Pesticides and Honey Bees: 

State of the Science

MAY 2012

PESTICIDE ACTION NETWORK NORTH AMERICA
Pes7cide Ac7on 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 ?ive PAN Regional Centers worldwide, we link local and international consumer, labor, health,

environment and agriculture groups into an international citizens’ action network. This network

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.

Written by Heather Pilatic, PhD with research assistance from PANNA interns Juli 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.

Pesticide Action Network

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Table of Contents

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

Overview :: Colony Collapse Disorder in context ...........................................................2

Understanding pes*cides as a causal factor in colony collapse.....................................4

Pes*cide Prevalence .................................................................................................................4

Neonico*noids :: Acute, subElethal & chronic effects...............................................................6

Synergis*c + Combined Effects ...............................................................................................11

“Chemical cocktails” :: Fungicides, pyrethroid insec;cides, mi;cides......................................11

Pathogen interac;ons :: Nosema + pes;cides.........................................................................13

Microbiota out of balance :: Gut cultures, immunity + nutri;on .............................................15

Research Challenges.....................................................................................................17

Equipment + detec*on sensi*vity ...........................................................................................17

Study design ............................................................................................................................18

Laboratory vs. field studies......................................................................................................18

Mul;ple exposure pathways....................................................................................................19

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

Structural Bias..............................................................................................................23

Appendix A :: Neonico*noid use paTerns in U.S. agriculture......................................24

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Introduc7on

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 hit the U.S., it was named “Colony

Collapse Disorder,” or CCD. Each year since, U.S. beekeepers have reported annual hive losses of 29% Y

36%. Commercial beekeepers tell us that their industry, which is the care and cultivation of an indicator

species, is on the verge of collapse.

Honey bees pollinate 71 of the 100 most common crops that account for 90% of the world’s food

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

2000 data from the U.S. Department of Agriculture (USDA) indicates that this industry was worth more

than $15 billion per year twelve years ago.ii With the subsequent growth of U.S. agriculture and decline

of natural pollinators, commercial beekeeping likely contributes much more than $15 billion to the U.S.

economy now.

Claims of imminent food system collapse are not supported by pollination biologists, but without

pollination, agriculture quickly becomes less ef?icient—requiring more land and water to grow the

same amount of food—and our diets lose nutritionally vital variety. For instance, most fruits and many

nuts rely on the pollination services of bees, including almonds, blueberries, apples and melons. Alfalfa,

which is a primary feed source for dairy cows, relies on bees for pollination as well. 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 alreadyYstressed food system, the public debate over what lies behind CCD is at this

point so polarized and confusing that concerned citizens ?ind it dif?icult to know how or where to

intervene. Indeed, the debate over the causes of CCD has become a case study in public, scienti?ic

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. While PANNA’s position

in this lineYup is clear enough, we have sought to hew to a commitment to nonYpartisan, scienti?ically

literate public discourse. We believe that engaged forms of

scienti?ic citizenship are a vital part of democratic civic life,

and a needed force in environmental decisionYmaking. In

cases such as this where the debate has become intractable

and positionYdriven, as is reported by participants and

scholars alike, the conversation between experts has

clearly broken down. Historically, these kinds of logjams

are broken either through concerted public demand, or

through a catastrophic focusing or “triggering” event that

compels policymaker action—or both in conjunction. With

oneYthird 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 identi?ied 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.

“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.”

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Overview :: Colony Collapse Disorder in context

Honey bee populations have steadily declined in the U.S. since 1947 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 speci?ically 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 broadYspectrum herbicides that accompanies herbicideYresistant,

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 con?irmed, 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 “...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.”xiParasitic 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 signi?icantly 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 largeYscale bee deaths since the early 1900s,

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

issue in CCD, although they do still happen. Regulations and phaseYouts 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. SubYlethal effects, less studied and

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

—present in small amounts throughout plant tissues from seed to harvest—have become an

important 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 to control

pathogens, and certain fungicides thought to be safe for bees which have recently been found to

act synergistically with some neonicotinoids, increasing the latter pesticides’ bee toxicity by

200Y to 1,000Yfold.xiv

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What is Colony Collapse Disorder?

