Document kDQjLg8E4Dpq8LrBpraMLGML0

Itfff Accepted number Study number S03-4114 14114 M M - *717 05 JUN 20 AH II: 5U FINAL REPORT ' ''i j Biodegradation study of ( cbi ) by microorganisms 63050000034/S 3 CI 00 tCC-==o>> or 'O rn ro -a c x Xe* en co September 18,2003 Kuiume Laboratory Chemicals Evaluation and Resear ch Institute, Japan Company Sanitized omom. ^^ 3 itiiiM ir STATEMENT 14114 Kururne Laboratoiy Chemicals Evaluation and Research Institute, Japan Sponsor DAIKIN INDUSTRIES, LID Title Biodgradation study of ( CBi ) by microoiganisms Study number 14114 I, the undersigned, hereby declare that this report provides a correct English translation of the Final Report (Study No 14114, issued on September 18, 2003, amended on October 10, 2003), Date Study Director D e c e m b e r -4 / ^ Yasuko Matsunobu 3 ooooo* 14114 GLP STATEMENT Kuiume Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor DAKIN INDUSTRIES, LTD Title Biodegradation study of ( cbi ) by microorganisms Study number 14114 This study was performed in compliance with "OECD Piinciples of Good Laboratory Practice" (November 26,1997) This final report reflects the raw data accurately and it has been confirmed that the test data are validity Date Study Director September 18,2003 Signed in original Yasuko Matsunobu 000003 O QOOOg - 14114 GLP STATEMENT Kurume Laboratoiy Chemicals Evaluation and Research Institute, Japan Sponsor DAIKIN INDUSTRIES, LTD. Title Biodegradation study of ( cbi ) by microorganisms Study number 14114 Amendment to the Final Report was performed in compliance with "OECD Principles of Good Laboratory Practice" (November 26,1997) This GLP statement was issued as supplement to the statement issued on September 18, 2003 because of the amendment of the Final Report Date Study Director October 10,2003 Signed in original Yasuko Matsunobu 000004 14114 QUALITY ASSURANCE STATEMENT Kumme Laboratoiy Chemicals Evaluation and Research Institute, Japan Sponsor Title DAIKIN INDUSTRIES, LTD. Biodegradation study of ( CBi ) by microorganisms Study number 14114 The inspections of this study weie carried out and the results were reported to the test facility management and the Study Director by Quality Assurance Unit of Kurume Laboratoiy, Chemicals Evaluation and Research Institute, Japan as follows. Item of inspection Study plan Test Conduct Raw Data and Final Report Date of inspection July 17,2003 August 11,2003 September 10,2003 July 18, 2003 August 1,2003 August 15,2003 August 18,2003 August 21,2003 August 25, 2003 August 26, 2003 September 18,2003 Date of report to Study Director July 18, 2003 August 11,2003 September 10,2003 July 18,2003 August 26,2003 August 26,2003 August 26,2003 August 26,2003 August 26,2003 August 26,2003 September 18,2003 Date of report to test facility management July 18, 2003 August 11,2003 September 10, 2003 July 18, 2003 August 26,2003 August 26, 2003 August 26, 2003 August 26, 2003 August 26,2003 August 26,2003 September 18,2003 It has been assured that the final report describes accurately the test method used, that details in the report are in compliance with the study plan and Standard Operating Procedures and that the final report reflects the raw data accurately Date September 18, 2003 Quality Assurance Unit, Head Signed in original Kyoshiro Hori 000005 14114 QUALITY ASSURANCE STATEMENT Kumme Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor DAIKIN INDUSTRIES, LTD Title Biodegradation study of ( cbi ) by microorganisms Study number 14114 Study inspection of the coirected parts in the Final Report was carried out and it was confirmed that the correction has no problem. The result was reported to the test facility management and the Study Director as follows Date of inspection October 10, 2003 Date of report to Study Director October 10,2003 Date of report to test facility management October 10, 2003 This statement was issued as a supplement to the quality assur ance statement issued on September 18, 2003. Date Quality Assurance Unit, Head October 10,2003 Signed in original Kyoshiro Hori 000006 CONTENTS Title Sponsor Test facility Objective Test method Applied GLP Dates Stoiage of test item, raw data, etc . Personnel Approval of final report SUMMARY 1. Test item 2 Item supplied by sponsor 3 Activated sludge 4 Performance of biodegradation test 5 Validity of test conditions 6 Factors possibly affecting accuracy 7. Results 8 Remarks 14114 page 1 1 1 1 1 1 2 2 3 3 4 5 6 7 9 21 21 21 24 000007 14114 Contents of tables and figures Contents of tables Table-1 Calculation table foi percentage biodegradation by BOD Table-2 Calculation table for amount of dissolved organic carbon by TOC Table-3 Calculation table for percentage biodegradation by weight measur ement Table-4 Calculation table for concentration of perfluorooctanoic acid by LC-MS Table-5 Calculation table for recovery rate by GC-MS ( 2-(perfluorooctyl)ethanol) Table-6 Calculation table for concentration of 2-(perfluorooctyl)ethanol by GC-MS Contents of figures Fig 1 Chart of BOD Fig. 2 IR spectrum of residue Fig 3 Mass fiagmentogram of LC-MS analysis for calibration curve and calibration curve ( perfluorooctanoic acid) Fig. 4 Mass fiagmentogram of GC-MS analysis for calibration curve and calibration curve ( 2-(perfluorooctyl)ethanol) Fig. 5 Mass fiagmentogram of GC-MS analysis for recovery and blank test ( analysis of 2-(perfluorooctyl)ethanol) Fig. 6 Mass fiagmentogram of LC-MS analysis for test solution ( perfluorooctanoic acid) Fig 7 Mass fiagmentogram of GC-MS analysis for test solution ( 2-{perfluorooctyl)ethanol) Fig 8 Mass spectrum of perfluorooctanoic acid Fig. 9 Mass spectrum of 2-(perfluorooctyl)ethanol Fig. 10-1 IR spectrum of test item measured before experimental start Fig. 10-2 IR spectrum o f test item measured after experimental completion Reference IR spectrum supplied by sponsor 000008 14114 Study No 14114 Title Biodegradation study of ( cbi ) by microorganisms Sponsor DAIKIN INDUSTRIES, LTD 1-1 Nishi-hitotsuya, Settsu, Osaka 566-8585, Japan Test facility Kurume Laboratory Chemicals Evaluation and Resear ch Institute, Japan 19-14 Chuomachi, Kurume, Fukuoka 830-0023, Japan Objective This study was performed to evaluate the biodegradability of ( cbi ) by microorganisms Test method This study was performed according to "Ready Biodegradability: Modified MITI Test (I) (Guideline 301C, Revised July 17, 1992)" in the OECD Guideline for Testing of Chemicals. Applied GLP This study was performed in compliance with "OECD Principles of Good Laboratory Practice" (November 26,1997) 1- - 000009 14114 Dates Study initiation date Expeiimental starting date Experimental completion date Study completion date July 17,2003 July 18,2003 August 15,2003 September 18,2003 Storage of test item, raw data, etc . (1) Test item About 5 g of the item supplied by the sponsor is sealed in a store vessel and stored in archives in this laboratory for ten years after the publication of the final report. Treatment of the item supplied by the sponsor after the storage period is discussed with sponsor . If it is not stable for the storage period, it is stored while it is kept stable and it is disposed with approval of sponsor (2) Raw data and materials, etc Raw data, the study plan, documents about the study presented by the sponsor, the final report and necessary materials are stored in archives in this laboratory for the same term as the test item. Treatment of raw data and materials, etc after the storage period is discussed with sponsor . 2- - OOOOIO Personnel Study Director Study per sonnel (Operation of biodegradation test) Staff for cultivation of activated sludge Approval o f final report Study Director Date Signature 14114 Yasuko Matsunobu Kazuhiro Oyama Takakazu Kayashima Hiroto Nishijima September 18,2003 Signed in original Yasuko Matsunobu -3 O O O O ll 14114 SUMMARY Title Biodegradation study of ( cbi ) by microorganisms Conditions of cultivation (1) Concentration of test item (2) Concentration of activated sludge (as the concentration of suspended solid) (3) Volume of test solution (4) Cultivation temperature (5) Cultivation duration 100 mg/L 30 mg/L 300 mL 251 C 28 days Measurement and analysis for percentage biodegradation (1) Measurement of biochemical oxygen demand (BOD) by means of a closed system oxygen consumption measuring apparatus (2) Determination of test item by means of weight measurement Other measurement and analysis (1) Determination of dissolved organic carbon by means of total organic carbon (IOC) analysis (2) Measurement of IR spectrum by means of a fouiier transform infrared spectrophotometer (3) Determination of perfluorooctanoic acid by means of liquid chromatography-mass spectrometry (4) Determination of 2-(perfluorooctyl)ethanol by means of gas chromatography-mass spectrometry Results (1) Percentage biodegradation by BOD 2 %, 4% , 6 % average 4% (2) Percentage biodegradation by weight measurement 0 %, 0 %, 0 % average 0 % Conclusion The test item was not biodegraded by microorganisms under the present test conditions 4- - 000012 1. Test item In this report, ( cbi ) has the following chemical name, etc 11 Chemical name*1 ( CBI 12 Chemical structure, etc.*1 Structural formula 14114 Molecular weight Weight-average ( cbi ) *1 Information supplied by the sponsor 5- - 000013 14114 2 Item supplied by sponsor 2 1 Supplier and lot number'1 (1) Supplier DAKIN INDUSTRIES, LTD (2) Lot number ( cbi ) 2 2 Purity'1 (1) Test item (2) Impurity < CBI ) ( cbi ) The test item was treated as 100 % in purity 2 3 Confirmation of test item Two infrared (IR) spectra of the test item provided by the sponsor and measured at this laboratory were confirmed to be identical (see Fig 10 and Reference) 2 4 Physicochemical property*1 Appearance ( CBI ) *1 Information supplied by the sponsor 2 5 Storage and stability (1) Storage condition Dark storage place at room temperature2 (2) Stability The test item was stable under the storage conditions, as shown by the finding that IR spectra of the test item before the experimental start and after the experimental completion were identical (see Fig 10) . 6- - 000014 14114 3 Activated sludge 3.1 Sludge sampling sites and date (1) Sampling sites On-site sludge sampling was carried out at the following 10 locations in Japan. Fushikogawa city sewage plant (Sapporo-shi, Hokkaido) Fukashiba industiial sewage plant (Kashima-gun, Ibaraki) Nakahama city sewage plant (Osaka-shi, Osaka) Ochiai city sewage plant (Shinjuku-ku, Tokyo) Kitakami River (Ishinomaki-shi, Miyagi) Shinano River (Niigata-shi, Niigata) Yoshino River (Tokushima-shi, Tokushima) Lake Biwa (Otsu-shi, Shiga) Hiroshima Bay (Hiroshima-shi, Hiroshima) Dookai Bay (Kitakyushu-shi, Fukuoka) (2) Date June, 2003 3 2 Sludge sampling (1) City sewage Return sludges from sewage plants were collected. (2) Rivers, lake and sea Surface water and surface soil which was in contact with the atmosphere were collected. 3 3 Preparation of activated sludge Activated sludge was prepared as follows to maintain its uniformity. The filtrate (5 L) of the supernatant of the activated sludge*2 cultivated about for 3 months was mixed with the mixed filtrate (5 L) of the supernatant o f a sludge collected newly at each location The mixed filtrate (10 L) was aerated*3after the pH value of the mixture was adjusted to 7 0+1,0 *2 The activated sludge cultivated the mixed filtrate (10 L) of the supernatant of sludge collected at the ten locations . *3 Prefiltered open air was used. 7- - 000015 14114 3 4 Cultivation Roughly 30 minutes after ceasing aeiation of the sludge mixture, supernatant corresponding to about 1/3 of the whole volume was removed Dechloiinated water was added to the remaining portion so that the total volume reached 10 L. This mixture was aerated, and then a predetermined amount of synthetic sewage*4 was added to the mixture so that the concentration of the synthetic sewage was 0.1 wt% in the volume of dechlorinated water added. This procedure was repeated once every day. Cultivation was carried out at 252 C *4 Synthetic sewage Glucose, peptone and potassium dihydrogenphosphate were dissolved in purified water to obtain 50 g/L of the solution for each component. The pH of the solution was adjusted to 7 01 0 with sodium hydroxide. 