AN ALDEHYDE CONTAINING COMPOSITION FOR INSECT CONTROL

Abstract

This invention relates generally to use of a stable aqueous carbonyl compound containing solution, or a mixture of different carbonyl compounds containing solutions, in a program of integrated vector management.

Claims

1. A method of insect control by reducing the surface tension of a body of water containing the egg stage of the insect, the method including the step of applying a stable aqueous carbonyl compound containing solution to the surface of the body of water, wherein the solution includes: a) at least one carbonyl compound; b) a surfactant or detergent; c) a pH modifier; and d) a buffer.

2. A method according to claim 1 wherein the solution is prepared, prior to application, by: (a) adding the surfactant to a volume of water, heated to between 30° C. and 70° C.; (b) adding the pH modifier to adjust the pH of the solution to within 6.0 to 8.5 (c) adding at least one carbonyl compound to the body of water, to allow the carbonyl compound and the surfactant to complex, whilst maintaining the temperature within the range 30° C. and 70° C., for at least 10 minutes; (d) reducing the temperature of the body of water to below 30° C. to slow further complexation of the carbonyl compound with the surfactant; and (e) adding the buffer to the solution to buffer the pH and to produce the stable aqueous carbonyl compound containing solution.

3. A method according to claim 1 wherein the carbonyl compound is at least one of the following: an aldehyde, a ketone, a terpenoid and a lactone.

4. A method according to claim 3 wherein the terpenoid is citral and the ketone is acetone.

5. A method according to claim 1 wherein the following are present in the solution in the following concentration ranges: a) the carbonyl compound—0.001% to 45% m/v; b) the surfactant or detergent—0.1% to 45% m/v; and c) the buffer—0.05% to 25% m/v.

6. A method according to claim 1 wherein the surfactant or detergent is one or more of the following: an alcohol ethoxylate surfactant, a nonylphenol surfactant, an alkyl glycoside, sulphonic acid, sodium lauryl ethyl sulphate, sodium lauryl sulphate, a twin chain quaternary ammonium compound, cocopropyldiamide (CPAD), alkyl sulphate esters, benzenesulfonic acid, C10-13-alkyl derivatives and their sodium salts, D-glucopyranose, oligomeric glycosides and sorbitan monostearate.

7. A method according to claim 1 wherein the buffer includes at least one of the following: calcium acetate, magnesium acetate, sodium acetate, sodium acetate tri-hydrate, potassium acetate, lithium acetate, propylene glycol, hexalene glycol, sodium phosphate, sodium tri-phosphate, potassium phosphate, lithium phosphate, zinc perchlorate, zinc sulphate, cupric chlorate and cupric sulphate.

8. A method according to claim 1 wherein when the at least one carbonyl compound is an aldehyde, the aldehydes is at least one of the following: formaldehyde, acetaldehyde, glyceraldehyde, proprionaldehyde, butraldehyde, pentanaldehyde, methyl pentanaldehyde, ethyl pentanaldehyde, tiglic aldehyde, valeraldehyde, iso-valeraldehyde, hexanaldehyde, heptanaldehyde, octanaldehyde, nonanaldehyde, 2-ethyl hexaldehyde, decanaldehyde, undecanaldehyde, dodecyl aldehyde, cuminaldehyde, benzaldehydes, iso-valeraldehyde, chloraldehyde hydrate, furfuraldehyde, paraformaldehyde, ethane dialdehyde, glyoxal, succinaldehyde, glutaraldehyde, adipaldehyde, iso-phthalaldehyde, ortho-phthalaldehyde, cinnamaldehyde, salicylaldehyde and malonaldehyde.

9. A method of insect control comprising the step of applying, to an environment containing an immature form of the insect, a stable aqueous carbonyl compound containing solution, the solution including: a) at least one carbonyl compound. b) a surfactant or detergent; c) a pH modifier; and d) a buffer.

10. A method according to claim 9 wherein the carbonyl compound is at least one of the following: an aldehyde, a ketone, a terpenoid and a lactone.

11. A method according to claim 9 wherein the solution is applied to the environment by spraying a dispersant.

12. A method according to claim 11 wherein the dispersant is a diluted form of the stable aqueous carbonyl solution, diluted either with distilled or potable water, an alcohol or a solvent.

13. A method according to claim 11 wherein the solution or the dispersant is applied in the form of a spray, a fog, a foam or mist.

14. A method according to claim 9 wherein the solution is applied as an additive to a granule or pellet of a compressed binding substance.

15. A method according to claim 9 wherein the following are present in the solution in the following concentration ranges: a) the carbonyl compound—0.001% to 45% m/v; b) the surfactant or detergent—0.1% to 45% m/v; and c) the buffer—0.05% to 25% m/v.

16. A method according to claim 9 wherein the surfactant or detergent is one or more of the following: an alcohol ethoxylate surfactant, a nonylphenol surfactant, an alkyl glycoside, sulphonic acid, sodium lauryl ethyl sulphate, sodium lauryl sulphate, a twin chain quaternary ammonium compound, cocopropyldiamide (CPAD), alkyl sulphate esters, benzenesulfonic acid, C10-13-alkyl derivatives and their sodium salts, D-glucopyranose, oligomeric glycosides and sorbitan monostearate.

17. A method according to claim 9 wherein the buffer includes at least one of the following: calcium acetate, magnesium acetate, sodium acetate, sodium acetate tri-hydrate, potassium acetate, lithium acetate, propylene glycol, hexalene glycol, sodium phosphate, sodium tri-phosphate, potassium phosphate, lithium phosphate, zinc perchlorate, zinc sulphate, cupric chlorate and cupric sulphate.

18. A method according to claim 9 wherein, when the at least one carbonyl compound is an aldehyde, the aldehyde is at least one or more of the following: formaldehyde, acetaldehyde, glyceraldehyde, proprionaldehyde, butraldehyde, pentanaldehyde, methyl pentanaldehyde, ethyl pentanaldehyde, tiglic aldehyde, valeraldehyde, iso-valeraldehyde, hexanaldehyde, heptanaldehyde, octanaldehyde, nonanaldehyde, 2-ethyl hexaldehyde, decanaldehyde, undecanaldehyde, dodecyl aldehyde, cuminaldehyde, benzaldehydes, iso-valeraldehyde, chloraldehyde hydrate, furfuraldehyde, paraformaldehyde, ethane dialdehyde, glyoxal, succinaldehyde, glutaraldehyde, adipaldehyde, iso-phthalaldehyde, ortho-phthalaldehyde, cinnamaldehyde, salicylaldehyde and malonaldehyde.

19. An insecticidal composition which includes: a) at least one carbonyl compound; b) a surfactant or detergent; c) a pH modifier; and d) a buffer.

20. An insecticidal composition according to claim 19 wherein the carbonyl compound is at least one of the following: an aldehyde, a ketone, a terpenoid and a lactone.

21. An insecticidal composition according to claim 20 wherein the terpenoid is citral and the ketone is acetone.

22. An insecticidal composition according to claim 19 wherein the following are present in the solution in the following concentration ranges: a) the carbonyl compound—0.001% to 45% m/v; b) the surfactant or detergent—0.1% to 45% m/v; and c) the buffer—0.05% to 25% m/v.

23. An insecticidal composition according to claim 19 wherein the surfactant or detergent is one or more of the following: an alcohol ethoxylate surfactant, a nonylphenol surfactant, an alkyl glycoside, sulphonic acid, sodium lauryl ethyl sulphate, sodium lauryl sulphate, a twin chain quaternary ammonium compound, cocopropyldiamide (CPAD), alkyl sulphate esters, benzenesulfonic acid, C10-13-alkyl derivatives and their sodium salts, D-glucopyranose, oligomeric glycosides and sorbitan monostearate.

24. An insecticidal composition according to claim 19 wherein the buffer includes at least one of the following: calcium acetate, magnesium acetate, sodium acetate, sodium acetate tri-hydrate, potassium acetate, lithium acetate, propylene glycol, hexalene glycol, sodium phosphate, sodium tri-phosphate, potassium phosphate, lithium phosphate, zinc perchlorate, zinc sulphate, cupric chlorate and cupric sulphate.

25. An insecticidal composition according to claim 19 wherein, when the at least one carbonyl compound is an aldehyde, the aldehyde is one or more of the following: formaldehyde, acetaldehyde, glyceraldehyde, proprionaldehyde, butraldehyde, pentanaldehyde, methyl pentanaldehyde, ethyl pentanaldehyde, tiglic aldehyde, valeraldehyde, iso-valeraldehyde, hexanaldehyde, heptanaldehyde, octanaldehyde, nonanaldehyde, 2-ethyl hexaldehyde, decanaldehyde, undecanaldehyde, dodecyl aldehyde, cuminaldehyde, benzaldehydes, iso-valeraldehyde, chloraldehyde hydrate, furfuraldehyde, paraformaldehyde, ethane dialdehyde, glyoxal, succinaldehyde, glutaraldehyde, adipaldehyde, iso-phthalaldehyde, ortho-phthalaldehyde, cinnamaldehyde, salicylaldehyde and malonaldehyde.

26-32. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0097] The invention is described with reference to the following drawings in which:

[0098] FIGS. 1, 2 and 3 are photographs under microscope, of bed bug eggs, taken before and 24 hours after the application of a insecticidal composition in accordance with the invention ; and

[0099] FIG. 4 is a photograph of a petri dish in which is placed in filter paper soaked with an insecticidal composition in accordance with the invention and onto which is placed instar nymphs.

DESCRIPTION OF PREFERRED EMBODIMENT

[0100] The biocidal efficacy of aldehydes resides in the aldehyde functional group. This functional group reacts with free amine groups of, for example, a cell membrane of an organism. Aldehydes have biocidal efficacy as they disrupt cellular process within target cells which ultimately kills the organism. However, prior to the invention, it was not known to use aldehydes, and in particular stabilized aldehydes, to control insects as the vectors of disease.

[0101] Without buffering and stabilizing, aldehydes (with the exception of formaldehyde and aldehydes with carbon chain lengths of 2 to 4 carbon atoms) have a tendency, especially at low concentrations, to adopt a cyclic molecular configuration, which results in the aldehyde molecule losing its biocidal efficacy and, at relatively higher concentrations over a period of time, aldehyde solutions tend to polymerize with other aldehyde molecules. Polymerization accelerates at temperatures greater than 50° C. (and at less than 4° C. for aldehydes that have chain lengths of less than 5 carbon atoms). Polymerization of aldehydes also results in a loss of biocidal effect. To overcome the problem of polymerization, it is known to dilute a product containing an aldehyde solution prior to use.

[0102] Raising the pH of an aldehyde solution activates the solution, which increases the reactivity of the aldehyde functional groups with amine groups and the associated biocidal effect upon cell membranes. The stability of the aldehyde solution, however, is compromised when the pH is raised. Higher pH aldehyde solutions are only stable for a matter of days.

[0103] With these inherent drawbacks in mind, the invention relates to the development of a novel array of biodegradable, insecticides and larvicides, and to methods of use of same, that are highly effective in their ability to kill eggs, larvae, nymphs, and pupae of many insect species, before developmental metamorphosis to an adult insect. The incidence and prevalence of diseases borne by insects can therefore be reduced due to the reduction of insect concentration and inherent transmission rates.

[0104] The insects that may be controlled in accordance with one or more aspects of the invention, include both flying and terrestrial insects, such as: ants, aphids, bed bugs, cicadas, cockroaches, fleas, flies, lice, mites, mosquitoes, moths, stink bugs, silverfishes and termites.

[0105] The diseases that can be indirectly controlled as a result of using relevant aspects of the invention as part of a IVC program include: Yellow Fever, Malaria, Dengue Fever, West Nile Virus, Eastern and Western Equine Encephalitis, Dog Heartworm and Myiasis.

[0106] However, to illustrate the full potential for the invention, a vector table is provided below which highlights a possible range of diseases that potentially can be controlled with the use of an insecticidal composition or a method of insect control in accordance with the invention.

TABLE-US-00001 TABLE 1 VECTOR DISEASE PATHOGEN TYPE Mosquitoes Filariasis Helminthes Malaria Protozoa Derigue fever Virus Yellow fever Virus St Louis enceptialites Virus Eastern equine enceptialites Virus Wesern enquine encepphatis Virus West nile Virus River valley fever Virus Ticks Lyme disease Bacteria Rocky mountain spotted fever Bacteria Q fever Bacteria Tularemia Bacteria Relapsing fever Bacteria Ehilichiosis Bacteria Colorado tick fever Virus Crimean haemorrhagic fever Virus Babesious Protozoa Mites Q fever Bacteria Rickeftsioses Bacteria Deer flies Tularemia Bacteria Tsetse flies Sleeping sickness (African Protozoa Trpanosonias Blackflies Orichoceriasis Helminthes Muscoid flies Yaws Bacteria Sandflies Leishmanasis Protozoa Sanfly fever Virus Vesicular stomatitis Virus Lice Epidemic typhus Bacteria Trench fever Bacteria Fleas Endemic typhus Bacteria Bubonicplague Bacteria Reduvids (also Chagas disease Protozoa known as bed bugs, (American kissing bugs, terypanosomiasis) cone-nose bugs)

[0107] The insecticidal composition of the invention is shown to be highly effective at controlling insects by disrupting one or more of the immature forms of the insect. The insecticidal composition controls insect infestation at these stages of development, without adversely impacting the environment; as the components of the compositions are readily biodegradable, non-caustic and non-corrosive.

[0108] It is thought that the insecticidal composition of the invention works in controlling insect infestation by: [0109] a) the fixation and reduction of proteins and other nitrogen sources in or on the surface of insect eggs, larvae, nymphs and pupae (immature stages of an insect) that come in contact with the stabilized active carbonyl solution of the composition, and [0110] b) in the case of insects laying their eggs on a water surface, the disruption of the surface tension of the water surface and the resultant destabilization and breaking apart of the floating “egg boat”.

[0111] With regards to this latter hypothesis, it is thought that the stable aqueous carbonyl solution, when produced by the method of preparation described below, disrupts the formation and integrity of the floating ‘egg boat’. Once the integrity is broken, then the cytoplasm of the eggs, and the emerging larvae and pupae, are fixed by the reducing carbonyl functional group. The pathogens hosted by these insects are also fixed. The result is death of the insect at its immature state and its hosted pathogen. The lifecycle is therefore interrupted, and there is a reduction in the concentration of viable vector insects, for example, mosquitoes. By reducing the concentration of viable insect vectors in an area, the incidence of new pathogenic infections is reduced as is the overall prevalence of the disease.

[0112] The stable aqueous carbonyl solution, according to the invention, is manufactured, in a concentrate solution preferably with the use of an aldehyde. The concentrate solution is, by definition, a solution in which the aldehyde concentration is in the range 2% to 20% m/v.

[0113] In participation a non-ionic surfactant, i.e. alcohol ethoxylate (of either 3, 5, 7 or 9 ethoxylate groups), is added to a predetermined volume of water. The mixture is heated to a temperature between 40° and 50° C. followed by an aldehyde or a mixture of aldehydes. Without limitation, single aldehydes from the following list were selected and stabilized using the methodology that follows to perform an array of tests that follow: glutaraldehyde, furfuraldehyde, nonanaldehyde, glyoxyl, succinaldehyde, or ortho-phthalaldehyde, iso-phthalaldehyde and adipaldehyde. Also, a carbonyl, being the terpenoid citral, was selected.

[0114] The selected aldehyde, lactone, ketone or terpenoid (hereinafter simply referred to as “aldehyde”) is allowed to complex with the chosen alcohol ethoxylate for a period of between 15 and 30 minutes whilst maintaining the temperature of the volume of water between 30° C. and 70° C. The result is an aldehyde-surfactant solution is produced. During this period of heating the aldehyde complexes with the alcohol ethoxylate substantially to completion.

[0115] Following this period, a further amount of water, at a temperature of less than 25° C., is added to the aldehyde-surfactant complex solution to reduce the temperature of the solution to below 30° C. thereby to slow and stop the complexing reaction of the alcohol ethoxylate with the aldehyde.

[0116] A pH modifier, such as potassium hydroxide, is then added in a sufficient quantity to adjust the pH of the aldehyde-surfactant complex solution to within 7.0 to 8.5. Potassium hydroxide is used in a one molar solution.

[0117] Finally a buffer mixture, preferably comprising sodium acetate, trihydrate and potassium acetate is added to the aldehyde-surfactant complex solution to produce a stable aqueous aldehyde solution in the concentrate solution. In Example 1 that follows, a buffer mixture of potassium acetate and sodium bicarbotrate is, however, used.

[0118] Sodium acetate trihydrate and potassium acetate each have a concentration in the buffer mixture of between 0.250 to 1.5 grams/liter. This concentrated solution is diluted when added to the aldehyde-surfactant complex solution to within a range 0.005% to 0.1% m/v.

[0119] It is thought that this method produces, in complexation, micelles of the aldehyde and surfactant in the aqueous solution.

[0120] As an insecticidal or larvacidal composition (hereinafter “insecticidal” and “larvacidal” are used interchangeably), the invention provides a method of preventing the hatching of insect eggs or killing of insect larvae, pupae or nymphs, by contact with a stable aqueous aldehyde solution of the composition.

[0121] The use of the insecticidal composition of the invention when added as a concentrate to a crop irrigation system, would address plant pathogens derived from, for example, spider mites, weevils, beetles and psyllids. In another application, the composition is useful in the treatment of laundry, mattresses and bedding to help eradicate nuisance insect infestations of bed bugs, fleas, mites and lice.

[0122] Further use of the insecticidal composition of the invention are in pre- and post-construction of homes and structures where subsequent possible invasions of ants, termites, bedbugs and other insects may be addressed and controlled at source i.e. at the nests of eggs. Application, in this use, can be in the form of a foam of the insecticidal composition.

[0123] The insecticidal composition also can be applied by ground spraying, aerial spraying, or by hand or mechanical dispersion, including but not limited to backpack or other hand held devices, hydraulic or air nozzles, granular applicators, electrostatic applicators, controlled droplet applicators (CDA), or ultra-low volume (ULV) applicators. Method of application will, of course, depend on the particular context. The composition is also suitable for application by low pressure spraying so that large areas including water or wetlands can be easily treated.

[0124] The composition can be applied in single or repeated applications until the target insect infestation is effectively inhibited. The conditions leading to effective insect inhibition depend, in part, on the environment. In some instances, a single application of the composition is sufficient, in another, a plurality of applications may be required. This is often dependent on climatic conditions.

[0125] In the examples that follow, various test protocols were followed in the application of the insecticidal composition in accordance with the invention to mosquitos and bedbugs. These two insect vectors were chosen due to the many diseases associated with, and topical issues surrounding, these particular insects. The choice is not intended to be limiting.

[0126] In the case of the bed bug tests, eggs were chosen as the immature stage of this particular insect, to set a high benchmark in insecticidal efficacy of the insecticidal composition as the eggs are known to be very difficult to kill due to their mineralized surface covering.

[0127] In the mosquito directed tests (Examples 1 and 2 in particular), the count of viable larvae and pupae in a liquid sample is used as a surrogate for the relative incidence of pathogenic disease in an area. In the case of Example 1, due to the choice of the mosquito species, the pathogenic disease is viral e.g. yellow fever. In the case of Example 2, again due to the choice of species, the disease is protozoal e.g. Malaria.

[0128] In these tests laboratory assays were carried out with a colony of insectary-reared larvae originally derived from wild-caught mosquitoes maintained at the South African Bureau of Standards (“SABS”). Larvae were fed by adding a pinch of crushed Tetramin® (Tetra, Germany) fish food spread evenly on the water surface twice daily.

[0129] Assays were performed to determine the minimum effective dosages of a 20% concentration stable aqueous aldehyde solution. Four groups of fifteen larvae each were selected for testing. The concentrate solution was diluted to five different test concentration, one dilution for each experiment. Each experiment was run in four concurrent replicates at the same time. Larvae were fed during the experiments and all tests were run at ambient temperature ranging between 21° C. and 34° C. After a 24 hour period larvae were counted and mortality scored.

[0130] A number of stable aqueous aldehyde solutions, differing in the aldehyde of choice, were studied for their relative efficacy by taking them through the same test or protocol described above. A representative of each of the following types of low molecular weight aldehydes (<12 carbons) was studied in this manner: a mono-aldehyde, a dialdehyde, a straight chain aldehyde, a branched chain aldehyde, a cyclic aldehyde, a halogen containing aldehyde and a water insoluble aldehyde.

[0131] Other components, notably a biodegradable twin chain quaternary ammonium compound and the surfactant, were also studied in isolation to understand their relative contribution to the insecticidal/larvicidal effect.

Example 1

[0132] This test was conducted to determine the biological efficacy of a sample (marked “20% Aqua Cure”) against Aedes aegypti and Anopheles arabiensis mosquito larvae. Aqua Cure is a trade name for a composition of glutaraldehyde, a tergitol 15S9 surfactant, a polymer (polyvinyl pyrrolidone (“PVPK”)), a potassium acetate and sodium bicarbonate buffer and Arquad®. Aqua Cure is manufactured in accordance with the invention.

[0133] The test was performed in the SABS laboratories. The first exposures commenced on Aedes aegypti last instar larvae. Fifteen larvae were used per container (replicate). Four replicates were used for each of the three concentrations used. They were diluted, 1:10 and 1:100. Deionized water was used as diluent and where this was used the larvae were placed in the water before the sample was added. A separate set of four containers with larvae in deionized water only served as untreated controls. The larvae were supplied with laboratory diet as food. Mortality counts were made the next day.

[0134] A second set of exposures on Aedes larvae commenced the next day using dilutions of 1:500, 1:1000 and 1:2000 in the same manner as the first. Using the dilutions above, exposures were also carried out with 30 Anopheles arabiensis larvae per replicate.

[0135] The results are tabulated below:

TABLE-US-00002 TABLE 2 MORTALITY COUNT REPLICATES OUT OF 15 TOTAL OUT MOSQUITO DILUTION 1 2 3 4 OF 60 Aedes 0 15 15 15 15 60 Aegypti 1:10 15 15 15 15 60 (Yellow 1:100 15 15 15 15 60 Fever) 1:500 15 15 15 15 60 1:1000 15 15 15 15 60 1:2000 15 15 15 15 60 OUT OF 30 OUT OF 120 Anopholos 1:500 30 30 30 30 120 Aerobionsis 1:1000 30 30 30 30 120 (Malaria) 1:2000 30 30 30 30 120 NOTE: All the untreated control larvae were alive after the exposure period.

Example 2

[0136] This test was conducted to determine the biocidal efficacy of each of the samples listed below against Aedes aegypti larvae.

[0137] Samples Tested: [0138] 1. Original Product #1012 30/11/08 aged 6 month coded “1” (glutaraldehyde +PVPK+sodium acetate trihydrate +sodium bicarbonate); [0139] 2. 2-furfuraldehyde 10% complexed 16/3/9 coded “2” (furfuraldehyde+Tergitol 15S9+sodium acetate trihydrate+sodium bicarbonate); [0140] 3. N-Nonanal complexed 16/3/9 coded “3” (nonanaldehyde+Tergitol™ 15S9 +sodium acetate trihydrate+sodium bicarbonate); [0141] 4. Glyoxyl complex 16/3/9 coded “4” (glyoxyl+Tergitol™ 15S9+sodium acetate trihydrate+sodium bicarbonate); [0142] 5. Arquad® Q.A.L 4001094749 coded “5” (twin chain quaternary ammonium compound); [0143] 6. GK 10 BB 1060 coded “6” (glutaraldehyde+Tergitol™ 15S9+potassium acetate+sodium bicarbonate); [0144] 7. 20% Aqua Cure (glutaraldehyde+PVPK+Arquad®+Tergitol™ 15S9+potassium acetate+sodium bicarbonate).

[0145] Each of the samples subjected to this test were manufactured in accordance with the methodology described above.

[0146] The test was performed in the SABS laboratories. The exposure commenced on last instar larvae. Fifteen larvae were placed in each of 60 plastic containers (each a “replicate”) filled with 500 mg deionized water. The contents of the sample containers were shaken prior to adding the correct volume to the containers with deionized water and larvae to obtain dilutions of 1:2000 and 1:4000 respectively. Four replicates were used for each treatment. The remaining four containers with larvae served as untreated controls. The larvae were supplied with laboratory diet as food. Morality counts were made after 48 hours.

TABLE-US-00003 TABLE 3 MORTALITY COUNT SAMPLE REPLICATES OUT OF 15 TOTAL OUT OR CODE DILUTION 1 2 3 4 OF 60 1 1:2000 15 15 15 15 60 1:4000 15 15 15 13 58 2 1:2000 12 9 7 6 34 1:4000 0 1 0 0 1 3 1:2000 14 10 12 15 51 1:4000 2 1 2 0 5 4 1:2000 0 0 0 0 0 1:4000 0 0 0 0 0 5 1:2000 15 15 15 15 60 1:4000 15 15 15 15 60 6 1:2000 0 0 0 0 0 1:4000 0 0 0 0 0 7 1:2000 15 15 15 15 60 1:4000 15 15 15 15 60

Example 3

[0147] All the untreated control larvae were alive after the exposure period.

[0148] This test was conducted to determine the biological efficacy of the samples listed below against mosquito larvae, pupae and eggs.

[0149] Samples tested: [0150] 1. a 200 ml plastic bottle with approximately 30 ml liquid coded “7”; [0151] 2. a 200 ml plastic bottle with approximately 60 ml liquid coded “8”; and [0152] 3. 20% Aqua Cure™.

[0153] This test was performed at the SABS laboratories and started with exposure on Aedes aegypti larvae (+/−10 mm) commenced 30 Mar. 2009. Ten larvae were placed in each of 28 plastics containers (“replicate”) filled with 500 ml deionized water. The contents of the sample containers were shaken prior to adding the correct volume to the containers with deionized water and larvae to obtain dilutions of 1:2000 and 1:4000 respectively. Four replicates were used for each treatment. The remaining four containers with larvae served as untreated controls. The larvae were supplied with laboratory diet as food. Morality counts were made after 48 hours.

[0154] A second part of the test involved Anopheles arabiensis pupae were 5 pupae were placed in each of the first two replicates of each treatment. The number of adults that hatched were counted after 48 hours.

[0155] A third part of the test involved a rafter of Anopheles arabiensis eggs being placed in replicates, three per treatment. Food was supplied in each container with the eggs. Three days later, each container was examined for live larvae.

[0156] The results are tabulated below:

TABLE-US-00004 TABLE 4 MORALITY COUNTS OF AEDES AEGYPTI LARVAE SAMPLE REPLICATES OUT OF 10 TOTAL OUT OR CODE DILUTION 1 2 3 4 OF 40 7 1:2000 10 10 10 10 40 1:4000 10 10 10 10 40 8 1:2000 10 10 10 10 40 1:4000 10 10 10 10 40 AQUA 1:2000 10 10 10 10 40 CURE 1:4000 10 10 10 10 40

TABLE-US-00005 TABLE 5 NUMBER OF ANOPHELAS ARABIENSIS ADULTS THAT EMERGED FROM PUPAE REPLICATES SAMPLE OUT OF 5 TOTAL OUT OR CODE DILUTION 1 2 OF 10 7 1:2000 5 5 10 1:4000 0 0 0 8 1:2000 0 0 0 1:4000 0 0 0 AQUA 1:2000 0 0 0 CURE 1:4000 0 0 0

TABLE-US-00006 TABLE 6 NUMBER OF LIVE ANOPHELES ARABLENSIS LARVAE REPLICATES SAMPLE OUT OF 10 TOTAL OUT OR CODE DILUTION 3 4 OF 40 7 1:2000 0 0 0 1:4000 0 0 0 8 1:2000 0 0 0 1:4000 0 0 0 AQUA 1:2000 0 0 0 CURE3 1:4000 0 0 0

Example 4

[0157] Bed bug eggs were collected five days after the bed bugs had been fed. The eggs that were used were white and smooth in appearance as seen in FIG. 1. 10 bed bug eggs were placed into a petri dish containing 1 ml of either a control or a Microbidex-G solution. All eggs were immersed under the solution for 24 hours. After a 24 hour incubation period at 25° C. (60% relative humidity) the bed bug eggs were placed onto dry filter paper and left to incubate for a further 14 days.

[0158] Microbidex-G is a tradename for a composition, manufactured in accordance with the invention, which includes glutaraldehyde, tergitol 15S9 and a buffer of sodium acetate tri-hydrate and potassium acetate.

[0159] FIG. 2 shows the bed bug eggs following 24 hours of incubation with the control while FIG. 3 shows the bed bug eggs after 24 hours of incubation with concentrated (10%) Microbidex-G.

[0160] As can be seen in FIG. 3, the bed bug eggs incubated with Microbidex-G changed to a brown colour when compared to the eggs incubated with the control. This indicates that the eggs are non-viable.

Example 5

[0161] Ten first instar nymphs bedbugs were placed onto filter paper soaked with 1 ml of either a control or 10% Microbidex-G, a 1/100 and dilution or a 1/1000 dilution, for 24 hours at 25° C. (60% relative humidity). After 24 hours the first instar nymphs were checked for viability by prodding with a set of forceps.

[0162] The above graph details the number of dead and alive first instar nymphs following 24 hours of incubation with either the control or Microbidex-G solution. Incubation with Microbidex-G has increased the morality if first instar nymphs when compared to the control.

Example 6

[0163] This test involved the count of the number of surviving bed bugs 24 hours after a 1 minute exposure to a number of test solutions of 30% Microbidex-G at different dilutions.

[0164] The samples studied were on instar nymphs. The nymphs had a human blood feed the week before.

TABLE-US-00007 Sample description PPM Survival % Deionized water — 90% Microbidex-G (3.0% stabilized activated glutaraldehyde) = neat 30,000  3% Microbidex-G (3.0% stabilized activated glutaraldehyde) = 1:100 dilution 300 53% Microbidex-G (3.0% stabilized activated glutaraldehyde) = 1:1000 dilution 30 77% Tide HE ® (1 cap in 75 liters = working solution) 80% Tide HE ® + Microbidex-G (1:1)  7%

[0165] What is notable is the high mortality rate, in the concentrate and Tide HE® samples and this rate is achieved only after a minute of exposure.

Example 7

[0166] In this test, 120 mated, female (lab strained) bed bugs were ordered. The transit time for shipment was between 7-10 days during which time the female bed bugs laid eggs on a piece of white, corrugated paper. The paper that contained all the bed bugs, nymphs, and eggs were removed and placed on a disposable petri dish (60 mm×15 mm). All nymphs and adult bed bugs were removed using flexible forceps and placed back into a vial. Using forceps, bed bug eggs were carefully scraped from the paper and collected in the petri dish.

[0167] Five Microbidex formulations were used in this study (Microbidex “C”, Microbidex, “G”, Microbidex “I”, Mlcrobidex “N”, Microbidex “S”). Each formulation is a composition, manufactured in accordance with the invention, containing citral, glutaraldehyde, iso-phthalaldehyde, nonanoldehyde and succindaldehyde respectively.

[0168] Microbidex “C”, “G”,“N”, and “S” were tested at 100%, 50%, and 10% of the sample concentrations provided. Formulations were diluted using acetone and an acetone only solution was used as a control. Mlcrobidex “I” did not stay in solution, so it was diluted to 10%, 5%, and 1% of the sample concentration provided.

[0169] Whatman #1 5.5 cm filter paper (Cat No Whatman, 1001-055) were placed inside a petri dish and 25 μL of each concentration was dispensed onto the filter paper using a pipette to ensure complete saturation of the filter paper. Each sample and the acetone control were replicated three times. Bed bug eggs were checked under the microscope to determine their viability. Viable eggs can be identified by their pearly grey color and the eggs should appear round and smooth with the red eyes of the developing nymph visible. Eggs that were collapsed or dented were non-viable and hatched eggs were white and transparent. Three to five, viable eggs were collected and placed in the center of each filter paper and lids placed back over the Petri dish. The number of initial eggs for each sample was recorded.

[0170] Each sample was examined under the microscope daily for 6 days to determine egg mortality. Eggs were recorded either as viable, dead, or hatched (nymphs). At the end of the experiment, samples and supplies were placed in the freezer at −40° C. to kill off all surviving eggs and nymphs. Tables, tray, and equipment were sprayed with Ortho® Home Defense Dual-Action Bed Bug Killer after each day of testing.

[0171] The results are tabulated below:

TABLE-US-00008 TABLE 8 Treatment active ingredient Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Control 0 7.69 7.69 7.69 7.69 15.38 (Acetone Only) Microbidex “C” 10% citral 0 28.57 28.57 50.00 50.00 50.00 5% citral 0 18.18 18.18 36.36 36.36 36.36 1% citral 0 9.09 9.09 9.09 9.09 9.09 Microbidex “G” 3.0% glutaraldehyde 0 9.09 9.09 18.18 18.18 18.18 1.5% glutaraldehyde 0 7.69 7.69 7.69 15.38 15.38 1.0% glutaraldehyde 0.3% glutaraldehyde 0 0.00 0.00 8.33 8.33 8.33 Microbidex “I” (10%) 1.0% isophthalaldehyde 0 9.09 9.09 9.09 36.36 36.36 0.5% isophthalaldehyde 0 25.00 25.00 33.33 33.33 41.67 0.1% isophthalaldehyde 0 20.00 20.00 30.00 30.00 30.00 Microbidex “N” 10.0% nonanal 0 7.69 7.69 15.38 46.15 69.23 5.0% nonanal 0 9.09 9.09 9.09 9.09 9.09 1.0% nonanal 0 16.67 16.67 16.67 25.00 25.00 Microbidex “O” Microbidex “S” 10.0% succindialdehyde 0 0.00 0.00 0.00 0.00 0.00 Microbidex “S” 5.0% succindialdehyde 0 0.00 0.00 13.33 13.33 13.33 Microbidex “S” 1.0% succindialdehyde 0 0.00 0.00 0.00 0.00 0.00

[0172] Notably in this test is the adaptation of a high hurdle of insecticidal efficacy in that eggs were chosen as the immature stage and the relevant insecticidal composition was applied to the filter paper before the introduction of the eggs to the paper. The composition was not applied directly to the eggs by soaking or dipping.

[0173] This test mimicked a real life application in which the composition would be applied to, for example, bedding onto which the bed bugs would thereafter infect.