Patent Publication Number: US-2020275651-A1

Title: Bioinsecticidal compositions containing monoterpenes and methods of use thereof

Description:
INTRODUCTION 
     This application claims the benefit of priority of U.S. Provisional Application No. 62/581,225, filed Nov. 3, 2017, the content of which is incorporated herein by reference in its entirety. 
    
    
     BACKGROUND 
     Synthetic insecticides raise concerns about food contamination, environmental pollution and ecological adverse effects. In addition, heavy use of synthetic insecticides has led to the development of insect resistance. Such problems and concerns require development of effective natural insecticides. Botanicals offer an alternative to synthetic insecticides, as they may exhibit lower mammalian toxicity. 
     Studies have reported that essential oils and their components are toxic to insect pests at different life stages. In this respect, several studies have focused on the potential use of essential oils in biological control of different insect pests. For example,  Artemisia sieberi  essential oil can be a control agent against stored-product insects.  Citrus sinensis  essential oil can control  Musca domestica  adults and inhibit P450 . Cymbopogon citratus  essential oil and its major components, citral and 1,8-cineole, are insecticidal to housefly. Thyme and lemongrass essential oils are also active against larvae and an ovarian cell line of the cabbage looper  Trichoplusia ni . However, the results indicated a weak correlation between insecticidal activity and cytotoxicity, which the latter limits them as insecticides. Essential oil from  Alpinia purpurata  inflorescences can control  Sitophilus zeamais  (maize weevil) and kill  S. zeamais  adults with a median lethal concentration (LC 50 ) of 41.4 mL/L in air.  Eucalyptus  essential oils have fumigation toxicity against  Ectomyelois ceratoniae  under various conditions. Further, essential oil nanoformulations have been used to control the storage pests  Tribolium castaneum  and  Rhizopertha dominica.    
     While the chemical composition and insecticidal properties of essential oils have been investigated, the effectiveness of individual terpenoids against specific pest insects remains species-specific. For example, linalool and (−)-pulegone have been shown to exhibit fumigant toxicity against palm  thrips  (Kim, et al. (2015)  Pest. Manag. Sci.  71:1292-1296); pulegone, carvacrol, trans-anethole and linalool are toxic to mosquitoes (Dias, et al. (2014)  Parasitol. Res.  113:565-592; Waliwitiya, et al. (2009)  Pest Management Sci.  65(3):241-248); trans-anethole has been shown to exhibit fumigant activity against  Blattella germanica  (Chang &amp; Ahn (2001)  Pest Management Sci.  58:161-166); carvacrol, thymol and citral exhibit toxicity against Pochazia shantungensis (Park, et al. (2017)  Sci. Rep.  7:40902); trans-anethole, estragole, and (+)-fenchone are active against  Sitophilus oryzae, Callosobruchus chinensis  and  Lasioderma serricorne  in a fumigation test (Kim &amp; Ahn (2001)  Pest Manag. Sci.  57(3):301-6); and one or more of pulegone, linalool and 1-fenchone have fumigation toxicity against rice weevil, red flour beetle, sawtoothed grain beetle, housefly and German cockroach (Lee, et al. (2003)  J. Stored Prod. Res.  77-85). Further, oxygenated monoterpenes have been suggested to have a high potential for control of  S. zeamais  adults. Moreover, basil oil and its three major active constituents (trans-anethole, estragole, and linalool) exhibit insecticidal activity against the tephritid fruit fly species  Ceratitis capitata  (Wiedemann),  Bactrocera dorsalis  (Hendel), and  Bactrocera cucurbitae  (Coquillett) (Chang, et al. (2009)  J. Econ. Entomol.  102(1):203-209). 
     SUMMARY OF THE INVENTION 
     A method for controlling an insect pest by exposing at least one insect pest to a bioinsecticidal composition containing an effective, insect controlling amount of (+)-pulegone, carvacrol, linalool, trans-anethole, or estragole is provided. In some embodiments, the bioinsecticidal composition is a fumigant. In other embodiments, the bioinsecticidal composition further includes piperonyl butoxide. A bioinsecticidal composition containing an effective, insect controlling amount of (+)-pulegone and piperonyl butoxide is also provided. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Botanical insecticides are needed to selectively and effectively control one or a number of insect pests. It has now been shown that certain monoterpenes are of use in selectively controlling agricultural, storage, and household insect pests. The fumigation toxicity of these monoterpenes appears to be related to their structures. Most of the monoterpenes tested exhibit good-to-moderate toxicity against the test pests and low toxicity to the insect natural enemy species. In particular, (+)-pulegone and estragole are highly toxic to adult pests even at low concentrations. Carvacrol is effective against household pests, and linalool was found to control both  thrips  and leafhopper in field tests. The action of these monoterpenes is fast and shows a steep dose-response relationship, indicating a distinct action site in the neuron system. 
     Accordingly, the present invention provides bioinsecticidal compositions and methods for combating or controlling one or more insect pests in a location susceptible to the presence or incursion thereof using an effective, insect-controlling amount of a monoterpene, in particular (+)-pulegone, carvacrol, linalool, trans-anethole and/or estragole. The term “bioinsecticide” or “bioinsecticidal” as used herein refers to a chemical substance of natural origin that can be optionally synthesized and used in the control of agricultural, natural environmental, storage, and/or domestic/household pests. In accordance with the present invention the bioinsecticide is at least one of (+)-pulegone, carvacrol, linalool, trans-anethole or estragole. In particular embodiments, the bioinsecticide is (+)-pulegone or linalool. In certain embodiments, the monoterpene used in accordance with the present invention is pure. More specifically, the monoterpene used is at least 95%, 96%, 97%, 98%, 99% or 99.9% homogenous to the monoterpene of interest. 
     The amount of monoterpene present in the bioinsecticidal composition is an amount as low as 1 wt. %, in an amount as low as 5 wt. %, or in an amount as low as 10 wt. %. The monoterpene can be included in an amount that is as high as 70 wt. %, in an amount as high as 80 wt. %, or in an amount as high as 99 wt. %. The monoterpene may further be present within any range delimited by any pair of the foregoing values, such as between 1 wt. % and 99 wt. %, or between 10 wt. % to 70 wt. %, for example. 
     An insecticide is rarely suitable for application in its pure form. It is usually necessary to add other substances such as carriers or diluents so that the insecticide can be used at the required concentration and in an appropriate form, permitting ease of application, handling, transportation, storage, and maximum pesticide activity. Thus, the bioinsecticidal compositions of this invention are formulated to include at least one monoterpene (e.g., (+)-pulegone, carvacrol, linalool, trans-anethole, estragole or a combination thereof) in admixture with a carrier or diluent. The carrier or diluent of this invention is preferably an inert material, organic or inorganic, with which an active ingredient can be mixed or formulated to facilitate its application, storage, transport, and/or handling, or improve various product characteristics such as its odor. Commonly used carriers and diluents include, but are not limited to, ethanol, isopropanol, other alcohols, water, nanoparticles and nanotubes. Exemplary carriers that can be used include inert carriers listed by the U.S. EPA as a Minimal Risk Inert Pesticide Ingredients (4A), Inert Pesticide Ingredients (4B) or under EPA regulation 40 CFR 180.950, and include for example, citric acid, lactic acid, glycerol, castor oil, benzoic acid, carbonic acid, ethoxylated alcohols, ethoxylated amides, glycerides, benzene, butanol, 1-propanol, hexanol, other alcohols, dimethyl ether, and polyethylene glycol. In particular embodiments, the at least one monoterpene is formulated as the core material of a nanoparticle or nanotube. 
     The bioinsecticidal compositions of this invention can be formulated into solid, gaseous or liquid forms, for example, concentrated emulsions, dusts, emulsifiable concentrates, fumigants, gels, granules, microencapsulations, seed treatments, suspension concentrates, suspoemulsions, tablets, water soluble liquids, water dispersible granules or dry flowables, wettable powders, and ultralow volume solutions. For further information on formulation types, see “Catalogue of pesticide formulation types and international coding system,” Technical Monograph n° 2, 7 th  Edition by CropLife International (March 2017). In certain embodiments, the bioinsecticidal composition of this invention is in a liquid or gaseous form. 
     A bioinsecticidal composition can be applied as an aqueous suspension or emulsion prepared from a concentrated formulation containing a monoterpene. Such water-soluble, water-suspendable, or emulsifiable formulations, are either solids, usually known as wettable powders, or water dispersible granules, or liquids usually known as emulsifiable concentrates, or aqueous suspensions. Wettable powders, which may be compacted to form water dispersible granules, include an intimate mixture of the monoterpene, a carrier, and one or more surfactants. The concentration of the monoterpene is usually from about 10% to about 90% by weight. The carrier is usually chosen from among the attapulgite clays, the montmorillonite clays, the diatomaceous earths, the purified silicates, nanoparticles or nanotubes. Effective surfactants, constituting from about 0.5% to about 10% of the wettable powder, are found among sulfonated lignins, condensed naphthalenesulfonates, naphthalenesulfonates, alkylbenzenesulfonates, alkyl sulfates, and nonionic surfactants such as ethylene oxide adducts of alkyl phenols. 
     Emulsifiable concentrates provide a convenient concentration of a monoterpene, such as from about 50 to about 500 grams per liter of liquid dissolved in a carrier that is either a water miscible solvent or a mixture of water-immiscible organic solvent and emulsifiers. Useful organic solvents include aromatics, especially xylenes and petroleum fractions, especially the high-boiling naphthalenic and olefinic portions of petroleum such as heavy aromatic naphtha. Other organic solvents may also be used, such as the terpenic solvents including rosin derivatives, aliphatic ketones such as cyclohexanone, and complex alcohols such as 2-ethoxyethanol. Suitable emulsifiers for emulsifiable concentrates are chosen from conventional anionic and nonionic surfactants. 
     Aqueous suspensions include suspensions of water-insoluble monoterpenes dispersed in an aqueous carrier at a concentration in the range from about 5% to about 50% by weight. Suspensions are prepared by combining the monoterpene and vigorously mixing it into a carrier composed of water and surfactants. Ingredients, such as inorganic salts and synthetic or natural gums, may also be added, to increase the density and viscosity of the aqueous carrier. 
     Monoterpenes may also be applied as granular compositions that are particularly useful for applications to the soil. Granular compositions usually contain from about 0.5% to about 10% by weight of the monoterpene, dispersed in a carrier such as clay or a similar substance. Such compositions are usually prepared by dissolving the monoterpene in a suitable solvent and applying it to a granular carrier which has been pre-formed to the appropriate particle size, in the range of from about 0.5 to 3 mm. Such compositions may also be formulated by making a dough or paste of the carrier and monoterpene and crushing and drying to obtain the desired granular particle size. 
     Dusts containing a monoterpene are prepared by intimately mixing the monoterpene in powdered form with a suitable dusty agricultural carrier, such as kaolin clay, ground volcanic rock, and the like. Dusts can suitably contain from about 1% to about 10% of the monoterpene. They can be applied as a seed dressing, or as a foliage application with a dust blower machine. 
