Talanta 148 (2016) 1–6

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Determination of selected environmental contaminants in foraging

honeybees
A.I. García-Valcárcel n, Encarnación Molero, J.L. Tadeo, M.D. Hernando
Department of Environment, INIA, Ctra. de La Coruña Km 7, 28040 Madrid, Spain

art ic l e i nf o a b s t r a c t

Article history: Colony losses of honeybees have been of great concern in the last years. To explain these losses, several
Received 22 June 2015 studies have been reported, and various factors, such as pathogens and pesticides, have been considered
Received in revised form as possible causes. Nevertheless, organic contaminants, rather than pesticides, are continuously released
20 October 2015
to the environment, and can be intercepted by honeybees during foraging with the possible consequent
Accepted 23 October 2015
Available online 24 October 2015
damage. Azoles and organophosphorus esters have been selected in this work as environmental con-
taminants to be monitored in honeybees. A fast and robust method has been developed to determine
Keywords: these organic pollutants in honeybees. It is based on matrix solid phase dispersion (MSPD), which
Organophosphorus esters performs sample dispersion with extraction and clean up in the same step, followed by LC-ESI-MS/MS
Azoles
determination. Recoveries of the method varied between 73% and 119% and MQLs ranged from 0.8 to
Matrix solid phase dispersion
4 ng g
1. Honeybee samples from ten apiaries located in different regions were analyzed applying the
LC–MS/MS
Honeybee developed method. Azole compounds were found at low levels, but not in all samples, while organo-
phosphorus esters were found in most samples whatever location. Tris-(2-chloroisopropyl) phosphate,
TCPP, and tributyl phosphate, TBP, were detected in all honeybees samples at levels higher than the rest
of organophosphates analyzed.
& 2015 Elsevier B.V. All rights reserved.

1. Introduction surrounding environment. OPs have been found in the environ-

Honeybees (Apis melifera L.) are vital as pollinators of crops and
they are bred commercially for their abilities to produce honey and
pollinate crops. Nevertheless, annual losses of honeybee colonies
averaged about 33 percent each year since the winter of 2006,
which could threaten the economic viability of the bee pollination
industry. Among the factors that could be the cause of honeybees
disappearance, the pesticides, the infection by pathogens such as
viruses, bacteria, fungi and parasites; poor nutrition or the global
warming, are the most studied [1–5]. Furthermore, bee popula-
tions may also be vulnerable to contaminants in the environment,
generated by the industrialization, housing development and
agricultural practices (i.e. via sewage sludge amendments).
Organic environmental contaminants such as organopho-
sphorus (OPs) and azole compounds have been selected for this
study. OPs are widely used as flame retardants and plasticizers in
different types of materials (building materials, electronic equip-
ment, plastics….). They are used as additives and are not chemi-
cally bound in the material, therefore they can be released to the
n
Corresponding author. Department of Environment I.N.I.A, Ctra de la Coruña
Km 7, 28040 Madrid, Spain. Fax: þ34 91 357 2293.
E-mail address: aigarcia@inia.es (A.I. García-Valcárcel).
ment, mainly in water and in air [6–10]. Triphenyl phosphate
(TPhP) and tributyl phosphate (TBP) are suspected to be neuro-
toxic [11,12] while others such as tris-(2-chloroethyl) phosphate
(TCEP), tris-(1,3-dichloro isopropyl) phosphate (TDCPP) or tris-(2-
chloro- isopropyl) phosphate (TCPP) are carcinogenic [13,14].
Azoles are used as fungicides in agriculture, as biocides for
commodities and material protection, and as antimycotic phar-
maceuticals for animals and humans. These compounds are con-
sidered as a new group of endocrine-active agents in humans and
animals, disturbing the biosynthesis of steroids by inhibition of
enzymes like aromatase (CYP19) [15,16]. Azoles may enter the
wastewater after use in households, agricultural fields and farms,
and runoff into sewage. They can be found in water or in sewage
sludge, which is other route of entrance into the environment [17–
21].
OPs and azoles may be present in soil, water and air. Honeybees
forage around their hive areas, collecting water, nectar and pollen
from flowers: during this activity, honeybees can intercept en-
vironmental contaminants in their dense body hair, via inhalation
or by intake of the pollutants present in pollen or water. Honey-
bees or even beehive products have been used as bioindicadors of
environmental pollution, but in relation to presence of pesticide
residues, heavy metals and radionuclides. To our knowledge, there
are very few studies concerning environmental contaminants in

