APPENDIX B.

ST A T E O F T H E SC I ENC E REPORT
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Pesticides!and!Honey!Bees:

State!of!the!Science

March!21,!2012

Submitted)to)the)U.S.)Environmental)Protection)Agency

as)a)supporting)appendix)to)

Emergency)Petition)to)U.S.)E.P.A.)Seeking)Suspension)of)Registration)for)Clothianidin
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Pesticide Action!Network!North!America

Pesticide!Action!Network!North!America!(PAN!North!America)!works!to!replace!the!use!of!hazardous!

pesticides!with!ecologically!sound!and!socially!just!alternatives.!

As!one!of!five!PAN!Regional!Centers!worldwide,!we!link!local!and!international!consumer,!labor,!health,!

ǯǤ

challenges!the!global!proliferation!of!pesticides,!defends!basic!rights!to!health!and!environmental!

quality,!and!works!to!ensure!the!transition!to!a!just!and!viable!society.

Pesticide!Action!Network!North!America

49!Powell!St.,!Suite!500

San!Francisco,!CA!94102

Tel ::!(415)!981P1771

Web ::!www.panna.org

Written!by!Heather!Pilatic!with!research!assistance!from!PANNA!interns!Julie!Chamberlain!and!Neva!

Jacobs.!Colleagues!at!Beyond!Pesticides!provided!important!annotations.!Dr.!Susan!Kegley,!consulting!

scientist!for!PANNA,!and!Dr.!James!Frazier,!professor!of!entomology!at!Pennsylvania!State!served!as!

helpful!readers!of!early drafts.!PANNA!colleagues!Kristin!Schafer!and!Sara!Knight!contributed!careful!

copyediting.!Errors!belong!to!the!main!author!alone.!Support!for!this!work!provided!by!the!Ceres!

Foundation!and!PANNA!supporting!members.
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Contents

I. Introduction!

II. Overview!::!Colony!Collapse!Disorder!in!context

III. Understanding!pesticides!as!a!causal!factor!in!colony!collapse

A. Pesticide!prevalence

B. Neonicotinoids!::!Acute,!subPlethal!&!chronic!effects

C. Synergistic!&!combined!effects

 Dzdzǣǣ	ǡethroids,!miticides

 Pathogen!interactions!!::!Nosema &!pesticides

 Microbiota!out!of!balance!::!Gut!cultures, immunity!& nutrition!

IV. Research!challenges

A. Equipment!&!detection!sensitivity

B. Study!design

 Lab!vs.!field

 Multiple!exposure!pathways

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

Oral!toxicity!(ingestion)!::!Pollen,!nectar!&!guttation!droplets

 Time!&!timing!

Hive!&!bee!lifecycles

V. Structural!bias!
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I!@ Introduction!

Honey!bees!and!other!pollinators!are!dying!off!at!unprecedented!rates!around!the!world. First!in!France,!

then!in!the!U.S.!and!elsewhere,!colonies!have!been!mysteriously!collapsing!with!adult!bees!abandoning!

their!hives.!In!2006, two!years!after!this!phenomenon ǤǤǡDz

ǡdzǤEach!year!since, U.S.!beekeepers!have!reported!annual hive losses!of!29%!P 36%.!

Commercial!beekeepers!tell!us!that!their!industry,!which!is!the!care!and!cultivation!of!an!indicator!

species,!is!on!the!verge!of!collapse.!

While!claims!of!imminent!food!system!collapse!are!not!supported!by!pollination!biologists,!we!do!know!

that honey bees!pollinate!71!of!the!100!most ͻͲΨǯ

supply,!making!managed!honey!bees!the!most!economically!important!pollinator.i In!the!U.S. alone, the!

U.S.!Department!of!Agriculture!(USDA)!indicates!this!industry!is!worth!more!than!$15!billion!per!year.ii

Without!pollination,!agriculture!quickly!becomes!less!efficientȄrequiring!more!land!and!water!to!grow!

the!same!amount of!foodȄand!our!diets!lose!nutritionally!important variety.!For!instance,!most!fruits!

and!many!nuts!rely!on!the!pollination!services!of!bees,!including!almonds,!blueberries,!apples!and!

melons.!Altogether,!more!than one!in!every!three!bites!of!food!depends!on!honey!bees!for!pollination.

While!few!contest!that!the!recent,!dramatic!decline!of!honey!bee!populations!presents serious!

challenges!to!an!alreadyPstressed!food!system,!the!public!debate!over!what!lies!behind!CCD is!at!this!

point!so!polarized!and!confusing!that!concerned!citizens!find!it!difficult!to!know!how!or!where!to!

intervene.!Indeed,!the debate!over!the!causes!of!CCD!has!become!a!case!study!in!public,!scientific!

controversyiii.!As!with!other recent!matters!of!public!and!environmental!health, such!as!the!link!between!

tobacco!and!cancer!and!anthropogenic!climate!change,!this!issue!has!become characterized!by!

policymaker!inaction!in!the!face!of!irreducibly!complex!science.!

Two!increasingly!intractable!sides!have!emerged!in!this controversy:!beekeepers!and!environmental

health!advocates vs.!pesticide!companies!and!the!scientists!supported!by!them. ǯ

in!this!linePup!is!clear!enough,!we!have!sought to!hew!to!a!commitment!to!nonPpartisan,!scientifically!

literate!public!discourse.!We!believe!that!engaged!forms!of!scientific!citizenship!are!a!vital!part!of!

democratic!civic!life, and!a!needed!force!in!environmental!decisionPmaking.!In!cases!such!as!this!where!

the!debate!has!become!intractable!and!positionPdriven,!as!is!reported!by!participants!and!scholars!alike,!

the!conversation!between!experts!has!clearly!broken!down.!Historically,!these!kinds!of!logjams!are!

ǡDzdz

event!that!compels!policymaker!actionȄor!both!in!conjunction. With!onePthird!of!our!bees!dying!off!

each!winter!and!wild!pollinators!facing!similarly!catastrophic!declines,!it!would!seem!that!we!have!

before!us!the!focusing!event.!Needed!now!is!public!demand!for!policy!action.

Thus,!the!purpose!of!this!document!is!to!inform!public!debate!and!build!national!will!for!policy!action!on!

a!timeline!that!will!be!meaningful!to!bees!and!beekeepers.! Our!consideration!of!the!evidence!for!the!

causes!of!bee!decline!and!CCD!is!focused!on!pesticides!as!one!of!three!leading!factors!identified!by!

researchers.!By!our!analysis,!the!weight!of!evidence!demonstrates!that!pesticides!are!indeed!key!in!

explaining!honey!bee!declines,!both!directly!and!in!tandem!with!the!other!two!leading!factors,!

pathogens!and!poor!nutrition.!The!science!supporting!this!conclusion!is!presented!in!this!report.
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2!@ Overview!::!Colony!Collapse!Disorder!in!context

Honey!bee!populations!have!steadily!declined!in!the!U.S.!since!1947,!but!at!a!gradual!rate!averaging!1%!

per!yeariv.!Steeper!declines!have!been!recorded!since!1987,!but!the!last!four!winters!have!seen!

extraordinary!losses!averaging 29!to!36%!per!year.v,vi,vii,viii

Most!scientists!agree!that!there!is!no!single!cause!of!CCD.!

Rather,!recent!population!declines!are!likely!caused!by!a!

combination!of!factors acting!in!concert!to!weaken!bee!

colonies!to!the point!of!collapse;ix and!emerging!science!

points!specifically!to!impaired!immunity.!Lead!suspects

in!this!causal!complex!include:!nutritional!stress,!

pathogens and!pesticides.

Nutritional*stress :: Nutritional!stress!

undermines!colony!health!through!a!variety!of!

mechanisms,!including!immune!system!harm!and!

reduction!in!reproductive!viability.!One!key!

component!of!nutritional!stress!for!honey!bees!

includes!habitat!loss!that!results!in!a!less!varied!

and!therefore!less!nutritious!diet.!Habitat!loss!

has!been!occurring!steadily!for!the!last!50!years!with!measurable!effects!on!bee!health.!For!

instance,!regional!differences!in!ratios!of!open!to!developed!land!have!been!traced!to!higher!

colony!losses. One!key!driver!of!recent!habitat!loss!is!the!increased!use!of!broadPspectrum!

herbicides!that!accompanies!herbicidePresistant,!genetically!engineered!crops.x

Pathogens :: Pathogens!like!parasitic!mites,!viruses!and!a!gut!fungus!have!garnered!the!most!

media!attention!as!causal!factors!in!CCD.!Multiple!studies!have!confirmed,!however,!that!there!is!

no!single!pathogen!associated!with!the!disorder.!In!an!analysis!of!studies!published!as!of!early!

2009,!two!leading!U.S.!researchers!noted!that!Dzǥno!single!pathogen!found!in!the!insects!could!

explain!the!scale!of!the!disappearance.!In!other!words,!the!bees!were!all!sick,!but!each!colony!

seemed!to!suffer!from!a!different!combination!of!diseasesǤdzxiParasitic!mites!of!the!genus!Varroa

are!the!most!important!pest!to!honey!bees globallyxii and!act!as!vectors!to!transmit!a!number!of!

viruses!that!significantly!weaken!colonies.!Deformed!wing!virus!and!a!trio!of!related!paralysis!

viruses!have also emerged!as!important!to!colony!losses,!as!has!a!fungal!gut!pathogen!of!the!

genus!Nosema.!Emerging!microbiota!research!points!to!the!possible!disruption!of!normal,!

symbiotic!bee!gut!cultures!by!a!combination!of!stressors!resulting!in!increased!susceptibility!to!

pathogens.!

Pesticides :: Pesticides!have!been!known!to!cause!largePscale!bee!deaths!since the!early!1900s,!

many!through!direct!poisoning!during!aerial!sprays. These!types!of!acute!bee!diePoffs!are!not!at!

issue!in!CCD,!although!they!do!still!happen.!Regulations!and!phasePouts!of!acutely!toxic!

pesticides!have!reduced!the!number!of!acute!poisonings!in!most!of!Europe!and!North!America,!

but!bee!exposure!to!multiple!pesticides!continues.!SubPlethal!effects,!less!studied and!

understood!than!acute!effects,!have!become!a!key!concern as!systemic!neonicotinoid!

pesticidesȄpresent!in!small!amounts!throughout!plant!tissues!from!seed!to!harvestȄhave!

become!an!importantant!and!rapidly!growing!segment!of!the!global!insecticide!market!since!

their!introduction!in!the!1990s.xiii Other!pesticides!of!concern!include!those!used!by!beekeepers!

What*is*Colony*Collapse*Disorder?

Colony!Collapse!Disorder,!or!CCD,!was!first!

described!in!the!U.S.!in!2006.!Its!symptoms!

are!distinct!from!other!loss!epidemics and!

include!the!following:!

3⁄4 Colonies!found!suddenly!empty!of!adult!

bees,!leaving!their!brood!unattended

3⁄4 No!sign!of!dead!bees

3⁄4 No!hive!pests!or!food!robbers,!despite!

surplus!honey!and!pollen!stores

3⁄4 Common!parasites!not!present!at!levels!

thought to!cause!population!decline
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to!control!pathogens,!and!certain!fungicides!thought!to!be!safe!for!bees!which!have!recently!

ǡǯ

toxicity!by!200P to!1,000Pfold.xiv

The!following!four!studies!provide!an!overview!of!the!factors!involved!in!recent!honey!bee!declines!as!

well!as!a!description!of!Colony!Collapse!Disorder.!!

1)!vanEngelsdorp!D,!Meixner,!MD.!2010.!A(historical(review(of(managed(honey bee(populations(in(

Europe(and(the(United(States(and(the(factors(that(may(affect(them. J!Invertebr!Patholology!103:!

S80PS95.

This!article!is!the!most!recent!and!comprehensive!review!of!historical!loss!patterns!and!the!data!

gaps!that!make!these!patterns!difficult!to!describe. Researchers!reviewed!110!years!of!census!

surveys,!experimental!studies,!technical!reports and!review!studies to!arrive!at!an!analysis!of!

global!population!trends!and!the factors!that!affect!managed!honey!bee!populations.!Various!

data!inconsistencies!notwithstanding,!they!determined that populations!have!increased!

worldwide!over!the!last!50!years,!but!severe!declines!have!occurred!in!the!U.S.!(61%),!Mexico,!

and!Europe!(27%!continentPwide) in!the!same!time!frame.!In!all!of!these!declines,!several!

interacting!factors!are!likely.!These!include!longPterm!reductions!in!bee!foraging!habitat,!

changing!weather patterns,!a!contraction!of!the!gene!pool,!weak!queen!bees,!pathogens,!

pesticides!and!socioeconomic!factors!that!affect!beekeeping!popularity!and!profitability.

2)! vanEngelsdorp!D,!Evans!JD,!Saegerman!C,!Mullin!C,!Haubruge!E, Nguyen!BK,!et!al. 2009. Colony(

collapse(disorder:(A descriptive(study. PLoS ONE 4(8):!e6481.

This!study!looked!at 91!managed!honey!bee!colonies!from!13!apiaries!in!California!and!Florida!

to!confirm!the!definition!of!CCD,!identify!its!potential!causes and!inform!future!research.!Over!

200!variables!were!quantified!and!compared!between CCDPafflicted!colonies!and!apiaries,!and!

those!not!afflicted.!While!61!of!the!200!variables!were!found!frequently!enough!to!make!

worthwhile!comparisons,!no!single!one!stood!out!as!being!significantly!linked!to!CCD.!The!

comparative!results!did!suggest!some important!trends:!1)!compromised!immunity!is!likely!

playing!a!role;!and!2)!CCD!is!either!a!contagious!condition!or!caused!by!exposure!to!a!common!

risk!factor.!Recommendations!for!future!research!include:!1)!longitudinal!studies!that!monitor!

parasite,!pathogen and!pesticide!loads!while!quantifying!pesticide!tolerance!in!the!study!

populations;!2)!studies!on!the!interactions!among!pesticides!and!pathogen!loads.

3)!vanEngelsdorp!D,!Speybroeck!N,!Evans!JD,!Nguyen!BK,!Mullin!C,!Frazier!M,!et!al.!2010.!Weighing(risk(

factors(associated(with(bee(colony(collapse(disorder(by(classification(and(regression(tree(

analysis. J!Econ!Entomol 103(5):!1517P1523.

To!better!understand!the!relative!importance!and!relationships!among!different!risk!factors!in!

explaining!CCD,!researchers!performed!a classification!and!regression!tree!(CART)!analysis on!

the!data!set!of!the!above epidemiological!study!(vanEngelsdorp!et!al.!2009). This!was!the!first!

case!of!CART!analysis!being!used!to!understand!bee!pathology.!Overall,!the!results!of!the!

analysis!provide!further!evidence!for!the!prevailing!consensus!that!CCD!is!caused!by!multiple!

factors!acting!together!to!decrease!colony!fitness!and!increase!susceptibility!to!disease.!

Pesticides!accounted!for!6!of!the!19!variables!having!greatest!discriminatory!power.!Of!note!is!

the!fact!that,!at!a!level!above!66!ppb!in!developing!bees,!the!Varroa miticide,!coumaphos,!was!

positively!correlated!to!healthy!colonies;!this!was!the!most!predictive!factor!between!the!two!

populations.!Results!indicate!that!pesticides!are!very!likely!involved!in!the!CCD!causal!complex.!
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Among!several!areas!recommended!for!further!study,!two!were!highlighted:!the!effect!of!subP

lethal!pesticide!exposure!on!pathogen!prevalence,!and!the!relationship!between!varying!

tolerance!to!pesticides!and!colony!survival.

3!@ Understanding!pesticides!as!a!causal!factor!in!colony!collapse

Pesticide(Prevalence

Pesticides!are!a!prominent!part!of!the!honey!bee!environment,!both!in!the!hive!and!in!the!larger!

environment. This!is!especially!true!in!the!U.S.,!where!1,200!different!pesticide!active!ingredients!are!

approved!and!in!use!in!18,000!different!product!combinations.!By!contrast,!France!and!Britain!have!

each!registered!around!500!and!300,!respectively.xv

Multiple!surveys!in!the!U.S.!and!Europe!have!shown!that!a!mixture!of!pesticide!formulations!and!types!

are!present in!bees,!wax,!stored!food!and!the!pollen!and!nectar on!which!bees!forage.!Field!studies!have!

found!neonicotinoid!pesticides!in!particular!in!soil,!dust,!planter!exhaust,!water!(guttation)!droplets!

exuded!by!treated!plants!and!on!nearby,!untreated!plants!and!fields.!A!crossPsection!of!these!studies!

follows.

1)!Krupke!C,!Hunt!G,!Eitzer!B,!Andino!G,!Given!K.!2012.!Multiple(routes(of(pesticide(exposure(for(

honey(bees(living(near(agricultural(fields. PLoS!ONE!7(1).

This!field!study!established!that!bees!near!agricultural!fields!are!exposed!to!a!variety!of!

pesticides!via!multiple!routes!at!harmful!levels!throughout!the!foraging!period.!Pesticides!found!

include!the!neonicotinoids!clothianidin!and!thiomexotham,!atrazine!(an!herbicide)!and!

fungicides,!including one known!to!synergize!with!neonicotinoids!(propiconazole).!Soils,!pollen!

(beePcollected!and!directly!from!plants),!dandelions,!dead!and!healthy!bees,!and!planter!waste!

products!were!all!examined!as!potential!exposure!routes.!The!authors!looked!specifically!at!

corn,!which!occupies!more!arable!land!in!North!America!than!any!other!cropȄ88!million!acres.!

Corn!is!planted!throughout!the!U.S.!Midwest!from!midPApril!through!early!May when!the!

energetic!requirements!of!bees!are!increasing!rapidly!as!hives!prepare for!colony!growth,!

requiring!increased!foraging.!Virtually!all!corn!(excepting!the!0.2%!cultivated!organically)!in!the!

U.S.!is!grown!from!treated!seeds,!and!this!study!found!that!bees!forage heavily!on!corn:!corn!

pollen!made!up!over!50%!of!the!pollen!collected!by!bees,!by!volume,!in!10 of!20!samples.!

Authors!also!sampled!dandelions,!which!are!a!preferred!nectar!and!pollen!source!during!this!

period.!Dandelions!in!nearby,!untreated!fields!were contaminated!with!clothianidin.!Soil!from!

fields!which!had!not!been!planted!with!treated!seeds!in!over!two!growing!seasons!tested!

ǡǯ

persistence and!mobility.!

Pollen!collected!from!the!treated!plants!was!contaminated!by!clothianidin!as!expected,!but!beeP

collected!pollen!samples!showed!higher!levels,!indicating!additional!pathways!of!exposure.!

Levels!of!contamination!in!beePcollected!corn!pollen!in!this!study!were!10Pfold!higher!than!

reported!from!an!experiment!on!clothianidinPtreated!canola.!This!is!significant!because!

clothianidin!was!approved!for!use!on!corn!and!canola!simultaneously!based!on!the!canola!field!

test.!The!finding!that!beePcollected!pollen!contained!neonicotinoids!is!of!particular!concern!

because!clothianidin!is!even!more!toxic!when!ingested!orally by!a!bee,!and!because!of!the!

potential!for!harm when!developing!bees!are!exposed!to!pesticides!within!the!hive!through!

stored!pollen.!During!the!period!observed,!nurse!bees!were!emerging!and!they!must!feed!on!
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pollen!reserves!in!the!form!of!royal!jelly.!Authors!calculated!that,!at!the!levels!observed,!a!new!

bee!would!consume!50%!of!the!oral!LD50!during!the!10!days!it!spends!as!a!nurse!bee.!

In!sum,!the!study!established!multiple!exposure!routes,!at!harmful!levels,!at!a!critical!time,!on!

the!most!common!crop.!The!authorsǯ most!salient!finding,!however,!is!the!establishment!of!a!

new,!especially!mobile!and!toxic!exposure!routeȄplanter!exhaust!material.!Corn!seeds!are!sown!

using!an!automated!planting!system!that!relies!on!air/vacuum!mechanisms!to!space!the!seeds;

in!order!to!keep!seeds!treated!with!pesticides!from!sticking!to!one!another,!talc!is!used.!This!talc!

becomes!contaminated!and!is!then!exhausted!during!planting,!either!down!with!the!seed!or!into!

ǤDzdztinoids!and!fungicides!in!planter!

exhaust!material.!

2)!Mullin!CA,!Frazier!M,!Frazier!J,!Ashcraft!S,!Simonds!R,!vanEngelsdorp!D,!et!al.!2010.!High(levels(of(

miticides and(agrochemicals(in(North(American(apiaries:(implications(for(honey(bee(health. PLoS!

ONE 5(3):!e9754.

Researchers!conducted!the!most!extensive!North!American!survey!of!pesticide!residues!in!

managed!honey!bee!colonies!to!date!in!23!states!and!one Canadian!province!during!the!2007P

2008!growing!season.!They!used!conventional!(gas!chromatographyPmass!spectrometry)!and!

recently!developed!(liquid!chromatographyPtandem!mass!spectrometry)!analytical!techniques!

to!detect!any!of!200!pesticides!and!their!metabolites!at!concentration!levels!as!low!as!0.1!ppb!in

a!representative!crossPsection!of!bees,!pollen and!wax.!A!total!of!121 pesticides!and!metabolites

comprising!5,519!total!residues were!detected!and!quantified!in!887 samples.!Wax!samples!

averaged!8,!pollen!samples!averaged!7.1,!and!bee!samples!averaged!2.5!different!pesticide!

residues!each,!with!at!least!two pesticides!detected!in!92%!of!all!samples!analyzed.!Pyrethroids,!

a!group!of!widely!used!pesticides!that!are!toxic!to!bees, were!the!dominant!class!of!insecticides!

detected!in!all!samples.!Nearly!half (49.9%) of!all!samples!contained!at!least!one systemic!

pesticide. Pollen!contained!high!levels!of!fungicides,!which!tended!to!coPoccur!with!low!levels!of!

systemic!pesticides,!implicating!possible!synergistic!effects.!

DzThe!widespread!occurrence!of!multiple!residues,!some!at!toxic!

levels!for!single!compounds,!and!the!lack!of!any!scientific!literature!on!the!biological!

consequences!of!combinations!of!pesticides,!argues!strongly!for!urgent!changes!in!regulatory!

policies!regarding!pesticide!registration!and!monitoring!procedures!as!they!relate!to!pollinator!

safety.!This!further!calls!for!emergency!funding!to!address!the!myriad!holes!in!our!scientific!

understanding!of!pesticide!consequences Ǥdz

3)!vanEngelsdorp!D,!Evans!JD,!Donovall!L,!Mullin!C,!Frazier!M,!Frazier!J,!et!al.!2009.!Dzdzǣ

a(new(condition(in(honey(bee(colonies(associated(with(increased(risk(of(colony(mortality.))J!

Invertebr!Pathol 101(2): 147P149.

Two!U.S.!longitudinal!studies!that!sought!to!uncover!causes!of!poor!colony!health (and!CCD!in!

particular),!were!begun!in!spring!of!2007.!During!both!studies,!researchers!discovered!a!

phenomenon known!as!entombed!pollen,!in!which!bees!responsible!for!managing!food!stores!in!

the!hive!seal!off!some!pollen!with!propolis!and!wax.!This!behavior!is!generally!used!by!bees!to!

quarantine!microbial!threats,!such!as!an!invading lizard!or!mouse!that!dies!inside!the!hive.!

Entombed!pollen!had!much!higher!levels!of!three!specific!pesticides!(two!miticides!and!one!

fungicide)!than!typical pollen,!and!notably!had!no!detectable!microbes.!Hives!with!such!pollen!in!

late!spring!were!twice!as!likely!to!die!in!midPfall!as!hives!with!normal!pollen.!Incidence!of!

entombed!pollen!was!notably!greater!in!reused!wax!comb!regardless!of!any!disinfection!
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treatments!applied,!which!suggests!there!is!a!transmittable!factor!common!to!both!entombing!

behavior!and!colony!death.!Researchers!urged!further!study.!

4)!Bonmatin!JM,!Marchand!PA,!Charvet!R,!Moineau!I,!Bengsch!ER,!Colin!ME. 2005. Quantification(of(

imidacloprid(uptake(in(maize(crops. J!Agr!Food!Chem 53: 5336P5341.

Researchers!in!France!conducted!a!threePyear,!randomPsample!field!study!of!environmental!

contamination!by!the!most!prevalent!neonicotinoid,!imidacloprid,!in!corn!fields!with!active!bee!

colonies.!This!study!was!part!of!a!national!research!effort!seeking!to!evaluate!the!environmental!

risk!of!imidacloprid!to!honey!bees.!Using!a!recently!developed!method!capable!of!quantifying!

imidacloprid at!1!ppb and!detecting!it!at!0.1!ppb!(high!pressure!liquid!chromatographyȂmass!

tandem!spectrometry,!HPLC/MS/MS),!researchers!established!that!imidacloprid!is!habitually!

present!in!flowering,!treated!corn!at!levels!known!from!previous!dose!studies!to!induce!a!variety!

of!harmful!effects,!including!eventual!death,!for!honey!bees.!Average!levels were!6.6!ppb!for!

pollenPproducing!flowers,!4.1!ppb!for!stems!and!leaves,!and!2.1!ppb!for!pollen!itself.!These!levels!

are!similar!to!those!previously!studied!for!sunflower!and!canola.!ImidaclopridPcontaminated!

corn!pollen!made!up!54%!of!pollen!samples!collected!at!hive!entrances,!reflecting!a!mixture!of!

pollen!sources!and!with!a!correspondingly!lower!average!contamination!of!0.6!ppb.!

Neonicotinoids ::(Acute,(sub5lethal(&(chronic(effects

Neonicotinoids!are!a!relatively!new,!and!very!widely!used!class!of!insecticides!that!work!on!the!central!

nervous!system!of!sucking!insects!such!as!fleas!and!aphids.xvi They!were!introduced!in!the!1990s!and!

have!since!become!the!fastestPgrowing!class!of!insecticides!in!the!history!of!synthetic!pesticides.!By!

2005!neonicotinoids!had!gained ͳ͸Ψ̀ ͅǡ

nearPȋ͹͹ΨȌ̀ͳͷͷ

̀ͷ͵ͷǤxvii Among!their!approved!uses!in!the!U.S.!are!topical!flea!treatments!for!pets,!lawn!

and!garden!uses,!and!a!variety!of!agricultural!uses!including!stone!fruits,!nuts,!canola,!sunflowers!and!

corn.!!

Neonicotinoids!fall!into!two!subclasses:!nitroguanidines!and!cyanoamidines.! The!nitroguanidines,!

which!are!highly!acutely!toxic!to!honey!bees,!include!imidacloprid,!clothianidin,!thiamethoxam!and!

dinotefuran.!The!cyanoamidines!are!not!as!acutely!toxic!to!honey!bees!and!include!thiacloprid!and!

acetamiprid. Neonicotinoids!are!known!to!persist!in!soil!for!years!and!have!the potential!to!accumulate!

in!soil.!These!chemicals are also highly!water!soluble,!and!are!present!throughout!treated!plants!from!

seed!to!harvest.!

To!date,!most!U.S.!regulatory!decisionmaking!addressing!the!risks!posed!to!honey!bees!by!

neonicotinoids!has!hinged,!by!default,!on!the!establishment!of!acute!toxicity!exposure!scenarios!without!

requiring!tests!for!subPlethal!effects.xviii The!standard!laboratory!method!for assessing!pesticide!risk!is

determine!the!median!lethal!dose!(LD50)!required!to!kill!half!the!tested!population!over!a!certain!

timeframe.!In!the!U.S. this!protocol!remains!the!primary!basis!for!risk!assessment!in!pesticide!

registration. However,!this!approach!to!risk!assessment!only!takes!into!account!the!survival!of!adult!

honey!bees!exposed!to!pesticides!over!a!short!time!frame:!the!typical!acute!toxicity!test!is!48!hours!

although!it!may!be!extended!to!96!hours.!Acute toxicity!tests!establishing!LD50 levels!on!adult!honey!

bees!may!be!particularly!illPsuited!for!the!testing!of!systemic!pesticides!because!of!the!ways!bees!are!

exposed!to!systemics!in!the!fieldȄin!smaller!doses,!over!a!long!period!of!time,!through!a!variety!of!

routes.!Chronic!feeding!tests!using!whole!colonies!have!been!suggested!better!way!to!quantify!the!

effects!of!systemics.xix
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Despite repeated!calls!for!a!reevaluation!of!pesticide!testing!protocols,!regulatory!processes!in!the!U.S.!

and!Europe!have not!been!adapted!to!consider!subPlethal,!chronic!or!synergistic!effects!of!pesticides!on!

pollinators. xx,

Key(terms(for(describing(pesticide impacts

Acute*toxicity*:: The!acute!toxicity!of!a pesticide to!bees!(either!by!contact!or!ingestion) is!

quantified!by!noting!the!dose!at!which half!of!the!insects!die!within!a!specific!time!period.!This!is!

known!dose!that!is!lethal!to!50%!of!the!test!population!or!LD50.!DzdzDzdz

used!synonymously,!but!a range!of!effects,!such!as!general!agitation,!vomiting,!wing!paralysis,!

arching!of!the!abdomen!similar!to!a!sting!reflex!and!uncoordinated!movement, have!been!

observed!in!association!with!acute!doses .

xxi The!acceptable!risk!for!each!pesticide!is!set!by!

regulatory!agencies!based!on!acute!toxicity!tests!and!the!expected!rate!and!mode!of!pesticide!

application,!both!of!which!are!determined!by!the!manufacturer.!The!LD50 values!of!

neonicotinoids!are!low!compared!to!older!classes!of!insecticides;!they!are!considered!highly!

toxic!to!honey!bees.!

Sub=lethal*toxicity*::*The!subPlethal!toxicity!of!neonicotinoid!pesticides!is!of!particular!concern!

because!the!most!common!field!

exposure!scenarios!are!likely!at!the!

subPlethal!rather!than!acute!level.!SubP

lethal!effects!of!neonicotinoids!on!

honey!bees!include!behavioral!

disruptions!such!as!disorientation,!

reduced!foraging,!impaired!memory!

and!learning,!and!shifts!in!

communication!behaviors.!Other!

important!subPlethal!effects!might!

include!compromised!immunity,!

delayed!development!and!a!host!of!

indirect,!potentially!cascading!effects!

ǯ

itself.!!

Cumulative and*chronic effects*::

Neonicotinoids!function!by!binding!to!

niconitic!acetylcholine!receptors!in!

ǯǡ

particularly!abundant!in!bees,!

increasing!during!development!from!

larval!to!adult!stages.

xxii This!binding!

leads!to!an!overPaccumulation!of!

acetylcholine,!resulting!in!paralysis!

and!death. The!most!recent!scientific!

observations!point!to!a!longPlasting!effect!in!which!molecules!unbind!from!receptors,!but!remain!

in!the!bee!brain,!possibly!rebinding!multiple!times!before!metabolization!occurs.

xxiii Whether!

this!constitutes!effectively!irreversible,!cumulative!toxicity!remains!unclear;!but!chronic!toxicity!

effects!over!time!are!a!likely!result.!

Neonicotinoids*at=a=glance

Neonicotinoids!are!a!widely!used!class!of!systemic!

insecticides!introduced!in!the!early!1990s!that!have!been!of!

particular!interest for!their effects!on!honey!bees.!

They!can!be!applied!as!a!spray (foliar)!or,!more!commonly,!

used!as!systemics.!Systemic!pesticides!are!applied!as!seed!

coatings!or!soil!drenches!and!are!taken!up!through!the!

ǯǡ

the!plant,!including!pollen!and!nectar.!Neonicotinoids!are!

very!persistent!and!therefore!accumulate!over!time!in!the!

environment.!

Most neonicotinoids are!classified!as!acutely!toxic!to!bees.!

But!single,!highPdose!(i.e.!acute)!exposures!are!likely!less!

common!than!are!the!chronic,!subPlethal!exposure!levels!

faced!by!bees!as!they!forage!in!the!field.!

Honey!bee!colony!collapses!in!France!in!1999 were!the!first!

to!implicate!imidacloprid,!the!most!widely!used!

neonicotinoid,!in colony!loss.!Researchers have!since!found!a!

range!of!subPlethal!effects!caused!by!neonicotinoids: altered!

foraging!and!feeding!behavior and!impaired!orientation,!

social!communication,!undermined!immunity!and!delayed!

larval!development.!
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Many!independent!studies!in!the!U.S.!and!in!Europe!have!shown!that!small!amounts!of!neonicotinoidsȄ

both!alone!and!in!combination!with!other!pesticidesȄcan!cause!impaired!communication,!

disorientation,!decreased!longevity,!suppressed!immunity and!disruption!of brood!cycles!in!honey!bees.

A!selection!of!these!studies!follow.

1)!Medrzycki!P,!Montanari!R,!Bortolotti!L,!Sabatini!AG,!Maini!S,!Porrini!C.!2003. Effects(of(imidacloprid(

administered(in(subKlethal(doses(on(honey(bee(behaviour.(Laboratory(tests. B!Insectol 56(1):!59P

62.

In!this!study,!researchers!monitored!the!behavior!of!adult!forager!bees!in!highly!controlled,!

laboratory!conditions!to!observe!effects!of!imidacloprid!fed!at!subPlethal doses!in!sugar!solution.!

Four!different!contamination!concentrations!were!tested:!100!ppb!and!500!ppb!as!a!single!dose!

(20!microliters),!and!each!concentration!as!a!continuously!available!food!source!for!24!hours.!

Three!replications!of!the!experiment!were!performed.!Researchers!concluded!that!imidacloprid!

at!all!doses!given!caused!significant!reductions!in!mobility!that!lasted!for!one!to!several!hours,!

with!bees!remaining!stationary!for!longer!periods!as!well!as!moving!more!slowly.!They!also!

noted!that!bees!seemed!to!lose!their!communicative ability!at!all!doses,!failing!to!coordinate!

their!activity!with!other!bees.!They!recommended!further!study!to!better!understand!the!length!

of!time!that!these!effects!last,!and!to!investigate!the!effects!on social!behavior!essential!to!the!

proper!functioning!of!a!hive.

3)!Colin!ME,!et!al.!2004.!A(method(to(quantify(and(analyze(the(foraging(activity(of(honey(bees:(

relevance(to(the(subKlethal(effects(induced(by(systemic(pesticides.!Archives!of!Environmental!

Contamination!and!Toxicology!47:!387P395.

This!study!investigated!the!subPlethal!effects!of!two!insecticides!in!semiPfield!conditions!on!the!

foraging!behavior!of!honey!bees.!Imidacloprid!and!fipronil!were!chosen!because!both!behave!

systemically,!were!recently!introduced,!considered!highly!toxic!to!bees,!had!shown!subPlethal!

effects!on!bees!in!lab!conditions!and!had!been!implicated!in!honey!productivity!declines!in!

Europe.!The!primary!aim!was!to!address!a!gap!in!environmental!assessment!of!systemic!

pesticides!by!improving!on!the!methods!used!to!quantify!foraging!behavior!changes.!Bee!

colonies!were!placed!in!enclosed!tunnels!and!their!feeding!behavior!video!recorded!over!a!

period!of!five!days,!constituting!a!cumulative!effects!study!much!shorter!than!a!bee!or!hive!

lifecycle!study!would!be.!With!imidacloprid!at!6.0!μg/kg,!inactive!beesȄthose!visiting!the!

feeder,!but!not!feedingȄincreased!over!time!in!relation!to!active!bees.!With!fipronil!at!2.0!μg/kg,!

most!bees!stopped!coming!to!the!feeder!by!the!last!day,!and!the!few!that!did!tended!to!be!

inactive.!Convulsions!and!paralysis!were!also!observed!in!bees!feeding!on!fipronilPcontaminated!

Ǥǯǡǡ

subPlethal!levels!70!times!below!the!referenced!LD50s.!They!also!concluded!that!their!

Dzprovided!an indispensable!interface!between!controlled!conditions!in!

the!laboratory!and!the!field,dz which!suggests!its!adoption!in!regulatory!testing!of!subPlethal!

effects.!

4)!Decourtye!A,!Armengaud!C,!Renou!M,!Devillers!J,!Cluzeau!S,!Gauthier!M,!et!al.!2004.!Imidacloprid(

impairs(memory(and(brain(metabolism(in(the(honeybee((Apis(mellifera(L.). Pestic!Biochem!Phys

78:!83P92.

This!laboratory!study!shows!subPlethal effects!at!low!concentrations!of!imidacloprid!using!four

different!methodologies. Researchers!sought!to!build!from!prior!studies!that!clarified!the!role!of!
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nicotinic!acetylcholine receptors!(nAChR)!in!honey!bee!learning!and!memory!capacities!to!

investigate!the!specific!effects!of!the!neonicotinoid!imidacloprid!on!those!capacities.!Bees!were!

conditioned!to!respond!to!a!specific!floral!scent!in!association!with!food,!a!behavior!that!

demonstrates!associative!learning!and!memory!formation.!Behavioral!results!led!researchers!to!

conclude!that!imidacloprid!at!a!dose!of!12!ng/bee!significantly!inhibited!associative!learning!as!

well!as!retention!of!successfully!learned!associations,!whether!learned!before,!during!or!after!

exposure.!This!impairment of!retention!notably!affected mediumPterm,!but!not!longP or!shortP

term!memory,!appearing!as!a!temporary!amnesia.!Metabolic!activity!in!specific!parts!of!the!bee!

brain!were!also!analyzed!for!changes!in!response!to!imidacloprid.!!These!results!indicated!that!

at!both!doses,!imidacloprid!was!acting!detrimentally!in!the!parts!of!the!bee!brain!involved!with!

associative!and!contextual!memory.!

5)!Yang!EC,!Chuang!YC,!Chen!YL,!Chang!LH.!2008.!Abnormal(foraging(behavior(induced(by(sublethal(

dosage(of(imidacloprid(in(the(honey(bee((Hymenoptera:(Apidae).!J!of!Econ!Entomol 101(6):!1743P

1748.

Researchers!conducted!a!semiPfield!experiment!to!follow!on!results!of!laboratory!studies!that!

had!shown!a!number!of!detrimental!effects!from!ingestion!of!imidaclopridPcontaminated food.!

They!tested!the!time!intervals!between!individual!worker!bee!visits!to!a!feeder!after!ingestion!of!

imidaclopridPcontaminated!sugar!solution!at!concentrations!ranging from!40!μg/L!to!6,000!

μg/L.!Bees!showed!abnormal!foraging!behavior!beginning!at!50!μg/L!(41.6!ppb)!and!worsening!

with!higher!contamination.!Effects!ranged!from!a!statistically!significant!time!delay!between!

feeder!visits,!to!disappearance!(from!feeder!and!hive)!for!a!full!day!without!return.!At!1600!

μg/L,!more!than!90%!of!bees!went!missing!for!a!full!day,!but!all!returned!the!following!day.!

Above!this!concentration,!a!portion!of!bees!did!not!return,!and!above!800!μg/L,!bees!that!

returned!from!long!delays!continued!to!show!abnormal!foraging!behavior.!Based!on!an!

estimation!of!average!meal!size!per!feeder!visit,!abnormal!foraging!behavior!began!a!dose!per!

bee!of!1.82P4.33!nanograms.!Researchers!concluded!that!abnormal!foraging!behavior!could!

occur!in!the!field!through!multiple!visits!to!imidaclopridPcontaminated!flowers.

Synergistic(+(Combined(Effects

Synergism!is!a!phenomenon!in!which!two!or!more!factors!produce!a!combined!effect!that!is!greater!than!

the!sum!of!their!separate!effects.!As!investigations!into!the!causes!of!CCD!have!continued!to!point!

toward!multiple!factors!working!in!concert!to!increase!bees!susceptibility!to!disease,!synergism!and!

combined!effects!have!emerged!as!a!critical!area!of!research.!

In!2004,!a!lab!study!(see!#!1!below)!showed!that!the!acute!toxicity!of!two!neonicotinoid!pesticides!on!

honey!bees!dramatically!increases!when!combined!with!either!of!two!common!fungicides.!Four!years!

after!this!finding!was!published,!researchers!established!that!these!types!of!combinations!are!prevalent!

in!bee!hives.xxiv Between!2010!and!2012,!three!separate!studies!demonstrated!synergism between!the!

common!parasite!Nosema)and!pesticide!exposure!(thiacloprid,!imidacloprid!and!the!nonPneonicotinoid!

systemic!fipronil).!Hives!exposed!to!these!pesticides!were!significantly!more!susceptible!to!infection.

͞ŚĞŵŝĐĂůĐŽĐŬƚĂŝůƐ͗͗͟&ƵŶŐŝĐŝĚĞƐ͕Ɖyrethroid(insecticides,(miticides
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Neonicotinoids!are!but!one!class!of!pesticides,!honey!bees!are!exposed!to!dozens of!different!pesticides

ȋDz,dz esp.!Mullin!2010).!Included!among!these!are!a!mix,!or!

Dzǡdz of!insecticides,!herbicides!and!fungicides!as!well!as!the!miticides!used!by!

beekeepers!to control!pathogens!in!the!hive.!

NonPneonicotinoid!pesticides!of!special!concern!for!their!impacts!on!bees,!and!potential!role!in!the!

causal!complex!of!CCD!are!fungicides!and!pyrethroidsȄparticularly!in!combination.!Fungicides!have!

long!been!thought!to!be!relatively!harmless!to!bees,!but!their!recent,!dramatic!uptick!in!use!(especially!

with!corn,!beginning!around!2007)xxv coupled!with!their!synergizing!effect!on!certain!insectides!

(including!neonicotinoids!and!pyrethroids)!have!brought!renewed!attention.!Pyrethroids!are!highly!

variable!in!their!toxicity!to!bees,!but!have!come!under!recent!scrutiny!both!because!their!high!fat!

solubility!means!that!pyrethroids!persist!and!bioaccumulate!in!bee!wax,!and!because!they!are!known!to!

synergize!with!certain!fungicides.!

ǡDzdz

composed!of!certain!fungicides,!pyrethroids!and!neonicotinoids!are!slowly!coming!into!resolution.!

ǯ

relatively!few!genes!that!encode!detoxification!enzymes.!For!example,!one!such!group!of!enzymes!

(P450s)!mediate!detoxification!pathways!in!ways!that!can!be!inhibited!by!certain!widely!used!fungicides!

ȋǤǤȌǡǯ

miticide,!fluvalinate.xxvi Although!current!data!are!unclear,!another!hypothesis!as!to!the!physiological!

mechanisms!by!which!synergistic!effects!between!pathogens!and!pesticides!may!operate!is!that!

pathogen!metabolites!may!interfere!with!the!detoxification!process.xxvii

1)!Iwasa!T,!Motoyama!N,!Ambrose!JT!and!Roe!M.!2004. Mechanism(for(the(differential(toxicity(of(

neonicotinoid(insecticides(in(the(honey(bee,(Apis(mellifera.!Crop!Protection!23(5):!371.

This!laboratory!study!established!a!synergistic!effect!between!neonicotinoids!and!fungicides.!A!

neonicotinoid!was!found!to!be!up!to!1,141!times!more!toxic!to!bees!when!combined!with!a!

common!fungicide.!Researchers!performed!laboratory!tests!for!acute!toxicity!of!several!

neonicotinoid!pesticides!and!metabolites,!both!alone!and!combined!with!each!of!several!

fungicides!commonly!used!in!crop!production.!These!tests!were!for!contact!toxicity,!rather!than!

oral!ingestion.!They!found!that!three!combinations!between!a!neonicotinoid!and!a!fungicide!

were highly synergistic.!Acetamiprid,!a!neonicotinoid!that!is!much!less!acutely!toxic!than!

imidacloprid,!becomes!244!times!more!so when!combined!with!the!fungicide!triflumizole.!

Thiacloprid,!also!much less!toxic!than!imidacloprid,!becomes!559!times!more!so when!combined!

with!the!fungicide!propiconazole,!and!1,141!times!more!toxic!when!combined!with triflumizole.!

Though!plants!treated!with!the!maximum!recommended!levels!for!agricultural!use!did!not!

exhibit!a!statistically!significant!effect!on!honey!bee mortality!after!three!and!24!hours, further!

study!was!suggested before!reaching!conclusion!as!to!inPfield,!synergistic!toxicity.

2)!Smodis!Skerl!MI,!Kmecl!V,!Gregorc!A. 2010. Exposure(to(pesticides(at(sublethal(level(and(their(

distribution(within(a(honey(bee((Apis(mellifera)(colony. Bulletin!of!Environmental!Contamination!

and!Toxicology 85(2):!125P8.

This!study!examined!the!role!and!prevalence!of!pesticides!in!honeybee!colonies,!both!those!

introduced!intentionally!by!beekeepers!to!control!mites!(acaricides)!as!well!as!agricultural!

chemicals!found!incidentally!in!the!hive. The!goal!was!to!determine!whether,!and!if!so,!how!

much,!pesticides!accumulated!in!the!bodies!of!bees!in!the!hive,!in!the!royal!jelly,!and!in!bee!

larvae!as!well!as!how!the!chemicals!are!spread!throughout!the!hive. The!acaricides!introduced!
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into!the colony!were!amitraz,!coumaphos and!fluvalinate,!while!the!organophosphate!diazinon!

served!as!a!representative!agricultural!chemical.!The!acaricides!were!applied!following!

commonly!accepted!regimes!to!treat!against!the!Varroa)destructor,!and!the!diazinon!was!

introduced!in!accordance!with!a!treatment!plan!that!would!be!applicable!to!an!apple!orchard.!

Coumaphos!and!fluvalinate!were!the!most!prevalent!throughout!the!colony.!No!diazinon!was!

found!in!the!samples!tested!and!amitraz!only!in!amounts!below!the!level!of!detection.!

Coumaphos!was!found!in!royal!jelly!from!nurse!bees,!while!fluvalinate!was!found!in!the!bodies!

of!bees!and!in!bee!larvae.!The!presence!of!fluvalinate!throughout!the!colony,!especially!in!larvae,!

demonstrate!that!chemicals!in!the!hive!can!be!transmitted!from!bee!to!bee!as!well!as!to!food!and!

thus!to!larvae,!spreading!throughout!the!entire!colony.

3)!Dai!PPL,!Wang!Q,!Sun!JPH,!Liu!F,!Wang!X,!Wu!YPY!and!Zhou!T. 2010. Effects(of(subKlethal

concentrations(of(bifenthrin(and(deltamethrin(on(fecundity,(growth,(and(development(of(the(

honeybee(Apis(mellifera(ligustica. Environmental!Toxicology!and!Chemistry 29: 644Ȃ649.

This!study!examined!the!subPlethal effects!of!two!pyrethroid!insecticides,!bifenthrin!and!

deltamethrin,!on!honeybee!health.!The!study!was!performed!on!colonies!in!the!laboratory!that!

were!fed!small!doses!of!the!chemicals.!Effects!measured!included!fecundity,!growth and!the!

development!of!individual!bees.!The!importance!of!these!particular!issues!for!overall!health!of!

the!colony!and!general!bee!population!was!emphasized.!Data!was!taken!over!several!years!and!

results!measured!against!control!colonies!not!fed!the!insecticides.!The!authors!found!that!

DzdzDzdzǤ

findings!that!exposure!to!the!insecticides!significantly!reduced!colony!fecundity!due!to!reduced!

rates!of!egg!laying!and!impaired ability!of!the!colony!to!transition!to!a!new!queen.!

Developmental!effects!on!honeybee!larvae!were!also!observed.!In!comparing!their!findings!to!

ǡǯ

bifenthrin!application!rates!are!higher!than!the!ǯ50 for!honey bees!and!

deltamethrin!application!rates!are!about!the!same!as!the!LC5.

Pathogen(interactions(::(Nosema(+(pesticides

Nosema,!a!family!of!fungal!gut!parasites, and!the!Varroa)destructor mite!are!two!relatively!recent!honey!

bee!pathogens.!A!particularly!virulent!and!newly!emergent!(ca.!2005)!strand!of!Nosema,!Nosema)

ceranae,)has!become!an!area!of!research!and!concern!around!the!world,!especially!in!Spain.!Both!

pathogens!have!been!shown!to!interact!with!pesticides!to!weaken!colony!health!more!than!either!does!

alone.!Nosema)is!a!fastPǯ

ability!to!absorb!nutrients!(infected!bees!consume!significantly!more!calories),!and!known!to!suppress!

immune!response.xxviii Varroa)mites!act!as!vectors,!transmitting!disease!across!and!within!colonies.!

With!the!relatively!recent!observation!that!CCDPaffected!hives!are!marked!by!an!overall!increased!and!

variable!pathogen!load,!but!with!no!one!pathogen!found to!consistently!correlate!with!hive!loss,!

researchers!have!begun!looking!for!what!is!making!the!bees!susceptible!to!disease!to!begin!with.xxix

Three!separate!studies!between!2010!and!2012!(below)!have!demonstrated!a!synergistic!effect!between!

pesticides!and!the!pathogen!Nosema.)The!most!recent!study!by!leading!USDA!bee!researchers!found!that!

bees!with!undetectable!levels!of!imidaclopridȄto!!which!they!were!exposed!only!indirectly!in!brood!

food!as!developing!larvaȄfaced significantly!more!Nosema)infections!than!did!their!control!

counterparts.!
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The!overall!pattern!for!bees!exposed!both!to!systemic!pesticides!(neonicotinoids!and!fipronil)!and!

Nosema)infection!in!these!studies!is!that!bees!get!sick!more!easily!and!die!sooner!as!a!result!of!both!

stressors!in!combination!than!either!in!isolation.!

1)!Pettis!JS,!vanEngelsdorp!D,!Johnson!J,!Dively!G.!2012.!Pesticide(exposure(in(honey(bees(results(in(

increased(levels(of(the(gut(pathogen(Nosema. Naturwissenschaften.

This!study!demonstrated!increased!pathogen!growth!among individual!bees!reared!in!colonies!

exposed!to!imidacloprid!at!levels!below!those!considered!to!have!sublethal!impacts.!Researchers!

exposed!honey!bee!colonies during!three!brood!generations!to!subPlethal!doses!of!a!widely!used!

pesticide,!imidacloprid,!and!then!challenged!newly!emerged!bees!with!Nosema.!They!used!

GC/MS!(gas!chromatographyPmass!spectrometry)!with!a!limit!of!detection!of!0.1!ppb!to!analyze!

the!bees,!and!verified!pesticide!exposure!to!colonies!by!measuring!the!weekly!consumption!of!

the!treated!protein!patties!and!by!analyzing!imidacloprid!in!stored!bee!bread.!Nosema infections!

increased!significantly!in!the!bees!from!pesticidePtreated!hives!when!compared!to!bees!from!

control!hives.!Newly!emerging!bees!which!tested!negative!for!imidacloprid,!but!had!been!

exposed!in!the!hive,!were!also!significantly!lighter in!weight. As!noted!by!the!authors,!this!study!

is!distinct!from!previous!studies!establishing!this!synergistic!effect!(esp.!Vidau!et!al.!and!Alaux!et!

al.,!below)!in!its!focus!on!larva!exposed!only!indirectly!via!brood!food!tended!by!nurse!bees!that!

had!eaten!imidaclopridPǤDz

exposure!durinǤdz

The!finding!that!individual!bees!with!undetectable)levels of!imidacloprid,!after!being!reared!in!a!

subPlethal!pesticide!environment!within!the!colony,!had!higher!Nosema)is!significant in!itself.!It!

also!has!suggestive!implications!that!the!authors!do!not!tease!out!beyond!noting!that!future!

research!should!be!conducted!at!the!hive!level!over!multiple!generations!rather!than!at!the!

individual!bee!level.!Authors!do!Dz

incorporate!inǤdzǣ

Dz

mortality!of!honey!bee!colonies,!including!colony!collapse!disorder,!and!other!pollinator!declines!

Ǥdz

2)!Vidau!C, Diogon!M, Aufauvre!J, Fontbonne!R, Viguès!B, et!al. 2011. Exposure(to(sublethal(doses(of(

fipronil(and(thiacloprid(highly(increases(mortality(of(honeybees(previously(infected(by Nosema*

ceranae. PLoS!ONE 6(6): e21550.

In!this!laboratory!study!individual!bees!were!infected!with!Nosema)ceranae,!exposed!10!days!

later!to!systemic!pesticides!fipronil!and!thiacloprid!(a!neonicotinoid).!The main!finding!was!that!

infected!bees!have!a!much!higher!mortality!rate!than!uninfected!bees!when!exposed!to!the!same!

sublethal!level!of!fipronil!or!thiacloprid!(71%!P 82%!vs.!47%).!Infected!bees!also!appeared!to!be!

generally!more!sensitive!to!sublethal!pesǡDzǡ

uninfected!honey!bees!did!not!display!any!signs!of!intoxication.!By!contrast,!at!this!level!of!

exposure,!insecticides!triggered!aggressiveness!and!tremors!in!infected!honey!bees!during!the!

first!days!of!exǤdz

main!symptom!of!N.)ceranae)infection!in!itself:!infected!bees!consumed!much!more!sucrose!than!

uninfected!bees.!Authors!note!that!while!the!synergistic!effect!observed!by!Alaux!et!al.!(2010)!

seemed!to!be!linked!to!increased!sucrose!consumption,!their!findings!were!not.!

A!secondary,!and!by!this!data!unconfirmed,!hypothesis!pursued!by!this!study!was!that!the!

mechanism!of!the!synergistic!effect!between!Nosema and!these!pesticides!was!linked!to!a!
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on!the!mechanisms!underlying!synergistic!effects!between!pesticides!and!pathogens!is!poorly!

ǡǡDzects!to!pesticides!is!a!more!complex!phenomenon!

than!previously!thought.!The!influence!of!parasitism!in!the!ecosystem!must!be!considered!in!

ǥǤȏȐN.)ceranae)spreads!rapidly!and!can!affect!more!than!

80%!of!honey!bee!colonǤdz

3)!Alaux!C,!Brunet!J,!Dussaubat!C,!et!al.!2010.!Interactions(between(Nosema(microspores(and(a(

neonicotinoid(weaken(honeybees((Apis(mellifera). Environmental!Microbiology 12(3):!774Ȃ782.!

Researchers!conducted!a!laboratory!experiment!to!test!an!emerging,!but!unstudied hypothesis!

that!high!colony losses might!be!attributable!to!a!combination!of!two!factors!each!known!to!have!

effect!on!honey!bee!colony!health:!a!fungal!gutPpathogen!(Nosema)ceranae)!and!a!neonicotinoid!

(imidacloprid).!Their!results!indicate!that!a!synergistic!interaction!is!occurring!that!significantly!

weakens bees,!both!individually!and!socially.!Concentrations!of!orally!administered!imidacloprid!

were!0.7,!7.0,!and!70!μg/kg,!made!available!for!10!hours!per!day!over!the!study!period!of!10!

days.!These!concentration!levels!are!based!on!several!studies!that!showed!environmental!levels!

of!imidacloprid!in!the!honey!and!pollen!of!treated!crops!reached!5.0 μg/kg. The!highest!

individual!death!rates!and!energetic!stress!occurred!with!the!combination!of!both!agents!

Ǥǯ

ability!to!sterilize!the!hive!food!for!adults!and!larvae!significantly!decreased!only!when!the!two!

Ǥǯ

ability!to!withstand!a!broad!range!of!pathogens!in!the!longPterm.!

4)!Wu!JY,!Anelli!CM,!Sheppard!WS.!2011.!SubKlethal(effects(of(pesticide(residues(in(brood(comb(on(

worker(honey(bee((Apis(mellifera)(development(and(longevity.!PLoS!ONE 6(2):!e14720.

This!was!the!first!study!to!establish!subPlethal!effects!on!worker!bees!from!pesticide!residues!in!

contaminated!brood!comb.!Observed!effects!in!this!laboratory!study!included: delayed!

development!of!larval!worker!bees,!delayed!adult!emergence!and!reduced!adult!longevity in!

larvae!reared!in!cells!contaminated!with!the!miticides!fluvalinate!(a!pyrethroid)!or!coumaphos.!

These!effects!can!impact!colony!viability!indirectly!by!causing!premature!shifts!in!hive!roles,!

foraging!activity!and!population!dynamics,!and!by!creating!increased!developmental!time!for!

Varroa)mites!which!in!turn!can!render!a!hive!more!susceptible!to!this!common!parasite!and!

disease!vector.!Pesticide!residues!rapidly!migrated!from!treatment!to!control!comb,!with!a!

corresponding!change!in!developmental!and!longevity!effects:!broods!raised!in!treatment!comb!

gradually!had!reduced!effects!while!those!raised!in!control!comb!(successively!more!

contaminated!with!each!brood!cycle)!had!increased!effects.!Brood!deaths followed!this!trend,!

with!high!rates!occurring!after!multiple!cycles!in!progressively!more!contaminated!control!

comb,!and!significantly!lower!rates!occurring!in!treatment!comb!as!its!contamination!level!

lessened.

Microbiota(out(of(balance(::(Gut(cultures,(immunity(+(nutrition(

Unintentional!disruption!of!natural,!symbiotic!bee!microbial!cultures!is!one!way!in!which!hive!health!

may!be!critically!undermined!by!pesticides!as!well!as!other!stressors!in!the!contemporary,!commercial!

beekeeping!environment.!

Honey!bee!microbiota!(including!fungi,!bacteria,!viruses,!etc.)!exists!at!two!major!levels:!within!the!

DzdzǤ
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ǯǡ

enough!to!describe!a!coPevolveǡǡDzǡdz

well!as!hypothesize!about!key!functions!susceptible!to!disruptionȄspecifically!nutrition!and!immunity.!

For!example,!within!the!larger!hive!environment,!beebread!is!the!most!microbially!active,!although!

whether!this!activity!serves!primarily!to!preserve!or!process!nutrients!is!not!well!understood.!Scientists!

studying!CCD!have!also!recently!found!a!consistent!difference!in!the!microbial!abundance!profile!of!

affected!vs.!healthy!hives.xxx

Emerging!research!in!this!area!has!been!made!possible!in!part!through!recent!breakthroughs!in!new,!

highPthroughput!metagenomic!sequencing!technologies.!These!tools!allow!scientists!to!both!better!

characterize!insect!microbial!life!in!ways!that!focus!on!how!hosts!and!symbionts!interact!functionally,!at!

the!epigenetic!level.!!

1)!Anderson,!K.E.,!Sheehan,!et al.)2011. An(emerging(paradigm(of(colony(health:(microbial(balance(

of(the(honey(bee(and(hive((Apis*mellifera).(Insectes!Sociaux 58:!431P444.

Taking!CCD!as!its!critical!context,!this!review!article!argues!that!research!attention!should!be!

paid!to!the!symbiotic!microbial!communities!that!play!critical!roles!in!bee!nutrition!and!

pathogen!defense.!Authors!note!that!most!important!immunePrelated!function!of!a!gut

microbiota!may!be!the!ability!to!obstruct!colonization!by!pathogens,!thereby!preventing!

infections.!In!this!context,!the!wellPknown!pathogen!Nosema)ceranae, is!listed!as!an!infectious!

microbe!that!interferes!with!digestion!midPgut.!

Dz dzPthroughput!metagenomic!

sequencing!technologies,!the!authors!review!existing!literature!and!then!argue!for!increased,!

systemsPoriented!research!into!the!role!of!microbiota!in!bee!colony!health.!With!specific!regard!

to!pesticides,!the!authors!note!that!broad!spectrum!antibiotics!and!fungicides!applied!directly!to!

control!disease!are!also!known!to!destroy!beneficial,!nonPtarget!fungi!and!bacteria!in!ways!that!!

ǯe.!Quoting!Mullin!et!al.,!(2010),!they!flag!

ǡǯ

DzǤdzǣDz

pollination!may!eventually!require!detoxification!of!agricultural!systems!and!in!the!short!term,!

Ǥdz
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4!@ Research!Challenges

In!the!context!of!multiple,!interacting!factors,!methodological!challenges!are!expected.!Some!are

endemic!to!the!task!of!epidemiological!research!and!therefore!unavoidable.!Others!are!the!result!of!

equipment!limitations,!poor!study!design!or!regulatory!framework!failures.

Equipment!+!detection sensitivity

Until!2003,!analytical!techniques!were!not!sensitive!enough!to!detect!systemic!pesticide!residues!in!

plant!tissue!below!a!level!of!20P50!ppbȄmuch!higher!than!the!levels!now!known!to!be!typical.!Pollen!

had!also!never!been!analyzed.!Detection!of!pesticides!at!very!low!levels!is!key!for!our!understanding!of!

the!actual!pesticide!load!in!bee!hives,!bees and!foraging!habitat,!including!soil.!This!challenge!remains!an

issue even!in!the!most!extensive!studies,!with!limits!of!detection!achieved!between!1.0 Dzdz

while!chronic!effects!have!been!observed!at!concentrations as!low!as!0.1 ppb.!Over!the!last!nine!years!

more!sensitive!analytical!techniques!and!tools!such!as!high!performance!liquid!chromatography!coupled!

with tandem!mass!spectrometry!(HPLC/ACPIPMS/MS!or!LCPMS/MS)!have!been!developed,!allowing

sublethal!and!chronic!exposure!via!pollen!and!beebread!to!be!measured.xxxi

1)!Bonmatin!JM,!Moineau!I,!Charvet R,!Colin!ME,!Fleche C,!Bengsch!ER.!2005.!Behaviour(of(

Imidacloprid(in(Fields.(Toxicity(for(Honey(Bees.((Environmental!Chemistry:!Green!Chemistry!and!

Pollutants!in!Ecosystems;!SpringerPVerlag!Berlin!Heidelberg,!483P494.

Using!the!recently!developed!method!for!detecting!imidacloprid!at!levels!as!low!as!0.1!ppb!(high!

pressure!liquid!chromatographyȂmass!tandem!spectrometry,!HPLC/MS/MS),!researchers!

showed!the!long!persistence!and!slight!accumulation!of!imidacloprid!in!soils!(treated!and!

untreated)!as!well!as!its!uptake!in!nonPtreated!crops.!Sunflowers!were!shown!to!be!particularly!

capable!of!recovering!imidacloprid!from!untreated,!contaminated!soils,!as!were!corn!and!several!

other!adventitious!plants.!Untreated!wheat,!barley!and!rape!recovered!less!imidacloprid!from!

contaminated!soils.!The!average!values!of!imidacloprid!found!in!sunflower!and!corn!pollen!

corresponds!to!a!range!of!concentrations!in!which!subPlethal!effects!in!bee!foraging!behavior!

had!been!observed.!

2)!Bonmatin!JM,!Moineau!I,!Charvetet!R,!et!al.!2003.!Method(for(analysis(of(imidacloprid(in(soils,(

plants(and(pollens. Analytical!Chemistry!75(9):!2027P2033.

In!this!study,!researchers!set!out!to!address!a!limitation!in!thenPcurrent!research!methods!by!

developing!a!technique!to!quantify!systemic!pesticide!residues!in!the!field!at!levels!known,!in!the!

lab,!to!have!subPlethal effects!on!honey!bees.!Using!rigorous!protocols,!they!developed!a!method!

to!consistently!detect!imidacloprid!and!similar!pesticides!at!levels!below!1.0ppb.!A!new!

extraction!method!was paired!with!a!known!analytic!method!already!in!recent!use,!high!

performance!liquid!chromatography!coupled!to!tandem!mass!spectrometry!(HPLC/ACPIPMS/MS!

or!LCPMS/MS).!Their!limit!of!detection!was!0.1!ppb!in!soil!and!plants!(stems,!leaves!and!flowers)!

and!0.3!ppb!in!pollen,!which!had!never!undergone!analysis!for!imidacloprid.!To!validate!the!

method,!researchers!conducted!small!field!tests!in!sunflower!and!corn!fields.!They!found!that!

most!samples!from!treated!fields,!as!well!as!fields!that!had!been!treated!a!full!year!earlier,!had!

detectable!levels,!most!of!which!were!high!enough!to!have!subPlethal effects!on!bees!according!

to!studies!cited.
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Study!design

ǯȋǤǤȌ

conditions!is!especially!difficult!because!of!the!wide!variety!of!factors!in!the!natural!environment.!

Multiple!exposure!pathways,!synergistic!and!combined!effects!from!multiple!chemicals!(i.e.!the!

DzdzȌǡǡsting!pathogens,!variabilities!of!weather!and!

genetic!predispositions!all!run!the!risk!of!confounding!any!experiment!designed!to!measure!pesticide!

exposure!and!toxicity!in!the!honey!bee!environment.!

Laboratory(vs.(field(studies

Studies!seeking!to!determine!the!effects!of!pesticides!on!honey!bees!typically!begin!in!the!lab!with!a!

single!pesticide!and!a!sample!of!adult!honey!bees.!Once!several!studies!achieve!similar!results,!the!

relationship!between!the!tested!substance!and!the!organism!is!informed!with!an initial!understanding!of!

potential!effect.!Conditions!in!the!lab!are!highly!controlled!to!eliminate!the!possibility!that!observed!

effects!might!actually!be!caused!by!some!other!factor!than!the!tested!substance.!To!further!understand!

that!relationship,!subDzPdz

the!natural!bee!environment,!but!still!partially!control!the!parameters!to!limit!the!possibility!of!errors!in!

the!results.!Full!field!experiments!are!used!to!assess!the!effǯ

environment,!but!tend!to!have!less!consistent!results!because!conditions!are!not!as!readily!controllable.!

It!tends!to!take!a!larger!population!of!subjects!and!a!longer!period!of!experiment!time!to!achieve!results!

that!correlate!to!either!semiPfield!or!lab!studies.!Lab!results!and!semiPfield!results!are!not!always!

replicable!in!fullPfield!studies!even!with!these!necessary!allowances.!

It!is inherently!more!difficult!to!track!an!individual!beeȄto!measure!the!pesticide!concentrations!to!

which!each!bee!is!exposed!and then!its!subsequent!behavior.!Many!other!factors!also!play!a!role!in!bee!

behavior!and!colony!health,!such!as!the!presence!of!other!pesticides!in!the!hives!and!the!foraging!

environment!(the!latter!of!which!can!be!several!to!many!tens!of!square!miles),!weather!conditions,

genetic!predispositions, the!age!and!health!of!the!queen,!and!the!relative!presence!of!parasites!and!

pathogens. One!of!these!many!variabilities!specific!to!field!conditions!that!has!recently!come!into!focus!

is!the!impact!of!relative!humidity!on!the!toxicity!of!neonicotinoids!to!bees.!

1)!Decourtye!A,!Devillers!J,!Aupinel!P,!Brun!F,!Bagnis!C,!Fourrier!J,!Gauthier!M.!2011.!Honeybee(

tracking(with(microchips:(a(new(methodology(to(measure(the(effects(of(pesticides. Ecotoxicology

20:429P437.

This!study!shows!disorientation!by!pesticides!and!illustrates!the!difficulties!of!measuring!such!

effects!under!field!conditions.!Few!studies!have investigated!the!impact!of!pesticides!on!homing!

flight!due!to!the!difficulty!of!measuring!the!flight!time!between!the!food!source!and!the!hive.!The!

aim!of!this!study!was!to!show!how!the!RFID (radio!frequency!identification) device!can!be!used!

to!study!the!effects!of!pesticides!on!both!the!behavioral!traits!and!the!lifespan!of!bees.!

Researchers developed!a!method!to!automatically!record!the!disorientation!of individual

foragers!and!to!detect!the!alteration!of!the!flight!pattern!between!an!artificial!feeder!and!the!

hive.!Fipronil!was!selected!as!test!substance!due!to!the!lack!of!information!on!the!effects!of!this!

insecticide!on!the!foraging!behavior!of!freePflying!bees.!It!was!shown!that!oral treatment!of!0.3!

ng!of!fipronil!per!bee!(LD50/20)!reduced!the!number!of!foraging!trips.
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Multiple(exposure(pathways(

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

Noting!the!correlation!between!bee!losses!and!corn!planting!season!in!Italy!and!Europe,!scientists!there!

began!exploring!the!possibility!that!bees!were!being!poisoned!by!the!dust!emitted!from!pneumatic!

drilling!machines!used!to!plant!neonicotinoidPcoated!seeds!around!2003.xxxii More!recent!studies!have!

confirmed!that!this!route!of!exposure!is!indeed!lethal,!and!is!exacerbated!by!humidity.!The leading!

Dzdz

neonicotinoids!as!their!abdomens!collect!airborne!fragments!of!treated!seed!coating.!!!!

1)!Tapparo!A,!Marton!D,!Giorio C,!et.!al.!2012.!Assessment(of(the(environmental(exposure(of(

honeybees(to(particulate(matter(containing(neonicotinoid(insecticides(coming(from(corn(coated(

seeds. Environmental!Science!&!Technology.

This!field!study!investigates!planter!exhaust!during!corn!sowing!as!an!exposure!pathway!for!

bees!foraging!in!and!around!those!fields.!The!core!finding!is!that!bees!flying!over!sowing!fields!

are!directly!exposed!to!neonicotinoids!at!lethal!levels!significantly!higher!than!the!contact!LD50

values!(18, 22,!and!30!ng/bee!for!imidacloprid,!clothianidin!and!thiamexotham,!respectively).!

Also!confirmed!are recent!findings!that!a)!high!quantities!of!neonicotinoids!from!seed!coating!

particles!are!emitted!by!drills!used!during!corn!sowing;!b)!lethal!levels!of!neonicotinoidP

contaminated!planter!exhǯǢȌ

mortality!is!higher!under!humid!conditions,!supporting!the!hypothesis!that!particles!are!more!

ǯnditions.!Study!authors!note!that!bees!seem!to!

remove!the!seed!coating!particles!during!subsequent!foraging!or!in!the!hive!under!normal!

ǡDzdz

sampled!after!death.!They!hypothesize!that!metabolic!degradation!(probably!also!effective!postP

mortem)!may!affect!concentrations!found!in samples.!!

ǯ two!methods!of!harm!reduction!proposed!by!

industry:!thicker,!more!adhesive!seed!coating,!and!modifications!of!the!drills!intended!to!reduce!

airborne!release!of!contaminated!planter!dust.!They conclude!that!neither!presents!a!likely!

solution.!In!drawing!these!conclusions,!they!point!to!the!fact that!following!the!European!

introduction!of!seed!coatings!meant!to!be!more!resistant!to!abrasion!in!2009P2010,!Austrian,!

Slovenian!and!German!(prior!to!the!ban)!beekeepers!continued!to!report!colony!loss!in!

conjunction!with!corn!sowing.!No!such!colony!losses!were!observed!in!Italy!after!the!ban!there.!

These!conclusions!were!also!supported!by!direct!observation differences!in!toxic!emissions!

between!modified!and unmodified!drills:!!modified!drilling!machines emit!large!amounts!of!

contaminated!dust!at!acute!levels.!!

2)!Marzaro,!M.,!Vivian,!L.,!et!al.!2011.!Lethal(aerial(powdering(of(honey(bees(with(neonicotinoids(

from(fragments(of(maize(seed(coat. Bulletin!of!Insectology!64!(1):!119P126.

This!field!study!investigated!two!possible!mechanisms!through!which!bees!can!come!into!lethal!

contact!with!neonicotinoidPcontaminated!fragments,!or!dust,!emitted!as!treated!seeds!are!sown:!

ͳȌDzdzǢǡʹȌ

through!the!nearby!contaminated!vegetation, dew!and!guttation!droplets.!Authors!also!tested!

the!synergistic!effect!of!relative!humidity!levels!on!bee!mortality.!Conducting!chemical!analyses!

of!contaminated!dew,!guttation!droplets,!dust!from!abraded!seed!coatings,!and!dead!bees,!they!

found!that!bees were!not!lethally!poisoned!by!drinking!dew!and!guttation!droplets,!but!that!

direct!contact!with!airborne,!contaminated!dust!is!acutely!toxic!to!bees!under!conditions!of!high!
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humidity.!Dust!from!abraded!seed!coatings!expelled!during!sowing!were!shown!to!contain!more!

than!20%!neonicotinoid,!a!concentration!at!least!2,600!times!greater!than!what!is!used!in!spray!

applications.!Study!authors!also!conducted!trials!with!seeds!treated!with!a!fungicide!but!not!a!

neonicotinoid,!under!both!humidity!conditions.!They!found!that!the!fungicidePtreated!seed!

coating!dust!was!not!lethal!to!bees!under!either!condition.!Using!these!trials!as!de!facto!controls,!

they!concluded!that!humidity!alone!does!not!cause!mortality;!rather,!humidity!has!a!synergistic!

influence!on!the!contact!toxicity!of!neonicotinoids.!

3)!APENET.!2010!&!2011.!Effects(of(coated(maize(seed(on(honey(bees.(Reports based!on!results!

obtained!from!the!second!and!third!yearǯ activity!of!the!APENET!project.!Consortium!for!research!and!

experimentation!in!agriculture.

A!group!of!Italian!scientists!from!various!institutions!began!publishing!annual!results!from!an!

ongoing!monitoring!network!(APENET)!in!2009.!Their!purpose!has!been!to!investigate!the!

DzȏȐǤdzconsist!in!establishing!that!

bees!are!exposed!to!acutely!toxic!levels!of!pesticidePcontaminated!dust!from!automatic!planters!

depositing!treated!seed!in!the!field,!and!that!relative!humidity!has!a!synergistic!effect!mortality.!

These!studies!look!at!the!neonicotinoids!clothianidin,!imidacloprid,!and!thiamexotham,!as!well!

as!fipronil,!which!is!a!systemic!insectide!commonly!used!in!treated!corn!seed.!They!have!also!

conducted!field!trials!in!methods!for!dust!and!drift!abatement.!2010!and!2011!reports!are!97!

and!123!pages!respectively,!and!are!structured!as!six!and!eight!chapters.!Each!of!these!chapters!

can!be!treated!as!a!distinct!study!with!its!own!research!agenda,!methods!and!results!section.!

ǯbelow.!

Dz maize!seed!sowing!and!estimated Ǥdz

In!field!trials,!dispersal!of!pesticidePcontaminated!dust!was!found!to!depend!on!a!number!of!

procedures:!seed!coating!procedure;!use!of!a!modified!drill!(deflector)!to!reduce!dust!dispersal!

ǢǤʹͲͳͲǡǯ

pneumatic!drills!with!an!air!deflector!succeeded!in!reducing!the!dispersal!of!pesticideP

contaminated!dust!by!around!50%.!These!results,!however,!depended!on!seed!quality.!In!2011,!

experimenters!devised!a!filter!to!attach!to!air!deflectors!with!the!intention!of!further!reducing!

dustiness.!This!combined!modification!reduced!dust!dispersal!by!90%!P 95%,!excepting!very!fine!

dust!particles.!In!both!situations!bees!die!at!higher!rates:!30%P60%!with!the!filter!+!deflector,!

85%!with!deflectors!alone.

DzǤdz

Researchers!confirmed!a!hypothesis!that!bees!flying!over!a!seeder!that!is!sowing!treated!seed!

may!be!exposed!to!a!lethal!dose!in!a!single!flight!via!contact!toxicity!without!the!poisoning!being!

mediated!by!ingestion!of!contaminated!food.!Humidity!was!further!found!to!significantly!

synergize!with!the!pesticideǤǯȋȌ

to!catch!pollen!and!are!thus!extremely!likey!to!trap!dust,!and!that!dust!had!an!exceptionally!20%!

(by!weight)!concentration!of!the!active!ingredient!when!compared!to!spray!formulations.!!!

DzSubPlethal!effects!of!neonicotinoids!and!fipronil!on!learning!and!memory!of!odors!and!spatial!

Ǥdz

Studying!the!proboscis!extension!reflex!(PER)!as!an!index!of!certain!cognitive!processes,!

researchers!found!subPlethal!effects!of!neonicotinoids!and!fipronil!on!learning!and!memory!of!
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ǤǯǤ

exposed!to!clothianidin!at!0.7!and!0.47!ng/bee!showed!marked!impairment!in!homing!ability!

and!foraging!frequency!after!a!single!dose!at!either!level!in!2010.!Disorientation!and!disrupted!

ǯ

both!are!mediated!heavily!by!scent.!Results!from!2011,!although!not!yet!complete,!appear!to!

confirm!2010!findings.!

Oral!(ingestion)!toxicity!::!Pollen,!nectar!+!guttation!droplets

Established!oral!toxicity!levels!of!neonicotinoids!for!bees!are!significantly!higher!than!are!contact!

toxicity!levels.!Potential!oral!exposure!routes!that!have!been!recently!studied!include!pollen,!nectar!and!

guttation!droplets.!Guttation!droplets!are!a!kind!of!dew!exuded!by!plants!during!the!night!and!in!the!

early!morning;!they!have!been!shown!to!contain!lethal!levels!of!neonicotinoid!pesticides.!While!bees!

readily!consumed!guttation!droplets!in!lab!conditions,!subsequent!field!studies!have!failed!to!establish!

that!bees!readily!use!these!droplets!as!a!water!source.!Field!studies!have!shown!that!bees!collect!and!

bring!back!to!the!hive!pollen!and!nectar!contaminated!with!neonicotinoid!pesticides!both!from!directly!

treated!crops!(corn),!and!from!nearby!untreated!plants!known!to!serve!as!nutrition!sources!for!bees!

(dandelions).

1)!Girolami!V,!Mazzon!L,!Squartini!A,!et!al.!2009!Translocation(of(neonicotinoid(insecticides(from(

coated(seeds(to(seedling(guttation(drops:(a(novel(way(of(intoxication(for(bees.!Journal!of!Economic!

Entomology 102(5):!1808P1815.

Researchers!investigated!the!levels!of!pesticides!present!in!the!droplets!exuded!by!plants!grown!

from!treated!seeds,!and!the!toxicity!effects!of!those!droplets!to!honey!bees!that!consumed!them.!

Study!showed!that!the!concentration!of!imidacloprid!in!guttation!drops!can!be!near!those!in!the!

active!ingredient!that!is!applied!in!field!sprays.!Bees!die!within!minutes!after!consuming!

guttation!drops!from!imidaclopridPtreated!seeds.!Four!different!pesticides!were!tested,!all!

grown!from!treated!corn!seed,!both!openPfield!and!inPlab:!three!neonicotinoids!(imidacloprid,!

clothianidin!and!thiamexotham)!and!one!nonPsystemic!pesticide!(fipronil).!All!treated!seeds!also!

included!two!fungicides,!fludioxonil!and!metalaxylPm,!reflecting!commercially!available!seeds.!

The!experimental!controls!consisted!of!untreated!seeds!and!seeds!treated!only!with!fungicides.!

The!neonicotinoid!pesticides!were!found!to!be!consistently!present!in!guttation!drops.!Levels!of!

contaminant!varied!by!pesticide!and!by!replication!as!expected,!but!in!all!cases!were!above!

1,000!μg/L.!ImidaclopridPtreated!corn!was!the!most!efficient!translocator,!concentrating!up!to!

200,000 μg!of!active!substance!per!liter,!but!clothianidin!and!thiamexotham!were!more!toxic!

(maximum!concentrations!found!at!100,000!μg/L).!Drops!remained!on!the!young!plants!

throughout most!of!the!day!due!to!collecting!in!the!cup!formed!at!the!base!of!the!young!plants.!

Bees!readily!consumed!fieldPgrown!guttation!drops!presented!to!them!in!lab!conditions.!In!all!

cases!for!neonicotinoids,!bees!experienced!acute!toxicity,!dying!within!two to!44!minutes!

(concentrationPdependent).

2)!Greatti!M,!et!al.!2006. Presence(of(the(a.i.(imidacloprid(on(vegetation(near(corn(fields(sown(with(

Gaucho®(dressed(seeds.!Bulletin!of!Insectology!59!(2):!99P103.

Following!European!beekeeper!assertions!of!a!link!between!imidaclopridPdressed!corn!seed!and!

largePscale!honey!bee!deaths,!research!efforts!were!made!to!determine!whether!the!pesticide!

was!becoming!airborne!during!seedPsowing.!This!study!showed!that!imidacloprid!can!be!

released!at!levels that!are!toxic!to!bees!during!sowing!operations!and!that!nearby!plants!can!be!

contaminated!by imidacloprid!in!dust!from!sowing!operations. Three!different seed!treatments!
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containing!imidacloprid,!one!with!a!new!ingredient!intended!to!improve!pesticide!adherence,!

and!two!without!imidacloprid!were!tested.!Results!clearly!demonstrated!that!imidacloprid!from!

all!three!treated!seeds!can!contaminate!nearby!grasses!and!flowers!on!the!day!of!sowing!and!for!

at!least!four!days!afterward,!in!amounts!relevant!to!both!subPlethal!and!lethal!effects!on!honey!

bees.!Sunny,!warm!weather!was!observed!to!correlate!to!higher!amounts!of!imidacloprid!

detected,!and!for!longer!periods!compared!to!cold!and!rainy!weather.!The!amount!of!soil!dust!

generated!by!pneumatic!seed!drills!used!in!the!trial!was!also!implicated!in!potentially!wide!

dispersal!of!imidacloprid!particles!through!wind.!Based!on!known!patterns!of!cornPseed!sowing!

in!Northern!Italy,!researchers!concluded!that!imidacloprid!could!accumulate!on!vegetation!

surrounding!corn!fields!during!successive!periods!of!sowing!for!a!period!of!3P4!weeks,!

representing!a!longerPterm!contamination!than!was!observed!in!the!study.!

Time(+(Timing

Understanding!the!effects!of!pesticides!and!other!stressors!on!hive!health!is!complicated!by!issues!of!

time:!duration,!sequencing!and!developmental!stages!of!a!bee can!all!play!a!role.!Studying!the!effects!of!

pesticide!exposure!over!too!short!a!time!scale!is!perhaps!the!most!critical!blindspot!of!most!research!to!

date.!Current!U.S.!regulatory!guidelines!specify!that!honey!bee!toxicity!testing!be!done!within!a!

timeframe!of!48!P 96!hours,!which!is!too!short!to!observe!many!chronic!or!sublethal!effectsȄ

particularly!when!those!effects!are!indirect!or!cascading. Recent!research!into!synergistic!effects!of!

pesticides!and!Nosema)has!surfaced!a!potential!sequencing!issue!whereby!bees!exposed!first!to

infection,!then!to!pesticides!show!signs!of!poisoning!at!sublethal!levels!when!pesticide!exposure!alone!

(without!previous!infection)!at!the!same!levels!do!not!appear!to!have!a!toxic!effect.xxxiii

Hive!+!Bee!Lifecycles!

Hives!contain!overlapping!generations!in!which!each!plays!a!critical!and!interdependent!role!in!

maintaining!hive!health.!The complex!social!structure of!the!hive!is!an!ecosystem!(some!scientists!

characterize!it!as!an!organism!or!superorganism) in!which!individual!bees!can!be!categorized!by!a!

combination!of!age!and!hive!function.!Each!category!of!bee!consumes!different!types!and!amounts!of!

food:!pollen,!nectar,!and!beePmade!food!containing!one!or!both,!such!as!bee!bread,!honey and!royal!jelly.!

They!also!differ in!their!activities,!some!spending!most!of!their!lives!in!the!hive!while!others!spend!a!

majority!of!time!foraging!in!flowers!a!few!miles!away!from!the!hive.

Contact!exposure!to!pesticides!accordingly variesȄlarvae!spend!their!first!three!days!of!life!floating!in!

nectar,!and!foraging!bees!are!in!contact!with!pollen!for!many!hours. Larvae!and!adult!bees,!like!human!

infants!and!adults,!have!differing!detoxification!capacities!and!nutritional!needs.!The!queen,!nurse!bees!

and!drones!all!likewise!exist!in!different!milieus.!These!variations!all!make!a!difference!in!how!a!hive!or!

individual!bee!would!be!affected!by!pesticides.!

1)!Rortais!A,!et!al.!2005. Modes(of(honeybee(exposure(to(systemic(insecticides:(estimated(amounts(

of(contaminated(pollen(and(nectar(consumed(by(different(categories(of(bees.!Apidologie!36:!71Ȃ

83.

In!this!study,!researchers!demonstrated!the!importance!of!bee!life!cycle!to!the!study!of!pesticide!

effects.!Researchers!used!wellPestablished!bee!biology!data!to!quantify!imidacloprid!ingestion!by!

bees!whose!hives!are!near!treated sunflowers,!according!to!each!bee!category.!Their!estimates!

focus!on!those!categories!that!consume!the!highest!amounts!of!pollen!(nurse!bees)!and!nectar!
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(waxPproducing,!broodPattending,!winter and!foraging!bees).!Individuals!within!these!categories!

potentially!consume!between!0.5!to!3.8!nanograms!of!imidacloprid,!over!a!period!of!time!that!is!

most!relevant!to!their!age!and!activities!(from!5!to!90!days).!These!cumulative!doses!are!

consistent!with!amounts!now!known!from!both!prior!and!subsequent!feeding!experiments!to!be!

lethal,!or!to!cause!a!variety!of!subPlethal effects!that!can!result!in!premature!death!within!hours!

or!days.

5(5 Structural(Bias

Bias,!sources!of!which!scientists!seek!to!minimize!and!eliminate,!appears!to!be!playing!a!role!in!our!

collective!understanding!of!pesticide!effects!on!honey!bees.!The!prominent!role!of!pesticide!

manufacturers!in conducting!and!funding!studies!has generated!controversy!and!concern!among!

independent!researchers,!beekeepers!and!citizen!groups.

The!following!critical!reviews!examine!how!conflicts!of!interest!in!honey!bee!research!impact!research!

findings,!yield!citation!bias!(where!contradictory!studies!are!excluded!from!introductory!literature!

reviews),!and!exert!undue!influence!on!pesticide!policymaking!decisions.

1)!Maini!S,!Medrzycki!P,!Porrini!C.!2010.!The(puzzle(of(honey(bee(losses:(a(brief(review.!B!Insectol

63(1):!153P160.

The!authors!reviewed!84!studies!that!address!the!question!of!whether!pesticides!are!adversely!

affecting!honey!bees!and!other!nonPpest!insects,!within!the!context!of!scientific!and!public!

controversy!around!CCD.!They!provide!a!focused,!critical!review!of!the!most!recent!experimental!

and!review!studies!addressing!CCD,!especially!the!effects!of!imidacloprid!in!the!field!(the!mostP

used!neonicotinoid!that!has!been!banned!to!varying!degrees!in!four!European!countries).!They!

also!critically!discuss!venues!for!research!publication!and!presentation.!They!compellingly!

demonstrate!that!science!funded!by!agrochemical!companies!(including!Bayer!CropScience,!the!

maker!of!several!neonicotinoids!including!imidacloprid),!have:!1)!focused!CCD!research!more!on!

parasites!and!pathogens!than!on!pesticides;!2)!published!the!most!favorable!among!all!results!

on!studies!of!pesticide!effects!on!honey!bees!(no!significant!effects!or!effects!at!dose!levels!that!

do!not!correlate!to!environmental!levels);!and!thus!3)!potentially!influenced!policy!decisions!

made!to!protect!bees!from!pesticides!toward!less!rigorous!risk!assessments!and!less!cautious!

regulations.

2)!Kindemba!V.!2009. The(impact(of(neonicotinoid insecticides(on(bumblebees,(honey(bees and(

other(nonKtarget(invertebrates (revised!version).!Buglife: The!Invertebrate!Conservation!Trust.

This!report!presents!a!critical!analysis!of!scientific!research,!technical!reports!and!regulatory!

process!documents!relevant!to!the!effects!on!nonPtarget!organisms!of!all!five!neonicotinoids!

ǤǤǯ

to!the!bulk!of!research!available,!the!author makes!a!strong!case!for!the!existence!of!structural!

bias!and!regulatory!inadequacy.!Among!studies!showing!that!imidacloprid!has!negligible!subP

lethal!or!chronic!toxicity!to!honey!bees,!or!that!the!effects!seen!are!not!relevant!to!amounts!

found!in!the!bee!environment,!most!were!funded!or!carried!out!by!the!manufacturer.!

Conversely,!a!longer!list!of!industryPindependent!research!tends!toward!opposite!results:!

imidacloprid!being!subPlethally!and!chronically!toxic!at!lower!amounts,!which!are!indeed!

relevant!to environmental!levels.!In!some!cases,!this!was!noted!to!be!related!to!equipment!

sensitivity!for!pesticide!detection,!but!the!overall,!patterned!discrepancy!indicates!that!results!

were!influenced!by!factors!related!to!the!agendas!of!those!who!funded!and!conducted!the!
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studies!as!well!as!the!regulatory!reviewers.!The!regulatory!process!is!found!to!be!deficient!in!its!

assessment!for!a!variety!of!other!reasons:!lack!of!standard!methodology!for!investigating!subP

lethal!effects,!failure!to!investigate!longPterm,!seasonal,!conditional,!or!synergistic!effects!in!the!

face!of!compelling!evidence!for!doing!so,!negligence!in!requiring!studies!on!larvae,!lack!of!

validation!criteria!for!reviewing!study!methodologies!and!failure!to!investigate!all!possible!

routes!of!bee!exposure.

Works(Cited(In(Text

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