Document 71a8NaQ0K3LeroQNrBmYZ5k0a

0 //5A R M - I BIOACCUMULATION TEST SUBSTANCE Identity: [2-(N-Ethylperfluorooctanesulfonamido) ethyl acrylate: may also be referred to as B1228, D-1, EtFOSEA, or FX-13. (2-Propenoic acid, 2[ethyl[heptadecafluorooctyl)sulfonyl]amino]ethyl ester, CAS # 423-825) Remarks: Material is an amber solid. Report states that sample purity is 99% or more based on information supplied by Sponsor. The purity/identity of 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: MITI described in "Chemical Substances Control Law" (Japanese Law No. 117, 1974). OECD 305C. GLP (Y/N): Yes Year: 1995 Exposure period: 8-weeks Exposure concentrations: 0.01 and 0.1 mg/L Test species: Cyprinus carpio RESULTS Bioconcentration Factors: Week 2 0.01 mg/L exposure: 0.4 duplicate 0.4 0.1 mg/L exposure duplicate 3 3 Week 4 0.6 0.7 4 3 Week 6 0.6 0.4 2 2 Week 8 0.5 0.5 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, Environmental Laboratory, P.O. Box 33331, St. Paul Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 3. The purity/identity of the test substance cannot be substantiated. The extraction/analytical methodology has not been validated therefore it is not possible to know if the test compound did not bioconcentrate in the fish or simply was not extractable by the methodology of the experiment. The report states that the chromatographic peaks eluting from " 1 to 3 minutes" are considered to be metabolites. While this statement does not change the conclusions of the report, this assumption is cannot be justified. OTHER Last changed: 5/22/00 000546 M. S. I . R E P O R T N o . 2 B 1 2 1 G Bioaccumulation Study of Sample D-l with Carp ( Cyprinus carpio ) Submitted to : SUMITOMO 3M LTD. Prepared by : Mitsubishi Chemical Safety Institute Ltd. 000547 Nov. 30, 1995 ,HflflVV7l TRANSLATION OF THE TEST REPORT Study No. 2B121G Study Title Bioaccumulation Study of Sample D-l with Carp ( Cyprinus carpio) Study Period From May 18, 1992 to Sept. 24, 1992 Sponsor SUMITOMO 3M LTD. This test was conducted in Yokohama Laboratory, Mitsubishi Chemical Safetylnstitute Ltd.(M.S. I. ), 1000 Kamoshida-cho, Aoba-ku, Yokohama 227, JAPAN. The original test report was written in Japanese and this report was trans lated 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 Tadayosm SHIGEDKA, Date Chief Research Scientist of Environmental Sciences Division Shoko TANIMOTO, B.Sc. Date Scientist of Environmental Sciences Division Approved by Head of Yokohama Laboratory Date 000548 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARD Study No. 2B121G Study Title Bioaccumulation Study of Sample D-l with Carp ( Cyprinus carpio) Sponsor SUMITOMO 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). Yokohama Laboratory, Mitsubishi Chemical Safety Institute Ltd. * (M.S.I.) * The company's name was changed on October 1, 1994. ( Former name : Mitsubishi-kasei Institute of Toxicological and Environmental Sciences ) Test Facility Management Sakae KOIKE, B.Sc. Head of Yokohama Laboratory Sealed Date : Sept. 24, 1992 Study Management 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 Yokohama Laboratory, Mitsubishi Chemical Safety Institute Ltd. Study Title Study No. Bioaccumulation Study of Sample D-l with Carp (Cyprinus carpio) 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 (MITI). Dates of inspections and findings reported to the Study Director and the Facility Management are as follows: In-progress study Final report Dates of Inspection May 26, 1992 June 23, 1992 July 21, 1992 Sept. 24, 1992 Dates of Reporting May 26, 1992 June 23, 1992 July 22, 1992 Sept. 24, 1992 Quality Assurance Staffs Junko KATOH, Ph.D. Yumiko MIURA, B.Sc. Fumio YAMAUCHI, Ph.D. Sealed Date : Sept. 24, 1992 Sealed Date : Sept. 24, 1992 Sealed Date : Sept. 24, 1992 000551 T a b l e of C o n t e n t s page Summary ..................................................................... 8 1 Introduction ............................................................ 9 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 9 2 Test Substance .......... -................................... -.......... 10 2.1 Chemical name, Structural formula, Molecular formula and weight 10 2.2 Physico-chemical properties 10 2.3 Source 11 2.4 Confirmation of test substance 11 2.5 Stability of test subsance under storage conditions 11 3 Materials................................................................ 12 3.1 Apparatus for bioaccumulation test 12 3.2 Apparatus for analysis 12 3.3 Reagents 12 4 Analytical methods ..................................................... 13 4.1 Gas chromatographic (GC) conditions 13 4.2 Calibration curve 13 4.3 Analytical method of test substance in test water 14 4.4 Analytical method of test substance in test fish 15 4.5 Blank test 17 4.6 Recovery test 17 4.7 Detection limit 18 5 Acute toxicity to fish .................... 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.................. 21 6.1 Stability in water 21 6.2 Stability in feed solution 21 7 Bioaccumulation test method.............................................. 23 7.1 Test fish 23 7.2 Methods and conditions 0 0 5 S 2 23 8Results and Discussion .................................................... 26 8.1 Circumstances that may have affected the reliability of the test results 26 8.2 Biological observations of test fish 26 8.3 Concentrations of test substance in test water 26 8.4 Concentrations of test substance in test fish and bioconcentration factors(BCFs) 26 Table 1 ~ 7 and Figures 1 -- 18 Appendix-1 Infared spectrum of test substance (Data provided by the sponsor) Appendix-2 GC chromatograms (Analysis of water) 28-55 1 page 17 pages Sponsor S u mm a r y :SUMITOMO 3MLTD. Study Title : Bioaccumulation study of Sample D-l with carp ( Cyprinus carpio ) Study No. :2B121G Test Substance : Sample D-l 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). (OECD testing guideline 305C) Supply of test water : Continuous flow-through diluter system flow rate : 1600 L/ day Nominal concentration of Sample D-l in test water : High exposure level ; 0.1 g/mL Low exposure level ; 0.01 g/mL Exposure period : 8 weeks Analytical method : GC analysis after pre-treatment Results : Bioconcentration factors (BCFs) obtained in this study were as follows: Bioconcentration factors (BCFs ) l e v e l ^ \ ^ e x p o s u r e period (weeks) 2 46 High exposure level,sample fish No.1 No. 2 0.4 0.4 0.6 0.6 0.7 0.4 Low exposure level,sample fish No.1 No. 2 3 3 42 32 8 0.5 0.5 2 2 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. 