WO2000051433A1 - Low toxicity insecticides - Google Patents

Abstract

Insecticide formulations incorporating oil, diatomaceous earth (DE) includes an organophosphate insecticide are synergystically efficacious at reduced application rates of organophosphate insecticide. One formulation includes approximately 75 % DE, 21 % oil and not more than 3 % organophosphate insecticide. Another formulation incorporates oil, DE, an organophosphate insecticide, a pyrethroid insecticide and a synergistic compound co-operating with the pyrethroid insecticide in an insecticidally efficacious formulation including approximately 75 % DE, 21 % oil, not more that 1 % organophosphate insecticide and not more than 0.05 % pyrethroid insecticide.

Description

LOW TOXICITY INSECTICIDES

Field of the Invention

This invention relates to insecticide formulations and, in particular, insecticide formulations incorporating diatomaceous earth (DE).

Background of the Invention

Insect infestation is a major contributor to physical, nutritional, and deterioration of cereals, pulses, roots and tubers which are primary dietary constituents of many countries. Substantial quantities of stored foods are destroyed on an annual basis by storage pests. A number of the disinfestation technologies which are effectively applied in the developed world cannot be applied in the developing world owing to high costs coupled with socioeconomic factors. The use of traditional storage protectants (plants and inert materials) therefore continues to be increasingly explored and exploited for the control of pests in stored food products in the developing world. Research into the use of traditional protectants applied in stored food protection is currently focused on:

• Methodologies used in the application of traditional storage protectants

• The potentials and constraints of effectively using storage protectants at farm, domestic, and commercial levels

• The potential for further development of traditional storage protectants to enhanced their efficacy and facilitate their more widespread use.

The grain industry in many countries operates under regulations stating that stored grain must be free from detectable insect pests. The presence of pests in grain or on the premises, or the detection of insects pests in grain being loaded for export can results in extreme time delays and expenses for the companies involved. In order to minimize qualitative and quantitative losses of stored cereals, pulse crops, oilseeds and their by-products, effective methods of prevention, detection and control of pests must be used. Infested grain is typically treated with pesticides to control pest (usually insects) populations.

Two categories of traditional protectants are utilized to combat insect infestation in stored foods. These include: (i) plant extracts (botanical insecticides) which effect their pest control activity through antifeedant, repellent, ovicidal and pesticidal mechanisms; and (ii) inert materials such as diatomaceous earth, ashes, charcoal, fine sand, soaps and oil which inhibit insects by physical mechanisms.

The most significant botanical insecticides include nicotine, rotenone, sabadilla, ryania, limonene, neem and pyrethrum types. Some are particularly interesting from many points of view such as efficacy, spectrum of activity, safe usage, etc. However, because of the high price of plant extracts and the uncertainty of their production on yearly basis, for the time being we excluded them as potential synergists in the new formulations.

Some alternative low cost, low toxicity compounds to control stored -product insects rely on diatomaceous earths (DE) - very likely the most effective naturally occurring insecticides. DE has long been known as a potentially useful grain protectant because it is safe to use, does not affect grain end-use quality, provides long-term protection and is comparable in cost to other methods of grain protection. Over the years DE use had been limited because the required dose rates of 1000 to 3500 ppm (parts per million or mg per kg, or grams per tonne) for most DE products significantly reduced grain bulk density, flowability and left visible dust residues. In US Patent 5,773,017, 1 describe a diatomaceous earth insecticidal composition that can be used at lower concentrations with acceptable efficacy against insects, and has reduced adverse effects on grain handling and bulk density.

Oils are one of the oldest natural pesticides. Petroleum oil (also called mineral oils) have long been used as a dormant spray to protect plants against scale insects, aphids, mites and other pests. Today, most commercial horticultural oils continue to be petroleum based. However, recent research with vegetable oils indicates a promising future for plant oils as well.

For many years, throughout most of the world, insect pests in stored grain have been primarily controlled by direct application of protective chemicals as the grain was loaded into the storehouse. Pesticide concentrations were sufficient to provide residual control during most of storage period. This preventive approach for insect pest management was favored because of its simplicity, the sensitivity of stored grain to insect infestation during the first few months in storage, the difficulty of adequate sampling in large storage granaries (it is difficult to determine the extent of insect infestations), and the requirements of stored grain marketing channels. Supplemental control during the storage season was mainly accomplished by a fumigation which is supposed to kill the existing pest populations but provides no residual and/or extended protection.

Currently there are only a few synthetic chemical products that are permitted for controlling insects in stored grain products. Some chemicals used to control stored- product insects (e.g. malathion, chlorpy fos-methyl, pirimiphos-methyl, fenitrothion) must be applied directly to the grain, and hence, residues can be ingested by consumers. Given that the effects of consuming low levels of insecticide residues for many years are unknown, and the effects may only appear after years of exposure, there is a desire to have foods free of pesticide residues or with as low as possible residue levels.

Compounds such as malathion (which has been used for 50 years) are becoming ineffective due to insect resistance requiring unacceptably high application rates (and hence residues) for control.

Only those insecticides that have been specifically approved for use on and around grain should be used. Therefore, to ensure absolute safety of food commodities, very strict requirements limit the actual choice of insecticides permitted for commercial use. To qualify as a grain protectant candidate, a given insecticide must comply with the llowing requirements prescribed by the Food and Agricultural Organization (FAO) of he United Nations in 1982: it must be effective at economical application rates it must be effective against a wide variety of insect pests it must be capable of being used without hazard to operators its use must be acceptable to health authorities it must present no hazard to consumers of grain and grain products • it must not effect the quality, flavor, smell, or handling of grain it must not be flammable, explosive, or corrosive its method of use must be compatible with established grain-handling procedures.

The number of insecticides registered for application on stored grain through the world is limited. Compounds that are currently being used for treating stored grain and other stored commodities are known through their generic names such as bioresmethnn, bromophos, carbaryl, chlorpyήfos-methyl, dichlorvos, deltamethrin, et mfos, fenitrothion, fenvalerate, malathion, methacrifos, methoprene, permethrin, phenthhn, pirimiphos-methyl, piperonyl butoxide, and pyrethrins. Certain other compounds have been subject to extensive studies and appear to fulfill the stringent criteria for approval as grain protectants.

The application of pesticides is under scrutiny due to problems related to the toxicity and possible carcinogenicity to humans, contamination of the work space and environment (in particular by ozone-depleting substances such as methyl bromide and other halocarbons), resistance of pests to insecticides, public concerns about chemical residues in commodities, and general market/consumer aversion to chemicals. This presents opportunities for the development of low-toxicity chemicals and physical pest control strategies. Physical methods of pest control in grain storage are becoming increasingly important. Physical control of insect pests involves the manipulation of physical factors to eliminate pests or reduce their populations to a tolerable level. Temperature, relative humidity, atmospheric composition, impact, desiccation, physical exclusion, removal, and ionizing radiation all may be employed separately or in combination . However, in comparison to chemical methods, these alternative methods of pest control had a minor role until recently.

The United States Environmental Protection Agency (EPA), Office of Pesticide Programs, published a notice 98-7 (8/24/98) to manufacturers, formulators, producers and registrants of pesticide products about the changes to registration priority system involving organoposhpate (OP) alternatives and reduced risk candidates. The EPA Registration Division indicated that registration actions would prioritized in the following order: (i) methyl bromide alternatives, (ii) reduced-risk candidates. The newly developed formulations described herein have constituent components that belong to these two groups.

Therefore, there is an increasing need for a broader use of alternative pest control methods and/or the application of naturally occurring insecticidal compounds, as well as a need for the development of new low-toxicity (to mammalians) synthetic insecticides having a specific activity against stored product pests.

Summary of the Invention

The invention provides a new type of insecticide compatible with the following principles: safe, low toxicity, easy to apply, with minimal problems with residues, with high efficacy against stored grain insect adults pests and their progeny at very low concentrations, wide spectrum of efficacy against stored grain insect pests, low adverse effect on grain handling and quality properties, and price that is acceptable in terms of efficacy and economic viability. In one of its aspects the invention provides, an insecticidal composition comprising a mixture of diatomaceous earth, oil and an insecticide compound selected from an organophosphate insecticide group combined in a synergistic insecticidally effective amount. In another aspect, the invention includes a second insecticide compound selected from the pyrethroid insecticide group and a synergizing compound for synergizing said second insecticide compound, combined in a synergistic insecticidally effective amount.

