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Pesticide treatments before and during the flowering of honey bee forage crops may lead to residues in honey. In northern regions oilseed rape belongs to the main forage crops that is mostly cultivated by means of intensive agriculture, including several pesticide treatments. However, in addition to the focal forage crops, pesticides from non-forage crops can spread to wild flowers around fields, and thus the residues in honey would reflect the whole range of pesticides used in the agricultural landscape. The aim of our study was to clarify which currently used pesticides are present in honey gathered from heterogeneous agricultural landscapes after the end of flowering of oilseed crops. Honey samples (N = 33) were collected from beehives of Estonia during 2013 and 2014, and analysed for residues of 47 currently used agricultural pesticides using the multiresidue method with HPLC-MS/MS and GC-MS and a single residue method for glyphosate, aminopyralid and clopyralid. Residues of eight different active ingredients with representatives from all three basic pesticide classes were determined. Although no correlation was detected between the cumulative amount of pesticide residues and percent of oilseed crops in the foraging territory, most of the residues are those allowed for oilseed rape treatments. Among all pesticides, herbicide residues prevailed in 2013 but not in 2014. Despite the relatively small agricultural impact of Estonia, the detected levels of pesticide residues sometimes exceeded maximum residue level; however, these concentrations do not pose a health risk to consumers, also acute toxicity to honey bees would be very unlikely.
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541
Research Article
Received: 9 May 2017 Revised: 7 September 2017 Accepted article published: 9 October 2017 Published online in Wiley Online Library: 4 December 2017
(wileyonlinelibrary.com) DOI 10.1002/ps.4756
Synergistic interactions between a variety of
insecticides and an ergosterol biosynthesis
inhibitor fungicide in dietary exposures of
bumble bees (Bombus terrestris L.)
Risto Raimets,a* Reet Karise,aMarika Mänd,aTanel Kaart,bSally Ponting,c
Jimao Songcand James E Cresswellc
Abstract
BACKGROUND: In recent years, concern has been raised over honey bee colony losses, and also among wild bees there is
evidence for extinctions and range contractions in Europe and North America. Pesticides have been proposed as a potential
cause of this decline. Bees are exposed simultaneously to a variety of agrochemicals, which may cause synergistically detri-
mental impacts, which are incompletely understood. We investigated the toxicity of the fungicide imazalil in mixture with
four common insecticides: fipronil (phenylpyrazoid), cypermethrin (pyrethroid), thiamethoxam, and imidacloprid (neonicoti-
noids). Ergosterol biosynthesis inhibitor (EBI) fungicides like imazalil can inhibit P450 detoxification systems in insects and
therefore fungicide insecticide co-occurrence might produce synergistic toxicity in bees. We assessed the impact of dietary
fungicide insecticide mixtures on the mortality and feeding rates of laboratory bumble bees (Bombus terrestris L.).
RESULTS: Regarding mortality, imazalil synergised the toxicity of fipronil, cypermethrin and thiamethoxam, but not imidaclo-
prid. We found no synergistic effects on feeding rates.
CONCLUSION: Our findings suggest that P450-based detoxification processes are differentially important in mitigating the
toxicity of certain insecticides, even those of the same chemical class. Our evidence that cocktail effects can arise in bumble
bees should extend concern about the potential impacts of agrochemical mixtures to include wild bee species in farmland.
© 2017 Society of Chemical Industry
Supporting information may be found in the online version of this article.
Keywords: bumble bees; ergosterol biosynthesis inhibitor fungicide; insecticides; synergy
1 INTRODUCTION
Recently, concern has been raised over pollinator declines in
Europe and North America.1In some regions, beekeepers have
experienced severe losses among colonies of managed honey
bees (Apis mellifera L.)2and among some wild bees3there is
evidence for extinctions4and range contractions.5Bee declines are
of concern because of the valuable pollinator services that they
provide to crops and wildflowers.6,7 The declines probably have
various anthropogenic causes, including the use of pesticides in
intensively cultivated farmland.8
In farmland, pollinators may be exposed to several pesticides
during their lifetime because numerous pesticide residues are
present in bee forage plants9and in various hive matrices of man-
aged honey bees.10 For example, Mullin et al.11 found 118 different
pesticides and their metabolites among the various matrices (e.g.
