Document VJdZ3GzZJL4Jjarv71jYzVngq

BIODEGRADATION (WARBURG) TEST SUBSTANCE Identity:Perfluorooctanesulfonylamido(ethyl)acemtaytea;lso be referredtoas PFOSAA, FC-1 28 oras the major component of FC-1 27, FC-1 29, FC-1 09, or FC-1 09-X. (Glycine,N-ethyl-N[(heptadecafluorooetyl)sulfopnoytla]s-s,iumsalt,CAS # 299151-7) Remarks: The testsample isFCt-128C.urrentinformatioinndicateistis a mixtureof86% testsubstanceand 14% C4:-C7 fluoroalkcyalrboxylate compounds. METHOD: Method: Warburg Determination. Type: Aerobic GLP: No Year completed: 1976 Contact time (units)7: hours lnoculum: Activatedsludgemixed liquorcollectefdrom the Metropolitan Wastewater Treatment Plant,St.Paul,Minnesota. Liquorwas washed, suspended ina basalsaltsmedium, and used ata concentrationof2000 mg ofbiologicaslolidsper liter. Analyticalmonitoring: Oxygen uptake Temperature: 300C Test Concentrations: 1667 mg/L glucosecontrolE,ndogenous control, 20 mg/L testsubstance and 1667 mg/L glucosecombined solution2,0, 100 and 500 mg/L testsubstance solutionsa,nd 500 mg/L and 1667 mg/L glucosecombined solution, Test Solution Preparation: Dissolved1.5grams ofFC-128 in500 mL of DI waterto make a 3000 mg/L stocksolution.A 5 mL aliquotofthestock solutionwas added to20 mL ofDI watertomake a 600 mg/L sample. A 5 mL aliquoteofthe600 mg/L sample was added to20 mL of DIwater to make a 120 mg/L sample. One halfmL aliquotsofone oftheabove 3000, 600 and 120 mg/L solutionsisthen added to2 mL ofthemixed liquorplus 0.5mL Di wateror0.5 mL glucosecontrolsolutiontomake thefinaltest substance concentrationosf500, 100,and 20 mg/L. Remarks: Described method lacksspecificiotny testprocedures. RESULTS Approximately70% ofthetheoreticamlaximum oxygen uptake occurred withinthe 7 hour experimentalperiodifbiodegradationfollowedthe theoreticadlegradationroute: CSF17S02N(C2Hs)CH2COO- K+ + 4.2502 + H+ -> C,3F17SO2NH2 + 4CO2 + 3H20 + K+ Remarks: Oxygen uptake appeared tobe continuingatthe end ofthe experiment. CONCLUSIONS Thistestingindicatedthatthehydrocarbon moietyofthetestsubstance potentiallbyiodegraded inaerobicenvironmentscapableofsupporting biodegradation. Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St.Paul,Minnesota,55133 DATA QUALITY ReliabilitKyl:imischranking3. Testingindicatead highlevelof biodegradationb,utonlyfora theoreticaplartiadlegradationofthe compound. Sample puritywas notproperlycharacterizedI.mpuritiemsay have significantalfyfectedrecordedvalues.Test method, raw data,and an accuratedescriptioonftheprocedurefollowedduringtestingare lackingspecificirteyquiredto insurevaliditoyfthe study. REFERENCES Thisstudywas conducted by the3M Company, Environmental Laboratory,1976. OTHER Last changed: 5/16/00 TO: T*chnical Coormunications Cent&,.201-2S 3coluMmTECHNICALREPORT SUMMARY DATG.. August 12, 1976 Environmental --- LABORATORY, DEPT. NUMBER 0222 - Titi* ), - BiodetradationStudies of Flwroc 'azbons toiCROFCOORPMIES: orojee$2 Fate of Fluorochomicalsin the Environmental ProjecNtwpabor: I R*portNo- : (3 digift) 75-6398-29 T&t R. L. Bohon syl E. A. Reiner 47816 Objective: To determine the biodegradability of SECURITY selected 3M fluorocarbon compounds. Company Confweatiol(open) Noteb*ok Reference: 40671 Pgs. 29-37, 41-SO --SpeciaAlutherination(Closog) IF SUM14ARY REPORT "as informatioInn thisreport been coverebdyothereportsABSTRACT subm;"ed to TCC? and Conclusions(.Sysf*mcanaccommodateM250 words) ofpages lnc6d,rics6vergt#eet: 12 Partially Biodegradation