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S T A T E O F T H E S C I E N C E R E P O R TPesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

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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)ClothianidinPesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

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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!loca l!and!international!consumer,!labor,!health,! ǯǤ challenges!the!global!proliferation!of!pesticides,!defends!basic!rights!to!health!and!environmental! quality,!and!wo rks!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)!981

P 1771

Web : :!www.panna.org

Written!by!Heather!Pilatic!with!research!assistance!fro m!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!read ers!of!earl y 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.Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

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## Contents

I. Introduction!

II. O verview!::!Colony!Collapse!Disorder!in!context

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

A. Pesticide!prevalence

B. Neonicotinoids!::!Acute,!sub

P lethal!&!chronic!effects

C. Synergistic!&!combined!effects

 Dzdzǣǣ	ǡ ethroids,!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!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

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## I!

@ Introduction!

Honey!bees!and!other!pollinators!are!dying!off! at!unprecedented!rate s! around!the!world. First!in!France,! then!in!the!U.S.!and!elsewhere,!colonies!have!been!mysteriously!collapsing!with!adult!bees!abandoning!

their!hives.!I n!2006, two!years!after!this!phenomen on ǤǤǡDz ǡdzǤ Each!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!syste m!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!efficient

Ȅ requiring!more!land!and!water!to!grow!

the!same!amount of!food

Ȅ and!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!poll ination.

While!few!contest!that!the!recent,!dramatic!decline!of!honey!bee!populations!present s serious! challenges!to!an!already

P stressed!food!system,!the!public!debate!over!what!lies!behind!CCD is!at!this! point!so!polarized!and!confusing!that!concerned!ci tizens!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!

controversy iii .!As!with!other recent!matters!of!public!and!environmental!health

, such!as!the!l ink!between!

toba cco!and!cancer! and!anthropogen ic!climate!change,!this!issue!has!become characterized!by! policymaker!inaction!in!the!face!of!irreducibly!complex!science.!

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

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

in!this!line

P up!is!clear!enough,!we!have! sought to!hew!to!a!commitment!to!non

P partisan,!scientifically! literate!public!discourse.!We!believe!that!engaged!for ms!of!scientific!citizenship!are!a!vital!part!of!

democratic!civic!life

, and!a!needed!force!in!environmental!decision

P making.!In!cases!such!as!this!where! the!debate!has!become!intractable!and!position

P driven,!as!is!reported!by!participants!and!scholars!alik e,! the!conversation!between!experts!has!clearly!broken!down.!Historically,!these!kinds!of!logjams!are! ǡDzdz event!that!compels!policymaker!action

Ȅ or!both!in!conjunc tion. With!one

P third!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,!t he!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!fac tors!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.Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

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## 2!

@ Overview!::!Colony!Collapse!Disorder!in!c ontext

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

per!year iv .!Steeper!declines!have!been!rec orded!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 : : Nutritio nal!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!theref ore!less!nutritious!diet.! Habitat!loss! has!been!occurring!steadily!for!the!last!50!years!with!measurable!effects!on!bee!health.! For!
instance,!r egional!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!broad

P spectrum! herbicides!that!accompanies!herbicide

P resistant,!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! Dzǥ no!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!diseases Ǥdz xi Parasitic!mites!of!the!genus! Varroa

are! the!most!important!pest!to!honey!bees globall y xii 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!importan t!to!colony!losses,!as!has!a!fungal!gut! pathogen!of!the!

genus! Nosema

.! Emergin g!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!large

P scale!bee!deaths!since the!early!1900s,! many!through!direct!poisoning!during!aerial!sprays. These!types!of!acute!bee!die

P offs!are!not!at! issue!in!CCD,!although!they!do!still!happen.! Regulations! and!phase

P outs!of!acutely!toxic! pesticides! have!reduced!the!number!of!acute!poisonin gs!in!most!of!Europe!and!North!America,! but!bee!exposure!to!multiple!pesticides!continues .!Sub

P lethal!effects,!less!studied and!

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

Ȅ present!in!small!amounts!throughou t!plant!tissues!from!seed!to!harvest

Ȅ have! 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!lo ss!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!declinePesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

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### 7 to!control!pathogens,!and!cer tain!fungicides!thought!to!be!safe!for!bees!which!have!recently! ǡǯ toxicity!by!200

P to!1,000

P fold. xiv

T he!following!four!studies!provide!an!overview!of!the!facto rs!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!Pathol ology! 103:!

