Ecotoxicology and Environmental Safety 114 (2015) 1–8
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Ecotoxicology and Environmental Safety
journal homepage: www.elsevier.com/locate/ecoenv
Organophosphorus insecticides in honey, pollen and bees (Apis mellifera L.) and their potential hazard to bee colonies in Egypt
Yahya Al Naggar a,b,n, Garry Codling b, Anja Vogt b, Elsaied Naiem a, Mohamed Mona a,
Amal Seif a, John P. Giesy b,c,d,e,f,g
a
Department of Zoology, Faculty of Science, Tanta University, 31527 Tanta, Egypt
Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK S7N 5B3, Canada
c
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada
d
Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA
e
Department of Biology & Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong, SAR, China
f
School of Biological Sciences, University of Hong Kong, Hong Kong, SAR, China
g
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China
b
art ic l e i nf o
a b s t r a c t
Article history:
Received 26 September 2014
Received in revised form
25 December 2014
Accepted 26 December 2014
There is no clear single factor to date that explains colony loss in bees, but one factor proposed is the
wide-spread application of agrochemicals. Concentrations of 14 organophosphorous insecticides (OPs) in
honey bees (Apis mellifera) and hive matrices (honey and pollen) were measured to assess their hazard to
honey bees. Samples were collected during spring and summer of 2013, from 5 provinces in the middle
delta of Egypt. LC/MS–MS was used to identify and quantify individual OPs by use of a modified Quick
Easy Cheap Effective Rugged Safe (QuEChERS) method. Pesticides were detected more frequently in
samples collected during summer. Pollen contained the greatest concentrations of OPs. Profenofos,
chlorpyrifos, malation and diazinon were the most frequently detected OPs. In contrast, ethoprop,
phorate, coumaphos and chlorpyrifos-oxon were not detected. A toxic units approach, with lethality as
the endpoint was used in an additive model to assess the cumulative potential for adverse effects posed
by OPs. Hazard quotients (HQs) in honey and pollen ranged from 0.01–0.05 during spring and from 0.02–
0.08 during summer, respectively. HQs based on lethality due to direct exposure of adult worker bees to
OPs during spring and summer ranged from 0.04 to 0.1 for best and worst case respectively. It is concluded that direct exposure and/or dietary exposure to OPs in honey and pollen pose little threat due to
lethality of bees in Egypt.
& 2014 Elsevier Inc. All rights reserved.
Keywords:
Hazard quotient
OPs
Risk assessment
Africa
Bees
1. Introduction
Honey bees (Apis mellifera L.) fulfill important ecological and
economic roles as pollinators of crops and produce honey that can
be harvested for consumption. Approximately 35% of arable crops
depend directly on pollinators (Klein et al., 2007), accounting for
an annual value of 153 billion Euros (Gallai et al., 2009). Egypt has
1.3 million hives, 7700 are mud hives and approximately
270,000 beekeepers (The first international Forum for the Egyptian Beekeepers, 2009). There is limited statistical information on
the beekeeping industry in Egypt, such as its annual revenue and
production volumes, but it is estimated to be one of the most influential in the Middle East and Africa (http://www.beekeeping.
n
Corresponding author at: Toxicology Center, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada. Fax: þ 306 966 4796.
E-mail address: Yehia.elnagar@science.tanta.edu.eg (Y. Al Naggar).
http://dx.doi.org/10.1016/j.ecoenv.2014.12.039
0147-6513/& 2014 Elsevier Inc. All rights reserved.
com/articles/us/arab_countries.htm). Egyptian beekeepers based
along the Nile River have reported increased colony losses over
winter with no clear cause for this phenomenon (Hassan, 2009).
There is a global concern about the decline of populations of
the honey bee (UNEP, 2010; Van Engelsdorp and Meixner, 2010;
Fairbrother et al., 2014). The term ‘Colony Collapse Disorder’ (CCD)
has been coined to identify this issue (Cox-Foster et al., 2007;
Williams et al., 2010). During the 1990s 15–20% of hives failed this
loss was considered manageable and losses were attributed to a
range of factors such as disease, pathogens and pesticides, in the
mid-2000s losses have risen to 430% in some locations. Although
causes of CCD are still unclear, results of some studies suggest that
extensive use of insecticides might be responsible or a significant
co-factor for increased colony losses. Genome sequencing of the
honeybee provides a possible explanation for their sensitivity to
pesticides. Relative to other insects, the honeybee genome is deficient in a number of genes encoding detoxification enzymes
(Claudianos et al., 2006). A strong association between disease,
2
Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–8
pathogens and pesticides have been cited as potential causes of
CCD (Cox-Foster et al., 2007; Ratnieks and Carreck, 2010). This has
led to the theory that the syndrome might be the result of multiple
stressors (Van Engelsdorp and Meixner, 2010). The syndrome is
most commonly associated with infections of the Varroa mite and
the diseases it carries (Fairbrother et al., 2014).
