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BIOACCUMULATION (ANALYTICAL ONLY) TEST SUBSTANCE Identity: Perfluorooctanoiaccid,ammonium salt;may alsobe referredto as PFOA ammonium salt,Ammonium perfluorooctanoateF,C-1 16,FC-1 26, FC-169, or FC-143. (Octanoicacid,pentadecafluoro-a,mmonium salt,CAS 3825-26-1) Remarks: The 3M productionlotnumber was 37. The testsample isFC-143. It'psuritywas notsufficientclhyaracterized, though currentinformationindicatesitisa mixtureof96.5 - 100% test substance and 0 - 3.5% C6, C7, and Cg perfluoroanalogue compounds. METHOD Method: Devised by 3M Company todetermine ifbluegilslunfish bioaccumulatefluorochemicalfsrom 3M's Decatur,AL plant effluen(tSee Remarks) GLP (Y/N): No Year completed: 1978 Remarks: The followingmethod was devisedand used: 1 . Two lotsof30 fisheach were used. 2. One lotwas exposed toDecatur planteffluenftrom Oct.5 to Oct.25. The otherlotwas exposed toTennessee Riverwater onlyfrom Oct.3 to Oct. 25. 3. Twelve fishirom the effluenetxposed lotwere collectedead (diedin test)and the remainingeighteenfishwere sacrificeidnDecatur.The riverwater exposed fishsurvivedthroughthe exposure periodand all were sacrificeidnDecatur. 4. Effluent-exposedand riverwater-exposedfishwere allpreservedand shipped indry icetothe 3M Environme ntalLaboratoryinSt.Paul,MN foranalysis. 5. Fishwere homogenized usinga Tekmar Model SDT tissuemizer. 6. Two 2 g.portionswere weighed and extractedO.ne portionusingethyl acetateand tolueneon the other. 7. Samples were analyzed by GC, TLC, and GC/MS. RESULTS AnalyticalObservations: The effluent-exposefdishweighed 25.7 grams. The riverwater-exposed fish weighed 33.0 grams. GC/MS analysis0fethylacetateand tolueneextractedfishexposed to plant effluenctontainedno detectableamounts ofammonium perfluorooctanoate. No fluorochemicalwsere detectedintheriverwater-exposedfish. Remarks: Study was conducted because 3M's Decaturplant manufacturesfluorochemicalsW.ater used inmanufacturingislatetrreated and thetreatedeffluendtischargeseventualliyntotheTennessee River. CONCLUSIONS No reliablceonclusionscan be derivedfrom thisstudy. Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul,Minnesota,55133- DATA QUALITY ReliabilityK:limischranking3. Study was conductedatthe manufacturing facilirtaytherthanat3M's EnvironmentalLaboratory.Effluenctoncentrations ofsubjectfluorochemicalwsere notcharacterizeadnd thespecifipcrotocolfor exposureofthefishwas notfound. There was alsono informatioonn analysisoftheTennessee Riverwateror effluenutsed inthestudy(e.g.N,H3N ineffluenitsknown tocause deathduringNPDES testingo,r low DO, high TDS). Additionalliyti,snotknown iftherewas any opportunitfyordepuration ofthefishpriortosacrificeP.resence ofthetestsubstancecouldhave been a normalfunctionofthefishconcentrationhsomogenizingwiththeeffluent concentrationsN.o explanatioinsattemptedas to what was thecause of the 12 dead fishintheeffluent-exposegdroup. No differentiatwiaosn givento whether thesubjectchemicalwas actuallayccumulatedinthefishor simply sorbedon thesurfaceofthefishbody. Sampling locationwsere not described. REFERENCES 3M TechnicalReport"AnalysisforFluuorochemicalsinBluegilFlish"G. A. Vraspir/ArthurMendel, Project9970612600, Fate ofFluorochemicals, Report Number 14,May 1,1979 3M requestedexpertoverview,"BioaccumulationStudies",Dr.James Gilleft, CornellUniversityM,arch 8, 1993 OTHER Last changed: 5/25/00 3MForm 6747-11 -A TO: TECHNICAL TECHNICAL REPORT- COMMLINICATIONS CENTER - 201-=N SUMMARY timportmt- Ifreport isprintmd on bog A*sofpipw, swdtw copiesto TCC.) T'aiect@ Environmental Laboratory Pate of Fluorochomicals (BE & PC) Reg. No. 48765--- Analysis for Fluorochemieals in Bluezill Fish D. L. Bacon Dept. Pdumbw 0535 rraftet Nwnbw 9 6u6oo rwoo" murn 014 G. A. Vrasp@_r/Arthur Mendel Wo-tmock vqofweme 48838-18 SECURITY )o 13ocm AT 06kd KEYWORDS: (Selowcmte from3M TheaurusS.ugmt ottw CURRENT ONACTIVI: JOACHERMAL I ago v StatusReport 76416Z43939 M06 M rM ln@tUM"e @@vftnhom 7 t4sw Ch"kok Row%d E3 Yo 0 "a REPORT AMTRACT, 1200-25w0ordsT)hisdorectinforrmdiosndioftbutbeyddw TachnimCionwnuaketioCnosnfttro &art3Mws toOwroanyR&D. cc:R.A.Prokop-236-3B V-Pothapragada-236-3A A.N.Welter-21-2W R.L'.Bobon-21-2W information Lie.i_sog. Initial%: PC (Req. 48768)/'GIAV/AU -2- 5/l/79 OBJECTIVE The purpose of this investigation was to determine if bluegill fish, after exposure to our Decatur plant effluent. bioaccumulated fluorochemicals. SUMMARY Extracts from bluegill fish exposed to Decatur plant effluent (PE) contained 10 ppm (ug/g) of FU 3923 and 7 ppm of FU 3422. TLC indicated the presence of PC-95, while FC-143 and FC-128 were not detected. Extracts from bluegill fish exposed to river water (Rff)contained ethanol, ethyl other,- othoxyethyl acetate, toluene, and xylene. No :fluorochomicals were detected. A copy in attached of CRL report 7124 which detailed GC/US experiments. INTRODUCTION The uptake and clearance of specific radiolabeled fluorochemicals by fish were reported (1). The current report details the analyses for fluorochemicals in fish previously exposed to our Decatur plant effluent and in fish exposed only to Tennessee River water (2). Thin-layer chromatography (TLC), gas chromatography, and gas chromatography /mass spectroscopy (GC/NS) were used. DISCUSSION Our Decatur plant manufactures fluorochemicals. Water used in manufacturing in later treated and the treated effluent discharges eventually into the Tennessee River. Bluegill fish, chosen for these bioassay experiments, were exposed to P.R. Another lot of fish were exposed only to the Tennessee River water (2), The fish were then analyzed for fluorochemicals by an involved three-route procedure as outlined in Flow Diagram 1 and detailed in the experimental section. Briefly, fish were homogenized, and some of the homogenate was extracted withtoluene (Route 1), while some of the homogenate was extracted with ethyl acetate (Route 2). A portion of the latter extract was concentrated Route 3) for GC/MS and TLC. Results are given in the summary section. -3- FLOW DIAGRAM I ANALYSIS OF BLUEGILL FISH 30 Fish (1) Weigh (2) "Tissuemizell 0 -e (1)Weigh 2 g. (2)Extract (ETOAC) (3)Centrifuge (4)Isolate ETOAC Layer Route 3 GC For FM 3923 FU 3925 FU 3422 Concentrate GC/HS TLC For FC-95 FC-128 FC-143 (1) Weigh 2 a. (2)Acidify (3)Extract (Toluene) (4)Centrifuge (5)Isolate Toluene Layer (6)Methylate GC/MS GC For FC-143 (Ester) FC (Req. 4876S)/qAV/Ag -4- 5/l/79 EXPERIMENTAL Two lots of thirty fish each, wrapped in aluminum toil, respectively, and shipped in dry lee, were received on November 3, 1978, by the Environmental Laboratory. The fish were kept frozen until analyzed (Nov. 3, 1978). The effluent-exposed lot (Oct. 5 to Oct. 25) weighed 25.7 g. Twelve fish were collected dead and eighteen fish were sacrificed in Decatur. The river-exposed lot (Oct. 3 to Oct. 25) weighed 33.0 grams and were all sacrificed at Decatur. To simplify,the experimental one lot is discussed. The other was analyzed in exactly the same manner. The following codes were used: River River Plant Plant Water Holdin-g Tank Exposed Fish - ethylacetate extract (R.W.-EtAc) Water Holding Tank Exposed Fish - toluene extract (R.W.-Tol.) Effluent S-1 Exposed Fish - ethylacetate extract (P.E.-EtAc) Effluent B-1 Exposed Fish - toluene extract (P.B.-Tol.) The fish were homogenized using a Tekmar Model SDT tissuemizer. Two 2 g. portions were weighed into separate polyethylene centrifuge tubes. Two ml's of pesticide grade ethylacetate were added to one and 2 ml cone. NCI plus 2 ml of AR grade toluene were added to the other. Bach sample was mixed approximately 1 min. an a Vortex Genie mixer. The samples were centrifuged for 15 min. at 10,000 rpmls. The ethylacetate and toluene extracts were drawn off and placed in glass vials, respectively. The ethylseetate extract was ex.49ned using the following GC conditions: lostr. HP 5713 with Ni electron capture detector. Collimn - Slx 1/81' S.S. 10% Carbowax 20 U on 60/80_mesh Chromosorb W-AW.* Ing. Port - - Temp - 200 C., Def. Temp 0 300 C., Oven Temp - 150 C isothermal, Flow -.4.0 cc/min. Argon/Metbane (95/5). After GC analysis of EtAc extract, a portion was concentrated under N purge for two dimensional TLC analyses. The following TLC coniitions were used. The concentrated EtAc extracts were spotted alongside standards of PC-95, PC-143, and FC-128 on E. Merck silica gel plates. They were first developeg in 10% ETOH in EtAc and dried. Then the plates were turned 90 and developed in 50% ETOB in EtAc. The dried plates were visualized by spraying with 0.01% New Fuchsin in water (3). The toluene extract was methylated with diazomethane and then exgtined using the following GC conditions: Instr. HP 5713 with Ni electron capture detector. Column - 12'x 1/811 S.S. 20% DC-GOO and 10% Bentone 3a or 80/90 mesh Anakrom PA. lnj. Port 200 C. Det. temp - 300 C., Oven - 110 C Isothermal, Flow 40 cc/min. Argon/Methane (95/5). The ethylseetate and methylated toluene extracts were submitted to Central Research Analytical for GC/MS analysis (Req. No. C-50687). 8ee attached CR Report No. 7124 for GC/MS conditions. FC (Req. 4876S)/GAV/AM 5/l/79 PE/EtAc extract showed approximately 10 mg/g of FM 3422. RW & PE-Tol. showed no difference. No FC-143 or FC-128 was detected by TLC of the PE EtAc, but a spot was noticed whose Rf was that of reference FC-95. REFERENCES (1) a) U. T. Elnabzrawy, "Bioconcentration of FM 3422 in Bluegill 3untish and In Channel CoLtfish,11Eg & PC Report, 5/17-/77. b) A. N. Welter, 'Aquatic Fate of A Fluorochemical, FU 3422," EE & PC Report, 10/14/77. (2) Decatur Effluent Fish Toxicity Test Protocols, dated Oct. 18, 1978 and Feb. 5, 1979; M. T. Elnabarawy. (3)J. Sliwiok and A. Macioszczyk, minr-nhAmLr.Li iauxn&ll 23t 121 (1978). VAP.@ #,bq GAV/AM/cen Attachment -rogNTitAL ANALYTICAL LABORATOTY Report No. __-7124 ------------.!I@.rch-2-8,-1-9-79---------- Subject-. Decatur fish &toasty. Requestor.@.._ Vrasp Ir A. ftrael Request No. C50687 -------Report: INTRODUCTION Dept. Name Dated Novemb-e-r--16-.- 1976 Proj. 140.-9-1S-o-l-61- S&Wles werereceived-frotmherequestorfsroma fishbloossytakenat the Decatur Plant. Fish wer4F*xposedto ba differentwater syst@* the Tennessee River which flows Just outside the plant &W the plant effluent running Into the river. Ethyl acetate extractsof the fish were analyzed using the GC/NS system. Toluene.extractsof the fish war* iothylatedwith diazomethans and analyzed. DISUCSSION The s&Vlas were labeled R.W. 01 and P.E. 01 (notivyistodtoluene extracts) and R.W. #2 and P.E. #I (ethylacetate*xtrects). Sm*les R.Y. 01 OW P.E. it were analyzedusing conditionsfor column A below &rW samples R.W. 12 *rW P.E. #2 were analyzed using conditions for column 0 belaw. instrument: Varian 2740 gas chromtograph Column A: 121 S.S. 20% DC-200 + 10% lentone 34 on Anakrco PA Oven: 110*C isothermal for 11 minutes then 110-170*Cat 10*C/sin. InJecto r: 165*C Column 6: 6, s.S. CW 20H 15% on Chromosorb W Oven: 50 - 225*C at 10*C/sin. Injector: 220*C Detector: Varian flam Ionizationat 290*C Carrier Gas: Helium at 25 cc/min. Instrument: dupont 21-4918 mass spectrowtor Source: Chemical Ionization Po": all sampleswere run In Electron Ionization,sm*le was also run In Chemical Ionizationwith M3 9" Mass Range: 18 - 617 m/e . Helium Separator: Single StageAL*t Multiplier; londix ChanneltronMg P.E. 12 Hu. -U-1*4.#t d.LO. RESULTS SamPlos R.W. ift and P. E. ift shamed by CC/14S only the solvents used In extracting and wthylating: othenolo ethyl other. othwq ethyl watate toluene and xylones. it was necessary to air concentrate the ethyl watate "tracts In order to obtain sufficient date to Interpret the mass spectra. The Tennessee River exposed f lsh ethyl acetate extract contained @rous G.C. peaks none of which appeared to contain fluarim. The its appear to correspo#W to references of 2-mthyl-3-ectenow. &="Aetc ocid, a phthalate, and high molecular alkenes from about C to at least C 240 The plant effluent exposed fish ethyl watate "tract ootalmd which appear to correspond to referencesof 2-mftl-3-"to@ **tic acid, flucrochanicalswhose highestmass frogmts saw "ft 43 We -arA 582 a/a, nitrogen containing flucrocohmicals(Wwso highest =so fragments seen were 448 a/* and $84 0/0@ PH 3923 (POSE&Olds) OW PW )4n (FM alcohol). SUMMARY This fish bigessy showed that extracts from the fish owuw to the Tennessee River water contained such cowpounds as ethanol# ethyl other, othm ethyl acetate, toluene and xylons. The extracts from the fish oxpaood to the Decatur plant effluent contained FOSE mide, alcohol, other related fluerachmicals, 2-mathyl-3-octenons, and acetic acid. J. N. Schroop,@'or /ch .1 -70 PRO JECR N%,Subject:- MJOOICC(Ar-OtAla-@OK PAC@ 3M TECUMCA@ NO oQx No. 4JL947 21 OkFVN-34*2-t te: 1/1,61-74 . 6t( -k@- . . 4.,@- v ' Object: ot Reference: T@ s 1#'tePtteX.AJfL, cI s Ar (d e c@ I @( A lo is6. c@4M& 4o / (, F'A -3 f q 400mi 4 00,v c,,[ 'OK 4 -m-e- C k 4-@v 3"2 Jay -e@c@sv-,re t&w--re et'ed *4 CU4 in..VtAt@- f, @65@t 477 ;ZA e" C.C,f i 4c w A-@ OC Les LUtq-ep 4@ @ w,4L ic) l@m --si-zlz 0 Lo c(,t-v4c f 4o p i@444. .9..e 4txul a-w wjos opw-4ec4c( 25 6 s f".$I A se-e F y w clt4.r "J/.t FA-31z-z 10 C@ft4va I - AJ.-1 A c.-eJv^ e - Ali'l FPA-'3(z-z- 0-17oTil -ko yf e*l,dl R% dro@d laISW!? 17 otwo 117/@- 0,1(ot L AOL* Date: 3L6/76 PROJI *Ecr No. 1 Subiect: Object: f2 L*oo 3M TECHNICAL NOTEBOOK PAGE NO. 419417 j@t Date1 . Z/ $ '7-70 7$ 5 Reference: L wo.c, 4 dgw to* 4it t&r4vk.-e Iq ir&ovoo%r.oe,(444i'ii 1* 4 1160 ff Ik Is 41 @! .-&.,k 'Z I c') 3-34,tl Fxfc,54we rv,.64 0 17, Tesf wo-4-,Of 40 sftv%fcp t 0A j A 4,0 cl ect t4;lt )6.41 DC4 PLe tomi 020 16v* I ric@ c k ot 04-S-048 rf lit 4,@l .30 4w4 o-A&-Lor w ite% X;(+or+4 4,r t Al.-tiperd-.c271, r\@rd4 rTw4 64q OS-1 i4ef6,O,f/07/f. Signature-.-.. Date3:j- /76 PIIQJEC@ NO.--!I!l-'7O'@"2 Subject: 3M TECHMCAL NOTEBOOK NO. Date PACE 9@7 2 Object: Reference*. L P 4 -211 'r;q seror, pa.,4 o-C -A,* o s PAR r-em ov I- or 5A pro L\ o LA-c btoeskl, gwf@p@ S tJ&Lr OV.&I k U41,w ct4. s@ fxIst *,-( IjiLyim- @,am@f o.,Jroft-4 2.01 000, // . eotfrili:rg.r^l Lie /g7 16 OKI S'@vwpro Y, cf r, 0&/- 647 e- OIA.v i r7roi do 0 (#'7 10 oleo31 Signature-- Dn*tc: PROJECR No.- 1 Subject: Object: 5 Reference: A lml PAGT, 00 3M TECHrilCAL NOTEBOOK No. 41947 2 Date: -4-f-/,-,-7s- 27/-?4?76 eAk L-Ve racvj 40 va 1;4 a -ti cf-r-et,41.$^. i-4 FM -34?Z *,,Pl-,0 6s irOL .t0 0 vt4Ga 04,78 ww go-ot *JUN wo 0 f -2 ,c( -3 30 r-TOY-1i 0, 84,IU -.@o"K .35 Signature-. Date:- PROJECT NO._ej'-l7 (o17.(,00 I Subject: 001% Object: 5 Reference: 3 -ki.@ k , % @eis as 3M TECHNICAL NOTEBOOK PAGE NO. 4J.947 41 Date: 3/8/77 AO 15 Al rdi@ 6,9-77 -3 1 3//11-77 Lo,4.:1.431 E4Foso4 *3//IJ'77 -?I-.I Uj4,-t1,1771. 4 -504% 110 ww., 00.^0 Oct 4@% p ex4oeted -2 -.to^ r toitt 2./Ooi ;t ex fo@ligot, of ef (,+AL d t e I - -4 @ h, Tt. 30 41 AJ,,6- rOL4*,C% ,rouliol(.,rn4iii T041 OA 16/77- 0.477k 0.11sro@ L61I.laft, .35 ON 14 c4aT.53/J4/77 - 1.1721. Mal e.1171/lF,@ Date:-l-L71--@i- PROJETT NO. Subject: F)k-347'6' (APk6t eafM TECHNICAL *--ek*qtwl T.-S@ NOTEBOOK No. 46269 Date: Z/@/76 PACE :3.5 Object: Of' vima-or #,.A -fit@ saofl^ -tct&o:O#J leI A) we 14--w Fvt - 3,4-2-1 - I&t Ef-e(ir- Ccf4ue@f Rcfcrcnce: CO-CF-41bhg tAS-t@ fol $4A4' COIUVAVI Ckv&^,o,.o-6W A.W. 60/A6 IR.-iA e 0 20-* T.ewp.- :Ewq.Por4 'zo**C t>,4. 30o"e-, Ov-w (7o*e- - /07. OP Cdu0.01 Flto-Aw, telL--" S46vtA*,srAru vt -eoc K)MA-tv SC.vvPles e-*4,r*,c4,e@ no wo t4d- W/V 01 e+i"f 4Ce@,2@* sal k6et tootal UA4,01F dc&wfle @eitraci-e@ -$- 8.*.I,-Cle,'-40 toot. T, --,v p sa. @r-C. 4 %'ai.t^w %,x4 ;A ef@yl scoi6tf @(@ &x e4-@f areJ%L-e wa5 -1-o @Rtf 4y 4o -jolu,*,LL4,,c fiark-. *,..4 rIv -c 4-4 WO r.@ ^ PI-0 Cl -0 +4^tp" A;(IA4-d t rat4.e.-A%(4 jo@,,d ele6ravt4 co (4,Oti Ve.4 ik).All 20@,r+ -Tofo rilsL 20@.. k)@1o-2c FOK-34-2"2 1-,2e.i.3s 4 Fit& @le PIVIC.,401. pfw, tl. t-7 342 6.5 1"@ Fft -"342'z z.,o -75.2 60.(o 73-0 io6)0-z 9 ?41 Si,Lnaturc_?@4!. 3M REPORT: BIOACCUMULATION STUDIIES March 8, 1"3 4. ReportNa 14 (511179-)AnalysiosfFluomchendcalsinBluegilFlishAltboughthisreportoffersome detaileedxplanatioonfanalyticpamlcedures,thesamplesouva and data arepresentedina very marginalizedmanner. Itgivestheimpressionthatdes-(hydroxycthyl)atoifoNn-Et FOSE alcoholomors in the enviroaMCnL 3