Synergistic and Sublethal Effects of Pesticides on Honey Bees

<strong>Synergistic</strong> <strong>and</strong> <strong>Sublethal</strong> <strong>Effects</strong>
<strong>of</strong> <strong>Pesticides</strong> on Honey Bees
Pesticide/Pollinator Symposium
Alfred, NY

PSU; Pesticide
Investigating Group
PSU; Cox-Foster Lab
Roger Simonds <strong>and</strong>
staff
USDA-AMS National
Science Laboratory
Pesticide Database
Program (PDP)
CCD working team

Exposure to <strong>Pesticides</strong>

Samples Analyzed 2007-’08
• > 900 samples
• From
– CCD study
– Apple orchard study
– Migratory study
– Beekeeper submitted samples
• Cost-sharing program

Honey Bees - Excellent Indicator
<strong>of</strong> Environmental <strong>Pesticides</strong>
Acephate
Acetamiprid
Aldicarb sulfone
Aldicarb sulfoxide
Allethrin
Amicarbazone
Amitraz (2,4-DMA)
Amitraz (2,4-DMPF)
Atrazine
Azinophos-methyl
Azoxystrobin
Bendi ocarb
Bifenthrin
Boscalid
Captan
Carbaryl
Carbary l (1-Naphthol)
Carbendazim
Carb<strong>of</strong>uran , 3-hydroxy
Carfentrazone
Chlorfenapyr
Chlorothalonil
Chlorpyrifos
Coumaphos
Coumaphos -Chlorferone
Coumaphos oxon
Cyfluthrin
Cyhalothr in (total)
Cype rmethrin
Cyprodi nil
Deltamethrin
Diazinon
Dic<strong>of</strong>ol
Difenoconazole
Diflubenzuron
Dimethomorph
Diphenylamine
Endos ulfan 1
Endos ulfan 2
Endos ulfan sulfate
Esfenval erate
Etoxazole
Fenbuconazole
Fenhexamid
Fenpropath rin
Fluva linate
Heptac hlor
Hexachlorobenzene
Imida cloprid
Imida cloprid olefin
Imida cloprid, 5-hydroxy
Indoxacarb
Malathion
Methidathion
Methoxyfeno zide
Methyl par athion
Metolachlor
Metribuzin
Myclobuta nil
Norflurazon
Oxamyl
Oxyfluorfen
p,p' -DDE
Pendi methalin
Permethrin
Phosmet
Pyraclostrobin
Pyrethrins
Pyrimethanil
Quintozene (PCNB)
Sethoxydim
Simazine
Tebuconazole
Tebufenozide
Tebuthiuron
Tetramethrin
Thiabendazole
Thiacloprid
Tribufos
Trifloxystrobin
Trifluralin
Vinclozolin

Multiresidue Pesticide Analysis on US Wax,
Pollen <strong>and</strong> Bees Samples 2007-08
• Very few samples lacked detections
• Found 121 different pesticides <strong>and</strong> metabolites
pyrethroids, organophosphates, carbamates, neonicotinoids
insect growth regulators, organochlorines, chlorinated cyclodienes,
‣20 fungicides, 12 herbicides, 2 acaricides, 1 synergist,
‣At least 14 <strong>of</strong> these are systemic pesticides
• On average 6 different pesticides per pollen sample
- Up to 31 in a single sample

Selected <strong>Pesticides</strong> fond in WAX, POLLEN <strong>and</strong> BEES
Pesticide or
Metabolite
Class
LOD
Samples
Analyzed
Total
Detections
% <strong>of</strong>
Samples
Mean
(ppb)
SEM
(ppb)
Lowest
Value
Highest
Value
LD50
(ppb)
Carbaryl
PS
CARB 5 836 44 5.3 170.2 9.9 4.5 1010.0 10500
Coumaphos OP 1 887 668 75.3 1454.6 174.8 1.0 91900.0 46300
Fluvalina te PYR 1 887 749 84.4 2947.6 330.7 1.1 204000.0 15860
Imidacloprid NEO 2 836 14 1.7 77.5 5.9 2.4 646.0 280
Imidacloprid
olefin NEO 25 836 2 0.2 597.0 2.1 554.0 640.0 280
Imidacloprid,
5-hydroxy NEO 25 836 2 0.2 151.0 0.0 150.0 152.0 280
Chlorpyrifos OP 0.1 886 396 44.7 40.1 4.0 0.1 890.0 1220
Chlorothalonil FUNG 1 797 316 39.6 1860.4 286.3 1.0 98900.0 1110000