S80

P S95.

This!article!is!the!most!recent!and!comprehensive!review!of!historical!loss!patterns!and!the!data! gaps!that!make!these!patterns!d ifficult!to!describe. R esearchers!reviewed!110!years!of!census! surveys,!experime ntal!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%!continent

P wide) in!the!same!time!frame .!In!all!of!these!declines ,!several! interacting!factors!are!likely.!These!include!long

P term!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!popular ity!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.

T his!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 CCD

P afflicted!colonies!and!apiaries,!and!

those!no t! 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)!l ongitudinal!studies! that!monitor! parasite,!path ogen and!pesticide!loads!while!quantifying!pesticide!toleranc e!in!the!study! populations;!2)!s tudies!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):!1517

P 1523.

To!better!understand!the!relative!importance!and!re lationships!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.!

Pes ticides!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,!c oumaphos ,!was! positively!correlated!to!healthy!colonies;!this!was!the!most!predict ive!factor!between!the!two! populations.!Results!indicate!that!pesticides!are!very!likely!involved!in!the!CCD!causal!complex.!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

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### 8 Among!several!areas!recommended!for!further!study,!two!were!highlighted:!the!effect!of!sub

P lethal!pesticide!exposure!on!pathogen!pr evalence,!and!the!relationship!between!varying! tolerance!to!pesticides!and!colony!survival.



## 3!

@ U nderstanding!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!an d!300,!respectively. xv

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

are! present in!b ees,!wax,!stored!food!and!the!pollen!and!nectar on!which!bees!forage.! Field!studies!have! found!neonicotinoid!pesti cides!in!particular!in!soil,!dust,!planter!exhaust,!water!(guttation)!droplets! exuded!by!treated!plants!and!on!nearby,!untreated!plants!and!fields.!A!cross

P section!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!exp osed!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!n eonicotinoids!(propiconazole).!Soils,!pollen! (bee

P collected!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!mor e!arable!land!in!North!America!than!any!other!cro p

Ȅ 88!million!acres

.! Corn!is!plant ed!throughout!the!U.S.!Midwest!from!mid

P April!through!early!May when!the! energetic!requirements!of!bees!are!increasing!rapidly!as!hives!prepare for!colony!growth,! requiring!i ncreased!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 heavil y!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!i n!over!two!growing!seasons!tested! ǡǯ persistenc e and!mobility.!

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

P collect ed!pollen!samples!showed!higher!levels,!indicating!additional!pathways!of!exposure.!

Levels!of!contamination!in!bee

P collected!corn!pollen!in!this!study!were!10

P fold!higher!than! reported!from!an!experiment!on!clothianidin

P treated!canola.!This!is!significant! because! clothianidin!was!approved!for!use!on!corn!and!canola!simultaneously!based!on!the!canola!field! test.!The!finding!that!bee

P collected!pollen!contained!neonicotinoids!is!of!particular!concern! because!clothianidin!is!even!more!toxic!when!ingested!orally by!a!bee,!and!because!of!the!

po tential!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!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

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### 9 pollen!reserves!in!the!form!of!royal!jelly.!Au thors!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!cr op.!The!author s ǯ most!salient!finding,!however,!is!the!establishment!of!a! new,!especially!mo bile!and!toxic!exposure!route

Ȅ planter!exhaust!material.!Corn!seeds!are!sown! using!an!automated!planting!system!that!relies!on!air/vacuu m!mechanisms!to!space!the!see ds; 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! ǤDzdz tinoids!and!fungicid es!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 Canadi an!province!during!the!2007

P 2008!growing!season.!They!used!conventional!(gas!chromatography

P mass!spectrometry)!and! recently!developed!(liquid!chromatography

P tandem!mass!spectrometry)!analytical!techniques! to!detect!any!of!200!pesticides!and!their!metabolit es!at!concentration! levels!as!low!as!0.1!ppb!in a!representativ e!cross

P section!of!bees,!pollen and!wax.! A!total!of!121 pesticides!and!metabolites comprising!5,519!total!residues wer e!detected!and!quantified!in!887 samples.!Wax!samples! averaged!8,!pollen!sa mples!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!inse cticides! detected!in!all!samples .!N early!half (49.9%) of!all!samples!contained!a t!least!one systemic! pesticide. Pollen!contained!high!levels!of!fungicides,!which!tended!to!co

P occur!with!low!levels!of! systemic!pesticides,!implicating!possible!synergistic!ef fects.!

