Biopesticide emulsion comprising polysorbate 20

Abstract

Biopesticide emulsion system having a first continuous phase comprising water and a second discontinuous phase comprising a biopesticide derived from concentrated liquid garlic juice, a synthetic poly

Description

Field of the Invention The present invention relates to improved formulations for the delivery of a biopesticide, said biopesticide being primarily derived from garlic and synthetic analogues thereof. In addition, methods of producing and uses of said formulations are disclosed. Background to the Invention Given the continued increase in world population, the requirement to increase food output becomes more pressing. This leads usually to more intensive farming methods in which crop rotation and fallow agricultural land become used less and less, which in turn give rise to difficulties of a higher risk of infestation from pests which are adapted, especially in a parasitic relationship, to a particular crop. One crop with which the present disclosure is concerned, although not exclusively, is the potato. This can become infested with roundworms of the genus globodera, and which are commonly referred to as potato cyst nematodes (PCN). Serious infestations can result in up to 90% crop loss and the PCN is estimated to cause losses of around £40,000,000 in the UK alone. A number of methods and products can be employed, which are only partially successful in combating PCN. Firstly, as mentioned above crop rotation is used, although this normally means a gap of at least six years between plantings of a susceptible crop. Alternatively, catch crops can be used which aim to attract the pests and which are then subsequently, at the height of infestation, destroyed. This is quite an expensive method to carry out and is only relatively effective. Thirdly, chemical agents have been developed based, for example, around carbamates. As examples of these can be mentioned Aldicarb (marketed under the trade name “Temik”(RTM)) and Oxamyl (marketed under the trade name “Vydate”(RTM)). A number of problems attach to this form of control, however. There is evidence that the degradation time of the carbamate decreases with repeated short-time use, and hence the effectiveness is reduced. Secondly, legislation is increasingly restricting the use of pesticides and it is expected that the use of some will become illegal in the near future. There is therefore both a need and a desire for pest control products which are derived from or based on naturally occurring materials. One such set of products are those derived from garlic, such as garlic oil and also synthetic analogues of active compounds in garlic, such as the polysulphanes (also commonly referred to as polysulphides) disclosed in WO 08 / 59217. With these compounds, the main problems to be overcome are delivery of the active at the correct time; and that said delivery is sustainable. One means of addressing these criteria is to incorporate the active compounds within a granule. The compounds are then eluted from the granule by rainwater or applied water. However, in the case of some synthetic analogues the problem is encountered that compounds including a sulphur chain of four or more atoms are relatively water insoluble and so are not readily eluted. In addition, typically only 15% of product can be eluted with the remainder being relatively strongly held within the granule and unavailable for use. It is an object of the current invention to provide a biopesticide formulation and delivery means therefor which addresses the above problems. Summary of the Invention According to a first aspect there is provided a biopesticide emulsion system having a first continuous phase comprising water and a second discontinuous phase comprising a biopesticide derived from a concentrated liquid garlic juice, a synthetic polysulphane or mixture thereof, and including an emulsifying agent to retain the two phase system. The emulsion enables the pesticide to be more effectively and efficiently delivered. The diameters of the emulsion droplets of the discontinuous phase are preferably less than 2.4 pm, more preferably less than 2.0 pm, less than 1.5 pm, less than 1.3 pm or about 1 pm. These particular diameters provide increased mortality when employed against a target pest. The emulsifying agent is advantageously an esterified carbohydrate. Particularly advantageously, the carbohydrate is sorbitan. Especially advantageously, the sorbitan ester is polyethoxylated to increase the HLB value. A preferred emulsifying agent is Tween (RTM) 20 which produces, when using the above pesticides, emulsions having the desired droplet size. Tween 20 (RTM) is an established product used in biological applications with which the skilled reader would be familiar. It is a polyoxyethylene sorbitol ester that belongs to the polysorbate family. It is a nonionic detergent having a molecular weight of 1,225 daltons, assuming 20 ethylene oxide units, 1 sorbitol, and 1 lauric acid as the primary fatty acid. The ethylene oxide subunits are responsible for the hydrophilic nature of the surfactant, while the hydrocarbon chains provide the hydrophobic environment. Sorbitol forms the backbone ring to which the ethylene oxide polymers are attached. In an alternative embodiment, the emulsifying agent is