Ecotoxicology and Environmental Safety 114 (2015) 1–8

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Ecotoxicology and Environmental Safety

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Organophosphorus insecticides in honey, pollen and bees (Apis melli-fera 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, Egyptb Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK S7N 5B3, Canadac Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canadad Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USAe Department of Biology & Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong, SAR, Chinaf School of Biological Sciences, University of Hong Kong, Hong Kong, SAR, Chinag State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, China

a r t i c l e i n f o

Article history:Received 26 September 2014Received in revised form25 December 2014Accepted 26 December 2014

Keywords:Hazard quotientOPsRisk assessmentAfricaBees

x.doi.org/10.1016/j.ecoenv.2014.12.03913/& 2014 Elsevier Inc. All rights reserved.

esponding author at: Toxicology Center, UnivSK S7N 5B3, Canada. Fax: þ306 966 4796.ail address: Yehia.elnagar@science.tanta.edu.e

a b s t r a c t

There is no clear single factor to date that explains colony loss in bees, but one factor proposed is thewide-spread application of agrochemicals. Concentrations of 14 organophosphorous insecticides (OPs) inhoney bees (Apis mellifera) and hive matrices (honey and pollen) were measured to assess their hazard tohoney bees. Samples were collected during spring and summer of 2013, from 5 provinces in the middledelta of Egypt. LC/MS–MS was used to identify and quantify individual OPs by use of a modified QuickEasy Cheap Effective Rugged Safe (QuEChERS) method. Pesticides were detected more frequently insamples 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 asthe endpoint was used in an additive model to assess the cumulative potential for adverse effects posedby 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 toOPs during spring and summer ranged from 0.04 to 0.1 for best and worst case respectively. It is con-cluded that direct exposure and/or dietary exposure to OPs in honey and pollen pose little threat due tolethality of bees in Egypt.

& 2014 Elsevier Inc. All rights reserved.

1. Introduction

Honey bees (Apis mellifera L.) fulfill important ecological andeconomic roles as pollinators of crops and produce honey that canbe harvested for consumption. Approximately 35% of arable cropsdepend directly on pollinators (Klein et al., 2007), accounting foran annual value of 153 billion Euros (Gallai et al., 2009). Egypt has�1.3 million hives, 7700 are mud hives and approximately270,000 beekeepers (The first international Forum for the Egyp-tian Beekeepers, 2009). There is limited statistical information onthe beekeeping industry in Egypt, such as its annual revenue andproduction volumes, but it is estimated to be one of the most in-fluential in the Middle East and Africa (http://www.beekeeping.

ersity of Saskatchewan, Sas-

g (Y. Al Naggar).

com/articles/us/arab_countries.htm). Egyptian beekeepers basedalong the Nile River have reported increased colony losses overwinter with no clear cause for this phenomenon (Hassan, 2009).

There is a global concern about the decline of populations ofthe 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 thisloss was considered manageable and losses were attributed to arange of factors such as disease, pathogens and pesticides, in themid-2000s losses have risen to 430% in some locations. Althoughcauses of CCD are still unclear, results of some studies suggest thatextensive use of insecticides might be responsible or a significantco-factor for increased colony losses. Genome sequencing of thehoneybee provides a possible explanation for their sensitivity topesticides. Relative to other insects, the honeybee genome is de-ficient in a number of genes encoding detoxification enzymes(Claudianos et al., 2006). A strong association between disease,

Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–82

pathogens and pesticides have been cited as potential causes ofCCD (Cox-Foster et al., 2007; Ratnieks and Carreck, 2010). This hasled to the theory that the syndrome might be the result of multiplestressors (Van Engelsdorp and Meixner, 2010). The syndrome ismost commonly associated with infections of the Varroa mite andthe diseases it carries (Fairbrother et al., 2014).

In Egypt several classes of pesticides including organochlorine(OC), organophosphorus (OP), carbamates, ureas, anilides andpyrethroids are used. OPs have become the major compoundgroup used in pest control, while OC use has declined. In 1995 over80% of all insecticide used in Egypt were OPs (Badawy, 1998 andMansour, 2004). Contamination of food with OPs and their re-sidues is of concern; the widespread application has been ques-tioned as a potential risk to human health (Pico et al., 1996).

To address potential effects of pesticides on pollinators, severaltools have been developed. These tools range from relativelysimple hazard assessments, such as evaluating lethality, to moresophisticated assessments of risk (Fairbrother et al., 2014). As-sessments of risk integrate probabilities of response based onhazard or potency with probability of exposure (Giesy et al., 2014).If sufficient information is available, it is generally considered to bemore relevant for estimation of potential adverse effects than thesimpler hazard quotient (HQ). However, since all the informationon 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 ofpesticides in bee matrices and their potential risks to bees (Rissatoet 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 thepotential effects of pesticides on colonies of bees in Egypt. Thisstudy of 14 OPs pesticides in honey and bee matrices from 5 dif-ferent agricultural governances in Egypt and their potential forlethality from direct and dietary exposure represents the firststudy of its kind in Egypt.

