Saltzbart.Ilana@epamail.epa. gov 11/15/2006 04:16 PM To Lori E Stephans/AE/DuPont@DuPont cc Andrea V Malinowski/AE/DuPont@DuPont bcc Subject Fifth Extension Request granted in full History: This message has been replied to. With this e-mail, EPA grants, in full, DuPont's request for a fifth extension to reach agreement with EPA on the procedure(s) that DuPont will use to purify the Fluorotelomer Products, as required by Section I.E.2.a of Appendix A (conditionally granted by EPA on November 9, 2006)). DuPont will provide EPA with the data by March 7, 2006, and the parties will reach agreement by March 30, 2006. Ilana Saltzbart Attorney-Advisor, US EPA, OECA, OCE 1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460 Phone: 202 564 9935 Fax: 202 564 0035 NOTICE: This communication may contain privileged or other confidential information. If you are not the intended recipient, or believe you have received this communication in error, please delete the copy you received, and do not print, copy, retransmit, disseminate, or otherwise use the information. Thank You.

Ilana Saltzbart/DC/USE PA/US To Lori E Stephans 11/09/2006 08:50 <Lori.E.Stephans@USA.dupont.com> AM cc Andrea V Malinowski <Andrea.V.Malinowski@USA.dupont.c om> Subject Conditional Grant for Fifth Extension(Document link: Ilana Saltzbart)

Lori, In light of the Nov. 11 deadline, this e-mail will serve to conditionally grant your request for an extension. I will speak with Cathy regarding the new date that you have proposed and EPA either accept that date or identify a different date. Thanks. Ilana SaltzbartAttorney-Advisor, US EP A , OECA , O CE 1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460 Phone: 202 564 9935 Fax: 202 564 0035 NOTICE: This communication may contain privileged or other confidential information. If you are not the intended recipient, or believe you have received this communication in error, please delete the copy you received, and do not print, copy, retransmit, disseminate, or otherwise use the information. Thank You. Lori E Stephans <Lori.E.Stephans @USA.dupont.com> To Ilana Saltzbart/DC/USEPA/US@EPA 11/08/2006 04:08 cc PM Andrea V Malinowski <Andrea.V.Malinowski@USA.dupont.c om> Subject Re: deadline Biodegradation SEP-advice requested

IlanaI will be submitting via over night mail tomorrow a 5th request for extension for agreement to reach a procedure for purification. I hope the date I chose will give us plenty of time to complete the path forward experiments and come to agreement on a procedure. The goal of course would be to complete the work as soon as possible. Please let me know if you have any questions or concerns. We look forward to talking next week. Regards, Lori (See attached file: CD 2006 30 5th Request for Extension.pdf) Lori E. Stephans, Ph.D. Manager, Supplemental Environmental Programs Rm 4110, Jackson Laboratory Route 130, Chambers Works Deepwater, NJ 08023 856-540-3748 (office) 856-540-4077 (fax)856-341-0255 (cell) lori.e.stephans@usa.dupont.com This email message is for the sole use of the intended recipient(s) and may contain privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply email and destroy all copies of the original message.
Saltzbart.Ilana@e pamail.epa.gov To 11/06/2006 03:34 Lori E Stephans/AE/DuPont@DuPont PM cc Andrea V Malinowski/AE/DuPont@DuPont Subject Re: deadline Biodegradation SEP-advice requested

Lori, please submit a request for extension and use your best judgment regarding proposed extension dates. Ilana Saltzbart Attorney-Advisor, US EPA, OECA, OCE 1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460 Phone: 202 564 9935 Fax: 202 564 0035 NOTICE: This communication may contain privileged or other confidential information. If you are not the intended recipient, or believe you have received this communication in error, please delete the copy you received, and do not print, copy, retransmit, disseminate, or otherwise use the information. Thank You.Lori E Stephans <Lori.E.Stephans @USA.dupont.com> To Ilana Saltzbart/DC/USEPA/US@EPA 11/03/2006 09:46 cc AM Andrea V Malinowski <Andrea.V.Malinowski@USA.dupont.c om> Subject deadline Biodegradation SEP-advice requested

IlanaIn our path forward agreement dated 08.23.06, the following deadlines are expressed: "The deadline for the parties to reach agreement on the procedure(s) that DuPont will use to purify the fluorotelomer products is November 27, 2006. On or before November 11, 2006, DuPont must provide EPA with all reports generated pursuant to this proposal and all data associated with the work performed pursuant to this proposal." Since we are not complete even with the equivalency studies, how would you like me to proceed with the November 11 and November 27 deadlines. Shall I write a 5th request for extension? November 11 is actually next Saturday and I am in all day site safety training Tuesday, Wednesday and Thursday. I will need to handle a formal request for extension on Monday or Friday. Thanks, Lori

Lori E. Stephans, Ph.D. Manager, Supplemental Environmental Programs Rm 4110, Jackson Laboratory Route 130, Chambers WorksDeepwater, NJ 08023 856-540-3748 (office) 856-540-4077 (fax) 856-341-0255 (cell) lori.e.stephans@usa.dupont.com This email message is for the sole use of the intended recipient(s) and may contain privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply email and destroy all copies of the original message. This communication is for use by the intended recipient and contains information that may be Privileged, confidential or copyrighted under applicable law. If you are not the intended recipient, you are hereby formally notified that any use, copying or distribution of this e-mail, in whole or in part, is strictly prohibited. Please notify the sender by return e-mail and delete this e-mail from your system. Unless explicitly and conspicuously designated as "E-Contract Intended", this e-mail does not constitute a contract offer, a contract amendment, or an acceptance of a contract offer. This e-mail does not constitute a consent to the use of sender's contact information for direct marketing purposes or for transfers of data to third parties. Francais Deutsch Italiano Espanol Portugues Japanese Chinese Korean http://www.DuPont.com/corp/email_disclaimer.html This communication is for use by the intended recipient and contains information that may be Privileged, confidential or copyrighted under applicable law. If you are not the intended recipient, you are hereby formally notified that any use, copying or distribution of this e-mail, in whole or in part, is strictly prohibited. Please notify the sender by return e-mail and delete this e-mail from your system. Unless explicitly and conspicuously designated as "E-Contract Intended", this e-mail does not constitute a contract offer, a contract amendment, or an acceptance of a contract offer. This e-mail does not constitute a consent to the use of sender's contact information for direct marketing purposes or for transfers of data to third parties. Francais Deutsch Italiano Espanol Portugues Japanese Chinese Korean http://www.DuPont.com/corp/email_disclaimer.html [attachment "CD 2006 30 5th Request for Extension.pdf" deleted by Ilana Saltzbart/DC/USEPA/US]Saltzbart.Ilana@epamail.epa. gov 11/22/2006 04:09 PM To Lori E Stephans/AE/DuPont@DuPont cc Lynch.David@epamail.epa.gov bcc Subject Next Steps in the Purification Path Forward For Follow Up: Normal Priority History: This message has been forwarded. Lori, At this time, EPA believes that the most prudent course of action is for Exygen to perform all of the analytical work for the control experiments and the sparge for Group 1, Substance 3 and Group 3, Substance 2, under the terms of the August 23, 2006 Purification Path Forward Agreement. However, based upon the information and data that EPA has received thus far pertaining to measuring PFOA in the control systems, EPA believe it is necessary for Exygen to demonstrate its accuracy in measuring PFOA. Towards that end, we would like Exygen to propose a design for an experiment that demonstrates Exygen's accuracy in measuring PFOA. Exygen should submit this to EPA (through you) by COB Friday, December 1. Because Exygen agreed that it would perform a second round of equivalence testing at no extra charge to EPA, it is EPA's expectation that the same term would apply to this experiment to demonstrate accuracy since EPA is not requesting RAS and Exygen to go through a second round of equivalence testing. With regard to spike recovery, EPA does not have sufficient data from Exygen to conclusively demonstrate that with the SPE procedure, that Exygen has achieved acceptable spike recovery (i.e., within a range of 75% -- 110%). In order to conclusively demonstrate that Exygen has achieved acceptable spike recovery, EPA requests either: 1) additional data from the work that Exygen has already performed or 2) additional work that clearly demonstrates that Exygen has achieved acceptable spike recovery. I will be out of the office until Wednesday, November 29. If you have any questions pertaining to this e-mail before I return, please contact David Lynch at lynch.david@epa.gov. Have a wonderful holiday. Ilana Saltzbart Attorney-Advisor, US EPA, OECA, OCE 1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460 Phone: 202 564 9935 Fax: 202 564 0035 NOTICE: This communication may contain privileged or other confidential information. If you are not the intended recipient, or believe you have received this communication in error, please delete the copy you received, and do not print, copy, retransmit, disseminate, or otherwise use the information. Thank You.RESEARC H Precis e Research . Prove n Results . Novembe r

