Analytical Challenges and Environmental Relevance of ... QUASIMEME PFC... · Analytical Challenges and Environmental Relevance of Branched and Linear Isomers of Perfluorooctane Sulfonate

Analytical Challenges and Environmental Analytical Challenges and Environmental Relevance of Branched and Linear Isomers of Relevance of Branched and Linear Isomers of

Perfluorooctane Sulfonate and Related Perfluorooctane Sulfonate and Related Fluorinated Compounds in BiotaFluorinated Compounds in Biota

Robert J. LetcherRobert J. Letcher1,21,2, Shaogang Chu, Shaogang Chu11, , Wouter A. GebbinkWouter A. Gebbink22, Jason O, Jason O’’BrienBrien33

and Sean Kennedyand Sean Kennedy11

QUASIMEME Workshop, “Analysis of Perfluorinated Compounds”, Amsterdam, The Netherlands, Oct. 14-15, 2009

1 Wildlife Toxicology Section, Ecotoxicology and Wildlife Health Division, Wildlife and Landscape Science Directorate, Science and Technology Branch, Environment Canada, National Wildlife Research Centre, Carleton University, Ottawa, Ontario, Canada

2 Department of Chemistry, Carleton University, Ottawa, Ontario, Canada3 Department of Biology, University of Ottawa, Ottawa, Ontario, Canada

(contact e(contact e--mail: mail: [email protected]@ec.gc.ca))

C

O

OHS

O

OH

O

CF3(CF2)n S

O

N

O

Perfluorocarboxylic acids(e.g., PFCAs, n = >7 - 14) Perfluorosulfonic acids

(e.g., PFSAs, n = >5 - 9)

Perfluorosulfonamides(PFOSA, R=H, CH3, CH2CH3; e.g., n=7)

S

O

N

O

CH2CH2OH

Perfluorosulfonamidoethanols(PFOSE, R=CH3, CH2CH3; e.g., n=7)

CF3(CF2)nCF2CH2CH2OH CF3(CF2)n P

O

OH

OH

Examples of Fluorinated Surfactants Reported in (Biotic and Abiotic) Environmental Compartments

S

O

OPerfluorosulfonamidoacetic acids

(PFOSAA, R=H, CH3, CH2CH3; e.g., n=7)

Fluorotelomer alcohols(e.g., FTOH, n=4, 6 and 8)

Perfluorinated phosphonic acids(PFPAs, n = 5,7 and 9)

NCH2COH

O

CF3(CF2)n

CF3(CF2)n

CF3(CF2)n CF3(CF2)nR R

R

R

(M. Houde, J.W. Martin, R.J. Letcher, K. Solomon, D.C.G. Muir. 2006. Environ. Sci. Technol. 40:3463-3473) (C. Lau, K. Anitole, C. Hodes, D. Lai, A. Pfahles-Hutchens, J. Seed. 2007. Toxicol. Sci. 99:366-394)

Manufacture of PFCs

(Prevedouros et al., 2006 Environ. Sci. Technol. 40:32-44)

PFCs and Sources to the Environment

(Butt et al., 2009 Sci Total Environ. in review)

e.g., N-Et-FOSE (N-ethyl-perfluorooctane sulfoamidoethanol)aliphatic or branched hydrocarbon

(e.g. aliphatic alkyl sulfonamide alcohol)

Synthesis, Production and Environmental Fate of Perfluorinated Sulfonic Acids (PFSAs)

Electrochemical Fluorination, e.g., PFOS (perfluorooctane sulfonic acid)

F3C SO

NCH2CH2OH

F F

F F

F F F F

F F F F

F F O

CH2CH3

F3C SO

OH

F F

F F

F F F F

F F F F

F F O

PFOS

PFOSAA (perfluorooctane sulfonamido acetic acid)

F3C SO

F F

F F

F F F F

F F F F

F F O

fluorine; electric current; replacement of all H by F

+linear and branched isomers and shorter

chain length impurities

H3C SO

NCH2CH2OH

H H

H H

H H H H

H H H H

H H O

F3C SO

O-

F F

F F

F F F F

F F F F

F F O

PFOS (conjugate base)

CH2CH3

PFOSE (perfluorooctane sulfoamidoethanol)

