Document J30vKx7MojzRz482V5rGo4JJ2

M u t - 3300 482 Accession No. Study No. S03-4102 44102 3360 Study Report Study of cumulation in Carp September 22,2003 Chemicals Evaluation and Research Institute, Japan --Kurume Laboratory- Company Sanitized. Does no! contain TSCA CBI Figures Fig. 1 Mass fragmentograms of LC-MS analysis for recovery and blank tests (analysis of test water). Fig. 2 Mass fragmentograms of LC-MS analysis for recovery and blank tests (analysis of test fish). Fig. 3 Calibration curves Study No. 44102 2 Company Sanitized. Does not oontaln TSC Title: Study o f M H H P A c c u m u l a t i o n in Carp Commissioned by: DuPont KK 1-8-1 Shimo-meguro, Meguro-ku, Tokyo 153-0064 Testing Facility: Kurume Laboratory Chemicals Evaluation and Research Institute, Japan (CERI) 19-14 Chuo-machi, Kurume-shi, Fukuoka Prefecture 830-0023 Purpose of Study: To obtain Endings on the accumulation of f l H H H V in carp. Personnel: Study Coordinator: Naoaki YAGATA,* Testing Section 2 Researchers: Yoshitaka KOGA Masanori WATASH3MA Approval of Study Report: [signed] Naoaki YAGATA Study Coordinator September 22, 2003 *Translator's Note: Spellings of personal names may need to be verified. Study No. 44102 3 Company Sanitized. Does not containTSC CB 1. Test Name: Lot No. Purity: 2. Preparation of Stock Solution A stock solution having a test substance concentration of 500 mg/L was prepared by first grinding together in a mortar the test substance and a 20-fold amount of crystal sugar, subsequently adding a 20-fold amount (relative to the test substance) of Megafac F-142D and again grinding the ingredients together in a mortar, then adding ionexchanged water. 3. Preliminary Test of Acute Toxicity 3.1 Test Conditions (1) Test fish: Himedaka (Oryzias latipes) (2) Number of test fish: 2 fish per concentration group (3) Period of exposure: 96 hours (4) Method of exposure: semi-stagnant water method (change of water every 8 to 16 hours) (5) Preliminary test results: preliminary LC50 over 96 hours was >30.0 mg/L* *TN: Japanese name of fish. English name is not known. Study No. 44102 4 Company Sanitized. Does not contain TSCA CB1 4. Investigation of Analysis Conditions Based on the preliminary LC50value of the test substance over 96 hours and the sensitivity of analysis, the test concentrations were set as follows First concentration group: 50 fig/L Second concentration group: 5 fig/L, and an examination of the analysis conditions was carried out. 4.1 Test Substance Analysis in Test Water and Test Fish When the test substance was analyzed by liquid chromatography-mass spectrometry (LC/MS), three peaks were detected. Quantitative determinations were thus carried out on each of the peaks. The respective peak concentrations are indicated as the test substance concentrations shown in section 4.2.2 ("Preparation of Standard Solution"), regardless of the constituent compositions. The peaks were named Peak 1, Peak 2 and Peak 3, in the order of elution. 