studies using a Warburg respirometer were conducted on FC-GS, FM 3422, FC-128, and hydrogen analogs of FC-9S and Pleasekeyword iniot:;@ov,, FM 3422. No biodegradability was observed on FC-95, although an n*IA@nciudedInotherr4arts approximate hydrogen analog of FC-95 Was readily degradable. andgivepagenun**rosfnow FM 3422 and FC-128 both were demonstrated to undergo some biodegradation. Attempts to isolate degradation products of FM 3422, from the Warburg studies, and from a subsequent activated 3M CHEMICAL REGISTRY #Y-,,Chemlcols reported? sludge study were unsuccessful. me i Yes KEYWORDS Select rm*fal. specific, and 3M product forms from 3M rnsiscurus. Enclose suggested terms in parentheses. EE & PC Div. Envron Assess Biology Bacteria Bioscreening Fluorochemical Biodegradable SPECIFIC PROBLEMS remainintgoreachobjective. Continued attempts will be made to isolate and identify the biodegradation products of Ri 3422 and other 3M fluorocarbons. Work with radioactivity labeled FM 3422 is being considered. Sc,ent-st BIODEGRADATION STUDIES OF FLUOROCARBONS SUMKARY AND RECOMMODATION No biodegradation was observed in Warburg studies on FC 9S. Biodegradation of FC.9S is improbable because it is completely fluorinated. The resistance of this compound to biodegradation by an acclimated microbial culture, however, has not yet been demonstrated. Warburg studies on FM 3422 and PC 128 both indicated that 3omi,b-i@odegradation occurred. The productsof this biodegradationare not known. Semicontinuous activatedsludgestudies on FM 3422 did not confirm or disprovethe Warburg findings. Future investigationof the biodegradabilityof the fluorocarboncompounds would be greatlyfacilitatedby'the developmentof an analyticalprocedure for FC 9S. Warburg studies using purified PC 128 shouldbe made to confirm the present findings. Studiig-onthe biodegradabilityof FM 3422 were hinderedby its low water solub*113'.tTyh.is -problemcould be overcome using FM 3422 radioactivity labeledon its hydrocarb,6pnortion providedthis materialhad a high specific activity(>Smci/m mole)and purity. Biodegradationof saturatedsolution of the labeled compound could be measured by detecting itC02 evolution. INTRODUCTION 7be susceptibilityto microbialmodificationis an importantparameterin the study of the environmentalfate of any ciass of compounds. It'is the most importantform of degradationfor organic compounds. A vast array of organic compounds can be completely degradedby microorganisms. So vastin factthatit was once believed by some that given enough time and the proper conditions,microorganismscould degrade any organicmaterial. This doctrineof microbialinfallibilityis stilla commonmiscmception(l) Perfluorinatedcompoundsare extremelyresistantto biodegradation(2). Although compoundswith single fluorineshave been shown-torelease fluoride ions as a resultof biodegradation,perfluorinatedcompoundshave rarely or never been shown to undergonatural degradation. For this reason,no modification of the perfluorocomponentsof compoundsin this study was anticipated. However., modificationof its hydrocarboncomponents seemed possible. An understandingof the partial degradationproductsis importantsince the environmentwill be exposed to these products in addition'to the undegraded materials. NET HODS AND MATERIALS Chemicals The chemicals used in these experiments are shown in Table I. -2TABLE 1. CHEMICALS USED IN BIODEGRADATICK EXPERIMENTS FM 3422 Hydrogen Analog of FM 3422 C2HS C8Fl7SO@C2H40H C.2HS C8Hl7SO2NC2H40H FC 95 CgFl7SO3 x 4L Sipex-ols FC 128 CSH 170SOpa C12HS C8Fl7SO2NCH2COOK They were obtained from Don Ricker of the Commercial Chemical Division in eptember, 197S. FM 3422 (N-et Fose alcohol) was identified as 788 CC 74-5-2. The PC 9S.used was from lot S83. Lot numbers were not given for the FM 3422 hydrogen analog or the sipex-ols (RM 26442). - . These chemicals were selected for a number of reasons. 