Document mqLVzzmqnBQL03aqmqM2J7owb

/)R2K-03<($ BIODEGRADATION (WARBURG) TEST SUBSTANCE Identity: Perfluorooctanesulfonylamido(ethyl)acetate; may also be referred to as PFOSAA, FC-128 or as the major component of FC-127, FC-129, FC-109, or FC-109-X. (Glycine, N-ethyl-N[(heptadecafluorooctyl)sulfonyl]-, potassium salt, CAS # 299151-7) Remarks: The test sample is FC-128. Current information indicates it is a mixture of 86% test substance and 14% C4-C7 fluoroalkyl carboxylate compounds. METHOD: Method: Warburg Determination. Type: Aerobic GLP: No Year completed: 1976 Contact time (units): 7 hours Inoculum: Activated sludge mixed liquor collected from the Metropolitan Wastewater Treatment Plant, St. Paul, Minnesota. Liquor was washed, suspended in a basal salts medium, and used at a concentration of 2000 mg of biological solids per liter. Analytical monitoring: Oxygen uptake Temperature: 30C Test Concentrations: 1667 mg/L glucose control, Endogenous control, 20 mg/L test substance and 1667 mg/L glucose combined solution, 20, 100 and 500 mg/L test substance solutions, and 500 mg/L and 1667 mg/L glucose combined solution, Test Solution Preparation: Dissolved 1.5 grams of FC-128 in 500 mL of Dl water to make a 3000 mg/L stock solution. A 5 mL aliquot of the stock solution was added to 20 mL of Dl water to make a 600 mg/L sample. A 5 mL aliquote of the 600 mg/L sample was added to 20 mL of Dl water to make a 120 mg/L sample. One half mL aliquots of one of the above 3000, 600 and 120 mg/L solutions is then added to 2 mL of the mixed liquor plus 0.5 mL Dl water or 0.5 mL glucose control solution to make the final test substance concentrations of 500, 100, and 20 mg/L. Remarks: Described method lacks specificity on test procedures. RESULTS Approximately 70% of the theoretical maximum oxygen uptake occurred within the 7 hour experimental period if biodegradation followed the theoretical degradation route: C 8F 17S 0 2N(C2H5)CH2C 0 0 ' K+ + 4.2502 + H+^ C 8F 17S 0 2NH2 + 4 C 0 2 + 3H20 + K+ Remarks: Oxygen uptake appeared to be continuing at the end of the experiment. CONCLUSIONS This testing indicated that the hydrocarbon moiety of the test substance potentially biodegraded in aerobic environments capable of supporting biodegradation. Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 3. Testing indicated a high level of biodegradation, but only for a theoretical partial degradation of the compound. Sample purity was not properly characterized. Impurities may have significantly affected recorded values. Test method, raw data, and an accurate description of the procedure followed during testing are lacking specificity required to insure validity of the study. REFERENCES This study was conducted by the 3M Company, Environmental Laboratory, 1976. OTHER Last changed: 5/16/00 OOO Fo*' TO: Technical Communications Canter, 201-2S TECHNICAL REPORT SUMMARY DATE: August 12, 1976 Environmental________ LABORATORY, D EPT. NUMBER 0222 Title MICROFORM COPIES: Biodegradation Studies of Fluorocarbons Project: Fate of Fluorochemicals in the Environmental To: R. L. Bohon By: ProjectNumber: IReport No: ! (3digits) 75-6398-29 ! Employee Number: E. A. Reiner 47816 S E C U R IT Y ,Company Canfidantial(Opan) Objective: To determine the biodegradability of selected 3M fluorocarbon compounds. Notebook Roforonco: 40671 Pgs. 29-37, 41-50 [ Spaciol Authorise!ian(Closed) IF SUMMARY REPORT H o t inform ation in th is raport boon covered by other reports subm itted to TCC? No. ofpoQOB Including eovorshoot 12 ABSTRACT and Conclusions. (System con accommodate 200*250 words) S * ' . P a rtia lly : C om pletely 1 P lease keyw ord in fo rid ^tio n not^included in other repdrts and give page numbers of new m aterial: Biodegradation studies using a Warburg respirometer were conducted on FC-95, FM 3422, FC-128, and hydrogen analogs of FC-95 and FM 3422. No biodegradability was observed on FC-95, although an approximate hydrogen analog of FC-95 was readily degradable. FM 3422 and FC-128 both were demonstrated to undergo some biodegradation. Attempts to isolate degradation products of FM 3422, from the Waiburg studies, and from a subsequent activated 3M