1. Evaluation of Permethrin-treated Uniforms Kristin Rury Armed Forces Pest Management Board The George Washington University

2. Uniform to be tested (permethrin treatment by factory impregnation, IDA kit, aerosol spray can, or air compressed sprayer methods) 1. Each uniform is washed 0, 20 or 50 times 2. Two samples are cut from each washed uniform 5A. The untested sample of the same uniform is sent to CMAVE after passing step 4 3. Using an ASE, the permethrin is extracted from one of the cut samples 5B. If the concentration of permethrin in sample is not within the minimum to maximum range (in milligrams/ cm 2 ) a single retest is allowed 4. A GC is used to analyzed the permethrin concentration in the sample* 6. Female mosquitoes are collected 7. Volunteer arm with uniform sample is placed in a chamber with live mosquitoes 8. Testing the efficacy of bite protection from the permethrin treatment is conducted by counting number of bites in each 0, 20 or 50 washing sample Soldier Systems Technology Research Laboratories Center for Medical, Agricultural and Veterinary Entomology * Testing the concentration of permethrin is important to ensure the concentration left in the garment is not too high (a potential health hazard) or too low (ineffective in protecting against vector bites) GC: Gas Chromatograph ASE: Accelerated Solvent Extractor

3. Soldier Systems Technology Engineering Research Laboratories Contracted textile lab where permethrin is tested for binding and retention to the fabric after a set number of washings Lab receives pre-treated fabric from each company

4. Instruments/products used for testing one uniform sample Analytical Balance 10-100 mL volumetric flasks Funnel Pipettes Automatic Die Cutter Three inch cutting die Extraction Process – ASE – Soxhlet Gas Chromatograph Mass Spectrometer Reagents – Permethrin analytical standard – Solvent mixture – Helium carrier gas

5. Sample Preparation The uniforms are washed using AATCC 135, 3, V, III to the specified number of cycles (0, 20, or 50) A three inch diameter circle is cut from the uniform using a 3 inch cutting die from single ply areas only

6. Sample Preparation – Samples are weighed to the nearest milligram – A second sample is cut from the same uniform for bite protection and insect resistance testing at a separate lab

7. Standard Preparation Standards are used to compare the amount of permethrin in the uniform – Six concentrations of permethrin standards which are 20, 50, 75, 100, 150, and 200ng/µL – The concentrations of the standards are calculated based on the weight of the permethrin.

8. Accelerated Solvent Extractor (ASE) Preparing the Specimens – Each circular uniform sample is rolled and placed into an ASE cell fitted with a cellulose filter – To ensure procedural quality, a control is used during each run of the ASE to detect any permethrin distribution to other vials

9. Extraction Procedure The permethrin is extracted from the treated specimens for 6 hours or until and extraction recovery of 95% or greater has been achieved. The extract is then re- concentrated to a final volume of 40mL. – A 1mL aliquot (a small portion of the solution) is prepared from every specimen extraction for GC analysis. – Permethrin recovery must be 95% or greater.

10. Quality Control “Blank vials” containing no permethrin or solution are used to ensure samples are contaminant free and to ensure there is no cross contamination during a run. – If any blank, after multiplying concentration by five, is greater than any specimen concentration result, the data becomes invalid and the system is checked.

11. Gas Chromatograph (GC) Procedure One µL of the solution from the ASE is placed into the Gas Chromatograph equipped with Mass Spectrometer. – Helium carrier gas is used – Rinse vials are used and contain 80% acetonitrile and 20% methanol The concentration of permethrin in each sample is found by: – Extracting the ion chromatograms 183 and 163 with the GC.

12. Concentration Calculation The concentration of permethrin is determined through: – Area under the chromatographic curve expressed in mass of permethrin per surface area (mg/cm2) If the concentration of permethrin in a sample is not within the minimum to maximum range (in milligrams/cm2) a single retest is allowed, otherwise the sample fails testing and is not distributed to troops.

13. USDA Center for Medical Agricultural and Veterinary Entomology (CMAVE)

14. Bite Protection Testing USDA Center for Agricultural and Veterinary Entomology (CMAVE) lab in Gainesville, FL The fabric is tested against live mosquitoes after a certain # of washings at the Soldier Systems Technology Engineering Research Laboratories – 0, 20, 50 times

15. CMAVE Mosquito Collection and Testing Figure 1 Figure 2 Figure 3

16. Efficacy of Bite Protection The process is repeated for samples of each washed uniform set with the same volunteer to ensure consistency of results Control – no permethrin; heavy biting and blood-feeding Permethrin treatment (0 washings) – very little biting and therefore blood-feeding