1. Leaves were sprayed with the pesticides dinotefuran or Safari and honey bees were exposed to the leaves periodically over several weeks. The bees and leaves were then analyzed using LC-MS/MS to measure the amount of pesticide residues. 2. Safari resulted in higher pesticide residues on the bees compared to dinotefuran alone after the same exposure period, and caused bee mortality for a longer time. 3. When bees were immediately exposed to freshly treated leaves, dinotefuran caused more rapid bee death, but Safari residues persisted longer on the bees over time.

1. Sample Preparation Method
Leaves were sprayed with dinotefuran or Safari and left outside to be exposed to the elements. Periodically, a set of leaves
were picked and honey bees were exposed for 24 hours. To extract dinotefuran or Safari on the exterior of bees in preparation
for analysis, 5 bees were placed in 5 mL of distilled water. 1 µg/mL of d3-dinotefuran (Toronto Research Chemicals, Toronto,
Canada) was included as an internal standard. The bees were vortexed using a Multi-Tube Holder attached to a Vortex Genie 2
(Scientific Industries, Bohema, NY) for 4 minutes at maximum power. 1 mL of the water was transferred to a 1.5 mL plastic
tube, which was vortexed at 5,000 rcf for 5 minutes. 100 µL of the supernatant was transferred to a 300 µL polypropylene
autosampler vial (MicroSolv Technology Corp., Eatontown, NJ).
LC-MS/MS Method
• Instrumentation: AB Sciex 3200 QTRAP Mass Spectrometer (Foster City, CA), Shimadzu Prominence Series HPLC (Columbia, MD)
• LC Column: GL Sciences (Tokyo, Japan) Inertsil Phenyl 3 (4.6 mm x 150 mm x 5 µm) with Opti-Solv 2 micron guard column
(Optimize Technologies - Portland, OR)
• Internal Standard: d3-dinotefuran
• Mass Spec Conditions: Multiple reaction monitoring (MRM) masses: dinotefuran m/z 203>129, m/z 203>114
d3-dinotefuran m/z 206>132, m/z 206>117
• Mobile Phase: Solvent A: H2O with 10 mM ammonium formate and 0.1% formic acid
Solvent B: Acetonitrile with 0.1% formic acid
Results
Future Work
• Investigation into additional exposure pathways that may
be facilitated by nanotechnology based neonicotinoids.
• Study more pesticides and NBPs that may pose risks to
honey bees.
Mass Spectrometry-based Detection of Neonicotinoids on Honey Bees
Trevor Shear†, Jeff Morré†, Louisa Hooven‡
† Department of Chemistry, Oregon State University, Corvallis, Oregon 97331
‡ Department of Horticulture, Oregon State University, Corvallis, Oregon 97331
D ays
%Mortality
5 1 0 1 5 2 1 2 6 3 1 3 6
0
2 5
5 0
7 5
1 0 0
W a te r
D in o te fu ra n
S a fa ri
Figure 3. Concentrations of Safari and dinotefuran on honey bees after 24 hours of exposure
to treated leaves. Leaves were weathered from 5 to 39 days prior to bee exposure. Results
are from three samples of five bees at four time intervals, analyzed by LC-MS/MS.
Figure 4. Residual toxicity of Safari compared to dinotefuran. Honey bees were exposed to
treated leaves for 24 hours and mortality counted. The results are from ten time points with
up to six replicates, consisting of up to 30 bees at each time point.
Figure 5. Concentration of Safari and dinotefuran on honey bees after immediate exposure
to treated leaves. After one hour of exposure, six samples of five bees each were analyzed
with LC-MS/MS.
0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5
0
2 5
5 0
7 5
1 0 0
H o u r s
Percentsurvival
D in o te fu ra n
S a fa r i
C o n tro l
Figure 6. Toxicity of Safari and dinotefuran after leaves were sprayed and bees were
immediately introduced. Dead or dying bees were then counted at half hour intervals.
Introduction
With increasing use of new pesticide formulations worldwide to repel and kill unwanted organisms, negative consequences on
non-target species such as honey bees and other pollinators are of increasing concern and not well studied. This study
compares the nanotechnology based pesticide (NBP) Safari and its base neonicotinoid, dinotefuran, and their effect on bee
death using liquid chromatography tandem mass spectrometry (LC-MS/MS). This research examines whether nanotechnology
based pesticides, with particles smaller than pollen, can transfer more readily to the bees than the active ingredient alone and
does the NBP have a longer residual toxicity to bees. This transfer can occur in the same manner that pollen is transferred
from flowers to bees, or the particles may cling to pollen which is then collected by bees and returned to the hive (Figure 1).
What are Nanotechnology Based Pesticides (NBPs)?
Particulate pesticide formulations are already on the market containing engineered particles in the nano to micro size scale
and are commonly applied in agricultural environments. The pesticide active ingredient may be; a) adsorbed onto the particle,
b) attached to a particle via ligands, c) encapsulated by or d) entrapped within a polymer matrix (Figure 2). When the
pesticides active ingredients are associated with particles, their behavior in the environment changes.
Figure 2. On the left, a schematic representation of different nanodevices for delivery of pesticides, fertilizers or nucleic acids (a) adsorption on nanoparticle;
(b) attachment on nanoparticle mediated by different ligands; (c) encapsulation in nanoparticulate polymeric shell; (d) entrapment in polymeric nanoparticle.
Ghormande et al, Biotechnology Advances 29 (2011) 792–803. On the right, a scanning electron microscopy image of the nanotechnology based pesticide,
Safari.
Safari is an NBP formulated from the neonicotinoid dinotefuran and other proprietary ingredients. The dinotefuran in Safari is
associated with irregular particles that vary in size from nanometers to micrometers (Figure 2). Safari has been associated
with bumble bee death.
Figure 1. Bees are perfectly designed to attract and collect particles such as
pollen. In addition to actively collecting pollen, positively charged bees induce a
charge in flowers, which are already negatively charged. This facilitates the
transfer of pollen to bees, which are covered with hairs that trap and hold the
pollen. Bees may inadvertently collect many other types of particles, some of
which have potential adverse effects.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Time,min
0.0e0
100%
Intensity
11.31
60 70 80 90 100 110 120 130 140 150 160 170 180 190 200
Mass/Charge,Da
129.2
203.2
114.2
87.1
157.2
100.173.1
Figure 7. LC-MS/MS spectrum showing retention time and fragmentation pattern of
dinotefuran. Limit of quantitation was 0.01 µg/mL.
MRM quantification ion
m/z 203.2>129.2
MRM qualification ion
m/z 203.2>114.2
dinotefuran parent mass
m/z 203.2 (M+H)
Acknowledgement
• OSU Research Office
• USDA NIFA Nanotechnology
• Oregon State University General Research Fund
• Agricultural Research Foundation
• National Honey Board
• The OSU Mass Spectrometry Center, Dr. Claudia Maier
• Montana Miller
Conclusion
Utilizing LC-MS/MS, we investigated whether Safari,
which is a particulate formulation made from the
neonicotinoid dinotefuran and other ingredients, poses
more risk to bees than dinotefuran alone. Compared to
dinotefuran, it is clear that Safari persists longer in the
environment. Safari can then transfer from foliage to
bees, and its residues remain lethal to bees for a longer
period of time. Interestingly, when bees are exposed to
leaves immediately after treatment with Safari or
dinotefuran, the bees exposed to dinotefuran die more
quickly. We are continuing to investigate how the
particulate formulation of Safari plays a role in these
differences.
0.00
0.10
0.20
0.30
0.40
5 8 12 39
Concentration(µg/ml)
Exposure Length of Leaves (Days)
Concentration after 24 hours of
exposure
Dinotefuran
Safari
0.000
0.020
0.040
0.060
0.080
0.100
0.120
1
Concentration(µg/ml)
Averaged Sample
Concentration after initial exposure
Dinotefuran
Safari