IMPROVEMENT OF U.S.

EPA MINIMUM RISK ESSENTIAL OILS’ PESTICIDE

ACTIVITY THROUGH SURFACTANT ENHANCEMENT AND SYNERGY

A Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctorate of

Philosophy in the Graduate School of The Ohio State University

By

JoAnna Gillilan

Graduate Program in Horticulture & Crop Science

The Ohio State University

2012

Committee:

Dr. David S. Gardner, Advisor

Dr. T. Karl Danneberger

Dr. Brian McSpadden Gardener

Dr. Dave Shetlar

Dr. Rami Soufi

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Copyrighted by

JoAnna Gillilan

2012

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 Abstract

Plant essential oils have been used since antiquity for many purposes, including

pest control in agriculture and against nuisance pests such as mosquitoes, flies and ticks.

In recent years, consumers have increasingly expressed interest in purchasing organically

grown foods, as well as using natural and naturally derived materials to eradicate pests in

their lawn, garden and homes. In 1996, the U.S. Environmental Protection Agency

established a list, referred to as the 25(b) list, containing 31 active ingredients deemed

minimum risk. Companies developing pesticides using active ingredients from the 25(b)

list are exempt from federal registration requirements, providing a financial incentive to

develop such products as well as fill growing consumer demand. There are 14 plant

essential oils and essential oil constituents on the 25(b) list with proven pesticide

properties, but there is little public information comparing efficacy of these ingredients.

This study compared 14 essential oils and essential oil constituents from the 25(b) list in

insecticidal and herbicidal contact bioassays: cedar oil, cinnamon oil, citronella oil, clove

oil, eugenol, garlic oil, geraniol, geranium oil, lemongrass oil, mint oil, peppermint oil,

rosemary oil, thyme oil, and 2-phenethyl propionate. Their performance as single active

ingredients and as pairwise combinations with one another within liquid formulations

was evaluated for additive or synergistic benefits. In contact insecticidal assays using 9th

instar American cockroach (Periplaneta americana) nymphs, there was a significant

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difference in knockdown (KD) time in response to the different essential oils tested, but

no synergistic effect was observed when two essential oils were combined . There was a

positive correlation of insect KD time and contact angle of co-formulants that were

combined with essential oils. Cinnamon, lemongrass and rosemary oils had slow KD

times (>1 minute), but were significantly enhanced when paired with the surfactant

system having the lowest contact angle of 60°. The 25(b) oils that should be considered

for insecticide development based on performance are clove, eugenol, geraniol,

geranium, peppermint and thyme oils. The same essential oils and surfactant

combinations were tested for herbicide burndown on dandelions (Taraxacum officianale)

and smooth crabgrass (Digitaria ischaemum). There were significant differences in

herbicidal activity of the essential oils, but again no synergistic effect was observed when

two essential oils were combined. A novel discovery of synergy was made when

herbicidal activity was greatly enhanced by combining essential oils with a surfactant

system containing a non-herbicidal rate of sodium caprylate, a fatty acid salt. The 25(b)

oils with the highest weed injury were cinnamon, citronella, eugenol, geraniol, geranium,

peppermint and thyme oils. The findings of these experiments suggest that all 25(b)

essential oils except garlic oil can be formulated into effective pesticides. Targeting a

low contact angle surfactant system can enhance insecticidal activity of these oils, while

the use of sodium caprylate can speed phytotoxic symptoms when used as herbicides.

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 Acknowledgments

I would like to express my sincere appreciation to The Scotts Miracle-Gro

Company for the financial support of this project and for giving me the opportunity to

pursue my degree while serving as an employee. While there are many at “Scotts” who

have provided support, patience and friendship along the way, I would like to specifically

thank Dr. Rami Soufi for encouraging me to pursue this dream. You have been an

excellent mentor over the years, and thank you for taking time to serve on my advisory

committee. To Robert Radabaugh, Stuart Falk, Tiffany Stegner, Jean-Francois Bayle and

Jim Essinger, thanks to each of you for your advice, patience, and overall support of this

project. I would also like to extend many thanks to Amy Boone, Addaleigh Rife and

Michelle Green for the wonderful assistance in the lab and field. You made all the hard

work fun. I also thank my major advisor, Dr. David Gardner, as well as my other

advisory committee members Dr. Karl Danneberger, Dr. Brian McSpadden Gardener, Dr.

Dave Shetlar, and Dr. Rami Soufi. Your excellent guidance, support and confidence in

me helped make this journey a fulfilling and productive experience.

My deepest gratitude is reserved for the two that sacrificed the most while I

pursued a dream: my husband John and daughter Josie. Because of you and the Lord

Jesus Christ, I was able to accomplish what many said would just be too hard for a

working mommy.

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 Vita

May 1997 .......................................................JoByrns High School

2002................................................................B.S. Plant Science, Middle Tennessee State
University
2004................................................................M.S. Plant Science, University of Kentucky
2004 to present ..............................................Biologist, The Scotts Miracle-Gro Company
2009 to present………………………………Graduate Student, Ohio State University

Field of Study

Major Field: Horticulture

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 Table of Contents

Abstract ..............................................................................................................................ii

Acknowledgements...........................................................................................................iv

Vita..................................................................................................................................... v

List of Tables ...................................................................................................................vii

List of Figures ...................................................................................................................xi

Chapter 1: Literature review on the use of essential oils as pesticides.............................. 1

Chapter 2: Physical and chemical characteristics of U.S. EPA minimum risk essential oils
influencing their insecticide and herbicide activity ......................................................... 28

Chapter 3: Contact toxicity of U.S. EPA minimum risk plant essential oils on American
cockroach (Periplaneta americana) and the influence of surfactants on knockdown speed
.......................................................................................................................................... 57

Chapter 4: Post-emergent herbicide activity of U.S. EPA minimum risk plant essential
oils on dandelion (Taraxacum officianale) and crabgrass (Digitaria ischaemum) and the
influence of surfactants on their herbicidal activity......................................................... 84

Appendix A: Essential oil chromatograms from gas chromatographic analysis ........... 119

Appendix B: Photographs of essential oil HLB experiment.......................................... 134

Appendix C: Photographs from tensiometer for contact angle measurement ............... 141

Appendix D: Phase 1 photographs of weed injury 1 and 24 hours after application..... 145

Appendix E: Phase 2 photographs of weed injury 6 hours after application, greenhouse &
field trials…………………………………………………………………………….154

Comprehensive Bibliography…………………………………………………………..160

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 List of Tables

Table 1.1 The 25(b) list.................................................................................................... 20

Table 1.2. Common & botanical names of 25(b) essential oils, plant parts used, raw
material origin and trade quantities.................................................................................. 21

Table 1.3. Primary constituents of 25(b) essential oils…….……………………………22

Table 2.1. Guideline for required hydrophilic-lipophilic balance (HLB) to emulsify

various classes of materials………………………………………………………….…..45

Table 2.2 Nonionic HLB kit used in determining required HLB for 10 essential oils…..46

Table 2.3. Formulation components in the 3 surfactant systems tested in phase 2 insect
knockdown and dandelion herbicide experiments…………………...…………………..47

Table 2.4. Supplier list and lot codes for essential oils used in insect and weed control
experiments…………………………………………………………..…………………..48

Table 2.5. Constituent standards used in the GC-MS analysis of essential oils………....49

Table 2.6. Required HLB to emulsify the noted oils in distilled water………………….49

Table 2.7. Contact angle of essential oils, 5% v/v, combined with 3 unique surfactant
systems and the corresponding knockdown time of 9th instar American cockroach
nymphs…………………………………………………………………………………...50

Table 2.8. P values of essential oils + surfactant system C, compared to systems A and
B…………………………………………………………………….……………………51

Table 2.9. Mean knockdown time of surfactant systems A, B and C on 9th instar
American cockroaches, averaged across all essential oils……………….………………51

Table 2.10. Percent injury of dandelions 1 hour, 6 hours, 24 hours and 5 days after
application of 10 essential oils formulated with surfactant systems A, B and C in 2
greenhouse experiments………………………………………………………………….52

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Table 2.11. Percent injury of dandelions 1 hour, 6 hours, 24 hours and 6 days after
application of 10 essential oils formulated with surfactant systems A, B and C under field
conditions………………………………………………………………………………...53

Table 2.12. GC-MS constituent analysis, retention times and the corresponding chemical
structures and molecular weights of 25(b) essential oils…………..…………………….54

Table 3.1. EPA-exempt insect control products currently sold or state-registered for sale
in the market……………………………………………..…............................................76

Table 3.2. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% cedar oil alone compared to combinations with other
oils.……………………………………………………………………………………….78

Table 3.3. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% clove oil alone compared to combinations with other oils…………………..78

Table 3.4. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% eugenol alone compared to combinations with other oils……………………79

Table 3.5. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% garlic oil alone compared to combinations with other oils…………………..79

Table 3.6. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% geraniol alone compared to combinations with other oils…………………...80

Table 3.7. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% peppermint oil alone compared to combinations with other oils…………….80

Table 3.8. Essential oils in order of KD speed at 10% w/w concentration on American
cockroach………………………………………………………………………………...81

Table 3.9. Main effect of essential oil on KD speed of 9th instar American cockroaches at
a 5% w/w concentration, average KD across three surfactant systems..………………...81

Table 3.10. Mean knockdown time of 9th instar American cockroaches with eugenol,
geraniol and geranium oil combined with surfactant systems A & C, averaged across 3
test dates………………….………………………………………………………………82

Table 4.1. Non-selective EPA-exempt weed control products currently sold in the
market……………………………………………………………………………….….105

Table 4.2. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing cedar oil ……………………………………………………………….……106

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Table 4.3. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing cinnamon oil………………..……………………………………………….107

Table 4.4. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing eugenol…………………………………..………………………………….108

Table 4.5. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing garlic oil…………………………………………………………………….109

Table 4.6. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing geraniol……………………………………………………………………...110

Table 4.7. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing geranium oil……………….………………………………………………..111

Table 4.8. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing mint oil……………………………………………………………………...112

Table 4.9. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing peppermint oil……………………………………….……………………...113

Table 4.10. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing rosemary oil……………………………….………………………………...114

Table 4.11. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing 2-phenethyl propionate……………………………………………………..115

Table 4.12. Percent injury of dandelions 1 and 24 hours after foliar application with 10%
w/w essential oil formulations………………………………………………………….116

Table 4.13. Percent injury of crabgrass 1 and 24 hours after foliar application with 10%
w/w essential oil formulations………………………………………………………….116

Table 4.14. Herbicide synergy of ten essential oils mixed at 5% w/w concentration with
surfactant system A six hours after application to dandelions………………..………..117

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 List of Figures

Figure 2.1. Application of treatment to cockroach inside knockdown tube using a

pipette…………………………………………………………………………………..45

Figure 2.2. Figure 2.2. Positive linear relationship between knockdown time of American
cockroaches with increasing contact angle of essential oil formulations containing
surfactant systems A, B or C…………………………………………..………………..46

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 Chapter 1: Literature review on the use of essential oils as pesticides

1. Essential oil-based pesticides

The pesticide properties of natural ingredients, specifically essential oils, have

been known for years, perhaps centuries for some. Evidence for the production of

essential oils via water distillation dates back to ca. 3500 BC, and the ancient equipment

is on display at the Texila museum in Pakistan (Brud 2010). While the use of essential

oils for pest control has been in practice for many years, there are few commercialized

agricultural or homeowner lawn and garden essential oil-based products available on the

market. In the 1940’s, botanical insecticides were widely used on farms for insect control

purposes, but were eventually replaced with synthetic pesticides (Isman 2000).

Agriculture chemical companies have traditionally focused their discovery and

development efforts on synthetic pesticides. However, in recent years, the continuous

use of pesticides in agriculture is a growing concern for many researchers, government

agencies, and general public. There has been a renewed interest in the use of botanical

pesticides for agriculture in the last decade, and that trend is mirrored in the consumer

market (Butterfield & Baldwin 2011). With the rising demand for natural based pest

control options, public and private sector organizations have re-focused their discovery

programs by looking back to natural sources for new biologically active compounds.

Choices for insect control in organic food production are limited primarily to biological

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control agents and a few botanicals such as neem oil, spinosad and some essential oils

(OMRI Policy and Standards Manual 2010). Additionally, organic agriculture does not

allow the use of synthetic herbicides for weed control, which leads to labor issues in

having to control weeds by mechanical means, as well as reduced yields due to resource

competition between the crop and weeds (Dayan et al. 2009). Similarly, backyard

gardeners who desire to grow fruits, vegetables and ornamentals organically are left with

few commercially available product options for controlling pests. In addition to their low

mammalian toxicity, their effectiveness of controlling pests with certain synthetic

insecticide resistances, and overall reduced ecological hazards, there is much literature to

support the benefits of using essential oils as part of an integrated pest management

(IPM) program due to the various means by which they can reduce pest populations

(Regnault-Roger 1997). The challenge for industry is to meet this demand for natural

and organic products while matching the efficacy performance of synthetic pesticides

with as little economic tradeoff to the grower or consumer as possible.

In 1996, the U.S. Environmental Protection Agency (EPA) established a 25(b)

active ingredient list containing 31 ingredients which were deemed to be “minimum risk”

to the environment. Along with the 25(b) active ingredient list, a “4(a)” minimum risk

list of inert ingredients was also established. The purpose of the minimum risk

designation was meant to encourage companies, particularly small companies, to develop

“natural” pesticides by providing the financial incentive of avoiding the costly federal

registration process. A pesticide formulation developed in compliance with 25(b)/4(a)

requirements is allowed an exemption from the EPA registration process, but is still

required to register with some states (40 CFR Part 152, Vol. 61, No. 45, March 6, 1996).

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Table 1.1 is a complete list of the 25(b) minimum risk pesticides as listed on the EPA

website www.epa.gov (Feb 1, 2010).

A few lawn and garden companies have commercialized exempt products. Most

of these products utilize the 11 essential oils and the 3 essential oil constituents from the

25(b) list as the active ingredient(s). The consumer products that are available often have

poor emulsions that require the user to “shake well”, can cause significant phytotoxic

effects to desirable plants, and have a reputation among consumers as having weaker

performance against control of pests (Scotts unpublished data). There are several

explanations of why exempt products containing botanical insecticides have been fairly

unsuccessful in the market, including variable performance, lack of persistence and

inconsistent availability (Isman 2008).

2. What are essential oils?

Essential oils are mixtures of complex, highly volatile plant secondary products

which rapidly break down in the environment and are non-persistent when used on food

products as a means of pest control (Machial et al. 2010; Sampson et al. 2005; Isman

2000; Misra et al. 1996). Dayan et al. (2009) state that the short half life of essential oils

is primarily due to their lack of “unnatural” ring structures, as well as the presence of

very few halogen substituents, such as Cl, F, and Br, that are sometimes found in

synthetic pesticides. The most widely recognized plant essential oils having pesticide

activity come from the families Myrtaceae, Lauraceae, Rutaceae, Lamiaceae,

Asteraceae, Apiaceae, Cupressaceae, Poaceae, Zingiberaceae, and Piperaceae

(Regnault-Roger 1997; Tisserand et al. 1995; Tarelli et al. 2009; Isman & Machial 2006).

3
These oils are extracted via steam distillation of the foliage or other plant parts (Isman &

Machial 2006). Plants that produce essential oils do so as a means to provide self-

protection from insects, herbivores and diseases. Others have the ability to excrete

allelopathic chemicals, primarily from their roots, as a means to suppress growth of

neighboring plants, while yet others use their secondary compounds as semio-chemicals

to attract pollinators. There are numerous essential oils not included on the 25(b) list that

have well documented insecticidal, herbicidal, antifungal, antimicrobial, and repellent

properties. Many other oils have been widely studied and used for medicinal and

therapeutic purposes and are also used throughout the world as fragrances and food

flavoring agents. Significant cost differences exist among essential oils, driven by global

market demand and trade quantities available (Table 1.2).

3. Constituents of essential oils

There is published literature with supporting evidence of pesticide activity of the

14 essential oils on the 25(b) list (Table 1.1). Many researchers have elucidated the

constituents within the essential oils primarily responsible for the biological activity.

Ngoh et al. (1998) classify the volatile components of essential oils into 4 groups:

terpenes, benzene derivatives (including phenols), hydrocarbons and miscellaneous

compounds. The combinations of these constituents within the plant are responsible for

the characteristic flavor and odor of plants. Functional groups include aromatic phenols,

oxides, ethers, alcohols, esters, aldehydes, and ketones (Batish et al. 2008).

Monoterpenes, which contain 10 carbon atoms, and sesquiterpenes, which contain 15

carbon atoms, are major constituents of essential oils. These are lipophilic molecules

4
with high vapor tension. Linalool, menthyl acetate, limonene, menthone, geraniol, citral,

eucalyptol (1,8-cineole), pulegone, citronellal, thymol, carvacrol, eugenol, geraniol, and

trans-anethole are well known examples of pesticide compounds (Phillips et al. 2010;

Tarelli et al. 2009; Isman & Machial 2006; Isman 2000; Sampson et al. 2005). Species

within the Lamiaceae family are particularly well documented as having acute toxicity to

insects. Four of the 25(b) essential oils are in this family: mint, peppermint, rosemary,

and thyme oil. These particular oils are rich in the mono- and sesquiterpenes carvacrol,

cymene, and thymol, and are primarily responsible for the efficacy of these oils (BJ

Sampson 2005). Other monoterpenes present in 25(b) essential oils include geraniol,

limonene, linalool, menthol, menthone, citronellol, citronellal, pinene, citral (cis-geranial;

trans- neral), carvacrol, menthyl-acetate, camphene and camphor. The chemical structure

of these compounds, among others, is presented in Table 1.3 to illustrate the various

functional and structural features of essential oil constituents.

4. Essential oil pesticide mode of action

There are various documentations describing exactly how essential oils work as

insecticides. The means of delivery include fumigant and topical applications, and may

have antifeedant and repellent properties, ovicidal activity, cause reproductive delays or

modifications in development (Isman 2000; Regnault-Roger 1997; Rice & Coats 1994;

Singh et al. 1989; Choi et al. 2002; Choi et al. 2004; Huang et al. 1998; Petrakis et al.

2005). Lipophilic plant oils are known to kill soft-bodied insects by penetrating their

waxy cuticle, leading to desiccation. There is also evidence that once penetrated through

the cuticle, certain components within essential oils may begin to block

5
neurotransmitters, digestive enzymes or growth hormones (BJ Sampson et al. 2005;

Tarelli et al. 2009; Tisserand & Balacs 1995). In addition to the physical effects of

disrupting cellular tissue, the rapid toxic effects of essential oils indicate that there is a

neurotoxic effect on pests (Enan 2001, Isman & Machial 2006). Through experiments on

volatile oil constituents’ insecticidal and repellent properties against the American

cockroach (Periplaneta americana), Ngoh et al. (1998) concluded that “contact toxicity

decreases when the double bond is closer to the aromatic ring in isoeugenol compared to

eugenol and methyl-eugenol”. Furthermore, the additional methoxy group in methyl-

eugenol increases knockdown capability without significantly affecting its lethal

capacity. In a study by Phillips et al. (2010), there was negative correlation between

essential oil constituent density and its toxicity to German cockroaches (Blattella

germanica). The authors concluded that the density may effect the penetration of the

compounds through the cuticle. They also found that aromatic compounds such as

carvacrol, thymol, eugenol, and trans-cinnamaldehyde were more toxic than the aliphatic

compound geraniol. Their conclusion was that the benzene ring present in aromatic

compounds is not easily metabolized and detoxified within the insect. Rice and Coats

(1994) also found that aromatic compounds were more efficacious to the house fly

(Musca domestica) than aliphatic compounds. In a study by Isman (2000) to determine

pesticide activity of phenolic versus aliphatic terpenes, the extent of toxicity of these

compounds was highly dependent on the test species. At least one of the phenols eugenol

or carvacrol was more active on fruit fly (Drosophila melanogaster), tobacco cutworm

(Spodoptera litura), house fly and western corn rootworm beetle (Diabrotica virgifera

virgifera) than the terpenes a-terpineol and terpinen-4-ol. However, for twospotted

6
spider mites (Tetranychus urticae), terpinen-4-ol was much more active than either

phenol. Grodnitzky and Coats (2002) utilized QSAR (Quantitative Structure-Activity

Relationship) models as a means of predicting insect toxicity of thirty monoterpenoids.

They found that as the electron accessibility increased in the monoterpenoids, there was a

corresponding increase in toxicity, suggesting a change in receptor(s) binding affinity.

The QSAR derived from the acyclic monoterpenoids as compared to the aromatic

monoterpenoids suggested there may be a different mode of action.

In recent years, it has been discovered that there is a relationship between

octopamine and tyramine receptors and essential oil mode of action in insects. The role

of octopamine and tyramine, neuroactive ligands present in invertebrates, is well

understood in terms of their physiological functions as neurotransmitters,

neurohormones, and neuromodulators (Enan 2004; Bischof & Enan 2004). In Enan’s

(2004) research on essential oil constituents and insecticidal mode of action in D.

melanogaster, thymol and carvacrol induced a decrease in receptor binding activity,

while significantly increasing cyclic AMP (cAMP) and Ca2+ levels. The presence and

location of a hydroxyl group was critical in the tyramine receptor mediation of

insecticidal activity, with the 2 or 3 ring positions being most favorable. Kostyukovsky

et al. (2002) observed a similar response of significantly increased cAMP levels in

abdominal epidermal tissue of the Cotton bollworm moth (Helicoverpa armigera) in

response to two unnamed essential oil terpenes belonging to the Labiatae family. This

reaction is similar to the effect of octopamine on cAMP levels, indicating that there may

be competitive activation of the octopaminergic receptors by these terpenes. Activity of

these constituents was not found to correlate with acetyl cholinesterase activity.

7
 Several of the plant essential oils listed on the EPA 25(b) minimum risk pesticide

list are also sold commercially as contact, non-selective herbicides. The mode of action

is typically attributed to a rapid loss of cell membrane integrity (Copping & Duke 2007).

5. Pesticide activity of the essential oils and their constituents

Cedar oil (Cupressus & Juniperus spp): The major components of cedar oil

(Virginia) are α- and β-cedrene at 80% concentration, cedrol at 3-14% and cedrenol.

Other sesquiterpenes, such as thujopsene, b-elemene, caryophyllene, cuparene and

acorene, are present, as well as monoterpenoids such as sabinene and sabinyl acetate

(Khan & Abourashed 2010). Choi et al. (2003) included cedar oil (Cedrus atlantica) in a

fumigant bioassay test of 53 essential oils and their toxicity to 3 stages of greenhouse

whitefly (Trialeurodes vaporariorum). The mortality rate was less than 40% at all life

stages with cedar oil at the highest tested concentration of 19 x 10-3 µl/ml air, making it

one of the least effective oils in the trial.

Cinnamon oil (Cinnamomum spp): In Cassia bark oil from trees grown in China,

65.5% is comprised of (E)-cinnamic aldehyde, 8.7% coumarin, 3.6% cinnamyl acetate

and 2-methoxycinnamaldehyde. Trace amounts of benzyl benzoate, cinnamyl alcohol, 2-

phenylethyl acetate, eugenyl acetate, (Z)-cinnamic aldehyde, eugenol and others may also

be found (Khan & Abourashed 2010). Wijesekera et al. (1974) reported that oils

extracted from the root stem and leaves of Cinnamomum zeylanicum were significantly

different in terms of constituent levels, with the stem bark containing the highest amount

of cinnamaldehyde at 70%. Root bark oil consisted mostly of camphor and linalool, and

8
leaf oil consisted mostly of eugenol. In a study conducted by Lee et al. (2008), cinnamon

oil was found to have an LD50 value of 0.016 mg cm2 on rice weevil (Sitophilus oryzae),

a common destructive pest of stored grains. This study also tested fumigant activity of

(E)-cinnamaldehyde and 41 structurally related compounds, of which allyl cinnamate was

the most toxic and comparable to that of dichlorvos, with 83% mortality using the closed

container fumigant method at a 0.013 mg/cm3 dosage. Other compounds included in the

test that displayed potent insecticidal activity include cinnamonitrile, (E)-4-

hydroxycinnamic acid, and hydrocinnamyl acetate. Activity against S. oryzae in this test

was via the vapor phase due to the fumigant mode of delivery. However, there is still not

a clear understanding of cinnamon oil’s insecticide mode of action. Cinnamaldehyde has

been registered with the U.S. EPA since 1994 as an active ingredient for use as an

agricultural fungicide. It was later registered as a cat and dog repellent.

