Richard Grace , Coreen Hamilton, Million Woudneh

1. “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 17025 CERTIFIED

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

3. PURPOSE (s) of ANALYSIS • Analysis of Wide Variety of Pesticides • Legacy (OC) Pesticides • Current Use Pesticides (Canada / U.S. Focus) • “Multi-Residue Pesticide Test” or “MRES” • 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 • Reduction of False Positives and Matrix Effects by Use of HRMS and Serial Isotope Dilution / RRF Quantification

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

5. HISTORY of DEVELOPMENT • 2003 - Initial Method Development for Environment Canada (Pacific Region) for Aqueous Samples • 2003 – 2005 – Groundwater / Surface Water Survey for Environment Canada in Lower Fraser Valley • 2005 – Application to Soils / Sediments • 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 • 2007 – Further Method Refinement and Validation Under Contract to U.S. EPA Office of Water • 2007 – Application of Method to Tissue and Serum

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

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

8. OVERVIEW of ANALYSIS

9. EXTRACTON DETAILS (Aqueous) • 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 • Add surrogates in acetone (1mL), include rinses • Extract through shaking ( 3 x 100 mL DCM) • 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 • Makeup to 1mL in 1:2:1 ethyl acetate, acetonitrile, toluene

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

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

12. MICROSILICA COLUMN CLEANUP DETAILS • Makeup extract to 1mL in Hexane • Wet microsilica column (Silica AR 100-200 mesh from Mallinckrodt) with 5-10 mLs Hexane • 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 • Add 5 mL acetone and 1 mL iso-octane, rotovap to 1 mL • Transfer to centrifuge tube with hexane rinses, concentrate to 20 - 300 uL, transfer to microvial, add recovery standards

13. ANALYSIS DETAILS • 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 • 8,000 static mass resolution in EI mode • Multiple Ion detection – two ions for each target* and surrogate • 20 – 400 uL typical extract volume, 1-2 uL injection volume

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

15. AQUEOUS DETECTION LIMITS • 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, • OP Pesticides - 2.6 to 98.9 pg/L, • Triazines – 1.5 to 12.7 pg/L, • Pyrethroids – 22 to 45 pg/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

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

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

18. METHOD QC CRITERIA • Max. Batch Size (including QC) is 20 samples (8 to 14 is more common) • Each Batch contains: • method blank • duplicate • Laboratory Control Sample (LCS) • MS/MSD, SRM,CRM included by request and applicability • LCS Recovery 50-150 most common, some wider specs. if reproducibility high

19. QA ACCEPTANCE CRITERA • 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 • Bracketing Cal. – 40% RSD for targets detected • 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

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

21. APPLICATIONS and RESULTS (cont.) • “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. • Compounds detected vary widely dependant on region • Urban vs. rural • Type of Agricultural Activity • Regulatory Action

22. NEXT STEPS • Peer Review of draft method by EPA – Q3/4 2007 • Further processing of samples to develop further statistical information • Tissue / Serum Application • Multi-Lab Validation? • Questions?