Document 1gMNG579XdJMvqMoMZqgy5Zno
AR226-3338
FINAL REPORT
Bioaccumulation test ofPerftooroalkylcarboxylie acid (C--7-13) (This test 1$ performed using Periluorooctanoic add (Test substance number K-l 51!?)] in carp
(Test No. S1519)
Kurume Laboratory Chemicals Evaluation and Research Institute, Japan
STATEMENT
51519
Kumme Laboratory Chemicals Evaluation and Research Institute, Japan
Sponsor
Ministry of Internationa) Trade and Industry
Title Bioaccmnulalinn test oFPerHiinrnalkylcarhoxyltc acid (0=7-1 !5) {This test is performed using PerSuorooelanoic. add (Test substance number K-1519)]incarp
Test number
51519
J, the-undersigned, hereby declare that this report provides a correct English translation ofthe Final Report (Test No.51519, issued on December 18.2000}.
Date Study director
Brim Kilnishimn
K 51519
STATEMENT
Kunrnic Laboratory Chemicals Evaluation and Research Institute, Japan
Sponsor ' Title
Ministry of International Trade and Industry
Bioaccumulation test of Perflunmalkylcarboxylic acid (C=7-l.l) [This test is perfijtined using Perfluoroocianoie acid (Test substance number K-1519)]incarp
Test number
51519
This final report (copy) was made the copy accurately of the final report on above the test.
t
Date Management
December 18,2000 Signed in original Hiroshi Tadokoro
GLP STATEMENT
51519
Kumme LaboratoryChemicals Evaluation and Research Institute, Japan
Sponsor
Ministry of International Trade and Industry
Title Bioaccumulation test of Perfluoroalkylcarboxylic add (C-7-13) [This test is performed using Perfluorooctanoic acid (Test substance number K-1519)] in carp
Test number
51519
This test was conducted in compliance wilh Good Laboratory Practice Standards for industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests stipulated in article 4 of the Order Prescribing Those Items of the Test Relating to the New Chemical Substances and Study on Harmful Effects of Designated Chemical Substances" (March 31, 1984, Revised March 1, 2000, Kanpogyo No.39, Planning and Coordination Bureau, Environment Agency, Yakuhatu Tin 229, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 59 Kikyoku No.SS, Basic Industries Bureau, Ministry of I-niRrnatinnnl Trade, and Industry. Japan) and "OKCn Principles of Good Laboratory Practice" (November26,1997).
Jt has been confirmed that this final report reflects the raw data accurately and the test
data are valid.
Date Study director
December IS, 2000 Signed in original Jenna RUaishnna
4
51519
QUALITY ASSURANCE STATEMF.NT
Kunime Laboratory Chemicals Evaluada and Research Institue, Japan
Spisor Tille
Ministry uPintnational Trade and Industry
Bioaccumulation test of Perfluoroalkylcarboxylic add (C=7-13) [Hus to t is ptaformed using Perfluorooctanuie add (Test substance number TC-1519)] in carp
Test number
51519
Study audits and inspections of this test were earned out and the results were reported to the management and the study director by Quality Assurance Unit of Kurum Laboratory, Chemicals Evaluation arid Research Institute, Japan as follows.
Item of audit or inspection : . Protocol
Test Conduct
Raw Data and Final Report
Date of audit or inspection September?, 2000 September IR 2000 September 28,2000 November 28,2000 September 12,2000 October 16,2000 October 17,2000 October27,2000 November 14,2000
Date ofreport to study director September 7, 2C0 September IS, 2000 September 2S, 2000
November 26, 2000 September 18,2000
October 17,2000 October 17,2000
' November 15, 2000 "Novemhei* 15. 2000
December 18,2000 j TW.emlw'r 18. 2000
Date ofreport to management September 8,2000 September 18,2000 Septembei 28, 2000 November 28,2000 September 21,2000 October 23,2000 October 23,2000 November 16, 2000 November 16,2000
December 18.2000
It has been assured that the final report describes accurately the lest 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 Quality Assurance Unit, Head
December 18,2000 Signed in original Kyoshlrol-Iori
CONTENTS
Title
Sponsor
Testing facility Objective Test method Applied GLP Test schedule Storage of test substance, taw data, etc, Personnel Preparation of final report SUMMARY 1. Test substance 2. Performance of acme toxicity test
5. PerfonnSin.ce ofbioaocum uiation tact
4. Factors possibly affecting accuracy 5. Results 6. Remarks
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page 1 1 1 1 1 2 'A
2
3 3 4 5 ? in 23 24 26
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Contents of tables and figures
Contents oftables
Table-1
Measured concentrations of test substance in testwater (see test)
Table-2 JVbla 3 Tablc-4
BCFs (seetext) Concentrations ofti-ii <aiW a/ims in test wafra at a steady-state (see text!) Calculation table for analysis of test water (Level 1)
Table-5
Calculation table for analysis oftest water (Level 2)
Table-6
Calculation table lot recovery and blank test (analysis oftest fish)
Table-7
Calculation table for'analysis of test fish (Level 1)
Table-8
C alculation table for analysis ofrest fish (Level 2)
Table-9
Calculationtable fut analysis oftest fish (Control)
Reference 1 Analytical results ofdilution waLer
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Contents o f figures
Pig. 1
Correlation between exposure period and bioconcentration factor (Level 0
Fig. 2
Correlation between exposure period and bioeoncentration factor (Level 2)
Fig. 5 Fig. 4 Fig. 5 Fig. 6 Fig. 7
Fig. 8 Fig.9 Fig. 10 Fig.ll Fig 12 Fig-12 Fig.14-1 Fig. 14-2
Concentration mortality curve
Calibration curve for T.C-MS analysis (analysis oftest water) Mass chromatogram ofLC-MS analysis for analysis of test water Calibration curvefor LC-MS analysis (analysis ol test fish) Mass chromatogram ofLC-MS analysis for recovery and blank test (analysis oftest fish) Mass chromatogram oFLC-MS analysis for test fish (Level 1) Mass chromatogram ofLC-MS analysis for test fish (Level 2) Mass chromatogram ofLC-MS analysis for test fish (Control) Concentration of dissolved oxygen (Level 1) Concentration of dissolved oxygen (Level 2) Concentration of dissolved oxygen (Control) [R spectrum oftest substance measured before the experimental start IR spectrum of test substance measured after the experimental completion
Fig-15 Reference 2
Mass spectrum of test substance KMr. spectrum oftest substance
51519
Title
Bioaccumulalioa test of Perftuoroalkyicarboxylic add <0=7-13) [lhis test is performed usingPetiloorooctamne acid (Test substance number K-1519)] m carp
Sponsor
'
Minfatry of International Trade and Industry
1-3-1 Kasurtiigaseki, Chiyoda-ku. Tokyo !00-8901, japan
Testing facility
Kumme Laboratory
_
Chemicals Evaluation and Research institute, Japan
19-14 Chuomachi, Kurume-shi, Fukuoka 83(J4X)23, Japan
Objective This lest was performed to evaluate the bioaccumuktion potential ofK-3519 in carp.
Test method
This test was conducted according to the "Method for Testing the Degree of Accumulation of Chemical Substances m Fish Body" stipulated in the 'Tesmtg Methods for New Chemical Substances" (July 13, 1974, Revised October 8, 1998, Kanpogyu No.5, - m ir n m 2nd CaofdmtionDuraiu, Environment Agwcy, Yakuham No 613, Phannaceuticat Affairs Bureau, Ministry- of Health and Welfare, and 49 Kikyoku No.392. Basic Industries Bureau MhiisUy of International Trade and Industry, Japan). This test method is anticniiciny the me a* that in the OFfD Guidelines for Testing of Chemicals. ''Biocoucentration: Flow-through Fish Test (Guideline 305, June 14,1996) .
