Report No. NCAS 10-221


## Final Report StudyTitle 

## Biodegradation Test of

Author Kenji Miya



### Study Completion Date December 13, 2010



### Test Facility Nisso Chemical Analysis Service Co., Ltd. 345 Takada, Odawara, Kanagawa 250-0216, Japan



### Sponsor

Study No. NCAS 10-221 Page 1of27Report No. NCAS 10-221 Page 2 of27 

## GLP Compliance Statement Study No.: NCAS 10-221 Study Title: Biodegradation Test of

This study was performed in accordance with the Good Laboratory Practice Regulations of "Standard for the test facility conducting tests concerning new chemical substances, etc." (Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, No. 1121003, November 21, 2003; Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, No. 3, November 17, 2003; Environmental Policy Bureau, Ministry of the Environment, No. 031121004), Final Amendment: July 4, 2008, and OECD Principles of Good Laboratory Practice [OECD ENV/MC/CHEM (98) 17, 1998].

This study was carried out in accordance with the following procedure and the final report was prepared faithfully and consistentlywith the raw data obtained.

Study Director: (signature) (seal) Kenji Miya Odawara Laboratory Nisso Chemical Analysis Service Co., Ltd. (December 13, 2010) Date

The original signature page of GLP Compliance Statement is shown in page 3.

### Report No. NCAS 10-221 Page 3 of 27 

## Original GLP Compliance Statement
The English translation of GLP Compliance Statement is shown in page 2.

NCAS 10-221 Page 2 of25

NCAS 10-221**:.o)ifit~t";J:****f#Jfmil:;'¥:4t1~~~::.{*6W~~~Jit[i".O~lt!!jj(1Jli!~l::.l~:l-<5]!;$t::.--:>v\-cJ** (3Jl.nt 15 &f. n YJ 21 fl***1-t5e:~** 112100315-, 3JliJX.15 ·11· 17**!i"lJl'iJ~** 3%,**~i!f<:JE;e~**031121004 %),**f&~rii::iE:** stz.JtX: 20&f. 7Yl 4 13 :JOJ:U OECD Principles ofGood Laboratory Practice [OECD ENVtrvlC/CHEM (98) 17, 1998] tZ::Vf:-:>""(;%Jii)i;1.-I'C.a

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### i!lf7J-.Vr-z :/7-**,HBJ.JR*~m**Report No. NCAS 10-221 Page 4 of27 

## Quality Assurance Statement Study No.: NCAS 10-221 Study Title: Biodegradation Test of

Quality Assurance inspections of the study referred above were conducted according to the appropriate GLP regulations and the standard operating procedures (SOPs) of the Quality Assurance Unit (QAU). The results ofthe inspections were reported to the study director and the facility management on the following dates. Dates (Month/Day/Year) Items inspected Inspected Reported to SD Management Study plan 9/16/2010 9/16/2010 9/16/2010 Study plan amendment No.1 10/7/2010 10/7/2010 10/7/2010 Study plan amendment No.2 11/15/2010 11/16/2010 11/16/2010 Experimental operation

·Receiptand maintenance ofthe activated 9/24/2010 9/24/2010 9/24/2010

sludge ·Receipt and maintenance ofthe activated 10/18, 19/2010 10/19/2010 10/19/2010

sludge (new lot) ·Infrared resonance spectral measurement 9/17, 24/2010 9/24/2010 9/24/2010 ·Preparation ofthe test systems for BOD 9/21,24/2010 9/24/2010 9/24/2010

measurement ·Set up ofthe enclosed respirometer and

BOD measurement (1 51 trial) 9/21, 24/2010 9/24/2010 9/24/2010 ·Setup ofthe enclosed respirometer and

BOD measurement (3rd trial) 10/18, 19/2010 10/19/2010 10/19/2010

·BOD measurementand observation 9/28/2010 9/28/2010 9/28/2010 ·BOD measurement and observation (2nd 10/7/2010 10/7/2010 10/7/2010 trial)
·BOD measurement and observation (3rd 10/25/2010 10/25/2010 10/25/2010 trial) ·Sampling and treatment ofthe bottle 11/15, 16/2010 11/16/2010 11/16/2010 contents ·Preparation ofthe standard solutions 11/15, 16/2010 11/16/2010 11/16/2010

(LC/MS/MS) ·Preparation ofthe standard solutions (AA) 11/15, 16/2010 11/16/2010 11/16/2010 ·Analysis (LC/MS/MS) 11/15, 16/2010 11/16/2010 11/16/2010 ·Analysis (AA) 11/15, 16/2010 11/16/2010 11/16/2010 Raw Data 11/26-30/2010 11/30/2010 11/30/2010 Draft Report 11/26-30/2010 11/30/2010 11/30/2010 Final Report 12/13/2010 12/13/2010 12/13/2010

The QAU found that the study was performed according to the study plan and SOPs, the reported methods and procedures were actually used and the results accurately reflect the recorded data. QAU Manager: (signature) (seal) Ken Watabe Nissa Chemical Analysis Service Co., Ltd. (December 13, 2010) Date
The original signature page of Quality Assurance Statement is shown in page 5.

### Report No. NCAS 10-221 Page 5 of 27 

## Original Quality Assurance Statement
The English translation of Quality Assurance Statement is shown in page 4.
Page 3of25**~Yl't;'!l:"%** : NCAS 10·221**J:~c~~vHiHrit!Eiii'JiEcr)~'J1f.;£ icl:i:'Th':~:i"** i@Jf1 GLP .toJ::**U{H~t!f:f!i';:~iEffilP~** (QAU) (f) SOP**t;::Ji¥--:5~ \""(~~iff** L-1co**\%''lit~ icl'i'ilt~O)$ti:ti!::l:i:,**  J;J,""f<7) 13ft<::it>\!!i,bl(ff:ff·;fJJ::**G~)!J!E"ili'1!Jl~·;;::** ¥fl.-a: 

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.**[J,'i~*~*7l1J;.iii:iil.1J/E~~** 9/21, 2412010 9/2412010 9/2412010**(f)~JE ~** iJl.U/Emlft€1

