Yahya Al Naggar1,2, Anja Vogt2, Garry Codling2, El Saied Naiem1, Mohamed Mona1, Amal Seif1 and John P. Giesy2 1 Department 2 Toxicology

of Zoology, Faculty of Science, Tanta University 31527, Tanta, Egypt.

Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK, S7N 5B3, Canada.

 Provide an overview about OPs residues in honey, pollen and bees

in Egypt.

 Characterization of 14 current use OPs

in Bee Matrices

 Estimate potential hazard of OPs to bee hives.

 Are there OPs residues in samples of honey, pollen and bees collected from Egypt during spring and summer 2013?  Are

there

concentrations

differences between

spring

in and

summer?  Is there a potential hazard from direct and/or dietary exposure to these OPs to bee colonies?

Toxicology Centre Oxon Diazinon

Malathion

Chlorpyrifos

Methyl

Profenofos

Ethoprop Fenamiphos

Dicrotophos

Dimethoate

Coumaphos Title or place of presentation

Dichlorvos

Phorate

Fenthion

Date of presentation

Toxicology Centre

 35% of all arable crops need honey bees to pollinate and 90% of wild plants need bees too [1].

 Egypt has about 1.3 million hives[2] :  7,700 are mud hives  270,000 beekeepers

 Stats on Bee keeping in Egypt are limited but it is considered to be one of the most influential in the Middle East and

Africa [3].

Title or place of presentation

Date of presentation

 Colony Collapse Disorder (CCD):  Normally 15% of hives fail over winter, today its ~30%  Egypt has reported incidence of CCD along River Nile [4].  To

date,

no

clear

single

explains colony loss in bees [5]

factor

 Parasites and Pathogens.  Malnutrition and Loss of habitat.  Genetic factors and Beekeeping practice.

 Pesticides  Combination of all factors.

Toxicology Centre

 There are no clear guidelines for use in Egypt  OPs have become the major compound group used in pest control, over 80% of all insecticide used in Egypt are OPs [6]

World market share of insecticides, by class (2008) Title or place of presentation

Date of presentation

 Study areas

Map of study sites (S1-15) in the Nile Delta governorates of Egypt.

[7]

Quick Easy Cheap Effective Rugged Safe

Weigh 3g of matrix in a 50 ml falcon tube

Fortified with 100 µL of Dimethoate d6 (PCS)

Extraction Add 27 mL of extraction solution (44% deionized water, 55% acetonitrile, and 1% glacial acetic acid).

Salting-out Add 6 g MgSO4 + 1.5 g NaAc, centrifuge (4000 rpm, 10 min)

Clean-up C18 SPE cartridges + PSA

Quantification LC-MS/MS

Shaking (1 min)

Assessment of Hazard 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.[8]

 Uncertainty was assessed by calculating :

Best case

In the best case scenario concentrations of OPs less than the limit of detection (LOD) were set to zero (0.0)

Worst case

In the worst case scenario concentrations of OPs less than the LOD were set to LOD

Total daily intake (TDI) of pesticides received by bees via food was calculated (Equation 1): TDI = OP concentration detected in honey and pollen x Proposed total food consumption rate of adult workers (292 mg d-1)



(1)

HQs for individual OPs were calculated (Equation 2): HQs = TDI (honey + pollen )/ Acute oral LD 50

(2)

 HQs based on lethality of bees exposed to OPs directly were estimated (Equation 3): HQs = OPs detected in bee body burden /Acute oral LD 50

(3)

Organophosphorous pesticides (OPs) (ng/g, wm) detected in honey, pollen and honey bees Spring No. of positive samples

Mean Conc.

Dimethoate

4/19

3.36

Dichlorvos

1/19

2.8

OP

Honey

Summer Mean Conc.

Diazinon

1/20

0.25

Dicrotophos Profenofos

1/20 2/20

0.34 0.28

Chlorpyrifos

1/20

3.27

OP

Malathion Profenofos

6/14 5/14

0.61 1.45

Diazinon Malathion

5/17 6/17

0.16 2.91

Chlorpyrifos

1/14

23.63

Dimethoate

1/17

0.43

Profenofos

16/17

11.56

Chlorpyrifos

11/17

26.44

Ch. Methyl Fenthion Diazinon

2/17 217 5/18

17.5 5.74 0.19

Fenamiphos

2/18

0.36

Profenofos

2/18

6.85

Chlorpyrifos

1/18

31.04

Pollen

Bees

No. of positive samples

Diazinon

4/16

0.42

Chlorpyrifos

1/16

32.72

Toxicology Centre

Proportion of positive samples (%)

Diazinon 100

Chlorpyrifos

90

Malathion

80

Profenofos

Spring honey

Summer honey Spring pollen

70 60 50 40

0

10

20

30

Frequencies of detections (%) (n=104)

Summer pollen Spring bees Summer bees

30 20 10 0

Percentages (%) of positive samples of OPs detected in honey, pollen and honey bees Title or place of presentation

Date of presentation

Tier-1 HQs for lethality of bees exposed to OPs in honey and pollen consumed by bees during spring

OP

Ref.LD50 (ng. bee-1)

Total Daily Intake (TDI) (ng. bee-1 day-1) Honey Honey Pollen Pollen (best case) (worst case) (best case) (worst case) 0.00 0.04 0.00 0.02

HQs (honey & pollen) best case 0.000

worst case 0.000

Diazinon

168.0

Dicrotophos

137.6

0.00

2.10

0.00

1.26

0.000

0.024

Ethoprop

5560.0

0.00

0.09

0.00

0.17

0.000

0.000

Malathion

335.2

0.00

0.13

0.18

0.18

0.001

0.001

Dimethoate

129.6

0.98

0.98

0.00

3.56

0.008

0.035

Coumaphos

14390.0

0.00

0.13

0.00

0.13

0.000

0.000

Phorate

196.0

0.00

0.01

0.00

0.58

0.000

0.003

Dichlorvos

218.4

0.82

0.82

0.00

0.16

0.004

0.004

Fenamiphos

1870.0

0.00

0.04

0.00

0.11

0.000

0.000

Profenofos

95.0

0.00

0.04

0.42

0.42

0.004

0.005

Chlorpyrifos

67.8

0.00

0.04

6.90

6.90

0.102

0.102

Ch. methyl

110.0

0.00

0.82

0.00

0.91

0.000

0.016

Fenthion

251.2

0.00

0.43

0.00

1.17

0.000

0.006

0.1 10

0.2 5

Sum Margin of Exposure (MOE)

Tier-1 HQs for lethality of bees exposed to OPs in honey and pollen consumed by bees during summer.

pesticide

Ref.LD50 (ng. bee-1)

Total Daily Intake (TDI) (ng. bee-1 day-1) Honey (worst case) 0.07

Pollen (best case) 0.05

Pollen (worst case) 0.05

HQs (honey & pollen) best worst case case 0.00 0.00

Diazinon

168.0

Honey (best case) 0.07

Dicrotophos

137.6

0.10

0.10

0.00

1.26

0.00

0.01

Ethoprop Malathion Dimethoate Coumaphos Phorate Dichlorvos

5560.0 335.2 129.6 14390.0 196.0 218.4

0.00 0.00 0.00 0.00 0.00 0.00

0.09 0.13 0.99 0.13 0.01 6.29

0.00 0.85 0.13 0.00 0.00 0.00

0.17 0.85 0.13 0.13 0.58 0.16

0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.01 0.00 0.00 0.03

Fenamiphos

1870.0

0.00

0.04

0.00

0.11

0.00

0.00

Profenofos

95.0

0.08

0.08

3.38

3.38

0.04

0.04

Chlorpyrifos

67.8

0.95

0.95

7.72

7.72

0.13

0.14

Ch. methyl Fenthion

110.0 251.2

0.00 0.00

0.82 0.43

5.12 1.68

5.12 1.68

0.05 0.01

0.05 0.01

0.2 5

0.3 3.3

Sum Margin of Exposure (MOE)

Tier-1 HQs for lethality of bees from direct exposure to OPs during spring and summer. Conc. (ng g-1, wm) Ref.LD50 (ng. bee-1)

spring

summer

168

0.42

Dicrotophos

137.6

Ethoprop

HQs

0.19

spring best worst case case 0.000 0.000

summer best worst case case 0.000 0.000

ND

ND

0.000

0.002

0.000

0.002

5560

ND

ND

0.000

0.000

0.000

0.000

Malathion

335.2

ND

1.11

0.000

0.000

0.000

0.000

Dimethoate

129.6

ND

ND

0.000

0.007

0.000

0.007

Coumaphos

14390

ND

ND

0.000

0.000

0.000

0.000

196

ND

ND

0.000

0.000

0.000

0.000

Dichlorvos

218.4

ND

ND

0.000

0.001

0.000

0.001

Fenamiphos

1870

ND

0.36

0.000

0.000

0.000

0.000

95

ND

6.85

0.000

0.000

0.006

0.006

67.76

32.72

31.04

0.039

0.039

0.037

0.037

110

ND

ND

0.000

0.003

0.000

0.003

251.2

ND

ND

0.000

0.001

0.000

0.001

0.04 25

0.1 10

0.04 25

0.1 10

OP

Diazinon

Phorate

Profenofos Chlorpyrifos Ch. methyl Fenthion

Sum Margin of Exposure (MOE)

0.6

Best case

Worst case

Summer

Spring

0.5 0.4 0.3 0.2 0.1 0 Spring

Summer

Comparison of HQs for lethality of bees from direct exposure or and dietary exposure to OPs .

 Samples collected in summer were more contaminated with OPs.  Pollen was most contaminated with OPs.  Profenofos, chlorpyrifos, malathion and diazinon were the most frequently detected OPs.  Coumaphos, the most common OP used directly on hives was not detected.  The OPs investigated

pose a minimal threat to bees in Egypt at

measured concentrations, higher-tier assessments (Tier II and Tier III)

were not indicated by the results of this study.

 Literature derived concentrations of OP insecticides daizinon, malathion, profenofos and chlorpyrifos in honey were compiled and were tested as a mixture at two different concentrations, the median and the 95th percentile for best and worst case to assess their effects on: 1.

Learning behavior and memory of bee foragers after 24 h exposure.

2.

Survival, AChE, detoxification and immunity genes transcripts of honey bee workers after 5 days exposure.

[1] Klein, A.M., Vaissie`re, B.E., Cane, J.H., Steffan-Dewenter, I., Cunningham, S.A., Kremen, C., Tscharntke, T., 2007. Importance of pollinators in changing landscapes for world crops. Proc. R. Soc. B 274,303–313 [2] The first international Forum for the Egyptian Beekeepers, 2009. (For the Best Future for Beekeeping Industry). http://pcela.rs/1st_forum_Egyptian.htm [3] http://www.beekeeping.com/articles/us/arab_countries.htm [4] Fairbrother, A., Purdy, J., Anderson, T., Fellk, R., 2014. Risks of Neonicotinoid Insecticides to Honeybees. Environ. Toxicol. Chem. 33(4), 719-731. [5] Hassan, A.R., 2009. “Proceedings of the 4th COLOSS Conference”. http://www.unep.org/dewa/Portals/67/pdf/Global_bee_colony_disorder_and_threats_insect_pollinators.pdf. [6] Mansour, S.A., 2004. Pesticides exposure- Egyptian scene. Toxicol. 198, 91–115 [7] Al Naggar, Y., Vogt, A., Codling, G., Naeim, E., Mona, M., Seif, A., Giesy, J (In press). Organophosphorus insecticides in honey, pollen and bees (Apis mellifera L.) and their potential hazard to bee colonies in Egypt. Ecotoxicology and Environmental Safety. [8] USEPA, 2012. White Paper in Support of the Proposed Risk Assessment Process for Bees. Office of Chemical Safety and Pollution Prevention, Office of Pesticide Programs, Environmental Fate and Effects Division, United States Environmental Protection Agency, Washington, DC.

Contact

PhD researcher, Toxicology center, Saskatchewan University 44 campus drive, Saskatoon, S7N 5B3 Phone: 3067156328 Assistant Lecturer Zoology Department, Faculty of Science Tanta University 31527 Egypt. E-mail:[email protected] :[email protected] https://www.researchgate.net/profile/Yahya_Al_Naggar https://usask.academia.edu/yahyaAlNaggar