AU3018599A - Tea tree oil pesticidal compositions - Google Patents

Description

WO 99/46993 PCT/AU99/00198 1 "TEA TREE OIL PESTICIDAL COMPOSITIONS" THIS INVENTION relates to pesticidal compositions containing tea tree oil (TTO). The use of TTO as a component of a pesticidal composition 5 is known in International Publication WO96/28032. This reference refers to a in a disinfectant and insecticidal composition comprising 0.25-14% TTO, 0.25-14% of eucalyptus oil together with 10-40% by volume of an alcohol and 5-25% by weight of a wetting agent or surfactant. In this reference it is stated that when TTO and eucalyptus oil are used together 10 that there is provided a synergistic activity wherein the composition has a broad spectrum range of activity both as a disinfectant and insecticide. It is also stated that there are miscibility problems with this composition having regard to the nature of the components and this is why the inclusion of the surfactant is necessary to provide a homogenous 15 solution. Thus, it is important to first dilute the surfactant with water, eucalyptus oil and finally TTO. The composition is used to control or repel cockroaches, ants, fleas, mosquitos, beetles, spiders, silverfish and moths. A similar composition is described in International Publication WO96/28033. 20 Reference may also be made to International Publication WO93/17558 which refers to a disinfectant composition comprising stable aqueous solutions of (a) a blend of biocidally active terpenes such as TTO, (b) one or more biocidally active surfactants, (c) one or more proton donor type biocides, and (d) a salt of mono-, di-, or trihydroxy aliphatic or 25 aromatic acid. The TTO contains terpinen-4-ol and 1,8-cineole and acts as a carrier for the control of biological fouling. Fabric may be treated by cleaning with a surfactant and applying a preferred composition comprising linear alkyl benzene sodium sulfonate, sodium dodecyl sulfate, anhydrous sodium citrate, TTO, glycoxal, perfume and water. 30 There is also included KATHON WT which is a surfactant. Reference is also made to J6310495-A which describes a WO 99/46993 PCT/AU99/00198 2 formulation for preventing infestation of house mites comprising an extract of plants such as Myrtaceae or their active components, such as mono-, sesqui- and diterpene compounds and an essential oil which are applied to a support for the house mites, such as wood veneer. 5 Reference may also be made to AT9101755-A which describes a composition of a solution of ethereal oil from plants of Myrtaceae and Labiatae species together with a vegetable oil carrier which is applied to the skin and hoofs of horses to inhibit the effects of biting insects, such as horseflies as well as parasites inclusive of fungi 10 and mites. US5449517 refers to a formulation for killing fleas on fur bearing animals which comprises TTO, aloe vera gel, soap and water which may be used to treat dogs, cats, hamsters and other household pets. 15 AU666834 describes TTO vapours as an insecticide against dust mites wherein the vapour is introduced into an air conditioning system. Reference is also made to EP734727 which describes the use of TTO for controlling ectoparasites inclusive of fleas, ticks, mites and 20 lice on animals, such as cats and dogs. The TTO may be applied in pure form where it is rubbed into the skin to obtain entry to the bloodstream. TTO may also be used in compositions containing essential oils or fragrant oils, such as cedarwood oil, lemon oil, geranium oil and lavender oil and macadamia oil. A preferred composition is 90-95% water, a 25 vegetable emulsifier and 5-10% by volume of TTO. It is already known from several publications, which include: (i) In-Cosmet. Exhib. Conf. Proc. (1995) 405-411 entitled "Preserving Naturally with Tea-Tree Oil"; and 30 (ii) SOFW J. (1995) 121(7) 486, 488-9 entitled "Preserving Naturally with Tea Tree Oil"; WO 99/46993 PCT/AU99/00198 3 that tea tree oil (TTO) is used currently in a wide range of personal care products inclusive of medicated soaps, liquid soaps, disinfectants, deodorants, antiseptics, hair care products inclusive of shampoos and conditioners, oral hygiene products inclusive of toothpaste and 5 mouthwashes and skin care products inclusive of acne creams, hand creams and sunscreens. TTO is currently included within such products within a range of 0.5-3.0% by weight. It is considered as a broad spectrum anti-microbial substance. It is also known in a variety of references, for example, in J. 10 Appl. Bacteriol (1995) 78(3) 264-9, that TTO is active against micro organisms inclusive of S. aureus, E. coli, P. aeruginosa and C. albicans. Linalool with alpha-terpineol which are components of TTO were found to be active against all of these organisms with the exception of P. aeruginosa. Another component, i.e. terpinen-4-ol, was found to be active 15 against all of the above micro-organisms. In Japanese Patent J04041407, reference is made to melaleuca oil when diluted with water as having effective anti-microbial activity against P. aeruginosa, E. coli, S. aureus, S. faecalis, C. albicans and A. niger. The resulting anti-microbial preparations were useful in 20 relation to protection of vegetables and flowers from the above micro organisms. Thus, from the foregoing, it will be appreciated that TTO is known to be an effective anti-microbial substance as well as having insecticidal properties. This is established in EP734727 wherein it is 25 established that TTO in pure form is active against blood-sucking parasites, such as fleas, ticks, mites and lice in dogs and cats. Reference is also made in this reference to the fact that TTO is active against aphids in plants although no exemplification or support is provided for this statement. 30 In this regard, it is very difficult to provide any predictions as to responses of arthropods inclusive of insects to potential pesticides or WO 99/46993 PCT/AU99/00198 4 insecticides until appropriate testing has been carried out under reproducible and controlled scientific conditions. Some responses may be broad spectrum and induce similar responses in most species of insect, such as DDT as discussed in "Plant Pests and their Control" by P. 5 G. Fenemore (Butterworths). Another example of a broad spectrum insecticide is pyrethrum flowers or pyrethrins. In other cases, some potential insecticides may be narrow spectrum insecticides, such as the carbamate ester known as "Pirimicarb" as its activity is primarily against aphids. 10 Unexpectedly, it has now been discovered that TTO has insecticidal activity against termites. The term "termites" as used herein refers to termites generally or insects of the order Isoptera which also includes Nasutitermes sp, which includes hardwood termites, Coptotermes sp, 15 which includes softwood termites and Mastotermes sp, and, in particular, Mastotermes darwiniensis which is a major tropical termite species. In another aspect of the invention, it has been discovered that TTO also has activity in inhibition of tunnelling by termites. Therefore, the invention provides a method of inducing 20 mortality in termites by application of TTO to plants or habitats populated with these pests. The invention also provides a method of inhibition of tunnelling by termites wherein TTO is applied to plants or habitats infected or populated with these pests. 25 The TTO is preferably utilized in a composition which may have at least 0.05% of TTO in a suitable vehicle or solvent. Preferably the composition is a clear aqueous solution which may comprise from 1 20% surfactant, emulsifier or solubilizing agent. Any suitable surfactant may be utilized such as an ionic amphoteric or non-ionic surfactant. 30 Preferably a non-ionic surfactant is utilized which may comprise a polyoxyethylene surfactant such as sorbitan mono-9-octadecenoate WO 99/46993 PCT/AU99/00198 5 poly(oxy-1,2-ethanediyl) derivatives which are marketed under the trade mark POLYSORBATE 80 or TWEEN. Alternatively, TTO may be dissolved totally in the surfactant. TTO may also be dissolved in suitable solvents inclusive of 5 polar organic solvents such as ethanol, methanol, propylene glycol, butylene glycol, ethylene glycol or other polyhydroxy alkanes. The preferred concentrations of TTO in the vehicle or solvent may comprise from 0.05-10% and more preferably from 0.1-5.0% and most preferably 1.0-5.0%. 10 Such compositions of the invention may be applied to the insect habitat or plants infected with the insects in the form of a spray or fine droplets which is expelled from a pressurized container as is known in the art. The invention also includes within its scope the use of 15 termite barriers which may be formed from soil, sand or other finely divided material which may be used for propagation of termites. Such termite barriers may be arranged within or around termite infested habitats for the purpose of eradication of termites or alternatively, such termite barriers may be used for prevention or control 20 of locations which are susceptible to termite infestation. Suitable habitats include ground mounds of termites, buildings inclusive of dwellings or even crops. Usually the TTO treated barriers will have limited penetration by termites and termites after initial contact with the TTO will 25 experience mortality within 24-48 hours. Usually the barrier may provide appropriate protection for 30-150 days or more. The barriers may be provided with manually operated or automatically operated pressurized injection or distributions systems which may continually apply TTO at suitable concentrations at spaced 30 intervals along the length of barrier or intermediate the height of the barrier. Such pressurized injection or distribution systems can operate on WO 99/46993 PCT/AU99/00198 6 a stand alone basis or may be topped up by outside contractors. The pressurized injection or distribution points may include jets, atomized sprays or spargers. In relation to use of TTO solutions that may be sprayed or 5 applied in the form of a fog onto a particular location infested with termites, it will be appreciated that TTO solutions may be dissolved in a liquefied gas which has the dual role of solvent and propellant and one example of such a product is a range of products marketed under the ENVIROSOL trade mark by BOC Gases which use liquid carbon dioxide 10 at a high pressure, e.g. 5000 kPa. Alternatively, use may be made of propellants including dimethylether chlorinated fluorocarbons, hydrocarbons inclusive of propane, butane and isobutane and nitrous oxide. Preferably, however, such propellants include low pressure gaseous nitrous oxide and carbon dioxide. 15 Preferably use may be made of the BACTIGAS composition of 0.3 wt% TTO, 0.9 wt% odour absorber, 1.8 wt% ethanol and 97 wt% liquid carbon dioxide. Alternatively, use may be made of a BACTIGAS concentrate comprising 10 wt% TTO, 30 wt% odour absorber and 60 wt% ethanol. In this regard, BACTIGAS is a trade mark of BOC Gases. 20 Use may also be made of encapsulated TTO preparations wherein, for example, a cyclodextrin is complexed with TTO which reduces its strong odour and makes it less susceptible to oxidation. Other suitable materials that can be used to encapsulate TTO either in its pure form or at a suitable concentration are zeolites and polyamides, which are 25 used as millicapsules. It will also be appreciated that the term "tea tree oil" as used herein refers to natural forms of TTO which are obtained from any appropriate Melaleuca species or Leptospermum species, such as, for example, M. alternifolia, M. linariifolia and M. dessitafolia as well as 30 modified extracts thereof. The term "tea tree oil" also includes constituents such as terpinen-4-ol or other constituents which WO 99/46993 PCT/AU99/00198 7 demonstrate the relevant activity of the invention per se. EXPERIMENTAL SUMMARY (Investigations 1-3) 5 The study reported here in Investigations 1-3 were carried out to investigate under laboratory conditions insecticidal activity of tree oil. Both high and low cineole formulations were assessed for their efficacy in inducing mortality in two key arthropod pests, i.e. ground mound termites and tomato fruit borer. 10 Investigations were also conducted to determine the effect of both tea tree oil formulations on inhibition of tunnelling by ground mound termites. Both high and low cineole formulations were highly efficacious against ground mound termites. The low cineole formulation 15 produced 50 and 90% mortality after 48 hour exposure at concentrations of 1.2 and 5.7, 2.3 and 11.7, 0.8 and 2.9 g/L respectively whereas the high cineole formulation produced the same mortalities at concentrations of 1.5 and 8.6, 2.1 and 11.7, 0.6 and 1.6 g/L respectively. Little insecticidal activity was, however, found against tomato fruit borer. The 20 mortality dose levels obtained are comparable with a number of insecticides, and compare favourably with products such as petroleum spray oils. Behavioural studies with ground mound termites showed reduced tunnelling activity with high and low cineole formulations 25 concentrations greater than 0.1 g/L. Mortality also occurred in the termites after seven days at rates of 1 g/L for the high cineole and 0.1 g/L for the low cineole formulation. From these results, it is concluded that both low and high cineole formulations process good insecticidal activity against termites. 30 There appears to be little difference in efficacy between the two formulations (i.e. high and low cineole). Tea tree oil was also shown to WO 99/46993 PCT/AU99/00198 8 restrict tunnelling in termites. These behavioural responses may be as important as direct mortality for pest control. INVESTIGATION 1: Laboratory studies to determine the bioefficacy of tea tree oil against ground 5 mound termites (Nasutitermes exidiosus) Study Obiective To investigate the insecticidal activity of tea tree oil formulations against ground mound termites. Test species and life stages 10 Ground mound termites Nasutitermes exidiosus Soldiers + Workers Test products and formulation Tea tree oil (low cineole) 10% EC Tea tree oil (high cineole) 10% EC Study conditions 15 Insecticide free laboratory, maintained at 27 + 2 0 C temperature and 60 70% relative humidity. Study duration 2 months Post treatment assessments 20 24 hours 48 hours Treatments and replications Treatment Repictionr Mean Number 25 (/L) Soier Workers) 0.50 3 20 0.75 3 20 1.00 3 20 2.50 3 20 30 5.00 3 20 10.00 3 20 WO 99/46993 PCT/AU99/00198 9 Treatment Replication Mean Number (Soldiers + Workers) Blank 3 20 Control (Water) 3 20 Both products were tested at two different occasions in triplicate and 5 means were taken of 120 termites. Materials and Methods Formulation Desired concentrations were obtained by serial dilution of 10% formulations of both low and high cineole samples of tea tree oil on 10 v/v basis by mixing with distilled water. The serial dilutions were made to obtain responses between 10-99% mortality of the test species. Test Species Soldiers and workers of ground mount termites (Nasutitermes exidiosus) were initially collected from a nest in the field 15 and maintained under laboratory conditions. Equipment Potter's Spray Tower, laboratory bio-assay equipment. Methods Twenty termites (10 soldiers + 10 workers) were taken for 20 each replicate to both low and high cineole treatments in petri dishes onto the base of which was placed moist filter paper. The termites were maintained on moistened filter paper in the petri dishes until they were transferred to new petri dishes, which were then placed on the spray tower stage and sprayed. 25 All the emulsions/serial dilutions were thoroughly agitated immediately before spraying. 5 mL aliquots of each concentration were sprayed on each group of termites by the Potter's Spray Tower at 120 kPa inlet and 5 psi outlet air pressure. Ten minutes after spraying, treated termites were placed 30 back into the petri dishes contained moistened filter paper and WO 99/46993 PCT/AU99/00198 10 maintained in a constant environment room (27 + 2 0 C temperature and 60-70% relative humidity) until all post treatment observations had been completed. Each sample was tested at six different concentrations in 5 triplicate on two different occasions and included water as control. In addition to water, three replications of a blank (consisting of all ingredients except tea tree oil) were also run equivalent to the highest test concentration, i.e. 10 g/L to assess any effect of the blank on the test species. 10 Observations on mortality were assessed 24 and 48 hours after treatment. Termites which did not move their legs when gently prodded were counted as dead.. Results were analysed using SPSS statistical software which calculated the probit regression and fiducial limits of concentrations 15 and illustrated graphically by plotting graphs with log concentrations and probit transformed mortality using Fig. P software. Results and Discussion Table 1 gives the average percentage mortality of termites for each treatment. Dose mortality data show that ground mound termites 20 Nasutitermes exidiosus are susceptible to both low and high cineole samples of tea tree oil. The mortality increased with increasing dose and there was linear correlation between log dose and transformed mortality (FIGS. 1 and 2). High cineole oil was found to be slightly more efficacious than low cineole oil at both 24 and 48 hours mortality 25 assessments. In low cineole, 50 and 90% mortality of termites were obtained at the dose rate of 1.19 and 5.45 g/L at 24 hours and 0.834 and 2.88 g/L at 48 hours exposure respectively. The high cineole resulted in 50 and 90% mortality at 0.771 and 2.31 g/L at 24 hours and 0.624 and 1.64 g/L at 48 hours exposure respectively. The relative median potential 30 for high cineole compared to low cineole was 0.70. From the bio-assay results, it is concluded that both samples of tea tree oil have very good WO 99/46993 PCT/AU99/00198 11 biocidal activity towards termites and further studies on their efficacy and persistence should be carried out against different species of termites under natural and semi-natural field conditions. INVESTIGATION 2: Laboratory studies to determine the 5 bioefficacy of tea tree oil against tomato fruit borer (Helicoverpa armigera) Study Obiective To investigate the insecticidal activity of tea tree oil formulations against larvae of tomato fruit borer. 10 Test species and life states Tomato fruit borer Helicoverpa armigeras (Hubner) 2nd Instar larvae Test products and formulation Tea tree oil (low cineole) 10% EC Tea tree oil (high cineole) 10% EC 15 Study conditions Insecticide free laboratory, maintained at 27 ± 20C temperature and 60 70% relative humidity. Study duration 2 months 20 Post treatment assessments 24 hours 48 hours Treatments and replications 25 Treatment Repic n Mean Nube of (9L)(Number of Leaf Adult Thrips/Leaf Discs) Dise 0.25 3 20 0.50 3 20 1.00 3 20 30 2.50 3 20 5.00 3 20 WO 99/46993 PCT/AU99/00198 12 Treatment Replication Mean Number of (g/L) (Number of Leaf Adult Thrips/Leaf Discs) Ds 10.00 3 20 Blank 3 20 Control (Water) 3 20 5 Both products were tested at two different occasions in triplicate and means were taken of 36 larvae. Materials and Methods Formulation Desired concentrations were obtained by serial dilution of 10 10% formulations of both low and high cineole samples of tea tree oil on v/v basis by mixing with distilled water. The serial dilutions were made to obtain responses between 10-99% mortality of the test species. Test Species Second instar larvae of tomato fruit borer used for testing 15 were taken from a culture reared for several generations at the Cotton Research Institute, Narrabri. Equipment Potter's Spray Tower, laboratory bio-assay equipment. Methods 20 Six second instar larvae were taken for each replication. All the emulsions/serial dilutions were thoroughly agitated immediately before spraying. 5 mL aliquots of each concentration were sprayed on larvae in petri dishes by a Potter's Spray Tower at 120 kPa inlet and 5 psi outlet air pressure. 25 After 30 minutes of spraying, each larvae was transferred to a separate cup containing larval food and maintained in a constant environment room (27 + 2 0 C temperature and 60-70% relative humidity) until all post treatment observations had been completed. Each sample was tested at six different concentrations in WO 99/46993 PCT/AU99/00198 13 triplicate on two different occasions and included water as control. In addition to water, three replications of a blank (consisting of all ingredients except tea tree oil) were also run equivalent to the highest test concentration, i.e. 10 g/L to assess any effect of the blank on 5 the test species. Observations on mortality were assessed 24 and 48 hours after treatment. Larvae which did not move when gently prodded were counted as dead. Results and Discussion 10 Table 2 gives the average percentage mortality of larvae for each treatment. Both low and high cineole samples of tea tree oil were not found to be effective in causing mortality to Helicoverpa armigera larvae. Only 16.7 and 22.2% mortality was obtained at the highest concentration, i.e. 10 g/L of low and high cineole respectively. From 15 these assay results, It is concluded out that tea tree oil has little insecticidal potential in killing Helicoverpa larvae. INVESTIGATION 3: Laboratory studies to determine the repellency of tree oil against ground mound termites (Nasutitermes exidiosus) 20 Study Obiective To investigate the repellency activity of tea tree oil against ground mound termites. Test species and life stages Ground mound termite Nasutitermes exidiosus Soldiers + Workers 25 Test products and formulation Tea tree oil (low cineole) 10% EC Tea tree oil (high cineole) 10% EC Study conditions Insecticide free laboratory, maintained at 27 ± 2 0 C temperature and 60 30 70% relative humidity. Study duration WO 99/46993 PCT/AU99/00198 14 2 months Post treatment assessments Length of Tunnelling 1, 3, 7,14 and 21 days Mortality Assessment 4, 8 and 21 days 5 Treatments and replications Treatment Replication Number (% ad. Conc.) (Number of Tubes) (Workers +~ Soldiers) 0.01 4 25 + 5 10 0.1 4 25 + 5 1.0 4 25+5 10.00 4 25 + 5 Blank 4 25 + 5 Control (Water) 4 25 + 5 15 Both products were tested at two different occasions in quadruplicate. Materials and Methods Formulation Desired concentrations were obtained by serial dilution of 20 10% formulations of both low and high cineole samples of tea tree oil on v/v basis by mixing with distilled water. The serial dilutions were made for obtaining the repellency response of the test species. Test Species Soldiers and workers of ground mound termite 25 (Nasutitermes exidiosus) were collected from the field and maintained in a laboratory until required for testing. Methods Thirty termites (25 workers + 5 soldiers) were taken for each replicate for all the treatments and were maintained on moistened filter 30 paper in petri dishes until they were transferred into tubes. Clean fine (< 1 mm) moist sand particles were used as substrate. Ten grams of air dry sand were thoroughly mixed with 1 mL WO 99/46993 PCT/AU99/00198 15 aliquots of each concentration to obtain a final moisture content of approximately 10%. This treated sand was placed in the base of plastic tubes (SIZE) to a height of 10 mm and tamped down firmly. A liquid 2% agar solution was added to each tube on top of 5 the sand to a depth of 35 mm, then left overnight at 40C for the agar to set. The 25 worker and 5 soldier N. exidiosus were introduced from the petri dishes into each tube in the remaining space at the top of the tube. A narrow strip of folded filter paper was provided as source of 10 termite food. Caps were loosely screwed on all tubes, to prevent escape, but allow limited gas exchange. The tubes were then placed upside down in racks in a constant environment room (25 + 20C temperature and 6 70% relative humidity) until all post treatment observations had been completed. 15 Each sample was tested at four different concentrations in quadruplicate and included water as control. In addition to water, four replications of a blank (consisting of all ingredients except tea tree oil) were also run equivalent to the highest test concentration, i.e. 10 g/L or 1% concentration to assess any 20 effect of the blank on termites. Observations on the length of the tunnel made in the agar were recorded 1, 3, 7, 14 and 21 days after treatment. Termite mortality was assessed 4, 8 and 21 days after treatment. Data were analysed using the general linear model SPSS 25 (1993) statistical software. When significant treatment differences were detected, comparisons between means were made using the Least Significant Difference (LSD) test. The data were also illustrated graphically by plotting number of days against length of tunnel using Fig. P software. The distance between the end of the tunnelling and the lower 30 surface of the sand containing the oil treatments were compared, as this gave the best estimate of repellency.

WO 99/46993 PCT/AU99/00198 16 Results and Discussion Low cineole Table 3 gives the mean distance between the end of the termite tunnel and the treated substrate. Within 24 hours of being placed 5 in the tubes, the termites had commenced tunnelling in all treatments, with the distance of tunnelling from the substrate between 21.3 to 26.2 mm. In the highest concentration treatment (i.e. 10%), tunnelling practically stopped after three days and all termites died within four days 10 of treatment. The mean distance between the end of the tunnel and the substrate was 23.8 mm. In the next highest concentration treatments (i.e. 1% and 0.1%), termites ceased tunnelling after seven days and all termites died within four to eight days of treatment. The mean distance between the end of the tunnel and the substrate was 22 and 21 mm 15 respectively. The termites in the 0.01% concentration rate of treatment all survived the assessment period and were observed tunnelling until the last assessment at 21 days. The mean distance between the end of the tunnel and the substrate was only 2 mm. 20 The termites in the blank (i.e. surfactant) treatment were all alive and produced multiple tunnels until the last assessment at 21 days. Mean minimum distance at this time between the end of tunnels and the substrate was 9.5 mm. In the nil treatment control, termites achieved maximum tunnelling and reached the substrate within 14 days of 25 commencement of the experiment. All were alive and active at the last assessment at 21 days. Although differences were observed in the distance of tunnels from the substrate between different treatments, there were no significant differences found between the 0.1, 1.0 and 10% 30 concentrations treatments. All of these treatments were significantly superior to all other treatments in preventing termites from reaching the WO 99/46993 PCT/AU99/00198 17 substrate. No significant differences were found between the nil treatment control, the surfactant only and 0.01% tea tree oil treatments. From these results, it is concluded that the low cineole formulation of tea 5 tree oil possesses good repellent qualities against the ground mound termite when used at concentrations of 0.1% and above. Higqh cineole Table 4 gives the mean distance between the end of the termite tunnel and the treated substrate. Within 24 hours of being placed 10 in the tubes, the termites had commenced tunnelling in all treatments, and the distance of tunnels from the substrate between 14.5 to 25.5 mm. At the three day assessment, termites in the 10% and 1% concentration treatments of the high cineole formulation had practically stopped tunnelling and all termites died within four and eight days 15 respectively. The mean distance between the end of the tunnel and the substrate was 24.5 and 22.5 mm respectively. In the 0.1% and 0.01% treatments, tunnelling had stopped by 14 days and all termites died within 21 days of treatment while several were still alive in the 0.01% treatment. The mean distance between the 20 end of the tunnel and the substrate was 14.3 and 6.8 mm respectively. The termites in the blank (i.e. surfactant) treatment and the nil treatment control were all alive on the last assessment at 21 days. Although differences were observed in the distance of tunnels from the substrate between different treatments, there were no 25 significant differences found between the 0.1, 1.0 and 10% concentrations treatments. These treatments were significantly superior to all other treatments in preventing termites from reaching the substrate. The 10% and 1% treatments were both significantly superior (p s 0.05) to all other treatments, however, there was no significant difference between 30 them. There were no significant differences were found between WO 99/46993 PCT/AU99/00198 18 the 0.1% and the 0.01% tea tree oil and the surfactant (blank) treatments but the 0.1% treatment was significantly superior to the untreated control in reducing termite tunnelling. CONCLUSION 5 From these results, it is concluded that both low and high cineole oil formulations of tea tree oil possess significant termite repellent activity. In addition, these trials confirm the previously reported effect of tea tree oil on termite mortality. The high cineole formulation demonstrated greater repellency. It can also be demonstrated that the 10 activity against termites could not be predicted based on the non-activity against tomato fruit borer. INVESTIGATION 4: Acute Toxicity Studies Materials and Methods Termite species 15 Acute toxicity studies were carried out against three termite species. They were: (i) Coptotermes sp; (ii) Nasutitermes sp; and (iii) Microtermes sp. 20 Concentrations of Tea Tree Oil (TTO) for evaluation A series of TTO solutions with concentrations of 0.1, 0.25, 0.5, 0.75 and 1% were prepared from 10% TTO solution to test against Coptotermes, Nasutitermes and Microtermes. The control treatment was distilled water. 25 Application of TTO A Potter's precision spray tower was used to apply TTO at a constant pressure (125 psi) using 5 ml of each treatment to spray a petri dish (90 mm diameter) containing 10 termite workers on wet filter paper (i.e. wetted with 1 ml distilled water). Each treatment was replicated four 30 times and each study was carried out twice. Elimination of fumiqation effects WO 99/46993 PCT/AU99/00198 19 Immediately following application of treatments, petri dishes were closed with specially designed lids to eliminate possible fumigation effects of TTO. Assessment of mortality 5 The number of dead termites (i.e. termites that did not move when they were disturbed with the fine paint brush) were counted at 24 and 48 hours after the application of TTO using a dissecting microscope. Results Knock down effects 10 All termites were knocked down within 20 minutes after the application of TTO compared with no knock down with the distilled water control treatment. The knock down effect was even noticed 48 hours after the treatments. Acute toxicity 15 Tables 5-7 show the percentage mortality of all termite species. Even at 0.1%, TTO caused significantly higher mortality in Coptotermes, Nasutitermes and Microtermes when compared with the distilled water control treatment. However, acute toxicity varied with the termite species, with Microtermes showing highest mortality (up to 100%) 20 when sprayed with concentrations between 0.5-0.75% TTO. INVESTIGATION 5: Assessment of possible fumigant effects Possible fumigant effects were observed by using filter paper application techniques and petri dishes with lids that eliminated fumigation effects. This study was carried out using two species of 25 termites, Coptotermes sp. and Microtermes sp. Method Fifty termites were introduced into each of 90 mm petri dishes containing a filter paper which was treated with 1 ml of TTO just before the introduction of termites. The petri dishes were then closed with 30 two different types of lid (viz fumigation effect eliminated or not eliminated). This treatment was repeated twice. In the control treatment, WO 99/46993 PCT/AU99/00198 20 50 termites were also transferred into each petri dishes containing filter paper which was treated with 1 ml of distilled water. Two different types of lids were again used in the controls with two replicates, as occurred in the treatment. All petri dishes were sealed with paraffin. 5 Results Knock down effect All treated termites were knocked down by TTO 20 minutes after their introduction. Acute toxicity 10 Mortality in all treatment replicates after 24 hours was 100%, whereas the control mortality was only 1%. INVESTIGATION 6: Soil Barrier Treatments Results of the initial investigations of soil barriers treated with 2% TTO (i.e. 200 ppm) were promising. For this study, we used clear 15 plastic tubes 40 mm diameter and 210 mm length to observe termite tunnelling. Materials and Methods Soil treatment for barrier evaluation 100 g samples of air dried soil were each treated with 10 ml 20 of 2% TTO and 10 ml of water to bring the moisture level up to 20% to ensure adequate mixing of TTO with soil particles used to provide optimal conditions for termite activity. Initially, 2 cm of soil barrier was created in the middle of the plastic tube and a 2% agar solution was used to hold the soil barrier in 25 place. Introduction of termites Finely diced decayed wood particles where Coptotermes sp. had been previously feeding were placed in one side of the barrier and 100 Coptotermes workers with few soldiers were introduced into the other 30 side of the barrier. Both ends of the tubes were closed with aluminium foil. In a second experiment, we used 4 cm barriers with freshly treated soils WO 99/46993 PCT/AU99/00198 21 with 2% TTO. Results 2 cm soil barrier In the water only control, 100% of termites crossed into the 5 decayed wood within 24 hours after their introduction. In the 2% TTO freshly treated soil, termites commenced tunnelling, but they did not completely penetrate the barrier to the decayed wood. In addition, 100% mortality occurred 48 hours after the introduction of termites in these tubes. 10 4 cm soil barrier In the water only control, 100% of termites made tunnels across the soil barrier and crossed into the decayed wood within 24 hours after their introduction. In the 2% TTO freshly treated soil, no tunnelling was observed and more than 95% mortality occurred 48 hours after the 15 introduction of termites. SUMMARY (Investigations 7-10) Additional toxicity studies showed TTO concentrations between 0.5-1.0% caused 100% mortality in economically important 20 termite species Nasutitermus exidiosus, N. walkeri, N. magnus (a pest species seen in grave vines) and Coptotermes acinoaciformes. TTO also showed fumigant activity causing 100% mortality when termites were confined in petri dishes containing freshly treated filter papers with 1% TTO. However, fumigant activity did not persist on filter papers after 25 drying and subsequent wetting with water. TTO knocked down all termite species even at lowest concentration of 0.1% 20 minutes afer the application. There was no feeding deterrent effect observed when tested on filter papers treated with 1% TTO. Termites fed on both treated and 30 untreated filter papers. Further trials with treated and untreated diced timbers are currently being undertaken under semi-field conditions.

WO 99/46993 PCT/AU99/00198 22 Barrier treatments under semi-field conditions using TTO treated soil and sand produced promising results. The termite species N. walkeri did not cross test 4 cm soil barriers treated with 1000 ppm TTO in experimental tubes one day after the treatment and proximity to the 5 barrier caused 100% mortality within 24-48 hours after the introduction of the termites. However, this activity did not persist 30 days after treatment. Soil treated with 2000 ppm of TTO showed promising barrier effects up to 30 days after the initial soil treatment and 100% mortality in termites occurred after 24-48 hours exposure to treated soil barriers 30 days after 10 initial treatment. At this concentration, persistence of activity did not continue to 90 days after treatment. For soil treated with 4000 ppm TTO, however, the barrier has remained effective for at least 150 days (the latest tested) and 100% mortality in termites occurred within 24-48 hours following exposure to the treated soil. 15 Sand barriers showed even more promising results with low concentrations of TTO. At 1000 ppm, an effective barrier has been maintained for at least 180 days (the latest tested) after the initial treatment. Exposure to the treated sand also resulted in 100% termite mortality within 24-48 hours. 20 These results suggest termite control applications involving the use of TTO which may comprise: (a) the direct application of formulated TTO sprays, fogs or aerosols to control existing termite incursions in dwellings; 25 (b) as a barrier treatment; (c) using slow release formulations, such as encapsulation; and (d) using horizontal or vertical perimeter treatments made of TTO treated sand as a sandwich barrier. 30 INVESTIGATION 7: Further Acute Toxicity Studies Materials and Methods WO 99/46993 PCT/AU99/00198 23 Termite species Investigations were undertaken to determine the effect of direct application of TTO on termites. Acute toxicity studies were carried out against three species. They were: 5 (i) Coptotermus acinaciformes; (ii) Nasutitermes walkeri; and (iii) N. magnus. C. acinaciformes is an important pest primarily of softwood, N. walkeri is a major pest of hardwood in the Sydney region, whereas N. 10 magnus is a prominent species in Central Australia (e.g. Alice Springs). Concentrations of Tea Tree Oil (TTO) for evaluation A series of TTO solutions with concentrations of 0.1, 0.25, 0.5, 0.75 and 1% were prepared from 10% TTO solution to test against Coptotermus, N. magnus and N. walkeri. The control treatment was 15 distilled water. Application of TTO A Potter's precision spray tower was used to apply TTO at a constant pressure (125 psi) using 5 ml of each treatment to spray a petri dish (90 mm diameter) containing 10 termite workers on wet filter paper 20 (i.e. wetted with 1 ml distilled water). Each treatment was replicated four times and each study was carried out twice. Elimination of fumigation effects Immediately following application of treatments, petri dishes were closed with specially designed lids to eliminate possible fumigation 25 effects of TTO. Assessment of mortality The number of dead termites (i.e. termites that did not move when they were disturbed with the fine paint brush) were counted at 24 and 48 hours after the application of TTO using a stereo microscope. 30 Results Knock down effects WO 99/46993 PCT/AU99/00198 24 All termites were knocked down within 20 minutes after the application of TTO compared with no knock down with the distilled water control treatment. The knock down effect was even noticed 48 hours after the treatments. 5 Acute toxicity Tables 8-10 show the percentage mortality of all termite species. Even at 0.1%, TTO caused significantly higher mortality in Coptotermes, N. walker and N. magnus when compared with the distilled water control treatment. However, acute toxicity varied with the termite 10 species, with N. magnus showing highest mortality (up to 100%) when sprayed with concentrations between 0.5-0.75% TTO. Conclusion TTO concentrations between 0.5-1% can cause up to 100% mortality after 48 hrs in termites such as Nasutitermus exidiosus, N. 15 walker and Coptotermes acinaciformes when applied as a direct spray. INVESTIGATION 8: Assessment on Fumigant Effect of TTO Possible fumigant effects were investigated by using a filter paper application technique and specially designed petri dishes with lids. This study was carried out using two species of termites, Coptotermus sp. 20 and N. magnus. Method Fifty termites were introduced into each of 90 mm petri dishes containing a filter paper which was treated with 1 ml of TTO solution just before the introduction of termites. The petri dishes were 25 then closed with two different types of lid (viz. fumigation effect eliminated or not eliminated). This treatment was replicated twice. In the control treatment, 50 termites were also transferred into individual petri dishes containing filter paper which was treated with 1 ml of distilled water. Two different types of lids were again used in the controls with two replicates, 30 as occurred in the treatment. All petri dishes were sealed with parafilm. Results WO 99/46993 PCT/AU99/00198 25 Knockdown effects All treated termites were knocked down by TTO 20 minutes after their introduction. Acute toxicity 5 Mortality in all treatment replicates after 24 hrs was 100%, whereas the control mortality was only 1%. Conclusion TTO showed lethal fumigation effects on termites within 24 hours after application in a confined space. 10 INVESTIGATION 9: Soil Barrier Treatments Results of initial investigations of soil barriers treated with 2% TTO (i.e. 2000 ppm) were promising. For this study, we used clear plastic tubes 40 mm diameter and 210 mm length to observe termite tunnelling. 15 Materials and Methods Soil treatment for barrier evaluation 100 g samples of air dried soil were each treated with 10 ml of 2% TTO and 10 ml of water to bring the moisture level up to 20% to ensure adequate mixing of TTO with soil particles and to provide optimal 20 conditions for termite activity. Initially, 2 cm of soil barrier was created in the middle of the plastic tube and a 2% agar solution was used to hold the soil barrier in place. Introduction of Termites 25 Finely diced decayed wood particles where Coptotermus sp. had been previously feed were placed in one side of the barrier and 100 Coptotermus workers with few soldiers were introduced into the other side of the barrier. Both ends of the tubes were closed with aluminium foil. In a second experiment, we used 4 cm barrier with freshly treated soils with 30 2% TTO. Results WO 99/46993 PCT/AU99/00198 26 2 cm soil barrier In the water only control, 100% of termites crossed into the decayed wood within 24 hours after their introduction. In the 2% TTO freshly treated soil, termites commenced tunnelling, but they did not 5 completely penetrate the barrier to the decayed wood. In addition, 100% mortality occurred 48 hours after the introducing of termites in these tubes. 4 cm soil barrier In the water only control, 100% of termites made tunnels 10 across the soil barrier and crossed into the decayed wood within 24 hours after their introduction. In the 2% TTO freshly treated soil, no tunnelling was observed and more than 95% mortality occurred 48 hours after the introduction of termites. INVESTIGATION 10: Soil Barrier Treatments under Semi-field 15 Conditions SOIL BARRIER TREATMENTS (PERSISTENCE OF TTO IN SOIL AND SAND) Introduction Acute toxicity studies showed that TTO is toxic to termites. 20 Concentrations between 0.5-1% produced 100% mortality in most termite species. However, in termite control programs, direct application of termiticides on termites cannot be used as the only method of control. Most economically significant termites are subterranean species and utilize soil as the medium of transport between the colony and the 25 destination of food sources. Therefore, any candidate product for a termiticide should persist in the soil for a long period of time, eliminating and controlling termite populations and their activity through the soil. Materials and Methods Soil treatments 30 Three different soil concentrations of TTO (i.e. 1000, 2000 and 4000 ppm) were prepared using 1, 2 and 4% TTO solutions to form WO 99/46993 PCT/AU99/00198 27 soil barriers. For each treatment, 800 g of dried air top soil was mixed with 80 ml of TTO (1, 2 and 4%) and 80 ml of distilled water to bring the moisture level around 20%. In the control treatment, no TTO was used and soil was mixed with 160 ml of distilled water to maintain moisture 5 levels at 20%. The treated soils were stored in glass jars with air tight lids for future use to assess the termite barrier properties periodically at 30 day intervals. The treatments were: (1) 1000 ppm TTO; 10 (2) 2000 ppm TTO; (3) 4000 ppm TTO; and (4) Control. Sand barrier treatments Two different concentrations of TTO in washed air dried 15 sand (i.e. 1000 and 2000 ppm) was prepared using 1 and 2% TTO solutions. For this purpose, 400 g of air dried white sand was mixed with 40 mi of 1 or 2% TTO for each treatment maintaining the moisture levels around 10%. In the control treatment, 400 g of sand was mixed with 40 ml distilled water without TTO. The treated sands were stored in glass jars 20 with air tight lids for future use to assess the termite barrier properties in sand treated with TTO. The treatments were: (1) 1000 ppm TTO; (2) 2000 ppm TTO; and 25 (3) Control. Creation of the barrier in perspex tubes: soil or sand A compacted soil or sand barrier (40 mm) was created in the middle of perspex tubes (30 mm in diameter and 200 mm in length) using two plastic disks (30 mm diameter) attached to wooden handles. These 30 barriers were further stabilized by pouring a layer (2 mm) of Agar (Agar Bacteriological No. 1, Oxoid, Oxoid Ltd., Basingstoke, Hampshire, WO 99/46993 PCT/AU99/00198 28 England) solution at 600C on the surface of the barrier. Introduction of termites One side of the soil barrier was filled with diced decayed wood that termites (N. walker) had been feeding on. Fifty worker termites, 5 including five soldiers, were introduced to the other side of the soil or sand barrier in the tubes and both ends of the perspex tubes were then tightly closed using perforated aluminium foil which allowed aeration. After the introduction of termites, tubes were placed in the dark under laboratory conditions at 220C for 20-24 hours. 10 Replication of treatments and periodical assessment of barriers Each treatment was repeated three times and trials were conducted at every 8-10 week intervals with treated soil to assess the efficacy. Experiments with treated sand were carried out 180 days after the initial treatment. 15 Assessment of efficacy of barriers treated with TTO After 24 and 48 hours, perspex tubes were inspected for: (1) tunnelling through soil barrier into decayed wood through the barriers; (2) mortality of termites; 20 (3) presence of termites on the decayed wood in the tubes. In addition to this, close-up photographs were taken 24 hours after the introduction of termites into the perspex tubes. Results 25 Under semi-field conditions, TTO at 1000 ppm in the soil showed promising barrier effects 1 day after the treatment of soil. All termites in the tube dies within 24 hours after their introduction, and there were no signs of tunnelling. However, these soils did not show any residual activity against termites when tested 30 days after the initial 30 treatment of soil (Table 11). At this time, termites made tunnels through the soil barrier into decayed wood within 24 hours. These results were WO 99/46993 PCT/AU99/00198 29 consistent with what was observed in controls where no TTO was used. Soil with 2000 ppm of TTO caused 100% mortality in termites between 24-48 hours even 30 days after the initial treatment and there was no evidence of tunnelling (Table 12). However, when tested 5 after 90 days, these soils (i.e. 2000 ppm TTO) did not show any significant residual activity and were similar to water treated controls (Table 13). Soil treated with 4000 ppm of TTO showed promising results even 150 days after the treatment (Table 14). This concentration of TTO 10 killed all termites within 24-48 hours after their introduction, and there was no evidence of tunnelling or tunnel initiation. Further trials will be required to determine the duration of the persistence of TTO at this concentration in soil. In sand, TTO persisted for a longer period than in soil even 15 at low concentrations. TTO at 1000 ppm level showed 100% mortality of termites in tubes even 180 days after treatment (Table 15). There was no evidence of initiation of tunnelling in tubes treated with sand barriers containing TTO at 1000 ppm. In control treatments, termites crossed to the wood food source within 24-48 hours after their introduction. 20 Discussion and conclusions Under semi-field conditions, soil treated with low TTO concentrations (1-2%) demonstrated termite barrier properties only for a short time. High concentrations such as 4000 ppm in soil, however, persisted with termiticidal activity for more than 180 days after the initial 25 treatment. Low concentrations lasted for a shorter period, and this may be due to factors such as degradation or adsorption in the soil. This was further supported by the results obtained from the barrier studies with TTO treated sand, in which much lower concentrations (1000 ppm) maintained high activity for more than 180 days. 30 There are three possibilities for use of TTO in termite soil barrier treatments:- WO 99/46993 PCT/AU99/00198 30 * Using formulations of TTO that have more persistent activity in soils, or alternatively, regular applications of more concentrated solutions to soils. * Using slow release formulations, such as 5 encapsulations as described above. * Using horizontal or vertical perimeter treatments made of TTO treated sand as a sandwich barrier.

WO 99/46993 PCT/AU99/00198 31 TABLES TABLE 1 Mean Percentage Mortality of Nasutitermes exidiosus Treatment Exposure Average Perce Itage Mortait~y~ (g) ... Peiod (Hr) 0.25 24 5.3 9.5 48 11.5 14.6 0.50 24 13.5 26.7 48 24.6 32.5 0.75 24 40.5 48.5 48 42.7 53.3 1.00 24 60.5 70.3 48 70.8 81.6 2.50 24 73.6 85.5 48 85.3 93.7 5.00 24 83.0 100.0 48 95.5 100.0 Blank 24 0.0 0.0 48 0.0 0.0 Control 24 0.00 0.0 48 0.0 0.0 WO 99/46993 PCT/AU99/00198 32 TABLE 2 Mean Percentage Mortality of Helicoverpa armigera Treatment Exposure Average Percentage Mortality (Lrd.Low C .neole High Cineole 0.25 24 0.0 0.0 48 0.0 0.0 0.50 24 0.0 0.0 48 0.0 0.0 1.0 24 0.0 0.0 48 0.0 0.0 2.5 24 0.0 0.0 48 0.0 0.0 5.0 24 5.5 0.0 48 5.5 0.0 10.0 24 16.7 22.2 48 16.7 22.2 Blank 24 0.0 0.0 48 0.0 0.0 Control 24 0.0 0.0 48 0.0 0.0 WO 99/46993 PCT/AU99/00198 33 TABLE 3 Mean Distance of Termite Tunnel below Substrate: Low Cineole Formulation 1 day.*>~ 3:: day 7 days 14da? 2dy Control 25.00 22.5 3.00 0.75 0.00" Surfactant 26.25 17.75 12.50 9.75 9.50b 0.01 21.75 17.75 13.25 4.50 2.00" 0.1 22.50 21.00 21.00* 21.00 21.00c 1.0 22.75 22.00 22.00* 22.00 22.00c 10.0 23.75 23.75 23.75* 23.75 23.75c TABLE 4 Mean Distance of Termite Tunnel below Substrate: High Cineole Formulation ,. ......... ........ ...... .'." . . .:'. "::. . . .. . .

....

iii ........ N. .... 1 a as 7 das 14dy 21dy Control 14.50 11.50 11.75 6.75 6.75a Surfactant 19.00 14.5 13.75 13.50 13.25ab 0.01 24.50 19.00 13.25 9.75 9.75ab 0.1 24.50 22.00 16.00 15.00 15.00b 1.0 23.25 22.50 22.50* 22.50 22.50c 10.0 25.50 24.50 24.50* 24.50 24.50C WO 99/46993 PCT/AU99/00198 34 TABLE 5 Percentage morality of Coptotermus sp. 24 and 48 hr after spraying with TTO at different concentrations. The percentage mortality in each column of Experiments I and II are mean values from 4 replicates. Experiment I Experinent II Experiment i %mortality %meality % mortality 24r48hr 24hr 48hir 24hr 48hr Control 10 22.5 2.5 7.5 6.25 15 0.1% TTO 40 52.5 5 7.5 22.5 30 0.25% TTO 66.6 66.6 40 67.5 53.3 67 0.5% TTO 75 82.5 45 52.5 60 67.5 0.75% TTO 77.5 77.5 90 100 83.7 88.75 1.0% TTO 10 100 92.5 100 96.2 100 TABLE 6 Percentage morality of Microtermes sp. 24 and 48 hr after spraying with TTO at different concentrations. The percentage mortality in each column of Experiments I and II are mean values from 4 replicates. ntExpernnt Exper ent % mertalty me taliy mortalityy 24 . X r br 48 hr 24 r rI Control 0 7.5 0 9.05 0 8.3 0.1% TTO 17.5 40 65.8 74.8 41.6 57.4 0.25% TTO 95 100 56 75 75.85 87.48 0.5% TTO 100 100 72.1 86.2 86.4 93 0.75% TTO 100 100 100 100 100 100 1.0% TTO 100 100 100 100 100 100 WO 99/46993 PCT/AU99/00198 35 TABLE 7 Percentage morality of Nasutitermus sp. 24 and 48 hr after spraying with TTO at different concentrations. The percentage mortality in each column of Experiments I and II are mean values from 4 replicates. Treatment Experiment 1 Experiment II Experiment III .. .... % mc rtality % mortality % mortality 24. 48.r 24hr 48r 24hr 48 hr Control 0 10 0 0 0 5 0.1% TTO 30 37.5 55 80 42.5 58.75 0.25% TTO 42.5 72.5 65 87.5 53.75 80 0.5% TTO 65 75 72.5 95 68.75 85 0.75% TTO 67.5 75 80 100 73.75 87.5 1.0% TTO 95 100 85 100 90 100 TABLE 8 Percentage morality of Coptotermus sp. 24 and 48 hr after spraying with TTO at different concentrations. The percentage mortality in each column of Experiments I and 11 are mean values from 4 replicates. SEx I Experinnt II Experinont 10 % ertlty mortalityy % met tailty 24 br 48 hr 24 hr 48 hr 24 hr 48 br Control 10 22.5 2.5 7.5 6.25 15 0.1% TTO 40 52.5 5 7.5 22.5 30 0.25% TTO 66.6 66.6 40 67.5 53.3 67 0.5% TTO 75 82.5 45 52.5 60 67.5 0.75% TTO 77.5 77.5 90 100 83.7 88.75 1.0% TTO 100 100 92.5 100 96.2 100 WO 99/46993 PCT/AU99/00198 36 TABLE 9 Percentage morality of N. magnus sp. 24 and 48 hr after spraying with TTO at different concentrations. The percentage mortality in each column of Experiments I and II are mean values from 4 replicates. Te e Eeriment I Experi ent II Experiment III % tlty % motality % motality 24r 8hr 24hr 48hr 24hr 48hr Control 0 7.5 0 9.05 0 8.3 0.1% TTO 17.5 40 65.8 74.8 41.6 57.4 0.25% TTO 95 100 56 75 75.85 87.48 0.5% TTO 100 100 72.1 86.2 86.4 93 0.75% TTO 100 100 100 100 100 100 1.0% TTO 100 100 100 100 100 100 TABLE 10 Percentage morality of N. walker sp. 24 and 48 hr after spraying with TTO at different concentrations. The percentage mortality in each column of Experiments I and II are mean values from 4 replicates. % mertalty mo taliy %mt tality 24 br 48 hr 24 hr 48 hr 24 br 48 hr Control 0 10 0 0 0 5 0.1% TTO 30 37.5 55 80 42.5 58.75 0.25% TTO 42.5 72.5 65 87.5 53.75 80 0.5% TTO 65 75 72.5 95 68.75 85 0.75% TTO 67.5 75 80 100 73.75 87.5 1.0% TTO 95 100 85 100 90 100 WO 99/46993 PCT/AU99/00198 37 TABLE 11 Soil persistence of TTO treated with different concentrations, and effectiveness as a barrier against termites 1 day after the treatment Tretmet bsevaton Obevtos Presence of (contrationr 24 hours 48 hours termite tunnel in of TTO in soi the barriers or sand barrier) 1000 ppm all dead all dead no 2000 ppm all dead all dead no Control alive alive yes, within 24 hours TABLE 12 Soil persistence of TTO treated with different concentrations, and effectiveness as a barrier against termites 30 days after the treatment Treatmet ObservatiosOrons 0s Presence of (cncntration 24 hours 4$ hours termite tunnelJs in of TTO in soil the barrers or sand ... . . . . . .. .... . . . . . . . . . . .. w w -.. . . . barrier) 1000 ppm alive alive yes, within 24 hours 2000 ppm all dead all dead no Control alive alive yes, within 24 hours WO 99/46993 PCT/AU99/00198 38 TABLE 13 Soil persistence of TTO treated with different concentrations, and effectiveness as a barrier against termites 90 days after the treatment Treatment Observations. O Presence .f. (concentration 24 hours 48 hours termite tunnels in of iTO in soil the barriers or sand barrier) 2000 ppm alive alive yes, within 24 hours 4000 ppm all dead all dead no Control alive alive yes, within 24 hours TABLE 14 Soil persistence of TTO treated with different concentrations, and effectiveness as a barrier against termites 150 days after the treatment .. . ........

***.... :: T r e a tm e n t O b s e r v a t io ionP r e s e n e o f....... concentrationn 24 hours 48 hours termite tunnels in of TTO in soil the barriers or sand barrier) 4000 ppm all dead all dead no Control alive alive yes, within 24 hours TABLE 15 Soil persistence of TTO treated with different concentrations, and effectiveness as a barrier against termites 180 days after the treatment Treatment Observations Observations resence of (concntrtin 24 hours 48 hours terinite tnnels in of TTO in suit the barrers or sand barrier) 1000 ppm all dead all dead no Control alive alive yes, within 24 hours WO 99/46993 PCT/AU99/00198 39 LEGENDS TABLE 3 * Denotes that all introduced termites dead. Means followed by the same letter are not significantly different at 5 ac = 0.05 TABLE 4 * Denotes that all introduced termites dead. Means followed by the same letter are not significantly different at ac =0.05 10 FIGS. 1 and 2 Dose-response regression for ground mound termites (Nasutitermes exidiosus) mortality against tea tree oil at 48 hours exposure. (Dashes represent 95% fiducial limits.)

Claims (24)

1. A method of inducing mortality in termites by introduction of a composition containing a morticidal amount of tea tree oil in a suitable solvent or vehicle to a termite infested habitat. 5

2. A method as claimed in Claim 1 wherein the composition is a clear aqueous solution which comprises from 1-20% v/v surfactant, emulsifier or solublizing agent.

3. A method as claimed in Claim 1 wherein the surfactant is a non-ionic surfactant. 10

4. A method as claimed in Claim 3 wherein the non-ionic surfactant is a polyoxyethylene surfactant.

5. A method as claimed in Claim 4 wherein the surfactant is sorbitan mono-9-octadecenoate.

6. A method as claimed in Claim 1 wherein the solvent is 15 selected from ethanol, methanol, propylene glycol, butylene glycol or other polyhydroxy alkanes.

7. A method as claimed in Claim 1 wherein the concentration of tea tree oil is 0.05-10%.

8. A method as claimed in Claim 1 wherein the concentration 20 of tea tree oil is 0.1-5%.

9. A method as claimed in Claim 1 wherein the concentration of tea tree oil is 1-5%.

10. A method as claimed in Claim 1 wherein tea tree oil is mixed with sand or soil to provide a barrier to a termite infested habitat so as to 25 control or reduce termite populations.

11. A method as claimed in Claim 1 wherein the tea tree oil is applied in the form of a spray fog or aerosol to control existing termite incursions in dwellings.

12. A method as claimed in Claim 10 wherein the barrier is used 30 in the form of a horizontal or vertical perimeter barrier.

13. A termite mortality composition comprising a morticidal WO 99/46993 PCT/AU99/00198 41 amount of tea tree oil in a suitable solvent or vehicle.

14. A termite mortality composition as claimed in Claim 13 wherein the composition is a clear aqueous solution which comprises from 1-20% v/v surfactant, emulsifier or solublizing agent. 5

15. A termite mortality composition as claimed in Claim 13 wherein the surfactant is a non-ionic surfactant.

16. A termite mortality composition as claimed in Claim 13 wherein the non-ionic surfactant is a polyoxyethylene surfactant.

17. A termite mortality composition as claimed in Claim 13 10 wherein the surfactant is sorbitan mono-9-octadecenoate.

18. A termite mortality composition as claimed in Claim 13 wherein the solvent is selected from ethanol, methanol, propylene glycol, butylene glycol or other polyhydroxy alkanes.

19. A termite mortality composition as claimed in Claim 13 15 wherein the concentration of tea tree oil is 0.05-10%.

20. A termite mortality composition as claimed in Claim 13 wherein the concentration of tea tree oil is 0.1-5%.

21. A termite mortality composition as claimed in Claim 13 wherein the concentration of tea tree oil is 1-5%. 20

22. A method of controlling termite infestations in a habitat comprising introduction of barriers to soil habitat comprising sand, soil or other finely divided material suitable for termite passage therethrough which has been treated with 0.05-10% tea tree oil.

23. A method as claimed in Claim 22 wherein the tea tree has a 25 concentration of 0.1-5%.

24. A method as claimed in claim 22 wherein the concentration of tea tree oil is 4-10%.