In Egypt several classes of pesticides including organochlorine
(OC), organophosphorus (OP), carbamates, ureas, anilides and
pyrethroids are used. OPs have become the major compound
group used in pest control, while OC use has declined. In 1995 over
80% of all insecticide used in Egypt were OPs (Badawy, 1998 and
Mansour, 2004). Contamination of food with OPs and their residues is of concern; the widespread application has been questioned as a potential risk to human health (Pico et al., 1996).
To address potential effects of pesticides on pollinators, several
tools have been developed. These tools range from relatively
simple hazard assessments, such as evaluating lethality, to more
sophisticated assessments of risk (Fairbrother et al., 2014). Assessments of risk integrate probabilities of response based on
hazard or potency with probability of exposure (Giesy et al., 2014).
If sufficient information is available, it is generally considered to be
more relevant for estimation of potential adverse effects than the
simpler hazard quotient (HQ). However, since all the information
on probabilities of exposure is not yet available for the honey bee,
a HQ approach was used as an initial assessment.
Although there have been several studies on concentrations of
pesticides in bee matrices and their potential risks to bees (Rissato
et al., 2007; Mullin et al., 2010; Wiest et al., 2011; Chauzat et al.,
2011; Cutler et al., 2014) there have been no such studies of the
potential effects of pesticides on colonies of bees in Egypt. This
study of 14 OPs pesticides in honey and bee matrices from 5 different agricultural governances in Egypt and their potential for
lethality from direct and dietary exposure represents the first
study of its kind in Egypt.
2. Materials and methods
2.1. Study areas
Within Egypt the main region of agriculture is the Nile River
Valley, particularly the Nile Delta region. During spring and
summer 2013 honey, bees, and pollen stored in the comb were
collected from 15 locations (3 apiaries per location) from the
5 primary agricultural governorates in the Nile Delta of Egypt: Kafr
El-Sheikh, Al Gharbiya, Al-Menofiya, Al-Beheira and Al-Dakahlia
(Fig. 1). In Egypt, there are three main seasons for pollination; 1)
Citrus season during the first two weeks in April; 2) Clover season
from May until the first week of June and 3) Cotton season during
August and September. Egyptian clover, berseem, is the major
forage crop cultivated in the Nile Valley and Delta and occupies
1.2 million hectares. Egyptian clover is planted between the 1st of
September and 1st of June and flowering starts by the 1st of April.
European honeybees forage largely on clover from the beginning
of spring until the 1st week of June, after which cotton, maize,
vegetables and pumpkins represent the predominant sources of
nectar and pollen during summer in these locations. Samples were
collected during clover and cotton growing seasons of 2013.
2.2. Experimental
2.2.1. Beehive samples
Three hives were selected at random in each apiary. Pollen was
collected by cutting a 6 cm2 piece of comb containing stored pollen using a disposable plastic knife and placed in a 15 mL Falcon
tube (Fisher Scientific). Fresh honey was squeezed from the comb
into a 50 mL polyethylene Falcon tubes. Worker bees were brushed into disposable polyethylene bags. Worker bees were collected from the honey combs located on the farthest side walls of
the hive from the entrance. These were normally older worker
bees, which were more likely to have accumulated pesticides, and
should represent similar ages. All samples were transported in a
cool box with ice packs and frozen at 20 °C in the laboratory
until extraction (Al Naggar et al., 2013). The total number of
samples collected was 39, 31 and 34 for honey, pollen and bees
respectively.
2.2.2. Chemicals and reagents
Standards used for quantification of pesticides were all technical grade (4 98% purity, Accu Standard, New Haven, CT, USA). All
solvents (Hexane, MeOH, MeCN, etc.) were of HPLC grade or better
and tested for OP contamination prior to use, (VWR supplier).
