1502

Journal of Food Protection, Vol. 65, No. 9, 2002, Pages 1502–1511

Copyright Q, International Association for Food Protection

Review

Analytical Methods for Pesticide Residue Determination in

Bee Products
M. FERNÁNDEZ,* Y. PICÓ, AND J. MAÑES

Laboratori de Bromatologia i Toxicologia, Facultat de Farmácia, Universitat de València, Av. Vicent Andrés Estellés s/n, 46100 Burjassot,
València, Spain
Downloaded from jfoodprotection.org by 146.185.202.52 on 09/06/18. For personal use only.

MS 01-485: Received 21 December 2001/Accepted 25 March 2002

ABSTRACT
Monitoring pesticide residues in honey, wax, and bees helps to assess the potential risk of these products to consumer
health and gives information on the pesticide treatments that have been used on the Ž eld crops surrounding the hives. The
present review seeks to discuss the basic principles and recent developments in pesticide analysis in bee products and their
application in monitoring programs. Consideration is given to extraction, cleanup, chromatographic separation, and detection
Journal of Food Protection 2002.65:1502-1511.

techniques.

Honeybees (Apis mellifera) perform the vital task of
pollinating agricultural crops and native species and are im-
portant for the commercial production of honey and bees-
wax. Every day, 10,000 to 25,000 honeybee workers make
an average of 10 journeys to explore roughly 7 km2 in the
area surrounding their hive, gathering nectar, water, and
pollen from  owers. During this process, diverse microor-
ganisms, chemical products, and particles suspended in the
air are intercepted by these workers and retained in the hair
of their body surface or inhaled and attached in their tra-
chea. Because of this, bees can be used as one bioindicator
to monitor environmental stress caused by biological,
chemical, and physical factors, such as parasites, industrial
contaminants, or pesticides (44, 45).
Pesticide residues are among the most signiŽ cant caus-
es of mortality in the bee colony and can lead to important
economic losses to the apiarist and the farmers in the sur-
rounding area, owing to the decreases in pollination. The
European Union regulation of plant protection products
takes into account the potential ecological effects of any
new insecticide by assessing hazard to bees before the sale
and distribution of these products (18). Although in some
cases pesticides do not affect bees, the bees transport pes-
ticide residues from nectar or pollen of contaminated blos-
som to the honey or the wax of the comb (14, 46). There-
fore, the pesticide contents in apiarian samples serve as a
good indication of pesticidal pollution.
The sudden widespread diffusion of the parasitic mite
Varroa jacobsoni and Ascophera apis, which affect hon-
eybee colonies and cause serious disease, has forced the
use of chemical therapeutic treatments to combat the pest
inside beehives (65, 66). The most important substances
used in European countries to prevent and control varroa-
tosis are amitraz, bromopropylate, coumaphos, cymiazole,
 uvalinate, and malathion (27). Their application in the
beehive causes a direct contamination of bee products. Pes-
ticide determination in honey, wax, and bee samples is nec-
essary to monitor contamination and guarantee consumer
health. However, these samples pose substantial analytical
problems mainly because of their complexity. Bees and
waxes contain a large number of fatty compounds and hon-
ey has a high percentage of sugars.
The purpose of this review is to examine the latest
analytical methods developed to determine pesticide resi-
dues in apiarian samples. An overview of monitoring pro-
grams performed in different countries is given, and the
pesticide residue levels reported in honey, pollen, and pro-
polen are discussed to evaluate if the pesticide residue
could be a human health risk.
ANALYTICAL PROCEDURES
Most methods currently used for pesticide analysis are
based on the traditional liquid-liquid extraction (LLE) pro-
cedures developed for determining pesticides in nonfatty
and fatty foods. However, solid-phase extraction (SPE), sol-
id-phase microextraction (SPME), and supercritical  uid
extraction (SFE) have been shown to be a good alternative
to LLE because of their simplicity, robustness, low quan-
tities of solvent used, and potential for automation. The
determination is performed using gas chromatography (GC)
or liquid chromatography (LC). A scheme of the methods
applied to wax, honey, and bees is illustrated in Figure 1.
Table 1 summarizes the pesticides, extraction procedures,
and detection techniques reported in analysis of bee prod-
ucts.