Colony Collapse Disorder, or CCD, was first

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

dis*nct from other loss epidemics and include

the following:

! Colonies found suddenly empty of adult

bees, leaving their brood unaTended

! No sign of dead bees

! No hive pests or food robbers, despite

surplus honey and pollen stores

! Common parasites not present at levels

thought to cause popula*on decline
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:

S80YS95.

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

gaps that make these patterns dif?icult 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% continentYwide) in the same time frame. In all of these declines, several

interacting factors are likely. These include longYterm 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 pro?itability.

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 con?irm the de?inition of CCD, identify its potential causes and inform future research. Over

200 variables were quanti?ied and compared between CCDYaf?licted colonies and apiaries, and

those not af?licted. While 61 of the 200 variables were found frequently enough to make

worthwhile comparisons, no single one stood out as being signi?icantly 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 classi@ication and regression tree

analysis. J Econ Entomol 103(5): 1517Y1523.

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

explaining CCD, researchers performed a classi?ication and regression tree (CART) analysis on

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

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 ?itness 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.

Among several areas recommended for further study, two were highlighted: the effect of subY

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

tolerance to pesticides and colony survival.

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Understanding pes7cides as a causal factor in colony collapse

Pes$cide 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 At least 143 million of the 442 million acres of U.S.

cropland is planted with crops treated with one of three neonicotinoid pesticides known to be highly

toxic to bees: clothianidin, imidacloprid and/or thiamethoxam. This is a conservative estimate derived

from crossYreferencing USDA ARS data, industry reports and available pesticide use data. It does not

begin to account for nonYagricultural uses. (See appendix A for more complete use data and analysis.)

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 ?ields. A crossYsection 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 @ields. PLoS ONE 7(1).

This ?ield study established that bees near agricultural ?ields 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

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

products were all examined as potential exposure routes. The authors looked speci?ically at

corn, which occupies more arable land in North America than any other

crop—88 million acres. Corn is planted throughout the U.S. Midwest

from midYApril 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 ?ields

were contaminated with clothianidin. Soil from ?ields which had not

been planted with treated seeds in over two growing seasons tested

positive for clothianidin as well, which authors interpreted as a feature

of the chemical’s persistence and mobility.

Pollen collected from the treated plants was contaminated by

clothianidin as expected, but beeYcollected pollen samples showed

higher levels, indicating additional pathways of exposure. Levels of

contamination in beeYcollected corn pollen in this study were 10Yfold higher than reported from

an experiment on clothianidinYtreated canola. This is signi?icant because clothianidin was

approved for use on corn and canola simultaneously based on the canola ?ield test. The ?inding

that beeYcollected 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

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developing bees are exposed to pesticides within the hive through stored pollen. During the

period observed, nurse bees were emerging and fed on 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 authors’ most

salient ?inding, however, is the establishment of

a new, especially mobile and toxic exposure

route—planter 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 the air. Authors found “extremely high” levels of neonicotinoids 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

2007Y2008 growing season. They used conventional (gas chromatographyYmass spectrometry)

and recently developed (liquid chromatographyYtandem 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 crossYsection of bees, pollen and wax. A total of 121 pesticides and

metabolites comprising 5,519 total residues were detected and quanti?ied 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 coYoccur with low

levels of systemic pesticides, implicating possible synergistic effects.

The authors concluded that “The widespread occurrence of multiple residues, some at toxic

levels for single compounds, and the lack of any scienti?ic 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 scienti?ic

understanding of pesticide consequences for pollinators.”

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

pollen”: a new condition in honey bee colonies associated with increased risk of colony

mortality. J Invertebr Pathol 101(2): 147Y149.

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

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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 speci?ic 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

midYfall as hives with normal pollen. Incidence of

entombed pollen was notably greater in reused wax comb regardless of any disinfection

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. Quanti@ication of

imidacloprid uptake in maize crops. J Agr Food Chem 53: 5336Y5341.

Researchers in France conducted a threeYyear, randomYsample ?ield study of environmental

contamination by the most prevalent neonicotinoid, imidacloprid, in corn ?ields 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–mass

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

present in ?lowering, 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 pollenYproducing ?lowers, 4.1 ppb for stems and leaves, and 2.1 ppb for pollen itself. These

levels are similar to those previously studied for sun?lower and canola. ImidaclopridY

contaminated corn pollen made up 54% of pollen samples collected at hive entrances, re?lecting

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

Neonico$noids :: Acute, sub6lethal & 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 ?leas and aphids.xvi They were introduced in the 1990s and

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

2005 neonicotinoids had gained a 16% total market share of the nearly € 8 billion global market, and a

nearYlock (77%) on the global seed treatment market which itself grew from a niche € 155 million to a

€ 535 million market.xvii Among their approved uses in the U.S. are topical ?lea treatments for pets, lawn

and garden uses, and a variety of agricultural uses including stone fruits, nuts, canola, sun?lowers and

corn.

