Ecotoxicology and Environmental Safety 97 (2013) 210–222

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

The development, validation and application of a GC-dual detector

(NPD-ECD) multi-pesticide residue method for monitoring bee
poisoning incidents
Bożena Łozowicka
Plant Protection Institute—National Research Institute, Laboratory of Pesticide Residues, Chełmońskiego 22, Postal Code: 15-195 Bialystok, Poland

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

Article history: A simple multiresidue method based on matrix solid phase dispersion (MSPD) combined with clean-up
Received 22 February 2013 has been developed for the simultaneous screening of 153 pesticides in honeybees suspected of suffering
Received in revised form from pesticide poisoning during field spraying. Extraction and clean-up were carried out in a glass
8 July 2013
column containing anhydrous sulphate, 2.0 g of octadecyl (C18) and a 2.0-g sample of bees (23 insects on
Accepted 9 July 2013
Available online 31 July 2013
average) macerated with 4.0 g of Florisil. An additional layer of anhydrous sodium sulphate was added,
and acetonitrile was used as the elution solvent. This combination of clean-up steps ensured an efficient
Keywords: purification. A gas chromatograph with dual selective detectors for electron capture and nitrogen–
Pesticide phosphorous was used. The results presented in this paper demonstrate that matrix solid-phase
Bees
dispersion (MSPD) with the one-step clean-up procedure is the most effective extraction technique.
Poisoning incidents
MSPD method recoveries ranged from 70 to 118%, with precision values expressed as a relative standard
Gas chromatography
Multiresidue method of o20%, except for 10 pesticides that had recoveries of 50–70% and two with 120–130%. Low limits of
detection (0.003–0.04 μg/g) and quantification (0.005–0.05 μg/g) were readily achieved with this method
for all tested pesticides. A “top down” empirical model was used to estimate the expanded uncertainty at
28% on average (coverage factor k ¼2, confidence level 95%). The MSPD method was successfully used on
real bee samples to analyse four acaricides, 55 fungicides, 16 herbicides and 78 insecticides from various
regions of Poland. A total of 33 honeybee samples from suspected pesticide poisoning incidents were
analysed, in which 17 different pesticides were determined (14 insecticides and three fungicides). The
pesticides most often found in honeybees were cypermethrin (in 51% of the samples, 0.008–0.563 mg/bee),
chlorpyrifos (27%, 0.001–51.5 mg/bee) and biphentin (21%, 0.002–0.012 mg/bee).
& 2013 Elsevier Inc. All rights reserved.

1. Introduction animal pollinator (Williams, 1994). Chemical pesticides (insecticides,

Bees constitute a very important group of insects that are
exposed to both natural toxins and environmental contaminants
(Krupke et al., 2012; Detzel and Wink, 1993; Chauzat et al., 2009).
Throughout history, bees have come into contact with natural
toxins from nectar (Tan et al., 2012), honeydew and plant pollen
(Enan, 2001). In Europe, natural toxins represent no major danger
for bees. Thus, all bee poisoning cases on record can be traced to
human activity. Pesticide bee poisoning is a serious problem
throughout the world (Barnett et al., 2007; Genersch et al., 2010).
Honey bees are important in agriculture, in which growers of a
wide variety of crops rent hives from beekeepers to pollinate plants
and help increase yields (Garibaldi et al., 2011). It was estimated that
84% of European crop species depend at least to some extent on
animal pollination, with honey bees being the most important
E-mail addresses: B.Lozowicka@iorpib.poznan.pl, biuro@ior.bialystok.pl
herbicides and fungicides) are also important for providing the
optimal level of agricultural crop protection (Aktar et al., 2009).
Over the past decade, several health problems have affected the
beekeeping sector in different countries worldwide (Chauzat et al.,
2006; Cox-Foster et al., 2007; Genersch et al., 2010; Highfield et al.,
2009; Johnson et al., 2010; Mullin et al., 2010; van Engelsdorp
et al., 2007). In recent years, there have been several reports of
increased mortality in bees both in the EU and elsewhere (EC,
2010). This issue has caused serious concern throughout the world,
but scientific studies have not yet determined the exact cause or
extent of this increased mortality.
Most pesticides are toxic to honey bees to some extent; however,
the degree of toxicity varies considerably from product to product.
Insecticides are generally the most likely to cause bee death (Bernal
et al., 2011; Rortais et al., 2005), and herbicides, fungicides, and
defoliants present a minor danger. Severe bee losses may be expected
if particular pesticides are used when bees are present, if the product
is applied near beehives (Bonmatin et al., 2005; Chauzat and Faucon,