Sample pretreatment. Samples are processed in dif-

* Author for correspondence. E-mail: Monica.Fernandez@uv.es. ferent ways to facilitate the extraction procedure. Honey is
 J. Food Prot., Vol. 65, No. 9 PESTICIDE RESIDUE DETERMINATION IN BEE PRODUCTS
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Journal of Food Protection 2002.65:1502-1511.
FIGURE 1. Scheme of analytical procedures for pesticide analysis in bees, honey, and wax.
diluted with water, miscible organic solvent, or a mixture
of both and heated to reduce viscosity and to facilitate the
homogenization process (3, 5, 59). Honeycombs are melted
and the wax obtained is cut into pieces and ground in a
food processor. Pollen, larvae, and bees are homogenized
in a glass mortar (8).
LLE. This technique is the classic approach to pesti-
cide extraction from honey, wax, and bee samples. The liq-
uid partitioning is performed with water-immiscible sol-
vents, such as hexane (7, 13, 29, 33, 34, 54, 57, 58, 62,
64), dichloromethane (3, 47), ethyl acetate (4, 8), petroleum
ether (22, 26), or a mixture such as benzene and isopro-
panol (6) or methanol and ethyl acetate (21). This process
is assisted by sonication, shaking, and homogenization or
performed with a soxhlet apparatus (11, 43). Hexane gives
the cleanest extract, but it does not fully extract polar pes-
ticides. The addition of acetone (7, 40) increases hexane
polarity. Other factors that have in uence on the extraction
are the addition of salts, such as sodium sulfate (4) or so-
dium chloride (3, 52), which tend to ‘‘salt out’’ pesticides
from aqueous solutions. The pH of water affects the solu-
bility of ionizable pesticides. Acetic acid has been used to
reduce  uvalinate water solubility (64), whereas basic pes-
ticides such as cymiazole have been isolated from honeybee
samples using sodium hydroxide (12). The fungicides, ben-
omyl and carbendazim are extracted with acid solution from
honey, bee, and larvae samples and partitioned into ethyl
acetate after initial acidiŽ cation and again after subsequent
alkalinization of the extract. During acidiŽ cation, benomyl
1503
was hydrolyzed and determined as carbendazim (8). Final-
ly, the organic extracts obtained are evaporated under a
gentle nitrogen stream at 40 to 508C and redissolved with
acetone (7) or hexane (22, 34, 40, 54, 64) for GC deter-
mination or with acetonitrile (13) or methanol (8) for LC
analysis.
Some studies concentrate their effort on determining
the residue of a single compound, such as vinclozolin (7),
cymiazole (12, 13), acrinathrin (54, 61),  uvalinate (64,
58), amitraz (40), or  umethrin (53) in honey; cymiazole
(12) or carbaryl (43, 52) in bees; and  uvalinate (58) or
 umethrin in beeswax (53). Other studies have focused on
developing multiresidue methods, which are suitable for
screening for a large number of pesticides in a single anal-
ysis. Several types of pesticides, including organochlorine,
organophosphorus, pyrethroid, and carbamate, in honey
have been extracted by LLE with ethyl acetate (4), dichlo-
romethane (3), or hexane (29, 33, 34). Although an ade-
quate separation of the pesticides from matrix is achieved
using these methods, the methods are time-consuming and
require large amounts of solvent. However, a miniaturized
LLE method has been proposed for the analysis of 11 or-
ganochlorine pesticides in honey using 15 ml of petroleum
ether with 10% of diethylether. This procedure requires less
solvent than the traditional ones and a small amount of
sample (4 g), with the consequent reduction in coextracts
(22).
Bee wax is a complex sample to analyze because it is
composed by fatty esters and long-chain hydrocarbons,
 1504 FERNÁNDEZ ET AL. J. Food Prot., Vol. 65, No. 9

TABLE 1. Extraction and determination of pesticides in bees, honey, and waxa

Technique Matrix Pesticides Determination References

LLE Honey Organochlorine Dicarboximide GC-ECD 3, 4, 6–8

Organophosphorus Amitraz GC-NPD 10, 12, 13
Nitrogenated Cymiazole GC-AED 20–22, 26
Compounds Carbendazim GC-MS 28, 33, 34
Pyrethroid Benomyl LC-MS 37, 39, 47
Carbamates Tolyl uanid LC-UV 53–56
Triadimefon Rotenone LC-Fluor 60–62, 64
Wax Fluvalinate Flumethrin LC-Fluor 10, 54, 58
Acrinathrin Coumaphos GC-ECD 60, 62
Vinclozolin GC-MS
Bee Organophosphorus Benomyl LC-Fluor 7, 8, 12
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Carbaryl Cymiazole LC-UV 32, 43, 52

Carbendazim Vinclozolin LC-MS
GC-NPD
Pollen Carbendazim Benomyl LC-Fluor 8
LC-MS
SFE Honey Fluvalinate LC-UV 5
Beeswax Organophosphorus GC-FPD 42
Carbamate GC-NPD
Journal of Food Protection 2002.65:1502-1511.