C H E M IC A L R E G IS TR Y sludge study were unsuccessful. '' * \ w c h e m ic a ls re p o rte d ? i'llTes KEYWORDS Select general, s p e c ific , and 3M p ro d u c t te rm s from 3M T hesaurus. E n clo se suggested term s in parentheses. EE 5 PC - Div. Envron - Assess Biology Bacteria Bioscreening Fluorochemical Biodegradable SPEC IFIC PROBLEMS remaining to reach objective. Continued attempts will be made to isolate and identify the biodegradation products of FM 3422 and other 3M fluorocarbons. Work with radioactivity labeled FM 3422 is being considered. fo" " 'jt io n jc ie n t* s 00 BIODEGRADATION STUDIES OF FLUOROCARBONS SUMMARY AND RECOMMENDATION No biodegradation was observed in Warburg studies on FC 95. Biodegradation of FC 95 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 FC 128 both indicated that some biodegradation occurred. The products of this biodegradation are not known. Semicontinuous activated sludge studies on FM 3422 did not confirm or disprove the Warburg findings. Future investigation of the biodegradability of the fluorocarbon confounds would be greatly facilitated by the development of an analytical procedure for FC 95. Warburg studies using purified FC 128 should be made to confirm the present findings. Studied* on the biodegradability of FM 3422 were hindered by its low water solubility. This problem could be overcome using FM 3422 radioactivity labeled on its hydrocarbon portion provided this material had a high specific activity (>5 mci/m mole) and purity. Biodegradation of a saturated solution of the labeled compound could be measured by detecting 14C02 evolution. INTRODUCTION The susceptibility to microbial modification is an important parameter in the study of the environmental fate of any class of compounds. It is the most important form of degradation for organic compounds. * ,%* A vast array of organic compounds can be completely degraded by microorganisms. So vast in fact that it was once believed by some that given enough time and the proper conditions, microorganisms could degrade any organic material. This doctrine of microbial infallibility is still a common misconception(1). > Perfluorinated compounds are extremely resistant to biodegradation (2). Although compounds with single fluorines have been shown to release fluoride ions as a result of biodegradation, perfluorinated compounds have rarely or never been shown to undergo natural degradation. For this reason, no modification of the perfluoro components of compounds in this study was anticipated. However, modification of its hydrocarbon components seemed possible. An understanding of the partial degradation products is important since the environment will be exposed to these products in addition' to the undegraded materials. METHODS AND MATERIALS Chemicals The chemicals used in these experiments are shown in Table I. 000290 -2TABLE I. CHEMICALS USED IN BIODEGRADATION EXPERIMENTS FM 3422 Hydrogen Analog of FM 3422 c2h5 i C8H 17SO2NC2H4OH FC 95 ^8F173^ Sipex-ols ' 9. FC 128 CgH17OSO a They were obtained from Don Ricker of the Commercial Chemical Division in September, 1975. FM 3422 (N-et Fose alcohol) was identified as 788 CC 745-2. The FC 95 used was from lot 583. 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 95 is essentially the* fluorocarbon constituent of a large number of 3M fluorocarbon compounds. FM 3422 is an intermediate in the production of 3M fluorocarbons, and FC 128 is a finish fluorocarbon product. Sipex-ols and the Hydrogen Analog of FM 3422 were selected for comparison to the fluorocarbons. Sipex-ols is an approximate hydrogen analog of FC 95. These hydrogen analogs were tested because biologically labile fluorocarbons have frequently been found to be gratuitously defluorinated 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 completely degrade fluorocarbons. WARBURG DETERMINATION Warburg studies were conducted according to the attached standard procedures. (Attachment) Microogranisms 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 g/1 glucose solution or deionized water in this side arm. The second side arm contained either glucose or deionized water. -3- Oxygen uptake