplied were discussed during this contct s ws the proximity of residentil res to these fields. If the frmer ws willing to prticipte nd considered suitble cndidte for inclusion in the study, n in-person meeting ws rrnged t the frm. The community reserchers recruited owners/mngers of frms nd orchrds (herefter both referred to s frms) tht reported when they were likely to spry their griculturl crops with certin specified pesticides nd which hd residentil res within 100 m of these fields. Informed consenting frmers were sked to provide detils of their pesticide usge throughout the spry seson. The spry informtion included informtion on the strt nd finish times of sprying, product nd ctive ingredients used, spry method nd wether conditions. In instnces where frmers demonstrted tht they lredy mintined comprehensive records of their pesticide usge, the resercher requested copies of these to be mde vilble. Where detiled records were not lredy mintined, prticipting frmers were sked to record the relevnt informtion using n dpttion of the spry record form recommended by the Deprtment of Environment, Food nd Rurl Affirs. 12 Tble 1 provides detils of the pesticides considered in the study nd reported in this mnuscript. Inclusion ws restricted to those pproved pesticides where nlyticl methods for the ssocited urinry biomrkers were vilble to the project tem. No vlidted method ws vilble for determining penconzole or its metbolites in urine, nd so novel method ws developed during this project, detils of which, s well s the penconzole results from this study re reported elsewhere. Residents (dults ged 18 yers nd over nd children in their cre ged 4 12 yers) living within 100 m of the edge of field belonging to recruited frm were pproched to prticipte in the study. Frmers nd their fmily members residing within 100 m of the fields were lso invited to prticipte providing they were not directly involved in pesticide spry or crop re-entry ctivities nd they fulfilled the other eligibility criterion. Informed consenting prticipnts completed bckground questionnire. This included questions concerning their ge, sex, weight, typicl consumption of orgnic food nd home-grown produce, occuption/ eduction, occuptionl nd pr-occuptionl exposure to pesticides nd typicl pesticide usge round the home or grden. The children s questionnire ws shorter nd ws completed by the consenting dult. Urine Smple Collection nd Accompnying Activity Questionnire Following due considertion of preliminry modelling (not presented), the peer-reviewed literture 13 nd the need to obtin smples over sustined period of time, first-morning void smples were chosen s the most pproprite urine smple to request from prticipnts. First-morning void urine smples nd ccompnying smple-relted questionnires were collected using two strtegies. 1. Residents were sked to provide first-morning void urine smples nd complete ccompnying questionnire once week on designted dy during the sprying seson nd lso for 3 weeks fter the sprying seson. 2. In instnces when community resercher ws dvised of relevnt spry event occurring on given field, prticipnts living within 100 m of the field were contcted nd sked to collect dditionl first-morning void urine smples one nd two dys following the dy of the spry event s well s complete smple-relted questionnires on both dys. Ech smple-relted questionnire completed by dult prticipnts requested informtion on their time spent indoors nd outdoors t home nd other loctions, domestic use nd pr-occuptionl exposure to pesticides, s well s their dietry consumption of home-grown produce within the previous 48-hour period. The smple-relted questionnire completed by the dult on behlf of child prticipnt ws shorter nd included questions concerning the time spent by the child in both outdoor nd indoor environments. The sprying seson ws considered to be between the months of Mrch August, coinciding with the min crop growing seson, nd stopping before hrvest. First-morning void smples were requested nd the time the urine smple collection ws recorded by the prticipnt. Urine smples (~70 ml) were collected in polypropylene continers (Strplex Scientific, Cnd) long with the completed smple-relted questionnire were collected t greed times by the community resercher on the dy of smple provision. The urine smples were stored in cool bg before collection by the community resercher. The smples were frozen s soon s possible (o6 h fter collection) nd couriered to the lbortory, being stored t temperture rnging from 15 to 20 C before nlysis. The community reserchers checked the completeness of the smple-relted questionnires. Frmers spry records were reviewed longside the dtes tht prticipnts urine smples were provided. Urine Smple Anlysis Urine smples were selected for nlysis s follows, with relevnt spry event referring to those involving the ppliction of pesticides including the ctive ingredients cptn, chlormequt, chlorpyrifos nd cypermethrin: Journl of Exposure Science nd Environmentl Epidemiology (2015), Nture Americ, Inc.

4 Tble 2. Description of the prticipnts (bckground questionnire responses). Descriptor N % Adult Child Sex Mle Femle Loction Est Lothin Kent Norfolk Employment (dults) Full time employment Prt time employment Full time eduction 1 1 Prt time eduction Retired 8 7 Non-pid employment 1 1 Missing or no nswer Eduction (children) Secondry 2 9 Primry Full time nursery 0 0 Prt time nursery 2 9 Smoke 5 4 Own pet Job involves trvelling round the locl re Use pesticides t work 6 4 Fmily use pesticides t work 7 5 Use pesticides in the home t lest once yer Men Rnge Age (dult) 55 (18 83) Age (child) 8 (4 12) 1. Urine smples collected within 2 dys of relevnt pesticide spry events tking plce in fields within 100 m of the prticipnt s residence (spry event-relted smples). 2. For ech prticipnt providing t lest one relevnt pesticide spry event smple, up to three rndomly selected bckground smples obtined within the sprying seson which did not coincide with relevnt spry event (within spry seson bckground smples). 3. For ech prticipnt providing t lest one relevnt spry event smple, up to three rndomly selected bckground smples collected outwith the sprying seson (during November December) (outwith spry seson bckground smples). Urine smples collected within 2 dys of relevnt sprying event were nlysed only for the relevnt pesticide(s) spryed during the event. Bckground smples, both within nd outwith the spry seson, were nlysed for ll the relevnt pesticides of interest to the study. Smples were nlysed ccording to estblished methods by the Helth nd Sfety Lbortory (HSL), which prticiptes in externl qulity ssurnce schemes for chlorpyrifos nd cypermethrin (G-EQUAS, The nlysts were blind to whether the urine smples were relted to spry events or were bckground smples. Detection limits were comprble to previous reported studies looking t generl popultion levels. All nlytes were quntified using multi-point mtrix-mtched clibrtion curves nd qulity control smples were run every five smples. Smples were nlysed in duplicte nd the men vlue reported. Aliquots of positive smples were renlysed throughout the project to evlute smple stbility. There ws no evidence of smple degrdtion for ny biomrker studied throughout the ssessment period (see Supplementry Informtion). The nlyticl method for chlormequt ws bsed on tht reported by Lindh et l. 14 mesuring chlormequt itself. The nlyticl method for cptn ws bsed on tht reported by Berthet et l. 15 mesuring cis- 1,2,3,6-tetrhydrophthlimide (THPI). The nlyticl method for chlorpyrifos ws bsed on tht reported by Sms et l. 16 mesuring 3,5,6- trichlorpyridinol (TCP). The nlyticl method for cypermethrin mesured cis- nd trns- 2,2-dichlorovinyl-3,3-dimethylcyclopropne-1-crboxylic cid (DCVA). 17 Further nlyticl methodology detils re provided s Supplementry Informtion. Dt Anlysis Urine smples with cretinine concentrtion below 2 or 430 mmol/l (0.23 g/l nd 3.39 g/l, respectively) were excluded from the nlyses. 18,19 Next, prticipnts for which either no spry event-relted smples or no bckground (either within or outwith) smples were vilble were excluded. The descriptive chrcteristics of the prticipnts were summrised, long with summries of their responses to the bckground questionnire, in terms of percentge in ech ctegory or men nd rnge for continuous vribles (Tble 2). Urinry mesurements were corrected for cretinine by dividing the urinry biomrker concentrtion by the cretinine concentrtion nd re reported s μg biomrker per g cretinine. A very high proportion of smples were below the nlyticl limit of detection (LOD) for cptn (89%) (LOD = 0.1 μg/l) nd cypermethrin (93%) (LOD = 1.0 μg/l) nd for these two pesticides only the proportion of detects, 95th percentile nd the mximum levels re reported. For chlormequt nd chlorpyrifos rndom imputtion procedure ws used to replce the vlues below the LOD (0.6 μg/l nd 0.8 μg/l, respectively). The geometric men (GM) nd geometric stndrd devition (GSD) of concentrtions bove the LOD were determined, ssuming lognorml distribution. Ech of the vlues below LOD ws then replced by vlue between 0 nd the LOD which ws rndomly generted from this distribution. As is typicl of dt of this nture, concentrtions were not normlly distributed. Trnsforming by tking the nturl logrithm improves the normlity of the dt significntly nd therefore the smple results re summrised in terms of GM nd GSD. 20 For ech individul the GM of their smples ws determined seprtely for smples provided fter spry event nd for within nd outwith spry seson bckgrounds. The GM rtio of the spry event smple to the bckgrounds ws then clculted by tking the log of ech individul rtio, verging nd exponentiting. Results re reported s GM nd ssocited 95% confidence intervl. A confidence intervl contining the vlue 1 mens tht the rtio is not significntly different from 1, tht is, spry event smples re not significntly higher or lower thn bckgrounds. The dt were further exmined to investigte fctors from either the bckground questionnire (person-specific) or the smple-relted questionnires (smple-specific) tht might explin ny differences between the biomrker concentrtions. Generl liner mixed models were used with the log of the biomrker level s the response. The prticipnt ws treted s rndom vrible, to ccount for repeted mesures from the sme individuls. Smple type ws treted s fixed effect of priori interest, where the levels of smple type were defined s; outwith nd within spry seson bckgrounds, smples provided the dy fter sprying (24 h), smples provided the second dy fter spry event (48 h) or smple ws provided fter 2 consecutive dys of sprying (24 nd 48 h). In step-wise mnner vribles were considered for inclusion in the model where they significntly improved the fit of the model, bsed on the likelihood rtio test or ffected the coefficients of terms lredy in the model (by 410%). Imputtion ws crried out using R v3.1.0, 21 while ll sttisticl nlyses were crried out using Genstt v16 (ref. 22) nd plots were prepred using Sigmplot v10). 23 RESULTS A totl of 13 prticipting frms during 2011 nd 17 frms during 2012 reported spryed events using t lest one of the relevnt pesticides. The brekdown by region is provided in Tble 3, long with detils of the number of relevnt spry events fter which urine smples were collected. All prticipting Kent frms were orchrds while the frms in Est Lothin nd Norfolk were rble frms Nture Americ, Inc. Journl of Exposure Science nd Environmentl Epidemiology (2015),

5 626 Tble 3. Prticipting frms nd relevnt spry events by pesticide nd geogrphicl re. Are No. frms Pesticide No. spry events Est Lothin 7 Chlormequt 57 Cypermethrin 2 Kent 9 Cptn 214 Chlorpyrifos 51 Norfolk 4 Chlormequt 15 Cypermethrin 3 Spry event numbers derived by multiplying the number of frms pplying the pesticide by the number of relevnt fields on which the pesticide ws pplied. For exmple if frm A spryed cptn in six fields on four occsions, the number of spry events for this frm would be 24. Tble 4. Urinry biomrker concentrtions (μg/g cretinine). Pesticide N NoLOD %olod Mx GM GSD 95th % ile Cptn Outwith Within Spry Cypermethrin Outwith Within Spry A totl of 296 prticipnts were recruited to the study (238 dults, 58 children), providing 3275 urine smples. One hundred forty-one people provided t lest one smple tht ws relted to relevnt spry event involving cptn, chlormequt, chlorprifos or cypermethrin nd hd t lest one bckground, resulting in 1565 smples results. Altogether, 73% of prticipnts hd the full complement of outwith spry seson bckgrounds nd 76% hd the full complement of within spry seson bckgrounds. Appliction of the cretinine exclusion criterion resulted in 40 smples being excluded from the nlysis (12 sprys nd 28 bckgrounds). Following the ppliction of this exclusion criterion, one further person ws removed due to no spry event-relted smples remining nd s such were no longer eligible to be included in the dt nlysis (six bckgrounds). The finl dt set for sttisticl nlysis contined 1518 smple results for 140 people, consisting of 995 bckground smples (440 outwith nd 555 within seson) (Figure 1) nd 523 spry event-relted smples. The spry smples included 255 cptn, 63 chlorpyrifos, 46 cypermethrin nd 197 chlormequt spry event results, respectively (some spry events involved multiple relevnt pesticides) b b Chlormequt Outwith Within Spry Chlorpyrifos Outwith Within Spry Abbrevitions: GM, geometric men; GSD, geometric stndrd devition; LOD, limit of detection; Mx, mximum; N, number; 95% ile, 95th percentile. GM nd GSD were not clculted due to the high proportion of vlues below LOD. b 95th percentile not clculted s over 95% of the smples were below LOD. 5.1 Figure 1. Prticipnt numbers by re, sex. Tble 4 summrises the urine smple results for spry events nd bckgrounds (within nd outwith spry seson). For cptn nd cypermethrin, the proportion of spry event vlues below the LOD ws 91 nd 98%, respectively, wheres the proportion of bckground results below the LOD ws 88% for cptn nd in excess of 90% for cypermethrin. The cptn biomrker concentrtions rnged from below the LOD to lmost 4 μg/g cretinine. There is slightly higher proportion of non-detects in the spry event-relted smples with the lrgest individul result being ssocited with bckground smple collected within the spry seson. The cypermethrin biomrker concentrtions follow similr pttern, with the mximum concentrtion (15.4 μg/g cretinine) found to be in bckground smple obtined outwith the spry seson. Figure 2 shows boxplots of the cretinine djusted urinry biomrker concentrtions (μg/g) for chlorpyrifos nd chlormequt for the spry event-relted smples s well s the within nd outwith spry seson bckground smples. Both figures show tht spry event-relted smples did not pper to contin elevted concentrtions of urinry biomrkers compred with the bckground levels. Spry event smple concentrtions for chlormequt re sttisticlly significntly higher thn outwith spry seson bckground, with the GM of the individul rtios being 1.22 (95% CI: 1.09, 1.38) but there is no significnt difference between spry event-relted smple concentrtions nd within spry seson bckgrounds (GM rtio 0.96 (95% CI: 0.87, 1.06)) (Tble 5). Interestingly, the verge spry event-relted chlorpyrifos urinry biomrker concentrtions re lower thn both the outwith nd within spry seson bckground smple results (GM rtio: 0.80, 95% CI: 0.74, 0.87 nd 1.01; 95% CI: 0.92, 1.11, respectively). There were minor differences in chlormequt biomrker concentrtions mesured for mles nd femles, with mles hving slightly higher urinry biomrker levels thn femles (Tble 6). This difference ws not significnt for outwith seson bckgrounds nd spry event-relted smples but ws significnt for within spry seson bckgrounds (P = 0.036). There were no sttisticlly significnt differences in the chlorpyrifos concentrtions mesured in smples provided by mles nd femles (Tble 6). The levels of chlormequt nd chlorpyrifos were higher in smples provided by children thn dults, lthough not significntly so; the vrition in the children s smple results lso tended to be greter. To ensure these ptterns were not the result of vrition in cretinine levels, which tend to be higher in mles thn femles nd in dults thn children, the uncorrected Journl of Exposure Science nd Environmentl Epidemiology (2015), Nture Americ, Inc.

6 Tble 6. Urinry biomrker concentrtions (μg/g cretinine) for mles nd femles nd for dults nd children for chlormequt nd chlorpyrifos. 627 Pesticide Femle Mle N NoLOD GM GSD N NoLOD GM GSD Chlormequt Outwith Within Spry Chlorpyrifos Outwith Within Spry Child Adult N NoLOD GM GSD N NoLOD GM GSD Chlormequt Outwith Within Spry Chlorpyrifos Outwith Within Spry Abbrevitions: GM, geometric men; GSD, geometric stndrd devition; LOD, limit of detection; N, number. Figure 2. Cretinine djusted urinry biomrker levels for spry event nd within nd outwith spry seson bckground smples for () chlorpyrifos nd (b) chlormequt. Tble 5. GM rtio of cretinine corrected concentrtions following spry events to the GM of bckgrounds. Outwith spry seson Within spry seson Pesticide GM rtio 95% CI GM rtio 95% CI Chlormequt Chlorpyrifos Abbrevitions: CI, confidence Intervl; GM, geometric men. cretinine urinry biomrker concentrtions were nlysed nd similr ptterns were observed. There were no sttisticlly significnt differences in chlormequt or chlorpyrifos concentrtion for the smples collected t 1 nd 2 dys fter the spry events (Tble 7). In ddition, we investigted if there is ny reltionship between biomrker concentrtions nd timing of spry event nd no significnt reltionship ws found. We investigted whether other fctors such s ge, sex, loction, smoking sttus, typicl consumption of orgnic food nd typicl use of pesticides (s nswered in bckground questionnire), whether the subject hd used pesticides in the previous 48 h, time spent indoors nd outdoors in the previous 48 h (bsed on the smple-relted questionnires) might explin ny differences in the biomrker concentrtions between when the smple ws collected. For chlormequt, fter including smple type (i.e., spry event or bckground) in the model, the only significnt fctor, bsed on Wld test for ddition to the model, ws the level of orgnic food consumption reported, where higher levels of orgnic food consumption were ssocited with higher levels of chlormequt (where orgnic food consumption ws recorded by prticipnts s; none, little, some nd most). However this fctor did not significntly improve the fit of the model. As shown before, the sttisticlly significnt differences in the urinry biomrker levels between smple type is driven by the fct tht outwith seson bckground levels re lower thn spry eventrelted smple levels nd within seson bckgrounds. The results of the modelling re very similr for chlorpyrifos. Although some of the fctors were significnt (including ge, sex nd time spent outdoors), ccording to the Wld test, they did not significntly improve the fit of the model nd therefore were not included in the finl model. The significnce of the smple type vrible in the model for chlorpyrifos is likely being driven by the levels in the spry event-relted smples being lower thn bckground levels, both outwith nd within. DISCUSSION We report on study tht imed to ssess exposure to pesticides for UK dults nd children living within 100 m from the edge of griculturl lnd, nd identify whether or not exposures were elevted following spry events. A distnce of 100 m ws selected s blnce between proximity to griculturl lnd nd vilble potentil study prticipnts. In ddition, reserchers modelling vpour dispersion reported tht vpour concentrtion is significntly reduced fter distnce of 100 m (dependent on field size) Nture Americ, Inc. Journl of Exposure Science nd Environmentl Epidemiology (2015),

