Document Oz471519j9QELROj1BYnMy3zj
ARRARE --3512
404
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ARG _ 33%
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TRANSLATION
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SUMMARY OF BIOCONCENTRATION STUDY REPORT
1. General items
SA `uNbasmteanocfen(ovTeUlPAC
nomenclature)
_
_
Other name
a)
1
Structural formula
`Main component
or rational
Sifoerkmsuola,n(igfponoeiitsher Subcomponent
IMolecular formula
_
Purity of the novel
Lot No. of the novel 1-11-00
1
chemical substance
~~
used in the stud
|_
chemical substance
used in the stud
Name and
~
[ Topun TT concentration of
Partition coefficient | 570.37 (Main
Molecular weight
[B[oilM ingpeoinl t t[n~[ c~ogmpop nento )| mt| tPreomppeerrtaytuarteroom Brown viscous solid
St`Saobliulbiitlity in various Unknown
Stability in solvent
[[A[ mcestoene [-|
oe]
Other
3
--
[1 Solubiliitnyother solvents: soliunebthlaneol (unknown solubility)
--_
(Bioconcentation Report] 1 Lr --------
~
2. Acute toxicity test
= potdeot LCS0 value (96 h) ----1[>3c5Y 0 omg omimr to substance studied
we
[soumessmoon
|
3. Study method Method
Bioconcentration of Chemical Substance in Fish Body> specified in "Method for Testing New Chemical Substance, etc." (Environmental Protection Notification No. 5, Notification No. 615ofPharmaceutical Affairs Bureau and MITI Basic Industry
1998) and "Bioconcentation: Flow-through Fish Test (Guideline 305, June 14,
1996)" specifiedin"OECD GuidelinesforTestingofChemicals."
4 =Ei (%)
(ug)
level
Second
T|r aweveoce omm an) | Na.me ofaviandaproprortyion
to subnce suid
[Bioconcentration Report] 2
Company Saniizod. Dosnotcontain TSCA Cat
~ 4.(1R)eTsaublltesfor the result of bioconcentration test
Namcomponent
|Measuremednaty
| Aterd Gays
|After? [days
|After | After | After| Afr [00 [17 (24 (28
days | days | days da
pal WONclll Al level
concentration(1
=" Bale proportion
<s1 ssa |ssa |ssalssa
a Gl lll level
concentration (1g
=F GEE] proportion
st ss [ssi |ssi [ss
Subcomponent
Mommementday
| Afterd|After? ays [days
| After | After | After| After 0 |r | |
--
days [days | days |days
SIE level
concentration (12 Concentration
2d 360
30 [a0 [410 [470
proportion
370
0 [510 [410 [430
G10 350) | en) | @10)| eso)
evel
concentration (ug Concentration
|e 30 | 430
20
proportion :
320 EE
210
350 340) | 380)
210)
Emm EERE e Concentration propoarttstieoadny state orupperorlowerlimitofconcentration proportion
Slevele | vow [eee
--_~
[Bioconcentration Report] 3
Company Sanitized. Does not contain TSCA CBI
~
ion
_Oow aa [dows]
Femend sonpemraonievel | GOR BOF[0s
J
5. Test water and fish body analytical methods
(1) Test water and fish body analysis flow
Test
water analysis Column chromatography
(SepPackCis)
=>
Analysis
@ Test f`iHsohmaongaelnyisizsation extraction(methanol"formic acid (S00/0.5 vA) > Dilution -- Analysis +2: containing 10 mmol/L ammonium acetate
(2) Analytical instruments andconditionsused
"Instrument: liquid chromatography-mass spectrometer
`Pump: Nippon Bunko, Model PU-980
--~
Autosampler: Nippon Bunko, Model AS-950
`Mass spectrometer: Micromass, Model QuattrIo
Colum: Lo<olumn ODS (prepared by Chemical Evaluation and Research Insitute), 15 cm x 2.1mm LD.
Column temperature:
`Elution solution:
25C
Solution
A
(75%):methanol
*/formic
acid
(S00/0.5
v/v)
Solution B (25%): waterformic acid (500/0.5 vA)
Flow rate: 02 mL/min
Amount to be injected: 20 kL.