(Cinnamaldehyde fact sheet: http://www.epa.gov/pesticides/biopesticides).

Citronella oil (Cymbopogon spp): There are two main types of citronella oil,

referred to as Ceylon and Java. Both types contain citronellal, citronellol and geraniol as

the major components, but the proportions of these vary greatly depending on source and

type, with Java having a higher percentage made up of these 3 components. Other

constituents found in citronella oil include esters of geraniol, citronellol and linalool,

monoterpenes such as limonene, pinene and camphene, various sesquiterpenes and

alcohols, phenols and free acids. The Ceylon type contains a higher percentage of

monoterpenes than the Java type (Khan &Abourashed 2010). Citronella is widely used

as a mosquito repellent. Waliwitiya et al. (2009) found that citronellal, as well as other

9
essential oil constituents, was highly effective against all larval stages of Aedes aegypti

(Diptera: Culicidae) at LC50 of 40 to 263 mg/L. Citronellal also showed 88% oviposition

deterrence. Citronella is currently sold in the UK as an herbicide under the name Barrier

H™, a ready-to-use formulation containing 22.9% citronella oil. Its herbicidal activity

was tested by Clay et al. (2005) as a treatment for clearing area for tree establishment. At

a high dose of 229 kg a.i. ha-1, citronella oil killed foliage of all weed species within one

day of application, but re-growth was reported. Choi et al. (2003) included citronella

(Cymbopogon nardus) in a study of 53 essential oils’ toxicity to greenhouse whitefly. It

was highly effective on both adult and egg stages, but only at the highest dose tested. As

a result, it was not ranked among the most toxic of oils on this particular species.

Clove oil (Eugenia spp, Syzygium aromaticum): Clove bud oil contains 60-90%

eugenol, 2-27% eugenol acetate, 5-12% b-caryophyllene and other minor constituents

such as methyl acetate, methyl eugenol, benzaldehyde, methyl amyl ketone, a-ylangene

and chavicol. Constituents found in clove leaf and clove stem oils are very similar, but

may differ in ratio. Napthalene may be present in leaf and stem oils, but does not occur

in the bud oil (Khan & Abourashed 2010). Tworkoski (2002) showed that clove oil and

eugenol increased cell membrane permeability in common lambsquarter (Chenopodium

album), common ragweed (Artemesia artemisiifolia), and johnsongrass (Sorghum

halepense), which led to significant injury to the plant tissue. In Bainard et al. (2006),

clove leaf oil (2.5% v/v) inhibited growth of broccoli seedlings by 64%, and 67% when

leaf epicuticular wax was removed prior to spraying, with clove leaf oil proving more

effective than eugenol (1.5% v/v). Clove oil is a commonly used active ingredient in

10
commercial products sold for organic agriculture. Dayan et al. (2009) list the products

Burnout™, Bioorganic™, Weed Zap™, Weed-A-Tak™, Matran II™, and Eco-

Exempt™ as non-selective herbicides containing clove oil as the active ingredient. Boyd

and Brennan (2006) reported that a 90% reduction of burning nettle and commom

purslane required rates from 12-61 L/ha, with an estimated price of $880 to $2140 per

hectare. While clove may be cost prohibitive as a broadcast-applied herbicide for

agriculture, homeowners desiring an organic solution for weed control may find the costs

within reason, particularly if weeds are being spot treated. In Choi et al. (2003), clove oil

provided 98% mortality to greenhouse whitefly nymphs at 9.3 x 10-3 concentration, and

clove (bud) oil provided 94% mortality to eggs at the same concentration. Among the 53

essential oils included in this study, only 8 including clove, were considered highly

effective against all 3 stages (adult, nymph and egg) of greenhouse whitefly. The mode

of delivery was via vapor phase. Other 25(b) oils included in this study were cedar

(Cedrus atlantica), citronella (Cymbopogon nardus), geranium (Pelargonium

graveolens), lemongrass (Cymbopogon citratus), thyme red and thyme white (Thymus

vulgaris). Of these, only peppermint oil was equally as effective as clove on the three life

stages.

Eugenol: Eugenol is an aromatic monoterpenoid compound commonly found in

many essential oils. It is the primary constituent of clove oil, but is also found in

significant amounts in cinnamon oil. Eugenol is known for its herbicidal, insecticidal and

antifungal activity. Bainard et al. (2006) reported on the ability of both eugenol 1.5% and

clove oil 2.5% to reduce seedling growth of common lambsquarter (Chenopodium album

11
L.), redroot pigweed (Amaranthus retroflexus), and sprouting broccoli (Brassica oleracea

var. italica). Overall, clove oil was more effective as an herbicide than eugenol, and both

worked better on redroot pigweed due to its lack of leaf epicuticular wax. Ngoh et al.

(1998) tested the insecticidal and repellent properties of several volatile constituents of

essential oils against American cockroaches, and found that the most toxic compounds in

the contact toxicity test were eugenol, methyl-eugenol, safrole and isosafrole. Methyl-

eugenol was a better knockdown agent than eugenol, but both constituents had the same

mortality effect. In conclusion, Ngoh et al. state that the benzene derivatives tested are

better toxicants and repellents to American cockroaches than the monoterpenes included

in the trials, which were limonene, cineole, and p-cymene. Enan (2005) determined

eugenol LD50 values of 1.9 and 47 µg/insect for fruit fly (Drosophila melanogaster) and

American cockroach (Periplaneta americana), respectively. In the same study, cinnamic

acid LD50 values were 1.65 and 98 µg/insect for D. melanogaster & P. americana, and

trans-anethole LD50 values were 6 and 1300 µg/insect for D. melanogaster & P.

americana. In addition, Enan found that eugenol had lower LC50 values on American

cockroaches, German cockroaches and carpenter ants in comparison to α-terpineol and

cinnamic acid.

Garlic oil (Allium sativum L): Most published literature cites that diallyl disulfide

is the main compound in garlic oil, at 60%, with diallyl thiosulfinate, allylpropyl

disulfide, diallyl disulfide and diallyl trisulfide also being major components. Other

commonly found components in lesser amounts include dimethyl sulfide, dimethyl

disulfide, dimethyl trisulfide, allylmethyl sulfide, 2,3,4-trithiapentane, bis-2-propenyl tri-,

12
tetra-, and pentasulfides and other related sulfur compounds. Other non-sulfur

compounds that may be found in garlic oil include citral, geraniol, linalool and b-

phellandrene (Khan &Abourashed 2010). Machial et al. (2010) reported garlic oil to be

the most effective of 17 tested essential oils on 1st instar cabbage looper larvae

(Trichoplusia ni), with LC50 = 3.3 µl/mL and LD50 = 22.7 µg/insect. It ranked 5th out of

17 in terms of its toxic effect to the oblique-banded leaf roller (Choristoneura rosaceae),

with 22% mortality of 1st instar larvae at 5 µL/mL. Major constituents of the garlic oil

sample used in this experiment were 35.2% diallyl disulfide, 26.2% di-2-propenyl

trisulfide, 20.7% 3,3’ thiobis-1-propene, 6.6% methyl 2-propenyl trisulfide and 4.5%

methyl 1-propenyl disulfide.

Geranium oil (Pelargonium spp): Geranium oil consists of 60-70% alcohols,

primarily l-citronellol and geraniol, with linalool and phenethyl alcohol also present. The

esters geranyl tiglate, geranyl acetate, citronellyl formate and citronellyl acetate comprise

20-30% of the oil. Many other minor constituents may also be found in geranium oil,

such as sesquiterpene hydrocarbons and alcohols, acids, dimethyl sulfide, cis- and trans-

dehydrocitronellol, menthol, citronellyl-diethylamine and others (Khan &Abourashed

2010). In Tarelli et al. (2009), toxicity of multiple essential oils including geranium,

mint, lavender, eucalyptus and orange oils were tested on the common house fly. A

topical application of 0.09 µg mint oil per insect was required to reach LD50, as compared

to 0.07 µg geranium oil. In the same publication, fumigant toxicity was conducted using

the same essential oils. Knockdown times of 50% (KT50) were 10.4 minutes for mint and

17.7 minutes for geranium oil, with the fastest being eucalyptus at 3.3 minutes. geranium

13
oil was tested for toxicity against three life stages of greenhouse whitefly, among 53

other essential oils by Choi et al. (2003). All 3 stages were effectively controlled (>90%

mortality) by geranium oil, but at the highest dose only. There were several other oils,

including clove and peppermint, which proved much more effective at lower doses.

Geraniol: Geraniol is an acylic monoterpenoid alcohol occurring in essential oils

from several plants. It is widely used in the flavor and fragrance industries and is

described as having a sweet floral and citrus fragrance (Chen & Viljoen 2010). Both

geraniol and eugenol have been used as attractants in Japanese beetle (Popillia japonica)

lures. Phillips et al. (2010) included geraniol in a toxicity study on German cockroaches.

The resulting LD50 values ranged from 0.05 to 0.83 mg per cockroach on small nymphs

and adult females, respectively. While certainly found to be toxic, other essential oil

constituents such as thymol, trans-cinnamaldehyde, eugenol, and carvacrol had lower

LD50 values at most life stages. Joen et al. (2009) found that the acaricidal effects of

geraniol on storage food mites (Tyrophagus putrescentiae) were more effective than the

industry standard benzyl benzoate, with LD50 values of 1.27 µg/cm3 and 1.95 µg/cm3,

respectively. Geraniol also displayed highly toxic acaricidal properties when in direct

contact with Otodectes cynotis in a study by Traina et al. (2005). Three other

monoterpenes (α-pinene, limonene and p-cymene) were included in this study, but were

not as toxic as geraniol.

Lemongrass oil (Cymbopogon citratus): West Indian lemongrass oil contains 65-

85% citral, whereas Cameroonian C. citratus contains geranial as its major component,

14
comprising 33% of the oil. Other compounds that may be present in lemongrass oil

include myrcene (12-20%), dipentene, methylheptenone, b-dihydropseudoionone, neral,

β-pinene, linalool, methylheptenol, a-terpineol, geraniol, nerol, farnesol, citronellol, and

volatile acids such as isovaleric, geranic, caprylic, citronellic and others (Khan &

Abourashed 2010). Lemongrass oil is known to have herbicidal activity, and a

commercial organic herbicide GreenMatch EX™ contains 50% lemongrass oil as the

active ingredient (Dayan et al. 2005). Lemongrass oil is also known for its insecticidal

properties and was included, among 52 other essential oils, in a test against greenhouse

whitefly by Choi et al. (2003). Lemongrass oil was highly effective against the adult

stage, but only at the highest rate tested; nymphs were only suppressed at this rate.

However, on the egg stage, lemongrass oil was the most effective (98% mortality) of all

53 oils at the lowest tested rate of 0.0023 µl/ml air. A fumigant toxicity study of essential

oils and constituents on 4 stored-product beetle species showed that lemongrass oil had

little to no toxic effect. However, linalool and terpinen-4-ol, found in lemongrass oil as

minor constituents, were found to be highly effective (Shaaya et al. 1991). In a study by

Machial et al. (2010) on the toxicity of 17 essential oils on two lepidopteran species,

lemongrass oil was the second most toxic to 1st instar cabbage looper larvae, with 53%

mortality at a concentration of 5 µL/mL, an LC50 of 7.2 µL/mL and an LD50 of 60.5

µL/mL. However, it was found to be one of the least effective on 1st instar oblique

banded leafroller. A GC-MS analysis found the major constituents of this lemongrass oil

sample to be citral (47.1%), trans-verbenol (32.1%) and camphene (10.7%).

Mint oil (Mentha spp.): “Mint” generically covers multiple mint species within

the Mentha genus. Major constituents of mint oil include menthol, menthone, etc, and

15
vary according to species. In Tarelli et al. (2009), a study of toxicity of multiple essential

oils including geranium, mint, lavender, eucalyptus and orange oils, to house fly was

conducted. A topical application of 0.09 µg mint oil per insect was required to reach

LD50, compared to 0.07 µg geranium oil. In the same publication, a fumigant trial was

conducted using the same essential oils. Knockdown times of 50% (KT50) were 10.4

minutes for mint and 17.7 minutes for geranium oil, with the fastest time of 3.3 minutes

obtained with eucalyptus oil. Koschier and Sedy (2003) tested repellency and oviposition

effects of mint oil, along with several other essential oils, against onion thrips (Thrips

tabaci). Mint oil concentrations of 0.1% and 1% significantly reduced egg-laying

activity of females on leaves (0.8 eggs per leaf disc treated with 1% mint oil compared to

7.2 eggs per leaf disc on untreated).

Peppermint oil (Mentha piperita): The volatile components of peppermint oil are

primarily menthol (29-48%), menthone (20-31%), menthyl acetate (31%), menthofuran

(1-7%) and limonene. Other compounds commonly found in peppermint include

viridiflorol, pulegone, 1,8-cineol, piperitone, caryophyllene, bisabolene, isomenthone,

isomenthol, α- and β-pinene, neomenthol, ledol, d-trans-sabinene hydrate, and

bicycloelemene (Khan & Abourashed 2010). Peppermint was included in a study by

Choi et al. (2003) that tested the efficacy of 53 essential oils on 3 life stages of

greenhouse whitefly. Peppermint oil, along with clove oil, was ranked as one of the eight

most toxic oils on all three life stages. The vapor phase was responsible for mortality. In

a separate study by Choi et al. (2004), peppermint oil was considered highly toxic in a

diffusion bioassay on twospotted spidermites (Tetranychus urticae), as well as adult

16
Phytoseiulus persimilis, a natural predator of T. urticae. Harwood et al. (1990)

discovered that peppermint monoterpenes resulted in reduced growth and molting

abnormalities in cutworm. Peppermint oil was also highly effective on the red flour

beetle (Tribolium castaneum) as a fumigant in a study evaluating toxicity of essential oils

and constituents against four stored-grain pests. Of the constituents tested, 1,8-cineole,

which is commonly found in peppermint oil and is also known as eucalyptol, was one of

the most toxic (Shaaya et al. 1991). Conversely, a peppermint oil sample containing 29%

menthol and 17% menthone was ranked least toxic to turnip aphids (Lipaphis erysimi) in

a study of 23 essential oils by Sampson et al. (2005). Samarasekera et al. (2008) studied

the toxicity of menthol derivatives against three mosquito species: Culex

quinquefasciatus, Aedes aegypti and Anopheles tessellates. The authors concluded that

the optimum activity of the derivatives was related to their volatility, degree of saturation,

position of the hydroxyl group and type of functional group.

Rosemary oil (Rosmarinus officinalis): Rosemary oil consists mainly of monoterpenes

such as α- and β-pinene, camphene, limonene, cineole, borneol, camphor, linalool,

verbenol, verbenone, terpineol, 3-octanone and isobornyl acetate (Khan & Abourashed

2010). Koschier and Sedy (2003) tested rosemary oil, among other essential oils, for its

repellency and deterrence capabilities against Thrips tabaci. When tested for repellency

in olfactory bioassays (glass Y-tube), 10% rosemary oil performed the best with 77%

repellency of female thrips. Additionally, 1% rosemary oil significantly reduced female

thrip settling on the leaf surface. Choi et al. (2003) evaluated rosemary oil, among 52

other essential oils, for toxicity of greenhouse whitefly at three life stages. Rosemary oil

17
was one of the least toxic oils in the study, with less than 40% mortality of adults and

eggs. Rosemary oil was also ranked low in toxicity to turnip aphids (Lipahis erysimi) in

a study of 23 essential oils by Sampson et al. (2005). The major constituents of this

particular rosemary sample were 38% 1,8-cineole and 19% α-terpineol. However, in a

fumigant toxicity study on stored grain pests, both rosemary oil and linalool proved to be

highly effective in controlling the lesser grain borer, Rhyzopertha dominica (Shaaya et

al., 1991).

Thyme oil (Thymus vulgaris): Thyme oil consists mainly of the phenols (20-

80%) carvacrol and thymol, with thymol typically being the dominant compound present.

Up to 51% monoterpene hydrocarbon content has been reported, being made up of p-

cymene and g-terpinene. Alcohols such as linalool, a-terpineol and thujan-4-ol are also

present (Khan & Abourashed 2010). Choi et al. (2003) tested efficacy against

greenhouse whitefly adults, nymphs and eggs. At the highest rate of 9.3 x 10-3

concentration, thyme oil provided 100% mortality to adults and 88% mortality to eggs.

However, there were many other oils in this study, including clove and peppermint,

which provided greater mortality when tested at lower rates. In a study by Shaaya et al.

(1991), thyme oil had highly toxic fumigant toxicity against the stored-grain pest,

sawtoothed grain beetle (Oryzaephilus surinamensis). Among constituents tested in the

same study, carvacrol, linalool, and a-terpineol were also highly toxic. Machial et al.

(2010) tested 17 essential oils for toxic effect on two Lepidopteran species, oblique-

banded leafroller (Choristoneura rosaceana), and cabbage looper (Trichoplusia ni), in

which thyme oil was the second most toxic on 1st instar C. rosaceana larvae, with 64%

18
mortality at a concentration of 5.0 µL/mL, superseded only by the 97% mortality with

patchouli oil at the same concentration. Despite thyme oil’s potency on C. rosaceana, it

was one of the least toxic essential oils on 1st instar T.ni. A GC-MS analysis identified

the two primary constituents of thyme oil were thymol (57.8%) and p-cymene (28.6%),

with all other constituents making up <5% of the total. Sampson et al. (2005) evaluated

insecticidal activity of 23 essential oils, including three species of thyme (Coridothymus

capitatus, Thymbra sintenisii and Thymbra spicata) against turnip aphids (Lipaphis

pseudobrassicae). They found that plant species containing high amounts of carvacrol,

present at 20-27% in the three thyme species, were highly active against the turnip aphid.

Only two constituents (E)-2-tridecental and (E)-20-tetradecenal found in the essential oil

of wild bishop (Bifora radians) at 47% and 23%, respectively, were more toxic than the

carvacrol-containing essential oils. The constituent thymol was found to be highly

larvicidal to Aedes agypti (Diptera: Culicidae) with LC50 values ranging from 17.3 to

53.5 mg/L on 1st to 4th instar larvae, respectively (Waliwitiya et al. 2009). It also had

significant oviposition repellency, with 80% deterrence at 5 days in a binary choice

assay.

2-phenethyl propionate: 2-phenethyl propionate is a propanoid compound

naturally present in many plants. In 2000, it was patented as an herbicide and is currently

sold as an herbicide, in combination with eugenol, in the consumer lawn and garden

market. It is also frequently used as a lure for insect traps and as an active ingredient in

contact spray insecticides in combination with geraniol and eugenol (Copping & Duke

2007).

19
6. Tables

Corn gluten Sesame &

Castor oil Geraniol Mint & mint oil
meal sesame oil
Peppermint & Sodium
Cedar oil Corn oil Geranium oil
peppermint oil chloride
Cinnamon & 2-phenethyl Sodium lauryl
Cottonseed oil Lauryl sulfate
dinnamon oil propionate sulfate
Potassium
Citric acid Dried blood Lemongrass oil Soybean oil
sorbate
Putrescent
Citronella &
Eugenol Linseed oil whole egg Thyme oil
citronella oil
solids
Clove & clove Garlic & garlic Rosemary &
Malic acid White pepper
oil oil rosemary oil
Zinc metal
strips
Table 1.1. The 25(b) list.
http://www.epa.gov/oppbppd1/biopesticides/regtools/25(b)_list.htm

20
Trade name Species Plant Family Used Wild/Cultivate Qua
Plant d Collection ntitie
Part(s) sa
Cedarwood, Cupressus CupressaceaeWood Wild MQ
Chinese funebris Endl
Cedarwood, Juniperus Cupressaceae Wood Wild MQ
Texas mexicana
Schiede
Cedarwood, Juniperus Cupressaceae Wood Wild MQ
Virginia virginiana L.
Cinnamon Cinnamomum Lauraceae Bark Cultivated LQ
bark, Ceylon zeylanicum Nees
Cinnamon Cinnamomum Lauraceae Bark Cultivated LQ
bark, Ceylon cassia Blume
Cinnamon leaf Cinnamomum Lauraceae Leaf Cultivated LQ
zeylanicum Nees
Citronella, Cymbopogon Poaceae Leaf Cultivated HQ
Ceylon nardus (L.) Wats
Citronella, Cymbopogon Poaceae Leaf Cultivated HQ
Java winterianus
Clove buds Syzgium Myrtaceae Leaf/bud Cultivated LQ
aromaticum
Clove leaf Syzgium Myrtaceae Leaf Cultivated HQ
aromaticum
Garlic Allium sativum Alliaceae Bulb Cultivated LQ
L.
Geranium Pelargonium spp. Geraniaceae Leaf Cultivated MQ
Lemongrass, Cymbopogon Poaceae Leaf Cultivated LQ
West Indian citratus
Peppermint Mentha piperita Lamiaceae Leaf Cultivated HQ
Rosemary Rosmarinus Lamiaceae Leaf Cultivated/Wil LQ
officinalis L. d
Thyme Thymus vulgaris Lamiaceae Herb Cultivated LQ
Table 1.2. Common & botanical names of 25(b) essential oils, plants parts used, raw
material origin, and trade quantities. Excerpt from Table 3.7 (C.Franz and J.Novak,
Handbook of Essential Oils, Chapter 3: Sources of Essential Oils, 2010)
a
HQ=high quantities (>1000 t/a); MQ=medium quantities (100-1000 t/a); LQ=low
quantities (<100 t/a)

21
Geraniol Eugenol 2-phenethyl propionate

Limonene Linalool α-cedrene

Menthol Menthone Citronellol

Citronellal α-pinene Trans-cinnamaldehyde

Table 1.3. Representative constituents of 25(b) essential oils displaying various
functional and structural features.
(images from www.ncbi.nlm.nih.gov)

continued

22
 Table 1.3 continued

Coumarin Cedrol Diallyl disulfide

Citral Thymol Carvacrol

Menthyl acetate Camphene Camphor

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properties in essential oils against rice weevil (Coleoptera: Curculionidae) in stored
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Tarelli, G, EN Zerba, and RA Alzogaray. 2009. Toxicity to vapor exposure and topical
application of essential oils and monoterpenes on Musca domestica (Diptera: Muscidae).
J. Econ. Entomol. 102: 1383-1388.

Tisserand, R, and T Balacs. 1995. Essential oil safety. Livingstone, London,United

Kingdom.

Traina, O, C Cafarchia, G Capelli, NS Iacobellis, and D Otranto. 2005. In vitro

acaricidal activity of four monoterpenes and solvents against Otodectes cynotis (Acari:
Psoroptidae). Experimental and Applied Acarology 37: 141-146.

Tworkoski, T. 2002. Herbicide effects of essential oils. Weed Science 50: 425-431.

Waliwitiya, R, CJ Kennedy, and CA Lowenberger. 2009. Larvicidal and oviposition-

altering activity of monoterpenoids, transanethole and rosemary oil to the yellow fever
mosquito Aedes aegypti (Diptera: Culicidae). Pest Mgmt Sci. 65: 241-248.

Wijesekera, R., AL Jajewardene, and LS Rajapakse. 1974. Composition of the essential

oils from leaves, stem bark and root bark of two chemotypes of cinnamon. J. Sci. Food
Agric. 25: 1211-1218.

27
Chapter 2: Physical and chemical characteristics of U.S. EPA minimum risk essential oils

and relationship to their insecticide and herbicide activity

1. Introduction

In 1996, the U.S. Environmental Protection Agency (EPA) established the 25(b)

list of active ingredients which were deemed minimum risk to the environment. The

establishment of the 25(b) list allows companies to develop alternative pesticides with a

financial incentive of avoiding the costly federal registration process. Some states still

require “exempt” products to be state-registered but most do not (40 CFR Part 152, Vol.

61, No. 45, March 6, 1996). In addition to exempting these products from the registration

process, the EPA will also allow the manufacturer to use such claims as “safe to use

around kids and pets” on the label. This is the only category of pesticides in which the

EPA will allow any type of safety claim to be made. This is obviously an appealing

marketing aspect for companies who wish to sell to a certain segment of consumers who

may be leery of using pesticides, particularly around children and pets.

