Report No. NCAS 10-221 

Final Report 

Study Title 

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 1 of 27 
Report No. NCAS 10-221 Page 2 of 27 

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 consistently with 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 

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(98) 17, 1998] tZ::Vf:-:> ""(;%Jii)i; 1.-I'C.a 

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Report No. NCAS 10-221 Page 4 of 27 

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 of the 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 

·Receipt and maintenance of the activated 9/24/2010 9/24/2010 9/24/2010 sludge 

·Receipt and maintenance of the 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 of the test systems for BOD 9/21,24/2010 9/24/2010 9/24/2010 measurement 

·Set up of the enclosed respirometer and 

BOD measurement (1 51 trial) 9/21, 24/2010 9/24/2010 9/24/2010 

·Set up of the enclosed respirometer and 

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

· BOD measurement and 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 of the bottle 11/15, 16/2010 11/16/2010 11/16/2010 contents 

·Preparation of the standard solutions 11/15, 16/2010 11/16/2010 11/16/2010 (LC/MS/MS) 

·Preparation of the 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 11/26-30/201 0 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, 201 0) 

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 3 of25 

~Yl't;'!l:"% : NCAS 10·221 

J:~c~~vHiHrit!Eiii'JiEcr)~'J1f.;£ icl:i:'Th':~:i" i@Jf1 GLP .to J:: 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) 13 ft<::it>\!!i,bl(ff:ff·;fJ J:: G~)!J!E"ili'1!Jl~·;;:: 

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f';Jt~fl-Hffii~: 9/16/2010 9/16/2010 9/16/2010 

-:;xt~lltilliii!:Hl'k::il!Jffi 1 1017/2010 10/7/2010 10!7/2010 

1A~llti®i!ill£:!l!$2 11115/2010 11116/2010 11/16/2010 

~~illl!f"F 

• r&t1.:ll5t16(f)}\.-'F ~ ~{r:t!l! 9/24/2010 9/24/2010 9/24/2010 

• 1i!it1!'5rJt;(f) .A-¥~~::::-¥. 10/18, 19/2010 10/19/2010 10/19/2010 (!ifro;; 1-) 

• iiF~~~Il}.kJJlZ.A-":7 )-;VffiiJJE 9/17, 24/2010 9/24/2010 9/24/2010 

·BOD ~~1JE(f)1::10(f)l{;;j~fVf,J!)l1t 9/21, 2412010 9/24/2010 9/24/2010 

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

. llf1i~*~*¥l~!C?JiiJliJJE~fiT 

(f)fi9:JE ~ ffi!JJE!#lftil 10/18, 19/2010 10/19/2010 10/19/2010 

(f!j:;%~ 2@] !§) 

• BOD i!ltlZi:":iO J: V~~ 9/28/2010 9/28/2010 9/28/2010 

• BODiJliJJE:foJ::·amt~ 1017/2010 1017/2010 1017/2010 (W:~~) 

·BOD iUIJif: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&Jt 51-: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 of the Test Substance 

Confirmation of the Stability of the 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 

Experimental Start Date: September 17, 2010 

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. 

1121002 of MHLW, Manufacturing Industries Bureau No.2 of METI and 

Environment Policy Bureau No. 031121002 of MOE (Amended on 

Archiving: 

November 20, 2006), and OECD Guidelines for the Testing of Chemicals No. 

301C (1992). 

All the documents of this study will be retained for 10 years after the end of 

this study in the archives of the test facility. The management of the 

documents after that will be determined by mutual consultation between the 

Sponsor and Nisso Chemical Analysis Service Co., Ltd. at that time. The 

test substance will be returned after the end of this study. 

Deviation from SOPs and Study Plan: 

There was no deviation from SOPs and the study plan. 

Circumstance/matter that Affect the Reliability of the Test Result: 

There was no environmental agent which influenced the reliability of the 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 Quality Assurance 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 of the Test Substance ............................................................................... 10 

4. Stability of the Test Substance ...................................................................................... 10 

5. Reagents and Apparatus ............................................................................................... 10 

6. Experimental Methods ................................................................................................... 11 

6.1 Conditions of the Biodegradation Test .................................................................... 11 

6.2 BOD Measurement and Observation of the Bottle Contents ................................... 12 

6.3 pH Measurement .................................................................................................... 12 

6.4 Analytical Procedures after the End of the BOD Measurement ............................... 12 

6.5 LC/MS/MS Measurement ........................................................................................ 12 

6.6 AA Measurement .................................................................................................... 13 

6.7 Measurement of the Dissolved Organic Carbon ...................................................... 14 

Results and Discussion ............................................................................................................ 15 

1. Confirmation of the Test Substance ............................................................................... 15 

2. Confirmation of the Stability of the Test Substance ....................................................... 15 

3. Confirmation of the Degradation Activity of the Activated Sludge ................................... 15 

4. Observation Results of the Contents ............................................................................. 15 

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

Contents of Tables and Figures 

Table 1 Degradability of the test substance based on the BOD measurement ....................... 17 

Table 2 Observation results of the contents ............................................................................ 17 

Table 3 pH values of the test solutions ................................................................................... 17 

Table 4 Degradability of the test substance based on the LC/MS/MS measurement .............. 18 

Table 5 Degradability of the test substance based on the AA measurement .......................... 18 

Figure 1 Outline of the analytical procedure after the BOD measurement .............................. 19 

Figure 2 IR spectrum of the test substance (supplied from the sponsor) ................................ 19 

Figure 3 IR spectrum of the test substance measured before the experiment ........................ 20 

Figure 4 lR spectrum of the test substance measured after the 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 of the LC/MS/MS measurement .................................................... 23 

Figure 10 LC/MS/MS chromatogram of the calibration solution at 0.03 !Jg/ml. ....................... 23 

Figure 11 LC/MS/MS chromatogram of the calibration solution at 1 !Jg/ml. ............................ 24 

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

Figure 13 LC/MS/MS chromatogram of the calibration solution at 5 !Jg/ml.. ........................... 25 

Figure 14 LC/MS/MS chromatogram of the test solution for 

the [Water+ Test Substance] System (Bottle No.1) ............................................... 25 

Figure 15 LC/MS/MS chromatogram of the test solution for 

the [Sludge+ Test Substance] System (Bottle No. 2) ............................................. 26 

Figure 16 LC/MS/MS chromatogram of the test solution for 

the [Sludge+ Test Substance] System (Bottle No. 3) ............................................. 26 

Figure 17 LC/MS/MS chromatogram of the test solution for 

the [Sludge+ Test Substance] System (Bottle No. 4) ............................................. 27 

Figure 18 Calibration curve of the AA measurement. .............................................................. 27 
Report 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, 201 0). 

Introduction 

The biodegradation test of 

biodegradability. 

was performed to evaluate the 

1. Test Substance 

Name: 

Rational formula: 

Molecular formula: 

Molecular weight: 

CAS number: 

Lot No.: 

Purity: 

NCAS retrieval No.: 

Supplier: 

Received weight: 

Received date: 

Expiry date: 

Storage condition: 

Materials and Methods 

STD-1180-4 (Preparation of the test systems, Identification and 

stability of the test substance) 

STD-1180-6 (Preparation of the 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: 

Molecular formula: 

Chemical structure: 

Aniline 

C6H7N o-NH2 
Report No. NCAS 10-221 

Molecular weight: 

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 1 00% 

ANI-013 

Wako Pure Chemical Industries, Ltd. 

12-mL ampoule, 25 ampoules 

August 25, 2008 

August 25, 2011 

Stored in a freezer (about -20°C) 

Pale yellow and transparent liquid 

Page 10 of 27 

3. Confirmation of the 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 sponsor to confirm the identity. 

4. Stability of the Test Substance 

TheIR spectrum of the 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 of the experiment to 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: 

Membrane filter: 

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) 

10-100 f.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-31 OOA (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 of the 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 of the test substance. 

6.1 Conditions of the 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 of the 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 the water. 

Bottle No. 2-No. 4: [Sludge +Test substance] system 

Mineral medium· (297 .1 ml) was transferred into 3 bottles each and 

then 30 mg of the 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 of the mineral medium (ml) 

300 =Volume of the test solution (ml) 

Vs =Addition volume of the standard activated sludge (ml) 
Report No. NCAS 10-221 Page 12 of 27 

**) 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 of the 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 of the test solution (ml) 

6.2 BOD Measurement and Observation of the 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) I TOO} 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 of the test substance or aniline (mg) 

<Calculation of the 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 

Test substance: TOO was calculated by having assumed that 30 mg of the test 

substance was 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 of the test solutions was measured at the start and the end of the 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 of the exposure. At the end of the exposure, the pH was measured using the 

test solution which remained in the bottle after the treatment according to section 6.4. 