     It is equally practical to apply a monoterpene in the form of a solution in an appropriate organic solvent, such as petroleum oil, corn oil, or peanut oil, which are widely used in agricultural chemistry. 
     Monoterpenes can also be applied in the form of an aerosol composition. In such compositions the monoterpene is dissolved or dispersed in a carrier, which is a pressure-generating propellant mixture. The aerosol composition is packaged in a container from which the mixture is dispensed through an atomizing valve. 
     Fumigant forms of insecticides have a relatively high vapor pressure and hence can exist as a gas in sufficient concentrations to kill insects in soil or enclosed spaces. The toxicity of the fumigant is proportional to its concentration and the exposure time. They are characterized by a good capacity for diffusion and act by penetrating the pest&#39;s respiratory system or being absorbed through the pest&#39;s cuticle. Fumigants are applied to control insects under gas proof sheets, in gas sealed rooms or buildings or in special chambers. 
     Monoterpenes can be microencapsulated by suspending the monoterpene droplets in plastic polymers of various types. By altering the chemistry of the polymer or by changing factors in the processing, microcapsules can be formed of various sizes, solubility, wall thicknesses, and degrees of penetrability. These factors govern the speed with which the active ingredient within is released, which in turn, affects the residual performance, speed of action, and odor of the product. 
     Oil solution concentrates are made by mixing a monoterpene in a solvent that will hold the monoterpene in solution. Advantages of oil solutions include better storage stability, better penetration of crevices, and better adhesion to greasy surfaces. 
     Another embodiment is an oil-in-water emulsion, wherein the emulsion includes oily globules which are each provided with a lamellar liquid crystal coating and are dispersed in an aqueous phase. In accordance with this embodiment, each oily globule includes at least one monoterpene, and is individually coated with a monolamellar or oligolamellar layer including: (1) at least one non-ionic lipophilic surface-active agent, (2) at least one non-ionic hydrophilic surface-active agent and (3) at least one ionic surface-active agent, wherein the globules having a mean particle diameter of less than 800 nanometers. Further information on the embodiment is disclosed in US 2007/0027034. 
     In any of these forms, the bioinsecticidal composition can include a propellant, so that they can be aerosolized. Such aerosolized compositions can be used in fumigation applications, such as ship/air cargo or food containment areas. The bioinsecticidal composition can also be used as a pesticide treatment prior to transporting items. In one embodiment, the bioinsecticidal composition is converted to an aerosol composition by adding nitrogen to the formula, and keeping the contents in a pressurized container, such as a metal can. Nitrogen is an inert additive, and not a green-house gas, and can be preferred over other propellants, such as low molecular weight hydrocarbons. When present, the nitrogen is present in an amount of up to 10 percent, though is typically in the range of about 0.5% by weight of the bioinsecticidal composition. The aerosol composition can also include a propellant other than nitrogen, such as n-propane, n-butane, iso-pentane, iso-butane, n-pentane or hydrofluorocarbons. 
     In addition to at least one monoterpene and a suitable carrier or diluent, the bioinsecticidal composition of this invention can contain other components, which may or may not contain insecticidal activity. These components include, but are not limited to, wetters, spreaders, stickers, penetrants, buffers, sequestering agents, drift reduction agents, compatibility agents, anti-foam agents, cleaning agents, dispersants or surfactants, thixotropic agents, and emulsifiers. The amount of the additional component used can be in an amount as low as 0.01 wt. %, in an amount as low as 0.1 wt. %, or in an amount as low as 1 wt. %. The additional component can be included in an amount that is as high as 15 wt. %, in an amount as high as 30 wt. %, or in an amount as high as 50 wt. %. The additional component may further be present within any range delimited by any pair of the foregoing values, such as between 0.01 wt. % and 30 wt. %, or between 0.1 wt. % to 15 wt. %, for example. 
     A wetting agent is a substance that when added to a liquid increases the spreading or penetration power of the liquid by reducing the interfacial tension between the liquid and the surface on which it is spreading. Wetting agents are used for two main functions in agrochemical formulations: during processing and manufacture to increase the rate of wetting of powders in water to make concentrates for soluble liquids or suspension concentrates; and during mixing of a product with water in a spray tank to reduce the wetting time of wettable powders and to improve the penetration of water into water-dispersible granules. Examples of wetting agents used in wettable powder, suspension concentrate, and water-dispersible granule formulations include sodium lauryl sulphate, sodium dioctyl sulphosuccinate, alkyl phenol ethoxylates, and aliphatic alcohol ethoxylates. 
     A dispersing agent is a substance which adsorbs onto the surface of a particle and helps to preserve the state of dispersion of the particle and prevents it from reaggregating. Dispersing agents are added to formulations to facilitate dispersion and suspension during manufacture, and to ensure the particles redisperse into water in a spray tank. They are widely used in wettable powders, suspension concentrates and water-dispersible granules. Surfactants that are used as dispersing agents have the ability to adsorb strongly onto a particle surface and provide a charged or steric barrier to reaggregation of particles. The most commonly used surfactants are anionic, non-ionic, or mixtures of the two types. For wettable powder formulations, the most common dispersing agents are sodium lignosulphonates. For suspension concentrates, very good adsorption and stabilization are obtained using polyelectrolytes, such as sodium naphthalene sulphonate formaldehyde condensates. Tristyrylphenol ethoxylate phosphate esters are also used. Non-ionics such as alkylarylethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersing agents for suspension concentrates. Very high molecular weight polymeric surfactants have also been developed as dispersing agents. These have very long hydrophobic ‘backbones’ and a large number of ethylene oxide chains forming the ‘teeth’ of a ‘comb’ surfactant. These high molecular weight polymers can give very good long-term stability to suspension concentrates because the hydrophobic backbones have many anchoring points onto the particle surfaces. Exemplary dispersing agents used in the formulations of this invention include sodium lignosulphonates, sodium naphthalene sulphonate formaldehyde condensates, tristyrylphenol ethoxylate phosphate esters, aliphatic alcohol ethoxylates, alky ethoxylates, EO-PO block copolymers, and graft copolymers. 
     An emulsifying agent is a substance which stabilizes a suspension of droplets of one liquid phase in another liquid phase. Without the emulsifying agent the two liquids would separate into two immiscible liquid phases. The most commonly used emulsifier blends contain alkylphenol or aliphatic alcohol with 12 or more ethylene oxide units and the oil-soluble calcium salt of dodecylbenzene sulphonic acid. A range of hydrophile-lipophile balance (“HLB”) values from 8 to 18 will normally provide good stable emulsions. Emulsion stability can sometimes be improved by the addition of a small amount of an EO-PO block copolymer surfactant. 
     A solubilizing agent is a surfactant which will form micelles in water at concentrations above the critical micelle concentration. The micelles are then able to dissolve or solubilized water-insoluble materials inside the hydrophobic part of the micelle. The type of surfactants usually used for solubilization are non-ionics such as sorbitan monooleates, sorbitan monooleate ethoxylates, and methyl oleate esters. 
     Surfactants are sometimes used, either alone or with other additives such as mineral or vegetable oils as adjuvants to spray-tank mixes to improve the biological performance of the insecticide on the target. The types of surfactants used for bioenhancement depend generally on the nature and mode of action of the insecticide. However, they are often non-ionics such as alky ethoxylates, linear aliphatic alcohol ethoxylates, or aliphatic amine ethoxylates. 
     Organic solvents are used mainly in the formulation of emulsifiable concentrates, ULV formulations, and to a lesser extent granular formulations. Sometimes mixtures of solvents are used. The first main groups of solvents are aliphatic paraffinic oils such as kerosene or refined paraffins. The second main group, and the most common, includes the aromatic solvents such as xylene and higher molecular weight fractions of C 9  and C 10  aromatic solvents. Chlorinated hydrocarbons are useful as cosolvents to prevent crystallization of insecticides when the formulation is emulsified into water. Alcohols are sometimes used as cosolvents to increase solvent power. 
     Thickeners or gelling agents are used mainly in the formulation of suspension concentrates, emulsions and suspoemulsions to modify the rheology or flow properties of the liquid and to prevent separation and settling of the dispersed particles or droplets. Thickening, gelling, and anti-settling agents generally fall into two categories, namely water-insoluble particulates and water-soluble polymers. It is possible to produce suspension concentrate formulations using clays and silicas. Examples of these types of materials, include, but are limited to, montmorillonite, e.g., bentonite; magnesium aluminum silicate; and attapulgite. Water-soluble polysaccharides have also been used as thickening-gelling agents. The types of polysaccharides most commonly used are natural extracts of seeds and seaweeds or are synthetic derivatives of cellulose. Examples of these types of materials include, but are not limited to, guar gum, locust bean gum, carrageenan, alginates, methyl cellulose, sodium carboxymethyl cellulose (SCMC), and hydroxyethyl cellulose (HEC). Other types of anti-settling agents are based on modified starches, polyacrylates, polyvinyl alcohol and polyethylene oxide. Another good anti-settling agent is xanthan gum. 
     Microorganisms cause spoilage of formulated products. Therefore, preservation agents can be included to eliminate or reduce their effect. Examples of such agents include, but are limited to, propionic acid and its sodium salt, sorbic acid and its sodium or potassium salts, benzoic acid and its sodium salt, p-hydroxy benzoic acid sodium salt, methyl p-hydroxy benzoate, and 1,2-benzisothiazalin-3-one (BIT). 
     The presence of surfactants, which lower interfacial tension, often causes water-based formulations to foam during mixing operations in production and in application through a spray tank. In order to reduce the tendency to foam, anti-foam agents are often added either during the production stage or before filling into bottles. Generally, there are two types of anti-foam agents, namely silicones and non-silicones. Silicones are usually aqueous emulsions of dimethyl polysiloxane while the non-silicone anti-foam agents are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the anti-foam agent is to displace the surfactant from the air-water interface. 
     In one embodiment, the bioinsecticidal compositions include a thixotropic agent. In this embodiment, mechanical agitation, such as that which occurs when the compositions are sprayed, liquefies the compositions and allows them to be applied in aerosol form. When the mechanical agitation is stopped, the compositions then return to their original state, for example, a gel. The use of a thixotropic agent can enable the formulations to be prepared without using any oil, and enables the active components to stick on plant surfaces, and protect the plants from insect damage. 
