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
Center for Research in Palynology, Institute of Botany, Department of the Environment of Sa~o Paulo, SP, Brazil

h ig hl ig ht 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.

articleinfo
abstract
Article history:
Received 2 June 2016 Honeybees and bee products are potential bioindicators of the presence of contaminants in the envi-
Received in revised form ronment, enabling monitoring of large areas due to the long distances travelled by bees. This work
2 August 2016 evaluates the use of bee pollen as a bioindicator of environmental contamination by pesticides. A GC-
Accepted 3 August 2016 MS/ MS analytical method for multiresidue determination of 26 different pesticides in pollen was
developed 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
Handling Editor: I. Cousins
beehives
in the experimental apiary of Embrapa in Jaguariúna (Sa~o Paulo State, Brazil). Bioallethrin and
Keywords:
pendi-
Bee pollen
methalin were identified in four and eighteen samples, respectively, at concentrations below the LOQ of
Bioindicator
Pesticides the method (25 ng g—1). Passive sampling with polyurethane foam discs was used as a control, and no
Contamination pesticides were found. The detection of pesticide residues in seven samples (33%) from commercial
Multiresidues apiaries in Ribeira~o Preto (S~ao Paulo State) confirmed the efficiency of the analytical method and
GC-MS/MS the
need for environmental monitoring for the presence of pesticide residues. The results demonstrated the
potential of bee pollen as a bioindicator of environmental contamination by pesticides.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
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).
constant application of pesticides poses risks to human health, due
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.
 R.C. de Oliveira et al. / Chemosphere 163 (2016) 525e534
52
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 mL—1) 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 mL—1 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
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.
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 min—1, and then to 250 ○C (hold for 6 min) at
5 ○C min—1. 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 min—1. 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 min—1. 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 g—1), with
lindane as a surrogate internal standard (at 200.0 ng g —1). 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 mL—1) and the calibration
curves obtained at the same concentration levels using solvent.
Lindane (200 ng mL—1) was used as a surrogate internal standard.
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 g—1).
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 g —1), 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 g—1), 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
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 g—1. 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
amount of analyte sorbed by the pollen (Cpads, mg g—1 pollen). The
concentrations of the analytes in the aqueous solutions (C ads, m
aqg mL
—1
) were calculated from the difference between the initial
concentrations in the aqueous solutions and Cpads. 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 Kf ads þ log Caadsðeq:Þ [1]
n q
where Cads and Cads are the concentrations of analyte in the pollen
p aq

Ten bee pollen samples collected in the region of Ribeir~ao tained (in mg g pollen) were used to calculate the amounts of
—1

Preto (Sa~o Paulo State) were sent for palynological analysis at the sorbed analyte for each equilibration time (qt). All the procedures
Center
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 g—1 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-
and the aqueous phase, respectively, K f (mg g1—1/n (cm3)1/n g—1) 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 g—1. 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
monitor different ions in each method. The development of two
chromatography coupled to tandem mass spectrometry (Vazquez
programs for ion monitoring enabled determination of all the
et al., 2015). Although this procedure is simple, it does not elimi-
selected pesticides. Lindane was used as the surrogate internal
nate matrix effects caused by the co-extraction of undesired com-
standard in both methods.
pounds from the sample. Some of the parameters that affect
After defining the experimental parameters of the GC-MS/MS
pesticide analysis by gas chromatography-ion trap mass spec-
technique for the determination of multiresidue pesticides in bee
trometry were discussed by Przybylski and Hommet (2008).
pollen, the sample preparation procedure was evaluated. For this
Optimization of the sample preparation process took into
purpose, the QuEChERS approach and a simple solid-liquid
consideration the extraction efficiencies (Fig. 1) and matrix effects
extraction procedure using acetonitrile were assessed. The
(Fig. 2).
QuEChERS approach (Anastassiades et al., 2003), which consists of
In general, the QuEChERS approach resulted in higher
solvent partitioning under buffered conditions and dispersive solid
extraction efficiencies than simple solvent extraction of the
phase extraction, was successfully used previously for the deter-
pesticides from the bee pollen using acetonitrile. The addition of a
mination of 253 pesticides in pollen samples by gas and liquid
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 g —1.

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

the sorption percentage against time (0, 1, 2, 4, 5, 7, 20, and 24 h). Aldrin 0.86 934 0.998
The initial concentrations of the analytes in contact with pollen Malathion 0.62 501 0.999
Abamectin 0.78 482 0.997
were
1.0 mg g—1. The graphs indicated that 20 h was long enough to reach
the apparent sorption equilibrium for all the analytes evaluated,

Table 1
Validation parameters of the analytical method.

Pesticide Matrix effect (%) Linear range (ng g—1) LOD (ng g—1) LOQ (ng g—1) 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).
Table 3
Parameters of sorption of the pesticides on bee pollen.
Pesticide Log Kow Freundlich
KF (mg1—1/n (cm3)1/n g—1) 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 (r¼0.98) 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 10 e40% 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 K ow
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.
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
r disease or population extremes, and all the colonies had access to
0.9856 the same type and amount of food. The differences in productivity
0.9903 were therefore attributed to the genetic characteristics of each hive.
0.9911 The 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 g—1.
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
from producers in the city of Ribeira~o Preto (S~ao Paulo State).
Seven of the samples (33%) were contaminated with at least one
pesticide,
Table 4
Pesticide residues (ng g—1) determined in bee pollen samples collected in commercial apiaries from Ribeir~ao Preto.
Sample Concentration, ng g —1 (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 g—1. In the European
Union, MRL values
for trifluralin are between 10.0 and 20.0 ng g—1 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 g—1, as established for unregulated substances and
products (EC-396/2005). In Brazil, MRLs for trifluralin vary
between
20.0 and 50.0 ng g —1 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 g—1). According to the US EPA (United States
Environmental Protection Agency), bioallethrin presents a low risk
to bees by oral exposure or contact, with an LD 50 value of 3400 ng
bee—1 (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
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 g—1. In other work conducted in France, Wiest et al.
(2011) found one honey sample contaminated with endosulfan at
31 ng g—1, 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 g—1, and
endosulfan in 19 samples at levels ranging from 1 to 33 ng g—1.
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 g—
1
. 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 g—1. The pyrethroids deltamethrin,
cyfluthrin, cypermethrin, and permethrin were found in 41
sam- ples (59%). The concentrations of permethrin ranged
from 1 to 272 ng g—1, and the sum of the pyrethroids found in
one sample reached 715 ng g—1. Aldrin and endosulfan residues
were also found at concentrations ranging from 1 to 21 ng g—1
in honey samples
collected in the region of Bauru (Sa~o Paulo State) between 1999 and
2004 (Rissato et al., 2006).
4. Conclusions
The GC-MS/MS ion trap method developed enabled the
determination of 26 pesticides in bee pollen with adequate preci-
sion and accuracy. The detection and quantitation limits varied
from 5.0 ng g—1 to 50.0 ng g—1 and from 10.0 ng g—1 to 100.0 ng g—1,
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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