GC-MS
SPE Honey Organochlorate Fluvalinate GC-NPD 1, 5, 7, 20
Organophosphorus Bromopropylate GC-ECD 23, 29, 30
Carbamate Rotenone GC-MS 35, 36, 42, 49, 51
Flumetrhin Amitraz LC-UV 53, 55, 57
Cymiazole Vinclozolin GC-FID 59
Bees Vinclozolin GC-MS 7
GC-NPD
Wax Fluvalinate Flumethrin GC-ECD 53, 58
SPME Honey Organochlorine Dicarboxamide GC-MS 38, 63
Organophosphorus Amitraz
Pyretroids
Bees Organophosphorus GC-NPD 24
a LLE, liquid-liquid extraction; SFE, supercritical  uid extraction; SPE, solid-phase extraction; SPME, solid-phase microextraction; GC,
gas chromatography; ECD, electron capture detector; NPD, nitrogen-phosphorous detector; AED, atomic emission detector; MS, mass
spectrometry; LC, liquid chromatography; Fluor,  uorescence; FPD,  ame photometric detector; FID,  ame ionization detector.

which are readily extracted by organic solvents commonly
used in residue analysis. This causes important extraction
interferences because these lipids cannot be fully separated
from pesticides, requiring long and tedious cleanup meth-
ods.
Bogdanov et al. (10) proposed pesticide separation
from wax by repeated freezing and centrifugation to cause
wax crystallization and eliminate compounds of high mo-
lecular weight. More laborious procedures are described for
the extraction of  umetrim (53) and coumaphos (60), which
include some additional steps as two solvent-solvent parti-
tions.
SPE. Trace enrichment of pesticides from bee samples
is provided by SPE. Before SPE can be used with bees or
bee products, extraction with water or water-miscible sol-
vents, such as methanol, acetone, and acetonitrile, is nec-
essary to obtain an aqueous solution. The most traditional
approach, the reversed-phase C18 cartridge, has been used
to preconcentrate a single compound, such as rotenone (39),
bromopropylate (57), vinclozolin (7), and  umethrin (20),
or a group of pesticides, such as organophosphates (51),
organochlorines (59), or carbamates (1), and the acaricides
bromopropylate (10, 28, 30), coumaphos (10, 28, 30, 35),
 uvalinate (10, 28, 35), amitraz (28, 30),  umethrin (10),
4,4-dibromobenzophenone (35), and cymiazole (30). The
solvent used to elute the retained pesticides depends on the
partitioning coefŽ cient of the pesticide. The solvent most
often chosen was hexane (51), dichloromethane (36), ethyl
acetate (10), or ethyl acetate followed by hexane (10), a
mixture of hexane and methylene chloride (28, 30), or a
mixture of hexane and toluene (20). Besides the typical
extraction cartridge device, the use of the Empore disk in
honey was advantageous due to the higher  ow rate and
shorter extraction times achieved (49). However, SPE pro-
cesses have the disadvantage of being ineffective for many
pesticides that are not retained in the solid phase and also
are unable to handle large sample volumes.
Recently, developed solid-phase sorbents have been
tested to extract pesticides from bee products. The National
Food Administration of Sweden has proposed a new poly-
meric sorbent for analysis of 29 pesticides in honey, poly-
styrene-divenylbenzene phase, which has been demonstrat-
 J. Food Prot., Vol. 65, No. 9 PESTICIDE RESIDUE DETERMINATION IN BEE PRODUCTS 1505

ed to provide good recoveries and precision for all pesti- TABLE 2. Maximum residue limits (MRLs) of pesticides (mg
cides except the more polar ones, such as trichlorfon, di- kg2 1) in honey (66)
methoate, and pirimicarb (36). European United Switzer-
Phases such as Florisil have been used for the deter- Pesticide Union States Germany Italy land
mination of 23 pesticides in honey. The use of adsorbents
such as Florisil were demonstrated to be a fast, reliable, Amitraz 0.2 1 0.01 10 10
Bromopropylate — — 0.1 10 100
and reproducible alternative to classic procedures and pro-
Coumaphos 0.1 0.1 0.01 10 50
vided simpler chromatograms. Matrix-standard calibration
Cymiazole 1 — 0.01 10 500
is required for quantitative analysis in honey to reduce the Flumethrine — — 0.01 10 5
matrix in uence on pesticide response, particularly for the Fluvalinate — 0.05 0.01 10 50
lowest spiking levels (41).
Recently, a multiresidue method for the determination
of 15 pesticides in various commercial honeys was devel-
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to the sample and the pesticides adsorbed in the stationary