4.1.1 Investigation of Method for Pretreating Test Water Analysis Specimens Stock solution (containing 2 fig of the test substance) was added to 400 mL of test water for the recovery test, and pretreatment operations were carried out according to the flow scheme shown below to give a liquid chromatography-mass spectrometry (LC-MS) specimen. Flow Scheme Study No. 44102 5 Company Sanitized. Does not contain TSCA CBt 00 Ut *1: Column chromatography conditions: SepPack Qg (Cleaning method: 10 mL each of methanol*Vwater*2,3 (8/2 V/V) and water*3) Loading method: Entire amount was loaded. Elution method: First eluting solvent: water Second eluting solvent: methanol*2/water*2'3 (8/2 V/V) The second eluting solvent was furnished for analysis. *2: Contains 10 mmol/L of ammonium acetate *3: Water obtained by treating tap water using an ultrapure water production system. The percent recovery in each of the above operations are given below (see Fig. 1). Percent recovery (n = 1) Peak 1: 85.1% Peak 2: 90.5% Peak 3: 74.2% Blank (n = 1) Peak 1: not detected Peak 2: not detected Peak 3: not detected Study No. 44102 6 Company Sanm **. 3 ,.,5no: 00nta,,, TSCA CB 4.1.2 Investigation of Method for Pretreating Test Fish Analysis Specimens Stock solution (containing 5 fig of the test substance) was added to 5 g of a finely ground sample of the test fish for the recovery test, and pretreatment operations were carried out according to the flow scheme shown below to give a liquid chromatographymass spectrometry (LC-MS) specimen. Flow Scheme Test fish analysis specimen -Methanol*2, 15 mL (measuring cylinder) Homogenize (Polytron, about 1 minute) Wash (methanol*2, 3 mL) Centrifugal separation (700xg, 5 minutes) Residue Supernatant Filtration (absorbent cotton) Bring up to 25 mL (methanol*2, measuring flask) Take aliquot sample of 5 mL (transfer pipette) -Water*2,3, 2 mL (transfer pipette) Bring up to 10 mL (methanol*2, measuring flask) LC-MS sample (peak 3) Take aliquot sample of 2.5 mL (transfer pipette) Bring up to 10 mL (methanol*2/water*2,3 (8/2 V/V, measuring flask) LC-MS samples (peaks 1, 2) The percent recovery in each of the above operations are given below (see Fig. 2). Percent recovery (n = 1) Peak 1 104% Peak 2 119% Peak 3 (not measurable due to insufficient LC-MS sensitivity) Blank (n = 1) Peak 1 not detected Peak 2 not detected Peak 3 not detected Study No. 44102 Company Sanitized. Does not contain TSCA CB) 4.2 Quantitative Analysis 4.2.1 Quantitative Determination Conditions: Apparatus: Liquid chromatograph-mass spectrometer Pump: PU-980 (JASCO) Autosampler: AS-950 (JASCO) Mass Spectrometer: Quattro II (Micromass) Liquid Chromatography Conditions Column: L-column ODS (CERI); 15 cm x 2.1 mm I.D. Column temperature: 35C Eluting solvent: A (80%): methanol*2 B(20%): water*2'3 Flow rate: 0.2 mL/min Injection rate: 20 fxh Mass Spectrometer Conditions Ionization method: Detected ions: Detection method: Selected ions: Ion source temperature: Cone voltage: Electrospray (ESI) Positive ions Selected ion monitoring (SIM) Peak 1 m/z571 Peak 2 m/z671 Peak 3 m/z771 120C 50 V Study No. 44102 8 Company Sanitized. m conah7A CB 4.2.2 Preparation of Standard Solution: Standard solutions for determining the test substance concentration in the analytic sample were prepared as described below. (1) Test water analysis: One hundred milligrams of the sample furnished was precisely weighed out and dissolved in methanol to form a 1,000 mg/L test substance solution. This solution was then dissolved in methanol*2/water*2,3 (8/2 V/V) to form a 2.00 mg/L solution. Pretreatment operations were carried out on the resulting solution in accordance with the flow scheme shown below, thereby forming 0.0500 mg/L (for Peaks 1 and 2) and 