1. For: Minnesota Pollution Control Agency April 16, 2015

2. OUTLINE Overview of AXYS Analytical Services Significance of Chlorinated Paraffins (CPs) AXYS CPs Method MLA-020 Qualitative and Quantitative Determination Considerations for the Analysis of CPs Questions

3. AXYS Analytical Services 100+ employees located in Sidney, BC One location, no internal subcontracting required 19 Ph.D. and Masters-level scientists on staff Largest commercial ultra-trace laboratory in North America Client-assigned Project Managers, min. B.Sc. and lab trained Focus – Multi Target Ultra Trace Organic Analysis (pg or ng levels) POPs CECs HRMS, LC MS/MS, GC/MS instrumentation 4 Analytical Areas Environmental (All matrices associated with bioaccumulation cycle) Biomonitoring Method Development (i.e. EPA 1668, 1614, 1694) Targeted Metabolomics

4. AXYS - Markets Served 65% U.S., 30% Cdn., 5% International CECs – pre-regulatory or early stages Government and Regulatory Authorities (60% of work) Federal - EPA, DoD, USGS, USFWS, DOI, EC, TC State, Municipal, and Provincial Clients Includes consultants on behalf of government TMDL Studies, Ambient/Baseline Levels Legal and RP issues Method Development Environmental Consultants on Behalf of Industry (30%) Assessment and Remediation Regional and Ambient/Background Work Risk Assessment/Litigation/RP

5. AXYS Industry Leading QA/QC Capabilities Strong internal data validation processes and quality system Dual data validation-every batch of data through primary and secondary data validation Rigorous ISO 17025 compliant Quality System Full documentation of operations Participation in national and international performance evaluation studies Ongoing internal monitoring and process improvement Routine internal and external auditing of procedures On-spec data reduces validation time for our clients AXYS ACCREDITATION * North American Accreditation ACLASS to ISO/IEC 17025:2005 National Environmental Laboratory Accreditation Program (NELAP) held through the following states: Minnesota Department of Health New York State Department of Health State of Florida Department of Health California Department of Public Health State of New Jersey Department of Environmental Protection Virginia Division of Consolidated Laboratory Services Washington State Department of Ecology State of Maine Department of Health & Human Services Canadian Association for Laboratory Accreditation (CALA) to ISO/IEC 17025:2005 standards International Accreditation Cooperation Agreements International Laboratory Accreditation Cooperation (ILAC) Asia Pacific Laboratory Accreditation Cooperation (APLAC) *Operation of a laboratory quality management system for specific parameters, methodologies, and fields of accreditation per each Scope of Accreditation.

6. AXYS - Method Development AXYS develops new analytical methods for leading North American organizations. Some examples: US EPA Office of Water Reference Methods including: Minnesota Pollution Control Agency For MPCA, PFAAs method development was focused mostly on applying existing methods to new matrices. Examples include high volume ambient air, unique matrices (e.g. gasoline), and the development of an FTS method for chrome plating sites. DuPont AXYS and DuPont collaborated on several method development and validation projects. Examples include PFCs in urine, hold time studies, and telomer alcohols. PCBs by HRMS (USEPA 1668A/C) PBDEs by HRMS (USEPA 1614) Current Use Pesticides by HRMS (USEPA 1699) PPCPs by LC-MS/MS (USEPA 1694) Sterols & Hormones by HRMS (USEPA 1698) Draft Procedure for Analysis of PFAAs in Sewage Sludge and Biosolids by HPLC/MS/MS

7. Significance of Chlorinated Paraffins (CPs) What are CPs? Polychlorinated n-alkanes; Defined as short-chain (SCCPs of C10-C13), medium chain (MCCPs of C14-C17), and long-chain (LCCPs of C18-C20); Complex mixtures with chlorine content of 30% to 70%; There are longer chain alkanes, C21+, but they are not considered for CPs analysis in this methodology, due to lower toxicity; Predominantly anthropogenic compounds; and SCCPs have been detected world-wide in the environment, wildlife and humans.

8. Chemical Structure of Polychlorinated Paraffins 2,5,6,7,8,11,15-heptachloroheptadecane (C17, MCCP) 2,3,4,5,6,8- hexachlorodecane (C10, SCCP) Figure 1 Figure 2

9. Significance of Chlorinated Paraffins (CPs) Use of CPs Used as a component of lubricants and coolants in metal cutting/forming operations, as plasticizers, and as flame retardants in plastics, rubber formulations, paints/coatings, and adhesives and sealants (CPIA, 2009); In the United States, Dover Chemical Corp. is the sole manufacturer of chlorinated paraffins while several companies import SCCPs (EPA, 2006); The current use of chlorinated paraffins (SCCPs, MCCPs, and LCCPs) in the United States is approximately 150 million pounds per year. (EPA)

