- Email: [email protected]
Evaluation of pesticide residues in honey from different geographic regions of Colombia
Accepted Manuscript Evaluation of pesticide residues in honey from different geographic regions of Colombia Danny Rodríguez López, Diego Alejandro Ahumada, Amanda Consuelo Díaz, Jairo Arturo Guerrero PII:
S0956-7135(13)00457-X
DOI:
10.1016/j.foodcont.2013.09.011
Reference:
JFCO 3453
To appear in:
Food Control
Received Date: 10 April 2013 Revised Date:
1 September 2013
Accepted Date: 5 September 2013
Please cite this article as: Rodríguez LópezD., Alejandro AhumadaD., DíazA.C. & GuerreroJ.A., Evaluation of pesticide residues in honey from different geographic regions of Colombia, Food Control (2013), doi: 10.1016/j.foodcont.2013.09.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Evaluation of pesticide residues in honey from different geographic regions of
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Colombia
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Danny Rodríguez Lópeza; Diego Alejandro Ahumadab; Amanda Consuelo Díazc; Jairo
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Arturo Guerrerod*
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Laboratorio de Análisis de Residuos de Plaguicidas. Departamento de Química, Facultad
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de Ciencias, Universidad Nacional de Colombia, Carrera 30 No. 45-03, Edificio 451, Of.
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206, Bogotá, Colombia. Email: [email protected]
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Subdirección de Metrología Química y Biomedicina. Instituto Nacional de Metrología de Colombia. Carrera 50 26-55. Int. 2. Centro Administrativo Nacional, Bogotá, Colombia.
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Email: [email protected]
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c
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Colombia, Carrera 30 No. 45-03, Edificio 401, Of. 206, Bogotá, Colombia. Email:
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[email protected]
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Laboratorio de Análisis de Residuos de Plaguicidas. Departamento de Química, Facultad
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Instituto de Ciencia y Tecnología de Alimentos- ICTA, Universidad Nacional de
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de Ciencias, Universidad Nacional de Colombia, Carrera 30 No. 45-03, Edificio 451, Of.
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206, Bogotá, Colombia. * Corresponding author: Telephone: 57-1-3165000 Ext 14412, fax:
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57-1-2826197. Email: [email protected]
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Abstract
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To assess potential exposure of bees to chemicals contaminants, pesticides and other
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management practices of local beekeepers and farmers, four Colombian regions were
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surveyed and residue concentrations were determined on some pesticides used and others
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banned for honey. A total of 61 honey samples were collected and analyzed during 2011.
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Residual levels of selected insecticides, fungicides and acaricides were determined by a
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multiresidue method, using gas chromatography with nitrogen phosphorous detector /
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micro electron capture detector for the analysis and gas chromatography coupled to mass
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spectrometry detector to confirmation. In this study, pesticide residues were identified in 32
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samples (52.4% incidence), where organochlorine and organophosphorus pesticides were
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frequently found. The main detected compounds were chlorpyrifos (36.1% incidence),
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followed by profenofos (16.4% incidence), DDT (6.6% incidence), HCB, γ-HCH (4.9%
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incidence) and fenitrothion (1.6% incidence).
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However, the found concentrations found were low and just 4.9% of the samples exceeded
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the MRL concentration established in Regulation (EC) No 396/2005 by European
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Parliament. According to the survey results, it is highly probable that the honey
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contamination produced in Colombia beekeeping regions under study, is caused by
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agricultural practices developed around of the hives installed.
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Keywords: honey, pesticide residues; organochlorine pesticide; food safety.
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1. Introduction
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Honey is a natural product of bees and is recognized as a food with nutritional properties
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and is known like a food with valuable therapeutic applications. Most of the honey is
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produced by domesticated bees (Apis mellifera L.) from the nectar of flowers or from the
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sugar secretions from the leaves of arboreal essence. Honey is composed primarily of
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carbohydrates, proteins, minerals, vitamins and other substances, but its composition
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mainly depends on the floral origin of the nectar (Ball, 2007).
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However, bee products can also be a source of toxic substances, such as antibiotics,
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pesticides and heavy metals due to environmental pollution and misuse of beekeeping
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practices. Honey bees collect pollen and nectar from the surrounding flowers (over very
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large areas) and then they may return to hives collecting significant amounts of toxic
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contaminants, therefore their hives and products can result contaminated with many
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different kinds of pollutants (Bogdanov, Imdorf, Charrière, Fluri, & Kilchenmann, 2003;
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Morgano, et al., 2010; vanEngelsdorp & Meixner, 2010). At environmental level, honey
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bees pick contaminants through a wide range of pathways: (i) by consumption of pollen and
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contaminated nectar, (ii) by contact with plants and soil from crops in which farmers apply
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pesticides (iii) by inhalation during flight and recollection, (iv) by ingestion of polluted
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surface water and also v) by direct overspray or flying through spray drift, among others
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(Bogdanov, 2006; Colin, et al., 2004). Thus, some studies related to the presence of
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contaminants in samples from the bees’ products, provide information about the
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environmental compartments near to the hives and can serve to indicate anomalies in the
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environment in time and space (Conti, & Botrè, 2001). In addition the honeybees are also
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exposed to pesticides and antibiotics administered by beekeepers as part of the hive to
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control some infestations such as Varroa destructor, Acarapis wood and Paenibacillus
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larvae (Fell & Cobb, 2009; Genersch, Evans, & Fries, 2010); unfortunately the
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conventional commercial beekeepers frequently apply agrochemicals whether there are
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healthy or sick bees (Kevan, 199; Blacquière, Smagghe, Gestel, & Mommaerts, 2012),
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therefore, the presence of pesticides and antibiotics in bees’ products has become
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commonplace. Due to this situation, the European Union has established maximum residue
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limits (MRLs) for a large number of pesticides used in agricultural and beekeeping
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practices, through the Regulation (EC) No 396/2005.
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Pesticides in both bees and in bees’ products have been the subject of many studies, in
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recent years the presence of pesticide residues in honey were determined in some countries
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such as Belgium (Pirard, et al., 2007), Brazil (Rissato, Galhiane, de Almeida, Gerenutti, &
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Apon, 2007), China (Jin, et al., 2006), France (Wiest, et al., 2011), India (Choudhary &
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Sharma, 2008), Poland (Bargańska, Ślebioda, & Namieśnik, 2013), Portugal and Spain
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(Blasco, et al., 2003), and Turkey (Erdogrul, 2007). Table 1 shows the commonly reported
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pesticide residues in honey produced in these countries.
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It is noteworthy that in most of these studies the pesticide detected/quantified do not present
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a risk to human health but in many cases whether they pose a risk to bees. Finally in
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Colombia there is only one published study about the development of a method for
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pesticide residues determination in bees products, specifically in bee pollen (Rodriguez,
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Díaz, Zamudio, & Ahumada, 2012).
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The lack of information about pesticide residues in honey in Colombia implies the
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necessity to determinate the pollution of those bees' products in the country. In that way,
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the aims of this study were to (1) identify currently used pesticides and common pesticide
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management practices through interviews for beekeepers, (2) validate a multiresidue
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analysis method based on a liquid-liquid extraction followed by clean up with a classic
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chromatographic column, and (3) assess the presence of residues of selected currently
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applied pesticides and some widely used and/or persistent in the nearly areas at four honey
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productive areas in Colombia.
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2. Materials and Methods
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2.1.
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During 2011, a total of 61 samples of multifloral honey were collected from individual
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beekeepers in four regions of Colombia: Cundinamarca, Boyacá, Santander and Magdalena
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states; the geographical position of the regions is shown in Figure 1. These states represent
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about 50% of honey production in the country and 35% of the population of beekeepers
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identified by the Ministry of Agriculture and Rural Development of Colombia (Martínez,
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2006).
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Upon collection, all honey samples were placed into clean glass bottles, labeled, placed in
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an ice-chest kept at 4 °C, transferred to the laboratory and kept at -20°C until analysis. The
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sample size was at least 500 g and the minimum weight was 250 g.
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Identification of pesticides
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2.2.
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The beekeepers and farmers (around the hives of interest.) were selected by the Instituto de
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Ciencia y Tecnología de Alimentos (ICTA), Universidad Nacional de Colombia to
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represent the beekeeping and/or agricultural practices in each area. They were interviewed
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individually using a questionnaire on pesticide management prepared by the Laboratorio de
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Análisis de Residuos de Plaguicidas (LARP), Universidad Nacional de Colombia. The
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questions were directed to know the bee type, diseases in apiaries, type of crops planted
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around the hives, environmental conditions, and the pesticides or drugs applied to control
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specific pests and/or diseases on the hives or crops. A total of 110 beekeepers and farmers
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were visited as follow: 20 in Boyacá, 30 in Cundinamarca, 30 in Magdalena and 30 in
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Santander.
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Pesticide residues analysis
2.3.
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2.3.1. Reference materials, reagents and solutions
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Pesticide reference standards, all >95% purity, were obtained from Dr. Ehrenstorfer GmbH
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(Augsburg, Germany) and Chemservice (West Chester, USA). Stocks solutions were
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prepared in a concentration around 500 µg mL-1, using ethyl acetate as solvent, and they
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were stored in amber glassware under appropriate conditions such as -20°C, exclusion of
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moisture and light. All the solvents and reagents were HPLC grade and analytical grade,
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respectively (J.T. Baker, Deventer, The Netherlands).
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2.3.2. Extraction and clean up
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Pesticide residues in honey samples were simultaneously determined using a multi-residue
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method developed in laboratory. Briefly, 1 g sample was weighed in a 50 mL
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polypropylene centrifuge tube with screw caps. Then, 1.5 mL of methanol and 1.0 mL of
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citrate solution (pH 5.6 and 0.17 g/mL) were added, and samples were homogenized by
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mixing and shaking for 15 min.
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Afterwards, 10 mL of ethyl acetate were added, and the sample was shaken vigorously in a
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horizontal shaker for 15 min. Then, the tubes were centrifuged for 5 min at 4500 rpm.
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On the other hand, with the aim to improve the sensitivity of the methodology a
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concentration step upper layer (ethyl acetate) was needed, so this extract were left in a 50
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mL polypropylene centrifuge tube and a second extraction was performed with 1.5 mL of
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methanol and 5 mL of ethyl acetate. The extracts were combined and evaporated to 5 mL in
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a rotary evaporator at 35 °C.
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A clean up procedure was performed using a chromatographic column, packed with 2.5 g
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of a mixture of florisil previously activated for 5 h in an oven at 130ºC and silica deactivate
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15.5%, in proportion (1:1), and anhydrous sodium sulfate (all rinsed with 10 mL of hexane)
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was used. The extract was transferred to the column. One fraction was obtained after
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elution with 13 mL of a mixture of ethyl acetate and hexane (1:1.25). Maximal flux rate of
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elution was 1 mL/ min. The eluate was evaporated, quantitatively transferred into a 1 mL
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volumetric flask, 20 µL of I.S were added (Mix of PCB 52 (2, 2', 5, 5'-Tetrachlorobiphenyl
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for Electronic Capture Detector µECD and Sulfotep for Nitrogen Phosphorus Detector
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NPD), and finally, ethyl acetate was employed to complete to volume. Finally the extracts
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were injected into a gas chromatographic system for identification and quantification of the
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pesticides.
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The surrogate compounds, PCB 103 (2, 2', 4, 5’, 6-pentachlorobiphenyl) for µECD and
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TPP (triphenyl phosphate) for NPD, were used to evaluate the performance of the analytical
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method by calculating the recovery percentages of these compounds in each of the samples.
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Figure 2 illustrates the full scheme of multi-residue method used for the analysis of honey.
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2.3.3. Equipment
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Identification and quantification of the studied pesticides were performed by gas
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chromatography on a HP6890 plus with auto injector 7683 Agilent Technologies ® (Palo
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Alto, CA, US). Detection was performed in parallel with micro electron capture detector
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(µ-ECD63Ni) and NPD. Split/splitless injector was connected via Y-piece to a column HP 5
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(30 m, 0.32 mm id, 0,25 µm df) coupled to an electron-capture detector and a column HP
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50 (30 m, 0.32 mm id, 0.25 µm) coupled to a nitrogen-phosphorous detector. All data were
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stored and reprocessed with PC HP Chemstation. Pulsed splitless injection mode with a
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pulse pressure of 65 psi to 0.8 min, purge time of 0.6 min, purge flow 40 mL/min, 2 µL
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injection volume and injector temperature 256 º C was carried out.
