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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
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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 ::
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