Document rxKOZBeJ4kbx11nOXXNpkQq0E

BIOACCUMULATION TEST SUBSTANCE Identity: [2-(N-Ethylperfluorooctanesulfonamido) ethyl acrylate; may also be [reetfheyrleldhetpotaadsecBa1f2l2u8o,roDo-c1t,yljEstuFlfOoSnEyAlj,amoirnoFlXe-t1h3y. e(s2t-erP,roCpAenSoi#c 4a2ci3d-,822-- 5) Remarks:`mMoarteerbiaasleids aonn ainmfboerrmastoiloind.sRupepploiretdsbtyatSepsotnhsaotrs.amTphleeppuurriittyyiiidse9nt9it%y oofr the test substance cannot be substantiated. As presented in the report, the structural formula indicates that "purity" cannot be assigned as "99% or more. Lot number 101 METHOD Method/guideline followed: MIT described in "Chemical Substances Control Law' (Japanese Law No. 117, 1974). OECD 305C. GLP (Y/N): Yes YEexapro:su1r9e95period: 8-weeks Exposure concentrations: 0.01 and 0.1 mg/L Test species: Cyprinus carpio RESULTS Bioconcentration Factors:Week2 0.01 mg/L exposure: 04 duplicate 04 0.1 mg/L exposure 3 duplicate 3 ~~ Week4 06 07 4 3 Week 06 04 2 2 Weeks 05 05 2 2 CONCLUSIONS Neither mortality nor abnormalities in appearance or behavior were observed in the treated and control fish during the 8-week test period. The test substance is not bioaccumulative in carp under the prescribed test conditions. 000545 Submitter: 3M Company, Minnesota, 55133 Environmental Laboratory, P.O. Box 33331, St. Paul, DATA QUALITY Reliability: Kiimisch ranking 3. The purity/identityof the test substance cannot be substantiated. The pexotsrsaicbtlieont/oankanloytwiciaf lthmeettehsotdcoolmogpyouhnads not did been validated therefore not bioconcentrate in the itis fish not or simply twhaescnhortomeaxttroagcrtaapbhliecbpyetahkesmeeltuhtoindgolforogmy "o1f ttohe3emxipneurtiemse"nta.reTchoensriedpeorrtedsttaotebes.that metabolites. While this statement does this assumption is cannot be justified. not change the conclusions of the report, OTHER Last changed: 5/22/00 000546 M.S. I. REPORT No. 2B1216G Bioaccumulation Study of Sample D-1 with Carp ( Cyprinus carpio ) Submitted to : SUMITOMO 3M LTD. Prepared by : Mitsubishi Chemical Safety Institute Ltd. Nov. 30, 1995 000547 DECEIVE 1C] ww 2c ne TRANSLATION OF THE TEST REPORT Study No. Study Title Study Period Sponsor 2B121G carpio) Bioaccumulation Study of Sample D-1 with Carp ( Cyprinus From May 18, 1992 to Sept. 24, 1992 SUMITOMO 3M LTD. This test was conducted in Yokohama Laboratory, Mitsubishi Chemical SafetyInstitute Ltd. (M.S.1.), 1000 Kamoshida-cho, Aoba-ku, Yokohama 227, JAPAN. The original test report was written in Japanese and this report was translated into English. The persons, the undersigned, hereby declare that this report reflects faithfully the original report as much as possible to our Knowledge. Translator from original Japanese version ; Fodor Sagerlls Mov 30,1275. = ; | /7 Tadayoshi SHI , Ph.D. Date Chief Research Scientist of Environmental Sciences Division Sele [inimie. Shoko TANIMOTO, B.Sc. Date Scientist of Environmental Sciences Division Approved by me 36908 7E odS asze. JHeuand of IYVAo,kohama Laboratory Me de 1250 Date 000548 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARD Study No. Study Title Sponsor 2B121G Bioaccunulation Study of Sample D-1 with Carp ( Cyprinus carpio) SUMITONO 3M LTD. To the best of our knowledge and belief, the study described in this report was conducted in compliance with the Good Laboratory Practice (GLP) regulations recognized by Basic Industries Bureau, Japanese Ministry of International Trade and Industry (MITI). Yokohana Laboratory, Mitsubishi Chemical Safety Institute Ltd. * (M.S.I.) * The co(mpFaonrym'esr nnaamnee w:asMicthsaunbgiesdhio-nkaOscetiobeIrnst1,itu1t9e84.of Toxicological and Environmental Sciences ) Test Facility Management Sakae KOIKE, B.Sc. Head of Yokohana Laboratory Sealed Date : Sept. 24, 1992 Study Managenent Tadayoshi SHIGEOKA, Ph.D. Sealed Date : Sept. 24, 1992 Chief Research Scientist of Environmental Sciences Division Study Director Ayao NISHIKAWA Sealed Date : Sept. 24, 1992 Scientist of Environmental Sciences Division 000549 We, the undersigned, declare that the work was performed according to the procedures herein described, and this report provides a correct and faithful record of the results obtained. Experimental Scientist Yasuo SATO Sealed Date : Sept. 24, 1992 Scientist of Environmental Sciences Division Naomi SAEKI, B.Sc. Sealed Date : Sept. 24, 1992 Scientist of Environmental Sciences Division 000550 `QUALITY ASSURANCE STATEMENT Yokohana Laboratory, Mitsubishi Chemical Safety Institute Ltd. Study Title Bioaccumulation Study of Sample D-1 with Carp (Cyprinus carpio) Study No. 2B121G We hereby certify that the above study was performed in compliance with the Good Laboratory Practice (GLP) regulations recognized by the Basic Industries Bureau, Japanese Ministry of International Trade and Industry rT). Dates of inspections and findings reported to the Study Director and the Facility Managenent are as follows: In-progress study i re Final reportreese IDnastpeesctoifon ReDpaotretsinogf JMuanye 2236,, 11999922 July 21, 1992 JMauyne 2236,, 11999922 July 22, 1992 Sept. 24, 1e 992 eeSept. E24,e1992 Quality Assurance Staffs Junko KATOH, Ph.D. Yuniko MIURA, B.Sc. Funio YAMAUCHI, Ph.D. Sealed Date : Sept. 24, 1992 Sealed Date : Sept. 24, 1992 Sealed Date : Sept. 24, 1992 000551 Table of Contents Sumary ester page an Sts 8 1 Introduction -- soonest essa m--------_ 1.1 Study title 9 1.2 Objective 9 1.3 Testing Method 9 1.4 GLP compliance 9 1.5 Study Period 9 1.6 Storage and retention of records and sample 2 Test Substance -.....