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Tracing the Migration of Pesticidesthrough the Production of Southwestern
Connecticut Honey
Eliza McNittGreenwich High School
March 2008
INTEL International Science and Engineering Fair, Grand Award,Environmental Management
Connecticut Science Fair, Overall Winner, Life Sciences
Connecticut Junior Science and Humanities Symposium, Overall Winner
Audubon Award for Environmental Science Research
Pfizer Life Sciences Research Award
MIT Lincoln Laboratory Award
Connecticut Academy of Science Research Award
Connecticut United for Research Excellence (C.U.R.E.)Award for Excellence in Scientific Research
Science Education Center of Fairfield County, Marianne Smith Award for Initiative in Science
Greenwich Science Center,Jose Manual Otero Award for Outstanding Research
2
Table of Contents
Table of Contents 2
Abstract 3
Introduction 4
Purpose 5
Hypothesis 5
Materials 6
Methods 6
Conclusion 20
Further Research 20
Acknowledgments 20
Works Consulted 21
3
Abstract:
Continued use of pesticides and herbicides throughout both the agricultural and
private sector has led to concern about the effects of these chemicals on common fruits
and vegetables, and whether these often harmful chemicals are still present when the food
is consumed. Most research has focused on the growth and retail of typical agricultural
products, however little work has been done to identify the presence of residual pesticides
in the honey of Apis mellifera (the typical honey bee). This investigation seeks to verify
the migration of five known pesticides, applied within a 30 mile radius within the Bartlett
Arboretum (in Stamford, CT), to the honey product of a controlled bee-hive located
within the Arboretum grounds. The five pesticides applied within the Arboretum are
Dipel, Neem Oil, Insecticidal Soap, Imidacloprid, and Pyrethrins. Initial experiments
based on ultrasonic-solvent extraction of honey, and the analysis of the organic layer
against known samples of the five pesticides, with Attenuated Total Reflectance (ATR)
Fourier Transform Spectroscopy (FTIR), proved to be non-specific and lacked sufficient
sensitivity since all pesticides, and the neat honey samples exhibited an asymmetric C-O
stretch at 1058 cm–1. High Pressure Liquid Chromatography based analysis, with UV
detection at 255 nm, indicates that components of Pyrethrins and Imidacloprid are not
detectable in the organic honey extract. This same analysis points out, however, that
components of BioNeem Oil are present in the final product of the Arboretum 2007 Fall
Honey.
4
Introduction:
The use of pesticides is an essential aspect of modern agriculture. These pesticides
can be used to control insects, weed growth, plant diseases, along with worm and rodent
infestations. Fruit and flower growers in the state of Connecticut use pesticides primarily
to control insect infestation. Although pesticides are very effective in controlling the
population growth of unwanted insects, they are usually non-toxic to honeybees. In part,
the lack of toxicity to honeybees can be attributed to the timing of the pesticide
application. Farmers methodically spray their crops at night, cover or even move
beehives to minimize the direct contact with honeybee populations until the pesticides
have become less hazardous. The pesticides remains on the plant surfaces, and although
the pesticide does not kill the honeybee, those that feed on the nectar and pollen from
these treated flowers can easily carry the residues into the hive and introduce the
pesticide into the honey that is produced.
Establishing a working relationship with the Bartlett Arboretum in Stamford, CT, a
wildlife preserve possessing 30 acres of gardens and woodland, I was fortunate enough to
have the opportunity to monitor the production of honey from a specific beehive located
on the arboretum grounds.
Figure 1. The location of the beehives on the Arboretum Grounds
5
The honeybees within the centrally located controlled hive are known to feed off of
flowers in a five-mile radius surrounding the arboretum according to Mr. James Kaechel
(the Director of Education, and bee specialist) and Mr. Andrew Coté (the Stamford
Arboretum Beekeeper). The pesticides used within this five mile radius are limited to
Dipel, Neem Oil, Insecticidal Soap, Imidacloprid, and Pyrethrins, the most hazardous of
the pesticides used on the grounds. With the Assistance of Mr. James Kaechel and Mr.
Andrew Coté I collected samples of fall honey harvest from the controlled beehive and
located samples of the five commercial-grade pesticides, used in the neighboring
locations where the bees feed.
Purpose:
The investigation of the subsequent transmission/migration of applied pesticides
into the final honey product.
Hypothesis:
In a controlled environment, where the applied pesticides in a five-mile radius are
known, and the bee colony feeding is limited to these same locations, trace levels of
pesticides will be found in the final honey product.