MaIosniszsatpieocntrmoemtehtoedr:ceolnedcittriosopnray ionization (ESD
Lon detected: cation
Detection method: selective fon monitoring (STM)
Measuremention: main component m/z 571
subcomponent: m/z 513
~
Ton source temperature: 120C
Cone voltage: S0V
[Bioconcentration Report] 4
`Company Sanitized. Doesnotcontain TSCA CBI
-- 6. Recovery rate (mean `Misincomponent84.0 Recovery fromfishbodsLO mpmmo92
`Subcomponent 87.1 s`eSubmcomeponnent 89.9 |
7. Discu"shtseioofhnleocnrueasutlitonboeffotrhee eTxipedricmoemnetns,ta(nmdeiat)wafsieosvceorm2p5l%e.tiOonneoofefxtpheerciamuesnetssis choisnsstiuddyetvroaebsde1% ctoamtpaablrettohduecetdaitnigomneitnhpohdy,sieocl,ogricealocboendsintdiioensdefduirngts0thasteie,rm tfcor.icmapursionvgemieindtscoantleinptidHcuocnttueanttion. dena, changes and fluctuations.
--~
8. Other [i
Comor`Evaltuatis onand ReserarhchnInististut,e,EKrurnumeeLoLraboirateo | [19-14Chuo cho, Kurumesb, Fuknokarken PCS30.0023
Sudy implementation
|[TeAledphdoneraendss| Tel:0942-34-1500, FAX: 0942:39.6804
Study manager FAX facil |
om Texo perienr ce:17,r 2003p -Decee mbern 25.2003
--
(Bioconcentration Report] 5 Company Sanitized. Doesnot contain TSCA CB
Study requester: Du Pont KK.
Statement Chemical Evaluation and Research Institute: Kurume Laboratory
Study tie: Bioconcentration studyof
ER 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
--_ [Bioconcenization Report] 6
`Company Saniized. Doss not contain TSCA Cai
-
Final Report
Biconcentzion Sty -- cam
~
December 25, 2003 Chemical Evaluation and Research Institute
Kurume Laboratory
ps
[Bioconcentration Report] 7 Company Sanitized. Does not contain TSCA CBI
r~
Statement
Chemical Evaluation and Research Institute Kurume Laboratory
`Study requester: Du Pont KX. Study tie: Bioconcentration study oPin c=
"The above study was carried out according to the following GLPt(1e)m"sS,teatncd,arodfstefsotr rteelsattiendg tfoacnileitwiecshsepmeicciafliesduibnstMainnciesstrayndOredvianluaantcieonArotfictloexi4cisttyipruellaattiendgto NdoetsiifgincaatteidonchNeom.ic2a2l9soufbsPthaanrcmea"ce(utGiLc)al(AEfnfvaiirrosnBmuernetaaul ParnodteMcItTiIonBNaostiicfiIcnadtuisotnr3y9B,ureau Notification No. 85 dated March 31, 1984, amended on March 1, 2000) I(n2)a"ddOitEiConD, PtrhiincfiipnlaelsreopfoGrtooredflLecatbsorthaetorraywPrdaicttaiaccec"ur(aNtoevlye,mabnedrt2h6e,t1e9st97d)ata are confi rmed tobe valid. |,
December 25, 2003 Study manager: Naoaki Yakata
{Bioconcentration Report] 8 `Company Sanitized. Does not contain TSCA CBI
[an
Reliability Guarantee Certificate
Chemical Evaluation and Research Institute Kurume Laboratory
Study requester: Du Pont KX.
Study tte: Bioconcentration studyof
in carp
~ S| departm"eTnhte oafudtihteinCgheomriicnaspleEcvtaiolnuaotfitohneasnbdoRveessetaurdcyhwIansstictaurtre,ieKduoruutmbeyLtahbeorrealtiaobriyl,itayngduatrhaentee content and date of auditing or inspection as well as dates reported to the study manager and operation manager are as follows.