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All positive samples were confirmed on an Agilent Technologies GC model 7890A
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coupled to a 5975 mass-selective detector equipped with a PTV and an Agilent 7673
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autoinjector. A HP-5MS (30 m x 0.25 mm ID x 0.25 µm) capillary column was used. The
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acquisition, control and data processing were performed using the MSD ChemStation
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version E02.00.493 software. Acquisition conditions were published in previous works
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(Ahumada & Guerrero, 2010).
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2.3.4. Validation and Uncertainty
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The validation procedure was performed following some parameters of SANCO guidance
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“Method Validation and Quality Control Procedures for Pesticide Residues Analysis in
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Food and Feed” (SANCO, 2011). Linear dynamic range, precision, recovery, lower limits,
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selectivity, ruggedness and uncertainty, were evaluated for the analytical methodology
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developed. For linear dynamic range, the calibration samples were prepared by appropriate
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dilution of the stock solution in blank matrix extract in order to avoid matrix effects.
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Calibration solutions, at concentrations ranging between 0.005 and 2536 mg/mL, were
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used.
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Intra-assay precision and recovery were assessed using spiked blank samples. On a set of
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replicated (n= 5) samples the R.S.D. and recovery values were calculated. The limit of
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detection (LOD) and quantification (LOQ) were calculated according to EPA
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recommendation and were verified (Lee, 2003). For most pesticides was possible to have
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the LOQ at a value equal to or greater than the MRL, but for other pesticides such acephate,
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alachlor and azoxistrobin, it not was possible because the sensitive method was
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insufficiently.
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The presence of potential interferences in the chromatograms from the analyzed samples
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was monitored by running control blank samples on each calibration. The absence of any
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chromatographic components at the same retention times as target pesticide suggested that
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no chemical interferences occurred.
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Uncertainty on pesticide measurements was evaluated based on a bottom-up approach.
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Uncertainty was further divided into the following sources: (i) preparation of solutions, (ii)
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analysis (volumetric material, weigthing), (iii) regression and (iv) validation data. After the
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estimation of all sources of uncertainty, they were combined according to the law of
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propagation of uncertainties, obtaining the combined standard uncertainty. The expanded
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uncertainty, U, is obtained by multiplying relative uncertainty by a coverage factor k (2),
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assuming a normal distribution of the measurand.
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3. Results and Discussion
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3.1.
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The interviews results shown that most beekeepers use bee Apis Mellifera (around 80%)
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and Santander has the largest number of native stingless bees (almost 35%). Furthermore,
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interviews showed that about 50% of hives have had some type of disease (Mainly Varroa,
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American and European foulbrood), where Boyacá and Cundinamarca areas were the most
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affected, and an average 11% of beekeepers apply chemical pesticides as: τ-fluvalinate and
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flumethrin for the control of these diseases. Moreover, all beekeepers reported that the
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hives were less than 1 km of agricultural crops.
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Farms producing agricultural crops near to the hives were included in the survey and these
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interviews showed that agricultural practices conducted in Boyacá and Cundinamarca are
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similar to each other, this is given by the climatic conditions of these regions, which allow
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to the farmers planting the same crops. For this reason, the pesticides used around the hives
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are the same and only dose and frequencies of pesticide application vary. A similar
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situation occurs between Magdalena and Santander. Table 2 shows the principal pesticides
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used by the farmers in the four regions under study.
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Other pesticides used were aldicarb, atrazine, carbofuran, mancozeb, propamocarb,
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thiametoxan, 2, 4-D and glyphosate. However, these pesticides were not included in our
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multi-residue analysis because our laboratory does not have the necessary instrumental for
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these pesticides. Finally, it is important to mention that Instituto Colombiano Agropecuario
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(ICA), who is the competent authority to fix the policies about to the importation,
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manufacture, use and disposal of pesticides in Colombia, authorizes the use of all pesticides
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reported by beekeepers and farmers in the study areas.
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3.2.
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Once known the pesticides applied in the study areas, the analytical methodology was
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validated for 53 pesticides, which were mainly organophosphorus, pyrethroids,
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organochlorines, azoles and some pesticides such as dimetomorph, folpet, metalaxyl,
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among others (see Table 3). Thus, in order to investigate specificity of the methodology, 5
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different honey samples produced in hives installed in native forest (no pesticide
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application) were extracted and analyzed. All samples had low intensity peaks in the region
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of interest. Given the efficient separation obtained from GC and the high selectivity of
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NPD, the absence of significant interferences was not surprising for the 53 analytes.
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Linearity was evaluated by the calculation of a five-point linear plot with three replicates
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(Table 3 shows the linear range), based on linear regression and squared correlation
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coefficient, r2, which should be >0.9800. Average recovery and the highest RSD were
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obtained in repeatability studies from samples of spiked honey at three different
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concentration levels (LOQ, 2xLOQ and 5xLOQ); Table 3 shows the results for two
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fortification levels. Recoveries were in the range of 73.4% to 120%, which is a satisfactory
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performance, except for acephate, metamidophos and monocrotophos where recovery
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values were 34.5%, 47.8% and 46.6% respectively. The RSD values were less than 20% for
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all the concentration levels tested. Table 3, shows the validation results for pesticides under
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study. In this Table were flagged pesticides for which it was not possible to achieve the
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MRL.
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3.3.
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The confirmation of all pesticides was performed by GC-MS, including samples with a
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concentration below the limit of quantification. Retention time, molecular weight, target ion
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and qualifier ions of some pesticides analyzed by gas chromatography-mass spectrometry
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(GC-MS) are listed in the Table 4. Table 5 shows the frequencies and concentrations of
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detected pesticides in honey samples from the four regions studied. It was found that 32
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(52.8%) samples were contaminated, mainly with organophosphorus (47.5%) and
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organochlorine (9.8%) pesticides. From 61 samples analyzed only five samples were
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contaminated with more than one pesticide, of these five three samples contained three
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pesticides and two samples contained four pesticides. Of the 32 positive samples, 28
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(87.5%) samples contained pesticide residues at or below MRLs established by the
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European Union (EU) and 3 samples (9.4%) contained pesticide residues above MRL, these
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pesticides were: gamma-HCH, HCB, chlorpyrifos and fenitrothion (MRL for these four
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pesticides is 0.01 mg/kg).
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It is noteworthy that other pesticides as flumetrine and τ-fluvalinate reportedly used by
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beekeepers in the hives were not detected in the samples of honey.
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Chlorpyrifos was the most abundant pollutant (68.8% of positive samples, although only
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two samples contained concentrations above LOQ) followed by profenophos (31.2% of
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positive samples), DDTs (12.5% of positive samples), HCB (12.5% of positive samples),
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gamma-HCH (9.3% of positive samples) and fenitrothion that was detected in only one
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sample and this concentration was above LOQ. The major presence of chlorpyrifos was not
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surprising since they are the most abundant in commercial pesticide mixtures and most
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commonly used for crop protection in colombian agriculture, including in the four areas of
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study (Arias, Bojacá, Ahumada, & Schrevens, 2014). Comparing the results with published
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data, it was found that chlorpyrifos seems to be one of the most frequently detected
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pesticides in foods and environment (Cox, 1995 ; Angioni, et al., 2011). In addition, other
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studies have found that chlorpyrifos is one of the most volatile pesticides (Rice, Nochetto,
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& Zara, 2002) and also seems to undergo a slow degradation rate in field conditions
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(Baskaran, Kookana, & Naidu, 1999). For those reasons and in accordance to the
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contamination mechanism of bee products, the probability of finding this pesticide in honey
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is high.
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Profenofos is highly toxic to honey bees and unfortunately it is the second most detected
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pesticide in honey (EPA, 2000). However, this molecule only was detected in samples from
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Magdalena state, where cotton is cultivated; none of these concentrations exceeded the
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LOQ (0.005 mg/kg). According to table 2, the presence of this pesticide in honey may be
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attributed to its intense use by the farmers for cotton crop; this affirmation is based on that,
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first, the distance between the hives and cotton crops is less than 1 km, second, the honey
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bees may be foraging in a cotton crop before, during and for a short time after the crop has
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flowers, and third, the ICA authorized the use of this insecticide for the control of mites in
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cotton, therefore its application becomes more intense. Other authors also showed
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contamination by pesticides in honey because the hives were located close to an area
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extensively cultivated with cotton, sunflowers and citrus trees (Balayiannis & Balayiannis,
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2008).
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Organochlorine pesticides have been banned for decades in most of the countries including
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Colombia, but their residues are still present as pollutants in water, soil, air and food. Table
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5 shows that the highest concentration of pesticide residue was 0.038 mg/kg of DDTs. As
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DDT is metabolized to DDE, the DDE/DDT ratio is used to assess the chronology of DDT
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entering to the ecosystems (Bordajandi, et al., 2003). The concentration of DDE for the
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samples M3 and M11 were 0.020 mg/kg and 0.038 mg/kg respectively, thus the ratio
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between DDE and DDT averages across the quantified samples was less than 1 (0.1), this
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ratio suggested a recent and direct use of such pesticide in the fields along the study areas,
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which is prohibited. Furthermore, Table 5 shows that the most active and important isomer
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of HCH, gamma-isomer (lindane) was detected in three samples with the concentration
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range between 0.008 to 0.016 mg/kg. The MRL of 0.010 mg/kg, as designated for lindane
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(EU), surpassing it only in one sample. The concentrations of other pesticides were
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generally low comparing with DDT. It should be noted that the detection frequency of HCB
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was 12.5% and the max value of concentration was as high as 0.028 mg/kg in Magdalena.
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Figure 3 shows the distribution of pesticide contamination in honey samples at the areas
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evaluated. This figure shows that Boyacá was the zone with the least number of
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contaminated samples with pesticide residues, followed by Magdalena and Santander.
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Honey samples from Magdalena, presented the highest incidence of organochlorine
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pesticides, which were widely used in coffee crops, until that they were totally banned in
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Colombia, while in Cundinamarca and Boyacá did not detect the presence of these
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compounds. Cundinamarca presented the greatest number of pesticide residues in samples,
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however for all the cases only chlorpyrifos were detected and its concentration never
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exceeded the MRL.
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On the other hand, when the results obtained for each state were compared, it was found
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that chlorpyrifos is the only compound that was detected in all the states and its
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concentration was generally similar, except for one sample in Santander (S-9) where the
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concentration was at least three times greater than the highest concentration in
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Cundinamarca (C-7). Profenofos, γ –HCH, and DDT were detected only in two
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departments (Magdalena and Santander) and its concentrations too were comparable.
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Cundinamarca and Boyacá were the states that did not have organochlorine pesticides in
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their samples, and only organophosphorus pesticides were detected. In adittion,
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Cundinamarca and Boyacá do not have high concentrations of pesticide compared to
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Magdalena and Santander. Finally the honey samples from Magdalena are the most
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worrying case, because in these samples the largest number of compounds and the highest
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concentrations were found.
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3.4 Comparison with previous works
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A few works on the monitoring of pesticide residue levels in South American honey have
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been published in the literature; nevertheless the results obtained are consistent with
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investigations from Brazil whose beekeeping and agricultural practices are similar to those
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in Colombia. For instance, for honeys produced in Sao Paulo-Brazil (Rissato, et al., 2007),
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it was reported the presence of four organochlorine pesticides (Aldrin, endosulfan sulfate,
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HCB, and tetradifon) in a concentration range between 0.008 to 0.027 mg/kg,
328
organophosphorus (chlorpyrifos and malathion) in a concentration range between 0.010 to
329
0.243 mg/kg, among other pesticides. Furthermore, the results obtained are similar to
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Argentina (Fontana, Camargo, & Altamirano, 2010) where it was reported the presence of
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methidathion (organophosphorus pesticide) at concentrations less than 2.3 mg/kg in honey
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produced in Mendoza.