10. Significance of Chlorinated Paraffins (CPs) Toxicity and Restrictions EPA defines SCCPs as persistent, bioaccumulative, and toxic to aquatic life; EPA is further evaluating MCCPs and LCCPs as part of its Work Plan for Chemical Assessments; In 2014 EPA issued a Significant New Use Rule (SNUR) under the Toxic Substances Control Act (TSCA) for chlorinated alkanes C12- 13 an SCCP; A 90-day notification by manufacturers/processors to the EPA under the SNUR for this SCCP, allows EPA the to evaluate the intended uses and, if necessary, to prohibit or limit that activity;

11. Significance of Chlorinated Paraffins (CPs) Toxicity and Restrictions Cont’d. SCCPs have been recommended to be added, as persistent organic pollutants, to the relevant annexes of the Stockholm Convention on POPs; SCCPs, MCCPs, and LCCPs (up to C20) may have, according to Environment Canada, “an immediate or long-term harmful effect on the environment or its biological diversity, and thus meet the definition of ‘toxic’ under paragraph 64(a) of CEPA 1999”; and It has also been stated by Environment Canada that SCCPs and MCCPs “constitute or may constitute a danger to human life or health.”

12. Considerations for the Analysis of CPs There are presently no recognized reference or standard methods for determination of CPs. Little info on occurrence / fate / behaviour Cart and Horse issue for Stockholm treaty evaluation of CPs Technical standards only, no product evaluation, 1 labelled standard (C10) Efforts to develop and validate methodology and reference standards for CPs are on-going within the environmental analysis industry. AXYS method MLA-020 was developed as a reconnaissance method and is not fully validated for detection limit or for accuracy against multiple external sources.

13. AXYS Experience with CPs Circa 2000 – Interest in CPs Technical standards (C10 to C20) available Method developed based on literature (GC ECNI MS), calibration from C10-20 technical standard, no analysis by product Limited early projects, high vol. for water and fish Environment Canada High Vol. work (water / solids) in Great Lakes (AXYS) Tissue work (low level) Internal method development by EC (multiple platforms and methods, not complete) Circa 2012 to Present On Environment Canada CMP List. AXYS method de-archived for PTOW biosolids work (POTW solids is suspected largest source) Released by approval for MPCA and WA DOE for fish tissue analysis 2012/13 Further studies underway @ MPCA / WA DOE – fish, sediments, water

14. AXYS Experience with CPs - WWTP Biosolids from some wastewater treatment facilities have been found to contain significant concentrations of CPs. Example from biosolids analyzed Client ID 1Lab Blank Spiked Matrix Matrix Spike Matrix Spike Duplicate Description Field BlankQC Conc Unitsng/g %Recng/g METHOD NAME MLA-020 Rev 3.01 SAMPLE SIZE0.7520 g0.7500 g0.6760 g10.11 g1.000 g0.1000 mL0.7830 g0.7780 g TOTAL CHLOROPARAFFIN- SUM37,870.0031,510.0036,910.00ND 494.6ND 5,000.0092.0301,100.00286,000.00 TOTAL SHORT-CHAIN CHLOROPARAFFIN14,050.0012,620.0014,900.00ND 247.3ND 2,500.0018.868,990.0065,640.00 TOTAL MEDIUM-CHAIN CHLOROPARAFFIN16,080.0013,350.0016,360.00ND 197.8ND 2,000.0044.3140,400.00135,200.00 TOTAL LONG-CHAIN CHLOROPARAFFIN7,724.006,639.007,948.00ND 148.4ND 1,500.0029.291,910.0087,390.00

15. AXYS Method MLA-020 for CPs Sample Handling and Storage Conditions 1 Glass containers should be organically clean; i.e. purchased certified clean, baked or solvent-rinsed. All containers must be tightly sealed with screw cap lids (PTFE or foil-lined) to prevent loss of volatiles or contamination from volatiles. Samples must be protected from the light. 2 Samples stored at a nominal temperature of 4°C are permitted a variance of ±2°C and samples stored at -20°C are permitted a variance of ±4°C. 3 Hold times quoted are recommended guidelines; longer hold times do not invalidate results.