a sucrose ester, such as sucrose stearic acid SP70. Preferably the sucrose ester has a hydrophilic-lipophilic balance (HLB) value of at least 15 or at least 16. The level of emulsifying agent present is conveniently less than 3% w / w, and especially conveniently less than 2%w / w of the emulsion system. The level is further conveniently greater than o.25%w / wto ensure good emulsion droplet size of the discontinuous phase. The percent w / w ratio of continuous to discontinuous phase is preferably greater than 20:1 and especially preferably greater than 30:1. According to a second aspect there is provided a granular pesticide comprising a granular matrix, said granular matrix retaining a biopesticide emulsion having a first continuous phase comprising water and a second discontinuous phase comprising a biopesticide derived from a concentrated liquid garlic juice, a synthetic polysulphane or mixture thereof, and including an emulsifying agent to retain the two phase system. The diameters of the emulsion droplets of the discontinuous phase are preferably less than 2.4 pm, less than 2.0 pm, less than 1.5 pm, less than 1.3 pm or about 1 pm. The emulsifying agent is advantageously an esterified carbohydrate. Particularly advantageously, the carbohydrate is sorbitan. Especially advantageously, the sorbitan ester is polyethoxylated to increase the HLB value. A preferred emulsifying agent is Tween 20 (RTM) which produces, when using the above pesticides, emulsions having the desired droplet size. The level of emulsifying agent present is conveniently less than 3% w / w, and especially conveniently less than 2%w / w of the emulsion system. The level is further conveniently greater than o.25%w / wto ensure good emulsion droplet size of the discontinuous phase. The percent w / w ratio of continuous to discontinuous phase is preferably greater than 20:1 and especially preferably greater than 30:1. The emulsion is preferably present at a ratio of o.5%w / w - i.o%w / w to the granular matrix. The granular matrix is advantageously selected from a diatomaceous earth or alternatively from a cellulosic material, especially advantageously a wood cellulose. According to a third aspect there is provided a method of manufacture of a granular biopesticide comprising the steps of: • producing an emulsion of a biopesticide in water; • selecting a granular matrix material and; • mixing said emulsion with the granular matrix material to absorb the emulsion within the matrix, wherein the biopesticide is derived from a concentrated liquid garlic juice, a synthetic polysulphane or mixture thereof. In some embodiments, production of the emulsion comprises a step of blending at a speed of at least 2000 rpm, at least 3000 rpm, at least 4000 rpm, at least 6000 rpm or at least 8000 rpm; or at about 3000 rpm, about 4000 rpm, about 6000 rpm or about 8000 rpm. The diameters of the emulsion droplets of the discontinuous phase are preferably less than 2.4 pm more preferably less than 2.0 pm, less than 1.5 pm, less than 1.3 pm or about 1 pm. According to a fourth aspect there is provided the use of a granular biopesticide composition, wherein the granular biopesticide composition includes a granular matrix and an emulsified biopesticide absorbed within said matrix wherein the biopesticide is derived from a concentrated liquid garlic juice, a synthetic polysulphane or mixture thereof. Brief Description of the Drawings The description is illustrated with respect to the drawings which illustrate results obtained is in the preparation and effectiveness of the disclosed invention. In the drawings: Figure i illustrates the relationship between droplet size of an emulsion and mortality rate; Figure 2 illustrates the effect of blending speed on the mortality rate of the produced emulsion; Figure 3 illustrates droplet diameter produced in a synthetic garlic oil emulsion system; Figure 3a illustrates effect of homogeniser speed on emulsions form from natural garlic using 1% Tween 20 (RTM); Figure 4 illustrates the effect of surfactant concentration on droplet size; Figure 5 illustrates the effect of emulsification speed on mortality rate using a synthetic garlic oil; Figures 6 and 7 illustrate the elution profile of granules prepared; Figure 8 illustrates mortality rate of nematodes using the granules of Figures 6 and 7; Figure 9 illustrates the mortality rate of elution fractions from the material illustrated in Figure 6. Figure 10 illustrates the concentration of sulphur-containing compounds in the elution cycle of a synthetic polysulphide-containing granule; and Figure 11 illustrates the elution pattern obtained from a soil sample containing granules. Detailed Description of the Invention The use of granules containing concentrated garlic oil as an active biopesticide is known in the art. In the soil, when applied to combat a perceived pest, the granules are able to release the biopesticide - in the case of garlic oil an active sulphur-containing compound - into the soil, which active compounds either kill, weaken or act as a deterrent towards the pest. The granules are most effective when exposed to water, either through natural rainfall or irrigation, which water acts to transport the active compounds from the granule into the soil. This is believed to be primarily through dissolution of the active