was first observed in each flash for a period up to 1.5 hrs. with readings at 10-15 min. intervals to establish the endogenous activity. This was followed by addition of the first side arm and continued oxygen monitoring for approximately 2 hrs. Addition of the second side arm containing glucose, a readily degradable material, allowed a further evaluation of 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 ml of activated sludge was added to 3 SCAS reactors and tap water as a control to a fourth. FM 3422 was added to 3 reactors below the water surface in 1/2 ml of absolute alcohol. Each addition increased the FM 3422 concentration by 33 mg/1. Pure ethanol was added to one sludge-containing reactor as a control. The operation of the semi continuous reactors is shown in Figure 1. The SCAS reactors were aerated for 23 hrs. with 500 ml/min. of air while the contents of each reactor were stirred with a magnetic stirrer to prevent settling. After the aeration period, the sludge was settled for an hour and one titer of supernatant was replaced with primary effluent from the Pigs Eye Plant. FM 3422 was added at the.beginning of the Test Cycles 1, 2, and 4. Samples were taken at the start and end of each test cycles and from the supernatant after settling. The aeration chambers used in the SCAS studies were plexiglass cylinders 13" high with a 4" internal diameter. A side arm allowed drainage of the supernatant leaving the 500 ml with the.settled sludge undisturbed. Analytical ,v. Samples taken at the termination of the first Warburg study on FM 3422 were evaluated by thin layer chromatography (Central Research analytical work req. No. A59412). The samples were extracted into dichloromethane, dried to a small volume, and separated on Woelm silica plates. The developing solvent system was 10:90 ethanol, chloroform (V:V). The developed plates were visualized by the iodine starch technique and compared to known standards with a detection limit of one yg of FM 3422. Samples for the SCAS study were extracted into n-octanol and separated by gas chromatography with an electron capture detector. Extractions were performed in capped 50 ml polypropylene centrifuge tubes and phases separated by centrifuging at 26,700XG for 10 minutes. RESULTS AND DISCUSSION Warburg - FM 3422 Results from the Warburg study on FM 3422 are summarized in Figure 2. This experiment was performed by first sonicating FM 3422 and its analog in water to make emulsions of approx. 24,000 mg/1 of the FM 3422 and 11,000 mg/1 of the FM 3422 analogy Since FM 3422 and its hydrogen analog are not very soluble water, it was felt that forming an emulsion would put more of these compounds in contact with the microorganisms in the Warburg study. 000292 ~4- STEP 4: Drain supernatant. STEP 3: Stop aeration and mixing. Let sludge settle. FIGURE 1: Test cycle for semicontinuous activated sludge reactor. 000^93 -5FM 3422 FM 3422 -6- While the FM 3422 analog was relatively easily emulsified and stable once emulsified, the FM 3422 was not. Approximately one hour was required to put 75% 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 which had not been used in the experiment, turned into a semi solid gel. Complete chemical oxidation of the hydrocarbon component of the FM 3422 at the highest concentration (^16 p moles) would require 87 y moles of 02 based on the following equation: C8F17S02N(C2H5)C2H4OH + 5 *502 "* C8F17S02NH2 + 4C02 + 4H2 Microbial oxidation 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 of this experiment. Addition of glucose to the FM 3422 culture also produced increased oxygen uptake, confirming that the FM 3422 emulsion was not inhibitory to the microbial culture. On the other hand, the hydrogen analog of FM 3422 showed significant toxicity. Upoi^addition of the most concentrated emulsion of the analog, endogenous oxygen uptake ceased and was not restored even after the addition of glucose to the culture. The negative slope of the hydrogen analog's oxygen uptake curve (Figure 2) is due to the endogenous correction and not oxygen evolution. Similar results were obtained in a second Warburg experiment with FM 3422 and its hydrogen