Selected <strong>Pesticides</strong> fond in WAX, POLLEN <strong>and</strong> BEES
Pesticide or
Metabolite
Class
LOD
Samples
Analyzed
Total
Detections
% <strong>of</strong>
Samples
Mean
(ppb)
SEM
(ppb)
Lowest
Value
Highest
Value
LD50
(ppb)
Carbaryl
PS
CARB 5 836 44 5.3 170.2 9.9 4.5 1010.0 10500
Coumaphos OP 1 887 668 75.3 1454.6 174.8 1.0 91900.0 46300
Fluvalina te PYR 1 887 749 84.4 2947.6 330.7 1.1 204000.0 15860
Imidacloprid NEO 2 836 14 1.7 77.5 5.9 2.4 646.0 280
Imidacloprid
olefin NEO 25 836 2 0.2 597.0 2.1 554.0 640.0 280
Imidacloprid,
5-hydroxy NEO 25 836 2 0.2 151.0 0.0 150.0 152.0 280
Chlorpyrifos OP 0.1 886 396 44.7 40.1 4.0 0.1 890.0 1220
Chlorothalonil FUNG 1 797 316 39.6 1860.4 286.3 1.0 98900.0 1110000

Selected <strong>Pesticides</strong> fond in WAX, POLLEN <strong>and</strong> BEES
Pesticide or
Metabolite
Class
LOD
Samples
Analyzed
Total
Detections
% <strong>of</strong>
Samples
Mean
(ppb)
SEM
(ppb)
Lowest
Value
Highest
Value
LD50
(ppb)
Carbaryl
PS
CARB 5 836 44 5.3 170.2 9.9 4.5 1010.0 10500
Coumaphos OP 1 887 668 75.3 1454.6 174.8 1.0 91900.0 46300
Fluvalina te PYR 1 887 749 84.4 2947.6 330.7 1.1 204000.0 15860
Imidacloprid NEO 2 836 14 1.7 77.5 5.9 2.4 646.0 280
Imidacloprid
olefin NEO 25 836 2 0.2 597.0 2.1 554.0 640.0 280
Imidacloprid,
5-hydroxy NEO 25 836 2 0.2 151.0 0.0 150.0 152.0 280
Chlorpyrifos OP 0.1 886 396 44.7 40.1 4.0 0.1 890.0 1220
Chlorothalonil FUNG 1 797 316 39.6 1860.4 286.3 1.0 98900.0 1110000

Selected <strong>Pesticides</strong> fond in WAX, POLLEN <strong>and</strong> BEES
Pesticide or
Metabolite
Class
LOD
Samples
Analyzed
Total
Detections
% <strong>of</strong>
Samples
Mean
(ppb)
SEM
(ppb)
Lowest
Value
Highest
Value
LD50
(ppb)
Carbaryl
PS
CARB 5 836 44 5.3 170.2 9.9 4.5 1010.0 10500
Coumaphos OP 1 887 668 75.3 1454.6 174.8 1.0 91900.0 46300
Fluvalina te PYR 1 887 749 84.4 2947.6 330.7 1.1 204000.0 15860
Imidacloprid NEO 2 836 14 1.7 77.5 5.9 2.4 646.0 280
Imidacloprid
olefin NEO 25 836 2 0.2 597.0 2.1 554.0 640.0 280
Imidacloprid,
5-hydroxy NEO 25 836 2 0.2 151.0 0.0 150.0 152.0 280
Chlorpyrifos OP 0.1 886 396 44.7 40.1 4.0 0.1 890.0 1220
Chlorothalonil FUNG 1 797 316 39.6 1860.4 286.3 1.0 98900.0 1110000

Selected <strong>Pesticides</strong> fond in WAX, POLLEN <strong>and</strong> BEES
Pesticide or
Metabolite
Class
LOD
Samples
Analyzed
Total
Detections
% <strong>of</strong>
Samples
Mean
(ppb)
SEM
(ppb)
Lowest
Value
Highest
Value
LD50
(ppb)
Carbaryl
PS
CARB 5 836 44 5.3 170.2 9.9 4.5 1010.0 10500
Coumaphos OP 1 887 668 75.3 1454.6 174.8 1.0 91900.0 46300
Fluvalina te PYR 1 887 749 84.4 2947.6 330.7 1.1 204000.0 15860
Imidacloprid NEO 2 836 14 1.7 77.5 5.9 2.4 646.0 280
Imidacloprid
olefin NEO 25 836 2 0.2 597.0 2.1 554.0 640.0 280
Imidacloprid,
5-hydroxy NEO 25 836 2 0.2 151.0 0.0 150.0 152.0 280
Chlorpyrifos OP 0.1 886 396 44.7 40.1 4.0 0.1 890.0 1220
Chlorothalonil FUNG 1 797 316 39.6 1860.4 286.3 1.0 98900.0 1110000

