Science of the Total Environment 485486 (2014) 633642

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Science of the Total Environment

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Pesticide residues in honeybees, honey and bee pollen by LCMS/MS

screening: Reported death incidents in honeybees
Konstantinos M. Kasiotis a, Chris Anagnostopoulos b, Pelagia Anastasiadou a, Kyriaki Machera a,
a
Benaki Phytopathological Institute, Department of Pesticides Control and Phytopharmacy, Laboratory of Pesticides' Toxicology, 8 St. Delta Street, Kissia, 14561 Athens, Greece
b
Benaki Phytopathological Institute, Department of Pesticides Control and Phytopharmacy, Laboratory of Pesticides' Residues, 8 St. Delta Street, Kissia, 14561 Athens, Greece

H I G H L I G H T S

Reported cases of honeybee death incidents were investigated.

An LCESI-MS/MS pesticide multiresidue method was therefore developed.
Pesticide residues were detected in honeybees, honey and bee pollen.
Concentrations in honeybees ranged from 0.3 to 81.5 ng/g.
The reported method is sufcient as a monitoring tool for pesticide residues.

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

Article history: The aim of this study was to investigate reported cases of honeybee death incidents with regard to the potential
Received 9 January 2014 interrelation to the exposure to pesticides. Thus honeybee, bee pollen and honey samples from different areas of
Received in revised form 11 March 2014 Greece were analyzed for the presence of pesticide residues. In this context an LCESI-MS/MS multiresidue
Accepted 11 March 2014
method of total 115 analytes of different chemical classes such as neonicotinoids, organophosphates, triazoles,
Available online 17 April 2014
carbamates, dicarboximides and dinitroanilines in honeybee bodies, honey and bee pollen was developed
Keywords:
and validated. The method presents good linearity over the ranges assayed with correlation coefcient values
Pesticides r2 0.99, recoveries ranging for all matrices from 59 to 117% and precision (RSD%) values ranging from 4 to
Multiresidue method 27%. LOD and LOQ values ranged for honeybees, honey and bee pollen from 0.03 to 23.3 ng/g matrix weight
LCMS/MS and 0.1 up to 78 ng/g matrix weight, respectively. Therefore this method is sufcient to act as a monitoring tool
Honeybee for the determination of pesticide residues in cases of suspected honeybee poisoning incidents. From the analysis
Bee pollen of the samples the presence of 14 active substances was observed in all matrices with concentrations ranging for
Honey honeybees from 0.3 to 81.5 ng/g, for bee pollen from 6.1 to 1273 ng/g and for honey one sample was positive to
carbendazim at 1.6 ng/g. The latter conrmed the presence of such type of compounds in honeybee body and
apicultural products.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Honeybee'sdeath incidents are of great concern, because declines in
bee populations in the USA mainly (Burkle et al., 2013; Pettis and
Delaplane, 2010; Tylianakis, 2013a,b) and in Europe in lesser extent
might have detrimental impact on agriculture and environment. They
could affect for some crops, pollination and disturb the stability of the
agricultural ecosystems. Research organizations and governments
have therefore inaugurated national monitoring schemes and conduct-
ed numerous studies, to explain the losses. Honeybees forage in a wide
Corresponding author at: Laboratory of Pesticides Toxicology, Benaki Phytopathological
Institute, 8 St. Delta Street, Kissia, 14561 Athens, Greece. Tel.: + 30 210 8180339;
fax: + 30 210 8180223.
E-mail address: k.machera@bpi.gr (K. Machera).
range, a fact that enables them to come in contact with contaminated food
sources, such as pollen and nectar as well as water (Bromenshenk and
Preston, 1986; Porrini et al., 2003; Raeymaekers, 2006).
The use of pesticides in agricultural cropping systems is often
discussed as a factor inuencing bee health (Johnson et al., 2010). Single
events of poisonings by spray applications have been reported in many
countries [indicatively in Greece (Bacandritsos et al., 2010)], usually due
to misuse of products resulting in contamination of nectar and pollen.
Additionally beehives are treated with insecticides such as coumaphos
to protect honeybee against parasites as Varroa jacobsoni (Elzen et al.,
2000). There are different ways through which honeybees can be ex-
posed to these substances (Krupke et al., 2012); through pollen and
nectar collected and stored in the hive (Chauzat and Faucon, 2007;
Chauzat et al., 2006), through the exudation excreted from the plants
(Girolami et al., 2009), through surface water (Van Dijk, 2010), through