stored honey and bee bread) of hone bee hives. Contemporary
intensive agriculture involves protecting crop plants with a variety
of pesticides, including fungicides and insecticides, and bees will
almost certainly encounter these residues in mixture when they
forage in agrochemically treated bee-attractive crops.12,13
The existence of disproportionate, or non-additive, toxicity of
pesticides in mixture is known as a ‘cocktail effect’, ‘synergistic
interaction’,14 or ‘potentiation’.15 Our focal example arises from
the capacity of certain fungicides, which typically have low tox-
icity to insects, to greatly increase the toxicity of an insecti-
cide by inhibiting the insect’s capacity to metabolically degrade
the insecticide. Specifically, the widely used group of fungicides
known as ergosterol biosynthesis inhibitors (EBIs) are well known
Correspondence to: R Raimets, Department of Plant Protection, Institute of
Agricultural and Environmental Sciences, Estonian University of Life Sciences,
Kreutzwaldi 1, 51014, Tartu, Estonia. E-mail: ristorai@gmail.com
aDepartment of Plant Protection, Institute of Agricultural and Environmental
Sciences, Estonian University of Life Sciences, Tartu, Estonia
bDepartment of Animal Genetics and Breeding, Institute of Veterinary Medicine
and Animal Sciences, Estonian University of Life Sciences, Tartu, Estonia
cBiosciences, College of Life and Environmental Sciences, University of Exeter,
Exeter, UK
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542
www.soci.org R Raimets et al.
to increase toxicity to honey bees of pyrethroid insecticide in
mixture.16 However, while mixture effects have been tested widely
in honey bees,17,18 the susceptibility of wild bees to these syn-
ergistic interactions has not been fully explored. We therefore
investigated the potential for an EBI fungicide, imazalil, to syn-
ergise (or, more strictly, potentiate) the toxicity to bumble bees
of environmentally relevant insecticides from a varied range of
chemical families, namely the neonicotinoids (thiamethoxam and
imidacloprid), pyrethroids (cypermethrin) and phenylpyrazoles
(fipronil).
The four focal insecticides that we studied all target the insect
nervous system. The neonicotinoids block the ligand-gated ion
channels of the nicotinic acetylcholine receptors. In bees, dietary
exposure to neonicotinoids can impair a wide range of behavioural
and life history-related characteristics19 including homing
behaviour,20 colony performance21 and foraging activity.22 The
pyrethroid cypermethrin affects insect sodium channels23 and
has been demonstrated to affect longevity24 and respiratory
patterns25 in bees. The phenylpyrazole fipronil blocks receptors
that respond to the neurotransmitter gamma-aminobutyric acid
(GABA)-gated chloride channels in the insect central nervous
system and can affect longevity in bees.26
We chose imazalil to represent the EBI fungicides. Imazalil is envi-
ronmentally relevant because its residues can occur in combina-
tion with imidacloprid in fruit orchards27 and it is water soluble,
which facilitates dose preparation. In view of their low toxicity
to insects in pure exposures, EBI fungicides are not considered
harmful to farmland bees provided that the ‘good practice’ label
rates and prescriptions are followed.28 However, the EBI fungi-
cides can detrimentally affect bees’ tolerance for other pesticides
because of effects on metabolic detoxification pathways. A cer-
tain degree of insecticide tolerance in bees is possible as a con-
sequence of metabolic detoxification of the active ingredients by
enzymes of the cytochrome P450 system.17 Impairment of the
P450 system by EBI fungicides can result in an increase of insecti-
cide toxicity for bees.29 Therefore, the principal aim of our present
study was to establish the involvement of metabolic detoxifica-
tion in bumble bee pesticide interactions by testing whether
imazalil synergises various insecticides representing some of the
major chemical families that are widely used in farmland crop
protection.
2 MATERIALS AND METHODS
2.1 Bee provenance and husbandry
Bumble bees (Bombus terrestris L. ssp audax) were purchased
as boxed queen-right colonies from commercial suppliers (Kop-
pert Biological Systems, Berkel en Rodenrijs, the Netherlands and
BioBest, Westerlo, Belgium). For each of five separate experiments,
adult workers were collected from a single colony under red light
and individually allocated to a wooden cage (0.07 x 0.05 x 0.04 m)
whose two largest faces were covered by ventilating mesh. Each
cage was supplied with a small ad libitum syrup feeder. During
experiments, the bees were kept in a semi-controlled environment
(24 ±1C, 47% relative humidity and 12:12 h dim light:darkness).