F3C SO

NHCH2CH2OH

F F

F F

F F F F

F F F F

F F O

PFOSA (perfluorooctane sulfonamide)

F3C SO

NH2

F F

F F

F F F F

F F F F

F F O

- environmental release- atmospheric transport- environmental degradation (e.g., metabolism)

NHCH2COH

O

pKa = -3.27 (cal.)strong acid

PFSA and precursor production:- 3M Company from the 1950s to 2001- 3M voluntary phase-out of its PFOSF-based chemicals in favour of shorter chain-length compounds- PFOS has been directly produced in China since 2003 - influence on global emission patterns

PFOS: Precursor Transformation/Degradation

Degradation of Perfluorosulfonamides to PFOSAbiotic degradation as well as in vivo & in vitro biotransformation in dogs, rats & rainbow trout

(Arrendale et al., 1989; Grossman et al., 1992, Tomy et al., 2004, Xu et al., 2004)

No furtherdegradation

N-Et-FOSA (N-ethyl-perfluorooctane sulfoamide)

F3C SO

NH

F F

F F

F F F F

F F F F

F F O

CH2CH3

F3C SO

O-

F F

F F

F F F F

F F F F

F F O

L-PFOS?

PFOSA (perfluorooctane sulfonamide)

F3C SO

NH2

F F

F F

F F F F

F F F F

F F O

What about isomer-specific transformation (metabolism) and degradation?

e.g., N-Et-FOSE (N-ethyl-perfluorooctane sulfoamidoethanol)

F3C SO

NCH2CH2OH

F F

F F

F F F F

F F F F

F F O

CH2CH3

PFOS and Precursors Are Not a Singles Compound But Composed of Various Isomers

production of PFOS derivatives from linear alkyl precursors using ECF is not a clean process

commercial PFOS claimed to be generally >98% pure, but based on purity assessments using e.g., acid/base titration method after ion exchange with a strong acid resin

commercial PFOS contained approximately 86% PFOS; 70% L-PFOS and ~30% branched isomers (by 19F NMR):

(G. Arsenault, B. Chittim, A. Mclees, R. McCrindle, N. Riddell, B. Yeo. 2008. Chemosphere 70:616-625)

F3C SO

O-

F F

F F

F F F F

F F F F

F F OL-PFOS

P1MHpS P2MHpS P3MHpS

P4MHpS P5MHpS P6MHpS

F3CSO

F F

F F

F F F F

F F F

O

F3CSO

F F

F F

F F F

FF F FO

F3CSO

F F

F F

F F F

FF

F

FCF3

OCF3

F3C

F3CSO

F F

F F

F FF

FF F F

O

F3C

F3CSO

F F

F F

F F F

FF

F

FCF3

O

F3CSO

F F

F F

F FF

FF F F

O

F3C

O-O- O-

O-O-O-

P35DMHxSP44DMHxS P45DMHxS P55DMHxS

F3C SO

F

F F

F FF

F F

O

F3C

F3C

F3C SO

F F FF FF

F F

O

CF3

CF3

F3C SO

F

F

FF FF

F F

O

CF3

F3C

F3C SOFF

FF FF

F F

O

F3C

F3C

O- O- O- O-

(M. Houde, G. Czub, J.M. Small, S. Backus, X. Wang, M. Alaee, D.C.G. Muir. 2008. Environ. Sci. Technol.)

Limited Analytical and Environmental Study on Isomer-Specific PFOS; None For Other PFSAs

Up to this study, little known about environmental presence and fate of PFOS isomers

total of 6 isomers, 3 M-PFOS and 3 DM-PFOS isomers as well as linear L-PFOS detected in technical mixture, water, sediment and biota

L-PFOS >88% of total PFOS in all compared to technical PFOS (77%)

T-PFOS; 76% L-PFOS, 20% M-PFOSs and 3.6% DM-PFOSs

High L-PFOS suggested a reduced uptake of branched isomers, more rapid elimination of the branched isomers and/or a selective retention of the L-PFOS

Trophic magnification factor of L-PFOS (4.6) was greater than M-PFOS isomers (1.3), whereas DM-PFOS isomers showed no biomagnification

