Sludge t lesf item -1 

Study No . 14019 

a--1 

Operating date : Dec. 12, 2002 

PKNO Time Area Height Conc. 

(min) (~uV " sec) ( ;uV) ( o) 

1 12. 56 1671930 14814 100. 00 

Total 1671930 100,00 

0.0 5.0 10.0 15 .0 20.0(min) Operator : Kaoru Kondo 

Sludge f test item -2 Operating date : Dec. 12, 2002 

PKNO Time Area Height Conc. 

(min) (UV-sec) (,u V) (?/.) 

1 12. .57 1624782 14144 100. 00 

Total 1624782 100.00 

0. 0 5.0 10.0 15.0 20 .0(min) Operator : Kaoru Kondo 

Control blank 

Peak position 

n. d. 

Operating date : Dec. 12, 2002 

PKNO Time Area Height Conc. 

(min) (uV~sec) (uV) (V-) 

Total 0 0. 00 

0.0 5 .0 10.0 15.0 20.0(min) Operator : Kaoru Kondo 

Fig. 3 - 2 Chromatogram of HPLC analysis for recoyery test . 

Date : Dec. 12, 2002 Name 
Study No. 14Q19 

Standard solution 30 .0 mg/L 

Total 1582026 100.00 

, 

0.0 5 . 0 10. 0 15 . 0 20. 

0(min) Operator, : Kaoru Kondo 

[11 Wafer + test item Operating date : Feb. 19, 2003 

PKNO Time Area Height Conc. 

1 (min) (uV " sec) ()tV) (ib) 

1 12 . 57 1528540 13524 100.00 

Total 1528540 100.00 

0.0 5. 0 10 .0 15.0 20. 0(min) Operator : Kaoru Kondo 

Operating date : Feb. 19, 2003 

PKNO Time Area Height Conc. 

(min) (llV " sec) ( ;tV) (?/.) 

1 12. 56 1582026 14347 100. 00 

PKNO Time Area Height Conc. 

(min) (uV " sec) (uV) 0/) 

1 12 .55 1522509 13244 100. 00 

Total 1522509 100.00 

Chromatogram of HPLC analysis for test solution . 

Date : Feb. 20, 2003 Name : , 

~a .rj 

000202 
Study No. 14019 P" 3 

( 3 ) Sludge + test item Operating date : Feb. 19, 2003 

PKNO Time Area Height Conc. 

(min) (ltV " sec) (UV) (%) 

1 12 . 55 1498059 13032 100.00 

Total 1498059 100.00 

0. 0 5. 0 10.0 15.0 20, 0(min) Operator : Kaoru Kondo 

f 5 ] Control blank 

Peak position 

n. d. 

Operating date : Feb. 19, 2003 

PKNO Time Area Height Conc. 

(min) (uV " sec) UtV) (%) 

Total 0 C . 00 

0.0 5 .0 10.0 15.0 20.0(min) Operator : Kaoru Kondo 

Fig . 4 - 2 Chromatogram of HPLC analysis for test solution . 

Date : Feb. 20, 2003 Name : 
MEASURING MODE ; ABS 

SAMPLING PITCH(Delta ~) ; AUTO(0 .5 nm) 

SCAN SPEED ; P~IIDDLE 

SLIT !~IIDTH ; 2 .0 nm 

SAMPLE NAME ; Test item (14019) 

ANALYST ; ~( . XLn,.Elo 

COMMENT ; St d,1e .Unig/L 

--------------------------------------------------------------------------- 

° o 0 0 ° QD o 0 0 

O 1= i 

- I 

p. 4 

0 0 

_o ° 

O 

I 

I I ' i 

i 

I . 

O 

O 

O 

M 

O 

O 

O 

O 

M 

O 

O 

O 

CV 

O 

CD 

m 

O 

CV 

I I I ! > . I + N0, ABSCISSA PEAK 

C ] 

O 

O 

I 

O 

O 

O O 

O 

O 

E 

O C 

Fig. 5 UV spectrum of test item. (9-VIS 

Study No . G 1 Date z0oz, It, / q Instrument _ Shu V-.zzo04 

Sample 7e5-t~ ~ tem Slit(Spectral Band)Width n,*n 

Solvent 7-e f/'CL l't

,

un'l-Di UYGL!'1 Scan Speed _ M i (,(~~( ( e 

Reference Sampling Pitch ,~nryr (A (UT 0 ) 

Photometric Mode Abs Note. U V absorption soectrum 

Cell lOmm x 10mm(Quartz) 

Wavelength / q o - ,j¢Q rIm 000 .0 

Scale Limit 0, g' . X - 1~~Lzzl 6, 

O 

O 

Qi 

O 

O 

O 

Gr 

O 

O 

O 

O 

M 

O 

HEIGHT ABSCISSA VALLEY HEIGHT 
100.0 -

%T 

80 .0 -i 

60 .0 - 

40.0 

20 .0 --4-- --- - ---~ --- - - ~ - ~ -- 

/ 

0.0 ' 

4000 .0 3000.0 2000.0 1500 .0 

L 

~y 

1000 .0 500.0 

1 /cm 

F i g . s-1 I R spectrum of test item measured before 

experimental start. 

Study No . : 14019 

Sample : Test item 

Method : KBr tablet 

Date : November 19, 2002 

Name : X . }Co,,,." 

1 

000205 
100 .0 -= 

i 

%T 

90 .0 -~r80 .0 - 

70.0 -i 

60.0 - 

50 .0 -F 

40 .0 --, 

' 

4000 .0 3000 .0 2000.0 1500 .0 

i ~ -- 

1000 .0 

Fig.s-2 IR spectrum of test item measured after 

experimental completion . 

Study No . : 14019 

Sample : Test item 

Method : KBr tablet 

Date : February 21, 2003 

Name : 

I 

i 

500.0 

1 /cm 

oooZos 
60 .0 -r 

%T - 

40.0 - 

20 .0 -, 

0 .0 - 

4000 .0 3500.0 3000 .0 2500.0 2000.0 1750.0 1500.0 1250.0 1000 .0 750.0 500.0 GPH821RS : GPH82 Lot No. G-6182 1/c m E11: 2002/11/01 P1l : 16:02:16 r) It: 40 T -9(31`nIl : HYPER 1R 1-q'-: g*~t: ;MI-t°=.jf Ift ;: standard 41l9M 1/cm 3VF!<f."A': %T 7V9~1x~PdI'1', : Happ wZIN}M: 401 .17 F1* ;g('j,¢ ; 3998 .18 MM®: 1/crn -T-6z A $L ; 1866 7--9r.M: 1 .92868 3j/ ; n; 4.0 auto 7/1-ft-: auto' E7-A 1: 2.8(10w) 

Reference IR spectrum supplied by sponsor 
p. 8 

~~"Iuu~yL~f~~-~- 

'~~ ~-Jo) 

70 

%7n~' .` 

FQl ~ L j 

-T-A 1 2 * 1 1 A 1 7 Q 

ggkj~ Q 
Accepted number I S02-4020 

Study number 1 44020 

FINAL REPORT 

Bioconcentration study of[ Jin carp 

July 31, 2003 

Kurume Laboratory 

Chemicals Evaluation and Research Institute, Japan 
STATEMENT 

Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor 1 

Title Bioconcentration study of F j in carp 

Study number 44020 

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

of the Final Report (Study No.44020, issued on July 31, 2003). 

The Study Director was changed from Yuka Koga to Naoaki Yakata, because Yuka 

Koga is absent from our laboratory now on maternity leave. 

Date 

Study Director 

, Se.~~rt~~~~c~~~ 

Naoaki Yaka.ta 

000210 
GLP S TATEMENT 

Kurume Laboratory 

' Chemicals Evaluation and 

Research Institute, Japan 

Sponsor 

Title Bioconcentration study of L ]in carp 

Study number 44020 

This study was performed in compliance with Good Laboratory Practice Standards for 

industrial chemicals, "Standard Concerning Testing Facility Provided in Article 4 of 

Ministerial ordinance Prescribing Test Items Relating to New Chemical Substances and 

Toxicity Research of Designated Chemical Substances" (March 31, 1984, Revised March 1, 

2000, No.39, Planning and Coordination Bureau, Environment Agency; No.229, 

Pharmaceutical Affairs Bureau, Ministry of Health and Welfare; and No.85, Basic Industries 

Bureau, Ministry of International Trade and Industry, Japan) and "OECD Principles of Good 

Laboratory Practice" (November 26, 1997) . 

It has been confirmed that this final report reflects the raw data accurately and the test 

data are valid. 

Date July 31, 2003 

Study Director Signed in original 

Yuka Koga 

00023.1. 
GLP STATEMENT 

Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor ) 

Title Bioconcentration study of( j in carp 

Study number 44020 

This study was performed in compliance with Good Laboratory Practice Standards for 

industrial chemicals, "Standard Concerning Testing Facility Provided in Article 4 of 

Ministerial ordinance Prescribing Test Items Relating to New Chemical Substances and 

Toxicity Research of Designated Chemical Substances" (March 31, 1984, Revised March 1, 

2000, No.39, Planning and Coordination Bureau, Environment Agency; No.229, 

Pharmaceutical Affairs Bureau, Ministry of Health and Welfare; and No.85, Basic Industries 

Bureau, Ministry of International Trade and Industry, Japan) and "OECD Principles of Good 

Laboratory Practice" (November 26, 1997). 

This GLP statement was issued as supplement to the statement issued on July 31, 2003 

because of the amendment of the Final Report. 

Date August 7, 2003 

Study Director Signed in original 

Naoaki Yakata 

000212 
QUALITY ASSURANCE STATEMENT 

Kururne Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor E -j 

Title Bioconcentration study of F I in carp 

Study number 44020 

The inspections of this study were carried out and the results were reported to the test 

facility management and the Study Director by Quality Assurance Unit of Kurume Laboratory, 

Chemicals Evaluation and Research Institute, Japan as follows. 

Item of inspection Date of inspection Date of report to 

Study Director 

Date of report to test 

facility management 

May 1, 2003 May 1, 2003 May 1, 2003 

Study plan 

May 15, 2003 May 16, 2003 May 16, 2003 

May 13, 2003 May 27, 2003 May 27, 2003 

May 20, 2003 May 27, 2003 May 27, 2003 

May 26, 2003 May 27, 2003 May 27, 2003 

Test Conduct 

May 27, 2003 May 27, 2003 May 27, 2003 

June 3, 2003 June 25, 2003 June 25, 2003 

June 24, 2003 June 25, 2003 June 25, 2003 

Raw Data and 

Final Report July 31, 2003 July 31, 2003 July 31, 2003 

It has been assured that the final report describes accurately the test method used, that 

details in the report are in compliance with the study plan and Standard Operating Procedures 

and that the final report reflects the raw data accurately . 

Date July 31, 2003 

Quality Assurance Unit, Head Signed in original 

Kyoshiro Hori 

19 

000213 
QUALITY ASSURANCE STATEMENT 

Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Sponsor 

Title Bioconcentration study of I ]in carp 

Study number 44020 

Study inspection of the corrected parts in the Final Report was carried out and it was 

confirmed that the correction has no problem. The result was reported to the management 

and study director as follows. 

Date of inspection Date of report 

to study director 

Date of report 

to management 

August 7, 2003 August 7, 2003 August 7, 2003 

This statement was issued as a supplement to the quality assurance statement issued on 

July 31, 2003 . 

Date August 7, 2003 

Quality Assurance Unit, Head Signed in original 

Kyoshiro Hori 

e Cnh 000214 
CONTENTS 

Title 

Sponsor 

Test facility 

Objective 

Test method 

Applied GLP 

Dates 

Storage of test item and raw data 

Personnel 

Approval of final report 

SUIvIMARY 

1 . Test item 

2. Item supplied by sponsor 

3 . Performance of acute toxicity test 

4. Performance of bioconcentration test 

5. Factors possibly affecting accuracy 

6. Results 

7. Remarks 
Contents of tables and figures 

Contents of tables 

Table-1 

Table-2 

Table-3 

Table-4 

Table-5 

Table-6 

Table-7 

Table-8 

Table-9 

Table-10 

Measured concentrations of test item in test water (see text) 

BCFs (see text) 

Concentrations of the test item in test water at a steady-state (see text) 

Calculation table for recovery and blank test (analysis of test water) 

Calculation table for analysis of test water (Level 1) 

Calculation table for analysis of test water (Level 2) 

Calculation table for recovery and blank test (analysis of test fish) 

Calculation table for analysis of test fish (Level 1) 

Calculation table for analysis of test fish (Level 2) 

Calculation table for analysis of test fish (Control) 

Reference 1 Analytical results of dilution water 

0002JL6 
Contents of figures 

Fig. 1 

Fig. 2 

Correlation between exposure period and bioconcentration factor 

(Level 1) 

Correlation between exposure period and bioconcentration factor 

(Level 2) 

Fig. 3 Concentration-mortality curve 

Fig. 4 

Fig. 5 

Fig. 6 

Fig. 7 

Fig. 8 

Fig. 9 

Fig.10 

Fig. 11 

Fig. 12-1 

Fig. 12-2 

Chromatogram and calibration curve of HPLC analysis 

Chromatogram of HPLC analysis for recovery and blank test 

(analysis of test water) . Chromatogram of HPLC analysis for analysis of test water 

Chromatogram of HPLC analysis for recovery and blank test 

(analysis of test fish) 

Chromatogram of HPLC analysis for test fish (Level 1) 

Chromatogram of HPLC analysis for test fish (Level 2) 

Chromatogram of HPLC analysis for test fish (Control) 

Fluorescence spectrum of test item 

IR spectrum of test item measured before experimental start 

IR spectrum of test item measured after experimental completion 

Reference 2 IR spectrum supplied by the sponsor 

11212 000217 
Study No. 44020 

Title 

Bioconcentration study of E Jin carp 

Sponsor 

C 

Test facility 

Kurume Laboratory 

Chemicals Evaluation and Research Institute, Japan 

19-14 Chuo-machi, Kurume-shi, Fukuoka. 830-0023, Japan 

Objective 

This study was performed to evaluate the bioconcentration potential ofE- ]in carp. 

Test method 

This study was performed according to the "Method for Testing the Degree of 

Accumulation of Chemical Substances in Fish Body" stipulated in the "Testing Methods for 

New Chemical Substances" (July 13, 1974, Revised October 8, 1998, No.5, Planning and 

Coordination Bureau, Environment Agency; No.615, Pharmaceutical Affairs Bureau, 

Ministry of Health and Welfare; and No.392, Basic Industries Bureau, Ministry of 

International Trade and Industry, Japan), and "Bioconcentration : Flow-through Fish Test 

(Guideline 305, June 14,1996)" in the OECD Guidelines for Testing of Chemicals. 

r. .7

000218 
Applied GLP 

4+uzu 

p. 19 

(1) Chemical GLP 

This study complied with "Standard Concerning Testing Facility Provided in 

Article 4 of Ministerial ordinance Prescribing Test Items Relating to New Chemical 

Substances and Toxicity Research of Designated Chemical Substances " (March 31, 

1984, Revised March 1, 2000, No .39, Planning and Coordination Bureau, 

Environment Agency; No.229, Pharmaceutical Affairs Bureau, Ministry of Health 

and Welfare; and No.85, Basic Industries Bureau, Ministry of International Trade and 

Industry, Japan) . 

(2) OECD-GLP 

This study complied with "OECD Principles of Good Laboratory Practice" 

(November 26, 1997). 

Dates 

Study initiation date May l, 2003 

Experimental starting date May 23, 2003 

Experimental completion date July 22, 2003 

Study completion date July 31, 2003 

Storage of test item and raw data 

(1) Test item _ 

About 5 g of the item supplied by the sponsor is sealed in a store vessel and 

stored in archives in this laboratory for ten years after the receipt of notice that the 

test item comes under No.l, No.2 or No.3 in Clause 1, Article 4 of "Law Concerning 

Examination and Regulation of Manufacture, etc. of Chemical Substances". 

Treatment of the item supplied by the sponsor after the storage period is discussed 

with sponsor: If it is not stable for the storage period, it is stored while it is kept 

stable and it is disposed with approval of sponsor. 

(2) Raw data and materials, etc. 

Raw data, the study plan, documents about the study presented by the sponsor, 

the final report and necessary materials are stored in archives in this laboratory for 

the same term as the test item. Treatment of raw data and materials, etc. after the 

storage period is discussed with sponsor. 

000219 
Personnel 

Study Director Yuka Koga 

Study personnel Yuka Koga 

(Operation of bioconcentration test) Yoshiyuki Inoue 

Naoaki Yakata 

Yasuro Kawashima 

Chitose Fukuda 

Staff for fish care Yasuro Kawashima 

Person to conduct of fish acute toxicity test Yasuro Kawashima 

Tadayoshi Tonai 

Approval of final report 

Study Director Date July 31, 2003 

Signature Signed in original 

Yuka Koga 
SUMMARY 

Title 

Bioconcentration study of [ _I in carp 

Test conditions 

Acute toxicity test 

(1) Test fish Orange-red killifish (Oryzias latipes) 

(2) Duration of exposure 96 hours 

(3) Exposure method Semi static system 

(Renewal of test water, at every 8 -16 hours) 

Bioconcentration test 

(1) Test fish Carp (Cyprinus carpio) 

(2) Nominal concentrations of test item 

High exposure level (Level 1) 20 ~ig/L 

Low exposure level (Level 2) 2 ~ig/L 

(3) Duration of exposure 60 days 

(4) Exposure method Continuous flow system 

(5) Analytical method High-performance liquid chromatography 

Results 

(1) 96-hour LC50 value 28.7 mg/L 

(2) Bioconcentration factors at a steady state 

Peak 1 Level 1 Not calculated 

Level 2 Not calculated 

Peak 2 Level 1 57 

Level 2 110 

(3) Bioconcentration factors 

Peak 1 Level 1 c 0.49 

Level 2 c 4.8 

00022JL 
1 . Test item 

In this report,[ _] has the following chemical name, etc. 

1 .1 Chemical name`1 

1 .2 Chemical structure, etc. `1 

Structural formula 

Molecular weight L .J 

* 1 Information supplied by the sponsor 
4020

2. Item supplied by sponsor

2.1 Supplier and lot number# ~

(1) Supplier ~

(2) Lot number 0-6182

2.2 Purity*'

(1) Test item 99.9 wt%

(2) Impurity Water 0 .1 wt%

The test item was treated as 100 % in purity.

2.3 Confirmation of test item

Two infrared (IR) spectra of the test item provided by the sponsor and measured at

this laboratory were confirmed to be identical (see Fig. 12 and Reference 2).

2 .4 Physicochemical properties* '

Appearance Tawny solid

Melting point 48 °C (DSC)

Solubility Water 1 mg/L

Dimethylsulfoxide _> 10 %

Acetone 10 %

Chloroform 10 %

* 1 Information supplied by the sponsor

2.5 Storage and stability

(1) Storage condition

A dark storage place at room temperature

(2) Stability

The test item was stable under the storage condition as shown by the finding

that IR spectra of the test item before and after the experiment were identical (see

Fig. 12).

2.6 Stability under testing conditions

Prior to the bioconcentration test, a stability of the test item under the testing

conditions was confirmed by a preliminary test.

rT, °

0002~.3

-6-
3. Performance of acute toxicity test 

3.1 Test method 

The test was performed in accordance with Japanese Industrial Standard (JIS K 

0102-1998-71 .), "Testing methods for industrial waste water, Acute toxicity test with 

fish". 

3.2 Test fish 

(1) Species Orange-red killifish (Oryzias latipes) 

Reason for selection : This species is similar in sensitivity to carp and 

readily available as test fish. 

(2) Supplier Nakashima fish farm . 

(Address : 2029 Ooaza Nagasu Nagasu-cho Tamana-gun, Kumamoto 

869-0123, Japan) 

(3) Conditions for fish care before acclimatization 

Period 

The fish were checked visually at receipt and those with any abnormalities 

were removed. The fish were reared for 56 days in a flow-through system 

following an external disinfection for sick prevention and parasitic extermination. 

External disinfection 

The first external disinfection for sick prevention was carried out in an aqueous 

solution containing 20 mg/L ELBAZIU and 7 g/L sodium chloride for 24 hours. 

The second external disinfection for parasitic extenrunation was carried out two 

times in an aqueous solution containing 30 ~tLIL formalin for 24 hours . 

(4) Conditions of acclimatization 

Period 

After rearing, the fish were transferred to an acclimatizing aquarium and 

acclimatized there after the second external disinfection . The fish showing any 

abnormalities during this period were removed and the remainder of the fish were 

reared for 8 days in a flow-through system at the temperature of 25 f 2 °C. The 

fish were checked for health conditions and reared for 23 days or 29 days*Z after the 

external disinfection. 

External disinfection 

The first external disinfection was carried out in an aqueous solution containing 

20 mg/L ELBAZI[1 and 7 g/L sodium chloride for 24 hours, followed by the second 

external disinfection in the same manner after abnormal fish were removed. 

0 oo .z4 
44U1U p.24 

(5) Weight average 0.22 g, 0.19 g"2 

(6) Length average 2.9 cm, 2 .8 cm`2 

(7) Certification 

The 48-hour LC50 value of the reference substance'3 for the fish of the same 

lot (TFO-030408) was 0.919 mg/L. 

*2 Since mortality test fish was confirmed in the test concentration (70 .0 mg/L), 

therefore the test concentrations (4.38 - 35.0 mg/L) as an additional test 

conditions were carried out. 

*3 PCP-Na (pentaclhlorophenol sodium salt, Tokyo Kasei Kogyo Co., Ltd.) 

3.3 Dilution water for test 

(1) Origin 

Groundwater from the premises of Kunune Laboratory . 

(2) Water quality assessment 

The dilution water for test was taken out on May 8, 2003, and it was analyzed 

and measured (once every six months in this laboratory). The results are shown in 

Reference 1 . 

It was confirmed that the dilution water met the ministerial ordinance of the 

Ministry of Health and Welfare (December 21, 1992), water quality criteria for 

fisheries (Shadanhozin Nihon Suisansigen Hogokyokai, March 1983), OECD 

Guidelines for Testing of Chemicals, "Fish, Early-life Stage Toxicity Test" 

(Guideline 210, July 17, 1992) and environmental quality standards for water 

pollutants No. 14 (Revised February 22, 1999, Environment Agency) or OECD 

Guidelines for Testing of Chemicals, "Bioconcentration: Flow-through Fish Test 

(Guideline 305, June 14,1996)". 

3 .4 Test conditions 

(1) Test tank Round glass vessel 

(2) Volume of test water 4 L / level 

(3) Temperature of test water 

At initial exposure 24.5 °C 

Before renewal of test water 24 .8 - 25.0 °C 

.,, 

000225 
(4) Concentration of dissolved oxygen in test water 

At initial exposure 8 .1 mg/L 

Before renewal of test water 6 .7 - 7 .3 mg/l . 

(5) pH of test water 

At initial exposure 8 .0-8.2 

Before renewal of test water 7.8-8.1 

(6) Number of fish 10 l level 

(7) Duration of exposure 96 hours 

(8) Exposure method Semi static system 

(Renewal of test water, at every 8-16 hours) 

3.5 Preparation of stock solution 

(1) Dispersants 

Dimethylsulfoxide 

HCO-40 (Hydrogenated castor oil) 

(2) Preparation 

The test item and HCO-40 (10 times amount of the test item) were dissolved 

in dimethylsulfoxide to prepare 20 g/'L solution. The solution was diluted with 

ion-exchanged water to prepare 1000 mglL stock solution. 

3.6 Performance of test 

(1) Place 214 LC50 room 

(2) Date May 12, 2003 - May 22, 2003 

3 .7 Estimation of 96-hour LC50 value 

The 96-hour LC50 value was estimated by the Doudoroff method. 

3 .8 Result of test 

96-hour LC50 value 28.7 mg/L (see Fig. 3) 

. . . .a~~, 00022G 
p. 26 

4. Performance of bioconcentration test 

4.1 Test fish 

(1) Species Carp (Cyprinus carpio) 

Reason for selection : The previous data conducted with this species 

can be compared and the size of this species is 

adequate for handling. 

(2) Supplier Kurume Laboratory Chemicals Evaluation and Research Institute, Japan 

(Address : 19-14 Chuo-machi, Kurume-shi, Fukuoka 830-0023, 

Japan) 

Date of hatch February 24, 2003 

Starting date of acclimatization March 29, 2003 

(3) Conditions for acclimatization 

Period 

After rearing, the fish were transferred to an acclimatizing aquarium and 

acclimatized there after the second external disinfection . The fish showing any 

abnormalities during this period were removed and the remainder of the fish were 

reared for 18 days in a flow-through system at the temperature of 25 f 2 °C. 

The test fish were acclimatized in the well water for 35 days after the external 

disinfection . 

External disinfection 

The external disinfection in the acclimatizing aquarium was carried out in an 

aqueous solution containing 20 mg/L ELBAZIU and 7 g/L sodium chloride for 24 

hours. The external disinfection in the test tanks was carried out in an aqueous 

solution containing 20 mg/L ELBAZIU and 7 g/L sodium chloride for 24 hours. 

(4) Length 6.3 - 10.6 cm 

(5) Lot No. TFC-030224 

(6) Age Yearling fish 

(7) Feeding 

Feed Feed for fry of carp 

Composition Proteins content ? 43 .0 % 

Lipid content >_- 3 .0 % 

Manufacturer Nippon Formula Feed Mfg. Co., Ltd. 

Feeding amount and interval 

Amount corresponding to about 2 % of total body weight was fed twice a 

day in halves . 

The fish were starved for 24 hours before sampling. 

00022'7 

-10- 
p. 27 

4.2 Dilution water for test 

The same as described in Section 3 .3 . 

4.3 Conditions of test and circumstances 

(1) Supply of test water 

Flow-through system assembled at this laboratory was used. 

(2) Test tank 

100-L glass tank 

(3) Flow rate of test water 

At 2 mL/min for stock solution and 800 mL/min for dilution water; 

1155 L/day of test water were supplied. 

(4) Stock solution bottle 25-L glass bottle 

(Frequency of renewal 1 - 2 times/week) 

(5) Temperature of test water 

Level 1 25 .2 - 25 .5 °C 

Level 2 25 .3 - 25 .6 °C 

Control 25 .0 - 25 .4 °C 

(6) Concentrations of dissolved oxygen in test water 

Level 1 7.7 - 8 .1 mg/l, 

Level 2 7.9 - 8 .1 mg/L 

Control 7.8 - 8 .1 mg/L 

(7) pH of test water 

Level 1 8 .0-8 .2 

Level 2 8 .0-8 .2 

Control 8 .0-8 .2 

(8) Time of irradiation with light 

Artificial light of white fluorescent lamp (14 hours/day) 

(9) Number of fish (at the beginning of exposure) 

Level 1 and 2 29 

Control 12 

_11- 0002ti8 
(10) Duration of exposure 60 days 

Reason : A steady-state has been reached after 60 

days. 

(11) Place 213 Aquatron room 

4.4 Preparation of stock solutions 

(1) Dispersant 

The same as described in Section 3 .5 (1) . 

(2) Preparation 

" Level 1 

20 gfL solution was prepared in the same way as described in Section 3 .5 (2). 

The solution was diluted with ion-exchanged water to prepare 8 mglL stock 

solution. 

" Level 2 

20 g/L solution was prepared in the same way as described in Section 3.5 (2). 

The solution was diluted with ion-exchanged water to prepare 0.8 mg/L stock 

solution. 

" Control 

HCO-40 were dissolved in dimethylsulfoxide to prepare 200 g/L solution. 

The solution was diluted with ion-exchanged water to prepare 80 mg/L stock 

solution . 

"z` 

-, ->-b 

4%ffl 911 - 

-12- 0 002 .9 
44U2U 

p. 29 

4.5 Test concentrations 

Based on preliminary test results for the 96-hour LC50 value and analytical 

detection limits, test concentrations of the test item were decided as follows . The 

control was set as a blank test . 

Level 1 20 [tg/L 

Level 2 2 ~Lg/L 

4.6 Observation, measurement and cleaning of test tank 

(1) Observation of test fish 

Condition of test fish was observed visually twice a day. 

(2) Flow rate of test water 

Flow rate of stock solution and dilution water were measured with graduated 

cylinder and recorded once a day. 

(3) Temperature of test water 

Temperature of test water was measured with alcohol thermometer and 

recorded once a day. 

(4) Concentration of dissolved oxygen in test water 

Concentration of dissolved oxygen in test water was measured with dissolved 

oxygen probe and recorded twice a week. 

(5) pH of test water 

pH of test water was measured with pH meter once a week or more. 

(6) Cleaning of test tank 

In experimental period, excreta of carp, dirt on test tank, etc. were removed 

about once a day. 