0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ecoenv.2013.07.010
 B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222
2007), or if bees forage in the pesticide application area within one
day after application (Barnett et al., 2007).
It is very difficult to quantify pesticide residues in samples of
poisoned bees. Sample preparation is one of the most critical parts of
pesticide analysis in bees because of their large amount of beeswax,
proteins and other substances, which are readily extracted by
solvents typically used in residue analysis. Most published methods
involve liquid–liquid extraction (LLE) followed by a clean-up step
(Fernandez et al., 2001a, 2001b, 2003; Bernal et al., 1997). However,
this technique is time-consuming, laborious, and requires large
volumes of both the sample and the organic solvent. Alternative
procedures analysing pesticides in honeybees include solid-phase
microextraction (SPME), gel permeation chromatography (GPC) or
supercritical fluid extraction (SFE).
To date, only a few reports have detailed the analytical metho-
dology for determining over 100 pesticides from various chemical
groups in the bodies of poisoned bees (Genersch et al., 2010; Mullin
et al., 2010; Walorczyk and Gnusowski, 2009).
Marconi et al. (2001) described a method for determining 18
organophosphorus pesticide residues in honeybee samples based on
solid-phase microextraction (SPME). The honeybee sample was homo-
genised by shaking it with an acetone/water solution and diluted with
water. Then, the pesticides were extracted by using two types of fibre
polyacrylate and poly(dimethyl)siloxane and analysed by gas chroma-
tography with nitrogen–phosphorus detection. Rossi et al. (2001)
analysed 28 organophosphorus and carbamate pesticide residues
using gas chromatography (GC) and gel permeation chromatography
(GPC) clean-up. The freeze-dried or lyophilised insect sample was
blended with diatomaceous earth (ExtraElut) and then eluted with
methylene chloride. The clean-up step was conducted by GPC using a
Bio Beads SX-3 column and a cyclohexane/ethylacetate eluent. Orga-
nophosphorus and carbamate compounds were quantified using
capillary gas chromatography with a flame ionisation detector (FID)
or an electron capture detector (ECD), and these results were
confirmed by GC–MS. Morzycka (2002) described two sample pre-
paration methods for analysing 12 insecticides in honeybees. The
methods are based on matrix solid phase dispersion using Florisil and
silica as dispersing agents, alumina and silica as clean-up adsorbents,
and n-hexane/ethyl ether/acetate as the elution mixture. The final
determinations were carried out by capillary gas chromatography with
a nitrogen–phosphorus detector (NPD). For the simultaneous deter-
mination of six pesticides in honeybees, Totti et al. (2006) developed a
matrix solid phase dispersion technique using C18 as the dispersant
and dichloromethane/methanol as the eluent in liquid chromatogra-
phy–atmospheric pressure chemical ionisation-mass spectrometry
(LC-APCI-MS). They compared this method with liquid–liquid
extraction (LLE) combined with LC-APCI-MS analysis. To quantify
the residues of 11 imidazole and triazole ergosterol-biosynthesis-
inhibiting (EBI) fungicides, Charlton and Jones (2007) developed an
analytical method with a high-performance gel permeation chro-
matography (HPGPC) clean-up and solid-phase extraction (SPE) on
Florisil cartridges. Detection was performed by gas chromatogra-
phy–mass spectrometry (GC–MS) in selected ion monitoring (SIM)
mode. To analyse imidacloprid and its main metabolite (6-chloroni-
cotinic acid), Garcia et al. (2007) proposed using liquid chromato-
graphy with post-column photochemical derivatisation in an
alkaline medium along with fluorescence detection. The compounds
were extracted from honeybees with acetone under ultra-sonic
conditions prior to liquid–liquid partitioning with dichloromethane.
Wiesta et al. (2011) described a multi-residue method based on a
modified ”QuEChERS method” for determining 80 environmental
contaminants, pesticides and veterinary drugs. Analysis was carried
out by gas chromatography coupled with time-of-flight mass
spectrometry (GC-ToF) and liquid chromatography coupled with
tandem mass spectrometry (LC–MS/MS). The “QuEChERS method”
combines salting-out liquid–liquid extraction with acetonitrile and
211
dispersive-SPE clean-up. A small fraction of hexane in acetonitrile
was added to honeybee samples to eliminate lipid interference with
mass spectrometry analysis. Walorczyk and Gnusowski (2009)
optimised this analytical method by employing gas chromatogra-
phy–tandem quadrupole mass spectrometry (GC–MS/MS) for the
simultaneous screening of approximately 150 pesticides in honey-
bees. Honeybees were subjected to homogenisation with an acet-
onitrile/water mixture followed by salting out with citrate buffer,
magnesium sulphate and sodium chloride. The amount of matrix
constituents in the extract with limited solubility in acetonitrile was
reduced by precipitation at a low temperature (freezing-out clean-
up). SPE clean-up was carried out by using primary secondary amine
(PSA), octadecyl (C18) and graphitised carbon black (GCB). This
combination of clean-up steps ensured efficient extract purification.
The two instrumental techniques cited above (GC–MS/MS or LC–MS/
MS) require very expensive equipment.
The objectives of this work were to develop a robust and
interference-free analytical method for the simultaneous extrac-
tion and determination of a much broader range of 153 pesticides
from various chemical groups associated with honeybees poison-
ing and to apply this method for routine analysis.
In this article, we present the validation and analytical features
of one of the most promising techniques. This procedure is based
on matrix solid-phase dispersion (MSPD) and uses Florisil as a
sorbent with a subsequent clean-up step by column chromato-
graphy with C18 as a cleaning agent, followed by gas chromato-
graphy with an NPD (nitrogen–phosphorous detector) and/or ECD
(electron capture detector), determination and confirmation. The
aim of this work was to maximise pesticide sensitivity and to
minimise the presence of interfering compounds in the extract. To
propose a reliable and robust method, parameters such as the type
and weight of sorbents, type and volume of solvent, spiking level
and amount of extracted sample were optimised. Moreover, the
method was widely tested using real honeybee samples. This
article also contains unique reports about the most frequent
incidental cases of honeybee poisoning in Poland.
2. Experimental
2.1. Chemicals and reagents
All reagents used were residue analysis grade. Acetone, acetoni-
trile and n-hexane for pesticide residue analysis were provided by J.T.
Baker (Deventer, Holland), along with Florisil (60–100 mesh), which
was activated at 600 1C. Anhydrous sodium sulphate was purchased
from Fluka (Seelze-Hannover, Germany). Octadecyl gel C18 (40 μm
Prep LC) was obtained from J.T. Baker (USA).
2.2. Pesticide standards
Pesticides (153 active substances) were obtained from Dr.
Ehrenstorfer Laboratory (Germany). Standard stock solutions of
various concentrations were prepared in acetone and stored at
4 1C (purity 495%). Standard working solutions were prepared by
dissolving 1 ml of stock solutions in n-hexane/acetone (9:1, v/v)
mixture (concentration range 0.001–2.5 μg/g).
2.3. Sample preparation procedure
An amount of 2 g of bee (23–29 insects) sample was put in a
mortar with 4 g of solid support (Florisil). This was manually
blended using a pestle to produce a homogeneous mixture which
was packed into a glass macro column (300  12 mm I.D.) with
anhydrous sodium sulphate (2.0 g) and C18 (2.0 g). An additional
212 B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222
Fig. 1. Steps of isolation, extraction and purification of 153 pesticide residues in bees
by MSPD and determination by GC with dual system EC/NP detectors. 1—glass wool, 2
—anhydrous sodium sulphate, 3—C18, 4—anhydrous sodium sulphate, 5—bee samples
with Florisil, 6—acetonitrile, 7—evaporation and dissolving in hexane/acetone 9:1, v/v.
layer of anhydrous sodium sulphate (2.0 g) was put on the bottom of
column. The adsorbed analytes were eluted using 25 ml acetonitrile.
The obtained extracts were evaporated to dryness using a
rotary evaporator at a temperature of about 40 1C, and dried
residue was dissolved in the 2 ml of hexane/acetone (9:1, v/v)
and then transferred to 2 ml vials for further GC analysis. The
sample preparation procedure is shown on Fig. 1.
2.4. Preparation of spiked bee samples
Honeybee samples were obtained during the winter from environ-
mental monitoring stations located in the Podlasie region. Winter bees
are pesticide-free, as bees are not active during this period; this fact
was confirmed before the validation study through the analysis of
blank samples. Samples were stored at  20 1C until analysis. Spiked
samples of bees were prepared by spraying 2 g of bees with the 1 ml
of spiking solution. The spiked samples were left for 1 h. Bee samples
were extracted by MSPD extraction according to the scheme presented
in Fig. 1. The main purpose of this step was to calculate the average
recovery percentage of investigated pesticides achieved by MSPD
extraction techniques.
2.5. GC–ECD/NPD conditions
An Agilent 7890 gas chromatograph (Santa Clara, CA, USA)
equipped with a Model HP 7683 automatic split–splitless injector, a
63
Ni micro-electron capture detector (mEC), and a nitrogen phosphor-
ous detector (NP) were used. The flux at the end of the GC column was
divided into two branches by means of a “Y” press-tight connector
connected at one end to the GC column and on the other to the two
detectors (Fig. 1). Data acquisition and processing were performed
with Chemstation (Hewlett-Packard, version A.10.2) software.
The DB-35, a midpolarity column (35%-phenyl)-methylpolysi-
loxane) with low bleed (30 m  0.32 mm I.D.  0.5 mm film thick-
ness), supplied by Agilent (Little Falls, DE, USA), was employed.
The operating conditions were as follows:
– injector temperature: 210 1C; carrier gas: helium at a flow-rate of
1.9 ml/min; detector temperature: 300 1C (ECD and NPD); make
up gas: nitrogen at a flow-rate of 57 ml/min (ECD) and 8 ml/min
(NPD), hydrogen 3.0 ml/min, air 60 ml/min.
– oven—initial temperature: 120 1C increased to 190 1C at 13 1C/min,
then to 240 1C at 8 1C/min and finally to 295 1C at 168 1C/min and
held for 20 min (ECD and NPD).
The 2 ml volume of the final sample extract was injected at
210 1C in splitless mode (split off time 2 min). Total time of
analysis: 35 min and equilibration time 2 min. Quantification was
performed by comparing the height of peaks obtained in samples
with those found in matrix-matched calibration standards for the
mixture ( 70.005 min for positive match).
In the case of positive peaks of pesticides detected above LODs,
the results were confirmed by analysis on a different polarity
column. A fused silica capillary column, HP-5, with 5% diphenyl,
95% dimethyl polysiloxane the nonpolar stationary phase
(30 m  0.32 mm I.D.  0.25 mm film thickness) was found to be
ideal for confirmatory analysis under the following conditions:
– injector temperature: 210 1C; carrier gas: helium at a flow-rate
of 3.0 ml/min; detector temperature: 300 1C (ECD and NPD);
make up gas: nitrogen at a flow-rate of 57 ml/min (ECD) and
8 ml/min (NPD), hydrogen 3.0 ml/min, air 60 ml/min.
– oven—initial temperature: 120 1C increased to 190 1C at 16 1C/
min, then to 230 1C at 8 1C/min and finally to 285 1C at 18 1C/min
and held for 10 min (ECD and NPD).
2.6. Estimation of uncertainty
The steps taken during uncertainty estimation of the analytical
result were according to the Guide to Quantifying Uncertainty in
Analytical Measurements (EURACHEM/CITAC, 2000). These steps
were: defining the measuring procedure and determining the
measured value, developing a mathematical model to be used
for calculating analytical results based on the measured para-
meters, finding values for all possible parameters that can influ-
ence the final results, estimating the associated standard
uncertainties, and applying the law of propagation of uncertainty
in order to calculate the combined standard uncertainty of the
final results. The combined standard uncertainty was determined
by using ProNP3 (PROLAB) software.
3. Results and discussion
3.1. Matrix solid phase dispersion
Sample preparation represented a critical point in the devel-
opment of the method because of the high complexity of the
honeybee matrix, which contained large amounts of beeswax,
proteins and other substances readily extractable by organic
solvents (Fernandez et al., 2002). The isolation and clean-up
procedure for the 153 pesticides in bee samples was assayed to
develop a quick, easy and efficient procedure, which could be
adapted for routine analysis at high throughput and low cost.
The method for isolating, extracting and purifying pesticide
residues in bee samples by MSPD is shown on Fig. 1. Kadenczki
et al. (1992) have developed an MSPD technique using activated
Florisil at 600 1C as a dispersing sorbent and a mixture of
dichloromethane/acetone to elute analytes from fruits and vege-
tables. That method was not applied in this study, but it provided a
basis for a number of modifications.
In our work, different parameters that affect MSPD extraction,
such as the dispersing sorbent, clean-up adsorbent, matrix/sorbent
ratio, eluent solvents and sample weight were studied. Analyte
recoveries were calculated against the sample weight. Sample weight
increases of up to 10.0 g did not affect compound recoveries.
The efficiency of MSPD depends on the type and quantity of the
sorbent. First, three different types of dispersing materials such as
Florisil, silica gel, and basic alumina are activated and deactivated
(by adding water), then tested. The use of deactivated sorbents
such as 12% basic alumina and 5% silica gel yielded recoveries
 B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222
below 50% (first activated at a temperature of 130 1C and then
deactivated by the addition of 12 and 5% water). The increase in
the sorbent activation temperature to 600 1C was necessary to
increase the pesticide recovery. Florisil gave better results than silica
and alumina. Moreover, Florisil produced the cleanest chromato-
graphic profiles, probably because of its preferential adsorption of
polar sample components, which interfered with compounds on the
Florisil surface. This sorbent has several functions, as follows: (1) it
works as an abrasive compound by breaking the physical structure of
the bee sample, (2) it adsorbs the compounds of the matrix, (3) it
works as a\solid support for filling the column and (4) it allows for
the fractionation of the sample. Therefore, Florisil was chosen as the
solid support for subsequent experiments.
The main challenge in developing the reported method was the
separation of the beeswax such as cuticular lipids (n-alkanes, fatty
acids, and alkenes) (Kather et al., 2011), pigments, or other com-
pounds from the analytes of interest. Then, five adsorbents with very
diverse surface characteristics and selectivity such as neutral alu-
mina, silica, activated carbon, Florisil, silica-based octadecyl (C18) or
their combinations were tested to purify the extract. Although
pesticide recoveries were not similar for all clean-up adsorbents,
C18 provided a cleaner extract because lipids showed a greater
affinity for this phase. Using 2.0 g of C18 as the cleaning adsorbent at
the bottom of the column yielded the best recoveries.
Based on the results of our experiments, we decided that the
combination of Florisil and C18 sorbents at a proportion of 4.0 and
2.0 g, respectively, would ensure efficient and robust simultaneous
extraction and remove interferences from the matrices for good
results. Florisil, a form of magnesium silicate, is a normal-phase
sorbent used to remove polar interferences from the extract prior
to instrumental analysis. The remarkable advantages of this clean-
up technique are its simplicity of operation, rapid sample through-
put, and no need for specialised equipment.
We also tested four solvents of different polarities, individually or
in mixtures, at different elution volume ratios. The pesticides of
interest covered a wide range of polarities from the polar, with pro-
poxur (logKow 0.14) to the non-polar, with lambda-cyhalothrin (logKow
6.9) (PPDB, 2013). The extraction and elution of these pesticides was
carried out in one step with the same single solvent or mixture of
solvents to establish the best procedure. The selected solvents were
15 ml of hexane/acetone (8:2, v/v) and hexane/acetone/diethyl ether
(1:2:2, v/v/v). Elution with different proportions of hexane and ethyl
ether or acetone provided high recoveries of R4120% for some
compounds and low recoveries of Ro50% for other compounds, but
the resulting extracts were dirty with waxes and pigments. In contrast,
the best results were found for an elution with 100% of acetonitrile.
Among all the tested solvents, a small volume (25 ml) of acetonitrile
appeared to be optimal, yielded a good mean recovery of 70–118%, and
reduced the amounts of co-extractives.
Finally, this method employed a simultaneous extraction and
purification step using a glass column with (starting from the
bottom of the column) the following: a layer of anhydrous sodium
sulphate (2.0 g), 2.0 g of organic phase C18 as a clean-up material,
a layer of anhydrous sodium sulphate (2.0 g), 2.0 g of bees blended
with 4.0 g of Florisil as a supporting material and acetonitrile as
the extraction and elution solvent, which represented the best
compromise for all analytes. An additional purification step was
not necessary. This technique was successfully assessed for the
extraction of pyrethroids, OCPs, OPPs, and acaricides from bees.
Moreover, the extraction and clean-up are performed in the same
step, thus saving analysis time and organic solvent.
3.2. GC instrumental analysis
Analyte identification was carried out by comparing the
peak retention times within a sample to the retention times for
213
standards; quantification was based on the height of the sample
extract peak compared to the height of the pesticide standard
solution in the matrix (70.005 min for positive confirmation).
In gas chromatography analysis, some applications simulta-
neously use two GC detectors connected to two columns contain-
ing different stationary phases (Tse et al., 2004) or the same
stationary phase (Morello et al., 1996). Applications in which the
flux of a single column is divided between two different GC
detectors, in which the splitter is placed after the injection port,
or in which the precolumn and sample run in parallel on two gas
chromatographic columns of different polarities may be used to
analyse substances with different chemical structures after inject-
ing the sample only once.
Our proposed instrumental method allows for the determina-
tion of pesticides in bees by GC using two simultaneously
functioning selective detectors. In this work, we used a configura-
tion with a “Y” piece at the end of the GC column to divide the flux
at the end of the GC column into two equal flowing branches
(Fig. 1) (one to the NPD and the other to the ECD), thereby
allowing different pesticides to be quantified in the same run;
43 pesticides were detected by the ECD, whereas 34 were analysed
by NPD, although ECD and NPD also provided a discernible signal
for 76 of them.
Table 1 summarises the pesticides from this study. All pesti-
cides were satisfactorily separated with a high degree of sensitivity
and selectivity. The absence of co-extracted compounds was
confirmed by blank sample analysis. The developed MRM (Multi
Residue Method) provides extracts without interference during
the GC run.
Fig. 2 shows representative chromatograms for selected stan-
dard mixtures of 39 pesticides as prepared in the matrix. Among
them, 32 were determined on the ECD (Fig. 2a and b), 21 on the
NPD (Fig. 2c and d) and 15 were simultaneously determined on
both detectors.
In the case of co-eluting pesticides, the application of a dual
detection system allows for their determination. For example, the
co-eluted peaks of pyrimethanil and gamma-HCH were observed.
However, this result was not a problem because pyrimethanil was
only detected with NPD and gamma-HCH was only with ECD (HP-
35 column) (Table 1). In that situation, a capillary column with
different polarity (HP-5) was also used in the same detection
system. This is very important when compound identification is
only possible on a single detector. In this way, the presence or
absence of the compound can still be confirmed.
3.3. Validation method
The capacity of the analytical method to determine a particular
analyte, metabolite or known additives was investigated. A lack of
interference was demonstrated by the analysis of the concentrated
blank formulations and concentrated sample extracts. The use of a
second capillary column with a different polarity also confirmed
the interference-free separation (HP-5). The following parameters
were determined during the validation of the analytical method:
linear range, LOD, LOQ, accuracy, precision, and matrix effects. All
validation procedures were performed using bee extracts from
samples with no pesticides.
Calibration curves were obtained from matrix-matching solu-
tion calibrations. The lowest concentration level in the calibration
curve was established as a practical determination limit. Calibra-
tion standards were prepared by adding spiking solutions into a
blank matrix of the bees to produce a final concentration with a
1st range of 0.005 to 0.05 μg/g, a 2nd range of 0.05 to 0.5 μg/g and
a 3rd range of 0.5 to 2.5 μg/g.
Recovery data were obtained at three range concentrations in
the matrix each day by using blank bee samples in accordance
Table 1

214
Validation parameters for bees samples fortified with 153 pesticides (sorted by retention time, DB-35 and DB-5 column).