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51519
Applied GLP
fl) Chemical GIP
. ..... -
This te. complied with "Basic standards to be observed by testmg facilities in
conducting tests stipulated in article 4 of the Order Prescribing Thosei lions of the
Test Relating to the Mew Chemical Substances and Study on Harmful Effects oi
Designated Chemical Substances (henatilci id ared to as "OLP standards")1' ^farc
3L 1984, Revised March 1, 2000, Kanpogyo No.39, Planning and Coordination
B uieau, Environm ent AgHWy, VakuhMu No.3?.P, Pharmaceutical Afihir* J - * " ',
Ministry of Health and Welfare, and 59 Kifcyofcu No.85, Basic Industries Bureau.
Ministry of International Trade and Industry', Japan).
(2)OtCD-^rLP^ C(>mp,{el wilh -OECD Principles ur Quud Labcatory Fractioc" (November 2 6 ,19P7), ` .
Test schedule Start of test Experimental starting date Experimental completion date End of test
September 7,2000 October 17,2000 November 14,2000 December 18, 2O0
Storage oftest substance, raw data, etc.
(J) Test substance Because the test
substance of the
same
lot
is
already
stored
atier
the
end
of
UiwdstnTulntforLlesL, lliu lost su bottinaci io not otorod oflar-thic tect
(2) Raw' data and materials, etc. Raw data used for the preparadon of the final report, the protocol, documents
about the test presented by the sponsor, the copy ol'Lhe final report and necessary
materials are stored in a storage room Inthis laboratory.
51519
Personnel Studydirector . Personal (Operation ofbioacarniulaticra test)
Stafffor fish " "' Person to conduct of fish acute toxicity lest
Preparation of final report Study director
Date Signature
Frina KikushiniR RjeObo Yuji Kusune Kazuomi Katagiri Ymurn Kawashima Yasuro Kawashima Katsumi Kiyono
December 18,2000 Signed in original BrajaKiJnisbiffia
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SUMMARY
51519
Title.
Bioaccumulation test of Perfluoroalkylcatboxylic acid (C--7-13) [This lest is perbiiitt*d using Peifh.ie>'-ctcinriinie nr.id (Th<3-siihyannft numher K-1519)] in carp
Test conditions
Acute toxicity test (1) Test fish (2) Duration of exposure (3) Exposure method
Orange-red I
96hrs, Semi static system (KenewaJ of test water, at every - JOhr$.)
Bioaccumulation test (1) Test fish .
Harp (flyprirms carpin')
(2) Nominal concentrations oftest substance High exposure level (Level 1) 50 pgL
Low exposure level (Level 2) 5 pg/L
(3) Duration of exposure (4) Exposure method (5) Analytical method
28 days Continuous flow system High-performance liquid chromatography-mass
spectrometry
Results
(1) 96-hour LC50 value
100 mg/L
(2) B iocunw autaduu Cautui at a steady state
Level 1
(3) Bioconcentrarion factors Level 2
3.1 5.1 - 9.4
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1. Test substance In this report, K-1519 has the following chemical name, etc.
1.1 Chemical name Perfluoronctanoic acid 1.2 Chemical structure, etc.
SliuOtural fonmila.
C.Fa-- (C.Fz)s--- 0 ft OH
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Molecular fujinyla
CsRFisCh
Molecular weight
414.07
1.3 Manufacturer, name and lot number 1 (1) Manufacturer . DAIK-IN i-inectiemical Laooratojy, .Lid.
{2) Name (3) Lot number
C-1700 C17009201
*1 Information supplied bythe manufacturer
1.4 Purity'3 Test substance
98 % (GC)
The test substance was treated as 100 % in purity.
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1.5 Confirmation oftest substance The structure of the lesL substance is uonliinsed by mfiaied (1R) spectrum (sec
Tig. 14), Mass spectrum (see Fig.15) and NMR spectrum (see Reference 2).
1.6 Storage and stability
(1) Storage condition Cold storage place
(2) Stability
.. , ,
The test suhstau.ee was stable under the storage condition as shewn hy the
finding that IR spectra of the test substance before and after the experiment were
identical (see Fig.14).
1.7 Stability undertesting conditions Prior to the bioaccumulation tea, a stability of the test substance under the testing
conditions was confirmed by a preliminary tea.
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51519
2. Performance of acute toxicity test
2.1 was performed in accordance with Japanese I tid TM i SntodQ]IS K 0IO2-J998-71.), "Testing methods for industrial waste water, Acute toxi ity ffiirs,lh,'l1
2.2 Test fish (1) Species
0 1anac-redkillifish(OrymasMpes)
..
Reason for selection :This species is similar in nativity to carp and
readily available as test fish.
(2) Supplier
Nakashima fish {aim
t
(Address: 2029 Ooaza Naensu, Kagasu-oho, Tamana-gjjn, Kumamoto
869-0123., Japan)
n ) Conditions ibr fish care before acclimatization The fish were checked visually at the receiving and those demonstrating any
abnormality were removed. The fish were reared for 39 days m a flow through
system following an external disinfection.
{4 )Caad1Z S t f i w
c re
tra n s fe rre d to a n
^ and
acclimatized there after the second external disinfection.
any abnormality duringthis period were removed and the " df 2f ^ ^ Z reared for 21 days in a flow through system at the temperature o fish were checked for health conditions and reared another 4 1 -4 days afteT fit
external disinfection.
(5) Weight
average 0.20 g {for additional test 0.19 g)
(6) Length
average 2.9 cm
(7) C ertification^
0f tbc reference substance*' * r the fish ofthe same lot
(TFO-1)00706)was nin/T.
*2 PCP-fra (pentachlorophenol sodium salt, Tokyo Kasei Kogyo Co., Ltd.)
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51519
2.3 Dilution water for test
(1) Origin TInrfprBTrwmrfwater from the premises of Kurame Laboratory.
(2) Water quality assessment
_.
The dilution water for test was taken out on August S, 2000, and it was
analyzed and measured (once every six months in this laboratory). The results are
abuwulullcfuicnec 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 lest" {Guideline 210, July 17, 1992) and environmental quality standards for water
pollutants No, 14 (Revised February 22, 19D9, Environment .Agency) or OECD
Guidelines for Testing of Chemicals, "Bioconcentration: Flow-through Fish Test
(Guideline 31)5, June 14,1996)"
2.4 Test conditions (1) Test tank
Round glass vessel
C2) Volume oftest water 4 L / level
(3) Temperature oftest water At initial exposure
Before renewal oftest water
24.4-24,6 C 24.3 - 24.7 C
(4) Concentration of dissolved oxygen in test water
At initial exposure
7.9 - 8.1 mgZL
Before renewal oftest water 7.1 -7.3 mg/L
(5) p H u f Lcsl waLia
At initial exposure Befare renewal of rest water
7.1 -8.0 7.5 -7.9
(6) Number of fish
10/level
(7) Duration of exposure 96 hra.
(8) Exposure method
Semi static system (Renewal oftest water, at every R*16 hrs )
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5)519
2.5 Preparation of srock solution
(1) Dispersant HCO-Q (Hydrogenated castor oil)
(2) Preparation The test substance and HCO-60 (50 times amount of the test substance) were
dissolved with acetone. After the acetone was evaporated from the solution, ion-
bsmgod wotor woo ddtd to tb* mktvu-0, to papato lOUft ntgfl. norie solulTM
2.6 Performance oftest
(1) Place (2) Date
2l4LC50room September L1,2000 - Scptember 11,2000
2.7 Estimation of96-howLC50 value The 96-hourLC50 value was estimated by the Doudoroffmethod.
2:8 Result of test 96-hour LC50 value
100 mgA- (see Fig. 3)
S
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3. Performance ofbioaccumulation test
3.1 Test fish (t) Species
Carp (Cvprinus tarpig) Reason for selection : The previous data conducted -with this species
can be compared and the size ofthis species is
adequate for handling.