, r~-~t~J'\.-0 

0002u0 

-13- 
4 .7 Analysis of test water and fish 

Analysis of the test item in test water and test fish was performed with 

high-performance liquid chromatography (HPLC) analysis . 

Two peaks were detected with high-performance liquid chromatography (HPLC) 

analysis of the test item. The each peak on the chromatogram was determined. The 

concentration of each peak was described using the test item concentration shown in 

Section 4.7 .3 (2) without consideration of the constituent ratio. The peaks were named 

peak 1 and 2 in the elution order, respectively. 

4.7 .1 Frequency of analysis 

(1) Test water analysis 

The test water of each level was analyzed once before first analysis of test fish 

and at the same time as analysis of test fish thereafter. 

(2) Test fish analysis 

Analysis of test fish was performed five times at each level in duration of 

exposure. Four fish were taken out at each sampling time and divided into two 

groups, then both were analyzed individually'4 . 

Analysis of control fish was performed before the experimental starting and 

after the experimental completion. Four fish were taken out at each sampling time 

and divided into two groups, and then both were analyzed individually . 

In addition, two fish were taken out and three groups (two fish per group) were used 

for measurement of lipid contents . 

*4 Because one fish was too small to take out the stored sample for the 

measurement of lipid content, two fish a group were employed. 

0002a,1 

-14- 
4.7 .2 Pretreatment for analysis 

(1) Test water 

An aliquot of the test water, 

Level 1 10 ML 

Level 2 100 ML 

was taken from each test tank, and pretreated for high-performance liquid 

chromatography (HPLC) analysis as follows : 

Test water 

F--Water for recovery test 90 mL 

(graduated cylinder) (only Level 1) 

" Column chromatography *5 (see the following details) - 

" Filling up to 10 mL (tetrahydrofuran, volumetric flask) 

*5 Conditions of column chromatography 

Bond Elut JR Cis 

Conditionings Tetrahydrofuran 10 ML 

Water`6 10 mL 

Loading Whole volume of the solution was loaded . 

Elution Eluent 1 Waterib 5 ML 

Nitrogen purge 

Eluent2 Tetrahydrofuran 8 ML 

Test item was eluted with eluent 2. 

*6 City water was treated by Ultra pure water system. 

n~ T\ ^~ 4 1 .I 

000232 

-15- 
44020 p.32 

(2) Test fish 

Test fish were taken from each test tank and pretreated for high-performance 

liquid chromatography (HPLC) analysis as follows : 

Test fish 

" Measurement of weight and body length 

" Chopping into pieces (scissors) 

" Refinement (polytron, 2 min or more, 

on ice water) 

Fine sample 

- Taking out 1 - 5 g 

(analytical balance) 

Sample for storage 

Residue 

Residue 

" Evaporation to dryness (rotary evaporator, about 40 °C) 

" Filling up to 10 mL (tetrahydrofuran, volumetric flask) 

r'NI ;J 

0002 :3 

" Taking out 4 - 5 g (analytical balance) 

E-Tetrahydrofuran 15 mL (graduated cylinder) 

" Homogenization (polytron, about 1 min.) 

" Washing (tetrahydrofuran 3 mL) 

" Centrifugation (7000xg, 5 min.) 

" Filtration (absorbent cotton) 

" Filling up to 25 mL (tetrahydrofuran, volumetric flask) 

" Taking out 2.5 mL (transfer pipet) 

E-Acetonitrile 10 mL (measuring pipet) 

E-Tetrahydrofuran 2.5 mL (measuring pipet) 

F-Hexane 100 mL (graduated cylinder) 

" Shaking for 10 min 

Supernatant 

-16- 
p. 33 

4.7 .3 Quantitative analysis for test item 

The samples for HPLC analysis in pretreatment were analyzed by 

high-performance liquid chromatography under the following analytical conditions. 

When the concentration of the test item in the sample for HPLC analysis exceeded the 

range of the calibration curve in test fish analysis, the sample was analyzed after it was 

diluted to a concentration within the range of the calibration curve. The 

concentration of the test item in each sample solution was determined on the basis of a 

comparison of the peak area on the chromatogram of the sample solution with that of a 

standard solution (see Tables-S, 6, Fig. 6 and Tables-8, 9, 10, Figs. 8, 9, 10) . 

(1) Analytical conditions 

Instrument High-performance liquid chromatograph 

Pump Shimadzu Corporation type LC-10ADvp 

Detector Shimadzu Corporation type RF-lOAxi. 

Column oven Shimadzu Corporation type CTO-l0ACvr 

Column Asahipak GF-310HQ (Showa Denko K.K.) 

30 cm x 7.6 mm I.D. 

Temperature 40 °C 

Eluent Tetrahydrofuran 

Flow rate 0 .8 mL/min 

Measurement wave length Excitation wave length 278 nm 

Fluorescence wave length 322 nm 

(see Fig. 11) 

Sample size 20 ~tL 

Detector output 1 VlFS 

(2) Preparation of standard solution 

The standard solution to determine the concentration of the test item in the 

sample solutions was prepared as follows. 

100 mg of the item supplied by the sponsor was accurately weighed and 

dissolved in tetrahydrofuran to prepare 1000 mg/L solution of the test item. 20.0 

~ig/L standard solution was then prepared from this solution by dilution with 

tetrahydrofuran. 

(3) Calibration curve 

10.0, 20 .0 and 40.0 pg/L standard solutions were prepared by the same 

method as described in (2). These solutions were analyzed according to the 

analytical conditions described in (1). A calibration curve was drawn on the basis 

of the relation between the peak area on the chromatograms and the respective 

concentrations . 

The lowest detectable peak areas of the test item were regarded as 150,000 

p.V ~ sec for peak 1 and 100,000 p.V ~ sec for peak 2 considering the noise level, 

which corresponded to the test item concentrations of 0.38 4g/L for peak 1 and 

0.49 [IglL for peak 2, respectively (see Fig. 4) . 

" 7"4 

PS ~ 

7NSA+ 

-17- 000234 
4.7 .4 Recovery and blank test 

(1) Method 

Water and fine sample of fish (10 g) spiked a specified amount of the test 

item for the recovery test were prepared in the same way as described in section 

4.7 .2 . The blank tests were also performed in the same manner without the test 

item. All the recovery and blank tests were performed in duplicate, 

(2) Results of recovery test 

In the blank test of test water (peak 2) and test fish, the chromatogram of 

HPLC had no peaks interfering with determination of the test item concentration . 

On the other hand, in the blank test of test water (peak 1), the chromatogram of 

HPLC had peak interfering with deternunation of the test item concentration . 

Therefore recovery rate for analysis of test water (peak 1) was revised by 

subtracting average (5 .47 ng) of two blanks . 

The duplicate recovery rates and the average of them in the pretreatment are 

shown below (see Tables-4, 7 and Figs.5, 7) . The average recovery rate was used 

as correction factors for the determination of the test item concentrations in the 

analytical samples. 

For analysis of test water (200 ng test item added) 

Peak 1 93 .0%, 95.6% average 94 .3% 

(subtract blank) 

Peak 2 82.9%, 85.3% average 84 .1% 

For analysis of test fish (4000 ng test item added) 

Peak 1 77.8%, 80.7% average 79 .2% 

Peak 2 71 .6%, 72.1% average 71 .8% 

4.7 .5 Lipid content in test fish 

Lipid contents in the sample for storage of the control test fish were determined 

after chloroform-methanol extraction with gravimetric analysis. 

: . 

0002i5 -18- 
't'+ULU 

p. 35 

4.7 .6 Calculation of the test item concentration in sample and minimum limit of determination 

(1) Calculation of the test item concentration in test water 

The equation in Tables-5, 6 was used to obtain the concentrations, and they 

were rounded off to 3 figures. In the blank test of test water (peak 1), the 

chromatogram of HPLC had peak interfering with determination of the test item 

concentration (see 4.7 .4(2)) ; therefore, the test item concentration in test water 

(peak 1) was revised by subtracting the blank which was determined in analysis of 

test water for recovery test . However, no blanks in analysis of test water for this 

study were detected (see Tables-5, 6 and Fig. 6) . 

(2) Determination limit of the test item in test water 

The determination limit'6 of the test item in test water was calculated on the 

basis of that obtained from the calibration curve in Section 4.7 .3 (3) as follows. 

Peak 1 Level 1 0.41 l.tg/L 

Level 2 0.041 pg/L 

Peak 2 Level 1 0.59 ~Lg/L 

Level 2 0.059 ~igIL 

(3) Calculation of the test item concentration in test fish 

The equations in Tables-7, 8 and 9 were used to obtain the concentrations, 

and they were rounded off to 3 figures. 

(4) Determination limit of the test item in test fish 

Assuming the fish to be 5 g, the determination limit* b of the test item in test 

fish was calculated to be 9.7 ng/g for peak 1 and 14 nglg for peak 2 on the basis of 

that obtained from the calibration curve in Section 4.7 .3 (3). 

*6 Minimum determination limit of the test item (ug/L or ng/g) 

A 

_B CXE 

100 " D 

where 

A : Minimum determination limit of the test item on the calibration curve 

(n[L) 

B : Recovery rate (°lo) 

C : Sampling volume of test water (mL) or weight of fine sample of fish 

(g) 

D : Final volume of sample solution (mL) 

E : Ratio of the portion, used for analysis to whole volume 

Results were rounded to 2 figures. 

~l T1 4 ] R +N 

{ .l1J 000236 

-19- 
4 .7.7 Calculation of average concentration of the test item in test water (duration of 

exposure) 

Cwt = { Cw(1) + . . . . . . . + Cw(n) } / n 

where 

Cwt : The average concentration of the test item in test water (n/L) 

n : Number of analysis for test water (measurement times) 

Cw(1) : Concentration of the test item in 1st analysis of test water (~ig/L) 

Cw(n) : Concentration of the test item in n-th analysis of test water (~LglL,) 

4.7 .8 Calculation of bioconcentration factor (BCF) 

Bioconcentration factor (BCF) was calculated as follows. 

(1) Calculation of average concentration of the test item in test water for calculating 

BCF 

_Cw= { Cw(n-1) + Cw(n) } / 2 (only lst analysis of test fish) 

Cw = { Cw(n-2) + Cw(n-1) + Cw(n) } / 3 (from 2nd analysis of test fish) 

where 

Cw : The average concentration of the test item in test water for calculating 

BCF ([ig/L) 

Cw(n) : Concentration of the test item in n-th analysis of test water (ug/L) 

(2) Calculation of bioconcentration factor 

BCF = 

Cf 

Cw 

where _ 

BCF : Bioconcentration factor 

Cf : Concentration of the test item in test fish (ng/g) 

_ (FB is subtracted if it exists) 

Cw : The average concentration of the test item in test water for calculating 

BCF (~Lg/L) 

FB : Average concentration of the test item or blank in the control test fish 

analyzed before and after the experiment (ng/g) 

000237 

-20- 
(3) The average bioconcentration factor in m-th analysis 

BCFm = (BCFa + BCFb) / n 

where 

BCFm : The average bioconcentration factor in m-th analysis (number of 

individual or group 2 (a,b)) 

BCFa,b : Each bioconcentration factor in m-th analysis of test fish 

n : Number of individual or group in m-th analysis of test fish 

The data of the analysis for test fish containing value when were not more than 

calculable BCF, BCFm is not caluculated. 

4.7 .9 Definition of the steady-state 

The steady-state of BCF is defined to reach when the variation of BCFs in three 

successive analyses at intervals of more than 48 hours is within -h20 %. When 

BCFs are less than 100, it is evaluated that a steady-state has been reached after 60 

days even if the variation of BCFs are over t 20 %. 

Criterion of the steady-state was reached : V(m-2), V(m-1), V(m) c 20(%) 

V(m-2) _ 

~ 

BCF(m-2) - BCF ` x 100 

BCF 

V(m-1) _ 

~ 

BCF(m-1) - BCF , X 100 

BCF 

V(m) - 

I BCF(m) - BCF 

x ~100 

BCF 

V(m-2), V(m-1), V(m) : Variation of bioconcentration factor (%) 

BCF(m-2), BCF(m-1), BCF(m) : The average bioconcentration factor in m-2, 

m-1, m-th analysis of test fish 

BCF : { BCF(m-2) + BCF(m-1) + BCF(m) } / 3 

0002i8 

-21- 
4 .7 .10 Calculation of bioconcentration factor at a steady-state (BCFss) 

Bioconcentration factor at a steady-state (BCFss) was calculated as follows . 

(1) Calculation of the average concentration of the test item in test water for calculating 

BCFss 

Cws = { Cw(n-2) + Cw(n-1) + Cw(n) } / 3 

where 

Cws : The average concentration of the test item in test water for calculating 

BCFss, which is calculated from three successive analyses of test 

water before last analysis of test fish as a general rule (~tg/L) 

Cw(n) : Concentration of the test item in n-th analysis of test water (~tgiL) 

(2) Calculation of the average concentration of the test item in test fish at a steady-state 

Cfs = { Cf(m-2) + Cf(m-1) + Cf(m) } / 3 

where 

Cfs : The average concentration of the test item in test fish at a steady-state 

(ng/g) 

Cf(m) : The average concentration of the test item, from which FB is 

subtracted, in m-th analysis of test fish (ng/g) 

FB : The arithmetical average concentration of the test item or blank in the 

control test fish analyzed before and after the experiment (ng/g) 

(3) Calculation of BCFss 

BCFss = Cfs / Cws 

where 

_BCFss : Bioconcentration factor at a steady-state 

Cfs : The average concentration of the test item in test fish at a steady-state 

(ng/g) 

Cws : The average concentration of the test item in test water for calculating 

BCFss (ugfL) 
'+'+UL U 

p. 39 

4.7 .11 Calculable BCF 

On the basis of the minimum determination limit of the test item in Section 

4.7 .6 (4), BCF can be obtained when BCF exceeds the following. The average 

concentration of the test item in test water obtained from all the analyzed sample was 

used to calculate the following calculable BCF. 

Peak 1 Level 1 0.49 

Level 2 4.8 

Peak 2 Level 1 0.74 

Level 2 8 .0 

4.7 .12 Calculation of lipid content 

Lipid contents were calculated with the following formula. 

Lipid content (%) = ( T - To) / S x 100 

where 

To Weight of vessel (g) 

T Weight of sample for gravimetric analysis (containing vessel) (g) 

S Weight of fine sample taken out for analysis of lipid content (g) 

4.8 Treatment of numerical values 

Values were rounded in accordance with SIS Z 8401 :1999 rule B. The each 

value used for calculation was used without rounding on the way of the calculation. 

