for: minnesota pollution control agency april 16, 2015

AXYS Analytical Services Ltd. Analytical Procedure for the Determination of Chlorinated Paraffins in Aqueous, Solid, Biosolid, and Tissue Samples

For: Minnesota Pollution Control Agency

April 16, 2015

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

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

65%

30%

5%

Sales GeographyUS Canada International

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

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.

AXYS - Method DevelopmentAXYS develops new analytical methods for leading North American organizations. Some examples:

• US EPA Office of Water Reference Methods including:

• Minnesota Pollution Control AgencyFor 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.

• DuPontAXYS 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

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.

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


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)


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;


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.”

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.

AXYS Experience with CPsCirca 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

AXYS Experience with CPs - WWTPBiosolids from some wastewater treatment facilities have been found to contain significant concentrations of CPs.

Example from biosolids analyzed

Client ID 1 Lab Blank Spiked

Matrix Matrix Spike Matrix Spike Duplicate

Description Field Blank QC QC QC QCConc Units ng/g ng/g ng/g ng/g ng/g %Rec ng/g ng/g

METHOD NAMEMLA-020 Rev 3.01

MLA-020 Rev 3.01

MLA-020 Rev 3.01

MLA-020 Rev 3.01

MLA-020 Rev 3.01

MLA-020 Rev 3.01

MLA-020 Rev 3.01

MLA-020 Rev 3.01

SAMPLE SIZE 0.7520 g 0.7500 g 0.6760 g 10.11 g 1.000 g 0.1000 mL 0.7830 g 0.7780 gTOTAL CHLOROPARAFFIN-SUM 37,870.00 31,510.00 36,910.00 ND 494.6 ND 5,000.00 92.0 301,100.00 286,000.00TOTAL SHORT-CHAIN CHLOROPARAFFIN 14,050.00 12,620.00 14,900.00 ND 247.3 ND 2,500.00 18.8 68,990.00 65,640.00

TOTAL MEDIUM-CHAIN CHLOROPARAFFIN 16,080.00 13,350.00 16,360.00 ND 197.8 ND 2,000.00 44.3 140,400.00 135,200.00TOTAL LONG-CHAIN CHLOROPARAFFIN 7,724.00 6,639.00 7,948.00 ND 148.4 ND 1,500.00 29.2 91,910.00 87,390.00

AXYS Method MLA-020 for CPsSample Handling and Storage Conditions

MatrixSample Size

(per analysis)

Sample Container 1

Condition Upon

ReceiptStorage

Conditions 2

Sample Hold Time

Guideline 3

Extract Holding Time 3

Preservation

Bio-solids 2.5 g wet Glass < 4°C -20°C, dark 1 year 1 year none required

Solids(Sediment/Soil)

Maximum10-15 g wet Glass < 4°C -20°C, dark 1 year 1 year none required

Tissue 5-10 g wet Glass or foil wrapped < 4°C -20°C, dark 1 year 1 year none required

Aqueous 0.5 -1 litre Brown glass 0 – 4°C 4°C, dark 1 year 1 year none required

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.

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 Group Carbon Chain

Short Chain (SCCPs) C10 to C13

Medium Chain (MCCPs) C14 to C17

Long Chain (LCCPs) C18 to C20

AXYS Method MLA-020 for CPs Summary

Matrices: Aqueous, Solids, Biosolids, Tissue

Solids

Extraction:DCM:Hexane

Cleanup:

KOH/H2SO4/Cu/Alumina

Solvent exchange to HexaneAliquot of Quant. Std.

Analysis by GC/MS

Aqueous

Extraction:DCM

Cleanup:

KOH/H2SO4/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

AXYS Method MLA-020 for CPsGC 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

• 13C12-PCB 153 is used as a quantification standard.

GC Column General GC Conditions

DB-5 capillary column (60 m, 0.25 mm i.d., 0.10 μm film thickness)

Injector Temp (°C) 240Injector SplitlessCarrier Gas HeliumMaximum Temp (°C) 325

GC Temperature Program MS ConditionsTemp (oC) 60 Source Temp (°C) 142Hold time (min) 1 Electron Energy (eV) VariableRate (°C/min) 15 Emission Current (µA) VariableTemp (°C) 320 Mass Resolution unitHold time (min) 13.7 Detector Voltage (V) Variable

Ionization gas Methane

AXYS Method MLA-020 for CPsQualitative 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

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 CPsAnalyte Identification

Analyte group Ion Sets 1 Quantification Reference

C10 to C13 Ion Set 1 13C12-PCB 153C14 to C17 Ion Set 2 13C12-PCB 153C18 to C20 Ion Set 3 13C12-PCB 153Total CPs Ion set 1,2 and 3 13C12-PCB 15313C12-PCB 153 372/374 External standard

C Cl H m/z % Cl M-Cl M-Cl+2 M-Cl+4 M-Cl+6 M-Cl+8

10 3 19 244 43.0 209 211 213 215 217

4 18 278 50.4 243 245 247 249 251

5 17 312 56.1 277 279 281 283 285

6 16 346 60.7 311 313 315 317 319

7 15 380 64.5 345 347 349 351 353

8 14 414 67.6 379 381 383 385 387

9 13 448 70.3 413 415 417 419 421

10 12 482 72.6 447 449 451 453 455

11 3 21 258 40.7 223 225 227 229 231

4 20 292 47.9 257 259 261 263 265

5 19 326 53.7 291 293 295 297 299

6 18 360 58.3 325 327 329 331 333

7 17 394 62.2 359 361 363 365 367

8 16 428 65.4 393 395 397 399 401

9 15 462 68.2 427 429 431 433 435

10 14 496 70.6 461 463 465 467 469

12 3 23 272 38.6 237 239 241 243 245

4 22 306 45.8 271 273 275 277 279

5 21 340 51.5 305 307 309 311 313

6 20 374 56.1 339 341 343 345 347

7 19 408 60.0 373 375 377 379 381

8 18 442 63.3 407 409 411 413 415

9 17 476 66.2 441 443 445 447 449

10 16 510 68.6 475 477 479 481 483

13 4 24 320 43.8 285 287 289 291 293

5 23 354 49.4 319 321 323 325 327

6 22 388 54.1 353 355 357 359 361

7 21 422 58.1 387 389 391 393 395

8 20 456 61.4 421 423 425 427 429

9 19 490 64.3 455 457 459 461 463

10 18 524 66.8 489 491 493 495 497

11 17 558 69.0 523 525 527 529 531

12 16 592 70.9 557 559 561 563 565

Quantification Mass Ions for SCCPs (Ion Set 1, C10-C13)