“The presence of entombing is the 

biggest single predictor of colony 

loss. It's a defence mechanism that 

has failed." 

- Dr. Jeffrey Pettis, in the UK Guardian

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Table 1 :: Concentrations of highly toxic neonicotinoid insecticides known to cause harm to honey bees. 

Sources :: EPA; Aliouane et al. 2009; Iwasa et al. 2004; Krupke 2012.

Toxicity to 

bees

Oral acute toxicity 

48-hr LD 50 

(a.i./bee)

Contact acute 

toxicity 48-hr LD 

50 

(a.i./bee)

Sub-lethal 

effects range 

(a.i./bee)

Persistence 

(half-life in soil)

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

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

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

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

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 subYlethal 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 illYsuited for the testing of systemic pesticides

because of the ways bees are exposed to systemics in the ?ield

—in smaller doses, over a long period of time, through a variety of routes. Chronic feeding tests using

whole colonies have been recommended as a better way to quantify the effects of systemics.xix

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

and Europe have not been adapted to consider subYlethal, 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

quanti?ied by noting the dose at which half of the insects die within a speci?ic time period. This is

known dose that is lethal to 50% of the test population or LD50. “Acute” and “lethal” are often

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

arching of the abdomen similar to a sting re?lex 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

“U.S. regulatory decisionmaking ... 

has hinged, by default, on the 

establishment of acute toxicity 

exposure scenarios without 

requiring tests for sub-lethal effects.”

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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.

• Sub8lethal toxicity :: The subYlethal

toxicity of neonicotinoid pesticides is

of particular concern because the

most common ?ield exposure

scenarios are likely at the subYlethal

rather than acute level. SubYlethal

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 subYlethal

effects might include compromised

immunity, delayed development and

a host of indirect, potentially

cascading effects that impact the

hive’s ability to sustain itself.

• Cumulative and chronic effects ::

Neonicotinoids function by binding to

nicotinic acetylcholine receptors in

insects’ brains, receptors which are

particularly abundant in bees,

increasing during development from larval to adult stages.xxii This binding leads to an overY

accumulation of acetylcholine, resulting in paralysis and death. The most recent scienti?ic

observations point to a longYlasting 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.

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

both alone and in combination with other pesticides—can cause impaired communication,

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

A selection of these studies follow.

1) Henry, M., Reguin, M., Requier, F., et al. 2012. A Common Pesticide Decreases Foraging Success

and Survival in Honey Bees. Science (20): 348 Y 350.

Researchers attached radio frequency devices (RFIDs) to honey bees to test the impact of

sublethal doses of thiamethoxam (a neonicotinoid) on foraging, homing and survival. They

then plugged these ?indings into colony population dynamics models to assess the extent to

which “homing failure” may contribute to colony collapse. The study con?irms the hypothesis

that sublethal, ?ieldYrealistic doses of thiamethoxam undermine bees’ foraging and homing

abilities at rates signi?icant enough to increase risk of colony collapse. Intoxicated bees were up

to twice as likely as control bees to fail to return home to their hives.

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Neonico7noids atLaLglance

Neonico*noids are a widely used class of systemic insec*cides

introduced in the early 1990s that have been of par*cular

interest for their effects on honey bees.

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

as systemics. Systemic pes*cides are applied as seed coa*ngs or

soil drenches and are taken up through the plant’s vascular

system, and then transmiTed to all parts of the plant, including

pollen and nectar. Neonico*noids are very persistent and

therefore accumulate over *me in the environment.

Most neonico*noids are classified as acutely toxic to bees. But

single, highEdose (i.e. acute) exposures are likely less common

than are the chronic, subElethal exposure levels faced by bees

over *me as they forage in the field.