The concentration values of the test item in test water and fish were rounded to 3 

figures. BCFss values were rounded to 2 figures. 

5. Factors possibly affecting accuracy 

No adverse effects on the reliability of this test were noted. 
6. Results 

6.1 Concentration of the test item in test water 

The measured concentrations of the test item in test water are shown in Table-1 . 

Each concentration of the test item was maintained at more than 82 % of each the 

nominated concentration. The variation of the concentrations of the test item was 

within t 20 % of the average of the measured concentrations . 

Table-1 Measured concentrations of the test item in test water 

(Unit ~Lg/L) 

After After After After After Af ter 

Average 

Peak Level 5 days 11 days 25 days 32 days 46 days 60 days (Standard Table Fig. 

deviation) 

1 18.2 20 .0 19.7 19.7 21 .0 19.8 19'7 5-1 1 (0 .90) 

2 1 .95 1 .98 2.07 1 .97 2.13 2 .07 

2'03 6-1 (0.071) 6 

1 17.4 18 .7 18.5 18 .7 19.6 18 .8 

18.6 5-2 

(0.69) 1 

1 .72 

- 2 -- I 1 .64 L 1 .72 l 1 .78 I 1 . 68 I 1 .88 I 1 .64 I 6-2 (0- 094) 

Lti kJr 

0002,411 

-24- 
6.2 Bioconcentration factors 

BCFs are shown in Table-2. 

BCFs in Table-2 plotted against the duration of exposure are shown in Figs. 1 and 2. 

BUS during the exposure period are as follows. 

Peak 1 Level 1 50 .49 

Level 2 ~ 4.8 

Peak 2 Level 1 49-73 

Level 2 72-280 

Table-2 BCFs 

Peak Level After After Table Fig. 11 days 25 days 32 days 46 days da 60 days 

-_<0 .49 :-S 0.49 -:S 0.49 :-S 0.49 :-5 0.49 

1 < 0.49 ~ 0 .49 ~ 0 .49 ~ 0.49 ~ 0 .49 8-1 8 

1 

c4.8 ~4.8 s4.8 ~4.8 ~4.8 

2 s4.8 <4.8 s4.8 ~4.8 54.8 9-1 9 

75 71 60 49 49 

1 49 65 68 73 49 8-2 8 

1 (62) (68) (64) (61) (49) 

140 240 120 72 100 

2 280 110 120 120 110 9-2 9 

(210) (170) (120) (98) (110) 
~+4v/-u p.42 

6.3 BCFs at a steady-state 

6.3 .1 Peak 1 

Because the all measured concentrations of the test item in test fish were not more 

than minimum determination limit of test item at the last three successive analyses, 

BCFs at a steady-state were not calculated . However, it is evaluated that a steady-state 

was reached within 60th day because all BCFs were less than 100. 

6.3 .2 Peak 2 

Because the variation of BCFs (average value) after 32, 46 and 60 days from the 

initiation of exposure were within t 20 % of the average for these days' BCFs (see 6.2), 

it was evaluated that a steady-state was reached after 60 days. BCFss were calculated 

on the basis of these result. 

(1) Concentrations of test item in test water at a steady-state 

The average concentrations of the test item in test water at a steady-state are 

shown in Table-3 . The average concentrations were 95 % at Level 1 and 87 % at 

Level 2 of their respective nominated concentrations . 

Table-3 Concentrations of the test item in test water at a steady-state 

(Unit : ug/L) 

Peak Level 32 

days 

~ ~ Average Table Fig. 

1 18 .7 19.6 18.8 19.0 5-2, 8-2 

2 6 

2 1 .68 L 1 .88 I 1 .64 I -1-73- 6-2,9-2 

(2) BCFs at a steady-state (BCFss) 

BCFss were calculated as follows. 

Level 1 57 

Level 2 110 

6 .4 Lipid content in test fish 

The measured lipid contents in the test fish are shown as follows . 

Before initiation of exposure 3 .07% 

After termination of exposure 3 .72% 

6.5 Results of test fish observation 

No abnormality in behavior or appearance was noted. ,t. .G~DTr~ oooz43 

-26- . . 3 
p. 43 

7. Remarks 

Instruments, apparatuses, special apparatuses and reagents, etc . for the test 

(1) Instruments for fish care 

Micro quantitative pump for supplying stock solution : 

Tokyo Rika Kikai Co., Ltd. type GivIW 

Instrument for measuring concentration of dissolved oxygen : 

Iijima Seimitsu Industries Co., Ltd. 

type F-102 

pH meter Toa Electronics Ltd. type HM-14P 

(2) Instruments, apparatuses, special apparatuses and reagents 

Instruments and apparatuses 

High-performance liquid chromatograph : 

see page 17 

Electronic analytical balance : Sartorius AG type LP4200S 

Sartorius AG type CP324S 

Sartorius AG type BP301S 

Metler Toledo type PB602 

Infrared spectrophotometer : Shimadzu Corporation type FTIR-8200PC 

Fluorescence spectrometer : Hitachi, Ltd. type F-2000 

Rotary evaporator : Tokyo Rika Kikai Co., Ltd. type N-NJ 

Mechanical shaker : Taiyo Kagaku Industries Co., Ltd. 

type SR-IIW 

Homogenizer (polytron) : Kinematica type PT3000 

Kinematica type PT3100 

Centrifuge : Hitachi Koki Co., Ltd. type CR21 G 

Special apparatuses 

Bond Elut JR C18 : Varian 

Reagents 

Acetonitrile (HPLC grade) : Wako Pure Chemical Industries, Ltd. 

Tetrahydrofuran (HPLC grade) : Kanto Chemical Co., Inc . 

Hexane (residual pesticide analytical grade) : 

Sigma-Aldrich Japan Corporation. 

Dimethylsulfoxide (extra pure) : NACALAI TESQUE, INC. 

HCO-40 : Nikko Chemicals Co., Ltd. 

-27- 

000244 

Corrected page 
(3) Instruments, apparatuses and reagents for gravimetric analysis of lipid content in test fish 

Instruments and apparatuses 

Electronic analytical balance : 

Rotary evaporator : 

Homogenizer (polytron) : 

Homogenizer (autocellmaster) : 

Vacuum pump : 

Vacuum desiccator : 

Sartorius type BP301 S 

Tokyo Rika Kikai Co ., Ltd. type N-1 

Kinematica type PT3100 

Iuchiseieido Co., Ltd. type CM-200 

Sink-u. Kiko Co., Ltd. type DA-20D 

Sinku Kiko Co., Ltd. type DAH-20C 

Iuchiseieido Co., Ltd. type VL 

Reagents 

Purified water : Takasugi Seiyaku Co., Ltd. 

Methanol (extra pure) : Wako Pure Chemical Industries, Ltd. 

Chloroform (guaranteed reagent) : Kishida Chemical Co., Ltd. 

Anhydrous sodium sulfate (extra pure) : 

Sigma-Aldrich Japan Corporation. 

Kanto Chemical Co., Inc. 
Table- 4-1 Calculation table for recovery and blank test 

(Analysis of test water, Peak 1) 

Study No. 44020 

Sample description A B C D E F 

Standard 20.OUg/L 7347062 

Recovery a 7031466 1 10 - 186 93.0% 

Recovery b 7223098 1 10 - 191 95 .6% 

Average 

94.3% 

Standard 20.O,tg/L 7291910 

Blank a 197052 1 10 5.40 

Blank b 202056 1 10 5 .54 

Average 

( a, b : individual sample ) 5 .47 

A : Peak area (ttV " sec) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 

C : Final volume (mL) 

D : Amount of blank in test water (ng) 

E : Amount of test item recovered (ng) 

E = P x(A(t) / A(std) ) / B xC - D 

F : Recovery rate (%) 

F=E/Qx100 

P : Concentration of test item in standard solution 

Q : Amount of test item added (200ng) 

See Fig. 5 

20.0ug/i. 

~ July 25, 2003 Name 

. 

0002,016 

G 
p. 46 

Table- 4- 2 Calculation table for recovery and blank test 

(Analysis of test water, Peak 2) 

Study No. 44020 

Sample description A B C D E F 

Standard 20.Oyg(L 4059936 ` 

Recovery a 3364690 1 10 - 166 82.9 

Recovery b 3461420 1 10 - 171 85.3 

Average 

84.1 

Standard 20.O,ug/L 4107864 

Blank a n.d . 1 10 

Blank b n.d . 1 10 

(a, b : individual sample) 

A : Peak area (ftV " sec) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 

C : Final volume (mL) 

D : Amount of blank in test water (ng) . 

E : Amount of test item recovered (ng) 

E = P x( A(t) / A(std) ) / B xC - D 

F : Recovery rate (%) 

F=E/Qx100 

P : Concentration of test item in standard solution 20.O#g/L 

Q : Amount of test item added (200ng) 

See Fig. 5 

Average 

000247 

July 24, 2003 Name L D Gt/ 

61 
Table- 5-1 Calculation table for analysis of test water 

(Level 1, Peak 1) 

Study No. 44020 

Sample description A I 

Standard 20.O,tg/L 6989609 

Test water after 5 days 5985860 18 .2 

Control water after 5 days n.d . 

Standard 20.O,ugiL 6769777 

Test water after 11 days 6367757 20.0 

Control water after 11 days n.d . 

Standard 20.O,ug/L 6655936 

Test water after 25 days 6196205 19.7 

Control water after 25 days n.d . 

Standard 20.O,ug/L 6953120 

Test water after 32 days 6470086 19.7 

Control water after 32 days n.d. 

Standard 20.O,ug/L 7135768 

Test water after 46 days 7054884 21.0 . 

Control water after 46 days n.d . 

Standard 20.Oyg/L 6846297 

Test water after 60 days 6405320 19.8 

Control water after 60 days n.d . 

Average concentration of test item in test water 19 .7 (S.D . 0.90) 

A : Peak area (,IV " sec) 

A(std) : Standard solution A(t) : Sample A(c) : Control blank 

B : Ratio of portion used for analysis 1 

C : Final volume lOmL 

E : Amount of blank (ng) 

E = P x( A(c) / A(std) ) / B xC _ 

F : Recovery rate 943% 

H : Volume of test water taken out lOmL 

I : Concentration of test item in test water (Ug/L) 

I=(Px(A(t)/A(std))/BxC- E)/Fx100/H 

I : Average concentration of test item in test water (/.ig/L) 

J = ( I(1) + .. . + I(n) ) / n 

n : Number of test water analyses ( n = 6 ) 

I (1) : First analysis of test water I (n) : Last analysis of test water 

S.D . - lI 

. 2 

n x ~ I(i)2 - ` . ~I(i) I 

i . 1 ` t - Jl 

nx(n-1) 

P : Concentration of test item in standard solution 20.O,ug(L 

See Fig . 6 

MO 

qI July 25, 2003 Name t 

248 
Table- 5- 2 Calculation table for analysis of test water 

(Level 1, Peak 2) 

Study No. 44020 

Sample description A I 

Standard 20.0,ug/L 3634999 

Test water after 5 days 2662182 _ 17.4 

Standard 20.0,ug/L 3568494 

Test water after 11 days 2806912 18.7 

Standard 20.0,ug/L 3581146 

Test water after 25 days 2782285 18.5 

Standard 20.0,ug/L 3647864 

Test water after 32 days 2869343 18.7 

Standard 20.0,ug/L 3763784 

Test water after 46 days 3094176 19 .6 

Standard 20.O,ug/L 3731389 

Test water after 60 days 2944528 18.8 

Average concentration of test item in test water 18.6 ( S.D. 0.69) 

A : Peak area (~eV -see) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 1 

C : Final volume 10mL 

F : Recovery rate 84.1% 

H : Volume of test water taken out IOmL 

I : Concentration of test item in test water (/.cg/L) 

I = P x( A(t) / A(std) ) / B xC / F x100 / H 

J : Average concentration of test item in test water (~cg/L) 

J = ( I(1) + . .. + I(n) ) / n 

n : Number of test water analyses ( n = 6 ) 

I (1) : First analysis of test water I (n) : Last analysis of test water 

)2 

n x ~I(i)2 - ~I(i) 

S

.D . 

n x (n -1) 

P : Concentration of test item in standard solution 

See Fig. 6 

July 24, 2003 

20.0,ug/L 

. . ..< 0002f 9 
p. 49 

Table- 6- 1 Calculation table for analysis of test water 

(Level 2, Peak 1) 

Study No. 44020 

Sample description A I 

Standard 20.0,ug/L 6989609 

Test water after 5 days 6414494 1.95 

Controi water after 5 days n.d . _ 

Standard 20.0,ug/L 6769777 

Test water after 11 days 6332407 1 .98 

Control water after 11 days n.d . 

Standard 20.OFcg/L 6655936 

Test water after 25 days 6490121 2.07 

Control water after 25 days n.d. 

Standard 20.0,ug/L 6953120 

Test water after 32 days 6454658 1.97 

Control water after 32 days n.d . 

Standard 20.Opg/L 7135768 

Test water after 46 days 7153482 2.13 

Control water after 46 days n.d . 

Standard 20.0,ug/L 6846297 

Test water after 60 days 6678865 2.07 

Control water after 60 days n.d. 

Average concentration of test item in test water 2.03 ( S.D. 0.071) 

A : Peak area (~,cV -sec) 

A(std) : Standard solution A(t) : Sample A(c) : Control blank 

B : Ratio of portion used for analysis 1 

C : Final volume lOmL 

E : Amount of blank (ng) 

E = P x ( A(c) / A(std) ) / B xC . 

F : Recovery rate 94.3% . 

H : Volume of test water taken out 100mL 

I : Concentration of test item in test water (Ug/L) 

I=(P)(A(t)/A(std))/BxC- E)/Fx100/H 

J : Average concentration of test item in test water (ug/L) 

J = ( I(1) + . . . + I(n) ) / n 

n : Number of test water analyses ( n = 6 ) 

1(1) : First an alysis of t est water I (n) : Last analysis of test water 

S.D . - lI 

2 

n x ~I(i)2 - ~I(i) 

nx(n-1) 

P : Concentration of test item in standard solution 

See Fig. 6 

20.0,ug/L 

July 25, 2003 Name O~ 
Table- 6- 2 Calculation table for analysis of test water 

(Level 2, Peak 2) 

Sample description A I 

Standard 20.OFcg/L 3634999 

Test water after 5 days 2505678 _ 1 .64 

Standard 20.O~CgiL 3568494 

Test water after 11 days 2579918 1.72 

Standard 20.0/Cg/L 3581146 

Test water after 25 days 2683500 1 .78 

Standard ZO.Oyg/L 3647864 

Test water after 32 days 2576558 1.68 

Standard 20.OFcg/L 3763784 

Test water after 46 days 2974105 1.88 

Standard 20.Ofcg/L 3731389 

Test water after 60 days 2570048 1 .64 

Average concentration of test item in test water 1.72 (S.D . 0.094) 

A : Peak area (uV -sec) 

A(std) :Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 1 

C : Final volume lOmL 

F : Recovery rate 84.1% 

H : Volume of test water taken out 100mL 

I : Concentration of test item in test water (Ug/L) 

I = P >c( A(t) / A(std) ) / B xC / F x100 / H 

J : Average concentration of test item in test water (Ug/L) 

J = ( I(1) + .. . + I(n) ) / n 

n : Number of test water analyses ( n = 6 ) 

1(1) : Fi rst analysis of test water I (n) : Last analysis of test water 

Si) . - }, 

2 

n x ~I(i)2 - I I(i) 

e - 1 i - 1 

n x (n -1) 

P : Concentration of test item in standard solution 20.OEtg/L 

See Fig. 6 

July 24, 2003 

Study No. 44020 

000251. 

p,m~(` 

-~ :a 
Table- 7-1 Calculation table for recovery and blank test 

(Analysis of test fish, Peak 1) 

Study No. 44020 

Sample description A C D E F G 

Standard 20.O,ug/L 7365228 

Recovery a 5730200 2.5/25 10- 3110 77.8 

Recovery b 5942725 2.5/25 10 - 3230 80.7 

Average 

79.2 

Standard 20.O,ug/L 7490804 

Blank a n.d . 2.5/25 10 

Blank b n.d . 2S/25 10 

( a, b : individual sample) 

A : Peak area (,cV " sec) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis (fine sample) 5/10 

C : Ratio of portion used for analysis (extracted solution) 

D : Final volume (mL) 

E : Amount of blank in test fish (ng) 

F : Amount of test item recovered (ng) -

F=Px(A(t)/A(std))/B/CxD- E 

G : Recovery rate (%) 

G=F/Q X100 

P : Concentration of test item in standard solution 20.O,ug/L 

Q : Amount of test item added (4000ng) 

See Fig. 7 

July 24, 2003 

Average 

0()052 40 
p. 52 

Table- 7- 2 Calculation table for recovery and blank test 

(Analysis of test fish, Peak 2) 

Study No. 44020 

Sample description A C D E F G 

Standard 20.O,ug/L 3685230 

Recovery a 2637743 25/25 10 - 2860 71.6 

Recovery b 2655230 2.525 10 - 2880 72.1 

Standard 20.Oftg/L 3738736 

Blank a n.d . 2.5/25 10 

Blank b n.d . 2.5/25 10 

Average 

( a, b : individual sample) 

A : Peak area (fqV -sec) 

A(std) : Standard solution A(t) : Sample , 

B : Ratio of portion used for analysis (fine sample) 5/10 

C : Ratio of portion used for analysis (extracted solution) 

D : Final volume (mL) 

E : Amount of blank in test fish (ng) 

F : Amount of test item recovered (ng) -

F=Px(A(t)/A(std))/B/CxD- E 

G : Recovery rate (%) 

G=F/Qx100 

P : Concentration of test item in standard solution 20.O,Cg/L 

Q : Amount of test item added (4000ng) 

See Fig. 7 

July 24, 2003 Name GI , ~ p Cj~ , 

U 

Average 

71.8 

000253 
p. 53 

Table-8-1 Calculation table for analysis of test fish 

(Level 1, Peak 1) 

Study No. 44020 

Sample description A G K H J M 

Standard 20.O,Cg/L 6645451 

Test fish after 11 days a n.d . 5 5.00 - 19.1 

Test fish after 11 days b n.d. 5 5.00 - 19.1 

Standard 20 .Oftg/L 6644804 

Test fish after 25 days a n.d . 5 5.00 - 19.3 

Test fish after 25 days b n.d . 5 5.00 - 19.3 

Standard 20.O,ug/L 6844800 

Test fish after 32 days a n.d. 5 5.00 - 19.8 

Test fish after 32 days b n.d. 5 . 5.00 - 19.8 

Standard 20.O,ug/L 7083014 

Test fish after 46 days a n.d . 5 5.00 - 20.2 

Test fish after 46 days b n.d . 5 5.00 - 20.2 

Standard 20.0,ug/L 7025127 

Test fish after 60 days a n.d . 5 5 .00 - 20.2 

Test fish after 60 days b n.d . 5 5 .00 - 20.2 

( a, b : individual sample ) 

A : Peak area( uV " sec) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 25125 

C : Final volume 10mL 

D : Dilution factor 

E : Average concentration of blank in analysis of control Ong/g 

F : Recovery rate 79.2% 

G : Weight of fine sample (g) 

K : Concentration of test item in test fish (ng/g) 

K={ Px(A(t)/A(std))/BxDxC/G- E}/Fx100 

H : Average concentration of test item in test water (/cg/L) 

H = { I(n- 2) + I(n-1) + I(n) } / m ; n : Number of test water analyses ; m = 2 when n = 2, m = 3 when n ? 3 

1 : Concentration of test item in test water (lug/L) 

J :BCF 

J=K/H 

M :Average value of BCF(a) and BCF(b) 

M = { BCF(a) + BCF(b) } / 2 

P : Concentration of test item in standard solution 20.O,ug/L 

See Fig. 8 

July 24, 2003 Name a= 

, r ooozs4 

,~ ~ 
p. 54 

Table-8-2 Calculation table for analysis of test fish 

(Level 1, Peak 2) 

Study No. 44020 

Sample description A D G K H J M O 

Standard 20.0,ug/L 3571133 

Test fish after 11 days a 1738277 5 5.00 1360 18.1 75 62 

Test fish after 11 days b 1126496 5 5.00 ~879 18.1 49 

Standard 20.0,ug/L 3648453 

Test fish after 25 days a 1685938 5 5.00 1290 18.2 71 68 

Test fish after 25 days b 1556703 5 5.00 1190 18 .2 65 

Standard 20.0,ug/L 3617316 

Test fish after 32 days a 1452173 5 5.00 1120 18.6 60 64 65 

Test fish after 32 days b 1653630 5 . 5 .00 1270 18.6 68 

Standard 20.0,ug/L 3728526 

Test fish after 46 days a 1228399 5 5.00 918 18 .9 49 61 64 

Test fish after 46 days b 1854876 5 5.00 1390 18.9 73 

Standard 20.0,ug/L 3849895 

Test fish after 60 days a 1284747 5 5.00 929 19.0 49 49 58 

Test fish after 60 days b 1279788 5 5.00 926 19.0 49 

( a, b : individual sample) 

BCFss: 57 

A : Peak area (~cV -sec) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 2S/25 

C : Final volume lOmL 

D : Dilution factor ~ 

E : Average concentration of blank in analysis of control Ong/g 

F : Recovery rate 71.8°1o 

G : Weight of fine sample (g) 

K : Concentration of test item in test fish (ng/g) 

K = { P x( A(t) / A(std) ) / B xD xC / G - E } / F x100 

H : Average concentration of test item in test water (ugIL) 

H = { I(n- 2) + I(n-1) + I(n) } / m ; n : Number of test water analyses 

I : Concentration of test item in test water (4g/L) 

J :BCF ' 

J=K/H 

M : Average value of BCF(a) and BCF(b) 

M = { BCF(a) + BCF(b) } / 2 

O : Average value of BCF 

O = { M(n- 2) + M(n-1) + M(n) } I 3 

P : Concentration of test item in standard solution 20.0,ug/L 

m=2whenn=2,m=3whenn Z3 

BCFss = ( { K(n- 3) + K(n- 2) + K(n-1) } / 3 J / [ { I(n- 2) + I(n-1) + I(n) } / 3 ] 

K : Average value of K (ng/g) 

K = { K(n-1)a + K(n-1)b } / 2 

See Fig. 8 

0002 5 
p. 55 

Table-9-1 Calculation table for analysis of test fish 

(Level 2, Peak 1) 

Study No. 44020 

Sample description A D G K H J M 

Standard 20.O,ug/L 6719426 

Test fish after 11 days a n.d . 1 5.00 - 1 .97 

Test fish after 11 days b n.d . 1 5.00 - 1 .97 

Standard 20.0/Cg/L 6506544 

Test fish after 25 days a n.d. 1 5.00 - 2.00 

Test fish after 2.5 days b n.d . 1 5.00 - 2.00 

Standard 20.Opg/L 6912338 

Test fish after 32 days a n.d . 1 5.00 - 2.01 

Test fish after 32 days b n.d . 1 . 5.00 - 2.01 

Standard 20.0jug/L 7182742 

Test fish after 46 days a n.d . 1 5.00 - 2.05 

Test fish after 46 days b n.d . 1 5.00 - 2.05 

Standard 20.Opg/L 7187156 

Test fish after 60 days a n.d . 1 5.00 - 2.06 

Test fish after 60 days b n.d . 1 5.00 - 2.06 

( a, b : individual sample ) 

A : Peak area (uV ~ sec) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 2.5125 

C : Final volume lOmL 

D : Dilution factor 

E : Average concentration of blank in analysis of control Ong/g 

F : Recovery rate 79.2% 

G : Weight of fine sample (g) 

K : Concentration of test item in test fish (ng/g) _ 

K={Px(A(t)/A(std))/BxDxC/G-E}/Fx100 

H : Average concentration of test item in test water (fag/I,) 

H = { I(n- 2) + I(n-1) + I(n) } / m ; n :Number of test water analyses 

1 : Concentration of test item in test water (Ug/L) 

J : BCF 

J=KlH 

M :Average value of BCF(a) and BCF(b) 

M = { BCF(a) + BCF(b) } / 2 

P : Concentration of test item in standard solution 20.O,ug/L 

See Fig. 9 

m=2whenn=2,m=3whenn z3 

July 24, 2003 Name ~°v~ 

ooozss 
p. 56 

Table-9-2 Calculation table for analysis of test fish 

(Level 2, Peak 2) 

Study No. 44020 

Sample description A D G K H J M p 

Standard 20.0,ug/L 3617019 

Test fish after 11 days a 1497601 1 5.00 231 1.68 140 210 

Test fish after 11 days b 3019422 1 5.00 -465 1 .68 280 

Standard 20.0,ug/L 3576519 

Test fish after 25 days a 2591942 1 5.00 404 1.71 240 170 

Test fish after 25 days b 1239790 1 5.00 193 1.71 110 

Standard 20.0,ug/L 3686050 

Test fish after 32 days a 1427639 1 5.00 216 1 .73 120 120 170 

Test fish after 32 days b 1411997 1 5 .00 213 1 .73 120 

Standard 20.0,ug/L 3750505 

Test fish after 46 days a 863849 1 5.00 128 1 .78 72 98 130 

Test fish after 46 days b 1485337 1 5.00 221 1 .78 120 

Standard 20.OFcg/L 4003544 

Test fish after 60 days a 1305774 1 5 .00 182 1 .73 100 110 110 

Test fish after 60 days b 1363693 1 5.00 190 1 .73 110 

. ( a, b : individual sample ) 

BCFss: 110 

A : Peak area (/uV ~ sec) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 2.5/L5 

C : Final volume lOmL 

D : Dilution factor 

E : Average concentration of blank in analysis of control Ong/g 

F : Recovery rate 71.8% -

G : Weight of fine sample (g) 

K : Concentration of test item in test fish (ng/g) 

K={Px(A(t)/A(std))/BxDxC/G- E}/Fx100 

H : Average concentration of test item in test water (r,g/L) 

H = { I(n- 2) + I(n-1) + I(n) } / m ; n : Number of test water analyses ; m = 2 when n = 2, m = 3 when n ~ 3 

1 : Concentration of test item in test water (~Ug/L) 

J :BCF 

J=K/H 

M : Average value of BCF(a) and BCF(b) 

M = { BCF(a) + BCF(b) } / 2 

O : Average value of BCF 

O = { M(n- 2) + M(n-1) + M(n) } ( 3 

P : Concentration of test item in standard solution 20.0,ug/L 

BCFss = [ { K(n- 3) +'F-(n- 2) + K(n-1) } / 3 ) / [ { I(n- 2) + I(n-1) + I(n) } / 3 ] 

K : Average value of K (ng/g) 00015,1, 

K = { K(n-1)a + K(n-1)b 112 ._, 

, See Fig. 9 
p. 57 

Table-10- 1 Calculation table for analysis of test fish 

(Control, Peak 1) 

Study. No. 44020 

Sample description A E G I 

Standard 20.O,ug/L 

Before the experimental start a 

Before the experimental start b 

Standard 20.OEcgIL 

After the experimental completion a 

After the experimental completion b 

( a, b : individual sample) 

A : Peak area (f,tV " sec) 

A(std) :Standard solution A(t) :Sample 

B : Ratio of portion used for analysis 2S/25 

C : Final volume lOmL 

E : Amount of blank in analysis of control (ng) 

E= P x( A(t) / A(std) ) / B xC 

G : Weight of fine sample (g) 

I : Concentration of blank in test fish (ng/g) 

I=E/G 

P : Concentration of test item in standard solution 20.Oug/L 

See Fig. 10 

July 24, 2003 Name 

7047044 - 

n.d . - 4.00 -

n.d . - 4.00 - 

6721196 

n.d . - 5 .00 

n.d . - 5.00 

Average 

000258 
p. 58 

Table-10- 2 Calculation table for analysis of test fish 

(Control, Peak 2) 

Study No. 44020 

Sample description 

Standard 20.Ofcg/L 

Before the experimental start a 

Before the experimental start b 

Standard 20.O,ttg/1. 

After the experimental completion a 

After the experimental completion b 

A E G I 

3853554 ' 

n.d . - 4.00 

n.d . - 4.00 

3618246 

n.d . - 5.00 

n.d . - 5.00 

Average 

( a, b : individual sample) 

A : Peak area (E.tV -sec) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 2.5/25 

C : Final volume lOmL 

E : Amount of blank in analysis of control (ng) 

E= P x( A(t) / A(std) ) J B xC 

G : Weight of fine sample (g) 

I : Concentration of blank in test fish (ng/g) 

I=E/G . 

P : Concentration of test item in standard solution 20.Ofcg/L 

See Fig. 10 

000259 

July 24, 2003 Name -- - 
rnnaiyzical results of dilution water 

p.59 

Sampling date May 8, 2003 

Item Unit Measured value Standard value Detection limit 

Total hardness (Ca, Mg) mg/L 78.5 < 300 " 0. 1 

Suspended solid mg/L < 1 < 20 '2 1 

pH - 8 .o 6. 5 - 8. 5 '~ 

Total organic carbon mg/L 0.3 < 2 "Z 0. 1 

Chemical oxygen demand mg/L 3 .0 < 5 '3 0 . 1 

Residual chlorine mg/L < 0.-01 < 0.02 " 0.01 

Ammonia nitrogen ~ mg/L 0.01 < 1 " 0.01 

Total cyan mg/L < 0.01 n. d. " 0.01 

Alkalinity mg/L 158 - 1 

Electric conductivity pS/cm ] 492 - 

Organic phosphorus mg/L < 0.1 n. d.'3 0. 1 

A1kylmercury mg/L < 0.0005 n. d.'3 0 .0005 

Mercury mg/L < 0 .0005 < 0.0o05 }3 0.0005 

Cadmium . mg/L < 0 .001 < 0. 01 '3 0.001 

Crfi' mg/L < 0.02 < 0.05 *3 0.02 

Lead ~ mg/L < 0.005 < 0. 1 "3 0.005 

Arsenic mg/L 0.003 < 0. 05 '3 0.001 

Iron mg/L < 0.01 < 1 .0 *' 0.01 

Copper mg/L < 0 .005 < 0. 005 '3 0.005 

Cobalt mg/L < 0.001 < 0.001 'S 0.001 

Manganese mg/L < 0.01 < 0.05 " 0.01 

Zinc mg/L < 0.01 < 1 .0 *` 0.01 

Aluminium mg/L < 0 .001 < 0.2 " 0 .001 

Nickel mg/L < 0 .001 < 0. 01 '' 0.001 

Silver mg/L < 0.0001 < 0.0001 "5 0.0001 

Organochlorine pesticides 

1, 2-Dichloropropane mg/L < 0.0001 < 0 . 06 '~ 0.0001 

Chlorothalonil mg/L < 0.0001 < 0.04 " 0.0001 

Propyzamide mg/L < 0.0001 < 0. 008 '4 0.0001 

Chlornitrofen mg/L < 0 .0001 < 0. 0001 " 0.0001 

Simazine mg/L < 0.001 < 0 . 003 '" 0.001 

Thiobencarb mg/L < 0.0001 < 0. 02 '~ 0.0001 

Organophosphorous pesticides 

Diazinon mg/L < 0.0001 < 0. 005 '4 0.0001 

Isoxathion mg/L < 0.0001 < 0.008 "4 0.0001 

Fenitrothion mg/L < 0.0001 < 0.003 '4 0.0001 

EPN mg/L < 0.0001 < 0. 006 '4 0.0001 

Dichlorvos mg/L < 0.0001 < 0.01 " 0.0001 

Iprobenfos mg/L < 0 .0001 < 0. 008 0.0001 

~ PCB 

_ 

mg/L < 0. 0005 n. d. ~' 0.0005 

*1 Ministerial ordinance of the Ministry of Health and Welfare No . 69 (Revised 

December 21, 1992) 

*2 OECD Guidelines for Testing of Chemicals, Fish, Early-life Stage Toxicity Test 

(Guideline 210, July 17, 1992) 

*3 Water quality criteria for fisheries (Shadanhozin Nihon Suisansigen Hogokyokai, 

,^ °a ~ z--'y 

-,_> :~ 

March 1983) 0002so 

*4 Environmental Quality Standards for Water Pollutants No. 14 (Revised February 

22, 1999, Environment Agency) 
p. 60 

Study No. 44020 

U 

w 

C 

. 

O 

C 

U 

r0 U 

O 

q 

101 

i 

I 

10° 

The minimum calculation limit of bioconcentration factor . 

10-t 1 

0 5 10 15 20 25 30 35 40 45 50 55 60 

Exposure period (days) 

Fig. 1-1 Correlation between exposure period and bioconcentration factor (Level 1,Peak 1) 

Ten data after 11, 25, 32, 46 and 60 days were lower than detection limit . 

101 

- The minimum calculation limit of bioconcentration factor. 

0 .r 

U 

W 

C 

O 

. 

C 

U 

U 

C 

O 

U 

q 

100 

10' 1 

0 5 10 15 20 25 30 35 40 45 50 55 60 

Exposure period (days) 

F irzcy . 2-1 Correlation between exposure period and bioconcentration factor (Level 2,Peak 1) 

Ten data after 11, 25, 32, 46 and 60 days were lower than detection limit. 

000261 

July 24, 2003 Name c . ~~ 
Study Na. 44020 

0 

U 

C 

O 

. 

C 

U 

U 

C 

O 

U 

_O 

103 

101 

O a 8 ' O 0 

The minimum calculation limit of bioconcentration factor. 

10-i 

0 5 10 15 20 25 30 35 40 45 50 55 60 

Exposure period (days) 

Fig. 1-2 Correlation between exposure period and bioconcentration factor (Level 1,Peak 2) 

0 

.~ 

U 

w 

C 0 

c 

U 

C 

O 

U 

O_ 

Ga 

103 

O 

O 

101 

10-1 

O 

O O 

The minimum calculation limit of bioconcentration factor . 

O 

0 

0 

0 5 10 15 20 25 30 35 40 45 50 55 60 

Exposure period (days) 

Fig. 2-2 Correlation between exposure period and bioconcentration factor (Level 2,Peak 2) 

July 24, 2003 Name , G7 Gt~ 

ooozs2 
Study No. 44020 

\° 0 

. 

0 

100 

50 

0 

96- hour LC50= 28 .7 mg/L (Doudoro 

1 10 

Concentration (mg/L) 

100 

Concentration Cumulative Mortality (%) 

rn 24 hours 48 bours 72 hours 96 hours 

Control 0 0 0 0 

4.38 0 0 0 0 

8.75 0 0 - 0 0 

17.5 0 0 0 0 

35.0 10 60 70 70 

70.0 40 100 100 100 

Fig. 3 Concentration - Mortality Curve. 

Date : May 22, 2003 

000263 
Study P0.644020 

Solvent 

PKNO 

Operating date : Apr. 3,2003 

Time Area Height Conc. 

(min) (uV " sec) (uV) (I) 

Total 

-

0. 0 

Peak position 

1 z 

~n.d .l n.d . 

---~- 

-~ --, 

5. 0 10.0 15. 0 

Standard solution 10 .0 Fg/L 

Operator : Yuka Koga 

PKNO 

1 

2 

Time 

(min) 

9. 77 

10 . 29 

Total 

, 

0. 0 5.0 10.0 15.0 

Standard solution 20 .0 pg/L 

Operator : Yuka Koga 

PKNO 

1 

2 

2 

1 

0. 0 5.0 10. 0 15. 0 

Standard solu tion 40 . 0 Fg/L 

2 

h 

Time 

(min) 

9 . 77 

10 . 29 

Total 

0 0.00 

Operating date : Apr. 3, 2003 

Area Height Conc. 

(0-sec) (uV) M) 

3791182 79732 65.37 

2008272 110101 34.63 

5799454 100.00 

Operating date : Apr. 3, 2C03 

Area Height Conc. 

(cuV~sec) (uV) (%) 

7444029 160069 64.22 

4146930 223232 35.78 

11590959 100.00 

Operator : Yuka Koga 

PKNO 

1 

2 

Operating date : Apr . 3, 2003 

Time Area Height Conc. 

(min) (0-sec) (uV) (%) 

9.77 15914547 320641 66.34 

10.29 8074811 439193 33.66 

Total 23989358 100.00 

r----- ---r- - 

0. 0 5. 0 10. 0 15, 0 Operator 

Fig. 4 - 1 Chromatogram of HPLC analysis for calibration curve. 

Date : Apr. 3, 2003 Name : 

~ . 

3 

: Yuka Koga 

'' /' _^ 000264 

- 
Study No. 44020 

U 

N 

a, 

18000000 ; 

9000000 

/o 

0 22 .5 

Concentration (~ig/L) 

y =392083x 

r = 0.999 

Concentration 

(Ag/L) 

Peak area 

(~iV " sec) 

10.0 3791182 

20.0 7444029 

40.0 15914547 

r" --1 

Fig. 4 - 2 Calibration c 

July 8, 2003 Name 

0 

45 

ooo2ss 
Study No. 44020 

9000000 

0 

U 

U 

a 

4500000 

I 0 ~ . ' 

0 22 .5 45 

Concentration (~LglL) 

y = 202864x 

r= 1 .00 

Concentration 

(pgIL,) 

Peak area 

(iV ~ sec) 

10.0 2008272 

20.0 4146930 

40.0 8074811 ' 

Fig. 4 - 3 Calibration curve of E I (Peak 2) . 

July 8, 2003 Name _ 

---~ - - 

% 

ooo2ss 
Study No. 44020 p.66 

Standard solution 20 .0 pg/L Operating date : Apr. 3, 2003 

PKNO Time Area Height Conc. 

(min) (l1V~ sec) (aV) (/) 

1 9 .77 7341062 157347 64.41 

2 10.29 4059936 218862 35.59 

2 

Total 11406998 100.00 

, 

0.0 5. 0 10. 0 15. 0(min) Operator : Yuka Koga 

Recovery test from test water a Operating date : Apr. 3, 2003 

PKNO Time Area Height Conc. 

(min) (0-sec) (p.V) (/) 

1 9.77 7031466 144252 67.64 

, 2 10.29 3364690 183528 32.36 

2 

tA 

A 

0. 0 5 . 0 10.0 

Recovery test from test water b 

Total 10396156 100.00 

15' 0(min) Operator : Yuka Koga 

Operating date : Apr. 3, 2003 

PKNO Time Area Height Conc. 

(min) (UV-sec) (uV) M 

1 9.77 7223098 146004 67.60 

2 10.29 3461420 188690 32.40 

0.0 5.0 

2 

10 .0 

Total 10684518 100.00 

15.0(min) Operator : Yuka Koga 

Fig. 5 - 1 Chromatogram of HPLC analysis for recovery and blank test (analysis of test water) . 

Date : Apr. 3, 2003 Name : 

1 
Study 40. M20 

Standard solution 20 .0 }Ig/L Operating date : Apr. 3.2003 

PKNO Time Area Height Conc. 

(min) (1tV " sec) (0) (%) 

1 9 .77 7291910 157779 63.97 

2 10 .28 4107864 221353 36.03 

2 

s 

0.0 5. 0 10. 0 

Blank test of test water a 

Total 11399775 100.00 

5

.0(min) Operator : Yuka Koga 

Operating date : Apr. 3.2003 

PKNO Time Area Height Conc. 

(min) (1tV " sec) (gV) (%) 

1 9. 71 197052 16209 100.00 

Total 197052 100.00 

Peak position 

z 

1 

. 

0. 0 5.0 10.0 

Blank test of test water b 

15. 

0(min) Operator : Yuka Koga 

Operating date : Apr. 3. 2003 

PKNO Time Area Height Conc. 

(min) (0-sec) ('UV) M) 

1 9.70 202056 16319 100.00 

Total 202056 100.00 

Peak position 

z 

1 ~n.d. 

0.0 5.0 10. 0 15.0(min) Operator : Yuka Koga 

Fig. 5 - 2 Chromatogram of HPLC analysis for recovery and blank test (analysis of test water) . 
Study No. 44020 

Standard solution 20 .0 pg/L 

2 

- 

0.0 5 .0 10 .0 

Test water after 5 days (Level 1) 

15.0 

p. 68 

Operating date : May. 27, 2003 

PKNO Time Area Height Conc. 

(min) ( ;tV " sec) (uV) 

1 9.81 6989609 139349 65.79 

2 10.33 3634999 203455 34.21 

Total 10624608 100.00 

Operator : Yuka Koga 

Operating date : May. 27, 2003 

PKNO Time Area Height Conc. 

(min) (y.V " sec) (uV) (Y) 

1 9.80 5985860 130773 69.22 

2 10.31 2662182 153557 30.78 

Total 8648042 100 .00 

< < 

0.0 5.0 10.0 15.0 

Test water after 5 days (Level 2) 

Operator : Yuka Koga 

Operating date : May. 27, 2003 

PKNO Time Area Height Conc. 

(min) (LaV " sec) (,uV) (96) 

1 9.80 6414494 143115 71 .91 

2 10 .32 2505678 139932 28.09 

Total 8920172 100.00 

1 2 

0.0 5.0 10 .0 15.0 

Test water after 5 days (Control) 

Operator : Yuka Koga 

Operating date : May. 27.2003 

PKNO Time Area Height Conc. 

(min) (AV " sec) (uV) (l) 

Total 0 0.00 

Pezk position 

1 z 

In. din. d. 

0. 0 5~ 0 10. 0 S5. 0 Operator : Yuka Koga 

Fig. fi - 1 Chromatogram of HPLC analysis for test water . 

Date : May. 27, 2003 Name : 

0002G9 

U 
Study (Dio .M020 

Standard solution 20 .0 pa/l, Operating date : Jun. 3, 2003 

PKNO Time Area Height Conc. 

(min) (tuV " sec) (uV) (%) 

1 9 . 68 6769777 139560 65. 48 

2 10.40 3568494 193134 34.52 

r---- - 

0.0 5 .0 

2 

10. 0 15 .0 

Test water after 11 days (Level 1) 

Total 10338271 100.00 

Operator : Yuka Koga 

Operating date : Jun. 3, 2003 

PKNO Time Area Height Conc. 

(min) (,tV " sec) (,u V) (%) 

1 9.89 6367757 132218 69.41 

2 10.41 2806912 153614 30.59 

Total 9174669 100.00 

I 

0.0 5.0 10.0 15 .0 

Test water after 11 days (Level 2) Operating date : Jun. 3, 2003 

PKNO Time Area Height Conc. 

(min) (gV " sec) (uV) (%) 

1 9.89 6332407 132983 71 .05 

2 10.41 2579918 137836 28.95 

Total 8912326 100.00 

1 

-- -- -r 

0.0 5.0 10.0 15.0 

A1 2 

Operator : Yuka Koga 

Test water after 11 days (Control) Operating date : Jun. 3,2003 

PKNO Time Area Height Conc. 

(min) (uV~sec) (1tV) (%) 

Total 0 0.00 

Peak position 

1 21 

.tn.d. 

t 

n. d. 

0. 0 5. 0 10. 0 1~ 0 Operator : Yuka Koga 

Fig. 6 - 2 Chromatogram of HPLC analysis for test water . 

Date : Jun. 3, 2003 Name : 

Operator : Yuka Koga 
~ t.uuy IVO . 44VGV 

St andard so l ution 20 .0 pg/L Operating date : Jun. 17,2003P

. 

PKNO Time Area Height Conc. (min) (ttV " sec) (UV) (/) 

1 9.85 6655936 140456 65.02 

2 10.37 3581146 198339 34.98 

Total 10237081 100 00 2 

1 

. 

0.0 5. 0 10.0 15.0 Operator : Yuka Koga 

Test water after 25 days (Level 1) Operating date : Jun. 17,2003 

PKNO Time Area Height Conc. 

. (min) (aV" sec) (uV) (/) 

1 9.84 6196205 136604 69.01 

2 10.36 2782285 156444 30.99 

Total 8978490 100.00 

. 

0.0 5. 0 10.0 15. 0 Operator : Yuka Koga 

Test water after 25 days (Level 2) Operating date : Jun. 17,2003 

PKNO Time Area Height Conc. 

(min) (ttV " sec) (UV) M) 

1 9.85 6490121 140272 70.75 

2 10.37 2683500 148071 29.25 

Total 9173620 100.00 

12 

0.0 5.0 10.0 15. 0 Operator : Yuka Koga 

Test water after 25 days (Control) Operating date : Jun. 17,2003 

PKNO Time Area Height Conc. 

(min) (1tV " sec) (UV) (/) 

Total 0 0.00 

Peak position 

1 z 

0.0 5.0 

------------------ 

i n n ,G n Operator : Yuka Koaa 

Fig. 6 - 3 Chromatogram of HPLC analysis for test water . 000271 

Date : Jun. 17, 2003 Name : 

. .-~ .r. - 

.'r ..`:a 67 
Study IL 71$020 

Standard solution 20 .0 p;lL 

2 

7 ---f~

. . 

-l 

0.0 5.0 10.0 15.0 

Test water after 32 days (Level 1) 

2 

0.0 5.0 10.0 15 .0 

Test water after 32 days (Level 2) 

0.0 5.0 10 .0 15.0 

Test water after 32 days (Control) 

Peak position 

1 z 

~ in. d. d. 

Total 0 0. 00 

- 'I Operator : Yuka Koga 0. 0 5.0 10.0 15. 0 

Fig . 6 - 4 Chromatogram of HPLC analysis for test water . 

Date : Jun. 24, 2003 Name : 

Operating date : Jun. 24, 2003 

PKNO Time Area Height Conc. 

(min) (uV~sec) (u.V) (°/a) 

1 9.83 6953120 145119 65.59 

2 10 .34 3647864 205219 34.41 

Total 10600984 100.00 

Operator : Yuka Koga 

Operating date : Jun.24.2003 

PKNO Time Area Height Conc. 

(min) (uV" sec) (1cV) M 

1 9.83 6470066 138682 69.28 

2 10.35 2869343 160886 30.72 

Total 9339429 100.00 

Operator : Yuka Koga 

Operating date : Jun. 24, 2003 

PKNO Time Area Height Conc. 

(min) (AV-sec) (uV) (I) 

1 9.84 6454658 139838 71 .47 

2 10.36 2576558 146945 28.53 

Total 9031216 100.00 

Operator : Yuka Koga 

operating date : Jun. 24.2003 

PKNO Time Area Height Conc. 

(min) (ltV " sec) (0) (%) 

0002'72 
Study No. 44020 p' 72 

Standard solution 20 .0 pg/L Operating date : Jul. 8, 2003 

PKNO Time Area Height Conc. 

(min) (uV~sec) (UV) (Y-) 

1 9 .96 7135768 149532 65.47 

2 10 .48 3763784 207617 34.53 

2 

0.0 5 .0 10.0 15 .0 

Test water after 46 days (Level 1) Operating date : Jul. 8, 2003 

PKNO Time Area Height Conc. 

(min) (ltV~sec) (uV) (%) 

1 9.95 7054884 149089 69.51 

2 10.47 3094176 173078 30.49 

Total 10149060 100.00 

Test water after 46 days (Level 2) Operating date : Jul . 8, 2003 

PKNO Time Area Height Conc. 

(min) (ltV " sec) (uV) (%) 

1 9.96 7153482 150360 70.63 

2 10.48 2974105 164449 29.37 

Total 10127588 100.00 

2 

-

0.0 5 . 0 10.0 15. 0 

Test water after 46 days (Control) 

Operator : Yuka Koga 

Operating date : Jul . 8, 2003 

PKNO Time Area Height Conc. 

(min) (9V- sec) (uV) (%) 

Total 0 0.00 

Peak position 

t z 

~n.d.I4 n.d . 

0.0 5.0 10.0 15 .0 Operator : Yuka Koga 

Fig. 6 - 5 Chromatogram of~HPLC analysis for test water. 

Date : Jul. 8, 2003 

1 f n t1 

Total 10899552 10O.CO 

Operator : Yuka Koga 

Operator : Yuka Koga 

UUC 
Study 

Standard solution 20 .0 p g/L 

Afi20 

Operating date : Jul. 22, 2003 

PKNO Time Area Height Conc. 

(min) ( ;iV~sec) (uV) 

1 9 .86 6846297 145656 64.72 

2 10 .38 3731389 211218 35.28 

Total 10577686 100.00 

---T- - -, 

0.0 5. 0 10 . 0 15. 0 

Test water after 60 days (Level 1) 

Operator : Yuka ~K ;-..--'a. }(o~a 

Operating date : Ju1 .22.2003 

PKNO Time Area Height Conc. 

(min) (9V " sec) (uV) (?/.) 

1 9.86 6405320 138904 68.51 

2 10.37 2944528 167428 31 .49 

Total 9349848 100.00 

- --r - 

0. 0 5.0 10.0 15 . 0 

Test water after 60 days (Level 2) 

Operator : Yuka ~ "a 

Operating date : Jul . 22, 2003 

PKNO Time Area Height Conc. 

(min) (/IV " sec) (gV) ( 

1 9.86 6678865 144640 72.21 

2 10 .38 2570048 140837 27.79 

Total 9248913 100.00 

1 2 

0.0 5.0 10.0 15. 0 

Test water after 60 days (Control) 

Operator : Yuka Ktdl a hngLL 

Operating date : Jul . 22. 2003 

PKNO Time Area Height Conc. 

(min) (GtV " sec) (uV) (%) 

Total 0 0.00 

Peak position 

1 

tn . d. .tn.d . 

r. - 

---i_- ~- --i 

0. 0 5. 0 10 . 0 15 . 0 

Fig. 6 - 6 Chromatogram of HPLC analysis for test water. 

Operator : Yuka ~zz:a Kc~~a 

'fri~J~t~:~ 171c Y~, 3fi'} 

Date : Jul. 22, 2003 Name : ~ 

al

, 

''~ -~i0002'74 
stUdY NO. 44020 

Standard solution 20 .0 POOL Operating date : Apr. 16, 2003 . 

PKNO Time Area Height Conc. 

(min) (uV " sec) (UV) ( 

1 9 .79 7365228 147085 66.65 

2 10 . 31 3685230 201945 33.35 

0. 0 5. 0 10. 0 

Recovery test from test fish a 

2 

L, 

Total 11050458 

. . 

15.0(min) Operator : Yuka Koga 

100.00 

Operating date : Apr. 16, 2003 

PKNO Time Area Height Conc. 

(min) (ltV " sec) (uV) (?/.) 

1 9.81 5730200 116567 68.48 

2 10.33 2637743 147274 31 .52 

Total 8367944 100.00 

2 

A-,,j 

0.0 5 .0 1 0, 0 15 . 0(min) Operator : Yuka Koga 

Recovery test from test fish b Operating date : Apr. 16, 2003 

PKNO Time Area Height Conc. 

(min) (uV" sec) (gV) (V 

1 9.81 5942725 115300 69. 12 

2 10.33 2655230 146834 30.88 

Total 8597956 100.00 

r, 

2 

0. 0 5.0 10, 0 15. 0(min) Operator : Yuka Koga 

Fig . 7 - 1 Chromatogram of HPLC analysis for recovery and blank test (analysis of test fish) . 

Date : Apr. 16, 2003 Name : 

74 
Study Po.7FX020 

Standard solution 20.0 ~.~g/L Operating date : Apr. 16, 2003 

PKNO Time Area Height Conc, 

(min) (uV~sec) (kv) M.) 

1 9 .78 7490804 149021 66.71 

2 10 .30 3738736 203729 33.29 

, -~ -

0.0 5.0 10.0 

2 

Total 11229540 100.00 

15 0(min) Operator : Yuka Koga 

Blank test of Operating date : Apr. 16, 2003 test fish a 

PKNO Time Area Height Conc. 

(min) (uV " sec) (uV) (Y-) 

Total 0 0. 00 

Peak position 

I z 

0.0 5. 0 10.0 

Blank test of test fish b Operating date- : Apr. 16 . 2003 

PKNO Time Area Height Conc. 

(min) (y.V~sec) (ttV) (W 

Total 0 0. 00 

Peak position 

1 z 

' d.`n .d. 

0.0 5.0 10. 0 15.0(min) Operator : Yuka Koga 

Fig. 7 - 2 Chromatogram of HPLC analysis for recovery and blank test (analysis of test fish) . 

Date : Apr. 16, 2003 Name : 

djn.d. 

15.0(min) Operator : Yuka Koga 
p. 76 Study No .44020 

Standard solution 20 .0 pg/L Operating date ; Jun. 3, 2003 

PKNO Time Area Height Conc. 

(min) (AV-Sec) (uV) (V.) 

1 9.89 6645451 138543 65.05 

2 10.41 3571133 195078 34.95 

2 

, 

0.0 

----r5. 0 10. 0 

Test fish after 11 days (Level 1-a) D.5 

Total 10216584 

15 .0 (min) Operator : Yuka Koga 

100. 00 

Operating date : Jun. 3, 2003 

PKNO 

2 

Time Area Height Conc. 

(min) (ltV " sec) (uV) (96) 

10. 49 1738277 113977 100.00 

Total 1738277 100.00 

Peak position 

1 

. ~n. d. 2 

.,A 

-

0. 0 5. 0 10. 0 15, 0(min) Operator : Yuka Koga 

Test fish after 11 days (Level 1-b) D.5 Operating date : Jun. 3, 2003 

PKNO Time Area Height Conc. 

(min) (gV " sec) (ItV) ( 

2 10.48 1126496 72318 100.00 

Total 1126496 100.00 

Peak position 

. 

1 

f n.d . 2 

0.0 5.0 10. 0 15 . 

0(min) Operator : Yuka Koga 

Fig. 8 - 1 Chromatogram of HPLC analysis for test fish (Level 1) . 
Study IGo. 44020 

Standard solution 20 .0 pg/L Operating date : Jun. 17. 2003 

PKNO Time Area Height Conc. 

(min) (AV-sec) (uV) ( a) 

1 9 .85 6644804 141746 64.55 

2 10.37 3648453 199114 35.45 

2 

I 

Total 10293258 100. 00 

0.0 5.0 10.0 

Test fish after 25 days (Level 1-a) D .5 

PKNO 

2 

1510(min) Operator : Yuka Koga 

Operating date : Jun. 17, 2003 

Peak position 

t 

~n.d . 2 

0.0 5.0 10.0 

Test fish after 25 days (Level 1-b) D.5 

Time Area Height Conc. 

(min) (,UV-sec) (1tV) (0/6) 

10.44 1685938 110269 100. 00 

Total 1685938 100.00 

15.0(min) Operator : Yuka Koga 

Operating date : Jun. 17, 20C3 

PKNO Time Area Height Conc. 

(min) (EaV " sec) (uV) ( 

2 10.43 1556703 100211 100. 00 

Total 1556703 100.00 

Peak position 

1 

~n.d . 

A 

,- 

0.0 5.0 10.0 15. 0(min) Operator : Yuka Koga 

Fig . 8 - Z Chromatogram of HPLC analysis for test fish (Level 1) . 

. Date : Jun. 17, 2003 Name : 
Study No.44020 

Standard solution 20 .0 pg/L 

p. 78 

Operating date : Jun. 24,2003 

PKNO Time Area Height Conc. 

(min) (uV " sec) (uV) (90) 

1 9 .84 6844800 143211 65.42 

2 10 .36 3617316 202938 34.58 

2 

r0.0 5.0 10 .0 

Test fish after 32 days (Level 1-a) D.5 

100.00 

Operating date : Jun. 24, 2003 

PKNO Time Area Height Conc. 

(min) (tcV~sec) (uV) (I) 

2 10 .43 1452173 96767 100.00 

Total 1452173 100.00 ' 

Peak position 

t 

t". 

d. 2 

0. 0 5.0 10.0 

Total 10462116 

15 0(min) Operator : Yuka Koga 

15.0(min) Operator : Yuka Koga 

Fig. 8 - 3 Chromatogram of HPLC analysis for test fish (Level 1? . 

Date : Jun. 24, 2003 Name : 
StudypJo7.94020 

Standard solution 20 .0 pglL Operating date : Jul . 8, 2003 

PKNO Time Area Height Conc. 

(min) (ltV~sec) (uV) 0/0) 

1 9.98 7083014 147593 65.51 

2 10.51 3728526 208564 34.49 

2 

Y 

0.0 ~ 5. 0 10.0 

Test fish after 46 days (Level 1-a) D.5 Operating date : Jul. 8, 2003 

PKNO Time Area Height Conc. 

(min) (aV ~ sec) ( ~aV) 0/6) 

2 10.60 1228399 78519 100.00 

Total 1228399 100.00 " 

Peak position 

--n 

t 

in

. d. 2 

0.0 5.0 10.0 

Total 1081 1541 100.00 

15. 0(min) Operator : Yuka Koga 

j 0 (min) Operator : Yuka Koga 

Test fish after 46 days (Level 1-6) D .5 Operating date : Jul. 8, 2003 

PKNO Time Area Height Conc. 

(min) (0-sec) (uV) ( 

2 10 . 62 1854876 116738 100.00 

Total 1854876 100.00 

Peak poaition 

I 

I 

n-d 2 

I 

0.0 5.0 10.0 15 .0(min) Operator t Yuka Koga 

Fig. 8 - 4 Chromatogram of HPLC analysis for test fish (Level 1) . 

Date : Jul. 8, 2003 Name : 
Study Nc .44020 

Standard solution 20 .0 pg/L 

p. 80 

Operating date : Jul. 22, 2003 

PKNO Time Area 

(min) (,UV-sec) 

1 9 .85 7025127 

2 10 .37 3849895 

1 

0.0 5. 0 10.0 

Total f0875022 

Height 

( uV) 

150306 

216963 

15~ 0(min) Operator : Yuka .#~a }CqR 

Canc. 

(9a) 

64. 60 

35. 40 

100 . 00 

Test fish after 60 days (Level 1-a) D .5 Operating date : Ju1 .22.2003 

PKNO Time Area Height Conc. 

(min) (0-sec) (UV) (I) 

2 10.45 1284747 85370 100.00 

Total 1284747 100 .00 

Peak position 

1 

In.d.2 

~L'_"`-~l l 

0.0 5.0 10.0 

Test fish after 60 days (Level 1-b) D.5 Operating date : Ju1.22, 2003 

PKNO Time Area Height Conc. 

(min) (laV " sec) (uV) (%) 

2 10.46 1279788 86716 100.00 

Total 1279788 100.00 

Peak position 

1 

IILd

. 2 

, . -,A 

0. 0 5. 0 10. 0 15. 0(min) Operator : Yuka #~ }(cga 

Fig . 8 - 5 Chromatogram of HPLC analysis for test fish (Level 1) . 

a rrfi),Viz~r~ y ,p : 

2 

15. 

0(min) Operator : Yuka i~. . Kqa. 

Date : Jul. 22. 2003 Name : ( 

U 
Study Ta, a1020 

Standard solution 20 .0 }lg/L Operating date : Jun. 3, 2003 

PKNO Time Area Height Conc. 

(min) [9V-sec) ( ;tV) (%) 

1 9 .89 6719426 139376 65.01 

2 10 .41 3617019 197390 34.99 

- r 

0. 0 5.0 ~ 10.0 

Test fish after 11 days (Level 2-a) 

2 

Total 10336444 

15.0(min) Operator : Yuka Koga 

100. 00 

Operating date : Jun. 3, 2003 

PKNO 

2 

Peak position 

1 

`n .d.2 

A 

, - T0.0 5.0 10.0 

Test fish after 11 days (Level 2-b) Operating date : Jun. 3, 2003 

PKNO Time Area Height Conc. 

(min) (0-sec) (11V) (N 

2 10.48 3019422 182853 100.00 

Peak position 

2 

1-11 

Total 3019422 100.00 

0.0 5.0 10.0 15. 0(min) Operator : Yuka Koga 

Fig. 9 - 1 Chromatogram of IIPLC analysis for test fish (Level 2) . 

Date : Jun. 3, 2003 Name : 

Time Area Height Conc. 

(min) (ItV " sec) (AV) M) 

10.49 1497601 100682 100.00 

Total 1497601 100.00 

15~0(min) Operator : Yuka Koga 
Study No, 44020 

p.82 

Standard solution 20 .0 pg/L Operating date : Jun. 17, 2003 

PKNO Time Area Height Conc. 

(min) (uV" sec) (uV) (%) 

1 9 .85 6506544 140142 64.53 

2 

2 10.27 3576519 198781 35. 47 

Total 10083062 

0.0 5.0 10.0 

Test fish after 25 days (Level 2-a) 

100. 00 

15.0(min) Operator ; Yuka Koga 

Operating date : Jun. 17, 2003 

PKNO Time Area Height Conc. 

(min) (0-sec) (itV) (%) 

2 10.44 2591942 166101 100.00 

Peak position 

1 2 

~n.d . ~I 

Total 2591942 100.00 -~ 

~'`-') t 

0.0 5.0 10.0 

Test fish after 25 days (Level 2-b) 

15.0 (min) Operator : Yuka Koga 

Operating date : Jun. 17, 2003 

PKNO Time . Area Height Conc. 

(min) (4V-sec) (tcV) (%) 

2 10.45 1239790 63920 100.00 

Peak position 

i 

I 

d. 2 

n. A 

Total 1239790 100.00 

-

0.0 5.0 10.0 15. 

0(min) Operator : Yuka-Koga 

Fig. 9 - 2 Chromatogram 4f HPLC analysis for test fish (Level 2) . 

Date : Jun. 17, 2003 Name : 
StudyR044020 

Standard so lulion 20 .0 pg/L Operating date : Jun. 24, 2003 

PKNO Time Area Height Conc. 

(min) (gV " sec) (uV) M) 

1 9 .84 6912338 144518 65.22 

2 10 .36 3686050 204588 34.78 

0. 0 5 .0 10.0 

2 

Total 10598388 

15 . 

0(min) Operator : Yuka Koga 

100. 00 

Test fish after 32 days (Level 2-a) Operating date : .1un . 24, 2003 

PKNO Time Area Height Conc. 

(min) (0-sec) (uV) (/) 

2 10.44 1427639 91276 100.00 

Peak position 

, n. d. 2 

~l, n 

Total 1427639 100.00 

0.0 5 .0 10.0 15. 0(min) Operator : Yuka Koga 

Test fish after 32 days (Level 2-b) Operating date : Jun.24,2003 

PKNO Time Area Height Conc. 

(min) (AV-sec) (uV) (96) 

2 10.44 1411997 93415 100.00 

Peak position 

1 

in. d. 2 

Total 1411997 100.00 

0. 0 5T 0 10 . 0 15. 0(min) Operator : Yuka Koga 

Fig. 9 - 3 Chromatogram of HPLC analysis for test fish (Level 2) . 

Date : Jun. 24, 2003 Name : 
Study No . 44020 p' $4 

Standard solution 20.0 ylg/L 

PKNO Time Area 

(min) (1tV-sec) 

1 9.97 1182742 

2 10.50 3750505 

Operating date : Jul . 8, 2003 

Height Conc. 

0-, V) 0/6) 

149477 65.70 

207619 34.30 

2 

0.0 5.0 10. 0 

Test fish after 46 days (Level 2-a) 

Total 10933248 

15. 0(min) Operator : Yuka Koga 

100.00 

Operating date : Jul . 8,2003 

PKNO Time Area Height Conc. 

(min) (0-sec) (uV) (/) 

2 10.58 863849 56757 100.00 

Peak position 

t 

Total 863849 1 00. 00 1 

I 

d. 

IL 

0 

.0 

5 

0 10.0 

Test fish after 46 days (Level 2-b) 

~5 0(min) Operator : Yuka Koga 

Operating date : Jul. 8, 2003 

PKNO Time Area Height Conc. 

(min) (jtV " sec) (uV) (%) 

2 10.60 14B5337 98675 100.00 

Peak position 

1 

n. d. 2 

Total 1485337 100.00 

0.0 5 . 0 10. 0 15, O(min) Operator : Yuka Kaga 

Fig. 9 - 4 Chromatogram of HPLC analysis for test fish (Level 2) . 

Date : Jul. 8, 2003 Name : 
Studyp Na544020 

Standard solution 20 .0 pglL Operating date : Jul. 22, 2003 

PKNO Time Area Height CCnc. 

(min) (uV " sec) (j1V) (%) 

1 9 .64 7187156 150273 64, 22 

2 10 .36 4003544 226057 35.78 

Total 11190701 100.00 

---

0. 0 5.0 10.0 

Test fish after 60 days (Level 2-a) 

15 0( min) Operator : Yuka #tz'a }Cc~aOperating date : Ju1.22,2003 

PKNO Time Area Height Conc. 

(min) (uV~sec) (,tV) (%) 

2 10.44 1305774 88401 100.00 

Peak position 

1 

, 

( n. d. 2 

r - - - - -~ --- - ~ - T0.0 5.0 10.0 

Test fish after 60 days (level 2-b) 

Total 1305774 100.00 ' 

15~ 0(min) Operator : Yuka k~+s Yaaa. 

Operating date : Ju1 .22.2003 

PKNO 

2 

Time Area Height Conc. 

(min) (UV-sec) (uV) (%) 

10.45 1363693 93725 100.00 

Total 1363693 100.00 

Peak position 

1 

ln,d .2 

0. 0 5~0 10. 0 15. 

0(min) Operator : Yuka ~ }Co~n, 

Fig. 9 - 5 Chromatogram Of HPLC analysis for test fish (Level 2) . 

Date : Jul . 22, 2003 Name : 

3 i%7 ~~'z t7s1: %» =P~ 

c 

U 
Study No. 44020 p,86 

Standard solution 20 .0 pg/L Operating date : May. 23, 2003 

PKNO Time Area Height Conc. 

(min) (u.V~sec) (uV) ( 

1 9 .85 7047044 149043 64.55 

2 10 .36 3853554 212334 35.35 

2 

Total 10900598 100.00 

- - --r0.0 5.0 10.0 

Before the experimental start ( Control-a. ) 

15 .0(min) Operator : Yuka Koga 

Operating date : May. 23, 2003 

PKNO Time Area Height Conc. 

(min) (uV " sec) (uV) 0/6) 

Total 0 0.00 

Peak position 

t z 

in .d.`n.d . 

,~---,-- ^ 

0. 0 5. 0 10.0 15 .0(min) Operator : Yuka Koga 

Before the experimental start ( Control-b ) Operating date : May. 23,2003 

PKNO Time Area Height Conc. 

(min) (icV " sec) (uV) (I) 

Total 0 0.00 

Peak position 

i 

In. d.In.d. 

I --

0.0 5.0 10.0 

Fig.10 - 1 Chromatogram of 1PLC analysis for test fish (Control) . 

Date : May. 23, 2003 Name : 

15.0(min) Operator : Yuka Koga 
Standard solution 20 .0 pg/L 

azuay NC . 44020 --_Q 7 N. "'----

Operating date : Jul. 23, 2003 

PKNO Time Area Height Conc. (min) (UV-sec) (uV) (/) 

1 9.87 6721196 139908 65 . 0 1 

2 10.39 3618246 201018 34. 99 

0.0 5. 0 10.0 

2 

Total 10339442 100 . 00 

15 . 

0(min) Operator : Yuka Koga 

After the experimental completion ( Control-a ) Operating date : Ju1 .23,2003 

PKNO Time Area Height Conc. (min) (uV" sec) (,tV) (/) 

Total 0 0.00 

Peak position 

I 

In. d. n. d. 

. 

0.0 - 5. 0 10. 0 

After t he experimental completion ( Control-b ) 

15.0(min) Operator : Yuka Koga 

Operating date : Jul. 23, 2003 

PKNO Time Area Height Conc . 

(min) (uV " sec) (uV) (/) 

Total 0 0.00 

Peak position 

z 

In. d. 

In

. d. 

-

0' 0 5. 0 10. 0 15, 0(min) Operator : Yuka Koga 

Fig.10 - 2 Chromatogram of HPLC analysis for test fish (Control) . 
c 

\ 

U 

0 

a1~ e 

V~ trn c 

T 

2 

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r\ 

\ 

m 

0 

0 

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O 

Q 

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F i g. 11 Fluorescence spectrum of test item. 

1 

+~yoio .~on3. i" / 7 Yu u 1c; vl11Ll 

5~'>`Oyo .3oo,y. l" /7 ~, f`Dy+.v 

p. 88 

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Study No. Az4z0_2o Date .1 .,v,3, Instrument HI TAGNI 

Sample C 3 Slit ex /0 nm 

Solvent_ _ _ 7etrzt.kvdt-_o f ,.a ~ em / o nm 

Reference -Te trA A, yd m+u rczr,, Scan Speed .~~Id nm/min 

Cell /0 mm x /D mm 

Wave Length ~ ex 178 nm A em ~$~o n m, y,Sa n m 

A em 3-1-2 nm A ex .~S-a nm^" ~z~v nm Note : 

i Operator 

n 

----- 

T , 
60 .0 -

-I 

%T 

40 .0 -- 

20 .0 

0.0 -J 

-20.0 

~~v I 

3000 .0 2000 .0 1500 .0 1000.0 500.0 

1 /cm 

Fig. i 2-1 I R spectrum of test item measured before 

experimental start . 

Study No . : 44020 

Sample : . C 

Method : KBr tablet 

Date : Jan.10, 2003 

Name : Y-koP., 
60.0 

i 

%T 

50 .0 - 

40 .0 --i 

30 .0 - 

20.0- 

10 .0-1 

I 

- ---° --f 

0.0 

3000.0 2000 .0 1500.0 

r-i r I I r i --i -

1000 .0 500.0 

I /Cm 

Fig. Y 2-2 IR spectrum of test item measured after 

experimental completion. 

Study No . : 44020 

Sample : 

Method : K8r tablet 

Date : Ju f . 23, 2003 

Name : 
60.0 

s s 

i 

1 %T - 

I 

I 

! 

0.0 .... ._~ . 

t 

4 

, 

20.0 J 

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I ! 

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--rI 

i 

I 1 1 i 

0 .0 i s s, ~s f i 

4000.D 3500 .0 3000.0 . 2500.0 2000.0 1750,0 1500.0 ' 1250 .0 1000.0 750.0 500.0 

GPH82.IRS : GPH82 Lot No . 0-8182 1/cm 

Bit: 2002/11/01 Of 111_: 18:02218 TA#RlM 40 . 

T -MRi1: HYPER 1R standard , 

49P$M 1/cm %T T*g~x~'~'t: }iapp ' ' -

iall~~~: 401.17 x`1s"ft: 3998.18 AIMEE 1/am , 

AM 1888 T--~rr11~ : 1 .92888 3MAE: 4 .0 

''72-(J : auto 7J%*-ft-: auto' ?.7-1f°'1: 2 .8(low) 

Reference 2 IR spectrum supplied by sponsor 
Amendment to Final Report 

Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

1 . Title Bioconcentration study ot ]in carp 

2. Study number 44020 

3 . Content 7.(2) Instruments, apparatuses, special apparatuses and reagents 

Special apparatuses (page 27) 

4. Reason Miswriting . 

5 . Content of correction " Bond Elut JR C18 : GL Science " is corrected to 

" Bond Elut JR Cts : Varian ". 

6. Approval 

Date August 7, 2003 

Study Director Signed in original 

Naoaki Yakata 
Change of Study Director 

Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

1 . Title Bioconcentration study of ~ ]in carp 

2. Study number 44020 

3 . Content Study director changed from " Yuka Koga " to "Naoaki Yakata". 

4. Reason Because Yuka Koga is absent from our laboratory now on 

maternity leave . 

5 . Date of correction August 4, 2003 

6. Preparation 

Date August 5, 2003 

Management Signed in original 

Hiroshi Tadokoro 
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