analog. Analysis of FM 3422 has shown it to be quite pure. The 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 hydrocarbon components of FM 3422 molecule were degraded. However, using thin layer chromatography we were unable,-to detect any materials formed as a result of the biodegradation of FM 3422. It is not known if the hydrogen analog of FM 3422 itself is toxic. Thin layer and gas chromatography showed this material to be impure. Gas chromatograph* showed the analog to be 90% pure with two major contaminants. The contaminants may have been the cause of the observed toxicity. v SCAS - FM 3422 The semicontinuous activate sludge (SCAS) study was a second attempt to isolate the hypothesized degradation products of FM 3422. This study was conducted over a period of 1 week with samples taken at the initiation and 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 significant microbial degradation. n-octanol extracts of 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. The sulfonic acid is not sufficiently volatile to pass through the gas chromatography column. * performed by Commercial Chemicals Division 000295 -7- The n-octanol extracts could not be separated by thin layer chromatography because of the low volatility of this solvent. Frozen nonextracted samples still exist at this date and could be extracted into a more volatile solvent for thin layer analysis. Three additions of FM 3422 in 33 ppm increments were made during the SCAS experiment. The FM 3422 settled with the solids and for the most part remained in the reactor when the supernatant was withdrawn. The final concentration (although not in solution) was approximately 100 mg/1. This material was not homogeneously distributed and accumulated on the sides of the reactors. WARBURG FC-95 The results of Waxburg studies with FC-95 are graphed in Figure 3 . No oxygen uptake was observed as a result of the addition of FC-95. This material also caused no toxic effects. Sipel-ol, an approximate hydrogen analog of FC-95, was shown to be readily biodegradable and to have no toxic effects. '# * The Sipel-ols was soluble at all concentrations tested (as high as 1700 mg/1). FC-95 was incompletely soluble at 4000 mg/1, but was completely in solution at 400 mg/1. The lack of degradation with FC-95 was expected since periluorinate compounds are characteristically nonbiodegradable. 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 biodegradation occurs as is shown below, approximately 70% of the theoretical maximum oxygen uptake occurred within the 7-hr. experimental period. ,rThis oxygen uptake is greater than expected and appeared to be continuing at the end of the experiment. These results are somewhat in question since this FC-128 is known to be an impure chemical. v * ^2^5 CgF17S02NCH2C00K + 4.2502 + H+ * C gF17S02NH2 + 4C02 i 3H20 + K+ 000296 S 7.; -8- 6. w 3 O C 41 00 In 2 T<3U VO 4) fi fi * UO V +3-> (N O / </> O --4 O 6 5~ f 2- glucose / addition j 1_ 0 X '* substrate addition 4, I J. 1 glucose addition FIGURE 3: Warburg study of FC 95 and Sipex-ols. 000297 nioitib 81 7-- -9FC 128 audition ) " 1 i i i ^I I 12 i i t 11 l 3 i time hours FIGURE 4: Warburg study of FC 128. 000298 -70 .60 50 L40 30 V 20 10 /im oc t /9m to t avr?liT n ^C \ TT?3Tld.IOin. REFERENCES: (1) Alexander, M.; Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility. Adv. Appl. Microbial 7: 35-80, 1965. (2) Chapman, P. J.; Department of Biochemistry, University of Minnesota, St. Paul, Minnesota, Personal Communications 2/24/76. 000299 ATTACHMENT I STANDARD PROCEDURE FOR WARBURG DETERMINATIONS 7/10/75 E. A. Reiner 1. Design experiment and calculate concentrations of materials to add. 2. Fill water bath (DI water if left in bath). 3. Adjust temp, of bath (several hrs. or overnight). 4. Place manometers in desired order. S.. Prepare thermobarometer. Add about 3ui of 1^0 to 1 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 . Prepare samples in Dl water (or according to request) to add to side arms. Keep''refrigerated until used. 9. Prepare cells (keep cells cold at all times but avoid freezing). A. Centrifuge 0 C. B. Wash with cold BSM - centrifuge. C. Resuspend in cold BSM. D. Determine concentration of an aliquot with the spectronic 20 at 600 nm. Adjust remainder to desired cone. C basic salts medium. Refrigerate until use. E. Take sample of final adjusted sludge for standard MLSS analysis. $. 