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(f)fi9:JE**~**ffi!JJE!#lftil 10/18, 19/2010 10/19/2010 10/19/2010**(f!j:;%~**2@] !§) •BODi!ltlZi:":iOJ:**V~~** 9/28/2010 9/28/2010 9/28/2010 •**BODiJliJJE:foJ::·amt~** 1017/2010 1017/2010 1017/2010**(W:~~)** ·BODiUIJif:t:>**J:V~t~** 10/25/2010 10/25/2010 10/25/2010**(N;%~** 2 @10) •BOD**jj!IJ;E,j;~**Tfii:(f) ll/15, 16/2010 11/16/2010 11116/2010**5)-1'ffj)ijj,11~** •**t\l'!i$~10Zil)i't11J~~** 11/15, 16/2010 11/16/2010 11116/2010

(LC/1vfS/Iv1S) •**ti'i1!3m~1(0)W!J!J\:!** 11/15, 16/2010 11116/2010 11116/2010

(@-i'·P&J't7)-1'JT) •:5-Hfr (LC/JVIS/MS) 11/15, 16/2010 11116/2010 11/16/2010 ·£-iff (fjj\

### =t-P&Jt51-:t.lf) 11115, 16/2010 11116/2010 llil6/2010 1:.-T-7 11/26·30/2010 11/30/2010 11/30/2010**¥~~:-i!}:f!i.3R** 11126·30/2010 11/30/2010 11/30/2010**m:~~115~** 12/13/2010 12113/2010 12/13/2010 QAU ii,**;:(f);;i,~jt~>;;t:Wtilti@:;;ti:!OJ::**"LJ SOP**l::::Vf:-:>-c~'Tbh, ¥a\lr2:'ht:.:/Ji'&-'\='-¥J;]:~>~itr'H:** **{~b;h,t;:** 1_,ci)-z"J? fJ,**1:il:frH~J:llcifik2EiL1c7-:?** :i".iEJ;"!i!::.&B,"<: L--n\0::!::**Z.liJli~:g**L-tco

liltffll R1E (:!*)**rl\!N~J':tJr·t>7-**Report No. NCAS 10-221

Study No.: Study Title: Report No.: Sponsor: Client:
Test Facility:

Study Director: Experimenter: 

## Study Information
NCAS 10-221 Biodegradation Test of NCAS 10-221

Nisso Chemical Analysis Service Co., Ltd. Odawara Laboratory 345 Takada, Odawara, Kanagawa 250-0216, Japan TEL 0465-42-8207 FAX 0465-42-3586 Kenji Miya Page 6 of 27

Confirmation ofthe Test Substance Confirmation ofthe Stability ofthe Test Substance Cultivation (BOD measurement) Shinpei Tsushima, Hiroyuki Ueda Hiroyuki Ueda Hiroyuki Ueda LC/MS/MS measurement Atsushi Utsugi AA measurement Junko Tashiro Study Initiation Date: September 16, 2010 September 17, 2010 Experimental Start Date: Exposure Period: Experimental Completion Date: October 18, 2010- November 15, 2010 November 17, 2010 Study Completion Date: December 13, 2010 Testing Guideline: "Biodegradation Test of Chemical Substances" prescribed in "Test Methods for New Chemical Substances", Pharmaceutical and Food Safety Bureau No. 1121002of MHLW, Manufacturing Industries Bureau No.2 of METI and Environment Policy Bureau No. 031121002 of MOE (Amended on
Archiving: November20, 2006), and OECD Guidelines forthe Testing ofChemicals No. 301C (1992). All the documents ofthis study will be retained for 10 years afterthe end of this study in the archives ofthe test facility. The management ofthe documents afterthatwill be determined by mutual consultation between the Sponsor and Nisso Chemical Analysis Service Co., Ltd. at thattime. The test substance will be returned afterthe end ofthis study. Deviation from SOPs and Study Plan:

There was no deviation from SOPs and the study plan. Circumstance/matterthat Affectthe Reliability ofthe Test Result:

There was no environmental agent which influenced the reliability ofthe test result.

### Report No. NCAS 10-221 Page 7 of 27


## Table of Contents
Title Page
.................................................................................................................................. 1 GLP Compliance Statement.

......................................................................................................2 Original GLP Compliance Statement

......................................................................................... 3 Quality Assurance Statement.

....................................................................................................4 Original QualityAssurance Statement

.......................................................................................5 Study Information

.......................................................................................................................6 Table of Contents

......................................................................................................................7 Summary

...................................................................................................................................9 Introduction
................................................................................................................................9 Materials and Methods
............................................................................................................... 9 1. Test Substance

................................................................................................................9 2. Standard Substance

........................................................................................................9 3. Confirmation ofthe Test Substance

............................................................................... 10 4. Stability ofthe Test Substance

...................................................................................... 10 5. Reagents and Apparatus
............................................................................................... 10 6. Experimental Methods
................................................................................................... 11 6.1 Conditions ofthe Biodegradation Test
.................................................................... 11 6.2 BOD Measurement and Observation ofthe Bottle Contents

................................... 12 6.3 pH Measurement
.................................................................................................... 12 6.4 Analytical Procedures afterthe End ofthe BOD Measurement
............................... 12 6.5 LC/MS/MS Measurement
........................................................................................ 12 6.6 AA Measurement
.................................................................................................... 13 6.7 Measurement ofthe Dissolved Organic Carbon

...................................................... 14 Results and Discussion
............................................................................................................ 15 1. Confirmation ofthe Test Substance

............................................................................... 15 2. Confirmation ofthe Stability ofthe Test Substance

....................................................... 15 3. Confirmation ofthe Degradation Activity oftheActivated Sludge

................................... 15 4. Observation Results ofthe Contents

............................................................................. 15 5. Results ofthe pH Measurement.
.................................................................................... 15 6. Degradability Based on the BOD Measurement.
............................................................ 16 7. Degradability Based on the LC/MS/MS Measurement
................................................... 16 8. Degradability Based on the AA Measurement..
.............................................................. 16 9. Statistical Work
.............................................................................................................. 16 Conclusions

............................................................................................................................. 16Report No. NCAS 10-221 Page 8 of 27 

## Contents ofTables and Figures

Table 1 Degradability ofthe test substance based on the BOD measurement
....................... 17 Table 2 Observation results ofthe contents
............................................................................ 17 Table 3 pH values ofthe test solutions
................................................................................... 17 Table 4 Degradability ofthe test substance based on the LC/MS/MS measurement
.............. 18 Table 5 Degradability ofthe test substance based on the AA measurement
.......................... 18 Figure 1 Outline ofthe analytical procedure afterthe BOD measurement
.............................. 19 Figure 2 IR spectrum ofthe test substance (supplied from the sponsor)

