perfluorinated compounds: an overview of …giesy/pfcs/hamburg-presentation-4-30-04.pdfzoology dept....

Zoology Dept. & National Food Safety and Toxicology CenterMichigan State University

Perfluorinated Compounds: An Overview of Environmental

and Analytical Issues

John P. Giesy*, P. D. Jones & J. L. Newsted, K. Kannan

Zoology DepartmentNational Food Safety and Toxicology Center

Institute of Environmental ToxicologyMichigan State University


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 • e-mail message to the docket staff at [email protected] or

call 202-566-0280. The docket staff will send the index and anydocuments to you via e-mail.

• Additional details regarding the Pollution Prevention and Toxics Docket Can be Obtained online at http://www.epa.gov/epahome/dockets.htm.

• Send a blank CD to the public reading room at the following address:

Public Reading Room, Room B102EPA West Building1301 Constitution Avenue, NWAttn: OPPT Docket Staff

Web Site: http://www.msu.edu/user/giesy


SAMPLING LOCATIONS


Previously Published Papers-I• Giesy, J. P. and K. Kannan. 2001. Global Distribution of Perfluorooctane

Sulfonate and Related Perfluorinated Compounds in Wildlife. Env. Sci. Technol. 35:1339-1342.

• Kannan, K., et al. 2001. Perfluorooctane Sulfonate and Related Fluorinated Organic Chemicals in Marine mammals. Environ. Sci. Technol. 35:1593-1598.

• Kannan, K., et al. 2001. Perfluorooctane Sulfonate in Fish-Eating Water Birds Including Bald Eagles and Albatrosses. Environ. Sci. Technol.35:3065-3070.

• Kannan, K., et al. 2002. Perfluorooctane Sulfonate in Oysters (Crassostreavirginica), from the Gulf of Mexico and Chesapeake Bay, USA. Arch. Environ. Contamn. Toxicol. 42:313-318.

• Kannan, K., et al. 2002. Perfluorooctane Sulfonate and Related Fluorinated Hydrocarbons in Marine Mammals, Fish and Birds from Coasts of the Baltic and The Mediterranean Seas. Environ. Sci. Technol. 36:3210-3216.


Previously Published Papers II• Kannan, K., et al. 2002. Concentrations of Perfluorinated Acids in Livers of

Birds from Japan and Korea. Chemosphere, 49:225-231.• Kannan, K., J.L. Newsted, R.S. Holbrook and J.P. Giesy. 2002.

Perfluorooctanesulfonate and Related Fluorinated Hydrocarbons in Mink and River Otters from the United States. Environ. Sci. Technol. 36:2566-2571.

• Giesy, J.P. and K. Kannan. 2002. Perfluorochemical Surfactants in the Environment. Environ. Sci. Technol. 36:146A-152A.


Structures of Sulfonated Fluorochemicals

POSF: Perfluorooctane sulfonylfluoride

O

C8F17 S - F

O

PFOSA: Perfluorooctane sulfonamide

O

C8F17 S - NH2O


Structures of Sulfonated Fluorochemicals

PFOA or POAA: Perfluorooctanoic acid

O

C7F15 CO-

Also referred to as C8, Other homologues include C7and C9-12

O

C8F17 S - O-

O

PFOS: Perfluorooctane sulfonate

C C C C C C C C SO

O O

F

FF

F F

FF

F F

FF

F F

F F

F F


Compounds of Interest

C8F17SO3-Perfluorooctane sulfonate (PFOS)

C12F23COO-Perfluorododecanoate (C12)C11F21COO-Perfluoroundecanoate (C11)C9F19COO-Nonadecafluorodecanoate (C10)C8F17COO-Heptadecafluorononoate (C9)C7F15COO-Pentadecafluorooctanoate (C8; PFOA)C6F13COO-Tridecafluoroheptanoate (C7)FormulaCompound


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


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


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


Methods - 2• HPLC is good for separation, but PFCs do

not contain chromophores• HPLC followed by fluorescence detection

has been applied to perfluorocarboxylicacids but sensitivity is poor

• 19F - NMR has been used, but is not very sensitive and is not quantitative– Requires pre-concentration and cleanup


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, 766-770.


Ion Pairing Methods For PFOS/PFOA

Ion-pair extraction (TBA/MTBE) & Filter

Betasil- C 18 column separation

HPLC-ESMS/MS (triple quadrapole ; ES-ve)

Sample-Homogenize

: Florisil/carbon column

Monitor ions: PFOS: 499 80, 99, 130PFOA: 413 369, 219, 169, 119

TBA=tetrabutylammonium hydrogen sulfate; MTBE=methyl tert-butyl etherInternal standard, 1H,1H,2H,2H-perfluorooctane sulfonate (THPFOS)


Criteria for the Selection of Analytical Methods

• Sufficiently Sensitive• Accurate• Reproducible• Transportable• Simple• High Throughput• Minimize Interferences


SPE Methods For PFOS/PFOA in Water50 mL H20

C18 SPE Cartridge

Elute with 0.5 mL Methanol

Spike with Recovery Internal Standard


Compounds Studied

499Perfluorooctane sulfonate (PFOS)613Perfluorododecanoate (C12)563Perfluoroundecanoate (C11)513Nonadecafluorodecanoate (C10)463Heptadecafluorononoate (C9)413Pentadecafluorooctanoate (C8; PFOA)363Tridecafluoroheptanoate (C7)

Molecular Weight

Compound


Instrumentation

• Liquid Chromatography– HP Series 1100– Keystone Betasil C18 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 .


Matrix Spikes For PFOS/PFOA in Water

• 50 pg/mL• 80 pg/mL• 500 pg/mL


Unextracted Standard

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

Time (min)

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

PFHS (399 > 99) 6.43

PFOSA (498 > 78) 7.42

PFOS Quantification (499 > 99) 6.80

PFOS Confirmation (499 > 80) 6.806.74

PFOA (413 > 169) 6.63


SPE Standard Extracted from Water

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

Time (min)

0

100

%

0

100

%

0

100

%

0

100

%

PFOSA (498 > 78) 7.22


PFOS Confirmation (499 > 80) 6.83

PFOA (413 > 169) 6.69


Spiked Water Sample Extracted by SPE

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

Time (min)

0

100

%

0

100

%

0

100

%

0

100

%

PFOSA (498 > 78) 7.16

5.563.15

7.227.488.09

7.74 9.408.57 9.75


0.750.36 2.86

1.391.682.60

6.166.064.654.243.28 5.39

7.67

7.479.278.668.08

9.729.78

PFOS Confirmation (499 > 80) 6.746.67

2.091.290.59 3.212.92 5.873.82 4.814.33 5.33

6.80

6.877.797.19 8.858.05

9.04 9.75

PFOA (413 > 169) 6.63

6.315.705.030.58 4.68 7.827.08 8.118.74 9.55 9.74


SPE Methods For PFOS/PFOA in WaterRecoveries (%)

125 (27)[94-147]

122 (31)[67-151]

103 (13.4)[77-146]

PFOA PFOSAPFOS

Mean (SD)

[Range]


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


SPE Methods For PFOS/PFOA in WaterResults

• 67 Samples analyzed from N. A. Great Lakes and Tributaries

• No Detectable Concentrations of PFOS, PFOSA, PFOA


SPE Methods For PFOS/PFOA in Biota

Extract 8 mL H20 + 40 mL Acetonitrile

Sample-2.0 mL Serum or 1 g tissue Homogenize

C18 SPE Cartridge (0.5 g)

Dilute with 350 ml H20

Extract 20 min; Centrifuge 20 min 3,500 rpm

Elute with 0.5 mL Methanol


SPE-Extracted Mink Liver

2.00 4.00 6.00 8.00 10.00

Time (min)

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

PFHS (399 > 99) 6.656.42

6.306.010.15 1.21 3.322.231.40 2.52 4.413.51 5.34

7.197.387.77 9.698.677.99 9.59

PFOSA (498 > 78) 7.44

6.80

PFOS Quantification (499 > 99)6.77


PFOA (413 > 169)6.43

6.376.18

6.055.732.050.45 1.700.67 5.153.553.072.85 4.35

6.536.636.767.307.84 9.329.168.10 9.51


SPE-Extracted Amphipod

2.00 4.00 6.00 8.00 10.00

Time (min)

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

PFHS (399 > 99) 7.07

8.06 9.53

PFOSA (498 > 78) 8.18

6.81 9.34

PFOS Quantificaqtion (499 > 99) 7.50

7.22


PFOA (413 > 169) 7.30

6.606.310.51

8.787.498.58 9.51 9.70


SPE Sensitivity for PFOA and PFOSBlood Plasma and Serum

1.0PFOS0.01C120.01C110.1C100.25C9

0.1 (0.52 min obs)C8 (PFOA)0.01C7

LOQ (ng/mL) Compound


SPE Methods Blood-Precision

5.90.9996950.1-30.7C72.80.9997360.52-31.0C8 (PFOA)5.10.9998470.25-30.4C92.80.9996950.1-30.3C101.20.9995850.1-30.9C115.90.9997590.1-31.0C122.20.99972211.0-30.0PFOS

