Analytical Summary Report

Transcription

1 Food and Environmental Quality Laboratory Page 1 of 43 Analytical Summary Report Insecticide Residues in/on Blueberry Foliage Authors Jane LePage and Vince Hebert Testing Facility Food and Environmental Quality Laboratory Department of Entomology Washington State University 2710 Crimson Way Richland, WA FEQL Study No.: 0313B Principle Field Investigators Lynell K. Tanigoshi 1, Beverly S. Gerdeman 1 and Wei Yang 2 1 Washington State University Mount Vernon Northwestern Washington Research & Extension Center (WSU- NWREC), State Route 536 Mount Vernon, WA North Willamette Research and Extension Center (OSU-NWREC), NE Miley Rd., Aurora, OR Collaborators Steve Erikson, CEO, Pan-American Berry Growers, Salem OR Study Timetable Study Initiation Date: 7/15/13 Experimental Termination Date: 2/20/14 Report Date 5/01/2014

2 Food and Environmental Quality Laboratory Page 2 of 43 Certification The undersigned hereby declares that this study was performed according to the procedures described herein, and that this report provides a true and accurate record of the results obtained. Associate Research Scientist: Date: 05/01/2014 Dr. Vince Hebert, Food and Environmental Quality Laboratory Washington State University, Tri-City Campus, Richland WA Analytical work performed by: Jane LePage Research Analyst Archives (Location of Raw Data) The original raw data, correspondence logs, and all relevant information for the study titled: Insecticide Residues in/on Blueberry Foliage, FEQL project number 0313B, along with certified originals of the signed analytical summary report will be maintained by the testing facility for a period of 3 years. Exact copies of the analytical summary report and relevant information for the construction of this study can be made available to the principle field investigators or collaborators on request. Associate Research Scientist Testing Facility: Dr. Vincent Hebert Food and Environmental Quality Laboratory Washington State University 2710 Crimson Way Richland, WA

3 Food and Environmental Quality Laboratory Page 3 of 43 Table of Contents Page Certification 2 Archives (location of raw data) 2 Table of Contents 3 Analytical Summary 4 I. Objective/Introduction 4 II. Sample Inventory & History 4 III. Standard Preparation 10 IV. Analytical Procedure 11 A. Residue Method 11 B. Analytical Limits 12 C. Instrumentation 12 D. Quantitation 13 E. Interferences 15 F. Confirmatory Techniques 15 G. Time Required for Analysis 15 H. Modifications or Potential Problems 15 V. Results 16 A. Method Validation and Recovery Results 16 B. Storage Stability 18 C. Residue Results 18 Appendix A: Project Protocol 25 Appendix B: Working Method 31 Appendix B: Sample Chromatograms 34

4 Food and Environmental Quality Laboratory Page 4 of 43 I. Objective/Introduction Analytical Summary Report Pesticides were applied weekly on high bush blueberries (Vaccinium corymbosum) at Pan- American Berry Growers (PBG), Salem, OR as part of a program to control spotted wing drosophila, Drosophila suzukii (Matsumura; SWD). An extensive study to monitor pesticide decline on marketable fruit was conducted during the 2013 growing season (See FEQL project 0313A). In conjunction with that study, leaf samples were collected for SWD bioassays by WSU-NWREC. This study provides residue data for malathion and fenpropathrin to support the bioassay data. From July through September, 2013, malathion, zeta-cypermethrin (Mustang Max ) and fenpropathrin (Danitol ) were applied to high bush blueberry plots by either mistigation or airblast sprayer. Leaf samples were collected at approximately -1, 0, 1, 3, 5, and 7 days after treatment (DAT) to evaluate pesticide residues in/on the leaves. Only malathion and fenpropathrin were successfully quantified from the complex foliage matrix for this study. High interference and matrix enhancement, coupled with unreliable control samples precluded the analysis for zeta-cypermethrin. II. Sample Inventory & History A separate control block of Aurora high bush blueberries was designated at the start of this study. This plot received no malathion, zeta-cypermethrin or fenpropathrin pesticide applications during the course of the study. Control blueberry leaves were collected from the control block on each sampling day. At the treatment blocks, leaves were picked at mid and high locations from each side of the blue berry bush row at ca. 10 randomly selected plants for the composited sample. Foliage samples were labeled according to protocol with M, AB, or C to designate treatment block (mistigation, airblast or control), the sampling date and F for Foliage. The control and treated foliage samples for subsequent chemical analysis were stored in freezers at PBG or the North Willamette Research and Extension Center (OSU-NWREC) until transport to the FEQL. Samples were shipped by ACDS freezer truck on 8/12/13, 9/13/13 and 10/15/13. On arrival at the laboratory all samples were placed in a freezer (ID Dasher) until analysis. Table 1 provides the foliage sample inventory and dates for subsequent handling of the samples in frozen storage at FEQL. The sample names represent the name on the sample upon arrival appended with the FEQL project number, 0313.