During experimental exposures, the caged bumble bees were
fed on dose-appropriate syrup ad libitum and their feeders were
weighed before and at the end of the experiment (after 48 h of
exposure) in order to measure syrup consumption. We recorded
mortality at 24 and 48 h of exposure. Bees were considered dead
when they did not move their legs or antennae and did not
respond to stimulation.
2.2 Exposure to agrochemicals
In order to test for synergistic interactions between the fungicide
and a single insecticide, each experiment comprised four treat-
ments: (1) undosed controls; (2) fungicide alone; (3) insecticide
alone; and (4) fungicide insecticide mixture. At the University of
Exeter laboratory, we conducted four separate experiments (one
per focal insecticide) in which we delivered sublethal dietary doses
of the four agrochemical treatments in feeder syrup (Attraker; Kop-
pert Biological Systems). At the Estonian University of Life Sciences
laboratory, Tartu, we repeated the experiment conducted at Exeter
(12 bees per treatment) with imidacloprid using both a larger num-
ber of replicates (i.e. 20 per treatment) and also the local proce-
dures for dose preparation in order to validate the result previ-
ously obtained at Exeter. Except for the imidacloprid experiment
at Exeter, each treatment was replicated in at least 20 bumble bee
individuals in every experiment.
For each agrochemical, we used experimental doses (see below)
that aimed to produce approximately 20% mortality in exposures
to single dietary substances. The purpose of this level of dosing
was both to demonstrate that the fungicide and insecticide were
physiologically active in the exposed bees and also to provide
enough capacity for the dietary mixture to reveal a statistically
detectable synergistic interaction between the test substances, if it
should exist. Specifically, if the two test substances each separately
cause 20% mortality in treatment groups of 20 bees (i.e. 4 deaths
per treatment), then their mixture is expected to cause 36%
mortality (i.e. approximately 7 deaths) if they act independently
(see Eqn 1 below) and a statistically significant non-independence
(synergy) is detected when mortality exceeds 65% (13 deaths) in
the mixture (see statistical testing below).
Before incorporation into diets, the active substances were dis-
solved initially in small volumes of acetone, which was subse-
quently adjusted so that syrup in each treatment group contained
1% acetone, including the undosed control diet, according to the
method described by Thompson et al.24 The dietary concentra-
tions of the active substances in the feeder syrups were as follows:
imazalil (Sigma Aldrich), 300 mg L-1 ; fipronil (Sigma Aldrich, Poole,
UK), 20 𝜇gL
-1; thiamethoxam (Sigma Aldrich), 13 𝜇gL
-1; imida-
cloprid (Sigma Aldrich), 500 𝜇gL
-1; cypermethrin (Sigma Aldrich),
7mgL
-1. The doses were established based on data from the lit-
erature and pilot experiments. The relatively high ratio of fungi-
cide:insecticide concentrations in our diets facilitates the manifes-
tation of synergistic interactions.16
2.3 Statistical analyses
We tested statistically for synergistic interactions between the
fungicide and a single insecticide with a modified binomial pro-
portion test for additivity (BPA).38 The BPA test uses the ‘Bliss inde-
pendence criterion’,30 whose basis is that:
pexp
AB =pA+pBpA·pB(1)
where pAand pBdenote the probabilities of mortality attributable
to dietary substances A and B, respectively, and pexp
AB denotes the
expected probability of mortality attributable to a dietary mixture
of A and B if they act independently. If pobs
AB denotes the observed
proportion of bees that die by consuming the dietary mixture of A
and B, then the null hypothesis of an absence of interaction is:
H0D=(pobs
AB pexp
AB )=0(2)
An expression that evaluates the sampling distribution of D
under H0as a z-score has been produced by Sgolastra et al.,31
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Synergistic interactions between a variety of insecticides and fungicide www.soci.org
Figure 1. Mortality [proportion (%) dying] after 24 h in three exposure treatments: A, dietary imazalil; B, insecticide (Fip, fipronil; Tmx, thiamethoxam; Imi,
imidacloprid; Cyp, cypermethrin); and AB, imazalil insecticide mixture. In the AB column, the grey fill indicates the expected mortality if the components
of the dietary mixture act independently (H0) and the dashed horizontal line indicates the upper 95% confidence interval on the sampling distribution
under H0. An asterisk indicates that the mixture has produced a statistically significant synergistic effect (one-tailed binomial proportion test). A column
is blank (has no bar) if no mortality occurred.