6
Materials:
• Pesticides obtained from Bartlett Arboretum including:
o Dipel
o Neem Oil
o Insecticidal Soap
• Imidacloprid Standard (Riedel-de Haën Item 37894)
• Pyrethrins extracted from chrysanthemum daisies
• Raw Honey obtained from Bartlett Arboretum
• Pipettes
• Perkin Elmer Spectrum 1 FTIR With A Diamond ATR Sampling Accessory
• Water Module 1 HPLC System with Acetonitrile & Water as the Mobile Phase
Methods:
The initial procedure involved the characterization of the pesticides used at the
Bartlett Arboretum using Diamond Attenuated Total Reflectance (ATR) Fourier
Transform Infrared (FTIR) Spectroscopy at the Greenwich High School Labs.
Figure 2. ATR FTIR spectrometer
This technology helps to understand the interactions between the different atoms within a
molecule and produces a spectral profile of the specimens being examined collecting high
7
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quality data within a minute or less. The procedure began by sampling the five pesticides
with the Diamond ATR FTIR by placing liquids on the metal plate and isolating solids
with the high pressure clamp, providing close contact between the sample and the ATR
crystal.
Figure 3. ATR FTIR Spectrum Insect Killing Soap Figure 4. ATR FTIR Spectrum of BiNeem Oil
Figure 5. ATR FTIR Spectrum of Garden Dust Dipel Figure 6. ATR FTIR Spectrum of Pyretherin
The ATR FTIR spectra of the five
pesticides all exhibited a peak at 1058
cm-1, which is attributed to an
asymmetric C-O stretch within a
molecule contained in the pesticide.
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Figure 7. ATR FTIR Spectrum of Imidacloprid Standard
8
Figure 8. All Pesticides Share a C-O stretch at 1058 cm-1
Using the Ultrasonic Extraction of Pesticide from honey, a commercial honey
product was spiked with Dipel pesticide to check the detection of the pesticides in
honey. 1.0 grams of honey were dissolved in 1.25 grams water mixed with 2.5 grams of
Heptane. An ultrasonic mixer was used to combine the two layers for 60 minutes at
room temperature. This distinguished between the water soluble, hydrophilic
components that stayed in the aqueous layer and the non-polar, hydropholic components
that migrated into the Heptane layer.
Figure 9. The arrows indicate the presence of the heptane peaks
Taking an ATR FTIR Spectra of Honey Extract (Wet Heptane Layer) and Dry Film after
evaporation, demonstrated after the evaporation of the Heptane layer pesticides were
undetectable via ATR FTIR. Therefore it was concluded that the FTIR methodology
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Heptane Peaks
9
lacked the sensitivity required to distinguish between the components in the scans by
looking at the Heptane layer.
Analyzing a sample of “neat” Arboretum Honey, the FTIR indicated that the raw
honey also produced a C-O asymmetric stretch at 1058 cm-1.
Figure 10. A “Neat” Sample of Arboretum Honey
This peak coincided with a peak in all other FTIR of the pesticides used
on/around the Arboretum and concluded that the FTIR technique was non-selective.
This specified that it was not possible to identify any of the pesticides independent of
one another.
Deciding to utilize a new approach, a more careful
investigation of the presence of Pyretherins was chosen because
they are the most hazardous of the five pesticides. Pyretherin is a
pesticide commonly found in low quantities in foggers and garden sprays. Use of
Pyretherins from one of these foggers or sprays, would introduce many other
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unidentifiable compounds that would complicate the analysis. Therefore, to create pure
Pyretherin, the pesticide was extracted directly from Chyrsanthemums (i.e. the
pyrethrum daisy) flower heads using isopropyl alcohol (91%). Leaving the solution
overnight, the flower heads excreted the natural pesticide.
Previous literature by Kasaj, D., et al suggests that Pyrethrins can be detected via
HPLC (High Pressure Liquid Chromatography). All HPLC work was performed in the
labs at Greenwich High School.
My new approach involves the
examination of the Heptane-layer of the
Arboretum Honey extract, and the comparison of
it to the HPLC of the chrysanthemum Pyrethrin
standard. A new possible method was devised to
extract the Pyrethrins from honey by taking 1.5 g
of Chrysanthemum Heads in 20 ml IPA. 10 ml of
Pyretherin/IPA Extract with 10 ml was mixed
with 1:1 Methanol:Water and loaded onto a C18
Prep Column.
Figure 11. HPLC Chromatography at GHS Labs
The function of the column is to separate the water soluble substances from the
organic components that are hydrophobic. After the Pyrethrins are passed onto a C18
cartridge, a 5 ml MeOH/Water wash is used to remove any remaining water soluble
components and 10 ml of Heptane is used to remove the Pyrethrins from the C18 Prep
Column.
11
Figure 12. Pyretherin Extraction & Purification from Chyrsanthemum
The UV/Vis Spectrum of the Pyrethrin (in Heptane) showed significant
absorbance in the UV region. 255 nm was selected as the wavelength of detection for
HPLC analysis.