oped based on matrix solid-phase dispersion. This method
phase are thermally desorbed in the injector of a gas chro-
involves sample dispersion onto a free- owing adsorbent,
matograph. Bee products, which are mostly solid and het-
in this case, a mixture of Florisil and anhydrous sodium
erogeneous, do not allow for similar direct extraction. How-
sulfate, which is eluted from the glass column with a few
ever, the homogenized sample can be suspended in water,
milliliters of an organic solvent. By this method, materials
which acts as a transfer medium between matrix and the
such as waxes and pigments are retained on the surface of
surface of the SPME Ž ber with the concurrence of a three-
the adsorbent (2).
phase equilibrium. SPME offers advantages over conven-
LLE and SPE on C18 cartridges have been compared
Journal of Food Protection 2002.65:1502-1511.

tional methods with respect to the amount of organic sol-

for the extraction of vinclozolin in honey and bee larvae
vent used and operational simplicity. The SPME technique
(7). In both cases, bee larvae extracts contain more coeluted
was applied to analyze amitraz in honey (63). The homog-
compounds such as fatty acids than seen in honey samples.
enized sample suspended in water was extracted by a poly-
SPE recovery from honey samples was similar (98%) to
dimethylsiloxane Ž ber. The results obtained in this study
LLE, but for larvae was somewhat lower (91%). In terms
showed that SPME is an interesting alternative to the tra-
of selectivity and precision, SPE proved to be more accu-
ditional methods.
rate and provide better cleanup, resulting in cleaner chro-
Jiménez et al. (38) studied the viability of SPME Ž bers
matograms, than LLE.
based on polyacrylate and polydimethylsiloxane for deter-
Acrinathrine and its major metabolite 3-phenoxyben-
mining 21 pesticides of different chemical families in hon-
zaldehyde were isolated from honey by means of LLE us-
ey. The detection limits reached using electron capture de-
ing benzene-isopropanol and by SPE with Florisil and C18
tector (ECD) are basically of the same magnitude as those
cartridges. Although C18 separations lead to very low re-
achieved after extraction with organic solvent or SPE,
coveries due to the low polarity of the pesticides, both LLE
whereas the reproducibility obtained is slightly lower com-
and Florisil give reliable, precise determination; the latter
pared with the aforementioned methods.
was recommended because recoveries were higher, the
Fernández et al. (24) reported an SPME method to de-
chromatogram was cleaner, and the use of benzene was
termine 18 organophosphorous pesticides in honeybees.
avoided (6).
The method involves a honeybee preextraction with a mix-
SFE. A general trend in the isolation of pesticide res- ture of acetone and water followed by a dilution in water
idues is to reduce the consumption of expensive and/or tox- before Ž ber extraction. The use of small amounts of acetone
ic organic solvent and to increase the availability of a broad seemed to improve extraction efŽ cacy with biological sam-
range of analytes and matrices. A possible solution is to ples. The SPME procedure was demonstrated to be quicker,
use SFE. This technique requires a supercritical  uid, usu- simpler, and more effective than the LLE method common-
ally CO2 , with or without an organic modiŽ er and without ly used.
further cleanup steps. There is a rapid mass transfer and
Cleanup. The complexity of the cleanup procedure de-
faster solute extraction because supercritical  uid diffusiv-
pends on the matrix, the detection limit required, and the
ities are higher and its viscosity is lower in comparison with
detection technique used. The most common interferences
organic solvents. Problems appear when direct SFE is used
that are present in apiarian extracts are lipids, pigments, and
to extract analytes from an aqueous matrix because of the
carbohydrates. Pollen extracts required further cleanup with
solubility of the supercritical  uid in water. In these cases,
hexane to decrease the excessive extract pigmentation and
the use of an additional organic modiŽ er or sample lyoph-
reduce the high solvent front observed in the chromatogram
ilization should be considered. SFE has been used to mea-
(8). Sugar constitutes 82% of honey content, and as car-
sure organophosphate and carbamate in bees (42) and  u-
bohydrates are partially soluble in polar organic solvents,
valinate in honey (5) and has proven to be an alternative
coextractive problems appear mostly in analysis of high po-
to the classic method.
lar pesticides (32). In contrast, beeswax contains fatty esters
SPME. SPME is a rapid, solvent-free technique that and long-chain hydrocarbons that have been identiŽ ed in
concentrates the organic compounds from aqueous samples. larva extracts with gas chromatography–mass spectrometry
A fused silica Ž ber coated with polymeric Ž lm is exposed (GC-MS) (7).
 Journal of Food Protection 2002.65:1502-1511.
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TABLE 3. Monitoring programs of pesticides residue in bee productsa