     While it can be preferred in some embodiments for the bioinsecticidal compositions to be capable of being organically certified, as including only natural ingredients, in another embodiment, an additional repellent or cidal agent is included, which is a non-naturally occurring substance. Representative repellent and cidal agents include compounds or compositions that are used as acaricides, insecticides, insecticide synergists, and ixodicides. Chemical classes of insecticides include 2-dimethylaminopropane-1,3-dithiol, 2-dimethylaminopropane-1,3-dithiol analogs, amidines, arylpyrroles, avermectin, benzoylureas, carbamates, carbamoyl-triazoles, cyclodienes, diacylhydrazines, dinitrophenols, fiprole, METI, neonicotinoids, non-ester pyrethroids, organochlorines, organophosphates, oxadiazines, oximes, carbamates, pyrethroids, and spinosyns. Suitable insecticides include 1,1-bis(4-chlorophenyl)-2-ethoxyethanol, 1,1-dichloro-1-nitroethane, 1,1-dichloro-2,2-bis(4-ethylphenyl)ethane, 1,2-dichloropropane with 1,3-dichloropropene, 1-bromo-2-chloroethane, 2-(1,3-dithiolan-2-yl)phenyl dimethylcarbamate, 2-(2-butoxyethoxy)ethyl thiocyanate, 2-(4,5-dimethyl-1,3-dioxolan-2-yl)phenyl methylcarbamate, 2-(4-chloro-3,5-xylyloxy)ethanol, 2,2,2-trichloro-1-(3,4-dichlorophenyl)ethyl acetate, 2,2-dichlorovinyl 2-ethylsulfinylethyl methyl phosphate, 2,4-dichlorophenyl benzenesulfonate, 2-chlorovinyl diethyl phosphate, 2-isovalerylindan-1,3-dione, 2-methyl(prop-2-ynyl)aminophenyl methylcarbamate, 2-thiocyanatoethyl laurate, 3-bromo-1-chloroprop-1-ene, 3-methyl-1-phenylpyrazol-5-yl dimethylcarbamate, 4-chlorophenyl phenyl sulfone, 4-methyl(prop-2-ynyl)amino-3,5-xylyl methylcarbamate, 4-methylnonan-5-ol with 4-methylnonan-5-one, 5,5-dimethyl-3-oxocyclohex-1-enyl dimethylcarbamate, 6-methylhept-2-en-4-ol, abamectin, acephate acequinocyl, acrinathrin, alanycarb, aldicarb, aldoxycarb, aldrin, allethrin [(1R)-isomers], allyxycarb, alpha-cypermethrin, amidithion, amidothioate, aminocarb, amiton, amiton hydrogen oxalate, amitraz, anabasine, aramite, athidathion, azadirachtin, azamethiphos, azinphos-ethyl, azinphos-methyl, azocyclotin, azothoate, barium polysulfide, Bayer 22/190, Bayer 22408, bendiocarb, benfuracarb, bensultap, benzoximate, beta-cyfluthrin, beta-cypermethrin, bifenazate, bifenthrin, binapacryl, biopermethrin, bis(2-chloroethyl)ether, bistrifluoron, bromfenvinfos, bromocyclen, bromophos, bromophos-ethyl, bromopropylate, bufencarb, buprofezin, butacarb, butathiofos, butocarboxim, butonate, butoxycarboxim, cadusafos, calcium polysulfide, camphechlor, carbanolate, carbaryl, carbofuran, carbophenothion, carbosulfan, cartap hydrochloride, CGA 50 439, chinomethionat, chlorbenside, chlorbicyclen, chlordane, chlordecone, chlordimeform; chlordimeform hydrochloride, chlorethoxyfos, chlorfenapyr, chlorfenethol, chlorfenson, chlorfensulphide, chlorfluazuron, chlormephos, chloro-benzilate, chloromebuform, chloropropylate, chlorphoxim, chlorprazophos, chlorpyrifos, chlorpyrifos-methyl, chlorthiophos, chromafenozide, cloetho-carb, clofentezine, clothianidin, codlemone, coumaphos, coumithoate, crotoxyphos, crufomate, cryolite, CS 708, cyanofenphos, cyanophos, cyanthoate, cycloprothrin, cyfluthrin, cyhalothrin, cyhexatin, cypermethrin, cyphenothrin [(1R)-trans-isomers], cyromazine, DAEP, dazomet, DCPM, DDT, decarbofuran, deltamethrin, demephion; demephion-O; demephion-S, demeton; demeton-O; demeton-S, demeton-S-methyl, demeton-S-methylsulphon, diafenthiuron, dialifos, diazinon, dicapthon, dichlorvos, dicofol, dicrotophos, dicyclanil, dieldrin, dienochlor, diethyl 5-methyl-pyrazol-3-yl phosphate, diflubenzuron, dimefox, dimethoate, dimethrin, dimethylvinphos, dimetilan, dinex; dinex-diclexine, dinobuton, dinocap, dinocton, dinopenton, dinoprop, dinosulfon, dinotefuran, dinoterbon, dioxabenzofos, dioxacarb, dioxathion, diphenyl sulfone, disulfoton, dithicrofos, DNOC, dodec-8-enyl acetate, dofenapyn, DSP, EI 1642, emam ectin benzoate, EMPC, empenthrin [(EZ)-(1R)-isomers], endosulfan, endothion, endrin, ENT 8184, EPBP, EPN, esfenvalerate, ethio-fencarb, ethion, ethiprole, ethoate-methyl, ethoprophos, etofenprox, etoxazole, etrimfos, famphur, fenazaflor, fenazaquin, fenbutatin oxide, fenchlorphos, fenethacarb, fenfluthrin, fenitrothion, fenobucarb, fenothio-carb, fenoxacrim, fenoxycarb, fenpirithrin, fenpropathrin, fenpyroximate, fenson, fensulfothion, fenthion, fentrifanil, fenvalerate, fipronil, flonicamid, fluacrypyrim, flubenzimine, flucofuron, flucycloxuron, flucythrinate, fluenetil, flufenoxuron, flufenprox, flumethrin, fluorbenside, fluvalinate, FMC 1137, fonofos, formetanate, formothion, formparanate, fosmethilan, fospirate, fosthiazate, fosthietan, furathiocarb, furethrin, gamma-cyhalo-thrin, gamma-HCH, glyodin, GY-81, halfenprox, halofenozide, heptachlor, heptenophos, hexadecyl cyclopropanecarboxylate, hexaflumuron, hexythiazox, hydramethylnon, hydroprene, hyquincarb, imidacloprid, imiprothrin, indoxacarb, iprobenfos, IPSP, isazofos, isobenzan, isodrin, isofenphos, isolane, isoprocarb, isopropyl O-(methoxyaminothio-phosphoryl)salicylate, isothioate, isoxathion, jodfenphos, kelevan, kinoprene, lambda-c yhalothrin, leptophos, lirimfos, lufenuron, lythidathion, m-cumenyl methylcarbamate, malathion, malonoben, mazidox, MB-599, mecarbam, mecarphon, menazon, mephosfolan, mercurous chloride, mesulfenfos, metam, methacrifos, methamidophos, methanesulfonyl fluoride, methidathion, methiocarb, methocrotophos, methomyl, methoprene, methoquin-butyl, methothrin, methoxychlor, methoxyfenozide, methyl isothiocyanate, metolcarb, metoxadiazone, mevinphos, mexacarbate, milbemectin, mipafox, mirex, MNFA, monocrotophos, morphothion, naled, naphthalene, nicotine, nifluridide, nitenpyram, nithiazine, nitrilacarb; nitrilacarb 1:1 zinc chloride complex, nornicotine, novaluron, noviflumuron, O,O,O′O′-tetrapropyl dithiopyrophosphate, O,O-diethyl O-4-methyl-2-oxo-2H-chromen-7-yl phosphorothioate, O,O-diethyl O-6-methyl-2-propylpyrimidin-4-yl phosphorothioate, O-2,5-dichloro-4-iodophenyl O-ethyl ethylphosphonothioate, oleic acid (fatty acids), omethoate, oxabetrinil, oxamyl, oxydemeton-methyl, oxydeprofos, oxydisulfoton, parathion, parathion-methyl, pentachlorophenol, permethrin, petroleum oils, phenkapton, phenothrin [(1R)-trans-isomer], phenthoate, phorate, phosalone, phosfolan, phosmet, phosnichlor, phosphamidon, phosphine, phoxim, phoxim-methyl, piperonyl butoxide, pirimetaphos, pirimicarb, pirimiphos-ethyl, pirimiphos-methyl, polychlorodicyclopentadiene isomers, polynactins, prallethrin, primidophos, proclonol, profenofos, promacyl, promecarb, propaphos, propargite, propetamphos, propoxur, prothidathion, prothiofos, prothoate, pymetrozine, pyraclofos, pyresmethrin, pyrethrins (pyrethrum), pyridaben, pyridalyl, pyridaphen thion, pyrimidifen, pyrimitate, pyriproxyfen, quinalphos, quinalphos-methyl, quinothion, quintiofos, R-1492, RA-17, resmethrin, rotenone, RU 15525, RU 25475, S421, sabadilla, schradan, silafluofen, SN 72129, sodium fluoride, sodium hexafluorosilicate, sodium selenate, sophamide, spinosad, spirodiclofen, spiromesifen, spirotetramat (BYI8330), SSI-121, sulcofuron-sodium, sulfluramid, sulfosulfuron, sulfotep, sulfur, sulprofos, SZI-121, taroils, tazimcarb, TDE, tebufenozide, tebufenpyrad, tebupirimfos, teflubenzuron, tefluthrin, temephos, TEPP, terallethrin, terbufos, tetrachlorvinphos, tetradifon, tetramethrin, tetramethrin [(1R)-isomers], tetrasul, theta-cypermethrin, thiacloprid, thiamethoxam, thicrofos, thiocarboxime, thiocyclam, thiodicarb, thiofanox, thiometon, thionazin, thioquinox, thiosultap-sodium, tolfenpyrad, tralomethrin, transfluthrin, transpermethrin, triamiphos, triarathene, triazamate, triazophos, trichlorfon, trichloronat, trifenofos, triflumuron, trimedlure, trimethacarb, vamidothion, XMC, xylylcarb, zeta-cypermethrin, zolaprofos, and ZXI 8901. In particular embodiments, the present invention provides a synergistic bioinsecticidal composition comprising or consisting essentially of (+)-pulegone and piperonyl butoxide. 
     The bioinsecticidal compositions of the invention can be formulated for indoor or outdoor use, for application to a surface (e.g., skin, apparel, furniture, personal accessories, plastic products, food containers, and the like), or for broadcasting by misting systems or other distribution equipment. Aerosol formulations typically include the monoterpene and a suitable propellant (as carrier). Alternatively, the monoterpene can be dissolved or dispersed in a suitable medium, such as water or a water-miscible liquid, such as n-propanol, to provide compositions for use in non-pressurized, hand-actuated spray pumps. Ideally, aerosol formulations provide a quick kill and rapid knockdown of insects. 
     The bioinsecticidal compositions of this invention find particular application in controlling agricultural, storage and household pests. Therefore, this invention also provides methods for controlling insect pests by exposing one or more insect pests to an effective, insect controlling amount of (+)-pulegone, carvacrol, linalool or estragole. The efficacy of the bioinsecticidal compositions of the present invention may be monitored by determining the mortality of or damage to the insect population, i.e., by determining its adverse effect upon treated insects. This includes damage to the insects, inhibition or modulation of insect growth, inhibition of insect reproduction by slowing or arresting its proliferation, or complete destruction/death of the pest, all of which are encompassed by the term “controlling”. 
     The term “effective, insect controlling amount” is an amount of a monoterpene of the invention, or a composition containing the monoterpene, that has an adverse effect on at least 25% of the insects treated, more preferably at least 50%, most preferably at least 70% or greater. Preferably, an “effective, insect controlling amount” is an amount of the monoterpene of the invention, or a composition containing the monoterpene, where 25% or greater mortality against insects is achieved, preferably 50% or greater, more preferably 70% or greater mortality. Accordingly, the amount of monoterpene used in the methods of the invention, meets the mortality criteria above, and preferably has minimal or no adverse effect on ornamental and agricultural plants (such as phytotoxicity), wildlife and humans that may come into contact with the monoterpene. 
     The compositions and methods described herein can be used to treat insect infestations in crop loci, storage areas, property, and the like, and to treat animals and humans exposed to, or likely to be exposed to, one or more insect. In some embodiments, the bioinsecticidal composition is used in agricultural applications, and can be non-phytotoxic or nearly so. When used in agricultural applications, bioinsecticidal composition decreases the likelihood of damage to a plant and/or a plant crop, and decreases the likelihood of adverse side effects to workers applying the bioinsecticidal composition, or to animals, fish or fowl which ingest the tissues or parts of treated plants. The methods of use of the bioinsecticidal composition of the invention will depend at least in part upon the insect to be treated and its feeding habits, as well as breeding and nesting habits. While minor dosage rates of the bioinsecticidal composition will have an adverse effect on insects, adequate control usually involves the application of a sufficient amount to either eliminate the insects entirely or significantly deter their growth and/or rate of proliferation. Dosage rates required to accomplish these effects, of course, vary depending on the target insect, size, and maturity, i.e., stage of growth. More mature pests may be more resistant to insecticides and require higher dosage rates for a comparable level of control. Dose response experiments using different dilutions (for example, about 1:5, 1:10, and 1:20) of the bioinsecticidal composition of the present invention on target insect and on plants are performed to determine the optimal concentration of the monoterpene that shows insecticidal activity without phytotoxicity or dermal sensitivity. 
     The compositions can also be applied to cargo holds, food containers, and storage areas, for example, by fumigation, to control insect pests. Formulae described herein are capable of being organically certified, and, when the compositions are intended for application to food containers, it is preferable that such compositions are applied. The compositions can be used to protect stored products such as grains, fruits, nuts, spices or tobacco, whether whole, milled or compounded into products, from moth, beetle, mite or grain weevil attack. Also protected are stored animal products such as skins, hair, wool or feathers in natural or converted form (e.g., as carpets or textiles) from moth or beetle attack as well as stored meat, fish or grains from beetle, mite or fly attack. In these embodiments, the naturally-occurring and safe nature of the active agents is particularly relevant. 
     When used as a fumigant, the bioinsecticidal composition may be used on soil, plants or structures as an aqueous solution or dispersion applied via shank injection or drip irrigation, aerosol or mist. For structural fumigation the bioinsecticidal composition may be heated to a gas before introduction within a building, chamber, vehicle or other space or structure. The space or structure is preferably sealed with a tarpaulin, fumigant tape or gas impermeable sheeting. Stacked commodities may also be treated by draping the commodities with a gas-impermeable tarp or sheet that can be sealed to an impermeable surface (such as a concrete floor) using, for example, sand-filled tubes. 
     Suitable means of applying the bioinsecticidal composition to growing crops include as foliar sprays (for example as an in-furrow spray), fogs or foams. Suitable means of applying the compositions to soil or roots include liquid drenches, smokes or foams. When an insect is soil-borne, the composition is distributed evenly over the area to be treated (i.e., for example broadcast or band treatment). Suitable means of applying the compositions to crop seeds include application as seed dressings, e.g., by liquid slurries. The bioinsecticidal composition can be applied to plants or areas under cultivation in the form of droplets, drop-like areas or thin, defined layers by using conventional devices. Fruit-bearing trees or vines can advantageously be treated by applying the bioinsecticidal composition using dosing dispensers, pipettes or syringes, brushing devises, or surface nozzles to distribute the compositions over a substantial area. Suitable means of applying the bioinsecticidal composition to the environment in general or to specific locations where insects may lurk, including stored products, timber, household goods, or domestic or industrial premises, include sprays, fogs, dusts, smokes, or lacquers. 
     The bioinsecticidal compositions can be used to control insects that are injurious to, or spread or act as vectors of diseases in domestic animals. Suitable means of applying the bioinsecticidal compositions to animals infested by or exposed to an insect, include topical application, such as by using pour-on formulations, sprays, baths, dips, showers, jets, dusts, greases, shampoos, creams, wax smears or livestock self-treatment systems. 
     Methods of using the instant bioinsecticidal composition offer several advantages over existing methods of pest control. The formulations disclosed herein provide for effective control of a variety of insect pests. As used herein, the term “insect pest” or “pest” refers to an insect that negatively affect plants or animals by colonizing, attacking or infecting them. This includes insects that spread disease and/or damage the host and/or compete for host nutrients. In addition, plant pests are insects known to associate with plants and which, as a result of that association, cause a detrimental effect on the plant&#39;s health and vigor. 
     In general, the bioinsecticidal composition of the present invention is useful for control of insects such as fleas, mosquitoes, bees, wasps, cockroaches, termites, houseflies, fruit flies, whiteflies, leafhoppers, cabbage looper, ants,  thrips , aphids, spider mites, beetles and the like. In certain embodiments, the bioinsecticidal composition and method of this invention are used to control one or more insects including, but not limited to, Examples of pest species which may be controlled include:  Myzus persicae  (aphid),  Aphis gossypii  (aphid),  Aphis fabae  (aphid),  Lygus  spp. (capsids),  Dysdercus  spp. (capsids),  Nilaparvata lugens  (planthopper),  Nephotettixc incticeps  (leafhopper),  Nezara  spp. (stinkbugs),  Euschistus  spp. (stinkbugs),  Leptocorisa  spp. (stinkbugs),  Frankliniella occidentalis  ( thrips ),  Thrips  spp. ( thrips ) such as  T. simplex  and  T. palmi, Megalurothrips usitatus  ( thrips ),  Leptinotarsa decemlineata  (Colorado potato beetle),  Tribolium castaneum  (red flour beetle),  Haptoncus picinus, Anthonomus grandis  (boll weevil),  Aonidiella  spp. (scale insects),  Trialeurodes  spp. (white flies),  Bemisia tabaci  (white fly),  Ostrinia nubilalis , (European corn borer),  Spodoptera littoralis  (cotton leafworm),  Heliothis virescens  (tobacco budworm),  Helicoverpa  amigera (cotton bollworm),  Helicoverpa zea  (cotton bollworm), Sylepta derogata (cotton leaf roller),  Pieris brassicae  (white butterfly),  Plutella xylostella  (diamond back moth),  Agrotis  spp. (cutworms),  Chilo suppressalis  (rice stem borer),  Locusta migratoria  (locust),  Chortiocetes terminifera  (locust),  Diabrotica  spp. (rootworms),  Panonychus ulmi  (European red mite),  Panonychus citri  (citrus red mite),  Tetranychus urticae  (two-spotted spider mite),  Tetranychus cinnabarinus  (carmine spider mite),  Phyllocoptruta oleivora  (citrus rust mite),  Polyphagotarsonemus latus  (broad mite),  Brevipalpus  spp. (flat mites),  Boophilus microplus  (cattle tick),  Dermacentor variabilis  (American dog tick),  Ctenocephalides felis  (cat flea),  Liriomyza  spp. (leafminer),  Musca domestica  (housefly),  Bactrocera cucuribitae  (melon fly),  Drosophila melanogaster  (fruit fly),  Aedes aegypti  (mosquito),  Anopheles  spp. (mosquitoes),  Culex  spp. (mosquitoes), Lucillia spp. (blowflies),  Blattella germanica  (cockroach),  Blattella lateralis  (cockroach),  Periplaneta americana  (cockroach),  Blatta orientalis  (cockroach), termites of the Mastotermitidae (for example  Mastotermes  spp.), the Kalotermitidae (for example  Neoterines  spp.), the Rhinotermitidae (for example  Coptotermes formosanus, Reticulitermes flavipes, R. speratu, R. virginicus, R. hesperus , and  R. santonensis ) and the Termitidae (for example  Globitermes sulphureus ),  Solenopsis geminata  (fire ant), Monomoriumn  pharaonis  (pharaoh&#39;s ant),  Damalinia  spp. and  Linognathus  spp. (biting and sucking lice). 
     The preferred bioinsecticidal compositions of the present invention have a lethal effect on pest targets. Unlike the bulk of currently available insecticides on the market, the preferred bioinsecticidal compositions have an active ingredient that is substantially non-toxic to man, domestic animals, and honeybees and has minimal adverse effects on wildlife and the environment. In this respect, the bioinsecticidal compositions of this invention are advantageous in that the bioinsecticidal compositions control insects without introducing a notable amount of harm to the surrounding environment of which the provided bioinsecticidal compositions are being used. Accordingly, the bioinsecticidal compositions can be used to treat plant pests on food crops up to and immediately before the harvesting period, a practice that generally is avoided when using conventional methods of pest control. The compositions also can be used to control the growth of pest organisms on harvested crops. Thus, the instant bioinsecticidal compositions can be incorporated into an integrated pest management (IPM) program. 
     The bioinsecticidal compositions of the invention have insecticidal activity against one or more insects. However, it is understood that certain bioinsecticidal compositions may be more effective on some insects than others, and may even be ineffective against some insects. In this respect, certain embodiments of this invention provide particular bioinsecticidal compositions and methods for controlling particular insect pests. In some embodiments, the bioinsecticidal composition includes (+)-pulegone and is used in a method for controlling  thrips , beetles and/or fruit flies. In further embodiments, the bioinsecticidal composition is a fumigant containing (+)-pulegone, which is used in a method for controlling  thrips , flour beetles and/or fruit flies. In certain embodiments, the bioinsecticidal composition includes (+)-pulegone and is used in a method for controlling  thrips  of the genus  Megalurothrips  (e.g.,  M. basisetae, M. distalis, M. equaletae, M. flaviflagellus, M. formosae, M. guizhouensis, M. Haopingensis, M. mucunae, M. peculiaris, M. sinensis, M. sjostedti, M. typicus  and  M. usitatus ). In yet another embodiment, the bioinsecticidal composition includes (+)-pulegone and is used in a method for controlling fruit fly of the genus  Drosophila . In yet further embodiment, the bioinsecticidal composition includes (+)-pulegone and is used in a method for controlling beetles, in particular flour and sap beetles, of the genera  Tribolium  (e.g.,  T. castaneum  and  T. confusum ) and  Haptoncus  (e.g.,  H. picinus  and  H. luteolus ). In some embodiments, the bioinsecticidal composition includes (+)-pulegone in combination with piperonyl butoxide and is used in a method for controlling  thrips , e.g., of the genus  Megalurothrips.    
     In other embodiments, the bioinsecticidal composition is a fumigant containing carvacrol and is used in a method for controlling mosquitoes. In certain embodiments, the bioinsecticidal composition is a fumigant containing carvacrol and is used in a method for controlling mosquitoes of the genus  Aedes  (e.g.,  A. aegypti  or  A. albopictus ). 
     In yet other embodiments, the bioinsecticidal composition includes trans-anethole and is used in a method for controlling rusty red cockroaches ( Blatta lateralis ). 
     In still other embodiments, the bioinsecticidal composition includes estragole and is used in a method for controlling mosquitoes. In certain embodiments, the bioinsecticidal composition is a fumigant containing estragole and is used in a method for controlling mosquitoes, e.g., mosquitoes of the genus  Aedes  (e.g.,  A. aegypti  or  A. albopictus ). 
     According to other embodiments, the bioinsecticidal composition includes linalool and is used in a method for controlling  thrips  and/or leafhoppers. In further embodiments, the bioinsecticidal composition is a fumigant containing linalool, which is used in a method for controlling  thrips  and/or leafhoppers. In certain embodiments, the bioinsecticidal composition includes linalool and is used in a method for controlling  thrips  of the genus  Megalurothrips  (e.g.,  M. basisetae, M. distalis, M. equaletae, M. flaviflagellus, M. formosae, M. guizhouensis, M. Haopingensis, M. mucunae, M. peculiaris, M. sinensis, M. sjostedti, M. typicus  and  M. usitatus ). In yet another embodiment, the bioinsecticidal composition includes linalool and is used in a method for controlling leafhoppers of the genus  Cicadella  (e.g.,  C. viridi, C. lasiocarpae, C. longivittata, C. lunulata  and  C. transversa ). 
     The following non-limiting examples are provided to further illustrate the present invention. 
     Example 1: Materials and Methods 
     Monoterpenes included (+)-pulegone (95% purity), (−)-pulegone (98%), trans-anethole (98%), estragole (98%), 1,8-cineole (98%), carvacrol (95%), eugenol methyl ether (98%), linalool (97%), (−)-linalool (95%), (+)-limonene (96%), (−)-limonene (96%), ethyl formate (98%) and myrcene (90%) (Table 1). These chemicals were purchased from Fisher Scientific (Pittsburgh, Pa.) or Sigma-Aldrich (St. Louis, Mo.). 2,2-Dichlorovinyl dimethyl phosphate (DDVP, or dichlorvos) was commercially purchased. 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Monoterpene 
                 Structure 
                 Vp (Pa)/T (° C.) 
               
               
                   
               
             
            
               
                 (+)-Pulegone 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 16.3/25 
               
               
                   
               
               
                 (−)-Pulegone 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 16.3/25 
               
               
                   
               
               
                 Estragole 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 27.93/25 
               
               
                   
               
               
                 trans-Anethole 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 5.45/21 
               
               
                   
               
               
                 Linalool 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 21.28/22-25 
               
               
                   
               
               
                 Carvacrol 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 3.99/25 
               
               
                   
               
               
                 Eugenol Methyl Ether 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 1.60/25 
               
               
                   
               
               
                 1,8-Cineole 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 253.31/25 
               
               
                   
               
               
                 (+)-Limonene 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 263.97/25 
               
               
                   
               
               
                 (−)-Limonene 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 206.65/25 
               
               
                   
               
               
                 Myrcene 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 278.64/25 
               
               
                   
               
               
                 (−)-Linalool 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 21.20/23.5 
               
               
                   
               
            
           
         
       
     
     
       Thrips.  
     
     The  thrips Megalurothrips usitatus  (Bagnall, 1913) (Thysanoptera: Thripidae) were used in preliminary screening tests and LC 50  determinations. Adult  thrips  were used in both cases, but differed in crops where they were collected and collection areas and rearing. 
     The colony of  thrips  used in preliminary tests was maintained in the laboratory to provide adults for the mortality experiment to screen monoterpenes. Adult  thrips  were obtained from hyacinth bean plants ( Lablab purpureus ) grown on the University of Hawaii at Manoa campus to initiate the colony. The  thrips  used to determine 24-hour LC 50  were also  M. usitatus . The adult  thrips  were obtained from a cowpea ( Vigna unguiculata ) field. The adult  thrips  were placed inside a ventilated 500-mL glass spawn bottle with cowpea pods and maintained at 2510° C. and a photoperiod of 12:12 hours (Light:Day). The colony of adult  thrips  was artificially reared for 19 generations and used for fumigation tests. This process was repeated for colony maintenance and future collection of adults. 
     Spider Mites. 
     Carmine spider mites,  Tetranychus cinnabarinus  (Boisduval, 1867) (Acari: Tetranychidae) were collected from an eggplant field. The  Tetranychus  mites that were healthy and had no previous exposure to pesticides were used for fumigation tests. 
     Melon Fly. 
       Bactrocera cucuribitae  (Coquillet, 1849) (Diptera: Tephritidae), melon fly, were reared in accordance with known methods (Follett, et al. (2013)  J. Econ. Entomol.  106(5):2020-2026). The flies were emerged for 3 days and were in a sensitive stage. 
     Nitidulidae. 
       Haptoncus picinus  (Grouvelle, 1906) adults were collected from an eggplant field. The insects were healthy and had no previous exposure to pesticides. 
     Red Flour Beetle. 
       Tribolium castaneum  (Herbst, 1797) (Insecta, Coleoptera), a storage insect commonly called red flour beetle, was reared with wheat bran. 
     House Fly. 
     Pupae of  Musca domestica  (Linnaeus, 1758) (Diptera: Muscidae) were purchased from a commercial source. Three-day-old adults (after emergence) were used for fumigation tests. 
     Cockroach. 
     Larvae of rusty red cockroaches  Blatta lateralis  (Walker, 1868) ( Blattaria : Blattidae) were purchased from a commercial source). Second instar nymphs (0.9-1.3 cm) that were reared with peanut oil on a piece of paper for one week were used in fumigation tests. 
     Mosquito. 
     Yellow fever mosquitos  Aedes aegypti  (Linnaeus, 1762) (Diptera: Culicidae) were reared with 8% glucose solution. Three-day-old mosquito adults were used in fumigation tests. 
     Insect Egg Parasitic Wasp (Natural Enemy). 
     Two species of insect natural enemy parasitoids were analyzed including  Trichogramma chilonis  (Ishii, 1941) (Hymenoptera: Trichogrammatidae) and  Anastatus japonicus  (Ashmead, 1904) (Hymenoptera: Eupelmidae). Both species were purchased from a commercial source. One- to two-day-old adults (after eclosion) were used for fumigation tests. 
     Preliminary Screening Tests. 
     Monoterpenes applied at varying concentrations on pieces of moist tissue paper were tested against thirty, 5-day-old adult  Megalurothrips usitatus  in 1-L (1000 cm 3 ) flasks for 24 hours of fumigation. No monoterpene was the control. Acetone and chloroform were the solvent controls and ethyl formate was used as a positive fumigant control. Each treatment was replicated four times. Mortality data were used to preliminarily select monoterpenes for further studies. The preliminary tests indicated that six out of 11 monoterpenes were active and acted fast. 
     Median Lethal Concentration Tests. 
     Median lethal concentrations (LC 50 ) of (+)-pulegone, estragole, trans-anethole, linalool, carvacrol, and eugenol methyl ether were determined according to the pesticides guidelines for laboratory bioactivity tests and the laboratory efficacy test methods and criteria of public health insecticides for pesticide registration. The species tested included  thrips  ( M. usitatus ), spider mite ( T. cinnabarinus ), melon fly ( B. cucuribitae ), cockroach ( B. lateralis ), housefly ( M. domestica ), mosquito ( A. aegypti ), parasitic wasp ( T. chilonis ) and fruit fly ( Drosophila melanogaster ). After being exposed to the test chemicals for one hour, adult  thrips , spider mites, and roaches were transferred to another flask. In addition to fresh water, adult  thrips , spider mites, and roaches were then respectively fed with cowpea, fresh blade of eggplant, and peanut oil via filter. Mortality of  thrips  was observed in 24 hours. Mortality of melon fly was observed after three hours of exposure. Mortality of fruit fly was observed after 280 minutes. After adult housefly and melon fly were exposed for one hour and mosquitoes were exposed for 30 minutes (Manimaran, et al. (2012)  Adv. Biosci. Biotechnol.  3:855-862), they were fed with fresh water and food (milk powder for housefly; 1:1 yeast powder and sugar mix for melon fly; water alone for mosquito) in the same flask. The mortality was observed in 24 hours. Mortality of the parasitic wasps was observed after 15 minutes of exposure. 
     Median Lethal Time (LT 50 ) and Median Knockdown Concentration (KC 50 ) Tests. 
     Five species of adult insects or natural enemies were tested for LT 50  values including melon fly ( B. cucuribitae ), red flour beetle ( T. castaneum ), Nitidulidae beetle ( H. picinus ), parasitic wasp ( A. japonicus ) and housefly ( M. domestica ). For each treatment and control, 20 insects at the developmental stages described above were placed individually without food in a 1-L wide-mouth glass bottle (height 19.5 cm, bottom diameter 10.5 cm, mouth i.d. 5.5 cm) containing pieces of tissue paper. The bottle was incubated at 25±1° C. under a 12:12-hour light:dark regime. Five different concentrations of the test chemicals were placed on pieces of cellulose-based qualitative filter paper. Each of the filter papers was placed in a bottle to have 5 appropriate concentrations in serial dilutions. DDVP was the positive control and no chemical treatments were the negative control. All treatments and controls were in triplicate. Mortality was observed periodically in a 10-30 minute interval for 2 days or until all insects died. The death was defined as no movement when the insects were touched. 
     LC 50  Determinations for a Mixture of Monoterpenes and Linalool Mixed in Corn Oil. To investigate the potential use of the monoterpenes in a mixture, two and three monoterpenes were mixed and then tested for LC 50  values against the  thrips, M. usitatus.    
     Fumigation Toxicity. 
     To confirm the fumigation toxicity, laboratory bioassays were conducted to evaluate the toxicity of linalool mixed in edible peanut oil. Larvae of  M. usitatus thrips  were maintained in a rearing bottle and fed with cowpea at 25±1° C. under a 12:12-hour light:dark regime. Larvae in the second instar were used in this test. The  thrips  were placed individually without food in an RNA purification tube above the filter and 1 mL of linalool/corn oil mixture was placed at the bottom of the tube. Thirty insects were used in each treatment in triplicate. Linalool was in 19.2-, 13.7-, 9.8-, 7.0- and 5.0-fold dilutions with edible peanut oil. 
     Field Tests. 
     Linalool exhibited moderate laboratory toxicity among all tested monoterpenes. Therefore, it was determined whether this compound could control  thrips  in the field. Linalool was in 20-, 10- and 5.0-fold dilutions with edible peanut oil. The positive control was spinetoram (Dow AgroSciences) in 3000-fold dilution. 
     The field experimental plots were 15 m 2  in size, in triplicate. A designated sprayer (Dia spray No. 3530, Japan Furlpla Co., Japan) was used to spray the solvent only negative control in this experiment. Grub density was calculated at every plot prior to application. During the experiments, the field was also infested with leafhoppers, so that the control efficacy of leafhoppers was also assessed. The number of adult  thrips  and leafhoppers was assessed in the treatment and control areas at 1, 3, 5, 7, 14 and 21 days after application. The decline of the adult  thrips  and leafhoppers was calculated to assess the control efficacy of linalool. 
     Statistical Analysis. 
     Mortality percentage was calculated based upon the total number of insects died from the monoterpene treatment. Mortality was transformed to logit (L=In[(m+0.5)/(100.5−m)], where m is mortality percentage. Regression analysis of mortality records included only the final record of zero mortality and the first record of 100% morality. All other occurrences of 0% and 100% in the time data matrix were omitted. 
     The co-toxicity coefficient (Sarup, et al. (1980)  J. Entomol. Res.  4:1-14) and synergistic factor (Kalayanasundaram &amp; Das (1985)  Ind. J. Med. Res.  82:19-21) for the monoterpene mixtures were calculated (Chenniappan &amp; Ayyadurai (2012)  Parasitol. Res.  110:381-388) after LC 50  and LC 90  for each mixture were determined. 
     Co-toxicity coefficient (CTC)=Co-toxicity of Compound A (alone)/toxicity of Compound A with B×100. 
     Synergistic factor (SF)=toxicity of Compound A (alone)/toxicity of Compound A with B. A value of SF&gt;1 indicates synergism, while SF&lt;1 indicates antagonism. 
     The LC 50  values were calculated with GraphPad Prism version 6.05 (GraphPad Software, La Jolla, Calif., USA). The concentration-mortality data were used to estimate LC 50  values and associated 95% confidence limits for each treatment. 
     Example 2: Preliminary Screening Tests 
     Numerous monoterpenes were screened for potency against flower  thrips . Four monoterpenes, including 1,8-cineole, (+)-limonene, (−)-limonene and myrcene, were not active against flower  thrips  and thus were not further analyzed. Notably, both linalool and (−)-linalool had similar toxicity to flower  thrips . In addition, ethyl formate, acetone and chloroform were tested for their potency against flower  thrips . No mortality was observed at a concentration of 40 nL/cm 3  for acetone and chloroform (approximately 32 μg/cm 3  for acetone and 60 μg/cm 3  chloroform), which was consistent with previous studies (Chang, et al. (2009)  J. Econ. Entomol.  102(1):203-209). The LC 50  of ethyl formate was greater than 9.2 μg/cm 3 . These data indicated that (+)-pulegone, trans-anethole, estragole, 1,8-cineole, carvacrol and eugenol methyl ether exhibit insecticidal activity. 
     Example 3: Toxicity of Monoterpenes to Agricultural Pests 
       Thrips . The legume  thrips, M. usitatus , is one of the most serious insect pests of leguminous plants including cowpea in tropical Africa (Ekesi, et al. (1998)  J. Afr. Crop. Sci.  6:49-59; Karungi, et al. (2000)  Crop. Prot.  19:343-347). The repellency of 24 plant extracts was evaluated against adult female  thrips  of  M. sjostedti  in the laboratory, and extracts from  Piper nigrum, Cinnamomum  zeylanicum and  Cinnamomum cassia  were identified as strong repellents. Another report studied the fumigation toxicity of basil oil compounds and related compounds to  Thrips palmi  and Orius strigicollis. This study indicated that linalool was the most toxic fumigant of adult  T. palmi  (LC 50  5.5 μg/cm 3 ), and was 15.2-fold more effective than dichlorvos (83.7 μg/cm 3 ). Strong fumigation toxicity was also observed for (−)-pulegone (9.5 μg/cm 3 ), (±)-camphor (9.7 μg/cm 3 ) and 1,8-cineole (16.7 μg/cm 3 ) (Kim, et al. (2015)  Pest Manag. Sci.  71:1292-1296). 
     Spider Mite. 
       Tetranychus  is one of the most economically important genera of mites, due to its high potential to destroy agriculture. It contains over 140 species, the most significant of which is  Tetranychus cinnabarinus. Solanum  sarrachoides essential oils and their monoterpene constituents have been shown to play a role in regulating oviposition in the tomato spider mite  Tetranychus evansi  (Lucy, et al. (2013)  Industr. Crops Prod.  46:73-79). 
     Melon Fly. 
     Melon fly is an important economic and quarantine pest of tropical fruits and one of the most radiation-tolerant tephritid fruit flies known (Follett &amp; Armstrong (2004)  J. Econ. Entomol.  97:1254-1262; Follett, et al. (2011)  J. Econ. Entomol.  104:1509-1513). 
     LC 50  (Table 2), LC 90  (Table 3), and dose-response slopes (Table 4) of six monoterpenes and DDVP for these three agricultural pest species ( M. usitatus, T. cinnabarinus , and  B. cucuribitae ) were determined. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                   
                   
                 LC 50  (fiducial limits) 
                 Chi- 
               
               
                   
                 Compound 
                   
                 (μg/cm 3 ) 
                 square 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                   Megalurothrips usitatus  (Bagrall) (thrips) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 DDVP 80% 
                 0.68 
                 (0.43-1.43) 
                 3.91 
               
               
                   
                 (+)-Pulegone 
                 0.20 
                 (0.11-0.27) 
                 0.352 
               
               
                   
                 Estragole 
                 0.24 
                 (0.09-0.38) 
                 1.67 
               
               
                   
                 trans-Anethole 
                 0.36 
                 (0.00-1.86) 
                 0.037 
               
               
                   
                 Linalool 
                 2.50 
                 (1.72-3.36) 
                 0.202 
               
            
           
           
               
               
               
               
            
               
                   
                 Carvacrol 
                 &gt;19.2 
                   
               
               
                   
                 Eugenol methyl ether 
                 &gt;19.2 
               
            
           
           
               
            
               
                   Tetranychus cinnabarinus  (Boisduval) (spider mites) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 DDVP 80% 
                 0.43 
                 (0.33-0.58) 
                 1.81 
               
               
                   
                 (+)-Pulegone 
                 2.52 
                 (1.31-5.23) 
                 0.064 
               
               
                   
                 Estragole 
                 3.28 
                 (2.03-5.89) 
                 5.23 
               
               
                   
                 trans-Anethole 
                 5.73 
                 (2.77-19.8) 
                 4.002 
               
               
                   
                 Linalool 
                 550 
                 (46.3-5940) 
                 0.103 
               
               
                   
                 Carvacrol 
                 37.5 
                 (14.1-770) 
                 0.708 
               
            
           
           
               
               
               
               
            
               
                   
                 Eugenol methyl ether 
                 &gt;48.6 
                   
               
            
           
           
               
            
               
                   Bactrocera cucuribitae  (Coquillett) (melon fly) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 DDVP 80% 
                 0.0014 
                 (—) 
                 0.005 
               
               
                   
                 (+)-Pulegone 
                 112 
                 (65.4-430) 
                 1.34 
               
               
                   
                 Estragole 
                 483 
                 (212-261) 
                 0.050 
               
               
                   
                 trans-Anethole 
                 417 
                 (217-18700) 
                 1.15 
               
            
           
           
               
               
               
               
            
               
                   
                 Linalool 
                 &gt;243 
                 &gt;243 
               
               
                   
                 Carvacrol 
                 &gt;243 
                 &gt;243 
               
               
                   
                 Eugenol methyl ether 
                 &gt;243 
                 &gt;243 
               
               
                   
                   
               
               
                   
                 (—), Fiducial limits were larger than 1000-fold. 
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Compound 
                 LC 90  (fiducial limits) (μg/cm 3 ) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                   Megalurothrips usitatus  (Bagrall) (thrips) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 1.85 
                 (1.36-31.6) 
               
               
                   
                 (+)-Pulegone 
                 0.67 
                 (0.46-3.15) 
               
               
                   
                 Estragole 
                 1.29 
                 (0.69-7.48) 
               
               
                   
                 trans-Anethole 
                 8.53 
                 (3.98-1850) 
               
               
                   
                 Linalool 
                 6.4 
                 (4.51-11.7) 
               
            
           
           
               
               
               
            
               
                   
                 Carvacrol 
                 &gt;19.2 
               
               
                   
                 Eugenol methyl ether 
                 &gt;19.2 
               
            
           
           
               
            
               
                   Tetranychus cinnabarinus  (Boisduval) (spider mites) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 0.80 
                 (0.59-1.58) 
               
               
                   
                 (+)-Pulegone 
                 40.8 
                 (14.5-415) 
               
               
                   
                 Estragole 
                 21.0 
                 (10.1-86.6) 
               
               
                   
                 trans-Anethole 
                 144 
                 (33.1-8588) 
               
               
                   
                 Linalool 
                 4330 
                 (—) 
               
               
                   
                 Carvacrol 
                 510 
                 (—) 
               
            
           
           
               
               
               
            
               
                   
                 Eugenol methyl ether 
                 &gt;48.6 
               
            
           
           
               
            
               
                   Bactrocera cucuribitae  (Coquillett) (melon fly) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 0.0014 
                 (0.0014-0.0028) 
               
               
                   
                 (+)-Pulegone 
                 657 
                 (—) 
               
               
                   
                 Estragole 
                 953 
                 (—) 
               
               
                   
                 trans-Anethole 
                 5130 
                 (—) 
               
            
           
           
               
               
               
            
               
                   
                 Linalool 
                 &gt;243 
               
               
                   
                 Carvacrol 
                 &gt;243 
               
               
                   
                 Eugenol methyl ether 
                 &gt;243 
               
               
                   
                   
               
               
                   
                 (—), Fiducial limits were larger than 1000-fold. 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                   
                 Regression 
                   
               
               
                   
                 Compound 
                 equation 
                 Slope ± SE 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                   Megalurothrips usitatus  (Bagrail) (thrips) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 0.976x + 0.94  
                 3.0 ± 0.5 
               
               
                   
                 ( + )-Pulegone 
                  2.3x + 1.61 
                 2.3 ± 0.5 
               
               
                   
                 Estragole 
                 1.71x + 1.07 
                 1.7 ± 0.5 
               
               
                   
                 trans-Anethole 
                 0.942x + 0.40  
                 0.94 ± 0.39 
               
               
                   
                 Linalool 
                 3.11x − 1.43 
                 3.1 ± 0.6 
               
               
                   
                 Carvacrol 
                 — 
                 — 
               
               
                   
                 Eugenol methyl ether 
                 — 
                 — 
               
            
           
           
               
            
               
                   Tetranychus cinnabarinus  (Boisduval) (spider mites) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 4.82x + 2.50 
                 4.8 ± 1.2 
               
               
                   
                 (+)-Pulegone 
                 1.06x + 0.46 
                 1.1 ± 0.2 
               
               
                   
                 Estragole 
                 1.59x − 0.85 
                 1.6 ± 0.3 
               
               
                   
                 trans-Anethole 
                 0.193x + 0.69  
                 0.91 ± 0.23 
               
               
                   
                 Linalool 
                 0.442x − 1.24  
                 0.44 ± 0.22 
               
               
                   
                 Carvacrol 
                 1.14x + 1.83 
                 1.1 ± 0.4 
               
               
                   
                 Eugenol methyl ether 
                 — 
                 — 
               
            
           
           
               
            
               
                   Bactrocera cucuribitae  (Coquillett) (melon fly) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                  6.90 + 21.9 
                 6.9 ± 2.3 
               
               
                   
                 (+)-Pulegone 
                 1.70x − 3.56 
                 1.7 ± 0.5 
               
               
                   
                 Estragole 
                 4.36x − 11.8 
                 4.4 ± 1.8 
               
               
                   
                 trans-Anethole 
                 1.18x − 3.09 
                 1.1 ± 0.4 
               
               
                   
                 Linalool 
                 2.17x − 9.03 
                 2.2 ± 2.3 
               
               
                   
                 Carvacrol 
                 2.35x − 7.89 
                 2.3 ± 2.8 
               
               
                   
                 Eugenol methyl ether 
                 3.93x − 11.0 
                 3.9 ± 0.9 
               
               
                   
                   
               
            
           
         
       
     
     In general, the six monoterpenes varied from being inactive to very toxic to the test organisms. (+)-Pulegone, for example, was very toxic to the  thrips , whereas carvacrol and eugenol methyl ether were generally not toxic to any of the test organisms. Specifically, (+)-pulegone, estragole and trans-anethole were highly toxic to  thrips  with LC 50  values of 0.196, 0.241 and 0.356 μg/cm 3 , respectively (Table 2). The activities of (+)-pulegone, estragole and trans-anethole were 3.5-, 2.8- and 1.9-fold greater (smaller LC 50  values) than that of the positive control, DDVP. 
     The LC 50  of all six compounds for spider mites,  T. cinnabarinus , were at least 5.8-fold greater than that of DDVP (Table 2). Among the six monoterpenes, (+)-pulegone was the most toxic to  T. cinnabarinus  with an LC 50  of 2.52 μg/cm 3 . 
     The monoterpenes tested were not very active against  B. cucuribitae  (Tables 2 and 3). Among the six terpenoids, (+)-pulegone had the lowest LC 50  (112 μg/cm 3 ) and linalool was inactive against  B. cucuribitae.    
     To assess the impact of exposure time on efficacy, LC 50  and LC 90  values of (+)-pulegone and linalool were determined against  thrips  ( M. usitatus  (Bagrall)) after a 24-hour exposure to the compounds. The results of this analysis are presented in Table 5. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 LC 50  (fiducial limits) 
                 Chi- 
                 LC 90  (fiducial limits) 
               
               
                 Compound 
                 (μg/cm 3 ) 
                 square 
                 (μg/cm 3 ) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 (+)-Pulegone 
                      0.032 × 10 −3   
                 8.56 
                        0.121 × 10 −3   
               
               
                   
                 (0.025-0.04 × 10 −3 ) 
                   
                 (0.086-0.199 × 10 −3 ) 
               
               
                 Linalool 
                        0.24 × 10 −3   
                 34.07 
                       0.60 × 10 −3   
               
               
                   
                  (0.20-0.29 × 10 −3 ) 
                   
                  (0.46-0.94 × 10 −3 ) 
               
               
                   
               
            
           
         
       
     
     Example 4: Activity of Monoterpenes Against Household Insect Pests 
     Housefly.  M. domestica  is a major domestic pest that causes irritation, spoils food, and acts as a vector for more than 100 pathogens of medical and veterinary significance (Kumar, et al. (2012)  Parasitol. Res.  110:1929-1936). In spite of this, the control aspect of the common housefly,  M. domestica , is often neglected (Kumar, et al. (2013)  Parasitol. Res.  112:69-76). It has been shown that citral and 1,8-cineole are highly active against housefly (Kumar, et al. (2013)  Parasitol. Res.  112:69-76), and that  Citrus sinensis  essential oil is effective in a fumigation assay (Rossi &amp; Palacios (2013) Acta  Tropica  127(1):33-37). Further, efficacy tests against housefly adults revealed high repellent activities by menthol (96%) and menthone (83%) (Kumar, et al. (2014)  Ecotoxicol. Environ. Safety  100:1-6). 
     Cockroach. 
       B. lateralis  is an important insect pest in urban environments. Cockroaches not only spoil food, but also transfer pathogens and cause allergic reactions and psychological distress. Notably, larvae of rusty red cockroach,  B. lateralis , are prevalent in hotels and food outlets. 
     Mosquito. 
       A. aegypti  is known to mediate that spread of dengue as well as many other diseases. As a consequence, controlling the spread of dengue requires that mosquitoes be directly targeted (Konishi (2011)  Trop. Med. Health  39(4 Suppl.):63-71; Gupta &amp; Reddy (2013)  Parasitol. Res.  112(4):1367-78). Essential oil compounds from 269 plant species have been tested for their larvicidal activity against mosquito larvae, including pulegone, linalool, trans-anethole and carvacrol (Dias, et al. (2014)  Parasitol. Res.  113:565-592). Preliminary screening of 25 essential oils against the mosquito  Culex quinquefasciatus  indicated that mentha oil and calamus oil were the most promising larvicides and orange oil had potent knockdown activity (Manimaran, et al. (2012)  Adv. Biosci. Biotechnol.  3:855-862). 
     Fruit Fly. 
       Drosophila melanogaster  is the common fruit fly found in homes with ripe, rotting, or decayed fruit and produce. Aside from being a nuisance, fruit flies have the potential to contaminate food with bacteria and other pathogens. 
     LC 50  (Table 5), LC 90  (Table 6), and dose-response slopes (Table 7) of monoterpenes and DDVP for these four household pest species ( M. domestica, B. lateralis, A. aegypti  and  D. melanogaster ) were determined. 
     
       
         
           
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                   
                 LC 50  (fiducial limits) 
                 Chi- 
               
               
                 Compound 
                 (μg/cm 3 ) 
                 square 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                   Musca domestica  (Linnaeus) (housefly) 
               
            
           
           
               
               
               
               
            
               
                 DDVP 80% 
                 0.21 
                 (0.00-0.29) 
                 3.59 
               
               
                 (+)-Pulegone 
                 7.38 
                 (5.14-10.3) 
                 0.118 
               
               
                 Estragole 
                 5.89 
                 (0.00-9.36) 
                 0.053 
               
               
                 trans-Anethole 
                 83.0 
                 (57.3-158) 
                 1.04 
               
               
                 Linalool 
                 30.6 
                 (17.5-65.5) 
                 5.07 
               
            
           
           
               
               
               
            
               
                 Carvacrol 
                 &gt;243 
                 6.04 
               
               
                 Eugenol methyl ether 
                 &gt;243 
                 3.61 
               
            
           
           
               
            
               
                   Blatta lateralis  (Walker) (cockroach) 
               
            
           
           
               
               
               
               
            
               
                 DDVP 80% 
                 4.99 
                 (3.42-14.6) 
                 0.015 
               
               
                 (+)-Pulegone 
                 15.0 
                 (6.8-47.7) 
                 1.23 
               
               
                 Estragole 
                 24.1 
                 (14.5-37.6) 
                 0.738 
               
               
                 trans-Anethole 
                 4.25 
                 (0.09-9.19) 
                 3.02 
               
               
                 Linalool 
                 345 
                 (—) 
                 0.642 
               
               
                 Carvacrol 
                 84.5 
                 (62.9-103) 
                 0.829 
               
            
           
           
               
               
               
            
               
                 Eugenol methyl ether 
                 &gt;243 
                 0.705 
               
            
           
           
               
            
               
                   Aedes aegypti  (Linnaeus) (mosquito) 
               
            
           
           
               
               
               
               
            
               
                 DDVP 80% 
                 0.0014 
                 (0.00-0.00142) 
                 0.141 
               
               
                 (+)-Pulegone 
                 10.3 
                 (1.30-187) 
                 0.142 
               
               
                 Estragole 
                 0.25 
                 (0.0676-0.936) 
                 0.647 
               
               
                 trans-Anethole 
                 2.67 
                 (1.28-10.9) 
                 5.24 
               
            
           
           
               
               
               
            
               
                 Linalool 
                 &gt;243 
                 16.3 
               
            
           
           
               
               
               
               
            
               
                 Carvacrol 
                 0.77 
                 (0.19-1.88) 
                 1.69 
               
            
           
           
               
               
               
            
               
                 Eugenol methyl ether 
                 &gt;243 
                 0.602 
               
            
           
           
               
            
               
                   Drosophila melanogaster  (fruit fly) 
               
            
           
           
               
               
               
               
            
               
                 DDVP 80% 
                 0.001 × 10 −3   
                 (0.000-0.001 × 10 −3 ) 
                 115 
               
               
                 (+)-Pulegone 
                 0.043 × 10 −3   
                 (0.034-0.055 × 10 −3 ) 
                 53.3 
               
               
                 (−)-Pulegone 
                 0.106 × 10 −3   
                 (0.087-0.144 × 10 −3 ) 
                 20.8 
               
               
                   
               
               
                 (—), Fiducial limits were larger than 1000-fold. 
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                 Compound 
                 LC 90  (fiducial limits) (μg/cm 3 ) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                   Musca domestica  (Linnaeus) (housefly) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 0.53 
                 (0.33-3.39) 
               
               
                   
                 (+)-Pulegone 
                 11.9 
                 (9.06-13.4) 
               
               
                   
                 Estragole 
                 15.3 
                 (6.85-5530) 
               
               
                   
                 trans-Anethole 
                 137 
                 (98.2-2800) 
               
               
                   
                 Linalool 
                 176 
                 (76.4-2970) 
               
            
           
           
               
               
               
            
               
                   
                 Carvacrol 
                 &gt;243 
               
               
                   
                 Eugenol methyl ether 
                 &gt;243 
               
            
           
           
               
            
               
                   Blatta lateralis  (Walker) (cockroach) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 9.51 
                 (6.54-26.9) 
               
               
                   
                 (+)-Pulegone 
                 173 
                 (52.4-6920) 
               
               
                   
                 Estragole 
                 15.3 
                 (9.65-5530) 
               
               
                   
                 trans-Anethole 
                 25.8 
                 (12.5-411) 
               
               
                   
                 Linalool 
                 1700 
                 (—) 
               
               
                   
                 Carvacrol 
                 118 
                 (95.7-204.0 
               
            
           
           
               
               
               
            
               
                   
                 Eugenol methyl ether 
                 &gt;243 
               
            
           
           
               
            
               
                   Aedes aegypti  (Linnaeus) (mosquito) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 0.0028 
                 (0.00-1.83) 
               
               
                   
                 (+)-Pulegone 
                 16.8 
                 (—) 
               
               
                   
                 Estragole 
                 0.91 
                 (0.44-8.59) 
               
               
                   
                 trans-Anethole 
                 10.6 
                 (4.36-869) 
               
            
           
           
               
               
               
            
               
                   
                 Linalool 
                 &gt;243 
               
            
           
           
               
               
               
               
            
               
                   
                 Carvacrol 
                 3.92 
                 (1.69-162) 
               
            
           
           
               
               
               
            
               
                   
                 Eugenol methyl ether 
                 &gt;243 
               
            
           
           
               
            
               
                   Drosophila melanogaster  (fruit fly) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 0.002 × 10 −3   
                 (0.001-0.004 × 10 −3 ) 
               
               
                   
                 (+)-Pulegone 
                 0.140 × 10 −3   
                 (0.096-0.292 × 10 −3 ) 
               
               
                   
                 (−)-Pulegone 
                 0.359 × 10 −3   
                 (0.236-0.715 × 10 −3 ) 
               
               
                   
                   
               
               
                   
                 (—), Fiducial limits were larger than 1000-fold. 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                   
                 Regression 
                   
               
               
                   
                 Compound 
                 equation 
                 Slope ± SE 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                   Musca domestica  (Linnaeus) (housefly) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 2.73x + 2.46 
                 2.7 ± 1.1 
               
               
                   
                 (+)-Pulegone 
                 6.25x − 5.62 
                 6.2 ± 2.2 
               
               
                   
                 Estragole 
                 3.09x − 7.43 
                 3.1 ± 1.6 
               
               
                   
                 trans-Anethole 
                 5.84x − 11.2 
                 5.8 ± 2.5 
               
               
                   
                 Linalool 
                 1.69x − 2.61 
                 1.7 ± 0.5 
               
               
                   
                 Carvacrol 
                 0.365x − 0.976 
                 0.37 ± 0.43 
               
               
                   
                 Eugenol methyl ether 
                 0.784x − 2.56  
                 0.78 ± 0.70 
               
            
           
           
               
            
               
                   Blatta lateralis  (Walker) (cockroach) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 4.63x − 2.53 
                 4.6 ± 1.4 
               
               
                   
                 (+)-Pulegone 
                 1.29x − 1.46 
                 1.2 ± 0.3 
               
               
                   
                 Estragole 
                 3.57x − 4.97 
                 3.6 ± 1.0 
               
               
                   
                 trans-Anethole 
                 1.64x − 1.05 
                 1.6 ± 0.6 
               
               
                   
                 Linalool 
                 1.86x − 4.82 
                 1.9 ± 0.8 
               
               
                   
                 Carvacrol 
                 8.81x − 17.2 
                 8.8 ± 2.8 
               
               
                   
                 Eugenol methyl ether 
                 1.81x − 5.43 
                 1.8 ± 1.4 
               
            
           
           
               
            
               
                   Aedes aegypti  (Linnaeus) (mosquito) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 0.861x − 2.29  
                 0.86 ± 0.42 
               
               
                   
                 (+)-Pulegone 
                 5.89x − 6.11 
                 5.9 ± 2.9 
               
               
                   
                 Estragole 
                 2.33x + 1.38 
                 2.3 ± 0.8 
               
               
                   
                 trans-Anethole 
                  2.16x − 0.945 
                 2.2 ± 0.8 
               
               
                   
                 Linalool 
                 1.86x − 4.82 
                 1.9 ± 0.8 
               
               
                   
                 Carvacrol 
                  1.81x − 0.157 
                 1.8 ± 0.7 
               
               
                   
                 Eugenol methyl ether 
                 2.15x − 7.99 
                 3.4 ± 2.2 
               
            
           
           
               
            
               
                   Drosophila melanogaster  (fruit fly) 
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 2.56x + 8.49 
                 8.49 ± 0.69 
               
               
                   
                 (+)-Pulegone 
                 2.50x + 3.41 
                 3.41 ± 0.36 
               
               
                   
                 (−)-Pulegone 
                 2.42x + 2.36 
                 2.36 ± 0.41 
               
               
                   
                   
               
            
           
         
       
     
     The LC 50  of (+)-pulegone, trans-anethole, estragole, 1,8-cineole, carvacrol and eugenol methyl ether against house flies, roaches and mosquitoes were higher than those of DDVP (Table 5). These monoterpenes were at least 28 times less toxic to  M. domestica  than DDVP (Table 5). Estragole was the most active compound against  A. aegypti  (LC 50 , 0.25 μg/cm 3 ) and  M. domestica  (LC 50 , 5.89 μg/cm 3 ); linalool was moderate; and eugenol methyl ether was not active against the three household insects (Table 5). Trans-anethole was potent against rusty red cockroaches with an average LC 50  value of 4.25 μg/cm 3 , i.e., an amount which was slightly more toxic than of DDVP (4.99 μg/cm 3 ) (Table 5). Moreover, this analysis indicated that the (+) isomer of pulegone is significantly more toxic to fruit fly than the (−) isomer. 
     Example 5: Monoterpenes as Fumigants 
     Linalool was used as a representative chemical to confirm that the monoterpenes act as fumigants rather than contact poisons. Bioassays were conducted in which  thrips, M. usitatus , were exposed to the headspace above a solution containing linalool in peanut oil. The LC 50  value was 4.7% for linalool mixed in peanut oil. The results indicate that linalool is indeed a fumigant to the flower  thrips.    
     Example 6: Toxicity of Monoterpenes as Fumigants 
     Notably, the varying slopes of the dose-response curves (Tables 4 and 7) suggest different modes of action among the monoterpenes. Therefore, to determine how fast the monoterpenes act as fumigants, it was necessary to determine their LT 50  values for the target organisms. Table 8 shows LT 50  values of six test compounds and DDVP to the pest species  B. cucuribitae  (melon fly),  T. castaneum  (red flour beetle),  H. picinus  (Nitidulid beetle), and  M. domestica  (housefly). 
     
       
         
           
               
               
             
               
                   
                 TABLE 8 
               
             
            
               
                   
                   
               
               
                   
                 LT 50  (fiducial limits) (hour) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 
                   B. 
                 
                 
                   T. 
                 
                 
                   H. 
                 
                 
                   M. 
                 
               
               
                 Compound 
                 
                   cucuribitae 
                 
                 
                   castaneum 
                 
                 
                   picinus 
                 
                 
                   domestica 
                 
               
               
                   
               
               
                 80% DDVP 
                 0.89 
                 2.70 
                 0.89 
                 0.14 
               
               
                   
                 (0.28-1.27) 
                 (2.14-3.46) 
                 (0.70-1.40) 
                 (0.13-0.16) 
               
               
                 (+)- 
                 0.87 
                 3.47 
                 0.50 
                 0.33 
               
               
                 Pulegone 
                 (0.67-1.00) 
                 (2.50-4.41) 
                 (0.42-0.56) 
                 (0.25-0.39) 
               
               
                 Estragole 
                 1.69 
                 8.23 
                 0.60 
                 1.04 
               
               
                   
                 (0.49-2.22) 
                  (5.87-10.8) 
                 (0.48-0.72) 
                 (0.91-1.15) 
               
               
                 trans- 
                 2.28 
                 7.97 
                 1.07 
                 2.01 
               
               
                 Anethole 
                  (1.62-2350) 
                 (7.42-8.46) 
                 (0.85-1.34) 
                 (1.82-2.27) 
               
               
                 Linalool 
                 2.05 
                 35.1 
                 1.58 
                 1.28 
               
               
                   
                 (1.11-2.76) 
                 (25.6-60.6) 
                 (1.37-1.95) 
                 (1.04-1.70) 
               
               
                 Carvacrol 
                 2.49 
                 9.95 
                 1.94 
                 2.30 
               
               
                   
                 (2.05-2.92) 
                 (0.002-23.4)  
                 (1.47-7.29) 
                 (2.01-2.86) 
               
               
                 Eugenol 
                 2.96 
                 &gt;48 a        
                 0.61 
                 2.56 
               
               
                 methyl 
                 (2.14-16.2) 
                   
                 (0.35-0.80) 
                 (2.32-3.13) 
               
               
                 ether 
               
               
                 Acetone 
                 &gt;48 a        
                 &gt;48 a        
                 &gt;48 a        
                 &gt;48 a        
               
               
                 Chloroform 
                 &gt;48 a        
                 &gt;48 a        
                 &gt;48 a        
                 &gt;48 a        
               
               
                   
               
               
                   a Less than 50% mortality were observed 48 hours after exposure. 
               
            
           
         
       
     
     LT 50  Values for Agricultural Pests. 
     Relative to the other five monoterpenes, (+)-pulegone acted at least 1.9- and 1.2-fold faster against  B. cucuribitae  and  H. picinus , respectively (Table 8). The LT 50  of (+)-pulegone and DDVP were comparable to each other against  B. cucuribitae . Notably, (+)-pulegone acted on  H. picinus  approximately twice as fast as DDVP (LT 50  0.500 hour vs 0.894 hour). Fast toxicity of (+)-pulegone indicates probable action on nerve systems. The LT 50  of (+)-pulegone against  H. picinus  was approximately half of DDVP (0.500 hour vs 0.894 hour). On the basis of LC 50  and LT 50  values, (+)-pulegone was the most active compound amongst the monoterpenes tested. 
     LT 50  Values for Storage Pests. 
       T. castaneum  is a serious insect pest of stored products. LT 50  values of the six monoterpenes against  T. castaneum  were at least 1.3-fold greater than that of DDVP (Table 8). The LT 50  of (+)-pulegone was comparable to DDVP against  T. castaneum  (LT 50  3.47 hours vs 2.70 hours). 
     LT 50  Values for Household Pests. 
     Evaluation of LT 50  valued indicated that houseflies responded to carvacrol within 10 minutes; to (+)-pulegone, estragole, linalool and DDVP within 20 minutes; and to trans-anethole within 30 minutes. Eugenol methyl ether was not insecticidal to the housefly by 30 minutes. The LT 50  of (+)-pulegone to  M. domestica  was approximately twice of DDVP (0.331 hour vs 0.143 hour) (Table 8).  Aedes aegypti  responded to (+)-pulegone, estragole, trans-anethole, and carvacrol within 10 minutes, to linalool within 20 minutes, and to DDVP and eugenol methyl ether within 30 minutes (Table 8). 
     Example 7: Knockdown Activity of Monoterpenes 
     In addition to the rate of killing as an assessment of toxicity, the median knockdown dose (KD 50 ) was also determined for (+)-pulegone, estragole, linalool, trans-anethole, eugenol methyl ether, and carvacrol as compared to DDVP. When tested against  A. aegypti  and  M. domestica , the results indicate that estragole and (+)-pulegone were significantly insecticidal to houseflies (Tables 5-7 and 9). 
     
       
         
           
               
               
             
               
                   
                 TABLE 9 
               
             
            
               
                   
                   
               
               
                   
                 KC 50  (fiducial limits) (μg/cm 3 ) 
               
            
           
           
               
               
               
               
            
               
                 Compound 
                 10 Minutes 
                 20 Minutes 
                 30 Minutes 
               
               
                   
               
            
           
           
               
            
               
                   Aedes aegypti (mosquito) 
               
            
           
           
               
               
               
               
            
               
                 DDVP 80% 
                 . . . 
                 . . . 
                     &gt;0.081 
               
            
           
           
               
               
               
               
               
               
            
               
                 (+)-Pulegone 
                 19.6(8.41-35.5) 
                 1.40 
                 (0.67-2.52) 
                 1.4 
                 (0.67-2.52) 
               
            
           
           
               
               
               
               
               
            
               
                 Estragole 
                 &gt;8.1 
                 2.32 
                 (1.06-4.44) 
                    &gt;8.1 
               
            
           
           
               
               
               
               
               
               
            
               
                 trans- 
                 &gt;8.1 
                 4.84 
                 (2.87-10.9) 
                 4.05 
                 (1.88-35.6) 
               
               
                 Anethole 
               
            
           
           
               
               
               
               
               
            
               
                 Linalool 
                 . . . 
                 &gt;243 
                 69.0 
                 (32.3-14800) 
               
            
           
           
               
               
               
               
               
            
               
                 Carvacrol 
                 &gt;243    
                 6.0 
                 (—) 
                 &gt;243 
               
            
           
           
               
               
               
               
            
               
                 Eugenol 
                 . . . 
                 . . . 
                 &gt;243 
               
               
                 methyl ether 
               
            
           
           
               
            
               
                   Musca domestica (housefly) 
               
            
           
           
               
               
               
               
            
               
                 DDVP 80% 
                 . . . 
                    &gt;2.7 
                    &gt;2.7 
               
            
           
           
               
               
               
               
               
            
               
                 (+)-Pulegone 
                 . . . 
                 &gt;243 
                 12.2 
                 (8.97-15.0) 
               
            
           
           
               
               
               
               
            
               
                 Estragole 
                 . . . 
                 &gt;502 
                 &gt;243 
               
               
                 trans- 
                 . . . 
                 . . . 
                 &gt;243 
               
               
                 Anethole 
               
            
           
           
               
               
               
               
               
            
               
                 Linalool 
                 . . . 
                 &gt;243 
                 192 
                 (122-393) 
               
            
           
           
               
               
               
               
            
               
                 Carvacrol 
                 &gt;7600    
                 &gt;243 
                 &gt;243 
               
               
                 Eugenol 
                 &gt;7600    
                 &gt;243 
                 &gt;243 
               
               
                 methyl ether 
               
               
                   
               
               
                 (—), Fiducial limits were larger than 1000-fold. 
               
            
           
         
       
     
     Example 8: Activity of Monoterpenes Against Parasitic Wasps 
     Egg parasitoids are among the best studied natural enemies and have been successfully used in biological control of various lepidopteran agricultural pests. They are easy to rear and kill their hosts before they hatch, thereby preventing crop damage. Previous analyses have indicated that essential oils from  Ferula assafoetida  can affect some biological and behavioral traits of  Trichogramma embryophagum  and  T. Evanescens  (Poorjavad, et al. (2014)  BioControl  59:403-413). The LC 50  and LC 99  values of  Prangos ferulacea  essential oils against the egg stages of  T. embryophagum  were 2.12-5.66 μL/L air (Sumer Ercani, et al. (2013)  Turk. J. Agric. Forest.  37:719-725). 
     Toxicity in terms of LC 50  (Table 10), LC 90  (Table 11) and dose-response slopes (Table 12), or KD 50  (Table 13) of the monoterpenes to non-target species was assessed with two natural insect egg parasitic wasp species ( T. chilonis  and  A. japonicus ). The LT 50  values of all six compounds against  A. japonicus  were greater than DDVP (Table 13). In particular, the LT 50  of (+)-pulegone was approximately 1.2-fold greater than that of DDVP for  A. japonicus  (Table 13). (+)-Pulegone was also quite safe to  T. chilonis  with an LC 50  of 2.02 μg/cm 3 , which was greater than that of estragole (1.64 μg/cm 3 ) and trans-anethole (0.62 μg/cm 3 ), but lower than linalool (2.88 μg/cm 3 ) (Table 10). 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 10 
               
               
                   
                   
               
               
                   
                   
                   
                 LC 50  (fiducial limits) 
                 Chi- 
               
               
                   
                 Compound 
                   
                 (μg/cm 3 ) against  T. chilonis   
                 square 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 DDVP 80% 
                 &gt;3.75 
                 3.91 
               
            
           
           
               
               
               
               
               
            
               
                   
                 (+)-Pulegone 
                 2.06 
                 (0.20-3.83) 
                 0.142 
               
               
                   
                 Estragole 
                 1.64 
                 (0.00-3.19) 
                 3.94 
               
               
                   
                 trans-Anethole 
                 0.62 
                 (0.00-3.16) 
                 3.72 
               
               
                   
                 Linalool 
                 2.88 
                 (1.22-3.84) 
                 0.310 
               
               
                   
                 Carvacrol 
                 826 
                 (0.00-2.53) 
                 3.71 
               
            
           
           
               
               
               
               
            
               
                   
                 Eugenol methyl ether 
                 &gt;60    
                 0.197 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 11 
               
               
                   
                   
               
               
                   
                   
                 LC 90  (fiducial limits) (μg/cm 3 ) 
               
               
                   
                 Compound 
                 against  T. chilonis   
               
               
                   
                   
               
             
            
               
                   
                 DDVP 80% 
                 &gt;3.75 
               
            
           
           
               
               
               
               
            
               
                   
                 (+)-Pulegone 
                 12.1 
                 (7.46-35.2) 
               
               
                   
                 Estragole 
                 34.9 
                 (17.2-762) 
               
               
                   
                 trans-Anethole 
                 19.6 
                 (5.60-2.25E11) 
               
               
                   
                 Linalool 
                 6.24 
                 (4.74-13.4) 
               
               
                   
                 Carvacrol 
                 7.08 
                 (1.55-60.8) 
               
            
           
           
               
               
               
            
               
                   
                 Eugenol methyl ether 
                 &gt;60    
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 12 
               
               
                   
                   
               
               
                   
                   
                 Regression 
                   
               
               
                   
                 Compound 
                 equation 
                 Slope ± SE 
               
               
                   
                   
               
             
            
               
                   
                 DDVP 80% 
                 0.787x + 0.576 
                 0.79 ± 0.55 
               
               
                   
                 (+)-Pulegone 
                 0.963x + 0.202 
                 1.7 ± 0.5 
               
               
                   
                 Estragole 
                 0.963x + 0.220 
                 0.96 ± 0.36 
               
               
                   
                 trans-Anethole 
                 0.855x + 0.173 
                 0.86 ± 0.43 
               
               
                   
                 Linalool 
                 3.81x − 1.97 
                 3.8 ± 1.2 
               
               
                   
                 Carvacrol 
                  1.34x + 0.106 
                 1.3 ± 0.6 
               
               
                   
                 Eugenol methyl ether 
                 0.326x − 0.863 
                 0.33 ± 0.31 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 13 
               
               
                   
                   
               
               
                   
                   
                 LT 50  (fiducial limits) (hour) 
               
               
                   
                 Compound 
                 against  A. japonicus   
               
               
                   
                   
               
             
            
               
                   
                 80% DDVP 
                 0.41 (0.07-0.49) 
               
               
                   
                 (+)-Pulegone 
                 0.47 (0.00-0.68) 
               
               
                   
                 Estragole 
                 1.19 (1.02-1.37) 
               
               
                   
                 trans-Anethole 
                 0.933 (0.73-1.11)  
               
               
                   
                 Linalool 
                 0.788 (0.71-0.86)  
               
               
                   
                 Carvacrol 
                 3.32 (2.05-7220) 
               
               
                   
                 Eugenol methyl ether 
                 1.77 (1.49-2.23) 
               
               
                   
                 Acetone 
                 &gt;48 a   
               
               
                   
                 Chloroform 
                 &gt;48 a   
               
               
                   
                   
               
               
                   
                   a Less than 50% mortality were observed 48 hours after exposure. 
               
            
           
         
       
     
     Example 9: Activity of Monoterpenes Against Honey Bees 
     The honey bee ( Apis mellifera ) is the most frequent floral visitor of crops worldwide. Unfortunately, many of the same pesticides used to control unwanted insects also have a negative effect on honey bees. Accordingly, the toxicity of (+)-pulegone on honey bee  Apis mellifera  was assessed in a 24-hour fumigation assay. The results of this analysis are presented in Table 14. 
     
       
         
           
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                 Amount (μL/dm 3 ) 
                 No. of total bees 
                 Mortality ± STD (%) 
               
               
                   
               
             
            
               
                 0 (control) 
                 60 (20 × 3) 
                 5.0 ± 5.0 
               
               
                 0.012 
                 60 (20 × 3) 
                 5.0 ± 8.7 
               
               
                 0.024 
                 60 (20 × 3) 
                 3.3 ± 5.8 
               
               
                 0.048 
                 60 (20 × 3) 
                 5.0 ± 5.0 
               
               
                 0.096 
                 60 (20 × 3) 
                 5.0 ± 5.0 
               
               
                 0.192 
                 60 (20 × 3) 
                 8.3 ± 7.6 
               
               
                   
               
            
           
         
       
     
     The results indicate that (+)-pulegone failed to have a negative impact on honey bees. Given that monoterpenes have also been shown to inhibit disease in honey bees, e.g., mites, bacteria and fungi (Tutun, et al. (2018)  Turkish J. of Agricult.  6(1):34-45), there is an additional benefit to using the monoterpenes of this invention over conventional insecticides. 
     Example 10: Toxicity of Monoterpenes Mixtures 
     A mixture of (+)-pulegone and estragole (1:1) had an LC 50  and LC 90  of 0.287 μg/cm 3  (0.223-0.351 μg/cm 3 ) and 0.443 μg/cm 3  (0.362-0.723 μg/cm 3 ), respectively. The co-toxicity coefficient (CTC) and synergistic factor (SF) were 67.9 and 0.679, respectively, which indicated antagonism as a bi-mixture (Table 15). A mixture of (+)-pulegone, estragole and trans-anethole (1:1:2) had LC 50  and LC 90  values of 0.654 μg/cm 3  (0.539-0.771 μg/cm 3 ) and 0.993 μg/cm 3  (0.833-1.25 μg/cm 3 ), respectively. The CTC and SF were 29.8 and 0.298, respectively, which indicated an antagonism of the monoterpene mixture (Table 15). 
     
       
         
           
               
               
               
             
               
                 TABLE 15 
               
               
                   
               
               
                   
                 (+)-Pulegone:Estragole 
                 (+)-Pulegone:Estragole:trans- 
               
               
                 Analysis 
                 (1:1) 
                 Anethole (1:1:2) 
               
               
                   
               
             
            
               
                 Regression 
                 7.124x + 3.707 
                 7.398x + 1.202 
               
               
                 equation 
               
               
                 LC 50  (fiducial 
                 0.287 (0.223-0.351) 
                 0.654 (0.539-0.771) 
               
               
                 limits) (μg/cm 3 ) 
               
               
                 Chi-square 
                 0.075 
                 0.009 
               
               
                 LC 90  (fiducial 
                 0.443 (0.362-0.723) 
                 0.993 (0.833-1.25) 
               
               
                 limits) (μg/cm 3 ) 
               
               
                 CTC 
                 67.9   
                 29.8   
               
               
                 SF 
                 0.679 
                 0.298 
               
               
                 Type of action 
                 Antagonist 
                 Antagonist 
               
               
                   
               
            
           
         
       
     
     Example 11: Field Applications 
     To assess the use of monoterpenes in the field, linalool was tested for control of flower  thrips  in cowpea fields. Field control efficacy of linalool against  thrips  indicated that linalool diluted 10-fold in peanut oil was the most efficacious application compared to 5- and 20-fold dilutions in peanut oil. At 1 and 3 days after treatment, control efficacy respectively reached 98% and 100% for the 10-fold dilution (Table 15). By comparison, the positive control, a 3000-fold dilution of spinetoram, reached 70% and 94% at 1 and 3 days after treatment, respectively (Table 16). 
                             TABLE 16                          Control Efficacy (%)                                             1   3   5   7   14   21       Treatments   DAT   DAT   DAT   DAT   DAT   DAT                         Thrips                                         Spinetoram   70   94   37   33   62   0       20X Linalool   74   61   67   20   39   0       10X Linalool   98   100   34   38   6.4   0       5X Linalool   71   97   37   2.2   49   0                 Leafhopper                                         Spinetoram   68   87   36   49   66   25       20X Linalool   77   83   24   47   60   17       10X Linalool   93   67   39   96   98   23       5X Linalool   82   64   65   55   74   5.1                    
DAT, days after treatment.
 
     A 10-fold dilution of linalool was also the most efficacious application, compared to 5- and 20-fold dilutions, in controlling leafhoppers,  Cicadella viridi  linne (Hemiptera: Cicadellidae). At 7 and 14 days after treatment, the control efficacy of the 10-fold dilution of linalool reached 96% and 98%, respectively. By comparison, at 7 and 14 days after treatment, the 3000-fold dilution of spinetoram reached 49% and 66%, respectively (Table 16). 
     Example 12: Dose-Dependent Toxicity of (+)-Pulegone 
     To assess the specificity and dose-dependency of (+)-pulegone, mortality of  thrips M. usitatus  was determined for (+)-pulegone and (−)-pulegone in a 24-hour fumigation assay over a range of concentrations (0, 0.012, 0.024, 0.048, 0.096 and 0.192 μL/dm 3 ). The results of this analysis are presented in Table 17. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 17 
               
               
                   
               
               
                   
                   
                 LC 50   
                   
                 fiducial 
               
               
                 Compounds 
                 equations 
                 (μL/dm 3 ) 
                 R 2   
                 limits 
               
               
                   
               
             
            
               
                 (+)-pulegone 
                 y = 2.110 x + 2.325 
                 0.079 
                 0.928 
                 0.066-0.097 
               
               
                 (−)-pulegone 
                 y = 2.958 x + 2.738 
                 0.119 
                 0.794 
               
               
                   
               
            
           
         
       
     
     Example 13: Representative Bioinsecticidal Compositions 
     Formula I is prepared as an oil-in-water emulsion (Table 18). 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 18 
               
               
                   
                   
               
               
                   
                 Ingredient 
                 Amount (% of total) 
               
               
                   
                   
               
             
            
               
                   
                 Water 
                 78.8%  
               
               
                   
                 Vegetable Oil (Peanut Oil or Corn Oil) 
                 8.0% 
               
               
                   
                 Linalool or (+)-pulegone 
                 10.0%  
               
               
                   
                 Soy Lecithin or polysorbate 20 
                 2.0% 
               
               
                   
                 Sodium Bicarbonate 
                 1.0% 
               
               
                   
                 Benzoic Acid, Sodium Salt 
                 0.2% 
               
               
                   
                 Total: 
                 100%  
               
               
                   
                   
               
            
           
         
       
     
     Formula II is prepared as an oil-in-water emulsion (Table 19) 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 19 
               
               
                   
                   
               
               
                   
                 Ingredient 
                 Amount (% of total) 
               
               
                   
                   
               
             
            
               
                   
                 Water 
                 60% 
               
               
                   
                 Vegetable Oil (Peanut Oil or Corn Oil) 
                 30% 
               
               
                   
                 Linalool or (+)-pulegone 
                 10% 
               
               
                   
                 Total: 
                 100%