Crop Pollination Exposes Honey Bees to Pesticides Which
Alters Their Susceptibility to the Gut Pathogen Nosema
Informative, descriptive title in sentence form with a verb.
ceranae
Please note that not all publications accept sentence titles.
Jeffery S. Pettis1, Elinor M. Lichtenberg2, Michael Andree3, Jennie Stitzinger2, Robyn Rose4,
Dennis vanEngelsdorp2*
1 Bee Research Laboratory, USDA-ARS, Beltsville, Maryland, United States of America, 2 Department of Entomology, University of Maryland, College Park, College Park,
Maryland, United States of America, 3 Cooperative Extension Butte County, University of California, Oroville, California, United States of America, 4 USDA-APHIS, Riverdale,
Maryland, United States of America
Overall, abstract is

Use of "We"with active
voice

informative and concise
Abstract

Recent declines in honey bee populations and increasing demand for insect-pollinated crops raise concerns about
pollinator shortages. Pesticide exposure and pathogens may interact to have strong negative effects on managed honeybee colonies. Such findings are of great concern given the large numbers and high levels of pesticides found in honey beecolonies. Thus it is crucial to determine how field-relevant combinations and loads of pesticides affect bee health. We
Introductorycollected pollen from bee hives in seven major crops to determine 1) what types of pesticides bees are exposed to whensentences
Principal
rented for pollination of various crops and 2) how field-relevant pesticide blends affect bees’ susceptibility to the gut
provide parasite Nosema ceranae. Our samples represent pollen collected by foragers for use by the colony, and do not necessarily
objectives
backgroundare clearly
indicate foragers’ roles as pollinators. In blueberry, cranberry, cucumber, pumpkin and watermelon bees collected pollen
almost exclusively from weeds and wildflowers during our sampling. Thus more attention must be paid to how honey bees
and context.
identified
are exposed to pesticides outside of the field in which they are placed. We detected 35 different pesticides in the sampledwithpollen, and found high fungicide loads. The insecticides esfenvalerate and phosmet were at a concentration higher thannumbers.
their median lethal dose in at least one pollen sample. While fungicides are typically seen as fairly safe for honey bees, weResults concisely
found an increased probability of Nosema infection in bees that consumed pollen with a higher fungicide load. Our resultssummarized.
highlight a need for research on sub-lethal effects of fungicides and other chemicals that bees placed in an agricultural
setting are exposed to.

Principal conclusion places paper incontext
withBeesotherstudiesCitation: Pettis JS, Lichtenberg EM, Andree M, Stitzinger J, Rose R, et al.appropriate(2013) Crop Pollination
Exposes Honeyto Pesticides
Whichand
Alters Their Susceptibility
to the Gut Pathogen Nosema ceranae. PLoS ONE 8(7): e70182. doi:10.1371/journal.pone.0070182
highlights areas for future research

Editor: Fabio S. Nascimento, Universidade de São Paulo, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Brazil
Received March 25, 2013; Accepted June 16, 2013; Published July 24, 2013This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone forany lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: Funding for this study was provided by the National Honey Board (http://www.honey.com/) and the USDA-ARS Areawide Project on Bee Health (http://
www.ars.usda.gov/research/projects/projects.htm?accn_no = 412796). Neither the Honey Board nor USDA-ARS Program Staff had a role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Dennis vanEngesldorp is a PLOS ONE Editor. All other authors have declared that no competing interests exist. This does not alter theauthors’ adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: dennis.vanengelsdorp@gmail.com

As appropriate, introduction
has a funnel shape.
Introduction
Introduction begins broadly
Honey bees, Apis mellifera, andare oneof the backgroundmost important
providespollinators of agricultural cropsand[1]. Recent
declinesin honeybee
context:
in thiscase,
populations in many North AmericanandEuropeancountries[2–
the importance of4] and increasing cultivation of crops that require insects forhoneybees to agriculture.
pollination [5] raise concerns about pollinator shortages [5,6].

Foreshadowsbehavior, learning and immune function [9,13,14,16–22].
Reduced immune functioning is of particular intereststudy's
becauseresearchofrecent disease-related declines of bees including honeybees [3,23]. andquestions
Pesticide and toxin exposure increases susceptibility
to andobjectives
mortality from diseases including the gut parasite Nosema spp.
[14,15]. These increases may be linked to insecticide-induced
alterations to immune system pathways, which have been found
Habitat destruction, pesticide use, pathogens and climate change
for several insects, including honey bees [22,24–26].
are thought to have contributed to these losses [2,7,8]. Recent
Surveys of colony food reserves and building materials (i.e. wax)
research suggests that honey bee diets, parasites, diseases andhave found high levels and diversity of chemicals in managed
pesticides interact to have stronger negative effects on managed
colonies [18,27,28]. These mixtures have strong potential to affectbegins[11,12]
to narrowhoney bee colonies [9,10]. Introduction
Nutritional limitationand inindividual and colony immune functioning. However, almost all
here,with areview researchof
exposure to sub-lethal dosesscope;of pesticides[13–16],in brief
particular,
to-date on pesticides’ effects on pathogen susceptibility
may alter susceptibility to orthe
severityof diverse bee parasites andliterature.
fed a single chemical to test bees [16]. Because pesticides may havepathogens.
interactive effects on non-target organisms (e.g. [29]), it is crucial
Recent research is uncovering diverse sub-lethal effects of
to determine how real world combinations and loads of pesticides
pesticides on bees. Insecticides and fungicides can alter insect andaffect bee health.
Topic sentence clearlyspider enzyme activity, development,
oviposition behavior,
Introduction
One pathogen of major concern to beekeepers is Nosema spp.
states the main point ofoffspring sex ratios, mobility, navigationand orientation,
feeding
The endoparasitic fungal infections of N. apis and N. ceranaecontinues
to

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narrow; here, therelevance of
pathogens.

the paragraph.

