NZ242613A

NZ242613A - Microcapsuled agriculturally active agents encapsulated in lignin or a lignin compound

Info

Publication number: NZ242613A
Application number: NZ242613A
Authority: NZ
Inventors: Richard D Moss; Franklin Lim
Original assignee: Lim Lab Inc
Priority date: 1991-05-06
Filing date: 1992-05-05
Publication date: 1994-09-27
Also published as: IL101774A0; IE921425A1; EP0584210A4; AU1905092A; CA2102707A1; ZA923275B; WO1992019102A1; EP0584210A1

Description

<div class="application article clearfix" id="description"> 242613 Priority Dato(is): . Compicls Cpsrii'ic?■::; r £'£'.9?r*. Class: Publication Dcic: . . ?.f P. P.O. Journsl, No: .......$?Jf: No.: Date: NEW ZEALAND PATENTS ACT, 1953 COMPLETE SPECIFICATION MICROENCAPSULATED AGRICULTURALLY ACTIVE AGENTS AND METHOD OF PRODUCING SAME We, LIM LABORATORIES, INC., a corporation of the State of Virginia, USA, of 409 E. Main Street, Richmond, Virginia 23219, USA, hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- - 1 - (followed by page la) ?42613 BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to agriculturally active preparations and, more particularly, to microencapsulated biological and chemical agriculturally active preparations, such as pesticides and fertilizers having improved stability upon application in a given environment. Microencapsulation of pesticides has been used as a means for providing controlled-release preparations, for reducing the toxicity to persons handling the pesticides, and for protecting the active ingredient from deactivation by exposure to environmental conditions. Exposure of non-encapsulated insecticides to these conditions can lead to leaching and run-off by rainfall, loss by volatilization, degradation by bacterial inactive metabolites, and sunlight, especially ultraviolet inactivation. Selection of an appropriate carrier material for use in an interactive delivery system can not only reduce this deactivation, but also produce specific desired affects when combined with the active insecticidal material. In an effort to provide a delivery system for an active ingredient meeting the criteria set forth above, the art has propospd i number of encapsulation materials. Methods for preparing microencapsulated insecticidal pathogens have been described in which a capsule material itself shields the pathogen from sunlight-induced inactivation. US Patent No. 4,328,203 describes a microbial insecticide composition which comprises a microbead of a nucleic acid and a 242613 proteinaceous material which partially protects the insect pathogen from UV inactivation. However, because these substances are not environmentally stable, the encapsulating material is easily broken down, and thus cannot efficiently protect the insecticide. The use of sunscreens in controlled release preparations has also been described. Diisophorone derivatives are utilized in US Patent No. 3,839,561 to protect insecticidally active cyclopropane carboxylic acid compounds from UV~induced degradation. In US Patent No. 4,094,969, there is described the use of a sulfonated copolymer of catechin and leucocyanidin which stabilizes a pesticide by retarding degradation upon exposure to sunlight. However, these formulations do not maintain the sunscreen and insecticidal preparation in close enough contact to be effective against UV irradiation. Ignoffo and Batzer, in "Microencapsulation and Ultraviolet Protectants to Increase Sunlight Stability of an Insect Virus," Journal of Economic Entomology. Vol 64, pp. 850-853 (1966), attempt to solve this problem through the use of Buffalo Blacky Carbo-Jet Black, carbon and aluminum oxide and powder with microencapsulated Heliothis nucleopolvhedrosia virus to protect preparations from UV irradiation. However, the microencapsulating materials used by Ignoffo, namely gelatin and ethylcellulose, are not environmentally stable and are thus readily broken down by the environment. Similarly, US Patent No. 2,090,109 discloses the use of chlorophyll green as a protectant of various insecticides in a gelatinous vehicle from UV light. Fogle, et al. describe the use of UV absorbing materials such as carbon blacx* in a polymer matrix composition containing 242613 an insect virus in US Patent No*. 