10. Add samples to side arms (usually l ml if one side arm, h ml to each side arm-if 2 side arms). 11. Add 2 ml of washed cells to flasks. 12. Add filter paper strip to alkali in center cup. 13. Attach flasks to the correct manometer. 14. Retighten flasks after about 5 min. shaking in bath. 15. Leave stopcock open to atmosphere, and let temp, adjust for an additional 10 min. 16. Adjust level in manometer to 150 with stopcock open (close stopcock). 17. Begin readings (always adjust closed arm of manometer to 150 mm before reading). 18. Add contents of side arms according to experiment design requirements. J 000300 9. Take readings periodically (on open arms) throughout course of experiment. 0. Disconnect and clean flasks. A. Rinse with water. B. Wash off grease with acetone. ('. Acid wash. L>. Rinse with DI Water. v 000301 Ksrcopr/iMiuat ^-- 7 WARBURG DATA FORM uo _ Title: & la Run N o .: Date 75- Cell Cone. : Temp. : 3c> <- Strokes/Min. : j j Content_s__o_f__I_n_ne_r__W_el_l_:_________ Flask Time 9/5 >I'Q1*tfoks ks ks boI'^lz/zl/fol^oM/^M, |iwUo5|jvc^ No. t 68921J27W-^f9r7 2f-0w*--j;//^Ff^CcC.--*-r-//^trvzv--'r[/JILIPSfp<e77/)/,11I11%f1??1f1l?7l ///fSS5t-1Op?o//A</3?*?// //iSiiitb ///AS//7O /5 k /5oVA _. !?/Y~ 1110^2/5 /ooV*^/A --" ffj - )3oo 0 o ff3s HZ*? 3 /Zoz A,2t9~Z< 4 / O I 'XZHXH 'A 5 tvoi Z, -K30Z 7 Z37P ?3o-i Z,37f &L2 f a '32.7. 727*} /oo lo o t c 0 *.W w * -- fc uk a Fc~/z f" 12 13 2'Z,^Z9'J/s7fy**S5^ o %V1? ^ !^ /A 14 F c-iL *' f^c-i -r- e l$ l & Iso /so /SQ M m \ fttl jm ft p<\ M !?1m IS< ISo 1so & IBS ! $ H i O I40 fsp f l 'i 17 n s m m toH tOf. n )9 l$3 IS? M 150 IS I \SL / & m m /37 -vtA1 w h \ 137 M m I f l ! % Ho 137 V7 m f(A 93 z s t i f H i H i /So I f /tjj 14s < 3 f p . no in V I m k ] /0S 01 # ? n i in Ub H? fto 9 i n 11 H I H i IIC ? f m 131 MS u iso 11% n s '1/ l$ H i i n vs fb m t t i in H i iw It m f> 7t> r> ISO H i m '}1 flk w 17 fp(Ht rn If ft fz W tp *1 w /p . 'n \an ( I t t m (30 ft 3i m 10 if? > / / $ fQ(> faz (VtM<* nlntosvi UH7%CSH ?>j?t/1oiz1 1 37% 70 97 53 & $1 77 7 / toi m ll 70/ft ?5 fi tH V- 10 j\ \ / HI Hi Hi Hi f5o w no 61 N5ita>\ HW** iK i 3K/of %r 1-ii ^iV <7 JXJL-VVO > Notes: C l J j U l ^ycJU . #-w-~ 1Fi- (p^-e-tr^ cn- l( f /ft 7 t*K>> SoUh*x> ) U~ 9 7 # *J -A .(***1 4% 5~ lSr-^r f b 0 |C 7. K, ^ P C c F<- WARBURG DATA FORM Title: -o Cv~aM4&, U>KZC./W 4. j, frC. ' J z e & ___________________5 _______ P**?.:, 7.~ 2 > V & , Cell Cone. : JZQOQny/fiemp. : 3 >*<2 Strokes/Min.: / A 5 rFlask No Time /5 3oo *30k^> M(20 1 2 3 4 f*9t<\N9*Co1i3.s2A75^-*,. /.SifO??X, /&W0iZe + Flask Contents + ^^4srJibaut-/r7rujd**-> Citi<sfi.)aiac.-oeAutJcLuLOOtohn^lLllcod'! * 5 -sir M fh /fl /7Z!?} )1D 6 92.239f7i. ' 7 22l3x*'7. Zo Zn^jJ FCJ?? /57 r^f 2Lx Mo m-m (<& 9 10 11 12 13 42J3<?/#3 23 IS *233X?T7E-?-*<?/ a*232i.122z-^/9/sS7-17 /o^ /J Fc-yzr //-e#--a-.-n--yc//J<?F/nAct^/.Zrxtrf _ ^ y^c/zv 0&<?>Au>y^/=yz/ C !& / / i tfi JZs m n // -- *(# in. (01f t ?7 /5o3y?f V //5 til --1 ih 11 ^ Contents of Inner Well:,/? I Reading in mm. --- /% k it ! 14 Notes: / 5 $ - o 5 0 0 0 3 0 3 PROJECT NO. j Subject. ! /J Cu> , BEST COPY AVAILABLE TECHNICAL NOTEBOOK f r, V 1 .,,,, i & x t c j f * t~- *f f NO. PatC: 40671 C} ' 2 ' S ~ 7 '3~ PAG 33 Reference: 6*X M j~*~ 'f _ < k d y * * * * & -A-- r I? -; Read and Understood 000304 >*^ i PROJECT NO .V. f - T ` ' d FC -f- &- fT ' + U S ' ---->ji 1 ' j ' . I' " . . -[-- -fy T - T * '--l - 3M TECHNICAL NOTEBOOK NO. 40671 34 1 "i --4--- -- i &r r e - t u r ~J * ~t^r >1 irr-u Signature. Dl>flil oaiA>lwA>itiir>><- Ux+fsj ^ j k ^ T W ' i_______ y > __ .p. . 4. / . *j> ** W X . -"t--- f-- T~i" "T ~L~.. 000305 Date:- \.l 1 I BEST COPY AVAILABLE P e- a r mumbest copy ") 'oo 3x/o X * ($6 0 Ar^- F C~ l ^ y 2 a *, y I,/' ^ f ;, s O , C . //* V /1 > u Sf A/-- A V ? 0 K + Y.2 S 0 . Vi >! *r to w - PA H ' /7 ^ z A ^ - a A j, ^ V ^5> 5 < K?H k^ c.