Document LpXmdXqga4Q43a212ExXMqVwd

AR226-2688 AR226-2688 A i- U itf f . v r - ! ----- - !. * 5 . i BIODEGRADATION STUDY REPORT R e visio n 1 W ednesday, November 06,2002 Biodegradation Screen Study fo r Telom er-Type Alcohols project Number 3M Projects ID: E01-0684 Pace C ontract A nalytical Projects ID: CA085 Stu d y D irector C leston C. Lange, Ph.D. C l ie n t Jam es K. Lundberg, Ph.D., 3M Environm ental Laboratory Bldg 2-3E-09, P.O. Box 33331, St-Paul, MN 55133-3331 CONTRACT laboratory Pace Analytical Services, Inc. B io-Analytical Services Group 1700 Elm Street, Suite 200 M inneapolis. M innesota 55414 Project Dates P roject Initiation: A ug usts, 2000 Project C om pletion: Septem ber 18,2001 A u thor C leston C. Lange, Ph.D. Page 1 b f 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols' -R evision 1 Study Director Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group O rtM T A IM O L J In IM U v Wednesday, November 06,2002 .!/-% In LJ CBI 0#0003 EID839548 Table of Contents T itle P age....................................... Table o f C o nten ts......................... List o f Tables & F ig u re s.............. List o f A p p e n d ic e s ................ Executive Sum m ary............................. 1.0 P roject P e rso n n e l.......................... 2.0 D ata Requirem ents and Revision 3.0 P roject O bjective............................ 4.0 T e st A rtic le ...................................... 5.0 Reference A rtic le s ........................ B.O R eceipt/G eneration o f Sam ples 7.0 M ethods..................................................... 7.1 Sam ple P rep ara tion......................... 7.1.1 C ollection o f S ludge................ 7.1.2 C ulture P reparation................. 7.1.3 S olid Phase Extraction (SPE) 7 .2 Instrum ental A nalysis (HPLC/M S).. 7.2.1 Instrum ent P aram eters.......... 7.2.2 Q ualitative A nalysis o f Parents and Products 7.2.3 Q uantitative A na lysis......................................... 7.3 D ata T ransform ations and C alculations........ 7.3.1 M olar C a lculatio ns... 7.3.2 C onversion o f ng/m L to m icrom olar and 7.3.3 M olar M ass Balance 7.4 Softw are V e rsio n s............. 8.0 R esults........................................ 8.1 Sludge C haracterization.... 8.2 Q uality C ontrol/S am ple M atrix Spike Results 8.3 A nalytical B lanks R esults.................................................. 8.4 HPLC/M S A na lysis............................................................. 8.4.1 Q ualitative HPLC/MS and HPLC/M S/M S Results 8.4.2 Q uantitative Analysis Results fo r PFO A......... 9.0 C onclusions.......................................................................... 10.0 Literature C ite d ...................... 11.0 Sam ple and D ata Retention . ... 1 ... 2 .... 3 .4 .5 .6 .6 ...6 7-8 ...9 ...9 ...9 ...9 .1.91--1102 12-13 13 ..13-14 ..1 4 -1 5 .......1 6 .......16 ,! IT,.--ft C a ta iia liw s .. .17 18 ,,1 8 .1 8 .18 .19 ....19-24 :iiS fui i ....2 4 -2 5 .27 .28 .29 Page 2 o f 38 Pace Project CAQ85: "Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Blo-Aralytical Services Group Wednesday, November 06,2002 000004 EID839549 List of Tables & Figures T ables T a b le 1. Parent telom er alcohol expected io n s ...................................................................................................14 T a b le 2. Expected product fluorlnated fa tty acid products io n s ....................................................................... 15 T a b le 3. HPLC/MS D ata T able fo r Parent and Product Ions o b se rve d ......................................................... 23 T a b le 4. HPLC/M S/M S D ata fo r fi-O xIdation Product Ions ob se rve d .............................................................24 Fig ures F ig u re 1. C hem ical R epresentation o f Telom er A lco h o ls...................................................................................8 F ig u re 2. C hem ical S tructure o f perfluorooctanoate (PFO A)........................................................................... 9 F ig u re 3. Plotted HPLC/M S integrated Peak Areas fo r Ca, C ,0and C 12 Telom er A lco ho ls........................20 F ig u re 4. Plotted HPLC/M S Integrated Peak Areas fo r C * C M and C 18 Telom er A lco ho ls........................20 F ig u re 5. Plotted HPLC/M S Integrated Peak Areas fo r P erfluorinated F atty Acids D e te cte d ................... 22 F ig u re 6. Plotted P eak A reas fo r Transient P olyfluorinated F atty Acids D etected.......................................22 F ig u re 7. P lotted PFO A C oncentrations M easured in B iodegradation Sam ples (SPE elua te 2 ) ............. 25 F ig u re 8. Proposed Biodegradation Pathway fo r Telom er A lco h o ls....................................................... ....... 26 `v , i-i ss? M e" . o ` J >.' Page 3 of 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 000005 EID839550 List of Appendices A p p e n d ix A : A pproval S ignatures...................................................................................................................... 30 A p p e n d ix B : F inal Q ua ntitative Results fo r PFOA (ng/m L) T a b le 1 . T e st C ultures Final R esults .......................................................................................... 31 T a b le 2 . A b io tic C ultures Final R esults........................................................................................31 T a b le 3 . B lank Cultures Final R e su lts.........................................................................................31 A p p e n d ix C : Integrated Chrom atogram Peak A rea D ata fo r the Telom er Alcohols T a b le 1. T elom er Alcohols Peak A re a s .......................................................................................32 T able 2. Percentage o f Peak Areas For Telom er A lcohols Rem aining a t T im e P oints........32 A p p e n d ix D : T a b le 1. Integrated LC/MS (SIR ) P eak A rea Data fo r Perfluorinatecl F atty A c id s............. 33 A p p e n d ix E: T a b le 1. P eak Area Data fo r T ransiently Form ed P olyfluorinated Fatty A c id s .................... 34 A p p e n d ix F: T a b le 1. T otal Ion Chrom atogram (TIC ) fo r SIR D ata fo r Biodegradation S am ples........... 35 A p p e n d ix G : T a b le 1. T IC s fo r SIR Data fo r Day 0 and Day 16 Biodegradation Sam ples....................... 36 A p p e n d ix H : T a b le 1. TIC s fo r SIR Data fo r D ay 0 and Day 16 A biotic C ontrol S am ples........................ 37 A p p e n d ix I: T able 1. TIC s fo r Extracted Ion D ata fo r Transient Polyfluorinated F atty A cid s................... 38 Page 4 o f 38 Pace Project CAQ85: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange. Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 000006 EID839551 Executive Summary A screening study w as undertaken to determ ine w hether the fluorochem ical telom er interm ediate (telom er alcoh ol) biodegrades when exposed to m unicipal w astew ater treatm ent sludge. The study included the preparation o f cultures fo r a six sam ple-point com parative study, and included te st cultures, blank cultures, and control cultures. The m icrobial inoculum fo r cultures was sludge obtained from the Tw in C ities M etro W astew ater Treatm ent Facility. Telom er alcohol test substance w as added to cultures a t approxim ately 5 pM concentrations. C ultures were incubated w ith shaking a t 25C. Solid phase extraction o f cultures fo r recovery o f biodegradation products and parent analytes was em ployed. A n HPLC/M S analytical m ethod w as developed fo r analysis o f the telom er alcohols and expected products. The HPLC/MS analysis o f cultu re extracts provided strong evidence that the telom er alcohols were biodegraded. T he observed loss o f telom er alcohols occurred concom itant w ith the appearance o f several expected perfluorinated carboxylic acids. Unexpectedly, transiently form ed polyfluorinated p-oxidation interm ediates w ere observed. The p-oxidation pathw ay was suspected to be the m ajor route o f biodegradation resulting in the observed even-num bered carbon chain length perfluorinated fa tty acids. M inor end products, as odd-num bered carbon chain