1506

No. of Positive
Country Year Matrix type Analytical method samples samples (%) Pesticides type Range References

Germany 1997 Wax — 226 55 Bromopropylate 0.5–10 ppm 66

61 Coumaphos
37 Fluvalinate
Germany 1997 Honey — 1000 11 Bromopropylate 2–10 ppb 66
28 Coumaphos 2–15 ppb
1 Fluvalinate 2–7 ppb
European Union 1997 Wax — 158 21 Bromopropylate 0.5–20 ppm 66
FERNÁNDEZ ET AL.

19 Coumaphos
55 Fluvalinate
Spain 1993 Honey SFE/LC-UV 16 100 Fluvalinateb 0.029–0.750 mg kg2 1 5
6 67 Fluvalinate 0.055–0.215 mg kg2 1
Spain 1988/1990 Honey LE/GC-NPD(ECD) 177 39 Organophosphates 15 1–116 ppb 34
Jordan 1995 Honey LLE/GC-NPD (ECD) 26 86 Multiresidue (50) 0.001–0.373 mg kg2 1 3
Greece 2000 Beeswax LE/GC-ECD 66 100 Fluvalinate 0.44–30.1 mg kg2 1 58
India 1993/1995 Honey LLE/GC-NPD (ECD) 27 100 Organochlorines (10) 0.01–1.1 mg g2 1 4
11/14 Pyrethroids (2) 0.18–6.64 mg g2 1
96/100 Organophosphates (8) 0.02–3.9 mg g2 1
92/100 Carbamates (3) 0.1–4.84 mg g2 1
Spain 1988/1990 Honey LLE/GC-NPD 177 14 Coumaphos 1–5 ppb 26
4 Diazinon 13–35 ppb
n.d. Malation n.d.
Tunisia 1994 Honey LLE/GC-ECD 28 86 Organochlorine 12 ng/g–0.35 ng/g 22
Switzerland 1996 Wax LE/GC-ECD Bromopropylate 2.4 mg kg2 1 10
Coumaphos
Fluvalinate
Switzerland 1996 Propolis LE/GC-ECD 27 27 Bromopropylate 0.5–38.7 mg kg2 1 10
Coumaphos
Fluvalinate
Spain 1990 Honey SPE/GC-ECD (FID) 101 n.d. Amitraz — 30
16 Bromopropylate 5–60 mg/kg
n.d. Coumaphos —
11 Fluvalinate 10–40 mg/kg
United States 1995 Pollen LLE/GC-MS 23 17 Parathion methylc 0.013–2.5 ppm 50
Killed bees 28 57 Parathion methyl 0.388–001 ppm
Greece 2000 Honey SPE/GC-ECD 93 100 Fluvalinate 1–39 mg kg2 1 48
32 90 Fluvalinateb 1–4 mg kg2 1
Austria 1995 Honey 105 13 Bromopropylate 2–33 ppb 48
84 12 Coumaphos 2–12 ppb
32 22 Cymiazole 15–17 ppb
33 n.d. Flumethrin n.d.
J. Food Prot., Vol. 65, No. 9

94 3 Fluvalinate 2–7 ppb

 J. Food Prot., Vol. 65, No. 9 PESTICIDE RESIDUE DETERMINATION IN BEE PRODUCTS 1507

SFE, supercritical  uid extraction; LC, liquid chromatography; LE, liquid extraction; GC, gas chromatography; ECD, electron capture detector; NPD, nitrogen-phosphorous detector; LLE,
liquid-liquid extraction; SPE, solid-phase extraction; AED, atomic emission detector; MS, mass spectrometry; Fluor,  uorescence; FID,  ame ionization detector; n.m., not mentioned; n.d.,
References
Two lengthy methods have been proposed for carbaryl
extraction in bees previous to GC or LC determination. The

31
Ž rst one basically consisted of two partitioning steps be-
tween two solvents of different polarity and silica gel chro-
matography (43), whereas the other method introduces a

0.0001–0.534 mg kg2 1
coagulation solution that precipitates wax particles, which
63.3 ppb (max) can then be removed by a simple Ž ltration (52).
63.5 ppb (max)
Range

15 ppb (max)
The most common cleanup technique is chromato-
graphic cleanup. The use of shorter Sep Pack cartridges,
including Florisil (1, 7, 22, 26, 29, 33, 34, 54) and silica
gel (20) for honey and Florisil (10) and silica gel (53) for
wax and Propolis, reduced the time used for the analysis.
Various methods using a column packed with diatomaceous
Downloaded from jfoodprotection.org by 146.185.202.52 on 09/06/18. For personal use only.

earth material (Extrelut) were recently described for wax

and honeybees. This sorbent material appeared to be suit-
able for the rapid removal of the wax content from the
Pesticides type

Bromopropylate

extracted solutions (16, 19, 58).

Multiresidue
Coumaphos

Gel permeation chromatography is generally known to

Flumethrin

be effective in separating pesticides from complex matrices.

Because of its stearic exclusion mechanism, gel permeation
Journal of Food Protection 2002.65:1502-1511.

chromatography allows the separation of the analytes from

the waxes as a result of the different molecular size. This
puriŽ cation approach has been used for the analysis of pes-
samples (%)

ticides in honey and honeybees (19, 23).

Positive

n.m.

n.m.
71

96

Instrument analysis: GC. GC is the most widely used

technique for multiresidue screening because of the number
of pesticides that are amenable to this technique, the high
samples

separation power, and the availability of selective detectors.

No. of

41

683
n.m.

n.m.

These detectors respond to the compounds containing a

c Parathion methyl was the only pesticide detected after a multiresidue method of 100 pesticides.

speciŽ c or deŽ ned range of groups or elements, such as

ECD, nitrogen-phosphorous detector (NPD),  ame ioniza-
tion detector, atomic emission detector (AED), or MS de-
b Honey obtained from known treated beehives instead of being commercial honeys.

tector. Although an ECD has been applied extensively for

Analytical method

halogenated compounds (1–5, 7, 10, 19–22, 24, 28, 29, 32,

34, 36, 38, 41, 42, 47, 53, 54, 56, 58, 59, 61, 63, 64)
because it is very sensitive to molecules that contain elec-
tronegative atoms, its lack of speciŽ city requires subsequent
GC-MS

conŽ rmation of positive Ž ndings. The NPD enhances the

detection of organophosphorous, carbamate, urea, and tri-
azine pesticides (3–5, 16, 24, 26, 33, 34, 43, 47, 51, 55,
56), but conŽ rmation is essential because of possible inter-
ferences of high levels of N-containing compounds.
Matrix type

Beeswax

GC-MS is the reliable approach for identiŽ cation and

Honey

conŽ rmation of unknown compounds in multiresidue

screenings. The most widely used ionization technique is
electron impact (7, 40) in which sample molecules are bom-
barded by high energy (70 eV), but molecular ions are not
1992/1996

always seen in electron impacts spectra. A complementary

Year

ionization technique is chemical ionization, which has been

used in a multiresidue approach to search for the presence
of more than a hundred pesticides (50).
Several studies included the simultaneous determina-
TABLE 3. Continued

tion of acaricide residues in honey using various detection

systems of different selectivities to enhance better infor-
Country

not detected.

