STOTEN-21999; No of Pages 16

Science of the Total Environment xxx (2017) xxxxxx

Contents lists available at ScienceDirect

Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Review

The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to

pesticides: Room for improvement in research
Johanna Benuszak a, Marion Laurent b, Marie-Pierre Chauzat a,b,
a
Unit of Coordination and Support to Surveillance, ANSES, Scientic Affairs Department for Laboratories, Maisons-Alfort, France
b
Unit of Honeybee Pathology, ANSES, European Union and National Reference Laboratory for Honeybee Health, Sophia Antipolis, France

H I G H L I G H T S G R A P H I C A L A B S T R A C T

PEO and PRISMA standardised methods

used for bibliographic analysis.
264 articles analysed to formulated
recommendations for scientic experi-
mentations.
Sampling methods, matrices and units
should be better described and
harmonized to some extent.
Beeswax and beebread should be more
studied.
Interactions between stresses and
bioavailability of pesticides should be
further investigated.

a r t i c l e i n f o a b s t r a c t

Article history: Losses of honey bees have been repeatedly reported from many places worldwide. The widespread use of syn-
Received 19 October 2016 thetic pesticides has led to concerns regarding their environmental fate and their effects on pollinators. Based
Received in revised form 7 February 2017 on a standardised review, we report the use of a wide variety of honey bee matrices and sampling methods in
Accepted 7 February 2017
the scientic papers studying pesticide exposure. Matrices such as beeswax and beebread were very little
Available online xxxx
analysed despite their capacities for long-term pesticide storage. Moreover, bioavailability and transfer between
Editor: D. Barcelo in-hive matrices were poorly understood and explored. Many pesticides were studied but interactions between
molecules or with other stressors were lacking. Sampling methods, targeted matrices and units of measure
Keywords: should have been, to some extent, standardised between publications to ease comparison and cross checking.
Acaricide Data on honey bee exposure to pesticides would have also benet from the use of commercial formulations in
Active substance experiments instead of active ingredients, with a special assessment of co-formulants (quantitative exposure
Air and effects). Finally, the air matrix within the colony must be explored in order to complete current knowledge
Metabolites on honey bee pesticide exposure.
Plant protection products
2017 Elsevier B.V. All rights reserved.
Standardisation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.1. Identication of the key concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Corresponding author at: Unit of Coordination and Support to Surveillance, ANSES, Scientic Affairs Department for Laboratories, Maisons-Alfort, France.
E-mail address: marie-pierre.chauzat@anses.fr (M.-P. Chauzat).

http://dx.doi.org/10.1016/j.scitotenv.2017.02.062
0048-9697/ 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Benuszak, J., et al., The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for
improvement in research, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.02.062
2 J. Benuszak et al. / Science of the Total Environment xxx (2017) xxxxxx

2.2. Investigation methods for bibliographic databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.3. Protocol for scientic paper review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3.1. General information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3.2. Pesticides and investigations on interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3.3. Protocols implemented in scientic experimentations and on-site investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Author contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Additional information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction
Honey bees (Apis mellifera) have an important economic and ecolog-
ical role in crop pollination and ecosystem services (Ghazoul, 2005;
Klein et al., 2006). Across the world, many research teams have worked
to explain honey bee colony losses (Vanbergen and Initiative, 2013;
Goulson et al., 2015). Potential drivers for bee losses have been identi-
ed and can be grouped into pests and pathogens, environmental
stressors (e.g. malnutrition, exposure to agrochemicals and apicultural
mismanagement) and lack of genetic diversity and vitality (Potts et al.,
2010). According to the European wide EPILOBEE programme, beekeep-
er knowledge and apicultural practices are key elements in avoiding
honey bee colony losses during winter (Jacques et al., 2016). Bees are
not exposed to one risk factor but to a plethora of stresses that can inter-
act amongst themselves, pesticide residues being one of them (Goulson
et al., 2015). The effects of a number of active substances and formula-
tions on A. mellifera cognition and mortality are well known. Recently
much attention has been paid to the insecticide class of neonicotinoids
(Lundin et al., 2015; SimonDelso et al., 2015). Because of their large
spectra of action and their efcacy, neonicotinoid insecticides became
the most commonly used class of insecticides on a large variety of
crops. Therefore, their use has expanded rapidly since they were intro-
duced on the market (Elbert et al., 2008; Limay-Rios et al., 2016). Since
the extensive use of neonicotinoid insecticides in the 1990s, beekeepers
have reported losses of honey bee populations rst in France (Pham-
Delgue, 2001) and subsequently in other parts of the world
(Vanengelsdorp et al., 2009; Codling et al., 2016). For any pesticide, in-
cluding neonicotinoid insecticides, the risk assessment of their effects
on non-target species is required in the dossier submitted for marketing
authorisation. However, for honey bees in particular, the sublethal ef-
fects of pesticides are not totally covered by the tests currently required
(EFSA, 2012).
Detrimental effects of pesticides at sublethal doses have been largely
documented in animal species (Frampton et al., 2006; Luzardo et al.,
2014). The effects of pesticides on benecial arthropods have been the
subject of an increasing number of studies reviewed several times
(Desneux et al., 2007). For honey bees, negative impacts of sublethal
doses of pesticides have been shown in laboratories on various biologi-
cal traits such as bee longevity and survival rates (Wu et al., 2011;
Cresswell et al., 2012), learning and memory abilities (Ciarlo et al.,
2012; Farooqui, 2013) and the detoxication processes (Berenbaum
and Johnson, 2015). Pesticides at low doses can also interact with infec-
tious and parasitic agents affecting honey bees (Pettis et al., 2012;
Retschnig et al., 2014). More recently, studies in the eld have demon-
strated the negative effect of pesticides at sublethal doses on the forag-
ing activity of the honey bee colony (Henry et al., 2012; Schneider et al.,
2012). Semi-eld studies have also reported effects on the development
of brood (Thompson et al., 2014b) and on the fertility and survival of
queens (Williams et al., 2015; Dussaubat et al., 2016). Although insecti-
cides are largely studied, other active substances or commercial prepa-
rations have also been tested in the experimentations conducted on
honey bees and showed effects on honey bees (fungicides, agricultural
spray adjuvants for instance).
Literature reviews on pesticides have focused on concentrations of a
specic substance or group of substances found in the environment
(Blacquire et al., 2012), the effects of pesticides on honey bees
(vanderSluijs et al., 2013) or which research approaches have been
used to study neonicotinoids (Lundin et al., 2015). In this bibliographic
review, we enlarged our research to the exposure of honey bees to pes-
ticides in general without restricting our approach to one group of ac-
tive ingredients. In the eld, environmental contamination occurs via
a number of routes including drift of sprayed pesticides, dust generated
during drilling of dressed seeds, contamination and accumulation in ar-
able soils and soil water, runoff into waterways, and uptake of pesticides
by non-target plants via their roots or dust deposition on leaves
(Bonmatin et al., 2015). In laboratory experiments, honey bees are ex-
posed to pesticides through topical or oral exposures. For topical expo-
sure, the use of drops of solutions applied on the bee thorax is the most
common method (EPPO, 2010). However other routes of exposure can
be used such as the use of contaminated plants or parts of plants on
which bees walk. For oral exposure, the methodologies include the
use of various contaminated foods (honey, pollen or treated plants)
(EPA, 2016). Therefore, there is a need for indexing, classication and
selection of the means reported in the scientic papers concerning
honey bee exposure to pesticides.
Using a standardised method, we reviewed how this exposure was
assessed and reported in the published literature and divided the stud-
ies into three major groups: scientic experimentations, on-site investi-
gations and bibliographic reviews. This review aims to (a) provide a
better understanding of the different types of matrices associated with
pesticide exposure, (b) clarify the range of methods used to address
pesticide exposure and permit new insights into the development of
better experimental approaches, (c) pinpoint the lack of connections
and cross-references between studies, and (d) identify current knowl-
edge gaps in the results and formulate recommendations for future
research.
2. Methods
2.1. Identication of the key concepts
The Population, Exposure, Outcomes (PEO) method was used to
identify the key concepts of our question. The PICO method (Population,
Intervention, Comparison, Outcomes) is widely used in quantitative re-
search. We chose the PEO method because it is most useful for qualita-
tive research questions. Initially developed to respond to health
questions (University Campus Suffolk, 2016), the PEO method was ap-
plied to our review to dene the honey bee colony as the population
studied and the pesticides as the nature of honey bee exposure. The out-
comes of the study are the classication of matrices and methods used
in the literature and the formulation of recommendations for future sci-
entic experiments. Three subsections were identied: the studied