LC-ESI(-)-MS/MS;MRM: m/z = 499-99 (SO3F-), m/z = 499-80 (SO3

-)Confirmation by LC-Q-ToF-MS

- homogenized sample in polypropylene centrifuge tube- IS spike: sodium perfluoro-1-[1,2,3,4-13C4]octane sulfonate (MPFOS); 100 ng/mL

Vortex Mixing and Filtration

- KOH (10 mM) acetonitrile/water (80/20 v/v) extraction- centrifuge, add water and vortex- pH = ~4 with 2% aq. formic acid- Oasis SPE-WAX (weak anion exchange) clean-up; pre-conditioned

Egg, Liver or Plasma sample

GC-MS(ECNI)

SIM m/z 137, 480, 499, 503

- 0.5 g tetrabutyl ammonimum hydroxide (TBAH) in 5 mL diethyl ether- sonication for 0.5 h, centrifugation, upper phase isolation- 100 uL of TBAH mixture added to sample fraction- addition 1 mL of ether added, centriguged and frozen at -20C- unfrozen ether phase transferred, to dryness and reconstituted in 100 uL ether

Isomer-Specific PFOS Derivatization, GC-MS-Based Methodology

(S.G. Chu, R.J. Letcher. 2009. Anal. Chem. 81:4256-4262)(R.J. Letcher, S.G. Chu. 2009. Anal. Chem. 81:7856-7857)

- sample loaded on SPE- SPE washed with 2% aq. formic acid (1 mL), 2×1 mL H2O, 2×1 mL MeOH- PFOS isomer elution with 2×1 mL NH4OH (28-30%) in aq. MeOH (1/9 v/v)

GC-MS in-port derivatization

C8F17SO3H + (C4H9)4N+OH- → C8F17SO3-(C4H9)4N+ + H2O

C8F17SO3-(C4H9)4N+ → C8F17SO3C4H9 + N(C4H9)3

In-Port TBAH Derivatization, GC-MS(ECNI) Determination of Isomer-Specific PFOS


[C4H9SO3]-[C8F17SO3-F]-

[C8F17SO3]-[C8F17SO3+ C4H9]- [C5F11]-

[C8F17-SO3-F]-


Mass Chromatograms (In-Port TBAH Derivatization GC-MS(ECNI)) of Isomer-Specific PFOS

GREEN = m/z 137BLUE = m/z 480RED = m/z 499YELLOW = m/z 503

Butyl ether-PFOS technical mixture (potassium T-PFOS salt; Wellington Labs, Guelph, ON, Canada)

Butyl ether-PFOS isomer fraction from polar bear liver

Isomer-Specific PFOS in Representative Biological Environmental Samples


~65% L-PFOS + ~35% branched isomers

K+PFOS- from 3 suppliers by LC-ESI(-)-MS/MS: L-PFOS consistently 67-79%(G. Arsenault, B. Chittim, A. Mclees, R. McCrindle, N. Riddell, B. Yeo. 2008. Chemosphere 70:616-625)

Isomer-Specific PFOS Determination: LODs, MDLs and Recovery Efficiency


Unspiked

GC-MS(ECNI) mass chromatograms of spiked and unspiked pork fat with T-PFOS

Spiked


GC injection port temperature effect: MS(ECNI) response of L-PFOS after pyrolytic

alkylation of L-PFOS using TBAH

Isomer-Specific PFOS Determination: Effects of Injection Port Temperature and Volume Injected

Effects of the sample injection volume: MS(ECNI) response of L-PFOS after pyrolytic alkylation

of L-PFOS using TBAH

PFCs and Trends in Colonial Herring Gulls From the Great Lakes

Canada

L. Ontario

L. Erie

L. HuronL. Michigan

L. Superior

U.S.A.

AgawaAgawa RockRock

Fighting Is.Fighting Is.

ChannelChannel--Shelter Is.Shelter Is.

Chantry Is.Chantry Is.

TorontoToronto

Niagara R. (above the falls)Niagara R. (above the falls)

Gull Is.Gull Is.

2007-collected, individual eggs; completed characterization and spatial trends of 21 PFCs and precursors in herring gull eggs

Granite Is.Granite Is.

Big Sister Is.Big Sister Is.

Middle Is.Middle Is.

Double Is.Double Is. Strachan Is.Strachan Is.