5 extraction for Malathion extraction for fenpropathrin Washington State University Food and Environmental Quality Laboratory Page 5 of 43 Table 1 FEQL Foliage Sample Inventory Sample IDs Sample date Treatment Status F1 Arrival at FEQL PFPD analysis Days in frozen storage 0313-C F 7/27/13 8/13/13 2/20/14 2/20/ set15re NA NA 0313-M F-T 7/27/13 M(-1) 8/13/13 2/5/14 2/6/ set12re NA NA 0313-M F-TD 7/27/13 M(-1) 8/13/13 2/5/14 2/6/ set12re NA NA 0313-AB F-T 7/27/13 M(-1) 8/13/13 2/5/14 2/6/ set12re NA NA 0313-AB F-TD 7/27/13 M(-1) 8/13/13 2/5/14 2/6/ set12re NA NA 0313-C F 7/28/13 8/13/13 12/17/13 12/18/ SET1RE 0313-M F 7/28/13 M0 9/14/13 1/30/14 2/3/ set10 NA NA 0313-AB F 7/28/13 9/14/13 1/30/14 2/3/ set10 NA NA Set ECD Days in frozen storage 0313-C F 7/29/13 8/13/13 1/7/14 1/11/ set2re 1/7/14 1/8/ set2re 0313-C F 7/29/13 2/3/14 2/4/ set11 NA NA 0313-M F-T 7/29/13 M1 8/13/13 2/3/14 2/6/ set11 NA NA 0313-M F-TD 7/29/13 8/13/13 2/3/14 2/4/ set11 NA NA 0313-AB F-T 7/29/13 9/14/13 2/3/14 2/6/ set11 NA NA 0313-AB F-TD 7/29/13 9/14/13 2/3/14 2/6/ set11 NA NA 0313-C F 7/30/13 8/13/13 1/30/14 1/30/ set10 NA NA 0313-M F 7/30/13 M2 8/13/13 1/30/14 2/3/ set10 NA NA 0313-AB F 7/30/13 8/13/13 1/30/14 2/3/ set10 NA NA 0313-C F 7/31/13 8/13/13 2/10/14 2/10/ set13 NA NA 0313-M F 7/31/13 M3 8/13/13 2/10/14 2/10/ set13 NA NA 0313-AB F 7/31/13 8/13/13 2/10/14 2/10/ set13 NA NA 0313-C F 8/2/13 8/13/13 1/28/14 1/28/ set9 NA NA 0313-M F 8/2/13 M5 8/13/13 1/28/14 1/28/ set9 NA NA 0313-AB F 8/2/13 8/13/13 1/28/14 1/28/ set9 NA NA Set

6 extraction for Malathion extraction for fenpropathrin Washington State University Food and Environmental Quality Laboratory Page 6 of 43 Sample IDs Sample date Treatment Status F1 Arrival at FEQL PFPD analysis Days in frozen storage 0313-C F 8/3/13 8/13/13 2/3/14 2/4/ set11 NA NA 0313-M F 8/3/13 MM(-1) 8/13/13 2/10/14 2/10/ set13 NA NA 0313-AB F 8/3/13 8/13/13 2/10/14 2/10/ set13 NA NA 0313-C F 8/4/13 8/13/13 1/30/14 1/30/ set10 NA NA 0313-M F 8/4/13 M7, MM0 8/13/13 1/28/14 1/28/ set9 NA NA 0313-AB F 8/4/13 8/13/13 1/28/14 1/28/ set9 NA NA Set ECD Days in frozen storage 0313-C F 8/5/13 8/13/13 2/17/14 2/19/ set16 2/17/14 F2 196 set M F 8/5/13 MM1 8/13/13 1/28/14 1/28/ set9 NA NA 0313-AB F 8/5/13 8/13/13 1/28/14 1/28/ set9 NA NA 0313-C F 8/7/13 8/13/13 2/4/14 2/4/ set12 NA NA 0313-M F 8/7/13 M10, MM3 8/13/13 2/11/14 2/12/ set14 NA NA 0313-AB F 8/7/13 8/13/13 2/11/14 2/12/ set14 NA NA 0313-C F 8/9/13 8/13/13 2/4/14 2/4/ set12 NA NA 0313-M F 8/9/13 MM5 8/13/13 2/10/14 2/10/ set13 NA NA 0313-AB F 8/9/13 8/13/13 2/10/14 2/10/ set13 NA NA 0313-C F 8/10/13 8/13/13 2/4/14 2/4/ set12 NA NA 0313-M F 8/10/13 M(-1) 8/13/13 2/11/14 2/12/ set14 NA NA 0313-AB F 8/10/13 8/13/13 2/11/14 2/12/ set14 NA NA 0313-C F 8/11/13 9/14/13 1/15/14 1/16/ set4 NA NA 0313-C F 8/11/13 2/5/14 2/6/ set12re NA NA 0313-M F 8/11/13 M0, MM7 9/14/13 2/11/14 2/12/ set14 NA NA 0313-AB F 8/11/13 9/14/13 2/20/14 2/24/ set15re NA NA 0313-C F 8/12/13 9/14/13 1/23/14 1/23/ set 8 NA NA 0313-M F 8/12/13 M1 9/14/13 1/23/14 1/23/ set 8 NA NA Set

