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Bioconcentration test o f 2-perfluoroalkyl (C=4-16) ethanol [This test was performed using 2-{perfluorooctyl) ethanol (Test substance number K-1518)] in carp
(Test number : 51518)
Kurume Laboratory Chemicals Evaluation and Research Institute, Japan
000001*1
STATEMENT
51518
Kumme Laboratory Chemicals Evaluation and Research Institute, Japan
Sponsor
ASAHI GLASS Co., Ltd.
Title
Bioconcentration test o f 2-perfluoroaIkyl (C=4-16) ethanol [This test was performed using 2-(perfluorooctyl) ethanol (Test substance number K-1518)] in carp
Test number
51518
I, the undersigned, hereby declare that this report provides a correct English translation o f the Final Report (Test N o.51518, issued on February 22,2001).
Date Study Director
1 "eIffV'U (AV-y ^ , * - D C X
Erina Kikushima
000002
GLP STATEMENT
51518
Kurume Laboratory Chemicals Evaluation and Research Institute, Japan
Sponsor
Ministry o f Economy, Trade and Industry
Title Bioconcentration test of 2-perfluoroalkyl (C=4-16) ethanol [This test was performed using 2-(perfluorooctyl) ethanol (Test substance number K-1518)] in carp
Test number
51518
This test was performed in compliance with Good Laboratory Practice Standards for industrial chemicals, "Standard Concerning Testing Facility Provided in Artiicle 4 o f 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 o f Health and Welfare, and No.85, Basic Industries Bureau, Ministry o f International Trade and Industry, Japan) and "OECD Principles o f 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 Study Director
February 22,2001 Signed in original Erina Kikushima
000003
51518
QUALITY ASSURANCE STATEMENT
Kurume Laboratory Chemicals Evaluation and Research Institute, Japan
Sponsor
Ministry o f Economy, Trade and Industry
Title Bioconcentration test o f 2-perfiuoroalkyl (C=4-16) ethanol [This test was performed using 2-(perfluorooctyl) ethanol (Test substance number K -1518)] in carp
Test number
51518
The inspections o f this test were carried out and the results were reported to the management and the Study Director by Quality Assurance Unit o f Kurume Laboratory, Chemicals Evaluation and Research Institute, Japan as follows.
Item o f inspection Protocol
Test Conduct
Raw Data and Final Report
Date o f audit or inspection October 10,2000 November 24,2000 January 30,2001 October 17,2000 October 23,2000 October 24,2000 October 30,2000 October 31,2000 November 2,2000 November 13,2000 November 14,2000
February 22,2001
Date of report to study director October 10,2000 November 29,2000 February 5,2001 November 1,2000 November 1,2000 November 1,2000 November 1,2000 November 1,2000 November 14,2000 November 14,2000 November 14,2000
February 22,2001
Date of report to management October 10,2000 November 29,2000 February 5,2001 November 2,2000 November 2,2000 November 2,2000 November 2,2000 November 2,2000 November 16,2000 November 16,2000 November 16,2000
February 22,2001
It has been assured that the final report describes accurately the test method used, that details in the report are in compliance with the protocol and Standard Operating Procedures and that the final report reflects the raw data accurately.
Date
February 22,2001
Quality Assurance Unit, Head
Signed in original
Kyoshiro Hori
000004
CONTENTS
Title Sponsor Testing facility Objective Test method Applied GLP Dates Storage o f test substance, raw data, etc. Personnel Approval o f final report SUMMARY 1. Test substance 2. Performance of acute toxicity test 3. Performance o f bioconcentration test 4. Factors possibly affecting accuracy 5. Results 6. Reference test 7. Discussion 8. Remarks
51518
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2 2 2 3 3 4 5 7
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Contents of tables and figures
Contents of tables
Table-1
Measured concentrations o f test substance in test water (see text)
Table-2
BCFs (see text)
Table-3
Concentrations and BCFs o f the test substance in all the parts (see text)
Table-4
Residual rate of test substance in depuration test (see text)
Table-5
Calculation table for recovery and blank test (analysis o f test water)
Table-6
Calculation table for analysis o f test water (Level 1)
Table-7
Calculation table for analysis of test water (Level 2)
Table-8
Calculation table for recovery and blank test (analysis o f test fish)
Table-9
Calculation table for analysis of test fish (Level 1)
Table-10
Calculation table for analysis of test fish (Level 2)
Table-11
Calculation table for analysis o f test fish (Control)
Table-12
Calculation table for analysis in parts o f test fish (Level 1)
Table-13
Calculation table for analysis in parts of test fish (Level 2)
Table-14
Calculation table for analysis of test fish (Level 1) (depuration test)
Table-15
Calculation table for analysis of test fish (Level 2) (depuration test)
Reference 1 Analytical results of dilution water
oooooa
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Contents of figures
Fig. 1
Correlation between exposure period and bioconcentration factor (Level 1)
Fig. 2
Correlation between exposure period and bioconcentration factor (Level 2)
Fig. 3
Concentration-mortality curve
Fig. 4
Mass fragmentogram and calibration curve o f GC-MS analysis for test water
Fig. 5
Mass fragmentogram of GC-MS analysis for recovery and blank test (analysis o f test water)
Fig. 6 Fig. 7
Mass fragmentogram o f GC-MS analysis for analysis o f test water Mass fragmentogram and calibration curve o f GC-MS analysis for test fish
Fig. 8
Mass fragmentogram of GC-MS analysis for recovery and blank test (analysis o f test fish)
Fig. 9
Mass fragmentogram of GC-MS analysis for test fish (Level 1)
Fig.10
Mass fragmentogram of GC-MS analysis for test fish (Level 2)
Fig.11 Mass fragmentogram of GC-MS analysis for test fish (Control)
Fig.12
Concentration o f dissolved oxygen (Level 1)
Fig. 13 Concentration o f dissolved oxygen (Level 2)
Fig. 14 Concentration of dissolved oxygen (Control)
Fig. 15
Mass fragmentogram o f GC-MS analysis for test fish (Level 1) (analysis in parts of test fish)
Fig. 16 Fig. 17
Mass fragmentogram o f GC-MS analysis for test fish (Level 2) (analysis in parts o f test fish)
Mass fragmentogram of GC-MS analysis for test fish (Level 1) (depuration test)
Fig. 18
Mass fragmentogram of GC-MS analysis for test fish (Level 2) (depuration test)
Fig. 19-1 IR spectrum o f test substance measured before the experimental starting
Fig.19-2 IR spectrum o f test substance measured after the experimental completion
Fig.20
Mass spectrum of test substance
Reference 2 NMR spectrum o f test substance
000007
51518
Title Bioconcentration test o f 2-perfluoroalkyi (0 4 -1 6 ) ethanol [This test was performed
using 2-(perfluorooctyl) ethanol (Test substance number K-1518)] in carp
Sponsor Ministry of Economy, Trade and Industry 1-3-1 Kasumigaseki, Chiyoda-ku, Tokyo 100-8901, Japan
Testing facility Kurume Laboratory Chemicals Evaluation and Research Institute, Japan 19-14 Chuomachi, Kurume-shi, Fukuoka 830-0023, Japan
Objective This test was performed to evaluate the bioconcentration potential o f K-1518 in carp.
Test method This test was perfomed according to the "Method for Testing the Degree o f
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 o f Health and Welfare, and No.392, Basic Industries Bureau, Ministry o f International Trade and Industry, Japan), and "Bioconcentration : Flow-through Fish Test (Guideline 305, June 14,1996)" in the OECD Guidelines for Testing o f Chemicals.
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Applied GLP
(1) Chemical GLP This test complied with "Standard Concerning Testing Facility Provided in
Artiicle 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, PJiarmaceutical Affairs Bureau, Ministry of Health and Welfare, and No.85, Basic Industries Bureau, Ministry o f International Trade and Industry, Japan).
(2) OECD-GLP This test complied with "OECD Principles o f Good Laboratory Practice"
(November 26,1997).
Dates Study initiation date Experimental starting date Experimental completion date Study completion date
October 10,2000 October 23,2000 December 22,2000 February 22,2001
Storage of test substance, raw data, etc.
(1) Test substance Because the test substance o f the same lot is already stored after the end o f
biodegradation test, the test substance is not stored after this test.
(2) Raw data and materials, etc. Raw data, the protocol, documents about the test presented by the sponsor, the
final report and necessary materials arc stored in a storage room in this laboratory.
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Personnel Study Director Study personnel (Operation of bioconcentration test)
Staff for fish care Person to conduct o f fish acute toxicity test
Approval o f final report Study Director
Date Signature
Erina Kikushima Kazuma Fujimoto Yuji Kusune Emi Yamaguchi Yasuro Kawashima Yasuro Kawashima Katsumi Kiyono
February 22,2001 Signed in original Erina Kikushima
-3-
OOOOiO
SUMMARY
51518
Title
Bioconcentration test of 2-perfluoroalkyl (C=4-16) ethanol [This test was performed using 2-(perfluorooctyl) ethanol (Test substance number K -1518)] in carp
Test conditions
Acute toxicity test (1) Test fish (2) Duration o f exposure (3) Exposure method
Orange-red killifish CQryzias latipes) 96 hrs. Semi static system (Renewal of test water, at every 8 - 1 6 hrs.)
Bioconcentration test
(1) Test fish
Carp tCvprinus carpio)
(2) Nominal concentrations o f test substance
High exposure level (Level 1) 10 )ig/L
Low exposure level (Level 2) 1 pg/L
(3) Duration o f exposure
60 days
(4) Exposure method
Continuous flow system
(5) Analytical method
Gas chromatography-mass spectrometry
Results
(1) 96-hour LC50 value (2) Bioconcentration factors
> 125 mg/L
Level 1 200- 1100
Level 2
8 7 - 310
-4 O O O O ll
1. Test substance In this report, K-l 518 has the following chemical name, etc.
1.1 Chemical name 2-(perfluorooctyl) ethanol 1.2 Chemical structure, etc.
Structural formula
CF3-- (CF2)6-- CF2CH2CH2--OH
Molecular formula
C ioHsFitO
Molecular weight
464.12
1.3 Manufacturer, name and lot number*1
(1) Manufacturer
DAUCIN Finechemical Laboratory, Ltd.
(2) Name
A-1820
(3) Lot number
A 18207X01
*1 Information supplied by the manufacturer
1.4 Purity*1 Test substance
98 % (GC)
The test substance was treated as 100 % in purity.
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51518 1.5 Confirmation of test substance
The structure o f the test substance is confirmed by infrared (IR) spectrum (see Fig.19), Mass spectrum (see Fig.20) and NMR spectrum (see Reference 2). 1.6 Storage and stability (1) Storage condition
Cold storage place (2) Stability
The test substance was stable under the storage condition as shown by the finding that IR spectra o f the test substance before and after the experiment were identical (see Fig.19). 1.7 Stability under testing conditions
Prior to the bioconcentration test, a stability o f the test substance under the testing conditions was confirmed by a preliminary test
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2. Performance of acute toxicity test
2.1 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".
2.2 Test fish
(1) Species
Orange-red killifish (Qryzias latipcs) Reason for selection : This species is similar in sensitivity to carp and
readily available as test fish.
(2) Supplier Ogawa shoten (Address: 181 Ooishi-machi Kurume-shi, Fukuoka 830-0049, Japan)
(3) Conditions for fish care before acclimatization The fish were checked visually at the receiving and those demonstrating any
abnormality were removed. The fish were reared for 23 days in a flow through system following an external disinfection.
(4) Conditions for acclimatization After rearing, the fish were transferred to an acclimatizing aquarium and
acclimatized there after the second external disinfection. The fish demonstrating any abnormality during this period were removed and the remainder of the fish were reared for 40 days in a flow through system at the temperature o f 25 2 "C. The fish were checked for health conditions and reared another 34 days after the external disinfection.
(5) Weight
average 0.30 g
(6) Length
average 3.1cm
(7) Certification The 48-hour LC50 value o f the reference substance'1for the fish of the same lot
(TFO-000801) was 0.596 mg/L.*
*2 PCP-Na (pentachlorophenol sodium salt, Tokyo Kasei Kogyo Co., Ltd.)
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2.3 Dilution water for test
(1) Origin Groundwater from the premises of Kumme Laboratory.
