“ANALYSIS OF CURRENT USE PESTICIDES IN ENVIRONMENTAL AND WASTEWATER SAMPLES BY HIGH RESOLUTION GC WITH HIGH RESOLUTION MASS SPECTROMETRIC DETECTION” Richard Grace, Coreen Hamilton, Million Woudneh AXYS Analytical Services Ltd., Sidney, BC, Canada AXYS ANALYTICAL SERVICES LTD. NELAP ACCREDITED / ISO CERTIFIED

AGENDA  Purpose (s) of Analysis  Development History  Details of the Method  Detection Limits and Method QC  Applications and Results

PURPOSE (s) of ANALYSIS Analysis of Wide Variety of Pesticides Analysis of Wide Variety of Pesticides Legacy (OC) Pesticides Legacy (OC) Pesticides Current Use Pesticides (Canada / U.S. Focus) Current Use Pesticides (Canada / U.S. Focus) “Multi-Residue Pesticide Test” or “MRES” “Multi-Residue Pesticide Test” or “MRES” Improvements in Sensitivity and Selectivity Improvements in Sensitivity and Selectivity Improvement in Detection Limits vs. Reference Methods (10 – 1000X lower detection limits vs. non 1600 series EPA methods) – meet ambient criteria where established Improvement in Detection Limits vs. Reference Methods (10 – 1000X lower detection limits vs. non 1600 series EPA methods) – meet ambient criteria where established Reduction of False Positives and Matrix Effects by Use of HRMS and Serial Isotope Dilution / RRF Quantification Reduction of False Positives and Matrix Effects by Use of HRMS and Serial Isotope Dilution / RRF Quantification

PURPOSE (s) of ANALYSIS Applicable to Wide Variety of Matrices Applicable to Wide Variety of Matrices Platform for Analysis in Most Matrices (1600 series) Platform for Analysis in Most Matrices (1600 series) Water – Surface, Ground, Effluent, Potable, High Volume Sampling on XAD Resin Water – Surface, Ground, Effluent, Potable, High Volume Sampling on XAD Resin Solids – Soil, Sediment, POTW Biosolids Solids – Soil, Sediment, POTW Biosolids Tissue / Serum (current development work) Tissue / Serum (current development work) Reduction of Pre-Laboratory Work Reduction of Pre-Laboratory Work Reduction in number / size of samples Reduction in number / size of samples Reduction in compositing / homogenization for broad reconnaissance of Pesticides Reduction in compositing / homogenization for broad reconnaissance of Pesticides

HISTORY of DEVELOPMENT Initial Method Development for Environment Canada (Pacific Region) for Aqueous Samples Initial Method Development for Environment Canada (Pacific Region) for Aqueous Samples 2003 – 2005 – Groundwater / Surface Water Survey for Environment Canada in Lower Fraser Valley 2003 – 2005 – Groundwater / Surface Water Survey for Environment Canada in Lower Fraser Valley 2005 – Application to Soils / Sediments 2005 – Application to Soils / Sediments 2006 – Application / Validation to POTW Influent / Effluent / Biosolids for U.S. EPA Office of Water 2006 – Application / Validation to POTW Influent / Effluent / Biosolids for U.S. EPA Office of Water 2006 – High Volume Water Application / Collection on XAD Resin Developed 2006 – High Volume Water Application / Collection on XAD Resin Developed 2007 – Further Method Refinement and Validation Under Contract to U.S. EPA Office of Water 2007 – Further Method Refinement and Validation Under Contract to U.S. EPA Office of Water 2007 – Application of Method to Tissue and Serum 2007 – Application of Method to Tissue and Serum

ANALYTES CURRENT LIST CURRENT LIST 35 OC Pesticides and Metabolites 35 OC Pesticides and Metabolites 20 Organophosphorous Compounds 20 Organophosphorous Compounds 7 Triazine Compounds 7 Triazine Compounds 2 Pyrethroid Groups 2 Pyrethroid Groups 14 Organonitrogen Compounds 14 Organonitrogen Compounds