2. Materials and methods

2.1. Study areas

Within Egypt the main region of agriculture is the Nile RiverValley, particularly the Nile Delta region. During spring and

Fig. 1. Map of study sites (S1-15) in the main agric

summer 2013 honey, bees, and pollen stored in the comb werecollected from 15 locations (3 apiaries per location) from the5 primary agricultural governorates in the Nile Delta of Egypt: KafrEl-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 seasonfrom May until the first week of June and 3) Cotton season duringAugust and September. Egyptian clover, berseem, is the majorforage crop cultivated in the Nile Valley and Delta and occupies1.2 million hectares. Egyptian clover is planted between the 1st ofSeptember and 1st of June and flowering starts by the 1st of April.European honeybees forage largely on clover from the beginningof spring until the 1st week of June, after which cotton, maize,vegetables and pumpkins represent the predominant sources ofnectar and pollen during summer in these locations. Samples werecollected during clover and cotton growing seasons of 2013.

2.2. Experimental

2.2.1. Beehive samplesThree hives were selected at random in each apiary. Pollen was

collected by cutting a 6 cm2 piece of comb containing stored pol-len using a disposable plastic knife and placed in a 15 mL Falcontube (Fisher Scientific). Fresh honey was squeezed from the combinto a 50 mL polyethylene Falcon tubes. Worker bees were bru-shed into disposable polyethylene bags. Worker bees were col-lected from the honey combs located on the farthest side walls ofthe hive from the entrance. These were normally older workerbees, which were more likely to have accumulated pesticides, andshould represent similar ages. All samples were transported in acool box with ice packs and frozen at �20 °C in the laboratoryuntil extraction (Al Naggar et al., 2013). The total number ofsamples collected was 39, 31 and 34 for honey, pollen and beesrespectively.

2.2.2. Chemicals and reagentsStandards used for quantification of pesticides were all tech-

nical grade (498% purity, Accu Standard, New Haven, CT, USA). Allsolvents (Hexane, MeOH, MeCN, etc.) were of HPLC grade or betterand 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

ultural governorates in the Nile Delta of Egypt.

Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–8 3

were prepared by dissolving 10 mg of each compound in methanoland mixed compound calibration solutions, were prepared fromthe stock. Matrix-matched standards were prepared in the sameconcentration as that of calibration solutions, by adding appro-priate amounts of standards to the control matrix including honey,pollen and bees. Pesticides investigated in this study were selectedbased on previous studies (Rissato et al., 2007; Mullin et al., 2010;Wiest et al., 2011; Chauzat et al., 2011) and their use pattern andtoxicity in Egypt (Mansour, 2004; Malhat and Nasr, 2013).

2.2.3. Extraction and cleanupIndividual OPs were identified and quantified by use of the

QuEChERS method described by Lehotay et al. (2005) and adaptedfor the lesser masses of samples used in this study. Briefly 3 g(70.5) of sample was weighed into a 50 mL falcon tube and for-tified 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) wasadded, followed by 1.5 g NaAc and 6 g MgSO4. Tubes were sealedand vortexed for 1 min, centrifuged for 10 min at 4000 g, 15 mL ofthe supernatant transferred to a 15 ml falcon tube and evaporatedunder N2 to �4 mL. This was then passed through C18 SPE car-tridges (1000 mg supplier) under gravity and collected in a 15 mLfalcon tube, 10 ml of 1% acetic acid/MeCN was added to the C18after the sample had passed through. The C18 was preconditionedwith 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 aliquottransferred to a 2 mL tube containing 0.15 g MgSO4 and 0.05 gprimary secondary amine (PSA). Samples were vortexed for 1 minand centrifuged at 10000 g. for 1 min, and filtered through 13 mm0.2 mm nylon syringe filter, (Whatman UK), into a 2 mL amber GCvial.

2.2.4. Quantification of OPs by LC.MS.MSA 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. Se-paration of target analytes was performed by HPLC (AgilentTechnologies) 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 mmin-1. The solvent gradientwas 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. Massspectra were collected by use of a triple quadruple, tandem massspectrometer fitted with an electrospray ionization source (Ap-plied Biosciences SCIEX 3000) operated in positive ionizationmode (MRM), by using the following operations parameters:temperature 500 °C; capillary voltage, 5.5 kV; collision gas, nebu-lizer gas, curtain gas were 4, 8 and 10 respectively. Quantificationwas by use of Analyst 1.4.1 software (SCIEX, Applied BiosciencesFoster City, CA). A list of target ions is given in Table S1 (SeeSupplementary material)

2.3. Assessment of hazard

Data on toxic potency of OPs to honey bees (A. mellifera, L.),expressed as acute oral LD50, were obtained from the literature asillustrated in Table S2 (see Supplementary material). The hazardcharacterization scheme applied was based on methods proposedby the USEPAs Office of Chemical Safety and Pollution Preventionfor assessing risks of foliar sprayed pesticides to pollinators(USEPA, 2012). Uncertainty was assessed by calculating the max-imum possible exposure (worst case) and least possible exposure(best case) scenarios because of some concentrations of some OPswere oLOQ. Since, pollen and honey represent primary sources ofexposure for both larval and adult stages of bees, both were

included in the investigation. Based on positive detections of OPsresidues in honey and pollen, median and 95th centile values wereused for calculating total daily intake (TDI) for best and worst caserespectively. HQs were calculated based on total daily intake (TDI)of OPs in honey and pollen for which surrogate values for samplesfor which concentrations were less than the limit of detection wasset to the LOD. In the best case scenario concentrations of OPs lessthan the LOD were set to zero (0.0). Total daily intake (TDI) ofpesticides received by bees via food was calculated based on totalfood consumption rate (TFR) for adult worker bees estimated to be292 mg d�1 for nectar (USEPA, 2012) and 9.5 mg d�1 for pollen fornurse bees (Crailsheim et al., 1992) (Eq. (1)).