16. AXYS Method MLA-020 for CPs AXYS Method MLA-020 is applicable for the analysis of polychlorinated paraffins in aqueous, solid, biosolid and tissue samples. The term polychlorinated paraffins (a.k.a. chlorinated paraffins or CPs) refers to a group of chlorinated alkanes with carbon content, for purposes of this method, ranging from C10 to C20 and with a chlorine content of 30% to 70%. These compounds are grouped for quantification and reporting as Total Short Chain (SCCPs), Medium Chain (MCCPs), Long Chain (LCCPs) and Total Chloroparaffins. CPs GroupCarbon Chain Short Chain (SCCPs)C10 to C13 Medium Chain (MCCPs)C14 to C17 Long Chain (LCCPs)C18 to C20

17. AXYS Method MLA-020 for CPs Summary Matrices: Aqueous, Solids, Biosolids, Tissue Solids Extraction: DCM:Hexane Cleanup : KOH/H 2 SO 4 /Cu/Alumina Solvent exchange to Hexane Aliquot of Quant. Std. Analysis by GC/MS Aqueous Extraction: DCM Cleanup : KOH/H 2 SO 4 /Cu/Alumina Solid samples are extracted by column elution, while aqueous samples are extracted by liquid-liquid extraction; Extracts are cleaned up using a rigorous 4-step process, if necessary; The final extract is reduced, spiked with quantification standard and analyzed by GC-MS. Quantification: the entire window within a certain retention time range and mass range is integrated. The MS is operated in an open scan mode acquiring masses in the range 200 to 650 mass units in the electron- capture negative ionization (ECNI) mode. Figure 3

18. AXYS Method MLA-020 for CPs GC Condition Details Instrument Linearity and Calibration A minimum of five calibration solutions are run initially to demonstrate instrumental linearity of response; An average calibration factor (RRF) is calculated from the mid-level calibration solution run at the beginning and end of the analysis bracket; and 13 C 12 -PCB 153 is used as a quantification standard.

19. AXYS Method MLA-020 for CPs Qualitative and Quantitative Determination Open Scan of Mid-level Calibration Solution (at 116550 ng/500 µL) - Clear polychlorinated paraffins pattern. Chromatogram notes: Peaks at approx 18.30 and 20.25 are internal standards, which are subtracted from the polychlorinated paraffins calculations. Figure 4

20. A chromatographic peak is identified as a target compound if the following criteria are met: 1. Peak responses must be at least 3X higher than the background noise level. 2. The detected concentration must be 2X higher than the background concentration detected in the laboratory blank for positive detection in samples. 3. Each group of CP peaks is broad; the peak width and retention times are defined based on the opening calibration solution that is run at the beginning of the analysis bracket. AXYS Method MLA-020 for CPs Analyte Identification

21. CClHm/z% ClM-ClM-Cl+2M-Cl+4M-Cl+6M-Cl+8 1031924443.0209211213215217 41827850.4243245247249251 51731256.1277279281283285 61634660.7311313315317319 71538064.5345347349351353 81441467.6379381383385387 91344870.3413415417419421 101248272.6447449451453455 1132125840.7223225227229231 42029247.9257259261263265 51932653.7291293295297299 61836058.3325327329331333 71739462.2359361363365367 81642865.4393395397399401 91546268.2427429431433435 101449670.6461463465467469 1232327238.6237239241243245 42230645.8271273275277279 52134051.5305307309311313 62037456.1339341343345347 71940860.0373375377379381 81844263.3407409411413415 91747666.2441443445447449 101651068.6475477479481483 1342432043.8285287289291293 52335449.4319321323325327 62238854.1353355357359361 72142258.1387389391393395 82045661.4421423425427429 91949064.3455457459461463 101852466.8489491493495497 111755869.0523525527529531 121659270.9557559561563565 Quantification Mass Ions for SCCPs (Ion Set 1, C 10 -C 13 ) Green fields indicate quantifying and alternate m/z acquisition ions. Bold type indicates m/z used for quantification

22. Quantification Mass Ions for MCCPs (Ion Set 2, C 14 -C 17 ) Green fields indicate quantifying and alternate m/z acquisition ions. Bold type indicates m/z used for quantification CClHm/z% ClM-ClM-Cl+2M-Cl+4M-Cl+6M-Cl+8 1442633441.9299301303305307 52536847.6333335337339341 62440252.2367369371373375 72343656.2401403405407409 82247059.6435437439441443 92150462.5469471473475477 102053865.1503505507509511 111957267.3537539541543545 121860669.3571573575577579 131764071.1605607609611613 1542834840.2313315317319321 52738245.8347349351353355 62641650.5381383385387389 72545054.4415417419421423 82448457.9449451453455457 92351860.8483485487489491 102255263.4517519521523525 112158665.7551553555557559 122062067.7585587589591593 131965469.6619621623625627 1643036238.7327329331333335 52939644.2361363365367369 62843048.8395397399401403 72746452.8429431433435437 82649856.2463465467469471 92553259.2497499501503505 102456661.8531533535537539 112360064.2565567569571573 122263466.2599601603605607 132166868.1633635637639641 142070269.8667669671673675 1753141042.7375377379381383 63044447.3409411413415417 72947851.3443445447449451 82851254.7477479481483485 92754657.7511513515517519 102658060.3545547549551553 112561462.7579581583585587 122464864.8613615617619621