Outcomes <strong>of</strong> Honey Bee Pesticide Analysis
• No “commodity” has had as many detections at such high amounts in so few samples over
such a short time as has bee pollen
• Highest detections were in-house miticides, fluvalinate <strong>and</strong> coumaphos, but well over 100
other pesticides <strong>and</strong> metabolites found
• Pyrethroids dominate: known to impact foraging behavior,
• No individual chemical is likely to explain CCD
• Systemic or other fungicides occur at levels that may synergize with pyrethroids,
organophosphates or neonicotinoids.
• Role <strong>of</strong> pesticides <strong>and</strong> diseases like IAPV in CCD remains to be reconstituted in lab
bioassays at relevant doses
• Impacts <strong>of</strong> multiple pesticide residues in bee food most likely will be via synergistic
interactions at sublethal levels on key behaviors/physiology

Key Concerns
• Exposure (kinds, amounts, combinations)
• Systemic pesticides
• Fungicides
• Adjuvents/synergists
• Metabolites
• Labeling - lack <strong>of</strong> adequate warning

Herbicides
• Reduce forage
• Reduce nesting habitat

Insecticides
• Lethal/Toxic
– Depending on Class/Mode <strong>of</strong> Action
– Toxic effect (multiple pesticides) (Sara Ashcraft)
• <strong>Sublethal</strong>
– Behavioral effects
• Learning <strong>and</strong> retention (Daniel Schmehl)
• Foraging Behavior
– Physiological effects
• Immune system function (Diana Cox-Foster)
• Longevity
- Larval development
- adult behavior <strong>and</strong> physiology
• Acute vs Chronic Toxicity

Interactions (joint action) -
• similar (additive) - combining effects (same or similar target site)
• independent - no interaction (unrelated sites <strong>of</strong> action)
• synergistic - one chemical increases the biological activity <strong>of</strong> the
other (> sum <strong>of</strong> each effect)
• antagonistic - one chemical decreases the biological activity <strong>of</strong>
the other (< sum <strong>of</strong> each)

Adult Bee Toxicity Bioassay
5 day bioassays
Oral ingestion in sugar
syrup 24 hrs

Probit Units
Assail-Nova Probit at 24 Hours
10
8
6
4
Assail
Nova
Assail/Nova
2
0
0 1 2 3
Dose*
Dose*:
Assail 1: 10ppm
Nova 1: 50ppm
Assail/Nova 1: 5/25ppm
2: 100ppm
2: 500ppm
2: 50/250ppm
3: 200ppm
3: 1000ppm
3: 100/500ppm

Percent Mortality
Delayed Mortality Feeding on Pesticide at 95% Pollen Level
100
90
80
70
60
50
treatment
Solvent Control
40
30
20
10
0
1 2 3 4
Days Since Ingestion

Percent Mortality
Delayed Mortality Feeding on Pesticide at 95% Pollen Level
100
90
80
70
60
50
Solvent Control
Fluvalinate
Coumaphos
40
30
20
10
0
1 2 3 4
Days Since Ingestion

Percent Mortality
Delayed Mortality Feeding on Pesticide at 95% Pollen Level
100
90
80
70
60
50
40
Solvent Control
Fluvalinate
Coumaphos
Chlorpyrifos
30
20
10
0
1 2 3 4
Days Since Ingestion

Percen Mortality
Delayed Mortality Feeding on Pesticide at 95% Pollen Level
100
90
80
70
60
50
40
30
20
10
0
1 2 3 4
Days Since Ingestion
Fluvalinate/Coumaphos

Percent Mortality
Delayed Mortality Feeding on Pesticide at 95% Pollen Level
100
90
80
70
60
50
40
30
20
10
0
1 2 3 4
Days Since Ingestion
Fluvalinate/Coumaphos
Fluvalinate/Chlorothalonil

Percent Mortality
100
Delayed Mortality Feeding on Pesticide at 95% Pollen Level
90
80
70
60
50
40
30
20
10
0
1 2 3 4
Days Since Ingestion
Fluvalinate/Coumaphos Fluvalinate/Chlorothalonil Coumaphos/Chlorpyrifos

Percent Mortality
Delayed Mortality Feeding on Pesticide at 95% Pollen Level
100
90
80
70
60
50
40
30
20
10
0
1 2 3 4
Days Since Ingestion
Fluvalinate/Coumaphos
Fluvalinate/Chlorothalonil
Coumaphos/Chlorpyrifos
Fluvalinate/Coumaphos/Chlorpyrifos/Chlorothalonil