7 extraction for Malathion extraction for fenpropathrin Washington State University Food and Environmental Quality Laboratory Page 7 of 43 Sample IDs Sample date Treatment Status F1 Arrival at FEQL PFPD analysis Days in frozen storage 0313-AB F 8/12/13 9/14/13 1/23/14 1/23/ set 8 NA NA 0313-C F 8/13/13 9/14/13 1/21/14 1/21/ set6 NA NA 0313-M F 8/13/13 M2 9/14/13 1/21/14 1/21/ set6 NA NA 0313-AB F 8/13/13 9/14/13 1/21/14 1/21/ set6 NA NA 0313-C F 8/14/13 9/14/13 1/22/14 1/22/ set7 NA NA 0313-M F 8/14/13 M3, MM10 9/14/13 1/22/14 1/22/ set7 NA NA 0313-AB F 8/14/13 9/14/13 1/22/14 1/22/ set7 NA NA 0313-C F 8/16/13 9/14/13 2/5/14 2/6/ set12re NA NA 0313-M F 8/16/13 M5 9/14/13 1/16/14 1/17/ set5 NA NA 0313-AB F 8/16/13 9/14/13 1/16/14 1/20/ set5 NA NA 0313-C F 8/17/13 9/14/13 2/5/14 2/6/ set12re NA NA 0313-M F 8/17/13 MM(-1) 9/14/13 1/21/14 1/21/ set6 NA NA 0313-AB F 8/17/13 9/14/13 1/21/14 1/21/ set6 NA NA 0313-C F 8/18/13 9/14/13 1/16/14 1/17/ set5 NA NA 0313-M F 8/18/13 M7, MM14, MM0 9/14/13 1/16/14 1/17/ set5 NA NA 0313-AB F 8/18/13 9/14/13 1/16/14 1/17/ set5 NA NA 0313-C F 8/19/13 9/14/13 2/11/14 2/12/ set14 NA NA 0313-M F 8/19/13 MM1 9/14/13 1/21/14 1/21/ set6 NA NA 0313-AB F 8/19/13 9/14/13 1/21/14 1/21/ set6 NA NA 0313-C F 8/21/13 9/14/13 NA NA NA 0313-M F 8/21/13 M10, MM3 9/14/13 1/16/14 1/17/ set5 NA NA 0313-AB F 8/21/13 9/14/13 1/16/14 1/20/ set5 NA NA 0313-C F 8/23/13 9/14/13 NA NA NA Set ECD Days in frozen storage Set

8 extraction for Malathion extraction for fenpropathrin Washington State University Food and Environmental Quality Laboratory Page 8 of 43 Sample IDs Sample date Treatment Status F1 Arrival at FEQL PFPD analysis Days in frozen storage 0313-M F 8/23/13 MM5 9/14/13 1/15/14 1/16/ set4 NA NA 0313-AB F 8/23/13 9/14/13 1/15/14 1/16/ set4 NA NA 0313-C F 8/24/13 9/14/13 1/14/14 1/14/ set3re NA NA 0313-M F 8/24/13 D(-1) 9/14/13 1/14/14 1/14/ set3re NA NA 0313-AB F 8/24/13 9/14/13 1/14/14 1/14/ set3re NA NA 0313-C F 8/25/13 9/14/13 1/15/14 1/16/ set4 NA NA 0313-C F 8/30/13 9/14/13 1/15/14 1/16/ set4 NA NA 0313-M F 8/30/13 D5 9/14/13 1/14/14 1/14/ set3re NA NA 0313-AB F 8/30/13 9/14/13 1/14/14 1/14/ set3re NA NA Set ECD Days in frozen storage 0313-M F 9/1/13 9/14/13 1/7/14 1/11/ set2re 1/7/14 1/8/ set2re 0313-C F 9/1/13 9/14/13 1/15/14 1/16/ set4 NA NA 0313-AB F-A 9/1/13 D7, MM14 9/14/13 1/7/14 1/11/ set2re 1/7/14 1/8/ set2re 0313-AB F-B 9/1/13 D8/D0 9/14/13 1/7/14 1/11/ set2re 1/7/14 1/8/ set2re 0313-C F 9/2/13 9/14/13 1/15/14 1/16/ set4 NA NA 0313-AB F 9/2/13 D9/D1 9/14/13 1/7/14 1/11/ set2re 1/7/14 1/8/ set2re 0313-C F 9/3/13 9/14/13 2/17/14 2/19/ set16 2/17/14 F2 167 set AB F 9/3/13 D10/D2 sample not found in shipment 0313-C F 9/5/13 9/14/13 2/17/14 2/19/ set16 2/17/14 F2 165 set AB F 9/5/13 D4 9/14/13 1/7/14 1/11/ set2re 1/7/14 1/8/ set2re 0313-C F 9/9/13 9/14/13 2/17/14 2/19/ set16 2/17/14 F2 161 set AB F 9/9/13 D8 9/14/13 12/17/13 12/18/13 99 set1re 12/17/13 12/19/13 99 set1re 0313-C F 9/13/13 9/14/13 2/17/14 2/19/ set16 2/17/14 F2 157 set AB F 9/13/13 D12 9/14/13 12/17/13 12/18/13 95 set1re 12/17/13 12/19/13 95 set1re 0313-C F 9/16/13 10/17/13 2/17/14 2/19/ set16 2/17/14 F2 154 set16 Set

9 extraction for Malathion extraction for fenpropathrin Washington State University Food and Environmental Quality Laboratory Page 9 of 43 Sample IDs Sample date Treatment Status F1 Arrival at FEQL PFPD analysis Days in frozen storage Set ECD Days in frozen storage 0313-AB F 9/16/13 D15 10/17/13 12/17/13 12/18/13 92 set1re 12/17/13 12/19/13 92 set1re 0313-C F 9/25/13 10/17/13 2/17/14 2/19/ set16 2/17/14 F2 145 set AB F 9/25/13 D24 10/17/13 12/17/13 12/18/13 83 set1re 12/17/13 12/19/13 83 set1re NA=not analyzed for this compound F1 Treatment status: M=malathion; MM=Mustang Maxx; D=Danitol application. Numbers represent days after treatment F2: These samples were analyzed for fenpropathrin on a GC/MS because ECD was not available. Sample set not accepted for quantification due to high recovery from matrix enhancement, but samples were qualitatively observed to have small fenpropathrin peaks. See section IV.H. Set

10 Food and Environmental Quality Laboratory Page 10 of 43 III. Standard Preparation Linearity standards in control matrix were prepared with each sample set to compare sample residues to a linear regression calculated from four standard concentrations. When necessary, samples were diluted to fit the range of linearity standards. Table 2 lists the test substances, spiking solutions, and standard dilutions used throughout this study. Table 2: Test Substances and Standards Use Reference Expiration Compounds No. Purity Source Date Malathion Test Substance % ChemService 5/31/16 Zeta-cypermethrin Test Substance ChemService 3/31/18 Fenpropathrin Test Substance ChemService 6/30/18 Compound Malathion Zeta-cypermethrin Fenpropathrin Solutions & Dilutions of Test Substances Use Solution Expiration Conc. Solvent No. Date Spiking mg/ml acetonitrile 5/22/14 Spiking ug/ml acetonitrile 5/22/14 Spiking ug/ml acetonitrile 5/22/14 Std verification mg/ml acetonitrile 6/10/14 Std verification ug/ml acetonitrile 6/10/14 Std verification ug/ml acetonitrile 6/10/14 Spiking mg/ml acetonitrile 5/22/14 Spiking ug/ml acetonitrile 5/22/14 Spiking ug/ml acetonitrile 5/22/14 Std verification mg/ml acetonitrile 5/22/14 Std verification ug/ml acetonitrile 5/22/14 Std verification ug/ml acetonitrile 5/22/14 Spiking mg/ml acetonitrile 5/22/14 Spiking ug/ml acetonitrile 5/22/14 Spiking ug/ml acetonitrile 5/22/14 Std verification mg/ml acetonitrile 5/22/14 Std verification ug/ml acetonitrile 5/22/14 Std verification ug/ml acetonitrile 5/22/14

11 Food and Environmental Quality Laboratory Page 11 of 43 Mixed Solutions Compounds Solution No. Conc. Solvent Expiration Data Malathion; Zeta-cypermethrin; M0313T µg/ml each acetonitrile 5/22/14 Fenpropathrin; Phosmet Malathion; Zeta-cypermethrin; M0313T µg/ml each toluene 5/22/14 Fenpropathrin; Phosmet Malathion; Zeta-cypermethrin; M0313T-22 1 µg/ml toluene 5/22/14 Fenpropathrin; Phosmet Malathion; Zeta-cypermethrin; M0313T µg/ml toluene 5/22/14 Fenpropathrin; Phosmet Malathion; Zeta-cypermethrin; M0313T µg/ml toluene 5/22/14 Fenpropathrin; Phosmet Malathion; Zeta-cypermethrin; M0313T µg/ml toluene 5/22/14 Fenpropathrin; Phosmet Malathion; Zeta-cypermethrin; Fenpropathrin; Phosmet M0313T µg/ml toluene 5/22/14 All standard solutions were stored in the freezer at <-15 C (I.D. Prancer). The dilutions made during the course of the study are recorded in the FEQL analytical laboratory standards book. Matrix standards were created on each day of analysis using an extra control leaf extract diluted to the same level as the samples. Generally the matrix standards were prepared in the range of 0.2 to 2 µg/ml by spiking matrix with one of the mixed standards listed in Table 2. IV. Analytical Procedure A. Residue Method i. Sample control and fortification For each analytical set, at least one control and one recovery fortification sample were extracted and analyzed in the same manner as the residue samples. The recovery fortification sample was prepared by administering a known volume of mixed spiking solution into the pre-weighed foliage. ii. Procedure The working method used in this study was a variation of the QuEChERS method. Analytical quantification was performed by gas chromatography (GC)-mass spectrometry (GC-MS), GC with micro electron capture detection (GC-µECD) and GC with pulsed flame photometric detector (GC-PFPD). The working method for this study is provided in Appendix A. Matrix extraction: 10 g of blueberry leaves were weighed and cut to approximately 1/4-inch strips into a 50 ml centrifuge tube. To each sample was added ca. 8 ml of water and 10 ml extraction solvent (1% acetic acid in acetonitrile). Samples were allowed to soak for 30 minutes or more and then blended for 3 minutes with a Pro Homogenizer until relatively smooth.

12 Food and Environmental Quality Laboratory Page 12 of 43 The organic solvent was separated from the aqueous phase by addition of 4 g anhydrous magnesium sulfate and 1 g sodium acetate. The sample was shaken vigorously for 1 minute, then placed in a centrifuge for 5 minutes. Next approximately 8 ml of the upper organic layer was transferred to a second, 15 ml, centrifuge tube containing PSA and magnesium sulfate. This tube was then mixed and centrifuged again. A second cleanup step was achieved by draining a measured 5 ml aliquot of sample though conditioned Extract Clean Carbograph 300 mg SPE cartridge, followed by a ca. 3 ml acetonitrile rinse. The eluate was collected; the solvent was exchanged to toluene and brought to final volume for analysis. B. Analytical Limits The working method was validated by fortifying and recovering the compounds of interest from control blueberry leaves. For malathion analysis by GC-PFPD, the method was validated in triplicate at 0.1 µg/g, 0.5 µg/g and 5 µg/g. Fenpropathrin was validated in triplicate at 0.5 and 5 µg/g. Results from the method validation are presented in section V. C. Instrumentation Pesticide residue concentrations were determined using one of three instruments. Instrument conditions are presented below. A Varian 3400 CX gas chromatograph with pulsed flame photometric detection (GC/PFPD) and 8200 CX autosampler was used for malathion determination. Integration of chromatographic data was performed using the Star Workstation software. Column: Alltech EC-1, 15m x 0.53mm, 1.2 μm film thickness Carrier gas: Temperatures: Detector: 300 C Injection volume: Detector: Retention time: Ultrapure helium, flow ca. 12 ml/min. Injector port: 225 to 250 C at 250 C/min Oven program 2 μl Initial: 100 C ramp 30 C/min to 300 C, hold 1 min. Air Hydrogen Nitrogen makeup gas Based on the observed retention times of external calibration standards in each set. An Agilent 6890N gas chromatograph with micro-electron capture detection (GC/µECD) and 7683 Autosampler was used for fenpropathrin detection and quantification. Integration of chromatographic data was performed using Chemstation software. Column: DB-5ms, 30m x 0.32mm, 0.25 μm film thickness Carrier gas: Ultrapure helium, constant flow 2.2 ml/min.

13 Food and Environmental Quality Laboratory Page 13 of 43 Temperatures: Injector port: splitless, 250 C Injection volume: Detector: Retention time: Oven program 2 μl Initial: 100 C ramp 25 C/min to 150 C, ramp 10 C/min to 300 C, hold 2 min. µecd 250 C; hydrogen 2 ml/min; air 60 ml/min; nitrogen makeup 30 ml/min Based on the observed retention times of external calibration standards in each set. An Agilent 6890N gas chromatograph with mass spectrometry detection (GC/MS) and 7683 Autosampler was used for zeta-cypermethrin detection and quantification. Integration of chromatographic data was performed using Chemstation software. Column: Carrier gas: DB-5ms, 25m x 0.25mm, 0.25 μm film thickness Temperatures: Injector port: 250 C Injection volume: Detector: SIM Retention time: Ultrapure helium, constant flow 2.2 ml/min. Oven program 2 μl Initial: 100 C ramp 25 C/min to 150 C, ramp 10 C/min to 300 C, hold 2 min. Source 230 C, Quad 150 C Zeta-cypermethrin: 163 m/z, and 181 m/z Based on the observed retention times of external calibration standards in each set. To verify the reliability of the GC instrument, calibration standards were inserted into the sample set during each GC analysis. The peak area counts and retention times for the compound were assessed for reproducibility and accuracy. Additionally, extracts were injected in duplicate. If extract peak areas agreed within 20% then the average of the two results were used to calculate concentrations. D. Quantification The quantification of pesticide residues in 2 g blueberry leaf sample was performed by electronic peak area measurement of each pesticide and comparison to the linear regression from at least four standards in the concentration range of sample extracts. To assure high quality during GC operation, all samples were bracketed with external calibration standards during the analytical set. For each analytical set, linearity and calibration standards were used to construct the calibration curves using a spreadsheet program (Excel ).

14 Food and Environmental Quality Laboratory Page 14 of 43 The residue concentration in the foliage samples is calculated by multiplying the calculated concentration by the equivalent final volume of the sample extract, and then dividing by the weight of foliage sample (dilution factor). Results are reported as total concentration of each individual pesticide in blueberry leaves. For example, set-2re, sample 0313-F-FS8, the malathion linear regression line of best fit, computed from the combined linearity and calibration standards of this set (n=11, R 2 =0.994) is expressed by equation 1: Eq. 1 Y =m X + B Where Y is peak area, m is the slope of the line, X is detected concentration, and B is the intercept. Y = x X ml of the acetonitrile extract was exchanged to toluene for analysis and brought to a final volume of 5 ml. This is equivalent to 1 g of crop in 5 ml. The average peak area count from two injections of the sample was Because this is a fortified sample, the peak area of the control is subtracted from the peak area of the fortified sample. However, there was no malathion detected in the corresponding control sample in this set. The concentration (in µg/ml) of malathion in the extract is calculated according to Eq. 1: = x X X=1.073 µg/ml malathion The concentration of malathion in the foliage sample is then figured by multiplying by the ratio of the final volume to the sample weight: (1.073 µg/ml malathion)(5 ml/1 g) = 5.36 µg/g malathion To assess overall analysis precision and percent recovery on a per-set basis, control samples were fortified with a known amount of standard prior to extraction. For each analytical set, percent recovery for the fortified sample was calculated according to Equation 2. When necessary, control-sample peak area was subtracted from fortified recovery sample peak of similar dilution for fortified-residue determination. Eq. 2 % Recovery = (amount detected*) x 100 Amount fortified * amount detected is calculated from fortified peak area minus control peak area. For example, sample 0313-F-FS8 was a fortified sample spiked with 10 µg malathion (5 µg/g in foliage). The percent recovery of malathion in the extract is then calculated by eq. 2. % Recovery = (5.36 µg/g) x 100 = 107.3% 5 µg/g The same calculation method was used for each of the pesticides in this study.

15 Food and Environmental Quality Laboratory Page 15 of 43 E. Interferences The GC/PFPD analysis for malathion was straight forward and sensitive. The zeta-cypermethrin peak is a cluster of four isomers of the compound. To adequately quantify the compound the integration program was set to force the peak and sum all peak areas within the retention time window. Although zeta-cypermethrin was validated at the 0.5 µg/g level, analysis of sample sets was unacceptable due to the lack of reliable control leaf matrix for use in matrix standards. Results were not reliable and often exhibited high matrix enhancement even with matrix standards. Additionally, there was a large interfering peak at the retention time of the compound which prevented use of µecd for quantitation. Zetacypermethrin was not evaluated for this project. Typical for ECD chromatography, the µecd chromatograms have some interfering peaks at the lower concentration levels attempted for validation, making integration of the peak of interest difficult. However, sample analysis indicated that these low levels were not necessary for this project. Additionally, matrix enhancement was present which required the use of matrix-matched standards for the foliage sample analyses. F. Confirmatory Techniques Matrix-matched analytical standards were used to confirm the presence of malathion and fenpropathrin by retention times. G. Time Required for Analysis The time required for an experienced person to work up a set of samples (generally six samples, one control, and one fortified sample) was approximately 6 hours. The duration of the GC analyses of each set depended on which compounds were of interest in the samples. Sample sets were analyzed on multiple instruments after extraction. Each sample set was injected using the auto sampler associated with the instrument. H. Modifications or Potential Problems High-quality reagents, pesticide grade or better, must be used throughout the study to avoid chromatography problems and contamination in the control. Method development work indicated a significant matrix enhancement effect characterized by improved peak shape and peak response in the presence of matrix compared to the analytical standards made in pure solvent. To overcome the influence of the matrix, matrixmatched standards were created for each set. The QuEChERS method was quick and easy and yielded reliable extraction results for malathion and fenpropathrin. However the method introduces a lot of salt and co-extractable components to the sample extracts. The consumption of disposable supplies, need for matrix standards and the greatly increased maintenance requirements makes this method less than ideal for a project of this magnitude.

16 Food and Environmental Quality Laboratory Page 16 of 43 Due to laboratory project schedules, the fenpropathrin analysis for control samples in set 16 was performed by GC/MS. The fortified recovery for this set was outside the range of acceptability however a qualitative review of the control foliage samples in the set (see table 1) indicated a persistent background concentration of fenpropathrin in all samples at less than 0.5 µg/g but observable on the chromatograms. These samples are marked less than limit of quantitation (<LOQ) in the results. V. Results A. Method Validation and Recovery All reagents and instruments used during the course of this study are commercially available and typical of what would be present in most analytical laboratories. The working method was validated at three concentration levels for malathion, 0.1 µg/g, 0.5 µg/g and 5 µg/g and two levels for fenpropathrin, 0.5 µg/g and 5 µg/g. The low end fortifications of 0.1 or 0.5 µg/g, respectively, were designated as the method s limit of quantitation (LOQ) for malathion and fenpropathrin. The method limit of detection (LOD) was empirically estimated at approximately 0.05 µg/g for malathion and 0.1 µg/g for fenpropathrin. Table 3 lists the method validation results. Table 4 provides the concurrent recovery results for malathion and fenpropathrin in the fortified samples extracted with each set. Table 3 Method Validation Percent Recoveries ID Fortification Level (µg/g) SET Malathion Fenpropathrin 0313-FMV MV % 0313-FMV MV % 0313-FMV MV % Zetacypermethrin 0313-F-FS1 0.1 Set1 90.3% 60.8% Rejected 0313-F-FS2 0.1 Set1 97.1% 74.3% Rejected 0313-F-FS3 0.1 Set1 91.9% 76.2% Rejected 0313-FMV7 0.5 MV2 76.3% 90.7% 98.1% 0313-FMV8 0.5 MV2 78.7% 87.2% 98.9% 0313-FMV9 0.5 MV2 86.4% 88.4% 99.3% 0313-F-FS4 5 set1re 84.6% 78.8% 118.3% 0313-F-FS5 5 set1re 84.8% 83.7% Rejected 0313-F-FS6 5 set1re 88.7% 98.6% Rejected

17 Food and Environmental Quality Laboratory Page 17 of 43 Table 4 Concurrent Fortified Samples Percent Recoveries ID Fortification Level (µg/g) SET Malathion Fenpropathrin 0313-F-FS1 0.1 Set1 90.3% 60.8% 0313-F-FS2 0.1 Set1 97.1% 74.3% 0313-F-FS3 0.1 Set1 91.9% 76.2% 0313-F-FS9re 0.5 set3re % NA 0313-F-FS10re 0.5 set3re 129.3% NA 0313-F-FS set % NA 0313-C F-FS set5 93.2% NA 0313-C Fre 0.5 set15re 123.3% NA 0313-C F-FS set % NA 0313-F-FS4 5 set1re 84.6% 78.8% 0313-F-FS5 5 set1re 84.8% 83.7% 0313-F-FS6 5 set1re 88.7% 98.6% 0313-F-FS7 5 set2re 90.9% F-FS8 5 set2re 107.3% C F-FS16 5 set9 93.9% NA 0313-C F-FS17 5 set % NA 0313-C F-FS18 5 set % NA 0313-C F-FS20 5 set % NA 0313-C F-FS21 5 set % NA 0313-F-FS7 20 set2re % 0313-F-FS8 20 set2re % 0313-C F-FS13 20 set6 94.9% NA 0313-C F-FS14 20 set7 83.4% NA 0313-C F-FS15 20 set8 94.7% NA 0313-C F-FS19re 20 set12re 83.0% NA Overall Average 96.9% 81.8% Standard deviation 15% 12% Count (includes validation) re = these sets were re-extracted due to one or more poor recoveries. 2 Different fortification level for this spike.

18 Food and Environmental Quality Laboratory Page 18 of 43 B. Storage Stability To verify stability of malathion and fenpropathrin on blueberry leaves during frozen storage, FEQL fortified control foliage at 0.5 µg/g on 12/12/2013. These storage stability samples are maintained in the freezer (ID Dasher) with study samples. If necessary, these stability samples can be extracted according to the analytical method to verify the stability of the pesticides of interest in frozen storage. The longest storage interval for malathion on foliage was 208 days. The longest interval for fenpropathrin treated sample was 128 days in frozen storage before analysis. C. Residue Results This study reports the malathion residue on foliage from both air blast and mistigation application methods one day before application through approximately 7 days after application, and the residues of fenpropathrin after a single air blast application. See appendix A for the project protocol. Figures 1 & 2 illustrate the individual pesticide concentrations in/on blueberry leaves after application. Table 5 provides the results for the analyzed leaf samples. All residues are expressed micrograms of pesticide per gram of foliage (µg/g or ppm). Results below the method limit of detection are listed as <LOD. Residues that were detectible but less than the method limit of quantitation are listed as <LOQ. Example chromatograms which have been rescaled to illustrate the peak of interest are provided in Appendix B.

19 Food and Environmental Quality Laboratory Page 19 of 43 Figure 1 Malathion applications on 7/28/2013 and 8/11/2013 Figure 2 Danitol (fenpropathrin) application by airblast sprayer on 9/1/2013

20 Food and Environmental Quality Laboratory Page 20 of 43 Table 5 Analysis Results Sample IDs Sample date Treatment Status extraction for Malathion Total Malathion (ug/g) 0313-C F 7/27/2013 2/20/2014 ND set15re NA 0313-M F-T 7/27/2013 M(-1) 2/5/2014 ND set12re NA 0313-M F-TD 7/27/2013 M(-1) 2/5/2014 ND set12re NA 0313-AB F-T 7/27/2013 M(-1) 2/5/2014 ND set12re NA 0313-AB F-TD 7/27/2013 M(-1) 2/5/2014 ND set12re NA Set extraction for fenpropathrin Fenpropathrin (ug/g) 0313-C F 7/28/ /17/2013 ND SET1RE ND 0313-M F 7/28/2013 M0 1/30/ set10 NA 0313-AB F 7/28/2013 1/30/ set10 NA 0313-C F 7/29/2013 1/7/2014 ND set2re 1/7/2014 ND set2re 0313-C F 7/29/2013 2/3/2014 ND set11 NA 0313-M F-T 7/29/2013 M1 2/3/ set11 NA 0313-M F-TD 7/29/2013 2/3/ set11 NA 0313-AB F-T 7/29/2013 2/3/ set11 NA 0313-AB F-TD 7/29/2013 2/3/ set11 NA 0313-C F 7/30/2013 1/30/2014 ND set10 NA 0313-M F 7/30/2013 M2 1/30/ set10 NA 0313-AB F 7/30/2013 1/30/ set10 NA 0313-C F 7/31/2013 2/10/2014 ND set13 NA 0313-M F 7/31/2013 M3 2/10/ set13 NA 0313-AB F 7/31/2013 2/10/ set13 NA 0313-C F 8/2/2013 1/28/2014 ND set9 NA 0313-M F 8/2/2013 M5 1/28/ set9 NA 0313-AB F 8/2/2013 1/28/ set9 NA Set

21 Food and Environmental Quality Laboratory Page 21 of 43 Sample IDs Sample date Treatment Status extraction for Malathion Total Malathion (ug/g) 0313-C F 8/3/2013 2/3/2014 ND set11 NA 0313-M F 8/3/2013 MM(-1) 2/10/ set13 NA 0313-AB F 8/3/2013 2/10/ set13 NA 0313-C F 8/4/2013 1/30/2014 ND set10 NA 0313-M F 8/4/2013 M7, MM0 1/28/ set9 NA 0313-AB F 8/4/2013 1/28/ set9 NA Set extraction for fenpropathrin Fenpropathrin (ug/g) 0313-C F 8/5/2013 2/17/2014 ND set16 2/17/2014 set M F 8/5/2013 MM1 1/28/ set9 NA 0313-AB F 8/5/2013 1/28/ set9 NA 0313-C F 8/7/2013 2/4/2014 ND set12 NA 0313-M F 8/7/2013 M10, MM3 2/11/ set14 NA 0313-AB F 8/7/2013 2/11/ set14 NA 0313-C F 8/9/2013 2/4/2014 ND set12 NA 0313-M F 8/9/2013 MM5 2/10/ set13 NA 0313-AB F 8/9/2013 2/10/ set13 NA 0313-C F 8/10/2013 2/4/2014 ND set12 NA 0313-M F 8/10/2013 M(-1) 2/11/ set14 NA 0313-AB F 8/10/2013 2/11/ set14 NA 0313-C F 8/11/2013 1/15/2014 ND set4 NA 0313-C F 8/11/2013 2/5/2014 ND set12re NA 0313-M F 8/11/2013 M0, MM7 2/11/ set14 NA 0313-AB F 8/11/2013 2/20/ set15re NA 0313-C F 8/12/2013 1/23/ set 8 NA 0313-M F 8/12/2013 M1 1/23/ set 8 NA Set

22 Food and Environmental Quality Laboratory Page 22 of 43 Sample IDs Sample date Treatment Status extraction for Malathion Total Malathion (ug/g) 0313-AB F 8/12/2013 1/23/ set 8 NA 0313-C F 8/13/2013 1/21/ set6 NA 0313-M F 8/13/2013 M2 1/21/ set6 NA 0313-AB F 8/13/2013 1/21/ set6 NA 0313-C F 8/14/2013 1/22/ set7 NA 0313-M F 8/14/2013 M3, MM10 1/22/ set7 NA 0313-AB F 8/14/2013 1/22/ set7 NA 0313-C F 8/16/2013 2/5/ set12re NA 0313-M F 8/16/2013 M5 1/16/ set5 NA 0313-AB F 8/16/2013 1/16/ set5 NA 0313-C F 8/17/2013 2/5/2014 ND set12re NA 0313-M F 8/17/2013 MM(-1) 1/21/ set6 NA 0313-AB F 8/17/2013 1/21/ set6 NA 0313-C F 8/18/2013 1/16/ set5 NA 0313-M F 8/18/2013 M7, MM14, MM0 1/16/ set5 NA 0313-AB F 8/18/2013 1/16/ set5 NA 0313-C F 8/19/2013 2/11/ set14 NA 0313-M F 8/19/2013 MM1 1/21/ set6 NA 0313-AB F 8/19/2013 1/21/ set6 NA 0313-C F 8/21/2013 NA NA 0313-M F 8/21/2013 M10, MM3 1/16/ set5 NA 0313-AB F 8/21/2013 1/16/ set5 NA 0313-C F 8/23/2013 NA NA 0313-M F 8/23/2013 MM5 1/15/ set4 NA Set extraction for fenpropathrin Fenpropathrin (ug/g) Set

23 Food and Environmental Quality Laboratory Page 23 of 43 Sample IDs Sample date Treatment Status extraction for Malathion Total Malathion (ug/g) 0313-AB F 8/23/2013 1/15/ set4 NA 0313-C F 8/24/2013 1/14/2014 ND set3re NA 0313-M F 8/24/2013 D(-1) 1/14/ set3re NA 0313-AB F 8/24/2013 1/14/ set3re NA 0313-C F 8/25/2013 1/15/ set4 NA 0313-C F 8/30/2013 1/15/ set4 NA 0313-M F 8/30/2013 D5 1/14/ set3re NA 0313-AB F 8/30/2013 1/14/ set3re NA Set extraction for fenpropathrin Fenpropathrin (ug/g) 0313-M F 9/1/2013 1/7/ set2re 1/7/ set2re 0313-C F 9/1/2013 1/15/ set4 NA 0313-AB F-A 9/1/2013 D7, MM14 1/7/ set2re 1/7/ set2re 0313-AB F-B 9/1/2013 D8/D0 1/7/ set2re 1/7/ set2re 0313-C F 9/2/2013 1/15/ set4 NA 0313-AB F 9/2/2013 D9/D1 1/7/ set2re 1/7/ set2re 0313-C F 9/3/2013 2/17/ set16 2/17/2014 set AB F 9/3/2013 D10/D2 NA 0313-C F 9/5/2013 2/17/ set16 2/17/2014 set AB F 9/5/2013 D4 1/7/ set2re 1/7/ set2re 0313-C F 9/9/2013 2/17/ set16 2/17/2014 set AB F 9/9/2013 D8 12/17/ set1re 12/17/ set1re 0313-C F 9/13/2013 2/17/ set16 2/17/2014 set AB F 9/13/2013 D12 12/17/ set1re 12/17/ set1re 0313-C F 9/16/2013 2/17/ set16 2/17/2014 set AB F 9/16/2013 D15 12/17/ set1re 12/17/ set1re Set

24 Food and Environmental Quality Laboratory Page 24 of 43 Sample IDs Sample date Treatment Status extraction for Malathion Total Malathion (ug/g) Set extraction for fenpropathrin Fenpropathrin (ug/g) 0313-C F 9/25/2013 2/17/ set16 2/17/2014 set AB F 9/25/2013 D24 12/17/ set1re 12/17/ set1re ND=None detected <LOD=less than the limit of detection (0.05 µg/g for malathion, 0.1 µg/g for fenpropathrin) <LOQ=Less than the limit of quantitation (0.1 µg/g malathion, 0.5 µg/g fenpropathrin) but greater than the limit of detection NA=Not analyzed. Sample not analyzed for particular compound Set

25 Food and Environmental Quality Laboratory Page 25 of 43 Appendix A: Project Protocol

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31 Food and Environmental Quality Laboratory Page 31 of 43 Appendix B: Working Method

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34 Food and Environmental Quality Laboratory Page 34 of 43 Appendix C: Sample Chromatograms Figure C1 - Malathion 1 ug/ml standard in leaf extract matrix, malathion 1 ug/ml

35 Food and Environmental Quality Laboratory Page 35 of 43 Figure C2 Malathion: Control leaf extract, 0313-C F in 5 ml malathion expected retention time ~4.3 min

36 Food and Environmental Quality Laboratory Page 36 of 43 Figure C3 Malathion: fortified recovery sample, 0313-F-FS8 in 5 ml malathion, ug/ml in 5 ml extract, equivalent to 5.36 ug/g;

37 Food and Environmental Quality Laboratory Page 37 of 43 Figure C4 Malathion: mistigation sample, 0313-M F in 5 ml malathion, 0.06 ug/ml in 5 ml extract, equivalent to 0.3 ug/g

38 Food and Environmental Quality Laboratory Page 38 of 43 Figure C5 Malathion: airblast sample, 0313-AB F-A in 5 ml malathion, ug/ml in 5 ml extract, equivalent to 3.49 ug/g

39 Food and Environmental Quality Laboratory Page 39 of 43 Figure C6 Fenpropathrin 1 ug/ml standard in extract matrix, Fenpropathrin 1 ug/ml

40 Food and Environmental Quality Laboratory Page 40 of 43 Figure C7 - Fenpropathrin Control leaf extract, 0313-C F in 5 ml Fenpropathrin, expected retention time min

41 Food and Environmental Quality Laboratory Page 41 of 43 Figure C8 Fenpropathrin fortified recovery sample, 0313-F-FS8 in 5 ml Fenpropathrin ug/ml in 5 ml extract, equivalent to ug/g

42 Food and Environmental Quality Laboratory Page 42 of 43 Figure C9 Fenpropathrin mistigation field sample, 0313-M F in 5 ml Fenpropathrin ug/ml in 5 ml extract, equivalent to 4.12 ug/g

43 Food and Environmental Quality Laboratory Page 43 of 43 Figure C10 Fenpropathrin airblast sample, 0313-AB F-A in 5 ml Fenpropathrin ug/ml in 5 ml extract, equivalent to 6.41 ug/g