[utyproocsl | November 17.2003
Satoef mplemenaion | November 15,2003
November 17,2008 Deconber 25,2003
November 26,2003
November 26,2008
Novemb2e7r, 2003 November 28, 2003
December 2,2003 December 2.2003
December 2.2003 December 2.2003
Rawdataand final `December 25, 2003
December 25,2003 | December25,2003
repo
h"Tehisstfuidnaylprreoptoorctolisagnudarsatnatnedeadrdfoprrothceedtuertesmentdhoadstbheeisnagmaecctuirmaet,erlyawdedsactraibbeedi,ngcornetfelnetctbeediancgcucroantfeolyr.med (0
~~
DReelciaebmiblietry2G5u,ar2a0n0t3ee Department Manager: Shiro Horikoshi
(Bioconcentration Report] 9
.
company Saniti]zed. Doss not contain TSCA Cat
~
Table of Contents
Page
Title
Study requester
Study facility
Study object
Study method
GLP applied]
Study schedule
Storageof 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
e
8. Notes
~~
[Bioconcentration Report] 10 Company Saniized. Doss not contain TSCA CBI
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44133
Tables
Table] Test substance concentration in test water (shown in main text)
Table2 Concentration proportion (shown in main text)
Table3 Test substance concentration in test water at steady state (shown in main text)
Table4 Calculation table for recovery test and blank test (test water analysis)
Table5 Calculation table for analysis of test water in the first concentration level
Table6 Calculation table for analysis of test water in the second concentration level
Table7 Calculation table for recovery test and blan test (test fish analysis)
Tables Calculation table for analysis of test fish in the first concentration level
Table9 Calculation table for analysis of test fish in the second concentration level
Table 10 Calculation table for analysis of test fish in the control group.
Reference 1 Table for the results of quality test for test water
Figures
Figuel Correlation between exposure period and bioconcentration factor (first
concentration level)
~
Figure2 Correlation between exposure period and bioconcentration factor (second
concentration level)
i
Figure Study substance concentration -- mortality curve in acute toxicity test
Figore4 Test water analysis calibration curve:
FigureS LC-MS mass fragmentogram in recovery test and blank test (test water analysis)
Figure6 LC-MS mass fragmentogram in test water analysis
Figure 7 Calibration curve for test fish analysis
Figure LC-MS mass fragmentogram in recovery test and blank test (test fish analysis)
Figure9 LC-MS mass fragmentogram in test fish analysis at the first concentration level
Figure 10 LC-MS mass fragmentogram in test fish analysis at the second concentration level
Figure 11 LC-MS mass fragmentogram in test fish analysis in the control group
Figure 12 Mass spectrumoftest substance
Figure 13-1 Infrared absorption spectrum of test substance (before startofexperiment)
Figure 13-2 Infrared absorption spectrum of test substance (after completion of experiment)
Reference 2 Infrared absorption spectrum provided by study requester
--_
[Bioconcentration Report] 11 `Company Sanitized. Does not contain TSCA CBI
Title: Bioconcentration TestofSNin Corp
Study requester:
DuPont KX. 1-8-1 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 oN 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, te." (Environmental
Protection Notification No. 5, Notification No. 615 of Pharmaceutical
Affairs Bureauand MITI Basic Industry Bureau 1974 Notification No. 302
~~
dated July 13, 1974 and ameonnOcdtoeberd8, 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 for testing facilities specified in Ministry Ordinance Article 4 stipulating items, etc, of test elated to new chemical substances and evaluation of toxicity related to designated chemical substance" (GLP) (Environmental Protection Notification 39, Notification No. 229 of Pharmaceutical Affairs Bureauand MITI Basic Industry Bureau Notification No. 85 dated March 31, 1984, amended on March 1, 2000) (2) "OECD PrinciplesofGood Laboratory Practice" (November 26, 1997)
~~
[Bioconeentration Report] 12 Company Sanitized.Doesnot contain TSCA CEB
--
Study scheduleStudy stant date
November 17, 2003
=
EExxppeerriimmeenntt cstoamrptldeattieon date NDeocveemmbbeerr 2191,,22000033
Study completion date
December 25, 2003
Storage(o1f) sSttuuddyydmoacteurmieanltss and materials "cTohnetasianmeprlsefsorpr1o0viyedaerdsbfyrtohm ttheestnroteiqfuiecsatteironarceortroebseposnodriendg,taoftehresAcratliicnlgei4n,sCtloaruasgee c1h,eNmoi.ca1l,2suobrs3taonfcteshe(albabwrerveilatteeddttoo r"eCghuelmatiicoanls efvoarleuvaatliuoantiloanw,,"prboedluocwt)i,oni,n etthce, of sthaempalcteiosntsoroangethreoosammopfletshearKeutroubmeedLeatbeorrmaitnoreyda.fAtfetrecrotnhseulptraetsicornibweidthsttohreagteestterm, `mraerqukeesdtelry.dHuorwienvgtehre,stiforthaegeprtoedrumc,ttqhueatleirtmy oisfsaestaompbleetihseeoxnpeectfoerdthtoepcrhoandguect qGubatlaiitnyewdiftrhosmtatnhdeinsgtusdtyorraegqeu,esatnedr.at the timeofdisposal, an approval is o be
(2) Raw"Tdhaetar,adwocdautmae,nsttsu,dyetpc.rotocol, study request, study substance survey form and other nfercoemssthaerynodtoicfiucmaetniotnsctoorgreetshepronwdiitnhgtthoetfhienAartriecploert4,arCelatousbee 1s,tNoroe.d 1f,or2.100r y3eoafrtshe "CThheemaicctailonEsvoalnutahteisoenmLataewriianlstahfetdroctuhemesnttorsatgoeratgeerrmoaorme otofbeKduertuemremiLnabeodraafttoerry. consultation with the tes requester.