http://dx.doi.org/10.1016/j.talanta.2015.10.064
0039-9140/& 2015 Elsevier B.V. All rights reserved.
2 A.I. García-Valcárcel et al. / Talanta 148 (2016) 1–6
honeybees, such as the determination of polycyclic aromatic hy-
drocarbons (PAHs) in honeybees [22,23] and the analysis of poly-
chlorinated byphenyls (PCBs) [24] and brominated flame re-
tardants (BFRs) [25,26] in honey samples.
In this work, a method to analyze OPs flame retardants and
azole compounds in honeybees was developed to improve
knowledge about potential exposure of honeybees to environ-
mental contaminants. These compounds, which are widespread in
the environment, were chosen. Azole compounds are endocrine-
active agents inhibiting the synthesis of ergosterol that might af-
fect honeybees. OP compounds have a chemical structure similar
to that of OP insecticides and might affect the nervous system of
honeybees by inhibiting the cholinesterase enzyme.
The difficulty for the extraction of those chemicals from hon-
eybee samples is the content in wax, proteins and other sub-
stances that can interfere in the analytical determination. Re-
ported methods that deal with the extraction of pesticide residues
from honeybees are based on several modifications of the QuE-
ChERS method, [27], followed by GC or LC determination [28–31].
Only few reported studies make use of MSPD (matrix solid phase
dispersion) method to extract pesticides from honeybees [32–34].
MSPD has the advantage of using low amount of sample and sol-
vent and, in addition, homogenization, extraction and purification
are combined in the same step. Disruption of the honeybee
structure is achieved without using freeze drying or blending with
a disperser or mixer which requires a higher amount of sample. In
order to provide a miniaturized method, we developed this pro-
cedure for the extraction of azoles and OPs in honeybees.
The aim of this work was to optimize a rapid and robust
method for the simultaneous extraction of OPs and azole com-
pounds from honeybees and subsequent quantitation by LC–MS/
MS. MSPD based method was carried out for the extraction and
purification. The method was applied for the determination of
concentration levels of OPs and azoles in honeybees collected at
various locations.
2. Experimental
2.1. Chemicals and standards
Tris-(2-butoxyethyl) phosphate (TBEP), tris- (2-chloroethyl)
phosphate (TCEP), tributyl phosphate (TBP), triphenyl phosphate
(TPhP), tris-(1,3-dichloro) isopropyl phosphate (TDCPP) and tris-
(2-chloro)- isopropyl phosphate (TCPP) and fluconazole, clo-
trimazole, propiconazole and tebuconazole were provided by Dr.
Ehrenstorfer GmbH (Augsburg, Germany).
Trace analysis quality solvents, acetonitrile and n-hexane were
supplied by AppliChem Panreac (Darmstadt, Germany) and
Scharlab Chemie S.A. (Barcelona, Spain), respectively.
Florisil (60–100 mesh), aluminium oxide 90 standarized, silica
(35–70 mesh), C18 (40–60 mm) and anhydrous sodium sulphate,
were obtained from Acròs Organics (New Jersey, USA), Merck
KGaA (Darmstandt, Germany), Panreac (Darmstandt, Germany),
Scharlab Chemie S.A. (Barcelona, Spain) and Probus, S.A. (Badalo-
na, Barcelona), respectively.
Individual stock standard solutions were prepared in acetoni-
trile (ACN) at 500 ng mL
1. Working standard mixtures were
prepared in ACN by dilution of stock standard solutions.
To avoid possible contamination of glass materials and solvents,
due to the presence of OPs in indoor air, all glassware was pre-
viously cleaned with acetone and, samples and glassware were
covered with aluminum foil.
2.2. Sample preparation
Foraging honeybees (Apis melifera L.) were sent to the labora-
tory thanks to the collaboration of beekeepers (Sample locations
from different regions of Spain are shown in Fig. S1). Samples were
stored at
80 °C until analysis. Approximately 0.5 g (5–8 insects)
were mixed with 0.5 g of anhydrous sodium sulphate and dis-
persed with 1.5 g of Florisil in a glass mortar, with a pestle, until
homogenization of sample was obtained. The mixture was trans-
ferred to a glass syringe barrel (glass column) closed with one way
stopcock that contained two paper filters (Whatman Grade 1) and
a layer of 1.0 g of co-sorbent (alumina). After the addition of the
sample, another two paper filters were placed on top applying a
slight compression with a syringe plunger. To eliminate the lipids
and waxes, 3.5 mL of n-hexane were added to the sample located
in the glass column and the eluate was discharged opening the
stopcock. The stop valve was closed, 3 mL of ACN were added and
the column was placed in an ultrasonic bath (Branson; Carouge,
Switzerland) of 1 L capacity operating at 290 W, 40 kHz, at ambi-
ent temperature for 10 min. After extraction, the column was
placed on a multiport vacuum manifold (Supelco/Sigma Aldrich-
Chemie Gmb; Steinheim, USA) where the solvent was eluted from
the column by opening the stopcock and collected in a glass
graduated tube by gravity flow applying vacuum at the end of
elution. This extraction procedure was repeated with 2 mL of ACN
and the yielded extracts were combined and concentrated to 2 mL
in a centrifugal vacuum concentrator (Genevac Limited, UK). Fi-
nally, the extracts were filtered through a 0.22 μm polypropylene
syringe filter before LC–MS/MS analysis.
2.3. LC–MS/MS conditions
An Agilent 1200 (Waldbronn, Germany) liquid chromatograph
equipped with an autosampler, a quaternary pump and a ther-
mostatic column compartment was used. Separations were carried
out using a Luna-C8 (150 mm  4.6 mm i.d., 5 m particle size)
analytical column with a C8 security guard cartridge supplied by
Phenomenex (Torrance, CA, USA). Eluent flow rate was set at
0.35 mL min
1 and the column was kept at 40 °C. The gradient
elution program was performed with 0.15% formic acid in water as
mobile phase A and ACN as mobile phase B. The gradient was as
follows: 0 min, 90% A; 3 min, 50% A; 4 min, 40% A; 6 min, 20% A;
7 min, 5% A; 8 min, 5% A; 10 min 0% A, 13 min, 0% A; 16 min, 90% A.
After recovering initial conditions, 9 min of equilibration time was
included between runs resulting in a total analysis time of 25 min.
An Agilent 6410 triple quadrupole mass spectrometer equipped
with an electrospray ionization interface (ESI), operating in posi-
tive ion (PI) mode was used. Drying and nebulizing gases for the
ESI source were produced in situ by a nitrogen generator fed by
compressed air at 7 bars. The optimized ESI parameters were:
drying gas flow rate 9 L min
1; drying gas temperature 350 °C;
nebulizer gas pressure 40 psi and capillary voltage 4500 V.
Two different suitable transition pairs (precursor/product ion
pair) were selected for each compound in multiple reaction
monitoring (MRM) mode after direct injection to the interface. The
optimized settings for fragmentor and collision energies were
tested for each compound (Table 1).
3. Results and discussion
3.1. Optimization of MSPD conditions
The efficiency of MSPD method was based in the adequate
selection of dispersant, co-sorbent and eluent solvent depending
on the properties of target compounds and matrix complexity.
 A.I. García-Valcárcel et al. / Talanta 148 (2016) 1–6 3

Table 1 140%
CID-MRM conditions for the simultaneous analysis of azoles and organopho-
sphorus compounds by LC–MS/MS.
120%

100%
Chemical MRM transitiona Fragmentor (V) Collision Energy (V)
80%
Fluconazole 3074238 100 15
60% Alumina
3074169 100 15
Clotrimazole 2774165 120 30 C18
40%
2774241 120 30
PSA
Propiconazole 342 4 159 90 35 20%
342 4 69 90 10
Tebuconazole 308 4 70 120 25 0%
308 4 125 120 45
TCEP 285 4 63 80 25
285 4 99 80 30
TBP 267499 90 15
2674155 90 5 Fig. 2. Percentage recoveries as a function of co-sorbent used in the MSPD pro-
TDCPP 431 499 80 10 cedure using Florisil as dispersant (three replicates).
433499 80 30
TBEP 399 4 199 60 15
399 4 299 60 10 capability of Florisil to retain liphophilic compounds in compar-
TPhP 3274215 150 30 ison with alumina. Therefore, the more apolar OPs, such as TPhP
3274153 130 30 (with a log Kow 4.6 versus log Kow from 1.8 to 3.7), may be par-
TCPP 329 4 99 90 25
tially eliminated by washing with hexane when alumina was
327499 90 25
employed as dispersant [39]. Then, Florisil was selected as dis-
a
Bold type mean transitions used for quantification. persant sorbent.
After sample homogenization with Florisil and in order to ob-
140% tain cleaner extracts, a second assay using a co-sorbent, such as
alumina, C18 or PSA, was carried out. Co-sorbent was loaded into
120% the column before the addition of the homogenized sample, and
100% ACN was the elution solvent. Fig. 2 shows the percentage re-
coveries obtained with the different co-sorbents. PSA gave the
80%
Alumina lowest recoveries for the azole compounds, whereas better re-
60% coveries were obtained for OPs. Alumina and C18 yielded accep-
Florisil
40% table recoveries for all compounds. As somewhat lower recoveries
Silicagel
were obtained, in general, with C18, alumina was selected as the
20% C18 co-sorbent. In addition, this clean-up step with alumina improved
0% the recoveries obtained when Florisil was used as both dispersant
and co-sorbent (Fig. 1). Good recoveries (from 77% to 120%) were
obtained using ACN as the extracting solvent; therefore, no further
solvents were assayed. With this solvent, evaporation to dryness of
the ACN extract before injection in LC–MS/MS was not required as
Fig. 1. Percentage recovery of selected analyte as a function of the different sor-
bents used in the MSPD procedure (three replicates).
ACN is used as mobile phase in LC. It saves time in sample
preparation.
In order to improve recovery, the application of ultrasound in
Preliminary experiments were carried out to evaluate the effect the extraction procedure was assayed. A comparison of extraction
of the dispersant on the selectivity and recoveries of OPs and with or without application of ultrasound before elution with ACN
azoles from honeybees. Sorbents commonly used in MSPD pro- was tested (Fig. 3). Recoveries without application of ultrasound
cedures were tested as dispersant: C18, Florisil, silica-gel, and
were acceptable, but somewhat higher recoveries were obtained
alumina [35,36]. Approximately, samples of 0.5 g of honeybees and
when ultrasonic extraction was employed, then ultrasound was
0.5 g of anhydrous sodium sulfate were fortified at 40 ng g
1 bee
applied in the extraction procedure.
with the mixed OPs-azoles solution; the samples were left for
30 min to allow evaporation of solvent, and then, samples were
140%
dispersed with 1.5 g of sorbent in a glass mortar, with a pestle, and
transferred to a glass column containing 1 g of the same sorbent 120%
(co-sorbent). To remove lipids, as matrix interferences, hexane was 100%
used to rinse the MSPD column [37]. Acetonitrile was employed as
extraction solvent and a final volume of 4 ml of extract was ob- 80%
tained. Recoveries were calculated by comparison with a blank 60%
with US
extract fortified after the corresponding extraction procedure.
40% without US
Fig. 1 shows the recoveries for three replicates obtained by using
these sorbents. A low recovery was obtained for both azole and 20%
OPs, when C18 was used as dispersant sorbent. Although silica gel
0%
showed somewhat better recoveries than C18, low yields were
obtained with these dispersants. Recoveries between 75% and
102% were obtained using Florisil and, between 70% and 108%
using alumina except for TPhP that showed a low recovery (38%).
This anomalous value for TPhP is in accordance with Garcia et al. Fig. 3. Effect of ultrasound application in the recoveries percentage (three
[38] for OPs in dust samples. This could be due to the better replicates).
4 A.I. García-Valcárcel et al. / Talanta 148 (2016) 1–6

3.2. Instrumental performance and matrix effect Table 3

Means recoveries of azole and organophosphorus compounds (%) from honeybee
samples (four replicates).
ESI source and MS/MS were optimized in order to maximize
the response of precursor ion (protonated molecule) and to yield Added 40 ng g
1 10 ng g
1 5 ng g
1
two intense transition ions for each compound. A C8 chromato-
graphic column was used for the simultaneous analysis of azole % RSD % RSD % RSD
and OP compounds and to obtain the lowest LOD. Chromato-
Fluconazole 86 13 109 18 99 7
graphic separation was optimized using formic acid in Milli-Q Clotrimazole 88 10 108 15 110 4
water as the aqueous phase and ACN or methanol as the organic Propiconazole 84 13 119 12 82 15
phase. Tebuconazole 98 7 102 10 110 3
ACN was chosen as the organic mobile phase because honeybee TCEP 90 11 107 14 86 7
TBP 96 8 79 2 73 7
extracts were obtained in ACN and no improvement was obtained
TDCPP 91 10 100 8 111 14
when methanol was used as organic mobile phase. As aqueous TBEP 82 18 94 6 81 11
mobile phase, different concentrations of formic acid were assayed TPhP 96 14 119 7 76 4
and the best results, good peak intensity and resolved symmetrical TCPP 99 13 108 16 107 16
peak, were obtained using 0.15% of formic acid in Milli-Q water.
Evaluation of matrix effect on signal response was carried out
by comparing the slope of a standard calibration in neat solvent Recovery through the method was studied with four replicates
(from 0.5–10 ng mL
1) with a matrix-matched standard calibra- of honeybees samples (0.5 g) fortified before extraction at levels of
tion (from 0.5–10 ng mL
1) containing 0.25 g bee mL
1. Values of 5, 10 and 40 ng g
1 with the azole and OPs mixture and analyzed
slopes ratio lower than 100% indicated signal suppression, following the procedure described above.
whereas those higher than 100% indicated signal enhancement. Recoveries were quantified using matrix-matched standards
Matrix-matched standards with honeybees from five different and are presented in Table 3. Acceptable recoveries, from 73% to
locations were evaluated (Table 2). This table shows signal sup- 119% with RSD o18%, were obtained in the range of the assayed
pression for TDCPP in all honeybee samples analyzed and, for TBP concentrations.
and TPhP in almost all of them. The rest of compounds showed Method detection limits (MDLs) for OPs and azoles were de-
acceptable responses (between 119% and 73%). Variation between termined by spiking a blank matrix with the lowest concentration
honeybee matrices from different geographical origins was, in from the linear range of the calibration curve (from 1 to
general, not very remarkable. Nevertheless, due to the matrix ef- 120 ng g
1) and calculated as MDL¼ 3.143  SD, where 3.143
fect obtained for TDCPP, TBP and TPhP, the standard addition corresponds to Student’s value for a 99% confidence level and six
method was used in the quantitation of these compounds in degrees of freedom and SD is the standard deviation of seven re-
honeybees. In this procedure, 100 mL of a standard solution in neat plicate analysis. Method quantitation limits (MQL), established as
solvent, containing azole and OPs in increasing concentration, 10  SD, varied from 0.8 to 4 ng g
1 (Table S1). Precision of the
were added to 900 mL aliquots of each honeybee extract obtained method was calculated by determining the average coefficient of
by the developed MSPD method. variation of the replicate analysis (n¼ 8) of a spiked extract, during
Blanks (quality control sample) were analyzed for each sample the same day for repeatability and on different days for reprodu-
batch to take into account the contamination caused during cibility. The precision of the method (RSD) showed good repeat-
sample preparation and analysis. Peaks corresponding to TBP and ability, between 1% and 8%. Reproducibility was from 2% to 12% for
TCPP were obtained even for simulated injections of empty vials all compounds (Table S1). Calibration curves in ACN showed good
whatever was the organic mobile phase used (methanol or ACN). correlation coefficients R2 40.98 in the range of 0.25–30 ng mL
1
Background contamination in the analysis of OPs, introduced as a for the different honeybee samples.
consequence of the leaching from LC-system and during sample The obtained results indicate that the developed MSPD method
preparation, has been reported by several authors analyzing these followed by LC–MS/MS quantitation is robust and sensitive en-
compounds [40–43]. Accordingly, concentrations of target com- ough for the determination of OPs and azole compounds in hon-
pounds in honeybee were reported after blank reagents subtrac- eybee samples. Although MSPD methods have been applied for the
tion in this work. extraction of organic compounds, such as pesticide residues, the
extraction of organic pollutants from honeybees by MSPD has not
3.3. Method validation been previously reported. To the best of our knowledge, this is the
first multiresidue method for the determination of OPs and azole
The optimized extraction method was validated in terms of compounds in honeybees. The good results obtained suggest that
accuracy, precision and limits of quantitation. the developed method can be used for the determination of other
organic contaminants belonging to those chemical groups, after an
Table 2 adequate validation.
Matrix effect (%) in honeybees extract from different Spanish locations (S, T, H, B, A)
showed in Fig. 1. 3.4. Application to real samples
S T H B A
The developed method was applied to the determination of OPs
Fluconazole 114 119 107 99 93 and azole levels in different honeybee samples collected from
Clotrimazole 118 114 98 77 99 various Spanish locations (Fig. S1). As shown in Table 4, higher
Propiconazole 73 89 78 114 99
levels of OPs than azole compounds were detected in honeybees.
Tebuconazole 113 108 104 114 115
TCEP 114 103 83 85 96 Among azole compounds, propiconazole followed by tebucona-
TBP 38 85 56 83 56 zole, which are fungicides applied in agriculture, were the com-
TDCPP 36 47 32 57 53 pounds most frequently found, although at low concentrations
TBEP 56 88 90 110 101 (o7 ng g
1). In comparison with the analysis in honeybee sam-
TPhP 38 85 56 83 61
TCPP 78 82 83 78 98
ples from western France [31], propiconazole and fluconazole were
found in our study in a higher percentage of bee samples.
 A.I. García-Valcárcel et al. / Talanta 148 (2016) 1–6 5

Table 4
Levels found in honeybees (ng g
1) and standard deviation of two replicates from different Spanish hives locations.

Bu B A C H T S So Co G

TCEP 2.3 (0.34) 3.7 (0.3) 8.4 (1.7) 6.7 (1.2) 2.2 (0.2) o MLQ 3.4 (1.2) 2.3 (0.9) 1.8 (0.7) 2.1 (0.2)
TCPP 27.9 (0.3) 35.4 (1.9) 52.4 (0.5) 64.8 (5.1) 34.6 (4.4) 9.6 (1.6) 35.1 (3.6) 14.0 (2.1) 10.2 (1.8) 37.7 (3.8)
TDCPP n.d n.d o MQL 6.1 (1.0) n.d o MQL MQL 7.8 (1.2) n.d MQL
TPhP 3.1 (0.4) 1.4 (0.2) 6.6 (1.1) 13.1 (1.8) 8.4 (2.9) 1.5 (0.2) o MQL 3.0 (0.9) 2.6 (0.7) 8.0 (0.7)
TBP 16.8 (3.0) 44.6 (0.7) 113.0 (13.7) 47.3 (8.1) 14.6 (3.9) 6.2 (1.0) 7.6 (1.5) 20.4 (2.3) 3.3 (0.7) 6.1 (1.0)
TBEP 3.5 (1.4) o MQL 3.2 (0.9) 6.2 (1.9) 9.3 (1.3) 20.1 (4.2) o MQL 5.4 (1.1) o MQL 2.2 (0.6)
Fluconazole 1.1 (0.002) o MQL 2.0 (0.05) o MQL o MQL n.d n.d n.d n.d o MQL
Clotrimazole o MQL o MQL o MQL 2.0 (0.06) n.d n.d o MQL 2.5 (0.2) o MQL o MQL
Tebuconazole o MQL o MQL n.d 7.0 (1.9) o MQL 1.3(0.4) 1.1 (0.01) 1.8 (0.07) 1.5 (0.06) n.d
Propiconazole 1.7 (0.8) o MQL 1.2 (0.3) 2.3 (0.7) o MQL 1.1 (0.2) 1.5 (0.07) 0.9 (0.069) o MQL 2.4 (0.9)

Nevertheless, the LOQs for tebuconazole and propiconazole,
17.9 ng g
1 and 17.0 ng g
1, respectively, of that study were higher
than the ones obtained in the present work (Table 4).
Fluconazole and clotrimazole, fungicides of animal and human
use, were, in general, below the quantitation limit, being only
quantified in two samples. To the best of our knowledge, this is the
first time that these compounds are detected in honeybees.
Except TDCPP, all the OPs under study were found in most of
the samples analyzed. TBP and TCPP were quantified in all samples
with concentration varying between 3.3 and 113 ng g
1. TCEP, and
TPhP were also found in almost all samples at similar low
levels,o13.1 ng g
1. TBEP was quantitated in eight of ten samples
analyzed, with a range of concentration of 2.2–9.3 n g
1.
Honeybees from two locations (A and C in Fig. S1) presented
higher contamination levels probably due to a higher source of
contamination around their hives.
In general, higher levels of TCPP than TBP were found in fora-
ging bees from the different locations; it is in accordance with the
higher concentration levels of these compounds in air samples in
comparison with other OPs [44,45]. Though the production of
TCEP is not forbidden, this compound has been replaced by TCPP
in Europe. From 6 to 18 folds more TCPP than TCEP was found in
honeybee samples. TBP, the other OPs found in all analyzed hon-
eybees, is also prevalent in the atmosphere, especially when traffic
is heavy, due to the emission of hydraulic fluids from cars [46].
Variation of concentration levels of TBP in honeybees could be due
to the varying distances of hives to highways.
In this study, low levels of TPhP in bees were found (from
oMQL to 13.1 ng g
1), and it was detected in the 90% of samples,
whereas Lambert et al. [31] using a modified QuEChERS method
followed by GC-ToF, reported levels up to 61.6 ng g
1 with a mean
of 1.95 ng g
1 in the 24.8% of the bees analyzed from western
France. No data of levels of the other OPs selected in this work
were found in the available literature.
In conclusion, OPs are ubiquitous pollutants in the atmosphere
that can contaminate honeybees, probably by deposition in the
dense hair on their bodies.
This study shows that the developed method is suitable to
determine azoles and OP flame retardants in bees. The application
of the method showed that OPs compounds are prevalent con-
taminants in honeybees and due to a possible similar behavior to
other OP pesticides as inhibitors of cholinesterase further studies
should be carried out to determine potential effects in bees as well
as possible synergism with other substances such as pesticides. In
addition, bees seem to be a good bioindicator to monitor these
compounds in the environment associated to industrial uses and
their long-range air transport.
4. Conclusion
In the present work a fast and cost-effective analytical method,
based in MSPD, for the simultaneous determination of azoles and
organophosphate esters in honeybees was developed. The best
results were obtained using Florisil as dispersant, alumina as co-
sorbent and acetonitrile as extracting solvent, assisted by sonica-
tion. In comparison with QuEChERS method, usually employed in
sample preparation for the analysis of organic compounds (mainly
pesticides) in bees, the developed method uses a lower amount of
sample (0.5 g versus 5–10 g), lower volumes of solvent (5 mL
versus 10 mL) and the sample homogenization, extraction and
clean-up is carried out in one single step, reducing time of ana-
lysis. The method was successfully applied for the determination
of azoles and OPs in honeybees from different Spanish locations.
OPs were detected in honeybees at levels higher than azoles, with
values for TCPP and TBP varying from 9.6 to 64.8 ng g
1 and from
3.3 and 113 ng g
1, respectively.
Acknowledgments
This study is part of the INIA (National Institute for Agricultural
and Food Research and Technology) project number “RTA 2013-
00042-C10-01” and the financial support of the Ministry of
Economy and Competitiveness is gratefully acknowledge.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.talanta.2015.10.
064.
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