Control of arthropod infestation

Abstract

Use of particles in controlling a population of arthropod pests, wherein the particles comprise i) a hydrophobic exterior that adheres to the cuticle of at least one species of an arthropod pest; and ii) at least one pesticide associated with the said particles, wherein the pesticide is present at a weight of no more than 2% of the weight of the particles, populations of particles, methods of producing populations of particles and methods of application of such populations.

Claims

1. A method for controlling a population of arthropod pests on growing crops and/or on produce thereof with dry particles, wherein each of the particles has a hydrophobic exterior at which the particle adheres to the cuticle of at least one species of an arthropod pest and wherein the particles include at least one pesticide, the pesticide being present in a weight of no more than 2% of the weight of the particles, the method comprising the steps of: i) collecting the particles in a dusting apparatus; and ii) applying the particles to the growing crop and/or to produce thereof.

2. The method according to claim 1, wherein the particles potentiate the action of the pesticide on target arthropod pests.

3. The method according to claim 1, wherein the pesticide is present at a weight from ≧0.1% to ≦2% of the weight of the particles.

4. The method according to claim 1, wherein the hydrophobic exterior of each of the particles is formed by a wax that has a melting point ≧40° C. and is selected from one or more species of waxes selected from the group consisting of natural waxes, synthetic waxes and mineral waxes.

5. The method according to claim 1, wherein the particles are made up of solid wax particles that are selected from one or more species of waxes selected from the group consisting of natural waxes, synthetic waxes and mineral waxes.

6. The method according to claim 5, wherein the wax is selected from carnauba wax, montan wax and a mixture thereof.

7. The method according to claim 1, wherein the pesticide is selected from the group consisting of pyrethroids, spinosyns, carbamates, gamma amino butyric acid (GABA) inhibitors, neonicotinoids, anthranilamides, formonetins, essential oils, insect growth regulators, organophosphates and a mixture of two or more thereof.

8. The method according to claim 1, wherein the at least one species of arthropod pest is selected from the group consisting of Saw-toothed Grain Beetle (Oryzaephilus surinamensis); Grain Weevil (Sitophilus granarius); Common Flour Mite (Acarus siro); Warehouse Moth (Ephestia elutella); Flour or Mill Moth (Ephestia kuhniella); Flour Beetles (Tribolium spp.); Rust-red Grain Beetle (Cryptolestes ferrugineus); Cosmopolitan Food Mite (Glycyphagus destructor); codling moth (Cydia pomonella); oriental fruit moth (Grapholita molesta); armyworms from Spodoptera spp.; cabbage looper (Trichoplusia ni); thrips (Frankliniella spp.), onion thrips (Thrips tebeciv); leafminers (Uriomyza spp.); Corn earworm (Helicoverpa zea); European cornborer (Ostrinie nubilalis); Agrotis spp.; Cotton Bollworms(Helicoverpa spp.); Soybean looper (Pseudoplusia includes); Apple maggot (Rhagoletis pomonella); Pear psylla (Cacopsylla spp.); Navel orangeworm (Amyelois transitella); Tree Nuts Codling moth; Crucifers Diamondback moth (Plutella xylostella); cabbage worms (Pieris spp.); Grape berry moth (Lobesia botrana); Cucurbits Pickleworm (Diaphania spp.); and psocids.

9. The method according to claim 1, wherein the one or more species of arthropod pest is selected from the group consisting of Saw-toothed Grain Beetle (Oryzaephilus surinamensis), Grain Weevil (Sitophilus granarius), Common Flour Mite (Acarus siro), Warehouse Moth (Ephestia elutella), Flour or Mill Moth (Ephestia kuhniella), Flour Beetles (Tribolium spp.), Rust-red Grain Beetle (Cryptolestes ferrugineus) and Cosmopolitan Food Mite (Glycyphagus destructor).

10. A population of dry particles in the form of a powder for controlling populations of arthropod pests, wherein each of the particles has a hydrophobic exterior at which the particle adheres to the cuticle of at least one species of an arthropod pest and wherein the particles include at least one pesticide, the pesticide being present at a weight ≧0.1% to ≦2% of the weight of the particles.

11. The population of particles according to claim 10, wherein the hydrophobic exterior of each of the particles is formed by a wax that has a melting point ≧40° C. and is selected from one or more species of waxes selected from the group consisting of natural waxes, synthetic waxes and mineral waxes.