6.4 Analytical Procedures after the End of the 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 of the 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 (1 0.69 mg) of the test substance was dissolved in distilled water to make 100 

ml solution. 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 of the 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 of the calibration curve was good when the 

square of the correlation coefficient (R2

) was more than 0.99. 

6.5.3 Measurement of the Test Solutions 

The test solutions (bottle Nos. 1, 2, 3 and 4) were treated in accordance with section 

6.4 and 1.5 ml of each 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 of the Degradability 

The amount of the residual test substance was calculated from the concentration of the 

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 =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) 

6.5.6 Limit of Quantification 

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 of the 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 of air: 

Flow rate of acetylene: 

Wavelength: 

Slit width: 

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 of Calibration Curve 

Page 14 of 27 

A lithium standard solution (1 004 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 of the test substance to 

confirm the linearity. It was considered that the linearity of the calibration curve was 

good when the square of the correlation coefficient (R2

) was more than 0.99. 

6.6.3 Measurement of the Test Solutions 

The test solutions (bottle Nos. 1, 2, 3 and 4) were treated in accordance with section 

6.4. Aliquots of the 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 of the 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 =Amount of the residual lithium in the [Sludge+ Test substance] system 

(mg) 

C9 =Amount of the residual lithium in the [Water+ Test substance] system 

(mg) 

6. 7 Measurement of the 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 of the 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 I R spectra 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 3 was obtained. Therefore, no difference was recognized on the property of the 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 of the Stability of the Test Substance 

Figure 4 shows the IR spectrum of the test substance measured after the experiment. 

The IR spectra obtained before and after the 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 of the Degradation Activity of the Activated Sludge 

Table 1 ([Sludge+ Aniline] system) shows the results of the 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.: 201 006R, received 

date: July 15, 2010) was tested before using the activated sludge of this lot. However, BOD 

degradability was less than 40% after 7 days. Thus, it was considered that this test was 

invalid. 

4. Observation Results of the Contents 

Table 2 shows the observation results of the contents. The test solutions of the [Sludge + 

Test substance] system (Bottle Nos. 2- 4) and the [Sludge blank] system (Bottle No. 6) were 

slightly muddy white. The test solution of the [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 of the 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 of each 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 of the 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 of the 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 degradability was calculated to be 2%. The difference 

of the degradation rates between maximum and minimum was less than 20% so the validity 

criterion of the 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 of the 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 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 degradability was calculated as 

0%. The difference of the degradation rates between maximum and minimum was less than 

20% so the validity criterion of the biodegradation test was fulfilled. 

No unknown peak which had larger area than the LOQ was observed on the LC/MS/MS 

chromatogram of the [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 of the 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 

of the [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 of the 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 for the Designation of Monitoring Chemical Substances, April 12, 201 0). 
Report No. NCAS 10-221 Page 17 of 27 

T a e bl 1 D e Jra d b a 11ry Tt o f th e t t b t es su s ance b ase d on th e BOD 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) I TOO} 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 of the 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 muddy white muddy white muddy white muddy white 

Sludge+ slightly slightly slightly slightly 3 Test None None None None 

substance muddy white muddy white muddy white muddy white 

Sludge+ slightly slightly slightly slightly 4 Test None None None None 

substance muddy white muddy white muddy white muddy white 

5 Sludge+ observed muddy white observed muddy white observed muddy white observed muddy white Aniline 

6 Sludge None slightly None slightly None slightly None slightly 

blank muddy white muddy white muddy white muddy white 

T a e bl 3 H p va ues o f th e t es t sou I f tons 

pH 

Bottle No. Sample at the start 

at the 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 18 of 27 

T a e bl 4 0 egra d b a 11ry Tt o f th e t t es su b s t ance b d ase on th e LC/MS/MS measuremen t 

Bottle Concentration (mg/L) Residual Degradability (%) 