There are 31 ingredients listed on the 25(b) list, 14 of which are plant essential

oils (cedar, cinnamon, citronella, clove, garlic, geranium, lemongrass, mint, peppermint,

rosemary and thyme oils) or major essential oil constituents (eugenol, geraniol and 2-

phenethyl propionate) (Table 1.1). Exempt products must only contain inert ingredients

that are listed on the 4(a) list or are considered 4b food-grade or “generally regarded as

28
safe” (GRAS). This restriction severely limits the formulator’s toolbox in terms of being

able to create highly stable emulsions, restricting microbial contamination of the product

with antimicrobial agents or increasing performance using certain surfactants. Many

exempt products being sold in the consumer lawn and garden category use essential oils

as the active ingredients and are marketed for several uses, including deer and rabbit

repellents, indoor insecticides, plant protectant insecticides, herbicides, fungicides, lawn

insecticides, and personal tick/mosquito repellents. These products often have poor

emulsions that require the user to “shake well,” can cause significant phytotoxic effects to

desirable plants, and are typically short-lived in the environment, requiring frequent re-

application.

There is a need for better understanding of how surfactant technology can be used

to increase the performance of essential oil-containing products. Surfactants are widely

used in the formulation and enhancement of pesticides. There is a wide range of

materials categorized under the term surfactant, including emulsifiers, detergents and

wetting agents. They are also designated by their electrical charge, being ionic (anionic

or cationic) or nonionic. Surfactants have dual character, allowing them to be compatible

with lipids and water (Behrens 1964; Tadros 1994). When surfactants are added to a

liquid that exceeds the critical micelle concentration, the coalescence of oil droplets is

hindered in an oil-in-water (o/w) formulation. This process slows the separation of oil

and water phases (Behrens 1964), thus creating an emulsion. Two tools that enable

formulators to narrow their surfactant options based on the functionality of the end-

product are the measurement of contact angle (θ) and the hydrophilic-lipophilic balance

(HLB).

29
 The HLB system is a widely used method that was developed as a means of

selecting appropriate surfactants for emulsion purposes (Griffin 1949; Tadros 1994). The

HLB system is a scale that is based on the percentage of hydrophilic to lipophilic groups

in a molecule and can only be directly applied to nonionic surfactants. A low HLB is

characteristic of materials that are predominantly lipophilic such as plant oils, whereas a

higher HLB is characteristic of hydrophilic materials such as alcohols (Table 2.1). The

typical range of HLB for an oil-in-water (o/w) emulsifier is 8 to 18 (Tadros 1994), and

the HLB values for non-ionic surfactants are typically available from suppliers. There is

also a large amount of information readily available on the HLB required to emulsify

many different types of materials. For example, surfactants with an HLB range of 15-20

are known to be the most effective in enhancement of glyphosate efficacy for weed

control (Wyrill & Burnside 1977). The fairly high required HLB for glyphosate is

indicative of its water-soluble nature. Foliar nutrient uptake can also be influenced by

surfactants. Nonionic surfactants with HLB of 13-16 most significantly increased iron

uptake in chrysanthemum foliage (Nelson & Garlich 1969).

It is a fairly common industry practice to mix ionic and non-ionic surfactants

together as proprietary blends. One benefit of doing so is that using the non-ionic

material allows for the formulator to adjust the HLB of the formulation as needed

(Behrens 1964). Despite the long history of pesticide enhancement through use of

surfactants and the HLB system, there is very little information available on the required

HLB for essential oils. Essential oils are not water soluble, and therefore require

emulsifiers to achieve homogenization in an o/w formulation. Eucalyptus, lippia and

peppermint oils have required HLB values of 9.8, 12.1 and 12.3, respectively (Oradifiya

30
& Oladimeji 2002). Required HLB values for the following 25(b) listed essential oils are

not currently reported in the literature: cedar, cinnamon, citronella, clove, eugenol,

garlic, geraniol, geranium, lemongrass, mint, rosemary and thyme oils and 2-phenethyl

propionate.

Contact angle is the direct result of the interaction between a liquid droplet and a

particular type of surface. As the surface tension is decreased, the contact angle at the

liquid/surface interface is lowered. This helps the pesticide to spread more evenly,

increase retention on leaf surfaces, and enhance cuticle penetration of the active

ingredient, increasing the effectiveness of the pesticide. Cowles et al. (2000) reported a

strong correlation between trisiloxane surfactants with low surface tension as being very

toxic to twospotted spider mites (Tetranychus urticae), even though these surfactants are

registered as inert ingredients with the U.S. EPA. Along a similar vein, a patent was filed

in 2008 claiming that liquid formulations having a contact angle of less than 40 degrees

would result in rapid knockdown of arthropods (Hollis et al. 2008). Herbicides can also

be dramatically enhanced by lowering contact angle to provide better adhesion to leaf

surfaces (Singh et al. 1984). In one example, barnyardgrass control was increased from

30% with Diuron alone to up to 70% when treated with a formulation containing Diuron

plus an organosilicone surfactant. The contact angle was decreased from 94° with Diuron

alone to 25° when the surfactant was added (Singh et al. 2002). Gaining an

understanding of the interaction of contact angle of formulations containing essential oils

with their pesticide activity may be a useful tool in their commercial development.

The objective of this study was to determine the usefulness of required HLB

values and contact angle in formulating essential oil-based pesticides. This objective was

31
achieved by 1) determining the required HLB of the 25(b) essential oils cinnamon,

citronella, clove, eugenol, geraniol, geranium, lemongrass, peppermint, rosemary and

thyme; 2) measuring the contact angle of essential oil formulations made with different

surfactant systems; and 3) determining whether a significant correlation exists between

surfactant system being used with essential oils and the resulting pesticide activity. A

GC-MS analysis of the essential oil samples was conducted as a means of quantifying

primary constituents that are likely drivers for pesticide properties of the oils.

2. Materials and methods:

2.1. HLB required to emulsify essential oils

Required HLB values of cinnamon, citronella, clove, eugenol, geraniol, geranium,

lemongrass, peppermint, rosemary and thyme oils were determined following the

instructions outlined in Croda’s Guide to Emulsifier Selection (2008). Using the

nonionic surfactants Span 80, Tween 80, and Tween 20, solutions with HLB values of 6,

8, 10, 12, 14 and 16 were created (Table 2.2). The mixtures were prepared in 50 ml glass

tubes sealed with #4 rubber stoppers. In individual tubes, 16 g of essential oil and 1.6 g of

surfactant solution were added, and then tubes were vigorously shaken for 30 seconds.

Next, 22.4 g deionized (DI) water was added, and then tubes were inverted and shaken 20

times. The tubes were set aside in racks to allow for phase separation. Photographs were

taken at approximately 10, 20 and 60 minute intervals. The HLB solution that separated

into phases last was considered to be within + 1 HLB unit of the required HLB for that

particular essential oil. To validate the method, soybean oil was included since its

required HLB is known to be six. For rosemary oil, the test was repeated, and surfactant

32
to essential oil ratio was reduced by 50%, using 0.8 g, in order to get a better

determination of HLB. For Thyme oil, only 4 g of essential oil 0.4 g surfactant solution,

and 5.6 g water were used due to limited quantity of the thyme oil.

2.2 Contact angle

2.2.1 Preparation of samples

Ten essential oils were each formulated with the three unique surfactant

combinations described in Table 2.3. Contact angle was measured for all essential

oil/surfactant combinations created. Surfactant system A is compliant with 25(b)/4(a)

minimum risk regulations. System B utilizes non-ionic ethoxylated sorbitan esters,

which are commonly used as agro-chemical surfactants. These two particular surfactants

were chosen based on the determination of HLB required to emulsify the essential oils of

interest (Table 2.3). Surfactant system C is a combination of ingredients considered to be

“softer chemistry” components. Agnique® is an alkyl polyglucoside that is produced

from renewable raw materials and is readily biodegradable. Stepanol® (sodium lauryl

sulfate) is one of the most commonly used anionic surfactants across many industries,

including personal care products. It is also listed on the 25(b) list as a minimum risk

active ingredient.

System A: Na Caprylate and xanthan gum were individually added to a water

phase while on magnetic stir plate. Caprol® MPGO was added to essential oil in a

separate vial and shaken until homogenized. After xanthan gum had fully hydrated in

water for 15 to 20 minutes, the oil phase was added while the water was still on the stir

plate. After the oil phase was added, the mixture was stirred for an additional 15 minutes.

33
 System B: Tween 20 and then Tween 40 were added into a vial containing the

essential oil. The vial was shaken until mixture was homogenized, then poured into

water while on a stir plate. After the oil phase was added, the mixture was stirred for an

additional 15 minutes.

System C: Sodium lauryl sulfate was added to water while on stir plate. In a

separate vial, Agnique® was added to the essential oil and shaken until homogenized,

then poured into the water phase while stirring. After the oil phase was added, the

mixture was stirred for an additional 15 minutes.

2.2.2 Contact angle determination using tensiometer

Contact angle of the samples was determined using a Kruss tensiometer DSA 100.

The tensiometer test chamber was set to 50-60% relative humidity temperature ranged

from 21.0 to 22.6°C and gas carrier flow was 5 to 10 psi. The test surface used was

parafilm. Samples were thoroughly mixed with no visible sign of phase separation prior

to being pulled into the syringe. The time that the solution was in the syringe before it

was dropped onto the parafilm for measurement was minimized to reduce risk of phase

separation inside the syringe. The sample was dispensed at 200 micro liters per minute

until a drop fell onto the parafilm. The video was recorded at a rate of 12-25 frames per

second. The contact angle of the droplet was measured 240 milliseconds after the drop

had made contact with the surface, allowing enough time for the droplet to stabilize. The

contact angle was measured using the sessile drop method, which takes into effect the

properties of the droplet, as well as the solid surface and the atmosphere inside the

chamber (www.kruss.de/fileadmin/kruss-website/brochures/kruss-bro-dsa100-en.pdf.).

34
2.3 Gas chromatography/Mass spectrometry (GC/MS) analysis of essential oils

Cedar, cinnamon, citronella, clove, garlic, geranium, lemongrass, mint,

peppermint, rosemary and thyme oils were analyzed for their major constituents using

GC/MS. A list of essential oil suppliers is presented in Table 2.4. A list of constituent

standards used in the analysis is presented in Table 2.5. Computerized GC/MS analysis

was carried out on an Agilent 7890A gas chromatograph coupled to an Agilent 5975C

quadrupole mass spectrometer. The GC was fitted with a fused silica capillary column

(HP-5MS: 0.250 mm i.d._30 m) coated with a mobile phase consisting of (5% phenyl)-

methylpolysilicone (0.25 μm film thickness). During the course of the chromatographic

run, the column temperature was held at 35° C for 3 minutes, ramped at 2° C/min from

35° C to 90° C, and then ramped for a second time at 5° C/min to a final temperature of

140° C. The total time for the chromatographic separation was 45 minutes. Solutions of

essential oils and standard compounds were prepared in methanol for quantitative

purposes. All data were collected and quantified using Agilent ChemStatation®

software. The solutions were introduced into the GC via split injection (split ratio 1/100).

The temperature of the injector as well as the mass spectrometer detector was held at

250° C. Hydrogen was used as the carrier gas and the flow rate was 1.0 mL/min.

Generation of ions for mass spectrometric analysis was performed using 70 eV electron

ionization (EI) and analysis was performed in full scan mode (m/z 50-550). A solvent

delay of 4 minutes was used at the beginning of the separation in order to protect the EI

filament. The 2011 NIST (National Institute of Standards and Technology) mass spectra

35
library, version 2.0 g, was used to identify the compounds associated with the major

peaks in the chromatograms, which are presented in Appendix A.1.

2.4 Insecticide testing

An experiment measuring speed of knockdown of American cockroaches

(Periplaneta americana) was conducted to determine whether the contact toxicity of

essential oils could be influenced by the surfactant systems used. This experiment

consisted of a factorial treatment design that allowed for testing the effects of 10 essential

oils, each combined with three unique surfactant systems. The experimental unit

consisted of one lab-reared 9th instar American cockroach nymph, with 10 replicates per

treatment (n =10). The measured variable was speed of knockdown, recorded in seconds.

Knockdown is defined as the insect’s inability to control its movements in a coordinated

fashion, typically as a result of being treated with a pesticide. The experiment was a

completely randomized design and was conducted twice. A maximum time limit for

observations was set at 7 minutes (420 seconds). For observations that did not end in

mortality, a time of 420 seconds was recorded. Due to time constraints, it was not

possible to mix all of the 33 treatments in the laboratory and test them on the same day.

Therefore, for each day of testing, 6-10 samples were randomly selected, formulated in

the lab, and tested for cockroach knockdown within 24 hours.

Cockroaches were reared in glass tanks in a room maintained at 20-25°C with 30-

40% relative humidity. The rearing room was kept under dark conditions and

cockroaches were fed a diet of apples and dog food on a weekly basis to maintain colony

vigor. The cockroach nymphs were gathered from rearing tanks by anesthetizing with

36
carbon dioxide (CO2), individually placed into knockdown tubes, and randomly assigned

to treatments. Knockdown tubes were made of 10 inch diameter PolyVinylChloride

(PVC) tubes with an aluminum drain cup in the bottom. Food and water was not

provided to cockroaches after being removed from rearing tanks since tests were initiated

approximately 30 minutes afterwards. For each treatment, a 100 ml sample was placed

on a magnetic stir plate to maintain homogeneity of the formula. A 4.8 ml aliquot of the

sample was placed directly on the cockroach inside the knockdown tube using a pipette

(Figure 2.1). The knockdown tube was held over a beaker to catch excess sample as it

ran off the insect and through the drain cup in the bottom of the knockdown tube.

Immediately after application, the cockroach was transferred into an individual 10 cm

polypropylene bowl with a lid and observed for knockdown time. A timer was started as

soon as the application from the pipette was completed. Knockdown was determined

based on the insect’s inability to propel itself forward, control its movements in a

coordinated effort to move in a particular direction, or to upright itself.

2.5 Herbicide testing

Two greenhouse experiments and one field experiment were conducted to test for

foliar burn potential from essential oil formulations mixed with three different surfactant

systems. Fresh samples were made in the lab following the procedure described in

section 2.2.1 within 24 hours of the greenhouse and field applications. Applications were

made in late morning hours, and subjective % leaf injury ratings were measured at 1 hour

and 24 hours after application. A two week evaluation was also taken as a means of

measuring weed re-growth.

37
 The greenhouse experiments were a completely randomized design, and the

experimental unit was one individually potted six week old dandelion, with four

replicates per treatment (n=4). Dandelion seeds were germinated in flat trays filled with

MetroMix® potting media, then transplanted into individual 10 cm pots filled with the

same type of potting media within approximately one month of germination. The

transplants were allowed an additional two to three weeks to establish in the pots before

treatment applications were made. Treatments were randomly assigned to weeds.

Greenhouse temperatures ranged from 15° to 21° C at night and 21° to 32° C during the

day. Overhead irrigation was provided daily to maintain moisture in the weed pots.

Fertilization was provided through the irrigation system at the time of transplanting at an

end-rate dilution of 450 ppm N (174 ppm NH4 and 276 ppm Urea), 901 ppm P2O5, and

450 ppm K2O. Weeds were trimmed weekly to simulate effects of lawn mowing.

Applications were made using hand-pump bottles, and approximately one gram of spray

was applied to each dandelion, ensuring full coverage of the foliage.

The field experiment was conducted on a natural population of mature dandelions

growing in a low maintenance turf area that was mowed weekly to 7 cm height. The

experimental unit consisted of one dandelion, with four replicates per treatment (n=4).

The weeds were individually marked and randomly assigned to treatments in a

completely randomized block design. Spot applications to the dandelions were made

using the same hand-pump bottles used in the greenhouse trials. Approximately 2.5 g of

spray was foliar-applied to each dandelion, fully coating leaf tissue.

38
2.5 Data Analysis

The correlation of contact angle with insect knockdown speed was determined

using PROC CORR procedure (SAS 9.2 version). The dandelion data were subjected to

statistical analysis using PROC MIXED (SAS 9.2) and treatment mean separation was

based on Fisher’s LSD, with α =0.05. Data for runs 1 and 2 were treated as blocks within

the statistical model.

3. Results

3.1 HLB Results:

The majority of essential oils had a required HLB of 16, with the exceptions being

eugenol (6), peppermint (10), rosemary (12-14), and thyme (6) oils (Table 2.6). The

required HLB was determined at different time points after oils were mixed with

surfactants. Eugenol, geraniol, cinnamon, clove and thyme oils immediately began

separating into phases within the glass tubes; therefore required HLB was determined

within approximately 3 hours. Citronella, geranium, lemongrass, peppermint, and

rosemary oils held emulsions longer, and HLB was determined approximately 24 hours

after mixing with the surfactant systems. Rosemary oil held its emulsion with the

surfactants longer than all other oils tested, and the phase separation in the tubes

containing surfactant blends with HLB values of 12 and 14 were indistinguishable. The

required HLB for rosemary oil can not be more precisely determined without repeating

the experiment at lower surfactant rates. Soybean oil had a required HLB of 6,

confirming the validity of the method. Required HLB of peppermint had previously been

determined as 12.3 by Oradifiya & Oladimeji (2002), but results from this experiment

39
showed peppermint oil to have a required HLB of 10, with an error of +/- 1. Inherent

differences in constituents of essential oils based on environmental influence, harvest

methods, and geographic location may influence HLB needed to emulsify different

batches of an essential oil such as peppermint.

3.2 Influence of surfactant system on essential oils used as insecticides

Contact angle of all mixtures ranged from 47.5° to 81.6° (Table 2.7). All

surfactant systems reduced contact angle when mixed with water compared to water

alone, which had a contact angle of 111.2°. When added with water, System A surfactant

solution θ = 80.3°, System B surfactant solution θ = 81.6°, and System C surfactant

solution θ = 60.0° (Table 2.7). The addition of essential oils to the surfactant mixtures

further reduced contact angle in all formulations. It is worth noting that the surfactant

system with the lowest contact angle, C, was toxic to cockroaches when tested without

being combined with an essential oil, with a knockdown time of 309 seconds (Table 2.7).

Surfactant systems A and B were not toxic to cockroaches when tested without an

essential oil, so the maximum time of 420 seconds was recorded as the knockdown time.

When averaged across all essential oils, surfactant system C treatments had significantly

faster KD time than treatments containing systems A or B (Table 2.9). Some essential

oils were very fast KD agents regardless of surfactant system (clove, eugenol, geraniol,

geranium, peppermint and thyme oils), and KD speed was not significantly increased

with system C for these oils, except for thyme oil (Table 2.8). It is not understood at this

time why thyme oil KD speed was significantly faster when combined with surfactant

systems A or B, compared to C. Cinnamon, lemongrass and rosemary oils’ KD speed

40
were significantly increased with system C (Table 2.8). The strength of the relationship

between knockdown time across all essential oils to contact angle was dependent on the

surfactant system used. Treatments containing surfactant system A best fit the regression

line, with an R2 = 0.74 (Figure 2.2). A correlation of contact angle and knockdown time

could not be determined with treatments containing surfactant systems B and C, with R2

values of 0.52 and 0.45, respectively. A PROC CORR analysis of the correlation of KD

time with all treatments indicated a Pearson correlation coefficient of 0.67. However,

because of the significant differences in KD of the essential oils as a main effect, future

studies should limit the study of contact angle influence on KD to one essential oil at a

time.

3.2 Surfactant system effects on efficacy of essential oil herbicides

There was no correlation of contact angle to herbicide activity (p=0.11); however,

there was a highly significant increase in herbicide injury to dandelions when essential

oils were mixed with surfactant system A at 1, 6, and 24 hours after application (p

<0.0001 at all three data collection periods) (Tables 2.10 and 2.11). All essential oils

combined with system A, except rosemary oil, burned greater than 90% dandelion foliage

within 24 hours in greenhouse experiments. Most essential oil formulations containing

systems B and C burned 25% or less foliage within 6 hours and 50% or less foliage

within 24 hours. One exception is thyme oil which injured nearly 90% foliage with

system C by 24 hours. It is worth noting that system A, in the absence of an essential oil,

is the only system that had herbicide activity on its own, with 23% injury by 24 hours

41
(Table 2.10). The validity of the results obtained in the greenhouse was validated with a

field experiment, which yielded similar results (Table 2.11).

3.4 GC-MS analysis of essential oils

Citronella, clove, geranium, lemongrass, mint, peppermint, rosemary and thyme

oils primarily consisted of monoterpenes, which were both cyclic and acyclic in structure.

cedar oil was the only 25(b) essential oil that was composed of cyclic sesquiterpene

compounds, a-cedrene, cis-thujopsene, and (+)-cedrol (Table 2.12). Since not all

components could be quantified, it is not possible to determine percent of the oils made

up of aromatic versus aliphatic terpenes. However, one could reasonable argue that clove

and thyme oils consisted of a larger percentage of aromatic compounds than the other

oils. Furthermore, it is well known that clove oil typically consists of 60-80% eugenol,

and thyme oil consists of 60% or more thymol, both of which are aromatic monoterpenes

(Khan & Abourashed 2010). No peaks were observed for garlic oil, and it is assumed

that the known major constituent, diallyl sulfide, also known as “oil of garlic” was lost in

the first few minutes along with the methanol solvent. For cinnamon oil, there were three

distinct peaks with retention times of 14.366, 20.294, and 26.860, and two distinct peaks

in lemongrass oil with retention times of 17.242 and 18.252. These were labeled as

unidentified since they could not be matched within the library at a threshold of 85% and

we did not have standards at those retention times (Table 2.12). However, the

compounds in the standard mix with retention times of 20 minutes or greater were

bicyclic compounds camphene, (+)-cedrene, coumarin and cedrol. This indicates that the

42
two unidentified peaks with retention times of 20.294 and 26.860 in cinnamon oil are also

likely to be bicyclic compounds. Trans-cinnamaldehyde had a retention time of 14.345

in the standards analysis, so this is most likely the identification of the peak in cinnamon

oil which had a retention time of 14.366 minutes. It was not specified on the sample of

cinnamon oil from The Essential Oil company which plant parts were used for the oil

extraction. Wijesekera et al (1974) describe significant variations in cinnamon oil

constituents depending on whether the oil is extracted from stem bark, leaves or root stem

bark. It is understood that if these experiments had included multiple sources of

cinnamon oil, or any of the other oils for that matter, the pesticide efficacy could vary

significantly due to constituent differences.

4. Conclusions

Determination of required HLB values was shown to be a useful tool for

formulating oil-in-water emulsions with essential oils. Regardless of HLB, the essential

oils eugenol, cinnamon, clove, geraniol and thyme were less inclined to emulsify in water

than others and phase separation occurred very quickly. It is interesting that these oils

were also the most effective insecticides and herbicides. While citronella, geranium,

lemongrass, peppermint, and rosemary oils held emulsions longer, these oils were still

highly lipophilic.

One hypothesis as to why eugenol, cinnamon, clove, geraniol and thyme oils are

most difficult to emulsify with water is that their main constituents are terpenoids

containing an aromatic ring (Table 2.12). These compounds are not water soluble.

Eugenol is the primary constituent in clove oil, comprising around 80%. Cinnamon oil

43
primarily consists of cinnamaldehyde, as well as eugenol and coumarin, all of which

contain an aromatic ring. Thyme oil consists primarily of thymol, and can also contain

significant amounts of carvacrol, both of which are also aromatic monoterpenes.

Geraniol, an acyclic terpene alcohol may be the exception in this hypothesis. In this

study, geraniol was highly effective as a contact insecticide as well as herbicide.

However, a study conducted by Phillips et al. (2010) concluded that while geraniol was

found to be toxic to German cockroaches, other essential oil constituents such as thymol,

trans-cinnamaldehyde, eugenol, and carvacrol had lower LD50 values at most life stages.

These components also had higher densities than linalool, limonene, menthone and

geraniol, and toxicity to German cockroaches was significantly positively correlated to

density and boiling point. Rice and Coats (1994) also reported that aromatic compounds

were generally more toxic than aliphatic compounds to the house fly, Musca domestica.

The higher potency of constituents containing aromatic structures compared to acyclic

compounds is that they are generally more difficult to metabolize once inside the insect,

leading to slower detoxification. One explanation as to why geraniol treatments in the

current study were equivalent to aromatic terpenes is that, being a pure constituent,

geraniol was actually higher in percent monoterpene content when added at 5% in

formulations as compared to the essential oils, which are blends of many types of

constituents. This also applies to eugenol and 2-phenethyl propionate treatments.

Although there was a positive relationship to knockdown time and contact angle

of the formulations containing surfactant system A, none had contact angles less than

40°. In a patent filed by Hollis et al. (2008), there is a significant increase in KD speed of

surfactant mixtures which have a contact angle less than 40°. Further exploration of the

44
contact angle and knockdown speed relationship using “super-spreader” surfactants could

be investigated as a means of improving activity of some of the other essential oils.

