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BIODEGRADATION TEST SUBSTANCE Identity:[2-(N-Ethylperfluorooctanesulfonameitdhoy)lacrylatem;ay alsobe referredtoas Bl 228, D-1, ETFOSEA, or FX-13.(2-Propenoicacid,2[ethyl[heptadecafluorooctyl)sulfonyl]aminoe]settehry,ClAS # 423-825) Remarks: Materialisan amber solid.Sample puritynot recorded.3M lot number 13. METHOD Method: Not noted. Type: Aerobic GLP: No Year completed: 1984 Test solutionpreparation:Sample weighed onto a coverglass,dissolvedin acetone and washed intoa bottlewithacetone. Bottlecoated withacetone solution.Acetone was then purged from the bottlewithnitrogen. Contact time:20 days Inoculum: Not noted RESULTS Test substance: 5-day BOD: 10-day BOD: 20-day BOD: COD: Not measurable 18,990 mg/kg 23,000mg/kg 240,000 mg/kg Reference substance: The glucose-glutamiaccid standardachieveda BOD of 201 mg/L in5-days,which iswithinthe recommended range forthe Standard Methods BOD testmethod. CONCLUSIONS This testingindicatedthatthe testsubstance isnot capable ofreadily biodegradinginaerobicenvironments. DATA QUALITY ReliabilitKyl:imiscrhankin3g.Thisstudyw,hilewellconductedusingthe methodology acceptableatthe time,lacksanalyticadlata fordeterminationof the sample purity.Testingwas alsodone wellabove the solubilitlyimiotfthe test substance withthe use ofacetone as a solubiliziangent. REFERENCES This study was conducted by the3M Company, EnvironmentalLaboratory,Lab Request number B1228, 4/26/84 OTHER Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St.Paul, Minnesota,55133 Last changed: 5/22/00 eWsiolooWrO"I6*nsammaUtbW-aMrUa&wIr.y 2 Controls, and Standwds Lab Request No. =p'y Analyst S Type & So Nonowded Olluom - Wow '" 12@o,0)23@ q),.,f @ Dot SUr"d ;@772 ow con v uon Owk BOC) Run No. I;r Analyst e- - Day SOD Readinp Battle No. 70 Initial DAL (Al) g. Ye) YO' pmmtda. (A2) D.O. DoOotion (Al.A2) 6.30 Awrap 0.0. Depletion (Al X2) C) SeedeDddutlo-iWtow Mmk (opdond) 0.'20 oo"m fie. b*W DA. ICI) Fo@l 0.0. (C@) 0.0.Dqftdon (Cl.C2) AvenW 04. I -F-2) SendCon!!@ 8.00 YO 0.30 0, Y2 now* No. Sur4lbOu :20 yo % lnkw DXL lei) Y, :t p @ tD.O. 182) DA).Dopktion (Bl-B2) ?0 Glucose - Olutwnic Aidd sotft NO. ssn*b Stu TO 8,3 62 0 Wu DAL (Dil Phowt 0.0. (02) YO' . (0.,00. ve . q.60 9--go 1 3.30 i-a Aw 1./. -.e TOlW @@Z DA 401 -D2) a-lo DwMdan Due Togandwd SiFo-* c- oev Day C- SOD I-lq 1?7 3 3,39 .--2o'3 BattleNo. smwb $in T3 9 Farm 19756 FM laidwD.& (Oil Yo 9- vo 1-Y how tD.C. (021 a./o 9 -/0 Er-I"r ToldDA. D*Wan IDI 021 0.30 0.30 o- Ze Otmdmd par" -AvwsW Day BOB-- Laboremy Shwt.Blanks, wW Sandar* RLoaqbbARueeqsute'sNtNo$L.otl Analyst 14-Ola4l@- S"d Type & Boww 123o,,2O3)7 81.212 600 RunNo. Dem A"V$t 3M Day800 Readings Date Nons*WW DOwdon-waisrBlm*limaim IAI) YC) 0 --?.yo C> SO*" Dkdm.. waow No* %P"W) @sub pa@.@ bdu DAL ICI) Sewcontrds I'sM& dy 6NP4d@ Nu 20 !io how- D.O. IA2) ?o FO (C-2) r c) MNW DA). 0 (Al -A2) Avempoo.,* (Xi -X2) O.:Fo DA).a""" fr-I'C2) 0.(70 0 -(eo A"relp 046 -5?L:r o pe@ D.O. 462) 7--oo.ol :z0 /.yo -@.0 0 --Glucm - OluUmle Add Botdo thi6 I%mt D.CL 93 (a @l-- 1 I. POMWum Hy*.oM M*Wft $Unftd WpdwW) Bonk N& kd" DAL IDII h"Mt 0.0. W2) Told D.CL DIPI@don 101-%) D"kdom Do* TO UWWwd 53 'raW DAL D"Man owletwo D" To gtwwwd /o Awnp to Day ow Aw@w @-Ow Form19756;z- ~IW Labwatory ,Ark ShM -Monks, lo,iowlWs, Stmdw& 3M Lab RequestNo. Analyst :er@ S"d TYPO A BOURM 1230.0123 18,1@2 DmBtwUd corammation BOD Run No. yet -Day BOD Readinp Date 1/..2 Nonne" DOuti-mWotwvia* I*h bhw D416 (All YC) 0 S,,-Vo C) 6- SSWW DNUIIMWow sidwra Pnowi0t.0. L%l 21.0 t- DA)D."bdon (Al-A2) 0.k:r 0 o - C/o AvwopD.O.'' (Ai Aal 0. kdd0d.06 runnDtAL (C2) 0.00."6"4 (Cl-C2) Avemp04. *don SndConUds gotdo $""men 20 Yo so hdd0e4l. 01) t.Yo. o -yl' o.t!r PMUM 0.0. (121 ---6,.1.0 0.93 D.OD.spbdon -tsi.32) .2-3C) 0 Gluam -OluomkAM SardW Baum Me. smwb WAM bdw D& IDI) pman DAL 021 vd, q.t. o -.g,'IiD ?0 PotnWum ilydia" Phomift Slmmkrd WpdonMI 1wod DAL Plawt D.O. Tod DA. Dopildm (1)1'02) .3.-To t t) -- D""M ow To INWAwd 1 2 29 D" C BOD 1 113-2- '2.'37-;r Aomop. O-WO 237 2 Y49 Teed 04. ompwdan (01 D2) D""don Due To lbwmiord Day A,, qp cwv goo Fortn 19755 PWO sonmental Labofatory ,JDWork Sheet-Somplos Lab Roq. No. Analyst atestarted BOD Run No.* Analyst y -Day SOD Readings 3M' Date Sample couription InitiaDlilution InitiDaillutioFneeW 3 1-.25-/ 9) f of Sample dilutetdo 13 mi withD Iwater fdilutiofnactoor I ifundiluted) 60"10 CGIW. of NO. IMPISI ommas Sin2 bdim 0.0-1011 h@t 0-0- ID2) TOIM 0.0. Dq"don D*Won Due TO somm Doy moo Day (01-%) 0. 00-fi@ o o m. qf 0.03 17 1!@) q ai, o. )O@IV, YC-7 )-13 o.qy Y/ 31'0 0 1-10- r -If 0-39 23 votr) 1 Y-2 I' Note: 1. Conc. Concentratioonfsample(mlA OsOzi.AA BOD bottles. 2. Sample Size - mi fsample or dilutedsample added to 800 Bottle. 0& Comm*nts e ol z tW Laboratory ,/Oorrkk S%hIoot-Samples ^LLraob bRR:eqo.qN:o:.