RATIONALE for ANALYTE SELECTION Current or Historic Use Current or Historic Use Availability of Standards and Appropriate Surrogates Availability of Standards and Appropriate Surrogates Capture by SPE cartridge Capture by SPE cartridge Analyte Stability (Matrix and Instrument) Analyte Stability (Matrix and Instrument) Appropriate monitoring ions Appropriate monitoring ions Validation of Positive Identification Validation of Positive Identification

OVERVIEW of ANALYSIS Sample 1L Water or 1-5 g (dry wt.) soil, Sediment, or Biosolid Surrogate Addition followed by DCM Extraction (3X) Dry, Filter, Sample Reduction to 100uL Make-up to 1mL in 1:2:1 ethyl acetate, acetonitrile, toluene SPE Cartridge Cleanup (1 gram aminopropyl bonded silica –Varian) SPE Steps – condition, load, rinse, elute, reduce, transfer to hexane Microsilica Column Cleanup – wet, load, rinse, elute, reduce ANALYSIS by HRGC / HRMS Run 1 – OC, OP, Triazine, and Pyrethroid Analytes Run 2 – ON Analytes

EXTRACTON DETAILS (Aqueous) Weigh 1L bottle, and all steps in transfers Weigh 1L bottle, and all steps in transfers Transfer water to 2L sep. funnel, rinse sample container with DCM, transfer DCM to 2L sep. funnel Transfer water to 2L sep. funnel, rinse sample container with DCM, transfer DCM to 2L sep. funnel Add surrogates in acetone (1mL), include rinses Add surrogates in acetone (1mL), include rinses Extract through shaking ( 3 x 100 mL DCM) Extract through shaking ( 3 x 100 mL DCM) Combine extracts, then dry with anhydrous NaSO4 Combine extracts, then dry with anhydrous NaSO4 Transfer to round bottom with 1 ml keeper toluene and roto vap to 1 mL tube, concentrate to 100ul by N2 Transfer to round bottom with 1 ml keeper toluene and roto vap to 1 mL tube, concentrate to 100ul by N2 Makeup to 1mL in 1:2:1 ethyl acetate, acetonitrile, toluene Makeup to 1mL in 1:2:1 ethyl acetate, acetonitrile, toluene

EXTRACTION DETAILS (Solid) 1 -5 g dry weight in beaker, add anhydrous powdered sodium sulfate, equilibrate 30 min g dry weight in beaker, add anhydrous powdered sodium sulfate, equilibrate 30 min. Load to soxhlet thimble, add surrogates Load to soxhlet thimble, add surrogates Add 300 mL DCM to soxhlet, add thimble Add 300 mL DCM to soxhlet, add thimble Soxhlet 16 hours Soxhlet 16 hours RotoVap to 1 ml, dry with sodium sulfate if necessary, filter through glass wool column RotoVap to 1 ml, dry with sodium sulfate if necessary, filter through glass wool column Continue as per aqueous procedure Continue as per aqueous procedure

SPE CLEANUP DETAILS SPE Cartridge Type – 1 g aminopropyl bonded silica column SPE Cartridge Type – 1 g aminopropyl bonded silica column Solvent – 1:2:1 ethyl acetone, acetonitrile, toluene Solvent – 1:2:1 ethyl acetone, acetonitrile, toluene Condition using 2 X 3 mL (column volume) Condition using 2 X 3 mL (column volume) Load sample followed by 1 mL rinse Load sample followed by 1 mL rinse Elute with 11 mLs Elute with 11 mLs Concentrate to 1 mL by RotoVap Concentrate to 1 mL by RotoVap Transfer to centrifuge tubes with hexane rinse Transfer to centrifuge tubes with hexane rinse Concentrate to 100 uL using gentle nitrogen Concentrate to 100 uL using gentle nitrogen