Sample Peak Area 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 a bl e 5 0 egra d a bTt tny o e f th t es t su b t s ance b d ase on th e PV\ measuremen t 

Concentration of Li 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 of the residual lithium in the [Sludge+ Test substance] system (mg) 

C8 : Amount of the residual lithium in the [Water+ Test substance] system (mg) 

Mean 

-

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

Test solution (300 ml) 

I 

Take 1 00 m L of the test solution 

I 

Centrifuge (at 4000G for 15 minutes) 

I 

Take the supernatant 

I 

LC/MS/MS measurement M measurement 

Figure 1 Outline of the analytical procedure after the 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 1 400 1 200 1 000 800 

Wavenuml:ers Ccm-1) 

Figure 2 IR spectrum of the 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 I R spectrum of the test substance measured before the experiment 

3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 cm-1 

Figure 4 IR spectrum of the test substance measured after the experiment 

600 400.0 

600 400.0 
Report 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 of 27 

21 days 

t 

28 days 
Report 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. 

(/) 

(!) 

0:::: 1000 

500 

0 . I ug/ml 

0.00 1.00 2.00 3.00 4.00 5.00 

Figure 9 Calibration curve of the 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 of 2 channels,ES- 111 o-4 MRM of 2 channels,ES100l 

Ann -::.~ ..... "77 .84 

102 

% 

j '"I" " o' "'11 ' ' , 'I "' , , ' I mm 

i 

0 I 'I'''' i .. . ; . . 'tj \ ) J l j I ' , 'I·· .. 1 min 

2.00 4.00 6.00 2.00 4.00 6.00 

I # N•mo Trace RT Area 

for quantification for qualification 

ug/mL 

Figure 10 LC/MS/MS chromatogram of the calibration solution at 0.03 1-19/ml 
Report 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 of 2 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 1 min 

4.00 6.00 2.00 4.00 6.00 

#Name Trace RT Area 

: for quantification, for qualification 

Page 24 of 27 

· ug/mL 

Figure 11 LC/MS/MS chromatogram of the 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 

MRM of 2 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 of the calibration solution at 3 !Jg/mL 
Report 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 of 2 channels,ESi.90 

004 

Trace 

1115-7 

100··· 

-I 

% 

l 'i 

0 i . 

for quantification 

MRM of 2 channels,ES33 > 77.84 

.309e+004 

. ,. ,,,,,,,\,/,, ., .. ''1''''1''''1 min 

2.00 4.00 6.00 

RT Area 

for qualification 

Page 25 of 27 

ug/mL %R1 -~, 

I 

Figure 13 LC/MS/MS chromatogram of the calibration solution at 51Jg/ml 

Name: 1115-8, Date: 15-Nov-2010, Time: 17:02:12, Description: No.1_221Water T 

1115-8 

100l 

% 

0 . I ' I·' 

2.00 

#Name 

MRM of 2 channels,ES36.90 

:+003 

1115-8 

100 

% 

MRM of 2 channels,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 of the test solution for 

the [Water+ Test Substance] System (Bottle No. 1) 

ug/mL 
Report 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 of 2 channels,ES-

• 96.90 

)e+003 

1115-9 

100 

%l 

I 

MRM of 2 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, for quallttcatton 

Figure 15 LC/MS/MS chromatogram of the 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 

MRM of 2 channels,ES-

-~6.90 

1'003 

1115-10 MRM of 2 channels,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 of the test solution for 

the [Sludge+ Test Substance] System (Bottle No. 3) 

Page 26 of 27 

·· ugfinL · · 

ug/mL 
Report 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 of 2 channels,ES-

·3 > 96.90 

523e+003 

1115-11 

100-, 

% 

MRM of 2 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 

for quantification, for qualification 

Figure 17 LC/MS/MS chromatogram of the 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 

R2 = 0.9985 

0.0000 '------~-----------------' 

0.00 0.50 1.00 1.50 

iF.iffi'(mg/L) 

Figure 18 Calibration curve of the AA measurement 

Page 27 of 27 

ug/ml 
Authenticity of Translation 

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

English consistently. 

Translated by : 

Kenji Miya 

Nisso Chemical Analysis Service Co., Ltd. 

Odawara Laboratory 

Date