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 

745 
PROJECT 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 of test 

very small 

globules of test 

very small 

globules of test 

very small 

globules of test 

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 of test globules of test globules of test 

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. 


746 
PROJECT 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 

0 

o~ -----­

/

/ • 

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) 

747 
PROJECT 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 

NH4CI 0.50 gIl 

pH =7.4 

Solution b CaCI2 27.50 g/l 

Solution c MgS04.7H2O 22.50 gIl 

Solution d FeCI3.6H2O 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) 

748 

PROJECT NUMBER: 0145/0215 PAGE 28 

Appendix 2 Statement of GLP Compliance in Accordance with Directive 

2004191EC 

'Dk) Department ~ of Health 

THE DEPARTMENT OFHEALtllOF THE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

IN ACCORDANCE WITH DIRECTIVE lDII4i9/EC 

TESTFACILITY 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 

19th August ,2008 

A generlll inspection for compliance witb the :Principle! of Good Laboratory Practice 

\\'as carried our at the above lest faciJily as part of: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 


IReceipt 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 of PE3MB 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 of Economy, 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 of this 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 of report to 

Item of inspection I audit Date of inspection 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 of cultivation 

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 of test 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 of test 21 

7. Results 21 

8. Remarks 29 

Contents of tables and figures 

Contents oftables 


Table-l Calculation table for percentage biodegradation by BOD 


Tablc-2 Calculation table for percentage detection of DOC 


Table-3 Calculation table for recovery rate of test item 


Table~4 Calculation table for percentage decrease of test item 


Table~5 Calculation table for percentage production of3 MBA 


Table-6 Calculation table for percentage production of SBA 


Tablc-7 Calculation table for percentage production of pentacrythritol 


754 

15089 


Contents offigures 

Fig. 1 Chart ofEOD 

Fig. 2-] Chromatograms of HPLC analysis for calibration curvc 

(test item, recovery test) 

Fig. 2-2 Calibration curve of test 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 of 3 MBA 

Fig. 4-1 Chromatograms ofHPLC analysis for calibration curve (SBA) 

Fig. 4-2 Calibration curve of SBA 

Fig. 5-1 Chromatograms of LC-MS analysis for calibration curve (pentaerythritol) 

Fig. 5-2 Calibration curve of pentaerythritol 

Fig. 6 Chromatograms ofHPLC analysis for recovery test (test item) 

Fig. 7 Chromatograms of HPLC analysis for test solution (test item) 

Fig. 8 Chromatograms ofHPLC analysis for test solution (3MBA and SBA) 

Fig. 9 Chromatograms of LC-MS analysis for test solution (pentaerythritol) 

Fig. 10 PDA chromatograms of LC-MS analysis for test solution 

(LC-MS sample -2, qualitative analysis of other converted products, 

1 205 - 300run) 

Fig. It Total ion chromatogrdffis of LC-MS analysis for test solution 

(LC-MS sample -2, qualitative analysis of other 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 of LC-MS analysis for test solution 

(LC-MS sample -3, qualitative analysis ofother converted products, 

1 205 - 300run) 

Fig. 18 Total ion chromatograms of LC-MS analysis for test solution 

(LC-MS sample -3, qualitative analysis of other converted products) 

Fig. 19 UV spectrum oftest item 

Fig. 20 UV spectrum of3MBA 

Fig. 21 UV spectrum of SBA 

Fig. 22 Mass spectrum of pentaerythritol 

Fig. 23-1 JR spectrum of test item measured before experimental start 

Fig. 23-2 IR spectrum of test item measured after experimental completion 

Reference IR spectrum supplied by sponsor 

755 

15089 

Title 

Biodegradation study ofPE3MB 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 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 of Health, 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 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 of the 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 of notice 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 of Manufacture, ctc. of Chemical Substances". If it is 

not stable for the storage period, it is stored as long while it is kept stable. 

Treatment of the 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 of raw data and materials, etc. after the storage period will 

be discussed with sponsor. 

757 

Personnel 

Study Director 

Study personnel 


(Operation ofbiodegradation test) 


Staff for cultivation of activated sludge 


Approval offmal report 

Study Director Date 

Signature 

15089 


Takakazu Kayashima 

(1 st Chemical Safety Section) 

Naoki Onozawa 

Yuko Kanetake 

Takakazu Kayashima 

Hirokazu Kiuchi 

Yuko Shimogawa 

Kimiko Tanaka 

Sayuri Nakata 

Ryosuke Nabeoka 

Koichi Hiromatsu 

Noriko Miyaura 

Hiroko Mori 

Masako Yunoki 

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 of PE3MB by microorganisms 

Conditions of cultivation 

(1) Concentration of test item 

(2) Concentration of activated sludge 

(as the concentration of suspended solid) 

(3) Volume of test solution 

(4) Cultivation temperature 

(5) Cultivation duration 

(under the conditions of darkness) 

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 decrease oftest 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 of the 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 of the test item was changed or biodegraded on 3-mercaptobutylate group under the 

test conditions of this 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. 
 r·.·---·····----····--··.·-.······--·. w ••••• ---•••• --- .-•• - •• --.-••••• --••• - •• - ••• - •• --•••• --•••• ~--- ••: 

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


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 

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15089 

3. Activated sludge 

3.1 	 Preparation of activated 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 of activated 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) of the supernatant of sludge 

collected at the ten loeations according to the cultivation methods described in 

Section 3.2. 

*4 Prefiltered open air was used. 

162 

-7­
15089 

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 of the 

synthetic sewage was 0.1 % in the volume of dechlorinated 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 of the solution for each component. The pH of the 

solution was adjusted to 7.0±1.0 with sodium hydroxide. 

3.3 Control and use 

During cultivation, the appearance of the 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 of activity and date of initiation ofuse of activated sludge 

(1) Inspection of activity 

Activity of the sludge was assessed using standard items before initiation of use. 

(2) Date ofinitiation ofuse 


January 15,2008 


763 

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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 of basal culture medium 

Each 3 mL of solutions 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 

-9­
15089 

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 of the test sample was accurately weighed and added to 

300 mL of purified water, so that the concentration of the 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) of activated sludge inoculatcd], so that the concentration of the 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 of aniline 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) of activated 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 of the suspended 

solid reached 30 mglL. 

