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1. (WO1997016972) PESTICIDAL COMPOSITION AND PROCESS FOR PREPARATION THEREOF
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Pesticidal composition and process for preparation thereof

The invention relates to a pesticidal composition containing pesticidal components preferable for use in an aqueous environment. The composition comprises a pesticidal component derivable from a microorganism, a solid fat, a disintegrant , and optionally a detergent. The invention also relates to a process for the preparation of a pesticidal composition and to a method of controlling or combating insects by use of the composition. The invention also relates to a composition in the form of a controlled release composition.

Background of the invention

A number of different insects cause substantial problems as vectors and transmitters of infectious diseases affecting both humans as well as animals and as major pests in agricul-ture and forestry, and tremendous efforts are invested in controlling and combatting these insects. Efforts have been concentrated on controlling insects belonging to the order Diptera, including Brachycera and Nematocera covering black flies and mosguitos, the transmitter of e.g. malaria. Previ-ous methods of combatting and controlling insects have been through the use of chemical insecticides whereas more recent approaches taking environmental protection into consideration when applying insecticides have focus on the use of biologically derived insecticides specifically targeting the insects in question.

Included in this approach has been the use of bacteria of the Bacillus type which produce a substance which is toxic to mosquito larvae.

Accordingly, microbial pesticides have shown to be very effective in the control of the larvae of mosquitoes and blackflies (L. Lacey and Undeen, 1986). Microorganisms, such as bacteria and fungi (Closϋridium Jbi-ermenta-αs, I Thiery, 1992) , have been used experimentally, and with regard to bacteria also commercially, in the control of mosquitos.
Bacteria have been used in programs as the only mosquito control measure (Becker, N.F. Rettich, 1994) or in combination with chemical pesticides, for larvicidal or adulticidal effect.

Among the current formulations of pesticides containing microorganisms, including bacteria, most products offer only a short term effect. Such formulations include concentrates, flowables, soluble powders and primary powders mixed with sand and oil by the user. Commercial products with sustained release also exist on the market such as large, pressurized briquettes which have a deferred, but very local effect.
Recently, a granule product incorporating sand and Bti in a matrix has been launched commercially.

There are several patents describing slow release pesticide products which require a rather high technology to produce. With a late exemption, none of these have appeared on the market due to the fact that in general, reapplication is a more economic solution rather than the use of expensive formulations. The release mechanisms described in these patents are based on elements such as polymers, micro-encapsulation etc., e.g. as disclosed in WO 94/16561.

US 5 484 600 describes a composite granule based on a heavy-article such as sand as a sinking agent and a light agent such as cork powder as a floating agent. Fat is not mentioned as a floating agent. The commercial product based on this patent contains a low concentration of B. thuringiensis var israelensis (Bti) and is an alternative to the traditional sand granule (sand, oil and Bti primary powder) . As for this simple granule, the weight of the sand will bring the granule to pass through foliage and to the bottom of the water below. Once in the water, the adhesive agent is dissolved and cork particles with the bacteria float to the surface. At the surface, these particles are further disintegrated by the dis-solution of the adhesive agent. The sustain release mechanism is based on the dissolution of this adhesive agent. The equipment for the production method is expensive (micro-encapsulating with a prill technique) , but once this investment is passed, the ingredients used are quite cheap. It is stressed that the adhesive agent must be a water-soluble or water-sensitive adhesive agent. This excludes fat as the adhesive agent.

US 5 283 060 is an example of a patent relating to a slow release mechanism and describes a water floatable granular form of a pesticide (such as Bacillus) with a core of wax on which a biological pesticide in a matrix of proteinaceous adhesive material is secured. The system is a non-homogenous system where each particle is a two- component particle consisting of the wax core with or without a base material as a floating agent and an outer layer containing the biological pesticide in the matrix. The matrix containing the biological pesticides may be water dispersive, water soluble and water insoluble. Accordingly, the release of the biological pesticides from the granules takes place either by wetting of the water dispersive particle portion of the surface of the granule and the resulting release into the water or - in the case of a water dispersive or water soluble matrix - via release of the matrix system from the wax core. It is mentioned that an important factor is that the matrix material is capable of providing an inert and stable medium and does not comprise any fat or wax.

US 5 326 560 discloses a composition comprising an insecticide such as Bacillus thuringiensis , petrolatum and
diatomaceous earth. The composition may further comprise a crop oil, an emulsifier to vary overall viscosity and to obtain any desired dilution ratio. However, the compound is considered most effective when free of emulsifiers, since the purpose of the compound is to adhere on plant surfaces and avoid wash off.

More simple, slow release formulations based on the attachment of the pesticide on grounded corncob and slow release therefrom are known, including "Bactimos Granules". However, these granules are very light and can not be applied from e.g. a helicopter as the granules are easily carried off the target by the wind.

Accordingly, only US 5 484 600 provides a composition which is heavy enough to be applied from e.g. a helicopter and -after decomposition - to stay in the feeding zone for a sufficient time so that the pesticide component is available for the insects in an active form.

The formulation disclosed in US 5 484 600 is different from the present formulation in that it requires (expensive) equipment for micro-encapsulation, uses the dissolution of an adhesive as the basis for the slow release mechanism, and does not give protection to the released particles with spores of the Bacillus toward germination and hostile microorganisms in the environment. In long standing water in nature, it is therefore doubtful that the bacteria and their toxin will remain at a toxic level for a long time.