Honey bee colony collapses in France in 1999 (called “mad bee

disease”) were the first to implicate imidacloprid, the most

widely used neonico*noid, in colony loss. Researchers have

since found a range of subElethal effects caused by

neonico*noids: altered foraging and feeding behavior, impaired

orienta*on and social communica*on, undermined immunity

and delayed larval development.
This semiY?ield study was carried out in an agricultural area in western France and in a suburban

area in southern France. Bees were given a ?ieldYrealistic, sublethal dose of thiamethoxam (1.34

ng in a 20Yμl sucrose solution), then released up to 1 km away from their hives with an RFID

glued to their thorax. RFID readers were placed at the hive entrance to track the return of tagged

bees. “PostYexposure homing failure was then derived from the proportion of nonYreturning

foragers. To discriminate against other potential causes of homing failure in treated foragers (e.g

natural mortality, handling stress, predation), control foragers were fed with untreated sucrose

and tracked in the same way. The authors conclude that sublethal, commonly encountered doses

of thiamethoxam can impact forager survival at suf?icient rates to contribute to colony collapse

and that this impact increases when homing is more dif?icult. One consequence of this ?inding,

according to authors, is that “impact studies are likely to severely underestimate sublethal

pesticide effects when they are conducted on honey bee colonies placed in the immediate

proximity of treated crops.”

2) Aliouane Y., El Hassani Athiamethoxam. K., Gary V., Armengaud C., Lambin M., Gauthier M., 2009.

Subchronic exposure of honey bees to sublethal doses of pesticides: effects on behavior. Environ.

Toxicol. Chem. 28(1): 113–122.

In this laboratory study, emergent honey bees received a daily dose of insecticide ranging from

1/5th to 1/500th of the median lethal dose (LD50) of three pesticides over 11 days. Two of these

pesticides were neonicotinoids, thiamethoxam and acetamiprid; the other was ?ipronil. Fipronil is

another systemic pesticide that is banned in part of Europe because it is highly toxic to bees. The

authors sought to test the effects of sublethal, chronic exposure to these pesticides on honeybee

behavior. Fipronil, used at the dose of 0.1 ng/bee, induced mortality of all honey bees after one

week of treatment. FipronilYtreated bees also exhibited impaired o?lactory memory, and spent

more time immobile. Thiamethoxam by contact induced either a signi?icant decrease of olfactory

memory 24 h after learning at 0.1 ng/bee or a signi?icant impairment of learning performance

with no effect on memory at 1 ng/bee. The experiments with thiamethoxam show that repeated

exposure to a dose that has no behavioral effect when applied in acute conditions results in the

appearance of some behavioral de?icits.

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

administered in subNlethal doses on honey bee behaviour. Laboratory tests. B Insectol 56(1):

59Y62.

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

laboratory conditions to observe effects of imidacloprid fed at subYlethal 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 signi?icant 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.

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

relevance to the subNlethal effects induced by systemic pesticides. Archives of Environmental

Contamination and Toxicology 47: 387Y395.

This study investigated the subYlethal effects of two insecticides in semiY?ield conditions on the

foraging behavior of honey bees. Imidacloprid and ?ipronil were chosen because both behave

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

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 ?ive 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 bees—those visiting the

feeder, but not feeding—increased over time in relation to active bees. With ?ipronil 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 ?ipronilYcontaminated

food. Researchers concluded that both insecticides disturb the hive’s primary activity, feeding,

at subYlethal levels 70 times below the referenced LD50s. They also concluded that their

experimental protocol “provided an indispensable interface between controlled conditions in

the laboratory and the ?ield,” which suggests its adoption in regulatory testing of subYlethal

effects.

5) 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: 83Y92.

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

different methodologies. Researchers sought to build from prior studies that clari?ied the role of

nicotinic acetylcholine receptors (nAChR) in honey bee learning and memory capacities to

investigate the speci?ic effects of the neonicotinoid imidacloprid on those capacities. Bees were

conditioned to respond to a speci?ic ?loral 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 signi?icantly inhibited associative learning as

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

exposure. This impairment of retention notably affected mediumYterm, but not longY or shortY

term memory, appearing as a temporary amnesia. Metabolic activity in speci?ic 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.

6) 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):

1743Y1748.

Researchers conducted a semiY?ield experiment to follow on results of laboratory studies that

had shown a number of detrimental effects from ingestion of imidaclopridYcontaminated food.