12. The population of particles according to claim 10 or claim 11, wherein the particles are solid wax particles selected from one or more species of waxes selected from the group consisting of carnauba wax, bees wax, Chinese wax, shellac wax, spermaceti wax, candelilla wax, montan wax, castor wax, ouricury wax and rice bran wax.

13. The population of particles according to claim 10, wherein the particles are selected from carnauba wax, montan wax and a mixture thereof.

14. The population of particles according to claim 10, wherein the pesticide is selected from the group consisting of pyrethroids, spinosyns, carbamates, gamma amino butyric acid (GABA) inhibitors, neonicotinoids, anthranilamides, formonetins, essential oils, insect growth regulators, organophosphates and a mixture of two or more thereof.

15. A method for controlling an infesting population of grain storage arthropod pests in a grain storage area with dry particles, wherein each of the particles has a hydrophobic exterior at which the particles adhere to the cuticle of at least one species of an arthropod pest and wherein the particles include at least one pesticide, the pesticide being present in a weight of no more than 2% of the weight of the particles, the method comprising the steps of: i) collecting the particles in a dusting apparatus; and ii) applying the particles to the grain storage area.

Description

FIGURES

(1) FIG. 1: Percentage mortality of Oryzaephilus surinamensis in treatments of 1% Pirimiphos-methyl in Entostat (w/w) after 48 hours of exposure. The results indicate that 100% mortality was achieved in all treatments containing pirimiphos-methyl with no variance and a standard deviation of 0%. The control shows a mean mortality of 8% and a standard deviation of 6.71%.

(2) FIG. 2: Graph to show the percentage mortality of O. surinamensis in different concentration treatments of pirimiphos-methyl in Entostat (w/w) after 48 hours of exposure. Control 1 represents the untreated control and Control 2 represents the vehicle control. The results indicate that 100% mortality was achieved in treatments containing 1% and 0.5% pirimiphos-methyl, with no variance and a standard deviation of 0% for both. The 0.25% treatment has a mean mortality of 51.75% with a standard deviation of 9.68%. The untreated control shows a mean mortality of 6.16% and a standard deviation of 6.80%, whilst the vehicle control shows a mean mortality of 11.33% with a standard deviation of 8.18%.

EXAMPLES SECTION 1

Efficacy of Different Application Rates of 1% Pirimiphos-Methyl in Entostat (w/w) when Used Against Oryzaephilus surinamensis in Grain

1. OBJECTIVE

(3) The purpose of the study was to examine the influence of application rate in grain on the efficacy of 1% Pirimiphos-methyl in Entostat (w/w) against Oryzaephilus surinamensis (sawtooth grain beetle). Rates examined were equivalent to 200 g, 100 g, and 20 g of powder per 1000 kg of grain.

2. STUDY OUTLINE

(4) Actellic is an organophosphate insecticide originally developed by Imperial Chemical Industries (now Syngenta) in 1967. It uses pirimiphos-methyl as its active ingredient. It is highly efficacious against a range of insect species and up until recently has been successfully used for the control of insect pests in stored grain and a range of public health situations. New regulations have resulted in the withdrawal of support for Actellic due to concerns over mammalian toxicity, environmental persistence and operator overexposure. Therefore any new formulations that are able to maintain a high efficacy whilst reducing the concentration of active ingredient are sought after.

(5) The present study examined the influence of application rate in grain on the efficacy of 1% Pirimiphos-methyl in Entostat (w/w) against O. surinamensis. To gather this information, quantities of grain were treated with four separate treatments of powder (inclusive of negative control), homogenised, and split into samples before the addition of the beetles. The beetles were to be maintained within the grain for two weeks or until ˜95% mortality was achieved, with mortality checks at 48 hour time points. However, in actuality only one time point check was required.

(6) Methods

3. TEST ITEM DETAILS

(7) 1% concentrate of Pirimiphos-methyl in Entostat (w/w) was produced at Exosect. Wax was heated in a copper pan and added to an over-head stirrer set to 600 rpm. Pirimithos-methyl was then added slowly under stirring. Stirring was continued for 5 min before pouring the liquid onto a metal tray and allowing to cool. Generally, the particles of wax of use in a dry powder composition of the invention possess a volume mean diameter of a selected size. To obtain particles of wax loaded with pirimithos-methyl of a volume mean diameter applicable for use in the invention, wax in the form of cooled down blocks, for example, 1 to 5 kilogram blocks or tablets may be broken up or kibbled into small millimeter-sized pieces (such as from 2 mm-8 mm approximate diameter in size, for example from 4 mm to 6 mm) in a kibbling machine. The millimeter-sized pieces can then be passed through a comminuting means such as a standard mill, e.g. an Apex Comminuting mill, and milled or comminuted into particles having an approximate diameter in the range from 100 μm-500 μm, for example from 250 μm-300 μm. The micron-sized comminuted particles can then be passed through a micronising apparatus, such as an AFG micronising air mill to obtain particles of any desired VMD range, such as up to 40 μm, that is of use in the present invention. The skilled addressee will appreciate that such procedures for obtaining small particles are well known in the art. Dry powder compositions of the invention typically comprise particles having a volume mean diameter of ≧5 μm, for example of 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm up to 200 μm or any value there in between, for example from ≧10 μm to 100 μm; or from ≧10 μm to 40 μm; or from ≧10 μm to 30 μm or any desired volume mean diameter value in between.

(8) 3a. QC Analysis was Carried Out Alongside the Study Following the Protocol:

(9) Summary

(10) The active, Pirimiphos-Methyl is extracted from the wax matrix by ultrasonication into n-hexane and analysed by gas liquid chromatography utilising a 100% dimethyl polysiloxane phase column to achieve separation from the non-actives. Detection is by Micro Electron Capture Detector (μECD) and quantitation is by internal standard.

(11) 1. Reagents

(12) TABLE-US-00001 1.1. n-hexane HPLC grade 1.2. internal standard Pentachloronitrobenzene 1.3. actives Pirimiphos-Methyl analytical reference material
2. Equipment and Apparatus
2.1. Gas Chromatograph

(13) Capable of operating over the range 100 to 350° C. with a μECD and an inlet which can accommodate a fused silica capillary column and a deactivated glass liner for split injections.

(14) 2.2. Column

(15) Fused silica 15 m, 0.25 mm id, 100% dimethyl polysiloxane film thickness 0.25 μm (Rxi-1 ms or equivalent). The column should be conditioned according to the manufacturer's recommendations before use.

(16) 2.3. Ultrasonic Bath

(17) Heated to 40° C. to improve extraction.

(18) 2.4. Dispenser Capable of Repeatable Dispensing of 50 ml of Organic Solvent

(19) 2.5. General Laboratory Glassware

(20) Volumetric flasks, bottles, beakers, pipettes, etc.

(21) Chromatographic Conditions

(22) Equilibrate the system using the following conditions until a steady baseline is achieved:

