Apidologie 41 (2010) 312–331 Available online at:
c
INRA/DIB-AGIB/EDP Sciences, 2010 www.apidologie.org
DOI: 10.1051/apido/2010018
Review article
Pesticides and honey bee toxicity – USA*
Reed M. Johnson
1
,MarionD.Ellis
1
, Christopher A. Mullin
2
, Maryann Frazier
2
1
Department of Entomology, 202 Entomology Hall, University of Nebraska, Lincoln, NE 68583, USA
2
Department of Entomology, 501 Ag Sciences and Industry Building, The Pennsylvania State University,
University Park, PA 16802, USA
Received 12 October 2009 – Revised 26 January 2010 – Accepted 17 February 2010
Abstract – Until 1985 discussions of pesticides and honey bee toxicity in the USA were focused on pes-
ticides applied to crops and the unintentional exposure of foraging bees to them. The recent introduction
of arthropod pests of honey bees, Acarapis woodi (1984), Varroa destructor (1987), and Aethina tumida
(1997), to the USA have resulted in the intentional introduction of pesticides into beehives to suppress
these pests. Both the unintentional and the intentional exposure of honey bees to pesticides have resulted
in residues in hive products, especially beeswax. This review examines pesticides applied to crops, pesti-
cides used in apiculture and pesticide residues in hive products. We discuss the role that pesticides and their
residues in hive products may play in colony collapse disorder and other colony problems. Although no
single pesticide has been shown to cause colony collapse disorder, the additive and synergistic effects of
multiple pesticide exposures may contribute to declining honey bee health.
pesticide / honey bee / toxicity / wax residue / CCD
1. PESTICIDES APPLIED TO CROPS
Despite the dependence on honey bees
for the pollination of crops in the USA,
colony numbers have declined by 45% over
the past 60 years (NAS, 2007). Most honey
bee losses from 1966–1979 were attributable
to organochlorine, carbamate, organophospho-
rus, and pyrethroid pesticide exposure (Atkins,
1992). Efforts to restrict pesticide applica-
tion during bloom provided some relief; how-
ever, the residual activity of some pesticides
was never effectively addressed (Johansen and
Mayer, 1990). Previous reviews and extension
publications are available concerning the pro-
tection of honey bees from these 4 classes of
pesticides (Johansen, 1977; Crane and Walker,
1983;Adeyetal.,1986; Johansen and Mayer,
1990; Ellis et al., 1998).
Corresponding author: M.D. Ellis,
mellis3@unl.edu
* Manuscript editor: Yves Le Conte
Colony losses were especially severe from
1981 to 2005 with a drop from 4.2 million
to 2.4 million (NAS, 2007) although some
of the decrease is attributable to changes in
how colony numbers were estimated. The
introduction of parasitic honey bee mites,
Acarapis woodi (1984) and Varroa destruc-
tor (1987), contributed to dramatic bee losses.
At the same time, the control of crop pests
in USA agriculture was rapidly changing. Ge-
netically engineered (GE) crops were devel-
oped and extensively deployed, and two new
classes of systemic pesticides, neonicotinoids
and phenylpyrazoles, replaced many of the
older pesticides described above.
The rapid development and deployment of
these 2 new insect control techniques dis-
tinguish USA agriculture from agriculture in
other regions of the world. In Europe a more
cautious approach to the adoption of new agri-
cultural practicices has been taken. Since the
registration and regulation of GE crops and
neonicotinoid and phenylpyrazole pesticides
Article published by EDP Sciences
Honey bee toxicity – USA 313
are major shifts in insect control in USA agri-
culture, they are emphasized in this section of
our review.
The recent sequencing of the honey bee
genome provides a possible explanation for
the sensitivity of bees to pesticides; rela-
tive to other insect genomes, the honey bee
genome is markedly deficient in the num-
ber of genes encoding detoxification enzymes,
including cytochrome P450 monooxygenases
(P450s), glutathione-S-transferases, and car-
boxylesterases (Claudianos et al., 2006).
1.1. GE plant varieties
GE (genetically engineered) plant varieties
that have herbicide tolerance or insecticidal
properties were first introduced into the USA
in 1996. Soybeans and cotton are genetically
engineered with herbicide-tolerant traits and
have been the most widely and rapidly adopted
GE crops in the USA, followed by insect-
resistant cotton and corn. In 2007 these GE
crops were planted on more than 113 mil-
lion hectares worldwide (USDA-Biotech Crop
Data, 2009), and the United States leads the
world in acres planted with GE crops with
most of the plantings on large farms (Lemaux,
2008). Insect resistance is conferred by in-
corporating genes coding for insecticidal pro-
teins produced by Bacillus thuringensis (Bt), a
widespread soil bacterium (ISB, 2007). While
Bt is also delivered through traditional spray
application, plants benefit from continuous
production of Bt toxins through genetic en-
gineering. Bt δ-endotoxins are activated in
the insect gut where they bind to receptor
sites on the midgut epithelium to form pores.
These pores allow gut contents to leak out
of the lumen and cause osmotic stress to
midgut cells, leading to the eventual destruc-
tion of the midgut and the death of the insect
(Soberon et al., 2009). To date, Bt genes have
been incorporated into corn (Zea mays), cot-
ton (Gossypium hirsutum), potato (Solanum
tuberosum) and tomato (Lycopersicon escu-
lentum), and GE seeds of these crops are avail-
able to producers (ISB, 2007). Precommercial
field tests of 30 different plant species with Bt
genes were conducted in 2008 including ap-
ples, cranberries, grapes, peanuts, poplar, rice,
soybeans, sunflowers and walnuts (ISB, 2007).
Numerous studies have been conducted to
determine the impact of GE crops on honey
bees. Canadian scientists found no evidence
that Bt sweet corn affected honey bee mor-
tality (Bailey et al., 2005). Studies conducted
in France found that feeding Cry1ab protein
in syrup did not affect honey bee colonies
(Ramirez-Romero et al., 2005). Likewise, ex-
posing honey bee colonies to food containing
Cry3b at concentrations 1000 times that found
in pollen resulted in no effect on larval or pupal
weights (Arpaia, 1996). Feeding honey bees
pollen from Cry1ab maize did not affect larval
survival, gut flora, or hypopharyngeal gland
development (Babendreier et al., 2005–2007).
A 2008 meta-analysis of 25 independent stud-
ies concluded that the Bt proteins used in GE
crops to control lepidopteran and coleopteran
pests do not negatively impact the survival of
larval or adult honey bees (Duan et al., 2008).
There is no evidence that the switch to Bt
crops has injured honey bee colonies in the
USA. To the contrary, it has benefited bee-
keeping by reducing the frequency of pesticide
applications on crops protected by Bt, espe-
cially corn and cotton. On the other hand, the
switch to GE crops with herbicide resistance
has eliminated many blooming plants from
field borders and irrigation ditches as well as
from the crop fields themselves. The reduction
in floral diversity and abundance that has oc-
curred due to the application of Round-UP
Herbicide (glyphosate) to GE crops with her-
bicide resistance is difficult to quantify. How-
ever, there is a growing body of evidence that
poor nutrition is a primary factor in honey
bee losses. Eischen and Graham (2008) clearly
demonstrated that well-nourished honey bees
are less susceptible to Nosema ceranae than
poorly nourished bees. Because honey bees are
polylectic, the adoption of agricultural prac-
tices that provide greater pollen diversity has
been suggested, including the cultivation of
small areas of other crops near monocultures
or permitting weedy areas to grow along the
edges of fields (Schmidt et al., 1995). A de-
tailed review of management of uncropped
farmland to benefit pollinators by Decourtye
et al. (2010) is included in this special issue.
314 R.M. Johnson et al.
1.2. Neonicotinoid and phenylpyrazole
pesticides
Another major shift in USA agriculture
has been the development and extensive de-
ployment of neonicotinoid and phenylpyra-
zole pesticides. These pesticides are exten-
sively used in the USA on field, vegetable,
turf, and ornamental crops, some of which
are commercially pollinated by bees (Quarles,
2008). They can be applied as seed treatments,
soil treatments and directly to plant foliage.
Neonicotinoids are acetylcholine mimics and
act as nicotinic acetychloline receptor ago-
nists. Neonicotinoids cause persistent activa-
tion of cholinergic receptors which leads to hy-
perexcitation and death (Jeschke and Nauen,
2008). One neonicotinoid, imidacloprid, was
applied to 788 254 acres in California in 2005
(CDPR, 2006), making it the 6th most com-
monly used insecticide in a state that grows
many bee-pollinated crops. The phenylpyra-
zoles, including fipronil, bind to γ-amino bu-
tyric acid (GABA)-gated chloride ion chan-
nels and block their activation by endogenous
GABA, leading to hyperexcitation and death
(Gunasekara et al., 2007).
Neonicotinoid and phenylpyrazole insecti-
cides differ from classic insecticides in that
they become systemic (Trapp and Pussemier,
1991) in the plant, and can be detected in
pollen and nectar throughout the blooming pe-
riod (Cutler and Scott-Dupree, 2007). As a
consequence, bees can experience chronic ex-
posure to them over long periods of time.
While some studies have shown no negative
effects from seed-treated crops (Nguyen et al.,
2009), acute mortality was the only response
measured. Desneux and his colleagues (2007)
examined methods that could be used to more
accurately assess the risk of neonicotinoid and
phenylpyrazole insecticides including a test on
honey bee larvae reared in vitro to test for lar-
val effects (Aupinel et al., 2005), a proboscis
extension response (PER) assay to access as-
sociative learning disruption (Decourtye and
Pham-Delegue, 2002), various behavioral ef-
fects (Thompson, 2003), and chronic exposure
toxicity beyond a single acute dose exposure
(Suchail et al., 2001;Decourtyeetal.,2005;
Ailouane et al., 2009). Pesticide exposure may
interact with pathogens to harm honey bee
health. Honey bees that were both treated with
imidacoprid and fed Nosema spp. spores suf-
fered reduced longevity and reduced glucose
oxidase activity (Alaux et al., 2010).
1.3. Registration procedures and risk
assessment
In the USA risk assessment related to agro-
chemical use and registration follow specific
guidelines mandated by the Federal Insecti-
cide Fungicide and Rodenticide Act (EPA,
2009a). Despite the importance of honey bees,
the effect of pesticide exposure on colony
health has not been systematically monitored,
and the Environmental Protection Agency
(EPA) does not require data on sublethal ef-
fects for pesticide registration (NAS, 2007).
For many years, the classical laboratory
method for registering pesticides was to de-
termine the median lethal dose (LD
50
)ofthe
pest insect. In a second step, the effects of
pesticides on beneficial arthropods were ex-
amined by running LD
50
tests on the benefi-
cial species to identify products with the low-
est non target activity (Croft, 1990; Robertson
et al., 2007). The honey bee has often served as
a representative for all pollinators in the reg-
istration process, though the toxicity of pes-
ticides to non-Apis species may be different
(Taséi, 2003; Devillers et al., 2003). In the
USA this protocol remains the primary ba-
sis for risk assessment in pesticide registra-
tion. However, this approach to risk assess-
ment only takes into account the survival of
adult honey bees exposed to pesticides over
a relatively short time frame (OEPP/EPPO,
1992). In Europe, where the standard proce-
dures do not provide clear conclusions on the
harmlessness of a pesticide, additional stud-
ies are recommended; however, no specific
protocols are outlined (OEPP/EPPO, 1992).
Acute toxicity tests on adult honey bees may
be particularly ill-suited for the testing of sys-
temic pesticides because of the different route
of exposure bees are likely to experience in
field applications. Chronic feeding tests using
whole colonies may provide a better way to
Honey bee toxicity – USA 315
quantify the effects of systemics (Colin et al.,
2004).
Registration review is replacing the
EPA’s pesticide re-registration and tolerance
reassessment programs. Unlike earlier re-
view programs, registration review operates
continuously, encompassing all registered
pesticides. The registration review docket for
imidacloprid opened in December 2008. To
better ensure a “level playing field” for the
neonicotinoid class as a whole and to best
take advantage of new research as it becomes
available, the EPA has moved the docket
openings for the remaining neonicotinoids on
the registration review schedule (acetamiprid,
clothianidin, dinotefuran, thiacloprid, and thi-
amethoxam) to fiscal year 2012 (EPA, 2009b).
The EPA’s registration review document states
that “some uncertainties have been identified
since their initial registration regarding the
potential environmental fate and effects of
neonicotinoid pesticides, particularly as they
relate to pollinators (EPA, 2009b)”. Studies
conducted in Europe in the late 1990s have
suggested that neonicotinoid residues can
accumulate in pollen and nectar of treated
plants and represent a potential risk to polli-
nators (Laurent and Rathahao, 2003). Adverse
effects on pollinators have also been reported
in Europe that have further heightened con-
cerns regarding the potential direct and/or
indirect role that neonicotinioid pesticides
may have in pollinator declines (Suchail
et al., 2000). Recently published data from
studies conducted in Europe support concerns
regarding the persistence of neonicotinoids.
While the translocation of neonicotinoids into
pollen and nectar of treated plants has been
demonstrated, the potential effect that levels of
neonicotinoids found in pollen and nectar can
have on bees remains less clear. Girolami et al.
(2009) report high levels of neonicotinoids
from coated seeds in leaf guttation water and
high mortality in bees that consume it. While
the frequency of guttation drop collection by
bees under field conditions is not documented,
the authors describe the prolonged availability
of high concentrations of neonicotinoids in
guttation water as “a threatening scenario
that does not comply with an ecologically
acceptable situation”. The pending EPA
review will consider the potential effects of
the neonicotinoids on honey bees and other
pollinating insects, evaluating acute risk at
the time of application and the longer-term
exposure to translocated neonicotinoids (EPA,
2009b; Mullin et al., 2010).
2. PESTICIDES USED IN
APICULTURE
The Varroa mite, Varroa destructor, is one
of the most serious pests of honey bees in the
USA and worldwide. It injures both adult bees
and brood, and beekeepers are frequently com-
pelled to use varroacides to avoid colony death
(Boecking and Genersch, 2008). Varroacides
must be minimally harmful to the bees, while
maintaining toxicity to mites, which is a chal-
lenge given the sensitivity of honey bees
to many pesticides (Atkins, 1992). The var-
roacides used in the USA can be broadly di-
vided into three categories: synthetic organic,
natural product and organic acid pesticides.
2.1. Synthetic organic pesticides
The pyrethroid tau-fluvalinate, a subset of
isomers of fluvalinate, was the first synthetic
varroacide registered for use in honey bee
colonies in the USA. It was first registered
as a Section 18 (emergency use label, state
by state approval) in 1987 (Ellis et al., 1988).
The Section 18 label allowed plywood strips
to be soaked in an agricultural spray formu-
lation of tau-fluvalinate, (Mavrik
), and treat-
ment was made by suspending the strips be-
tween brood frames. In 1990 plastic strips
impregnated with tau-fluvalinate (Apistan
)
replaced homemade plywood strips (PAN,
2009) with a Section 3 label (full registration
for use in all states). According to the label,
a single strip contains 0.7 g tau-fluvalinate,
as much as 10% of which may diffuse from
the plastic strip formulation into hive ma-
trices over the course of an 8 week treat-
ment (Bogdanov et al., 1998; Vita Europe
Ltd., 2009). While the agricultural spray for-
mulation of tau-fluvalinate (Mavrik
)isno
longer legal to use in the USA, its low cost
316 R.M. Johnson et al.
and history of legal use in beehives make it
vulnerable to misuse and may contribute to
tau-fluvalinate residues detected in beeswax
(Bogdanov, 2006; Wallner, 1999; Berry, 2009;
Mullin et al., 2010).
As a pyrethroid, tau-fluvalinate kills mites
by blocking the voltage-gated sodium and cal-
cium channels (Davies et al., 2007). While
most pyrethroids are highly toxic to honey
bees, tau-fluvalinate is tolerated in high
concentrations due in large part to rapid detox-
ification by cytochrome P450 monooxyge-
nases (P450s) (Johnson et al., 2006). How-
ever, tau-fluvalinate is not harmless to bees
and does affect the health of reproductive
castes. Queens exposed to high doses of tau-
fluvalinate were smaller than untreated queens
(Haarmann et al., 2002). Drones exposed to
tau-fluvalinate during development were less
likely to survive to sexual maturity relative to
unexposed drones, and they also had reduced
weight and produced fewer sperm (Rinderer
et al., 1999). However, the practical conse-
quence of tau-fluvalinate exposure on drones
may be limited, as drones exposed to tau-
fluvalinate produced as many offspring as un-
exposed drones (Sylvester et al., 1999).
Tau -fluvalinate was initially very effective
at controlling Va rro a mites, but many Var ro a
populations now exhibit resistance (Lodesani
et al., 1995). Resistance to tau-fluvalinate in
Var ro a is due, at least in part, to a mutation in
the voltage-gated sodium channel which con-
fers reduced binding affinity for tau-fluvalinate
(Wang et al., 2002). Despite diminished effec-
tiveness, tau-fluvalinate continues to be used
for Varro a control in the USA (Elzen et al.,
1999; Macedo et al., 2002).
As the efficacy of tau-fluvalinate against
Var ro a was beginning to wane, coumaphos,
an organophosphate pesticide, was granted
Section 18 approval in the USA in 1999
as varroacide (Federal Register, 2000), and
as a treatment for the small hive beetle,
Aethina tumida Murray. Coumaphos is admin-
istered as Checkmite+
strips, each contain-
ing approximately 1.4 g coumaphos, which
are hung between brood frames for 6 weeks.
Coumaphos, or its bioactivated oxon metabo-
lite, kills through the inactivation of acetyl-
cholinesterase, thereby interfering with nerve
signaling and function. While coumaphos ini-
tially proved effective at killing tau-fluvalinate
resistant Varro a populations (Elzen et al.,
2000), coumaphos resistant mite populations
were found as early as 2001 (Elzen and
Westervelt, 2002). The mechanism of resis-
tance to coumaphos in Va r ro a is unknown,
though esterase-mediated detoxification may
be involved (Sammataro et al., 2005). Re-
sistance likely follows the mechanisms of
coumaphos resistance observed in the south-
ern cattle tick, Boophilus microplus,which
include acetylcholinsesterase insensitivity and
enhanced metabolic detoxification (Li et al.,
2005). Honey bees tolerate therapeutic doses
of coumaphos, at least in part, as a conse-
quence of detoxicative P450 activity (Johnson
et al., 2009). However, honey bees can suf-
fer negative effects from coumaphos exposure;
queens exposed to coumaphos were smaller,
suffered higher mortality and were more likely
to be rejected when introduced to a colony
(Haarmann et al., 2002; Collins et al., 2004;
Pettis et al., 2004). Drone sperm viability was
lower in stored sperm collected from drones
treated with coumaphos (Burley et al., 2008).
Amitraz, a formamidine pesticide, was once
registered (1992–Section 18 label) in the USA
under the trade name Miticur
with the active
ingredient incorporated in a plastic strip that
was suspended between brood frames (PAN,
2009). However, the product was withdrawn
from the market in 1994 when some beekeep-
ers reported colony losses following treatment
(PAN, 2009). While no conclusive evidence
was presented that the product had harmed
bees, the registrant decided to withdraw the
product from the market (PAN, 2009). Amitraz
is available in the USA as a veterinary miticide
(Taktic
), but the label does not allow for use
in honey bee colonies; however, the frequency
with which amitraz metabolites are found in
beeswax suggests that it continues to be used
(Mullin et al., 2010; Berry, 2009). Amitraz
strips (Apivar
) were granted an emergency
registration for Va rroa control by the Canadian
PMRA for 2009 (PMRA, 2009), but they are
not available to beekeepers in the USA.
Amitraz is an octopaminergic agonist in
arthropods (Evans and Gee, 1980)andas
such has the potential to influence honey