................................ 19 Figure 3 IR spectrum ofthe test substance measured before the experiment
........................ 20 Figure 4 lR spectrum ofthe test substance measured afterthe experiment
........................... 20 Figure 5 BOD measuring chart (start- 7 days)

....................................................................... 21 Figure 6 BOD measuring chart (7 days- 14 days)

.................................................................. 21 Figure 7 BOD measuring chart (14 days- 21 days)

................................................................ 22 Figure 8 BOD measuring chart (21 days- 28 days)

................................................................ 22 Figure 9 Calibration curve ofthe LC/MS/MS measurement
.................................................... 23 Figure 10 LC/MS/MS chromatogram ofthe calibration solution at 0.03 !Jg/ml.

....................... 23 Figure 11 LC/MS/MS chromatogram ofthe calibration solution at 1 !Jg/ml.

............................ 24 Figure 12 LC/MS/MS chromatogram ofthe calibration solution at 3 !Jg/ml.

............................ 24 Figure 13 LC/MS/MS chromatogram ofthe calibration solution at 5 !Jg/ml..

........................... 25 Figure 14 LC/MS/MS chromatogram ofthe test solution for the [Water+Test Substance] System (Bottle No.1)

............................................... 25 Figure 15 LC/MS/MS chromatogram ofthe test solution for the [Sludge+Test Substance] System (Bottle No. 2)

............................................. 26 Figure 16 LC/MS/MS chromatogram ofthe test solution for the [Sludge+Test Substance] System (Bottle No. 3)

............................................. 26 Figure 17 LC/MS/MS chromatogram ofthe test solution for the [Sludge+Test Substance] System (Bottle No. 4)

............................................. 27 Figure 18 Calibration curve ofthe AA measurement.
.............................................................. 27Report No. NCAS 10-221 Page 9 of 27 

## Summary The biodegradation test (28 days) of was conducted and the mean degradability calculated from the biological oxygen demand (BOD) was 2% (n=3). Furthermore, the mean degradabilities calculated from the residual amount of the test substance obtained by measurement with the liquid chromatograph mass spectrometer and the atomic absorption spectrophotometer were 0% (n=3) and -4% (n=3), respectively. It was considered that is not ready biodegradable because the degradabilities for all vessels were less than 60% (Test Methods and Judgment Criteria for the Designation of Monitoring Chemical Substances, April 12, 2010). 

## Introduction The biodegradation test of biodegradability. was performed to evaluate the

1. Test Substance Name: Rational formula: Molecularformula: Molecularweight: CAS number: Lot No.: Purity: NCAS retrieval No.:
Supplier: Received weight: Received date: Expiry date: Storage condition: 

## Materials and Methods

STD-1180-4 (Preparation ofthe test systems, Identification and stability ofthe test substance) STD-1180-6 (Preparation ofthe standard solutions for LC/MS/MS measurement) 0.5 g x 5 bottles and 7.5 g x 1 bottle July 6, 2010 May 27, 2011 The substance in a glass bottle was covered with the aluminum lamination bag, and was stored at room temperature (dark). Physicochemical properties: Soluble in DMSO, acetone and water, Deliquescence The test substance was weighed as 100% of purity. 2. Standard Substance Name: Molecularformula: Chemical structure: Aniline 

### C6H7N 

# o-NH
2Report No. NCAS 10-221

Molecularweight: CAS No.: Lot No.: Purity: NCAS retrieval No.: Supplier: Received weight: Received date: Expiry date: Storage condition: Appearance: 93.13 62-53-3 ALL9668 Regarded as 100% ANI-013 Wako Pure Chemical Industries, Ltd. 12-mL ampoule, 25 ampoules August 25, 2008 August 25, 2011 Stored in afreezer (about -20°C) Pale yellow and transparent liquid Page 10 of 27

3. Confirmation ofthe Test Substance The infrared (IR) spectrum of the test substance (NCAS retrieval No.: STD-1180-4) was measured (using KBr pellet) at the start of the experiment. This spectrum was compared with the IR spectrum obtained from the sponsorto confirm the identity. 4. Stability ofthe Test Substance TheIR spectrum ofthe test substance (NCAS retrieval No.: STD-1180-4) was measured at the end of the experiment. This spectrum was compared with the IR spectrum measured at the start ofthe experimentto confirm the stability. 5. Reagents and Apparatus Potassium dihydrogenphosphate, disodium hydrogenphosphate dodecahydrate, ammonium chloride, magnesium sulfate heptahydrate, calcium chloride (anhydrous), iron (Ill) chloride hexahydrate and nitric acid: Guaranteed (Wako Pure Chemical Industries, Ltd.) Dipotassium hydrogenphosphate: Guaranteed (Junsei Chemical Co., Ltd.) Distilled water and acetonitrile: for HPLC (Wako Pure Chemical Industries, Ltd.) Soda lime: No.1 (Wako Pure Chemical Industries, Ltd.) Potassium bromide: KBr Powder (PIKE TECHNOLOGIES) Lithium standard solution: 1004 mg/L (Lot No. KWJ3984, Expiry date: August 2011) Deionized water: Distilled water prepared with an automatic water distiller (WDA-15S, G-487, lsuzu Seisakusho Co., Ltd.) was purified with a pure water manufacturing device (LV-08, Balance: Stirrer: Membranefilter: Micropipette: pH meter: Centrifuge: Drier (for MLSS measurement): Standard thermometer: G-70, Toray Industries, Inc.). AG285 (G-549, Mettler-Toledo International Inc.) EB-330H (G-3, Shimadzu Corp.) IS-34N (G-440, IKEDA SCIENTIFIC Co., Ltd.) Stirrer/Hotplate (G-418, CORNING) GL Chromatodisc (25A, 0.45 f.Jm, KURABO INDUSTRIES L TO.) 500-5000 f.JL (MP-1-15, eppendorf Research) 10-100f.JL (MP-1-20, eppendorf Research) 720A (G-677, Thermo Fisher Scientific K.K.) F-23 (G-254, HORIBA, Ltd.) H-9R (G-674, KOKUSAN Co., Ltd.) D0-300A (G-822, AS ONE Corp.) 0-50°C (G-588-77, ANDO KEIKI CO., LTD.)

