Perfluorinated Compounds: An Overview of Environmental and Analytical Issues

Transcription

1 Perfluorinated Compounds: An Overview of Environmental and Analytical Issues John P. Giesy*, P. D. Jones & J. L. Newsted, K. Kannan Zoology Department National Food Safety and Toxicology Center Institute of Environmental Toxicology

2 Availability of Information Reports containing detailed information are available at docket EPA AR226. Information is available electronically from the EPA docket staff in the form of pdf files. To request the index or any document listed on the docket, send an message to the docket staff at or call The docket staff will send the index and any documents to you via . Additional details regarding the Pollution Prevention and Toxics Docket Can be Obtained online at Send a blank CD to the public reading room at the following address: Public Reading Room, Room B102 EPA West Building 1301 Constitution Avenue, NW Attn: OPPT Docket Staff Web Site:

3 SAMPLING LOCATIONS

4 Previously Published Papers-I Giesy, J. P. and K. Kannan Global Distribution of Perfluorooctane Sulfonate and Related Perfluorinated Compounds in Wildlife. Env. Sci. Technol. 35: Kannan, K., et al Perfluorooctane Sulfonate and Related Fluorinated Organic Chemicals in Marine mammals. Environ. Sci. Technol. 35: Kannan, K., et al Perfluorooctane Sulfonate in Fish-Eating Water Birds Including Bald Eagles and Albatrosses. Environ. Sci. Technol. 35: Kannan, K., et al Perfluorooctane Sulfonate in Oysters (Crassostrea virginica), from the Gulf of Mexico and Chesapeake Bay, USA. Arch. Environ. Contamn. Toxicol. 42: Kannan, K., et al Perfluorooctane Sulfonate and Related Fluorinated Hydrocarbons in Marine Mammals, Fish and Birds from Coasts of the Baltic and The Mediterranean Seas. Environ. Sci. Technol. 36:

5 Previously Published Papers II Kannan, K., et al Concentrations of Perfluorinated Acids in Livers of Birds from Japan and Korea. Chemosphere, 49: Kannan, K., J.L. Newsted, R.S. Holbrook and J.P. Giesy Perfluorooctanesulfonate and Related Fluorinated Hydrocarbons in Mink and River Otters from the United States. Environ. Sci. Technol. 36: Giesy, J.P. and K. Kannan Perfluorochemical Surfactants in the Environment. Environ. Sci. Technol. 36:146A-152A.

6 Structures of Sulfonated Fluorochemicals POSF: Perfluorooctane sulfonylfluoride O C 8 F 17 S - F O O PFOSA: Perfluorooctane sulfonamide C 8 F 17 S - NH 2 O

7 Structures of Sulfonated Fluorochemicals PFOS: Perfluorooctane sulfonate F F F F F C C C C F C C F F C C F O S F F F F F F F F O O O C 8 F 17 S - O - O PFOA or POAA: Perfluorooctanoic acid O C 7 F 15 CO - Also referred to as C8, Other homologues include C7and C9-12

8 Compounds of Interest Compound Tridecafluoroheptanoate (C7) Pentadecafluorooctanoate (C8; PFOA) Heptadecafluorononoate (C9) Nonadecafluorodecanoate (C10) Perfluoroundecanoate (C11) Perfluorododecanoate (C12) Perfluorooctane sulfonate (PFOS) Formula C 6 F 13 COO - C 7 F 15 COO - C 8 F 17 COO - C 9 F 19 COO - C 11 F 21 COO - C 12 F 23 COO - C 8 F 17 SO - 3

9 Relationship between PFOS and PFOA Based on chemical physical properties methods for PFOS and PFOA should be similar Environmental fate profiles should be similar Source patterns may be similar or different, depending on use Toxicological profiles should be similar

10 Relationship between PFOS and PFOA Based on the fact that there are similarities between PFOS and PFOA, knowledge about PFOS should be useful in developing analytical procedures and assessing toxic potential and assessing potential risks of PFOA For this reason, I will present some historical information of PFOS and discuss how it relates to PFOA