Study personneSltudy manager S(itmupdlyeimnevnetsattiigoatnorosf bioconceniration test) .
RAecaurtientgoxciocnittryoltemstanager investigators
NAfafoialikaitiYoan:kaSteacond Testing Department MYoaskhiiySuakcikiInoue YSohirni-kiachMiorroiKaku A`AkkiekmoiHiIrnoosuheige YChaisruirsoe FKuakwuadsahima YYaassuurroo KKaawwaasshhiimmaa Tadayoshi Fujivchi
--
Approval of finSatlurdeypomratnager:
December 25, 2003 Naoaki Yakata
[Bioconcentration Report] 13 `Company Sanilized. Does not contain TSCA CBI
--
Summary
Study tite
Bioconcentration est ofSENN in carp
Study conditions Acute toxicity test (1)Fish used (2) Exposure time: (3) Exposure method
Killifish 9%h Semistatic type (water exchange every 8-16 h)
Bioconcentration test
(1) Fish used.
Cap
(2) Concentration tested First level: 50 ug/L
Second level: 5 pg/L
(3) Exposure time
28 days
(4) Exposure method Continuous water flow system
~~ (5) Analytical method Liquid chromatography-mass spectrometer combination
Results
(1)96hLCS0
5350 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 WIIG i cor hs the following name and other elated items.
11 Name"!
1.2 Structural formulas and others Structural formulas
a -- cMoslevcu.lar weight (
*1 From documents provided by the study requester
2. Sample provided by study requester
:
In the following the sample provided by the study requesteri called the sample provided.
2.1 Provider and Lot No."
(1) Provider: Du Pont K.K.
22 Purity"!
[Bioconcentration Report] 15 Company Senitized. Does not contain TSCA CBI
om
Opry
----
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"
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 Storageat 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.
5. Acstst 31 Test"ihneg mmeetthhoodd usd was the method of "Indust wstowaer esting method. - sete axis
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 Suiza,Dossntcontain TSGACBI
~
(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 amedicinal bath for preventionofdiseases and extermination of parasites and rearedfor44 days in a state of flowing water.
Medicinal bath: For disease prevention, a mixtureof20 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 K/L of formalin for24 h was used twice.
(4) Conditioning conditions `Term: after rearing and storing, the fish were transferred to aconditioning 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 mixtureof20 mg/L Elverju and 7 g/L sodium chloride was used to
carry outmedicinal 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 caseof test fish of the same
10t.(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 Sanitized. Does notcontain TS ren:
~~
`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 TestingofChemicals," "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 TestingofChemicals," "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 Liconcentration level
~
(3) Test temperature
Start of exposure:
28C
Before water exchange ~~ 25.1C
(4)Dissolvedoxygen concentration Start of exposure
8.1mgL
Before water exchange 7.8 mg/L
)pH
Startofexposure
80
Before water exchange 7.8
(6) No. of fish tested.