% RSDR2Cal Range(ng/mL)

Analyte


SPE Methods Blood-Spike Recoveries

<LOQ75102C7<LOQ8981C8 (PFOA)<LOQ8097C9<LOQ8391C10<LOQ7890C11<LOQ84101C12<LOQ84102PFOS

Matrix Blank

Spike (#2)(%)

Spike (#1)(%)

Analyte


Concentrations in Blood 1Plasma (ng mL)

0.290[0.016]0.100<0.010C73.873.072.61<0.52C8 (PFOA)

0.5350.5850.435<0.025C90.1600.170[0.0127]<0.010C10

[0.071]0.135[0.049]<0.010C11[0.024][0.022]3[0.036]<0.010C12

18.315.611.12<1.0PFOS

Golden West(TCR-684)

Innovative(TRC-683)

Lampire(TCR-685)

Chinese(TCR-674)

Analyte

1Plasma centrifuged; 2 < LOQ; ng/ml = ppb; 3[ ] = Concentrated SPE method;


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

• PFOS 4X greater than PFOA 4.04.8PFOS/PFOA

0.25(0.067)

0.21(0.016)1

PFOA/PFOS

SerumPlasma

1 MEAN (SD)

Ratios


Comparison of Solid Phase (SPE) and Ion Pairing (IP) Techniques

Great Lakes Trophic Level Study


LOQ for SPEGreat Lakes Trophic Levels

• LOQ = 2X SNR and <30% RSD for MSD• PFOS 499 80Da transition is most

sensitive, but often affected by an interference

• Use 499 99 Da transition; Less sensitive but no interference


7.57.4419IP

0.2 - 1.00.2 - 5.00.2 - 1.00.2 - 2.0SPE

PFHSPFOAPFOSAPFOSMethod

LOQ Comparison SPE vs. IP

ng/g, ww (ppb)


Trophic-Level Study-PFOS

IPSPE

<LOQ99 (87)Zebra Mussel89 (1)<LOQS.M. Bass Muscle

113 (3)76 (19)Round Goby Muscle<LOQ110 (33)Pseudo Feces204 (2)154 (5)Mink Liver

53136 (26)L.M. Bass Muscle<LOQ133(35)Crayfish Muscle<LOQ68 (13)Benthic Algae<LOQ125 (3)Amphipod

Recovery (%) (+/-RSD)Matrix


Trophic-Level Study-PFOA

IPSPE

<LOQ<LOQZebra Mussel<LOQ<LOQS.M. Bass Muscle47 (3)<LOQRound Goby Muscle<LOQ<LOQPseudo Feces120 (3)22 (7)Mink Liver<LOQ<LOQL.M. Bass Muscle<LOQ432 (143)Crayfish Muscle<LOQ76 (43)Benthic Algae<LOQ<LOQAmphipod



Trophic-Level Study-PFHS

IPSPE

655 (120)125 (22)Zebra Mussel232 (8)<LOQS.M. Bass Muscle184 (13)65 (18)Round Goby Muscle<LOQ150 (10)Pseudo Feces176 (0)53 (3)Mink Liver17503<LOQL.M. Bass Muscle304 (5)66 (4)Crayfish Muscle322 (35)99 (1)Benthic Algae774 (53)155 (12)Amphipod



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)


2,518(5,667)

84(37)

98(14)

114.8(56)

IP

102(39)

177(181)

28(7.5)

112.6(28)

SPE

PFHSPFOAPFOSAPFOSMethod

Mean RecoveriesComparison SPE vs. IP

Mean % (SD)


• Critical Mechanism of toxic action of PFOS is unknown

• Preferentially retained in plasma and liver– binds to protein

Toxicity


Toxicity Summary - 1

Uncouple oxidativephosphorylation

PFOS, PFOSA

Inhibit Gap junctionalintercellular communication

PFOA, PFDA, PFOS

Peroxisome proliferation andorgan-specific DNADammage

PFOA, PFDA, PFBA

Induce liver microsomalcarboxylase RL4

PFOA, PFDA, PFOS

PFOA: Perfluorooctanoate; PFDA Perfluorodecanoate; PFBA: Perfluorobutyricacid; PFOS: Perfluorooctane sulfonic acid




Hypophagia, weight loss,bradychardia, hypothermia andreduced motor activity

PFDA

Reduced T3 and T4 concentrations PFDAInduction of cytochrome P450;ECOD,AcylglycerophosphocholineAcyltransferase, acyl CoA-hydrase

PFOA

Induced liver phospholipase CInhibited phosphocholine

PFDA



Dechreased serumcholesterol

PFOA, PFOS

Increased faty acid and acylCoA binding proteins in ratliver

PFDA

Increased proteinkinase Cactivity, decreased Acyl CoAsynthase activity

PFOS



1. Effects on Membranes1. Fluidity2. Mitochondrial potential3. Gap Junctions

2. Binding to Proteins1. Hormone displacement2. HPLC/MS

3. Effects on Gene Expression1. Differential display2. Gene chip technology

4. Aromatase activity5. In vivo testing

1. Rats, Monkeys, Mallard, Quail

Toxicity TestingToxicity Testing


Alterations in Membrane Properties Exposed to PFC can be used to Determine

Relative Potencies


Why Study Membrane Effects?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.


What is Gap Junction Intercellular Communication?


Observe under fluorescent microscope

Analyze imagewith gelexpert

Cell grow to confluence(monolayer)

Expose to PFC Apply lucifer yellow dye

Make scrapes with blade

Discard dye after 3min

GJIC measured by scrape loading dye technique

Experimental Design I


Experimental Design IIExperimental 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 experimenttime course experimentrecovery experiment


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


A3A3 C1C1A3A3 C1C1

In vivo inhibition of GJIC by PFOS

Control PFOS, 5mg/kg/d for 21 days


Effects of Perfluorinated Compounds on GJIC

Compound NOEL EC 50

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 50PFOS (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

Dol

phin

CD

K C

ells

Rat

WB

Cel

ls


Structure-ActivityGJIC-In Vitro

020406080

100120140160180200

NOEL (WB) EC-50 (WB) NOEL (DK) EC-50 (DK)

PFOSPFOSAPFHSPFBS

* Infinite (No Effects Observed)

*

**

*

888 88 8 8 84 4 4 46 66 6

Rat Dolphin


•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 tissue-

specific.•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.

GJIC Summary


TEQ Approach

Relative Potency (ReP)Relative Accumulation

{ }TEQ ReP Coni

n

= ∗∑TEQ = Toxic Equivalents of mixturen = number of components in the mixture


RePs

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


Relative PotenciesToxic Units approach

NA (Infinite)PFBS

3.16PFHS

0.7PFOSA

1.0PFOS

TEFCompound

Based on EC-50 of PFOS in GJIC Assay DKC(Hu et al., 2002 . Tox. Sci. 68:429-436.


Risk Assessment-Mink & Otter


Average Concentrations-Mink

<0.14<0.020.14Lousiana

<0.012<0.021.7S. Carolina

0.040.0080.2Mass.

0.0140.021.4Illinois

RatioPFOA/PFOS

PFOA(mg/Kg, ww)

PFOS(mg/Kg, ww)

Location(State, USA)

Kannan et al., 2002, Environ. Sci. Technol. 36:2566-2571


Risk Assessment-PFOS Mink

83 – 5000.002 – 0.012Louisiana

7 – 410.024 – 0.14S. Carolina

50 – 3330.003 - 0.02Mass.

8.3 - 500.02 - 0.12Illinois

MOSHQLocation(State USA)

TRV = 12-72.5 mg/Kg, ww liver, Depending on Uncertainty Factors


Average Concentrations-River Otter

0.008*<0.008-0.0190.23(0.034-1.0)

Oregon(10)

0.10*<0.008-0.0190.19(0.025-0.42)

Washington(8)

Ratio(PFOA/PFOS)

PFOA(mg/Kg, ww)

PFOS (mg/Kg, ww)

Location(State, USA)

*Maximum possible


Risk Assessment- PFOS River Otter

50 -3330.003 – 0.02Oregon

63 – 3330.003 – 0.02Washington

MOSHQLocation(State, USA)

TRV = 12-72.5 mg/Kg, ww liver, depending on Uncertainty Factors


ACKNOWLEDGEMENTS

3M Company, MinnesotaProvided Funding & Analytical Support

Several Individuals Provided Field and Lab Assistance

Data have been submitted to US EPA and OECD


Questions ???????

S

E

T

A

C


Thank You• John P. Giesy• Dept. Zoology• Michigan State University• East Lansing, Michigan, 48824, USA• Tel: (517) 353-2000• Fax: (517) 432-1984• Email: [email protected]

Web Site: http://www.msu.edu/user/giesy

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