TDI OPH and OPP TFR (1)= ×

where OPH refers to concentrations of OPs detected in honey, OPPrefers to concentrations of OPs detected in pollen and TFR refers torate of consumption of nectar and pollen.

Because the mode of toxic action of OPs is mainly via inhibitionor 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. Theoverall HQ was the sum of HQs for individual OPs (Eq. (2)).

HQ TDI (honey) TDI (pollen)/Acute oral LD50 (2)= +

To assess potential effects of OPs to worker bees during springand summer, Tier-1 screening-level assessments (worst case) HQs,were based on concentrations of OPs in bees (body burden) withthe limits of detection (LODs) used as a surrogate for concentra-tions of OPs that were not detected. The range of uncertainty wasalso assessed by calculating HQs (best case) based on OPs in beebody burden with concentrations that were less than the LOD setto a value of 0.0. Median and 95th centile values of OPs detected inbee body burden were also used to calculate HQs. Since, totalconcentrations of OPs in bees could be due to dietary exposure andor direct contact (Uncertainty factor), HQ for individual OPs werecalculated as the ratio of measured concentrations of individualOPs in bee’s body burden divided by the LD50 for each OP,respectively.

If the sum of HQs of individual OPs exceeded the levels ofconcern (LOC) of 0.4 set by US EPA for acute lethality (USEPA,2012) or 0.1 over a one-day consumption period set by EuropeanFood Safety Authority (EFSA, 2013) then higher-tier assessments(Tier II and Tier III) would be evaluated to obtain a more realisticmeasure of the risk of OPs to honey bees as indicated in Fig. S1(see supplementary material). The margin of exposure (MOE) isthe inverse of the HQ. An HQ of 0.1 would have an MOE of 10. Thatis, concentrations would need to be 10-fold greater than LD50 tocause 50% lethality of bees. This “margin” is essentially the es-tablished “safety buffer” between the toxicity effect level dose andthe predicted exposure dose.

2.4. Quality control and assessment

Precision and accuracy of the modified QuEChERS method wereassessed by use of analysis of replicates of six different con-centrations each spiked into the matrices; bees, honey or pollen.Samples were spiked at concentrations ranging from 600 to1800 ng/g, wet mass (wm) which represented 30 to 90 ng/g, wetmass (wm) at final injection. Recoveries of individual OPs weredetermined by use of external standards. Mean recoveries werebetween 86–106% with a relative standard deviation (RSD)o14%for honey, 75–117% with RSDo18% for pollen and 64.5–102% withRSDo19% for bees. Limits of detection (LODs) of pesticides weredefined based on a signal 3 times the background noise (S/N¼3),

Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–84

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 ngmalathion/g to 10.7 ng dimethoate/g for bees. Limits of quantifi-cation (LOQs) of pesticides were calculated based on a signal tentimes the background noise (S/N¼10) as illustrated in Table S3(see Supplementary material).

Statistical analysis was not applied in this study due to thelimited number of samples and limited detection of compounds inall 3 matrices however, the range of each OP residue detected(maximum, minimum, mean, median and 95th centile) in eachmatrix was reported based on positive detections.

3. Results and discussion

3.1. Residue analysis of OP Insecticides

OP insecticides were frequently detected in samples collectedduring summer (Fig. 2a). Dimethoate and dichlorvos were the onlyOPs detected in honey collected during spring with mean con-centrations of 3.4, 1.9 ng/g, wet mass (wm), respectively. In honeycollected during summer the only OPs detected were diazinon,dicrotophos, profenofos and chlorpyrifos with mean

Fig. 2. Frequencies of detections (%) of (a) organophosphorus (OPs) detected in honey, pfrequently detected OPs: Profenofos, malathion, chlorpyrifos and diazinon in total samp

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 andchlorpyrifos were detected with mean concentrations of 0.6,1.5 and 23.6 ng/g, wm, respectively (Table 1). Concentrations ofOPs were greater in pollen collected during summer with meanconcentrations of 0.2, 2.9, 0.4, 11.6, 26.4, 17.5 and 5.7 ng/g, wm, fordiazinon, malathion, dimethoate, profenofos, chlorpyrifos, chlor-pyrifos methyl and fenthion, respectively. The only OPs detected inadult worker bees during spring were diazinon and chlorpyrifoswith mean concentrations of 0.42 and 32.7 ng/g, wm, respectively(Table 1). Bees collected during summer contained detectableconcentrations of diazinon, malathion, fenamiphos, profenofosand chlorpyrifos with mean concentrations of 0.2, 1.1, 0.4, 6.9,31 ng/g, wm, respectively (Table 1). These results could be attrib-uted to wide application of OPs insecticides for controlling agri-cultural pests mainly, cotton, maize, vegetables and rice in sum-mer (Mansour, 2004).

Organophosphorus insecticides represent more than 80% oftotal insecticides used in Egypt during 1995 (Badawy, 1998;Mansour, 2004; Malhat and Nasr, 2013). Greater frequencies ofdetections of OPs in pollen than in honey might be due to the

ollen and honey bees collected from Egypt in spring and summer 2013 and (b) mostles of honey, pollen and bees (n¼104) in spring and summer2013.

Table

1Organ

ophosphorusinsecticides

(OPs)(ng/g,w

m),detectedin

hon

ey,p

ollenan

dbe

esco

llected

from

Egyp

tin

springan

dsu

mmer

2013

.

Matrix

Spring

Summer

OP

No.o

fposi-

tive

samples

Minim

um

(ngg�1)

Max

iumum

(ngg�1)

Med

ian

(ngg�1)

95th

centile

(ngg�1)

Mea

n(n

gg�1)

OP

No.o

fposi-

tive

samples

Minim

um

(ngg�1)

Max

iumum

(ngg�1)

Med

ian

(ngg�1)

95th

centile

(ngg�1)

Mea

n(n

gg�1)

Honey

Dim

ethoate

4/19

1.4

5.2

3.4

5.0

3.4

Diazinon

1/20

0.3

0.3

0.3

0.3

0.3

Dichlorvos

1/19

1.9

1.9

1.9

1.9

1.9

Dicro

tophos

1/20

0.3

0.3

0.3

0.3

0.34

Pro

fenofos

2/20

0.2

0.4

0.3

0.3

0.28

Chlorp

yrifos

1/20

3.3

3.3

3.3

3.3

3.3

Pollen

Malathion

1/14

0.4

0.4

0.6

0.6

0.6

Diazinon

5/17

0.1

0.2

0.2

0.2

0.2

Pro

fenofos

5/14

0.7

3.2

1.1

2.8

1.5

Malathion

6/17

0.3

7.7

1.4

7.2

2.9

Chlorp

yrifos

1/14

23.6

23.6

23.6

23.6

23.6

Dim

ethoate

1/17

0.4

0.4

0.4

0.4

0.4

Pro

fenofos

16/17

1.2

57.4

3.7

45.2

11.6

Chlorp

yrifos

11/17

4.1

71.9

12.9

62.2

26.4

Ch.M

ethyl

2/17

3.3

31.7

17.5

30.4

17.5

Fenth

ion

2/17

3.2

8.3

5.7

8.0

5.7

Bee

sDiazinon

4/16

0.3

0.5

0.4

0.5

0.4

Diazinon

5/18

0.2

0.3

0.2

0.3

0.2

Chlorp

yrifos

1/16

32.7

32.7

32.7

32.7

32.7

Malathion

2/18

0.7

1.8

1.0

1.7

1.1

Fenam

iphos

2/18

0.2

0.5

0.4

0.5

0.4

Pro

fenofos

1/18

6.6

7.1

6.9

7.1

6.9

Chlorp

yrifos

1/18

31.0

31.0

31.0

31.0

31.0

Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–8 5

anatomy of flowers which enables nectar to be more protectedthan pollen from exposure to spray droplets (Willmer, 2011).Nectar, water, and honeydew are carried internally in the “honeystomach” by bees (Gary, 1975; Snodgrass, 1975), where residues ofpesticides are more likely to be absorbed and metabolized, redu-cing the amount transferred to the hive. Chemical compositions ofhoney (water and sugar) and pollen (more lipophilic) might beanother reason for the greater frequencies of detections of OPs inpollen, 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 consistentwith results of previous studies (Ghini et al., 2004; Rissato et al.,2007). In general, concentrations of OPs detected in this study-rthose detected in previous studies as shown in Table S4 (seeSupplementary material), though to the authors knowledge thereis no previous publications on OPs in bees and their matricesavailable from Egypt. OPs identified in this study are consistentwith those of Malhat and Nasr (2011) who found Chlorpyrifos,Cadusafos, diazinon, prothiphos and malathion in fish from theNile tributaries and residues of chlorpyrifos, diazinon, malathionand profenofos have been detected in Egyptian herbs, fruits andvegetables (Farag et al., 2011).

The greatest frequency of detection of OPs was for profenofoswhich was found in 24% of the 104 samples. In contrast, ethoprop,phorate, coumaphos and chlorpyrifos oxon were never detected inany samples of honey, pollen and bees (Fig. 2a). This result mightbe due to wide application of profenofos for control of variouscaterpillars, white fly and mites on cotton and vegetable crops inEgypt (Tomlin, 2004). Malathion was detected in (19%) of totalsamples of this study (n¼104) (Fig. 2b). Use of malathion forcontrolling pests affecting agricultural crops, ornamentals, greenhouses, livestock, stored grain, buildings, household and gardens(Abou El Ella, 2008), might be a factor of its greater frequency ofdetection.

In Egypt, chlorpyrifos is one of the most widely applied OPinsecticides, it is classified as a general use pesticide (GUP) and it isregistered for agriculture uses with 64 crops in the Egypt (El-Marsafy, 2004). It represents the third most detected OP in 14% oftotal samples (Fig. 2b). Its greatest frequency 65% of total samples(n¼17) was observed in pollen during summer (Fig. 2a). Europeanhoneybees in Egypt are widely exposed to chlorpyrifos despiteregulatory statements that risks to bees are minimized by labelrestrictions on time of application (WHO, 2009). Chlorpyrifos wasthe 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 in63.2% 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 ornamentalplants, and on fruits and vegetable field crops (Tomlin, 2004). Italso has veterinary uses against fleas and ticks, to eliminate cropand cattle plagues, as well as in household pest control (ATSDR,1997). It was detected in 14% of total samples (n¼104) in thisstudy (Fig. 2b).

Colony collapse occurs mainly in winter, when pesticide use islimited. Larva and the queen would be exposed to OPs mainlythrough diet since there is little chance for direct exposure. Thus,stored honey and pollen could be a sink in the summer and asource of OPs to the hive (Faucon et al., 2005; Chauzat et al., 2006).During winter pollen acts as the primary protein source and itsconsumption could cause toxic effects. Therefore in winter in-vestigations of oral exposure to a hive should be studied as ex-ternal exposure is limited and most dietary intake is from storedbee bread and honey (Seeley, 1985). In this study, greater con-centrations of OPs insecticides were observed in stored pollencollected during summer. While it is unlikely that reported colony

Table 2Tier-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 Ref. LD50 (ng bee�1) Total Daily Intake (TDI) (ng bee�1 day�1) HQs (honey&pollen)

Honey Honey Pollen Pollen Best Worst(best case) (worst case) (best case) (worst case) Case Case

Diazinon 168.0 0.00 0.04 0.00 0.00 0.00 0.00Dicrotophos 137.6 0.00 2.10 0.00 0.04 0.00 0.00Ethoprop 5560.0 0.00 0.09 0.00 0.01 0.00 0.00Malathion 335.2 0.00 0.13 0.00 0.01 0.00 0.00Dimethoate 129.6 0.99 1.45 0.01 0.01 0.01 0.012Coumaphos 14390.0 0.00 0.13 0.00 0.00 0.00 0.00Phorate 196.0 0.00 0.01 0.00 0.02 0.00 0.00Dichlorvos 218.4 0.55 0.55 0.01 0.03 0.00 0.003Fenamiphos 1870.0 0.00 0.04 0.00 0.00 0.00 0.00Profenofos 95.0 0.00 0.04 0.00 0.00 0.00 0.001Chlorpyrifos 67.8 0.00 0.04 0.22 0.22 0.003 0.004Ch-methyl 110.0 0.00 0.82 0.00 0.03 0.00 0.01Fenthion 251.2 0.00 0.43 0.00 0.04 0.00 0.002

Sum 0.01 0.05

Margin of Exposure (MOE) 73 22

Table 3Tier-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 Ref. LD50 (ng bee�1) Total Daily Intake (TDI) (ng bee�1 day�1) HQs (honey&pollen)

Honey Honey Pollen Pollen Best Worst(best case) (worst case) (best case) (worst case) Case Case

Diazinon 168.0 0.07 0.07 0.002 0.002 0.00 0.00Dicrotophos 137.6 0.10 0.10 0.00 0.041 0.00 0.001Ethoprop 5560.0 0.00 0.09 0.00 0.01 0.00 0.00Malathion 335.2 0.00 0.13 0.013 0.07 0.00 0.00Dimethoate 129.6 0.00 0.99 0.004 0.004 0.00 0.01Coumaphos 14390.0 0.00 0.13 0.00 0.004 0.00 0.00Phorate 196.0 0.00 0.01 0.00 0.02 0.00 0.00Dichlorvos 218.4 0.00 6.29 0.00 0.01 0.00 0.03Fenamiphos 1870.0 0.00 0.04 0.00 0.004 0.00 0.00Profenofos 95.0 0.08 0.10 0.04 0.43 0.00 0.01Chlorpyrifos 67.8 0.95 0.95 0.12 0.59 0.02 0.02Ch-methyl 110.0 0.00 0.82 0.17 0.29 0.00 0.01Fenthion 251.2 0.00 0.43 0.05 0.08 0.00 0.00

Sum 0.02 0.08

Margin of Exposure (MOE) 50 13

Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–86

losses of the Egyptian beekeepers along the Nile River (Hassan,2009) can be attributed to chronic exposure of bees to pesticidesresidues in stored honey and pollen collected during summer itmight be a contributing factor in a complex of multiple etiologiesor possibly interactions between and among stressors, includingnutrition, changes in climate, infestations with mites, and viraldiseases (Cornman et al., 2012). It has been suggested that someinsecticides can affect immune competence and thus render beesmore susceptible to infections (Di Prisco et al., 2013).