In addition, most of other current formulations containing microorganisms, including bacteria, offer only short term effect. This also includes formulations which comprise Bacil lus sphaericus (Bs) which by nature often has a long term effect in clean water. Accordingly, it has been shown that some slow release formulations used commercially for Bti do not provide extra residual effect when formulated with Bs as compared to unformulated Bs ( acey, 198) . On the contrary, it has been found that in sewage water and other water types with organic content, the effect of Bs formulations is often short and this can only be compensated for with very high dosages (Hougard et al, 1992) .

Summary of the present invention

Many insects living in or closely related to water, in particular insect larvae, feed at or near the surface of the water. This implies that a pesticidal composition aimed at controlling or combating such insects must be supplied in that region and/or must be capable of floating and releasing pesticidal components at or near the surface of the water where the insects search for food, thereby rendering inges-tion of the pesticidal component by the insects possible. The pesticidal composition therefore must exhibit a suitable degree of buoyancy and dispersibilitity together with presenting the pesticide in an active form to the insect or insect larvae.

The present invention relates to such a pesticidal compo-sition of increased persistence and stability with ingredients and production methods that will not increase the price of the product to a non-commercial level even if just a minor production is established. Release of the active pesticidal component is slow, and the density of the composition is sufficiently high to allow the composition to be dispersed from e.g. , a helicopter. At the same time the composition is water floatable, preventing the pesticidal component from disappearing on the bottom of the aqueous environment.

Other ingredients may be added to the pesticidal composition according to the invention, e.g. a UV- filter or a UV- reflecting material which protects the product against UV-degrada-tion. Bitter, non-toxic ingredients may be added to prevent the product from being eaten by birds, small inammals or non-target insects in the water or on the ground in situations where the composition is applied on the ground before an expected flooding of the treated area results in an aqueous environment.

The invention also relates to the process for preparing a composition according to the present invention. This process may of course include large scale production using high industrial technology, but an interesting aspect of the invention is that, in a tropical country, the composition may be formulated by the process using for example a dried powder form of the pesticide component which is more stable to store under tropical conditions.

Air may be entrapped during any stage of the process, such as in a drying step, to reduce the specific gravity of the final product. It is characteristic for the pesticide composition that the fat phase (eventually with air entrapped) carries the insecticide phase near to the water surface where it floats constantly or periodically or moves up and down in the water due to a constant change of gravity of the composition.

It has now surprisingly been found that spores of Bs may germinate fast in sewage water during with process the toxic parasporal body disappears .The germination may take place among free spores applied to the water from a fluid concentrate of Bs or from spray dried particles when agents wetting the spores are added to the formulation. Spores may be reformed, but then without toxin and they are therefore not toxic to the larvae. This is illustrated in Tables 1-3.

Further, it has been shown that toxicity is lost quickly from dead (radiated) spores in sewage water, but not from dead spores in tap water or sterile filtered sewage water
(Table 2) .

Accordingly, a very interesting aspect of the invention relates to the prevention of spore germination and protection of the spore against a hostile environment in a composition which will prolong the effect of a Bs based product. This prevention may include encapsulation, e.g. resulting from embedding the active substance in relative hydrophobic components in the composition.

Detailed description of the invention

The pesticidal composition according to the invention relates to a pesticidal composition comprising an insecticide phase which comprises one or more ingestible pesticidal components effective against insects of the order diptera in an aqueous environment and being derivable from a microorganism, the insecticide phase being dispersed in a fatty phase comprising a lipid or a mixture of lipids which is solid at ambient temperature, the composition further comprising at least one disintegrant, and optionally a detergent.

The amount of fat in the fatty phase is preferably such that it renders it possible for the product to float either permanently or periodically as balanced by the other ingredients which are dissolved from the matrix of the formulation. These other ingredients are the pesticidal component, the disinte-grant to disintegrate the composition in smaller parts, and optionally the detergent. A UV-protecting agent, anti-feeding agents to prevent the product from being eaten by non-target organisms, a filler to change gravity of the composition, and an anti-oxidizing agent may also be included in the composition.

In the present context the term "pesticidal component" means any component having a pesticidal effect and being active in controlling or combating insects such as water living
insects.

According to the present invention, the pesticidal component can be a biological pesticide or a chemical component derivable from a microorganism, or any combination thereof. The chemical pesticide can be synthesized chemically or derived directly from a biological microorganism producing the component. The term chemical component should encompass components which have been modified, including chemical derivatives of the pesticide. In a further aspect, any other pesticides could be mixed with the pesticidal component or added to the composition.

The pesticidal component may include particles of the pesticide with or without an attachment to or encapsulation in a small amount of the original microorganism.

Examples of a chemical pesticidal component are a larvicide as diflubenzuron urea and the hormone mimic methoprene.

Preferred pesticidal components for use in the present invention are biological pesticide components. Examples of biolo-gical pesticidal components are biological components such as microorganisms, e.g. a bacterium, virus and fungi or a part derived therefrom. A number of such biological components are already proved in the field such as those based on the bacteria Bacillus thuringiensis , Bacillus thuringiensis var israelensis (H14) and Bacillus sphaericus, including suitable serotypes (especially for Bacillus thuringiensis serotype 8a, 8b, 10, 14, 30, and the toxins characterised as CryIV (A, B, C or D) , Cyt A, Cyt B whether contained in the microorganism, purified and/or genetically transferred to other Bacillus thuringiensis strains or to other microorganisms.

One very important aspect of the invention therefore relates to a pesticidal composition containing bacteria of the type Bacillus, in particular Bacillus sphaericus or Bacillus thuringiensis var israelensis, var morrisoni or var medellin or from the anaerobic bacterium Clostridium bifermentans and/or the toxin derived from the bacteria. Accordingly, the pesticidal composition may comprise a microbial organism as the active ingredient in which the microbe is toxic, comprises a toxic component (toxin) , a parasporial body with toxic components, or mixtures thereof.