10/concentration level
(7) Exposure time
96h
(8) Exposure method
`Semistatic type (water exchange in every 8-16 h)
3.5 Stock solution preparation method
,
(1) Dispersant
Crystallized sugar
Megafuc F-142D
(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 sontain TSCA cay
~
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
(W) Testsite
Room 214LC50
(2) Testdate
November 17, 2003 ~ Novem21b,2e00r3
3.7 Calculationof96 h LCS0 `The Doudoroff method was used.
3.8 Test result
96 h LCS0 of test substance
>35.0 mg/L" (see Figure 3)
*2: The concentration of the dispersant (Megafac F-142D) used in the above case was 700 mg/L, the 96 h LCS0 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 CarpCyprinuscarpio
Reason for selection: for considering consistency with past findings and size being easy to handle
(Source
Sugishima Fish Farm (123-2 Gunchikuichiban-cho, Yatushiro-shi, Kumamoto-ken 866-0024) Date of receiving tested fish August 14, 2003
(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 L/L formalin for 24 h was used twice.
[Bioconcentration Report] 19
Company Sanitizod. Does NOtcontain TREAA mm.
--
(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 toa test tank, and medicinal bathing, the selected fish were.
conditioned at the same temperature as above in a tateofflowing water for 42 days.
OTCanMded7igc/iLnaslodbaituhm: cahfleorritdreanwsafesrruisnegdttooaccaornrdyiotuitonmiendgictiannka,labamtihxitnugrefoorf2540h.mgoff/ishLery
In the test tank, a mixture of20 mg/L of Elverju and 7 g/L sodium chloride was used to
carry out medicinal bathing for 24 h.
(5) Bodytotal length 7.0-9.0 cm
Lot
TFC-030814
Age
Less than one year old
() Feed
Kind
Compounded feed for young carp
Composition
Protein content of43.0% or higher
~~
Fat contentof 3.0% or higher
Manufacturer Nippon Haigo Shiryo KK.
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 24h prior to sampling of study fish.
4.2 Water used for the test Same asin 33.
43 Test and environment conditions
+ ()Testwater feeding method The feeding was carried out by using a flowing water device -
assembled at the Kurume Laboratory
(@) Test tank
100-L volume glass water tank
(3) Amount of test water
With 2proportion of2 mL/min of the stock solution and
800 mL/min of test water, 1155 Liday were fed to the test
tank
(4) Stock solution tank.
25 L glass bottle
--~
Replacement frequency ~~ Oncelveek
(5) Test temperature
First concentration level ~~ 25.0-25.2C
[Bioconcentration Report] 20 `Company Sanitzad. Does not contain TscA car
~~
Second concentration level 25.0-25.2C
Control group
25025.1C
(6) Dissolved oxygen concentration First concentration level ~~ 7.6-8.1 mg/L.
Second concentration level 7.88.1 mg/L
Control group
8.1mgL
pH
First concentration level ~~ 7.7-8.0
Second concentration level 7.7-8.0
Control group
7.680
(8) lumination time
Astificial illumination by white fluorescent lamps (14 h
light10 h dark)
(9) No. of fish tested
First and 2* concentration levels: 38 (startof 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 concentrationof20 mg/L was prepared by using the same procedures as those in section 3.5 (2).
+ Second concentration level
A stock solution having atest substance concentrationof 2 mg/L was prepared by using the
same procedures as those.n 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 LCS0 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 pg/L.
Second concentration level 5 pg/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, tc., observed twice aday 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 onorcmoere 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.
47 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 outforeach peak.
However, the concentrationofeach peak shown doesnottake into account the component
composition asa test substance concentration shown in the standard solution preparation in section
4730).
477.1 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 est fish analysis. One sample per
analysis was used.
) Test fish
For both the first and second concentration levels during exposure, the test fish analysis
was carried out times, the number of fish used per analysis was 4, and they were divided into 2
groups (2 per group)".
~~
In the control group, analysis was carried out before and afer the experiment, 4 per analysis were used, and they weredivided into 2 groups (2 per group). In addition, for fat
measurement, 2 were selected making the total of 3 groups (2 per group).
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#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
G=iTstesctonwcaettafioronrelceovveelroyntleys)t 360 mL (graduated cylinder) Column chromatography (ses below for etl) --~
+ Constant volume make-up 10mL eluate", volume flask)
*4 Column chromatography conditions
Sep-Pak Cig
(Washing method: eluate, water"
10 mL each)
.
Loading method: whole amount loaded
Elution method: first solution water 5 mL
second solution eluateTM mL
Second solution used for analysis
*5 Seequantitativedetermination 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. Doos not contain TSCA CBI
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(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
++ BCuotdiynwgeuipgh(tscainsdsolrse)ngth measurements + Pulverization (i ce water, Polytron, 2minor longer)
+(Slaemctpolneidc 1sn5syical balance) [pSreemsperlveaftoiron
SamemtphlaneoSdlg feolrecmtircoanciicda(nSal0y0t/i0c.a5lvbva)lan1c5e)mL. +(gHroamdougaetneidzcaytliinodner()Polytron, about 1 min) ++ CWeansthiifnugg(amteotnha(n7o0l00fXorgm,i5cmaicni)d (S0/0.5 vA) 3 mL)
Frm] -- +FiClotnrsattainotnv(oalbsuomrebemnatkceo-tutpon2)5 mL (methanol formic acid (+ 5B0a0t1c0h5ovf14),mLvo(luamnesttreirc pfilpaestk)) `+WCaotnesrt*a6n7tfvfoorlmuimceamcaidke(-Sup00100 mVL./V(m)et2h.5manLol(ifroarnmsitecpaicpiedt) (5000.5 vi), volumetric flask)
"7: containing 10 mmol ammonium scctats
47.3 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, ifthe test
~~
substance concentration was outside the range of the calibration curve, the sample was diluted
[Bioconcentration Report] 24 `CompanySanitzed.Doesnotcontain TSCA Ca
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before analysis to bring the concentration within range. The test substance concentration in an LC-
MS 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 Rescarch Institute)
15cm x 2.1 mm LD. Column temperature: 25C
Elution solution: Solution A (75%): methanol "/formic acid (500/0.5 viv)
Solution B (25%): water'*"/formic acid (S00/0.5 v/v)
~ Flow rate: 0.2 mL/min Amount injected: 20 4L
Mass spectrometer conditions
Tonization method: electrospray (ESD
Ton detected:
cation
Detection method: selective ion monitoring (SIM)
Ton measured: Main component mz S71
Subcomponent m/z 513
Ton source temperature:
120C
. Cone voltage:
s0v
.
(2) Standard solution preparation `The standard solution for determining the test substance concentration of an analytical
sample was prepared as follows.
(@) Test water analysis
~
100mgof 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 solutionTM to prepare a
[Bioconcentration Report] 25 `Company Sanitized. Doss not contain TSCA cay
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2.00mLsolution of the test substance, which was used to carry out a pretreatment according to
the following scheme to obtain a 200 pg/L standard solution.
Scheme
Column chromatography (details describedonpage 13)
2.00 mg/L solutionof test substance 1 mL (ransfer pipet) + Constant volume make-up 10 mL (eluate, volumetric flask)
[200 g/L Standard solution For test water analysis)
(b) Test fish analysis
m~
100 mg ofthe provided sample weighed accurately were dissolved in methanol to obtain a
1000 mg/L solutionofthe test substance. It was diluted with the elution solution" to prepare a
2.00 mg/L solution of the test substance, which was ustoecardry out a pretreatment according to the following scheme to obtain a 200 g/L standard solution.
[Bioconcentration Report] 26 `Company Sanitized. Does not contain TSCA CBI
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Scheme
[Pulverized sample similar that in the overy testand blanktest of 7.4 Sg
Metha(ngorlad"ufaotremdiccylaicnidder()S00/0.5 v/v) 15 mL. HWoamsohgienngiz(amettihoanno(Plo"lfyotrromni,cabaociudt (1S0m0i/n0).5 v/v) 3 mL) Centrifugation (7000xg for 5 min)
[seman|
Filtration (absorbent cotton)
+ Constant volume make-up 25 mL (methanol"formic acid
+(5S0a0m0p0l5edvv4),mLv.ol(urmaentsrfiecr fpliapsekt))
Water"formic acid (S00/0.5 v/v) 2.5 mL.
(wansfer pipet)
+ Co2n.s0t0anmtg/vomlLutmeestmsaukbes-tuapnc1e0smolLuti(omnet|hmanLol("rfaonrsfmeircpiapceitd)
--
(5000.5 vv), volumetric flask)
200 g/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) (2) were carried out to prepare 100, 200 and 400 ug/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 pg/L) Subcomponent 1000 (test substance concentration of 12 g/L)
by considering the noise level (see Figure 4).
(b) Test fish analysis
~
`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 ug/L standard solutions. These standard solutions
[Bioconcentration Report] 27 Company Sanitized. Doss not contain TSCA CBI
~~
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 concentrationof20 pg/L)
Subcomponent 1000 (test substance concentration of 19 pg/L)
by considering the noise level (see Figure 7),
4.7.4 Recovery test and blank test (1) Method
To determine the recovery rateofthe 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 testswere 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.Forthe recovery and blank tests, the measurements were carried out for 2 items each.
(2) Results
~~
Asa result of measurement carried out by the methodof(1), no peaks were observed at the
positions of the test substance in the mass fragmentograms obtained intheblank tests. The
recovery rates and meanrecoveryrate fo2r items each in the analytical procedures were as
follows, and the meanrecoveryrate was used as a correction in the case of determinitnhge
concentrationofthe test substance in an analytical sample (se Tables 4 and7 and Figures 5 and
8).
Recovery rate in the analytical procedures
`Test water analysis (2000 ng of test substance added)
Main component 82.7%, 85.3% Mean 84.0%
Subcomponent 84.7%, 89.6% Mean 87.1%
.
`Test fish analysis (25000 ng of test substance added)
Main component 99.5%, 98.9% Mean 99.2%
Subcomponent 90.3% 89.5% Mean 89.9%
477.5 Fat content of test fish A pulverized test fish sampleofthe control group was extracted with chloroform/methanol,
~~ and the fat content was determined by weight analysis.
[Bioconcentration Report] 28 `Company Sanltized. Does not contain TSCA CBI
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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 concentrationoftest 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 were calculated as follows.
Main component First concentration level 7.5 ug/L.
Subcomponent
Second concentration level 0.75 pg/L. First concentration level 3.5 pg/L Second concentration level 0.35 g/L
(3) Calculation of test substance in tet fish analytical sample `The caleulation 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 concentrationoftest substance in test fish From the quantitative lower limit determined in the preparationofthe calibration curve in
section 4.7.3 (3) (b), the qualitative lower limit concentrations" 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: Quanitative lower limit concentration of test substance (ug/L or ng/g) =a
B/100 x CXE/D
A :. calibration curve quantitative lower limit concentration (g/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.
--~
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4.7.7 Calculation method for total mean test substance concentration in test water during exposure
term
Tat = (Ce) Het Orn) Sn
Cw: total mean test substance concentration in test water (ug/L)
ni: No. of test water analyses (frequency of measurement) Cw(1): test substance concentration in test water measured for the 1 time (ug/L)
Cw(n): test substance concentration in test water measuredfor the n time (ug/L)
4.7.8 Calculation method for bioconcentration factor (BCF) `The bioconcentration factor (BCE) was calculated according to the following formulas.
(1) Calculation of mean test substance concentration in test water for calculation of bioconcentration factor
Cw = [Cwin - 1)+ Cw(@)l2 (first test fish analysis)
Cw = [Cw(n - 2) + Cw(n - 1) + Cw(m)}/3 (2 or later test fish analysis)
~
Cw: mean test substance concentration in test water for calculation of bioconcentration
factor (ug/L)
Cw(n): test substance concentration in the n' test water analysis determined at the same.
time as test fish analysis (ug/L)
(2) Bioconcentration factor calculation
BCF=Cf/Cw
BCF : bioconcentration factor
.
Cf test substance concentration in test fish (value with FB subtracted) (ng/g) ~~ -
Cw: mean test substance concentration in test water for calculation of
bioconcentration factor (ug/L)
FB
mean concentrationoftest substance in est fish before and after experiment
in the control group or apparent (blank) concentration of test substance
(ng/g)
--
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(3) Mean of bioconcentration factor of m"" time
BCFm = (BCFa + BCFb/n
BCFm : mean of bioconcentration factor of mTM time (No. of individuals or group
BCFab :
2(b) bioconcentration factorof each individual or group of m time
n
number of individuals or group analyzed in the m time
`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 intervalor 48 h or longer.Ifthe bioconcentration factor is less than 100 times, it is considered to have reached a steady state after 28 days evenifthe bioconcentration factor fluctuation is over 20%.
Standard for attainmentof a steady state: V(m - 2), V(m - 1, Vm) S20 (%)
~
Va--2) = --| -- Bor--@-2--) ---- B--F--| X 100 .
BOF
vp = PBEOREGDD)C--EBGEF-.L| gg
BF
| BeFw) -- BCF |
Vm) = ----B-- OFm-- X: 100
Vm -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)", (m - 1)" and mTM
bioconcentration factorofn individuals or groups
BCF : {BCF (m - 2) + BCF (m - 1) +BCF (m)}/3
--_~
[Bioconcentration Report] 31 Company Sanlized. Does not contain TSCA cay
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4.7.10 Method for calculation of steady state bioconcentration factor (BCFss)
formulas`.The steady state bioconceniration factor (BCFss) was calculated using the following
(1)Calculation ofmean test substance concentration in test water for calculation of steady state bioconcentration factor
Tos = {on-2) + oval) + Cxw)) /3
Cws: mean test substance concentration in test water for calculationofsteady 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) (ug/L)
Cw(n): test substance concentrationof nTM test water analysis determined simultaneously
with test fish analysis (ug/L)
(2) Calculationofmean test substance concentration in test ish in a steady state TE = (ct) + Ctl) + C2) /3
--
CTs: mean steady sate test substance concentration in test fish (ng/g)
(Cf(m): mean test substance concentration in m test 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
BCRss = Cfs / Cvs BCFss: steady state bioconcentration factor
CFs: 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 (ug/L)
47.11 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 caseofal test water analyses.
[Bioconcentration Report] 32 Company Sanitized. Does not cantain TSCA ay
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Main component First concentration level 5.1 times
`Subcomponent
First concentration level 5.6 times
Second concentration level 55 times
4.7.12 Method for calculation of fat content
The fat content was determined from the following formula.
Fat content (%) = (T-To)/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 ofJIS 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 reliabilityof 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%ofthe values set. The fluctuations in test substance `concentrations were maintained within + 20%ofthe mean measurement result.
Table 1. Test substance concentration in test water
. Init: pg
t mm=aT l. wTo deRFrAr TrEEAoS p drT d=FS o lT orr eA e=edald --
om | |
[Bioconcentration Report] 33
`Company Saniized. Doss ot containTSCA G1
2
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
Subcomponent
Second concentration level less than 51 times
First concentration level 350-510 times
Second concentration level 270-430 times
TE e C E C[ T EEREEe EE f EfEBe EplEEE e TpB ETTT aT component
`Table 2. Bioconcentration factor
Mean inside
S51 | S51
S51 S51
S51
6.3 Steady state bioconcentration factor
6.3.1 Main component
As a result of section 6.2, allofthe final consecutive 3 analyses showed no detection, and
consequently, no steady state bioconcentration factor could be calculated. However, the results on
the bioconcentration factorwere all less than 100 times, and thus, it was concludedthatthe steady
state had been attainedafter 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.
`CompanySanitead. Does not contain TSCA C81
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(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
[eConcmenvation T|aAfer 17|[aAfier2d [|aAfier 8 Po [oTabele |rFniiegt:uere|pg/L)
fetefo fuLe Te) Ce Toon[sm [ase[as |ao |
(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 contentoftest fish
"The results on the mean fat contentofthe 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 `CompanySantee.Dees not containTSCA CBI
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(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 lijima Denshi Kogyo
pH meter:
Model HM - 14P manufactured by Toa Denpa Kogyo
`Thermometer for water:
Model ALCO-50 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 (Cerzpany Saniizad. Does notcontain TSCA CBI
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Homogenizer (Autocellmaster): Model CM - 200 manufactured by Iuchi Seieido
Vacuum pump:
Model DA-20D manufactured by Shinku Kiko
Vacuum desiccator:
Model VL manufactured by Tuchi Seieido
Reagents
Purified water:
`Pharmacopoeiajaponica, 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 7 Samira. Doss notcontain TSCA CB