Alternatively, using low contact angle surfactants may allow for lower concentrations of

essential oils while still maintaining good KD activity.

5. Figures

Figure 2.1. Application of treatment to cockroach inside knockdown tube using a pipette

45
 85
A
B
80 C
Linear (C)
Linear (B)
75
Linear (A)

70
System A:
Contact Angle

y = 0.0604x + 54.072
65
R2 = 0.7411

60
System B:
y = 0.0375x + 50.312
55 2
R = 0.5165

50
System C:
y = 0.0521x + 58.203
45 2
R = 0.4518

40
0 50 100 150 200 250 300 350 400
KD time (s)

Figure 2.2. Positive linear relationship between knockdown time of American

cockroaches with increasing contact angle of essential oil formulations containing
surfactant systems A (89.57% DI water, 2% w/w sodium caprylate, 0.33% Caprol®
MPGO, 0.1% xanthan gum), B (90% DI water, 2.5% w/w Tween 20, 2.5% Tween 40) or
C (92.4% DI water, 2.5% w/w alkyl polyglucoside, 0.1% sodium lauryl sulfate)

6. Tables

Class Required HLB

Vegetable oil family 5-7

Silicone oils 8-12
Petroleum oils 9-11
Typical ester emollients 10-12
Fatty acids; Alcohols 14-15
Table 2.1. Guideline for required HLB to emulsify various classes of materials, from The
HLB System: Croda's time-saving guide to emulsifier selection

46
 HLB Kit
HLB % Material Span 80 (g) Tween 80 (g) Tween 20 (g)
83% Span 80/
6 17% Tween 80 41.5 8.5 -
65% Span 80/
8 35% Tween 80 32.5 17.5 -
46% Span 80/
10 54% Tween 80 23.0 27.0 -
28% Span 80/
12 72% Tween 80 14.0 36.0 -
9% Span 80/
14 91% Tween 80 4.5 45.5 -
60% Tween 20/
16 40% Tween 80 - 20.0 30.0
Table 2.2. Nonionic HLB kit used in determining the required HLB for 10 essential oils

Surfactant system A Surfactant system B Surfactant system C

5% w/w Essential oil 5% w/w Essential oil 5% w/w Essential oil
89.57% w/w DI water 90% w/w DI water 92.4% w/w DI water
2% w/w Sodium caprylate 2.5% w/w Tween 20 2.5% w/w Alkyl polyglucoside
0.33% w/w Caprol®MPGO 2.5% w/w Tween 40 0.1% w/w Sodium lauryl sulfate
0.1% w/w Xanthan gum
Table 2.3. Formulation components in the 3 surfactant systems tested in phase 2 insect
knockdown and dandelion herbicide experiments

47
Ingredient Supplier Genus species
Cedar oil Cedarcide Industries Juniperus virginiana
Cinnamon oil The Essential Oil Co. Cinnamomum cassia
Citronella oil The Essential Oil Co. Cympogon nardus
Clove (bud) oil The Essential Oil Co. Eugenia caryophylatta
Garlic oil Brenntag Mid-South Not specified
Geranium oil The Essential Oil Co. Pelargonum graveolens
Lemongrass oil The Essential Oil Co. Cympogon citratus
Mint oil Brenntag Mid-South Not specified
Peppermint oil The Essential Oil Co. Mentha piperita
Rosemary oil Brenntag Mid-South Rosmarinus officinalis
Thyme oil The Essential Oil Co. Thymus vulgaris
Eugenol (>95% purity) Acros Organics n/a
Geraniol (>95% purity) Acros Organics n/a
2-phenethyl propionate Acros Organics n/a
(>95% purity)
40% Sodium caprylate Calvary Industries n/a
solution
Agnique® PG264 Cognis n/a
Caprol® MPGO Abitec n/a
Soybean oil Archer Daniels Midland Glycine max
Stepanol® WA-100 Stepan n/a
Tween 20 Croda n/a
Tween 40 Croda n/a
Xanthan gum Jungbunzlauer Austria AG Xanthomonas campestris
Table 2.4. Supplier list for raw materials used in phases 1 and 2 insecticide and herbicide
experiments
(n/a = not applicable)

48
Standards Purity Supplier Product code/#
Allyl sulfide 97% Sigma Aldrich A35801
Camphene 95% Sigma Aldrich 456055
Camphor 96% Sigma Aldrich 148075
(+) Citronellal >95% Sigma Aldrich 27470
Β-Citronellol 95% Sigma Aldrich C83201
Coumarin >99% Sigma Aldrich C4261
Eucalyptol 99% Sigma Aldrich C80601
Eugenol 99% Alfa aesar A14332
d-Limonene 95% Sigma Aldrich UC101-26
Linalool 97% Alfa aesar A14424
Menthone 97% Sigma Aldrich 63680
Menthyl acetate 99% Sigma Aldrich 441031
Methyl cinnamate 99% Alfa aesar A15975
Α-Pinene 98% Sigma Aldrich 147524
Table 2.5. Constituent standards used in the GC-MS analysis of essential oils

Essential oil Required HLB

(+/- 1)
Cinnamon oil 16
Citronella oil 16
Clove oil 16
Eugenol 6
Geraniol 16
Geranium oil 16
Lemongrass oil 16
Peppermint oil 10
Rosemary oil 12-14
Thyme oil 6
Soybean oil 6
Table 2.6. Required HLB to emulsify the noted oils in distilled water.. (HLB values in
the table are based on one experiment).,

49
 Surfactant system Oil θ (degrees) KD time in seconds
None (water + surfactants) 80.3 420.0
Cinnamon 76.8 349.2
Citronella 63.5 79.2
Clove 60.3 43.2
A
Eugenol 57.2 30.0
(89.57% DI water
Geraniol 51.5 28.5
2% sodium
Geranium 50.8 30.4
caprylate, 0.33%
Caprol® MPGO, Lemongrass 64.6 129.0
0.1% xanthan gum) Peppermint 64.0 42.8
Rosemary 56.0 69.2
Thyme 80.3 27.1
113.5
Mean 62.8 (nlog 4.04, SE 0.039)
None (water + surfactants) 81.6 420.0
Cinnamon 69.6 312.1
Citronella 49.9 49.7
Clove 57.5 53.0
Eugenol 60.2 51.9
B
Geraniol 50.2 35.2
(90% DI water, 2.5%
Geranium 54.0 42.4
w/w Tween 20, 2.5%
Tween 40) Lemongrass 61.6 96.0
Peppermint 60.7 57.9
Rosemary 61.1 117.0
Thyme 65.0 33.3
115.3
Mean 61.1 (nlog 4.17, SE 0.039)
None (water + surfactants) 60.0 309.5
Cinnamon 57.7 141.2
Citronella 52.7 62.0
Clove 55.9 41.5
C
Eugenol 48.9 32.6
(92.4% DI water,
Geraniol 47.5 25.9
2.5% w/w alkyl
Geranium 49.7 51.3
polyglucoside, 0.1%
sodium lauryl Lemongrass 52.6 70.7
sulfate) Peppermint 49.9 32.3
Rosemary 52.8 44.2
Thyme 59.2 82.2
81.2
Mean 53.3 (nlog 3.86, SE 0.039)
Table 2.7. Contact angle of essential oils, 5% v/v, combined with three unique surfactant
systems labeled A, B and C and the corresponding knockdown time of 9th instar
American cockroach nymphs. Statistical analysis of KD time using Scheffe’s for treatment mean
separation, α = 0.05.

50
Essential oil System A System B System C
Cinnamon <.0001 0.0083 -
Citronella NS NS -
Clove NS NS -
Eugenol NS 0.07 -
Geraniol NS NS -
Geranium NS NS -
Lemongrass <0.001 <.0001 -
Peppermint NS 0.03 -
Rosemary NS <.0001 -
Thyme <.0001 0.01 -
Blank (surfactants 0.02 0.02 -
only- no EO)
Table 2.8. P values of knockdown time comparisons of essential oils + surfactant system
C compared to systems A (89.57% DI water, 2% w/w sodium caprylate, 0.33% Caprol®
MPGO, 0.1% xanthan gum) and B (90% DI water, 2.5% w/w Tween 20, 2.5% Tween
40). Fisher’s LSD., NS = not significant, with p value > 0.10.

Surfactant System N Mean KD Time in

seconds (nlog)
A 220 114 (4.04) a
B 220 115 (4.17) a
C 220 81 (3.86) b
Table 2.9. Mean KD time of surfactant systems A (89.57% DI water, 2% w/w sodium
caprylate, 0.33% Caprol® MPGO, 0.1% xanthan gum), B (90% DI water, 2.5% w/w
Tween 20, 2.5% Tween 40), and C (92.4% DI water, 2.5% w/w alkyl polyglucoside,
0.1% sodium lauryl sulfate) on 9th instar American cockroaches, averaged across ten
essential oils. Means followed by same letter within a column are not significantly different at
α=0.05, according to Scheffe’s.

51
Essential oil Surfactant system 1 hr 6 hr 24 hr 5 Days
None A 1 7 23 35
B 0 0 0 1
C 0 0 0 0
Cinnamon A 40 94 100 100
B 2 18 43 65
C 4 22 51 72
Citronella A 38 85 93 96
B 4 5 8 23
C 1 6 11 22
Clove A 49 96 99 100
B 14 18 35 53
C 12 18 44 71
Eugenol A 46 91 97 100
B 19 22 44 60
C 19 38 63 74
Geraniol A 48 89 99 100
B 20 28 51 82
C 16 24 49 90
Geranium A 43 69 97 100
B 9 11 15 32
C 9 11 24 5
Lemongrass A 43 72 99 100
B 9 10 28 59
C 8 13 28 75
Peppermint A 36 59 93 99
B 7 6 10 18
C 11 11 11 28
Rosemary A 7 24 60 68
B 1 1 5 5
C 3 3 4 0
Thyme A 46 70 99 99
B 8 11 18 35
C 34 46 89 97
Std Error 2.16 4.05 5.19 6.60
Table 2.10. Percent injury of dandelions 1 hour, 6 hours, 24 hours and 5 days after
application of 10 essential oils formulated with surfactant systems A (89.57% DI water,
2% w/w sodium caprylate, 0.33% Caprol® MPGO, 0.1% xanthan gum), B (90% DI
water, 2.5% w/w Tween 20, 2.5% Tween 40), and C (92.4% DI water, 2.5% w/w alkyl
polyglucoside, 0.1% sodium lauryl sulfate) in 2 greenhouse experiments, Fisher’s LSD, α
= 0.05, n = 8.

52
Essential oil Surfactant system 1 hr 6 hr 24 hr 6 days
None A 4 123 23 25
B 0 0 0 0
C 0 0 0 0
Cinnamon A 66 99 98 95
B 9 20 31 28
C 5 20 21 29
Citronella A 60 100 100 99
B 0 3 3 11
C 0 0 0 0
Clove A 70 95 100 97
B 8 11 26 46
C 0 0 0 0
Eugenol A 61 98 93 84
B 0 0 10 0
C 10 13 16 53
Geraniol A 68 100 98 93
B 19 21 23 44
C 10 24 61 85
Geranium A 60. 46 100 98
B 0 1. 13 24
C 0 6 19 33
Lemongrass A 40 91 100 95
B 3 5 19 45
C 0 3 16 33
Peppermint A 21 58 74 57
B 0 0 11 14
C 0 0 0 18
Rosemary A 0 23 43 29
B 0 0 0 0
C 0 0 0 3
Thyme A 50 85 100 94
B 0 0 0 9
C 9 16 36 74
Std Error 2.50 3.26 4.23 8.08
Table 2.11. Percent injury of dandelions 1 hour, 6 hours, 24 hours and 6 days after
application of 10 essential oils formulated with surfactant systems A (89.57% DI water,
2% w/w sodium caprylate, 0.33% Caprol® MPGO, 0.1% xanthan gum), B (90% DI
water, 2.5% w/w Tween 20, 2.5% Tween 40), and C (92.4% DI water, 2.5% w/w alkyl
polyglucoside, 0.1% sodium lauryl sulfate) under field conditions, , Fisher’s LSD, α =
0.05, n = 4.

53
Essential Retention Compound Structure Mol wt % of oil
oil time (g/mol)
(minutes)
Cedar 20.267 α-cedrene Cyclic ST 204.35 4.06
20.986 Cis-thujopsene Tricyclic ST 204.35 -
25.687 (+)-cedrol Cyclic ST 222.37 5.11
Cinnamon 14.366 Unidentified - - -
20.294 Unidentified - - -
26.860 Unidentified - - -
Citronella 8.946 Citranellal Acyclic MT 154.25 23.96
12.617 Citranellol Acyclic MT 156.27 7.18
13.995 Geraniol Acyclic MT 154.25 9.05
17.143 Isopulegol Cyclic MT 154.25 -
18.713 Citranellyl formate Acyclic MT 184.28 -
19.838 β-Myrcene Acyclic MT 136.23 -
22.531 β-Copaene Tricyclic MT 204.35 -
23.836 β-Cadinene Bicyclic ST 204.35 -
24.520 Elemol Cyclic ST 222.37 -
Clove 18.784 Eugenol Aromatic MT 164.20 25.46
20.580 β-caryophyllene Bicyclic ST 204.35 -
24.152 Eugenol acetate Aromatic MT 206.24 -
acetate
Garlic No peaks - - - -
observed
Geranium 6.740 Isogeraniol Acyclic MT 154.25 -
8.758 Menthone Cyclic MT 154.25 1.78
9.198 Menthone Cyclic MT 154.25 -
12.710 Citranellol Acyclic MT 156.27 14.78
13.990 Geraniol Acyclic MT 154.25 6.31
15.081 Citranellyl formate Acyclic MT 184.28 -
16.453 β-Myrcene Acyclic MT 136.23 -
26.231 α-Gurjunene Tricyclic ST 204.35 -
Lemongrass 13.076 Menthol derivative Cyclic MT 156.27 -
13.943 Geraniol Acyclic MT 154.25 4.13
14.710 β-Citral Acyclic MT 152.23 -
17.242 Unidentified - - -
18.252 Unidentified - - -
19.852 β-Myrcene Acyclic MT 136.23 -
Table 2.12. GC-MS constituent analysis, retention times and the corresponding chemical
structures and molecular weights of 25(b) essential oils
MT = monoterpenoid; ST = sesquiterpenoid

54
 continued
Table 2.12 continued

Mint 8.825 Menthone Cyclic MT 154.25 18.21

9.238 Unidentified - - -
9.327 Isomenthone Cyclic MT 154.25 -
9.837 Menthol Cyclic MT 156.27 26.68
15.905 Menthyl acetate Cyclic MT 198.30 3.39
Peppermint 8.792 Menthone Cyclic MT 154.25 18.01
9.205 Unidentified - - -
9.269 Isomenthone Cyclic MT 154.25 -
9.741 Menthol Cyclic MT 156.27 15.69
15.888 Menthyl acetate Cyclic MT 198.30 2.53
acetate
Rosemary 4.493 Eucalyptol Cyclic ether 154.25 28.98
MT
8.283 Camphor Bicyclic MT 152.23 6.65
20.563 β-caryophyllene Bicyclic ST 204.35 -
Thyme 4.332 Cymene Aromatic MT 134.22 -
6.742 Linalool Acyclic MT 154.25 3.07
16.140 Thymol Aromatic MT 150.22 -
*Compounds in italics are proposed based on 2011 NIST, version 2.0 g, mass spectral
library matching.

References

Behrens, RW. 1964. The physical and chemical properties of surfactants and their
effects on formulated herbicides. Weeds. 12: 255-258.

Colby, SR. 1967. Calculating synergy and antagonistic responses of herbicide

combinations. Weeds. 15: 20-22.

Cowles, RS, EA Cowles, AM McDermott, and D Ramoutar. 2000. Inert formulation

ingredients with activity: toxicity of trisiloxane surfactant solutions to two-spotted spider
mites (Acari: Tetranychidae). J. Econ. Entomol. 93(2): 180-188.

Griffin, WC. 1949. Classification of surface active agents by HLB. J. Cosmet.

Chemists. 1: 311-326.

Hollis, S, C McDonald, and J Rader. 2008. Methods for treating arthropods. U.S. Patent
Application. Pub. No. US2008/0221223 A1. Pub Date: September 11, 2008.

55
Khan, IA and EA Abourashed. 2010. Leung’s Encyclopedia of Common Natural
Ingredients- Used in Food, Drugs, and Cosmetics (3rd edition). John Wiley and Sons.

Nelson, PV and HH Garlich. 1969. Relationship of chemical classification and

hydrophile-lipophile balance of surfactants to enhancement of foliar uptake of iron. J. Ag.
and Food Chem. 17: 148-152.

Orafidiya, LO and FA Oladimeji. 2002. Determination of the required HLB values of

some essential oils. Int’l J. Pharma. 237: 241-249.

Singh, M, JR Orsenigo, and DO Shah. 1984. Surface tension and contact angle of
herbicide solutions affected by surfactants. J. Am. Oil Chemists’ Soc. 61: 596-600.

Singh, M, S Tan, and SD Sharma. 2002. Adjuvants enhance weed control efficacy of
foliar-applied Diuron. Weed Tech. 16: 74-78.

Tadros, T. 1994. Surfactants in agrochemicals. Surfactant Science series. Vol. 54. ISBN
0-8247-9100-2.

The HLB System: Croda’s time-saver guide to emulsifier selection. Edison: Croda,
2008. Print

www.kruss.de/fileadmin/kruss-website/brochures/kruss-bro-dsa100-en.pdf. Drop shape

analysis system DSA-100 manual.

Wyrill, JB and OC Burnside. 1977. Glyphosate toxicity to common milkweed and hemp
dogbane as influenced by surfactants. Weed Sci. 25: 275-287.

56
Chapter 3: Contact toxicity of U.S. EPA minimum risk plant essential oils on American

cockroach (Periplaneta americana) and the influence of surfactants on knockdown speed

1. Introduction

Cockroaches are a major household pest worldwide. Of the 4,000 known species

of cockroaches, the American cockroach, Australian cockroach (Periplaneta

australasiae), brownbanded cockroach (Supella longipalpa), German cockroach

(Blattella germanica) and oriental cockroach (Blatta orientalis) are listed as public health

pests by the U.S. EPA due to transmission of Salmonella, allergens, fecal deposition and

hepatitis (PR Notice 2002-1). German cockroaches are the most common pest for which

indoor pesticide applications are made in public housing (Greene & Breisch 2002, Miller

& Meek 2004). Not only are these pests transmitters of etiological diseases, but there is

also a strong emotional response to cockroach infestations, which are most commonly

found in food preparation areas and bathrooms. There are several product forms used to

control cockroach infestations indoors, including baits, aerosols, trigger-sprayer Ready-

to-Use (RTU), and traps. The liquid control products typically work either by contact

spray or by residual control through treating pest-frequented surfaces.

The most common insecticides reportedly used for residual control of pests in

public buildings include dichlorvos, chlorpyrifos, propoxur, and pyrethroids (Wei et al.

2001). All of these, except for pyrethroids, have been removed from residential use by

57
the U.S. EPA. Unfortunately, insect resistance to these synthetic compounds is common.

To manage resistance, integrated pest management (IPM) strategies are often

recommended (Miller & Meek 2004). Use of essential oils from aromatic plants may be

a viable tool for the adopter of IPM strategies, which can offer quick contact kill of pests

on sight, with no residual control left behind, minimizing the risk of resistance build-up.

There is also a need for these types of products in the consumer market for pest control.

An estimated 14 million pounds of insecticides were used in home and garden in 1999,

and the vast majority was used towards controlling public health pests such as flies,

cockroaches, ticks and mosquitoes, as well as termites (Ware & Whitacre 2004, Yu

2008). Worldwide, natural products make up 7.6% of the insecticides used across all

sectors (Nauen 2006).

Essential oils vary in their potency as insecticides, and concentrations within a

formulation can greatly influence performance in terms of speed of knockdown (KD).

Knockdown is defined as the insect’s inability to control its movements in a coordinated

fashion, typically as a result of recently coming in contact with a pesticide. Some

pesticide products in the consumer market combine multiple essential oils within liquid

formulations (Table 3.1), but there is no support in the scientific literature showing a

performance benefit of using combinations of essential oils compared to individual oils as

stand-alone active ingredients. The majority of pesticide products containing essential

oils sold in the U.S. market are formulated with 25(b)-listed essential oils so that they are

exempt from EPA federal registration. Many of these products also use other 25(b)

active ingredients, such as soybean oil, sesame oil, corn oil, malic acid and sodium lauryl

sulfate (Table 3.1). With significant price differences among essential oils, it is critical to

58
understand which oils perform best alone, and whether efficacy performance of a

formulation can be enhanced by adding additional essential oils.

There are varied reports as to exactly how essential oils work as insecticides. The

means of application techniques for essential oils include fumigants and contact sprays.

They have been reported as having antifeedant properties, repellent properties, and

ovicidal activity, and can cause reproductive delays and modifications in development

(Isman 2000; Regnault-Roger 1997; Rice & Coats 1994; Singh et al. 1989; Choi et al.

2002; Choi et al. 2004; Huang et al. 1998; Petrakis et al. 2005). The lipophilic plant oils

are known to kill soft-bodied insects by disrupting their waxy cuticle, leading to

dessication. There is also evidence that, once penetrated through the cuticle, or entered

via the spiracles, certain components within essential oils may begin to block

neurotransmitters, digestive enzymes or growth hormones (Sampson et al. 2005; Tarelli

et al. 2009; Tisserand & Balacs 1995). In addition to the physical effects of disrupting

cellular tissue, the rapid toxic effect of essential oils indicates that there is a neurotoxic

affect on pests (Enan 2001, Isman & Machial 2006). Through experiments of volatile oil

constituents’ insecticidal and repellent properties against the American cockroach, Ngoh

et al. (1998) concluded that “contact toxicity decreases when the double bond is closer to

the aromatic ring in isoeugenol compared to eugenol and methyl-eugenol. Furthermore,

the additional methoxy group in methyl-eugenol increases KD capability without

significantly affecting its lethal capacity”. In a study by Phillips et al. (2010) there was a

negative correlation of essential oil constituent density and its toxicity to German

cockroaches (Blattella germanica). The authors concluded that the density may effect

the penetration of the compounds through the cuticle. It was also found that aromatic

59
compounds such as carvacrol, thymol, eugenol, and trans-cinnamaldehyde were more

toxic than the aliphatic compound geraniol. The conclusion was that the benzene ring

present in aromatic compounds is not easily metabolized and detoxified within the insect.

Rice and Coats (1994) also found that aromatic compounds were more efficacious to

house fly than aliphatic compounds. In a study by Isman (2000) to determine pesticide

activity of phenols versus monoterpenes, the extent of toxicity of these compounds was

highly dependent on the test species. At least one of the phenols eugenol and carvacrol

were more active on fruit fly (Drosophila melanogaster), tobacco cutworm (Spodoptera

litura), housefly and western corn rootworm beetle (Diabrotica virgifera virgifera) than

the terpenes a-terpineol and terpinen-4-ol. However, for twospotted spider mites

(Tetranychus urticae), terpinen-4-ol was much more active than either phenol.

Grodnitzky and Coats (2002) utilized QSAR (Quantitative Structure-Activity

Relationships) models as a means of predicting insect toxicity of thirty monoterpenoids.

They found that as the electron accessibility increased in the monoterpenoids, there was a

corresponding increase in toxicity, suggesting a change in receptor(s) binding affinity.

The QSAR derived from the acyclic monoterpenoids as compared to the aromatic

monoterpenoids suggest there may be a different mode of action.

In recent years, it has been discovered that there is a relationship between

octopamine and tyramine receptors and essential oil mode of action in insects. The role

of octopamine and tyramine, neuroactive ligands present in invertebrates, is well

understood in terms of their physiological functions as neurotransmitters,

neurohormones, and neuromodulators (Enan 2005; Bischof & Enan 2004). In Enan’s

(2005) research on essential oil constituents and insecticidal mode of action in D.

60
melanogaster, thymol and carvacrol induced a decrease in receptor binding activity,

while significantly increasing cyclic AMP (cAMP) and Ca2+ levels. The presence and

location of a hydroxyl group was critical in the tyramine receptor mediation of

insecticidal activity, with the 2 or 3 ring positions being most favorable. Kostyukovsky

et al. (2002) observed a similar response of significantly increased cAMP levels in

abdominal epidermal tissue of the cotton bollworm moth (Helicoverpa armigera) with

two un-named essential oil terpenes belonging to the Labiatae family. This reaction is

similar to the effect of octopamine on cAMP levels, indicating that there may be a

competitive activation of the octopaminergic receptors by these terpenes. Activity of

these constituents was not found to correlate with acetyl cholinesterase activity.