'FC 9S is essentially@the@, fluorocarbon constituent of a latgo'number of 3M fluorocarbon con@o*-tn.ds. FM 34@22is an intermediate in the production of 3M fluorocarbonsan d FC 128 is. a finish fluorocarbon product. Sipex-ols and.the Hydrogen Analog of FM 3422 were..selected.for comparison to the fluorocazbons. Sipex-ols is an approximate hydrogen analog of FC 9S. These hydrogen analogs were tested because biologically .labilefluorocarbons have frequently-been found to be gratuitously.defl!uotinated by enzymes which normally remove a hydrogen.' Thus, it seemed probable that microbial growth on hydrogen analogs could select populations-of organisms which .could more-coipletel:y*degrade fluorocarbons. WARBURG DETERMINATION Warburg studies were conducted according to the attached standard procedures. (Attachment) Hicroogranisms were collected from the mixed liquor of the Pigs Eye treatment system,washed, and suspended in a basal salts medium and used at a concentration of 2000 mg of biological solids per liter. Water insoluble substrates were emulsified in water prior to addition to the Warburg flasks. Emulsions were made using a Blackstone model EP;,-2 ultrasonic probe, base 1/2 inch, at 100% power. Logarithmic dilutions in water were made of the test substrates, and 1/2 ml was placed in the first side arm of the.Warburg flasks. Controls contained 1/2 ml of 10 gil glucose solution or deionized watet in this side arm. The second side arm contained either glucose or deionized water. Oxygen uptake was first observed in each flash for a period up to l.S hrs. with readings at 10-15 min. intervals to establish the endogenousactivity. This was followedby addition of the first side arm and continued oxygen monitoring for approximately2 hrs. Addition of the second side arm' containing glucose,a readily degradablematerial, allowed a further evaluationof the toxicity of the previously added material. Semicontinuous Activated Sludge Studies A week-long semicontinuous activated sludge (SCAS) study was conducted on FM 3422. The microorganisms used were obtained, as before, from Pigs Eye Treatment Plant. One Hundred Fifty =I of activated sludge was added to 3 SCAS reactors and tap water as a control to a fourth. FM 3422 was added to 3 reactorsbelow the water surface in 1/2 al of absolutealcohol. Each addition increased the FM 3422 concentration by 33 mg/l. .Pure ethanol was added to one sludge-containingreactor as a control. The operation of the semicontinuousreactors is shown in Figure 1. The SCAS reactorswere aerated for 23 hrs. with 500 al/min. of air while the contentsof each reactor were stirred with a magnetic stirrer to prevent setti.ing.After the aeration period, the sludge was settled for an hour and one*@kter of supernatant was replaced with primat@ effluent from the Pigs Eye Plant. FM 3422 was added at the,beginn3*Lnogf the Test Cycles 1, 2, and 4. Samples were taken at the start and end of -each test cycles and from the supernatant