Document rB3RQgVMoKoNJvNGRYK48DBaE
P
1R A 6 - 3 3 / ^
494
TRANSLATION
SUMMARY OF BIOCONCENTRATION STUDY REPORT
1. General items Name of novel chemical substance (IUPAC nomenclature)
Other name
HBHHHI
Structural formula
Main
or rational
formula (if neither
is known, outline of preparation
Subcomponent
method)
Molecular formula
Purity of the novel
Lot No. of the novel
chemical substance
chemical substance
used in the study
used in the study
Name and
concentration of
impurity
CAS No.
MHP* Vapor pressure
Partition coefficient 570.37 (Main
Molecular weight
component)
Melting point
~
Property at room
Boiling point
--
temperature
Stability
Unknown
Solubility in various Solvent
Solubility
solvents
Water
--
DMSO
--
Acetone
~
Other
*1
[Note]
*1 Solubility in other solvents: soluble in ethanol (unknown solubility)
1-11-00
-- --
Brown viscous solid
Stability in solvent
-- -- -- --
[Bioconcentration Report] 1 Company Sanitized Does not contain TSCA CBI
2. Acute toxicity test Fish used (Latin name): LC50 value (96 h) Use of auxiliary
Name of auxiliary and proportion to substance studied
killifish (Oryzias latipes) >35.0 mg/L
(Yes) Crystallized sugar Megafac F-142D
No 20 times amount 20 times amount
3. Study method
<Bioconcentration of Chemical Substance in Fish Body> specified in "Method for Testing New Chemical Substance, etc." (Environmental Protection Notification No.
Method
5, Notification No. 615 of Pharmaceutical Affairs Bureau and MITI Basic Industry Bureau 1974 Notification No. 392 dated July 13,1974 and amended on October 8,
1998) and "Bioconcentration: Flow-through Fish Test (Guideline 305, June 14,
1996)" specified in "OECD Guidelines for Testing of Chemicals."
Fish used (Latin carp (Cyprinus carpio)
name):
Lipid content Start: 2.94
End: 4.32
(%) Test substance concentration
First concentration
50
(Mg/L)
level
Second
5
concentration
level
Use of auxiliary
(Yes)
No
Name
Auxiliary concentration (/xg/L)
Name of auxiliary and proportion to substance studied
Crystallized sugar Megafac F-142D
First concentration level: 1000 Second concentration level: 100 First concentration level: 1000
Second concentration level: 100
|
[Bioconcentration Report] 2
Company Sanitized. Does not contain TSCA CBI
4. Results (1) Table for the result of bioconcentration test
Main component
Measurement day
After 4 days
First concentration level
Second concentration level
Water tank concentration (fig/L) Concentration proportion Water tank concentration (fig/L) Concentration proportion
46.0 4.43
After 7 days
45.7
<5.1 <5.1 4.87
<51 <51
After 10 days 51.7
After 17 days 53.4
After 24 days 53.1
After 28 days 51.5
<5.1 <5-1 <5.1 <5.1 <5.1 <5.1 <5.1 <5.1 5.08 5.47 5.27 4.75
<51 <51 <51 <51 <51 <51 <51 <51
Subcomponent
Measurement day
After 4 After 7 days days
First concentration level
Water tank concentration (fig/L) Concentration proportion
45.7
Second concentration Water tank level concentration (fig/L)
Concentration proportion
4.39
45.3
360 370 (370) 5.01
390 320 (350)
After 10 days 51.7
After 17 days 46.5
After After 24 28 days days 50.7 48.4
350 360 (350) 5.20
410 510 (460) 5.17
410 410 (410) 5.20
470 430 (450) 4.56
370 320 (340)
430 340 (380)
350 270 340 270 (340) (270)
(2) Concentration proportion at steady state or upper or lower limit of concentration proportion
Main component
Concentration proportion
First concentration
BCFss -(BCF)
Less than 5.1 times
level
Second concentration
BCFss -(BCF)
Less than 51 times
level
[Bioconcentration Report] 3
Company Sanitizad. Does not contain TSCA CBI
Subcomponent
First concentration level Second concentration level
(BCFss) BCF (BCFss) BCF
Concentration proportion 440 times
340 times
5. Test water and fish body analytical methods
(1) Test water and fish body analysis flow
Test water analysis
Column chromatography (SepPack Ci8) --Analysis
Test fish analysis Homogenization extraction (methanof2/formic acid (500/0.5 v/v)) -->Dilution --Analysis *2: containing 10 mmol/L ammonium acetate
(2) Analytical instruments and conditions used_______________ _____ ___________________ Instrument: liquid chromatography-mass spectrometer Pump: Nippon Bunko, Model PU-980 Autosampler: Nippon Bunko, Model AS-950 Mass spectrometer: Micromass, Model Quattro 13
Liquid chromatography conditions used
Column: L-column ODS (prepared by Chemical Evaluation and Research Institute), 15 cm x 2.1 mm I.D.
Column temperature: 25C
Elution solution:
Solution A (75%): methanof2/formic acid (500/0.5 v/v)
Solution B (25%): water*2/formic acid (500/0.5 v/v)
Flow rate: 0.2 mL/min
Amount to be injected: 20 iL
Mass spectrometer condition Ionization method: electrospray ionization (ESI) Ion detected: cation Detection method: selective ion monitoring (SIM) Measurement ion: main component m/z 571
subcomponent: m/z 513 Ion source temperature: 120C Cone voltage: 50 V________________________________________________________________
[Bioconcentration Report] 4 Company Sanitized. Does not contain TSCA CBI
6. Recovery rate (mean) Recovery from test water (%) Main component 84.0 Recovery from fish body (%) Main component 99.2
Subcomponent 87.1 Subcomponent 89.9
7. Discussion ______________________________________________________________ The fluctuation of the lipid content (mean) after completion of experiments in this study was 47%
compared to the result before experiments, and it was over 25%. One of the causes is considered to be attributable to fluctuations in physiological conditions, feeding state, etc., causing lipid content fluctuation. Incidentally, the feeding method, etc., are to be studied further to aim for improvements in lipid content changes and fluctuations.