mation. AED has been compared with the detection by

ECD and NPD in parallel. The selection of a characteristic
France

wavelength of radioactive emission in AED for the analysis

of chlordimeform, bromopropylate, amitraz, and couma-
a
 1508 FERNÁNDEZ ET AL.
phos allowed better sensitivity, whereas higher selectivity
is achieved with the ECD/NPD system (37). The AED and
MS detector have been included in a method for determin-
ing amitraz and degradation products in honey. The Ž rst
detector allows a rapid screening of nitrogen-containing
compounds before MS identiŽ cation of the ambiguous
peaks (40).
Those compounds that cannot be analyzed directly by
GC can be derivatized. Derivatization reactions are essen-
tial to increase volatility, overcome the thermal instability,
and enhance the selectivity or sensitivity of the detector.
Methylation of acid pesticides (50) and carbaryl (43) ana-
lyzed by GC-MS and GC-NPD, respectively, and butyryl
Downloaded from jfoodprotection.org by 146.185.202.52 on 09/06/18. For personal use only.
derivatives of amitraz (55), which is sensitive to ECD, have
been reported in honey and bee analysis. However, deriv-
atization decreased reproducibility by adding more sample-
handle steps.
LC. In comparison with GC analysis, LC has proven
to be effective in separating organic compounds regardless
of their volatility, polarity, and thermal stability. Normally,
Journal of Food Protection 2002.65:1502-1511.
LC separations are performed using reversed-phase chro-
matography with C18 or C8 columns and aqueous mobile
phases (8, 12, 13, 52). Increased separation is achieved us-
ing stationary phases with small particles and narrowbore
and microbore columns (35). UV detection,  uorescence,
and MS are common detectors. The most widely used LC
detector has been the UV detector because of its stability
and low cost (5). However, conŽ rmation becomes difŽ cult
for pesticides due to the large amount of interferences from
the matrix. The  uorescence detector is more selective and
sensitive than the UV detector. Its use is restricted to an-
alytes that are  uorescent or are converted to  uorescent
derivatives. Only carbaryl in honeybees and pollen (52) and
carbendazim in honey, wax, and pollen (8) have been de-
rivatized to  uorescent products.
Different possibilities of coupling LC to MS are now
well established and commercially available. Particle beam
has been applied to conŽ rm vinclozolin in honey and bee
larvae, but its poor sensitivity constitutes a drawback to its
use in honey analysis (8). The development of new inter-
faces, such as atmospheric pressure ionization, could lead
to a signiŽ cant increase in LC-MS utility for pesticide anal-
ysis in bee products. Twenty-two organophosphorous pes-
ticides in honeybees have been determined by LC–atmo-
spheric pressure chemical ionization–MS with positive and
negative ion modes. Detection limits ranged from 1 to 5
mg kg2 1. The selectivity of this technique using selected
ion monitoring solves problems of coeluting pesticides and
avoids most of the coextractive compounds from the hon-
eybee matrix (25).
MAXIMUM RESIDUE LIMITS
The maximum concentration of pesticide residues per-
mitted in honey is not included in the Codex Alimentarius
(9, 15), and the absence of maximum residue limits (MRLs)
makes it difŽ cult to ascertain whether a product is safe for
consumers (27). Individual European countries (66) have
established tolerance limits either for total pesticide resi-
J. Food Prot., Vol. 65, No. 9
dues in honey or for individual pesticides. Table 2 shows
MRLs established for pesticides in honey. The discrepan-
cies between the regulations of the different countries cause
much confusion. The European legislation has regulated the
MRLs for amitraz, coumaphos, and cymiazole in honey as
0.2, 0.1, and 1 mg kg2 1 , respectively (17, 48). The U.S.
Environmental Protection Agency has established MRLs
for amitraz (1 mg kg2 1 ), coumaphos (0.1 mg kg2 1 ), and
 uvalinate (0.05 mg kg2 1 ).
Regulation of pesticides in wax is important because
signiŽ cant amounts of beeswax are processed for pharma-
ceutical purposes and cosmetic industries. Furthermore,
acaricides used for beehive treatment are highly lipophilic;
consequently their residues accumulate in wax. Surprising-
ly, residues of pesticides in beeswax are not regulated, ex-
cept in the United States, which has established an MRL
of 6 mg kg2 1 for  uvalinate and amitraz and 100 mg kg2 1
for coumaphos.
APPLICATIONS OF THE METHOD
Pesticide residue control programs performed in dif-
ferent countries are shown in Table 3. Some of them are
focused on pesticides applied for crop protection, such as
organochlorides, organophosphate, pyrethroids, and carba-
mates, whereas others monitor acaricides used for varroa
control in beehives, such as amitraz, coumaphos, malathion,
bromopropylate, and  uvalinate. Amitraz and malathion are
not detected in high levels because they are unstable in
honey and degrade quickly. In contrast, lipophilic and non-
volatile acaricides, such as bromopropylate and  uvalinate,
are stable in honey and together with coumaphos are often
detected (65).
Every year, long-term quality control studies are per-
formed in Germany in which up to 1,000 honey, 220 Ger-
man wax, and 158 imported wax samples are analyzed.
During 1997, the most frequent varroacides found in honey
were coumaphos (28%), bromopropylate (11%), and  u-
valinate (1%). These pesticides were at ppb levels. These
relatively low levels of contamination were due to the high
lipophilic character of these acaricides (66). German wax
was shown to be contaminated with a large percentage of
bromopropylate (55%) and coumaphos (61%), with
amounts in the range of 1 to 5 mg kg2 1 and 0.5 to 10 mg
kg2 1 , respectively. On the other hand, in the same study,
more than 55% of the imported samples were contaminated
with  uvalinate in the range of 0.5 to 3.5 mg kg2 1 . Since
treatment practices by beekeepers change in each country,
the residue proŽ le of German honey was different from
imported honey (66).
Of 101 honey samples analyzed in Spain, 73 samples
were free of detectable residues; bromopropylate was found
in 16 samples (average, 21 mg kg2 1 ) and  uvalinate in 11
samples (average, 22 mg kg2 1). No residue of amitraz and
coumaphos was detected. Fluvalinate was the most widely
applied acaricide in beehives during the sampling period,
whereas the higher bromopropylate detection is attributed
to the acaricide application in previous years and its accu-
mulation in beeswax (28).
Routine sample analysis performed in France from
 J. Food Prot., Vol. 65, No. 9 PESTICIDE RESIDUE DETERMINATION IN BEE PRODUCTS
1986 to 1996 showed that 36% of the honey samples were
contaminated with  uvalinate, bromopropylate, and/or cou-
maphos, with average amounts of 2, 1.08, and 0.017 mg
kg2 1 , respectively (31).
Studies in Switzerland showed that the residue levels
found in wax samples contaminated with bromopropylate
and coumaphos decreased from 1991 to 1996. Residues of
bromopropylate diminished from levels of 5.3 to 2.4 mg
kg2 1 ; coumaphos residues slowly declined from 1.3 to 0.7
mg kg2 1 . However, since  uvalinate registration in 1991,
the levels of  uvalinate residues rapidly reached 2.6 to 2.9
mg kg2 1 between 1994 and 1996. Flumethrin was the only
pesticide that was not detected (10). In accordance with
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these results, all the beeswax samples analyzed in Greece
contained  uvalinate at concentrations that ranged from
0.44 to 30.1 mg kg2 1 (58).
Propolis has an afŽ nity to accumulate fat-soluble var-
roacides, and therefore it is susceptible to pesticide contam-
ination. In the 27 samples studied, all contained residues of
at least one acaricide. Fluvalinate was found in 26 samples
at an average level of 9.8 mg kg2 1, 10 samples contained
Journal of Food Protection 2002.65:1502-1511.
bromopropylate at 1.17 mg kg2 1 , and in two samples  u-
metrin at 2.54 mg kg2 1 was detected (66).
Recently, a review (48) related to residues in honey
and beeswax caused by acaricides in European countries
discussed the effect of different treatment procedures, the
migration of these substances from wax to honey, and the
fate of  uvalinate during recycling of wax. Wallner (65)
reviewed the accumulation of various varroacide residues
in different hive compartments, according to their chemical
characteristics.
Monitoring programs also focus on pesticides used for
plant protection and introduced into hives by contaminated
bees. Fifty pesticides in 26 samples of locally produced
honey and imported honey in Jordan were investigated dur-
ing 1995. Approximately 86% of the samples were contam-
inated with organochlorine pesticides, most commonly b-
HCH, a-HCH, and lindane. The organophosphorus levels
observed were lower than expected, probably because of
the rapid decomposition of these pesticides (3). In contrast
to a previous report, a screening of 27 samples in India
from 1993 to 1997 showed that the levels of organophos-
phorus were relatively high in comparison with organo-
chlorine and carbamates. This parallels the pattern of
changes in pesticides application that have taken place in
the last decade (4).
More than 100 pesticides in 52 samples of bees and
pollen samples were examined in New Jersey apiaries dur-
ing 1995. Of the samples, 42% had detectable levels of
methyl parathion, mostly coming from encapsulated for-
mulations. This was attributed to the pollen-gathering bees,
which may inadvertently pick up microcapsules, approxi-
mately the same size as a grain of pollen, and incorporate
them into the hive (50).
The concentrations of pesticides found in these reports
are far too low to cause toxic effects in humans, especially
if we consider that the daily average consumption of honey
is rarely higher than 50 g. Nevertheless, pesticide levels
should be periodically surveyed in honey to ensure that as
1509
long as the amount found does not rise above certain levels,
there will not be a risk for consumers. The possibility of
pesticide accumulation in wax and its migration to honey
should be also considered. In the near future, it is hoped
that some natural products, such as oxalic acid and formic
acid, would minimize the use of synthetic acaricides in bee
colonies.
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