Assessment of Hazard of Organophosphorus Pesticides to Honey bees (Apis mellifera L.) in Saskatoon,
Saskatchewan, Canada
Al Naggar Yahya1,2, Vogt Anja2, Codling Garry2, Naiem Elsaied1, Mona Mohamed1, Seif Amal1, Albert Robertson4, Giesy P. John2, 3
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.4Meadow Ridge Enterprises LTD, Saskatoon,
Saskatchewan, S7K 3J9.Canada.
Background
Results
Methods
Colony Collapse Disorder (CCD) poses a serious threat to apiculture and agriculture
Over the winter of 2012–2013, the average loss of honey bee colonies across Canada
was 28.6% and the average rate of loss in Saskatchewan was 27%.
chlorpyrifos
Quant.
ion
Although the putative causes of CCD are still unknown and currently being studied,
results of some studies have suggested that extensive use of insecticides might be
Chlropyrifos
Confirm.
ion
responsible for increased rates of CCD.
ND
ND
ND
0.42
Dicrotophos
ND*
ND
ND
ND
ND
ND
Ethoprop
ND
ND
ND
ND
1
1.36
Malathion
ND
ND
ND
ND
1
3.74
Dimethoate
1
1.5
ND
ND
ND
ND
Coumaphos
ND
ND
ND
ND
ND
ND
Phorate
ND
ND
ND
ND
ND
ND
Dichlorvos
ND
ND
ND
ND
1
899.19
Fenamiphos
ND
ND
4
0.29
ND
ND
Profenofos
ND
ND
ND
ND
ND
ND
Chlorpyrifos
ND
ND
3
2.69
ND
ND
Ch. methyle
ND
ND
5
15.82
ND
ND
Ch. oxon
2
0.21
ND
ND
ND
ND
Fenthion
ND
ND
ND
ND
ND
ND
called for, and in particular the need to better understand the response of pollinator
communities to complex environmental stressors.
Objectives:
Extraction procedures:
i. Optimize and validate an extraction method for 14 OP insecticide in honey, pollen and bees
using liquid chromatography mass spectrometry (LC-MS/MS).
ii. Assess the potential risks to Saskatchewan beehives from exposure to OPs pesticides.
Spike with 100 µL of
Dimethoate d6 (PCS)
Methods
Salting-out
pesticide
Shaking (1 min)
Centrifugation
(4000 rpm, 10 min)
Hazard quotients (HQs) calculated based
on ratios of estimated exposure by oral
uptake of OPs-contaminated nectar and
pollen only.
(SPE with C18 column)
B. Worst case
•HQs based on lethality of bees exposed to OPs in honey and pollen consumed by bees
was estimated (Equation 1):
HQs oral = Total daily intake (TDI) of honey or pollen / Acute oral LD50
Diazinon
Dicrotophos
Ethoprop
Malathion
Dimethoate
Coumaphos
Phorate
Dichlorvos
Fenamiphos
Profenofos
Chlorpyrifos
Ch- methyle
Fenthion
Hazard quotients (HQs) calculated based on
ratios of estimated exposure by oral uptake of
OPs-contaminated nectar and pollen and on
limit of detections (LODs) of OPs that never
detected in honey and pollen .
•Total daily intake (TDI) of pesticides received by bees via food was calculated based on
total food the consumption rate of nectar and pollen for adult worker bees (292 mg d-1 ).
TDI
(ng. bee-1
day-1)
Worst case
(Honey)
TDI
(ng. bee-1
day-1)
Best case
(pollen)
TDI
(ng. bee-1
day-1)
Worst case
(Pollen)
HQs
honey &
pollen
( best case)
Clean-up
with PSA
LC-MS/MS analysis
Figure 2. Sample preparation strategy based on the modified QUECHERS method.
Acknowledgments:
HQs
honey &
*MOE (best
MOE (worst
pollen
case)
case)
( worst case)
Ref.LD50
(ng. bee-1)
2.1
1.72
70.625
4.19
1.62
179.875
2.45
2.73
23.375
1.1875
0.847
1.375
168
137.6
5560
335.2
129.6
14390
196
218.4
1870
95
67.76
110
0.073
0
0
0
0.438
0
0
0
0
0
0
0
0.073
2.1024
0.09052
0.12556
0.438
0.12556
0.0146
6.28968
0.03504
0.09052
0.04088
0.8176
0
0
0
0
0
0
0
0
0.0876
0
0.78548
4.61944
0.02044
1.2556
0.16644
0.219
3.5624
0.13432
0.584
0.15768
0.0876
0.33288
0.78548
4.61944
0.0004
0.0000
0.0000
0.0000
0.0034
0.0000
0.0000
0.0000
0.0000
0.0000
0.0116
0.0420
2301.4
0.0
0.0
0.0
295.9
0.0
0.0
0.0
21347.0
0.0
86.3
23.8
0.001
0.024
0.000
0.001
0.031
0.000
0.003
0.030
0.000
0.004
0.012
0.049
1797.9
41.0
21637.6
972.8
32.4
55371.7
327.4
33.9
15247.9
224.4
82.0
20.2
3.14
251.2
0
0.42632
0
1.168
0.0000
0.0
0.006
157.6
0.1
24054
0.2
95947
*MOE, margin of exposure
Concentration step by Liquid N2
(1)
Figure 4. Percentages (%) of positive samples for
OPs pesticides detected in honey, pollen and
honey bees.
TDI
(ng. bee-1
day-1)
Best case
(Honey)
Total
Centrifugation
and filtration
0
Ref. LD50
(µg. g-1)
Add 6 g MgSO4 + 1.5 g NaAc
Clean-up
A. Best case
5
Table 2. Tier-1 Hazard quotients (HQs) for lethality of bees exposed to OPs in honey and pollen consumed by bees
in Saskatchewan, Canada during summer 2013.
Add 27 mL of extraction solution (44% deionized
water, 55% acetonitrile, and 1% glacial acetic acid.
Tier-1 screening-level
assessments
10
*ND, none detected
Extraction
•Samples of honey, pollen and bees were collected in summer of the year 2013 from seven
independent apiaries that represent seven different locations in east and north east of Saskatoon in
the Aberdeen and Blucher municipalities. (Figure 1)
•LC/MS-MS was used to identify and quantify 14 OP insecticide in 67 samples of bees, honey and
pollen by use of a modified (QUECHERS) method, the procedure outlined in Figure2.
•The hazard characterization scheme applied was that used by the USEPA Office of Chemical Safety
and Pollution Prevention for assessing risks of foliar sprayed pesticides to pollinators .
•The scheme incorporates :
Honey, pollen
and bees
Weigh 3g of matrix in 50 mL centrifuge tube
15
Fenthion
0.25
Ch. oxon
1
Ch. methyle
Diazinon
20
Chlorpyrifos
Recently the need for further research on the drivers of the ‘pollinator crisis’ was
Bees
Profenofos
Saskatchewan.
Pesticide
Fenamiphos
Honey bees (n=16)
No. of
positive mean conc.
samples
Dichlorovos
Pollen (N=21)
No. of
positive
mean conc.
samples
Phorate
Honey (n=30)
No. of
positive
mean conc.
samples
Pollen
25
Coumaphos
(Lorsban 4E) and dimethoate (Cygon) were implicated in lethality of bees in
Honey
Dimethoate
There are some reports of incidents in 2012 for which OPs including chlorpyrifos
Table 1. Organophosphorous pesticides (ng/g, wm) detected
in honey, pollen and honey bees collected from
Saskatchewan, Canada in summer 2013.
Malathion
pesticide poisoning; during the pollination of agricultural crops.
Figure1. Sampling sites of beehives represented with red triangles, Saskatoon, Saskatchewan,
Canada.
Diazinon
Dicrotophos
Ethoprop
Honeybees (Apis mellifera) are subjected to an intensive and continuous hazard from
Figure 3. Extracted ion chromatograms (EIC) of OPs standers at 40 ng/ml (left) and a pollen sample
contaminated with chlorpyrifos (right).
Proportion of positive samples (%)
Organophosphates (OPs) are a class of insecticides, which are highly toxic to bees.
Conclusion :
Concentrations of 14 OP pesticides were measured in bees, honey and pollen collected from
different locations near Saskatoon, Saskatchewan, Canada during summer 2013.
Cumulative potential for adverse effects of dietary exposure to OPs insecticides was determined.
Of the OP insecticides studied, fenamiphos, chlorpyrifos, its active metabolite CYPO and
chlorpyrifos methyl are the most frequently detected OPs.
Bee bread (stored pollen) was the most contaminated while; honey was the less contaminated with
OP.
 HQs based on lethality of bees exposed to OPs in the diet via consumption of honey and pollen
were less than the level of concern (0.4), which suggested minimal potential lethality from dietary
exposure bee hives in Saskatchewan to OPs. However, one uncertainty is the lack of information
on cumulative effects of the various OP insecticides on immune function and bees behavior.