3.2. Assessment of hazard

To address potential effects that pesticides might have onpollinators several tools have been developed. The potential ha-zard to bee hives from exposure to OPs was evaluated by using asimplified hazard assessment approach. The scheme incorporatedTier-1 (worst case) screening-level assessments that calculate HQsbased on ratios of estimated exposure by dietary uptake of OPs inhoney and pollen. Hazard quotients based on toxic units to cause

lethality of all of the OPs present in honey and pollen ranged from0.01 to 0.05 during spring and from 0.02 to 0.08 during summer,which were the best- and worst-case exposure scenarios respec-tively Tables 2 and 3. Whereas, HQs based on toxic units to causelethality of all of the OPs present in total body burden of bee fromdirect exposure in spring and summer separately ranged from 0.04to 0.1 for best and worst case respectively as shown in Table 4. Theproposed 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 notexceeded the UFSA LOC of (0.1) for both best and worst case, in-dicating a little threat from OPs pesticides to bee colony in Egyptas indicated in Figure S2 (See supplementary material). Therefore,those higher-tier assessments (Tier II and Tier III) were not re-quired or justified based on the results of this study. However, oneuncertainty is the lack of information on cumulative effects of thevarious OP insecticides on immune function and bee behavior.

Table 4Tier-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 Summer Spring Summer

Best case Worst case Best case Worst case

Diazinon 168 (0.4–0.5) (0.2–0.3) 0.00 0.00 0.00 0.00Dicrotophos 137.6 ND ND 0.00 0.00 0.00 0.00Ethoprop 5560 ND ND 0.00 0.00 0.00 0.00Malathion 335.2 ND (1–1.7) 0.00 0.00 0.00 0.00Dimethoate 129.6 ND ND 0.00 0.01 0.00 0.01Coumaphos 14390 ND ND 0.00 0.00 0.00 0.00Phorate 196 ND ND 0.00 0.00 0.00 0.00Dichlorvos 218.4 ND ND 0.00 0.00 0.00 0.00Fenamiphos 1870 ND (0.4-0.5) 0.00 0.00 0.00 0.00Profenofos 95 ND (6.9–7.1) 0.00 0.00 0.01 0.01Chlorpyrifos 67.76 (32.7–32.7) (31-31) 0.04 0.04 0.04 0.04Ch- methyl 110 ND ND 0.00 0.00 0.00 0.00Fenthion 251.2 ND ND 0.00 0.00 0.00 0.00

Sum 0.04 0.1 0.04 0.1

Margin of Exposure (MOE) 25 10 25 10

Y. Al Naggar et al. / Ecotoxicology and Environmental Safety 114 (2015) 1–8 7

3.3. Conclusion

Information on concentrations of OPs in honey, pollen and beessamples collected from different locations during spring andsummer in a developing country like Egypt, where resources arelimited and pesticides application is wide spread are few. Samplescollected during summer were more contaminated with OPs.Pollen was most contaminated with OPs, especially during sum-mer. In this study, profenofos, chlorpyrifos, malathion and diazi-non were the most frequently detected OPs. Hazard quotientsestimated as the total toxic units of individual OP indicate thatlethality due to cumulative exposure to OPs detected in honey andpollen and consumed by bees via food were less than the levels ofconcern. Hazard quotients, based on lethality of bees from directexposure to OPs, not exceeded the levels of concern. It is con-cluded that direct exposure and or dietary exposure to OPs inhoney and pollen pose little threat to colonies of bees in Egypt.Results of the present study provide useful background informa-tion that can be used directly or indirectly in designing morecomplex studies based on environmentally relevant concentra-tions of OPs. Since OPs all act through the same mechanism ofaction with different potencies, this might not be the case. Thus,future studies of interactions should be focused on those OPs thatoccur at greater concentrations.

Acknowledgment

The authors wish to thank Egyptian beekeepers for their help incollecting samples. This study was financially supported by Grantsfrom the Egyptian Fellowship and Missions Sector, Science andEngineering Research Council of Canada (Project # 326415-07),Western Economic Diversification Canada (Project # 6578 and6807) and the support of an instrumentation grant from the Ca-nada Foundation for Infrastructure. Prof. Giesy was supported bythe Canada Research Chair program, a Visiting DistinguishedProfessorship in the Department of Biology and Chemistry andState Key Laboratory in Marine Pollution, City University of HongKong, the 2012 “High Level Foreign Experts” (#GDW20123200120)program, funded by the State Administration of Foreign ExpertsAffairs, the P.R. China to Nanjing University and the Einstein Pro-fessor Program of the Chinese Academy of Sciences. The research

was supported by a Discovery Grant from the Natural Science andEngineering Research Council of Canada (Project # 326415-07) anda grant from the Western Economic Diversification Canada (Project# 6578, 6807 and 000012711). The authors wish to acknowledgethe support of an instrumentation grant from the Canada Foun-dation for Infrastructure.

Appendix A. Suplementary materials

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.ecoenv.2014.12.039.

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1

Title: Organophosphorus insecticides in honey, pollen and bees (Apis mellifera L.) and their potential hazard to bee colonies in Egypt

Journal: Journal of Ecotoxicology and Environmental Safety.

Authors: Yahya Al Naggar1. 2, Garry Codling2, Anja Vogt2, Elsaied Naiem1, Mohamed Mona1, Amal Seif1, John P. Giesy2, 3, 4, 5, 6, 7.