11 Methods - 1 Fluorine-carbon bond is very strong Organo-fluorine determined by difference between organic and inorganic F PFOS not volatile - GLC not useful Derivatization followed by gas chromatography with either electron capture or mass-specific detection have been applied Due to excellent leaving group properties, derivatives are unstable

12 Methods - 2 HPLC is good for separation, but PFCs do not contain chromophores HPLC followed by fluorescence detection has been applied to perfluorocarboxylic acids but sensitivity is poor 19 F - NMR has been used, but is not very sensitive and is not quantitative Requires pre-concentration and cleanup

13 Methods - 3 Recent methods have used ion pairing with separation by HPLC and quantification by negative ion, electro-spray, tandem mass spectrometry Compound-specific Sensitive with detection limits of approximately 5 ng/ml (ppb) in plasma Hansen et al. 2001, Environ. Sci. Technol., 35,

14 Ion Pairing Methods For PFOS/PFOA Sample-Homogenize Ion-pair extraction (TBA/MTBE) & Filter Betasil- C 18 column separation : Florisil/carbon column HPLC-ESMS/MS (triple quadrapole ; ES-ve) Monitor ions: PFOS: , 99, 130 PFOA: , 219, 169, 119 TBA=tetrabutylammonium hydrogen sulfate; MTBE=methyl tert-butyl ether Internal standard, 1H,1H,2H,2H-perfluorooctane sulfonate (THPFOS)

15 Criteria for the Selection of Analytical Methods Sufficiently Sensitive Accurate Reproducible Transportable Simple High Throughput Minimize Interferences

16 SPE Methods For PFOS/PFOA in Water 50 ml H 2 0 Spike with Recovery Internal Standard C 18 SPE Cartridge Elute with 0.5 ml Methanol

17 Compounds Studied Compound Tridecafluoroheptanoate (C7) Pentadecafluorooctanoate (C8; PFOA) Heptadecafluorononoate (C9) Nonadecafluorodecanoate (C10) Perfluoroundecanoate (C11) Perfluorododecanoate (C12) Perfluorooctane sulfonate (PFOS) Molecular Weight

18 Instrumentation Liquid Chromatography HP Series 1100 Keystone Betasil C 18 2X100 mm, 5 um particles Mass Spectrometry Micromass Quatro Ultima Triple Quadrapole MassLynx Ver. 3.5 Sciex API 4000 with collision energy of -10 (ev) for PFOA results in approximately 10X lesser LOQ.

19 Matrix Spikes For PFOS/PFOA in Water 50 pg/ml 80 pg/ml 500 pg/ml

20 Unextracted Standard 100 PFHS (399 > 99) 6.43 Time (min) % % PFOSA (498 > 78) PFOS Quantification (499 > 99) 6.80 % PFOS Confirmation (499 > 80) % PFOA (413 > 169) 6.63 %

21 SPE Standard Extracted from Water 100 PFOSA (498 > 78) 7.22 Time (min) % PFOS Quantification (499 > 99) 6.80 % PFOS Confirmation (499 > 80) 6.83 % PFOA (413 > 169) 6.69 %

22 Spiked Water Sample Extracted by SPE 100 PFOSA (498 > 78) 7.16 Time (min) % PFOS Quantification (499 > 99) % PFOS Confirmation (499 > 80) % PFOA (413 > 169) %

23 SPE Methods For PFOS/PFOA in Water Recoveries (%) Mean (SD) [Range] PFOS 103 (13.4) [77-146] PFOSA 122 (31) [67-151] PFOA 125 (27) [94-147]

24 LOQ for PFOS/PFOA in Water LOQ 2X Signal to Noise, <30% Precision LOQ = 15 ng/l (pptr) Can lower LOQ to approximately 10 pg/l by using more water May need to clean-up Can lower ph to improve extraction eficiency