3.5 Control and use During cultivation, the appearance of the supernatant, sedimentation of the sludge, formation of flock, pH, dissolved oxygen concentration in the solution and temperature were checked to maintain a normal state of sludge. It was confirmed that these were within the scope of the control standard stipulated in the " Testing Methods for New Chemical Substances", and these results were stored as raw data. Mictoflota in the activated sludge was microscopically observed and sludge with no abnormal symptoms was used for the test , 3 6 Inspection of activity and date of initiation of use of activated sludge (1) Inspection of activity Activity of the sludge was assessed using a reference item2 (2) Date of initiation of use July 15,2003 8- - 000016 14114 4 Peifoimance of biodegradation test 4 1 Preparations for test (1) Measurement of concentiation of suspended solid The concentiation of suspended solid was measured to determine the amount of activated sludge to add. Method Date Result In accordance with Japanese Industrial Standards (JIS) K 0102-1998 section 14.1 July 15, 2003 Concentiation of suspended solid in the activated sludge was 3200 mg/L (2) Preparation of basal culture medium Each 3 mL of solutions (a), (b), (c) and (d) shown below wer e made up to 1L with purified water. The following stock solutions were prepared by use of analytical grade regents: (a) Potassium dihydrogenphosphate, KH2PO4 8.50 g Dipotassium hydrogenphosphate, K2HPO4 21.75 g Disodium hydrogenphosphate dodecahydrate, Na2HPC>4 12H2O Ammonium chloride, NH4CI 1 70 g Dissolved in water and filled up to 1 L 44 60 g (b) Magnesium sulphate heptahydrate, MgSCU7H2O 22 50 g Dissolved in water and filled up to 1 L (c) Calcium chloride anhydrous, CaGh 27 50 g Dissolved in water and filled up to 1 L (d) Iron(IH) chloride hexahydrate, FeC13 6H2O 0 25 g Dissolved in water and filled up to 1 L3 (3) Reference item Aniline (reagent grade, Showa Chemicals Inc Lot No HO-2729D) was used as a reference item to confirm that the sludge was sufficiently active This test is effective when the percentage biodegradation calculated from the BOD of aniline after 7 and 14 days exceeds 40 % and 65 %, respectively, according to regulation of the OECD Guideline for Testing of Chemicals. 9- - 000017 14114 4 2 Preparation of test solutions Test solutions for analysis of the test item and peifluorooctanoic acid and test solutions for analysis of 2-(perfluorooctyl)ethanol were prepared, because the test item and converted products (petfluofooctanoic acid and 2-(perfluorooctyl)ethanol), which was expected to form in test solutions, could not be analyzed by the same pretreatment. Eleven test vessels were prepared The following test solutions were prepared and cultured under the conditions described in section 4 3. (1) Addition of test item or aniline (a) Test solution (water + test item) (n=2: Vessel No 6 and a test solution for analysis of 2-(peifluorooctyl)ethanol) In one test vessel, 30 mg of the item supplied by the sponsor was accurately weighed and added to 300 mL of purified water, so that the concentration of the test item reached 100 mg/L, (b) Test solution (sludge + test item) (n=6: Vessel No.l, 2, 3 and three of test solutions for analysis of 2-(perfluorooctyl)ethanol) In each test vessel, 30 mg of the item supplied by the sponsor was accurately weighed and added to the basal culture medium (the volume was less than 300 mL by the volume (2 81 mL) of activated sludge inoculated), so that the concentration of the test item reached 100 mg/L (c) Test solution (sludge + aniline) (n= l: Vessel No .4) In one test vessel, 29 5 pL [30 mg = 29.5 pL x 1 022 g/cm3 (density)] of aniline was added into the basal culture medium (the volume was less than 300 mL by the volume (2.81 mL) of activated sludge inoculated), so that the concentration reached 100 mg/L (d) Test solution (control blank) (n=2: Vessel No 5 and a test solution for analysis of 2-(perfluorooctyl)ethanol) In one test vessel, nothing was added to the basal culture medium (the volume was less than 300 mL by the volume (2.81 mL) of activated sludge inoculated),2* (2) Inoculation of activated sludge The activated sludge cultivated under the conditions described in section 3 was added to each test vessel, (b), (c) and (d), so that the concentration of the suspended solid reached 30 mg/L - 10- 000018 14114 4 3 Instruments and conditions of cultivation (1) Instruments for cultivation (a) Test solution for analysis of test item and perfluorooctanoic acid Closed system oxygen consumption measuring apparatus Temperature controlled bath and measuring unit : Ohkura Electric C o , Ltd. Data sampler : Asahi Techneion C o , Ltd Vessel 300 mL in volume (improved type) Absorbent for carbon dioxide Soda lime No.l (for absorption of carbon dioxide, Wako Pure Chemical Industries, Ltd ) (b) Test solution for analysis o f 2-(perfluorooctyl)ethanol Closed system oxygen consumption measuring apparatus Temperature controlled bath and measuring unit (Only the temperature controlled bath was used) : Ohkura Electric C o L t d Vessel 300 mL in volume (sealed improved type) (2) Conditions of cultivation Cultivation temperature Cultivation duration Stirring method 251 C 28 days Each test solution was stirred by a magnetic stirrer (3) Room Apparatus room No 511 4 4 Observation and measurement of test conditions (1) Observation of test solution During the cultivation period, the appearance of the test solution was observed periodically and conditions of the instruments were checked properly.2 (2) Measurement of biochemical oxygen demand (BOD) During the cultivation period, the change in BOD of the test solutions was measured by autorecording using a data sampler . Cultivation temperature was measured and recorded once a day. BOD of the test solutions for analysis of 2-(perfluorooctyl)ethanol was not measured -11 - 000019 14114 4 5 Analysis of test solution After the teimination of the cultivation, dissolved organic carbon, the test item and converted products (perfluorooctanoic acid and 2-(perfluorooctyI)ethanol), which was expected production in test solutions, were determined. 4.5.1 Pretreatment of test solutions for analysis (1) Pretreatment for analysis of test item and perfluorooctanoic acid After the termination o f the cultivation, the test solution (water + test item), the test solutions (sludge + test item) and the test solution (control blank) for analysis of the test item and perfluorooctanoic acid were pretreated for' total organic carbon (TOC) analysis on dissolved organic carbon and liquid chromatography - mass spectrometry (LC-MS) analysis of perfluorooctanoic acid as follows. Weight measurement of the test item was performed IR spectrum of the residue was measured. Test solution 300 mL Taking out 10 mL (measuring pipet) Centrifugation (1000 x g, 10 min) Super natant Residue Taking out 2 5 mL (transfer pipet) Filling up to 5 mL (methanol, volumetric flask) Filtration (The test solutions were filtered by use of membrane filter*5 with suction The residue in the vessels was transferred on the filter and washed with purified water.) Sample for TOC analysis Sample for LC-MS analysis Determination of perfluorooctanoic acid Res idue Filtrate Drying (vacuum oven, 40 C, 137 his ) Cooling (desiccator, room temper ature, about 30 min) Sample for weight measurement MILLIPORE type HA Sample for measurement of ERspectrum - 12- 000020 14114 (2) Pretreatment fox analysis of 2-(perfIuorooctyl)ethanol After the termination of the cultivation, the test solution (water + test item), the test solutions (sludge + test item) and the test solution (control blank) fox analysis of 2-(perfluorooctyl)ethanol were cooled at 5 C for 66 hrs. and preheated fox gas chromatography - mass spectrometry (GC-MS) analysis as follows. Test solution 300 mL --Ammonium sulfate 200 g (even balance) *--Chloroform 90 mL (graduated cylinder) Shaking (10 min) * Filtration and dehydration (No.2 filter paper, sodium sulfate 30 g) Filling up to 200 mL (chloroform, volumetric flask) Sample for GC-MS analysis - 13- 000021 14114 4.5.2 Quantitative and qualitative analysis (1) Determination of dissolved organic carbon by means of TOC analysis The dissolved organic carbon (DOC) in the samples for TOC analysis was analyzed under the following conditions. The concentration of DOC was calculated by subtracting concentration of the inorganic carbon (IC) from concentration of the total carbon (TC). The concentration of TC and IC in the test solutions was proportionally calculated from the peak area of the test solution by comparison with that of 80.0 mgC/L of a standard solution for TC analysis and 80.0 mgC/L of a standard solution for IC analysis, respectively (see Table-2). The standard solution for TC analysis was prepared by dissolving potassium hydrogenphthalate in purified water. The standard solution for IC analysis was prepared by dissolving sodium hydrogencarbonate and sodium carbonate in purified water. The concentration of dissolved organic carbon corresponding to the minimum determination limit was regarded as 1.0 mgC/L. Analytical conditions Instrument Temperature of furnace Flow rate Injection volume Sensitivity Total organic carbon analyzer Shimadzu Corporation type TOC-5000 680 C 150 mL/min 33 pL Range 5 (2) Determination of test item by means of weight measurement Weight of the samples for weight measurement was measured under the following conditions (see Table-3). Analytical conditions Instrument Electronic analytical balance Mettler Toledo type AT201 Minimum measuring weight 0.1 mg (3) Measurement of IR spectrum by means of fourier transform infrared spectrophotometer IR spectrum of the samples for measurement of IR spectrum was measured under the following conditions (see Fig.2). Analytical conditions Instrument Measuring method Fourier transform infrared spectrophotometer Shimadzu Corporation type FTIR-8200PC KBr tablet - 14- 000022 14114 (4) Deteimination of peifluorooctanoic acid by means o f LC-MS The samples for LC-MS analysis were analyzed for peifluorooctanoic acid under the following conditions. The concentration of peifluorooctanoic acid in the sample for LC-MS analysis was proportionally calculated by comparing the peak area on the mass ffagmentogram of the sample for LC-MS analysis with that on the mass fiagmentogram of 0.500 mg/L standard solution (see Table-4 and Fig 6) The lowest detectable peak area was regarded as 2000 considering the noise level, which corresponded to 0 0098 mg/L of peifluorooctanoic acid . (a) Analytical conditions Instrument LC MS Liquid chromatograph-mass spectrometer JASCO Corporation type PU-980 Mictomass type Quattro II Conditions of LC Column Eluent Flow rate Sample size L-colunm ODS 15 cm x 4.6 mm ID. Methanol*6/ purified water*6(80/20 V/V) 1.0 mL/min 10 pL Conditions of MS Ionization method Detection mode Monitoring method Monitoring m/z Source temperature Cone voltage Electrospray (ESI) Negative ion Selected ion monitoring (SIM) 413 (see Fig 8) 150 C 20 V *6 Containing 0 1 % formic acid -15 - 000023 14114 (b) Preparation of standard solution The standard solution to determine the concentration of perfluorooctanoic acid in the sample for LC-MS analysis was prepared as follows. 100 mg of the standard substance 7supplied by the sponsor was accurately weighed and dissolved in methanol to obtain 2000 mg/L solution of perfluorooctanoic acid 0 500 mg/L standard solution was then prepared from this solution by dilution with methanol / purified water (1/1 V/V). *7 Name, lot number and purity Name Perfluorooctanoic acid (C-1700) Lot number C l7001401 Purity Perfluorooctanoic acid was treated as 100 % in purity (c) Calibration curve 0 125, 0 250 and 0.500 mg/L standard solutions were prepared by the same method as described in (b) These solutions were analyzed according to the analytical conditions described in (a). A calibration curve was drawn based on the relation between the peak area on the mass fiagmentograms and the respective concentrations (see Fig 3). - 16- 000024 (5) Determination of 2-(perfluorooctyl)ethanol by means of GC-MS The samples for GC-MS analysis were analyzed for 2-(peifluorooctyl)ethanoI under the following conditions The concentration of 2-(perfluorooctyl)ethanol in the sample for GC-MS analysis was proportionally calculated by comparing the peak area on the mass fiagmentogram of the sample for GC-MS analysis with that on the mass fiagmentogram of 0.500 mg/L standard solution (see Table-6 and Fig.7) The lowest detectable peak area was regarded as 300 considering the noise level, which corresponded to 0 021 mg/L of 2-(perfluorooctyl)ethanol. (a) Analytical conditions Instrument Gas chromatograph-mass spectrometer Shimadzu Corporation type QP-5000 Conditions of GC Column Column temperature Carrier gas Pressure of carrier gas Injection temperature Sample size Sampling method Sampling time HP-FFAP (Agilent) 25 m x 0 32 mm I D , made of fused silica 50 C (4 min)--170 C (Rate 30 C/min) Herium 10.0 kPa 200 C 2 pL Splitless 2.0 min Conditions of MS Ionization method Detection mode Monitoring method Monitoring m/z Interface temperature Electron ionization (El) Positive ion Selected ion monitoring (SIM) 95 (see Fig 9) 230 C i H- -0 1 000025 14114 (b) Preparation of standard solution The standard solution to determine the concentration of 2-(peifluoiooctyl)- ethanol in the sample for GC-MS analysis was prepared as follows 100 mg of the standard substance*8 supplied by the sponsor was taken out accurately and dissolved in chloroform to obtain 2000 mg/L solution of 2-(peifluorooctyl)ethanol. 1.50 mg/L standard solution was then prepared from this solution by dilution with chloroform. *8 Name, lot number and purity Name 2-(perfluorooctyl)ethanol (A-1280) Lot number A18201X01 Purity 2-(peifluorooctyl)ethanol was treated as 100 % in purity (c) Calibration curve 0 375, 0 750 and 1 50 mg/L standard solutions were prepared by the same method as described in (b). These solutions were analyzed according to the analytical conditions described in (a). A calibration curve was drawn based on the relation between the peak area on the mass fiagmentograms and the respective concentrations (see Fig.4) - 18- 000026 14114 4 5.3 Recovery test and blank test Each two test solutions (water + 2-(perfluorooctyl)ethanol) and two test solutions (sludge + 2-(peifluorooctyl)ethanol) for recovery test were prepared according to the methods described in section 4 2 0 300 mg of 2-(perfluorooctyl)ethanol was added to each test vessel, so that the concentration of it reached 1 mg/L The test solutions were pretreated in accordance with the method described in section 4 5 1(2), then analyzed according to analytical conditions described in section 4 .5 2(5). A test solution for blank test was prepared according to the method described in section 4.2 . The test solution for blank test was analyzed in the same way as the recovery test. As for the blank test, no peaks were appeared around the peak of the 2-(perfluorooctyl)ethanol on the mass fiagmentogiam Two individual recovery rates and their averages on the analytical procedure are shown below. The average recovery rates were used as correction factor, for the determination of 2-(perfluorooctyl)ethanol in the test solutions (see Table-5 and Fig 5) Recovery rate of the test solutions (water + 2-(perfluorooctyl)ethanol) 99 6 %, 98 2 % average 98 9 % Recovery rate of the test solutions (sludge + 2-(perfluorooctyl)ethanol) 80.5%, 8 1 5 % average 8 1 0 % - 19- Corrected page 000027 14114 4 6 Calculation of percentage biodegradation The percentage biodegradation was calculated by the following equations and expressed in whole numbers (1) Percentage biodegr adation by BOD BOD-B Percentage biodegradation (%) = TOD'y x 100 BOD B IO D *9 Biochemical oxygen demand in the test solution (sludge + test item) (experimental) (mg) : Biochemical oxygen demand in the control blank (experimental) (mg) Theoretical oxygen demand required when the test item was completely oxidized (theoretical) (mg) *9 The purity was regarded as 100 % and TOD was calculated from composition formula ( CBl ) which was calculated by use of n=8, monomer values of (CBl) regarded as mol% in section 1 2 (2) Percentage biodegradation by weight measurement Sw - Ss Percentage biodegradation (%) = Sw x 100 Ss : Residual amount of the test item in the test solution (sludge + test item) (experimental) (mg) Sw : Residual amount of the test item in the test solution (water + test item) (experimental) (mg) 4.7 Treatment of numerical values Values were rounded off'in accordance with JIS Z 8401:1999 rule B - 20- 000028 14114 5 Validity of test conditions Percentage biodegradations of aniline calculated by the BOD values were 68 % and 72 % after 7 and 14 days, respectively It was concluded that this test conditions were valid (see Table-1 and Fig 1). 6 Factors possibly affecting accuracy No adverse effects on the reliability of this test were noted. 7 Results 7 1 Appearances of test solutions Appearances of test media in cultivation vessels were as follows Test solution Appearance At the start of cultivation Water + test item The test item was not dissolved . Sludge + test item The test item was not dissolved pH - Water + test item At the termination of cultivation Sludge + test item Insoluble compound was observed. Insoluble compound except for the sludge was observed Growth of the sludge was not observed. - -21 - 000029 14114 7 2 Analytic results o f test solutions Analytic results of the test solution after 28 days were as follows Water + test item Sludge + test item Theoretical Table amount Vessel-6 Vessel-1 Vessel-2 Vessel-3 Fig BOD*10 mg 0 1 06 1.1 1.8 ( CBI ) 1 1 *10 mgC 0 0 0.3 0.3 ( CBI ) Detection amount and percentage 2- detection of DOC % 0 0 3 3 - Residual amount and percentage residue of test item (Weight measurement) *11 Concentration of perfluorooctanoic acid (LC-MS) mg % mg/L 28 5 95 2*0.020 300 100 2*0.020 29.6 99 0 020 289 96 0.020 30 0 - - 346 Water + test item *12 Concentration of 2-(perfluorooctyl) mg/L 0 0 1 4 ethanol (GC-MS) Sludge + test item 12 3 0.017 0017 0017 Theoretical amount Table -6 Fig 7 *10 The value of control blank was subtracted from the values of the test solutions (sludge + test item) *11 Minimum determination limit of perfluorooctanoic acid in test solution = (Minimum determination limit in sample for LC-MS analysis) x {(final volume) / (volume of test solution)} / (ratio of portion used for analysis) = 0 0098 mg/L * (5 mL/300 mL) / (2 5/300) = 0 020 mg/L *12 Minimum determination limit of 2-(perfluorooctyl)ethanol in test solution = (Minimum determination limit in sample for GC-MS analysis) * {(final volume) / (volume of test solution)} / {(average recovery rate) / 100} / (ratio of portion used for analysis) Test solution (water + test item) : 0 021 mg/L x (200 mL / 300 mL) / (98 9/100) / 1 = 0 014 mg/L Test solution (sludge + test item): 0 021 mg/L x (200 mL/300 mL) / (81 0/100) /1 =0.017 mg/L - 22- 000030 14114 7.3 Percentage biodegradation Percentage biodegr adations after 28 days were as follows Method Percentage biodegradation (%) Vessel-1 Vessel-2 Vessel-3 Average BOD 24 6 4 Weight measurement 0 0 0 0 Table T 3 7 4 Results of measurement of IR spectrum No significant difference between IR spectrum o f the test item and that of the residue after weight measurement was observed (see Figs 2,10). 7 5 Discussion Determination of the test item by chromatography was difficult, because the test item was not completely dissolved in water and organic solvents (tetrahydrofuran and chroloform) Therefore, the test item was determined by means of weight measurement. As a result, the percentage residues of the test item were 95 % in the test solution (water + test item) and 100, 99 and 96 % in the test solutions (sludge + test item). The percentage detections of DOC were 0 % in the test solution (water + test item) and 0, 3 and 3 % in the test solutions (sludge + test item) These results indicate that the test item was not dissolved in the test solution No significant difference between DR. spectra o f the test item before and after the cultivation was observed Converted products (perfluorooctanoic acid and 2-(perfluorooctyl)ethanol), which was expected to foim in test solutions, was not detected These results indicate that the test item was not converted during the cultivation. It is considered that the test item was not biodegraded by microorganisms, because the average percentage biodegradation by BOD was 4 % and no significant change in the weight of the test item before and after the cultivation was observed 7 6 Conclusion The test item was not biodegraded by microorganisms under the present test conditions. -23 - 000031 14114 8. Remarks 8.1 Instruments used for test Fourier transform infrared spectrophotometer : Shimadzu Corporation type FTIR-8200PC Closed system oxygen consumption measuring apparatus : see page 11 Total organic carbon analyzer : see page 14 Liquid chromatograph- mass spectrometer : see page 15 Gass chromatograph- mass spectrometer : see page 17 Electronic analytical balance : Mettler Toledo type AT201 Sartorius AG type CP324S Sartorius AG typeBP210S Refrigerated centrifuge : Shimadzu Corporation type CST-060LF Mechanical shaker : TIETECH Co., Ltd. type SR-2w Vaccume oven : SIBATA SCIENTIFIC TECHNOLOGY LTD. type VS-300 8.2 Reagents used for analysis Methanol (HPLC grade) : Wako Pure Chemical Industries, Ltd. Purified water : Takasugi Seiyaku Co., Ltd. Chloroform (reagent grade) : Kishida Chemical Co., Ltd. Ammonium sulfate (reagent grade): Kanto Chemical Co., Inc. Sodium sulfate (reagent grade): Kanto Chemical Co., Inc. Formic acid (reagent grade): Wako Pure Chemical Industries, Ltd. Sodium hydrogencarbonate (reagent grade): Wako Pure Chemical Industries, Ltd. Sodium carbonate (reagent grade): Wako Pure Chemical Industries, Ltd. Potassium hydrogenphthalate (reagent grade) : Wako Pure Chemical Industries, Ltd. Potassium bromide (for measurement of infrared absorption) : NACALAI TESQUE, INC. Perfluorooctanoic acid : Standard substance supplied by the sponsor 2-(perfluorooctyl)ethanol : Standard substance supplied by the sponsor - 24- 000032 Table 1 Calculation table for the percentage biodegradation by BOD Test No. 14114 . Cultivating Duration: 28 days Vessel No . 0 1 7th day BOD Deg. (nig) ( %) 63.6 68 2.0 2.7 2 21 0 3.0 3 0.0 - 14th day BOD Deg. (mg) (%) 67,8 72 2.6 33 2 2.7 0 3.6 3 0-0 - 21st day BOD Deg.. (mg) (%) 69.6 73 3.4 4.0 2 3,7 1 4.3 3 0.0 - 28th day BOD Deg. (mg) ( %) Mean Deg % 71.8 75 4.5 - 5.1 2 4 5.6 4 6.3 6 01 - Deg. : Percentage biodegradation Vessel no. SI Vessel no. 0 Vessel no,[D 0 0 Vessel no. [6] :Sludge :Control : Sludge :Water + Aniline blank [B] + Test substance + Test substance Test substance of 30 mg was added. Chart of BOD : Fig 1 Deg. = [ BOD - B ] / [ TOD ] x 100 (*) TOD of test substance : ( CBi ) (mg) TOD = 30.0 X (CBI) = (CBI) (mg) TOD of aniline : 90.3 (mg) Cs HtN + 8.75 0a -- 6 C0a + 3.5 H a0 + NOa 8,75 0a / C s H tN = 279.99 / 93.13 = 3.01. TOD = 30.0 x 3.01 = 90,3 (mg) 2003.08.15 Name 000033 Table-2 Calculation table for amount o f dissolved organic carbon by TOC Sample description Water blank [6] Water + test item [1] Sludge + test item [2] Sludge + test item [3] Sludge + test item [5] Control blank A B CD n.d n.d. 0 n.d, 0 00 0 1.15 0 3 3 2 1 02 0 3 3 nd Amount o f test item added : 30 (mg) Volume o f test solution : 300 (mL) Iheoretical amount o f carbon :(CBI) (mgC) (30) * ( CBI A : Measured value (mgC/L) B : Amount of DOC Bw = (A(Water + test item) - A(Water blank)) * 300 /1 0 0 0 (mgC) Bs = (A(Sludge + test item) - A(Control blank)) x 300 /1 0 0 0 (mgC) C : Percentage detection (%) C = B / (Iheoretical amount of carbon) x 100 (%) D : Average percentage detection (%) ) August 26,2003 Name 000034 Table-3 Calculation table for percentage biodegradation by weight measurement Sample description Study No. 14114 A B C DE [6] Water + test item [1] Sludge + test item [2] Sludge + test item [3] Sludge + test item [5] Control blank 28 5 28.5 95 33 9 3 0 0 100 33.5 2 9 6 99 328 28.9 96 39 0 00 0 Amount o f test item ad d ed : 30 (mg) Volume of test solution: 300 (mL) A : Weight of residue (mg) 5 : Residual amount of test item (mg) Bw = A(Water + test item) Bs = A(Sludge + test item) - A(Control blank) C : Per centage residue (%) C = B / (Amount of test item added) x 100 D : Percentage biodegradation (%) D * (Bw - Bs) / Bw x 100 E : Average percentage biodegradation (%) August 26,2003 Name 000035 Table-4 Calculation table for concentration of perfluorooctanoic acid by LC-MS Sample description Study No. 14114 A DE Standard solution 0. 500mg/L [6] W ater + test item [1] Sludge + test item [2] Sludge + test item [3] Sludge + test item [5] Control blank 102018 n.d. n.d. n d.. n i. n.d.. 0 ( 0 020) 0 ( ^ 0 020) 0 (g0 020) 0 ( ^ 0 020) - Volume of test solution : 300 (mL) A : Peak area ( - ) B : Final volume : 5 (mL) C : Ratio o f portion used for analysis: 2 5/300 D : Concentration o f perfluorooctanoic acid in test water (mg/L) D * F x ( A / A(Standard)) x ( B / 300 ) / C Minimum determination lim it: 0.020 (mg/L) E : Aveiage concentration o f perfluorooctanoic acid (mg/L) F : Concentration o f standard solution: 0 .500 (mg/L) See Fig. 6 September 18,2003 Name 000036 Table-5 Calculation table for recovery rate by GC-MS ( 2-(perfluorooctyl)ethanol) Sample description AD Standard solution 1.50mg/L Water + 2-(perfluorooctyl)ethanol -1 Water + 2-(perfluorooctyl)ethanol -2 Sludge + 2-(perfluorooctyI)ethanol -1 Sludge + 2-(perfluorooctyl)ethanol -2 Control blank 26329 26222 25861 21196 21459 nd 0 299 0295 0242 0.245 StudvNo. 14114 EF 9 9 6 98 9 982 80.5 81,0 81.5 Amount o f 2-(perfluorooctyl)ethanoI a d d ed : 0.300 (mg) Volume of test solution : 300 (mL) A : Peak area ( - ) B : Final volum e: 200 (mL) C : Ratio of portion used for analysis : 1 D : Recovery amount (mg) D = G x ( A / A(Standard)) * ( B / 300) / C E : Recovery rate (%) E = D / 0 300 (mg) x 100 F : Average recovery rate (%) See Fig. 5 September 11,2003 Name 000037 Table-6 Calculation table foi concentration of 2-(perfluorooctyI)ethanol by GC-MS Sample description Study No. 14114 A EF Standard solution 1 50mg/L Water + test item Sludge + test item -1 Sludge + test item -2 Sludge + test item -3 Control blank 20965 nd. n..d. n d. n.d. n.d 0 (SO 014) 0 (Sa 0 017) 0 (SO 017) 0 (SO 017) - Volume of test solution: 300 (mL) A : Peak area ( ) B : Final volum e: 200 (mL) C : Ratio o f portion used for analysis r 1 D : Recovery rate : 98.9 (%) (Water + test item) 81 0 (%) (Sludge + test item) E : Concentration of 2-(perfluorooctyl)ethanol in test solution (mg/L) E ** G * ( A / A(Standard)) * ( B / 3 0 0 ) / ( D / 1 0 0 ) / C Minimum determination lim it: 0 .014 (mg/L) (Water + test item) 0 017 (mg/L) (Sludge + test item) F : Average concentration of 2-(perfluorooctyl)ethanol (mg/L) G : Concentration o f standard solution : 1 50 (mg/L) See Fig 7 September 18,2003 Name jc. ()yci***cS 000038 Fig.l Chart of BOD Test No, 14114 ( Test substance Apparatus Cultivating conditions: Concentration Test substance Reference substance! Activated sludge Temperature Duration aniline ) Note: Regular condition CBI No. C M - 17 100 (mg/tf) 100 (mg/ID 30 (mg/lD 25 l-C 2 8 d a y s (J u l .18- Vug 15,2003) Vessel no. m m a s m Sample description Sludge + Test substance Sludge + Test substance Sludge + Test substance Sludge + Aniline Control blank [B] Water + Test substance 100- 7thday 2.7 2.1 3.0 63.6 2.0 0.0 B 0 D (mg) 14thday 3.3 2.7 3.6 67.8 2.6 0.0 2Lstday 4.0 3.7 4.3 69.6 3.4 0.0 28thday 5.1 5.6 6.3 71.8 4.5 0.1 80- 60- BO >> 40aD o S 20- -m i --i-- i--i-- i-- r n-- i--i--i-- 14 21 Cultivation Time ( 0~28thday) 2003.08 15 Name - -[] --l-- i-- 28 000039 Fig. 2 - 1 IR spectrum of residue. Study No. :14114 Sample :[6] Water + test item Method :KBr tablet Date :August 26,2003 Name :A 000040 Fig.2 -2 IR spectrum of residue Study No Sample Method Date Name :14114 :[1] Sludge :KBr tablet :August 26, jc + test 2003 item 000041 Fi g. 2 -3 IR spectrum of residue. Study No. Samp Ie Method Date Name 14114 [2] Sludge + test item KBr tablet August 26, 2003 000042 Fig.2 - 4 IR spectrum of residue. Study No .: 14114 Sample :[3] Sludge + test item Method :KBr tablet Date :August 26, 2003 Name :k 000043 Fig 2 -5 IR spectrum of residue. Study No Samp Ie Method Date Name 14114 [5] Control blank KBr tablet August 26, 2003 jc 000044 Solvent 14114d021 ' 15-Aug203 SIR of 1 Channel ES- Fi&.3 - 1 Mass fiagmentogram of LC-MS analysis for calibration cuive ( perfluorooctanoic acid). 000045 120000 r Study No 14114 Peak area ( - ) y = 201414x r** 1.00 Concentration (mg/L) 0.125 0.250 0.500 Peak ar ea (-) 23889 50521 100945 Fig. 3 - 2 Calibration curve of LC-MS analysis for peifluorooctanoic acid. August 22,2003 Name 000046 Datafile : 14114d063 Sample : Solvent Study No 14114 Sample : Standard solution 0.375mg/L Datafile :14114d065 Sample : Standard solution 0 750mg/L Datafile : 14114d066 Sample : Standard solution 1 50mg/L Peak R T. Peak area 1 6 357 6136 Peak R I . Peak area 1 6 353 12339 Peak R I . Peak area 1 6 352 25069 F ig.4-1 D ate: August 7,2003 Name : Mass fragmentogram of GC-MS analysis for calibration ctnve ( 2-(perfluorooctyl)ethanol) 000047 Study No 14114 Peak area ( - ) y = 16646x r * 1.00 Concentration (mg/L) 0.375 0.750 1.50 Peak area (-) 6136 12339 25069 Fig. 4 - 2 Calibration crave o f GC-MS analysis for 2-{peifluorooctyl)ethanol August 11,2003 Name_____ . 0 ___________ 000048 Datafile : 14114d067 Sample : Standard solution 1,50mg/L intensity 300001 Study No 14114 9J.OO Peak R.T Peak area 1 6353 26329 Datafile : 14114d069 Peak R.T Peak area 1 6,355 26222 Peak R..T, Peak area 1 6.357 25861 Date : August 7,2003 TName : k. Fig-5 -1 Mass fragmentogram of GC-MS analysis for recovety and blank test ( analysis of 2-(peifluorooctyl)ethanol ). 000049 Datafile : 14114d070 Sample : Sludge + leaf item -1 intensity 30000' 25000 - 20000-^ 15000: 10000- 5000: 0- Datafile : I4114d071 Study No 14114 95.00 Peak FU Peak area 1 6358 21196 mm Peak R.T Peak area 1 6355 21459 Datafile :14114d072 Sample : Control blank intensity _______________________________________________________ 95.00 30000 25000- 200001500010000-H 5000: Peak position n.4. '* 0- .............. ' 5 ..I1- 1" 1 1 ...................--r 1 ..... i 1 1 1 " 67 min D ate: A ugust7,2003 Name : Jc. iyV**-v-iK^ Fig.5 - 2 Mass fiagmantogiam of GC-MS analysis for recovery and blank test ( analysis of 2-(perfluorooctyl)ethanol ). 000050 Fig.6 -1 Mass fragmentogram of LC-MS analysis for test solution ( peifluorooctanoic acid ). 000051 [2J'Sludge + test item 14114d018 100-, 15-Aug-20Q3 SIR of 1 Channel ES TIO 8 00e5 1.98 A1 1T" 0.00 ' 2.b i[3l Sludge + test item i14114d019 ! 100-, Peak position n.d. 4.00 s.bo 8.bo ..............i Time 10.00 S-Aug-2003 SIR o f 1 Channel ES TIO 8 00e5 %- 1 99 A0 0.00 zoo |5] Control blank 14114d020 i 100-, I Peak position n.d. 15o" .bo .bo r--t--, Time 10.00 15-Aug-2Q03 SIR of 1 Channel ES TIO 800e5 %00 1 98 A 20 Peak position n.d. Too .b Time _8 00_ ^ r ' ' io 'oo Fig.6 - 2 Mass fiagmentogiaxn of LC-MS analysis for test solution ( peifluorooctanoic acid ). 000052 Datafile : 14U4d074 Sample : Standard solution 1 50mg/L Study No 14114 Peak R T. Peak area 1 6.290 20965 Datafile : 141I4d075 Sample : Water + test item Datafile : 14114d076 Sample : Sludge + test item -1 intensity ________________________________________________________ 95.00 30000; 25000- 20000- 15000: 10000-] 5000- 05 Peak position n.cL '1 A . . < -----1i -- i-- |-- i-- i 1 I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-- -- -- 67 min Date : August 18,2003 Name : K Fig 7 -1 Mass fragmentogram of GC-MS analysis for test solution ( 2-(perfluorooctyl)ethanol ). 000053 Datafile : 14114d077 Sample : Sludge + test item -2 intensity 30000 ; Study No. 14114 95.00 Datafile :14114d078 Sample : Sludge + test item -3 intensity __________________ ________________________________ 30000 25000: 95,00 20000- 15000: Peak position tJ d loooo: 5000- \ * A. 0^ ----------1---1---1---.--i---1-------1--i---r ---,--i---1--,--r- i------i--t---------i--J--i----i---- 5 67 min Datafile : 141144079 Sample : Control blank Date : August 18,2003 Name : Fig..7 - 2 Mass ftagmentogram of GC-MS analysis for test solution ( 2-{peifluorooctyI)ethanol ) 000054 Study No. 14114 Instrument MS : Micromass Quattro H. HPLC : Jasco PU-980. AS-950 Sample ____ Perfluorooctanoic acid________ HPLC Conditions Column L-oolumn OPS Size 15 cm x 4.6 mm I D Eluent Methanol*1/purified water** ( 8/2 V/V) *1 containing 0,1% formic acid Flow rate 1.0 mL/min Sample size 20 uL (Solvent Methanol ) MS Conditions Ionization mode ESI Detection mode Negative Function MS Source conditions ( Capillary 3.0 kV. Cone Mass range (m/z) 100 - 1000 20 V. Temperature 150 C ) Date June 17. 2003 Operator jc , B y x u * u * . / Kurume Laboratory, Chemicals Evaluation and Research Institute, Japan Fig.8 - 1 Mass spectrum of perfluorooctanoic acid ( analytical condition ). 000055 std 20.0mg/L 14114d001 ' 17-Jun-2003 2: Scan ES- Fig.8 - 2 Mass spectrum ofperfluorooctanoic acid ( mass spectrum ) 000056 stcT20.0mg/L 141I4d00i 100-, 3 86 %-1 0.89 1.49 2 65 3.04 l7 -J u 'n-2003 2: Scan ES- TIC 1 99e6 14114d001 100-, m/z = 369 %- 3 86 2: Scan ES368.90 3 60e5 0- 141l4d001 ; 100- m /z = 413 %- 3.86 i 1111i 2: Scan ES412.89 6 43e5 t 141l4d001 100-, m /z = 459 i 1111i 3.86 %- 2: Scan ES458.86 2.08e5 141I4d001 100valz - 827 %- "r"T 3.86 \'' ''r 2: Scan ES- 826.54 7 07e4 Time 0.00 2.56 4.56 slo " 8.56 10.00 Fig.8 - 3 Mass spectrum of perfluorooctanoic acid ( mass chromatogram ) 000057 Study Na. 14114 Instrument Shimadzu QP-5000 Sample _____ 2-(perfluorooctyl)ethanol___________ GC Conditions Column HP-FFAP____ ( Fused silica ) Size 25 m x 0.32 mm LD.., Film thickness 0.52 u m Column temp. 50 C ( 3 min) -* 170 C. Tamp, rate 30 C/min Carrier gas He Column head Dressure 10.0 kPa Iru'ection temp. 200 C Sample size 1 U L (Solvent Methanol Inlet mode Splitiess Sampling time 2.0 min MS Conditions Ionization Mode El Detection Mode Positive Mass ranee (m/z) 50 - 600 Interface temp. 230 C Date June 11. 2003 Operator Kuruma Laboratory, Chemicals Evaluation and Research Institute, Japan Fig,.9 - 1 Mass spectrum of 2-(perfluorooctyl)ethanol ( analytical condition ).. 000058 StudyNo 14114 Datafile: 141 MdOOI Sample: standard solution 10 Omg/L Total ion chromatogram Peak#:! R.Iime:5.5(Scan#:235) MassPeaks:104 BasePeak:95(704356) intensity MS spectrum Date : June 11, 2003 Name : Fig.9 - 2 Mass spectrum of 2-(perfluorooctyl)ethanol ( mass spectrum ). 000059 Datafile: 14114d001 Sample: standard solution 10 Omg/L Study No 14114 Mass chromatogram Date: June 11, 2003 Name: Fig 9 - 3 Mass spectrum of 2-(perfluorooctyl)ethanol ( mass chromatogram) 000060 Fig,10-1 IR spectrum of test item measured before experimental start. Study No. Sample Method Date Name 14114 Test item KBr tablet July 10, 2003 000061 Fig.10-2 IR spectrum of test item measured after experimental completion. Study No .: 14114 Sample :Test item Method :KBr tablet Date :August 27, 2003 Name :jc 000062 Reference IR spectrum supplied by sponsor 000063 Amendment to Final Report Kurume Laboratory Chemicals Evaluation and Research Institute, Japan 1 Title Biodegradation study of ( cbi ) by microorganisms 2. Study number 14114 3. Content 4.5 3 Recovery test and blank test (page 19) 4 Reason Writing error 5. Content of correction " Recovery rate of the test solutions (water + test item) 99 6%, 98.2% average 98.9% Recovery rate of the test solutions (sludge + test item) 80 5% , 81.5% average 81.0%" is corrected to " Recovery rate of the test solutions (water + 2-(perfluorooctyl)ethanol) 99 6% , 98 2 % average 98.9% Recovery rate of the test solutions (sludge + 2-(perfluorooctyl)ethanol) 80 5% , 8 1 5 % average 81.0%" 6. Approval Date Study Director October 10,2003 Signed in original Yasuko Matsunobu 000064 Accepted number Study number S03-4115 14115 RECEIVED 05J U . V m i l : SU u 'j" D FINAL REPORT 97 7 7 ^ Biodegradation study of ( cbi ) by microorganisms hCZOZ.' Xt* j r *_ cn OO September 18,2003 Kurume Laboratory Chemicals Evaluation and Research Institute, Japan Company Sanitized tv & # n 3 3 000065 tMUfUSI'i STATEMENT 14115 Kuiume Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor DAKIN INDUSTRIES, LTD Title Biodegradation study of ( CBl ) by micr oorganisms Study number 14115 I, the undersigned, hereby declare that this report provides a correct English translation of the Final Report (Study No.14115, issued on September 18, 2003, amended on October 10, 2003). Date Study Director Pece//1.1 e r , l e v 0cZUPP&ii Yasuko Matsunobu 000066 14115 GLP STATEMENT Kurume Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor DAKIN INDUSTRIES, LID. Title Biodegradation study of ( CBl ) by microorganisms Study number 14115 This study was performed in compliance with "OECD Principles o f Good Laboratory Practice" (November 26,1997). This final report reflects the raw data accurately and it has been confirmed that the test data are validity Date Study Director September 18, 2003 Signed in original Yasuko Matsunobu 000067 14115 GLP STATEMENT Kururne Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor DAIKIN INDUSTRIES, LTD Title Biodegradation study of ( CBl ) by microorganisms Study number 14115 Amendment to the Final Report was performed in compliance with "OECD Principles of Good Labor atory Practice" (November 26,1997) This GLP statement was issued as supplement to die statement issued on September 18, 2003 because of the amendment of the Final Report. Date Study Director October 10,2003 Signed in original Yasuko Matsunobu 000068 14115 QUALITY ASSURANCE STATEMENT Kumme Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor DAKIN INDUSTRIES, LTD. Title Biodegradation study of ( CBl ) by microorganisms Study number 14115 The inspections of this study were carried out and the results were reported to the test facility management and the Study Director by Quality Assurance Unit of Kurume Laboratory, Chemicals Evaluation and Research Institute, Japan as follows Item of inspection Study plan Test Conduct Raw Data and Final Report Date of inspection July 17,2003 August 11,2003 September 10,2003 July 18, 2003 August 1,2003 August 15, 2003 August 18,2003 August 19,2003 August 21,2003 August 26,2003 September 18,2003 Date of report to Study Director July 18,2003 August 11,2003 September 10,2003 July 18,2003 August 27,2003 August 27,2003 August 27,2003 August 27,2003 August 27,2003 August 27,2003 September 18,2003 Date of report to test facility management July 18,2003 August 11,2003 September 10,2003 July 18,2003 August 27,2003 August 27,2003 August 27,2003 August 27,2003 August 27,2003 August 27,2003 September 18,2003 It has been assured that the final report describes accurately the test method used, that details in the report are in compliance with the study plan and Standard Operating Procedures and that the final report reflects the raw data accurately Date September 18, 2003 Quality Assurance Unit, Head Signed in original Kyoshiro Hoii 000069 14115 QUALITY ASSURANCE STATEMENT Kuiume Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor DAIKIN INDUSTRIES, LTD Title Biodegradation study of ( CBi ) by microorganisms Study number 14115 Study inspection of the corrected parts in the Final Report was carried out and it was confirmed that the correction has no problem. The result was reported to the test facility management and the Study Director as follows. Date o f inspection October 10,2003 Date of report to Study Director October 10,2003 Date o f report to test facility management October 10,2003 This statement was issued as a supplement to the quality assurance statement issued on September 18, 2003 . Date Quality Assurance Unit, Head October 10,2003 Signed in original Kyoshiro Hori 000070 CONTENTS Title Sponsor Test facility Objective Test method Applied GLP Dates Stoiage of test item, raw data, etc. Peisonnel Approval of final report SUMMARY 1. Test item 2 Item supplied by sponsor 3 Activated sludge 4. Performance of biodegradation test 5 Validity of test conditions 6 Factors possibly affecting accuracy 7. Results 8. Remarks 14115 page 1 l 1 1 1 1 2 2 3 3 4 5 6 7 9 21 21 21 24 000071 14115 Contents of tables and figures Contents of tables Table-1 Calculation table for percentage biodegradation by BOD Table-2 Calculation table for amount of dissolved organic catbon by TOC Table-3 Calculation table for percentage biodegradation by weight measurement Table-4 Calculation table for concentration of peifluorooctanoic acid by LC-MS Table-5 Calculation table for recovery rate by GC-MS ( 2-(perfluorooctyl)ethanol) Table-6 Calculation table for concentration of 2-(perfluorooctyl)ethanol by GC-MS Contents of figures Fig. 1 Chart o f BOD Fig- 2 IR spectrum of residue Fig.. 3 Mass fiagmentogram of LC-MS analysis for calibration curve and calibration curve ( perfluorooctanoic acid) Fig 4 Mass fiagmentogram o f GC-MS analysis for calibration curve and calibration curve ( 2-(perfluorooctyl)ethanol) Fig. 5 Mass fiagmentogram of GC-MS analysis for recovery and blank test ( analysis of 2-(perfluoiooctyl)ethanol) Fig 6 Mass fiagmentogram of LC-MS analysis for test solution ( perfluorooctanoic acid ) Fig 7 Mass fiagmentogram of GC-MS analysis for test solution ( 2-(perfluorooctyl)ethanol) Fig. 8 Mass spectrum of perfluorooctanoic acid Fig 9 Mass spectrum of 2-(perfluorooctyI)ethanol Fig. 10-1 IR spectrum of test item measured before experimental start Fig. 10-2 IR spectrum of test item measured after experimental completion Reference IR spectrum supplied by sponsor 000072 14115 Study No . 14115 Title Biodegradation study of ( cbi ) by microorganisms Sponsor DAIKIN INDUSTRIES, LTD 1-1 Nishi-hitotsuya, Settsu, Osaka 566-8585, Japan Test facility Kurume Laboratory Chemicals Evaluation and Research Institute, Japan 19-14 Chuomachi, Kurame, Fukuoka 830-0023, Japan Objective This study was performed to evaluate the biodegiadability of ( cbi ) by microorganisms Test method This study was performed according to "Ready Biodegradability: Modified MITI Test (I) (Guideline 301C, Revised July 17, 1992)" in the OECD Guideline for Testing of Chemicals Applied GLP This study was performed in compliance with "OECD Principles o f Good Laboratory Practice" (November 26,1997) -1 - 000073 14115 Dates Study initiation date Expeiimental starting date Experimental completion date Study completion date July 17,2003 July 18,2003 August 15, 2003 September 18,2003 Stoiage of test item, taw data, etc (1) Test item About 5 g of the item supplied by the sponsor is sealed in a store vessel and stored in archives in this laboratory for ten years after the publication of the final report Treatment of the item supplied by the sponsor after the storage period is discussed with sponsor, If it is not stable for the storage period, it is stored while it is kept stable and it is disposed with approval of sponsor (2) Raw data and materials, etc . Raw data, the study plan, documents about the study presented by the sponsor , the final report and necessary materials are stored in archives in this laboratory for the same term as the test item. Treatment of raw data and materials, etc. after the storage period is discussed with sponsor. -2- 000074 Personnel Study Director Study personnel (Operation of biodegradation test) Staff for cultivation of activated sludge Approval of final report Study Director Date Signature 14115 Yasuko Matsunobu Kazuhiio Oyama Takakazu Kayashima Hiroto Nishijima September 18,2003 Signed in original Yasuko Matsunobu -3 - 000075 14115 SUMMARY Title Biodegradation study of ( cbi )by microorganisms Conditions of cultivation (1) Concentration of test item (2) Concentration of activated sludge (as the concentration of suspended solid) (3) Volume of test solution (4) Cultivation temperature (5) Cultivation duration lOOmg/L 30 mg/L 300 mL 251 C 28 days Measurement and analysis for percentage biodegradation (1) Measurement o f biochemical oxygen demand (BOD) by means of a closed system oxygen consumption measuring apparatus (2) Determination of test item by means of weight measurement Other measurement and analysis (1) Determination of dissolved organic carbon by means of total organic carbon (TOC) analysis (2) Measurement of IR spectrum by means of a fouiier transform infrared spectrophotometer (3) Determination of perfluorooctanoic acid by means of liquid chromatography-mass spectrometry (4) Determination of 2-(perfluorooctyl)ethanol by means o f gas chromatography-mass spectrometry Results (1) Percentage biodegradation by BOD 0 %, 0 %, 0 % average 0 % (2) Percentage biodegradation by weight measurement 0% , 0 %, 0 % average 0 % Conclusion The test item was not biodegraded by microorganisms under the present test conditions. -4 - 000076 1 Test item In this report, ( cbi ) has the following chemical name, etc 1 1 Chemical name*1 ( cbi 1 2 Chemical structure, etc *' Structural formula 14115 ) Molecular weight Weight-average ( cbi ) *1 Information supplied by the sponsor -5 - 000077 14115 2 Item supplied by sponsor 2.1 Supplier and lot number*1 (1) Supplier (2) Lot number DAKIN INDUSTRIES, LTD. ( cbi ) 2 2 Purity*1 (1) Test item (2) Impurity ( CBI ( 1 cbi ) The test item was treated as 100 % in purity 2.3 Confirmation of test item Two infrared (IR) spectra of the test item provided by the sponsor and measured at this laboratory were confirmed to be identical (see Fig. 10 and Reference) 2 4 Physicochemical property*1 Appearance ( cbi ) *1 Information supplied by the sponsor 2 5 Storage and stability (1) Storage condition Dark storage place at room temperature (2) Stability The test item was stable under the storage conditions, as shown by the finding that IR spectra of the test item before the experimental start and after the experimental completion were identical (see Fig .10) . -6- 000078 14115 3 Activated sludge 3 1 Sludge sampling sites and date (1) Sampling sites On-site sludge sampling was canied out at the following 10 locations in Japan. Fushikogawa city sewage plant (Sapporo-shi, Hokkaido) Fukashiba industrial sewage plant (Kashima-gun, Ibaiaki) Nakahama city sewage plant (Osaka-shi, Osaka) Ochiai city sewage plant (Shinjuku-ku, Tokyo) Kitakami River (Ishinomaki-shi, Miyagi) Shinano River- (Niigata-shi, Niigata) Yoshino River (Tokushima-shi, Tokushima) Lake Biwa (Otsu-shi, Shiga) Hiroshima Bay (Hiroshima-shi, Hiroshima) Dookai Bay (Kitakyushu-shi, Fukuoka) (2) Date June, 2003 3.2 Sludge sampling (1) City sewage Return sludges from sewage plants were collected (2) Rivers, lake and sea Surface water and surface soil which was in contact with the atmosphere were collected. 3 3 Preparation of activated sludge Activated sludge was prepared as follows to maintain its uniformity . The filtrate (5 L) of the supernatant of the activated sludge*2 cultivated about for 3 months was mixed with the mixed filtrate (5 L) of the supernatant of a sludge collected newly at each location The mixed filtrate (10 L) was aerated*3 after the pH value of the mixture was adjusted to 7 01 0 *2 The activated sludge cultivated the mixed filtrate (10 L) of the supernatant of sludge collected at the ten locations *3 Prefiltered open air was used -7 - 000079 14115 3 4 Cultivation Roughly 30 minutes after ceasing aeration of the sludge mixture, supernatant corresponding to about 1/3 of the whole volume was removed. Dechlorinated water was added to the remaining portion sb that the total volume reached 10 L. This mixture was aerated, and then a predetermined amount of synthetic sewage*4 was added to the mixture so that the concentration of the synthetic sewage was 0.1 wt% in the volume of dechlorinated water added. This procedure was repeated once every day. Cultivation was carried out at 252 C *4 Synthetic sewage Glucose, peptone and potassium dihydrogenphosphate were dissolved in purified water to obtain 50 g/L of the solution for each component The pH of the solution was adjusted to 7 01.0 with sodium hydroxide 3 5 Control and use During cultivation, the appearance of the supernatant, sedimentation of the sludge, formation of flock, pH, dissolved oxygen concentration in the solution and temperature were checked to maintain a normal state of sludge. It was confirmed that these were within the scope of the control standard stipulated in the "Testing Methods for New Chemical Substances", and these results were stored as raw data. Microflora in the activated sludge was microscopically observed and sludge with no abnormal symptoms was used for the test 3 6 Inspection of activity and date of initiation of use of activated sludge (1) Inspection of activity Activity o f the sludge was assessed using a reference item2 (2) Date o f initiation of use July 15,2003 -8- 000080 14115 4 Perfoimance of biodegiadationtBSt 4 1 Preparations for test (1) Measurement of concentration of suspended solid The concentration of suspended solid was measured to determine the amount of activated sludge to add Method Date Result In accordance with Japanese Industrial Standards (JIS) K 0102-1998 section 14.1 July 15,2003 Concentration of suspended solid in the activated sludge was 3200 mg/L. (2) Preparation of basal culture medium Each 3 mL o f solutions (a), (b), (c) and (d) shown below were made up to 1L with purified water The following stock solutions were prepared by use of analytical grade regents: (a) Potassium dihydrogenphosphate, KH2PO4 8 50 g Dipotassium hydrogenphosphate, K2HPO4 21 75 g Disodium hydrogenphosphate dodecahydrate, Na2HP04 12H2O Ammonium chloride, NH4CI 1.70 g Dissolved in water and filled up to 1 L 44 60 g (b) Magnesium sulphate heptahydrate, MgSCU7H2O 22.50 g Dissolved in water and filled up to 1 L (c) Calcium chloride anhydrous, CaCk 27.50 g Dissolved in water and filled up to 1 L (d) Iron(III) chloride hexahydrate, FeC13 *6H2O 0.25 g Dissolved in water and filled up to 1 L3 (3) Reference item Aniline (reagent grade, Showa Chemicals Inc Lot No. HO-2729D) was used as a reference item to confirm that the sludge was sufficiently active This test is effective when the percentage biodegradation calculated from the BOD of aniline after 7 and 14 days exceeds 40 % and 65 %, respectively, according to regulation of the OECD Guideline for Testing of Chemicals -9 - 000081 14115 4.2 Preparation of test solutions Test solutions for analysis of the test item and peifluorooctanoic acid and test solutions foi analysis of 2-(perfluorooctyl)ethanol were prepared, because the test item and converted products (perfluorboctanoic acid and 2-(perfluorooctyl)ethanol), which was expected to form in test solutions, could not be analyzed by the same pretreatment Eleven test vessels were prepared The following test solutions were prepared and cultured under the conditions described in section 4 3 (1) Addition of test item or aniline (a) Test solution (water + test item) (n=2: Vessel No 6 and a test solution for analysis of 2-(perfluorooctyl)ethanol) In one test vessel, 30 mg of the item supplied by the sponsor was accurately weighed and added to 300 mL of purified water, so that the concentration of the test item reached 100 mg/L (b) Test solution (sludge + test item) (n=6: Vessel No 1, 2, 3 and three of test solutions for analysis of 2-(perfluorooctyl)ethanol) In each test vessel, 30 mg of the item supplied by the sponsor was accurately weighed and added to the basal culture medium (the volume was less than 300 mL by the volume (2.81 mL) of activated sludge inoculated), so that the concentration of the test item reached 100 mg/L (c) Test solution (sludge + aniline) (n-1 : Vessel No 4) In one test vessel, 29.5 pL [30 mg = 29 5 pL * l 022 g/cm3 (density)] of aniline was added into the basal culture medium (the volume was less than 300 mL by the volume (2 .81 mL) of activated sludge inoculated), so that the concentration reached 100 mg/L (d) Test solution (control blank) (n=2: Vessel No.5 and a test solution for analysis of 2-(perfluorooctyl)ethanol) In one test vessel, nothing was added to the basal culture medium (the volume was less than 300 mL by the volume (2 81 mL) of activated sludge inoculated)2* (2) Inoculation of activated sludge The activated sludge cultivated under the conditions described in section 3 was added to each test vessel, (b), (c) and (d), so that the concentration of the suspended solid reached 30 mg/L. -10- 0000S2 14115 4.3 Instruments and conditions of cultivation (1) Instruments for cultivation (a) Test solution for analysis of test item and perfluorooctanoic acid Closed system oxygen consumption measuring apparatus Temperature controlled bath and measuring u n it: Ohkura Electric Co , Ltd. Data sampler: Asahi Techneion C o , Ltd. Vessel 300 mL in volume (improved type) Absorbent for carbon dioxide Soda lime No 1 (for absorption o f carbon dioxide, Wako Pure Chemical Industries, Ltd.) (b) Test solution for analysis of 2-(perfluorooctyl)ethanol Closed system oxygen consumption measuring apparatus Temperature controlled bath and measuring unit (Only the temperature controlled bath was used) : Ohkura Electric Co , Ltd. Vessel 300 mL in volume (sealed improved type) (2) Conditions of cultivation Cultivation temperature Cultivation duration Stirring method 251 C 28 days Each test solution was stirred by a magnetic stirrer. (3) Room Apparatus room No ,511 4 4 Observation and measurement of test conditions (1) Observation of test solution During the cultivation period, the appearance of the test solution was observed periodically and conditions of the instruments were checked properly.2 (2) Measurement of biochemical oxygen demand (BOD) During the cultivation period, the change in BOD of the test solutions was measured by autorecording using a data sampler Cultivation temperature was measured and recorded once a day BOD of the test solutions for analysis of 2-(perfluorooctyl)ethanol was not measured - 11 - 0000S3 14115 4 5 Analysis of test solution After the teimination of the cultivation, dissolved organic carbon, the test item and converted products (perfluorooctanoic acid and 2-(perfluorooctyl)ethanoI), which was expected production in test solutions, were determined 4.5.1 Pretreatment of test solutions for analysis (1) Pretreatment for analysis o f test item and perfluorooctanoic acid After the termination of the cultivation, the test solution (water + test item), the test solutions (sludge + test item) and the test solution (control blank) for analysis of the test item and perfluorooctanoic acid were pretreated for total organic carbon (TOC) analysis on dissolved organic carbon and liquid chromatography - mass spectrometry (LC-MS) analysis of perfluorooctanoic acid as follows. Weight measurement of the test item was performed. IR spectrum of the residue was measured. Test solution 300 mL Taking out 10 mL (measuring pipet) Centrifugation (1000 x g, 10 min) Supernatant ~[ Residue Taking out 2 .5 mL (transfer pipet) Filling up to 5 mL (methanol, Filtration (The test solutions were filtered by use of membrane filter*5 with suction The residue in the vessels was transferred on the filter and washed with purified water ) Sample for TOC analysis Sample for LC-MS analysis Determination of perfluorooctanoic acid Residue Filtrate Drying (vacuum oven, 40 C, 137 his.) Cooling (desiccator, room temperature, about 30 min) Sample for weight measurement MELLIPORE type HA As( n A r a P i 7 f l f l x Sample for measurement ofTR spectrum - 12- 0000S4 14115 (2) Pretreatment for analysis of 2-(perfluorooctyl)ethanol After the termination of the cultivation, the test solution (water + test item), the test solutions (sludge + test item) and the test solution (control blank) for analysis of 2-(perfhiorooctyl)ethanol were cooled at 5 C for 66 his and preheated for gas chromatography - mass spectrometry (GC-MS) analysis as follows. Test solution 300 mL --Ammonium sulfate 200 g (even balance) --Chloroform 90 mL (graduated cylinder) Shaking (10 min) Filtration and dehydration (No 2 filter paper, sodium sulfate 30 g) Filling up to 200 mL (chloroform, volumetric flask) Sample for GC-MS analysis -13- 000085 14115 4.5.2 Quantitative and qualitative analysis (1) Determination of dissolved organic carbon by means of TOC analysis The dissolved organic carbon (DOC) in the samples for TOC analysis was analyzed under the following conditions. The concentration of DOC was calculated by subtracting concentration of the inorganic carbon (IC) from concentration of the total carbon (TC). The concentration of TC and IC in the test solutions was proportionally calculated from the peak area of the test solution by comparison with that of 80.0 mgC/L of a standard solution for TC analysis and 80.0 mgC/L of a standard solution for IC analysis, respectively (see Table-2). The standard solution for TC analysis was prepared by dissolving potassium hydrogenphthalate in purified water. The standard solution for IC analysis was prepared by dissolving sodium hydrogencarbonate and sodium carbonate in purified water. The concentration of dissolved organic carbon corresponding to the minimum determination limit was regarded as 1.0 mgC/L. Analytical conditions Instrument Temperature of iumace Flow rate Injection volume Sensitivity Total organic carbon analyzer Shimadzu Corporation type TOC-5000 680 C 150 mL/min 33 pL Range 5 (2) Determination of test item by means of weight measurement Weight of the samples for weight measurement was measured under the following conditions (see Table-3). Analytical conditions Instrument Electronic analytical balance Mettler Toledo type AT201 Minimum measuring weight 0.1 mg (3) Measurement of IR spectrum by means of fourier transform infrared spectrophotometer IR spectrum of the samples for measurement of IR spectrum was measured under the following conditions (see Fig.2). Analytical conditions Instrument Measuring method Fourier transform infrared spectrophotometer Shimadzu Corporation type FTIR-8200PC KBr tablet - 14- 0000S6 14115 (4) Determination of peifluorooctanoic acid by means of LC-MS The samples for LC-MS analysis were analyzed for peifluorooctanoic acid under the following conditions. The concentration of peifluorooctanoic acid in the sample for LC-MS analysis was proportionally calculated by comparing the peak area on the mass fiagmentogram of the sample for LC-MS analysis with that on the mass fiagmentogram of 0.500 mg/L standard solution (see Table-4 and Fig.6) The lowest detectable peak area was regarded as 2000 considering the noise level, which corresponded to 0.0097 mg/L of peifluorooctanoic acid. (a) Analytical conditions Instrument LC MS Liquid chromatograph-mass spectrometer JASCO Corporation type PU-980 Micromass type Quattro II Conditions of LC Column Eluent Flow rate Sample size L-column ODS 15 cm x 4.6 mm I..D. Methanol'6/ purified water'6(80/20 V/V) 1 OmL/min 10 pL Conditions of MS Ionization method Detection mode Monitoring method Monitoring m/z Source temperature Cone voltage Electrospray (ESI) Negative ion Selected ion monitoring (SIM) 413 (see Fig 8) 150 C 20 V *6 Containing 0 1 % formic acid -15- 000087 14115 (b) Preparation of standard solution The standard solution to determine the concentration of perfluorooctanoic acid in the sample for LC-MS analysis was prepared as follows. 100 mg of the standard substance*7 supplied by the sponsor was accurately weighed and dissolved in methanol to obtain 2000 mg/L solution of perfluorooctanoic acid. 0 500 mg/L standard solution was then prepared fiom this solution by dilution with methanol / purified water (1/1 V/V) *7 Name, lot number and purity Name Perfluorooctanoic acid (C-l700) Lot number C17001401 Purity Perfluorooctanoic acid was treated as 100 % in purity (c) Calibration curve 0 125, 0 250 and 0.500 mg/L standard solutions were prepared by the same method as described in (b) These solutions were analyzed according to the analytical conditions described in (a) A calibration curve was drawn based on the relation between the peak area on the mass fiagmentogtams and the respective concentrations (see Fig 3) , -16- 000088 14115 (5) Deteimination of 2-(perfluorooctyl)ethanol by means of GC-MS The samples for GC-MS analysis were analyzed foi 2-(perfluorooctyl)ethanol under the following conditions. The concentration of 2-(peifluorooctyl)ethanol in the sample for GC-MS analysis was pioportionally calculated by comparing the peak area on the mass fiagmentogiam of the sample for GC-MS analysis with that on the mass fiagmentogiam of 0 500 mg/L standard solution (see Table-6 and Fig .7). The lowest detectable peak area was regarded as 300 considering the noise level, which corresponded to 0 021 mg/L of 2-(perfluorooctyl)ethanol. (a) Analytical conditions Instrument Gas chromatograph-mass spectrometer Shimadzu Corporation type QP-5000 Conditions of GC Column Column temperature Carrier gas Pressure of carrier gas Injection temperature Sample size Sampling method Sampling time HP-FFAP (Agilent) 25 m x 0 32 mm I D , made of fused silica 50 C (4 min)--170 C (Rate 30 C/min) Hetium 10.0 kPa 200 C 2 pL Splitless 2.0 min Conditions of MS Ionization method Detection mode Monitoring method Monitoring m/z Interface temperature Electron ionization (El) Positive ion Selected ion monitoring (SIM) 95 (see Fig.9) 230 C -17- 0000S9 14115 (b) Preparation of standard solution The standard solution to deteimine the concentration of 2-(perfluorooctyl)- ethanol in the sample for GC-MS analysis was prepared as follows 100 mg of the standard substance*8 supplied by the sponsor was taken out accurately and dissolved in chloroform to obtain 2000 mg/L solution of 2-(perfluorooctyl)ethanol. 1.50 mg/L standard solution was then prepared from this solution by dilution with chloroform *8 Name, lot number and purity Name 2-(peifluorooctyi)ethanol (A-1280) Lot number A18201X01 Purity 2-(perfluorooctyl)ethanol was treated as 100 % in purity (c) Calibration curve 0 375, 0 750 and 1.50 mg/L standard solutions were prepared by the same method as described in (b) These solutions were analyzed according to the analytical conditions described in (a) A calibration curve was drawn based on the relation between the peak area on the mass fiagmentograms and the respective concentrations (see Fig.4), - 18- 000090 14115 4 5 3 Recovery test and blank test Each two test solutions (water + 2-(perfluorooctyl)ethanol) and two test solutions (sludge + 2-(perfluorooctyl)ethanol) for recovery test were prepared according to the methods described in section 4 2 0 300 mg of 2-(perfIuorooctyl)ethanol was added to each test vessel, so that the concentration of it reached 1 mg/L . The test solutions were pretreated in accordance with the method described in section 4 5.1(2), then analyzed according to analytical conditions described in section 4 5 2(5). A test solution for blank test was prepared according to the method described in section 4 2 The test solution for blank test was analyzed in the same way as the recovery test. As for the blank test, no peaks were appeared around the peak of the 2-(perfluorooctyl)ethanol on the mass fiagmentogram. Two individual recovery rates and their averages on the analytical procedure are shown below. The average recovery rates were used as correction factor, for the determination of 2-(perfluorooctyl)ethanol in the test solutions (see Table-5 and Fig.5) . Recovery rate of the test solutions (water + 2-(perfluorooctyl)ethanol) 99.6%, 9 8 2 % average 98.9% Recovery rate of the test solutions (sludge + 2-(perfluorooctyl)ethanoI) 80.5%, 81.5% average 81.0% -19- Corrected page 000091 14115 4 6 Calculation of percentage biodegradation The percentage biodegradation was calculated by the following equations and expressed in whole numbers. (1) Percentage biodegradation by BOD B O D -B Percentage biodegradation (%) = TOD'45' x 100 BOD B TOD*9 : Biochemical oxygen demand in the test solution (sludge + test item) (experimental) (mg) : Biochemical oxygen demand in the control blank (experimental) (mg) Theoretical oxygen demand required when the test item was completely oxidized (theoretical) (mg) *9 The purity was regarded as 100 % and TOD was calculated from composition formula ( CBl ) which was calculated by use of n=8, monomer values of (CBl) regarded as mol% in section 12 (2) Percentage biodegradation by weight measurement Sw - Ss Percentage biodegradation (%) = Sw x 100 Ss : Residual amount of the test item in the test solution (sludge + test item) (experimental) (mg) Sw Residual amount of the test item in the test solution (water + test item) (experimental) (mg) 4 7 Treatment of numerical values Values were rounded off in accordance with JIS Z 8401:1999 rule B -20- 000092 14115 5. Validity of test conditions Percentage biodegradations of aniline calculated by the BOD values were 65 % and 69 % after 7 and 14 days, respectively . It was concluded that this test conditions were valid (see Table-1 and Fig.l). 6 Factors possibly affecting accuracy No adverse effects on the reliability o f this test were noted. 7. Results 7 1 Appearances of test solutions Appearances of test media in cultivation vessels were as follows Test solution Appearance At the start of cultivation Water + test item The test item was not dissolved Sludge + test item The test item was not dissolved. PH - At the termination of cultivation Water + test item Sludge + test item Insoluble compound was observed. Insoluble compound except for the sludge was observed Growth of the sludge was not observed. - -21 - 000093 14115 7 2 Analytic results of test solutions Analytic results of the test solution after 28 days were as follows Water + test item Sludge + test item Theoretical amount Vessel-6 Vessel-1 Vessel-2 Vessel-3 Table Fig. BOD*10 mg 0 02 0 0 ( CBI ) 1 1 no mgC 0 0 0 0 ( CBI ) Detection amount and percentage 2- detection of DOC % 0 0 0 0 - Residual amount and percentage residue of test item (Weight measurement) ni mg % 28 2 94 Concentration of perfluorooctanoic mgL 2*0 019 acid (LC-MS) 301 100 ^0.019 308 103 ^0.019 292 97 ^0.019 300 - - 346 Water + test item *12 Concentration o f 2-(perfluorooctyl) mgL ia 0.014 ethanol (GC-MS) Sludge + test item 12 3 2*0.017 ^0.017 ^0.017 Theoretical Table amount -6 Fig 7 *10 The value of control blank was subtracted fiora the values of the test solutions (sludge + test item) *11 Minimum determination limit of perfluorooctanoic acid in test solution = (Minimum determination limit in sample for LC-MS analysis) x {(final volume) / (volume of test solution)} / (ratio of portion used for analysis) = 0.0097 mg/L x (5 mL / 300 tnL) / (2.5/300) = 0 019 mg/L *12 Minimum determination limit of 2-(perfluorooctyl)ethanol in test solution = (Minimum determination limit in sample for GC-MS analysis)x {(final volume) / (volume of test solution)} / {(average recovery rate) / 100} / (ratio of portion used for analysis) Test solution (water + test item) : 0 021 mg/L x (200 mL / 300 mL) / (98.9/100) / 1 = 0 014 mg/L Test solution (sludge + test item) : 0 021 mg/L x (200 mL / 300 mL) / (81.0/100) / 1 = 0,017 mg/L -22- 000094 14115 7.3 Percentage biodegradation Percentage biodegradations after 28 days were as follows Method Percentage biodegradation (%) Vessel-1 Vessel-2 Vessel-3 Average BOD 000 0 Weight measur ement 0 0 0 0 Table 1 3 7.4 Results of measurement of DRspectrum No significant difference between IR spectrum of the test item and that of the residue after weight measurement was observed (see Figs.,2,10). 7.5 Discussion Determination of the test item by chromatography was difficult, because the test item was not completely dissolved in water and organic solvents (tetrahydrofuran and chroloform). Therefore, the test item was determined by means of weight measurement As a result, the percentage residues of the test item were 94 % in the test solution (water + test item) and 100, 103 and 97 % in the test solutions (sludge + test item). The percentage detections of DOC were 0 % in the test solution (water + test item) and 0 % in all test solutions (sludge + test item) These results indicate that the test item was not dissolved in the test solution. No significant difference between IR spectra of the test item before and after the cultivation was observed. Converted products (perfluorooctanoic acid and 2-(perfluorooctyl)ethanoI), which was expected to form in test solutions, was not detected. These results indicate that the test item was not converted during the cultivation It is considered that the test item was not biodegraded during microorganisms, because the average percentage biodegradation by BOD was 0 % and no significant change in the weight of the test item before and after the cultivation was observed. 7 6 Conclusion The test item was not biodegraded by microorganisms under the present test conditions. -23 - 000095 14115 8. Remarks 8.1 Instruments used for test Fourier transform infrared spectrophotometer : Shimadzu Corporation type FTIR-8200PC Closed system oxygen consumption measuring apparatus : see page 11 Total organic carbon analyzer : see page 14 Liquid chromatograph- mass spectrometer : see page 15 Gass chromatograph- mass spectrometer : see page 17 Electronic analytical balance : Mettler Toledo type AT201 Sartorius AG type CP324S Sartorius AG type BP21OS Refrigerated centrifuge : Shimadzu Corporation type CST-060LF Mechanical shaker : TIETECH Co., Ltd. type SR-2w Vaccume oven : SIBATA SCIENTIFIC TECHNOLOGY LTD. type VS-300 8.2 Reagents used for analysis Methanol (HPLC grade) : Wako Pure Chemical Industries, Ltd. Purified water : Takasugi Seiyaku Co., Ltd. Chloroform (reagent grade) : Kishida Chemical Co., Ltd. Ammonium sulfate (reagent grade) : Kanto Chemical Co., Inc. Sodium sulfate (reagent grade) : Kanto Chemical Co., Inc. Formic acid (reagent grade) : Wako Pure Chemical Industries, Ltd. Sodium hydrogencarbonate (reagent grade) : Wako Pure Chemical Industries, Ltd. Sodium carbonate (reagent grade) : Wako Pure Chemical Industries, Ltd. Potassium hydrogenphthalate (reagent grade) : Wako Pure Chemical Industries, Ltd. Potassium bromide (for measurement of infrared absorption) : NACALAI TESQUE, INC. Perfluorooctanoic acid : Standard substance supplied by the sponsor 2-(perfluorooctyl)ethanol : Standard substance supplied by the sponsor -24- 000096 Table 1 Calculation table for the percentage biodegradation by BOD Test No. 14115 Cultivating Duration: 28 days Vessel No. S SI m m m m 7th day BOD Deg. (mg) (9 0 61.9 65 2.9 - 2.8 0 2.1 0 3.1 0 0..0 - 14th day BOD Deg.. (mg) (9 0 67.0 69 4.8 - 4.4 0 3.6 0 4.7 0 0.0 - 21st day BOD Deg.. (mg) (9 0 68.9 70 6.0 - 5.8 0 5.4 0 5.9 0 0.0 - 28th day BOD Deg.. (mg) (9 0 Mean Deg. % 70 0 70 7.2 - 7.4 0 0 7,0 0 7.1 0 0.0 - Deg. : Percentage biodegradation Vessel no. 3] : Sludge + Aniline Vessel no. ED :Control blank [B] Vessel no.QQ 3 @1 : Sludge + Test substance Vessel no. El :Water + Test substance Test substance of 30 mg was added.. Chart of BOD : Fig. 1 Deg. = [ BOD - B ] / [ TOD ] x 100 (%) TOD of test substance : ( cbi ) (mg) TOD - 30.0 x ( cbi ) = ( CBI ) (mg) TOD of aniline : 90.3 (mg) CeHrN + 8.75 02 - 6 C0a + 3.5 HaO + NOa 8.75 Oa / CeHrN - 279.99 / 93.13 3.01. TOD = 30.0 x 3.01 = 90.3 (mg) 2003.08.15 Name k. _^ 000097 Table-2 Calculation table for amount o f dissolved organic carbon by TOC Sample description StudvNo. 14115 A B CD Water blank [6) Water + test item [1] Sludge + test item [2] Sludge + test item [3] Sludge + test item [5] Control blank n.d. n.d. n.d. 1.01 1 02 1.14 0 0 0 0 00 0 00 0 Amount o f test item added: 30 (mg) Volume of test solution: 300 (mL) Theoretical amount o f carbon :(CBI) (mgC) (30) ( CBI A : Measured value (mgC/L) B : Amount of DOC Bw = (A(W ater + test item) - A(Water blank)) x 300 /1 0 0 0 (mgC) Bs " (A(Sludge + test item) - A(Control blank)) x 300 /1 0 0 0 (mgC) C : Percentage detection (%) C * B / (Theoretical amount o f carbon) * 100 (%) D : Average percentage detection (%) ) September 3,2003 Name 000098 Table-3 Calculation table for percentage biodegradation by weight measurement Sample description Study No. 14115 A B C DE [6 ] Water + test item [1] Sludge + test item [2] Sludge + test item [3] Sludge + test item [5] Control blank 28 2 28.2 94 33.7 30.1 1 0 0 0 34.4 30.8 103 00 32.8 2 9 2 97 0 3.6 Amount o f test item added: 30 (mg) Volume o f test solution: 300 (mL) A : Weight o f residue (mg) B : Residual amount o f test item (mg) Bw * AfWater + test item) Bs - A(Sludge + test item) - A(Control blank) C : Percentage residue (%) C = B / (Amount of test item added) * 100 D : Percentage biodegradation (%) D (Bw - Bs) / Bw x 100 E : Avetage percentage biodegradation (%) September 3,2003 Name 000099 I able-4 Calculation table for concentration of perfluorooctanoic acid by LC-MS Sample description Study No. 14115 A DE Standard solution 0 500mg/L [6 ] Water + test item [1] Sludge + test item [2] Sludge + test item [3] Sludge + test item [5] Control blank 103449 n.d n.d. n.d n.d ad. 0 (^ 0 .0 1 9 ) 0 ( ^ 0 019) 0 ( ^ 0 019) 0 ( g 0 019) - Volume of test solution: 300 (mL) A : Peak area ( - ) B : Final volum e: 5 (mL) C : Ratio o f portion used for analysis: 2 5/300 D : Concentration of perfluorooctanoic acid in test water (mg/L) D - F x ( A / A(Standard)) x ( B / 300) / C Minimum determination lim it: 0 .019 (mg/L) E : Average concentration of perfluorooctanoic acid (mg/L) F : Concentration o f standar d solution: 0 500 (mg/L) See Fig. 6 September 18,2003 Name 000100 Table-5 Calculation table for recovery rate by GC-MS ( 2-(perfluoiooctyl)ethanol ) Sample description AD Standar d solution 1 50mg/L W ater + 2-(perfluorooctyl)etbanol -1 Water + 2-(perfluorooctyl)ethanol -2 Sludge + 2-(perfluorooctyl)ethanol -1 Sludge + 2-{perfluorooctyl)ethanol -2 Control blank 26329 26222 25861 21196 21459 n d. 0.299 0295 0.242 0245 Study No. 14115 EF 9 9 6 98 9 982 80.5 810 81 5 Amount o f 2-(perfluorooctyl)ethanol added: 0 300 (mg) Volume o f test solution: 300 (mL) A : Peak area ( - ) B : Final volume : 200 (mL) C : Ratio o f portion used for analysis: 1 D : Recovery amount (mg) D =*G x ( A / A(Standard)) x ( B / 3 0 0 ) / C E : Recovery rate (%) E - D / 0 3 0 0 (m g )x 100 F : Average recovery rate (%) See Fig. 5 September 11,2003 Name O O O lO l Table-6 Calculation table for concentration o f 2-(peifIuorooctyl)ethanol by GC-MS Sample description Study No. 14115 A EF Standard solution 1 50mg/L Water + test kern Sludge + test item -1 Sludge + test item -2 Sludge + test item -3 Control blank 21672 n.d ad n.d. n..d. n.d. 0 (0.014) 0 ( 0 017) 0 (0.017) 0 (0.017) - Volume o f test solution: 300 (mL) A : Peak area ( - ) B : Final volume : 200 (mL) C : Ratio o f portion used for analysis: 1 D : Recovery rate : 98.9 (%) (Water + test item) 81.0 (%) (Sludge + test item) E : Concentration of 2-(perfluorooctyl)ethanol in test solution (mg/L) E - Q * ( A / A(Standard)) x ( B / 300 ) / ( D /1 0 0 ) / C Minimum determination lim it: 0 014 (mg/L) (Water + test item) : 0 017 (mg/L) (Sludge + test item) F : Average concentration o f 2-(pfluorooctyl)ethanol (mg/L) G : Concentration of standard solution: 1 50 (mg/L) See Fig. 7 September 18,2003 Name 000102 Fig. 1______ Chart of BOD_______________ Test No.. 14115 ( Test substance Apparatus ................ _ Cultivating conditions: Concentration Test substance Reference substance( aniline Activated sludge Temperature Duration ) Note: Regular condition CBI No CM-23 ) 100 (mg/!>) 100 (mg/U) 30 (mg/lD 25 1" C 28days( Jul .1 8 ~ A u g 15,2003) Vessel no. ffi 123 a 33 a S3 Sample description Sludge + Test substance Sludge + Test substance Sludge + Test substance Sludge + Aniline Control blank [B] Water + Test substance 7thday 2.8 2.1 3.1 61.9 2.9 0.0 BOD 14thday 4.4 3.6 4.7 67.0 4.8 0.0 (rag) 21stday 5.8 5.4 5.9 68.9 6.0 0.0 28thday 7.4 7.0 7.1 70.0 7.2 0.0 2003.08.15 Name 000103 Fig,2 -1 IR spectrum of residue. Study No : 14115 Sample : [6] Water + test item Method : KBr tablet Date : August 26,2003 Name : Jc 1/om 000104 Fig 2 -2 IR spectrum of residue. Study No. Sample Method Date Name 14115 [1] Sludge + test item KBr tablet August 26, 2003 k 0y 1/cm 000105 Fig.2 -3 IR spectrum of residue. Study No .: 14115 Sample :[2] Sludge + test item Method :KBr tablet Date :August 26, 2003 Name C 1/cm 000106 Fig 2 -4 IR spectrum of residue. Study No Sample Method Date Name :14115 :[3] Sludge + test item :KBr tablet :August 26, 2003 :A -- ' 1/cm 000107 Fig.2 -5 IR spectrum of residue.. Study No Sample Method Date Name 14115 [5] Control blank KBr tablet August 26, 2003 000108 ?-n- Fig.3 -1 /y ///1 M 'O ) 5>-(S r f : D ^ c c ^ . Mass ffagmentogiam of LC-MS analysis for calibration curve ( perfluorooctanoic acid ).. 000109 Peak area ( - ) 120000 Study No. 14114 60000 0 y *201414x r =* 1 .0 0 Concentration (mg/L) 0.125 0.250 0.500 Peak area (-) 23889 50521 100945 Fig. 3 - 2 Calibration cuive o f LC-MS analysis for perfluorooctanoic acid August 22,2003 Name /f. O O O llO Study No. 14114 Datafile : 14114d063 Sample : Solvent intensity 30000 : 250001 2 0 0 0 0 -| 15000-3 IOOOO-I 50003 Peak position T o-T 1 1-.....>..........1..' "Tl~-T''~l*' '1 1 ' "' ' ' I 1 567 Datafle 14114d064 Sample : Standard solution 0 375mg/L 95.00 A*R) p 7 ?"/"S~ min Peak RT. Peak area 1 6357 6136 Datafile : 14114d065 Sample : Standard solution 0 750mg/L intensity 30000: 25000- 95.00 20000-: 150003 R<n 100003 50003 _________ .___ L______ 0^1 I . I 1J-- rV--'* r - r-- r-- 11 ' ' * --' ' * f.......................J . . , 56 7 min Datafile :14114d066 Sample : Standard solution 1 50mg/L Peak R T Peak area 1 6353 12339 Peak R.T Peak area 1 6 352 25069 Fig.4 -1 min Date : August 7,2003 Name : d/ Mass fragmentogram of GC-MS analysis for calibration curve ( 2-(peifluorooctyl)ethanol ). O O O lll  c ' 'l f- I 1 2'1' /w r Study No. 14114 y =* 1664X r-1.00 Concentration (mg/L) 0.375 0.750 1.50 Peak area (-) 6136 12339 25069 Fig,, 4 - 2 Calibration curve of GC-MS analysis for 2-(peifluorooctyl)ethanol. August 11,2003 Name je. _________ 000112 Datafile : 14114d067 Sample : Standard solution 1 SOmg/L intensity 30000"; Study No. 14114 j tc , ) / / ne'- k 95 00 ~ Peak R T Peak area 1 6353 26329 Datafile : 14U4d068 Sample : Water + teat item-I intensity 30000' ' 95.00 Peak R .I Peak area 1 6 355 26222 Datafile : I4U4d069 Peak R.T. Peak area 1 6357 25861 Date : August 7,2003 Name : jt, d- Fig.5 -1 Mass fragmentogram of GC-MS analysis for recovery and blank test ( analysis of 2-(perfiuorooctyl)ethanoI ). 000113 Datafile : 14114d070 Sample : Sludge + iww iea >1 intensity 30000 ;i 25000-m 20000^ 15000: 10000- 5000 0 Datafile : 14114d07t Sample : Sludge + teatataoi -2 intensity 30000 Study No. 14114 m*fa tri, V . *20 .?r>3e'ft" ? 95.00 Peak R..T. Peak area 1 6358 21196 min 95.00 Peak R.T Peak area 1 6.355 21459 Datafile : 14U4d072 Sample : Control blank Date : August 7,2003 Name : (jyew?*-/ Fig 5 -2 Mass fragmentogram of GC-MS analysis for recovery and blank test ( analysis of 2-{perhioiooctyl)ethanol ).. 000114 Standard solution 0.500mg/L 14115dOQ4 i5-Aug-2003 SIR of 1 Channel ES- Fig 6 - 1 Mass fragmentogram of LC-MS analysis for test solution ( perfluorooctanoic acid ). 000115 /fr r r Fig 6 - 2 Mass fagmentogiam of LC-MS analysis for test solution ( perfluorooctanoic acid ). 000116 Datafile : 14115d003 Sample : Standard solution 1 JOmg/L Study No. 14115 Peak R I Peak area 1 6.290 21672 Datafile :14113d004 Sample : Water + test item Datafile : 14114d005 Sample : Sludge + test item -1 min Date : August 18,2003 Name : Fig-7 - 1 Mass ffagmentogram of GC-MS analysis for test solution ( 2-(perfluorooctyl)ethanol ). 000117 Study No 14115 Datafile : 14115d006 Sample : Sludge + test item -2 intensity ____________________________________________________________95.00 30000 25000: 2000015000- 10000- PeaJt position n.d. 5000- 0-], 5 , I . I I , I ...........I ' ' I I I .......... 67 Datafile :14115d007 Sample : Sludge + test item -3 min Datafile : 14115d008 Sample : Control blank intensity _______________________________________________________ 95.00 30000; 25000: 20000- 1500010000: 5000: Peak position a. d.. '< 1"........ . ' 1 1 1 I 56 1 I 7 min Date : August 18,2003 Name : Fig 7 - 2 Mass fragmentogram of GC-MS analysis for test solution ( 2-(perfluorooctyl)ethanoI ), 000118 p*>cK( n ->tr- tv n r Study No. 14114 Instrument MS : Micromass Quattro II. HPLC : Jasco PU-98Q. AS-950 Sample Pcrfluorooctanoic acid________ HPLC Conditions Column L-column ODS Size 15 cm x 4.6 mm I.D. Eluent Methanol*Vpurified water*1 ( 8/2 V/V) *1 containing 0.1% formic acid Flow rate 1.0 mL/min Sample size 20 uL (Solvent Methanol ) MS Conditions Ionization mode ESI Detection mode Negative Function MS Source conditions ( Capillary 3.0 kV. Cone Mass range (m/z) 100 - 1000 20 V. Temperature 150 C ) Date June 17. 2003 Operator__ Kurume Laboratory, Chemicals Evaluation and Research Institute, Japan Fig 8 - 1 Mass spectrum of peifluorooctanoic acid ( analytical condition ). 000119 6.a t- ^ / vtti' 000120 std 2.0mg/L 141I4d001 x&) n tY '/r fMn-2003 2: Scan ESTIC 1 99e6 Fig 8 - 3 Mass spectrum of peifluorooctanoic acid ( mass chromatogram ).. 000121 t<fra- Study No. 14114 Instrument Shimadzu QP-5000 Sample _____ 2-(perfluorooctyl)athanol___________ GC Conditions Column HP-FFAP ( Fused silica ) Size 25 m x 0.32 mm ID., Film thickness 0.52 u m Column temp.. 50 C ( 3 min) --* 170 C, Temp, rate 30 C/min Carrier gas He Column head Dressure 10.0 kPa Injection temo. 200 C Sample size 1 u L (Solvent Methanol Inlet mode Solitless Sampling time 2.0 min MS Conditions Ionization Mode El Detection Mode Positive Mass range (m/z) 50 - 600 Interface temp 230 C Date June 11. 2003 Operator K . Kurume Laboratory, Chemicals Evaluation and Research Institute, Japan Fig.9 -1 Mass spectrum of 2-(perfluorooctyl)ethanol ( analytical condition ).. 000122 Datafile: 14114d001 Sample: standard solution 10 Omg/L Study No 14114 <> -it"' fct* Peak#:l R.Tlme:5.5(Scsm#:235) MassPeaks:104 BasePeafc95(7043S6) intensity MS spectrum Date: Tune 11,2003 Name: Fig.9 - 2 Mass spectrum of 2-(perfluorooctyl)ethanol ( mass spectrum ) 000123 Datafile: 14114d001 Sample: standard solution 10 Omg/I Study No . 14114 Mass chromatogram t " -!C kty* Date : June 11, 2003 Name : a a Fig.9 - 3 Mass spectrum of 2-(peifluorooctyl)etbanol ( mass chromatogram ) 000124 Fig. 10 -1 IR spectrum of test item measured before experimental start. Study No.: 14115 Sample :Test item Method :KBr tablet Date :July 10. 2003 Name :jc 1/cm 000125 Fig. 10-2 IR spectrum of test item measured after experimental completion. Study No. Sample Method Date Name 14115 Test item KBr tablet August 27, 2003 h& 1/cm 000126 Reference IR spectrum supplied by sponsor 000127 Amendment to Final Report Kmurne Laboratory Chemicals Evaluation and Research Institute, Japan 1 Title Biodegradation study of ( cbi ) by microor ganisms 2 Study number 14115 3 Content 4.5 3 Recovery test and blank test (page 19) 4. Reason Writing error 5 Content of correction " Recovery rate o f the test solutions (water + test item) 99.6%, 98 2 % average 98.9% Recovery rate of the test solutions (sludge + test item) 80 5% , 81.5% average 8 1 0 % " is corrected to " Recovery rate of the test solutions (water + 2-(perfluorooctyl)ethanol) 99 6% , 98.2% average 98 9 % Recovery rate o f the test solutions (sludge + 2-(perfluorooctyl)ethanol) 80 5 %, 81 5 % average 81,0 % ". 6. Approval Date Study Director October 10,2003 Signed in original Yasuko Matsunobu 000128