Anhydrous sodium sulfate (NaAc) and Magnesium sulfate (MgSO4)
were from (Sigma Aldrich, Ca.). Individual stock standard solutions
Fig. 1. Map of study sites (S1-15) in the main agricultural governorates in the Nile Delta of Egypt.
Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–8
were prepared by dissolving 10 mg of each compound in methanol
and mixed compound calibration solutions, were prepared from
the stock. Matrix-matched standards were prepared in the same
concentration as that of calibration solutions, by adding appropriate amounts of standards to the control matrix including honey,
pollen and bees. Pesticides investigated in this study were selected
based on previous studies (Rissato et al., 2007; Mullin et al., 2010;
Wiest et al., 2011; Chauzat et al., 2011) and their use pattern and
toxicity in Egypt (Mansour, 2004; Malhat and Nasr, 2013).
2.2.3. Extraction and cleanup
Individual OPs were identified and quantified by use of the
QuEChERS method described by Lehotay et al. (2005) and adapted
for the lesser masses of samples used in this study. Briefly 3 g
(7 0.5) of sample was weighed into a 50 mL falcon tube and fortified with 100 uL of 100 ng mL 1 process control spiking solution
(PCS) containing dimethoate-D6, 27 mL of extraction solution (55%
acetonitrile, 44% deionized water and 1% glacial acetic acid) was
added, followed by 1.5 g NaAc and 6 g MgSO4. Tubes were sealed
and vortexed for 1 min, centrifuged for 10 min at 4000 g, 15 mL of
the supernatant transferred to a 15 ml falcon tube and evaporated
under N2 to 4 mL. This was then passed through C18 SPE cartridges (1000 mg supplier) under gravity and collected in a 15 mL
falcon tube, 10 ml of 1% acetic acid/MeCN was added to the C18
after the sample had passed through. The C18 was preconditioned
with 5 ml 1% acetic acid/MeCN), and 2 g MgSO4 added to the top.
Samples concentrated to 2 mL under N2, and a 1 mL aliquot
transferred to a 2 mL tube containing 0.15 g MgSO4 and 0.05 g
primary secondary amine (PSA). Samples were vortexed for 1 min
and centrifuged at 10000 g. for 1 min, and filtered through 13 mm
0.2 mm nylon syringe filter, (Whatman UK), into a 2 mL amber GC
vial.
3
included in the investigation. Based on positive detections of OPs
residues in honey and pollen, median and 95th centile values were
used for calculating total daily intake (TDI) for best and worst case
respectively. HQs were calculated based on total daily intake (TDI)
of OPs in honey and pollen for which surrogate values for samples
for which concentrations were less than the limit of detection was
set to the LOD. In the best case scenario concentrations of OPs less
than the LOD were set to zero (0.0). Total daily intake (TDI) of
pesticides received by bees via food was calculated based on total
food consumption rate (TFR) for adult worker bees estimated to be
292 mg d 1 for nectar (USEPA, 2012) and 9.5 mg d 1 for pollen for
nurse bees (Crailsheim et al., 1992) (Eq. (1)).
TDI = OPH and OPP × TFR
(1)
where OPH refers to concentrations of OPs detected in honey, OPP
refers to concentrations of OPs detected in pollen and TFR refers to
rate of consumption of nectar and pollen.
Because the mode of toxic action of OPs is mainly via inhibition
or acetylcholinesterase activity, a toxic units approach was used.
HQs for individual OPs were calculated based on total daily intake
(TDI) of OPs in honey and pollen divided by the LD50 for each OP.
The total HQ was the sum of the HQs for individual OPs. The
overall HQ was the sum of HQs for individual OPs (Eq. (2)).