No. Active substance Pesticides R2 LOD LOQ Retention time tr [min] First fort. level Mean recovery 7 RSD Second fort. Mean recovery 7 RSD Third fort. level Mean recovery 7RSD
type [μg/kg] [μg/g] [μg/g] [%] (n¼ 3) level [μg/g] [%] (n¼ 3) [μg/g] [%] (n¼3)
DB-35 HP- 5

EC NP EC NP

1. Dichlorvos I/A 0.99983 0.009 0.010 5.291 5.288 2.951 2.961 0.030 61.9 7 8.00 0.300 62.17 4.80 1.500 71.17 8.80
2. Cymoxanil F 0.99866 0.040 0.050 6.250 6.242 3.125 3.159 0.050 50.3 7 2.57 0.500 55.0 7 3.97 2.500 62.8 7 2.47
3. Dichlobenil H 0.99994 0.009 0.010 6.848 6.840 3.680 3.678 0.010 70.2 7 4.29 0.100 75.2 7 6.50 0.500 74.17 4.58
4. Propham H 0.99947 0.009 0.010 7.428 4.309 0.010 108.4 7 1.35 0.100 90.0 7 2.27 0.500 95.17 3.06
5. Mevinphos I/A 0.99967 0.008 0.010 7.652 7.648 4.103 4.107 0.010 78.4 7 0.76 0.100 70.8 7 4.26 0.500 84.6 7 2.07
6. Metacriphos I/A 0.99939 0.005 0.010 8.086 8.078 4.623 4.625 0.010 76.4 7 1.89 0.100 76.9 7 2.94 0.500 78.4 7 1.67
7. Trifluralin H 0.99999 0.005 0.010 8.754 8.746 5.940 5.937 0.010 94.8 7 2.05 0.100 106.2 7 3.87 0.500 92.5 7 2.50
8. DEET I 0.99996 0.010 0.030 9.157 5.242 0.030 110.8 7 2.48 0.300 116.5 7 0.85 1.500 105.4 7 4.06
9. Heptenophos I 0.99913 0.008 0.010 9.479 5.228 0.010 110.7 7 3.74 0.100 98.4 7 2.51 0.500 75.9 7 3.65

B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222

10. Tecnazene F 0.99742 0.010 0.010 9.618 5.539 0.010 67.5 7 1.12 0.100 68.4 7 1.66 0.500 78.4 7 0.76
11. Propachlor H 0.99999 0.005 0.010 9.772 9.764 5.538 5.537 0.010 105.0 7 2.59 0.100 113.5 7 2.07 0.500 104.4 7 2.17
12. Ethoprophos I 0.99754 0.005 0.010 9.805 9.796 5.678 5.679 0.020 91.0 7 2.48 0.200 91.7 7 10.11 1.000 97.0 7 2.32
13. Chlorpropham H 0.99706 0.008 0.010 9.764 5.999 0.020 99.7 7 3.34 0.200 95.7 7 2.63 1.000 86.9 7 2.02
14. Captan F 0.99917 0.009 0.010 16.064 9.823 0.010 52.7 7 12.15 0.100 55.17 6.46 0.500 57.5 7 5.26
15. Folpet F 0.99889 0.009 0.010 16.146 9.948 0.010 62.6 7 2.06 0.100 69.17 2.86 0.500 71.6 7 1.86
16. Propoxur I/A 1.00000 0.020 0.030 9.960 5.478 0.030 82.6 7 2.15 0.300 95.17 2.86 1.500 87.6 7 1.86
17. Diphenylamine F 0.99507 0.009 0.010 10.128 5.619 0.010 75.9 7 8.58 0.100 88.0 7 2.67 0.500 72.3 7 5.34
18. Phorate I/A 0.99991 0.006 0.010 10.645 10.634 6.211 6.211 0.010 64.5 7 0.36 0.100 68.6 7 2.46 0.500 73.6 7 0.44
19. Cadusafos I 0.99562 0.005 0.010 10.720 6.129 0.010 71.9 7 5.89 0.100 70.8 7 4.26 0.500 75.9 7 10.9
20. HCB I 0.99761 0.005 0.010 10.780 6.514 0.010 76.0 7 2.43 0.100 73.7 7 3.30 0.500 75.5 7 2.57
21. Alpha-HCH I 0.99963 0.004 0.005 11.087 6.365 0.005 89.5 7 6.06 0.050 78.7 7 1.23 0.250 93.6 7 3.39
22. Propyzamide H 0.99937 0.008 0.010 11.243 11.234 6.965 6.962 0.010 53.9 7 1.19 0.100 54.8 7 2.07 0.500 61.9 7 3.04
23. Diazinon I/A 0.99934 0.010 0.010 11.560 11.548 7.078 7.079 0.010 71.0 7 2.61 0.100 87.57 2.56 0.500 78.2 7 2.33
24. Atrazine H 0.99898 0.010 0.010 11.566 6.639 0.010 84.6 7 2.07 0.100 87.17 1.08 0.500 85.3 7 1.15
25. Simazine H 0.99956 0.005 0.010 11.711 6.556 0.010 97.0 7 3.04 0.100 98.8 7 1.21 0.500 73.7 7 2.20
26. Dicloran F 0.99568 0.005 0.008 11.762 6.540 0.010 83.17 2.84 0.100 95.8 7 4.52 0.500 93.5 7 6.04
27. Quintozene F 0.99794 0.010 0.030 11.820 7.009 0.030 95.8 7 1.12 0.300 92.4 7 6.65 1.500 91.9 7 2.95
28. Gamma-HCH I 0.99779 0.003 0.005 12.099 6.922 0.005 93.5 7 3.17 0.050 94.6 7 2.80 0.250 85.8 7 3.50
(lindane)
29. Pyrimethanil F 0.99998 0.008 0.010 12.104 7.081 0.010 102.5 7 3.30 0.100 107.4 7 3.66 0.500 116.17 2.97
30. Carbofuran I/A 0.99997 0.010 0.020 12.190 6.577 0.020 65.2 7 0.96 0.200 65.2 7 0.15 1.000 72.3 7 4.88
31. Beta-HCH I 0.99967 0.010 0.010 12.203 6.885 0.010 93.0 7 2.63 0.100 97.3 7 1.59 0.500 89.7 7 2.47
32. Dimethoate I/A 0.99989 0.005 0.008 12.305 12.295 6.524 6.524 0.010 101.4 7 0.88 0.100 103.0 7 2.57 0.500 94.7 7 3.95
33. Paraoxon-methyl I 0.99945 0.007 0.010 12.833 12.822 7.225 7.225 0.010 70.8 7 8.24 0.100 78.5 7 5.03 0.500 74.17 6.04
34. Vinclozolin F 0.99995 0.009 0.010 12.881 12.871 8.002 7.999 0.010 82.4 7 6.00 0.100 98.17 2.59 0.500 97.3 7 1.75
35. Acetochlor H 0.99890 0.010 0.020 12.905 12.895 7.969 7.966 0.020 64.7 7 1.62 0.100 67.4 7 2.60 0.500 75.4 7 4.06
36. Chlorothalonil F 0.99653 0.008 0.010 12.924 7.418 7.416 0.010 49.3 7 5.89 0.100 52.5 7 6.28 0.500 53.4 7 5.89
37. Heptachlor I 0.99920 0.005 0.010 13.089 8.250 0.010 72.8 7 0.83 0.100 74.17 2.48 0.500 73.7 7 1.15
38. Chlorpyrifos- I 0.99990 0.005 0.010 13.364 13.352 8.037 8.037 0.010 104.9 7 4.62 0.200 106.17 3.50 1.000 93.4 7 0.64
methyl
39. Prometrine H 0.99771 0.009 0.010 13.394 8.199 0.010 106.7 7 3.35 0.100 106.4 7 3.15 0.500 97.0 7 3.77
40. Fenchlorphos I 0.99989 0.008 0.010 13.448 13.437 8.314 8.314 0.010 54.8 7 1.72 0.100 71.2 7 1.78 0.500 75.4 7 7.02
41. Parathion-methyl I/A 0.99964 0.008 0.010 13.573 13.562 8.034 8.034 0.010 98.9 7 2.44 0.100 109.6 7 2.72 0.500 95.6 7 3.29
42. Metalaxyl I/A 0.99999 0.007 0.010 13.630 8.242 0.010 64.6 7 1.19 0.100 76.5 7 2.50 0.500 89.4 7 2.40
43. Pirimiphos- I/A 0.99997 0.009 0.010 13.662 8.539 0.010 71.3 7 0.62 0.100 70.2 7 0.67 0.500 75.8 7 2.66
methyl
44. Paraoxon-ethyl I 0.99997 0.007 0.010 13.618 13.581 8.231 8.231 0.010 74.7 7 6.56 0.100 72.3 7 5.03 0.500 75.8 7 3.19
45. Tolclofos-methyl F 0.99998 0.008 0.010 13.666 13.655 8.132 8.132 0.010 104.8 7 3.78 0.100 97.9 7 2.80 0.500 96.6 7 0.23
46. Metribuzin H 0.99999 0.006 0.010 13.705 13.694 7.880 7.877 0.010 53.8 7 3.57 0.100 58.17 11.97 0.500 58.17 2.87
47. Aldrine I 0.99999 0.004 0.005 13.856 8.934 0.005 101.0 7 3.56 0.050 93.17 1.79 0.250 98.17 0.15
48. Tetraconazole F 0.99990 0.006 0.010 13.953 13.941 9.077 9.074 0.010 76.5 7 2.57 0.100 87.5 7 2.49 0.500 84.6 7 2.21
49. Triadimefon F 0.99995 0.009 0.010 13.991 13.978 9.016 9.013 0.010 64.4 7 2.16 0.100 77.4 7 3.29 0.500 98.5 7 3.05
 50. Malathion I/A 0.99999 0.005 0.010 14.013 14.001 8.704 8.704 0.010 78.8 7 3.78 0.100 94.7 7 2.57 0.500 94.7 7 3.31
51. Chlorpyrifos I/A 0.99995 0.005 0.010 14.060 14.049 8.958 8.958 0.010 106.6 7 1.79 0.100 106.6 7 2.99 0.500 105.7 7 2.80
52. Fenitrothion I 0.99998 0.005 0.010 14.065 14.052 8.542 8.544 0.010 76.7 7 2.47 0.100 70.8 7 3.06 0.500 74.8 7 3.22
53. Carbaryl I 0.99993 0.020 0.030 14.160 8.133 0.030 54.9 7 3.79 0.300 67.8 7 15.87 1.500 77.8 7 2.29
54. Parathion-ethyl I/A 0.99644 0.005 0.010 14.197 14.184 8.977 8.978 0.010 113.7 7 1.15 0.100 94.8 7 2.78 0.500 95.2 7 1.32
55. Pirimiphos-ethyl I/A 0.99997 0.009 0.010 14.233 9.343 0.010 71.7 7 6.24 0.100 76.3 7 5.03 0.500 75.4 7 3.90
56. Dicofol A 0.99965 0.005 0.010 14.424 9.049 0.010 55.2 7 1.51 0.100 53.3 7 5.16 0.500 73.7 7 2.45
57. Fenthion I 0.99933 0.010 0.010 14.493 8.918 0.010 65.17 3.33 0.100 65.6 7 3.25 0.500 73.7 7 2.36
58. Pendimethalin H 0.99983 0.008 0.010 14.625 14.607 9.603 9.601 0.020 81.2 7 2.20 0.200 88.9 7 5.84 1.000 83.0 7 1.40
59. Bromophos I 0.99977 0.008 0.010 14.652 14.639 9.340 9.341 0.010 86.8 7 1.36 0.100 76.8 7 3.01 0.500 84.8 7 2.50
methyl
60. Isofenphos I 0.99992 0.007 0.010 14.659 14.646 9.473 9.473 0.010 103.4 7 1.36 0.100 93.7 7 0.31 0.500 97.7 7 1.01
methyl
61. Isofenphos I 0.99995 0.008 0.010 14.790 14.777 9.758 9.757 0.020 100.4 7 0.26 0.200 105.3 7 4.08 1.000 93.8 7 2.65
62. Cyprodinil F 0.99974 0.009 0.010 14.791 9.477 0.010 96.6 7 2.74 0.100 96.5 7 2.90 0.500 96.8 7 0.64
63. Penconazole F 0.99996 0.007 0.010 14.807 14.794 9.635 9.632 0.010 77.9 7 1.72 0.100 74.7 7 3.05 0.500 77.17 3.12
64. Triadimenol F 0.99960 0.010 0.020 14.846 14.833 9.832 9.829 0.020 76.3 7 2.13 0.200 74.0 7 0.44 1.000 72.4 7 6.32
15.002 14.989 9.956 9.953
65. Chlorfenvinphos I/A 0.99999 0.008 0.010 15.010 14.996 9.756 9.758 0.010 103.0 7 0.96 0.100 95.4 7 12.45 0.500 91.3 7 5.29