1. I n t r o d u c t i o n 1.1 Study Title Bioaccumulation Study of Sample D-l with Carp ( Cyprinus carpio ) 1.2 Objective The objective of this study was to evaluate bioaccumulation potential of D-l for application of a new chemical substance in Japan. 1.3 Testing Method Testing method of Ministry of International Trade and Industry (MITI) described in "Chemical Substances Control Law" (Japanese Law No.117, 1974). (OECD testing guideline 305C) 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 From May 18, 1992 to Sept. 24, 1992 ( exposure period : From May 26, 1992 to July 21, 1992) 1.6 Storage and retention of records and sample 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 ammendment record. 2) The final report. 3) Raw data. 4) Quality assurance reports. 000555 5) Test substance ( ca. 2g ) 6) Other documents required by MITI GLP regulations. 2. Test s u b s t a n c e 2.1 Chemical name, Structural formula, Molecular formula and weight. Chemical name *: 2-[N-Ethyl-N-perfluoroalkyl (C= l~8)sulfonylamino]ethylacrylate [Abbreviated name: Sarnie D-l, identification No.2B121G] Structural formula : Ca Ha 0 C ,, F a n+i S O a N - C H C H a 0 - C - C H = C H a n = 1-- -8, n = 8 : ca. 78%, n =: T-- '7 : ca. 21% Molecular formula : C n H u O i N S F n ( n = 8 ) Molecular weight * : 6 2 5 ( n = 8 ) ( * Data from the sponsor) 2.2 Physico-chemical properties Appearance :amber likewax Melting point * :27 ~42 C Boiling point * :ca.150 C ( ImmHg) Solubility : water : < 0. 1 % ( 25 C) methanol > 5 % ( 25 C) acetonitrile : > 5 % ( 25 C) acetone n-hexane > 5 0% > 5 % ( 25 C) THF > 5 % ( 25 C) DMSO : insoluble Infrared spectrum* : shown in Appendix-1 ( * Data from the sponsor) 000556 2.3 Source (1) Supplier SUMITOMO 3M LTD. (2) Supplied quantity* : 50 g (3) Date of receipt : April 1, 1992. (4) Lot. No. * : 101 (5) Purity * : > 99 % 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-l 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-l 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 c o n f i r m e d to be stable under s t o rage c o n d i t i o n s stored in the refrigerator during the test period of ca.2.5 months. 000557 3, M a t e r i a l s 3.1 Apparatus for bioaccumulati >n test The flow-through test system is shown in Fig.3 (p.29 ). [ For test aqruarium ] Dilution water supply pump : Yamazen (FMI) Co., QD-2CSC Feed solution supply pump : Yamazen (FMI) Co., QG6-RH00STY [ For control aqruarium ] Dilution water supply pump : Yamazen (FMI) Co., RP-D-2CKC Feed solution supply pump : Tokyo Rika Kikai Co., GMW-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., S R - n : Buchi Ltd., RE-111 : Yamato Kagaku Co., BP-51 : Mettler Ltd., PL1200 : Shimadzu Ltd., GC-14A 3.3 Reagents Acetonitrile Acetone Chloroform Ethyl Acetate Tetrahydrofuran (THF) n-Hexane Methanol 000558 Sodium chloride Sodium sulfate, anhydrous Kokusan Chemical Co., Guaranteed Reagent Junsei Chemical Co., Guaranteed Reagent Junsei Chemical Co., Guaranteed Reagent Kokusan Chemical Co., Guaranteed Reagent Kishida Chemical Co., Guaranteed Reagent Kishida Chemical Co., Guaranteed Reagent Junsei Chemical Co., Guaranteed Reagent Kishida Chemical Co., Guaranteed Reagent Kokusan Chemical Co., Guaranteed Reagent 4. A n a l y t i c a l m e t h o d s 4.1 Gas chromatographic(GC) conditions Column : Shimadzu Ltd. .Wide Bore column, CBP20-W25-100 0 O . 5 3 m m x 2 5 m Temperature : Column ; 130C Injector ; 200C Detector ; 240c Flow rate of N2 20mL/min Range Attenuation 101 7 Chart speed 5mm/min Injection volume 3//L 4.2 Calibration curve Standard solution of 1000 #g/ m L was prepared by dissolving 100 mg of D-l in 100 mL of n-hexane and this standard stock solution was diluted with n-hexane to prepare 0.25, 0.50, 1 . 0 0 ^ g / m L n-hexane solutions of D-l. These solutions were injected into GC described above. The GC peak areas( V sec) were plotted against the concentrations of D-l. 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-l 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-l was determined with the ratio of area on GC chromatograms against the standard solution (0.5 ugM L) per measurement. 000559 4.3 Analytical method of test substance in test water Analysis was performed according to the following procedure. Filter ( 11G3 glass filter ) Evaporate to dryness *- N-Hexane Dilute to 20mL GC ( ) * High exposure level 000S60 4.4 Analytical method of test substance in test fish Analysis was performed according to the following procedure. Test Fish (ca. 25g ) T Wei gh Discard 0S6l Evaporate to dryness n-Hexane 5mL Dissolve [ Continued on next page] n-Hexane solution 5 mL I Column chromatography S E P - P A K TM cartridge(Florisi1) * Elute * Waters Cq . Dilute to 20mL r GC 000562 4.5 Blank test (1) Mater The blank tests were performed twice according to the procedure shown in 4.3, using 1000 mL of the test water in the absence of D-l(Control aquarium). There was no peak which disturbed the analysis of D-l in GC chromatograms from water(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-l (Control aquarium) ,at the biginning and the termination of the exposure period. There was no peak which disturbed the analysis of D-l in GC chromatograms from fish(Fig.7(p.33, 34)). 