765 

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15089 

4.3 Instruments and conditions of cultivation 

(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 of carbon 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 

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15089 


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 on the next page 

(measuring pipet) 

--Ammonium sulphate 200 g (even balance) 

+-Tetrahydrofuran 90 tnL (graduated cylinder) 

• Shaking for 10 minutes 

I

l Water layer I ITetrahydrofuran 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 

TOCsample • Taking out 2 mL (transfer pipet) I I 

• Filling up to 5mL (purified water. 

volumetric flask) I HPLC sample -2 I 


Determination of3MBA and SBA 


I LC-MS sample -1 1 

Determination of pentaerythrito) 

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 1water"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 

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15089 

4.5.2 Quantitative and qualitative anaJysis 

(1) Determination of dissolved 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 of test item, 3MBA and SBA 

The HPLC sample -1 were analyzed under the following conditions. The 

concentration of the test item in the HPLC sample -1 was calculated proportionally by 

comparing the peak area on the chromatogram of the HPLC sample -1 with that on the 

chromatogram of 300 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 of the 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 of the 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 IlV • see considering the 

noise leve1, which corresponded to the test item concentration of 1.4 mg/L, 3MBA 

concentration of 0.70 mgfL and SBA concentration of 0.49 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 

-15­
15089 


(b) Preparation ofstandard solution 

The standard solution to detcnnine the concentration of the 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. 


® SBA 

100 mg of SBA 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 

-16­
15089 


(3) Detennination of pentaerythritol 

The LC-MS sample -1 were analYLed under the following conditions. The 

concentration of pentaerythritol in the LC-MS sample -1 was calculated proportionally 

by comparing the peak area on the chromatogram of the 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 of 0.15 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. 120ClC 


Desolvation temp. 400°C 


Cone voltage 15 V 


(b) Preparation of standard 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 

-17­
15089 


(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 

-18­
15089 

(b) Preparation of solutions 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 of the 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 Sec lion 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) of the test 

item on the chromatogram. 

Two individual recovery rates and their averages of the 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~20AV 

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 

-19­
15089 

4.6 	 Calculation ofpercentage biodegradation 

The percentage biodegradations were calculated by the follovving equations and 

rounded off to 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 in the 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 of test 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 ofthe test 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 840 I: 1999 rule B. 

775 

-20­
15089 

5. Validity oftest conditions 

The validity criteria of the 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 

of aniline by 

BOD 

After 

14 days 

76% 2::60% 

Table-l 

Fig. 1 

6. Factors that affected reliability oftest 

No adverse effects on the reliability of this 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 

of cultivation 

Water + test item 

Sludge + test item 

Insoluble compound could not be 

confirmed. 

Insoluble compound except the

sludge could not be continned. 

C'.rrowth ofthe sludge was observed. 

-

-

776 

-21­
15089 


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 of DOC % 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 

-22­
15089 

*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 of test item added, was calculated instead of the percentage biodegradation ofthe 

test item (percentage biodegradation by the chemical analysis ofthe test item) because the 

percentage residue of the 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 

-23­
15089 

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 of the 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 Presumed structural formula Detected (0) / Not detected ex)

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 of determination oftest item 

Decrease of the test item was confirmed in the test solution (water + test item) 

because of the percentage residue of the 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 

of the percentage residue of the 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 of the test item and the percentage productions of pentaerythrito), 

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 of the percentage biodegradation by BOD 

described in (1) suggested that some of the test item or the converted products were 

biodegraded by microorganisms. 

781 

-26­
15089 


(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 of the 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 of discussion 

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 

-28­
15089 

7.6 Conclusion 

All of the 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 of cultivation: 28 days 

7th day 14th day 21st day 28th day Mean Vessel 

BOD Deg. BOD Deg. BOD Deg. BOD Deg. Deg. No. (%) (mg) (%) (mg) (%) (mg) (%) (mg) (%) 


(6] 
 59.3 62 
 75.8 76 
 80.4 79
 82.3 79 


[5] 3.1 - 7.1 -
 9.0 . 10.9 - [2] 14.2 21 
 23.8 32 
 27.7 36
 29.9 36 


[3] 13.7 20 
 23.6 32
 27.4 35 
 29.8 36 


[4] 12.3 22.8 30 
 26.7 34
 29.0 35 


(1] 


18 


0.6 1.7 2.8 4.2 - ­ - -

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 of BOD: Fig. 1 


Deg. =[BOD· B ] / [TOO] x 100 (%) 


TOD of test 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 of aniline: 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 

Water blank 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 of test item added: 30 (mg) 

Volume oftest solution: 300 (mL) 

Theoretical amount ofcarbon : 13.8 (mgC) (30) x ( 17C 1C11H,oO~ ) 

A: Measured value (mgCIL) 

B: Detected amount ofDOC 

Bw = (A(Water + test item) - A{\Vater blank) ) )( 300/1000 (mgC) 

Bs =­ ( A(Sludge +test item) - A(Conttol blank) ) )( 300 11000 (mgC) 

C: Percentage detection (%) 

C '" B I ( Theoretical amollIlt of carbon»( 100 

D: Average percentage detection (%) 

April 24. 2008 

786 

-31­
Table~3 Calculation table for recovery rate of test 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 of portion 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 of test 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 

[4JSludge' +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 == D124.4 (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 

Stutly d 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: 1513 00 

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 	 tUly N o. 15089

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 of test item added: 30 (mg) 

Theoretical amount ofpenta erythritol : 9.2 (mg) 

(30) x ( CSHI20J C17H3007S3) 

A: Peak area ( - ) 

B: Final volume: 5 (mL) 

c: Ratio ofportion used for analysis: 151300 x2/15 

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 
Stud 'I No. 15089 Solvent blank Operatin, Date: Feb.OB.2008 . 

PkNO TillI8 Area !MIsfit (jnfn) <II V'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 Operet I", 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 

-38­
Study 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 oftest item (recovery test). 

February 10, 2008 

-39­

794 
Solvent blank Study No. 15089 

Operating Oats: Apr.25,2008 

PKHO Tillie

6n i n) 

Area 

(p V-seo) Height(/I V'J 

Cone. 

(I) 

I 20. 73 217376 8361 100. 00 

Total 217375 100.00 

Peak position of test 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 

Opent Ing Date : APr. 25, 2008 

PKHO Th. "ra He Ililt 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 

-40­
Study 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 of standard 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 < 

0.0 10.0 20.0 30,0 Operator : Haokl OnotaWi

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 • . :HlO 30.0 Operator : Naoki Onezawa

Frg. 3 - 1 Chromatograms of HPLe analYSIS for calibration curve 

(3MBA). 

797 


-42­
Study 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) (iJ V'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 

-44­
Study 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 of SBA. 

April 28. 2008 

-45­

800 
1 

Solvent blank 23-Apr..20 15089-021 SIR of 1Channel 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 of LC-MS analysis for calibration curve (pentaerythritol). 

-46­

801 
Study 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 

0.0 

I 

~ 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 

0.0 10.0 :0.0 	 30. 0 Operator : Neaki OnoZ8W8 

Waier + Operating Date: Feb.09.2008 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. 