UDSA/PDP
• Honey added as a commodity to PDP
• 2007-2008
• 744 samples
• Processed <strong>and</strong> bottled
– May mitigate residues
found in raw honey

HONEY
Pesticide Name
# <strong>of</strong>
Samples
# <strong>of</strong>
Detects
% <strong>of</strong>
Detects
Min
Concen
Max
Concen
LOD
(PPB)
Coumaphos 744 263 35.3 1 8.2 1 100
Fluvalinate 744 88 11.8 1 14.5 1 50
2,4-dimethylphenyl formamide (2,4-DMPF) 744 81 10.9 4.1 85.1 4 1000
Dic<strong>of</strong>ol p,p' 744 35 4.7 1 90 1 NT
Piperonyl butoxide 744 10 1.3 6.3 9.6 6 NT
Vinclozolin 744 5 0.67 1.3 2.1 1 NT
Azoxystrobin 744 3 0.4 3.3 4 2 NT
Dicloran 744 3 0.4 1 1.6 1 NT
Carbendazim (MBC) 744 1 0.13 27.3 27.3 5 NT
Chlorpyrifos 744 1 0.13 1.3 1.3 1 NT
Methoxyfenozide 744 1 0.13 2.8 2.8 2 NT
Permethrin Total 744 1 0.13 11 11 10 NT
EPA
Tolerance

USDA
Pesticide Data Base Program
• http://www.ams.usda.gov/AMSv1.0/ams.fetchTemplateDat
a.do?template=TemplateC&navID=Science<strong>and</strong>Laboratorie
s&leftNav=Science<strong>and</strong>Laboratories&page=PesticideDataP
rogram&description=Pesticide%20Data%20Program&acct
=pestcddataprg

SUMMARY
• Honey (processed) for human
consumption contains few residues,
most below allowable EPA tolerance
• Pesticide residues in wax <strong>and</strong>
pollen in general are numerous <strong>and</strong>
some are at high levels, including
- cappings wax
- tapped pollen
http://www.nhb.org/
• Beekeepers should consider
having hive products tested,
especially pollen <strong>and</strong> wax, if selling
for human consumption

Recommendations
• Monitor <strong>and</strong> control varroa mites using “s<strong>of</strong>t” chemicals.
– formic acid (Mite-Away II®, Apiguard®, <strong>and</strong> Apilife var®.
• Reduce pathogen (<strong>and</strong> pesticide) build-up by regularly
culling old comb, recycling comb <strong>and</strong>/or irradiation <strong>of</strong> old
comb.
– Especially for dead-out colonies.
• Based on wide-spread resistance <strong>and</strong> these findings
fluvalinate is not recommended.
– Use <strong>of</strong> <strong>of</strong>f-label product should NOT be considered.

Recommendations
• If coumphos must be used, only the registered
product, CheckMite+® should be considered.
• Communicate with growers where bees are used for
pollination to minimize colony exposure to
agricultural-use pesticides.
• Monitor <strong>and</strong> control Nosema disease using fumagilin.

Pesticide Analysis
Cost-sharing Program
• Funding from PAm <strong>and</strong> Foundations for the
Preservation <strong>of</strong> Honey Bees
• Grant pays half the cost <strong>of</strong> analysis ($134) <strong>and</strong>
beekeepers pays half ($134)
• Results are confidential
– Data added to our over all data base
• To participate:
MAAREC.psu.edu <strong>and</strong>/or email: Mfrazier@psu.edu

Date 9/17/08 9/17/08
Compound name 75030 89543
Pollen Pollen Average* Range*
2,4 Dimethylphenyl formamide (DMPF) ND 40 54 42 -66
Chlorothalonil ND 13.1 534.1 1.1 -4200
Chlorpyrifos ND 6.7 51.5 0.1 -830
Coumaphos 193 8.1 249.9 2.4 -5828
Cyfluthrin ND 6 4.5 4.5
Fenpropathrin 9 9.2
Fluvalinate 13.6 ND 139.6 2.4 -2670
Pendimethalin 11.656 ND 9.4 2.8 -16

FUNDING SOURCES
‣ National Honey Board
‣USDA
‣ Haagen-Dazs
‣Wyman’s Growers
‣Penn State University College <strong>of</strong> Agricultural Sciences
‣PA Department <strong>of</strong> Agriculture
‣ PAm
‣ Foundation for the Preservation <strong>of</strong> Honey Bees
‣ Florida Department <strong>of</strong> Agriculture
‣ NC508 Sustainable Solutions to Problems Affecting Honey Bee Health
‣Beekeeping Associations (FL state <strong>and</strong> Tampa Bay)

MAAREC.psu.edu