(23) TABLE-US-00002 Column Fused silica 15 m, 0.25 mm i.d. Rxi-1 ms or equivalent, film thickness 0.25 μm. Detector μECD 330   C. nitrogen make up gas: 60 ml/min (or as recommended by the manufacturer) Inlet System Split/Splitless Inlet at 250   C. Spit Ratio 40:1 Incorporating a pre-treated Split/Splitless liner Syringe Wash Solvent A: n- hexane Syringe Wash Solvent B: acetone Injection Volume 1 μl 3. C Procedure Work in a fume hood and away from other equipment, use Benchkote to protect the work surface from spillages of Pirimiphos- Methyl solutions and to contain the active and sample powder. Solution volumes and weighing amounts may be reduced in ratio if required but the resulting concentration must not change. 3.1. Preparation of internal standard solution Weigh 0.01 g of pentachloronitrobenzene Initial: 60   C. (PCNB) into a 100 ml volumetric flask, Hold for 2 min. dissolve in and make to volume with 40   C. min.sup.−1 to 350   C. extraction solvent. Transfer 10 ml into a Hold for 10 min. 100 ml volumetric flask and make to volume with extraction solvent (0.01 mg/ml internal standard solution). NB the detector response to PCNB is approx 100 x that of pirimiphos-methyl hence the amount of IS added is approx 100 times lower. 3.2. Preparation of calibration standard solution NB The values stated below are based on a nominal loading of 10 mg/g of active with a 100% purity reference material. The calibration range is calculated to be approx. 80-120% of this value. If the purity of reference material is less than 100% or if the nominal loading of active in the sample is different or uncertain (e.g. in weathered samples) the calibration and AQC must be adjusted accordingly. Not also that the detector response to PCNB is approx 100 that of pirimiphos- methyl hence the amount of IS added is approx 100 times lower. Calibration Standard Solution 1 Weigh accurately 0.1 g (to the nearest 0.1 mg) of the Pirimiphos-Methyl reference material into a 100 ml volumetric flask, dissolve in and make to volume with extraction solvent (1 mg/ml standard solution). Calibration Standard Solution 2 Into a series of 50 ml volumetric flasks pipette 0.7 ml of 0.01 mg/ml internal standard solution and add to each sucessive flask 0.56, 0.63, 0.7, 0.77 and 0.84 ml of Calibration Standard Solution 1. Make up to volume with extraction solvent. Correct for the purity of the reference material and to calculate the exact concentration of the standard solution in ug/ml. Preparation of Analytical Quality Control (AQC) solution. Weigh acurately 0.1 g (to the nearest 0.1 mg) of the Pirimiphos-Methyl ( ideally this should be from a difference source to the technical material but due to the limited sources of actives this is not always possible, and made up by a different operator on a different day) into a 100 ml volumetric flask, dissolve in and make to volume with extraction solvent (1 mg/ml AQC solution). 3.3. Linearity A linearity check may be made by examination of the calibration curve for the standards. Examine the curve and the correlation coefficient. Review the data if the r.sup.2 value is <0.98. 3.4. Sample Extraction Weigh accurately 70 mg (to the nearest 0.1 mg) prepared sample into a tared 60 ml bottle and record its weight. Add by dispenser 50 ml extraction solvent. Add by 0.7 ml of 0.01 mg/ml internal standard solution. Cap the bottle and shake vigourously for 5 seconds. Place in an ultrasonic bath heated to 40° C. and sonicate. Turn off the bath after 5 minutes, remove the bottles and shake each bottle vigourously to re-disperse the product. Return to the bath and continue to sonicate. Repeat every 5 mins until the 15 mins has elapsed. 3.5. Extraction AQC 3.6. On an analytical balance tare a 60 ml bottle, and weigh in 70 mg bare micronised carnuba wax (to the nearest 10 mg). Add by dispenser 50 ml of extraction solvent. Add by pipette 0.7 ml of AQC solution and 0.7 ml of 0.01 mg/ml internal standard solution. Cap the bottle and shake vigourously for 5 seconds. Place in an ultrasonic bath heated to 40° C. and sonicate. Turn off the bath after 5 minutes, remove the bottles and shake each bottle vigourously to re-disperse the product. Return to the bath and continue to sonicate. Repeat every 5 mins until the 15 mins has elapsed. Remove bottles from the bath and leave to stand for a minimum of 2 hours for the wax to settle. Correct for the purity of the reference material and to calculate the exact concentration of the aqcsolution in ug/ml. 3.7. Chromatographic Analysis The chromatographic injection sequence should be as follows: Blank run —extraction solvent Calibration standards x 5 Sample solutions (max. 12) AQC x 3 Calibration standard Low Calibration standard High Blank run (no injection) 30 min hold @ 350   C. Calculation 3.7.1. Calibration Construct a graph of peak area ratio PAR (Al peak area/peak area IS) (y axis) vs concentration (x axis) for the calibration standards use a linear trendline to find the line of best fit and display the coefficient of determination r.sup.2 and the equation for the line y = mX + c. Review the data if the r.sup.2 value is <0.98. olumn oven Helium Carrier Gas Constant pressure, 10 psi Run time Approximately 20 min. Approximate Retention Times Internal standard 6.6 min. Active 7.1 min.
3.7.2. Samples

(24) $\mathrm{Pirimiphos}\text{-}\mathrm{Methyl}\mathrm{concentration}\mathrm{mg}\text{/}g=\frac{\left(PAR-c\right)\times 50}{m\times \mathrm{sample}\mathrm{wt}\left(\mathrm{mg}\right)}$
Wherein:
PAR=peak area ratio
c=constant
m=slope
50=extraction solvent volume ml
3.8. AQC

(25) $AQC\mathrm{Al}\mathrm{concentraion}\mathrm{mg}\text{/}\mathrm{ml}=\frac{\left(PAR-c\right)}{m}$$\mathrm{Check}\mathrm{that}\text{:}\frac{AQC\mathrm{Al}\mathrm{concentration}\mathrm{mg}\text{/}\mathrm{ml}*100}{\mathrm{Calculated}AQC\mathrm{conc}.}=100+\text{/}-5\%$
If the AQC value lies outside the range 95-105%, review the data.

4. TEST SYSTEM

(26) The beetles tested in this study were sawtoothed grain beetle Oryzaephilus surinamensis. All beetles were taken from a lab-susceptible strain maintained at Exosect, which were set up from cultures supplied by the Food and Environment Research Agency (FERA) in 2011.

(27) Oryzaephilus surinamensis were maintained in cultures reared on rolled oats and wheatgerm in a ratio of 3:1, w/w at 22±1° C. and 40±5% relative humidity (RH). Unsexed adult beetles were used for the trial. Mating status and age were unknown.

(28) The untreated grain used for the experiment was supplied by Herbiseed, Twyford, England.

5. TEST LOCATION

(29) Bioassay room 2: Temperature was maintained between 25±1° C. (Howe, R. W., 1956). Temperature and relative humidity (RH) were measured hourly using a Lascar data logger.

6. EXPERIMENTAL DESIGN

(30) The treatments applied to the grain are listed as follows. There were 5 replicates for each treatment: 1) 10 mg of Entostat containing 0.1 mg of Pirimiphos-methyl (1%) per 50 g of grain. This is equivalent to 200 ppm of the formulation in grain. 2) 5 mg of Entostat containing 0.05 mg Pirimiphos-methyl (0.5%) per 50 g of grain. This is equivalent to 100 ppm of the formulation in grain. 3) 1 mg of Entostat containing 0.01 mg Pirimiphos-methyl (0.1%) per 50 g of grain. This is equivalent to 20 ppm of the formulation in grain. 4) Untreated control

7. APPLICATION DETAILS AND REGIME

(31) The treatments (adjusted based on sample size) were added to the grain in 250 g samples which were homogenised by hand in a glass conical flask sealed with parafilm to ensure a uniform coating of the grain. 5×50 g lots of each 250 g grain sample were placed into 5 clean glass pots per treatment.

(32) The different quantities of 1% Pirimiphos-methyl in Entostat (w/w) used in the protocol were based upon the standard 200-500 g/1000 kg of 2% Actellic dust (also expressible as 200-500 ppm in grain) used in a standard grain silo (Actellic, 1982, p. 43), in order to give comparable results. The quantity of Entostat added to each 50 g sample of grain equates to 10 mg, 5 mg and 1 mg respectively, for each treatment.

(33) Once the treatments were added to the grain, 20 mixed-sex adult beetles were added to each pot and covered with a piece of gauze held in place with an elastic band.

8. SAMPLING/MEASUREMENT REGIME

(34) The beetles were monitored for mortality every 48 h for two weeks, or until ˜95% mortality had been achieved. During analysis, each pot was tipped out onto a white tray and the number of dead beetles recorded. Death was defined as when beetles failed to respond to continuous physical agitation with forceps or a fine-tipped paintbrush. All beetles, grain and loose powder were tipped back into the sample pots using a glass funnel and the white trays. After the final mortality check, the contents of each pot were frozen then disposed of in the chemical waste bin.

9. STATISTICAL ANALYSIS

(35) Due to the nature of the results, the LT.sub.50 (time to kill 50% of individuals) could not be determined using univariate general linear models. One-way ANOVA and Tukey's HSD post-hoc tests were used to determine significant differences between the treatments.

(36) Results

10. EXPERIMENTAL RESULTS

(37) Analysis of the data across groups via one-way ANOVA (F.sub.(3 16)=3.0069, p=0.05) indicates that there are significant differences in the data at the 5% level between treatments (F=835.9506).

(38) Pair-wise comparison across groups using Tukey's HSD post-hoc tests indicate that the significant differences at the 5% level (q.sub.(4 16)=4.05, α=0.05) occur between the control and each pirimiphos-methyl treatment (q=61.3333).

(39) There are absolutely no significant differences between the 10 mg, 5 mg, and 1 mg pirimiphos-methyl treatments (q=0).

(40) Raw Data

(41) TABLE-US-00003 Mean S. dev Treatment 1 2 3 4 5 Mean S. dev (%) (%) Control 1 1 1 1 4 1.60 1.34 8 6.71 1 mg/50 g 20 20 20 20 20 20.00 0.00 100 0.00 5 mg/50 g 20 20 20 20 20 20.00 0.00 100 0.00 10 mg/50 g  20 20 20 20 20 20.00 0.00 100 0.00
QC Analysis

(42) TABLE-US-00004 Analysis Result for Batch: W2491-3 Product : 1% Pirimiphos Methyl in Entostat (w/w) Total Nominal Loading (mg/g): 10 Formulation Code: n/a Date of Analysis: 24 Jul. 2012 Sequence File Name: 120723 Pirimiphos-methyl Result Result of Nominal (mg/g) (%) Average 11.52 112

(43) Results from the QC analysis show that the concentration of Pirimiphos-methyl is well within the expected range in the Entostat Powder used for the study.

(44) Discussion

11. DISCUSSION

(45) This study demonstrates that 1% Pirimiphos-methyl in Entostat (w/w) is highly efficacious over an exposure duration of 48 hours when uniformly distributed at 10 mg, 5 mg and 1 mg in 50 g of wheat grain (equivalent to 200 g, 100 g, and 20 g per 1000 kg of wheat grain).

(46) The 1 mg/50 g samples in particular demonstrate the potential of 1% Pirimiphos-methyl in Entostat (w/w), as the application rate in grain is a tenth of that recommended for use with 2% conventionally applied Actellic Dust (Actellic, 1982, p. 43).

12. REFERENCES

(47) Howe, R. W. (1956). The Biology of the two common storage species of Oryzaephilus (COLEOPTERA, CUCUJIDAE). Annals of Applied Biology 44: 341-355. Imperial Chemical Industries. (1982). Actellic: Pirimiphos-methyl Broad Spectrum Insecticide (2.sup.nd ed.). Surrey, England: ICI.

EXAMPLES SECTION II

(48) The protocols described in Examples Section I above are used in determining the efficacy of different concentrations of Pirimiphos-methyl (0.25% and 0.5%) in Entostat (w/w) when used against Oryzaephilus surinamensis in grain. Similar results are observed.

Efficacy of Different Concentrations of Pirimiphos-Methyl (0.25% and 0.5%) in Entostat (w/w) when Used Against Oryzaephilus surinamensis in Grain

Study Outline

(49) Actellic is an organophosphate insecticide originally developed by Imperial Chemical Industries (now Syngenta) in 1967. It uses pirimiphos-methyl as its active ingredient. It is highly efficacious against a range of insect species (Actellic, 1982, p. 44) and up until recently has been successfully used for the control of insect pests in stored grain and a range of public health situations. New regulations have resulted in the withdrawal of support for Actellic due to concerns over mammalian toxicity, environmental persistence and operator overexposure. Therefore, any new formulations comprising active ingredient e.g. an insecticide such as Actellic, that demonstrate high efficacy at low concentration levels would prove desirable. It is an object of the present invention to provide formulations for treating seed that contain lower amounts of active ingredient than formulations described in the prior art.

(50) Preliminary tests at Exosect have demonstrated that 286 mg of 1% pirimiphos-methyl in Entostat (w/w) achieved 100% mortality against O. surinamensis when evenly distributed onto a filter paper (90 mm Ø) within a petri dish. This initial work served as evidence that pirimiphos-methyl was still active within the Entostat formulation.

(51) Following on from preliminary work, the influence of application rate in grain on the efficacy of 1% pirimiphos-methyl in Entostat (w/w) against O. surinamensis was examined. To gather this information, quantities of grain were treated with various amounts of powder and homogenised before the addition of the beetles. The beetles were maintained within the grain for two weeks or until ˜95% mortality was achieved, with mortality checks at 48 hour time points. This study demonstrated that 1% pirimiphos-methyl in Entostat (w/w) is highly efficacious, after achieving 100% mortality across all treatments over an exposure duration of 48 hours when uniformly distributed at 200 ppm, 100 ppm, and 20 ppm in wheat grain in sub-samples of 50 g.

(52) The purpose of the present study is to examine the influence of pirimiphos-methyl concentration in Entostat (w/w) on the mortality of Oryzaephilus surinamensis in grain. An application rate of 20 ppm in wheat grain is used.

(53) Having established that an application rate of 20 ppm in wheat grain provided an acceptably uniform coating and resulted in 100% mortality at a 1% concentration (w/w), this study looked to investigate the influence of active ingredient concentration at the same rate. 0.25%, 0.5% and 1.0% were tested.

(54) Methods

(55) Test Item Details

(56) 0.25%, 0.5% and 1% pirimiphos-methyl was added to liquid carnauba wax and particles were micronised therefrom as described herein, resulting in particles wherein the pesticide is present at a weight of 0.25%, 0.5% and 1% of the weight of the particles. Wax is heated in a copper pan and added to an over-head stirrer set to 600 rpm. Pirimithos-methyl is then added slowly under stirring. Stirring continues for 5 min before pouring the liquid onto a metal tray and allowing to cool.

(57) Batch numbers were: W2491-1 (0.25%), W2491-2 (0.50%), W2491-3 (1.00%)

(58) Test System

(59) The beetles tested in this study were sawtoothed grain beetle Oryzaephilus surinamensis. All beetles were taken from a lab-susceptible strain maintained at Exosect, which were set up from cultures supplied by the Food and Environment Research Agency (FERA) in 2011.

(60) Oryzaephilus surinamensis were maintained in cultures reared on rolled oats and wheatgerm in a ratio of 3:1 w/w at 22±1° C. and 40±5% relative humidity (RH). Unsexed adult beetles were used for the trial. Mating status and age were unknown.

(61) The untreated grain used for the experiment was supplied by Herbiseed, Twyford, England.

(62) Test Location

(63) Bioassay room 2: Temperature was maintained between 25±1° C. (Howe, R. W., 1956). Temperature and relative humidity (RH) were measured hourly using a Lascar data logger.

Experimental Design

(64) 0.25%, 0.5% and 1% pirimiphos-methyl in carnauba wax (Entostat) (w/w) and the blank Entostat were applied at a rate of 20 ppm in wheat grain (20 g/tonne). Therefore the quantity of Entostat added to each 50 g sample equates to 1 mg. The treatments applied to the grain are listed as follows. There were 5 replicates for each of the following treatments: 1) 1 mg of carnauba wax (Entostat) containing 10 μg of Pirimiphos-methyl (1% w/w) in 50 g of grain 2) 1 mg of carnauba wax (Entostat) containing 5 μg Pirimiphos-methyl (0.5% w/w) in 50 g of grain 3) 1 mg of carnauba wax (Entostat) containing 2.5 μg Pirimiphos-methyl (0.25% w/w) in 50 g of grain 4) 1 mg of blank carnauba wax (Entostat) in 50 g of grain (Vehicle control) 5) 50 g of grain (Untreated control)
Application Details and Regime

(65) The treatments (adjusted based on sample size) were added to the grain in 250 g samples and homogenised by hand in a glass conical flask sealed with parafilm to ensure a uniform coating of the grain. 50 g sub-samples of grain were placed into 5 clean glass pots per treatment.

(66) Once the treatments had been divided into sub-samples, 20 mixed-sex adult beetles were added to each pot and covered with a piece of gauze held in place with an elastic band.

(67) Sampling/Measurement Regime

(68) The beetles were monitored for mortality after 48 hours of exposure to the treatments. During analysis, each pot was tipped out onto a white tray and the number of dead beetles recorded. Death was defined as when beetles failed to respond to continuous physical agitation with forceps or a fine-tipped paintbrush. All beetles, grain and loose powder were tipped back into the sample pots using a glass funnel and the white trays were wiped down with 5% decon between checks. After the final mortality check, the contents of each pot were frozen for 24 h then disposed of in the chemical waste bin.

(69) Statistical Analysis

(70) One-way ANOVA and Tukey's HSD post-hoc tests were used to determine significant differences between treatments. Probit analysis was performed on mortality results, with adjustments for control mortality using Abbott's Formula (up to 20%), to estimate the LC.sub.50.

(71) Results

(72) Experimental Results

(73) Analysis of the data across groups via one-way ANOVA (F.sub.(4 20)=2.8661, p=0.05) indicates that there are significant differences in the data at the 5% level between treatments (F=252.2817).

(74) Pair-wise comparison across groups using Tukey's HSD post-hoc tests indicate that there is no significant difference at the 5% level (q.sub.(5 20)=4.23, α=0.05) between the untreated control and the vehicle control (q=1.7975). There are however significant differences between the 0.25% treatment and the untreated control (q=15.8543), and the vehicle control (q=14.0567).

(75) The untreated control is also significantly different to the 0.5%, 1.0% treatments (q=32.6293), as is the vehicle control against the same treatments (q=30.8321). There is no significant difference between the 0.5% and 1% pirimiphos-methyl in Entostat (w/w) treatments (q=0).

(76) QC Analysis

(77) TABLE-US-00005 Analysis Results for Batch: W2491-1, 2, 3 Comment: W2491-1 = Pirimiphos-Methyl in Entostat 0.25% Product: (w/w) W2491-2 = 0.50% W2491-3 = 1.00% Pirimiphos-methyl Result Result (mg/g) (%) W2491-1 2.47 96 W2491-2 5.52 110 W2491-3 11.53 115

(78) Results from the QC analysis show that the concentration of pirimiphos-methyl in Entostat (w/w) is well within the expected range for all treatments used in the study.

(79) Probit Analysis

(80) Probit analysis returned probit values of 4.98 and 4.9 for the 0.25% pirimiphos-methyl in Entostat (w/w) treatment after correcting for both the untreated control and the vehicle control respectively. No other probit values could be determined due to the nature of the results, therefore the 0.5% treatment result was regarded as a 99% mortality to give a probit value of 7.33.

(81) Using the regression method the LC.sub.50 was calculated to give a Log.sub.10 concentration of 0.407069 and therefore a concentration of 2.55 mg/g (0.255%) when using the mortality percentage corrected for the vehicle control. When using the untreated control, the regression method returned a Log.sub.10 concentration of 0.395669 and therefore a concentration of 2.49 mg/g (0.249%). Obtained LC.sub.50 values should be considered as marginal underestimations due to the methods used.

(82) Discussion

(83) This study shows that 0.249-0.255% pirimiphos-methyl in Entostat (w/w) is a good estimation of the LC.sub.50 against Oryzaephilus surinamensis over an exposure duration of 48 hours (LT.sub.50) when the powder is uniformly distributed at a rate of 20 ppm in wheat grain (LD.sub.50).

(84) This demonstrates the potential of ˜0.25% pirimiphos-methyl in Entostat (w/w), as the application rate in grain is a tenth of that recommended for use with 2% Actellic Dust (Actellic, 1982, p. 43), which is eight times more concentrated in formulation. This unexpectedly represents an overall 80 fold reduction over conventional application rates in the field.

(85) The LT.sub.50 for the 0.5% and 1% treatments is well within the 48 hour exposure period (given an LD.sub.50 of 20 ppm in grain).

REFERENCES

(86) Howe, R. W. (1956). The Biology of the two common storage species of Oryzaephilus (COLEOPTERA, CUCUJIDAE). Annals of Applied Biology 44: 341-355. Imperial Chemical Industries. (1982). Actellic: Pirimiphos-methyl Broad Spectrum Insecticide (2.sup.nd ed.). Surrey, England: ICI.
Raw Data

(87) TABLE-US-00006 Mean S. dev Treatment 1 2 3 4 5 Mean S. dev (%) (%) Control 1 0.00 3.16 1.00 2.00 0.00 1.23 1.36 6.16 6.80 Control 2 1.00 2.11 2.22 1.00 5.00 2.27 1.64 11.33 8.18 0.25% 8.42 11.00 13.33 10.00 9.00 10.35 1.94 51.75 9.68 0.50% 20.00 20.00 20.00 20.00 20.00 20.00 0.00 100.00 0.00 1.00% 20.00 20.00 20.00 20.00 — 20.00 0.00 100.00 0.00
NB: Control 1 represents the untreated control. Control 2 represents the vehicle control. In some cases data has been corrected to account for missing beetles. Also, due to a lack of insects available, 1 rep has been omitted from the 1.0% pirimiphos-methyl group. An artificial pseudo-result of 20.00 was used for rep 5 in that group in order to generate statistical data. This estimation for rep 5 was based on the mean results and variance in the other 4 reps in the data set.
Probit Analysis with Corrected Mortality (%) Using Abbott's Formula

(88) TABLE-US-00007 Corrected Corrected Conc. Conc. Total Mean % Mortality Mortality Probit Probit (mg/g) Log.sub.10 Number Mortality Mortality (C1) (C2) (C1) (C2) (C1) 0 — 20 1.23 6.158 — — — — (C2) 0 — 20 2.27 11.33 — — — — 2.47 0.39 20 10.35 51.75 48.5885212 45.591242 4.98 4.9 5.52 0.74 20 20 100 100 100 7.33* 7.33* 11.53  1.06 20 20 100 100 100 7.33* 7.33* *As mentioned in the report, 0.5% and 1.0% pirimiphos-methyl in Entostat (w/w) were treated as giving a corrected mortality of 99% in order to return Probit values of 7.33. This allowed for the calculation of the LC.sub.50, which as such should be considered as a marginal underestimation of the true LC.sub.50.
NB: C1 represents Control 1, the untreated control. C2 represents Control 2, the vehicle control. Probit values for the controls were not generated due to the concentration (mg/g) being 0. Probit values for 0.5% and 1.0% Primiphos-methyl in Entostat (w/w) could not be generated due to the corrected mortality being 100% in both cases.