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Regarding other monitoring studies, it has been reported that honey from Spain and
334
Portugal shows a high frequency of organochlorine pesticides such as HCH (isomers α, β
335
and γ), HCB and 4, 4 '-DDT (Along with the metabolites 4, 4 '-DDD and 4, 4 '-DDE),
336
however, the authors specified that concentrations in the majority of cases are low and do
337
not represent a health risk to the consumers (Blasco, et al., 2003; Herrera, et al., 2005). In a
338
monitoring study conducted to determine pesticide residues in 17 honeys from Greece the
339
results indicated contamination by diazinon, chlorpyrifos-ethyl, procymidone,
340
bromopropylate, and endosulfan (Tsiropoulos & Amvrazi, 2011). Other study in India
341
determined that HCH and its isomers were the pesticides most frequently detected followed
342
by DDT and its isomers (Choudhary & Sharma, 2008). Finally, in Poland was found
343
pesticide residues of bifenthrin, fenpyroximate, methidathion, spinosad, thiamethoxam and
344
profenophos, being the last one the most abundant (Bargańska, et al., 2013).
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4. Conclusions
347
The proposed multi-residue method by GC-µECD/NPD and confirmation by a GC-MS for
348
the analysis of 53 pesticides has been successfully applied in 61 honey samples produced in
349
Colombia. The data obtained in this study indicate that only a total 47.3% of the honey
350
samples collected in apiaries of the regions under study, showed concentrations below the
351
MRLs, 4.9% of the honey samples exceeding the MRLs and 47.8% the samples were free
352
from measurable pesticide residues. The pesticides exceeding MRLs were γ-HCH (0.016
353
mg kg-1), HCB (0.019 mg kg-1 to 0028 mg kg-1), chlorpyrifos (0.021 mg kg-1) and
354
fenitrothion (0.054 mg kg-1).
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The data obtained from this study and the high frequency of detection of pesticide residues
356
in honey samples are probably an indication of the widespread use of pesticides in the area
357
of study. In addition, this study revealed, for the first time that the bees and/or hives in the
358
study areas are exposed to chemical contaminants, including some insecticides such as
359
organophosphorus and organochlorine pesticides, which represents a risk to bees.
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360
5. Acknowledgements
362
The present study was funded by the Ministerio de Agricultura y Desarrollo Rural as part of
363
the initiative project ‘Selección de Indicadores Fisicoquímicos Mediante Aplicación de
364
Nariz Electrónica para la Catalogación de Productos Apícolas’.
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365 366
6. References
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Journal of Environmental Science and Health, Part B: Pesticides, Food
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Balayiannis, G., & Balayiannis, P. (2008). Bee Honey as an Environmental Bioindicator of
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Pesticides’ Occurrence in Six Agricultural Areas of Greece. Archives of
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Environmental Contamination and Toxicology, 55(3), 462-470.
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Ball, D. W. (2007). The Chemical Composition of Honey. Journal of Chemical Education,
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Bargańska, ś., Ślebioda, M., & Namieśnik, J. (2013). Pesticide residues levels in honey
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from apiaries located of Northern Poland. Food Control, 31(1), 196-201.
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Baskaran, S., Kookana, R. S., & Naidu, R. (1999). Degradation of bifenthrin, chlorpyrifos and imidacloprid in soil and bedding materials at termiticidal application rates.
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Blacquière, T., Smagghe, G., Gestel, C. M., & Mommaerts, V. (2012). Neonicotinoids in
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Blasco, C., Fernandez, M., Pena, A., Lino, C., Silveira, M. I., Font, G., & Pico, Y. (2003). Assessment of Pesticide Residues in Honey Samples from Portugal and Spain.
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(2003). Study on PCBs, PCDD/Fs, organochlorine pesticides, heavy metals and
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arsenic content in freshwater fish species from the River Turia (Spain).
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Colin, M. E., Bonmatin, J. M., Moineau, I., Gaimon, C., Brun, S., & Vermandere, J. P. (2004). A Method to Quantify and Analyze the Foraging Activity of Honey Bees:
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Relevance to the Sublethal Effects Induced by Systemic Insecticides. Archives of
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heavy metals contaminations. Environmental Monitoring Assessment,69, 267–282. Cox, C. (1995). Chlorpyrifos, Part 2: Human Exposure. Journal of Pesticide Reform, 15(1),
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Conti, M. E., & Botrè, F. (2001). Honeybees and their products as potential bioindicators of
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Choudhary, A., & Sharma, D. (2008). Pesticide Residues in Honey Samples from Himachal
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Pradesh (India). Bulletin of Environmental Contamination and Toxicology, 80(5),
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417-422.
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EPA. (2000). Interim Reregistration Eligibility Decision (IRED) Profenofos Case No. 2540 (http://www.epa.gov/oppsrrd1/REDs/2540ired.pdf). In. Washinton D.C. (Visited
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September 2013).
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Erdogrul, Ö. (2007). Levels of selected pesticides in honey samples from Kahramanmaras, Turkey. Food Control, 18(7), 866-871.
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spectrometry. Journal of Chromatography A, 1217(41), 6334-6341.
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Genersch, E., Evans, J., & Fries, I. (2010) Honey bee disease overview. Journal of
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Fontana, A. R., Camargo, A. B., & Altamirano, J. C. (2010). Coacervative microextraction ultrasound-assisted back-extraction technique for determination of organophosphates pesticides in honey samples by gas chromatography-mass
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Herrera, A., C., P.-A., P., C., Bayarri, S., Lazaro, R., Yagüe, C., & Ariño, A. (2005). Determination of pesticides and PCBs in honey by solid-phase extraction cleanup
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followed by gas chromatography with electron-capture and nitrogen-phosphorus
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detection. Analytical & Bioanalytical Chemistry, 381(3), 695-701.
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Jin, Z., Lin, Z., Chen, M., Ma, Y., Tan, J., Fan, Y., Wen, J., Chen, Z., & Tu, F. (2006). Determination of Multiple Pesticide Residues in Honey Using Gas
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Chromatography-Electron Impact Ionization-Mass Spectrometry. Chinese Journal
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of Chromatography, 24(5), 440-447.
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Kevan, P. G. (1999) Pollinators as bioindicators of the state of the environment: species, activity and diversity. Agricultural Ecosystem Environmental, 74, 373–393 Lee, P. W. (2003). Handbook of residue analytical methods for agrochemicals (Vol. 1). New Jersey: John Wiley & Sons.
Martínez, A. T. (2006). Diagnóstico de la Actividad Apícola y de la Crianza de Abejas en
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Colombia
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(http://corpomail.corpoica.org.co/BACFILES/BACDIGITAL/55617/55617.pdf ). In
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Bogotá D.C - Colombia (Visited September 2013).
441
Morgano, M. A., Teixeira Martins, M. C., Rabonato, L. C., Milani, R. F., Yotsuyanagi, K., & Rodríguez-Amaya, D. B. (2010). Inorganic Contaminants in Bee Pollen from
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Soil. Journal of Agricultural and Food Chemistry, 50(14), 4009-4017.
443 444
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Southeastern Brazil. Journal of Agricultural and Food Chemistry.
Rice, C. P., Nochetto, C. B., & Zara, P. (2002). Volatilization of Trifluralin, Atrazine, Metolachlor, Chlorpyrifos, α-Endosulfan, and β-Endosulfan from Freshly Tilled
Rissato, S. R., Galhiane, M. S., de Almeida, M. V., Gerenutti, M., & Apon, B. M. (2007). Multiresidue determination of pesticides in honey samples by gas chromatography-
ACCEPTED MANUSCRIPT 20 449
mass spectrometry and application in environmental contamination. Food
450
Chemistry, 101(4), 1719-1726.
451
Rodriguez, L. D., Díaz, A. C., Zamudio, A. M., & Ahumada, D. A. (2012). Evaluation of Pesticide Residues in Pollen From Cundiboyacense High-Plateau (Colombia).
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Vitae, 19(Supl. 1), S303-S305.
456 457
Analysis in Food and Feed, Document No. SANCO/12495.
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SANCO. (2011). Method Validation and Quality Control Procedures for Pesticide Residue
Tsiropoulos, N., & Amvrazi, E. (2011). Determination of pesticide residues in honey by single-drop microextraction and gas chromatography. J AOAC Int, 94(2), 634-644.
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458
vanEngelsdorp, D., & Meixner, M. D. (2010). A historical review of managed honey bee
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populations in Europe and the United States and the factors that may affect them.
460
Journal of Invertebrate Pathology, 103(1), S80-S95.
Wiest, L., Buleté, A., Giroud, B., Fratta, C., Amic, S., Lambert, O., Pouliquen, H., &
462
Arnaudguilhem, C. (2011). Multi-residue analysis of 80 environmental
463
contaminants in honeys, honeybees and pollens by one extraction procedure
464
followed by liquid and gas chromatography coupled with mass spectrometric
465
detection. Journal of Chromatography A, 1218, 5743–5756
467 468 469 470 471 472
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Tables
474
Table 1. Frequently detected pesticide groups in honey.
475
Table 2. Pesticides applied in the study regions
476
Table 3. Validation parameters obtained for pesticide multiresidue methodology in honey
477
bee.
478
Table 4. Retention time (RT), Molecular Weights (MW), Target ion (T) and Qualifier Ions
479
(Q1, Q2 and Q3) of some pesticides analyzed by gas chromatography-mass spectrometry
480
(GC-MS).
481
Table 5. Positive samples detected by screening and confirmatory method for honey
482
samples from four study areas.
483
Figures:
484
Figure 1. Map showing sampling locations of honeybee analyzed in this study.
485
Figure 2. Procedure schematically for pesticide analysis in honey.
486
Figure 3. Distribution of positive samples per study area.
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62 honey samples were analyzed for pesticides, 52.2% were positive and 47.8% the samples were free from pesticide residues.
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Main organochlorine and organophosphorus pesticides were found.
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Less than 5% of the samples had residue levels above the MRL.
ACCEPTED MANUSCRIPT 19 1
Tables
2
Table 1. Pesticide residues reported most frequently in honey. Concentration µg/kg
Organohalogens
Country of origin of honey
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Pesticides
0.1 - 4310
Brazil, Turkey, Spain, Portugal and India.
Organophosphates
2.4 – 243
Brazil, China, France, India, Portugal, Spain and Turkey.
Organonitrogen
0.05 – 116
Brazil, Belgium and France.
Pyrethroids
1 - 92
Brazil, China, India and poland.
Carbamates
1 - 645
China, Portugal and Spain.
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Table 2. Pesticides applied in the study regions State
Applied pesticides
B, C
Acephate
B, C, M, S
λ-Cihalotrin
B, C, S B, C, M, S B, C, M, S B B, C, S B, C, S B, C B, C, M, S
Cipermethrin Chlorpyrifos Deltamethrin Dimetomorph Fenitrothion Metalaxyl Parathion-methyl Profenophos
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5
Crops
Tomato. Cotton, rice, carnations, beans, corn, potatoes, pasture, pompom, roses and tomato. Rice, beans, potatoes. Rice, coffee, potatoes, bananas and other fruits. Cotton, rice, corn, potato, pasture, pompom and soy. Rice, onion, carnation, potatoes, roses, and tomato. Coffee Rice, onion, carnation, potatoes and roses. Rice and cotton. Cotton, carnation and potatoes.
EP
B: Boyacá; C: Cundinamarca; M: Magdalena; S: Santander.