(2) Supplier Fuktiokaksn
__ cooperative association
(Address: 748 Yamauchi, Yame-shi, Fukuoka 834-0012, Japan)
Date received
September ], zoro
(3) Conditions for fish care before acclimatization The fish were checked visually in the receiving and those demonstrating any
abnormality were removed. The fish were reared for 8 days in a flow through
system following an external disinfection
(4) Conditions foracclimatization After rearing, the fish were medicaid lu eliminate parasites and iianslencd to
an acclimatizing aquarium. After the second external disinfection, they were acclimatized, The fish demonstrating any abnormality during this period were
removed and the remainder of the fish were reared for 15 days in a flow through system at the temperature of 25 = 2 *C. The fish were then transferred to test tanks and reared at the same temperature in the flow through system for another 19 days, follow ing th< external distnfVrntm
(5) Length 6.8 -S.6cm
(6) Lot No. TFC-QOQ901
(7) Age
Yearling fish
(8) Feeding Feed Composition
Feed for &y of carp Proteins content s= A3.U%
Lipid content = 3.0%
Manufacturer Nippon Formula Feed Mfg. Co., Ltd.
Feeding amount and interval Amount corresponding to 2 % oftotal body weight was fed twice a day in
halves. The fish wert sawed 14 boats birforesampling.
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f
% I
51519
3.2 Dilution water for test The same aSdescribed in 2.3.
3 3 Conditions oftest and circumstances
(1) Supply of test water
_
Flow through system assembled at this laboratory' wasused.
2( ) Tesi tank
100-L glass tank
(3) Flow rate of Lest water
_
2 mUmin for stock solution and 800 mL/min for drlulion votes
-.< for tM water were supplied.
1155
(4) Stock solution bottle
25-1. glass bottle (Frequency of renewal about 1 fimea/vveek)
(5) Temperature oftest water
Level 1
24J -25.6 C
Level 2
24.9-25.4 C
Control
25.2 - 25.8 C
(6) Concentrations of dissolved oxygen intest water
Level 1
7 9 - 8.1 me/L (see Fig. 11)
Level 2
7.9 - 8.1 ingfL (see Fig. 12}
Control
8.0 - 8.1 mg/L (see fig. 13)
(7) pH of test water Level 1 Level 2 Control
7.7 7,8 7.7 -7 8 7.6-7,8
(S) Time of irradiation with light Artificial fight of whins fluorescent lamp (14 hrs./day)
(9) Number offish (at the beginning of exposure)
Level 1 anil 2 2S
Control
8
(10) Duration of exposure Reason for decision:
28 days
..
The time fui leaching a steady-state was estimated m
28 days from preliminarytest results.
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(11) Place
213 Aquatron room
3A Preparation of stock solutions
(1) Dispersant The same as described in 2.5 (1).
(2) Preparation
Level 1
.
20 ma'L stock solution was prepared inthe same way as described in 2.5 (2)
Level 2
_.
2 mg/L stock solutionwas prepared in the same way as described in 2.5 (2),
Control HCO-60 was dissolved with ion-exchanged water to prepare 1000 mg/I.
stock solution.
3.5 Test concentrations
,
Based on preliminary test results for the 96-hDur LC50 value and analytical
detection limits, test concentrations of the test substance were decided as follows. The
control was set as a blank test
Level 1 Level 2
50 jig/L 5 pg/L
f
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3.6 Observation, measurement, etc.
(1) Observation of test fish
_
Condition oftest fish m s observed visually tw ee a day.
(2) Flow rate oftest water Flow rate of stock solution and dilution water were measured with graduated
cylinder and recorded once a day.
(3) Temperature of test water Temperature of tesL water was measured with alcohol thermometer and
recorded once a day.
(4} Concentration of dissolved oxygen in test water
_
Concentration of dissolved oxygen in test water was measured with dissolved
oxygen probe and recorded twice a week.
(5) pH oftest water pH of test water was measured with pH meter once a week or more.
(6) Cleaning of test tank In experimental period, excreta of carp, dirt on test tank, etc. were removed
about once a day.
3.7 Analysis of test -water and fish
. ...
Analysis of the test substance in test water and test fish was performed with high
performance liquid Chromatography-mass spectrometry (LC-MS) analysis.
3.7.1 Frequency of analysis
(1) Test water analysis The test water of each level was analyzed once before first analysis of test
fish at the autna time as analyem UJ' tost fish. On* sample of each level w analyzedateach samplingtime,
(2) Test fish analysis Analysis of test fish was performed five rimes at each level m duration of
exposure Analysis of control fell was performed befute die experimental
starting and at the experimental completion.
_
Four fish were taken out at each sampling time and divided into two groups,
then both were analyzed individuallyJ.
*3 Because one fish was loo small to take out the stored sample for the measurement oflipid content, two fish a group were employed.
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3.7.2 Pretreatment foranalysis
(1) Test water
An Aliquot ofthe test water,
Level 1
) jnL
Level 2 2.5 mL
,
was taken from each test tank, and pretreated for high-performance liquid
ohTomuUijp'npliy-mnns spectrometry (LC MS) analysis as follows .
| Test water
Filling up to 10 mL (water for rectjvtay lest,
volumetric flask) (only for Level 1)
Taking out 2.5 mL (transfer pipette)
{only for Level 1)
_
Fillingup to 10mL (water'", volumetric flask )
Sample forLC-MS analysis |*
*4 r.iiy water was treated by Ultra pure water system.
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{*y\ {jsJi Test fish were taken from each test tank and pretreated for high-performance
liquid chromatography-mass spectrometry (LC-MS) analysis as follow-s.
Measurement of weight and body length Chopping into pieces - Making sample fine (polytron, 2 min. or more,
on ice water)
- Taking out 1- 5 g (analytical balance)
| Sample for storage
Taking out 1 g (analytical balance) --Acetonitrile 20 mL (graduated cylinder) *Homogenization (polytron, about 1 min.)
- Washing (acetonitrile 7 mL) - Centrifugation (7100 x <j, 5 min,)
*Filtration (absoibent cotton) - Filling up to 50 mL {acetonitrile, volumetric
flask) - Taking out 1 mL (transferpipette) Filling up to 25 mL (water*4, volumetric
flask)
Sample for LC-MS analysis
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3.7.3 Quantitative analysis for test substance The samples for LC-MS analysis in piclreatment were analyzed by high
performance liquid chromatography-mass spectrometry under the following analytical conditions The concentration of the lest substance in each sampfe solution was determined on the baas of a comparison of the peak area on the mass chromatogram of the sample solution with that of a standard solution (see TabJcs-4, 5. Fig.5 and Tables-
7,8,9, Figs.8,9,10).
( 1) A nalytical conditions
Instrument
High-performance liquid chromatograph-
mass spectrometer
High-performance liquid chromatograph
Waters Corporation type 2690
Mass spectrometer
Waters Corporation typeZMD
r n priitinm ofhigh-performance liouid chromalagraEh
Column
L-colunui ODS 15 cm * 2.1 mm f.D. stainless steel
Column temp. Eluent
40 CC A (60%): Acetonitrile B (40%): 5 mtnoL'L Di-fi-butylamine acetate
Flow rate
0.2 mL-'min.
Sample size
20 uL
rntuTninin)! n f m ass spectrometer
Ionization mode
TSenrospray
Detection bn
Negative ion
Cone voltage
1ft V
Source block temp.
120 C
Desolvation temp.
300 "C
Desolvation gas flow rate 3 50 L/hr
Monitoring ion
m/n 413
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(2) Preparation of standard solution
.
The standard solution to determine the concentration of the test substance ;n
tie sample solutions was prepared as follows.
(a) Analysis oftest watei 100 mg ofthe substancewas dissolved in acetonitrile to prepare 1000 rrwL.
solution of the rest substance 2.50 ug/L- standard solution was then prepared
from this solution by dilution with water">water for recovery lest (1/1 V/V).
(b) Analysis of test fish ' 100 mg ofthe substance was dissolved in acetonitrile to prepare 1000 mg/L
solution of the test substance. 2.00 pg/h standard solution was then prepared
from this solution by dilution with water
(3) Calibration curve
(a) Analysis oftest water
.,
1.25, 2.50 and 5.00 pg/L standard solutions were prepared by the same
method as described in (2)(a). These solutions were analyzed according to the
analytical conditions described in (1). A calibration curve was drawn on the
basis ofthe relation between tire peak ansa on the mass chromatograms and the
respective concentrations. In consideration of the noise level, the bwesL detectable peak area of the
test substance was regarded as 250, which correspond! to 0.10 pg/L of the test
tiiihmjiriccconcentration (see 11s.4),
(b) Analysis oftest fish
.,
1 00, 2 00 and 4.00 psi/L standard solutions were prepared by the same
method as described in (2)(b). These solutions were analyzed according to the
analytical conditions described in (.!]. A calibration curve was drawn on t e
baas of the relation between the peak area on the mass chromatograms and the
respective concentrations In conoidoration nf the note* level, the lowest rifitftr.tflhte peak area fit the
test substance was regarded as 300 for analysis of test water and test fish, winch
corresponded to 0.092 itg/L ofthe test substance concentration (see Fig.6),
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3.7.4 Recovery and blanktest
(1) Method Chopped fish (10 g) spiked a specified amount of 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. The recovery
and blank tests were performed in duplicate.
(Z) R esults o f recovery lcat
,
in the blank test, the mass chromalogtam of LC-MS had no peaks interfering
with determination of the test substance concentration. The duplicate recovery
rates and the average of them in the pretreatment are shown below (see Table-6
and Fig.7). The average recover)' rate was used as correction factors for the
determination, ot the test substance concentrations in me analytical samples,
For analysis oftest fish (5 ng test substance added) 96.4 %, 94.3 % average 95.3 %
3.7.5 Lipid content in test fish Lipid contents in the sample for storage of the control test fish (see 3.7 2(2))
were determined after chloroform-methanol extraction with gravimetric analysis.
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3.7.(5 Calculation of the test substance concentration in sample and minimum hmu of determination
(1) Calculation of the test substance concentration in test water
i
Tlie aquation in Tables-4, 5 was used to obtain the concentrations, and they
were rounded offto 3 figure.
(2) Determination limit ofthe test substance in test water The determination limit** of the test substsmr.#* in n-st water was calculated on
the basts ofthat obtained fromthe calibration curve in 3.7.3 (3) as follows.
Level 1 Level 2
'3-1 ug /L 0.21 pg/L
(3) Calculation o f the leal substance concentration in test fish The equation.? in Tables-7, 8, 9 were used to obtain the concentrations, and
they were rounded off to 3 figures.
(4) Determination limit of the test substance in test fish
__
Assuming the fine sample of fish to be 5 g, the determination limit ofthe tost
substance in test fish was calculated to "be 24 ng/g, on the basis of that obtained
from the calibration curve in 3 7 3 (3).
*5 .
Minimum determination limit ofthe test substance (pgtL or ng/g)
__ A " _B_ C *E
100 * D
where
_ ._ _
A ; Minimum determination limiL of the test substance on the calibration
curve (pg/L)
B : Recovery rate (%) C : Sampling volume of test water (mL) or weight Of fine sample of fish
(g) D : Final volume of sample solution (mL) E : Ratio of the portion, used for analysis to whole volume
Resulte were ouinlcd offto 2 figures.
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3.7.7 Calculation of mean concentration of the test substance; in test water {duration of exposure)
C w t={C w (l)+ ......... + G ain)}/ n
where Cwt
n , Cn<1>
Cw(n)
Tire mean concentration o f llietest substance in test water
Numberofanalysis for test water(fnesurecneni times) Concentration nfthe lest substance in 1st analy-sis oftest water Concentration ofthe test substance in n-th analysis oftea water
3.7.8 Calculation cfbiocortcentralbn factor (BCF) Bioconcentration factor (BCF) was calculated as follows.
(1) Calculation of mean concentration of the test substance in test water for calculating BCF
Cw = { Cw(n-1) + Cw(n)}/ 2 (only 1st analysis of test fish) "Gw1- ( Cw(n 2) Cw(n 1) + Cw(n) } / 3 {from 2nd analysis o f test fish)
where Cw
Cw(a)
., The mean concentration or the test substance in test water for
calculating BCF Concoritrscticm o f the tost subetatte in f-th analysis o f test avatar
(2) Calculation of bioconcentration factor f
B C F - -- -- ----Cw
where BCF
Cf Cw
Bioconcentration factor Concentration oftbe test substance intest fish iijc mean cunccmrarion of the test -suitstuncc: in iesi water ii
calculating BCF
(3) The mean, bioconcentratldm factor in Kh analysis
BCFm = (BCFa + BCFb) / n
BCFrn ; The mean bioconcentration factor in m-Lh analysis (number of individual or group 2 (a,b))
BCFa.b ; Each bioconcentration factor in nr-th analysis oftest fish n ; Number of individual or group in w-th analysis of test fish
-20-
51519
.17 9 Confirmation ofthe steady-state was reached It was evaluated that a steady-slate had been reached when three successive
analyses ofBCFs made on samples taken ai intervals of at least 48 hours were within 7ft % nfearii other When BCFs were less than 100, it was evaluated that a steadystate had bees reached after 2Sdays evm ifBCFs were over J- 20 % ofeacli other.
Ciiieiion of the steady-state was reached: V{m-2), V(m-1), V{tn)
I RCftm-2)-BCF V(m-5.) = -
"
100
20 {%)
| BCF(rti-1)-BCF |
V(m-i) =
13
x 100
V(m)
| BCF(rn) - BCF | x 100 BCF
V(m-2),
V(m)
3CFim-2>, S o W l ) , BCF(iri)
gCF
: Variatiem ofbioccmcentiation factor : The mean bioconcentration factor in m-Z, m-1,
777-th analysis of lest fish { BCF(m-2) * BCF(m-l) + ftCF(m)}/ 3
-23 -
51519
3.7.30 Calculation ofhincnncentration factor at a steady-state (BCFss) Bioconcentratron factor at a steady-state (BCFss) was calculated as follows.
(1) Calculation of tHe mean concentration of the test substance its lest water for calculating BCFss
Cws = { Cw(n-2) + Cw(ij- 1) + Cw(n)}1 3
where
Cws The jpegn concentration of the test substance jn test water for calculating BCFss, which is calculated From tines successive analyses of test water before last analysis oftest fish as a general rale
Cw(n) Concentration ofthe test substance in n-tb analysis of test water
(2) Calculation of the mean concentration of the test substance in test fish at a steadystate
Cfe ~ { Cfl(m-2) + Cftm 1) + Ci(m) }/ 3
where "Cfe The mean concentration of Ihe test substance in test fish at a steadystale Cffiri) The mean concentration of the test substance, from which FB is subtracted, in m-th analysis of lest fish FB The arithmetical mean concentration of the test substance or blank in the control test fish analyzed before and afterthe experiment
(?) Odrariatinn nf RC.Fnk
BCFss = CfF/ Cws
where BCFsa Bioconcontration. factor ot o stssmly-statc Cfs Tlic mean concentration of the test substance in test fish at a steadystate Cws The mean concentration of thr: test substance in test water for calculating BCFss
-22-
51519
3.7.11 CaICU^ ^ BbCasis Lheminifflurn determination limit o f the test substance m
BO -5 can be obtained when BCF exceeds the Mowing. The mean ooncwitratio the test substance in test water obtained from all the analyzed samp e was _tk frtlrtw5n<yralimhtbl SCF.
Level 1 Level 2
0-51 *!
3.7.12 Calculation oflipid uuirtent
_
Lipid contents were calculated according to the following equation.
Lipid content (14) = (T -T ti)/S * 100
where
To Weight of vessel (g)
..
T Weight of sample for gravimetric analysis (containing vessel; is;
S Weight offine sample taken out for analysis of lipid content (g)
Treatment of numerical values
pc Z 84Q1-1*90 rule B. The each
Values were rounded offm accordance with JTS Z tWuJ.ijW *>
value used for calculation was used without rounding off on The concentration values of the test substance tn test water and fish were rounded
offto 3 figures. BCFss values were rounded offto 2 figures.
4 Pstunra possibly affecting accuracy No adverse effects nn the reliability of this test were noted.
51519
5. Results
5.1 Concentration of test substance in test water The measured concentrations of lire test substance in test water are shown in
Table-1. Each concentration of the test substance was maintained at more than 92 % of each the nominated concentration. The variation of ihe concentrations of the test substance was within 20 % ofthe mean o f the measured concentrations.
T able' 1 Mcosui cd concentrations o f the test substance in test -water
(Unit. Jlg/L)
Level 1 2
After j After 1 day i 3 days
1
50.2 j 47.6 1 l
4 83 ' 469 i
Average After After Mei j After (Standard Table 10days 16days 23 days ! 28 days deviation)
46.9 48.1
46.7 45.8
47.6 (1.51)
4
S
4.61
4.66
4.H3 1, 4.61
4.71 (0,101)
5
Fig. 5
5.2 Bioconcentration factors
R C F s art* ahmvn m Tshln-3
BCFs in Table-2 plotted against duration of exposure are shown in Figs. 1and 2. These BCFs ofthe test substance ranged from 2.0 to 4.2 at Level 1 and from less than 5.1 to 9.4 at Level 2.
Tablc-2 BCFs
Level
1 !
After 3 days
2.9
2.1
After 10 days
2,4
2,5
After 16 days
3.0
3.0
After After 23 days 2S days
3.0 4.2 \
2.0 3.3
Table 7
Fig. 8
6.0 5,1
7.7 6.1
68
21
89
S 5., 5.1 9.4 5.1 5.1
-24-
51519
5.3 Calculation ofBCF at a steady-stale (BCFss)
5.3.1 Level 1 The variation of BCFs (average value) after 16,23 and 28 days were more than
20 % of each other. But because all BCFs were less than 300, it was evaluated that a steady-state was reached within 28th days. BCFss was calculated on the basis of
these result.
(1) Conecntrutiontii'Ujiil. ubclans* in lest Jter ai a ctoiidy-ernt*
The mcan concentrations of the test snbstance in lest water at a steady-state are
shown in Table-3. The mean concenrrations was about 94 % o f nominated
concentration at Level 1.
'
I able- i Concentrations tsr ihc lust substance in Ul w*tc.r nt n .iteady- itatc (Unit: jtg/L)
Level 1
After 16 davs
48.1
After 23 days
46.7
After 28 days
45.8
Average 46.9
Table 4 ,7
Fig5
(2) BCF at a steady-state (BCFss) BCFss al Level ! was calculated as iullows.
Level 1 3,1
5.3.2 Leva! 2
Because the measured concentrations of the test substance in test fish were
containing value which were not more than minimum determination limit of test
substance, BCFss was not calculated.
Because all BCFs were less than 100, it was evaluated that a steady-state was
reached within 28th davs.
5.4 Lipid content in test fish The measured lipid contents in the test fish are shown as follows,
Bocr<> Smtiation oe:pm:u3i: T.lfl %
After termination of exposure 2.82 %
5.5 Results of test fish observation No abnormality in behavior or appearance was noted.
-25-
51519
6. Remarks
Instruments, apparatuses and reagents, etc. for the test
(I) Instruments for fish care
Micro quantitative pump for supplying stock solution :
Tokyo Rika Kilcai Co., Ltd. type GMW
Instrument ft measuring concentration of dissolved oxygen :
Itj-irru Scimitsu 3nctvsfrian Co., -td
type F-302
pH meter :
Toa Electronics Ltd. typeHM-14P
{2} Instruments, apparatuses and reagents fbr analyxis and preparatietn o f stock solution
Instruments and apptu Iuss
High-performance liquid chromatograph-mass spe.ctrom&er
seepagel
Electronic analytical balance : Shimadzu Corporation type AEX-200B
Metier Toledo type AE163
Sartorius type 1404MP&
Sai lonus type BP301S
A and D type FA-200
Rotary evaporator ;
Tokyo Ttika Kikai Co,, Ltd. type N
Homogenker (polytran) :
Vamato Scicntic Co., Ltd. type RE5Q Kinematica type PT3100
Homogenizer (autocellmaster) : lucMseieido Co., Ltd. type CM-200
Centrifuge :
Kubuta Svouj! Co., Hd. type 6000
1ftMgRTltC
Acetonitrile (HPLC grade):
Wafco"Pure Chemical Industries, Ltd.
Purified water:
'fakasugi Sciyaku Co., Ltd.
Methanol (extra pure):
Wako Pure Chemical Industries, Ltd.
Acetone (extra, pure):
Wako Pure Chemical Industries, Lid.
Cldci-isform (gtuiranlccd reagent) : Ki3hido Chemical Co., Ltd.
Anhydrous sodium sulfate (extra pure):
TCaiayama Chemical Industries Co., Ltd,
Di-w-butylamitie acetate (Jon-PairReagent forLC-MS):
HCU-6U:
Tokyo Kasei Kogyo Co., Ltd is'ikko Chemicals Co., Ltd.
(3) Instruments and apparatuses for confirmation of the test substance Instruments and apparatuses Infrared spectrophotometer: Shimadzu Corporation type FTIR-82GQPC High-performance liquid chromatograph-mass spectrometer: Waters Corporation type ZMD
-26-
Tabte-5
Calculation table fur analysis of test water (Level 2)
Sample description
A
Standard 2^0|X&'L Test water after 1 day
Slartdatd 2.50 a g/L Test water after 3 days
Standard 2,50^ g/L Test water after 10 days
Standard 230#tg/L Test water aer Id days
Standard 2.50 g/L Ttis. watet afi 23 days
Standard 2-50 g/L Test water after 28 days
6Z57 6050
6405 6003
6243 5756
6234 5814
6123 5910
6144 5670
Average concentration of test substance m lest water 4.71 (ju gO.)
Test No. 5151
i
4.83 4.5?
4.61 4.66 4.83 4.61 (S.D. 0.101)
A : Peak area Afstd) t Standard solution A (t): Sample
; Kudo of portion used for analysis 25/2.5 C : Final volume 5mL H ; Volume of test water taken out Z5mL 1 1 Coaontt^wn ftf uhtnw. in fwt WSfir (s: g/1.)
I P X( A V ) l ACstd) ) / B * C 1 K J : Average concentration oftest substance to test water {i gfU)
J =(1(1)+ ...+ 1(0))/ft
n : Number of tcsl water analyses ( n = 6 ) I (1): First analysis ot test water i (o ): Last analysts or test water
. * 1 !(->*-[ St(o]
- n 1 1-1
\J
K n x{u -1)
/
' ? : CcrqOfentmtirto i ctwicfard colvtfon
See Fig. 5
? sn /i
-
November 28,2000
Name R i E 0/30
Table-4
Calculation table for analysis of test water (Level l )
Sample description
A
Standard 2.50 g/L Test water after I day
Standard 2.50y g/L i esc water after 3 nays
Standard 2.50 p g/L Test water after 10 days
Standard 2.50 *i g/L Test water after 16 days
Standard 2 5 0 ^ g/L Test water after 23 days
Standard- 2.50 (i g/L Test water after 28 days
6257 6284
6405 OSO
6243 5862
6234 5992
6123 5720
6144 5629
Average concentration oftest substance ittest water 47-$ ( u g*1!*)
TestNo. 51519 1
S02
+7.6 46.9 45.J 46,7 45.8 (S.D. 1.51)
A : Peak area A(std): Standard solution A (t): Sample
.
B : Ratio of portion used for analysis 2_5/10
C : Flail volume 5mL H : Volume o f Mil water taken out ImL I rrirKv-iUrftrtnn o f test substance 5n test watei ( a s/L ) '
l - V * (A (l)/ A(sitl}}/B * C /H ] : Average concentration o f lest ibsiaitee in test water {y g/L)
J " (I(I)-t-... + i(n))/n
n ; Number oftesl water analyses ( n = 6 ) 1(1); Fitst analysis of test water r to ): Last analysis or teai waiei
vZ
I*i x 2{tV 2-!(!i>
5X). It x (a - 1)
n : r-nm-fnirti! !nn n-f1S-.I nHctance in standard solnlUus
2.50 u/L
See Fig. 5
November 29,2000
Name P * t Ogf?
Table-6
Calculation table for recovery and Want test (Analysis of lest iisk)
Sample description
A
c
D
E
Test No. 51519 FG
Standard 2.(ftp g/L kFrnvp.Ty a Recovery b
6905 6654 6511
1/30 1/50
23 25
-
Standard 2,00 figO. Btank a Blank b
mi
a d . 1/50 a d . 1/50
25 25
-
( a, b ; individual sample)
A : Peak area Afstd); Standard sedutien A(c); Sample
B : Ratio o f portion used for analysis (fish homogenate) 3/; [> C : Ratio o f portion used for analysis (extracted solution) D : Huai volume (mi-) E ; Amount of blank in test fish (ng) F : Amount o f test substance recovered (ng)
F - P - ( A(t)/A(ttd))/E/C *D-E O : Recovery rate (94)
O -F /Q x 1Q0
P : Concentration of test substance in standard solution 2.00 p g/L Q : Amount ofiesi substance added (5M0ng) See Fig. 7
4820 4710
96.4% 94.3 % Average 953 %
November 18,2000
Name. ...
Table-7 C a lcu la tio n table for analysis ot test fish
(Level 1)
Tesi N o. 5131?
Sample description
A
a
KH
J
MO
Standard 2.00 grL Test fish after 3 days a Test fish after 3 daysb
6810 1811 1346
5.00 5.00
139 48.9 104 48.9
2.9
2.1
25
-
Standard 2.00 a fi/L
6900
Test fish after 10 days a 1499 5.00 114 48.2
24
2.4
Test fish after 10 days b 1586 5.02 120 48,2 2Jj
*
Standard 2,00P $-
6949
Test fish after 16 days a 1862
5.D1
140 47.5
3.0
3-i> 2.6
Test fish after Id daysb 1909
5.03
143 47.5
3-0
Standard 2130 pg/L
6804
Test fish after 23 days a 3863
5.03
143 47.2
3.0
25
2.6
Test fish after 23 daysb 1253 5.02 96.1 47.2
2,0
Standard 2.00 g/L
6756
Test fish after 28 days a 2597
5.07
199 46.9
4.2
3.8
3.1
Test fish after 28 days b 1989 5.02 154 46.9
33
( a, b ; individual
)
BCEss:
3.1
A ; l'ea.1: area
Afstd): Standard solution A (l]: Sample
B : Ratio of portion used for analysis t/50
C iPinal volume 25niL.
D : Dilution Eactoi 1
_
B : Average concentration of blank in analysis of control
Qng/g
p : Recovery rate 95.3%
G : Weight offins sample (g) K . Cui.viL<iotlou uTleal iuUstiuc.c in icrffiah (>5/5}
K. (PxC A (t)/A fsid) )7B x D x C f G - E } / FxlOO
H : Average concentration of test substance in test water
_ , h_n n > 3
H={Kn-2) + l(n-l) + I(n)}/m ; a : Number of test water analyses ; m = 2 w h e n a = 2 ,m -3 hen
1: Ccmeentretion of test substance in test water ( g/I.)
r- u rn
J ttX /H M : Average value of BCF(a) and BCF(b)
M = {BCF{a)4- BCF(b) } /2
O : Average value OfBCK
0 = f M(n*2>+ M(n-1) + M(n)} / 3
,, nn .
P : Cbncenaation of test substance nt ^ da,d S0,ullQa "W l i
Tfn n , T fn U /3 I
BCFs! = [ ( K(n-2)a + jqn-2)b 4 K(o-l)a + Hfn-ljb 4 K(n> 4 K(n)b >; 6 ] 1 [ { l(n-2> 4 I(n-l) 4 1(n) } /3 1
See Fig. 8
November 28,2000
Name
/?lg
Iable-B
Calculation tabic for analysis Of test fish (Level 2}
Sample description
A
0
K
Test No. 51519 11 J
Standard 200/ig/L
6832
Test fish aflet 3 days a
401
5.00 30.8 4.76 o.5
Test fish after 3 daysb
n.d_
5.00
*
4*70
Standard 2 .0 0 p g /l-
6S63
Test fish after 10 days a
n.d.
5.02
-
4.71
Test fish after 10 daysb
n.d.
5.01
"
4.71
'
Standard 2.00 P g/L
6919
Test fish after 16 days Test fish aft 1 6 diys b
47$ 5B5
5.04 35.8 4.65 5.05 43.9 4.65
7,7 9.4
Standard 2.00 it u/L.
67-17
Test fish after 23 days a
374
5.04 28.8 4.70 6.1
Test fish after 23 Jays b
311
5.04 24.0 4.70
5.1
Standard 2.00/10/1.
6743
Test fisll after 28 days a
417
5,07 320 4.70 6.8
Test fish after 28 daysb
n-cL
5.07
- . 4.70
t u b : individual samole 1
A ; Peak area Afsldl : Standard soiutiuiL A(t) : Sample
R t Ratio Ofportion used for analysis 1/50
C i Pinal volume 25tnL
D ; Dilution factor 1 13 : Average CMieeatratiuuofWank in amlyebmreiml.nl
Ong/g
F : Recovery rate 95.3 0 : 'Weight of fine sample (g) K. : Concentration of tea substance in lest fish (ng/g)
Px{A(t)M(std))/BxDxC/G-E}/FxlOO
H : Average concentration of test substance in lest water (n g/L) H = fIfo-2.) t I{n-1) + IW } / m ; n : Number nf test water analyses ; m = 2 when fl =2, in =3 when It 3
1 : Concentration oftest substance tu test water { p gIL)
] :BCF
J = K /H P : Concentration of test substance in standard solution
2.00 P g/L
S t Kg. S7
December 5. 2000
Name
E 0B0
Tflbic-9
Calculation table for analysis of test fish (Control)
Sample description
A
Slanciarci 2.00u g/L Before the initiation of exposure a Before the initiation of exposure b
6952 n.d n.d. -
Test Mo, 5153L9 O1
J.UU 5.00 -
Standard 2.00 pg/L After the termination o f exposure a After the leimitiaiion o f CuQSUIft b
( a, b : indrvidusi sample )
6701 n.d. - 5.08 n.d. 5.01
A :Peak area
A{stt3) : Standard solution A(t) : Sample
B : Ratio oCportion used for analysis C : Final volume 2SmX.
}/jo
E : Amount o f Hank in analysis OFcontai (ng)
E P { A{t]f A(std) )/U x C
0 ;Weightoffinesample(g)
1 : Concentraiion of blank in test fish (ng/g)
I*EfG
F ; Concentration a r teat suoslanee in stiaidatUsolution
See Fig. 10
. 2.0;; go.
-
November 28.2000
Name
M E 8$i>
Reference 1
Analytical results of dilution water
Itom
Total hardness (Ca, Hi)
Suspended solid
pH
Total organic carbon
ateuical oxygen demand
Residual chiorIds
Ammonia nitrogen
Total cyan
Alkalinity Electric; conductivity
Organic phosphorus
i Alkylmercury
! Mercury
; Cadmium i Ur*
1Lead
Arsenic
Iron
Copper
Cobalt
'
Manganese
Zinc
Aluminium Nickel Silver Orgsricichlorina pasticides
1,2-Di chioreprapsma Chlorothalonil
Propytaoido CPlorn1trufen
Simay.ino Thiobencarb
Organophosphorous pesticides
Diazinon
Ftnitrothien EPfi Dichlorvos Iprobenfos FCH
Sampling dace August 8, 2008
I Unit Meacvu-vd vain* Srsmrfarrt vaIiir
mg/L i mg/L
i" mg/L s/t. liig/L mg/L
<>
V*
in <l
7.8 1.8
< 0.01 < Q, 01
< 300 *l
<20 ,
6.5 '8.5 ^
i 2* < G
)
: < o. 02 *
. <i *
mg/L mg/L ps/cm mg/L mg/L
mg/L mg/L mg/L
mg/L mg/L mg/L
< 0. 01 96.1 3T4 < 0.1
< 0.0005 < 0.0Q0S
. < 0.005 .V0. wt
<0.005 0. 002
< 0.01
; ;
i I I : * i I i
n. d.13 -
t___
*-* *___ n.d.`=
< 0.0005 <3
< . 01 ** <: o. oa *'
< 0.1 15
< 0. 05 *5
< 1 , 0 *
mg/L mg/L
mg/L mg/L
mg/L mg/L rnR/j.
< 0.005 < O. GDI
< 0.91 < 0.51
< 0.001 < 0 .001 <O, >9<3t
i
< 0.005 ** < . OOt **
< 0,05 "
< 1.0 *'
< 0.2 'l <0, 01 " < e.ooii
mg/L <a. m
mg/L C6. 004 mg/L < 0. 0008 mg/L < <t.0<K)JL
mg/L < 0. 0003 mg/L < 0. 002
< 0.06 * <0. 04 ** < 0.008 * < o, oooi
< 0.003 * < 0.02 *
mg/!. < 0. 0005 mg/L < o. oonn
mg/L < 0, 0003 mg/L < 0. 0006 mg/L < 0. 001 mg/L < 0. 0005 mg/L <O. 0000
<0.005 * < O. 008 **
< 0.003 ** < 0.006 ** < 0.01 M < 0.008 *4
. U.*
limit.
i
o.i 0.6 0.01 0,01 0.01
0. 1
o. aoos 0.0005 0.005 Qr02 0,005 0,002
0.01 0.005 O. aci 0.01 0.01
0.0J 0.005 ft
/
0.006 0. 04
0.0008 o. ooai 0. 0003 0.002
0.0005 O. 9000 0.0003 0.0006 0.001 0.0008 0. 0005
*1 Ministerial ordinance of the Ministry of Health and Welfare So, 69 (Revised December 21, 1592)
*7 OEr.1) Guidelines for Tooling of Chemicals, Fish, Early-Life Stage Toririty Tost ffinideline 210. Tulv IT. 1992)
*3 Water quality criteria for fisheries {Sbadanhozitt Nihon Suisensigsn Hogokyofcai, March 1BB3)
*4 Enviioiunetita) Quality Standards for Water Pollutants Xu. 14 (Revised February 22,1099, Envi rotiioent Agency)
*5 GECD O u id tfli tie* ft* T<,i.iB w ^linmwnl.>, Qi.Sncntx'tftinn i R o ^ `hlnv.uRh P i* h
Tost {Guideline 305, June 14, 1996)
\nr ,t 2 a Pi ECBg
3ioconcentratlon 'actor
10'
o o
0 6
Test No. 51519
o 00 o
. TJrt minimum calculation irntit of bioeonceniratictn factor.
10'1 ----- 1---- 1-----1------------ 1--- 1----- 1---- 1------------ ' >--- .-- ----------0 Z 4 6 5 to 12 14- IB IS 20 Z3 24 20 28
Exposure period (days)
Fig, 1 Correlation between exposureperiod anti biuconcentialion factor (Level 1).
Bioconcentraton factor
o
o
o
0
--o
0 2 4 6 & 10 12 14 16 18 20 22 24 26 28
Fig. 2
Exposure period (days)
Correlation between exposure period and bioconcentratioc factor (Level 2).
Four date after 3, 10 and 28 days were lower than detection limit.
December 18,2000
Name R l c C ' B D
96-huui LC50- 100 mg/L 300 r
T est H o. 51519
M ortality (%)
Concentration fmg/Ll Control 3.13 6.25 12.5 25.0 50.0 100
24 hours 0 0 0 0 0 0 10
Cumulative Mortality {%)
48 houts 72 hours 96 hours
0no
0 00
0 00
0n0
0
o ...
o
000
30- 40
50
Fig. 3 Concentration - Mortality Curve.
Date : September 19,2000 Name
Tesi No. 51518
14000
Test No. 51319
Peak atea
7ono -
/
/
y
y = 2-438 x r = a.ooD
zs Concentration (g/L)
Conditions of LC-MS analysis
Instrument
Sample
Solvent
: Wafers ZMD.26SO
: K-1519
Water/Test weter for Tecovcry test (1/ V/V)
HFLC Condition Injection vol. Column (Size,Temp.} Eluent Polwrafc
20fiL
L-column ODS( 1Sem >r2.1mm I.D., 4(fC) (O&):Ac=tonittiJ,2(4%):5inmol/L Di-n-buyUfflte. acetate
0-2mUiHiil
MS Condition Ionization mode Cone voltage Source block temp.
Monitoring ion
Pssah'slion
Temperature Gas flow talc
Electrospmy (negative) ldV
natfc m/z413
300V 3501/ht
Fig. 4 - 2 Calibration curve and conditions of LC-MS analysis for K-1519 (test walei).
December 7 ,200fl
Name R j S Q B E
Teal Nu, 31513 Dale :aesO. H , j 1 hiame:
"test Mo. 5 lStfl
Dato - iliiif U . i4~ Marne r
0& &
Toot Ho, S1S19
OalB ,
u . fti- Na me : & *'. 0 8 Q
TwstWn S151S
Dato : SQff. l) . |4 Mamo
0$ ff
Tsi NO. 5151S
Fig. 5-5 Mass chromatogram of LC-MS analysis 1er test water. Dote :'.ote it , /tf Nonno: ?J E Q
TsiatNin. fi-m i
D a ta :JlieP. U . i Mame*
'fit'
Teat No. 51510
I4QOO
Test No. 51519
/
/
Peak area
7000
/ 0^
0
/
y = 3274 Jf r O.SSS
C oncentration (wgfQ
C onditions o f LC-MS analysis
Fig. 6 - 2
Insliamwi
Sample Solvcui HPLC Condition
injection voi. Column (5izc,l>.nip.) lilncnt Foiw rate MS Condition Ionization mode Cons voltane Soutcc block temp.
Monitoring Ion Desohation Temperature
Gas Uow rale
Waters 2M D , 2690 K-151S Water
20L
l. -column ODS( 15cm * 2.1mm L D ., 4<fC) A(G0%1:AefitonicrileIB(4!)%);5nanol/,L Di-n-butylamine acetate
0.2inliiain
Eleclrospray (negative) 16V L2CfC m. 'z 413
SOO'C
350I*itr
Calibration curve and conditions of LC-MS analysis for K-1519 (test fisb)
Decwnb 7,2000
Name fcl-F
,,,.
Test No. 51510
fanafysis of test fish).
Dato : oca. >/ . i 'f- Marna ;
C'RD
Tosi No. 1519
Standard solution 2.00 (ig/L
FA03F01
lOO-i
Teat No. E1S1Q
20-Oct*2tHJ0
SIR of 1 Channel ES413,001.00Db 2.00s5 &KU)
-o -r'T 0.00 1,00 2.00 3.00 Test fish after 3 days {level t - a) FA03F03 lOCh
", "i ...................... ...... -i Time 5.00 6.00 7.00 8-(
2Q-Oct-200D
- SIR of 1 CharmefES-
4 1 3 .0 0 "I.OODa
2.00b5 Area
%-
3.75
-> --i-t--11'i'i' <--11' 11.111 0,00 1,00 2.00
1811 __2j-- ___ 11 *I ` | I1't 'I 1:' I!; I` i i, i . i i ,' " ..................... iTroe 3.00 4.00____ 5,00 6.00 7.00 3,00
T est Fish afte r 3 day: {level 1 b)
2O-Oet-20Q0
FA03F04
100n
SIR of 1 Channel ES413.001.000a 2,00e5
Ares
%'
-5 0.00
Fig. 3-1
3.75
1346
A
S Sl*| I V `
T1
1.00 3.00 a.uu T o tT 6.00 6.00 7.00
Mass chromatogram of LC-MS analysis for test fish (level 1).
n Time 8.00
Dale-JCpO. I I , l Name r A?*e & & &
TfistNo. 51519 n a ta o n G . i t . f MatnS-L Rj-t P &
TatN. C1S1 r>nte.Jtimr. It ,/V N 'g ^Xpp>P
Standard solution 2,QQ pg/L
FA23H01
JOOi
Toot Ho. 6-1610
O9-MOV-200Q
SIR ot 1 Channel E5413.001 .OODa 2,O0e5 Area
%'
3.7U 6B04
. , i | t i ; t , r r i . n-] TllTlft
Q,QO
1,00
2.00
30 r ~4.bo
5.00
6,00
T.QQ S.QQ ag-Mov-200c|
Test fish after 23 days (level 1 - a}
SIR of 1 Channel ES- ,
FA23H02
413001.000
100-1
2.00e5
Area
3.78
13S3
A
- 5 ~ ........... i i . " l " ' - i " ' ' ' i.7~Y'
0.00 1.00 2-00 T est fish after 23 days (level t - t>V FA23HQ3
4.D0
100-]
i ' 11' 5.00
.-pi .......Time 6-00 7.00 6.00
' 09-NOV-200C
SIR of 1 Channel ES413.00 i.OOQa 2.00e5 Area
%
F,g. 8-4 Mass chromatogram of LC-MS analysis for test fish (level 1). Date : COP, n ^ J lL Nlatrts ; OHO-
Standard solution 2.00 pg/L
Test No. 5S1S
li-Nov-ZOOO
Rg. 8-5 Mass chromatogram of LG-MS analysis for test fish (level 1).
Date :
I I , ! V Klamp1 f f i - f 0 & 0 ___
Test No. 51519 fiaiB : 2 g . a . /V N a m e j _ - M i
Toot Wo. 61610 Dato : rf . f V Slama ! /? 7 g C /?f?
TftstNn R1519
.Jr.i-.ff ti . HL Msnfi-L f r t C
Standard solution 2.00 pg/L
FA23L01
100-]
Test Mo. S1610
09-NoV20tKl
SIR of 1 Channel ES4-13.001.00Da 2,0QeS At03
3.78 6747
ft
! JL *"i .!,r. i 0.00 1.00 2.00 3.00 4.00
Test fish after 23 days (level 2 - a)
FA23L02 100-j
\
--T-4-' 1;
-n Time
5,00 6.00 7,00 S.Q0
' 09-Mov-2000
SIR of 1 Channel E5-
413.00 t.OODa
2.00e5 Area
3.79 374
-s o.ao
1.00
2.00
n -I' M' '
3.00 4.00
5.00
6.00
r-n Time
7.Q0 a.oo
T eel fish after S3 d a y s (lovl 2 - b) FA23L03 100|
SIR of 1 Channel S4lS.001.00Da .00e5
Area
%'
-5 0.00 Fig. 9-4
a.77 311 1.00 2.00 8.00 4.00 rr5v.0i 01"''P 6.DO 7.00
Mass chromatogram of LG-M5 analysis tor test fish (level 2).
rr-i Time
8.00
Hate ;-?cQg-.V . W. Mnme : CfiO_____ ,,
Standard solution 2.00 pg/L
FA2SL06
100-.
TPstNo. 51*19
14>Nov*2000 SIR of 1 Channel ES-
413.001.00Da 2.00e5 Area
Test fish after 28 days {level 2 - dj FA2BL1
100
. H-Nou-2000
SIR of 1 Channel ES413.001.00Da 2.00eS
%-
-3 |-rr 0.00 Fig. 9-5
Peak poaitlaa u.
;
11.00 " 1T2.0 ` 3.00 ' 4.0 ' s!o r 6.00 ` 7.00
Mass chromatogram of LC-MS analysis tor test fsh (level 2).
--i Tim-* s.oo___
nato after i t Ui Mama- /?4fc. Q
Test No. 51519
Test Na. S1S10
Concentralljji of dissolved okygenimg/L)
10 -
--------------------- *------------ *.................. *
5
TestNo. 51519
Concentration of dissolved oxygenjingfl.)
0 10 20
Exposureperiod (days]
Fig. 11 Concentration of dissolved oxygen (Level 1).
10 -
4f. 'ft1 ....>
*----- --
5-
30
Cuoccnlralcn of dissolved osygenfmg/L)
0 10 20 30 Exposure period (days)
Fig. 12 Concentration o f dissolved oxygen (Level 2). 10 r s 11 --ts-------#--------- ---ft.-..- ........n ^
5-
0 ---------------------------- 1-------------------------- 1---------------------------- :
0
10 20
30
Exposure period (days)
Fig. 13 Concentration ofdissolved oxygen (Control).
November 22,2000
Name
fcxE. 0C
Test No. 51519
IR conditions Instrument . shlmadzu FTIR-8200PC Sample : K--1519 Method : KBr Tablet
Fig . 14 - 1 IR aleuti um o f te s t substance measured before the experimental start. Date : Aug.31.2000 Name :
T e st Mo. 51619 100.0
> i i i i ! ;
4000.0
3000.0
; iii;jii
20M.0
1SOO.O
i | i *|i !:
1000.0
SBO1./Om
!R conditions Instrument : Shimadzu FTIR-8200PC Sample : K-1519
Method : K8r Tablet
hii. 14- 2 IK spectrum ot test substance measured atter the experimental completion.
Date : Nov, 20,2000 Marne : O'PO
Test No. S7 t i f
tnctrumonl MR W aters .7140
HPLG : Waters aS90
t ~ f r/ 9
f .C ^ /
HPLC Conditions
Inletsystem j
Flow injection
Sample size_ Column____
fsjffi PFF&
Column size / f cm x J-X w m I.D., Column temp.
Eluent A(Pt>*&) AectbMtillL . B (W6)Si*t*efcF g'/jn/y/AucW a t t*foTe
Row ta te fr.^ mLAnio
Post column eluent
--
Post column flowrate -- mUmtn
Split ratio --
________ ______________ _-- --------------- --
MS Conditions Innivafinn mrafe <ESj) APcl Deteetioh modg Positive. Negatfek Source Capillary2JW, Cone to V. Eidractor/f? V, FIF Lens i f f i V Ion source temp, rlo C. Desoluation gas <2 Uhr, Desclvalion temp. gfei' C Corona ~~ kV. APcl proba (emp. 450. ^ G MS loti energy 4)3. V. Multiplier^sji>. V Monitoring ion m/? ____ ___________
Note
Kurume Laboratory, Chemicals Valuation and Research Instlute, Japan
F ig .1 5 - iM S spectrum of test substance. Dataitic i 6 /** Operator Yj!
F ig . 1 5 - 2
MS spectrum of t*et stitctanee.
P i g . i 5-* 3 MS spectrum of te st substance.
U V < ro?& Y & tl\K 294l^M m 47tU S2t .*1S X-1H9
a1cCti ? a p f i x e r e a c 75,45 BK*
1 2 4 , ffiz
154&.fl 23
203420734,19 2 112.,3510M.04 VMKMBd
n 25,5 0..f3lQfl Spsn, 24
stri?
i t ,z X-
teereace 2
SW R Bpectrnm d t e s t . s u b s te a e .'