MICROSILICA COLUMN CLEANUP DETAILS Makeup extract to 1mL in Hexane Makeup extract to 1mL in Hexane Wet microsilica column (Silica AR mesh from Mallinckrodt) with 5-10 mLs Hexane Wet microsilica column (Silica AR mesh from Mallinckrodt) with 5-10 mLs Hexane Load sample followed by 500uL hexane rinse, 2 x 300uL rinse 10% MeOH/ 90% DCM Load sample followed by 500uL hexane rinse, 2 x 300uL rinse 10% MeOH/ 90% DCM Elute with 5 mL 10% MeOH/90% DCM, combine with rinses Elute with 5 mL 10% MeOH/90% DCM, combine with rinses Add 5 mL acetone and 1 mL iso-octane, rotovap to 1 mL Add 5 mL acetone and 1 mL iso-octane, rotovap to 1 mL Transfer to centrifuge tube with hexane rinses, concentrate to uL, transfer to microvial, add recovery standards Transfer to centrifuge tube with hexane rinses, concentrate to uL, transfer to microvial, add recovery standards

ANALYSIS DETAILS All Analysis performed with Micromass Ultima HRMS with Agilent 6890 GC, CTC autosampler, alpha data system running Opus 6.3 software All Analysis performed with Micromass Ultima HRMS with Agilent 6890 GC, CTC autosampler, alpha data system running Opus 6.3 software DB-17 capillary GC column (30m, 0.25 mm i.d., 0.25 um film thickness, direct couple to MS source DB-17 capillary GC column (30m, 0.25 mm i.d., 0.25 um film thickness, direct couple to MS source 8,000 static mass resolution in EI mode 8,000 static mass resolution in EI mode Multiple Ion detection – two ions for each target* and surrogate Multiple Ion detection – two ions for each target* and surrogate 20 – 400 uL typical extract volume, 1-2 uL injection volume 20 – 400 uL typical extract volume, 1-2 uL injection volume

INSTRUMENT DETAILS (cont.) 2 runs 2 runs Run 1 – OC compounds, OP compounds, Triazine compounds, Pyrethroids Run 1 – OC compounds, OP compounds, Triazine compounds, Pyrethroids Run 2 – Organonitrogen compounds Run 2 – Organonitrogen compounds Injector temperature and ramping is key difference Injector temperature and ramping is key difference 220C for Run 1 injector temp. 220C for Run 1 injector temp. 260C for Run 2 injector temp. 260C for Run 2 injector temp. Bracketed calibration during run Bracketed calibration during run Calibration vs. 6 point linearity Calibration vs. 6 point linearity

AQUEOUS DETECTION LIMITS AQUEOUS DETECTION LIMITS Determined by Federal Register (USA) 40 CFR Part 136, Appendix B, October 26,1984 Determined by Federal Register (USA) 40 CFR Part 136, Appendix B, October 26,1984 OC Pesticides – 0.4 pg/L to 60.7 pg/L, OC Pesticides – 0.4 pg/L to 60.7 pg/L, OP Pesticides to 98.9 pg/L, OP Pesticides to 98.9 pg/L, Triazines – 1.5 to 12.7 pg/L, Triazines – 1.5 to 12.7 pg/L, Pyrethroids – 22 to 45 pg/L, Pyrethroids – 22 to 45 pg/L, Organonitrogen – 0.53 to 9.41 ng/L, Organonitrogen – 0.53 to 9.41 ng/L, Sample Detection Limits (similar to EPA 1613b) often used to reduce DLs 2-5X from calculated MDLs in clean matrices Sample Detection Limits (similar to EPA 1613b) often used to reduce DLs 2-5X from calculated MDLs in clean matrices

DETECTION LIMIT NOTES Clean Matrix MDLs follow Aqueous patterns, units in pg/g Clean Matrix MDLs follow Aqueous patterns, units in pg/g POTW Effluent generally raises DLs by 10X POTW Effluent generally raises DLs by 10X POTW Influent generally raises DLs by 40X POTW Influent generally raises DLs by 40X POTW Biosolids generally raises DLs by to 0.2 to 70 ng/g POTW Biosolids generally raises DLs by to 0.2 to 70 ng/g