Snake Is.Snake Is.

HamiltonHamilton

Port ColbornePort Colborne

Completed analysis:- 10-13 individual eggs from selected years and from all 15 CWS monitoring sites to be analyzed for 21 PFCs and precursors

Photo: Craig Hebert

- homogenized sample in polypropylene centrifuge tube- IS spikes: 18O- and 13C-labeled PFCs- 3 mL MeOH/H2O (50/50) with 2 nM NH4OAc

Vortex Mixing and Filtration

- KOH (10 mM) acetonitrile/water (80/20 v/v) extraction- centrifuge, add water and vortex- pH = ~4 with 2% formic acid- SPE-WAX (weak anion exchange) cartridge clean-up

Liver or Plasma sample

LC-ESI(-)-MS/MS (tandem quad.)

Quantification of:10 PFCAs (C6 – C15)4 PFSAs (C4, C6, C8,C10)3 FTUCAs (6:2, 8:2, 10:2)

Acidic Fraction Neutral fraction- reconstitute in MeOH- centrifugal filter (0.2 µm Nylon) at 6000 rpm for 20 min.

Quantification of:PFOSA, N-Me-FOSA3 FTOHs (6:2, 8:2, 10:2)

LC-APPI(-)-MS/MS (tandem quad.)

13C PFHxA, PFOA, PFNA, PFDA, PFUnA, PFDoA13C/18O PFHxS, PFOS

d-N-Me-FOSA13C 6:2, 8:2, 10:2 FTOH 13C 6:2, 8:2, 10:2 FTUCA

Non-Isomer-Specific PFC, LC-MS-Based Methodology

(S.G. Chu, R.J. Letcher. 2008. J. Chromatogr. A 1215:92-99)

(W.A. Gebbink, C.E Hebert, R.J. Letcher. 2009. Environ. Sci. Technol. 43:7443-7449)

Arithmetic Mean Total PFOS Concentrations (±SE) (LC-ESI(-)-MS/MS) in the Eggs of Herring Gulls From the

Great Lakes of North America

0100200300400500600

PFO

S (n

g/g

ww

)

ChicagoChicago DetroitDetroit ClevelandCleveland

BuffaloBuffalo

HamiltonHamilton

TorontoToronto

CANADACANADA

USAUSA

±SE (n=10-13 individual eggs per site)

p<0.03

northern colonies

x

xxx

x

x

x

x

xx

xx

x x

x

northern gull colonies


Arithmetic Mean Total PFOS Concentrations (±SE) (LC-ESI(-)-MS/MS) in the Eggs of Herring Gulls From the

Great Lakes of North America

0100200300400500600

PFO

S (n

g/g

ww

)


BuffaloBuffalo

HamiltonHamilton

TorontoToronto

CANADACANADA

USAUSA


p<0.03

northern colonies

0

200

400

600

800

1000

2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2

p < 0.009r = 0.65002

PFO

S (n

g/g

ww

)

(δ15N) Trophic Position

n = 15

Trop

hic

Posi

tion

0

1

2

3

4

5

Gra

nite

Is

Aga

wa

Roc

ks

Big

Sis

ter I

s

Gul

l Is

Dou

ble

Is

Cha

nnel

-She

lter I

s

Cha

ntry

Is

Figh

ting

Is

Mid

dle

Is

Port

Col

born

e

Nia

gara

Riv

er

Ham

ilton

Har

bour

Toro

nto

Har

bour

Snak

e Is

Stra

chan

Is

Aquatic food web

Mammals

Birds Garbage

Terrestrial inputin the diet

(C.E. Hebert et al. 2008. Ecol. 89:891-897)

Terrestrial and Aquatic Dietary Inputs Into Herring Gull Explain PFOS (and Other PFC)

Exposure

(δ15

N)

Arithmetic Mean Total PFOS (±SE) in the Eggs of Herring Gulls From the Great Lakes:

LC-ESI(-)-MS/MS vs. GC-MS(ECNI) Quantification

0100200300400500600

PFO

S (n

g/g

ww

)


BuffaloBuffalo

TorontoToronto

CANADACANADA

USAUSA

(W.A. Gebbink and R.J. Letcher. 2009. In preparation)