6
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Table 3. Validation parameters obtained for pesticide multiresidue methodology in honey
9
bee. Pesticide
Detector
LOD
LOQ
LMR
(mg kg-1)
(mg kg-1)
Recovery % (RSD)n=5, LOQ
Recovery % (RSD)n=5, 5xLOQ
Linearity range
34.5 (6.9)
(mg kg-1)
r2
Acephate †
NPD
0.005
0.040
(mg kg-1) 0.02
30 (8.4)
0.400-2.132
0.999
Alachlor †
µECD
0.015
0.030
0.01
95.9 (10.3)
119.6 (17.4)
0.030-0.136
0.998
Azoxistrobin †
µECD
0.004
0.014
0.01
83.8 (6.3)
79.5 (14.2)
0.015-0.491
0.998
Captan
µECD
0.005
0.016
0.05
106.5 (13.5)
99.2 (13.0)
0.020-0.152
0.994
ACCEPTED MANUSCRIPT 20 β-cyfluthrin †
µECD
0.005
0.015
0.01
108.0 (6.5)
92.1 (9.3)
0.015-0.088
105.2 (6.8)
0.993
Cyhalothrin
µECD
0.003
0.009
0.02
99.8 (7.0)
0.010-0.048
0.995
Cymoxanil
µECD
0.094
0.310
0.05
86.8 (13.8)
108.4 (4.4)
0.310-1562
0.997
Cypermethrin
µECD
0.014
0.050
0.05
86.3 (5.7)
87.2 (4.2)
0.050-0.255
0.993
Cyproconazole †
NPD
0.125
0.250
0.05
92.6 (5.6)
83.3 (7.5)
0.250-1240
0.999
89.4 (6.4)
84.3 (1.0)
Chlorpyrifos
NPD
0.001
0.005
0.01
Coumaphos
NPD
0.003
0.010
0.1
90.6 (2.9)
90.7 (1.8)
0.010-0.293
0.993
4´,4´-DDD
µECD
0.002
0.005
0.05
89.7 (5.7)
98.8 (8.0)
0.010-0.058
0.999
4´,4´-DDT
µECD
0.003
0.010
0.05
92.1 (6.7)
99.7 (7.2)
0.010-0.024
0.997
99.4 (3.7)
RI PT
0.010-0.100
0.999
µECD
0.003
0.010
91.3 (8.0)
0.010-0.050
0.999
Deltamethrin
µECD
0.008
0.028
0.03
107.0 (12)
106.6 (11.5)
0.030-0.064
0.992
Dichlofluanid †
µECD
0.007
0.021
0.01
100.4 (4.8)
94.8 (13.2)
0.025-0.128
0.999
Dieldrin
µECD
0.002
0.006
0.01
109.0 (7.9)
128.1 (3.1)
0.006-0.024
0.998
90.0 (8.8)
87.8 (11.4)
0.050-0.256
0.995
84.8 (4.6)
0.010-0.074
0.999
88.8 (13.4)
0.200-2056
0.991
83.0 (15.2)
0.015-0.062
0.999
µECD
0.02
0.050
Dimethoate
NPD
0.003
0.010
0.01
83.0 (7.9)
Dimetomorf †
µECD
0.050
0.167
0.05
101.9 (4.3)
α -endosulfan
µECD
0.007
0.012
0.01
97.2 (18.3) 84.9 (9.4)
M AN U
Difenoconazole
0.05
SC
4´4´- DDE
0.05
β -endosulfan
µECD
0.007
0.012
0.01
92.6 (10.1)
0.015-0.062
0.999
Epoxiconazole
µECD
0.008
0.016
0.05
117.4 (17.6)
106.5 (14.4)
0.016-0.080
0.995
Fenamiphos †
NPD
0.006
0.020
0.01
84.1 (5.8)
69.6 (3.8)
0.020-0.248
0.998
Fenitrothion
NPD
0.001
0.005
0.01
88.3 (6.1)
85.2 (4.7)
0.010-0.064
0.999
91.4 (5.0)
NPD
0.004
0.001
85.1 (7.5)
0.010-0.120
0.999
Fenthoate
NPD
0.001
0.005
0.01
93.3 (10.1)
85.8 (8.3)
0.010-0.088
0.999
Flumethrin
µECD
0.005
0.01
0.01
110.7 (5.1)
111.1 (5.6)
0.015-0.422
0.987
τ-fluvalinate
µECD
0.004
0.01
0.01
104.4 (10.3)
77.8 (12.8)
0.015-0.283
0.994
0.087
0.01
73.4 (16.2)
73.1 (19.9)
0.087-0.436
0.996
0.006
0.01
119.6 (10.9)
89.1 (11.8)
0.006-0.032
0.997
0.01
0.01
80.4 (6.0)
88.1 (13.3)
0.015-0.058
0.997
0.010
0.01
97.7 (5.7)
85.4 (18.0)
0.010-0.050
0.987
0.002
0.003
0.01
108.1 (1.8)
99.1 (13.3)
0.003-0.016
0.998
0.010
0.020
0.01
80.1 (20.0)
79.6 (18.4)
0.020-0.115
0.999
0.003
0.012
0.01
80.1 (1.3)
73.5 (1.4)
0.015-0.128
0.994
0.047
0.01
108.8 (4.6)
98.1 (7.8)
0.050-0.480
0.999
94.2 (4.8)
µECD
0.025
HCB
µECD
0.003
α-HCH
µECD
0.005
β-HCH
µECD
0.004
µECD
Hexaconazole †
µECD
Iprodione
NPD
Isofenphos † Malathion Metalaxyl † Metamidophos † Parathion-methyl
AC C
γ- HCH
EP
Folpet †
µECD
TE D
Fenthion
0.01
0.016
NPD
0.001
0.004
0.02
94.1 (4.7)
0.010-0.114
0.999
NPD
0.140
0.286
0.05
93.6 (12.2)
84.5 (2.2)
0.286-1432
0.999
NPD
0.008
0.050
0.01
47.8 (6.9)
46.8 (3.3)
0.050-0.622
0.986
NPD
0.002
0.008
0.01
81.1 (4.4)
82.3 (3.2)
0.010-0.081
0.999
46.7 (4.8)
Monocrotophos †
NPD
0.034
0.200
0.01
44.9 (5.7)
0.200-0.999
0.999
Oxadixil †
NPD
0.130
0.250
0.01
103.1 (8.2)
100.1 (9.6)
0.250-1256
0.998
Permethrin †
µECD
0.030
0.060
0.01
111.0 (1.3)
99.9 (5.5)
0.060-0.296
0.994
Pyrazophos †
NPD
0.005
0.017
0.01
92.4 (5.3)
83.2 (4.4)
0.010-0.496
0.999
95.5 (11.9)
87.8 (4.9)
0.226-1128
0.999
92.5 (16.0)
92.5 (18.4)
0.005-0.024
0.999
Pyrimethanil †
NPD
0.101
0.226
0.05
Profenophos
NPD
0.001
0.005
0.01
ACCEPTED MANUSCRIPT 21 Propargite †
µECD
0.792
2.467
0.01
76.6 (4.1)
69.0 (7.3)
2.467-1.233
0.999
119.9 (3.5)
Propiconazole †
µECD
0.015
0.030
0.01
102.6 (2.1)
0.030-0.152
0.992
Tebuconazole †
NPD
0.051
0.163
0.05
91.1 (4.4)
82.1 (5.5)
0.163-2536
0.998
Tetradiphon
µECD
0.003
0.008
0.05
104.5 (5.1)
88.4 (13.5)
0.008-0.040
0.997
Tiabendazole †
NPD
0.370
0.730
0.01
86.2 (19.1)
84.2 (12.0)
0.730-3652
0.994
0.026
0.1
100.7 (7.4)
102.6 (12.9)
0.026-0.128
0.989
µECD
0.013
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Triadimephon
† The methodology was validated for these pesticides but LOQ> MRL. However these pesticides were included in the monitoring.
10 11
SC
12
Table 4. Retention time (RT), Molecular Weights (MW), Target ion (T) and Qualifier Ions
14
(Q1, Q2 and Q3) of some pesticides analyzed by gas chromatography-mass spectrometry
15
(GC-MS).
RT (min)
MW
T
Q1
Q2
Q3
Metamidophos
8.55
141.1
94
95
141
126
Acephate
10.41
183.2
136
94
95
125
Cymoxanil
11.81
191.2
167
183
184
-
Monocrotophos
12.50
223.2
127
192
223
-
α-HCH
12.80
290.8
219
217
181
183
HCB
13.15
284.8
284
286
282
288
Dimethoate
13.35
229.2
93
125
143
229
13.56
290.8
217
181
183
219
13.65
290.8
219
216
181
183
13.71
199.25
198
199
184
-
Parathion-methyl
15.00
291.3
263
125
109
-
Alachlor
15.48
269.8
160
188
237
238
Metalaxyl
15.59
279.3
249
279
220
234
Fenitrothion
16.20
277.2
278
260
125
109
Dichlofluanid
16.60
333.2
123
167
224
226
Malathion
16.87
330.4
173
158
125
127
Fenthion
17.00
278.3
278
279
169
153
Chlorpyrifos
17.24
350.6
314
316
258
260
Triadimephon
17.78
293.8
208
210
181
183
Tiabendazole
18.20
201.2
149
264
266
270
Captan
18.32
300.6
79
107
106
182
γ- HCH
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345.4
213
255
185
121
Fenthoate
18.62
320.4
274
246
157
-
Folpet
18.88
296.5
260
262
297
-
α -endosulfan
19.00
406.9
238
339
337
341
Fenamiphos
19.10
303.4
303
288
304
260
Hexaconazole
19.14
314.2
214
231
256
-
Profenophos
19.24
373.6
337
339
374
372
4´,4´-DDT
20.15
354.5
235
236
165
-
Dieldrin
20.21
380.9
263
265
277
318
Cyproconazole
20.47
291.0
222
224
139
141
β -endosulfan
20.69
406.9
237
265
339
341
4´,4´-DDD
21.13
320.0
235
237
165
199
Oxadixil
21.38
278.3
163
233
278
132
Propiconazole
21.60
342.2
259
261
173
175
4´4´- DDE
21.65
318.0
246
248
318
316
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Table 5. Positive samples detected by screening and confirmatory method for honey
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samples from four study areas.
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Concentration mg kg-1 ± Uncertainty (k=2)
a-d
Sample
HCB
ND ND ND ND ND ND ND ND ND ND ND 0.020 + 0.02 ND ND ND ND ND ND 0.038 + 0.004
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND † 0.016 + 0.001 0.010 + 0.001 ND ND
ND ND ND ND ND ND ND ND ND
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DDT e
Organophosphorus Chlorpyrifos Fenitrothion Profenofos
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
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S-3 S-4 S-5 S-6 S-7 S-8 S-9 S-10 S-11 S-12
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Figure 1. Map showing sampling locations of honeybee analyzed in this study.
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Figure 2. Procedure schematically for pesticide analysis in honey. 1 g of honey
Dissolution: 1.5 mL of methanol + 1.0 mL of citrate solution (pH 5.6, 0,17 g/mL) Shaking (1 min)
Extraction 1: 10 mL of ethyl acetate.
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Shaking (15 min) Centrifuge for 5 min a 4500 rpm Combine the two organic fractions
Concentration: in a rotary evaporator at 35 C (approx. 5mL).
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Clean up: Transfer the extract to the column and eluted with 13 mL of ethyl acetate: hexane (1:1.25).
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Transfer into a 1 mL volumetric flask
17 18 19
GC-NPD/µECD analysis
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Spike with 20 µ L of mixture of PCB 103 (15µ g/g) and TPP 25 (µ g/g)
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Figure 3. Distribution of positive samples per study area.
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S0956-7135(13)00457-X
DOI:
10.1016/j.foodcont.2013.09.011
Reference:
JFCO 3453
To appear in:
Food Control
Received Date: 10 April 2013 Revised Date:
1 September 2013
Accepted Date: 5 September 2013
Please cite this article as: Rodríguez LópezD., Alejandro AhumadaD., DíazA.C. & GuerreroJ.A., Evaluation of pesticide residues in honey from different geographic regions of Colombia, Food Control (2013), doi: 10.1016/j.foodcont.2013.09.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Evaluation of pesticide residues in honey from different geographic regions of
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Colombia
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Danny Rodríguez Lópeza; Diego Alejandro Ahumadab; Amanda Consuelo Díazc; Jairo
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Arturo Guerrerod*
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6 a
Laboratorio de Análisis de Residuos de Plaguicidas. Departamento de Química, Facultad
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de Ciencias, Universidad Nacional de Colombia, Carrera 30 No. 45-03, Edificio 451, Of.
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206, Bogotá, Colombia. Email: [email protected]
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Subdirección de Metrología Química y Biomedicina. Instituto Nacional de Metrología de Colombia. Carrera 50 26-55. Int. 2. Centro Administrativo Nacional, Bogotá, Colombia.
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Email: [email protected]
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c
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Colombia, Carrera 30 No. 45-03, Edificio 401, Of. 206, Bogotá, Colombia. Email:
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[email protected]
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Laboratorio de Análisis de Residuos de Plaguicidas. Departamento de Química, Facultad
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Instituto de Ciencia y Tecnología de Alimentos- ICTA, Universidad Nacional de
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de Ciencias, Universidad Nacional de Colombia, Carrera 30 No. 45-03, Edificio 451, Of.