B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222

66. Mecarbam I/A 0.99994 0.009 0.010 15.032 15.018 10.012 9.759 0.010 98.7 7 0.80 0.100 97.2 7 0.75 0.500 109.4 7 2.54
67. Heptachlor- I 1.00000 0.006 0.010 15.074 9.786 0.020 118.0 7 4.60 0.200 94.3 7 1.72 1.000 94.3 7 3.50
epoxide
68. Bromophos ethyl I 0.99993 0.007 0.010 15.124 15.110 10.132 10.132 0.010 100.0 7 2.35 0.100 88.4 7 0.72 0.500 97.5 7 0.40
69. Tolylfluanid F 0.99987 0.009 0.010 15.136 9.724 0.010 59.8 7 8.55 0.100 69.3 7 3.70 0.500 73.17 13.40
70. Procymidone F 0.99998 0.009 0.010 15.164 15.151 9.946 9.943 0.010 78.7 7 2.40 0.100 78.4 7 10.14 0.500 75.6 7 2.06
71. Metazachlor H 0.99407 0.005 0.010 15.222 15.209 9.602 9.600 0.010 65.4 7 1.02 0.100 64.3 7 2.80 0.500 75.2 7 8.76
72. Quinalphos I/A 0.99987 0.005 0.010 15.256 15.241 9.832 9.832 0.010 76.5 7 8.10 0.100 70.6 7 2.48 0.500 72.2 7 1.05
73. Paclobutrazol F 1.00000 0.010 0.020 15.376 15.361 10.193 10.190 0.020 77.17 13.76 0.200 78.8 7 4.29 1.000 76.3 7 5.14
74. Alpha-endosulfan I/A 0.99998 0.005 0.010 15.653 10.365 0.010 89.4 7 3.59 0.100 89.6 7 3.16 0.500 90.3 7 3.15
75. Tetrachlorvinphos I/A 0.99999 0.008 0.010 15.717 15.702 10.260 10.260 0.010 90.2 7 6.88 0.100 99.8 7 3.94 0.500 85.0 7 1.04
76. Hexaconazole F 0.99979 0.008 0.050 15.750 15.734 10.531 10.528 0.050 74.3 7 4.60 0.500 71.2 7 1.68 2.500 78.6 7 3.15
77. Picoxystrobin F 0.99995 0.009 0.010 15.776 15.762 10.393 10.391 0.010 58.7 7 2.35 0.100 55.6 7 2.31 0.500 63.9 7 1.53
78. p,p′DDE I 0.99959 0.005 0.010 15.869 10.705 0.010 102.5 7 5.54 0.100 103.17 4.54 0.500 101.5 7 3.86
79. Profenfos I/A 0.99999 0.005 0.010 16.003 15.988 10.622 10.621 0.010 79.4 7 0.17 0.100 77.8 7 3.96 0.500 76.2 7 6.52
80. Buprofezin I/A 0.99996 0.008 0.010 16.022 10.833 0.010 62.0 7 2.90 0.100 77.5 7 0.89 0.500 62.7 7 1.96
81. Mepanipyrim F 0.99972 0.020 0.030 16.028 10.279 0.030 64.3 7 1.35 0.300 63.5 7 4.52 1.500 77.17 1.74
82. Napropamide H 0.99999 0.010 0.020 16.045 10.505 0.020 97.6 7 6.46 0.200 84.17 0.23 1.000 83.7 7 2.90
83. Flusilazole F 0.99976 0.005 0.010 16.095 10.854 0.010 83.6 7 0.84 0.100 92.8 7 2.52 0.500 93.5 7 0.64
84. Methidathion I/A 0.99975 0.008 0.010 16.107 16.092 10.102 10.104 0.010 96.8 7 2.69 0.100 108.17 0.90 0.500 95.3 7 2.86
85. Fipronil I 0.99997 0.009 0.010 16.140 9.749 9.746 0.010 59.5 7 1.25 0.100 72.5 7 5.68 0.500 78.5 7 5.23
86. Flutriafol F 0.99985 0.010 0.020 16.166 10.402 0.020 91.9 7 0.38 0.200 83.7 7 3.11 1.000 85.5 7 2.22
87. Bupirimate F 0.99994 0.006 0.010 16.219 16.204 10.857 10.855 0.010 85.5 7 4.49 0.100 93.17 3.61 0.500 93.7 7 1.92
88. Dieldrin I 0.99971 0.007 0.010 16.258 10.793 0.010 98.6 7 0.72 0.100 97.4 7 1.62 0.500 104.6 7 2.42
89. Myclobutanyl F 0.99990 0.009 0.010 16.377 16.363 10.791 10.789 0.010 65.8 7 0.20 0.100 87.6 7 2.54 0.500 72.3 7 13.12
90. Kresoxim-methyl F 0.99993 0.009 0.010 16.602 16.588 10.867 10.864 0.010 75.7 7 1.34 0.100 75.7 7 1.36 0.500 72.3 7 2.80
91. Cyproconazole F 0.99992 0.009 0.010 16.787 11.296 0.010 77.6 7 4.12 0.100 78.0 7 1.96 0.500 75.6 7 3.47
92. Nitrofen H 0.99995 0.005 0.010 16.763 11.046 0.010 76.8 7 2.50 0.100 93.17 6.27 0.500 90.9 7 4.15
93. Diniconazole F 0.99946 0.005 0.010 16.814 16.797 11.298 11.296 0.010 79.6 7 2.15 0.100 77.3 7 1.35 0.500 73.9 7 2.16
94. Fludioxonil F 0.99977 0.008 0.010 17.010 10.625 0.010 55.8 7 4.48 0.100 106.6 7 0.87 0.500 51.8 7 1.16
95. p,p′DDD I 0.99990 0.005 0.010 17.033 11.353 0.010 95.6 7 3.10 0.100 92.8 7 2.61 0.500 84.9 7 2.23
96. Endrin I 0.99987 0.004 0.010 17.037 11.142 0.010 83.7 7 2.10 0.100 91.9 7 4.91 0.500 88.8 7 2.44
97. Ethion I/A 0.99996 0.007 0.010 17.049 17.033 11.384 11.384 0.010 71.17 2.52 0.100 74.7 7 5.46 0.500 89.17 5.04
98. Azaconazole F 0.99995 0.008 0.010 17.062 17.048 10.895 10.893 0.010 74.9 7 1.97 0.100 75.17 2.60 0.500 78.9 7 1.62
99. o,p′DDT I 0.99608 0.008 0.010 17.088 11.409 0.010 95.4 7 3.26 0.100 99.0 7 4.53 0.500 97.3 7 1.52
100. Beta-endosulfan I/A 0.99999 0.006 0.010 17.413 11.269 0.020 107.8 7 0.72 0.200 93.0 7 12.04 1.000 92.5 7 2.65
101. Trifloxystrobin F 0.99956 0.009 0.010 17.456 17.440 11.779 11.777 0.010 67.5 7 1.73 0.100 66.5 7 3.59 0.500 78.6 7 5.37
102. Propiconazole F 0.99996 0.005 0.010 17.721 17.705 11.789 11.786 0.010 71.17 4.51 0.100 78.6 7 3.36 0.500 75.5 7 2.79
17.829 17.814 11.877 11.875
103. p,p′DDT I 0.99987 0.005 0.010 17.810 11.878 0.010 98.5 7 2.44 0.100 91.2 7 3.97 0.500 85.17 4.67
104. Benalaxyl F 0.99984 0.020 0.030 17.954 11.737 0.030 54.7 7 13.15 0.300 64.6 7 4.46 1.500 79.0 7 4.61
105. Bifenthrin I/A 0.99979 0.008 0.010 18.017 12.497 0.010 69.8 7 4.21 0.100 67.5 7 1.27 0.500 72.9 7 0.59

215
106. Fenhexamid F 0.99965 0.009 0.010 18.085 18.071 11.882 11.879 0.010 61.17 2.66 0.100 71.9 7 3.40 0.500 75.9 7 2.19
 216
Table 1 (continued )

No. Active substance Pesticides R2 LOD LOQ Retention time tr [min] First fort. level Mean recovery 7 RSD Second fort. Mean recovery 7 RSD Third fort. level Mean recovery 7RSD
type [μg/kg] [μg/g] [μg/g] [%] (n¼ 3) level [μg/g] [%] (n¼ 3) [μg/g] [%] (n¼3)
DB-35 HP- 5