### Report No. NCAS 10-221 Page 11 of 27

Enclosed respirometer: Incubator OM-31OOA (G-281, Ohkura Electric Co., Ltd.) Recorder RM18G (G-281-1, Ohkura Electric Co., Ltd.) Liquid chromatograph mass spectrometer (LC/MS/MS): Acquity/Q micro (G-722, Waters) Atomic absorption spectrophotometer (M): M 240 (G-708, Varian, Inc.) Infrared spectrophotometer: Spectrum One (G-641, PerkinEimer Inc.) 6. Experimental Methods Biological oxygen demand (BOD) which occurred by the degradation of the test substance in the presence of the standard activated sludge was measured periodically (28 days) with the enclosed respirometer. After the last measurement of BOD, the concentration ofthe test substance was determined with LC/MS/MS and 

### M. And then the biodegradability of the test substance was evaluated from the results of the BOD and the concentration ofthe test substance. 6.1 Conditions ofthe Biodegradation Test 6.1.1 Standard Activated Sludge Supplier: Chemicals Evaluation and Research Institute, Japan Received date: October 14, 2010 Lot No.: 201009R Suspended solid concentration (MLSS): 3100 mg/L (October 18, 2010) 6.1.2 Conditions Temperature: Exposure period: Volume: Concentrations: 24.7 - 24.9°C (actual measurement) 28 days (BOD measurement) 300 ml Test substance and aniline (standard substance) Standard activated sludge (MLSS) 100 mg/L 30 mg/L 6.1.3 Experimental Design and Addition ofthe Test Substance Bottle No.1: [Water+Test substance] system Deionized water (300 ml)was transferred into a bottle and then the test substance (30 mg) was added to thewater. Bottle No. 2-No. 4:[Sludge +Testsubstance] system Mineral medium· (297.1 ml) was transferred into 3 bottles each and then 30 mg ofthe test substance was added into each bottle, followed by the addition of the standard activated sludge
.. (2.9 ml) to each bottle. Bottle No. 5: [Sludge +Aniline] system Mineral medium· (297.1 ml) was transferred into a bottle and then 29 !JL (30 mg) of aniline was added into the bottle with a micropipette. After mixing it well, the standard activated sludge·· (2.9 ml) was added to the bottle. Bottle No. 6: [Sludge blank] system Mineral medium· (297.1 ml) was transferred into a bottle and the standard activated sludge
.. (2.9ml) was added to the medium. *) 

### A, B, C and D solutions (each 6 ml, defined in JIS K 0102-1998 21) were added to deionized water to make a 2-L solution. The addition volume of the mineral medium was calculated by the following equation. Vm = 300- Vs where, Vm =Addition volume ofthe mineral medium (ml) 300 =Volume ofthe test solution (ml) Vs =Addition volume ofthe standard activated sludge (ml)Report No. NCAS 10-221 Page 12of27
**) The addition volume of the standard activated sludge was calculated by the following equation based on the MLSS value. Vs = Cs 

### I MLSS x Vo where, Vs =Addition volume ofthe standard activated sludge (ml) Cs = Sludge concentration in the test solution (mg/L) MLSS =Suspended solid concentration (mg/L, see section 0.1)) Vo =Volume ofthe test solution (ml) 6.2 BOD Measurement and Observation ofthe Bottle Contents BOD of each bottle was measured for 28 days with the enclosed respirometer. The growth of the activated sludge and color of the test systems were observed during the exposure period (after 7, 14, 21 and 28 days from the experimental start). The BOD degradability was calculated by the following equation (7, 14, 21 and 28 days). Degradability (%) = {(BOD - B) 

### ITOO} x 100

where, BOD: Oxygen demand in the [Sludge + Aniline] or [Sludge + Test substance) systems (mg) B: Oxygen demand in the [Sludge blank] system (mg) TOO: Theoretical oxygen demand ofthe test substance or aniline (mg) <Calculation ofthe TOO>

Aniline: TOO was calculated by having assumed that 30 mg of aniline was decomposed as follows. CeH1N + 8.75 Oz --t 6 COz + 3.5 HzO + NOz TOO= (30/93.13) x 8.75 x 32 = 90.2 mg Testsubstance: TOO was calculated by having assumed that 30 mg of the test substancewas decomposed as follows.

6.3 pH Measurement FzliN04S2 + 2 Oz --t 2 S03 + NOz + 2 F + 

### Li TOO= (30/187.07) x 2 x 32 = 10.3 mg
The pH ofthe test solutions was measured atthe start and the end ofthe exposure. For the [Water+ Test substance] and the [Sludge + Test substance] systems, another solution for each system was prepared and the pH measurement of these solutions was conducted at the start ofthe exposure. At the end of the exposure, the pH was measured using the test solution which remained in the bottle afterthe treatment according to section 6.4. 6.4 Analytical Procedures afterthe End ofthe BOD Measurement Figure 1 shows the outline of the analytical procedure after the end of the BOD measurement. An aliquot (100 ml) of each solution (bottle No. 1 to No.4) was taken into a centrifugal tube and then it was centrifuged for 15 minutes at 5000 rpm (3944 g). The supernatant was filtered with a membrane filter and the filtrate was analyzed in accordance with the procedures described in sections 6.5 and 6.6. For bottle No. 5, the above-mentioned operation was not carried out since only pH was measured. 6.5 LC/MS/MS Measurement The concentration ofthe test substance in the test solution was determined in accordance with the procedure described in the next page.Report No. NCAS 10-221 Page 13 of 27

6.5.1 LC/MS/MS Conditions Column: Ascentis Express C18, 2.1 mm i.d. x 100 mm, 2.7!Jm (SUPELCO) Column temperature: 40°C Mobile phase: Distilled water