In addition to studying the intrinsic efficacy of 25(b) essential oils as stand-alone

active ingredients and in combination with one another, there is also a critical need to

understand how to best use surfactant chemistry to improve efficacy. The influence of

surfactants and inerts in liquid formulations can significantly alter performance of a

pesticide active ingredient. Some surfactants have pesticide activity of their own and

have even been recommended as an alternative means of controlling pests to mitigate

resistance issues with conventional active ingredients (Liu & Stansly 2000; Butler et al.

1993; Imail et al. 1994; Imail & Tsuchiya 1995). Cowles et al. (2000) reported a strong

correlation of trisiloxane surfactants with low surface tension being very toxic to

twospotted spider mites even though these surfactants are registered as inert ingredients

with the U.S. EPA. Along a similar vein, a patent was filed in 2008 claiming that liquid

formulations having a contact angle of less than 40 degrees would result in rapid KD of

arthropods (Hollis et al. 2008).

61
 The fourteen 25(b) essential oils and essential oil constituents selected for the

current study are 2-phenethyl propionate (2PP), cedar (CED), cinnamon (CIN), citronella

(CIT), clove (CLO), eugenol (EUG), garlic (GAR), geranium (GER), geraniol (Giol),

lemongrass (LEM), mint (MIN), peppermint (PEP), rosemary (ROS) and thyme (THY)

oils.

2. Materials and methods

2.1. Phase 1: Pair-wise combinations of essential oils tested for KD of American

cockroach nymphs

To determine whether there is a benefit to combining essential oils to increase

speed of KD, 14 essential oils were tested at 10% w/w concentrations in an aqueous

solution, as well as combined with one another, each at 5% + 5% w/w. All pair-wise

combinations of the 14 oils were made, for a total of 105 combinations. Preliminary

formulation work was conducted to identify the inert materials, rates and combinations

which provided a minimum of two minutes physically stable emulsion.

All ingredients used throughout the experiments were used from the same batch/lot to

avoid batch to batch variability which is common to naturally sourced materials. All

treatments in the Phase 1 experiment contained the same inert system, which consisted

of, by weight:

5% soybean oil – a plant oil (triglyceride) to aid in emulsion stability

3% sodium caprylate - a fatty acid salt used for emulsion purposes

1% Caprol® MPGO- a polyglycerol oleate used for emulsion purposes

0.1% xanthan gum - a thickening agent to prolong emulsion stability

62
 American cockroaches were selected as the model pest for these experiments

because of their widespread distribution, notoriety as household pests, and large size

makes them a favorable model for toxicological experiments. The experimental unit

consisted of one lab-reared 9th instar nymph, n = 4. Cockroaches were reared in glass

tanks in a room maintained at 20-25°C with 30-40% relative humidity. The rearing room

was kept under dark conditions and cockroaches were fed a diet of apples and dog food

on a weekly basis to maintain colony vigor. The measured variable was speed of KD,

recorded in seconds. Knockdown time was determined to be the point at which the insect

was unable to propel itself forward, control its movements in a coordinated effort to

move in a particular direction or to upright itself. A maximum time limit for observations

was set at seven minutes (420 seconds), so 420 seconds was recorded for cockroaches

that were not knocked down within seven minutes.

The experiment was completely randomized and conducted twice. Due to time

constraint, 105 treatments could not be made in the laboratory and tested on the same

day. For each day of testing, 5-10 formulations were randomly selected and tested for

KD within 48 hours of being made. The assumption that temporal separation of

treatments does not significantly affect KD is addressed later in this chapter (Phase 2.5).

Cockroaches were gathered from rearing tanks by anesthetizing with carbon dioxide

(CO2), and placed individually into KD tubes and randomly assigned to treatments.

Knockdown tubes were made of 10 inch diameter PolyVinylC tubes, with an aluminum

drain cup in the bottom. Food and water were not provided to cockroaches after being

removed from rearing tanks since tests were initiated approximately 30 minutes

afterwards. For each treatment, a 100 ml sample was placed on a magnetic stir plate to

63
maintain homogeneity of the formula. A 4.8 ml aliquot of the sample was placed directly

on the cockroach inside the KD tube using a pipette (Figure 2.1). The KD tube was held

over a beaker to catch excess sample as it ran off the insect and through the drain cup.

Immediately after application, the cockroach was transferred into an individual 10 cm

diameter polypropylene bowl with a lid and observed for KD time. The clock was started

as soon as the application from the pipette was completed.

2.2. Phase 2: Effect of surfactant system on KD of American cockroach nymphs

A second experiment was conducted to determine whether efficacy of essential

oils on cockroach KD could be influenced by the surfactant system used. Phase 2

experiments consisted of a factorial treatment design that allowed for testing the effects

of 10 essential oils, each combined with three unique surfactant systems. This provides

critical information as to how to best design a full formulation using essential oils based

on whether it is for insecticidal or herbicidal use (see Chapter 4 for herbicide data). Four

essential oils from Phase 1 experiments were not included in Phase 2. Cedar oil, garlic

oil, and 2-phenethyl propionate were excluded based on poor KD performance in Phase

1. Mint oil was excluded due to the supplier’s inability to provide a second batch of

material consistent with the lot tested in Phase 1. All ingredients used throughout the

experiments were used from the same batch/lot to avoid batch to batch variability which

is common to naturally sourced materials. Preliminary formulation work was conducted

to identify the inert materials, rates and combinations which provided at least some

emulsion of the essential oils with the water content of the sample.

64
 All formulations contained 5% essential oil, a blend of surfactants and deionized

(DI) water (Table 2.3). Surfactant system A is compliant with 25(b)/4(a) minimum risk

regulations and is similar in composition to the inerts used in all formulations tested in

Phase 1. System B utilizes non-ionic ethoxylated sorbitan esters, which are commonly

used as agro-chemical surfactants. Preliminary work on determining hydrophilic-

lipophilic balance (HLB) required to emulsify the essential oils of interest was conducted

prior to developing this particular system (see Chapter 2). Surfactant system C is a

combination of ingredients considered to be “softer chemistry” components. Agnique®

is an alkyl polyglucoside product that is formulated using renewable raw materials and is

readily biodegradable. Stepanol® (sodium lauryl sulfate) is one of the most commonly

used anionic surfactant across many industries, including personal care products. It is

also listed on the 25(b) list as a minimum risk active ingredient. Formulations with

system C also had the lowest contact angle (see Chapter 2).

2.2.1 Preparation of samples for Phase 2 insect KD experiments

System A: Na Caprylate and xanthan gum were individually added to water while

on magnetic stir plate. Caprol® MPGO was added to essential oil in a separate vial and

shaken until homogenized. After xanthan gum had fully hydrated in water for 15-20

minutes, the oil phase was added while water was still on the stir plate. The mixture was

then stirred for an additional 15 minutes.

System B: Tween 20 and then Tween 40 were added into a vial containing the

essential oil. The vial was shaken until the mixture was homogenized, then poured into

65
water while on a stir plate. After the oil phase was added, the mixture was stirred for an

additional 15 minutes.

System C: Sodium lauryl sulfate was added to water while on stir plate. In a

separate vial, Agnique was added to the essential oil and shaken until homogenized, then

poured into the water Phase while stirring. After the oil phase was added, the mixture was

stirred for an additional 15 minutes.

All samples were stored in a refrigerator until ready for contact angle

determination.

The experimental unit in Phase 2 consisted of 1 lab-reared 9th instar American

cockroach nymph, n =10. The measured variable was speed of KD, recorded in seconds.

The experiment was a completely randomized design, and was conducted twice. A

maximum time limit for observations was set at seven minutes (420 seconds). For

observations that did not end in mortality, a time of 420 seconds was recorded. Due to

time constraint, it was not possible to mix all of the 33 treatments in the laboratory and

test them on the same day. Therefore, for each day of testing, 6-10 samples were

randomly selected, formulated in the lab, and tested for cockroach KD within 24 hours.

2.3. Experiment to test for significance of day to day variation on KD speed of

formulations

To confirm that there was minimal variation from day to day that would

significantly impact the KD data, an additional experiment was conducted in which six

formulas from Phase 2 were tested for KD on 9th instar American cockroaches (n=10) on

66
three non-consecutive days. The method for generating the KD data was the same as the

Phase 1 insect experiments using the KD tubes. To account for cockroach and

formulation variability, cockroaches from the insectary were randomly selected and

assigned to treatments and a new batch of formulations for each treatment were mixed on

each day of testing. Temperature and humidity of the room during the KD experiment

was recorded on each day of testing: Day 1 (June 2, 2011): 80°F, 31% RH, Day 2 (June

7, 2011): 81°F, 49% RH, Day 3 (June 10, 2011): 81°F, 45% RH.

2.4. Data analysis:

2.4.1 Phase 1 experiment

The alternate hypothesis is that KD time of essential oil combination treatments

on American cockroach nymphs will differ in comparison to a single essential oil when

used at the same concentration within a liquid formulation applied topically to a pest.

Data for the total 105 treatments were separated into fourteen groups based on essential

oil content. All 14 treatments containing cedar oil, for example, composed a group. All

groups were subjected to statistical analysis using PROC MIXED (SAS 9.2). For

treatment mean separation of essential oil groups, Dunnett’s was implemented with α =

0.05. Data were transformed using natural log, due to non-normal distribution of error.

Data for runs 1 and 2 were treated as blocks within the statistical model. To compare

treatment means of formulas composed of a single essential oil at 10% concentration,

PROC ANOVA was used, and treatment mean separation was determined with Fisher’s

LSD, with α=0.05.

67
2.4.2. Phase 2 experiment:

The alternate hypothesis is that utilizing different chemistries of inert ingredients

in a liquid formulation with essential oils will significantly influence KD speed of

cockroaches. Data were transformed using natural log, due to non-normal distribution of

error. Data for runs 1 and 2 were treated as blocks within the statistical model. To

compare main effects of essential oils and main effects of surfactant systems used, data

were analyzed using PROC ANOVA, treatment mean separation using Scheffe, α=0.05.

2.4.3. Experiment to confirm low variability from day to day on cockroach KD

Time period (day of testing) was used as the blocking criteria in the statistical

model. Treatments were structured within a randomized complete block design. PROC

GLM was used, with “day” set as a random variable. Treatment means were separated

using Fisher’s LSD, α=0.05.

2.4 Material suppliers

Table 3.3 lists the raw material suppliers for essential oils and inert materials used

in phase 1 and phase 2 experiments.

3. Results

3.1 Phase 1: KD with essential oil combinations

There were significant differences in KD speed among cedar oil containing

treatments (p<0.0001). Knockdown speed was significantly increased to less than 30

seconds when cedar oil was combined with citronella, clove, eugenol, geranium, geraniol,

mint or thyme oils as compared to cedar oil alone (Adjusted p values = <0.01for the

68
mentioned combinations) (Table 3.2). The only treatments that had slower KD on

average than 10% cedar oil within this treatment group were the cedar + garlic and cedar

+ peppermint combinations, but these treatments were not significantly different from

10% cedar oil.

None of the cinnamon oil combination treatments were significantly different

from 10% cinnamon oil alone. Combinations with clove, eugenol, geraniol, mint or

peppermint oils had less than thirty second KD, whereas 10% cinnamon oil alone had a

KD time of 46.25 seconds.

There were significant differences among citronella oil treatments (p<.0001).

Knockdown speed was increased when clove oil or eugenol were added, compared to

citronella oil alone (Adjusted p values of 0.01 and 0.01, respectively). The only

treatments with average KD time greater than thirty seconds (nlog of 30 seconds = 3.40)

were citronella + garlic oils and citronella + lemongrass oils.

There were significant differences among clove oil treatments (p<0.0001).

Treatments which had significantly slower KD compared to 10% clove oil alone were

treatments that contained garlic oil, lemongrass oil, peppermint oil or 2-phenethyl

propionate (adjusted p values of <0.01, <0.001, 0.05, and <0.0001, respectively). There

were no clove oil combination treatments that had significantly faster KD time than 10%

clove oil alone (Table 3.3).

Although all treatments containing eugenol had less than 30 second KD time,

there were significant differences among treatments as well as blocks (p<0.0001 and

p=<0.001, respectively) within the eugenol treatment group. Knockdown time was

significantly slower when cedar, cinnamon, citronella, garlic, geranium, lemongrass,

69
peppermint oils or 2-phenethyl propionate were combined with eugenol, compared to

10% eugenol alone (adjusted p values of <0.01, <0.001, <0.01, 0.02, 0.01, 0.03, 0.04, and

<0.0001, respectively). There were no eugenol combination treatments that had

significantly faster KD speed than 10% eugenol alone (Table 3.4).

There were significant differences among treatments containing garlic oil

(p<0.0001). The speed of KD was significantly increased when any of the other oils

were added. The difference between 10% garlic oil and garlic+cedar was marginally

significant, with p value = 0.07 (Table 3.5). Garlic oil was not toxic as a contact pesticide

to the cockroaches at a 10% w/w concentration. Insects were not monitored for KD

beyond 7 minutes, so KD time for cockroaches treated with 10% garlic oil was recorded

as 7 minutes.

All treatments containing geraniol resulted in less than thirty second KD time of

cockroaches. However, there were significant treatment differences (p<0.0001). When

cedar, cinnamon, citronella, mint oils or 2-phenethyl propionate were combined with

geraniol, KD time was significantly slowed (adjusted p values =0.03, 0.01, <0.0001, 0.01,

and <0.0001, respectively) (Table 3.6).

There were statistically significant differences among treatments containing

geranium oil (p<0.0001). Knockdown speed was significantly increased when geranium

oil was combined with eugenol or mint oils, compared to 10% geranium oil alone (p

value = 0.01 and 0.03, respectively). For the other geranium oil combinations, there were

no statistically significant differences in KD speed compared to 10% geranium oil alone.

70
 Statistically significant differences existed among treatments containing

lemongrass oil (p<0.0001). The oils most antagonistic to KD were garlic oil and 2-

phenethyl propionate, whereas eugenol, geraniol and mint oil combinations with

lemongrass oil were the fastest treatments, with KD speed well under 30 seconds.

The only treatment significantly different from 10% mint oil within the mint oil-

containing treatment group was mint + 2-phenethyl propionate (p<0.0001). Most mint oil

treatments had less than 30 second KD time except when mint oil was combined with

garlic, thyme oils or 2-phenethyl propionate oils.

Most peppermint oil-containing treatments, including 10% peppermint oil, had

less than 30 second KD time. Treatments containing cedar, garlic, lemongrass, rosemary

oils or 2-phenethyl propionate had greater than 30 second KD time. Of these five, cedar,

garlic and 2-phenethyl propionate oil treatments were significantly slower than 10%

peppermint oil (adjusted p values of <0.0001, 0.01 and <0.001, respectively) (Table 3.7).

Compared to 10% rosemary oil, the only treatment that was significantly different

was the rosemary + 2-phenethyl propionate treatment (adjusted p value of <0.001).

Knockdown speed was less than 30 seconds when rosemary oil was combined with

citronella, clove, eugenol, geranium, geraniol, lemongrass, mint or thyme oils, but these

treatments were not significantly faster than 10% rosemary oil alone which had 34

second KD.

All treatments containing thyme oil had KD close to or under 30 seconds, with the

exception of thyme oil + 2-phenethyl propionate, which was the only treatment

significantly different from 10% thyme oil alone (adjusted p value <0.0001).

71
 The majority of treatments containing 2-phenethyl propionate resulted in KD

speed greater than 30 seconds, with 10% phenethyl propionate over two minutes. Due to

high variation among 2-phenethyl propionate treatments, only one treatment, 2-phenethyl

propionate + geraniol, was significantly faster than 10% 2-phenethyl propionate alone

(adjusted p = 0.01). However, less than 30 second KD time was still achieved when 2-

phenethyl propionate was combined with citronella oil, eugenol or geraniol.

Eugenol, geraniol, clove and thyme oils were superb KD agents on their own at

10% concentration, each having a mean KD time well below 30 seconds (Table 3.8).

Peppermint, mint, geranium, lemongrass, citronella, rosemary and cinnamon oils were

also good KD agents at 10% concentration, with mean KD times very similar to one

another at close to 30 seconds. Cedar oil and 2-phenethyl propionate were poor KD

agents in comparison to these other oils (Table 3.8). While slower in KD time than most

of the other oils, both were able to provide mortality, whereas garlic oil was non-toxic at

10% w/w concentration. While KD time of several of the oils were significantly

improved when combined with other oils, this does not imply synergy. The improved

performance was driven by the added oil which had better activity on its own (Table 3.8).

An example of this is when 10% cedar alone was significantly improved when mixed

with citronella, clove, eugenol, geraniol, geranium, mint and thyme oils, which all had

much faster KD as stand-alone actives (Table 3.2). The KD times of these other oils

were 31, 20, 13, 15, 28, 30 and 18 seconds, respectively, compared to 10% cedar alone

with KD time of 161 seconds (Table 3.8).

72
3.2. Phase 2 experiment results

The only essential oil to have less than 30 second KD at a 5% w/w concentration,

averaged across all three surfactant systems was geraniol. Other oils showing fast KD

potential, and not significantly different from geraniol in speed, were eugenol, geranium,

thyme, clove, and peppermint oils (α=0.05). As expected, liquid formulations containing

only inert ingredients were significantly less able to control pests than all formulations

containing an essential oil (Table 3.9). Of the samples containing an essential oil,

cinnamon oil was the least effective oil in knocking down cockroaches. When KD times

were averaged across surfactant systems, System C was shown to significantly enhance

essential oil speed of KD (Table 2.9).

3.3 Phase 2.5

There were significant differences in KD time of the six treatments tested

(p<0.0001), but “day of testing” was not significant in the model (p=0.43). Eugenol and

geraniol treatments had faster mean KD time than geranium treatments, regardless of

surfactant system (Table 3.10).

4. Conclusions:

Eugenol, geraniol, clove and thyme oils at 10% w/w concentration exhibited

excellent KD of less than 30 seconds on American cockroach nymphs. Peppermint, mint,

geranium, lemongrass, citronella, rosemary and cinnamon oils were also toxic to

cockroaches, with KD times similar to one another, around 30 seconds. Cedar and garlic

73
oils and 2-phenethyl propionate were poor KD agents. Of all treatment groups in Phase 1

experiment, garlic oil and 2-phenethyl propionate combination treatments had the largest

standard error. The garlic oil sample tested in this experiment was not toxic to the

cockroaches at 10% w/w, but when many of the other oils such as clove and eugenol

were added to garlic oil, KD was significantly improved. Based on the performance of

these other oils when tested alone, it is clear that any level of KD achieved when mixed

with garlic oil was due to the effect of the other oil. There was no performance benefit to

combining multiple essentials together within a formulation.

Based on the data presented in Table 3.9, the top performing 25(b) essential oils

that should be considered for insecticide product development include thyme oil, clove

oil, eugenol and geraniol. These essential oils, other than geraniol, primarily consist of

aromatic compounds. Thymol is the primary constituent of thyme oil, and eugenol is the

primary constituent of clove oil. These results are consistent with previous findings that

aromatic compounds such as thymol and eugenol are more potent contact toxicants than

the aliphatic and bicyclic compounds such as limonene, citronellal, menthone, linalool,

camphor, camphene, and pinene (Phillips et al. 2010, Rice & Coats 1994). The aromatic

ring structure is typically more stable than the aliphatic compounds, making it more

difficult to metabolize inside an organism. This, coupled with the possibility of these

compounds interfering with the neurotransmitters, octopamine and tyramine, provide

strong evidence as to why the aromatic compounds are more toxic to insects (Bischof &

Enan 2004, Enan 2001, Enan 2005).

Surfactant systems were also found to significantly impact KD speed of essential

oils on American cockroaches. The faster KD of essential oils when mixed with

74
surfactant system C is thought to be due to the lower contact angle of this system (see

Chapter 2). The primary factor affecting KD speed in these experiments, however, was

the essential oil. In all experiments, eugenol and geraniol are the fastest KD agents.

Other essential oils that have potential as insect KD agents include citronella, clove,

geranium, peppermint and thyme oils, which have KD times close to or less than 1

minute on American cockroach nymphs at 5% w/w dilution (Table 3.9). Since many of

them display excellent KD capability, choosing which oil to develop commercially will

depend on cost, intellectual property, and ease of formulating into a stable product.

Given that control of urban pests such as cockroaches often elicits strong emotion

for the home resident, providing a fast knockdown of the pests assures them of success in

their pesticide application. This research has shown that many of the 25(b) essential oils

can provide this fast KD effect. The relationship of contact angle and fast KD with

essential oil formulations is one avenue of research that should be explored further,

particularly with oils primarily composed of aromatic ring structures similar to

octopamine. Future development of 25(b) insecticides for urban pest management should

focus on the use of eugenol and geraniol with surfactants that can lessen the contact angle

of the formulation. Odor of these essential oils, while often considered pleasant, could be

an issue for some, particularly when applied indoors. Encapsulation techniques to reduce

volatility may be one route of exploration to address this issue.

75
5. Tables

Product Name Active Ingredients Company

All Natural General 0.05% rosemary oil Monterey
Purpose Garden Spray 0.04% peppermint oil
RTU 0.03% clove oil
0.02% malic acid
0.01% sesame oil
0.01% thyme oil
0.01% cinnamon oil
All Natural Home Pest 0.4% rosemary oil Monterey
Control 0.4% peppermint oil
0.3% sesame oil
0.3% cinnamon oil
0.2% clove oil
0.1% garlic oil
0.1% thyme oil
0.1% mint oil
All Natural Mite and 0.04% rosemary oil Montery
Insect Control 0.04% sesame oil
0.03% peppermint oil
0.03% thyme oil
0.03% cinnamon oil
0.02% malic acid
EcoSmart Ant & Roach 5% rosemary oil EcoSmart
Killer 3% cinnamon oil
EcoSmart Bed Bug Killer 2% 2-phenethyl propionate EcoSmart
for cracks & crevices
EcoSmart Bed Bug Killer 3% 2-phenethyl propionate EcoSmart
for mattresses & carpets 1.5% peppermint oil
1.5% rosemary oil
EcoSmart Garden Insect 0.25% rosemary oil EcoSmart
Killer 0.25% peppermint oil
0.25% thyme oil
0.25% clove oil
EcoSmart Home Pest 2% 2-phenethyl propionate EcoSmart
Control 1% clove oil
1% rosemary oil
1% peppermint oil
0.5% thyme oil
Table 3.1. EPA-exempt insect control products currently sold or state-registered for sale
in the market.

continued

76
Table 3.1 continued

EcoSmart Flying Insect 5% peppermint oil EcoSmart

Killer 1% cinnamon oil
1% sesame oil
EcoSmart Lawn Insect 3.5% 2-phenethyl EcoSmart
2% sodium lauryl sulfate
1% eugenol
1% thyme oil
1% sesame oil
EcoSmart Spider Blaster 5% rosemary oil EcoSmart
EcoSmart Wasp & Hornet 1% peppermint oil EcoSmart
Killer 0.5% 2-phenethyl
propionate
Elementals Home Insect 18.75% soybean oil The Scotts Miracle-Gro
Company
Essentria IC3 10% rosemary oil Envincio, LLC
5% geraniol
2% peppermint oil
Finito 0.2% citric acid EcoLAB
Hot Shot Natural Ant & 3% lemongrass oil Spectracide
Roach Killer
Hot Shot Natural Flying 3% lemongrass oil Spectracide
Insect Killer
Hot Shot Natural Home 3% lemongrass oil Spectracide
Insect Control
Hot Shot Natural Wasp & 3% lemongrass oil Spectracide
Hornet Killer
Mother Earth Exempt 6% geraniol BASF
Contact Insecticide 0.5% lemongrass oil
Natria Home Pest Control 3% soybean oil Bayer
0.25% eugenol
Organics Avenger Natural 0.03% clove oil Cutting Edge Formulations
Bed Bug Killer 1% peppermint oil
TyraTech Naturals 3.1% thyme oil TyraTech
Crawling Insect Spray

77
Treatment Knockdown (seconds, Adjusted p
nlog) value
10% cedar oil 4.59 -
5% cedar oil + 5% cinnamon oil 3.77 NS
5% cedar oil + 5% citronella oil 3.17 <0.01
5% cedar oil + 5% clove oil 2.99 <0.001
5% cedar oil + 5% eugenol 2.97 <0.001
5% cedar oil + 5% garlic oil 5.13 NS
5% cedar oil + 5% geraniol 3.05 <0.001
5% cedar oil + 5% geranium oil 3.26 <0.01
5% cedar oil + 5% lemongrass oil 3.62 0.08
5% cedar oil + 5% mint oil 3.27 <0.01
5% cedar oil + 5% peppermint oil 4.62 NS
5% cedar oil + 5% rosemary oil 4.29 NS
5% cedar oil + 5% thyme oil 3.29 <0.01
5% cedar oil + 5% 2-phenethyl propionate 4.51 NS
Standard error 0.36
Table 3.2. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% cedar oil alone compared to combinations with other oils. NS = not significant
from 10% cedar oil alone, with p value > 0.10, using Dunnett’s for treatment mean separation.

