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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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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
( 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
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
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:
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
Pest Manag Sci 2018; 74: 541 –546 © 2017 Society of Chemical Industry
542 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
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
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),
-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:
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:
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 © 2017 Society of Chemical Industry Pest Manag Sci 2018; 74: 541 –546
Synergistic interactions between a variety of insecticides and fungicide
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.
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.
4.1 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,
Pest Manag Sci 2018; 74: 541 –546 © 2017 Society of Chemical Industry
544 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 © 2017 Society of Chemical Industry Pest Manag Sci 2018; 74: 541 –546
Synergistic interactions between a variety of insecticides and fungicide
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.
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
Supporting information may be found in the online version of this
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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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Fungicides account for more than 35% of the global pesticide market and their use is predicted to increase in the future. While fungicides are commonly applied during bloom when bees are likely foraging on crops, whether real-world exposure to these chemicals – alone or in combination with other stressors – constitutes a threat to the health of bees is still the subject of great uncertainty. The first step in estimating the risks of exposure to fungicides for bees is to understand how and to what extent bees are exposed to these active ingredients. Here we review the current knowledge that exists about exposure to fungicides that bees experience in the field, and link quantitative data on exposure to acute and chronic risk of lethal endpoints for honey bees (Apis mellifera). From the 702 publications we screened, 76 studies contained quantitative data on residue detections in honey bee matrices, and a further 47 provided qualitative information about exposure for a range of bee taxa through various routes. We compiled data for 90 fungicides and metabolites that have been detected in honey, beebread, pollen, beeswax, and the bodies of honey bees. The risks posed to honey bees by fungicide residues was estimated through the EPA Risk Quotient (RQ) approach. Based on residue concentrations detected in honey and pollen/beebread, none of the reported fungicides exceeded the levels of concern (LOC) set by regulatory agencies for acute risk, while 3 and 12 fungicides exceeded the European Food Safety Authority (EFSA) chronic LOC for honey bees and wild bees, respectively. When considering exposure to all bees, fungicides of most concern include many broad-spectrum systemic fungicides, as well as the widely used broad-spectrum contact fungicide chlorothalonil. In addition to providing a detailed overview of the frequency and extent of fungicide residue detections in the bee environment, we identified important research gaps and suggest future directions to move towards a more comprehensive understanding and mitigation of the risks of exposure to fungicides for bees, including synergistic risks of co-exposure to fungicides and other pesticides or pathogens.
... Generally, honey bee and all its products such as pollen, wax, and particularly honey are potential natural indicators of environmental pollution, and might be regularly used for biomonitoring of contamination, since they provide miniature samplers (Malhat et al., 2015;Rodríguez López et al., 2014). Being used as a universal food, there would be a high demand on the quality of honey among consumers (Karise et al., 2017). In this context, not only the nutritional aspect of honey as a natural food is crucial, but also the levels of pesticide residues would be of great importance (Rahman et al., 2021;Song et al., 2018). ...
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A multi-residue method was developed for 45 pesticides from different groups, encompassing the most widely used pesticides. 30 samples of honey were randomly collected from different regions of Iran and extraction was performed using validated QuEChERS method. Next, pesticide residues were identified and measured using optimized UHPLC-MS/MS and GC-MS analysis. All validation assays were performed with a blank sample. For the evaluation of linearity, matrix-matched calibration curves were prepared with spiked samples at seven concentration levels, ranging from 0.005 to 0.5 mg L⁻¹ for pesticides in UHPLC-MS/MS and from 0.025 to 2 mg L⁻¹ for GC-MS analysis. Recovery and precision were certified through extraction of spiked samples at concentration levels of 0.1 and 0.25 mg L⁻¹. The limit of quantification (LOQ) was fixed as the lowest concentration level of the calibration curve, which presented adequate recovery and precision. Results indicated that the observed pesticides in honey samples were approximately 44.4% of the total studied pesticides, of which 90% belonged to the group of insecticides. Eventually, the human health risk assessment was performed for children and adult consumers based on Monte Carlo simulation. The rank order of pesticides based on HQ was lindane > diazinon > trichlorfon > permethrin for honey samples. In our study, the calculated hazard index (HI) for adults (0.18) and for children (0.57) lower than 1, suggested no potential health risks to the honey consumers. All pesticides except for lindane did not pose a cancer risk to humans, unfortunately due to lindane residue as a Persistent Organic Pollutants (POPs), CR (2.5E-5) was higher than 1.0E-6 and controlling plans should be conducted to decrease the concentration of this pesticide in Iran.
... For each pesticide, five concentrations were tested: 0 (control), 0.01, 0.1, 1 and 10 µg/L (equivalent to 0, 0.0083 0.083, 0.813 and 8.130 µg/kg, respectively, calculated with a sucrose solution density of 1.23 ± 0.02 g/L (n = 10)). Concentrations were consistent with the residual contamination found in honey, pollen and wax [11,15,[44][45][46][47][48]. Six experimental groups of exposure were investigated: the control group (C); the insecticide alone (I); the fungicide alone (F); the herbicide alone (H); the binary mixtures of insecticide + fungicide (IF), insecticide + herbicide (IH) and herbicide + fungicide (HF); and the ternary mixture of insecticide + herbicide + fungicide (IHF). ...
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To explain losses of bees that could occur after the winter season, we studied the effects of the insecticide imidacloprid, the herbicide glyphosate and the fungicide difenoconazole, alone and in binary and ternary mixtures, on winter honey bees orally exposed to food containing these pesticides at concentrations of 0, 0.01, 0.1, 1 and 10 µg/L. Attention was focused on bee survival, food consumption and oxidative stress. The effects on oxidative stress were assessed by determining the activity of enzymes involved in antioxidant defenses (superoxide dismutase, catalase, glutathione-S-transferase, glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase) in the head, abdomen and midgut; oxidative damage reflected by both lipid peroxidation and protein carbonylation was also evaluated. In general, no significant effect on food consumption was observed. Pesticide mixtures were more toxic than individual substances, and the highest mortalities were induced at intermediate doses of 0.1 and 1 µg/L. The toxicity was not always linked to the exposure level and the number of substances in the mixtures. Mixtures did not systematically induce synergistic effects, as antagonism, subadditivity and additivity were also observed. The tested pesticides, alone and in mixtures, triggered important, systemic oxidative stress that could largely explain pesticide toxicity to honey bees.
... The main task of a mated queen is to sustain colony development and survival via laying eggs [16]. During their lifetime, queens are fed pure royal jelly (RJ) secreted by nurse bees [17] that feed on nectar, pollen, and beebread, which can be contaminated by various pesticides [3,[18][19][20]. Contaminated pollen and nectar can lead to contamination of beeswax, which absorbs lipophilic compounds well [3,5,21,22]. ...
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Various pesticide residues can be found in different bee colony components. The queen larvae of honey bee (Apis mellifera L.) receive non-contaminated food from nurse bees. However, there is little knowledge about how pesticide residues affect developing bees. Additionally, little is known about the migration of lipophilic pesticides between bee matrices. While wax, royal jelly (RJ), and bee larvae are chemically distinct, they all contain lipids and we expected the lipophilic fungicide tebuconazole to be absorbed by different contacting materials. Our aim was to analyze the translocation of tebuconazole residues from queen cell wax to RJ, queen larvae, and newly emerged queens and to evaluate its potential risk to queens. We demonstrated the potential for the migration of tebuconazole from wax to RJ, with a strong dilution effect from the original contamination source. No residues were detected in queen bee larvae and newly emerged queens, indicating that the migration of tebuconazole probably did not directly endanger the queen bee, but there was some risk that tebuconazole might still affect the homeostasis of developing bees.
... Studies in Canada [19] and Switzerland [47] detected the presence of glyphosate in almost all samples, but at values below the MRL of 50 µg/kg [77]. In the Estonian study, although glyphosate was detected in a small number of samples, there were two samples that contained glyphosate levels above the MRL up to 62 µg/kg [79]. In the USA, residues were detected in about 30% of the samples, more than half at levels that were much higher than the MRL, including a sample that was seven times higher than allowed (342 µg/kg) [92]. ...
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Glyphosate is a systemic, broad-spectrum and post-emergent herbicide. The use of glyphosate has grown in the last decades, and it is currently the most used herbicide worldwide. The rise of glyphosate consumption over the years also brought an increased concern about its possible toxicity and consequences for human health. However, a scientific community consensus does not exist at the present time, and glyphosate’s safety and health consequences are controversial. Since glyphosate is mainly applied in fields and can persist several months in the soil, concerns have been raised about the impact that its presence in food can cause in humans. Therefore, this work aims to review the glyphosate use, toxicity and occurrence in diverse food samples, which, in certain cases, occurs at violative levels. The incidence of glyphosate at levels above those legally allowed and the suspected toxic effects of this compound raise awareness regarding public health.
... OSR is regularly treated with fungicides and insecticides during the period of flowering to prevent yield losses by the pathogen Sclerotinia sclerotiorum (Lib.) de Bary and by seed damaging insects like cabbage seedpod weevil, Ceutorhynchus obstrictus Marsham (Coleoptera: Curculionidae), or brassica pod midge, Dasineura brassicae Winnertz (Diptera: Cecidomyiidae). The active substances used for these applications reflect the most frequent residues found in honey (Karise et al. 2017) and bee bread (Rosenkranz et al. 2019), since OSR is an important forage crop for honeybees and other pollinators such as bumble bees or solitary wild bees (Garratt et al. 2014;Hayter and Cresswell 2006;van Reeth et al. 2018;Westphal et al. 2003Westphal et al. , 2009. In flowering OSR, below-canopy spraying with droplegs avoids direct exposure of pollinators with pesticides in the field. ...
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Dropleg sprayers apply pesticides below the flower horizon of oilseed rape plants and thus reduce unwanted side effects on pollinating insects. Whether this technique benefits parasitoids of seed and pollen feeding insect pests has not been studied earlier. To answer this question, we first assessed the vertical distribution of pests and parasitoids using a portable aspirator. In addition, parasitism rates of pollen beetle, Brassicogethes aeneus Fabricius (Coleoptera: Nitidulidae) , by the larval parasitoid Tersilochus heterocerus Thomson (Hymenoptera: Ichneumonidae) were compared in conventional and dropleg sprayed fields over four years (2016–2019), using the neonicotinoids thiacloprid and acetamiprid. Our results show that seed and pollen feeders were mainly found in the flowering canopy, while the predominant location of parasitoids was species-specific. Among pollen beetle parasitoids, Phradis interstitialis Thomson (Hymenoptera: Ichneumonidae) was more abundant below flowering canopy (63% of total catch), whereas T. heterocerus was mainly caught in the flowering canopy (84% of total catch). In the spraying experiments, average parasitism rates of pollen beetles by T. heterocerus ranged between 55 and 82% in the untreated controls. In the dropleg spray treatments, parasitism rates did not differ significantly from control levels, with the exception of thiacloprid application in 2019. In contrast, conventional spray applications resulted in a reduction of parasitism rates by up to 37% compared to the control for at least one of the insecticides in three out of four years. The impact of conventional application differed between years, which may be explained by the temporal coincidence between spray application and the immigration of parasitoids into the crop. We conclude that dropleg spraying exerts lower non-target effects on the main biological control agent of pollen beetle.
Clopyralid is a systemic herbicide used in oilseed rape and other crops. It was found in Danish honey from 2026 in concentrations exceeding the maximum residue level (MRL) of 0.05 mg kg⁻¹. About 50% of the Danish honey is based on nectar from winter oilseed rape. In 2019 and 2020, winter oilseed rape fields were sprayed with clopyralid just before the assigned spraying deadline. At flowering, nectar and pollen samples were collected and the content of clopyralid was measured. Honey and pollen samples were also collected from beehives next to ten conventional winter oilseed rape fields sprayed with clopyralid. Clopyralid was found in nectar and pollen from the experimental fields, and in honey and pollen from beehives next to the conventional fields. For most samples the content in nectar and honey exceeded the MRL. The concentrations found, may not pose any health risk for consumers, as the MRL is based on the original detection limit and not on toxicological tests. However, it can have a significant economically consequence for the beekeepers, who are not allowed to sell the honey if the concentration of clopyralid exceeds 0.1 mg kg⁻¹. Reducing the acceptable applicable rate of clopyralid or implement an earlier deadline for spraying of clopyralid may reduce the risk of contaminating bee food products. However, if it is not possible to obtain a satisfactory effect of clopyralid on the weed flora under these conditions, spraying with pesticides containing clopyralid should be restricted in winter oilseed rape. Determination of an MRL value based on toxicological tests might result in a higher value and makes it acceptable selling the honey containing higher levels of clopyralid.
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The aim of this article is to discuss the phenomenon of food terrorism along with identifying threat factors and determining the potential consequences of its occurrence. Food terrorism is a new phenomenon which poses a challenge for the food industry in the 21st century. It consists in intentional contamination of food using biological, chemical and physical substances. Intentional food contamination may occur at any stage of food production. In order to achieve the goal, a literature study was conducted, on the basis of which it was stated that the issue of food terrorism is a new phenomenon in the economy, while the methods used by terrorists and the potential consequences of the threat are not fully known.
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Experiments linking neonicotinoids and declining bee health have been criticized for not simulating realistic exposure. Here we quantified the duration and magnitude of neonicotinoid exposure in Canadatextquoterights corn-growing regions and used these data to design realistic experiments to investigate the effect of such insecticides on honey bees. Colonies near corn were naturally exposed to neonicotinoids for up to 4 monthstextemdashthe majority of the honey beetextquoterights active season. Realistic experiments showed that neonicotinoids increased worker mortality and were associated with declines in social immunity and increased queenlessness over time. We also discovered that the acute toxicity of neonicotinoids to honey bees doubles in the presence of a commonly encountered fungicide. Our work demonstrates that field-realistic exposure to neonicotinoids can reduce honey bee health in corn-growing regions.
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Honey bees provide critical pollination services for many agricultural crops. While the contribution of pesticides to current hive loss rates is debated, remarkably little is known regarding the magnitude of risk to bees and mechanisms of exposure during pollination. Here, we show that pesticide risk in recently accumulated beebread was above regulatory agency levels of concern for acute or chronic exposure at 5 and 22 of the 30 apple orchards, respectively, where we placed 120 experimental hives. Landscape context strongly predicted focal crop pollen foraging and total pesticide residues, which were dominated by fungicides. Yet focal crop pollen foraging was a poor predictor of pesticide risk, which was driven primarily by insecticides. Instead, risk was positively related to diversity of non-focal crop pollen sources. Furthermore, over 60% of pesticide risk was attributed to pesticides that were not sprayed during the apple bloom period. These results suggest the majority of pesticide risk to honey bees providing pollination services came from residues in non-focal crop pollen, likely contaminated wildflowers or other sources. We suggest a greater understanding of the specific mechanisms of non-focal crop pesticide exposure is essential for minimizing risk to bees and improving the sustainability of grower pest management programs.
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Cytochrome P450 monooxygenases (P450) in the honey bee, Apis mellifera, detoxify phytochemicals in honey and pollen. The flavonol quercetin is found ubiquitously and abundantly in pollen and frequently at lower concentrations in honey. Worker jelly consumed during the first 3 d of larval development typically contains flavonols at very low levels, however. RNA-Seq analysis of gene expression in neonates reared for three days on diets with and without quercetin revealed that, in addition to up-regulating multiple detoxifying P450 genes, quercetin is a negative transcriptional regulator of mitochondrion-related nuclear genes and genes encoding subunits of complexes I, III, IV, and V in the oxidative phosphorylation pathway. Thus, a consequence of inefficient metabolism of this phytochemical may be compromised energy production. Several P450s metabolize quercetin in adult workers. Docking in silico of 121 pesticide contaminants of American hives into the active pocket of CYP9Q1, a broadly substrate-specific P450 with high quercetin-metabolizing activity, identified six triazole fungicides, all fungal P450 inhibitors, that dock in the catalytic site. In adults fed combinations of quercetin and the triazole myclobutanil, the expression of five of six mitochondrion-related nuclear genes was down-regulated. Midgut metabolism assays verified that adult bees consuming quercetin with myclobutanil metabolized less quercetin and produced less thoracic ATP, the energy source for flight muscles. Although fungicides lack acute toxicity, they may influence bee health by interfering with quercetin detoxification, thereby compromising mitochondrial regeneration and ATP production. Thus, agricultural use of triazole fungicides may put bees at risk of being unable to extract sufficient energy from their natural food.
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Background: Neonicotinoid insecticides have been identified as an important factor contributing to bee diversity declines. Nonetheless, uncertainties remain about their impact under field conditions. Most studies have been conducted on Apis mellifera and tested single compounds. However, in agricultural environments, bees are often exposed to multiple pesticides. We explore synergistic mortality between a neonicotinoid (clothianidin) and an ergosterol-biosynthesis-inhibitor fungicide (propiconazole) in three bee species (A. mellifera, Bombus terrestris, Osmia bicornis) following oral exposure in the laboratory. Results: We developed a new approach based on the binomial proportion test to analyze synergistic interactions. We estimated uptake of clothianidin per foraging bout in honey bees foraging on seed-coated rapeseed fields. We found significant synergistic mortality in all three bee species exposed to non-lethal doses of propiconazole and their respective LD10 of clothianidin. Significant synergism was only found in the first assessment times in A. mellifera (4 and 24 h) and B. terrestris (4 h), but persisted throughout the experiment (96 h) in O. bicornis. Osmia bicornis was also the most sensitive species to clothianidin. Conclusion: Our results underscore the importance to test pesticide combinations likely to occur in agricultural environments, and to include several bee species in environmental risk assessment schemes.