28 , 200 6 Lor i E . Stephans , Ph.D . Manager , Supplemental Environmenta l Program s Rm4110 , Jackso n Laborator y Rout e 130 , Chamber s Work s Deepwater , N J 0802 3 Dea r Lori : I n respons e t o lian aSaltzbart' s emai l date d11/22/0 6 concernin g Exyge n Research' s nee d t o demonstrat e accurac y i n recoverin g PFO A fro mth e po t sample s b y SPE , th e followin g i s proposed : Exyge n propose s t o d o a1-da y study , fre e o f

charg e t o th e EPA , t o verif y recoverie s i n th e po t sampl e solutions . Thi s wil l satisf y th e EPA' s reques t

fo r additional wor

k that clearl y demonstrate s tha t Exyge n can achiev e acceptabl e spike recoveries . Th e verificatio n stud y wil l b e performe do n th e secon dshipment o f po t sample s receive dat Exyge n Researc h o n Augus t 15 , 2006 . Thes e sample s are th e one s dilute db y Pete r Stchur 100 0 time s an dkep t

at refrigerate dcondition s fo r th e entir e stud y (includin g shipping) . TL F1009 4 E l 11184-48 A LC/MS/M S (L9440-l)--Exyge n Result=No t Detecte d TL F1009 4 E l 11184-48 B LC/MS/M S (L9440-2)--Exyge n Resul t = 2. 1 ng/mL TL F1009 4 E l 11184-48 C LC/MS/M S (L9440-3)»Exyge n Resul t = 2 1 ng/m L Eac h sampl e abov e wil l b e spike di n triplicat e at 3 level s (at

-leve l foun di n sample , -1/ 2 leve l foun di n sampl e an d~2 x th e leve l foun di n th e sample) . Th e contro l sampl e (48A ) wil l b e spike dat

th e same level s a s 48B . A portio n o f th e sampl e wil l the n b e dilute dt o 4 0 mL an db e pu t

throug h th e SP E extractio n an dLC/MS/M S analysi s procedur e liste di n Exyge n Metho d01M-008-04 6 Revisio n 2 . Thi s wil l giv e 2 7 spik e recovery value s to demonstrat e accuracy . I f th e 2 7 spike recovery value s obtaine dar e betwee n th e EPA' s acceptabl e rang e o f

75-11 0 % the n Exyge n wil l hav e prove n tha t acceptabl e spike recoverie s can b e obtaine dusin g th e SP E procedur e o n th e po t samples . Al l result s an doriginal ra w dat afro mth e 1-da y verificatio n stud y wil l b e presente di n a repor t at th e conclusio n o f

th e trial .

p; 0 u Q ,^^305 8 Researc h Drive Kjsn a ^ m ^ ^ College , PA 16801 , US A J : 814.272.103

9 F : 814.231.158 0 exygen.co mRESEARC H ^.Precis e Research . Prove n Results . TITL E Metho do f

Analysi s fo r

th e Determinatio n o f

Perfluorooctanoi c Aci d(PFOA ) i n Wate r Revisio n 2 AUTHOR S Joh n Flaherty an dKare n Rish a DAT E ISSUE D Augus t 15 , 200 5 SPONSO R DuPon t Corporatio n P.O . BO X 8030 2 Wilmington , D E 19880-030 2 PERFORMIN G LABORATOR Y Exyge n Researc h 305 8 Researc h Driv e Stat e College , P A 1680 1 METHO D NUMBE R 01M-008-04 6 Revisio n 2 (V0001013 ) TOTA L NUMBE RO FPAGE S

2 8

J^305 f ^T^Sta l 

## V 305 8 Researc h Drive ' Stat e College , P A 16801 , US A .7:800.281.321 9 F : 814.272.101 9 exygen.co mExyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### MANAGEMEN T APPROVA L

6h n Flahert y Dat e Vice-Presiden t Exyge n Researc h



# ^ Richar dA . Grazzini , Ph.D . (j*&&* j Dat e I Presiden t Exyge n Researc h

Exyge n Researc h Pag e 2 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) 

### TABL E O FCONTENT S TITL E 1 MANAGEMEN T APPROVA L 2 TABL E O FCONTENT S 3 LIS T O FFIGURE S 4 1 . SUMMAR Y 5 2 . EXPERIMENTA L COMPOUND S 6 3 . CHEMICAL S AN D SUPPLIE S 6 

### 3.1 . CHEMICAL S 6 3.2 . STANDARD S 6 3.3 . EQUIPMEN TAN D SUPPLIE S 7 3.4 . SOLUTION S 8 

### 3.5 . PREPARATIO NO FSTANDARD S AN D FORTIFICATIO NSOLUTION S 8 3.5.1 . Stoc k

solutio n 8 3.5.2 . Fortificatio n Solution s 9 3.5.3 . Calibratio n Standard s 9 3.5.4 . Controllin g Standar dBia s 1 0 4 . METHO D 1 1 

### 4.1 . FLO WDIAGRA M 1 1 4.2 . SAMPL EPROCESSIN G1 1 4.3 . SAMPL EPREPARATIO N1 2 4.4 . EXTRACTIO N1 2 4.5 . QUANTITATIO N1 3 4.5.1 . LC/MS/M S Syste man dOperatin g Condition s (Electrospray ) 1 3 4.5.2 . Exampl e M S Operatin g Parameter s 1 4 4.5.3 . Calibratio n Curv e Procedure s 1 5 4.5.4 . Sampl e Analysi s 1 5 

### 4.6 . ACCEPTANC ECRITERI A1 6 4.7 . MAS S CALIBRATIO NCRITERI A1 7 4.8 . PERFORMANC ECRITERI A1 8 4.9 . LIMI TO FDETECTIO N(LOD ) AN D LIMI T O FQUANTITATIO N(LOQ ) 1 8 4.10 . TIM EREQUIRE D FO RANALYSI S 1 9 5 . CALCULATION S 1 9 6 . SAFET Y 2 0 7 . REFERENCE S 2 0

Exyge n Researc h Pag e 3 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) 

### LIS T O FFIGURE S Figur e 1 . Calibratio n curv e forPFO A 2 1 Figur e 2 . Representativ e Chromatogra mo f a5 0 ng/L PFO A Standar d2 2 Figur e 3 . Representativ e Chromatogramo f a10 0 ng/L PFO A Standar d2 2 Figur e 4 . Representativ e Chromatogramo f a25 0 ng/L PFO A Standar d2 3 Figur e 5 . Representativ e Chromatogra mo f a50 0 ng/L PFO A Standar d2 3 Figur e 6 . Representativ e Chromatogra mo f 100 0 ng/L PFO A Standar d2 4 Figur e 7 . Chromatogra mo f PFO A i n Reagen t

Contro l Sampl e 2 4 Figur e 8 . Chromatogramo f PFO A i n aDrinkin g Wate r Contro l Sampl e 2 5 Figur e 9 . Chromatogra mo f PFO A i n aDrinkin g Wate r Contro l Sampl e Fortifie dat

10 0 ng/ L 2 5 Figur e 10 . Chromatogra mo f PFO A i n aStreamWate r

Contro l Sampl e 2 6 Figur e 11 . Chromatogra mo f PFO A i n aStreamWate r

Contro l Sampl e Fortifie dat

10 0 ng/L 2 6 Figur e 12 . Chromatogra mo f PFO A i n aWel l Wate r

Contro l Sampl e 2 7 Figur e 13 . Chromatogra mo f PFO A i n aWel l Wate r

Contro l Sampl e Fortifie dat

10 0 ng/L 2 7 Figur e 14 . Chromatogra mo f PFO A i n aTa

p Wate r Contro l Sampl e 2 8 Figur e 15 . Chromatogramo f PFO A i n aTap Wate r

Contro l Sampl e Fortifie dat

10 0 ng/L 2 8

Exyge n Researc h Pag e 4 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )

Reaso n fo r

revision : Adde dchange s a s resul t o f discussion s betwee n DuPon t an dEPA . 1 . SUMMAR Y Thi s report

detail s ametho dof

analysi s for

residue s of

ammoniu m perfluorooctanoat e (APFO ) i n water . APF O form s perfluorooctanoi c aci d(PFOA ) i n solution . PFO A i s extracte d fro mwate r

usin g Ci s soli dphas e extractio n (SPE ) cartridges . Quantificatio n o f PFO A i s accomplishe db y liqui dchromatography/tande mmas s spectrometr y (LC/MS/MS ) analysi s usin g selecte dreactio n monitorin g (SRM) . Residue s o f PFO A foun dar e mathematicall y converte dan dreporte da s APFO . Th e chemical formul ao f

APF O i s give n i n Sectio n 2 o f thi s method . Th e anticipate dlimi t

o f quantitatio n (LOQ ; th e lowes t fortificatio n specifie db y th e metho dwhic h give s adequat e recover y accordin g t o EP A guidelines ) fo r thi s metho di s 5 0 ng/L (parts-per-trillion ) [o r 0.0 5 micrograms/liter ] fo r APFO . Quantificatio n i s performe dusin g calibratio n standard s prepare di n filtere d HPL C wate r

an dprocesse dthroug h th e extractio n procedure , identica l t o samples . Durin g th e extractio n procedure , al l sample s (includin g standards , controls , fortification s etc. ) ar e concentrate db y afacto r o f eight ; therefore , th e smalles t standar d(2 5 ng/L ) injecte ddurin g th e chromatographi c ru n i s equivalen t t o 20 0 ng/L (concentrate d8x) . Th e metho ddetectio n limi t (MDL ) i s approximatel y 6 t o 1 5 ng/L dependin g o n th e instrumen t use dfo r analysis . Thi s valu e i s base do n procedure s foun di n 4 0 CF RPart 13 6 Appendi x B . Thi s metho dwa s develope dusin g strea mwater , wel l water , tap water , an d drinkin g wate r

(bottle dwater) . Typica l percen t recoverie s ± standar ddeviation s (at 10 0 an d50 0 ng/L ) fo r

APF O i n tap wate r

wer e 112 % ± 4.1% ; i n wel l wate r wer e 104 % ± 2.3% ; i n strea mwate r wer e 108 % ± 3.3% ; an di n drinkin g wate r were 110 % ±3.3% . A representativ e calibratio n curv e i s show n i n Figur e 1 . Representativ e chromatogram s are show n i n Figure s 2 t o 15 .