\V < r**i " " 3 9 "7 t' 2- c O LV " A * t \>H " ) / y P /H i * 6 ;AA >vv r "7 y tV TC-rxr /t ci w 66 A M 1 'v 3 ^~/- ^ f" 000309 * * . && j'V--'.1 :V-xV WARBURG 0, UPTAKE CALCULATION SHEET Title: ^ *hX^> Tine h<^ Elapsed Beading Tine T.B.1 5 330 * 3*0 ICC /9 _JW o.. m 7 V20 173 Date: ^ - ^ '7 S Flask No.: -3 Contents: C o ^ri Reading Flask (ml . Change2 in an. T.B. Correction Actual C_hange4 Endogenous:Corrected Upta*ke5 0 < u2 Uptake j Uptake6 fan) Can) j (uaoJ.esi i. wgU*JL, (Mrdggl.. /3i - 7 ^ W 1 -75 +v*6 -9 5 --/o l 7,9? ----- -7 9 -+?9 - />g q.oi + 3 0 - IO 1.H1 -- .-- >-- - --~ ----- -- -**. * ................ ----- -- -- ---- J t'rf . W\ V-*"?v . A.At Footnotes over. a ------ - '-V 1 . . . -Tj P ...-.a -'s-> '' "' - ,; i '"1-- "" '" ...-... - v.--',' &*Kk~A r _ . ..V,u A ' - ltr WARBURG 02 UPTAKE CALCULATION SHEET . O S r 3 5 R r<- Title: FC-/AS-, rov'fy 4 ---&-i----d----r~ftc---m-l-a--i--'-b7y~ Time Hr S Elapsed Time yv\.^ Reading T. B .1 Date: Reading Flask (mm) A i -75 Flask No.: Flask Contents : Change^ in mm. T.B. Correction^ Actual CTMhange4 Onm) . . fmm) 2 s Uptake fumles] rat Endogenous' Corrected Upt2ake Up5t2ake6 fumles] Jym . 0O ,167 ;o -33 2 o -67 ' Ho 75 HS /SI /5o O l$o H i -/ tSO H I - 3 150 / i l -<9 / S o /3 7 - I S 00 -1 0 - 1 - 7, -1 - S - 1 - IX 0 L ._ . .... _x 0 0 , / 7 JT.-n , l l 7,50 - .3 3 ' t b-c~ . . i j y . . . - . 5 2 1,00 /. 3 ^ - 3 4 ),Q% /. A5 1.5 K75 i.n 5 75 ...._ _ 9 o ____ m IS) /SI IS1 is) iso /3 /> 7 ----- -- ll !U - /? -A3 0 r>l J b 0 -A C ~ 3 3 ] " ... ~ -3 1 -3 V 1 _ i 1 -33 ).$# ).M 2 ,il 2 **7/ 2,o*> ? .A 7 .....2 .5 2 2. S'd . 3,/'. -H H - .3 - .36. -.* $ r -, 37 X 2.33 xsr 7 .0 3 . ol 3 IS )}0 wo IS5 )10 US in ISO ISO so lS3 iSt ISO m . 93 ?5 . Hi -ir " -V ' -5 7 -LS -w -fri -i -i hX 4-1 +7 -3 * -4 $ -5 -7 . - L i * .. . 3. U 3-79 9.64 5.57 7 .3 1 7.3*1- . 2 ^ . - . i X 3 ,5 3 ,xi 3 .t no nnr m i $,oi J:.? K S, 0 5 7 , 7 7 3-92 H 4 '0 3" 2 o5 HO ?%Q 300 15) \Si m US HI -Sr9 + ? - 97 s .7 S'- 7-7 r ni -- j - 1*7 iO'Si . i n-H2> -M l + M - LO 1 1 , 3 0 7, / H i . u I X - e t . . . +-X - n o H 4 7 1 7.Si 7'i! Footnotes over. OO0 3 1 2 Footnotes over 000313 T itle . Time KrS 0 U? 33 . -O <75 ;,or /> 5 15 lis /.n a 2.33 ;.$ r 7.iT3 3 , otf i/3 3. Y 2 H n<n s WARBURG 02 UPTAKE CALCULATION SHEET \ X{r' t .u 3 1* d * tir*c(al3;H'hy/ 7^ Elapsed Reading Time T.B.1 pv\ ^ J fmml o ISI /o . i s o 2 0 ISO ISO MS (So 5 /$ / IS * io 105 IS I IS I ISI m 15 0 u o ISO HO l$ 5 170 . ISO ... /S0 /3 1*5 /*T } OS /i 1 iso 151 v id 15 i > to 300 u i. ..J -fe l Date / 5 Flask N o .: t> tla s k Contents: Reading Flask 00 J Change^ in mm. T.B. Actual 3 C o rre c tio n C_hange4 co (mmY a 5 Uptake Cumoles') 1-50 mv tH -2 c 0 - V 1.77 6 -u 0 -3 ~ js s ~ i< 1 //V - 33 -3 6 6 -) -J3 _J5 ~5W iii -3 7 -i *34 III - 3*i , 10 7 __ - H 3 -/ -3 - 'V -Y 7 3 *! 2> S ) i /0 1 - + 7 - T c ~ ~ Y..73 150 . -YY ^ H h T * -5L nTf lY i _ - 5 ) - S7 rrS H - - 67 JY L / Yo -S it- . _ + J i . - 7 7 ~S7 J 3 .' -* i 6 *4 4 .5 o J *! J F c -/^ Endogenous Correct 2 2 < Uptake | Uptake C a s t e ) ; ium<te 0 0,17 0 ,5 0 -.0 * >t% , /.3Y 7-11 7 ,5 ^ 2 .tC U 'J $ .7 ? 3.53 3 .*6 M .ir 5, o V - ,2* ~ . o7 1 -.0 7 - ,oS - 03 - . 7? 1 -/vz - .31 S'. 2 ? '3 7J1 l$ C ~'3i -.7 ^ Footnotes over. *>.v u FC " /S-P Tby T it le : r d f7 o 390 i ?0 Reading T .B . 1 ICC 10 m 173 WARBURG 02 UPTAKE CALCULATION SHEET fit Date: 9 - > f- 7S F la sk N o .: / 6 Fla sk C o n te n ts: /00 v *;/e Reading F la s k 2 Change T.B. in am. C o rrectio n A ctu a l C_ hange4 2 Uptake^ Endogenous Correct Upt2ake 1Uptake6 (ran) (mm) (mm) (ym o lesl (u n cle s) O* ^63 -K O -u 7 ,7 4 7,9 v , . - 0 . -4 7 li* - 7 / 1+ 6 + ?9 -7 3 - / oO 7 , 9-5 9.07T s . H i 63 - '6 3" -7 3 +30 - /03 * .0 *?.? .... . .-- . - Footnotes o ver. *Vr- . ----_ ^ % *.** --- -- - - OOO3 1 5 w* ;S-.` ''. '*-*i.V* *! V.v'"S' 'r* ;v.: '''X*<&U.X WARBURG 02 UPTAKE CALCULATION SffiET T it le . F C A- jl>*r&*1rf*bevta.ct*/;ifCvh*+/ 7 Tine Elapsed Reading HrS Tine *V\l^ T .B . 1 fu i Date: 5*P-i'?S Flask No. : ~ 7 t& Siits: FW lr Reading Flask (M l) Change2 in on. T.B. Correction Actual C_ hange4 M )........ (mm) Endogenous corrected Uptakes Upt2ake 4Upta2ke6 funoles) L .lM asisLjj:uB ai$?j. 0 0 ! $ \ IS) 0 0 i n /o So H i / . 0 * 0,17 - . 0 ^ 133 o ISO ih S C -1 5 0 0,50 - . 