Dz The!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!urge nt!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(colonie s(associated(with(increased(risk(of(colony(mortality

.)) J!

Invertebr!Pathol 101(2) : 147

P 149.

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,!resea rchers!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!mid

P fall!as!hives!with!normal!pollen.!Incidence!of! entombed!pollen!was!notably!greater!in!reused!wax!comb!regardless!of!any!disinfection!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 10



### 10 treatments!applied,!which!suggests!there!is!a!transmittable!factor!common!to!both!entombing!

behavio r!and!colony!death.!R esearchers!urged!further!study.!

4 )!Bonmatin!JM,!Marchand!PA,!Charvet!R,! Moineau!I,!Bengsch!ER,!Colin!ME

. 2005. Quantification(of( imidacloprid(uptake(in(maize(crop s. J!Agr!Food!Chem 53: 5336

P 5341.

Researchers!in!France!conducted!a!three

P year,!random

P sample!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!chromatography

Ȃ mass! tandem!s pectrometry,!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! pollen

P producing!flowers,!4.1!ppb!for!stems!and!leaves,!and!2.1!ppb!for!pollen!itself.!The se! levels!

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

P contaminated! corn!pollen!made!up! 54%! of!pollen!samples!coll ected!at!hive!entrances,!reflecting!a!mixture!of! pollen!sources!and!with!a!correspondingly!lower!average!contamination!of!0.6!ppb.!



## Neonicotinoids ::(Acute,(sub

5 lethal(&(chronic(effects

Neonicotinoids!are!a!relatively!new,!and!very!widely!used!class!of!i nsecticides!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!fastest

P growing!class!of!insecticides!in!the!history!of!synthetic!pesticides.!By!

2005!neonicotinoi ds!had!gained ͳ͸Ψ̀ ͅǡ

near

P ȋ͹͹ΨȌ̀ͳͷͷ

̀ͷ͵ͷǤ xvii Among!their!approved!uses!in!the!U.S.!are!topical!flea!tr eatments!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!acu tely!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!kno wn!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!h inged,!by!default,!on!the!establishment!of!acute!toxicity!exposure!scenarios!without! requiring!tests!for!sub

P lethal!effects. xviii T he!standard!laboratory!method!for assessing!pesticide!risk!is

determine!the!median!lethal! dose!(LD 50 )!required!to!kill!half!the!t ested!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!pest icides!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!LD 50 levels! on!adult!honey! bees!may!be!particularly!ill

P suited!for!the!testing!of!systemic!pesticides!bec ause!of!the! ways!bees!are! exposed!to!systemics!in!the!field

Ȅ in!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. xixPesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 11



### 11 D e spite repeated!calls!for!a!reevaluation!of!pesticide!testing!protocols,!re gulatory!process es!in!the!U.S.! and!Europe!have not!been!adapted!to!consider! sub

P lethal,!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!pop ulation! or!LD 50

.! 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! w ith!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!LD 50 values!of!

neonicotinoi ds!are!low!compared!to!older!classes!of!insecticides;!they!are!considered!highly! toxic!to!honey!bees.!

Sub

= lethal*toxicity*::* The!sub

P lethal!toxicity!of!neonicotinoid!pesticides!is!of!particular!concern!

because!the!most!common!field! exposure!scenarios!ar e!likely!at!the!

sub

P lethal!rather!than!acute!level.! Sub

P 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! impor tant!sub

P lethal!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!over

P accumulation!of! acetylcholine,!resulting!in!paralysis!

and! death. The!most!recent!scientific! observations!point!to!a!long

P lasting!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!irrev ersible ,!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.!Neonicotino ids!are! very!persistent!and!therefore!accumulate!over!time!in!the!

environment.!