@ Dow Surted SOD Run No. /0 y y -Day SOID Readings 3M SamOowp&@ltieo" lnitw DOution InitiaOlRmOm Fww act" cora.Of Me. %mofti I I 1 low** $Igo of Sampledilutedto (Dilutifownw 13 ml with D Iwater I ifundiluted) po@.t TOIW 0.0. 0""ti"D" 10 cw I cipktim TO alvoo e@wo oav 401 - D2) I Ic 0.01(t.')Lg YO 5- -50 q3 30) a. ye 6. o ().3 -o 10. 5.30 3 v3 0.ot /'7 -7.41 C-) 0 0, q 3 0 <0. @ro 17 3'70 0 Not$: 1. Cong,a C@mmntrationof umple (miA or mgA) In8'00 bottles. 2. SWPIS Size- ml or0 of wnple or dilutedsompl*added to BOD Bottle. Coinonents 0,0 [as$ t e-/Ilo OL &A,-%5/ t 77 Form 19754-PWO tW Laboratory 401,19humotSon pla .f /ab Req.No. Analyst om Swtsd % Boo Run No. 0 y -IL3@ y Oey1300Rt&dinp lAnalVit Dow 131 laidDdowd"F gold$ COrA680 ofSempldeilutOed 13 miwit0h1water (IDiluftaicmw Iifundiluted) DAL(DII D.O(.Da) TowDAL DN"tl" (DI-D2) .10 1.3 2 X. YC.7 ly. Yo 50. 1 0 ou.0 Dev Tobm* Q- O.y 2 3 :1 (3'1000) '3 0 0 -2300d > ?.I-f -76. 0 . Note: COM 4!C.@r@tratioonfswnple(rniolrlci%gai)n800 bottl*L 2. mi OF9 Ofsunp'eordl'UW swnp'*addsdtoBoo Bottw. 0,0 rep 2 Moto low o 4e e&"LitL#74t Foo,1m975P6WO BIODEGRADATION (ModifiedMITI Test) TEST SUBSTANCE Identity:[2-(N-Ethylperfluorooctanesulfonamiedtoh)ylacrylate;may also be referredtoas Bl 228, D-1, ETFOSEA, or FX-13.(2-Propenoicacid,2[ethy[lheptadecafluorooctyl)sulfonyl]amino]eesttheyrl,CAS # 423-825) Remarks: Materiiaslan ambersolidR.eportlistssampleas> 99%,as provided by sponsor.The pudty/identiotfythetestsubstance cannot be substantiated.As presentedinthe report,thestructuraflormula indicatesthat"purityc"annot be assigned as "99% or more". Lot number 4. METHOD Method: OECD 301C Type: Aerobic GLP: Yes Year completed: 1995 lnoculum source: Chemical BiotestinCgenter,Chemical Inspectioannd TestingInstitutJea,pan Remarks: Itappears thatthe inoculumwas obtainedfrom onlyone source, ratherthan theten sources requiredinthe method. RESULTS Test substance: After28-days,the percentbiodegradabilitoyfthe test substance was determinedby threemethods: BiochemicalOxygen Demand (BOD), DissolvedOrganicCarbon (DOC), and ResidualTest Substance Concentration.The resultsareas follows: Percent Biodegradability(of3 replicates): BOD: 4,-1,and 6% DOC: Not calculatedt;estsubstance not dissolvedinsolution Test Substance Concentration:5 and 1% (onlytwo determined) Reference substance: The anilineachieved68% biodegradabilitiyn28-days. Measurement of testsubstance: The testsubstance concentrationwas only measured atthe end ofthestudy. Although thislaboratorfyollowedthe procedures as outlinedinOECD 301 C, the startincgoncentrationshould also be determined ratherthan relyingon nominalconcentrations.Inaddition, subsamples were removed from the bioreactorfsorpH and DOC determinations atthe end ofthe study. The sample used forpH determinationsand the remainingsample aftercentrifugatifoonrDOC were returnedtothe bioreactor priortosamplingforthe measurement ofthetestsubstance concentration. These samples should have been removed afterthe bioreactorswere sampled fortestsubstance concentrationdetermination. Calculationsoftheoreticaolxygen demand should have made which account for possibleoxygen consumption due to nitrification. CONCLUSIONS This testingindicatedthatthe testsubstance isnot capable ofreadily biodegradinginaerobicenvironments. DATA QUALITY ReliabilityK:limischranking3.There isno referenceto thesolubilitiynformation providedinthe study. The quoted purityfidentoifttyhe testsubstance isnot substantiated.Measurement oftestsubstance concentrationwas only performed atthe end ofthe study.The bioreactorwsere potentially contaminated by the practiceofreturningsubsamples afterpH and DOC determinations.The calculatioonftheoreticaolxygen demand should have been perfon-nedusingthe correctionfornitdfication. REFERENCES Study conducted atthe requestof Sumitomo 3M LTD by MitsubishiChemical Safety InstitutLetd.,Yokohama, Japan OTHER Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St.Paul, Minnesota, 55133 Last changed: 5/22/00 M.S.I. REPORT No.lB363(2)G Ready Biodegradability Test of D-1 Submitted to SUMITOMO 3M LIMITED Prepared by Mitsubishi Chemical Safety Institute Ltd. October 31, 1995 Ready Biodegradability Test of D-1 (English version) Study No. Study Title Sponsor : lB363(2)G : Ready Biodegradability Test of D-1 : SUMITOMO 3M LIMITED This report was conducted in the Yokohama Laboratory of the Mitsubishi Chemical Safety Institute Ltd., 1600 Kamoshida-cho, Aoba-ku, Yokohama 227, Japan. This report is an English version of the orginal, which was written in Japanese. The underesigned hereby declare that this version faithfully reflects the original report to the best of our knowledge. Mitsubishi Chemical Safety Institute.Ltd. Date Kikuo Yoshida, Ph.D. Environmental Science Division of Yokohama Laboratory Approved by . Jun Toriya, B si@@ Head of Yokohama Laboratory Date COMPLIANCE WITH THE GLP STANDARDS Study No. Study Title Sponsor : IB363(2)G : Ready Biodegradability Test of D-1 : SUMITOMO 3M LIMITED To the best of our