3,541,203. The^ microencapsulating process as well as the microcapsules obtained by Fogle et al. suffer from a jiunber of disadvantages. The polymers forming the walls of the capsules are not always capable of retaining the sunscreening agent within the interior of- the capsule. This diminishes the stability of the insecticidal preparation because the loss o2 sunscreening agent makes the pathogen more susceptible to the damaging effects of ultraviolet light. In addition, the microcapsules of Fogle, et al. are prepared using highly toxic materials, and cumbersome washing steps are required for their removal. A particularly preferred group of encapsulating polymers is disclosed in US Patents No. 4,844,896 and 4,948,586 by Bohm and Friend, of common assignee. These polymers are synthesized from acrylic and methacrylic acid esters with a low content of quaternary ammonium groups, and are known as Eudragit L, S, RL and RS. They are useful because they can yield capsules with strong walls which can be made soluble at a pH of 6 or 7.5, depending on the particular formulation used. The Eudragit capsules can be used effectively to retain sunscreening agents within the walls of the capsule until ingested by an insect. The use of lignin in certain types of controlled release formulations is also known in the art. US Patent No. 3,929,453 relates to controlled release preparations of lignin and biological materials which are produced via coprecipitation-inclusion from an aqueous alkaline lignin solution, or via the elimination of a common solvent from a lignin-biologically active organic agent mixture. US Patent No. 4,244,729 describes a reversibly swellable carrier gel for the incorporation of - 3 - 24 2 6 1 pesticides. The&e gels are forfaed by crosslinking lignin with epichlorohydrin, leading to the formation of gels with differing surface chemical characteristics, which are able to sustain controlled release of water-soluble and water-insoluble pesticides. The use of other cross-linking reagents such as formaldehyde, paraformaldehyde, glyoxal and glutaric dialdehyde is described in related US Patent No. 4,244,728. Methods for microencapsulation and protection from UV irradiation are not disclosed in any of these references. One of the disadvantages of prior art lignin-based pesticidal preparations and to microencapsulated preparations usinj other known polymers, is that the insecticidal agent is subject to autocatalytic breakdown because of its partial exposure on the exterior of both the lignin gel matrices and the polymeric microcapsules. Thus, even in the presence of sunscreens added to certain polymeric microcapsules, insecticide which is present near the surface of the microcapsule which i3 not protected by sunscreen can undergo UV degradation. As a result, this degradation produces an autocatalytic degradation of neighboring molecules of insecticide toward the interior of the microcapsule. These interior molecules might have otherwise been protected by sunscreen which was present within the microcapsule. Therefore, prior art preparations, even those which seemed to effectively retain sunscreening agents within the microcapsule, were still not capable of protecting the insecticidal preparation from autocatalytic degradation which began at the surface of the microcapsule. In addition, microencapsulated insecticidal formulations characterizing the prior art typically required use of tox'c materials in their preparation. - 4 - 242613 Another problem confronting, the prior art was the inability to load sufficiently large amounts of active agent into a , capsule. Difficulties were also encountered in tailoring the microcapsule sizes to the particular application. SUMMARY AND OBJECTS OF THE INVENTION In view of the aforementioned disadvantages attendant with prior art formulations and processes as well as other disadvantages not specifically mentioned above, it should be apparent that there still exiats a need in the art for microencapsulated active agents which (1) include no toxic components in either the final product or in the materials used to form such microcapsules, (2) provide protection for an active agent against environmental effects, such as sunlight, which could degrade an active agent and (3) break down to release the active agent once it has reached its desired target. It is therefore a primary objective of the present invention to fulfill that need by providing an active agent, such as a biological or chemical insecticide, which is microencapsulated in lignin. Another object of the present invention is to provide an active agent, such as a microencapsulated insecticide, which provides controlled release of the active agent when contacted with the desired target. yet another object of the present invention is to load sufficiently large< amounts of an active agent into a microcapsule. still another object of the present invention is to provide a process for preparing microcapsules which enables the size of - 5 - 2^2613 the microcapsule to be tailored to the particular end use contemplated. In particular, these and -other objects of the present invention are achieved by providing a controlled-releasa preparation of an active agent which uses lignin not only as the capsule material, but also as a sunscreening agent which protects the agent from the harmful effects of light. In a first aspect, the present invention relates to a microencapsulated active agent comprising: (i) an active agent such as an insecticidal agent, and (ii) a polymeric encapsulating agent comprising lignin or a lignin compound. In a second aspect, the present invention relates to a process for preparing a microencapsulated active agent comprising the steps of: (i) mixing (1) an encapsulating agent comprising lignin or a lignin compound, (2) an active agent in a form compatible with microcapsule formation and (3) an aqueous solvent or cosolvent system which is miscible with water and which dissolves lignin or the lignin compound to form a first mixture; (ii) preparing an emulsifying solution comprising a surfactant and a liquid which is immiscible with said aqueous solvent or cosolvent system; (iii) preparing a solution to harden the capsules; (iv) mixing the first mixture and the emulsifying solution to form an emulsion; (v) mixing the resultant emulsion with the hardening solution to harden emulsion droplets to form capsules; and 242613 (vi) concentrating and separating the capsules from the water insoluble liquid. With 1ihe foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure l is a graph depicting the percent mortality of cabbage looper larvae as a function of hours of exposure to ultraviolet radiation of encapsulated and free Bacillus thuringiensls at different dosages? and - • Figure 2 is a graph depicting the percent mortality of cabbage looper larvae as a function of hours of exposure to ultraviolet radiation of an encapsulated and free viral biopesticide. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 1. Starting Materials The basic starting materials for preparing the microencapsulated active agents of the present invention are the active agents themselves. In this regard, while the preferred embodiment of the present invention which is described relates to microencapsulation of insecticidal agents, it will be appreciated that other agriculturally effective agents may be utilized. Such agents include, but are not limited to, fertilizers and plant regulators, e.g.,plant hormones. Suitable insecticidal agents include insecticidal viruses, bacteria, fungi or chemical toxins. In addition, other starting materials include the lignin polymer used to encapsulate the insecticide, and optionally, the Eudragit •t7 polymer which can b* used in combination with thfc"lignin to form the microcapsule. Polymers such as Eudragit L, Eudragit Sr Eudragit RL and Eudragit RS can be used in combination with the lignin to form the microcapsule. By Eudragit is meant a group of modified acrylic acid polymers which can be made soluble at various pH levels, depending on the modifying groups employed* Such polymers, as well as the techniques for modification thereof to obtain a desired pH solubility, are known in the art* Any of the pathogens currently known to infect insects, including viral, bacterial and fungal pathogens or any chemical toxins known to act on insects, can be encapsulated by and protected from UV light by lignin. Such viruses include, but are not limited to the nuclear polyhedrosis virus (NPV) of the bullworm, Heliothls zea. of the gypsy moth Lvmantria dlsoar. of the Douglas fir tossock moth, Praia pseudotsugata. of the European pine saw fly Neodiprlon sertifer or of Autoarapha californica. Bacteria known tO' be pathogenic to target insects, including but not limited to Bacillus thuringiensls. Bacillus Sphaerlcus. Bacillus Popllllaa may also be encapsulated. Insecticidal chemical toxins including, but not limited to amidino hydrazonas such as hydramethylnon (Amdro*) may also be used. Finally, it will fcNt appreciated that a number of recombinant active agents which are now available may also be encapsulated in accordance with the present invention. The insecticidal agents are employed in combination with a lignin. The lignin component is discussed first. By NligninM is meant any lignin or modified lignin derived from the alkaline digestion of lignocellulosic material, including sulfated lignins, hydrolysis lignins, and lignin amines. Thus, any of the lignins may be employed tc make the carrier used in this invention. Most commonly, they are obtained as byproducts from _ Q mm. A-' * '"O O N A- 242613 the alkaline process of papermaking where sodium hydroxide alone or in combination with sodium sulfide is employed. These lignins are generally referred to as soda or sulfate lignins after the pulping process used. In the preferred practice of this invention, the lignin employed is a kraft pine lignin. Likewise, lignins known as "hydrolysis lignins" obtained by enzymatic or acidic reactions with lignocellulosic materials may be used. Also, sulfite waste liquor lignins and sulfonated alkali lignins may be used if the degree of sulfonation is controlled. Thus, mixtures of alkali lignin and sulfite or sulfonated lignin may be used if an immediate release of pesticide is desired. Lignin amines may also be used to form capsules. Lignin amine? are soluble at a pH <3.5 or >8.5, and are generally supplied at about pH 10. Lignin amine at pH 6 is not soluble in water, but will dissolve in solutions of polyethylene glycol and water, for example, a solution of 70% PEG/30% water. The present inventors have discovered that lignin is effective for use as a microencapsulating material as well as a sunscreening agent for* agriculturally active agents such as pesticides. In some instances, the lignin and active agent can be formed into a microcapsule which concentrates the active agent towards the center of the capsule. Depending on the nature of the interaction between the lignin carrier and the pesticide, the effects demonstrated by the composite can vary from simple dilution of the active ingredients to sustained release., The use of these types of microcapsules, whether interactive or not, also allows for the reduction or elimination of some of the environmentally undesirable side effects of pesticide use such as contamination of non-target areas caused by toxicant run-off, - 9 - 24 2 6 leaching or evaporation. In addition, incorporation of the pesticide into the delivery system of the. invention offers potential for decreasing chemical breakdown into biologically inactive compounds following exposure to light and extremes of pH. Thus, the active ingredient is shielded, its initial activity is reduced, and its release is sustained. Lignin can be easily modified due to its phenolic hydroxyl, carboxylate and aliphatic hydroxyl groups, as well as its high aromatic content. Matrices of varying pore structure, polarity and solubility can be created by blocking certain groups on the lignin unit, and by crosslinking with reagents such as epichlorchydrin, formaldehyde, ammonia and formaldehyde, and hexamethylenetetramine, among others. It will be appreciated that the ability to modify lignin enables the use thereof against different targets. Thus, for example, when it is an insecticide which is encapsulated in th« lignin, the lignin is designed to be insoluble at approximately neutral or acidic pH levels as encountered outdoors' and soluble at alkaline pH levels as encountered within the gut of an insect. The high aromatic content of lignin also gives it the property of excellent ultraviolet radiation absorption. This characteristic, along with its inert properties, makes lignin highly suitable for use with pesticides which are sensitive to UV-initiated or -catalyzed degradation. Finally, the good dispersant qualities, the low cost and the environmentally benign nature of lignin compared to other carriers such as high surface area, porous plastics, makes lignin highly desirable for use in virtually all pesticide formulations* The microencapsulated preparations of the present invention, through the process of — 10 — 242613 emulsification, maintain their activity due to the physical-chemical protection and intrinsic sunscreening activity of the microencapsulating pclyw" 'gnin. The capsules formed are spherical and can be made to have a large range of diameters depending on the final-application-. Thus, for example, when insects are the target, they can be formed with average diameters of 10-50/j which allows the capsules to form free flowing suspensions which can be sprayed-and are small enough that they are easily ingested by the target insect. Thus, lignin provides an ideal encapsulating agent for an insecticide because it is insoluble in water at normal environmental pH values and thus protects the pesticidal agent while present on vegetation, but will dissolve in the alkaline pH of the insect*s gut, releasing the pesticidal agent. In a preferred embodiment, microcapsules are formulated with a mixture of lignin and the polymer Eudragit which, depending on the active agent, can prevent autocatalysis and degradation until the time of ingestion. In this regard, it will be appreciated that Eudragits can be manufactured so as to have either a positive or a negative charge. Accordingly, depending on what charge the active agent has, an Eudragit having the opposite charge can be selected. One particularly preferred combination is the chemical insecticide Amdro*, which has a positive charge, and a negatively charged Eudragit. In fact, without being limited to theory, it is believed that such combination has been found to result, during microcapsule formation with the lignin encapsulating agent, in a partial phase separation as the droplet forms and a pulling of the active agent into the core of the microcapsule where it is better rotected in the final product. - 11 - 242613 f This, in turn, increases the resistance of the microcapsule to autocatalysia. A water-immiscible liquid.such as vegetable oil, kerosene or liquid hydrocarbon containing a surfactant can be used to form an ^ emuxsion with the encapsulating polymer and insecticide. •Solutions such as soybean oil and other vegetable oils, non-polar liquids such as kerosene and other liquid hydrocarbons such as hexane, in combination with a surfactant can be used as the continuous phase for making the emulsion of the capsule material. The emulsifying agent can consist of a SPAN-80/Tween-80 mixture. For soybean oil, 20% of a mixture of 30% Tween-80/70% SPAN-80 has been found to be effective. For other, continuous phase liquids, the ratio of SPAN to Tween is adjusted to optimize the formation of the emulsion. Surfactants other than SPAN and Tween may also be used. Volume ratios between 1.5 and 2.0 of oil to capsule material have been successfully used. Lower ratios are possible but viscosity increases at the lower ratios and the capsules which result are not as uniform in size and shape. In addition, lower ratios may reverse the phase of the emulsion, resulting in oil droplets in the capsule material and formation of distorted capsules or lack of capsules. Tween-20 can be used a3 a surfactant in the hardening solution for hardening lignin capsules. Concentrations between 2 and 10% have been used, but too little Tween results in poor separation of the capsules from the oil. 2.5% Tween is sufficient in most cases. Capsules made from alkaline solutions of lignin can be hardened using a water solution of an acid such as acetic acid or an acid buffer. Capsules made with lignin » - 12 - 242 6 1 3 dissolved in organic solvent/va'ter can be hardened in water alone. 2. PreparaUgn of Mi<?r«?<?apgvil^g A number of schemes for preparing microencapsulated active chemical toxins are now described. It will be appreciated that such techniques are also applicable to the encapsulation of f insecticidal viruses, bacteria and fungi as well as other active agents. The first step of the process involves mixing the encapsulating agent comprising lignin, an active agent in a form compatible with microcapsule formation and a solvent or cosolvent system which is miscible with water and which dissolves lignin. The lignin component, and the modifications which can be made thereto, were discussed previously. The active agent in a form which is compatible with microcapsule formation is one which does not interfere with the solvents employed to prepare the microcapsules. In general, such active agent is in the form of either a solid in an aqueous suspension or it is dissolved in an organic solvent which is not miscible with the microcapsule forming solvents. The solvent or cosolvent system includes water and is one which is miscible with water and which dissolves lignin and includes a solvent comprising ethylene glycol, polyethylene glycol, isopropyl alcohol, acetone or mixtures thereof. Other solvents known in the art and meeting the above criteria can be used. An emulsifying solution is also prepared including a surfactant and a liquid which is immiscible with the solvent system described above used to dissolve lignin. Suitable cosolvent immiscible liquids include vegetable oils such as - 13 - v 2 4 2 613 soybean oil, kerosene or a liquid hydrocarbon such as hexane. The surfactant is selected from those known in the art such as Tween-80, Span-80 or mixtures thereof. The ratio of these surfactants can be adjusted to optimize the formation of the emulsion, depending on the particular materials selected. A solution for hardening the capsule is also prepared. Such solution comprises water and a surfactant when lignin is the encapsulating agent. For lignin capsules, water with 2 to 10% by weight of Tween-20 has been found to be suitable. Capsules made from alkaline solutions of lignin may be hardened with 0.1 M acetate buffer, pH 4.5. Acetic acid will also work. For lignin amine capsules, the final pH should be near neutral, such as by using a 0.1M phosphate buffer of pH €.8. The emulsion prepared above is then combined with the hardening solution to harden the emulsion droplets to form capsules. Such capsules are then concentrated and separated from the from the oil and hardening solution either'by allowing the capsules to settle or by centrifugation. The present method enables the preparation of microcapsules having a lignin content above 10% by weight, preferably between about 10 and 30% and most preferably between about 20 and 30%. Typically, below about 10%, it is possible to form microcapsules. However, at such low levels, an effective sunscreening effect is not obtained. At levels above about 30%, lignin solutions can become quite viscous and difficult to handle. The present invention, by employing a solvent or cosolvent system which is miscible with water and which dissolves lignin, enables handling of lignin solutions with such high percentages of lignin therein. 14 - 24 2 6 The apparatus for preparing microcapsules is one used conventionally in the art. To prepare the microcapsules including Eudragit*, Eudragit* is dissolved in a solution containing PEG, the pH is adjusted with NaOH, and the solution mixed until dissolved. Separately, lignin is dispersed in water to form a thick paste. Because lignin is insoluble in water, PEG is added to the solution, and the lignin is allowed to dissolve. Alternatively, ammonium or sodium salts of lignin can be used which are soluble in water. Lignin has only limited solubility in acetone and isopropanol, but it is soluble in mixtures of such with water. Lignin is soluble in PEG-400 solutions of greater than 50% PEG, but if adjusted to pH 7.3, can become soluble in 35% PEG. Therefore, capsules have been successfully made from lignin dissolved in 50% PEG/water or 70% isopropanol/water, the aramonium salt of lignin dissolved in water, and from lignin adjusted to pH 7.3 dissolved in 35% PEG/water. The Eudragit-PEG solution is added and mixed until the lignin is completely dissolved. To this mixture is added the suspension of the insecticide. If it is desired to add an additional sunscreening agent, it can be added to the Eudragit-PEG solution. After adding the suspended biopesticide to the lignin-Eudragit solution, SPAN-85/Wesson oil (1:1) is added and stirred at maximum speed for about one minute to form the emulsion of capsule material in oil. This is poured rapidly into a hardening solution of Tween-20 in acetic acid and stirred for an additional minute to form capsules. The mixture is centrifuged in a 50ml centrifuge tube to separate the oil and the capsules. The capsules are washed several times to remove as - 15 - 242 613 much oil as possible, and stored in water. The average diameter of the capsules is 25(m. For lignin. amine capsules, the final pH should be near neutral. O.IK phosphate buffer pH 6.8 has been used to maintain the pH in this range. 3. Application of Microcapsules to Vegetation. The microcapsules prepared by any of the above-described methods can generally be applied anywhere a conventional insecticide or other active ingredient, such as a feirtilizer could be applied. Generally, the concentration of the microcapsules and the rate of application depend on the nature of the pathogen and on the nature of the vegetation being, treated. Such are readily ascertainable by persons skilled in the art. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be .construed,;,however, as limiting the broad scope of the invention. EXAMPLE I Microencapsulated Amdro* in Lignin-Eudragit was prepared by dissolving 14.4g Eudragit S-10Q in I20g 40% PEG-400, adding lg NaOH and mixing until dissolved. In a separate beaker, 20g lignin was added to 15g water and 15g of PKG and mixed to form a thick paste. The mixture was stirred for 15 minutes on magnetic stirrer to improve mixing. 50g of the Eudragit-PEG solution was added and stirred 20-30 minutes until all of the lignin was dissolved. 333/il micronized. Amdro* was added to lOg of the resulting mixture and; stirred for about a minute. 12.5g 50% SPAN-85/50% soybean oil was added and stirred one minute to form the emulsion of capsule material in oil. 32ml of 7% Tween-20 in acetate buffer was poured rapidly into the emulsion, and stirred 94 2 6 1 3 for one minute to form capsule?. The capsules were separated by centrifugation at lOOOrpm. The capsules were then washed to remove the remaining oil. Cotton leaves were treatod with capsules with and without sunscreen, and with Amdro* in a solution of acetone/water. Leaves were excised and bioassayed as above. Amdro* formulations were used at 100 ppm at (1 ml per leaf.) Treated leaves were excised at various intervals and bioassayed for control of first-instar larvae of Heliothis virescens. Plants received seven hours of direct sunlight in the first 24 hours post-treatment, followed by a 14 hour photo period exposure to metal halide lamps in the greenhouse. The above-described encapsulated product was compared with nonencapsulated Amdro*. After days of exposure to the sun, the micrencapsulated active agent of the invention retained most of its activity whereas the activity of the unprotected agent was mostly lost. EXAMPLE 2 Bacillus thurlnqiensls was encapsulated in lignin. First, the capsule material was prepared by mixing 135g of lignin and 135g of PES-400 and 8.1g of 25% NaOH in a 5 quart bowl thereby forming a thick paste. There were then added 240g of Bacillus thurinoiensis slurry in 4 aliquots, mixing thoroughly after each addition. After the last addition, mixing was carried out for an additional 5 minutes to assure complete mixing and dissolution of the lignin. An emulsifying solution was prepared by mixing together 84Og soy bean oil, 47g of SPAK-80 and 63g of TWEEN-80. 17 - 9 4 2 6 1 3 A hardening solution was prepared by nixing together 1388g water, 75g TWEEN-20 and 4.5ml acetic acid. The emulsifying solution yas then added to the capsule material in the mixing bowl and emulsified by mixing with a mixer. The mixing was continued for 15 minutes. The hardening solution was then poured in followed by mixing for another minute to harden and separate the capsules from the oil. The capsules were then separated form the oil and concentrated followed by pouring off the oil and excess water. Bioassays were done by first exposing a dilute water suspension of capsules or free Bacillus thurlnqiensia to ultraviolet light for a given period of time followed by spreading aliquots of the sample on the surface of food in small cups. Next several cabbage looper larvae were placed in each cup and allowed to faed for three days. The living and dead larvae were then counted and the results expressed as percent mortality, (dead larvae)/(totaly. The capsules prepared above at doses of 2/ig, 1/ig, and 0.5/xg were compared to the free Bacillus at doses of 2ng and 4/ig„ As is shown in Figure 1, after only a few hours of exposure to ultraviolet radiation, the encapsulated products of the invention provided a much higher mortality against cabbage loopers as compared to the nonencapsulated products. EXAMPLE 3 Microencapsulated viruses were prepared. The lignin component was Lignin AT from Westvaco. Polyethylene glycol having a molecular weight of 400 was employed. As the emulsifying solution, there was used 10% of (60% Span-80/40% Tween-80) by weight in soybean oil. A stock Eudragit S-100 was employed including 14.4g Eudragit s-100 and 120g PEG and lg NaOH. w 6 » A working Eudragit S-100 solution was prepared by adding 25g of the stock Eudragit S-100 solution and 15.og PEG and 10.Og water, to make a 5% Eudragit in 50% PEG/water. A 25% NaOH w/w solution was prepared by adding 25g NaOH to 75g water. The virus used was A. cal from American Cyanamid of approximately 1010 PIBs/g. A hardening solution was prepared from 20g acetate buffer, 20g Tween-20, 160g water and lOOmg CaCl2 dihydrate; To prepare the microcapsules, 2g of Lignin-AT were added to a 50ml beaker with 1.5g of PEG, 2g of 5% Eudragit S-100 in 50% PEG, and 180^1 of 25% NaOH. The liquids were next mixed into the lignin to form a paste. Then, there was added Ig of water followed by mixing. The mixture was warmed slightly in order to speed the dissolution of the Lignin. After all of the lignin was dissolved, the capsule solution was allowed to cool. Then, there were added lOOmg of virus dispersed in 0.3g water followed by mixing. There were then added lOg of emulsifying solution. An emulsion was formed by stirring with a stir bar at maximum speed on a magnetic stir plate for about 1 minute. Next, 30ml of the hardening solution was poured in and stirring continued for another minute. The mixture was then poured into a 50cc centrifuge tube, shaken, and centrifuged for 3 minutes at 1000 rpm. The oil and supernatant were removed and the capsules were retained and resuspended in water. The encapsulated and nonencapsulated virus was again applied against the cabbage looper larvae as described in Example 2. The results, shown in Figure 2, demonstrate the dramatic difference between the encapsulated and nonencapsulated product in terms of - 19 - 242 6 the percent mortality after only a few hours of exposure to ultraviolet radiation. While the invention has been described and illustrated herein by references to various specific materials, procedures and examples it is understood that the invention is not restricted to the particular material combinations of material, and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. - 20 - </div>

Claims

<div class="application article clearfix printTableText" id="claims"> WHAT4JWE CLAIM IS

1. A microencapsulated agriculturally active agent comprising (i) an agriculturally active agent, and (ii) a polymeric encapsulating agent comprising lignin or a lignin compound.

2. The microencapsulated active agent of Claim 1 wherein said active agent is an insecticidal agent.

3. The microencapsulated active agent of Claim 2 wherein said insecticidal agent is an insecticidal pathogen including a virus, bacterium or fungi which infects insects.

4. The microencapsulated active agent of Claim 3 wherein said insecticidal pathogen is a nuclear polyhedvosis virus.

5. The microencapsulated active agent of Claim 3 wherein said insecticidal agent is Bacillus thurlnaiensis Bacillus sphaericus. Bacillus Popilliae or a recoaMnantly produced pathogenic microorganism.

6. The microencapsulated active agent of Claim 4 wherein said nuclear polyhedrosis virus is a nuclear polyhedrosis virus of H$li9thlg Z2&, Lvmantria difiEOE, Org la psmlptgyqata, Hertforign Bert if ar. or Apographs aslltsuaiSA'

7. The microencapsulated active agent of Claim 2 wherein said insecticidal agent is a chemical toxin. 21 1

8. The microencapsulated active agent of Claim 1 wherein said encapsulating agent further comprises a modified acrylic acid polymer.

9. The microencapsulated active agent of Claim 8 wherein said modified acrylic acid polymer has a positive or negative charge and said active agent has a charge opposite to that of said polymer.

10. The microencapsulated active agent of Claim 9 wherein said active agent is hydramethylnon and wherein said polymer has a negative charge.

11. The microencapsulated active agent of Claim 1, wherein said polymeric encapsulating agent further comprises a polymer synthesized from acrylic and methacrylic acid esters.

12. The microencapsulated active agent of claim 11 wherein said acrylic/methacrylic acid ester polymers are Eudragit L, Eudragit S, Eudragit RL and Eudragit RS.

13. Tha microencapsulated active agent of Claim l comprising substantially 10-30% by weight of said lignin or lignin compound. 14..

The microencapsulated active agent of Claim 13 comprising substantially 20-30% by weight of said lignin or lignin compound.

15. A process for preparing & microencapsulated agriculturally active agent comprising the steps of: 22 - 242613 f (i) nixing (1) an encapsulating agent conprising lignin or a lignin compound, (2) an agriculturally active agent in a form compatible with microcapsule formation and (3) an aqueous solvent or cosolvent system which is miscible with water and which dissolves lignin or * ... the lignin compound to form a first nixture; p (ii) preparing an emulsifying solution comprising a surfactant and a liquid which is immiscible with said aqueous solvent or cosolvent system; (Hi) preparing a solution to harden the capsules; (iv) mixing the first mixture and the emulctifying solution to form an emulsion; (v) mixing the resultant emulsion with the hardening solution to harden emulsion droplets to form capsules; and (vi) concentrating and separating the capsules from the water insoluble liquid.

16. The process of Claim 15 wherein said aqueous solvent or cosolvent system which is miscible with water comprises ethylene glycol, polyethylene glycol, isopropyl alcohol, acetone or a mixture thereof. I

17. The process of Claim 15 wherein said active agent is an insecticide.

18. The process of claim 15 wherein said encapsulating polymer is lignin amine or lignin amine sulfonate.

19. The process of claim 15 wherein said solvent is polyethylene glycol, isopropyl alcohol, or acetone. - 23 - °>\ ijuzyli

20. The process of claim 15 wherein said emulsifying agent is a liquid hydrocarbon.

21. The process of claim 19 wherein said liquid hydrocarbon is vegetable oil or kerosene.

22. The process of Claim 15 wherein said encapsulating agent is lignin and said hardening solution comprises water and a surfactant.

22. /\n agriculturally active agent as claimed in any one of claims 1 to substantially as hereinbefore described with reference to any example thereof.

24. A process as c<aimed ir. any one of claims 15 to 22. substantially as hereinbefore described with reference to any example thereof.

25. A microencapsulated agriculturally active agent when_produced by a process as claimed in any one of the claims 15 to 22 and 24substantially as hereinbefore described with reference to any example thereof. itm LabOffttdnts Int By the authorised agente A J PARK & SON t- ft 24 </div>