Please cite this article as: Benuszak, J., et al., The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for
improvement in research, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.02.062
 J. Benuszak et al. / Science of the Total Environment xxx (2017) xxxxxx 3

honey bee matrices, the type of exposure and the target populations
(Fig. 1).
2.2. Investigation methods for bibliographic databases
The search for scientic papers was based on the methods proposed
by the guidelines of Preferred Reporting Items for Systematic Reviews
and Meta-Analyses (PRISMA) (Moher et al., 2009). Key words were
listed after reading publications related to honey bee exposure to pesti-
cides. We searched in Cab Abstract, Pub Med, Science Direct databases
and a personal research set of bibliographic references for studies in-
cluding this set of key words. The search strings are provided in Appen-
dices A. The selected articles were written in English and available in full
text. Grey literature was not included in our work. References published
prior to 2005 were not included. The last database query was performed
on April, 4th 2016.
To be included in this review, scientic papers must have had in-
volved honey bee exposure to one or several active substances, quanti-
cation of pesticides in bee matrices or bibliographic review on bees
and pesticides. Publications on analytical methods to identify and quan-
tify pesticide residues in honey bee matrices were also included in the
review if results on honey bee matrices were reported. We also took
in consideration studies on genetically modied organisms (GMO)
only when the genetic modication aimed at producing a pesticide mol-
ecule, e.g. GM maize to produce insect and herbicide resistance
(Hendriksma et al., 2012). Studies concerning interactions between
pesticides and interactions between pesticides and other stressors
such as diseases, predators or parasites were also included in the re-
view. Heavy metals were not included in the analysis. Only the species
Apis mellifera was considered in this work.
We focused the review on pesticides; therefore publications
reporting exposure to essential oils and organic acids (e.g. formic acid)
were discarded from the analysis. Book chapters or congress summaries
were not included in this work to ensure easy access to full text.
2.3. Protocol for scientic paper review
Each publication was reviewed according to a standardised protocol.
General information related to authors, title, year of publication, journal
and afliation country of the primary author was checked. The publica-
tions were classied into three categories: scientic experimentations
(i.e. exposure was controlled) whether they were performed in the lab-
oratory, in semi-eld or eld conditions (using cages or tunnels in the
eld), pesticide search in honey bee matrices (on-site investigations)

Fig. 1. Classication of the outcomes for the literature review of honey bee exposure to pesticides following the PEO method. Three subsections were identied: bee matrices studied, type
of exposure and target population.

Please cite this article as: Benuszak, J., et al., The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for
improvement in research, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.02.062
4 J. Benuszak et al. / Science of the Total Environment xxx (2017) xxxxxx

Fig. 2. Number of scientic papers (n = 264) related to honey bee exposure to pesticides published between 2005 and 2016 (last access: April, 4th 2016).

and bibliographic reviews. For scientic experimentations, more specif-
ically, we reported information on how pesticides were used: which
type of application (e.g. oral, topical - or both - applications), the pesti-
cide doses, which units were used, whether interactions between pesti-
cides or with other stressors were studied, matrices under study,
sampling method (location and way of sampling), sample size and
whether cages were used. Additionally for on-site investigations the
number of sites was reported together with the number of colonies in-
volved, if the test was done on full colonies and if the landscape was
described.
3. Results
3.1. General information
A total of 1482 articles were identied after database inquiries. After
removal of duplicates, 264 articles were included and analysed in this
review (see Appendices B, the PRISMA ow diagram). An increase in
the number of papers published in 2010 is noted (Fig. 2). Papers were
published in 91 different journals; Plos One, Journal of Chromatography
A and Pest Management Sciences being the three most frequent
journals (Table 1).
Studies were conducted in 39 countries located in Europe (51.1% of
studies), North America (26.7%) and South America (22.1%). Half of
the studies originated from four countries: the United States (n = 59),
France (n = 31), the United Kingdom (n = 21) and Italy (n = 17)
(Fig. 3).
Three types of studies were identied: scientic experimentations
(54%), on-site investigations (35%) and bibliographic reviews (11%).
Scientic experimentations exposed honey bee to pesticides to study
their effects on cognition, behaviour or mortality; on-site investigations
looked at the quantity of pesticides found in matrices sampled from the
eld and bibliographic reviews studied the existing literature related to
our question.
3.2. Pesticides and investigations on interactions
The majority of pesticides studied in scientic experimentations
over the last decade were neonicotinoids especially imidacloprid
(39%), thiamethoxam (22%) and clothianidin (17%) (Fig. 4). Other
main chemicals studied were the miticides coumaphos (10%) and tau-
uvalinate (9%). In total more than 50 different pesticides have been
studied in scientic experimentations. Commercial formulation were
used in 47.8% of the scientic papers (n = 142).
Most of the scientic experimentations (69%) did not investigate any
interaction. The co-exposure of honey bees to pesticides and pathogens
was the interaction the most studied (10%), with a special interest in the
parasitic mite Varroa destructor (Acari: Mesostigmata). Interaction be-
tween different insecticides and between insecticides and fungicides
were explored respectively in 6% and 7% of the studies. Other interac-
tions included insecticides and miticides, insecticides and antibiotics
and between GMOs (Table 2).
3.3. Protocols implemented in scientic experimentations and on-site
investigations