knowledge and belief, the study described in this report was conducted in compliance with the Good Laboratory Practice (G.LP)standards applied to industrial chemicals under the Chemical Substances Control Law. Facility management Sakae Kpike, B.Sc. Facility management Head of Yokohama Laboratory Sealed date :April 28, 1992 Study director Kikuo Yoshida, Ph.D. Study Director Environmental Science Division Sealed date :April 28, 1992 QUALITY ASSURANCE STATEBENT Study No. Study Title Sponsor : lB363(2)G : Ready Biodegradability Test of D-1 : SUMITOMO 3M LIMITED We hereby. certify that the above study has been performed in accordance with the Good Laboratory Practice (GLP) standards applied to industrial chemicals under the Chemical Substances Control Law. Dates of inspections and reportings to the Study Director and the Facility Management are as follows,: In-progress study Final report Date of Inspection March 18, 1992 April 15, 1992 April 16, 1992 April 28, 1992 Date of Reporting_ March 30, 1992 April 15, 1992 April 16, 1992 April 28, 1992 Quality Assurance Staff Junko Katoh, Ph.D. Yumiko Miura, B.Sc. Sealed date :April 28, 1992 Sealed date :April 28, 1992 We, the undersigned declare that the work was performed according to the procedures herein described, and that this report provides a true and correct record of the results obtained. Experiment Staff Ayumi Okuda, M.Sc. Sealed date :April 28, 1992 Environmental Science Division Junko Koyasu, B.Sc. Environmental Science Division Sealed date :April 28, 1992 Yukiko Mizoguchi Sealed date :April 28, 1992 Environmental Science Division Staff for cultivation of activated sludge Yasuo Sato Sealed date :April 28, 1992 Environmental Science Division CONTENTS Page EXPLANATORY REMARKS ...............................................- .7 SUMMARY ...........................................................8... 1 STUDY TITLE .......................................................9... 2 PURPOSE ..........................................................9... 3 TEST GUIDELINES ...................................................9... 4 GLP COMPLIANCE .............................................. 9 5 STUDY PERIOD .............................................. 9 9 6 SPONSOR ..............................................*-.,. 9 7 TESTING FACILITY ......................................... 8 STORAGE AND RETENTION OF TEST SUBSTANCE AND RECORDS .................10 9 TEST SUBSTANCE ....................................................11.. 10 CONFIRMATION OF IDENTITY AND STABILITY .............................1.2 10.1 . IDENTITY ...........................................*.**.**** 12 10.2 STABILITY ...........................................*.-.-*** 12 11 TEST 11.1 11.2 11.3 11.4 11.5 11.6 11.7 13 METHODS .......................................... ACTIVATED SLUDGE .................................... 13 EXPOSURE CONDITIONS .....................................I.-.13 14 BOD MEASUREMENT .................................... 14 pH MEASUREMENT .................................... DOC MEASUREMENT ................................... 14 MEASUREMENT OF RESIDUAL TEST SUBSTANCE CONCENTRATION ........15 EQUATIONS FOR CALCULATION OF DEGRADABILITY ..................1.6 17 12 RESULTS ............................................... 12.1 12.2 12.3 12.4 12.5 OBSERV4TION pH MEASUREMENT ACTIVITY OF DEGRADABILITY DEGRADABILITY OF TEST BOTTLES AFTER EXPOSURE PERIOD ...........17 ................................*.*****'****** 17 SLUDGE ................................*.***,*,'' 17 BASED BASED ON BOD .................................1.7 ON DOC .................................17. 12.6 DEGRADABILITY TEST SUBSTANCE BASED ON THE RESIDUAL ...... 18 CONCENTRATION ........................... 13 CONCLUSIONS TABLES AND 18 ...................................*.-.,**,******'*''*''* FIGURES 19-33 ................................................ EXPLANATORY REMARKS Study No. Study Title lB363(2)G Ready Biodegradability Test of D-1 The ready biodegradability test of D-1 was originally started on January 29, 1992, and the BOD measurement of the test'substance was initiated on February 13, 1992 (Study No. IB363G) . However, the degradability value of aniline based on the BOD measurements did not exceed 40%. Therefore, the test was terminated on February 20, 1992 and the new test (Study No. lB363(2)G) was started with the same test substance and the same test method after approval of the sponsor. Some of data used in this report (IR chart, calibration curve and recovery) are those obtained in the former study (Study No. lB363G). SU14MARY STUDY TITLE Ready Biodegradability Test of D-1 METHOD The method described in the Order, 3ahich prescribes the procedure of testing new chemical substances as required by the Chemical Substances Control Law(Japanese Law 117, 1973). ("Ready biodegradability, Modified MITI test" in OECD Guidelines for Testing of