Document x10j278NpJx6zpd7kXngzvLLb
"ARRKG -- 137/
ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF
PLANT TISSUES PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN
`WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125
3M Environmental Lab Project No. U2723
AUTHORS:
RayJmoonAnd.LM.aVcGarneHgoovre,nB,.SP.h.D.
`Willard B. Nixon, Ph.D.
STUDY INITIATION DATE: August 28, 2001 STUDY COMPLETION DATE: October 22, 2001
`Submitted to:
3M Corporation
555 uh Ave Environmental Laboratory
St. Paul, Minnesota 55106
Wildlife International, Ltd.
3598 Commerc Drive
Easton, Maryland 21601 (410) 822-8600
Pagel ofsi
002374
Wildlife International, Ltd. 2
Pues Number454.125
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
SPONSOR: 3M Corporation
TITLE:
AnalyticalMethod ValidationfortheDetermination of Perflucrooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues
`WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125
3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723
STUDY COMPLETION: October 22, 2001
"Thisstudywasconducted in compliancewithGoodLaboratoryPractice Standards as publisbhytehde: U.S. Environmental Protection Agencyin 40 CFR Part 160, 17 August 1989 and OECDPrinciopflGoeosd Laboratory Practice (ENV/MC/CHEM (98) 17) with the following exceptions:
Thestablyofthetetsubstanceunderstoragecondiion ate et. icwisnotdeterminedin
accordance with Good Laboratory Practice Standards.
STUDY DIRECTOR:
/
`Van Hoven, Ph.D. Scientist `Wildlife International, Ltd.
1020-0
DATE
SPONSOR APPROVAL:
ee
Sr
lft---- 002375
Wildlife International, Ltd. 2
project Number 454.125
QUALITY ASSURANCE STATEMENT
`Thisstudy wasexaminedforcompliancewith Good LaboratoryPractice Standards as publishedbythe
USS. Environmental Protection Agency in 40 CFR Part 160, 17 August 1989 and OECD PrinciopfGloeods.
aLnabyonrdationrgysPrwaectricree(poErNteVd/oMtCh/eCSHtuEdMy(D9i8e)t1o7)r. aTnhdeLadbatoersaotfoarlylMiannspaegcteimoensnawnedraeudsitaslalnodwst:he dates that
scm, Mori FifsionPrepenion RawDr,Dt ep J
DATECONDUCTED: STUDYDIDRAECTTEORREPORTMEADNTAOGEMENT:
Aus 36,200
J
Oct 1, 12000 15,2001 uber15.2001 Over 16,2001
[----
Cum 00 Ouse
Yn [ 1. 3
Je H. Coleman, B.S. Quality Assurance Representative
1033-01
DATE
002376
Wildlife International, Ltd. a
Pret Number 454.125
REPORT APPROVAL
SPONSOR: 3M Corporation
TITLE: Analytical MethodValidation fortheDeterofmPeriflunorooactatnesiulfoonatne, Potassium Salt (PFOS) in Plant Tissues
`WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125
3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723
STUDYDIRECTOR:
tffo Los L. Van Hoven, Ph.D.
MANAGEMENT:
Hl tr
`Willard B. Nixon, Ph.IJ. DiroefAcnatlytoicral Chemistry
10-21-00
DATE
wh
DATE'
002377
Wildlife International, Ltd. se
TAOFB COL NTEE NTS
Project Number454C.125
TaofbColnteents...
i.
nS
ObECHVe......c
_--
SR
--
9
`ExperimentalDesiga.......
"
S-- --
9
"Tisoue Sump
Fruit Samples
Primary andSecondary StockSolutions.
sn----
ter
2
LimitofQuantitation ......
EH
----
wn NE
Reagentand Matrix Blank Samples ----
Method Validation Samples.
nee
-
Conclusions.
wera
o"--
-
ree 13
inn}
esr
13
002378
Wildlife International, Ltd. i"
pies Number454C.125
"TABLE OF CONTENTS
-Continued-
TABLES
Table 1 - Method Validation Recoveries for PFOS in Onion.
--
16
Table 2 - Method Validation Recoveries for PFOS in Alfalfa..
rw
--_--
Table-:Method ValidationRecoveries fox PFOSin
Tomato..
a
Tabl4e- MethodValidationRecoveriesfor PF inSO oybS ean
APPENDICES
Appendix 1- Protocol.
SI
Appendi2.x Cotifiateof nays
Appendix3 AnaMletyhodtVAiIAconal DBA..
"
19
2
rn
2
Appendix3.1 - AnalyticalMethodFlowchartfortheAnalysisof PFOSin PlantTissueand
Fruit.....33
Appendix 3.2- Typical HPLC/MS/MS Operational PATBMCLETS ...........cvvvrssrssssmssssssssses 38
Appendix 3.3- A Typical Calibration Curve for PFOS.
wwnss----------35
Append3.i4s ExampleCalultonsfor 8Representative Sample...
3%
Append3i5x- Ion ChromaotfoaLgowr-laevmelPFOSStandard...
31
Appendis 3.6. Ton ChromaotfoHgigrhlaevmelPFOS Standard.
3
Appendix 3.7 Ion ChromatogramofanOnion Matrix Blank ......
39
Appendis 33 - Ton Chromatogram of Low-level Onion Fortification Sample.
nd
Appendix 3.9- lon Chromatogramofa High-level Onion Fortification Sample.
werner]
Appendix 3.1-0 lon ChromatogramofanAlfalfa Matrix Blank.
.2
002379
Wildlife International, Ltd. 2.
Priest Number 54C.125
`TABLE OF CONTENTS - Continued -
Appendix 3.11 -lon ChromaotfoaLgowr-laevmel Alfalfa Fortification Sample ................... 43
Appendix 3.1-2 lonChromaotfaoHgigrh-laevmel Alfalfa Fortification Sample..
44
Appendix 3.13 - Ion Chromatogram ofa Tomato Matrix Blank.................
wero
45
Appendix 3.1-4 TonChromatogramof aLow-level Tomato Fortification Sample.....oevrerrsernrserss 46.
Appendix 3.15 - lon Chromatogramof a High-level Tomato Fortification Sample.
47
Appendix 3.16 - IonChrom ofa aSotybo eang Matr rixam Bank...........covvvvv
48
Appendix 3.17 Ton ChromtogramofaLowlevel Soybean FOricaon SSP... 49
Appendix 3.18- Ion Chromatogram ofa High-levelSoybeanFortification Sample...
50
Appendix 4- Personnel Involved in the Study..........
pr
I
.
002380
Wildlife International, Ltd. 5
Prost Number 454.125
SUMMARY
SPONSOR: 3M Corporation
TITLE:
Analytical Method ValifordthaeDetteriminoatinonofPerfluorooctanesulfonate, Potassium
Salt (PFOS) in Plant Tissues
`WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125
3M ENVIRONMENTAL LAB PROJECT NUMBER: Uz723
TEST DATES:
Study Initiation: August 28, 2001
`Experimental Start (OECD): August 30, 2001 Experimental Start (EPA): August 30, 2001 Experimental Termination: October 4, 2001
TEST SYSTEM:
`Tissue Samples (Onion and Alfalfa)andFruit Samples (Tomatoand Soybean)
FORTIFIED TEST CONCENTRATIONS:
OnionandAlfalfa `Tissue Samples
00s (mg ai/kg) 0S50000
50.0
`Toanmd Saoyt beaon: Fruit Samples
(m0g0a5i0/0kg) 0S.o50o0
500
RESULTS:
Samplesoftissuematrix (onionandalfalfa)andfruitmatrix (tomatoandsoybean)were
`each fortifiiendtriplicatewith PFOStoreflectnominalconcentrationsof 0.0500, 0.500,
5.0020d 50.0 mg 2./Kg. Thesampleswere extractedwith methanol,agitated,centrifuged,
dilutedinto theinstrumentalcalibrationrangewithdilution solvent (50%methanol : 50%
NANOpurc water) and analyzed by high performance liquid chromatography triple
`quamdasrssupepcto romlete ry (HPLC/MS/MS). Recoveries ofPFOS inonionand alfalfa `yieldedoverallmeanpercentrecoveriesof96.7% (SD = 6.34; CV=6.56N%=; 12)and
94.6%(SD=4.1CV3=;4.37%; N = 12),respectively. RecoveorfPiFeOsSintomatoand
`soybeansyielded overallmeanpercentrecoveriesof 90.5% (SD= 5.68;CV = 6.28%;
N
co
=
nsi
12) and 95.3%
stingofsolvent
s(,SaDnd=
m4a.t9r2i;x
CblV a=nk
5s.a1m6p%l;es,Nc
o=n
s1i2s)t,i
nrgeospfeuctnifvoerltyi.fieRdeoangieonnt,
ballfaanlkfsa,,
tomato or soybean,weredevoidofinterfering components. Therelativeprecision (CV) of
triplicate fortificoaftPiFoOnSs in plant tissue matrix taken thtr hemeothoudolg ogywhas
typically 5 percent, or better for the 0.500, 5.00, and 50.0 mg a.i/Kg fortification levels.
pec 002381 The relative precision at the 0.0500 mg a.i /Kg fortification level, ranged from 2.28 to 11.4.
Wildlife International, Ltd. 9.
Buses Number 454.125
INTRODUCTION
`This study was conducted by Wildlife Intemational, Ltd. for 3M Corporation at the Wildiife
International, Ltd. analyticalchemistryfacilityinEaston, Maryland. Validation samples werefortifiedwith
Perfluorooctanesulfonate, PotassiumSalt (hereafter referredtoas PFOS)andanalyzedwithhighperformance.
licqeuitdcehmroimnataooftgrPiaFpoOhnyimnapslsanstptecitsruocmse.trVyal(iHdPatLiCo/nsMaSm/pMleSs) wtoeervaplrueaptaetrehdep ndenlryfoboer ftawmemeetaAhuon gdufsocrtte0h,e
2001andOcto4b,2e001r. RawdatageneratedbyWildlife Intemational,Ltd.and acopyof th finalreport
are filed under Project Number 454C-125 in archives located on the Wildlife International, Ltd. site.
`OBJECTIVE
analyseTshoefoPbjRecOtSivieonftplhainsttsitsusduyewsa.sTthoevevraifiydttheedpmeerftohromdawnciellofbauHsPLeCi/nMsS/MuS mpoefthdpoedfoioliogvyerfpolrtatnhte ccotoncologytests 0b conductedwith he estsubstance,
EXPERIMENTAL DESIGN `Tissue samples, defined herein asthe entire plantsystem including leaves and stems,and fruitsamples, definedhereinastheseed-bearing structureof the floweringplant, wereeachfortified atfourdifferent
`cIonntceernntartaitoino]n,s iLindt.ripTlhiceatfeoratnifdiacnatailoynzleedvbelassredaocnkeHdPLtChe/MnSd/eiMpSameetdhcoodnocleongtyzdteovnesloofpesdaamtplWeisldfliifoe
antic plaintp toxaicittyetesdts. Areagentblanksample andmatrixblanksamplesineachmatrixwere analyzedconcurrentlytoevaluatepotentialanalyticalmethodinterferences. Acalibrationcurvewas generated fromanalysesof standard solutions of PFOSwith cachseriesofsamples analyzed.
MATERIALS AND METHODS `Thisstudywasconductedaccordingtotheproceduresoutlinedintheprotocol,"Analytical Method Validation for the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues" (Appendix 1).
`Test Substance
The test substance was received from 3M Corporation on October 29, 1998, assigned Wildlife
International, Ltd. identification number 4675, and stored under ambient conditions. The test substance
002382
Wildlife International, Ltd. "0.
Proict Number454C.125
`wasdescasr awhiitb epoewdedr. Itwasidentifiedas FC-95fromlotnumber 217, withanexpirationdateof
August 31, 2006. Information from the most recent Certificate of Analysis provided by the Sponsor indicated apurity of 86.9% (Append2i)x.
Reag ane d Sn olvtens ts
AllsolventsusedinthisstudywereHPLCgradeorequivalent. AllreagentswereACSreagentgrade.
orequivalent. NANwO aterp(equu ivalr enttc oAS TM Type IIDesignation D1193-91)wasused(1).
Test Systems
TissueSamples:
Onion tissuewasobtainedfromthe WildlifeIntemational,Ltd. GreeinnEahstoon,Muaryslaned,
`on October 2, 2001and identified as lot number 09-07-01. Upon receipt, the bulk onion tissue was initiallyplacedin a-4Cfreezeruntilfurtherprocessing. Thebulkonion tissuewasplacedin a
`cyrogenic (-80C) freezer for approximately 30 minutes, transferred to a Waring blender and blended
until homogenous. Anappropriatenumber of 1.00 galiquotswereweighedfromtheonionhomogenate.
`Theremainderofthe homogenizedonionwasthen transferredto azip-lockbag, and placedintothe4Cfreezerforuseascontrolmatrixinanticipatedplantstudics.
Alfalfatissuewasobtained fromthe WildlifeInternational, Ltd.GreeinnEahstoon,uMarsylaend,
`onSeptember 21, 2001,andidentifiedaslotnumber 12-18-01. Uponreceipt,thebulkalfalfatissuewas
placedina 4 freszruntilfurtherprocessing.Theafl waremovedfromfozenstorage nd
`manuallychoppedusinganEkco 8" chef'sknifeandplasticcuttingboard andthenretumedtothe -4C
frocasr. Thefrozentissu wasthe fhehomogenizedusing a Waringblender. Anappropriate
numberof1.00 galiquotswereweighedfromthealfalfa homogenate.Theremainodftehre homogenizedalfalfawasthentransferredto azip-lockbag,andplacedintothe -4Cfreezerforuscas controlmatrix in anticiplpanat sttuedieds.
ETromiattSoaemspulsese.dwer purchase omAugust 30, 2001 fom Wal-Mart SuperCenter, Milford, Dela, and identified as lot umber 3151 with an expiration date of 53002. The tomatosswereqarierd,
002383
Wildlife International, Ltd. au.
Priest Number 454.125
`placedin aWaringblenderandblendeduntilhomogenous.Anapprnuombperorf1i.00agaltiqueots `wereweighedfromthetomato homogenate. Theremainderof the homogteomnatiowazsethden
transfteoar1r6-e0zd. French-squarebottleandplacedintothe-4Cfreezerforuseas controlmatrixin
anticpilapntasttudeicds.
`Soybeanswereobtainedfrom theWildlifeIntemational,Ltd. GreeinnEashtono,Maurylsandeon `September 6, 2001, and identified as lot number 05-15-01, withanexpiration datcof 9/6/02. The
soybeanswereplacedin Waringblenderand blendeduntilhomogenous.Anappropristcnumberof
1.00 galiquotswereweighedfromthesoybeanhomogenate.Theremoftahehiomognenidzedseoybr ean
ticipated plant studies. `wasthentransferredto azip-lockbag,andplacedintothe-4Cfreezerforuseascontrolmatrixin
Analytical Validation Procedures Wild Ineratona, Lid. conducted a validation tril usin procedures developed at Wide
International, Ltd. (Appendix 3). Matrix blanksandmatrix fortification sampleswere prepiantrisesude
`samples (onion and alfalfa) and in fruit samples (tomato and soybean). A reagent blank (containing
`everythingexcept PFOSandmatrix)wascarriedthroughtheentireprocedureforeachof thefourmatrices.
{`aTbhleteiastsaue spamppleswrere2oe5x0tvrapctmiedawnimdtthhmaeetnhcteanntorlti,faguyigteadt.eTdhfeorfamtinsaimmpulmeosfwe30rmineuxttreascotendawgiyrtahtmoertyhsahnaokle,r
`agitatedfor aminimumofoneminutemanuallyandthencentrifuged.Dilutionsintothecalibraratnigeoonf `the HPLC/MS/MSmethodologywereperformedwith asolutionof 50% methanol (HPLCgrade, 99.94%)
and 50% NANOpurwaeter. Sampleswere thenanalyzedbydirectinjection. ConcentrofaPtFOiSownerse: detebyrrevmersie-pnhasee hidgh performanceliquidchromautsinogagHerwleattp-PahckayrdModel 1100 High Performance Liquid Chromatograph (HPLC) witah Perkin-Elmer API 3000LC Mass Spectrometer
`equippedwith a Perkin-Elmer TurbolonioSnpsoruarcye. Chromaseptaraotiogns r wera eacp hich vedi usic ng
c`aK olue mn 2y 0Bems tmasit lCy2so .0cmo)ln .umAn(me e5t0hmomdlxow2.0chmamti,s3p-ruomvipadretdiiclneAspizpee)nfidtitexdw.itahndatKheyesitnosnteruJmaevneltipnarCyasmgeutearrsd are summainrApipenzdiex3d.2.
002384
Wildlife International, Ltd. 2.
Project Number 654.125
Primary and Secondary Stock Slutons
Alprimaryandsecondarystockpreparationswereadjustedforthepurity ofthetest substance
(86.9%). A 100 mg a.i/mL primary stock solutionof PFOS in methanol was prepared by weighing. 1.1508 gofthetestsubsatndbarinngicngeto afinalvolume of 100 milliliterswithmethanol. Secondary stocksolutions (1000, 100, 10.0, 1.00,and0.100mg a.i.L)of PFOSinmethanolwereprepbayrseeridal
`volumetric dilution from the primary stock.
Calibration Standards and Curves
Calistanbdarrdswaereptrepiareodinn50:50methanol: NANwatO erbypappru opriar tedilc utio nsof
the 1.00 and 0.100 mg a.i./L stock solutionsof PFOSinmethanol. Calibration staonfPd FOa S,rr angd ingsin
concentfrraomt0i.o40n0to 5.00 pg a.i/L, wereanalyzed with cach sampleset. The saanmdmoest
prominentpe responsefo PFOS was uletomonitor PROS inal calibrationandsidy sample. No attempt was made to quay PFOS on the basis of individual isomers components, The calbraion standardserieswas njctd athebeginningandendofcachru,andonestandard was jctd, at a `eiimum, aftr ovry five sample. Liaregrssonequationswre generated using peak ara responses
versus the respective concentrationsof thecalibration standards. A typicalcalibrationcurveispresented in
reAspppoennsdeixi3n.3o. thTehapepcloinccaebnlteratciaorn roefgPreFsOsSionienqutahteisoanmp(lAepspweandsidxete4)r.miRnedebypsurbsteituistoinnceghtrhtoempaaetaokigraarvmesa oflow andhigh calibration standards ar preci in Appendices 3.5 and 3., spectively.
Matrix Blanks and Matrix Fortifications
Selected1.00-galiquotsofeachmatrixtypewere fortified withtheappropriatesecondarystock
solutionsof PFOS preparedinmethanolusing agas-tightsyringe.Thematrixblanksforeachmatrixtype.
------------
Limitof Quantitation
`The method limit of quantitation (LOQ) in each matrix was 0.0400 mg a.i/Kg calculated as the
`prodouftchte lowestcalibration standard (0.000400 mg a.i./L) andthe overall dilution faocftt hemo atrr ix
.
blank samples (100 LIKg).
002385
Wildlife International, Ltd. 1-
Prost Number 454C.125
RESULTS
Reagent and Matrix Blank Samples
Areagentblankand thrematrixblanksamplesincach txweeanalyzedto determinepossible
interferences. Nointerferenceswere observedator abovethe LOQ(Tables 1,23,and 4). Representative ionchromatogramsofonion,alfalfa,tomatoand soybeanmatrixblanksamplesarepresentedin Appendices.
3.7,3.10,3.13 and 3.16, respectively.
Method Validation Samples
`Oniontissue wasfortifiedin triplicateat0.0500, 0.500, 5.00and 50.0 mga.i/Kgresultinginmean
recoveriesof100,910, 7.6, nd98.2,rspestively (Tale 1). Regreionschreomattogaramts oiflvow
`and high-level fortifications in onion are presented in Appendices 3.8 and 3.9, respectively. Alfalfa tissue
`was fortifiedintriplicaatte 0.0500,0.500,5.00and 50.0 mga.i/Kg resultinginmeanrecoveorfi9e6.s4
94.4 92.8, and 94.8%, respectively (Table 2). Representative ion chromatograms of low and high-level
fortificationsinalfalfaarepresented in Appendices 3.11 and3.12, respectively. Tofrm uitwa asfort tifieo din.
triplicate at 0.0500, 0.500, 5.00 and 50.0 mg a.i/Kgresultinginmeanrecoveriesof 87.5, 90.6, 93.4 and
90.6%, respectively (Table 3). Representative ion chromatogramsoflow andhigh-level fortifications in tomatoarepresienAnppetndeicdes3.14and3.15,respectively. Soybeanfruitwasfortifiedintriplicateat
0.0500, 0.500, 5.00 and 50.0 mg a.i/Kg resulting in
espetively(Table4). Representative onchromatogra
mean recoveries
ofowandig
oefv8e7.f9,or9i5i.s9i,o9n8s.i5nanedy99e.2%re,
presineApnpentdiecesd3.17and3.18,respectively.
`Therelativeprecision (CV)ofthe methodology,asdemonstratbeyd the anaoftlripylicastesaimplses.
was percent,orbetterforthe0.500,5.00,and 50.0 mga.i/Keg fortificationlevelsinalltesting matrices. Therelativeprecaittshei0.o05n00 mg 2.1 /Kg fortification level,rangedfrom 2.28to 11.4 perciennthte
four testing matrices.
`CONCLUSIONS
Onion, alfalfa, tomato and soybean were fortified with PFOS to reflect nominal concentrations of
0.0500, 0.500, 5.00 and 50.0 mg a.i/Kg. The samples were extracted with methanol, agitated for the
`appropriate amount of time, and centrifuged. Dilutions into thecalibrationrange of the HPLC/MS/MS
`methodology were performed witha solutionof 0%methanol and 50% NANOpurcwater. Samples were.
002386
Wildlife International, Ltd. a
Proict Number 54C.125
`then analyzed by high performanceliquidchromatography massspectrometry(HPLC/MS/MS). Recoveries
of PFOS inonionyielded anoverallmeanpercentrecoveofr96y.7%(SD=6.34;CV = 6.56%; N = 12).
RecoveorfPiFeOSs inalfalfayieldedanoverall meanpercentrecoveryof94.6% (S=D 4.13;
CV =4.37% N= 12). Recoveries of PFOS intomatoyieldedanoverall mean percent recovery of 90.5%
(SD = 5.68; CV = 628% N= 12). Recoveries of PEOS in soybean yilded an oveal mean percent
recoveryof 95.3% (SD = 4.92; CV = 5.16%; N = 12). Reagent blanks, consiosfstolivenntgs, and matrix `blanksamples,consistingof unfortifiedplantmatrix,weredevoidofinterferingcomponents.
002387
Wildlife International, Ltd. se
Projet Number 454C.125
REFERENCES
I. American SocietyforTesting and Materials. 1991. StandardSpecification forReagent Water.
D1193-91, ASTM Section II Water and Environmental Technology, Vol. 11.01:45-47.
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Wildlife International, Ltd.
-16-
ProstNumber 454-125
Tabl1e
EETo Senge (454C-125)
Method Validation Recoveries for PFOS in Onion
mpi Pct MVeeand Type
Fottified Measured! Recovered' (mgai/Kg)
Pveemo
Recovery
VREB-4
Reagent Blank
<LoQ*
-
-
-
WWAABB-1I0 VAD:
WMeMamebrblxeBekk
000 ls<lloo0ggQ
-
.
-
VVAASS MMuarrFiFfoesaiiomn 000000 000054% 518192 SD-o200o0567 lm VAS3 MuFfesion 000 0OMS 2 Celle
WWAASS 4401 MMauFFoodufelstimn 0055000 00470 WAS2 MaaFediim 0500 ode
5330 sD~osdoostel si 056 ovis
WWVAAAS4SS5 MMaMaFanoFFuofuoeiusaiiinnn S5S000000 444s3%t3 51962s scpv-ewso0ss0e0m3 os
WWVAASA-S4S46S MMMuaaFrmexnPFiooftteesasitiomnn 0500000 9S407714 9410470 cSupweizaolm wa
-- TTFe Re
Sum duiDeve ion- w634 CNE eh
ST og aenSe Vo ey
500 pm ho o tt fbe tl e th: gis 1005 varyslightly.
Thelimitofquantitation(LOG)was 0.0400 mg8../Kgbasedupontheproduct ofthelowestcalibrationstandard
002389
Wildlife International, Ltd.
vr.
Project Number 454C-125
Table2
MethodValidation Recoverifeosr PFOS in Alfalfa
RE To seme (454C-125.)
Type
VREB-3 RegBek
WWWaAabb_sss
MMeumeBlkek Mune
VVMMAASS-4390 MMasmikFFoaficssiioonn
VMASSI MaFafain
nevi -- Pecan Meeawnt ovne
Fortified
0
00 0
Measured'
<iq
<<ii0qq <ioq
Recovered'
--
- (mgai/Kg) -
-Recovery -
000050000 0O0OsF 0050 00M
915043 SD~oo00m036 564 897 Cv-7sime
VWWAASS-2285 MMaaxFrootftetsian 0O8S000 004487 VMAS30 MumFoufaicn 050 04% WVAASS3.I MMauxmFxoFdoensiifoinn S30000 444M0 WAS Muon 300 4m
9946 sp=oam00is su m0 Cv-35me 84159 sp-wswoms ms 953 Ov-dem
WVVWWAAASSS-I3H%5 MMMiuumniFrFoeoudulffsaiiiooonnn | 000000 443s5 92s5e51s scoe sverosws
SwdudDovnsievonors a4W1wm3
gy Re
I i FaNeenh1 Fam ley
coma1 dove Gon robe tt kis100 Lg The limit ofquantitation(LOQ)was 0.0400mg 2./Kgbasedupontheproductofthelowestcalibrationstandard
002390
Wildlife International, Ltd. Le
Proict Number 454C.125
Table3
Method ValidationRecoversfor PROSinTomato
-- e Yo sump (ogew e Posen MeNaemmed PMaecme
(454C-125.)
Type
Fortified Measured! Recovered! (mgai/Kg) Recovery
VREB-1
VWAADB-21 WAB-3 VVMMAASS-?1 VMAS3
ReagentBlank
MMaxsiBBlkak MamBlak MuMFaFroiftofsiionn MsFodfien
o <10Q*
o0 G<loqg oo
0000500 00004439 00X0 0041
-
-
-
--
=
988165 SD-0000008879 8S 821 CV-1loN
WWAAS-SS# MMuucmFFootueasiionn 0055000 004400 WAS6 MumFouaion 0500 045 WWAASS MMuuFmoPtofuifsaiioonn SS0000 442% VMAS MarxPoifation S00 450
WVWAWAASSS_-1I1zI0 MMMauirnrxFaFeooiuffcfssmiiicomnn S0S0000 44455397
855599 sD-0s40s0s2 %06 898 CV-55T %#093 sp-wsozw m e %00 CU=S3m
9@o1l78e csup5e-3isossmm ss
-- Sum ndarie Dotar n w36m8 vNe e`em
TTeeReeioswe edwieVaanven 15 Var
500i0eyenecv tin eofbettkfe100Lg varyslightly.
Thelimitof quantitation(LOQ)was0.0400mga.i/Kgbasedupontheproductofthelowestcalibrationstandard
002391
Wildlife International, Ltd.
"19.
Project Number 454C.125
Tabled
MethodValidationRecoveriesfo PFO in Soybean
--T_o-- samp
@sac125) Type
ening Pct MVeeard PMeetnt
Fonified _ Messed! _ Recovered! __(mgai/Kg) _ Recovery.
VREB-2 ReagentBlank
0 <LoQ* -
-
-
WVaBB-S4 MMaammbBiakk 00 <<QQ --
--
WaB-6 Mumbwk 0 <q -
VVMWAASS-1143 MMwahxFFaoilffsaiioonn 00003000 0000MM) 8999 SD0-400m0)0100 19
VWAS15 MuhFaifsion 000 00 857 Ov-2amh
WVMAAS-S16 MMaaFFooutfaetseisn 00550000 00448 957437 sp0-saomcoms 959 VMASIE MuFoucsion 0300 O18 956 vole
VV VMMAMASSA-221S09 MMMaturaiFxFuFooritiafficiiatimnon 53S.000000 5440B%1 %51060 oSvDe=4l9330s0e0w7 985 AVAATSSRR MMMeaoenfreoFieieiinon 003000 0817 91j40m sopvwsisoemm om
T-- iE y -- Re m STwdeigDov nsivodNneae-n vnsw4iie m35e Fa e r y Thelimit of quantitation(LOQ)was0.0400mg .i./Kgbasedupontheproductofthelowestcalibrationstandard 0013p 113d cv ton Ft ofbet okas (ODL 3Measuredvalueisthemeanofonere-dilution,duplicateinjection, 454C-125-a1nd91R9R12,.
002392
Wildlife International, Ltd. 20.
Appendix 1 Protocol
Prost Number 454C.125
002393
Wildlife International, Ltd. ES
PriestNumber 54C-125
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002394
Wildlife International, Ltd. 2.
Proiest Number 454C-125
Wildlife International, Ltd.
a PEREANOAOLRYSTOILCTAALNMEESUTLHOODNNVTAEL,IFDOATTIAOSNSFTOMRSTAEFD(EFT7E05RMINIPNLAATTITOIFNSSUES
seouson
MFSearogameantmmamesr3y52
SPONSORS REPRESENTATIVE RacesRoto
Tm: E$W5l8mCavomtmone1tt,011.
STUDYDIRECTOR:
p[oeri---- Ray L.VmasHo ovenn , Phd .D.
LABORATORYMANAGEMET VWE ia8tNhelsS ee,idAnDiC.hemie
Fri FORLABORAUTSEOORYLYY
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PROTOCONOL: 4OSCIOIMVFTSUBMSK Evin Libor ReptNorUF2S
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002395
Wildlife International, Ltd. .
Priest Number 54.125
Wildlife International, Ltd.
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PROTOCOLNO: 450STAIMY-FTSUBISH Enema Labor Rep Nore E73
002396
Wildlife International, Ltd. 24
ProsNtumber 54C.15
Wildlife International, Ltd.
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PROTOCOLNO: 450SOUMY-FTSUBS Eminem bryRsNoss 23
002397
Wildlife International, Ltd. 2s.
Priest Number454C.125
Wildlife International, Ltd. -
FriahStoeckiodonkn gm i edwih sick)of ROS cl ittbec vl heron
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------
SUMMARY OF ATYPICALVERIFICATIONANALYSISSCHEME
rire Comes _ TRehmro _ pFiarmaet
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PROTOCOLINO. 4SU0MTOIMYPSUS En abriri ResNes er 2723 002398
Wildlife International, Ltd.
2%.
Project Number 454C-125
Wildlife International, Ltd.
. IrneptroselsdGrivebibesptornoifbbee1yiinss.een serwl SomNefifbio cnosdecieteevdfoedntirvenogi.nomvrdsdexvecire, Sectrtwei hodveeeiennvsehi Soymre.eUfpmsmybobfbnsmsdofvmeln,,Se mctin trnr.edovae dis.Toe epi gsofrei 0 0of [--On.lion uswil clei sti. peinct fe [(oeEselns ilidsrtby vito----rep--os--pka--e --i epsi--ns--i Saninii moedit or il ce 0 kr ion cho
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PROTOCOLNO: 4SUUSIOINYPSUS Exons Labora Ri NoesUE 002399
Wildlife International, Ltd. a.
Project Number 454.125
Wildlife International, Ltd.
Theo mi Fogcsa.navrorrer i,prs crissb, rt ein mc ids ed ee nd it nl fo spypr ie to and themethodsandinstrumentemaptlioyoend. Uponreceiptofthesefindings, theSponsorwill [ ThmR pe ih bo--i --,vi: 3[Ovlietn--pr--t--nd--nh--pe--rlpric, dnty hes 0
Ttgtmemprsentedoosydom spdteinchin a, hen sn mnbo,cr ot bae,nrch,,psya,ycoemdpnetibond,oafpofrmat,otote oioen, 5. prdoeatd eoty--of Bh sil olny Adi of n pei ects,creclio,alpanic(srswigs ed em) Mcdmuepiaonfnmtoe ot ay aefc hogy eg ne. instrumentation employed,reagentsandsolvestsused,classofwaterused,andanymajor 7. TtoahmpeifenSpyeDee,vemt enof er nispont, od nn 5. TAhsdpaesdnuedrritsno,faccohoifn,mpiilonspmtscsa rord.ics 10, Tc hoinh lsryropwt ndF pe esd ocho 1. mtrr bnCotyAsseUk ing hedaety pei
weremade ndthedatosofany findings reportedtotheStudyDirector/Management.
PRO NO.:T 454/0O 50201C /MV-PO T/SUBL A4S4
.
EnvLiaborratooryaReqmueset Nn umbterUa27l 23
002400
Wildlife International, Ltd. a.
Project Number 454.125
Wildlife International, Ltd.
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PROTOCOLNO: 45K FTSUBASA Enicmm uberiryRest Name 723 002401
ildlife International, Ltd. -
projec Number 540.125
Appendi2x CertifofiAcnaalytsies
INTERIM CERTIFICATE OF ANALYSIS
CentsAntaPLoRRaeeavtibas:imooancP1:erRC(9OO/SSs7AB,/0L00tRo1)t8eo21i7ins: 02318
ry
[Ff oeEr erieo iiee eeseem iwwwooewaal) |
[Appearance 7
WhiteCrystallinePowder |Coulorms |
Niae s(CPS) l IAs rs SIRT E----
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orm
B[EEEGLEEERRIEREERiOE 3Loovmeet
i = Een [HROeUSiSEnL ESIES]E; toemk
[T[oo topt iOOe M)J| TRaE TrapE a eaLtTos wn
Phme CESSES [ResidualSolvents (TOA) roe =Rte SGT ST ==|
[Puomyi1c. ByCDhliSoreiCdse 10) l|(gBEaRrTiBeEraReEle baee erRe PateEhrTa!T
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NotAppliable' |
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consaina
rte 002402
Wildlife International, Ltd.
-30-
Prsect Number 54.125
CeInNtsTAEnRaIlMyC aaEbRoTrGI sOAF tROefI oeFrerC AncNesA ALT YCSZE IS0S10A
DateofLastAnalysis: 08/31/00 `ExpirationDate: 08/31/01
StorageComins: Fron10C Re-useDe: 043101
Fo"iPeu,=1r00S0i%-At(sDyumofpmaetialnim:puroitsieso, 1.45% +sLdC/MmSpimuptuirieti0es0,89 .P+O4AInRor1,gan%ic:
033%)
"Total iiympu=ri1t0y0f%rom1a3l.l0t7e%sts=$6135.509,%
Potassiumisexpectedinthissai form and isthereforenotconsideredanimpurity. PuritybyDSCisgenerallynotsppicabletomaterialsoflow purity.Noendothermwas
`obsfoer thrisvsameplde.
{C"oSeouorlfmuirimnathtoeosnatmpelheadpmpceeaortnstfsoible.cyTon,oveardtoeindntorceSsOe4aanerdehsetnwoceelsdewteeictnpeduissinrg,theBased ono eth S07 ot ordsp.
STWTFPAB.AAA "HR PTrpifeulutoe raooanceatbrcie cdisiocd id
"Theoreticalvaluecalculationsbased oatheempiricalformula, CoF17SOyK*(MW=538)
ThisworkwasconductedunderEPAGood LaboratoryPracticeStandards (40 CFR160).
conmann
J
002403
Wildlife International, Ltd. a.
Prost Number 54.125
enItrNeTAEnRaIyMaCLEaRboTaiIoFriIcsCCAOTAEReOfFerAeNeA:LYG2S3I.0S10A Lpour s rosie:
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SiSE,EI Cars Anyi-- Lab-- orsors ReviewedBy: Ty ______
aP aa
conmsorsa
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002404
Wildlife International, Ltd. a.
Appendi3a Ansyical Method ValidationData
Priest Numb 454.125
002405
Wildlife International, Ltd. a.
Price Number 454.125
Appendix 3.1 Analytical Method Flowchart for the Analysis of PFOS in Plant Tissue and Fruit
`wei`ghWtesi.gFhoarpmpartorxiixmaftoerltyifi1c.a0t0iognrsaammpolfehso,mfoorgteinfyiezaecdhplsaanmtpmlaetwriitxhptehresaapmpprloepriinattoeavtoalruedmevioalf. PFROeScorsdtock
`solution in methanol with a gas-tight syringe. The matrix blank is unfortified plant matrix.
4
For each sample, measure 10.0 millilitersofmethanol with a Class A volumetric pipette and transfer into thevial. Add 10.0millilofimteteharnoslintoanemptyvialandprocessasfortheplantsamples. The
`additional sample is the reagent blank. 1
ForOpl3an0t miissnuutseamatplspep,rcoaxpimvaailcslsyn25d0pplmc.onFoarshhaekeirttabslaem.plAelsl,ocwtphvesiasmpaldessthoaskhekmeaonruasmfionimum niumofoo mime
+
Centrifuge samples for approximately ten minutes at 2000 rpm.
4
Dilute samples into the calibration rangeofthe PFOS LC/MS methodology: Partially fill Class A
`volumetric flasks with dilution solvent (50% methanol: 50% NANOpurewater). Add appropriate `volumeofsamplewith a gas-tightsyringe andbringtovolumewithdilutionsolvent.Processmatrixblank
`samples using thesamedilution and aliquot volumes as for the lowest fortification level. 4
Ampulate samples and submit for HPLC/MS/MS analysis.
002406
Wildlife International, Ltd.
4
Project Number454C.125
INSTRUMENT:
Appendix 32
`Typical HPLC/MS/MS Operational Parameters
`Hewlett-Packard Model 1100HighPerformanceLiquid Chromatograph
`with aPerkin-Elmer API 3000 Mass Spectrometerequippedwith aPerkinElmer TurbolonSpray ion source. Operated in multiple ion reaction `monitoring (MRM) mode.
ANALYTICALCOLUMN: KeystoneBetasil Cycolumn (50mm x 2mm LD.,3-um particlesize)
`GUARD COLUMN:
Keystone Javelin Cy column (20mm x 2mm LD.)
`OVEN TEMPERATURE:
STOP TIME:
40C
500mimes
FLOW RATE:
MOBILE PHASE:
250 pL/minute
70.0%Methanol
30.0% NANOpure Water containing 0.1% Formic Acid
INJECTION VOLUME:
PROSRETENTIONTIME: PFOS MONITORMAESDS:
10.0 ul.
Approxim4.atmienulteys:
499.0smu 99. amu
002407
Wildlife International, Ltd.
-35.
BuseNsumber 54.125
Append3i.x3
ATypical Calibration Curvefor PFOS
a05io499.0:55r.1eNoo n=emssazSot4asn1den ESorPe RsoE158R315 asescs
ao sooo 4000 sooo 2000 1000
eR TR Re eR ih kk ww Govng aien.
002408
Wildlife International, Ltd.
-36.
Project Number454-125
Appendix 34 Example Calculations for a Representative Sample
Sample number 454C-125n-omVinaMlAconScen-tr1ati,onof0.0500mg ./Ke YPeeianktArrceeapt==8348653.6709 PSrliompaer=y D1i2l3ut4i0o8n:760
InOirtiigailnaWleiFignhatl(VWolu=me1(0V,05)= 10.0 mL. Secondary Dilution:
FIniintailalVVoolluummee((VV))== 110.000mmLL Overall itionFactor(VW x ev) ~9k -- 1k
in Tomato
PROS at i. nstrument (ug 8/1) _ (peak areaS--eyx-nteroept)
_ (846152-324308837.660709 _ 04s2BL gai PROS in sample (mg ./Keg) = cPoFnOveSrsaitoinnsftacrtuomren(t0.(0u0g1.m.g/Ls))x overal dilutionfactor (Lg) x
PFOSinsample (mgaig)- L273 182 100, 000m1g
_ 0045278mga
Kg
PercentofNomin. al Concentratiio on Po FOS (mR g/L) imnssaammppllee 1g
SooS m 100=98.6%
Instrument Software: MacQuan, version 1.6
002409
Wildlife International, Ltd. x.
Proect Number 454.125
Appendi3.x5
on Chromatogram ofa Low-level PFOS Standard
oPronstoalGmak0ug ein
Astipatnos
Rtoanna Sancars
L4o37de 23 can
NomhSSisoehEmievees. 21880
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=
W8T7
TeeOuia ton
"
nant: 6000con
.
poe
p
a:
p o
Bsa uss maAs s
To ta Sh Sh Th da Them
`Nominalconcentration: 0.00m0g4a.0i/0L.
002410
Wildlife International, Ltd. 8.
Project Number 454.125
Appendi3.x6 lon Chromatogramof a High-level PFOS Standard
rpcossI amin
rete.
t|o-- n
im fJunpa.ry--
FnAh
Fo EEolTnoen i0n
=E,So.we
wm i
Hie a
Tie ouzmoron
a:on
a
p
5h
;:
ouwg
i"
Th Th Th To TE
Nominal concentration: 0.00500 mg a.i/L
002411
Wildlife International, Ltd. 3.
Prec Number 54.125
Appendix3.7
Ion ChromofaantOnioonMgatrrixaBlamnk
wnossie VAT
Loam tps
Rneinnsantas
TSf: o47gmn
0wre 2303
a ven8
ASSSorhiroeo i130n
fam
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000
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EALERTS a,
Samplenumber454C-125-VMA11B.Thearrowindictstheapproxima rtcon tieof EOS.
002412
Wildlife International, Ltd. "0.
ProjectNumber 54.125
Append3i.x8 TonChromatogram ofa Low-level Onion Fortification Sample
orosnie ASB
ffoEksSantos
fa L1:4o7oshe
8sn 20
i m m a i l135
ESaruatneng in
pilwe den 5087
ee
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I"
[p----
a
,.
.
:
:.
LE ono gs ee 2
Ohh SS Les
S1a0mpUlKegumber 454C-125-VMAS-38, 0.0500mga./Kgninal concentration, vealdluonfactor =
002413
Wildlife International, Ltd. a
Proje Number 454-125
Appendix 3.9
lon Chromsiogorfaam ighlevel Onion Forication Sample
verosaas VMASAT
A asin pores
tom
P: 4rd527s,re
Noise Thves.
mnmee
2333320
MTTaimenees 3i10n
Hawersrs
Slant Time.
ass
Ffooreteian 33X081
tograton Type
WedOua
met a
" . . pp s
k*yOEPT
Go She a Wn
SN
pe
i re as
Bm Us Thm
2`5Sa0m0pl0eLugm.ber454C-125-VMAS-4,50.0mg Kg nominalconceniration, overall lution ia=
002414
Wildlife International, Ltd. a.
Pres Number454.125
Appendix3.10
Ton Chromatogramofan Alfalfa Matrix Blank
pmoistoN WAM
-Agni tol
us ----
L+: 4a rrw,258m
Nopm E sisaemvhr1heesi . 2i20
HH [myeg-i J3
FN
Stan Timo.
000
-- egWa 8G0%0
isgraicn Type.
Tue.aomu0zs
, p
he
p p
=
52
' ww
the
os_1s3 tes
the Hh ho
ay, 0 50
| spe ans
By We Wm
Samplenumber 454C-125-VMAB-8. Thearrowindicatestheapproximateretentiontimeof PFOS.
002415
Wildlife International, Ltd.
a
Project Number 454.125
Appendixaan lon Ciomsograof aLowlve Alfa Fortification Sample
mposes vse gon sus Saebe-- r 1b: 3d7o,20cn fEtami NoiseTrves. 2200 W TDraotewi 3i10n fo
aout2m v0 . :be Ppp a:
[----
oe etagesion,Whit asls
22 To T9e5 Th S1h68 1B98h 4265ThE
`Sample number 454C-125-VMAS-49, 0.0500 mg a.i/Kg nominal concentration, overall dilution factor =
100L/Kg.
002416
Wildlife International, Ltd.
a
ois Number 454-125
Append 312
lon Chromatogramof a High-level Alfalfa Fortification Sample
muoahn vss Twoassono
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fEnmHmiaiareye]n0
; :
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Th
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ue Pe Bh UW dh EE
Toto Samplenumber454C-125-VMAS-34, 50 mg. a.i0 /Kg nominal concentration,overalldilution facto=r
002417
Wildlife International, Ltd.
ws.
Project Number 454C.125
m=oen was: JA iarm in ca sF amooYu NoiseThres.. 20 [FB ihBeaeTe 1|5 Start oeTims pAase [Eo {ctageaton.Wigs
Appendix 3.13 lon Chromatogorfaam Tomato Matrix Blank
swmoreee
..
o
: p 3;
Gh 1241
2
Rh__ 135
th159 184
[------
l 27OW A285 m
`Samplenumber 454C-125-VMAB-2. Thearrow indicatestheapproximateretention time of PFOS.
002418
Wildlife International, Ltd. Le
Pues Nommber 454.125
Appendix 3.14 lon Chromatogram ofa Low-level Tomato Fortification Sample:
[ nn EO --
mnt
J nl
\
Jop se
.
Mof E[[ise-aTihr v-esN. a210
,of
Fives 1
s
sy00 5
ESxoepaecwtneedArTos
m2
s3w
Enwon 332
"a: :
I.
Le we mis who
iagruion Type A-88
the fhe Hh Hh FH We Uh
100Uxg `Sample number 454C-125-VMAS-1, 0.0500 mg a.i./Kg nominal concentration, overall dilution factor =
002419
Wildlife International, Ltd. a.
ProjectNumber 854.125
Append 3.15
Ton Chromatogramof a High-level Tomato Fortification Sample
pwosos mast swept, zn
Loni us
kF=am sans
\
os0,con
1:Lo7 prw,258wn
.
NSSmoisoemThhmrses. 211023
a; .
AaSroinhmee+ 11d0n
s: a
a
jor
Ste StartTime.
=ass.
iB
ogratonWha 054
oh2e 7393 120 tse KX 01 2
Inegrton Tipe A-VE
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2S5am0p0l0eUnKum.ber 454C-125-VMAS-11, 50.0 mg :/Kgnominalconceiraton, overall lution ctor =
}
002420
Wildlife International, Led.
"as-
Appendi1x6
IonChrom ofa aSot ybeoang Matr rixa Blamnk
moa was osrmu.sses
mf fnsia er
"
n7y oe
NsgmmFoimsremaThuvnh-s. 20
hhpd p
LEa---pem in
: .
Sine as
=
E[eEeEian 50
bTh he BE
Project Numberasic 125
ry00 |
dm
`Sample number 454C-125-VMAB-4. Thearrowindicates the approximate retention time of PFOS.
vuz4z1
Wildlife International, Ltd.
5.
Project Number 454-125
Appendix 3.17
Ton Chromatogram ofa Low-level Soybean Fortification Sample
mrosHs was
fi-hsoeompi
emwg5. 4nnaoe57a,08
nw
ETHiniernTs i36n
= StanTime:
bi350
Te
monroe
nt 0.00
p
.
.
p:5
2:
Wh
EEE EEE
firs Samplenumber454C-125-VMAS-13,0.0500mg 8.i./Kgnominal `concentration, overall dilution factor =
vue
Wildlife International, Ltd. "50.
Buses Number 834.125
Appendis 3.18 lonChromoafatHioghglevrelSoybeanForfcation Sample
jmossasA Masa Fa sew mon vem
pr
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"
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p
She
2
EreKlin ian 3
3 2039 75 102124 154175, 250
InrtegirantionTypeee AS85
060
TR
138
TE
203
a
270 337
2H se
404 471 Time
2Sa5m0p0l0eLsgum,ber 434C-125-VMAS-24, 50.0 mg Kgnominalconcntation, overall dt cor =
<u2423
.
Wildlife International, Ltd. sie
. Projet Number 454C.125
Appendix 4
Personnel Involved in the Study
Th folowingkeyWildentermational, Li, personne wee involved nthe cono dmanuagcmetnt
ofthis study:
21.. RWaiyllmaordndB.L.NiVxaonn,HoPvh.eDn,,,DiPrheDc,toSrc,ieAnntailsyttical Chemistry
3. Jon MacGregor, B.S., Scientist 4. Frank J. Lezotte, B.S., Chemist
002424