EC NP EC NP

107. Tebuconazole F 0.99971 0.009 0.010 18.135 12.044 0.010 71.3 7 3.43 0.100 75.9 7 1.15 0.500 86.7 7 5.09
108. Triazophos I/A 0.99986 0.008 0.010 18.270 11.571 0.020 104.5 7 0.92 0.200 93.9 7 0.32 1.000 76.3 7 2.91
109. Oxadixyl F 0.99995 0.020 0.030 18.410 18.395 11.393 11.391 0.030 93.2 7 5.42 0.300 112.2 7 4.52 1.500 123.17 6.70
110. Endosulfan- I/A 0.99988 0.005 0.010 18.556 11.870 0.010 97.5 7 2.13 0.100 106.8 7 2.01 0.500 96.5 7 0.58
sulfate
111. Iprodione F 0.99908 0.009 0.010 18.661 18.647 11.293 11.290 0.010 88.9 7 0.50 0.100 86.17 1.34 0.500 92.4 7 2.15
18.851 18.865 12.353 12.351
112. Bromopropylate A 0.99988 0.008 0.010 18.787 12.521 0.010 86.9 7 4.87 0.100 78.17 1.86 0.500 85.17 2.61
113. Fenpropathrin I/A 1.00000 0.007 0.010 18.927 12.600 0.010 91.17 1.75 0.100 102.8 7 1.61 0.500 82.8 7 0.58
65.5 7 3.30 65.3 7 3.31 71.8 7 1.09

B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222

114. Tebufenpyrad A 0.99917 0.009 0.010 18.911 12.641 0.010 0.100 0.500
115. Epoxiconazole F 0.99889 0.005 0.010 19.116 19.100 12.273 12.515 0.010 76.9 7 0.87 0.100 78.17 0.86 0.500 75.17 0.61
116. Lenacil H 0.99998 0.030 0.040 19.275 11.805 0.040 94.8 7 0.40 0.400 98.5 7 2.38 2.000 90.17 3.20
117. Lambda- I 0.99976 0.008 0.010 19.322 19.308 13.080 13.078 0.010 70.17 6.86 0.100 75.4 7 4.95 0.500 76.2 7 4.00
cyhalothrin
19.675 19.656 13.237 13.235
118. Metconazole F 0.99892 0.008 0.010 19.967 12.759 0.010 62.6 7 12.15 0.100 55.17 2.86 0.500 77.6 7 1.86
119. Methoxychlor I 0.99962 0.008 0.010 19.954 12.595 0.010 95.9 7 8.58 0.100 108.0 7 2.67 0.500 92.3 7 5.34
(DMDT)
120. Dimoxystrobin F 0.99980 0.009 0.010 19.731 19.715 12.462 12.459 0.010 64.5 7 0.36 0.100 54.6 7 2.46 0.500 63.6 7 0.44
121. Bromuconazole F 0.99920 0.008 0.010 19.938 19.924 12.483 12.481 0.010 71.9 7 0.27 0.100 78.8 7 0.80 0.500 89.4 7 2.80
21.043 21.026 12.808 12.806
122. Pyriproxyfen I 0.99993 0.020 0.030 20.815 13.049 0.030 65.17 13.31 0.300 68.8 7 3.45 1.500 72.3 7 3.10
123. Tetradifon A 0.99986 0.009 0.010 20.885 12.904 0.010 52.5 7 3.30 0.100 67.4 7 3.66 0.500 76.17 2.97
124. Phosalone I/A 0.99983 0.008 0.010 21.068 21.051 13.036 13.035 0.010 95.2 7 0.96 0.100 95.2 7 0.15 0.500 109.3 7 4.88
125. Phosmet I/A 0.99998 0.009 0.010 21.125 21.109 12.515 12.515 0.010 103.0 7 2.63 0.100 97.3 7 1.59 0.500 89.7 7 2.47
126. Fenamidon F 0.99600 0.010 0.020 21.170 21.153 12.686 12.683 0.020 101.4 7 0.88 0.200 102.9 7 2.57 1.000 94.3 7 3.95
127. Pyrazophos F 0.99994 0.010 0.020 21.556 21.539 13.472 13.472 0.030 74.3 7 2.34 0.300 70.6 7 5.46 1.500 77.0 7 3.65
128. Acetamiprid I 0.99980 0.010 0.020 22.255 12.481 0.020 95.5 7 3.52 0.200 80.0 7 2.37 1.000 79.0 7 11.95
129. Permethrin I 0.99738 0.010 0.020 22.477 13.932 0.020 112.2 7 3.92 0.200 97.7 7 5.55 1.000 90.5 7 5.41
22.771 14.052
130. Bitertanol F 0.99998 0.010 0.020 22.987 13.878 0.020 71.9 7 0.55 0.200 76.4 7 4.29 1.000 77.3 7 4.10
131. Fenarimol F 0.99988 0.009 0.010 22.748 22.729 13.477 13.474 0.030 63.5 7 2.18 0.300 66.9 7 3.16 1.500 77.5 7 2.21
132. Azinphos-methyl I/A 0.99994 0.009 0.010 23.115 23.099 13.063 13.064 0.050 94.1 7 3.89 0.500 83.7 7 1.75 2.500 75.5 7 3.86
133. Pyridaben I/A 0.99686 0.010 0.020 23.247 23.231 14.108 14.106 0.020 71.6 7 3.15 0.200 75.3 7 12.05 1.000 78.9 7 2.96
134. Pyraclostrobin F 0.99545 0.020 0.030 23.314 23.307 16.307 16.304 0.030 95.8 7 1.12 0.300 92.4 7 6.65 1.500 91.9 7 2.95
135. Prochloraz F 0.99706 0.009 0.010 23.469 23.416 14.254 14.252 0.010 72.9 7 2.43 0.100 82.17 5.25 0.500 71.0 7 2.46
136. Beta-cyfluthrin I 0.99876 0.009 0.010 23.667 14.525 0.020 95.5 7 3.52 0.200 80.0 7 2.37 2.000 79.0 7 1.95
23.825 14.618
24.133 14.753
137. Cyfluthrin I 0.99997 0.009 0.010 23.670 14.528 0.010 122.2 7 3.92 0.100 119.7 7 5.55 0.500 90.5 7 5.41
23.821 14.619
24.131 14.752
138. Azinphos-ethyl I/A 0.99998 0.009 0.010 23.993 23.978 13.582 13.582 0.010 81.9 7 0.55 0.100 96.4 7 4.29 0.500 77.3 7 4.10
139. Coumaphos I 1.00000 0.010 0.020 24.498 24.482 14.202 14.202 0.030 73.5 7 2.18 0.300 76.9 7 3.16 1.500 77.5 7 2.21
140. Fluquinconazole F 0.99999 0.010 0.020 24.822 24.803 14.203 14.200 0.050 74.17 3.89 0.500 73.7 7 1.75 2.500 75.5 7 3.86
141. Alpha- I 0.99908 0.008 0.010 25.086 14.903 0.020 72.6 7 3.15 0.200 85.3 7 2.05 1.000 98.9 7 2.96
cypermethrin
25.675 15.119
142. Zeta- I 0.99953 0.009 0.010 25.092 14.904 0.010 71.9 7 10.27 0.100 78.8 7 0.80 0.500 79.4 7 2.80
cypermethrin
25.310 15.013
25.668 15.141
 B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222 217

with European Commission (EC) guidelines (SANCO, 2011). After

homogenisation, blank samples (2.0 g) were sprayed with 1 ml of
the pesticide standard mixture in a solution of hexane/acetone

81.3 7 13.70
(9:1, v/v) and left for 1 h (equilibration times) and then prepared
77.0 7 3.65
75.5 7 6.80
73.7 7 2.20

84.3 7 2.34

72.4 7 4.68
71.5 7 8.56
76.2 7 4.86

70.2 7 5.89
75.7 7 1.86
92.3 7 3.10

according to the procedure described above.

The pesticide recoveries should be in a range of 70–120% with
relative standard deviations (RSDs) of o20% (SANCO, 2011). In this
study, recovery experiments for 153 pesticides at three spiking
levels between 0.005 and 2.5 μg/g were performed for a period of
five days. Each pesticide was fortified to its LOQ level, or 0.005–
0.05 μg/g, at 10 times the LOQ level, or 0.05–0.5 μg/g, and at a third
1.000

2.000

1.000

1.000

1.000
1.500
0.500
0.500

0.500

2.500

1.500
level, or 0.5–2.5 μg/g.
The recoveries obtained for most pesticides were satisfactory
and ranged from 70 to 118% with the exception of cymoxanil,
dichlorvos, dicofol, dimoxystrobin, fenarimol, fenchlorphos,
fenthion, phorate, propyzamide and picoxystrobin (40–70%), and
88.8 7 3.45

70.6 7 5.46
72.8 7 6.22

88.8 7 3.45

72.4 7 5.62
68.3 7 4.59
82.5 7 8.25

75.5 7 3.86
71.17 4.35
78.8 7 1.21

78.7 7 1.11

cyfluthrin and oxadixyl (120–130%) for RSDs of 0.15 to 13.76%.

A—acaricides; F—fungicides; H—herbicides; I—insecticides; EC—electron capture; NP—nitrogen phosphorus; R —correlation coefficient; RSD—relative standard deviation.

Table 1 presents the recoveries along with the RSDs obtained by a

matrix-standard calibration in bees spiked with three concentra-
tion levels. However, a range of 60–140% may be used in routine
multi residue analysis. A variable matrix influence on the calcu-
lated recoveries was observed, depending on the physicochemical
properties of each pesticide and its concentration in the sample.
0.200

0.300

0.400

0.200

0.500
0.200
0.300

0.200
0.100
0.100

0.100

These matrix effects may be observed as an increase or

decrease, compared with those produced by analyte solvent
solutions. The effect of the matrix can be variable and unpredict-
able in the occurrence of measurable effects. The effect of the
matrix on the detectors’ (ECD and NPD) response to the studied
71.9 7 12.53
74.3 7 2.34

77.0 7 3.04

70.2 7 5.89

69.5 7 5.40
59.4 7 8.25
71.9 7 0.27

75.3 7 4.27

80.0 7 2.37
95.17 3.31

79.9 7 1.80

pesticides and matrices was evaluated in this work. Matrix-matched

standards were used to determine if there is a different response
between the matrix-matched and solvent standards. Acetamiprid
(recovery in solution—308%), diazinon (137%), heptenofos (131%),
iprodione (132%), cyhalothrin lambda (161%), malaoxone (189%),
mecarbam (132%), methidathion (150%), p,p′-DDT (142%), paraoxone
2

methyl (193%), parathion ethyl and methyl (137%), permethrin

0.020

14.596 0.030

16.914 0.040

0.020

0.050
18.048 0.020
18.148 0.030

19.378 0.020
0.010
0.010

0.010

(135%), propazine (163%) and pyrazophos (161%) were found to have

significant response differences between the solvent and matrix
17.038

18.771

prepared standards.
The accuracy and precision of this method was tested via recovery
17.042
14.907

15.053

16.540
16.220

17.367
17.345

18.775
19.382
16.541

18.051
15.014

16.915
16.218

18.148
15.131

17.112

experiments with fortified samples. Method precision was expressed

as the repeatability (ten replicates) of the recovery determinations at
given spiked levels and the RSDs for all compounds have been
26.631

defined (≤20%). Accuracy can be measured by analysing samples

with known concentrations and comparing the measured values
25.326

29.260

30.265
25.675

30.248
29.278

32.819
25.100

29.121

with the true values. These results indicate that the pesticide
recoveries and accuracies were good. Consequently, the pesticides
were determined to a satisfactory degree by using this method.
0.020

0.040

0.020

0.050
0.020
0.030

0.020
0.010

0.010
0.010

0.010

The instrument limits of detection (LODs) and limits of quanti-

fication (LOQs) were estimated for signal-to-noise ratios of 3 and
10, respectively, and measured by the peak-to-peak method at the
0.99976 0.008

0.99984 0.009
0.99996 0.009

0.99970 0.009

0.99989 0.030

0.99568 0.040

0.99743 0.020
0.99998 0.010

0.99653 0.010

0.99942 0.010

0.99642 0.010

lowest calibration level. The real LOQ estimation was based on the
trueness of the method and on precision data and was defined as
the lowest validated spiking level.
Linearity was evaluated by calculating a five-point linear plot
with three replicates, based on a linear regression and squared
correlation coefficient (R2), which should be above 0.9950. Pesti-
cides had a linear range from 0.002 to 2.5 μg/g. The results are
summarised in Table 1. The detector response to certain pesticides
F
F

F
F

F
I

may be affected by the presence of co-extractives from the sample.