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

imidaclopridYcontaminated 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 signi?icant 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 1.82Y4.33

nanograms. Researchers concluded that abnormal foraging behavior could occur in the ?ield

through multiple visits to imidaclopridYcontaminated ?lowers.

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Synergis$c + 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 ?inding 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 nonYneonicotinoid

systemic ?ipronil). Hives exposed to these pesticides were signi?icantly more susceptible to infection.

“Chemical cocktails” :: Fungicides, pyrethroid insec$cides, mi$cides

Neonicotinoids are but one class of pesticides, honey bees are exposed to dozens of different pesticides

on a daily basis (see “Pesticide Prevalence,” esp. Mullin 2010). Included among these are a mix, or

“chemical cocktail,” of insecticides, herbicides and fungicides as well as the miticides used by

beekeepers to control pathogens in the hive.

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

causal complex of CCD are fungicides and pyrethroids—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 certain insecticides

(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.

As bee detoxi?ication mechanisms are increasingly understood, the potential threats posed by

particularly toxic “chemical cocktails” composed of certain fungicides, pyrethroids and neonicotinoids

are slowly coming into resolution. Scientists believe that part of bees’ increased vulnerability to

pesticides comes from their having relatively few genes that encode detoxi?ication enzymes. For

example, one such group of enzymes (P450s) mediate detoxi?ication pathways in ways that can be

inhibited by certain widely used fungicides (e.g.

propiconazole), and are thought to be important for

bees’ ability to tolerate the common pyrethroid

miticide, ?luvalinate.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 detoxi?ication 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

“Honey bees have fewer genes involved in 

detoxification than other insects.”

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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 tri?lumizole.

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 tri?lumizole.

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

exhibit a statistically signi?icant effect on honey bee mortality after three and 24 hours, further

study was suggested before reaching conclusion as to inY?ield, 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): 125Y8.

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

into the colony were amitraz, coumaphos and ?luvalinate, 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 ?luvalinate 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 ?luvalinate was found in the bodies

of bees and in bee larvae. The presence of ?luvalinate 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 PYL, Wang Q, Sun JYH, Liu F, Wang X, Wu YYY and Zhou T. 2010. Effects of subNlethal

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

honeybee Apis mellifera ligustica. Environmental Toxicology and Chemistry 29: 644–649.

This study examined the subYlethal 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

bifenthrin is “highly toxic” and deltamethrin “moderately toxic” to honey bees. This was based

on ?indings that exposure to the insecticides signi?icantly 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 ?indings to

pesticide applications in the ?ield, the study’s authors note that commonly recommended

bifenthrin application rates are higher than the insecticide’s LC50 for honey bees and

deltamethrin application rates are about the same as the LC5.

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Pathogen interac$ons :: Nosema + pes$cides

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 fastYspreading fungal gut pathogen that is thought to interfere with honey bees’

ability to absorb nutrients (infected bees consume signi?icantly 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 CCDYaffected 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—to which they were exposed only indirectly

in brood food as developing larva—faced signi?icantly more Nosema infections than did their control

counterparts.

The overall pattern for bees exposed both to systemic pesticides (neonicotinoids and ?ipronil) 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 subYlethal doses of a

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

They used GC/MS (gas chromatographyYmass spectrometry) with a limit of detection of 0.1 ppb

to analyze the bees, and veri?ied 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 signi?icantly in the bees from pesticideYtreated 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 signi?icantly 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 imidaclopridYspiked protein. “Our test bees

could have only received pesticide exposure during larval development.”

The ?inding that individual bees with undetectable levels of imidacloprid, after being reared in a

subYlethal pesticide environment within the colony, had higher Nosema is signi?icant 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 “suggest new pesticide testing standards be devised that

incorporate increased pathogen susceptibility into the test protocols.” Their conclusion:

“Interactions between pesticides and pathogens could be a major contributor to increased

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

declines worldwide.”

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

@ipronil and thiacloprid highly increases mortality of honey bees 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 ?ipronil and thiacloprid (a neonicotinoid). The main ?inding was that

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

sublethal level of ?ipronil or thiacloprid (71% Y 82% vs. 47%). Infected bees also appeared to be

generally more sensitive to sublethal pesticide poisoning, “After exposure to insecticides,

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

?irst days of exposure.” This study also con?irmed previous ?indings that energetic stress was the

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 ?indings were not.