25 SPE Methods For PFOS/PFOA in Water Results 67 Samples analyzed from N. A. Great Lakes and Tributaries No Detectable Concentrations of PFOS, PFOSA, PFOA

26 SPE Methods For PFOS/PFOA in Biota Sample-2.0 ml Serum or 1 g tissue Homogenize Extract 8 ml H ml Acetonitrile Extract 20 min; Centrifuge 20 min 3,500 rpm Dilute with 350 ml H 2 0 C 18 SPE Cartridge (0.5 g) Elute with 0.5 ml Methanol

27 SPE-Extracted Mink Liver 100 PFHS (399 > 99) Time (min) 7.19 % PFOSA (498 > 78) 7.44 % PFOS Quantification (499 > 99) 6.77 % PFOS Confirmation (499 > 80) 6.77 % PFOA (413 > 169) %

28 SPE-Extracted Amphipod 100 PFHS (399 > 99) 7.07 Time (min) % % PFOSA (498 > 78) % % PFOS Quantificaqtion (499 > 99) PFOS Confirmation (499 > 80) PFOA (413 > 169) 7.30 %

29 SPE Sensitivity for PFOA and PFOS Blood Plasma and Serum Compound C7 C8 (PFOA) C9 C10 C11 C12 PFOS LOQ (ng/ml) (0.52 min obs)

30 SPE Methods Blood-Precision Analyte Cal Range R 2 % RSD (ng/ml) PFOS C C C C C8 (PFOA) C

31 SPE Methods Blood-Spike Recoveries Analyte Spike (#1) (%) Spike (#2) (%) Matrix Blank PFOS <LOQ C <LOQ C <LOQ C <LOQ C <LOQ C8 (PFOA) <LOQ C <LOQ

32 Concentrations in Blood 1 Plasma (ng ml) Analyte Chinese (TCR-674) Lampire (TCR-685) Innovative (TRC-683) Golden West (TCR-684) PFOS 2 < C12 < [0.036] [0.022] [0.024] C11 <0.010 [0.049] [0.071] C10 <0.010 [0.0127] C9 < C8 (PFOA) < C7 < [0.016] Plasma centrifuged; 2 < LOQ; ng/ml = ppb; 3 [ ] = Concentrated SPE method;

33 Conclusions Human Blood Total PFOS and PFOA in serum and plasma from 2.5 to 27 and 0.65 to 5.6 ng/ml, respectively Linear isomers C7, C9-C12 from <0.01 to 0.9 ng/ml Branched isomers from < LOQ to low levels PFOA/ PFOS Ratios Plasma 0.21 (0.016) 1 PFOS/ PFOS 4X greater than PFOA PFOA MEAN (SD) Serum 0.25 (0.067)

34 Comparison of Solid Phase (SPE) and Ion Pairing (IP) Techniques Great Lakes Trophic Level Study

35 LOQ for SPE Great Lakes Trophic Levels LOQ = 2X SNR and <30% RSD for MSD PFOS Da transition is most sensitive, but often affected by an interference Use Da transition; Less sensitive but no interference

36 LOQ Comparison SPE vs. IP ng/g, ww (ppb) Method PFOS PFOSA PFOA PFHS SPE IP

37 Trophic-Level Study-PFOS Matrix Amphipod Benthic Algae Crayfish Muscle L.M. Bass Muscle Mink Liver Pseudo Feces Round Goby Muscle S.M. Bass Muscle Zebra Mussel Recovery (%) (+/-RSD) SPE IP 125 (3) <LOQ 68 (13) <LOQ 133(35) <LOQ 136 (26) (5) 204 (2) 110 (33) <LOQ 76 (19) 113 (3) <LOQ 89 (1) 99 (87) <LOQ

38 Trophic-Level Study-PFOA Matrix Amphipod Benthic Algae Crayfish Muscle L.M. Bass Muscle Mink Liver Pseudo Feces Round Goby Muscle S.M. Bass Muscle Zebra Mussel Recovery (%) (+/-RSD) SPE IP <LOQ <LOQ 76 (43) <LOQ 432 (143) <LOQ <LOQ <LOQ 22 (7) 120 (3) <LOQ <LOQ <LOQ 47 (3) <LOQ <LOQ <LOQ <LOQ