4 .6 Recovery test (1) Water Two mL of acetone solution of D-l (5//g/mL) was added to 1000 mL of the test water in the absence of D-l. D-l disperse solution of 0.01 g/mL (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 l(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 Two mL of acetone solution of D-l (5/zg/mL) were added to the untreated test fish with D-l 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 c o n c e n t r a t i o n of D-l in fish would be 0.3/z g / g and correspond to 30 times of that in the test water. As shown in Table 2 000563 (p.35) and Fig.9 (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 T h e r e was no peak which d i s t u r b e d the a n a l y s i s of D-l in GC chromatograms from blank water and blank fish. Therefore, the area of the minimum detectable peaks, which can be recognized visually, was set to be 30,000 /V Sec.. This value corresponds to ca. 0.02 g/mL(D- 1) on the calibration curve. Detection limit is calculated according to following calculation formula and conditions. The results are as follows; Fish : 0.02#g/g Water : High exposure level 0.004 g/mL 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. l ^ g / m L ) : > 0.2 times Low exposure level ( 0.01 g/mL ) : > 2 times [Calculation formula] Detection limit = Concentration in x Volume of the /Amount of the final solution final solution sample [Conditions] Fish : Volume of the final solution 20 mL Amount of sample (fish weight) 20 g Water : High exposure level ; Volume of the final solution 20 mL ; Amount of sample 100 mL Low exposure level ; Volume of the final solution 20 mL Amount of sample WOS64 1000 mL 5. A c u t e t o x i c i t y t o f i s h 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 r e f e r e n c e for d e t e r m i n i n g the n o m i n a l c o n c e n t r a t i o n s to be u s e d 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-LC50, respectively. 5.1 Preparation of test solutions Four mL of 400mg/mL THF solution of HCO-20 was mixed with 4mL of 50mg/mL THF solution of D-l. After evaporation of THF in the resultant solution by blowing N 2 gas,it was mixed with HC0-20 solution(6.4g in lOOmL water) and dispersed. This dispersed solution was diluted to 2L with declorinated water (Yokohama municipal tap water was treated with activated charcoal)to prepare 100 #g/ m L solution of D-l. Ccontrol level ) Eight g of HCO-20 was dissolved with 100 mL of deionized water and diluted to 2L with dechlorinated water. 5.2 Test conditions (1) Test fish : Orange killifish (Oryzias latipes ) Source ; Miyazawa Fish Farm(l323, Suenaga, Takatsu-ku, Kawasaki) Date of receipt ; March 3, 1992. Lot No. 92-H-0303 Body length ; approx. 2 cm Body weight ; approx. 0.2 g (2) Acclimation : Aquarium No. ; C-5 Water temperature 252 C . Period Over one week Feed ; Tetramin (Teti 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 ; 10 per one aquarium Volume of water ; 2 L Temperature ; 24. 4--- 25- 4C Duration ; 48 hours Dissolved oxygen ; 4. 2~7. 4 ppm Aeration ; Test water was aerated during the test period. Renewal of test water ; non 5.3 Results The 48hr-LC5o value of test substance was more than 100 ^ g / m L as shown in Table-3 (p 35). OC05C& 6. S t a b i l i t y the test of t e s t s u b s t a n c e conditions under 6.1 Stability in water (1) Preparation of test solution The feed solution of high exposure level (concentration of D-l: 4.0mg/mL) was prepared by the procedure in 7.2(1). Then, 2.5mL of this solution was diluted to lOOOmL with deionized water. I t (concentrtion of D-l: 10 #g/mL) was used for the stability test of D-l in water. (2) Method of measurement The solution prepared by the procedure shown in 6.1 was left under room conditions, and 5mL of this solution was withdrawn and diluted with 100 mL of water on days 0, 1, and 2. Each of them 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 time was evaluated. (3) Results Typical GC chromatograms 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 g/mL -> 9 . 9 # g / m L ) of initial concentrations after 2 days, it is concluded that D-l 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-l: 400# g / mL) was prepared by the procedure in 7.2(1). 0 00 5 6 7 (2) Method of measurement The test solution prepared by (1) was left under the room conditions. At days 0, 3, 7, and 14, 2.5mL of the test solution was withdrawn and was diluted to 50 mL with acetone. Then each of 2.5mL of these solution was withdrawn and was diluted to lOOmL with acetone, and analyzed by GC. Variation of the test substance concentration with time was evaluated. (3) Results Typical GC chromatograms 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% (394 # g / m L - 3 9 0 Jug/m L) of initial concentrations after 14 days, it is concluded that D-l is stable in feed stock solution. 000S68 7. B i o a c c u m u l a t i o n t e s t m e t h o d 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). .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.ll cm. Mortal fish were less than 5. % during the acclimation of a week prior to the introduction of fish into the test system. .2 Methods and conditions (1) Preparation of. feed solution High exposure level The stock solution was prepared by dissolving 2.0 g of D-l and 80 g of HCO-20 in 500mL of THF. ( Cone, of D-l : 4.0 mg/mL, Cone, of HC0-20: 160 mg /mL ) Low exposure level Fifty mL of the feed solution of high exposure level was diluted to 500 mL with THF (Cone, of D-l : 0.40 mg /mL, cone, of HCO-20 : 16 mg/mL). Control aquarium The 160g of HCO-20 was dissolved in 1L of THF(stock solution). The 370mL of this solution was diluted to 11 L with deionized water. 000569 (Cone, of HCO-20 : 5.4 mg /mL ). The feed solutions were renewed every 7 -10 days. (2) Preparation of test solution The feed solution prepared by the procedure shown in 7.2(1) was supplied to mixing tube by feed solution supply pump and dilution water (dechlorinated tap water) was also supplied to the same tube. Then these were mixed and the concentration-of test substance reached nominal value. The test solution in mixing tube was supplied to an aquarium. Dilution water is the tap water of Yokohama city which was dechlorinated with activated charcoal. (Fig.3 (p.29)) (3) Conditions Flow rate of feed solution : 40m L/day for high and low exposure levels and 1.2 L/day for control level Flow rate of dilution water : 1600 L/day (The turnover rate for each aquarium : 20 times/ day) Test fish : Carp (Cyprinus carpio, Lot.No. 91-K-1206, aquarium No. B5) Body weight 31-- 40 g Body length 11-- 12 cm Fat content of fish 3.7 % ( n=4, 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 (Minipet(Kyorin Co. Ltd.)) once a day except holidays. Nominal concentration of D-l in water : High exposure level 0.1/zg/mL (cone, of HC0-20, 4mg/mL) Low exposure level 0.01 /g/mL (cone, of HCO-20, 0.4mg/mL) Control 0 fjig/mL (cone, of HCO-20, 4mg/mL) Population density (at the biginning of the exposure period) : 0 5 7 o High exposure level Low exposure leve 15 fish / 80L test water 15 fish / 80L test water Control 12 fish / 80L test water  Exposure period : 8 weeks (4) Introduction of fish, biological observation and environmental control The test system was operated for 4 days prior to the introduction of fish to confirm the stability of the concentration of D-l in water. After that, fish were introduced into the test system. Fish were 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-l 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-l 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 water was 800 L/day in first 3 days , but because of concentration decrease of D-l due to uptake by fish, it was changed to 1600 L/day after the day for each aquaria. 000571 8. R e s u l t s a n d D i s c u s s i o n 8.1 Circumstances that may have affected the reliability of the test results. There was no significant matter that may have affected the \ reliability of 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 ^ g/mL ( high exposure level ) and 0.0082 g/mL ( low exposure level ). The decreases of D-l concentration in both levels were observed at 3 days from the initiation of test. This phenomena will be attributable to uptake of D-l by fish. Therefore, flow rates of the feed solution for both aquaria were increased by a factor of two, and as a result, e a c h c o n c e n t r a t i o n is a p p r o a c h e d to e a c h nominal value. Th e coefficients of variation are 8.4 % ( high exposure level ) and 13.5 % ( low exposure level ), respectively. 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 ~ 1 8 (p.47~ 55). Concentrations of D-l in fish are 0 . 0 3 2 ~ 0 . 0 6 2 # g/g ( high 000572 exposure level ) and 0.013 ~ 0 . 0 3 4 # g / g (low exposure level ). The BCFs 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 as metabolites of D-l. On the basis of the above results, it can be concluded that sample D-l is not bioaccumulative in carp under the prescribed test conditions. #00573 Fig.1 Infrared spectrum of D-l (before starting the study) -looo..0. <100.00 cm- 1: 0 . 06. 6 5 . 92 XT 4 s c a n s : mode r a t i o : r e s o l 4 . 00 cm-1: 28 121G 0 - 1 L o t . N o .101 t h r e s h o l d 3.00%; band cm-1 % cm-1 X cm- 1 2986.8 46.13 1730.3 13.67 1638.4 1<168.6 <16.61 1392. 0 1 6. 90 12 0 2 . 5 1 0 6 2 . 0 2 9 . 3 3 10 17. 7 2 8 . 3 7 904 .6 808.0 35.17 784.1 40.20 746.7 706.4 44.98 653.5 33.43 18 peaks found 9 2/ 05/ 20 11: 00 apod s trong N . Saek i X 46.05 6.36 27.24 44.54 cm- 1 1619.8 1148.9 857.9 735.7 % 53.28 7.71 57.01 45.63 Fig. 2 -Infrared spectrum of D-l (at the termination of the study) O IoD 0 1 Eu :: 4000. 0. 4 0 0 . 0 0 4 s c a n s : mode r a t i o : . r 23. eso 7 l 6 .43.000XTc m- 1 : 2E3121G 0 - 1 Lo t . N o . 101 t h r e s h o l d 3.00%: band cm-1 X cm- 1 X cm-1 3854.2 70.59 3751.9 71.78 3676.2 3630.0 71.45 3587.7 71.30 2907.1 1638.4 60.89 1467.8 1392.9 1062.4 46.04 1018.3 44 . 44 985.2 784.3 59.95 746.9 56.19 735.7 19 peak s found 92/ 08/07 09: 57 apod :s t r o n g N . Saek i X 71.50 64.35 31.11 42.65 56.30 cm- 1 3650.1 1731.7 1202.3 B08.2 % 71.29 29.76 23.91 49. 13 000574 -28- Fig- 3 Bioconcentration test system feed solution supply pump dilution water supply pump 000575 Fig.4 Gas chromatograms of D-l(Standard solution) for calibration curve 0 U g/m I (n-hexane only) 0. 25 // g/m1 0.50 // g/a 1 . o 7 7_____ 1 17 9 4 7 6 1.00// g/m 1 filO D O O Fig.5 Calibration curve of D-l Y = 3 .8 3 3 8 X I O 3 + 1 .4 4 7 3 X 1 0 6 X X 1 r = 0.99995 X 1000000 Calibration Curve ( 3 3 S A r i ) ea JV 'Head 000577 Fig. 6 Gas chromatograms (Blank test from water) Std. 0. 50 p. g/m 1 OJ o k in .* TT 7*` T CO --t N . * bO fN. bO ^ o . .* OJ --1 *:o r*. o fN. o o R OJ <1 f '.. UJ *T\ o j m OJ r-. :* oj til' r*-. OJ U b" *' io oj iT* ( V J iT \ OJ '.V <x H- a: OJ ^ <x <X H A R M I N G NO P E A K START 0OO5T4 Fig.7-1 Gas chromatograms (Blank test from fish) (before starting the study) Std. 0. 50/ z g/ml COMC :j < 0 f-N. * T l CO CO uo O J 1 Ml* 0 K U 0 1 '-O CO r f 11 . . 1 CO Y-- CO f s . 1 o Oi~) T v y-H l CD o r.. AREA MK ID MO TI ME *v.. 'X U O 1T 1 CO i T . 1 UO ~~t o CO CO 1 IT . a : CO t T -- 01* 1 UO uo<x -- ^ UO 1 CO CO CO 1 CT. y-4 r - . 1 1 1 UJ C O U O K ( V i *f* C O iT , i T , |VJ ij- , UO <1 .. . t-- C O C O CO -'T 0 h~ co co o. c<ei: to PKM 0 -- 0> rt C O l'l'l i j j Ko OJ OJ UO O -- OJ t I 1 1 1 CD 1o rs. c o co c o \r> co OJ >z< to o m OJ z co OJ co 4i JL* 0? ITT* -- IS. . OJ OJ co <x f- i-- C <X ID UO iT-i OJ ") co H T'. 0 Z" co r*. CO o:> O'* co T-- T'.. 1 1 1 1 K 10 Z. 0 T-, \ `Z ` OJ -- OJ --t -- 1 c * o =i iX -- z> UO (X- K iT> L ^ r OJ UO uO K -tf* CSC iT \ 4-4 r-'. ` O -I* <x CO CO uo IC* OJ -- LU -- h- . f--\. CKO. K*r7T>' OOyJJ.. Kce* z.* OJ OJ CO CO UO ho-1IH- o t OJ 0*i O bLooo Fig.7-2 Gas chromatograms (Blank test from fish) T, co fN. r\ z * :o *7*' OJ `7** '.V j7 I O JoN. i co co <e-x OJ CO T a: <x OJ Q oj OJ o fN. *3- O'1 jj co T~ o "7" l**l in o T **r iM o ^ `O *' <Cx d* i*o -- in co mTO CtoO UJ m co to CO OJ >z* to >T> OJ -- -- ** o OJ CO START oooS^ 10 O cu -a* 1 cu X' r*. 1 U".' T* io r |V| C'J co f o 1 S I --< tT\ T\ I *X` o * r co 1 'X* o" o~r~ =i ?874i TOTAL L<XJ alO-. .*>* if> Ki <X fvJ.> ccuo t*oJ> '' fs. 00 CU UJ Cc*oJ o? CoU _ J tO 'T> CU <X -- ''T c O -- CU <x 2 Table 1 Recovery test from water w amount of water mL 1 1000 2 1000 A added amount uZ 10 10 BC peak area n V sec Sample Std. - 723856 762903 720337 762903 D cone, in final volume n g/mL 0.474 0.472 E measured amount R recovery Fig. No. uZ % 9.48 94.8 8 9.44 94.4 8 Average : 94.6 % Concentration of standard solution(Cstd. ) Final volume (V) : 0.56 mg/mL :20 mL Calculation D = Cstd X B / C ; E = D x V ; R = E/Axl00 Table 2 Recovery test from fish WA BCD ER Fish weight added amount peak area t i V - sec cone.in final volume measured amount recovery Fig. No. Z 1 36.92 HZ 10 sample 803266 Std. 813996 uZ/id 0.493 HZ 9.86 % 98.6 9 2 34.25 10 813149 813996 0.499 9.98 99.8 9 Concentration of standard solution(CStd. ) Final volume (V) Average : 99.2 % : 0.5 /zg/mL :20.0mL Calculation D = Cstd x B / C ; E = D x V ; R = E / A x l 0 0 Table 3 Acute toxicity test ( 48 hrs) D-1 /zg/mL number of dead fish test fish 0 ( control ) 10 0 000581 100 10 0 mortality % 0 0 48hr-LC5o = >100 u g/mL Gas chromatograms (Recovery test from water) Std. 0. 