AXYS ANALYTICAL SERVICES Ltd “Ultra Trace Organic Analysis, POPs, and Emerging Contaminants” For ASU April 8, 2011 By Richard Grace – Director – Sales, Marketing and Service – AXYS Analytical Services Ltd. rgrace@axys.com 1-888-373-0881 Or 905-484-2314

AGENDA About AXYS Ultra- Trace POPs and Emerging Contaminant Analysis Basics Selecting and Developing Methods Attributes Accreditation + Quality Systems Detection + Reporting Limits Applicable Levels + Blank Levels Analyte Lists + Time Some topical items on CECs

About AXYS 100 employees located in Victoria B.C. (Vancouver Island) ISO 17025 Certified, Multiple NELAP accreditations Focus – Multi Target Ultra Trace Organic Analysis (pg or ng levels) POPs Emerging Contaminants HRMS, LC MS/MS some GC/MS, GC-ECD options High barrier to entry 3 Analytical Areas Environmental (All matrices associated with Bioaccumulation cycle) Bio-monitoring Method Development and Validation (i.e. EPA 1668, 1614, 1694, 1698, 1699) Big “D” , little “R”

Where is Victoria?

AXYS HRMS Analysis (AXYS Developed EPA Methods in brackets) Chlorinated and Brominated Dioxins and Furans PCBs (EPA 1668A and 1668C) OC, OP, ON Pesticides (EPA 1699) PBDEs (EPA 1614) Hormones and Sterols (EPA 1698) Acid Extractable Herbicides Chlorinated Naphthalenes PBBs Pyrethroids

AXYS LC MS/MS Analysis (EPA Reference Methods in Brackets) Perfluoronated Compounds (pending) Personal Care and Pharmaceutical Products (EPA 1694) Hormones (EPA 1645) Phthalate Esters Metabolites Parabens and metabolites Bisphenol A and metabolites Nonyl Phenols and Nonyl Phenol Ethoxylates Variety of Pesticide Panels

Who do we work for? 70% U.S., 30% Cdn. + International Government and Regulatory Authorities (50% of work) National and State Governments POP and CEC Assessment Legal issues Method Development Private Sector Industry + Consultants (40% of work) Assessment and Remediation Regional and Ambient / Baseline Work / TMDLs POP and CEC Risk Assessment / Litigation Academic, Legal, NGO – 10%

AXYS Analysis Early Days (1980 to 2000) Occurrence of POPs in all environmental and bioaccumulation matrices Arctic Regulatory Framework, Litigation and Settlements POPs in humans, NRD assessments Early Reconnaissance Studies and NGOs Awareness of CECs Epidemiology Studies and CECs Explosion of programs 2007-8 CEC focus AXYS does not publish studies, our clients do 2-4 year lag vs. time of work

AXYS Analytical Process (75 Staff)

Expanded AXYS Analytical Process (15 client services staff)

AXYS Analysis All analysis performed with multiple mass or mass fragment detection per target HRMS (EI or CI) GC/MS (EI or CI in SIM) LC MS/MS in MRM mode (multiple transitions where possible) All analysis use labelled standards added at start of analytical process, recovery corrected vs. recovery standard All “active” analysis EPA Tier 1 validation (IPR, MDL, MS/MSD by EPA Federal Register 40 CFR Part 136, Appendix B, no iteration

GCMS Scan Chromatogram in Urine Matrix Ultra Trace Detection Not Required for Targets

GC/MS with Ultra Trace DLs Not Met pp-DDT by GC EI MS SIM

Meeting Ultra Trace Requirements 13C12 pp DDT and pp DDT by HRMS

LC / MS vs. LC MS/MS TIC (Total Ion Chromatogram) vs. MRM Monitoring Dehydronefidipine

Ultra Trace Analysis Principles Positive Identification and Quantification Performance of multiple ions monitored and reported to meet response, ratio, and retention criteria Recovery correction required Max. chromatographic separation and selectivity achieved through cleanup, columns, instrument type Selectivity achieves low detection limits, ruggedness Common to all CDC and EPA 1600 series methods for POPs and CECs Basis for EU 657 standard for positive identification and quantification of targets with no specified reference method Many bio-monitoring methods would be considered a “non-positive screen by this standard

# of Ions (native plus labeled surrogate) EU 657 Point Criteria for Positive Identification if Using Isotope Dilution (4 Points Required for a Positive) Technique # of Ions (native plus labeled surrogate) # of Points GC/MS (EI or CI) N LC/MS GC MS/MS 1 precursor, 2 daughter 4 LC MS/MS 2 precursor, each with one daughter 5 HRMS 2N