(S.G. Chu, R.J. Letcher. 2009. Anal. Chem. 81:4256-4262)GC-MS(ECNI)

LC-ESI(-)-MS/MS

HamiltonHamilton



Arithmetic Mean Concentrations (±SE) of Monotrifluoromethyl-Branched PFOS Isomers in the

Eggs of Herring Gulls From the Great Lakes

ChicagoChicago DetroitDetroitClevelandCleveland

BuffaloBuffalo

HamiltonHamilton TorontoToronto

CANADACANADA

USAUSA


ng/g

ww

0

2

4

6

8

10

12

14

16

P1MHpSP2MHpSP3MHpSP4MHpSP5MHpSP6MHpS


ChicagoChicago DetroitDetroitClevelandCleveland

BuffaloBuffalo

HamiltonHamiltonTorontoToronto

CANADACANADA

USAUSA


Arithmetic Mean Concentrations (±SE) of Ditrifluoromethyl-Branched PFOS Isomers in the Eggs of

Herring Gulls From the Great Lakesng

/g w

w

0.00

0.05

0.10

0.15

0.20

0.25

0.30

P35DMHxSP44DMHxSP45DMHxSP55DMHxS

~95% L-PFOS~4% M-PFOS~0.1% DM-PFOS

T-PFOS:~65% L-PFOS~33% M-PFOS~2% DM-PFOS

in vivo biotransformation to PFOS?

PFOSA

N-Et-FOSAN-Me-FOSEN-Et-FOSE

N-Et-FOSA PFOSA

PFOSA

in vivo biotransformation to PFOS?

?

PFOSA

Degradation of PFOSA as an Additional Source of PFOS In Herring Gull and Food Chain


* Martin et al., 2004; Furdui et al., 2007

0

1

2

3

4

Log

[PFO

S/PF

OSA

]

Superior

Michigan

Huron

ErieOntar

io

smelt

alewife

Superior

Michigan

Huron

ErieOntar

io

Herring gull eggs Lake TroutSpatial Trend*

Lake OntarioFood web*

InvertebratesMysis, Diporeia

Prey FishSlimy Sculpin, Alewife, Rainbow Smelt

Predator FishLake Trout

(Isomer-Specific) PFOS in Circumpolar Polar Bears

Arctic OceanCanada

USA Russia

Norway

Greenland

60°N

70°N

80°N

Iceland55°N

East Greenland (EG)(n=20)

Svalbard (SVAL)(n=10)

South HudsonBay (SHB)

(n=14)

Lancaster / Jones Sound (LJS)

(n=13)

Davis Strait (DS)(n=8)

Beaufort Sea (BS) (n=38)

Bering-Chukchi Sea (BCS) (n=10)

Western HudsonBay (WHB)

(n=11)

Baffin Bay (BB)(n=14)

Foxe Basin / Gulf of Boothia (FB/GB)

(n=6)

E. Peacock

• Years: 2006-2008 (2005-2007 Bering-Chukchi) • n: 6–38 per zone (total 134)• Sex: ♀ and ♂; Age: 3+ yr• Season: Fall/Winter• liver (plasma for SVAL bears): plasma-liver conversion of

PFC levels, and males/females treated as a single group

• Years: 2006-2008 (2005-2007 Bering-Chukchi) • n: 6–38 per zone (total 134)• Sex: ♀ and ♂; Age: 3+ yr• Season: Fall/Winter• liver (plasma for SVAL bears): plasma-liver conversion of

PFC levels, and males/females treated as a single group

(Letcher et al. 2009. In preparation)

Photo: E. Peacock

Isomer-Specific PFOSs in Polar Bears(Letcher et al. 2009. In preparation)

Arctic OceanCanada

USA Russia

Norway

Greenland

60°N

70°N

80°N

Iceland55°N

East Greenland (EG)(n=8)

Svalbard (SVAL)(n=5)

Nunavut (NU)(n=10)

Beaufort Sea (BS) (n=17

Bering-Chukchi Sea (BCS) (n=5)

P1MHpSP2MHpSP3MHpSP4MHpSP5MHpSP6MHpS

~99% L-PFOS~<1% M-PFOS0% DM-PFOS

T-PFOS:~65% L-PFOS~33% M-PFOS~2% DM-PFOS

a) Sex/Reproductive status

b) Age

c) Seasonal fasting/nutritional status/body condition (lipid content)

d) Diet

a) Sex/Reproductive status

b) Age

c) Seasonal fasting/nutritional status/body condition (lipid content)

d) Diet

Factors Affecting PFOS Trends in Polar BearsFactors Affecting PFOS Trends in Polar BearsFactors Affecting PFOS Trends in Polar Bears

- Other genetic and physiological (e.g. metabolic) variation, environmental or ecological factors (e.g., associated with Arctic warming)

- Other genetic and physiological (e.g. metabolic) variation, environmental or ecological factors (e.g., associated with Arctic warming)

1. Inter-population variation: 1. Inter-population variation:

AMAP, 2004

2. Inter-individual variation: 2. Inter-individual variation:

a) Regional contamination-emissions + LRT (atmos. + oceanic) to the Arctic

a) Regional contamination-emissions + LRT (atmos. + oceanic) to the Arctic

Norstrom et al. 1998; Henriksen et al. 2001; Polischuk et al. 2002Norstrom Norstrom et al.et al. 19981998; Henriksen ; Henriksen et al.et al. 20012001; Polischuk ; Polischuk et al.et al. 20022002

Various concentrations of PFOS technical mixture were injected into the air cell of domestic chicken eggs prior to incubation

Eggs were incubated until pipping, at which time embryos were euthanized and livers were excised

Hepatic PFOS concentration was determined by liquid chromatography-mass spectrophotometry

RNA was extracted from liver tissues using Trizol and reverse transcribed into cDNA

Relative quantity of PPARα regulated genes was determined using real-time reverse transcription PCR

PPARα regulated genes were:Peroxisomal acyl-CoA oxidase (ACOX)Bifunctional enzyme (BIEN)

Liver fatty acid binding protein (L-FABP)Peroxisomal 3-ketoacyl thiolase (PKT)

(J.M. O'Brien, A.C. Carew, S.G. Chu, R.J. Letcher, S.W. Kennedy. 2009. Comp. Biochem. Physiol. Part C.149:524-530)

Regulates genes involved in:-peroxisome proliferation-lipid metabolism-cholesterol metabolism-cell cycle

PFOS Isomer-Specific Toxicokinetics and Effects on Gene Expression in Chicken Eggs Injected With Technical PFOS

Liver PFOS Concentration of Exposed Embryos Increases With Dose

0.01

0.1

1

10

100

untreated DMSO 0.1 5 100Dose Group (ug/g)

Tota

l PFO

S (u

g/g

ww

)

(J.M. O'Brien, A.C. Carew, S.G. Chu, R.J. Letcher, S.W. Kennedy. 2009. Comp. Biochem. Physiol. Part C. 149:524-530)

Significant increase (p<0.05)±SE (2×3 sets of replicates)

Conclusions

New in-port GC-MS based method allows for complete quantitative assessments of all geometrical isomers known to be present in T-PFOS mixtures

Isomer-specific processes that favour L-PFOS occur in abiotic and more so in biotic compartments (e.g., wildlife and fish); selective uptake, bioconcentration, degradation, metabolism and/or bioaccumulation

As typified by Great Lakes herring gulls and circumpolar polar bears; highly conserved PFOS isomer pattern and enriched L-PFOS suggest isomer-specific processes occurring lower on the food web

PFOSA metabolism appears to occur in gulls and bears as a source of bioaccumulative PFOS: Isomer-specific?

Isomer-specific PFOS, as well as other precursors and PFCs (e.g., PFCAs and PFPAs), are currently not or under studied

Acknowledgements and Funding

Natural Sciences and Engineering Research Council (to R.J.L.)

Environment Canada’s Chemicals Management Plan (CMP)

Northern Contaminants Program (to R.J.L.)

Canadian International Polar Year (to R.J.L.)

Molson Foundation (to R.J.L.)

Danish Department of the Arctic Environment; Norwegian Polar Institute

Thank you

F3C SO

O-

F F

F F

F F F F

F F F F

F F O

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Analytical Challenges and Environmental Relevance of ... QUASIMEME PFC... · Analytical Challenges and Environmental Relevance of Branched and Linear Isomers of Perfluorooctane Sulfonate