7 628 Tble 7. Description of spry smple results by whether the smple ws the dy fter spry event (24 h) or 2 dys fter (48 h). 24 h Spry smples 48 h Spry smples N NoLOD GM GSD 95th % N NoLOD GM GSD 95th % Chlormequt Chlorpyrifos Abbrevitions: GM, geometric men; GSD, geometric stndrd devition; LOD, limit of detection; N, number; 95%, 95th percentile. Level of Urinry Biomrker Levels The urinry biomrker levels for cptn nd cypermethrin were very low in this popultion with ~ 90% of smples hving undetectble concentrtions. For chlormequt nd chlorpyrifos, the GM urinry biomrker concentrtions following spry events were 15.4 μg/g cretinine nd 2.5 μg/g cretinine, respectively, compred with 16.5 μg/g cretinine nd 3.0 μg/g cretinine for within seson bckground. For chlormequt, we hve only been ble to identify one other, relevnt, study. Lindh et l. 14 reported generl popultion study in southern Sweden where the chlormequt concentrtions rnged from μg/g cretinine (medin 2.9, 95th percentile o17.3 μg/g cretinine, N = 100). These vlues re somewht lower thn our reported concentrtions for ll the chlormequt spry event nd bckground smples combined (medin 15.1, 95th percentile 79.8 μg/g cretinine). Despite these higher concentrtions, UK exposures re two orders of mgnitude below the vlues obtined from n orl dose t hlf the ADI (Lindh et l.). 14 The only other report of mesuring chlormequt in humn urine ws in n ccidentl poisoning cse, 25 but levels were not quntified. Excluding the different time periods of smple collection in our nd Lindh et l. s 14 study, it is possible tht we observed higher chlormequt urinry concentrtions due to different frming prctices for cerel crops nd consumption of foods nd beverges derived from cerel crops for which this growth regultor ws pplied. For exmple, the men dily per cpit consumption for bred nd rolls, bkery products, cerel nd products in the UK ws reported s 103, 44 nd 36 g, respectively, compred with 96, 21 nd 25 g for Sweden. 26 Approximtely 90% of the cptn urinry biomrker concentrtions in this study were below the LOD, with the mximum vlue of 3.9 μg/g cretinine detected in bckground smple. There re very few dt vilble on environmentl exposures to cptn. Berthet et l. 27 reported men pre-seson urine biomrker concentrtions of 0.2 μg/l (~0.15 μg/g cretinine) but this ws bsed on only four smples. Verberk et l. 28 reported results below the LOD (8, ~ 5.9 μg/g cretinine) for six unexposed controls. Recent occuptionl studies hve determined post-exposure THPI levels of o5 μg/l (~3.1 cretinine). 27 No other published generl popultion studies could be found. There is n extensive literture on the use of urinry trichloro-2- pyridinol (TCP) s biomrker of chlorpyrifos exposure. The US CDC NHANES study reports 95th percentiles for dults s 6.4 μg/g cretinine, N = 832 nd 7.4 μg/g cretinine, N = 1,113 for /2000 nd 2001/2002, respectively. 29 A study of 100 pregnnt women in the Netherlnds found 95th percentile of 6.4 μg/l (~4.7 μg/g cretinine) with mximum of 158 μg/l (~116 μg/g cretinine). 30 Studies in Germny 31 nd Itly 32 showed similr vlues. For Germny, the 95th percentile ws 11.3 μg/l (~8.3 μg/g cretinine, N = 50) nd for Itly, the estimted 95th percentile ws 6.5 μg/g cretinine (N = 42). Our dt for ll the spry nd bckground smples combined (95th percentile 10.1 μg/g cretinine) re comprble with these dt from other generl popultion studies, despite the vried geogrphicl sources of the dt sets. A study of residents exposure to chlorpyrifos fter tretment inside the home showed tht potentil exposures to the dult residents, s indicted by urinry 3,5,6-TCP biomonitoring, did not increse s result of the ppliction. 33 Another study reported urinry TCP concentrtions of μg/g cretinine in 41 residents from houses where chlorpyrifos hd been detected in collected indoor ir smples. 34 Alexnder et l. 35 reported study of frm fmily members (spouses, nd children ged 4 17 yers) from Minnesot nd South Crolin. Five consecutive 24-h urine smples were obtined from 34 fmilies of licensed pesticide pplictors from one dy before to three dys fter chlorpyrifos ppliction. The spouses GM exposure ws reported s being 3.6 μg/g cretinine pre-ppliction, 3.8 μg/g cretinine on the dy of ppliction nd then 4.2 μg/g μg/g cretinine on dys 1 nd 2 post ppliction. The reported children s exposure ws 5.1 μg/g μg/g cretinine pre-ppliction, 6.0 μg/g cretinine on dy of ppliction nd 5.0 nd 5.9 μg/g cretinine for dys 1 nd 2 post ppliction. These re ll higher thn the GMs tht we observed. A number of generl popultion studies hve been reported for cypermethrin exposure. The US CDC NHANES study reports 95th percentiles for dults s 0.9 μg/g cretinine (N = 1128) nd 2.5 μg/g cretinine (N = 1123) for cis-dcva nd trns-dcva respectively in 2001/ Detection rtes were reported s less thn 50% for cis- DCVA nd o25% for trns-dcva. A study of 1149 pregnnt women in Chin found medin levels of 0.7 μg/g cretinine for cis- DCVA nd 1.9 μg/g cretinine for trns-dcva. 36 In the UK the 95th percentiles for dults (on the voting register) in the generl popultion were reported s 0.7 μg/g cretinine (N = 405) nd 1.8 μg/g cretinine (N = 404) for cis-dcva nd trns-dcva, respectively nd 2.3 μg/g cretinine combined s totl-dcva. 37 In comprison, the 95th percentile ws 5.8 μg/g cretinine for the spry event smples nd 5.2 μg/g cretinine for our within spry seson smples (which includes both dults nd children). Although the 95th percentile results for our dt re greter thn reported by Bevn et l., 37 the number of smples bove the LOD ws fr lower despite the sme detection limit. These differences my reflect regionl differences in exposure (our study ws conducted in three regions compred with cross the whole of the UK for Bevn et l. 37 ) or temporl differences in pesticide use nd food residues of pyrethroids (our smples were collected in 2011 nd 2012 wheres those of Bevn et l. 37 were collected in 2005/06). Do Spry Events Result in Elevted Exposures to Residents Living Ner Agriculturl Lnd? The results presented in this pper provide no evidence tht, in this study popultion, the spry events resulted in elevted exposures compred with bckground smples tken within the seson. For chlormequt, the biomrker levels (both spry event nd bckground) were higher within the spry seson compred with outwith the seson. Chlormequt is plnt growth regultor, which cts by inhibiting cell elongtion hence shortening nd strengthening the stem producing sturdier plnt. It lso influences the developmentl cycle, leding to incresed flowering nd hrvest. 38 Growth regulting products contining chlormequt re widely used by the UK. The Pesticide Usge Survey Tems of the Food & Environment Reserch Agency (FERA) conducted surveys of pesticide usge in rble crops in 2011/12 Journl of Exposure Science nd Environmentl Epidemiology (2015), Nture Americ, Inc.

8 by visiting holdings throughout the UK. In this survey they reported tht chlormequt, pplied lone or in mixtures, ccounted for 59% of the re of rble crops treted with specific growth regultors: in ddition there ws 13% increse in re treted using chlormequt since the previous survey conducted in Although not the primry im of the study nd reporting on different pesticides, Jones et l. 40 reported sttisticlly significnt difference for dilkylphosphte levels (orgnophosphte) in young children ( o5 yers) during different sesons, with utumn resulting in the highest levels. No sesonl effect ws however observed for pirimicrb or crbryl. Our chlormequt findings could be due to other sources of exposure. The most recent publiction presenting results of pesticide residues in food commodities (including both rw nd processed) smpled during the clendr yer 2010 in the 27 Europen Union Member Sttes nd two Europen Free Trde Assocition countries (Icelnd nd Norwy) reported chlormequt/ots to be the pesticide/crop combintion for which residue concentrtions were most frequently bove the reporting level (64.6% of the smples). In ddition, the highest percentge of mximum residue limit (MRL) exceednces in foods ws found for chlormequt in ots (8.1% of ll smples). In rye, the most frequently found pesticide residue ws lso chlormequt (35.9%). 41 Of the 178 pesticides included in the 2010 EU-coordinted progrmme, the most frequent MRL exceednces were detected for chlormequt (3.6% of the smples). Chlormequt ws lso detected in smll number of orgnic food smples nlysed (13 out of the 3571 smples), with mesured residue levels rnging from mg/kg. 41 Whilst in most instnces these dt relte to unprocessed food commodities nd residue levels my decrese during food production, it is considered tht diet is the primry source of exposure. For chlorpyrifos, the biomrker levels were very similr for the vrious smple types. Finlly, for cptn nd cypermethrin very lrge proportion of the mesurements were below the LOD, whether or not these smples were collected following spry events. The reltively short biologicl hlf-life of pesticide compounds nd their biomrkers in the humn body presents mjor chllenge to linking biologicl monitoring dt to specific spry events