Affiliations: 1 Department of Zoology, Faculty of Science, Tanta University 31527, Tanta, Egypt. 2 Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK, S7N 5B3, Canada. 3 Department of veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan 4 Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA 5 Department of Biology & Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong, SAR, China 6 School of Biological Sciences, University of Hong Kong, Hong Kong, SAR, China 7 State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China.

Corresponding Author:

Yahya Al Naggar,

Toxicology center

University of Saskatchewan

Saskatoon, SK, S7N 5B3, Canada

Yehia.elnagar@science.tanta.edu.eg

Tel: 306-715-6328

Fax: 306-966-4796

2

Figure S1. A simplified proposed tiered approach for assessing risk to honey bees from

foliar spray applications (USEPA 2012).

3

Figure S2. Comparison of calculated HQs due to lethality of bees from dietary exposure or

and direct exposure to OPs during spring and summer 2013 with the levels of concern (LOC)

of 0.4 and 0.1 for acute lethality from foliar sprayed pesticides set by United Stated

Environmental Protection Agency (USEPA 2012) and European Food Safety Authority (

EFSA 2013) respectively.

4

Table S1. Primary and transition ions and quantification ion of organophosphorus insecticides (OPs) identified and quantified by LC-MS/MS.

Pesticides

Ions monitored (m/z)

Quantification

ion (m/z) Diazinon 305.1; 168.1; 153.1 168.1

Dicrotophos 238; 112.1; 127 112.1

Ethoprop 243; 173; 131 173

Dimethoate d6 (PCS) 236; 131; 177 131

Malathion 331; 127; 99 127

Dimethoate 230.3; 125.1; 171 125.1

Coumaphos 363; 227; 211 227

Phorate 261; 75; 171 75

Dichlorvos 221; 109; 127 109

Fenamiphos 304.3; 217; 234 217

Profenofos 374.9; 304.8; 346.8 304.8

Chlorpyrifos 349.9; 97; 197.9 97

Chlorpyrifos methyl 323.5; 125; 291.8 125

Chlorpyrifos-oxon 336; 280; 308 280

Fenthion 279; 169; 102.2 169

Malathion d10 (IS) 343.3; 132; 100 132

5

Table S2 Toxicity of organophosphorus pesticides (OPs) to the European honey bee (Apis

mellifera, L.), expressed as the acute, oral LD50 from previous literatures.

OP LD50 (µg/g, wm) LD50 (ng/bee) Reference

Diazinon 2.1 168 (Hardstone and Scott 2010)

Dicrotophos 1.72 137.6 (Hardstone and Scott 2010)

Ethoprop 51.125 5560 (PPDB 2009)

Malathion 4.19 335.2 (Hardstone and Scott 2010)

Dimethoate 1.62 129.6 (Hardstone and Scott 2010)

Coumaphos 179.875 14390 (Klochko et al. 1994)

Phorate 2.45 196 (Hardstone and Scott 2010)

Dichlorvos 2.73 218.4 (Hardstone and Scott 2010)

Fenamiphos 23.375 1870 (Atkins et al. 1975)

Profenofos 1.1875 95 (Winter 1990)

Chlorpyrifos 0.847 67.76 (Hardstone and Scott 2010)

Ch- methyl 4.75 380 (Chlorpyrifos-methyl SANCO/3061/99 – rev. 1.6, 2005)

Fenthion 3.14 251.2 (Hardstone and Scott 2010)

6

Table S3. Recoveries, Relative Stander Deviations (RSDs), Limits of Detections (LODs), Limits of Quantifications (LOQs) of organophosphorus insecticides (OPs) for honey, pollen and honey bees analyzed by LC-MS.MS.

OP Sample

Spiked (ng/g)

Honey

pollen honey bees

Recovery ± RSD

LOD

(ng/g)

LOQ

(ng/g) Recovery ± RSD

LOD

(ng/g)

LOQ

(ng/g)

Recovery ± RSD

LOD

(ng/g)

LOQ

(ng/g)

Diazinon 600 91 ± 6.8 0.13 0.44

87.7 ± 6.6 0.07 0.2

78 ± 9.4 0.1 0.6

Dicrotophos 1800 89.8 ± 5.8 7.2 25.9

101 ± 16.8 4.3 12.3

76.4 ± 10 3.2 11.9

Ethoprop 600 98 ± 8.6 0.31 1.7

81.8 ±7.4 0.57 2.1

67.5 ±16.1 0.5 2.5

Dimethoate. d6 1800 85.9 ± 6.7 1.31 8.2

78.9 ± 9.2 1.5 5.9

67.6 ± 15.6 2 6

Malathion 600 96.7 ± 11 0.43 2.2

101 ± 8.9 0.75 1.9

70.8 ± 9.6 0.02 1.9

Dimethoate 1800 88.6 ± 14 3.4 11.4

78.8 ± 11.6 12.2 41.2

85.1 ± 7.2 10.7 27.3

Coumaphos 600 94.8 ± 13.7 0.79 2.9

79.7 ± 14.7 0.46 1.7

72 ± 10.3 0.1 0.9

Phorate 600 106.2 ± 10.6 0.05 0.2

68.9 ± 23.7 2 4.8

72.7 ± 18.8 0.7 2.3

Dichlorvos 8000 101.2 ± 9.2 21.54 56.4

86.2 ± 14.4 0.54 47

101.9 ± 11 2.4 52.8

Fenamiphos 600 102.6 ± 15.1 0.12 0.8

85.57 ± 8.1 0.37 1

73 ± 13.4 0.1 0.6

Profenofos 600 92.4 ± 4.7 0.31 2.3

77.2 ± 12.4 1.14 2.9

74 ± 4.4 0.1 1.9

Chlorpyrifos 1800 109.6 ± 9.3 0.14 5.4

81 ± 12.5 5.8 27.8

73.3 ± 16.8 4.9 41.3

Ch. methyl 1800 90.4 ± 9.1 2.8 13.5

117 ± 13.8 3.1 8.8

72.4 ± 5.7 4.4 12.9

Ch. oxon 600 95.9 ± 10.5 0.26 1.3

99.8 ± 17.9 0.64 1.7

64.5± 3.6 0.1 0.6

Fenthion 600 95.1 ± 12.9 1.46 3.9

75.2 ± 17.6 4 8.5

97.5 ± 12.4 2.1 9.1

7

Table S4. Concentrations of orgnophosphorus (OPs) insecticides detected in honey, pollen and bees in previous studies and the present study. (ND, not detected)

pesticicde concentration (ng/ g, wm)

honey Pollen bees

Diazinon

ND (Rissato et al. 2007) 23.5 ( bernal et al 2010) 3 (Ghini et al. 2004)

35 (Johnson et al.2010) 29 (Mullin et al. 2010) (0.42-0.19 )(Spring-Summer; present study)

14 (Wiest et al. 2011) ND (Wiest et al. 2011) - - (ND-0.25 )(Spring-Summer; present study)

(ND-0.16 )(Spring-Summer; present study) - -

Ethoprop ND ( present study) ND ( present study) 1.36 ( present study)

Malathion

0.24 (Rissato et al. 2007) 61 (Mullin et al. 2010) 360 (Ghini et al. 2004)

243 (Johnson et al.2010) 37.7 ( bernal et al 2010) ND (Chuazat et al.2011)

ND (Chuazat et al. 2011) ND (Chuazat et al. 2011) ( ND-1.11 )(Spring-Summer; present study)

ND ( present study) ( 0.61-2.91 )(Spring-Summer; present study) - -

Dimethoate

9 (Johnson et al.2010) ND (Chuazat et al. 2011) 19 (Ghini et al. 2004)

ND (Wiest et al. 2011) 5828 (Mullin et al. 2010) ND (Chuazat et al. 2011) ( 3.36-ND )(Spring-Summer; present study

( ND-0.43 )(Spring-Summer; present study) ND ( present study)

coumaphos

2020 (Mullin et al. 2010) 79.6 ( bernal et al 2010) 208 (Ghini et al. 2004)

29 (Wiest et al. 2011) 40 (Wiest et al. 2011) 8 (Mullin et al. 2010)

934 (Chuazat et al. 2011) 423.5 (Chuazat et al. 2011) ND (Chuazat et al. 2011)

60 (Pareja et al. 2011) ND ( present study) ND ( present study)

ND ( present study) - - - -

Phorate 0.9 (Johnson et al.2010) ND ( present study) ND ( our study)

ND ( present study) - - - -

Dichlorvos

ND (Rissato et al. 2007) ND (Wiest et al. 2011) 899.19 ( present study)

8 (Johnson et al.2010) ND ( present study) - -

ND (Wiest et al. 2011) - - - - ( 1.9-ND )(Spring-Summer; present study) - - - -

Fenamiphos ND ( present study) 0.29 ( present study) ( ND-0.36 )(Spring-Summer; present study)

Profenofos ND (Rissato et al. 2007) ( 1.45-11.56 )(Spring-Summer;

present study) 17 (Ghini et al. 2004)

( ND-0.28 )(Spring-Summer; present study) - - ( ND-6.85 )(Spring-Summer

;present study)

Chlorpyrifos

0.01 (Rissato et al. 2007) 830 (Mullin et al. 2010) 2.2 (Mullin et al. 2010)

15 (Johnson et al.2010) 87.4 ( bernal et al 2010) ( 32.72-31.04)(Spring-Summer ;present study

80 (Pareja et al. 2011) 140 (Wiest et al. 2011) - -

ND (Wiest et al. 2011) ( 23.63-26.44 )(Spring-Summer; present study) - -

( ND-3.27 )(Spring-Summer; present study) - - - -

Ch. Methyl 0.2 (Johnson et al.2010) ( ND-17.5 )(Spring-Summer;

present study) 9 (Ghini et al. 2004)

ND ( present study) ND ( present study)

Fenthion

ND (Rissato et al. 2007) ND (Chuazat et al. 2011) 16 (Ghini et al. 2004)

ND (present study) ( ND-5.74 )(Spring-Summer; present study ND (Chuazat et al. 2011)

- - - - ND (present study)

8

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