0.200 mg/L (for Peak 3) standard solutions. Flow Scheme (2) Test fish analysis: One hundred milligrams of the sample furnished was precisely weighed out and dissolved in methanol to form a 1,000 mg/L test substance solution. This solution was then diluted with methanol*2/water*23 (8/2 V/V) to form 0.0500 mg/L standard solutions for Peaks 1 and 2 and a 0.200 mg/L standard solution for Peak 3. (3) Preparation of calibration curve: Following the same procedure as that used to prepare the standard solutions in (2) above, 0.0250,0.0500 and 0.100 mg/L standard solutions for Peaks 1 and 2, and 0.100, 0.200 and 0.400 mg/L standard solutions for Peak 3 were prepared. These were analyzed under the quantitative determination conditions given in section 4.2.1 above, and a calibration curve was prepared from the respective concentrations and the peak areas on the chromatogram (see Fig. 3). Study No. 44102 9 Company Sanitized. Doq$ m \ nanTSCA CBf Fig.1-1 M ass fragmentogram of LC-M S analysis for recovery end blank test (analysis of test water) Study No. 44102 10 Company Sanitized. Doos not contain TSCA CB1 oto u.Lfumg/ 44102B2S 100- i :% ------- -- jjSJ u|pcl03 SIR of3 Channels ES+ TIC 1.0064 Area ! 43 z s r ^ n a b 1......1" 12 k n (l 14.00 >TI" stR o fs te " ' TIC l,00s4 Area Peak 3 -J' 12.45 548 Time ' ' Atn ' ' ' a i 11111 abo ' ' 1 io! ' 12:00 1 foo OS-J-2003 Blank te s t o f te a t water SIR of3 Channafs ES+ TIC 1.00e4 peakposition PeakS NJ>. -Wv-WX / 11 M a^A /^V V 45` ....... "?hr>" ' 1' Ahn ' " W 1 8.bo ' " il" 1 12:00 ' C ; fi. fi Fig.1-2 Mass fragmentogram of LC-MS analysis for recovery and blank test (analysis of test water) Study No. 44102 Company Sanitized. Doss not contain TSCACBf 11 Flg.2 M ass fragmentogram of LC-M5 analysis for recovery end blank test (analysis of test fish) Study No. 44102 Company Sanitized. Does not contain YSCACSS! 12 44102804 100- %- Peakposition Peak! 4i-iviay-iiutM SIR of 3 Channels ES+ TIC 3,S0e4 0.00 rn" m ...r,'m .........b e ..' " ' w e W pr ' II II*"i 12.00 IS T 11" Standard solution .2fmg/L *2i'-May-2$03 44102811 S IR of 3 Channels ES+ Fjg,3- 1-1 M ass fragmenfognerr of LC-M S analysis for calibration curve (Peak 1) Study No. 44102 13 Company Sanitized. Does not containTSC CBS Study No. 44102 Peakarea /**`*N. y - 15249bc r*lM Conceotr&ttaa fasti) 0.0230 0.0500 0.100 PeakKrca 3810 7782 1.5171 Fig. 3 - 1 - 2 Calibration, curve ofLC-MS analysis (Peak 1). September 5,2003 Kanae . {ft Study No. 44102 14 Company Sanitized Does not contain TSCA CBI 44102a04 SIR of 3 ChannelsES+ Flg.3-2-1 Mass fregmentcgram of LC-MS analysis for calibration curve (Peak 2) Study No. 44102 15 Company Sanitized. Doos not contain TSCA CBI Study No. 44102 y - 39958* r * 1.00 (kmwntottfori (mg/L) 0.0230 0.0500 0.100 Peakare 1018 2030 3975 Fig. 3 - 2 - 2 Calibration curve ofLC-MS analysis (Peak 2). September 5,2003 Nam* <j\ - Study No. 44102 16 Company Sanitized. Doss not contain TSCA CBfl ooivent 44102a04 2 i May2003 SIR of 3 Channels S5+ /"N Study No. 44102 17 Company Sanitized. Does noi contain TSCA CW StaiyNo. 44J02 ty*-*400.94999* Con(0mc.e1sn0tf0rtattion 0o.a4o0o0 M kent 384748 1609 Fig. 3 - 3- 2 CalibraiioncurveofLC-MSanalysis (Teak3). September S, 2003 Name / f i tfH o JU Translation: Language Services F. Metreaud December 3, 2003 Study No. 44102 18 Company Sanitized. Does not contain T5CA CBf