8. Other Study implementation Study manager
Study term
Name: Address:
Chemical Evaluation and Research Institute, Kurume Laboratory 19-14 Chuo-cho, Kurume-shi, Fukuoka-ken PC830-0023
Telephone and
Tel: 0942-34-1500, FAX: 0942-39-6804
FAX facility Name:
Naoaki Yakata
Years of
15
experience:
November 17, 2003 -December 25, 2003
[Bioconcentration Report] 5 Company Sanata,* D o ., ,,o, conUt,,, rs c A c m
Study requester: Du Pont K.K.
Statement
Chemical Evaluation and Research Institute Kurume Laboratory
Study title: Bioconcentration study o
in carp
This final report (copy) is hereby certified to be an accurate copy of the final report of the above-titled study.
December 25, 2003 Operation manager: Hiroshi Tadokoro
[Bioconcentration Report] 6 Company Sanitized, Does not contain TSCA CBI
Acceptance No.: Test No.:
Final Report
Bioconcentration Study o:
'in Carp
December 25, 2003 Chemical Evaluation and Research Institute
Kurume Laboratory
[Bioconcentration Report] 7
Company Sanitizod. Does not contain TSCA CB
Statement
Study requester: Du Pont K.K. Study title: Bioconcentration study o
Chemical Evaluation and Research Institute Kurume Laboratory
in carp
The above study was carried out according to the following GLP.
(1) "Standards for testing facilities specified in Ministry Ordinance Article 4 stipulating items, etc., of test related to new chemical substances and evaluation of toxicity related to designated chemical substance" (GLP) (Environmental Protection Notification 39, Notification No. 229 of Pharmaceutical Affairs Bureau and M ill Basic Industry Bureau Notification No. 85 dated March 31,1984, amended on March 1, 2000)
(2) "OECD Principles of Good Laboratory Practice" (November 26,1997) In addition, this final report reflects the raw data accurately, and the test data are confirmed to be valid.
December 25, 2003 Study manager: Naoalci Yakata
[Bioconcentration Report] 8 Company Sanitized. Does not contain TSCA CBf
Reliability Guarantee Certificate
Study requester: Du Pont K.K. Study title: Bioconcentration study o
Chemical Evaluation and Research Institute Kurume Laboratory
in carp
The auditing or inspection of the above study was carried out by the reliability guarantee department of the Chemical Evaluation and Research Institute, Kurume Laboratory, and the content and date of auditing or inspection as well as dates reported to the study manager and operation manager are as follows.
Audit or inspection content Date of audit or inspection
Date reported (study manager)
Study protocol
November 17,2003
November 17, 2003
State of implementation of study
December 25,2003 November 18,2003 November 25,2003
December 25,2003 November 26,2003 November 26,2003
November 26,2003
November 26,2003
November 27,2003
December 2, 2003
November 28,2003
December 2,2003
December 1, 2003
December 2,2003
December 2,2003
December 2, 2003
Raw data and final
December 25, 2003
December 25, 2003
report
Date reported (operation manager)
November 17, 2003 December 25, 2003 November 26, 2003 November 26, 2003 November 26, 2003 December 2,2003 December 2, 2003 December 2, 2003 December 2, 2003 December 25,2003
This final report is guaranteed for the test methods being accurately described, content being conformed to the study protocol and standard procedures and at the same time, raw data being reflected accurately.
December 25, 2003 Reliability Guarantee Department Manager: Shiro Horikoshi
[Bioconcentration Report] 9
Company Saniti**. Dona conte|,, TSCA 0J(
Table of Contents
Title Study requester Study facility Study object Study method GLP appliedl Study schedule Storage of study documents and materials Study personnel Final report approval Summary 1. Test substance 2. Sample provided by study requester 3. Acute toxicity test 4. Implementation of bioconcentration test 5. Environmental factors considered to affect the reliability of test results 6. Test results 7. Discussion 8. Notes
Page
[Bioconcentration Report] 10 Company Sanitized. Does not contain TSCA CBI
Tables Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Reference 1
44133
Test substance concentration in test water (shown in main text) Concentration proportion (shown in main text) Test substance concentration in test water at steady state (shown in main text) Calculation table for recovery test and blank test (test water analysis) Calculation table for analysis of test water in the first concentration level Calculation table for analysis of test water in the second concentration level Calculation table for recovery test and blank test (test fish analysis) Calculation table for analysis of test fish in the first concentration level Calculation table for analysis of test fish in the second concentration level Calculation table for analysis of test fish in the control group Table for the results of quality test for test water
Figures Figure 1
Figure 2
Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13-1 Figure 13-2 Reference 2
Correlation between exposure period and bioconcentration factor (first concentration level) Correlation between exposure period and bioconcentration factor (second concentration level) Study substance concentration - mortality curve in acute toxicity test Test water analysis calibration curve LC-MS mass fragmentogram in recovery test and blank test (test water analysis) LC-MS mass fragmentogram in test water analysis Calibration curve for test fish analysis LC-MS mass fragmentogram in recovery test and blank test (test fish analysis) LC-MS mass fragmentogram in test fish analysis at the first concentration level LC-MS mass fragmentogram in test fish analysis at the second concentration level LC-MS mass.fragmentogram in test fish analysis in the control group Mass spectrum of test substance Infrared absorption spectrum of test substance (before start of experiment) Infrared absorption spectrum of test substance (after completion of experiment) Infrared absorption spectrum provided by study requester
[Bioconcentration Report] 11 Company Sanitized. Does not conla'm TSCA CBf
Title: Bioconcentration Test o:
in Carp
Study requester:
Du Pont K.K. IrB-l Shimomeguro, Meguro-ku, Tokyo-to PC 153-0064
Study facility:
Chemical Evaluation and Research Institute, Kurume Laboratory 19-14 Chuo-cho, Kurume-shi, Fukuoka-ken PC 830-0023
Study objective:
obtaining information on the extent of bioconcentration oij carp
Study method:
this study was carried out according to the following testing methods. (1) <Bioconcentration of Chemical Substance in Fish Body> specified in "Method for Testing New Chemical Substance, etc." (Environmental Protection Notification No. 5, Notification No. 615 of Pharmaceutical Affairs Bureau and MITI Basic Industry Bureau 1974 Notification No. 392 dated July 13,1974 and amended on October 8,1998) (2) "Bioconcentration: Flow-through fish test (Guideline 305, June 14, 1996)" specified in "OECD Guidelines for Testing of Chemicals."