M ost neonicotinoids are!classifie d!as!acutely!toxic!to!bees.! But!single,!high

P dose!(i.e.!acute)!exposures!are!likely!less! common!than!are!the!chronic,!sub

P lethal!exposure!leve ls! 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! sub

P lethal!effects!caused!by! neonicotinoids: altered! foraging!and!feeding!behavior and!impaired! orientation,! social!communication,! undermined! immunity!and! delayed! larval!development.!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 12



### 12 Many!independent!studies!in!the!U.S.!and!in!Europe!have!shown!that!small!amounts!of!neonicotinoids

Ȅ both!alone!and!in!combination!with!other!pesticid es

Ȅ can!cause!i mpaired!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.!2 003. Effects(of(imidacloprid(

administered(in(sub

K lethal(doses(on(honey(bee(beha viour.(Laboratory(tests

. B!Insectol 56(1):!59

P 62.

In!this!study,!researchers!monitored!the!behavior!of!adult!forager!bees!in!highly!controlled,! laboratory!conditions!to!observe!effects!of!imidacloprid!fed!at! sub

P lethal doses!in!sugar!solution.!

Four!diffe rent!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!th at!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!functioni ng!of!a!hive

.

3)! C olin!ME,!et!al.! 2004

.! A(method(to(quantify(and(analyze(the(foraging(activity(of(honey(bees:(

relevance(to(the(sub

K lethal(effects(induced(by(systemic(pesticides

.! Archives!of!Environmental! Contamination!and!Toxicology! 47:!387

P 395.

This!stu dy!investigated!the!sub

P lethal! effects!of!two!insecticides!in!semi

P field!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,!h ad!shown!sub

P lethal! 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!for aging!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!a t!6.0!μg/kg,!inactive!bees

Ȅ those!visiting!the! feeder,!but!not!feeding

Ȅ increased!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.!Convulsion s!and!paralysis!were!also!observed!in!bees!feeding!on!fipronil

P contaminated! Ǥǯǡǡ

sub

P lethal!levels!70!times!below!the!referenced!LD 50 s.!They!also!concluded!that! their! Dz provided!an indispensable!interface!between!controlled!conditions! in! the!laboratory!and!the!field, dz which!suggests!its!adoption!in!regulatory!testing!of!sub

P lethal!

effects.!

4)!Decourtye!A,!Armengaud!C,!Renou!M,!Devillers!J,!C luzeau!S,!Gauthier!M,!et!al.!2004.! Imidacloprid( impairs(memory(and(brain(metabolism(in(the(honeybee((Apis(mellifera(L.)

. Pestic!Biochem!Phys

78:!83

P 92.

This!laboratory!stu dy!shows! sub

P lethal effects!at!low!concentrations!of!imidacloprid!using!four different!methodologies. Researchers!sought!to!build!from!prior!studies!that!clarified!the!role!of!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 13



### 13 nicotinic!acetylcholine receptors!(nAChR)! in!honey!bee!learning!and!memory!capac ities!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.!B ehavioral!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 medium

P term,!but!not!long

P or!short

P 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!tha t! 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( f oraging( b ehavior( i nduced(by( s ublethal(

d osage(of( i midacloprid(i n(the( h oney( b ee(( H ymenoptera:( A pidae)

.! J!of!Econ!Entomol 101(6):!1743

P 1748.

Researchers!conducted!a!semi

P field!experiment!to!follow!on!results!of!laboratory!studies!that!

had!shown! a!number!of! detrimental!effects!from!ingestion!of!imidacloprid

P contaminated food.! They!tested!the!time!intervals!between!individual!worker!bee!visits!to!a!feeder!after!ingestion!of! imidacloprid

P contaminated!sugar!solution!at!concentrations!rang ing 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!missi ng!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!1.82

P 4.33!nanograms.!Researchers!concluded!that!abnormal!foraging!behavior!could! occur!in!the!field!through!multiple!visits!to!imidacloprid

P contaminated!flowers.



## Synergistic(+(Combi ned(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!concer t!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!incre ases!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!non

P neonicotinoid! systemic! fipronil).!Hives!exposed!to!these!pesticides!were!significantl y!more!susceptible!to!infection

.



### ͞ŚĞŵŝĐĂůĐŽĐŬƚĂŝůƐ͗͗͟&ƵŶŐŝĐŝĚĞƐ͕Ɖ yrethroid(insecticides,(miticidesPesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 14



### 14 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! Dz ǡdz of!insecticides,!herbicides!and!fungicides!as!well!as!the!miticides!used!by! beekeepers!to control!pathogens!in!the!hive.!