Exyge n Researc h Pag e 5 of 28Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### 2 . EXPERIMENTA L COMPOUND S Th e structur e o f

ammoniu mperfluorooctanoat e i s give n below . APF O Chemica l Nam e Molecula r weigh t Ammoniu mPerfluorooctanoat e

43 1 FFFO O " NH 4 FFFF Note : Th e PFO A (anion ) (formul aweigh t 413 ) i s presen t

i n solutio n an di s use d fo r th e assay . 

### 3 . CHEMICAL S AN D SUPPLIE S 3.1 . CHEMICAL S Chemica l Methano l (MeOH ) Ammoniu mAcetat e Wate r Sodiu mThiosulfat e Grad e Sourc e HPL C EM D Reagen t Sigma-Aldric h HPL C EM D Reagen t J T Bake r Catalo g No .

MX0475- 1 A-733 0 WX0004- 1 

### STANDARD S Standar d Lo t Numbe r Purit y (% ) Sourc e Pentadecafluorooctanoi c 08316D O Aci d Perfluorooctanoi c Aci dU14 D 9 6 Aldric h Che m 9 6 Fluoroche mUS A

Exyge n Researc h Pag e 6 of2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) 

### 3.3 . EQUIPMEN T AN D SUPPLIE S

Equipmen t

Supplie r Balance , analytica l (displa y at leas t 0.000 1 g ) Mettle r Vacuu mpump Buch i Visipre p vacuu mmanifol dSupelc o Se p Pak Va c 6 c c (Ig ) tC1 8 cartridge s Water s (part #WA T 036795 ) 5 0 m Ldisposabl e polypropylen e centrifug e tube s VW R 1 5 mLdisposabl e polypropylen e centrifug e tube s VW R Disposabl e glas s micropipet s (50-10 0 uL , 100-20 0 Drummon d(VWR ) uL ) 

### 2 5 mmdiamete r

glas s fibr e acrodis c 0.4 5 u (cat Gelman #4523 ) Clas s A pipet s an dvolumetri c flask s variou s supplier s Hypercarb drop-i n guar dcolum n ( 4 mm ) Keyston e (part #844017-400 ) Stand-alon e drop-i n guar dcartridg e holde r Keyston e 125-m LLDP E narrow-mout hbottle s Nalgen e HPL C pum p (LC10AD ) Shimadz u 2 m L clear

HPL C via l ki t

(cat #518 1 -3400 ) H P Standar dlab equipmen t

(graduate dcylinders , variou s supplier s disposabl e tube s etc. ) LC/MS/M S an dHPL C system s A s describe di n sectio n 4.5 . Note : Equivalen t

material s ma y b e substitute dfo r thos e specifie di n thi s metho d i f the y ca n b e show n t o produc e satisfactor y results . Notes : 1 . I n orde r

t o avoi dcontamination , th e us e o f disposabl e labwar e i s highl y recommende d(tubes , pipets , etc.) . 2 . PTFE o r

PTF E line dcontainer s o r equipment , includin g PTFE-line d HPL C vial s fo r

th e HPL C autosample r

mus t no t b e used . 3 . HPL C grad e wate r

use ddurin g th e sampl e an dstandar dextractio n shoul d b e filtere dthroug haHypercarb guar dcolum n usin g aHPL C pump , a s describe di n sectio n 4.2 . Thi s wate r i s referre dt o a s "filtere dHPL C water" , here afte r

i n thi s report . 4 . I t i s necessary t o chec k

th e solvent s (methanol ) fo r th e presenc e o f contaminant s b y LC/MS/M S befor e use . Certai n lo t number s hav e bee n foun dt o b e unsuitabl e fo r

use . 5 . Us e disposabl e micropipet s o r pipet s t o aliquo t

standar dsolution s t o mak e calibratio n standard s an dsampl e fortifications .

Exyge n Researc h Pag e 7 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) 

### 3.4 . SOLUTION S (1 ) 5 0 mM ammoniu macetat e solutio n i s prepare db y weighin g 3.8 6 g o f ammoniu macetat e an ddissolvin g i n 1 L o f HPL C water . Dilut e th e 5 0 m M solutio n b y afacto r o f 2 5 t o mak e th e 2 m M ammoniu macetat e solutio n use dfo r mobil e phas e A . (2 ) 40 % methano l i s mad e b y measurin g 40 0 m L methano l an dadjustin g th e volum e t o 1 L wit h filtere dHPL C water . (3 ) 25 0 mg/m L sodiu mthiosulfat e solutio n i s mad e b y dissolvin g 2 5 g i n 10 0 m L filtere dHPL C water . Note : Th e aforementione dexample s ar e provide dfo r guidance , alternativ e volume s ma y b e prepare da s lon g a s th e ratio s o f th e solven t

t o solut e ratio s ar e maintained . 

### 3.5 . PREPARATIO NO FSTANDARD S AN D FORTIFICATIO NSOLUTION S Analytica l standard s ar e use dfo r thre e purposes : 1 . Calibratio n Standard s - Thes e standard s ar e prepare di n filtere dHPL C wate r an dar e use dt o calibrat e th e respons e o f th e detecto r

use di n th e analysis . 2 . Laborator y Contro l Spike s - Thes e fortification s ar e prepare da t concentration s correspondin g t o th e LO Q an dlO x LO Q an dar e use dt o determin e analytica l recovery . Laboratory contro l spike s ar e prepare di n filtere dHPL C water . 3 . Matri x Spike s - Thes e fortification s ar e prepare db y spikin g int o th e fiel dsample s a t

concentration s rangin g fro m50 0 ng/L u p t o 500,00 0 ng/L . Matri x spike s ar e use dt o evaluat e th e effec t o f th e sampl e matri x o n analytica l recovery . Th e absolut e volume s o f

th e standard s ma y b e varie db y th e analys t a s lon g a s th e correc t

proportion s o f

solut e t o solven t

ar e maintained . 3.5.1 . Stock

solutio n Prepar e astoc k

solutio n o f

10 0 ug/m L o f

PFO A b y weighin g ou t

1 0 m g o f analytica l standar d(correcte dfo r purity ) an ddilut e t o 10 0 m L wit h methano l i n a100-m

L volumetri c flask . Th e stoc k solutio n (i n 125-mL LDP E bottles ) i s t o b e store di n arefrigerato r at 2° C t o 6° C an di s stabl e fo r amaximu mperio do f

6 month s fro mth e dat e o f preparation .

Exyge n Researc h Pag e 8 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) 3.5.2 . Fortificatio n Solution s a . Prepar e afortificatio n standar do f 1. 0 ug/m L (100 0 ng/mL ) o f PFO A b y addin g 1. 0 mL o f

th e 10 0 ug/m L stoc k

solutio n t o a100-m

L volumetri c flas k

an dbrin g u p t o volum e wit h methanol . b . Prepar e afortificatio n standar do f 0. 1 ug/m L (10 0 ng/mL ) b y dilutin g 10. 0 mL o f

th e 1. 0 ug/m L solutio n t o 10 0 mL wit h methano l i n a volumetri c flask . c . Prepar e afortificatio n standar do f 0.0 1 ug/m L (1 0 ng/mL ) b y dilutin g 10. 0 mL o f

th e 0. 1 ug/m L solutio n t o 10 0 mL wit h methano l i n a volumetri c flask . Example : tw o hundre dmicroliter s o f th e 1 0 ng/m L solutio n spike dint o 4 0 m L o f wate r i s equivalen t

t o a0.0 5 pp b (5 0 ng/L ) fortification . Stor e al l fortificatio n standar dsolution s i n arefrigerato r (i n 125-mL LDP E bottles ) a t

2° C t o 6° C fo r

amaximu mperio do f

3 month s fro mth e dat e o f preparation , afte r

whic h tim e i t

i s necessary t o mak e ne w standard s usin g th e stoc k solution . Not e als o that additiona l concentration s ma y b e prepare di f necessary . 3.5.3 . Calibratio n Standard s LC/MS/M S calibratio n standard s ar e prepare di n HPL C wate r filtere d throug h ahypercarb drop-i n guar dcartridge . Th e calibratio n standard s are processe dthroug h the extractio n procedure , identica l to samples . The extracte dstandard s ma y b e use dfo r aperio do f

tw o week s whe n store d refrigerate d(at

2° C t o 6°C) . Th e followin g i s atypica l example : additiona l concentrations ma y b e prepare da s needed . Concentratio n Fortificatio n Volum e o f o f Fortificatio n Volum e Fortifie dContro l Solutio n (ng/mL ) (uL ) Sampl e (mL ) 0 1 0 1 0 1 0 10 0 10 0 100 0

10 0 20 0 40 0 100 20 0 40 0 4 0

4 0 4 0 4 0 4 0 4 0 4 0 Fina l

Concentratio n o f

Calibratio n Calibratio n Standar dI D Standar d(ng/L) * (example ) 0 2 5 5 0 10 0 25 0 50 0 100 0 Xcmmddyy- 0

Xcmmddyy- 1 Xcmmddyy- 2 Xcmmddyy- 3 Xcmmddyy- 4 Xcmmddyy- 5 Xcmmddyy- 6 * Th e extracte dconcentratio n o f th e calibratio n concentration , du e t o th e concentratio n o f th e (SPE) . X C = extracte dcalibratio n standard . standar di s equa l t o 8 x it s initia l standar ddurin g th e extractio n A zer o standar dsolutio n (reagen t blank ) mus t

b e prepare dwit h eac h se t o f standard s extracted . Thi s standar di s use donl y t o acces s th e reagent s Exyge n Researc h Pag e 9 of2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )

use dt o prepar e th e standard s an di s no t include da s par t

o f th e calibratio n curve . Stor e al l extracte dcalibratio n standard s i n 15-m L polypropylen e tube s at 2° C t o 6°C , u p t o tw o weeks . 3.5. 4 Controllin g Standar dBia s Prepar e aseparate stoc k

solutio n o f

10 0 ug/m L fro madifferen t PFO A nea t standar dfro madifferen t

sourc e b y weighin g ou t

1 0 m g o f analytica l standar d(correcte dfo r

purit y only ) an ddilut e t o 10 0 m L wit h methano l i n a100-m

L volumetri c flask . Th e stoc k solutio n (i n 125-m

L LDP E bottles ) i s t o b e store di n arefrigerato r at 2° C t o 6° C an di s stabl e fo r a maximu mperio do f

6 month s fro mth e dat e o f preparation . Prepar e afortificatio n standar do f 1. 0 jig/m L (100 0 ng/mL ) o f PFO A b y addin g 1. 0 mL o f

th e 10 0 ug/m L stoc k

solutio n t o a100-mL volumetri c flas k an dbrin g u p t o volum e wit h methanol . Fro mtha t solution , prepar e afortificatio n standar do f

0. 1 ng/m L (10 0 ng/mL ) b y dilutin g 10. 0 m L o f th e 1. 0 jig/mL solutio n t o 10 0 mL wit h methano l i n avolumetri c flask . Prepar e a25 0 ng/L extracte dcalibratio n standar dwit h eac h se t o f extracte dstandards . Ad d10 0 u L o f th e 10 0 ng/m L fortificatio n solutio n t o 4 0 mL o f

hypercarb filtere dHPL C wate r an dproces s throug h th e extractio n procedure , identica l t o samples .

Note : Thi s standar di s referre dt o a s th e "chec k standard " fro mthi s poin t

forward . Th e chec k

standar dshoul db e include dat leas t onc e wit h eac h se t

o f extract s analyzed . Th e chec k standar dshoul dgiv e arespons e withi n ± 15 % o f th e averag e respons e fro mth e 25 0 ng/L extracte dcalibratio n standards i n th e set .

Exyge n Researc h Pag e 1 0 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### 4 . METHO D 4.1 . FLO WDIAGRA M Th e flo w diagra mo f

th e metho di s give n below , followe db y adetaile d descriptio n o f

eac hstep . Metho dFlo w Diagra m Measur e 4 0 mL o f

sampl e (fortif y sample s designate da s calibratio n standard s an dfres hfortifications )



# 4 4 - Ci 8 Solid-Phas e Extractio n Fina l Volum e ( 5 mL ) LC/MS/M S 4.2 . SAMPL EPROCESSIN G N o sampl e processin g i s neede dfo r wate r samples . However , froze n sample s mus t b e allowe dt o completel y thaw , un-aided , a t roo mtemperature . Sample s store drefrigerate dshoul dals o b e allowe dt o equilibrat e t o roo mtemperature . All sample s mus t

be thoroughl y mixe dandany visibl e solid s remove dby filterin g th e sampl e throug ha25-mmdiamete r glas s fibr e acrodis c 0.4 5 u filte r befor e bein g sample dfo r

extraction . Initia l studie s sho w tha t

th e metho di s no t

effecte db y th e p H o f

th e sample . Sampl e p H i s recorde dprio r t o extraction . Contro l HPL C water , use dfor the preparatio n of

sample s andcalibratio n standard s a s wel l a s wate r

use dfo r equilibratin g an dwashin g th e SP E cartridges , mus t b e prepare db y filterin g HPL C wate r throug hahypercarb guar dcartridg e usin g aHPL C pump . Befor e use , was hth e guar dcartridg e wit h~ 2 5 m l HPL C grad e acetonitrile , the n -2 5 mL o f HPL C grad e methanol , followe db y ~ 2 5 mL o f HPL C wate r

usin g th e HPL C pum p a t ~ 2 mL/mi n flo w rate . The n start collectin g th e filtere dHPL C wate r eluat e fro mth e cartridge . I t i s recommende d tha t th e cartridg e b e washed , a s describe dabove , afte r filterin g ~ 2 liter s o f HPLC water . A flo w rat e o f 2 t o 3 mL/mi n i s recommended . Exyge n Researc h Pag e 1 1 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### 4.3 . SAMPL EPREPARATIO N a . Eac hbatc ho f

sample s extracte d(typicall y 2 0 o r less ) mus t

includ e at

leas t on e reagen t

contro l (metho dblan k

usin g filtere dHPL C water ) an dtw o reagen t control s fortifie da t

know n concentration s t o verif y procedura l recover y fo r

th e batch . b . Eac hsampl e mus t

b e extracte di n replicat e unles s th e sampl e i s marke da s a fiel dblank . Fiel dblank s d o no t nee dt o b e extracte di n replicate . c . Eac hsampl e extracte d(excep t fo r fiel dblanks ) mus t

als o b e separatel y fortifie da t aknow n concentratio n an dcarrie dthroug hth e procedur e t o verif y recovery . Matri x spike s shoul drang e fro m50 0 ng/Lt o 500,00 0 ng/L . I f th e sampl e residu e i s foun dt o b e significantl y highe r tha n th e leve l a t

whic h th e sampl e wa s initiall y fortified , th e sampl e shoul db e fortifie da t ahighe r concentratio n (u p t o 500,00 0 ng/L ) an dre-extracted . 4.4 . EXTRACTIO N 1 . Measur e 4 0 mLo f sampl e int o 5 0 mLpolypropylen e centrifug e tube s (fortif y a s needed , replac e li dan dmi x well) . Note : Fo r

ta p water sample s an dan y wate r sample s suspecte dt o contai n chlorine , ad d20 0 ju Lo f a25 0 mg/mLsodiu mthiosulfat e solutio n t o th e 4 0 mL sampl e an dmi x wel l t o de-activat e chlorine , befor e fortification . 2 . Conditio n th e d g SP E cartridge s ( 1 g , 6 mL ) b y passin g 1 0 mL methano l followe db y 5 m Lfiltere dHPL C wate r ( ~ 2 drop/sec) . D o no t

le t colum n ru n dry . 

### Note : Fo r th e followin g steps , maintai n a~ 7 drop / se c flo w rate . D o no t allo w th e colum n t o ru n dr y a t an y time . 3 . Loa dsampl e o n conditione dCi g SP E cartridge . Discar deluate . 4 . Was hwit h~ 5 mL 40 % methano l i n water . Discar deluate . 5 . Elut e wit h~ 5 m L100 % methanol . Collec t 5 m Lo f eluat e int o graduate d 1 5 m Lpolypropylen e centrifug e tube s (fina l volum e = 5 mL) . 6 . Analyz e sample s usin g electrospra y LC/MS/MS . 

### NOTE : SAMPLE S AR ECONCENTRATE D B Y AFACTO RO FEIGH TDURIN GTH E EXTRACTION ; INITIA LVO L= 4 0 M L- > FINA LVOL . = 5 ML .

Exyge n Researc h Pag e 1 2 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) 4.5 . QUANTITATIO N 4.5.1 . LC/MS/M S Systeman dOperatin g Condition s (Electrospray ) Mas s Spec : Micromas s Quattr o Ultim a(Micromass ) Interface : Electrospray (Micromass )

Harvar dinfusio n pum p (Harvar dInstruments) , fo r tunin g Computer : COMPA Q Professiona l Workstatio n AP20 0 Software : Window s NT , MassLyn x 3. 3 HPLC : Hewlett

Packar d(HP) Serie s 1100 H P Quat Pum p H P Vacuu mDegasse r H P Autosample r H P Colum n Ove n Note : A 4 x 1 0 m mhypercarb drop-i n guar dcartridg e (Keystone , par t #844017

- 400 ) i s attache don-lin e afte r th e purg e valv e an dbefor e th e sampl e injecto r port t o trap an y residu e contaminant s tha t ma y b e i n th e mobil e phas e and/o r HPL C system . HPL C Column : Genesi s C g (Jone s Chromatography) , 2. 1 mmx 5 0 mm , 4|t i Colum n Temperature : 35 ° C Injectio n Volume : 1 5 fi L Mobil e Phas e (A) : 2 mM Ammoniu mAcetat e i n HPL C wate r Mobil e Phas e (B) : Methano l

Tim e % A 0. 0 6 0 0. 4 6 0 1. 0 1 0 7. 0 1 0 7. 5 0 9. 0 0 9. 5 6 0 13. 5 6 0 14. 0 6 0 15. 0 6 0 I t ma y b e necessary t o adjus t th e HPL C gradien t

i n orde r t o optimiz e instrumen t performance . Column s wit h differen t dimension s (e.g . 2. 1 x 30 ) an dals o column s fro mdifferen t

manufacturer s (Keyston e Betasi l Ci g etc. ) coul db e used , provide dequivalen t

chromatograph y i s obtained . Ion s monitored : Approximat e Analvt e Mod e Transitio n Monitore dRetentio n Tim e PFO A Negativ e 41 3 - > 36 9 ~ 5 min . % B 4 0 4 0 9 0 9 0 100 100 4 0 4 0 4 0 4 0 Flo w Rat e CmL/min )

0. 3 0. 3 0. 3 0. 3 0. 3 0. 4 0. 4 0. 4 0. 3 0. 3

Exyge n Researc h Pag e 1 3 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) Th e retentio n time s ma y vary , o n ada y t o da y basis , dependin g o n th e batc h o f mobil e phas e etc . Drif

t i n retentio n time s (u p t o ± 2 % ) i s acceptabl e withi n a n analytica l run , a s lon g a s th e drif t continue s throug h th e entir e analysi s an dth e standard s ar e include dat

th e beginnin g an den do f

th e analytica l run . Note : A n alternativ e LC/MS/M S syste mma y b e use donc e demonstrate dt o b e equivalent . 4 . 5 . 2 . Exampl e M S Operatin g Parameter s Th e followin g M S operatin g value s ar e provide da s a n exampl e only . Actua l value s wil l mos t

likel y var y fro minstrumen t t o instrument . Thes e value s ma y als o chang e ove r

tim e eve n whe n analyzin g sample s o n th e sam e instrument . Th e value s ar e change dt o optimiz e fo r greates t sensitivity . Fo r

example , hexapol e 1 voltag e ma y b e se t t o 2 V ; however , a s tim e goe s on , hexapol e 1 voltag e may b e increase do r decreased . Th e exac t

value s use deac h tim e ase t i s analyze dwil l b e documente dwit h eac h dat aset . Th e mas s spectromete r

i s tune dfo r

th e analyt e b y infusin g a~ 0. 2 standar dsolutio n o f

PFO A (at 1 0 fiL/min , usin g an infusio n pump ) vi aa"T " int o astrea mo f

mobil e phas e containin g 40 % methano l an d60 % 2m M ammoniu m acetat e a t 0. 2 mL/mi n flo w rate . Th e analyt e i s initiall y tune dfo r th e paren t

io n an dthe n tune dfo r

th e produc t

ion . Onc e th e instrumen t i s tuned , th e optimize d parameter s ar e save da s atun e file . Thi s tun e fil e i s the n use ddurin g routin e analysis . Analvt e Dwel l (s ) Collisio n Energ y (eV ) Con e (V ) PFO A 0. 2 1 0 1 0

Sourc e Se t Capillar y 3. 0 k V Hexapol e 1 0 V Apertur e 1 0 V Hexapol e 2 0 V Sourc e Bloc k

Temp . 1 00° C Desolvatio n Temp . 300° C Analyze r Se t LMRes l 14.0 V H M Re s 1 14. 0 V lEnerg y 1 I V Entranc e - 2 V Exi t 2 V LM Re s 2 14. 0 V HMRes 2 14.0 V lEnerg y 2 2. 0 V Multiplie r 70 0 V Exyge n Researc h Pag e 1 4 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) Ga s Flow s Se t Con e Ga s ~130L/h r Desolvatio n -75 0 L/hr Pressure s Rea dbac k Ga s Cel l ~3.0e-3mba r 4.5.3 . Calibratio n Curv e Procedure s a . Injec t th e sam e aliquo t

(betwee n 1 0 t o 2 5 uL ) o f eac h extracte dcalibratio n standar dprocesse di n matri x (rangin g fro mth e lowes t leve l standar dt o th e highes t leve l prepared) , int o th e LC/MS/M S (th e fina l concentration s o f thes e calibratio n standard s wil l b e equivalen t t o 8 x th e concentratio n o f th e initia l standar ddu e t o th e concentratio n durin g SPE) . b . Us e linea r

standar dcurve s fo r

quantitation . Th e zer o calibratio n standar di s no t include di n th e calculatio n o f th e calibratio n curve . Linea r standar d curve s ar e generate dfo r eac h analyt e b y linear regressio n usin g 1/x weightin g o f

peak are aversu s calibratio n standar dconcentratio n usin g MassLyn x 3. 3 (o r

equivalent ) softwar e system . An y calibratio n standar d foun dt o b e astatistica l outlie r b y usin g th e Hug e Erro r Test , ma y b e exclude dfro mth e calculatio n o f th e calibratio n curve . However , th e tota l numbe r o f extracte dcalibratio n standard s tha t ma y b e exclude dmus t

no t excee d20 % o f

th e tota l numbe r

o f extracte dstandard s injected . c . Th e correlatio n coefficien t (R ) fo r calibratio n curve s generate dmus t b e >0.99 2 (R 2 >0.985) . I f

calibratio n result s fal l outsid e thes e limits , the n appropriat e step s mus t

b e take n t o adjus t

instrumen t

operation , an dth e standard s o r

th e relevan t

se t o f sample s shoul db e reanalyzed . A typica l calibratio n curv e fo r PF O A ca n b e foun di n Figur e 1 . 4.5.4 . Sampl e Analysi s a . Injec t th e sam e aliquo t

(betwee n 1 0 t o 2 5 uL ) o f eac h standard , sample , recovery , control , etc . int o th e LC/MS/M S system . b . Standard s correspondin g t o a t leas t fiv e o r mor e concentratio n level s (startin g wit h th e LO Q leve l o r

below ) mus t

b e include di n a n analytica l se t (not e tha t th e calibratio n standard s ar e prepare db y spikin g filtere dHPL C wate r an d processing similar

t o actua l samples) . c . A n entir e se t

o f extracte dcalibratio n standard s shoul db e injecte dat th e beginnin g o f

ase t followe db y extracte dcalibratio n standard s intersperse d Exyge n Researc h Pag e 1 5 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) 

### approximatel y every 5-1 0 sample s (t o accoun t fo r asecon dse t

o f extracte d standards) . A s a n alternative , a n entir e se t o f extracte dcalibratio n standard s may b e include dat

th e beginnin g an dat

th e en do f

asampl e set . I n eithe r case , extracte dcalibratio n standard s mus t b e th e firs t

an dlas t injectio n i n a sampl e set . d . Injec t th e chec k standar donc e wit heac hse t o f extracts . e . Th e concentratio n o f

eac hsample/fortification/contro l i s determine dfro mth e standar dcurve , base do n th e peak are ao f eac hanalyte . Th e standar d response s shoul dbracke t

response s o f

th e residu e foun di n eac hsampl e set . I f necessary , dilut e th e sample s t o giv e arespons e withi n th e standar dcurv e range . f . Fortificatio n recoverie s fallin g withi n 7 0 t o 130 % ar e considere dacceptable . g . Th e tota l holdin g tim e betwee n sampl e collectio n an danalysi s mus t no t excee d1 4 days . Extracte dsample s mus t b e store drefrigerate dbetwee n 2° C t o 6° C unti l analysis . h . Fiel dsample s i n whic heithe r n o peak s o r

peak s les s tha n th e MD L ar e detecte da t

th e correspondin g analyt e retentio n tim e wil l b e reporte da s N D (no t detected) . Sample s i n whic hpeak s ar e detecte dat th e correspondin g analyt e retentio n tim e tha t

ar e les s tha n th e LO Q an dgreate r tha n o r equa l t o th e MD L wil l b e reporte da s N Q (no t quantifiable) . Al l chromatogram s ar e considere dra w dat aan da s such , wil l b e include di n th e dat apackag e wit h eac hreport . Th e analysi s performe ddurin g th e metho ddevelopmen t include dfortification s a t 10 0 an d50 0 ng/ Lo f

PFO A i n wel l water , tap water , an dstreamwate r (collecte d locally , Stat e College , PA ) an dbottle ddrinkin g wate r (fro maloca l grocery store) . 4.6 . ACCEPTANC ECRITERI A Th e followin g criteri amus t b e me t t o ensur e th e presenc e o f

PFOA : 1 . Chromatogra mmus t

sho w apeak

o f adaughte r

io n at 36 9 amu fro ma paren t o f 41 3 amu . Th e 41 3 amu paren t correspond s t o th e PFO A anion , whil e th e daughte r

io n (36 9 amu ) represent s th e los s o f carbo n dioxide . Thi s transitio n woul dno t

discriminat e betwee n linea r an dbranche dform s o f PFOA ; therefore , both , i f

present , woul db e include di n th e calculation . I f th e branche disomer

peak s are resolve do r

partiall y resolve dfro mth e linea r peak , th e summe dpeak

area s wil l b e use di n th e calculation .

Exyge n Researc h Pag e 1 6 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) 

### 2 . Metho dblank s mus t

no t contai n PFO A a t

level s greate r

tha n th e LOD . I f ablan k contain s PFO A at

level s greate r

tha n LOD , the n ane w blan k sampl e mus t

b e obtaine dan dth e entir e se t mus t b e re-extracted . 3 . Recoverie s o f

contro l spike s an dmatri x spike s mus t b e betwee n 70-130 % o f thei r know n values . I f

acontro l spik e fall s outsid e th e acceptabl e limits , th e entir e se t

o f sample s shoul db e re-extracted . I f amatri x spik e bein g use dt o evaluat e an entire extractio n se t fall s outsid e 70-130% , th e entir e se t mus t b e re-extracted . I f

amatri x spik e bein g use dt o evaluat e a n individua l sampl e fall s outsid e 70-130% , th e matri x spik e alon g wit h it s correspondin g sampl e an dreplicat e mus t b e re-extracted . 4 . An y calibratio n standar dfoun dt o b e astatistica l outlie r b y usin g th e Hug e Erro r

Test , may b e exclude dfro mth e calculatio n o f th e calibratio n curve . However , th e tota l numbe r o f extracte dcalibratio n standard s that coul db e exclude dmus t

no t excee d20 % o f

th e tota l numbe r

o f extracte d standard s injected . 5 . Th e correlatio n coefficien t (R ) fo r calibratio n curve s generate dmus t b e >0.99 2 (R 2 

### >0.985) . I f

calibratio n result s fal l outsid e thes e limits , the n appropriat e step s mus t

b e take n t o adjus t

instrumen t

operation , an dth e standard s o r

th e relevan t

se t o f sample s shoul db e reanalyzed . 6 . Th e respons e fro mth e chec k standar dshoul db e withi n ± 15 % o f th e averag e respons e fro mth e 25 0 ng/ Lcalibratio n standardconcentrations . I f th e standar drespons e fall s outsid e thes e limits , appropriat e step s mus t b e take n t o adjus t

instrumen t

operatio n an dth e se t

o f sample s shoul db e reanalyzed . I f

th e standar drespons e i s stil l outsid e th e ± 15 % the n standar dpreparation s shoul db e reviewed , an di f warranted , ne w stock , fortificatio n an dextracte dcalibratio n solution s shoul db e prepared . Both set s o f dat awoul dthe n b e include di n th e dat apackage . 7 . Retentio n time s betwee n standard s an dsample s mus t no t drif t mor e tha n ± 2 % withi n a n analytica l run . I f retentio n tim e drif

t exceed s thi s limi t withi n a n analytica l ru n the n th e se t mus t b e reanalyzed . 4.7 . MAS S CALIBRATIO NCRITERI A I n orde r t o verif y mas s resolution , acalibratio n chec k i s performe dweekl y o n th e mas s spectromete r

usin g aNal/Rb/C s tunin g solution . The tunin g solutio n is infuse donto th e mas s spe c usin g an infusio n pum p se t t o aflo w rat e o f

1 0 uL/min . A n M S sca

n fro m8 0 t o 138 0 amu i s performe do n bot hMS I an dMS2 . Th e calibratio n i s considere dacceptabl e i f th e followin g 1 1 peak s ar e foun d withi n ± 0. 2 am

u o f their know n masse s o n bothMS I an dMS2 : 84.9 , 132.9 , 172.8 , 322.7 , 472.6 , 622.5 , 772.4 , 922.3 , 1072.2 , 1222. 1 an d1372.0 . I f an y o f Exyge n Researc h Pag e 1 7 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) th e masse s ar e outsid e th e ± 0. 2 amu , the n th e instrumen t wil l b e recalibrate dan d anothe r calibratio n chec k

performed . 

### 4.8 . PERFORMANC ECRITERI A Th e followin g tw o criteri amus t b e performe da s asyste msuitabilit y test , befor e th e commencemen t

o f analysi s whe n usin g a n instrumentatio n set-u p tha t ha s no t bee n use dfo r

thi s method . Firs t Criterion : Ru n astandar dsolutio n o n LC/MS/M S correspondin g t o th e estimate dLO Q (5 0 ng/L ) i n matri x an dobtai n asigna l t o nois e rati o o f th e 41 3 -> • 36 9 transitio n o f at leas t 9:1 , compare dt o areagen t blank . I f

thi s criterio n canno t

b e met , optimiz e an dchang e instrumen t operatin g parameter s (o r increas e th e injectio n volume , i f appropriate) . Secon dCriterion: Ru n ase t o f standard s o f

fiv e o r mor e concentratio n levels , fro m1/ 2 LOQ , u p t o th e highes t

concentratio n leve l t o b e include di n th e analysis . Generat e a calibratio n curv e fo r

PFO A an dobtai n alinea r regressio n wit h acoefficien t o f determinatio n (R 2 ) o f

at leas t 0.98 5 fo r

PFOA . Onc e thi s criterio n i s met , sample s ma y b e analyze dwit h standard s interspersed . 

### 4.9 . LIMI T O FDETECTIO N(LOD ) AN D LIMI T O FQUANTITATIO N(LOQ ) A Limi t o f Detectio n (LOD ) an dLimi t o f Quantitatio n (LOQ ) wil l b e reporte d fo r eac h se t o f sample s analyzed . Th e LO D i s define dusin g th e metho dblan k (o r reagen t blank) , an di s th e concentratio n (a s woul db e expresse di n th e origina l samples ) that

woul dcorrespon dt o 3 time s th e peak-to-peak amplitud e o f

th e baselin e nois e i n th e vicinit y o f th e PFO A peak . I f

laboratory o r

instrumenta l backgroun dcontaminatio n o r interferenc e i s present , th e LO D wil l b e th e concentratio n tha t

correspond s t o thre e time s th e amplitud e o f th e backgroun d peak a t th e retentio n tim e o f

PFOA . Th e LO Q wil l b e define da s 5 time s th e LOD . PFO A level s i n th e sample s belo w th e LO D wil l b e reporte da s "Not Detected " (ND) . PFO A level s i n sample s greate r tha n LO D bu t

les s tha n LO Q wil l b e reporteda s "No t

Quantifiable " (NQ) . Value s at o r greate r tha n LO Q wil l b e reporte da s numerica l values , rounde dt o tw o significan t figures . Al l ra w dat a wil l b e include di n th e dat apackage , includin g peak area s belo w LO D o r

LOQ . LOD/LO Q wil l b e determined , a s above , at leas t quarterly .

Exyge n Researc h Pag e 1 8 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) 

### 4.10 . TIM EREQUIRE D FO RANALYSI S A se t o f 2 4 sample s ( 6 standard s an d1 8 samples ) can b e take n throug h th e extractio n procedur e i n approximatel y 2- 3 hour s b y on e person . Th e LC/MS/M S analysi s (standard s an d1 8 samples ) wil l tak e approximatel y 7 hours . 

### 5 . CALCULATION S a . Us e Equatio n 1 t o calculat e th e amoun t o f PFO A foun d(i n ng/L , base do n peak area ) usin g th e standar dcurv e (linear regressio n parameters ) generate d b y th e Mas s Lyn x softwar e program . Equatio n 1 : PFO A foun d(ng/L ) = (Peak are a- intercept ) x D F slop e D F= facto r

b y whic hth e fina l volum e wa s diluted , i f necessary . b . Fo r sample s fortifie dwit hknow n amount s o f PFO A prio r

t o extraction , us e Equatio n 2 t o calculat e th e percen t recovery . Equatio n 2 : Recover y (% ) = [ tota l analyt e foun d(ng/L ) - analyt e foun di n contro l (ng/L) ]

analyt e adde d(ng/L ) Note : Subtrac t

analyt e foun di n contro l (ng/L ) fro manalyt e foun d(ng/L) , i f applicable . c . I f necessary , us e Equatio n 3 t o calculat e th e amoun t o f APF O foun d(i n ng/L ) usin g th e molecula r

weigh t rati o o f APFO/PFOA : Equatio n 3 : M WAPF O (431 ) APF O foun d(ng/L ) - PFO A Foun dx M WPFO A (414 )

Exyge n Researc h Pag e 1 9 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 ) Note : calculation s coul dals o b e performe di n pp b o r ng/mL i f concentration s wer e assigne di n ng/m L instea do f ng/L whe n performin g th e analysis . Fo r reportin g purposes , fiel dsample s i n whic h eithe r n o peak s o r

peak s les s tha n th e MD L ar e detecte da t th e correspondin g analyt e retentio n tim e wil l b e reporte da s N D (no t

detected) . Sample s i n whic h peak s ar e detecte dat th e correspondin g analyt e retentio n tim e tha t ar e les s tha n th e LO Q an dgreate r tha n o r equa l t o th e MD L wil l b e reporteda s N Q (no t quantifiable) . 

### 6 . SAFET Y Th e analys t

shoul drea dth e materia l safet y dat asheet s fo r al l standard s an d reagent s befor e performin g thi s method . Us e universa l precaution s whe n handlin g standards an dreagents, includin g workin g i n fum e hood s an dwearin g laborator y coats, safet y glasses , an dgloves . 

### 7 . REFERENCE S Ellis , D.A. ; Martin , J.W. ; Muir , D.C.G. ; Mabury , S.A . Developmen t o f a n 19 F NM RMetho dfo r

th e Analysi s o f

Fluorinate dAcid s i n Environmenta l Wate r Samples . Anal . Chem . 2000 , 72 , 726-731 . Moody , C.A. ; Kwan , W.C. ; Martin , J.W. ; Mui r D.C.G. ; Mabury , S.A . Determinatio n o f

Perfluorinate dSurfactant s i n Surfac e Wate r Sample s b y Tw o Independen t

Analytica l Techniques : Liqui dChromatography/Tande mMas s Spectrometr y an d 19 FNMR . Anal . Chem . 2001 , 73 , 2200-2206 .

Exyge n Researc h Pag e 2 0 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### Figur e 1 . Calibratio n curv e fo r PFO A

Sompcuw S 1 rang ; APF O CwffidBlrtc f

Otntmanatom ; 0,99133 8 Caitjrartc n euro * W.772

4 ' X pofa e ffl:n : Ejeemo l $W , A m Di m typo : L'rear , CrtplxExcktda . 5.ec»+- | 1

Rc*pa-ac

Exyge n Researc h Pag e 2 1 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### Figur e 2 . Representativ e Chromatogra mo f a5 0 ng/L PFO A Standar d

50pptCl21S39- 2 1 £3199-200 2 S m (33,2x2 ) 21-DeC'19& & 23:34:0 4 LC/MS/M S uni t 6 M.ft M o f 3 ChannEl s E5 -

Area '

1,0 0 2.C O 3JO O 4.0 0 S.C O .6.0 0 7.Q O T\m e



### Figur e 3 . Representativ e Chromatogra mo f a10 0 ng/ L PFO A Standar d

tOOpptCl21S9^- 3 1*2199-100

3 Sm(£G , 2X2 )

1 1 , . 1 1.GG 5,t 6 21-Dec»199 9 23:49:42 ! LC/MS/M S uni t 6 hCRM«f3Channala£S 2.0 0 ' 3.0 0 4.0 0 5.P O 6.0 0 7.0 0 5 ^ Tmn e s.o a

Exyge n Researc h Pag e 2 2 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### Figur e 4 . Representativ e Chromatogra mo f a25 0 ng/L PFO A Standar d

122l99-ZG04Sm(SG.2*2 } 100" ! S.0 9 36. 1 tQ- 1

1.Q O 2,0

0 1 1 , ' I ' " I 3,0 0 22-Pec M 99( 9 00:05:3 5 LCJMSJM S unlt

€ MR M fr f 3 Channel s E S

Are a

5,0 0 6.0 0



### Figur e 5 . Representativ e Chromatogra mo f a50 0 ng/L PFO A Standar d

500'ppt.Cl21539- 5 122-19^200 6 Sm{SQ , 2x2 )

1,0 0 2.0 0 22-Deu- l 99 9 00:21:2 2 5.C- 9 MR M o f 3 Channel s ES 413>3G 9 Are a

•4.0 0 5,0 0 6,0 0 i,.. . i .'i^^ypri' i Tim e

Exyge n Researc h Pag e 2 3 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### Figur e 6 . Representativ e Chromatogra mo f 100 0 ng/L PFO A Standar d

10QOp33tCl;21599- 5 123199-2006 Si n (SG r ass2. ) Ofc37MS | LCWS/MSlinfc e UR M

'I i t I L I If I HP«Y' ' ' J ''•;-!- • ' I ! ! l''i"l"<'" » UaO 2.0 0 3sCO 413>G6 9 Affi a

«.0 0 5:O Q 6.0

0 7.0 0 j ,!>^-vTim B



### Figur e 7. Chromatogra mof PFO A in Reagen t

Contro l Sampl e

Reagen t Corvtrd l

1.Q Q ' ' £0 0 'I' 1 I ' ' ? 3;DO 5-0 0 LCftfl$/M S uris i $ , Mfl M o f 3 Channe l ES *

USBe4

7.0 0 Tim o

Exyge n Researc h Pag e 2 4 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### Figur e 8 . Chromatogra mo f PFO A i n aDrinkin g Wate r Contro l Sampl e

Drinkin g wate rConfrc i B LG/MS/M S unit # 122188-201 1 Sro (SO . 2*2 ) MR M o f 3 Channel s E S 100- i TM*x7,3 9 41 3 > 36 9 3*0 3 Lv*w i i. ' " ' n ' • ' • < • • -"-I. ' -•'i' ' • ] i ' " ' i ' *' ' i ' •' ' i' ' ' i •'' ' i ' ""*-v11.111 1 p i 1111 1 Tim e 1.C O 2.0 0 3,0 0 4.g Q &.CO 6.0 0



### Figur e 9 . Chromatogra mo f PFO A i n aDrinkin g Wate r Contro l Sampl e Fortifie dat

10 0 ng/L

Drinkin g wate r10 0 pp t Spf o A LC/RAS/NI S uni t 6 1 y^ M e H 3 Channel s ES - £.0 9 15477 I "llSe S Are a 0 i^-j-.- . ,.•!,, , n „ . ;,,, . 11 1 ,-i...,.... , .< , c...,•.. . i |"'i=^rT77 1 i^yyn-.' . j.. . 11 1 iTr" Tim e

1,0 0 2.S O 3.Q & 4jQ O S.O O 6.Q O 7jC O

Exyge n Researc h Pag e 2 5 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### Figur e 10 . Chromatogra mo f PFO A i n aStrea mWate r Contro l Sampl e

Strea m wate rC&niro l A 121199-101 0 Sm(SG , 2x2 } 1-Oft i 19-D«-199916;17!4 B LGMS/M S unit e MP M a ! 3 Chaniwts ES 7,107-1 6 413 > 36 9

s.o o -CLOT?.t

o



### Figur e 11 . Chromatogra mo f PFO A i n aStrea mWate r Contro l Sampl e Fortifie dat

10 0 ng/L

wate r10 0 pp t Sp k

A &0 5 uni t 6 MR M o f 3 Channel s ES * % i 1.00 2J3 0 '..9.0 0 ... ; .-4.0.0 . 5.0 Q "^iTbb ' ' ' T^h n """ ^

Exyge n Researc h Pag e 2 6 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### Figur e 12 . Chromatogra mo f PFO A i n aWel l Wate r

Contro l Sampl e

Wel l Wate roantro U 2 . 121599- 1 mS m (SO , 2x2 3 lOC h 00:49:36 1 MRMof3ChaimelaE S



### Figur e 13 . Chromatogra mo f PFO A i n aWel l Wate r

Contro l Sampl e Fortifie dat

10 0 ng/L

WeKWateMQOpptSpk A ta i 593*111 8 SratSG,ax2 ) 10(h 5.15

- 2S-41 3 16-Dec-199 9 02:40:1 1

MR M o l 3 Channel s ES 41 3 > 33 9

1.00 2.0 0 4.6 0

Exyge n Researc h Pag e 2 7 o f 2 8Exyge n Metho dNo : 01M-008-04 6 Revisio n 2 (V0001013 )



### Figur e 14. Chromatogra mof PFO A in aTap Wate r

Contro l Sampl e

Ta p wate rCentre ! A 23-Oec*19& & 17:34:1 1 LOWS/M S uni t < 122.399^20-11Sm . (35,2X2 ) 41 3 > 36 9

JC^^^^

V .„ . '-f ] -!"'T"~. , r'. ' . , t . . I , ' I'), " ...,!' " ' ' ! i ''•'. : ' ! ! I ' ' • P^ 1 I ' • • • I ' , ! • • • , I • I I , . . ! ! I |i|m S 1,00 2.&D 3rO O 4I..Q O 5.0 0 6.& 0 7.0 0



### Figur e 15 . Chromatogra mo f PFO A i n aTap Wate r

Contro l Sampl e Fortifie dat

10 0 ng/ L

12239&-3Q2 Q S m [SG . 2x2 ) 1QG H .^?L- i ' 41 3 > 36 9 t,95c 5 Are a ,11. 1 !["•,»-.".-.,.,,. , |,,.,. , .1...;. . . : --|

- ;." , "•"."--'• ||

. 1

. •.'. . tw i . ^ . i-Tt-qi^. , , u , 11 1 11., " Tima 1 l.DQ • g.Q Q 3.0 0 4.0 0 S.Q O 6.0 0 7,0 0

Exyge n Researc h Pag e 2 8 o f 2 8Saltzbart.Ilana@epamail.epa. gov 12/08/2006 02:09 PM To Lori E Stephans/AE/DuPont@DuPont cc bcc Subject Comments to Exygen's Proposed Verification Study and Method of Analysis History: This message has been forwarded. Lori, EPA has had an opportunity to review Exygen's proposed "verification study" as described in the November 28, 2006, letter from Karen Risha to you, and transmitted to me on November 30, 2006. EPA has also had an opportunity to review Exygen's "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water, Revision 2 ("Method of Analysis)". 1. Overall, EPA found the Method of Analysis to be quite good. However, Exygen should include a mass-labeled extraction surrogate and a different mass-labeled internal standard. 2. Additionally, EPA seeks clarification from Exygen regarding how the values for LOQ, LOD, and MDL relate to the calibration standards. Additionally, please address whether, based upon the calibration standards, there is the potential for reported concentrations below the lowest standard concentration. 3. Finally, to eliminate uncertainties, for purposes of the verification study, EPA has a strong preference that Exygen prepare a fresh batch of the high, low, and blank controls as described in DuPont's October 24, 2006 First Progress Report. In particular, Exygen should follow the Control Sample Recipes in Section 3.1 for the high, low, and blank. We hope that DuPont will provide any raw materials that Exygen does not have to avoid any delay in ordering such raw materials. EPA is providing a table that Exygen should use in reporting the data to EPA. Exygen should include any additional information in the table that it would report as standard operating procedure. (See attached file: Data Quality Summary Table for Verification Study.xls) Ilana Saltzbart Attorney-Advisor, US EPA, OECA, OCE 1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460 Phone: 202 564 9935 Fax: 202 564 0035 NOTICE: This communication may contain privileged or other confidential information. If you are not the intended recipient, or believe you have received this communication in error, please delete the copy you received, and do not print, copy, retransmit, disseminate, or otherwise use the information. Thank You.Saltzbart.Ilana@epamail.epa. gov 12/13/2006 05:35 PM To Lori E Stephans/AE/DuPont@DuPont cc chas.simons@exygen.com, j ay.bernarding@exygen.com bcc Subject Re: Exygen feedback on cost issue. My client has authorized the additional $2,500 expenditure for Exygen to prepare the high, low, and blank control systems for the verification study. Ilana Saltzbart Attorney-Advisor, US EPA, OECA, OCE 1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460 Phone: 202 564 9935 Fax: 202 564 0035 NOTICE: This communication may contain privileged or other confidential information. If you are not the intended recipient, or believe you have received this communication in error, please delete the copy you received, and do not print, copy, retransmit, disseminate, or otherwise use the information. Thank You. Lori E Stephans <Lori.E.Stephans @USA.dupont.com> To Sent by: Ilana Saltzbart/DC/USEPA/US@EPA Lori.E.Stephans@ cc USA.dupont.com Subject Re: Exygen feedback on cost 12/08/2006 07:49 issue. PM

Thanks Ilana. I'll give Exygen the Okay to proceed when I get the green light from you.

----- Original Message ----- From: Saltzbart.Ilana Sent: 12/08/2006 05:06 PM To: Lori E Stephans/AE/DuPont@DuPont Subject: Re: Exygen feedback on cost issue. Thanks Lori. I'm waiting to hear back from my client regarding the $2,500, and I'll follow-up with Exygen next week (with a cc to you).Have a great vacation. Ilana Saltzbart Attorney-Advisor, US EPA, OECA, OCE 1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460 Phone: 202 564 9935 Fax: 202 564 0035 NOTICE: This communication may contain privileged or other confidential information. If you are not the intended recipient, or believe you have received this communication in error, please delete the copy you received, and do not print, copy, retransmit, disseminate, or otherwise use the information. Thank You. Lori E Stephans <Lori.E.Stephans @USA.dupont.com> To Ilana Saltzbart/DC/USEPA/US@EPA 12/08/2006 04:43 cc PM jay.bernarding@exygen.com Subject Exygen feedback on cost issue.

cc Jay Bernarding Exygen

IlanaHere was Exygen's response to the cost question you posed. Exygen will agree to perform the verification study described in Karen's letter of November 28, 2006 at no cost to EPA however, if EPA wants Exygen to prepare fresh blank, low control and high control samples in our laboratory with ingredients provided by DuPont, the charge for this task will be $2,500 as previously stated. I hope this will work for EPA. Please let me know if I need to follow up with Exygen. Not a problem. Regards, Lori Lori E. Stephans, Ph.D.Manager, Supplemental Environmental Programs Rm 4110, Jackson Laboratory Route 130, Chambers Works Deepwater, NJ 08023 856-540-3748 (office) 856-540-4077 (fax) 856-341-0255 (cell) lori.e.stephans@usa.dupont.com This email message is for the sole use of the intended recipient(s) and may contain privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply email and destroy all copies of the original message. This communication is for use by the intended recipient and contains information that may be Privileged, confidential or copyrighted under applicable law. If you are not the intended recipient, you are hereby formally notified that any use, copying or distribution of this e-mail, in whole or in part, is strictly prohibited. Please notify the sender by return e-mail and delete this e-mail from your system. Unless explicitly and conspicuously designated as "E-Contract Intended", this e-mail does not constitute a contract offer, a contract amendment, or an acceptance of a contract offer. This e-mail does not constitute a consent to the use of sender's contact information for direct marketing purposes or for transfers of data to third parties. Francais Deutsch Italiano Espanol Portugues Japanese Chinese Korean http://www.DuPont.com/corp/email_disclaimer.html This communication is for use by the intended recipient and contains information that may be Privileged, confidential or copyrighted under applicable law. If you are not the intended recipient, you are hereby formally notified that any use, copying or distribution of this e-mail, in whole or in part, is strictly prohibited. Please notify the sender by return e-mail and delete this e-mail from your system. Unless explicitly and conspicuously designated as "E-Contract Intended", this e-mail does not constitute a contract offer, a contract amendment, or an acceptance of a contract offer. This e-mail does not constitute a consent to the use of sender's contact information for direct marketing purposes or for transfers of data to third parties. Francais Deutsch Italiano Espanol Portugues Japanese Chinese Korean http://www.DuPont.com/corp/email_disclaimer.htmlSaltzbart.Ilana@epamail.epa. gov 12/28/2006 01:00 PM To Lori E Stephans/AE/DuPont@DuPont cc bcc Subject Request for DuPont to Resubmit proposed RFPs for Characterization and Biodegradation Laboratories History: This message has been replied to and forwarded. Lori, On February 28, 2006, EPA received DuPont's submission of the proposed RFPs for the Characterization and Biodegradation laboratories. In light of changes that EPA requested DuPont make to certain documents that are part of the Panel Administrator package that are also part of the proposed RFP packages, EPA is requesting that DuPont resubmit the proposed RFPs with the same modifications that were made to such documents in the Panel Administrator package. In particular, such documents include Attachments J, L, and M. Also, please ensure that the scope of work in each proposed RFP is an exact replication of the SEP language. Please resubmit these documents to Tony Ellis on or before February 1, 2006. Thanks. Ilana Saltzbart Attorney-Advisor, US EPA, OECA, OCE 1200 Pennsylvania Avenue, NW, MC 2245A, Washington, DC 20460 Phone: 202 564 9935 Fax: 202 564 0035 NOTICE: This communication may contain privileged or other confidential information. If you are not the intended recipient, or believe you have received this communication in error, please delete the copy you received, and do not print, copy, retransmit, disseminate, or otherwise use the information. Thank You.