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Justification forresearch model

Funnel shape continues as
introduction becomes more specific.

Words like "then"readers
crops via a Kruskal-Wallis test followed by a help
post-hoc
nonunderstandparametric Tukey-type test (using the R package nparcomp thesequence[33]). We then divided each sample into three portions.
A 5 gofsubsample was sorted by color and then each groupofsimilarly
experimental
colored pollen pellets were identified (see below);
a 3g
procedures.

adversely affect honey bee colony health, and can result incomplete colony collapse [30]. Infection with Nosema in theautumn leads to poor overwintering and performance thefollowing spring [31], and queens can be superseded soon after
becoming infected with Nosema [32]. We chose Nosema as a model
pathogen because earlier work [13,14] had demonstrated aninteraction with pesticide exposure.
This study addresses two important questions. 1) What types ofpesticides might bees be exposed to in major crops? While multiple
Researchquestionsstudieshave characterized the pesticide profile of various materials
clearly identified
inside a honey bee nest [27,28], few have looked at the pollen
with numbers.
being brought back to the nest. 2) How do field-relevant pesticides
Manybyjournals
blends affect bees’ susceptibility to infection
the Nosema
parasite?
require an

subsample was sent to the USDA’s Agricultural Marketing
Service Laboratory in Gastonia, NC for pesticide analysis; and a
10 g subsample was sent to the USDA-ARS Bee ResearchLaboratory (Beltsville, MD) for the Nosema infection study.
Past tense used, as
Because almond pollen was collected after all other pollens, we
is appropriate
were unable to include it in the pesticide analysis
and Nosema in amethods
section.
infection study. In cases where the total amount of pollen
collected from a single colony was less than 6 g all the pollen
was used for pesticide analysis.

EthicsStatement.

Methods

Crop Pollens Affect Bee Health

Pollen Identification
Each 5 g pollen subsample was dehydrated in a drying oven at
40uC. We considered a sample to be dry when its weight did notchange between two consecutive time points (measured every 4–6 h). Typically pollen dried in 12–18 h. To identify pollen types
collected by the bees, we sorted the pollen in each subsample byAddresses methodcolor, quantified each color by comparing to Sherwin-WilliamsH
color palettes, re-weighed after color separationlimitations
and fixed eachcolor from each subsample on a separate slide. We prepared eachslide by grinding 2 pollen pellets in 2 mL water and letting themdissolve to form a slurry. We placed a small amount of slurry on aslide with a drop of silicon oil, and covered slides and sealed withclear nail polish after letting air bubbles escape for 48 h. We
visually identified each pollen type under 400x magnification bycomparing with published reference collections [34–36]. Visual
identification of pollen grains through comparison with voucher orreference specimens is standard in pollination Genus
ecology [37,38].
and species
Similarities between closely related pollens, however,sometimes
names
provided.
prevent identification to genus or species with this method [39].
Because of this limitation, we assumed that all pollen collected inapple (Malus domestica) orchards that was identified as Malus sp. wasfrom apple trees, and that all pollen in the Cucurbitaceae familycollected in cucumber (Cucurbitaceae, Cucumis sativus) fields wasfrom cucumber flowers.
For each subsample, we estimated pollen diversity as thenumber of different pollen colors collected from that bee hive. We
also calculated the proportion, by weight, of the pollen that was
identified as belonging to the target crop’s genus. Many samples
could only be identified to genus, so assessing target genus rather
than target crop permitted a more inclusive analysis. We used

Ethics Statement
Pollen was collected from honey bees with permission of thebeekeepers and the land owners.

Hive Selection and Pollen Collection
We collected pollen carried by foraging honey bees returning to
the hive for nine hives in seven crops: almond, apple, blueberry,
cranberry, cucumber, pumpkin, and watermelon (Table 1). For
each crop,
Use of "we"withwe selected three fields that were separated by at least
3.2 km.makes
Hives were deployed in these fields for pollination services
active voice
based on growers’ needs. Within each selected field, we chose themethods
sectionthree honey bee hives with the strongest foraging forces by
easier to
read. flight in the bee yard for 5–10 min, and attached plastic
observingpollen traps (Brushy Mountain Bee Farm, Moravian Falls, NC) tothese hives. Pollen traps collect the pollen pellets bees carry on
their hind tibiae in flattened regions called corbiculae. Bees use
this pollen to make food for larvae inside the nest. We checkedof whyat least
traps after three days, and removedExplanation
them if they contained
5 g of pollen. Traps with less than 5methods
g remained wereon hives until they
contained 5 g of pollen or for 10 days.
We placed pollen removed
appropriate
from traps in 50 mL centrifuge tubes and stored the samples on ice
until they could be transferred to a 229uC freezer in the lab.
Because our first round of pollen trapping in cranberry fields
yielded little pollen, we collected pollen from each hive in
cranberry fields twice: early in the flowering season and late in theseason. We separate these samples in data analyses, referring tothem as ‘‘Cranberry early’’ and ‘‘Cranberry late.’’We measured the wet weight of each pollen sample, andcompared the quantity of pollen collected by hives in different

Table 1. Quantity and diversity of pollen collected in pollen traps on individual honey bee hives.

Crop

Location

Mean grams of pollen collected (se)Mean number of pollen types (se)42.0 (9.1)

a,b

1.7 (0.2)a,bYork Springs, PA; Adams County

26.7 (2.6)

a4.9 (0.5)c

Blueberry

Deblois, ME; Washington County

4.1 (1.5)b

Cranberry (early season)

Hammonton, NJ; Atlantic County

13.0 (2.5)a,b

4.0 (1.0)b,c

Cranberry (late season)

Hammonton, NJ; Atlantic County

13.9 (3.8)a,b

4.1 (0.6)b,c

AlmondApple

CucumberWatermelon
Pumpkin

Rosedale, CA; Kern County

Cedarville, NJ; Cumberland County
Seaford, DE; Sussex County
Kutztown, PA; Berks County

8.1 (2.7)

Table title isinformative; table isunderstandable
without reference tothe text.

6.0 (1.0)cb

5.5 (1.3)b,c

27.1 (11.2)a,b

7.1 (1.2)c

98.6 (29.0)a,b3.7 (0.6)b,c

Letters indicate statistically different groups.
doi:10.1371/journal.pone.0070182.t001

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Crop Pollens Affect Bee Health

Words
like
"furtheron infection prevalence, the number of individuals
with
Nosematested"help
orientspores.
thereaderandTo look for potential effects of individual pesticides on
susceptibility to Nosema infection, we calculated
the relative
explainwhyriskand its 95% confidence interval for bees becoming
infectedafter
methods wereconsuming pollen with a specific pesticide. Relativerisk
measuresappropriate.
the chance of developing a disease after a particular exposure [41],here each pesticide. A relative risk value of one indicates that theprobability of infection is equal between exposed and non-exposedgroups.
We further tested effects of pesticides in pollen on measured
Nosema prevalence using a generalized linear mixed model with abee’s Nosema status as the response variable, the source hive andpesticide variables as fixed effects, and the pollen sample fed to thebee as a random effect. Collinearity prevented developing a fullmodel to investigate in detail how pesticides and pollen source
affect bees’ susceptibility to Nosema infection. We thus selected foranalysis two measures that vary with crop and are not nested: total
pesticide diversity and fungal load. To graph logistic regression
results in a meaningful manner, we followed Table
recent recommenis referenced
dations [42,43] and a modification of the logi.hist.plot
function in andin parentheses
the R popbio package [44] that shows our mixed
output.
notmodeldirectly
in text.

Kruskal-Wallis tests to determine whether either of these measuresdiffered with the crop in which sampled bee hives were placed.

Pesticide Analysis

Subheadings guidereaders
throughthe
We determined the identity and loadof pesticide
residues
present in pollen samples collected frompaper.
all crops (except almond).
For each field sampled (n = 19), we pooled pollen from the threehives for analysis. One early-season cranberry field and one
cucumber field did not yield sufficient pollen in traps for pesticide
analysis. Methods follow the LC/MS-MS and GC/MS methods
for pollen analysis described in Mullin et al. [27]. We used thesedata to determine the total number of pesticides detected in eachsample, each sample’s total pesticide load, and the diversity andpesticides in each of 10 categories: insecticides, fungicides,
Citationload
of ofanotherherbicides, and several insecticide types (carbamates, cyclodienes,
paper formethodsneonicotinoids, organophosphates, oxadiazines andformamidines,
description
pyrethroids). To permit comparison between categories withdifferent numbers of elements, we calculated diversity as the
proportion of pesticides from a category found in a given sample,
and load as the total load divided by the number of chemicals inthat category. We only calculated diversity for categories with atleast three chemicals.
The total number of pesticides present and total load did notmeet parametric assumptions. We thus analyzed how thesevariables differ between crops using non-parametric KruskalWallis tests. When separated by category and log-transformed,
pesticide loads did meet parametric assumptions. We thus
determined whether load varied by pesticide category using ageneral linear mixed model with sample as a random effect, tocontrol for the fact that our regression included one data point per
category from each sample. Insufficient degrees of freedom
prevented us from expanding this model to include crop. We
thus asked whether the pesticide load and diversity varied withcrop for each category using one Kruskal-Wallis test per category
and applying a sequential Bonferroni correction [40] acrosspesticide categories to control for multiple comparisons.

Results
Pollen Collection
Bee colonies collected different amounts of pollen in the
different crops (Table 1; Kruskal-Wallis test: H7 = 29.6,
Statistical values
p = 0.0001). Pollen diversity, estimated by quantifying theare in inparentheses,
number of differently colored pollen pellets collected
pollentraps, varied by crop (Table 1; Kruskal-Wallismaking
test: H7 results= 23.5,p = 0.0014). The proportion of pollen that bees
collectedeasierfrom tosectionthe target crop, except for almond and read.
apple, was low(mean6se = 0.3360.05; Table S1). Like pollen weights, this
proportion dramatically differed between crops (Fig. 1;
H7 = 44.86, p,0.0001). Notably, none of the pollen trappedfrom hives in blueberry, cranberry (early and late), pumpkin or
watermelon fields was from the target crop.

Nosema Infection
The Nosema infection experiment is similar to published methods[26]. We obtained 210 disease-free honey bees from each of three
Includesmaterial
healthy colonies at the Bee Research Laboratory. Each bee wasinformationsuch
as21 groups upon emergence, with the ten bees inplaced into
one ofcompany
andthe samegroup and from the same colony housed together in alocationwooden hoarding cage (12612612 cm). Each group of bees was
fed 1 g of pollen mixed with 0.5 mL of syrup (1:1 sucrose to waterby weight), which they fully consumed in 2–4 days. These pollen
cakes were placed in small petri dishes with the laboratory cages.
Pollen from either one of the crop fields or one of two control diets
were used. The pollen control group (‘‘BRL’’) was fed a mixed
pollen diet prepared by the USDA-ARS Bee Research Laboratory. This pollen was collected in the desert Southwest (Arizona
Bee Products, Tucson, AZ) and tested as pesticide-free by the
USDA Agricultural Marketing Service prior to use. A protein
control group was fed an artificial honey bee pollen substitute,
MegaBeeH. The Nosema inoculum was freshly prepared by mixing
Nosema spores isolated from an infected colony (details provided in[26]) with 50% sucrose solution to obtain a concentration of ca. 2
million spores per 5 mL. We fed 5 mL of the Nosema inoculum toeach cage during the first two days of adult life, then provided bees
with ad libitum access to clean 50% (w/v) sucrose solution. We
collected bees 12 days after infection and examined them for thepresence or absence of N. ceranae spores by homogenizing
individual abdomens in 1 mL distilled water. Here we focus only
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Pesticide Analysis
All pollen collected in this study contained pesticides (Table 2;mean 6 se = 9.161.2 different chemicals, range 3–21). Pesticide
loads ranged from 23.6 to 51,310.0 ppb (11,760.063,734.2 ppb).
The maximum pesticide concentration in any single pollen sample
exceeded the median lethal dose (LD50, the dose required to kill
half a population within 24 or 48 h) for esfenvalerate and phosmet(Table 2). The number of pesticides detected in trapped pollen
varied by the crop in which the bee hives were located (KruskalWallis test: H6 = 12.96, p = 0.04), but the total pesticide
load didSentencesarenot (H6 = 11.21, p = 0.08)(Fig. 2).
short and concise,
We found insecticides and fungicides in all 19,
and herbicides
allowing
results toin 23.6% of, pollen samples. Insecticides present in pollen
beclearly
collected by the bees came from seven categories. We foundunderstood.
oxadiazines in 10.5%, neonicotinoids in 15.8%,
carbamates in
31.6%, cyclodienes in 52.6%, formamidines in 52.6%, organophosphates in 63.2%, and pyrethroids in 100% of pollen
samples. Both neonicotinoids and oxadiazines were present onlyin pollen collected by bees in apple orchards (Figs. 3, S1).
Within a sample, pollen fungicide loads were significantly higher
than loads of herbicides or any of the insecticide categories(Fig. 4; GLMM, likelihood ratio test: x2 = 121.9, df = 8,p,0.0001).
After adjusting for multiple comparisons, pesticide loads did notvary by crop for any pesticide category (Fig. S1). We calculated
3

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Discussion begins
Crop Pollens
Bee results,
HealthwithAffectstudy

Discussion

then broadens in
scope.

The results from this study highlight several patterns that meritfurther attention. First, despite being rented to pollinate specificcrops, honey bees did not always return to the nest with corbicularpollen from those crops. These findings support other researchwith honey bees and native bees indicating that in some crops
Results arenative bees may be more efficient pollinators [45]. Second,
discussed
fungicides were present at high levels in both cropand non-crop
meaningfullypollen collected by bees. Third, two fungicides (chlorothalonil
andandpyraclostrobin), and two miticides used by beekeepers
to control
not simply
varroa infestation (amitraz and fluvalinate) had
a pronounced
repeated;in thisPlaces this paper in
effect on bees’ ability to withstand parasite infection.
on arecase,Researchpatterns
pesticides’ effects on bee health has focused almost exclusively on
context with otheranalyzed.
insecticides (e.g. fipronil [15] and the neonicotinoids imidacloprid
research.
[13,14] and thiacloprid [15]). Finally, several individual pollen
samples contained loads higher than the median lethal dose for a
specific pesticide. While multiple studies have shown negative
effects of specific pesticides on honey bee individual and colony
health [14,15,22,26] and high pesticide exposure [27,28], ours isthe first to demonstrate how real world pollen-pesticide blends
affect honey bee health.
Our results show that beekeepers need to consider not only
pesticide regimens of the fields in which they are placing theirbees, but also spray programs near those fields that may contributeto pesticide drift onto weeds. The bees in our study collected
pollen from diverse sources, often failing to collect any pollen from
Figure 1. Pollen collection from the crop where a hive wasClearly
identifies
the target crop (Fig. 1). All of the non-target pollenthat we
werelocated was low for most crops. Bars show mean 6 se. Letters
Important,practical
able to identify to genus or species was from wildflowers (Table
indicate statistically significant differences (p,0.05).
representative
S1), suggesting the honey bees were collecting significant
amounts
applications
doi:10.1371/journal.pone.0070182.g001
results are clearlyof pollen from weeds surrounding our focal fields. The two
exceptions to this were hives placed in almond and apple orchards.
identified;pesticide
repetitive
diversity within only those categories containing three or
Almond flowers early in the year, and almond orchards are large,
chemicals.
Fungicide and neonicotinoid diversities varied bydata are moreshown
inthus providing honey bees with little access to other flowers.
tables. crop, but diversities of other pesticide categories did not (Fig. 3).
Honey bees rarely collect pollen from blueberry or cranberry
flowers, which only release large quantities of pollen after being
Nosema Infection
vibrated by visiting bees (buzz pollination) [46,47]. Honey bees are
147 of the 630 bees (23.3%) fed Nosema spores became infected.
Addresses
study
not capable of buzz pollination and thus are unlikely
to collect
22 of the 35 pesticides (62.9%) found in our pollen
samples
had
Unexpected results
limitations
large
amountsofpollenfromtheseplants
to
bring
back
to
the
relative risk values significantly different from are
1 (Table2). 8 andidentified
colony. Bumble bees, which can buzz pollinate, collect mainly
pesticides (22.9%) were associated with increased Nosema prevadiscussed.
blueberry pollen when placed in blueberry fields [48]. Interestlence, while the remaining 14 were associated with decreased
ingly, the two crops that saw high levels of pollen collection by
Results Nosema
are prevalence. Two of the three detected pesticides applied byhoney bees are Old World crops that evolved with honey bees as
beekeepers
tocontrol
hive
mites(marked
with
a
*
in
Table
2)
had
a
provided but notnatural pollinators. Crops native to the New World, where honeyrelative risk larger than two, indicating Nosema prevalence in bees
discussed;
bees have been introduced, yielded little or no pollen in our
fed pollen containing those chemicals (DMPF and fluvalinate) was
discussion
saved
samples.
moreisthan
double the Nosema prevalence in bees that did not
Transition words
for the next
section.
It is possible that bees were exposed to pesticides while
consumethese chemicals. Of the seven pesticides found in pollen
provide flow
collecting nectar from our focal crops, even when we detectedfrom over half, or at least four, of the crops, the majority were
betweenno pollen from those crops. Because pollen traps
collect only
associated with higher Nosema prevalence in bees that consumed
sentences.
corbicularpollenintendedfor
consumptionbythe colony, ourthem. Both control diets had relative risk values not significantly
data indicate only flowers from which bees are actively collecting
different from one.
pollen and not all flowers they visited. Several studies haveA pollen sample’s fungicide load significantly affected Nosema
detected pesticides in floral nectar and pollen [49,50], sometimes
prevalence among bees fed that pollen (Fig. 5; GLMM, likelihoodin concentrations with sublethal effects on honey and bumble bees
ratio test: x2 = 5.8, df = 1, p = 0.02), but pesticide diversity did not[51,52]. Honey bees may collect nectar from blueberry and(x2 = 1.7, df = 1, p = 0.19). A bee’s source colony, included as acranberry flowers via legitimate visits or ‘‘robbing’’ through slits
blocking variable, also did not affect Nosema prevalence (x2 = 2.0,
cut at the base of flower corollas [53]. However, exposure to
df = 2, p = 0.36). Replacing fungicide load with chlorothalonil load
pesticides via nectar may be unlikely in cucumber, pumpkin andobtained the same result (chlorothalonil load: x2 = 5.3, df = 1,
watermelon. Beekeepers often report poor honey production whenp = 0.02; pesticide diversity: x2 = 1.5, df = 1, p = 0.23; source
their hives are placed in these crops (pers. obs.).
colony: x2 = 2.0, df = 2, p = 0.36; fungicide load model
The combination of high pesticide loads and increased Nosema
AIC = 612.71, chlorothalonil load model AIC = 613.15). Chlorinfection rates in bees that consumed greater quantities of theothalonil was also the most abundant fungicide in our samples,
fungicides chlorothalonil and pyraclostrobin suggest that someand comprised 50.0610.2% (mean 6 se) of the per sample total
fungicides have stronger impacts on bee health than previously
fungicide load.
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Information reads from left toright, making informationeasier to follow.

Crop Pollens Affect Bee Health

Table 2. Pesticides found in pollen trapped off honey bees returning to the nest.

Pesticide

Insecticide family

LD50 (ppm)a

Crops in which
detectedc

Detections

Quantity detected,
mean6se (max) (ppb)

Relative risk (95%
CI)

Fungicides
Azoxystrobin.1,562.5 [64]Cr, Cu, Wa

10

60.3625.6 (332)0.75 (0.56, 1.02)

Captan.78.13 [65]Ap, Cr, Cu, Wa

9

976.96734.4 (13,800)

0.59 (0.42, 0.81){

Chlorothalonil.1,414.06 [66]Ap, Bl, Cr, Cu, Pu, Wa17

4,491.262,130.7 (29,000)

2.31 (1.35, 3.94){

Cyprodinil.6,125 [67]Ap

996.96707.5 (12,700)

0.31 (0.15, 0.65){

3

171.46119.4 (2,110) Alternating
0.31 (0.15,gray0.65){

Difenoconazole.781.25 [68]Ap3Fenbuconazole.2,282.65 [69]Ap, Cr, Cu

10

Pyraclostrobin573.44 [70]Cr, Pu4

Quintozene (PCNB).0.78 [71]Cr

2

Cr, Cu3

and whitelines
0.33 (0.23,
0.48){helpseparate
2,787.161,890.1 (27,000)
2.85 (2.16, 3.75){
informationa0.360.3 (4.7)
0.97 (0.59, in
1.61)
long
table.
832.16531.8 (9,470)
0.42 (0.21, 0.82){

THPI

Captan metabolite

227.3689.2 (1,420)

Herbicides
Carfentrazone ethyl.217.97 [72]Cr

10.160.08 (1.6)

1.05 (0.54, 2.05)

Pendimethalin.388.28 [73]Ap, Cr, Pu

55.163.7 (69.5)

1.47 (1.08, 1.99){

Bl, Cu, Pu, Wa

10

171.56117.0 (2,060)

2.13 (1.56, 2.92){

Insecticides
2,4 Dimethylphenyl
formamide (DMPF)*

Amitraz (formamidine)
metabolite

Acetamiprid

Neonicotinoid

55.47 [60]Ap359.1632.2 (401)0.31 (0.15, 0.65){

Bifenthrin

Pyrethroid

0.11 [74]Pu, Wa3

6.663.8 (53.1)

2.08 (1.53, 2.83){

Carbaryl

Carbamate8.59 [75]Ap, Cu, Wa

6

57.8630.0 (403)0.42 (0.27, 0.66){

Chlorpyrifos

Organophosphate

0.86 [16]Ap, Cr, Cu, Pu

7

3.161.1 (15.5)

0.89 (0.64, 1.23)

Coumaphos*

Organophosphate

35.94 [16]Bl, Cr, Cu

6

2.261.0 (17.5)

0.62 (0.43, 0.91){

Cyfluthrin

Pyrethroid,0.31 [76]Cr, Wa20.660.4 (5.4)

1.31 (0.85, 2.02)

Cyhalothrin

Pyrethroid

0.30 [77]Ap, Pu, Wa

7

14.667.9 (131)0.94 (0.69, 1.29)

Cypermethrin

Pyrethroid

0.18–4.38 [78]Cr

10.460.4 (6.9)

1.05 (0.54, 2.05)

Deltamethrin

Pyrethroid

0.39 [79]Cr

1

4.564.5 (85.3)

1.05 (0.54, 2.04)

DiazinonOrganophosphate

1.72 [80]Ap, Cr3

1.461.0 (19.8)

0.56 (0.32, 0.97){

Endosulfan I

Cyclodiene

54.69 [16]Ap, Cr, Cu, Pu, Wa

8

1.560.7 (12.9)

1.60 (1.20, 2.14){

54.69 [16]Endosulfan II

Cyclodiene

Endosulfan sulfate

Endosulfan metabolite

Esfenvalerate

Pyrethroid

Fluvalinate*

Pyrethroid

Heptachlor epoxide

Heptachlorb (cyclodiene)
metabolite

Ap, Cr, Cu, Pu

60.860.3 (5.3)

1.41 (1.04, 1.91){

Cr, Cu40.360.2 (2.1)

0.79 (0.52, 1.19)

0.13 [81]Ap, Cr, Cu

7

16.9612.0 (216)0.51 (0.35, 0.75){

1.56 [82]Bl, Cr, Cu, Pu, Wa

16

42.4629.7 (570)

2.43 (1.49, 3.96){

Cr1

0.660.6 (12)1.05 (0.54, 2.04)
0.31 (0.15, 0.65){

Imidacloprid

Neonicotinoid

0.23 [83]Ap

32.862.0 (36.5)

Indoxacarb

Oxadiazine

1.41 [84]Ap20.560.5 (9)0.28 (0.11, 0.73){

Methidathion

Organophosphate

1.85 [85]Cr

1

1.661.6 (31)1.05 (0.54, 2.04)

Methomyl

Carbamate,3.91 [86]Wa1

13.6613.6 (259)

1.54 (0.91, 2.61)

PhosmetOrganophosphate

8.83 [85]Ap, Cr, Cu

5

798.76772.4 (14,700)

0.36 (0.21, 0.61){

Pyrethrins

Pyrethroid

0.16 [16]Cr

1

5.165.1 (97.4)

1.05 (0.54, 2.05)

Thiacloprid

Neonicotinoid

114.06 [60]Ap21.160.8 (12.4)

0.35 (0.15, 0.82){

BRL

NA

NA

NA

NA

NA

0.58 (0.23, 1.48)

MegaBee

NA

NA

NA

NA

NA

0.74 (0.33, 1.67)

Control dietsa

We divided LD50 values given as mg/bee (g) by 0.128 (equivalent to multiplying by 7.8) to obtain ppm when necessary [85]. If multiple values have been published, we
include only the smallest.
Heptachlor has been banned for use on cranberries since 1978 [87], but can persist in the soil for extended periods of time.
cAp = apple, Bl = blueberry, Cr = cranberry, Cu = cucumber, Pu = pumpkin, Wa = watermelon.
Legend allows*Used by beekeepers within the hive for parasitic mite control.
{
readers toRelative risk different from 1 at the 95% confidence level.
NA indicates information that is not relevant to control diets.
understanddoi:10.1371/journal.pone.0070182.t002babbreviations usedin the table.

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Crop Pollens Affect Bee Health

Figure 2. Pesticide diversity found in pollen samples, but notpesticide load, varied by crop. White bars show pesticide diversity,
gray bars show pesticide load (mean 6 se). Post-hoc testing found thefollowing groups, where letters indicate statistically significant differences: apple a, b; blueberry c; cranberry_early d; cranberry_late b, d, e, f;
cucumber e; pumpkin c, d, f; and watermelon d.
doi:10.1371/journal.pone.0070182.g002

Transition words suchthought. Nosema infection was more than twice asas likely(relative "While,""However,"risk .2) in bees that consumed these fungicides andthan inbees that increase
Figure 4. Load varied by pesticide category. Letters indicate"Indeed"statistically significant differences. The total load for each category is
did not. Research on the sub-lethal effects of pesticides
on honey sentences.
flow between
weighted by the number of chemicals in that category, to facilitate
bees has focused almost entirely on insecticides, especiallycomparison across categories.
neonicotinoids [54]. In our study, neonicotinoids entered the nest
doi:10.1371/journal.pone.0070182.g004
only via apple pollen. However, we found fungicides at high loads
in our sampled crops. While fungicides are typically less lethal to
make bees more sensitive to acaricides, reducing medial lethal
bees than insecticides (see LD50 values in Table 2), these chemicals
doses [58]. In our study, consuming pollen with higher fungicide
still have potential for lethal [55] and sub-lethal effects. Indeed, theloads increased bees’ susceptibility to Nosema infection. This result
fungicides chlorothalonil (found at high concentrations in ouris likely driven by chlorothalonil loads. The pesticide with thepollen samples) and myclobutanil increases gut cell mortality to thehighest relative risk was the fungicide pyraclostrobin. Bees thatsame degree as imidacloprid [56], an insecticide with numerous
consumed pollen containing pyraclostrobin were almost three
sub-lethal effects (e.g. [21,57]). Exposure to fungicides can also
times as likely (relative risk = 2.85, 95% CI 2.16–3.75; Table 2)

Study conclusions
are discussed inrelation to previous
research.

Figure 3. Fungicide and neonicotinoid diversities varied by crop. Bars show the total number of pesticides in each category found in eachcrop. Kruskal-Wallis test statistics comparing pesticide diversity between crops are: fungicides, H6 = 16.1, p = 0.01; cyclodienes, H6 = 6.9, p = 0.33;
neonicotinoids, H6 = 17.9, p = 0.007; organophosphates, H6 = 14.3, p = 0.03; pyrethroids, H6 = 7.8, p = 0.26. We only compared pesticide diversities for
categories containing at least three chemicals. Sequential Bonferroni adjusted critical values are: 0.01, 0.0125, 0.0167, 0.025, 0.05. A * indicates thatthe total number of pesticides varied between crops within that pesticide category.
doi:10.1371/journal.pone.0070182.g003

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Use of "First," "Second,"
Crop Pollens Affect Bee Healthand"Third" guide readers
through the paper.
however, additional sampling is necessary to determine how robust
this pattern is.
The large numbers of pesticides found per sample and the highconcentrations of some pesticides are concerning. First, two pollen
samples contained one pesticide each at a concentration higher
than the median lethal dose. Esfenvalerate (LD50 = 0.13 ppm) was
measured at 0.216 ppm in pollen collected by bees in a cucumber
field, and phosmet (LD50 = 8.83 ppm) at 14.7 ppm in one apple
orchard. While the mean loads for these pesticides are well below
their respective median lethal doses (0.0169 ppm for esfenvalerate,
0.7987 ppm for phosmet), our data indicate some bee colonies arebeing exposed to incredibly high levels of these chemicals. Second,
research suggests that simultaneous exposure to multiple pesticides
decreases lethal doses [58,60] or increases supersedure (queen
replacement) rate [61]. Our pollen samples contained an averageof nine different pesticides, ranging as high as 21 pesticides in one
cranberry field. Thus published LD50 values may not accurately
indicate pesticide toxicity inside a hive containing large numbersof pesticides. Research looking at additive and synergistic effects
between multiple pesticides is clearly needed. Third, pesticides canhave sub-lethal effects on development, reproduction, learning andmemory, and foraging behavior. The mean and maximum
imidacloprid loads in our samples (0.0028 and 0.0365 ppm,
respectively) are higher than some published imidacloprid
concentrations with sub-lethal effects on honey and bumble bees
(0.001–0.0098 ppm [21,54,62]).
It is not surprising that total pollen collection varied by crop.
Bee foraging activity levels vary with weather [63], thus outcomesof short-term measurements may be sensitive to temperature,cloud cover or humidity during data collection. Because wecollected pollen samples from different parts of the country and on
different days, weather conditions undoubtedly differed between
crops. Crop flowering timing and landscape-level floral availability
can also affect bee activity levels. We focused our analyses onvariables less affected by these factors, such as the diversity ofpollen types found in samples and the proportion of a sample thatwas from the target crop. Conclusions are
Our results are consistent with previously published pesticide
clearly stated andanalyses of pollen collected by honey bees or honey bee nest
significanceofgeographically morematerial [16,18,27]. The more
intensive andpaper
discussed.
diverse sampling of Mullin et al. [27] resulted in almost triple thenumber of pesticides we found, but the average number ofpesticides per sample (7.1) is slightly lower than our 9.1. In our
Citationsare placed
study and those listed above, pesticides appliedby beekeepers
tocontrol hive pests were present in a large
proportionof thewithinthe sentence
samples, often in quantities higher than most(andof thenot
pesticides
thatat theend)
are applied to crops.
when appropriate.
Our results combined with several recent studies of specificpesticides’ effects on Nosema infection dynamics [13–15] indicatethat a detrimental interaction occurs when honey bees are exposed
to both pesticides and Nosema. Specific results vary, and may
depend on the pesticide or dose used. For example, bees exposed
to imidacloprid and Nosema can have lower spore counts than bees
only infected with the pathogen but also exhibit hindered immune
functioning [13]. Our study improves on previous methodologies
by feeding pollen with real-world pesticide blends and levels that
truly represents the types of exposure expected with pollination ofagricultural crops. The significant increase in Nosema infection
following exposure to the fungicides in pollen we found therefore
Significanceindicates a pressing need for further researchon lethal and of
subwork
in relation
lethal effects of fungicides on bees. Given the diverseroutes ofto
exposure to pesticides we show, and increasing
evidence that
other researchpesticide blends harm bees [16,18,58], there is a pressing need for

Figure 5. Probability of Nosema infection increased withfungicide load in consumed pollen. Histograms show the numberof bees with (top) and without (bottom) Nosema spores as a function of
the fungicide load in the pollen they were fed. The curve shows thepredicted probability of Nosema infection.
doi:10.1371/journal.pone.0070182.g005

than bees consuming pollen without this chemical to become
infected after Nosema exposure. Our results show the necessity of
testing for sub-lethal effects of pesticides on bees, and advocate fortesting more broadly than the insecticides that are the targets ofmost current research.
A similarly large increased risk of Nosema infection was
associated with consumption of DMPF and fluvalinate, miticides
applied by beekeepers to help control the highly-destructive Varroa
mite [3]. The path from in-hive application of these miticides topollen on foragers returning to the hive is unclear. An increasingly
Significancepopular ofpractice, rotating combs out of hives to remove
resultsaccumulated
in relationpesticides,tois expected to reduce miticide levels inhives,
andwill
hopefully
decrease spread of these chemicals to thebroader research
environment.
Potential extra-nest sources, however, would slow
questions;however,effortstoreducemiticide accumulation and slow the development
the word "suggests"of resistance to these chemicals.
avoids overstating
Insecticide relative risk values showed an interesting pattern:
the implications.
directional separation by insecticide family. Within a family,
relative risk values significantly different than one were almost allin the same direction. The formamidine (DMPF) and two of thethree the pyrethroids (bifenthrin and fluvalinate, but not
esfenvalerate) were associated with an increased risk of Nosema
infection. The carbamate (carbaryl), all neonicotinoids (acetamiprid, imidacloprid and thiacloprid), organophosphates (coumaphos, diazinon and phosmet) and the oxadiazine (indoxacarb)
were associated with reduced risk of Nosema infection. Esfenvalerate and coumaphos have previously been found to be associatedwith apiaries without Colony Collapse Disorder [59]. These
patterns suggest that insecticides’ modes of action have differential
effects on honey bee immune functioning. Because of the relatively
small number of pesticides we found in each insecticide family,

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July 2013 | Volume 8 | Issue 7 | e70182

Acknowledgments
Crop Pollens
Affect Bee HealthSome journals
allow supplements.

are courteous andspecific.

Table S1 Plant sources of pollens collected by bees placed infurther research on the mechanisms underlying pesticide-pesticideand pesticide-disease synergistic effects on honey bee health.

seven crops.
(DOCX)

Supporting Information
Acknowledgments

Figure S1 Pesticide loads did not differ by crop for any

We thank David Hackenberg and David Mendes for letting us work with
their bee hives, John Baker and Rob Snyder for field assistance, Roger
Some
Simonds for pesticide identification, and Vic Levi andNathanjournals
Rice forrequire author
assistance with Nosema assays.
The views expressed in this article are those of the contributions.
authors and do notnecessarily represent the policies or positions of the US Department ofAgriculture (USDA).

pesticide category. Kruskal-Wallis test statistics comparing
pesticide loads between crops are: fungicides, H6 = 10.6, p = 0.10;
herbicides, H6 = 8.3, p = 0.22; carbamates, H6 = 13.4, p = 0.04;
cyclodienes, H6 = 6.7, p = 0.35; formamidines, H6 = 13.6, p = 0.03;
p = 0.007;
organophosphates,
neonicotinoids,
H6 = 17.8,
H6 = 14.5, p = 0.02; oxadiazines, H6 = 11.3, p = 0.08; pyrethroids,
H6 = 9.6, p = 0.14. Sequential Bonferroni adjusted critical values
are: 0.0055, 0.0063, 0.0071, 0.0083, 0.01, 0.0125, 0.0167, 0.025,
0.06.
(DOCX)

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