# + acetonitrile = 1 + 1 (v/v, 0.1 v/v% formic acid was Flow rate: Injection volume: Ionization mode: Measuring mode: Monitored ion: contained) 0.3 mllmin 5 !JL ESI, negative MRM 180.33>96.90 (for quantification), 180.33>77.84 (for confirmation) 6.5.2 Preparation of Calibration Curve An aliquot (10.69 mg) ofthe test substance was dissolved in distilled waterto make 100 mlsolution. This solution was diluted with 50% acetonitrile in water to prepare 0.03, 1, 3 and 5 !Jg/ml calibration solutions. These calibration solutions (0.03- 5 !Jg/ml) and 50% acetonitrile in water were injected into the LC/MS/MS described in section 6.5.1 in order to determine the peak areas ofthe test substance. The calibration curve was prepared by plotting the peak areas vs. the concentrations of the test substance to confirm the linearity. It was considered that the linearity ofthe calibration curve was good when the square ofthe correlation coefficient (R2) was more than 0.99. 6.5.3 Measurement ofthe Test Solutions The test solutions (bottle Nos. 1, 2, 3 and 4) were treated in accordance with section 6.4 and 1.5 mlofeach solution was diluted with 50% acetonitrile in water to make 50 ml solution. The diluted solutions were measured on the LC/MS/MS conditions described in section 6.5.1 (n=1) and the peak areas of the test substance were determined. The concentrations of the test substance in the test solutions were calculated by using the calibration curve. 6.5.4 Recovery Test The recovery of the analytical method was not carried out because no extraction procedure was included in the analytical procedure described in section 6.5.3. 6.5.5 Calculation ofthe Degradability The amount ofthe residual test substance was calculated from the concentration ofthe test substance in the test solutions obtained from section 6.5.3. The degradability was calculated by the following equation. Degradability (%) = { (Cs - CA) 

### I Cs} x 1 00 where, CA =Amountofthe residual test substance in the [Sludge +Test substance] system (mg) C8 =Amount ofthe residual test substance in the [Water+ Test substance] system (mg) 6.5.6 Limit ofQuantification The lowest concentration (0.03 !Jg/ml) of the calibration solution prepared by section 6.5.2 was regarded as the limit of quantification. This concentration is equivalent to 1 !Jg/ml as the concentration ofthe test substance in the test solutions. 6.6 

### M Measurement The concentration of lithium in the test solutions was determined in accordance with the procedure described in the next page.Report No. NCAS 10-221

6.6.1 

### AA Conditions Hollow cathode lamp: Fuel/ Support : Electric current: Flow rate ofair: Flow rate of acetylene: Wavelength: Slitwidth: Lithium Air 

### I acetylene 5.0 mA 13.5 Llmin 2.00 Llmin 670.8 nm 1.0 nm Burner height: 0.0 mm Number of replications: 5 Measuring time: 3 seconds 6.6.2 Preparation ofCalibration Curve Page 14of27

A lithium standard solution (1004 mg/L) was diluted with 0.2 moi/L nitric acid to prepare 0.08, 0.2, 0.4, 0.8, 1.2 and 2 mg/L calibration solutions. These calibration solutions (0.08 - 2 mg/L) were injected into the 

### AA conditions described in section 6.6.1 and the mean absorbance of five repetitive measurements was calculated. The calibration curve was prepared by plotting the mean absorbance 

### vs. the concentrations ofthe test substance to confirm the linearity. It was considered that the linearity of the calibration curve was good when the square ofthe correlation coefficient (R2) was more than 0.99. 6.6.3 Measurement oftheTest Solutions The test solutions (bottle Nos. 1, 2, 3 and 4) were treated in accordance with section 6.4. Aliquots ofthe each solution (4 ml) and 0.4 moi/L nitric acid (4 ml) were taken into a 20-ml volumetric flask. In addition, they were diluted with 0.2 moi/L nitric acid. The diluted solutions were measured on the 

### AA conditions described in section 6.6.1 (n=1) and the mean absorbance of five repetitive measurements was calculated. The concentrations of lithium in the test solutions were calculated by using the calibration curve. 6.6.4 Calculation ofthe Degradability The amount of the residual lithium was calculated from the concentration of lithium in the test solutions obtained from section 6.6.3. The degradability was calculated by a following equation. Degradability (%) = {(C9 - CA) 

### I Cs} x 100

where, CA =Amountofthe residual lithium in the [Sludge+ Test substance] system (mg) C9 =Amount ofthe residual lithium in the [Water+ Test substance] system (mg) 6.7 Measurement ofthe Dissolved Organic Carbon The measurement of the amount of the dissolved organic carbon was not carried out since no carbon was contained in the test substance.

### Report No. NCAS 10-221 Page 15 of 27 

## Results and Discussion 1. Confirmation ofthe Test Substance Figure 2 shows the IR spectrum of supplied from the sponsor and Figure 3 shows the IR spectrum of the test substance (NCAS retrieval No. STD1180-4) measured before the experiment. Since both IRspectra coincided with each other, it was considered that the test substance was However, the peak width became large because the test substance had deliquescence. Furthermore, IR spectrum of another test substance (NCAS retrieval No. STD-1180-3, the same lot) was measured and the same spectrum as Figure 3was obtained. Therefore, no difference was recognized on the property ofthe test substance among the NCAS retrieval Nos. Thus, the calibration solutions for LC/MS/MS were prepared by using NCAS retrieval No. STD-1180-6 which had the same lot number the others. 2. Confirmation ofthe Stability ofthe Test Substance Figure 4 shows the IR spectrum ofthe test substance measured afterthe experiment. The IR spectra obtained before and afterthe experiment coincided with each other, so that it was considered that the test substance was stable during the experimental period under room temperature (dark place). 3. Confirmation ofthe Degradation Activity ofthe Activated Sludge Table 1 ([Sludge+ Aniline] system) shows the results ofthe BOD degradability of aniline to confirm the degradation activity of the activated sludge. The BOD degradabilities of aniline were 51% (7 days after the exposure) and 65% (14 days after the exposure). Therefore, it was considered that the activated sludge had good activity. Thus, the validity of this study was confirmed. By the way, the activated sludge of another lot (lot No.: 201006R, received date: July 15, 2010) was tested before using the activated sludge ofthis lot. However, BOD degradability was less than 40% after 7 days. Thus, it was considered that this test was invalid. 4. Observation Results ofthe Contents Table 2 shows the observation results ofthe contents. The test solutions ofthe [Sludge + Test substance] system (Bottle Nos. 2- 4) and the [Sludge blank] system (Bottle No. 6) were slightly muddy white. The test solution ofthe [Sludge + Aniline] system (Bottle No. 5) was muddy white and that of the [Water + Test substance] system (Bottle No. 1) was colorless and transparent. The multiplication of activated sludge was observed in the [sludge + aniline] system only. 5. Results ofthe pH Measurement Table 3 shows the pH values measured at the start and end of the cultivation. At the start of the cultivation, the pH values of the [Water + Test substance] system, the [Sludge + Test substance] system, the [Sludge + Aniline] system and the [Sludge blank] system solutions were 5.3, 7.3, 7.3 and 7.3, respectively. At the end of the cultivation, the pH values ofeach system solution were 6.4, 7.3 (mean value), 8.4 and 7.7, respectively.Report No. NCAS 10-221 Page 16 of 27