Chemicals). Test bottle Measurements Bottle 1 Bot tle 2 Bott* les 3-5 Bottle 6 : aniline + sludge + basal medium : sludge + basal medium : test substance + sludge + basal medium : test substance 4 water Biochemical oxygen demand (BOD) Dissolved organic carbon (DOC) Residual test substance concentration (for 28 days) (at day 28) (at day 28) RESULTS Measured values (day 28) BOD, DOC, Test value mg mg/l substance, mg/l corrected with BOD Bottle No. 3 4 5 6 1.0* -0.3* 1.6* 0.0 0.7* 0.3* 1.6* 2.0 101.3 105.2 91.5 106.7 or DOc value of Bottle 2. Theoretical value 27.7 28.8 100.0 Degradabilities Bottle No. 3 4 5 BOD DOC Test substance 4 -1 6 NA* NA* NA* 5 1 NA** not calculated because the test substance was not dissolved in the test solution. not calculated because the test substance might be lost accidentally during condensation of the solution under vacuum. CONCLUSION From the degradability results based on the BOD and the residual test substance concentration , it can be concluded that the test substance is not readily biodegradable and is not transformed under the conditions prescribed by the Modified MITI test. 1 STUDY TITLE Ready Biodegradability Test of D-1 2, PURPOSE This study was conducted substance for notification to evaluate the ready biodegradability of the test under the Chemical Substance Control Law of Japan. 3 TEST GUIDELINES The test was conducted according to the method described in the Order, which prescribes the procedure of testing new chemical substances as required by the Chemical Substances Control Law (Japanese Law 117, 1973). This method is also known as the "Ready biodegradability, Modified MITI test" in the OECD Guidelines for the Testing of Chemicals No. 301C. 4 GLP CM4PLIANCE The test was conducted according to the Good Laboratory Practice (GLP) standards applied to industrial chemicals under the Chemical Substances Control Law. 5 STUDY PERIOD March 17, 1992 to April 28, 1992 (BOD measurement : March 18, 1992 to April 15, 1992) 6 SPONSOR SUMITOMO 3M LIMITED 8-8 Minamihashimoto 3-chome, Sagamihara 229, Japan 7 TESTING FACILITY Yokohama Laboratory Mitsubishi Chemical Safety Institute Ltd. (M.S.I.) 1000 Kamoshida-cho, Aoba-ku, Yokohama 227, Japan Head Office: Mitsubishi Chemical Safety Institute Ltd. (M.S.I.) 1-30 Shiba 2-chome, Minato-ku, Tokyo 105, Japan 8 STORAGE AND RETENTION OF TEST SUBSTANCE AND RECORDS The following records, the raw data, and a small sample of the test substance will be stored in the archives of M.S.I. for 10 years after the submission of the final report. After this period, the sponsor will be contacted for the approval of the disposal of data. Data can be stored in the archives for a further specified period at the sponsor's request with an additional fee. 1) Protocol. 2) Final report. 3) Raw data. 4) Quality assurance reports. 5) Test substance (approx. 2 g). 6) other documents required under the GLP standards. 9 TEST 1)Name* SUBSTANCE 2)Chemical name* :D-1 (M.S.I. identification No. lB363(2)G) :2-(N-Ethyl-N-perfuloroalkyl(C=1-8)sulfonylaminolethylacrylate 3)Structural formula* C2H5 0 -1 11 Cn F2n+l@02-N-CH2CH20-C-CH=CH2 4)Elemental composition (n.-@::1-8,main component; n=B) :C;28.8%, H; 1.9%, N; 2.3%, 0; 9.8%, F;52.0% S; 5.2% (Measured by M.S.I.) 5)Physico-chemical Solubility* properties insoluble in water insoluble in DMSO >50% in acetone Melting point* Boiling point* B)Batch* 9)Purity* 10)Appearance ll)Date of receipt 12)Supplied quantity* :27-420C :ca. 150*C/lmmHg :Lot No. 4 :>99% (n=1-7;ca.21 %(total), n=8;ca.78%) :amber solid :September 25, 1991 :100 g provided by the sponsor. 10 CONrlRb&kTION OF IDENTITY AND STABILITY 10.1 IDENTITY For the identification of the test substance, the infrared absorption spectrum of the test substance was measured. The measured spectrum was consistent with that supplied by the sponsor. 1)Apparatus : Perkin-Elmer model 1640 infrared spectrophotometer. [Figure I (P-23)] 10. 2 STABILITY After the exposure period, the infrared absorption spectrum of the test substance that had been stored in a refrigerator during the course of the study was measured. The spectrum was consistent with that shown in Fig. 1, indicating that the test substance was stable under the storage condition. I)Apparatus Perkin-Elmer model 1640 infrared spectrophotometer. [Figure 2 (P-24)] 11 TEST METHODS The test was performed according to the method described in the Order, which prescribes the procedure of testing new chemical substances as required by the Chemical Substances Control Law (Japanese Law 117, 19'73). This method is also known as the "Ready biodegradability, Modified MITI test" in OECD Guidelines for Testing of Chemicals No.301C (1981). The test substance was exposed to the activated sludge in a closed-system oxygen consumption measuring apparatus. The biochemical oxygen demand (BOD) was measured over a 28 day period. After this period, the concentrations of the dissolved organic carbon (DOC) and the residual test substance in the test bottles were measured. The biodegradability of the test substance was evaluated from these results. 