compounds, although other mechanisms may be at work. However, a large proportion of active compounds can remain inside the granule, where it has almost no effect. Moreover, release can be so slow and low that the granules effectiveness is reduced. The present invention therefore acts to mobilise the active compounds through their emulsification, using water as the continuous phase prior to granulation. Moreover, it has also been found that the emulsified systems bring enhanced effectiveness of the biopesticide, even when the biopesticide is employed without granulation. General Preparation of an Emulsion An example of a method of producing the emulsions the following is provided. It will be recognised by the skilled person, however, that other methodologies for the preparation of an emulsion can be used without departing from the scope of the invention. For example, the emulsifier can be mixed with water followed by addition of the biopesticide. In addition, desired emulsion droplet size can be obtained using any suitable means, for example homogenization, or high shear mixing using, for example, a Silverson (RTM) mixer. A solution of an emulsifier was produced by dissolving the emulsifier in the biopesticide at 58°C, with stirring. Emulsions were then prepared by pouring the mixture into a small Waring cup and homogenising using a Waring blender. Atypical preparation cycle involves the following cycle: blend (low speed) 15s, rest 30s, blend (high speed) 15s, rest 30s, blend (high speed) 20s. In some embodiments, preparation of the emulsion involves at least one step of blending at a speed of least 2000 rpm, at least 3000 rpm, at least 4000 rpm, at least 6000 rpm or at least 8000 rpm; or at about 3000 rpm, about 4000 rpm, about 6000 rpm or about 8000 rpm. The finished product was stored in a sealed vial until required. In order to determine the particle size, approximately 0.5 ml was removed from each sample and diluted to 1.5 ml. The particle size distribution was then determined using a Beckmann Coulter LS13320 Laser Diffraction Particle Size Analyzer. The importance of the disperse phase within the emulsion formed can be clearly seen from Figure 1. The results shown were obtained from in vitro experiments on the mortality of the emulsion towards nematodes. In the experiments, test solutions (0.9ml) were dispensed into small tubes. Water (0.1ml) containing 2-300 nematodes was added to the tube to complete the test volume (i.oml). Tests were then run at room temperature at various time intervals: 50 pl aliquots of test solution were removed from the solution for inspection of the nematodes. Figure 1 shows emulsions formed from a concentrated garlic oil obtained from natural sources. The relationship of droplet size of the dispersed phase to its effect on mortality of the nematodes can be clearly seen in that smaller droplet size produces a higher rate of and absolute value for mortality. The effect of garlic oil itself, with no added emulsifier is also shown (columns second from right, marked T.2pm’). The background mortality rate, under the test conditions, without garlic oil present is shown in the final columns on the far right-hand side of the results. In respect of a synthetic garlic oil, in which the active ingredient is a mixture of diallyl polysulphanes having differing sulphur chain lengths, the effect of Tween (RTM) concentration is shown in Figure 2. Emulsions for data shown in column sets 3-7 of Figure 2 were prepared by blending at 8000 rpm. The bioactivity of systems containing 0.5 to 2.0 % w / w Tween 20 (RTM) can be seen to be greater than when the system contains 0.1 % w / w Tween 20 (RTM), and all are improved over an emulsion created using natural garlic oil (columns marked ‘clail’). Figure 2 also contains data from two samples of emulsions of synthetic garlic oil prepared using 1% Tween 20 (RTM) as surfactant: the first using a homogeniser speed of 8000 rpm (syn8ooo) (column 1 in Figure 2) and the second a speed of 3000 rpm (synsooo) (column 2 in Figure 2). The size distribution of the emulsion produced using a synthetic oil is more varied than that produced using natural garlic oil. Figure 3 demonstrates this point for the synsooo and syn8ooo emulsions prepared as discussed above. The droplet size of the disperse phase was measured and the results are shown in Figure 3. As can be seen, the droplet size at 3000 rpm is far greater than that produced using 8000 rpm. This contrasts with the emulsions formed from natural garlic oil which showed little effect of homogeniser speed when using 1% Tween 20 (RTM), as shown in Table 1 below, and in Figure 3a. Without being bound by theory, this may be caused by the natural garlic oil including compounds (such as carbohydrates) which can act as surfactants in their own rights. Table 1: Emulsion formed from natural garlic oil using 1% Tween 20 (RTM) Homogeniser speed 2000 Mean Droplet Diameter (pm) 1-25 4000 8ooo 1.24 1.32 Moreover, Tween 20 (RTM) is effective at producing an emulsion over a range of concentrations. Figure 4 illustrates droplet size distribution for emulsions prepared using 0.1 % w / w - 2% w / w Tween 20 (RTM). Figure 4 illustrates that from 0.5 to 2.0 % w / w Tween (RTM), the droplet size distribution is relatively constant. There is, however a shift in the position of the maximum peak and particularly when 0.1% w / w Tween 20 (RTM) is used, although the mean droplet diameter actually decreases. In producing the emulsion, the hydrophile / lipophile balance (HLB) of the surfactant needs to be borne in mind. In general, more lipophilic surfactants, having a low HLB value, tend to be more efficient at producing water in oil emulsions whereas more hydrophilic surfactants tend to produce oil in water emulsions. The importance of the physical structure of the active compound is again shown in Figure 5. A 3% w / w synthetic garlic oil was emulsified using a sucrose stearic acid SP70 (obtained from S. Black limited), and having an HLB value of 15, as surfactant and a range of mixing rates. It is to be expected that other sucrose esters, including mono-, di- and tri-esters would also be applicable as emulsifiers and this would be apparent to the skilled person. In addition, other fatty acids such as lauric, myristic and palmitic, again well known in the art, can be contemplated. The resultant emulsions were then tested as nematicides. An emulsion having processed natural garlic oil (also referred to herein as clail or clailo2i) was also utilised for comparison (CL00021). Results are shown in Figure 5. At lower mixing speeds, the bioassays show that the effectiveness of the active against nematodes was relatively local. Above 4000 rpm, however, the effectiveness became almost 100% after 24 hours showing a stepped improvement. Given the effects shown above, in relation to emulsification conditions and droplet size it is likely that an effective size of droplet was produced by the higher rate mixing. The emulsions produced herein can be incorporated into solid formulations, such as granules, which provides advantages over purely liquid spray systems. For example, unlike liquid spray systems, granules themselves are not washed away with water and remain active within the soil over a long period of time. Secondly, the granule composition can be tailored to provide a release rate suitable for the particular active ingredient and target pest. It has been surprisingly found that emulsions described above retain their activity even when incorporated into a granule. Moreover, the provision of the active compounds enhances the performance of granules containing an equivalent amount of non-emulsified active. In summary, an emulsion in accordance with the description above is prepared. The emulsion is then mixed into the carrier matrix of the granule by sequential addition of the emulsion to the carrier matrix in a coating pan until the desired volume of emulsion has been added and a well formed pellet results. The ratio of emulsion to carrier matrix varies with the particular carrier matrix used. For example, where wood flour is the carrier matrix then the %w / w mixture ratio can be 45:55 emulsion:wood flour. For a diatomaceous earth carrier, then a ratio of 25:75 is typical. A typical granule matrix is wood flour having a granule size of from i.5-2.5mm. However the carrier matrix can be selected from wood flour, diatomaceous earth or Biodac (RTM), a cellulosic material (47-53%) containing predominantly calcium carbonate (14-20%) and kaolin clay (28-34%), along with titanium dioxide (less than 1%). The incorporation of garlic oil into granules is known but the percentage of active ingredient released by water flow is usually no higher than 15% of the total amount contained in the granule even after repeated elutions. Again, without being bound by theory, it is likely that the polysulphides remain in the granule, which tend to be those of higher sulphur chain length, are quite strongly bound within the granule and are also relatively insoluble in water. An object of including emulsions of the active is therefore to improve the overall percentage release. In general, results achieved show a 10 to 20-fold increase in release of active polysulphide from granules prepared from emulsions. This is exemplified in the results below in Figures 6 and 7. As elsewhere DAS in the specification stands for diallylsulphane and the numeral designates the S chain length within the molecule. In these experiments, the release profile of polysulphide from granules was explored. Granules were compared and water passed repeatedly therethrough. In the experiment the granules were mixed in equal weight proportions with Celite 545 (RTM) (a silica carrier made from diatomaceous earth calcined with flux material) and the mixture loaded into a syringe barrel. The lower end of the syringe barrel has a layer of glass wool to precent granules falling through the hub of the syringe. Once the mixture is loaded further glass wool is inserted over this. Water in discrete 15ml aliquots is eluted through the barrel (test cell) and the fractions collected for bioassay. The first aliquot is not usually recovered due to absorption. The eluted water was analysed by HPLC to determine the relative amount and type of polysulphide released into the water. The granules employed in Figure 6 were a single core wood cellulose granule, mixed with a processed garlic oil (prepared from whole plant extract) emulsified into the disperse phase, with water as the continuous phase. The emulsifying agent was 2% Tween 20 (RTM): the Tween 20 (RTM) being added to the garlic oil prior to emulsification. The granules employed in Figure 7 utilise a liquid matrix comprising 25% w / w sucrose and 5% w / w polysulphide. These granules were included in order to investigate the effect of using a non-esterified carbohydrate. Comparison of Figures 6 and 7 shows a tenfold greater release of polysulphide from the granules formed using the emulsion. These results are reflected in the nematicidal effects of the eluted fraction taken from cycle 2 of each granule type above, as shown in Figure 8. In these experiments, bioassays were carried out whereby 0.9ml of eluted material from cycle 2, was transferred to an Epindorf (RTM) tube. Water (toopl), containing 250 to 350 fresh nematodes, was then added to the Epindorf (RTM) tube which was then closed and gently mixed. Immediately, and at hourly intervals thereafter, 50 pl aliquots were removed and tested to determine the mortality of the nematodes. A control using water was also included. The results presented in Figure 8 show mortality rate versus time. The granules prepared using a polysulphide emulsion (B28) can be seen to have a vastly increased effect on the mortality of the nematodes, presumably related to the greater release. As further evidence of the efficacy of the eluted material from the granules prepared from emulsified material, the nematicidal effects of the first five elutes (as detailed in Figure 6) are shown in Figure 9. In this experiment the eluted material was in contact with the nematodes for 18 hours. In a further experiment, granules were prepared using a synthetic polysulphide oil emulsified in water using a sucrose ester (SP70) at a level of 3% weight for weight and a level of 4% weight for weight polysulphide oil. The elution patterns for this compound are shown in Figure 10. The efficacy of a granular carrier for emulsified active in soil was also determined. An emulsion was formed using moderate sheer of garlic oil concentrate in water using 1% w / w Tween 20 (RTM) as added surfactant. The emulsion thereby produced was sprayed into the granule build process to produce the carrier. Granules produced were mixed with soil and eluted once per day over a fifteen day period. The sequential fractions were collected and analysed for polysulfide concentration. The elution profile is shown in Figure 11. As can be seen, almost all of the polysulfide released appears by the end of day 4. In vitro tests with this granule through Celite™545 indicated high direct nematicidal on vermiform Nematodes (J2s). It should be noted that the concentrations of polysulfides at cycles 2 to 4 are tenfold more than seen for granules of a commercially available granule containing concentrated garlic oil, which has been shown to have demonstrable activity in soil. A field trial was carried out on behalf of the applicant by ORETO (Official Recognition of Efficacy Testing Organisations and facilities) certified contract trials agents. The trial was carried out in Lincolnshire, in a typical silt soil. An emulsion according to the present disclosure was made using natural garlic extract. In addition, an emulsion was made according to the present disclosure comprising a synthetic formulation with the same gravimetric polysulfide oil composition as the natural garlic oil product 12L of the natural garlic extract. This product was based around water, polysorbate 20 and polysulfide oil mixed to an emulsion. The treatments were applied, over a comparable area in each case, to a field with potato crop nematode present, as follows: - Control (no application); - Device control (injector with water); - 6L / ha dose of natural garlic emulsion delivered via injector; - i2L / ha dose of natural garlic emulsion delivered via injector; - 24L / ha dose of natural garlic emulsion delivered via injector; - i2L / ha dose of synthetic emulsion delivered via injector; - Nemathorin® (Syngenta), a commercially available registered nematicide containing fosthiazate for the control of potato cyst nematode, was applied at the recommended dose. Results Gross yield recovered from the field; and mean final PCN population (determined as eggs / gram of soil) is shown in Table 2, below. Table 2 Mean Final PCN Population Yield Untreated 594-76 57-61 Device Control 297-59 55-4 6L natural garlic emulsion 307.02 55-91 12L natural garlic emulsion 293-5 54-14 24L natural garlic emulsion 180.7 58.21 12L Synthetic emulsion 183.56 58.09 Nemathorin® 67-99 56.44 These results demonstrate the effectiveness of emulsions according to the present disclosure. The application of 24L of natural emulsion and 12L of synthetic emulsion increased yield, by a comparable amount, relative to the untreated control, the device control and the commercially available nematicide, Nemathorin®. In addition, the application of 24L of natural emulsion and 12L of synthetic emulsion decreased final PCN population, by a comparable amount, relative to the untreated control and the device control, thus demonstrating a nematicidial effect. These results indicate that on a dose to dose comparison, the synthetic emulsion may demonstrate greater efficacy than the natural product. Various modifications to the embodiments of the present invention described herein will be readily apparent to those skilled in the art and such modifications are included within the scope as defined in the appended claims.

Claims

1. A biopesticide emulsion system having a first continuous phase comprising water and a second discontinuous phase comprising a biopesticide derived from a concentrated liquid garlic juice, a mixture of synthetic diallylpolysulphanes having different sulphur chain lengths, or mixture thereof, and including polysorbate 20 to retain the two phase systemwherein the mean diameter of the emulsion droplets of the discontinuous phase is less than 2.4 pm; andwherein the polysorbate 20 is present at 0.5% w / w to less than 2.0% w / w.

2. An emulsion system according to claim 1, wherein the mean diameter is less than 2.0 pm, less than 1.5 pm, less than 1.3 pm or about 1 pm.

3. An emulsion system according to any preceding claim, wherein the percent w / w ratio of continuous to discontinuous phase is greater than 20:1, optionally, wherein the ratio is greater than 30:1.

4. A granular pesticide comprising a granular matrix, said granular matrix retaining a biopesticide emulsion having a first continuous phase comprising water and a second discontinuous phase comprising a biopesticide derived from a concentrated liquid garlic juice, a mixture of synthetic diallylpolysulphanes having different sulphur chain lengths, or mixture thereof, and including polysorbate 20 to retain the two phase system;wherein the mean diameter of the emulsion droplets in the discontinuous phase is less than 2.4 pm; andwherein the polysorbate 20 is present at 0.5% w / w to less than 2.0% w / w.

5. A granular pesticide according to claim 4, wherein the mean diameter is less than 2.0 pm, less than 1.5 pm, less than 1.3 pm or about 1 pm.

6. A granular pesticide according to claim 4 or 5, wherein the percent w / w ratio of continuous to discontinuous phase of the emulsion is greater than 20:1, or is greater than 30:1.7- A granular pesticide according to any of claims 4-6, wherein the emulsion ispresent at a ratio from 0.5 - 1.0 % w / wto the granular matrix.

8. A granular pesticide according to any of claims 4-7, wherein the granular matrix5 is selected from a diatomaceous earth or a cellulosic material preferably, wherein the granular matrix is a wood cellulose.

9. A method of manufacture of a granular biopesticide comprising the steps of: •producing a biopesticide emulsion system according to any one of claims 1-3;10 ‘selecting a granular matrix material and;• mixing said emulsion with the granular matrix material to absorb the emulsion within the matrix.

10. The method of claim 9, wherein production of the emulsion comprises a step of 15 blending at a speed of at least 2000 rpm, at least 3000 rpm, at least 4000 rpm, atleast 6000 rpm or at least 8000 rpm; or at a speed of about 3000 rpm, about 4000 rpm, about 6000 rpm or about 8000 rpm.

11. Use of a granular biopesticide composition according to any one of claims 4-8..

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