In another of its aspects, the invention provides an insecticidal composition comprising a mixture of diatomaceous earth, oil, a first insecticide compound selected from the organophosphate insecticide group, a second insecticide compound selected from the pyrethroid insecticide group and a synergizing compound for synergizing said second insecticide compound combined in a synergistic insecticidally effective amount.

In yet another of its aspects, the invention provides: a method for producing an insecticidal composition comprising mixing oil and an insecticide compound selected from an organophosphate insecticide group to form a first intermediate mixture, mixing diatomaceous earth with said first intermediate mixture to produce a second intermediate mixture; and screening said second intermediate mixture to produce said insecticidal composition.

And in yet another of its aspects, the invention provides: a method for producing an insecticidal composition comprising mixing oii, a first insecticide compound selected from an organophosphate insecticide group, a second insecticide compound selected from a pyrethroid insecticide group and a synergistic compound to form a first intermediate mixture, mixing diatomaceous earth with said first intermediate mixture to produce a second intermediate mixture, and screening said second intermediate mixture to produce said insecticidal composition. It is an object of the invention to minimize pest infestation and to maximize safety when it is necessary to use grain protectants on various stored commodities. This will improve the storage quality of cereals, pulses, oilseeds and their products.

In order to control all stored -product insects and mites for at least 7 to 21 days, even in grain having a relatively high moisture content (up to 15%), compounds acting as synergists are included to improve the efficacy of DE-based insecticidal formulations. In accordance with the invention, new DE-based formulations, containing preferably 70 to 80%, w/w of DE with additives, such as very low concentrations of selected substances, are disclosed which have a combined mode of action against insects. The combined mode of action of these new formulations provides a synergistic mode of action which combines desiccation and poisoning. Because of this combined action, the required concentrations of DE and other substances used in these mixtures are much less than if any one component were used alone.

In accordance with the invention, formulations of diatomaceous earth and oil include substances selected from the group of organophosphate insecticides or pyrethroid insecticides and may include the synergist piperonyl butoxide (PBO). The organophosphate, chlorpyrifos methyl, the pyrethroid detamethrin and the synergist PBO are registered grain protectants in many countries of the world.

In a preferred embodiment of the invention, chlorpyrifos methyl and PBO are used at 6 ppm, 0.5 ppm and 5 ppm, respectively, for the long term protection of stored grain. In most cases, diatomaceous earth is registered to be used at concentrations of 500 to 3500 ppm. Various vegetable oils are efficient against insects when used at 5 to 10 ml of oil per kg of grain ml (approximately 5, 000 to 10,000 ppm).

Results from two different formulations are disclosed referred to as formulations F 1 and F 2. Formulation F 1 contains DE, chlorpyrifos methyl and vegetable oil. F 2 contains DE, chlorpyrifos methyl, deltamethrin, PBO and vegetable oil. Due to synergism, the formulations were found to exhibit very high to complete mortality of tested stored grain insects and their progeny at application rates of 10-20% of the rates used for control by a single active compound. For example, the highest recommended concentration of 100 ppm of the new formulations, with the best ratio of substances, contains 75.4 ppm of DE, 2 ppm of chlorpyrifos and 21.2 ppm of vegetable oil (F 1 ), or 77.4 ppm of diatomaceous earth, 0.945 ppm of chlorpyrifos methyl, 0.033 ppm of deltamethrin, 0.4 ppm of PBO, and 21.2 ppm of vegetable oil (F

2).

The new approach and the advantages of use of these new formulations are: • pronounced synergism with regards to the efficacy against insects. When the individual components of the formulations are used alone at the same concentrations used in the formulation, the mortality of test insects on treated grain is very low or not significantly different with the mortality on untreated grain. However, when the same concentrations are used in the newly developed formulations, the mortality of test insects is very high;

• when used at the recommended dosages for grain protection, the concentrations of the individual components used in the formulations are well below the tolerance residue levels set by many countries;

• the concentrations of selected synthetic substances used in new formulations for grain protection are about 16 to 30% of the recommended dosage for chlorpyrifos methyl when used alone (F 1 ) or 8 to 16% of the recommended concentrations for chlorpyrifos methyl and 6 to 15% for deltamethrin when used alone (F 2);

• the cost of using F 1 or F 2 is less than the cost of using deltamethrin or clopyrifos methyl or DE alone;

• the mixing of all substances is performed in a way that insure the long life of very low concentrations of the synthetic substances, while preserving the desiccating properties of DE; and • the role of vegetable oil is very important because (i) oil acts as a carrier of the synthetic substances, (ii) it facilitates the penetration of the synthetic substances into the body of insects, (iii) it reduces the adverse effects of DE on the handling properties of grain (test weight and flowability), and (iv) it increases the density of DE-based formulations (much less airborne dust and lower transportation cost).

The invention provides effective, environmentally safe, and economically acceptable formulations with enhanced efficacy against insects. The insecticide formulations are based on a diatomaceous earth (DE) selected for demonstrating the highest insecticidal activity mixed with very low concentrations of several selected substances including synthetic grain protectants, synergists and edible oil. The preferred formulations contain constituents selected from the following, namely: (i) diatomaceous earth, preferably, the most insecticidally efficacious diatomaceous earth having a low crystalline silica component; (ii) a substance from the organophosphate insecticide group, in particular, chlorpyrifos methyl; (ii) a substance from the pyrethroid insecticide group, in particular, deltamethrin; (iii) a synergist, piperonyl butoxide (PBO) to enhance the action of deltamethrin; and (iv) vegetable oil.

Brief Description of the Drawings

Figure 1. is a graph depicting mean loss of body mass of red flour beetle and lesser grain borer at 26 and 6 hours post-treatment by varying insecticides.

Figure 2. is a graph of depicting mean percent mortality mortality of red flour beetle and lesser grain borer at 26 and 6 hours post-treatment by varying insecticides.

Detailed Description of the Preferred Embodiments

In accordance with the invention, we prepared 2 different formulations and labeled them as F 1 and F 2. F 1 contains DE, chlorpyrifos methyl and vegetable oil. F 2 contains DE, chlorpyrifos methyl, deltamethrin, PBO and vegetable oil. Due to synergism, our formulations gave very high to complete mortality of tested stored grain insects and their progeny at application rates of 10-20% of the rates used for control by a single active For example, the highest recommended concentration of 100 ppm of new formulations with the best ratio of substances contains 75.4 ppm of DE, 2 ppm of chlorpyrifos and 21.2 ppm of vegetable oil (F 1 ), or 77.4 ppm of diatomaceous earth, 0.945 ppm of chlorpyrifos methyl, 0.033 ppm of deltamethrin, 0.4 ppm of PBO, and 21.2 ppm of vegetable oil (F 2). The following describes characteristics and the mode of action of the constituent components of these formulations.

Diatomaceous earth

Diatomaceous earth (DE) is formed from fossilized diatoms, a family of single celled algae. DE is probably the most efficacious natural dust used as an insecticide. Chemicaly, DE is 80 to over 90% amorphous silicon dioxide ("silica" or SiO₂) plus other inorganic oxides and salts. It is well known as a non-toxic inorganic compound registered as a food additive in the U. S. A., Canada and in many other countries.

Due to its drying and anti-caking properties, amorphous silica is used in many industrial products. This property also makes amorphous silica a very mild mechanical irritant of the human skin, eyes and upper respiratory tract. Crystalline silica (also known as "quartz"), in addition to acting as an irritant, is associated with a lung disease (silicosis) and is also believed to be a low-risk carcinogenic compound. Depending on the origin, diatomaceous earth may contain only <1 % (fresh water DE), or up to 7% (marine DE), crystalline silica. Based of the results of numerous tests with DE samples from different geological deposits around the world, we selected the most active DE sample against insects which contains only 0.1 to 0.3% of crystalline silica.

The primary mode of action of DE's insecticidal activity is desiccation. DE particles adhere to the body of the insect and damage the protective waxy layer of the insect cuticle by sorption. The degree of dessication largely depends on relative air humidity, the moisture content of treated grains, and the DE's sorption power for the lipids that form the water-impermeable layer covering the cuticle of insects. Depending on the rate of water loss, the insect dies when it looses about 25 to 35% of its total weight. In addition to sorption, DE is thought to cause abrasion, suffocation, repellency, etc.