nd so urine smples hve to be collected idelly within 24 h, nd no lter thn 48 h following the spry events (depending on the urinry biomrker hlf-life). Frming ctivities, nd in prticulr spying with pesticides, re inherently unpredictble becuse of the chnging wether nd the presence of insects or other potentilly dmging infesttions which influence pesticide selection nd ppliction. Along with the plnned weekly urine smple collections, there ws need for effective enggement with frmers to elicit this spry informtion t short notice nd with residents to obtin the required spry event smples rectively. Through the use of community reserchers, locted in nd knowledgeble of the geogrphicl res nd fmilir with locl frming prctices, effective enggement ws chieved nd over 3000 urine smples were collected. 11 The methodology pplied in this study is robust but is not without some limittions. The prticipting frmers my not be representtive of ll frmers within the study res lthough there is no reson to suggest tht their sprying prctices re ny different to those of the wider frming communities. In the UK, Government Ministers must pprove ll pesticides before they cn be mrketed or used nd everyone who uses pesticides must hve dequte guidnce, instruction or trining for their correct use nd must ensure tht ll resonble precutions re tken to prevent spry drift. 12 It ws cler from the frmers spry records tht number of pesticide products were pplied throughout the sprying seson (dependent on crops, infesttion, wether conditions nd other fctors) nd so the number of relevnt spry events observed my differ from yer to yer. Spry event strt nd finish times were obtined nd residents were sked to provide detils of their ctivities in the 48 h period before provision of ech urine smple. However, it ws not possible to estblish from this when, where or how the residents potentil exposure to the ssessed pesticides my hve occurred. Following due considertion of preliminry modelling nd the peerreviewed literture, 13 first-morning void smples were requested from prticipnts. There is the possibility tht the urine void with the highest biomrker concentrtion ws missed however no significnt reltionship between biomrker concentrtions nd timing of spry event ws found for ny of the pesticides. Wether informtion, whilst obtined by both the frmer nd the reserch tem, ws typiclly reported from wether sttions locted in some instnces severl miles wy from where the spry took plce. It is possible tht the vilble wind speed nd direction informtion my not reflect wht ctully occurred during the given spry event nd so ws not considered further. Good spry event informtion ws obtined from the frmers prticipting in the study. However it is not possible to estblish whether residents were potentilly exposed to ny dditionl relevnt spry pplied by non-prticipting frmers in the loclity. Given the scope of the study, the number of dietry-relted questions ws kept to minimum, restricted to the consumption of food from the residents grden nd orgnic food (s reported in the bckground questionnire). In retrospect, the inclusion of dditionl food-relted questions my hve been useful in explining some of the results observed. Residents (dults ged 18 yers nd over nd children in their cre ged 4 12 yers) were recruited, with infnts ( o4 yers old) nd dolescents (13 17 yers) being excluded from the study. This prgmtic decision ws tken due to likely difficulties being encountered with obtining relevnt urine smples from bbies nd toddlers nd dolescents potentilly being less engged with the study nd so the exposures of these sub groups ws not determined. All lbortory methods for the urine smples showed good dyto-dy repetbility nd were bsed, where possible, on vlidted methods (see Supplementry Informtion) with comprble LODs to those lredy reported nd used in environmentl exposure studies. Where known, the primry or most bundnt biomrker ws mesured; mesuring multiple biomrkers for the sme pesticide would not hve incresed the sensitivity of mesurement unless ll metbolites re converted to single biomrker. All chlorpyrifos nd chlormequt smples were nlysed within the timefrme of the stbility trils. The length of ech nlyte stbility tril ws determined by vilble smples within the dt set. Some smples for cptn nd cypermethrin were nlysed outside of the evluted stbility time frme. These smples hve not necessrily degrded but extended smple stbility ws not evluted due to insufficient smple volume. It is considered tht the biomrkers nd methodology used were demonstrbly fit for purpose. When considering the generlisblity of the study results to other pesticides, considertion should be given to the spry techniques used nd their potentil to distribute the pesticide beyond the trget re nd