HQ = TDI (honey) + TDI (pollen)/Acute oral LD50
(2)
2.2.4. Quantification of OPs by LC.MS.MS
A subsample (100 uL) was transferred to an auto-sampler vial
fitted with glass insert and internal standard (IS) (malathion-D10)
was introduced into extracts prior to analysis by LC–MS/MS. Separation of target analytes was performed by HPLC (Agilent
Technologies) on a Kinetex C18 100A column (Phenomenex,
100 4.6 mm, 5 mm particle size), using water (A) and methanol
(B) as solvents at a flow rate of 250 m min-1. The solvent gradient
was 10% B increasing to 85% B over 15 min, then increasing to 95%
B at 25 min, before returning to 10% B and holding for 5 min. Mass
spectra were collected by use of a triple quadruple, tandem mass
spectrometer fitted with an electrospray ionization source (Applied Biosciences SCIEX 3000) operated in positive ionization
mode (MRM), by using the following operations parameters:
temperature 500 °C; capillary voltage, 5.5 kV; collision gas, nebulizer gas, curtain gas were 4, 8 and 10 respectively. Quantification
was by use of Analyst 1.4.1 software (SCIEX, Applied Biosciences
Foster City, CA). A list of target ions is given in Table S1 (See
Supplementary material)
To assess potential effects of OPs to worker bees during spring
and summer, Tier-1 screening-level assessments (worst case) HQs,
were based on concentrations of OPs in bees (body burden) with
the limits of detection (LODs) used as a surrogate for concentrations of OPs that were not detected. The range of uncertainty was
also assessed by calculating HQs (best case) based on OPs in bee
body burden with concentrations that were less than the LOD set
to a value of 0.0. Median and 95th centile values of OPs detected in
bee body burden were also used to calculate HQs. Since, total
concentrations of OPs in bees could be due to dietary exposure and
or direct contact (Uncertainty factor), HQ for individual OPs were
calculated as the ratio of measured concentrations of individual
OPs in bee’s body burden divided by the LD50 for each OP,
respectively.
If the sum of HQs of individual OPs exceeded the levels of
concern (LOC) of 0.4 set by US EPA for acute lethality (USEPA,
2012) or 0.1 over a one-day consumption period set by European
Food Safety Authority (EFSA, 2013) then higher-tier assessments
(Tier II and Tier III) would be evaluated to obtain a more realistic
measure of the risk of OPs to honey bees as indicated in Fig. S1
(see supplementary material). The margin of exposure (MOE) is
the inverse of the HQ. An HQ of 0.1 would have an MOE of 10. That
is, concentrations would need to be 10-fold greater than LD50 to
cause 50% lethality of bees. This “margin” is essentially the established “safety buffer” between the toxicity effect level dose and
the predicted exposure dose.
2.3. Assessment of hazard
2.4. Quality control and assessment
Data on toxic potency of OPs to honey bees (A. mellifera, L.),
expressed as acute oral LD50, were obtained from the literature as
illustrated in Table S2 (see Supplementary material). The hazard
characterization scheme applied was based on methods proposed
by the USEPAs Office of Chemical Safety and Pollution Prevention
for assessing risks of foliar sprayed pesticides to pollinators
(USEPA, 2012). Uncertainty was assessed by calculating the maximum possible exposure (worst case) and least possible exposure
(best case) scenarios because of some concentrations of some OPs
were oLOQ. Since, pollen and honey represent primary sources of
exposure for both larval and adult stages of bees, both were
Precision and accuracy of the modified QuEChERS method were
assessed by use of analysis of replicates of six different concentrations each spiked into the matrices; bees, honey or pollen.
Samples were spiked at concentrations ranging from 600 to
1800 ng/g, wet mass (wm) which represented 30 to 90 ng/g, wet
mass (wm) at final injection. Recoveries of individual OPs were
determined by use of external standards. Mean recoveries were
between 86–106% with a relative standard deviation (RSD) o14%
for honey, 75–117% with RSD o18% for pollen and 64.5–102% with
RSDo 19% for bees. Limits of detection (LODs) of pesticides were
defined based on a signal 3 times the background noise (S/N ¼3),
4
Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–8
ranged from 0.05 ng phorate/g to 21.5 ng dichlorvos/g for honey,
0.07 ng diazinon/g to 12.2 ng dimethoate/g for pollen and 0.02 ng
malathion/g to 10.7 ng dimethoate/g for bees. Limits of quantification (LOQs) of pesticides were calculated based on a signal ten
times the background noise (S/N ¼10) as illustrated in Table S3
(see Supplementary material).
Statistical analysis was not applied in this study due to the
limited number of samples and limited detection of compounds in
all 3 matrices however, the range of each OP residue detected
(maximum, minimum, mean, median and 95th centile) in each
matrix was reported based on positive detections.