Non

P neonicotinoid!pesticides!of!special!concern!for!their!impacts!on!bees,!and!potential!role!in!the! causal!complex!of!CCD!are!f ungicides!and!pyrethroids

Ȅ particularly!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!cer tain!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!neonicoti noids!are!slowly!coming!into!resolution.! ǯ relatively!few!genes!that!encode!detoxification!enzymes.!For!example,!one!such!group!of!enzymes! (P450s)!mediate!det oxification!pathways!in!ways!that!can!be!inhibited!by!certain!widely!used!fungicides! ȋǤǤȌǡǯ miticide,!fluvalinate. xxvi Although!current!data!are!unclear,!anothe r!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,!A mbrose!JT!and!Roe!M.! 2004

. Mech anism(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!u p!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!

imi dacloprid,!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!i n

P field,!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)(colon y

. Bulletin!of!Environmental!Contamination! and!Toxicology 85(2):!125

P 8.

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!agricultura l! 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!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 15



### 15 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! Va rroa)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!amit raz!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!P

P L,!Wang!Q,!Sun!J

P H,!Liu!F,!Wang!X,!Wu!Y

P Y!and!Zhou!T. 2010. Effects(of( sub

K lethal concentrations(of(bifenthrin(and(deltamethrin(on(fecundity,(growth,(and(development(of(the( honeybee(Apis(mellifera(ligustica

. Environmental!Toxicology!and!Chemistry 29: 644

Ȃ 649.

This!study! examined!the! sub

P lethal 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!fecundi ty,!growth a nd!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!insecti cides.!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! ǯ 50 for!honey bees!and! deltamethrin!application!rates!are!about!the!same!as!the!LC 5

.



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

Nosema ,!a!family!of!fungal!gut!parasites, and!the! Varroa)destructor mite!are!two!rel atively!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!pe sticides!to!weaken!colony!health!more!than!either!does!

alone.! Nosema) is!a!fast

P ǯ ability!to!absorb!nutrients!(infected!bees!consume!significantly!more!calories),!and!known!to!suppres s! immune!response. xxviii Varroa) mites!act!as!vectors,!transmitting!disease!across!and!within!colonies.!

With!the!relatively!recent!observation!that!CCD

P affected!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!imidacloprid

Ȅ to!!which!they!were!exposed!only!indirectly!in!brood! food!as!developing!larva

Ȅ faced significantly!more! Nosema) infections!th an!d id!their!control!

counterparts.!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 16



### 16 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!comb ination!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(g ut(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!sub

P lethal!doses!of!a!widely!used! pesticide,!imidacloprid,!and!then!challenged!newly!emerged!bees!with! Nosema .!They!used! GC/MS!( gas!chromatography

P mass!spectrometry)! with!a!limit!of!detection!of!0.1!ppb!to!analyze!

the!bee s,!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!pesticide

P treated!hives!when!com pared!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!th is!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!imidacloprid

P ǤDz exposure!durin Ǥdz

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

sub

P lethal!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!in Ǥdzǣ Dz mortality!of!honey!bee!colonies,!including!colony!collapse!disorder,!and!other!pollinator!d eclines!

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!pes ǡDzǡ 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!ex Ǥdz 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!Ala ux!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!pesticid es!was!linked!to!a!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 17



### 17 ǯǤ on!the!mechanisms!underlying!synergistic!effects!between!pesticides!and!pathogens!is!poorly! ǡǡDz ects!to!pesticides!is!a!more!complex!phenomenon! than!previously!thought.!The!influence!of!parasitism!in!the!ecosystem!must!be!considered!in! ǥǤȏȐ N.)ceranae) spreads!rapidly!and!can!affect!more!than! 80%!of!honey!bee!colon Ǥdz

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):!7 74

Ȃ 782.!

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!gut

P pathogen! ( Nosema) ceranae )!and!a!neonicotinoid! (imidacloprid).! Their!results!indicate!that!a!synergistic!interaction!is!occurring!that! significantly!

weaken s bees,!both!individually!and!socially.! C oncentrations!of!orally!administered!imidacloprid!
were!0.7,!7 .0 ,!and!7 0!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!decr eased!only!when!the!two! Ǥǯ ability!to!withstand!a!broad!range!of!pathogens!in!the!long

P term.!

