1. Analysis of PCB Congeners in SRRTTF Program – Analytical Limits and Considerations SRRTTF PCB Workshop Spokane WA, Jan.13-14, 2015 Presented by Richard Grace Director – Sales, Service, Marketing AXYS Analytical Services Ltd.

2. Agenda  About AXYS  Some PCB Basics – Methods, Aroclors, Congeners – Total PCBs  Basics on HRMS, and 1668 PCBs  Limits of PCBs Measurements – Identification, blanks

3. AXYS Focus  Focus – Multi Target Ultra Trace Organic Analysis (pg or ng levels) – POPs (all matrices) – Emerging Contaminants – HRMS, LC MS/MS, GC/MS, GC-ECD instrumentation – Targeted Metabolomics  3 Analytical Areas – Environmental (All matrices associated with Bioaccumulation cycle – LifeScience – Bio-monitoring and Metabolomics – Method Development (i.e. EPA 1668,1614,1694,1698,1699)

4. AXYS Analytical Services Ltd  Located Victoria British Columbia  100 Employees  Largest Ultra-Trace Lab in NA  65% in U.S.  ISO 17025 + NELAP Accreditation

5. AXYS and PCB Congeners  Since early eighties in Arctic “baseline” investigations (all matrices in bioaccumulation, high volume sampling), HRMS capable in 1984.  Full 209 congener analysis using Isotope Dilution / DB-1 column (HRMS and GC/MS platforms) in early 90s.  EPA Method 1668A Source – Process and Validation (2001 to 2003) – Published 2003 by U.S. EPA Office of Water  Active in 1668B review.  1 of 2 labs to review / validate EPA 1668C for publication 2009

6. Some PCB Basics Biphenyl Rings with CL substitution 209 possibilities or “congeners” 10 homologue possibilities indicating # of Cl (eg. Mono to deca) per PCB or level of chlorination 12 congeners are “WHO Toxic” congeners with Dioxin Like AH receptor affects

7. Some PCB Basics  Aroclors – variety of technical PCB products sold by Monsanto (now Solutia) from early 30s to early 80s, each composed of 1-7 primary congeners + trace levels of congeners  Named by wt.% Cl – Aroclor 1242 (12 carbons, 42% chlorination by weight) – Lightest by MW – Aroclor 1221, 1232 – Heaviest by MW Aroclor 1268,1270,1272 (primarily 209,206 congeners)  Used as technical standards for calibration and QC control in many PCB methods, primarily GC/ECD

8. Conversion of EPA 1668C Congener Values to Aroclors (Basis for NELAP Accreditation as Domestic PE Providers Use Aroclor Standards)  Aroclor 1016 = the sum* of PCBs 8, 18/30, 31, 28/20 concentrations multiplied by 2.7  Aroclor 1221 = the sum of PCBs 1, 3, 8 concentrations multiplied by 1.4  Aroclor 1232 = the sum of PCBs 1, 3, 18/30 concentrations multiplied by 3.4  Aroclor 1242 = the sum of PCBs 8, 18/30, 31, 28/20 concentrations multiplied by 3.0  Aroclor 1248 = the sum of PCBs 44/47/65, 49/69, 66 concentrations multiplied by 6.1  Aroclor 1254 = the sum of PCBs 86/87/97/108/119/125, 99 concentrations multiplied by 8.0  Aroclor 1260 = the sum of PCBs 183/185, 180/193, 170 concentrations multiplied by 5.0

9. PCBs – Multiple Production Sources – More Technical Products Than Aroclors

10. Widespread Use (1930s to 1980s) plus Global Transport -Ubiquitous

11. Properties of PCBs  Stable, resistant to breakdown, half-life of 8-20 yrs.  Breakdown largely by de-chlorination to lower MW congeners  High KOW, low water solubility. Lypophyllic and bioaccumulative  Many toxicological end points  In solids, tend to partition to solids. Increasing affinity for solids as MW increases.  Low volatility (varies by MW) but are transported long range  Stockholm POP, banned, targeted for elimination  General trend in environment – decreasing prevalence, shift to lower MW

12. The Geometric Means of 35 PCBs (whole-weight in ng/g) from NHANES 2003-2004 by Age Group for all Sex, Race/Ethnicity in the U.S. Population

13. The Mean Percent Congener Contribution to the Sum of the 35 PCBs (Whole-Weight in ng/g) for NHANES 2003-2004 by Age Group – Shift to Lower MW PCBs Over Time

14. “Total PCBs” and Analytical Measurements  Sum of PCBs measured  Aroclor measurements – total of selected Aroclors only – Not all Aroclors measured (1268,1270, 1272) – Aroclors that degrade or weather, not measured – Aroclor pattern required for identification? Aroclors overlap – Many other PCB sources  Congener Measurements – Often short list of congeners present in Aroclors  PCB Metabolites (OH, ME) not included in totals