3. Results and discussion
3.1. Residue analysis of OP Insecticides
OP insecticides were frequently detected in samples collected
during summer (Fig. 2a). Dimethoate and dichlorvos were the only
OPs detected in honey collected during spring with mean concentrations of 3.4, 1.9 ng/g, wet mass (wm), respectively. In honey
collected during summer the only OPs detected were diazinon,
dicrotophos,
profenofos
and
chlorpyrifos
with
mean
concentrations of 0.3, 0.34, 0.28 and 3.3 ng/g, wm, respectively
(Table 1). Pollen contained the greatest concentrations of OPs,
while honey contained lesser concentrations (Fig. 2a).
In samples collected during spring, malathion, profenofos and
chlorpyrifos were detected with mean concentrations of 0.6,
1.5 and 23.6 ng/g, wm, respectively (Table 1). Concentrations of
OPs were greater in pollen collected during summer with mean
concentrations of 0.2, 2.9, 0.4, 11.6, 26.4, 17.5 and 5.7 ng/g, wm, for
diazinon, malathion, dimethoate, profenofos, chlorpyrifos, chlorpyrifos methyl and fenthion, respectively. The only OPs detected in
adult worker bees during spring were diazinon and chlorpyrifos
with mean concentrations of 0.42 and 32.7 ng/g, wm, respectively
(Table 1). Bees collected during summer contained detectable
concentrations of diazinon, malathion, fenamiphos, profenofos
and chlorpyrifos with mean concentrations of 0.2, 1.1, 0.4, 6.9,
31 ng/g, wm, respectively (Table 1). These results could be attributed to wide application of OPs insecticides for controlling agricultural pests mainly, cotton, maize, vegetables and rice in summer (Mansour, 2004).
Organophosphorus insecticides represent more than 80% of
total insecticides used in Egypt during 1995 (Badawy, 1998;
Mansour, 2004; Malhat and Nasr, 2013). Greater frequencies of
detections of OPs in pollen than in honey might be due to the
Fig. 2. Frequencies of detections (%) of (a) organophosphorus (OPs) detected in honey, pollen and honey bees collected from Egypt in spring and summer 2013 and (b) most
frequently detected OPs: Profenofos, malathion, chlorpyrifos and diazinon in total samples of honey, pollen and bees (n¼ 104) in spring and summer2013.
0.3
0.34
0.28
3.3
0.2
2.9
0.4
11.6
26.4
17.5
5.7
0.2
1.1
0.4
6.9
31.0
0.3
0.3
0.3
3.3
0.2
7.2
0.4
45.2
62.2
30.4
8.0
0.3
1.7
0.5
7.1
31.0
0.3
0.3
0.3
3.3
0.2
1.4
0.4
3.7
12.9
17.5
5.7
0.2
1.0
0.4
6.9
31.0
0.3
0.3
0.4
3.3
0.2
7.7
0.4
57.4
71.9
31.7
8.3
0.3
1.8
0.5
7.1
31.0
0.3
0.3
0.2
3.3
0.1
0.3
0.4
1.2
4.1
3.3
3.2
0.2
0.7
0.2
6.6
31.0
1/20
1/20
2/20
1/20
5/17
6/17
1/17
16/17
11/17
2/17
2/17
5/18
2/18
2/18
1/18
1/18
0.4
32.7
0.5
32.7
0.4
32.7
0.5
32.7
Diazinon
4/16
Chlorpyrifos 1/16
Bees
0.3
32.7
Malathion
1/14
Profenofos
5/14
Chlorpyrifos 1/14
Pollen
0.4
0.7
23.6
0.4
3.2
23.6
0.6
1.1
23.6
0.6
2.8
23.6
0.6
1.5
23.6
Diazinon
Dicrotophos
Profenofos
Chlorpyrifos
Diazinon
Malathion
Dimethoate
Profenofos
Chlorpyrifos
Ch. Methyl
Fenthion
Diazinon
Malathion
Fenamiphos
Profenofos
Chlorpyrifos
3.4
1.9
5.0
1.9
3.4
1.9
5.2
1.9
1.4
1.9
4/19
1/19
Dimethoate
Dichlorvos
Honey
No. of positive samples
OP
OP
No. of positive samples
Minimum
(ng g 1)
Maxiumum
(ng g 1)
Median
(ng g 1)
95th centile Mean
(ng g 1)
(ng g 1)
Summer
Matrix Spring
Table 1
Organophosphorus insecticides (OPs) (ng /g, wm), detected in honey, pollen and bees collected from Egypt in spring and summer 2013.