Treatment Knockdown Adjusted p

(seconds, nlog) value
10% clove oil 2.88 -
5% clove oil + 5% cedar oil 2.99 NS
5% clove oil + 5% cinnamon oil 2.77 NS
5% clove oil + 5% citronella oil 2.96 NS
5% clove oil + 5% eugenol 2.66 NS
5% clove oil + 5% garlic oil 3.33 <0.01
5% clove oil + 5% geraniol 2.75 NS
5% clove oil + 5% geranium oil 3.09 NS
5% clove oil + 5% lemongrass oil 3.39 <0.001
5% clove oil + 5% mint oil 2.94 NS
5% clove oil + 5% peppermint oil 3.16 0.05
5% clove oil + 5% rosemary oil 3.09 NS
5% clove oil + 5% thyme oil 3.04 NS
5% clove oil + 5% 2-phenethyl propionate 3.47 <0.0001
Standard error 0.14
Table 3.3. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% clove oil alone compared to combinations with other oils. NS = not significant
from 10% clove oil alone, with p value > 0.10, using Dunnett’s for treatment mean separation.

78
Treatment Knockdown Adjusted p
(seconds, nlog) value
10% eugenol 2.49 -
5% eugenol + 5% cedar oil 2.97 <0.01
5% eugenol + 5% cinnamon oil 3.06 <0.001
5% eugenol + 5% citronella oil 2.95 <0.01
5% eugenol + 5% clove 2.66 NS
5% eugenol + 5% garlic oil 2.90 0.02
5% eugenol + 5% geraniol 2.59 NS
5% eugenol + 5% geranium oil 2.94 0.01
5% eugenol + 5% lemongrass oil 2.89 0.03
5% eugenol + 5% mint oil 2.74 NS
5% eugenol + 5% peppermint oil 2.87 0.04
5% eugenol + 5% rosemary oil 2.82 NS
5% eugenol + 5% thyme oil 2.65 NS
5% eugenol + 5% 2-phenethyl propionate 3.24 <0.0001
Standard error 0.13
Table 3.4. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% eugenol alone compared to combinations with other oils. NS = not significant
from 10% eugenol alone, with p value > 0.10, using Dunnett’s for treatment mean separation.

Treatment Knockdown Adjusted p

(seconds, nlog) value
10% garlic oil 6.04 -
5% garlic oil + 5% cedar oil 5.13 0.07
5% garlic oil + 5% cinnamon oil 4.31 <0.0001
5% garlic oil + 5% citronella oil 3.50 <0.0001
5% garlic oil + 5% clove 3.33 <0.0001
5% garlic oil + 5% eugenol oil 2.90 <0.0001
5% garlic oil + 5% geraniol 2.95 <0.0001
5% garlic oil + 5% geranium oil 3.06 <0.0001
5% garlic oil + 5% lemongrass oil 4.29 <0.0001
5% garlic oil + 5% mint oil 3.71 <0.0001
5% garlic oil + 5% peppermint oil 4.06 <0.0001
5% garlic oil + 5% rosemary oil 4.14 <0.0001
5% garlic oil + 5% thyme oil 3.18 <0.0001
5% garlic oil + 5% 2-phenethyl propionate 4.88 <0.01
Standard error 0.34
Table 3.5. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% garlic oil alone compared to combinations with other oils, using Dunnett’s for
treatment mean separation.

79
Treatment Knockdown Adjusted p
(seconds, nlog) value
10% geraniol 2.69 -
5% geraniol + 5% cedar oil 3.05 0.03
5% geraniol + 5% cinnamon oil 3.09 0.01
5% geraniol + 5% citronella oil 3.26 <0.0001
5% geraniol + 5% clove 2.75 NS
5% geraniol + 5% eugenol oil 2.59 NS
5% geraniol + 5% garlic oil 2.95 NS
5% geraniol + 5% geranium oil 3.01 0.07
5% geraniol + 5% lemongrass oil 2.88 NS
5% geraniol + 5% mint oil 3.09 0.01
5% geraniol + 5% peppermint oil 2.86 NS
5% geraniol + 5% rosemary oil 2.97 NS
5% geraniol + 5% thyme oil 2.97 NS
5% geraniol + 5% 2-phenethyl propionate 3.24 <0.0001
Standard error 0.12
Table 3.6. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% geraniol alone compared to combinations with other oils. NS = not significant
from 10% geraniol alone, with p value > 0.10, using Dunnett’s for treatment mean separation.

Treatment Knockdown Adjusted p

(seconds, nlog) value
10% peppermint oil 3.1357 -
5% peppermint oil + 5% cedar oil 4.62 <0.0001
5% peppermint oil + 5% cinnamon oil 3.04 NS
5% peppermint oil + 5% citronella oil 3.20 NS
5% peppermint oil + 5% clove 3.16 NS
5% peppermint oil + 5% eugenol oil 2.87 NS
5% peppermint oil + 5% garlic oil 4.06 0.01
5% peppermint oil + 5% geraniol 2.86 NS
5% peppermint oil + 5% geranium oil 3.29 NS
5% peppermint oil + 5% lemongrass oil 3.44 NS
5% peppermint oil + 5% mint oil 3.01 NS
5% peppermint oil + 5% rosemary oil 3.57 NS
5% peppermint oil + 5% thyme oil 3.18 NS
5% peppermint oil + 5% 2-phenethyl propionate 4.36 <0.001
Standard error 0.28
Table 3.7. Time (seconds, nlog transformed) to knock down American cockroach nymphs
using 10% peppermint oil alone compared to combinations with other oils. NS = not
significant from 10% peppermint oil alone, with p value > 0.10, using Dunnett’s for treatment mean
separation.

80
Essential Oil, 10% w/w N Mean KD in seconds (nlog)
Garlic 8 420 (6.04) a
Cedar 8 161 (4.59) b
2-phenethyl propionate 8 137 (4.34) bc
Cinnamon 8 46 (3.54) bcd
Rosemary 8 35 (3.46) bcd
Citronella 8 31 (3.41) bcd
Lemongrass 8 29 (3.35) bcd
Geranium 8 28 (3.33) bcd
Mint 8 30 (3.32) bcd
Peppermint 8 23 (3.14) cd
Thyme 8 18 (2.88) d
Clove 8 20 (2.88) d
Geraniol 8 15 (2.69) d
Eugenol 8 13 (2.49) d
Table 3.8. Essential oils in order of KD speed at 10% concentration on American
cockroach Means followed by same letter within a column are not significantly different at the
α=0.05 level according to Fisher’s LSD.

Essential Oil, 5% w/w N Mean KD in seconds (nlog)

Inert blank 60 383 (5.89) a
Cinnamon 60 267 (5.27) b
Lemongrass 60 98 (4.10) c
Rosemary 60 77 (3.95) cd
Citronella 60 69 (3.88) cd
Peppermint 60 44 (3.64) de
Clove 60 46 (3.59) de
Thyme 60 48 (3.58) de
Geranium 60 41 (3.53) de
Eugenol 60 38 (3.50) de
Geraniol 60 30 (3.34) e
Table 3.9. Main effect of essential oil on KD speed of 9th instar American cockroaches at
a 5% w/w concentration, average KD across three surfactant systems, Means followed by
same letter within a column are not significantly different at the α=0.05 level according to Scheffe’s.

81
 Treatment N Mean KD Time in Seconds
Eugenol + System A 30 23 a
Eugenol + System C 30 24 a
Geraniol + System A 30 25 a
Geraniol + System C 30 24 a
geranium + System A 30 34 b
geranium + System C 30 40 b
th
Table 3.10. Mean KD time of 9 instar American cockroaches with Eugenol, Geraniol,
and Geranium oil combined with surfactant systems A (89.57% DI water, 2% w/w
sodium caprylate, 0.33% Caprol® MPGO, 0.1% xanthan gum) & C (92.4% DI water,
2.5% w/w alkyl polyglucoside, 0.1% sodium lauryl sulfate), averaged across three test
dates. Means followed by same letter within a column are not significantly different at the α=0.05
level, Fisher’s LSD.

References:

Bischof, L.J. and E.E. Enan. 2004. Cloning expression and functional analysis of an
octopamine receptor from Periplaneta americana. Insect Biochem and Molec Biol. 34:
511-521.

Butler, G.D. Jr, T.J. Henneberry, P.A. Stansly and D.J. Schuster. 1993. Insecticidal
effects of selected soaps, oils and detergents on the sweetpotato whitefly (Homoptera:
Aleyrodidae). Florida Entomol. 76: 161-167.

Greene, A. and N.L. Breisch. 2002. Measuring integrated pest management programs for
public buildings. Journal of Econ. Entom. 95: 1-13.

Enan, E.E. 2001. Insecticidal activity of essential oils: octopaminergic sites of action.
Comp. Biochem. Physiol. 130: 325-337.

Enan, E.E. 2005. Molecular response of Drosophila melanogaster tyramine receptor

cascade to plant essential oils. Insect Biochem. & Mol. Biol. 35: 309-321.

Hollis, S., C. McDonald, J.Rader. 2008. Methods for treating arthropods. U.S. Patent
Application. Pub. No. US2008/0221223 A1. Pub Date: September 11, 2008.

Imail, T., S. Tsuchiya, K. Morita, and T. Fujimori. 1994. Surface tension-dependent

surfactant toxicity on the green peach aphid, Myzus persicae (Sulzer) (Hemiptera:
Aphididae). Appl. Entom. Zool. 29: 389-393.

Imail, T. and S. Tsuchiya. 1995. Aphicidal effects of Silwet L-77, organosilicone

nonionic surfactant. Appl. Entom. Zool. 30: 380-383.

82
Liu, T-X. and P.A. Stansly. 2000. Insecticidal activity of surfactants and oils against
silverleaf whitely (Bemisia argentifolii) nymphs (Homoptera: Aleyrodidae) on collards
and tomato. Pest Mgmt. Sci. 56: 861-866.

Miller, D.M. and F. Meek. 2004. Cost and efficacy comparison of Integrated Pest
Management strategies with monthly spray insecticide applications for German
cockroaches (Dictyoptera: Blattellidae) control in public housing. J. Econ. Entomol. 97
(2): 559-569.

Mosson, H.J., J.E. Short, R. Schenker and J.P. Edwards. 1995 Pesticide Sci. 45: 237-246.

Nauen, R. 2007. Insecticide resistance in disease vectors of public health importance.

Pest Mgmt. Sci. 63: 628.

Pesticide Registration (PR Notice) Notice 2002-1. www.epa.gov. Feb 2, 2012.

Phillips, A.K., A.G. Appel, and S.R. Sims. 2010. Topical toxicity of essential oils to the
German cockroach (Dictyoptera: Blattellidae). J. Econ. Entomol. 103: 448-459.

Rice, P.J. and J.R. Coats. 1994. Insecticidal properties of several monoterpenoids to the
housefly (Diptera: Muscidae), red flour beetle (Coleoptera: Tenebrionidae), and southern
corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 87: 1172-1179.

Schal, C. and R.L. Hamilton. 1990. Integrated suppression of synanthropic cockroaches.

Annual Rev. Entomol. 35: 521-551.

Ware, G.W. and D.M. Whitacre. 2004. The Pesticide Book, 6th ed. Willoughby, OH:
MeisterPro Information Resources.

Wei, Y., A.G. Appel, W.J. Moar, and N. Liu. 2001. Pyrethroid resistance and cross
resistance in the German cockroach, Blattella germanica (L). Pest. Mgmt. Sci. 57: 1055-
1059.

Yu, S.J. 2008. The toxicology and biochemistry of insecticides. Boca Raton, FL. CRC Press,
1st edition. ISBN: 978-1-4200-5975-5.

83
 Chapter 4: Post-emergent herbicide activity of U.S. EPA minimum risk plant essential

oils on dandelion (Taraxacum officianale) and crabgrass (Digitaria ischaemum) and the

influence of surfactants on their herbicidal activity

1. Introduction

Weed management is a major input for crop production and herbicides make up

the largest portion of pesticides sold in agriculture. The EPA reported a total of 2 billion

pounds of herbicides used globally in 2007, with the U.S. making up 25% of the world

market (www.epa.gov/pesticides/pestsales/07pestsales/table_of_contents2007.htm). In

the 2011 National Gardening survey, it was reported that a total of 44 million American

homeowners used pesticides in 2010, of which 9.6 million specifically used herbicides

(Butterfield & Baldwin 2011). Typical areas of herbicide application by consumers vary

widely, from gravel paths, patio cracks, around edible vegetable gardens and ornamental

landscape beds to selective treatment of weeds in the lawn. Dandelion is the most

common weed species in residential lawns and gardens. Its notoriety as a pest can be

attributed to its wide distribution across North America, its perennial nature enabling it to

re-grow from rootstock, prolific seed production at multiple times within the growing

season, and the ability to thrive under mowing conditions in the lawn. Another weed

species that consumers spend a great deal of effort trying to eradicate is crabgrass, a

monocot annual weed that is common in lawns and gardens.

84
 Homeowners who wish to grow organic edibles or use certified organic materials

for lawn care are restricted to a small number of products that are compliant with the

practices outlined by the National Organic Program (NOP). The certified organic

herbicide materials that have been approved for use in organic food production, which are

available to both large-scale growers and homeowners, include corn gluten meal, plant

oils and essential oils, and the essential oil component limonene. Soaps may also be used

as herbicides in organic production, but only on non-food crop areas (2011 OMRI

Products List). Regardless of these limited herbicide options for the organic grower,

over 20% of consumers active in the lawn and garden category (8.8 million users) report

using natural or less toxic chemicals (Butterfield & Baldwin 2011). Several plant

essential oils have documented herbicide capabilities, but cost and weed re-growth are

limiting factors in their widespread adoption. However, the plight of weed control for the

homeowner differs from agriculture managers in that it is often not necessary for the

homeowner to broadcast apply herbicides in order to reduce weeds below an acceptable

threshold. Homeowners are also typically treating weeds on a much smaller scale than

production growers, with the average U.S. home lawn estimated at 0.08 hectare or 8,600

square feet (Zirkle 2010). These differences in use patterns may present better economics

using natural compounds in residential weed control as opposed to crop production.

According to current U.S. EPA guidelines, it is unlawful for any registered

pesticide to make any claim of safety on the label, regardless of toxicity profile of the

active ingredient (ww.epa.gov/oppfead1/labeling/lrm/chap-12.pdf). The establishment of

the U.S. EPA’s 25(b) minimum risk pesticide list presents an opportunity for industry to

develop pesticides for the consumer that can be marketed as “safe to use around kids and

85
pets”. Being able to use such language on a pesticide label can address health and safety

concerns that consumers may have about using pesticides in and around their home.

Additionally, many schools are now promoting the sole use of exempt pesticides for

insect and weed control because of favorable toxicity profiles and reduced risk of residual

pesticides in areas where children could be exposed. Several exempt herbicide products

are currently being sold that use essential oils as the active ingredients (Table 4.1). Many

of the products listed in Table 4.1 use multiple essential oils as active ingredients, with

various blends of 2-phenethyl propionate, eugenol, lemongrass, clove and cinnamon oils.

U.S. patents have been filed or issued for lemongrass, citronella and 2-phenethyl

propionate oils as herbicides (Fernandez et al. 2008, Bessette, SM 2000, Ryan & Morris

2002).

Despite three patents and several commercially available products on the market,

there is very little reported information in the scientific literature on the use of essential

oils as herbicides, and no publicly accessible scientific literature to support their

increased performance by adding multiple essential oils together. Tworkoski (2002)

screened 25 essential oils for herbicide activity using dandelion leaf disks, and reported

that cinnamon, clove, summer savory and red thyme oils were highly phytotoxic. When

these oils were mixed 5 to 10% w/w in an aqueous concentration and applied to shoots of

lambsquarter, common ragweed and johnsongrass, plant death occurred within 1 to 24

hours after the application. Eugenol was found to be the active component within the

cinnamon oil. It is well known that eugenol is the primary constituent within clove oil as

well. Others have also reported on the burn-down effects of clove oil on plant tissue

(Evans et al. 2009, Bainard et al. 2006). A ready-to-use herbicide formulation containing

86
22.9% citronella oil is currently sold in the UK under the name Barrier H™. Its herbicide

activity was tested by Clay et al. (2005) as a treatment for clearing vegetation for tree

establishment. At a high dose of 229 kg a.i. ha-1, citronella oil killed the foliage of all

weed species within one day of application, but re-growth was reported.

It is generally agreed that essential oils damage plant tissue by increasing cell

membrane permeability, resulting in electrolyte leakage and significantly injuring plants

within a short time after application (Tworkoski 2002, Bainard et al. 2006). The presence

of leaf epicuticular wax on leaf surface can reduce the severity of injury caused by

essential oils such as clove oil and eugenol by reducing the retention of the spray on the

leaf surface (Bainard et al. 2006). In 2000, Romagni et al. reported that 1,4-cineole and

related monoterpene structures, which are common essential oil components, inhibited

plant asparagine synthetase. This was the first indication that there was a possible

molecular mode of action site for essential oils’ herbicide activity. However, in 2005, the

article was retracted by the author due to an inability to duplicate the original results.

The current study is designed to 1) determine whether multiple essential oils have

performance benefits over a single essential oil within a liquid formulation and 2)

evaluate performance of essential oils when formulated with different surfactant systems.

This study will be the first to compare all 25(b) essential oils and essential oil

constituents for herbicide activity, as stand-alone ingredients and in combination with one

another. It will also provide useful information on the effects of essential oils’ herbicide

activity when paired with various inert ingredients. The fourteen 25(b) essential oils and

essential oil constituents selected for the current study are 2-phenethyl propionate (2PP),

cedar (CED), cinnamon (CIN), citronella (CIT), clove (CLO), eugenol (EUG), garlic

87
(GAR), geranium (GER), geraniol (Giol), lemongrass (LEM), mint (MIN), peppermint

(PEP), rosemary (ROS) and thyme (THY) oils.

2. Materials and methods:

2.1 Phase 1 weed control: Pairwise combinations of essential oils tested for herbicide

activity on dandelion and smooth crabgrass

To determine whether there is a benefit to adding essential oils together to

enhance herbicide activity, 14 essential oils were tested individually and in combination

with one another in an aqueous solution at a total of 10% w/w. All pairwise

combinations of the 14 oils were made, for a total of 105 combinations.

All ingredients used throughout the experiments were used from the same batch/lot to

avoid batch to batch variability which is common to naturally sourced materials.

Preliminary formulation work was conducted to identify the inert materials, their rates

and combinations which provided a physically stable emulsion for a minimum of two

minutes. All formulation treatments in phase 1 experiment contained the same inert

system, which consisted of:

5% soybean oil - a triglyceride plant oil to aid in emulsion stability

3% sodium caprylate – an organic compound for emulsion purposes

1% Caprol® MPGO- polyglycerol esters of oleic acid, used for emulsion

purposes

0.1% xanthan gum - a thickening agent to prolong emulsion stability

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 Samples were prepared in the described process: sodium caprylate and xanthan

gum were individually added to a water phase while on magnetic stir plate. Caprol®

MPGO was added to the essential oil and soybean oil in a separate vial and shaken until

homogenized. After xanthan gum had fully hydrated in the water phase for 15-20

minutes, the oil phase was added to the water phase while stirring on the stir plate. After

the oil phase was added, the mixture was allowed to stir for an additional 15 minutes.

Phase 1 weed control experiments were conducted July-October 2010 in a

greenhouse using dandelions (Taraxicum officianale) and smooth crabgrass (Digitaria

ischaemum). An individual weed, planted into a 10 cm pot, was the experimental unit,

n=3 for each species. The experiment was a completely randomized design, and was

conducted two times. Dandelions and crabgrass were grown from seed in flat trays, and

then individual plants were transplanted into 10 cm pots filled with MetroMix® potting

media within approximately one month of germination. Transplants were allowed an

additional two to three weeks to establish in the pots before treatment applications were

made. Weeds were trimmed to approximately 10 cm height weekly to stimulate mowing

in a lawn setting. Treatments were not all applied on the same day due to time

restrictions. Treatments being applied on a particular day were randomly selected and

assigned to weeds. Since there were multiple application dates, weed planting dates were

staggered accordingly so that at each application date the dandelions and crabgrass plants

were approximately six weeks old. Greenhouse temperatures ranged from 15° to 21° C at

night and 21° to 32° C during the day. Overhead irrigation was provided daily to

maintain adequate moisture in the weed pots. Fertilization was provided through the

irrigation system at time transplanting, at an end-rate dilution of 450 ppm N (174 ppm

89
NH4 and 276 ppm Urea), 901 ppm P2O5, and 450 ppm K2O. Formulations were applied

using hand-held pump sprayers which provided a fine mist spray, ensuring full coverage

of the sample onto the leaf tissue of each weed with approximately 1 g of spray.

Applications were made in late morning hours, and subjective percent leaf injury ratings

were measured at 1 hour and 24 hours after application. A two week evaluation was also

taken to determine weed re-growth or recovery.

2.2 Phase 2 weed control

Two greenhouse experiments and one field experiment were conducted to

determine the influence of differing surfactant systems on the herbicide activity of

essential oils. All three experiments were of a factorial treatment design that allowed for

testing the effects of ten essential oils (cinnamon, citronella, clove, eugenol, geraniol,

geranium, lemongrass, peppermint, rosemary and thyme oils), each combined with three

unique surfactant systems (Table 2.3). This provides critical information as to how to

best develop a full formulation using essential oils based on whether it is for insecticidal

or herbicidal use, which is discussed in Chapter 1. Four essential oils, cedar, garlic, mint

and 2-phenethyl propionate, from phase 1 experiments were not included in phase 2. All

essential oils and inert ingredients used throughout the experiments were used from the

same batch/lot to avoid batch to batch variability which is common to naturally sourced

materials. Preliminary formulation work was conducted to identify the inert materials,

rates and combinations which provided at least a short term (5 minutes) emulsion without

phase separation of the essential oils and inert ingredients from the water content in the

sample.

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 All formulations contained 5% essential oil, a blend of surfactants and deionized

(DI) water. Surfactant system A is compliant with 25(b)/4(a) minimum risk regulations,

and is similar in composition to the inert ingredients used in phase 1. System B utilizes

non-ionic ethoxylated sorbitan esters, which are commonly used as agro-chemical

surfactants. These two particular surfactants were chosen based on the preliminary work

on determining hydrophilic-lipophilc balance (HLB) required in order to emulsify the

essential oils of interest, which is discussed in Chapter 2. Surfactant system C is a

combination of ingredients considered to be “softer chemistry” components. Agnique®

is an alkyl polyglucoside product that is formulated using renewable raw materials and is

readily biodegradable. Stepanol® (sodium lauryl sulfate) is one of the most commonly

used anionic surfactant across many industries, including personal care products. It is

also listed on the 25(b) list as a minimum risk active ingredient. Formulations with

system C also had the lowest contact angle, indicating that these had better spreading

properties than formulations containing surfactant systems A and B (see Chapter 2).

2.2.1 Preparation of samples

Table 2.3 provides a list of the components in surfactant systems A, B and C used

in Phase 2 experiments.

System A formulas were prepared in the following manner: sodium caprylate and

xanthan gum were individually added to a water phase while on magnetic stir plate.

Caprol® MPGO was added to essential oil in a separate vial and shaken until

homogenized. After xanthan gum had fully hydrated in the water phase for 15-20

91
minutes, the oil phase was added to the water phase while stirring on the stir plate. After

the oil phase was added, the mixture was stirred for an additional 15 minutes.

System B formulas were prepared in the following manner: Tween 20 and then

Tween 40 were added into a vial containing the essential oil. The vial was shaken until

mixture was homogenized, then poured into water while on a stir plate. After the oil

phase was added, the mixture was stirred for an additional 15 minutes.