Several methods have been used in the scientic papers (n = 69) to

Table 1 expose honey bees to pesticides. Pesticides were mostly applied orally
Scientic journals that have published more than 10 papers related to honey bee exposure (51%) using pollen (in 30 experiments), nectar (9 experiments) or
to pesticides between 2005 and 2016 (last access: April, 4th 2016). honey (3 experiments). Contact exposure (26%) was implemented
Scientic journals Number of publications (% of the total, through topical application (using drops on thoraxes or Potter tower
n = 264) in 37 experiments) or using plants (e.g. seed coating or spraying in 35
Plos One 26 (9.7)
experiments).
Journal of Chromatography A 21 (7.8) The majority of scientic experimentations (the exposure was con-
Journal of Economic Entomology 17 (6.4) trolled) were conducted in laboratories (57%). Only 29% of experimen-
Pest Management Sciences 12 (4.5) tations were conducted in eld conditions. Interestingly, this
Environmental Toxicology and 11 (4.1)
information whether the study was performed in eld or laboratory,
Chemistry
Ecotoxicology and Environmental Safety 11 (4.1) was not reported in 14% of the papers. Half of the scientic experimen-
Talanta 11 (4.1) tations included the use of cages to expose a group of honey bees or an
Journal of Insect Physiology 10 (3.7) entire colony to pesticides (in 12% of the scientic papers). In scientic
Other journals with less than 10 147 (55.6) experimentations, the mean number of sites (i.e. the location where the
publications each
apiary stands) was 1.6 whereas it was 17.7 in on-site investigations

Please cite this article as: Benuszak, J., et al., The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for
improvement in research, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.02.062
 J. Benuszak et al. / Science of the Total Environment xxx (2017) xxxxxx
Fig. 3. Geographical distribution of the origin of publications on honey bee exposure to pesticides, based on the afliation of the rst author (n = 264).
(search of pesticides in bee matrices). The mean number of colonies in-
volved in on-site investigations (12.6) was higher than the one in scien-
tic experimentations (7.07; meaning 7 colonies under study per
experimentation).
In the scientic experimentations, 35 different units were used to
express pesticide quantities in contaminated vectors (e.g. food, plants,
and solutions) (Appendices C). Units were discriminated in 3 catego-
ries: units used to express quantities in general, units used to quantify
pesticides in food to orally exposed honey bees and units used to quan-
tify plant treatments to orally and/or by contact exposed honey bees. In-
terestingly, different units were used to express the same quantity (i.e.
parts per billion [ppb], g/kg and ng/g).
Fig. 4. Pesticides studied in scientic experimentations (n = 142) related to honey bee
exposure to pesticides published in the last decade. Miscellaneous included more than
fty pesticides. Imid = Imidacloprid, Thiam = Thiamethoxam, Cloth = Clothianidin,
Fipro = Fipronil, Coum = Coumaphos, Tau-F = Tau-Fluvalinate, GMO = Genetically-
Modied Organism, Acetam = Acetamiprid, Thiacl = Thiacloprid, Miscel Pest =
Miscellaneous pesticides (about 50 molecules).
Please cite this article as: Benuszak, J., et al., The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for
improvement in research, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.02.062
5
A large range of matrices has been studied in the scientic experi-
mentations (n = 33) and on-site investigations (n = 23) (Fig. 5). Scien-
tic experimentations focused mostly on worker bees (32.3%),
emergent bees (26%), foragers (22.5%) and pollen (21.1%). Honey
(61.2%), pollen (23.6%), worker bees (22.5%) and beeswax (19.3%)
were mostly sampled during on-site investigations (Fig. 5).
For each matrix a large variety of sampling methods were reported
in scientic experimentations and on-site investigations (Table 3). In-
terestingly, the sampling methods were not often reported in the pa-
pers. The lowest rate of missing information was for soil sampling
(10% of missing information). The honey bee matrix (including dead
or alive bees but also different bee life stages) was the most frequently
sampled in scientic papers (reported 196 times). The sampling meth-
od (which honey bee age was taken, from where and the methodology
when available) was not reported in 21.4% of the papers. Adult honey
bees were sampled from colonies in 15% of the papers. In 13 of the pa-
pers, adult honey bees were obtained after emergence of brood in an in-
cubator in the laboratory. Living honey bees were sampled in front of
the hive in 13.2% of the papers. The 25 other honey bee sampling
methods included different ages of bees (eggs, larvae, pupae), different
Table 2
Interactions between stresses studied in scientic experimentations (n = 142) published
between 2005 and 2016 (last access: April, 4th 2016).
Type of interactions Percentage of studies (%)
No interaction studied 69
Between different insecticides 7
Between insecticides and fungicides 6.3
Between pesticides and pathogens 7.7
Between insecticides and miticides 3.5
Between pesticides and Varroa destructor 2.1
Between insecticides and antibiotics 0.7
Between GMOs 0.7
6 J. Benuszak et al. / Science of the Total Environment xxx (2017) xxxxxx

Fig. 5. Matrices studied in scientic experimentations (black, n = 142) and on-site investigations (white, n = 93) reported in scientic papers on honey bee exposure to pesticides
published between 2005 and 2016 (last access: April, 4th 2016).