4 )! Wu!JY,!Anelli!CM,!Sheppard!WS.!2011.! Sub

K lethal(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!sub

P lethal!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!c ontaminated!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!rai sed!in!treatment!comb! gradually!had!reduced!effects!while!those!raised!in!control!comb!(successively!more! contaminated!with!each!brood!cycle)!had!increased!effects.!Brood!death s followed!this!trend,! with!high!rates!occurring!after!multiple!cycles!in!progre ssively!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!mic robial!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!ma jor!levels:!within!the! DzdzǤPesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 18



### 18 ǯǡ enough!to!describe!a!co

P evolve ǡǡDzǡdz well!as!hypothesize!about!key!functions!susceptible!to!disruption

Ȅ specifically!nutrition!and!immunity.! For!example,!within!the!larger!hive!environment,!beebread!is!the!most!microbially!a ctive,!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

Eme rging!research!in!this!area!has!been!made!possible!in!part!through!recent!breakthroughs!in!new,! high

P throughput!metagenomic!sequencing!technologies.!These!tools!allow!scientists!to!both!better! characterize!insect!microbial!life!in!ways!that!focus!on!how!ho sts!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:!431

P 444.

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!immune

P related!function!of!a!gut microbiota!may!be!the!ability!to!obstruct!colonization!by!pathogens,!thereby!preventing! infections.!In!this!context,!the!well

P known!pathogen! Nosema)ceranae, is!listed!as!an!infectious! microbe!that!interferes!with!digestion!mid

P gut.!

Dz dz

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

systems

P oriented!research!into!the!role!of!microbiota!in!bee!colony!health.!With!specific!rega rd! to!pesticides,!the!authors!note!that!broad!spectrum!antibiotics!and!fungicides!applied!directly!to! control!disease!are!also!known!to!destroy!beneficial,!non

P target!fungi!and!bacteria!in!ways!that!! ǯ e.!Quoting!Mullin!et!al.,!(2010),!they!flag! ǡǯ DzǤdzǣDz pollination!may!eventual ly!require!detoxification!of!agricultural!systems!and!in!the!short!term,! ǤdzPesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 19



### 19 

## 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!se nsitive!enough! to! detect!systemic!pesticide!residues!in! plant!tissue!below!a!level!of!20

P 50!ppb

Ȅ much!higher!than!the!levels!now!known!to!be!typical.!Polle n! had!also!never!been!analyzed.! Detection!of!pesticides!at!very!low!levels!is!key!for!our!understandin g!of!

the! actual! pe sticide!load!in!bee!hives,!bees and!foraging!hab itat,!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!observ ed!at!concentration s 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!spectrometr y!(HPLC/ACPI

P MS/MS!or!LC

P MS/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,! Bengsc h!ER.!2005.! Behaviour(of( Imidacloprid(in(Field s.(Toxicity(for(Honey(Bees.(( Environmental!Chemistry:!Green!Chemistry!and!

Pollutants!in!E cosystems ;!Springer

P Verlag!Berlin!Heidelberg,! 483

P 494

.

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

Ȃ mass!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!non

P treated!crops.!Sunflowers!were!shown!to!be!particularly!

c apable!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!sunf lower!and!corn!pollen! corresponds!to!a!range!of!concentrations!in!which!sub

P lethal!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):!2027

P 2033.

In!this!study,!researchers!set!out!to!address!a!limitation!in!then

P current!research!methods!by! developing!a!technique!to!quantify!systemic!pestici de!residues!in!the!field!at!levels!known,!in!the! lab,!to!have! sub

P lethal effects!on!honey!bees.!Using!rigoro us!protocols,!they! developed!a!method! to!consistently!detect!imidacloprid!and!similar!pesticides!at!levels!below!1 .0 ppb.!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/ACPI

P MS/MS!

or!LC

P MS/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! sub

P lethal effects!on!bees!according!

to!studies!cited.Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 20



### 20 

## 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! DzdzȌǡǡ sting!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!determi ne!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,!sub Dz

P dz 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!eff ǯ 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!res ults!

that!correlate!to!either!semi

P field!or!lab!studies.!Lab!results!and!semi

P field!results!are!not!always! replicable!in!full

P field!studies!even!with!these!necessary!allowances.!