153. Imibenconazole
152. Dimethomorph
148. Difenoconazole
144. Fenbuconazole
143. Cypermethrin

149. Deltamethrin
146. Esfenvalerate

151. Azoxystrobin

3.4. Honey-bee poisoning in real samples

147. Fenvalerate

150. Indoxacarb
145. Boscalid

The danger of bee poisoning by pesticides still exists. Bees

are still at risk of poisoning by organophosphates, carbamates,
pyrethroids, organochlorines, and, in particular, with the newly
introduced neonicotinoid pesticides.
218 B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222

In cases of incidental bee poisoning, it is important to provide
scientifically defensible evidence that the honeybees could be
killed by pesticides and not by other factors (Barnett et al., 2007).
Pesticide toxicity is generally measured in acute contact toxi-
city values of LD50, or the exposure level that causes 50% of the
exposed population to die. Toxicity thresholds are generally set at
highly toxic (acute LD50 o2 μg/bee), moderately toxic (acute LD50
2–10.99 μg/bee), slightly toxic (acute LD50 11–100 μg/bee), and
nontoxic (acute LD50 4100 μg/bee) levels to adult bees. The most
highly toxic pesticides to bees are abamectin, acephate, aldicarb,
avermectin, azinphos-methyl, bifenthrin, carbaryl, carbofuran, carbo-
sulfan, chlorpyrifos, clothianidin, cyfluthrin, cyhalothrin, cyperme-
thrin, deltamethrin, diazinon, dicrotophos, dieldrin, dimethoate,
fenpropathrin, famoxadone, imidacloprid, indoxacarb, malathion,
methamidophos, methidathion, methiocarb, methomyl, methoprene,
parathion-methyl, naled, permethrin, phosmet, propoxur, spinosad,
thiamethoxam and zeta-cypermethrin. The list of compounds tested
in this work is a precise reflection of the compounds tested in crops
within the framework of official inspections. These are compounds
that have been selected based on the sales of pesticides that are
widely used in Polish agriculture, using results from previous years of
monitoring tests for pesticide residues in crops, compounds exceeding
the MRL, and information from the Inspectorate of Plant Health and
Seed Inspection on the application of pesticides in a way that does not
conform to the Polish act on plant protection, persistent compounds.
Insecticides pose the most direct risk to pollinators, and the
negative impacts of these compounds have been demonstrated for
the honeybee Apis mellifera (Carrasco-Letelier et al., 2012; Greig-
Smith et al., 1994; Shires et al., 2006) and several non-Apis bees
(Tasei, 2001). Pollinators can be exposed to insecticides during

Fig. 2. Chromatograms obtained on column HP-5 from ECD detector: (a) at 2nd fortification level (concentration 0.05–0.5 μg/g), (b) at 1st fortification level (concentration
0.005–0.05 μg/g), and NPD detector: (c) at 2nd fortification level (concentration 0.05–0.5 μg/g), (d) at 1st fortification level (concentration 0.005–0.05 μg/g) (see Table 1).
1. mevinphos; 2. propachlor; 3. trifluralin; 4. alpha-HCH; 5. dimethoate; 6. beta-HCH; 7. diazinon; 8. parathion-methyl; 9. heptachlor; 10. fenitrothion; 11. malathion;
12. chlorpyrifos; 13. chlorfenvinphos; 14. bromophos-ethyl; 15. tetrachlorvinphos;16. α-endosulfan; 17. p,p′DDE; 18. dieldrin; 19. nitrofen; 20. endrin, 21. p,p′DDD;
22. o,p′DDT; 23. p,p′DDT; 24. acetamiprid; 25. fenpropathrin; 26. phosalone; 27. azinphos-ethyl; 28. permethrin; 29. cyfluthrin; 30. cypermethrin; 31. deltamethrin;
32. imibenconazole; 33. propham; 34. heptenophos; 35. cadusafos; 36. cyprodinil; 37. napropamide; 38. flusilazole; 39. triazophos.
 B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222 219

application, through contact with residues, or from the ingestion
of pollen, nectar or guttation fluid (drops of xylem sap on the tips
or edges of leaves) containing insecticide (Mullin et al., 2010).
Based on our analytical studies of dead bee samples, our
research has concluded that the most frequent cause of bee deaths
was poisoning with pyrethroids and organophosphate insecti-
cides, which are groups of highly toxic pesticides.
Other Polish studies (Walorczyk and Gnusowski, 2009)
found exceptional amounts of the organophosphates dimethoate
(4.9 μg/g), fenitrothion (1 μg/g), and omethoate (1.2 μg/g), and up
to 1.2 μg/g of the systemic fungicide tebuconazole in honey
bees from other poisoning incidents. Fletcher and Barnet (2003)
report that some 38 different agricultural compounds have been
identified in bee poisoning cases in the UK, and insecticides are
the most likely cause. They identified 15 different organopho-
sphates, two organochlorines, five carbamates and seven pyre-
throid compounds, rather than herbicides and fungicides. Some of
these compounds are found together with others in bee poisoning
incidents. Anderson and Wojtas (1986) linked dead honey bees to
high residues of the insecticides carbaryl (5.8 μg/g), chlordane
(0.7 μg/g), diazinon (0.35 μg/g), endosulfan (4.4 μg/g), malathion
(4.2 μg/g), methomyl (3.4 μg/g), methyl parathion (3.6 μg/g), and
the fungicide captan (1.7 μg/g). Similarly, elevated residues of the
organophosphates bromophos methyl (1.7 μg/g) and fenitro-
thion (10.3 μg/g) were associated with high honey bee mortality
(Ghini et al., 2004). In contrast to systemic fungicides, systemic

Table 2
Pesticide detected in poisoning bees.

Bee sample Pesticide Detected concentration LD50 [mg/bee] % LD50

[mg/g] [mg/bee]

Sample 1 Lambda-cyhalothrin 0.110 0.010 0.038 25.8

Sample 2 Tebuconazole 0.538 0.048 83.05 0.0
Sample 3 Dichlorvos 0.302 0.027 0.29 9.3
Sample 4 p,p′-DDE 0.090 0.008 5 0.2
o,p′-DDT 0.030 0.003 0.1
p,p′-DDT 0.080 0.007 0.1
DDT-sum 0.200 0.018 0.4
Gamma-HCH 0.010 0.001 0.011 8.0
Zeta-cypermethrin 5.910 0.528 0.002 26 385.0
Sample 5 Zeta-cypermethrin 0.291 0.026 0.002 1 300.0
Sample 6 Dimethoate 0.011 0.001 0.12 0.8
Sample 7 Phosalone 0.235 0.021 4.4 0.5
Sample 8 Bifenthrin 0.130 0.012 0.015 77.3
Zeta-cypermethrin 2.540 0.227 0.002 11 340.0
Sample 9 Bifenthrin 0.040 0.004 0.015 24.0
Zeta-cypermethrin 0.771 0.069 0.002 3 440.0
Sample 10 Bifenthrin 0.080 0.007 0.015 47.3
Zeta-cypermethrin 0.990 0.088 0.002 4 420.0
Sample 11 Bifenthrin 0.020 0.002 0.015 12.0
Zeta-cypermethrin 0.390 0.035 0.002 1 740.0
Sample 12 Bifenthrin 0.090 0.008 0.015 53.3
Zeta-cypermethrin 0.460 0.041 0.002 2 055.0
Sample 13 Bifenthrin 0.020 0.002 0.015 12.0
Zeta-cypermethrin 0.090 0.008 0.002 400.0
Sample 14 Fipronil 0.008 0.001 0.004 17.5
Sample 15 Fipronil 0.015 0.001 0.004 32.5
Sample 16 Cypermethrin 0.029 0.003 0.02 13.0
Bifenthrin 0.069 0.006 0.015 41.3
Sample 17 Dimethoate 0.627 0.056 0.12 46.7
Sample 18 Chlorothalonil 0.011 0.001 40 0.0
Chlorpyrifos 0.011 0.001 0.059 1.7
Alpha-cypermethrin 0.040 0.004 0.03 12.0
Sample 19 Alpha-cypermethrin 1.344 0.120 0.03 400.0
Sample 20 Fipronil 0.017 0.002 0.004 37.5
Sample 21 Dimethoate 0.258 0.023 0.12 19.2
Chlorpyrifos 0.038 0.003 0.059 5.8
Sample 22 Chlorpyrifos 0.056 0.005 0.059 8.5
Sample 23 Chlorpyrifos 4.278 0.382 0.059 647.5
Sample 24 Chlorpyrifos 3.270 0.292 0.059 494.9
Sample 25 Dimethoate 7.280 0.650 0.12 542.0
Sample 26 Chlorpyrifos 576.576 51.480 0.059 87 254.0
Sample 27 Permethrin 15.650 1.397 0.029 4 817.0
Zeta-cypermethrin 0.170 0.089 0.002 4 450.0
Tebuconazole 0.430 0.038 63 0.0
Sample 28 Zeta-cypermethrin 0.020 0.002 0.002 89.0
Tebuconazole 0.060 0.005 63 0.0
Sample 30 Zeta-cypermethrin 0.380 0.034 0.002 1 696.0
Tebuconazole 1.780 0.159 63 0.0
Sample 31 Cypermethrin 6.3 0.563 0.02 2812,5
Chlorpyrifos 0.03 0.0027 0.059 4.5
Sample 32 Cypermethrin 3.56 0.318 0.02 1589,3
Chlorpyrifos 0.01 0.0009 0.059 1.5
Sample 33 Cypermethrin 1.32 0.118 0.02 589.3
Chlorpyrifos 0.01 0.0009 0.059 1.5
220 B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222
neonicotinoid residues are generally absent from bee samples,
although they are present in pollen and wax (Blacquiere et al.,
2012; Mullin et al., 2010).
Our results are presented in Table 2, and information regarding
pesticides detected in bees (their description, status, type, and
mode of action) are widely available. Pesticide concentrations from
the bodies of dead bees have been expressed as a percentage of
the LD50 dose.
Pyrethroids are frequently associated with honey bee deaths
(Mineau et al., 2008). Our results confirmed that pyrethroids are
the most prevalent causes of bee poisonings. In the dead bee
samples, the greatest number of beehive extinctions has been
caused by cypermethrin and its isomers (over 50% of incidental
bee poisonings). Zeta-cypermethrin, the most toxic insecticide
among the cypermethrin isomers (LD50 ¼0.002 μg/bee), has been
detected in 33% of bee samples at concentrations ranging from
0.002 to 0.528 μg/bee, which constitutes from 89 to 26 385% of the
LD50, respectively. Zeta cypermethrin is present in the plant
protection products Ammo Super 100 EW, Fury 100 EW, Minuet
100 EW, Rage 100 EW, and Titan 100 EW. In the case of samples
27–30, poisoning was the result of improper Fury 100 EW applica-
tion for the preparation of rape crops. Alpha-cypermethrin,
another isomer, is 10 times less toxic and was detected in two
samples at concentrations ranging from 0.004 to 0.012 μg/bee, or
12 and 400% of the LD50. Cypermethrin has been found in four bee
samples in concentrations from 12 to 2 812% of the LD50. In
samples 30–33, cypermethrin concentrations ranged from 0.12 to
0.56 μg/bee, and chlorpyrifos was also present at concentrations
from 0.001 to 0.03 μg/bee.
Sample 16 also contained bifenthrin at 0.015 μg/bee, constitut-
ing 42% of LD50. In this instance, the toxic effects of these active
substances could have overlapped and been amplified. Bifenthrin
is an insecticide/acaricide belonging to the pyrethroid group and
has been found in 18% (6) of samples at a concentration of 0.02–
0.13 μg/g (0.0018–0.0116 μg/bee), which constitutes 12–77% of the
LD50. The bee samples (nos. 8–13) came from southern Poland, an
area subject to flooding that had been sprayed with the Aqua
K-Othrine preparation to destroy mosquitoes. According to the EU
Commission′s decision from November 30th, 2009, bifenthrin was
not included as an active substance in Annex I for directive 91/414/
EEC. As a result, the application of bifenthrin pesticide has been
severely limited because the use of this substance is almost
completely prohibited, and permits for pesticides containing this
substance have been revoked.
Chlorpyrifos, an organophosphate insecticide, has been detected in
27% of samples, with the highest concentration reaching 51.5 μg/bee
(254% of LD50). Dimethoate, which belongs to the organophosphate
group of insecticides, is characterised by very potent toxic properties
(LD50 ¼0.12 μg/bee) and has been detected in three samples of
poisoned bees in concentrations from 0.023 to 0.65 μg/bee, which
constituted 19.2 and 542% of the LD50, respectively. Indicative chro-
matograms of real bee samples are presented in Fig. 3a–c.
Three cases of bee poisoning with fipronil (samples 14, 15
and 20), a phenylpyrazole that affects the nervous system
(s.a. Regent 200), have been recorded. The neurotoxic action of
fipronil is based on the disruption of the passage of chlorine ions
through gamma-aminobutyric acid (GABA) receptors and highly
specific glutamic acid receptors (GluCl) in their nervous system,
which causes the overexcitation of nerves and muscles, leading to
their permanent paralysis and death. The exceptional effectiveness
of fipronil against arthropods is explained by the fact that they
have both types of receptors. Higher order animals (e.g., birds and
mammals) do not have GluCl receptors. There are reports in the
literature on the action of sub-lethal doses (Bonmatin et al., 2005)
with a residual efficacy period of 21 days. In these three studied
cases, the LD50 was not exceeded, and doses ranged from 17.5 to
37.5%, which may rule out direct bee poisoning but not their
weakening condition and chronic poisoning. In the Czech Republic
(Modra and Svobodova, 2009), the largest number of mass bee
poisonings has been caused by the insecticide Regent WP 50. The
use of this substance on oilseed rape to control the rape blossom
beetle has been banned since 2006.
However, some fungicides have been found to increase the
toxicity of pyrethroid insecticides to A. mellifera (Pilling and
Jepson, 1993). Three fungicides have been detected from the tested
range in 5 samples, namely tetraconazole, tebuconazole and
chlorothalonil. These pesticides are slightly toxic and their LD50
percentages are 63, 83 and 40%, respectively. Among these
fungicides, chlorothalonil had the lowest concentration at
0.001 μg/bee, and tetraconazole had the highest at 0.159 μg/bee.
Fungicides are classified as less harmful to bees (based on LD50
values) and are often applied to bee pollinated crops whilst the
bees are foraging in the field. Thus, fungicides have been the
predominant pesticide detected in pollen collected by A. mellifera.
Multiple pesticide residues have repeatedly been detected in
the bodies of bees (13 samples of bees containing two pesticides,
two samples with three and one sample of bees containing five
pesticides). Poisoning has occurred when tank mixes of pyre-
throids and fungicides have been applied to crops. This mixture
has resulted in an apparent increase in the toxicity of the
pyrethroids to bees and confirms laboratory experiments in which
this toxicity has been demonstrated (Pilling and Jepson, 1993).
Pyrethroids were detected in addition to fungicides in four
samples (18, 27–30). The direct toxic effect of fungicides on bees
is generally lower than that of insecticides and varies depending
on the fungicide, the bee and the mode of exposure (Ladurner
et al., 2005; Malone et al., 2007; Huntzinger et al., 2008; Everich
et al., 2009).
Instances of poisoned bees (from the southeastern part of Poland)
in which organochlorine insecticides are present (p,p′-DDE;
o,p′-DDT; p,p′-DDT; HCH-gamma) along with the pesticides respon-
sible for beehive extinctions, such as zeta-cypermethrin, have also
been recorded. Organochlorine insecticides have been prohibited
from use in Poland for over a decade. This ban shows that DDT and
lindane are still present in the environment and that bees are a
perfect bio-indicator of environmental pollution (Porrini et al., 2003;
Ghini et al., 2004; Haarmann, 1997). Products of DDT degradation
(p,p′-DDE, o,p′-DDT and p,p′-DDT) were determined to have a total
concentration of 0.2 μg/g, which constitutes only 0.4% of the
LD50, along with lindane (gamma-HCH), with a concentration of
0.010 μg/g, for 8% of the LD50. The detected organochlorine sub-
stances did not cause bee poisonings and are indicative of environ-
mental pollution. In this case, bees can be considered as bioindicators
of environmental pollution. The direct cause of the demise of bee
families in sample no. 4 was determined as zeta cypermethrin with a
concentration of 0.528 μg/bee, which constitutes 26,385% of the LD50.
In this case, the Fury 100 EW preparation was used on a rape crop in
a way that was not compliant with its label.
Additionally, the presence of individual compounds, namely
lambda-cyhalothrin, permethrin, dichlorvos and fozalon has been
detected, and their doses did not exceed 25.8% of the LD50.
Pesticide bee poisonings still happen every year and they cause
significant losses. However, there are always a number of incidents
for which there is no evidence available from field data, which in
turn demands improved analytical methods so that a greater number
of pesticides can be detected at lower concentration levels.
4. Conclusions
In this study, we presented an analytical method for the trace
quantitative determination of widely used plant protection products,
 B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222 221

Fig. 3. Chromatograms of samples poisoning bees by insecticides: (a) chlorpyrifos (trECD¼8.959; 4.28 μg/g, sample 23), (b) dimethoate (trNPD¼ 6.524; 0.627 μg/g, sample 17),
(c) permethrin (trECD¼13.934; 14.048; 15.65 μg/g) and zeta-cypermethrin (trECD¼14.907; 15.014; 15.130; 0.17 μg/g, sample 27).

specifically, three acaricides, 49 fungicides, 15 herbicides and 75
insecticides, in honeybees. Pesticides belonging to different chemical
classes including organohalogens, organophosphorus, carbamates,
pyrethroids, strobilurins, and triazoles were efficiently extracted by
MSPD using Florisil as the dispersion phase and C18 as the clean-up
phase and then determined and identified by gas chromatography
with dual system detection. Honeybees are complex because of their
relatively large and variable amounts of wax residues. However, the
MSPD procedure did not require additional clean-up steps because
the sample extraction and clean-up were carried out during the same
step. MSPD is superior to liquid methods because of its simplicity and
rapidity (15 min) and it also requires small samples (2.0 g) and
solvent volumes (25 ml). In addition, the application of MSPD
combined with GC NPD/ECD for multiresidue screening allows for
the unequivocal identification of the studied pesticides and solves
problems with co-eluting pesticides. To ensure the quality of results
when the proposed method is applied to routine analyses, various QC
criteria have been established. The first one is a blank extract to
control for contamination in the extraction, clean-up processes and
used instruments or chemicals. One blank sample was processed in
each set of experiments. The second criterion is to check the
extraction efficiency. Recoveries will be accepted if the majority of
recoveries are within the 70–120% range. This method is in agree-
ment with the technical criteria of Document N1 SANCO/12495/2011
(SANCO, 2011) and is compliant with ISO/IEC 17025 requirements.
The proposed method was sensitive, reliable and successfully applied
to the analysis of real samples, and it is currently employed for
routine analysis in our laboratory.
Moreover, not only EU-prohibited DDT and their metabolites were
detected, but pyrethroids (cypermethrin) and organophosphates
(chlorpyrifos) were also frequently found in the real poisoning
samples of bees.
However, to establish the cause of bee poisonings, we must
have a sensitive analytical method that allows for the determina-
tion of a wide range of pesticides; the method presented in this
paper fulfils this requirement. Furthermore, knowledge about the
contamination and poisoning of bees by pesticides is very impor-
tant and still needed.
222 B. Łozowicka / Ecotoxicology and Environmental Safety 97 (2013) 210–222
References
Aktar, W., Sengupta, D., Chowdhury, A., 2009. Impact of pesticides use in
agriculture: their benefits and hazards. Interdiscip. Toxicol. 2, 1–12.
Anderson, J.F., Wojtas, M.A., 1986. Honey bees (Hymenoptera, Apidae) contaminated
with pesticides and polychlorinated biphenyls. J. Econ. Entomol. 79, 1200–1205.
Barnett, E.A., Charlton, A.J., Fletcher, M.R., 2007. Incidents of bee poisoning with
pesticides in the United Kingdom, 1994–2003. Pest Manag. Sci. 63, 1051–1057.
Bernal, J.L., del Nozal, M.J., Toribio, L., Jimenez, J.J., Atienza, J., 1997. High-
performance liquid chromatographic determination of benomyl and carbenda-
zim residues in apiarian samples. J. Chromatogr. A 787, 129–136.
Bernal, J.L, Martin-Hernandez, R., Diego, J., Nozal, M., Gozalez-Porto, A., Bernala, J.,
Higesb, M., 2011. An exposure study to assess the potential impact of fipronil in
treated sunflower seeds on honey bee colony losses in Spain. Pest Manag. Sci.
67, 1320–1331.
Blacquiere, T., Smagghe, G., van Gestel, C.A., Mommaerts, V., 2012. Neonicotinoids
in bees: a review on concentrations, side-effects and risk assessment. Ecotox-
icology 21, 973–992.
Bonmatin, J.M., Marchand, P.A., Charvet, R., Moineau, I., Bengsch, E.R., Colin, M.E.,
2005. Quantification of imidacloprid uptake in maize crops. J. Agric. Food Chem.
53, 5336–5341.
Carrasco-Letelier, L., Mendoza-Spina, Y., Branchiccela, M.B., 2012. Acute contact
toxicity test of insecticides (Cipermetrina 25, Lorsban 48E, Thionex 35) on
honeybees in the southwestern zone of Uruguay. Chemosphere 88, 439–444.
Charlton, A.J.A., Jones, A., 2007. Determination of imidazole and triazole fungicide
residues in honeybees using gas chromatography–mass spectrometry. J. Chro-
matogr. A 1141, 117–122.
Chauzat, M.P., Carpentier, P., Martel, A.C., Bougeard, S., Cougoule, N., Porta, P.,
Lachaize, J., Madec, F., Aubert, M., Faucon, J.P., 2009. Influence of pesticide
residues on honey bee (Hymenoptera: Apidae) colony health in France. Environ.
Entomol. 38, 514–523.
Chauzat, M.P., Faucon, J.P., 2007. Pesticide residues in beeswax samples collected
from honey bee colonies (Apis mellifera l.) in France. Pest Manag. Sci. 63,
1100–1106.
Chauzat, M.P., Faucon, J.P., Martel, A.C., Lachaize, J., Cougoule, N., Aubert, M., 2006.
A survey of pesticide residues in pollen loads collected by honey bees in France.
J. Econ. Entomol. 99, 253–262.
Cox-Foster, D.L., Conlan, S., Holmes, E.C., Palacios, G., Evans, J.D., Moran, N.A.,
Quan, P.L., Briese, S., Hornig, M., Geiser, D.M., Martinson, V., van Engelsdorp, D.,
Kalkseitn, A.L., Drysdale, L., Hui, J., Zhai, J., Cui, L., Hutchison, S., Simons, J.F.,
Egholm, M., Pettis, J.S., Lipkin, W.I., 2007. A metagenomic survey of microbes in
honey bee colony collapse disorder. Science 318, 283–287.
Detzel, A., Wink, M., 1993. Attraction, deterrence or intoxication of bees (Apis
mellifera). Chemoecology 4, 8–18.
Enan, E., 2001. Insecticidal activity of Essentials oils: octopaminergic sites of action.
Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 130, 325–337.
EURACHEM/CITAC, 2000. Guide quantifying uncertainty in analytical measure-
ments, second ed. 〈http://www.eurachem.org/guides/pdf/QUAM2000-1.pdf〉.
European Commission Brussels, 2010. 714 Final Communication from the Commission
to the European Parliament and the Council on Honeybee Health. 6.12.2010.
Everich, R., Schiller, C., Whitehead, J., Beavers, M., Barrett, K., 2009. Effects of Captan
on Apis mellifera brood development under field conditions in California
almond orchards. J. Econ. Entomol. 102, 20–29.
Fernandez, M., Padron, C., Marconi, L., Ghini, S., Colombo, R., Sabatini, A.G.,
Girottic, S., 2001a. Determination of organophosphorus pesticides in honeybees
after solid-phase microextraction. J. Chromatogr. A 922, 257–265.
Fernandez, M., Pico, Y., Girotti, S., Manes, J., 2001b. Analysis of organophosphorous
pesticides in honey bee by liquid chromatography–atmospheric pressure
chemical ionization-mass spectrometry. J. Agric. Food Chem. 49, 3540–3547.
Fernandez, M., Pico, Y., Manes, J., 2002. Analytical methods for pesticide residue
determination in bee products. J. Food Prot. 65, 1502–1511.
Fernandez, M., Pico, Y., Manes, J., 2003. Simultaneous determination of carbamate
and organophosphorus pesticides in honeybees by liquid chromatography–
mass spectrometry. Chromatographia 58, 151–157.
Fletcher, M., Barnett, L., 2003. Bee pesticide poisoning incidents in the United
Kingdom. Bull. Insectol. 56, 141–145.
Garcia, M.D.G., Galera, M.M., Valverde, R.S., Galanti, A., Girotti, S., 2007. Column
switching liquid chromatography and post-column photochemically fluores-
cence detection to determine imidacloprid and 6-chloronicotinic acid in
honeybees. J. Chromatogr. A 1147, 17–23.
Garibaldi, L.A., Aizen, M.A., Klein, A.M., Cunningham, S.A., Harder, L.D., 2011. Global
growth and stability of agricultural yield decrease with pollinator dependence.
Proc. Nat. Acad. Sci. 108, 5909–5914.
Genersch, E., von der Ohe, W., Kaatz, H., Schroeder, A., Otten, C., Buchler, R., Berg, S.,
Ritter, W., Muhlen, W., Gisder, S., Meixner, M., Liebig, G., Rosenkranz, P., 2010.
The German bee monitoring project: a long term study to understand
periodically high winter losses of honey bee colonies. Apidologie 41, 332–352.
Ghini, S., Fernandez, M., Pico, Y., Marin, R., Fini, F., Manes, J., Girotti, S., 2004.
Occurrence and distribution of pesticides in the province of Bologna, Italy,
using honeybees as bioindicators. Arch. Environ. Contam. Toxicol. 47, 479–488.
Greig-Smith, P.W., Thompson, H.M., Hardy, A.R., Bew, M.H., Findlay, E., Stevenson, J.H.,
1994. Incidents of poisoning of honey bees (Apis mellifera) by agricultural pesticides
in Great Britain 1981–1991. Crop Prot. 13, 567–581.
Haarmann, T.K., 1997. Honey bees as indicators of radionuclide contamination:
exploring colony variability and temporal contaminant. J. Apic. Res. 36, 77–87.
Highfield, A.C., El Nagar, A., Mackinder, L.C.M., Noel, L.M.L.J., Hall, M.J., Martin, S.J.,
Schroeder, D.C., 2009. Deformed wing virus implicated in overwintering honey
bee colony losses. Appl. Environ. Microbiol. 75, 7212–7220.
Huntzinger, C.I., James, R.R., Bosch, J., Kemp, W.P., 2008. Fungicide tests on adult
alfalfa leafcutting bees (Hymenoptera: Megachilidae). J. Econ. Entomol. 103,
1088–1094.
Johnson, R.M., Ellis, M.D., Mullin, C.A., Frazier,M., 2010. Pesticides and honey bee
toxicity—USA. Apidologie, available online INRA/DIB-AGIB/EDP Sciences, 〈www.
apidologie.org〉.
Kadenczki, L., Arpad, Z., Gardi, I., Ambrus, A., Gyorfi, L., Reese, G., Ebing, W., 1992.
Column extraction of residues of several pesticides from fruits and vegetables:
a simple multiresidue analysis method. J. AOAC Int. 75, 53–61.
Kather, R., Drijfhout, F.P., Martin, S.J.J., 2011. Task group differences in cuticular
lipids in the honey bee Apis mellifera. Chem. Ecol. 7, 205–212.
Krupke, C.H., Hunt, G.J., Eitzer, B.D., Andino, G., Given, C.H., 2012. Multiple routes of
pesticide exposure for honey bees living near agricultural fields. PLoS One 7,
e29268.
Ladurner, E., Bosch, J., Kemp, W.P., Maini, S., 2005. Assessing delayed and acute
toxicity of five formulated fungicides to Osmia lignaria Say and Apis mellifera.
Apidologie 36, 449–460.
Malone, L.A., Scott-Dupree, C.D., Todd, J.H., Ramankutty, P., 2007. No sub-lethal
toxicity to bumblebees, Bombus terrestris, exposed to Bt-corn pollen, captan and
novaluron. N. Z. J. Crop Hortic. Sci. 35, 435–439.
Marconi, L., Ghini, S., Colombo, R., Sabatini, A.G., Girottic, S., 2001. Determination of
organophosphorus pesticides in honeybees after solid-phase microextraction.
J. Chromatogr. A 922, 257–265.
Mineau, P., Harding, K.M., Whiteside, M., Fletcher, M.R., Garthwaite, D., Knopper, L.D.,
2008. Using reports of bee mortality in the field to calibrate laboratory-derived
pesticide risk indices. Environ. Entomol. 37, 546–554.
Modra, H., Svobodova, Z., 2009. Incidence of animal poisoning cases in the Czech
Republic: current situation. Interdiscip. Toxicol. 2, 48–51.
Morello, M., Previale, L., Quaglino, P.J., 1996. Gas chromatographic system equipped
with a mass detector and a selective nitrogen–phosphorous detector operating
simultaneously in the analysis of pesticide residues. J. Chromatogr. A 740, 263–271.
Morzycka, B., 2002. Simple method for the determination of trace levels of
pesticides in honeybees using matrix solid-phase dispersion and gas chroma-
tography. J. Chromatogr. A 982, 267–273.
Mullin, C.A., Frazier, M., Frazier, J.L., Ashcraft, S., Simonds, R., van Engelsdorp, D.,S.,
Pettis, J.D., 2010. High levels of miticides and agrochemicals in north American
apiaries: implications for honey bee health. PLoS One 5, e9754.
Pilling, N.A., Jepson, P.C., 1993. Synergism between EBI fungicides and a pyrethroid
insecticide in the honey bee (Apis meliffera). Pest. Sci. 39, 293–297.
Porrini, C., Sabatini, A.G., Girotti, S., Fini, F., Monaco, L., Celli, G., Bortolotti, L., Ghini, S.,
2003. The death of honey bees and environmental pollution by pesticides: the
honey bees as biological indicators. Bull. Insectol. 56, 147–152.
PPDB, 2013. Pesticide Properties DataBase. University of Hertfordshire 〈http://sitem.
herts.ac.uk/aeru/footprint/en/〉.
Rortais, A., Arnold, G., Halm, M.P., Touffet-Briens, F., 2005. Modes of honeybees
exposure to systemic insecticides: estimated amounts of contaminated pollen
and nectar consumed by different categories of bees. Apidologie 36, 71–83.
Rossi, S., Dalpero, A.P., Ghini, S., Colombo, R., Sabatini, A.G., Girotti, S., 2001.
Multiresidual method for the gas chromatographic analysis of pesticides in
honeybees cleaned by gel permeation chromatography. J. Chromatogr. A 905,
223–232.
SANCO, 2011. Method Validation and Quality Control Procedures for Pesticide
Residues Analysis in Food and Feed. SANCO 〈http://ec.europa.eu/food/plant/
protection/pesticides/docs/qualcontrol_en.pdf〉. (12495/2011).
Shires, W., Murray, A., Debray, P., Le Blanc, J., 2006. The effects of a new pyrethroid
insecticide WL-85871 on foraging honey bees (Apis mellifera L.). Pest. Sci. 15,
491–499.
Tan, K., Wang, Z., Yang, M., Fuchs, S., Luo, L., Zhang, Z., Li, H., Zhuang, D., Yang, S.,
Tautz, J., Beekman, M., Oldroyd, B., 2012. Asian hive bees, Apis cerana, modulate
dance communication in response to nectar toxicity and demand. Anim. Behav.
84, 1589–1594.
Tasei, J.N., 2001. Effects of insect growth regulators on honey bees and non-Apis
bees. A review. Apidologie 32, 527–545.
Totti, S., Fernandez, M., Ghini, S., Pico, Y., Fini, F., Maes, J., Girotti, S., 2006.
Application of matrix solid phase dispersion to the determination of imidaclo-
prid, carbaryl, aldicarb, and their main metabolites in honeybees by liquid
chromatography–mass spectrometry detection. Talanta 69, 724–729.
Tse, H., Comba, M., Alaee, M., 2004. Method for the determination of organopho-
sphate insecticides in water, sediment and biota. Chemosphere 54, 41–47.
Van Engelsdorp, D., Underwood, R., Caron, D., Hayes, J., 2007. An estimate of
managed colony losses in the winter of 2006–2007: a report commissioned by
the apiary inspectors of America. Am. Bee J. 147, 599–603.
Walorczyk, S., Gnusowski, B., 2009. Development and validation of a multi-residue
method for the determination of pesticides in honeybees using acetonitrile-
based extraction and gas chromatography–tandem quadrupole mass spectro-
metry. J. Chromatogr. A 1216, 6522–6531.
Wiesta, L., Buletea, A., Girouda, B., Fratta, C., Amica, S., Lambertb, O., Pouliquenb, H.,
Arnaudguilhema, C., 2011. Multi-residue analysis of 80 environmental con-
taminants in honeys, honeybees and pollens by one extraction procedure
followed by liquid and gas chromatography coupled with mass spectrometric
detection. J. Chromatogr. A 1218, 5743–5756.
Williams, I.H., 1994. The dependences of crop production within the European
Union on pollination by honey bees. Agric. Zool. Rev. 6, 229–257.