11.1 ACTIVATED SLUDGE 1)Mixed liquor suspended solid (MLSS) 3300 mg/l 2)Source Chemical Biotesting Center, Chemical Inspectibn and Testing'Institute, Japan 3)Date of receipt.: January 23, 1992 11.2 EXPOSURE CONDITIONS 1)Temperature 25 1 *C 2)Exposure period 28 days 3)Test volume 4)Concentration 300 ml test substance (bottles 3-6) aniline*' (bottle 1) activated sludge (bottles 1-5) : 100 mg/i : 100 mg/i 30 mg/l Reference substance : Syowa Chemicals, Lot No.SC-2726 5)Test bottle Bottle 1 contents : : Aniline + activated sludge + basal medium 29Rl(30 mg) of aniline was added to the basal mediUM*2, then activated sludge was added. Bottle 2 : Activated sludge + basal medium Activated sludge was added to the basal mediUM*2. Bottles 3-5 : Test substance + activated sludge + basal medium 30 mg of the test substance was added to the basal medium*2, then activated sludge was added. Bottle 6 Test substance + deionized water 30 mg of the test substance was added to 300 ml of deionized water (purified by Milli-Q). *2 The total volume of the basal medium and the sludge was held fixed at 300 ml.- 11.3 BOD MEASUREMENT The BOD was measured for 28 days. 1)Apparatus closed system oxygen consumption measuring apparatus Ohkura Electric Co., Model OM-2001 (M.S.I. ID No.B). 11.4 pH MEASUREMENT After the exposure period, 20 ml-of the test solution in each test bottle was transferred into a 20 ml glass beaker for pH measurement. After pH measurement, the test solution was returned to each bottle for measurement of residual test substance concentration. 1)Apparatus pH meter, Denki Kagaku Keiki, Model COM-10 11.5 DOC MEASUREMENT The concentration of the DOC was measured as follows. 1)Apparatus TOC analyzer, Shimadzu Co., model TOr--5000 2)Conditions Furnace temperature Air flow rate Sensitivity Injection volume : 680*C(TC) : 150 ml/min. :x 5 : 50 ILl 3)Calibration curve The following standard solutions were injected into the TOC analyzer. The calibration curve was prepared by the data processor of the analyzer. standard solutions TC(total carbon) 20 and 50 mg C/1 aqueous solutions of potassium biphthalate. IC(inorganic carbon) 0 mg C/1 deionized water (purified by Milli-Q) and 10 mg C/1 aqueous solution of sodium hydrogen carbonate. 4)Measurement of DOC in the bottles Ten ml of the test solution in each bottle was transferred into a 10 ml centrifuge tube and centrifuged at 3000 rpm for 10 minutes. Five ml of supernatant was used for the measurement of DOC. The remaining supernatant and the precipitate were returned to each bottle for the measurement of the residual test substance concentration. 11.6 MEASUREMENT OF RESIDUAL TEST SUBSTANCE CONCENTRATION The concentration of the residual test substance was measured by the gas chromatography (GC) as follows: 1)Apparatus Gc Integrator :Hewlett-Packard. Model 5890A (No.2) with FID detector :Hewlett-Packard. Model 3392A 2)Conditions Column Temperature Carrier gas Detector flow Attenuation Injection volume :J & W. DB-17(50%Phenyl 50%methyl polysiloxane), 30 m x 0.25 mm i.d., 0.25@= (film thickness) (Column oven) 100*C (lmin.) -10OC/min. -200*C (lmin.) (Injector]250*C, (Detector]280*C :He 30 ml/min. :H2 40ml/min., air 400 ml/min. :[GCI 1 V :1 Kl 3)Calibration curve The standard solutions of the test substance with 0, 250, 500, and 1000 mg/l in ethyl acetate were analyzed. The total peak area of test substance was calculated by adding the area of the peaks with retention time of about 5.6, 6.2 and 6.5 min. The total area was plotted against the concentration. The correlation coefficient was calculated to be 1.00 by the method described in Japanese Industrial Standards Z9041-1966. (Figure 3 (P-25) and Figure 4 (P-26)] 4)Recovery test Duplicate test bottles (identical to the test bottles 3-5 described in 11.2) were kept in a closed-system oxygen consumption measuring apparatus at 251*C for 30 minutes. Each test solution was treated by the procedures described in 11.6.6. The concentration of the test substance was measured by GC. The average recovery of the test substance was 97%. (Table 3 (P-21) and Figure 5 (P-27)] The concentration of the residual test substance measured for bottles 3 -6 were corrected with the average recovery value. 5)Detection limit The detection limit of the test substance was calculated to be 3 mg/l based on the minimum detectable peak area of 100000 @LV- sec for the largest peak (R.T. 6.2 min.) in the GC chromatogram. (Table 3 (P-21) and Figure 5 (P-27)] 6)Measurement of test substance in test bottles The contents in each bottle was separately transferred into a 500 ml glass separatory funnel, which was then extracted twice with 100 ml of ethyl acetate. The extract was filtered through a grass funnel containing 50 g sodium sulfate, anhydrous to remove water. Then filtrate was condensed to ca. 20 ml under vacuum (<500C). The solution was brought up to 50 ml with ethyl acetate. The ethyl acetate solution was then analyzed by GC. The concentration of the residual test substance was determined as described in 11.6.2. 11.7 EQUATIONS FOR CALCULATION OF DEGRADABILITY Equations for calculation of degradability based on BOD, DOC, and residual test substance concentration are as follows: I)Degradability based on BOD Degradability (%) = (BODS- BODb)/ThOD X 100 where BODs BODB THOD Oxygen consumption Oxygen consumption Theoretical oxygen test substance. (mg) in bottles 1, 3, 4, and 5. (mg) in bottle 2. demand (mg) of aniline and the THOD aniline : CGH7N THOD + 35/402 - 6CO2 + 7/2H20 : 90.2 Mg 02/30 mg-aniline + N02 test substance : C+02-CO2, H+1/402-1/2H20, N+02-NO2, S+02-S02, F-F THOD : 27.7 Mg 02/30 mg-test substance 2)Degradability based on DOC Degradability (%) = (1 - (DOCs-DOCb)/DOCc)XlOO where DOCs DOCB DoCc DOC (mg/1) in bottles 3, 4, and 5. DOC (mg/1) in bottle 2. DOC (mg/1) in bottle 6. 3)Degradability based on residual test substance concentration Degradability (%) = (1 - Cs/Cc) X 100 where Cs Cc : concentration (mg/1) in bottles 3, 4, and S. : concentration (mg/1) in bottle 6. 12 RESULTS There was no specific circumstances bility of the test results. which might have affected the relia- 12.1 OBSERVATION OF TEST BOTTLES AFTER EXPOSURE PERIOD The solutions in the bottles were colorless except the bottle 1, in which the solution was cloudy. Growth of the sludge was observed in bottle 1 in contrast with the bottle 2. No growth was observed in the bottles 3, 4 and S. 12.2 pH MEASUREMENT After 28 days of exposure, the pH was determined to be 7.4, 7.4, 7.5 and 7.4 for bottles 3, 4, 5 and 6, respectively. [Table 1 (P-19)) 12.3 ACTIVITY OF SLUDGE The degradability value of aniline based on the BOD measurements was 59% after 7 days. The activity of the sludge was thus shown to be satisfactory. (Table 1 (P-19) and Figure 6 (P-26-31)] 12.4 DEGRADABILITY BASED ON BOD BOD*L in bottles 3, 4, and 5 (as corrected with the value in bottle 2) were 1.0, -0.3 and 1.6 mg, respectively. BOD in bottle 6 was 0.0 mg. (*' Maximum theoretical value = 27.7 mg) - The degree of degradability based on the BOD measurements were 4, -1 and 6% for bottles 3, 4, and 5, respectively. [Table 1 (P-19) and Figure 6 (P-28-31)] 12.5 DEGRADABILITY DOC*2 in bottles BASED ON DOC 3, 4 and 5 (as corrected with the value in bottle 2) were 0.7, 0.3, and 1.6 mg/l, respectively. DOC in bottle 6 was 2.0 mg/l. (*2 Maximum theoretical value = 59.8 mg/1) Degradability based on the DOC measurements was not calculated because the test substance was not dissolved in the test solution. (Table 1 (P-19) and Table 2 (P-20)] 12.6 DEGRADABILITY BASED ON THE RESIDUAL TEST SUBSTANCE CONCENTRATION The test substance*3 was detected at concentrations of 101.3, 105.2, 91.5 and 106.7 mg/l in bottles 3, 4, 5 and 6, respectively. (*2 Initial concentration = 100 mg/1) The degree of degradability based on the residual test substance concentration were 5 and 1 % for the bottle 3 and 4, respectively. [Table 1 (P-19), Table 4 (P-22), and Figure 7 (P-32-33)] During condensation of the filtrate of the bottle 5 under vacuum, a part of the filtrate spouted from an evaporating flask. The spouted filtrate was recovered and then condensed with the remaining filtrate. However, the residual test substance concentration in the bottle 5 was obviously lower than those in the bottle 3 and 4 and it would impair reliability of biodegradability of the test substance in the bottle 5. Therefore, the degree of degradability based on the residual test substance concentration was not calculated for the bottle 5. 13 CONCLUSIONS From the degradability results based on the BOD and the residual test substance concentration, it can be concluded that the test substance is not readily biodegradable and is not transformed* under the conditions prescribed by the Modified MITI test. ible 1 Summaryof the test results Degradabilitieswere calculated by the equations shown in Sec. 11.7. De adability based on BOD ittle Test THOD Na substance mg 1. aniline 90.2 2 --- 3. D-1 27.7 4 D-1 27.7 5, D-1 27.7 6 D-1 27.7 Where % degradability was da 7 da 14 BOD % Degra- BOD % Degra- mg dability mg dability 56.2 3.1 3.0 2.4 3.5 59 --- 0 -3 1 63.7 5.0 5.0. 4.11 5.71 T5 --- 0 -31 111 0.0 O-Of calculated to be negative, this value is day 21 BOD % Degra- mg dability 66.3 67 5.5 - - - 6.1 2 4.81 -3 6.71 4, shown in brackets. da BOD mg 67.4 5.8 6.8 5.5 7.4 0.0 28 % Degradability 68 --- 4 -1 6 --- I Oegradability base )tileOrganic carbon No. added, mg/l 3 28.8 4 28.8_ 5 28.8 6 28.8 on DOC TC mg/l 1.7 2.5 2.11 3.31 2.0 ic mg/l 0.0 0.1 0.1 0.0 0.0 DOC mg/l 1.7 -2.4 2.0 3.3 2.0 d) Degradabilitybased on residuhl t Bottle Concentration Degra No. mg/i 2 <3 3 101.3 4 105.2 g -- 91.5 6 106.7 not calculated because the test substance mi accidentallyduririgcondensationof the solu Table 2 Resultsof DOC measurements Standgrdsolution TC 50.0 mc/L vlal :81 ------------- A arer ------------- 1 346737 2 34 3 346490' ------------- MN 34690 TC 20.0 mg/L vial :a2 ------------- # arer ------------- 1 14107 2 139941 3 140 9 ------------- MN 14045 IC .10.0 mg/L vial :63 ------------- # arer ------------- 1 7185 -2 7107 3 7187 ------------- MN 7159 IC 0.0 mg/L vial :&4 ------------- # aror ------------- 1 0 2 0 3 0 ------------ MN 0 Bottle2, TC ---------------------------- # arer mg/L RKK ---------------------------- 1 1178 1.71 2 1157 1.68 3 1135 1.65 --- ------------------------ MN 1156 1.68 Bottle3 TC ------------------------------#---a-r-e-r-----m-e/-L-------RM-K---- 1 1743 2.53 2 1.743 2.53 3 1713 2.49 --------------------------- b(N 1733 2.52 Bottle4. TC ---------------------------- # arer mc/L RMK ---------------------------- 1 1470 2.14 2 1418 2.06 3 1374 2.00 ---------------------------- MN 1420 2.06 lc ---------------------------- # aror mg/L RMK ---------------------------- 1 0 0.00 2 0 0.00 3 0 0.00 ---------------------------- btN 0 0.00 lc ------------------------------0---a-r-o-r-.----m-s/-L-------RM-K---- 1 0 0.00 2 0 0.00 3 166 0.23 --------------------------- MN 56 0.09 ic ---------------------------- # aror me/L RMX ---------------------------- 1 157 0.22 2 0 0.00 3 0 0.00 ---------------------------- MN 52 0.07 .Boftl5e TC ---------------------------- # arer mg/L RMK ---------------------------- 1 2340 3.40 2 2302 3.35 3 2215 3.22 ---------------------------- MN 2285 3.32 Bottle6 TC ---------------------------- # arer mg/L RMK ---------------------------- 1 1442 2.10 2 1326 1.93 3 1354 1.97 ---------------------------- MN 1374 2.00 ic ---------------------------- # arer mg/L RMK ---------------------------- 1 0 0.00 2 0 0.00 3 0 0.00 ---------------------------- MN 0 0.00 ic ---------------------------- # arer mg/L RMK ---------------------------- 1 0 0.00 2 0 0.00 3 0 0.00 ---------------------------- MN 0 0.00 ---------------------------------------------------- SPL TC.me/L RMK IC.sx/L RMK TOC.ne/L Bottle2 2 1.68 0.00 1.68 Bottle3 3 2.62 4 2.06 0.08 0.07 2.44 1.99 <------ < Bottle4 5 3.32 6 2.00 0.00 0.00 3.32 2.00 ( < ---- @@-@Boftle 5 ---------------------------------------------------- Bottle6 Table 3 Calculationof recoveryand detectionlimit Recovery test 1 Recovery test 2 ,Blank Peak area AV-sec (A) 4805210 4815940 <132800 Concentration in solution, mg/l for GC in bottle analysis (B) (C) 578.8 96.5 580.1, 96.7 <16.01 <2. 7 Recovered Added mass mass mg mg (D) (E) 29.0 30.0 29.0 ---- 30.0 0.0 Average recovery: Recovery Detection limit % (F) mg/l (G) 97 - - - - 97 - - - - ---- 3 97 % Standard solution Concentration Peak area Volume of solution for GC analysis Volume of test solutionfor GC analysis Equation :B=HXA-* I C=BXJ-* K D=CXK (H) (1) (1): (K): F=D 500 mg/l 4150910 gv-sec 0.050 1 0.300 1 EX100 G=C raise decimal fractions to unit. Calculationof detection limit Retention time, min -5.6 6.2 65 500 mg/l std. Peak area Rate of pea@ tLV-sec area, % 298720 7.2 3127000 75.3 725190 17.5 4150910 100. Blank Peak area a V- see --- <100000, -- <132800 The minimum detectable peak area of the'test substance was calculatedby the following equation based on the minimum detectable peak area of 100000 uV-see for the largest peak (retentiontime 6.2 min.) in GC chromatogram. Minimum detectable peak area (,aV -sec) 100000 (.uV-sec) 4150910 X - - - - - - - - - (jLV-sec) 3127000 (g V-sec) < 132745 132800 (,uV-see) (,uV-sec) Table 4 Residualtest substanceconcentrationin test bottles Bottle No. 2 3 4 5 6 Concentration in Peak area liv-see solution, mg/l for GC in bottle analysis (A)__ _ <132800 5149880 -5349150 654900 54281201 (B) <15. 2 589.3 612.1 532.7 621.2 (C) <3 101.3 105.2 91.5 106.7 Standard solution Concentration (D) Peak area (E) Volume of solution for GC analysis Volume of test solution for GC analysis Average recovery (F): (G): (H): Equation :B=AXD -E C--AXD EXF G--!--HX100 500 mg/l 4369340 4V-see 0.050 1 0.300 1 97 % Figure 1 Infraredabsorptionspectraof the testsubstance Measure by the-P.Pns-or 0.71 .-.963 $5.239 37,-X.00 79.M 7-@ 3A4.- nu. 0Do0 EM7. 00 0 "-g.co.0000 7A 512 SLZDD U. 997 74A 2 .-p 4 --. 17=. 0: - 1139. At.37: 4Z.7'-' 34.203 27.021 IAIM.:: !A&g.S5a17 sA4s:di r.n7.-0 !-.".00 in 1207.:: s4.oo i 260 .7.7 9:495 U.113 n.u7-s7 EM.00 54. Do 24.710 31-M 21C.00 ts sic.::: 757 a* 7-C7.0: 57.350 =.so =.Me AE.W? A-- 9!3 739-.D M.A'B5 'S.ZV?ss ll.AF7 709.00 W-.Do 5= 254:.:1,:0 g3-n-..0 32.DCZ =.540 -IV amt: ff7 0 3X.200 3.=5 Acoo me 2300 2000 Igoe 1200 goo DotrZtorl 400 Measure@in M.S.I. X@ 4n(In.0.4no.nii cm-t; o.ni, fit).5%3r 2: 02/ni/13 .11)"ClStrulig 00.31 4 scisnu: saild i*atiu-. I'@llut 4.Go L: 8363U 0-i Lut No.4 I threstiuld 6.0n%*. tianct cis -I % cm-1 ,988.3 24.t7 1619.13 2U.11 itirl;1!. 7.-I-l 808.1 U.87 2364.1 i4sH.8 It)ty.A ld4.1 50.05 18.77 i.sn 22.2ti t729.3 13U2.0 C)IIA..4 '14Y.4 4.34 5.27 7. tf) id.J4 A. Okuda co-I 1639.4 1202.0 nifl.1 853.7 19.44 2.186 36.1 11.30 16 paeka found T --.r--i H STt C-H stretch tc'. T C-C so sttfC,e.hli@cch 5.0 0.07 $Goo 500 .000 3500 2000 2340 mto C--O stretch isoo S--OstretchC-F stretch C-0 stretch Figure 2 Infraredabsorptionspectrum of the testsubstancestored in a refrigerator X: 4000.0. 400.00 Cm-l-. 0. 14. 77.66 xr 4 scans: made ratio: resal 4.00 cm-1; 13363(2)0 0-1 Lot No.4 thr1edliald 5.OGX: bamd cm7-1 co-I 21961 .2 51.57 9.8 54.81 2353.0 1468.8 1062.3 20.58 lotO.0 784.2 46.'59 747.2 x 68.77 40.04 17.95 40.04 cm-1 1731 .1 1392.9 985.1 735.8 16 peaks round aped 92104116 strong A. 3.85 5.36 16.75 43.83 cm-1 1636.8 1205,5 808.1 653.6 16.39 OkUde x 42.99 1.32 25.32 25.40 C-H stretch t stretch 4000 35@o 3000 T 20(1 C--O stretch ISO 1000 co@ S--o stretch CF stretch C,o stretch Figure3 CalibrationCurve ofthe Test Substance CalibrationCurve. Concentration X(mg/1) 0 250 500 1000 Peak Area Y(YV-sec) 0 1.92785E+06 3.89009E+06 8.12341E+06 Y= -7.552xlo4 + 8.139xlo3 X r= 0.999707 X106 10 8 L(U) a) L- 41 CL- 2 oiz 0 500 1000 eoncent-rati-en(mg/1) ir IF 0 c-6.499 to (D CD 0 AREA% R7 5 60 5.88 6.29 6.32 6.48 11.09 AREA TYPE pgt7ea n pp 156388 0 py 2885188 0 VY 188128 0 VY 713218 D Vg 2819600 PY AR/HT A-ago 0.898 9,090 0.964 R@871 1.335 CD AREA% 4 13(; 2.217 48,999 2.660 le@tio 39.9/19 3 Ati IF ct IF -----I 6.24 co 1.63 0 T 11.09 AREA% Ri AREA TYPE AR/IIF 4. tl3 5.06 5.32 .1-61 5.89 100228 0 VV 127970 0 va, 117448 D PP Out.@ PF 318488 0 PV 9.090 0.886 0.676 0,103 0.997 6-50 fi.99 1412506-0 YO 8@876 534090 0 BY 0.26, CD =r < ID 0 CD AREA% 1.154 1.988 1.252 6-IBB 3.396 15-1 5 5.695 51ARI IF l@IFF Nd RUH PEAKS STORED s ARI IF et I IF .46 to -4 11...884969- AREA% R7 6,176.46 lt.09 AREA TYPE 145 pa 0 BP 143 08 D fly 2998400 By AR/HT 0 896 0-078 --29.02JL 1.678 60.865 IF rt 0 ft 6.46 0 < 7613 (D (D co AREA7 Ic@- RI AREA TYPE AR/HT AREA% 5. t35 144480 D VV 9.185 1.606 5@60 362918 0 VP 0 - 100 4,035 5.08 194960 0 PV 0.892 2.167 6.21 3565700 D VY Oz 895 39,641- 6.33 232290 0 VV 0.864 2.598 9-48 826680 0 VB 0.072 9.ZI5. -f.63 618298 VY 9.756 6.874 lt.88 29993tio VY 1.182 33.344 SIARI IF -71 0rt, IF a I0[D C()D i@s6o cc ko 1 IL 0 AREA% RT AREA TYPE AR/HT AREA% 5.96 104890 0 Po 0.084 1.269 5.60 360210 p pp 8 R99 4-3'iq 5.88 195250 0 PV 8.092 3572709 n vy 0 R99 2.363 43-315 6.33 -E.48 232248 0 VY 9.064 8768 n n yn 0-023 2.818 IR 6AR 11.88 2915908 BY 1.258 35.284 SIARI IF IF 11*.09 6.41 AREA% RI 5.06 5 68 11.09 AREA TYPE 105999 .0 vy 296720 D yp 1617 0 py AR/HT 0.896 e.e99 9.091 AREA% 1.664 4.609 -F53-8 3juni-ELit L-JLSM---12-u--L t9tiz/vo . ry 871 1.091 38.648 SIARI IF F 110 RUH PEAKS STOREO Figure6 BOD chart .0 E -4 5-- U) 0 -3 0- CYL--IM 7- MeaSUTement of BOD Test substance: D-2 Study nwaber IB363(2)G Period 3/18/1992 to 4/15/1992 Temperat ure 2.5t I 'C Apparatus Ohkura Electric Co. Model OH-2001(l.D.No.:B) Chart speed : 2mm/hour bottl Subs tance Cone. isludmgge/clonc. No. mg/l 1 Aniline 100 30 2 ------ ---- 30 3 D-1 100 30 4 D-1 loo 30 5 D- I loo 30 256 D-1 Operator 100 Ayumi Okuda -------. ........ ---.. .... 0 -- 0 1 da) --7 (hour) Li :Bottl .. ........... Bottle Figure6 (Continued) Bottle1 !Percentagedegradabilityof anilineat day 7 BOD in bottle1 BOD in bottle2 56.2 mg - 3.1 mit X 100 59% 90.2 mx t THOD ofaniii - --------------------------------- Bottle5 Bottle2 Bottle3 Bottle4 Bo@le 6 'igur6e (Continued) 14 1 I .(336) . I; . I Bottle1 17 .........-... Bottle5 Bottle3 ':Bottl2e Bottle4 Bo@le 6 .'igu6re(Continued) ......... 28 Figure7 GC chromatograms of the testsubstance - Measurement of residualtestsubstance concentration The concentrationsofthe residualtestsubstance in the bottlesare shown inTable 4. 500 mqA in-ethylacetate uVajc-eoM:;- r-n0-Iinn IX . IV:o@ 0Ch0C0%U NON%wo-CmU m CD 0 co co c co c@ W, c@ ui CL Z-.2-. m 0 c:i a CD m C'D M crS,) LLir, (U = = &p w -w CD 4= 0 OD:; = CU cu N cu m r.)- cu w C%i cc C%i @ 04 'i@c@IU li L61 W@%n cr Cc .total peak.areat436934ORv*sec Bottle2 xx 40 I.U) U) LU ix Rottl63 Umi r@ am-WMM C@ ri L,5CD eu m m wt c@ 'cr c@ to' c@ c@ c@ CD' > CL CL >. >I--ca CgTwg:ww W% a tn 12.@ll> cc ON V) -,a- co m m ad 4 vlo CP w . 0 63 01 cu C@ in total peak area:51498801LV*Sec ccC%i%a --r 0 N C%J %a r.)Ch X) I.- co ru c@ c@ c@ > CL.> -@- c- c= = C2 0 = co CwD CD w cc r, w P,*Pcm ON CD co Ch m cr. r, W) cCoj CD C%i C0D, V'j Lfo C@ %D C-ul t6talpeak areat5349150@tv'sec STARI IF IF 3 6.49 6.22 AREA% RI 4.73 5.03 5.62 5.98 6.22 6.34 6.49 AREA TYPE 322608 0 PY 164988 0 VY 33 193;19: s ;yp 347271, E26410 0a VyVy 846999 D VO AR/HT 8.985 0.183 '181 :.99Z .093 10.864 0.072 AREA% 5.818 .2.971 6.g@19 i.ili! 6Z.546 4.U77 15.251 5 ARI IF et F@IIF Im co AREA% RT S.62 5.90 6.22 c qA AREA 361488 zuuuiu 4159208 907448 TYPE 0 PP u ry VV u Yo AR/HT AREA% 0-100 5.850 F.093-3.258 0.163 67.386 8.872 14.bU@ 18 8.!)FB-