The bacteria may be of a naturally occurring type but interesting aspects of the invention relate to the use of
bacteria, in particular of the Bacillus type, in which the pesticidal activity of e.g. the toxin has been improved as compared to the natural type. Such improvement may be performed by selective breeding or by genetic engineering, including transfer of genes from one microorganism to the other. Genetic engineering may be performed by inserting one or more copies of the gene encoding the toxin into the genome of the bacteria using e.g. methods generally known in the field of genetic engineering.

The gene to be inserted may be derived directly from a bacte-rium of the bacillus type, in particular Bacillus sphaericus or Bacillus thuringiensis var israelensis, var morrisoni or var medellin or from the Clostridium bifermentans , or the gene may be modified so as to improve the pesticidal activity of the toxin produced by the gene.

In another interesting aspect of the invention, a microorganism which does not by nature carry a gene encoding a toxin with pesticidal effect is modified by providing the microorganism with one or more genes encoding the toxin. Such modification may be performed using well known techniques for transferring genes. Suitable microorganisms include algae such as blue-green algae (Cyanophyta) .

Of specific interest in connection with genetic engineering is with respect to Bacillus sphaericus the DNA homology group IIA, or their toxins purified or genetically transferred to other microorganisms.

Thus, one embodiment of the invention is directed to a composition wherein the microorganism from which the pesticide is derivable is genetically engineered with respect to expression and/or production of the pesticidal component resulting in increased activity or increased amount of the pesticidal component as compared to the non-engineered microorganism.

In a further embodiment, the microorganism expresses or produces the pesticidal component after ingestion of the compo- sition by the insects. This may occur by the contact to a trigger mechanism such as a specific pH range or enzymes within the insect.

By water living insects is meant any insect living in the water, independent of the stage of the life- cycle of the insect. Insects of the order Diptera selected from brachycera and nematocera, such as culicidae are within the scope of insects which can be treated by the composition according to the present invention.

Water living insects are most commonly in the larval stage, accordingly, larvae of mosquitoes, midges and blackflies, may, in particular, be controlled by the composition according to the invention.

By the term "aqueous environment" is meant an environment providing humidity to such an extent that it is sufficient for the disintegrant to exert its effect and disintegrate the composition by swelling, e.g. by uptake of water. However, more or less dry areas, wherein rain or flooding is expected to occur, are also included in the term as long as the compo-sition exhibits its effect in a aqueous environment as defined.

As used herein, the term "fat phase" refers to one or more of the following components (including mixtures thereof) : a fat (including triglycerides) , an oil, a wax or a waxy detergent and a fat soluble detergent. Also, an oil soluble UV- filter may be included in the fat phase.

As used herein, the term "insecticide phase" refers to the pesticide component or a mixture of the pesticide component, a detergent, and optionally one or more fillers. The pesti-cide component, in particular those derived from microorganisms or fungi, may be in a powder form such as a dried powder form which is more stable when storing under tropical conditions .

As used herein, the term "percentage by weight" refers to percentage of total composition of fat phase and insecticide phase.

The pesticidal composition of the present invention may be in the form of a granule or a powder. Granules are made from the matrix by cutting, crushing, milling or homogenising a solid formulation or by an extrusion process in which the premixed separate phases are mixed, homogenized and extruded as pellets or granules in one, continuous process. A solid formula-tion may be obtained by immersing a cooled cylinder in the melted formulation whereby a layer of the formulation solidify on the cylinder. The solidified layer is peeled off from the cylinder. The temperature difference between the cylinder and the melted formulation, the contact period and the soli-difying temperature of the formulation affect the thickness of the solidified layer on the cylinder. A preferred thickness of the layer is about 2 mm.

Grinding the granules or sieving the ground matrix after crunching and using the fine particle fraction provides a powder that can be used as a wettable powder which is able to exhibit the slow release properties.

In this invention, the effect of the disintegrant is
optimally combined with the effect of a detergent that cannot dissolve or emulsify the fat components into water. Contact with water disintegrates the product into minor parts from which the other ingredients are released by the disintegrant. The detergent thus functions primarily to spread the granules and subparts of granules on the surface of the water by repulsion. As a final result of this process, the active component is released in small particles comprising fat in a size small enough to be ingested or caught and macerated by the insect such as the insect larvae. As will appear from the following, the fat of these small particles will protect the active ingredient from degradation.

The mechanically treated matrix may be fractionated according to particle size or processes may be used to obtain different particle sizes.

As used herein, a granule is a dry fraction with a particle size of 0.5 mm or greater, a preferred range is about 0.5 to 2 mm.

A powder form is produced be grinding the granules or sieving the ground matrix after crunching and using the fine particle fraction. A powder according to the invention is a dry frac-tion with a particle size less than 0.5 mm. Milling or similar mechanical treatment will result in a wide range of particle size. These particles can be separated with simple equipment such as a series of sieves, shaking sieves or by air centrifugation.

A typical amount of fat phase is 15-99.999 percentage by weight (compared to the total dry weight of the composition of fat phase and insecticide phase) . A preferred amount of fat phase is 25 to 90 percentage by weight. Suitable ingredients of the fat phase are waxes, oils and detergents where the combined gravity of those and optionally air entrapped are less than 1.0 g/cubic centimetre. The wax has to be a hard wax, lipophilic and preferably cheap. Preferred wax is a combination of hydrogenated ox- tallow and petrolatum. The fat or mixture of fat should be solid at the ambient temperature corresponding to the area of use and storage, such as a melting point of 45°C or higher.

A typical amount of insecticide phase is 0.001-85 percentage by weight (compared to the total composition of fat phase and insecticide phase) . A preferred amount of insecticide phase is 0.1-75 percentage. The pesticidal component is of course a part of this phase. Other optional and suitable ingredients are water soluble detergents, inert fillers, left over components of the fermentation process from the production of the microbial insecticide, and a disintegrant. Suitable disinte- grants are such that increase volume by uptake of water like methyl and ethyl celluloses, starch, gelatine powder, guar gum. The most preferred disintegrants are modified celluloses used at a concentration between 5 and 50% of the final compo-sition, preferred range 5 to 30 percentage by weight, or a combination of modified cellulose and starch at the same concentration ranges.

The preferred type of derived cellulose is a methyl cellulose (such as Sigma Methyl cellulose or Metolose from ShinEtsu) with a viscosity at 2% aqueous solution at 25 °C of between 500 and 10,000 centipoise (cps), preferably 1000 to 4000 cps and most preferably 12000 to 2000 cps (Table 5) . This range may also be obtained by mixing different lots of methyl cellulose to obtain a mean viscosity in the preferred range. Various types of methyl cellulose may have different cohesion effect, despite having the same viscosity classification, and the range is therefore product dependent. However, the person skilled in the art will be able to prepare a suitable mixture having the desired viscosity.

The detergents used generally serve two purposes: to distribute the granules on the water surface and to facilitate the disintegration of the active ingredient from the wax-disintegrant (eg. wax/cellulose) matrix of the granule. The latter detergent may be omitted if a slightly hydrophilic wax is mixed into the fat phase. Ionic and non- ionic detergents may be used including ampholytic detergents. A preferred combination is a powder form of a non- ionic or anionic detergent to be premixed with the fat phase and melted herein and a anionic or non- ionic to be mixed as a powder with the insec-ticide phase or as a flowable or powder to be mixed with the mixture of fat phase and insecticide phase. The most preferred is sodium salt of a polycarboxylic acid to be mixed in the fat phase and monoethanolamine laurylsulphate to be added during mixing of the two phases. A typical amount of deter-gents is 0.1-10 percentage by weight. The most preferred range is 3-5 %. It is contemplated that a detergent in the insecticide phase helps this phase to be mixed with the fat phase. A detergent of the fat phase also enables this mixing, but the detergent also enables the final release of the active component from the matrix when in contact with water. However, when germination should be avoided, the detergents are selected so that the active ingredient is protected, such as encapsulation in the fatty components.

The insecticide phase and the fatty phase may both comprise detergents selected to optimise spreading of granules in water after application and slow dissolution of the fat phase after dispersion caused by the disintegrant.

In one embodiment, the fat phase comprises a lipophilic solid (wax) , a non- ionic or ionic detergent, the insecticide phase may beside the microbial insecticide comprise a detergent, and the final formulation contains one or more disintegrants . The detergents are selected in such a way that their combined effect does not provoke spore germination or wetting of toxin components that are inactivated by contact with the water or microorganisms in the water. In this situation, the role of the detergents are primarily to spread the granules on the water surface .

In a further embodiment, the fat phase comprises a lipophilic solid (wax) , a non- ionic detergent, a liquid detergent and a small amount of a linear primary alcohol ethoxylate (non-ionic detergent) which is added to increase the fat solubility, the insecticide phase comprises an anionic detergent and soluble starch as the disintegrant. The final formulation is a very cheap formulation.

In an interesting embodiment, the pesticidal composition com-prises a spore-toxin complex which is prevented from germination either by adding inhibitive agents or by preventing the contact between the spore and the water e.g. by a waxy encapsulation preventing wetting of the complex. Accordingly, the invention also relates to formulations where the active com- ponent is entrapped in a water protecting phase as e.g. wax and released from this phase in small particles without wetting agents. The release mechanism is then based on the disintegrant and should contain detergents that will not wet the spores or particles with other spores and toxin components.

In addition, to obtain a long residual effect of a pesticidal composition comprising a spore-toxin complex, the toxin should also be protected against the influence of other microorganisms or their metabolites or enzymes. Accordingly, an interesting aspect of the present invention is to provide a formulation wherein the spore-toxin complex is embedded in a fatty component which in addition to preventing the spores from germination also protects the complex from degradation due to the presence of the above mentioned factors in the aqueous environment.

Depending on the use of the final product, a UV- filter may be added to prevent or reduce the inactivation of the sunlight of the product. Such UV- filters may be para-aminobenzoic acid (PABA) and the like, micronized metal salts such as microni-zed zinc white. It has been shown (Pozsgay et al . , 1987) that the UV- inactivation of the toxins of Bacillus thuringiensis is indirect via an electron transport from other molecules reacting with the sunlight. Antioxidants can therefore also be used for UV protection and for the prevention of oxidation of the fat phase.

Mineral and inorganic fillers can be silica based, alumina or carbonates, such as bentonite, attapulgite, pyrophyllite, talc, kaolin, diatomaceous earth, synthetic silicas, celite, vermiculite, ground silica or sand, clay, chalk, calcium carbonate, starch, modified starch, flours.

The compositions of the invention may contain other additives. Such additives include fragrances for the attractant of the insect larvae and fragrances for the repellency or antifeeding of non- arget organisms. An antifeeding agent may be "Bitrex", most commonly used in household cleaning agents to prevent babies or dogs ingesting the product. It is included in the composition of the invention to prevent birds insects or small mammals from eating or carrying away the product when applied on the earth before flooding or on the water.

The present invention also relates to the process of processing the pesticidal composition comprising melting the fat phase, which should have a combined solidifying point above the use and storage temperature, e.g. above 35 °C. The insecticide phase is mixed separately and then added to the fat phase. This combination process may be as simple as a mechanical mixing of the two phases, but it may also be carried out in a process whereby the fat phase more or less encapsu-lates the insectieidal phase. To entrap air, the composition is homogenized at high speed at a temperature slightly (e.g., 10'C) above setting point for the mixture with a suitable propeller for air- trapping. Air encapsulating may also be obtained when producing the insectieidal powder in a drying process, e.g., by adding slightly hydrophilic wax to the heated bacterial sludge before spray drying. The melting point of the wax must be below the inlet temperature of the spraydryer to obtain a liquid form before cooling down in the spraying tower.

In a further embodiment the invention relates to a process for the preparation of a pesticidal composition described above comprising:

i) dispersing an insecticide phase optionally comprising a detergent and/or a disintegrant in a fatty phase which op-tionally comprises a detergent and/or a disintegrant, provided that at least one of the phases comprises at least one disintegrant, or provided that at least one disintegrant are added during or after dispersing the insecticide phase in the fatty phase;

ii) subjecting the dispersion of i) to a treatment for obtaining a granular or a powder composition,
the method optionally comprising
iii) entrapping air into any of the fatty and insecticide phases and/or into any of the compositions obtained by i) and ii) .

In one embodiment, a detergent is added to the insecticide phase and/or the fatty phase or alternatively, is added after dispersing the insecticide phase in the fatty phase

The detergents involved may be selected thus to avoid or to prevent spore germination when the formulation is subjected to disintegration in the aqueous environment.

A simple test for selecting a suitable detergent when protection of spore germination is desired, is mixing the detergent with the relevant fat component, e.g. a wax to a fat phase. The fat phase is then added to a bottle of water having a temperature of 50°C. The bottle is then shaken for a time sufficient to disperse the fat phase in the water. If the liquid of the bottle hereafter has a clear and transparent appearance, the fat phase is suitable for preventing germination. If the appearance of the liquid is turbid and whitish (milky) , the detergent has solubilized the fat to such a degree that wetting of the spores can be expected.

It is believed that detergents having a HLB value of above 7 such as above 9 or above 12 is suitable detergents. In general, detergents being more hydrophillic than lipophilic are preferred.

In a further embodiment the pesticidal component is intact Bacillus sphericus spores and/or crystals (parasporal
bodies) . In such cases the detergent may comprise a cationic detergent .

A filler may be added at any step of the process. Also an antioxidant as well as a UV protecting agent may be added at any step of the process.

The invention also relates to a method of controlling or combating insects by the use of the pesticidal composition described above comprising applying the composition to the aqueous environment to be controlled.

More specific, the invention relates to a process for controlling or combatting larvae of the insects of the order diptera in an aqueous environment comprising applying a composition described above to an aqueous environment harbouring said larvae in such a way as to prevent the active principles (spores, protoxins, toxins) to be inactivated by contact with aqueous environment.

In one embodiment the process includes applying to the aqueous environment a composition designed to disintegrate in an aqueous environment in a substantially gradual rate during a period of from about 3 minutes to about 50 hours after application. It is contemplated that some minor particles of the composition will disintegrate within 3 minutes whereby the active ingredient is immediately available to the insects, and other particles do not disintegrate for a period up to weeks, still other particles will gradually disintegrate during a period of 48 hours or 72 hours depending on the difference of temperature of the water and melting point of the fats used in formulation and also on the detergent composition.

In a further embodiment the process includes applying to the aqueous environment a composition designed to disintegrate in an aqueous environment and wherein the disintegrated particles start to sink towards the bottom whereafter the particles reappear at the surface due to a substantial graduate degradation. In a still further embodiment the reappearing particles during the next about 24 hours to about two weeks continuously travel up and down between the bottom and the surface of the aqueous environment or at any distance there between due to further graduate degradation.

The composition according to the invention may be applied simply by throwing by hand or with the aid of mechanical devices usually applied to spread granules in agriculture or in mosquito control programs. Most granules will for a time float on the surface of the water, but some granules will immediately sink and reappear later. After a period of time, most granules will sink and then reappear on the surface, moving up and down as they are slowly disintegrated.

Polycarboxyl may also be used as a detergent. In such formulations comprising polycarboxyl it has surprisingly been found that the polycarboxyl is gradually released from the formulation. Since the density of the detergent is higher than the density of the water, the remaining formulation moves toward the surface. As part of the fat in contact with the water subsequently dissolves the formulation again sinks towards the bottom until additional small fractions or beads of polycarboxyl are released from the formulation whereafter the movement of the formulation towards the surface is repeated. Accordingly, the formulation continuously moves up and down in the water while it disintegrate, whereby the active ingredient is available for insects living at completely different levels in the water. The same behaviour may be obtained by using inert fillers such as talc, diatomaceous earth, etc. that are heavier than water.

A granular form and especially a powder form of the composition may first be mixed with water for more than 1 minute and preferably 30 minutes optionally under gently stirring. The particles of the composition will then be equal dispersed in the water phase or be so at light stirring or shaking and can be applied as a wet product with apparatus that allow the distribution of particles up to 2-3 mm. After distribution on the water surface, these particles will behave as described for the granules, but the whole process will take less time. The duration of the disintegration process is regulated by the effective amount of fat, detergents and disintegrant. This duration is balanced considering the amount applied to the surface of the treated area, the concentration of the pesticide component in the composition and the desired concentration in the water. Once dissolved to the level where the spores of Bacillus sphaericus or the crystals of Bacillus thuringiensis are freed from other ingredients, they sink quite rapidly.

In a series of experiments with Bs suspensions in bottles kept at 30°C in dark, it was shown that Bs germinate in sewage water and even spores may reform, they do not reform the toxin. Only in cases where other microorganisms are removed by ultrafiltration or by heat treatment, the vegetative cells can proliferate and reform spores with toxins (Table 1-3) . These tables shows that in sterilised sewage water, Bs will often proliferate and more toxin was produced as seen in the bioassays (Table 2) . When the sewage water is not treated to remove other microorganisms, Bs will also germinate and may even proliferate, but after 4 days, the nuumber of spores and vegetative cells were diminished in most cases (Table 1 and 3) , and toxicity vastly reduced (Table 2) . So, even the number of Bs spores did not always diminish, the toxicity disappeared. In these tests, this can be explained by the germination of spores and resporulation without toxin formation or by the direct loss of the toxin without loss of the spores. The possibility of UV inactivation did not exist since the bottles were kept without light.

Tests were also carried out with radiated spores (Cells dead, toxicity remained, although at a lower level) . These tests showed that toxicity was lost or vastly reduced in sewage water without the germination of the spores (since the cells were dead) . Accordingly, to obtain a long residual effect, the spores must be protected not only against germination, but also against the influence of other microorganisms or their metabolites or enzymes.

The only way to obtain a prolonged effect in sewage water is therefore to protect the spore- toxin complex from the sewage water by a form of encapsulation which will also prevent germination. According to the present invention, this encapsulation may be performed by a fatty component forming a coat on the active principle of the invention.

In a second series of experiments, the granule disintegrating component was chosen to be either a detergent or a disintegrant like methyl cellulose. The products were tested in 0.5 1 glass tubes. These tests showed that after two days, water turned turbid where a detergent was used but remained clear in some of the samples where a disintegrant was used.

Microscopic examinations of the three groups showed that where water was turbid, the water was full of vegetative B. Sphaericus cells. Where the water was clear, most or all B. Sphaericus cells were still found in the tiny particles of the primary powder and with a protective coat of fat even that coat in some cases were very thin. The ratio of spores to vegetative cells was found to be 100:1 in the original primary powder and 1:100 in a formulation that did not produce turbidity, 1:1 to 1:100 in products where another dispersing detergent was used, and 1:1000 or more, where a fat soluble detergent was used to disintegrate the granules and where the water quickly became turbid.

Semifield test of these formulations in 75 1 containers
(Table 4) showed that two formulations which left a clear suspension for days in the glass tubes, had a long residual effect (column 3) . These formulations had the disintegration of the granules based on disintegrants only, not in detergents. One of these contained a detergent dissolved in glycol. Two formulations that gave milky suspension after a day or two, had a median residual effect (column 2) . In these formulations, disintegration was based on a mixture of disintegrants and detergents. Finally, two formulations that gave a milky solution of the granules within a day, had a short residual effect (Table 4, column 1).

These effects may be due to the wetting or dissolving of the fatty component of the granules by detergents and thus a function of their HLB potential. The use of glycol may also reduce the germination of the Bs spores just as the use of certain detergents with bacterial inhibiting abilities.

These formulations were tested in semifield tests. Two formulations with detergent as disintegrating principle had a short residual effect. These samples gave in the technical test a turbid solution within a day and the ratio of spores to vegetative cells were very low (Table 4, column l) . Two formulations with disintegration based on mixed effect of detergent and disintegrant had a medium residual effect, and these were those with medium turbidity and spore:vegetative cell ratio. Two formulations with disintegration based solely on disintegrant and (for one) with a content of glycol had a long residual effect, produced clear suspensions in the technical test and a high spore-vegetative cell ratio.

These effects may be due to the wetting of the granules by the detergents and thus a function of their wetting abilities. But some detergents are also known to have bactericidal or bacterial growth inhibiting effect, which may give the same results.

What is claimed here is also the principle of using a disintegration mechanism of the granule primarily based on disintegrants and not on detergents and of using dispersing agents which do not promote germination of the spores.

The type and amount of disintegrant influence the cohesion of the granule and of the subparticles of the granule. If the disintegrant has a too long chain length (e.g. as measured by the viscosity property) , particles remain as cloudy, loose conglomerates and do not release smaller particles and thus do not move up and down in the water as described, but remain either at the surface or at the bottom. If the disintegrant has a too short chain length, the granules disintegrate too fast into very small particles where spores are exposed to the water environment (Table 5) .

Table 1


x) Non-heated/heated refers to sample treatment. By heating o 80°C for 10 minutes, vegetative cells are killed.
Plating of non-heated samples gives numbers of vegetative cells and spores. This count may be lower than the spore count when spores are dormant and thus only appear after heat treatment.

#) Sterilised sewage water without Bs is a negative control, whereas sewage water alone is a control of the presence or not of Bs - the samples from this (untreated) sewage station never gave positive plaques.

o) the mixing of non- radiated and radiated Bs to obtain 1% live Bs, but full toxicity is explained in the text.

The table shows the development of Bs spore and total counts in different water types. The development in their numbers was dependent on water type/quality. In sterilised tap water, there was nearly no development. In sterilised sewage water (autoclaved or ultrafiltrated) , cell counts increased. In sewage water, cell count initially increased then to decline.

Table 2

X) 1 or 5 ml of the solution of Bs (or untreated water) was added to 99 or 95 ml water in two cups with 20 L4 larvae of Culex quinquefasciatus.

/@ assays made on days where LC50 of C. quinquefasciatus against the Bs standard SPH88 was 0.0049 mg/l and 0.0027 mg/l, respectively, except for the three tests series with Radiated powder only where LC50 was 0.0071 mg/l.

The samples tested in the bioassay are the same samples used for the countings in Table 1, except that the radiated solutions were not shown since they were all 0.

The table shows an increase in toxicity in the two samples where live Bs was mixed into sterilised sewage water (row 2, 6, and 12) , a stable toxicity where Bs live were mixed into tap water or radiated Bs mixed into tap water or sterilised sewage water, and a declining toxicity where Bs (live or dead) were mixed into sewage water.

Table 3. Development in spores counts from day 0 to day 4.

8 tests in bottles, 6 +/- aeration number of number of number of
spores spores stable spores
increased *) decreased

Sterilised sewage water +Bs 4 0 4 do, aerated 3 2 1

Sewage water + Bs 0 2 6 do, aerated 0 1 5 tap water + Bs 1 7 1 do, aerated 0 6 0

*) Increased indicates more than 100% increase. Stable
means changes between +100% and -75%, decreased means less than 25% remained.

This summary table of 8 tests shows that the fate of spores are dependent on water quality, but also that sewage water is a variable source. Sewage water samples are taken from January till July, thus periods without algae to periods with water green of algae. In two trials, sewage water were used a month after sampling and Bs behaved like in tap water, indicating that nutrients provoking germination was no longer present. In the other samples, Bs spore counts diminished.

Table 4

Product and υ DiissssooilUubDilee w waax and Fat and Dispersing detergents
detergents fat-dissoluble no fat dissolving

Effect x days after detergents detergents appplication
Mortality of larvae 82 % 90 % 100 % day 2 *)
Mortality of larvae 88 % 98 % 100% day 6
Mortality of larvae 94 % 98 % 98%
day 9
Mortality of larvae 100 % 100 % 100% day 16
Mortality of larvae 100 % 100 % 100% day 25

The increase in mortality during time is influenced by recycling of the bacterium also.

Table 5

Type of methyl "Pendule behaviour" Residual efficacy cellulose in field trials

1500 cps > 7 days > 28 days
4000 cps 0.5 day 2 days
30% 1500 cps
70% 4000 cps 1 day 4 days

The type of methyl cellulose is described by its viscosity at a 2% aqueous solution at 25°C.

The pendulum behaviour indicates the number of days where the granules will sink and reappear at the surface under slow disintegration.

Residual efficacy is defined as the number of days where the product gave more than 95% control of mosquito larvae in cesspits in an African town.

The present invention is further illustrated by the following examples. All percentages are by weight unless otherwise indicated

Example 1

Bacillus sphaericus primary powder (a powder containing spores and crystals of Bacillus sphaericus) was mixed with methyl cellulose and a non-ionic detergent 40%, 40%, 20% to form an insecticide phase. The insecticide phase is thoroughly mixed and eventually ground if containing any larger particles. Hydrogenated ox tallow and an anionic detergent is mixed 90%, 10% after melting of the fat to form a fat phase. The fat phase is cooled down to 60-70°C and the insecticide fraction is added. The two phases are mixed 50% to 50% and the composition poured into a plastic bag which is then left on the table to solidify as a 1-3 mm thick plate. When cooled down to room temperature, the plate is broken into particles with a paste roller. The particles are then sieved with a kitchen sieve to separate the powder from the granule.

Example 2

Bacillus thuringiensis var israelensis primary powder was mixed with starch and a non- ionic detergent 30%, 55%, 15% to form an insecticide phase. The fat phase consisted of modified plant oil wax (tradename Vegeol, Aarhus Oliemoelle, Denmark) 90% and an anionic detergent 10% which were mixed. The fat phase is melted in an industrial steam kettle at 70 'C under homogenisation. With the homogeniser still going, the powder phase is slowly poured in. After a few minutes of homogenising, the content of the kettle is poured onto a cooled steel plate transport belt forming a 2 mm thick plate.

The transport belt ends under a steel roller which breaks the plate into particles. Particles are sieved through a shaking sieve. Unwanted particle fractions may be recycled to the fat phase of the next batch.

Table A

A product based on Bacillus sphaericus according to Example 1 was tested in clean water on 4th instar larvae of Culex pipiens. The product of the present invention was compared with a commercially available product based on the same bacteria.

Days Example 1 Spherimos Example 1 Spherimos after 3.0 g/m2 FC 0.3 g/m2 0.3 g/m2 application 3.0 g/m2
0 100% 100% 100% 100%
(100%) (96%) (100%) (96%)

2 100% 100% 100% 100%
(87%) (57%) (72%) (96%)

4 100% 100? 95% 80%
(72%) (39? (45%) (18%)

7 100% 66% 90% 74%
(82%) (19%) (40%) (26%)

18 66% 8?
(48%) 8?

Table A shows the mortality of mosquito larvae after 2 days (in brackets) and cumulative death until hatching of the adult mosquitoes. The test shows that the granular formulation based on the present invention has a more persistent control of mosquito larvae in pure water fully exposed to sun in the mediterranean climate of ontpelier .

Table B

A product based on Bacillus sphaericus according to Example 1 was tested in polluted (organic rich) water on 4th instar larvae of Culex pipiens quinquefasciatus . The product of the present invention was compared with a commercially available product based on the same bacterium.

Days Control 40C2 Spherimos 40C2 Spherimos after 3.0g/m2 3.0g/m2 .3g/nT .3g/m2 application
0 12% 100% 100% 100% 100%
(8%) (98%) (96%) (24%) (38%)

2 6% 100% 100% 66% 100%
(0%) (100%) (100%) (34%) (98%)

6 10% 100% 78% 56% 56%
(0%) (90%) (32%) (24%) (20%)

11 4% 86% 12% . 16%
(0% (66%) (6%) (2%)

Table B shows the mortality of mosquito larvae after 2 days exposure to the test products (numbers in brackets) and the accumulated death of the mosquito larvae or pupae till hatch-ing of the adult mosquitoes. The table shows that also in polluted water, fully exposed to sun, the product described in the present invention is more persistent than the reference, commercially available product based on the same bacterium (0. Skovmand and S. Bauduin, in print).

Table C

A product based on Bacillus sphericus according to Example 1 was tested in the field against larvae of Anopheles gambiae . The product of the present invention was compared with a commercially available product based on the same bacterium.

Days 40C2 40C2 Spherimos Spherimos after 0.3g/m2 0.3g/m2 0.3g/m2 0.3g/m2 application
0 0% 0% 0% 0%
3 93% 94% 100% -8%
6 66% 100% 97% 91%
10 88% 96% 50% 0%
15 0% 98% 0% 0%

Table C shows the percentage of control compared to the number of mosquito larvae before the application. There are 5 repetitions for the product 40 C at both test levels, 4 for Spherimos 3.0 g/m2 and just 1 for Spherimos 0.3 g/m2 - the latter figure is therefore not significant compared to the others. It is obvious that 40 C at the low level was not easy to distribute and the effect is not stable. The test sites were fully sun exposed and the prolonged effect obtained for 40 C at 3.0 g/m2 is probably due to the sun protection of the product compared to the non-protected Spherimos.

Example 3

Bacillus sphaericus or Bacillus thuringiensis var israelensis powder was mixed with methyl cellulose and a non- ionic detergent in a ratio 50%, 40%, 10%. The methyl cellulose is cha-racterized by having a viscosity of a 2% solution at 2000 cps at 25°. The insecticide phase was thoroughly mixed and ground. Hydrogenated ox tallow, paraffinic wax and anionic detergent were mixed in a ratio of 60%, 30% and 10% after melting of the two waxes. The paraffinic wax is characterized by having a melting point at 46-48°C. The fat phase was cooled down to 60°C and the insecticide phase was added and composition was thoroughly mixed and then left to solidify in a 2 mm thick plate. The plate is broken down to particles that are sieved. Particles between 0.5 and 2 mm were used as a granule.

When this granule is added to water it will initially float and disperse on the water surface. Within 10 minutes at 20°C and 5 minutes at 30°C the particles started to sink to the bottom. Within the next hour, particles from the bottom reappeared at the surface under degradation. During the next two days at 20°C and one day at 30°C, particles continuously travelled up and down from the bottom of a 20 cm deep container to the surface and released smaller particles on their way in this process. With deeper containers, not all particles will reach the bottom before returning toward the surface again. A part of the active substance reaching the surface remains there attached to the fat.

This product is aimed to be used under circumstances where a simple floating formulation is not suitable because it will be removed by e.g. wind movement of water, run off of surface water from the treated area or when subject to fast sun-inactivation, or where the mosquito species composition contains mosquito larvae living at the bottom, near surface and underside surface eating mosquito larvae or just two of such species. These problems showed to be important when testing the commercial granular product Vectolex® (Karch et al, J Amer Mosq Contr Ass 1992, 8 (4 (, 376-380) in rice fields and swamps .

References

1. Becker, N. , Rettich, F. 1994: Protocol for the introduction of new Bacillus thuringiensis israelensis products into the routine mosquito control program in
Germany, J American Mosquito Control Ass, 10 (4) : 527-533

2. Hougard, J.-M., Mbetntengam, R. , ochouarn, L., Escaf- fre, H., Darriet, F., Barbazan, P., and Quillevere, D.
1993: Lutte contre Culex quinquefasciatus par Bacillus sphaeri cus : resultats d'une campagne d'Afrique equatori- ale. WHO Bulletin 71: 367-375

3. Karch, S., and Charles, J.-F. 1987: Toxicity, viability and ultrastructure of Bacillus sphaericus 2362
spore/crystal complex used in the field. Ann. Inst.
Pasteur/Microbiol . 138, 485-492

5. acey, L.A., Urbina, M. J. , and Heitzman, C. M. 1984:
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6. Lacey L.A. , H Undeen, 1986: Microbial control of black flies and mosquitoes, Ann Rev Entomol, 31:265-296.

7. Becker, N. , Rettich, F. 1994: Protocol for the introduction of new Bacillus thuringiensis israelensis products into the routine mosquito control program in Germany, J American Mosquito Control Ass, 10 (4) : 527-533

8. Pozsgay M, P Fast, H Kaplan, P R Carey, 1987: The effect of sunlight to the protein Crystals from Bacillus thu ringiensis var kurstaki HD1 and NRD12 : A Raman Spectroscopic study, J Inv Pathol 50,246-253.

9. Skovmand, 0. (in prep) Different behaviour of Bacillus sphaericus in various types of water.

10. Skovmand, 0 and Bauduin, S: Efficacy of a granular
formulation of Bacillus sphaericus against Culex quin - quefasciatus and Anopheles ga biae in West African
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11. Thiery I, S Hamon, B Gaven H de Barjac, 1992: Host range of Clostridium bifermentans serovar malaysia, a mosqui- tocidal anaerobic bacterium, J Am Mosq Control Ass
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