Non Technical Summary
Previous work has established that the C8910, a mixture of volatile fatty acids (octanoic, nonanoic, and decanoic acids), has both repellent and insecticidal activity against a variety of biting flies, including stable and horn flies. Existing EPA registered formulations of C8910 for livestock fly control are oil solutions or adsorptions onto common clays that have been at relatively high concentrations of fatty acids (15% total fatty acids). While the dust formulation is cost competitive with traditional pesticide dusts when used in dust bags, this form of treatment is not suitable for many producers and the oil formulations are too expensive for cattle fly control. Therefore, we are proposing to develop an emulsifiable concentrate formulation of C8910 that can be field diluted with water and applied to cattle with manual or automated sprayers. User cost estimates show that a C8910 EC is cost-competitive with traditional pesticide ECs and initial tests have shown that an aqueous dilution of C8910 EC will kill stable flies and horn flies at relatively low concentrations (1% or less). We propose to optimize the C8910 EC formulation in the laboratory to insure that the EC and aqueous dilution are physically and chemically stable, while maintaining a high level of pesticidal activity at low actives concentration. Follow on field tests at Arkansas and Nebraska will optimize spray variables (dilution level, spray nozzle and volume, and spray frequency) to achieve and maintain horn fly numbers below the economic impact level. Successful completion of Phase I will be followed by Phase II activities to include additional field testing against horn flies and stable flies, which will be necessary for EPA registration and ultimately to market entry. Stratacor's collaboration with companies for raw material manufacture (Emery Oleochemicals) and marketing (Ecovet) have resulted in the successful market entrance of an oil formulation of C8910 (Ecovet Fly Repellent) we are eager to bring other formulations to market.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
121	0199	1130	100%

Knowledge Area
121 - Management of Range Resources;

Subject Of Investigation
0199 - Soil and land, general;

Field Of Science
1130 - Entomology and acarology;

Goals / Objectives
The first aim will be the optimization of emulsifiable concentrate (EC) formulations of C8910, using the E8700 formulation as a starting point to assure the physical and chemical stability of the concentrate and aqueous dilution, while preserving the insecticidal efficacy against both sensitive and resistant stable flies and horn flies. The second aim will be verification of laboratory studies with field testing of the optimized formulation in automated sprayers against natural populations of the horn fly only in Arkansas (stable flies not abundant) and horn fly and stable fly in Nebraska during the 2020 fly season. The variables of spray volume and nozzle type, spray frequency, and dilution level of the C9010 concentrate will be investigated for their effect on fly toxicity and duration of fly protection.

Project Methods
Using the E8700 formulation as a baseline, we will evaluate variations of this formulation (to include the ratio of the fatty acids, total fatty acid concentration, and surfactant and other formulation components) for their effect on the physical (40c, 200C and 400C storage) and chemical stability (GC assay) of the concentrate and aqueous dilution, and efficacy in killing stable and horn flies (Reifenrath, 2014). Permethrin sensitive and resistant stable fly and horn fly pupae will be obtained from the University of Florida and New Mexico State University, respectively. Pupae will be raised to adults at Stratacor and graded concentrations of C8910 in aqueous emulsion will be tested for knockdown and mortality using published procedures . A dose range is achieved by serial dilution of the 1% C8910 experimental formulations. Typically, fifteen flies (stable or horn flies in separate runs) are introduced into the covered Petri dish plate. Fly status (incapacitation) was tested immediately after treatment, at 15, 30, 60 minutes and 24 hours post-treatment. The assessment of knock-down was made within 60 minutes post-exposure. Mortality is measured at 24 hours post-exposure. A fly is classified as dead if it was immobile or unable to stand or fly in a coordinated way at 24 hours. For each formulation, positive and negative controls, tests will be replicated 4 times. An approximate LD50 and LD95 will be determined as calculated by Probit analysis.The skin disposition (evaporation, penetration, skin surface residue) of the fatty acids in aqueous dilutions of emulsion formulations will be conducted according to published procedures using radiometric or GC assays. A validated GC assay for the fatty acids in this formulation will be developed, necessary for EPA registration data requirements (OPPS 830.6317). These studies are important to understand the effect of formulation on the disposition of the actives, as skin penetration reduces fly exposure (contact is necessary for insecticidal effect). Studies will be conducted with freshly excised pig skin obtained under a tissue sharing arrangement with local laboratories, so no additional animal use will take place. Previous comparative studies between pig and cattle skin have shown that cattle skin is approximately 2x more permeable than pig skin and further that relative differences found between formulations tested on pig skin are likewise seen or correlated with cattle skin (r2 = 0.88). Data, expressed as percent of applied dose, is analyzed by ANOVA/Newman-Keuls Multiple Range Test at the 0.05 level of significance. Comparing the different concentrate dilutions, significant differences in evaporation are determined (approximately 60% for octanoic acid and 40% for decanoic acid). Likewise, significant differences in percutaneous penetration (about 20% for octanoic acid and 15% for decanoic acid) and residue remaining on the skin surface (approximately 5% for octanoic acid and 20% for decanoic acid) are determined at 24 hours. For comparison purposes, the skin dispositions of octanoic and decanoic acid in the concentrate dilutions will be compared to those obtained in previous in vitro pig skin studies with a mineral oil formulation, as this formulation has been registered with the US EPA for livestock fly control. The results for nonanoic acid can be interpolated between the results for octanoic and decanoic acids, as its physical properties are intermediate and previous studies with radiolabeled nonanoic acid support this approximation.Based on physical/chemical stability, laboratory pesticidal efficacy, and skin disposition studies, the best formulation (and a back up) will be prepared for field testing.Field Trials at Nebraska Objective: The overall objective of this study is to evaluate the efficacy of an aqueous (water-based) formulation of C8910, applied with an automated sprayer, on native horn and stable fly populations of pastured cattle in Nebraska. Duration is a 12 week period, May at onset of fly season into September, 2020. Location is North Platte, Nebraska. Pastures and treatments include 1) C8910, 2) Positive control (permethrin 10%/10% PBO insecticide, Pyrahna Stock Guard®) and 3) negative (untreated) Control.The study will be conducted with producer owned cattle, pastured in Lincoln County, NE. All pastures are within a ~3 kilometer distance of each other and we have data (unpublished) showing that fly numbers among pastures in that range of proximity are similar. All treatments will have a minimum of 15 cow/calf pairs with water and mineral supplements supplied ad libitum. Insecticide impregnated ear tags will not be applied to any cattle included in this study.Stage I: Insecticide treatments will be applied with a 3-D Quick Hand™ Cattle Sprayer made by 3-D Cattle Equipment, LLC, Pine Ridge, Arkansas 71961. The unit is an automated sprayer which applies a pre-determined amount of insecticide per animal as it passes through the sprayer entrance. Sprayers will be located in an entrance to a fenced-in water station. A pre-determined formulation of C8910 (Treatment 1) and Pyrahna Stock Guard (Treatment 2) will be applied to cattle as they enter and exit through the entrance of the water station. Frequency of spray applications will be determined by the number of times cattle visit the water tank. As a rule, cattle normally require water twice a day. In theory, animals could receive four spray treatments per day. The following factors will be evaluated to achieve maximum fly reduction for both treatments: 1) C8910 will be applied at a 1% concentration, and 2) Permethrin will be applied at labeled rate (0.05%). For Stage Ia, both sprayers will utilize current factory supplied nozzles, Tee Jet® 8008 flat fan nozzles. For Stage Ib, both sprayers will utilize Tee Jet® 8004 flat fan nozzles. Each nozzle package will be evaluated over a 2 week period.Efficacy of test products and nozzle packages will be determined by maintaining horn flies numbers below the Economic Injury Level (EIL) of 200 flies per animal and 3.6 flies/leg for stable flies.Stage II: Upon successful completion of Stage I, a longer-term study (4 - 6 weeks) will be initiated to directly compare the long-term effectiveness of treatments 1 and 2 on native horn flies and stable flies. The most effective nozzle from the initial stage Ia or Ib trials will be used.Fly Counts: A pre-count fly load (total flies per animal) will be recorded 1 hour prior to the spray application. Post-treatment fly counts will be recorded 2 hr. post-treatment and on days 1, 2, 3, 4 and 5 between the hours of 08:00 and 11:00 AM.Assessment of horn fly numbers will be made from digital images of one side of individual animals. Images will be recorded with a Nikon Model D7000 digital camera with a NIKKOR 28-300 lens. Archived images will later be processed using GIMP 2.6.11 GNV Image Manipulation Program. For horn flies, the count per image will be doubled to express the total number of flies per animal.Statistical Analysis: Fly numbers will be log transformed and analysis performed on the transformed variable. Repeated Measures and Least Square Means in GLIMMIX (SAS Institute 9.4) will be used to determine effects of fly treatment and fly population numbers. A P-value ≤ 0.05 will be considered significant.B2. Field Trials at Arkansas Field work in Arkansas will be similar to that outlined above for Nebraska field trials.