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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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

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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

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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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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.