(2) Water quality assessment The dilution water for test was taken out on August 8, 2000, 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 o f 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 o f Chemicals, "Bioconcentration: Flow-through Fish Test (Guideline 305, June 14, 1996)".
2.4 Test conditions
(1) Test tank
Glass gallon vessel
(2) Volume of test water
3.85 L x 2 / level
(3) Temperature o f test water At initial exposure Before renewal of test water
24.3 C 24.1 - 24.2 C
(4) Concentration of dissolved oxygen in test water
At initial exposure
8.0mg/L
Before renewal of test water
5.2 - 5.7 mg/L
(5) pH o f test water At initial exposure Before renewal o f test water
7.9 7.7
(6) Number o f fish
10/level
(7) Duration o f exposure
96 hrs.
(8) Exposure method
Semi static system (Renewal o f test water, at every 8 -1 6 hrs.)
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2.5 Preparation o f stock solution
(1) Dispersant HCO-20 (Hydrogenated castor oil)
(2) Preparation The test substance and HCO-20 (40 times amount of the test substance) were
dissolved with acetone. After the acetone was evaporated from the solution, ionexchanged water was added to the mixture to prepare 1000 rag/L stock solution.
2.6 Performance of test
(1) Place
214 LC50 room
(2) Date
October 16,2000 - October 20,2000
2.7 Estimation o f 96-hour LC50 value The 96-hour LC50 value was estimated by the Doudoroff method.
2.8 Result o f test 96-hour LC50 value
> 125 mg/L*5(see Fig. 3)
*3 The concentration o f the dispersant at this time was 5000 mg/L. Taking into account o f the toxicity o f the dispersant, the test at higher concentration was not performed, because the 96-hour LC50 value of the dispersant was more than 50000 mg/L.
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3. Performance of bioconcentration test
3.1 Test fish
(1) Species
Reason for selection : The previous data conducted with this species can be compared and the size o f this species is adequate for handling.
(2) Supplier Fukuokaken yabegawa fishermen's cooperative association (Address : 748 Yamauchi, Yame-shi, Fukuoka 834-0012, Japan)
Date received
September 1,2000
(3) Conditions for fish care before acclimatization The fish were checked visually in the receiving and those demonstrating ar.y
abnormality were removed. The fish were reared for 8 days in a flow through system following an external disinfection.
(4) Conditions for acclimatization After rearing, the fish were medicated to eliminate parasites and transferred :c<
an acclimatizing aquarium. After the second external disinfection, they w ere acclimatized. The fish demonstrating any abnormality during this period w ere removed and the remainder o f the fish were reared for 15 days in a flow through system at the temperature o f 25 2 C. The fish were then transferred to test ta x is and reared at the same temperature in the flow through system for 25 days, following the external disinfection.
(5) Length 6.5 - 9.4 cm
(6) Lot No. TFC-000901
(7) Age
Yearling fish
(8) Feeding
Feed
Feed for fry o f car )
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.
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3.2 Dilution water for test The same as described in section 2.3.
3.3 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 for volatile substance
(3) Flow rate o f test water 2 mL/min for stock solution and 1600 mL/min for dilution water ; 2307
liters/day for test water were supplied.
(4) Stock solution bottle
10-L teddlar bag (Cooling in refrigerator) Frequency o f renewal 2 times/week
(5) Temperature o f test water
Level 1
25.0-25.6 C
Level 2
24.8 - 25.2 C
Control
25.2-25.6 C
(6) Concentrations o f dissolved oxygen in test water
Level 1
7.6 - 7.9 mg/L (see Fig. 12)
Level 2
7.7 - 7.9 mg/L (see Fig.13)
Control
7.8 - 8.0 mg/L (see Fig. 14)
(7) pH o f test water Level 1 Level 2 Control
7.6 -7.7 7.6 -7.7 7.6 -7.8
(8) Time o f irradiation with light Artificial light o f white fluorescent lamp (14 hrsVday)
(9) Number o f fish (at the beginning of exposure)
Level 1 and 2 40
Control
8
(10) Duration o f exposure Reason for decision :
60 days The time to reach a steady-state was not estimated to be within 28 days from preliminary test results.
(11) Place
213 Aquatron room
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3.7.2 Pretreatment for analysis
(1) Test water
An aliquot o f the test water,
Level 1
100 mL
Level 2
1000 mL
was taken from each test tank, and pretreated for gas chromatography-mass
spectrometry (GC-MS) analysis as follows:
\ Test water
-Water for recovery test 900 mL (graduated cylinder) (only Level l)
-Sodium chloride 300 g (even balance)
-Ethyl acetate 80 mL (graduated cylinder) Shaking for 10 min
Water layer
| Ethyl acetate layer
Filtration and dehydration (20 mmI.D. column, anhydrous sodium sulfate about 20 g)
Evaporation to about 5 - 9 mL (rotary evaporator, about 35 C)
Filling up to 10 mL (ethyl acetate, volumetric flask)
Sample for GC-MS analysis
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*5 Conditions o f column chromatography
Sep-Pak Cu Loading Elution
Whole volume of the solution was loaded. Eluent Acetonitrile/purified water (4/1 V/V) 1 mL
Test substance was eluted with the loaded solution and eluent.
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3.7.3
Quantitative analysis for test substance The samples for GC-MS analysis in pretreatment were analyzed by gas
chromatograph-mass spectrometry under the following analytical conditions. When the concentration o f the test substance in the sample for GC-MS analysis exceeded the range o f the calibration curve in test fish analysis, the sample was analyzed after it was diluted to a concentration within the range o f the calibration curve. The concentration o f the test substance in each sample solution was determined on the basis of a comparison o f the peak area on the mass fragmentogram o f the sample solution with that o f a standard solution (see Tables-6, 7, Fig.6 and Tables-9, 10, 11, Figs.9,10,11).
(1) Analytical conditions Instrument
Gas chromatograph-mass spectrometer Shimadzu Corporation type QP-5000
Conditions of gas chromatograph
Column
30 m x 0.25 mml.D. Fuzed silica
Liquid phase
HP-5MS film thickness 0.25 pm
Column temp.
5 0 C ( 2 min) --9 0 'C -* 200C
Temperature rate
4 C/min, 30 C/min
Injection temp.
220 "C
Interface temp.
250 C
Carrier gas
Helium
Total flow rate
lOmL/min
Column head pressure
40kPa
Injection method
Splitless
Sampling time
2 min
Sample size
1 pL
Conditions of mass spectrometer
Ionization mode
El
Ionization voltage
70 eV
Monitoring ion
m/z95
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(2) Preparation of standard solution The standard solution to determine the concentration o f the test substance in
the sample solutions was prepared as follows.
(a) Analysis of test water 100 mg o f the substance was dissolved in ethyl acetate to prepare 1000
mg/L solution o f the test substance. 100 pg/L standard solution was then prepared from this solution by dilution with ethyl acetate.
(b) Analysis of test fish 100 mg o f the substance was dissolved in acetonitrile to prepare 1000 mg/L
solution of the test substance. 100 pg/L standard solution was then prepared from this solution by dilution with acetonitrile/purified water (4/1 V/V).
(3) Calibration curve
(a) Analysis of test water 50.0, 100 and 200 pg/L standard solutions were prepared by the same
method as described in (2Xa). These solutions were analyzed according to the analytical conditions described in (1). A calibration curve was drawn on the basis o f the relation between the peak area on the mass fragmentograms and the respective concentrations.
In consideration o f the noise level, the lowest detectable peak area o f the test substance was regarded as 200, which corresponded to 2.7 pg/L of the test substance concentration (see Fig.4).
(b) Analysis of test fish 50.0, 100 and 200 pg/L standard solutions were prepared by the same
method as described in (2Xb). These solutions were analyzed according to the analytical conditions described in (1). A calibration curve was drawn on the basis o f the relation between the peak area on the mass fragmentograms and the respective concentrations.
In consideration o f the noise level, the lowest detectable peak area o f the test substance was regarded as 200, which corresponded to 4.2 pg/L o f the test substance concentration (see Fig.7).
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3.7.4 Recovery and blank test
(1) Method Water and fine sample of fish (10 g) spiked a specified amount o f the test
substance for the recovery test were prepared in the same way as described in 3.7.2. The blank test was also performed in the same manner without the test substance. All the recovery and blank tests were performed in duplicate.
(2) Results of recovery test In the blank test, the mass fragmentogram o f GC-MS had no peaks interfering
with determination o f the test substance concentration. The duplicate recovery rates and the average o f them in the pretreatment are shown below (see Tables-5, 8 and Figs.5, 8). The average recovery rate was used as correction factors for the determination of the test substance concentrations in the analytical samples.
For analysis of test water (1 pg test substance added) 78.4%, 82.8% average 80.6%
For analysis of test fish (5 pg test substance added) 79.5%, 81.4% average 80.5%
3.7.5
Lipid content in test fish Lipid contents in the sample for analysis o f lipid content o f Level 1, Level 2
and the control test fish (sec 3.7.2(2)) were determined after chloroform-methanol extraction with gravimetric analysis.
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3.7.6 Calculation o f the test substance concentration in sample and minimum limit o f determination
(1) Calculation of the test substance concentration in test water The equation in Tables-6, 7 was used to obtain the concentrations, and they
were rounded off to 3 figures.
(2) Determination limit o f the test substance in test water
The determination limit** o f the test substance in test water was calculated on
the basis o f that obtained from the calibration curve in section 3.7.3 (3Xa) as
follows.
Level 1
0.33 pg/L
Level 2
0.033 pg/L
(3) Calculation o f the test substance concentration in test fish The equations in T ables-9,10,11 were used to obtain the concentrations, and
they were rounded off to 3 figures.
(4) Determination limit o f the test substance in test fish Assuming the fine sample o f fish to be 5 g, the determination limit'5 o f the
test substance in test fish was calculated to be 26 ng/g on the basis o f that obtained from the calibration curve in section 3.7.3 (3Xb).
*5 Minimum determination limit of the test substance (pg/L or ng/g)
A " _B_ C * E
100 * D
where A : Minimum determination limit of the test substance on the calibration curve (pg/L) B : Recovery rate (%) C : Sampling volume of test water (mL) or fine sample o f fish (g) D : Final volume o f sample solution (mL) E : Ratio o f the portion, used for analysis to whole volume
Results were rounded off to 2 figures.
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3.7.7 Calculation o f mean concentration of the test substance in test water (duration of exposure)
Cwt { Cw (l) + .......... + C w (n)} / n
where Cwt n Cw(l) Cw(n)
The mean concentration o f the test substance in test water (pg/L) Number o f analysis for test water (mesurement times) Concentration o f the-test substance in 1st analysis o f test water (pg/L) Concentration o f the test substance in n-th analysis o f test water (pg/L)
3.7.8 Calculation of bioconcentration factor (BCF) Bioconcentration factor (BCF) was calculated as follows.
(1) Calculation o f mean concentration o f the test substance in test water for calculating W BCF
Cw = { Cw(n-1) + C w (n)) / 2 (only 1st analysis o f test fish) Cw = { Cw(n-2) + Cw(n-l) + Cw (n)} / 3 (from 2nd analysis o f test fish)
where Cw
Cw(n)
The mean concentration o f the test substance in test water for calculating BCF (pg/L) Concentration o f the test substance in n-th analysis of test water (pg/L)
(2) Calculation o f bioconcentration factor
Cf BCF = -----= -----
Cw
where BCF Cf Cw
Bioconcentration factor Concentration of the test substance in test fish (ng/g) The mean concentration of the test substance in test water for calculating BCF (pg/L)
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3.7.9 Calculable BCF On the basis of the minimum determination limit o f the test substance in section
3.7.6 (4), BCF can be obtained when BCF exceeds the following. The mean concentration of the test substance in test water obtained from all the analyzed sample was used to calculate the following calculable BCF.
Level 1 Level 2
2.8 . 30
3.7.10 Calculation of lipid content Lipid contents were calculated according to the following equation.
Lipid content (%) = ( T - To) / S * 100
where To Weight o f vessel (g) T Weight o f sample for gravimetric analysis (containing vessel) (g) S Weight o f fine sample taken out for analysis o f lipid content (g)
3.8 Treatment of numerical values Values were rounded o ff in accordance with JIS Z 8401:1999 rule B. The each
value used for calculation was used without rounding off on the way of the calculation. The concentration values o f the test substance in test water and fish were rounded
off to 3 figures. BCFss values were rounded off to 2 figures.