aftersettling. The aeration chambers used in the SCAS studies were plexiglass cylinders 1311high with a 411internal diameter. A side a= allowed drainage of the supernatant leaving the SOO al with the-settled sludge undisturbed. Analytical ..Sampletsaken at the termination of the first Warburg study on FM 3422 .wereevaluatedby thin layer chromatography (Central Research analytical work req. No. AS9412). The samples were extracted into dichloromethane,d'ried to a small volume, and separated on Noels silica plates. The developing solvent system was 10:90 ethanol chloroform (V:V). @Me developedplates were visualized.,byt.he iodine si#archtechnique and comp!Lredto.known standardswith a detection limit of one ug of FM 3422. Samples for the SCAS study were extracted into n-octanol and separatedby gas chromatographywith an electron capture detector. Extractionswere performed in capped SO al polypropylene centrifuge tubes and phases separated by centrifugingat 26,70OXG for -10minutes. RESULTS AND DISCUSSION Warburg - FM 3422 Results from the Warburg study on FM 3422 are summarized in_Eig=e 2. This experimentwas performed by first sonicating FM 3422 and its analog in water to make emulsions of approx. 24,000 mg/l of the FM 3422 and 11,000 mg/i of the FM 3422 analog.. Since FM 3422 and its hydrogen analog are not very soluble in water, it was felt that forming an emulsionwould put more of these compounds in contact with the microorganisms in the Warburg study. -4- STEP 1: Add test compomdt media, and microorganisms STEP 2: Aerate and mix for 23 hours STEP s 'Re-add test compound/ and media. Repeat cycle. /00, magnetic sti.xringbar air sparger r supernatant 4- drain . SIEP 4: Drain supernatant. f sludge STEP 3: Stop aeration and mixing. Let sludge settle. FIGURE-1: Test cycle for semicontinuousactivated sludge reactor. FM 3422 4 32 FM 3422 11,3000 mg/ /"'T"(16ju mole- FM 3422 f@,300mg/l (1.6 Ai mole 00 4JO r,.i3421_@@ An log 0 Addit11ion 0 -2 glucose agddlddujition -3 1..- L .25 .5 .75 1.0 12.@25 1. 1.75 2.0 time hours tlon FIGURL-2: Warburg study of FM 3422 and its5hydrogecLnoseanalog. Hydrogen Analog @-1860.mtii -.._(21u moles) -6- While the FM 3422 analog was relatively easi@lyemulsified and stable once emulsified, the FM 3422 was not. Approximately one hour was required to put 7S% of the FM 3422 into emulsion, and this material proceeded to slowly come back out of emulsion. -In about two to three hours,.excess FM 3422 emulsion,whichhad not been used in the experiment, turned into a semisolid gel. Complete chemical oxidation of the hydroca-rboncomponent of the FM 3422 at the highest concentration (1@16p moles) would require 87 u moles of 02 based on the following equation: c 8F 17S02 NI(C2H 5)C2H 4OH + S.50 2 -1@C 8F 17So2NH2 + 4CO 2 + 4H2 0 Microbialoxidation rarely exceeds 60% of the chemical oxidation. In this experiment, only 2-3 micro moles of oxygen uptake was observed. However, oxygen uptake was continuing at the end--ofthis experiment. Addition of glucose to the FM 3422 culture also produced increased oxygen uptake, confirmingthat the FM 3422 emulsion was not inhibitory to the microbial culture. On the other hand, the hydrogen analog of FM 3422 showed significant toxicity. l@pbiotil-additioofn the most concentrated emulsion 6f the analog, endogenous oxyieiiuptake ceased and was not restored evetkafter the addition of glucose to* th* culture. The negative slope of the.hydrogen analog's oxy