It!is inherently!more!difficult!to!track!an!individual!bee

Ȅ to!measure!the!pe sticide!concentrations! to! which!each!bee!is!exposed! and then!its!subsequent!behavior.!Many!o ther!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!w hich!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!rec ently!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.! Honey bee( tracking(with(microc hips:(a(new(methodology(to(measure(the(effects(of(pesticides

. Ecotoxicology

20:429

P 437.

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

effects!under!field!conditions.!Few!studies!h ave 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!feed er!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!free

P flying!bees.! It!was!shown!that!oral treatment!of!0.3! ng!of!fipronil!per!bee!(LD 50 /20)!reduced!the!nu mber!of!foraging!trips.Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 21



### 21 

### 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!possibilit y!that!bees!were!being!poisoned!by!the!dust!emitted!from!pneumatic! drilling!machines!used!to!plant!neonicotinoid

P coated!seeds!around!2003. xxxii More!r ecent!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 ,!Gi orio C ,!et.!al.!2012.! Assessm ent(of(the(environmental(exposure(of( honeybees(to(particulate(matter(containing(neonicotinoid(insecticides(coming(from(corn(coated(

s eeds

. Environmental!Science!&!Technology

.

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

be es!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!LD 50 values!(18, 22,!and!30!ng/bee!for!imidacloprid,! clothian i din!and!thiamexotham,!respe ctively).!

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!neonicotinoid

P contaminated!planter!exh ǯǢȌ mortality!is!higher!under!humid!conditions,!supporting!the!hypothesis!that!particles!are!more! ǯ nditions.!Study!authors! note!that!bees!seem!to!

r emove!the!seed!coating!particles!during!subsequent!foraging!or!in!the!hive!under!normal! ǡDzdz sampled!after!death.!They!hypothesize!that!metabolic!degradation!(probab ly!also!effective!post

P mortem)!may!aff ect!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!r educe!

airborne!release!of!contamin ated!planter!dust.!They conclude!that!neither!prese nts!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!abrasio n!in!2009

P 2010,!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!th e!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):!119

P 126.

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

contact!with!neonicotinoid

P contaminated!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!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 22



### 22 humidity.!Dust!from!abraded!seed!coatings!expelled!during!sowing!were!shown!to!co ntain!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!f ound!that!the!fungicide

P treated!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!conta ct!toxicity!of!neonicotinoids.!



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

.( Report s based!on!results!

obtained!from!the!second! and!third! year ǯ activity!of!the!APENET!project .!Consortium!for!research!and! experimentation!in!agricult ure.

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! DzȏȐǤdz consist!in!establishing!that! bees!are!exposed!to!acutely!toxic!levels!of!pesticide

P contaminated!dust!from!automatic!planters! depositing!treated!seed!in!the!field,!and!that!relative!humidity!has!a!synergistic!effect!mortality.!

These!studies!look!at!the!neon icotinoids!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.! ǯ below.!

Dz maize!seed!sowing!and!estimated Ǥdz

In!field!trials,!dispersal!of!pesticide

P contaminated!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!pesticide

P contaminated!dust!by!around!50%.!These!results,!ho wever,!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!bo th!situations!bees!die!at!higher!rates:!30%

P 60%!with!the!filter!+!deflector,!
85%!with!deflectors!alone.

DzǤdz

Researchers!confirmed!a!hypothesis!that!bees!flying!ov er!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!pesticide ǤǯȋȌ 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.!!!

Dz Sub

P lethal!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!sub

P lethal!effects!of!neonicotinoids!a nd!fipronil!on!learning!and!memory!of!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 23



### 23 ǤǯǤ exposed!to!clothianidin!at!0.7!and!0.47!ng/bee!showed!marked!impairment!in!homing!ability! and!foraging!frequency!after!a!s ingle!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!drop let s!are!a!kind!of!dew!exuded!by!plants!during!the!night!and!in!the! early!mor ning;!they!have!been!shown!to!contain!lethal!levels!of!neonicotinoid!pesticides.!While!bees! readily!c onsumed!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!wit h!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(f rom( coated(seeds(to(seedling(guttation(drops:(a(novel(way(of(intoxication(for(b ees

.! Journal!of!Economic! Entomology 102(5):!1808

P 1815.

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.!

S tudy!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!consum ing! guttation!drops!from!imidacloprid

P treated!seeds.! Four!different!pesticides!were!tested,!all! grown!from!treated!corn!seed,!both!open

P field!and!in

P lab:!three!neonicotinoi ds!(imidacloprid,!

clothianidin! and!thiamexotham)!and!one!non

P systemic!pesticide!(fip ronil).!All!treated!seeds!also! included!two!fungicides,!fludioxonil!and!metalaxyl

P m,!reflecting!commercially!available!seeds.! The!experimental!controls!consisted!of!untreated!seeds!and!seeds!treated!only!with!fungicides.! The!neonicotinoid!pesticides!were!f ound!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.!Imidacloprid

P treated!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!field

P grown!guttation!drops!presented!to!them!in!lab!conditions.!In!all! cases!for!neonicotinoids,!bees!experienced!acute!toxicity,!dying!within! two to!44!minutes! (concentration

P dependent).

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):!99

P 103.

Following!European!beekeeper!assertions!of!a!link!between!imidacloprid

P dressed!corn!seed!and! large

P scale!honey!bee!deaths,!res earch!efforts!were!made!to!determine!whether!the!pesticide! was!becoming!airborne! during!seed

P sowing.! 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!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 24



### 24 containing!imidacloprid,!one!with!a!new!ingredient!intended!to!improve!pesticide!adherence,! and!two!without!imidacloprid!were!tested.!Results!clearly!demonstrated!that!imidaclopr id!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!sub

P lethal!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!thr ough!wind.!Based!on!known!patterns!of!corn

P seed!sowing! in!Northern!Italy,!researchers!concluded!that!imidacloprid!could!accumulate!on!vegetation! surrounding!corn!fields!during!successive!periods!of!sowing!for!a!period!of!3

P 4!weeks,! representing!a!longer

P te rm!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!de velopmental!stages!of!a!bee can!all!play!a!role.!Stud ying!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,!whic h!is!too!short!to!observe!many!chronic!or!sublethal!effects

Ȅ 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!

c ombination!of!age!and!hive!function.!Each!category!of!bee!consumes!different!types!and!amounts!of! food:!pollen,!nectar,!and!bee

P made!food!containing!one!or!both,!s uch!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.

Con tact!exposure!to!pesticides!accordingly varies

Ȅ larvae!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(bee s .!Apidologie!36:! 71

Ȃ 83.

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

effects.!Researchers!used!well

P established!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!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 25



### 25 (wax

P pro ducing,!brood

P attending,!winter and!foraging!bees).!Individuals!within!these!categories!

potent ially!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!exp eriments!to!be! lethal,!or!to!cause!a!variety!of! sub

P lethal 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!

collect ive!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!critic al!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):!153

P 160.

The!authors!reviewed!84!studies!that!address!the!question!of!whether!pesticides!are!adversely! affecting!honey!bees!and!other!non

P pest!i nsects,!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!most

P used!neonic otinoid!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!agr o chemical!companies! ( including!Bayer!CropSci ence,!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!signifi cant!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)! Kindemb a!V.!2009. The(impact(of(neonic otinoid insecticides(on(bumblebees,( honey(bees and(

other(non

K target(invertebrates (revised!version).! Buglife: The!Invertebrate!Conservation!Trust.

This!report!presents!a!critical!analysis!of!scientific!research,!technical!re ports!and!regulatory! process!documents!relevant!to!the!effects!on!non

P target!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!sub

P 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!industry

P independent!research!tends!toward!opposite!results:! imidacloprid!being!sub

P lethally!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!con ducted!the!Pesticides(and(Honey(Bees:( State(of(the(Science ,(v.1

Pesticide!Action!Network!North!America (PANNA) 26



### 26 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!sub

P lethal!effects,!failure!to!investigate!long

P term,!s easonal,!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!exp osure.



### Works(Cited(In(Text!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! i Gallaia!N,!Sallesc!JM,!Setteled!J,!Vaissierea!BE.!2009.!Economic!valuation!of!the!vulnerability!of!world!agriculture! confronted!with!po llinator!decline.!Ec ological!Economics!68:!810

Ȃ 821;! United!Nations!Environmental!Programme! (UNEP).!2010.!Emerging!Issues:!Global!Honey!Bee!Colony!Disorder!and!Other! Threats!to!Insect!Pollinators.!

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