39 Trophic-Level Study-PFHS Matrix Amphipod Benthic Algae Crayfish Muscle L.M. Bass Muscle Mink Liver Pseudo Feces Round Goby Muscle S.M. Bass Muscle Zebra Mussel Recovery (%) (+/-RSD) SPE IP 155 (12) 774 (53) 99 (1) 322 (35) 66 (4) 304 (5) <LOQ (3) 176 (0) 150 (10) <LOQ 65 (18) 184 (13) <LOQ 232 (8) 125 (22) 655 (120)

40 Results- Matrix Spike Recoveries Matrix at 2-3 times the concentration of analyte (PFOS) originally detected in the extracted sample for each matrix listed. For the SPE extraction most of the PFOS spike recoveries were > LOQ while one recovery was <LOQ. For the Ion Pair extraction, the majority of matrix spike recoveries were <LOQ)

41 Mean Recoveries Comparison SPE vs. IP Mean % (SD) Method PFOS PFOSA PFOA PFHS SPE (28) (7.5) (181) (39) IP ,518 (56) (14) (37) (5,667)

42 Toxicity Critical Mechanism of toxic action of PFOS is unknown Preferentially retained in plasma and liver binds to protein

43 Toxicity Summary - 1 Uncouple oxidative phosphorylation Inhibit Gap junctional intercellular communication Peroxisome proliferation and organ-specific DNA Dammage Induce liver microsomal carboxylase RL4 PFOS, PFOSA PFOA, PFDA, PFOS PFOA, PFDA, PFBA PFOA, PFDA, PFOS PFOA: Perfluorooctanoate; PFDA Perfluorodecanoate; PFBA: Perfluorobutyric acid; PFOS: Perfluorooctane sulfonic acid

44 Toxicity Summary - 2 Hypophagia, weight loss, bradychardia, hypothermia and reduced motor activity Reduced T3 and T4 concentrations Induction of cytochrome P450; ECOD, Acylglycerophosphocholine Acyltransferase, acyl CoA-hydrase Induced liver phospholipase C Inhibited phosphocholine PFDA PFDA PFOA PFDA PFOA: Perfluorooctanoate; PFDA Perfluorodecanoate; PFBA: Perfluorobutyric acid; PFOS: Perfluorooctane sulfonic acid

45 Toxicity Summary - 3 Dechreased serum cholesterol Increased faty acid and acyl CoA binding proteins in rat liver Increased proteinkinase C activity, decreased Acyl CoA synthase activity PFOA, PFOS PFDA PFOS PFOA: Perfluorooctanoate; PFDA Perfluorodecanoate; PFBA: Perfluorobutyric acid; PFOS: Perfluorooctane sulfonic acid

46 Toxicity Testing 1. Effects on Membranes 1. Fluidity 2. Mitochondrial potential 3. Gap Junctions 2. Binding to Proteins 1. Hormone displacement 2. HPLC/MS 3. Effects on Gene Expression 1. Differential display 2. Gene chip technology 4. Aromatase activity 5. In vivo testing 1. Rats, Monkeys, Mallard, Quail

47 Alterations in Membrane Properties Exposed to PFC can be used to Determine Relative Potencies

48 Why Study Membrane Effects? Structure of Perfluorinated compounds: Similarity to endogenous fatty acids Physico-chemical Properties of Perfluorinated Compounds: Surface Active Agents Effects of Perfluorinated Compounds: GJIC Bioassay interactions all suggest membrane related effects.