Minimum
(ng g 1)
Maxiumum
(ng g 1)
Median
(ng g 1)
95th centile Mean
(ng g 1)
(ng g 1)
Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–8
5
anatomy of flowers which enables nectar to be more protected
than pollen from exposure to spray droplets (Willmer, 2011).
Nectar, water, and honeydew are carried internally in the “honey
stomach” by bees (Gary, 1975; Snodgrass, 1975), where residues of
pesticides are more likely to be absorbed and metabolized, reducing the amount transferred to the hive. Chemical compositions of
honey (water and sugar) and pollen (more lipophilic) might be
another reason for the greater frequencies of detections of OPs in
pollen, and explain the variation in OPs seen in each matrix.
In this study the most frequently detected OPs were profenofos,
chlorpyrifos, malathion and diazinon (Fig. 2b), which is consistent
with results of previous studies (Ghini et al., 2004; Rissato et al.,
2007). In general, concentrations of OPs detected in this studyrthose detected in previous studies as shown in Table S4 (see
Supplementary material), though to the authors knowledge there
is no previous publications on OPs in bees and their matrices
available from Egypt. OPs identified in this study are consistent
with those of Malhat and Nasr (2011) who found Chlorpyrifos,
Cadusafos, diazinon, prothiphos and malathion in fish from the
Nile tributaries and residues of chlorpyrifos, diazinon, malathion
and profenofos have been detected in Egyptian herbs, fruits and
vegetables (Farag et al., 2011).
The greatest frequency of detection of OPs was for profenofos
which was found in 24% of the 104 samples. In contrast, ethoprop,
phorate, coumaphos and chlorpyrifos oxon were never detected in
any samples of honey, pollen and bees (Fig. 2a). This result might
be due to wide application of profenofos for control of various
caterpillars, white fly and mites on cotton and vegetable crops in
Egypt (Tomlin, 2004). Malathion was detected in (19%) of total
samples of this study (n ¼104) (Fig. 2b). Use of malathion for
controlling pests affecting agricultural crops, ornamentals, green
houses, livestock, stored grain, buildings, household and gardens
(Abou El Ella, 2008), might be a factor of its greater frequency of
detection.
In Egypt, chlorpyrifos is one of the most widely applied OP
insecticides, it is classified as a general use pesticide (GUP) and it is
registered for agriculture uses with 64 crops in the Egypt (ElMarsafy, 2004). It represents the third most detected OP in 14% of
total samples (Fig. 2b). Its greatest frequency 65% of total samples
(n ¼17) was observed in pollen during summer (Fig. 2a). European
honeybees in Egypt are widely exposed to chlorpyrifos despite
regulatory statements that risks to bees are minimized by label
restrictions on time of application (WHO, 2009). Chlorpyrifos was
the most frequently detected insecticide in honey in Uruguay (42%
of samples at up to 80 μg/kg), and propolis (78% at up to 111 μg/
kg) (Pareja et al., 2011). In North America, it was found in wax in
63.2% of colonies (at up to 890 μg/kg), in 43.7% of pollen samples
(at up to 830 μg) (Mullin et al., 2010).
Diazinon, which is used to control insects in soil on ornamental
plants, and on fruits and vegetable field crops (Tomlin, 2004). It
also has veterinary uses against fleas and ticks, to eliminate crop
and cattle plagues, as well as in household pest control (ATSDR,
1997). It was detected in 14% of total samples (n ¼104) in this
study (Fig. 2b).
Colony collapse occurs mainly in winter, when pesticide use is
limited. Larva and the queen would be exposed to OPs mainly
through diet since there is little chance for direct exposure. Thus,
stored honey and pollen could be a sink in the summer and a
source of OPs to the hive (Faucon et al., 2005; Chauzat et al., 2006).
During winter pollen acts as the primary protein source and its
consumption could cause toxic effects. Therefore in winter investigations of oral exposure to a hive should be studied as external exposure is limited and most dietary intake is from stored
bee bread and honey (Seeley, 1985). In this study, greater concentrations of OPs insecticides were observed in stored pollen
collected during summer. While it is unlikely that reported colony
6
Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–8
Table 2
Tier-1 Hazard quotients (HQs) for lethality of bees exposed to organophosphorus (OPs) in honey and pollen consumed by bees in Egypt during spring 2013.
pesticide
Diazinon
Dicrotophos
Ethoprop
Malathion
Dimethoate
Coumaphos
Phorate
Dichlorvos
Fenamiphos
Profenofos
Chlorpyrifos
Ch-methyl
Fenthion
Ref. LD50 (ng bee 1)
168.0
137.6
5560.0
335.2
129.6
14390.0
196.0
218.4
1870.0
95.0
67.8
110.0
251.2
Total Daily Intake (TDI) (ng bee 1 day 1)
HQs (honey&pollen)
Honey
(best case)
Honey
(worst case)
Pollen
(best case)
Pollen
(worst case)
0.00
0.00
0.00
0.00
0.99
0.00
0.00
0.55
0.00
0.00
0.00
0.00
0.00
0.04
2.10
0.09
0.13
1.45
0.13
0.01
0.55
0.04
0.04
0.04
0.82
0.43
0.00
0.00
0.00
0.00
0.01
0.00
0.00
0.01
0.00
0.00
0.22
0.00
0.00
0.00
0.04
0.01
0.01
0.01
0.00
0.02
0.03
0.00
0.00
0.22
0.03
0.04
Sum
Margin of Exposure (MOE)
Best
Case
Worst
Case
0.00
0.00
0.00
0.00
0.01
0.00
0.00
0.00
0.00
0.00
0.003
0.00
0.00
0.00
0.00
0.00
0.00
0.012
0.00
0.00
0.003
0.00
0.001
0.004
0.01
0.002
0.01
0.05
73
22
Table 3
Tier-1 Hazard quotients (HQs) for lethality of bees exposed to organophosphorus (OPs) in honey and pollen consumed by bees in Egypt during summer 2013.
pesticide
Diazinon
Dicrotophos
Ethoprop
Malathion
Dimethoate
Coumaphos
Phorate
Dichlorvos
Fenamiphos
Profenofos
Chlorpyrifos
Ch-methyl
Fenthion
Ref. LD50 (ng bee 1)
168.0
137.6
5560.0
335.2
129.6
14390.0
196.0
218.4
1870.0
95.0
67.8
110.0
251.2
Total Daily Intake (TDI) (ng bee 1 day 1)
HQs (honey&pollen)
Honey
(best case)
Honey
(worst case)
Pollen
(best case)
Pollen
(worst case)
0.07
0.10
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.08
0.95
0.00
0.00
0.07
0.10
0.09
0.13
0.99
0.13
0.01
6.29
0.04
0.10
0.95
0.82
0.43
0.002
0.00
0.00
0.013
0.004
0.00
0.00
0.00
0.00
0.04
0.12
0.17
0.05
0.002
0.041
0.01
0.07
0.004
0.004
0.02
0.01
0.004
0.43
0.59
0.29
0.08
Sum
Margin of Exposure (MOE)
losses of the Egyptian beekeepers along the Nile River (Hassan,
2009) can be attributed to chronic exposure of bees to pesticides
residues in stored honey and pollen collected during summer it
might be a contributing factor in a complex of multiple etiologies
or possibly interactions between and among stressors, including
nutrition, changes in climate, infestations with mites, and viral
diseases (Cornman et al., 2012). It has been suggested that some
insecticides can affect immune competence and thus render bees
more susceptible to infections (Di Prisco et al., 2013).
3.2. Assessment of hazard
To address potential effects that pesticides might have on
pollinators several tools have been developed. The potential hazard to bee hives from exposure to OPs was evaluated by using a
simplified hazard assessment approach. The scheme incorporated
Tier-1 (worst case) screening-level assessments that calculate HQs
based on ratios of estimated exposure by dietary uptake of OPs in
honey and pollen. Hazard quotients based on toxic units to cause
Best
Case
Worst
Case
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.02
0.00
0.00
0.00
0.001
0.00
0.00
0.01
0.00
0.00
0.03
0.00
0.01
0.02
0.01
0.00
0.02
0.08
50
13
lethality of all of the OPs present in honey and pollen ranged from
0.01 to 0.05 during spring and from 0.02 to 0.08 during summer,
which were the best- and worst-case exposure scenarios respectively Tables 2 and 3. Whereas, HQs based on toxic units to cause
lethality of all of the OPs present in total body burden of bee from
direct exposure in spring and summer separately ranged from 0.04
to 0.1 for best and worst case respectively as shown in Table 4. The
proposed Tier-1 scheme includes an acute oral level of concern
(LOC of 0.4) HQ for adult and larval honey bees (USEPA, 2012) and
(LOC of 0.1) over a one-day consumption period (UFSA 2013).
Calculated HQs were less than the US EPA LOC of (0.4) and not
exceeded the UFSA LOC of (0.1) for both best and worst case, indicating a little threat from OPs pesticides to bee colony in Egypt
as indicated in Figure S2 (See supplementary material). Therefore,
those higher-tier assessments (Tier II and Tier III) were not required or justified based on the results of this study. However, one
uncertainty is the lack of information on cumulative effects of the
various OP insecticides on immune function and bee behavior.
Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–8
7
Table 4
Tier-1 Hazard quotients (HQs) for lethality of bees from direct exposure to organophosphorus (OPs) in Egypt during spring and summer 2013.
OP
Ref. LD50 (ng bee 1)
Conc. (ng g 1, wm) (Median-59th centile)
HQs
Spring
Spring
Summer
Best case
Diazinon
Dicrotophos
Ethoprop
Malathion
Dimethoate
Coumaphos
Phorate
Dichlorvos
Fenamiphos
Profenofos
Chlorpyrifos
Ch- methyl
Fenthion
168
137.6
5560
335.2
129.6
14390
196
218.4
1870
95
67.76
110
251.2
(0.4–0.5)
ND
ND
ND
ND
ND
ND
ND
ND
ND
(32.7–32.7)
ND
ND
(0.2–0.3)
ND
ND
(1–1.7)
ND
ND
ND
ND
(0.4-0.5)
(6.9–7.1)
(31-31)
ND
ND
Sum
Margin of Exposure (MOE)
3.3. Conclusion
Information on concentrations of OPs in honey, pollen and bees
samples collected from different locations during spring and
summer in a developing country like Egypt, where resources are
limited and pesticides application is wide spread are few. Samples
collected during summer were more contaminated with OPs.
Pollen was most contaminated with OPs, especially during summer. In this study, profenofos, chlorpyrifos, malathion and diazinon were the most frequently detected OPs. Hazard quotients
estimated as the total toxic units of individual OP indicate that
lethality due to cumulative exposure to OPs detected in honey and
pollen and consumed by bees via food were less than the levels of
concern. Hazard quotients, based on lethality of bees from direct
exposure to OPs, not exceeded the levels of concern. It is concluded that direct exposure and or dietary exposure to OPs in
honey and pollen pose little threat to colonies of bees in Egypt.
Results of the present study provide useful background information that can be used directly or indirectly in designing more
complex studies based on environmentally relevant concentrations of OPs. Since OPs all act through the same mechanism of
action with different potencies, this might not be the case. Thus,
future studies of interactions should be focused on those OPs that
occur at greater concentrations.
Acknowledgment
The authors wish to thank Egyptian beekeepers for their help in
collecting samples. This study was financially supported by Grants
from the Egyptian Fellowship and Missions Sector, Science and
Engineering Research Council of Canada (Project # 326415-07),
Western Economic Diversification Canada (Project # 6578 and
6807) and the support of an instrumentation grant from the Canada Foundation for Infrastructure. Prof. Giesy was supported by
the Canada Research Chair program, a Visiting Distinguished
Professorship in the Department of Biology and Chemistry and
State Key Laboratory in Marine Pollution, City University of Hong
Kong, the 2012 “High Level Foreign Experts” (#GDW20123200120)
program, funded by the State Administration of Foreign Experts
Affairs, the P.R. China to Nanjing University and the Einstein Professor Program of the Chinese Academy of Sciences. The research
Summer
Worst case
Best case
Worst case
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.04
0.00
0.00
0.00
0.00
0.00
0.00
0.01
0.00
0.00
0.00
0.00
0.00
0.04
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.01
0.04
0.00
0.00
0.00
0.00
0.00
0.00
0.01
0.00
0.00
0.00
0.00
0.01
0.04
0.00
0.00
0.04
0.1
0.04
0.1
25
10
25
10
was supported by a Discovery Grant from the Natural Science and
Engineering Research Council of Canada (Project # 326415-07) and
a grant from the Western Economic Diversification Canada (Project
# 6578, 6807 and 000012711). The authors wish to acknowledge
the support of an instrumentation grant from the Canada Foundation for Infrastructure.
Appendix A. Suplementary materials
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.12.
039.
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