A secondary, and by this data uncon?irmed, hypothesis pursued by this study was that the

mechanism of the synergistic effect between Nosema and these pesticides was linked to a

decrease in bees’ detoxi?ication capacity as mediated by two groups of enzymes. Noting that

data on the mechanisms underlying synergistic effects between pesticides and pathogens is

poorly understood, authors note, “susceptibility of insects to pesticides is a more complex

phenomenon than previously thought. The in?luence of parasitism in the ecosystem must be

considered in toxicological studies....[especially] since N. ceranae spreads rapidly and can affect

more than 80% of honey bee colonies.”

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

neonicotinoid weaken honey bees (Apis mellifera). Environmental Microbiology 12(3): 774–782.

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 gutYpathogen (Nosema ceranae) and a

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

that signi?icantly 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 compared to each alone and a control group. The enzymatic activity

that correlates to bees’ ability to sterilize the hive food for adults and larvae signi?icantly

decreased only when the two agents were combined. This suggests a synergistic interaction that

could threaten the colony’s ability to withstand a broad range of pathogens in the longYterm.

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

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

This was the ?irst study to establish subYlethal 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 ?luvalinate (a pyrethroid) or coumaphos.

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

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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 signi?icantly lower rates occurring in treatment comb as its contamination level

lessened.

Microbiota out of balance :: Gut cultures, immunity + nutri$on

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

individual bee “gut” culture and throughout the hive considered as an extended organism. While very

little is understood about the honey bee’s complex and diverse microbial community, scientists do

know enough to describe a coYevolved, minimally

functioning, or “core,” honey bee microbial community as

well as hypothesize about key functions susceptible to

disruption—speci?ically 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

pro?ile of affected vs. healthy hives.xxx

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

highYthroughput 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: 431Y444.

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 immuneYrelated function of a gut

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

infections. In this context, the wellYknown pathogen Nosema ceranae, is listed as an infectious

microbe that interferes with digestion midYgut.

Noting the “microbial frontier” opened by recent advances in highYthroughput metagenomic

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

systemsYoriented research into the role of microbiota in bee colony health. With speci?ic regard

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

control disease are also known to destroy bene?icial, nonYtarget fungi and bacteria in ways that

may disruption of the hive’s bene?icial microbial balance. Quoting Mullin et al., (2010), they ?lag

“The road to sustainable honey bee 

pollination may eventually require 

detoxification of agricultural systems 

and in the short term, the integrated 

management of honey bee microbial 

systems.”

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as a concern the fact that synergistic effects of multiple, commonly found pesticides on the

hive’s microbiota are “entirely unknown.” Authors conclude: “The road to sustainable honey bee

pollination may eventually require detoxi?ication of agricultural systems and in the short term,

the integrated management of honey bee microbial systems.”

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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 + detec7on sensi7vity

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

plant tissue below a level of 20Y50 ppb—much 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 and “a few”

ppb 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/ACPIYMS/MS or LCYMS/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; SpringerYVerlag Berlin Heidelberg, 483Y494.

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

pressure liquid chromatography–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 nonYtreated crops. Sun?lowers 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 sun?lower and corn pollen

corresponds to a range of concentrations in which subYlethal 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): 2027Y2033.

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

developing a technique to quantify systemic pesticide residues in the ?ield at levels known, in

the lab, to have subYlethal 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/ACPIYMS/

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

?lowers) and 0.3 ppb in pollen, which had never undergone analysis for imidacloprid. To

validate the method, researchers conducted small ?ield tests in sun?lower and corn ?ields. They

found that most samples from treated ?ields, as well as ?ields that had been treated a full year

earlier, had detectable levels, most of which were high enough to have subYlethal effects on bees

according to studies cited.

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Study design

Designing studies that accurately assess pollinators’ exposure to pesticides under ?ield (i.e. outdoor)

conditions is especially dif?icult because of the wide variety of factors in the natural environment.

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

“chemical cocktail” effect), timing, relative levels of existing 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, subsequent studies typically create “semiY?ield” conditions that more closely resemble

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

in the results. Full ?ield experiments are used to assess the effects of the substance as it occurs in the

bee’s 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 semiY?ield or lab studies. Lab results and semiY?ield results are

not always replicable in fullY?ield studies even with these necessary allowances.

It is inherently more dif?icult to track an individual bee—to 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 speci?ic to ?ield 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

20:429Y437.

This study shows disorientation by pesticides and illustrates the dif?iculties of measuring such

effects under ?ield conditions. Few studies have investigated the impact of pesticides on homing

?light due to the dif?iculty of measuring the ?light time between the food source and the hive. The

aim of this study was to show how the RFID (radio frequency identi?ication) 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 ?light pattern between an arti?icial 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 freeY?lying bees. It was shown that oral treatment of 0.3

ng of ?ipronil per bee (LD50/20) reduced the number of foraging trips.

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Mul$ple 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 neonicotinoidYcoated seeds around 2003.xxxii More recent studies have

con?irmed that this route of exposure is indeed lethal, and is exacerbated by humidity. The leading

hypothesis is that bees ?lying through contaminated dust are “powdered” with acutely toxic levels of

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 honey

bees to particulate matter containing neonicotinoid insecticides coming from corn coated seeds.

Environmental Science & Technology.

This ?ield study investigates planter exhaust during corn sowing as an exposure pathway for

bees foraging in and around those ?ields. The core ?inding is that bees ?lying over sowing ?ields

are directly exposed to neonicotinoids at lethal levels signi?icantly higher than the contact LD50

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

Also con?irmed are recent ?indings that a) high quantities of neonicotinoids from seed coating

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

contaminated planter exhaust can land on bees’ abdomens as they ?ly through the dust; c) bee

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

likely to stick to bees’ abdomens in humid conditions. Study authors note that bees seem to

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

humidity conditions, and ?ind a “signi?icant decrease in the insecticide content” when bees are

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

mortem) may affect concentrations found in samples.

The study’s authors also explicitly address two methods of harm reduction proposed by

industry: thicker, more adhesive seed coating, and modi?ications 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 2009Y2010, 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 modi?ied and unmodi?ied drills: modi?ied 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): 119Y126.

This ?ield study investigated two possible mechanisms through which bees can come into lethal

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

1) direct aerial “powdering” of the bees as they come into contact; or, 2) indirect exposure

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

humidity. Dust from abraded seed coatings expelled during sowing were shown to contain more

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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 fungicideYtreated 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

in?luence 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 years’ 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

“effects of coated maize [corn] seed on honey bees.” Main ?indings consist in establishing that

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

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

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

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

conducted ?ield 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.

Three relevent chapters published in both years’ reports are treated below.

“Dust dispersal during coated maize seed sowing and estimated effects on bees.”

In ?ield trials, dispersal of pesticideYcontaminated dust was found to depend on a number of

procedures: seed coating procedure; use of a modi?ied drill (de?lector) to reduce dust dispersal

during planting; weather and environmental conditions. In 2010, modi?ication of the seeders’

pneumatic drills with an air de?lector succeeded in reducing the dispersal of pesticideY

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

experimenters devised a ?ilter to attach to air de?lectors with the intention of further reducing

dustiness. This combined modi?ication reduced dust dispersal by 90% Y 95%, excepting very

?ine dust particles. In both situations bees die at higher rates: 30%Y60% with the ?ilter +

de?lector, 85% with de?lectors alone.

“Effects in bees by contact with dust during ?light over a ?ield sown with coated maize seed.”

Researchers con?irmed a hypothesis that bees ?lying over a seeder that is sowing treated seed

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

mediated by ingestion of contaminated food. Humidity was further found to signi?icantly

synergize with the pesticides. Authors noted that bees’ abdomens (or integuments) are shaped

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.

“SubYlethal effects of neonicotinoids and ?ipronil on learning and memory of odors and spatial

orientation.”

Studying the proboscis extension re?lex (PER) as an index of certain cognitive processes,

researchers found subYlethal effects of neonicotinoids and ?ipronil on learning and memory of

odors. Researchers also designed a testing protocol to measure bees’ orientation capacity. Bees

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

olfactory memory and learning can signi?icantly impact bees’ foraging abilities and social life as

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

con?irm 2010 ?indings.

Oral (inges7on) toxicity :: Pollen, nectar + gu]a7on droplets

Established oral toxicity levels of neonicotinoids for bees are signi?icantly 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 ?ield 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): 1808Y1815.