4. Factors possibly affecting accuracy No adverse effects on the reliability of this test were noted.
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5. Results
5.1 Concentration o f test substance in test water The measured concentrations o f the test substance in test water are shown in
Table-1. Each concentration o f the test substance was maintained at more than 75 % of each the nominated concentration. The variation o f the concentrations o f the test substance was within 20 % o f the mean o f the measured concentrations.
Table-1 Measured concentrations of the test substance in test water (U nit: ng/L)
After Level
3 days
After 7 days
After 14 days
After 21 days
After 28 days
After 42 days
After 60 days
Average (Standard deviation)
Table
Fig.
1 8.63 8.70 9.54 8.77 9.05 10.1
9.74 9.22 (0.584)
6
0.844 2 0.769 0.759 0.866 0.861 0.827 0.947 0.876 (0.0652) 7
6
5.2 Bioconcentration factors BCFs are shown in Table-2. BCFs in Table-2 plotted against duration o f exposure are shown in Fig.l and 2. These BCFs o f the test substance ranged from 200 to 1100 at Level 1 and from 87
to 310 at Level 2.
Table-2 BCFs
( ) : average value
Level 1
After 7 days
200 560 (380)
After 14 days
890 210 (550)
After 21 days
290 550 (420)
After 28 days
1000 900 (950)
After 42 days
1100 450 (780)
After 60 days
460 820 (640)
Table 9
Fig. 9
130 180 310 210 130 150
2 170 140 120 98 87 190 10 10
(150)
(160)
(210)
(150)
(110)
(170)
-23-
000027
51518
5.3 Calculation.of BCFs at a steady-state (BCFss) Because the variation o f BCFs (average values) for three successive analyses
containing maximum value was over 20 %, BCFss were not calculated. The range o f all BCFs (maximum - minimum) in the period of exposure were reported.
5.4 Lipid content in test fish The measured lipid contents in the test fish are shown as follows.
Before initiation o f exposure After termination o f exposure
4.20 % 3.97 %
5.5 Results o f test fish observation No abnormality in behavior or appearance was noted.
6. Reference test
6.1 Objective Those tests were performed to clarify the clearance process o f the test substance
from test fish and to determine the distributions of test substance in all the parts.
6.2 Analysis in parts of test fish
6.2.1
Test method The fish which were exposed for 64 days were separated into parts; integument,
head, viscera and remaining matter were weighed separately. The integument consisted of the skin except head, scales, fin, alimentary canal or gills. The viscera consisted o f internal organs except alimentary canal. The test substance in all the parts was determined in the same manner as section 3.7.
6.2.2
Results BCFs was calculated according to the equation in Table-12, 13. The results were
rounded off to 3 figures at the concentration of test substance and 2 figures at BCFs, respectively.
In case of no detection, the concentration of the test substance was calculated on the basis o f the equation of section 3.7.6(4).
The fine sample of fish (C : the equation of section 3.7.6(4)) was calculated using the mean o f the weight in every parts, subsequently calculated the calculatable BCFs.
Concentrations and BCFs o f the test substance in all the parts were shown in Table-3.
-24-
000028
51518
Table-3 Concentrations and BCFs of the test substance in all the parts
Level
Parts
Concentration in each part (ng/g)
Integument
4840 3700
BCF
500 380
Head 1
Viscera
5560 3830^
8330 8850
580 400
860 920
Remainder Parts
2940 1860
300 190
Integument
<152 <152
<170 <170
Head 2
Viscera
<70.9 167
<462 <462
<80 190
<520 <520
Remainder Parts
66.5 67.1
75 76
Table 12
13
Fig. 15
16
-25 -
00009
51518
6.3 Depuration test
6.3.1 Test method After the test fish were exposed for 64 days, this depuration test was started by
transferring them to another test tank (without test substance and dispersant).
<Test condition> Test tank Flow rate o f test water
Test temperature
100-L glass tank for volatile substance 1600 mL/min for dilution w a te r; 2304 liters/day for test water were supplied. 25 2 C
6.3.2 Residual rate o f the test substance Residual rate o f the test substance in the test fish were calculated as follows :
Fn W PFn CFo x 100
PFn : Residual rate of the test substance in test fish on n day from start of this test (%)
Fn : Absolute quantity of the test substance on n day from start o f this test (ng)
W : Weight o f test fish (g) CFm : The average concentration of the test substance in test fish on 60
days from start of this test (ng/g)
-26-
000030
51518
6.3.3 Results The residual rates (%) o f the test substance in test fish after 2 and 7 days from the
initiation of depuration were calculated by comparison with the average concentration (two fish) o f the test substance in test fish after 60 days (see Tables-14,15, Figs. 17,18).
Table-4 Residual rate o f test substance
(Unit : %)
Level 1 2
After 2 days
0.87 20
"6 *6
After 7'days
_*6
6 6
Table
Fig.
14 17
15 18
*6 Undetectable
Because the concentration of the test substance was undetectable after 7 days at Level 1,2 and 7 days at Level 2, the depuration half-value (tin) were not calculated.
-27-
000031
51518
7. Discussion (1 ) BCF After 60 days, the variation o f BCF is over 20 % in analysis o f last three times. It is considered that the cause of change of BCF is an individual difference. This change was beyond 20 % in spite o f the correction of BCF by lipid content In the exposure periods between 60 days, the maximum value of BCFs was obtained after 42 days at Level 1, 21 days at Level 2. Afterward the increase o f BCFs was not obserbed. Because BCFss at steady-state were not calculated, the range o f all BCFs (maximum minimum) in the period o f exposure were reported. (2) Reference materials Analysis in parts o f test fish and depuration test was performed as reference materials. BCF of the test substance in viscera was higher than that in other parts. Because the residue of test substance in analysis o f test fish after 2 days was less than 20 %, it is considered that the rate o f depuration of the test substance is extremely fast. (3) Metabolites Analysis of expected metabolite (carboxylic acid) a-carbon o f test substance oxidized was performed by using o f test fish after 60 days. Consequently, a peak was detected that was not found in the control. It was difficult to identity the stracture of this peak by LC-MS. BCF o f the metabolite calculated using the concentration o f the test substance was approximately from 64 to 94.
-28-
000032
51518
8. 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 GMW
Instrument for measuring concentration o f dissolved oxygen :
* Iijima Seimitsu Industries Co., Ltd.
pH m eter:
typeF-102 Toa Electronics Ltd. type HM-14P
(2) Instruments, apparatuses, special apparatuses and reagents
Instruments and apparatuses
Gas chromatograph-mass spectrometer:
seepage 17
Electronic analytical balance: Sartorius typeBP30IS
Sartorius type 1216MP
Shimadzu Corporation type AEX-200B
Metier Toledo type AE163
A&D CO., LTD type FA-2000
Rotary evaporator :
Tokyo Rika Kikai Co., Ltd. type N-l
Tokyo Rika Kikai Co., Ltd. type N
Mechanical shaker :
Taitec Co., Ltd. type SR- IIW
Homogenizer (polytron) :
Kinematica. type PT300
Centrifuge :
Kubota Syouji Co., Ltd type 6900
Special apparatuses Sep-PakCu :
Nihon Waters K. K.
Reagents
Acetonitrile (HPLC grade):
Wako Pure Chemical Industries, Ltd.
Purified w ater:
Takasugi Seiyaku Co., Ltd.
Ethyl acetate (extra pure) :
Kanto Chemical Co., Inc.
Sodium chloride (extra pu re): MANAC Incorporated
HCO-20:
Nikko Chemicals Co., Ltd.
Anhydrous sodium sulfate (extra pure) :
Katayama Chemical Industries Co., Ltd.
-29-
000033
51518
(3) Instruments, apparatuses and reagents for gravimetric analysis of lipid content in test fish
Instruments and apparatuses
Electronic analytical balance: Metier Toledo type AE 163
Sartorius type BP301S
Rotary evaporator:
Tokyo Rika Kikai Co., Ltd. type N -1
Tokyo Rika Kikai Co., Ltd. type N
Homogenizer (autocellmaster): Iuchiseieido Co., Ltd. type CM-200
Vacuum pum p:
Sinku Kiko Co., Ltd. type DA-20D
Sinku Kiko Co., Ltd. type DAH-20C
Vacuum desiccator:
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) :
Katayama Chemical Industries Co., Ltd.
(4) Apparatuses and instruments for stracanai confirmation of test substance Fourier transform infrared spectrometer : Shimadzu Corporation type FTIR-8200PC Gas chromatograph-mass spectrometer : JEOLLTD. type JMS-700QQ Nuclear Magnetic Resonance : Hitachi, Ltd. type R-3000
-30-
000034
Table-5
Calculation table for recovery and blank test (Analysis of test water)
Sample description
A
B
C
D
TestNo. 51518 EF
Standard 100;r g/L Recovery a Recovery b
7232 5668 5986
1 . 10 1 10
Standard 100 u g/L Blank a Blank b
7048 n.d. n.d.
( a, b : individual sample )
1 1
10 10
784 78.4 % - 828 82.8 /.
Average 80.6 /.
-.
A : Peak area A (std): Standard solution A (t): Sample
B : Ratio of portion used for analysis C : Final volume (mL) D : Amount o f blank in test water (ng) E : Amount of test substance recovered (ng)
E -P * (A(t)/A(std))/B*C-D F : Recovery rate (%)
F - E / Q * 100 P : Concentration of test substance in standard solution Q : Amount o f test substance added ( lOOOng) See Fig. 5
100 m g/L
January 26,2001 Name /f . T-i/j 'ih e h e
00003S
Tablc-6 Calculation tabic for analysis of test water (Level 1)
Sample description
Standard 100 mg/L Test water after 3 days
Standard 100 mg/L Test water after 7 days
Standard 100 mg/L Test water after 14 days
Standard 100 mg/L Test water after 21 days
Standard 100 mg/L Test water after 28 days
Standard 100 mg/L Test water after 42 days
Standard 100 mg/L Test water after 60 days
A
6252 4345
5457 3*826
6121 4703
5703 4029
7015 5114
3681 3004
8339 6542
Test No. 51518 I
8.63 8.70 9.54 8.77 9.05 10.1 9.74
Average concentration of test substance in test water 9.22 (S.D. 0.584)
A : Peak area A(std) : Standard solution A (t): Sample
B : Ratio o f portion used for analysis
1
C : Final volume lOmL
F : Recovery rate 80.6%
H : Volume of test water taken out
lOOmL
1: Concentration of test substance in test water ( it g/L)
I Px( A(t )/ A(s td)) /B*C/Fx 100/H
J : Average concentration of test substance in test water J - ( I ( l ) + ... + n ) ) / n
n : Number of test water analyses ( n " 7 )
1(1): First analysis o f test water I (n) : Last analysis of test water
n* 2 0 2 - f 2 K o f
S.d. - l -- i - i i --------1 ' " 1- l | a x (a - 1)
P : Concentration of test substance in standard solution
See Fig. 6
100 it g/L
February 1,2001
Name / f - F ^ J ' u i0^~e
000036
Table-7
Calculation table for analysis of test water (Level 2)
Sample description
Standard 100 u g/L Test water after 3 days
Standard 100 mg/L Test water after 7 days
Standard lOOng/L Test water after 14 days
Standard 100 mg/L Test water after 21 days
Standard 100 mg/L Test water after 28 days
Standard 100 mg/L Test water after 42 days
Standard 100 mg/L Test water after 60 days
A
6252 3876
5457 3336
6121 4272
5703 3958
7015 4674
3681 2808
8339 5883
Test No. 51518 I
0.769 0.759 0.866 0.861 0.827 0.947 0.876
Average concentration of test substance in test water 0.844 (S.D. 0.0652)
A : Peak area
A(std): Standard solution A(t) : Sample
B : Ratio of portion used for analysis
1
C : Final volume lOmL
F : Recovery rate 80.6%
H : Volume o f test water taken out
1,000mL
I : Concentration of test substance in test water ( mg/L)
I " P * ( A(t) / A(std) ) / B * C / F * 100/ H
J : Average concentration of test substance in test water J ( I(l) + ... + I(n) ) / n
n : Number of test water analyses ( n - 7 )
I (1): First analysis of test water I (n ): Last analysis o f test water
W -f S.D. . 1 -- !-=J_____ U -.L . L
1 n >. (n -1)
P : Concentration of standard solution
See Fig. 6
100 n g/L
February 1,2001
Name
'f U( Q 'to
000037
Table-8
Calculation table for recovery and blank test (Analysis of test fish)
Sample description
A
c
DE
Test No. 51518 FG
Standard 100 mg/L Recovery a Recovery b
4138 3289 3370
4/20 4/20
5 3
Standard 100 m g/L Blank a Blank b
4336 n.d. n.d.
4/20 4/20
5 5
-
( a, b : individual sample )
A : Peak area
A(std): Standard solution A(t) : Sample
B : Ratio of portion used for analysis (fish homogenate) 1/2
C : Ratio of portion used for analysis (extracted solution)
D : Final volume (mL)
: Amount of blank in test fish (ng)
F : Amount of test substance recovered (ng)
F * P { A(t) / A(std) ) / B / C * D - E
G : Recovery rate (%)
G - F / Q * 100
P : Concentration of test substance in standard solution
100 m g/L
Q : Amount o f test substance added (5000ng)
See Fig. 8
3970 4070
79.5 % 81.4% Average 80.5 %
-
January 26,2001
Name K . F o j h n - h
00003$
Table-9-2
Calculation table for analysis of test fish (Level 1)
Sample description
Test No. 51518 A DGKH J
Standard 100 ii g/L Test fish after 60 days a Test fish after 60 days b
5771 4012 4690
10 4.88 4430 9.64 460 15 4.79 7910 9.64 820
( a, b : individual sample )_____________________________________________________________
.
A : Peak area( n V 'saty ^ yo>'
-*i*
A(std): Standard solution A (t): Sample
B : Ratio of portion used for analysis 4/20
C : Final volume 5mL
D : Dilution factor
E : Average concentration of blank in analysis o f control Ong/g
F : Recovery rate SO.3%
G : Weight of fine sample (g)
K : Concentration of test substance in test fish (ng/g)
K - { P * ( A ( t ) / A ( s t d ) ) / B * D * C / G - E } / F * 100 H : Average concentration of test substance in test water ( n g/L)
H " { I(n-2) + I(n-1) + I(n) } / m ; n : Number of teat water analyses ; m 2 when a * 2, m - 3 when n 2 3
I : Concentration of test substance in test water ( n g/L)
J : 8CF
J-K/H
P : Concentration o f test substance in standard solution 100 ^ g/L
See Fig. 9
January 26,2001
Name
000039
Table-10-1
Calculation table for analysis of test fish (Level 2)
Sample description
A D G K.
T ea No. 5IS 18 HJ
Standard 100 m g/L Test fish after 7 days a Test fish after 7 daysb
3484 543 697
1
4.83
100 0.764
130
1 4.91 127 0.764 170
Standard 100 m g/L Test fish after 14 days a Test fish after 14 days b
4837 1097 873
1 4.84 146 0.798 180
1
4.91
114 0.798
140
Standard 100 m g/L Test fish after 21 days a Test fish after 21 days b
3610 1407 581
1
4.79
253
0.829
310
1 4.85 103 0.829 120
Standard 100m g/L Test fish after 28 days a Test fish after 28 daysb
4989 1387 644
1 4.92 176 0.852 210
1
4.82 83.2 0.852
98
Standard 100 m g/L Test fish after 42 days a Test fish after 42 daysb
2240 410 269
1 4.87 117 0.878 130 1 4.87 76.6 0.878 87
( a, b : individual sample )___________________________________________________________________
A : Peak area.( jt Vi-sccfc-
8JT
A(std) : Standard solution A(t) : Sample
B : Ratio of portion used for analysis 4/20
C : Final volume 5mL
D : Dilution factor
E : Average concentration of blank in analysis o f control Ong/g
F : Recovery rate 80.3%
G : Weight o f fine sample (g)
K : Concentration of test substance in test fish (ng/g)
K - { P * ( A ( t ) / A ( s t d ) ) / B * D * C / G - E } / F * 100
H : Average concentration of test substance in test water ( mg/L) H { l(n-2) + l(n-l) + I(n) } / m ; n Number of test water analyses ; m - 2 when n - 2, m - 3 when n 3
I : Concentration of test substance in test water ( mg/L)
J : BCF
J-K/H
P : Concentration of test substance in standard solution 100 mg/L
See Fig.10
January 2d. 2001
Name
000040
Table-10-2
Calculation table for analysis o f test fish (Level 2)
Sample description
A D GK
Test Ko. 51518 HJ
Standard 100 mg/L Test fish after 60 days a Test fish after 60 daysb
5766 1203 1492
1
4.84
134 0.883
150
1 4.86 165 0.883 190
( a. b : individual sample )________________________________________________________ _
A : Peak area<B3s*e
f. & *
A(std) : Standard solution A(t) : Sample
B : Ratio o f portion used for analysis 4/20
C : Final volume SmL
D : Dilution factor
E : Average concentration of blank in analysis of control Ong/g
F : Recovery rate 80.5%
G : Weight of fine sample (g)
K : Concentration of test substance in test fish (ng/g)
K - { P * ( A (t )/ A (std) ) / B * D * C / G - E } / F x 100
H : Average concentration of test substance in test water ( u g/V)
H " { I(n-2) + I(n-l)+ l(n) } /m ; n : Number of test water analyses ; m " 2 when n - 2, m - 3 when 3
I : Concentration of test substance in test water ( u g/L)
J : BCF
J-K/H
P : Concentration of test substance in standard solution
100 n g/L
SeeFig.10
January 26,2001
Kme
000041
T a b le - 11
Calculation table for analysis of test fish (Control)
Sample description
AE
Standard 100 u g/L Before the experimental start a Before the experimental start b
4451 n.d. n.d.
Test No. 51518 G1
5.02 5.02 *
Standard 100 n g/L
5929
After the experimental completion a n.d. - 4.80
After the experimental completion b n.d. - 4.80
( a, b : individual sample )
A : Peak arca fri A(std) : Standard solution A(t) : Sample
B : Ratio of portion used for analysis 4/20 C : Final volume 5mL E : Amount of blank in analysis o f control (ng)
E" P * ( A(t) / A(sCd) ) / B C G : Weight of fine sample (g) I : Concentration of blank in test fish (ng/g)
I-E/G P : Concentration of test substance in standard solution See Fig. 11
100 p g/L
-
January 30,2001
Name
000042
Table-12
Calculation table for analysis in parts o f test fish (Level 1)
Sample description
Standard 100;; g/L Teguments* a Teguments* b 'Including intestine and gill
Standard 100^ g/L Head a Head b
A
5250 3806 3627
4794 5249 3025
DG K
3.333 1.55 4840 . 2 1.16 3700
5 3.06 5560 5 2.56 3830
Test No. 51318 HJ
9.64 500 9.64 380
9.64 580 9.64 400
Standard 100 u g/L Viscera a Viscera b
4720 6833 4384
1
0.540
8330
9.64
860
1
0.326
8850
9.64
920
Standard 100 u g/L Remainder parts a Remainder parts b
5074 4010 2490
5
4.18 2940 9.64
300
5
4.09 1860 9.64
190
( a, b : individual sample )
A : Peak area
A(std) : Standard solution A(t) : Sample
B : Ratio o f portion used for analysis 4/20
C : Final volume SmL
D : Dilution factor
E : Average concentration of blank in analysis of control Ong/g
F : Recovety rate
80.5%
G : Weight of part (g)
K : Concentration of test substance in test fish (ng/g)
K" {P*A{t)/A(std)/B*DxC/G-E}/F*100
H : Average concentration of test substance in test water ( u g/L)
J : BCF
J-K/H
P : Concentration of test substance in standard solution
100 u g/L
See Fig. 15
February 5,2001
Name A'- F w i Ml f o
000043
Table-13
Calculation table for analysis in parts of test fish (Level 2)
Sample description
Standard lOOug/L Teguments* a Teguments* b ` Including intestine and gill
Standard 100 m g/L Head a Head b
A
5848 n.d. n.d.
5096 n.d. 496
DG K
1 0.84 1 0.87 *
-
1 1.87 1 1.81 167
Test No. 51518 HJ
0.883 0.883
.
-
0.883 0.883
-
190
Standard 100 m g/L Viscera a Viscera b
4871 n.d. n.d.
1 0.274 - 0.883 1 0.290 - 0.883 -
Standard 100 p g/L Remainder parts a Remainder parts b
5199 316 291
1 2.84 66.5 0.883 75 1 2.59 67.1 0.883 76
( a, b : individual sample )
A : Peak area
A(std) : Standard solution A(t) : Sample
B : Ratio of portion used for analysis
4/20
C : Final volume 5mL
D : Dilution factor
E : Average concentration of blank in analysis of control Ong/g
F : Recovery rate 80.5%
C : Weight of part (g)
K : Concentration of test substance in test fish (ng/g)
K _ { P x A ( t ) / A ( s t d ) / B * D x C / G - E } / F * 100
H : Average concentration of test substance in test water ( p g/L)
J : BCF
J-K/H
P : Concentration of test substance in standard solution 100 p g/L
See Fig. 16
February i,2001
Name M
(K jfv
000044
Table* 14
Calculation table for analysis of test fish Depuration test (Level 1)
Sample description
A DG
Standard 100 /x g/L Test fish after 2 days a
Standard 100 m g/L Test fish after 2 daysb
Standard 100 mg/L Test fish after 7 days a Test fish after 7 days b
4332 372
4951 1896
7820 n.& n.d.
1 4.95
5 4.87
1 4.76 1 4.88
TestNo. 515IS KM
53.9 0.87
1220 20
--*
( a, b : individual ample )
A : Peak area
A(std) : Standard solution A(t) : Sample
B : Ratio ofportion used for analysis 4/20
C : Final volume 5mL
D : Dilution factor
E : Average concentration of blank in analysis of control Ong/g
F : Recovery rate 80.2%
G : Weight of fine sample (g)
K : Concentration of test substance in test fish (ng/g)
K ", { P x A ( t ) / A ( s t d ) / B * D * C / G - E } / F * 100
L : Average concentration of test substance in test fish after 60 days 6 170ng/g
M : Residual rate (/)
M - K / L * 100
P : Concentration of test substance in standard solution
100 mg/L
See Fig. 17
February 6,2001
Name
000045
Table-15
Calculation table for analysis of test tish Depuration test (Level 2)
Sample description
Standard 100 u g/L Test fish after 2 days a Test fish after 2 days b
Standard 100 u g/L Test fish after 7 days a Test fish after 7 days b
A
4662 n.d. n.d.
3866 n.d. n.d.
D
1 1
1 1
G
4.98 4.81
4.82 4.71
TestNo. 51518 KM
-.
--
-.
-
( a, b : individual sample )
A : Peak area
A (std): Standard solution A (t): Sample
B : Ratio of portion used for analysis 4/20
C : Final volume 3mL
D : Dilution factor
: Average concentration of blank in analysis of control Ong/g
F : Recovery rate SO.3%
C : Weight of fine sample (g)
K : Concentration of test substance in test fish (ng/g) K " { P x A ( t ) / A ( s t d ) / B * D * C / G - E } / F x 100
L : Average concentration of test substance in test fish after 60 days
M : Residual rate (%)
M - K / L 100
P : Concentration of test substance in standard solution
100 n g/L
See F ig 18
ISOng/g
February 6,2001
Name A '. F y jt in y f o
000046
Reference 1
Analytical results of dilution water
Item Total hardness (Ca, Mg) Suspended solid pH Total organic carbon Chemical oxygen demand Residual chlorine Ammonia nitrogen Total cyan Alkalinity Electric conductivity Organic phosphorus Alkylmercury Mercury Cadmium Crs* Lead Arsenic Iron Copper Cobalt Manganese Zinc Aluminium Nickel Silver Organochlorine pesticides
1, 2-Dichloropropane Chlorothalonil Propyzamide Chlornitrofen Simazine Thiobencarb Organophosphorous pesticides Diazinon Isoxathion Fenitrothion EPN Dichlorvos Iprobenfos PCB
Sampling date August 8, 2000
Unit mg/L mg/L
mg/L mg/L mg/L mg/L mg/L mg/L p.s/cm mg/L mg/L rag/L mg/L mg/L mg/L mg/L rag/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L
Measured value 111 <1 7.8 1.8 < 0.5 < 0.01 < 0.01 < 0.01
- 96. 1 374 < 0.1 < 0.0005 < 0. 0005 < 0.005 < 0.02 < 0.005 0. 002 < 0.01 < 0.005 < 0.001 < 0.01 < 0.01 < 0.001 < 0.001 < 0.0001
Standard value < 300 ,l < 20 6.5 ' 8. 5 43 < 2 42 < 5 43 < 0.02 43 < 1 43 n. d.*3 -- -
n. d.43 n. d.43 < 0. 0005 *3 < 0.01 *3 < 0. 05 *3 < 0. 1 43 < 0.05 *3 < 1.0 *3 < 0. 005 43 < 0.001 " < 0.05 ,l < 1.0 41 < 0.2 41 < 0.01 " < 0.0001 48
Detection limit
1
0. 1 0.5 0.01 0.01 0.01
0. 1 0. 0005 0. 0005 0. 005 0. 02 0.005 0. 002 0.01 0. 005 0. 001 0.01 0. 01 0. 001 0. 001 0. 0001
mg/L mg/L mg/L mg/L mg/L mg/L
< 0. 006 < 0.004 < 0.0008 < 0.0001 < 0.0003 < 0.002
< 0.06 44 < 0. 04 44 < 0. 008 *4 < 0.0001 41 < 0.003 44 < 0. 02 44
0. 006 0.004 0. 0008 0. 0001 0. 0003 0. 002
rag/L mg/L mg/L mg/L mg/L mg/L rag/L
< 0.0005 < 0.0008 < 0.0003 < 0.0006 < 0.001 < 0.0008 < 0.0005
< 0. 005 44 < 0. 008 *4 < 0.003 *4 < 0.006 44 < 0.01 *4 < 0. 008 44
n. d.44
0. 0005 0. 0008 0. 0003 0. 0006 0. 001 0. 0008 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,
March 1983)
*4 Environmental Quality Standards for Water Pollutants No. 14 (Revised February
22,1999, Environment Agency)
*5 OECD Guidelines for Testing of Chemicals, Bioconcentration Flow-through Fish
Test (Guideline 305, June 14, 1996)
2 f f o i- ^ f-yjiM bo
000047
Bioconcentration factor
10'
i<r o o
10"
o 0
Test No. 51318
o o
o0
10'
'Hie minimum calculation limit of bioconcentration 10' _i____ i____ i i_____i____ i_____i____ i_
0 5 10 15 20 25 30 35 40 45 50 55 60 Exposure period (days)
Fig. 1 Correlation between exposure period and bioconcentration factor (Level 1).
10'
ioJ
10*
10`
CD
o
8
8
o
o o
o o
The minimum calculation limit of bioconcentntion
iow L------ * 0 5 10 15 20 25 30 35 40 45 50 55 60
Exposure period (days)
Fig. 2 Correlation between exposure period and bioconcentration factor (Level 2).
February 21,2001
Name /(. F ^ J ' W C'f
itralion factor
000048
96-hour LC5Q > 125 mg/L
Test No. 51518
Concentration (mg/L) Control 62.5 125
24 hours 0 0 10
Cumulative Mortality (%) 48 hours 72 hours 00 00 10 10
96 hours 0 0 10
Fig. 3 Concentration - Mortality Curve.
Date : October 30, 2000 Name
000049
t'-flrn : 1-1.020
977*3
: Solt*dt b lu k
IV 9-9
: k.fujiooto
T u t NO. 51511
n w w iN /irc*
"lu4u9.A.V.
ID : 1 S S * : 91.00
9f7* : T arnt 0 * : SlSll
attun : T :0
15.00 IHK : 0.000(1.0.)
aa. r-*
P ,, .ia/.dlioo.. irt
T u t No. 51511
t ' - t 7 * 3 * : 9-3.021
777' 9 4
: Standard aolutlon SO.Oot/l
KHi
: k.fujiooto
2000/01/W 4
uftl
10 : 1 K U 9<7* : T arnt 3 * 0 * : SlSll
tl*n : T.I27
SB : 3393
13.00 K
: 0.000
: 9S.00
an
h
K- F<\/
*+5."
T u t Ko. SlSll
r-97W 3* : 9-3.022
977*3
: Standard aolotton lOOuf/l
94*9-9*
: k.fttjinoto
2000/09/0 04
"U.M
10 9* 0
to n n a sw
1 K U : 95.00 Tarnt SlSll 7.130 71S5 0.000
Test No. 51518
X. F \f* i* o
Taat Ko. SlSll
9*-97rf9 : 9-3.023
977*94
: Standard tolutIon ZOCut/1
24*9-9
: k.fnjinoto
2000/09/0 o t
i.ttau NI
10 : 1 K U
9<7* : Tortot
0 * : SlSll
SS""95.00 aus
T.S27
01.S000900
: 93.00
3a. f 3**'' u2n* rtX
X- rtfavtl
Fig. 4-1
Mass fragmentogram of GC-M S analysis for calibraon curve (test water).
000050
Test No. 51513
Peak area
Concentration (jip/L)
Conditions o f GC-MS analysis
Instrument
Shimadzu QP-5000
Sample
K-1518
Solvent
Ethyl acetate
GC Condition
Injection vol.
UL
Column (Size) : HP-5MS (30m x 0.25mm LD-Jilm thickness 0 2 5 u
Col. temp.
: 5Qt:(2min.) - 90X3- 2001;
Col. temp, rate
4 t/m in ., 30*C/min.
Interlace temp.
250X3
Injection temp. : 220X3
Carrier Oas
He
- Total flow rate
lOmLAnin.
Column head press: 40kPa
Injection method : Splitless
Sampling time
2min.
MS Condition Ionization mode : El
Ionization vo lt : 70V
Monitoring ion
m/z 95
Calibration curve and conditions o f GC-MS analysis for K-1518
Febnuiy 21,2001
% F e lla h
000051
Test No. 51518
r 77-01 : 9-3. D25
Vj7'%%i
: Standard solution 100ug/L
fV l/-J
; k.fujiaoto
2000/09/88* of lW \u .tt fVS Test No* 51518
ID nr
Smi*m8W
95.00 * *
1 * * : 95.00 Target 51518 7.830 7232 0.000
*>* i 4 -an n - fr-x. ?~n 'T ^
K fi u 'rfb
Test No. 51518 2000/09/ o f
?*-*7rl : 9-3.D25
iW 'l
: lecovery test froa test eater a
TV : k.fujiaoto
ID nr fi2^
DB 95.00 * *
1 K * t t : 95.00 Target 51518 7.820 5668 0.000
se.***
Test No. 51518 2000/09/M Of
UT. AJ
T*-*7rMS : 9-3.D27
*'***
W *45
: lecovery te st from test water b
TVH i
: k.fujiioto
ID W
mm
95.00 P &
JCSft : 95.00 Target
51518
7.823 5986 0.000
K - F^ i+o av{>I
Fig. 5-1
Mass fragmentogram of GC-MS analysis for recovery and blank test (analysis of test water).
000052
Test No. 51518 2000/09/' ^
f*-*7r<45 : 9-3.D29
Ts7` &
: Standard solution lOOug/L
KHi
: k.fujiioto
54* : J a . M T" tN0, 51518
nIDr mmmmmm m
i X f tf t : 95.00 Target 51518 7.833 7048 0.000
jw > . jjM . \ 1l
~ --A J --n t, j L i.11 n
^ ).|.3 0 W
Test No. 51518
r - 9 7 T < * % : 9-3.D30
W7*ft:S
: (lank test for test water a
: k.fujisoto
2000/09/ 0*t ft*
ID HT
fmtm# #
95.00 * *
i X S f t : 95.00 Target 51518 0 0.000(N.D.)
Test No. 51518
r - t 7 T < V & : 9-3.D31
fV7*JkS
: (lank test for teat water b
W V-f&
: k,fujioto
2000/09/ 0<t v AXh. *N* t o
ID HT
ft} * # * 95.00 *
1 It f t : 95.00 Target 51518 0.000(N.D.)
Iff & itorfr.
K, f t y c.'*<eb
Fig. 5-2
Mass fragmentogram of GC-MS analysis for recovery and blank test (analysis of test water).
000053
Test No. 51518 2000/10/27
f '- W r : 10-26.D04
: Standard solution lQQug/l
11-9%,
: kfujimoto
Test No. 51518
ID 1 * R HV Targieet
5158
{ * : 7.794 * : 6252 95.00 mat : 0.000
95.00
-2 /?. J 7 li- Fi(j!cio
r - n r i t : 10-26.005
W IV
: A3B
1V H *
: Icfujimoto
U st No. 51518 2000/10/27
w aiev
r 3
o
ID : 1 * * R : 95.00 W : Target J#45 : 51518
fcWM : 7.793
EBflt 4345
00 * *
0.000
1<TV .
a 7 /< F c y lx c r f- o
WM5 KH4
: 10-26.006 : WA3L : kfujimoto
Test No. 51518 2000/10/27 T e s i wert OV a ^ t e r
el 2 )
ID : 1 X S ft : 96.00 H V : Target
: 51518 MWM : 7.793 Bf : 3876 95.00 mat : 0.000
3 0 2 TP.
R g. 6-1
Mass fragmentogram of G C-M S analysis for test water.
K- F<<' 'K0f
000054
r-i77< % : 10-30.004
?JT1t%
: Standard solution lQ0ug/l
KHi
: Icfujiioto
7 *V/ Wi
Test No. 51518
ID : 1 M R : 95.00 9<T : Target
: 51518
* : 7.792 OB* : 5457 95.00 S K : 0.000
20et> /t>. 3 / K - F ty '.'u ttfo
PV.*% .
: 10-30.005 : NA7H : kfujimoto
Test No. 51518 2000/10/31
_ TS"t uwtev* crt-t** T a y s ( l e v e l
`
l)
1$ f 1 M R : 95.00 M7 : Target J * * : 51518
R fcW M : 7.790
QD4H : 3828
95.00 f t
: 0.000
aczv-/ff.3i. K - f y J W i
* '* : lw?-8Si
tVW &
: kfujiaoto
Test No. 51518 2000/10/31
r.rt *
11
c
'1 )
ID HT
OB* 95.00 * *
1 M R : 95.00 Target 51518 7.790 3336 0.000
2 cez>` /D .3 i K .F y jY u O 'fa
Fig. 6-2
Mass fragmentogram of G C-M S analysis for test water.
000055
Test No. 51518 2000/11/07 r ' - W r : 11-6.D04
: Standard solution 100ug/l : kfujiioto
Test No. 51518
ID MV &&
mmmv m
95.00 tide
1 * * : 95.0C Target 51518
7.806 6121 0.000
2 0 7 * . /A 7 K- F ^j''u' t ^
WV-9V7&r< KH
: 11-6:.DW05A14H : kfujinoto
Test No. 51518 2000/11/07 T e s^t lUA,'te' o ,j,W lIfHl- fVlor (/ ,
, l, )\
ID MV A
1 KMifc : 95.00 Target 51518
SS*M : 7.803 IBA : 4703 95.00 : 0.000
JT.//. 7 K% **
*V7*** a` H
: 11-6.D06 : VA14L : kfujinoto
Test No. 51518 2000/11/07
T est wotev- d'fcev 14 d t y s ( L e v e l Z J)
ooeoss
VjT%S, iVlr-fcg
Test No. 51518 11-13.D04
: Standard solution lOOug/l : kfujlioto
2000/11/14
Test No. 51518
ID 94V A ft*
95.00
SB* ax
1 X ftft : 95.00 Tarsot 51518
7.777 5703 0.000
2cv*- n m
rn r~r*j7*^
: 11-1; '^ 1H : kfujiioto
Tost No. 51518 2000/11/14
- ^ s - t wa-fc*v
-2-1 etay * ( L** I 0
ID 94T 95.00 5 5
1 K * & : 95.00 Tarjet 51518 7.778 4029 0.000
2G<&- tf- 14' Fyj'>i*cr}0
fW"~T9%7Sr4t tSi : 11-1:3.V0A0261L
V W 45
: kfujiaoto
Test No. 51518 2000/11/14 J s\ uW.tw* n0i,"Pt eev*r 2.1 otoy-s (
ID : 1 XftSt : 95.00 94T : Target J*dM5 : 51518
ftftftM : 7.774
,, SB*
95.00 g g
: 3958
; o.OOO
2')
****> / / /<* K. F ty w o + o
Fig. 6 -4
Mass fragmentogram of G C-M S analysis for test water.
000057
Test Ho. 51518
?*-*7WS : 11-22.003 : Standard solution lOOug/L : kfttjixoto
2C00/U/23
Test No. 51518
IO UT
SMtK : 95.00 Target
51513
7.780 m a 7015 u t 5.009
20** th y K
r-977<i% : 11-22.D04
W I45
: WA28H
; Irfujiioto
Test Ho. 51518 2000/11/23
,,
M10T
.. _
, , Jl -x
J**- 3 " d a y t ( Lev!
levi. J. it
ia u. ft
1
Target 51513
oo mst.
7.780
501.01040
2-*. i>- l - (<
*^#
f*** V H
: 11-22.D05 HA281
: kfujiaoto
Test No. 51518 2000/11/23
,,,, , , / . . ,, \
T&t im** 4^'t*v 2 8
( Levtl 2 j
000058
Test Ho. 51518 2000/12/05
r'- W : 12-5.Dll
TJ'J'M
: Standard solution ICOug/I.
r : kiajincto
Test No. 51518
TOOOj
0
nr
' a * 95.00
ftuts. : 95.00
Target 51513 T.793 3631 C.OOC
5trrt>. (2. i f
r-T?7<A& : 12--5,1312
: A.42K
h*H i
: Irfujinoto
Test No. 51513 2000/12/05
Test
4 2 oto^<S C Leu I I )
ID 1 XX : 5.00 n r Target
51513
SUB : 7.791
mm : 3004
95.00 X
: 0.000
5 ot* . / 2-X / < - ^ '> d f o
Test No. 51518 2000/12/05
i r S * 6 : 12'5;Dwa42l
T es1 w f' tf * * ^ t y * A ''/ O '
I a*v->45
: kfujiioto
ID nr m nw a mm 95.00 mm
1 X X : 95.00 Target 51518 7.787 2808
0.000
ZM-p.lxcS & F tf* * t*
Fig. 6-6
Mass fragmentogram of GC-M S analysis for test water.
000053
Test No. 51518 2000/12/23
r - W W : 12-22.D08
W*45
: Standard solution 100u*/L
Vlr-
: kfujiaoto
Test No. 51518
S--
1
f. !;.
Ik ...
r
OOOs.
ID 9<T A*
1 R : 95.00 Target 51513
89*181 ; 7.773
9D 3329
95.00 * *
0.000
' ' " I ---------6 ? 8.
>03 . la- 3 3 K -F tyV -rf
r-97rW & : 12-22.D0
W **
: NA60K
4*W
: kfiijinoto
Test No. 51518 2000/12/23 T fl WftieV' a ^ -i* w' 0 J a y # ( L e u ,1 f )
7000 ID W
m m ! S9 55.00 *
! K ft& : 95.00 Target 51513 .
7.780
6542
3.000
! 0?
aw. io. 3S K-
r-477-M : 12-22.DIO : NMOL : Itfujinoto
Test No. 51518 2000/12/23 T esi
'3- )
ooooeo
t'-nrtt* W
24*9-9*
T u t h . 5151 9-21.021
loliw Uuk
: ijiMta
2000/09/21
nior 99.00 e s
BB
tut
:: T1iri! X B B : 99.00
: $1511
?
:0 : 0.000(9.0.)
9c 1-
/. r.jc
X- s<g\'yh IR-"1-
T ot 9. 51911 2000/09/22
f- f 7 r it* : 9-22.022
tW'i S
:: Iktfiuod'laioMtoaliti o 50.0uc/l
MIDV
95.00 * * *
1 USB
Tunt 51519
N M : 1.707 B B : 2342 S B : 0.000
92.00
y*. f.O*
w o it i,
x-
31 ' *x
Test 9. 61512 2000/09/29
r-w* . 9-22.023
1tiV*7V*-I9**
:: 2UttutjdiMudUsalutloc 100si/l
n10r 95.00 * *
: 1 XBK
: Tutet : 51619
aWmBB
BK
7.717
405.03030
95.00
g.1# 3l. H i *
KF t f `
T ut 9>. 51512
f - n r ih * : 9-22.024
t>7*M5
: StudirO l o l i t iu 200s*/l
2V9-9
: bfillMte
2000/09/22
ID : 1 K S B : 95.00
n r : Tunt
95.00 f t * : 51519
aBKmKB B
: 7.707
:: 095.03010
Test No. 5151B
T'U'.-'V e 3Tr*j
Fig. 7-1
Mass fragmentogram of GC-M S analysis for calibration curve (test fish).
ooootii
Test No. 51518 2000/09/28
r - 9 : 9-28.DOS
VJ7'h%t
: Standard solution 100ug/L
A*l/-g
; kfujiaoto
Test No. 51518
ID HT B9C5.0n0n S#
E* WkX.
: 1 KMS : 95.00 : Target : 51518
: 7.723 : 4138 : 0.000
5TJ0. - J i t ....y a t. t
/<. f i y , /w iTa
Test No. 51518 2000/09/28
'-*7rM'* : 9-28.D07
WK
: Recovery test froa te st fish a
V : kfujiaoto
ID MT
95.00 #I*W OB *X
1 It MR : 95.00 Target 51518 7.713 3289 0.000
y&so. f. 7-S
O / I* J t * T
. f t `t'nififo
Test No. 51518 2000/09/28
r-97ri%4k : 9-28.D08
f77*J45
: Recovery test iroa te st fish h
fVI'-MS
: kfujiaoto
ID 9<T 95.00 * *
* ft*
: 1 KMS : 95.00 : Target
: 51518 : 7.697 : 3370 : 0.000
Fig. 8-1
Mass fragmentogram of GC-MS analysis for recovery and blank test (analysis of test fish).
000062
Teat No. 51518 2000/09/28
V-977-i : 9-28.:D1S0tandard. so,lution lQOug/L
KW
: kfujinoto
TastN o. 51518
ID nr 62- 95.00 ft* * U K*
: 1 ** : Target : 51518
: T.7Q0 :' i0H.00L0
: 35.00
JCBO . ?
2.0 I * 5 'L * . jIX '2-TM v
Test No. 51518 2000/09/28
?r'7-7**7j|7r&-f : 9-28:.DBlllank test Tor test fish a
VV-f
: kfujinoto
nIDr
95.00 Ad
1 M f c : 95.00 Target 51518
a* ax
0.00Q(N.D.)
ysm . *?- ^
/C- ^
j-/
i'? .r
Test No. 51518 2000/09/28
r - n n m .- 9-28.012
f>7**
: Blank test for te st fish b
*VI<-dg
: kfujisoto
nIDr
95.00 JttfrS
tmomw n
ax
1 x a -. 95.00 Target 51518
0.000(N.D.)
Fig. 8-2
305 >f.UE=B=
/<. f t'i'wofa
3J/- f>
Mass fragmentogram of GC-MS analysis for recovery and blank test (analysis of test fish).
000063
Test Ko. 51518 2000/10/31
r - J i h i : 10-31.D04
W 'US
: Stenderti solution 100uc/L
2A*W4
: kfujiioto
Test No. 51518
ID
mt
52#
aw i
it*
1 * * : 95.00 Target 51518
7.892 3908 0.000
I f p X t - / D - d I- K - F i ' ' * < f H )
T*-*77-r* : 10-31.005
VjV ,
: FA7HS.D2.5
: kfujiioto
Test No. 51518 2000/10/31 i `>u
ID : 1 K * : 95.00 H T : Target
: 51518 : 7.689 X * : 4214 a t * : 0.000
- ) . D`
20V- f >31 '
r-* 7 rf* S : 10-31.DOS
tin i
: FA7Hb.D10
jVIr-Ml
: kfujiioto
Test No. 51518 2000/10/31
7 t
i 't * '1' o-ys
i~l),D'lO
ID HV
95.00 ft# * BBS
: 1 It* : Target : 51518 : 7.682 : 2926
: 0.000
: 95.00
jcnw -
16 Fiy'** * f
Fig. 9-1
Mass fragmentogram of GC-M S analysis for test fish (level 1).
000064
fjTt KH4
Test No. 51518 2000/11/07
: 11-7.D03 : Standard solution 10Qug/L : icfujiaoto
Test No. 51518
ID 9iT A ft 95.00
DO m at
i K ftft : 95.0G Target 51518
7.698 4372 0.000
2&rv `>'7
HPM
r-97rft% : 11-7.D04
W *
: FA14Ha.D20
iV l-*
: kfujiioto
Test No. 51518 2000/11/07 ji* h jt I4
ID 9<T A ft 95.00
mm
:i : Target : 51518 : 7.696
2740 0.000
r-9 W * % : 11-7.D05
tV7*
: FA14Hb.D5
W -
: kfujiioto
Test No. 51518 2000/11/07 Jes\ JU h ft*' d*y*(Lev*l
3
ID : 1 KftlK : 95.00 9<7` :Target
\
A f t :51518 95.00
f t : 7.705
9m0at
::
2509
o.ooo
p 's
, T r-
zavo-i/.'P
Fig. 9-2
Mass fragmentogram of GC-M S analysis for test fish (level 1).
000065
Tost No. 51518 f-9l7U% : 11-H.D04
: Standard solution 100uc/L : kfujiioto
2000/11/14
Test No. 51518
ID 9<r 95.00 A f t
: 1 K ftft : 95.00 : Tarret : 51518
8 * 1 : 7.672 mm : 3797
: 0.000
2F&0- f / ' K '
*-7r-f* : 11-14.005
W J
: FA21Ha.D5
tVV-^
: kfujiioto
Test No. 51518 2000/11/14
T it jisK
2 l a-fs ( L t * !
ID : 1 * * K : 95.00 9i V : Target
:; 51518 JM IftH : 7.685 OB* : 3009 UK : 0.000
2 CTS A /V /- Fty'*ofc>
W `18 a* H 4
: 11--14.D06 : FA21Hb.D10 : kfujiioto
Tost No. 51518 2000/11/14 TevV
L e v e l ( ' 0 O ' (0
ID 9<T 95.00 # em m n mm *
1 Jtftfe : 95.00 Tarnt 51518 7.676 2891 0.000
jtrzr 11 W K
Fig. 9-3
Mass fragmentogram of G C-M S analysis for test fish (level 1).
000066
Test No. 51518
r-*7r-f : 11-23.003
VjTitQk
: Standard solution lOOuf/L
KH
: kfujiioto
2000/11/23
Test No. 51518
ID 947*
95.00 a* i* ms
1 H i f t : 95.00 Target 51518 7.677 5014 0.000
Test No. 51518 2000/11/23
f > 7 ' 11 2:'?A28Ha.D12
iVk-FI5
: kfujiaoto
sil
3 8 cJo/S (
ID 947* # 95.00
W M B *&
A1 K * f i : 95.00 Target 51518 7.678 6124 0.000
l~ <0. P' P
20W .//.23-
Test No. 51518 2000/11/23
W7**4i*^ ' 11 2? FA28Hb.D12
A*\i-9%
: kfujiioto
4lah
d& fs ( L v v J I - D `(2
ID 947* &&% 95.00
sm M mB s
i Target 51513 7.677 5395 0.000
Fig. 9 -4
Mass fragmentogram of GC-M S analysis for test fish (level 1).
0000G7
'J l't *v*/-&
Test Ho. 51518 : 12-5.DOS
: Standard solution ICOug/L : kfujisoto
20C0/12/Q5
Test No. 51518
l ____ -----1
5000 ID
wr
a# 95.00
mD0x
: 1 * * B : 95.00 : Target : 51518 : 7.693 : 2507 : 0.000
*
r - ---------- i------ T 678
M r t . a . t S K-
r~ 9 VJVhtk
: 12-5.D04 : FA42Ha.D2Q : kfuj inoto
Test No. 51518 T est
2000/12/05 4-i ch-ys(Lvt. l-
)< D O O
ID HT
95.00 81 *
: 1 X X K : 95.00 : Target : 51518 : 7.695 : 2023 : 0.000
i ! ........................................... 678
aojo, a- eS K .F t f m t h
r-*7W ** : 12-5.D05
W *
: FA42Hb.D10
W -
: kfujiaoto
Test No. 51518 2000/12/05 a^tev*
ID HV 98.00 *
as
**
:1 : Target : 51518 : 7.702 : 1647 : 0.000
- b ) . P -/0
Jiff** t3`
fi.Fy'.'rn*
Fig. 9 -5
Mass fragmentogram of GC-M S analysis for test fish (level 1).
000068
2 V V-3;
Test Mo. 51518 : 12-23.D06
: Standard solution lQ0ug/L : kfujiaoto
2000/12/23
Test No. 51518
5000 ID HT
/ v 95* f t * H ft ft*
1 : * * R : 95.00 Target 51518 7.708 5771 0.000
.JL -
12.32. f<. Fty'.Vafo
r - W t : 12-23.D07
fJT>%
; mOHa.DIO
KV-M5
: kfujiioto
Test No. 51518 T*** M c
20cb/12/23 O a f A ( u v t | ~ a). 0-IO
i
: S55
ID : 1 ! J tftft : 95.00 n r : Target
: 51518
K 95.00 &j$g||B : 7.717
B ft : 4012: : 0.00Q i
78
* 12-23.D08
r/T l*
: FA6.0Hb.D15
2VW&
: tfujisoto
3*. 2. > i- K .F u j'l"* * 9
L. -
Test No. 51518 2000/12/23
. f \ i -i i> /\ tkH&( -i*sa* I *--b ) *
T w '
a*eT
1
ID : 1 i * * R . H V : Tercet
A : 51518
95.00 $ r| hb : 7.712 m*t : 4890: * a c .. : 0.000
: 95.00
3nre. 2->3- K -F i^'.'w ^o Fig. 9-6 Mass fragm entogram of GC-M S analysis for test fish (level 1).
000069
Test No. 51518 V-*7Mf : 10-30.016
9, : Standard solution 100u*/L : kfujinoto
2000/10/31
Test No. 51518
10 MV 95.00 MS
* D0 *3E
: 1 X * K : 95.00 : Tariet : 51518
: 7.680 : 3484
: 0.000
^L.oxst> -
3 ^ K- T iy'nto fo
r*-47r4M5 : 10-30.017
W J*
: FA7La
jV W S
: kfujiaoto
Test No. 51518 2000/10/31 ^ o^ s CL**! 2 - a - J )
ID MV 95.00 DO
: 1 X * R : 95.00 : Target : 51518
: 7.673 : 543 : 0.000
Uova-
3 A /X
Test No. 51518 2000/10/31
5 2 3 * * : IM O -U ^
^
Vk-F
: kfujiaoto
j , , , , ,, )+ .,, q c ty . f u . J 1 - U
ID 447*
95.00
rhw w
DO *&
1 K U R : 95.00 7.690 697 0.000
en- /&
Fig. 10-1 Mass fragmentograrn of G C -M S analysis for test fish (level 2 ).
000070
Test Ho. 51518 2000/11/07 f - W r U * : 11-8.D14
: Standard solution 100ug/L : kfujisoto
Test No. 51518
ID : 1 KM& : 95.00 W : Target A # : 51518 95.00
f tttf tN : 7.694 UH* : 4837
mat : o.ooo
2 J . / - 7 K.Fuf'HDfo
r-977<* : 11-6.D15
tV7***
: F14L
KH4
: kiujiaoto
Test Ho. 51518 2000/11/07 T e r f j U h a j * * * I* <#</*
2 -0
5000 ID : 1 M f t : 95.00 9<r : Target A : 51518
95.00 M : 7.704
m m : 1097
mat : o.ooo
67 8
2 ^ . /a 7 K . F t f ~ # h
r-W r-fVS : 11-6.D16
W *
: FA14Lb
|V |/-
: kfjiaoto
Test Ho. 51518 2000/11/07 T-M >'sh 4****
ID H7`
95.00 W M
m at
: 1 **R : Target : 51518
: 7.683 : 873
; o.ooo
: 95.00
2' 0
6 Fig. 10-2
. ft. 7 X ' F \j'^ rf"0 78 Mass fragmentogram of GC-MS analysis for test fish (level 2).
000071
Test No. 51518
r-nrihi : 11-13.D14
7>7`*
: Standard solution lQOug/L
iVV-$
: Wujinoto
2000/11/14
Test No. 51518
ID 1 * * * : 95.00 H T Target
51518 7.672 93* 3610 *3E 0.000
MSi. // /
r-?77< *-s 11-13.015^
w *
K !r**
: kfujinoto
Test No. 51518 2000/11/14
la /rC
ID MT fig#-
{%$5M mm mm
1 Target 51518
7.674 1407 0.000
Z- O
2csv if- IV K- toj'"-' # ^ c
Test No. 51518 2000/11/14
r*-i7r4)l' : 11-13.016
. i Aj j ^ j l loyi f~L-?
777*
: FA21Lb T w
'0/ 1
tV H 4
: kfujiaoto
ID MV fig*
an*
**
1 Target 51518 7.660 581 0.000
2 --b ) ^
jicm i - //< t #
Fig. 10-3 Mass fragmentogram of GC-MS analysis for test fish (level 2).
000072
Test No. 51518 2000/12/05
T'-Wr-f : 12-4.D16
tV7*A&
: Standard solution iOOug/1
W-*
: kiujiaoto
Test No. 51518
5000 ^ ___________ (/~^j________
ID W' & 95.00 fltftftN H S&
: 1 KftjK : 95.00 : Target : 51518 : 7.698 : 2240 : 0.000
j
678
V S 7rY4 : 12-4.D17
W JfcS
: FA42U
A*V-9&
; kfujisoto
k- Ftf ih
Test No. 51518 2000/12/05 T est iti t i a fU * <t d y * C
a)
ID
nr
95.00
mmmm w
mm
1 S t 8 : 95.00 Target 515.18 7.690 410 0.000
Saco o - tS K - F t f u i f e
Test No. 51518 2000/12/05
r S * * : 1 W " 42Lb T t - i U k
Vli-945
: kfujisoto
C 2" ^
ID M7* B#=S 95.00 81$ asm
**
1 J t f t t t : 95.00 Target 51518 7.697 269 0.000
g r t.w .e - T K* R g. 10-5 Mass fragmentogram of G C-M S analysis for test fish (level 2).
000073
Test No. 51518
r-*77-i : 12-22.017
>7'
: Standard solution lOOux/l
tVl-
: kiujiioto
2000/12/23
Test No. 51518
Test No. 51518 2000/12/23
?*-*77ttS : 12-22.019 : FAfiOLb
TH
: kiujiioto
ID 7*
95.00 ftttW M OB
1 ; K f ttt : 95.00 Tarjlet 51518 7.700 1492! 0.000
2-~ 0
o . /> 113 K-FiflVerifo Fig. 10-6 Mass fragmentogram of QC-MS analysis for test fish (level 2).
000074
Teat No. 51518 2000/10/23
r - 9 7 r : 10-23.D01
tV7`JI>45
: Standard solution 100ug/L
Vl-f4&
: kfujiaoto
Test No. 51518
nIDr
95.00
nnm m
mat
: 1 X*& : Target : 51518
: 7.890 : 4451 : 0.000
: 95.00
2CTC* f o . /< F t y ' '^ e f o
Test No. 51518 2000/10/23
T*-777-f : 10-23.D02 B-jcr- the **f*rlartl *1 a
V/7'H A 'H
: -IgflOTFthe tflit i afck ""* : kfujiaoto
aum t-il
Ai IX n *
ID HT 95.00
mmmw m
WA
1 X ft& : 95.00 Target 51518 0 0.000N.D.)
2.0V. / <- P t^ V <*oto
Test No. 51518 2000/10/23
f*-7f** : 10-23.D03 8 V '
** iaratol sVxft
f j T 545 V V-
: Iiifure-UartalUattw : kfujiaoto
-_K i
nIDr
# a* * **
1 ** Target 51518
0
00.0007(N.D.)V
K - P i/yV<tt o f O
Fig. 11-1 Mass fragmentogram of GC-MS analysis for test fish (control).
000075
Test No. 51518 2000/12/23
r-91rQ A : 12-22.D21
W 'I
: Standard solution i00ug/t
A*1<-JS
: kfuj inoto
Test No. .51518
ID : l XMfc : 95.00 H T : Target
: 51518
95.00 *W M : 7.701 U ff : 5929 f f : 0.000
Doten. /? 23 4~
K yMo+b
TD , *<r -1 95.00 *mam* m mm
i * * : 95.00 Target 51518 0.000(0 .)
2iT O .12.i3 '
Fig. 11-2 Mass fragmentogram of GC-M S analysis for test fish (control).
000076
Test No. 51518
Concentration of dissolved oxygen(/ng/L)
Concentration o f dissolved oxygenfmglL)
Concentration o f dissolved oxygen(mg/L)
Fig. 12 Concentration o f dissolved oxygen (Level 1). 10 r
5-
0 L,-- --
0
......-j -
10
-t ....... I ... .. , .... In- -- ...--
20 30 40 Exposure period (days)
.1 ..
30
Fig. 13 Concentration o f dissolved oxygen (Level 2). 10 r
-J
60
5-
0 --------------- 1----------------I---------------- 1----------------1----------------1----------------! 0 10 20 30 40 30 60
Exposure period (days)
Fig. 14 Concentration o f dissolved oxygen (Control).
February 1,2001
Name /X F u / ' 'fr O'h:
000077
Test No. 51518 2001/1/5
f*-*7rf* : 12-30.003
: Standard solution lOOuc/L
KHi
: kfujiioto
Test No. 51518
ID 7* A *
SS as
1 K ftft : 95.00 Target 51518
7.692 5250 O.OCO
2 | K- *>
r-*77'.%& : 12-30.D04
*77*6
: FPs84Ha.D3.33
tW -i
: kfujisoto
Test No. 51518
t 201/1/5
i
1-a ).D -3 .3 3
ID
W A #
t
1 j f t * * : 95.00 Target 515$ '
95-00 }#WM 7.691 s s 3806; a s 0.000
*W -*
2 ffW-./< ** ! fC- f 'i j 1' *'6^
Test No. 51518 201/1/5 ?FPs64Hb.D2 . TeJdWft'* * ^ Levi* l - k ) 0 * 2 : kfjinoto
ID : l : K S K : 95.00 W7* : Target Add- : 51518 # W : 7.705
: 362T a s : 0.000
a o rf-A -T K- F td<lu^
Fig. 15-1 Mass fragmentogram of GC-M S analysis In parts of test fish (level 1).
00007S
r-9 Wli
Test No. 51518 : 12-28.040
: Standard aolution 10Qug/L ; kfujiaoto
2000/12/30
Test No. 51518
IB UT 8i
95.00 IS S
: 1 f t * : 95.00 : Target : 51518
; 7.875 : 4794 : 0. 0C0
K* f ty V *
f*-#77a : 12-28.041
*77'*
: FPh84Ha.D5
iV k -ii
; kfujiaoto
Test No. 51518 2000/12/30
H ead (le u e t I - < 0.0*5
ID 7*
95.00 ms.
1 MJtiS Target : 51518 : 7.679 5249 0.000
;uwr< 7 > K - F `>JI>M?*0
r - n r n t , : 12-28.D42
W ii
: FPh64Hb.D5
IW -i^
: kfujiaoto
Teat No. 51518 2000/12/30 Head.CLetfel
ID *17*
95.00 OB* X
1 ft MR : 95.00 Target 51518 7.588 3025 0.000
itrm , 0 . 3 0
Fig. 15-2 Mass fragmentogram of GC-MS analysis in parts of test fish (level 1).
000079
Teat o. 51518
r-t7T<t : 12-28.028
77*l-
: Standard aolution 100u/L
: kfujinoto
2000/12/30
Test No. 51518
ID W 62^45
95.00 3D* *K
I X ftK : 9S.OO Tarjet 51518
; 7.868 : 4720 : 0.000
2 i w ? . f 2->o
r-*7r<K& : 12-28.029
77**
: FPi64Ha
A k-
: kiujinoto
Teat No. 51518 2000/12/30 V is c e r a ( L e vel 1 - 0
ID M7* J
95.00 ftftftlIS SB* **
1 K M * : 95.00 Tarjet 51518 7.693 6833 0.000
2 c 3
f*-7W* : 12-28.030
77*8-45
: FPi64Hb
tV k-
: kfujinoto
Teat No. 51518 2000/12/30 , . . , . -v
V is ce ra C L*v/l I-
ID >Y7* I2 # BB* **
1 K f t f t : 95.00 Tar*et 51518 7.695 4384 0.000
Fig. 15-3
78
TJ** . 1 2 - 5 0 K ~FeJl**f
Mass fragmentogram of GC-M S analysis in parts of test fish (level 1).
ooooso
Test No. 51518
r - m n z : 12-28.035
IW'JWS
: Standard solution 100ug/L
r\* : kfujiaoto
2000/12/30
Test No. 51518
ID 9<V
95.00 KttftjIB OD mac
1 XX : 95.00 Target 51518 7.666 5074 0.0C0
: 12-28.037
977*
: FPi64Ha.D5
iVk-g
: kfujiaoto
Test No. 51518 2000/12/30 ^ I W ^ d e r p aris L^eve\ I- Oj. D-S
ID 9<r A* 95.00 00 X
: 1 X * : 95.00 : Target :51518 : 7.677 : 4010 : 0.000
>fre . n - >> fC' F<>' -
r - 9 7/4* : 12-28.038
977'*
: FPa84Kb.D5
V V-4&
: kfujiaoto
Test No. 51518 2000/12/30 Re*i*oUr pari-* Ct-eirel I - O - S "
ID : 1 XX : 95.00 7* : Target MS : 51518
95.00
: 7.678
BOX . : 2490
XX : 0.000
6
Fig. 15-4
.U .* 78 Mass fragmentogram of QC-M S analysis in parts of test fish (level 1).
O0O0. 1
Test Ko. 51518 2000/12/30
r - V j i h Z : 12-28.D08
W 'H
: Standard solution lQOug/L
iVP-*g
; Irfujimoto
Test No. 51518
K.
lt
ID *<7*
mi
95.00
amw ir
31 *J
i H R : 95.00 Target 51513
7.878 5348 0.000
----- - I ................. .
678
r - t t r t t * : 12-28.D09
tV7**
: FPs64La
A'V-*
: kfujiaoto
2cw6< I3. i* fc-fy.
.............. ...........................v
Test No. 51518 .2000/12/30
-,
T e u i e e a ts C l-*vt 2 - a)
*
ID 1 K * R
W7` Target
NI V
mi
31518
95.00 & m n e
05 0
M X 0.00(S.D.)
Peak position
K-Fyfmtto 3
VjT I X gVi/-*S
Test No. 51518 2000/12/30
: 12-2:8.FDP1s084Lb ^T eju * * ^+4, Vf .iUi'velI 2} .. "u21 : kfujinoto *
5000 ID
1
*<r Target
mi
51513
A.
9S- fffW H
?
6D
>V
M X i.CC0(N
o.d.
Peak position
6 R g. 16-1
78
2on*< 17*Do K - f y 'W 0
Mass fragmentogram of GC-M S analysis in parts of test fish (level 2).
0000S2
Test Ho. 51518 2000/12/30
T ' - f t r U Z : 12-28.024
Vj V \ %
: Standard solution 100ug/L
A 'H S
: kfujiioto
Test No. 51518
13
nr
95.00 mat
1 X X : 95.00 Target 51518 7.671 5096 0.000
2 n * . n 3o
r-* 7 ri * : 12-28.025
: FPh64La
VV-*S
: kfujinoto
Test No. 51518 2000/12/30 , ... . . H ead ( L*v*l 2 -< 0
10 W7*
& t> .
95.00 05 XX
: 1 KX& : 95.00 : Target : 51518
:? :0 : 0.000(0.0.)
873
5 1 . 13-
f'-*7r<h% : 12-28.026
7**
: FPh64Lb
V H
: kfujinoto
Test No. 51518 2000/12/30
, ...
.,, .*
H eacU U v | 2 - V
ID
HT
95-00
mm X
: 1 X X X : 95.00 : Target : 51518
; 7.887 : 496 : 0.000
2ptre-/7-50 R g. 16-2 Mass fragmentogram of GC-MS analysis in parts of test fish (level 2).
000083
Test No. 51518 2000/12/30
f ' - W r * : 12-28.D32
: Standard solution 100ug/L
VS/-J
; Mujinoto
Test No. 51518
ID W7`
95.00
mmmat
: 1 K 1 & : 95.00 : Target : 51518
7.865 4871 0.000
2 .CT-12 JO (.
i^O
: 12-28.D33
r/ram
pp64l
1V H
: kfujiaoto
Test No. 51518 2000/12/30
Viscera ( Levl 2 - a )
ID : 1 K ft& : 95.00 H T : Target
: 51518
?
DmOaMt
:0 : 0.0Q0(N.D.)
ZtV0` t2~y0 f(-Fiy'i lutrfo
r - n r : 12-28.D34
t/r% m
: FPi64Lb
VV-*
: kfujinoto
Test No. 51518 2000/12/30
Viscera CLevel 2 - b )
ID : 1 K l f t : 95.00 9<V : Target A * * : 51518
95.00 ftttWIB :
7
DmBa*t
:0 : 0.000N.D.)
6
Fig. 16-3
78
2006 / j *3 iC* PtfV ***
Mass fragmentogram of GC-M S analysis in parts of test fish (level 2).
0000S4
S v 'l 1" A \
Test No. 51518
12-28.018 : Standard solution lOOui/1 ; kfujiioto
2000/12/30
Test NO. 51518
5000 IO
i ; X * B : 95.00
U T Target
51518
/V . ^
95l0 9M 7. eVs
OD 5199
x& o.oOo
20* r fji- >0 fc- ft^' HfH>
rf'-jF77*ri&*45 : 12-28: .0F1P7a64La kfujiaoto
Test No. 51518 2^00/12/30 ,,Remai, *i.* p a ri** |(, /L.e,,v*. |I o2 - On.'Jl
5000 ID UT
95.00 . * *
1 j K * & : 95.00 Target 51518 7.73 318: 0.000
2.0* . |2- >x> fc r-F fy # f H
t'/7*S V V-9%
Test No. :51518 2000/12/30
: 12-28:.DF1P8*64Lb R e m a, i*j *\ p a r t i (. Lev iI 01 - bOJ
: kfujisoto
i
la- IO a * K S B : 95.00 H T target JStiJ 515j8
95.00 a
7.683 291i 0.000
78
, ri- >
Fig. 16-4 Mass fragmentogram of GC-M S analysis in parts of test fish (level 2).
000085
t u t No. S i t u
'- H l * : 12-28.040
' * KW
: It u O a r l M lo tio a lOOuc/l : k fu jlu to
1000/12/
Test No. 51518
l KM : >5. STiutunt 7.170 0U.0M00 200. ix-
Fig. 17-1 Mass fragmentogram of GC-MS analysis for test fish on depuration fest (level 1).
000056
Test Na. 51518 2001/1/9
r - * 7 * * : 1-5.D03
Tj V%%
: Standard solution 10Qui/t
VW45
: kfujinoto
Teat No. 51518
ID
nr &&$<
95.00 um M am
at
1 X ftft : 95.00 Target 51518 Q.Q0KN.D.)
3o
W '
Vk-M9
: 1-5.D05
: FOTHb
: kfujinoto
Test No. 51518 2101/1/9
Test
.
J s H
f..t*r..
or
..
Level l .0
p: fcj.
I.-'
00008?
Teat No. 51518 2000/12/30
r-*7T<%% : 12-28.D52
W 'l
: Standard solution lOOug/L
: kfujiioto
Test No. 51518
ID HV m ra OB*
1 M R : 95.00 Target 51518 7.674 4662 0.000
2<j v - ii- 3
f*-#77i* 12-28.D53
7*3-
: FD2La
: kfujiioto
Test No. 51518 2000/12/30
T rt J
nj-fc**- 2 o U / s C U o * | 2 - a ) J
5000 ID : 1 M R : 95.00
9<V : Target 1B : 51518
/
95.00 OB
:? :0
RJK : 0.000(N.D.)
_Peak
ud, position
6' 7'8
3**.
f*-*7rf : 12-28.D54
W **
: FD2Lb
h 'W S
: kfujiaoto
Test No. 51518 2000/12/30
T'fe f i s 1
2 cloys Cl-evel 2 - O
OOOOS8
W IZ, VV-*
.Test No. 51518 2001/1/9 : 1-5.D07
: Standard solution 100ug/l : kfujinoto
Test No. 51518
r - W r i i * : 1-5.D08
>7*Jl
: FD7La
tvIr-fcg'
: kiujiioto
Test No. 51518 2|01/l/9 . 'di*" ^ d a /S
2-.)
!\
I-- 8
/s
n .d. Peak poaitlon
' i >| 78
5000 ID : 1 K * K : 95.00 ff7* : Target : 515l|l
95- f t f t l t n : ! ? f* : 0 I * : O.OOjHN.D.) }
|
\
\ 2 era/- / J U- Fty>'"d
r - 9 7t- : 1-5.D09
t>7' *
: FDTLb
IV : kfojimoto
Test Nj>. 51518 ZP01/1/9
T est
a ;fie i 7 ola/s
2 -0
0000S9
ICO. 0
st H
50 0
A-- - - - - ./ jutiV; * *'Z-~
'\ /
\/ :
O-H tr. :
8 0 .0
Test No. 51518
---*
!ri
l \r ..
$$ 1
I 1 .: 'i.
a11 I1'If1
.0 j
C F3-- (CF2)6-- CF2CH2CH2 OH ;
20.0
0.0 -T--T , ; I |-- r
4 0 0 0 ,0
3000. 0
2000.0
;0 -- ; D ef.
15 0 0 .0
1 -- :--7-- r 1000.0
5 00.0
1/CPl
!R conditions
Instrument : Shimadzu FTIR-8200PC
Sample
: K- 1519
Method
' KBr Tablet
Fig.195
IR spectrum of test substance measured before
mf.pi t h e e x o e r ! manta! s t a r t .
AfrlX
Date : Oct. 4, 2000 Name :K. Fujimcto
2mf . f.
K-ftf****
000090
Ho. SI 518
3 0 0
200 0 .0
2G C 0.8
' 5 00.:/
(0 0 0 .0
5 00.0
IR conditions
instrument : Shimadzu FTIR-8200PG
Sample
: K- IS1#
Method
: KBr Tablet
19
l-:g.2 ,m .1*
Iixsx.
!R spectrum of test substance measured after the experimental completion.
Date : Dec. 26,2000
Name :K, Fujimoto 2e v i - / - l 1 K.fujVifo
000091
Reference -4 - 1 'H-KMR sp ectru m of te s t su b stan ce .
tAS* ti*
000092
Eefereac - S *C-KMR spectrum of te s t su b sta n c e .
000093
i*.
A ;:' -
ooi
:r. ,i
^ ; s * * f - s ifcjp t
:
:;|t ;
-t
/ ';.
*UW fc*fcH t S * * * # t e * - ? X T
IH fc& K -^ T U U
5 i* .* * .
tftt it* * tu S * # tfc W i JS B S K a
S ^ -J U fcK M tS iM M lL ttK H + Sfc^U L
-L-CV>s i K f c t t l l , i t .
5 rt . * * 0 * * lfc U U
#L -C 3^H K tit.
V.
'' I
tC ;
til2 + lU 1 7 8
f*-
--
j
&
7 -;v*.,.
?">- r i
* tfflfc A 4 L # f *H W *U M * *#***#
i m m s t o f t & i t h * * # * * + * * ] 1 9 * 1 4 -f-
& W M l
& & & #**> ^ K # & tO fc
000094