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206, Bogotá, Colombia. * Corresponding author: Telephone: 57-1-3165000 Ext 14412, fax:
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57-1-2826197. Email: [email protected]
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Abstract
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To assess potential exposure of bees to chemicals contaminants, pesticides and other
25
management practices of local beekeepers and farmers, four Colombian regions were
26
surveyed and residue concentrations were determined on some pesticides used and others
27
banned for honey. A total of 61 honey samples were collected and analyzed during 2011.
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Residual levels of selected insecticides, fungicides and acaricides were determined by a
29
multiresidue method, using gas chromatography with nitrogen phosphorous detector /
30
micro electron capture detector for the analysis and gas chromatography coupled to mass
31
spectrometry detector to confirmation. In this study, pesticide residues were identified in 32
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samples (52.4% incidence), where organochlorine and organophosphorus pesticides were
33
frequently found. The main detected compounds were chlorpyrifos (36.1% incidence),
34
followed by profenofos (16.4% incidence), DDT (6.6% incidence), HCB, γ-HCH (4.9%
35
incidence) and fenitrothion (1.6% incidence).
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However, the found concentrations found were low and just 4.9% of the samples exceeded
37
the MRL concentration established in Regulation (EC) No 396/2005 by European
38
Parliament. According to the survey results, it is highly probable that the honey
39
contamination produced in Colombia beekeeping regions under study, is caused by
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agricultural practices developed around of the hives installed.
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Keywords: honey, pesticide residues; organochlorine pesticide; food safety.
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1. Introduction
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Honey is a natural product of bees and is recognized as a food with nutritional properties
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and is known like a food with valuable therapeutic applications. Most of the honey is
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produced by domesticated bees (Apis mellifera L.) from the nectar of flowers or from the
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sugar secretions from the leaves of arboreal essence. Honey is composed primarily of
48
carbohydrates, proteins, minerals, vitamins and other substances, but its composition
49
mainly depends on the floral origin of the nectar (Ball, 2007).
50
However, bee products can also be a source of toxic substances, such as antibiotics,
51
pesticides and heavy metals due to environmental pollution and misuse of beekeeping
52
practices. Honey bees collect pollen and nectar from the surrounding flowers (over very
53
large areas) and then they may return to hives collecting significant amounts of toxic
54
contaminants, therefore their hives and products can result contaminated with many
55
different kinds of pollutants (Bogdanov, Imdorf, Charrière, Fluri, & Kilchenmann, 2003;
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Morgano, et al., 2010; vanEngelsdorp & Meixner, 2010). At environmental level, honey
57
bees pick contaminants through a wide range of pathways: (i) by consumption of pollen and
58
contaminated nectar, (ii) by contact with plants and soil from crops in which farmers apply
59
pesticides (iii) by inhalation during flight and recollection, (iv) by ingestion of polluted
60
surface water and also v) by direct overspray or flying through spray drift, among others
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(Bogdanov, 2006; Colin, et al., 2004). Thus, some studies related to the presence of
62
contaminants in samples from the bees’ products, provide information about the
63
environmental compartments near to the hives and can serve to indicate anomalies in the
64
environment in time and space (Conti, & Botrè, 2001). In addition the honeybees are also
65
exposed to pesticides and antibiotics administered by beekeepers as part of the hive to
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control some infestations such as Varroa destructor, Acarapis wood and Paenibacillus
67
larvae (Fell & Cobb, 2009; Genersch, Evans, & Fries, 2010); unfortunately the
68
conventional commercial beekeepers frequently apply agrochemicals whether there are
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healthy or sick bees (Kevan, 199; Blacquière, Smagghe, Gestel, & Mommaerts, 2012),
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therefore, the presence of pesticides and antibiotics in bees’ products has become
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commonplace. Due to this situation, the European Union has established maximum residue
72
limits (MRLs) for a large number of pesticides used in agricultural and beekeeping
73
practices, through the Regulation (EC) No 396/2005.
74
Pesticides in both bees and in bees’ products have been the subject of many studies, in
75
recent years the presence of pesticide residues in honey were determined in some countries
76
such as Belgium (Pirard, et al., 2007), Brazil (Rissato, Galhiane, de Almeida, Gerenutti, &
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Apon, 2007), China (Jin, et al., 2006), France (Wiest, et al., 2011), India (Choudhary &
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Sharma, 2008), Poland (Bargańska, Ślebioda, & Namieśnik, 2013), Portugal and Spain
79
(Blasco, et al., 2003), and Turkey (Erdogrul, 2007). Table 1 shows the commonly reported
80
pesticide residues in honey produced in these countries.
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It is noteworthy that in most of these studies the pesticide detected/quantified do not present
82
a risk to human health but in many cases whether they pose a risk to bees. Finally in
83
Colombia there is only one published study about the development of a method for
84
pesticide residues determination in bees products, specifically in bee pollen (Rodriguez,
85
Díaz, Zamudio, & Ahumada, 2012).
86
The lack of information about pesticide residues in honey in Colombia implies the
87
necessity to determinate the pollution of those bees' products in the country. In that way,
88
the aims of this study were to (1) identify currently used pesticides and common pesticide
89
management practices through interviews for beekeepers, (2) validate a multiresidue
90
analysis method based on a liquid-liquid extraction followed by clean up with a classic
91
chromatographic column, and (3) assess the presence of residues of selected currently
92
applied pesticides and some widely used and/or persistent in the nearly areas at four honey
93
productive areas in Colombia.
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2. Materials and Methods
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2.1.
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During 2011, a total of 61 samples of multifloral honey were collected from individual
98
beekeepers in four regions of Colombia: Cundinamarca, Boyacá, Santander and Magdalena
99
states; the geographical position of the regions is shown in Figure 1. These states represent
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Study locations
about 50% of honey production in the country and 35% of the population of beekeepers
101
identified by the Ministry of Agriculture and Rural Development of Colombia (Martínez,
102
2006).
103
Upon collection, all honey samples were placed into clean glass bottles, labeled, placed in
104
an ice-chest kept at 4 °C, transferred to the laboratory and kept at -20°C until analysis. The
105
sample size was at least 500 g and the minimum weight was 250 g.
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Identification of pesticides
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2.2.
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The beekeepers and farmers (around the hives of interest.) were selected by the Instituto de
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Ciencia y Tecnología de Alimentos (ICTA), Universidad Nacional de Colombia to
110
represent the beekeeping and/or agricultural practices in each area. They were interviewed
111
individually using a questionnaire on pesticide management prepared by the Laboratorio de
112
Análisis de Residuos de Plaguicidas (LARP), Universidad Nacional de Colombia. The
113
questions were directed to know the bee type, diseases in apiaries, type of crops planted
114
around the hives, environmental conditions, and the pesticides or drugs applied to control
115
specific pests and/or diseases on the hives or crops. A total of 110 beekeepers and farmers
116
were visited as follow: 20 in Boyacá, 30 in Cundinamarca, 30 in Magdalena and 30 in
117
Santander.
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Pesticide residues analysis
2.3.
120
2.3.1. Reference materials, reagents and solutions
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Pesticide reference standards, all >95% purity, were obtained from Dr. Ehrenstorfer GmbH
122
(Augsburg, Germany) and Chemservice (West Chester, USA). Stocks solutions were
123
prepared in a concentration around 500 µg mL-1, using ethyl acetate as solvent, and they
124
were stored in amber glassware under appropriate conditions such as -20°C, exclusion of
125
moisture and light. All the solvents and reagents were HPLC grade and analytical grade,
126
respectively (J.T. Baker, Deventer, The Netherlands).
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2.3.2. Extraction and clean up
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Pesticide residues in honey samples were simultaneously determined using a multi-residue
130
method developed in laboratory. Briefly, 1 g sample was weighed in a 50 mL
131
polypropylene centrifuge tube with screw caps. Then, 1.5 mL of methanol and 1.0 mL of
132
citrate solution (pH 5.6 and 0.17 g/mL) were added, and samples were homogenized by
133
mixing and shaking for 15 min.
134
Afterwards, 10 mL of ethyl acetate were added, and the sample was shaken vigorously in a
135
horizontal shaker for 15 min. Then, the tubes were centrifuged for 5 min at 4500 rpm.
136
On the other hand, with the aim to improve the sensitivity of the methodology a
137
concentration step upper layer (ethyl acetate) was needed, so this extract were left in a 50
138
mL polypropylene centrifuge tube and a second extraction was performed with 1.5 mL of
139
methanol and 5 mL of ethyl acetate. The extracts were combined and evaporated to 5 mL in
140
a rotary evaporator at 35 °C.
141
A clean up procedure was performed using a chromatographic column, packed with 2.5 g
142
of a mixture of florisil previously activated for 5 h in an oven at 130ºC and silica deactivate
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15.5%, in proportion (1:1), and anhydrous sodium sulfate (all rinsed with 10 mL of hexane)
144
was used. The extract was transferred to the column. One fraction was obtained after
145
elution with 13 mL of a mixture of ethyl acetate and hexane (1:1.25). Maximal flux rate of
146
elution was 1 mL/ min. The eluate was evaporated, quantitatively transferred into a 1 mL
147
volumetric flask, 20 µL of I.S were added (Mix of PCB 52 (2, 2', 5, 5'-Tetrachlorobiphenyl
148
for Electronic Capture Detector µECD and Sulfotep for Nitrogen Phosphorus Detector
149
NPD), and finally, ethyl acetate was employed to complete to volume. Finally the extracts
150
were injected into a gas chromatographic system for identification and quantification of the
151
pesticides.
152
The surrogate compounds, PCB 103 (2, 2', 4, 5’, 6-pentachlorobiphenyl) for µECD and
153
TPP (triphenyl phosphate) for NPD, were used to evaluate the performance of the analytical
154
method by calculating the recovery percentages of these compounds in each of the samples.
155
Figure 2 illustrates the full scheme of multi-residue method used for the analysis of honey.
156
2.3.3. Equipment
157
Identification and quantification of the studied pesticides were performed by gas
158
chromatography on a HP6890 plus with auto injector 7683 Agilent Technologies ® (Palo
159
Alto, CA, US). Detection was performed in parallel with micro electron capture detector
160
(µ-ECD63Ni) and NPD. Split/splitless injector was connected via Y-piece to a column HP 5
161
(30 m, 0.32 mm id, 0,25 µm df) coupled to an electron-capture detector and a column HP
162
50 (30 m, 0.32 mm id, 0.25 µm) coupled to a nitrogen-phosphorous detector. All data were
163
stored and reprocessed with PC HP Chemstation. Pulsed splitless injection mode with a
164
pulse pressure of 65 psi to 0.8 min, purge time of 0.6 min, purge flow 40 mL/min, 2 µL
165
injection volume and injector temperature 256 º C was carried out.
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All positive samples were confirmed on an Agilent Technologies GC model 7890A
167
coupled to a 5975 mass-selective detector equipped with a PTV and an Agilent 7673
168
autoinjector. A HP-5MS (30 m x 0.25 mm ID x 0.25 µm) capillary column was used. The
169
acquisition, control and data processing were performed using the MSD ChemStation
170
version E02.00.493 software. Acquisition conditions were published in previous works
171
(Ahumada & Guerrero, 2010).
172
2.3.4. Validation and Uncertainty
173
The validation procedure was performed following some parameters of SANCO guidance
174
“Method Validation and Quality Control Procedures for Pesticide Residues Analysis in
175
Food and Feed” (SANCO, 2011). Linear dynamic range, precision, recovery, lower limits,
176
selectivity, ruggedness and uncertainty, were evaluated for the analytical methodology
177
developed. For linear dynamic range, the calibration samples were prepared by appropriate
178
dilution of the stock solution in blank matrix extract in order to avoid matrix effects.
179
Calibration solutions, at concentrations ranging between 0.005 and 2536 mg/mL, were
180
used.
181
Intra-assay precision and recovery were assessed using spiked blank samples. On a set of
182
replicated (n= 5) samples the R.S.D. and recovery values were calculated. The limit of
183
detection (LOD) and quantification (LOQ) were calculated according to EPA
184
recommendation and were verified (Lee, 2003). For most pesticides was possible to have
185
the LOQ at a value equal to or greater than the MRL, but for other pesticides such acephate,
186
alachlor and azoxistrobin, it not was possible because the sensitive method was
187
insufficiently.
188
The presence of potential interferences in the chromatograms from the analyzed samples
189
was monitored by running control blank samples on each calibration. The absence of any
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chromatographic components at the same retention times as target pesticide suggested that
191
no chemical interferences occurred.
192
Uncertainty on pesticide measurements was evaluated based on a bottom-up approach.
193
Uncertainty was further divided into the following sources: (i) preparation of solutions, (ii)
194
analysis (volumetric material, weigthing), (iii) regression and (iv) validation data. After the
195
estimation of all sources of uncertainty, they were combined according to the law of
196
propagation of uncertainties, obtaining the combined standard uncertainty. The expanded
197
uncertainty, U, is obtained by multiplying relative uncertainty by a coverage factor k (2),
198
assuming a normal distribution of the measurand.
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3. Results and Discussion
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3.1.
202
The interviews results shown that most beekeepers use bee Apis Mellifera (around 80%)
203
and Santander has the largest number of native stingless bees (almost 35%). Furthermore,
204
interviews showed that about 50% of hives have had some type of disease (Mainly Varroa,
205
American and European foulbrood), where Boyacá and Cundinamarca areas were the most
206
affected, and an average 11% of beekeepers apply chemical pesticides as: τ-fluvalinate and
207
flumethrin for the control of these diseases. Moreover, all beekeepers reported that the
208
hives were less than 1 km of agricultural crops.
209
Farms producing agricultural crops near to the hives were included in the survey and these
210
interviews showed that agricultural practices conducted in Boyacá and Cundinamarca are
211
similar to each other, this is given by the climatic conditions of these regions, which allow
212
to the farmers planting the same crops. For this reason, the pesticides used around the hives
213
are the same and only dose and frequencies of pesticide application vary. A similar
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situation occurs between Magdalena and Santander. Table 2 shows the principal pesticides
215
used by the farmers in the four regions under study.
216
Other pesticides used were aldicarb, atrazine, carbofuran, mancozeb, propamocarb,
217
thiametoxan, 2, 4-D and glyphosate. However, these pesticides were not included in our
218
multi-residue analysis because our laboratory does not have the necessary instrumental for
219
these pesticides. Finally, it is important to mention that Instituto Colombiano Agropecuario
220
(ICA), who is the competent authority to fix the policies about to the importation,
221
manufacture, use and disposal of pesticides in Colombia, authorizes the use of all pesticides
222
reported by beekeepers and farmers in the study areas.
223
3.2.
224
Once known the pesticides applied in the study areas, the analytical methodology was
225
validated for 53 pesticides, which were mainly organophosphorus, pyrethroids,
226
organochlorines, azoles and some pesticides such as dimetomorph, folpet, metalaxyl,
227
among others (see Table 3). Thus, in order to investigate specificity of the methodology, 5
228
different honey samples produced in hives installed in native forest (no pesticide
229
application) were extracted and analyzed. All samples had low intensity peaks in the region
230
of interest. Given the efficient separation obtained from GC and the high selectivity of
231
NPD, the absence of significant interferences was not surprising for the 53 analytes.
232
Linearity was evaluated by the calculation of a five-point linear plot with three replicates
233
(Table 3 shows the linear range), based on linear regression and squared correlation
234
coefficient, r2, which should be >0.9800. Average recovery and the highest RSD were
235
obtained in repeatability studies from samples of spiked honey at three different
236
concentration levels (LOQ, 2xLOQ and 5xLOQ); Table 3 shows the results for two
237
fortification levels. Recoveries were in the range of 73.4% to 120%, which is a satisfactory
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performance, except for acephate, metamidophos and monocrotophos where recovery
239
values were 34.5%, 47.8% and 46.6% respectively. The RSD values were less than 20% for
240
all the concentration levels tested. Table 3, shows the validation results for pesticides under
241
study. In this Table were flagged pesticides for which it was not possible to achieve the
242
MRL.
243
3.3.
244
The confirmation of all pesticides was performed by GC-MS, including samples with a
245
concentration below the limit of quantification. Retention time, molecular weight, target ion
246
and qualifier ions of some pesticides analyzed by gas chromatography-mass spectrometry
247
(GC-MS) are listed in the Table 4. Table 5 shows the frequencies and concentrations of
248
detected pesticides in honey samples from the four regions studied. It was found that 32
249
(52.8%) samples were contaminated, mainly with organophosphorus (47.5%) and
250
organochlorine (9.8%) pesticides. From 61 samples analyzed only five samples were
251
contaminated with more than one pesticide, of these five three samples contained three
252
pesticides and two samples contained four pesticides. Of the 32 positive samples, 28
253
(87.5%) samples contained pesticide residues at or below MRLs established by the
254
European Union (EU) and 3 samples (9.4%) contained pesticide residues above MRL, these
255
pesticides were: gamma-HCH, HCB, chlorpyrifos and fenitrothion (MRL for these four
256
pesticides is 0.01 mg/kg).
257
It is noteworthy that other pesticides as flumetrine and τ-fluvalinate reportedly used by
258
beekeepers in the hives were not detected in the samples of honey.
259
Chlorpyrifos was the most abundant pollutant (68.8% of positive samples, although only
260
two samples contained concentrations above LOQ) followed by profenophos (31.2% of
261
positive samples), DDTs (12.5% of positive samples), HCB (12.5% of positive samples),
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gamma-HCH (9.3% of positive samples) and fenitrothion that was detected in only one
263
sample and this concentration was above LOQ. The major presence of chlorpyrifos was not
264
surprising since they are the most abundant in commercial pesticide mixtures and most
265
commonly used for crop protection in colombian agriculture, including in the four areas of
266
study (Arias, Bojacá, Ahumada, & Schrevens, 2014). Comparing the results with published
267
data, it was found that chlorpyrifos seems to be one of the most frequently detected
268
pesticides in foods and environment (Cox, 1995 ; Angioni, et al., 2011). In addition, other
269
studies have found that chlorpyrifos is one of the most volatile pesticides (Rice, Nochetto,
270
& Zara, 2002) and also seems to undergo a slow degradation rate in field conditions
271
(Baskaran, Kookana, & Naidu, 1999). For those reasons and in accordance to the
272
contamination mechanism of bee products, the probability of finding this pesticide in honey
273
is high.
274
Profenofos is highly toxic to honey bees and unfortunately it is the second most detected
275
pesticide in honey (EPA, 2000). However, this molecule only was detected in samples from
276
Magdalena state, where cotton is cultivated; none of these concentrations exceeded the
277
LOQ (0.005 mg/kg). According to table 2, the presence of this pesticide in honey may be
278
attributed to its intense use by the farmers for cotton crop; this affirmation is based on that,
279
first, the distance between the hives and cotton crops is less than 1 km, second, the honey
280
bees may be foraging in a cotton crop before, during and for a short time after the crop has
281
flowers, and third, the ICA authorized the use of this insecticide for the control of mites in
282
cotton, therefore its application becomes more intense. Other authors also showed
283
contamination by pesticides in honey because the hives were located close to an area
284
extensively cultivated with cotton, sunflowers and citrus trees (Balayiannis & Balayiannis,
285
2008).
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Organochlorine pesticides have been banned for decades in most of the countries including
287
Colombia, but their residues are still present as pollutants in water, soil, air and food. Table
288
5 shows that the highest concentration of pesticide residue was 0.038 mg/kg of DDTs. As
289
DDT is metabolized to DDE, the DDE/DDT ratio is used to assess the chronology of DDT
290
entering to the ecosystems (Bordajandi, et al., 2003). The concentration of DDE for the
291
samples M3 and M11 were 0.020 mg/kg and 0.038 mg/kg respectively, thus the ratio
292
between DDE and DDT averages across the quantified samples was less than 1 (0.1), this
293
ratio suggested a recent and direct use of such pesticide in the fields along the study areas,
294
which is prohibited. Furthermore, Table 5 shows that the most active and important isomer
295
of HCH, gamma-isomer (lindane) was detected in three samples with the concentration
296
range between 0.008 to 0.016 mg/kg. The MRL of 0.010 mg/kg, as designated for lindane
297
(EU), surpassing it only in one sample. The concentrations of other pesticides were
298
generally low comparing with DDT. It should be noted that the detection frequency of HCB
299
was 12.5% and the max value of concentration was as high as 0.028 mg/kg in Magdalena.
300
Figure 3 shows the distribution of pesticide contamination in honey samples at the areas
301
evaluated. This figure shows that Boyacá was the zone with the least number of
302
contaminated samples with pesticide residues, followed by Magdalena and Santander.
303
Honey samples from Magdalena, presented the highest incidence of organochlorine
304
pesticides, which were widely used in coffee crops, until that they were totally banned in
305
Colombia, while in Cundinamarca and Boyacá did not detect the presence of these
306
compounds. Cundinamarca presented the greatest number of pesticide residues in samples,
307
however for all the cases only chlorpyrifos were detected and its concentration never
308
exceeded the MRL.
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On the other hand, when the results obtained for each state were compared, it was found
310
that chlorpyrifos is the only compound that was detected in all the states and its
311
concentration was generally similar, except for one sample in Santander (S-9) where the
312
concentration was at least three times greater than the highest concentration in
313
Cundinamarca (C-7). Profenofos, γ –HCH, and DDT were detected only in two
314
departments (Magdalena and Santander) and its concentrations too were comparable.
315
Cundinamarca and Boyacá were the states that did not have organochlorine pesticides in
316
their samples, and only organophosphorus pesticides were detected. In adittion,
317
Cundinamarca and Boyacá do not have high concentrations of pesticide compared to
318
Magdalena and Santander. Finally the honey samples from Magdalena are the most
319
worrying case, because in these samples the largest number of compounds and the highest
320
concentrations were found.
321
3.4 Comparison with previous works
322
A few works on the monitoring of pesticide residue levels in South American honey have
323
been published in the literature; nevertheless the results obtained are consistent with
324
investigations from Brazil whose beekeeping and agricultural practices are similar to those
325
in Colombia. For instance, for honeys produced in Sao Paulo-Brazil (Rissato, et al., 2007),
326
it was reported the presence of four organochlorine pesticides (Aldrin, endosulfan sulfate,
327
HCB, and tetradifon) in a concentration range between 0.008 to 0.027 mg/kg,
328
organophosphorus (chlorpyrifos and malathion) in a concentration range between 0.010 to
329
0.243 mg/kg, among other pesticides. Furthermore, the results obtained are similar to
330
Argentina (Fontana, Camargo, & Altamirano, 2010) where it was reported the presence of
331
methidathion (organophosphorus pesticide) at concentrations less than 2.3 mg/kg in honey
332
produced in Mendoza.
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Regarding other monitoring studies, it has been reported that honey from Spain and
334
Portugal shows a high frequency of organochlorine pesticides such as HCH (isomers α, β
335
and γ), HCB and 4, 4 '-DDT (Along with the metabolites 4, 4 '-DDD and 4, 4 '-DDE),
336
however, the authors specified that concentrations in the majority of cases are low and do
337
not represent a health risk to the consumers (Blasco, et al., 2003; Herrera, et al., 2005). In a
338
monitoring study conducted to determine pesticide residues in 17 honeys from Greece the
339
results indicated contamination by diazinon, chlorpyrifos-ethyl, procymidone,
340
bromopropylate, and endosulfan (Tsiropoulos & Amvrazi, 2011). Other study in India
341
determined that HCH and its isomers were the pesticides most frequently detected followed
342
by DDT and its isomers (Choudhary & Sharma, 2008). Finally, in Poland was found
343
pesticide residues of bifenthrin, fenpyroximate, methidathion, spinosad, thiamethoxam and
344
profenophos, being the last one the most abundant (Bargańska, et al., 2013).
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4. Conclusions
347
The proposed multi-residue method by GC-µECD/NPD and confirmation by a GC-MS for
348
the analysis of 53 pesticides has been successfully applied in 61 honey samples produced in
349
Colombia. The data obtained in this study indicate that only a total 47.3% of the honey
350
samples collected in apiaries of the regions under study, showed concentrations below the
351
MRLs, 4.9% of the honey samples exceeding the MRLs and 47.8% the samples were free
352
from measurable pesticide residues. The pesticides exceeding MRLs were γ-HCH (0.016
353
mg kg-1), HCB (0.019 mg kg-1 to 0028 mg kg-1), chlorpyrifos (0.021 mg kg-1) and
354
fenitrothion (0.054 mg kg-1).
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The data obtained from this study and the high frequency of detection of pesticide residues
356
in honey samples are probably an indication of the widespread use of pesticides in the area
357
of study. In addition, this study revealed, for the first time that the bees and/or hives in the
358
study areas are exposed to chemical contaminants, including some insecticides such as
359
organophosphorus and organochlorine pesticides, which represents a risk to bees.
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5. Acknowledgements
362
The present study was funded by the Ministerio de Agricultura y Desarrollo Rural as part of
363
the initiative project ‘Selección de Indicadores Fisicoquímicos Mediante Aplicación de
364
Nariz Electrónica para la Catalogación de Productos Apícolas’.
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6. References
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Ahumada, D., & Guerrero, D. J. (2010). Estudio del efecto matriz en el análisis de plaguicidas por cromatografía de gases. Vitae, 17(1), 51-58. Angioni, A., Dedola, F., Garau, A., Sarais, G., Cabras , P., & Caboni, P.
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Journal of Environmental Science and Health, Part B: Pesticides, Food
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213-217.
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Arias, L. A., Bojacá, C. R., Ahumada, D. A., & Schrevens, E. (2014). Monitoring of pesticide residues in tomato marketed in Bogota, Colombia. Food Control, 35(1),
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Balayiannis, G., & Balayiannis, P. (2008). Bee Honey as an Environmental Bioindicator of
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Pesticides’ Occurrence in Six Agricultural Areas of Greece. Archives of
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Environmental Contamination and Toxicology, 55(3), 462-470.
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Ball, D. W. (2007). The Chemical Composition of Honey. Journal of Chemical Education,
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Bargańska, ś., Ślebioda, M., & Namieśnik, J. (2013). Pesticide residues levels in honey
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from apiaries located of Northern Poland. Food Control, 31(1), 196-201.
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Baskaran, S., Kookana, R. S., & Naidu, R. (1999). Degradation of bifenthrin, chlorpyrifos and imidacloprid in soil and bedding materials at termiticidal application rates.
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Pesticide Science, 55(12), 1222-1228.
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Blacquière, T., Smagghe, G., Gestel, C. M., & Mommaerts, V. (2012). Neonicotinoids in
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bees: a review on concentrations, side-effects and risk assessment. Ecotoxicology,
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21(4), 973-992.
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Blasco, C., Fernandez, M., Pena, A., Lino, C., Silveira, M. I., Font, G., & Pico, Y. (2003). Assessment of Pesticide Residues in Honey Samples from Portugal and Spain.
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Journal of Agricultural and Food Chemistry, 51(27), 8132-8138.
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Bogdanov, S., Imdorf, A., Charrière, J.-D., Fluri, P., & Kilchenmann, V. (2003). The Contaminants of the Bee Colony. Bern, Switzerland: Swiss Bee Research Center, 1-
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Bogdanov, S. (2006). Contaminants of Bee Products. Apidologie, 37(1), 1-18.
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Bordajandi, L. R., Gómez, G., Fernández, M. A., Abad, E., Rivera, J., & González, M. J.
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(2003). Study on PCBs, PCDD/Fs, organochlorine pesticides, heavy metals and
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arsenic content in freshwater fish species from the River Turia (Spain).
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Chemosphere, 53(2), 163-171.
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Colin, M. E., Bonmatin, J. M., Moineau, I., Gaimon, C., Brun, S., & Vermandere, J. P. (2004). A Method to Quantify and Analyze the Foraging Activity of Honey Bees:
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Relevance to the Sublethal Effects Induced by Systemic Insecticides. Archives of
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Environmental Contamination and Toxicology, 47(3), 387-395.
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heavy metals contaminations. Environmental Monitoring Assessment,69, 267–282. Cox, C. (1995). Chlorpyrifos, Part 2: Human Exposure. Journal of Pesticide Reform, 15(1),
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Conti, M. E., & Botrè, F. (2001). Honeybees and their products as potential bioindicators of
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Choudhary, A., & Sharma, D. (2008). Pesticide Residues in Honey Samples from Himachal
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Pradesh (India). Bulletin of Environmental Contamination and Toxicology, 80(5),
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417-422.
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EPA. (2000). Interim Reregistration Eligibility Decision (IRED) Profenofos Case No. 2540 (http://www.epa.gov/oppsrrd1/REDs/2540ired.pdf). In. Washinton D.C. (Visited
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September 2013).
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Erdogrul, Ö. (2007). Levels of selected pesticides in honey samples from Kahramanmaras, Turkey. Food Control, 18(7), 866-871.
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Fell, R., & Cobb, J. (2009). Miticide Residues in Virginia Honeys. Bulletin of
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Environmental Contamination and Toxicology, 83(6), 822-827.
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spectrometry. Journal of Chromatography A, 1217(41), 6334-6341.
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Genersch, E., Evans, J., & Fries, I. (2010) Honey bee disease overview. Journal of
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Fontana, A. R., Camargo, A. B., & Altamirano, J. C. (2010). Coacervative microextraction ultrasound-assisted back-extraction technique for determination of organophosphates pesticides in honey samples by gas chromatography-mass
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Herrera, A., C., P.-A., P., C., Bayarri, S., Lazaro, R., Yagüe, C., & Ariño, A. (2005). Determination of pesticides and PCBs in honey by solid-phase extraction cleanup
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followed by gas chromatography with electron-capture and nitrogen-phosphorus
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detection. Analytical & Bioanalytical Chemistry, 381(3), 695-701.
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Jin, Z., Lin, Z., Chen, M., Ma, Y., Tan, J., Fan, Y., Wen, J., Chen, Z., & Tu, F. (2006). Determination of Multiple Pesticide Residues in Honey Using Gas
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Chromatography-Electron Impact Ionization-Mass Spectrometry. Chinese Journal
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of Chromatography, 24(5), 440-447.
435 436 437
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Kevan, P. G. (1999) Pollinators as bioindicators of the state of the environment: species, activity and diversity. Agricultural Ecosystem Environmental, 74, 373–393 Lee, P. W. (2003). Handbook of residue analytical methods for agrochemicals (Vol. 1). New Jersey: John Wiley & Sons.
Martínez, A. T. (2006). Diagnóstico de la Actividad Apícola y de la Crianza de Abejas en
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Colombia
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(http://corpomail.corpoica.org.co/BACFILES/BACDIGITAL/55617/55617.pdf ). In
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Bogotá D.C - Colombia (Visited September 2013).
441
Morgano, M. A., Teixeira Martins, M. C., Rabonato, L. C., Milani, R. F., Yotsuyanagi, K., & Rodríguez-Amaya, D. B. (2010). Inorganic Contaminants in Bee Pollen from
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Soil. Journal of Agricultural and Food Chemistry, 50(14), 4009-4017.
443 444
447 448
Southeastern Brazil. Journal of Agricultural and Food Chemistry.
Rice, C. P., Nochetto, C. B., & Zara, P. (2002). Volatilization of Trifluralin, Atrazine, Metolachlor, Chlorpyrifos, α-Endosulfan, and β-Endosulfan from Freshly Tilled
Rissato, S. R., Galhiane, M. S., de Almeida, M. V., Gerenutti, M., & Apon, B. M. (2007). Multiresidue determination of pesticides in honey samples by gas chromatography-
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mass spectrometry and application in environmental contamination. Food
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Chemistry, 101(4), 1719-1726.
451
Rodriguez, L. D., Díaz, A. C., Zamudio, A. M., & Ahumada, D. A. (2012). Evaluation of Pesticide Residues in Pollen From Cundiboyacense High-Plateau (Colombia).
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Vitae, 19(Supl. 1), S303-S305.
456 457
Analysis in Food and Feed, Document No. SANCO/12495.
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455
SANCO. (2011). Method Validation and Quality Control Procedures for Pesticide Residue
Tsiropoulos, N., & Amvrazi, E. (2011). Determination of pesticide residues in honey by single-drop microextraction and gas chromatography. J AOAC Int, 94(2), 634-644.
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458
vanEngelsdorp, D., & Meixner, M. D. (2010). A historical review of managed honey bee
459
populations in Europe and the United States and the factors that may affect them.
460
Journal of Invertebrate Pathology, 103(1), S80-S95.
Wiest, L., Buleté, A., Giroud, B., Fratta, C., Amic, S., Lambert, O., Pouliquen, H., &
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Arnaudguilhem, C. (2011). Multi-residue analysis of 80 environmental
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contaminants in honeys, honeybees and pollens by one extraction procedure
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followed by liquid and gas chromatography coupled with mass spectrometric
465
detection. Journal of Chromatography A, 1218, 5743–5756
467 468 469 470 471 472
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Tables
474
Table 1. Frequently detected pesticide groups in honey.
475
Table 2. Pesticides applied in the study regions
476
Table 3. Validation parameters obtained for pesticide multiresidue methodology in honey
477
bee.
478
Table 4. Retention time (RT), Molecular Weights (MW), Target ion (T) and Qualifier Ions
479
(Q1, Q2 and Q3) of some pesticides analyzed by gas chromatography-mass spectrometry
480
(GC-MS).
481
Table 5. Positive samples detected by screening and confirmatory method for honey
482
samples from four study areas.
483
Figures:
484
Figure 1. Map showing sampling locations of honeybee analyzed in this study.
485
Figure 2. Procedure schematically for pesticide analysis in honey.
486
Figure 3. Distribution of positive samples per study area.
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62 honey samples were analyzed for pesticides, 52.2% were positive and 47.8% the samples were free from pesticide residues.
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Main organochlorine and organophosphorus pesticides were found.
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Tables
2
Table 1. Pesticide residues reported most frequently in honey. Concentration µg/kg
Organohalogens
Country of origin of honey
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Pesticides
0.1 - 4310
Brazil, Turkey, Spain, Portugal and India.
Organophosphates
2.4 – 243
Brazil, China, France, India, Portugal, Spain and Turkey.
Organonitrogen
0.05 – 116
Brazil, Belgium and France.
Pyrethroids
1 - 92
Brazil, China, India and poland.
Carbamates
1 - 645
China, Portugal and Spain.
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Table 2. Pesticides applied in the study regions State
Applied pesticides
B, C
Acephate
B, C, M, S
λ-Cihalotrin
B, C, S B, C, M, S B, C, M, S B B, C, S B, C, S B, C B, C, M, S
Cipermethrin Chlorpyrifos Deltamethrin Dimetomorph Fenitrothion Metalaxyl Parathion-methyl Profenophos
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Crops
Tomato. Cotton, rice, carnations, beans, corn, potatoes, pasture, pompom, roses and tomato. Rice, beans, potatoes. Rice, coffee, potatoes, bananas and other fruits. Cotton, rice, corn, potato, pasture, pompom and soy. Rice, onion, carnation, potatoes, roses, and tomato. Coffee Rice, onion, carnation, potatoes and roses. Rice and cotton. Cotton, carnation and potatoes.
EP
B: Boyacá; C: Cundinamarca; M: Magdalena; S: Santander.
6
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Table 3. Validation parameters obtained for pesticide multiresidue methodology in honey
9
bee. Pesticide
Detector
LOD
LOQ
LMR
(mg kg-1)
(mg kg-1)
Recovery % (RSD)n=5, LOQ
Recovery % (RSD)n=5, 5xLOQ
Linearity range
34.5 (6.9)
(mg kg-1)
r2
Acephate †
NPD
0.005
0.040
(mg kg-1) 0.02
30 (8.4)
0.400-2.132
0.999
Alachlor †
µECD
0.015
0.030
0.01
95.9 (10.3)
119.6 (17.4)
0.030-0.136
0.998
Azoxistrobin †
µECD
0.004
0.014
0.01
83.8 (6.3)
79.5 (14.2)
0.015-0.491
0.998
Captan
µECD
0.005
0.016
0.05
106.5 (13.5)
99.2 (13.0)
0.020-0.152
0.994
ACCEPTED MANUSCRIPT 20 β-cyfluthrin †
µECD
0.005
0.015
0.01
108.0 (6.5)
92.1 (9.3)
0.015-0.088
105.2 (6.8)
0.993
Cyhalothrin
µECD
0.003
0.009
0.02
99.8 (7.0)
0.010-0.048
0.995
Cymoxanil
µECD
0.094
0.310
0.05
86.8 (13.8)
108.4 (4.4)
0.310-1562
0.997
Cypermethrin
µECD
0.014
0.050
0.05
86.3 (5.7)
87.2 (4.2)
0.050-0.255
0.993
Cyproconazole †
NPD
0.125
0.250
0.05
92.6 (5.6)
83.3 (7.5)
0.250-1240
0.999
89.4 (6.4)
84.3 (1.0)
Chlorpyrifos
NPD
0.001
0.005
0.01
Coumaphos
NPD
0.003
0.010
0.1
90.6 (2.9)
90.7 (1.8)
0.010-0.293
0.993
4´,4´-DDD
µECD
0.002
0.005
0.05
89.7 (5.7)
98.8 (8.0)
0.010-0.058
0.999
4´,4´-DDT
µECD
0.003
0.010
0.05
92.1 (6.7)
99.7 (7.2)
0.010-0.024
0.997
99.4 (3.7)
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0.010-0.100
0.999
µECD
0.003
0.010
91.3 (8.0)
0.010-0.050
0.999
Deltamethrin
µECD
0.008
0.028
0.03
107.0 (12)
106.6 (11.5)
0.030-0.064
0.992
Dichlofluanid †
µECD
0.007
0.021
0.01
100.4 (4.8)
94.8 (13.2)
0.025-0.128
0.999
Dieldrin
µECD
0.002
0.006
0.01
109.0 (7.9)
128.1 (3.1)
0.006-0.024
0.998
90.0 (8.8)
87.8 (11.4)
0.050-0.256
0.995
84.8 (4.6)
0.010-0.074
0.999
88.8 (13.4)
0.200-2056
0.991
83.0 (15.2)
0.015-0.062
0.999
µECD
0.02
0.050
Dimethoate
NPD
0.003
0.010
0.01
83.0 (7.9)
Dimetomorf †
µECD
0.050
0.167
0.05
101.9 (4.3)
α -endosulfan
µECD
0.007
0.012
0.01
97.2 (18.3) 84.9 (9.4)
M AN U
Difenoconazole
0.05
SC
4´4´- DDE
0.05
β -endosulfan
µECD
0.007
0.012
0.01
92.6 (10.1)
0.015-0.062
0.999
Epoxiconazole
µECD
0.008
0.016
0.05
117.4 (17.6)
106.5 (14.4)
0.016-0.080
0.995
Fenamiphos †
NPD
0.006
0.020
0.01
84.1 (5.8)
69.6 (3.8)
0.020-0.248
0.998
Fenitrothion
NPD
0.001
0.005
0.01
88.3 (6.1)
85.2 (4.7)
0.010-0.064
0.999
91.4 (5.0)
NPD
0.004
0.001
85.1 (7.5)
0.010-0.120
0.999
Fenthoate
NPD
0.001
0.005
0.01
93.3 (10.1)
85.8 (8.3)
0.010-0.088
0.999
Flumethrin
µECD
0.005
0.01
0.01
110.7 (5.1)
111.1 (5.6)
0.015-0.422
0.987
τ-fluvalinate
µECD
0.004
0.01
0.01
104.4 (10.3)
77.8 (12.8)
0.015-0.283
0.994
0.087
0.01
73.4 (16.2)
73.1 (19.9)
0.087-0.436
0.996
0.006
0.01
119.6 (10.9)
89.1 (11.8)
0.006-0.032
0.997
0.01
0.01
80.4 (6.0)
88.1 (13.3)
0.015-0.058
0.997
0.010
0.01
97.7 (5.7)
85.4 (18.0)
0.010-0.050
0.987
0.002
0.003
0.01
108.1 (1.8)
99.1 (13.3)
0.003-0.016
0.998
0.010
0.020
0.01
80.1 (20.0)
79.6 (18.4)
0.020-0.115
0.999
0.003
0.012
0.01
80.1 (1.3)
73.5 (1.4)
0.015-0.128
0.994
0.047
0.01
108.8 (4.6)
98.1 (7.8)
0.050-0.480
0.999
94.2 (4.8)
µECD
0.025
HCB
µECD
0.003
α-HCH
µECD
0.005
β-HCH
µECD
0.004
µECD
Hexaconazole †
µECD
Iprodione
NPD
Isofenphos † Malathion Metalaxyl † Metamidophos † Parathion-methyl
AC C
γ- HCH
EP
Folpet †
µECD
TE D
Fenthion
0.01
0.016
NPD
0.001
0.004
0.02
94.1 (4.7)
0.010-0.114
0.999
NPD
0.140
0.286
0.05
93.6 (12.2)
84.5 (2.2)
0.286-1432
0.999
NPD
0.008
0.050
0.01
47.8 (6.9)
46.8 (3.3)
0.050-0.622
0.986
NPD
0.002
0.008
0.01
81.1 (4.4)
82.3 (3.2)
0.010-0.081
0.999
46.7 (4.8)
Monocrotophos †
NPD
0.034
0.200
0.01
44.9 (5.7)
0.200-0.999
0.999
Oxadixil †
NPD
0.130
0.250
0.01
103.1 (8.2)
100.1 (9.6)
0.250-1256
0.998
Permethrin †
µECD
0.030
0.060
0.01
111.0 (1.3)
99.9 (5.5)
0.060-0.296
0.994
Pyrazophos †
NPD
0.005
0.017
0.01
92.4 (5.3)
83.2 (4.4)
0.010-0.496
0.999
95.5 (11.9)
87.8 (4.9)
0.226-1128
0.999
92.5 (16.0)
92.5 (18.4)
0.005-0.024
0.999
Pyrimethanil †
NPD
0.101
0.226
0.05
Profenophos
NPD
0.001
0.005
0.01
ACCEPTED MANUSCRIPT 21 Propargite †
µECD
0.792
2.467
0.01
76.6 (4.1)
69.0 (7.3)
2.467-1.233
0.999
119.9 (3.5)
Propiconazole †
µECD
0.015
0.030
0.01
102.6 (2.1)
0.030-0.152
0.992
Tebuconazole †
NPD
0.051
0.163
0.05
91.1 (4.4)
82.1 (5.5)
0.163-2536
0.998
Tetradiphon
µECD
0.003
0.008
0.05
104.5 (5.1)
88.4 (13.5)
0.008-0.040
0.997
Tiabendazole †
NPD
0.370
0.730
0.01
86.2 (19.1)
84.2 (12.0)
0.730-3652
0.994
0.026
0.1
100.7 (7.4)
102.6 (12.9)
0.026-0.128
0.989
µECD
0.013
RI PT
Triadimephon
† The methodology was validated for these pesticides but LOQ> MRL. However these pesticides were included in the monitoring.
10 11
SC
12
Table 4. Retention time (RT), Molecular Weights (MW), Target ion (T) and Qualifier Ions
14
(Q1, Q2 and Q3) of some pesticides analyzed by gas chromatography-mass spectrometry
15
(GC-MS).
RT (min)
MW
T
Q1
Q2
Q3
Metamidophos
8.55
141.1
94
95
141
126
Acephate
10.41
183.2
136
94
95
125
Cymoxanil
11.81
191.2
167
183
184
-
Monocrotophos
12.50
223.2
127
192
223
-
α-HCH
12.80
290.8
219
217
181
183
HCB
13.15
284.8
284
286
282
288
Dimethoate
13.35
229.2
93
125
143
229
13.56
290.8
217
181
183
219
13.65
290.8
219
216
181
183
13.71
199.25
198
199
184
-
Parathion-methyl
15.00
291.3
263
125
109
-
Alachlor
15.48
269.8
160
188
237
238
Metalaxyl
15.59
279.3
249
279
220
234
Fenitrothion
16.20
277.2
278
260
125
109
Dichlofluanid
16.60
333.2
123
167
224
226
Malathion
16.87
330.4
173
158
125
127
Fenthion
17.00
278.3
278
279
169
153
Chlorpyrifos
17.24
350.6
314
316
258
260
Triadimephon
17.78
293.8
208
210
181
183
Tiabendazole
18.20
201.2
149
264
266
270
Captan
18.32
300.6
79
107
106
182
γ- HCH
AC C
Pyrimethanil
EP
β-HCH
TE D
Pesticide
M AN U
13
ACCEPTED MANUSCRIPT 22 18.55
345.4
213
255
185
121
Fenthoate
18.62
320.4
274
246
157
-
Folpet
18.88
296.5
260
262
297
-
α -endosulfan
19.00
406.9
238
339
337
341
Fenamiphos
19.10
303.4
303
288
304
260
Hexaconazole
19.14
314.2
214
231
256
-
Profenophos
19.24
373.6
337
339
374
372
4´,4´-DDT
20.15
354.5
235
236
165
-
Dieldrin
20.21
380.9
263
265
277
318
Cyproconazole
20.47
291.0
222
224
139
141
β -endosulfan
20.69
406.9
237
265
339
341
4´,4´-DDD
21.13
320.0
235
237
165
199
Oxadixil
21.38
278.3
163
233
278
132
Propiconazole
21.60
342.2
259
261
173
175
4´4´- DDE
21.65
318.0
246
248
318
316
M AN U
SC
RI PT
Isofenphos
16
17
Table 5. Positive samples detected by screening and confirmatory method for honey
19
samples from four study areas.
TE D
18
Concentration mg kg-1 ± Uncertainty (k=2)
a-d
Sample
HCB
ND ND ND ND ND ND ND ND ND ND ND 0.020 + 0.02 ND ND ND ND ND ND 0.038 + 0.004
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND † 0.016 + 0.001 0.010 + 0.001 ND ND
ND ND ND ND ND ND ND ND ND
EP
Organochlorine γ -HCH
AC C
B-1 C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 M-1 M-2 M-3 M-4 M-5 M-6 M-7 M-8 M-9 M-10 M-11 S-1 S-2
DDT e
Organophosphorus Chlorpyrifos Fenitrothion Profenofos
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
ACCEPTED MANUSCRIPT 23 ND ND ND
RI PT
S-3 S-4 S-5 S-6 S-7 S-8 S-9 S-10 S-11 S-12
AC C
EP
TE D
M AN U
SC
20
ACCEPTED MANUSCRIPT 22
Figure 1. Map showing sampling locations of honeybee analyzed in this study.
M AN U
SC
RI PT
1
2 3 4
TE D
5 6
9 10 11 12 13 14 15
AC C
8
EP
7
ACCEPTED MANUSCRIPT 23 16
Figure 2. Procedure schematically for pesticide analysis in honey. 1 g of honey
Dissolution: 1.5 mL of methanol + 1.0 mL of citrate solution (pH 5.6, 0,17 g/mL) Shaking (1 min)
Extraction 1: 10 mL of ethyl acetate.
M AN U
Extraction 2: 1.5 mL of methanol + 5 mL of ethyl acetate.
SC
Shaking (15 min) Centrifuge for 5 min a 4500 rpm Collect the organic fraction
Shaking (15 min) Centrifuge for 5 min a 4500 rpm Combine the two organic fractions
Concentration: in a rotary evaporator at 35 C (approx. 5mL).
TE D
Prepare a column packed with: Anhydrous sodium sulfate (3 g) Florisil: silica deactivated 15.5% (1: 1) Rinse with 10 mL of hexane
EP
Clean up: Transfer the extract to the column and eluted with 13 mL of ethyl acetate: hexane (1:1.25).
AC C
Concentrate the eluate (approx. 0.5 mL) Spike with 20 µ L of mixture of PCB 52 (10µ g/g) and Sulfotep (10µ g/g)
Transfer into a 1 mL volumetric flask
17 18 19
GC-NPD/µECD analysis
RI PT
Spike with 20 µ L of mixture of PCB 103 (15µ g/g) and TPP 25 (µ g/g)
ACCEPTED MANUSCRIPT 24
Figure 3. Distribution of positive samples per study area.
M AN U
SC
RI PT
20
AC C
EP
TE D
21