0* n HO ISO /vo /! 1 - 1 10 . %5 . . i d * L , - , 3 * .75 HS t*2 /? IH -- / K ) .> 7 t . Z 'H i r . o 7 ' /.Off l.* 5 /5 I-Z5 X 5 IS 70 5 nx o ISI ISI ISI is] 1 j is o ^ ib'O V i! ni ni hv Mr 0 ^ 1 2- r 0 3^ 0 3S -1 " I T ' -1 /.Cl 7 .0 ^ ; o3 7-7-1 - . M W 2.37 7 . S ^ ^ 15 2>r 5-7/ 1 15 > i'% _ _ u ' . . - 7 3 3^ 3 30 3 . ^ - >o T. 23J wo ISO IO - --( MH J.7 3 .5 3 + , J-0 15 5 u 55 -1 S A . . . . H .57 3 .$ 6 . 71 7.ST3 170 S3 r 3 v -i. S-SCi H .ir /VJ9- 3.o? /r5 ISV 7L 9:0. 4* 1 7.37 5, OH 3/3 is o ISO 4 - 7 . S . . 7 . .. - " ' 7 ."S7... S'. 0 $ T.'i 7 o 5 ISI H I 4-9 9 7. . . _ S '.* / S'. 75 7 - n 2 id S m tI O _> /b . 1> 4 10,i l ' 3 4,5 H 'H >ZQ s 300 ?S IM S l , % 7. w L I* U5 +2 ! ir ? 1 'S ,2> 1 W7 Footnotes over. W A t) ` w";. Title: r ^dArtclcbXU Tin ss c 7 . Elapsed TTmim*e 3>30 360 330 H?o 1, Reading T.B.1 ICC 10 117 173 BEST COPY AVAILABLE WARBURG 0, UPTAKE CALCULATION SHEET r> , * ____________________ _________________ _______n-yc ..... ""PlasLc"~"""" .. .` - Date Flask No.: *7 Contents:2- -fby -b $&***' Reading Flask Change2 in an. T.B. Correction^ Actual C_hange4 Endogenous'Corrected Upta2ke5 Upta2ke |Upt5a*ke6 (ran] (ran) (ran) ... (umples] .WP.US.).Amds&l 33 /37 -+-3 >20 18,43 7,95- ~ '/0 ' 5- -3 2J2> f4 >5*7 71,93 'k'H? ...... + ?9 +30 9,07 ?.? , `. `\ Footnotes over. .iViy, % .. ''V-:' '.'/>>:; '!: ;V'i-'.i (00317 v~/ T itle : FC-J &i a Tovicir"vf 7v _ Time Hr S Elapsed )TfViMme Reading T .B .1 WARBURG 02 UPTAKE CALCULATION SHEET 0 /V 7 Date: *-S Flask No.: Reading Flask (mm) Change 2 T.B. in mm. Correction (mm) 9 cSStt: ** T " * Actual C_ hange4 (mm) Endogenous Corrected 02 5 | 0- 2 Uptake | Uptake i Uptake5 (umoles) j moles) I (umoles) . 0 .lO 33 O 75 >.o8T / >5 / 5 / 7J 0 151 ISi 10 /*/< ?o . ' 5 0 /Y3 HO . '5 7 / i V 4 5 " 0 IL5 . 5 iSi n - 75 / 5 l //8r 90 . 151 / )> /05 1 IS I 1 /?2- /SO /ov 0 5 S' /? 75 3> - 1. HS h\ > - -/ 1 -/ It -/ 3 V 0 o 1 3* 0 3^ 0 J4S - 1 Hr i _ S _, \ o I ./7 ,/7. 0 ,5 0 ,o f 1. I s /./* , J 7 -- r r r - /. 3 ^ ,H 3 , */5 2 .o 7 - mb ini i'}i h ,oT ~ 7.<77 2 .5 2 2 /ir ^H `1 / ,s i ,7 V .<w 65 Y 73$ ;.s r 7 .0 3 ;> * 3O 3 Y2 4 -0 s / 0 HO !j5 ; / ?5 ; > YD >?D 300 iso ISO /SO /SJ /5iT ISO 151 15 6 U 3L _ j_ o /O'0 f 7 ^ ....... 1*#t Hi mo i^b 13) 5 1 _____ 55 6/ 65 70 7o 73 7-5 $*: H_ -j -1 +.2. 4- 1 4-7 4? --ft + ll 5M 40 a 7? TT " 9r 1 ,9 ft. Ios ll 0 . H,li H,5L 5 1 5-5 '5o ity 7 ." <5 3.5J^ 3,53 3 .$6 H.7V 5>oH S'. ?5 5 ' 7? 7 , >1 7.5C ~ V 9*f i.oi t.b* i </< TYT 1-SS uv / .V 1. 7 b ' Footnotes over. . -iv-V;r::^v^c;vv ~T .,,. Title. Time K'-* C'S t-/P* T o y 'c < Elapsed Time 33 0 J60_ J1I 2O Reading T.B.1 M 1U H7 17 3 BEST COPY AVAILABLE WARBURG 02 UPTAKE CALCULATION SHEET Pt(<. Si- Date: Flask No.: Reading Flask (an) l%5 W in in Change' in an. cmm^ 4l . /oi jO^ +30 ^ Contents: +\JjL P<-dY Endog enous Correctecf Uptake 2 ; 2 6 Uptake i Uptake (uaoles) XMnp.lgsj_]tpaaLtel. Hi \3^ q.W lo^l 7,95" /. 9 0 S`ft i 3 l 9,0 7 l . o r U^4 J L v l l 7' l l Footnotes over. - ....... % JtiSSGies$i'- wB&fed ^ WARBURG 02 UPTAKE CALCULATION SHEET || : l A -.t# 'S*aftj*i/s '' V 7t ' fri f 7____ >t * * * 1 f i d eh fair- Tine Elapsed Reading J\rS Time T.B. h i n ^. fmml Date 5 - M - 7 S Flask No.: l l Contents: S * 0 pc / r Reading Flask (mm) Change2 in am. T.B. Actual Correction3 4 Change fmm) fnanT Endogenous corrected 7 ^ f. Uptake Uptake 1 Uptake funoles) _ (umoles1) rillBAl mcl o0 / s i /</v O 0 0 00 'M 7 10 _ /5<0 ' - / , 0 i 0.17 -- , 0ir 33 ? 0 o * HO .75 H S /So 150 t *So /^ v /3Y '3 0 -7 - is -IV . -/ -/ ~N - ir , 5 7),ll h5 6 0 .5 0 , /y , /, 3 Y ,0 V .03 . *2.^ ).0% h>$ IS h is . /.? 7 33 5 IS 3? io 5 - y ' A . ... lio n o :_ /S 5 /5/ /' J '5 / /S'/ '50 /5 0 /i'O JS0 i/y l/S 10 I - ^0 . JJL_ 77 '3 / - 3H -5 0 -S 5 -57 --6 5 - 7 2. 0 0 0 0 -I -/ H -31 - /V -s r - iM - 7) _ i,3 3 ti.it 5oi S'SH C 'IS 2 .0 ^ 7 ^1 7 .S ^ 2 . S'O .. ! " .. 3 .** 3,53 3 .$6 t 6 (0 16 7 / /v 1 i Y? /, ^ 7 I.7 V - 2.. 01 7.. 7 .0 J70 S3 7/ -7? -f-l. C hi </.7S" S.oit 3 /3 / r5 /srr l5 V /so 4 /yV - is +7 +- 7 ? ih ) 5.cv 7,rt 7?T7 S 0* 2. .^ v 3 .*/> ;o ^ 15/ / * * +? -^7 i.Y S- 75 f IS i /3) -1 ;0 3 + fb - nc / 0 , 0s ! 3_ 4 -0 > to 300 m j./C L j j J L .