6. Degradability Based on the BOD Measurement Table 1 shows the degradability based on the BOD measurement and Figures 5 to 8 show the charts ofthe BOD measurement. The BOD values after 28 days for the [Sludge +Test substance] system were 1.9380 mg for bottle No. 2, 2.0440 mg for bottle No. 3 and 1.9960 mg for bottle No. 4. The BOD values for the [Sludge blank] and [Water+ Test substance] systems were 1.7450 mg and 0.0000 mg, respectively. Since the TOO ofthe test substance was 10.3 mg, the BOD degradabilities of the [Sludge +Test substance] system were 2% for bottle No. 2, 3% for bottle No. 3 and 2% for bottle No.4. Therefore, the mean degradabilitywas calculated to be 2%. The difference ofthe degradation rates between maximum and minimum was less than 20% so the validity criterion ofthe biodegradation test was fulfilled. 7. Degradability Based on the LC/MS/MS Measurement Table 4 shows the degradability based on the LC/MS/MS measurement. Figure 9 shows the calibration curve and Figures 10 to 17 show the LC/MS/MS chromatograms of the standard solutions and the test samples. The amounts ofthe residual test substance in the [Sludge+ Test substance] system were 31.1 mg for bottle No. 2, 29.5 mg for bottle No. 3 and 30.9 mg for bottle No. 4. The amount of the residual test substance in the [Water +Test substance] system was 30.4 mg. The degradation rates ofthe [Sludge+ Test substance] system were -2% for bottle No. 2, 3% for bottle No. 3 and -2% for bottle No. 4. Therefore, the mean degradability was calculated as 0%. The difference ofthe degradation rates between maximum and minimum was less than 20% so the validity criterion ofthe biodegradation testwas fulfilled. No unknown peak which had larger area than the LOQ was observed on the LC/MS/MS chromatogram ofthe [Sludge+Test substance] system. 8. Degradability Based on the AA Measurement Table 5 shows the degradability based on the AA measurement, and Figure 18 shows the calibration curve. The amounts ofthe residual lithium in the [Sludge +Test substance] system were 1.13 mg for bottle No. 2, 1.09 mg for bottle No. 3 and 1.12 mg for bottle No. 4. The amount of the residual lithium in the [Water+ Test substance] system was 1.07 mg. The degradation rates ofthe [Sludge+ Test substance] system were -6% for bottle No.2, -2% for bottle No. 3 and5% for bottle No. 4. Therefore, the mean degradability was calculated as -4%. The difference of the degradation rates between maximum and minimum was less than 20% so the validity criterion ofthe biodegradation test was fulfilled. 9. Statistical Work For the LC/MS/MS measurement, the concentration of the test substance in the test solutions were calculated by using the LC/MS/MS software and the degradabilities were calculated by using the Microsoft Excel 2003 (SP3). The degradability was rounded off by the first decimal place. 

## Conclusions The biodegradation test of (28 days) was conducted. The mean BOD degradability was 2% (n=3). The mean degradabilities calculated from the results of the LC/MS/MS and AA measurements were 0% (n=3) and -4% (n=3), respectively. Therefore, it was considered that is not ready biodegradable because the degradabilities for all vessels were less than 60% (Test Methods and Judgment Criteria forthe Designation of Monitoring Chemical Substances, April 12, 2010).Report No. NCAS 

### 10-221 Page 

### 17 of

### 27

a e 

### T bl 

### 1 De Jra a 11ry o e es su s ance 

### d bTt 

### f th 

### t t b t 

### base

### d on th BOD e measuremen

### t Bottle 7 days 14 days 21 days 28 days Sample BOD DG* BOD DG* BOD DG* BOD DG* Mean No. (mg) 

### (%) (mg) 

### {%) (m_g) 

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

### (%)

1 Water+Test 0.000 - 0.000 - 0.000 

### - 0.0000 

### - - substance 2 Sludge +Test 0.594 2 1.653 3 1.938 5 1.9380 2 substance 3 Sludge +Test 0.256 -1 1.391 0 1.822 3 2.0440 3 2 substance 4 Sludge +Test 0.571 2 1.590 2 1.829 3 1.9960 2 substance 5 Sludge +Aniline 46.09 51 59.94 65 62.17 67 64.193 69 

### - 6 Sludge blank 0.353 - 1.389 - 1.473 - 1.7450 

### - -

**: DG (Degradability, %) ={(BOD- B) 

### ITOO} x 100 BOD: Oxygen demand in the [Sludge+Aniline] or [Sludge+ Test substance] systems (mg) B: Oxygen demand in the [Sludge blank] system (mg) TOO: Theoretical oxygen demand (aniline 

### = 90.2 mg, test substance 

### = 10.3 mg)

Table

### 2 Observation results ofthe contents Bottle ?days 14 days 21 days 28 days

No. Sample Proliferation Appearance Proliferation Appearance Proliferation Appearance Proliferation Appearance Water+ colorless colorless colorless colorless 1 Test 

### - - - - substance transparency transparency transparency transparency Sludge+ slightly slightly slightly slightly 2 Test None None None None substance muddywhite muddywhite muddywhite muddywhite Sludge+ slightly slightly slightly slightly 3 Test None None None None substance muddywhite muddywhite muddywhite muddywhite Sludge+ slightly slightly slightly slightly 4 Test None None None None substance muddywhite muddywhite muddywhite muddywhite 5 Sludge+ observed muddywhite observed muddywhite observed muddywhite observed muddywhite Aniline 6 Sludge None slightly None slightly None slightly None slightly blank muddywhite muddywhite muddywhite muddywhite

a e p 

### T bl 

### 3 Hva ues o e es sou tons 

### f 

### th t 

### t I 

### f pH Bottle No. Sample atthe start atthe end Individual Mean 1 Water + Test substance 

### 5.3 6.4
- 2 Sludge 

### + Test substance 

### 7.2 3 Sludge 

### +Test substance 

### 7.3 7.4 7.3 4 Sludge 

### + Test substance 

### 7.4 5 Sludge +Aniline 

### 7.3 8.4
- 6 Sludge blank 

### 7.3 7.7 

## -Report No. NCAS 10-221 Page 18of27



### T bl 4 0

a e egra

### d bTt 

### f

### th t t b 

### t a 11ry o e es su s ance 

### b d th LC/MS/MS ase on e measuremen

## t Bottle Concentration (mg/L) Residual Degradability (%) Sample PeakArea amount No. Measured Test (mg) Individual Mean solution solution 1 Water+ Test 1671.6 3.0357 101.2 30.4 

### - - substance 2 Sludge + Test 1713.5 3.1134 103.8 31.1 -2 substance 3 Sludge + Test 1626.0 2.9508 98.36 29.5 3 0 substance 4 Sludge + Test 1703.3 3.0944 103.1 30.9 -2 substance Calibration equation: y

# = 538.749 x

# + 36.1851, R2

# = 0.999247 Degradability (%)

# ={(C8-CA) 

### I C8} x 100

where, CA: Amount of the residual test substance in the [Sludge +Test substance] system (mg) C8: Amount of the residual test substance in the [Water + Test substance] system (mg)



### T bl 5 0

a e 

### d bTt f

### th t 

### t b t egra a 

### tny o e es su s ance 

### b d th 

### PV\ ase on e measuremen

## t Concentration ofLi Residential Degradability (%) Bottle Absorbance (m /L) Sample amount of No.

1
2
3
4 CAbs) Measured Test solution solution Li (mg) Individual Water+ Test 0.2548 0.7158 3.5791 1.07 

### -

substance Sludge + Test 0.2673 0.7529 3.7644 1.13 -6

substance Sludge+Test 0.2588 0.7277 3.6384 1.09 -2

substance Sludge + Test 0.2644 0.7443 3.7214 1.12 -5

substance Calibration equation: y

# =0.3373 x

# + 0.0133, R2

# =0.9985 Oegradability (%) 

### = {(C8-CA) 

### I C8} x 100

where, CA: Amount ofthe residual lithium in the [Sludge+ Test substance] system (mg) C8: Amount ofthe residual lithium in the [Water+Test substance] system (mg) Mean



### -

-4Report No. NCAS 10-221 Page 19 of 27

Test solution (300 ml) 

# I Take 100 m L ofthe test solution 

# I Centrifuge (at 4000G for 15 minutes) 

# I Takethe supernatant 

# I
LC/MS/MS measurement 

### M measurement Figure 1 Outline ofthe analytical procedure afterthe BOD measurement



### :1HI

92:..:**t:A'.~.:':E:~~ _;_~~~-,f~** **~.~:~,~:;:-:;~~·:·,c·\.~--l';~;·J;.:~l-;;:,~;~:!.;:~' ;\~** **L';;~:~-,~;::\;£~;i~112~~~>;':t:~-.f:v};;'fi:_~;_;_::~;_:;;,i;;;,:t,:,).l~":,;.~.::~,-.;t~::.,;_.,,:..J·-** ;_,..t'J,\''"' .1**<·•""'~-** ..**~-"".""''1**......"'··"·-•-
..a· "-"• •-- •. • ,. . • ·. • .," 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 Wavenuml:ers Ccm-1) Figure 2 IR spectrum ofthe test substance (supplied from the sponsor)%T Report No. NCAS 10-221 Page 20 of 27

80

50
40

10
0.0+-··-···----- 4000.0

50
40
30
20
10
0.0 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 em-! Figure 3 IR spectrum ofthe test substance measured before the experiment

3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 cm-1 Figure 4 IR spectrum ofthe test substance measured afterthe experiment 600 400.0

600 400.0Report No. NCAS 

### 10-221



### 75-

BOD (mg) ."'"'



### 0



### 75

BOD (mg) Start Figure 5 BOD measuring chart (start- 7 days) Page 

### 21 of 

### 27



### 7 days

### 0~~--~~====~====~====~====~====~~==~== i!d1t 7 days 14 days Figure 6 BOD measuring chart (7 days- 14 days)BOD (mg)

BOD (mg) Report No. NCAS 10-221

14 days Figure 7 BOD measuring chart (14 days- 21 days)

21 days Figure 8 BOD measuring chart (21 days- 28 days) Page 22 of27

21 days



# t 28 daysReport No. NCAS 10-221 

### Compound name: Correlation coefficient: r 

### =**u.~~9624,** 

### r/\2 

### = 0.999247 Calibration curve: 538.749 * x 

### + 36.1851 Response type: External Std, Area Page 23 of 27



### Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None

2500 2000 (!) (/) c: 1500 0 a. (/) (!) 1000 0:::: 500 0

. I 

### ug/ml 0.00 1.00 2.00 3.00 4.00 5.00 Figure 9 Calibration curve ofthe LC/MS/MS measurement

Name: 1115-4, Date: 15-Nov-2010,Time: 16:29:50, Description: STD 0.03 ug/mL

1115-4 1001

## ~

%- MRM of2 channels,ES- 111o-4 MRM of2channels,ES

# 100l Ann**-::.~** ..... "77.84 102

% 

# j
'"I" " o' "'1 1 ' ' , 'I "' , , ' I mm 

# i 0

# I 'I'''' i
..

. ; . . 'tj\ ) J l j I ' , 'I··
.. 1min 6.00 2.00 4.00 6.00 2.00 4.00 

# I # N•mo Trace RT Area
forquantification for qualification ug/mL

Figure 10 LC/MS/MS chromatogram ofthe calibration solution at 0.03 

### 1-19/mlReport No. NCAS 10-221
Name: 1115-5, Date: 15-Nov-2010,Time: 16:37:54, Description: STD 1.0 ug/ml



## - 1115-5 100-

% MRM of 2 channels.ES- 1115-5 10 13 100l 

# l l**~/ol** ! ·i 

# i MRM of2 channels,ES77.84 +003

o·....._,_,..,..,.,...,..,..,,"'T"r";, ,..,..,.**.-+rl-rn..,........,.~"MT'l"r.",..,...,..,** .• min 2.00 0•......_,_,...,...,.-,...,..,._,..,..,..,...,..,,-,..,1"'! 1 ••

.•• 1 1• , 1 • , • 1 , 1, 1 1 1 1min 4.00 6.00 2.00 4.00 6.00 #Name Trace 

### RT Area

:for quantification, for qualification Page 24 of27

· ug/mL

Figure 11 LC/MS/MS chromatogram ofthe calibration solution at 1 lJg/mL

Name: 1115-6, Date: 15-Nov-2010,Time: 16:45:59, Description: STD 3.0 ug/ml

1115-6 100



# J MRMof2 channels,ES- :16.90 1+003**OI_,_,_,...,..,..,H'M...,..,...,...,..,...,...,..,..,..,.-,..,...,..,-,....,.....~** .•.1 , •, .**~** min 4.00 6.00 #Name Trace 1115-6

# ~ MRM of 2 channe!s,ES- > 77.84 '5e+004

o.-;..,1

# ,...,..,...,..,-.,-,.-r-rr.,..,..,...,...,...,...,..,...,-rr-r~ . , 1 .. - .• 1 min 2.00 4.00 6.00 

### RT Area
for quantification, for qualification ug/ml

Figure 12 LC/MS/MS chromatogram ofthe calibration solution at 3 !Jg/mLReport No. NCAS 10-221
Name: 1115-7, Date: 15-Nov-2010, Time: 16:54:05, Description: STD 5.0 ug/mL

111:>-f 100-

#Name MRM of2 channels,ESi.90 004

Trace 1115-7 100··· -I

%

### l



## 'i

0 i .

for quantification MRM of 2channels,ES33 > 77.84 .309e+004

.,. ,,,,,,,\,/,, ., .. ''1''''1''''1 min 2.00 4.00 6.00 RT Area
for qualification Page 25 of27

ug/mL 

# %R1**-~,** I Figure 13 LC/MS/MS chromatogram ofthe calibration solution at 51Jg/ml

Name: 1115-8, Date: 15-Nov-2010, Time: 17:02:12, Description: No.1_221WaterT

1115-8 100l

%

0
. I ' I·' 2.00 #Name MRM of2 channels,ES36.90 :+003 1115-8 100

% MRM of2channels,ES77.84 •+004

! ''' i ! 1 min 

### J...__,r-r-n.,..,.,.,...,..,--:-+:-..,..,.,,,rn-,.,...,-,...,...,..,..,..,.,...,-,. , min 4.00 6.00 2.00 4.00 6.00 Trace RT Area
for quantification, for qualification Figure 14 LC/MS/MS chromatogram ofthe test solution for the [Water+ Test Substance] System (Bottle No. 1) ug/mLReport No. NCAS 10-221 Name:**1115~9,** Date: 15-Nov-2010, Time: 17:10:16, Description: No.2_221-1T

1115-9 100l 

# l

% MRM of2 channels,ES- •96.90 )e+003 1115-9 100



# %l

# I MRM of2 channels,ES- .33 > 77.84 .735e+004


# j
0 11.1 1 r,. i 1**~.,** 1 ,: ,. i,.,,**~** ll l(if 11 t min 2.00 4.00 6.00 RT · Area
for quantification, forquallttcatton Figure 15 LC/MS/MS chromatogram ofthe test solution for the [Sludge+Test Substance] System (Bottle No. 2)
Name: 1115-10, Date: 15-Nov-2010,Time: 17:18:23, Description: No.3_221·2T

1115-10 100

%

# i MRMof 2 channels,ES-**-~6.90** 1'003 1115-10 MRM of2channels,ES- >77.84 9e+004

0-''..,..,...,...,...,. 1 -,... • .,...,,**..,...,..,...,-ri-r-~-r,.,.,...,-,-.,.,..,.,-,...,._,..,,..,.,..,..,..,** min 6.00 0•.........,...,..,.,..,...,1...,..',...,..,..,..,...,.-r.,.,..,..,...,..,...l,...,...,...,'..,...''"""· ,.,...,,

...,.-,!'To.,..,..,.., min 2.00 #Name 4.00 2.00 4.00 6.00 Trace RT Area
for quantification, for qualification Figure 16 LC/MS/MS chromatogram ofthe test solution for the [Sludge+Test Substance] System (Bottle No. 3) Page 26 of27

·· ugfinL · ·

ug/mLReport No. NCAS 10-221
Name: 1115-11, Date: 15-Nov-2010, Time: 17:26:27, Description: No.4_221-3T

1115-11 

# 1001 J 

# I 1**%~** MRM of2 channels,ES- ·3 > 96.90 523e+003 1115-11 100-,

% MRM of2 channels,ES- .33 > 77.84 .729e+004

Qi...J..,-.,..,.,.'1"'t'T..,..,..,.-;-r"!'T"M'T''.,, , 1..,,

.....,..,, •"''

,-,-,**~** min 

# j 0 ',.'I·
.. 'I'' ,... ',,, 

### ··•''''i''·'• min 2.00 #Name



### L. 4.00 6.00 2.00 4.00 6.00 Trace RT Area
forquantification, for qualification Figure 17 LC/MS/MS chromatogram ofthe test solution for the [Sludge+Test Substance] System (Bottle No.4)

0.8000
0.7000

0.0000
0.5000

0.3000
0.2000
0.1000 

### m:.& IJ!k;'it& (mg/L) (Abs) 0.08032 0.0308 0.2008 0.0766 0.4016 0.1522 0.8032 0.2941 1.205 0.4309 2.008 0.6801

y

### = 0.3373x+ 0.0133 R 2=0.9985

0.0000**'------~-----------------'** 0.00 0.50 1.00 1.50 iF.iffi'(mg/L) Figure 18 Calibration curve ofthe AA measurement Page 27 of 27

ug/ml

## Authenticity ofTranslation

I declare that the original Japanese final report (Report No. NCAS 10-221) is translated into English consistently.

Translated by : Kenji Miya Nisso ChemicalAnalysis Service Co., Ltd. Odawara Laboratory Date