Chlorpyrifos methyl

Chlorpyrifos methyl belongs to the group of organophosphate insecticides. Organophosphate is usually used as a generic term to include all insecticides containing phosphorus. They are all derived from phosphoric acid and generally, they are very toxic to vertebrate animals. Chlorpyrifos methyl, belonging to heterocyclic derivates, is an exception because it has only moderate toxicity, with a LD₅₀ (dose that kills 50% experimental animals) of 1630 mg/kg for rats (well known malathion has 2000 mg/kg for rats, but dichlorvos has only 25 mg/kg). Chlorpyriphos methyl is used in many countries as a grain protectant, due to its low toxicity and the fact that this insecticide is relatively chemically unstable (non-persistent).

The recommended concentration for long-term grain protection is approximately 6 mg active ingredient per 1 kg of grain (6 ppm). Maximum limits for chlorpyrifos methyl residues in grain , processed grain or oilseeds set by the Food and Agriculture Organization and World Health Organization of the United Nations are: flour and whole meal bread - 2 ppm; maize, sorghum and wheat - 10 ppm; and wheat bran - 20 ppm. The tolerance for chlorpyrifos methyl residues in the United States and Canada for barley, oats and wheat is 6 ppm. The concentrations of chlorpyrifos methyl in F 1 and F 2 formulation used for the protection of grain are from 0.46 to 2.0 ppm, depending on the commodities and the insect species, that are well below tolerance limits;

Organophosphates in general, and chlorpyrifos methyl in particular, inhibit the activity of cholinesterase enzymes in the nervous sustem. This inhibition results in the accumulation of acetylcholine, which interferes with the neuromuscular junctions, producing rapid twitching of voluntary muscles and finally paralysis. The death of insects occurs very quickly.

Deltamethrin

Deltamethrin belongs to the fourth group of pyrethroid insecticides. It is a synthetic version of one of the six natural pyrethrins produced by the chrysanthum flower. The characteristics of this group include pronounced stability under exposure to ultraviolet light, and extremely low rates of application (making them safe to use). Deltamethrin belongs to the group of pyrethroids that have a negative relationship between temperature and toxicity. They are more effective when the temperature is lowered. In several countries, deltamethrin is used as a grain protectant at a recommended dosage of 0.5 mg/kg of grain (0.5 ppm) for the long term protection of grain. The LD₅₀ for rats (oral) is 128 mg per kg. However, the very low concentrations used for grain protection insure safe use of this insecticide. The maximum limit for deltamethrin residues in oilseeds is 0.1 ppm (FAO/WHO). The highest concentrations of deltamethrin in F 2 formulation needed for the protection of grain is 0.033 ppm. This substance does not belong in any of the hazard categories (immediate health hazard, delayed health hazard, fire hazard, reactive hazard and sudden pressure release hazard; according to the MSDS of the producer).

Deltamethrin affects both the peripheral and central nervous system of the insect. In general, pyrethroids initially stimulate nerve cells to produce repetitive discharges and eventually cause paralysis. It is likely that the toxic action of pyrethroids is primarily due to its blocking action on the nerve axon, since this action shows a negative temperature coefficient. The ganglia of the central nervous system appear to be affected. Death of the insect occurs after prolonged exposure to pyrethroids.

Piperonyl butoxide (PBO) Piperonyl butoxide (PBO), an insecticide synergist, enhances the activity of many insecticides, in particular pyrethrins and pyrethroids. PBO minimizes the amount of these relatively expensive insecticides required for efficient insect control. As it is not stable in air and light, PBO should be stored in the dark in the original package. Under normal use, PBO is considered to be relatively risk-free. The oral LD₅₀ for rabbits and mice is > 2,500 mg/ kg. Like pyrethroids, PBO is synthetic compound that was originally isolated from a plant extract (Agave).

The function of PBO is to optimize the effect of insecticides. Its mode of action is the inhibition of the activity of enzymes produced by microsomes. These enzymes are involved in the rapid degradation of the insecticide in the body of insects. The activity of PBO prevents the recovery of deltamethrin treated insects.

Vegetable oils

Horticultural oils appears to be an effective alternative to more toxic synthetic pesticides in some circumstances. The most widely used commercial products are currently petroleum based formulations. Potential disadvantages of horticultural oils include phytotoxicity, toxic impurities, re-registration requirements by the USA EPA, etc.

However, the insecticidal effect of different vegetable (edible) oils make them promising candidates for pest control. Mixing grain with plant oils is an ancient Indian and African method of protecting against insect attack. Recently an increasing number of plant oils (corn, soybean, groundnut, coconut, sunflower, sesame, flax, olive, etc.) have been screened for preventing post-harvest losses due to stored grain insects. Rates of 5 to 10 ml of oil per kg of grain have a good insecticidal and ovicidal effect.

The mode of action of oils is still not completely understood, but it is believed that the effect is both physical and chemical. For example, it is obvious that the action of oils is probably more complex than simple physical interference with adult (or egg) respiration. Adult insects deprived of oxygen survive longer than those treated with oils. When used as a carrier for other insecticides, oils also aid in the distribution of the active substances over the insects' surface, and they accelerate the penetration of the toxins through insect's cuticle. Lower viscosity oils show the greatest penetration of the insect's cuticle.

The role of vegetable oil is very important because (i) the oil acts as a carrier of the synthetic substances, (ii) it facilitates the penetration or sorption of the synthetic substances into the body of insects, (iii) it reduces the adverse effects of DE on the handling properties of grain (test weight and flowability), and (iv) it increases the density of DE-based formulations (much less airborne dust and lower transportation cost).

Processing technology of the new formulations

F 1 Formulation

Constituent Materials

(a) Diatomaceous earth (DE)

The diatomaceous earth is produced in the USA by Eagle-Picher Minerals, Inc. It is exempt from residue tolerance limits in most countries. This is a natural DE with following physical and chemical properties: Color Beige

G.E. Brightness 65

Sieve Analysis (Tyler)

% + 150 mesh ( < 105 microns) n/a

% + 325 mesh ( < 44 microns) 0.3 Median Particle Diameter (microns) 5.0 pH ( 10% slurry) 7.0

Free Moisture (maximum % H₂O) 5.0

(typical % H₂O) 3.0

Density (g/L) Wet bulk 530

Dry bulk 175

Specific gravity 2.00

Refractive index 1.46

Oil absorption (Gardener Coleman) % by wt 130 Water Absoprption (Gardener Coleman) % by wt 140

Chemical analysis

SiO₂ 83.7%

AI₂O₃ 5.6%

Fe₂O₃ 2.3% CaO 0.9%

MgO 0.3%

Other oxides 2.2%

Loss on Ignition 5.0%

(b) Chlorpyrifos methyl

The chlorpyrifos methyl compound investigated is sold under the trade name Reldan 6 Insecticidal Concentrate. Reldan Insecticidial Concentrate is produced by Dow AgroScience Ltd., United Kingdom. The concentrate contains 60.6% of active ingredient (a.i.) and the rest is xylene as a balance. It is a light amber liquid with relative density (water =1 ) of 1.2 g. cm³ (approximately). The boiling point is 138 °C. It is stable under normal storage conditions.

(c) Canola oil This vegetable oil, produced from canola seeds, was obtained from a local supermarket. As a food product, it is exempt from residue tolerance limits. Density is estimated to be 0.85 g/ml.

Preparation of 100 grams of F 1 formulation

The goal was to prepare a DE based formulation that can control stored grain pests rice weevil and red flour beetle at very low concentrations, from 50 to 100 ppm. This formulation is not recommended to control lesser grain bore which is extremely tolerant species against chlorpyripfos methyl. If F 1 is used for the control of lesser grain borer, the recommended concentration is >100 ppm.

The following procedure was used to produce 100 grams of F 1 :

REDO Formulation

a) 3.3 ml of Reldan^* Insecticidal Concentrate is put into a containerthat contains 11 ml of canola oil. The container is shaken by hand for few seconds.

b) After mixing Reldan with oil, 30 grams of DE is put into a container. Substances in the container are mixed with a spatula for 2 minutes.

c) The mixture described in b) is screened through a No. 30 laboratory sieve (600 microns). A brush is used to break apart clumps of the oil/DE mixture. The mixture is mixed by shaking in a closed container for 30 seconds.

d) DE is added to the container to bring the total mass of the mixture to 100 grams. The mixture is thoroughly shaken by hand for 1 minute.

e) The formulation described in d) is again screened through the lab sieve No. 30 by aid of a brush. f) After screening, the formulation is again mixed thoroughly by hand in a closed containerfor 1 minute.

The result of the described process is a new formulation, labeled as F 1. This procedure can be easily mechanized for commercial production.

The composition of 100 grams of F 1

Substance Procedure Density Composition (g) Grams a.i.

Reldan 6E 60.6% 2.8 ml 1.2 g/ml 3.36 2.03

Oil 25 ml 0.85 g/ml 21.25 21.25

DE 75.39 g - 75.39 75.39

Total 100.0

F 2 formulation

Materials

a) Diatomaceous earth (DE)

The diatomaceous earth used in Formulation 2 is the same as was described in relation to formulation F 1.

b) Chlorpyrifos methyl

This insecticide is the same as was described in relation to formulation F 1.

c) Canola oil

This substance is the same as was described in relation to formulation F 1.

d) Deltamethrin The Deltamethrin component is available as Deltamethrin Technical which is produced by Roussel Uclaf Corporation, USA. It contains 98% active ingredient and 2% inert ingredients. This is an odorless solid, white/beige in color. Melting point is 98 to 101 °C. It is insoluble in water.

For the preparation of F 2, we used 10% solution by weight (90% inert isoparaffinic oil) prepared by the supplier, with estimated density of 0.8286 g/ml (10% x 0.5 g/ml/0.98 g a.i.) + 90% x 0.864 g/ml) = 0.8286.

e) Piperonyl butoxide

Piperonyl butoxide, or PBO, is an insecticide synergist. There are several producers of PBO, including AgrEvo, Great Britain; and MGK, the USA. This is a pale yellow liquid with at least 85 % active ingredient and 15 % inert ingredients (typicaly 97% active ingredient). PBO has a density of 1.06 g/ml, is a chemically stable substance in the absence of UV light, and is insoluble in water.

Preparation of 100 q of F 2 formulation

The goal was to prepare a DE-based formulation that, depending on insect species and a type of commodity, will protect grains using 50 to 100 ppm per tone. This concentration is developed to control a wide spectrum of insect including lesser grain borer. This species is susceptible on the combination of deltamethrin with PBO.

The following procedure was used to produce 100 grams of F 2:

REDO Formulation:

a) 1.3 ml of Reldan^* Insecticidal Concentrate (60.6%), 0.4 ml of Deltamethrin Technical (10%) and 0.4 ml of PBO (97%) are put into a container that contains 25 ml of canola oil and container is shaken by hand for few seconds. b) after mixing added substances with oil, 30 grams of DE is put into a container and the mixing of substances in the container are done by aid of spatula for 2 minutes.

c) mixture described under b) is screened through laboratory sieve No. 30 by aid of a brush to break balls formed with oil and DE. The mixture in closed container is mixed by shaking the container by hand for 30 seconds.

d) the determined quantity of DE is put into container until the mixture contains 100 grams of the formulation. The container with 100 grams is thoroughly shaken with hand for 1 minute.

e) the formulation described under d) is again screened through the lab sieve No. 30 by aid of a brush.

f) after screening the closed container with a formulation is again mixed thoroughly by hand for 1 minute.

The result of a described process is a new formulation labeled as F 2. This process can be easily mechanixed for commercial production.

The composition of 100 grams of F 2

Substance Procedure Density Composition (9) Grams a.i.

Reldan 6E 60.6% 1.3 ml 1.2 g/ml 1.56 0.945

Oil 25 ml 0.85 g/ml 21.25 21.25

Deltamethrin 10% 0.4 ml 0.8286 g/ml^* 0.33 0.033

PBO 97% 0.4 ml 1.0 g/l 0.4 0.39

DE 76.46 g - 76.46 76.46

Total 100.0

Mode of action of the new formulations Several tests were carried out to determine the mode of action of the F 1 and F 2 formulations. The main goal was to determine the relative importance of desiccation

(DE) and toxicity (synthetic components) in the mode of action of F 1 and F 2. Also, monitored and considered was the critical level of body weight loss to determine when treated insects start to die. When insects are treated with DE only, they usually die when they lose 25 to 35% of their total body weight, mainly because of water loss from the body. If toxicity is the main cause of the death of the insects, then the insects should die without significant body weight loss, but may begin to lose water from the body after death.

Testing parameters

Twenty red flour beetle (RFB) or 30 lesser grain borer (LGB) were used in each replicate. Chlorpyrifos (4.3 x 10^"5 mg of active ingredient (a.i.)/adult) and deltamethrin (4.5 x 10^"6 mg of a.i. / adult) were applied individually and in combination. PBO was applied at 0.097 μg per replication. Chlorpyrifos, deltamethrin, PBO, and a chloropyrifos & deltamethrin mixture, were diluted in 98% ethanol. Ten μL of each solution was added to the bottom of a glass vial (3 cm diameter). Twenty red flour beetle, or 30 lesser grain borer, were then added to the vial and gently shaken to ensure that all insects were evenly coated with the solution. A light airflow was directed into the vial to accelerate evaporation of the ethanol. DE was applied at dosages that correspond to about 10% of the test insects body weight and F 1 and F 2 in dosages that contained the same quantity of the synthetic active ingredients applied in dilutions with ethanol oil 0.1 μg per replication and ethanol itself in 10 μl per replications. The tests were held at 25 ± 3 °C and 55 ± 10% relative humidity (r.h.).

The results of tests are presented in Tables 1 to 4 , and in Figures 1 and 2.

The results clearly indicate that the efficacy of new formulations is due to a combination of desiccation and toxicity. Insects treated with DE alone were dead when they lost about 29% (RFB) or 38% (LGB) of their body mass. These results are in agreement with the results of various researchers which proved that insects treated with substances with a physical mode of action such as inert dusts including DE are dead when they lose 25 to 35% of their body mass through desiccation.

In the contrary, chlorpyrifos methyl and deltamethrin cause insect death by chemical poisoning. Treated insects were dead when they lost only about 6% of their body mass (RFB). The mortality of lesser grain borer treated with chlorpyrifos methyl and deltamethrin was 66.6% and 51.1 %, respectively, when they lost 7.6% of their body mass. However, lesser grain borer treated with DE lost 19.6% of the body weight with 1.1 % mortality only, but when the body mass was reduced by 37.9%, the mortality was 96.6%.

The results achieved with the treatment of insects with F 1 and F 2 formulations show the synergy of dessication and poisoning. The insects treated with the new formulations lost body mass faster than those treated with DE alone and they were dead when they lost about 15% (RFB) or 12% (LGB, F 2) or 20% (LGB, F 1 ) of their body mass.

Table 1. Body mass loss in red flour beetle treated with the new formulations, and their components.

Red flour beetle body mass loss (%)

Hours post-treatment

Treatment 1 4 6 10 15 22 26 48

Untreated 0.1 b 0.6 d 07 e,f 1.5 d 1.6 d 3.5 d 3.5 e 4.c,d

(0.1 ) (0.2) (0.1 ) (0.7) (0.4) (0.6) (0.4) (0.8)

Ethanol 0.0 b 0.2 d 0.6 f 1.7 d 1.9 d 2.4 d 3.1 e 3.6 d

(0.0) (0.1 ) (0.4) (0.7) (0.7) (0.4) (0.4) (1.1 )

PBO 0.4 b 0.0 d 1.8 d,e,f 2.3 c,d 2.9 c,d 4.3 d 5.6 d,e 6.6 b,c,d

(0.5) (0.0) (0.5) (0.5) (0.5) (0.5) (2.0) (1.3)

Oil 0.0 b 0.1 d 0.5 f 1.4 d 1.7 d 3.2 d 3.8 e 7.9 b,c

(0.0) (0.8) (0.7) (0.7) (0.5) (0.7) (0.4) (1.3) DE 0.6 b 3.5 b 5.3 9.9 a 16.0 a 25.9 29.5 a 39.4 a

(0.1 ) (0.07) (0.3) (0.2) (0.5) b a

(0-7) (0.2) (0.2)

Reldan 0.6 b 2.4 b,c 3.1 c,d 5.7 b 5.9 b 8.8 c 9.6 c 16.1 b (0.2) (0.2) (0.2) (0.4) (0.4) (0.1 ) (0.05) (1.8)

Delta met 0.2 b 1 .1 c,d 2.0 c,d ,e 3.2 4.2 b,c 6.8 c 6.9 c,d 8.9 c hrin (0.3) (0.3) b,c,d (0.5) (1 -0) (0.8) (2.6) (0.3)

(0.3)

Reldan + 0.7 b 2.2 b,c 3.2 c 4.4 b,c 5.5 b 8.3 c 9.3 c 13.5 b deltamet (0.4) (0.4) (0.5) (0.5) (0.5) (0.9) (1 .1 ) (1.8) hrin

F 1 2.1 a 6.6 a 8.2 a 1 1 .2 a 14.9 a 22.3 b 26.0 b 40.3 a (0.1 ) (0.4) (0.2) (0.3) (0.1 ) (0.4) (0.7) (1.6)

F 2 1 .8 a 7.1 a 9.4 a 12.5 a 16.4 a 24.2 28.1 a,b 40.5 a (0.5) (0.7) (0.7) (1 .4) (1 .2) a,b (1 .5) (1 .2)

(1 .5)

ANOVA, Tukey. P = 0.050. Means in each column followed by the same letter are not significantly different. Standard deviation shown in brackets.

Table 2. The mortality of red flour beetle treated with new formulations, and their components

Red flour beetle mortality (%)

Hours post-treatment

Treatment 1 4 6 10 15 22 26 48

Untreated 0.0 d 1.7 d 1.7 e 1.7 d 1.7 d 1.7 d 1.7 c 3.3 c

(0.0) (2.8) (2.8) (2.8) (2.8) (2.8) (2.9) (2.8)

Ethanol 0.0 d 0.0 d 0.0 e 0.0 d 0.0 d 0.0 d 0.3 0.0 c

(0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)

PBO 0.0 d 0.1 d 0.0 e 1.7 d 1.7 d 3.3 d 3.3 c 3.3 c

(0.0) (0.0) (0.0) (2.8) (2.8) (2.8) (2.8) (0.0)

Oil 0.0 d 0.2 d 0.0 e 0.0 d 0.0 d 0.0 d 0.0 c 0.0 c

(0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0)

DE 0.0 d 0.3 d (0.0) 0.0 d 28.3 c 86.7 b 93.3 a 100.0 a

(0.0) (0.0) (0.0) (0.0) (7.6) (7.6) (5.7) (0.0) WO 00/5143: $ PCT/CAOO/00225

23

Reldan 16.7 65.0 a 78.3 91.7 a 91.7 100.0 a 100.0 a 100.0 a b (5.0) b (7.6) a,b (0.0) (0.0) (0.0) (2.8) (2.8) (5.7)

Deltamethri 6.7 c 15.0 c 16.7 16.7 c 8.3 d 15.0 c 13.3 b 23.3 b n (7.6) (8.6) d (5.7) (10.4) (5.0) (7.6) (5.7) (5.7)

Reldan + 18.3 53.3 a 90.0 93.3 a 96.7 a 96.7 a,b 100.0 a 100.0 a deltamethrin b (12.5) a (2.8) (2.8) (2.8) (0.0) (0.0) (5.7) (0.0)

F 1 38.3 68.3 a 75.0 86.7 a 91.7 98.3 a 100.0 a 100.0 a a (5.7) b (5.7) a,b (2.8) (0.0) (0.0)

(7.6) (5.0) (7.6)

F 2 15.0 31.7 b 43.3 68.3 b 75.0 b 93.3 a, b 98.3 a 100.0 a

ANOVA, Tukey. P = 0.050. Means in each column followed by the same letter are not significantly different. Standard deviation shown in brackets.

Table 3. Body weight loss and mortality of lesser grain borer treated with the new formulations, and their components

Hours post-treatment

2 6 18

Treatment Body Mortality Body weight Mortality Body Mortality weight loss weight loss loss

Untreated 0.9 d (0.1 ) 0.0 a (0.0) 3.3 f (0.1 ) 1.1 (1.9) 8.7 f (0.2) 1.1 d (1.9)

Ethanol 1.1 d (0.2) 1.1 a (1.9) 4.2 d,e,f (0.1 ) 1.1 d (1.9) 9.6 f (0.6) 1.1 d (1.9)

PBO 1.3 d (0.4) 1.1 a (1.9) 4.1 e.f (0.1 ) 3.3 d (3.3) 10.4 e,f (1.1 ) 3.3 d (3.3)

Oil 1.3 d (0.4) 0.0 a (0.0) 5.2 d,e,f (1.9) 3.3 d (5.7) 8.3 f (1.3) 3.3 d (5.7)

DE 19.6 a (0.8) 1.1 a (1.9) 37.9 a (1.2) 96.6 a (3.3) 52.4 a (0.6) 100.0 a (0.0)

Reldan 2.4 d (0.4) 1.1 a (1.9) 7.6 d (0.3) 66.6 b (12.0) 20.2 d (4.0) 73.3 c (17.6)

Deltamethrin 2.7 d (0.4) 1.1 a (1.9) 7.7 d (0.7) 51.1 c (6.9) 16.3 d,e (1.8) 80.0 b,c (8.8)

Reldan + 2.6 d (0.3) 0.0 a (0.0) 7.0 d,e (0.4) 87.7 a (1.9) 17.5 d (1.8) 95.5 a,b (3.8) deltamethrin

F 1 9.4 b (1.3) 5.5 a (5.0) 20.0 b (2.3) 100.0 a (0.0) 40.23 b (3.2) 100.0 a (0.0)

F 2 4.6 c (0.7) 3.3 a (3.3) 11.41 c (1.5) 97.7 a (3.8) 27.3 c (3.3) 100.0 a (0.0) ANOVA, Tukey. P = 0.050. Means in each column followed by the same letter are not significantly different. Standard deviation shown in brackets.

Table 4. Loss of body mass and mortality in red flour beetle and lesser grain borer, 26 and 6 hours post-treatment, respectively.

RFB LGB

Treatment Loss of body Mortality Loss of body Mortality mass (%) mass (%)

(%) (%)

Untreated 3.5 1.7 3.3 1.1

Ethanol 3.1 0.3 4.2 1.1

PBO 5.6 3.3 4.1 3.3

Oil 3.8 0.0 5.2 3.3

DE 29.5 93.3 37.9 96.6

Reldan 9.6 100.0 7.6 66.6

Deltamethrin 6.9 13.3 7.7 51.1

Reldan + 9.3 100.0 7.0 87.7 deltamethrin

F 1 26.0 100.0 20.0 100.0

F 2 28.1 98.3 11.4 97.7

Selected data from Table 1 and 2 (red flour beetle-RFB) and Table 3 (lesser grain borer- LGB)

The results of the bioassay carried out until today using all substances separately and in mixture show clearly the synergistic mode of action of the new developed formulations. Table 5. The synergistic effect of the new formulations against red flour beetle on Hard Red Spring wheat, after 2 days of exposure time.

Treatment Concentration (ppm) Red flour beetle mortality (%)

Untreated 0 0.0

Ethanol 2000 0.0

PBO 0.6 0.0

Oil 10 0.0

DE 50 0.0

Reldan 0.45 0.0

Deltamethrin 0.06 0.0

Reldan + 0.45+0.06 0.0 deltamethrin

F 1 25 66.6

F 2 50 30.6

The efficacy of the new formulations

Numerous test (bioassays) were conducted with F 1 and F 2 formulations with the following composition:

Table 6. The composition of F 1 and F 2 formulation used in bioassays of short and long term efficacy tests

Grams of active ingredients in 100 g of formulation

Substances F 1 F 2 chlorpyrimifos methyl 0.967 0.581 oil 17 11.305

DE 82.0 87.0 deltamethrin - 0.0082

PBO - 1.0 Short term efficacy tests

We conducted several bioassays in order to compare the efficacy of the new formulations with the efficacy of the DE Protect-It™, a comparative efficacy standard, against several grain insects and their progeny. All bioassay cultures were held at 30 °C and 70% relative humidity, which are optimal conditions for the proliferation of stored grain insects.

By conducting numerous bioassays with Protect-It™ and with different sources of DE, it was discovered that efficacy varied with the commodity treated. Ranked in order from the highest (more than 1500 ppm) to the lowest dose (100 ppm) required for control of red flour beetle on rice and rusty grain beetle on wheat, respectively, the commodities are: milled rice > sunflower > birdseed > sorghum > corn > paddy rice > oats > barley > wheat. There was also significant variation in the susceptibility of different species to Protect-It™. The order of the most susceptible grain insect to the least is: rusty grain beetle > saw toothed grain beetle > granary weevil > rice weevil > red flour beetle > lesser grain borer > larger grain borer.

Table 7. The efficacy of the new formulations and Protect-It against rice weevil (RW), lesser grain borer (LGB) and red flour beetle (RFB), on popcorn.

LD 100 900 900 700 40 50

Whole kernel popcorn with 12.9% m.α, no dockage. Concentrations tested (ppm): F 1 : 0 30 40 50 60

F 2: 0 25 50 75 100

Protect-It^*: 0 300 500 700 900

Protect-It is a trade name for the diatomaceous earth insecticide formulation described in US patent 5,773,017.

Table 8. The efficacy of F 1 and F 2 formulations against rice weevil (RW), lesser grain borer (LGB) and red flour beetle (RFB) on Hard Red Spring wheat with 15.2% moisture content. ppm or grams per tone

After 1 day After 7 days After 14 days

LD F 1 F 2 F 1 F 2 F 1 F 2

(%) RW LGB RW LGB RW LGB RW LGB LGB LGB

50 30 137 45 145 78 50 76 48 90 44 354 69 399 160 76 158 67 100 100 N/A 125 N/A 25 N/A 50 N/A N/A 125 Concentrations tested (ppm): F 1 : 25 50 75 100 F 2: 50 75 100 125

As mentioned before, the efficacy of DE is usually lowest on milled rice and highest on wheat. The bioassay results for the F 1 formulation for four insect pests on wheat and milled rice are shown in Table 10. F 2 generated excellent results on both commodities, particularly on wheat (Table 7 and 8). The concentrations necessary for 100% mortality of the four insect species, on both commodities, ranged from 50 to 200 ppm. When using DE (alone) against the adults and progeny of these four insect species, the concentrations required ranged from 400 to 2000 ppm. Table 9. The number of rice weevil progeny on wheat and milled rice after 35 days

Average number of adults per

Commodity ppm replication after 35 days

0 185

50 0

Wheat 75 0

100 0

125 0

0 144

75 1

Milled 100 1

Rice 125 0

150 0

*Same conditions and formulations as per Table 8.

Table 10. The efficacy of the F 1 formulation against rice weevil (RW), red flour beetle (RFB), rusty grain beetle (RGB) and lesser grain borer (LGB), on wheat and milled rice

Exposure ppm or grams per tone time LD HRSW Milled Rice

(days) RW RFB RGB LGB RW RFB RGB LGB

LD₅₀ 27 34 21 107 93 164 109

1 LD₉₀ 49 57 82 243 444 351 234 n/a

LD₁₀₀ 100 100 - - - - -

LD₅₀ - - - 54 27 95 40 231

3 LD₉₀ - - - 130 54 128 74 469

LD₁₀₀ 50 50 50 - 100 - 125 -

LD₅₀ - - - 17 - 89 39 116

6 LD₉₀ - - - 47 - 117 68 201

Hard Red Spring Wheat (HRSW) at 14% m.c; milled rice at 12.7% m.c. Concentrations tested (ppm): Wheat: 0 50 75 100 125

Rice: 0 75 100 125 150

Long term efficacy test

The goal was to determine the concentrations of F 1 and F 2 needed for the protection of grain up to 3 months. Brief description of the methodology:

Hard Red Spring wheat (15% m.c.) was dusted with F 1 as follows: 6 kg of wheat with 25 ppm 50 ppm 75 ppm 100 ppm and 0 ppm (control)

Hard Red Spring wheat (15% m.c.) was dusted with F 2 as follows: 6 kg of wheat with 50 ppm 75 ppm 100 ppm and 125 ppm

Every 45 days, a bioassay was conducted with treated grain to determine if there is a decline, over time, in the efficacy of F 1 and F 2 against four (4) test insects; rusty grain beetle, rice weevil, lesser grain borer and red flour beetle. Bioassays were conducted at 30 °C and 70% r.h., with 5 replicates per treatment. Every time a bioassay was conducted, Hard Red Spring wheat from the same source was treated with F 1 and F 2 to compare the efficacy of freshly treated grain with the efficacy of grain treated at the beginning of the test. Table 11. Biological impact and degradation of F 1 and F 2 on stored wheat in Ontario.

* after 14 days of exposure time - 75 ppm caused 90% mortality, 100 ppm 100% mortality.

Concentrations (ppm): F 1 (ppm): 25 50 75 100

F 2 (ppm): 50 75 100 125

Table 12. Biological impact and degradation of F 1 and F 2 on stored wheat in Ontario

N/A* means that by tested concentrations 90% or 100% mortality is not achieved. Concentrations (ppm): F 1 (ppm): 25 50 75 100

F 2 (ppm): 50 75 100 125

Table 13. Biological impact and degradation of F 1 and F 2 on stored wheat in Ontario

Concentrations (ppm): F 1 : 25 50 75 100 F 2: 50 75 100 125

Table 14. The efficacy of F 1 and F 2 against the progeny of grain insects on wheat. Results against parents presented in Tables 11 to 13.

25 4.2 (40) 0.7 (5.2) 0.0 (0.4)

50 1.0 (7.5) 1.3 (0.5) 0.0 (0.0)

July 10, 1998 75 0.0 (3.0) 0.7 (1.1 ) 0.0 (0.0)

100 0.0 (0.2) 0.5 (1.0) 0.0 (0.0)

50 0.6 1.0

75 0.0 0.5

May 26, 1998 100 0.0 0.0 N/A

F 2 125 0.0 0.0

50 0.8 (15.3) 0.9 (1.7) 0.0 (0.4)

75 0.4 (4.7) 0.1 (1.6) 0.0 (0.4)

July 10, 1998 100 0.0 (1.1 ) 0.0 (0.3) 0.0 (0.0)

125 0.0 (0.1 ) 0.0 (0.3) 0.0 (0.0)

The average number of adults on untreated wheat: RW at 0 day -646; at 45^th day 489.2; LGB at 0 day - 41.8, at 45^th day 251.2; RFB at 45^m day - 53.6.

Hard Red Spring wheat, stored in screen-topped, 1 1.4L plastic containers, was kept under ambient climatic conditions from May 25, 1998 to August 25, 1998. Grain temperature and moisture content of 0 ppm control was recorded weekly. The recorded grain temperature and moisture content is presented in Table 14.

Table 15. Recorded grain temperature and moisture content of Hard Red Spring wheat used in the test .

Date Grain Temp.(°C) Grain M.C. (%)

05/06/98 15 14.0

12/06/98 21 14.4

19/06/98 24 15.1

26/06/98 28 14.3

06/07/98 24 14.3

09/07/98 26 14.7

18/07/98 25 13.4 The results presented in Tables 11 to 14 show an excellent efficacy of the tested formulation with this extremely low concentration of a.i. The efficacy on treated grain was reduced over time (after 90 days) because of a low percentage of a.i., but still acceptable and in some cases, excellent.

These initial results of the long term efficacy test clearly indicate that F formulations with an increase percentage of synthetic a.i. (enhanced F formulations described in Case 1 and Case 2, Processing technology for the new formulations) will ensure the long-term protection of grain against insect infestation. The long term efficacy test with F 1 and F 2 described under Case 1 and Case 2 is ongoing and will terminate at the end of August 1999. The first results with these enhanced F 1 and F 2 formulations are presented in tables 16, 17, 18 and 19.

Table 16. Long term efficacy of ZP³ formulations 0-day treatment for rice weevil (RW)

Cone. RW mortality (%) ± S.D. Percent

Treatment (ppm) 3 days 7 days 14 days Progeny

Untreated 0 0.8 ± 1.8 1.6 ± 2.6 3.2 ± 3.0 100^*

50 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 0

F 1 75 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 0

100 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 0

50 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 0.2

F 2 75 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 0

100 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 0

mean (± S.D.) no. RW in untreated = 410.4 ± 161.2 .

Table 17. Long term efficacy of F formulations 0-day treatment for lesser grain borer (LGB)

Cone. LGB mortality (%) ± S.D. Percent

Treatment (ppm) 3 days 7 days 14 days Progeny Untreated 0 0.4 ±0.9 1.2 + 1.1 2.0 ±2.0 100^*

50 50.4 ± 8.4 56.0 ±5.8 58.8 ±5.9 0.3

F1 75 73.6 ±6.2 76.0 ±6.8 79.2 ±6.6 0.3

100 81.2 ±3.3 86.4 ±2.6 88.8 ±3.0 0.3

50 42.4 ±19.8 89.2 ± 5.0 97.2 ±1.8 0

F2 75 46.0 ±13.0 94.8 ±5.0 100.0 ±0.0 0

100 54.8 ±9.3 92.0 ±4.2 100.0 ±0.0 0

^* mean (± S.D.) no. LGB in untreated = 224.6 ± 44.1

Table 18. Long term efficacy of F formulations 0-day treatment for red flour beetle (RFB)

Cone, RFB mortality (%) ± S.D. Percent

Treatment (ppm) 3 days 7 days 14 days Progeny

Untreated 0 14.4 ±13.1 29.6 ±17.3 35.2 ±15.1 100^*

50 100.0 ±0.0 100.0 ±0.0 100.0 ±0.0 0

F1 75 100.0 ±0.0 100.0 ±0.0 100.0 ±0.0 0

100 100.0 ±0.0 100.0 ±0.0 100.0 ±0.0 0

50 95.2 ±1.8 100.0 ±0.0 100.0 ±0.0 0

F2 75 100.0 ±0.0 100.0 ±0.0 100.0 ±0.0 0

100 100.0 ±0.0 100.0 ±0.0 100.0 ±0.0 0 mean (± S.D.) no. RFB in untreated = 224.6 ± 44.1

Table 19. Long term efficacy of F formulations 0-day treatment for rusty grain beetle (RGB)

Cone. RGB mortality (%) ± S.D. Percent

Treatment (ppm) 3 days 7 days 14 days Progeny

Untreated 0 0.0 ±0.0 1.2 ±1.8 3.2 ±3.6 100^*

50 100.0 ±0.0 100.0 ±0.0 100.0 ±0.0 0

F1 75 100.0 ±0.0 100.0 ±0.0 100.0 ±0.0 0 100 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 0

50 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 0 F 2 75 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 0 100 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 0

** mean (± S.D.) no. RGB in untreated = 43.3 ± 17.5

These results show high efficacy of the tested formulations at concentrations of 25 to 125 ppm, depending on the insect species and commodity tested. The tested formulations contain 6 to 24 times less chlorpyrifos methyl and about 22 to 60 times less deltamethrin than the recommended dosages when these two substances are used alone. The results of tests on the mode of action of the new formulations indicate that synergism between the activity of their individual components (desiccation and toxicity) is probably the main reason for the high efficacy. Synergism means that the efficacy of the new formulations is greater than the sum of the efficacy of their components alone. Because of this combined action, the concentrations of DE and other substances used in these mixtures may be much lower than if any one component were to be used alone.

The influence of the new formulations on grain bulk density (test weight)

Although the first commercial DE formulations have been widely available since the 1950's, there are problems associated with its use in stored grains that have not yet been resolved. When DE is mixed with grain at the currently recommended dosages of 500 to 3500 parts per million (ppm or g/t), some physical and mechanical properties of the bulk commodity are adversely affected: flowability and bulk density are reduced, visible residues are evident on the grain, dielectric moisture and infra red meter readings are affected, and an excessive amount of dust is produced during handling.

The addition of DE to grains creates greater friction between kernels, which affects the bulk density and flow properties of the grain. Bulk density, or test weight, is an extensively used grading factor. For example, Canadian Western Red Spring wheat requires a minimum bulk density of 750 kg/m³ (10^"1 kg/hL) to be considered grade No.

1. Using DE at currently recommended dosages of 500 to 3500 would cause a sufficient reduction in bulk density to reduce grain grade, causing loss of value without causing a loss in quality. An application of only 10 ppm, significantly reduces the bulk density (about 1.3 to 1.8%, w/w, respectively) of clean (no dockage) wheat with a

13.9% m.c. The greatest changes in bulk density occur when the concentration of DE ranges from 50 to 200 ppm. At concentrations greater than 500 ppm, increased DE concentrations has little influence on bulk density.

The magnitude of the adverse effects of DE can be reduced using lower concentrations of DE. However, lower concentrations of currently available DE formulations cannot achieve acceptable levels of control of stored-grain insects. The new DE-based formulations, F 1 and F 2, have been developed specifically to minimize: (i) the reduction in bulk density caused by DE, (ii) the volume of air-borne dust generated during application, handling, and grain transportation, (iii) the effect of DE on grain flowability, and (iv) the problem of visible residue of dust on grains. Therefore, the objectives for this part of the study were a) to determine the influence of the new formulations, F 1 and F 2, on the bulk density of wheat (test weight), b) to compare the effects of DE alone, F 1 and F 2, on wheat bulk density, c) to determine the change in the density of F 1 and F 2, and their effect on wheat bulk density when oil is added to the new formulations.

The bulk density was measured using the protocol and equipment (Ohaus apparatus) listed in the Canadian Grain Handbook, 1994). The volume of the measuring cup was 500 mL.

In addition to the significant increase in efficacy, the advantages of using the F 1 and F 2 formulations are readily apparent. A significantly lower effect on test weight was observed as compared to DE alone. The new formulations also occupied a significantly lower volume than equivalent volumes of DE (alone) or Protect-It™. Therefore, the lower concentrations of F 1 and F 2 will greatly mitigate, or completely eliminate (aqueous suspension), the adverse effect of DE on grain test weight, flowability of grain and the appearance of dust in the air during handling of grain

Table 20. Comparison of DE Protect-It™ with F 1 and F 2 on the test weight of Hard Red Spring wheat with 15.2% m.c.

Test weight (kg/hL)

Ppm Protect-It F 1 F 2

0 79.21 a (0 kg) 79.21 a (0 kg) 79.21 a (0 kg)

25 77.69 c (1.52 kg) 78.75 b (0.46 kg) N/A (N/A)

50 76.30 f (2.91 kg) 77.50 d (1.71 kg) 77.80 c (1.31 kg)

75 75.54 g (3.67 kg) 76.75 e (2.46 kg) 77.31 d (1.90 kg)

100 75.0 h (4.21 kg) 76.27 f (2.94 kg) 76.73 e (2.48 kg)

125 74.57 i (4.64 kg) N/A (N/A) 76.45 e,f (2.76 kg)

ANOVA. P=0.050. Means followed by the same letter are not significantly different.

The reduction of tests weight in kg/hL, compared with untreated wheat, is shown in brackets.

Table 21. The effect of oil added to the F 1 formulation, applied as a dust or suspension, on the test weight of HRS wheat (12% m.c).

Mean kg/hi and the reduction of test weight (%)

DE alone F 1 dust F 1 dust F 1 with oil, Protect- ppm without oil with oil suspension^* It™ kg/hi red. kg/hi red. kg/hi red. kg/hi red. kg/hi red.

(%) (%) (%) (%) _(0/o)

0 76.0 a - 76.0 a - 76.0 a - 76.0 a - 76.0 a 1.4

75 75.2 be 1.1 75.0 c 1.3 75.7 a,b 0.4 76.0 a 0.0 74.8 c,d

ANOVA. P= 0.005. Means followed by the same letter are not significantly different.

* I liter of suspension per tone = 50 ppm (5% suspension) Table 22. Loose and tapped density of Protect-It™, DE used in F formulations and F 2

g/L

Difference Difference (%) in (%) in density of _{Densjty of}

Density Protect-It™ DE F 2

F 2 and F 2 and

Protect-It™ DE

Loose 184.1 f 201.5 e 227.3 d + 43.2 + 25.8

Tapped 252.1 c 268.2 b 285.7 a + 33.6 + 17.5

ANOVA, Tukey. P=0.050. Means followed by the same letter are not significantly different.

Table 23. Influence of F 2, DE and Protect-It™ on the test weight of Canada Prairie Spring Red wheat (13.5% m.c.)

Mean tesl t weii ght (kg/hL) and reduction of test weight (%) ppm Protect-It TM DE F 2 kg/hi red. (%) kg/hi red. (%) kg/hi Red. (%)

0 72.4 a - 72.4 a - 72.4 a -

50 70.3 def 2.9 71.3 c 1.5 72.0 ab 0.6

75 69.6 fgh 3.9 70.8 d 2.2 71.3 c 1.5

100 68.9 hi 4.8 70.6 de 2.5 71.3 c 1.5

ANOVA. P= 0.005. Means followed by the same letter are not significantly different. The Cost

In addition to safety, low toxicity to mammals, and very high efficacy against insects and their progeny, one of our primary concerns was to develop new formulations comparable in price with grain protectants currently registered and in use. By analyzing the cost of the raw materials of the formulations of F 1 and F 2 described in, it is obvious that the new formulations are really very cost-effective (Table 24 and 25). The costs of the F 1 and F 2 formulations are based on the highest dosages needed for long term protection and needed to control the most tolerant species of insect, lesser grain borer. When other species have to be controlled (weevils, red flour beetle, rusty grain beetle), or the protection of grain up to 3 months is needed, the lower concentration of 50 ppm is recommended. Ii means that the costs are half of total cost listed in Tables 24 and 25.

Table 24. Cost for 100 grams of F 1 (US cents)

Substance Cost/unit Cost/gram /cent/ Total cost/cent/substance

Reldan 6E 60.6% $181.70/gal. 4.0 13.44

Oil 65 cents/L 0.065 1 .38

DE $0.21/lb 0.05 3.77

Total 100 g 18.59

Table 25. Cost for 100 grams of F 2 (US cents)

Substance Cost/unit Cost/gram /cent/ Total cost/cent/substance

Reldan 6E 60.6% $181.70/gal. 4.0 6.24

Oil 65cents/L 0.065 1.38

Deltamethrin 10% &33.66/lb 7.41 2.44

PBO 97% $12.20/lb 2.69 1.1

DE $0.21/lb 0.05 3.82

Total 100 g 14.98 Now that the invention has been described, numerous substitutions modifications and equivalences will occur to those skilled in the art which are intended to be within the spirit and scope of the invention as defined in the claims appended hereto.

Claims

The claimed invention is:

1. An insecticidal composition comprising a mixture of diatomaceous earth, oil and an insecticide compound selected from an organophosphate insecticide group combined in a synergistic insecticidally effective amount.

2. The composition of claim 1 wherein said insecticidally effective amount of said insecticiadal composition is not more than about 100 parts per million by weight of the product to be treated.

3. The composition of claim 1 wherein said diatomaceous earth substantially comprises fresh water diatomaceous earth.

4. The composition of claim 1 wherein said diatomaceous earth contains less than 1 % crystalline silica.

5. The composition of claim 1 wherein said insecticide compound comprises less than 3% by weight of said composition.

6. The composition of claim 1 or claim 5 wherein said insecticide compound comprises chlorpyrifos methyl.

7. The composition of claim 1 wherein said diatomaceous earth is in a range of about 70% to about 80% by weight of said composition.

8. The composition of claim 1 wherein said oil is about 21 % by weight of said composition.

9. The composition of claim 1 wherein said oil is a vegetable oil.

10. The composition of claim 1 containing about 75% by weight diatomaceous earth, about 21 % by weight oil and not more than about 3% of an insecticide compound selected from the organophosphate insecticide group.

11. The composition of claim 10 wherein said insecticide compound is chlorpyrifos methyl.

12. The composition of claim 10 wherein said diatomaceous earth comprises fresh water diatomaceous earth.

13. The composition of claim 1 further including a second insecticide compound selected from the pyrethroid insecticide group and a synergizing compound for synergizing said second insecticide compound, combined in a synergistic insecticidally effective amount.

14. The composition of claim 13 wherein said insecticidally effective amount of said composition is not more than about 100 parts per million by weight of the product to be treated.

15. The composition of claim 13 wherein said second insecticide compound is deltamethrin and said synergizing compound is piperonyl butoxide.

16. The composition of claim 13 wherein said insecticidally effective amount of said insecticidal composition is not more than about 100 parts per million by weight of the product to be treated.

17. The composition of claim 13 wherein said diatomaceous earth substantially comprises fresh water diatomaceous earth.

18. The composition of claim 13 wherein said diatomaceous earth contains less than 1 % crystalline silica.

19. The composition of claim 13 wherein said insecticide compound comprises less than 1 % by weight of said composition.

20. The composition of claim 13 or claim 18 wherein said insecticide compound comprises chlorpyrifos methyl.

21. The composition of claim 13 wherein said second insecticide compound is less than 0.04% by weight of said composition and said synergizing compound is less than 0.5% by weight of said composition.

22. The composition of claim 13 or claim 20 wherein said second insecticide compound is deltamethrin.

23. The composition of claim 21 or 21 wherein said synergizing compound is piperonyl butoxide.

24. The composition of claim 13 wherein this ^isid diatomaceous earth is in a range of about 70% to about 80% by weight of said composition.

25. The composition of claim 13 wherein said oil is about 21 % by weight of said composition.

26. The composition of claim 13 wherein said oil is a vegetable oil

27. The composition of claim 13 containing by weight about 75% diatomaceous earth, about 21 % oil, not more than about 1 % of chlorpyrifox methyl, not more than about 0.04% of deltamethrin and not more than about 0.5% piperonyl butoxide.

28. An insecticidal composition comprising a mixture of diatomaceous earth, oil, a first insecticide compound selected from the organophosphate insecticide group, a second insecticide compound selected from the pyrethroid insecticide group and a synergizing compound for synergizing said second insecticide compound combined in a synergistic insecticidally effective amount.

29. The composition of claim 28 wherein said insecticidally effective amount of said insecticidal composition is not more than about 100 parts per million by weight of the product to be treated.

30. The composition of claim 28 wherein said diatomaceous earth substantially comprises fresh water diatomaceous earth.

31. The composition of claim 28 wherein said diatomaceous earth contains less than 1 % crystalline silica.

32. The composition of claim 28 wherein said first insecticide comprises less than 1 % by weight of said composition.

33. The composition of claim 28 or claim 32 wherein said first insecticide is chlorpyrifos methyl.

34. The composition of claim 28 wherein, by weight, said diatomaceous earth is in a range of about 70% to about 80% of said composition.

35. The composition of claim 28 wherein said oil is about 21 % by weight of said composition.

36. The composition of claim 28 wherein said oil is a vegetable oil.

37. The composition of claim 28 containing, by weight, about 75% diatomaceous earth, about 21 % by oil and not more than about 1 % of insecticide selected from the organophosphate insecticide group and not more than about 0.5% of insecticide selected from the pyrethroid insecticide group.

38. A method for producing an insecticidal composition comprising mixing oil and an insecticide compound selected from an organophosphate insecticide group to form a first intermediate mixture, mixing diatomaceous earth with said first intermediate mixture to produce a second intermediate mixture; and screening said second intermediate mixture to produce said insecticidal composition.

39. The method of claim 38 wherein said diatomaceous earth substantially comprises fresh water diatomaceous earth.

40. The method of claim 38 wherein said diatomaceous earth contains less than 1 % crystalline silica.

41. The method of claim 38 wherein said insecticide compound comprises less than 3% by weight of said composition.

42. The method of claim 38 or claim 41 wherein said insecticide compound comprises chlorpyrifos methyl.

43. The method of claim 38 wherein said diatomaceous earth is in a range of about 70% to about 80% by weight of said composition.

44. The method of claim 38 wherein said oil is about 21 % by weight of said composition.

45. The method of claim 38 wherein said oil is a vegetable oil.

46. The method of claim 38 wherein said composition contains, by weight, about 75% diatomaceous earth, about 21 % by oil and not more than about 3% of an insecticide compound selected from the organophosphate insecticide group.

47. The method of claim 46 wherein said insecticide compound is chlorpyrifos methyl.

48. The method of claim 46 wherein said diatomaceous earth comprises fresh water diatomaceous earth.

49. The method of claim 38 wherein said screening removes particles larger that 600 microns.

50. A method for producing an insecticidal composition comprising mixing oil, a first insecticide compound selected from an organophosphate insecticide group, a second insecticide compound selected from a pyrethroid insecticide group and a synergistic compound to form a first intermediate mixture, mixing diatomaceous earth with said first intermediate mixture to produce a second intermediate mixture, and screening said second intermediate mixture to produce said insecticidal composition.

51. The method of claim 50 wherein said diatomaceous earth substantially comprises fresh water diatomaceous earth.

52. The method of claim 50 wherein said diatomaceous earth contains less than 1 % crystalline silica.

53. The method of claim 50 wherein said first insecticide comprises less than 1 % by weight of said composition.

54. The method of claim 50 or claim 53 wherein said first insecticide is chlorpyrifos methyl.

55. The method of claim 50 wherein, by weight, said diatomaceous earth is in a range of about 70% to about 80% of said composition.

56. The method of claim 50 wherein said oil is about 21 % by weight of said composition.

57. The method of claim 50 wherein said oil is a vegetable oil.

58. The method of claim 50 containing, by weight, about 75% diatomaceous earth, about 21 % by oil and not more than about 1 % of insecticide selected from the organophosphate insecticide group and not more than about 0.5% of insecticide selected from the pyrethroid insecticide group.

59. The method of claim 50 wherein said screening removes particles larger that 600 microns.