en.uptake curve (Figur'sIe)-is due to the endogenous correct3.onana not oxygen evolutim. Similar results were obtained i@fta second Warburg experiment with FM 3422 and its hydrogen analog. Analysis of FM 3422 has shown it to be quite pure. 7he oxygen uptake observed was greater than would be expected from impurities in the compound. It is conceivable that sonication produced degradation products that were biodegradable,but not detectable by thin layer chromatography. It is also possible that some of the hydrocathon components*of FM 3422 molecule were degraded. However, using thin layer chromatography we were tmable,..tdoetect any materials formed as,a result of the biodegradation of FM 3422. It 3.snot known if the hydrogen analog.of FM 3422 itself is toxic. Ihin layer and gas chromatography showed this material to be impure. Gas chromatograph* showed the analog to be 90% pure with two major contaminants. 7he contaminants may have been the cause of the *observedtoxicity. SCAS - FM 3422.. The semicontinuous activate sludge (SCAS) study was a second attempt to 3Lsolatethe hypothesized degradation products of FM 3422. This study was conducted over.a period of l-week with samples taken at the initiationand end of each 24-hr. cycle. The FM 3422 samples added in an ethanol solution rapidly separated from the liquid phase, and as a result may have had too small a surface area to allow significantmicrobial degradation. n-octanol extractsof the samples were analyzed by gas chromatography. No new leaks were formed as a result of exposure of the FM 3422 to the microorganisms. If some of the FM 3422 had been degraded to the sulfonic acid, it would not have been detected. 7bd sulfonic acid is not sufficiently volatile to pass through the gas chromatography column. performed by Commercial t:hemicalsDivision -7- The n-octanol extracts could not be separated by thin layer chromatography because of the low volatility of this solvent. Frozen nmextracted samples still exist at this date and could be extractedinto a more volatilesolvent for thin layer analysis. Three additions of FM 3422 in 33 pps increments were made during the SCAS experiment. The FU 3422 settled with the solids and for the most part remained in the reactor when the supernatantwas withdrawn. The final concentration (althoughnot in solution)was approximately100 mg/l. This material was not homogeneously distributed and accumulated on the sides of the reactors. WARBURG FC-9S The results of Warburi studies with PC-9S are graphed in,Figure 3 No oxygen.uptakewas observed as a result of the addition of FC-9S. This material also caused no toxic effects. Sipol-ol, an approximatehydrogen analog of FC-9S, was shown to be readily biodegradable and to have no toxic effects. The Sipet-ols was soluble at all concentrationstested (as high as 1700 mg/1). FC-9S was incompletely soluble it 4000 mg/l, but was completelyin solution at 400 mg/l. The lack of degradation 'with FC-9S was expected since perfluorinate compounds are characteristicallynonbiodegradable. WARBURG FC-128 Oxygen uptake curves from Warburg studies on FC-128 as shown in'Figure.4. These results indicate that FC-128 is readily biodegradable. Assuming biodegradatim occurs as is shown below, approximately 70%.of the theoretical ran oxygen uptake occurred within the 7-hr. experimentalperiod.