Applying Point System to PBDE Analysis Method HRGC/HRMS (8+ points) HRGC/LRMS (1-2 points) HRGC/ECD (0 Points) Details EI (Electron Impact) / Voltage SIR NCI (Negative Chemical Ionization) /SIM Scan Quantification 13C-labeled standards for isotope dilution Labeled standards not possible Specificity High: Monitor two to six ions from parent ion cluster, compare to theoretical ratio Medium: Monitor Br- ions lost from PBDEs. Other brominated compounds e.g. PBBs may interfere if have same GC retention time Low: Identification based on GC retention time. Other halogenated compounds may interfere e.g. PBBs, PCBs, Cl pesticides if same GC retention time. Sensitivity Best (Lowest) detection limit Highest detection limits Medium

PFC Methods That Meet Criteria MRM Transitions and Surrogates Used Target Analyte MRM Transition IS MRM Surrogate PFBA (C4 Acid) 213>169 13C4 PFBA 217>172 13C4 PFOA 417>372 PFPeA (C5 Acid) 263>219 PFHxA (C6 Acid) 313>269, 313>119 13C2 PFHxA 315>270 PFHpA (C7 Acid) 363>319, 363>169 PFOA (C8 Acid) 413>369, 413>219, 413>169 13C2 PFOA 415>370 PFNA (C9 Acid) 463>419, 463>219, 463>169 13C5 PFNA 468>423 PFDA (C10 Acid) 513>469, 513>269, 513>219 13C2 PFDA 515>470 PFUnA (C11 Acid) 563>519, 563>269, 563>219 13C2 PFUnA 565>520 PFDoA (C12 Acid) 613>569, 613>319, 613>169 13C2 PFDoA 615>570 PFBS (C4 Sulfonate) 299>80, 299>99 303>84 13 C2 FOUEA 18 O2 PFOS PFHS (C6 Sulfonate) 399>80, 399>99 18O2 PFHS 403>84 PFOS (C8 Sulfonate) 499>80, 499>99, 499>130 13C2 PFOS 503>80 FOSA (C8 Sulfonamide) 498>70

CEC Surprises – Triggers for Change PFOS Pharmacokinetics and Distribution Well absorbed orally (95% within 24 h) Distributed mainly in serum and liver Not metabolized Fecal and urinary excretion Perception change in early 2000’s 30 ng/mL serum average in North Americans in 2004 Rat: Plasma t½ = 7.5 days Monkey: Serum t½ = 200 days Human: Estimated Serum t½ = ~8.7 years

Standard AXYS PFC In Water Method AXYS PFC Method Basics Standard AXYS PFC In Water Method MLA 060 For water, soils, sediments, tissues, serum, air Applies principles of EPA 1600 series performance based methods Key AXYS PFC Method Attributes Recovery Corrected Matrix Matched Calibration Pre-Concentration by SPE Cartridge Low detection limits and blank levels Big “D”, small “R” Benchmarked, multiple method validations to insure accuracy LOQ is LMCL point above MDL or client specific level Adjust pH to 5.5 ±0.5 With HCOOH Spike labeled surrogates SPE extraction with WAX Condition: 5 mL 0.3% NH4OH in MeOH and 5mL0.1 M HCOOH Wash: with 5 mL of H2O 5 mL & With 1:1 (0.1 M HCOOH: MeOH) Elute: 4 mL of 0.3% NH4OH in MeOH Microvial 300uL portion Analyze by (-ESI) LC-MS/MS

POPs and CECs Analyses Accreditation and Benchmarking POPs and CECs = Environmental Contaminants ISO 17025:2005 is correct quality standard ISO 17025: 2005 = ISO 9001:2000 + technical requirements (method and statistical QC) National Bodies NELAP not ISO 17025 Accreditation by test and environmental matrix NELAP offers regulatory method programs Human matrices not offered in past CALA / SCC has now made available Proficiency not available, must go to other forums

POPs and CECs – Benchmarking Accuracy No “regulatory” programs or methods Tools for establishing accuracy Inter-calibrations / Round Robins / PE Programs SRMs / CRMs Inter-calibrations available AMAP Dioxin Inter-calibrations PFC Inter-calibrations Some academic programs for endocrine disruption (BPA, PEMs) NIST developed Available for most legacy (regulated) POPs Some persistent CECs in development (PFCs, PBDEs) Not available for most other CECs (BPA, PEM, Parabens etc.)