- pert 9 ----------------l 2.1 Chemical nane, Structural fornula, Molecular formula and weight 10 2.2 Physicochemical properties 1 2.3 Source 1 2.4 Confirmation of test substance un 2.5 Stability of test subsance under storage conditions 3 Materials - I asses --- 1 onc? 3.1 Apparatus for bioaccunulation test 12 3.2 Apparatus for analysis 12 3.3 Reagents 12 4 Analytical methods ----- sn . WA -- 4.1 Gas chromatographic (GC) conditions 13 4.2 Calibration curve 13 4.3 Analytical method of test substance in test water 1 4.4 Analytical method of test substance in test fish 15 4.5 Blank test 1" 4.6 Recovery test 1 4.7 Detection limit 18 5 Acute tOXICity to Fish ewer ren 19 5.1 Preparation of test solutions 19 5.2 Test conditions 19 5.3 Results 20 6 Stability of test substance under the test CONditions.-wrwuorni2] 6.1 Stability in water 21 6.2 Stability in feed solution 21 7 BioACCUMUIALION test MEENO- ror vrorrevr evr <1 7.1 Test fish 2 7.2 Methods and conditions 000552 23 8 Results and Discussion remiss sesame iss ssstissmpstron ol 8.1 Circunstances that may have affected the reliability of the test results 8.2 Biological observations of test fish 2% 2% 8.3 Concentrations of test substance in test water 2% 8.4 Concentrations of test substance in test fish and bioconcentration factors (BCFs) 2% Table 1 ~7 and Figures 1 ~18 Appendix-1_ Infared spectrun of test substance (Data provided by the sponsor) Appendix-2 GC chromatograns (Analysis of water) 28-55 1 page 17 peges 000553 `Sponsor Study Title Study No. Summary + SUMITOMO 3M LTD. : Bioaccumulation study of Sample D-1 with carp ( Cyprinus carpio ) 1 2B121G Test Substance : Sample D-1 Test Period + From May 18, 1992 to Sept. 24, 1992 Testing Method : Testing method of Ministry of International Trade and Industry (MITI) described in "Chemical Substances Control Law" (Japanese Law No.117, 1974). re (OECD testing guideline 305C) Supply of test water : Continuous flow-through diluter flow rate : 1600 L/ day Nominal concentration of Sample D-1 in test water : High exposure level : 0.1 ug/ml Low exposure level ; 0.01 ug/ml Analytical method : GC analysis after pre-treatment Results + Bioconcentration factors (BCFs) obtained in thisstudy Bioconcentration factors (BCFs ) High exposure level,sample fish No.1 0.6 Low exposurelevel,sample fish No.1 3 No.2 3 Conclusion 000554 On the basis of the above results, it can be concluded that the test substance is not bioaccumulative under the prescribed test conditions. I. Introduction 1.1 Study Title Bioaccunulation Study of Sample D-1 with Carp ( Cyprinus carpio ) 1.2 Objective The objective of this study was to evaluate bioaccunulation potential of D-1 for application of a new chemical substance in Japan. 1.8 Testing Method Testing method of Ministry of International Trade end Industry (MITI) described in "Chemical Substances Control Law" (Japanese Law No. 117, 1974). (OECD testing guideline 305) 1.4 GLP Compliance The study was conducted in accordance with the Good Laboratory Practice (GLP) regulations recognized by Basic Industries Bureau, Japanese Ministry of International Trade and Industry (MITI) 1.5 Study Period Fron May 18, 1992 to Sept. 24, 1092 ( exposure period : From May 26, 1992 to July 21, 1992) 1.6 Storage and retention of records and sanple Following records, raw data, and sample will be retained in the archives of MITES for 10 years after submission of the final report. After this period, the sponsor will be contacted for approval of the disposal of any data. Data will be retained in the archives for a further specified period at the sponsor's request (additional fee). 1) The protocol and its amnendnent record. 2) The final report. 3) Raw data. 4) Quality assurance reports. 5) Test substance ( ca. 2g ) 000555 6) Other documents required by MITI GLP regulations. 2. Test substance : 2.1 Chenical nae, Structural formula, Molecular formula and weight. Chenical name *: 2-(N-Ethyl-t-perfluoroalk(yCl= 1~8)sulfonylaminolethylacrylate {Abbreviated nane: Semle D-1, identificationNo.281216] Structural foraula *: Ca Hs 0 Ce Fra $0s N=CHs CH: 0-C-CHmCHs n=1~8 n=8:ca 8% n= I~7: ca 21% Molecular formula: CisHi:0 NSFur (n=8) Molecular weight * : 625 (n=8) (+ Data fron the sponsor) 2.2 Physico-chenical properties Appearance + amber like wax Melting point +: 27 ~42C Boiling point * : ca.150 C ( lnalig) Solubility : vater $C 01% (BC) methanol i> 05 % (25) acetonitrile : > 5 % (25C) acetone i> 50% nhexame i> 5 % (25C) HE i> 5 % (57) 150 + insoluble Infrared spectrun shown in Appendix-1 (+ Data from the sponsor) 000556 2.3 Source (1) Supplier (2) Supplied quantity (3) Date of receipt + SUMITOMO 3M LTD. : 50 g April 1, 1992. (4) Lot. No. * 1101 (5) Purity * $299 % Impurity * (* : Phenothiazine 60 =20ppm Hydroquinone monomethylether 170 + 35ppm provided by the sponsor ) 2.4 Confirmation of test substance Before starting the study, infrared (IR) spectrum of Sample D-1 was measured. The spectrum(Fig. 1(p.28)) was compared with the IR data provided by the sponsor (Appendix-1) and was confirmed the identity. And it also corresponded to the structural formula shown by the sponsor. 2.5 Stability of test substance under storage conditions At the termination of the study, IR spectrum of Sample D-1 was measured and the spectrum (Fig.2(p.28)) was compared with the IR spectrum obtained at the start of this study (Fig.1). Test substance was confirmed to be stable under storage conditions stored in the refrigerator during the test period of ca.2.5 months. ! 000557 3. Materials 3.1 Apparatus for bioaccumulation test The flow-through test system is shown in Fig.3 (p.29 ). [ For test agruarium ] Dilution water supply pump Feed solution supply pump : Yamazen (FMI) Co., QD-2CSC : Yamazen (FMI) Co., QG6-RHOOSTY [ For control agruarium } Dilution water supply pump . : Yamazen (FMI) Co., RP-D-2CKC Feed solution supply pump : Tokyo Rika Kikai Co., GMK-8 3.2 Apparatus for analysis Infrared spectrophotometer Homogenizer Shaker Rotary evaporator Aspirator Electronic balance Gas chromatograph + Parkin Elmer Co., Model 1640 + Nihon Seiki Kaisha Ltd., AM-8 : Taiyo Kagaku Kogyo Co., SR-II Buchi Ltd., RE-111 Yamato Kagaku Co., BP-51 Mettler Ltd., PL1200 + Shimadzu Ltd., GC-14A 3.3 Reagents Acetonitrile + Kokusan Chemical Co., Guaranteed Reagent Acetone : Junsei Chemical Co., GuaranteedReagent Chloroform Junsei Chemical Co., Guaranteed Reagent Ethyl Acetate : Kokusan Chemical Co., Guaranteed Reagent Tetrahydrofuran (THF) Kishida Chemical Co., GuaranteedReagent n-Hexane : Kishida Chemical Co., GuaranteedReagent Methanol 000558 sodiun chloride Junsei Chemical Co., Guaranteed Reagent + Kishida Chemical Co., Guaranteed Reagent Sodium sulfate, anhydrous : Kokusan Chemical Co., Guaranteed Reagent 4. Analytical methods 4.1 Gas chromatographic(GC) conditions Column: Shimadzu Lt,dWi.de Bore colum, CBP20-H25-100 0. 53amx 25m Temperature : Colum; 130C Injector; 200C Detector 240C Flow rate of N + 20al/min Range 20 Attenuation 21 Chart speed + Sm/min Injection volune : 3ul 4.2 Calibration curve Standard solution of 1000 4g/nL was prepared by dissolving 100 mg of D-1 in 100 nl of n-hexane and this standard stock solution was diluted with n-hexane to prepare 0.25, 0.50, 1.004g/nL n-hexane solutions of D-1. These solutions were injected into GC described above. The GC peak areas( x V - sec) were plotted against the concentrations of D-1. The GC chromatograms and the calibration curve thus obtained are shown in Fig.4(p.30), and Fig.5(p.31), respectively. Quantity of D-1 was calculated from the total area of peaks at 3.9 and 4.5 min. on the chromatogram. The calibration curve is straight line through the origin and its correlation coefficient is 1.000. D-1 was determined with the ratio of area on GC chromatograms against the standard solution (0.5 g/nl) per measurement. 000559 4.3 Analytical method of test substance in test water Analysis was performed according to the following procedure. Test Water 1000 nl (100 ni) "--SCohdliourmofocrhnlor8i0denL25( 1g(0 n5tg) )*+ Extract with shaker (Sain. ) Soars Tr Chloroforn 80 aL( 40 )* Extract with shaker (snin.)* Aqueous layer Discard Chloroforn layer Sodium sulfate anhyd. 40 Filter ( 1163 glass filter ) Evaporate to dr N-Hiexane Dilute to 20aL [3 (7) + High exposure level 000560 4.4 Analytical method of test substance in test fish Analysis was performed according to the following procedure. Repeat once again [resi]s Test Fish (ca. 25g ) Ni Cut into pieces Acetonitrile 80 mL Homogenize, 5 min. ( 10,000 rpm) Filter by suction ( Filter paper S-60) Discard Deionized water 500mL Sodium chloride 25g <n-Hexane 100nL. Extract with shaker, 5 min esetT1eaamnes Tog bist [Caceromie aw Extract with shaker, 5 min. Astaire Tage Discard Evaporate to dryness n-Hexane Sal Dissolve 000561, [ amine) on next page] n-Hexane solution mL Column chromatograph; SEP-PAKTM cartridge(Florisil) * Elute * Waters Co. [Gor] +n-Hexane/Ethyl Acetate=8/2, 10mL Elute [er] ,n-Hexane Dilute to 20ml GC 000562 4.5 Blank test (1) Hater The blank tests were perforned twice according to the procedure shown in 4.3, using 1000 aL of the test water in the absence of D-1(Control aquarium). There was no peak which disturbed the analysis of D-1 in GC chronatograns fromwater(Fig.6 (p.32)). (2) Fish The blank tests were performed according to the procedure shown in 4-4, using the each two untreated test fish with D-1 (Control aquariun) +at the biginning and the termination of the exposure period. There was no peak which disturbed the analysis of D-1 in GC chromatograms from fish(Fig.7(p.33, 34)). 4.6 Recovery test (1) Water To ml of acetone Solution of D-1 (5 g/aL) was added to 1000 al of the test water in the absence of D-1. D-1 disperse solution of 0.01 ,g/nl (the same as Low exposure level concentration in the test water) in the test water was thus prepared and the recovery tests were performed twice according to the procedure shown in 4.3. As shown in Table 1(p-35)and Fig.8(p.36), the mean value of the recovery ratios was 94.6%. Analytical values of the test water were corrected by this value. (2) Fish 000563; Two mL of acetone solution of D-1 (5ug/aL) were added to the untreated test fish with D-1 and the recovery test was peformed twice according to the procedure shown in 4.4. In case that a fish body weight is 25g, the concentration of D-1 in fish would be 0.3 g/g and correspond to 30 times of that in the test water. As shown in Table 2 os) ana Fig. (p-37), the mean value of the recovery ratios was 99.2%. Analytical values of the test fish were corrected by this value. 4.7 Detection limit There was no peak which disturbed the analysis of D-1 in GC chronatograns from blank water and blank fish. Therefore, the area of the mininun detectable peaks, which can be recognized visually, was set to be 30,000 4V + Sec.. This value corresponds to ca. 0.02 ug/nL(D1) on the calibration curve. Detection limit is calculated according to following calculation formula and conditions. The results are as follows; Fish: 0.024g/g Water : High exposure level 0.004 g/L Low exposure level 0.0004 g/ml. It would be possible to calculate bioconcentration factors (BCFs) of following values in the case of fish weight 20g. High exposure level ( 0.1ug/il) : > 0.2 times Low exposure level (0.01 g/ml) : >2 times [Calculation formula) Detection limit = tChoencfeinnatlratsioolnutiionn ~x VfionlaulneSoofluttiheon ~Amsoaumpnlte of (Condi tions] Fish: Volume of the final solution Amount of sample (fish weight) Vater High exposure level ; Volume of the final i Amount of sample Low exposure level ; Volume of the final Anount of sample 000564 solution solution 20 al, 0g 20 nL 100 nl. 20 nl 1000 aL 5. Acute toxicity to fish The static acute toxicity test of the test substance to fish was conducted according to the Japanese Industrial Standard Method "JIS K 0102-1986, Industrial Waste Water Testing Method, 71. Acute toxicity study using fish". The result obtained in this test were used as a reference for determining the nominal concentrations to be used in the bioaccumulation test because the nominal concentrations of high and low exposure levels should be lower than 1/100 and 1/1000 of 48hr-LCS0, respectively. 5.1 Preparation of test solutions Four nL, of 400mg/nL THF solution of HCO-20 was mixed with 4nL of SOmg/mL, THE solution of D-1. After evaporation of THF in the resultant solution by blowing Na gas, it was mixed with HCO-20 solution(s4g. in 100nL, water) and dispersed. This dispersed solution was diluted to 2L with declorinated water (Yokohama municipal tap water was treated ith activatedcharcoal )to prepare 100 ug/ml solution of D-1. (control level ) Eight g of HCO-20 was dissolved with 100 nL of deionized water and diluted to 2L with dechlorinated water. 5.2 Test conditions (1) Test fish: Orange Source i Date of receipt i Lot Yo. i Killifish (Oryzias latipes ) Miyazawa Fish Farn(1323, Suenaga, Kawasaki) Marh 3, 1992. 00565 926-0303 Takatsu-ku, Body length i approx. 2m Body weight i approx. 0.2g (2) Acclination : Aquarium No. i C5 Water temperature : 25:2 C Period i Over one week Feed 3 Tetramin (Tetra Werke, Germany) Fish were fed once a day except holidays and not fed 48 hours prior to the test. Percentage of mortal fish during one week befor test less than 5% (3) Test conditions : The number of fish i 10 per one aquarium Volume of water i 2 L Temperature i 2U.4~25.4C Duration i 48 hours Dissolved oxygen : 4.2~7.4 ppm Aeration i Test water was aerated during the test period, Renewal of test water ; non 5.3 Results The 48hr-LCso value of test substance was more than 100 4 g/nl as shown in Table-3 (p 35). 000566. 6. Stability of test substance under the test conditions 6.1 Stability in water (1) Preparation of test solution The feed solution of high exposure level (concentration of D-1: 4.0ng/nl) was prepared by the procedure in 7.2(1). Then, 2.5nL of this solution was diluted to 1000nL with deionized water. It(concentration of D-1: 10 ug/aL) wes used for the stability test of D-1 in water. (2) Method of measurement The solution prepared by the procedure shown in 6.1 was left under room conditions, and Sal of this solution was withdrawn and diluted with 100 nl of water on days 0, 1, and 2. Each of then was pre-treated by the procedure in Sec.4.3 and diluted to 100 mL with n-hexane. The resultant solution was analyzed by GC and variation of the test substance concentration with tine was evaluated. (3) Results Typical GC chromatograns are shown in Fig. 10(p.38) and the variation of concentration is shown in Fig.11(p.39). Based on the result that test substance remained 104% (9.5 ug/ml -- 9.9g/nL) of initial concentrations after 2 days, it is concluded that D-1 is stable in water. 6.2 Stability in feed solution (1) Preparation of test solution The feed solution of low exposure level (concentration of D-1: 400 4g/ nl) was prepared: by the procedure in 7.2(1). 000567 (2) Method of neasurenent The test solution prepared by (1) was left under the room conditions. At days 0, 3, 7, and 14, 2.50L of the test solution was withdrawn and was diluted to 50 aL with acetone. Then each of 2.50L of these solution was withdrawn and was diluted to 100aL with acetone, and analyzed by GC. Variation of the test substance concentration with time was evaluated. (3) Results Typical GC chronatograns are shown in Fig. 12(p.40) and the variation of concentration is shown in Fig.13(p.41). Based on the result that test substance remained 99% (3944g/ml -- 3904g/mL) of initial concentrations after 14 days, it is concluded that D-1 is stable in feed stock solution. 000568 7. Bioaccumulation test method The study was performed in accordance with the testing method of Ministry of International Trade and Industry (MITI) described in "Chemical Substances Control Law" (Japanese Law No.117, 1974) 7.1 Test fish Carp( Cyprinus carpio ) were purchased from Sankyo Suisan Co. (1-1 Ichigayatamachi, Shinjuku-ku, Tokyo) on December 6, 1991. Prior to the initiation of the test, these fish were acclimatized over a week under the test condition (water temperature 25 +2 C. fed with commercial pellets once a day) and the mean body length of them is ca.11 cm. Mortal fish were less than 5 % during the acclimation of a week prior to the introduction of fish into the testsystem. 7.2 Methods and conditions (1) Preparation of feed solution - High exposure level The stock solution was prepared by dissolving 2.0 g of D-1 and 80 g of HC0-20 in 500nL of THF. ( Conc. of D-1 : 4.0 mg/mL, Conc. of HCO-20: 160 mg /mL ) + Low exposure level - Fifty mL of the feed solution of high exposure level was diluted to - Control aquariun 500 mL with THF (Conc. of D-1 : 0.40 mg /nL, conc. of HCO-20 : 16 mg/mL). 000569 The 160g of HCO-20 was dissolved in IL of THF(stock solution). The 3700L of this solution was diluted to 11 L with deionizedwater. (Conc. of H00-20 : 5.4 mg /aL ). 11, 1u04 sorutions were renewed every 7 -10 days. (2) Preparation of test solution The feed solution prepared by the procedure shown in 7.2(1) as supplied to mixing tube by feed solution supply pump and dilution water (dechlorinated tap water) wes also supplied to the same tube. Then these were mixed and the concentration of test substance reached roninal value. The test solution in mixing tube was supplied to an aquariun, Dilution water is the tap water of Yokohama city which was dechlorinated with activated charcoal. (Fig.3 (p.29)) ) Conditions Flow Flow Test 000579 rate of feed solution : 40n L/day for high and low exposure levels and 1.2 L/day for control level rate of dilution water : 1600 L/day (The turnover rate for each aquarium : 20 tises/ day) fish : Carp (Cyprinus carpio,Lot.No. 91-K-1206, aquarium No. 85) Body weight :ied0 g -Body length lie 12 en +Fat content of fish : 3.7 % ( n=, 3.3~ 4.3%) Water temperature: 24.2~24.7C Dissolved oxygen 4.9 ~ 8.2 mg/L -Feeding : Fish were fed with commercial pellets (Hinipet (kyorin Co. Ltd.)) once a day except holidays. Nominal concentration of D-1 in water : High exposure level 5 0.1ug/nl (conc. of HCO-20, dng/oL) Low exposure level ; 0.01 ug/ml (conc. of HCO-20, 0.dng/aL) Control i 0 ug/ml (conc. of H0-20, dmg/L) +Population density (at the biginning of the exposure period) : High exposure level : 15 fish / BOL test water Low exposure leve ; 15 fish / 80L test water Control i 12 fish / 80L test water Exposure period : 8 weeks (4) Introduction of fish, biological observation and environmental control The test system wes operated for 4 days prior to the introduction of fish to confirm the stability of the concentration of D-1 in water. After that, fish were introduced into the test system. Fish vere observed for mortality, behavior and some abnormalities twice a week. Dissolved oxygen and temperature of test water were monitored twice a week. These results were recorded. (5) Analysis of D-1 in test water Aliquot of test water was withdrawn twice a week and was analyzed by the procedure shown in 4.3. : (6) Analysis of D-1 in test fish Three fish were collected in 2, 4, 6 and 8 week after exposure. Two of them were analyzed by the procedure shown in 4.4, * Flow rate of dilution vater was 800 L/day in first 3 days , but because of concentration decrease of D-1 due to uptake by fish, it was changed to 1600 L/day after the day for each aquaria. 000571 8. Results and Discussion 8.1 Circunstances that may have affected the reliability of the test results. There was no significant matter that may have affected the reliabiliotfy the test results during the test period of 8 weeks. 8.2 Biological observations of test fish Neither mortality nor abnormality in appearance or behavior was observed in treated and control fish during the test period of 8 weeks. 8.3 Concentrations of test substance in test water The results are shown in Table 4, 5(p.42, 43) and Fig. 14, 15(p.44). (Appendix-2) The mean values during the exposure period were 0.092 &/1L ( high exposure level ) and 0.0082 g/nL ( low exposure level ). The decreases of D-1 concentration In both levels were observed at 3 days fron the initiation of test. This phenomena will be attributable to uptake of D-1 by fish. Therefore, flow rates of the feed solution for both aquaria were increased by a factor of two, and as a result, | each concentration is approached to each nominal value. The coefficients of variation are 8.4 % ( high exposure level ) and 13.5 % ( low exposure level ), respectively. 000572 8.4 Concentrations of test substance in test fish and bioconcentration factors(BCFs) The results are shown in Table 6, 7 (p.45, 46) and Fig. 16 ~18 (p.47~ 55). Concentrations of D-1 in fish are 0.002~0.062 ug/g ( high exposure level ) and 0.013 ~0.034 g/g (low exposure level ). The BOFS are 0.4~0.7 ( high exposure level) and 2~4 ( low exposure Level). Peaks observed at the retention time from 1 min. to 3min. will be considered es metabolites of D-1. On the basis of the above results, it can be concluded that sample D-1 is not bioaccunulative in carp under the prescribed test conditions. 900573 Fig.1 Infrared spectrum of D-1 (before starting the study) 286 win 0305 ule su wos isles saw ---- > 1 = i|Y) i Fig.2 Infrared spectrum of D-1 (at the termination of the study) Wz ols wis nie sed lm wie i [TY Tr w- eT ia Vert Veo ly 000574 Ve1 o 5 Vie Vio, Verso, Veet. Fig. 3 Bioconcentration test system feed solution supply pump dilution water supply pump 1 1 1 tfaenekd solution 1 EEE , mixing tank aeration [1h : oT acchtairvcaotaled dilution water tank (dechlorination) 25%2C slehon | glass aquariun 85 (amount of water 8 0 2) 000575 1 waste treatment branr usesirnG wo peak 5:34he sos Edxs 2 zg = &- g s8 5g3zi ow vie aes mc te cou EI oLenSasm esess [cjoeree = gsxs o 5 zn ee s ewes v 2T7i.eee0ss = iag stant oe sees neesests To "4sesseaneets zg g 3 ser 8 4.558 " s ow vine area mc me cone 5 F Less A asses e1a3r0s 2 H SiwmrarwrrliiTioy 7a855s06 fo. oa assess To 00 wo ser = Toe ow vine akea mc imo cone g3sg Fo S1R olg+ ea.9n2a 8 s m 4 4016y e 5 some tora reese li2T.o6te2s9e3 = ier Toe 000516 Fig.5 Calibration curve of D-1 Y= 3.8338X 10% + 1.4473X 106 Xx! r = 0.99995 X 1000000 Calibration Curve 1.6 1.5 1.4 1.3 1.2 S11 > 1.0 2o.9 30.8 < 0.7 Sos % 0.5 0.4 0.3 0.2 0.1 0.0 0.0 0.2 0.4 0.6 0.8 Concentration(u g/ml) 1.0 1.2 X11 . "000577 START 2.365 Ter 3.528 PKHO THE Vases 23 3zA.mi3ee5rn START TOTAL AREA 1K 1DNO eee eiea1sr37se2es1se wv v seers cone 2.5362 73.1.57713478 17.1855 100 TS < 20000 pose HARNING HO PEAK TART So < 30000pV ee MARNING 10 PERK aZ 2 i =a 3 z = g x 28 7 gz " 00057% start 3388 oes FxHD Tine AREA e5 sszelsser 4 isss BI TOTAL ereamaieasn ussess 296595 2.907 HK 1DHO wv el2s5dd0 S3--.4--98< s0000pVosree Fino Tine eLeealsses Salas start Tata AREA WK ammo aSc0e5s3s v seas rsa cone 751l14078537 17.0524 109 cone c122.07831431 2515s rs 3 [5.i087 -- < 20000puV see. Fxno Tine e [EeElT s5 0 3sllseesr s S.087 AREA ne Ime frse1zn0 v se87s525 3477 ToTaL esas con 2175..5300e5s E 203t0s 19.7373 100 2 g e2 t3gL7 zs sg F 22 z2g3 :8 g> & 8B i = 8 5 - 000579 START 1:34 :0 co Hes s Frio Ting AREA mK 1DH0 conc x ei a2.l0a8n8 i1z3s0e7s9 ou 5 Eames ersaseers sv 21..35528545 2 2175..15503334 START TOTAL 810706 180 143% 4.208 PKNO TINE e: 1i.l5s3z5 3 ales START Total AREA MK 10ND as1s4e4s ssere 8746 . - conc 4150..13540262 s4laerz 100 143% 4.208 S-- 30000< sec Fino Tine 1 1.53 3 sailzsae: ToTaL AREA MK 1DKO 332265359 esses 67470 conc 31.1.23986224 33.3574 106 . 8 3 2g sSs g zggB B <5 g = g z 0005%0 [wT+Te5e Tol T+] Table 1 Recovery test from water amount of added amount. peak area cone. in | measured| recovery| Fig. final amount No. [Tm[ovo [ve|or a]wa CFoinnaclenvtorlautmieon(Vo)f standardsolution(Cstd. ) Calculation D=Cstdx B/C i E=DXV : 0.56 mg/mL 120 mL ; R=E/AXI100 [TaTo]coTvTw] Table 2 Recovery test from fish Fish added peak area conc. in measured | recovery| Fig. amount uV sec volume amount [e[lJseewsT[Tno Joooom J[woomm || voomr][vwo [|wwsm i|7]]] CFoinnaclenvtorlautmieon (oV)f standard solution(CStd. ) Average : 99.2 % : 0.5 pg/al +20. 0nL Calculation D=Cstdx B/C ; E=DXxV ; R=E/Ax100 Table 3 Acute toxicity test ( 48 hrs) ooossa[ D-1 ug/ml number of| dead fish mortality test fish x [oo|] 48hr-LCso= >100 xg/mL stakT 23 5:r 31% 22 2 4.593 e = Fan Time ea mc iDwo conc 2=: 2g : selemies a1mesn v 3 sa sem v 7821..1547764525735 g=Bb mm ons ov 17.2165 g star Total 796413 100 3 5:31% Tene 2.83 Frno Tie amen mc 1D es aelwmes aesnaw ov 3 2W.m29s8 5s9e38w9s8 vV start Total 783786 conc 21.7212757 7187..73023576 100 2:337788 Tees 2.508 Fano Tine ame we 1mw0 : esslsaiea ssesess ow I adeass seesses v Total 7as2re conc e15a3s33 7157l.i00a2z7s 16 000 SL START 2:38 See e51 asl2e.e9s3s ee21v03e3sae2rrS wv 4 Zora vw start Total s4sase F2rs.la3eg9as6z1e 17.2078 100 Tere 2.088 Fen Tine :i eealssis 3 iessse Ss Ase start Total aren wc inno 20ta1s6e6 essosnaeeee vw useless ses032 conc 2a.l3r3a6s6e 7a5.l3s3e5 17.74 10 8 g . & 2 Zz <= g ZS g2 g - # ses 2.898 PRHO, TINE AREA MK 1DNO CONC ~ eteealrss sasessess v 2L6e5s3s7 3 slsss 1ss7s vw Leer 4Swi3.m8s93 69i2s6se2re3 vo 7147..87905811 : Total e7erze 100 0c5%3 cra F230 Fo. ew : z z oe = eno Time ake meio cone we EF d[ o RSlEs PaTves ov FER odeJri cfoime 2 .g insss 23 star Total 790445 100 gg 8 EE tose g war Z Frio Tine AREA mk Tmo cone nave & = PLSeEaseew eTaase E. I$0 odHEaEe Theenee ov Watliees 35 "rant ow ries Tos 238 a 2 Tew z po Tne sea me imo cone we a LesB s EweE nn 2i.55e00 = LI55o0 aen ms w 7easlisieee stat ora reese To Lu s.92 fo vnc axes me imo Ss OoLEEeEsSsSe wNoiEsn ov dE we rans cone we 2 eaahn tai Te Fig.11 Stability in water : 3 w-- & Residual 60 v1 o @ 0 ;Tise(days) Z stan bith Tn ipr :g = pow Time kes mc deme conc we % = po oDSeEEmEEs aa ssetsasvov 2p32ii.ev3e0re9se7 2g& . ies __sssiz vw 17.92 2 . ors 7s oe z5 stone g 22 Ty pre o 8zgE po Tne eke we ime cone we 5 z iFVeeSaarne esene Scplaerteen 8 5 DoEEEE a ora 7raene I 5 san = 2 srt or sve poe Tee aca mow coe :22 wwe s8 s$1 oo3E2n.59t92 19238 ane v odEEEE i2t.s2i8i7e1 = fe stant ow sens To Nn a. on pro Tine ARTs me IDNO cone wane = iF LoeER ast rwleenvw fcLraerles PO vr 2 aE ora green To 000536 g5 o - e------------ a .i Table 4 Analysis of D-1 in test water (High exposure level) ee men sdaamptleing Talslels]] period uV sec cone. [mean | Fig. in |water [conc | No.. [TEomm[ oo| {{o ommn[amrm ooT os[ooomma]+r| [eee o [r|oo[maamoomm[ooom] | [ero]o[[roonaann owomu]oooon|]+1] [ o J Jw a [mnr noo]m 1|| [[ oo[oT ornwn[sm|alo owoomm |woomm|0] soros[[eoor[rronss[[maoloom[ooer|| [I oo3|0EErCmnfrom Eom] | o o o| [[ooweme ne[ron wo ooomm | [oo[ov[ron om[oa] | Concentration of standard solution ~~ Cstd : 0.5 ug/ml(+ Appendix-2 ) FSianmapllevovloulnuene Wviioo1o2000 nabl Recovery Ri 846 % calculation C=0Cstd xA/BXF : (F= V/W/(R/100)) 000588 E= = Ci/i (i: number of analysis) Mean concentration in water + standard deviation : 0.092 x 0.0077 ug/ml Tee Table 5 Analysis of D-1 in test water (Low exposure level) Sadmaptleing | peexproisoudre peak area | cionnc. || mcoenacn. || FiMgo.. uvVesee [water [0 | + | 0 | a| | 2 |esoser|eeesr|o.0oss o.o2o| I CE EETel [fe ERE 515797| 759199| 0.0072| wom 1] 0.00437|] |[mlwen[om won|0] o oe emow[rnls [oowmslwewon|m1+]] [I o 26 | s| o |[o1r0 rows|sesose f|sooseasm0Ea.00E0[0 wo0o.mm0[m0180|n0| FI CCO CNNCCWE 8Ci CCedn aECT C | 7m [ | s | 1s [sows] wrsas 0.0081 |0.001e6e|| CoFnicnealntrvaotliuonne of standard solution RSeacmopvleeryvolune Cstd : 0.5 (+ugA/plpendix-2 ) vWie: o0020 anl Ri 946 5% Calculation C=Cstd xA/BXF (F= V/W/ (R/100)) Q00589E= = Ci/i (i: number of analysis) Mean concentration in water standard deviation : 0.0082 + 0.0011lng /nL Fig.14 Concentrations of test substance in water High exposure level 0.2: as if 0.1 0.12 Concentration gol... -- NAonmailnyazledvavlauleue | RE -- (u@/) oa [x3 . oo ee aeoe 7 ua wm x @ Exposure period (days) Fig.15 Concentrations of test substance in vater Low exsposure level 0.020 0.018. 0.016 [en 0.02 CONCENLEBLION gy kre (ug/l) oom [x .000 0.002 PWT --~. NAonnailnyazledvvaalvleve mmm wa = me ew Exposure period (days) 000590 Table 6 Analysis of D-1 in test fish (High exposure level) [i] | o [ome Joon | oe| [wacl JoTe e J] e]| peexrpioosdure|| wfeiisghht| peak area | fcoinnca.l in||cionncf.ish|| cmoenacn. | BCE| YFiog uV -sec | volume [measured| vianter [or ma] |v | wor ro a) [2| 9956 [192018| vouzro| 0.1 |o2om2| 0.001[0.7[18-2] [[[r+oorseowss em[|e o||oew|oowrror [fvoossuJ[ii]]] |2|onan] niaes|soseos| oom| oss| 0.00 [0-4[183] CFoinnaclentvroaltuimoen of standard solution(Std.) Recovery CVsoitd : 0.5aug/ml R i992 % Calculation C=CstdxA/B i D=CXV/Wx10/0R F=D/E 000591 Table 7 Analysis of D-1 in test fish (Low exposure level) Ce TeTE] pexeproisoudre|| wfeiisghht| peek area | cfoinnca.l in|| cionncf.ish|| mcoenacn.| BOF uV sec |volune |measured| wianter [[Frru]aoosea]] oovse||| svwoon [Tsooomusr]] >[oe] [1|3128] osoe [so|soe.0o24s| 0.013 |o.ooee| 2[ust] [F isnoelor] ocor|[ros [[oowo]m i CFoinnaclentvroaltuinoen of standard solution(Std.) Cv std 5i0.n5 g/l Recovery R 99.2% Calculation C=CstdxA/B i D=CxV/WxI00 SR F=D/E 000592 Fig.16 Bioconcentration factors (BFS) of D-1 High exposure level 0 2 4 6 5 Exposure period (weeks) Fig.17 Bioconcentration factors (BCFs) of D-1 Low exsposure level | .a1 2 4 6 o Exposure period (weeks) 000593 Pom gm A 3 Abe PDE ici SLrLBoe we me wee we we owe we {i dE. ted Pane iE L 5:39 a P PEo Oa . . Uw8 =z g T: E&3 FF Sf ` = = : ro Cpeed me ene 5% 1B oa, DoE Teta 271sen Be HE we 00054 { 2887 a or HE si Smee 2 a = oe ai 1.23 i. = = ome wea we me com wane Do am sa Pon own Sa ii 58 . -- 2 |g en E50 g Lo i. : PoE wh - wom sees TTT 000595 L 3:82 cs o . m 5.07 o23esm + AEiE ran BR aor cernIs" EHD Te oe aween wewe mea [ert Tom Iomm ons [I WopEe--et woamemaRy atlas 567 Jin im Ti na z = = g | 85 fs 5F5L 3 % oF 23 Iz : gE 2 HE aise = a TE cho Sare=moem a oe wo Te we wee ame enc zi gFEw iti wom aE oe as ppeeed Th TTR 000596 [b5i5t so CEE S PoymmEey HE eidn SE pao Tome aca wow I wy 1.27 wend HE [ Lio F0 4m 0 dE brat Lm Ex oe es - cms nan er] 39.2078 si i fIo pee tPo ZH daens Awdhi 2 L| e es C LE 5 - = 2B : g2& = s : 2 wn PR Ee F PoSE mew PEE zi iid ewhhe [0 0059% HE oe eens i TAT peti Sw ooCo Tae wwees we Pm. cone wane ul SE lg 3 Bs cTag g z CTE 373 YDoamdoMse HiE ; we i wo cone ag Lomo sa I 1h - Fi or 00059% =_3:89 pn PE so Tora ana wea 1 M2 om TE Be : pr S362 AREA aire wi wo fwoi t Le Jide g c1 ig zd s: 38 3T:E 2 8 2:02 svt L oanems rmemso ieitn 5 ME A a rer 160 FiEY 3 mo. Tine akeq mx ten cone. ang CR : : ar wn s2.9609 = EE HE ore sine Jee po Ta 000 5% 218 -- wa Coden a Sm dew a Azsaa0ie Vv BE Tale 7al6057 LE yi Eaoom ene fiiiia PI mm i w EERE Ss 55 55 = g $3 5g 8 2T:g a g i ar Ss CPoomdemry boSwh : The ae oviT4la,n OT 53. ' 1.078 Tinos 4346 hd doowa nn VG odie ant tri w SESE Shes 000GUO Lp . LE ro a ah _ wa ener = TTT rr = oe em ane sir BEN bi Domes Ta em SA [i or wo }{ 38, aoiin, 3 SeiieliB0 vFiaei Lene Ar o 3 Li DEE. IE Jy DoE worsens Ro [i TTT 0060\ z >= uo g 5lg 2 sz 3 23 Tg 2 g z 2 = 8 Appendix--1 Infrared spectrum of D-1 ( Data provided by the sponsor ) ( 1 page in all ) 000602 oL r a int pg EO EfvE] ---- an sem ww. 72.708 proved hE wulBn uiNiaEs apiEnnz Ss 103000 13.00 REfmsn mm amp 20 I meter" Jorlol pHreL 22488 H4.3.27 MER Kwoiewit ApPppPpendix--2 Gas chrosatograns (Analysis of test water) { 17 pages in all ) 000604 start 32.:855.5 "sos no Te akea Leas ees 2H we3s.a3en2e2 ers1e2ee34rr2s start Tora 7sesss sn mc iw vwv con e.seis 731210.5l9e55i64a4 100 5:38 I. wo : Pro Tine ARER HK 1DMO i[R1E 2.3% lar Hw: starr total 17757 ssSiseees oy seesss cone fr2io.4n5e8d 11s 10s 2:35 wer PHO TIE kes Less ase 23 3od.a3ea35Enan1s2i0ee61r rota Gasser ss me im cons e.sere P11ai.le5s6re8ss8 Tos 000605 2 2 =T= . g5 .g "ki <2 g= 25 2 5 ; 2 g 2 = 5g2 g5= g 2 32 &2 start i Tor2Toes Fa Frito Tine Aken me neo Leasle i18s5as v s alsse egress v 4 4.533 13661s v oral sesdse START cone eVessa: 2203038 17.0621 10 3.358 ser 3.375 Frito Tine AREA mC: 1bwo 1 z.5es resin e 3.295 9315 3 4 Sv84 2eS36i1 ese2 ss 4 start Tota s7a030 c. one 2.23 1.6232 2172.32888053 13 5:23 ise 2.478 s.54e PKG Tine :Vo Cmalmis 3 2.82. 4Som4.5e52 AREA UK TpNg 3s0e3i4s 91045 v a3r4m4e5n 9wv Teva sseoes cone eVassees 74.2463 17C.i48a1n 105 000606 2 & 3 = o 5 a = fe ii 2 8 i 3 . 8z= z2 g2: =g e e 5_ . 2 5a 3 =  bast $555 q.577 3.39 Frio Tine AREA nk 1DNO dtaemsss 3 sr 4 4.522 riaseaens wv esses ov 133236 v start Total 791334 $517 Tae os Puno Tine e 1 sel si 45 Jziams Total start AREA HK DD daesess asaispezeess v 632293 conc 2T.i4s3s8 78.9728 16.8935 10 - cone 2V.i2s7a5s4 7175.c5a2s3e5 100 5:3iT4s95 5.850 Pico Tine :Veealarn 3 0 Lisessn Total AREA WK Tho n5e5s3s v wioissiesre wvv 538533 conc 21i1s0r7e2s 7175..6248763 160 0007 & & a bd Tsole goe g2 g = s = F) 5 c z 5 23 g :g2g 2z & start 2:40 Toe Fro TRE akEa 3:1 ss2l.ia9mm3ss sEesLneIsnTesEs Fr arrrrra start TOTAL 737320 s.338 me immo v 3:45 os 2.508 Fre TINE area me imo 21 3mlsseess iassessees v 5 laisme sassisgeess start oral 71530 cows 7231.715546237131 17.0453 108 . come 21.175357813 17alleessses 10 & szg Ig sFolEgze = |Z 8 = &=g g 5% . gc 5ig 35 2-383 "ss sees 5ggz rns TIRE akea nk mie cone 2 eLaessess s slsss ruasveerss ow arsesy 4 2i.ia1aes 3z 26lenee + Iss inns 13.062 Tora seeses 100 000608 brart 2:37 T.o% fn Tine eLaesrss 3 3.907 4 4.598 starr ora akea iwaeds 615105 1344328 seni 550 mk ioe vw v Y :35 se +603 Pao Tine sLeaalsat $0 GGlleeanm stant eral ekea mk too aaveess seuscsaee oovv cases conc Vieseio 78.788 17.1382 196 . conc faraens [13E.dR0sse To gd we : 0) Tine AREA mK 1DMO 3:1 sa2l.ls9ee5s. aw1dm4ii7se3ts3 owou Soames etal sasees cone E2T.i4es8s08 ies 109 000609 2 && s Pk2olosa ag RC &| 2 = zg : 5 2Z 2 E g5 H g t 5 3e: start 2:38 IT 3.958 Frio Tine Less 23Hv 33..396533 stant Total akes ok 1mHO res grs1i3e1es2i1 rvv seerse 387 Tees s.357 Pri 1 23 4 start TIE e.987 33.l336sr 4.563 Total AREA HK 1DMD 17961 s1w2s4e3s1e vv 146109 273214 cone e.eise 71812..a75i78s75e6 hm : cone 2.3289 771..16615844 13.8952 190 2 a &= xz - = soa 2= 3 z 2 i ==g HS=s | z < = 3T 2:38 Tees 258s =5 0) Tine AREA MKC IDNO Come 3 e seass aseereess c1a5sse =3 3 3.965 459404 V 77.3198 3 4 4.865 112648 18.9583 = Taal saares 199 9o06|0 START START 2:38 3.98 4.652 PiHD Tine AREA 1K 1DHO cone velazs 1i3s3e0 v 5Siesss ecseiiamzaeiiesss vov start Total 326305 21.le2n8s7s5 Le147l.e 2e5a3024 199 5:38 we 5 s.93 Prin Tine J @3 aleasn 4 dks start Tota AREA KC 1DND 14527 esasaesc) vvw oma ov e395? cone 2.3224 771.l2e3es8s7 1307723 100 38 3.688 3.997 Po TIE kes me ime @1 weveers a1n5i5s1e8 wv 4i ns.essssr ssescrie wvv Total 36380 cone 21.l2ee6s0s9 7187.19878075 190 a2 == on 3 3% a sog ~ &g =g 5 52g =g 2=2 zg5= e 2g2 g 8z 0o06l| start 352 Teor Pwo Tee oases esi aals2m.e9a52 see Total START e2is0s3see9s3 yewes esis 2.913 nc too ow 2.52 2:32 anes 3.623 Pwo Te :Venalnsa 3 dE 3.3288 staRT aL sees wk mo imsrss ow nv 567817 WV rasan 2 s5 cone 55 22P5.P54300s8 - 170422 190 cone eE.sY0ss 7T7e.1e85s3 190 SL 55 2 2 22 = 2.555 = =Teer 3.927 2: PRO Tine AREA HK 1DHO cone t 5 e51 a2l.l9a6r5 Pv ral s1es3s6e6ss7 v rr s3ssar e2il.ce2a9s4n7 TH 15.2479 100 z =z g2 = =z g | 2 z a 5= a z5g 0061 Tart cae Te 3.39% sere PRHO THE @31 2al.le9e3e3ss% ime start Total AREA 1K 1DHO ce2as1eeszseesss vv | inn ow sesien cone 72i5.r55aa4ee2e5 1201s 100 2s:39 w.91 4.608 Poi Tine akea mk mo eLsessess eaewrsa vw 3$0 i 3.9A)he m n575e 386 start Total 7arass 2.238 *I 3.913 Pano Tine 1 eae F 25 v 3i.s3i4s7. eral Rea mK 1bmo sss sreesrc7er4s2s wvv reese cone e.Visr0esser 7163..50638838 ey con 2.5505 R137..A5776Y234 120 " & a=2 3--- 5 g 3 gs go F8 g5a g g = = ga 5z g3 = gz 2s sz 2 F 8 start 2.9F835%e "aes uo Tine. ake mc imo conc "zs &x:& 8 2g4 eS1o E Ze3.ll9a9:s8 Esa1es9E me7e4er8 vy 7T3eVrl.is3e54oe2ene2 = EgEg3 stant TOTAL 343045 190 Ez : [EH . : 233 Tees 3.963 g 8 5o a: ve Tie Les anes wm imo ees cone esse g 5 : 2 3.375 148 y 1.5893 3 Dome 2 5.963 SS26EE0 V T77a.k22e7s2 = stant ora ress To Te 2.558 Ton fp -g eve wee wom one g [EVRE oeSHaeEg ares aln VETERE 2e3Fi7sv5ee0s0 g3:& Ties we ries Tos staRT 2.3% Tas 3.923 Fino Tine AREA mc tomo conc : seases aasmseens ow 5 Aalmsieer oreisseessesse ovwv start Total Sedo passin Le2147el.6s809163 1650 Tees :882 Tes 2.32 PRD Tine AREA HK 1DHD cL e e3lsae eanawn ow i3 EA.m%dn 68i5sEorss wW stakt Total srizre pTeest 5:38 Tes 2.522 Fens Tine FLR ass 5 Taosess Total Rea me ioe zreass v ssiesness vwv 7esare cone aoenrae 17371.3038284 100 cons 21iessaies 7182..0873344 150 @ & ac -i 5 g. = CElg Z35 =z --- z4 s2 52 5 2 3 8 g& 5 2 3@ s 00065 start 31 4.683 3.372 PRED Tine AREA MK 1DNHD iLo a zser smeeses w : 3 o 3.97a2 s6ai3zszs or aise START Tove 3:08 oon Toe puto Tine AREA MK to :S 1 isS.hierms seTaaansengs oars start ora gener cone 2p2o7e4s 7132..55008313 I cons ra2Sl.ie2er1e7e5 Tair Too 2:3 ee 5.973 BHO TIE ake me imme e | G eas Soom easns w Gah . ODER eran senna cons feceaomns Sl Tos 2 g a = : g = lBg 8 gg z z zge = 5 3 2 2 g s5: z- 2g g:g3 00616 eraer 2.: 5T0oot dss PRID Tine AREA MK TDD Lens z5 3s.l3a5ms 4 4.64 sens es1t3i2z3s5 v 1372306 TeraL | es337% START LLeLs Liesr Frio Tine ARER HK 100 ese eens 2 3.332 12636 3 i3E.9ss58es5e334r58 ovVv start votaL | raasar Tose ig 4.757 ez Pieo Tine eeeA mknx mo d1 aissss 5 alse 110a316s41 aeess v Enea 4 Tonal essen cone esa re1l.s6i47e5e 17.1658 190 cone e330 1.8012 716a.e5i8e9s3 100 conc 22e7.2i11ss Tense 10 2 &zz r=i lg 322 5 22 2 = E = :> =5z g z 33z 3g 5 3 = 5- . %3 :g2 00067 stakt Loess 3:438 Tee "10s Poo TINE kes mc toe i51 aS3l.hl0ae9e8s sei1s9d2we0es2 ovv ama start ToTAL 540505 Ton 3:83 Cees Re Po vine area nk me 1 3.092 18955 ela e E ees wv 3 4.937 534606 WV start oral 774878 Toe L9 ers 4.037 wre en we mmo eUlne e So Lee spisesn 9 sin . Lm Cw sree conc 2eTi.52s8ie4s3 Tress 100 cone 2T.e4a4s87 7T6e.l8s1se5r 100 cons bcrpasss p1e7lb5eice Tao g = Z=7 gF 2= - s gao 52 -g 23 as5 >2 552 g: 3g = = 5g 2 58g Qo06lg starr Tee cs 34T443ov 4s ono Time ames me ime d1 as2.1s18 3 4.115 4 A347 1s2e1e3s6 wv 4531) v 146394 "START TOTAL 324665 Tost 32:.43383 ee 41 PKNO Tine AREA NK 1DHO eS1 laesnase stseeensssss ovu oo odnmeEEE starr ora rican Tous esas 3:88 wus 4.848 PRNO Tine AREA HK 1DWO :LosSelsses 5 afin rsesiees easde ov i Sanne ora szaser cone 2[.s1e9n9s2 78.2513 17.7277 108 conc P2Ti.0ns72e6 Tassie Teo cone cToeseen [135a0s Teo " 2 &2> s& x5 52 &g = 52 2g | 2 =3 za 3a = 22 5- Ld 2 i _ :2 24 3 g2 ooo 6lY START Pima e1 i3 START Tes 3:48% 3.848 an Tine AREA mk 1owe s3.l1s0?2 1az6e8e3s2 vy Laalmye esseeienss ovv TOTAL 04309 cons 2s.0a884 7137..03304938 189 g = =I 35 g2 - Ig<s B2E sg8 =z :488 Sean 4.108 = 7 2 a- Pius Tine aRga mk Towa conc 3 g :vo acloeess r2i7s6e4s v 5 ales sasic ov 21.307120s3 g 76.3407 4 Lae eesne 15.2714 START Total seasze 100 [ET 2.538 z TA.e0e 4.463 |2 Fiano Tine AREA WK oma conc 5 I mass isan 52 a2.l3e0s7 a1r3a7e5s4 ovvw 2.4539 g 727..22157458 2 4 ess sia v 13.0437 TOTAL seaeis 150 : 000620 START 3.898 a S707 @.988 FriD Tine AREA mK IDMO vo sees d 3 asyss ser START Total 17ess s1e1e8r5s8 vov iasren v sesest 133 382 ae "Fan : wm me sme momo :1 aaleass aasreszees wv 43 Tiasmsas suseesreiys vwwv 5 less jasers v START Total reser 1T7i75s 3:99 = 2.388 2) Tine AREA WK DMO evo asleavss zzoesases . +5 uiszsn a1s1e7a3i1l7 sv TOTAL eassa2 cone 276 751.44654031 relsars 100 ows 21.7325s22 7s2.a53r03 1817747 108 conc 33.12707631 7155..33108375 100 o a5 &0 > > = .2 Esg Eb 22 2 5: g = g : g 2 2s 3 H 2= 00062