Green fields indicate quantifying and alternate m/z acquisition ions. Bold type indicates m/z used for quantification

Quantification Mass Ions for MCCPs (Ion Set 2, C14-C17)



14 4 26 334 41.9 299 301 303 305 3075 25 368 47.6 333 335 337 339 3416 24 402 52.2 367 369 371 373 3757 23 436 56.2 401 403 405 407 4098 22 470 59.6 435 437 439 441 4439 21 504 62.5 469 471 473 475 47710 20 538 65.1 503 505 507 509 51111 19 572 67.3 537 539 541 543 54512 18 606 69.3 571 573 575 577 57913 17 640 71.1 605 607 609 611 613

15 4 28 348 40.2 313 315 317 319 3215 27 382 45.8 347 349 351 353 3556 26 416 50.5 381 383 385 387 3897 25 450 54.4 415 417 419 421 4238 24 484 57.9 449 451 453 455 4579 23 518 60.8 483 485 487 489 49110 22 552 63.4 517 519 521 523 52511 21 586 65.7 551 553 555 557 55912 20 620 67.7 585 587 589 591 59313 19 654 69.6 619 621 623 625 627

16 4 30 362 38.7 327 329 331 333 3355 29 396 44.2 361 363 365 367 3696 28 430 48.8 395 397 399 401 4037 27 464 52.8 429 431 433 435 4378 26 498 56.2 463 465 467 469 4719 25 532 59.2 497 499 501 503 50510 24 566 61.8 531 533 535 537 53911 23 600 64.2 565 567 569 571 57312 22 634 66.2 599 601 603 605 60713 21 668 68.1 633 635 637 639 64114 20 702 69.8 667 669 671 673 675

17 5 31 410 42.7 375 377 379 381 3836 30 444 47.3 409 411 413 415 4177 29 478 51.3 443 445 447 449 4518 28 512 54.7 477 479 481 483 4859 27 546 57.7 511 513 515 517 51910 26 580 60.3 545 547 549 551 55311 25 614 62.7 579 581 583 585 58712 24 648 64.8 613 615 617 619 621

Quantification Mass Ions for LCCPs (Ion Set 3, C18-C20)



18 5 33 424 41.3 389 391 393 395 397

6 32 458 45.9 423 425 427 429 431

7 31 492 49.8 457 459 461 463 465

8 30 526 53.2 491 493 495 497 499

9 29 560 56.3 525 527 529 531 533

10 28 594 58.9 559 561 563 565 567

11 27 628 61.3 593 595 597 599 601

12 26 662 63.4 627 629 631 633 635

13 25 696 65.4 661 663 665 667 669

14 24 730 67.1 695 697 699 701 703

15 23 764 68.7 729 731 733 735 737

16 22 798 70.2 763 765 767 769 771

19 5 35 438 40.0 403 405 407 409 411

6 34 472 44.5 437 439 441 443 445

7 33 506 48.4 471 473 475 477 479

8 32 540 51.9 505 507 509 511 513

9 31 574 54.9 539 541 543 545 547

10 30 608 57.6 573 575 577 579 581

11 29 642 60.0 607 609 611 613 615

12 28 676 62.1 641 643 645 647 649

13 27 710 64.1 675 677 679 681 683

14 26 744 65.9 709 711 713 715 717

15 25 778 67.5 743 745 747 749 751

16 24 812 69.0 777 779 781 783 785

17 23 846 70.3 811 813 815 817 819

20 5 37 452 38.7 417 419 421 423 425

6 36 486 43.2 451 453 455 457 459

7 35 520 47.1 485 487 489 491 493

8 34 554 50.5 519 521 523 525 527

9 33 588 53.6 553 555 557 559 561

10 32 622 56.3 587 589 591 593 595

11 31 656 58.7 621 623 625 627 629

• 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 Cn to a concentration and then summing those concentrations in order to report a concentration per Ion Set.

AXYS Method MLA-020 for CPsQualitative and Quantitative Determination

• 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

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.


Sample matrix Aqueous Solid Biosolid TissueBased on nominal sample size 1 L 10 g 2.5 g 10 gSCCPs 1000 ng/L 120 ng/g 1000 ng/g 125 ng/gMCCPs 700 ng/L 100 ng/g 800 ng/g 125 ng/gLCCPs 400 ng/L 120 ng/g 600 ng/g 22.5 ng/gTOTAL polychlorinated paraffins 400 ng/L 100 ng/g 600 ng/g 22.5 ng/g

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


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.


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)


• 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.

Questions and Follow Ups

Thank you!

AXYS Analytical Services Contacts:2045 Mills Road West Robert WalkerSidney, BC Office: 613-825-0672 (Ottawa)V8L 5X2 Cell: 613-790-7512Toll Free: 1-888-373-0881 E-mail: [email protected]

Richard GraceCell: 905-484-2314E-mail: [email protected]

for: minnesota pollution control agency april 16, 2015

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