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Recent efforts to evaluate the contribution of neonicotinoid insecticides to worldwide pollinator declines have focused on honey bees and the chronic levels of exposure experienced when foraging on crops grown from neonicotinoid-treated seeds. However, few studies address non-crop plants as a potential route of pollinator exposure to neonicotinoid and other insecticides. Here we show that pollen collected by honey bee foragers in maize- and soybean-dominated landscapes is contaminated throughout the growing season with multiple agricultural pesticides, including the neonicotinoids used as seed treatments. Notably, however, the highest levels of contamination in pollen are pyrethroid insecticides targeting mosquitoes and other nuisance pests. Furthermore, pollen from crop plants represents only a tiny fraction of the total diversity of pollen resources used by honey bees in these landscapes, with the principle sources of pollen originating from non-cultivated plants. These findings provide fundamental information about the foraging habits of honey bees in these landscapes.
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The present study on organophosphate deals with the reports on pollution and toxicity cases throughout India. The use of pesticides was introduced in India during the 1960s which are now being used on a large scale and represents the common feature of Indian agriculture. Use of organophosphates as a pesticide came as an alternative to chlorinated hydrocarbons due to their easy degradability. Although these xenobiotics degrade under natural condition, their residues have been detected in soil, sediments, and water due to their non-regulated usage practice. The over-reliance on pesticides has not only threatened our environment but contaminations of organophosphate residues have been also detected in certain agricultural products like tea, sugars, vegetables, and fruits throughout India. This paper highlights many of the cases where different organophosphates have been detected exceeding their respective MRL values. Some organophosphates detected are so hazardous that even WHO has listed them in class 1a and class 1b hazardous group. Presence of their residues in blood, milk, honey, and tissues of human and animals revealed their excessive use and bioaccumulating capabilities. Their intentional or unintentional uptake is causing thousands of deaths and severity each year. Most of the toxicity cases presented here are due to their uptake during a suicidal attempt. This shows how easily these harmful substances are available in the market.
The aim of the study was to provide a comprehensive overview of neonicotinoid pesticide residues in honey samples for a single country and compare the results with the import data for neonicotinoid pesticides. The levels of four neonicotinoid pesticides, namely thiamethoxam, imidacloprid, acetamiprid, and thiacloprid, were determined in 294 honey samples harvested from 2005 to 2013 from more than 200 locations in Estonia. For the analyzed honey samples, 27% contained thiacloprid, and its levels in all cases were below the maximum residue level set by the European Union. The other neonicotinoids were not detected. The proportion of thiacloprid-positive samples for different years correlates well with the data on thiacloprid imports into Estonia, indicating that honey contamination with neonicotinoids can be estimated based on the import data.
The presence of even tiny quantities of pesticide residues in honey, a traditional healthy product, is a matter of concern for producers, packers and consumers. This paper aims to quantify pesticides in retail brands of polyfloral honey, and to calculate the mixture risk assessment of honey for consumers according to the results obtained from the analysed samples. A LC-MS/MS multi-residue method was developed and validated for 13 compounds: 11 pesticides (chlorfenvinphos, coumaphos, tau-fluvalinate, amitraz, which are very common in veterinary treatments, and imidacloprid, acetamiprid, simazine, cyproconazole, tebuconazole, chlorpiryphos-methyl, chlorpiryphos, widely used in agricultural practices), and 2 metabolites of amitraz (2,4-DMA and 2,4-DMF). Results showed that the samples contained pesticide residues at different concentrations; however, the MRL in honey for each of the 11 pesticides was never exceeded. The most common were amitraz (from 1 to 50 μg/kg) present in 100% of the samples, and coumaphos (up to 14 μg/kg) in 63%. The hazard index (HI) for adults was less than 0.002 in all cases, a long way from 1, the value established as the limit of acceptability. Therefore, commercial honey does not represent any significant risk to health. However, considering that residue levels should be present “as low as reasonably achievable” it is deemed necessary to make an effort to reduce their presence by appropriate agricultural and, above all, beekeeping practices due to acaridae treatments.