15. Lake Ontario Mass Balance - % of PCBs by Congener Lake Ontario Mass Balance - % of PCBs by Congener PCB Congener IUPAC ID PCB-11 IADN & US/DS PCB-1 IADN PCB-37 US/DS % of Lake Ontario Surface Water PCBs

16. 209 Congener Method Needed – EPA 1668A  Released Aug. 2003  EPA 1600 Series method – performance based – analytical variation allowed provided performance specifications met  Multi-matrix HRGC / HRMS method, similar to EPA 1613B  Isotope Dilution / Recovery Correction Qunatification  Designed to meet EPA CWA criteria 128 pg/L Total PCBs  169 reported congeners and congener co-elutions  Default SPB Octyl GC Column  Analytical Result is Congener Data – where QC is applied  Updated 2009 – EPA 1668C

17. AXYS EPA 1668C Analysis Flow Diagram – Aqueous and High Vol. / Passive Matrices

18. EPA 1668A/C Reported Values  Congeners reported as “analytical” result, low pg absolute  Blank limited if DLs good  Mathematical Calculations from Congeners – Total PCBs, Homologue totals, TEQs  Requires isotope dilution / recovery correction quantification (C labelled surrogates) from spikes in each unknown and QC sample  Requires 2 masses (+/- 15%) from native and surrogate for positive identification (NDR may be EMPC)  General reporting structure – unflagged above LMCL, “J” flagged value below LMCL if 2.5 to 1 S:N ration met for ratioing congener

19. 1668 QC Notes  QC Criteria must meet method guidance for performance (DLs, blank criteria, many specific criteria)  SRRTTF work requires better DLs and blank control than method minimum levels. Not instrument limited.  Validated by lab performing analysis – EPA Tier 1 validation (IPR, MDL, MS/MSD) – Sets control ranges for recovery of natives and surrogates (what is normal at known accuracy)  External Confirmation of Proficiency / Accuracy – PE programs and CRMs/ SRMs limited – Requires participation in international round robins / calibrations for real matrices

20. AXYS MLA 010 (EPA 1668A/C) QA Specifications QC ParameterSpecification Analysis Duplicate Must agree to within ±20% of the mean (applicable to concentrations  10 times the DL) 1 Procedural Blank Analyte concentrations in blank samples for PCB congeners 77, 81, 114, 123, 126 and 169 must be less than 2 pg/congener/sample, and concentrations of PCB congeners 156, 157, 167 and 189 must be less than 10 pg/congener/sample. Concentrations of all other individual PCB congeners or coelutions must be less than 50 pg/congener/sample in blank samples. The sum of all 209 congeners should be less than 300 pg/sample. Higher levels are acceptable where sample concentrations exceed 10 times the blank levels. Sample Specific Detection Limit Typical sample specific detection limits, determined from chromatographic noise, are in the range of 0.5 to 2.0 pg. Initial Calibration For 6-point calibration, a relative standard deviation of the RRF’s  20% for all compounds Ion ratios for all congeners must be within ±15% of theoretical for CS 0.2. Minimum S:N ratio 10:1 for all calibration standards. For CS0.2, S:N ratio may be as low as 3:1 for di-PCBs and nona-PCBs. Continuing CAL VERRefer to the tables above. Analyte/Surrogate Ratios Response must be within the calibrated range of the instrument. Coders may use data from more than one chromatogram to get the responses in the calibrated range. Ion Ratios Ion ratios must fall within ±15% of the theoretical values for positive identification of all targets in the calibration standards and samples. Sensitivity Minimum S:N ratio 10:1 for all calibration standards. For CS0.2, S:N ratio may be as low as 3:1. for di-PCBs and nona-PCBs.

21. Comparisons of Common PCB Methods vs. Positive Identification Method + EU Point Rating HRGC/HRMS (8 points) HRGC/GCMS (0-4 points) HRGC/ECD (0 Points) Details EI (Electron Impact) / Voltage SIR MID detection by GCMS in EI mode Scan Quantification 13 C-labeled standards for isotope dilution Labeled standards possible not possible Specificity High: Monitor two to six ions from parent ion cluster, compare to theoretical ratio. Multiple cleanups. 10,000+ RP. Medium: Monitor 2-6 masses per congener. Compare to theoretical ratio. Less cleanup. 100-400 RP Low: Identification based on GC retention time and halogen response. Limited cleanup. Other halogenated compounds may interfere. Sensitivity Best (Lowest) detection limit Highest detection limits Medium

22. Technique # of Ions (native plus labeled surrogate using isotope dilution) # of Points GC/MS (EI or CI) NN LC/MSNN GC MS/MS 1 precursor, 2 daughter 4 LC MS/MS 1 precursor, 2 daughter 4 LC MS/MS 2 precursor, each with one daughter 5 HRMSN2N EU 657 – Analytical Method - Point Criteria for Positive Identification if Using Internal Standard (4 Points Required for Positive Identification)

23. Isotope Dilution / & Surrogate Recovery  Isotope dilution/recovery correction quantitation – Increased accuracy, avoids matrix bias – Allows more extensive clean-up process – Reduction of detection limits  Labelled standard addition at start of analysis means Isotope dilution/surrogate recovery correction throughout extraction and clean-up  Recovery standards added at instrument  This correction compensates for loss of target analytes during the extraction and cleanup process for every sample  Extra standards, not reported for diagnostic monitoring – clean-up standards, field standards 23

24. Low Level PCBs in 1L Effluent Sample – all specs. met (PCB 67 @ 1.2 pg/L, PCB 63 @ 1.6 pg/LL SDL @ 0.55 pg/L, Reported “J” flagged Primary and Confirmation Ion Channels

25. AXYS EPA 1668C Reporting Limits based on Low Method Calibration Limit (CS0.2) MatrixWATER/EFFLUENT Unitspg/L Sample size 2.36L4L60L CL1-PCB-11.69 (4@1L)1.000.07 CL1-PCB-21.691.000.07 CL1-PCB-31.691.000.07 CL2-PCB-41.691.000.07 CL2-PCB-51.691.000.07 CL2-PCB-61.691.000.07 CL2-PCB-71.691.000.07 CL2-PCB-81.691.000.07 CL2-PCB-91.691.000.07 CL2-PCB-101.691.000.07 CL2-PCB-111.691.000.07 CL2-PCB-12/131.691.000.07

26. AXYS 1668C EDLs or SDLs Prorated for Sample Sizes Considered in SRRTTFF Analysis MatrixWATER/EFFLUENT Unitspg/L Sample size 2.36L4L60L CL1-PCB-10.420.250.02 CL1-PCB-20.420.250.02 CL1-PCB-30.420.250.02 CL2-PCB-40.850.500.03 CL2-PCB-50.850.500.03 CL2-PCB-60.850.500.03 CL2-PCB-70.850.500.03 CL2-PCB-80.850.500.03 CL2-PCB-90.850.500.03 CL2-PCB-100.850.500.03 CL2-PCB-110.850.500.03 CL2-PCB-12/130.850.500.03

27. Surrogates PCB 1 and 3 @ 50 pg (CS-3) In 12 hr cal. ver. Primary and Confirmation Ion Channel

28. Interferences – Minor Dimer Issue Primary and Confirmation Ion Channel + PFK Channel – Quantitation Not Affected

29. Interferences - Major Dimer PCB 1 poor chromatography, PCB 3 pushed out of window (NQ)

30. 206 Interference @ 55.26 Inflates Surrogate Recovery to 237%, Result NQ’d, Re-Run diluted to Resolve. High Level Tissue Interference Unknown – PCB 207,208 Unaffected

31. Blanks in 1668A/C  Any 1668 analysis will be able to see blank levels for some congeners in EPA 1668A/C  Lower detection limits will increase amount of blank concentrations detected  Multiple Conventions for Reporting of Blank QC Method Acceptance Criteria – exceedences “B” flagged  All applied at congener level  Blank contamination from laboratory for method blanks  Method blank + field + container for field blanks  Method blank + media+ sampler for high vol. and passives

32. Blank Control Chart Basics

33. AXYS Method Blanks and SRRTTF  Supplied to SRRTTF in two forms – Mean plus 2 standard deviations of all method blanks (aqueous matrix) at LMCL (EPA 1668A convention) – Mean plus 2 standard deviationsof all method blanks (aqueous matrix)  Produces very different specs.  Congener specs. not added, below blank limits PCBs behave independently  AXYS default spec. 300 pg per sample, with maxs. Set on select congeners  SRRTTF Limit for 2.36L samples appears to be approx. 50 pg/L

34. Blank Limits for AXYS MLA 010 – Varying with Sample Size, Type of Reporting Limit Used in Data, ND = 0, Mean + 2 Sigma of Reporting Limit Used EPA 1668 Convention @ RL Cut- Off CLAM Samples Size (L)12.364556090 Method Blank Total PCB (pg/L)20.668.7545.1650.3760.3440.230 AXYS 1668 Convention @ SDL Cut-Off CLAM Samples Size (L)12.364556090 Method Blank Total PCB (pg/L)117.61649.83729.4042.1381.9601.307

35. PCB Total Control Chart – All Matrices

36. PCB 118 Control Chart – Solids

37. PCB 7 – Specific Contract

38. Matrix Values – PCB 7 Aqueous

39. PCB 7 - Tissue

40. PCB 7 – Solid

41. PCB 7 – All matrices