49 What is Gap Junction Intercellular Communication?

50 Experimental Design I GJIC measured by scrape loading dye technique Cell grow to confluence (monolayer) Expose to PFC Apply lucifer yellow dye Make scrapes with blade Discard dye after 3min Observe under fluorescent microscope Analyze image with gelexpert

51 Experimental Design II Chemicals: PFOS (C8), PFOSA (C8), PFHS (C6), PFBS (C4). Two cell lines: WB ---- rat liver epithelial cell line; CDK ---- dolphin kidney epithelial cell line; Three experiments: dose response experiment time course experiment recovery experiment

52 WB cell Solvent Control WB cell exposed to 12.5 um PFOS WB cell exposed to 50 um PFOS WB cell exposed to 160 um PFOS

53 In vivo inhibition of GJIC by PFOS A3 C1 Control PFOS, 5mg/kg/d for 21 days

54 Effects of Perfluorinated Compounds on GJIC Compound NOEL EC 50 Dolphin CDK Cells Rat WB Cells PFOS (C8) 6.25uM (3.125ppm) 20.96uM (10.48ppm) PFOSA (C8) 6.25uM (3.125ppm) 18.60uM (9.3ppm) PFHS (C6) 50uM (20ppm) 91.5uM (36.6ppm) PFBS (C4) >160uM (48ppm) none Compound NOEL EC 50 PFOS (C8) 6.25uM (3.125ppm) 25.98uM (12.94ppm) PFOSA (C8) 6.25uM (3.125ppm) 36.60uM (18.30ppm) PFHS (C6) 25uM (10ppm) 82.15uM (32.86ppm) PFBS (C4) >160uM (48ppm) none

55 Structure-Activity GJIC-In Vitro Rat * * Dolphin NOEL (WB) EC-50 (WB) NOEL (DK) EC-50 (DK) * * PFOS PFOSA PFHS PFBS * Infinite (No Effects Observed)

56 GJIC Summary PFOS inhibits GJIC in a dose-dependent fashion; The length of the carbon chain, but not the functional group determines the GJIC inhibitory potency. The inhibitory effect is neither species- nor tissuespecific. The inhibition of GJIC by PFOS occurred in a short period of time, and is rapidly reversible. Post-transcriptional modification of gap junctional protein(s) may be involved. No evidence that PFOS is a carcinogen.

57 TEQ Approach Relative Potency (ReP) Relative Accumulation n { } TEQ= ReP Con i TEQ = Toxic Equivalents of mixture n = number of components in the mixture

58 RePs Relative Potencies Species- and Endpoint-Specific Rank-orders Similar Within Classes

59 Relative Potencies Toxic Units approach Compound PFOS PFOSA PFHS PFBS TEF NA (Infinite) Based on EC-50 of PFOS in GJIC Assay DKC (Hu et al., Tox. Sci. 68:

60 Risk Assessment-Mink & Otter

61 Average Concentrations-Mink Location (State, USA) PFOS (mg/kg, ww) PFOA (mg/kg, ww) Ratio PFOA/PFOS Illinois Mass S. Carolina 1.7 <0.02 <0.012 Lousiana 0.14 <0.02 <0.14 Kannan et al., 2002, Environ. Sci. Technol. 36:

62 Risk Assessment-PFOS Mink Location (State USA) Illinois HQ MOS Mass S. Carolina Louisiana TRV = mg/kg, ww liver, Depending on Uncertainty Factors

63 Average Concentrations-River Otter Location (State, USA) PFOS (mg/kg, ww) PFOA (mg/kg, ww) Ratio (PFOA/PFOS) Washington (8) 0.19 ( ) < * Oregon (10) 0.23 ( ) < * *Maximum possible

64 Risk Assessment- PFOS River Otter Location (State, USA) Washington Oregon HQ MOS TRV = mg/kg, ww liver, depending on Uncertainty Factors

65 ACKNOWLEDGEMENTS 3M Company, Minnesota Provided Funding & Analytical Support Several Individuals Provided Field and Lab Assistance Data have been submitted to US EPA and OECD

66 Questions??????? S E T A C

67 Thank You John P. Giesy Dept. Zoology East Lansing, Michigan, 48824, USA Tel: (517) Fax: (517) Web Site: