PROJECT NUMBER: 014510215 PAGE 24 Mixture of PEMB and PE3MB: ASSESSMENT OF READY BIODEGRADABILlTYj CO2 EVOLUTION TEST 

### Table 5 pH Values of the Test Preparations on Day 28 TeslVessel 

### pH Control R1 

### 7.5 Control R2 

### 7.5 Sodium Benzoate 10 mg CII R1 Sodium Benzoate 10 mg ell R2 Test Material 10 mg CII R1 

### 7.6 7.7 7.4 7.4

R1 - R2 

### = Replicates 1 and 2

745PROJECT NUMBER: 014510215 PAGE 25 Mixture of PEMB and PE3MB: ASSESSMENT OF READY BIODEGRADABILITY; CO2 EVOLUTION TEST 

### Table 6 Observations on the Test Preparations Throughout the Test Period

Observations on Test Preparations Test Vessel Day 0 Day6 Day 13 Day 20 Day 27 Slightly cloudy Slightly cloudy Slightty cloudy Slightty cloudy Slightly cloudy Control 

### R1 light brown light brown light brown light brown light brown dispersion dispersion dispersion dispersion dispersion Slightty cloudy Slightly cloudy Slightly cloudy Slightly cloudy Slightly cloudy 

### R2 light brown light brown light brown light brown light brown dispersion dispersion dispersion dispersion dispersion Slightly cloudy Slightly cloudy Slightly cloudy Slightly cloudy Slightly cloudy

light brown light brown light brown light brown light brown Standard Material 

### R1 dispersion, no undissolved dispersion, no undissolved dispersion, no undissolved dispersion, no undissolved dispersion, no undissolved standard standard standard standard standard material visible material visible material visible material visible material visible Slightly cloudy Slightly cloudy Slightly cloudy Slightly cloudy Slightly cloudy

light brown light brown light brown light brown light brown 

### R2 dispersion, no undissolved dispersion, no undissolved dispersion, no undissolved dispersion, no undissolved dispersion, no undissolved standard standard standard standard standard material visible material visible material visible material visible material visible Cloudy Cloudy Cloudy Cloudy Cloudy dispersion with dispersion with dispersion with dispersion with dispersion with Test Material 

### R1 very small globules of test very small globules oftest very small globules oftest very small globules of test very small globules oftest material on material on material on material on material on

surface surface surface surface surface Cloudy Cloudy Cloudy Cloudy Cloudy dispersion with dispersion with dispersion with dispersion with dispersion with 

### R2 very small globules of test very small globules of test very small globules of test very small globules otlest very small globules of test material on material on material on material on material on

surface surface surface surface surface Cloudy Cloudy Cloudy dispersion with dispersion with dispersion with

very small very small very small globules oftest globules oftest globules oftest Toxicity Control material on material on material on 

## - 

# . surface. No surface. No surface. No undissolved undissolved undissolved

standard standard standard material visible material visible material visible

Rl - R2 

## =Replicates 1 and 2

- No observations made due to the toxicity control being terminated after 14 days.

746PROJECT NUMBER: 0145/0215 PAGE 26 Mixture of PEMB and PE3MB: ASSESSMENT OF READY BIODEGRADABILITY; CO2 EVOLUTION TEST 

### Figure 1 Biodegradation Curves

100

90

80

70

&0

c:

### ~ "0 I!! 50 

### ! en '#. 40

30

20

10

0 -"0- Sodium benzoate 

# • Test Material 0 ---6-­ Toxicity Control 

# -----­ 0

# o~ 0

# / 

# / •

6 ol ol


### ..... 

### ...­ ,/ 

# I / / 6 ,/

/ / / 

# I / 

### / / 

### / 

# // •

I 

# • I 

### I



# •**~**

# I I 0 2 4 6 8 10 12 14 16 18 20 22 24 "26 28 TIme (Days)

747PROJECT NUMBER: 014510216 PAGE 27 Mixture of PEMB and PE3MB: ASSESSMENT OF READY BIODEGRADABILITYj C02 EVOLUTION TEST 

### Appendix 1 Culture Medium Solution a KH2P04 8.50 gIl 

### K2H P04 

### 21.75 gIl 

### Na2HP04

### .2H20 33.40 gIl 

### NH4 CI 0.50 gIl 

### pH 

# = 7.4 Solution b CaCI 2 

### 27.50 g/l Solution 

## c MgS04

### .7H2 

### O 22.50 gIl 

### Solution 

### d FeCI 3

### .6H 2

### O 0.25 g/l To 1 litre (final volume) of purified water* was added the following volumes of solutions a-d. 10 rnl of Solution a 1 ml of Solution b 1 ml of Solution C 

### 1 ml of Solution d

• Reverse osmosis purified and deionised water (Elga Optima 15+ or Eiga Purelab Option R-1S BP)



### 748PROJECT NUMBER: 0145/0215 PAGE 28 

### Appendix 2 Statement of GLP Compliance in Accordance with Directive 

### 2004191EC 

# 'Dk) Department**~** ofHealth



### THE DEPARTMENT OFHEALtllOFTHE 

### GOVERNMENT OFTHE UNITED KINGDOM GOOD LABORATORY PRACTICE STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE lDII4i9/EC

TEST FACILITY T1!STTI'PE Harlan"Laboratories Ltd. AnlllytlCaVClinical Chemistry ShardJow Business Park Environmental Fate London Road Environmental Toxicology Shardlow Mutagenicity Derby PbyslCbem DE722GD Toxicology

DATE OTlNSPECT10N 19thAugust ,2008 A generlll inspection for compliance witb the :Principle! of Good Laboratory Practice \\'ascarried our at the above lestfaciJily as partof :the UK GLP Complia,nce Programme. At tbe time of inspection no deviations were found of sufficient magnitude to affect ·the validilyof non-cHoical studies perfonned at these facilities'.

Dr. Andrew J. Gray Head, UK GLP Monlto.rinl Authority

749

# coo I Receipt number 632-07-S-5089 I 

### Stu4Y number 15089



# FINAL REPORT

Biodegradation study ofPE3MB by microorganisms

June 16,2008


### Kurume Laboratory Chemicals Evaluation and Research Institute, Japan

750

### 15089 

# STATEMENT

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan
Sponsor SHOWA DENKO K.K.

Title 

### Biodegradation study ofPE3MB by microorganisms
Study number 

### 15089

I, 

### the undersigned, hereby declare that this report provides a correct English translation of the 

### Final Report (Study No. 15089, issued on June 16,2008).

Date

## ~C'-7 

# 7 J 

### u;o 

# I Study Director 

# /'**I<r~~c/** Takakazu Kayashima

751

### 15089 

# GLP STATEMENT

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan
Sponsor SHOWA DENKO K.K.



### Title Biodegradation study ofPE3MB by microorganisms
Study number 15089

The study described in this report was conducted 

### in compliance with the following GLP principle: "Standard Concerning Testing Facility Relating to New Chemical Substances" (November 21, 2003; No. 1121003, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 17, 2003, No.3, Manufacturing Industries Bureau, Ministry ofEconomy, Trade and Industry; No. 031121004, Environmental Policy Bureau, Ministry ofthe Environment) This final report reflects the raw data accurately and 

### it has been confinncd that the test data are valid.

Date Junc 16, 2008 Study Director Signed in original Takakazu Kayashima



### 752]5089 

# QUALITY ASSURANCE STATEMENT

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor SHOWA DENKO K.K.
Title Biodegradation study ofPE3MB by microorganism.') Study number 15089

I assure that the fmal report accurately describes the test methods and procedures, and that the reported results accurately reflect the raw data ofthe study. The inspections and audits ofthis study were carried out and the results were reported to the Study Director and the Test Facility Management by Quality Assurance Unit as follows. Date ofreport to Item ofinspection I audit Date ofinspection 

### I audit Study Director and Test Facili Mana ement

At the middle ofcultivation April 9, 2008 April 9,2008

____ -

_____..;...2_0_0_8
___ April 28,**200~._** ..___ At the end ofcultivation

___April 24, 2008 1

__ A
......

p_rU 28,**.200~** ____.._.**Apri12~, 200~._____**  April 28, 2008 Raw**~ta** and fmal report draft June 16, 2008 June 16, 2008 Final report June 16, 2008 June 16, 2008 Amendment to study plan At the start ofcultivation

Date June 16,2008 Head ofQuality Assurance Unit Signed 

### in original Keiji Shiraishi 

### 753

### 15089 

# CONTENTS

page Tille 1 Sponsor Test facility 1 

### Objective 1 Test method AppliedGLP 2 Dates 2 

### Storage oftest item, raw data, etc. 2 

### Personnel 3 

### Approval offmal report 3 SUMMARY 4 

### 1. Test item 5 

### 2. Test sample 6 3. Activated sludge 7 

### 4. Perfonnance ofbiodegradation test 9 

### 5. Validity oftest conditions 21 

### 6. Factors that affected reliability oftest 

### 21 7. Results 21 8. Remarks 29 

### Contents oftables and figures Contents oftables Table-l Calculation table for percentage biodegradation by BOD Tablc-2 Calculation table for percentage detection ofDOC Table-3 Calculation table for recovery rate oftest item**Table~4** 

### Calculation table for percentage decrease oftest item**Table~5** 

### Calculation table for percentage production of3MBA Table-6 Calculation table for percentage production ofSBA Tablc-7 Calculation table for percentage production ofpentacrythritol

754

### 15089

Contents offigures Fig. 1 

### Chart ofEOD Fig. 2-] 

### Chromatograms ofHPLC analysis for calibration curvc (test item, recovery test) Fig. 2-2 

### Calibration curve oftest item (recovery tcst) Fig. 2-3 Chromatograms ofHPLC analysis for calibration curve (test item, analysis for test solution) Fig. 2-4 Calibration curve oftest item (analysis for test solution) Fig. 3-1 Chl'Omatograms ofHPLC analysis for calibration curve (3MBA) Fig. 3-2 Calibration curve of3MBA Fig. 4-1 Chromatograms ofHPLC analysis for calibration curve (SBA) Fig. 4-2 Calibration curve ofSBA Fig. 5-1 Chromatograms ofLC-MS analysis for calibration curve (pentaerythritol) Fig. 5-2 

### Calibration curve ofpentaerythritol Fig. 6 Chromatograms ofHPLC analysis for recovery test (test item) Fig. 

### 7 Chromatograms ofHPLC analysis for test solution (test item) Fig. 8 

### Chromatograms ofHPLC analysis for test solution (3MBA and SBA) Fig. 9 

### Chromatograms ofLC-MS analysis for test solution (pentaerythritol) Fig. 10 PDA chromatograms ofLC-MS analysis for test solution (LC-MS sample -2, qualitative analysis ofother converted products, 1 205

- 

### 300run) Fig. 

### It Total ion chromatogrdffis ofLC-MS analysis for test solution (LC-MS sample 

### -2, qualitative analysis ofother converted products) Fig. 12 Mass spectra (LC-MS sample 

### -2, [1] Watcr 

### + test item) Fig. ] 3 Mass chromatograms (LC-MS sample -2, 

## fl1 Water 

### + test item) Fig. 14 Mass spectra (LC-MS sample -2, [2] Sludge 

### + lest item) Fig. 15 Mass chromatograms (LC-MS sample -2, 

## l2J Sludge 

### + test item) Fig. 16 Mass chromatograms (LC-MS sample -2, 

### [51 Control blank) Fig. 

### 17 PDA chromatograms ofLC-MS analysis for test solution (LC-MS sample -3, qualitative analysis ofother converted products, 1 205

- 300run) 

### Fig. 18 

### Total ion chromatograms ofLC-MS analysis for test solution (LC-MS sample -3, qualitative analysis ofother converted products) Fig. 19 UV 

### spectrum oftest item Fig. 20 UV 

### spectrum of3MBA Fig. 21 

### UV 

### spectrum ofSBA Fig. 22 Mass spectrum ofpentaerythritol Fig. 23-1 JR spectrum oftest item measured before experimental start Fig. 23-2 IR spectrum oftest item measured after experimental completion Reference IR spectrum supplied by sponsor



### 75515089

Title Biodegradation study ofPE3MB by microorganisms Sponsor SHOWA DENKO K.K. 1-13-9, ShibaDaimon, Minato-ku, Tokyo 105-8518, Japan

Test facility Kurume Laboratory Chemicals Evaluation and Research Institute, Japan 2-7, 3-chome, Miyanojin, Kurume-shi, Fukuoka 839-0801, Japan Objective This study. was performed to evaluate the biodegradability of PE3MB by microorganisms.

Test method This study was performed according to the following test method. "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chcmical Substances" (November 21, 2003; No. 1121002, Phannaceutical and Food Safety Bureau, Ministry ofHealth, Labour and Welfare; November 13,2003, No.2, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121002, Environmental Policy Bureau, Ministry ofthe Environment)

756 

### -1­

### 15089
Applied GLP This sIDdy was conducted in ,compliance with the following GLP principle: "Standard Concerning Testing Facility Relating to New Chemical Substances" (November 21, 2003; No. 1121003, Phannaceutical and Food Safety Bureau, Ministry ofHealth, Labour and Welfare; November 17,2003, No.3, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121004, Environmental Policy Bureau, Ministry ofthe Environment) Dates Study initiation date March 10, 2008 Experimental starting date March 26, 2008 Experimental completion date April 23,2008 Study completion date June 16,2008 Storage oftest item, raw data, etc. (1) Test item The test sample is sealed in a storage vessel and stored in archives in this laboratory for ten years after the receipt ofnotice specified under Clause I or Clause 2 in Article 4, Clause 2 or Clause 3 or Clause 8 in Article 4-2, and Clause 2 in Article 5-4 or Clause 2 in Article 24 or Clause 2 in Article 25-3 of "Law Concerning Examination and Regulation ofManufacture, ctc. ofChemical Substances". Ifit is not stable for the storage period, it is stored as long while it is kept stable. Treatment ofthe test sample after the storage period will be discussed with sponsor. (2) Raw data and materials, etc. Raw data, the study plan, documents concerning the study presented by the sponsor, the final report and necessary materials are stored in archives in this laboratory for ten years after the receipt of the notice specified under Clause 1 or Clause 2 in Article 4, Clause 2 or Clause 3 or Clause 8 in Article 4-2, and Clause 2 in Article 5-4 or Clause 2 in Article 24 or Clause 2 in Article 25-3 of "Law Concerning Examination and Regulation of Manufacture, etc. of Chemical Substances". Treatment ofraw data and materials, etc. after the storage period will be discussed with sponsor.



### 757

### Personnel Study Director Study personnel (Operation ofbiodegradation test)

Stafffor cultivation ofactivated sludge Approval offmal report Study Director Date Signature 15089

Takakazu Kayashima (1 st 

### Chemical Safety Section) Naoki Onozawa Y

### uko Kanetake Takakazu Kayashima Hirokazu Kiuchi Yuko Shimogawa Kimiko Tanaka Sayuri Nakata Ryosuke Nabeoka Koichi Hiromatsu Noriko Miyaura Hiroko Mori Masako Y

### unoki Tomohiko Yoshida Noriko Yoshida EriAbe AkikoKoga Aki Toda Kazuomi Katagiri

June 16, 2008 Signed in original Takakazu Kayashima



### 758 

### -3­

### 15089 

# SUMMARY

Title Biodegradation study ofPE3MB by microorganisms Conditions ofcultivation (1) Concentration oftest item (2) Concentration ofactivated sludge (as the concentration ofsuspended solid) (3) Volume oftest solution (4) Cultivation temperature (5) Cultivation duration (under the conditions ofdarkness) 100 mglL 30 

### mgIL 300mL 25±I°C 28 days

Measurement and analysis for calculation ofpercentage biodegradation (percentage decrease) (1) Measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus (2) Determination oftest item by high-performance liquid chromatography (HPLe) Other analysis (l) Determination of dissolved organic carbon (DOC) by a total organic carbon analysis (TOC) (2) Determination of 3-mercaptobutanoic acid, 3,3'-disulfandiyldibutanoic acid by high-performance liquid chromatography (HPLC) (3) Determination ofpentaerythritol by liquid chromatography-mass spectrometry (LC-MS) (4) Qualitative analysis of other converted products by liquid chromatography-mass spectrometry (LC-MS) Results (1) Percentage biodegradation by BOD 36%, 36%, 35% average 36% (2) Percentage decreaseoftest item "I 

### (HPLC) 100%, 100%, 100% average 100% 

## * 1 The percentage decrease of the test item, the ratio of decreased amount to the amount oftest item added, was calculated instead ofthe percentage biodegradation of the test item (percentage biodegradation by the chemical analysis of the test item) because the percentage residue of the test item in the test solution (water 

### + test item) (68%) was less than 90%. Conclusion All ofthe test item was changed or biodegraded on 3-mercaptobutylate group under the test conditions ofthis study, and then many converted products were produced 

### in the test solution. The converted products were not biodegraded by microorganisms and remained. 

### 759 

## -4­

### 15089

L Test item In this report, PE3MB has the following chemical name, etc.

w r·.·---·····----····--··.·-.······--·. ••••• --- •••• --- .- •• - •• --.- ••••• -- ••• - •• - ••• - •• -- •••• -- ••••**~---** •• :



# .

..... 

## -.- ...**-~** 

### ....

## --.--.- .....

## --- ... 

## -- .... 

## -- ..

# ---- ... 

## - .......

### _--_ ...... _-._----- ... --- .... --_ .. -.. --- .. ,

760



## -5­---- ----- 

### 15089

2. Test sample 

## .---- ....**-.-.-~----** 

### .. 

### -.....**---~.-.------------** 

## ..

## ------ -- ... _--_ ..... .... _- .... .. 

### --- .... 

### -.. 

### _-- .... _-._.-._-----_._. , ,



# .**~----** 

## ....

# -- .. -.----.-.---- 

## ....

## -- ....**----.-.--~** 

### ..... -.. .. _---._--_._ .. _--------_._---.------------_.-----_.-----_.-.-._----­ 

### 2.5 Storage and stability Storage condition Dark storage place at room temperature 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 compJetion were identical (see Fig. 23).



### 761 

### -6­15089

3. Activated sludge

3.1 Preparation ofactivated sludge Activated sludge used in the present test was prepared as follows. (1) Sampling sites On-site sludge sampling was carried out at the following ten locations in Japan. Fushikogawa city sewage plant (Sapporo-shi, Hokkaido) Fukashiba industrial sewage plant (Karnisu-shi, 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 Cfokushima-shi, Tokushima) Lake Biwa (Otsu-shi, Shiga) Hiroshima Bay (Hiroshima-shi, Hiroshima) Dokai Bay (Kitakyushu-sill, Fukuoka) (2) Sampling method Sewage plant Return sludge was collected. Rivers, lake and sea Surface water and surface soil which was in contact with the atmosphere were collceted. (3) Sampling date December, 2007 (4) Preparation method ofactivated sludge Activated sludge was prepared as follows to maintain its uniformity. The filtrate (5 L) of the supernatant of the activated sludge*3 cultivated about for three 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 aeratect'4 after the pH value ofthe mixture was adjusted to 7.0±l.O.


## *3 'fhe activated sludge cultivated the mixed filtrate (10 L) ofthe supernatant ofsludge collected at the ten loeations according to the cultivation methods described in Section 3.2. 

### *4 Prefiltered open air was used.



### 162 

## -7­15089

3.2 Cultivation After ceasing aeration of the sludge mixture for approximately 30 minutes, supernatant corresponding to about one 

### third of the whole volume was removed. Dechlorinated water was added to the remaining portion so that thc total volume reached 10 L. This mixture was aerated for 30 minutes or more, and then a predetermined amount of synthetic sewage"S was added to the mixture so that the concentration ofthe synthetic sewage was 0.1 % in the volume ofdechlorinated water added. TIlls procedure was repeated once every day. Cultivation was carried out at 25±2°C. 

### *5 Synthetic sewage Glucose, peptone and potassium dihydrogenphosphate were dissolved in purified water to obtain 50 

### gIL ofthe solution for each component. The pH ofthe solution was adjusted to 7.0±1.0 with sodium hydroxide. 

### 3.3 Control and use During cultivation, the appearance ofthe supernatant, sedimentation of the sludge, formation of flock, pH, dissolved oxygen concentration 

### in thc 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 th.e activated sludge was microscopically observed and sludge with no abnomlal symptoms was used for the test. The activated sludge, which was cultivated for 19 hours after it had been added the synthetic sewage, was used. 3.4 Inspection ofactivity and date ofinitiation ofuse ofactivated sludge 

### (1) Inspection ofactivity Activity ofthe sludge was assessed using standard items before initiation ofuse. (2) Date ofinitiation ofuse January 15,2008



### 763 

### -8­

### 15089

4. Perfonnance ofbiodegradation test

4.1 Preparations for test (1) 

### Measurement ofconcentration ofsuspended solid in activated sludge The concentration of suspended solid was measured to detennine the amount of activated sludge to add. Method In accordance with Japanese Industrial Standards (TIS) K 0102-1998 Section 14.1 Date March 24, 2008 Result Concentration of suspended solid in the activated sludge was 3190 mglL. 

### (2) Preparation ofbasal culture medium Each 3 mL 

### ofsolutions A, S, C and D, which are prescribed in JIS K 0102-1998 Section 21, were made 

### up to 1000 mL with purified water (Japanese Pharmacopeia, Takasugi Phannaceutical Co., Ltd.), and then the pH of this solution was adjusted to 

### 7.0. (3) 

### Reference item Aniline (rcagcnt grade, Wako Pure Chemical Industries, Ltd. Lot No. TSK3652) was used as a reference item to confinn that the sludge was sufficiently active.

764 

## -9­15089

4.2 Preparation oftest solutions The following test solutions were prepared and cultivated under the conditions described in Section 4.3. (1) Addition oftest item or aniline (a) Test solution (water 

### + test item) (n=l, Vessel No. 1) In one test vessel, 30 mg ofthe test sample was accurately weighed and added to 300 

### mL ofpurified water, so that the concentration ofthe test item reached 100 mgIL. (b) Test solution (sludge 

### + test item) (n=3, Vessel Nos. 2, 3 and 4) In each test vessel, 30 mg of the test sample was accurately weighed and added to the basal culture medium [the volume was less than 300 mL by the volume (2.82 mL) ofactivated sludge inoculatcd], so that the concentration ofthe tcst item reached 100 mgIL. (c) Test solution (sludge 

### + aniline) (n=l, Vessel No.6) In one test vessel, 29.5 f.lL [30 mg 

### = 29.5 f.lL x 1.022 g/cm3 (density)] of aniline was taken out by microsyringe and added to the basal culture medium [the volume was less than 300 

### mL by the volume (2.82 

### mL) of activated sludge inoculated], so 

### that the concentration ofaniline reached 100 mgIL. (d) Test solution (control blank) (n=1, Vessel No.5) In one test vessel, nothing was added to the basal culture medium [the volume was less than 300 mL by the volume (2.82 mL) ofactivated sludge inoculated]. (2) Inoculation ofactivated 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 ofthe suspended solid reached 30 mglL.



### 765 

## -10­15089

4.3 Instruments and conditions ofcultivation (1) Instruments for cultivation Closed system oxygen consumption measuring apparatus (Temperature controlled bath and measuring unit: Asahi Techneion Co., Ltd.) (Data sampler: Asahi Techneion Co., Ltd.) Vesse} 300 

### mL in volume (Improved type) Absorbent for carbon dioxide Soda lime No.1 (for absorption ofcarbon dioxide, Wako Pure Chemical Industries, Ltd.) (2) Conditions ofcultivation Cultivation temperature 

### 25±IOC Cultivation duration 

### 28 days (under the conditions ofdarkness) Stirring method Each test solution was stirred by a magnetic stirrer. (3) Room Apparatus room IA 4.4 Observation and measurement oftest conditions (1) Observation oftest solution During the cultivation, the appearance ofthe test solution was observed once a day and conditions ofthe instruments 

### were checked properly. (2) Measurement ofbiochemical 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 wac; measured and recorded onee a day.



### 766 

## -11­15089

4.5 Analysis oftest solution After the end of the cultivation, the test item and three converted products [3-mercaptobutanoic acid (hereafter referred to as 3MBA), 3,3'-disulfandiyldibutanoic acid (hereafter referred to as SBA) and pentaerythritol], which were expected from results ofthe preliminary test prior to this study and the authentic samples ofwhich were available, in the test solutions were determined. 

### In addition, other converted products, which were also expected from results of the preliminary test prior to this study and the authentic samples ofwhich were not available, were analyzed qualitatively. Dissolved organic carbon in the test solutions was determined 

### in order to confirm presence or absence ofwater-soluble converted products. 4.5.1 Pretreatment oftest solutions for analysis After the end of the cultivation, the test solution (water 

### + test item), the test solutions (sludge 

### + test item) and the test solution (control blank) were pretreated for total organic carbon (TOC) analysis of dissolved organic carbon, high-performance liquid chromatography (HPLC) analysis of the test item, 3MBA and SBA, and liquid chromatography-mass spectrometry (LC-MS) analysis of pentaerythritol and other converted products as follows. 

# I Test solution 300 

### mL 

# I 

# I --Purified water / phosphoric acid 

### (9/1 v/v) 2 mL Continued to 

### CD onthe next page (measuring pipet) --Ammonium sulphate 200 g (even balance) +-Tetrahydrofuran 90 tnL (graduated cylinder)

• Shaking for 10 minutes 

## I 

# l Water layer 

# I I Tetrahydrofuran layer 

# I

• filtration and dehydration (IPS filter paper) • Filling up to 100 

### mL (tetrahydrofuran, volumetric flask)

Continued to 

### ® on the next page

767 

## -12­

### 15089

Carried down 

## ill

• Taking out 15 

### rnL (measuring pipet) • Centrifugation (3000 x 

### g, 5 min) 

# I Supernatant 

# I I • Taking out 

### 2 mL 

### (transfer pipet) TOCsample 

# I I • Filling up to 5 mL 

### (purified water. volumetric flask) 

# I HPLC sample -2 

# I Determination of3MBA 

### and SBA 

# I LC-MS sample -1 

# 1 Determination ofpentaerythrito) 

# I LC-MS sample 

# -2\ Qualitative analysis of other converted products Carried down @



### . Taking out 3 mL (transfer pipet) • Filling up to 10 mL (acetonitrile 1

### water"o () 19 

### v/v), Determination oftest item volumetric flask)

LC-MS sample-3 Qualitative analysis of other converted products *6 City water was treated with ultrapure water system.



### 768 

## -13­15089

4.5.2 Quantitative and qualitative anaJysis (1) Determination ofdissolved organic carbon The dissolved organic carbon (DOC) in the TOe sample was analyzed under the following conditions. The concentration of DOe in the TOe sample was calculated proportionally by comparing the concentration of carbon dioxide of the TOC sample with that of 

### 80.0 mgCfL standard solution (see**Table~2).** The standard solution for TOC analysis was prepared by dissolving potassium hydrogenphthalate 

### in purified water. The concentration of dissolved organic carbon corresponding to the minimum determination limit was regarded as 1.0 mgClL. Analytical conditions Instrument Total organic carbon analyzer Hiranurna Sangyo Co., Ltd. type TOC-2000 Catalytic agent Titanium oxide suspension·7 Injection volume 400 J.1L *7 Prepared by mixing titanium oxide powder, 70% perchloric acid solution and water·6•

769 

## -14­

### 15089
(2) Determination oftest item, 3MBA and SBA The HPLC sample -1 

### were analyzed under the following conditions. The concentration ofthe test item in the HPLC sample -1 

### was calculated proportionally by comparing the peak area on the chromatogram ofthe HPLC sample -1 

### with that on the chromatogram of300 mgfL standard solution (see Table-4 and Fig. 7). The HPLC sample -2 was analyzed under the following conditions. The concentration of 3MBA in the HPLC sample -2 was calculated proportionally by comparing the peak area on the chromatogram ofthe HPLC sample -2 

### with that on the chromatogram of 100 mgfL standard solution (see Table-5 and Fig. 8). The concentration 

### of SBA in the HPLC sample -2 was calculated proportionally by comparing the peak area on the chromatogram ofthe HPLC sample -2 

### with that on the chromatogram of 100 mgfL standard solution (see Table-6 and Fig. 8). 

### The lowest detectab1e peak area was regarded as 6000 Il V •

### see considering the noise leve1, which corresponded to the test item concentration of 1.4 mg/L, 3MBA concentration of0.70 mgfL and SBA concentration of0.49 mg/I.. (a) Analytical conditions Instrument High-performance liquid chromatograph Shimadzu Corporation type LC-201OClIT (Built-in ultraviolet and visible detector) Column L-column ODS (15 em x 

### 2.1 mm 

### LD., Chemicals Evaluation and Research Institute) Column temperature 40°C Eluent A: 

### Acetonitrile I 

### phosphoric acid (1000/1 

### v/v) B: Water"61 phosphoric acid (1000/1 

### v/v) Gradient condition

Time (min) A 

### (%) B 

### (%) 

### 0.00 10 90 3.00 10 90 20.00 100 {} 

### 30.00 100 

## o Flow rate 0.2 mUmin Measurement wavelength 210 nm (see Figs. 19

- 21) Sample size 5 ilL Detector output 2V/AU

770 

## -15­15089
(b) Preparation ofstandard solution The standard solution to detcnnine the concentration ofthe test item, 3MBA and SBA in the HPLC samples were prepared as follows. 

## CO Test item 100 mg of the test sample was accurately weighed and dissolved in tetrahydrofuran to obtain 1000 mg/L solution of the test item. 300 mgIL standard solution was then prepared from this solution by dilution with tetrahydrofuran. 

### ® 3MBA 100 mg of 3MBA supplied by the sponsor"S was accurately weighed and dissolved in acetonitrile to obtain 1000 mg/L solution of3MBA. 100 mgIL standard solution was then prepared from this solution by dilution with purified water. *8 Purity >99% Lot number 50913 3MBA was treated as 100% in purity. 

## ® SBA100 mg ofSBA supplied by the sponso/9 was accurately weighed and dissolved in acetonitrile to obtain 1000 mgIL solution of SBA. 100 mgIL standard solution was then prepared from this solution by dilution with purified water. *9 Purity >99% Lot number 551601 SBA was treated as 100% in purity. (c) Calibration curve 

## CD Test item 75.0, 150 and 300 mgIL standard solutions were prepared by the same method as described 

### in (b)CD. 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 chromatograms and tbe respective concentrations (see Fig. 2). 

## ® 3MBA 25.0, 50.0 and 100 mgIL standard solutions were prepared by tbe 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 chromatograms and the respective concentrations (see 

### Fig. 3). @ SBA 25.0,50.0 and 100 mgIL standard solutions were prepared by thc same method as described in (b)@. lbese solutions were analyzed according to the analytical conditions described 

### in (a). A calibration eurve was drawn based on the relation between 

### the peak area on the chromatograms and the respective concentf'dtions (see Fig. 4). 

### 771 

## -16­15089
(3) Detennination ofpentaerythritol The LC-MS sample -1 were analYLed under the following conditions. The concentration ofpentaerythritol in the LC-MS sample -1 was calculated proportionally by comparing the 

### peak area on the chromatogram ofthe LC-MS sample -1 with that on the chromatogranl of 15.0 mgIL standard solution (see Table-7 and Fig. 9). The lowest detectable peak area was regarded as 130 considering the noise level, which corresponded to pentaerythritol concentration of0.15 mg/L. (a) Analytical conditions Instrument Liquid chromatograph-mass spectrometer HPLC system Waters Corporation type Alliance2690 Mass spectrometer Waters Corporation type ZMD LC conditions Column L-column ODS (15 (''II1 x 2.1 mm 

### 1.0., Chemicals Evaluation and Research Institute) Column temperature 35°C Eluent 5 mmollL Ammonium acetate solution Flow rate 0.2 mL/min Sample size 5 ilL Mass conditions Ionization mode Electrospray ionization (ESI) Detection ion Negaitive Detection method Selected ion monitoring (SIM) Monitoring ion (m/z) 195.3 (see Fig. 22) Ion source temp. 

### 120Cl

### C Desolvation temp. 400°C Cone voltage 15 V (b) Preparation ofstandard solution The standard solution to detennine the concentration of pentaerythritol in the LC-MS sample 

### was prepared as follows. 100 mg ofpenta erythritol (Tokyo Chemical Industry Co., Ltd.) "10 was accurately weighed and dissolved in purified water to obtain 1000 mg/L solution of pentaerythritoL 15.0 mgIL standard solution was then prepared from this solution by dilution with purified water. *10 Pentaerythritol was treated as 100% 

### in purity. (c) Calibration curve 3.75, 7.50 and 15.0 mgIL 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 chromatograms and the respective concentrations (see Fig. 5).
772 

## -17­15089
(4) Qualitative analysis ofother converted products The LC-MS samples -2 and -3 were analyzed by liquid chromatography-mass spectrometry (LC-MS) for qualitative analysis of other converted products (see Figs. 10·18). The solutions of the test item, 3MBA and SBA solution were also analyzed for the comparison with the LC-MS samples. (a) Analytical conditions Instrument HPLC system Mass spectrometer Photodiode array detector Pump (post-column) LC conditions Column
Column temperature Eluent

Flow rate Sample size Measurement wavelength Post-column Solution Flow rate Mass conditions Ionization mode Detection ion Detcction mode Mass range mJz Ion source temp. Desolvation temp. Cone voltage Liquid chromatograph-mass spectrometer Waters Corporation Waters Corporation Waters Corporation Shimadzu Corporation
L-column ODS type Alliance2690 type ZMD type 996PDA type LC-20AD

(15 cm x 2.1 mm J.D., Chemicals Evaluation and Research Institute) 40°C A :Acetonitrile I formic acid (100011 v/v) 

### B : Water*6 I formic acid (100011 v/v) Gradient condition Time (min) 

### 0.00 3.00 20.00 40.00 0.2 

### mUmin 15 

### ilL 205 -300nm 

### A (%) B (%) 

### 10 90 

### 10 90 JOO 0 100 0

Acetonitrile 

### I water"6 

### (111 V/V)"11 

### 0.05 

### mLlrnin Electrospray ionization (ESI) Positive Scan 

### 80'';-0 800 1200C 4000C 20V


## *11 Containing 25 mmollL ammonium acetate.

773 

## -18­15089
(b) Preparation ofsolutions oftest item, 3MBA and SBA The solutions of the test item, 3MBA and SBA used for**LC~MS** analysis were prepared as follows. 

## CD Test item 100 mg/L solution ofthe test item was then prepared from 1000 mg/L solution of the test item, which was prepared according to the method described 

### in Section 4.5.2(2)(b)CD, by dilution with acetonitrile / water*6 

### (Ill v/v). @ 3MBA 100 mg/L solution of 3MBA was prepared from 1000 mg/L solution of 3MBA, which was prepared according to the method described 

### in Section 4.5.2(2)(b)@, by dilution with acetonitrile 1water*6 (1/9 v/v). 

### ® SBA 100 mglL solution ofSBA was prepared from 1000 mg/L solution ofSBA, which was prepared according to the method described in Seclion 4.5.2(2)(b)@, by dilution with acetonitrile 1water*6 (1/9 v/v). 4.5.3 Recovery test and blank test Each two test solutions (water 

### + test item) and two test solutions (sludge 

### + test item) for recovery test were preparcd according to the methods described in Section 4.2. The test solutions were pretreated in accordance with the method described in Section 4.5.1, then analyzed according to the procedures and analytical conditions described in Section 4.5.2(2). 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 appeared around the peak(s) ofthe test item on the chromatogram. Two individual recovery rates and their averages ofthe test item on the analytical procedure are shown below. The average recovery rates were used as correction factor, for the detennination ofthe test item in the analytical samples (see Table-3 and Fig. 6). Recovery rate 

### in the test solutions (water 

### + tesl item) 96.6%, 95.7% average 96.2% Recovery rate in the test solutions (sludge 

### + test item) 94.8%, 94.1% average 94.5% Instrument used in recovery test [Other conditions: see Section 4.5.2(2)]

Instrument High-perfonnance liquid chromatograph Pump Shimadzu Corporation type LC-20AD UV-VIS detector Shimadzu Corporation type**SPD~20A** V Column oven Shimadzu Corporation type CTO-20AC Auto injector Shimadzu Corporation type SIL-20A System controlJer Shimadzu Corporation type SCL-10Avp Degasser Shimadzu Corporation type DOV-l OA) 774 

## -19­15089

4.6 Calculation ofpercentage biodegradation The percentage biodegradations were calculated by the follovving equations and rounded offto the whole number. The percentage decrease of the test item, the ratio of decreased amount to the theoretical amount, was calculated instead of the percentage biodegradation of the test item (percentage biodegradation by the chemical analysis of the test item) because the percentage residue ofthe test item in the test solution (water 

### + test item) (68%) was less than 90% (1) Percentage biodegradation by BOD BOD-B Percentage biodegradation (%) x 100 TOO BOD : Biochemical oxygen demand inthe test solution (sludge 

### + test item) (experimental) (mg) B : Biochemical oxygen demand in the control blank (experimental) (mg) TOD : Theoretical oxygen demand required when the test item 

### was completely oxidized (theoretical) (mg) (2) Percentage decrease oftest item'12 Sw- Ss Percentage decrease (%) 

### x 

### 100 Sw Ss : Residual amount ofthe test item 

### in the test solution (sludge 

### + test item) (experimental) (mg) Sw : Additional amount ofthetest item (mg) *12 The residual amount was fixed at zero and the calculation was carried out when the analytical value obtained was less than the lowest detectable value in the quantitative analysis described 

### in Section 4.5.2(2). 4.7 Treatment ofnumerical values Values were rounded offin accordance vvilh JIS Z 840I: 

### 1999 rule 

### B.

775 

### -20­15089

5. Validity oftest conditions The validity criteria ofthe test and the values in this test are shown in the following table. This test was valid because all ofthe values in this test met the criteria.



# --------­ Value in present test Value of criterion Sec Difference of extremes of replicate values ofpercentage biodegradation (decrease) Percentage biodegradation**~_ ~()D**  ___._ Percentage decrease oftest item 1%**~.---.** 0% <20% Section 7.3 Percentage biodegradation Percentage biodegradation ofaniline by BOD After 14 days 76% 

### 2::60% Table-l 

### Fig. 1

6. Factors that affected reliability oftest No adverse effects on the reliability ofthis test were noted.

7. Results

7.1 Appearances oftest solutions Appearances oftest media in cultivation vessels were as follows.
Test solution Appearance pH
Water 

### + test item The test item was not dissolved. 

# - At the start ofcultivation Sludge 

### + test item The test item was not dissolved. 

# -

At the end ofcultivation Water 

### + test item
Sludge 

### + test item Insoluble compound could not be confirmed. Insoluble compound the except sludge could notbe continned. C'.rrowth ofthe sludge was observed. 

# -



# -

776 

## -21­15089

7.2 Analytical results oftest solutions Analytical results oftlIe test solutions after 28 days were as follows.

# ~ Water 

### + Sludge 

### + test item Theoretical test item Table Fig. Vessel-l Vessel-2 Vessel-3 Vessel-4 amount
BOD' 13 mg 4.2 19.0 18.9 

### ]8.1 52.2 

### 1 1

·13 mgC 

### 13.1 9.2 9.2 9.1 13.8 Detected amount and percentage 

### .... --_ ..

### - ---_..... "'--

### ... "'­ 

### ......................... 

### ..........-_

### ......... 

## --.......... --_ .... "'

### .............._ 

### ............ 2**~** detection ofDOC 

### % 95 67 67 66**~** Decrease amount 30.0 and percentage mg 20.5 

### 0 0 0 decrease of 

### ..

### -... 

### -..**~** 

### -... 

### -..

### -..

### -..

### -..... 

### -............

### --­ ........ ....... "'
......... 4
......... '" ......................... 

### -........ "" "' ............. '"

_ ..... 4 7 test item 

### %CD 68 0 0 0 

# - (HPLC) Produced amoWlt 0 and percentage mg 0 0 0 24.4 production of ...._ 

### ........... ..........**-~** ..**~**

### ..

### -..

### ­

.. '"
_ .. "
.. __ • "**~** x ...........**~** .. " 

### ......

### -­ .........

### _. -.. ,. .. '""" ..
- ....... -<'
...**-.~** ...... --­ 5 

### 8 3MBA 

### %@ 0 

### 0 0 0 

# - (HPLC) Produced amount and percentage mg 0 1.0 0.9 0.8 24.2 production of 6 8 SBA 

### %@ 0 4 

### 4 3 

## - (HPLC) Produced amount and percentage mg 0 1.2 1.2 1.2 9.2 I production of 7 9 pen1aerythritoI 

### %@ 0 (LC-MS) 

### 13 13 

### 13 

# -
·14 Detection ofother 

### Detected Detected 

### 10-18 converted products 

### - 3 components (6 components) 

# - - (LC-MS) Mass balance"15 

### % 68 4 4 

### 3

(CD+®+@) 

# - - 

# - Mass balancc· 16 

### % 68 13 13 13 (CD+@)) 

# - - -

777 

## -22­15089



## * 13 The value of control blank was subtracted from the values of the test solutions (sludge 

### + test item). *14 Determinations of detected converted products except for 3MBA, SBA and pentaerythritol could not be performed, because those authentics samples were not available. *15 Mass balance for 3-mercaptobutylate group oftest item


## * 16 Mass balance for pentaerytritol group oftest item 

### 7.3 Percentage biodegradation Percentage biodegradations after 28 days were as follows. The percentage decrease of the test item, the ratio of decreased amount to the amount oftest item added, was calculated instead ofthe percentage biodegradation ofthe test item (percentage biodegradation by the chemical analysis ofthe test item) 

### because the percentage residue ofthe test item in the test solution (water 

### + test item) (68%) Vlas less than 90% The percentage biodegradation by DOC was not calculated because the test item is not dissolved in water at the test concentration (100 mglL) and more.

# ~ Sludge 

### + test item Table Vessel-2 VesseJ-3 Vessel-4 Average Percentage biodegradation by % 36 36 35 36 

### 1 BOD Percentage decrease of % 

### 100 100 100 100 4 test item (HPLC)

778 

### -23­15089

C 7.4 Result ofqualitative analysis for converted products Many converted products except for 3MBA and SBA were detected 

### in quantitative analysis of the test item, 3MBA, and SBA. As the result ofthe qualitative analysis of other converted products by LC-MS, many peaks corresponding to converted products were detected on PDA chromatograms ( Il 205-300 nm) and total ion chromatograms (TIC) 

### in the test solution (water 

### + test item) and the test solutions (sludge 

### + test item) (see Figs. 10-18). The converted products shown 

### in the following list were presrnned by analysis oftheir mass spectra. 

### In addition, all ofthe converted products were detected in the LC-MS sample -2, but the converted product A was not detected 

### in the LC-MS sample -3. List ofconverted products (elution order, see Figs. 10-18) Converted 

### Detected (0)/ Not detected 

### ex) Presumed structural formula products (M: presrnned molecular weight) Water 

### + test item Slud,[e 

### + test item 

# · · · A*17 

# · · 

# · [1] x 

# · 

# · · · · · · · 

# · B 

### fn x

[I] x

D 

### [1] 

### x



# · · 

# · · 

# · · 

# · E 

### ll] 

### x 

# · · · · ·
· ·
-- .. .........

## --.-.- ......

## --- ... -

### ..... 

### .... 

## -..... -.**-.~** 

### [2] 0 [3] 0 [4] 0 [2] 0 

### [3] 0 [4] 0 

### [2] 0 [3] 0 

### [4J 0 

### [21 0 [3J 0 [4] 0
[2] 0 [3] 0 [4] 0



## *17 Detected in only LC-MS sampJe-2



### 779 

## -24­

### 15089

Converted products Presumed structural fonnula (M: 

### presumed molecular weight) 

### Detected (o)/ Not detected (x) 

### Water 

### + test item Sludge 

### + test 

### item I



### F

G



### H

I 

### [1] x 

### [2] 0 [3] 0 

### [4] 0



### [1] 0 

### [2J x [3] x 

### [4] x



### [1] 0 

### [2] x [3] x 

### [4] x



### [1] 0 

### [2] x [3] x 

### [4] x

780 

## -25­

### 15089

7.5 Discussion (1) 

### Percentage biodegradation by BOD Percentage biodegradation by BOD was 35-36%. This result suggested that the some ofthe test item was biodegraded by microorganisms (see Fig. 1). (2) Results ofdetermination oftest item Decrease of the test item was confirmed in the test solution (water 

### + test item) because ofthe percentage residue ofthe test item (68%). In 

### the test solutions (sludge + 

### test item), the test item did not remain. Many peaks corresponding to converted products were detected on the HPLC chromatogram in the test solution (water 

### + test item) and the test solutions (sludge 

### + test item) (see Fig. 7). (3) Results ofdeterminations ofexpected converted products According to (2), 3MBA, SBA and pentaerythritol, whose productions were expected by hydrolysis etc. of the test item, were determined. As the results, these converted products were not detected in the test solution (water 

### + test item). On the other hand, SBA and pcntaerythritol were detected in the test solutions (sludge 

### + test item) and their percentage productions were 3-4% and 13%, respectively. Additionally, in the determination of 3MBA and SBA, many peaks corresponding to converted products except for 3MBA, SBA and pentaerythritol were detected on the HPLC chromatogram in the test solution (water 

### + test item) and the test solutions (sludge 

### + test item) (see Fig. 8). (4) Mass balances The mass balance for 3-mercaptobutylate group, which was calculated from sum ofthe percentage residue ofthe test item and the percentage productions of3MBA and SBA, was 68% in the**te~i** 

### solution (water 

### + test item) and 3-4% in the test solutions (sludge 

### + test item). The mass balance ror pentaerythritol group, which was calculated from stUn ofthe percentage residue ofthe test item and the percentage productions ofpentaerythrito), was 68% in the test solution (water 

### + test item) and 13% in the test solutions (sludge 

### + test item). It was considered for shortage of those mass balances that other converted products except for 3MBA, SBA and pentaerythritol described 

### in (3) were produced rrom the test item. 

### In addition, the values ofthe percentage biodegradation by BOD described in (1) suggested that some of the test item or the converted products were biodegraded by microorganisms.

781 

## -26­15089
(5) Qualitative analysis for other converted products According to (2) and (3), many peaks corresponding to unknown converted products were detected, and they might contribute to the shortage ofthe mass balances described in (4). Then, other converted products were analyzed qualitatively by LC-MS. As a result, the unknown converted products (converted product A-I) were presumed as compounds which were produced by hydrolysis of 3-mercaptobutylate group and/or sulfer-sulfer (s-s) polymerization. However, detenninations of these converted products could not be performed and then the mass balances could not be confirmed because the authentic samples ofthese were not available. (6) Biodegradation of3-mercaptobutylate group The converted products detected in the test solutions (sludge 

### + test item) were SBA, pentaerythritol and the converted products A-F, which were produced by hydrolysis and/or biodegradation on 3-mercaptobutylate group of the test item. Despite this, 3MBA was not detected in the test solutions (sludge 

### + test item). Here, as the average percentage biodegradation by BOD in "Biodegradation study of 3MBA by microorganisms (Study No.l4359)" was 79%, 3MBA is thought to be biodegradable. From this knowledge and the value ofthe percentage biodegradation by BOD described in (1), it was considered that the shortage of 3-mercaptobutylate group ofthe test item was biodegraded completely by microorganisms. (7) Summary ofdiscussion According to (1)-(6), it was considered that all of the lest ilem was changed or biodegraded on 3-mereaptobutylate group illto many converted products and that these products were not biodegraded and remained under the conditions of this study (see Degradation diagram on next page).



### 782 

## -27­

## • 

# •

• • 15089
Degradation diagram oftest item [in test solutions (sludge 

### + test item)] 1--·.······.----------------·.····.·.···.-----·. 

# · . 

# · 

# . · 

# . · · : 

# . . Test item 

# · (percentage decrease: 

### 100%) 

# · · · · · · ·

.**~--** ....---P-
....... ---.---.---.-.--.------- ... ­ 

### I . Disappearance oftest item by conversion

... • Partly biodegradation on 3-mercaptobutylate group Residual converted products

### 1.~?l!::~~~~.Et:<?9.l!~~~.**{p!~~~~~ .~?~t.** **~~~~.~~. <:~~<:~~).**  

## ..................................... . • I 

### I : 

## t I 

### I •

• I 

## t • · ! !
. 

## , · 

# . I**~** 

### I E 

### I 'k.-.....

______________ .._ .._._ .._ .. _ .._ ...._ ...__ ._ .. _ ..._ ...._ ....__ ._..___ .._. _____ ...

Converted products (produced amount could not be checked)

......

### --

## ..

# _._--- ...

## _--_.-.......

# _--_.....

# _---_...

# _ ...

# _-_ ..._----------_...

## _--._---_..

## _­ 

# · . · · ·

783 

## -28­15089

7.6 Conclusion All ofthe test item was changed or biodegraded on 3-mercaptobutylate group under the test conditions ofthis study, and then many converted products were produced in the test solution. The converted products were not biodegraded by microorganisms and remained.


### 8. Remarks

8.1 Instruments used for test Fourier transform infrared spectrophotometer: Shimadzu Corporation type IRPrcstige-21 Closed system oxygen consumption measuring apparatus : see page] 

### 1 Total organic carbon analyzer: see page 

### 14 High-performance liquid chromatograph: see page 15 Liquid chromatograph-mass spectrometer: see pages 17, 18 Electronic analytical balance: Sartorius AG type CP224S Sartorius AG type ME235P Ultraviolet and visible spectrophotometer: JASCO Corporation type V-660 Refrigerated centrifuge: KUBOTA Manufacturing Corporation type 5922 Shaker: Taitec Corporation 

### type SR-2w 8.2 Reagents used for analysis Acetonitrile (HPLC grade) : Wako Pure Chemical Industries, Ltd. Tetrahydrofuran (HPLC grade) : Kanto Chemical Co., Inc. Purified water (Japanese Pharmacopeia) : Takasugi Pharmaceutical Co., Ltd. Ammonium sulphate (reagent grade): Kanto Chemical Co., Inc. Phosphoric acid (reagent grade) : Wako Pure Chemical Industries, Ltd. Fonnic acid (reagent grade) : Wako Pure Chemical IndustIies, Ltd. Potassium hydrogenphthalate (reagent grade) : Wako Pure Chemical Industries, Ltd. Ammonium acetate (reagent grade) : Wako Pure Chemical Industries, Ltd. 70% Perchloric acid solution (reagent grade) : Kanto Chemical Co., Inc. Titanium oxide powder: Hiranuma Sangyo Corporation 3-mercaptobutanoic acid (3MBA): Supplied by the sponsor 3,3'-disulfandiyldibutanoic acid (SBA) : Supplied by the sponsor Pentaery!hritol : Tokyo Chemical Industry Co., Ltd. 

### 184 

## -29­

### Table· 1 Calculation table for percentage biodegradation by BOD Study No. 15089 Duration ofcultivation: 28 days 7th day 21st day 14th day 28th day Mean Vessel BOD Deg. BOD 

### Deg. Deg. BOD Deg. BOD 

### Deg. No. (%) (mg) 

### (%) (mg) (%) (mg) (mg) 

### (%) (%) (6] 80.4 79 59.3 82.3 62 75.8 76 79



# . 9.0 [5] 3.1 10.9 7.1 

# - - - 27.7 36 32 29.9 [2] 14.2 21 23.8 36 27.4 35 29.8 [3] 13.7 23.6 32 20 36 26.7 34 30 29.0 [4] 12.3 22.8 35 

### (1] 18 2.8 4.2 0.6 1.7 

# ­ - 

# - 

# - Deg. : Percentage biodegradation Vessel No. [6] : Sludge 

### + aniline Vessel No. [5J : Control blank [B] Vessel No. (2] [3J [4J : Sludge 

### + test item Vessel No. [1] : Water 

### + test item Test item of 30 mg was added. Chart ofBOD: Fig. 1 Deg. 

## =[ BOD· B ] / [TOO] x 100 (%) TOD oftest item: 52.2 (mg) CI1H3001S3 

### + 2402

- 17 CO2 

### + 15 H20 

### + 3 S02 2402/ C11H3007S3= 767.97 /442.61 

## = 1.74 TOO = 

### 30 X 

### 1.74 

### =52.2 (mg) TOO ofaniline: 90.3 (mg) C6H1N 

### + 8.7502 - 6 C02 

### + 3.5 H20 

### +N02 8.7502/ C6H7N::= 279.99/93.13 

## =3.01 TOO 

## =30 X 

### 3.01:::: 90.3 (mg)

785 

## -30­ 36

### Table-2 Calculation table for percentage detection ofDOC


## studyNo.15089 SamDle description A B C D Waterblank n.d. [I] Water + test item 43.54 13.1 95 [2] Sludge + test item 33.05 9.2 67 [3J Sludge 

### +test item 32.96 9.2 67 [4] Sludge 

### +test item 32.66 9.1 66 [5] Control blank 2.32 95 66

Amount oftest item added: 30 (mg) Volume oftest solution: 300 (mL) Theoretical amount ofcarbon : 13.8 (mgC) (30) x ( 17C 1**C11H,oO~** ) A: Measured value (mgCIL) B: Detected amount ofDOC Bw = (A(Water + test item)

- A{\Vaterblank) ) )( 300/1000 (mgC) Bs =­ ( A(Sludge 

### +test item)

- A(Conttol blank) ) )( 300 11000 (mgC) C: Percentage detection (%) C '" B I ( Theoretical amollIlt ofcarbon»( 100 D: Average percentage detection (%)

April 24. 2008



### 786



## -31­**Table~3** 

### Calculation table for recovery rate oftest item 

## studv No. 15089 Sample descriPtion A 

### D E F
Standard solution 300mg/L 1233965 Water + lest item.1 1192360 29.0 96.6 96.2 Water + test item -2 1180870 28.7 95.7 Sludge + test item-l 1170360 28.5 94.8 94.5 Sludge + lest item -2 1161618 28.2 94.1 Control blank lI.d.

Amount of lest item added: 30 (mg) A: Peak area (.uV·sec) B: Final volume: 100 (mL) 

## c: Ratio ofportion used for analysis: 300/300 

### D: Recovery amount (mg) Dw
.. G x ( A(Water + test item) I A(Standard) ) x ( B / C ) /1000 Ds

### =G x { ( A(Sludge + test item) • A(Control blank) ) / A(Standard) } )( ( B I C) 11000 E: Recovery rate (%) E 

## = D /30 (mg) x 100 F: Average recovery rate (%) G: Concentration of standard solution: 300 (mg/L) See Fig. 6
February 10, 2008

787 

## -32­

### Table-4 Calculation table for percentage decrease oftest item Study No. 15089

. Simple description A E F 

### G H Standard solution 300mg/L )243545 [1521409 (measw-ed value)

- 277864 (solvent blank)] Solvent blank 277864 [1] Water 

### +test item 1053078 20.5 68 [2] Sludge + test item 277341 

## o 

## o 100 [3] Sludge +test item 277786 

### o 

## o 100 100 [4] Sludge +test item 277142 

## o o 100 [5] Control blank 277810

Amount oftest item added: 30 (mg) A: Peakarea("V'sec) B: Final volmne : 100 (mL) C: Ratio ofportion used for analysis: 285/300 D: Recovery rate : 96.2 (%) (Water + test item) 94.5 (%) (Sludge +test item) E: Residual amount oHest item (mg) Ew =1 " { (A(Water + test item)

- A(Solvent blank) ) 1A(Standard) } ,,( B 1C) I (D 1100) 11000 Es 

### =I " { ( A(Sludge + test item)

- A(Controi blank) ) 1A(Standard) }
x (B 1C) 1 (D 1100) 11000 F: Percentage residue (%) F" E 130 (mg) x 100 G:Percentage decrease (%) G { (30· E(Sludge +test item) ) 130 } )C 100 H: Average percentage decrease (%) I: Concentration ofstandard solution: 300 (mgIL) See Fig. 7
April 28, 2008



### 188 

## -33­

### Table-5 Calculation table for percentage production of3MBA
S d Mv No. 15089 Sample descritltion A D E F Standard solution 100mg/L 852951 [I] Water +test item n.d. 0 0 0 [2J Sludge + test item n.d. 0 0 [3J Sludge +test item n.d. 0 0 0 [4

### JSludge' +test item n.d. 0 0 [5] Control blank n.d.

Amount oftest item added: 30 (mg) Theoretical amount of3MBA : 24.4 (mg) (30) x (**3C4H8~SI CI7H'007~**  )
A: Peak area (Jl.V·sec) B: Final volume: 15 (mL) C: Ratio ofportion used for analysis: 15/300 D: Produced amount of3MBA (mg) Dw= G x ( A(W&ter +test item) I A(Standard) ) )( ( B Ie) /1000 Ds G x { ( A(Sludge + test item)

- A(Control blank) ) / A(Standard) } x (B I C)/1000 E: Percentage production (%) E == D 124.4 (mg) x 100 

### F: Average percentage production ("/0) G: Concentration ofstandard solution: 100 (mg/L) See Fig. 8
April 28, 2008 Name**O~1w\ /v,.o¥~** 



### 789 

## -34­

### Table-6 Calculation table for percentage production ofSBA
d Stutly No. 15089 Sample description A D E P Standard solution 100mg/L [I] Water + test item [2] Sludge + test item [3] Sludge + test item (4] Sludge + test item [5J Control blank 1216558 n.d. 38563 37281 33340

n.d. 0 1.0 0.9 0.8 0 4 4 3 0 4

Amount oftest item added: 30 (mg) Theoretical amount ofSBA : 24.2 (mg) (30) x ( 1.5CaH1404S2/ CI7H]oO,S3 ) A: Peak area (JlY'sec) B; Final volume: 15 (mL) C: Ratio ofportion used for analysis: 151300 D:Produced amount ofSBA (mg) Dw 

### =G x ( A(Water +test item) / A(Standard) ) x (B / C ) / 1000 Ds"G )( { ( A(Sludge + test item)

- A(Control blank) ) / A(Standard) } x ( B I C ) / 1000 E: Percentage production (%) E 

### =D 124.2 (mg) x 100 F: Average percentage production (%) G : Concentration ofstandard solution: 100 (mg'L) See Fig. 8
April 28, 2008

790 

## -35­

### Table-7 Calculation table for percentage production ofpentaerythritol
S d o. 15089 tUly N Sample description A D E F Standard solution 15.OmgIL 13184 [1] Water + test item n.d. 0 0 0 [2] Sludge + test item 1394 1.2 13 (3] Sludge + test item 1404 1.2 13 13 [4] Sludge + test item 1386 1.2 13 [5] Control blank n.d.

Amount oftest item added: 30 (mg) Theoretical amount ofpentaerythritol : 9.2 (mg) (30) x ( CSHI20JC17H3007S3)
A: Peak area ( - ) B: Finalvolume: 5 (mL) 

### c: Ratio ofportion used for analysis: 151300x2/15 D:Produced amount ofpentaeIythritol (mg) Dw = G x (A(Water 

### + test item) / A(Standard) ) )( ( B / C ) / 1000 Ds
..G x { (A(Sludge 

### + test item)

- A(Controi blank) ) / A(Standard) } x (B / C)/1000 E: Percentage production (%) E ==D/9.2 (mg) x 100 F: Average percentage production (%) G: Concentration ofstandard solution: 15.0 (mgIL) See Fig. 9
April 23, 2008

791 

## -36­

### Study No. 15089 ( Test**item~PE~3<!..!.:MB~** 

### __________ ) Cultivating conditions:

Concentration Test item
............................................. ,................ . 100 

### (mgIL) 

### Reference item (aniline)
....................................... 100 

### (mgIL) 

### Activated sludge
................................................... 30 

### (mgIL) 

### Temperature
............................................................. .. 

### 25±1°C Duration
................................................................... .. 28 days (Mar.26,2008

- Apr.23,2008) Note:

-

Vessell

No. Sample Description 7th day BOD(mg) 

### 14th day 21st day 28th day [1] 

### Water 

### + test item 0.6 1.7 

### 2.8 4.2 [2] Sludge 

### +test item 14.2 23.8 27.7 29.9 (3] Sludge 

### + test 

### item 13.7 23.6 27.4 29.8 [4] 

### Sludge 

### +test item 12.3 22.8 26.7 29.0 [5] Control blank [B] 3.1 

### 7.1 9.0 10.9 [6] Sludge 

### + aniline 59.3 75.8 80.4 82.3 100 

# r 80 

# - ." 1:1 cos 

# e**~** 

### 60 Q 1:1**~** tu:I**~**


# - 0 40 I:'S U**.~** 

# -= u Q 20


# .... 

# = 0 - [1] 

### {2J [3] 

### -.----------. [61 [4] .".,.

### .....

# -_ ...

# _--_
... , [5]**-~.**

.. ,*,,,,,

# -' ."'"

# .­ [6J /'



# ..

# " ,



# . / /'



### • 0 14 21 

### 28 Cultivation Time (day) 

### Flg.1 

### Chart ofBOD.



## -37­ 792

## Stu d N

'I 

## o. 15089 

### Solvent blank Operatin, Date: Feb.OB.2008
. PkNO TillI8 

### Area !MIsfit (jnfn) 

### <IIV'leo) Cit V) 

# C{t

Peak position n.d.

"­

# ~ 

# 1

• I 0.'0 • 10. 0 20'.0 30.0 Operator : Naoki Dnozawa**Sta~rd** 

### solution 75.0 mill OperetI", Date : Feb. 08.2008 PKNO li. Arel 

### Hellht Cone. {mIn) (IlV'I8G) (pV) (I) 1 

### HI. 45 310$23 

### 3S062 100.00 Total 310923 100.00

# ~ 

# i
.J\. I 

### 0:0 10.0 

## H.G a.J.1I Operator : Nloki OnOZ8Wa



### Standard solution 150 

## mill Operating Date: Feb.OS.2008 PKHO Time Area 

### Heliht CorIo. (mIn)**(~V·seo)** (p. V) (I)



## . 1 19.42 609824 69403 100.00 Totll**60982~** 100.00

1

J<. II 

# ­

# ~ o.'e taO 20.0**~o** Operator : NaOkI Onozawa



### Standard solution 300 ms/L \)PentIne \)ate : Feb.08,20Q8 PrHO Ti.. Area Neilht CQl)O. Olin)**(PY~$8o) (,,~**  tI) 1 Ht35 1211153 13717 100
..00 Total 1214153 100.00 1

"­

# ~ • 0:0 lio 2IlD sail Operator : Nacki Onozawa Fig. 

### 2.

- 1 

### Chromatograms 

### of HPLC 

### analysIs for oallbratlon 

### curve (test item. reoovery test). Date : Feb. 

### 10. 2008 Name: Oill'lll"" Mok; 

### 793 

## -38­Study No. 15089

0.0 11S.0 350• .0 

### Coneentration(mgIL)

y=4055" r"1.00 (400000


# '[

# ~ 

# e 700000 

# 1

o L­

____________________________-J

Concentration PeakInB (mWL) 

## ("v· seQ) 75.0 310923 ISO 609824 300 1214153

Fig. 2 - 2 Calibration curve of

### test item (recovery test). February 10, 2008



## -39­ 794Solvent blank 

### Study 

## No. 15089 Operating Oats: Apr.25,2008 PKHO Tillie 

### 6n in) Area (p V-seo) Height (/IV'J Cone. (I) I 20. 73 217376 8361 100. 00 Total 217375 100.00 Peak position oftest item (blank peak) 

# 1
1 l\. I 0.0



### Standard solution 15.0 

## mill

0.'0 lio

Standar!1 solution 150 mg/L

Standard solution 

## 300 mgfL 20.8



### .sd.o

30.0 Operator : Haokl Ono:Bwe Operating Date: Apr.25.200B PfmO 

### n. Area Hatlht_. 6IJft) (pY·.aec) (II¥) 00 I 20.62 579338 33356 10Q,.00 Toul 579338 100.00

Operator : Naoki Onozawa Operating Dat. : Apr.25.2008 71118 Area ltelitlt**~~** flIln)("V·secl (Jill) .(1). 1 20.62 899282 tI0375 IOQ,.OO Tota! 8911282 100.00

Operator !NaokJOnozftl OpentI ng Date : APr. 25, 2008 PKHO Th. "ra HeIlilt Cone. (iIIln)**.(~** Y'aeo) (p V) (t)
, 2il.60 15t3480 113020 100.40 Totti**~S23480** 100.00

Operator : Naoki Onozawa 

### Fig. 2 - 3 

### Chromatograms of HPLC analYSIS 

### for cal Ibratloo curve (test item. analysis for test solution). Date : Apr. 28. 2008 Name:**Gl\~u.v!.\** / ••**~** 795 

## -40­Study No. 15089
]400000



### o**~------------~----------**

0.0 175.0 3S0.0**C~on(mgIL)**
y"'4146x r-1.00 Concantration Peak area· (mgIL) (p.V·sec)

75.0 301963 150 621907 300 1246105



### • The value ofsolvent blank

### was subtracted from the values ofstandard solutions.

Fig. 2·4 Calibration curve oftest item 

### (analysis for test solution). April 28. 2008



## -41­ 796

### Solvent blank Operating Date: Apr. 25,2008 

### Study No 15089 PKNO Tille Area Height Cono. (mIn) 

# 'u V,seo) (/1 V) 

### ,ti>

Peale position n.d.



# . 

# 1 -A 

# . , • • -----, D.C 10. 0 lO.O 30.0 Operator : Naoki Onotawa



### Stam:l8rd solution 25.0 

## mLl: Operating Date: Apr. 25. 2008 PKHO 

### Time Area Height Cono. (mIn) (/1 V·sao) (pV) 

# '") 1 9.25 212328 7051 

## too. 00 

### Total 212321 100.00

I 

### -"**~** 

## • 1110 

### 2Ilo 

## , 0.9 

# ., Operator : Naokl Onozewa**~t8hdardaolutioD** 60
.. 0 

### l1li1. Operating Date; Apr.25.200s PKNO TIme Area Hel,ht Cone. (mIn) (,ltV·ltc) (/1 V) (I) 1 9.23 423180 14175 100,00 Total 423180 

### 100.00



### A.. '" 

### • 

### , i < 

### 30,0 Operator : Haokl OnotaWi 0.0 10.0 20.0



### Standard solution 100 

### mfIl Operatlne Date: Apr. 26. 2008 fl(fI() Time Area Height Cone. CIIIn) (IJ V-seo) (/1 If) (!I) 1 9.23 853246 27478 100,00 Total 853248 

### too. 00

I 

# A .if'. 

### lio to. 0 

### • 30.0 Operator : Naoki Onezawa


### . :HlO Frg. 3 - 1 

### Chromatograms 

### of 

## HPLe analYSIS for 

### calibration curve (3MBA).

797 

## -42­Study No. lS089
9(}OOOO

'U'**~** 

# i 450000 

# 1

o**~--------------~--------------~** 0.0 60.0 120.0 Concentnltion (mgIL)

y=8S17x 

### r= 1.00 Concentration Peak area (mgIL)**~V'sec)**

25.0 21232& 50.0 423180 100 8S3246

Fig. 3 - 2 Calibration curve of3MBA. Aprl12S.2008



## -43­ 798

### • • Study 

### No
. 15089 

### Solvent bl!!llk Operating Date: Apr. 2G. 2008 

### PKNO Time Area Height Cono. (mIn) (iJY·sec) (iJ Y)**~)**

Peak position n.d.



# I
A 

## , , j 0.0 10.0 20.0 3D. i 0 Operator : Naokj OnOlawa .Standard 

### solution 25.0 mgjl

I 

# ! ..A I 0:0 10.0 20.0 30.0



### Standard solution 50.Q mgfL PKNO 1



# . 0.0 -.i 10.0 I

# ! -" i 20.11 i 30..0**StAD~!rd** 

### so!ulion 

### 1QO mILL PKNO 1
1 . 

# l

.J'. 0;'(1 

### .flo**2~O** 

### 34.0



# . .. Operating Date: Apr.26,200B 

### ftHO Time Area He'Ih.t Cono. IY) fIIhv {IlV'Mo) (1.1
. (J) 

# , 15.43 30021S 24862 100. 00

Total 300218 100.00

Operator : Naoki Onozawe Operating Date: Apr. 

### 2G. 2008 Time Area Heleht Cone. (min) (iJ V·sec) (iJV> (I) 15.43 601729 49506 100.00 Total 601128 100.00

Operator : Haokl Onozewa Operating Date: Apr.26.2008 Tillie Area Height Cone. (min) (iJV'sec) (11 V> (I) 15.42 1215875 100334 100.00 Total 1215875 

### 100.00

Operator : Naoki Onoz8Wa Ftg. 4 - 1 

### Chromatograms of HPLC analYSIS for calIbration curve (SBA). Date : Apr. 28, 2008 Name:**D~~lf."'4. No.o~;**  799 

## -44­Study No. 15089

1400000



## o**'-------------~--------------~**

0.0 60.0 110.0 Co.ocentratiOD (mgIL)

y""12128x r- 1.00 Coocenlration Peak area (JD&'L) (JLV'sec)

25.0 300278 SO.O 601729 100 1215875

Fig. 4 - 2 Calibration curve ofSBA. April 28. 2008



## -45­ 8001 

## Solvent blank 23-Apr ..20 15089-021 SIR of 1 Channel ES. TIC 1.25eS
Peak position n.d.

8.00 7.00 8.00

8.00 7.00 8..00

tUIO 7.00

1.00 2.00 3.00 700 

### l'Of' Fig. 5 - 1 Chromatograms ofLC-MS analysis for calibration curve (pentaerythritol).



## -46­ 801Study No. 1S089

16000 

# r

""'" '-' 

### • 

# ; 8000 

# l**~** 1M 

## o L---

____________ ______________

0.0 8.0 Concentration (mgIL)

y=90lx r-l.OO Concentration Pcakarea (mgIL) (-)

.

3.7S 3478 7.50 6827 IS.0 13470

Fig. 5 - 2 Calibration curve ofpentaerythritol. April 23. 2008



## -41­ 802

## Study No 

## 15089 Standard solution 300 mg/l Operating Date: Feb.09:2008 

### PKNO

I Time (min) 19.37 Area (IJ V'iIlC)

1233965 Height (/1 V) 1385&5 Conc. (l) 100.00 Total 1233965 100.00 I

# ~ I 0.0 I 10.0 

### , 20.0 ..A
• 30.0 Operator : Naoki Onozawa 

### Water + 

### test item -1 Operatinc Date: Feb.OS.ZooS PlCNO Time (min) Area (IJ Y'see) Height (p. Y) Cone. (I) 1 19.38 1192360 134988 100.00 Total 1192360 100.00 1

_10..

# ~ , t I 30. 0 0.0 10.0 

### :0.0 Operator : Neaki OnoZ8W8 Operating Date: Feb.09.2008 

### Waier 

## + test item 

### -2 PICHa Tille 

### Area Heidtt Cone. (min) (pY'IIIIO) (Jl V)**(~)** 1 19.35 1180870 133362 100.00 Total 1180870 100.00



### . I

_A

# ~ j I 

### flO 10.0 

### • a.o 30.0 Operator : Naoki OnoZ8W8 Fig. 6 - 1 

### Ohromatograms of 

## HPLC analysIs 

# . for recovery test (test 

### item). Date : Feb. 

### 10.2008 Name: tillOV.":J, 

### AlI.M

B03 

## -48­

### Study 

### No 15089 

### Sludge + 

### test item -1 Operatinc Date: reb.09,2008 

### PKNO Time Area Height Cone. <min) (/.I V-sec) (S/\I) (%) I 19.38 1170360 131805 100.00 Total "70360 100.00 1

# ~ 0.'0



### Sludge + 

### test 

### • 10.0 

### it!!! 

### -2 i 20.0

1 ..A
• SO.O
PKNO I Operator : Neokt Onolawa Operating Date: Feb.09.200a Time Area Height Cone. (min) (/./Y-Gee) (11 V) (l) 19.35 1161618 130860 100.00 Total 1161618 100.00

.A

# ~ I I 

### • 

### 0.'. 10.11 20.0 30.0 Operator : Naoki Onozawa



### Controj blank Operatlog Date : Feb. 09, 2008 PK/fO TiM Area l!eight Cono. (mIn) (pY'sec) (pY) (l)

Peak position n.d.

.A

# ~ 

# 1

, , I • 0.0 10.0 20.0 

### au Operator- : Naok! Onolawa 

### Fig. Ii - 

### 2 

### Chromatograms of 

## HPle analysIs 

### for recovery test (test item). 

### Date Feb. 10.**200~** 

### Name: Ot.!!'!t\l!4't: 

## 1140\:

804 

## -49­

### Study No 

### 15089 Solvent blank Operating Date: Apr.26.200a PKNO Time' Area Height Conc. (illin) ("V'sec) (11 V) (Il 1 20.73 277864 8363 100.00 Total 100.00**~** Peak position oftest item (blank peak)

1 b 

# -.l\- 1

I I 0.0 

### Itlo 20.0 30:0 Operator : Naoki Onozawa



### Standard solutlgn 300 mgjL Operating Date: Apr.25.2008 PlCHO Time Arl;l8 Height Cone. (min) (p.V·seo) (JlVl (I) 1 20.60 1621409 113170 100.00 Total 100.00**~** I

I

# --~ . • 

### • 0:0 31).0 10.0 211.0 Operator : Nsoki Onezlla 

# mWater + 

### test item Operating Date: Apr. 25. 2008 PKNO Tillie Area Height Cono. (min) (p V-sec) (li V) (I) Test item 1 20.22 169146 25668 10.33 

# 1 2 20.33 327686 34988 20.01 3 20.58**1053~8** 71362 64.31 Converted Converted 4 23.52 87 8 6118 productG products Total 5.35 1637449 100.00 3 Handl 

# L/

• 

# -A

• 

### , 

### , 0.0 

### to.o .20.0 30.0 Operator': Nacki Onozawa



### [2J Sludge + 

### test item Operatlng·Date : Apr.25.2008 Converted SBA PI<NO Time Area Height Cono. Oain) (li V'S8C) (p. V) CI) Converted products Converted 1 13.73 16794 1781 1.41 productB CandD product E 2 14.45 116970 10448 9.71 3 15.43 118292 8995 9.91 4 16.15 198833 25786 16.66 

# /**~,,-** 16.32 36700 35.32 productF 5 421614 5 17.87 18525 2904 1.55



### .

# .~ P""po,;tI" 17.98 26412 3006 2.21 8 20. 72 277341 8141 23.23 

# -A '. '"; / oitest item Total 1193780 100.00 

# !
..... 7 (blank peak) 

# dl.!**~** 0.0 

### • 

# '''0 ilo**3O~O** Operator : Nacki Onozawa



# . Flg. 

### 7 - 1 

### Chromatograms of 

### HPLO analYSIS for test solution (test j

### tern)
. Date: Apr. 

### 28. 2008 Name: Oil.,!!.:!" N".\1 

### 805 

## -50­

### Study No. 15089 

### [3] Slud&e + 

### test item Operating Date: Apr, 25. 2008 

### PKNO Time Converted SBA Area Height Cone, (min) (pV'$ee) (pV) (I) Converted products Converted 1 13.73 15458 1617 1,33 product B CandD productE 2 14,47 102668 9187 8,83 3 15,43 111621 B853 9060 

### / Coo,_ 4 16,13 192209 24986 16,53 5 16.30 417894 35220 35.93

## ~"O"dF 6 17,85 19620 2891 1.60 Peak position 7 17.97 26728 3038 2.30 

# -A 8 20. 72 7976 23.89 

## 5 oftest item 

## mZ§1 Total 1162984 100.00 16

# ilr**~~/** (blankpeak)



### , 

### , I • 0.0 10.0 20.0 30.0 Operator ; Nacki Onozawa



### (4] 

### Sludge + 

### test item Operating Date: Apr.25.2008 

### PKNO Tillie Area Converted SBA Height Cone. (mIn) (,11 Y·aee) (JlV)**~)** Converted products Converted I 13.73 13359 1401 1.19 productB CandD 2 14.45 91004 8166 8.07 

# /,ro&w:,... 3 15.43 100832 797.4 8.95 4 16.12 188796 24454 16.75 5 16.30 412810 34216 36.63 6 17.87 17595 2772 1.56

# ~-P 7 l7. 97 25577 2915 2.27 Peakposition 8 20. 72 7927 24.59
.**iZZl~g** 

## i . / oftest item Total 1127115 100.00

# ~ 1

# I :

# 1. i.A (blanlc peat)

j 30.11 a,'o 111.11**211~** 0 

## , Operator : Naoki OnoZ8wa

[5] 

### Control blank

Peak position oftest item. (blank peak) PKNO Operating Date :**A~r.25.2008** Time Area Height Cane, (min) (JiY·see) (pV) (%) 20. 72 277810 7916 100.00 Total 277810 100.00

# ~ 0."0 10.0 

# 1 1 (}.. 

### ill. 0 

### , 311;0 Operator : Naoki OnOZIW8 

### "Fig. 

### 7 - 2 

### Chromatograms of HPlC analysis for test solution (test item). 

### Oate : Apr.28,2008 

### Name : Olll:!!
.....**ftf".~**



### 806 

## -51­

### Standard solution 100 

### mgt'LQIlML_'_.

___

I 

# 1\ • ..A I I I 0.0 10. I) 20.0 Operating Qate : Apr. 26. 2008 PKNO Time Area Height Cone. (min) (pY·sec) (J1Vl (%) 1 9. 25 852951 27554 100.00 Totsl 852951 100.00**Operat~r** : Naoki'Onozewe 

### Standard solution 

### 100 mg/l (SBA)



### "**~.o** Operating Date: Apr.26.20OB PlCHO TiIlB Area He i ght Cone. CIIin) (Sly·sec) (p,V) 00 1 15.42 1216558 99299 100.00 Total 1216558 100.00

OperBtor : Neokl Onozawa 

### [1J Water + 

### test 

### item

Peak position Peak position of3MBA ofSBA n.d. n.d.

# ~\ 0,'0**1~O** Operatlnl Date: Apr. 26. 2008 flKNO Time Area He i gilt Cano. 6IIin) (SlV-sec) (11V) (I) 1 20.18 54167 8355 8.74 2 20. 32 106593 11526 17. 20 3 20. 57 425966 21901 66. 73 4 23.47 33075 2281 5.34 Total 619790 100.00

Operator : Naoki Onozewa 

### [2) Sludge + 

### test item Converted Converted SBA product B products CandD Converted productE

# :~f~d,"**~=** 

# =--\**.~ ~** **~~** zl!a 1 A

_____ 

### 38.0 Operatinl Date: APr.26.200a 

### PKNO Tills Area Height Cone. (/Rio) (Jl Y·sec) (IIVl (I) 1 8.67 61578 2283 17.09 2 13.75 7725 887 U5 3 14.47 42088 4111 10.65 4 15.43

## ~H~ 3122 9.76 5 16. 13 9104 18.30 6 16.30 151829 13819 '38.41 7 17. 85 6390 1004 1.62 8 17.97 8792 1016 2.22 Total 395312 100.00

Operator : Nloki Onozewa 

### Study No. 15089

Fig. 

## a - 1 

### Chromatograms of HPlC analYSIS 

### for test solution 

### (3MBA and SBA). Date : Apr. 28. 2008 Name: OM'l4.w" #D.o**~'I** 

## -52­ 807____ _ Converted SSA

Peak position of3MBA

# n~'d'\ Converted 

## tltSi

CandD

Converted n.d.



### . 2:Ht 

# i Study 

## No. 15089 [3] Sludge + 

### test item Operating Date: Apr.26.2008 PKNO Time Area Height Cone (min) (J./Y,sec) (p.V) (If Converted 1 8.67 71504 2398 18.32 product E 2 13.73 7122 821 1.82 3 14.45 36649 3496 9. 39 4 15.42 

## .nm 3077 9. 55 5 16.12 

### i2849 9669 18.61 

# .**v~::~** 6 16.28 149977 13580 38.42 7 17. 87 6374 994 1.63 8 17.97 8815 1014 2.25 product A Total 390371 100.00 A

_________ 

### zO.o Operator : Naoki 

### Onoz8wa 

### I4J Sludge + 

### test item Operating Date: Apr.25.2008 Converted Converted SBA PKNO Time Ar. Height Cone. product B products Converted product E 1 2 (mIn) 8.65 13.75 (J./Y·8ec) 73280 6402 (/iV) 2464 728 (I) 19.14 1.67 3 14. 45 32706 3194 

### e. 54

# I/~= 4 5 6 7 15. 43 15.12 16.30 17.85**~** 

### 7Hii 149973

6340 2763 9664 13581

977 8.71 18.89 39.18

1.56 

### e 17.97 8439 978 2.20 product A Total 382775 100.00 A I 0.'0 

### eri.o 30.0 Operator : Naokl Onozawa



### L5] 

### Control blank Operatinr Date: Apr.25,2008 PKNO Time Area Height Cone. (mIn) (yV·sec) (gV)**~)**
Peak position Peak position of3MBA ofSBA n.d. n.d. 

# j 

# \ -'" I**IO~O** 20.0 Operator : Nlokl Onozewa 

### Fig. 8 - 2 

### Chromatograms of 

## HPLC analysis for test solution (3NBA and SBA). 

### Date : Apr. 28, 2008 Name: OIlo3

..../l Nl1lk



### 808 

## -53­15089-025 

### tandard solution 15.0 

### mg/l 23-Apr.. 200 SIR or 1 CIIannal ESTIC 

### uSeS

Area

1

Peak position lI.d.



# 1 [1] 

### water 

### + test Item 15Oe$.028

3;()O 4.00 11.00 6 6.00**I~Ol~** 1 It, 'U Fig. 9 - 1 Chromatograms ofLC-MS analysis for test solution (pentaerythritol).

809 

## -54­1 

### [3] 

### Sludge + 

### test Item 23-Apr-20 15089-028 SIR of 1 Channel ES. TIC 1.25&8

Am

7.00 8.00

Peak.position n.d.



# 1

••00 11.00. 6.00 Fig. 9 • 2 Chromatograms ofLC·MS analysis for test solution (pentaerythritol).



## -55­ 8101 

### 100 mg/L solution of

## test ltam 15089-060
Test item 

# 1

100 mgIL solution 

### of3MBA 1&Oee-oer t

3MBA

-5.
. 0.00 5.00 10.00 15.0a 

### 100 

### mgIL aolutJon ofSBA '16tJ8e:.0ti9 1 1f.05 

# / 20.00 2S.00 30.00

20.00 25.00 30.00

SBA



## I,........ Nil,o\c; Fig. 10· 1 PDA chromatograms ofLC-MS analysis for test solution (LC-MS sample -2, qualitative analysis ofother converted products, 

### A. = 20S---300nm).



## -56­ 811

### [1] Water +

## teat ltam 15089-070 1 Test item Converted products HandI Converted 

# 1 productG

# ~20 .,..



### [2] Sludge + 

### teat Item 1S089-071 'I ConvtJted products CandD

Converted prQdUets CandD SBA

16.00 20.00

SBA 

# I**CODv~** productE 30.00 

### 2S-Apr-200 2: DiodeArray TIC 1.4Ga7**·~'lf.\~N'M** • Fig. 10 - 2 PDA chromatograms ofLC-MS analysis for test solution (LC·MS sample -2, qualitative analysis ofother converted products, A

## =**205~300nm).**



## -57­ 8122.82

20.00
. 25.00 

### [41 Sludge + test Item 15089-078 J Converted products SSA Converted 1.40.7 CandD product E

# ~ Converted Converted product B 

# I

# L**p"ooaF~** ______

1 Converted product A 

# \ [11 COntrol blank 15089-074 fU4

15.00 20.00 .00

30;00 

### Fig. 10 - 3 PDA chromatograms ofLC-MS analysis for test solution (LC-MS sample -2. 

### qualitative analysis ofother converted products, A. 

## =205-300nm).**l~~** 

### lIiIl.lf..**~**



## -58­ 8131

7.1Oe8

Fig. 11 - 1 Total ion chromatograms ofLC-MS analysis for test solution (LC-MS sample -2, qualitative analysis ofother converted products).



## -59­ 15.00 

### 2&-Apr-2 1: Scan E&f-

. TIC 

### 100 mglL solution of test Item 

### 24-Apr.2 1508Il-08O 1: Scan ES+ TIC 7.5088 20 7 Test item 

# /

0.00 5.00 

### to.oo 1 • 20.00 2500
. 30.00 

### 100 mg/L solution of3MBA 15089-06& 1



### 8141 

## 11]Water + 

### teSt Item 15088.Q70· 

### 2S-Ap.... 

### 200 1:80lIlest

• TIC

7.5Oet Test item

Converted products CandD

lMlO 5.00 10.00 

# III Sludge + 

## test rtam**1~** 1 Converted ptOdttcts .CandD Converted ConvelUd

16.00

Convened productS

15.00 20.00

20..00 Converted products Hand I

25.00

25.00

lUM 30.00

Fig. 11 - 2 Total ion chromatograms ofLC-MS analysis for test solution (LC-MS sample -2, qualitative analysis ofotherconverted products).



## -60­ 81510 

### [41 

### Sludge +

### test Item 2H\pr·200 16Ofi.073 1;ScanES+ TIC 7.50e6



### [11 Control blank 15089-074 1:ScanES+ TIC 1 7



# "

20.00 111.00 

### Fig. 11 - 3 Total ion chromatograms ofLC-MS analysis for test solution (LC-MS sample -2, qualitative analysis ofother converted products).



## -61­ 816

### [1] Water + 

## test Item 

### 26-Apr .. 2 150&9-070**~O.2fJ** 

# J

1 Totalion chromatogram

O. 6. 2: Diad. Array 10 PDA chromatogram (:t z 205-300nm) TIC Converted 1.4007 products Converted Hand I product

## a**-51:;:~~:;:;::;::;::':;:::;::;:;:;:;:'TTT..,.,..,..,..,.......,....,..,..,..,,..r~-'-.** I'!' 'I" i' Iii" Iii ii," i' , , 'i" i' i' i TIme 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 [1] 

### Water + 

## test ItaID 

### 25-Apr .. 20 15Q88.;070 1: Scan &1+ TIC 7.5Oei Converted

10.00 1: .09**2O~OO** 11.00 '30.00 [1] 

### Water + l8at 

### item 15089-070 1102 (20.231) em (1076:1102-(1102:1143+1040:1074»(1.500) t 1 Mass spectrum of converted product 

### a

Fig. 12 - 1 Mass spectra (LC-MS sample -2, [1

## JWater + test item).



## -62­ 817

### [1] Water + 

### test item 25-Apr-2 15089-070 1266 (23.238) 

### em (1244:12BS-(1285:1323+1214:1242)x1.500) 1: Scan ES+ 1 321 1.80&4 Mass spectrum of converted products H and I

900 90S 

### / 323 89B



### 818 Fig. 12 - 2 Mass spectra (LC-MS sample -2, [1)**~ater** + test item).



## -63­

### Study No.lS089 

### Converted product G ([ 1] Water 

## 7" test item)


## .- ....**-----.-.~.-------.-----------** 

### ...

## ----- -- ... ---_

### ....

## ------ , 

### .... , ,

,

, , , , , ,



### ...... _-_ ............. --_ 

### ...... ,:; 

## .. : 

### .... _---_ 

### ...**---.~-.-** 

## ..

# - ....

# -. mJz IODS 

## 187 Unknown 

## 321**(Mo-C~S-H20+Ht** 

## 339**(Mo-C4~OS+Ht** 

## 423 (Mo-H20+Ht 441 (Mo+Ht Fig. 12 - 3 Mass spectra (LG·MS sample -2, [1] Water 

## +test 

### item, 

### presumed ions ofconverted product G).



## -64­ 819

### Study No.l5089 Converted product H ([1] Water 

## +test item)

_. _____ • ______ • ______ • ____**~** _________**~** ___ • _______**~** _____ ______ • _______________________________ a ______ • __ _



# ....Converte<fprodiict·i·············:·················
..__ ._. --.....-....-.._ .......... _ ........ ([lJ Water 

### + test item)**-----~** 

## ...**----~.----** 

## ... --- 

## ....**-----------.------------.--------.--~--.--.-----.----.-----------** 

## ..

## ----­

Fig. 12 - 4 Mass spectra (LC-MS sample -2, [1] Water 

### + test item, presumed ions ofconverted products H and 

### I). Date ).lIlt.Ii.n



## -65­ 820

### Study No.15089



## mJz Ions 

## 187 Unknown 221 Unknown 289 Unknown 321 (339

- 

## H2

## 0t 323 See structural formulae 339 See**s~ctural** 

## fQrmu1ae 423 (441- H2

## 0t 441 See structural formulae 

## 543 See 

# structural fonnulae 

## 763 See structural formula (converted product H) 

## 863 ( 

## MI

- H 2

## 0 

# +H 

# t 865 (MH 

## -H2

## 0+Ht 881 (M1

## + 

# Ht 883 (MH+Ht 898 (M, 

## +

# N114 

# t 900 (MH 

## +NH4

# t 903 (M1+Nat 905 (MH+Na)+ 919 (Mr+K)+ 921 (MH 

## + K)+ Fig. 12 - 5 Mass spectra (LC-MS sample -2, [1] Water 

### + test item, 

### presumed ions ofconverted products H and I).



## -66­ 821

### [1] Water 

### +·test Item 28-Apf.i
. 15089-010 1 PDA chromatogram (A =**20S~300nm)** Converted products Hand I 2: DIOde Array TIC 1.4Oe7 Converted product G**~'1A1.'2f)**

15.00 20.00'



### 1J Water +

### teatltem

# ~.j.

89-010

# 2Il~1D J

25.00 30.00

Mass chromatogram of 1

m/z= 187 211.1l1 " , , j i , , 

### i,')i , I' 

### i " I' ' i , 

### I'" i 'I

# ,'t. ,II II , "I I I Ii' Ii jill iii •• 'I Ii , i jI ",

# ".1 ,I 150J9.070 f:Scan ES+ 

# 'j""""';"""""""""" :1.""

# I",.,,""
..

# ""~**~:,=,~f"**,'''::;

15009-0 

# · 70 • 

# I

1: Soan

# £::9 

# 11

46 :M.O& Mus chromatogram of 1.6714 t

# j '''''I'''' I" "r' 

## "I"'''''' 'r' ",,

# ~ i...

# ,J",
....**,,,,:~:**

# ..,;,, 001 

## I" " 15089-070 t:**~n** EG+ 1. 321 Mass chromatogram of 4.14415 mk-3l1

It'll" 'lin, 

### It.l.!- QIIo,.**~** .v ..**~"** Fig. 13**~** 1 Mass chromatograms (LC-MS sample -2, [1] Water 

### + test item).



## -67­ 822

### [1] Water + 

### test Item 25-Apr-20 15089-070 1: Scan ES+

# ~l 

# ..,"
.., .."',,
.., ...., ...., """".1. ,0""
.. ,,'

# ,,~~**~~~= ~~''1W~**  "'j70 '''' "-"l',;



# J
..."
...; ...., ....,.".,."" , ..,',
..

# 1
..r."",
..,:.

# ~',~.~**~~, ,~A1"**  "'570
..... ',
...,,!"



# J.,
..,, ...,.,.., ....,.,.., ..."" ,.,....

# ~,.

# I,,.,.:,..**~~~~~~ ~~** 

# ..**~** 15089·070 1: Scan ES+ 

# 'j
... '1 •••• " •• '1 "" " ••• ,,, •• ,,, •• , ••

# I
. . x",.,
.., ..

# ~~=~ 

# ..**~.:::** 16089-070 1: Scan ES+ 1] • 

# 23M Mass chromatogram of 

# 1.A: mh.=S43 

# 1

• i • I J I •**~** i , t Yl /, j 1 i • • i • I , , 1\ t i • If. , , , i , I , IAt, •ftl ,1 I i ( i I j i , , « (t , , ( i • I i 1 , • , , i j i i $ , t 

# '1:':.,:.",
....,,,..,,,,,,.,,,'"'' r'" ""

# "1,,,,,,,,

# .~,~~='.~~! 15089-070 

# J'
1: Scan

# E: 1] Mass chromatogram of 4.22e3 miz-= 863

...

# :~ ... , .... f ••• ',"'"

# ~)" ,'" /,'"'\''' ".,
........ ".,.,•• , •• "0
.. ", 

# .>0, 

# 1;oa fl.t1TO D[**1:s~6n.~;** Mass chromatogram: of ."""'" miz= 86S 

# j

, tuo t i', j i • , , •r,f, i • " 

# i' f j J • i', I t I i it . I \ "" • l • f ,'. , • '11" .• t titl '( , ,It. i •• , t '/#1 j j I •, , 1, , I. f 1 11me 0.00t5.0010.oo 15.00 20.00 26.00 30.00 35.00 40. 

### ISOfC\**"~Q" I.p~**  OIl.O)AIV,. PRot; Fig. 13 - 2 Mass chromatograms (LC-MS sample -2, (1] Water + test item).



## -68­ 823

### (1) Water 

### + 

## test Item 

### H-Apr40 1S089..()70 1: Scan ES+ Mass chromatogram of 5,76e3 

# I m/z:= 881 

# 'j , I**~**

•6 

### -"
.. ,,, "I"" 

# 1'"""",,,,,,,,,,,,, "'''''''''''' '" ,,,, ""'II,,,,,

# "I
...., .i 11 089-070 1: sean

## E~i

. Mass chromatogram of 7;02&3 mlz" 883 

# j

\ i i. t' ii, j , 

### 'j', j'" 

### 'i' I',

## II~~'" "1 n" j'"" 1,'1' ,II j\ i, i ,,', "1

### ,.r" I 

### ,''''I i I

# '1'.,**~r 'Soo'':~**  

# ...,,' J..""", ""f""." 

## ,i."'"'' "''''''''

# ,J"""

# .'''~**~~:~~~,O,~,,'_** 15089-07Q 1: Sea n E'S+ 

# 'i".""."".""'''''.''"''''''''"''''I,

""1"

# ,~~~,~:;~,~"**~,'=** 16089-070 1: SCln ES+ 

# '1", ''''''''''''q",'"" 'I"""""'"

# I",'"**'''~,~~:**

# ...'","':

15089-070 ' 1: Scan ES+ 

# '1.,""""",.,"""" ,.."."".""." 

# ,1""""

# ,~~.~,,...,,,, 15089-070 1: $(;an ES+ 

# '1""",""",',".' "'t'" '.1" '/'" 

## ,to'

# .1,,1,

# "",,~~:,o:, 

# . .',,::;

16089-070 

# 11ft 1: Sean

# e::1



# 1
.. """,.n"."" i'" ''''''. """" ,/,,,

### '"""'yo"'."

# ,~~**:~,::,** .': o. 11.00 10.0& 100 20.00 25,00 30.00 35..00 40.**~** l.nt, I.f;.**').~ N"b~i**  Fig. 13 - 3 Mass chromatograms (LC-MS sample -2, [1] Water 

### + test item).



## -69­ 824

### [2] Sludge 

### +test Item 

### 25-Apr-200 15089-071 2: Diode Array

10 Total ion chromatogram TIC

16089-071737 (13.536) em (711:7A16-(750:nS+666:707)x1.500) 1: Scan ES+ 1 .., ".1ge3 Brank ion Mass spectrum of converted product B


# ,

# 1



# ..

700.800 100 2**~~I\** 'Uti.

### I.t. 1.S 7.SOeS Converted Converted

20.00 25.00

Mass spectrum of converted product A 10 TIC 1.40e7



### [2] Sludge 

### + test 

### item2l-Apr-200 15089-071 1:scan E'S+

Fig. 14 - 1 Mass spectra (LC-MS sample ·2, [2] Sludge + test item).



## -70­ 825

### [2] Sludge 

### + test Item 25-Apr ..

### 20 15089·071 n2 (14.180) Cm (750:787-(781:809+740:759)x1.500) 1: Sean ES+



### ,0- $711 10 9.2Oe3 1121 Mass spectrum of converted products C and D m 

### las 

# •% .7 fP 221 

# ,.1 

# '.**I~** l J 

### Ild 

# 1 " 'I 15089-071 863 (15.84a) Cm**(856:9~(900:930+B21** :655))(1.500) ." 6ft

### IsI. 'T 1 321 '1 

## ., 

## ., 1: Sean ES+ 1A2e6

15089-l:l71 973 (17.BeS) em (972:985-{995:1031+947:971)X1.5OO) 1 1 7 Mass spectrum of converted product B

1: ScanES+**2.7~** Mass spectrum of converted product F

Fig. 14 - 2 Mass spectra (LC-MS sample -2, [2] Sludge + test item).



## -71­ 826

### Study No.15089 

### Converted product A ([2] Sludge 

## + test item)

....... "'
.... _ ...... _ ............... __ ........ oo .......................... - ......... -_ .....

## --I**L_~** ...... __ ..... _ ...... _ ..... __ •••__ ._ ........ _*
___ ......**_~** ___ .....**~** 

## mJz Ions 112**1Jnkno~** 

## 137 (MA - 

## CJ1:60S 

## + H

# t 160 Unknown
. 221 (MA 

## -H2

## 0+Ht 239 (M A 

## +Ht 261 (MA 

## +Na/ Fig. 14 - 3 Mass spectra (LC-MS 

### sample -2, [2] Sludge 

## + test item, presumed ions ofconverted product

## A).

-72­ 827

### Study No.15089 Converted product B ([2] Sludge 

## +test item)



## mlz 

## Ions 

## 457 ( MB

- 

## H2

## 0 

## + H

# t 475 (MB+Ht 

## 497 ( MB 

## +Na )+ 

## 513 (MB 

# + 

# Kt Fig. 14 - 4 Mass spectra (LC-MS sample -2, [2] Sludge 

## + test item, 

### presumed ions ofconverted product B).



## -73­ 828

### Study No. 15089 Converted product C ([2] 

### Sludge 

## +test item)**;------.~-------** 

### ... --------- 

## ...

## ------ ..**---------.~.-----_._--------_._-----------;** 

# · 

# . · 

# . · . 

# · : 

# · · · 

# ·

· _______**~** ______ ._._____ •______ •___ •__ ._._.___ ••______ --- ••_------ •••_----_ •••••-.1

# .**~~** 

### Converted product D ([2] 

### Sludge 

## + test 

### item)

Fig. 14 - 5 

### Mass spectra (LC-MS sample -2, [2J Sludge 

## +test item, presumed ions ofconverted products C and DJ.



## -74­ 829

### Study No. 15089



## mJz Ions 128 Unknown 187 Unknown 221 

## See structura1 fomula,(converted product C) 

## 32l (Mc-2H2

## 0+Ht 339 (Mc-H2

## 0+Ht 357**(~+Ht** 

## 379 (Me 

## +Na·t 395 (Me+Kt, 489 (Mo -H2

## 0+H)+ 529 (Mo+Nat 545 (Mo+Kt Fig. 

### 14 - 6 Mass spectra (LC-MS sample -2, [2] Sludge 

## + test 

### item, presumed ions ofconverted products C 

### and D).



## -75­ 830

## Study 

## No. 15089 Converted product E ([2J Sludge 

# +test item)

, ..**~** 

### ............. 

### ...........-.. 

### -- 

### ......................

## --_....... -_ ............... .**~** ,



## rnJz Ions 187 Unknown 321**(ME~H20+Ht** 

## 339 

## (ME+Ht Fig. 14 - 7 Mass spectra (LC-MS sample -2, [2] Sludge 

## + test item, presumed ions ofconverted product E)
.

. Date , •

### .,.!t 1.$'



## -76­ 831

### Study 

### No.15089 Converted product F ([2] Sludge 

## + test item)

• •• ____ •••••• _ ••• _ •••••••••• _ •••• _ •• *.A
___ •• __ • _____ • __ •• __**~._**



## mJz Ions 659 (MF-H20+Ht 677 

## (MF+H)+ 699 (MF+Nat 715 

## (MF+Kt Fig. 14**~** 

### 8 Mass spectra (LC-MS sample -2, [2] Sludge 

## +test item, presumed ions ofconverted product F).



## -77­ 832

### [2J Sludge 

### +

### test Hem 26-Apr-20 150$9-011 1

Tota) ion chromatogram COnverted Converted Convened. product B procJuctB produefS: 

# I CandO 

# \**,~**

i

'$

Fig. 15 - 1 2: DIede

## /lira., TIC

.. 1.4061**2Up~** 1:Sc:an ES+ TlO 1.s0.6

Mass chromatogram of mJz=- 112

Mass chromatogram of mJz= 128
31**~** 34.073$.88 1: ScanES+ 13 Mass chromatogram of 1.91.5 mJz= 137

1: SdanES+ 180 Mass chromatogram of 7.58M mJz"'l60



## -78­ 833

### [2] Sludge + 

### test Item 15089-011 1

jut 

### 28-.AprMass chromatogram of mJz 186

1: Scan ES+ 187 Mass chromatogram of '1,57.5 mlz= 187

1: Scan es+ 221 Mass chromatogram of 1.SgeS mIz-ll1

1: Scan ES+ 230 Mass chromatogram of 5.4B84 mJz=239

1: Scan es+ 261 Mass chromatogram of 2.0784 mlz=261

.1: Scan fS+ 321 Mass chromatogram of 5,32&5 mlz- 321

1: Scan ES+ Mass chromatogram of 1.$9 mlz 339

"llIo\.1f\ 1$­**n",~ VQ.~~'**  Fig.15 2 Mass chromatograms (LC-MS sample -2, [2] Sludge + test item).



## -79­ 834

## [2] Sludge + 

### test Item 25-Apr-200i! 1soeo.o71 1: Scan ES+ 1 351 Mass chromatogram of 1.40e4 mlz = 357

1:Sc:en ES+ 37t 14.111 Mass cbromatogralJl of 2.30e4 mlz-3?9

1: Sean ES+ 1401 396 Mass chromatogram of 1.18&4 m/z=395

\I

1: Scan ES+ 497 Mass chromatogram of , .3:284 mJz"'497
11.22 2688 5.00 10.00 15.00 20.00 25.00 30.00 Fig. 15 - 3 Mass chromatograms**(LC~MS** sample -2, [2] Sludge + test item).



## -80­ 835

## l Sludge + 

### test Item 15089-071 1 fl'O

21 

### 26-Apr-2OO 1: Scan ES+ MaSs chromatogram of ,

# .o!!: m.h 2 513

1; ScanES. 529 Mass chromatogram of 1.41114 mil. 529

':SC8n'ES+ '545 Mass chromatogram of 7.87. m/l.= 545

1: Scan ES+ &59 Mass chromatogram of 2.74e3 miz"'659

1: Sean ES+

,**~** Mass chromatogram of 7.65$3 mfl.;: 699

1: Scan ES+ 715 Mass chromatogram of 4.6663 m/z=715

10.00 15.00 20.00 Fig. IS - 4 Mass chromatograms (LC-MS sample -2, [2] Sludge 

### + test item).



## -81­ 836Fig. 16 - I Mass chromatograms (LC-MS sample -2, [5] Control blank).



## -82­ 837

## ------- ---

Mass chromatogram ofmlz "" 187

1: Soan ES+ 221 2.

1 1 . Mass chromatogram ofmlz = 323

Fig, 16 - 2 Mass chromatograms (LC-MS sample -2, [5J Control blank);



### 838 

## -83­

## 1~j089-0~~1 

# . Mass chromatogram ofmlz"" 321 1:

## sca:,:;~
lAO 11." 2Q.13 ''f i I I'" i I 'I ,'n I I' "

### ~ I " , I i

### ~ i , i 1 

## A
.. I t'd i

### '1',**~'"** II'" I Ii,' •• i I I 1/\ II' I ,,, t I

# ""'1 • '1 q i I hi 15*-074 1: Scan ES+

## [6I Control blank' 

### 26..,.1'-2 16Ol1&-O74 1: Scan ES+ 10 1
.. Mass chromatogram ofmlz '" 379 379 2.5883

1S8N74 1: Scan ES+ 

# '3.1
..'"",., I.'

# '~'" ".

# '~,~' "1

# x:',~:~:~::~::"r.'I'**A'~~** 1eG8N74 1: Scan es+ 1 1 Mass chromatogram ofmlz

### =457 457 1.11eS

Fig. ] 6 • 3 Mass chromatograms (LC-MS sample -2, [5] Control blank).



## -84­ 839

### [5] 

### Control blank 18081).1)74 t 1.$11 Mass chromatogram ofmlz" S29

Fig. 16 - 4 Mass chromatograms (LC-MS sample -2, [5J Control blank).



## -85­ 840

### [5] Control blank 15089-074 Mass chromatogram ofmlz = 86S 

### 2&-Apr-200 1: Scan es+ 21.59 865 1.74113 24.«1
1: Scan ES+ 17.t. Mass chromatogram ofmlz '" 881 881 596

900 708

1: SQn ES+

. 905 Mass chromatogram ofmlz >= 90S

. 884



# -**O~~').Illvo.** 

### No.o**~~** Fig. 16 - 5 Mass chromatograms (LC-MS sample -2, [5] Control blank).

841 

## -86­

### 100 mg/L solution of 

### test Item 1!OOO-G59 1 Test item 

# 1 20,37

3MBA

1 1.40.7 11;0

7 SBA 

# / 100 mgIL solution of

### 3MBA**1508~1** 1 .



### 100 mg/L solution 

### ofSBA**150~2** 20.00 25.00 SO.OO

Fig. 17 - 1 POA chromatograms ofLC-MS analysis for test solution (LC-MS sample -3, qualitative analysis ofotherconverted products, l = 205--300nm).



## -87­ 842

### [1] Wafer 

### +test Item 150&9-063

Test item Converted Convened

# ~~~ 

# 1**~~~G~~~ ~**  __----------­

Fig. 17 - 2 PDA chromatograms ofLC-MS analysis for test solution (LC-MS sample ·3, qualitative analysis ofother converted products, 1 

## =205-300nm).



## -88­ SBA

to.flO ....

1 • 21:1.00
Convened
prodIlctl B .coavened productF

# r~ .....

15;00 20.00 25.00

Converted pt«luctP

IS., 30.00



### 843

### [4] Sludge + 

### taat Item 

### 2a.Apr.200 16089-068 2: Diode Artay 1 TlO Converted 1.40.1 SSA product E

10.00 

# j Converted 

# r 

# /.."-P

......

15.00 20.00 26.00 30.00

15.00 20.00 25.00**t~1\.** Fig. 17 - 3 PDA chromatograms ofLC-MS analysis for test solution (LC-MS sample -3, qualitative analysis ofother converted products, A.:::: 205-300nm).



## -89­ 844

### 100 

### moll solution of 

### test Item 15089-0S9

2M

0.00 15.00 10.00 15.00 

### 100 

### mg/l solution of 3MBA 15089-061 1**~.OO** $..00 10.00 15.00 20.00 25.00 30.00 

### 100 

### mgllsolution of SBA 15089-062 1 

### 24-Apr-20 1: Scan ES+ TIC 7.SOe8

Fig. ]8 - 1



## -90­ 845

### [1] 

### Water 

### +

## teet ltam 150a9..0e3 1

Converted COnverted products 

# _\HC 24-Apr 1: ScanES+ lIC 7.5Oe6

21ft 

## .w.1a•••-­

0.00 5.00 00 

### [2J 

### Sludge 

### +

### teat If8m
.**15~4** . 1

Converted pnlducts CandD 15.00

Converted productB 

# 1

'"'" 11.01 20.00 25.00

20;00 25.00 30.

30.00 . 11me 3$;00 40.00 J4.A,PN:tJUII 1: ScaIIIS+ TiC 1.50e6

Fig. 18 - 2 Total ion chromatograms ofLC-MS analysis for test solution (LC-MS sample -3, qualitative analysis ofother converted products).



### -91 84610 

### [4] Sludge 

### +

## test Item .26-Apr-200 1508&.:.066

# ~~**~~W-~'~~"MR~~**
20.00 1: Scan ES+ TIC 1.60e6 Converted productE

# .~:~ 1

21.00 30.00

4.1.

26.00 SO.OO**~** Fig. 18-3 Total ion chromatograms ofLC-MS analysis for test solution (LC-MS sample -3, qualitative analysis ofother converted products).



## -92­ 8473 Chemicals Evaluation and Research Institute,lapan Kwume Laboratory

:2

Abs ).for

### HPLC (210nm) 1

-0.1 

## t-

__--I..-

__

# :::t::====:::::i::==::::::::r======:::t::==:::j

200 250 300 340 Wavelength (nm] Files 15089 200ppm Fig. 19 

## Uv spectrum oftest item. Study No. Date Sample Solvent**~** Cd! Instrument Photometric MQQC 15089 April. 08, 2008 Test item' Acetonitrile 10aunXIOnun,quanz V-660 Abs Wavelength Scale Limit Slit Width Scan Speed Sampling Pitch Analyst Note 200.00-340.00 ..(l.lo-3.00 (UVNIS)2.0 run 2000mlmin 0.200run Akfto**"D~o.** 200mg/L



## -93­ 848Chemicals Evaluation and Research Institute,Japan**Kururo~** 4borato:ry**3r-------------------------------------------~--~**

2

Abs A. 

### for HPLC (21Own) 

# 1

..0.1 

## L

__**--1-_~=C====:::::i::=====::r======::::i::=:j**

200 250 300 340 Wavelength [nm] lS0891S0ppm 

### Fig. 20 UV spectrum of3MBA. Study No. 15089 Date April. 08, 2008 Sample 3MBA**~o!vent** Acetonitrile**~I** 10000xlOnun, quartz**~** V-660 !Photometric Mode Abs Wavelength Scale Limit Slit Width Scan Speed Sampling Pitch Analyst Note 200.0()'340.00 -0.10-3.00 (UVNIS)2.0 nm 200nmlmin O.200nm 

### AI<i/:() tcotAA I'SOmgIL



## -94­ 849, Study No. 15089 Wavelength 200.00·340.00 Date February. 06,2008 Scale Limit -0.10-3.00 Sample Solvent SBA Acetonitrile Slit Width Scan Speed (UVIVIS)2.0 run 200nmlmin Reference Sampling Pitch O.200nm Cell Instrument 10nunxlOnun,quanz V-660 Analyst Note A )c,'!:/)**Ic.,,~** 200mg/L Photometric Mode Abs Chemicals Evaluation and Research Institute,1apan Kurume Laboratory**3r---------------------------------------------~**
A. for 

### HPLC (210run)

Abs 

# 1

1

·O.1l--.....---......I---_-l..

____L-

_____-L-

___**--L_~**

200 250 300 340 Wavelength [nm] Files IS089SBA-2 Fig. 21 UV spectrum ofSBA.



## -95­ 850Study No. 15089 Instrument MS: Waters ZMD, HPLC ; Waters AlJiance2690, PDA : Waters 996PDA Sample Standard solution 1 S.O mgfL (pentaerythritol)
HPLC Conditions

Inlet system Column Sample size**~L** (Solvent Purified water ) Column L-co!umn ODS

CoJumn size**~Cnl** x -kLmm I.D. Eluent Smmol/L AnnuoniUnl acetate Flow rate**~mUmin**
MS Conditions

Ionization mode ESI Detection mode Negative FWlction Scan Mass range (mlz) J 2S

- 300 Source block temp. 'C Desolvatioll gas 400 LIhr Source Cone _1_5_V Desolvation temp. 400 'C

Kurume Laboratory, Chemicals Evaluation and Research InstiMe, Japan


### Fig. 22 - 1 Mass spectrum of pentaerythritol (analytical conditions),

Date 1009, i. IQ



## -96­ 8511 

### tandanl solution 16.0 mgIL 

### 1O..feb-20 15089.Q()4 SCanfS.. 3.82 TIC i.86e4 Total ion chromatogram'
Peale 

# I

%



### StanclaWl8olution 11.0 

### mg 1O-Feb-200 15089-004 207 (3.820) Cm (195:233-(238:299+125:193)x1.500) Scan ESt ,195.3\ (M+CH,COO)o Mass spectrum '890

%

# ~**OB**..' . H .**O~** H CSH I20. Mass nwnber (M) : 136

140 160 Fig. 22 - 2 MS spectrum ofpentaerythritol.



## -97­ 85210 

### ndard solution 15.0 

### mUll 1O..f.1J..1Q0 1508g..Q04 ScanEs­ 3.12 

### TIC 1.8884 Total ion chromatogram 

# / Peak

Mass chromatogram of m/z"" 195.3



### at

6.00 8.00 

### 2.00 Fig. 22 - 3 MS spectrum ofpentaerythritol.



## -98­ 85375

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### No. Sample 

### Method Date Name 110m



### : Shimadzu IRPrestige-21 

### : 15089 

### :Test item 

### : Neat : December 21. 2007 

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### item measured before experimental start.



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### : Test item' Method 

### :Neat Date ; May 16, 2008 

### Name**:~** 

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### IR 

### spectrum oftest item measured after experimental completion.



## -100­ 855

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### sponsor.



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# cERl
Receipt number**632~06~S-4711** 

### 14711



# FINAL REPORT

Biodegradation study ofKarenzMT(pEMB) by microorganisms

Septe.mber28.2007
Kurume Laboratory Chemicals Evaluation and Research Institute, Japan

858

### 14711 

# STATEMENT

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan
Sponsor SHOWA DENKO K.K.

Title Biodegradation study ofKarenzMT(pEMB) by microorganisms
Studynumber 14711



### I, the undersigned, hereby declare that this report provides a 

### correct English translation of 1he Final Report 

### (Study No. 14711, 

### issued on September 28, 2007).



### Date 

# M 

# 0.../ I I 1 ;"00 

# J Study Director 

# /.

# f<~o./ Takakazu Kayashima



### 859

### 14711 

# GLP STATEMENT

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan
Sponsor SHOWA DENKO K.K.



### Title Biodegradation study ofKarenzMT(pEMB) by microorganisms
Study number 14711



### The study described in 

### this report was conducted 

### in compliance with the following GLP principle: "Standard Concerning Testing Facility Relating to New Chemical Substances" (November 21, 2003; No. 1121003, Phamlaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 17, 2003, No.3, Manufacturing Industries Bureau, Ministry ofEconomy, Trade and Industry; No. 031121004, Environmental Policy Bureau, Ministry ofthe Environment) This final report reflects the raw data accurately and it has been confinned 

### that the 

### test data are valid.

Date September 28, 2007 Study Director Signed in 

### original Takakazu Kayashima

860

# -- __



# -- 

# --

---- 14711 

# QUALITY ASSURANCE STATEMENT

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor 

### SHOWA DENKO K.K.

Title Biodegradation study ofKarenzMT(pEMB) by microorganisms

Study number 14711

I assure that the final report accurately describes the test methods and procedures, and that the reported results accurately reflect the raw data ofthe study. The inspections and audits ofthis study were carried out and the results were reported to the Study Director and the Test Facility Management by Quality Assurance Unit as follows. Date ofreport to Item ofinspection / audit Study Director and Test Facility Management Date ofinspection / audit

1----------------
. 

## . 

# -­ Study plan draft 1---"---,-.,,-- .------­ January 22, 2007 January 22, 2007

Study plan January 22, 2007 January**22,.~007**

. Amendment to study plan February 21, 2007 February 21,2007 February 23,2007 February 23, 2007 

### At the start ofcultivation January 25, 2007**~aI!uary** 24, 2007 !-" 

### -- 

### '-­

At the middle ofcultivation February 7, 2007 February 7, 2007 February 21, 2007 February 21, 2007 -.**-.~** '-----_

..

### _-------­ Atthe end ofcultivation February 23, 2007

___**~ebruary** 23, 2007 

# -_.. Raw data and final report draft September**?21_~gg ?_.**  __...**Septe~~X~~~2Q~** Final report September 28, 2007 September 28, 2007

Date September 28, 2007 Head ofQuality Assurance Unit Signed in original Keiji Shiraishi
861

### 14711 

# CONTENTS

page Title Sponsor 1 Test facility 1 Objective 1 Test method 1 Applied GLP 

### 2 Dates 2 

### Storage oftest item, raw data, etc. 

### 2 Personnel 3 Approval offinal report 3 

### SUMMARY 4 

### 1. Test item 5 

### 2. Test sample 6 

### 3. Activated sludge 7 4. Performance ofbiodegradation lest 9 5. Validity oftest conditions 

### 22 6. Factors that affected reliability oftest 

### 22 7. Results 22 8. Remarks 

### 29 Contents oftables and figures Contents oftables Table-l Calculation table for percentage biodegradation by BOD Table-2 Calculation tabJe for percentage detection ofDOC Table-3 Calculation table for recovery rate oftest item Table-4 Calculation table for percentage biodegradation oftest item Table-5 Calculation table for percentage production ofpentaerythritol Table-6 Calculation table for percentage production of3MBA Table-7 Calculation table for percentage production ofSBA



### 862

### 14711

Contents offigures Fig. I Chart ofBOD Fig.2-1 Chromatograms ofHPLC analysis for calibration curve (test item) Fig.2-2 Calibration curve oftest item FigJ-l Mass fragmentograrns ofLC-MS analysis for calibration curve (pentaerythritol) FigJ-2 Calibration curve ofpentaerythritol FigA-1 Total ion chromatograms ofLC-MS analysis for calibration curve (3MBA) FigA-2 Calibration curve of3MBA Fig.S-1 Total ion chromatograms ofLC-MS analysis for calibration curve (SBA) Fig.S-2 Calibration curve ofSBA Fig.6 Chromatograms ofHPLC analysis for recovery test (test item) Fig.7 Chromatograms ofHPLC analysis for test solution (test item) Fig.8 Mass fragrnentogranls ofLC-MS analysis for tcst solution (pentaerythritol) Fig.9 Total ion chromatograD1s ofLC-MS analysis for test solution (3MBA and SBA) Fig.10 UV 

### chromatograD1s ofLC-MS analysis for test solution (20S-300nm) (LC-MS sample -1, qualitative analysis ofoil-soluble converted products) Fig.11 Total ion chrornatograD1s ofLC-MS analysis for test solution (LC-MS sample -1, qualitative analysis ofoil-soluble converted products) Fig.12 Mass spectra ofoil-soluble converted products (LC-MS sample -1, water + test item) Fig.13 Mass spectra ofoil-soluble convelted products (LC-MS sample -1, sludge 

### + test item) fig. 14 Mass chromatograms ofoil-soluble converted products (LC-MS sample 

### -1, control blank) Fig.lS UV 

### chromatograD1s ofLC-MS analysis for test solution (20S-30Onm) (LC-MS sample -2, qualitative analysis ofwater -soluble converted products) Fig.16 Total ion chromatograms ofLC-MS analysis for test solution (LC-MS sample -2, qualitative analysis ofwater -soluble converted product,;) Fig. 17 Mass spectra ofwater-soluble converted products (LC-MS sample -2, sludge 

### + test item) Fig.I8 Mass chromatograms ofwater-soluble converted products (LC-MS sample -2, control blank) Fig.19 UV 

### spectnun oftest item Fig.20 Mass spectrum ofpentaerythritol Fig.21 Mass spectnun of3MBA Fig.22 Mass spectrum ofSBA Fig.23-1 IR spectrum oftest item measurcd before experimental start Fig.23-2 IR spectrum oftcst item measured after experimental completion Reference IR spectrum supplied by sponsor



### 863

### 14711

Title Biodegradation study ofKarenzMT(pEMB) by microorganisms Sponsor SHOWA DENKO K.K. 1-13-9, Shiba Daimon, Minato-ku, Tokyo 105-8518, Japan
Test facility Kurume Laboratory Chemicals Evaluation and Research Institute, Japan 2-7, 3-chome, Miyanojin, Kurume-shi, Fukuoka 839·0801, Japan Objective This study was performed to evaluate the biodegradability of Karenz.MT(PEMB) by microorganisms.
Test method This study was performed according to the following test method. "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances" (November 21, 2003; No. 1121002, Pharmaceutical and Food Safety Bureau, Ministry ofHealth, Labour and Welfare; November 13,2003, No.2, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121002, Environmental Policy Bureau, Ministry ofthe Environment)

864 

### -1­14711
AppliedGLP This study was conducted in compliance with the following GLP principle: "Standard Concerning Testing Facility Relating to New Chemical Substances" (November 21, 

### 2003; No. 1121003, Phannaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; November 17, 2003, No.3, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121004, Environmental Policy Bureau, Ministry ofthe Environment) Dates Study initiation date January 22,2007 Experimental starting date January 24, 2007 Experimental completion date February 21,2007 Study completion date September 28, 2007 Storage oftest item, raw data, etc. (1) Test item 'lbe test sample is sealed in a storage vessel and stored 

### in archives in this laboratory for ten years after the receipt ofnotice specified under Clause 1 or Clause 2 in Article 4, Clause 2 or Clause 3 or Clause 8 in Article 4-2, and Clause 2 in Article 5-4 or Clause 2 in Article 24 or Clause 2 in Article 25-3 of "Law Concerning Examination and Rcgulation ofManufacture, etc. ofChemical Substances". If it is not stable for the storage period, 

### it is stored as long while it is kept stable. Treatment ofthe test sample after the storage period will be discussed v./ith sponsor. (2) Raw data and materials, etc. Raw data, the study plan, documents concerning the study presented by the sponsor, the fmal report and necessary materials are stored in archives in this laboratory for ten years 

### weT the receipt of the notice specified under Clause 1 or Clause 2 in Article 4, Clause 2 or Clause 3 or Clause 8 in Article 4-2, and Clause 2 in Article 5-4 or Clause 2 in Article 24 or Clause 2 in Article 25-3 of "Law Concerning Examination and Regulation of Manufacture, etc. of Chemical Substances". Treatment ofraw data and materials, etc. after the storage period will be discussed with sponsor.



### 865 

## -2­

### 14711

Personnel Study Director Study personnel (Operation ofbiodegradation test)



### Stafffor cultivation ofactivated sludge Approval offmaI report Study Director Date Signature 

### Takakazu Kayashima (1st 

### Chemical Safety Section) 

### Masanori Taruki 

### Hirokazu Kiuchi Yuko Shimogawa 

### Kimiko 

### Tanaka Kana Komiya 

### Marl Tsujimura Koichi Hiromatsu 

### Noriko 

### Miyaura Hiromi 

### Yamashita EriAbe 

### Takakazu Kayashima Kazuomi Katagiri

September 28, 2007 Signed 

### in 

### original Takakazu Kayashima



### 866 

### -3­14711 

# SUMMARY

Title Biodegradation study ofKarenzMT(PEMB) by microorganisms
Conditions ofcultivation (1) Concentration oftest item 100 

### mgfL (2) Concentration ofactivated sludge 30 mg/L (as the concentration ofsuspended solid) (3) Volume oftest solution 300mL (4) Cultivation temperature 25±loC (5) Cultivation duration 28 days (under the conditions ofdarkness)
Measurement and analysis for calculation ofpercentage biodegradation (1) Measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus (2) Determination oftest item by high-performance liquid chromatography (HPLC)
Other analysis (1) Determination of dissolved organic carbon (DOC) by a total organic carbon analysis (TOC) (2) Determination ofpentaerythritol by liquid chromatography-mass spectrometry (LC-MS) (3) Determination of 3-mercaptobutanoic acid and 3,3'-disulfandiyldibutanoic acid by liquid chromatography-mass spectrometry (LC-MS) (4) Qualitative analysis of oil-soluble converted products by liquid chromatography-mass spectrometry (LC-MS) (5) Qualitative analysis ofwater-soluble converted products by liquid chromatography-mass spectrometry (LC-MS)
Results (1) Percentage biodegradation by BOD 1%, 3%, 3% average 2% (2) Percentage biodegradation oftest item (HPLC) 14%, 16%, 13% average 14% Conclusion Some ofthe test item was changed or biodegraded on 3-mercaptobutylate group under the test conditions ofthis study, and then many converted products wcre produced in the test solution. The converted products and the rest of the test item were not biodegraded by microorganisms and remained.
867 

## -4­

### 14711

1. 

### Test item**I-·---~~·-·····-··-------------··-·-···-----------·~·· --------**  

### ....... --.-- ..-.-------- .........-*.-- ..........•.•.. , ' ,

, , ._------ ....... 

### -_._--_ .........- .. 

### ----_ ... _--- .... _----._-_ ... _----_ ..

### _-----.- .... _-- .......

### _--- .. ---- •..

_..

### _._---_.

868 

## -5­

### 14711

2. Test sample



### 2.5 Storage and stability Storage condition 

### Dark storage place at room temperature Stability The test item was stable under the storage conditions, as shown by the finding that 

### lR spectra of the test item before the experimental start and after the experimental completion were identical (see Fig. 23).

869 

### -6­

### 14711

3. Activated sludge



### 3.1 Preparation ofactivated sludge Activated sludge used in the present test was prepared as follows. (1) Sampling sites On-site sludge sampling was carried out at the following ten locations in Japan. Fushikogawa city sewage plant (Sapporo-shi, Hokkaido) Fukashiba industrial sewage plant (Kamisu-shi, 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) Dokai Bay (Kitakyushu-shi. Fukuoka) (2) Sampling method Sewage plant Return sludge was collected. Rivers, lake and sea Surface water and surface soil which was in contact with the atmosphere were collected. (3) Sampling date December, 2006 (4) Preparation method ofactivated sludge Activated sludge was prepared as follows to maintain its uniformity. The filtrate (5 L) ofthe supernatant ofthe activated sludge+2 cultivated about for three 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-) after the pH value ofthe mixture was adjusted to 7.0±1.0. *2 The activated sludge cultivated the mixed filtrate (to L) ofthe supernatant ofsludge collected at the ten locations according to the cultivation methods described in Section 3.2. *3 Prefiltered open air was used.

870 

## -7­14711

3.2 Cultivation After ceasing aeration of the sludge mixture for approximately 30 minutes, supernatant corresponding to about one third of the whole volume was removed. Dechlorinated water was added to the remaining portion so that the total volume reached 10 L This mixture was aerated for 30 minutes or more, and then a predetennined amount of synthetic sewage*4 was added to the mixture so that the concentration of the synthetic sewage was 0.1 % in the volume ofdechlorinated water added. TIris procedure was repeated once cvery day. Cultivation was carried out at 25±2°C. 

### *4 Synthetic sewage Glucose, peptone and potassium dihydrogenphosphate were dissolved in purified water to obtain 50 

### gIL of the solution for each component. The pH ofthe solution was adjusted to 7.0±1.O with sodium hydroxide. 3.3 Control and use During cultivation, the appearance of the supernatant, sedimentation of the sludge, fonnation of flock, pH, dissolved oxygen concentration in the solution and temperature were checked to maintain 

### a nonnal state of sludge. It was confinned 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 \\'llS microscopically observed and sludge with no abnonnal symptoms was used for the test. 'Inc activated sludge, which was cultivated 

### for 19 hours after it had been added the synthetic sewage, was used. 

### 3.4 Inspection ofactivity and date ofinitiation ofuse ofactivated sludge (1 ) Inspection ofactivity Activity ofihe sludge was assessed using standard items before initiation ofuse. (2) Date ofinitiation ofusc January 16,2007

871 

### -8­14711

4. Pelformance ofhiodegradation test

4.1 Preparations for test (1) Measurement ofconcentration ofsuspended solid in activated sludge The concentration of suspended solid was measured to determine the amount of activated sludge to add. Method In accordance with Japanese Industrial Standards (JIS) K 0102-1998 Section 14.1 Date January 22,2007 Result Concentration of suspended solid in the activated sludge was 3380 mglL. (2) Preparation ofhasal culture medium Each 3 

### mL of solutions A, B, C and D, which are prescribed in 

# ns K 0102-1998 Section 21, were made up to 1000 mL with purified water (Japanese Pharmacopeia, Takasugi Phannaceutical Co., Ltd.), and then the pH of this solution was adjusted to 7.0. (3) Reference item Aniline (reagent grade, Wako Pure Chemical Industries, Ltd. Lot No. DPG0938) was used as a reference item to confirm that the sludge was sufficiently active.



### 872 

### -9­14711

4.2 Preparation oftest solutions The following test solutions were prepared and cultivated under the conditions described in Section 4.3. (1) Addition oftest item or aniline (a) Test solution (water 

### + test item) (n=l, Vessel No.1) In one test vessel, 30 mg ofthe test sample was accurately weighed and added to 300 

### mL ofpurified water. so that the concentration ofthe test item reached 100 mgIL. (b) Test solution (sludge 

### + test item) (n=3, Vessel Nos. 2,3 and 

### 4) In each test vessel, 30 mg of the test sample was accurately weighed and added to the basal culture medium [the volume was less than 300 

### mL by the volume (2.66 

### mL) of activated sludge inoculated], so that the concentration ofthe test item reached 100 mg/L. (c) Test solution (sludge 

### + aniline) (n=l, Vessel No.6) In one test vessel, 29.5 f.l.L [30 mg 29.5 f.l.L x 1.022 g/cm3 (density)] ofaniline was taken out by microsyringe and added to the basal culture medium [the volume was less than 300 

### mL by the volume (2.66 mL) of activated sludge inoculated], so that the concentration ofaniline reached 100 mgfL. Cd) Test solution (control blank) (n=l, Vessel No.5) In one test vessel, nothing was added to the basal culture medium [the volume was less than 300 

### mLby the volume (2.66 

### mL) ofactivated sludge inoculated]. (2) Inoculation ofactivated 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 ofthe suspended solid reached 30 mgIL.



### 873 

## -10­

### 14711

4.3 Instruments and conditions ofcultivation (I) Instruments for cultivation Closed system oxygen consumption measuring apparatus (Temperature controlled bath and measuring unit: Asahi Techneion Co., Ltd.) (Data sampler: Asahi Techneion Co., Ltd.) Vessel 300 mL 

### in volume (Improved type) 

### Absorbent for carbon dioxide Soda lime No.1 (for absorption ofcarbon dioxide, Wako Pure Chemical Industries, Ltd.) (2) Conditions ofcultivation Cultivation temperature 

### 2S±lOC Cultivation duration 28 days (under the conditions ofdarkness) Stirring method Each test solution was stirred by a magnetic stirrer. (3) Room Apparatus room 1A 4.4 Observation and measurement oftest conditions (1) 

### Observation oftest solution During the cultivation, the appearance ofthe test solution was observed once a day and conditions ofthe instrument" were checked properly. (2) Measurement ofbiochemical 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.

874 

### -11­14711

4.5 Analysis oftest solution After the end of the cultivation, dissolved organic carbon, the test item and the converted products fpentaerythritol, 3-mercaptobutanoic acid (hereafter referred to as 3MBA), and 3,3'-disulfandiyldibutanoic acid (hereafter referred to as SBA)], which were expected from results ofthe preliminary test prior to this study and the authentic samples of which were available, in the test solutions were detelmined. 

### In addition, oil-soluble and water-soluble converted product<;, which were also expected from results of the preliminary test prior to this study and authentic samples ofwhich were not available, were analyzed qualitatively. 4.5.1 Pretreatment oftest solutions for analysis After the end of the cultivation, the test solution (water 

### + test item), the test solutions (sludge 

### + test item) and the test solution (control blank) were pretreated for total organic carbon (TOC) sample analyzed for determination of dissolved organic carbon, high-performance liquid chromatography (HPLC) sample analyzed for determination of the test item, liquid chromatography-mass spectrometry (LC-MS) sample -1 analyzed for qualitative analysis of oil-soluble converted products, LC-MS sample -2 analyzed for qualitative analysis ofwater-soluble converted products, LC-MS sample -3 analyzed for determination ofpentaerythrito] and LC-MS sample -4 analy-.red for determinations of3l\.1BA and SBA as follows. Test solution 300 

### mL I 

## I 

# I +---Purified wa ter / phosphoric acid 

### (9/1 v/v) 

### 2 mL (measuring pipet) Continued to the next page -Ammoniurn sulphate 200 g (even balance) +---Tetrahydrofuran 90 

### mL (graduated cylinder)

• Shaking for 10 minutes 

### I 

# I Waler layer 

# I 

# l Tetrahydrofuran layer 

# I

I ·HPLCsantple 

# I etermination ofthe test item • Filtration and dehydration (IPS filter paper) • Filling up to 100 mL (tetrahydrofuran, volumetric flask)

• Taking out 3 

### mL (transfer pipet) • Filling up to 10 

### mL (acetonitrile / water"S (1/9 v/v),

volumetric flask) 

### D 

# I LO
...MS sample ·1 

# I

. .. 

# . QuabtativeanalySlS of oll-soluble converted products

'" 5 City water was treated with ultrapure water system.


### -12­ 875

# I 14711

Canieddown

• Taking out 15 mL (measuring pipet) • Centrifugation (4000 x 

### g, 

### 15 min) 

### I Supernatant 

### 1 

# I TOCsampie 

# I I LC-MS sample -2 

# I Qualitative analysis of water-soluble converted products • Taking out 1

### mL (transfer pipet) • Filling up to 10 

### mL

(purified water, volumetric flask) 

# I LC-MS sample 

### -4 

# I

Determinations of3MBA and SBA

• Taking out 2 mL 

### (transfer pipet) • Filling up to 5

### mL (purified water,

volumetric flask) 

# I LC-MS sample -3 Determination ofpentaerythritol



### 876 

## -13­

### 14711

4.5.2 Quantitative and qualitative analyses (1) 

### Detennination ofdissolved organic carbon The dissolved organic carbon (DOC) in the TOC sample was analyzed under the following conditions. The concentration of DOC was calculated by subtracting concentration of the inorganic carbon (IC) from concentration ofthe total carbon (TC). The concentration ofTC and IC in the test solutions was proportionally calculated from the peak area of the test solution by comparison with that of 80.0 mgCIL standard solution for TC analysis and 80.0 mgCIL standard solution for IC analysis, respectively (see Table-2). The standard solution for TC analysis was 

### prepared by dissolving potassium hydrogen phthalate in purified water. The standard solution for IC analysis was prepared by dissolving sodium hydrogen carbonate 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 Total organic carbon analyzer Shirnadzu Corporation type 

### TOC-5000A Temperature offurnace 680°C Flow rate 150mUmin Injection volmne 33,..L Sensitivity Range 5

877 

## -14­

### 14711
(2) Detennination oftest item The HPLC sample was analyzed under the following conditions. The concentration of the test item in the HPLC sanlple was calculated proportionally by comparing the peak area on the chromatogram of the HPLC sample with that on the chromatogram of300 mg/L standard solution (see Table-4 and Fig. 7). 

### The lowest detectable peak area was regarded as 4500 I-lV' 

### sec considering the noise level, which corresponded to the test item concentration of2.8 mgIL. (a) Analytical conditions Instrument High-perfonnance liquid chromatograph Shimadzu Corporation type LC-2010A Colunm L-colurnn ODS (15 cm x 

### 2.1 mm I.D., Chemicals Evaluation and Research Institute) Co]unm temperature 40°C Eluent A: Acetonitrile I 

### phosphoric acid (1000/] 

### v/v) B: 

### Water*5 I 

### phosphoric acid (100011 

### v/v) Gradient condition

Time (min) A (%) B (%) 

# o 10 90 

### 2 10 90 ]5 100 0 30 100 

## o Flow rate 0.2 mUmin Measurement wavelength 210 nrn 

### (see Fig. 19) Sample size 21-lL 

### Detector output 2V/AU (b) Preparation ofstandard solution The standard solution to detennine the concentration ofthe test item in the HPLC sample was prepared as follows. 100 mg of the test sample was accurately weighed and dissolved in tetrahydrofuran to obtain 1000 mgIL solution of the test item. 300 mglL solution was then prepared from this solution 

### by dilution with tetrahydrofuran. (c) Calibration curve 75.0, 150 and 300 mgIL standard solutions were prepared by the same method as described in (b). 

### lbese 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 chromatograms and the respective concentrations (sec Fig. 2).



### 878 

## -15­

### 14711
(3) Determination ofpentaerythritol The LC-MS sample -3 

### was analyzed under the following conditions. The concentration ofpentaerythritol 

### in the LC-MS sample -3 

### was calculated proportionally by comparing the peak 

### area on the mass fragmentogram ofthe LC-MS sample -3 

### with that on the chromatogram of 10.0 mgIL standard solution (see Table-5 and Fig. 8). 

### The lowest detectable peak area was regarded as 600 considering the noise level, which corresponded to pentaerythritol concentration of0.094 mgIL. (a) Analytical conditions Instrument Liquid chromatograph-mass spectrometer HPLC system Waters Corporation type Alliance2690 Mass spectrometer Waters Corporation type ZMD LC conditions Column L-column ODS (25 cm x 

### 2.1 mm 

### l.D., 

### Chemicals Evaluation and Research Institute) Column temperature 35°C Eluent Water*5 (Containing 5 mmol!L ammonium acetate) Flow rate 0.2mUmin Sample size 5 

### ilL Mass conditions Ionization mode Electrospray ionization (ESI) Detection ion Ncgaitivc Detection method Selected ion monitoring (SIM) Monitoring ion (m/z) 

### ]95.2 (see Fig. 20) Ion source temp. 1200C Desolvation temp. 400°C Cone voltage 15 V (b) 

### Preparation ofstandard solution Thc standard solution to determine the concentration of pentaerythritol in the LC-MS sample was prepared as follows. 100 mg ofpentaerythritol (Tokyo Chemical Industry Co., Ltd.)*6 was accurately weighed and dissolved in purified water to obtain 1000 mgIL solution of pentaerythritoL 10.0 mgIL standard solution was then prepared from this solution by dilution with purified water. *6 

### Pentaerythritol was treated as 100% in purity. (c) Calibration curve 2.50, 5.00 and 10.0 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 fragmentograms and the respective concentrations (see Fig. 3). 

### 879 

## -16­14711
(4) Detenninations of3MBA and SBA The LC-MS sample 

### -4 was analyzed under the following conditions. The concentration of 3MBA in the LC·MS sample -4 was calculated proportionally by comparing the peak area on the total ion chromatogram ofthe LC·MS sample 

### -4 with that on the chromatogram of 10.0 mglL standard solution (see Table·6 and Fig. 9). The concentration of SBA in the LC·MS sample -4 was calculated proportionally by comparing the peak area on 

### the total ion chromatogram of the LC-MS sample -4 

### with that onthe total ion chromatogram of 10.0 mgIL standard solution (see Table-7 and Fig. 9). The lowest detectable peak area wa') regarded as 450 considering the noise level, which corresponded to 3MBA concentration of0.092 

### mgIL and SBA concentration of 0.024mgIL. (a) Analytical conditions Instrument Liquid chromatograph-mass spectrometer HPLC system Waters Corporation type Alliancc2690 Mass spectrometer Waters Corporation type ZMD LC conditions Column L-column ODS (15 em x 2.1 mm l.D., Chemicals Evaluation and Research Institute) Column temperature 40°C Eluent A: Acetonitrile 

### I fonnie acid 

### (IODO/I v/v) B: Water'SI ronnie acid (1000/1 v/v) Gradient condition Time (min) A (%) B (%) 

# o 20 80 3 20 80 

### 7 60 40 13 60 40 Flow rate O.2mUmin Sample size 

### 2 JlL Mass conditions Ionization mode Electrospray ionization (ESI) Detection ion Negaitive Detection method Selected ion monitoring (SIM) Monitoring ion (m/z) 3MBA 119.2 (see Fig. 21) SBA 237.4 (see**~Fig.** 22) Ion source temp. l20°C Desolvation temp. 400°C Cone voltage 

### lOV

880 

## -17­14711
(b) Preparation ofstandard solution The standard solutions to dctennine the concentrations of3MBA and SBA in the LC·MS sample were prepared as follows. 

### CD 3MBA 100 mg of 3MBA (supplied by the sponsor)'? was accurately weighed and dissolved in acetonitrile to obtain 1000 mglL solution of3MBA. 100 mgIL solution of 3MBA was then prepared from this solution by dilution with acetonitrile, and then 10.0 mgIL standard solution was then prepared from this 100 mgfL solution by dilution with purified water. *7 Purity >99% Lot number 50913 3MBA was treated as 100% in purity. 

## ® SBA100 mg of SBA (supplied by the sponsor)*8 was accurately weighed and dissolved in acetonitrile to obtain 1000 mgfL solution of SBA. 100 mgIL solution of SBA was then prepared from this solution by dilution with acetonitrile, and then 10.0 mgtL solution was then prepared from this 100 mg/L solution by dilution with purified water.


## *8 Purity >99% Lot number 551601 SBA was treated as 100% 

### in purity. (c) Calibration curve 

### CD 3MBA 2.50, 5.00 and ]0.0 mg/L standard solutions were prepared by the same method as described in 

### (b)CD. 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 total ion chromatograms and the respective concentrations (see Fig. 4). SBA 2.50, 5.00 and 10.0 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 total ion chromatograms and the respective concentrations (see Fig. 5).

881 

## -18­14711
(5) Qualitative analysis ofoil-soluble and water-soluble converted products The LC-MS samples -1 and -2 were analyzed by liquid chromatography-mass spectrometry (LC-MS) for qualitative analysis of oil-soluble and water-soluble converted products (see Figs. 10-18). The solutions ofthe test item, 3MBA and SBA were also analyzed for the comparison 

### with the LC-MS samples. (a) Analytical conditions Instrument HPLC system Mass spectrometer Photodiode array detector Pump (post-column) LC conditions Column
Column temperature Eluent

Flow rate Sample size Measurement wavelength Post-column Solution Flow rate Liquid chromatograph-mass spectrometer Waters Corporation type Alliance2690 Waters Corporation type ZMD Waters Corporation type 2996PDA Shimadzu Corporation type LC-l

### OAD
L-column ODS (15 em x 2.1 rom LD., Chemicals Evaluation and Research Institute) 40°C A: Acetonitrile I formic acid (10001 

### I v/v) B: Water*sl formic acid (1000/1 v/v) Gradient condition Time (min) 

# o 3 25 40 0.2mUmin ] 5 ilL 205-300 

### run A(%) 

### B (%») 10 90 

### 10 90 100 

# o 100 

# o

Acetonitrile 

### I water*s 

### (1/1 v/v) *9 0.05 mUmin *9 Containing 25 mmollL ammonium acetate. Mass conditions Ionization mode Detection mode Monitoring method Mass range 

### mlz Jon source temp. Desolvation temp. Cone voltage Electrospray ionization (ESJ) Positive Scan 80-]800 120°C 400°C 20V



### 882 

## -19­

### 14711
(b) Preparation ofsolutions oftest item, 3MBA and SBA The solutions ofthe test item, 3MBA and SBA used for thc LC-MS analysis were prepared as follows. 

## CD Test item 100 mglL solution ofthe test item was prepared from 1000 mglL solution of the test item , which was prepared according to the method described in Section 4.5.2(2)(b), by dilution with acetonitrile / water·s (111 v/v). @ 

### 3MBA 100 mgIL solution of 3MBA was prcpared from 

### 1000 mglL solution of 3MBA , which was prepared according to the method described in Section 4.5.2(4)(b)Q), by dilution with acetonitrile / water· s 

### (1/9 v/v). @ 

### SBA 100 

### mgIL solution of SBA was prcpared from 1000 mgIL solution ofSBA, which was prepared according to the method described in Section**4.5.2(4)(b~"ID,** 

### by dilution with acetonitrile / water'S (119 v/v). 4.5.3 Recovery test and blank test Each two test solutions (water 

### + test item) and two test solutions (sludge 

### + test item) for recovery test were prepared according to the methods described 

### in Section 4.2. The test solutions were pretreated 

### in accordance with the method described in Section 4.5.1, then analyzed according to the procedures and analytical conditions described in Section 4.5.2(2). A test solution for blank tcst 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 appeared around the peak(s) of the test item on the chromatogram. Two individual recovery rates and their averages ofthe test item on the analytical procedure are 

### shown below. Ibe average recovery rates were used as correction factor, for the determination ofthe test item in the analytical samples (see Table-

### 3 and 

### Fig. 6). Recovery rate in the test solutions (water 

### + test item) 92.9%, 97.7% average 95.3% Recovery rate in the test solutions (sludge 

### + test item) 96.4%, 92.9% average 94.7%



### 883 -20­14711

4.6 Calculation ofpercentage biodegradation The percentage biodegradations were ca1culated by the following equations and rounded offto the whole number. (1) Percentage biodegradation by BOD BOD-B Percentage biodegradation (%) 

### = TOD'to x 100 BOD : Biochemical oxygen demand inthe test solution (sludge 

### + test item) (experimental) (mg) B 	 : Biochemical oxygen demand in the control blank (experimental) (mg) TOD"lO : Theoretical oxygen demand required whenthe test item was completely oxidized (theoretical) (mg)


## * 10 The purity was regarded as 100 

### % and TOD was calculated. (2) Percentage biodegradation oftest item Sw-Ss Percentage biodegradation (%) x 100 Sw Ss : Residual amount ofthe test item in the test solution (sludge 

### + test item) (experimental) (mg) Sw : Residual amount ofthe test item in the test solution (water 

### + test item) (experimental) (mg) 4.7 Treatment ofnumerical values Values were rounded offin accordance with JIS Z 840I : 1999 rule B.

884 

## -21­

### 14711

5. Validity oftest conditions The validity criteria ofthe test and the values in this test are shown in the following table. This test was valid because all ofthe values in this test met the criteria.



# -------­ Value in present test Value of criterion See Difference of extremes of replicate values ofpercentage biodegradation Percentage biodegradation by BOD ,­ 

### Percentage biodegradation oftest item 

### 2% 3% <20% Section 

### 7.3 Percentage biodegradation Percentage biodegradation ofaniline by After 14 days 

### 76% ;:;;;60% Table-l Fig. I 

### BOD I
6. 

### Factors that affected reliability oftest No adverse efiects on the reliability ofthis test were noted.

7. 

### Results

7.1 Appearances oftest solutions Appearances oftest media in cultivation vessels were as follows. Test solution Appearance pH

At the start Water 

### + test item The test item was not dissolved. 

## - ofcultivation Sludge 

### + test item The**te~i** 

### item was not dissolved. 

# -

At the end Water 

### + test item Insoluble compound was observed. 

# - ofcultivation Sludge 

### + test item Insoluble compound in 

### addition to the sludge was observed. Growth of the sludge was not observed. 

# -

885 

## -22­14711

7.2 Analytical results oftest solutions Analytical results ofthe test solutions after 28 days were as follows.

# ~ Water 

### + Sludge 

### + test item test item Theoretical Table 

### Fig. Vessel-l Vessel-2 Vessel-3 Vessel-4 amount BOD*ll mg 3.2 0.4 1.8 1.4 52.8 

### 1 1
OIl 

### mgC 0.6 

### 1.1 1.1 0.9 

### 13.9 Detected amount and percentage 

### -......'

### ..... '

### .. .-"'

### .....**-~-~-** ....... 

### ........ -"'

### ..-­ ..

### -.. .. '" .....................

### _­ ....

### _- ... ""' ... "' 

### ..--­ 

### -- ..

### -............ "' ... "' ..._ .. 2 

### - detection ofDOC % 4 8 8 7 

# - Residual amount mg 29.2 25.1 24.6 25.3 30.0 and percentage



### -----.. '" 

### .....**~--** 

### ....**--~** 

### .... ............. "'--_&­ 

### . ..................._ ..

### ­

.. ­ - .. 't'­
.. -_ ....... ...**-~-------** .....

## -..**~-** 4 7 residue oftest item (HPLC) %CD 97 84 82 84 

# - Produced amount 0 0.2 0.2 0.2 

### 7.5 and percentage mg production of 

### . "

### .......... "'

### .. ,. 

### ........... ""'

### .. ..

### ...........--

### ..**~,..**

### -.. ......................... 

### . ...

### .....................**~---** 

### ....... -.....

## ­ ..

## ­...... 5 

### 8 pentaerythritol %® 

### 0 3 3 3 

# . (LC-MS) 

# I Produced amount 0 0 

### 0 0 26.5 and percentage mg production of 

### 6 

### 9 3MBA %@ 0 0 0 

### 0 (LC-MS) 

# - Produced amount 

### 0 0 

### 0 

### 0 26.2 and percentage mg production of 7 

### 9 SBA %@ 

### 0 0 

### 0 0 (LC-MS) 

# - '12 Detection of oil-soluble 

# - Detected Detected 

# - 

# - 1()"14 converted products (LC-MS) -12 Detection of Not water-soluble 

# - detected Detected 

# - - 15-18 converted products (LC-MS) Mass balance*13 % 97 87 

### 85 87 (<D+®) 

# - - 

# - Mass balance*14 % 97 84 82 84

(Q)+@+@) 

### - - 

## -

886 

## -23­

### 14711



## *11 The value of control blank was subtracted from the values of the test solutions (sludge + 

### test item).


## * 12 Detenninations of detected converted products except for pentacrythritol, 3MBA and SBA could not be perfOImed because those authentic samples were not available (sec Section 7.4).



## * 13 Mass balance for pentaerythritol group oftest item


## * 14 Mass balance for 3-mercaptobutylatc group oftest item 7.3 Percentage biodegradation Percentage biodegradations after 28 days were as follows. The percentage biodegradation by DOC was not calculated because the test item is not dissolved in water at the test concentration (100 mgIL) and more.

# ~ Vessel-2 Sludge 

### + test item Vessel-3 Vessel-4 Average Table Percentage biodegradation by BOD Percentage biodegradation of test item (HPLC) %
% 

### 1
14 3
16 3 

### 13 2
14 

### I
4
7.4 Results ofqualitative anaysis by LC-MS (1) Oil-soluble converted products (LC-MS samplc -], see Figs. 10-14) No peak corrcsponding to converted products was detected on UV chromatogram (205-300 nm) in the test solution (water 

### + test item) and the test solutions (sludge + test item). On the other hand, many peaks corresponding to converted products were detected on total ion chromatograms (TIC) in these test solutions. The converted products shown in the list 

### in next page were presumed hy analysis oftheir mass spectra. (2) Water-soluble converted products (LC-MS sample -2, see Figs. 15-18) No peak corresponding to converted products was detected on UV chromatogram (205-300 nm) and TIC in the test solution (water 

### + test item). On the other hand, many peaks corresponding to converted products were detected on TIC in the test solutions (sludge 

### + test item). The converted products listed in (1) 

### except for the converted products F and G were detected and presumed by analysis of their mass spectra. In addition, converted product A was newly detected and presumed by analysis oHts mass spectmm (see the list in next page).



## -24­ 88714711

List ofconverted products (elution order, see 

### Figs. 10-18 

### Detected (0) 

### I Not detected 

### (x) Presumed structural formula Converted (M: presumed molecular weight) products Water 

### + test item



### .. 

# · · · ·
, 

# · A'15 

# · [1] x 

# · · · · 

# · · 

# · 

# · , 

# · · , 

# · · · 

# · : 

# · , 

## : [1] x 

### B 

# · · :

, 

# :
, ! 

# · 

# · : 

# · · 

# · · ; 

## : 

# · , ! 

### [1] 

### x C 

# · ,
, 

# · 

# · · · · , 

# · , 

# · , 

# · · ,

, , , D 

## fl]X 

# · , , , , , , 

# · , 

# · · · · · , 

# · , , 

# · · , , 

# · E 

## U]X 

# · , 

### : 

# · , 

### ; 

# · : 

# · : 

# · 

# · , 

# · , , 

# · · , , 

# · · · , , 

# · F'16 

# · , [1] 0 

# · , 

# · 

# · · · · · · , 

# · , 

# · , 

# · · · · 

# · · · · 

# · , , 

# · · · 0+ 16 

# · [1] 0 

# · · · Sludge 

### + test item

, 

### [2] 0 [3] 0 [4] 0 [2] 0 [3] 0 [4] 0 [2] 0 

### [3] 0 [4] 0


### [2] 0 [3] 0 [4] 0
[2] 0 [3] 0 [4] 0

[2] 0 [3] 0 [4] 0



### [2] 0 [3] 0

, 

# · , 

# · [4] 0



# · , 

# · , 

# · · · · 

# · *15 Detected only 

### in analysis for water-soluble converted products (LC-MS sample -2)


## * 16 Detected only in analysis for oil-soluble converted products (LC-MS sample -1) 

### BBB 

## -25­14711

7.5 Discussion (1) Percentage biodegradation by BOD Percentage biodegradations by BOD were 1%, 3(Yo and 3% (see Fig. 1). (2) Results ofdetennination oftest item The percentage residue ofthe test item in the test solution (water 

### + test item) was 97%, whereas the percentage redisues of it in the test solutions (sludge 

### + test item) were slightly low as 82-84%. In addition, many peaks corresponding to converted products were detected on the HPLC chromatogram in the test solution (water 

### + test item) and the test solutions (sludge 

### + test item) (see Fig. 7). (3) Results ofdeterminations ofexpected convcltcd products According to (2), pentaerythritol, 3-mercaptobutanoic acid (3MBA) and 3, 3'-disulfandieydibutanoic acid (SBA), whose productions were expected by hydrolysis etc. of the test item, were determined. As the results, the percentage production ofpenta erythritol in the test solution (water 

### + test item) 

### was 0% and the average percentage production ofit in the test solutions (sludge 

### + test item) was 3% (see Fig. 8). 3MBA and SBA were not detected in all ofthe test solutiol1.<; (see Fig. 9). (4) Mass balances The mass balances tor pentaerythrito! group, which was calculated from sum of the percentage residue of the tcst item and the percentage production of pentaerythritol, in the test solution (water 

### + test item) and the test solutions (sludge 

### + test itcm) was 97% and 85-87%, respectively. The mass balances for 3-mercaptobutylate group, which was calculated from sum of the percentage residue of the test item and the percentage productions of 3MBA and SBA, 

### in the test solution (water 

### + test item) and the test solutions (sludge 

### + test item) were 97% and 82-84%, respectively. 

### It was eonsidercd for shortage ofthose mass balances that the test item and/or the converted products described in (3) were biodegraded by microorganisms or other converted products, which arc compounds expect for the convcrted products described in (3), were produced. (5) Other converted products In order to confinn productions of other converted products described in (4), oil- and water-soluble converted products were analyzed qualitatively by LC-MS. As a result, the converted products A-G were detected and presumed as compounds which were produced by hydrolysis and/or sulfer-sulfer (s-s) polymerization (see Section 7.4). These converted products might contribute to the shortage of the mass balances described in (4). However, determinations of these products could not be perfonned and then the mass balances could not be continned because the authentic samples ofthese were not available.



### -26­ 889

### 14711
(6) Biodegradation of3-mercaptobuty1ate group Despite detections ofpentaerythritoI and the converted products A-E, 3MBA and SBA, whose productions were expected, were not detected in all of the test solutions. Here, as the average percentage biodegradation by BOD in "Biodegradation study of 3MBA by microorganisms (Study No.14359)" was 79%, 3MBA is thought to be biodegradable. From this knowledge, it was considered that pentaerythritol and the converted products A-E were produced by biodegradation of some of3-mercaptobutylate group. According to (1), 

### the percentage biodegradations by BOD were low. It is considered that the variation of basal respiration of activated sludge might significantly affect to the percentage biodegradations by BOD because of slightly biodegradation on 3-mcrcaptobutylate group. (7) Water-soluble converted products In the detennination ofDOC to confinn presence or absence ofwater-soluble converted products, the percentage detections of DOC in the test solution (water 

### + test item) and the test solutions (sludge 

### + test item) were 4% and 7-8%, respectively. 

### It is considered that some of the test item and the converted products A-E were dissolved in the test solutions and contributed to the detection ofDOC, because some ofthe test item and the converted products were detected on TIC in the qualitative analysis of water-soluble converted products by LC-MS and pentaerythritol is a**water~soluble** 

### compound. (8) Summery ofdiscussion According to (l 

### )-(7), it was considered that some ofthe test item was changed or biodegraded on 3-mercaptobutylate group into many converted products and that these products and rest ofthe test item were not biodegraded and remained under the conditions ofthis study (see Degradation diagram on next page).

890 

## -27­

### 14711


### Degradation diagran;.

## t?f.**!~! .~t~r:.t}**  •..••••••••••••••.••...••••••••• , , ' , ' , :

, ,

Test item 

### (percentage residue: 82-84%) 

# l' Partly conversion and biodegradation on 3-mercaptbutylate group Residual converted products Converted products ( produced amount could be checked) ---------_ .... 

### _------_ .. _-_ ... _-_ .... -----------_._--- 

### -.---.-------.---.-- 

## .. --------.--- 

## ....**-~---------** 

## ... -- -----­

891 

### -28­14711

7.6 Conclusion Some ofthe test item was changed or biodegraded on 3-mercaptobutylate group under the test conditions ofthis study, and then many converted products were produced in the test solution. The converted products and the rest of the test item were not biodegraded by microorganisms and remained.
8. Remarks

8.1 Instruments used for test Fourier transform infrared spectrophotometer: Shimadzu Corporation type IRPrestige-21 Closed system oxygen consumption measuring apparatus: see page II Total organic carbon analyzer: see page 14 High-performance liquid chromatograph: see page 15 Liquid chromatograph-mass spectrometer: see pages 16, 17, 19 Electronic analytical balance: Sartorius AG type BP21OS Mettler-Toledo International inc. type AE-163 Ultraviolet and visible spectrophotometer: JASCO Corporation type V-560 Refrigerated centrifuge : KUBOTA Manufacturing Corporation type 5922 Shaker: Taitec Corporation type SR-2w 8.2 Reagents used for analysis Acetonitrile (HPLC grade) : Wako Pure Chemical Industries, Ltd. Tetrahydrofuran (HPLC grade) : Kanto Chemical Co., Inc. Purified water (Japanese Pharmacopeia) : Takasugi Phannaceutical Co., Ltd. A.mmonium sulphate (reagent grade): Kanto Chemical Co., Inc. Phosphoric acid (reagent grade) : Wako Pure Chemical Industries, Ltd. Fornlic 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. Ammonium acetate (reagent grade) : Wako Pure Chemical Industries, Ltd. Pcntaerythritol: Tokyo Chemical Industry Co., Ltd. 3-mercaptobutanoie acid (3MBA) : Supplied by the sponsor 3,3'-disulfandiyldibutanoic acid (SBA) : Supplied by the sponsor 892 

## -29­Table-l Calculation table for percentage biodegradation by BOD Study No. 14711 Duration ofcultivation: 28 days
Vessel

No. 7th day 14th day 21st day 28th day 

### Mean Deg. 

### (%) BOD (mg) Deg. 

### (%) BOD (mg) Deg. 

### (%) BOD (mg) Deg. 

### (%) BOD (mg) Deg. (%) 

### [6] 56.1 59 74.6 76 77.3 76 79.1 77 

### [5] 3.1 

# - 6.1 

# - 8.4**~** 10.0 

# . [2] 2.4 -1 

### 5.8 -1 8.3 

### 0 10.4 1 2 [3] 2.7**~l** 6.9 2 9.8 3 11.8 3 [4] 

### 2.8 -1 6.6 

### 1 9.6 2 "11.4 3 

### [1] 0.0 

# - 0.3 

## - 2.0 

## . 3.2 

# -

Deg. : Percentage biodegradation Vessel No. [6] : Sludge 

### + aniline Vessel No. [5] : Control blank [B

### J Vessel No. [2] [3J [4] : Sludge 

### + test item Vessel No. 

### II] : Water 

### + test item Test item of 30 mg was added. Chart ofBOD : Fig. 1 Deg. = [BOD

- B] I [TOO] X 100 (%) TOO oftest item: 52.8 (mg) C21H360gS4 

### + 3002 -+ 21 CO2 

### + 18 H20 

### + 4 S02 

### 30021 C21H360gS4= 959.96/544.77 = 1.76 TOO 

### = 30 X 1.76 

### = 52.S (mg) TOD ofaniline: 90.3 (mg) C6H7N 

### + 8.7502 -+ 6 C02 

### + 3.5 H20 

### + N02 8.75 02 

### I C6H7N=279.99 

### 193.13 =3.01 TOO 

## =30 X 3.01 

## =90.3 (mg)

Feb.21,2007 Name 

# M**Ta.h.Ak~**

893 

## -30­Table-2 Calculation table for percentage detection ofDOC
Stud IV No. 14711 Sample description A B C D
Water blank n.d. [1] Water + test item 2.00 0.6 4 (2] Sludge + test item 5.59 1.1 8 [3] Sludge + lest item 5.30 1.1 8 [4] Sludge + test item 4.82 0.9 7 [5} Control blank 1.78 4
7

Amount oftest item added: 30 (mg) Volume of lest solution: 300 (mL) Theoretical amount ofcarbon: 13.9 (mgC) (30) x ( 21 C /**~lH)60sS4** ) A: Measured value (mgC/L) B: Amount ofDOC Bw 

## = (A(Water + test item)

- A(Water blank) ) x 300/ 1000 (mgC) Bs 

## =(A(Sludge + lest item)

- A(Control blank) ) x 300/1000 (mgC) C: Percentage detection (%) C = B / ( Theoretical amount of**carbo~** ) x 100 D: Average percentage detection (%)

Much 8, 2007 Name 

# h. 

# Jg

# rv.k:

894



## -31­Table-3 Calculation table for recovery rate oftest item
Stud ly No. 14711 Sample description A D E F Standard solution 300mg/L 484689 Water + test item·l 450387 27.9 92.9 95.3 Water + test item -2 473462 29.3 97.7 Sludge +test item-1 467398 28.9 96.4 94.7 Sludge + test item -2 450164 27.9 92.9 Control blank n.d.

Amount oftest item added: 30 (mg) A: Peak area (}lV'sec) B: Final volume; 100 (mL) C: Ratio ofportion uS¢ for analysis: 3001300 D: Recovery amount (mg) Dw 

## = G x (A(Waler +test item) / A(Stllldard) ) x (B I C)/1000 Ds 

## = G x { ( A(Sludge + test item)

- A(Control blank) ) I A(Standard) } x ( B / C )/1000 E: Recovery rate (%) E 

## = D/30 (mg) x 100 F: Average recovery rate (%) G: Concentration ofstandard solution: 300 (mgIL) See Fig. 6

March 13, 2007 Name

895 

## -32­Table-4 Calculation table for percentage biodegradation oftest item Study No. 14711 Sample descriptien A E -",,""" F 0 H
Standard solution 300mg!L 481034 [11 Water + test item 446638 29.2 97 [21 Sludge + test ilem 380832 25.1 84 14 [3) Sludge + test item 374147 24.6 82 16 14 [4] Sludge + test item 384687 25.3 84 13 [5) Control blank n.d.

Amount oftest item added: 30 (mg) A: Peak area (;.tV·sec) B: Final volume: 100 (roL) C: Ratio ofportion used for analysis: 300/300 D: Recovety rate: 95.3 (%) (Water + test item) 94.7 (%) (Sludge + test item) E: Residual amount of test item (mg) Ew '" I x ( A(Watcr + test item) / A(Slandard) ) x (B / C ) / ( D / 100 ) /1000 Es '" I x { (A(Sludge + test item) • A(Control blank) ) / A(Standard) } x (B / C ) 1( D /100 ) / 1000 

### F: Percentage residue (%) F:: E 130 (mg) x 100 0: Percentage biodegradation (%) G:: { (E(Water + test item)

- E(Sludge + test item» / E(Water + test item) } x 100 H: Average percentage biodegradation (%) I: Concentration ofstandard solution: 300 (mgIL) SeeFig. 7

March 8, 2007

896 

## -33­Table-S Calculation table for percentage production ofpentaerythritolStudv No 14711 Sample descriPtion A D E F Standard solution 10.Omg/L 63917 [1] Water + test item n.d. 0 0 0 (2] Sludge +test item 2120 0.2 3 [3] Sludge + test item 1909 0.2 3 3 [4] Sludge + test item 1868 0.2 3 [5} O:mtrol blank n.d.

Amount oftest item added: 30 (ms) Theoretical amount ofpentaerythrito] : 7.5 (mg) (30) x ( CsH1200tl C;UH360gS.)
A: Peakarea(-) B; F'mal volume: 5 (mL) C; Ratio ofportion used for analysis: 15{300x2115 D: Amount ofpentaerythrito] (mg) Dw '" G )( ( A(Water +test item) / A(Standard) ) x (B/ C)/1000 Ds:: G x { (A(Sludge +test item) • A(Control blank) ) / A(Standard) } x ( B Ie)/1000 E: Percentage production (%) E 

## = D/7.5 (mg) x 100 F: Average percentage production (%) G: Concentration of standard solution: 10.0 (mg/L) See Fig. 8

March 8, 2007



### 897 

## -34­

### Table-6 Calculation table for percentage production of3MBA Study No 14711 Sample description A D E F Standard solution lO.OmWL 49090 [1] Water +test item n,d. 0 0 0 [2] Sludge + test item n.d. 0 0 [3] Sludge +test item n.d. 0 0 0 [4] Sludge +test item n.d. 0 0 [5] Control blank n.d.

Amount oftest item added: 30 (mg) Theoretical amount of3MBA : 26.5 (mg) (3D) x ( 4C4 H

B 0 2S/CzIH360aS4 )
A: Peakarea(-) B: Final volume: 10 (mL) 

### c: Ratio ofportion used for analysis: 15!3ooxl/lS D:Amount of3MBA (mg) Dw 

## = G x ( A(Water + test item) / A(Standard) ) x (B/ C ) /1000 Ds 

## =G x { (A(Sludge + test item)

- A(Control blank) ) / A(StBndard) } x ( B / C)/1000 

### E: Percentage production (%) E=D/26.5 (mg) x 100 

### F: Average percentage production (%) G: Concentration ofstandard solution: 10.0 (mWL) See Fig. 9

March 8, 2007 Name



### 898 

## -35­Table-7 

### Calculation table for percentage production ofSBA
S d 0.14711 tUly N Sample descriPtion A D E F
Standard solution 10.0mg/L 190718 [1JWater + lest item n.d. 0 0 0 [2] Sludge + test item n.d. 0 0 [3J Sludge + lest item n.d. 0 0 0 [4] Sludge + lest item n.d. 0 0 [5] Control blank n.d.

Amount oftest item added: 30 (mg) Theoretical amount of SBA : 26,2 (mg) (30) x (2CsH140 4SJ Cz,H3,OSS4)
A: Peak area ( - ) B: Final volume: 10 (mL) C: Ratio ofportion used for analysis: 15/3ooxll15 D:Amount ofSBA (mg) Dw = G x ( A(Water -+- test item) / A(Standard) ) x ( B / C) 11000 Ds

## = G x { ( A(Sludge + test item)

- A(Control blank) ) J A(Standard) } x (B 1 C ) J1000 E: Percentage production (%) E 

## = D /26.2 (mg) x 100 

### F: Average percentage production (%) G: Concentration ofstandard solution: 10.0 (ms'L) SeeRg. 9

March 8, 2007



### 899 

## -36­

### Study No. 14711 

### (Test item :1Jv:y;('MTlPEMB) ) 

### Cultivating conditions: Concentration 

### Test item ."

## .............................. ,

# ......... ............................. ............ , 

### 100 (mgIL)**~., ~**  

### Reference item (am1ine) *
................. , ..............**,.~** ....' •••• 

### 100 (mgIL) Ac.tivated 

### sludge
................................................... 30 (mgIL) 

### Temperature 2S 

### ± 1

### t "1'
.......... , ......... ,."'
................. , , .... , •••**~** .................. ".

.. ,. ...................... " ."
.................. "
.. t ............ ,. t
..**~** .. "'
... 10
...................**~** ...... 

### Duration 28 days (Jan.24,2007 •Feb.21 ,2007) Note! 

# ­ BOD (mg)

Vessel 

### Sample Description

No. 7th 

### day 14th 

### day 21st 

### day 28th 

### day 

### Water 

### +test item 

### [1] 

### 0.0 2.0 

### 0.3 3.2 

### Sludge 

### + test item
. 2.4 8.3 

### 10.4 

### 5.8 

### [2J 

### Sludge 

### +test item 2.7 9.8 11.8 

### [3} 6.9 

### Sludge 

### + test 

### item 2.8 6.6 9.6 11.4 

### [41 3.1 

### Control blank 

### [B] 6.1 8.4 

### [5] 10.0 [6] 

### Sludge 

### + aniline 56.1 77.3 79.1 74.6 100



# - [1]



# - 

# - [2]



## -- ...

# - [3]

..... ........ 't)Ij 

### [4] 

### 80 .-- •• -----------.------ [6]



# - - [5] _...-

## .... ",..,.

### .......... 

# A

...

# -.,-.­ 

# .---- [6] 

# .."" 

# i ,/ , 

# ! 60 

### .'**~** 

# ..,..' "..' 

## 5 f 

### " I

## ~ , 0 40 I 

# , 1 

# 's I 

# 'H

• t**~ ~**  fj 

### 20 

### I 

### .2 t 

# , t 

# . [3] 

## := ,**.~.--.- -?-:':--;;;:'~';'::'~**  

### [5][2)[4]

, ;. ..**..-,.,:_~~** ....**~."::!'-~~-s:-;......-** -...- -...



# .**..1-.~--** 

# ...........-"'--'" [1] 

### 0 I



### 0 

### 7 14 

### 21 

### 28 Cultivation Time (day) 

### Fig. 1 Chart of

### BOD.



## -37­ 900Study No 1471 I So l\'en t blank

Peak position n.d

# i~ 

# 1.
. ----­ • I i O. 0 10.0 20. a 

### Standard solution 75.0 mg/L

1

# L~ 

# L

. 

## .--...­ • I i 0;0 10.0 20.0 --J-­
i 30.0



# .
i



### so. 0 Operating date : Sep. 13. 2005 PKNO Time Area Height Cone. (min) (/LV' sec) (/LV) (%) Total 0 O. 00

Operator: Hirokazu Kiuehi Ope rat ing date : Sep. 13. 2006 PKNO Time Area Haight Cone. (m!n) (/LV' sec) (/LV) (%) 1 19.80 123011 18736 100. 00 Tota! 123011 100.00

Operator: Hlrokazu Kiuehi 

### Standard solution 150 

### mg/L

I

# ~ 

# - . 

# L
• I I 0,0 10.0 20.0 ....A.-­

I 

### so. 0 Operating date: Sep. 13.2006 PKNO Time Araa Height Cone. (min) (/LV' sec) (/lV) (%) 1 19.80 242535 37133 100.00 Total 242535 100. 00

Operator: Hlrokazu Kiuehi**Stand~rd** 

### solution 300 mg/L Operating data :**Se~** 13,2006

1 PKNO Time (min) I 19.79 Total Area (/LV'see) 479957 479957 Height (/LV) 73406 Cone. (%) 100. 00 100.00

# ~ 

# . 

# l.
"---­
i {).O i 1.0. 0 I 20.0 i 30.0 Operator : Hirokazu K1uehi Fig. 2 - I Chromatograms of HPLC analysis [or calibration curve (test item). Date : Sep. 13. 2006 Name: 

# H (**k1lAc&.~**

901 

## -38­Study No. 14711

600000



## o**~--------------~--------------~**

0.0 1SO.0 300.0 Concentrati0D 

### (mgIL)

y 

## = 1605x r = 1.00 Concentration Peak area**(~)** (;lV'sec) 75.0 123011 ISO 242535 300 479957



### Fig. 2 - 2 Calibration curve oftest item. September 13, 2006 Name 

# H.**t~t**



## -39­ 9021 

### Solvent blank 21-Feb-200 14711b002 SIR of 1 Channel ESTIC 5.00e5

Area Peak position n.d.



# 1

8.00 10.00 12.00 14.00 

### Standard solution 2.50 

### mg/l 21·Feb-200 14711b003 SIR of 1Channel ESTIC 5.ooe5

Area

3.89 33041 8.00

S.OO 10.00

10.00 12.00 14.00 

### 21-Feb-200 SIR of 1 Channel ESTIC 5.00e5

Area

12.00 14.00 

### Standard solution 10.0 

### mg/l 14711b005 

### 21·Feb-200 SIR of 1Channel ESTIC 3.87 5.00e5 Area 1**p.~~~~~~~~~~~~~~~~~~~~~~~~~**

0.00 2.00 4.00 6.00 B.OO 10.00 12.00 14.00 Fig. 3 - 1 Mass fragmentograms of LC-MS analysis for calibration curve (pentaerythritol).


## -40­ 90370000 Study No. 14711

,0 /
, " 

# // /'"

,......

I 

### ...., 

# g 35000 

# /**~** /r 

### If 

## /

# / / 00.0 5.0 lO.t} Concentration (mgIL)

y=6453x r= 1.00 Concentration Peak area (mgIL) (0)

2.50 16547 S.OO 33041 10.0 64044



### Fig. 3 - 2 Calibration curve ofpentaerythritol. February 21, 2007 Name



## -41­ 904l 

### Solvent blank 23-Feb-2007 14711b023 SIR of 2 Channels ES

### 100- TIC 1.50e6

Area
Peak**I>o~itlon** %­ n.d. 

# 1

, I 

## , June 1 0.00 

# 5.00 10..00 15.00 20.00 25.00 

### Standard solution 2.50 mglL (3MBA) 23-Feb-200i 14711b024 SIR of2 Channels E8­ 100- TIC 1.5086

Area 

### %: 

### 4.'" 

# 12158 1 Time 0.00 5.00 10:00 15.00 20.00 25.00 

### Standard solution 5.00 mg/L (3MBA) 23-Feb·200i 14711b025 SIR of2 Channels ESTIC. 100­ 1.50eS Area
%­ 

# 4.76 24r1

, 1 Time 0.00 5.00 10.00 15.00 20.00 25.00 

### !Standard lolutlon 10.0 mg/L (3MBA) 23-Feb·2007 14711b026 SIR of2 Channels ESTIC 100­ 1.50e6 Area**%~** 4.60 

# 17 ·Tim$ 1 

# .**25~OO** 0.00 

### 5.00 10.00 15.00 20.00 . 

# ILtl"l \ ,**2oa~,** 

### 'L. '2) Fig. 4 - 1 Total ion chromatograms of LC-MS analysis for calibration curve (3MBA). 

## -42­ 905Study No. 14711

60000



### ,...., I 

# - ; 30000 

# !

o**~--------------~--------------~** 0.0 5.0 10.0 Concentration (mgIL)

y = 4861x r= 1.00 Concentration Peak area (mgIL) (-) 2.50 12158 5.00 24221 10.0 48657

Fig. 4 - 2 Calibration curve 

### of3MBA FebIUaI)' 24, 2607 Name



## -43­ 906

### Solvent blank 23·Feb·200 14711b017 SIR of 2 Channels ES·, TIC 1.50e6

Peak position n.d.



# 1
Standard solution 2.60 

### mg/l (SBA) 14711b018

SIR of 2 Channels Es.­ TIC 1.50e6

Area
11.22 104712 %**1~~~~~~~~~~~~~~~~~~~~~~~~~me** 0.00 5
..00 10.00 15.00 20.00 25.00 

### Standard solution 10.0 

### mg/l (SBA) 23--Feb·200 14711b020 SIR of 2 Channels ES· TIC 1 1.50& 196699 Area 11.23

Fig. 5 - 1 Total ion chromatograms of LC-MS analysis for calibration curve (SBA). 

## -44­ 907Study No. 14711

250000 

# r

: 

## j A) //'. 

# /
.-. '-' 

### • 

# B 125000**~** u J:I.. 

# ol::

0.0 S.O 10.0 Coacentmtioa (m&IL)

y

## =19993x r .0.998 CoDccntutioo Peakuca. 

### (ma1LJ 

### (.)

2.50 53308 5.00 104m 10.0 196699



### Fig. 5 - 2 Calibration curve ofSBA. February 24. 2007 Name



### -45­ 908Study No 14711 Standard solution 300 mg/L Operating date : Sep. IS. 2006

1 PKNO I Time (min) 19.79 Total Area (ttV- sec) 484689 484889 Height (llV) 73583 Cone. (%) 100.00 100.00



# . 

# if'....


# - 1.

. "'-­


### , j I 

### o. a 10;0 20.0**30~** O(min) Operator: Hlrokazu Kiuchi 

### Water 

### + test ilem -\ Operat ing date : Sap. 13. 2006 PKNO

I Time (!ninl 19.79 Area (uV'sec) 450387 Height Cone. (u.Vl (%) 67340 100. 00 1 Total 450S87 100.00



## • 

# J"­

- ..­ 

# t

. "---­ i 0.0 j 10.0 I 20.0 

## sO. Olmin) Operator : Hlrokazu Klucht 

### later 

### + test item -2 Operating date: Sap. 13,2006

1 PKNQ Time (min) 1 19.78 Total Area (ILV'sec) 473462 473462 Height Cooc. (ltV) (%) 71376 100.00 100. 00

# ~ , 0.0 

### • 10. 0 

# l.

I 20.0 '-­ 30: O(mln) Operator Hlrokazu Kiuchl : Fig. 6 - ] Chromatograms of HPLC analysis for recovery test (test item). Date : Sap. 14, 2006 Nama : 

# H,**t.n~\**



## -46­ 909Study No 14711 

### Sludge t test item -I OPil rating date ; Sep. '4. 2006 Pl(f.D

I Time (mIn) 19.77 Area (/,V'sec) 467398 Haight (ltV) 70581 Cone. (%) 100.00 1 Total 467898 100.00



### II 1


### , I 

### • 0.0 10. 0 20.0 

### .sO. o(mln) Operetor : Hirokazu Kiuchi Sludge t test item-2 Operating date :**Se~** 14.2006

1 PKNO Time Area Height Cone. (min) (/'Y'see) (JJ.V) (%) 1 19.78 450164 61299 100.00 Total 450164 100.00

## ~ 

# - l.

A 

### • 

## , 0.0 10.0 I 20. 0 

## 3d o{min) operator : Hirokazu Kiuchi 

### Control blank Operating date : Sep. 14. 2006
Peak position n.d. PKNO Time (min) Total Area (UY'$Ele) 0 Height (UV) Cone. (%) 0. 00

# ~ .



# - . 

# t

"--­


## , I I**30~** o(min) Operator : !iirDkazu Kiuehi 0.0 10. 0 20.0 Fig. 6 - 2 Chromatograms of HPLC analysIs 

### for recovery test (test item). Oate ; Sep. 14, 2006 Nellie: 

# H,**t.c~**



## -47­ 910Study No 14711 

### Siandard solution 300 mg/L Operating date : Feb. 21. 2007

1 PKNO Time (m!n) I 19.76 Total Area (IN· sec) 481034 1,81034 Height Cone. (/.IV) (%) 80313 100.00 100. 00

# ~ • 

### , 0.0 10.,0 [t) Waler 

### + test ilem ..II 

# 1.

, 20. 0 

# - 30: O(min) Operator : Hirokazu Kiuchi Operating date :**Fe~21,2007** f'Kt.K) Time Area Height Cone. (min) (ttY'sec) (jJ.V) (lib) Test item I '9.88**~** 70018 94.43 Oonverted 2 22.57 26347 3791 6.67 product F Total 472985 100.00 

# \' l.

# /

, 

## . ...­

# ~ ----"
, 

## , I sO.OCIIIl.n) Operator : Hlrokazu K!uchi 0.0 10: 0 20.0 (2] Sludge 

### + les I item Operating date: Feb. 21, 2007, PKHO Time Area Height Cone. (mIn) (jJ.V·sec) (/.IV) (,.;) 1 19. B4**~** 59086 94..13 Test Item 2 22.51 23764 3459 5. 87**Co~** product F Total 404596 100.00 

# \1 

# j l.
, .J,

# Jl~ , I 

### • sO. O(min) Operator : Hlroka:z:u Kluchi 0.0 If}. 0 20.0 Fig. 7 - I . Chromatograms of lIPLC analysIs for test solutIon (!est Item). Date : Feb. 22. 2007 Name 

# H. f:.:OClc£

-48­ 911Study No. 14711 (3) Sludge t test iIem Operating date: Feb. 21,2007 PKNO Time Area Height Cone.. (minJ (/LV· sec.) (uV) (%) 

### , 19.81 ..3UlAL 59291 93. 76 Teat item 2 22.4Ei 24996 3619 6.24 prcduc:t F Total 8911042 100. 00 Converted 

# \' l. 

# ./**~** .J. ! -"­ 

### • 0.0 10. I () 20.0 

## , 

# ao. OCmin) Operator: Hirokazu Kiuchi (4] 

### Sludge 

### + lesl it ell Operating date :**Fe~21.2007** PI<NO Time Area Height Oone. (min) (/LV •sec:) (1M (%) 1 19.18**~** 6090<1 93. 65 Test item 2 22.45 26012 3853 6.36 Convwted produc:t F Total 410759 1 ()(l 00

'A.-­

# .l~~ --" 

# \1 I. 

# /

, 1 I 30: O(min) Operator: Hirokazu Kluchi 0.0 1'0. 0 20.0 [5) 

### Con I

### fO 1 blank Doerating date : Feb. 22. 2007 PKNO Time Area Heillht Cone. (min) (lLV' sec) (I'\') (%, Peak position Total 0 0.00 n.d.



# 1

.. .A..­

# ~ • I 

## , 

## , 0.0 10.0 20.0 

# so: o(min) Operator : Hirokaru Kiuchi Fig. 7 - 2 

### Chromatograms of 

### HPLC analYSIS for 

### test solutIon (test Item). Date :**Fe~** 22. 2007 Name : 

# H

-49­ 912

### tandard solution 10.0 mglL 14711b006 10 3.88 63917



### [1] Water + 

### test Item**(LC~MS** 

### sample -3) 14711b007

Pellk position n.d.



# 1 10.00 

### 21-Feb-200 SIR of 1 Channel ESTIC 5.ooe5

Area

12.00 14.00 

### 21-Feb-ZOo SIR of 1 Channel ESTI 5.0085**o~~~~~~~~~~~~~~~~~~~~~~~~nme** 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 

### [2] Sludge +

### test Item (LC-MS sample -3) 14711bOO8 1 

### 21-Feb-2 SIR of 1 Channel ESTIC 5.00e5

Area

Fig. 8 - 1 Mass fragmentograms of LC-MS analysis for test solution (pentaerythritoi).


## -50­ 913[3] Sludge 

### + 

### test Item (LC-MS sample -3) 14711bOO9 1

[4] Sludge 

### +

## teat Item 

### (LC-MS sample-3) 

### 21",..,..20 14711b010**SlRof1~le~** TIC 10 S.00e5 Iwa

1

P••position n.d. (II

### Control bJnak (LC-MS sample -3) 14711b011 10.00 12.00 14.00

Fig. 8 - 2 Mass fragmentograms of LC-MS analysis for test solution (pentaerythritoO
. 

## -51­ 914

### Standard solution 10.0 mglL (SBA) 23·Feb·200i 14711b021 SIR of 2 Channels ES·' TIC 10(} 11.22 1.S0e6 190718 Area



### IStandard solution 10.0 mglL (3MBA) 23-Feb-2007 14711b027 SIR of2 Channels ES· 

### 10(}

### =r~ 1.SV<:!Q

Area

4.80 49090
1 0.00 5.00 10.00 16.00 20.00

# as~o~ [1] Water + 

## test Item (LCMS sample -4) 23·Feb·2007 14711b028 SIR of 2 Channels ES· TIC 

### 1QO­ ·1.50e6 Peak position Peak position n,d. n.d.



# 1 1 [2] Sludge + test 

### Item (LCMS sample -4) 23-Feb.2007 14711b029 SIR of 2 ehannels ES­**4~ 1~**  

### M**1~e6** Peak position n.d. Peak position n.d.

Fig. 9 - 1 Total ion chromatograms of LO-MS analysis for test solution (3MBA and SBA)
. 

## -52­ 915

### [3J Sludge + 

### test Item (LCMS sample 

### -4) 

### 23-Feb-200 14711b030 SIR of 2 Channels ES10

Peak positlon Peak position n.d. n.d. % 

# 1 1 [4] 

### Sludge + 

### test Item 

### (LCMS sample 

### -4) 14711b031
. 1

Peak position Peak ))Osition n.d. n.d. TIC 1.50e6

# 1~1**~1~.,....,.......,** 0.00 5.00 15.00 

### (6) 

## Control blank 14711b032

Peak position Peak position n.d. n.d. 

# 1 1 Time 1 

### 0.00 5.00 10.00 15.00 

### 20.00 25.00 

### I 

## 't**~** 

### II**2(J(1~,** 

### 'L. 

### llf**M~** 

### Ttl.ilAk,\

Fig. 9
.. 2 Total ion chromatograms of LC-MS analysis for test solution (3MBA and SBA)

.


## -53­ 916

### Solution oftest item (100 mg/L) 14711b029 

### 22.feb·200 2: Diode Array Range: 1.298e+1 1.0$+1 Test item 

# I
25.72



### olutlon of 3MBA (100 mg/L) 22-Feb-200 14711b031 2: Diode Array Range: 1.443e+1 1.0e+1 7. 5. 

# I 3MBA**~ ~~~~~~Tn~~~~~~~nT~~~~~~~~~~n~**  5. 10.00 15.00 20.00 25.00 30.00 35.00 40;00 

### olutlon of SBA 

### (100 mg/L) 22·FelJ..200 

# I 14711b030 SBA 2: Diode Array Range: 1.966e+1**~** 5.

.00 2$.00 2: Diode Array Range; 1.322e+1

O

.. Testttem 

# /

•• '12



# I

Ltflll 200'7,].., 

## 1 L
. Fig. 10-1 UV chromatograms of LC-MS analysis for test solution (205 - 300nm) (LC-MS sample -1, qualitative analysis of oil-soluble converted products). 

## -54­ 917

## i

6.0010.00 [2] Sludge 

### + test item (LC·MS sample .1)

### 22.Feb.200~ 147111>020_1 2: Diode Alray Range: 1.227e+1 1.08+1 Test item 

# I
25.76 33.15 S. 2. 

### o. 'L,..,.....,IJI.""""'''''''''''''''''''rM'''T'''''''TTT"T''T'T'''''''''''''''''''''''''''''''''!"'/'TT''rT'''''''''''''''''''''""",'TTT'''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' Time

..00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 t3] Sludge + test item (LC·MS sample ·i) 

### 22·Feb·200 14711b021
. 2: Diode Array Range: 1.33Se+1 1.0e+l Test item 7. 

# I S. 25.72**~** 2; O.**~~~,..,......~~~~~,......,.,,.,.,...,~.,....,..nT,..,......~~~.,....,..~,..,....,~~.,....,..~nme** 15.1l0 20.00 25.00 30.00 35.00 40.00 [4} Sludge 

### + test Item (LC·MS sample ·1) 

### 22·Feb·200 14711b022 2: Diode Array Range: 1.304&+1 1.0e+t Testltem 7. 

# I 5,**~** 2. O. 5. 10.00 15.00 20.00 25.00 30.00 [5] Control blank (LC.MS sample .1) 

### 22·Feb-200 14711b023 2: DIode Array Range: 1.1978+1 1.0e+1

..5

5.00 10.00 15.00 

# 14-1 [I .00 Fig. 10-2 UV chromatograms of LC-MS analysis for test solution (205 - 300nm) (LC-MS sample -1. qualitative analysis of oil-soluble converted products). 

## -55­ 918Solution of test item (100 mg/L) 14711b029 Test item 

# 25.82/**2~~~~~~~~T"~~~~~RTnT~"i~i~ij~'~"~i~li~i~"TI'~i~ii~l~iI~i~i'~i~"~iIDM** 6.00 10.00 15.00 20.00 2S.OO 30.00 35.00 40.00 Solution of 3MBA (100 mg/L)**22~eb-200** 14711b031 1: Scan ES+

U.18 20.00 25.00 30.00 

## nc 1.50e8

1: ScanES+ TtC 

### 1.so.s

30.00 22-Feb-200 1: Scan ES+ n 1.50e8 Test item 

# U.7Y

6.00 taOO 11'1.00 20.00 25.00 

### t

# Lt'l 

# t \**2oo~** 

## '- 1. L 2­ Fig. 11-1 Total ion chromatograms of LC-MS analysis for test solution (LC-MS sample -1, qualitative analysis of oil-soluble converted products). 

## -56­ 919

## o E 

### e c 

# \1 \! 2Ut**".-:-:-,,----------:~~---:--;-:-----".---** •...

### ----.----:-::-:::---......, 

## 121 Sludge 

### + test Item (LC-MS sample -1) 22-Feb-200"11 14711b020_1 1: Sean ES+



### 22..feb·200 1: Scan ES+ TIC 1.50e8 Test item 

# 2UY

TIC' 1.5Oe8 5.00 10.00 15.00 20.00 TIC 1.5Oe8



### [3] Sludge 

### + test Item 

### (LC-MS sample .1) 14711bQ21

.



### o e c \ E



# \/ ai
4) 

### Sludge 

### +test Item 

### (LC-MS sample -1) 22..feb·200 14711bQ22 1: Scan ES+,

5.00 10.00 1 [5] Control blank (LC-MS sample ·1) 14711b023 1: Scan E$+ TIC 1.50.6

29.43 30.8$ 33.12 

# {'t" \ \ lrJO

# '7, 1**~** 22... 

# M, 

# TO-r\.\\<\' Fig. 11-2 Total ion chrDmatograms of LC-MSanalysis for test solution (LC-MS sample -1, qualitative analysis of eil-solubteconverted products). 

## -57- 920

### 1[1] 

## Water + test item (LC·MS sample ·1) 14711b019**.~**

14711b019 UV chromatogram (205 - 300 nm)

5.00 10.00

80 Total Ion chromatogram 15.00 20:17

20.00 Test item 

# 25.72/

25.00 30.00

26.15 Test item



# /

&.00 :10.00 15.00 20.00 25.00 30.00 

## til Water 

### + test Item (LC-MS sample -1) 14711b0191593 (29.444) em (1688:1608-(1574:1586+1614:1635»(1.500) 1**1~** 

### 22·Feb·200 2: Diode Array Range: 1.322e+1

35.00 40.00 1:ScanES+ TIC 1.0798

1:SeanES+ 2.5Oei Mass spectrum of peak F

14711 b019 1616 (29.859) em (1613:1637-(1605:1613+1641:1650)x1.500)**1~**

200 4Q() 600 

## lLt'l t \ 1: Scan ES+**~~** Mass ofpeak G

1600 1



## 20017,**2_1~** Fig. 12-1 Mass spectra of oil-soluble converted products (LC-MS sample -1, water + test item). 

## -58­ 921

## 1[1] Water 

### +test item (LC·MS sample -1) 

### 22·Feb·200 114111b019 1: &:anES+ 25.15

6.00 10.00 16.00 20;00 25;00 40.00TIC Test item t.07eS Total ion chromatogram 

# I

14711b019 1: SeanES'" mfz =1104 1 5.91

10.00 15.00 20.00 25.00 30.00**~.OO** 40.00 14711b019 1: ScanES+ mfz =11 1 1.S986

5.0010.00 15.00 20.00 25.00 Fig. 12-2 Mass spectra of oil-soluble converted products (LC-MS sample -1, water + test item, mass chromatograms). 

## -59­ 922

## Study NO.14711 

## PeakF

__ ._._._ •• •••• _._._. _____ • __ w __ ._. _______ • ______ • ___ __ ••• _______ •• ________**~_~_.** ___**~** ___**.~ ~~** **~ _~_.~.**  

# . .

-............

### --- -.. _

### ...
_

### ..........
_

### .... -_ ..
_

### ..................

### -.......
_

### ............... _

### .................................

### -...................... _

### ................................ 

## m/z Fragment ion 

# 1104 (M 

# + NH4)+
Fig.12 - 3 Mass spectra of oil-soluble converted products (LC-MS sample -1, water 

### + test 

### item, presumed fragment ion of peak F). Date J..tID

### 1. i. 3 I 

### Name 

# 7. f:'

# {f~ c-"



## -60­ 923

## Study NO.14711 

## PeakG



# m/z Fragment ion 

## 1102

Fig.12 - 4 Mass spectra of oil-soluble converted products (LC-MS sample -1, water 

### +test item, presumed fragment Ion of peak G). Date 

## ;u;o 

## 7· t·} 

### I Name 

# 7.**j::e£'J1wt.~p./** V



## -61­ 924[3) Sludge + test item (LC.MS sample -1) 14711b021

3.



### o.

14711b021
80 UV Chromatogram (205 • SOO run)

5.00 10.00 Total ion chromatogram 15..00 20.71

20.00

.l!t 

### o B C \ /

\ /

# ~3
5.00 10.00 16.00 2.0.00 Test item 

# 25.71

25.00 30.00

26.78 Test item 

# I

E

25.00 30.00 

### 22-Feb-200 2: Diodefi.rray Range: 1,336e+1

35.00 40.00 1: Scan ES+ TIC 1.08e8



### [3] Sludge + test Item (LC-MS sample ·1) 14711b021 918 (16.967) 

### em (903:918-(872:880+931:9S9)x1.500) 1. :Q$

.ji.t

14711b021 945 (17.486) em (944:951-(934:938+964:969)x1.500) 1 341

1000 

### 22·Feb--200 1: ScanES+ 'lAW Mass spectrum of peak. B

1200 Mass spectrum of peak. C

1400
. 1800**1o(}~** 

## _ 1 _ 

## 1.1 Fig. 13-1 Mass spectra of oil-soluble converted products (LC-MS sample -1, sludge +- test item, vessel [3]). 

## -62­ 925

### 3] Sludge + 

### test item (LC-MS sample -1) 14711b0211189 (21.978) em (1176:1194·1182:1172xl.500) 100 4511 

# I

14711b0211208 (22.328) Crn(1203:1213-1221:1229x1.500) 1 4tI(I

14711 b0211593 (29,444) em (1591 :1603-(1573:1583+1S12:1625)x1.500) 1 1104 

### 22-Feb·200

Mass spectrum of peak D

1: Scan ES+**8.7~** Mass spectrum of peak E

1: Scan ES+ 3.44e6 Mass spectrum of peak F

14711 b021 1616 (29.869) 

### em (1613:1635-(1809:1612+1637:1841)x1.5OO) 1: Scan ES+ 1 1m 3.1.385 Mass spectrum of peak G

200 1000 1200 140D 

# 200

# 7, 1,12. 

# M

# \!a\1.Ak; Fig. 13-2 Mass spectra ofoil-s'oluble converted products (LC-MS sample -1, sludge + test item, vessel [3]). 

## -63­ 926

### 22-Feb-200 

## [21 Sludge 

### + test item (LC-MS sample .1) Test item 14711b020 1 

# 0

-. D 25.4e F

# .:r:~':~.:h::~'::::~,

# """""\,**/.",'\l)~;;""** i';'" 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b020_1 1: Scan ES+ 11 

# 1:'] 1 1.,3 

### 2":Z1· mlz

## :'2~!~ JI1t 

# J

.\ t''', . i 1'1 ' I" • "t'" ,'r,**~.,** , ItI"'t**I"~** J r

# 1 ,,", ii' " 'I r ; 'P
.... l rt,il

### \ I i

# 'f!,,,,.A~1 f

## ,..£4, rI't'

# "I''''''~' ,',,".' 5.00 10.00 15.00 20.00 2MO 39.00 35.00 40.00 14711b020_1 1: ScanES+ 1j 17.13 mlz 

### =339

### 1~l 5;()2'

"', j f I, • " .1 •• ' i I' f "1 i I H \1. I, j I

# .~~. "i'

# I''''~ 'I ."1111 HI. I' , "1' ,', 'I" , 1\ ' I. 'I 5.00 10.00 15.00 20.00 26.00 30.00 35.00 40.00 14711b020_1 1: Sean ES+ 

# 1:1 11.13 mlz 

## = 361 ".21 8.88&4 ;#I

# t~**;i''ll!tf''~ lI\'tlhi";I'I<\>t't''''~.~** **IAf~¥fltliA"'''''''M''~~~/'I'II'''f'''''''''1** 5.00 10,00 15.00 20.00 25.00 30.00 35.00 40.00 14711b020_1 1: Sean ES+

### 1~**~ ~=W**  

# I 4.3985

.,. " ,., pIt I I • I

# ~i I I q Ii' '\ ,'1"( I , r III

# ~ 'T I , tt l¥t t't if I 1"1'1' I", I'll I'll I I , ,'\, I I "I{ 5,00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b020_1 1: Sean ES+ 1j 17.71 mlz 

## = 274 17, 20,: U.24 aU$ S2. 5.1284



## .,.

# qIl""II'IP~'~,*\tA"'I"~I*,AI'ffl".~tft~Q,***t~~~.".'~;~** 5,00 10.00 16..00 20.00 25.00 30.00 35.00 40.00 14711b020 1 1: Sean ES+ 

# 1j

- '7.71 mlz =323 

# I 22.48 25.18 3.B1e6

..- , , , " "" " , " , " " , " " , " "'I " 

# ,J , , , 1 , ,

# ,..l,**,,~l.** 

# """'" ,,,,, I " " I " "':00 5.00 10.00 16.00 20.00 25.00 30,00 35.00 14711b020 1 1: SeanES+

# ~ 

# - T**~~**



# J
Ii I 'I' Ii i\" , j i' I Iii I. "I' II i l'

# !l"~i' ,'j i' II., I ill 

### r40t" '1 " ,111'1. 

# r, fl', 

### 't

# ""'if '" q S.OO 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b020 1 1: Scan ES+

## 1~ - 1'1. 74 mlz

## 2~J: 

# J , , ,', t' i r I I , , I 

### 'i' i , • I • I til I I

# ~ I , { • I .'1'
....,,\

# ,~ t _,"1'1 I'T' " t

# I~**11"1~'1'** I! v,'r n't

# l"'~; rv, 5.00 1000 15.00 20.00 25.00 30.00 35.00 40.00 14711b02C l' 1: Selin ES+ 

# 1:J

-**1T~.82** mlz:. 7::

# J~II"'i\ f,
.. ,,,, ... fl'l , .....1'.,

# 'Pl"fo)t,.n~wItJllllJL·H'tw""'i';'r4J~N!c¥~*f!r""".I.,tl\\I.4lPIP"v.~ Time 5.00 10.00 15.00 20.00 26.00 30.00 35.00 40.00 

## \4-**~** 

## \\ 

## lOlJry. 'L .1.1..

### h,~Y\\t..; Fig. 13-3 Mass spectra of oil-soluble converted products (LC·MS sample -1, sludge + test item, vessel (2), mass chromatograms), 

### 927 

## -64­[2] Sludge 

### + test item (LC-MS sample -1) 

### 22-Feb-200 147110020_1 1: Scan ES+ Test Item TIC 1.18e8 Total ion chromatogram 0 B ;I 

# \;E \! C
2 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b020_1 1: Scan ES+ 100 17.13 

### 22.11 mlz 

### =321 

# 1 

# 1 nl 1
...." ",
..""',,, I" 

# "',','r "''I''
.. .., I
....

# J..
....,rl,

# II,,,,,,,,,,,,,,,,,,,,,
.., UM
5.00 10;00 15.00 20.00 25.00 SO.OO 35.00 40.00 14711b020 1 1: Soan ES+ 100 1X4 - m/z =4 

# oj,,,,, ',,,,,,,,, ',",',,,, ....,, ',",',,, 

# ,1
.. t,

# [~ .. '''I''''' ,...., I " "" "..," 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

# ~S:020_'. 

# T**~.. '~;;~**  

# J"",,,,,,,,,,,,,,,,.,,.,,,,,,,, ,."".,

# "'~'" ,Jl.':i' t, , ... , ..

# A
..,,, "\ 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b020_1 1:ScanES+ 

# 'j,, """ ,:'

0

'" " " ,"0 ., ," ,,'" , I ' 

## "0' '

# "of",", ,,

."," M., ,".,.. "'"

# ,~~.= 5.00 10.00 15.00 20.00 26.00 SO.OO 35.00 40:00

# ~5-_1 t22.1.

# 1:~~:;:a 

# J

# ,,,,~,"", """, "I , ,.,....

# "M, '"

# "'~'''''t ...

# fI""~,"""""""'''' ' " 5.00 10.0015.0020.00 2&00 SO.OO 35.00 40.00 14711bG2O 1 1;ScanES+ 

# 1J

-**~1~'14** trrIZtti,79



# "1".1
... f'r'" 

## 'j'

# ,,,,,,,¥,,,,~,\... ''to
....

# ,-.""~~IIIII4""""~ Itoo 10.00 15.00 20.00 26.00 SO.OO 35.00 40.00 14111b02(U 1: Scan ES+ mlz =575 1.976& 

# 1:r'
. :/2,01

# ~.U 

# -J"" t
.. ,

# 'r'."."" ,"" '''''''' ,", I"

# ".,,1,."il\.,
..

# ,.~",,..,,,,..

# ~, " •.­ 8.00 10.00 15.00 20.00 2.5.00 .00 3500 40.00 

## I

# Lt'l t \ Fig. 13-4 Mass spectra of oil-soluble converted products (LC-MS sample -1, sludge + test item, vessel [2], mass chromatograms). 

### 928 

## -65­5.00 10.00 15.00 20.00 25.00 30.00 

### 22-Feb-2007!

35.00 1: Scan ES+ TIC 1.18e8

40.00 14711b020_1 1: Scan ES+ 

# 1:0

# 1 ,,,. "'"

# I,.,1
..1" ."
.**,~Z3~':~** • t ,t I iii It '1' ii' I 4 11 • t 1.' j it I 

### ','i i 

### i, iii'" pI' , " t 1 f i]'o i 5.00 10.00 15.00 20.00 26.00 30.00 35.00 40.00 14711b020_1 1: Scan ES+

### 10~j"6**~.23** m/z

## = 425 

# "J,,,"
.. "1;"'''' ., .... ,.. "''''.,"
.. ,.

# "I.". ,lL.
....".,""
.."
.."
..:....., s. 10.00 15.00 20.00 25.00 80.00 35.00 40.00 14711b020_1 l:ScanES+

# ~:I.'" ., ...., ....,"
..,,,.., ....,...., ....,...X
....

# 1.
........, ...., ....,.:

# .~.::l 5.00 10.00 15.00 20.00 26.00 30.00 35.00 40.00 14711 b020 1 1; Scan ES+



# J - 2244 m/z 

# = 460 

# ':J.
..., ....'"
.."
..,, ....,"
..,"
.."
..., ...

# J"., ...., .. <0, "
.. ,"
.. ,...• ,"
.. 

# ,7.82.' 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b020_1
. 1: Scan ES+**1~** 22.44 mlz 

# = 465 

# "'J.·..,.... ,·...,.... ,..·.,"
.."·.".,
..,..

# ,i.,"
.., ....., ...., ...."',.,,,,",.,
...... 5.00 10.00 15.00 20.00 25.00 SO.oo 35.00 40.00
22. 1 

### tt 1: Scan ES+ mlz 

## = 481 2.68&5

### ~4j711b020_1 22 

# r

" ,II iii i

### i~t;;'" i 

## I' i

### 11~OO**1'i"J'I'~~~;;:'''I'** 

# I**~~tl 2~:~'l·r't".** **f';~!:ItI't'9'+'~~~rio""lll""~o'oo** 14711b020 1 1: Scan ES+ 

# 'j

# ,."~'" "
...,'
..'."'".",
.." ,.,""
..'""·" ".4
....

# ,1""
...," .,.,**,~,~=** 5.00 10.00 15.00 20.00 26.00 30.00 85.00 40.00 14711b02O_1 1: Scan ES+ 

# 'j"
..,.""
...., ...., ...."
.. 'p'" , .... , .. """ "A"

# ""I"",
.. Ph"

# .~~,;: 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 

## I

## tt'1 \ \ 1007, 1. 

## .11­ 

# M
..1o.Y'fJ.k; Mass spectra of oil-soluble converted products (LC-MS sample -1, sludge + test item, vessel [2], mass chromatograms). 

### 929 

## -66­[3] Sludge + test item (LC·MS sample ·1) Test item 22-Feb-2007 14711b021**~** 1: ScanES+

# ":r::~**:~::~,':~::~~"",** 

# , "".\/'"**~v" l:~~,**  

# ,,;, ., i;,

# ~'~ 5.00 10.00 15.00 20.00 25.00 '30.00 35.00 40.00 14711b021 1: Scan ES+ 1

# j
16.91 2M1 mlz

# = 321 

# P·D2 1.24e5 

# 2'1

#. i " ,','1"1'1"1 II

# 'f'W, I
..h'l !

# ,"v,.,." 1"'1 ""

# ~" ""'I

# ~ I, ; '1'

# ~~**fiiI1!Mf,,·,~ ~4'1"/.'''~**  15.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b021 1: Scan ES+ 

# 1j
16.91 m/z =339**16.~** 3.2386 l!1! 

### j', I , I , I 'i'" , • i j , , i fl. r j·t ' 

### ,'I

# ~ I' .11.'1'1 I ., 1"+1 ' , ; I 1"\ '"" I'" I • I ' J f 'I j I I 

## ,1' I I I f'I , •." 5.00 10.00 15.00 20.00 25.00 

### ao.oo , \ 35.00 40.00 14711b021 1: Scan ES+ 

# '1 96 

# 1:]**mfz8~** it 

# J II\I,

# r'''''

# V~"+\'~j l't" f I

# '·+~rf;',·II''''''**IJ~ (.\,~Jt¥.Jlt~I~~.W.,..~~~.~J44't**  \ 5,00 10.00 15,00 20.00 25.00 30.00 35,00 40.00 14711b021 1:ScanES+ 

# 1:1 HAll mlz ;221

## ~~! a '# '. 

## ,''''''!'.'",

# I'~'II"'''I'III 't!

# ,'f.

# ~'t 'IT Iiff ,-4-1"1'1 i"i'nl ¥

# ,4,t"" ,',': r'f

# ,.,,.v,

# p,~,.,~ ... fl"l 5.00 10.00 15.00 20,00 25.00 30.00 35.00 40.00 14711b021 1: Scan ES+ 

# 1j 11.52 mfz =274



### ..

# .nO~'" I 

### ',.,. ,"nl' 

### ., " -, 

# ""f I.,. 

# .•

# J

Ii • I' 

# .."**~**

# "',,," ,"'f'" .,

# ,~ ............,. ..,: I" "i in I' I 1 5.00 10.00 16.00 20.00 25,00 30,00 35.00 40.00 14711b021 1: Scan ES+**1~** 11.48 2U3 mfz =323 

# 2I J",,,"" ,',,'I' " , I ' " , , " , , I'

# ~'J,

# ", I "

# ,1"**,'~** 

# ,"',, I' , " , '" , I " " " '"

# II~"" 5.00 10,00 15.00 20,00 25.00 30.00 35.00 40.00 14711b021 1: Scan ES+ 1:J 1U2 mfz 

### =341 

# j, ,",","" """ "'" "'"''

# ,.,J. ,,"" , 4., ",4. 'I t" ",",,, ",' I' ,,' I
.. ":·
... 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b021 1:ScenES+ 

# 1j l1l'47

# mlZ3~Se5 I , , I I ' " , , I" j , , i ' • i , I i I I , , f •j I il i 1\' lti**~t,** ij , , iI" I 4 I I , I •, I**~** I ( r• I • t Ii. iii i i

... .. 

# *ft**~'r',e""r''''tl** 6.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711 b021 1: Scsn ES+ 1

### ~ 'J7.50 m/z1

### ~2~~5

# ~J i,ll', '''/

# ,~

# it., 1'.,"" ,', ,', i I'P, ."1 1.''1''11

# "~''I''~.r,i .'1 

### ;1
...."

# t'''j~''f4'1**Jl'~"t'''M,AyojI~'''''''''''.1** Time 5,00 10.00 16.00 20.00 25.00 30.00 35.00 40.00 Fig. 13-6 Mass spectra of oil-soluble converted products (LC-MS sample -1. sludge + test item. vessel [3]. mass Chromatograms). 

## -67­ 930[3] Sludge 

### + test item (LC-MS sample -1) 

### 22-Feb-2007 14711b021 1: ScanES+**25.7~sti1em** 

## - TIC 1.OSeS

# .:~TTo~ta~li~on~Ch~roMm~a~to~g~ranmTr~~~~B~,\~~~C~~D~~~~~E~~~~S~~~F~~~~G~~~~~~~~ I 

## 5.00 10.00 15.00 I 20,00 25.00 31).00 35.00 40.00 14711b021 1; Scan ES+ 22 1 

# 13 

# t

8.91 21-7 

# :1

.01 m/z

# 1~~ 18.67 17.D2



## "

# -M'II""'~**"'~!'** ...,..,."" 1'IiWNI--l

# 'I~ ...

# *4</ • .N,
....

# ~~ 5.00 1MO 16.00 20.00 25.00 30.00 35.00 40.00 14711b021 1: Scan ES+ 

# 1j<X2**~'1I9** 

# m/Zto: 21." .06
" "" I ' "
..... '" ' " , I ' " 'I
.. " I " " I I " 'I • ,

# !",, I " , 

## "'\1"" M, " '
... , ..... , 5.00 10.00 10.00 20.00 26.00 30.00 35.00 40.00 14711b021 1: Scan ES+ 

# 1j 441 2s:?8 m/z

# =**21.~99** 

# r·· 5;27$5

... ,"'" "'''' " I" 'I'"." •••• ".,

### I"" 1•

# .J'\h"

# I~'''''''' '1''''', • 

# ..4
.. ",""1 5.00 10.00 15.00 20.00 25;00 30.00 35.00 40.00 14711b021 1: Scan ES+ 

# 'j

I , , , I • I , I I' I I , I I I I 'I " I , I ' • I I , I I I , I ' , , , I

# "I",, I'?' , I I , , , I " I , I ",...,.. , , I 

### I'

# ,~.:;:: 5.00 10.00 1l5.O0 20.00 26.00 30.00 3$.00 40.00 14111b021 1: Scan ES+ 

# 1"**Wz=~** 

# 'j
.., ...'**,..,.~. ~,** **r'~'I""'~~!\I':""** I 

# "t'" 'I;' I.,.", ,,.., .. 5.00 10.00 15.00 20.00 25.00 30.00 35,00 40.00 14711b021 '1: Scan ES+ 

# ''3 roo nVZ:'o:!:

# l"'\"~""",,, liot.,......"'.t>""••

# ".~ljo ...,

# i40~.~iII! 5.00 10:.00 15.00 20.00 25.00 30.00

## ~oo 40.00 141111:1021 .' • 1: Sean E$+ 

# 1j,tl:10 21·11G 

# k'"**m1Z2~e~**
'

, .,' !
... .., I
... 

### '-'1' ' .....,I.a.L

# ~~_W. __I'II"''1''·dl'''''_''· TIme



### .. , 'I'" r j ",. 

### r.'" I'" ", n ',j**dtFp'~'** I' ,-, H 

### -rr (if FH¥jYF V.I'" 'j, 

# . l

5.00 10.00 15.00 20:00 25.00 30.00 35.00 40.00 

## l 

# 't1 t 

## I 100t"l-. 'l..12 

## M.**l'o.rli\k~** Fig. 13-7 Mass spectra of oil-soluble converted products (LC-MS sample -1. sludge + test item, vessel [3]. mass Chromatograms). 

## -68­ 931[3] Sludge 

### + test item (LC-MS sample -1)

### 22-Feb-200~ 14711b021 Test item 1: Scan ES"'j 

### o 25.7VF TIC trotal ion chromatogram t08e8**.~** 

# \/ \! 

# r\!**2~~~~~~~~~~~~~~~~!\~~A~~~~~~~** 5.00

### 10~OO . I 15.00 20.00 25.00 30.00 35.00" 40.00 14711b021 1: Scan ES'" 

# 1j 323 lVI8 22.33 25.50 m/z = 

# 1 22."o 1 4.33e5
" ," , ,,, , 'I' ' , " " , " ' , , , " , , , "

# :'~'" , " 

# .4.', ,"A., ,"" "", ,'"""
.. f'l'tl, 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711bD21 1: Scan E8:" m/z =42
.. 5.57e6 22.35 

# 1jU4 

# [.21

" '''''''''''' '" ' " " " , , ! ' , , , , ' " , I' , , " '

# "J,", "",, ',,,, "".',,, .',".
5.00 10;00 15.0020.00 25.00 30.00 3S.00 40.00 14711b021 1: Scan ES+ 

# 'J'K4 22.31 mlz 

### =443



# "J ,,",',,,,",'""".",, I' , , '" " , , , , " , ,

# ,1",,,**ft~':"** ,"',,,, "

,,

I' , " , ' , " 

# :'<T" 5.00 10.00 15.00 20;00 25.00 30.00 35.00 40.00 14711b021 1: Scan ES+ 

# 'j,,,, .',,,, ',, '"',,,, "

,,

.',,, I' ' , , , ' , , , , '

# ,1:,, '.',,,, ',,, .,,,,,',,,, ',,,, '

# ~~.'~ 5.00 10;00 15.00 2Q.OO 25.0030.00 35.00 40.00 14711b021 1: Scan ES+**~=~** 

# 'j,,,,"","'"""" 1''''' ""'"''''''''''

# I "'I"" ,"'"

# "If,,,,,,,~,,,,, ,,""
.. 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b021 1: Scan ES+ 22.36 • m/z

# = 481 

# 'j,,,,,,,,,,,,,""""" .,'''''''
M \'

# ,,,,.J,,,, r''''''''"",
..., .... .-.,.,.....a.... 5.00 10.00 1&.00 20.00 25.00 30.00 35.00 40.00 14711b021 1: Scan ES+ 

# ,'j,,,, '''''.'" ,, '" , ,,,".'","', "I''' , .. " ,"' , , '" '" I'

# ,~l"",,,,, ,,, ",

# ,~~**~~** 5.00 10;00 18.00 20.00 25.0030.00 35.00 40.00 14711b021 1: Scan ES+ 20.81 m/z

# = 1102 

# '3"" 1'''

## '1iO'''''''' '" "1''' 

### '1'''' '''' 'I' ","' 

## ,,,.M ".'

# ,,~" 'I' '" 

### 1'",.,,, ,.': 5. 1tl.OO 15.00 20.00 2500 30.00 35.00 40.00 Fig. 13-8 Mass spectra of oil-soluble converted products (LC-MS sample -1, sludge + test item, vessel [3], mass chromatograms). 

### 932 

## -69­[4] Sludge + test item (LC-MS sample -1) Test item 22-Feb-200 14711b022 •• ;; 1: Scan ES+

# ·:C::~:~::~,',t~:~~""

# """ ,\/."**~v" r~;**  :,:::"""

# i"~~ 5.00 10,00 15,00 20.00 25.00 30.00 35.00 40.00 14711b022 1: Scan ES+ 

# 1j 2111* m/z

## =

# 321 18.93**t1.~** 1.3SeS

..

# ~" 1" 

# /Io't:' l' , 

### "YO'

# ~YO'

# ,,,,,-i.,**,~,J),.** ..

# '.~"I.t.~_I"""~"'" s. 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711 b022 1: Scan ES+ 1**~** 16.91 m/z

# = 339 

# *J, ,"1'" I, "",," 

### ",¥1ft 

### 1'''''''

# "t~ A 

## r" .., NOli "''1 '" 

### '1M'''"
.

# ~, ",,', 

### ,,,,'-"'OS 5.00 10,00 15.00 20.00 25.00 30.00 35.00 40.00 14711 b022 1: Scan ES+

## 1~**1~.OO mlZ6~~:~** 

# ~l"",**,t"\""~'i'll~** 1"'-'

# '·'**i'!M"ft'h'l'''''\>llt~I+~'~f.Ji '''** **~~~'~""'lltIf~"P\** 5.00 10.00 16.00 20.00 25.00 30,00 35.00 40.00 14711b022 1: Scan ES+

### 1~**~ ~=~**  

# 6.30 Us! 2.22e5



## .. ,'
... '"

# ~**~/,** M'too 

## ''''1''"
... r"
..

### 0\011\ , ,...... I' , I'

# .~"" , ." , "" '" , 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b02.2 1: Scan ES+ 17M m/z:: 274 

# 10j r,52 8,63t4 fI! ,

# 't"'+'>'M1""h~!i'''''t III i til'

# t'.'*1'¥'·f\I~'f'1 f't'M

# 1"t*M'r.~~t,'" I\Mt'1'f'1"I"

# i4"''#f''''''r+'~'~ 5.00 10,00 15.00 20.00 26,00 

### ao.oo 35.00 ' 40,00 14711b022 1: Scan ES+ 10j 22.13 mlz

# = 323 

# j ,,",,,,,,"",""""'" ".,"

# ~r. 

# ', "" !." 1" ","" '" ," ,,

I' , " • " ,
... '" 5.00 10,00 16.00 20.00 25.00 30.00 35.00 40.00 14711b022 1: Scan ES+

### 10~ 17r 22.3

### m/z3~ 341



# fl!J
.. "I' .} " ,,', t Iii II I' j I iii \ I ii' I

# i'4'~ I I iii i i /. i 

# ,lit" i i

# l"'I"'i~ rI j I }, ,'I rill "\ 6.00 10,00 16.00 20.00 25.00 30.00 35.00 40,00 14111b022 1: Scan ES+**1~** 17.47 mlz

# = 36! 

# ':1"",,,.,,

# ""~,, .,,,,

# '1""""',1.
.. ,1'" """",,,

# 140~".Itot ...... "

# .ro,'''',:­ 6.00 10.00 15.00 20.00 25.00 30.00**~,OO** 40.00 14711 b022 1: Scan ES+ 

# 1:1 17!,<18 m/z

### 8~3~!!**~ ~~** 

# ·"",,.,'t~I'i""~li'Ir"'~'tl~'l"If'lII!HI~~L~ll'lhll"(""~!"\~i~'"_~~"'~~Tlme s.bo 10,00 15.00 20. 25;(')0 30.00 .00 40.00

## tLt~\\**2uo~,** 1.,,1..2..

## M.,lo..r~~\ Fig. 13-9 Mass spectra of oil-soluble converted products (LC-MS sampJe -1, sludge + test item. vessel [4], mass Chromatograms). 

### 933 

## -70­,[4] Sludge 

### +test item (LC-MS sample ·1) 

### 22
..

## Feb·2007 1 14711b022 1: Scan ES+

### ,,~tit'm TIC Irotal ion chromatogram 9,B1e7

# :~~~~~\~/~C~\~/~E~\~\~~~~~~ 5.00 10.00 15.00 20,00 25.00 30:00

### 35~OO· I

### 40~OO 14711b022 1: Scan ES+ 

# 10] mlz 

### =

# 321 21.98 16.13 1,3585 

# 21.1

.... "
.. , .... "", ,••••• ,,..,

# ~\'M •

# .,..l,**""4...J..,./,,,~~** ........., 5.00 10.00 15.00 20.00 25.00 30.00 35,00 40.00 14711b022 1: Scan ES+

# 'j~ .,"., .,...., ...., ..,., ...., ...., ...., ..

# I,"",, "I., I, ,j , ....... ' , "

# .~:' " 6.00 1G.00 16.00 20.00 25.00 80.00 35.00 40.00 14711b022 1: Scan ES+ 

# 1j 21.118 25.11$ mlz =441 

# j"",
...,,,."""",'" .".,."
...,.'.,

# ,.,.!.".,

# t~" ',''''f'
.... :­ 5.00 10.00 15.00 20,00 25.00 30.00 35.00 40.00 14111b022 1: Scan ES+ 

# 1J 21.l18 m/z =458 

# "'J" "','

# '~bii" ·" ';OliO;"

# ''',S~OO'' "';o1iO

# t' 2"';; ," ";;':;;';'" 

# "SO:';;""

# ,~"" 14111b022 1:**Sca~ES+** 

# ':J 

# T· m1Z:.::



# "'J. 1', i

# r~1 ,', ", i' , ; 1""11\

# "".'~"'~i" I'll .·,.. 

# J....,.,,,"""
..

# ,·A'1·44.,111~ I ,'l'1t 1 )'1'1"1 • i' • ''i I'" i 5.00 10,00 15.00 20.00 25.00 30.00 35;00 40,00 14711b022 1: Scan ES+ 

# ':']
. .... m/,·.,. 

# '''J."
.....\."'" "t,. ,., ... 'T'

# ''''I"
..

# ~. ,..,1" ,\. M
.....

# ~~.'*'" "
....'
..... 6.0(l 10.00 15.00 20.00 25.00 30.00 35,0040.00 14711b022
. 1: S!:an ES..­ 100 4>(10**mfz~S::** 

# OJ""\'" "'" 'i ", , ,,,' ."" OJ
.. " , .. " 

### r'

# ,~["**"J~T'** ...\,.. "'r""" "', "
....:_ 6.00 10,00 '\5.00 20.00 215.00 30.00 36.00 4OJ,,0 Fig. 13-10 Mass spectra of oil-soluble converted products (LC-MS sample -1, sludge
... test item, vessel [4]. mass chromatograms). 934 

## -71­ 

# \Lf1\([4] Sludge 

### + test item (LC-MS sample .1) 

### 22·Feb·2007 1: ScanES+ TIC 9.81e7

# ~:~1::~;o",hrom"o,,,m \ / 

# "\! 

# ty:

A 2,
. I 5.00 10.{!O 15.00 20.00 25.00 30.00 35.00 40.00 14711b022 1: Scan ES+ 

# 1:J 2203$ m/z 323 

# J
..""'
H " '" , , , " , H '" ' "'" 

# ,,:r. 'I"

# ,1,,.

# I, ,.". I ' ,••'. 'H' OJ , ".,,,,

# ~'.4'" 5.QO 10.00 15.0020.00 25.00 30.00 35.00 40.00 14711 b022 1: Scan ES+

# 'j~,,, 

# , ..,"
..,,'" H," H'"'' , .. ,." 

# 1,,,,[',:,,.,
..,,, ..,."
..

# ,,,,~~'= .. ."
.. 5.00 10.00 15..00 2OJ)() 25.00 30.00 35.00 40.00 14711b022 1: Scan ES+

# 'j~ 

# ...",
.., .., ",
..""
..,"
.., ..,"
...., ..

# T,
..[:".,
.., ., ..,', ...'"**~~'~2:::** 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711 b022 1: Scan ES+ 

# 'j," ,,

',

.. 5.00



# , ..,,, .,"

15.00 

# " 

# "

30.00 

# '

" ' , ., , 40.00 

# "

"" "
..,...,..,, ',

# I.. .," ,,.., to::: " : 10;00 H , ,. 35.00 , 20.0025.00 14711b022 1: Scan ES+ 

# 1:J**~138 mJZ4~=**  

# "J ,.,,,,,, '1iO ',,',,,,' ,. " ',,,,•" ,,
.. ,,,,, ,-, ,,,' ,,,,,
.. , ,. ," 1, , "" , .. 1, , , , , 5. 10.00 16.00 20.00 25.00 30.00 35.00 40.00 14711b022 1: Scan ES+ 

# 1."'/""""""","" "I'" '" ".,

# ~ "i"

# t, .,.,11'
..,,'_'"

# ~"'1 ..

# :2.7~'.:. 5.00 10.00 15.0020.00 25.00 30.00 35.00 40.00 14711b022 1: Scan E6+ 

# ':], "'

,,

'.', .., ..',, ', "'· ,, ' '",.,, ',

,.' ' ,,,,, ..,'**,~t,** 

# " ,, "' ,,**,~ ~~**  5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b022 1: Scan ES+ 

# 'j,, .."
..,. ,.,,, ··

,·,',
..,'· ,,, ',,,, '·

,,, ..·· , ··· ,,

",·,
..

# 1· ,

.·

'.', ··· ,**.~: ,~~~**  5..00 10.00 15.0020.00 25..00 30.00 35.00 40.00 

## (Lt'l \\ 10-01,- 1.11
.. 

# h**J'o.~ k.~**  Fig. 13-11 Mass spectra of oil-soluble converted products (LC-MS sample -1. sludge + test item. vessel [4], mass Chromatograms). 

## -72­ 935

### Study No.14711 

## PeakS**-.---~-** 

### ..

## --- ..**-----------------~** 

### .. ---.---------.---- 

## ..**-----------~** 

# · · · · ·

........ _ ........ _ .... "'
......... "'
....... _ .... "'
.............................................. _ .. _ ....... ___ .......... I



## m/z Fragment ions 361 (M 

# + Na)+ 339 (M 

# + H)+ 321 (M - 

# H20 + H)+

Fig.13 - 12 Mass spectra of oil-soluble converted products (LC-MS sample -1, sludge 

### + test item, presumed fragment Ions of peak 8). Date 

### ..loo?,l1
..J J 

### Name 

# 7.**j(1~'tr~**

-73­ 936

### Study No.14711 

## Peake

I
............................... "'
......................................................................................................, 

# · . 

# · 

# . · . 

# · ' 

# · : 

# · · , 

# · · · , 

# ·
· ,
, 

# · · · · 

# · · · · , 

# · ,



# · , 

# · , 

# · · · · · · · · .**~_** .. __**~** .. _ •• _._._ ... _______ ... ___ ...... _ .. _____ w _____ .... __ ... __ ......... _ .... . 

# . ·

m/z Fragment ions 

# 379 (M 

# + K)+ 

# 363 (M 

# + Na)+ 

# 341 (M + 

# H)+ 

# 323 (M - H20 + 

# H)+ 

# 274 Unknown 

# 221 ( M - 3MBA + 

# H)+ Fig.13 - 13 Mass spectra of oil-soluble converted products (LC-MS sample -1, sludge + test item, presumed fragment Ions of peak C). Date 

## ->..007.S·' I Name /.

## ,c:-1~~-

-74­ 937

## PeakD Study No.14711

- •••• ---.----.-- ••**~** •• --.-.- •••• -- ••••• -- ••• - •••••••••••• __ ••••• _-j , ,

, ,



### .................... ..,

### ...... ,. ......

## -....... 

### -_ 

### ......

### -......

### -_.............................

### -...........

### -.............

### ­ 

## m/z Fragment ions 

# 575 479 463 458 441 423 321 Unknown (M 

# + K)+ 

# (M 

# + Na)+ (M 

# + NH4)+ (M 

# + H)+ (M - H20 

# + H)+ (M<- 3MBA 

# + H)+ Fig.13 • 14 Mass spectra of oil-soluble converted products (LC-MS sample -1, sludge 

### + test item, presumed fragment Ions of peak D).



## -75­ 938

## Study No.14711 

## PeakE**~_M_** .. __ .... ... _________________ · ____ .. _ .._······___ ·-_ ........ -_._ ..._...._-:


# · 

# · · · · · ·

m/z Fragment ions 

# 481 465 460 443 425 323 (M 

# + K)+ 

# (M 

# + Na)+ 

## (M 

# + NH4)+ 

## (M 

# + H)+ (M - H20 

# + H)+ 

## ( M - 3MBA 

# + H) +

Flg.13 -15 Mass spectra 

### of oil-soluble converted products (lC-MS sample -1. sludge 

### +test Item, presumed fragment Ions 

### of peak 

### E).



### -76­ 939

### Study No.14711 

## PeakF**~~-.-** 

## ...**-.--~** 

### .. ---.-.-----.---- 

## ..

# -.. 

## -..**-~** 

## .. -

## ...**---.-.-~---.----.---.------** 

### .. 

## -..**-~--** 

### ..

## -...**-~-** 

## .. ' 

# -m/z Fragment ion 1104
Fig.13 - 16 Mass spectra of oil-soluble converted products (LC-MS sample -1, sludge + test item, presumed fragment ion of peak F). 

### Date 

## ,u,o'7.G.3 J 

### Name 

# 7..**)::'71~¥**



## -77­ 940

### Study No.14711 

## PeakG

w ____________________________ • _____ ··_·· __**·~** __ • ___ • ___ ------------------------------------------------: 

# · · · · · · · · · 

# · · · · · · · · · · ·

m/z Fragment ion 

# 1102

Flg.13 - 17 Mass spectra of oil-soluble converted products (LC-MS sample -1, sludge 

### + test item, presumed fragment Ion of peak G). Date .leo

# 7

# ~.; IName

# 7~**f::1~~** Q/



## -78­ 941

## l

# t.t**~** 

## l \ 200'1 

## 1,1.:1. "". TC-rtA\.(\' Fig. 14-1 Mass chromatograms of oil-soluble converted products (LC-MS sample -1, control blank), 

## -79­ 942

# ..

# ~~,.''''l;t<'''''**"''''.'I''~I'>Io** 'I""",,
...

# ¥t""~ 5. 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b023 14711b023 

# 'j**~"**

I

# tt'l\i Fig. 14·2 Mass chromatograms of oil-soluble converted products (LC-MS sample -1, control blank). 

## -80­ 943

### [5] Control blank (LC·MS sample -1) 22.Feb.2007 14711b023 1: Scan ES+ 

# ."'1 To'" 'on oh"matog"m • •

# ~2.o!~

, .. ,

# 'f~"" "t<" """"", i, ;;;,,;; 

## ;j:;;:;

# ~"" .. ""
.. ,,, .. , , $,' 10.00 15.00 20,00 25.00 30.00 35.00 40.00 14711b023 1: Scan ES

..

# '~t.~~~,~.AA.~ll.~tU)~_~.I~ 5.00 10,00 15.00 20.0025.00 30.00 35.00 40.00 14711b023 1: Scan ES+ 

# 1°1 Z'lL't2 m/z1

# ;i::

.,. ,flf'l f .1r,"I'

# l'~1**11"''''r'''''''!''~'** n',1 , ""'.'1

### 4 /" \',·t·
.....'\'

# 1"I''!I'1~**i~ft** ..

# ~"4"_";;I't; 5.00 1 0.00 15.00 20:00 25.00 30.00 35.00 40.00 14711h023 1: Scan ES+ 

# 1:J 

# at' !

## m/Z5~7= 

# ".1'''''''''.'1'

# j,~\\**",,,~,,,,,,,** ••

# '\lI'I4''''''''\'¥J¥Io!~' 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 14711b023 1: Scan ES+ 

# 1:J 

# ,36[**m/Z3~J:**



# "J
..

# ~~~Ji"'.lj~~~'" S.oo 10.00 15.00 20.00 25.00 30.00 36.00 40.00 14711bD23



### 944 

### iLt t"J \ 

## 1 100

### ~, 

## 1, L2.. 

# M" l'

# lArv.k,, . Fig. 14-3 Mass chromatograms of oil-soluble converted products (LC-MS sample -1, control blank). 

## -81­