Chemosphere 163 (2016) 525e534

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Bee pollen as a bioindicator of environmental pesticide contamination

Renata Cabrera de Oliveira a, Sonia Claudia do Nascimento Queiroz b,
Cynthia Fernandes Pinto da Luz c, Rafael Silveira Porto a, Susanne Rath a, *
a
Institute of Chemistry, Department of Analytical Chemistry, University of Campinas, PO Box 6154, 13084-971 Campinas, SP, Brazil
b
EMBRAPA Meio Ambiente, Jaguariúna, SP, Brazil
c ~o Paulo, SP, Brazil
Center for Research in Palynology, Institute of Botany, Department of the Environment of Sa

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Bee pollen samples were used for

monitoring pesticide residues in the
environment.

Bee pollen is a bioindicator of envi-
ronmental pesticide contamination.

A multiresidue method was devel-
oped for analysis of pesticides in bee
pollen.

Pesticide sorption on bee pollen was
evaluated.

Pesticides were quantified by ion trap
mass spectrometry.

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

Article history: Honeybees and bee products are potential bioindicators of the presence of contaminants in the envi-
Received 2 June 2016 ronment, enabling monitoring of large areas due to the long distances travelled by bees. This work
Received in revised form evaluates the use of bee pollen as a bioindicator of environmental contamination by pesticides. A GC-MS/
2 August 2016
MS analytical method for multiresidue determination of 26 different pesticides in pollen was developed
Accepted 3 August 2016
Available online 24 August 2016
and validated in accordance with the recommendations of the European Union SANCO guide. Environ-
mental monitoring was conducted using the analysis of 145 pollen samples collected from ten beehives
Handling Editor: I. Cousins in the experimental apiary of Embrapa in Jaguariúna (Sa ~o Paulo State, Brazil). Bioallethrin and pendi-
methalin were identified in four and eighteen samples, respectively, at concentrations below the LOQ of
Keywords: the method (25 ng g1). Passive sampling with polyurethane foam discs was used as a control, and no
Bee pollen pesticides were found. The detection of pesticide residues in seven samples (33%) from commercial
Bioindicator ~o Preto (S~
apiaries in Ribeira ao Paulo State) confirmed the efficiency of the analytical method and the
Pesticides need for environmental monitoring for the presence of pesticide residues. The results demonstrated the
Contamination
potential of bee pollen as a bioindicator of environmental contamination by pesticides.
Multiresidues
© 2016 Elsevier Ltd. All rights reserved.
GC-MS/MS

1. Introduction constant application of pesticides poses risks to human health, due

The use of pesticides is essential in agriculture as it provides pest
control, increases productivity, and reduces costs. However,
* Corresponding author.
E-mail address: Susanne.rath@gmail.com (S. Rath).
to exposure to contaminated water, soil, air, animals, and plants.
Furthermore, pesticide use can lead to the disappearance of bene-
ficial insects (bees and wild pollinators, for instance) and may
promote the appearance of new pests and the evolution of pesticide
resistance among the pest population (Ferna ndez et al., 2001;
Tomita and Beyruth, 2002; Koifman and Hatagima, 2003;
Koifman and Koifman, 2003; Magalha ~es, 2005; Veiga, 2007).

http://dx.doi.org/10.1016/j.chemosphere.2016.08.022
0045-6535/© 2016 Elsevier Ltd. All rights reserved.
526 R.C. de Oliveira et al. / Chemosphere 163 (2016) 525e534
Although honeybees (Apis mellifera) are not the target of pesti-
cides, they are highly vulnerable to contamination, since they are
exposed to these substances while collecting nectar, pollen, and
water for maintenance of the colony. In cases of acute toxicity,
honeybees can die rapidly after the application of pesticides, while
chronic exposure to sub-lethal doses can injure their foraging
behavior and affect colony health and development (Johnston et al.,
2010; Gregorc et al., 2012; Pettis et al., 2012; Di Prisco et al., 2013;
Williamson and Wright, 2013). The declining number of pollinating
insects, usually due to the abuse of pesticides, is of concern in Brazil
and in many other countries, due to the damage it can cause to
agricultural production (Bacandritsos et al., 2010; Pettis and
Delaplane, 2010; Burkle et al., 2013; Castilho and Gonza lez, 2013;
Tylianakis, 2013a, b; Tomazela, 2014).
Bees cover a wide area (up to 7 km2) in their search for nectar
and pollen, and are also numerous and easily kept by humans (Celli
and Maccagnani, 2003). For these reasons, the use of honeybees
and bee products (pollen and honey) as bioindicators of environ-
mental contamination has been of great interest in recent years.
Studies have shown that the level of contamination of hives by
pesticides is closely related to the proximity of the contamination
source and the duration of exposure (García-Chao et al., 2010;
Mullin et al., 2010; Chauzat et al., 2011; Panseri et al., 2014;
Malhat et al., 2015). Among bee matrices, pollen has been indi-
cated as the best for assessment of the presence of environmental
pesticide residues, as it is easy to collect and is frequently
contaminated (Chauzat et al., 2006, 2011). Furthermore, Chiesa
et al., 2016 verified that the presence of persistent organic pollut-
ants in organic honey, affected by the geographical area, confirms
that honey bee and beehive matrices are appropriate sentinels for
monitoring environmental contamination.
In order to use bee pollen as a bioindicator, it is necessary to
develop a sensitive and selective analytical method with an
appropriate sample preparation step, as this matrix is highly
complex. Solid-liquid extraction and the QuEChERS (Quick, Easy,
Cheap, Effective, Rugged, and Safe) approach have been widely
employed for this purpose. Regarding analytical techniques, gas
chromatography coupled to mass spectrometry (GC-MS) and liquid
chromatography coupled to tandem mass spectrometry (LC-MS/
MS) are the techniques most commonly used for multiresidue
analysis of pesticides at low levels of detection (Berrada et al., 2010;
García-Chao et al., 2010; Mullin et al., 2010; Wiest et al., 2011;
Kasiotis et al., 2014).
The main objective of the present study was to evaluate the
potential of bee pollen as a bioindicator of environmental
contamination by pesticides. Sorption of pesticides on bee pollen
was performed in order to assess the affinity between the pesti-
cides and the matrix. The presence of 26 pesticides was monitored
by GC-MS/MS using bee pollen samples, with passive samplers as
controls.
2. Material and methods
2.1. Chemicals and reagents
Certified analytical standards of acetochlor, alachlor, disulfoton,
alpha-endosulfan, beta-endosulfan, etrinfos, fempropatrim, phos-
alone, heptachlor epoxide, hexachlorobenzene, oxifluorfem, ethyl
parathion, methyl parathion, pendimethalin, and terbufos were
purchased from Dr. Ehrenstorfer (Augsburg, Germany). Aldrin,
bifenthrin, bioallethrin, methyl chlorpyrifos, DDT (dichloro-
diphenyl-trichloroethane), phenthoate, fluazifop, lindane, mala-
thion, permethrin, pirimiphos-ethyl, and trifluralin were obtained
from Fluka (USA). Abamectin were purchased from Chem Service
Inc. (USA). All these compounds had purity of at least 98%.
Acetonitrile (residue analysis grade) was purchased from Tedia
(Brazil). Anhydrous magnesium sulfate, sodium chloride, sodium
citrate, and sodium hydrogen citrate sesquihydrate (all analytical
grade) were purchased from J. T. Baker (USA). Primary-secondary
amine (PSA, 40 mm Bondesil) sorbent was purchased from Varian
(USA).
2.2. Standard solutions
For preliminary studies, standard stock solutions
(1000 mg mL1) of alachlor, aldrin, bifenthrin, bioallethrin, etrinfos,
fluazifop, heptachlor epoxide, malathion, oxyfluorfen, pendime-
thalin, terbufos, and trifluralin were prepared in acetone. DDT,
alpha endosulfan, beta endosulfan, phenthoate, ethyl parathion,
methyl parathion, permethrin, pirimiphos-ethyl, and fempropa-
trim were prepared in hexane. Hexachlorobenzene was prepared in
chloroform. Abamectin, used in the sorption studies, was prepared
in methanol. All the stock standard solutions were stored in dark
glass vials at 16  C. Individual intermediate standard solutions
containing 10.0 mg mL1 of these pesticides and the internal stan-
dard lindane were prepared daily by diluting 0.10 mL of the stock
standard solution in 10 mL of acetonitrile.
Method validation employed two working standard solutions.
Mixture I contained aldrin, bifenthrin, bioallethrin, chlorpyrifos-
methyl, disulfoton, alpha-endosulfan, beta-endosulfan, fempropa-
trin, o'p'DDT, oxifluorfem, ethyl parathion, methyl parathion, pen-
dimethalin, trifluralin, and the lindane internal standard. Mixture II
contained acetochlor, alachlor, etrinfos, phenthoate, fluazifop, fos-
alone, heptachlor epoxide, hexachlorobenzene, malathion,
permethrin, pirimiphos-ethyl, terbufos, and the lindane internal
standard. Mixture I, containing fifteen standards (including the
internal standard), was analyzed using Method I, while Mixture II,
containing thirteen standards (including the internal standard),
was analyzed using Method II. All standard solutions were kept in a
freezer at 16  C for up to six months.
2.3. Environmental monitoring
An apiary containing 10 beehives was installed in a bushland
region of a protected natural reserve. This field station, in Tan-
quinho Velho (municipality of Jaguariúna, Sa ~o Paulo State), is
located between latitudes 22º420 4400 and 22º420 5500 S and longi-
tudes 47º000 5300 and 47º0100500 W, near km 127.5 of the SP340
highway (Campinas-Mogi Mirim), between the Atibaia and Jaguari
rivers, and has a total area of 131.0 ha.
The main crops in and around the experimental field were sugar
cane (Saccharum officinarum L.) pasture hay (several grass species),
coffee (Coffea arabica L.), citrus (mainly Citrus sinensis), corn (Zea
mays), sunflower (Helianthus annus), eucalyptus (Eucalyptus spp.),
and green manure (several species). There were also areas of pro-
tected native forest.
Collection of 145 pollen samples was made in the experimental
apiary between May 2012 and May 2013. The collection of the
pollen samples was performed directly in the ten hives using front
porch pollen traps. This type of collector has an entrance screen
with several 4.5 mm holes that allow the passage of worker bees
but are small enough to retain the pollen grains attached to their
hind legs. Subsequently, the pollen falls into a collection drawer.
The collection period with the screen installed was continued for
three successive days, after which the bees were given free access
to the hives for approximately 30 days, in order to prevent any
negative impacts on colony development.
As a control, the presence of pesticides in the environment was
also monitored using passive samplers containing polyurethane
foam (PUF) discs, as described by Shoeib and Harner (2002). The
 R.C. de Oliveira et al. / Chemosphere 163 (2016) 525e534
PUF discs had the following specifications: 14 cm diameter x
1.35 cm thickness; density 0.018 g cm3; surface area 324 cm2; mass
3.3 g; volume 184.7 cm3. Before use, all discs were previously
cleaned by Soxhlet extraction with dichloromethane and acetone
(60  C, 8 h with each solvent) to ensure the absence of contami-
nants and/or interferences. After cleaning, each disc was dried
under an extraction hood, wrapped in aluminum foil, and stored at
room temperature until use.
For environmental sampling, six samplers were installed in the
experimental field: one in the apiary and the others in the sur-
rounding bushland. The PUFs were left in place for about 30 days,
and were exchanged and analyzed for every pollen collection
period.
The samples were placed in glass vessels (bee pollen) or wrap-
ped in aluminum foil (PUFs) and stored in a freezer at 16  C until
analysis.
2.4. Commercial bee pollen samples
Twenty-one commercial bee pollen samples were obtained
from beekeepers in the region of Ribeira~o Preto (Sa
~o Paulo State) in
the autumn of 2013. Two of these samples were reported to be from
beehives with elevated bee mortality and suspected contamination
by pesticides. The samples were packed in glass vessels and stored
in a freezer at 16  C until analysis.
2.5. Survey of pesticides to be included in the study
The pesticides considered for inclusion in this study were those
used in the region surrounding the experimental field, as well as
substances monitored by the National Residues and Contaminants
Control Program (PNCRC) of the Brazilian Ministry of Livestock
Supply and Agriculture (MAPA) and by the Program for Pesticide
Residues Analysis in Food (PARA) of the National Health Surveil-
lance Agency (ANVISA). Banned substances were also included.
Information on the use of pesticides and the current crops in the
properties surrounding the experimental field was obtained by
means of interviews with the farmers. The use of pesticides in the
experimental field was reported on a monthly basis by the re-
searchers responsible.
2.6. Analytical method
2.6.1. Bee pollen sample preparation
Bee pollen samples were defrosted in the refrigerator (5  C),
brought to room temperature, dried in a circulating air oven
(40e42  C) for up to 12 h, ground, and homogenized.
Approximately 2 g of pollen and 15 mL of acetonitrile were
transferred to polypropylene tubes (50 mL) and agitated in a vortex.
Addition was made of 4.0 g of anhydrous magnesium sulfate, 1.0 g
of sodium chloride, 1.0 g of sodium citrate, and 0.5 g of sodium
hydrogen citrate sesquihydrate, followed by further agitation and
centrifugation at 13,440 g (5  C) for 5 min. An aliquot (1 mL) of the
supernatant was transferred to a polypropylene tube (50 mL)
containing 95 mg of PSA sorbent and 750 mg of anhydrous mag-
nesium sulfate, followed by shaking in a vortex for 1 min and
centrifugation for 10 min at 13,440 g (5  C). Finally, the supernatant
was transferred to a round-bottom flask and evaporated to dryness
in a rotary evaporator at 40  C. The residue was resuspended in
1.0 mL of acetonitrile and analyzed by GC-MS/MS.
2.6.2. PUF sample preparation
The PUF samples were defrosted in a refrigerator (5  C), brought
to room temperature, cut into small pieces, and then Soxhlet
extracted with dichloromethane at 60  C for 8 h.
527
The extracts were collected in round-bottom flasks and evapo-
rated to dryness in a rotary evaporator at 40  C. The residues were
resuspended in 5.0 mL of acetonitrile and transferred to 12 mL
polypropylene tubes. The extracts were evaporated to dryness and
the residues were resuspended in 1.0 mL of acetonitrile before GC-
MS/MS analysis.
2.7. Gas chromatography analysis
The pesticides were analyzed using a Varian 3900 GC gas
chromatograph equipped with a Saturn 2100 T ion trap mass
spectrometer (Varian Inc., Palo Alto, CA, USA). The data were ac-
quired and processed with MS Workstation v. 6.5 software (Varian
Inc., Palo Alto, CA, USA). The analytes were separated on an Rxi® 5
Sil (30 m, 0.25 mm id., 0.25 mm) capillary column (Restek Corpo-
ration, Bellefonte, PA, USA). The column oven temperature was
programmed from 100  C (hold for 2 min) to 150  C (hold for
10 min) at 10  C min1, and then to 250  C (hold for 6 min) at
5  C min1. The injection temperature and volume were 250  C and
1 mL, respectively, and the trap, manifold, and transfer line tem-
peratures were set at 220, 60, and 280  C, respectively. The carrier
gas was helium at a constant flow rate of 1 mL min1. Single ion
monitoring (SIM) was used for confirmation and quantitation of the
pesticides, as required by SANCO document 10684/2009 (SANCO/
10684/2009). The retention times, precursor ions, collision en-
ergies, and the selected quantitation and confirmation ions are
shown in Table S1.
2.8. High performance liquid chromatography analysis of
abamectin
Abamectin was determined in pollen using the method
described by Dionisio and Rath (2016). The chromatographic sys-
tem consisted of an Agilent Series 1200 HPLC (Agilent Technologies,
USA) equipped with a G1311A quaternary pump, a G1321A fluo-
rescence detector (FLD), a G1316A column oven, and a G1329A
autosampler. Abamectin was separated on a LiChroCART® 55-4
Purospher® STAR RP-18 column (4.0 mm  50 mm, 3 mm) (Merck,
Germany) at 30  C. The mobile phase was 98:2 (v/v) methanol:-
water, at a flow rate of 0.8 mL min1. The injection volume was 5 mL.
The excitation and emission wavelengths were set at 365 nm and
470 nm, respectively.
2.9. Method development and validation
Validation of the developed GC-MS/MS analytical method for
the determination of pesticide residues in bee pollen was carried
out based on SANCO guideline (SANCO/10684/2009).
Calibration curves were constructed using solvent, fortified
blank bee pollen samples, and fortified blank bee pollen extracts,
employing the ordinary least-squares method. The linearity and
linear range were determined from calibration curves obtained by
analysis of fortified blank bee pollen samples at six concentration
levels (10.0, 25.0, 50.0, 100.0, 200.0, and 300.0 ng g1), with lindane
as a surrogate internal standard (at 200.0 ng g1). Possible outliers
were evaluated by the Jacknife standardized residual test. The ho-
mogeneity of variance was evaluated by Levene's test.
The selectivity of the method was evaluated by the analysis of
six different commercial pollen samples in order to identify any
interference of co-extractives derived from the sample matrix.
Matrix effects were evaluated by comparing the calibration
curves obtained using the fortified blank bee pollen extracts (10.0,
25.0, 50.0, 100.0, 200.0, and 300.0 ng mL1) and the calibration
curves obtained at the same concentration levels using solvent.
Lindane (200 ng mL1) was used as a surrogate internal standard.
528 R.C. de Oliveira et al. / Chemosphere 163 (2016) 525e534
For each concentration level, the matrix effect was expressed as the
percentage of signal reduction or enhancement in the extract,
relative to the standard prepared in solvent.
The within-laboratory repeatability was evaluated using blank
bee pollen samples fortified with the pesticides at three different
concentration levels (25.0, 100.0, and 300.0 ng g1). Six indepen-
dent replicates of each sample were analyzed on the same day by
the same analyst and with the same equipment. The results were
expressed as the relative standard deviation.
Accuracy was assessed by analysis of blank bee pollen samples
fortified with the 26 pesticides at three concentration levels (25.0,
100.0, and 300.0 ng g1), using six independent replicates (n ¼ 6).
The accuracy was expressed as the percentage recovery, defined as
the ratio of the measured values to the values predicted by the
calibration graph regression. According to SANCO document 10684/
2009, acceptable recovery values are between 70 and 120%.
The limits of detection (LODs) and quantitation (LOQs) were
determined by analysis of blank bee pollen samples fortified at
decreasing analyte concentrations (300.0, 200.0, 100.0, 50.0, 25.0,
and 10.0 ng g1), evaluating the accuracy and precision of the re-
sults. The limit of detection (LOD) was equal to the minimum
detectable concentration of each analyte present in the fortified
sample, based on a signal-to-noise ratio of 3. The limit of quanti-
tation (LOQ) was equal to the minimum concentration of each
analyte present in the spiked sample, based on a signal-to-noise
ratio of 10, which could be quantified by the analytical method
with adequate precision (lower than 20%) and accuracy (recovery
between 70 and 120%).
2.10. Palynological analysis
Ten bee pollen samples collected in the region of Ribeir~
ao Preto
~o Paulo State) were sent for palynological analysis at the Center
(Sa
for Research in Palynology of the Institute of Botany, Department of
the Environment of Sa ~o Paulo. The method employed was the Eu-
ropean standard for honey samples described by Maurizio and
Louveaux (1965), without acetolysis and with the modifications
described by Barth et al. (2009) and Modro et al. (2009).
2.11. Sorption experiments
Sorption kinetics and sorption isotherms were determined for
aldrin, malathion, and abamectin. Since there are no guidelines for
determination of the sorption of pesticides by pollen, the experi-
ments were based on the method described in the OECD 106 guide
for adsorption/desorption tests of chemicals in soil samples (OECD,
2000).
The times taken for the pesticides to reach equilibrium were
determined using a pollen:water ratio of 1:100 (w/v). Suspensions
containing 100 mg of pollen and 10 mL of water were agitated for
12 h in glass (aldrin and abamectin) or plastic (malathion) Falcon
tubes (50 mL), using an orbital shaker with temperature control
(24  C). Subsequently, the analytes were added at concentrations of
1.0 mg g1 and the suspensions were agitated for 0, 1, 2, 3, 5, 7, 20,
and 24 h in an orbital shaker (24  C). After agitation, the suspen-
sions were centrifuged (13,440 g, 15 min) and the supernatants
were discarded. The pollen was extracted using 10 mL of acetoni-
trile, with vortex agitation, followed by centrifugation (13,440 g,
15 min). The extracts were filtered (0.22 mm) and analyzed by GC-
MS/MS for aldrin and malathion (Section 2.7) and by HPLC-FLD for
abamectin (as described by Dionisio and Rath (2016)). The quan-
titation of the analytes in the bee pollen was carried out using the
matrix-matched calibration curves, and the concentrations ob-
tained (in mg g1 pollen) were used to calculate the amounts of
sorbed analyte for each equilibration time (qt). All the procedures
were carried out in duplicate. The equilibration time was estimated
by plotting the sorption percentage as a function of time. The data
were fitted to pseudo-first order, pseudo-second order, and Elovich
models in order to establish the sorption kinetics mechanism, as
described by Peruchi et al. (2015).
For the sorption isotherms, suspensions containing 100 mg of
pollen and 10 mL of water were agitated for 12 h in glass (aldrin and
abamectin) or plastic (malathion) Falcon tubes (50 mL), using an
orbital shaker with temperature control (24  C). The individual
analytes were then added to give final concentrations of 0.5, 1.0, 2.0,
5.0, 10.0, and 20.0 mg g1. The mixtures were agitated for 20 h in the
orbital shaker (24  C), centrifuged (13,440 g, 15 min), and the su-
pernatant was discarded. The pollen samples were analyzed as
described in Section 2.10. The analyte concentrations in the extracts
were determined using the matrix-matched calibration curves
obtained by analysis of blank bee pollen samples fortified with the
respective analytical standards, followed by calculation of the
1
amount of analyte sorbed by the pollen (Cads p , mg g pollen). The
concentrations of the analytes in the aqueous solutions (Cads aq , mg
mL1) were calculated from the difference between the initial
concentrations in the aqueous solutions and Cads p . All the proced-
ures were carried out in duplicate. The results were modeled by
means of the Freundlich equation (Eq. (1)), as described by Peruchi
et al. (2015). This model proposes an empirical isotherm for a non-
ideal system, considering a heterogeneous surface, non-equivalent
bonding sites, and a multilayer sorption process.
1
log Csads ðeq:Þ ¼ log Kfads þ log Caq
ads
ðeq:Þ [1]
n
where Cads ads
p and Caq are the concentrations of analyte in the pollen
and the aqueous phase, respectively, Kf (mg g11/n (cm3)1/n g1) is
the Freundlich sorption coefficient, and 1/n is the Freundlich
exponent or linearity factor, a constant related to sorption intensity.
3. Results and discussion
3.1. Selection of pesticides and multiresidue method development
Data collected at the Embrapa Environment experimental field
in Jaguariúna, together with information available in ANVISA and
PNCRC reports and surveys of neighboring properties (where
farmers reported the sporadic use of abamectin, carbendazim,
mancozeb, spirodiclofen, imidacloprid, thiophanate-methyl,
dimethoate, beta-cyfluthrin, glyphosate, deltamethrin, and 2,4 D),
resulted in the selection of 26 pesticides that are commonly
determined by gas chromatography. The physico-chemical prop-
erties of these compounds are provided in Table S2. Among them,
25 pesticides (96%) are included in the Program on Pesticide Res-
idue Analysis in Food (PARA), coordinated by ANVISA, 18 (69%) are
included in the National Residue and Contaminant Control Program
(PNCRC), coordinated by MAPA, and 9 pesticides (35%) are banned
in Brazil.
3.2. Method development and validation
The aim was to establish a method to determine concentrations
at least as low as 100 ng g1. However, limiting factors were the low
detectability of the ion trap mass analyzer for determination of
most of the analytes at this concentration level, together with low
chromatographic resolution. The initial lack of resolution observed
was overcome by developing two monitoring methods (I and II),
which enhanced detectability by increasing the retention time
window for monitoring each ion. Both methods used the temper-
ature program described earlier, but the mass analyzer was set to
 R.C. de Oliveira et al. / Chemosphere 163 (2016) 525e534 529

monitor different ions in each method. The development of two
programs for ion monitoring enabled determination of all the
selected pesticides. Lindane was used as the surrogate internal
standard in both methods.
After defining the experimental parameters of the GC-MS/MS
technique for the determination of multiresidue pesticides in bee
pollen, the sample preparation procedure was evaluated. For this
purpose, the QuEChERS approach and a simple solid-liquid
extraction procedure using acetonitrile were assessed. The
QuEChERS approach (Anastassiades et al., 2003), which consists of
solvent partitioning under buffered conditions and dispersive solid
phase extraction, was successfully used previously for the deter-
mination of 253 pesticides in pollen samples by gas and liquid
chromatography coupled to tandem mass spectrometry (Vazquez
et al., 2015). Although this procedure is simple, it does not elimi-
nate matrix effects caused by the co-extraction of undesired com-
pounds from the sample. Some of the parameters that affect
pesticide analysis by gas chromatography-ion trap mass spec-
trometry were discussed by Przybylski and Hommet (2008).
Optimization of the sample preparation process took into
consideration the extraction efficiencies (Fig. 1) and matrix effects
(Fig. 2).
In general, the QuEChERS approach resulted in higher extraction
efficiencies than simple solvent extraction of the pesticides from
the bee pollen using acetonitrile. The addition of a significant
amount of salt to the media acted to increase mass transfer of the

Fig. 1. Extraction efficiencies using the QuEChERS approach and a solid-liquid extraction with acetonitrile. Sample amount: 2 g, concentration of pesticides: 200 ng g1.

Fig. 2. Matrix effect using the QuEChERS approach or solid-liquid extraction with acetonitrile. Sample amount: 2 g.
530 R.C. de Oliveira et al. / Chemosphere 163 (2016) 525e534

pesticides to the solvent. The QuEChERS approach also significantly
decreased matrix effects, compared to solid liquid extraction (SLE).
This technique was therefore selected for sample preparation.
Finally, the method was validated in-house. Due to the positive
matrix effects observed, calibration curves were constructed using
fortified blank bee pollen samples, compensating the matrix effects
and ensuring the reliability of the results.
The selectivity of the method was confirmed by the absence of
interfering substances appearing at the retention times of the
pesticides that could compromise the identification or quantitation
of the analytes or the internal standard in six blank bee pollen
samples from different sources.
The validation parameters considered were linear range, line-
arity, matrix effects, LOD, and LOQ (Table 1). The linearity was
higher than 0.91 for all the pesticides and the LOQ varied from 10 to
100 ng g1. The most pronounced matrix effect was observed for
pirimiphos-ethyl, while the lowest was for phosalone.
Precision, expressed as the relative standard deviation (RSD),
was lower than 20%. Accuracy, expressed as recovery, was between
70% and 120% for all the concentration levels of the 26 pesticides
tested (25.0, 100.0, and 300.0 ng g1, n ¼ 6).
3.3. Sorption experiments
In order to evaluate the potential of bee pollen as a bioindicator
of environmental contamination, it is essential to assess its affinity
for the substances investigated. For this purpose, three pesticides
with different log Kow were selected: aldrin, malathion, and aba-
mectin. Determination of the sorption equilibration time is a key
step required by OECD 106 for undertaking sorption studies (OECD,
2000). The equilibration times were obtained by plotting the
sorption percentage against time (0, 1, 2, 4, 5, 7, 20, and 24 h). The
initial concentrations of the analytes in contact with pollen were
1.0 mg g1. The graphs indicated that 20 h was long enough to reach
the apparent sorption equilibrium for all the analytes evaluated,
and this time was used to establish the sorption isotherms. After
20 h, the sorption percentages of aldrin, malathion, and abamectin
were 90%, 62%, and 82%, respectively.
For all the analytes evaluated, the pseudo-second order model
provided better fits to the data than the other models (pseudo-first
order and Elovich) (Table 2). This indicated that the sorption ca-
pacity of the pollen mainly depended on the availability of active
sites, rather than the analyte concentrations in solution.
Sorption processes are influenced by the sorbent properties
(polarity, presence of bonding groups, porosity, and surface area),
the solute properties (the nature of the compound and its chemical
structure, considering ionic charges, molecular size, presence of
rings, and polar groups), and by external factors such as pH, tem-
perature, and agitation intensity (Marshall et al., 1999). In order to
estimate the amount of each substance sorbed per gram of pollen,
as a function of analyte concentration in solution, sorption iso-
therms modeled using the logarithmic form of the Freundlich
equation (Eq. (1)) were constructed. The results are shown in
Table 3.
Aldrin, malathion, and abamectin presented Freundlich sorption
coefficients (KF) of 2.95, 0.52, and 2.06 mg11/n (cm3)1/n g1,
respectively. These values are of the same order of magnitude as
those reported by Thio et al. (2011) for magnetized ragweed pollen
interacting with the pesticides atrazine, diuron, and lindane
(KF ¼ 1.86, 1.14, and 1.22 mg11/n (cm3)1/n g1, respectively). Based
Table 2
Sorption kinetic parameters of aldrin, malathion and abamectin for bee pollen.
Pesticide Pseudo-second-order model
qe (mg g1) k2 (104 g mg1 min1) r
Aldrin 0.86 934 0.998
Malathion 0.62 501 0.999
Abamectin 0.78 482 0.997

Table 1
Validation parameters of the analytical method.

Pesticide Matrix effect (%) Linear range (ng g1) LOD (ng g1) LOQ (ng g1) Linearity (r) Classification

PNCRCa PARAb Banned

Acetochlor 105 25e300 10 25 0.9619 X

Alachlor 109 25e300 10 25 0.9738 X X
Aldrin 121 50e300 25 50 0.9981 X X
Alpha endosulfan 111 50e300 25 50 0.9801 X X X
Beta endosulfan 74 50e300 25 50 0.9719 X X X
Bifenthrin 71 100e300 50 100 0.9909 X X
Bioallethrin 104 25e300 10 25 0.9547 X
Disulfoton 96 50e300 25 50 0.9437 X X
Ethyl parathion 129 25e300 10 25 0.9649 X X
Phirimiphos-ethyl 156 25e300 10 25 0.9732 X
Etrinfos 112 10e300 5 10 0.9837 X X
Fempropatrim 70 25e300 10 25 0.9606 X X X
Fluazifop 80 100e300 50 100 0.9629 X X
Phosalone 42 100e300 50 100 0.9244 X
Heptachlor epoxide 116 50e300 25 50 0.9108 X X
Hexachlorobenzene 83 100e300 50 100 0.9603 X
Malathion 96 25e300 10 25 0.9748 X X
Methyl Chlorpyrifos 82 25e200 10 25 0.9829 X X
Methyl parathion 90 100e200 50 100 0.9838 X X X
o'p'DDT 103 25e300 10 25 0.9937 X X
Oxifluorfem 91 25e300 10 25 0.9712 X X
Pendimethalin 130 25e300 10 25 0.9945 X X
Permethrin 69 50e300 25 50 0.9529 X X
Phenthoate 143 100e300 50 100 0.9628 X X
Terbufos 100 10e300 5 10 0.9944 X X
Trifluralin 81 25e300 10 25 0.9939 X X
a
National Residues and Contaminants Control Program of the Brazilian Ministry of Livestock Supply and Agriculture (MAPA).
b
Program for Pesticide Residues Analysis in Food of the National Health Surveillance Agency (ANVISA).
 R.C. de Oliveira et al. / Chemosphere 163 (2016) 525e534
Table 3
Parameters of sorption of the pesticides on bee pollen.
Pesticide Log Kow Freundlich
KF (mg11/n (cm3)1/n g1) 1/n
Aldrin 6.5 2.95 0.9558
Malathion 2.75 0.52 1.2183
Abamectin 4.4 2.06 0.8463
on the KF values, it was suggested that pollen could be as effective
as activated carbon in sorbing pesticides and other contaminants
from water (Thio et al., 2011). The results therefore indicated the
potential of bee pollen as a bioindicator of environmental
contamination, confirming that there can be significant transfer to
pollen of pesticides such as aldrin, malathion, and abamectin pre-
sent in the surrounding atmosphere.
Additionally, a direct relationship (r ¼ 0.98) was observed be-
tween the affinity of the pesticides for bee pollen and their octanol-
water partition coefficients (log Kow). This suggests that the main
mechanism responsible for the sorption process was probably hy-
drophobic interaction between the pesticide and lipophilic sites of
pollen, which is composed of 10e40% proteins, 1e13% lipids,
13e55% total carbohydrates together with fibers, minerals, and
vitamins (Campos et al., 2008). This is potentially a very useful
finding, because it could enable estimation of the sorption capacity
of any pesticide in bee pollen simply by considering the log Kow
value. New tests employing different pesticides are needed to
confirm this hypothesis.
3.4. Environmental monitoring
Pollen samples were collected from the ten beehives of the
experimental field during five months. The productivity varied
among the beehives (Fig. 3), with the lowest amount (5 g) collected
in beehive #8 and the highest amount (29 g) in beehive #5.
Low pollen productivity can be influenced by many factors: low
availability of floral resources; low rates of input and storage of the
pollen in the hive; poor queen laying rate; adverse queen genetic
factors; lack of space or feed; excess wind; death of larvae due to
Fig. 3. Pollen production of each beehive. From bottom to top: March 2012; May 2012; March 2013; April 2013 and May 2013.
531
disease; very weak swarms; and populous swarms (Magalha ~es,
2005). In the present case, all the boxes and pollen collectors
were the same in the 10 hives, there were no problems of disease or
r
population extremes, and all the colonies had access to the same
type and amount of food. The differences in productivity were
0.9856
therefore attributed to the genetic characteristics of each hive. The
0.9903
0.9911 humidity of the pollen samples varied between 3 and 48%,
depending on the weather conditions at the time of collection.
The validated method was used to evaluate the presence of the
26 pesticides in the pollen samples, as well as in the 30 PUF control
samples that were collected during the same periods (6 PUF sam-
ples at each collection). None of the PUF samples showed detect-
able levels of the pesticides analyzed.
Among the 145 samples collected in the experimental apiary, 18
(12%) presented residues of pendimethalin, but at concentrations
below the limit of quantitation, while four samples (3%) showed
positive results for bioallethrin, also below the limit of quantitation.
For both compounds, the LOQ was 25 ng g1.
Pendimethalin is a dinitroaniline herbicide that is widely used in
Brazil for the control of annual grasses and most broad-leaved
weeds in crops of corn, potato, rice, cotton, soybean, tobacco, and
sunflower (Rodrigues and Almeida, 2005; Coutinho et al., 2006). In
humans, pendimethalin is slightly toxic and may be mildly irritating
to the mucous membranes of the mouth, nose, throat, and lungs.
Bioallethrin is a member of the pyrethroid class pesticides,
which are widely used in agriculture. Due to their low toxicity to
humans and their rapid and effective insecticidal action, pyre-
throids are also commonly used as household insecticides. In Brazil,
the use of bioallethrin is authorized for domestic purposes
(ANVISA, 2016). These two pesticides were not used in the apiary,
and their use was not reported by farmers in the properties sur-
rounding the experimental field.
3.5. Analysis of commercial samples
In addition to the bee pollen collected in the experimental area,
analysis was made of 21 commercial bee pollen samples obtained
~o Preto (S~
from producers in the city of Ribeira ao Paulo State). Seven
of the samples (33%) were contaminated with at least one pesticide,
532 R.C. de Oliveira et al. / Chemosphere 163 (2016) 525e534
Table 4
Pesticide residues (ng g1) determined in bee pollen samples collected in commercial apiaries from Ribeir~
Sample Concentration, ng g1 (RSD, %)
Trifluralin Bioallethrin Aldrin
RP59 5.0 (2.0)
RP62 432 (10.0)
RP64 <25
RP67
RP68 102 (9.0)
RP70 <25
RP74
and five (24%) contained residues of two or more substances
(Table 4).
Sample RP59, which was collected from a beehive with sus-
pected pesticide contamination, contained the herbicides trifluralin
and alachlor. This suggested that the contamination probably
occurred in citrus plantations, because trifluralin is approved for
use on citrus, and was corroborated by a predominance in this
sample of pollen grains of the genus Citrus (Table S3).
Although it is practically non-toxic to humans and bees, triflu-
ralin is highly toxic to fish and other aquatic animals, in which it can
accumulate, with chronic effects in fish fingerlings and crustaceans
at levels as low as 0.005 mg g1. In the European Union, MRL values
for trifluralin are between 10.0 and 20.0 ng g1 for fruits and veg-
etables. There are no regulations concerning pollen samples, so
according to the EU, the MRL should therefore be considered equal
to 10.0 ng g1, as established for unregulated substances and
products (EC-396/2005). In Brazil, MRLs for trifluralin vary between
20.0 and 50.0 ng g1 for vegetables and fruits (ANVISA, 2015).
The herbicide alachlor is used to control annual grasses and
weeds in corn, cotton, soybean, sugar cane, coffee, groundnut, and
sunflower crops. However, its use is under international discussion,
due to the risks it may pose to health (CETESB, 2014). A sample
collected in the same apiary, but with no suspected contamination,
showed a heterogeneous floral profile with a greater contribution
of pollen from weeds (Baccharis and Bidens) and Eucalyptus.
No evidence of the 26 pesticides was found in another sample
collected from a beehive with a history of bee deaths. In this case,
there was a predominance of pollen grains from the genus Alter-
nanthera, family Amaranthaceae, which are weeds in terrestrial or
aquatic environments (Table S3).
All the other contaminated samples showed monofloral profiles
of pollen from the genus Alternanthera, indicating a possible source
of contamination. Most were contaminated with insecticides
instead of herbicides. The Alternanthera pollen could have been
derived from the species Alternanthera tenella Colla, which is a
native invasive plant controlled by the use of glyphosate (Petter
et al., 2007).
The highest level of contamination was found for bioallethrin
(432 ng g1). According to the US EPA (United States Environmental
Protection Agency), bioallethrin presents a low risk to bees by oral
exposure or contact, with an LD50 value of 3400 ng bee1 (EPA,
2009), which explains the survival of the colony despite the high
level of contamination. This insecticide, also found in samples
collected during environmental monitoring, is inexpensive and is
used as a domestic insecticide.
Permethrin was detected in four samples. This pyrethroid
insecticide is slightly toxic to humans and is widely used in public
health campaigns, especially for the control of ectoparasites of
small animals. In Brazil, permethrin is authorized for use on cotton,
rice, coffee, citrus, cauliflower, bean, tobacco, corn, cabbage, soy-
bean, tomato, wheat, and grape crops (ANVISA, 2010). Despite be-
ing relatively safe for humans, it is highly toxic to bees, affecting
ao Preto.
Alpha endosulfan Fempropatrim Alachlor Permethrin
<50
145 (1.0) <50
<25 <50
<25 <50
<25 70 (2.0)
their behavior and causing negative impacts on their social inter-
action and food collection ability (Ingram et al., 2015).
Aldrin was widely employed in the 1970s, especially in cotton
and corn crops, but was banned due to its high environmental
persistence and bioaccumulation potential. Hence, even after being
banned, aldrin and dieldrin can still be found in the environment
(ONU, 2001).
The organochlorine compound endosulfan is classified as
moderately hazardous (Class 2) (WHO, 2009). It was introduced
commercially in 1954 for the control of insects in fruits, vegetables,
teas, and non-food crops such as tobacco and cotton. However, it
has been banned or restricted in many countries because of its risks
to human health and the environment. In 2010, Brazil cancelled the
registration of nine products based on endosulfan and scheduled
the withdrawal of this substance from the Brazilian market by July
2013, when it was finally banned (CETESB, 2015).
The alpha isomer of endosulfan was detected in one of the
samples. The alpha and beta isomers of the compound are both
biologically active, and in the final product are found in a propor-
tion of around 70% alpha and 30% beta. Under aerobic conditions in
neutral and acidic soils, these isomers persist for around 1e2
months (alpha-endosulfan) and 3e9 months (beta-endosulfan)
(EC-396/2005).
Chauzat et al. (2006) reported endosulfan contamination in 7%
of pollen samples in a monitoring study conducted over three years
in apiaries located in France. The average level of contamination
was 81 ng g1. In other work conducted in France, Wiest et al.
(2011) found one honey sample contaminated with endosulfan at
31 ng g1, among 142 samples collected from 16 apiaries
throughout the country between 2008 and 2009.
In Brazil, in a study with pollen samples from the five macro-
regions of the country, Santos and Funari (2005) detected aldrin in
one sample at a concentration of 2 ng g1, and endosulfan in 19
samples at levels ranging from 1 to 33 ng g1. Overall, among 69
samples, 34 (49%) were contaminated with endosulfan or other
banned pesticides such as zolone, dieldrin, endrin aldehyde, epoxy
heptachlor, endrin, p,p'-DDE, and p,p'-DDT, at concentrations
ranging between 1 and 94 ng g1. Organophosphorus compounds
such as coumaphos, fenthion, dichlorvos, diazinon, malathion,
zolone, and methyl parathion were also found in 31 samples (45%)
at levels between 1 and 25 ng g1. The pyrethroids deltamethrin,
cyfluthrin, cypermethrin, and permethrin were found in 41 sam-
ples (59%). The concentrations of permethrin ranged from 1 to
272 ng g1, and the sum of the pyrethroids found in one sample
reached 715 ng g1. Aldrin and endosulfan residues were also found
at concentrations ranging from 1 to 21 ng g1 in honey samples
~o Paulo State) between 1999 and
collected in the region of Bauru (Sa
2004 (Rissato et al., 2006).
4. Conclusions
The GC-MS/MS ion trap method developed enabled the
 R.C. de Oliveira et al. / Chemosphere 163 (2016) 525e534
determination of 26 pesticides in bee pollen with adequate preci-
sion and accuracy. The detection and quantitation limits varied
from 5.0 ng g1 to 50.0 ng g1 and from 10.0 ng g1 to 100.0 ng g1,
respectively. The sample preparation step is important for quanti-
tation at low concentration levels with high selectivity, and here
the QuEChERS approach was more appropriate for this purpose
than solvent extraction with acetonitrile.
Sorption studies indicated that the pesticides could be sorbed by
bee pollen. Direct relationships between the affinities of the pes-
ticides for bee pollen and their octanol-water partition coefficients
(log Kow) were observed for the three pesticides assessed (mala-
thion, abamectin, and aldrin). This behavior could provide a useful
indication of those pesticides with greatest potential to be found in
bee pollen, supporting the notion that bee pollen can be used as an
indicator of environmental contamination.
Low or undetectable levels of pesticides in the bee collected
pollen samples during the environmental monitoring could have
been due to reduced agricultural activity during the collection pe-
riods or the privileged location of the beehives in the bushland.
Analysis of the PUF samples used as control passive sampling de-
vices showed no detectable levels of the pesticides investigated,
suggesting non-use of these substances in the experimental field
and the wider region, or an absence of transport from neighboring
areas.
The presence of pesticides in 33% of the samples provided by
beekeepers from Ribeir~ ao Preto confirmed the efficiency of the
validated analytical method and the need for environmental
monitoring of the presence of pesticide residues. The concentra-
tions measured were similar to those found in other studies.
Acknowledgments
This work was supported by Fundaça ~o de Amparo a Pesquisa do
Estado de Sa~o Paulo (FAPESP, #2010/18253-4 and #2013/09543-7),
Conselho Nacional de Desenvolvimento Científico e Tecnolo gico
(CNPq, #476501/2013-0) and the Brazilian Agricultural Research
Corporation (Embrapa). We thank Dr. Ricardo Camargo, from
Embrapa, for all the support with the apiary and the bee pollen
collection.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.chemosphere.2016.08.022.
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