1st Worldwide Interlaboratory Study on PFCs in Environmental and Human Samples *Van Leeuwen et al. Environ. Sci. Technol. (2006) Vol. 40, p. 7854-7860. *Fish Fillet Results PFOS PFOA Spiked Concentration (ng/g) 37 10 Analytical Results (ng/g) Minimum 2.8 0.54 Median 40 13 Maximum 295 204 %RSD 125 201 Evaluation of Results %Satisfactory 17 25 %Questionable - 30 %Unsatisfactory 83 45

Limits of Accuracy in early POPs and CEC Analysis Phthalate Ester metabolites Standards accuracy issue i.e. MCPP standard used for most studies appears to be 17% of reported value PFCs in Eggs BPA 0.25 to 10 ng/mL gives 90% population coverage in urine (NHANES) Urine deconjugated BPA 10X blood values Some studies show much higher blood values (BPA instrument values)

LC MS/MS False Positive Example Analysis – PFCs by LC MS/MS Matrix – Eggs for human consumption Analytical Result – 120 mean ng/g PFOS per Egg detected using LC MS/MS in MRM mode (Carbon labelled PFOS surrogate employed, one transition monitored @ specified retention time, UPLC technology submitted for publication) Net Result – Paper withdrawn, results were identified as false positive (TDCA, a bile acid in many animals) Issue Identified – not enough positive identification criteria Identified need for monitoring multiple transitions Did not focus on chromatographic separation

LC MS/MS Interferences PFOS and Taurodeoxycholate Acid (TDCA) Same MW, Common Transition Compound MW Parent Ion 1 Ion 2 Ion 3 CE CV Taurochendeoxycholate 499.29 498.2 79.8 106.8 123.8 55 80 Taurodeoxycholate Tauroursodeoxycholate PFOS 499.93 498.9 79.9 98.9  N/A 45/40

PFOS / TDCA Interference TIC Chromatogram Separation PFOS – Top, 3 TDCA Isomers

Detection and Reporting Limits Select analytes, then required reporting limits, then determine sample size Define reporting limits by study objectives NHANES is an excellent resource Literature MAY be useful Be aware of very different languages Many Laboratories and Publications Reporting Limit < MDL < Blank Level ISO 17025 + Reference Laboratories Reporting Limit > MDL > Blank Level (ideal)

AXYS Reporting Limits Default Sample Reporting Limits (RL) HRMS and GC/MS – LMCL or 3:1 signal to noise ratio of peaks LC MS/MS – LMCL or 3:1 signal to noise ratio if above LMCL IDL not calculated or important, LMCL response 10-30X S/N MDL 3-5X < RL (ideally), not used for reporting Dilution increases RL proportionally No blank correction, blank levels may influence reporting limits by prior agreement Applied “flags” to data quality – e.g. estimated maximum and estimated minimum probable concentrations (EMPCs) Should always be discussed with client

Blank Levels – A dirty secret Many CECs are ubiquitous as in many consumer goods and lab materials Blanks limit detection capability in analysis such as; PBDEs PCBs PFCs BPA Cotinine Can control with sample size selection Lab control not in method, but related SOPs Ask for control charts (10 method blanks min.)

Blank Control Charts Use them to set large study stat. limits

Analyte Lists – Which Ones to Measure? AXYS experience that what gets measured continues to get measured Relevant POPs and CECs change over time Legacy POPs Replacement products for PFCs PBDEs Phthalates Aging (POPs congener distribution) Very little measurement of metabolites and degradation products All lack toxicity / epidemiological end point info for initial years of study All may be endocrine disruptors Studies should change over time

The Mean Percent Congener Distribution to the Sum of the 35 PCBs (Whole Weight in ng/g) for NHANES 2003-2004 by Age Group

Lake Ontario % of PCB Concentrations Not Part of IADN and US/DS Lists – Drives EPA 1668 A,B,C

Observed Concentration Are PFOS and PFOA the Only PFCs? 6 Months of Occurrence Data POTW Aqueous Streams Analytes Units No. of Samples No. of Detects Percent of Detects Observed Concentration Avg Conc. Found Min Conc. Found Max Conc. Found PFBA ng/L 339 234 69 65.5 1.15 8540 PFBS 84 24.8 568 2.19 21000 PFDA 109 32.2 3.79 1.1 29.1 PFDoA 2 0.6 1.45 1.23 1.66 PFHpA 233 68.7 52.9 1.03 9610 PFHxA 253 74.6 328 76200 PFHxS 117 34.5 1650 2.03 190000 PFNA 210 61.9 5.62 28.1 PFOA 276 81.4 74.2 1.38 15600 PFOS 175 51.6 445 2.02 25200 PFOSA 25 7.4 15.3 1.04 61.7 PFPeA 340 216 63.5 174 29200 PFUnA 5 1.5 2.57 1.18 4.73

6 Months of PFCs in POTW Biosolids Analytes Units No. of Samples No. of Detects Percent of Detects Observed Concentration Avg Conc. Found Min Conc. Found Max Conc. Found PFBA ng/g 123 11 8.9 7.26 1.14 16.2 PFBS 14 11.4 9.12 2.45 32.4 PFDA 91 74 14.6 0.712 57.5 PFDoA 56 45.5 8.08 0.292 58.9 PFHpA 12 9.8 6.54 2.01 28.1 PFHxA 22 17.9 0.727 105 PFHxS 25 20.3 32.3 1.82 147 PFNA 60 48.8 9.37 0.139 39.2 PFOA 55 44.7 17 0.585 256 PFOS 85 69.1 476 0.901 7480 PFOSA 39 31.7 40.8 0.194 343 PFPeA 21 17.1 12.4 0.567 69.6 PFUnA 52 42.3 6.44 1.12 35.2

Minimum. Concentration Maximum Concentration PBDEs from TNSSS – EPA 2008 Analyte Units Minimum. Concentration Mean Concentration Maximum Concentration % Solids % 0.43 93.53 BDE – 28 DryWt. ng/kg 2,200 160,000 BDE – 47 73,000 709,174 5,000,000 BDE – 66 1,800 110,000 BDE – 85 3,200 150,000 BDE – 99 64,000 716,362 4,000,000 BDE – 100 13,000 1,100,000 BDE – 138 1,900 40,000 BDE – 153 9,100 52,685 410,000 BDE – 154 7,700 440,000 BDE – 183 2,100 120,000 BDE – 209 3,462,942 17,000,000

PBDE’s by EPA 1614 – Moving to 46 congeners from Top 8 to 11 Congeners Theory – Penta and Octa changes will eliminate most toxic PBDEs, incoming Deca controls will eliminate 209 congener Current Situation – about 850M tons in environmental inventory in NA Degradation of PBDE containing products giving ongoing supply Deca plus new products production rising Deca degradation rapid – to other PBDEs May be in for a long PBDE story

Legacy POPs and New POPs PFC Beluga Study – Published ES&T June 2009 POPs = Persistency, Toxicity, Bioaccumulation + Transport Field work: Village of Umiujaq / Nastapoka River Estuary

Sample Collections: E. Hudson Bay Study Overview Sample Collections: E. Hudson Bay Sediment samples (n = 8) petit ponar grabs Macro-algae (n = 6) Harvested beluga whales (n=25); 12 females, 12 males, 1 calf liver, blubber, blood, milk Fish: capelin, arctic cod, sculpin, salmon (n = 6 per species) Chemical analysis: Previous Work: PCBs, PBDEs, OC Pesticides Current Study: PFCs AXYS Extended PFC List (Carboxylates, Sulfonates, Sulfonamides)

Mean Values of PFCs in Beluga Blood, Liver, Milk

PFCs (ng/g) in Arctic Tissue

PFC Levels in Trophic Magnification vs. Legacy Organohalogens ng.g-1 dry wt. PFC Levels in Trophic Magnification vs. Legacy Organohalogens = PCBs = OC Pest. = PBDEs = PFCs Beluga Liver

Contaminants of Emerging Concern – “CECs” Some New POPs + Many Endocrine Disruptors Many not persistent but exposure constant Many present through consumer goods – ubiquitous Many thought to be endocrine disruptors Endocrine System Regulates Changes in Body Response to environment Orchestrates body development and reproductive changes Hormones are “Messengers” to Cells Change cell internal and external chemistry Many hormones and receptors, many interactions and outcomes Endocrine Disruption Occurs when; Hormone receptors inhibited to not function Hormone receptors triggered by “outside” compound Hormone System “Out of Balance” Effect is not a “toxic” effect

Defining Endocrine Disruption End Points Toxicity Typical screening process Long term, subtle developmental effects not screened “Nonmonotonic Dose Response Curves” (more chemical does not equal more response – example BPA) Effect May be at Specific Stage of Life Cycle Requires study over long periods of life cycle More difficult with more complex species Low level studies required, statistically relevant study size Limited Tools Limited analytical and epidemiological methods at relevant levels # of studies / study size – inconclusive data in many cases Interpretation / vested interests

Topical End Points for POPs and CECs Reproductive Reduced fertility Reproductive tract abnormalities Male / Female Sex ratios Brain Development and Behavior Issues Thyroid impairment I.Q. reduction ADHD Immune System Issues Increased Illness “Weaker” organism Cancer, Diabetes, Heart Disease, Cholesterol variations, obesity etc. Synergistic and Antagonistic Effects

An Interesting Study Kidd et al 2007 Environmental End Point – hormonal effects / vitellogenin gene expression on Fathead Minnows Vitellogenin – egg yolk precursor protein expressed by female fish Hormonal triggers (synthetic hormones, NP etc.) produce fish vitellogenin production / feminization Study – Test (5-6 ng 17A ethinyl estradiol addition) vs. Control Lake Year 3 produces complete Fathead Minnow feminization Fathead population eliminated Notes on Interpretation End Point measured on Fathead Minnows In other species - effects not noted Tie to Humans?

Length frequency distributions of fathead minnow captured in trap nets in reference Lake 442 (A) and Lake 260 (B) (amended with 5–6 ng·L−1 of EE2 in 2001–2003) during the fall of 1999–2005 Kidd K. A. et.al. PNAS 2007;104:8897-8901

Purpose of AXYS “Client” Requests for CEC Analysis – Example PPCPs “Reconnaissance” or Screen in Water and Sediments based on “USGS”, 1694 or Other List Occurrence, Levels, and Fate of PPCPs in POTW and Drinking Water Processes Occurrence, Levels, and Fate in POTW and Water Treatment Systems for New Treatment Systems / Capital Projects Trace back / fingerprinting of sources of PPCPs Support of Toxicity, Fate, and End Point Studies

The List – Which PPCP Compounds to Analyze? 3000 potential PPCPs Starting Point – USGS Reconnaissance List 40 PPCPS + Hormones and Sterols Constant requests from clients for more compounds based on literature, other studies, media articles Input from National Regulatory Bodies Lists supplied based on volumes, persistence, toxicity / end point concerns (i.e. endocrine disruption, antibiotic resistance) Fit for analysis – standards, surrogates, stability, appropriate instrumentation Framework to manage addition of compounds, groups to maintain, validate, and improve

AXYS PHASE 3 PPCP METHOD (2008/9) Grouping of Compounds by Analytical Performance EPA Method 1694 Compounds + Extended List

Building the Methods Built From Back to Front Column Selection Instrument Infusion Response, Mode, Cone Voltage, Transitions Retain and Repeat !!!! Column Selection Phase 1 only Selection of SPE Cartridges Multiple tests in Phase 1 only Varying pre-wash, loading, elution Extraction Acidic, Basic Validation MDL, IPR, MS/MSD Troubleshooting + Re-Validation

EPA 1694 PPCPs – Frequency and Range of Detects EPA “Targeted National Sewage Sludge Survey” All Results in dry weight ug/Kg ANALYTE # Detect Minimum ug/Kg Maximum ug/Kg 4-Epioxytetracycline 8 35.7 54.9 4-Epitetracycline 80 47.2 4,380 Erythromycin-Total 77 3.1 180 Flumequine NA Fluoxetine 79 12.4 3,130 Gemfibrozil 76 12.1 2,650 Ibuprofen 54 99.5 11,900 Isochlortetracycline 1 3,140 Linomycin 3 13.9 33.4 Lomefloxacin 2 33.3 39.8 Metformin 6 550 1,160 Miconazole 14.2 9,210 Minocycline 32 351 8,650 Naproxen 44 20.9 1,020 Norfloxacin 29 99.3 1,290

Are We Measuring the Right Thing Are We Measuring the Right Thing? Conjugates, Degradation Products, Metabolites Current State Analytes primarily native compounds Most compounds “persistent” by constant presence Limited degradation products and metabolites No Chlorination byproducts No conjugates About Conjugates Many compounds exist as conjugates from urine Deconjugation may occur at some rate in POTW environments or on consumption Limited info but detected in studies Methods from Biomonitoring Available I.e. Triclosan, BPA, Parabens, mono-phthalate esters

Observed Concentration Looking for the Correct Analyte Form Triclosan Conjugates in Urine vs. Native Triclosan in Serum Analyte Units No. of Samples No. of Detects Percent of Detects Observed Concentration Avg Conc. Found Min Conc. Found Max Conc. Found Total Triclosan In Urine ng/mL 62 52 83.9 109 1.33 1980 Native In Serum 22 ND

Degradation Products / Metabolites Many Compounds not Stable Fit for TOF LC-MS/MS to identify degradation products Not certain degradation products of less concern Metabolites In Humans and in Environment Fit for TOF LC-MS/MS to identify degradation

Major Cl Transformation Products Triclosan + Free Chlorine

Open Forum Questions Follow-Ups