bees&pesticides sos final may2012

7/31/2019 Bees&Pesticides SOS FINAL May2012

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Pesticides and Honey Bees:

State of the Science

MAY2012

PESTICIDEACTIONNETWORKNORTHAMERICA


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Pes7cideAc7onNetworkNorthAmerica

PesticideActionNetworkNorthAmerica(PANNorthAmerica)workstoreplacetheuseofhazardouspesticideswithecologicallysoundandsociallyjustalternatives.

Asoneof?ivePANRegionalCentersworldwide,welinklocalandinternationalconsumer,labor,health,environmentandagriculturegroupsintoaninternationalcitizensactionnetwork.Thisnetworkchallengestheglobalproliferationofpesticides,defendsbasicrightstohealthandenvironmentalquality,andworkstoensurethetransitiontoajustandviablesociety.

WrittenbyHeatherPilatic,PhDwithresearchassistancefromPANNAinternsJuliChamberlainandNevaJacobs.ColleaguesatBeyondPesticidesprovidedimportantannotations.Dr.SusanKegley,consultingscientistforPANNA,andDr.JamesFrazier,professorofentomologyatPennsylvaniaStateservedashelpfulreadersofearlydrafts.PANNAcolleaguesKristinSchaferandSaraKnightcontributed

carefulcopyediting.Errorsbelongtothemainauthoralone.SupportforthisworkprovidedbytheCeresFoundationandPANNAsupportingmembers.

Pesticide Action Network

49PowellStreet,Suite500Sanfrancisco,CA94102

Tel:415-981-11::Fax:415-981-1991

www.panna.org::www.pes7cideinfo.org
http://www.pesticideinfo.org/http://www.pesticideinfo.org/http://www.panna.org/http://www.panna.org/


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TableofContents

....................................................................................................................Introduc*on 1

...........................................................Overview::ColonyCollapseDisorderincontext 2

.....................................Understandingpes*cidesasacausalfactorincolonycollapse 4

.................................................................................................................Pes*cidePrevalence 4

...............................................................Neonico*noids::Acute,sub-lethal&chroniceffects 6

...............................................................................................Synergis*c+CombinedEffects 11

......................................Chemicalcocktails::Fungicides,pyrethroidinsec;cides,mi;cides 11

.........................................................................Pathogeninterac;ons::Nosema+pes;cides 13

.............................................Microbiotaoutofbalance::Gutcultures,immunity+nutri;on 15

.....................................................................................................ResearchChallenges 17

...........................................................................................Equipment+detec*onsensi*vity 17

............................................................................................................................Studydesign 18

......................................................................................................Laboratoryvs.fieldstudies 18

....................................................................................................Mul;pleexposurepathways 19

..........................................................................................................................Time+Timing 22

..............................................................................................................StructuralBias 23

......................................AppendixA::Neonico*noidusepaTernsinU.S.agriculture 24

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Introduc7on

Honeybeesandotherpollinatorsaredyingoffatunprecedentedratesaroundtheworld.FirstinFrance,thenintheU.S.andelsewhere,colonieshavebeenmysteriouslycollapsingwithadultbeesabandoningtheirhives.In2006,twoyearsafterthisphenomenonhittheU.S.,itwasnamedColonyCollapseDisorder,orCCD.Eachyearsince,U.S.beekeepershavereportedannualhivelossesof29%-

36%.Commercialbeekeeperstellusthattheirindustry,whichisthecareandcultivationofanindicatorspecies,isonthevergeofcollapse.

Honeybeespollinate71ofthe100mostcommoncropsthataccountfor90%oftheworldsfoodsupply,makingmanagedhoneybeesthemosteconomicallyimportantpollinator.iIntheU.S.alone,2000datafromtheU.S.DepartmentofAgriculture(USDA)indicatesthatthisindustrywasworthmorethan$15billionperyeartwelveyearsago.iiWiththesubsequentgrowthofU.S.agricultureanddeclineofnaturalpollinators,commercialbeekeepinglikelycontributesmuchmorethan$15billiontotheU.S.economynow.

Claimsofimminentfoodsystemcollapsearenotsupportedbypollinationbiologists,butwithoutpollination,agriculturequicklybecomeslessef?icientrequiringmorelandandwatertogrowthesameamountoffoodandourdietslosenutritionallyvitalvariety.Forinstance,mostfruitsandmany

nutsrelyonthepollinationservicesofbees,includingalmonds,blueberries,applesandmelons.Alfalfa,whichisaprimaryfeedsourcefordairycows,reliesonbeesforpollinationaswell.Altogether,morethanoneineverythreebitesoffooddependsonhoneybeesforpollination.

Whilefewcontestthattherecent,dramaticdeclineofhoneybeepopulationspresentsseriouschallengestoanalready-stressedfoodsystem,thepublicdebateoverwhatliesbehindCCDisatthispointsopolarizedandconfusingthatconcernedcitizens?inditdif?iculttoknowhoworwheretointervene.Indeed,thedebateoverthecausesofCCDhasbecomeacasestudyinpublic,scienti?iccontroversyiii.Aswithotherrecentmattersofpublicandenvironmentalhealth,suchasthelinkbetweentobaccoandcancerandanthropogenicclimatechange,thisissuehasbecomecharacterizedbypolicymakerinactioninthefaceofirreduciblycomplexscience.

Twoincreasinglyintractablesideshaveemergedinthiscontroversy:beekeepersandenvironmental

healthadvocatesvs.pesticidecompaniesandthescientistssupportedbythem.WhilePANNAspositioninthisline-upisclearenough,wehavesoughttohewtoacommitmenttonon-partisan,scienti?icallyliteratepublicdiscourse.Webelievethatengagedformsofscienti?iccitizenshipareavitalpartofdemocraticciviclife,andaneededforceinenvironmentaldecision-making.Incasessuchasthiswherethedebatehasbecomeintractableandposition-driven,asisreportedbyparticipantsandscholarsalike,theconversationbetweenexpertshasclearlybrokendown.Historically,thesekindsoflogjamsarebrokeneitherthroughconcertedpublicdemand,orthroughacatastrophicfocusingortriggeringeventthatcompelspolicymakeractionorbothinconjunction.With

one-thirdofourbeesdyingoffeachwinterandwildpollinatorsfacingsimilarlycatastrophicdeclines,itwouldseemthatwehavebeforeusthefocusingevent.Needednowispublicdemandforpolicyaction.

Thus,thepurposeofthisdocumentistoinformpublicdebateandbuildnationalwillforpolicyactiononatimelinethatwillbemeaningfultobeesandbeekeepers.OurconsiderationoftheevidenceforthecausesofbeedeclineandCCDisfocusedonpesticidesasoneofthreeleadingfactorsidenti?iedbyresearchers.Byouranalysis,theweightofevidencedemonstratesthatpesticidesareindeedkeyinexplaininghoneybeedeclines,bothdirectlyandintandemwiththeothertwoleadingfactors,pathogensandpoornutrition.Thesciencesupportingthisconclusionispresentedinthisreport.

The weight of evidence demonstrates

that pesticides are indeed key inexplaining honey bee declines, bothdirectly and in tandem with the other

two leading factors, pathogens andpoor nutrition.

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Overview::ColonyCollapseDisorderincontext

HoneybeepopulationshavesteadilydeclinedintheU.S.since1947atagradualrateaveraging1%peryeariv.Steeperdeclineshavebeenrecordedsince1987,butthelastfourwintershaveseenextraordinarylossesaveraging29to36%peryear.v,vi,vii,viii

MostscientistsagreethatthereisnosinglecauseofCCD.Rather,recentpopulationdeclinesarelikelycausedbyacombinationoffactorsactinginconcerttoweakenbeecoloniestothepointofcollapse;ixandemergingsciencepointsspeci?icallytoimpairedimmunity.Leadsuspectsinthiscausalcomplexinclude:nutritionalstress,pathogensandpesticides.

Nutritionalstress::Nutritionalstressunderminescolonyhealththroughavarietyofmechanisms,includingimmunesystemharmandreductioninreproductiveviability.Onekeycomponentofnutritionalstressforhoneybees

includeshabitatlossthatresultsinalessvariedandthereforelessnutritiousdiet.Habitatlosshasbeenoccurringsteadilyforthelast50yearswithmeasurableeffectsonbeehealth.Forinstance,regionaldifferencesinratiosofopentodevelopedlandhavebeentracedtohighercolonylosses.Onekeydriverofrecenthabitatlossistheincreaseduseofbroad-spectrumherbicidesthataccompaniesherbicide-resistant,geneticallyengineeredcrops.x

Pathogens::Pathogenslikeparasiticmites,virusesandagutfungushavegarneredthemostmediaattentionascausalfactorsinCCD.Multiplestudieshavecon?irmed,however,thatthereisnosinglepathogenassociatedwiththedisorder.Inananalysisofstudiespublishedasofearly2009,twoleadingU.S.researchersnotedthatnosinglepathogenfoundintheinsectscouldexplainthescaleofthedisappearance.Inotherwords,thebeeswereallsick,buteachcolony

seemedtosufferfromadifferentcombinationofdiseases.xiParasiticmitesofthegenusVarroaarethemostimportantpesttohoneybeesgloballyxiiandactasvectorstotransmitanumberofvirusesthatsigni?icantlyweakencolonies.Deformedwingvirusandatrioofrelatedparalysisviruseshavealsoemergedasimportanttocolonylosses,ashasafungalgutpathogenofthegenusNosema.Emergingmicrobiotaresearchpointstothepossibledisruptionofnormal,symbioticbeegutculturesbyacombinationofstressorsresultinginincreasedsusceptibilitytopathogens.

Pesticides::Pesticideshavebeenknowntocauselarge-scalebeedeathssincetheearly1900s,manythroughdirectpoisoningduringaerialsprays.Thesetypesofacutebeedie-offsarenotatissueinCCD,althoughtheydostillhappen.Regulationsandphase-outsofacutelytoxicpesticideshavereducedthenumberofacutepoisoningsinmostofEuropeandNorthAmerica,

butbeeexposuretomultiplepesticidescontinues.Sub-lethaleffects,lessstudiedandunderstoodthanacuteeffects,havebecomeakeyconcernassystemicneonicotinoidpesticidespresentinsmallamountsthroughoutplanttissuesfromseedtoharvesthavebecomeanimportantandrapidlygrowingsegmentoftheglobalinsecticidemarketsincetheirintroductioninthe1990s.xiiiOtherpesticidesofconcernincludethoseusedbybeekeeperstocontrolpathogens,andcertainfungicidesthoughttobesafeforbeeswhichhaverecentlybeenfoundtoactsynergisticallywithsomeneonicotinoids,increasingthelatterpesticidesbeetoxicityby200-to1,000-fold.xiv

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WhatisColonyCollapseDisorder?

ColonyCollapseDisorder,orCCD,wasfirst

describedintheU.S.in2006.Itssymptomsare

dis*nctfromotherlossepidemicsandinclude

thefollowing:

Coloniesfoundsuddenlyemptyofadult

bees,leavingtheirbroodunaTended

Nosignofdeadbees

Nohivepestsorfoodrobbers,despite

surplushoneyandpollenstores

Commonparasitesnotpresentatlevels

thoughttocausepopula*ondecline


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ThefollowingfourstudiesprovideanoverviewofthefactorsinvolvedinrecenthoneybeedeclinesaswellasadescriptionofColonyCollapseDisorder.

1)vanEngelsdorpD,Meixner,MD.2010.AhistoricalreviewofmanagedhoneybeepopulationsinEuropeandtheUnitedStatesandthefactorsthatmayaffectthem.JInvertebrPatholology103:S80-S95.

Thisarticleisthemostrecentandcomprehensivereviewofhistoricallosspatternsandthedatagapsthatmakethesepatternsdif?iculttodescribe.Researchersreviewed110yearsofcensussurveys,experimentalstudies,technicalreportsandreviewstudiestoarriveatananalysisofglobalpopulationtrendsandthefactorsthataffectmanagedhoneybeepopulations.Variousdatainconsistenciesnotwithstanding,theydeterminedthatpopulationshaveincreasedworldwideoverthelast50years,butseveredeclineshaveoccurredintheU.S.(61%),Mexico,andEurope(27%continent-wide)inthesametimeframe.Inallofthesedeclines,severalinteractingfactorsarelikely.Theseincludelong-termreductionsinbeeforaginghabitat,changingweatherpatterns,acontractionofthegenepool,weakqueenbees,pathogens,pesticidesandsocioeconomicfactorsthataffectbeekeepingpopularityandpro?itability.

2)vanEngelsdorpD,EvansJD,SaegermanC,MullinC,HaubrugeE,NguyenBK,etal.2009.Colonycollapsedisorder:Adescriptivestudy.PLoSONE4(8):e6481.

Thisstudylookedat91managedhoneybeecoloniesfrom13apiariesinCaliforniaandFloridatocon?irmthede?initionofCCD,identifyitspotentialcausesandinformfutureresearch.Over200variableswerequanti?iedandcomparedbetweenCCD-af?lictedcoloniesandapiaries,andthosenotaf?licted.While61ofthe200variableswerefoundfrequentlyenoughtomakeworthwhilecomparisons,nosingleonestoodoutasbeingsigni?icantlylinkedtoCCD.Thecomparativeresultsdidsuggestsomeimportanttrends:1)compromisedimmunityislikelyplayingarole;and2)CCDiseitheracontagiousconditionorcausedbyexposuretoacommonriskfactor.Recommendationsforfutureresearchinclude:1)longitudinalstudiesthatmonitorparasite,pathogenandpesticideloadswhilequantifyingpesticidetoleranceinthestudypopulations;2)studiesontheinteractionsamongpesticidesandpathogenloads.

3)vanEngelsdorpD,SpeybroeckN,EvansJD,NguyenBK,MullinC,FrazierM,etal.2010.Weighingriskfactorsassociatedwithbeecolonycollapsedisorderbyclassi@icationandregressiontreeanalysis.JEconEntomol103(5):1517-1523.

TobetterunderstandtherelativeimportanceandrelationshipsamongdifferentriskfactorsinexplainingCCD,researchersperformedaclassi?icationandregressiontree(CART)analysisonthedatasetoftheaboveepidemiologicalstudy(vanEngelsdorpetal.2009).Thiswasthe?irstcaseofCARTanalysisbeingusedtounderstandbeepathology.Overall,theresultsoftheanalysisprovidefurtherevidencefortheprevailingconsensusthatCCDiscausedbymultiplefactorsactingtogethertodecreasecolony?itnessandincreasesusceptibilitytodisease.Pesticidesaccountedfor6ofthe19variableshavinggreatestdiscriminatorypower.Ofnoteisthefactthat,atalevelabove66ppbindevelopingbees,theVarroamiticide,coumaphos,waspositivelycorrelatedtohealthycolonies;thiswasthemostpredictivefactorbetweenthetwo

populations.ResultsindicatethatpesticidesareverylikelyinvolvedintheCCDcausalcomplex.Amongseveralareasrecommendedforfurtherstudy,twowerehighlighted:theeffectofsub-lethalpesticideexposureonpathogenprevalence,andtherelationshipbetweenvaryingtolerancetopesticidesandcolonysurvival.

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Understandingpes7cidesasacausalfactorincolonycollapse

Pes$cidePrevalence

Pesticidesareaprominentpartofthehoneybeeenvironment,bothinthehiveandinthelarger

environment.ThisisespeciallytrueintheU.S.,where1,200differentpesticideactiveingredientsareapprovedandinusein18,000differentproductcombinations.Bycontrast,FranceandBritainhaveeachregisteredaround500and300,respectively.xvAtleast143millionofthe442millionacresofU.S.croplandisplantedwithcropstreatedwithoneofthreeneonicotinoidpesticidesknowntobehighlytoxictobees:clothianidin,imidaclopridand/orthiamethoxam.Thisisaconservativeestimatederivedfromcross-referencingUSDAARSdata,industryreportsandavailablepesticideusedata.Itdoesnotbegintoaccountfornon-agriculturaluses.(SeeappendixAformorecompleteusedataandanalysis.)

MultiplesurveysintheU.S.andEuropehaveshownthatamixtureofpesticideformulationsandtypesarepresentinbees,wax,storedfoodandthepollenandnectaronwhichbeesforage.Fieldstudieshavefoundneonicotinoidpesticidesinparticularinsoil,dust,planterexhaust,water(guttation)dropletsexudedbytreatedplantsandonnearby,untreatedplantsand?ields.Across-sectionofthesestudiesfollows.

1)KrupkeC,HuntG,EitzerB,AndinoG,GivenK.2012.Multipleroutesofpesticideexposureforhoneybeeslivingnearagricultural@ields.PLoSONE7(1).

This?ieldstudyestablishedthatbeesnearagricultural?ieldsareexposedtoavarietyofpesticidesviamultipleroutesatharmfullevelsthroughouttheforagingperiod.Pesticidesfoundincludetheneonicotinoidsclothianidinandthiomexotham,atrazine(anherbicide)andfungicides,includingoneknowntosynergizewithneonicotinoids(propiconazole).Soils,pollen(bee-collectedanddirectlyfromplants),dandelions,deadandhealthybees,andplanterwasteproductswereallexaminedaspotentialexposureroutes.Theauthorslookedspeci?icallyatcorn,whichoccupiesmorearablelandinNorthAmericathananyothercrop88millionacres.CornisplantedthroughouttheU.S.Midwest

frommid-AprilthroughearlyMaywhentheenergeticrequirementsofbeesareincreasingrapidlyashivesprepareforcolonygrowth,requiringincreasedforaging.Virtuallyallcorn(exceptingthe0.2%cultivatedorganically)intheU.S.isgrownfromtreatedseeds,andthisstudyfoundthatbeesforageheavilyoncorn:cornpollenmadeupover50%ofthepollencollectedbybees,byvolume,in10of20samples.Authorsalsosampleddandelions,whichareapreferrednectarandpollensourceduringthisperiod.Dandelionsinnearby,untreated?ieldswerecontaminatedwithclothianidin.Soilfrom?ieldswhichhadnotbeenplantedwithtreatedseedsinovertwogrowingseasonstestedpositiveforclothianidinaswell,whichauthorsinterpretedasafeatureofthechemicalspersistenceandmobility.

Pollencollectedfromthetreatedplantswascontaminatedbyclothianidinasexpected,butbee-collectedpollensamplesshowedhigherlevels,indicatingadditionalpathwaysofexposure.Levelsofcontaminationinbee-collectedcornpolleninthisstudywere10-foldhigherthanreportedfromanexperimentonclothianidin-treatedcanola.Thisissigni?icantbecauseclothianidinwasapprovedforuseoncornandcanolasimultaneouslybasedonthecanola?ieldtest.The?indingthatbee-collectedpollencontainedneonicotinoidsisofparticularconcernbecauseclothianidinisevenmoretoxicwheningestedorallybyabee,andbecauseofthepotentialforharmwhen

!

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developingbeesareexposedtopesticideswithinthehivethroughstoredpollen.Duringtheperiodobserved,nursebeeswereemergingandfedonpollenreservesintheformofroyaljelly.Authorscalculatedthat,atthelevelsobserved,anewbeewouldconsume50%oftheoralLD50duringthe10daysitspendsasanursebee.

Insum,thestudyestablishedmultipleexposureroutes,atharmfullevels,atacriticaltime,onthemostcommoncrop.Theauthorsmostsalient?inding,however,istheestablishmentofanew,especiallymobileandtoxicexposurerouteplanterexhaustmaterial.Cornseedsaresownusinganautomatedplantingsystemthatreliesonair/vacuummechanismstospacetheseeds;inordertokeepseedstreatedwithpesticidesfromstickingtooneanother,talcisused.Thistalcbecomescontaminatedandisthenexhaustedduringplanting,eitherdownwiththeseedorintotheair.Authorsfoundextremelyhighlevelsofneonicotinoidsand

fungicidesinplanterexhaustmaterial.

2)MullinCA,FrazierM,FrazierJ,AshcraftS,SimondsR,vanEngelsdorpD,etal.2010.HighlevelsofmiticidesandagrochemicalsinNorthAmericanapiaries:implicationsforhoneybeehealth.PLoSONE5(3):e9754.

ResearchersconductedthemostextensiveNorthAmericansurveyofpesticideresiduesinmanagedhoneybeecoloniestodatein23statesandoneCanadianprovinceduringthe2007-2008growingseason.Theyusedconventional(gaschromatography-massspectrometry)andrecentlydeveloped(liquidchromatography-tandemmassspectrometry)analyticaltechniquestodetectanyof200pesticidesandtheirmetabolitesatconcentrationlevelsaslowas0.1ppbinarepresentativecross-sectionofbees,pollenandwax.Atotalof121pesticidesandmetabolitescomprising5,519totalresiduesweredetectedandquanti?iedin887samples.Waxsamplesaveraged8,pollensamplesaveraged7.1,andbeesamplesaveraged2.5differentpesticideresidueseach,withatleasttwopesticidesdetectedin92%ofallsamplesanalyzed.Pyrethroids,agroupofwidelyusedpesticidesthataretoxictobees,werethedominantclassofinsecticidesdetectedinallsamples.Nearlyhalf(49.9%)ofallsamplescontainedatleastonesystemicpesticide.Pollencontainedhighlevelsoffungicides,whichtendedtoco-occurwithlowlevelsofsystemicpesticides,implicatingpossiblesynergisticeffects.

TheauthorsconcludedthatThewidespreadoccurrenceofmultipleresidues,someattoxiclevelsforsinglecompounds,andthelackofanyscienti?icliteratureonthebiologicalconsequencesofcombinationsofpesticides,arguesstronglyforurgentchangesinregulatorypoliciesregardingpesticideregistrationandmonitoringproceduresastheyrelatetopollinatorsafety.Thisfurthercallsforemergencyfundingtoaddressthemyriadholesinourscienti?ic

understandingofpesticideconsequencesforpollinators.

3)vanEngelsdorpD,EvansJD,DonovallL,MullinC,FrazierM,FrazierJ,etal.2009.Entombedpollen:anewconditioninhoneybeecoloniesassociatedwithincreasedriskofcolonymortality.JInvertebrPathol101(2):147-149.

TwoU.S.longitudinalstudiesthatsoughttouncovercausesofpoorcolonyhealth(andCCDinparticular),werebeguninspringof2007.Duringbothstudies,researchersdiscovereda

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phenomenonknownasentombedpollen,inwhichbeesresponsibleformanagingfoodstoresinthehivesealoffsomepollenwithpropolisandwax.Thisbehaviorisgenerallyusedbybeestoquarantinemicrobialthreats,suchasaninvadinglizardormousethatdiesinsidethehive.Entombedpollenhadmuchhigherlevelsofthreespeci?icpesticides(twomiticidesandonefungicide)thantypicalpollen,and

notablyhadnodetectablemicrobes.Hiveswithsuchpolleninlatespringweretwiceaslikelytodieinmid-fallashiveswithnormalpollen.Incidenceofentombedpollenwasnotablygreaterinreusedwaxcombregardlessofanydisinfectiontreatmentsapplied,whichsuggeststhereisatransmittablefactorcommontobothentombingbehaviorandcolonydeath.Researchersurgedfurtherstudy.

4)BonmatinJM,MarchandPA,CharvetR,MoineauI,BengschER,ColinME.2005.Quanti@icationofimidaclopriduptakeinmaizecrops.JAgrFoodChem53:5336-5341.

ResearchersinFranceconductedathree-year,random-sample?ieldstudyofenvironmentalcontaminationbythemostprevalentneonicotinoid,imidacloprid,incorn?ieldswithactivebeecolonies.Thisstudywaspartofanationalresearcheffortseekingtoevaluatetheenvironmental

riskofimidaclopridtohoneybees.Usingarecentlydevelopedmethodcapableofquantifyingimidaclopridat1ppbanddetectingitat0.1ppb(highpressureliquidchromatographymasstandemspectrometry,HPLC/MS/MS),researchersestablishedthatimidaclopridishabituallypresentin?lowering,treatedcornatlevelsknownfrompreviousdosestudiestoinduceavarietyofharmfuleffects,includingeventualdeath,forhoneybees.Averagelevelswere6.6ppbforpollen-producing?lowers,4.1ppbforstemsandleaves,and2.1ppbforpollenitself.Theselevelsaresimilartothosepreviouslystudiedforsun?lowerandcanola.Imidacloprid-contaminatedcornpollenmadeup54%ofpollensamplescollectedathiveentrances,re?lectingamixtureofpollensourcesandwithacorrespondinglyloweraveragecontaminationof0.6ppb.

Neonico$noids::Acute,sub-lethal&chroniceffects

Neonicotinoidsarearelativelynew,andverywidelyusedclassofinsecticidesthatworkonthecentralnervoussystemofsuckinginsectssuchas?leasandaphids.xviTheywereintroducedinthe1990sandhavesincebecomethefastest-growingclassofinsecticidesinthehistoryofsyntheticpesticides.By2005neonicotinoidshadgaineda16%totalmarketshareofthenearly8billionglobalmarket,andanear-lock(77%)ontheglobalseedtreatmentmarketwhichitselfgrewfromaniche155milliontoa535millionmarket.xviiAmongtheirapprovedusesintheU.S.aretopical?leatreatmentsforpets,lawnandgardenuses,andavarietyofagriculturalusesincludingstonefruits,nuts,canola,sun?lowersandcorn.

The presence of entombing is thebiggest single predictor of colonyloss. It's a defence mechanism thathas failed."

- Dr. Jeffrey Pettis, in theUK Guardian

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Table 1 :: Concentrations of highly toxic neonicotinoid insecticides known to cause harm to honey bees.Sources :: EPA; Aliouane et al. 2009; Iwasa et al. 2004; Krupke 2012.

Toxicity to

bees

Oral acute toxicity

48-hr LD 50

(a.i./bee)

Contact acute

toxicity 48-hr LD

50

(a.i./bee)

Sub-lethal

effects range

(a.i./bee)

Persistence

(half-life in soil)

Clothianidin Highly 2.8 - 3.79 ng/bee 22-44 ng/bee 24 ng/bee 148 1,155 days

Imidacloprid Highly 3.8 ng/bee 78 ng/bee 24 ng/bee 40 - 997 days

Thiamethoxam Highly 5 ng/bee 24 ng/bee 50 ng/bee 25 - 100 days

Dinotefuran Highly 7.6 - 23 ng/bee 24 - 61 ng/bee Unknown 138 days

Neonicotinoidsfallintotwosubclasses:nitroguanidinesandcyanoamidines.Thenitroguanidines,whicharehighlyacutelytoxictohoneybees,includeimidacloprid,clothianidin,thiamethoxamand

dinotefuran.Thecyanoamidinesarenotasacutelytoxictohoneybeesandincludethiaclopridandacetamiprid.Neonicotinoidsareknowntopersistinsoilforyearsandhavethepotentialtoaccumulateinsoil.Thesechemicalsarealsohighlywatersoluble,andarepresentthroughouttreatedplantsfromseedtoharvest.

Todate,mostU.S.regulatorydecisionmakingaddressingtherisksposedtohoneybeesbyneonicotinoidshashinged,bydefault,ontheestablishmentofacutetoxicityexposurescenarioswithoutrequiringtestsforsub-lethaleffects.xviiiThestandardlaboratorymethodforassessingpesticideriskisdeterminethemedianlethaldose(LD50)requiredtokillhalfthetestedpopulationoveracertaintimeframe.IntheU.S.thisprotocolremainstheprimarybasisforriskassessmentinpesticideregistration.However,thisapproachtoriskassessmentonlytakesintoaccountthesurvivalofadulthoneybeesexposedtopesticidesovera

shorttimeframe:thetypicalacutetoxicitytestis48hoursalthoughitmaybeextendedto96hours.AcutetoxicitytestsestablishingLD50levelsonadulthoneybeesmaybeparticularlyill-suitedforthetestingofsystemicpesticidesbecauseofthewaysbeesareexposedtosystemicsinthe?ieldinsmallerdoses,overalongperiodoftime,throughavarietyofroutes.Chronicfeedingtestsusingwholecolonieshavebeenrecommendedasabetterwaytoquantifytheeffectsofsystemics.xix

Despiterepeatedcallsforareevaluationofpesticidetestingprotocols,regulatoryprocessesintheU.S.andEuropehavenotbeenadaptedtoconsidersub-lethal,chronicorsynergisticeffectsofpesticidesonpollinators.xx

Keytermsfordescribingpesticideimpacts

Acutetoxicity::Theacutetoxicityofapesticidetobees(eitherbycontactoringestion)isquanti?iedbynotingthedoseatwhichhalfoftheinsectsdiewithinaspeci?ictimeperiod.Thisisknowndosethatislethalto50%ofthetestpopulationorLD50.Acuteandlethalareoftenusedsynonymously,butarangeofeffects,suchasgeneralagitation,vomiting,wingparalysis,archingoftheabdomensimilartoastingre?lexanduncoordinatedmovement,havebeenobservedinassociationwithacutedoses.xxiTheacceptableriskforeachpesticideissetbyregulatoryagenciesbasedonacutetoxicitytestsandtheexpectedrateandmodeofpesticide

U.S. regulatory decisionmaking ...

has hinged, by default, on the

establishment of acute toxicityexposure scenarios without

requiring tests for sub-lethal effects.

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application,bothofwhicharedeterminedbythemanufacturer.TheLD50valuesofneonicotinoidsarelowcomparedtoolderclassesofinsecticides;theyareconsideredhighlytoxictohoneybees.

Sub-lethaltoxicity::Thesub-lethaltoxicityofneonicotinoidpesticidesisofparticularconcernbecausethemostcommon?ieldexposurescenariosarelikelyatthesub-lethalratherthanacutelevel.Sub-lethaleffectsofneonicotinoidsonhoneybeesincludebehavioraldisruptionssuchasdisorientation,reducedforaging,impairedmemoryandlearning,andshiftsincommunicationbehaviors.Otherimportantsub-lethaleffectsmightincludecompromised

immunity,delayeddevelopmentandahostofindirect,potentiallycascadingeffectsthatimpactthehivesabilitytosustainitself.

Cumulativeandchroniceffects::Neonicotinoidsfunctionbybindingtonicotinicacetylcholinereceptorsininsectsbrains,receptorswhichareparticularlyabundantinbees,increasingduringdevelopmentfromlarvaltoadultstages.xxiiThisbindingleadstoanover-accumulationofacetylcholine,resultinginparalysisanddeath.Themostrecentscienti?icobservationspointtoalong-lastingeffectinwhichmoleculesunbindfromreceptors,butremaininthebeebrain,possiblyrebindingmultipletimesbeforemetabolizationoccurs.xxiiiWhetherthisconstituteseffectivelyirreversible,cumulativetoxicityremainsunclear;butchronictoxicityeffectsovertimearealikelyresult.

ManyindependentstudiesintheU.S.andinEuropehaveshownthatsmallamountsofneonicotinoidsbothaloneandincombinationwithotherpesticidescancauseimpairedcommunication,disorientation,decreasedlongevity,suppressedimmunityanddisruptionofbroodcyclesinhoneybees.Aselectionofthesestudiesfollow.

1)Henry,M.,Reguin,M.,Requier,F.,etal.2012.ACommonPesticideDecreasesForagingSuccessandSurvivalinHoneyBees.Science(20):348-350.

Researchersattachedradiofrequencydevices(RFIDs)tohoneybeestotesttheimpactof sublethaldosesofthiamethoxam(aneonicotinoid)onforaging,homingandsurvival.They thenpluggedthese?indingsintocolonypopulationdynamicsmodelstoassesstheextentto whichhomingfailuremaycontributetocolonycollapse.Thestudycon?irmsthehypothesis thatsublethal,?ield-realisticdosesofthiamethoxamunderminebeesforagingandhoming abilitiesatratessigni?icantenoughtoincreaseriskofcolonycollapse.Intoxicatedbeeswereup totwiceaslikelyascontrolbeestofailtoreturnhometotheirhives.

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Neonico7noidsat-a-glance

Neonico*noidsareawidelyusedclassofsystemicinsec*cides

introducedintheearly1990sthathavebeenofpar*cular

interestfortheireffectsonhoneybees.

heycanbeappliedasaspray(foliar)or,morecommonly,used

assystemics.Systemicpes*cidesareappliedasseedcoa*ngsor

soildrenchesandaretakenupthroughtheplantsvascular

system,andthentransmiTedtoallpartsoftheplant,including

pollenandnectar.Neonico*noidsareverypersistentand

thereforeaccumulateover*meintheenvironment.

Mostneonico*noidsareclassifiedasacutelytoxictobees.But

single,high-dose(i.e.acute)exposuresarelikelylesscommon

thanarethechronic,sub-lethalexposurelevelsfacedbybees

over*meastheyforageinthefield.

HoneybeecolonycollapsesinFrancein1999(calledmadbee

disease)werethefirsttoimplicateimidacloprid,themost

widelyusedneonico*noid,incolonyloss.Researchershave

sincefoundarangeofsub-lethaleffectscausedby

neonico*noids:alteredforagingandfeedingbehavior,impaired

orienta*onandsocialcommunica*on,underminedimmunity

anddelayedlarvaldevelopment.


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Thissemi-?ieldstudywascarriedoutinanagriculturalareainwesternFranceandinasuburban areainsouthernFrance.Beesweregivena?ield-realistic,sublethaldoseofthiamethoxam(1.34 ngina20-lsucrosesolution),thenreleasedupto1kmawayfromtheirhiveswithanRFID gluedtotheirthorax.RFIDreaderswereplacedatthehiveentrancetotrackthereturnoftagged bees.Post-exposurehomingfailurewasthenderivedfromtheproportionofnon-returning foragers.Todiscriminateagainstotherpotentialcausesofhomingfailureintreatedforagers(e.g naturalmortality,handlingstress,predation),controlforagerswerefedwithuntreatedsucrose

andtrackedinthesameway.Theauthorsconcludethatsublethal,commonlyencountereddoses ofthiamethoxamcanimpactforagersurvivalatsuf?icientratestocontributetocolonycollapse andthatthisimpactincreaseswhenhomingismoredif?icult.Oneconsequenceofthis?inding, accordingtoauthors,isthatimpactstudiesarelikelytoseverelyunderestimatesublethal pesticideeffectswhentheyareconductedonhoneybeecoloniesplacedintheimmediate proximityoftreatedcrops.

2)AliouaneY.,ElHassaniAthiamethoxam.K.,GaryV.,ArmengaudC.,LambinM.,GauthierM.,2009.Subchronicexposureofhoneybeestosublethaldosesofpesticides:effectsonbehavior.Environ.Toxicol.Chem.28(1):113122.

Inthislaboratorystudy,emergenthoneybeesreceivedadailydoseofinsecticiderangingfrom 1/5thto1/500thofthemedianlethaldose(LD50)ofthreepesticidesover11days.Twoofthese

pesticideswereneonicotinoids,thiamethoxamandacetamiprid;theotherwas?ipronil.Fipronilis anothersystemicpesticidethatisbannedinpartofEuropebecauseitishighlytoxictobees.The authorssoughttotesttheeffectsofsublethal,chronicexposuretothesepesticidesonhoneybee behavior.Fipronil,usedatthedoseof0.1ng/bee,inducedmortalityofallhoneybeesafterone weekoftreatment.Fipronil-treatedbeesalsoexhibitedimpairedo?lactorymemory,andspent moretimeimmobile.Thiamethoxambycontactinducedeitherasigni?icantdecreaseofolfactory memory24hafterlearningat0.1ng/beeorasigni?icantimpairmentoflearningperformance withnoeffectonmemoryat1ng/bee.Theexperimentswiththiamethoxamshowthatrepeated exposuretoadosethathasnobehavioraleffectwhenappliedinacuteconditionsresultsinthe appearanceofsomebehavioralde?icits.

3)MedrzyckiP,MontanariR,BortolottiL,SabatiniAG,MainiS,PorriniC.2003.Effectsofimidaclopridadministeredinsub-lethaldosesonhoneybeebehaviour.Laboratorytests.BInsectol56(1):59-62.

Inthisstudy,researchersmonitoredthebehaviorofadultforagerbeesinhighlycontrolled, laboratoryconditionstoobserveeffectsofimidaclopridfedatsub-lethaldosesinsugarsolution. Fourdifferentcontaminationconcentrationsweretested:100ppband500ppbasasingledose (20microliters),andeachconcentrationasacontinuouslyavailablefoodsourcefor24hours. Threereplicationsoftheexperimentwereperformed.Researchersconcludedthatimidacloprid atalldosesgivencausedsigni?icantreductionsinmobilitythatlastedforonetoseveralhours, withbeesremainingstationaryforlongerperiodsaswellasmovingmoreslowly.Theyalso notedthatbeesseemedtolosetheircommunicativeabilityatalldoses,failingtocoordinate theiractivitywithotherbees.Theyrecommendedfurtherstudytobetterunderstandthelength oftimethattheseeffectslast,andtoinvestigatetheeffectsonsocialbehavioressentialtothe

properfunctioningofahive.

4)ColinME,etal.2004.Amethodtoquantifyandanalyzetheforagingactivityofhoneybees:relevancetothesub-lethaleffectsinducedbysystemicpesticides.ArchivesofEnvironmentalContaminationandToxicology47:387-395.

Thisstudyinvestigatedthesub-lethaleffectsoftwoinsecticidesinsemi-?ieldconditionsontheforagingbehaviorofhoneybees.Imidaclopridand?ipronilwerechosenbecausebothbehave

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systemically,wererecentlyintroduced,consideredhighlytoxictobees,hadshownsub-lethaleffectsonbeesinlabconditionsandhadbeenimplicatedinhoneyproductivitydeclinesinEurope.Theprimaryaimwastoaddressagapinenvironmentalassessmentofsystemicpesticidesbyimprovingonthemethodsusedtoquantifyforagingbehaviorchanges.Beecolonieswereplacedinenclosedtunnelsandtheirfeedingbehaviorvideorecordedoveraperiodof?ivedays,constitutingacumulativeeffectsstudymuchshorterthanabeeorhivelifecyclestudywouldbe.Withimidaclopridat6.0g/kg,inactivebeesthosevisitingthe

feeder,butnotfeedingincreasedovertimeinrelationtoactivebees.With?ipronilat2.0g/kg,mostbeesstoppedcomingtothefeederbythelastday,andthefewthatdidtendedtobeinactive.Convulsionsandparalysiswerealsoobservedinbeesfeedingon?ipronil-contaminatedfood.Researchersconcludedthatbothinsecticidesdisturbthehivesprimaryactivity,feeding,atsub-lethallevels70timesbelowthereferencedLD50s.Theyalsoconcludedthattheirexperimentalprotocolprovidedanindispensableinterfacebetweencontrolledconditionsinthelaboratoryandthe?ield,whichsuggestsitsadoptioninregulatorytestingofsub-lethaleffects.

5)DecourtyeA,ArmengaudC,RenouM,DevillersJ,CluzeauS,GauthierM,etal.2004.Imidaclopridimpairsmemoryandbrainmetabolisminthehoneybee(ApismelliferaL.).PesticBiochemPhys78:83-92.

Thislaboratorystudyshowssub-lethaleffectsatlowconcentrationsofimidaclopridusingfourdifferentmethodologies.Researcherssoughttobuildfrompriorstudiesthatclari?iedtheroleofnicotinicacetylcholinereceptors(nAChR)inhoneybeelearningandmemorycapacitiestoinvestigatethespeci?iceffectsoftheneonicotinoidimidaclopridonthosecapacities.Beeswereconditionedtorespondtoaspeci?ic?loralscentinassociationwithfood,abehaviorthatdemonstratesassociativelearningandmemoryformation.Behavioralresultsledresearcherstoconcludethatimidaclopridatadoseof12ng/beesigni?icantlyinhibitedassociativelearningaswellasretentionofsuccessfullylearnedassociations,whetherlearnedbefore,duringorafterexposure.Thisimpairmentofretentionnotablyaffectedmedium-term,butnotlong-orshort-termmemory,appearingasatemporaryamnesia.Metabolicactivityinspeci?icpartsofthebeebrainwerealsoanalyzedforchangesinresponsetoimidacloprid.Theseresultsindicatedthatatbothdoses,imidaclopridwasactingdetrimentallyinthepartsofthebeebraininvolvedwith

associativeandcontextualmemory.

6)YangEC,ChuangYC,ChenYL,ChangLH.2008.Abnormalforagingbehaviorinducedbysublethaldosageofimidaclopridinthehoneybee(Hymenoptera:Apidae).JofEconEntomol101(6):1743-1748.

Researchersconductedasemi-?ieldexperimenttofollowonresultsoflaboratorystudiesthathadshownanumberofdetrimentaleffectsfromingestionofimidacloprid-contaminatedfood.Theytestedthetimeintervalsbetweenindividualworkerbeevisitstoafeederafteringestionofimidacloprid-contaminatedsugarsolutionatconcentrationsrangingfrom40g/Lto6,000g/L.Beesshowedabnormalforagingbehaviorbeginningat50g/L(41.6ppb)andworseningwithhighercontamination.Effectsrangedfromastatisticallysigni?icanttimedelaybetweenfeedervisits,todisappearance(fromfeederandhive)forafulldaywithoutreturn.At1600g/

L,morethan90%ofbeeswentmissingforafullday,butallreturnedthefollowingday.Abovethisconcentration,aportionofbeesdidnotreturn,andabove800g/L,beesthatreturnedfromlongdelayscontinuedtoshowabnormalforagingbehavior.Basedonanestimationofaveragemealsizeperfeedervisit,abnormalforagingbehaviorbeganadoseperbeeof1.82-4.33nanograms.Researchersconcludedthatabnormalforagingbehaviorcouldoccurinthe?ieldthroughmultiplevisitstoimidacloprid-contaminated?lowers.

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Synergis$c+CombinedEffects

Synergismisaphenomenoninwhichtwoormorefactorsproduceacombinedeffectthatisgreaterthanthesumoftheirseparateeffects.AsinvestigationsintothecausesofCCDhavecontinuedtopointtowardmultiplefactorsworkinginconcerttoincreasebeessusceptibilitytodisease,synergismandcombinedeffectshaveemergedasacriticalareaofresearch.

In2004,alabstudy(see#1below)showedthattheacutetoxicityoftwoneonicotinoidpesticidesonhoneybeesdramaticallyincreaseswhencombinedwitheitheroftwocommonfungicides.Fouryearsafterthis?indingwaspublished,researchersestablishedthatthesetypesofcombinationsareprevalentinbeehives.xxivBetween2010and2012,threeseparatestudiesdemonstratedsynergismbetweenthecommonparasiteNosemaandpesticideexposure(thiacloprid,imidaclopridandthenon-neonicotinoidsystemic?ipronil).Hivesexposedtothesepesticidesweresigni?icantlymoresusceptibletoinfection.

Chemicalcocktails::Fungicides,pyrethroidinsec$cides,mi$cides

Neonicotinoidsarebutoneclassofpesticides,honeybeesareexposedtodozensofdifferentpesticides

onadailybasis(seePesticidePrevalence,esp.Mullin2010).Includedamongtheseareamix,orchemicalcocktail,ofinsecticides,herbicidesandfungicidesaswellasthemiticidesusedbybeekeeperstocontrolpathogensinthehive.

Non-neonicotinoidpesticidesofspecialconcernfortheirimpactsonbees,andpotentialroleinthecausalcomplexofCCDarefungicidesandpyrethroidsparticularlyincombination.Fungicideshavelongbeenthoughttoberelativelyharmlesstobees,buttheirrecent,dramaticuptickinuse(especiallywithcorn,beginningaround2007)xxvcoupledwiththeirsynergizingeffectoncertaininsecticides(includingneonicotinoidsandpyrethroids)havebroughtrenewedattention.Pyrethroidsarehighlyvariableintheirtoxicitytobees,buthavecomeunderrecentscrutinybothbecausetheirhighfatsolubilitymeansthatpyrethroidspersistandbioaccumulateinbeewax,andbecausetheyareknowntosynergizewithcertainfungicides.

Asbeedetoxi?icationmechanismsareincreasinglyunderstood,thepotentialthreatsposedbyparticularlytoxicchemicalcocktailscomposedofcertainfungicides,pyrethroidsandneonicotinoidsareslowlycomingintoresolution.Scientistsbelievethatpartofbeesincreasedvulnerabilitytopesticidescomesfromtheirhavingrelativelyfewgenesthatencodedetoxi?icationenzymes.Forexample,onesuchgroupofenzymes(P450s)mediatedetoxi?icationpathwaysinwaysthatcanbeinhibitedbycertainwidelyusedfungicides(e.g.propiconazole),andarethoughttobeimportantforbeesabilitytotoleratethecommonpyrethroidmiticide,?luvalinate.xxviAlthoughcurrentdataareunclear,anotherhypothesisastothephysiologicalmechanismsbywhichsynergisticeffectsbetweenpathogensandpesticidesmayoperateisthatpathogenmetabolitesmayinterferewiththedetoxi?icationprocess.xxvii

1)IwasaT,MotoyamaN,AmbroseJTandRoeM.2004.Mechanismforthedifferentialtoxicityofneonicotinoidinsecticidesinthehoneybee,Apismellifera.CropProtection23(5):371.

Thislaboratorystudyestablishedasynergisticeffectbetweenneonicotinoidsandfungicides.Aneonicotinoidwasfoundtobeupto1,141timesmoretoxictobeeswhencombinedwithacommonfungicide.Researchersperformedlaboratorytestsforacutetoxicityofseveralneonicotinoidpesticidesandmetabolites,bothaloneandcombinedwitheachofseveralfungicidescommonlyusedincropproduction.Thesetestswereforcontacttoxicity,ratherthan

Honey bees have fewer genes involved in

detoxification than other insects.

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oralingestion.Theyfoundthatthreecombinationsbetweenaneonicotinoidandafungicidewerehighlysynergistic.Acetamiprid,aneonicotinoidthatismuchlessacutelytoxicthanimidacloprid,becomes244timesmoresowhencombinedwiththefungicidetri?lumizole.Thiacloprid,alsomuchlesstoxicthanimidacloprid,becomes559timesmoresowhencombinedwiththefungicidepropiconazole,and1,141timesmoretoxicwhencombinedwithtri?lumizole.Thoughplantstreatedwiththemaximumrecommendedlevelsforagriculturalusedidnotexhibitastatisticallysigni?icanteffectonhoneybeemortalityafterthreeand24hours,further

studywassuggestedbeforereachingconclusionastoin-?ield,synergistictoxicity.

2)SmodisSkerlMI,KmeclV,GregorcA.2010.Exposuretopesticidesatsublethallevelandtheirdistributionwithinahoneybee(Apismellifera)colony.BulletinofEnvironmentalContaminationandToxicology85(2):125-8.

Thisstudyexaminedtheroleandprevalenceofpesticidesinhoneybeecolonies,boththoseintroducedintentionallybybeekeeperstocontrolmites(acaricides)aswellasagriculturalchemicalsfoundincidentallyinthehive.Thegoalwastodeterminewhether,andifso,howmuch,pesticidesaccumulatedinthebodiesofbeesinthehive,intheroyaljelly,andinbeelarvaeaswellashowthechemicalsarespreadthroughoutthehive.Theacaricidesintroducedintothecolonywereamitraz,coumaphosand?luvalinate,whiletheorganophosphatediazinonservedasarepresentativeagriculturalchemical.TheacaricideswereappliedfollowingcommonlyacceptedregimestotreatagainsttheVarroadestructor,andthediazinonwasintroducedinaccordancewithatreatmentplanthatwouldbeapplicabletoanappleorchard.Coumaphosand?luvalinatewerethemostprevalentthroughoutthecolony.Nodiazinonwasfoundinthesamplestestedandamitrazonlyinamountsbelowthelevelofdetection.Coumaphoswasfoundinroyaljellyfromnursebees,while?luvalinatewasfoundinthebodiesofbeesandinbeelarvae.Thepresenceof?luvalinatethroughoutthecolony,especiallyinlarvae,demonstratethatchemicalsinthehivecanbetransmittedfrombeetobeeaswellastofoodandthustolarvae,spreadingthroughouttheentirecolony.

3)DaiP-L,WangQ,SunJ-H,LiuF,WangX,WuY-YandZhouT.2010.Effectsofsub-lethalconcentrationsofbifenthrinanddeltamethrinonfecundity,growth,anddevelopmentofthehoneybeeApismelliferaligustica.EnvironmentalToxicologyandChemistry29:644649.

Thisstudyexaminedthesub-lethaleffectsoftwopyrethroidinsecticides,bifenthrinanddeltamethrin,onhoneybeehealth.Thestudywasperformedoncoloniesinthelaboratorythatwerefedsmalldosesofthechemicals.Effectsmeasuredincludedfecundity,growthandthedevelopmentofindividualbees.Theimportanceoftheseparticularissuesforoverallhealthofthecolonyandgeneralbeepopulationwasemphasized.Datawastakenoverseveralyearsandresultsmeasuredagainstcontrolcoloniesnotfedtheinsecticides.Theauthorsfoundthatbifenthrinishighlytoxicanddeltamethrinmoderatelytoxictohoneybees.Thiswasbasedon?indingsthatexposuretotheinsecticidessigni?icantlyreducedcolonyfecundityduetoreducedratesofegglayingandimpairedabilityofthecolonytotransitiontoanewqueen.Developmentaleffectsonhoneybeelarvaewerealsoobserved.Incomparingtheir?indingstopesticideapplicationsinthe?ield,thestudysauthorsnotethatcommonlyrecommendedbifenthrinapplicationratesarehigherthantheinsecticidesLC50forhoneybeesand

deltamethrinapplicationratesareaboutthesameastheLC5.

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Pathogeninterac$ons::Nosema+pes$cides

Nosema,afamilyoffungalgutparasites,

andthe

Varroadestructormitearetworelativelyrecenthoneybeepathogens.Aparticularlyvirulentandnewlyemergent(ca.2005)strandofNosema,Nosema

ceranae,hasbecomeanareaofresearchandconcernaroundtheworld,especiallyinSpain.Bothpathogenshavebeenshowntointeractwithpesticidestoweakencolonyhealthmorethaneitherdoesalone.Nosemaisafast-spreadingfungalgutpathogenthatisthoughttointerferewithhoneybeesabilitytoabsorbnutrients(infectedbeesconsumesigni?icantlymorecalories),andknowntosuppressimmuneresponse.xxviiiVarroamitesactasvectors,transmittingdiseaseacrossandwithincolonies.

WiththerelativelyrecentobservationthatCCD-affectedhivesaremarkedbyanoverallincreasedandvariablepathogenload,butwithnoonepathogenfoundtoconsistentlycorrelatewithhiveloss,researchershavebegunlookingforwhatismakingthebeessusceptibletodiseasetobeginwith.xxixThreeseparatestudiesbetween2010and2012(below)havedemonstratedasynergisticeffectbetweenpesticidesandthepathogenNosema.ThemostrecentstudybyleadingUSDAbeeresearchers

foundthatbeeswithundetectablelevelsofimidaclopridtowhichtheywereexposedonlyindirectlyinbroodfoodasdevelopinglarvafacedsigni?icantlymoreNosemainfectionsthandidtheircontrolcounterparts.

Theoverallpatternforbeesexposedbothtosystemicpesticides(neonicotinoidsand?ipronil)andNosemainfectioninthesestudiesisthatbeesgetsickmoreeasilyanddiesoonerasaresultofbothstressorsincombinationthaneitherinisolation.

1)PettisJS,vanEngelsdorpD,JohnsonJ,DivelyG.2012.PesticideexposureinhoneybeesresultsinincreasedlevelsofthegutpathogenNosema.Naturwissenschaften.

Thisstudydemonstratedincreasedpathogengrowthamongindividualbeesrearedincoloniesexposedtoimidaclopridatlevelsbelowthoseconsideredtohavesublethalimpacts.Researchersexposedhoneybeecoloniesduringthreebroodgenerationstosub-lethaldosesofawidelyusedpesticide,imidacloprid,andthenchallengednewlyemergedbeeswithNosema.TheyusedGC/MS(gaschromatography-massspectrometry)withalimitofdetectionof0.1ppbtoanalyzethebees,andveri?iedpesticideexposuretocoloniesbymeasuringtheweeklyconsumptionofthetreatedproteinpattiesandbyanalyzingimidaclopridinstoredbeebread.Nosemainfectionsincreasedsigni?icantlyinthebeesfrompesticide-treatedhiveswhencomparedtobeesfromcontrolhives.Newlyemergingbeeswhichtestednegativeforimidacloprid,buthadbeenexposedinthehive,werealsosigni?icantlylighterinweight.Asnotedbytheauthors,thisstudyisdistinctfrompreviousstudiesestablishingthissynergisticeffect(esp.Vidauetal.andAlauxetal.,below)initsfocusonlarvaexposedonlyindirectlyviabroodfoodtendedbynursebeesthathadeatenimidacloprid-spikedprotein.Ourtestbeescouldhaveonlyreceivedpesticideexposureduringlarvaldevelopment.

The?indingthatindividualbeeswithundetectablelevelsofimidacloprid,afterbeingrearedinasub-lethalpesticideenvironmentwithinthecolony,hadhigherNosemaissigni?icantinitself.Italsohassuggestiveimplicationsthattheauthorsdonotteaseoutbeyondnotingthatfutureresearchshouldbeconductedatthehivelevelovermultiplegenerationsratherthanattheindividualbeelevel.Authorsdosuggestnewpesticidetestingstandardsbedevisedthatincorporateincreasedpathogensusceptibilityintothetestprotocols.Theirconclusion:Interactionsbetweenpesticidesandpathogenscouldbeamajorcontributortoincreased

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mortalityofhoneybeecolonies,includingcolonycollapsedisorder,andotherpollinatordeclinesworldwide.

2)VidauC,DiogonM,AufauvreJ,FontbonneR,VigusB,etal.2011.Exposuretosublethaldosesof@ipronilandthiaclopridhighlyincreasesmortalityofhoneybeespreviouslyinfectedbyNosemaceranae.PLoSONE6(6):e21550.

InthislaboratorystudyindividualbeeswereinfectedwithNosemaceranae,exposed10dayslatertosystemicpesticides?ipronilandthiacloprid(aneonicotinoid).Themain?indingwasthatinfectedbeeshaveamuchhighermortalityratethanuninfectedbeeswhenexposedtothesamesublethallevelof?ipronilorthiacloprid(71%-82%vs.47%).Infectedbeesalsoappearedtobegenerallymoresensitivetosublethalpesticidepoisoning,Afterexposuretoinsecticides,uninfectedhoneybeesdidnotdisplayanysignsofintoxication.Bycontrast,atthislevelofexposure,insecticidestriggeredaggressivenessandtremorsininfectedhoneybeesduringthe?irstdaysofexposure.Thisstudyalsocon?irmedprevious?indingsthatenergeticstresswasthemainsymptomofN.ceranaeinfectioninitself:infectedbeesconsumedmuchmoresucrosethanuninfectedbees.AuthorsnotethatwhilethesynergisticeffectobservedbyAlauxetal.(2010)seemedtobelinkedtoincreasedsucroseconsumption,their?indingswerenot.

Asecondary,andbythisdatauncon?irmed,hypothesispursuedbythisstudywasthatthe

mechanismofthesynergisticeffectbetweenNosemaandthesepesticideswaslinkedtoadecreaseinbeesdetoxi?icationcapacityasmediatedbytwogroupsofenzymes.Notingthatdataonthemechanismsunderlyingsynergisticeffectsbetweenpesticidesandpathogensispoorlyunderstood,authorsnote,susceptibilityofinsectstopesticidesisamorecomplexphenomenonthanpreviouslythought.Thein?luenceofparasitismintheecosystemmustbeconsideredintoxicologicalstudies.[especially]sinceN.ceranaespreadsrapidlyandcanaffectmorethan80%ofhoneybeecolonies.

3)AlauxC,BrunetJ,DussaubatC,etal.2010.InteractionsbetweenNosemamicrosporesandaneonicotinoidweakenhoneybees(Apismellifera).EnvironmentalMicrobiology12(3):774782.

Researchersconductedalaboratoryexperimenttotestanemerging,butunstudiedhypothesisthathighcolonylossesmightbeattributabletoacombinationoftwofactorseachknowntohaveeffectonhoneybeecolonyhealth:afungalgut-pathogen(Nosemaceranae)andaneonicotinoid(imidacloprid).Theirresultsindicatethatasynergisticinteractionisoccurringthatsigni?icantlyweakensbees,bothindividuallyandsocially.Concentrationsoforallyadministeredimidaclopridwere0.7,7.0,and70g/kg,madeavailablefor10hoursperdayoverthestudyperiodof10days.Theseconcentrationlevelsarebasedonseveralstudiesthatshowedenvironmentallevelsofimidaclopridinthehoneyandpollenoftreatedcropsreached5.0g/kg.Thehighestindividualdeathratesandenergeticstressoccurredwiththecombinationofbothagentscomparedtoeachaloneandacontrolgroup.Theenzymaticactivitythatcorrelatestobeesabilitytosterilizethehivefoodforadultsandlarvaesigni?icantlydecreasedonlywhenthetwoagentswerecombined.Thissuggestsasynergisticinteractionthatcouldthreatenthecolonysabilitytowithstandabroadrangeofpathogensinthelong-term.

4)WuJY,AnelliCM,SheppardWS.2011.Sub-lethaleffectsofpesticideresiduesinbroodcombonworkerhoneybee(Apismellifera)developmentandlongevity.PLoSONE6(2):e14720.

Thiswasthe?irststudytoestablishsub-lethaleffectsonworkerbeesfrompesticideresiduesincontaminatedbroodcomb.Observedeffectsinthislaboratorystudyincluded:delayeddevelopmentoflarvalworkerbees,delayedadultemergenceandreducedadultlongevityinlarvaerearedincellscontaminatedwiththemiticides?luvalinate(apyrethroid)orcoumaphos.Theseeffectscanimpactcolonyviabilityindirectlybycausingprematureshiftsinhiveroles,

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foragingactivityandpopulationdynamics,andbycreatingincreaseddevelopmentaltimeforVarroamiteswhichinturncanrenderahivemoresusceptibletothiscommonparasiteanddiseasevector.Pesticideresiduesrapidlymigratedfromtreatmenttocontrolcomb,withacorrespondingchangeindevelopmentalandlongevityeffects:broodsraisedintreatmentcombgraduallyhadreducedeffectswhilethoseraisedincontrolcomb(successivelymorecontaminatedwitheachbroodcycle)hadincreasedeffects.Brooddeathsfollowedthistrend,withhighratesoccurringaftermultiplecyclesinprogressivelymorecontaminatedcontrol

comb,andsigni?icantlylowerratesoccurringintreatmentcombasitscontaminationlevellessened.

Microbiotaoutofbalance::Gutcultures,immunity+nutri$on

Unintentionaldisruptionofnatural,symbioticbeemicrobialculturesisonewayinwhichhivehealthmaybecriticallyunderminedbypesticidesaswellasotherstressorsinthecontemporary,commercialbeekeepingenvironment.

Honeybeemicrobiota(includingfungi,bacteria,viruses,etc.)existsattwomajorlevels:withintheindividualbeegutcultureandthroughoutthehiveconsideredasanextendedorganism.Whilevery

littleisunderstoodaboutthehoneybeescomplexanddiversemicrobialcommunity,scientistsdoknowenoughtodescribeaco-evolved,minimallyfunctioning,orcore,honeybeemicrobialcommunityaswellashypothesizeaboutkeyfunctionssusceptibletodisruptionspeci?icallynutritionandimmunity.Forexample,withinthelargerhiveenvironment,beebreadisthemostmicrobiallyactive,althoughwhetherthisactivityservesprimarilytopreserveorprocessnutrientsisnotwellunderstood.ScientistsstudyingCCDhavealsorecentlyfoundaconsistentdifferenceinthemicrobialabundancepro?ileofaffectedvs.healthyhives.xxx

Emergingresearchinthisareahasbeenmadepossibleinpartthroughrecentbreakthroughsinnew,high-throughputmetagenomicsequencingtechnologies.Thesetoolsallowscientiststobothbettercharacterizeinsectmicrobiallifeinwaysthatfocusonhowhostsandsymbiontsinteractfunctionally,attheepigeneticlevel.

1)Anderson,K.E.,Sheehan,etal.2011.Anemergingparadigmofcolonyhealth:microbialbalanceofthehoneybeeandhive(Apismellifera).InsectesSociaux58:431-444.

TakingCCDasitscriticalcontext,thisreviewarticlearguesthatresearchattentionshouldbepaidtothesymbioticmicrobialcommunitiesthatplaycriticalrolesinbeenutritionandpathogendefense.Authorsnotethatmostimportantimmune-relatedfunctionofagutmicrobiotamaybetheabilitytoobstructcolonizationbypathogens,therebypreventinginfections.Inthiscontext,thewell-knownpathogenNosemaceranae,islistedasaninfectious

microbethatinterfereswithdigestionmid-gut.

Notingthemicrobialfrontieropenedbyrecentadvancesinhigh-throughputmetagenomicsequencingtechnologies,theauthorsreviewexistingliteratureandthenargueforincreased,systems-orientedresearchintotheroleofmicrobiotainbeecolonyhealth.Withspeci?icregardtopesticides,theauthorsnotethatbroadspectrumantibioticsandfungicidesapplieddirectlytocontroldiseasearealsoknowntodestroybene?icial,non-targetfungiandbacteriainwaysthatmaydisruptionofthehivesbene?icialmicrobialbalance.QuotingMullinetal.,(2010),they?lag

The road to sustainable honey bee

pollination may eventually requiredetoxification of agricultural systemsand in the short term, the integrated

management of honey bee microbialsystems.

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asaconcernthefactthatsynergisticeffectsofmultiple,commonlyfoundpesticidesonthehivesmicrobiotaareentirelyunknown.Authorsconclude:Theroadtosustainablehoneybeepollinationmayeventuallyrequiredetoxi?icationofagriculturalsystemsandintheshortterm,theintegratedmanagementofhoneybeemicrobialsystems.

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ResearchChallenges

Inthecontextofmultiple,interactingfactors,methodologicalchallengesareexpected.Someareendemictothetaskofepidemiologicalresearchandthereforeunavoidable.Othersaretheresultofequipmentlimitations,poorstudydesignorregulatoryframeworkfailures.

Equipment+detec7onsensi7vity

Until2003,analyticaltechniqueswerenotsensitiveenoughtodetectsystemicpesticideresiduesinplanttissuebelowalevelof20-50ppbmuchhigherthanthelevelsnowknowntobetypical.Pollenhadalsoneverbeenanalyzed.Detectionofpesticidesatverylowlevelsiskeyforourunderstandingoftheactualpesticideloadinbeehives,beesandforaginghabitat,includingsoil.Thischallengeremainsanissueeveninthemostextensivestudies,withlimitsofdetectionachievedbetween1.0andafewppbwhilechroniceffectshavebeenobservedatconcentrationsaslowas0.1ppb.Overthelastnineyearsmoresensitiveanalyticaltechniquesandtoolssuchashighperformanceliquidchromatographycoupledwithtandemmassspectrometry(HPLC/ACPI-MS/MSorLC-MS/MS)havebeendeveloped,allowingsublethalandchronicexposureviapollenandbeebreadtobemeasured.xxxi

1)BonmatinJM,MoineauI,CharvetR,ColinME,FlecheC,BengschER.2005.BehaviourofImidaclopridinFields.ToxicityforHoneyBees.EnvironmentalChemistry:GreenChemistryandPollutantsinEcosystems;Springer-VerlagBerlinHeidelberg,483-494.

Usingtherecentlydevelopedmethodfordetectingimidaclopridatlevelsaslowas0.1ppb(highpressureliquidchromatographymasstandemspectrometry,HPLC/MS/MS),researchersshowedthelongpersistenceandslightaccumulationofimidaclopridinsoils(treatedanduntreated)aswellasitsuptakeinnon-treatedcrops.Sun?lowerswereshowntobeparticularlycapableofrecoveringimidaclopridfromuntreated,contaminatedsoils,aswerecornandseveralotheradventitiousplants.Untreatedwheat,barleyandraperecoveredlessimidaclopridfromcontaminatedsoils.Theaveragevaluesofimidaclopridfoundinsun?lowerandcornpollencorrespondstoarangeofconcentrationsinwhichsub-lethaleffectsinbeeforagingbehaviorhadbeenobserved.

2)BonmatinJM,MoineauI,CharvetetR,etal.2003.Methodforanalysisofimidaclopridinsoils,plantsandpollens.AnalyticalChemistry75(9):2027-2033.

Inthisstudy,researcherssetouttoaddressalimitationinthen-currentresearchmethodsbydevelopingatechniquetoquantifysystemicpesticideresiduesinthe?ieldatlevelsknown,inthelab,tohavesub-lethaleffectsonhoneybees.Usingrigorousprotocols,theydevelopedamethodtoconsistentlydetectimidaclopridandsimilarpesticidesatlevelsbelow1.0ppb.Anewextractionmethodwaspairedwithaknownanalyticmethodalreadyinrecentuse,highperformanceliquidchromatographycoupledtotandemmassspectrometry(HPLC/ACPI-MS/MSorLC-MS/MS).Theirlimitofdetectionwas0.1ppbinsoilandplants(stems,leavesand?lowers)and0.3ppbinpollen,whichhadneverundergoneanalysisforimidacloprid.Tovalidatethemethod,researchersconductedsmall?ieldtestsinsun?lowerandcorn?ields.They

foundthatmostsamplesfromtreated?ields,aswellas?ieldsthathadbeentreatedafullyearearlier,haddetectablelevels,mostofwhichwerehighenoughtohavesub-lethaleffectsonbeesaccordingtostudiescited.

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Studydesign

Designingstudiesthataccuratelyassesspollinatorsexposuretopesticidesunder?ield(i.e.outdoor)conditionsisespeciallydif?icultbecauseofthewidevarietyoffactorsinthenaturalenvironment.Multipleexposurepathways,synergisticandcombinedeffectsfrommultiplechemicals(i.e.thechemicalcocktaileffect),timing,relativelevelsofexistingpathogens,variabilitiesofweatherand

geneticpredispositionsallruntheriskofconfoundinganyexperimentdesignedtomeasurepesticideexposureandtoxicityinthehoneybeeenvironment.

Laboratoryvs.fieldstudies

Studiesseekingtodeterminetheeffectsofpesticidesonhoneybeestypicallybegininthelabwithasinglepesticideandasampleofadulthoneybees.Onceseveralstudiesachievesimilarresults,therelationshipbetweenthetestedsubstanceandtheorganismisinformedwithaninitialunderstandingofpotentialeffect.Conditionsinthelabarehighlycontrolledtoeliminatethepossibilitythatobservedeffectsmightactuallybecausedbysomeotherfactorthanthetestedsubstance.Tofurtherunderstandthatrelationship,subsequentstudiestypicallycreatesemi-?ieldconditionsthatmorecloselyresemblethenaturalbeeenvironment,butstillpartiallycontroltheparameterstolimitthepossibilityoferrorsintheresults.Full?ieldexperimentsareusedtoassesstheeffectsofthesubstanceasitoccursinthebeesenvironment,buttendtohavelessconsistentresultsbecauseconditionsarenotasreadilycontrollable.Ittendstotakealargerpopulationofsubjectsandalongerperiodofexperimenttimetoachieveresultsthatcorrelatetoeithersemi-?ieldorlabstudies.Labresultsandsemi-?ieldresultsarenotalwaysreplicableinfull-?ieldstudiesevenwiththesenecessaryallowances.

Itisinherentlymoredif?iculttotrackanindividualbeetomeasurethepesticideconcentrationstowhicheachbeeisexposedandthenitssubsequentbehavior.Manyotherfactorsalsoplayaroleinbeebehaviorandcolonyhealth,suchasthepresenceofotherpesticidesinthehivesandtheforagingenvironment(thelatterofwhichcanbeseveraltomanytensofsquaremiles),weatherconditions,geneticpredispositions,theageandhealthofthequeen,andtherelativepresenceofparasitesandpathogens.Oneofthesemanyvariabilitiesspeci?icto?ieldconditionsthathasrecentlycomeintofocusistheimpactofrelativehumidityonthetoxicityofneonicotinoidstobees.

1)DecourtyeA,DevillersJ,AupinelP,BrunF,BagnisC,FourrierJ,GauthierM.2011.Honeybeetrackingwithmicrochips:anewmethodologytomeasuretheeffectsofpesticides.Ecotoxicology20:429-437.

Thisstudyshowsdisorientationbypesticidesandillustratesthedif?icultiesofmeasuringsucheffectsunder?ieldconditions.Fewstudieshaveinvestigatedtheimpactofpesticidesonhoming?lightduetothedif?icultyofmeasuringthe?lighttimebetweenthefoodsourceandthehive.TheaimofthisstudywastoshowhowtheRFID(radiofrequencyidenti?ication)devicecanbeusedtostudytheeffectsofpesticidesonboththebehavioraltraitsandthelifespanofbees.Researchersdevelopedamethodtoautomaticallyrecordthedisorientationofindividualforagersandtodetectthealterationofthe?lightpatternbetweenanarti?icialfeederandthe

hive.Fipronilwasselectedastestsubstanceduetothelackofinformationontheeffectsofthisinsecticideontheforagingbehavioroffree-?lyingbees.Itwasshownthatoraltreatmentof0.3ngof?ipronilperbee(LD50/20)reducedthenumberofforagingtrips.

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Mul$pleexposurepathways

Contact (by touch) toxicity :: Dust, soil and planter exhaust/talc

NotingthecorrelationbetweenbeelossesandcornplantingseasoninItalyandEurope,scientiststherebeganexploringthepossibilitythatbeeswerebeingpoisonedbythedustemittedfrompneumatic

drillingmachinesusedtoplantneonicotinoid-coatedseedsaround2003.xxxii

Morerecentstudieshavecon?irmedthatthisrouteofexposureisindeedlethal,andisexacerbatedbyhumidity.Theleadinghypothesisisthatbees?lyingthroughcontaminateddustarepowderedwithacutelytoxiclevelsofneonicotinoidsastheirabdomenscollectairbornefragmentsoftreatedseedcoating.

1)TapparoA,MartonD,GiorioC,et.al.2012.Assessmentoftheenvironmentalexposureofhoneybeestoparticulatemattercontainingneonicotinoidinsecticidescomingfromcorncoatedseeds.EnvironmentalScience&Technology.

This?ieldstudyinvestigatesplanterexhaustduringcornsowingasanexposurepathwayforbeesforaginginandaroundthose?ields.Thecore?indingisthatbees?lyingoversowing?ieldsaredirectlyexposedtoneonicotinoidsatlethallevelssigni?icantlyhigherthanthecontactLD50values(18,22,and30ng/beeforimidacloprid,clothianidinandthiamethoxam,respectively).

Alsocon?irmedarerecent?indingsthata)highquantitiesofneonicotinoidsfromseedcoatingparticlesareemittedbydrillsusedduringcornsowing;b)lethallevelsofneonicotinoid-contaminatedplanterexhaustcanlandonbeesabdomensasthey?lythroughthedust;c)beemortalityishigherunderhumidconditions,supportingthehypothesisthatparticlesaremorelikelytosticktobeesabdomensinhumidconditions.Studyauthorsnotethatbeesseemtoremovetheseedcoatingparticlesduringsubsequentforagingorinthehiveundernormalhumidityconditions,and?indasigni?icantdecreaseintheinsecticidecontentwhenbeesaresampledafterdeath.Theyhypothesizethatmetabolicdegradation(probablyalsoeffectivepost-mortem)mayaffectconcentrationsfoundinsamples.

Thestudysauthorsalsoexplicitlyaddresstwomethodsofharmreductionproposedbyindustry:thicker,moreadhesiveseedcoating,andmodi?icationsofthedrillsintendedtoreduceairbornereleaseofcontaminatedplanterdust.Theyconcludethatneitherpresentsalikelysolution.Indrawingtheseconclusions,theypointtothefactthatfollowingtheEuropeanintroductionofseedcoatingsmeanttobemoreresistanttoabrasionin2009-2010,Austrian,SlovenianandGerman(priortotheban)beekeeperscontinuedtoreportcolonylossinconjunctionwithcornsowing.NosuchcolonylosseswereobservedinItalyafterthebanthere.Theseconclusionswerealsosupportedbydirectobservationdifferencesintoxicemissionsbetweenmodi?iedandunmodi?ieddrills:modi?ieddrillingmachinesemitlargeamountsofcontaminateddustatacutelevels.

2)Marzaro,M.,Vivian,L.,etal.2011.Lethalaerialpowderingofhoneybeeswithneonicotinoidsfromfragmentsofmaizeseedcoat.BulletinofInsectology64(1):119-126.

This?ieldstudyinvestigatedtwopossiblemechanismsthroughwhichbeescancomeintolethal

contactwithneonicotinoid-contaminatedfragments,ordust,emittedastreatedseedsaresown:1)directaerialpowderingofthebeesastheycomeintocontact;or,2)indirectexposurethroughthenearbycontaminatedvegetation,dewandguttationdroplets.Authorsalsotestedthesynergisticeffectofrelativehumiditylevelsonbeemortality.Conductingchemicalanalysesofcontaminateddew,guttationdroplets,dustfromabradedseedcoatings,anddeadbees,theyfoundthatbeeswerenotlethallypoisonedbydrinkingdewandguttationdroplets,butthatdirectcontactwithairborne,contaminateddustisacutelytoxictobeesunderconditionsofhighhumidity.Dustfromabradedseedcoatingsexpelledduringsowingwereshowntocontainmore

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than20%neonicotinoid,aconcentrationatleast2,600timesgreaterthanwhatisusedinsprayapplications.Studyauthorsalsoconductedtrialswithseedstreatedwithafungicidebutnotaneonicotinoid,underbothhumidityconditions.Theyfoundthatthefungicide-treatedseedcoatingdustwasnotlethaltobeesundereithercondition.Usingthesetrialsasdefactocontrols,theyconcludedthathumidityalonedoesnotcausemortality;rather,humidityhasasynergisticin?luenceonthecontacttoxicityofneonicotinoids.

3)APENET.2010&2011.Effectsofcoatedmaizeseedonhoneybees.ReportsbasedonresultsobtainedfromthesecondandthirdyearsactivityoftheAPENETproject.Consortiumforresearchandexperimentationinagriculture.

AgroupofItalianscientistsfromvariousinstitutionsbeganpublishingannualresultsfromanongoingmonitoringnetwork(APENET)in2009.Theirpurposehasbeentoinvestigatetheeffectsofcoatedmaize[corn]seedonhoneybees.Main?indingsconsistinestablishingthatbeesareexposedtoacutelytoxiclevelsofpesticide-contaminateddustfromautomaticplantersdepositingtreatedseedinthe?ield,andthatrelativehumidityhasasynergisticeffectmortality.Thesestudieslookattheneonicotinoidsclothianidin,imidacloprid,andthiamethoxam,aswellas?ipronil,whichisasystemicinsectidecommonlyusedintreatedcornseed.Theyhavealsoconducted?ieldtrialsinmethodsfordustanddriftabatement.2010and2011reportsare97and123pagesrespectively,andarestructuredassixandeightchapters.Eachofthesechapterscanbetreatedasadistinctstudywithitsownresearchagenda,methodsandresultssection.Threereleventchapterspublishedinbothyearsreportsaretreatedbelow.

Dustdispersalduringcoatedmaizeseedsowingandestimatedeffectsonbees.

In?ieldtrials,dispersalofpesticide-contaminateddustwasfoundtodependonanumberofprocedures:seedcoatingprocedure;useofamodi?ieddrill(de?lector)toreducedustdispersalduringplanting;weatherandenvironmentalconditions.In2010,modi?icationoftheseederspneumaticdrillswithanairde?lectorsucceededinreducingthedispersalofpesticide-contaminateddustbyaround50%.Theseresults,however,dependedonseedquality.In2011,experimentersdeviseda?iltertoattachtoairde?lectorswiththeintentionoffurtherreducingdustiness.Thiscombinedmodi?icationreduceddustdispersalby90%-95%,exceptingvery

?inedustparticles.Inbothsituationsbeesdieathigherrates:30%-60%withthe?ilter+de?lector,85%withde?lectorsalone.

Effectsinbeesbycontactwithdustduring?lightovera?ieldsownwithcoatedmaizeseed.

Researcherscon?irmedahypothesisthatbees?lyingoveraseederthatissowingtreatedseedmaybeexposedtoalethaldoseinasingle?lightviacontacttoxicitywithoutthepoisoningbeingmediatedbyingestionofcontaminatedfood.Humiditywasfurtherfoundtosigni?icantlysynergizewiththepesticides.Authorsnotedthatbeesabdomens(orinteguments)areshapedtocatchpollenandarethusextremelylikeytotrapdust,andthatdusthadanexceptionally20%(byweight)concentrationoftheactiveingredientwhencomparedtosprayformulations.

Sub-lethaleffectsofneonicotinoidsand?ipronilonlearningandmemoryofodorsandspatial

orientation.

Studyingtheproboscisextensionre?lex(PER)asanindexofcertaincognitiveprocesses,researchersfoundsub-lethaleffectsofneonicotinoidsand?ipronilonlearningandmemoryofodors.Researchersalsodesignedatestingprotocoltomeasurebeesorientationcapacity.Beesexposedtoclothianidinat0.7and0.47ng/beeshowedmarkedimpairmentinhomingabilityandforagingfrequencyafterasingledoseateitherlevelin2010.Disorientationanddisruptedolfactorymemoryandlearningcansigni?icantlyimpactbeesforagingabilitiesandsociallifeas

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botharemediatedheavilybyscent.Resultsfrom2011,althoughnotyetcomplete,appeartocon?irm2010?indings.

Oral(inges7on)toxicity::Pollen,nectar+gu]a7ondroplets

Establishedoraltoxicitylevelsofneonicotinoidsforbeesaresigni?icantlyhigherthanarecontacttoxicitylevels.Potentialoralexposureroutesthathavebeenrecentlystudiedincludepollen,nectarand

guttationdroplets.Guttationdropletsareakindofdewexudedbyplantsduringthenightandintheearlymorning;theyhavebeenshowntocontainlethallevelsofneonicotinoidpesticides.Whilebeesreadilyconsumedguttationdropletsinlabconditions,subsequent?ieldstudieshavefailedtoestablishthatbeesreadilyusethesedropletsasawatersource.Fieldstudieshaveshownthatbeescollectandbringbacktothehivepollenandnectarcontaminatedwithneonicotinoidpesticidesbothfromdirectlytreatedcrops(corn),andfromnearbyuntreatedplantsknowntoserveasnutritionsourcesforbees(dandelions).

1)GirolamiV,MazzonL,SquartiniA,etal.2009Translocationofneonicotinoidinsecticidesfromcoatedseedstoseedlingguttationdrops:anovelwayofintoxicationforbees.JournalofEconomicEntomology102(5):1808-1815.

Researchersinvestigatedthelevelsofpesticidespresentinthedropletsexudedbyplantsgrownfromtreatedseeds,andthetoxicityeffectsofthosedropletstohoneybeesthatconsumedthem.Studyshowedthattheconcentrationofimidaclopridinguttationdropscanbenearthoseintheactiveingredientthatisappliedin?ieldsprays.Beesdiewithinminutesafterconsumingguttationdropsfromimidacloprid-treatedseeds.Fourdifferentpesticidesweretested,allgrownfromtreatedcornseed,bothopen-?ieldandin-lab:threeneonicotinoids(imidacloprid,clothianidinandthiamethoxam)andonenon-systemicpesticide(?ipronil).Alltreatedseedsalsoincludedtwofungicides,?ludioxonilandmetalaxyl-m,re?lectingcommerciallyavailableseeds.Theexperimentalcontrolsconsistedofuntreatedseedsandseedstreatedonlywithfungicides.Theneonicotinoidpesticideswerefoundtobeconsistentlypresentinguttationdrops.Levelsofcontaminantvariedbypesticideandbyreplicationasexpected,butinallcaseswereabove1,000g/L.Imidacloprid-treatedcornwasthemostef?icienttranslocator,concentratingupto200,000gofactivesubstanceperliter,butclothianidinandthiamethoxamweremoretoxic

(maximumconcentrationsfoundat100,000g/L).Dropsremainedontheyoungplantsthroughoutmostofthedayduetocollectinginthecupformedatthebaseoftheyoungplants.Beesreadilyconsumed?ield-grownguttationdropspresentedtotheminlabconditions.Inallcasesforneonicotinoids,beesexperiencedacutetoxicity,dyingwithintwoto44minutes(concentration-dependent).

2)GreattiM,etal.2006.Presenceofthea.i.imidaclopridonvegetationnearcorn@ieldssownwithGauchodressedseeds.BulletinofInsectology59(2):99-103.

FollowingEuropeanbeekeeperassertionsofalinkbetweenimidacloprid-dressedcornseedandlarge-scalehoneybeedeaths,researcheffortsweremadetodeterminewhetherthepesticidewasbecomingairborneduringseed-sowing.Thisstudyshowedthatimidaclopridcanbereleasedatlevelsthataretoxictobeesduringsowingoperationsandthatnearbyplantscanbe

contaminatedbyimidaclopridindustfromsowingoperations.Threedifferentseedtreatmentscontainingimidacloprid,onewithanewingredientintendedtoimprovepesticideadherence,andtwowithoutimidaclopridweretested.Resultsclearlydemonstratedthatimidaclopridfromallthreetreatedseedscancontaminatenearbygrassesand?lowersonthedayofsowingandforatleastfourdaysafterward,inamountsrelevanttobothsub-lethalandlethaleffectsonhoneybees.Sunny,warmweatherwasobservedtocorrelatetohigheramountsofimidaclopriddetected,andforlongerperiodscomparedtocoldandrainyweather.Theamountofsoildustgeneratedbypneumaticseeddrillsusedinthetrialwasalsoimplicatedinpotentiallywide

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dispersalofimidaclopridparticlesthroughwind.Basedonknownpatternsofcorn-seedsowinginNorthernItaly,researchersconcludedthatimidaclopridcouldaccumulateonvegetationsurroundingcorn?ieldsduringsuccessiveperiodsofsowingforaperiodof3-4weeks,representingalonger-termcontaminationthanwasobservedinthestudy.

Time+Timing

Understandingtheeffectsofpesticidesandotherstressorsonhivehealthiscomplicatedbyissuesoftime:duration,sequencinganddevelopmentalstagesofabeecanallplayarole.Studyingtheeffectsofpesticideexposureovertooshortatimescaleisperhapsthemostcriticalblindspotofmostresearchtodate.CurrentU.S.regulatoryguidelinesspecifythathoneybeetoxicitytestingbedonewithinatimeframeof48-96hours,whichistooshorttoobservemanychronicorsublethaleffectsparticularlywhenthoseeffectsareindirectorcascading.RecentresearchintosynergisticeffectsofpesticidesandNosemahassurfacedapotentialsequencingissuewherebybeesexposed?irsttoinfection,thentopesticidesshowsignsofpoisoningatsublethallevelswhenpesticideexposurealone(withoutpreviousinfection)atthesamelevelsdonotappeartohaveatoxiceffect.xxxiii

Hive+BeeLifecycles

Hivescontainoverlappinggenerationsinwhicheachplaysacriticalandinterdependentroleinmaintaininghivehealth.Thecomplexsocialstructureofthehiveisanecosystem(somescientistscharacterizeitasanorganismorsuperorganism)inwhichindividualbeescanbecategorizedbyacombinationofageandhivefunction.Eachcategoryofbeeconsumesdifferenttypesandamountsoffood:pollen,nectar,andbee-madefoodcontainingoneorboth,suchasbeebread,honeyandroyaljelly.Theyalsodifferintheiractivities,somespendingmostoftheirlivesinthehivewhileothersspendamajorityoftimeforagingin?lowersafewmilesawayfromthehive.

Contactexposuretopesticidesaccordinglyvarieslarvaespendtheir?irstthreedaysoflife?loatinginnectar,andforagingbeesareincontactwithpollenformanyhours.Larvaeandadultbees,likehumaninfantsandadults,havedifferingdetoxi?icationcapacitiesandnutritionalneeds.Thequeen,nursebees

anddronesalllikewiseexistindifferentmilieus.Thesevariationsallmakeadifferenceinhowahiveorindividualbeewouldbeaffectedbypesticides.

1)RortaisA,etal.2005.Modesofhoneybeeexposuretosystemicinsecticides:estimatedamountsofcontaminatedpollenandnectarconsumedbydifferentcategoriesofbees.Apidologie36:7183.

Inthisstudy,researchersdemonstratedtheimportanceofbeelifecycletothestudyofpesticideeffects.Researchersusedwell-establishedbeebiologydatatoquantifyimidaclopridingestionbybeeswhosehivesareneartreatedsun?lowers,accordingtoeachbeecategory.Theirestimatesfocusonthosecategoriesthatconsumethehighestamountsofpollen(nursebees)andnectar(wax-producing,brood-attending,winterandforagingbees).Individualswithinthesecategoriespotentiallyconsumebetween0.5to3.8nanogramsofimidacloprid,overa

periodoftimethatismostrelevanttotheirageandactivities(from5to90days).Thesecumulativedosesareconsistentwithamountsnowknownfrombothpriorandsubsequentfeedingexperimentstobelethal,ortocauseavarietyofsub-lethaleffectsthatcanresultinprematuredeathwithinhoursordays.

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StructuralBias

Bias,sourcesofwhichscientistsseektominimizeandeliminate,appearstobeplayingaroleinourcollectiveunderstandingofpesticideeffectsonhoneybees.Theprominentroleofpesticidemanufacturersinconductingandfundingstudieshasgeneratedcontroversyandconcernamongindependentresearchers,beekeepersandcitizengroups.

Thefollowingcriticalreviewsexaminehowcon?lictsofinterestinhoneybeeresearchimpactresearch?indings,yieldcitationbias(wherecontradictorystudiesareexcludedfromintroductoryliteraturereviews),andexertunduein?luenceonpesticidepolicymakingdecisions.

1)MainiS,MedrzyckiP,PorriniC.2010.Thepuzzleofhoneybeelosses:abriefreview.BInsectol63(1):153-160.

Theauthorsreviewed84studiesthataddressthequestionofwhetherpesticidesareadverselyaffectinghoneybeesandothernon-pestinsects,withinthecontextofscienti?icandpubliccontroversyaroundCCD.Theyprovideafocused,criticalreviewofthemostrecentexperimentalandreviewstudiesaddressingCCD,especiallytheeffectsofimidaclopridinthe?ield(themost-usedneonicotinoidthathasbeenbannedtovaryingdegreesinfourEuropean

countries).Theyalsocriticallydiscussvenuesforresearchpublicationandpresentation.Theycompellinglydemonstratethatsciencefundedbyagrochemicalcompanies(includingBayerCropScience,themakerofseveralneonicotinoidsincludingimidacloprid),have:1)focusedCCDresearchmoreonparasitesandpathogensthanonpesticides;2)publishedthemostfavorableamongallresultsonstudiesofpesticideeffectsonhoneybees(nosigni?icanteffectsoreffectsatdoselevelsthatdonotcorrelatetoenvironmentallevels);andthus3)potentiallyin?luencedpolicydecisionsmadetoprotectbeesfrompesticidestowardlessrigorousriskassessmentsandlesscautiousregulations.

2)KindembaV.2009.Theimpactofneonicotinoidinsecticidesonbumblebees,honeybeesandothernon-targetinvertebrates(revisedversion).Buglife:TheInvertebrateConservationTrust.

Thisreportpresentsacriticalanalysisofscienti?icresearch,technicalreportsandregulatory

processdocumentsrelevanttotheeffectsonnon-targetorganismsofall?iveneonicotinoidsregisteredforuseintheU.K.Largelyfocusedonimidaclopridseffectsonhoneybeesaccordingtothebulkofresearchavailable,theauthormakesastrongcasefortheexistenceofstructuralbiasandregulatoryinadequacy.Amongstudiesshowingthatimidaclopridhasnegligiblesub-lethalorchronictoxicitytohoneybees,orthattheeffectsseenarenotrelevanttoamountsfoundinthebeeenvironment,mostwerefundedorcarriedoutbythemanufacturer.Conversely,alongerlistofindustry-independentresearchtendstowardoppositeresults:imidaclopridbeingsub-lethallyandchronicallytoxicatloweramounts,whichareindeedrelevanttoenvironmentallevels.Insomecases,thiswasnotedtoberelatedtoequipmentsensitivityforpesticidedetection,buttheoverall,patterneddiscrepancyindicatesthatresultswerein?luencedbyfactorsrelatedtotheagendasofthosewhofundedandconductedthestudiesaswellastheregulatoryreviewers.Theregulatoryprocessisfoundtobede?icientinits

assessmentforavarietyofotherreasons:lackofstandardmethodologyforinvestigatingsub-lethaleffects,failuretoinvestigatelong-term,seasonal,conditional,orsynergisticeffectsinthefaceofcompellingevidencefordoingso,negligenceinrequiringstudiesonlarvae,lackofvalidationcriteriaforreviewingstudymethodologiesandfailuretoinvestigateallpossibleroutesofbeeexposure.

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AppendixA::Neonico7noidusepa]ernsinU.S.agriculture

Table 2 ::