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 ?ield sprays. Bees die within minutes after consuming

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

grown from treated corn seed, both openY?ield and inYlab: three neonicotinoids (imidacloprid,

clothianidin and thiamethoxam) and one nonYsystemic pesticide (?ipronil). All treated seeds also

included two fungicides, ?ludioxonil and metalaxylYm, re?lecting 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. ImidaclopridYtreated corn was the most ef?icient translocator, concentrating up to

200,000 μg of active substance per liter, but clothianidin and thiamethoxam 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 ?ieldYgrown guttation drops presented to them in lab conditions. In all

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

(concentrationYdependent).

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

Gaucho® dressed seeds. Bulletin of Insectology 59 (2): 99Y103.

Following European beekeeper assertions of a link between imidaclopridYdressed corn seed and

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

was becoming airborne during seedYsowing. 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

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 ?lowers on the day of sowing and for

at least four days afterward, in amounts relevant to both subYlethal 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

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

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

surrounding corn ?ields during successive periods of sowing for a period of 3Y4 weeks,

representing a longerYterm 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 Y 96 hours, which is too short to observe many chronic or sublethal effects—

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 ?irst 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 beeYmade 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 ?lowers a few miles away from the hive.

Contact exposure to pesticides accordingly varies—larvae spend their ?irst three days of life ?loating in

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

infants and adults, have differing detoxi?ication 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: 71–

83.

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

effects. Researchers used wellYestablished bee biology data to quantify imidacloprid ingestion

by bees whose hives are near treated sun?lowers, according to each bee category. Their

estimates focus on those categories that consume the highest amounts of pollen (nurse bees)

and nectar (waxYproducing, broodYattending, 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 subYlethal effects that can result in

premature death within hours or days.

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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 con?licts of interest in honey bee research impact research

?indings, yield citation bias (where contradictory studies are excluded from introductory literature

reviews), and exert undue in?luence 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): 153Y160.

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

affecting honey bees and other nonYpest insects, within the context of scienti?ic 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

?ield (the mostYused 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 signi?icant effects or effects at

dose levels that do not correlate to environmental levels); and thus 3) potentially in?luenced

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 nonNtarget invertebrates (revised version). Buglife: The Invertebrate Conservation Trust.

This report presents a critical analysis of scienti?ic research, technical reports and regulatory

process documents relevant to the effects on nonYtarget organisms of all ?ive neonicotinoids

registered for use in the U.K. Largely focused on imidacloprid’s effects on honey bees according

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 subY

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 industryYindependent research tends toward opposite results:

imidacloprid being subYlethally 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 in?luenced by factors related to the agendas of those who funded and conducted the

studies as well as the regulatory reviewers. The regulatory process is found to be de?icient in its

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

lethal effects, failure to investigate longYterm, 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.

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Appendix A :: Neonico7noid use pa]erns in U.S. agriculture

Table 2 :: U.S. crop acreage treated with clothianidin, imidacloprid and/or thiamethoxam

Sources :: Bayer Crop Science; USDA.

Crop Total acreage in 2010

Percentage treated in 2010

with one or more of three

neonico7noids for which use

data pa]erns can be compiled

(clothianidin, imidacloprid,

thiamethoxam)

Total projected treated

acreage

Corn 88 million

(2011 – 92..3 million acres) 94% 82.72 million – 2010

Soy 77.4 million

(2011 – 75.2 million acres) 32% 24.77 million – 2010

Wheat, 53.6 million

(2011E 56.4 million acres) 42% (Cereals) 22.51 million – 2010

CoTon 11 million

(2011 – 13.7 million) 42% 4.62 million – 2010

Sorghum 5.4 million

(2011 – 5.3 million acres) 75% 4.05 million – 2010

Sugarbeets 1.17 million

(2011 – 1.24 million acres) 65% 0.76 million – 2010

Sunflowers 1.95 million

(2011 – 1.9 million acres) Unknown Unknown

Canola 1.4 million

(2011 – 1.1 million) 100% 1.4 million – 2010

Rice 3.6 million

(2.6 million acres) 51% 1.84 million – 2010

Alfalfa 20 million

(19.3 million acres) Unknown Unknown

Peanuts 1.3 million

(1.1.5 million acres) Unknown Unknown

Projected total at least 142.67 million

acres

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Source :: EPA
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