http://dx.doi.org/10.1016/j.scitotenv.2014.03.042
0048-9697/ 2014 Elsevier B.V. All rights reserved.
634 K.M. Kasiotis et al. / Science of the Total Environment 485486 (2014) 633642
air contamination via drift of dust in coated seeds during sowing
(Greatti et al., 2003) and through extra oral secretions that are pro-
duced from some plants (Mizell, 2009) such as sunower and cotton.
Besides the reduction of the quality of hive products, and the effects
on bees' behavior, the accumulation of insecticides in honeybee tissue
can also inuence honeybees' health and the colony's population devel-
opment, as they can act as stressors for the entire colony. The recent
turmoil on the neonicotinoid insecticides resulted in the proposal of
the European Commission to restrict the use of 3 neonicotinoids
(clothianidin, imidacloprid and thiamethoxam) for seed treatment,
soil application (granules) and foliar treatment on bee attractive plants
and cereals (Clothianidin, 2013; Imidacloprid, 2013; Thiamethoxam,
2013).
Research in attributing death incidents is really troublesome. The
latter is further explained considering that honeybee deaths and popu-
lation decline are a multifactorial issue (OPERA, 2013). As regard to
pesticides it is difcult to collect info on where bees were foraging and
any pesticide application in a logical radius from the incidence. The
degrading of residues or their washing off by rain is also parameters
which if not accurately evaluated might mislead researchers and in-
vestigators. Numerous studies have demonstrated that concentra-
tion of various insecticides can seriously affect honeybees' health
and behavior. Indicatively Williamson and Wright have proven that
exposure to sub-lethal doses of combined cholinergic pesticides
signicantly impairs important behaviors involved in foraging of
honeybees (Williamson and Wright, 2013). Gene expression in hon-
eybee larvae was also veried to be seriously affected by pesticide
exposure (Gregorc et al., 2012). recent work on pesticide exposure
of honeybees indicated that this exposure can lead to increased level
of Nosema pathogen (Pettis et al., 2012), implying strong synergistic
effects. All these ndings signify the necessity to use multi-residue
analytical methods, with substantial sensitivity so as to detect as
much as possible analytes.
A recent publication showed that over 150 different pesticides can
be found in colony samples (Mullin et al., 2010). Likewise, honeybees
are subjected to a large range of compounds, including pesticides and
also antibacterial substances that can be used by beekeepers. The ana-
lytical determination of pesticides although for many research groups
can be considered a routine procedure, it still constitutes a major chal-
lenge especially due to the increasing demand for low limits of detection
(LODs) and the complexity of the matrices. The requirement of low
LODs is linked to bee toxicity since the honeybee oral LD50 and contact
LD50 are in the ng/g scale for many pesticides. Thus liquid chromatogra-
phy tandem mass spectrometry (LCMS/MS) has been used by various
researchers (indicatively see Fidente et al., 2005; Garcia-Chao et al.,
2010; Kamel, 2010; Pirard et al., 2007; Schoning and Schmuck, 2003)
due to its proved efciency in pesticide residue analysis. One of the
most widespread extraction techniques to analyze an extensive range
of pesticides is QuEChERS (stands for, Quick Easy Cheap Effective Rug-
ged Safe) introduced by Anastassiades in 2003 (Anastassiades et al.,
2003). The QuEChERS technique and its various modications demon-
strated satisfactory recovery of imidacloprid and other neonicotinoids
from complex matrices such as honeybees, honey, and pollen (indica-
tively see Blasco et al., 2011; Kamel, 2010).
Specically, analysis of contaminants in honeybees is a challenging
issue due to the presence of beeswax, proteins and other interfering
compounds into the matrix. Hence, a good sample preparation is essen-
tial so as to suppress matrix effect and its provoking impact on analyses.
Until now some research groups have described multiresidue analytical
methods in honeybees. An exemplifying work was published by
Walorczyk and Gnusowski in 2009 where 150 analytes were moni-
tored by GCMS/MS (Walorczyk and Gnusowski, 2009). The procedure
involved a main dispersive solid phase extraction (SPE) step, using prima-
ry secondary amine (PSA), octadecyl (C18) and graphitized carbon black
(GCB). An additional important publication on a multiresidue method
monitoring 80 environmental contaminants including pesticides in
honeybees, pollens and honey was reported by Wiest et al. (2011). The
authors presented a method based on modied QuEChERS followed by
GCTOF and LCMS/MS analysis. The addition of a small fraction of
hexane to acetonitrile fraction as also reported by Przybylski and
Segard (2009) in other commodities was implemented for honey-
bees, honey and pollen to eliminate lipids. In 2010 Kamel presented a
rened methodology for the determination of neonicotinoids and
their metabolites in honeybees and bee products using LCMS/MS
(Kamel, 2010). The breakthrough of this paper was the concomitant
detection of neonicotinoids and their metabolites. The latter is be-
coming important since some metabolic products exhibited LD 50
values similar to their parent compounds rendering their detection
imperative.
Neonicotinoids have also been analyzed by Martel and Lair in
honeybees' bodies (Martel and Lair, 2011). Prior to LCMS/MS, ex-
traction of analytes was achieved with acetonitrile and partitioning
with hexane and a cleanup step with Florisil. LOQ values ranged
from 0.5 to 1 ng/g bw of honeybee. Optimized dispersive liquidliquid
microextraction combined with LCMS/MS was published recently
as an efcient method for the detection of neonicotinoid insecticides
in honey matrix (Jovanov et al., 2013). The method can be applied
under routine laboratory conditions for analyses of real honey
samples.
Analytical determination of neonicotinoids in honey has recently
been reported by Tanner and Czerwenka (2011). The method was
then applied in Austrian honeys showing that a substantial number of
samples contained 3 neonicotinoids (mainly thiacloprid and in lesser
extent acetamiprid and thiamethoxam). Recently Yanez et al. published
a work on the fast determination of imidacloprid in beeswax by LCESI-
MS/MS. The method was then applied to the analyses of beeswax sam-
ples collected from Spanish apiaries (Yanez et al., 2013a), with one real
sample being contaminated with traces of imidacloprid of 40 g/kg. The
latter indicates that this research eld is of great importance consid-
ering that hives can be contaminated with toxic substances. Last but
not least Lozowicka published a research study on the detection of
150 pesticides monitoring bee poisoning incidents. The author uti-
lized a dual GCNPDECD by using a diverter for simultaneous deter-
mination of compounds by two detectors. The latter is the rst
published work where two detectors in gas chromatography are
combined to monitor pesticide residues in honeybee bodies (Lozowicka,
2013).
The presented study was pursued in the frames of the necessity to
monitor pesticide residues in honeybee bodies, after relatively constant
incidents that have taken place in Greece since 2011 till the middle of
2013 and determine pesticide residues on bee products. The possibility
of detecting more than one substance especially in honeybee bodies in-
trigued us to include as much as possible analytes of various classes, a
fact that positions this LCMS/MS method amongst the studies with
the highest encompassed analytes, especially for honeybees. The latter
is important considering that the biological effects of two or more
toxic substances can be different in kind and degree from those of one
of the substances alone due to synergistic and potentiating effects
(Schmuck et al., 2003). The method was also applied in pollen and
honey due to the provision by individuals of respective samples in
order to be screened for pesticides.
Consequently, this paper presents the validation of quantitative de-
termination of various pesticides in honeybees, bee pollen and honey
by LCESI-MS/MS using modications of the QuEChERS technique,
based on combinations of bibliographical work and laboratory observa-
tions. The method for some pesticides which were previously ana-
lyzed by other groups in these matrices as demonstrated by the
results, exhibited slightly lower LOD and LOQ values that render it
amongst the most sensitive of its category. The developed validated an-
alytical method was used for the analysis of real honeybee, honey and
bee pollen samples revealing interesting results as regards the pesti-
cides' load of these matrices. The latter should be attentively regarded
 K.M. Kasiotis et al. / Science of the Total Environment 485486 (2014) 633642
by authorities so as to shield bee health and pollination by effective
measures that will also consider a sustainable plant protection program
assuring viable agricultural production.
2. Materials and methods
2.1. Chemicals and solutions
Certied pesticide standards (purity N 90%) were purchased from
Sigma-Aldrich (Bchs, Switzerland), Dr. Ehrenstorfer GmbH (Augsburg,
Germany), and ChemService (Milan, Italy). Active substances were:
acetamiprid, aclonifen, alachlor, ametryn, atrazine, azimsulfuron,
azoxystrobin, benalaxyl, benfuracarb, bensulfuron-methyl, bifenthrin,
boscalid, bupirimate, buprofezin, cadusafos, carbendazim, carbofuran,
carboxin, chlorpyrifos, chlorsulfuron, clethodim, clofentezine,
clothianidin, coumaphos, cyhalothrin lambda, cyproconazole 1,
cyproconazole 2, cyprodinil, demeton-S-methyl, demeton-S-methyl sulf-
oxide, desmetryn, diazinon, diethofencarb, difenoconazole, dimethoate,
dimethomorph, disulfoton, disulfoton sulfoxide, dodemorph, EPN, ethion,
ethoprophos, famoxadone, fenamidone, fenamiphos, fenamiphos sulfone,
fenazaquin, fenbuconazole, fenpropathrin, fenpropimorph, fenthion,
fenthion-sulfone, fenthion-sulfoxide, pronil, pronil sulfone, udioxonil,
usilazole, fosthiazate, haloxyfopethyl ester, imazalil, imidacloprid,
indoxacarb, iprodione, iprovalicarb, isoproturon, linuron, malaoxon,
malathion, mepanipyrim, metalaxyl M, metconazole, methidathion,
methoxyfenozide, metoxuron, metribuzin, metsulfuron methyl,
monocrotophos, monolinuron, myclobutanil, nicosulfuron, oxadiazon,
paraoxon methyl, penconazole, pencycuron, pendimethalin, phosalone,
phosmet, phosphamidine, phoxim, piperonyl butoxide, pirimicarb,
pirimiphos methyl, prometryn, propyconazole, prothioconazole desthio,
pyraclostrobin, pyrazophos, pyrimethanil, quinoxyfen, spinosad A,
spiroxamine, tebuconazole, tebufenozide, terbufos, terbuthylazine,
tetraconazole, thiabendazole, thiacloprid, thiamethoxam thifensulfuron
methyl, thiodicarb, triazophos, tricyclazole, trioxystrobin, triticonazole
and zoxamide.
All solvents, namely acetonitrile, methanol and water were of HPLC
grade. A single composite working standard solution at 100 g mL1
was prepared in methanol and stored at 20 C. From this working
standard solution, working solutions at different concentrations were
prepared by serial dilution in acetonitrile. Methanol, acetonitrile and
water were purchased from Merck (Darmstadt, Germany) and were
LCMS grade. Magnesium sulfate anhydrous (MgSO4) and primary
secondary amine (PSA) were purchased from Agilent Technologies
while sodium acetate (NaOAc) from Panreac Quimica SAU (Barcelona,
Spain).
2.2. Sample collection-regions with incidents
Control matrix samples (honeybees, bee pollen and honey) were
provided by individual beekeepers (from beehives of organic apiculture
origin) and previously checked for interferences of the analytes of the
method. The investigated samples were collected by individuals or pub-
lic authorities who evidenced the losses or bee death incidents. Honey-
bee samples were collected very near or at the entrance of the hives. Bee
pollen was collected from the hives. Areas of Greece where incidents
were reported and subsequent sampling was performed are presented
in Fig. 1 and in details in Table S12 (Supplementary material). In the ma-
jority of cases areas were rural with substantial agricultural activity.
Special precaution was given to the transportation of the samples espe-
cially for bees. The samples were immediately cooled at 0 C with ice-
packs or at 78 C with dry ice (if available) to avoid degradation of
active substances therein, packed and sent the same or early next day
depending on the distance of the incident from the laboratory. After
reaching laboratory, the samples were stored at 20 C until analysis.
635
2.3. LCMS/MS instrumentation, chromatographic and mass spectrometry
conditions
An Agilent Technologies 6410 Triple Quad LC/MS system was used,
equipped with a Parker Nitrogen Generator (NitroFlowLab). The LC sep-
aration was achieved after injecting 10 L of sample on a reversed phase
column (ZORBAX Eclipse XDB-C18 Agilent, 2.1 150 mm, 3.5 ) using a
gradient system consisting of: A) Water with 5 mM ammonium for-
mate, 0.1% formic acid, and 0.02% acetonitrile and B) methanol
with 5 mM ammonium formate and 0.1% formic acid. The ow rate
was set at 0.3 mL min 1 and the column gradient program consisted
of 02 min 20% B, 212 min ramped linearly from 20 to 60% B,
1230 min linearly from 60 to 100% B, and 3035 min 100% B. Then gra-
dient system was ramped linearly up to 20 min at 100% concentration. A
post-time of 10 min at initial conditions was applied. The mass spec-
trometer was operated in Multiple Reaction Monitoring (MRM) mode
with positive and/or negative Electron Spray Ionization (ESI). Pesticides
have been analyzed using one injection in the LCESI-QqQ-MS/MS sys-
tem except pronil, pronil sulfone and udioxonil which were identi-
ed/quantitated in the negative ionization mode (ESI) and needed
separate injection and analysis. Nitrogen was used as nebulizer and
collision gas. For instrument control, Agilent Mass Hunter data ac-
quisition Triple Quad B.01.04 and for data processing Agilent
MassHunter Workstation Qualitative Analysis B.01.04. were used.
2.4. Sample preparation
This part is described in detail in Supplementary part. Extraction and
purication steps were based on recently published works on honey-
bees, pollen and honey. The rst work was reported by Kamel in 2010
(Kamel, 2010) and the other reported by Wiest et al. in 2011 both mod-
ifying QuEChERS procedure (Wiest et al., 2011). Briey, in current work
the matrix was homogenized with acetonitrile (with and without Et3N),
deionized water and hexane [hexane added to remove lipids as in
Przybylski and Segard procedure (Przybylski and Segard, 2009), the
named triple partitioning extraction step]. Then the mixture was
vortex-mixed with MgSO4, NaOAc and PSA, and centrifuged. The aceto-
nitrile phase was again shaken with a PSA and MgSO4 mixture and nal-
ly solid-phase extracted. For honey matrix a mild preheating of the
matrix in a water-bath controlled at 30 C was applied for better ho-
mogenization during spiking procedure.
2.5. Validation procedure scheme
The developed method was validated following principally the In-
ternational Conference on Harmonization (ICH) guideline (ICH, 2005).
The validation scheme was conducted on three days with concentration
ranges between 0.5 and 700 ng/g regarding honeybees, honey and pol-
len. Specically each day 9 samples of control blank matrix with 9 levels
of concentration were extracted to determine linearity and intermedi-
ate precision. In the case of one sample where the concentration
exceeded the 700 ng/g the bracketing method and/or the single point
calibration approach was used. Two more samples of control blank
matrix spiked with one level in each day were extracted to determine
repeatability (n = 3). Three samples of blank matrix were spiked at
same levels as for repeatability to determine recoveries. Concentrations
given, in spiked and real samples, refer to fresh weight basis of each
matrix.
Other method validation schemes were also considered such as the
Bioanalytical Method Validation Guidance for Industry document
(US Department of Health and Human Services, FDA) (Validation) and
the Document No. SANCO/12495/2011(SANCO, 2011). The fundamen-
tal parameters assessed were accuracy, precision, selectivity, sensitivity
and reproducibility. It should be stressed that although an internal stan-
dard is recommended in most of the validation guidelines it was not
636 K.M. Kasiotis et al. / Science of the Total Environment 485486 (2014) 633642
Fig. 1. Distribution of incidents reported in Greece.
necessary in this study due to the precision and trueness achieved (see
also Yanez et al., 2013a).
The method limit of detection (LOD) was determined as the lowest
concentration tested in which the peak signal was three times the
background noise from the chromatogram in both transitions and the
method limit of quantication (LOQ) was determined as the lowest
concentration tested in which the peak signal was ten times the back-
ground noise from the chromatogram regarding the quantitation tran-
sition and the ion ratio is consistent with the respective ratio of a
standard.
Criteria of validation were as follows: regression coefcient higher
than 0.99 for linearity, recoveries between 60 and 120%, RSD lower than
20% for repeatability and lower than 25% for reproducibility. In
Table 2 a summary of calibration line parameters for the pesticides
targeted is presented. Results of validation are presented in Tables 23
(and Tables S2S5 in Supplementary material for pollen and honey
respectively).
2.6. Assessment of matrix effect
In order to estimate if the matrix inuences in a signicant degree
the peak area and therefore the sensitivity of the analytes, the slopes
of the calibration lines obtained for the 3 matrices (indicatively for
spiked honeybees see Tables 8 and 9, Supplementary material), and
the solvent (Tables 1011, Supplementary material) were compared
using a Student t-test. The tcal is dened in the following equation:
jb1 b2 j
tcal q
S2b1 S2b2
with b the slope of the calibration line and Sb the standard deviation of
the slope.
If the theoretical value (ttheo) of 2.145 (df = 9 + 9 4 = 14, for two-
sided critical region, at probability 95%) exceeds the calculated tcal value,
 K.M. Kasiotis et al. / Science of the Total Environment 485486 (2014) 633642 637

Table 1 3.1.2. The Et3N approach

Distribution of samples per year, frequency of detection of PPP and concentrations. The use of Et3N proved to be efcient for some pesticides which
2011 Samples Honeybees Pollen Honey were also detected using the non-Et3N method. According to Kamels'
work the use of 2% Et3N in ACN aids the elution of neonicotinoid metab-
Number of samples analyzed 17 10 7
Number of positive samples 9 4 olites. In our case considering that we have not included these metab-
olites we observed that this extraction/eluting mixture aids also the
PPP Frequency of PPP Concentration elution of parent neonicotinoids and some other analytes with better
detection (N0) range ng/g
chromatographic performance in respect to the non-basic medium
Clothianidin 8 4 0.714.7 bees (indicatively see Figures S1a, b and S2a, b for comparison). In this con-
6.169.04 pollen text we applied the experimental protocol with the addition of Et3N in
Imidacloprid 2 1 0.35.74 bees
72 pollen
the extractionelution steps when residues of these substances by the
primary method were detected. The Et3N approach worked satisfactory
2012 Samples Honeybees Pollen Honey for clothianidin, thiamethoxam, imidacloprid and for carbendazim. For
Number of samples analyzed 19 4 5 practical reasons we decided for the Et3N method to present only the
Number of positive samples 17 2 1 four compounds that are quantitated using this method, since all
other analytes are quantitated using the rst method. From the chro-
PPP Frequency of PPP Concentration matographic point of view it is evident that peak shape and symmetry
detection (N0) range ng/g
were improved when that approach was followed. Et3N is widely used
Clothianidin 11 2 2.739.9 bees as an additive in mobile phase that reduces peak tailing and as an ion-
308.31273 pollen
pairing agent that alters selectivity in reverse phase HPLC separations.
Chlorpyrifos ethyl 3 9.146
Imidacloprid 1 73.9 In our case Et3N is neither a mobile phase additive nor an ion-pair re-
Thiamethoxam 1 14.4 agent; its existence in the vial prior to injection provides basic pH con-
Indoxacarb 1 15.2 ditions. The latter is important for basic or weak basic compounds [e.g.
Thiacloprid 1 bLOQ
carbendazim is a weak base (pka = 4.2), clothianidin is also a strong
Penconazole 1 47.4
Carbendazim 1 1.62
base (pKa = 11.1); for pka values see http://sitem.herts.ac.uk/aeru/
Trioxystrobin 1 58 iupac/] that in acidic pH undergo protonation which has been shown
Dimethoate 1 144.5 to produce broad, tailing peaks. However for base-labile pesticides,
Fipronil 1 81.5 such as the organophosphorous pesticides, the principal method is se-
Fipronil sulfone 1 79.1
lected due to observed degradation of these compounds.
2013 Samples Honeybees Pollen Honey
3.1.3. Pesticide concentrations in samples
Number of samples analyzed 8 1
Number of positive samples 6 In total, 71 samples of honeybees, bee pollen and honey from indi-
vidual beekeepers or other authorities (e.g. veterinary institutes) were
PPP Frequency of PPP Concentration sent to Benaki Phytopathological Institute, Laboratory of Pesticides Tox-
detection (N0) range ng/g icology usually after the report of honeybee death incidents during
Chlorpyrifos ethyl 1 bLOQ 2011, 2012 and 2013 [63% honeybees (44), 19% bee pollen (14), and
Clothianidin 2 6.16.8 18% honey (13)]. The distribution of samples per year, frequency of
Phoxim 1 750
plant protection product (PPP) detection and concentration levels
Thiamethoxam 3 0.549.6
Coumaphos 2 bLOQ20.3 are presented in Table 1 and for honeybees in graphs 1ac. Scarcely
honey or pollen samples have been sent independently from honey-
bees' death incidents.
The analyses conrmed the presence of pesticide residues in the
the null hypothesis, that there is no signicant difference between the samples received. The results show that 73% of honeybees, 43% of pollen
two calibration lines, is accepted and therefore the matrix effect is not and 0.1% of honey samples are positive to at least one PPP. For bees, in-
signicant. dicatively 22 samples contained clothianidin (50% of all honeybee sam-
ples analyzed), 6 chlorpyrifos ethyl (14%), 4 thiamethoxam (9%), 2
imidacloprid (4.5%) and 2 samples of coumaphos (4.5%). In Fig. 2, indic-
3. Results ative graphs for honeybees' distribution of pesticides from 2011 to 2013
are depicted. In y axis the number of samples is presented while in
3.1. Method development x-axis the pesticides detected are presented. The rst two bars in x-axis
refer to the total number of samples examined and number of positive
3.1.1. Calibration and matrix effect samples respectively.
It is a common ground that LCESI-MS/MS is prone to strong matrix
effect due to ion suppression. In order to include the error due to matrix 4. Discussion
effect in measurements, matrix-matched calibrations standards (MMCS)
were employed, due to cost-effectiveness and limited availability of 4.1. Pesticide residues in honeybees and honeybee products
many isotope labeled standards of the studied analytes. Matrices used
as blanks were previously analyzed so as to ensure the absence of analytes In Greece, a limited number of intoxication reports for honeybees
in the samples. The correction of matrix effect accomplished by MMCS were encountered. In 1997, Antoniou et al. reported an overview of
implies the fact that ion suppression of the analytes is similar in both sam- fatal animal poisonings (including bees) in Northern Greece covering
ples and MMCS. The latter was veried by performing MMCS in various a period from 1990 to 1995 (Antoniou et al., 1997). The results showed
types of matrices. A signicant difference was observed in all cases, except that the highest percentage of bee poisoning was attributed to pesti-
trioxystrobin, between the slopes of the calibration lines (see Tables S6 cides, with carbamates and organophosphates being the most common
S9, Supplementary material) which means that matrix effect is present groups. In 2010 Bacandritsos et al. published results after sudden deaths
and that quantitation should be conducted with matrix matched calibra- and honeybee colony population decline (Bacandritsos et al., 2010),
tion standards in order to have reliable and accurate quantitation. that occurred in the region of Peloponnesus (Mt. Mainalo). A virus
638 K.M. Kasiotis et al. / Science of the Total Environment 485486 (2014) 633642

Table 2
Method performance and validation: limits of detection (LODs) and quantication (LOQ), recoveries (%), repeatability (RSD%) and inter-day (inter-d) precision (RSD%) obtained in
honeybees.

Analyte LOD (ng/g) LOQ (ng/g) Calibration range (ng/g) Recovery RSD% Inter-d precision

n=3 RSD% n = 3

4 ng/g 50 ng/g 700 ng/g 4 ng/g 50 ng/g 700 ng/g

Acetamiprid 0.3 1.1 0.5700 88 7 85 9 90 14 8 14 16

Azoxystrobin 0.1 0.3 1700 72 7 69 6 70 10 10 15 14
Ametryn 0.2 0.6 68 9 71 11 70 15 21 17 14
Benalaxyl 4.0 13.4 80 10 74 6 74 12 12 15 18
Cadusafos 0.5 1.7 82 6 79 11 80 8 6 8 7
Diazinon 3.4 11.3 102 14 84 8 78 10 7 11 14
Difenoconazole 0.3 1.1 71 10 68 8 90 11 8 12 10
Flusilazole 0.2 0.7 90 5 80 6 79 11 11 17 19
Methoxyfenozide 0.1 0.3 85 15 78 8 80 10 9 15 18
Thiacloprid 0.1 0.4 90 15 84 9 79 13 8 17 20
Triazophos 0.3 1.1 82 20 84 17 78 10 10 11 19
Azimsulfuron 0.4 1.2 2700 105 21 100 25 95 18 12 8 10
Clofentezine 0.9 3.1 85 19 69 10 65 10 9 10 9
Fenazaquin 9.3 30.9 88 20 79 10 65 10 12 20 20
Fenbuconazole 0.3 1 98 21 85 17 80 12 10 15 16
Fenthion-sulfoxide 0.1 0.3 70 15 85 10 83 22 17 15 18
Monolinuron 0.4 1.2 99 10 70 17 75 12 8 10 11
Penconazole 2.0 6.8 85 4 70 7 75 5 16 12 14
Pencycuron 3.3 11.1 83 4 73 5 80 11 15 18 19
Pendimethalin 13.4 44.8 77 4 65 5 67 11 9 10 7
Pirimicarb 0.8 2.6 79 10 70 5 72 8 12 11 14
Pirimiphos-methyl 0.4 1.3 90 11 78 17 82 10 6 5 14
Prometryn 0.03 0.1 78 5 67 10 65 9 5 6 6
Pyraclostrobin 1.7 5.8 85 4 80 16 81 8 9 9 15
Spinosad A 0.2 0.6 71 10 68 8 60 8 5 7 8
Spiroxamine 0.1 0.4 74 6 66 8 64 10 5 12 20
Tebufenozide 0.7 2.4 81 16 82 8 78 8 6 7 8
Thiabendazole 0.2 0.6 110 9 98 11 96 17 13 20 19
Trioxystrobin 8.6 28.7 85 18 74 8 73 4 13 10 21
Atrazine 4.0 13.3 4700 76 7 71 14 69 8 13 14 10
Boscalid 3.6 12.0 69 12 75 10 65 15 12 11 14
Bupirimate 2.2 7.5 77 7 72 5 80 8 10 8 20
Buprofezin 8.7 28.9 61 6 64 5 62 10 7 9 14
Carbofuran 0.4 1.4 70 11 71 14 68 21 5 5 8
Chlorsulfuron 1.7 5.8 109 7 85 7 80 7 17 14 11
Coumaphos 4 13.1 100 12 93 7 91 13 12 20 19
Cyprodinil 2.4 7.9 89 20 90 18 79 25 10 18 21
Ethion 6.6 22 71 13 65 10 73 9 10 9 14
Fenamiphos 3.0 10 80 6 82 5 77 4 13 15 11
Fenamiphos sulfone 0.1 0.5 91 12 87 8 90 15 20 17 22
Haloxyfopethyl ester 2.1 6.9 78 9 72 12 68 15 13 15 11
Imazalil 2.2 7.2 115 21 91 12 95 13 12 20 21
Iprodione 1.1 3.7 75 8 73 10 70 14 13 22 23
Iprovalicarb 0.6 2.0 87 8 75 14 77 20 10 14 21
Mepanipyrim 10.7 35.5 80 14 82 11 81 15 12 9 22
Metconazole 0.1 0.4 89 11 78 12 77 11 15 21 22
Methidathion 0.4 1.4 117 20 106 14 105 22 23 22 25
Metoxuron 1.0 3.3 80 14 71 18 71 14 7 6 5
Metsulfuron methyl 0.3 1.0 94 13 91 12 88 9 8 15 21
Oxadiazon 0.2 0.6 67 5 72 6 70 4 10 11 21
Paraoxon-methyl 5.2 17.2 65 4 79 13 65 9 15 15 16
Phoxim 1.5 4.9 80 8 72 5 82 13 12 21 22
Quinoxyfen 1.9 6.2 69 4 60 5 64 3 8 7 14
Tebuconazole 1.6 5.2 72 10 70 8 68 12 9 10 13
Thifensulfuron-methyl 0.9 3.1 108 21 100 17 97 11 13 17 13
Terbuthylazine 1.3 4.4 65 10 68 9 70 11 6 8 7
Zoxamide 1.3 4.4 92 9 78 20 80 14 7 14 23
10 ng/g 50 ng/g 700 ng/g 10 ng/g 50 ng/g 700 ng/g
CYPROCONAZOLE 2 6.5 21.7 10700 73 6 71 9 68 9 11 20 22
DIETHOFENCARB 1.9 6.2 78 5 69 9 72 10 5 15 12
FENTHION 3.4 11.3 75 10 83 11 81 16 13 10 18
INDOXACARB 0.6 2.0 78 8 75 6 74 9 8 7 14
MONOCROTOPHOS 0.05 0.2 64 8 76 12 75 14 4 9 10
PROPICONAZOLE 2.9 9.8 75 11 60 14 65 21 7 6 14
TERBUFOS 15.1 50.4 85 15 73 15 78 12 8 5 12
Tetraconazole 2.8 9.2 81 11 80 15 78 9 4 5 11
4 ng/g 50 ng/g 100 ng/g 4 ng/g 50 ng/g 100 ng/g
Bensulfuron-methyl 0.2 0.8 1100 63 11 60 11 65 11 15 14 20
Carboxin 0.4 1.2 99 17 89 15 91 20 22 19 21
Desmetryn 0.3 1.1 78 4 70 8 68 11 12 16 19
Fipronil sulfone (ESI) 0.3 1.0 85 10 83 21 82 9 9 9 19
 K.M. Kasiotis et al. / Science of the Total Environment 485486 (2014) 633642 639

Table 2 (continued)
Analyte LOD (ng/g) LOQ (ng/g) Calibration range (ng/g) Recovery RSD% Inter-d precision

n=3 RSD% n = 3

4 ng/g 50 ng/g 700 ng/g 4 ng/g 50 ng/g 700 ng/g

Fludioxonil (ESI) 2.5 8.3 78 10 75 23 80 8 8 12 11

Alachlor 0.1 0,5 2100 77 8 85 20 71 14 12 16 21
Benfuracarb 0.2 0.8 61 12 60 14 64 18 4 7 11
Bifenthrin 7.4 24.6 59 12 62 10 58 6 12 8 20
Dimethoate 2.8 9.5 90 21 96 17 84 14 17 20 24
Dodemorph 0.2 0.8 64 15 58 12 66 11 17 19 24
Ethoprophos 0.2 0.6 90 20 78 24 75 9 20 12 25
Metalaxyl M 0.1 0.4 79 19 81 12 82 27 22 9 21
myclobutanil 1.9 6.4 92 8 77 8 90 8 25 24 27
Chlorpyrifos 3.2 10.5 4-100 94 12 80 12 76 7 20 21 18
Clethodim 5.7 18.9 80 8 65 15 72 7 20 21 27
Cyproconazole 1 6.4 21.3 84 8 69 15 80 7 8 9 11
Demeton-S-methyl 0.2 0.5 88 6 81 5 78 9 10 12 8
Demeton-S-methyl sulfoxide 3.8 12.7 114 10 97 9 95 14 10 12 16
Disulfoton 1.7 5.7 92 21 77 20 75 14 21 16 26
Disulfoton sulfoxide 3.0 9.9 73 7 76 10 70 8 13 15 17
EPN 2.7 8.9 67 12 68 14 65 11 15 17 17
Famoxadone 0.04 0.1 103 9 85 8 83 6 12 10 21
Fenamidone 2.1 6.9 65 9 60 17 59 10 5 7 5
Fenpropimorph 2.6 8.8 82 9 73 9 74 9 19 12 14
Fosthiazate 0.4 1.4 79 12 79 9 82 20 8 13 15
Isoproturon 0.2 0.6 86 10 79 14 75 20 12 17 24
Linuron 0.1 0.4 85 7 68 9 66 10 19 17 22
Malaoxon 0.8 2.6 90 15 82 13 82 17 6 7 12
Piperonil butoxide 16.5 54.9 64 11 60 19 63 9 7 12 14
Pyrazophos 0.3 0.9 102 8 80 12 81 11 14 10 12
Pyrimethanil 0.8 2.6 75 8 65 6 60 9 5 6 8
Thiodicarb 0.2 0.8 70 14 68 12 70 8 10 12 14
Tricyclazole 0.2 0.7 108 6 94 8 95 18 4 4 12
Triticonazole 5.4 18.0 70 8 65 7 65 9 6 8 14
10 ng/g 50 ng/g 100 ng/g 10 ng/g 50 ng/g 100 ng/g
Aclonifen 11.4 37.9 10100 77 11 72 9 67 11 14 18 23
Cyhalothrin lambda 12.5 41.0 64 5 70 7 65 6 10 8 16
Fenpropathrin 23.3 77.5 60 11 78 20 71 15 6 5 16
fenthion-sulfone 8.9 29.7 85 20 95 22 97 18 10 8 16
Fipronil (ESI) 1.3 4.4 95 6 97 4 94 5 14 8 25
Malathion 6.9 23.1 80 12 70 8 72 15 9 12 24
Metribuzin 0.7 2.3 87 11 68 12 69 9 12 15 26
Nicosulfuron 0.2 0.6 92 22 85 20 79 19 9 7 12
Phosalone 2.1 7.2 71 14 70 27 74 20 21 13 15
Phosmet 0.2 0.6 70 15 58 12 60 11 5 14 8
Phosphamidine 2.0 6.8 89 10 83 19 85 22 21 13 15
Prothioconazole desthio 3.5 11.7 62 7 60 5 62 10 5 4 7

infestation of the honeybee colony and exposure to imidacloprid, at
concentration levels ranging from 14 to 39 ng/g in 60% of the samples
was evidenced. The respective percentage of positive bee samples in
imidacloprid residues from the current work is far less than our work
but in the same order of magnitude regarding concentrations. However
we have to note that our work refers to a larger number of samples thus,
from that standpoint, the two studies are not comparable.
Residues in bee products in Greece have been reported to derive
mainly from treatments of colonies with protection agents. In 2001,
Menkissoglu-Spiroudi et al. published a work reporting acaricides
found in honey and varroacides in beeswax (Menkissoglu-Spiroudi
et al., 2001). Royal jelly has also been studied as regards its residue
prole by researchers in Greece. One work regarded coumaphos and
t-uvalinate commonly used to control Varroa destructor (Balayannis,
2001) and a second, a multiresidue method for the detection of the
mentioned compounds plus 7 extra compounds by GC-micro-ECD
(Karazaris et al., 2008).
The use of bee honey as an environmental bioindicator of pesticide
occurrence has been reported in 2008 (Balayiannis and Balayiannis,
2008). The target compounds of the authors were organophosphorous
insecticides and coumaphos. The results of that study showed the pres-
ence of some organophosphates in bee honey matrix; the majority of
the samples were derived from honeybees foraging in citrus groves.
Other authors in Greece have concentrated their research on chemical

Table 3
Method performance and validation: limits of detection (LODs) and quantication (LOQ), recoveries (%), repeatability (RSD%) and inter-day (inter-d) precision (RSD%) obtained in hon-
eybees with Et3N approach.

Analyte LOD (ng/g) LOQ (ng/g) Calibration range (ng/g) Recovery RSD%a Inter-d precision

n=3 RSD% n = 3

Carbendazim 0.4 1.4 1100 82 25 83 14 84 15 10 9 11

Clothianidin 0.6 1.9 1700 75 9 74 5 70 7 5 7 11
Imidacloprid 0.4 1.5 1700 84 6 75 9 73 7 8 13 15
Thiamethoxam 0.06 0.2 10700 79 9 80 10 73 20 6 10 8
a
Recovery and inter-d-precision levels as in Table 2.
640 K.M. Kasiotis et al. / Science of the Total Environment 485486 (2014) 633642

a Pesticide presence in honeybee colony matrices in France was pro-

18 spectively studied by Chauzat and Faucon (2007) and Chauzat et al.
16 (2006, 2011). Minimum concentrations were attributed as NLOD or
Number of Samples

14 ND with an average higher content of coumaphos in honeybees at

12 1545 ng/g, whereas in our work the respective higher value was ap-
10 proximately 20 ng/g almost 77-fold lower. In bee pollen the respective
8 Series1 average concentration was 423 ng/g, noting that in our work we did
6
not detect coumaphos.
4
A contemporary work has been published by Giroud et al. on the de-
2
termination of pyrethroid and neonicotinoid insecticides in beebread
0
Number of Number of Clothianidin Imidacloprid (Giroud et al., 2013). In this study 32 samples were analyzed revealing
Samples Positive the presence of 7 of the target substances. The most frequently detected
Samples
pesticides were belonging to the neonicotinoids family. Concentrations
Honeybees 2011
were generally low, but in some cases exceeded 170 ng/g. Pyrethroids
were also detected but at very low levels. These concentrations are in
b general comparable to the ones obtained in our work with exceptions
20
for one bee pollen sample in our study where clothianidin was deter-
Number of Samples

18
16 mined at 1273 ng/g and one honeybee sample with phoxim concentra-
14
12 tion exceeding 700 ng/g.
10 Trace analysis of seven neonicotinoids was reported in bee pollen as
8
6
well by Yanez Yanez et al. (2013b). In this regard a solidliquid extrac-
4 Series1 tion procedure was developed using dichloromethane as the organic
2 solvent to extract the neonicotinoids from bee pollen. In this view the
0
experimental procedure of this paper is quite simple, involving only
an extraction step, circumventing the QuEChERS step that we also
used. The developed method was then applied to bee pollen samples
collected from apiaries located close to fruit orchards in two Spanish re-
gions, and traces of acetamiprid and imidacloprid were found in two
samples. Recently the work by Lozowicka utilizing a GC-dual detector
Honeybees 2012 (NPDECD) has included some of the pesticides used in this work
(Lozowicka, 2013). LODs for most of analytes were near to 0.005 ng/g.
c These LODs belong amongst the lowest reported in honeybees; howev-
Number of Samples

9
8
er LOQs uctuated at 1050 ng/g.
7 An overall picture of LODs and LOQs of our work and the published
6
5 papers is given in Tables S10 and S11, in honeybee, honey and bee pol-
4 len respectively. The comparison results clearly demonstrate the ef-
3
2 ciency and sensitivity of the proposed LCMS/MS method since the
1
0 Series1 LOQs are in the ng/g scale. The developed LCESI-MS/MS method en-
compasses above 110 active substances and has the potential to be
expanded to more pesticides (such as some pyrethroid insecticides
which were not included) and some of their metabolites (especially
for neonicotinoid insecticides) and even incorporate other types of pol-
lutants amenable to LCMS/MS instrumentation and methodology.

Honeybees 2013 4.2. Pesticide characteristics and behavior

Fig. 2. a) Number of 2011 Samples, Positive Samples and Distribution of Pesticides,

b) Number of 2012 Samples, Positive Samples and Distribution of Pesticides, and c) Number
of 2013 Samples, Positive Samples and Distribution of Pesticides.
control agents used to protect honeycombs from Wax moth (Harizanis
et al., 2008). Zacharis et al. have published a dispersive liquidliquid
microextraction for the determination of organochlorine pesticides in
honey by GCECD and ion trap mass spectrometric detection; however
the honey samples were free from the pesticides studied (Zacharis et al.,
2012).
On global scale, numerous monitoring schemes exist and various re-
search works are published on the determination of pesticide residues
in these matrices. An extensive work on North American Apiaries was
published by Mullin et al. as regards the residue analysis of beehive
samples (Mullin et al., 2010). These included adult bees (106 samples),
bee brood (34), and pollen (350 samples including beebread, trapped
pollen, and other samples). Multiple residues prevailed in the bee, pol-
len and wax samples, with 2 or more pesticides detected in 92.3% of the
samples (749 samples).
The physicochemical characteristics of pesticides detected are de-
scribed in detail in literature (indicatively see http://sitem.herts.ac.uk/
aeru/iupac/). In this report the predominant family of pesticides detect-
ed was the neonicotinoids. Neonicotinoids are highly soluble to water
and persistent in soil exhibiting half-life values (degradation half-life,
DT50) ranging from 16 (thiacloprid) to 545 days (clothianidin). Fipronil
and pronil sulfone belong to the phenyl pyrazole chemical class;
pronil has a DT50 in soil of 142 days. As regards the organophospho-
rous pesticides detected in our study, chlorpyrifos has a DT50 value of
50 days, while dimethoate 2.6 days. Coumaphos which was detected
in two samples is a non-volatile phosphorothioate, soil-persistent com-
pound with DT50 value of 152 days. Other pesticides such as phoxim or
trioxystrobin are not persistent. In this context, it is evident that apart
from the concentrations found in honeybees and related products, the
possible existence of some of these pesticides in the environment
could pose threat to other organisms as well. As regards the behavior
of these substances in plants, guttation seems to be a contributing factor
in the transfer of active substances to honeybees. Guttation is a natural
process seen in many vascular plants whereby drops of xylem sap exude
from leaf tips or margins. Specically neonicotinoid insecticides,
 K.M. Kasiotis et al. / Science of the Total Environment 485486 (2014) 633642
Table 4
Comparison of acute oral LD50 values with selected maximum concentrations obtained
in honeybees.
Active substance Acute oral LD50 Maximum concentration %LD50
(ng a.s./gbee bw) obtained in honeybees
(ng a.s./gbee bw)
Imidacloprid 37 (LD50-Imidacloprid) 73.9
Clothianidin 37.9 (LD50-Clothianidin, 2005) 39.9
Thiamethoxam 50 (LD50-Thiamethoxam, 2006) 49.6
Fipronil 41.7 (LD50-Fipronil, 2013) 81.5
Fipronil sulfone 64 (LD50-Fipronil, 2013) 79.1
translocated in guttated droplets of seed-treated maize and wheat have
been examined as a threat to honeybees (Girolami et al., 2009; Reetz et al.,
2011). Girolami et al. demonstrated that translocation of neonicotinoid
insecticides from coated seeds to seedling guttation drops, is a novel
way of intoxication for bees. In the work by Reetz et al. it was found
that guttated water of plants germinated from seeds dressed with
neonicotinoids contained neonicotinoids. Although the concentration
was rapidly decreased it was still detectable after weeks.
4.3. Risk assessment
Pesticides in general have been suspected for the death incidents
of bees. The latter was alleged or hypothesized by individuals who
sent the samples. However, there was no extra/enlightening info as
regards the causality of death, which could also be attributed to diseases
caused by pests and parasites such as the V. destructor or combination of
factors.
In the majority of cases, the concentrations of pesticides detected
in bee bodies were much below the LD50 values (see Table 1 and
Table S12, Supplementary material). However when comparing the max-
imum concentrations encountered in some incidents for imidacloprid,
clothianidin, thiamethoxam, pronil and its sulfone metabolite and the
acute oral LD50s it is apparent that it cannot be omitted that these inci-
dents could have been caused by these pesticides. However, for the ma-
jority of incidents (see Table S12) the concentration levels of pesticides
were below the acute oral LD50 values indicating that further research
should have been pursued to reveal the etiology of honeybee death
incidents.
Based on the above results and with knowledge of toxicity values for
bees such as the oral LD50 value (see Table 4) and the sublethal dose of
0.5 ng/bee (Schneider et al., 2012) it was attempted to carry out a pre-
liminary risk assessment for the main compound detected at high levels
in bee pollen, clothianidin (Clothianidin, 2013). Considering the oral
LD50 and the lowest observed effect concentration (LOEC) value for
honeybee larvae to be 40 ng a.s./g diet (see Clothianidin, 2013 and
references therein) plus the ndings (Table 1) of the present study, it
cannot be excluded that subjection of bees to polluted pollen at concen-
tration levels up to 1273 ng/g (in one case) can cause toxicity/mortality
in bees. Besides all, the potential for cumulative toxicity of the exposure
to several pesticides at the same time having the same target system
should also be considered. In our work we evidenced the concomitant
presence of more than one pesticide in some incidents (see Table S12,
Supplementary material). In a study published in 2003, under laborato-
ry conditions, a slight synergistic effect in honeybees was reported
for insecticide thiacloprid when it was sprayed in combination with
tebuconazole which belongs to triazole fungicides (Schmuck et al.,
2003). On aquatic crustaceans it was also demonstrated that pesti-
cide cocktails comprising of imidazolic, triazolic pesticides and alpha-
cypermethrin can interact synergistically and enhance the effect of
the pyrethroid insecticide towards Daphnia magna (Norgaard and
Cedergreen, 2010). Therefore there should be a central and coordi-
nated program for the monitoring of bee issue with emphasis on:
a) chemical analyses to identify the transport of pesticides on
641
honeybees and beehive products, b) consideration of general health
problems of bees and c) strategies on proper management of bees
and beehives.
5. Conclusions
199.7 In the current study the reported cases of honeybee death incidents
105.3
were investigated with regard to the potential interrelation to the expo-
99.2
195.4 sure to plant protection products. Thus honeybee, bee pollen and honey
123.6 samples from different areas of Greece were received and analyzed for
the presence of pesticide residues. For the analysis of the samples an
LCESI-MS/MS multiresidue method including a total of 115 analytes
of different chemical classes in honeybee bodies, honey and bee pollen
was developed and validated. The method showed excellent linearity,
precision and accuracy and found to be sufcient as to act as a monitor-
ing method for the investigation of residues in cases of suspected honey-
bee poisoning incidents. From the analysis of the samples the presence of
14 active substances was observed in all matrices, a fact which veried
the presence of such type of compounds in honeybee body and apicul-
tural products. Our method exhibited quite low LOQs for all analytes
and it is comparable to the majority of the published multiresidue
methods. Further inclusion of analytes and inclusion of other related api-
cultural products (i.e. beeswax, propolis) is highly considered by our lab
so as to extend our monitor capacity and provide an overview to apicul-
turists who are concerned for their honeybees' survival and the quality of
their products.
Acknowledgments
The authors would like to thank the beekeepers for the provision of
honeybees, honey and pollen with no detectable residues obtained from
non-treated experimental bee-hives raised by them and used for the
validation part of the method.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2014.03.042.
References
Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ. Fast and easy multiresidue method
employing acetonitrile extraction/partitioning and dispersive solid-phase extrac-
tion for the determination of pesticide residues in produce. J AOAC Int 2003;86:
41231.
Antoniou V, Zantopoulos N, Tsoukali H. Fatal animal poisonings in northern Greece:
19901995. Vet Hum Toxicol 1997;39:356.
Bacandritsos N, Granato A, Budge G, Papanastasiou I, Roinioti E, Caldon M, et al. Sudden
deaths and colony population decline in Greek honey bee colonies. J Invertebr Pathol
2010;105:33540.
Balayannis PG. Gas chromatographic determination of coumaphos and tau-uvalinate
residues in royal jelly produced under commercial conditions. J Apic Res 2001;40:
718.
Balayiannis G, Balayiannis P. Bee honey as an environmental bioindicator of pesticides'
occurrence in six agricultural areas of Greece. Arch Environ Contam Toxicol 2008;
55:46270.
Blasco C, Vazquez-Roig P, Onghena M, Masia A, Pico Y. Analysis of insecticides in honey by
liquid chromatographyion trap-mass spectrometry: comparison of different extrac-
tion procedures. J Chromatogr A 2011;1218:4892901.
Bromenshenk JJ, Preston EM. Public-participation in environmental monitoring A
means of attaining network capability. Environ Monit Assess 1986;6:3547.
Burkle LA, Marlin JC, Knight TM. Plantpollinator interactions over 120 years: loss of spe-
cies, co-occurrence, and function. Science 2013;339:16115.
Chauzat MP, Faucon JP. Pesticide residues in beeswax samples collected from honey bee
colonies (Apis mellifera l.) in France. Pest Manag Sci 2007;63:11006.
Chauzat MP, Faucon JP, Martel AC, Lachaize J, Cougoule N, Aubert M. A survey of pesticide
residues in pollen loads collected by honey bees in France. J Econ Entomol 2006;99:
25362.
Chauzat MP, Martel AC, Cougoule N, Porta P, Lachaize J, Zeggane S, et al. An assessment of
honeybee colony matrices, Apis mellifera (Hymenoptera Apidae) to monitor pesticide
presence in continental France. Environ Toxicol Chem 2011;30:10311.
Clothianidin EEFSA J 2013;11. http://dx.doi.org/10.2903/j.efsa.2013.3066.
Elzen PJ, Baxter JR, Spivak M, Wilson WT. Control of Varroa jacobsoni Oud. resistant to
uvalinate and amitraz using coumaphos. Apidologie 2000;31:43741.
642 K.M. Kasiotis et al. / Science of the Total Environment 485486 (2014) 633642
Fidente P, Seccia S, Vanni F, Morrica P. Analysis of nicotinoid insecticides residues in
honey by solid matrix partition clean-up and liquid chromatographyelectrospray
mass spectrometry. J Chromatogr A 2005;1094:1758.
Garcia-Chao M, Agruna MJ, Flores Calvete G, Sakkas V, Llompart M, Dagnac T. Validation
of an off line solid phase extraction liquid chromatographytandem mass spectrom-
etry method for the determination of systemic insecticide residues in honey and pol-
len samples collected in apiaries from NW Spain. Anal Chim Acta; 2010;672:10713.
Girolami V, Mazzon L, Squartini A, Mori N, Marzaro M, Di Bernardo A, et al. Translocation
of neonicotinoid insecticides from coated seeds to seedling guttation drops: a novel
way of intoxication for bees. J Econ Entomol 2009;102:180815.
Giroud B, Vauchez A, Vulliet E, Wiest L, Bulete A. Trace level determination of pyrethroid and
neonicotinoid insecticides in beebread using acetonitrile-based extraction followed by
analysis with ultra-high-performance liquid chromatographytandem mass spectrome-
try. J Chromatogr A 2013. http://dx.doi.org/10.1016/j.chroma.2013.09.088.
Greatti M, Sabatini A, Barbattini R, Rossi S, Stravisi A. Risk of environmental contamina-
tion by the active ingredient imidacloprid used for corn seed dressing. Preliminary re-
sults. Bull Insectology 2003;56:6972.
Gregorc A, Evans JD, Scharf M, Ellis JD. Gene expression in honey bee (Apis mellifera) lar-
vae exposed to pesticides and Varroa mites (Varroa destructor). J Insect Physiol 2012;
58:10429.
Harizanis PC, Alissandrakis E, Tarantilis PA, Polissiou M. Solid-phase microextraction/gas-
chromatographic/mass spectrometric analysis of p-dichlorobenzene and naphtha-
lene in honey. Food Addit Contam Part A Chem Anal Control Expo Risk Assess
2008;25:12727. [http://sitem.herts.ac.uk/aeru/iupac/].
ICH. Validation of analytical procedures: text and methodology Q2(R1). http://www.ich.
org/leadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_
R1__Guideline.pdf, 2005.
Imidacloprid EEFSA J 2013;11. http://dx.doi.org/10.2903/j.efsa.2013.3068. [55 pp.].
Johnson RM, Ellis MD, Mullin CA, Frazier M. Pesticides and honey bee toxicity USA.
Apidologie 2010;41:31231.
Jovanov P, Guzsvany V, Franko M, Lazic S, Sakac M, Saric B, et al. Multi-residue method for
determination of selected neonicotinoid insecticides in honey using optimized dis-
persive liquidliquid microextraction combined with liquid chromatographytan-
dem mass spectrometry. Talanta 2013;111:12533.
Kamel A. Rened methodology for the determination of neonicotinoid pesticides and
their metabolites in honey bees and bee products by liquid chromatographytandem
mass spectrometry (LCMS/MS). J Agric Food Chem 2010;58:592631.
Karazaris E, Menkissoglu-Spiroudi U, Thrasyvoulou A. New multiresidue method using
solid-phase extraction and gas chromatography-micro-electron-capture detection
for pesticide residues analysis in royal jelly. J Chromatogr A 2008;1209:1721.
Krupke CH, Hunt GJ, Eitzer BD, Andino G, Given K. Multiple routes of pesticide exposure
for honey bees living near agricultural elds. PloS One 2012;7:e29268.
LD50-Clothianidin. European Commission, review report for the active substance
clothianidin, SANCO/10533/05 nal; January 2005.
LD50-Fipronil. Peer review of the pesticide risk assessment for bees for the active sub-
stance pronil. EFSA J 2013;11:3158.
LD50-Imidacloprid. http://www.bee-quick.com/reprints/imd/BayerFAQ.pdf.
LD50-Thiamethoxam. European Commission, review report for the active substance
thiamethoxam, SANCO/10390/2002 rev. nal; July 2006.
Lozowicka B. The development, validation and application of a GC-dual detector (NPD
ECD) multi-pesticide residue method for monitoring bee poisoning incidents.
Ecotoxicol Environ Saf 2013. http://dx.doi.org/10.1016/j.ecoenv.2013.07.010.
Martel AC, Lair C. Validation of a highly sensitive method for the determination of
neonicotinoid insecticides residues in honeybees by liquid chromatography with
electrospray tandem mass spectrometry. Int J Environ Anal Chem 2011;91:97888.
Menkissoglu-Spiroudi U, Tsigouri AD, Diamantidis GC, Thrasyvoulou AT. Residues in
honey and beeswax caused by beekeeping treatments. Fresenius Environ Bull 2001;
10:44550.
Mizell R. Many plants have extraoral nectaries helpful to benecials. Consult le 08 01,
2010, sur EDIS. University of Florida IFAS Extension; 2009 [http://edis.ifas.u.edu].
Mullin CA, Frazier M, Frazier JL, Ashcraft S, Simonds R, Vanengelsdorp D, et al. High levels
of miticides and agrochemicals in North American apiaries: implications for honey
bee health. PLoS One 2010;5:e9754.
Norgaard KB, Cedergreen N. Pesticide cocktails can interact synergistically on aquatic
crustaceans. Environ Sci Pollut Res Int 2010;17:95767.
OPERA. Bee health group: bee health in Europe facts & gs; 2013.
Pettis JS, Delaplane KS. Coordinated responses to honey bee decline in the USA. Apidologie
2010;41:25663.
Pettis JS, vanEngelsdorp D, Johnson J, Dively G. Pesticide exposure in honey bees re-
sults in increased levels of the gut pathogen Nosema. Naturwissenschaften 2012;
99:1538.
Pirard C, Widart J, Nguyen BK, Deleuze C, Heudt L, Haubruge E, et al. Development and
validation of a multi-residue method for pesticide determination in honey using
on-column liquidliquid extraction and liquid chromatographytandem mass spec-
trometry. J Chromatogr A 2007;1152:11623.
Porrini C, Sabatini AG, Girotti S, Ghini S, Medrzycki P, Grillenzoni F. Honey bees and bee
products as monitors of the environmental contamination. Apiacta 2003;38:6370.
Przybylski C, Segard C. Method for routine screening of pesticides and metabolites in
meat based baby-food using extraction and gas chromatographymass spectrometry.
J Sep Sci 2009;32:185867.
Raeymaekers B. A prospective biomonitoring campaign with honey bees in a district of
upper-Bavaria (Germany). Environ Monit Assess 2006;116:23343.
Reetz JE, Zuhlke S, Spiteller M, Wallner K. Neonicotinoid insecticides translocated in
guttated droplets of seed-treated maize and wheat: a threat to honeybees?
Apidologie 2011;42:596606.
SANCO. Method validation and quality control procedures for pesticide residues analysis
in food and feed Document No. SANCO/12495/2011. http://www.crl-pesticides.
eu/library/docs/fv/SANCO12495-2011.pdf.
Schmuck R, Stadler T, Schmidt HW. Field relevance of a synergistic effect observed in the
laboratory between an EBI fungicide and a chloronicotinyl insecticide in the honey-
bee (Apis mellifera L, Hymenoptera). Pest Manag Sci 2003;59:27986.
Schneider CW, Tautz J, Grunewald B, Fuchs S. RFID tracking of sublethal effects of two
neonicotinoid insecticides on the foraging behavior of Apis mellifera. PLoS One
2012;7:e30023.
Schoning R, Schmuck R. Analytical determination of imidacloprid and relevant metabolite
residues by LC MS/MS. Bull Insectology 2003;56:4150.
Tanner G, Czerwenka C. LCMS/MS analysis of neonicotinoid insecticides in honey: method-
ology and residue ndings in Austrian honeys. J Agric Food Chem 2011;59:122717.
Thiamethoxam EEFSA J 2013;11. http://dx.doi.org/10.2903/j.efsa.2013.3067. [68 pp.].
Tylianakis JM. Ecology. The global plight of pollinators. Science 2013a;339:15323.
Tylianakis JM. Pollination decline in contextresponse. Science 2013b;340:9245.
Validation. Bioanalytical method validation Guidance for Industry. http://www.fda.gov/
downloads/Drugs/Guidances/ucm070107.pdf.
Van Dijk T. Effects of neonicotinoid pesticide pollution of Dutch surface water on non-
target species abundance [MSc Thesis] Utrecht University; 2010.
Walorczyk S, Gnusowski B. Development and validation of a multi-residue method for
the determination of pesticides in honeybees using acetonitrile-based extraction
and gas chromatographytandem quadrupole mass spectrometry. J Chromatogr A
2009;1216:652231.
Wiest L, Bulete A, Giroud B, Fratta C, Amic S, Lambert O, et al. Multi-residue analysis of 80
environmental contaminants in honeys, honeybees and pollens by one extraction
procedure followed by liquid and gas chromatography coupled with mass spectro-
metric detection. J Chromatogr A 2011;1218:574356.
Williamson SM, Wright GA. Exposure to multiple cholinergic pesticides impairs olfactory
learning and memory in honeybees. J Exp Biol 2013;216:1799807.
Yanez KP, Bernal JL, Nozal MJ, Martin MT, Bernal J. Fast determination of imidacloprid in
beeswax by liquid chromatography coupled to electrospraymass spectrometry. Curr
Anal Chem 2013a;9:495503.
Yanez KP, Martin MT, Bernal JL, Nozal MJ, Bernal J. Trace analysis of seven neonicotinoid
insecticides in bee pollen by solidliquid extraction and liquid chromatography
coupled to electrospray ionization mass spectrometry. Food Anal Methods 2013b.
http://dx.doi.org/10.1007/s12161-013-9710-9.
Zacharis CK, Rotsias I, Zachariadis PG, Zotos A. Dispersive liquidliquid microextraction
for the determination of organochlorine pesticides residues in honey by gas chroma-
tographyelectron capture and ion trap mass spectrometric detection. Food Chem
2012;134:166572.