50^ g/m I CO in fN. m CO tn in o? *T f \. *- *3* f'-. ID CO r_i . . . 7Z co y--t CO r*v o oo O o >-- v Z Z T -' Z > 2?* ' I 'X ' -* CO IT* 1 uo UJ -- .X CO iV *.i iT . fs. 1 ** a: O ' co in is . l -- -- CO CO 1 O'* v? < r-. 1 1 1 u j co m 0*1 iT. -1 --4 o> 1*0 o> in <x 1-- . . . 1-- CO CO CO o >-- Q CO CO " i* cu TART 1 F' KHO TIME A R E A MK I DHO COHC K 7' f-, -.o fN. OJ CO lf> -- IO Z` OJ o j r*. K co OJ T-4 :o K fs. ^ CO 7* f s . UO T. OJ OJ -- CO -3 OJ -O -- Z* eu ^ o- r*-. 000 5 % 1 k co *:*: -- *- U".' K* OeoJ OJ -H r>4 l*f> IO OJ O? '7' `t.H"O tij\ OJ CO CO <!O--X OJ CO f H- L<1 ua IO OJ 7-O 007J 'U7O' O OJ OJ -- T-- (0/' u 07 07 * '7 ' CO T' eu CO <- Jr eu CO -T Gas chromatograms (Recovery test from fish) St d. 0 . 50^ g/ml *. fN. COUC A R E A MK I DHO TIME <1 b~j Ul cu co C <x Z' co CU -- bO CO o r v *Js co C-J co CJ CO T <i PKHO START  oj eo eo CO co CO r*.. eu cueo* T eeoo f'=sf. eu o CO IO K is. il* 'T '' T eu ' UO ` Z' r-4 f.-J '.'J f Z eo o eo C*J eo m uo -o OJ eo CZOO1 CO "' OJ UO eo -- If>UO - h eo o? fs. 7> iVi uo eu eu eu eo -a- <1 -- eu eo T u' L i-- eo o p *. ' ^ co eo fs. ir> uo -- 7% CC T CO . CO CO T'. - CO -* T fN- r^. -- 7> U"' '' U"> ' * O eo rs . fs . (N. 7> r j- O `.J? tfO OJ ' K m -- CO -- UO IT 'O eo fs. ro ro v k .`o ro j* *10 -- Ts eo eo ir. . eu o j eo eo 1 CO i eu i~ 1 eo i 1 eo i i i _j <1 -- y- OJ CO T O 0O05B3 Fig.10 Gas chromatograms (Stability in water) S t d . 0.50 u g/m] *t !> --1' 75 no no T> a no T 70 r- >.'J T Z* OJ - 7> r*. r*. o (=4 J* OO t \r> v *r .;? vJ -- uo *>: --:o ---- v i> -- ? 00 *7' <c C <1 CL -i? ?* liO -- OJ 1 7z* t -:u oo T *jO -- . % CJ -- K 07 IN. ~ -- -- ~ UO no 7 OO ? START t at*i Std. 0 . 50 g/m] J <r fs tr> U T CU ij :u ') ti"> r -. ' 2> o C*J r s o fs zz > > > X IS .;? co L J -- IS *- f s iS f s O J I z* :> fS r* CU -- CU 0? UJ K o fS IT/ CU -- 2? ?' CO t-- . co ' co CO n* o -- O J CO T w . day 2 LJ -- 7' 7> is T CU -- ^r 7? T *5 T *1? - CO A ??, ia tothl ?m ? i Fig.11 Stability in water 000585 100 80 - Residual D-l (%) 60 40 - 20 * 0L 0 1 Time(days) 2 Fig. 12 Stabirity in feed solution in bioaccumulation test Std.0.50 u g/n1 r>. -* pzv>. ^;*> *:o m o - o p'.o j -< ? o day 0 Ll> CE 07 t/5 07 -T 3 77 T Pv T '4? ;% T O ti5 0J - 7? 77. Std. 0 . 5Qg/n 1 LJ <E fs .7 (S 'O ;? 5 o j o j OJ D N . 05 oo2: OJ -- fv:o. f--s.. tTt 7 0 7 T 'w 77 * o:oj -- *u>5 V-fr/ iT<rJ* 07 07 o j 05 07 t -- oj oj t WJ 75 4 wv x i -o pv .7 <Z> -- -- w*> iTT\\ \7Q5 OJ |T\ -- OJ .v) *T *:o -- o j OUJ 07 V OJ OJ ?> OJ I 7' 07 OJ M5 7-O V-frf O oj 7s p*. ' 7 OJ *7 - OJ 07 T X <C1 U 5 OJ f ' . 1/5 fS. m > 0 7 OJ 'i7 /N Ji7 !* tr t t OJ - P*. fN -- -- -.fi. 2 O? 0 J 3 3 OJ 0J -- rs . l/5 7 07 OJ 07 T vD V WO O o O Fig.13 Stability in feed solution ^SSO Q o 100 - 80 Residual D-l (%) 60 40 20 0 0 J______________________ L 24 68 Time(days) 10 Table 4 Analysis of D-l in test water (High exposure level) sampling date exposure period i AB peak area n V sec C cone. in water E mean cone. Fig. No*. day 5.26, '92 0 week 0 Sample Std. n g/mL u. g/mL 1 691668 761885 0.096 0.096 1-1 29 3 2 551911 768251 0.076 0.086 2 6. 2 7 1 3 665450 75999 0.093 0.088 3 5 10 4 684891 764544 0.095 0.090 4 9 14 2 5 655152 752597 0.092 0.090 5 12 17 6 742762 794882 0.099 0.092 6 16 21 3 7 600994 794596 0.080 0.090 7 19 24 8 705686 824875 0.090 0.090 8 23 28 4 9 716171 792816 0.095 0.091 9 26 30 7. 3 31 10 687002 809639 0.090 0.091 10 35 5 11 776203 831148 0.099 0.091 11 38 12 709193 787890 0.095 0.092 12 7 42 6 13 674771 771065 0.093 0.092 13 10 45 14 742351 808504 0.097 0.092 14 14 49 7 15 701150 791505 0.094 0.092 15 17 52 16 544487 775383 0.074 0.091 16 21 56 8 17 733512 779453 0.099 0.092 17 Concentration of standard solution Final volume Sample volume Recovery Cstd : V: W: R: (* 0.5 g/mL 20 mL 100 mL 94.6 % Appendix-2 ) Calculation C = Cstd x A / B x F ; ( F = V / W / C R / 1 0 0) ) 0 00 5 8 8 e = I c i / i (i: number of analysis) Mean concentration in water standard deviation : 0.092 0.0077 g/mL Table 5 Analysis of D-l in test water (Low exposure level) sampling date exposure period i AB peak area iiV - sec C cone. in water E mean cone. Fig. No. * day week 5.26, '92 0 0 Sample Std. u g/mL /g/mL 1 738542 761885 0.0102 0.0102 1-1 29 3 2 359497 768251 0.0049 0.0076 2 6. 2 7 1 3 515797 759199 0.0072 0.0047 3 5 10 4 599070 764544 0.0083 0.0077 4 9 14 2 5 570779 752597 0.0080 0.0077 5 12 17 6 572050 794882 0.0076 0.0077 6 16 21 3 7 659682 794596 0.0088 0.0079 7 19 24 8 572309 824875 0.0073 0.0078 . 8 23 28 4 9 676137 792816 0.0090 0.0079 9 26 31 10 689946 809639 0.0090 0.0080 10 30 35 5 11 701085 831148 0.0089 0.0081 11 7. 3 38 12 636904 787890 0.0085 0.0081 12 7 42 6 13 615794 771065 0.0084 0.0082 13 10 45 14 662294 808504 0.0087 0.0082 14 14 49 7 15 606422 791505 0.0081 0.0082 15 17 52 16 591058 775383 0.0081 0.0082 16 21 56 8 17 600028 779453 0.0081 0.0082 17 Concentration of standard solution Final volume Sample volume Recovery Calculation Cstd : V: W: R: ( * Appendix-2 ) 0.5 /g/mL 20 mL 1000 mL 94.6 % C = Cstd X A / B x F ( F = V / W / ( R / l 0 0) ) 000589E = E C i / i (i: number of analysis) Mean concentration in water standard deviation : 0.0082 O.OOlllng /mL Fig-14 Concentrations of test substance in water High exposure level Fig.15 Concentrations of test substance in water Low exsposure level 000590 Table 6 Analysis of D-l in test fish (High exposure level) WAB CD EF exposure fish period weight peak area u V sec week g Sample Std. cone, in cone, final in fish volume measured g/mL n g/g mean cone. in water //g/mL BCF FigNo. 2 1 34.14 90324 839568 0.054 0.032 0.090 0.4 18-1 2 34.80 94243 796908 0.059 0.034 0.090 0.4 18-1 4 1 37.18 168720 807176 0.105 0.057 0.091 0.6 18-2 2 39.96 192918 790270 0.122 0.062 0.091 0.7 18-2 6 1 36.95 161768 839289 0.096 0.052 0.092 0.6 18-3 2 37.47 113668 804605 0.071 0.038 0.092 0.4 18-3 8 1 35.54 132230 879847 0.075 0.043 0.092 0.5 18-4 2 36.77 145406 885954 0.082 0.045 0.092 0.5 18-4 Concentration of standard solution(Std. ) Final volume Recovery Cstd : 0.5//g/mL V : 20 mL . R : 99.2 % Calculation C=CstdxA/B ; D = C x V / W x 100 / R F = D/E 00591 Table 7 Analysis of D-l in test fish (Low exposure level) WAB CD EF exposure period week 21 fish weight peak area u V - sec g Sample Std. 34.73 72837 839568 cone, in cone, final in fish volume measured fi g/mL 0.043 u Z /g 0.025 mean cone. in water u g/mL 0.0077 BCF 3 Fig. No. 18-5 2 35.61 61435 796908 0.039 0.022 0.0077 3 18-5 4 1 34.23 93655 807176 0.058 0.034 0.0079 4 18-6 2 35.70 63936 790270 0.040 0.023 0.0079 3 18-6 6 1 37.23 38016 804605 0.024 0.013 0.0082 2 18-7 2 40.00 53795 798557 0.034 0.017 0.0082 2 18-7 8 1 35.27 45555 851984 0.027 0.015 0.0082 2 18-8 2 35.73 46433 864383 0.027 0.015 0.0082 2 18-8 Concentration of standard solution(Std.) Final volume Recovery Cstd V R :0.5//g/mL :20 mL :99.2 % Calculation C=CstdxA/B ; D = C x V / W x 100 / R F=D/E 000592 Fig.16 Bioconcentration factors (BCFs) of D-l High exposure level 10 BCF 0.1 46 Exposure period (weeks) Fig.17 Bioconcentration factors (BCFs) of D-l Low exsposure level to BCF 1 Exposure period 000593 (weeks) Fig.18-1 Gas chromatograms (Analysis of test fish) -- High exposure level -- Std. 0.501 g/ml week 2 No. 1 Std.0.50^g/m| week 2 No. 2 o o o 58 H > -z> T fS. XYn C--3D *VoO Tt -T) uVVO>i .iVtn>v V-Vi ~o is. rPvS. Ofs.J Oi*> ,\j CO (ifTS% v i -X> .;o 'Vi *>j .,> O' t:o i<ry>v c..Vo| cn mCO o -- OJ CO T v> O--O K'3 i----l -Ni3> .'.On >n V-O > OJ CO - jJ .> J O J U 'i r *'j r s -.'j t fs -- 0VO <*>vJ -D p . o --o O J VO -- ->J CO -T V") 'O is. ?> Fig.18-2 Gas chromatograms (Analysis of test fish) -- High exposure level -- Std. 0.50 u g/m1 week 4 No. 1 7* Cj *7 Ui <L 7 3i0n) t m --i3 **7o o^> t '--.\i -c7 t *Om.n -f.Snn. t 03 u't !/ n ar>?. *.n > -- ..n m in?? --O *i> *T.n oj aj -`703 - PP>JJ N - *3 - tT'J O-- `3J T " - 10 2 * M * 2 7 03 w(\t l^* | III Oa -) i\ 7* 'D> j T T fv. *.,,J *33 **) om> o**j>*'7O 7r3*) ---7:5> -o --o> -y j ? ' t o.Tv .n 0-- o j \-> T _ r S> ra-s* *\7j' a--* --wf'*./ :o -r - T03 in ?' n -- AJ -'j o j >J 03 T tl> ' 0 f ' . .33 -7* OJ 1. TOTAL OC <s V) STARI S td.0.50 u g/nI week 4 No. 2 LJ \n io N T . VI AJ -- O AJ '0 O Or -.'J --fVs) r--s O O N v*> f S A.JJ A J LJ fA--s .o3T' Al n *f-s3 ru.3o"7)' OfsT' u*) --T >T >0 V--.\'Jl .;>-- tN .fJSNA--J .T\J V") -r vi 'D A J A . Aj --f S A J -D f S A J n AJ oo > >>> in tr \n o O O & Yr \ A> iZ o> O " A J A ) T W> * 0 f S *75 7' - 3 -- * il. Fig.18 3 Gas chromatograms (Analysis of test fish) -- High exposure level -- Std. 0.50// g/mI week 6 No. 1 Std. 0.50// g/mI week 6 No. 2 f--s. ---\j u-> r>. 3 T ' V*/ t T IO - M - f'. \-- rs. OJ -- V*> j o> OJ oj 7 ' fs *>*i> O J -7 o AJ ^ *i3 OJ --K r. CO T -- T VJ i7> `0 CO X> OJ*3 >OJ OJ OJ f'. T bJ OJ T t o j ' j i - - ' D .13 o o j -- a j LJ <X v:o -ioo >nn m^ O--J m*i> ---r W JWS. N-- OJ oj o> 0D 2D J LJ OJ I o j * r fs. * r * r t '> t 'O rs. v oj i \ O- H > K 'OOJ T OJ -- W'i -73 OJ v\J WJ -7 o j r. *> -- o j -- -- ~ o j OJ OJ -- -- i -r* I I-- vP a" if' o OO A AA 'X fv.i> T' *'J I J* r> -- T' <7% Oto' I * O J -- >3 O OJ O' .'0 Ow*J) *--7) X 73 O'- OJ T y> T> N aj 'oJ> --I .'J fs. >7 . OJ '0 O' T -- -- -- -- OJ .'j OJ LJ OJ i T' v? r> X o o > m \ n ^ m i to -- -- V") OJ '0 Yy/ ^ t is. o *7* o> OJ rv >> >> > 7 * OJ 'I bJ 7* oj IS 7 -- 0 J .n -- to :u r u T TJ -- T i s .73 0 bJ *\J in 7 3 cZ -- 'j n 7> -7? -- OJ OJ 0 7 -- *1 OJ 7 * -- 7 * 7 * -- OJ oj Tv 7 OJ rs 57 OJ Ts. o> OJ 0 oj -- -- b*> OJ --o -- 0D .*> r. *\j " LJ r> 0 J in OJ . 0 J rs 0 ? WJ OJ zz o> v> fs -- 73 o' 3 *r CO -- -- o> OJ *> 33 O) T 7 0 J -- >-- -- -- -* OJ OJ OJ OJ OJ T T r bT n i Z -- J `O J *T m rs. . (Z I (/I a . co a. OJ _o OJ .v, T *> !? fS 7 ' 0J 2Z -- -- Fig.18-4 Gas chromatograms (Analysis of test fish) -- High exposure level -- S td.0.50 u g/mI week 8 No. 1 UJ r>. ..r0> vCOo *0 t-7.'*t 2, .7 . -.tv ,y j - i 7J '1 CO r, -c3u T c--o O-oj T-- N -i - T >> t CO >1? `*0 *7) wO TO -- P1, p>j *.'0 . p - r CO 100 p -- <\J *.'0 T VO 0 * 1. iZ J't ~T t> r>o. p*. --f0os -- <O\-v\J.J vO -x* C---Or T:7'! .7 -jn .rCCpsOO. C--O v.y1-o> *>PV.fs C-J .x '0 cur rp*\. v.-u? v:oo u-xo1 -r* -- -- -- . ' J V J -,'J CO t wo & rv. c? 7* -r -- T0 TAL iH 1 L S S t d . 0 . 5 0 u g/ml week 8 No. 2 tfeSO0 0 o orO*Ts. ***--.0'jJ %>O*.*3o)j .--rry>* r o j -- co o A ff T . iTrrr/n OJ *> O -7* CC* OJ N n CO 3 -7%V `T> T rs, fs. -0 *>? f\ <*5 *kj 0 7 .7 *7 u l <> -- -.0 *7* O N -- O J .3 00 ** > T O J "i 7' .V -- oj *> V>> OJ -- 'i>'0 i-7n<" oojj wm m.,>*:> m s oj:> oj g*/ .i? :> o ' -- ---- O J OJ O J T *c T * ) -- a j . > T trt 0 i s .75 Fig.18-5 Gas chromatograms (Analysis of test fish) -- Low exposure level-- Std .0.50 u. g/m I week 2 No. 1 Std. 0. 50 s/m ] week 2 No. 2 X* T- *o0j oO? N. nO' 7 \ O - rs* n o- o -- in o-- \O'? Uin1) ^9% 'ris). O--J N . i s O ' T '3 !* \J - m .\ j .r i 7* rs. oj OJ n ij I o j f \ in. \n tn -- 0 -- 09 *J> v . -- OJ t n ) w> o j o j = o j -r> o j r > m OP o~ in 9 SC0 ' S t -- oj tm P*.v> Of-9 -- iV K 09 O ' 'O t . rv. -u O -- oj "> \ ij N ^ "> .co-3- m - ^O-O'j' O:*' 0o9) Ii9 0. OJ Fig. 18-6 Gas chromatograms (Analysis of test fish) -- Low exposure level-- Std. 0.50,u g/ml week 4 No. 1 Std.0.50 u g/m1 -- c* co -- t N oj oj -- r* * -0 OJ oj CO >7* 0 OJ 07 > <n co -- in *.T\1(7 -T T<VI -75 rTs f'.''vJ *4 01 T flj N N ---O O' m b7 N ? T 1*0 O J -- -Z* OJ . 07 i_1 . , . , " oj -- rs week 4 No. 2 07 OJ W) -- J' *V> O? 00 f -- I* ??'O 0? 'u -- -r *0 `0 *i> oj :> v? r>. *?j 7"> .;o O' r* *n T '4 #1 `3 -- N T 07 C*>*> OJ T - * O -- i> 7 N CO so '3.5 0>7 i*. ``o m x* -i- w7 />is. .'7 .07 lo/>j rN. ^ t N (\ -- -- .'j O -- O J CO - r 1*7 'i ? N CO -- oj co T OJ CO *t w0 * 0 \ ?? START TART Fig.18-7 Gas chromatograms (Analysis of test fish) -- Low exposure level-- St d. 0 . 50,u g/m week 6 No. 1 LJ <i> i N O ' 1*U0 W>O <UO --I/O o o - V) 0) T \> -- icso. r--s. rs 100 731000 TOTAL rvcf. ''To -- OJ CO T *t V ) rs. w ? START Std.0.50 g/m week 6 No. 2 O O >0 O LJ a T 9 J 07 -- KK'i T'T* *XX** frvs. O ? T J O -- -- -O' rs fs. -- O a N <X f s -} f 1 'O ccU -- CO s s 1 u"> <"> <7 -X X 1 CO X r i r -- -- IT s i A j s 1 CO i i i m % n O ) W t 00 -- v> - 00 r> o T - T y-- h- rtn\ <v eax O. V/3 LJ 5 w CO n si> -- a T I O t i't (M 7? ,\ j ' ! - *M >? r s V K ( U *i -r C -- -7 s" ' 'T ' i r t co `X* T ?' W fs . -Z> *7 O`=J T Opj r fO -- c i n O A \l `7) T W*> ' P f \ W ?' O CL. Fig.18-8 Gas chromatograms (Analysis of test fish) -- Low exposure level-- Std. 0. 50//g/ra] week 8 No. 1 Std. 0.50// g/ml week 8 No. 2 oi*Vopx. 1 Z'i t Oj 'Hrrn'Yi N . T ' > ~ -- ti"> > co u i r s -- 03 ! *J* 03 tO o > ~ O J 'X* *7? -- OJ -n oj o OJ O T *r rs O j o t ' O >B N if) \ IO *0 O0) 0. *3 o> t *x* .r> *vi T ' OJ 03 IS g`# 03 .?? !> 2> O J -- i s N - *) *7 -- !> T T t o O 1 > f s . -- -- r s o ' f s *7> oo u*> .s o j 0J r s - "> i W> O J ` t i j 7 d ) " O J 1 2 03 7 - N '*> r s 1 O OO O O 1O | i 1 >> /> >* i. fs. > Oj T1) O J ' *' -- O J - * iO 7 ~ --T t o *> to OJ to T O J '0 r |> 'J `O *l> rr O J 0> *`J C* IS >j rs. -:o w o v> o rs. 03 *7? 03 Oj M 0 J *T T ? ,s Ul X ,^ \ s . . 0) .v .0 . -c-j ^ Ut* 'rs. `V A r-- ^/W n . > *> CO O J >J o ? O J T 1*3 ;o I* O J t o 0 1*3 to T 1*3 Tl tn 1 oj 1 i* X* | `i t rs 1 1 .'f v l OJ OJ S T gfS -- 73 T -T _i sX -- Oj t u3 .VI 0 hi r TVJ. fl `V. n -- h l\v ) i) - 5 T T 'Vi .n fs .--\i O--J r*,s3 7' > v-V3I o j o ? f s r s : > rs. i s . -t* is *?> -- Oj r s. *j s o j * . -- -- -- o j OJ 03 -- * \ l '3 - r w3 -I? K *73 O o > o A p p e n d i jc -- 1 Infrared spectrum of D-l ( Data provided by the sponsor ) ( 1 page in all ) 000602 'T* -Q ~l H/-D- Z/T'f '// / OI -0/ J -O n y i- \,- a $ nuy-U -H 3 009 0002 0092 No. : 009E OOOt' 000603 A p p e n d i >c -- 2 Gas chromatograms (Analysis of test water) ( 17 pages in all ) 000604 TART Fig.1-1 Gas chromatogram (Analysis of test water) day 0 Std.0.50#g/ml CO T -- -.0 u in CO in o O n :o oj L J OJ T *7' <1 f '* . CO ' U o j `-JL' -- -- \OcJ to rcu-. co 7> o j :*. OJ CO ' t <X JN. rx k ' I* in T f s . - --i fx. i;o K --i Tv U-.1 r-. k oj -- CO ?* o j :o -- OJ CO TOTAL 7 4 U3 1GG low exposure level OJ X *ro 1 .r i -- OJ 1 0 ID 1 CO IT.* CJ X* 1 . .1 z : OJ 7' o r^. o l `Z 1 1 X 1 1 I l 1 i o ix A \/ IX > ,*X .Tv in 1 OJ bJ *Ti r.. 1 m OC 1? CO -( 1 CO <x 01 OJ O'* X 1 CO X` OJ 1 o 0 \ r-. \ 1 1 LJ CO in r-. IT in CO V- i--< .Tv CO Tv o i-- * . OJ ro .*0 T _j <i I-- O 1-- o OJ 00 *T s/ Cl . 0 0 0 GO 5 Fig.1-2 Gas chromatogram (Analysis of test water) day 3 Std.0.50^ g/il High exposure level OJ -- U - -- T ?'* (N. ' U0 *Z' -.0 CO *.1 t , ,31 r r oj co r.. r*. --< O01J* C'ZO' u'.tof 0*.' OJ CO oj .0 oj co OJ -- r. i? i r*. -- TART 0 0 0 GOG OJ IO <x U"' --* Ui oj co ?\ OJ mI1 i5 uoo o ,:o r-. oii".j' 7s OJ 0! OJ -j -1 -- OJ CO T : *.x CO 'X -- .*0 LU CatC: CO CO OJ -- u U*> u" U" OJ 7% K OJ CO 'OJ CO CO ri-n. -- OJ 0:* Fig.1-3 Gas chromatogram (Analysis of test water) day 7 Std.0.50//g/m1 High exposure level 0 CC' in CC' in OJ CO CO T* fv 3 r f iT. ro OJ 01 u 0.. -- o o T ' D CO CO U1 ~ T 0 CO fv iTN 3 OJ OJ in M l in c^ OJ f \ co o TV low exposure level rv T T\ N . 0? r v O 0*. 'D in o CU o , . T~ OJ iV X o o rv -- o o y--4 o o o >1 10 Q iT CO CO co ro LU z> OJ co t ^ I iT> .75 in LJ OJ 0? CO 3 '?* ? ' CO co OJ 0? co hO<EHa--: <r 6 'i Z 3 $ <1 -- iT\ U"J m LJ in o i o j OJ r v 3 oj OJ DO - I fV CO OJ I/O c*LU OJ (V OJ ilS i'T) .T\ in OJ TOTAL OJ CO -T <1 01 X ,01 o i **o 1 'X* U co in -- *3 1 CO <x OJ t - o i in -- 01 o j ro i in 3 1 01 ' T i in " i i i u OJ CO in ^ c * cr* O ' PHl CO in . . tX i-- OJ co co o h- O -- (TJ .'O -Tj* 1s t A RT Fig.1-4 Gas chromatogram (Analysis of test water) day 10 Std. 0. 50// g/mI High exposure level low exposure level ooogo* -- iT \ iTS f '. CO CO UO Z> CO T b*> o K *Z' . , ZZ OJ T- CO f'.. id O rs -- o ( ji *- CO uo co IN. CO o UO m CO j? uo IN. z> o o .., T* OJ i-H r-'. 0? oN r^J or <x iCO TOTAL 715630 1GG CO OJ OJ --1 V OJ --* OJ 'Z ' TT fN CO o r_i . . OJ 1--1 NO iT i m K >- Z* r_) H O i=) CO CO " > r> CO <1 co m -- O ' \ CO o:Ul IN. UO bO T-l 1 *" *Z' fN. O ' *- 1 *7^ bO Z O ' iT 1 NT 1 iH f \ i *:>j T 1 'i> 1 1 1 co o-) co uo CO 3 co l \ O ' CO CO bO . OJ co co <1 1-- -- 1 2 4 o T" V 2 _ START Fig.1-5 Gas chromatogram (Analysis of test water) day 14 St d. 0 . 5 0 # g/ml High exposure level low exposure level -- r*. oj i '3 i 7-, t oj i T 3 fr. i o i ZI OJ r> 1 <z* o r>. 1 3 rj <V| *T. OJ OJ CO T CO '.0 3 ?' 'i? rji -7- f'% O' V ZI OJ 0? o o r>.. --* 3 fjl -~1 OJ *n l**7 z* *o iVi OJ CO IO .7* r-.. o. ZI OJ o 1--1 K _ 1 3 <500 6 0 ^ oL OJ '} t a: i-- H- ri*i CO OJ Fig.1-6 Gas chromatogram (Analysis of test water) day 17 Std. 0.50/zg/ral High exposure level co CO K in 'X* jZ' .V i K D CO U0 K CO , . z : CO >:* Is-. o o r-% O ' -s* OJ CO in tn CO --* sj> *x* CO i!* CO CO fs. IO CO UO X* T~ *'y. <X CO , 1T U i in OJ :o *z* 1 UO ce: k CO Si? 1 K <n CO CO -- CO 1 sj? --* in "X 1 CO ' 1 co 1 1 1 .. U i CO IO c CO ` in 'D r--i iT l CO 7'< L* <x h-- 1-- CO CO CO 'X l~ l 1-- in soO *'co < -- CO o xLd s j j <T> sjCi o .T> r j - . < f'*. OJ * S D w^ T in Ld Tn . k co :: si? in s j! |T> CO O ' i? CO CO CO y~ C<1ll cco OJ ** T H<*x CO O J CO low exposure level START 0006|0 CO U0 0? v* CO iT, 03 'tf' r*-. -- ID u*? CO Tv OJ fN. rn N. -- O O O =1 in X. iX in 03 fr ` O 1 CO LJ ` O -T z* *3* 1 OJ L il fs. CO 0 1 _ CO <1 0J *x* O' OJ 1 -X r-. *-< in 1 X r-. - * 1 in Lo 1 fVU TcO CTVj CUT.-. V^ - r r / LiJ n i--i h- 1 K OJ U" in iT, CO o .0 > CO 7' *> . OJ CO CO T _J <E H* l~ l -- v o OJ T Fig.1-7 Gas chromatogram (Analysis of test water) day 21 St d . 0 . 50# g/m High exposure level U".< 0? 0? fs* .T\ O'-. ro .7-. 3 O'* in OJ - CO OJ r_1 . OJ ^-1 0-. C ' o 3 r_'t -- r f o K 0> OJ to *:: o OJ >& 0*.' ^ . OJ r>. rj . T* OJ K 0? o fs. TART <r O ^ CO Oj rj- Of <r OJ " I" s/ O . CO G_ low exposure level O^Okll 1G G .6 5 '35 7 O'* IT*. K U*> ' I t CO 7* O 'Lt OJ CO r*. OJ -0 7** i'J i O OJ f\. 07 ID N. - i O O o -r~ Kb" <ur OKJ --DC Wf --. a 10 .7 '. OJ to D COo u~ 'C7O' '."0 OJ c<cx OJ 0'.* T CO C L TOTAL Fig.1-8 Gas chromatogram (Analysis of test water) day 24 St d. 0.50 u g/ml High exposure level T'. K l Z' CO CO o j 1 CO Z' CO OJ 1 0 UO T 1 11 OJ CO r-.. 1 *z I~1 CO \c r i*`" i r n 0 o > M 7 UJ O '* OJ -- u o l iT 'i cc CO CO f \ . 1 UO I Z ' CO u o i T . 1 O '* OJ fv . 'T 1 uo i7 1 CO l 1 1 UJ OJ OJ OJ 0 0 O'? z* *--1 O ' CO O ' o <x !-- h- OJ ;o CO T i~ i h- (_ OJ !*** u<1l CO CO CO *1? CO X' 00 w o *-o Z` OJ 00 r*-. ii OJ K X1 o fH o ~T~" oj cr. > > > CO X uo rs fN. rr. U l f-s. uo -- o Lt: --< uo CO CO < i O ' - fs. o*. -0 07 uo .. .. UJ CO 07 fs. uo OJ OJ r--' 7' CO 7' *i7 h- - . . . O j CO CO O J CO TART low exposure level -XI5000 00 1 V<F9Zd r^. T iT 'X* X* I'. .j-. O ' t T CO il* CO OJ 1o . . . CO 0 T* o fs. r~* Z4 o *- O i=J r> > 2?* K U"> M i CO 1 m > o i r - . co l ` O U" > m > J? 1 in CO 'T . 1m --< tn T*""* 1 <7> x i in 1 1 1 m k r>. O T C'J CO O ' CO O ' M? CO CO CO T _l <x f-- o h- -- cu rz }-- to D_ I t 11 0 1 (s t a r t START 000 611 Fig.1-9 Gas chromatogram (Analysis of test water) day 28 Std. 0. 50 f i g/m I High exposure level UO >T' CO OJ CO CO rr co co uo f... co z: co t\ CO (S. co co -O IT. co uo CO bO f\ . O ' OJ --. ' T'. Z ' 0J f.. 'X CO bO UJ .i 'X' Z OJ f'-* <X co co co co t* r^. oj co CO <x I0--? a: U J -tf- o*.' f\. 0% 'X uo CO fv . T r>. f'. rs bO OJ -- ij> o-? O' co co co H- OJ CO r r low exposure level T OJ r*. CO OJ bO iTv O ' fN. ` o UO bO -z. r-. i i i i CO 7? r - 1 CD Cj r-s. *-< 1 .31 2? 1<X CO CO u o CO 0 1U J CO T O ' -tf- J? O' 0 r . . <z< 0 1 bO a : CO u o CO CO 1 CD - I CO -J - OJ 1 CO bO 1 r - . 111 .. U J CO T~ ~T -- D __1 -- O ' ro O ' - < 1 1-- . Y~ CO CO CO T l" l h- O OJ r r N/ CL Fig.1-10 Gas chromatogram (Analysis of test water) day 31 Std. 0. 50/ / g/ml High exposure level OJ OJ OJ 1 o j 'Z ' OJ in l OJ 7-* 1 CO 00 'T* 1 I_1 . . , 1 zz OJ CO 1 Zi o K . 1 i~ i r_ 1 1 1 1 1 ! o zr i= \ CO OJ O' 0 iT. K -D CO uo OJ fv. CO uo CJ 07 l'_> . . . . z r OJ fN. CO o r>. O 2Z * " ** < r CO *- 1 tn U l 'rt ' r-.. 1 *T i : f '. ' 00 Z1 1 o I iT-. CO OJ r*.. 1 ro T-i i 'Sf 1 T 1 CO 1 1 1 UJ uo iv zz r*. _ j Ts 0! O ' o <i H- . . . I-- C'J CO CO 'T i-- c ' s/ O' - j. 7 IT; <1 CO 'Z* OJ LJ z* T ) C fs. 0 O' <1 -- C'J ~T UO uo ro j U tr* OJO .. n n V T K T Vo A / Y\ - n m -fn V LJ zz t--t h~ UO CO UO O' r \ 0 O ' p } T* *.D . OJ co CO h- o OJ CO TT C T" <z l h - LU co o -- OJ CO TT zz 1C TART OOOGiM low exposure level fN. O') N CO o ij -- 'U `I? CO r--. h : OJ 1*__T o fv. *- 100 714352 TOTAL <1 C'J O ' IjJ OJ CO -- OJ OJ OJ -- IT; UO iT. U OJ to o <r hjt<--Xr OJ CO 0 `J Fig.1-11 Gas chromatogram (Analysis of test water) day 35 Std. 0. 50 /u. g/ral High exposure level low exposure level o O O Fig. 1-12 Gas chromatogram (Analysis of test water) day 38 Std.0.50// g/mI High exposure level CO -- in 1 ~T CO r. 1 K *1* z rx . 1 OJ `X* . in to 1 1 z : C'J -- *10 n . 1 z? o N. -- 1 <_> l 1 1 1 1 o ~r~ i= i 11 s/ n :> > <x |V| is . 1*0 1 iY i u T in co l iV i L *T fN. 1 IJN <x 10 co CO 'i f 1 7'* -rr 1 1 1*1*1 1 I 1 UJ fN. C*J CO 7^ o.. fx. 01* O' CO 2i X <1 1-- f-- OJ r f r~ b~ r in O T CO *X` CTO fx. cr. CU Mn z> r. t-H o 'ro - TT- V \J \ \ o OJ CO ZI b~ Cd <1 b~ iti CO *T C'J OJ fx . CO '> -- OJ rs r-.. CO C'J x rj . , , T " C'J 0J o X o fx. ZI 1' N/ z r :> > > < r in co X ' fx . UJ X r. r \ Ld* co b : x> in < t z* b . 7' 7 * C-J -* X CO in UJ CO CO fx. T~ fx. in in x> - r *x* r-. I" CO co Tt* T o 0J T ~r~ - TART low exposure level T bO Z *0 *T i j j 'T D V .T> oj OJ CO o o N OJ v9 19 % 1co - i to T' CO 1r>. f^ . O-. OJ rv. ~T oj U0 - -- fN. 1(v. 0 > CD O ' r^. --1 1T- fS. uo ' 'T 11J1 -o 0bo co co ? O ' P '. N . CO _J 0<L 7> o? O ' <x h- . . . -- t- OJ co CO o START O oj iX Fig.1-13 Gas chromatogram (Analysis of test water) low exposure level ooo &n ir o j co m ' ^ O ' oj iT t iV i CO .IO U"; OJ z : oj ' O'4 10 0 '0 OJ OJ CO o in --* O ' r \. OJ 0 .T-. OJ i\ . CO O K Z r_j i_i i=4 7 U 4 3-3 7 CO co -.c o oj OJ CO CO 0 ! U"i cr% 0"? O ' OJ CO <x -- T --- ,Z , LU IT -O O'* z* <1l --* 01 OJ in 'i- c? f t-H -- ^ .T-, OJ -- CoO t -D U CO OJ L fj u~) -Ti TOTAL tle.' ~ OJ CO T -.1 iyyia Fig.1-14 Gas chromatogram (Analysis of test water) day 45 High exposure level 'O U~ fs. cr of OJ ic*0o OJ H I O'* OJ o j --. r\. rs K ` jO C O *t s I T ; - .0 r f -T --' CO z : co .> 0*. rs ^ IO O 7' |T. U"* OJ O -- *-< *.j.' J7 Z <x UO .T\ u*? CdUJ U' - -.o O'* K a : OJ |TV OJ CO *!i* <X u i OJ fN m n *7-* ir . 7- OJ f--* Z1 ,*T i i 07 1- . . . o:* co * * <1CC OJ 'T <E low exposure level O d O 6ii! U0 il' 07 o in ' 0 O J f-^ T OJ in O C`J in r. z* T'. -- o i*_> 1 i1 0 774Q75 TOTAL Fig. 1 15 Gas chromatogram (Analysis of test water) High exposure level ID ' - T OJ <N V h<CiZ ' co *> OJ IT\ --i \t f \ *.jD ID T O ' tD OJ K CO r x -- o 1=1 <1 ' CO UJ ' T'. <xcz Z< m? id OJ 0*. Ld ID OJ -- u"` CO OD 'f a UL *X low exposure level CO T CO in CO I? id T- CO ID O ' T i~n ;*j fx OJ rx o o 52 vi> B 0 >>> <1 OJ *0 'O 1 OJ UJ in cu ,ys 1 CO 'XGC -- OJ 1 CO T\ CO 1 OD CO 1 OJ 1 *jD 1 1 1 co in 05 -- 'r to -- 55 . CO ^ T <X O -- START Fig.1-16 Gas chromatogram (Analysis of test water) day 52 High exposure level CO CO ` O r*-. -- r*. Xt CO z: o j I S . 2 ? 14 1Q 9 7 4 V OJ CO <1 <x iTS u coOJ -. <X -- r-. or* it * O CO O'? OJ START low exposure level q -9 ooO 1O0 cr. *0 TC` rs TO f \ tT oo TO \ n T *rf OJ OJ ZI* o . . zz OJ OJ CO o is. x> o t-H o ZI ,--1 \y ZZ "5. > > <1 1 0? LU 0 id *-* 1 1-4 'Sf fs. --* ITS O J a in TO 7 > r-i 1 ,0 --< .-1 f s . 1 OJ ' t 1 1? t 1 1 vUJ .V r. CO z z CO O in _J r-i TO OJ <E h- .. K- CO CO ' T in O 1-- v o OJ *r 563530 TOTAL Fig.1-17 Gas chromatogram (Analysis of test water) day 56 High exposure level `- -- f^. T Z' rs. co u*> X -- * * 'if 10 . . OJ V-i O ' r*. --* i~ i ?* to U" ;; f \ ^ OJ KC' iJ n n *t * -- OJ hcn~ -x ; 0* OJ X* o CD to OJ CO -- co r*. ?' OJ r> z ; OJ OJ -x K o o *s<x 10 OJ _ in lJ CO OJ --4 OJ fs. L 7> OJ rv. OJ U' <x K K OJ CO in CO -x in UJ f'-. uo CO CO 2Z .T s 0? CO OJ T-. 7' CO X4 CO *.o i-- OJ 0? T OJ CO *T uo START yOOfeZ-l low exposure level oo f v T' 'll* k :o oo z* r\. o OJ T- oo oo t/'l rn U-) 05 rv z <z 1u G 7 i- 4 7 Z 1 T0 TAL <X 5' lO ..o iX LO 00 f\ . <x Z* O C'J :u oj ` 0 z* *T% *z* rr> cc: OJ T <x f-- O')