the propensity for the pesticide to redistribute post ppliction. The spry equipment nd techniques used by the prticipting frmers were not typicl nd were reportedly used when pplying other pesticide products to the given crops throughout the spry seson. The rnge of vpour pressures for the relevnt products considered in this study rnged from to 1.43 mp t 25 C, of which the highest ws for chlorpyrifos. Monitoring in the USA suggests tht chlorpyrifos is the worst known cse for vpour concentrtions 42 nd so we consider tht our study covers both the likely spectrum nd worst cse vpour pressures used in modern dy pesticides in the UK (nd perhps more widely). Orchrd spry techniques re usully considered s potentilly giving rise to higher levels of pesticide drift (nd therefore potentil exposure) in comprison to field crop Nture Americ, Inc. Journl of Exposure Science nd Environmentl Epidemiology (2015),

9 630 sprying prctices. For exmple, mesurements of bystnder exposure during UK field crop sprying nd orchrd sprying pplictions hve been reported. 43,44 For boom spryers the verge potentil derml exposure (PDE) for bystnder, positioned 8 m downwind from the spryer nd the verge mount of spry potentilly inhled in the brething zone were 0.1 ml spry/person nd ml spry/person, respectively. For orchrd spryer pplictions the equivlent PDE nd inhltion vlues were 3.7 ml spry/person nd ml spry/person. Given tht we collected in excess of 300 orchrd spry event smples, which lso included those relted to chlorpyrifos, we consider tht our study dequtely considers potentilly higher risk spry techniques, of which the mjority of the urinry biomrker results were observed to be less thn the nlyticl LOD. CONCLUSION This study reports urinry biomrker concentrtions for number of ctive ingredients both for spry event nd bckground smples mongst people living within 100 m of griculturl lnd, which hs not previously been undertken nd reported on such scle for UK popultion. It hs lso, s fr s we re wre, not been studied s systemticlly nywhere else. Our study did not set out to determine whether rurl residents hve greter exposure to pesticides thn non-rurl residents nd so is not directly comprble to other studies tht my hve sought to do this. The primry conclusion of the study is tht there is no evidence of incresed pesticide biomrker excretion in rurl residents following spry event within 100 m of their home, when compred with the urinry biomrker levels obtined when relevnt spry events did not tke plce. The levels of urinry biomrkers detected in our popultion were generlly comprble to other studies of exposure in the generl popultion, where such dt re vilble, nd this supports the view tht generl popultion exposures to pesticides re primrily from non-spry event sources such s diet. It ppers tht the popultion recruited to this study exhibits greter exposure to chlormequt thn detected in Swedish popultion sub-set. Whether this is chrcteristic of our prticulr rurl residents or is more widely pplicble to the entire UK generl popultion is not known nd my be n re of further work. Exposure levels for cypermethrin nd cptn were very low. CONFLICT OF INTEREST Dr. Gle is member of the UK Government Pesticide Incidents Apprisl Pnel nd Dr. Cocker ws member of the UK Government Advisory Committee on Pesticides (Jnury 2009-December 2014). The uthors declre no conflict of interest. ACKNOWLEDGEMENTS This project ws funded by the UK Government Deprtment for Environment Food nd Rurl Affirs (DEFRA), project code PS2620. We thnk the following individuls (in lphbeticl order) for their work s community reserchers in this project: S Attwood, J Cdzow, A Crr, D Dhrendorf, T Hogg, L Jewsbury nd F McIver. Their hrd work ws crucil to the success of the recruitment nd dt collection in this project. Thnks lso to the independent project Advisory Committee (Prof. Dvid Coggon, Pul Hmey, Prof. Len Levy nd Dr. Sen Semple) for their helpful comments throughout the project nd on erlier mnuscript drfts; Dr. Anne Sleeuwenhoek nd Dvid Todd (IOM) for their ssistnce with the storge of the urine smples nd relted dministrtion tsks; IOM personnel (Anne Sleeuwenhoek, Shhzd Rshid, Peter Ritchie, Yvonne O Neill, Mrlyn Dvis, Julie O Neill, Selim Argoub nd Arceli Snchez Jimenez) for ssistnce with dt storge, dt input nd other dministrtive tsks; nd HSL personnel (Drs Crig Sms, Lur Kenny, Shhwiz Iqbl nd Fion Grner) for the urine smple nlysis. We express our sincere grtitude to ll the frmers nd householders who prticipted in the study. REFERENCES 1 RCEP. Crop Sprying nd The Helth of Residents nd Bystnders. Royl Commission on Environmentl Pollution: London, UK, ACP. Crop Sprying nd the Helth of Residents nd Bystnders. 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