Applicable GLP:
the following standards were applied to this study. (1) "Standards fortesting facilities specified in Ministry Ordinance Article 4 stipulating items, etc., of test related to new chemical substances and evaluation of toxicity related to designated chemical substance" (GLP) (Environmental Protection Notification 39, Notification No. 229 of Pharmaceutical Affairs Bureau and MITI Basic Industry Bureau Notification No. 85 dated March 31,1984, amended on March 1, 2000) (2) "OECD Principles of Good Laboratory Practice" (November 26,1997)
[Bioconcentration Report] 12 Company Sanitized. Does not contain TSCA CB
Study schedule Study start date Experiment start date Experiment completion date Study completion date
November 17, 2003 November 21, 2003 December 19, 2003 December 25, 2003
Storage of study documents and materials (1) Study materials The samples provided by the test requester are to be stored, after sealing in storage containers for 10 years from the notification corresponding to the Article 4, Clause 1, No. 1,2 or 3 of the law related to regulations for evaluation, production, etc., of chemical substances (abbreviated to "Chemical evaluation law," below), in the sample storage room of the Kurume Laboratory. After the prescribed storage term, the actions on the samples are to be determined after consultation with the test requester. However, if the product quality of a sample is expected to change markedly during the storage term, the term is set to be the one for the product quality withstanding storage, and at the time of disposal, an approval is to be obtained from the study requester.
(2) Raw data, documents, etc. The raw data, study protocol, study request, study substance survey form and other necessary documents together with the final report are to be stored for 10 years from the notification corresponding to the Article 4, Clause 1, No. 1,2 or 3 of the Chemical Evaluation Law in the document storage room of Kurume Laboratory. The actions on these materials after the storage term are to be determined after consultation with the test requester.
Study personnel Study manager Study investigators (Implementation of bioconcentration test)
Rearing control manager Acute toxicity test investigators
Naoaki Yakata Affiliation: Second Testing Department Maki Saeki Yoshiyuki Inoue Shin-ichiro Kaku Yurika Mori Akiko Hiroshige Akemi Inoue Yasuro Kawashima Chirise Fukuda Yasuro Kawashima Yasuro Kawashima Tadayoshi Fujiuchi
Approval of final report Study manager:
December 25, 2003 Naoaki Yakata
[Bioconcentration Report] 13 Company Sanitized. Does not contain TSCA CB!
Summary Study title
Bioconcentration test ofH H H H H p i n carp
Study conditions Acute toxicity test (1) Fish used (2) Exposure time (3) Exposure method
Killifish 96 h Semistatic type (water exchange every 8-16 h)
Bioconcentration test (1) Fish used (2) Concentration tested
(3) Exposure time (4) Exposure method (5) Analytical method
Carp First level: 50 fig/L Second level: 5 pg/L 28 days Continuous water flow system Liquid chromatography-mass spectrometer combination
Results
(1) 96 h LC50
>35.0 mg/L
(2) Bioconcentration factor at steady state
Main component First level: -
Second level: -
Subcomponent
First level: 440 times
Second level: 340 times
(3) Bioconcentration factor
Main component First level: less than 5.1 times
Second level: less than 51 times .
Subcomponent
First level: 350-510 times
Second level: 270-430 times
[Bioconcentration Report] 14 Company Sanitized. Does not contain TSCA CBi
1. Test substance
this report has the following name and other related items.
1.2 Structural formulas and others Structural formulas
*1 From documents provided by the study requester 2. Sample provided by study requester
In the following the sample provided by fhe study requester is called the sample provided. 2.1 Provider and Lot No.*1
(1) Provider: Du Pont K.K.
2.2 Purity*1
[Bioconcentration Report] 15
Company
Does not contain TSCA CBI
(2) Impurity
2.3 Confirmation of study substance The infrared absorption spectrum provided by the study requester was confirmed to
coincide with the spectrum measured at the Kurume Laboratory (see Figure 13, Reference 2).
2.4 Physicochemical properties*1
State at room temperature Brown viscous solid
Solubility
Soluble in ethanol (unknown solubility)
Stability
Unknown
*1 Information in documents provided by the study requester
2.5 Storage conditions and stability (1) Storage condition Storage at a room temperature in a dark place (2) Stability confirmation The infrared absorption spectrum measurement was carried
out before and after experiments, and as a result, the two spectra coincided confirming the substance was stable under the storage conditions used (see Figure 13).
2.6 Stability under test conditions A preliminary study was carried out before starting experiments to confirm the substance
was stable under the study conditions used.
3. Acute toxicity test 3.1 Testing method
The method used was the method of "Industrial wastewater testing method - acute toxicity test in fish" (71 of JIS K 0102-1998).
3.2 Fish used
(1) Kind of fish
Killifish Oryzias latipes
Reason for selection: sensitivity similar to that of carp and readily available
[Bioconcentration Report] 16 Company Sanitized. Does not contain TSCA CBI
(2) Source Nakajima Fish Farm (2029 Ohaza Nagasu, Nagasu-cho, Tamana-gun, Kumamoto-ken 869-0123)
(3) Rearing and storing conditions Term: upon delivery, those observed to be abnormal by naked eye observation were
removed, the remaining fish were exposed to a medicinal bath for prevention of diseases and extermination of parasites and reared for 44 days in a state of flowing water.
Medicinal bath: For disease prevention, a mixture of 20 mg/L of Elverju [transliteration] and 7 g/L sodium chloride was used as a medicinal bath for 24 h. For parasite extermination, a bath of 30 pL/L of formalin for 24 h was used twice.
(4) Conditioning conditions Term: after rearing and storing, the fish were transferred to a conditioning tank for
medicinal bathing. Subsequently, they were conditioned in a state of flowing water at 25 2C for 7 days. If any abnormality was observed during that period, the individual was removed.
After reselection and medicinal bathing, the selected fish were conditioned in a state of flowing water for 18 days.
Medicinal bath: After transferring to a conditioning tank, a mixture of 20 mg/L Elverju and 7 g/L sodium chloride was used to carry out medicinal bathing for 24 h.
After reselection, a mixture of 20 mg/L Elveiju and 7 g/L sodium chloride was used to carry out medicinal bathing for 24 h.
(5) Body weight Mean of 0.17 g (6) Body total length Mean of 2.8 cm (7) Sensitivity test
LC50 with a standard substance PCP-Na (pentachlorophenol sodium, reagent, manufactured by Tokyo Kasei Kogyo) for 48 h was 0.755 mg/L in the case of test fish of the same lot.(TFO-031020).
3.3 Water used for the test (1) Kind of water
Underground water pumped up from the ground of Kurume Laboratory.
(2) Water quality confirmation The results of water quality analyses carried out by sampling on November 11, 2003 at the Kurume Laboratory are shown in Reference 1 (measurement frequency: once/6 months).
[Bioconcentration Report] 17
Company Sanitated. Does not contain r s r a
The items used in the analyses of the water used for the test were confirmed to conform to one of the following standards.
"Water quality standard based on the Water Works Law" (amended December 21, 1992, Ministry of Health and Welfare Ordinance No. 69) "OECD Guidelines for Testing of Chemicals," "Fish, Early-life Stage Toxicity Test (Guideline 210, July 17, 1992)" "Standard for Fishery Water" (Japanese Association of Aquatic Resources Protection, March 1983) "Environmental standards related to water pollution" (amended on February 22,1999, Environment Protection Agency Notification No. 14) "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 water tank
(2) Amount of test solution
5 L/concentration level
(3) Test temperature
Start of exposure
24.8C
Before water exchange
25.1C
(4) Dissolved oxygen concentration Start of exposure
8.1 mg/L
Before water exchange
7.8 mg/L
(5) pH
Start of exposure
8.0
Before water exchange
7.8
(6) No. of fish tested
10/concentration level
(7) Exposure time
96 h
(8) Exposure method
Semistatic type (water exchange in every 8-16 h)
3.5 Stock solution preparation method (1) Dispersant
Crystallized sugar Megafac F-142D2
(2) Preparation method The sample provided and a 20 times amount of crystallized sugar were stone-milled;
subsequently, Megafac F-142D in a 20 times amount of the amount of the sample provided was
[Bioconcentration Report] 18 Company Sanitized. Does not contain TSCA CBS
added, and the mixture was stone-milled further. Subsequently, ion-exchanged water was added to prepare a stock solution with a test substance concentration of 500 mg/L.
3.6 Test implementation
(1) Test site
Room 214LC50
(2) Test date
November 17, 2003 - November 21, 2003
3.7 Calculation of 96 h LC50 The Doudoroff method was used.
3.8 Test result 96 h LC50 of test substance
>35.0 mg/L*2 (see Figure 3)
*2: The concentration of the dispersant (Megafac F-142D) used in the above case was 700 mg/L, the 96 h LC50 of the dispersant was 7140 mg/L, and considering the effect of the toxicity of the dispersant, no test above this concentration was carried out.
4. Implementation of bioconcentration test 4.1 Fish used (1) Kind of fish Carp Cvprinus carpio
Reason for selection: for considering consistency with past findings and size being easy to handle
(2) Source
Sugishima Fish Farm
(123-2 Gunchikuichiban-cho, Yatushiro-shi, Kumamoto-ken 866-0024)
Date of receiving tested fish
August 14, 20033
(3) Rearing and storing conditions Term: upon delivery, those observed to be abnormal by naked eye observation were
removed, the remaining fish were exposed to a medicinal bath for prevention of disease and extermination of parasites and reared for 18 days in a state of flowing water.
Medicinal bath: for disease prevention, a mixture of 50 mg/L of fishery OTC (oxytetracycline hydrochloride) and 7 g/L sodium chloride was used as a medicinal bath for 24 h. For parasite extermination, a bath of 30 pcUL formalin for 24 h was used twice.
[Bioconcentration Report] 19 Company s* TM ,. mx contain T o r .
(4) Conditioning conditions Term: after rearing and storing, the fish were transferred to a conditioning tank for
medicinal bathing. Subsequently, they were conditioned in a state of flowing water at 25 2C for 36 days. If any abnormality was observed during that period, the individual was removed.
After reselection, transfer to a test tank, and medicinal bathing, the selected fish were conditioned at the same temperature as above in a state of flowing water for 42 days.
Medicinal bath: after transferring to a conditioning tank, a mixture of 50 mg/L of fishery OTC and 7 g/L sodium chloride was used to carry out medicinal bathing for 24 h.
In the test tank, a mixture of 20 mg/L of Elverju and 7 g/L sodium chloride was used to carry out medicinal bathing for 24 h.
(5) Body total length 7.0-9.0 cm
(6) Lot
TFC-030814
(7) Age
Less than one year old
(8) Feed
Kind
Compounded feed for young carp
Composition
Protein content of 43.0% or higher
Fat content of 3.0% or higher
Manufacturer
Nippon Haigo Shiryo K.K.
Feeding method Amount corresponding to about 2% of the body weight of test fish
was fed in two portions a day (once a day on holidays).
No feeding for 24 h prior to sampling of study fish.
4.2 Water used for the test Same as in 3.3.
4.3 Test and environment conditions
(1) Test water feeding method
The feeding was carried out by using a flowing water device
assembled at the Kurume Laboratory
(2) Test tank
100-L volume glass water tank
(3) Amount of test water
With a proportion of 2 mL/min of the stock solution and
800 mL/min of test water, 1155 L/day were fed to the test
tank.
(4) Stock solution tank
25 L glass bottle
Replacement frequency Once/week
(5) Test temperature
First concentration level 25.0-25.2C
[Bioconcentration Report] 20
Company Sanitized. Does not contain TSCA c m
Second concentration level 25.0-25.2C
Control group
25.0-25.1C
(6) Dissolved oxygen concentration First concentration level 7.6-8.1 mg/L
Second concentration level 7.8-8.1 mg/L
Control group
8.1 mg/L
(7) pH
First concentration level 7.7-8.0
Second concentration level 7.7-8.0
Control group
7.6-8.0
(8) Dlumination time
Artificial illumination by white fluorescent lamps (14 h
light/10 h dark)
(9) No. of fish tested
First and 2ndconcentration levels: 38 (start of exposure)
Control group: 12 (start of exposure)
(10) Exposure time
28 days
Reason: reached steady state after 28 days
(11) Experiment site
213 Aquatron Room
4.4 Stock solution preparation method (1) Dispersant
Same as that in section 3.5 (1).
(2) Preparation method First concentration level
A stock solution having a test substance concentration of 20 mg/L was prepared by using the same procedures as those in section 3.5 (2).
Second concentration level A stock solution having a test substance concentration of 2 mg/L was prepared by using the
same procedures as thosejn section 3.5 (2).
Control group Crystallized sugar and Megafac F-142D were dissolved in ion-exchanged water to prepare
a stock solution having concentrations of 400 mg/L.
4.5 Test concentrations Considering the 96 h LC50 result and test substance analytical sensitivity, the test
substance concentrations were set as follows.
[Bioconcentration Report] 21 Company Sanitized. Does not contain TSCA CBi
First concentration level 50 /u.g/L Second concentration level 5 ptg/L At the same time, a control group was set as a blank test.
4.6 Observation, measurement and cleaning (1) Observation of test fish: state of health, etc., observed twice a day by the naked eye. (2) Amount of test water: measured once a day with a graduated cylinder and the result recorded. (3) Test temperature: measured once a day with an alcohol thermometer and the result recorded. (4) Dissolved oxygen concentration: measured twice a week with a dissolved oxygen meter and the result recorded. (5) pH measurement: measured once or more a week with a pH meter and the results recorded. (6) Cleaning: during the experiment term, the carp excreta, dirt on the water tank, etc., were cleaned once a day.
4.7 Test water and test fish analyses The analyses of the test substance in the test water and fish were carried out by the liquid
chromatography-mass spectroscopy (LC-MS) method. When the test substance was analyzed by liquid chromatography-mass spectrometry, two
peaks were detected. Therefore, quantitative determination was carried out for each peak. However, the concentration of each peak shown does not take into account the component composition as a test substance concentration shown in the standard solution preparation in section 4.7.3 (2).
4.7.1 Frequency of analysis (1) Test water
For both the first and second concentration levels during exposure, the test water analysis was carried out once before and then throughout the first test fish analysis. One sample per analysis was used.
(2) Test fish For both the first and second concentration levels during exposure, the test fish analysis
was carried out 5 times, the number of fish used per analysis was 4, and they were divided into 2 groups (2 per group)*3.
In the control group, analysis was carried out before and after the experiment, 4 per analysis were used, and they were divided into 2 groups (2 per group). In addition, for fat measurement, 2 were selected making the total of 3 groups (2 per group).
[Bioconcentration Report] 22
Company Sanitized. Does not contain TSCA CBf
/"""S
*3: because the amount of sample reserved for fat content measurement from only 1 fish was insufficient, 2 per group were used.
4.7.2 Pretreatment of analytical sample (1) Test substance in test water
From the test tanks, First concentration level: 40 mL Second concentration level: 400 mL
were collected, and a pretreatment was carried out according to the following scheme to obtain liquid chromatography-mass spectroscopy (LC-MS) samples.
Scheme
/'""`N
*4 Column chromatography conditions
Sep-Pak Cj8
(Washing method: eluate*5, water*6
10 mL each)
Loading method: whole amount loaded
Elution method:
first solution water*6 5 mL
second solution eluate*5 8 mL
Second solution used for analysis
*5 See quantitative determination conditions in section 4.7.3 (1)
*6 Purified water prepared from tap water using a super-pure water system
[Bioconcentration Report] 23
Company Sanitized. Does not contain TSCA CBI
(2) Study substance from study fish The study fish were taken from the test tank, and a pretreatment was carried out according
to the following scheme to obtain a liquid chromatography-mass spectrometry (LC-MS) sample.
Scheme
Study fish analytical sample
Body weight and length measurements Cutting up (scissors) Pulverization (in ice water, Polytron, 2 min or longer)
Pulverized sample
Sampled 1-5 g (electronic analytical balance)
Sample for preservation
Sampled 5 g (electronic analytical balance) <--methanol*7/formic acid (500/0.5 v/v) 15 mL (graduated cylinder) Homogenization (Polytron, about 1 min) Washing (methanor7/formic acid (500/0.5 v/v) 3 mL) Centrifugation (7000 X g, 5 min)
Cake
Supern atant
Filtration (absorbent cotton) Constant volume make-up 25 mL (methanol*7/formic acid (500/0.5 v/v), volumetric flask) Batch of 4 mL (transfer pipet) Water *67/formic acid (500/0.5 V/V) 2.5 mL (transfer pipet) Constant volume make-up 10 mL (methanor7/formic acid (500/0.5 v/v), volumetric flask)
LC-MS sample
*7: containing 10 mmol/L ammonium acetate
4.7.3 Quantitative analysis of test substance LC-MS samples prepared by the above pretreatment were used in the test substance
analysis by combined liquid chromatography-mass spectroscopy based on the following quantitative determination conditions. Incidentally, in the case of test fish analyses, if the test substance concentration was outside the range of the calibration curve, the sample was diluted
[Bioconcentration Report] 24 Compaiqr Sanitized. Does not contain TSCA CBf
before analysis to bring the concentration within range. The test substance concentration in an LCMS sample was determined by comparing peak areas of mass fragmentograms of standard solutions and LC-MS sample and extrapolating (see Tables 5 and 6, Figure 6, Tables 8, 9 and 10 and Figures 9, 10 and 11).
(1) Quantitative determination conditions
Instrument:
combined liquid chromatographer-mass spectrometer
Pump:
Nippon Bunko Model PU-980
Autosampler:
Nippon Bunko Model AS-950
Mass spectrometer: Micromass Model Quattro II
Liquid chromatography conditions Column: L-column ODS (prepared by Chemical Evaluation and Research Institute)
15 cm x 2.1 mm I.D. Column temperature: 25C Elution solution: Solution A (75%): methanol*7/formic acid (500/0.5 v/v)
Solution B (25%): water*6'7/formic acid (500/0.5 v/v) Flow rate: 0.2 mL/rnin .Amount injected: 20 jlL
Mass spectrometer conditions
Ionization method: electrospray (ESI)
Ion detected:
cation
Detection method: selective ion monitoring (SIM)
Ion measured:
Main component m/z 571
Subcomponent
m/z 513
Ion source temperature:
120C
Cone voltage:
50 V
(2) Standard solution preparation The standard solution for determining the test substance concentration of an analytical
sample was prepared as follows.
(a) Test water analysis 100 mg of the provided sample weighed accurately were dissolved in methanol to obtain a
1000 mg/L solution of the test substance. It was diluted with the elution solution*5to prepare a
[Bioconcentration Report] 25 Cwnpany Sanitized. Does not contain TSCA CBf
2.00 mg/L solution of the test substance, which was used to carry out a pretreatment according to the following scheme to obtain a 200 /xg/L standard solution. Scheme
(b) Test fish analysis 100 mg of the provided sample weighed accurately were dissolved in methanol to obtain a
1000 mg/L solution of the test substance. It was diluted with the elution solution*5to prepare a 2.00 mg/L solution of the test substance, which was used to carry out a pretreatment according to the following scheme to obtain a 200 fig/L standard solution.
[Bioconcentration Report] 26 Company Sanitized. Does not contain TSCA CBf
Scheme
Pulverized sample similar to that in the recovery test and blank test of 4.7.4 5 g
<--Methanol^/formic acid (500/0.5 v/v) 15 mL (graduated cylinder)
Homogenization (Polytron, about 1 min) Washing (methanol^/formic acid (500/0.5 v/v) 3 mL) Centrifugation (7000xg for 5 min)
Cake Supernatant
Filtration (absorbent cotton) Constant volume make-up 25 mL (methanol^/formic acid (500/0.5 v/v), volumetric flask) Sampled 4 mL (transfer pipet) <--Water*6,7/formic acid (500/0.5 v/v) 2.5 mL (transfer pipet) <r- 2.00 mg/mL test substance solution 1 mL (transfer pipet) Constant volume make-up 10 mL (methanol^/formic acid (500/0.5 v/v), volumetric flask)
200 jug/L standard solution (For test fish analysis)
(3) Calibration curve preparation
(a) Test water analysis
The same procedures as those used for the preparation of the standard solution in (2) (a)
were carried out to prepare 100,200 and 400 /rg/L standard solutions. These standard solutions
were analyzed under the quantitative determination conditions of (1), and a calibration curve was
prepared from the peak areas on respective mass fragmentograms and concentrations.
The quantitative determination lower limits of the peak areas were set as
Main component: 7000 (test substance concentration of 25 fig/L)
Subcomponent
1000 (test substance concentration of 12 fig/L)
by considering the noise level (see Figure 4).
(b) Test fish analysis /"p*v The same procedures as those used for the preparation of the standard solution in (2) (b)
were carried out to prepare 100, 200 and 400 xg/L standard solutions. These standard solutions
[Bioconcentration Report] 27
Company Sanitized. Does not contain TSCA CBS
were analyzed under the quantitative determination conditions of (1), and a calibration curve was
prepared from the peak areas on respective mass fragmentograms and concentrations.
The quantitative determination lower limits of the peak areas were set as
Main component: 7000 (test substance concentration of 20 jttg/L)
Subcomponent
1000 (test substance concentration of 19 jug/L)
by considering the noise level (see Figure 7).
4.7.4 Recovery test and blank test (1) Method
To determine the recovery rate of the test substance in the analytical procedures for the test water and test fish described in section 4.7.2, the test substance stock solution was added to the recovery test water and thinly sliced fish (10 g), and recovery tests were carried out. Furthermore, blank tests were carried out for the recovery test water and thinly sliced fish with no test substance added by carrying out the same procedures as those used for the above recovery test. For the recovery and blank tests, the measurements were carried out for 2 items each.
(2) Results As a result of measurement carried out by the method of (1), no peaks were observed at the
positions of the test substance in the mass fragmentograms obtained in the blank tests. The recovery rates and mean recovery rate for 2 items each in the analytical procedures were as follows, and the mean recovery rate was used as a correction in the case of determining the concentration of the test substance in an analytical sample (see Tables 4 and 7 and Figures 5 and Si-
Recovery rate in the analytical procedures
Test water analysis (2000 ng of test substance added)
Main component 82.7%, 85.3%
Mean84.0%
Subcomponent . 84.7%, 89.6%
Mean 87.1%
Test fish analysis (25000 ng of test substance added)
Main component 99.5%, 98.9%
Mean99.2%
Subcomponent
90.3% 89.5%
Mean89.9%
4.7.5 Fat content of test fish A pulverized test fish sample of the control group was extracted with chloroform/methanol,
and the fat content was determined by weight analysis.
[Bioconcentration Report] 28 Company Sanitized. Does not contain TSCA CBI
4.7.6 Calculation of test substance concentration in analytical sample and quantitative lower limit (1) Calculation of test substance concentration in test water analysis sample
The calculation was carried out according to the formulas shown in Tables 5 and 6; the results shown were rounded to 3 significant figures.
(2) Quantitative lower limit concentration of test substance in test water
From the quantitative lower limit determined in the preparation of the calibration curve in
section 4.7.3 (3) (a), the qualitative lower limit concentrations*8were calculated as follows.
Main component First concentration level 7.5 fig/L
Second concentration level 0.75 fig/L
Subcomponent
First concentration level 3.5 gg/L
Second concentration level 0.35 /xg/L
(3) Calculation of test substance in test fish analytical sample The calculation was carried out according to the calculation formulas shown in Tables 8,9
and 10; the results shown were rounded to 3 significant figures.
(4) Quantitative lower limit concentration of test substance in test fish
From the quantitative lower limit determined in the preparation of the calibration curve in
section 4.7.3 (3) (b), the qualitative lower limit concentrations*8 were calculated as follows when
the amount of pulverized test fish sample was 5 g.
Main component 260 ng/g
Subcomponent
270 ng/g
*8: Quantitative lower limit concentration of test substance (/xg/L or ng/g)
= A_________
B/100 x CxE/D
A : . calibration curve quantitative lower limit concentration (/xg/L) B : recovery rate (%) C : amount of test water sampled (mL) pulverized test fish sampled (g) D : final amount of liquid (mL) E : Sampling ratio The calculation results were rounded to 2 significant figures.
[Bioconcentration Report] 29
Company Sanitized. Does not contain TSCA GBf
4.7.7 Calculation method for total mean test substance concentration in test water during exposure term
Cwt = { O i l ) + '..... + Cw(n)} / n
C w t: total mean test substance concentration in test water (jig/L) n: No. of test water analyses (frequency of measurement) Cw(l): test substance concentration in test water measured for the 1st time (jtig/L) Cw(n): test substance concentration in test water measured for the nthtime (jUg/L)
4.7.8 Calculation method for bioconcentration factor (BCF) The bioconcentration factor (BCF) was calculated according to the following formulas.
(1) Calculation of mean test substance concentration in test water for calculation of bioconcentration factor
Cw = [Cw(n -1) + Cw(n)]/2 (first test fish analysis) Cw = [Cw(n - 2) + Cw(n -1) + Cw(n)]/3 (2nd or later test fish analysis) C w : mean test substance concentration in test water for calculation of bioconcentration factor (fig/L) Cw(n): test substance concentration in the nthtest water analysis determined at the same time as test fish analysis (jtig/L)
(2) Bioconcentration factor calculation
BCF = Cf/ Cw
BCF : Cf : Cw :
FB :
bioconcentration factor test substance concentration in test fish (value with FB subtracted) (ng/g) mean test substance concentration in test water for calculation of bioconcentration factor (fxg/L) mean concentration of test substance in test fish before and after experiment in the control group or apparent (blank) concentration of test substance (ng/g)
[Bioconcentration Report] 30
Company Sanitized. Does not contain TSCA
(3) Mean of bioconcentration factor of mthtime BCFm = (BCFa + BCFb)/n BCFm : mean of bioconcentration factor of m* time (No. of individuals or group 2(a,b)) BCFa,b : bioconcentration factor of each individual or group of mthtime n : number of individuals or group analyzed in the mthtime
The mean of the bioconcentration factor for a measurement of no detection is not to be determined.
4.7.9 Method of confirming steady state The steady state is determined by observing a 20% or less fluctuation of the results of the 3
bioconcentration factor measurements in a row in an interval or 48 h or longer. If the bioconcentration factor is less than 100 times, it is considered to have reached a steady state after 28 days even if the bioconcentration factor fluctuation is over 20%.
Standard for attainment of a steady state: V(m - 2), V(m -1), V(m) <
I BCF(a-2) - BCF (a ~ 2 ) = ----------_ .IS 5 _ --------- x 100 '
BCP
20 (%)
I BCFCa-l) - i c P '.|
---------- X' 100 BCF
BCF(to) -- BCF |
(m)
= ---------------= ------------ X 100
BCP
V(m - 2), V(m -1), V(m): variation (%) of bioconcentration factor from the mean BCF(m - 2), BCF(m -1), BCF(m): mean of the results of the (m - 2)th, (m - l)th and mth bioconcentration factor of n individuals or groups BCF : {BCF (m - 2) + BCF (m -1) + BCF (m)}/3
[Bioconcentration Report] 31 Company SanitizBd. Does net contain TSCA CBf
4.7.10 Method for calculation of steady state bioconcentration factor (BCFss) The steady state bioconcentration factor (BCFss) was calculated using the following
formulas.
(1) Calculation of mean test substance concentration in test water for calculation of steady state bioconcentration factor
Cws * (Cw(n-2) + Cw(n-l) + Cw(n)l / S
Cws: mean test substance concentration in test water for calculation of steady state bioconcentration factor (as a rule, mean test substance concentration in test water measured 3 times in a row up to the final test fish analysis) (fig/L) Cw(n): test substance concentration of nthtest water analysis determined simultaneously with test fish analysis (/g/L)
(2) Calculation of mean test substance concentration in test fish in a steady state
Cfs ~ (Cf
+ Cf(tt-1) + Cf(nt)} /3
Cfs: mean steady state test substance concentration in test fish (ng/g) Cf(m): mean test substance concentration in mthtest fish analysis (value with FB subtracted) (ng/g) FB: mean concentration of test substance in test fish before and after experiment in the control group or apparent (blank) concentration of test substance (ng/g)
(3) Calculation of steady state bioconcentration factor
BCFss- - Cfs / Cws
BCFss: steady state bioconcentration factor C fs: mean steady state substance concentration in test fish (ng/g) Cws: mean test substance concentration in test water for calculation of steady state bioconcentration factor (/g/L)
4.7.11 Calculatable bioconcentration factor From the quantitative lower limit concentration of the test substances in test fish
determined in section 4.7.6(4), it is possible to calculate a bioconcentration factor when it exceeds the following values. The test substance concentration in test water used was the mean test substance concentration in the case of all test water analyses.
[Bioconcentration Report] 32 Company Sanitized. Does not contain TSCA CBf
Main component Subcomponent
First concentration level Second concentration level First concentration level Second concentration level
5.1 times 51 times 5.6 times 55 times
4.7.12 Method for calculation of fat content The fat content was determined from the following formula. Fat content (%) = (T-T0)/S x 100 To: weight of container (g) T: weight of sample for weight analysis (including container) (g) S: Sampled amount of pulverized test fish (g)
4.8 Handling of numbers The method of JIS Z 8401: 1999 Rule B was used for rounding. The results used in
calculation were not rounded afterward. The test substance concentration in the test water and that in the test fish were rounded to 3
significant figures, and the bioconcentration factor was shown by rounding to 2 significant figures.
5. Environmental factors considered to affect reliability of test results No corresponding factors were found.
6. Test results 6.1 Test substance concentration in test water
As apparent from the results shown in Table 1, the test substance concentrations in the test water were maintained above 88% of the values set. The fluctuations in test substance concentrations were maintained within 20% of the mean measurement result.
Component Main component
Subcomponent
Table 1. Test substance concentration in test water
Concentration level
1
After 4 days
46.0
2 4.43
1 45.7
2 4.39
After 7 days
45.7
48.7
45.3
5.01
After 10 days
51.7
5.08
51.7
5.20
After 17 days
53.4
5.47
46.5
5.17
After 24 days
53.1
5.27
50.7
5.20
After 28 days
51.5
4.75
48.4
4.56
(Unit: g/L)
M ean (standard Table deviation)
50.2 (3.47)
5
4.98
6
(0.376)
48.1 (2.69)
5
4.92 (0.357)
6
Fig 6
[Bioconcentration Report] 33 Company Sanitized. Does not containTSCACBI
6.2 Bioconcentration factor
Table 2 shows the bioconcentration factor results.
The correlation between the bioconcentration factor results shown in Table 2 and exposure
term is shown in Figure 1 and Figure 2. The bioconcentration results factor during the exposure
term are as follows.
Main component First concentration level less than 5.1 times
Second concentration level less than 51 times
Subcomponent
First concentration level 350-510 times
Second concentration level 270-430 times
Table 2. Bioconcentration factor
Component
Concentration After After level 7 days 10 days
Main component
1 Si 5.1 1 5 .1
=! 5.1 ^ 5.1 2 =1 51 3= 51
3s 51 3s 51
Subcomponent
1 360 350
370 360
(370) (350)
2 380 370
320 320
(350) (340)
After 17 After 24 days days
3s 5.1
3s 5.1
g51
3i 51 410 510 (460) 430 340 (380)
3s 5.1
^ 5.1
3s 51
3s 51 410 410 (410) 350 340 (340)
Mean inside ()
After 28 days Table Fig.
^5.1
3s 5.1
3i 51
3s 51 470 430 (450) 270 270 (270)
89 9 10 89 9 10
6.3 Steady state bioconcentration factor 6.3.1 Main component
As a result of section 6.2, all of the final consecutive 3 analyses showed no detection, and consequently, no steady state bioconcentration factor could be calculated. However, the results on the bioconcentration factor were all less than 100 times, and thus, it was concluded that the steady state had been attained after 28 days.
6.3.2 Subcomponent As a result of section 6.2, the results on the bioconcentration factor (mean) after 17, 24 and
28 days showed fluctuations within 20% of the mean bioconcentration factor of those 3 analyses, and consequently, it was concluded to have reached a steady state. Those results were used to calculate a steady state bioconcentration factor.
[Bioconcentration Report] 34 Does not contain TSCA CBI
S0"*'--
(1) Steady state test substance concentration in test water As shown in Table 3, the mean test substance concentrations in test water in a steady state
were 97% of the prescribed value in the first concentration level and 100% in the second concentration level.
Table 3. Steady state test substance concentration in test water
Concentration level
1
2
After 17 days
46.5
5.17
After 24 days
50.7
5.20
After 28 days
48.4
4.56
Mean Table
48.5 5 ,8 4.98 6 ,9
Figure 6
(2) Steady state bioconcentration factor The results on the steady state bioconcentration factor were as follows. First concentration level 440 times Second concentration level 340 times
6.4 Fat content of test fish The results on the mean fat content of the test fish were as follows. Before experiment 2.94% After experiment 4.32%
6.5 Observed appearance of test fish No abnormality was observed.
7. Discussion The fluctuation in fat content (mean) after the experiment of the present study was 47% and
exceeded 25% compared with the fat content before the experiment. The fluctuations in physiological conditions, feeding state, etc., are considered to be involved in the observed fat content fluctuation. The study on the feeding method, etc., will be continued for improvement with respect to fat content changes and fluctuations.
8. Notes Major instruments, equipment, reagents, etc., used in the study were are as follows.
[Bioconcentration Report] 35 CkKppan^'SmMndLltaee not contain TSCA CBi
(1) Devices and equipment used in the study system (rearing facility)
Microquantitative pump for
supplying stock solution:
GMW Model manufactured by Tokyo Rikakikai
Dissolved oxygen meter:
Model F-102 manufactured by Iijima Denshi Kogyo
pH meter:
Model HM - 14P manufactured by Toa Denpa Kogyo
Thermometer for water:
Model ALCO-500 manufactured by Andoh Keiki
(2) Instruments, devices, special equipment and reagents used for analysis and stock solution preparation
Instrument and equipment Combined liquid chromatographer-mass spectrometer: see page 15 Balance: Model LP4200S manufactured by Sartorius
Model 1216MP manufactured by Sartorius Model BP301S manufactured by Sartorius Model PB602 manufactured by Mettler-Toledo Fourier-transformed infrared spectrophotometer: Model FTIR-8200PC manufactured by Shimazu Homogenizer (Polytron): Model PT3000 manufactured by Kinematica
Model PT3100 manufactured by Kinematica Centrifuge: Model CR21G manufactured by Hitachi Koki Pulverizer: stirring pulverizer manufactured by Ishikawa Kojo Special equipment SepPak Cig: manufactured by Nippon Waters Reagents Methanol: for HPLC manufactured by Wako Junyaku Kogyo Formic acid: Grade 1 reagent manufactured by Wako Junyaku Kogyo Ammonium acetate: Research grade reagent manufactured by Wako Junyaku Kogyo Crystallized sugar: manufactured by Ohtori Hyoutou Megafac F-142D: manufactured by Dainippon Ink Kagaku Kogyo
(3) Instruments, equipment and reagents used for fat content measurement
Balance: Model BP301S manufactured by Sartorius
Rotary evaporator:
Model N -1 manufactured by Tokyo Rika Kikai
Model N - 1000K manufactured by Tokyo Rika Kikai
Homogenizer (Polytron): Model PT3100 manufactured by Kinematica
Model PT3000 manufactured by Kinematica
[Bioconcentration Report] 36 Ccmsam Sanitized. Oms not contain TSCA CBI
/ #*K'
Homogenizer (Autocellmaster): Model CM - 200 manufactured by Iuchi Seieido
Vacuum pump:
Model DA-20D manufactured by Shinku Kiko
Vacuum desiccator:
Model VL manufactured by Iuchi Seieido
Reagents
Purified water:
Pharmacopoeia japonica, manufactured by Takasugi Seiyaku
Methanol:
Grade 1 reagent, manufactured by Wako Junyaku Kogyo
Chloroform:
Research grade reagent manufactured by Wako Junyaku Kogyo
Sodium sulfate (anhydrous): Grade 1 reagent, manufactured by Kanto Kagaku
LANGUAGE SERVICES UNIT RALPH MCELROY TRANSLATION COMPANY FEBRUARY 20, 2004
[Bioconcentration Report] 37 r '': SanfibscL Does ns! ecmSalr? TSCA OBI