Figure 13. UV/Vis Detection of Pyrethrins
The Gradient HPLC method outlined by Kasaj provided poor separation for the
Pyrethrins extracted from the chrysanthemum daisy. A modification of this gradient
HPLC method outlined by Kasaj, et al, provided the best separation of the Pyretherin
isomers:
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Table 1: Gradient mobile phase parameters
Time (min) % Acetonitrile % Water0 58 425 58 42
35 75 2546 100 0
47 - 60 58 42
Additional HPLC parameters include:
• Column Type: Agilent Zorbax RX-C18, 250 x 4.6 mm
• Flow Rate: 1 ml/min
• Temperature: 23˚
• Wavelength: 255 nm
The HPLC chromatogram of the Pyrethrin Heptane extract and the Heptane
(solvent) blank uncovered the presence of a Pyretherin II peak, a solvent peak, and a
Pyretherin I peak. The chromatogram of the Heptane blank was collected to verify any
contributions of the solvent to the Pyrethrum chromatogram. The Heptane blank contains
a peak at a retention time of 9.2 minutes, so this peak can be assigned as a blank
component in the Pyrethrin chromatogram as well. Using previous chromatography by
Kasaj D., et al. Pyrethrin II and Pyretherin I were assigned retention times of 8.4 mm and
9.65 minutes respectively.
13
Figure 15. Chromatogram of Pyretherin Heptane Extract
Figure 16.Chromatogram of Heptane (solvent) Blank
A similar method to extract possible Pyrethrins from honey was devised. 5 grams
of a honey sample were mixed with 10 ml of 1:1 Methanol:Water. The organic
components Methanol/Water mix is loaded onto a C18 prep column. Then after a 5 ml
MeOH/Water wash, the organic components are later removed from the C18 Prep Column
with 5 ml of Heptane.
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Pyretherin II
Solvent Peak
Pyretherin I
Solvent Peak
Figure 17. Extraction of Organic Components from Honey
The HPLC chromatogram of the Honey Organics Extract were taken in Heptane
was collected and compared to the Chromatograms of Pyretherins I & II, and the Heptane
(solvent) blank.
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Chromatogram of Honey Organics Extract
Chromatogram of Pyrethrins I & II
Heptane Solvent Blank
The comparison of the chromatograms revealed the presence of a new peak in the
honey organics extract that did not correlate with the other chromatograms.
Figure 19. Overlay of Chromatograms of Pyretherin with Honey Extract & Solvent Blank
Of the five pesticides that were used at the Arboretum, Dipel and Insecticidal
Soap are both water soluble and could not be detected with our new C18 Heptane
Cartridge/Solvent Extraction method. Imidacloprid and Bio Neem Oil, however, both
cointain pesticides that are hydrophobic and would elute with Heptane, so that I could
compare their chromatography with that of the Arboretum honey organics extract.
Imidacloprid was used to replaced its counterpart, Diazanon, a more powerful and
effective pesticide used on field crops and around the home. With the recent ban of
Diazinon, the use of Imidacloprid rose significantly amongst crop growers. It has been
4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5
Unknown From Honey
Pyrethrin II
Solvent Peak
Pyrethrin I
16
theorized that perhaps this pesticide may be responsible for instigating Colony Collapse
Disorder (or CCD), a little-understood phenomenon in which worker bees from a beehive
abruptly disappear. CCD was originally termed in 2006, when nearly a quarter of U.S.
honeybee colonies disappeared within a few months. The increased use of Imidacloprid
coincides with the onset of CCD and many suggest that the pesticide may play a role in
CCD. The presence of Imidacloprid in honey would define the pesticides pathway I the
honey production, and shed light on its role in CCD. Imidacloprid is also harmful to
humans by ingestion or inhalation.
A UV/Vis scan of the Riedel-de Haën Imidacloprid standard (in Heptane) shows
an absorbance at 255 nm so Imidacloprid would be detectable with our current HPLC
method.
Figure 20. UV/Vis Spectrum of Imidacloprid
A chromatogram of the Imidacloprid was taken (in Heptane) revealed that the
Imidacloprid eluted at a retention time of 52 minutes using the same HPLC method
parameters.
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Figure 21. Chromatogram of Imidiclorpid with Overlay of Honey Extract
An overlay of the honey organics extract chromatogram (see figure above) with
that of the Imidacloprid reveals that the Arboretum honey does not contain Imidacloprid.
A similar method to extract organic components from commercial BioNeem Oil
pesticide was devised.
Figure 21. Extraction of Organic Components from Bio Neem Oil
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Unknown fromHoney
Solvent Peak
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Bio Neem oil contains the pesticide Azadirachtin and also exhibits absorbance in
the UV region at 255 nm.
Figure 22. UV/Vis Spectrum of BioNeem Oil – Heptane Extract
As such, the same HPLC methodology can be used to detect the organic
components of BioNeem in Heptane, after separation from any hydrophilic components,
using the same C-18 cartridge/Heptane Extraction technique. The chromatogram for the
organic components of BioNeem oil in shown below:
Figure 23. BioNeem Oil Chromatography
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19
The Chromatogram of the BioNeem oil revealed the Heptane (blank) peak, as well as
four other components with retention times of 6.25 min., 11.6 min., 16.4 min., and 17.4
minutes.
Figure 23. Neem Oil Chromotography vs. Honey Organics Extract
Figure 24. Comparison of BioNeem Oil Chromatogram and Honey ExtractChromatogram
The peak at a retention time of 6.25 min. coincides with the unknown component in the
honey extract indicating that the Arboretum Honey final product does contain a
component of Commercial BioNeem Oil pesticide.
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BioNeem Oilin Heptane
Honey Extractin Heptane
20
Figure 25. Commercial Honey Analysis
Preliminary analysis of commercial honey samples also reveal the presence of this same
Bio Neem Oil component at 6.25 minutes, as well as additional unknown components
with retention times of 2.85 and 3.95 minutes.
Conclusions:
Results from High Pressure Liquid Chromatography analysis of Bartlett
Arboretum Honey support my original hypothesis; a component of Bio Neem Oil is
present in the honey final product of the Bartlett Arboretum 2007 fall harvest. HPLC also
uncovered that Pyretherins and Imidacloprid pesticides are not detectable in the same
honey final product. The absence of the Imidacloprd from the Honey could support the
linkage of the pesticide to CCD, where worker bees are killed before returning to the
hive. The limited analysis of Commercial Honey Products of unknown geographical
21
origin also reveals the presence of the Bio Neem Oil component, as well as additional
organic compounds.
Future Work:
Additional research will include the continuation of the HPLC analysis of
commercially available honey samples to screen for the Bio Neem component with a 6
min. retention time. A hyphenated Liquid Chromatography technique, such as LC-MS,
will be used to identify this organic component of Bio Neem Oil. Finally, the HPLC
Analysis will be extended to the water soluble pesticides sprayed at the Arboretum (Dipel
& Insect Killing Soap).
Acknowledgements:
I would like to thank Mr. James Keachel, and Mr. Andrew Cote, at of the Bartlett
Arboretum, in Stamford, CT, for their assistance in obtaining Fall Harvest Honey
samples, and for providing the names/samples of pesticides used within the Bartlett
Arboretum. I would also like to thank my mentor, Mr. Andrew Bramante, at Greenwich
High School, for his guidance and support during my research and The Science
Education Center, of Fairfield County. CT, for their financial assistance.
22
Works Consulted
1. Kasaj, D., Rieder, A., Krenn, L., Kropp, B., “Separation and Quantitative Analysisof Natural Pyrethrins by High Pressure Liquid Chromatography.”Chromatographia, 50 (1999): 607-610
2. McBride, Dean K. "Protecting Honeybees From Pesticides." NDSU. June 1990.NDSU. 5 Oct. 2007 http://www.ag.ndsu.edu/pubs/plantsci/pests/e494w.htm.
3. Moreira, Naila. "Pesticides Makes Bees Bumble." Science News 167 (2005): 350-351. Proquest. 31 Aug. 2007.
4. Pirard, C., J. Wildart, B. K. Nguyen, C. Deleuze, L. Heudt, E. Haubruge, E. DePauw, and J. F. Focant. "Development and Validation of a Multi-Residue Methodfor Pesticide Determination in Honey Using on-Column Liquid-Liquid Extractionand Liquid Chromatography-- Tandem Mass Spectrometry." Chromatography A1152 (2007): 116-123. Science Direct. 9 Oct. 2007
5. Pirard, C., J. Wildart, B. K. Nguyen, C. Deleuze, L. Heudt, E. Haubruge, E. DePauw, and J. F. Focant. "Development and Validation of a Multi-Residue Methodfor Pesticide Determination in Honey Using on-Column Liquid-Liquid Extractionand Liquid Chromatography-- Tandem Mass Spectrometry." Chromatography A1152 (2007): 116-123. Science Direct. 9 Oct. 2007.
6. Rezic, I., A.j.m. Horvat, S. Babic, and M. Kastelan-Macan. "Determination ofPesticides in Honey by Ultrasonic Solvent Extraction and Thin-LayerChromatography." Ultrasonics Sonochemistry 12 (2004): 477-481. Science Direct.5 Oct. 2007.
7. Rial-Otero, R., E. M. Gaspar, I. Moura, and J. L. Capelo. "Chromotographic-BasedMethods for Pesticide Determination in Honey." Talanta 71 (2007): 503-514.Science Direct. 9 Oct. 2007.