which enabled us to obtain P-values by approximation to a stan-
dard normal distribution. For each insecticide, BPA tests were
performed separately for mortality at 24 and 48 h. For each focal
insecticide, variation among treatments in feeding rate was anal-
ysed with one-way analysis of variance (ANOVA) and Tukey post
hoc tests. In analysing feeding rates at 48 h, only data from bumble
bees alive at 24 h were used.
3RESULTS
No mortality was observed in any of the control exposures
(undosed syrup). When mortality was the response variable, we
detected synergistic interactions between imazalil and fipronil
(BPA test: 24 h, P<0.005; 48 h, not significant), thiamethoxam
(BPA test: 24 and 48 h, P<0.005) and cypermethrin (BPA test:
24 and 48 h, P<0.001) (Figs 1 and 2). Dietary exposure to imi-
dacloprid alone (500 𝜇gL
-1) caused little mortality and we did
not detect positive synergistic interactions between imazalil and
imidacloprid in the experiment at Tartu (Figs 1 and 2). Dietary
imidacloprid reduced the mortality rate resulting from dietary
imazalil in the Exeter experiment (BPA test: 24 h, P<0.005; 48 h,
P<0.001; Supporting Information Fig. S1).
Feeding rates varied among the dietary treatments (one-way
ANOVA: fipronil: F3,87 =17.1, P<0.001; thiamethoxam: F3,60 =15.6,
P<0.001; imidacloprid: F3,73 =5.2, P<0.01; cypermethrin:
F3,64 =25.3, P<0.001) and generally dietary agrochemicals
reduced syrup consumption (Tukey post hoc tests: P0.05;
Fig. 3), but no interactions were observed between insecticides
and the fungicide.
4 DISCUSSION
4.1 Synergistic effects – physiological implications
Our present study revealed that dietary exposure to the fungi-
cide imazalil increased the toxicity to bumble bees of three out
of the four insecticides that we tested, which indicates that it has
the capacity to cause a positive synergistic interaction, or cocktail
effect, in these insects. Our findings are consistent with those of
several previous studies of the effects on honey bees of fungicides
in mixture. In honey bees, prochloraz synergises both pyrethroid32
and pyrazole29 insecticides, and thiamethoxam (a neonicotinoid
insecticide) is synergised by both tebuconazole16 and boscalid.13
Fungicides that synergise the toxicity of insecticides in honey bees
act by inhibiting detoxification systems, such as the P450 enzyme
complex.33 Taken together with previous work, our results sug-
gest that the P450s could play an important role in both honey
bees and bumble bees in the detoxification of a chemically varied
group of active ingredients from three chemical families, namely
the phenylpyrazoles (i.e. fipronil), the pyrethroids (cypermethrin)
and the neonicotinoids (thiamethoxam). These findings have a
straightforward adaptive explanation because the season-long
activity of social bees makes them forage-generalists who must
subsist on nectar and pollen from a wide variety of plant species,
each of whose blooming period is shorter than the lifespan of
the colony. Many plants protect their pollen against consump-
tion by non-pollinating flower visitors with secondary chemicals,34
which vary in constitution among plant lineages. Social bees
therefore have evolved to cope with a broad spectrum of plant
secondary chemicals in their diet including metabolic detoxifica-
tion by active enzymes (e.g. P450 systems) in the digestive tract.
These considerations suggest that social bees, including bumble
bees, are pre-adapted for tolerating dietary insecticides that are
artificial analogues of naturally occurring plant toxins,35 such as
the nicotine- and pyrethrum-based toxicants used in the present
study. It also implies that oligolectic solitary bees could be more
susceptible to insecticides than their social counterparts.