length perfluorinated fatty acids, were also observed and w ere like ly the products-of fatly acid a-oxidation, a m inor pathway o f fatty acid m etabolism usually observed in;branched-chain fatty acids. A fte r the 16day test period, perflourinated carboxylic a d d s ranging from C5 to C 12 w ere observed in the test cultures. These com pounds w ere not in controls o r blank cultures. Perfluorooctanoate (PFOA), was the only com pound quantitatively analyzed and, based on m ass balance data accounted fo r approxim ately 6-7% o f the total telom er alcohols initially present in ttie te st cultures. Page 5 o f 38 Pace Project CA085: ` Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Claston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 000007 EID839552 1.0 P ro je ct P erso nn el 1.1 Sponsor Company 1.2 S ponsor Representative 1.3 C ontract Facility Personnel: 1.3.1 Study D irector 1.3.2 Laboratory Management: 1.3.3 Sam ple Preparation 1.3.4 1 .3 .5 1 .3 .6 HPLC/MS Analyst Sam ple Custodian: Report Author 3M Dr. Jam es K. Lundberg Dr. C leston C . Lange M r. Bruce E . W arden M s. Angela L. Schuler Dr. C leston C . Lange Ms. Angela L. Schuler Or. Cleston C. Lange D r. Cleston C . Lange 2.0 Data Requirem ents and Revision Justification The sponsor representative desired an aerobic biodegradability screening study to be conducted using the telom er alcohol m ixture as test substrate. The study was initiated on August 9 ,2 0 0 0 as a non-GLP study. The report issued Friday, Novem ber 16, 2001 is revised to substitute "D upont Zonyl BA type Telom er, o r Zonyl type Teiom er" with Telom er. The reason fo r the revision-jissto w *; tyj- m ore accurately reflect the im pact o f results to the general class o f telom er alcohdSicuraW y reflect to * 3.0 P ro je c t O b je ctive This study was conducted in order to elucidate w hether the fluoroteiom er interm ediate (teiom er alcohol) is biodegradable under aerobic conditions using a m icrobial rich inoculum o f m unicipal wastewater treatm ent sludge. This study was sim ila r to earlier studies conducted fo r 3M by Pace as Pace projects CA05B 1 and CA097 * and CA104 *, CA105 *. and CA132 s. The developm ent of an analytical m ethod and the analysis o f parent a n a lyte i and possible products were critical param eters for the determ ination of biodegradation. Page 6 of 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday. November 06.2002 000008 EID839553 4.0 T e s t A rtic le T he test a rticle used fa r this study w as the Zonyl BA-type telom er alcohol fluorochem ical surfactant interm ediate. Two sources o f te s t m aterial w ere used fo r this study. The firs t source w as provided by the sponsor com pany. 3M, as approxim ately 2.5 g o f crystalline solid test m aterial labeled Zonyl BA-N telom er alcohol, 3M tracab ility num ber TN-A-2186, on July 7, 2000. The sponsor did n o t provide an M SDS, a chain o f custody, the purity inform ation fo r the test article, nor an expiration date fo r the m aterial. T his m aterial was given a test, control, and reference (T C R ) num ber a t Pace as CA-TCR02-Q09 and was stored a t 4C . M aterial CA-TCR 02-009 was used prim arily fo r m ethod developm ent purposes o f the study, including the firs t se t o f cultures prepared and original LC/MS m ethod developm ent. A com m ercial vendor, Aldrich C hem ical Com pany, provided the second source o f telom er alcohol used. M aterial was purchased as Zonyl BA-L fluorotelom er interm ediate (1999 A ldrich C atalog num ber 42,151-0), also called perfluoroalkylelhanol. The m aterial characteristics w ere boiling point 145-245C, F(CF2)nCH2CH2OH w here n equals approxim ately 7 to 8, and m olecular w e igh t was reported as M ,, ca. 443.70 w t. An expiration da te and the purity, o r percent com position, o f the m aterial were no t provided. T he purchased m aterial was received a t Pace on Septem ber 22, 2000. Upon receipt a t Pace, th e m aterial was given tra c fb ility num ber CA-TNCOO-254 and was stored at 4C, o r less. A representative chem ical com pilation o f the Zonyl BA-type telom er alcohol is shown in Figure 1. *.- i ; g w ; recabjMty ft* >~m m chem ical com eitita Page 7 of 38 Pace Project CA085: 'Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday. November 06,2002 00000*1 EID839554 F *F -F F Molecular formula = C8H5F9O H + K _ cf f r f '-- o" Molecular W eight = 264.089 JM + CHjCOO "J ` = 323 FFFF FF Fl l F| F |F IF |F \ ' ----<* " I Molecular formula =C8Hs F130 Molecular W eight = 364.104 [M + CH3C O 0 1 ' = 423 FFFFFFFF FFFFFFFF Molecular formula = C10H5F ,, O Molecular W eight =464.119 {M * CH3COO T = 523 FFFFFFFFFF FFFFFFFF Molecular form ula C12H5F210 '1 2 Molecular W eight = 564.134 [M + CH3C O 0 1 9= 523 PFFFF FFFFFFF F F F F F F W tK o FFPFFFFFFFPF FFFFFFFFFF Molecular formula = C ,, H5FJ5O '1 4 Molecular W eight =664.149 [M + CHjCOO T = 723 Molecular formula = C1&H, F j, O Molecular W eight = 764.164 [M + CHjCOO T = 823 F ig u re 1. The tetom er alcohols. Based on the HPLC/MS average peak area response observed in the six abiotic control cultures, the com position o f the test m aterial w as 6.4% + 0.3% as CBtelom er alcohol. 39.3% + 1.5% as Cb, 27.7% + 2.1% as C 10, 17.5% + 1.2% as CI2, 7.2% + 1.0% as C 14, and 2.0% + 0.2% as C ,B- Page 8 o f 38 Pace Project CA085: ` Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Blo-Anatytlcal Services Group Wednesday. November 06,2002 OOOOjLO EID839555 5.0 R eference M a te ria ls The sponsor provided reference m aterial. The neat m aterial w as stored at -80C. NS: E xpiration date no t specified 5.1, P erfluorooctanoate Am m onium S alt (PFOA) P urity: 95.2 % ; 3M#: TCR-99131-37; Pace fk C A-TCR 02-001; Expires: NS F FFFFFF F ig u re 2 . P e rflu o ro o c ta n o a te a m m o n iu m s a it 8.0 R e ca io t/G e n e ra tio n o f S am ple Sam ples w ere not received, but were generated as an inherent part o f this study. A ll of the experim ental cultures prepared for each culture set w ere extracted by solid phase extraction (S P E ) m ethodology to generate three analytical sam ples per culture. The analytical sam ples, as SPE eiuatss collected for each culture, w ere labeled as SPE eluates 1, 2 o r 3. O f the three eluates generated fo r each sam ple, eluate 1 was the 25 m L aqueous sam ple eluate and eluates 2 and 3 w ere 25 m L m ethanol eluates-ifrorm the SPE cartrid ge . Q ualitative and sem i-quantitative HPLC/MS analysis was conducted fo r eluate 2 only, and the resulting data was evaluated to determ ine w hether biodegradation o c c u rre d . 7.0 M etho ds 7.1 Sam ple Preparation 7.1.1. C o lle c tio n o f S ludge. The sludge fo r this study was obtained from the Tw in C ities M unicipal waste treatm ent fa cility in a m anner consistent w ith the sludge collected fo r other studies listed in section 3.0. T he sludge used fo r the preparation o f the firs t se t o f cultures was collected on July 31, 2000 and Page 9 of 38 Pace Project CA085: "Biodegradation Screen Study forTelom ef Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 O O O O ll EID839556 delivered with Pace chain o f custody (CO C) form 465254. The sludge fo r the second set o f cultures was collected on Septem ber 18, 2000 and w as accom panied w ith COC # 528791. S ludge used fo r the second set o f cultures in this study was also used in preparation o f cultures fo r other florochem ical biodgradation studies (C A105 * and CA132 5), and was shown to be active fo r biodegradation o f oth er florochem ical com pounds in those studies. T o prepare cultures, sludge was obtained from the prim ary m unicipal w aste treatm ent fa cility in the Twin C ities are a. Arrangem ents were m ade fo r Pace personnel to retrieve fresh m ixed liquor suspended solids (M LSS) from the aeration units a t the Tw in C ites M etro W astew ater Treatm ent Facility located in St. Paul, M N. T ypically, Four lite rs o f MLSS was collected by Pace laboratory personnel and delivered as fou r 1-liter Nalgene polypropylene bottles containing M LSS, and w ere accom panied w ith a corresponding chain o f custody w ith date collected. Upon receipt a t Pace Science S olutions, the individual bottles w ere labeled #1 through #4. The suspended solids in the bottles w ere allow ed to settle at least 24 hours a t 4C + 3C. The settled solids "sludge" w ere then used to prepare M LSS plus sludge fo r use in preparing te st cultures. A sludge . , characterization analysis was not conducted as pa rt o f this screening jw e a r i28t<'. study. -'iiov. The settled sludge in each bottle constituted approxim ately 20% o f the volum e, o r approxim ately 200 m L volum e in a 1-lite r bottle, based on visual observations-and was consistent w ith observation o f earlier sludge c o lle c tio n s . Page 10 o f 38 Pace Project CA085: "Biodegradation Screen Study fo r Telomer Alcohols" -Revision 1 Study D irector Cleston C. Lange, Ph.O. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06.2002 000012 EID839557 7.1.2. Culture Preparation. The firs t set o f cultures w ere prepared A ugust 9, 2000 and incubation o f those cultures continued w ith interm ittent culture harvests u n til th e fina l------- harvest on Septem ber 13, 2000, fo r a to ta l incubation tim e o f 35 days. The second set o f cultures were prepared on Septem ber 27, 2000 and incubation o f those cultures continued w ith interm ittent culture harvests until the fin a l harvest on O ctober 12, 2000, fo r a total o f 18 days incubation. The culture preparation procedure described below was used, and was docum ented as Pace standard operating procedure (SOP) CAG -SP-03 8. C ultures w ere prepared using a m ineral salts m edium defined by EPA G uideline OPPTS 835.3200. The m ineral salts m edium pH w as 7.4 and contained per lite r, 0.334 g : NazH P 0<-2H 20 , 0.005 g NHCI, 0.2175 g K2H P 04, and 0.085 KH2P 0 4l 0.0275 g C aC lj-anhydrous, 0.0225 g M gS 04-7H20 , and 0.00025 g FeCls-6H20 . Tw o liters o f a m iners* salts m edium containing 100 m L o f settled sludge was prepared and contained 1 m L o f m ethanol. To each test culture w as added 25 m L o f this m ineral m edium containing sludge. uu A m ineral salts m edium , un-sterilized and w ithout sludge, was prepared. T his m ineral salts m edium w ithout sludge w as used to prepare the 25 m L no-sludge (abiotic) control cultures. A ll cultures w ere prepared by dispensing 25 m L o f appropriate m ineral salts m edium solution in to clear sterne 125 m L Nalgene polycarbonate culture flasks containing labels w ith appropriate identification inform ation. Note: The m ineral salts m edium that contained sludge had to be swirled regularly during dispensing in ord er to keep the m ixture hom ogenous and prevent the sludge from settling out o f the solution. The test substance, eith er Zonyl BA-N telom er alcohol fo r culture set one (Stock ID CA058-SS-004 at 10,280 pg/m L in m ethanol) or Zonyl BA-L Page 11 o f 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director. Cleston C. Lange. Ph.D. Pace Analytical Services, Bio-Anaiytical Services Group Wednesday, November 06,2002 000013 EID839558 ) ! telom er alcohol fo r culture set two (S tock ID CA085-SW-0Q1 a t 7,288 pg/m L in m ethanol), w as added to the te st cultures by transferring 5 pL and 8pL, respectively, to the appropriate te s t cultures. The fina l test concentration In cultures was 2.056 pg/m L o f Zonyl BA-N telom er alcohol fo r set one, and 2.332 pg/m L o f Zonyl BA-L telom er alcohol fo r s e t two. Blank control cultures received 25 m L o f m ineral m edium solution, as did the test culture, but w ithout addition o f the te st substrate. A ll of the day zero cultures were prepared and im m ediately frozen at -20' C fo r storage un til SPE preparation could be conducted. A ll other cultures were placed in tem perature controlled shaking incubators that w ere m aintained a t 25C + 3C. C ultures w ere rem oved from the incubators at designated tim e points. Upon rem oval from the incubator, cultures were e ith e r im m ediately frozen, o r im m ediately prepared fo r analysis by solid phase extraction. A ll culture preparation inform ation, including tim es, analyte additions, etc. were recorded in sam ple preparation w orksheets and signed and dated b y the preparation a n a lyst A ll original data sheets w ere m aintained in project specific binder labeled as P roject CA085. 7.1 .3 . Solid Phase Extraction o f Cultures The solid phase extraction procedure described below was docum ented as Pace standard operating procedure (SO P) C A G -S P -04T. A ll cultures and control cultures were prepared by solid-phase extraction m ethodology using SEP-VAC C18 6cc SPE cartridges from W aters Corporation (P art No. W AT036905). A sam ple label was applied to each SPE cartridge prior to use, and each cartridge was packed w ith plug o f quartz glass wool to de te r plugging. Each SPE cartridge was washed prior to use by draw ing 5 m L o f m ethanol and then 5 m L o f aqueous 1% acetic acid solution through the cartridge. These wash solution eluates were discarded to w aste. A ll o f the SPE eluates fo r this study were collected in clear l-Chem vials w ith labels tha t identified them as eiuate 1 ,2 or 3, as defined below. Page 12 o f 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols* -Revision 1 Study Director: Cieston C. Lange, Ph.O. Pace Analytical Services. Bio-Analytical Services Group Wednesday. November 06.2002 000014 EID839559 Frozen cultures w ere thawed a t am bient room tem perature before extraction. Following thaw ing, and prior to solid phase extraction, 0.25 m L o f glacial acetic acid w as added to each o f th e cultures yielding a fin a l concentration o f 1% acetic acid. The content o f each acidified culture was sw irled to m ix, and then drawn by vacuum through the appropriately labeled SPE cartridge by carefully pouring the contents o f d ie culture flask Into the SPE cartridge. T he aqueous eluate was collected in an t-Chem vial labeled eluate 1 , rem oved from the vacuum m anifold, and capped. Then, 25 m L o f m ethanol was added to the culture flask, the fla sk sealed, and vigorously shaken. The cap w as then rem oved from the flask, and the m ethanol content (25 m L) drawn through the SPE cartridge, collected in an l-C hem vial labeled eluate 2. Eluate 2 was expected to contain a m ajority o f th e analyte that w as in the original culture. As a precaution tha t som e analyte m ay be retained in the SPE cartridge o r in the culture flask, a second 25 m L m ethanol eluate was collected in a sim ilar fashion to tha t collected fo r eluate 2 , and w as labeled eluate 3. A liquots o f eluates 2 and 3 were transferred to aulovials, capped, and then quantitatively analyzed by HPLC/M S. T he rem aining volum e of each eluate was stored a t 4C + 2C. The final SPE extractions o f the day 7, 14 and 16 day cultures from set two occurred on Septem ber 24,2001. 7.2 In s tru m e n ta l A n a lysis (LC /M S) 7.2.1 In s tru m e n t P aram eters The HPLC/MS m ethod used w as a m odified version o f Pace m ethod CAGO R G -238, as described below. Analysis o f culture extracts from culture set one, prepared w ith Zonyl BA-N telom er alcohol, was conducted using Instrum ent " STING " w hich included an HP1100 HPLC pum p w ith G ilson 215 liquid handling system in line with a M icrom ass Q uattro II triple-quadrapole m ass spectrom eter detector. Page 13 o f 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 000015 EID839560 A nalysis o f culture extracts from culture set two, prepared with Z onyl BA-L telom er alcohol, were analyzed troth on instrum ent "STING" and instrum ent " 10LCMS03" which consisted o f a W aters 2690 HPLC system in line w ith a M icrom ass Q uattro II triple-quadrapole m ass spectrom eter detector. Typical conditions fo r analysis o f telom er alcohols and fluorochem ical acid products w ere as follow s: Instrument conditions for HPLC/MS analysis. Mass Spectrometer HPLC Ionization mode: API-ES negative Desolvation Temperature: 200*C Time (m ini. %A M Source Block Temperature: 150C 0.00 97.0 3.0 RF lens: 0.2 0.50 97.0 3.0 E xtractors 5.00 5.0 95.0 Cone: 8 11.00 5.0 95.0 Capillary: 3.50 11.50 97.0 3.0 LM Resolution: 14 15.00 97.0 3.0 HM Resolution: 14 Flow: 1 mL/min, splitter approx. 3:1 Ion Energy: 2.5 Solvent A= 2 mM ammonium acetate Lens 6 :2 Solvent B= Methanol MS1 Multiplier: 650 Column Temperature: ambient MS/MS: Collision Energy-varied from 10 to 40 V for individual Experiments, MS/MS Collision Gas: Argon MS2 M ultiplier 750______________ T ypical injection volum es fo r sam ples and calibration standards w ere 50 pL. The 4.6 x 150 mm B etasil C8 colum n used fo r the quantitation o f extracts from culture set two had serial num ber 1101567H and the 4 x 35 m m NG1 colum n used had serial num ber 15104. A pressure-regulated sp litte r w as used w ith an approxim ate s p lit ration o f 2:1 (W aste.M S) 7,2 .2 Q ualitative Parent Analyte and Products Analysis. P arent A n a lyte C8 telom er alcohol acetate adduct C i0telom er alcohol acetate adduct C i2 telom er alcohol acetate adduct C u telom er alcohol acetate adduct C ,8 telom er alcohol acetate adduct E xpected dons (m /z) 423 523 623 723 823 T a b le 1. Parent telom er alcohols analyzed. Page 14 o f 38 Pace Project CA085: " Biodegradation Screen Study for Telomer Alcohols* -Revision 1 Study D irector Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Anatytical Services Group Wednesday. November 06,2002 0 0 0 O 6 EID839561 T o conduct the analysis o f parent com pounds and exported biodegradation products, the HPLC/MS system was set up w ith the sam e chrom atographic configuration as that described in m ethod CAG-ORG23 *. The m ethod utilized two colum ns in tandem w ith flow to a pressure re lie f valve that serves as a flow through splitte r to the m ass spectrom eter 2 -spray source. S pecific MS conditions fo r m ass analysis o f the telom er alcohols and acid products were developed during this study. The m ass spectrom eter. M icrom ass triple-quadrapole m ass spectrom eter w ith 2-spray ion source, was operated with electrospray ionization in e ith e r selected ion-recording (SIR ) m ode o r w ith m ass-range (150-1000 m /z) scanning on MS1. D iscrete chrom atographic peaks w ith singly charged negative ions, (M + Acetate)- fo r parent telom er alcohols and (M - H*)~ fo r expected acid biodegradation products, w ere observed and m onitored: E xpe cted P ro d u ct E xpected a n io n stru ctu re P erfluorobutyraie (C4) CFafCFzfeCOO ` Perfluoropentanoate (Cs) CF3(CF2)3COO P erfluorohexanoate (C0) CF3(CF2}4COO ' P erfluoroheptanoate (C7) P erfluorooctanoate (Cs, PFOA) CF3(CF2)sCOOCF3(CF2)eCOO ' P erfluorononanoate (C> CF3(CF2)7COO" Perfluorodecanoate (C10) CF3(CF2)sCOO' Perfluoroundecanoate (Cnn) CF3(CF2)sCOO ` P erfluorododecanoate (C 12) 2H, 2H -perfluorooctanoate (Ca) 2H. 2H -perfluorodecanoate (C10) CF3(CF2)wCOO ' CF3(CF2)sCH2C O O ' CF3(CF2)tCH2COO 2H, 2H -perfluorododecanoate (C ,2) CFj(CF2)sCH2COO' 1. E xpected a n io n s (m/z) | : 2 1 3 ,1 6 9 2 6 3 ,2*8wro,'nteno 313 ;4 269 ofah iK iif sft? 413, 369 463, 419 513, 469 563, 519 61 3,56 9 377 477 577 1 T a b le 2. Expected products, and expected anions fo r each. Page 15 o f 38 Pace Project CA085: ` Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Director: Cleston C. Lange, Ph.C. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 000017 EID839562 7.2.3 Quantitative Analysis. ........... - Q uantitative analysis w as conducted only fo r perfluorooctanoate (PFOA) and only on extracts o f cultu re se t two. ` Q uantitative analysis was perform ed by the external standard m ethod using SPE extracted calibration standards (eluate 2 only) and quadratic calibration curves. The HPLC/MS analysis o f culture set one extracts was used prim arily fo r m ethod developm ent purposes. ............. Typical injection volum es fo r sam ples and calibration standards w ere 50 pL. T he 4 .6 x 150 m m Betas CB colum n used fo r the quantitation o f extracts from culture se t tw o had serial num ber 1101567H and the 4 x 35 m m NG1 colum n used had serial num ber 15104. The pressureregulated splitter had no identifying num ber to distinguish it. T he part num ber fo r ordering the pressure re lie f valve (splitte r) w as Alltech catalog part num ber 39025. 7.3 D ata Transform ations and C alculations 7.3.1 M olar Calculations: ' '*'-**' 'ateuiario Because all data was collected on an ng/m L basis (part pe r b illion , ppb). a transform ation from ng/m L to m olar concentrations had to be conducted to obtain m ass balance inform ation when applicable. The m ole conversion values fo r each analyte are as follow s: PFOA m olecular w eight, as am m onium salt, is 431.10 Zonyl BA-L telom er alcohol average m olecular w eight is 434.70. 7 .3 .2 C o n ve rsio n o f ng /m L to m ic ro m o la r (pM ) a n d n a n o m o la r (nM ). (W orking Exam ples): 2,232 ng/mL telom er alcohol = (2,232 ng/m L)*{Inm ole / 434.7 ng) = 5.135 nm ole/m L = 5.135 pm ole/llter = 5.135 pM-- assum ing 100% purity. 150 ng/m L PFOA (NH4+ sa lt) = (150 ng/m L) * (1 nm ole / 431.1 ng) = 0.3479 nm ole/m L = 0.3479 pm ole/L = 0.3479 pM - 347.9 nM Page 16 o f3 8 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 00003.8 EID839563 7.3 .3 M olar Mass Balance C alculations (Theoretical Yield): C onvert all ng/m L values to th e ir corresponding m olar concentrations as pM o r nM (see section 7.3.2, above). D ivide the sum o f the analyte concentrations by th e known concentration o f starting com pound and represent the fina l resu lt as a percentage o f the known starting c o n c e n tra tio n . (W orking Exam ple): If, the starting concentration o f BA-L telom er alcohol was a t 5.135 pM And, a fte r Incubation, the follow ing was determ ined: If, PFOA was detected a t 0.3479pM Then, the m ass balance Is as follow s: M ass balance = [{0.3479 pM ) / 5.135 pM] X 100% M ass balance - [0.0678] X 100% = 6.78% O r, 6.78% o f the telom er BA-L alcohol w as oxidized to form PFOA. 7 .4 S o ftw are Versions M icrosoftTM Excel 2000 was used fo r data processing and producing tables. M icrosoftTM W ord 2000 was used fo r processing the analytical report te x t A dobe A crobat 4.0 was used fo r generation o f the fin a l electronic report. M asslynx version 3.2 was used fo r data collection and peak integration. A C D Chem sketch version 4.0 was used fo r chem ical drawings. Page 17 o f 38 Paco Project CA085: 'Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study D irector Cleaton C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday. November 06,2002 000019 EID839564 8.0 Results 8.1 Sludge Characterization A chem ical analysis o f the m ixed liquo r suspended solids used fo r the sam ples ....... prepared on 'August'S; 2000 was conducted, and" characterization inform ation - can be found in the final report fo r project C A0581. The sludge fo r sam ples prepared on Septem ber 27, 2000 w as not characterized, bu t was obtained from the T w in C ities M unicipal waste treatm ent fa c ility in a m anner consistent w ith the slu dg e collected and used fo r project CA058. 8.2 Q u a lity C ontrol/Sam ple M atrix Spike Results. T he determ ination o f the analyte recoveries from sam ple m atrix spikes was not included as part o f this screening study. Recoveries are only reflected in the a b ility to achieve m olar m ass balance based on expected parent and product yields from sam ples and by the use o f a sludge-extracted curve fo r sem i quantitative analysis o f PFOA. 8 .3 A n alytical Blanks M ethanol solvent blanks were injected onto the HPLC/M S colum n and qu an tita tively analyzed to determ ine the instrum ent background analyte concentration and carry over during the analysis. T he no-sludge (abiotic) controls containing m ineral salts m edium w ith test analyte, and culture blanks, containing m ineral salts m edium w ith test analyte and no sludge, were prepared and analyzed. A biotic controls and blank cultures w e re prepared and incubated in an identical m anner to biodegradation test cu ltu re s. T he results w ere used to determ ine w hether the sam ple m atrix contained any o f the analytes o f interest and w hether biodegradation could be attribu ted to the bioactivity o f the sludge. Fluorochem ical analytes w ere not detected in blanks and biodegradation did not occur in abiotic controls. Page 18 o f 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 _______ 000020 EID839565 8.4 HPLC/MS Analysis Results S em i-quantitative analysis was conducted on ly fo r the SPE aiuate 2 extracts to m the secESniff set o f cultures, prepared Septem ber 27. 2000, and w ere perform ed on Septem ber o f 2001 as sequence CA085_0924Q1 b.spl (w ith fu ll scan MS da ta), and CA085_092701a.spl (SIR data) or instrum ent 10LCMS03 in the Bio A na lytica l services group at Pace. The qualitative HPLC/M S analyses and HPLC/M S m ethod developm ent fo r this study w ere conducted in Septem ber and O ctober o f 2000 as analytical sequence runs S09150G.spl, S091800.spl, S 092000.spl, S092100.spt, S092600.spl, S 1000200.spl, and S100400.spl on Pace instrum ent "STING ". 8 .4 .2 Q u a lita tiv e HPLC/M S a n d HPLC/M S/M S R e s u lts T he firs t set o f te st cultures containing sludge and BA-N telom er alcohol substrate provided evidence tha t biodegradation occurred. The HPLC/MS data showed near total loss o f all o f the telom er alcohol peaks in the HPLC/M S chrom atogram , w ith form ation o f chrom atographic peaks that had m ass spectra consistent w ith perfluorinated acids. However, a t that tim e, the analytical test m ethod was not com plete. C oncerns about the integrity o f the te st m aterial resulted In a second set o f te st cultures being i i . o prepared using com m ercially purchased Zonyl B A -L telom er interm ediate. Although each sam ple, control, and blank fo r the second set o f cultures w as prepared and incubated in duplicate, analysis was conducted fo r SPE eluate 2 extracts to m one sam ple, control and blank per tim e point. T he data showed that the sam ple test cultures underw ent rapid loss o f the C6. Ca, C ,0.and C -telom er alcohols, and m oderate loss o f the C u-telom er alcohol over the 16 days o f incubation, as determ ined by decreasing peak area response fo r each telom er alcohol ion a t the sequential sam pling tim e points (F ig u re s 3 and 4). The C 18-telom er alcohol w as degraded very slow ly and showed little loss. Page 19 o f 38 Pace Project CA085: "Biodegradation Screen Study for TelomerAlcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 000021 EID839566 35000 Figure 3. HPLC/MS integrated peak areas for the most abundant telomer alcohols (TA). Near complete degradation of all three was observed. Data plotted with the best manual fit of the data. c 6000 5000 3 4000 <? 3000 ! i f f l M W IBISfflHfBiaiiSBaMHi a 2000 & Jim r d h u h h h h 1000 a CSTA peak area |C 1 4 TA peak area C 16 TA peak area o 5 10 15 20 Incubaiton time (days) Figure 4. HPLC/MS Integrated peak areas for the less abundant telomer alcohols (TA). Near complete degradation was observed forth a C6-TA. Moderate degradation of the Cn-TA was observed and litfla degradation of the Cu-TA was observed. Data plotted with the best manual fit of the data. Page 20 of 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange. Ph.D. Pace Analytical Services. Bio-Analytical Services Group Wednesday, November 06,2002 0 0 0 0 22 EID839567 C oncom itant w ith the loss in telom er alcohols was the form ation o f com pounds w ith HPLC/MS ions and retention tim es consistent w ith perfluorinated acids (PFAs), including perfluorooctanoate (PFOA, C-PFA), w hich was confirm ed as an end product. The transform ation o f telom er alcohols to PFAS was rapid, w ith a large increase in alt o f the PFA peak area responses at day 1, and continuing increase through day-16 (F ig u re 5). No degradation to form PFAs was observed in the abiotic no-sludge controls. A lthough PFOA was accurately quantified, m any other perfluorinated acids w ere qualitatively observed and th e ir relative concentrations determ ined based on the observed increasing peak area response. The observed perfluorinated fa tty ad d end products were: perfluoropentanoic a d d (C 6 P FA ), perfluorohexanoic ad d (C-PFA), perfluoroheptanoic acid (C r PFA). perfiuorooctanoic acid (Ca-PFA, PFOA), perfiuorononanoic acid (C9-PFA), perfluorodecanoic add (C jo-PFA), perfiuoroundecanoic acid (C t,-P F A ) and perfluorododecanoic acid (C (2-PFA). T ransiently form ed interm ediate compounds w ere also observed a t e a rly tim e po in ts follow ing the initial exposure o f the sludge to the telom er alcohol substrate (F ig u re 6). The transient com pounds w ere suspected to be: 2H, 2 H -perfluorooctanoata;2 H, 2H -rperfIuorodecanoate;2H ,2H Perfluorododecanoate&nd the possible p-oxidation pathway interm ediates: 2H -perfluoro-2-octenoate, 2H -perfiuoro-2-decenoate, and 2H -perfiuoro-2- dodecenoate. T able 3 shows the MS ions and retention tim es observed fo r th e polyfluorinated and perfluorinated acid products and the telom er alcohol pa ren t substrates. HPLC/M S/M S Data (T able 4 ) support the identification o f the suspected m etabolites.. " -**?= , r .* 4 a w i "-.m danaate: $?, ~i* itsdecBnoate #1 <' f <^ "1 *4 ! kuoskk. w Page 21 o f 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 0 6 ,2C02 000023 EID839568 Figure 5. Integrated peak area responses for the perfluorinated fatty acids (PFAs) observed in test culture extracts. Even numbered carbon chain length carboxylic acids were the most abundant carboxylic acid peaks and were: perfluorohexanoate (Cb-PFA), perfluorooctanoate (Ca-PFA) and perfluorodecanoate (Cio-PFA). - 16000 14000 12000 1 10000 Q1. 6000 I 6000 c 4000 2000 0 0 1 2 3 4 *5 Incubition Time (days) Figure 6. Transient polyfluorinated fatly acid intermediates observed. The 2Hperfluorinated olefinic fatty acids are suspected f)-oxidation intermediates that formed rapidly upon initial exposure to telomer alcohols, and then were transformed to their corresponding perfluorinated carboxylic acids end products Page 22 o f 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director. Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November OS, 2002 000024 EID839569 Analytes Observed Chemical Form ula Retention Time LC/MS anions observed Cs-telomer alcohol* CF3(CF2bCH2CH2OH,..CH3COO ` 7.63 Ca-telomer alcohol* Cio-telomer alcohol* CF3(CF2)sCH2CH2OH... CHjCOO; ..... CF3(CF2)jC H 2e H 2O H ...C H 3C O O ' 8: 13 8.50 CiT-tetomer alcohol* CF3(CF2)9CH2CH20H...CHjC 0 0 ' 8.96 Cu-telomer alcohol* CF3(CF2)i ,CH2CH2OH...CH;jCOO ' 9.53 Cia-telom er alcohol* CF3(CF2),3CH2CH2OH... CHaCOO' 10.25 2H, 2H-perfluorooctanoate* CF3(CF2)5CH2COO 7.17 2H, 2H-perfluorodecanoate* CFjjCFibCHzCOO 7.38 2H, 2H Perfluorododecanoate* CFj(CF2)sCHiCOO ` 7.49 2H-perfluoro-2-octenoate* CF3(CF2).CF=CHCOO ` 7.14 2H-perfluo ra-2-decenoate* CF3(CF2)sCF=CHCOO ' 7.35 2H-Perfluoro-2-dodecenoate* CFj(CF2)eCF=CHCOO * 7.52 Perfluoropentanoate CF3(CF2)3COO 6.62 Perfluorohexanoate CF3(CF2)COO 6.88 Perfluoroheptanoate CFj(CF2)5COO` 7.07 Perfluorooctanoate CF3(CF2)6COO 7.22 Perfluorononanoate iPsrflporodecanoate CF3(CF2)jCOO j CFjtCFiJsCOO' 7.30 l rV'754Ji?:.;s7s . Perfluoroundecanoate CF3(CF2)COO .'`:.,7V48ri . Pefflunrododecanoate t CF2(CF2),oCOO - t PiijV.7i52rtrif>i s> * ; Only anions corresponding to M a n chain length molecules were observed. : 323 423 523 623 723 823 377 477 577 357293 457.393 557,493 263,219 313,269 363,319 413,369 463,419 513,469 563,519 613,569 v -* Table 3. Parent and products anions observed in by HPLC/MS analysis of SPE efuate 2 extracts from j test cultures containing sludge and telomer alcohol substrata. The parent anion signals decreased In test cultures over time concomitant with increases in product signals. Many of the telomer alcohol signals decreased below detection limits in test cultures. Page 23 o f 38 Pace Project CA086: ` Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Director: Cteston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 000025 EID839570 P roduct Analytes Observed 2H,2H-perfluorooctanoate* 2H,2H-perfluorodecanoate* 2H.2H Perfiuorododecanoate* 2H-perfluoro-2-octenoate* 2 H -parfluoro-2-decenoate* 2H-PerfluoiO-2-dodecenoate* Chemical Formula CF3(CF2)5CHjCOO CF3(CF2)?CHiCOO CF3(CFz)jCH2COO CF3(CF2)CF=CHCOOCF3(CF2)CF=CHCOOCF3(CF2).CF=CHCOO- Retention Time 7.17 7.38 7.49 7.14 7.35 7.52 LCfMSfMS Parent anion 377 477 577 357 457 557 LC/MS/MS daughter anions observed 333,313,293 413.393 -- 313.293,243.143.119 413,393,343 493 Table 4. HPLCiMS/MS data fo r suspected -oxidation products. Parent and daughter anions from the HPLC/MS/MS analysis of the SPE eluate 2 extract from the 1-day biodegradation sample (CA0850927-SA-033 E2). The parent anions were observed as transiently formed anions in early sample point biodegradation samples only, with foe most intense signals far (he tons observed at day onea Thef ; i> MS/MS data collected for each preduct was consistent with the predicted chemical formuiadSommorouK mass loss from the parent anion, attributed to loss ofC Q t (mass 44) and HF (mass 2O)*w0!fe|ecM$aren! u wm. 8.4.2 Q uantitative A nalysis o f PFOA T he HPLC/MS m ulti-com ponent calibration standards used in this study fo r product quantitation contained the target analytes: PFOA, PFO Sulfinate, PFOS, FOSA, W-MeFOSA. M556, M 570, and A/-MeFOSE alcohol. Fwe calibration standards w ere prepared by addition o f a known am ount o (. perfluorinated analytes to a culture medium w ith sludge, and then extraction by solid phase extraction in m anner identical to the treatm ent o f sam ples, The PFOA was the only targe t analyte o f that m ixture that was used fo r this study. The exact concentrations o f the standards w ere used in calibration curves fo r quantitative a n a ly s is . *-< >&/ Page 24 o f 38 Pace Project CA085: ` Biodegradation Screen Study for Telomer Alcohols* -Revision 1 Study Director Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 _. , _ O O O O Ic o EID839571 A ll calibration standards and sam ple eluates w ere stored a t 4 "C in refrigerator ID 0213 until analysis. A liquots w ere transferred to autovials and capped fo r use in HPLC/M S runs. The Instrum ent calibrations w ere perform ed fo r PFOA by use o f a fiv e point calibration w ith quadratic fit o f the data. The LLOQ was 1 1.0 ng/mL PFO A. and the coe fficien t o f determ ination (r) for- PFOA w a s 0:965. Q uantitative sequence runs contained calibration standards a t the beginning and end o f the T he biodegradation o f the telom er alcohols resulted in an increase in the m easured levels o f PFOA during the 16-day study (F ig u re 7). 180.0 160.0 140.0 3 " 1200 100.0 <o 80.0 a 60.0 40.0 20.0 0.0 F igure 7. The measured perfluorooctanoate (PFOA) In cultures containing sludge and fluorotalom er alcohol. The PFOA was not measurable in cultures that did not contain sludge. Data plotted with the best manual fit of the data and concentrations shown were not adjusted for reference material purity o f 95.2%. Page 25 o f 38 Pace Project CA085; ` Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 000027 EID839572 C l0 Telomer Alcohol dehpfroBflna r pr r r r r r dahyAoQortaM FFIfFFFfF HI r n n t y * Periluarononanoate + CO, I!lb3 41-k*tcocy<thk** 'W -ttK Perfluorooctanoate Acetate Figure 8. Proposed biodegradation pathway for telomer alcohols, shown for Cio-telomer alcohol as example. The (5-oxidation pathway branch (Hlb) involves primary formation of a fatty acyl-CoA thioester that is not shown, to cieariy depict the products observed by HPLC/MS. The fatty acyl-CoA thiol bond is readily hydrolyzed to yield the corresponding fatty acids observed. Page 26 o f 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director. Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 000028 EID839573 9.0 Conclusions T his study has dem onstrated that fluorochem ical telom er alcohols are biodegradable and are transform ed to perfluorinated acid end.products. A lthough the telom er alcohols are supplied only as even num bered carbon chain com pounds, the end product perfluorinated ad ds consist o f both even num ber and odd num ber carbon chains, w ith even num bered carbon chain length a d d s predom inating as the m ajor end products. T his observation suggests that, follow ing the initial oxidation o f the hydroxyl-carbon to form the prim ary carboxylic ad d (telom er carboxyiate). tw o oxidation m echanism s exist as shown in F ig u re 8, ill. The firs t, less utilized rouie (F ig u re 8, Ilia ), involves oxidation o f the a-carbon concom itant w ith decarboxylation (oxidative decarboxylation) to form an odd chain length perfluorinated carboxylic acid, which does not undergo further biotransform ation. The second observed route o f oxidation (F ig u re 8, lllb ) is the m ore com m on ro u te o f fatty acid p-oxidation, as evidenced b y the form ation o f detectable transien t p-oxidation polyfluorinated fa tty acid interm ediates and the m ore abundant even num ber carbon chain length carboxylic acids . A lthough to xicity o f the telom er alcohols w as not addressed as pa rt o f thiscstudy, the m icrobial degradation observed here ove r the entirety o f the 16-day stu d ysugg ests that m icroorganism s present in the sludge w ere not significantly inhibited by the tested concentration o f the test m aterial. Page 27 o f 38 Pace Project CA085: ` Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study D irector Cleston C. Lange, Ph.D. Pace Analytical Services. Bio-Analytical Services Group Wednesday. November 06,2002 000029 EID839574 8.0 Literature Cited 1 . Final Reports fo r Pace P roject CA058 (3M # E00-2252), "2-W eek W-EtFOSE A lcohol Biodegradation Screen Study Report" and "A erobic Biodegradation o f N-EIFO SE alcohol Study R eport1. Author: C leston C. Lange, Ph.D. 2. F inal rep ort fo r Pace Project CA097 (3M # EQ1-0415). "The 18-Day A erobic Biodegradation Study of P eriluorooctanesulfonyt-based C hem istrie s* Author. C leston c. Lange, Ph.D. 3. Final rep ort fo r Pace Project CA104 (3M # E01-0444), "T he 35-Day A erobic Biodegradation Study o f Perfluorooctanesulfonate (PFOS).' A u th o r C leston C. Lange, Ph.D. 4 . Final R eport fo r Pace Project CA105 (3M # E01-0683), " Fluonochem ical Adipate (L 15468) Biodegradation Screen Study." Final report issued July 10,2001. S tudy Director: Cleston C. Lange, Ph.D. 5 .F inal R eport fo r Pace project CA132 (3M # E01-0682). "The A erobic S.Finsti tm p irBiodegradation Study o t the Fluorochem ical FC807-Diester". Final report Issued May 29,2001. stud y D ire c to r Cleston C. Langs, Ph.D. 6. Pace m ethod CAG-SP-03. "C ulture Preparation fo r Assessm ent o f A erobic B iodegradability o f Fluorochem icals Using M unicipal o r Industrial Sludge as M icrobial Inoculum ." 7 . Pace m ethod CAG-SP-04. "C18 Solid Phase E xtraction P rocedure fo r Fluorochem icals Recovery from Aqueous/Sludge M atrices.' 8. Pace m ethod CAG-ORG-23 "Q uantitative Analysis o f Fluorochem icals by High Perform ance Liquid Chrom atography with M ass Spectrom trie D etection." Page 28 o f 38 Pace Project CA085: 'Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 000030 EID839575 9-0 S am le and D ata R e te ntio n A t a m inim um , one copy o f a ll pertinent raw data and one copy o f the signed fin a l report w ill be retained in the Pace A nalytical-T ier 2 data archives fo r a m inim um period o f 2 years a fte r com pletion o f the pro je ct: The rem aining sam ple extracts w ill be retained at the Pace A nalytical facility fo r a period o f 2 years a fte r com pletion o f the pro je ct a t 4 "C in the C arroll w a lk-in cooler (Pace ID 0140) located in the Pace A nalytical-T ier 2 facility. The follow ing w ill be provided to the sponsor (3M ): T he origin al signed analytical report and one copy o f the signed original. T he fin a l scanned rep ort (read only) and pertinent electronic data on a CD. A ll o rigin al Data, correspondence, chrom atogram s, sam ple & standards prep sheets, etc. Upon request before 2 years, the stored sam ples m ay be sent to the sponsor. A lt electronic copies o f the instrum ental raw data w ill be archived onto CD disks and one copy provided to 3M and one copy retained at Pace. F acility data w ill be retained fo r a period o f 10 years. F acility data is avajlabtesfoM i be inspection and includes the follow ing records: : and in o u i,1,` T rain ing records C ontrolled storage tem perature logs S tandard preparation logs C alib ratio n and m aintenance logs C hem ical and solvent traceability logs S tandard O perating Procedures M ethods pertaining to the conduct o f this project * Page 29 of 8 Pace Project CA085: ` Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study D irector Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06.2002 000031 EID839576 Appendix A Approval Signatures P ro je c t T itle : Biodegradation Screen Study fo r Telom er A lcohols C lie n t P ro je c t ID : E01-Q684 C o n tra c t A n a ly tic a l P ro je c t N um ber: CAD85 R eport R evised by: M ourad R ahl Laboratory M anagem ent: Bruce E W arden / S ignature*^ ' ' X" S ignature D ate -- uhii ^ Dtef Page 30 of 38 Pace Project CA0B5: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 000032 EID839577 Appendix B F inal Q uantitative Results fo r PFOA in sam ples, co n tro ls and blanks T a b le 1. The m easured PFO A concentrations in te st cultures during the 1 & d a y screening study for telo m er alcohol ae rob ic biodegradability. --- --- S a y 0 o M o o m ta fto * ta n w ta . C M M -O N T -S A -O S f. 2 D ay 2 M ta w n a iw i u r n * . C A O M 4 a 7 *C A -0 2 S . E 2 im wS a y * tw x M a ra a a w t t . C A a e -o w r-S A -g a r. 2 tD * r b x x M a ra d X iM ta rn s * . C A 0 4 S -0 I? -& A -0 M . C 2 f A s a n o te . C A M 0 0 0 2 7 -S A -0 4 1 , 2 D ay 1 ft k te fe e re d a fe n s w n e ts . C A O 0O C O 27-5A -O O . S 2 m C o n c ftn tra tk x t t a r t n L I fik t o t e 31 0 .0 25 2 ts o 13 0 .6 IS . 19 0 .0 T a b le 2. The m easured PFOA concentrations in the abiotic control cultures during the 16-day screening study fo r telo m er alcohol aerobic biodegradability. ew ele day One tiuda ecnral.CAOW0027-A-ow. C2 Da* 5 ns riutaa c o ita l. CAQftS-O0Z73A>06B.2 Doy 7 no <HOM t o * * . CMMfrOttNSA-OftO. S2 Day !< no dudoa antrot. CAO0ft-Q027-SA>O62 2 Bay 18 no tfutfoe conttiM. CAQ05*0t27*3A>0B4. Z G o n ca n tro tto i(n a im U IChMa 21 PFOA 0.0 0.0 OA 0.0 0 .0 0.0 0.0 T a b le 3. The fina l data results for sludge blanks during the 16-day screening study fo r telom er alcohol aerobic biodegradability. PFOA was no t detected in any o f the sludge blanks. Su m D ay 1 fu d Q a M a n H .G M O ftO a 3 r< $ A ^ O .E 2 D ay 2 s fc x fc W a r*. C A O ftft-O O i7 -$ M M 7 . E 2 C ay S tfg d o * M a * . C A 0 8 0 4 0 2 7 -& M H 9 . K 2 S a v 7 Ilu d a a U a r* . C A C 0 5 > fil3 7 *5 M M . E2 D ay >4 s h x fc to a n lt C AO 06-O O 7-3A-O 5Q . E 2 a y to n u d H u rn . C A o e e rt z r-ft* o 0 i. E2 C o m m ra M n 0 * m u m u m PFO A 0 .0 0 .0 00 0 .0 00 00 00 > . - H: Page 31 o f 36 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director. Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday. November 06.2002 000033 EID839578 A p p e n d ix C Integrated Chrom atogram Peak Area Data fo r telom er alcohols T a b le 1. Integrated LC /M S peak area raw data fo r the parent telom er alcohols substrates. Data acquired from sequence run CA085_092701a.spl as SIR data. __ S a n a to d m O fc rtM ra a to f) o rn o *, c a m w m b t.sa. q ji. e2 3 lv 1acdaeratau u n u ft. CA08S4BZ7SAa33. E2 39V 2 ttoOMiradakon m ote. CAOBMNQT-SA-OM. 82 3ay s voOMraoawn wmcM. CaoS-092T.S*-<07. C2 3*v 7 vOearedaton w nrot*. CAMOft2?-3A-630. C2 Day H bedwradaten u rn e *. CA0*SH57-aAHj4t.62 3*v n tN M m iU M IM O b . CMM&-Q427-3A-043. E2 R to lD CAO65 092701a 006 CAQ6S 092701 009 CA065 092701 010 CAOOS 092701 O il CA0B5 092701 012 CA095 092701 013 CA065 092701 014 3av 0 M luda* cm* L CA08iW W 7SASJ, E2 Dm 1m UudM cant**. CA0M-0827.6A-0S. E2 Dm 2 m s v fw OMISI, CA0M -M 37SA4M . 3M Sne V u j ocuot. CA6M497-BA486. K2 Dm 7 no aludae contrai. CAOOS M ir-S A ftM . E l Dm i* ne VOM coran*. CAM&82f>SA'06 2 U h b ('U W U W r.C lJ X l t t CA08S 092701 016 CA0S 092701 017 CA0S5 092701 016 CA06S 062701a 016 CA05 0 9 ^0 1 020 CA06S 092701 021 nA A M 009701 099 Dm 0 KtiOM Mam. CA0a5-0M7.SA-0*S 62 Dm f uose M ai*. CA0*S4)0?7-SA-M> 2 Di 3 aiuoae sum . CA088427.3A447. E2 DaySUu)M Mw , CA08S-09Z7-SA-34. 62 Dm 7 aiuOM Mark. CA08M 82T-SAO4* S2 Dm 14sluM e M nfc. CA5-C2T.3A-<0.62 DM 1SkJdMttank. CA0-0927.5A-C5f. 62 CA0SS 092701 044 CA065 092701 045 CA065 092701 046 CA065 082701 047 C A065 092701 046 CAOOS 092701 04 CA0S5 092701a 050 Ci 3903 NO NO 12 NO NO NO 5072 4891 S IM 4M * 1S 3018 0M NO NO NO MO 37 NO 17 I ; Ct 21704 Bf 288 800 31 684 43 90841 31308 31882 274M 21480 21284 1M 47 NO NO NO 182 211 M TitomefAlcohol IntegratedPwk Ama C lt C12 C14 76840 18446 5558 130 8823 ... ; < 4788 282 8877^ *, - . . 4483 103 3701 NO -AM3' < * 3248 NO .8T0 : . 1224 117 fc. 127 _ C 19 1398 1340 191* 1140* 1343 IM S 1137' 22584 20373 24414 22470 13821 143 t7B41 13734-ss't * > 5014 1127*5 ?* - 712 1*888*1. -*: V 5378 12868s A &m 4041 1 0 4 ' A 10226'-'c^vC tm * Q * * n 122(6tt&L& 1482 1477 1482 1383 <00' 12' 1478 I NO NO NO NO NO NO NO MO t f f NO NO V 7*. -, t o m o <r>. ,y ii . M3 M0 ^ NO -, NO t* N ft m V *086; .. NO NO ' NO NO NO NO i NO NO NO SUM 132 134 1213 i iq j 1343 1043 1 } .` W 1477 1442 1303 10 12 147 Mi W NO M2 i NO ! no : NO NO NO ssm ^ m sm ^ T able 2. P ercentage o f integrated peak area rem aining at different tim e points w ith respect to the peak area at tim e zero fo r their respective culture types. Near com plete degradation o f several telom er alcohols (T A ) was observed in test cultures only. Near 90% rem ained in control cultures for a ll except C ,0 T A . w hich was 79.4%. Sample D escription Day Day 0 biodegradation sample, CA085-0927-SA-031, E2 Day 1 biodegradation sample, CA085-0927-3A-G33. E2 Day 2 biodegradation sample, CA085-0927-SA-035, E2 Day S biodegradation sample. CA085-Q927-SA-037. E2 Day 7 biodegradation sample, CA085-0927-SA-039. E2 Day 14 biodegradation sample. CA0B5-0927-SA-041, E2 day 16 biodegradation sam ple, CA085-927-SA-043, t : 0 2 5 7 14 to Day 0 no sludge control. CA085-0927-SA-053. E2 Day 1 no sludge control. CA08S-0927-SA-055, E2 Dav 2 no sludge control. CA085-0927-SA-056. E2 Dav 5 no sludge control. CA085-0927-SA-059, E2 Dav 7 no studoe control. CA085-0927-SA-060. E2 Day 14 no sludge control, CA085-0927-SA-062 E2 Day 16 no sludge control, CA085-0927-SA-064, 2 0 1 2 5 7 14 16 C .T A 100.00.%0% 0.0% 0.3% 0.0% 00..00%% C tVTATA ReCmn TalnAlroB venue C,,TA D ay 0 Cm TA Ci, TA 100.0% 100.0% -100.0%-: ,100.0% <400.0% 0.4% 4.5% 46;33te IsSiSfc: TOI.1% 1.2% 0.9% 36:2%r* is97%- isSil .5% 3.2% 0.4% 16.6%rt feSfe6% 86.0% 2.9% 0.0% 6.8% 58.4% 101.3% 2.7% 0.0% 3.6% '23.8% 82.4% 3.0% . 0.4% _ 3.7% 23.0% as.7% ,, 100.0% 96.4% 102.5% 86.4% 69.3% 65.4% 90.7% 100.0% 101.5% 103.3% 89.1% 69.6% 69.0% 88.6% 100.0% 90.2% 108.1% 99.5% 60.3% 63.6% 79.4% 100.0% 82.1% 101.0% 93.6% 75.9% 74.5% 88.9% 100.0% 94.0% 107.3% 98.5% 84.8% 99.6% 104.3% 100.0% 99.0% 98.0% 93.4% 73.4% 64.6% 99.1% 5 5 ,., W " "SI 1 M tm tim m Page 32 o f 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange. Ph.D. Pace Analytical Services. Bio-Analytical Services Group Wednesday. November 06,2002 000034 !i~ EID839579 Appendix D Integrated Peak Area Data fo r Perfluorinated Acid End Products T a b le 1. Integrated LC /M S peak area raw date fo r the end products form ed. Date acquired from sequence run C A 085_09270fa.spl as SIR data................ ............... - ...... -.....- - a m ia . 3m O M xlM rad atkw sa m o *. C A O a X K F 'S A K a i, EZ 3 w 1 tw ow raO M M u m d *. C A0aM 827.S A^J33. 6 2 3m 2 tM id e o a d a lim s a rra *. CA0BS-S82?*SA*03S.EZ 3av 5 M d e arad alien M m if# . C A M 5-0827-6A 437. S3 D w 7 bodeoradatiCM u m o b . C A M 5C 8ZT.3A 49. E l 3n> H teOMOndabcm ia m o *. C AM 60827-SJW K1. E2 D w M 9 M d M i^ ttB n u m i^ .< ^ 0 d 8 ;;eaiZ7SA'<M3, U I W D ^ i *V . C Aoes o w T o ta .o o a CAOSS B T 01 1 OOB C AM S 082701 D ID CAOM 002731a 011 CACOS 062701a 013 c a c a s 06273ta o ta P e rflu o ro * o o n ta n o a i* 270 12730 4B3M HSN 72870 70876 PfgrA * * ( M L C A 0 a S ^a K 7 .a *4 U , 6 2 Dm t n> M m c on trai. C *ttS '0 8 Z 7 .*A 4 8 5 . 13 D M 2 no tiu O M S O flM i CAC8543Q77-SA-0M 2 S M S no tiu d M CDW Di C A0B8^eZ7-SA49. E2 DSV7 HOSUOM C M Itt. CA0efl-O9Z7-SA-tJCQ.1 2 Dm 1 no lw lw o o rH re l.C A M M W r.a A -0 ** C if t m x u tw m e a l e ta iitflM tJ ia a i n CACOS fi82701a 016 CAOSS 002701a 017 CACOS 0827012 018 CACOS 0827011 018 CMOS 0827010 020 c a m s a a tra u o t i n iM A m e m t* m * 183 380 991 479 7 Ml an 3 lv 0 li d o W ar*. CA085O827-SA-O 41 1 2 3 M 1 m o o c BU T*. CAOeSS-0827-SA-Otl. S2 Dm 2 vudoe w a r*. CAQQ*08Z7-SA<04?. E2 ir iw tn a t * C A o e u ta f i4 < i. u 3m r H u ta W ar* eA M & 0B 27.SA 4M . E 2 3 iv I * tfu d m wank. C A 0 8$ -*27 -JU Q . E l DM IB Mudoa M a rt. CAD05-0827-5A>061.E2 ''1 i i 1 C AM S 000701a OM CAOSS 082701a 049 CAOBS 082701a 040 CAOSS 082701a 047 CAOSS 082701a 046 c a m s o e z ra ia 049 CACOS 082701S 090 I I i O il 99 80S e ra nr 870 624 P a rflu o fo h c u in o o ta 70 am 127337 204221 3 10 S M 321540 ^ Z lg K B fil 1722 1407 1744 1043 1731 h h Kh 525 152 010 320 243 207 290 (n ta c ra to d P ta fc A n a s . i ftw fb o ro IW D tS M a te iar 15444 25641 30747 40327 47751 ^4 7 3 1 ^^ 2 4 4 io ta s 17904 1TM 6 13212 10078 tro to P o rffc io ro * o e tw ie a to 2719 4102 B05M 140408 in w > 100800 ^ 1 *2 0 7 3 ^ W 1089 140 1061 1003 1480 1837 P e ifk io r o - P M flu o ro * no n a a iH M M tt M C IM Ita u ie U B S . 233 8424 0287 ..fe 7 * * S M 8379 , f.^ A M I 13751 ** i u m s n 15302 P o rflu o ro * P ltflU B T O * im d 8 n o 8 to tfo d a c a n o a ta I9 60 373 1281 M 1370 11 1141 2844 2065 9047 0071 11078 4982 ..164 H 9810 s s & m MM 7420 * m m _____ 0320 . -i m a to - . 7.128,^, e,U 2 i* i- M M aS m t * s ; k HHM 1202 1082 1178 1228 15 1475 1342 h Kh h 40 51 21 91 2S SO 34 31 .1 117 78 1 18 91 228 00 223 82 s a t 00 701 r MS K T iO K S IM vaw m fta a b i.f- j ..> U 1sM , :4SU*.V-fe% ... 307 ^ & m : u O 0 0 74 2 21 ** - V. J 48 V* : ' . -..C42 .39 '" O' . Page 33 o f 3fl Pace Project CAO05: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study D irector cieston C. Lange, Ph.D. Pace Analytical Services, Bio-Analyticai Services Group Wednesday, November 06,2002 000035 EID839580 A p p e n d ix E Integrated Peak Area Data fo r Tran siently form ed Polyfluorinated Acid Products T able 1. Integrated LC/M S peak area data for transient products. Data from sequence CA085_092401b.spl asfu ll scan MS data from m /z100 to m /z 1000. ' ................. S a m e l* O ar 3 tH edM fadaliM M m o l*. C M M frO O Zr<SM l37. 6 2 Day 7 n o udcw o o n M . C M O M 0Z 7.S A 49O . E2 Day ta ailidfW bla nk. C AM M 817-SA.O SO . 82 oa v to u m m w ank. C A O M -o tw -S A -ta t. e z R to iD . C M M 002401b 008 C AOM 002401b 00 C U M 002401b 010 CAOM OOMOlb O il CMOS 062401b 012 c a n s o B z w ib 013 C AM S 0*24019 01 CAM S M 24C tb 017 CAM S 012401b 01 C AM S M 240ID 01 CAOM 002401b 020 c a m s o s3 4 d ib 021 CMOS 002401b OM CAOM 002401b 031 C AO M .062401b 032 CAOM 002401b 033 CAOM 002401b 034 CMOS 002401b 035 CAOM 082401b 03 * 2H * p rf(u o fO > 3 < o d M o a t io a o 3429 223 fO NO M3 In te g ra te P ak n n 2H * 2H - p a r flu o ro -2 p o r flu o io 4 s 1 H .1 H p c rftu o ro - d S O M N tt KO 780 NO NO NO NO d M to c o n c M t NO 340 NO ND NO m .... M ttn o tn MM MSS 1020 230 137 00 ft* p o fflu o r o p M flu o r o d o e s n o a te d o d tc a n o a t 211 TOSS NO Ml 2S3 NO .... x- vf, 4 ( b lH W U ND NO___ * M I NO M 3 NO M I NO ND NO NO NO NQ MI to ND MI NO NO - NO NO NO .... NO NO ND NO ' NO NO NO NO ND NO NO NO NO NO HO . NO ND NO ND M ) NO NO NO NO M2 NO ND _______ IS _______ ND MI . * ,, , f NO V / i- :; NO NQ NO NO ND NO m . v'SM D'. - D rift * M3 NO NO... f t i& .- >A' :Xm . .. NQ CM ' NO r: ND a? b <&84iSfJi . NO NO ' " M l ': Page 34 o f 38 Pace Project CA085: ` Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Olrector: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 O O O O ov EID839581 Appendix F Typical T o tal ion Chrom atogram s fo r SIR Data Showing Telom er Alcohols and Polyfiuorinated Fatty Acid Products from Day-0 to Day-16 fo r HPLC/M S Analysis of Biodegradation C ulture SPE eluate 2. S IR o f 17 anions: m /z 2 63,3 1 3 ,3 6 3 ,4 1 3 ,4 6 3 ,3 1 3 ,5 6 3 , a n tt 613 fo r p erfluorinated acids; m /z 3 7 7 ,4 7 7 and 577 fo r polyfiuorinated acids; and m/z 3 2 3 ,4 2 3 ,5 2 3 ,6 2 3 ,7 2 3 ,and 823 fo r telom er alcohols. CAU*4M7-CA-*43El.day1Modesample CM66.QV9roia.C14 too-. Day-16 * j IM CA066.CtQ70ta.C13 ' 166- D a y -1 4 ': . ____ ,.u t......... ............. ` CA0ftS.00a701S.0l2 1- Day-7 *1 1H CMa9.OB27Q1a.Ot1 160- Day-5 * j CAOM.onrQis.oro mo. Day-2 ' j CMSS.OWTSia.W* 100 ............,,,w t_ ........ ............ 100 Day-0 *; *$? 1*.21 10O 2.00 aoo 400 S.M Perfluorinated Fatty Acid Peaks [ JrL sJCHr........................ * 7| 7.40 f t V ftj . 1 131 w * ...................... r 72 S{$} < . " i. J U ... J?0,. ..V . . . 6.00 760 ft00 \ 10.00 1100 12.60 SIRoMrename1.6E4TeSO6 sir*it cnaen1AeTMaC-I <r. SUtcMrChsnneifctEMTSCSiftof17ChanneISlsETMSCsotoirtrCfWM10tE2e*s6 SAcrfirCtwmt?s3ETTSCrf,i StIM7r.mertt4lliETeSC! 1209 14.00' Telomer Alcohol Peaks Page 35 of 38 Pace Project CA085: ` Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002 ' 000037 EID839582 A p p e n d ix G S IR data Show ing Ion s fo r Telom er A lcohols a t Day-0 and D ay-16 from A nalysis o f th a Biodegradation C ulture SPE eluate 2 . CAM*M274A431 E2.dyQ Motig MffVf* Caow.ow/ou.owj ' w % C A0a.S...O--)??..0.u...')0--8 100 C A M S .C 8 3 7 O 1 1 J0 0 I 100 %' CAMJMTOiSjxa 100 5k: C A M 5 .0 Q 3 7 0 la _ 0 C 6 00 5b tooC A M S .O K 70ia.O M ..j s . ........................ . M M -A HL A sh. SWtofirS^r2je1r0w0lF1E00S01- " SOOeS SIR o f >7'C2r3vwWnU<Oi GOSO-a 500*5 SIRof 17fCltaumobistomoESo*i EOOrS $MoM7ChnfcS- S Z 9 JD 1 .0 0D O 500e5 SM o<17CazmoonwoisoooEoo3e-as SlRoMTCnsmaoCS* 32900 S1C00O0S o- ----------- C A M 5.0 92 70 la.O O B 100: 5b; 0----- ----- iqq yg _______ _____ CAMS-M2?SA-Q43 E3. day 1 * bicxteg scrngts tooC A3AS.gM 7nia.Q 14 . 7.11 I D SIR o f 7 Cftanw fcES- 13 DO 14.00 nmt SIR o> 7 C n a tra s E5- S33.COS:OOdMOAa CA0O-o-s-.-u-u-r-g-is-.-Q-M-- 109 5b 0------------ CA0OSJZ7O1O_OI 100 5k. OMBS.0Ktra!_014 too % 5AOMS'..O..U..7.0.i.a...0.l4 100 % 0- 5A *8.<H fi!70!a.0M 100 % .>tovrot5_0Q27>'a_3i'* * 9" m IOO 2.00 ~ Too 4 .0 0 5 .00 700 5.00 Page 36 o f 38 S lR oM ?C h3*lk6S 72100 1.000 500*9 S M s fi; Cnanras ES823.001000 SOM smofirouMriMCs* M M5 2 3 .0 0 1 .0 0 0 SIR o f 17*C2h1a9n0ne1l0E0S0-a SOM SiR o f 17.CVhiCamCtotSuuQGCmSS-S.l SIRaf 17Chsrmct50E0r*ie5 Tbit# Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Setvices Group Wednesday. November 06,2002 000008 EID839583 Appendix H SIR data Show ing Io n * fo r Pertluorinated Fatty Acids a t D ay-0 and Day-16 from A nalysis o f th e Biodegradation C ulture SPE eluate 2. C A0IS-0627-SA-M 1 E 2 ,tfa y S b to d g M in p l CAOSSjCOZTQfa.OOS too H C40fi6.092701.00a too % CAOE.(MZfO1.O09 100 % C A cU .iX S 70fA .406 100 % CA0ttJM 27O18.OW i to o %.. CAC6K.OB270tS.00 100; CA0e.0B27O1S_009 to o . CAM *. OOSTOtp.OOft too % CAMS 06Q701H.00U tosoi y.QO 2.00 3.00 4.00 -- ....................... ........................................................ ..... -- ........ -- ten T& 792 10.13 S.00 ' 6,00 r ju r t * ,i * y e 7.00 6.00 ' S.00 io!oo ` 1140 1240 S R i f 17C M nM feft$. 19001400 ' * 2.00*9 S R o f 17CftarvuHa S940.00 1OOD* 7M S R Of l7C m nnM E S 513001000 200*5 SR of 17Cnarauk ES- 4 *} M C S 200*9 S tR of iTC nsonw afiS 2 1 3 0 0 1 0 0 0 * .00*0 S R f tfC h tm M E S 3S3.OOl.OOOa 2.06*6 S R o f irC n sm M E S 313.001.000 200*9 SR o f f7C hSftA*ttS243 001000 2 00*9 S R flf 17C harge* E5- TiC 2 0 0 *3 13.00 14 00 7 liM C A 0 flM t2 f4 U U M S E Z d iy 1 ft U & d tg s a n d h i CAOOS.OftCTOla.OM 11 s CAO05_i27OI_Ot* Vtoo C AO _O a2K )l.O t* s ito o . C A 46S .002rota.O t4 too S' C A oeo.u6zrots.oi stoo CA0#9_Q627(H3J914 100 S. CAASS.06270ta.OI4 too S CA4K>.0027013 OH too % C A 4tti.0627ttta.0i4 too s 0 .........-----------100 2.00 3.00 4.00 s R o ri7 C h w ^ *e s 6130010601 2.00*9 732 S IR o t1 7 Q W !f**E S - 2543001000 00*8 74 7.30 X 707 (H ........... ........... - SR o f i7C .-w fitr.ueS - 2s m o to o c a 00*9 SiR ct 17 Channel* S463X01 COCa 200*5 . S R 0t17O w nn*E S 413.00 tOODa 200*9 SIR 01 tIC m n fo ttC S 2363 0 0 1000a .00*6 ' R e f irO unnaS tE S 313 441 060 260*6 *P ........ SCO &00 7.00 0.00 0.00 Page 37 of 38 SR itt i7C ?tiii<ri*aS- 2263 '*l OOD.I UCe-J 10.00 11.00 SR 4 l 17Crtanrufn E* TC Z d d rti 12 * ....... *-- - .. - Tin# .00* 1300 14 00 Pace Project CA085: 'Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director; Cleston C. Lange, Fh.D. Pace Analytical Services. Bio-Analytical Services Group Wednesday, November 06,2002 00003d EID839584 Appendix I Chrom atogram s o f Extracted ions m /z 3 7 7 ,4 7 7 , and 577 and M ass Spectra fo r Each' Peak fo r Polyfluorinated A cid Interm ediates Form ed Transiently in Biodegradation C ultures at Day-0, Day-1, D ay-2 and Day-5. Suspected p-O xidation o lefin ic acid interm ediates. Day : A Yw 2. " in * ' too " IflO" 00 700 too HOC 17.00 >9.03 Page 38 o f 38 Pace Project CA085: `Biodegradation Screen Study far Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday. November 06.2002 000040 EID839585