System C formulas were prepared in the following manner: Sodium lauryl sulfate

was added to water while on stir plate. In a separate vial, Agnique alkyl polyglucoside

was added to the essential oil and shaken until homogenized then poured into the water

phase while stirring. After the oil phase was added, the mixture was stirred for an

additional 15 minutes.

Phase 2 weed control experiments were conducted June through July 2011. Fresh

formulations were made in the lab within twenty-four hours prior to the greenhouse and

field applications. Applications were made in late morning hours, and subjective % leaf

injury ratings were measured at 1 hour and 24 hours after application. A two week

evaluation was also taken as a means of measuring weed re-growth.

The greenhouse experiments were a completely randomized design, and the

experimental unit was one individually potted 6 week old dandelion, with 4 replications

per treatment (n=4). Dandelion seeds were germinated in flat trays filled with

MetroMix® potting media, then transplanted into individual 10 cm diameter pots filled

with the same type of potting media within approximately one month of germination.

The transplants were allowed an additional two to three weeks to establish in the pots

92
before treatment applications were made. Treatments were randomly assigned to weeds.

Greenhouse temperatures ranged from 15° to 21° C at night and 21° to 32° C during the

day. Overhead irrigation was provided daily to maintain moisture in the weed pots.

Fertilization was provided through the irrigation system at time transplanting, at an end-

rate dilution of 450 ppm N (174 ppm NH4 and 276 ppm Urea), 901 ppm P2O5, and 450

ppm K2O. Weeds were trimmed weekly to simulate effects of lawn mowing.

Applications were made using hand-pump bottles, and, for all treatments, approximately

1 g of spray was applied to each dandelion, ensuring full coverage of the foliage.

The field experiment was conducted on a natural population of mature dandelions

growing in a low maintenance turf area that was weekly mowed to 7 cm height. The

experimental unit consisted of one dandelion, and there were 4 replications per treatment

(n=4). The weeds were individually marked and randomly assigned to treatments in a

completely randomized block design. Spot applications to the dandelions were made

using the same hand-pump bottles used in the greenhouse trials. Approximately 2.5 g of

spray was foliar applied to each dandelion, fully coating leaf tissue.

2.3 Data analysis

2.3.1 Phase 1 weed control

The alternate hypothesis is that herbicide burn-down of essential oil combination

treatments on dandelion and crabgrass will differ in comparison to a single essential oil

when used at the same concentration within a liquid formulation that is applied foliarly.

Data for the total 105 treatments were separated into 14 groups based on essential oil

content. All 14 treatments containing cedar oil, for example, composed a group. All

93
groups were subjected to statistical analysis using PROC MIXED (SAS 9.2). For

treatment mean separation of essential oil groups, Dunnett’s was implemented with α

=0.05. Data for runs 1 and 2 were treated as blocks within the statistical model. To

compare treatment means of formulas composed of a single essential oil at 10% w/w

concentration, PROC ANOVA was used, and treatment mean separation was determined

with Fisher’s LSD, with α=0.05.

2.3.2 Phase 2 weed control

The alternate hypothesis is that the rate of overall effectiveness of herbicide burn-

down of essential oils will vary when combined with different surfactant systems. The

treatment list is a factorial arrangement of ten essential oils and three surfactant systems.

The dandelion data were subjected to statistical analysis using PROC MIXED (SAS 9.2)

and treatment mean separation was based on Fisher’s LSD, with α = 0.05. Data for runs

one and two were treated as blocks within the statistical model. Synergy of the essential

oil/surfactant system A treatments were calculated based on the equation described by

Colby (1967). A two-tailed t test was conducted using Minitab 15 software to determine

if differences between the actual injury rating and the expected injury rating from the

combinations were significant. For the synergy analysis, data from all three phase 2

experiments were combined.

2.4 Material suppliers

Table 2.4 lists the raw material suppliers for essential oils and inert materials used

in phase 1 and phase 2 herbicide experiments.

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3. Results

3.1 Phase 1 dandelion and smooth crabgrass injury

Cedar oil was phytotoxic to weeds when tested alone at 10% concentration, but as

a stand-alone essential oil it was less effective than when tested in combination with most

of the other oils (Table 4.2). Herbicide injury on dandelions was significantly improved

by 24 hours when 2-phenethyl propionate, cinnamon, citronella, clove, eugenol, geraniol,

lemongrass or thyme oils were mixed with cedar oil, in comparison to 10% cedar oil

alone. However, there were only two pairwise combinations, cedar + clove and cedar +

thyme, that significantly improved herbicide injury to crabgrass, increasing percent injury

from 43% with 10% cedar to 79.2% with cedar + clove and 64% with cedar + thyme at

24 hours (Table 4.2). For all cedar oil-containing treatments, when comparing % weed

injury at 1 and 24 hours after treatment, damage was much more severe on dandelions

than crabgrass (Table 4.2).

Within the cinnamon oil-containing treatment group, 10% cinnamon oil was

numerically the best herbicidal formulation on dandelions, causing 72.5% injury to

dandelions one hour after application, but was not statistically different from

cinnamon+geranium, cinnamon + lemongrass, cinnamon + peppermint or cinnamon +

rosemary . By 24 hours, all cinnamon oil treatments injured dandelion foliage >90%. For

crabgrass, only cinnamon + eugenol and cinnamon + garlic were significantly less

effective than 10% cinnamon at 24 hours (39.2% and 40.8% injury, respectively,

compared to 65.8% injury from 10% cinnamon (Table 4.3).

95
 All treatments containing citronella oil were highly phytotoxic to dandelions and

crabgrass, with all treatments burning at least 80% dandelion leaf tissue and 45%

crabgrass leaf tissue within 24 hours following foliar application. Citronella oil alone at

10% w/w concentration was very effective as an herbicide, with 97.5% injury to

dandelions and 51% injury to crabgrass by 24 hours. None of the combination treatments

containing citronella oil were significantly different from 10% citronella oil alone, except

for the citronella + lemongrass combination. Considering that these two oils are very

similar in composition and come from plants within the same genus, this apparent

antagonism was unexpected. However, the 1 hour and 24 hour crabgrass injury from this

particular combination was not antagonistic. There were no significant differences

among treatments when tested on crabgrass.

Clove oil treatments were highly phytotoxic to dandelions and crabgrass, with

most treatments resulting in greater than 90% injury to dandelions and greater than 50%

injury to crabgrass within 24 hours following foliar application. The effectiveness of

clove oil on dandelions was significantly reduced when mixed with garlic and rosemary

oils, with 76% and 63%, respectively, 24 hours after application, compared to 93% injury

with 10% clove oil alone. On crabgrass, effectiveness was significantly reduced when

clove oil was mixed with garlic, rosemary, cinnamon or peppermint oils.

Eugenol was a very effective burndown agent on dandelions and crabgrass, with

most treatments resulting in greater than 90% injury on dandelions and >40% injury on

crabgrass within 24 hours after foliar application (Table 4.4). Similar to the clove oil,

mixtures of eugenol with garlic or rosemary oils significantly reduced injury to

dandelions by 24 hours, compared to eugenol alone. On crabgrass, the only treatment

96
that was significantly less effective than 10% eugenol at 24 hours after application was

eugenol + cinnamon (Table 4.4). This is an unexpected result since Cinnamon was very

phytotoxic on its own (66% injury at 24 hours on crabgrass) and in combination with

many other essential oils (Table 4.3).

Garlic oil was not effective as a stand-alone ingredient in herbicide mixtures.

When mixed with cinnamon, citronella, clove, eugenol, geranium, geraniol, lemongrass

or mint oils, injury to dandelions was at least 70% by 24 hours, but injury is attributable

to these other oils (Table 4.5). On crabgrass, mixtures of garlic oil with citronella,

eugenol, lemongrass or thyme oils burned at least 50% of leaf tissue by 24 hours, but

again, the injury is not attributable to the garlic oil component of these formulations

(Table 4.5).

Geraniol was highly effective at damaging dandelions and crabgrass. There was

no reduction of efficacy when geraniol was mixed with other oils and applied to

dandelions, and injury exceeded 95% by 24 hours with all treatments (Table 4.6). On

crabgrass, efficacy was decreased significantly when geraniol was mixed with 2-

phenethyl propionate, cedar, cinnamon, citronella, garlic or mint oils. However, all

treatments had acceptable performance on crabgrass, resulting >40% injury by 24 hours.

Geranium oil is highly phytotoxic, with the 10% geranium oil treatment reaching

99% and 78% injury to dandelions and crabgrass, respectively, within 24 hours (Table

4.7). Geranium oil mixtures with cedar or garlic oils were significantly less effective on

dandelions and crabgrass at 24 hours compared to 10% geranium oil (Table 4.7). In

addition, crabgrass injury was also significantly reduced by 24 hours when geraniol, mint

97
and rosemary oils were mixed with geranium oil. However, all treatments were

acceptable herbicidal mixtures for quickly injuring dandelions and crabgrass.

Lemongrass oil was a very effective herbicide at 10% concentration, with 96%

injury to dandelions and 55% injury to crabgrass 24 hours after foliar application. There

was no statistically significant difference between 10% lemongrass oil and the treatments

in which it was combined with the other oils, except for the lemongrass + citronella

treatment, which resulted in 82% injury to dandelions by 24 hours on dandelions

compared to 96% injury with 10% lemongrass oil. This reduction in efficacy was not

expected, since lemongrass and citronella oils are derived from plants within the same

genus, and typically contain similar constituents. All treatments were effective at burning

crabgrass, and there were no statistically significant differences among the treatments 24

hours after application.

Mint oil was an effective herbicide at 10% concentration, as well as when

combined with the other essential oils (Table 4.8). All treatments, except for mint +

cedar and mint + garlic, resulted in 90% injury or greater on dandelions within 24 hours

after application. On crabgrass, there was a significant reduction in injury when mint oil

was combined with cedar, garlic or geraniol oils (Table 4.8).

Peppermint oil was highly effective as an herbicide on its own at 10%

concentration, as well as in combination with other essential oils. In comparison to the

10% peppermint oil, efficacy was significantly reduced on dandelions when mixed with

cedar, garlic or rosemary oils. When tested on crabgrass, mixtures of peppermint oil with

clove or garlic oils were significantly less herbicidal at 24 hours, as compared to the 10%

98
peppermint oil treatment (Table 4.9). However, all combinations with peppermint oil

were phytotoxic to both weed species.

While 10% rosemary oil was phytotoxic to dandelions with 68% injury at 24

hours, its efficacy was significantly improved when mixed with cinnamon, geranium,

geraniol or mint oils. None of the essential oil combinations were significantly different

from 10% rosemary oil on crabgrass injury at 1 and 24 hours after application (Table

4.10). Rosemary was one of the least herbicidal essential oils tested, with performance

similar to that of cedar oil.

Thyme oil was a very effective herbicide, resulting in 98% injury to dandelions

and 68% injury to crabgrass within 24 hours of foliar application with 10% thyme oil.

All essential oil combination treatments with thyme oil resulted in good burndown. The

thyme + garlic combination was the only treatment that was significantly less effective

than 10% thyme oil on dandelions at 24 hours. For the crabgrass 24 hour data, none of

the combination treatments were significantly different from 10% thyme oil.

2-phenethyl propionate was an effective herbicide, injuring 90% of dandelion leaf

tissue by 24 hours after application (Table 4.11). Combinations of other essential oils

with 2-phenethyl propionate were equally effective on dandelions at 24 hours, with the

exceptions of 2-phenethyl propionate + garlic and 2-phenethyl propionate + rosemary

which were significantly less effective (Table 4.11). The 2-phenethyl propionate +

garlic combination was also significantly less effective on crabgrass at 24 hours

compared to 10% 2-phenethyl propionate alone. All other essential oil combinations

were similarly effective at burning down crabgrass (Table 4.11).

99
 On dandelions 1 hour after application, there was a significant difference between

runs in the model (p = <0.01), but not significant in the model for 24 hour data (p=0.52).

Geranium, cinnamon, mint, peppermint, geraniol, eugenol, citronella and thyme oils

burned over 50% of the dandelion foliage by 1 hour at 10% w/w concentration, and were

not significantly different from one another (Table 4.12). By 24 hours, these eight oils,

as well as lemongrass oil, clove oil and 2-phenethyl propionate had injured at least 90%

of dandelion leaf tissue. While rosemary and cedar oils were significantly less effective

than all the others, not including garlic, these two oils still damaged dandelion leaf tissue

by 60% or greater by 24 hours after application (Table 4.12). Garlic oil was the least

effective as an herbicide (Tables 4.12 and 4.13). Other than garlic oil, there were no

significant differences in herbicide activity of the essential oils on crabgrass 1 and 24

hours after foliar application (Table 4.13). Essential oil injury to dandelions was more

severe than crabgrass across all treatments, within each timeframe of injury rating

(Tables 4.12 and 4.13). See Appendix A.1 for photographs of dandelion and crabgrass

injury at 1 and 24 hours after application with 10% essential oil mixtures.

3.2 Phase 2: essential oils x surfactant systems: dandelion injury

An unexpected synergistic herbicide effect occurred when essential oils were

combined with surfactant system A and applied to dandelion foliage. In the greenhouse

experiments, surfactant system A, when tested without an essential oil, injured an average

of 0.63% dandelion leaf tissue by 1 hour. When essential oils were combined with non-

herbicidal inert ingredients (surfactant system B), the resulting injury ranged from 1.5%

to 20% depending on the essential oil. However, when essential oils at 5% w/w dilution

100
were combined with surfactant system A, 1 hour injury ranged from 7% to 49%

depending on the essential oil. By 6 hours after application, cinnamon, citronella, clove,

eugenol, geraniol, lemongrass and thyme oils combined with surfactant system A had

injured dandelion leaf tissue by 70% or more (Table 2.10). The results of the field study

were similar to the greenhouse in that when essential oils were mixed with surfactant

system A, the resultant weed injury was significantly enhanced compared to when

surfactant systems B or C were used (Table 2.11). Due to the hydrophobic nature of the

oils, it is not possible to test the oils at a 5% w/w dilution in water without adding

emulsifiers to create a homogenous solution. Therefore, any injury from the essential oils

+ surfactant system B is considered to be solely due to the essential oil since these inert

materials are extensively used surfactants in agrochemical formulations and have no

herbicide activity on their own. To determine synergy, the following equation from

Colby (1967) was used:

E = X + Y – XY/100,

Where E = Expected % control of ingredients A & B at p and q lb/Acre

X = % control of ingredient A at p lb/Acre,

Y = % control of ingredient B at q lb/Acre,

XY = % control of ingredients A+B at p and q lb/Acre

If the actual data is significantly greater than the expected value from the mixture,

then the mixture is considered to be synergistic. If the actual and expected values are

equal, the components of the mixture are considered to have an additive effect. If the

101
actual data from the mixture is significantly less than the expected value, there is an

antagonistic effect of the components at the given rate.

Data from the two greenhouse trials and the field trial from Phase 2 were

combined and a two-tailed t-test was conducted to determine if the difference between

the actual and expected percent injury at 6 hours after treatment were significant. The

synergy of all essential oils mixed with surfactant system A was very highly significant,

with p values of 0.0005 or less for all oils. The primary benefit of this particular

combination of ingredients in surfactant system A with essential oils is the early onset of

herbicide injury symptoms. The injury of essential oils to dandelions when mixed with

surfactant systems B and C were comparable in herbicide activity, except thyme oil and

eugenol, which were significantly enhanced with system C in greenhouse and field trials

(Tables 2.10 and 2.11). Through a series of experiments not shown here, it was deduced

that the fatty acid component, sodium caprylate, in surfactant system A was responsible

for the synergistic occurrence. See Appendix B.2 for photographs of dandelion injury

illustrating the synergy effect of essential oils and surfactant system A.

4. Conclusions

The following essential oils, all of which are listed on EPA’s 25(b) minimum risk

pesticide list, have non-selective contact herbicidal properties at a 10% w/w

concentration within an aqueous solution: cedar oil, cinnamon oil, citronella oil, clove

oil, eugenol, geraniol, geranium oil, lemongrass oil, mint oil, peppermint oil, rosemary

oil, thyme oil and 2-phenethyl propionate. The degree of injury was dependent on the

essential oil, with cedar and rosemary oils typically less injurious than the others. The

102
only essential oil included in the study that did not have contact toxicity to dandelions

and crabgrass was garlic oil. Across all treatments, dandelion leaf tissue was more

susceptible to injury than crabgrass at given time intervals. With the exceptions of cedar,

rosemary and garlic oils, all other essential oils at 10% w/w concentration burned 90% to

100% of dandelion foliage by 24 hours, as opposed to 50% to 82% of crabgrass leaf

tissue (Tables 4.12 and 4.13). To my knowledge, this is the first public documentation on

the herbicidal effects of geraniol, geranium, mint, peppermint and rosemary oils.

Herbicide activity of thyme, citronella, clove, eugenol, citronella, lemongrass and 2-

phenethyl propionate oils has previously been reported and have been used as active

ingredients in commercialized herbicide products (Table 4.1). The current study

confirms their potential as potent contact herbicides. There was no perceived

performance benefit to mixing two essential oils together. However, there may be a

financial benefit to blending essential oils for commercialization due to significant

differences in cost of essential oils, as well as constituent changes in supply from year to

year due to environmental, economic, political, and regulatory influences.

The enhancement of essential oil herbicide injury with surfactant system A in

Phase 2 was due to the presence of the fatty acid sodium caprylate. It is well known that

fatty acids with a carbon chain length of 8 to 12 are herbicidal when used at certain

concentrations. Several patents have been filed claiming the herbicidal use of certain

fatty acids, both stand-alone and in combination with synthetic herbicide active

ingredients (Puritch et al., 1990, Caulder et al. 2000, Sedun et al. 1999, Beste et al. 2003,

Pline et al. 1999). In the current study, surfactant system A was not herbicidal on its own

due to a sub-lethal concentration of sodium caprylate. It is assumed at this time that the

103
sodium caprylate caused the synergistic activity with the essential oils by dissolving the

waxy leaf cuticle and allowing the essential oil to more rapidly induce disruption of cell

membrane integrity. This theory is supported by the report of monoterpenes’ disruption

of leaf cuticles of Arabidopsis thaliana, as well as increased transpiration rates by

affecting stomatal closure (Shulz et al. 2007). These deleterious effects lead to

desiccation of leaf tissue. Further studies have been conducted by the author to determine

whether effective essential oil dilutions can be lowered when mixed with fatty acids of

varying chain length. This may allow essential oil formulations to be more cost-effective

by maintaining their fast burn-down while using lower rates than typically required of an

essential oil being used as an herbicide. This enhanced effect from the use of fatty acid

with an essential oil could also provide a visual cue to the applicator that the herbicide is

working at a faster rate than expected. There may also be potential for using an essential

oil plus fatty acid component as a fast-acting agent for other herbicides that have a slow

onset of visual symptoms. A provisional patent on the synergistic combination of certain

essential oils in combination with fatty acids of varying chain lengths has been filed by

the author and collaborators at The Scotts Miracle-Gro Company.

104
5. Tables

Product Active Ingredient(s) Company

Brush Block Brush & Weed 20% citric acid Greenergy
Killer
Burnout II Weed & Grass 30% citric acid St. Gabriel Organics
Killer 18% clove oil
C-cide citric acid Biological Solutions
Ecosmart Weed & Grass 5% 2-phenethyl propionate, EcoSmart
Killer 5% eugenol
0.05% sodium lauryl sulfate
EcoExempt HC 21.4% 2-phenethyl Avant Garde Organics
propionate
21.4% eugenol
Elementals Grass & Weed 18.75% soybean oil The Scotts Miracle-Gro
Killer Company
GreenMatch EX 50% lemongrass oil Marrone BioInnovations
Matran EC 50% clove (leaf) oil EcoSmart
Matratec 50% clove oil Brandt Consolidated
Weedzap 45% cinnamon oil JH Biotech Inc.
45% clove oil
Table 4.1. Non-selective EPA-exempt weed control products currently sold in the market

105
Treatment Dandelion 1 hr Dandelion 24 hr Crabgrass 1 hr Crabgrass 24 hr
% Adj P % Adj P % Adj P % Adj P
injury value injury value injury value injury value
10% Ced 23 - 61 - 10 - 43 -
Ced + 2PP 40 NS 91 <0.01 9 NS 51 NS
Ced + Cin 33 NS 98 <0.0001 8 NS 58 0.06
Ced + Cit 28 NS 89 <0.01 15 0.01 46 NS
Ced + Clo 64 <.0001 98 <0.0001 5 0.01 79 <0.0001
Ced +Eug 44 0.07 96 <0.001 12 NS 44 NS
Ced + Gar 4 NS 53 NS 3 <0.001 36 NS
Ced + Ger 28 NS 75 NS 9 NS 45 NS
Ced + Giol 55 <0.01 95 <0.001 8 NS 50 NS
Ced + Lem 34 NS 90 <0.01 13 NS 46 NS
Ced + Min 16 NS 70 NS 9 NS 43 NS
Ced + Pep 39 NS 82 0.07 10 NS 53 NS
Ced + Ros 19 NS 83 0.06 5 0.01 45 NS
Ced + Thy 58 <0.001 98 <.0001 8 NS 64 <.0001
Std Error 7.93 7.68 1.94 7.79
Table 4.2. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing cedar oil. Adjusted p values based on Dunnett's comparison of each treatment to 10%
cedar oil alone. NS = not significant from 10% cedar oil alone, with p value > 0.10.

106
Treatment Dandelion 1 hr Dandelion 24 hr Crabgrass 1 hr Crabgrass 24 hr
% Adj P % Adj P % Adj P % Adj P
injury value injury value injury value injury value
10% Cinn 73 - 99 - 11 - 66 -
Cin + 2PP 48 <0.01 95 NS 5 NS 45 NS
Cin + Ced 33 <.0001 98 NS 8 NS 58 NS
Cin + Cit 43 <0.001 98 NS 3 <0.01 66 NS
Cin + Clo 44 <0.01 97 NS 7 NS 48 NS
Cin +Eug 37 <.0001 93 0.02 8 NS 39 0.02
Cin + Gar 34 <.0001 95 NS 8 NS 41 0.03
Cin + Ger 68 NS 99 NS 9 NS 72 NS
Cin + Giol 47 <0.01 98 NS 8 NS 47 NS
Cin + Lem 65 NS 98 NS 9 NS 65 NS
Cin + Min 37 <.0001 97 NS 13 NS 78 NS
Cin + Pep 66 NS 100 NS 9 NS 68 NS
Cin + Ros 57 NS 97 NS 6 NS 65 NS
Cin + Thy 53 0.04 92 <0.01 5 NS 53 NS
Std Error 6.84 2.09 2.28 8.131
Table 4.3. Percent control of dandelion and crabgrass (LSMeans) with treatments
containing cinnamon oil. Adjusted P values based on Dunnett's comparison of each treatment to
10% cinnamon oil alone. NS = not significant from 10% cinnamon oil alone, with p value > 0.10.

1
Standard error for Cin+Min comparison to 10% cinnamon oil on crabgrass was 10.087. This
was due to only 3 replicates for the Cin + Min treatment in the 2nd experiment.

107
Treatment Dandelion 1 hr Dandelion 24 hr Crabgrass 1 hr Crabgrass 24 hr
% Adj P % Adj P % Adj P % Adj P
injury value injury value injury value injury value
10% Eug 50 - 98 - 15 - 62 -
Eug + 2PP 60 NS 98 NS 8 NS 62 NS
Eug + Ced 44 NS 96 NS 12 NS 44 NS
Eug + Cin 37 0.05 93 NS 8 NS 39 0.05
Eug + Cit 38 NS 94 NS 8 NS 50 NS
Eug +Clo 63 0.08 98 NS 9 NS 53 NS
Eug + Gar 37 0.05 75 <.0001 11 NS 50 NS
Eug + Ger 42 NS 93 NS 10 NS 68 NS
Eug + Giol 55 NS 98 NS 6 0.05 62 NS
Eug + Lem 51 NS 99 NS 18 NS 68 NS
Eug + Min 52 NS 98 NS 8 NS 58 NS
Eug + Pep 56 NS 93 NS 11 NS 58 NS
Eug + Ros 43 NS 83 0.01 8 NS 54 NS
Eug + Thy 47 NS 98 NS 5 0.02 57 NS
Standard 4.66 4.31 3.15 7.78
Error
Table 4.4. Percent control of dandelion and crabgrass (LSMeans) with eugenol
treatments. Adjusted p values are based on Dunnett's comparison of each treatment to 10% eugenol
alone. NS = not significant from 10% eugenol alone, with p value > 0.10.

108
Treatment Dandelion 1 hr Dandelion 24 hr Crabgrass 1 hr Crabgrass 24 hr
% Adj P % Adj P % Adj P % Adj P
injury value injury value injury value injury value
10% Gar 8 - 25 - 0 - 9 -
Gar + 2PP 18 NS 46 NS 3 NS 28 NS
Gar + Ced 4 NS 53 NS 3 NS 36 NS
Gar + Cin 34 0.01 95 <.0001 8 <0.001 41 0.04
Gar + Cit 46 <.0001 91 <.0001 5 0.03 59 <0.001
Gar +Clo 22 NS 76 <0.001 5 0.03 43 0.03
Gar + Eug 37 <0.01 75 <0.001 11 <.0001 50 <0.01
Gar + Ger 10 NS 65 <0.01 5 0.03 38 0.08
Gar + Giol 52 <.0001 97 <.0001 8 <0.001 42 0.03
Gar + Lem 53 <.0001 98 <.0001 7 <0.01 53 <0.01
Gar + Min 19 NS 73 <0.001 2 NS 33 NS
Gar + Pep 8 NS 55 0.09 2a NS 23b NS
Gar + Ros 3 NS 58 0.05 0 NS 43 0.03
Gar + Thy 36 <0.01 59 0.04 6 <0.01 52 <0.01
Standard 7.39 11.44 1.61 10.63
Error
Table 4.5. Percent control of dandelion and crabgrass (LSMeans) with garlic treatments.
Adjusted P values are based on Dunnett's comparison of each treatment to 10% garlic oil. NS = not
significant from 10% garlic oil alone, with p value > 0.10.

a
Std Error for Gar+Pep = 1.99 for Crabgrass 1 hour data set.
b
Std Error for Gar+Pep = 13.18 for Crabgrass 24 hour data set

109
Treatment Dandelion 1 hr Dandelion 24 hr Crabgrass 1 hr Crabgrass 24 hr
% Adj P % Adj P % Adj P % Adj P
injury value injury value injury value injury value
10% Giol 57 - 98 - 8 - 66 -
Giol + 2PP 58 NS 98 NS 8 NS 52 0.02
Giol + Ced 55 NS 95 NS 8 NS 50 <0.01
Giol + Cin 47 0.02 98 NS 8 NS 47 <0.001
Giol + Cit 55 NS 96 NS 9 NS 47 <0.001
Giol +Clo 53 NS 98 NS 8 NS 53 0.05
Giol + Eug 55 NS 98 NS 6 NS 62 NS
Giol + Gar 52 NS 97 NS 8 NS 42 <.0001
Giol + Ger 60 NS 98 NS 9 NS 54 0.09
Giol + Lem 43 <0.001 97 NS 8 NS 60 NS
Giol + Min 55 NS 98 NS 8 NS 42 <.0001
Giol + Pep 55 NS 98 NS 8 NS 58 NS
Giol + Ros 57 NS 97 NS 8 NS 56 NS
Giol + Thy 60 NS 98 NS 9 NS 56 NS
Standard 3.04 1.12 0.84 4.41
Error
Table 4.6. Percent control of dandelion and crabgrass (LSMeans) with geraniol
treatments. Adjusted p values based on Dunnett's comparison of each treatment to 10% geraniol
alone, NS = not significant from 10% geraniol alone, with p value > 0.10.

110
Treatment Dandelion 1 hr Dandelion 24 hr Crabgrass 1 hr Crabgrass 24 hr
% Adj P % Adj P % Adj P % Adj P
injury value injury value injury value injury value
10% Ger 78 - 99 - 14 - 78 -
Ger + 2PP 59 NS 97 NS 7 <.0001 62 NS
Ger + Ced 28 <.0001 75 <.0001 9 0.02 45 <0.01
Ger + Cin 68 NS 99 NS 9 0.02 72 NS
Ger + Cit 38a <0.01 99b <.0001 6c <0.01 76d NS
Ger +Clo 56 NS 96 NS 9 0.02 88 NS
Ger + Eug 42 <0.01 93 NS 10 0.07 68 NS
Ger + Gar 10 <.0001 65 <.0001 5 <.0001 38 <.0001
Ger + Giol 60 NS 98 NS 9 0.02 55 0.04
Ger + Lem 65 NS 100 NS 18 0.07 65 NS
Ger + Min 48 0.01 96 NS 10 0.07 50 0.01
Ger + Pep 38 <0.001 93 NS 10 0.07 57 0.09
Ger + Ros 63 NS 97 NS 8 <0.001 48 <0.01
Ger + Thy 54 0.09 96 NS 10 0.07 66 NS
Standard 9.10 4.0 1.51 8.24
Error
Table 4.7. Percent control of dandelion and crabgrass (LSMeans) with geranium oil
treatments. Adjusted p values based on Dunnett's comparison of each treatment to 10% geranium
oil alone. NS = not significant from 10% geranium oil alone, with p value > 0.10.

a
Std error for Ger+Cit treatment = 11.29 in Dandelion 1 hr data set.
b
Std error for Ger+Cit treatment = 5.06 in Dandelion 24 hr data set.
c
Std error for Ger+ Cit treatment = 1.88 in Crabgrass 1 hr data set.
d
Std error for Ger+Cit treatment = 10.22 in Crabgrass 24 hr data set.

111
Treatment Dandelion 1 hr Dandelion 24 hr Crabgrass 1 hr Crabgrass 24 hr
% Adj P % Adj P % Adj P % Adj P
injury value injury value injury value injury value
10% Min 72 - 98 - 7 - 72 -
Min + 2PP 62 NS 94 NS 9 <.0001 55 NS
Min + Ced 16 <.0001 70 <.0001 13 <0.01 43 0.04
Min + Cin 37 <.0001 97 NS 15 NS 75 NS
Min + Cit 38 <.0001 90 NS 6 NS 49 NS
Min +Clo 38 <.0001 96 NS 8 <.0001 60 NS
Min + Eug 52 0.05 98 NS 2 <0.001 58 NS
Min + Gar 19 <.0001 73 <0.001 10 <.0001 33 <0.01
Min + Ger 48 <0.01 96 NS 8 <0.01 50 NS
Min + Giol 55 NS 98 NS 9 <0.001 42 0.02
Min + Lem 52 0.05 95 NS 18 <0.01 60 NS
Min + Pep 61 NS 98 NS 16 NS 47 0.09
Min + Ros 45 <0.01 96 NS 6 <.0001 51 NS
Min + Thy 59 NS 99 NS 5 <.0001 75 NS
Standard 6.92 5.81 2.13 9.53
Error

Table 4.8. Percent control of dandelion and crabgrass (LSMeans) with mint oil
treatments. Adjusted p values based on Dunnett's comparison of each treatment to 10% mint oil
alone. NS = not significant from 10% mint oil alone, with p value > 0.10.

112
Treatment Dandelion 1 hr Dandelion 24 hr Crabgrass 1 hr Crabgrass 24 hr
% Adj P % Adj P % Adj P % Adj P
injury value injury value injury value injury value
10% Pep 68 - 99 - 15 - 68 -
Pep + 2PP 50 NS 88 NS 10 NS 53 NS
Pep + Ced 39 0.0041 82 0.05 10 NS 53 NS
Pep + Cin 66 NS 100 NS 9 NS 68 NS
Pep + Cit 58 NS 98 NS 8 0.08 77 NS
Pep +Clo 29 <.0001 89 NS 8 0.08 43 0.03
Pep + Eug 56 NS 99 NS 11 NS 58 NS
a b
Pep + Gar 8 <.0001 55 <.0001 1 <0.001 19 <.0001
Pep + Ger 38 <0.01 93 NS 10 NS 57 NS
Pep + Giol 55 NS 98 NS 8 0.08 58 NS
Pep + Lem 70 NS 98 NS 10 NS 75 NS
Pep + Min 61 NS 98 NS 16 NS 47 0.09
Pep + Ros 22 <.0001 78 <0.01 8 0.08 47 0.09
Pep + Thy 35 <0.001 90 NS 8 NS 54 NS
Standard 7.57 5.98 2.77 7.93
Error

Table 4.9. Percent control of dandelion and crabgrass (LSMeans) with peppermint oil
treatments. Adjusted p values based on Dunnett's comparison of each treatment to 10%
peppermint oil alone. NS = not significant from 10% peppermint oil alone, with p value > 0.10.

a
Std error for Pep+Gar = 3.44 in Crabgrass 1 hr data set.
b
Std error for Pep+Gar = 9.84 in Crabgrass 24 hr data set.

113
Treatment Dandelion 1 hr Dandelion 24 hr Crabgrass 1 hr Crabgrass 24 hr
% Adj P % Adj P % Adj P % Adj P
injury value injury value injury value injury value
10% Ros 11 - 68 - 6 - 48 -
Ros + 2PP 34 0.06 69 NS 5 NS 58 NS
Ros + Ced 19 NS 83 NS 5 NS 45 NS
Ros + Cin 57 <.0001 97 0.03 6 NS 65 NS
Ros + Cit 33 0.07 86 NS 8 NS 55 NS
Ros +Clo 21a NS 59b NS 8c NS 32d NS
Ros + Eug 43 <0.01 83 NS 8 NS 54 NS
Ros + Gar 3 NS 58 NS 0 0.02 43 NS
Ros + Ger 63 <.0001 97 0.03 8 NS 48 NS
Ros + Giol 57 <.0001 97 0.03 8 NS 56 NS
Ros + Lem 40 <0.01 93 0.09 9 NS 50 NS
Ros + Min 45 <0.001 96 0.04 6 NS 51 NS
Ros + Pep 22 NS 78 NS 8 NS 47 NS
Ros + Thy 38 0.02 88 NS 7 NS 53 NS
Standard 7.94 9.50 1.76 9.97
Error
Table 4.10. Percent control of dandelion and crabgrass (LSMeans) with rosemary oil
treatments. Adjusted p values based on Dunnett's comparison of each treatment to 10% rosemary
oil alone. NS = not significant from 10% peppermint oil alone, with p value > 0.10.

a
Std error for Ros+Clo = 9.87 in Dandelion 1 hr data set
b
Std error for Ros+Clo = 11.79 in Dandelion 24 hr data set
c
Std error for Ros + Clo = 2.19 in Crabgrass 1 hr data set
d
Std error for Ros + Clo = 12.37 in Crabgrass 24 hr data set

114
Treatment Dandelion 1 hr Dandelion 24 hr Crabgrass 1 hr Crabgrass 24 hr
% Adj P % Adj P % Adj P % Adj P
injury value injury value injury value injury value
10% 2PP 39 - 90 - 9 - 49 -
2PP + Ced 40 NS 91 NS 9 NS 51 NS
2PP + Cin 48 NS 95 NS 5 <0.01 45 NS
2PP + Cit 60 <0.001 95 NS 11 NS 65 0.03
2PP + Clo 55 0.01 93 NS 5 <0.01 58 NS
2PP + Eug 60 <0.001 98 NS 8 NS 62 NS
2PP + Gar 17 <0.001 46 <.0001 3 <.0001 28 <0.01
2PP + Ger 59 <0.001 97 NS 7 NS 62 NS
2PP + Giol 58 <0.01 98 NS 8 NS 52 NS
2PP + Lem 53 0.03 94 NS 5 <0.01 45 NS
2PP + Min 52 <.0001 94 NS 7 NS 55 NS
2PP + Pep 50 NS 88 NS 10 NS 53 NS
2PP + Ros 34 NS 69 <0.01 5 <0.01 58 NS
2PP + Thy 55 0.01 95 NS 3 <.0001 50 NS
Standard 4.63 5.05 1.11 5.06
Error
Table 4.11. Percent control of dandelion and crabgrass (LSMeans) with 2-phenethyl
propionate treatments. Adjusted p values based on Dunnett's comparison of each treatment to
10% 2-phenethyl propionate alone. NS = not significant from 10% 2-phenethyl propionate alone,
with p value > 0.10.

115
Essential Oil N % injury (1hr) % injury (24hr)
Geranium 6 78 a 99 a
Cinnamon 6 73 ab 99 a
Mint 6 72 ab 98 ab
Peppermint 6 68 abc 99 a
Geraniol 6 57 abc 98 ab
Citronella 6 57 abc 98 ab
Thyme 6 53 abc 98 ab
Eugenol 6 50 abcd 98 ab
Lemongrass 6 48 bcd 96 ab
Clove 6 42 cd 93 ab
2-phenethyl propionate 6 39 cde 90 abc
Cedar 6 23 def 61 c
Rosemary 6 12 ef 68 cd
Garlic 6 8 f 25 d
Table 4.12. Percent injury of dandelions at 1 and 24 hours after foliar application with
10% w/w essential oil formulations. Means followed by same letter within a column are not
significantly different at the α=0.05 level, Fisher’s LSD.

Essential Oil N % injury (1hr) % injury (24hr)

Mint 6 18 a 72 a
Lemongrass 6 17 a 55 a
Eugenol 6 15 a 62 a
Peppermint 6 15 a 68 a
Geranium 6 14 a 78 a
cinnamon 6 11 ab 66 a
Cedar 6 10 ab 43 ab
2-phenethyl propionate 6 9 ab 49 ab
Thyme 6 9 ab 68 a
Citronella 6 9 ab 54 ab
Clove 6 9 ab 82 a
Geraniol 6 8 ab 66 a
Rosemary 6 6 ab 48 ab
Garlic 6 0 b 9 b
Table 4.13. Percent injury of crabgrass 1 and 24 hours after application of 10% w/w
essential oil formulations. Means followed by same letter within a column are not significantly
different at the α=0.05 level, Fisher’s LSD.

116
Treatment Actual % Expected % P value Joint activity of
injury (6 injury (6 hours) essential oil +
hours), n = surfactant
12 system A
Cinnamon + A 95 18 <0.0001 synergistic
Citronella + A 90 18 <0.0001 synergistic
Clove + A 95 26 <0.0001 synergistic
Eugenol + A 93 22 <0.0001 synergistic
Geraniol + A 93 33 <0.0001 synergistic
Geranium + A 78 24 <0.0001 synergistic
Lemongrass + A 78 16 <0.0001 synergistic
Peppermint + A 59 12 <0.0001 synergistic
Rosemary + A 23 9 0.0005 synergistic
Thyme + A 75 15 <0.0001 synergistic
Table 4.14 Herbicide synergy of ten essential oils mixed at 5% w/w concentration with
surfactant system A (89.57% DI water, 2% w/w sodium caprylate, 0.33% Caprol®
MPGO, 0.1% xanthan gum) 6 hours after application to dandelions. Actual and expected
injury percentages are an average of 2 greenhouse trials and 1 field trial. Two-tailed t test to
determine if adjusted = expected.

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www.epa.gov/pesticides/pestsales/07pestsales/table_of_contents2007.htm. 2006-2007
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Zirkle, G. 2011. Modeling Carbon sequestration in home lawns. HortSci. 46: 808-814.

118
Appendix A: Essential oil chromatograms from mass spectrometry analysis

119
Figure A.1. Chromatogram of cedar oil sample used in Phase 1 and 2 insecticide and
herbicide experiments

120
Figure A.2. Chromatogram of cinnamon oil sample used in Phase 1 and 2 insecticide and
herbicide experiments

121
Figure A.3. Chromatogram of citronella oil sample used in Phase 1 and 2 insecticide and
herbicide experiments

122
Figure A.4. Chromatogram of clove oil sample used in Phase 1 and 2 insecticide and
herbicide experiments

123
Figure A.5. Chromatogram of eugenol sample used in Phase 1 and 2 insecticide and
herbicide experiments

124
Figure A.6. Chromatogram of garlic oil sample used in Phase 1 and 2 insecticide and
herbicide experiments

125
Figure A.7. Chromatogram of geraniol oil sample used in Phase 1 and 2 insecticide and
herbicide experiments

126
Figure A.8. Chromatogram of geranium oil sample used in Phase 1 and 2 insecticide and
herbicide experiments

127
Figure A.9. Chromatogram of lemongrass oil sample used in Phase 1 and 2 insecticide
and herbicide experiments

128
Figure A.10. Chromatogram of mint oil sample used in Phase 1 and 2 insecticide and
herbicide experiments

129
Figure A.11. Chromatogram of peppermint oil sample used in Phase 1 and 2 insecticide
and herbicide experiments

130
Figure A.12. Chromatogram of rosemary oil sample used in Phase 1 and 2 insecticide
and herbicide experiments

131
Figure A.13. Chromatogram of thyme oil sample used in Phase 1 and 2 insecticide and
herbicide experiments

132
Figure A.14. Chromatogram of 2-phenethyl propionate sample used in Phase 1 and 2
insecticide and herbicide experiments

133
Appendix B: Photographs of essential oil HLB experiment

134
 Photographic images of HLB determination of essential oils experiment were

taken at multiple intervals. The photographs presented below were selected based on the

timepoint at which phase separation had occurred and required HLB could be

determined. In each of the following images, HLB surfactants ranging from left to right:

6, 8, 10, 12, 14, and 16.

Figure B.1. Cinnamon oil with surfactants and water, photograph taken 25 hours after
mixing ingredients.

Figure B.2. Citronella oil with surfactants and water. Photograph taken 26 hours after
mixing ingredients.
135
Figure B.3. Clove oil with surfactants and water. Photograph taken 25 hours after mixing
ingredients.

Figure B.4 Eugenol with surfactants and water. Photograph taken 2.5 hours after mixing
ingredients.

136
Figure B.5. Geraniol with surfactants and water. Photograph taken 3 hours after mixing
ingredients.

Figure B.6. Geranium oil with surfactants and water. Photograph taken 18 hours after
mixing ingredients.

137
Figure B.7. Lemongrass oil with surfactants and water. Photograph taken 1 hour after
mixing ingredients.

Figure B.8. Peppermint oil with surfactants and water. Photograph taken 1 hour after
mixing ingredients.

138
Figure B.9. Rosemary oil with surfactants and water. Photograph taken 1 hour after
mixing ingredients.

Figure B.10. Thyme oil with surfactants and water. Photograph taken 1 hour after mixing
ingredients.

139
Figure B.11. Soybean oil with surfactants and water. Photograph taken 10 minutes after
mixing ingredients.

140
Appendix C: Photographs from tensiometer for contact angle measurement

141
 Water, θ = 111.2°

Blank A, θ = 81.0° Blank B, θ = 79.9° Blank C, θ = 60.8°

Cinnamon A, θ = 76.8° Cinnamon B, θ = 69.6° Cinnamon C, θ = 57.7°

Citronella A, θ = 63.5°
Citronella B, θ = 49.9° Citronella C, θ = 52.7°

Table C.1. Sessile droplets of formulations measured for contact angle using Kruss
tensiometer

continued
142
Table C.1 continued

Clove B, θ = 57.5° Clove C, θ = 55.9°

Clove A, θ = 60.3°

Eugenol A, θ = 57.2° Eugenol B, θ = 60.2° Eugenol C, θ = 48.9°

Geraniol A, θ = 51.5° Geraniol B, θ = 50.2° Geraniol C, θ = 47.5°

Geranium A, θ = 50.8° Geranium B, θ = 54.0° Geranium C, θ = 49.7°

143
Table C.1 continued

Lemongrass A, θ = 64.6°
Lemongrass B, θ = 61.6° Lemongrass C, θ = 52.6°

Peppermint A, θ = 64.0° Peppermint B, θ = 60.7° Peppermint C, θ = 49.9°

Rosemary A, θ = 56.0° Rosemary B, θ = 61.1° Rosemary C, θ = 52.8°

Thyme A, θ = 80.3° Thyme B, θ = 65.0° Thyme C, θ = 59.2°

144
Appendix D: Phase 1 photographs of weed injury 1 and 24 hours after application

145
 Photographs of 10% Essential oil formulations applied to dandelions and

crabgrass 1 and 24 hours after treatment (HAT). For each group of pictures, top left –

dandelion 1 HAT, top right- dandelion 24 HAT, bottom left- crabgrass 1 HAT, bottom

right- crabgrass 24 HAT.

Figure D.1. 10% cedar oil, 1 and 24 HAT on dandelion and crabgrass

146
Figure D.2. 10% cinnamon oil, 1 and 24 HAT on dandelion and crabgrass

Photo not available

Figure D.3. 10% citronella oil, 1 and 24 HAT on dandelion and crabgrass

147
 Photo not available

Figure D.4. 10% clove oil, 1 and 24 HAT on dandelion and crabgrass

Figure D.5. 10% garlic oil, 1 and 24 HAT on dandelion and crabgrass

148
Figure D.6. 10% geraniol, 1 and 24 HAT on dandelion and crabgrass

Figure D.7. 10% geranium oil, 1 and 24 HAT on dandelion and crabgrass

149
Figure D.8. 10% eugenol, 1 and 24 HAT on dandelion and crabgrass

Figure D.9.. 10% lemongrass oil, 1 and 24 HAT on dandelion and crabgrass

150
Figure D.10. 10% mint oil, 1 and 24 HAT on dandelion and crabgrass

Figure D.11. 10% peppermint oil, 1 and 24 HAT on dandelion and crabgrass

151
Figure D.12. 10% rosemary oil, 1 and 24 HAT on dandelion and crabgrass

Photo not available

Figure D.13. 10% thyme oil, 1 and 24 HAT on dandelion and crabgrass

152
 Photo not available

Photo not available

Figure D.14. 10% 2-phenethyl propionate, 1 and 24 HAT on dandelion and crabgrass

153
Appendix E: Phase 2 photographs of weed injury 6 hours after application, greenhouse
& field trials

154
 C
A B
Figure E.1. Cinnamon oil treatments in greenhouse trial:
Within each photograph (L to R): untreated, cinnamon oil + system A B or C, and
system A B or C alone

Figure E.2. Cinnamon oil treatments in field trial:

(L to R) cinnamon oil+ system A, cinnamon oil + system B, cinnamon oil+ system C

A B C
Figure E.3. Citronella oil treatments in greenhouse trial:
Within each photograph (L to R): untreated, citronella oil + system A B or C, and
system A B or C alone

Figure E.4. Citronella oil treatments in field trial:

(L to R): citronella oil + system A, citronella oil + system B, citronella oil + system C

155
 A B C
Figure E.5. Clove oil treatments in greenhouse trial:
Within each photograph (L to R): untreated, clove oil + system A B or C, and system A
B or C alone

Figure E.6. Clove oil treatments in field trial:

(L to R): clove oil + system A, clove oil + system B, clove oil + system C

C
A B
Figure E.7. Eugenol treatments in greenhouse trial:
Within each photograph (L to R): untreated, eugenol oil + system A B or C, and system
A B or C alone

Figure E.8. Eugenol treatments in field trial:

(L to R): eugenol + system A, eugenol + system B, eugenol + system C

156
 C
A B
Figure E.9. Geraniol treatments in greenhouse trial:
Within each photograph (L to R): untreated, geraniol + system A B or C, and system A
B or C alone

Figure E.10. Geraniol treatments in field trial:

(L to R): geraniol + system A, geraniol + system B, geraniol + system C

C
A B
Figure E.11. Geranium oil treatments in greenhouse trial:
Within each photograph (L to R): untreated, geranium oil + system A B or C, and system
A B or C alone

Figure E.12. Geranium oil treatments in field trial:

(L to R): geranium oil + system A, geranium oil + system B, geranium oil + system C

157
 B C
A
Figure E.13. Lemongrass oil treatments in greenhouse trial:
Within each photograph (L to R): untreated, lemongrass oil + system A B or C, and
system A B or C alone

Figure E.14. Lemongrass oil treatments in field trial:

(L to R): lemongrass oil + system A, lemongrass oil + system B, lemongrass oil + system
C

C
A B
Figure E.15. Peppermint oil treatments in greenhouse trial:
Within each photograph (L to R): untreated, peppermint oil + system A B or C, and
system A B or C alone

Figure E.16. Peppermint oil treatments in field trials:

(L to R): peppermint oil + system A peppermint oil + system B peppermint oil +
system C

158
 C
A B
Figure E.17. Rosemary oil treatments in greenhouse trial:
Within each photograph (L to R): untreated, rosemary oil + system A B or C, and system
A B or C alone

Figure E.18. Rosemary oil treatments in field trial:

(L to R): rosemary oil + system A, rosemary oil + system B, rosemary oil + system C

A B C
Figure E.19. Thyme oil treatments in greenhouse trial:
Within each photograph (L to R): untreated, thyme oil + system A B or C, and system A
B or C alone

Figure E.20. Thyme oil treatments in field trial:

(L to R): thyme oil + system A, thyme oil + system B, thyme oil + system C

159
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