- :Mis oxygen uptake is greater than expected and appeared to be continuing it.the end of the experiment. These results are somewhat in question since this FC-128 is known to be an impure chemical. @c 2HS C8F17So2NC H 2COOK + 4.2SO2 + Ht CF +4 8 17SOiM2 C02 + 3-H-20 + K 7@: 6- po 40. 4 0 3 ul 0 2 -8- . glucose i addition substrate addition 0 time hours 2 3 glucose addition FIGURE- 3: Waiturg study of FC 9S and Sipex-ols. 172 mg/l Sipex-ols 000 mg/l FC 95 400 mg/l FC 95 p;l 4 7-- 8 7@ 6-. S- 44- 2 -9FC 128 70 0 Soo mg/i SO -40 r+ 03 -Cc 30 20 100 ;Bg/l lo -0 Substrate addition 2 3 4 s 6 7 time hour; FIGURE- 4: Warburg study of FC 128. -10- REFERENCES: (1) Alexander, M.; Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility. Adv. Appl. Microbial.7: SS-80, 196S. (2) Chapman, P. J.; Department of Biochemistry, University of Minnesota,. St. Paul,.Minnesota, Personal Co=umications 2/24/76. STANDARD ATTACTSIC-NT I PROCEDURE FOR WARBURG DETERMINATIONS 7/10/75 E. A. Reiner I. Design experiment and calculate concentrations of materials to add. 2. Fill-waterbath (DI water if left in bath). 3. Adjust temp. of bath (several hrs. or overnight). 4. .111acemanometers in desired order. S.. Prepare thermobarometer. Add about 3wlof H 20 to I flask. 6. Set Out glassware in desired order* (to match the manometer with which., they were calibrated). 7. Lightly grease center-well top with stopcock grease that can be removed with solvent. Add 0.2 ml 10% KOH. 8. Pr6pol-resamples in DI water.(or acco. rding to reqtiest)'toadd to side arms. Keep,iefrigerated until used,, 9. Prepare ceIls (keep cells cold at all times but avoid freezing). 0 A. Centrifuge 0 C. B. Wash with cold BSM - centrifuge. C. Resuspend in cold BSM. D. Determine concentration of an aliqCuot with the spectronic 20 at 600 nm. Adjust remainder t,odesired conc. basic salts medium. Rafriterate. until use. E. I'akesample of-final idjusted sludge for standard MLSS analys 10. Add saniplesto side arir.@(@usually I ml if one side am, 11ml to each side'arm-if side ams). 11. Add 2 ml of washeticells to flasks. . 12. Add filter paper str-ipttoalka,fiin cionter cup-. 13. Attach flasks to the correct manometer. 14. ltetiglitefnlasks after ttl)oi5jtmin. shaking in bath. IS. Lcive stopcock open tt)utmo,.,ptveraen,d let temp. adjust for an additional 10 'min. 16. Adjust level in manometer to 150 with stopcock open (close stopcock). 1 1'. Begin readings (always idjtistclosed arm of manometer to 1SO mm before reading). 18. Add contents of side arias.according to experiment design requirements. -219. 'fakereadingsperiodically (on open arms) throughout course of experiment. "0. Disconnect and clean flasks. A. Rinse.withwater. B. Wash off grease with icetone. (*. Acid wash. D. Rinse with DI Water. 7-- WARBURG DATA FORM Title: Coll Cone. 2-w6 TME. 31> 17 r Stro es/Min.: Run No.: D Contents of-I.nnerWal Flask No, )@.0-- V%#V%%%Ik- Time 1,5 + Flask Contents + @1,; 7- - ill irls 2 it2t7 3 /SOZ I A,2tr4" ctz--,@ 4 I&ol_ -1@ 6) lio 0 Readine in mm. 4L1 /5 0 LP 01-0'cS>1',5,131-q /-50 0 ell /1-111)7 /@3 71t3/ 3 92 L7-1I-li ,@o /o'4102 9395 pm -2--l-97 z 37-7 :zlo3 @k 13 U2 Lk 1/2 /61 /O_S )Ok 71, @z 3 vli; 9 fr )0)%.-A-7/r--c - (z e - / zz 7z r=;-_iti- 10 ad;@@ - .Zl/7 1 5-00 2Y-IA-- *7- Z trtf@ oty,Z, v 12 ;z-osc@ r-c,-tF-t- '17 'if /-Io L 3 13 loalobL6 70197 'g-1 o li'l53 7lat-@ -- IOL7 Lls- oOL', qD 917 - 7:9'1 44 lzs )144 aL ILI -1-115 @L @.5 141 71 Ai ol") -7 K,-, Title: -D;L" Coll cone*: &Ooml-e-MEO: No, FlIasskk FNNloo,_ T:lw , * + Flask Contents. + 2 3 @4 jk&" c@ - -.-. --- 5 1 656 WARBURG DATA FORM Run No. Dat strokes/Min.: 11@5 Contents of I or Well: - 1741141 Readin in va. 11 ?f o ZA 9 z3o3 12 '371 inc 9 10 -2x.z1i0s1 11 2 'q1-7 112290 1 13 134 is soy". 14 Notes: 152 - (D -/Z 3?- Ile, 7- 9-1 IZ -L ; 4/., -'777-i PItOJECtNO.- 5 Reference: 3M TECHMCAL NOTEBOOK NO. PAGE 406,"f1 33 ...... 2%)- 30 315 OAO Signature Si.Read anti llnei,-r@tev@L4 3@-A Ot A ..... ... r . .. ....... 0 yi Zo,t) Ft- -7 Date: NO ..7 F lop! %w7 .4 riOTFABOOX' NO. Date: PAGE 40671 34 4- 7 aos,". co t T L '7 2L - ------- :0 hA lOr do signauire Date- lip '70- 34 10, IVV Li rp;4 - 3j Soo -,e3k/O' F,7Soz WARSURG,O t)PTAKE*CALMIATION SHEE T F c- w ac@l + Asa d f, 12;zp%,e Tim Elapsisd Reading Time T.S. I 0 Io -67 YO ..75 1.off 1-*15 .76 ISO /SO 150 1150 iS1 Date: ')-A-(?'S Flask No.: 3, CFI"K ontents: FrNdovtAsks Reading Flask (vn) Change 2 in m. (ii" T. D. tfAlcutuaal 3 4 Correction Change m@ m 02 s Uptake Cion's 1Efidosono-u-s-- 0 2ke ta !UP 1,001 0 0 all 1204 -is @i 0050 I,L;4- 1..7y p.o; 2 16;L :2.33 3.0 1)2. 155 )70 ISO ISO /so. ID 16J .':7q@O 7 1''. 31 -31 3 ir rr6 I ISO OJ5 Liz)' ..300-- 1 0. qgl @,- . -60 63 73' Li 5,>9 0 C.1,3 7. Footnotes,over. 4 Z. ..,VARSURG.0 :.:UPTAa..CALCTAATION SHUT 13 Contents: 0 'rbitic-t 4" Date 75. Fl"k No.': F "it lehorso c E@ 2 T.B. Actual 02 0 Tim Elapse asaong I Reading Flask Change in m. Correcti 4 Change s Uptake 2 Uptike T. S. I kimlm- (Val (M) IT -71, . -t-*13 .-95 71 330 360 310 /72 ++o33@0 -lot *,g Footwtes over,; ---.c -w- ..................... FC- 1> cl Title: edri-:rova't,";.ifr-i+- Time Elapsed Time 0 10 70 1/0 .75 i.oir 45 1.-,t5 7:@ Reading 1 T.B. URL... 151 0 ISI WARBURG 0 UPTAKE 2 Date: 9-;'C-75 Reading 2 Change Flask in mm. (MM) t.Loi!2. /.-@o CALCULATION SHEET 0&3/5 Flask No.: T.B. 3 Correction Flask ---Contents: Actual 4 Change 0 25 Uptake !tn@d@ inoCuoi 02 Uptake --(mm) - --IV- )Ies) 0 0 1*7 o.so /,37 1,00 3 7 0 ;2 5 2.- 7@5 21 )-t3 oe 3.)3 44 )05 117- 1,@0 J4 155 )70 Ilr5 I ) 015--@ :241Z) ;L9"L-) "300 150- L.@0---- 2 150 @3 61 95 158' e Is () --1-1 Is)--- ill _I 7.1 --414 -57 -4.5 -31 4-'l -45 -5(, 1;1'7 140 .71 3.1 - Li6.4 Is 7-It 3IS3 3.t6 q. 2-V .13 -30 7,i,l. Footnotes over. . .:is FC -/2I-r -ro Title: (314cd@t-,eIli2y,,1,,1-y Time Elapsed Reading hi s Time (Tm.mB). 330 1360 3 @7 /7; '/;D 173 WARBURG 0 UPTAKE CALCULATI(IN SliEET 2 Date:9-@4-75 FlaskNo. Flask Contents: Reading Change 2 Flask in mm. (MM) L (=R) T.B. Actual 3 4 Correction Change (mm) /g, 02 5 Uptake (immol .@i +30 -i@(f@g ous 02 Uptake -@u!lglss) It 5,07 'If Footnotes over. ............ A F C- i;@r. w oc@ + Title: d ei r,c(, Time s Elapsed Time Reading T.B.1 WARBURG-OL.UPTAKE I)ate:1-;L(- 7 Reading Flask ChanS02 in m. CALAILATION SHEET Flask No.: T.D. 3 Correction Flask Can tent'sP:'b Fc Actual Change 4 (mm) 02 Uptak as s Enaog&nO%Ll I''@li.kc I 10 :;o 1/0 -.75 .1 .45 OT 1-7.5 105 is,.* )2,0 1019 6o)7 '0150 3q -24 1.71 :2.0,-L- 3%) 150 ,qo 0' t3 At -'47 09 1 f.5 15 e 0 -)05:. - '115'-. At +1 14 1 11 45 0 300 Footnotes ove FC 121-V Title: FTT'iimtne Elapsed Time f-Y Reading T.B.I S.'S @,330 360 72 173 WARBURG 0 2 UPTAKE cALCulATION SHE ET Date 7 5 Flask No.:' :6.-cFolnasttesnt Re ading Flask Change2 T.B. -,AvAacmnct"g'ue't4aLl1 Up.0t0a22kesS Correction Change Uptakes HERO 3 -7.S-5 -73 Endos7enous. rl'j'pUi0Oop22att'kiinek'.e s 7t'9 Tr ,vp 9107' Af .77 FootnoteOs",r 1:c-1",Lri-o + Title: A rad Reading Time Elapsed I Time T.B. Li WARBURG 02 UPTAKE. CALCUIATI ON SHEET -t)ate1:-;Lt 7 5 0 Fl&sk'N 7 F ask Contents: Reading Flask Change 2 T.B. Actua. l 4 in m. Correction Change 2 *S Uptake Endogenous 1 02 1-Uptike 153 ;o /so 047 qo y0 .75 1 11 of 75 75 ISI r ,5q 33 2.sr 15-5 /.So 96 .)70 oe, Ilr.5 Ise 3.15 00 Fwtnotes over 5 10 o. 0. 0,50 3q '73 6.SO .53 5,oy Tcl 4F FC f-y TiLtle: 6 dA i -c/7, WARBM.G 02 TWTAKE CALCUIATION SHEET Date: Flask No.: Flask contents:'2-a rC -'t 'Time Elapsed kae 330 Reading T. B. Reading Flask 33 Change 2 in M. 15 7 T. B. .3 Correction Actual Change 4 (mm) :@:z0 . 0 2 S. Uptake LU Endogenou 02 Uptake 71 91 6z> /72 @,i-@93 + g,o7 ./73. T4 Foot3l6tes over. Title: FC- i>frde-iaL + Time Elapsed Time YA IV% Reading I T.B. f=l 151 10 67. YO 75 1 '45 ).08 75 IS/ WARBURG-0 2 UPTAKE CALCULATION SHEET Date:1-;L(-7 5 Flask'No.: Flask. contents:-.. Reading Flask (mm) 151 2 Change in M. T.B. Actual .3 4 Correction Change 0 2s Uptake Mmol s En4ogenous up 0 2 take j. 0.17 0150. 0 33, ..o to 1171 -7 0 /04 3-ir 1,50' /so t3 151 16 .300 Footnotes over @73. .. -77 .1os OIL@L IID 6, l@O ZFOI... -7,77 o!S 7,iy -i4, Title: FC. Time Elapsed Time ..30 --,360 31Z) WARBURG 0.2 UPTAKE CALCUIATION SHEET c/2"Y07,f,2, Reading T.B. (MM) Date: Reading Flask -)-1-75 Change 2 in m. Flask No. T.B., .3 Correction FliLsk ContiM"tS' IQA l@ Actual Change 4 0 -6dogenou 25 02 Uptake Uptake fumoles). /7@ 9,o7 173. Al 9. Footnotes over' A Tit o: FC- I)lr row c,;f- + Tiow Elapsed Time Reading T.B. C) 150 /so YO 150 WARBURG 02 'TWTAKE CALA)LATION SHEET Date -'75 Flask No.: F ask con tents: Reading Flask amse 2 T.B. Actual' in m. Correction Change 4 .0 2 Uptake s (uml Endogenous. 0@2 Uptike I o'i 6.17 0,50 /3y 75 0 033'. 155 o;j Io@ ;L 300. FmtnOtes.o.ver. si 31 1,91f @kJL*7 :91 ... !:..: 0 Ila ISO -o 0 ..-, so -5 I q,3- 2. 65. 3. @3 jSo -7@7 71 16,3. '7.1 _to oy 151 .. 3) @@t-9 +1 c1 /O.OS Title: FC,08120,0d-atirrogi-tcle Time Elapsed Reading 1 Time T.D. S..S '330 ... /72 7 73 WARBURG.0 UPTAKE CALCULATION SMET Date: Reading Flask If 9-7 2 Change. in am. (am) I @ lo;. qo 2.. Flask No. F ask contents,4. T.S. Actual 3 4 Correc.tiLon Change (UM-1 0 Endogenous 2 S..: Uptake 02 Uptake (uno 7,9 1 -lb6 35 @.q .174 9,07 91., Af Footnotes over. FC-1,'xir low + Iritle: TFTiimse e Reading Elapseedd] I Time T.B. ,VARBL IRC 02 UPTA'KE"*'CALCtJLATION SHEET Date:+I-N 75 F'I"k'-No. 4 2 T.B. Reading Change 3 Flask in m. Correction F I"k Conteniti.:5'dO Actual 4 Change 02 s Uptake f ;Eunpd0agenous.t 2 take lo qo 1 l15 1.0 65 76 F,l.t5.5511 so0 lqt og 6.17 .1;0- .ISO- )qL 53 oe 15 + '7 ISO 151. 14q ir0 300 +19 iy Footnotes aver. FC TiLii 6 dA ",Cvbe,1, Time Elapsed- Reading .,Time 1 T.B. S -.,330 360 77 -173 2 UPTAKE CALCUIATION SHEET Date: Reading 9-).1-F*71&53kNo'.:: Change 2 T.B. in m.,' Correctio@ 31. Flask /-L 'contenti:,56."o Actual Change4 0 Endogenot :2,..'-. .02 UptakL- Uptake. (mleal (Mies) /2:Zg -7-1,-91" -)q 7 07 15 5 3y Footnotes over.. -tieN F C-I;fLr + Title: 031 del f, a* Time Elapsed Time Reading 1 T.D. WARBURG Date: Reading Flask 02 UPTAKE CALCULATIC)N SHEET 9-;Lc - 7 Flask'NO. CFonatesnkts: 150otA;/eet Change 2 in m. T.B. Actual Correct3iLO]n 4 Change 0 IEr.idoge 2 0 Liptake I UP ta2k [humo 10 ISO q@ o !@)0 015 L/0 175 1.3 .75 1.77 ISO 0 -3 Li 2.33 q4) -51-0 A41 CY 150 i6,3 -44 lr5 15e log 15) -70' 71. + -7- 115 :-7@7 + O/q 300 Footnotes over. MRM 10620.0-pwo SAMPLE DESCRIPTION ENVIRONMENTAL File: Page No. ENGR. LAB -WORK SHEET Date: Anal yst: Hrs So V uv Reviewed by: