BACKGROUND AND RATIONALE FOR THE DEVELOPMENT OF

  HYDROCAPSULES^® FOR BIO-PESTICIDE APPLICATIONS

by Ara Manukian

 CEO/President of ARS, Inc.

The need to maintain global market competitiveness between the world’s agricultural producers has led to the increasing use of chemical pesticides throughout the world.  In the ten years alone between 1988 and 1998, the total world use of chemical pesticides has practically doubled.  In 1988, the world market value of insecticides used for crop protection was estimated at $16 billion dollars annually (Jutsum, 1988).  Recently, this world market value for the pesticide industry totaled $30.6 billion in 1996 and $31 billion in 1997 and is expected to continue to grow at a rate of 5.5% per year.  Between 1995 and 1996 the pesticide industry grew 3.6% with the highest growth in South America.  Most sales were in North America with approximately 30.6% of the world’s sales followed by 25% in Western Europe, and 22.9 % in East Asia (STAC, 2000).

Even though chemical pesticide use may be declining in several developed countries due to political and environmental legislation along with the growing use of alternative forms of pest control, such as transgenic plants, many of the emerging economies are still increasing their use of chemical pesticides. As an example, China has nearly doubled its pesticide usage in just four years.   Chinese  pesticide production went from 230,000 tons in 1995 to 424,000 tons in 1999.  Along with this increased use of pesticides has also come an increase in pesticide poisoning.  China alone has somewhere between 7,000-10,000 deaths each year  attributed to pesticide poisoning (STAC, 2000).  Besides the acute effects of direct contact with chemical pesticides, there are growing concerns of the long term effects that pesticide residues on foods have on people and animals in addition to concerns of contamination of drinking water sources and the environment.

Usage of traditional chemical-based toxic pesticides must obviously be decreased; however, this will not occur until cost effective alternatives become available.

In response to legislative and consumer pressures, the pesticide industry has been moving more towards the use of “green” bio-pesticides and away from the use of conventional chemical pesticides.  This trend has been helped by the increased development and use of bio-pesticides such as genetically modified plants, botanical pesticides and microbial organisms.  The U.S. bio-pesticide market alone is expected to grow to $2.95 billion by 2005.  The areas with the fastest growth will be for insect growth regulators (IGR; hormones that affect a pest-insects body functions or growth) and transgenic plants (genetically engineered plants that contain genes for toxic substances, which are secreted by the plant to kill the pests) mainly due to heavy development that has been done in these areas over the past several years (STAC, 2000).  Projected usage of microbial agents is quite low in comparison.  This is partially due to the difficulty and/or costs associated in mass producing several of the different organisms; however, probably one of the biggest common problems is the lack of ability to effectively deliver these microbial agents in a cost efficient manner.

As a part of the many ongoing efforts to achieve improved bio-rational pest management as a whole, ARS’s Hydrocapsule^® technology could help to increase to use of microbial entomopatogens as effective bio-pesticides by delivering entomopathogenic organisms, such as bacteria, fungi, viruses and nematodes,  in a more efficient and cost-effective manner than what is currently available for large scale agriculture use.

Current problems with using insect pathogens for insect control:

Currently, many microbial entomopathogens have had limited success when used as bio-pesticides in large scale agriculture.  This can be typically attributed to one or two reasons: (1) the inefficiency of infecting pests with the pathogen (i.e. low probability of an insect encountering or ingesting the pathogen); and (2) the short-time viability of the pathogen after application (“field-life”).  Many of these agents are unstable in the ambient environment and breakdown rapidly due to ultraviolet (UV) radiation exposure, extreme temperature and pH ranges, desiccation, nutrient depletion, or attack by other microbial or fungal agents  (Weeden et al.,1996; Hoffman and Frodsham, 1993)

Solutions to Problems:

ARS’s Hydrocapsule^® technology can address both these issues and attempts to remove these limitations for  two representative entomopathogenic bio-pesticides (fungi and nematodes) are being researched.  (1) By combining these entomopathogens with attractants and/or phagostimulants, a pest insect can be brought directly to the source of the pathogens, thus dramatically increasing the chances of infecting the targeted pest. (2) By concentrating specific entomopathogens near a pest infestation and maintaining this level or concentration of active pathogens for a sustained period of time by sequestering the active agents  from environmental degradation, the  probability of insect contact with a viable pathogenic agents is increased.

Should this approach be done at a cost which is comparable to or cheaper than traditional chemical pesticides, we now have a very powerful alternative to using chemical insecticides. This would constitute a true enabling technology, and could lead to the generation of several new “green” products that will be highly effective for pest control while eliminating  risks to both humans and the environment.

Benefits to Nation and the World:   

The development of environmentally sound and effective bio-insecticides will have great benefits to all Americans from government to the average citizen.  With the passage of the Food Quality Protection Act of 1996, it is anticipated that many currently registered pesticides will no longer be available or will have their uses limited, as evidenced by the recent banning of DURSBAN^® along with many other commonly used pesticides.   It is vital to the nation’s economy that farmers have a means of protecting their crops, while fulfilling the requirements of the new laws.  This can only be accomplished though the development of new types of insecticides to replace those that are based on chemicals that are known to be health and environmental hazards.  Entomopathogens delivered by an effective method to target pests can help do this and be very  safe.  They are also pest-specific in that they will not harm non-targeted beneficial species and have no affect on humans, pets, wildlife or fish.  The applications are unlimited.  Not only can these bio-rational insecticides have widespread agricultural use for numerous insect pests, they can also be used in urban settings such as homes, schools, food preparation facilities, hospitals, etc.  Consumers will benefit from less physical contact with toxins and have a safer drinking water and food supply free from chemical residues.

Protection of natural resources and the environment:

The widespread use of entomopathogenic insecticides would greatly reduce ground and surface water contamination and damage to native habitat by reduction in the use of traditional toxic chemical pesticides.  Another significant benefit to using pest-specific bio-pesticides such as entomopathogens is the elimination of the non-discriminatory killing of other beneficial insects and organisms from the environment.  Another equally import benefit in using bio-rational pesticides is the reduction in pest resistance to traditional chemical pesticides.   Compared to the early 1970's,  pesticides are now being applied two to five times more a year in order to accomplish what one application used to do.  “While pesticide use has risen, crop losses to pests have not declined, and clear signs have emerged that chemical approaches do not work as well as they used to.  More than 500 insect pests, 270 weed species and 150 plant diseases are now resistant to one or more pesticides” (Benbrook et al., 1996).

The creation of a safer food supply by reduction of toxic pesticide residues:

There is great concern about the cumulative amount of pesticides that Americans are receiving in the food supply.  Annual surveys by the USDA’s Agricultural Marketing Service (AMS) provide clear evidence that consumers are increasingly exposed to mixtures of pesticide residues in their diets (USDA, 1995, 1996; Benbrook et al., 1996).   In 1993, AMS examined 7,328 food samples from consumer markets and found residues of 58 different pesticides.  Almost all were insecticides and fungicides applied on fruit and vegetable crops.  Almost half of them contained two or more pesticide residues (Benbrook et al., 1996).   The concern is great enough that new legislation, The Food Quality Protection Act,  has been enacted into law to address this public health hazard.  The replacement of traditional chemicals with effective alternative bio-rational pesticides will help to achieve a safer food supply.

Enhancement of the quality of life, particularly for rural areas:

The use of safer bio-pesticides, will increase the quality of life for farmers, workers and their families who are constantly exposed to high levels of toxic chemical pesticides by reducing the health risks associated with their use.  Additionally, the handling and usage of entomopathogenic bio-pesticides will create new and convertible job opportunities in packaging, distribution, storage and application of bio-pesticides.  Finally, it would probably be safe to assume that a considerable amount of personal satisfaction would be felt by most persons who use and/or live in the surrounding area where bio-rational pesticides were applied as opposed to toxic chemical pesticides.

Estimated Costs of Approach Relative to Benefits:

If the development and utilization of entomopathogenic-based insecticides is ultimately effective, the benefits will far outweigh the costs; however, ideally they should be equal to or lower than the cost of traditional pesticides. The true cost of implementation  will largely depend on the methods used to mass produce the specific entomopathogens and not by the Hydrocapsule encapsulation process itself.  The registration of encapsulated entomopathogenic insecticides should fall under the EPA “fast-track” registration process for bio-insecticides which should be faster and less expensive to register than traditional toxic chemicals.  Liquid suspensions of these encapsulated insecticides can be applied using existing farm spray rigs and dry spreaders filled with capsules with only minor modifications, so that minimal additional costs would be incurred by growers for new equipment.  Indirect costs to the public from human health and environmental risk will be greatly reduced with the development of a new class of safe and effective insecticides.  The long term reduction of toxic residues from food supplies and drinking water is of immeasurable benefit to human and wildlife.

Specific Policy Issues Affected the Implementation of Bi-Pesticides:

Problems associated with the wide-spread use of chemical pesticides have become so significant in the last few years that several national policy issues concerning the use of these pesticides in U.S. agriculture (and throughout the world) have been the topic of many recent legislative actions. Stricter enforcement and implementation of these actions have failed mainly due to the lack of reasonable alternatives:

In November 1992, countries attending the Montreal Protocol meeting agreed to freeze production of all ozone-depletory materials and specifically methyl bromide; however, this action has been postponed due to lack of viable alternatives.  In June 1993, the Clinton administration announced a new national agricultural policy directly affecting the way growers deal with pest management which requires them to increase their use of biological control strategies.

In November 1993, the EPA issued a ruling under the US Clean Air Act (CAA) freezing all new production of methyl bromide in 1994 and requiring complete elimination by January 2001. This action is also being delayed for lack of assured alternatives.  In December 1994, the U.S. Department of  Agriculture  put into action a three year initiative calling for increased research and development and implementation of pesticide reducing technologies.

In August 1996, the  President signed into public law “The Food Quality Protection Act (FQPA)” (H.R. 1627 Public Law 104-170), which tightly regulates the use of all pesticides. 

In August 1999, the EPA announced eliminating the use of methyl parathion and azinphos-methyl (two commonly used organophosphate pesticides).  By the end of 2000, the EPA is scheduled to complete its reassessment of 39 more commonly used pesticides, including atrazine, aldicarb, and carbofuran in order to meet the goals stated in the 1996 FQPA (EPA, 1999). 

And just recently in 2000, the EPA has banned any future production and use of the pesticide chlorpyrifos (trade name DURSBAN^®) which is one of the most widely used agricultural and household lawn pesticides, and has left many consumers and businesses looking for suitable substitutes for their pest control applications.

As fewer pesticides are registered and the standards for use become more stringent, the need for new environmentally-sound (“bio-rational”) insecticides will increase dramatically.  The development of new, effective bio-rational insecticides has become a top priority for the EPA, which has the goal of doubling the number of registrations of safer bio-pesticides by the year 2005 (EPA, 1997).

If an entirely new class of insecticides based on entomopathogens becomes available in the marketplace that is competitive in cost and effectiveness to traditional chemical insect controls, this would have an enormous impact on current pesticide policy.  Effective bio-rational pesticides would make it possible for the EPA to reduce even further the number of registrations of new chemicals and severely limit the uses for currently registered toxic pesticides given a viable alternative.

Entomopathogens as bio-rational insecticides:

One good approach for chemical-alternative insecticides is the use of insect pathogens or “entomopathogens” as a bio-rational method of controlling pests.  Entomopathogens  are naturally-occurring disease-causing organisms such as protozoa, bacteria, fungi, and nematodes which specifically  infect or vector other harmful agents (such as endotoxins) into insects causing death or disruption of its life cycle.  They are very good candidates for use as bio-pesticides since most insect pathogens are specific to certain groups of insects or certain life stages of insects.  Additionally, microbial entomopathogens generally do not directly affect beneficial insects and are non-toxic to wildlife or humans (Weeden et al., 1996; Hoffmann and Frodsham, 1993).  Entomopathogens generally infect their host (pest) insects through ingestion or by direct contact with an organism.  In either case, once the pathogen has entered the insect, it will eventually lead to the insects demise.

Encapsulation - General Considerations:

Encapsulation refers to processes whereby an active ingredient is placed into a stabilized form in order to allow it to be conveniently stored, and protected from unfavorable conditions, until needed.  The active ingredient may be dispersed in a protective matrix, or it may be surrounded by a coating, a shell, or a membrane. The release of active ingredient from the protected form may be rapid (such as by crushing, or by ingestion), or gradual (such as by dissolution, or bio-degradation).  In this manner it is possible to maximize the effectiveness of the active ingredient by ensuring that it is released at the proper time.  This “controlled release” can also be made to occur over a programmed time interval (sustained release), or on demand (stimulated release).

The term "microcapsule" has been used to describe small particles or beads, which range in size from less that one micron, up to several millimeters, which may contain a wide variety of active ingredients (Thies, 1994; Thies, 1987; Goodwin,1974; Deasy, 1984; Hegenbart, 1993).  Microcapsules can be divided into two broad groups:  (1) “Aggregate” type microcapsules have the active ingredient dispersed uniformly throughout a continuous matrix.  The matrix may be a solid dry polymer or a gel swollen with solvent.  In the case where the gel is swollen with water, the term “hydrogel” is applied.  Hydrogel encapsulation systems of this type are generally based on cross-linked forms of water-soluble polymers such as alginate, gelatin, pectin, agar, gellan, or starch (Sanderson, 1989).  (2) “Mononuclear” microcapsules, on the other hand, consist of materials which show a true "shell-core" morphology.  These are similar to an egg in that they have a solid shell or flexible membrane surrounding a core which may be a liquid,  a solid, or even a gel.

Encapsulation of Entomopathogens using ARS, Inc.’s “HYDROCAPSULE^®” Technology:

Encapsulations done by ARS, Inc. produce capsules that are of the shell-core type, and consist of a polymer membrane surrounding a liquid center.  The key feature of ARS’s mononuclear microcapsules is that they contain a water-based core.  Other types of processes, such as the familiar “softgel” technology used to encapsulate vitamin E are not suitable for encapsulating aqueous liquids (US Pat#: 4,744,988; Rose, 1987).  The shell materials produced by ARS Inc's. unique encapsulation process (patent pending) are cross linked hydrophobic elastomeric networks.  These shells are produced via the ultraviolet (UV)-initiated free-radical copolymerization of functionalized prepolymers (silicones, urethanes, epoxies, polyesters, etc.) and vinyl monomers such as acrylates.  Because the structure of these types of capsules is very distinct from the softgels or  aggregate-type hydrogel microcapsules described above, they are referred to as “Hydrocapsule^®”.  This implies that they have an aqueous liquid core surrounded by a hydrophobic membrane. This method is also used with non-aqueous solutions such as oils.

Entomological HYDROCAPSULE^® Encapsulation Research done by ARS, Inc:

ARS, Inc. under previous USDA SBIR Phase I & Phase II funding (1996 & 1997) recently developed patent-pending methods to encapsulate various insect diets (Greany and Carpenter, 1999) using proprietary Hydrocapsule^® technology.  In this form, the encapsulated artificial diet (a suspension of nutrients containing proteins, carbohydrates, and lipids) remains stable and sterile despite its high water content (50-75%).   The size range of Hydrocapsules currently produced by ARS, Inc.'s. encapsulation technology is from 100 microns up to 2 cm diameter, with a typically size of approximately 2 to 3 mm.  Wall thicknesses generally range between 10 to 100 microns as measured by scanning electron microscopy (SEM).  The mechanical properties of ARS Hydrocapsule^® can range from hard and plastic-like, to soft and flexible, like silicone rubber.  Membranes thin and soft enough to be punctured by a human hair have been produced.  These softer membranes were developed to feed insects with piercing-sucking mouth parts (such as the spined soldier bug, Podisus maculiventris), and have shown the ability to penetrate through the capsule wall under laboratory conditions.  It has also been observed that many species of insects with chewing mouth parts are capable of eating through the harder/thicker shell formulations of ARS's capsules (ants, mites, cockroaches, and lady beetles).   Observations indicate that roaches and lady beetles actually consume the entire non-toxic ARS Hydrocapsule^® shell along with the contents. This is an unexpected benefit which has favorable environmental consequences.

These same capsules which have been successfully used to feed beneficial insects may be converted into lethal snacks for pest insects, simply by incorporating the entomopathogenic agents into the aqueous or oil-based solution core.  For applications such as cockroach and fire ant baits, it would not be necessary that the shell be soft to allow the entrapped organisms to escape on their own, since the shells are easily breached by the feeding insect.  In fact, the capsule can serve as a convenient package which allows the target insect to carry the infective agent directly into its nest.  This feeding behavior has been observed in studies involving wild fire ants feeding on encapsulated artificial diet at the USDA-ARS Center for Medical, Agricultural and Veterinary Entomology and the University of Florida (both located in Gainesville, Florida).  Incentive to feed on the capsules could also be provided by incorporation of essential nutrients, or by the addition feeding stimulants or kairomones.  For more passive delivery approaches, it is possible to adjust the shell formulation to the needs of a particular system, so that the entomopathogenic agent can emerge without direct contact between the insect and the capsule.  In fact, a combination of the two release mechanisms may serve to be the most effective approach.

Preliminary Encapsulation Studies of Entomopathogens:

ARS  Inc., has  conducted a several preliminary experiments during the past year in order to determine the suitability of our proprietary encapsulation process for delicate and environmentally-sensitive microorganisms.  Several samples of commercially-available beneficial nematodes, Steinernema feltiae, were successfully encapsulated by our process (See Figure 4 below).  These nematodes were supplied to us by Dr. Albert Pye from the BioLogic Company (Willow Hill, PA).  A water suspension of these nematodes having a concentration of 500 million active units (AU) / 3 Liters was used.   Initial observations made immediately after encapsulation showed almost zero mortality.   The encapsulated nematodes were then stored dry in a container kept in a refrigerator and checked every week.  Under these conditions, these the majority of nematodes remained alive encapsulated in water-filled Hydrocapsules^® for almost one year using oxygen permeable coatings.

Other successful preliminary experiments involved encapsulating a fungal pathogen, Beauveria bassiana 447 (ATCC 20872) given to us by Dr. Jerry Stimac of  the University of Florida Dept. of Entomology (Gainesville, FL), for use in controlling several pest ant species.  The fungi were encapsulated in Hydrocapsules^® and returned to Dr. Stimac’s lab to test the survivability of the encapsulation process.  Their results showed that over 80% percent of the fungi remained viable.  Additional tests are planned including the encapsulation of the fungi in various pure oil-based solutions and to test insect foraging behavior.

A similar initial experiment was conducted using a commercially available bacteria, Bacillus thuringiensis (Bt), suspension (Thurcide HPC).  Subsequent plate cultures done on blood/agar media revealed substantial proliferation of viable bacteria from both as-received, and encapsulated Bt  suspension.   These results; however premature, are very promising.  Follow-up insect bioassays need to be performed to test the pathogenicity  The initial bacteria growth tests where done in the pathology/microbiology  laboratories  at SHANDS Teaching Hospital at the University Florida, Gainesville, FL.   Our encapsulation process is currently undergoing bacteria survivability studies using several different UV-susceptible bacteria species by an independent outside laboratory.

Commercialization of ARS Inc.’s HYDROCAPSULE Technology:

There is high commercialization potential for this technology.  Many entomopathogens are currently being used as bio-insecticides in small applications (such as greenhouses, small organic gardens, and homes) with various degrees of success; however in all cases, if the targeted pest insect is actually infected by the pathogen, it will surely be killed or weakened.  What will allow this pest control strategy to become widely accepted and implemented in large scale agricultural (1,000+ acre farms)  is  increased efficiency and efficacy in treatment along with lower costs.  By encapsulating entomopathogens with controlled release and/or attractive  properties this could occur and a new form of bio-rational insecticides could be achieved.

Not only do entomopathogenic  insecticides have great potential for agricultural uses, but they also could be used for urban pest control applications where use of toxic chemicals would be undesirable such as in schools, hospitals, food preparation establishments, homes and public buildings.   Post-harvest applications are also likely.  Encapsulated entomopathogens with controlled release formations may have great utility for stored grains and other stored commodities where longer exposure periods are needed to infect pest populations.

Entomopathogenic insecticides will have great benefits over traditional pesticides for environmental and human health concerns.  There are few safe and effective insecticides on the market to replace chemical controls such as organophosphates and carbamates.  Farmers have few choices for insect control in minor crops and bio-rational pesticides could provide relief to growers.  Because of the mode of action, it is unlikely that insects will develop resistance to entomopathogens as they do traditional insecticides.

It is the aim of Analytical Research Systems (ARS), Inc., a private research and development laboratory, specializing in emerging new bio-technologies, to develop, patent  and license newly developed bio-insecticide encapsulation processes to other companies such as larger pesticide/chemical producing companies for the production, marketing and distribution phases of commercialization of these products to the consumer and agricultural markets.