casts (workers, drones, queens), specic parts of individuals (queen's
spermatheca, drone sperm, nervous ganglions) and various devices
(traps, vacuum cleaner, brush).
Pollen was mostly sampled with traps located at the entrance of the
hives (52.4%). Pollen was also sampled directly on owers (13.1%) and
in the colony. Honey came mostly from the market (37.1%) or directly
from the colonies. Beeswax was sampled mostly from brood combs. In
45.1% and 44% of the papers, the sampling method was not reported
for honey and beeswax collection. Other matrices reported in scientic
papers were beebread (15 times), nectar (12times), owers (6 times)
and water (7 times).
4. Discussion
In this review we assessed how the exposure of honey bees to pesti-
cides was reported in the published literature. Over the last decade, the
number of publications was the highest between 2013 and 2016, show-
ing that honey bee exposure to pesticides is a current subject of interest
for many research teams around the world. Concerns regarding the en-
vironmental fate and effects of chemicals on pollinators have highlight-
ed some of the pitfalls associated with the widespread use of synthetic
pesticides. Indeed, public attention has been drawn to the issue of haz-
ards posed by pesticides to pollinators by many scientic and citizen ini-
tiatives (Cressey, 2015). The use of three active substances from the
neonicotinoid family (clothianidin, imidacloprid and thiamethoxam)
is temporarily banned in the Commission Implementing Regulation
(EU) No. 485/2013 amending Implementing Regulation (EU) No. 540/
2011(European Commission, 2013). Furthermore, several non-govern-
mental bodies (Greenpeace, 2013) have initiated actions to obtain the
ban of neonicotinoids in several countries e.g. in the United-States of
America the exemption of coated seeds from regulation or mandatory
labelling by the EPA was the basis of the lawsuit led by Center for
Food Safety (CFS) in the public interest and on behalf of several
impacted beekeepers. All these measures urged the scientic communi-
ty to determine exposure of bees to pesticides and to assess the impact
of these substances on pollinator populations in the different European
countries and in Northern America (Garric and Valo, 2016).
Neonicotinoids specically have been the subject of many literature re-
views published in various journals (Bonmatin et al., 2015; Lundin et al.,
2015). There is no dedicated journal for the topic of honey bee exposure
to pesticides.
Most of the papers (77.8%) have been published by European and
North American teams. Indeed the use of pesticides in these areas of
the world is very important (OPECST, 2010). Yet, the relevance of pesti-
cide use is global because they are used in most countries worldwide.
Therefore, detrimental effects on pollinators can be observed in princi-
ple everywhere (Faucon et al., 2002; Nguyen et al., 2009; Farooqui,
2013; van Lexmond et al., 2015; IPBES, 2016). More studies on pesticide
exposure should be carried out by laboratories located in Asia and
Africa. Particular climatic conditions in these vast continents, together
with specicities in agricultural and apicultural husbandries may
produce results that most probably will differ from the ones originating
from Europe and Northern America (Mumoki et al., 2014;
FotsoKenmogne et al., 2015). However, some information might already
be available in papers written in languages other than English, pub-
lished in local scientic journals or in grey literature. If this is the case,
English translated summaries with sufcient information would help
to disseminate the information more widely and learn more on pesti-
cides and honey bee interactions in various environmental conditions.
The pesticides the most studied (e.g. neonicotinoids and miticides)
were also the most used in agriculture and apiculture respectively. For
years, residues of these chemicals have been found in bee matrices
(Bogdanov et al., 1998; Jimenez et al., 2005; Chauzat and Faucon,
2007; Mullin et al., 2010), and their effects on bees largely studied
(Johnson et al., 2010; Lundin et al., 2015). Our review has shown that
most of the research efforts were concentrated on a few families of

Please cite this article as: Benuszak, J., et al., The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for
improvement in research, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.02.062
 J. Benuszak et al. / Science of the Total Environment xxx (2017) xxxxxx
Table 3
Sampling methods reported for each matrix in scientic experimentations (n = 142) and on-site investigations (n = 93) on honey bee exposure to pesticides published between 2005 and
2016 (last access: April, 4th 2016).
Number
Matrices of Sampling methods
papers
Not specied
Sampling of adults from the
colony
Adults emerged from the brood
in laboratory
Living adult bees sampled in
front of the colony
Honey bees Dead adult bees collected in traps 4
(all 196
stages)
Egg-laying in boxes
Adult bees with RFID tracking
Larvae in piece of brood
Emerging adult bees from brood
combs in the colony
Adult dead bees in front of the
colony
Adult bees captured from an
articial feeder
Larvae sampled from cells in the
colony
Queen's spermatheca
Honey bee prepupae sampled in
the colony
Adult bees sampled with a brush
from the combs
Dead adult bees sampled from
eld plots
White-eyed pupae sampled from
combs
Adult bees sampled in a tunnel
Living adult bees from a trap
Living adult drones from a trap
Sperm sampled by manual
eversion of the endophallus
Sperm sampled with a Latshaw
instrument and syringe
Hive frames isolated with bags of
plastic mesh
Queens reared with the Doolittle
method
Egg sampled and reared in
Please cite this article as: Benuszak, J., et al., The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for
improvement in research, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.02.062
7
Publications with the
References
sampling methods (%)
(Attencia et al., 2005; Sharma and Abrol, 2005; Stanghellini et al., 2005; Rancan et al.,
2006; Totti et al., 2006; Charlton and Jones, 2007; Garca et al., 2007; Pirard et al., 2007;
Rose et al., 2007; Liu et al., 2009; Walorczyk and Gnusowski, 2009; Laurino et al., 2010;
Kadala et al., 2011; Ondo Zue Abaga et al., 2011; Wiest et al., 2011; Wu et al., 2011;
Hendriksma et al., 2012; Sgolastra et al., 2012; Biddinger et al., 2013; Degrandi-Hoffman et
21 al., 2013; Hendriksma et al., 2013; Hera et al., 2013; ozowicka, 2013; Palmer et al., 2013;
Tapparo et al., 2013; Wilkins et al., 2013; Costa et al., 2014; Lken et al., 2014; Retschnig et
al., 2014; Sandrock et al., 2014; Sharma and Abrol, 2014; Alburaki et al., 2015; Dawit et al.,
2015; Dively et al., 2015; Dolezal et al., 2015; Frazier et al., 2015; Koo et al., 2015; Tavares
et al., 2015; Zaluski et al., 2015; Zhu et al., 2015; Cabrera-Marin et al., 2016; Kiljanek et al.,
2016; Pettis et al., 2012)
15 (Shawki et al., 2006; Ramirez-Romero et al., 2008; Aliouane et al., 2009; Chauzat et al.,
2009; Vanengelsdorp et al., 2009; Bacandritsos et al., 2010; Han et al., 2010a; Mullin et al.,
2010; Rabea et al., 2010; Smodis Skerl et al., 2010; Bernal et al., 2011; Chauzat et al., 2011;
Hawthorne and Dively, 2011; Mondet et al., 2011; Zhou et al., 2011; Badiou-Beneteau et
al., 2012; Boncristiani et al., 2012; Pohorecka et al., 2012; Carvalho et al., 2013; Pohorecka
et al., 2013; Rossi et al., 2013; Rossi Cde et al., 2013; Cresswell et al., 2014; Poquet et al.,
2014; Thompson, Fryday, et al., 2014; Al-Naggar, Codling, et al., 2015; Al-Naggar,
Wiseman, et al., 2015; Da Silva et al., 2015; Dively et al., 2015; Li et al., 2015; Guseman et
al., 2016)
13 (Gregorc and Ellis, 2011; Vidau et al., 2011; Gregorc et al., 2012; Khler et al., 2012; Pettis
et al., 2012; Teeters et al., 2012; Cousin et al., 2013; Di Prisco et al., 2013; Garrido et al.,
2013; Roat et al., 2013; Aufauvre et al., 2014; Human et al., 2014; Schmehl et al., 2014; Zhu
et al., 2014; Degrandi-Hoffman et al., 2015; Ingram et al., 2015; Wang et al., 2015; Abbo et
al., 2016; Brandt et al., 2016; Campbell et al., 2016; Chaimanee et al., 2016; Cizelj et al.,
2016; Gregorc et al., 2016; Renzi et al., 2016; Tan et al., 2016; Thompson et al., 2016; Vlez
et al., 2016)
13 (El Hassani et al., 2005; Shawki et al., 2006; Bacandritsos et al., 2010; Laurino et al., 2010;
Smodis Skerl et al., 2010; Bernal et al., 2011; Marzaro et al., 2011; Mondet et al., 2011;
Cresswell et al., 2012; Henry et al., 2012; Krupke et al., 2012; Boily et al., 2013; Frost et al.,
2013; Hillier et al., 2013; Pilling et al., 2013; Williamson, Baker, et al., 2013; Williamson,
Moffat, et al., 2013; Williamson and Wright, 2013; Carayon et al., 2014; Fournier et al.,
2014; Kasiotis et al., 2014; SimonDelso et al., 2014; Stewart et al., 2014; Thompson, Levine,
et al., 2014; Blanken et al., 2015; Stanley et al., 2015)
(Cutler and Scott-Dupree, 2007; Belien et al., 2009; Hardstone and Scott, 2010; Tremolada
et al., 2010; Sgolastra et al., 2012; Chaskopoulou et al., 2014; Pistorius et al., 2015;
Calatayud-Vernich et al., 2016)
3 (Gregorc and Ellis, 2011; Gregorc et al., 2012; Kayode et al., 2014; Zhu et al., 2014; Wang et
al., 2015; Vlez et al., 2016)
2.5 (Decourtye et al., 2011; Schneider et al., 2012; Henry et al., 2014; Henry et al., 2015;
Thompson et al., 2016)
2 (Vanengelsdorp et al., 2009; Han et al., 2010b; Mullin et al., 2010; Wu et al., 2011)
2 (Badiou et al., 2008; Johnson and Percel, 2013; Wu et al., 2015; Cizelj et al., 2016)
2 (Faucon et al., 2005; Chauzat et al., 2010; Moores et al., 2012; Tapparo et al., 2012; Tapparo
et al., 2013)
1.5 (Decourtye et al., 2011; Abramson et al., 2012; Zhang and Nieh, 2015)
1.5 (Mullin et al., 2010; Derecka et al., 2013; Thompson, Levine, et al., 2014)
1 (Williams et al., 2015; Chaimanee et al., 2016)
1 (Kadala et al., 2011; Cizelj et al., 2016)
1 (Bernal et al., 2011; Codling et al., 2016)
1 (Tapparo et al., 2012; Tapparo et al., 2013)
0.5 (Cizelj et al., 2016)
0.5 (Shawki et al., 2005)
0.5 (Zaluski et al., 2015)
0.5 (Burley et al., 2008)
0.5 (Ben Abdelkader et al., 2015)
0.5 (Burley et al., 2008)
0.5 (Carrasco-Letelier et al., 2012)
0.5 (Kayode et al., 2014)
0.5 (Aupinel et al., 2007)
(continued on next page)
8 J. Benuszak et al. / Science of the Total Environment xxx (2017) xxxxxx

Table 3 (continued)

Number
Publications with the
Matrices of Sampling methods References
sampling methods (%)
papers

laboratory
Queen larvae grafted from 0.5 (Degrandi-Hoffman et al., 2013)
worker cells into queen cups
Forager bees collected with a 0.5 (Reetz et al., 2016)
vacuum cleaner
Swarm collected in nature 0.5 (Bacandritsos et al., 2010)
Sampling of nervous ganglions 0.5 (Shaqur, 2009)
Sampling of dead brood 0.5 (Chauzat et al., 2010)
(Rezi et al., 2005; Rortais et al., 2005; Pirard et al., 2007; Rial-Otero et al., 2007a; Jimnez
et al., 2008; Shamsipur et al., 2008; Chauzat et al., 2009; Zhu et al., 2010; Kujawski and
Namienik, 2011; Wiest et al., 2011; Economou et al., 2012; Pohorecka et al., 2012; Xu et
Not specied 45 al., 2012; Barganska et al., 2013; Chen and Mullin, 2013; 2014; Dubreil-Chneau et al.,
2014; Kasiotis et al., 2014; Rodrguez Lpez et al., 2014; Sanchez-Bayo and Goka, 2014;
Alburaki et al., 2015; Chen et al., 2015; Jovanov et al., 2015; Malhat et al., 2015; Williams et
al., 2015; Calatayud-Vernich et al., 2016; Souza Tette et al., 2016; Kujawski et al., 2014)
Bought from the market 37 (Fidente et al., 2005; Erdorul, 2007; Rial-Otero et al., 2007b; Choudhary and Sharma,
2008; Zhu et al., 2008; De Pinho et al., 2010; Del Carlo et al., 2010; Fontana et al., 2010;
Wang et al., 2010; Blasco et al., 2011; Zhang et al., 2011; Gmez-Prez et al., 2012;
Honey 62
Tomasini et al., 2012; Zacharis et al., 2012; Li et al., 2013; Eissa et al., 2014; Jovanov et al.,
2014; Panseri et al., 2014; Yuan et al., 2014; Fan et al., 2015; Juan-Borrs et al., 2016; Li et
al., 2016; Shendy et al., 2016)
Sampling of a piece of honey 14 (Cutler and Scott-Dupree, 2007; Nguyen et al., 2009; Chauzat et al., 2010; Chauzat et al.,
comb 2011; Pilling et al., 2013; SimonDelso et al., 2014; Al-Naggar, Codling, et al., 2015; Lu et al.,
2015; Codling et al., 2016)
Sampling of unpressed honey 1.6 (Balayiannis and Balayiannis, 2008)
Grinding of a comb and 1.6 (Garca-Chao et al., 2010)
separation of honey, wax, pollen
and larva
(Rortais et al., 2005; Cutler and Scott-Dupree, 2007; Jimnez et al., 2007; Rose et al., 2007,
Chauzat et al., 2009; Mullin et al., 2010; Bernal et al., 2011; Chauzat et al., 2011; Wiest et
al., 2011; Krupke et al., 2012; Pohorecka et al., 2012; Boily et al., 2013; Degrandi-Hoffman
et al., 2013; Johnson and Percel, 2013; Pettis et al., 2013; Pilling et al., 2013; Pohorecka et
Pollen traps 52
al., 2013; Stoner and Eitzer, 2013; Byrne et al., 2014; Sandrock et al., 2014; Stewart et al.,
2014; Thompson, Levine, et al., 2014; Zhu et al., 2014; Alburaki et al., 2015; Botias et al.,
2015; Degrandi-Hoffman et al., 2015; Dively et al., 2015; Frazier et al., 2015; Lu et al.,
2015; Williams et al., 2015; David et al., 2016; Thompson et al., 2016)
Not specied 14 (Liu et al., 2009; Han et al., 2010a; Chen and Mullin, 2013; 2014; Costa et al., 2014;
Pollen 61
Sanchez-Bayo and Goka, 2014; Chen et al., 2015; Dolezal et al., 2015; Vzquez et al., 2015)
Sampling of anthers 13 (Rose et al., 2007; Han et al., 2010b; Mullin et al., 2010; Krupke et al., 2012; Stoner and
Eitzer, 2012; Gillespie et al., 2014; Botias et al., 2015; Renzi et al., 2016)
Collecting pollen from brood 4 (Bernal et al., 2010; Kasiotis et al., 2014; Muli et al., 2014; Li et al., 2015)
combs
Collecting pollen from any combs 3 (Nguyen et al., 2009; Krupke et al., 2012; Codling et al., 2016)
Collecting pollen in beebread 3 (Mullin et al., 2010; Al-Naggar, Codling, et al., 2015; Pistorius et al., 2015)
Using craft bags around the 2 (Dively and Kamel, 2012; Gillespie et al., 2014)
owers
(Pirard et al., 2007; Chauzat et al., 2009; Chauzat et al., 2010; Serra-Bonveha and
Orantes-Bermejob, 2010; Wu et al., 2011; Chen and Mullin, 2013; Yez et al., 2013; Chen
Not specied 42
and Mullin, 2014; Sanchez-Bayo and Goka, 2014; Sandrock et al., 2014; SimonDelso et al.,
2014)
Sampling of beeswax from brood 33 (Cutler and Scott-Dupree, 2007; Vanengelsdorp et al., 2009; Adamczyk et al., 2010; Mullin
combs et al., 2010; Pilling et al., 2013; Carayon et al., 2014; Muli et al., 2014; Li et al., 2015)
Bees-wax 25
Sampling of beeswax from any 13 (Chauzat and Faucon, 2007; Chauzat et al., 2011; Lawrence et al., 2016)
combs
Sampling of beeswax from 8 (Nguyen et al., 2009; Adamczyk et al., 2010)
cappings
Sampling of beeswax from blank 4 (Adamczyk et al., 2010)
combs
(Chauzat et al., 2010; Bernal et al., 2011; Pohorecka et al., 2012; Pilling et al., 2013;
Sampling of a piece of bee bread 53 Pohorecka et al., 2013; SimonDelso et al., 2014; Al-Naggar, Codling, et al., 2015; Lawrence
et al., 2016)
Bee-bread 15 Not specied 20 (Pettis et al., 2012; Degrandi-Hoffman et al., 2013; Sandrock et al., 2014)
Collecting beebread from brood 20 (Vanengelsdorp et al., 2009; Dively et al., 2015; Mullin et al., 2010)
combs including beeswax
Sampling with pipette 7 (Yoder et al., 2013)
Sampling from the ower with a
25 (Dively and Kamel, 2012; Stoner and Eitzer, 2012; Byrne et al., 2014)
pipette
Sampling of the bee gut 25 (Pilling et al., 2013; Byrne et al., 2014; Thompson, Levine, et al., 2014)
Nectar 12
Not specied 25 (Rortais et al., 2005; Stewart et al., 2014; Botias et al., 2015)
Sampling from any combs 17 (Krupke et al., 2012; Pohorecka et al., 2012)
Sampling from brood combs 8 (Cutler and Scott-Dupree 2007)
Top layer 50 (Ondo Zue Abaga et al., 2011; Krupke et al., 2012; Stewart et al., 2014; Botias et al., 2015)
Soil 8 Sampling at a depth between 25 (Chen et al., 2015; Limay-Rios et al., 2016)
015 cm

Please cite this article as: Benuszak, J., et al., The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for
improvement in research, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.02.062
 J. Benuszak et al. / Science of the Total Environment xxx (2017) xxxxxx
Table 3 (continued)
Number
Publications with the
Matrices of Sampling methods
sampling methods (%)
papers
Not specied 12.5
Waste talc on sowing machine 12.5
Collected from a bee colony used
11
Royal jelly 9 for royal jelly production
Not specied 89
Not specied 67
Flowers 6
Flower cut above roots 33
Guttation drops sampled with a
57
pipette
Water 7 Water from honey sacs 14
Water from puddles 14
Not specied 14
pesticides (6 active substances were reported in 97% of the papers), de-
spite a large number of molecules studied in single experiments (50 ac-
tive substances in total). For risk assessment, it is of utmost importance
to gather extended knowledge on various chemicals. Available data in-
dicate no basis for extrapolation of the acute toxicity from honey bee
adults to larvae, and no conclusion can be drawn on the predictability
of the toxicity to larvae from the chemical family (Alix and Vergnet,
2007). Effects on a particular development stage are not necessarily re-
lated to a particular chemical family or mode of action (Alix and
Vergnet, 2007). On the opposite, only a few studies were dedicated to
GMO-producing pesticides, despite their wide use in North America,
South America and China. However, it should be acknowledged that
no adverse effects have been observed in laboratory tests using Bt-pro-
teins (Hendriksma et al., 2011). To date GMOs used in the eld are not
designed to kill hymenoptera (Sanahuja et al., 2011).
Nearly half of the scientic experimentations used commercial for-
mulations, amongst which half were seed coatings. To better know
honey bee exposure to pesticides, it is important to study commercial
preparations as typical formulations contain less than 50% of active sub-
stances, the rest being co-formulants - surfactants, penetrant enhancers,
spreaders, stickers, and co-solvents - used to optimise the pest control
efcacy and stability of the active ingredients (Mullin, 2015). Recently
it has been shown that honey bees were negatively impacted by
organosilicone surfactants, nonylphenol polyethoxylates and the sol-
vent N-methyl-2-pyrrolidone (Mullin, 2015; Mullin et al., 2015). There-
fore, tests on commercial formulations should not be restricted to the
regulatory context and should be included in scientic experimenta-
tions in order to assess their impacts on honey bees and the presence
of co-formulants in on-site investigations.
Our review has demonstrated that only a few studies have been per-
formed on a limited number of metabolites of active ingredients. Howev-
er, it is known that some metabolites have higher toxicities that the initial
molecules (Nauen et al., 2001). Therefore, it may be recommended to as-
sess metabolites in hive matrices on a regular basis to better know the
threat they pose to honey bees. Information on metabolites is currently
requested in the dossier for marketing authorisation (EFSA, 2012).
Some data on risk assessment are available in the European Food Safety
Authority (EFSA) journals as well as on the Draft Assessment Reports,
available on EFSA's website. However, many data, particularly on analyt-
ical methods are condential and not accessible to the scientic commu-
nity. Much time, money and effort could be saved if these data were
available for scientists. There is a need to nd a compromise between
commercial condentiality and open data to better assess honey bee pes-
ticide exposure in a holistic manner. Finally, interactions between pesti-
cides and pathogens should be better investigated in general and
particularly concerning the recently detected coleopteran Aethina tumida
(Coleoptera: Nitidulidae) in Italy (Mutinelli et al., 2014).
Interactions between insecticides and miticides were only studied in
3.5% of papers (Smodis Skerl et al., 2010; Williamson et al., 2013a;
Williamson and Wright, 2013; Palmer et al., 2013; Zhu et al., 2014)
Please cite this article as: Benuszak, J., et al., The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for
improvement in research, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.02.062
9
References
(Tapparo et al., 2012)
(Krupke et al., 2012; Pistorius et al., 2015)
(Karazaris et al., 2008)
(Smodis Skerl et al., 2010; Degrandi-Hoffman et al., 2013; Charpentier et al., 2014)
(Krupke et al., 2012; Stewart et al., 2014; Alburaki et al., 2015; Mullin et al., 2015)
(Stoner and Eitzer 2012; Pilling et al., 2013)
(Shawki et al., 2006; Reetz et al., 2011; Tapparo et al., 2011; Reetz et al., 2016)
(Reetz et al., 2016)
(Samson-Robert et al., 2014)
(Chen et al., 2015)
although honey bees are largely exposed to miticides used for V.
destructor treatment (Garrido et al., 2013) (Frost et al., 2013; ANSES,
2015). The development of studies on miticides alone or in co-exposure
with other pesticides should be considered, as recently recommended
by the conclusions in an ANSES opinion from an expert working group
(ANSES, 2015). Some combinations of pesticides including insecticides
and fungicides, whether they mix two or three active substances, are
known to generate synergetic or additional effects increasing toxicity
to honey bees (Iwasa et al., 2003; Schmuck et al., 2003; Thompson et
al., 2014a; Zhu et al., 2014). Moreover, if chronic toxicity at low concen-
trations of multiple chemicals is considered, antagonism effects might
happen (Zhu et al., 2014). Multiple (several molecules or preparations)
and repeated exposures (as likely to occur over time in the eld) should
be better investigated and could be studied using chronic and sublethal
exposures. The challenging issue of choosing the molecules included in
the pesticide cocktails could be based on the results of on-site investiga-
tions. One step further would also be to assess the co-exposure of pesti-
cide cocktails with other stress factors such as infectious and parasitic
agents (Gregorc et al., 2012; Pettis et al., 2013; Blanken et al., 2015) or
heavy metals (Hesketh et al., 2016). However, the combinations of mo-
dalities to mimic eld exposure of honey bees to multiple stressors is a
complex issue that has to be simplied to most probable scenarios ac-
cording to standardised methods. Testing all combinations is unrealistic.
Other means should be developed to screen the most relevant expo-
sures using mathematical modelling, in silico methods or other tools
yet to be developed.
Only 29.6% of the papers reported scientic experimentations run in
semi-eld or eld conditions. Field tests produce indications on the for-
mulation performance and on the behaviour of chemicals in ecosystems,
for example the level of volatilisation of the molecule (Houbraken et al.,
2016) or the toxicity of formulations in dry and wet soil conditions (Van
Straalen and Lkke, 1997). Our results show that only 11.3% of the exper-
imentations were carried out on full colonies. Colonies being able to
compensate for the losses of worker bees (Henry et al., 2015), it is cru-
cial to use appropriate measurement tools to detect slight differences in
performance of colonies exposed to insecticide treated and non-treated
crops (Stadler et al., 2003; Faucon et al., 2005; Henry et al., 2012). How-
ever using whole colonies imply technical difculties in terms of vari-
ability of results, reproducibility between sites and repeatability
between years. More eld work is needed as the current methods of
eld testing would need major improvements as recommended by
EFSA (2012).
A wide variety of units has been used in the scientic experimenta-
tions to quantify pesticides applied on crops or on honey bees. Some
units were different but expressed the same quantities. Units such as
ppb (parts-per-billion) or ppm (parts-per-million) should be avoided.
Instead gkg1 and mgkg1 should be used. Units expressing propor-
tion should always use the same unit of reference, for instance kg
(i.e. gkg1 instead of ng/g). Comparability of studies would benet
from the harmonisation of units, particularly in the view of comparing
10 J. Benuszak et al. / Science of the Total Environment xxx (2017) xxxxxx
the results produced by scientic experimentations and on-site
investigations.
This review has also shown the large variety of matrices used in
experiments. The differences in matrix nature made the cross checking
of data between assessment of exposure (on-site investigation sampled
mostly honey) and the effects of pesticides on honey bees (scientic ex-
perimentations used mainly worker bees to study behaviour, cognition
or mortality) difcult. Although beeswax is known for its pesticide stor-
age capacity (Chauzat and Faucon, 2007), this matrix was studied in only
8.7% of the papers. More work is needed to assess how beeswax could be
used to trace the history of hive exposure to pesticides. Efforts should
also be made to study the metabolisation of molecules in this complex
matrix particularly in the specic conditions of temperature within the
hive. Interactions between different molecules contained in beeswax
might have an effect on ingredient stability (Jimenez et al., 2005;
Chauzat and Faucon, 2007; Serra-Bonveha and Orantes-Bermejob,
2010). Similarly, little information about beebread was available; despite
this matrix being a signicant route of honey bee exposure to pesticide
(AFSSA, 2008; Giroud et al., 2013). In conclusion, the overall results of
experimentations would benet from the harmonisation of the matrices
used in studies for better relevance.
Amongst all the papers included in our review (n = 264), none stud-
ied the air within the hive. Contaminants may enter the hive through
four main ways: by the air ux, by external food sources, by returning
foraging bees and by chemical application within the hive (Tremolada
and Vighi, 2014). The air inux into the hive for hive thermoregulation
and for oxygen supply is an important parameter. The oxygen request
(30 kg/year) corresponds to an air ux of 150 m3/year which results
in a twice-daily exchange of the full air volume of the hive (Tremolada
and Vighi, 2014). The maturation from nectar to honey involves the re-
moval of water (Root and Morse, 1990) leading to possible in-hive air
contamination. Air is in contact with bees and beeswax and can act as
a redistribution medium inside the hive, especially for volatile com-
pounds (Tremolada and Vighi, 2014). Given the gures showing the
large inux of air through the hive and recent data on the persistence
of pesticides in atmospheric particulate phase (Socorro et al., 2016), fur-
ther analysis of this matrix is needed and air should be included in the
assessment of the honey bee exposome.
In the hive, all matrices are connected and can exchange pollutants,
some compartments being more persistent than others. In the cells
made of beeswax, honey and pollen are stored and larvae are bread.
The periods of contact between beeswax and other matrices enable
transfer of contaminants by diffusion (Tremolada and Vighi, 2014).
Thus, the study of combinations of contaminated matrices would take
into account these simultaneous routes of exposure. Beeswax and pol-
len would be an interesting combination because of their high level of
contamination by pesticides (Chauzat et al., 2009). Beeswax and
honey being in contact, chemical transfer between the two matrices is
of concern for human consumption and for honey bee health. It was
shown that from 10 mg/kg, the miticide coumaphos can migrate from
beeswax to honey (Kochansky et al., 2001). Beeswax might also be con-
sumed by humans when eating honey directly from honeycomb. More-
over, beeswax is included in food products through the E 901 food
additive. However, a risk analysis of consumer chronic exposure to
some pesticide residues through the consumption of contaminated
honey and beeswax was recently conducted. It was concluded that
the food consumption of honey and beeswax did not compromise
consumer health as the quantities of honey and beeswax ingested
are low when compared with the theoretical maximum daily intake
(Wilmart et al., 2016). In order to decrease the level of pesticide res-
idues in beeswax, it is recommended in good beekeeping practices
that beekeepers replace two old frames from the brood chamber by
new ones every year to ensure a complete frame turnover in the
hive after 5 years (ITSAP, 2014). It should be noted that this recom-
mendation is not applied for frames located in the suppers, collecting
honey.
Please cite this article as: Benuszak, J., et al., The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for
improvement in research, Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.02.062
Our review has shown that a lot of different types of sampling
methods have been reported in the scientic papers. The great number
of matrices studied and the large number of sampling methods used
made it difcult to compare and link data between experiments.
Harmonisation of sampling methods would be necessary to ease connec-
tions and cross references. For example nectar was sampled from owers,
in honey bee crops and within the hive. Honey studied in on-site investi-
gations was generally commercial honey and did not represent the real
pesticide exposure of honey bees. This honey is stored in supers and not
consumed by bees. Honey kept within the brood chamber is stored longer
and can be contaminated by other matrices or be repeatedly exposed to
pesticides and veterinary treatments (Bogdanov et al., 1998; Devillers,
2014). Not only for honey, but for all honey bee matrices, comparative
studies are needed to better know what kind of samples (number and lo-
cation) is representative of a colony exposure. On the same issue, it is still
unknown how many colonies should be sampled to be representative of
an apiary when assessing pesticide exposure.
5. Conclusion
This review reports the use of a wide variety of honey bee matrices
and sampling methods in the study of pesticide exposure. Based on
our systematic literature review, we found that there are several knowl-
edge gaps in assessing honey bee exposure to pesticides. There is a real
need to fully assess the honey bee colony exposure to pesticides, as a
whole. Studies should be deepened by investigating alternative matri-
ces, alone or in combination to reach representativeness of the honey
bee exposome. Matrices such as beeswax and beebread are very little
analysed despite their capacities for long-term pesticide storage. More-
over, bioavailability and transfer between hive components are poorly
explored and understood. Many pesticides are studied but interactions
between molecules or with other stressors are lacking. Laboratory ex-
periments and eld studies should be developed to answer these ques-
tions. In the ANSES report on honey bee co-exposure to different
stressors it was recommended that the regulatory procedures include
the interaction between the molecules to be tested and a fungicide, a
miticide or an insecticide with the same mode of action (ANSES,
2015). Scientic experimentations that also test these modalities are
needed. Sampling methods, targeted matrices and units of measure
should, to some extent, be standardised between publications to ease
comparison and cross checking. Data on honey bee exposure to pesti-
cides would also benet from the use of commercial formulations in-
stead of active ingredients, with a special assessment of co-formulants
(quantitative exposure and effects). Finally, the air matrix within the
colony should be explored in order to complete current knowledge on
honey bee pesticide exposure.
Author contributions
MPC conceived the study; JB carried out the review; JB, ML and MPC
analysed the data; JB prepared the manuscript; ML and MPC edited the
manuscript.
Additional information
Competing nancial interests: the authors declare no competing -
nancial interests.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2017.02.062.
Acknowledgments
This work received the nancial support of the pharmacovigilance
tax (ANSES). The authors wish to thank the Scientic watch Unit at
ANSES for its methodological and technical assistance and Mrs. Pittman
for English editing.
 J. Benuszak et al. / Science of the Total Environment xxx (2017) xxxxxx
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