23. Quantification Mass Ions for LCCPs (Ion Set 3, C 18 -C 20 ) Green fields indicate quantifying and alternate m/z acquisition ions. Bold type indicates m/z used for quantification CClHm/z% ClM-ClM-Cl+2M-Cl+4M-Cl+6M-Cl+8 1853342441.3389391393395397 63245845.9423425427429431 73149249.8457459461463465 83052653.2491493495497499 92956056.3525527529531533 102859458.9559561563565567 112762861.3593595597599601 122666263.4627629631633635 132569665.4661663665667669 142473067.1695697699701703 152376468.7729731733735737 162279870.2763765767769771 1953543840.0403405407409411 63447244.5437439441443445 73350648.4471473475477479 83254051.9505507509511513 93157454.9539541543545547 103060857.6573575577579581 112964260.0607609611613615 122867662.1641643645647649 132771064.1675677679681683 142674465.9709711713715717 152577867.5743745747749751 162481269.0777779781783785 172384670.3811813815817819 2053745238.7417419421423425 63648643.2451453455457459 73552047.1485487489491493 83455450.5519521523525527 93358853.6553555557559561 103262256.3587589591593595 113165658.7621623625627629

24. The entire window within a certain retention time range and mass range is integrated in order to include all CPs that could be present; The GC/MS open scan creates an additive chromatogram that allows for visual confirmation of the CPs pattern; For positive detection there must be a clear CPs pattern resembling that which is produced by the technical mixture contained in the standard solutions. Each Ion Set is quantified by converting the area responses detected at select masses for each C n to a concentration and then summing those concentrations in order to report a concentration per Ion Set. AXYS Method MLA-020 for CPs Qualitative and Quantitative Determination

25. Analyte quantification is performed using average response factors generated from the mid level calibration solutions run at the beginning and end of the analysis bracket; Response factors are generated using the total area of the polychlorinated paraffin peaks from the calibration solution; Results generated by this method are not recovery corrected for losses through the analytical procedure. AXYS Method MLA-020 for CPs Analyte Quantification

26. AXYS Method MLA-020 for CPs Reporting Criteria Sample-specific Detection Limits (SDLs) are determined individually for each sample; Limitation of the SDL concept in analysis of CPs is that the detection limit is based on detection of a single compound, not of a mixture; Results are reported to SDLs, but are flagged below the threshold at which a CPs pattern can be distinguished - the Minimum Concentration for Positive Identification (MCPI) and varies by matrix type; and The MCPI for CPs is the lowest concentration at which a chromatographic pattern for short, medium, long-chain, or Total CPs can be distinguished in a given matrix type.

27. AXYS Method MLA-020 for CPs Reporting Criteria Cont’d. Results below the MCPI but above the SDL are ‘J’ flagged; The Total CPs result is determined as a unique chromatographic integration, rather than as a sum of short/medium/long CPs concentrations; and A consequence of this can be that the short, medium, or long-chain CPs results may all be ‘J’ flagged but the Total CPs result is not. Minimum Concentrations for Positive Identification of Polychlorinated Paraffins

28. AXYS Method MLA-020 for CPs Figure 6: Additive chromatogram of Ion Set 2 for a sample with no polychlorinated paraffins pattern present (SDL = 9.6 ng/sample, Concentration = 214 ng/sample) Figure 5: Additive chromatogram of Ion Set 2 for a sample with a low level polychlorinated paraffins pattern (SDL = 12.8 ng/sample, concentration = 1002 ng/sample) The SDLs are determined from the additive chromatograms created for each Ion Set. In the example chromatograms above, noise estimates for SDL calculations were taken from the region of 17-18 minutes for the MCCPs, Ion Set 2.

29. AXYS Method MLA-020 for CPs Figure 8: Solid sample with no polychlorinated paraffin pattern present (concentration at 548 ng/sample, SDL = 19.1 ng/sample) Figure 7: Solid sample with low-level polychlorinated paraffin pattern present (concentration at 2245 ng/sample, SDL = 21.3 ng/sample)

30. AXYS Method MLA-020 for CPs Figures 7 and 8 above are meant to illustrate that, due to the presence of other compounds, a concentration which is above the SDL can still be determined for a sample even when there is no visible CPs pattern present; Figure 8 above demonstrates an instance where basing the detection decision on SDL alone could, in our judgment, lead to false identification of CPs in the sample; It was necessary to demonstrate the lower limit or concentration for which the CPs pattern could be recognized within the relevant matrix. This concentration would then become the reporting limit for positive identification of polychlorinated paraffins.

31. Questions and Follow Ups Thank you! AXYS Analytical ServicesContacts: 2045 Mills Road WestRobert Walker Sidney, BCOffice: 613-825-0672 (Ottawa) V8L 5X2Cell: 613-790-7512 Toll Free: 1-888-373-0881E-mail: rwalker@axys.com Richard Grace Cell: 905-484-2314 E-mail: rgrace@axys.com