~ 1 .A

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. 

(illi n) ("V'sec) (11 V) (Il 1 	 20.73 277864 8363 100.00 

Total ~ 100.00

Peak position oftest item 

(blank peak) 

1 -.l\-	 b

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) (Jl Vl (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. Converted Oain) 	 (li V'S8C) (p. V) CI) products Converted productB 1 	 13.73 16794 1781 1.41 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 

Converted SBA PKNO Time Area Height Cone, Converted (min) (pV'$ee) (pV) (I) products Converted 

product B 1 13.73 15458 1617 1,33 CandD productE 2 14,47 102668 9187 8,83 3 15,43 111621 B853 9060 

/ Coo,_ 4 16,13 192209 24986 16,53

~"O"dF 5 16.30 417894 35220 35.93

6 17,85 19620 2891 1.60 

Peak position 7 17.97 26728 3038 2.30 

-A 5 oftest item 8 20. 72 mZ§1 7976 23.89

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 

Converted SBA PKNO Tillie Area Height Cone.

Converted (mIn) (,11 Y·aee) (JlV) ~) products Converted 

productB I 13.73 13359 1401 1.19 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 

~-P 6 17.87 17595 2772 1.56

Peak position 7 l7. 97 25577 2915 2.27

8 20. 72 iZZl~g 7927 24.59 . i . / oftest item Total 1127115 100.00 

~ 1 I : 1. i.A (blanlc peat) 

j a,'o 111.11 211~ 0 30.11

, Operator : Naoki OnoZ8wa 

[5] Control blank 

Peak position 

oftest item. 

(blank peak) 

PKNO 

Operating Date : A~r.25.2008 

Time Area Height Cane, (min) (Ji Y·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 ight 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 igilt 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) (II Vl (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 : Ap r. 28. 2008 Name: OM'l4.w" #D.o ~'I 

-52­

807 
____ _ 

Converted SSA 

Peak position 

of3MBA 

Converted n~'d'\ 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 

.v~::~ 5 16.12 i2849 9669 18.61

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) (y V·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 

-53­
15089-025 

tandard solution 15.0 mg/l 23-Apr..200 

SIR or 1CIIannal 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 

-54­
1 

[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 of LC·MS analysis for test solution (pentaerythritol). 

-55­

810 
1 

100 mg/L solution of test ltam 

15089-060 


Test item 

1 


100 mgIL solution of 3MBA 

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 of other 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 of LC-MS analysis for test solution (LC·MS sample -2, 

qualitative analysis of other converted products, A=205~300nm). 

-57­

812 
2.82 

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 of LC-MS analysis for test solution (LC-MS sample -2. 

qualitative analysis ofother converted products, A. =205-300nm). 

l~~ lIiIl.lf.. ~ 

-58­

813 
1 

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 of 3MBA 

15089-06& 

1 

814 
1 

11]Water + teSt Item 

15088.Q70· 2S-Ap....200 

1:80lIl est • 	 TIC 

Test 7.5Oet 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 of LC-MS analysis for test solution 

(LC-MS sample -2, qualitative analysis of other converted products). 

-60­

815 
10 

[41 Sludge + test Item 2H\pr·200 16Ofi.073 1;ScanES+ 

TIC 

7.50e6 

[11 Control blank 

15089-074 1:ScanES+ 

1 TIC

7 

" 

111.00 20.00

Fig. 11 - 3 Total ion chromatograms of LC-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 

Converted products

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 of converted products H and I). 

Date ).lIlt.Ii. n 

-65­

820 
Study No.15089 

mJz Ions 

187 Unknown 

221 Unknown 

289 Unknown 

321 (339 - H20t 

323 See structural formulae 

339 See s~ctural fQrmu1ae 

423 (441- H20t 

441 See structural formulae 

543 See structural fonnulae 

763 See structural formula (converted product H) 

863 ( MI - H20 +H t 

865 (MH -H20+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 of converted 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 1146 :M.O& Mus chromatogram of 1.6714 

t

j

'''''I''''I" "r'"I"'''''' 'r' ",, ~ i... ,J",....,,,,:~:..,;,, 0 01 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 ..... ', ...,,!" 

15089·070 

J.,..,,...,., ..,....,., ..,... "" ,.,....~,. I,,., .:, .. ~~~~~~1: ~~Scan .. ~ ES+ 

16089-070 'j... '1 •••• " •• '1 "" " ••• ,,, •• ,,, •• , ••I..x",., ..,.. ~~=~1: Scan 

.. ~.::: ES+ 

1] • 23M Mass chromatogram of 1.A: 

mh.=S43 

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

15089-070 

'1:':.,:.",.... ,,,.. ,,,,,,. ,,,'"''r'" """1,,,,,,,, J' .~,~~='.~~! 1: Scan E: 

1] Mass chromatogram of 4.22e3 

miz-= 863 

... :~ ..., ....f ••• ',"'" ~)" ,'" /,'"'\'''".,........".,., •• ,•• "0 ..",.>0, 1;oafl.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 111me 

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 'j m/z:= 881 , I 	 ~ 

•

6 

-".. ,,, "I""1'"""",,,,,,,,,,,,,"'''''''''''''",,,, ""'II,,,,,"I...., . i 

11089-070 	 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'':~

15089-07Q J..""",""f""." ,i." '"''"'''''''',J" "" .'''~~~:~~~,O,~,,'_ 1: Sea n E'S+ 

16089-070 	

'i".""."".""'''''.''"''''''''"''''I,""1" ,~~~,~:;~,~" 1: SCln ~,'= ES+ 

15089-070 '1", ' 	 ''''''''''''q",'"" 'I"""""'"I",'" '''~,~~:1: ...'"Scan ,"': ES+ 

15089-070 	 '1.,""""",.,"""" ,.. "."".""." ,1"""" ,~~.~,,...,,,, 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.. 

100 2 700.800

~~I\ 'Ut i. 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 1121 9.2Oe3

Mass spectrum of 

converted products C and D 

m 

las •% 

.7 

fP 221 ,.1 

'. 
 J Ild 1 l I~ " '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 -H20+Ht 

239 (MA +Ht 

261 (MA +Na/ 

Fig. 14 - 3 Mass spectra (LC-MS sample -2, [2] Sludge + test item, 

presumed ions of converted product A). 

-72­

827 
Study No.15089 

Converted product B 

([2] Sludge +test item) 

mlz Ions 

457 ( MB - H20 + 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 of converted 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-2H20+Ht 

339 (Mc-H20+Ht 

357 (~+Ht 

379 (Me + Na·t 

395 (Me+Kt, 

489 (Mo -H20+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 

procJuct B CandO produefS: I

\ ,~ 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

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

-82­

837 
------- ---

Mass chromatogram of mlz "" 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 

''fi I I'" i I 'I ,'n I I' " ~ I " , I i ~ i , i 1A.. It'd i '1', ~'" II'" I Ii,'•• i I I 1/\ II' I ,,, tI ""'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 of mlz '" 379 379 

2.5883 

1S8N74 1: Scan ES+ 

1eG8N74 

'3.1..'"",., I.' '~'" ". '~,~' "1 x:',~:~:~::~::"r.'I'1: A'~~ Scan es+ 

1 1 Mass chromatogram of mlz =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 of mlz = 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;07 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 of LC-MS analysis for test solution (LC-MS sample -3, 

qualitative analysis ofother converted 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 mgll solution 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 of LC-MS analysis for test solution 

(LC-MS sample -3, qualitative analysis ofother converted products). 

-91 

846 
10 

[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

847 
3 

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 of test 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

848 
Chemicals Evaluation and Research Institute,Japan Kururo~ 4borato:ry 

3r-------------------------------------------~--~ 

2 

Abs 

A. for HPLC (21 Own) 

1 

..0.1 L __--1-_~=C====:::::i::=====::r======::::i::=:j 200 250 300 340 

Wavelength [nm] 

lS0891S0ppm 

Fig. 20 UV spectrum of3MB A. 

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 of SBA. 

-95­

850 
Study 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) J2S - 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

851 
1 

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 

CSHI20. 

Mass nwnber (M) : 136 

140 160 

Fig. 22 - 2 MS spectrum of penta erythritol. 

-97­

852 
10 

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 

2.00 6.00 8.00

Fig. 22 - 3 MS spectrum ofpenta erythritol. 

-98­

853 
75 

4000 

fOO-;:,."..._'+'t. - - - {- - - -. - - • - 4- ----. _. --:- -----•• -­ '~ , t

• 

--.~~~----.----­ Str.; 

.------~~~-.-.~--.-~-- ..---.-­ --1-- ,

, , 

, 

CJi..H 1 I 

.-------~---.~~--~ ----.----~---.-.-.~ 

I • d-H : , I • 

• ~tr. : . I

, , 25 ·~--*----r-k.------,--.-------~~-----~-­ , 

_________ £ ______ ~~~J __________L ________ _ 

o , I • 

! , 

,

,

• ---1­•I 

I 

, 

,

, , ---~·-----·-·~tfJj-·- I

­ •, 

, I 

, 

I 

• ....... c=o _ ... ___ ... J . r .. ______ ... _.J ... ___ .... __ .. _ 


Str. . I 

2000 IiOO 

Instrument 

Study No. 

Sample 

Method 

Date 

Name 

110m 

: Shimadzu IRPrestige-21 

: 15089 

: Test item 

: Neat 

: December 21. 2007 

:6d CJilFig. 23-1 IR. spectnun oftest item measured before experimental start. 

-99­

854 
• • 

100 -­ "" ... ---... '"'r--- _.. --­ -;--­ -.. ---.. "":- -­--...-_ ... I , 

I I r , 

IT I I 

75 

50 --·.·.---r------·· . .. -.. -.--~-------. 

•I 

J •

· , .......'...!---....... --,_• ... -... _- ...... ­ -------.. -~------~--~-~-~.---.~.------------ I 

I 


I 


I 


I 


. _ ..... ___ ,.. _ ... l.,. ... _ .......... ___ ......... _ .... _ ......... _f.____ ........ _ "'" - ............ ____ _ J_" ....... _ .... _ ............... __' .... __ _ 


t ,. I 1. I,.

, 

• 

400D 1000 500 

Vc:m 

Instrument : Shimadzu IRPrestige-21 

Study No. :15089 

Sample : Test item' 

Method : Neat 

Date ; May 16, 2008 

Name :~ at£, 

Fig. 23 - 2 IR spectrum oftest item measured after experimental completion. 

-100­

855 
I 

7$ 

.. 

70 

eo 

IS 

«I0Il lli110 tiXlrl 

Reference sp"ectrum supplied by sponsor. 

-101­

856 
001 


¥JJX: 1 9:¥ 1 2 F1 2 1 El 


~tffit£A 1~~!Im.~fJffiiJf~~#4 

CD!I\{i*JJ~m1k.lt®*gJJ~m 

K•••0m~.CD.M~!I\.*$gJ.=T§2.7~ 

®~3i~~tltMliW~P 1IJJ*~~~Jf 1 6 0 0 


~M~~., ~~~K~1k.lt~~~~~. 

857 
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 SHOW A DENKO K.K. 

Title Biodegradation study ofKarenzMT(pEMB) by microorganisms 

Study number 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 M0.../ I I 1 ;"00 J 

Study Director /. f<~o./ 

Takakazu Kayashima 

859 
14711 

GLP STATEMENT 


Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor SHOW A DENKO K.K. 

Title Biodegradation study of KarenzMT(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 of Economy, Trade and Industry; No. 031121004, Environmental Policy Bureau, 

Ministry of the 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 of KarenzMT(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 of the 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 of inspection / 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 of cultivation ~aI!uary 24, 2007 January 25, 2007 

!-" -- '-­

At the middle ofcultivation February 7, 2007 February 7, 2007 

February 21, 2007 February 21, 2007 

At '-----_.._-------­-. -.~ the end of cultivation 

-_ ___~ebruary 23, 2007 February 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 of tables 


Table-l Calculation table for percentage biodegradation by BOD 


Table-2 Calculation tabJe for percentage detection of DOC 


Table-3 Calculation table for recovery rate of test item 


Table-4 Calculation table for percentage biodegradation oftest item 


Table-5 Calculation table for percentage production ofpenta erythritol 


Table-6 Calculation table for percentage production of 3MBA 


Table-7 Calculation table for percentage production of SBA 


862 

14711 

Contents of figures 

Fig. I Chart of BOD 

Fig.2-1 Chromatograms of HPLC analysis for calibration curve (test item) 

Fig.2-2 Calibration curve of test item 

FigJ-l Mass fragmentograrns ofLC-MS analysis for calibration curve 

(pentaerythritol) 

FigJ-2 Calibration curve of pentaerythritol 

FigA-1 Total ion chromatograms of LC-MS analysis for calibration curve 

(3MBA) 

FigA-2 Calibration curve of 3MBA 

Fig.S-1 Total ion chromatograms of LC-MS analysis for calibration curve (SBA) 

Fig.S-2 Calibration curve of SBA 

Fig.6 Chromatograms ofHPLC analysis for recovery test (test item) 

Fig.7 Chromatograms of HPLC analysis for test solution (test item) 

Fig.8 Mass fragrnentogranls ofLC-MS analysis for tcst solution 

(pentaerythritol) 

Fig.9 Total ion chromatograD1s of LC-MS analysis for test solution 

(3MBA and SBA) 

Fig.10 UV chromatograD1s of LC-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 of oil-soluble converted products) 

Fig.12 Mass spectra of oil-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 of LC-MS analysis for test solution 

(LC-MS sample -2, qualitative analysis of water -soluble converted product,;) 

Fig. 17 Mass spectra ofwater-soluble converted products 

(LC-MS sample -2, sludge +test item) 

Fig.I8 Mass chromatograms of water-soluble converted products 

(LC-MS sample -2, control blank) 

Fig.19 UV spectnun of test item 

Fig.20 Mass spectrum ofpenta erythritol 

Fig.21 Mass spectnun of3MBA 

Fig.22 Mass spectrum of SBA 

Fig.23-1 IR spectrum of test item measurcd before experimental start 

Fig.23-2 IR spectrum of tcst item measured after experimental completion 

Reference IR spectrum supplied by sponsor 

863 

14711 

Title 

Biodegradation study of KarenzMT(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 of Health, Labour and Welfare; November 13,2003, No.2, Manufacturing 

Industries Bureau, Ministry of Economy, Trade and Industry; No. 031121002, 

Environmental Policy Bureau, Ministry of the Environment) 

864 

-1­
14711 


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 of test 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 of 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 Rcgulation of Manufacture, etc. of Chemical 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 of raw data and materials, etc. after the storage period will 

be discussed with sponsor. 

865 

-2­
14711 

Personnel 

Study Director 

Study personnel 


(Operation of biodegradation test) 


Staff for 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 


-3­
14711 


SUMMARY 


Title 

Biodegradation study of KarenzMT(PEMB) by microorganisms 

Conditions of cultivation 

(1) Concentration oftest item 	 100 mgfL 

(2) Concentration ofactivated sludge 30 mg/L 


(as the concentration ofsuspended solid) 


(3) Volume of test 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 of water-soluble converted products by liquid chromatography-mass 

spectrometry (LC-MS) 

Results 

(1) Percentage biodegradation by BOD 	 1%, 3%, 3% average 2% 

(2) Percentage biodegradation of test item (HPLC) 14%, 16%, 13% average 14% 

Conclusion 

Some of the test item was changed or biodegraded on 3-mercaptobutylate group under 

the test conditions of this 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 of activated sludge 

Activated sludge was prepared as follows to maintain its uniformity. 

The filtrate (5 L) of the supernatant of the activated sludge+2 cultivated about for 

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 of sludge 

collected at the ten locations according to the cultivation methods described in 

Section 3.2. 

*3 Prefiltered open air was used. 

870 

-7­
14711 

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 of dechlorinated 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 of activity and date ofinitiation of use of activated sludge 

(1 ) Inspection ofactivity 

Activity ofihe sludge was assessed using standard items before initiation of use. 

(2) Date ofinitiation ofusc 


January 16,2007 


871 

-8­
14711 


4. Pelformance ofhi ode gradation test 

4.1 Preparations for test 

(1) Measurement ofconcentration of suspended 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 

-9­
14711 


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 of purified 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 of the 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)] of aniline 

was taken out by micro syringe 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 of aniline 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 mL by the volume (2.66 mL) of activated sludge inoculated]. 

(2) Inoculation of activated sludge 

The activated sludge cultivated under the conditions described in Section 3 was 

added to each test vessel, (b), (c) and (d), so that the concentration of the suspended 

solid reached 30 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 of carbon dioxide, 

Wako Pure Chemical Industries, Ltd.) 

(2) Conditions ofcultivation 

Cultivation temperature 2S±lOC 

Cultivation duration 28 days (under the conditions of darkness) 

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 of the test solution was observed once a day 

and conditions ofthe instrument" were checked properly. 

(2) Measurement of biochemical oxygen demand (BOD) 

During the cultivation period, the change in BOD of the test solutions was 

measured by autorecording using a data sampler. Cultivation temperature was 

measured and recorded once a day. 

874 

-11­
14711 


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 of the 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 of water-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 water / 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 lTetrahydrofuran layer I 

I ·HPLCsantple I 

etermination ofthe test item 

• Filtration and dehydration (IPS filter paper) 

• Filling up t o 100 mL (tetrahydrofuran, volumetric flask) 

• Taking out 3 mL (transfer pipet) 

• Filling up t o 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 of dissolved 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 of the total carbon (TC). The concentration 

of TC and IC in the test solutions was proportionally calculated from the peak area of 

the test solution by comparison with that of 80.0 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 of furnace 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 of 300 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 of standard 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 

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14711 


(3) Determination ofpentaerythritol 

The LC-MS sample -3 was analyzed under the following conditions. The 

concentration of pentaerythritol in the LC-MS sample -3 was calculated proportionally 

by comparing the peak area on the mass fragmentogram of the 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 of 0.094 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 of standard solution 

Thc standard solution to determine the concentration of pentaerythritol in the 

LC-MS sample was prepared as follows. 

100 mg of penta erythritol (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 

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(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 of the 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 on the 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 of 0.092 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 

-17­
14711 


(b) Preparation of standard solution 

The standard solutions to dctennine the concentrations of 3MBA 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 of 3MBA. 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. 


® SBA 

100 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 

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(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 of the 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 of solutions oftest item, 3MBA and SBA 

The solutions of the test item, 3MBA and SBA used for thc LC-MS analysis 

were prepared as follows. 

CD Test item 

100 mglL solution of the 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 of SBA, 

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 of the test item on the analytical 

procedure are shown below. Ibe average recovery rates were used as correction factor, 

for the determination of the 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 

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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 in the test solution 

(sludge + test item) (experimental) (mg) 

B 	 : Biochemical oxygen demand in the control blank 

(experimental) (mg) 

TOD"lO 	 : Theoretical oxygen demand required when the 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 of the test item in the test solution 


(water + test item) (experimental) (mg) 

4.7 	 Treatment ofnumerical values 

Values were rounded off in accordance with JIS Z 840 I : 1999 rule B. 

884 

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14711 

5. Validity of test conditions 

The validity criteria of the test and the values in this test are shown in the following table. 

This test was valid because all of the values in this test met the criteria. 

-------­

Value in 

present test 

Value of 

criterion See 

Difference of 

extremes of 

replicate values 

of percentage 

biodegradation 

Percentage 

biodegradation 

by BOD ,­

Percentage 

biodegradation 

of test item 

2% 

3% 

<20% 

Section 7.3 

Percentage 

biodegradation 

Percentage 

biodegradation 

of aniline by 

After 

14 days 76% ;:;;;60% Table-l 

Fig. I 

BOD I 

6. Factors that affected reliability of test 

No adverse efiects on the reliability ofthis test were noted. 

7. Results 

7.1 	 Appearances oftest solutions 

Appearances of test media in cultivation vessels were as follows. 

Test solution Appearance pH 

At the start 

Water + test item The test item was not dissolved. -

of cultivation 

Sludge + test item The te~i item was not dissolved. -

At the end 

Water + test item Insoluble compound was observed. -

of cultivation 

Sludge + test item 

Insoluble compound in addition to 

the sludge was observed. 

Growth of the sludge was not 

observed. 

-

885 

-22­
14711 


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 of test 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 of qualitative 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 of their 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

887 
14711 


List of converted products (elution order, see Figs. 10-18 

Detected (0) I Not detected (x) Converted Presumed structural formula 

products (M: presumed molecular weight) Water + test item .. 

· · 

·

· 

A'15 , · · [1] x · · · · · · · · , · 

· , · · · 

· :

B · ·, : [1] x

· : , : , !

· 

· : · 

· 

·· ;

: ·, ! C ·, [1] x

, · 

· · · · , · , · , 

· 

· 

, 

, 

, , 

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 

-25­
14711 


7.5 Discussion 

(1) Percentage biodegradation by BOD 

Percentage biodegradations by BOD were 1 %, 3(Yo and 3% (see Fig. 1). 

(2) Results of detennination oftest item 

The percentage residue of the 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 of determinations 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 of penta 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 of those 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 of these were not available. 

-26­

889 
14711 


(6) Biodegradation of3-mercaptobuty1ate group 

Despite detections of pentaerythritoI 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 of DOC to confinn presence or absence of water-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 of DOC, because some 

of the 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 of discussion 

According to (l)-(7), it was considered that some of the test item was changed 

or biodegraded on 3-mercaptobutylate group into many converted products and that 

these products and rest of the 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 

-28­
14711 

7.6 Conclusion 

Some of the test item was changed or biodegraded on 3-mercaptobutylate group under 

the test conditions of this 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 BP21 OS 

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 

-29­
Table-l Calculation table for percentage biodegradation by BOD 

Study No. 14711 Duration of cultivation: 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 [BJ 

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 of aniline: 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 

-30­
Table-2 Calculation table for percentage detection of DOC 

StudIV 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 of test item added: 30 (mg) 

Volume of lest solution: 300 (mL) 

Theoretical amount of carbon: 13.9 (mgC) (30) x ( 21C / ~lH)60sS4 ) 

A: Measured value (mgC/L) 

B: Amount of DOC 

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 

-31­
Table-3 Calculation table for recovery rate of test item 

Studly 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 of test item added: 30 (mg) 

A: Peak area (}lV'sec) 

B: Final volume; 100 (mL) 

C: Ratio of portion 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 of standard solution: 300 (mgIL) 

See Fig. 6 

March 13, 2007 Name 

895 

-32­
Table-4 Calculation table for percentage biodegradation of test 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 of test item added: 30 (mg) 

A: Peak area (;.tV·sec) 

B: Final volume: 100 (roL) 

C: Ratio of portion 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 of standard solution: 300 (mgIL) 

SeeFig. 7 

March 8, 2007 

896 

-33­
Table-S Calculation table for percentage production of pentaerythritol 

Studv 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 of test item added: 30 (ms) 

Theoretical amount of pentaerythrito] : 7.5 (mg) 

(30) x ( CsH1200tl C;UH360gS.) 

A: Peakarea(-) 

B; F'mal volume: 5 (mL) 

C; Ratio of portion used for analysis: 15{300x2115 

D: Amount of pentaerythrito] (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 of 3MBA 

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 of test item added: 30 (mg) 


Theoretical amount of 3MBA : 26.5 (mg) 


(3D) x ( 4C4HB0 2S/ CzIH360aS4 ) 


A: Peakarea(-) 

B: Final volume: 10 (mL) 

c: Ratio of portion used for analysis: 15!3ooxl/lS 

D: 	Amount of 3MBA (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 of standard solution: 10.0 (mWL) 

See Fig. 9 

March 8, 2007 Name 

898 

-35­
Table-7 Calculation table for percentage production of SBA 

S d 	 tUly N 0.14711

Sample descriPtion 	 A D E F 

Standard solution 10.0mg/L 190718 

[1 JWater +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 of test 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 of portion used for analysis: 15/3ooxll15 

D: 	Amount of SBA (mg) 

Dw = G x ( A(Water -+- test item) / A(Standard) ) x ( B / C) 11000 

Ds =G x { ( A(Sludge +test item) - A(Control blank) ) JA(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 "1' .......... , .........,."' ................. , , .... , ••• ~ ..................". 
 2S ± 1 t


Duration .. ,. ...................... " .".................. " .. t ............ ,. t .. ~ .. "' ... 10 ................... ~ ...... 28 days (Jan.24,2007 • Feb.21 ,2007) 

Note! ­

Vessel BOD (mg) Sample Description No. 7th day 14th day 21st day 28th day 

[1] Water +test item 0.0 0.3 2.0 3.2 

[2J Sludge +test item . 2.4 5.8 8.3 10.4

[3} Sludge +test item 2.7 6.9 9.8 11.8

[41 Sludge +test item 2.8 6.6 9.6 11.4

[5] Control blank [B] 3.1 6.1 8.4 10.0 

[6] Sludge + aniline 56.1 74.6 77.3 79.1

100 - [1] 

- - [2] 

--... - [3] 

't)Ij ..... ........
 80 [4] .--••-----------.------ [6] A - - [5] _...-.... ",..,. ..........
 ... -.,-.­ .---- [6] .."" i ,/

, ! 60 ~ .' ..,..' 5 "..'

f " ~ I 

, 0 40 I 

,

1 

's 'H I

•

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 ofBOD. 

-37­

900 
Study 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 

-38­
Study 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 of test item. 

September 13, 2006 Name H. t~t 

-39­

902 
1 

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 1 Channel 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 1 Channel 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

903 
70000 

Study No. 14711 

,0 

/ 

, " 

// 

/'" 

,...... I

...., 

g 35000 

~ /r

/

If 

/ / 

0 /

0.0 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 of pentaerythritol. 

February 21, 2007 Name 

-41­

904 
l 

Solvent blank 23-Feb-2007 

14711b023 SIR of 2 Channels ES100- TIC 

1.50e6 

Area 

Peak I>o~itlon %­ n.d. 

1 , 1 I , June 0.00 5.00 10..00 15.00 20.00 25.00 

Standard solution 2.50 mglL (3MBA) 23-Feb-200i 

14711b024 SIR of 2 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 of 2 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 

1 . ·Tim$

0.00 5.00 10.00 15.00 20.00 25~OO . 

ILtl"l \ , 2oa~, 'L.'2) 


Fig. 4 - 1 Total ion chromatograms of LC-MS analysis for calibration curve (3MBA). 

-42­

905 
Study 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 of 3MBA 

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

907 
Study 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 of SBA. 

February 24. 2007 Name 

-45­ 908 
Study 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 -\ Ope rat 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

909 
Study 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

910 
Study 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 Co~ 2 22.51 23764 3459 5. 87

product F Total 404596 100.00 \1 j l. , Jl~ .J,

, 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

911 
Study No. 14711 

(3) Sludge t test iI em 	 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 

\' ~ .J. l. ./! -"­

•

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 Convwted 2 22.45 26012 3853 6.36

produc:t F Total 410759 1()(l 00 

.l~~ 'A.-­ --" 

\1 

I. /, 1 I 	

30: O(min) Operator: Hirokazu Kluchi 0.0 1'0. 0 20.0 

[5) Con I fO 1 blank 	 Doe rating 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~ 

10 TIC

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·' 

10(} TIC

11.22 1.S0e6 

190718 Area 

IStandard solution 10.0 mglL (3MBA) 	 23-Feb-2007 

14711b027 	 SIR of 2 Channels ES· 

10(} 1.SV<:!Q =r~

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~ M1~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 0.00 5.00 	~1~.,....,......., 15.00 

(6) Control blank 

14711b032 

Peak position Peak position 

n.d. n.d. 

1 1 	

1 Time

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 of test 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

918 
Solution 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 

tLt'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 of peak 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 40.00 

TIC 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 of oil-s'oluble converted products 

(LC-MS sample -1, sludge + test item, vessel [3]). 

-63­

926 
[21 Sludge + test item (LC-MS sample .1) Test item 22-Feb-200

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:'] 	 11.,3 2":Z1· mlz:'2~!~ 

JI1tJ.\ t''', . i 1'1 ' I" • " t'" ,'r, ~., , ItI"'t I"~ J r1,,", ii' " 'I r 'f!,,,,.A~1 f,..£4, ; rI't' 'P "I''''''~' .... l ,',,".' rt,il \ I 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''~ 5.00 lI\'tlhi";I'I<\>t't''''~.~ 10,00 15.00 20.00 IAf~¥fltliA"'''''''M''~~~/'I'II'''f'''''''''1 25.00 30.00 35.00 40.00 


14711b020_1 1: Sean ES+ 


1~ ~ I ~=W4.3985 

.,. " ,., pIt I I • I ~iI I q Ii' '\ ,'1"( I , rIII ~ '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,* 5,00 10.00 16..00 20.00 25.00 t~~~.".'~;~ 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 	 ~~ 

JIi 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 	

mlz2~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 B 
 0

;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 1nl 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,,"""

5.00 ,:'0'" " " , 10.00 "0 ., ," ,,'" , 15.00 I ' "0' 20.00 '"of" 26.00 ,",,,." ,"SO.OO M., ,"35.00 .,.. 

"'" ,~~.= 40:00 

~5-_1 t22.1. 1:~~:;:a 

J,,,,~, "",5.00 """, "I 10.0015.0020.00 ' " , ,.,.... "M, '""'~'''''t 2&00 ...fI""~,"""""""'''' SO.OO 35.00 40.00 


14711bG2O 1 1;ScanES+
 1J - ~1~'14 trrIZtti,79 

"1".1... f'r'" 

Itoo 'j' ,,,,,,,¥,,,,~,\ 10.00 15.00 ... ''to ....20.00 ,-.""~~IIIII4""""~ 26.00 SO.OO 35.00 40.00 


14111b02(U 1: Scan ES+ 


mlz =575 
 1:r' . 1.976&
 :/2,01 ~.U 

-J"" t .. , 'r'."."","" '''''''' ,",I" ".,,1,."il\.,..,.~",,..,,,, ..~, "•. ­ 8.00 10.00 15.00 20.00 2.5.00 .00 3500 40.00 

ILt'l t \ 

Fig. 13-4 Mass spectra of oil-soluble converted products 

(LC-MS sample -1, sludge + test item, vessel [2], mass chromatograms). 

928 

-65­
5.00 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+ 

1:01 • t ,t I iii,,,. It '1' ii' I 4 11 • t 1.' "'"

j I,it I','i 

.,1..1" i i, iii'" ." 

pI' , " t 

.

1 f i]'o 

,~Z3~':~ 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.'" .,....

5.00 

,....,".. 

10.00 

,,, .. ,... 

15.00 

., .... ,... 

20.00 

., ...X.... 26.00 1. ........

30.00 

,....

35.00 

,....,.:40.00 .~.::l 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 

" 

,IIiii 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,."~ '" 

5.00 

"... ,' .. '."'" 

10.00 .",..15.00 

",., "" 

20.00 

.. '""·"

26.00 

".4....,1" 30.00 "...

85.00 ,"., ., ,~,~= 40.00 

14711b02O_1 1: Scan ES+ 

'j" .. ,."" 5.00 ....,....

10.00 ,....

".. 

15.00 

'p'" , .... 20.00 , .. """ 

25.00 

"A" ""I

30.00 "", .. 35.00 

Ph" .~~,;: 40.00 

Itt'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::~ :~::~,':~::~~"", 5.00 10.00 ,"" 

15.00 

.\/'" 

20.00 ~v" 25.00 l:~~, '30.00 35.00 ,,; ,., i;,~'~ 40.00 

14711b021 1: Scan ES+ 

1 j 16.91 2M1 mlz =321 P·D2 	 2'1 1.24e5

#. 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+ '196 1:] 	 mfz8~ 

itJII \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 I iff ,-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 I" ',.,.,"nl' ., " "i -, ""fI.,. .•J .."~ in "', Ii ,," I' ,"'f'" • .,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 2I mfz =323

J",,,""

5.00 

,',,'I' " , 

10,00 I ' " , , " , , 15.00 

I'~'J, ",20,00 25.00 I " ,1",'~ ,"',,

30.00 

I' , " , '" 

35.00 

, I " " " '" 

40.00 

II~"" 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 , I •, , I , f •j I il ~ i I ( r•I • t Ii. 1\' iii i i ... lti ~t, ij.. , , iI" *ft ~'r',e""r''''tl I 4 I I

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 t8.91 18.67 17.D2 21-7 :1.01 m/z1~~

"-M'II""'~ 5.00 "'~!' 1MO ...,..,.""16.00 1'IiWNI--l 

20.00 'I~ 25.00 ...30.00 *4</•.N, 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 	 2s:?8 	 m/z = 441 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 

5.00 

I' I I , I I I I 

10.00 

'I " I , I ' • I I , I I I , 

1l5.O0 

I ' , , , I 

20.00 26.00 

"I",,I'?' , I I , , , 

30.00 3$.00 

I " I , I ",...,.. , , I I' ,~.:;:: 40.00 

14111b021 1: Scan ES+ 1" Wz=~ 

'j..,...',..,.~. 5.00 ~, I "t'"

10.00 

'I;' I.,.",

15.00 ,,..,.. 20.00 r'~'I""'~~!\I':"" 25.00 30.00 35,00 40.00 

14711b021 '1:Scan ES+ ''3 	 roo nVZ:'o:!: 

l"'\"~""",,, 5.00 10:.00 liot.,......"'.t>"" 15.00 ••

20.00 25.00 ".~ljo...,i40~.~iII! 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, 

. 	

5.00 10.00 15.00 20:00 

l

25.00 30.00 35.00 40.00 

l 't1 tI 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 lVI8 22.33 25.50 m/z = 323

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 1jU4 22.35 [.21 

" '''''''''''' 5.00 '" ' " " " , , ! ' , , , , ' " , 10;00 15.0020.00 I' , , " ' "J,",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,,,,.',,,,

5.00 

',,'" ',,,,

10;00 

",,.',,,

15.00 

I' ' , , , ' , , , , ' 

2Q.OO 

,1:,,

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,,,,'''''.

5.00 

'" ,,'" 

10;00 

,,,, ". '" 

18.00 

,"',"I''' 

20.00 

, .. " ,"' , , 

25.0030.00 

'" '" I' ,~l"",,,,,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~:~~"" 5.00 10,00 """ 

15,00 

,\/." 20.00 ~v" 25.00 r~;30.00 35.00 :,:::""" 

40.00 i"~~ 14711b022 1: Scan ES+ 1j 2111* m/z =321 

18.93 t1.~ 1.3SeS 

..~" 1" /Io't:' 

s. 

l' , "YO' 

10.00 ~YO' ,,,,,-i.,15.00 ,~,J),. 20.00 .. '.~"I.t.~_I"""~"'" 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~ Ar" .., 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~ 5.00 1"'-' 

10.00 16.00 

'·'i'!M"ft'h'l'''''\>llt~I+~'~f.Ji 20.00 ''' 25.00 ~~~'~""'lltIf~"P\ 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+ 
 10j 17M m/z:: 274 r,52 8,63t4 

fI! , 't"'+'>'M1""h~!i'''''t 5.00 10,00 III i til' t'.'*1'¥'·f\I~'f'1 15.00 20.00 f't'M 1"t*M'r.~~ 26,00 t,'" I\Mt'1'f'1"I" 

ao.oo i4"''#f''''''r+'~'~ 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/z8~3~!! ~ ~~ 

·"",,.,'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 114711b022 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] 21.98 mlz =321 16.13 21.1 
 1,3585

...." ..,.... "", ,••••• ,,.., ~\'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~ .,".,6.00 ., ....,....

1G.00 

,..,.,....

16.00 

,....,....

20.00 

,..I,""

25.00 

,,"I., 80.00 I, ,j, ....... 

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 

14111b022 

"'J" "',' '~bii" ·" ';OliO;" ''',S~OO'' "';o1iO t'2"';; ,"";;':;;';'" "SO:';;"" 

1: 

,~"" Sca~ES+ ':J T· m1Z:.:: 

"'J. 1', i I ,'l'1t 

5.00 

1)'1'1"1 •i' • ''i 

10,00 15.00 

I'" i r~1 ,', ", i' , ; 1""11\ 

20.00 "".'~"'~i" 25.00 

I'll .·,..J....,.,,,""" 30.00 ..,·A'1·44.,111~ 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" "\' "

6.00 "'" 'i ", 10,00 , ,,,' ."" '\5.00 OJ .. " , .. 20.00 " r' ,~["215.00 "J~T' ... 30.00 36.00 \,.. "'r""" "', ".... 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 

~:~1::~ 9.81e7 ;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 " 

5.QO 

'" , , , " , H 

10.00 

'" ' "'" 

15.0020.00 

,,:r.'I" ,1,,.

25.00 

I,,.".30.00 I ' ,••'. 'H' 

35.00 

OJ , ".,,,, 

40.00 

~'.4'" 14711 b022 1: Scan ES+ 

'j~,,, ,..,"

5.00 

.. ,,'" 10.00 H," H'"'' 

15..00 

,.. ,." .. 

2OJ)() 

." .. 1,,,,[',:,,., 25.00 30.00 .. ,, ,.. ,." 

35.00 

.. ,,,,~~'= 40.00 

14711b022 1: Scan ES+ 

'j~...", .. 

5.00 

,.. ,", .. 

10.00 

"" ..," ..

15.00 

,.. ," ....

20.00 

,.. T,25.00 .. [:".,30.00 ..,., .. ,'

35.00 

,... '" ~~'~2::: 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."'/""

5.00 

"" """,10.00 "" "I'" 

15.0020.00 

'" "., ~ "i"t,25.00 .,.,11' ..,

30.00 35.00 

,'_'" ~"'1..:2.7~'.:. 40.00 

14711b022 1: Scan E6+ 

':],"',,'.'

5.00 ,..,.. 

',,10.00 ',"'·,,15.00 ''" ,.,,20.00 ',,. ,,,,,' '

25.00 

..,',~t, 30.00 ",,35.00 "',,,~40.00 ~~ 14711b022 1: Scan ES+ 

'j,,.."..

5..00 

,.,.,,,··,·,',

10.00 

..,'·,,,

15.0020.00 

',,,,'·,,,..··,···

25..00 

,,",·,..1·

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 
lt.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<''''' 5. 10.00 "''''.'I''~I'>Io 15.00 20.00 'I""",,...25.00 ¥t""~ 30.00 35.00 40.00 

14711b023 

14711b023 'j ~" 

Itt'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+ ."'1To'" '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 ,""'.'14

5.00 10.00 15.00 

/" \',·t·

20:00 .....'\' 1"I''!I'1~ 25.00 30.00 

i~ft..~"4"_";;I't; 35.00 40.00 

14711h023 1: Scan ES+ 

1:J at' ! m/Z5~7= 

".1'''''''''.'1' 5.00 j,~\\ ",,,~,,,,,,, 10.00 15.00 ••'\lI'I4''''''''\'¥J¥Io!~' 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