STANDARD METHOD QC – Standards and Surrogates Non-labeled standards supplied by Accustandard (78 compounds) Non-labeled standards supplied by Accustandard (78 compounds) Carbon labeled standards supplied by Cambridge Isotope Laboratories Carbon labeled standards supplied by Cambridge Isotope Laboratories 21 C13 labeled OC Pesticides 21 C13 labeled OC Pesticides 1 permethrin (cis/trans) mix 1 permethrin (cis/trans) mix 2 ON Compounds (Alachlor, Metalochlor) 2 ON Compounds (Alachlor, Metalochlor) 1 OP Compound (Fonofos) 1 OP Compound (Fonofos) 1 Triazine Compound (Atrazine) 1 Triazine Compound (Atrazine) Deuterated surrogates (linuran, azinophos methyl, diazinon) supplied by Dr. Ehrenstorfer Gmbh Deuterated surrogates (linuran, azinophos methyl, diazinon) supplied by Dr. Ehrenstorfer Gmbh Recovery Standards (C13 PCB 52,138) supplied by Wellington Laboratories Inc. Recovery Standards (C13 PCB 52,138) supplied by Wellington Laboratories Inc.

METHOD QC CRITERIA Max. Batch Size (including QC) is 20 samples (8 to 14 is more common) Max. Batch Size (including QC) is 20 samples (8 to 14 is more common) Each Batch contains: Each Batch contains: method blank method blank duplicate duplicate Laboratory Control Sample (LCS) Laboratory Control Sample (LCS) MS/MSD, SRM,CRM included by request and applicability MS/MSD, SRM,CRM included by request and applicability LCS Recovery most common, some wider specs. if reproducibility high LCS Recovery most common, some wider specs. if reproducibility high

QA ACCEPTANCE CRITERA EPA Tier 1 Validation Protocols for Single Laboratory Method for IPR, MS/MSD, and MDL criteria EPA Tier 1 Validation Protocols for Single Laboratory Method for IPR, MS/MSD, and MDL criteria Duplicate Criteria – 40% RPD for Analytes 10X above MDL Duplicate Criteria – 40% RPD for Analytes 10X above MDL Bracketing Cal. – 40% RSD for targets detected Bracketing Cal. – 40% RSD for targets detected Cal. Ver – 25% RSD for native analytes with exact labeled standards, 35% for others Cal. Ver – 25% RSD for native analytes with exact labeled standards, 35% for others Linearity (ICAL) – 25% RSD for native analytes that have exact labeled standards, 35% for others Linearity (ICAL) – 25% RSD for native analytes that have exact labeled standards, 35% for others

APPLICATIONS and RESULTS To date N > 500 samples To date N > 500 samples 50 % Groundwater / Surface Water (diverse locations) 50 % Groundwater / Surface Water (diverse locations) 35% POTW influent/effluent/biosolids 35% POTW influent/effluent/biosolids 15% sediments / high volume water / other 15% sediments / high volume water / other Continuous Improvement has led to 5 method revisions. Key experiences in; Continuous Improvement has led to 5 method revisions. Key experiences in; Spiking levels and standards maintenance / grouping Spiking levels and standards maintenance / grouping Bench “technique” in extraction / cleanup Bench “technique” in extraction / cleanup Sample size and final extract options to limit matrix effects and maintain instrument cleanliness Sample size and final extract options to limit matrix effects and maintain instrument cleanliness Use of Method QC to update acceptance criteria Use of Method QC to update acceptance criteria

APPLICATIONS and RESULTS (cont.) “Detects” vary widely dependant on matrices “Detects” vary widely dependant on matrices Many compounds are readily degraded by biological activity, photolysis, or low persistency. Generally, metabolites are not the target of the analysis. Many compounds are readily degraded by biological activity, photolysis, or low persistency. Generally, metabolites are not the target of the analysis. Compounds detected vary widely dependant on region Compounds detected vary widely dependant on region Urban vs. rural Urban vs. rural Type of Agricultural Activity Type of Agricultural Activity Regulatory Action Regulatory Action

NEXT STEPS Peer Review of draft method by EPA – Q3/ Peer Review of draft method by EPA – Q3/ Further processing of samples to develop further statistical information Further processing of samples to develop further statistical information Tissue / Serum Application Tissue / Serum Application Multi-Lab Validation? Multi-Lab Validation? Questions? Questions?