Our present investigation found no evidence for a synergis-
tic interaction during dietary exposure to a mixture of a known
P450 inhibitor, imazalil, and imidacloprid in bumble bees. Sim-
ilarly, previous research that exposed honey bees to imidaclo-
prid using oral doses found little synergistic interaction with EBI
fungicides.16 Contact applications of active ingredients to the tho-
rax of honey bees also produced very weak synergistic effects of
piperonyl butoxide (PBO; another P450 inhibitor) on imidacloprid,
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544
www.soci.org R Raimets et al.
Figure 2. Mortality [proportion (%) dying] after 48 h in three exposure treatments: A, dietary imazalil; B, insecticide (Fip, fipronil; Tmx, thiamethoxam;
Imi, imidacloprid; and Cyp, cypermethrin); and AB, imazalil insecticide mixture. In the AB column, the grey fill indicates the expected mortality if the
components of the dietary mixture act independently (H0) and the dashed horizontal line indicates the upper 95% confidence interval on the sampling
distribution under H0. An asterisk indicates that the mixture has produced a statistically significant synergistic effect (one-tailed binomial proportion test).
A column is blank (has no bar) if no mortality occurred.
Figure 3. Variation in individual feeding rates (mg syrup consumed per bee per day) during 48 h of exposure among four dietary treatments: C, undosed
controls; A, dietary imazalil; B, insecticide (Fip, fipronil; Tmx, thiamethoxam; Imi, imidacloprid; and Cyp, cypermethrin); and AB, imazalil insecticide
mixture. Among the histogram columns, different lower case letters indicate significant differences in mean feeding rate (Tukey test, P<0.05). Error bars
indicate 1 standard error.
even though PBO strongly synergised the toxicity of two other
neonicotinoids, acetamiprid and thiacloprid.36 Based on these
results, we tentatively propose two hypotheses. First, it is conceiv-
able that separate detoxification systems deal with imidacloprid
and the other toxicants and that one hallmark of the proposed
imidacloprid-specific enzyme system is insensitivity to inhibition
by imazalil and PBO. However, it is unclear what detoxification
enzyme could be both specific to imidacloprid and also selectively
immune to interference from imazalil and PBO. Second, it is possi-
ble that imazalil suppresses the metabolic activation of imidaclo-
prid by a P450 enzyme system. Imidacloprid has toxic metabolites,
5-hydroxyimidacloprid and olefin, that are implicated in causing
mortality.37 Disruption of metabolic activation may also explain
why the synergistic effects of imazalil on fipronil that were evi-
dent at 24 h had disappeared by 48 h; specifically, inhibition of
P450 oxidative enzymes may reduce the production of fipronil’s
highly toxic sulfone metabolite.38 Consequently, we postulate that
complex mixture effects can arise when both detoxification and
metabolic activation of an insecticide are inhibited by a second
active substance, such as a fungicide.
In contrast to the effects on mortality that we observed in our
experiment, no synergism was detected in regard to feeding rate,
although the separate exposures to the fungicide and insecti-
cides decreased it. These results provide further confirmation of
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Synergistic interactions between a variety of insecticides and fungicide www.soci.org
differential sensitivity to pesticides among various endpoints such
as mortality and feeding rate.39 Despite the reductions in feeding
rates caused by dietary agrochemicals, it is unlikely that any of the
individuals in our experiments died from starvation within the 48-h
exposure, because dosed bumble bees can live for 35 days while
feeding at less than half the rate of undosed controls.40
We observed differences among our separate experiments in
the levels of mortality caused by exposure to dietary imazalil.
We expect that these differences originated in either intrinsic or
environmental variation in the bumble bee colonies used, because
our experiments were conducted at different times of year and
for each experiment new bumble bee colonies were purchased.
However,while the differences indicate that the severity of mixture
effects can be expected to vary among real-world instances, it is
unlikely that the existence of synergistic interactions (i.e. our main
conclusion) can itself be governed by environmental influences or
genetic variation among bees.
4.2 Synergistic effects – environmental relevance
Our results indicate that exposures to environmentally relevant
mixtures of pesticides could be potentially harmful to wild bees
even when the impacts of separate exposures to the mixture’s
single components are negligible. Specifically, our experiments
confirm that cocktail effects arising from agrochemical pesticides
are physiologically possible in bumble bees, but we recognize that
further research is needed to establish their potency when bees
are exposed to residues at environmentally realistic levels, which
are likely to be lower than those we studied here. Thus, further
empirical testing of pesticide mixtures is warranted and should be
taken into account in regulations that govern the use of fungicides
and insecticides in farmland.
4.3 Summary
Our present study revealed that certain insecticide fungicide
mixtures (except imidacloprid imazalil) positively synergised the
effect of the insecticide in bumble bees when assessed by levels
of mortality, but not when assessed by variation in feeding rates.
The efficacy of imazalil (an EBI fungicide) to synergise the toxic-
ity of chemically varied insecticides suggests that P450 systems
are involved in broad-spectrum detoxification in bumble bees. As
previously found, imidacloprid alone was weakly synergised and
the physiological basis of this differentiation is a target for future
research. Our evidence that cocktail effects can arise in bumble
bees should extend concern over the potential impacts of agro-
chemical mixtures to include wild bee species in farmland.
ACKNOWLEDGEMENTS
This research was supported by institutional research funding
(IUT36-2) of the Estonian Ministry of Education and Estonian
Science Foundation and by Dora Plus Action 1 contract no
36.9-6.1/959.
SUPPORTING INFORMATION
Supporting information may be found in the online version of this
article.
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... For acute toxicity to foragers: For acute toxicity to in-hive worker bees: Silva et al. 2015, Karise et al. 2017, Muli et al. 2018, Piechowicz et al. 2018a, Gawel et al. 2019, Raimets et al. 2020 Belgium, Canada, Italy, Poland 5.0 18.5 3.0* 151.4* NA (Simon-Delso et al. 2014, Chiesa et al. 2016, Tsvetkov et al. 2017, Piechowicz et al. 2018b (Lambert et al. 2013, Kasiotis et al. 2014, Valdovinos-Flores et al. 2017, Muli et al. 2018, Bommuraj et al. 2019, Gawel et al. 2019) Cyprocanozole ...
... France, Poland 6.0 11.3 3.0* 6.0 NA (Wiest et al. 2011, Lambert et al. 2013 (Wiest et al. 2011, Lambert et al. 2013, Irungu et al. 2016, Karise et al. 2017, Saitta et al. 2017, Gawel et al. 2019, Raimets et al. 2020 , Krupke et al. 2012, Pettis et al. 2013, Stoner and Eitzer 2013, Long and Krupke 2016, Roszko et al. 2016, Bohme et al. 2018b, Drummond et al. 2018, Muli et al. 2018, Tong et al. 2018a, Tosi et al. 2018, Ostiguy et al. 2019, Prado et al. 2019, Stoner et al. 2019, Zawislak et al. 2019, Raimets et al. 2020 , Stoner and Eitzer 2013, Frazier et al. 2015, David et al. 2016, Roszko et al. 2016, Simon-Delso et al. 2017, Tsvetkov et al. 2017, Böhme et al., 2018b, Drummond et al. 2018, Tosi et al. 2018, Favaro et al. 2019, Ostiguy et al. 2019, Prado et al. 2019 , Lambert et al. 2013, Stoner and Eitzer 2013, David et al. 2016, Tong et al. 2016, Calatayud-Vernich et al. 2018, Drummond et al. 2018, Muli et al. 2018, Tong et al. 2018a , Pettis et al. 2013, Stoner and Eitzer 2013, Frazier et al. 2015, Roszko et al. 2016, Hakme et al. 2017, Simon-Delso et al. 2017, Böhme et al., 2018b, Drummond et al. 2018 (Kubik et al. 2000, Smodiš Š kerlet al. 2009, Pettis et al. 2013, Stoner and Eitzer 2013, Frazier et al. 2015, Long and Krupke 2016, Roszko et al. 2016, Böhme et al., 2018b, Drummond et al. 2018, Tong et al. 2018a, Favaro et al. 2019, Ostiguy et al. 2019 (Kubik et al. 1999, Wiest et al. 2011, Lambert et al. 2013, Frazier et al. 2015 , Pettis et al. 2013, Stoner and Eitzer 2013, Frazier et al. 2015, David et al. 2016, Long and Krupke 2016, Tsvetkov et al. 2017, Böhme et al., 2018b, Drummond et al. 2018 For chronic toxicity to in-hive worker bees: ...
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