- ^ 1/7^ - 1 c -1 ^ ._ + i.< L ... -1 1(.4 ^ 7. * U-- f c ~17-V Vo*t* > Title: r V d * y c c ( < h f c Tine 5:s Elapsed Time K* 330 J 0 3^0 12JL Reading T.B. 1 ,im\... ICC t& / 77 173 WARBURG 0, UPTAKE CALCULATION SHEET f y - Z- Date: 9-*^"'?S'Flask No.: Reading I Change4 T.B. Flask in am. Correction t-3 />`L IJ V ;i - * 3 / Yo 9-7 ^6 +w if Contents: P^~(^ ^ Endogenous'Corrected i5 Uptake Upt2ake Upta2ke6 (ymol.es) ..lilBP.U.0. Xmaltsi -I55 7,9V S-> `ft lb b <S`H * J 1 0 - Ilb 9 ,0 7_ I 7 iZ i, >5*" +30 -lit. Y X L ~Xja BBSSEd c-. ir~ Footnotes over. 000.121 -rvf * * tfV y. .. 0J-. H, !y\ * Kt'.'M - U Wl. HARBURG 0, URTARE CALCULATION SHEET O ,o*i #Jr. fbv ic/t-- --------- 7 " Tine Elapsed Reading Kr S KTVi\m1e T.B. 1 Date: Flask Mo.: TsF Reading Flask (om) Change2 in o b . T.B. Actual Correction C_hange4 () (mm) Endogenous Correcte< Upt2akes Upt2ake i Upta2ke6 (umoles) ^-i9lssJLUuial?J 0 <? 151 f $ 0 0 ,167 /O ISO IH * - z --i 0 0 ,O ,oB 0. 7 - .07 33 2 0 ISO iH L -H -/ -3 . 7 5 0 , 5 0 - X5 7 HO 5 0 t H ' l - / - 7 .S9r -- 1C0 .75 1 4 5 50 u t - n - / - I l >lt /, 3 4 -%"'l/V' ;.o8T 1 5 J.*5 75 1SI / M - ! C /S I 31 - 1 9 -0 -/6 ),7>H 2.o~^ .4* 0 -19 1.5? 7 'l - ,4^ -3-5l < 5 9p.. 151 ns 0 .-2S ?,o7 2.52- - * 3 . I7J ;o5 ! IS'l n s ~ -~3? " -3 J. 84 2 . f r i - -0^ /.?? m - J 5 . ? in * > } 0 150 m -H i -1 - 3 r 3.17 *" f .C?6 - h 1 3-H 2.33 1 HO IS O Ibi -HI -/ - 5 3,%H i , 53 St 7%r 15 5 IS O <94 -5H - 1 -53 3 .$6 7 .0 ji SP: 3. otf 3 /3 3.92 H tf>0 w S3 I l 5 X /W iso ; >5 151 IS i >rd U3* 300 L l _ l l - 5 9 -F'i %x m. I11 - 73 4- 7 +* 1 1 * ....+ /b \U? js/ L. -H9 _ ri2 i,, .. r i t. -7 5 -75 h -S / - fo \ - />0 ~ rii L s-o H .r : J / ,'> s , O H ../,.. ? 2 S,7'Cf i.ii .i S 05 /. S 7 1 ...Ui 7 __1.5_ 2a3 * /0,.V 7 , i l j , k r SI?* J 2.Si .1-3.7 I . '"T-'-fT: '' V Footnotes over. Yc.* " :iy'^ K-f -I .;*'-Zv^_`i 'V".jii'jv$^.(L&'&$yt-iiSfc SB y-'-rliit- : ;; H t l i : K 'Z u T M h .te Tine 5: s 4 C .S 7 Elapsed Time N * 330 -.3 6 0 33 0 . V ? *5 Reading T .B . 1 ICC 10 m 173 . - .( * WARBURG 02 UPTAKE CALCULATION SHEET Date ' Reading ' Flask . (mml _ <?? 4/ 3 7/ Flask N o.: 7 Change^ in nn. (am) . T.B. 3 C o r r e c tio n n~ C o n t e n t s : ^ *-3 A ^ ^ A ctu a l 4 Change (mm) 2 5 Uptake (unoles) Endogenous C o rre cts Upt2ake 4 Upta2ke 6 Im p .lA .si ilM B a ls s i -131 - I 3I -HI + -3 j-Zlo + ?9 - 145 /2 ,**. /3- 77" /*/.<9 ' 7 ,9 9 .0 7 v . f S >23. ^6 2 - ' 3 4 ' +3>0 -iu /$ .5 ^ &.0 3 / : ':r ' ' .v V' .' V' Footnotes over. ""I: -> " *- . i.* ** V S p n v U :' f ' -T KSICOPUMIUME f("V .*-ri. Ti <1 `foY iCi^-y + T lt le ` -____6 i* -vj-- Time Elapsed Reading Lr\rSf Time T.B .1 * 'WARBURG 02 UPTAKE CALCULATION SHEET / V ' *f Date Reading Flask (ram) V -^ i'7 5 Change in mm. (mm) Flask N o.: a c E iiK !; : 5 o o '7 //f c '> '- T.B. Actual Correction3 Change4 (ram) . Endogenous Corrected Upt2ake5 Upt2ake Up5ta2ke6 (weoles) r UuB&l&) . 00 , i o . /O >$> \H/ i 5 o ..... IH? o -/ 0 -i 0 0 0 -- 0 0.17 .i --. /7 \Jl <f! 33 2 0 t$o . , Vi? .75 L V 5 /.or L_ 5 !2-5 .25 ! 5 J0 . '5 3 '2 0 /St /SI iSl M i )o5 \ is) /.?? n-z ISO ws 1VO 151 1*4 /%? |U m m -V ,,l, - it "57 -/ -/ 0 0 0 0 -1 -3 -n -to -3 V -36 .2-3 .67 ,71 1. 6? / 77 2-?7 3 . 0.50 "- n s i . / s r , -0 .5 ' /. 3 ^ - ,I/V 7 .o*> - . V / ? .7 - ,5 ?.5 '2 ' 2 . fr'6 - ,is . 0 "oofr 2-35 2-5*1 ,i 2 - 0 j 3 , o.tf 3 t3 ho Jv'O /55 J .*7 / 65 nt 15 0 , IS O ISO IS3 15X ISO id. I oi )% is 10 150 -V / _ - v* -7V " 74 -! -1 f-* +1 -h 1 -HO -S -*C 3^7 5fi ii.il 5.5C 7.^M 7.24 3-2^ 3 .5 3 3 .$6 H.lY _ j ? . <?v S'. ?s "*o .o r . V3 ns . a ? o .i.0 3 . VP- . ....... V 'O ;o ^ -' Vi? 300 151 13 * "5 }oH - n s /ft Cl ' - / s ' L _ u I _ Z ^ J l - - 1% .:+ *+~/ b + l! -bli - (?7 - 1* L i ii. 5. 7? 11.6 7- . . J L L k - I S 17S 7. >i 1 l V . 3 t | 7.sc 3. DS %<>! /ono Footnotes over. 000324 */* FORM1 0 *0 -e-PWO 1 SAMPLE DESCRIPTION F(L <Z> /v\ / File: Page No. ENVIRONMENTAL ENGR. LAB - W O R K S H E E T Date: Analyst Hrs.: (/ ^ ^ 7t ~V. cy - Z * Z /0/n/ 2 3.1 3 kD / s o ..*r / y ____ a 7^ 2 ^ > // o' ^ v ---S-'--O---- 3w c-}- / { 1 if! ? 3 S o 3 q // y^ 2- 0 / ? /1 j .i /Z. ? 3/7, tfj 0 ' ' /zy V s o c - 0/ / ) (d 1 '' ^ 4 .--------- J 000. ______________ Reviewed by: