Document XzgRKxVO4e70VpgpDZ20g11QB

Final Report Bioconcentration Test of S510 in Carp, Cyprinus carpio Report No. E4 -15022C277CH July 15, 2015 Institute of Ecotoxicology Co., Ltd. 1 Table of contents Page Statement i Quality Assurance Certificate ii Preface 1. Title 1 2. Sponsor 1 3. Test facility 1 4. Objective of the test 1 5. Test methods 1 6. GLP applied 1 7. Name of the test substance 2 8. Test periods 2 9. Test results 2 10. Evaluation and discussion of the results and conclusion 2 11. Storage of materials 3 12. Environmental factors that may have affected data reliability 3 13. Personnel related to the test and their assignments 3 14. Names and affiliations of experts involved in reporting the final report 4 15. Approval of the final report 4 Main Subject 1. Test substance 5 1.1 Name 5 1.2 Structural formula 5 1.3 Supplier and Lot No. 5 1.4 Identification 5 1.5 Physico-chemical properties 5 2. Test methods and analytical methods 6 2.1 Acute toxicity test (96-h LC50) in Zebrafish (Danio rerio) for selecting 6 the substance concentration 2.2 Performance of the bioconcentration test 7 2.3 Analyses of fish and test water 10 2 2.3.1 Analytical method for fish 10 2.3.2 Analytical method for test water 11 2.3.3 Quantification method 12 2.4 Calibration curve by high performance liquid chromatography (HPLC) 14 2.4.1 Preparation of standard solutions 14 2.4.2 Preparation of calibration curve 14 2.5 Calibration curve for water analysis 14 2.5.1 Preparation of standard solutions 14 2.5.2 Preparation of calibration curve 14 2.6 Recovery test and blank test for the test substance 15 2.6.1 Recovery test from fish 15 2.6.2 Recovery test from test water 16 2.7 Quantification limit 17 2.7.1 Quantification limit for fish 17 2.7.2 Quantification limit for test water 18 2.8 Preliminary test 18 2.9 Schedule of analyses for fish and test water 18 3. Test results 19 3.1 Concentration of the test substance in test water 19 3.2 Bioconcentration factor (BCF) 20 3.3 Evaluation, discussion of the test results and conclusion 21 Table of contents for references 22 This report includes 91 pages 3 Statement Institute of Ecotoxicology Co., Ltd. Sponsor: JNC Corporation Title of the Test: Bioconcentration Test of S510 in Carp, Cyprinus carpio Test No.: C-277 The aforementioned test was conducted in compliance with following GLP principles: "Standards Concerning Testing Laboratories Implementing Tests Concerning New Chemical Substances" on Japanese GLP (Yakushokuhatsu 0331 No. 8, Director-General, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare, Heisei 230331; Seikyoku No. 6, Director-General, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; and Kanpokihatsu No. 110331010, Joint Notice from the Director General of the Environmental Policy Bureau, Ministry of the Environment, Heisei 230329), and "OECD Principles of Good Laboratory Practice" (as revised in 1997). July 15, 2015 Study Director: Signed in original Takashi Sato (seal) i Report No. E4 -15022 C277 CH Quality Assurance Certificate Test No. C-277 Title: Sponsor: Study Director: Study Personnel: Test Facility: Bioconcentration Test of S510 in Carp, Cyprinus carpio JNC Corporation Takashi Sato Takashi Sato Institute of Ecotoxicology Co., Ltd. We certify that the present final report describes the test methods accurately and the contents were performed according to the test protocol and the standard operation procedures, and the original data are accurately presented. The inspection and audit were performed as follows. Inspection of test performance Audit of test protocol Audit of final report Water/fish analysis on Day 7 of exposure Water/fish analysis on Day 14 of exposure Water/fish analysis on Day 21 of exposure Protocol Revised protocol Alteration in revised protocol Final report Date of Inspection/audit June 17, 2015 June 24, 2015 July 1, 2015 May 11, 2015 June 8, 2015 July 13, 2015 July 9, 2015 July 15, 2015 Report date to Test Facility Management and Study Director June 17, 2015 June 24, 2015 July 1, 2015 May 11, 2015 June 8, 2015 July 13, 2015 July 9, 2015 July 15, 2015 Institute of Ecotoxicology Co., Ltd., Department of Quality Assurance Unit Quality Assurance Director : Signed in original Mikiyasu Goto (seal) Signature date: July 15, 2015 (Inspection/audit of study conduct, protocol and final report) Quality Assurance : Signed in original Mitsuko Takamatsu (seal) Signature date: July 15, 2015 (Inspection/audit of study conduct, protocol and final report) Quality Assurance : Signed in original Miyuki Kuribara (seal) Signature date: July 15, 2015 (Audit of protocol and final report) ii Final Report -- Preface -- 1. Title Bioconcentration Test of S510 in Carp, Cyprinus carpio 2. Sponsor JNC Corporation 2-2-1 Otemachi, Chiyoda-ku, Tokyo 100-8105, Japan 3. Test facility Institute of Ecotoxicology Co., Ltd. 2-28-1 Yoshino-cho, Kita-ku, Saitama City, 331-0811 Japan Phone Representative Mikiyasu Goto Test Facility Management Tadayoshi Kan 4. Objective of the test The objective of this test is to determine the degree of bioconcentration of the test substance in fish. 5. Test methods The methods used in this test were based on "Test Methods Concerning New Chemical Substances < Bioconcentration Test of Chemical Substances in the Bodies of Fish and Shellfish>" (Yakushokuhatsu 0331 No.7, Director-General, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare, Heisei 23.03.31; Seikyoku No.5, DirectorGeneral, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; and Kanpokihatsu No.110331009, Joint Notice from the Director General of the Environmental Policy Bureau, Ministry of the Environment, Heisei 23.03.29); and OECD Guidelines for the Testing of Chemicals, "No. 305 Bioconcentration: Flow-through Fish Test" (adopted on October 2, 2012). 6. GLP applied Japanese Chemical Substance GLP: "Standards Concerning Testing Laboratories Implementing Tests Concerning New Chemical Substances" (Yakushokuhatsu 0331 No. 8, Director-General, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare, Heisei 23.03 31; Seikyoku No. 6, Director-General, Manufacturing Industries Bureau, Ministry of Economy, 1 Trade and Industry; and Kanpokihatsu No. 110331010, Joint Notice from the Director General of the Environmental Policy Bureau, Ministry of the Environment, Heisei 230329). OECD GLP: "OECD Principles of Good Laboratory Practice" (as revised in 1997). 7. Name of the test substance Chemical name : trans - 4-{[3,5-difluoro- 4-(trifluoromethyl)phenoxy] (difluoro)methyl}-4'- trans -propyl-1,1'- bicyclohexyl Abbreviation : S510 (Lot No. 14NA007) CAS No. : 208338-86-7 8. Test periods (1) Test initiation date : May 11, 2015 (2) Duration of the test Arrival date of fish : May 18, 2015 Acclimation completion date : June 10, 2015 Experimental starting date : June 10, 2015 Experimental completion date : July 8, 2015 (3) Test completion date : July 15, 2015 9. Test results A bioconcentration test of the test substance S510 in carp, Cyprinus carpio, was conducted. The bioconcentration factor (BCF) for 28 days of exposure was summarized in the following table. Concentration level of exposure Bioconcentration factor (BCF) High (Level 1) 0.02 mgL-1 12 to 30-fold Low (Level 2) 0.002 mgL-1 10 to 29-fold 10. Evaluation and discussion of the results and conclusion The acute toxicity (96-h LC50) of the test substance S510 in Zebrafish (Danio rerio) was greater than 25.6 mgL-1. Since the solubility in water was 2.3 10-2 mgL-1, concentrations of the test substance in water were set at 0.02 mgL-1 for the high exposure level (Level 1) and 0.002 mgL-1 for the low exposure level (Level 2). According to the results, the bioconcentration factor (BCF) was less than 50-fold both in Levels 1 and Level 2. Thus, steady state was considered to have reached by day 28 of exposure in both levels. As a result, bioconcentration of the test substance in fish was confirmed to be low. 2 11. Storage of materials The following records concerning the tests and samples will be stored for 10 years after receiving notification based on the provisions of CSCL (No. 117, 1973) Article 4, paragraphs 1 or 2, Article 5, paragraph 2, 3, or 8, Article 10, paragraph 3, or Article 14, paragraph 2, and the test substance, reference substance and other item will be stored for 10 years after receiving notification based on the provisions of the CSCL: Article 4, paragraph 1 or 2, Article 5, paragraph 2, 3, or 8, Article 10, paragraph 3, or Article 14, paragraph 2, or the period during which they can be stored stably without affecting their quality, whichever is shorter, at the Institute of Ecotoxicology Co., Ltd.. Storage after the period will be determined after separate discussion. (1) Protocol, and its modifications and changes (2) Records concerning the test substance, etc. (3) Experimental notes, raw data, and charts (4) Test substance, reference substance, and other items (at approximately 20C in the dark) (5) Inspection and audit records by quality assurance personnel (6) Final report (7) Other necessary items 12. Environmental factors that may have affected data reliability No particular abnormalities were observed. 13. Personnel related to the test and their assignments Study Director: Takashi Sato Study Personnel: Takashi Sato 96-h LC50 test Measurement of water solubility Establishment of analysis method for test water Establishment of analysis method for fish Preparation of calibration curves Recovery tests from test water Recovery tests from test fish Investigation of dispersion method of the test substance in water Preliminary test to set the test schedule Analysis of test water and fish during the experiment period Preparation and maintenance of test solution during the experiment period Measurement of aquatic environment during the experiment period Measurement of lipid contents in fish body 3 Measurement of IR Measurement of molar absorption coefficient Observation of fish rearing conditions Measurement of dissolved organic carbon concentration Preparation of final report: Takashi Sato 14. Names and affiliations of experts involved in reporting the final report None 15. Approval of the final report Study Director: Signed in original Takashi Sato (seal) Signature date: July 15, 2015 4 Main Subject 1. Test substance In the present report, the test substance has the following name and structural formula (based on the documents provided by the sponsor). 1.1 Name Chemical name: Abbreviation: CAS No.: trans - 4-{[3,5-difluoro- 4-(trifluoromethyl)phenoxy] (difluoro)methyl}-4'- trans -propyl-1,1'- bicyclohexyl S510 208338-86-7 1.2 Structural formula Molecular formula: C23H29F7O (MW 454.46) F O F F F F F F 1.3 Supplier and Lot No. (1) Supplier JNC Corporation (2) Lot No. 14NA007 1.4 Identification The IR spectrum of the test substance obtained before the test was compared with that from the sponsor, and their agreement was confirmed. Further, stability of the test substance during the test period was confirmed by the agreement of the IR spectrum obtained after the test with that measured before the test (see, Ref. 13). 1.5 Physico-chemical properties (*based on the data provided by the sponsor) Appearance*: White powder Purity*: 99.9% Impurities unknown0.1% Melting point*: 83C Solubility (our measurements at 25C) water 2.3 10-2 mgL-1 (Flask method) n-hexane > 1.0 105 mgL-1 dichloromethane > 1.0 105 mgL-1 acetone > 1.0 105 mgL-1 5 acetonitrile dimethylsulfoxide 1.0 104 mgL-1 1.2 103 mgL-1 2. Test methods and analytical methods This test was based on "Test Methods Concerning New Chemical Substances < Bioconcentration Test of Chemical Substances in the Bodies of Fish and Shellfish>" (Yakushokuhatsu No. 0331-7, Heisei 23/3/31; Seikyoku No. 5, Kanpokihatsu No. 110331009, Heisei 23/3/29; and OECD Guidelines for Testing of Chemicals, "No.305, Bioaccumulation in Fish: Aqueous and Dietary Exposure" (adopted on October 2, 2012). 2.1 Acute toxicity test (96-h LC50) in Zebrafish (Danio rerio) for setting the substance concentration (SOP 2.6) A 96-h LC50 value was obtained at concentrations set based on the results of preliminary test (performed with reference to the method of OECD Test Guideline 203). (1) Test fish Zebrafish (Danio rerio) Lot No. : 150310 Supplier: Kamihata Fish Ind., Ltd. (292-1 Chigusa-cho, Hanamigawa-ku, Chiba City, 262-0012 Japan) Date received: March 10, 2015 Mean total length: 3.4 0.1 cm Mean bodyweight: 0.25 0.02 g Acclimation period: from March 10 to May 12, 2015 Receiving and acclimation procedures were based on SOP 2.1 and SOP 2.5. (2) Test solution Vehicle (Dispersant): Polyoxyethylene Sorbitan Monooleate (Tween 80) Preparation of a stock solution: A test substance and 4 times the amount of Tween 80 as the test substance were weighed into a glass sample tube and mixed while heating. Boiling ultrapure water was added to the mixture. After cooling to room temperature with stirring, ultrapure water was added to prepare a 1000 mgL-1 stock solution. This stock solution was diluted with test for water (see (6)) to obtain test water. (3) Test temperature 25.1 0.1C (4) Test aquarium Glass with 4 L capacity (5) Exposure method Semi-static method (replacing water after 48 hours) (6) Water for test (water for dilution) Dechlorinated city water of Saitama (hereafter, referred to as "water") 6 As a result of the water quality test, it was confirmed that the items shown in Reference 10 comply with the "water quality standards for fisheries water". (7) Number of fish 10 fish/4 L test aquarium (8) Experiment period from May 12 to May 16, 2015 (9) Sensitivity test of Danio rerio A sensitivity test was conducted using CuSO45H2O as a standard substance, and fish from the same lot that passed our specified value (96-h LC50: 0.08 to 0.3 mgL-1) were used as test fish. (10) Test results The test substance was dispersed using a 4-times amount of Tween 80. When the concentration of the test substance should be set so that Tween 80 concentration does not exceed 100 mgL-1, the upper limit of test substance concentration is 25 mgL-1. Since the 96-h LC50 value in the preliminary test was greater than 25 mgL-1, a limit test with a maximum concentration of 25 mgL-1 was applied. As a result, the 96-h LC50 value was confirmed to be greater than 25.6 mgL1. The concentration of the test substance is the initial measured value determined by HPLC (see the table below). Test water Mortality (%) Compound Con(mcegnLtr-a1)tion 24 h 48 h 72 h 96 h CuSO45H2O (sensitivity test) 0.5 50 80 100 100 0.05 0 0 0 0 Test substance 25.6 0 0 0 0 Control (water with Tween 80) 100 Control (water) - 0 0 0 0 0 0 0 0 2.2 Performance of the bioconcentration test (1) Test fish To confirm the bioconcentration in fish, the carp (Cyprinus carpio), representative fish species in Japan, was selected (SOP 2. 1-2) as the test fish. Common carp: Cyprinus carpio (Lot No. 150518 No.3) Supplier: Kitamura Carp Breeding Farm (12-388 Gunchiku, Yashiro-shi, Kumamoto Prefecture, Japan) Date purchased: May 18, 2015 Feeding and acclimation: After being received by the Institute of Ecotoxicology Co., Ltd., the fish with a total length of approx. 8.0 4.0 cm were selected (SOP 2.4). After treatment with Elevage (4-[2-(5-nitro-2-furanyl)ethenyl]benzoic acid, sodium salt) and rock salt, acclimation was started in acclimation tank No. 1 (SOP2.3, SOP2.5). 7 Test fish: Finally, they were acclimatized at water temperature 25 2.0C for 7 days and used for the test, after confirming that the mortality rate was less than 5%. Mean body weight 4.45 0.33 g (SOP 2.8) Mean total length 7.8 0.1 cm (SOP 2.8) Mortality of fish during the experiment (SOP 2.7) High exposure level 0% Low exposure level 0% Control 0% Lipid contents (by chloroform/methanol extraction method) Before experiment 4.1% (Mean value, n=2) (SOP 2.9) After experiment 3.8% (Mean value, n=2) (SOP 2.9) (The conditions of the test fish were shown in Ref. 11) (2) Feeding pellet Feed type: Maruha-jirushi compound feed for juvenile carp C-2 Crude proteins 42.0% Crude lipids 3.0% Manufacturer: Hayashikane Sangyo Co., Ltd. Feeding method: fed every day in an amount of approximately 1.5% of the bodyweight, except the day before sampling on which no feed was given. (3) Conduct of the test Water for test: as described in Section 2.1 (6). Solution of the test substance Preparation of a stock solution: A test substance and 4 times its amount of Tween 80 were weighed into a glass sample tube and mixed while heating. Boiling ultrapure water was added to the mixture. After cooling to room temperature with stirring, ultrapure water was added to prepare a 1000 mgL-1 stock solution. This stock solution was diluted with water to obtain test water. High exposure level (Level 1): The stock solution was diluted with water to 2.0 mgL-1. This was further diluted with water and continuously supplied to the test aquarium. Low exposure level (Level 2): The stock solution was diluted with water to 0.2 mgL-1. This was further diluted with water and continuously supplied to the test aquarium. 8 Control: A 8 mgL-1 solution of Tween 80 was diluted with water and continuously supplied to the test aquarium. Bioconcentration test equipment: see Ref. 1 Test aquarium: "Flow-through system" Glass with 100 L capacity (amount of test water 50 L) Flow rate: 300 mL min-1 (432 L day-1) No. 1: High exposure level (Level 1), No. 2: Low exposure Level (Level 2), No. 3: Control Test concentration: The concentration of the test substance was set below its solubility in water i.e. 2.3 10-2 mgL-1. Since the 96-h LC50 in the zebrafish was greater than 25.6 mgL-1, 1/1280 and 1/12800 were used for safety factors, and the concentrations were set as shown in the following table. Exposure level Concentration of the test substance Concentration of Tween 80 High (Level 1) 0.02 mgL-1 0.08 mgL-1 Low (Level 2) 0.002 mgL-1 0.008 mgL-1 Control - 0.08 mgL-1 Test condition: Temperature of test water: 24.7 0.3C pH of test water: 7.6 0.1 DO of test water: 7.36 0.22 mgL-1 (Temperature, pH and DO on each analytical day were shown in Ref. 2-2) Total organic carbon concentrations in the test water (SOP 2.12): High exposure level (Level 1) 0.4886 mgL-1 Low exposure level (Level 2) 0.1364 mgL-1 Control (with dispersant) 0.2262 mgL-1 (10 mgL-1 or less in all 3 groups). Light type: UV- cut fluorescent lamp Photoperiod: Approximately 10 hours per day Experiment period: from June 10 to July 8, 2015 Analysis schedule: shown in Section 2.8. Number of test fish: High exposure level (Level 1) 45 Low exposure level (Level 2) 45 Control 15 Number of analyzed fish and number of repetitions: 2 fish were paired for a single analysis with 2 repetitions (n=2) 2 fish were paired for analysis of the Control Group (n=1) 9 2.3 Analyses of fish and test water 2.3.1 Analytical method for fish (SOP 2.11) Pretreatment: On each analysis day, 6 fish each from Level 1 and Level 2, and 2 fish from Control are sampled using a landing net. After immersing them in ice water and suspending them, the surface of the fish was washed with tap water and then lightly wiped with gauze. Body weight was measured to the nearest 0.01 g on a balance (SOP/INS/4.2.2), and the total length, from the tip of the mouth to the tip of the tail fin, to the nearest 0.1 cm. In each test group, 2 fish were used as one sample in duplicate analysis. Remaining fish are put into a plastic container and stored in a freezer after wrapped separately with aluminum-foil and packed in a plastic bag (SOP 2.15). Fish were put into a homogenizer cup after slicing. After adding 70 mL of acetonitrile, the mixture was homogenized at 9000 r.p.m. for 6 min (SOP/INS/4.17.2). The homogenate was transferred to a centrifuge tube. The homogenizer cup was rinsed twice with 5 mL of acetonitrile and the washings were added to the centrifuge tube. After centrifugation for 15 min (9000 r.p.m., at approximately 10C) (SOP/INS/4.16.3), the supernatant was filtered (Advantech Toyo glass filter GC50) into a 100 mL measuring flask. The filter paper was rinsed with 5 mL of acetonitrile, and the volume was adjusted to 100 mL with acetonitrile. 10 mL was aliquoted to a 50 mL round-bottom flask and dried under reduced pressure using a rotary evaporator (SOP/INS/4.14.3.2) (water bath 40C). The residue was dried by blowing air. Separately, a Sep-Pak Florisil (Waters, Milford, MA, USA) was conditioned by eluting 5 mL of n-hexane. The fish extract in the round-bottom flask was dissolved in 3 mL of n-hexane and injected into the conditioned Sep-Pak florisil, and processed in the following order. (1) The inside of the round-bottom flask was washed with 5 mL of n-hexane, and the washing was injected. (2) 5 mL of n-hexane/dichloromethane (2:1, v/v) was injected into the Sep-Pak, and the eluates (1) and (2) were received in a 50 mL round-bottom flask. The flow rate was approximately 3 mL/min throughout (1) and (2). The eluate was dried using a rotary evaporator (water bath 40C). The dried content was dissolved in a small amount of acetonitrile, and transferred to a 1 mL measuring flask while washing. The volume was adjusted to 1 mL and used for high performance liquid chromatography (HPLC) measurement. The pretreatment process is shown in the following flowsheet. 10 Residue Sep-Pak Florisil Conditioning n-Hexane 5 mL Test fish (1 sample with two fish) Measurements of the total length and bodyweight Slicing Transferred to a homogenizer cup Acetonitrile 70 mL Homogenized (9000 r.p.m., 6 min.) Acetonitrile 25 mL (Washing the homogenizer cup) Centrifuge (9000 r.p.m., at approximately 10C, 15 min.) Supernatant Filter (Advantech Toyo glass filter GC50) into a 100 mL volumetric flask Acetonitrile 5 mL (wash the filter paper) Adjust to 100 mL with Acetonitrile Fixed volume solution Take 10 mL by a hole pipette Evaporate under reduced pressure (Water bath 40C) n-Hexane 3 mL Development (1) n-Hexane 5 mL) (2) n-Hexane/dichloromethane (2:1) 5 mL Eluates (1), (2) Evaporate under reduced pressure (Water bath 40C) Acetonitrile Adjust to 1 mL with Acetonitrile Fixed volume solution HPLC 2.3.2 Analytical method for test water (SOP 2.10) Pretreatment: Test water, approximately 200 mL and 300 mL from Level 1 and 2, respectively, was sampled from the central area of each aquarium. After waiting for sedimentation of floating dust and etc., a supernatant, 100 mL and 200 mL for Level 1 and 2, respectively, was measured using a measuring cylinder, and transferred to a 200 mL and a 300 mL separatory funnel, respectively. The cylinder was rinsed with a small amount of ultrapure water and the washings were added to the separatory funnel. Dichloromethane (50 mL each for Level 1 and Level 2) was added and the contents were shaken vigorously for 5 min (SOP/INS/4.15). After standing until the two layers become transparent, the lower layer (dichloromethane) was transferred to a 100 mL Erlenmeyer flask. 10 g of anhydrous sodium sulfate was added and allowed to stand for 30 min or longer to 11 remove water. After that, sodium sulfate was removed by filtering (Advantech Toyo glass filter GC50) into a 100 mL round-bottom flask. The filter paper and the flask were washed three times with 5 mL of dichloromethane, and washings were also filtered to the round-bottom flask. The filtrate was evaporated to dryness (water bath 40C) using a rotary evaporator (SOP/INS/4.14.3.2). The dried content was dissolved in a small amount of acetonitrile and transferred to the measuring flask (10 mL for Level 1 and 2 mL for Level 2) while washing. After adjusting to each volume, resulting solution was used for HPLC measurement. The pretreatment process is shown in the following flowsheet. Upper phase (Aqueous layer) Test water Sampling: 200 mL for Level 1, 300 mL for Level 2, (wait for sedimentation of floating dust, etc.) Measurement: 100 mL for Level 1, 200 mL for Level 2 (with a measuring cylinder) Transfer to a separatory funnel Wash the cylinder with a small amount of ultrapure water Dichloromethane 50 mL Shake at Gauge 7 for 5 min Stand until the 2 layers became clear Lower phase (Dichloromethane) Transfer to a 100 mL Erlenmeyer flask Anhydrous sodium sulfate 10 g (Dried for more than 30 min) Filter (Advantech Toyo glass filter GC50) Dichloromethane 5 mLx3 (wash the Erlenmeyer flask and filter paper) Filtrate (Level 1) Filtrate (Level 2) Evaporate to dryness (Water bath 40C) Acetonitrile Adjust to 10 mL with Acetonitrile Fixed volume solution Evaporate to dryness (Water bath 40C Acetonitrile Adjust to 2 mL with Acetonitrile HPLC The water analysis for Control at the start of the test followed the method for Level 2. 2.3.3 Quantification method [for fish analysis] <Apparatus> High performance liquid chromatography LC-10A (Shimadzu Corporation) System No. 6 (SOP/INS/4.11.5) 12 <Analytical conditions> Column Asahipak C8-50 4E 4.6 250 mm (5 m) (Lot No. P103321) Showa Denko K.K. Mobile phase Acetonitrile/water (85:15) Flow rate 1.0 mL/min Temperature 40C Detector UV-VIS 214 nm Injection rate 20 L <Quantitative calculation> Instrument LCsolution (Shimadzu Corporation) Calculation method 2-point calibration method (Absolute calibration method) The quantification of samples was performed on the analysis day by calibration using two standard solutions. [For water analysis] < Apparatus > High performance liquid chromatography LC-10A (Shimadzu Corporation) System No. 6 (SOP/INS/4.11.5) <Analytical conditions> Column InertSustain C18 4.6 250 mm (5 m) (Lot No. 1BR98005) GL Sciences Inc. Mobile phase Acetonitrile Flow rate 1.0 mL/min Temperature 40C Detector UV-VIS 214 nm Injection rate 20 L <Quantitative calculation> Instrument LCsolution (Shimadzu Corporation) Calculation method Two-point calibration method (Absolute calibration method) The quantification of samples was performed on the analysis day by calibration using two standard solutions. The UV/visible absorption spectrum of the test substance is shown in Ref. 12. 13 2.4 Calibration curve by HPLC for fish analysis 2.4.1 Preparation of standard solutions The test substance, each 4.90 mg and 3.10 mg, was weighed with a balance, Mettler Toledo XS105DU (SOP/INS/4.1.4), into a 50 mL measuring flask, and the volume was adjusted with acetonitrile. The concentrations of these solutions were 97.9020 mgL-1 and 61.9380 mgL-1, respectively, when considering the purity. Each solution was diluted with acetonitrile to prepare standard solutions for calibration curve. 2.4.2 Preparation of calibration curve A calibration curve was prepared using the standard solutions prepared in Section 2.4.1 to confirm the linearity for quantitative measurement. Solution No. 1 2 3 4 5 Concentration [X] (mg L-1) 0.0979 0.6194 0.9790 6.1938 9.7902 Peak area [Y] 2151 15037 23500 154396 237859 Peak area [Y] 240000 120000 Correlation formula: Y = 24459.1955 X + 99.4172 (by least square method) Correlation coefficient: r = 1.000 0 [X] 0 5 10 Concentration (mgL-1) The chromatograms for preparing the calibration curve is shown in Ref. 6-1. 2.5 Calibration curve by HPLC for water analysis 2.5.1 Preparation of standard solutions The test substance, each 4.82 mg and 3.16 mg, was weighed with a balance, Mettler Toledo XS105DU (SOP/INS/4.1.4), into a 50 mL measuring flask, and the volume was adjusted with acetonitrile. The concentrations of these solutions were 96.3036 mgL-1 and 63.1368 mgL-1, respectively, when considering the purity. Each solution was diluted with acetonitrile to prepare standard solutions for calibration curve. 2.5.2 Preparation of calibration curve A calibration curve was prepared using the standard solutions prepared in Section 2.5.1 to confirm the linearity for quantitative measurement. 14 Solution No. 1 2 3 4 5 Concentration [X] (mg L-1) 0.0963 0.6314 0.9630 6.3137 9.6304 Peak area [Y] 2745 18011 27867 178107 275278 Peak area [Y] 280000 140000 Correlation formula: Y = 28482.0941 X53.6066 (by least square method) Correlation coefficient: r = 1.000 0 0 5 10 [X] Concentration (mgL-1) The chromatograms for preparing the calibration curve is shown in Ref. 6-2. 2.6 Recovery test and blank test for the test substance 2.6.1 Recovery from fish (SOP 2.11) Fish bodies (two fish for one sample) were crashed with a blender after mincing. After adding the acetonitrile solution (0.2 mL) of the test substance, pretreatment was processed according to "2.3.1 Analytical method for fish" for HPLC analysis. The recovery rates were shown in the following table. The amount of test substance added, 1.964 g, was calculated to be 0.376 gg-1 for a fish weight of 2.61 g (5.22 g/2 fish), which is equivalent to a bioconcentration factor (BCF) of approximately 188-fold in Level 2 (0.002 mgL-1) group. Added group Sample No. Fish weight (g) 1 22..7473 2 22..5545 3 22..6569 Fixed volume (mL) Concentration factor 100 1/10 100 1/10 100 1/10 Amount added Measured area Calculated concentration (mgL-1) 4594 0.1964 Amount recovered (measured area) 3936 Recovery rate (%) 85.7 4594 0.1964 3912 85.2 4594 0.1964 3613 78.6 Mean recovery rate: 83.2 3.2% <Background of the fish> After measuring weight and total length, fish bodies (two fish as one sample) were crashed with a blender after mincing. After adding 0.2 mL of acetonitrile, pretreatment was processed 15 according to "2.3.1 Analytical method for fish" for HPLC analysis. No peak corresponding to the test substance was detected. Control group Sample No. Fish bodyweight (g) 1 32..0416 2 22..4703 3 22..8444 Fixed volume (mL) 100 100 100 Concentration factor 1/10 1/10 1/10 Measured area N.D. N.D. N.D. The chromatograms for calculating the recovery rates from fish are shown in Ref. 7-1. 2.6.2 Recovery from test water (SOP 2.10) Water for fish breeding was collected, and the test substance solution (dispersed with Tween 80) was added. Pretreatment was processed according to "2.3.2 Analytical method for water" for HPLC analysis. As shown in the following table, the mean recovery rates of the test substance were 91.8 1.2% and 97.8 1.4% for Level 1 and Level 2, respectively. Recovery rate for Level 1 (Added group) Sample No. Amount of water sampled (mL) Fixed volume (mL) Amount added Measured area Calculated concentration (mgL-1) 1 100 10 5521 0.1932 Amount recovered (measured area) 5027 Recovery rate (%) 91.0 2 100 10 5521 0.1932 5005 90.7 3 100 10 5521 0.1932 5160 93.5 Mean recovery rate: 91.8 1.2% Recovery rate for Level 2 (Added group) Sample No. Amount of water sampled (mL) Fixed volume (mL) Amount added Measured areas Calculated concentration (mgL-1) Amount recovered (measured area) Recovery rate (%) 1 200 2 5590 0.1975 5394 96.5 2 200 2 5590 0.1975 5433 97.2 3 200 2 5590 0.1975 5578 99.8 Mean recovery rate: 97.8 1.4% 16 <Background of the test water> Water for fish breeding was collected, and Tween 80 was added. Pretreatment was processed according to "2.3.2 Analytical method for water" for HPLC analysis. No peak corresponding to the test substance was detected for both Level 1 and Level 2. Sample No. 1 2 3 Control for Level 1 Sampling volume (mL) Fixed volume (mL) 100 10 100 10 100 10 Measured area N.D. N.D. N.D. Sample No. 1 2 3 Control for Level 2 Sampling volume (mL) Fixed volume (mL) 200 2 200 2 200 2 Measured area N.D. N.D. N.D. The chromatograms for calculating the recovery rates of the test substance from water are shown in Ref. 7-2. 2.7 Quantification limit 2.7.1 Quantification limit for fish In the HPLC chromatogram of fish analysis, no background peak was observed at the position of the test substance. The following table shows the quantifiable bioconcentration factor when the peak area of 300 was set as the detection limit. Concentration set for exposure Level 1 0.02 mgL-1 Level 2 0.002 mgL-1 Detection limit concentration1) (mgL-1) Quantification limit2) (gg-1) Bioconcentration factor3) Level 1 Level 2 (0.02 mgL-1) (0.002 mgL-1) 0.0082 0.0189 1-fold or greater 9-fold or greater 1) The detection limit concentration was calculated from the calibration curve in Section 2.4.2. 2) The quantification limit for fish was calculated by the following formula. Calculations were performed with a fish weight of 5.22 g (2.61 g per fish, 2 fish for 1 sample), a recovery rate of 83.2%, a fixed volume of 100 mL, and a concentration factor of 1/10. Detection limit (mgL-1) Fixed volume (mL) Concentration factor Quantifiable limit (gg-1) = 100 Recovery rate (%) Fish weight (g) 3) Quantification limit (gg-1) Bioconcentration factor (BCF) = Concentration set for exposure (theoretical value) (mgL-1) 17 2.7.2 Quantification limit for test water In the HPLC chromatogram of water analysis, no background peak was observed at the position of the test substance. The following table shows the quantification limit when the peak area of 300 was set as the detection limit. Concentration set for exposure Level 1 Level 2 0.02 mgL-1 0.002 mgL-1 Detection limit concentration1) (mgL-1) 0.0124 Quantification limit2) (mgL-1) Level 1 (0.02 mgL-1) Level 2 (0.002 mgL-1) 0.00135 0.000127 1) The detection limit concentration was calculated from the calibration curve in Section 2.5.2. 2) The quantification limit for the test water was calculated by the following formula. Calculations were performed with fixed volume: 10 mL for Level 1 and 2 mL for Level 2, recovery rate: 91.8% for Level 1 and 97.8% for Level 2, and amount of water sampled: 100 mL for Level 1 and 200 mL for Level 2. Detection limit (mgL-1) Fixed volume (mL) Quantification limit (mgL-1) = 100 Recovery rate (%) Amount of water sampled (mL) 2.8 Preliminary test Test fish (Cyprinus carpio) were exposed to the concentration of Level 1 (0.002 mgL-1) under the flow-through system. On day 7 of exposure, test water and fish (2 fish for 1 sample, n=2) were collected and analyzed according to ``2.3.1 Analytical method for fish'' and ``2.3.2 Analytical method for water''. As a result, BCFs in duplicate of the test substance were found to be 62-fold and 51-fold. 2.9 Schedule of analyses for fish and test water Analyses were conducted according to the following schedule: Sampling date Days after exposure Analysis for Test group Exposure June 10, 2015 0 period June 17, 2015 7 (28 days) water water and fish June 24, 2015 14 water and fish Analysis for Control group water and fish lipid contents - - July 1, 2015 21 water and fish - July 6, 2015 26 water and fish - July 8, 2015 28 water and fish fish lipid contents On the day of water analysis, pH, DO (set to approximately 7 mgL-1) and water temperature in each aquarium were measured and recorded (pH meter: SOP/INS/4.3.3, DO meter: SOP/INS/4.4.4). 18 Water analysis was performed according to ``2.3.2 Test water analysis method'' in compliance with SOP 2.10 (measurement for concentration of the test substance in test water). The variation was confirmed to be within 20% of the average measured value during the test period. Fish analysis was performed according to "2.3.1 Analysis method for fish" in compliance with SOP 2.11 (measurement of concentration of test substance in test fish). The lipid content was determined by the "chloroform-methanol extraction method" in compliance with SOP 2.9 (Determination of lipid content of test fish). The variation at the start and end of the experiment was confirmed to be within 25%. Storage of fish before analysis and disposal of waste during analysis were performed according to SOP 2.15 (storage before analysis of fish) and SOP 2.16 (disposal of waste during analysis of fish). 3. Test results 3.1 Concentration of the test substance in test water Concentrations of the test substance in test water (mgL-1) are shown in the following table. Test group Days after exposure 0 7 14 21 26 28 Level 1 0.0179 0.0201 0.0203 0.0181 0.0202 0.0198 Level 2 0.00176 0.00200 0.00198 Analytical data for test water are shown in Ref. 5. 0.00175 0.00185 0.00185 Based on the above results, the mean concentration and standard deviation during the test period were calculated as follows. Level 1: 0.0194 0.00100 mgL-1 Level 2: 0.00186 0.0000967 mgL-1 Calculation was performed by the following formula. Mean Standard deviation n xi x x = i=1 = n n n n (xi - x)2 i =1 = n x2 - ( x)2 / n n The time-dependent changes in concentration in water, water temperature, pH and DO are shown in Ref. 2. The chromatograms of analysis on days 0, 7, 14, 21, 26 and 28 of exposure are shown in Ref. 9-1, and those for Control group are shown in Ref. 9-2. 19 3.2 Bioconcentration factor (BCF) Results of this test confirmed that the bioconcentration factor (BCF) of the test substance reached a steady state by 28th day of exposure in both Level 1 and Level 2. Therefore, the exposure was terminated on day 28. The following table shows the BCF of the test substance for each analysis day. Days after exposure Level 1 Sample No. (n=2) Bioconcentration factor (BCF) Level 2 Sample No. (n=2) Bioconcentration factor (BCF) 7 NNoo.. 12 14 NNoo.. 12 21 NNoo.. 12 26 NNoo.. 12 28 No. 1 No. 2 22 No. 1 20 16 No. 2 23 14 No. 1 14 18 No. 2 14 12 No. 1 14 18 No. 2 10 15 No. 1 29 21 No. 2 20 12 No. 1 22 30 No. 2 < 7 (Note 1) Analysis was performed in duplicate (n=2), No.1 and No.2, respectively, where 2 fish were used as 1 sample. (Note 2) The fish weight and other data of analysis are shown in Ref. 4, and the chromatograms of analysis on days 7, 14, 21, 26 and 28 are shown in Ref. 8-1. The chromatograms for Control group are shown in Ref. 8-2. (Note 3) The time-dependent change in the BCF is shown in Ref. 3. (1) Mean and standard deviation of bioconcentration factor (BCF) The mean and standard deviation of BCF during the experiment period are: Level 1: 18 5 (n=10) Level 2: 18 6 (n=9) Calculated as: Concentration of the test substance in fish (gg-1) BCF = Concentration of the test substance in the aquarium (mgL-1) Mean n xi x x = i=1 = n n Standard deviation n n (xi - x)2 i =1 = n x2 - ( x)2 / n n (2) Confirmation of steady state Since the BCFs were less than 50-fold throughout the exposure period, both Level 1 and Level 2 groups were considered to have reached steady state, and the exposure was terminated on day 28. 20 Variation (%) of the BCF in three measurements at intervals of 48 h or longer in Level 1 group was as follows: 6 Variation (%) = 100 = 33 18 (Calculated from the BCFs on 21, 26, and 28 days after exposure) In Level 2, variation (%) of the BCF was not calculated, because the concentration in fish on day 28 (1 out of 2 samples) was below the detection limit. Standard deviation of BCF for 3 consecutive time points Variation (%) = Mean BCF for 3 consecutive time points < Reference items> The test fish in Control group (n=1) were analyzed on day 0, before the start of exposure, and on day 28, after the end of exposure. No peaks were detected at the position of the test substance in the HPLC chromatograms, as observed in the recovery test for Control group. The chromatograms are shown in Ref. 8-2. 3.3 Evaluation and discussion of the results and conclusion The 96-h LC50 of the test substance S510 in Zebrafish (Danio rerio) was greater than 25.6 mgL-1. Since the solubility in water was 2.3 10-2 mgL-1, concentrations of the test substance in water were set at 0.02 mgL-1 for the high exposure level (Level 1) and 0.002 mgL-1 for the low exposure level (Level 2). According to the results, the bioconcentration factor (BCF) was less than 50-fold both in Levels 1 and 2. Thus, steady state was considered to have reached by day 28 of exposure in both levels. As a result, bioconcentration of the test substance in fish was confirmed to be low. 21 Table of contents for references Reference Page 1 Outline of equipment for bioconcentration test 23 2 Aquatic environment (concentration, temperature, pH and DO) 24 2-1 Concentration of the test substance in water 24 2-2 Measurement of temperature, pH and DO in water 24 3 Time-dependent change in bioconcentration factor (BCF) 25 4 Fish analysis data 26 5 Water analysis data 29 6 Chromatograms for preparing calibration curve 32 6-1 For fish analysis 32 6-2 For water analysis 35 7 Chromatograms for recovery rate of the test substance 38 7-1 For recovery from fish 38 7-2 For recovery from water 43 8 Chromatograms of fish analysis 52 8-1 Test group 52 8-2 Control group 67 9 Chromatograms of water analysis 70 9-1 Test group 70 9-2 Control group 82 10 Water quality test 84 11 Observation record of the test fish (carp) 86 12 UV/VIS absorption spectrum of the test substance 88 13 IR spectra of the test substance 89 22 Reference 1 Outline of equipment for bioconcentration test A: Tap water B: Filter/Activated charcoal tank (Dechlorination equipment) C: Water tank for water (Dechlorinated water) D: Regulator for flow rate and mixing ratio D-1: Water for test D-2: Test substance stock solution E: Tank for stock solution of test substance (10L) F: Mixing tank (2L) (Mechanical stirrer) G: Test aquarium (60L) G-1: Aeration G-2: Temperature regulator/heater 23 Reference 2 Aquatic environment (concentrations, water temperature, pH, and DO) 2-1 Concentration of the test substance in water 0.1000 Level 1 Level 2 Test substance concentration (mg/L) 0.0100 0.0010 0.0001 0 7 14 21 28 Days after exposure 2-2 Measurement of water temperature, pH and DO in water The aquatic environment was measured at a fixed time (10 a.m.). For pH and DO, water was sampled from the center of the aquarium and measured with a pH meter (SOP/INS/4.3.3) and a DO meter (SOP/INS/4.4.4), respectively. The results are shown in the following table. Days of exposure 0 7 14 21 26 28 Level 1 Water temperature pH (C) 24.5 7.5 24.8 7.6 25.3 7.6 24.8 7.7 25.2 7.6 24.6 7.6 [DO in water] 9.00 DO (mgL-1) 7.11 7.11 7.14 7.16 7.49 7.54 Level 2 Water temperature pH (C) 24.5 7.5 24.3 7.5 24.9 7.6 24.5 7.7 25.0 7.6 24.7 7.6 DO (mgL-1) 7.07 7.29 7.32 7.04 7.60 7.61 Control Water temperature pH (C) 24.5 7.5 24.4 7.6 24.9 7.6 24.4 7.7 24.7 7.6 24.6 7.7 Level 1 Level 2 Control 8.00 DO [mgL-1] 7.00 6.00 5.00 0 7 14 21 28 Days after exposure DO (mgL-1) 714 7.59 7.57 7.49 7.61 7.69 24 Reference 3 Time-dependent changes in bioconcentration factor [Level 1] 1000 Bioconcentration factor (BCF) 100 10 1 0 [Level 2] 1000 7 14 21 28 Days after exposure : Detection limit or below Bioconcentration factor (BCF) 100 10 1 0 7 14 21 28 Days after exposure 25 Reference 4 Fish analysis data [Level 1] Item A B C Sample (n=2) Fixed volume (mL) Concentration factor in preparation Peak area Day 7 No.1 100 Day 7 No.2 100 Day 14 No.1 100 Day 14 No.2 100 Day 21 No.1 100 Day 21 No.2 100 Day 26 No.1 100 Day 26 No.2 100 Day 28 No.1 100 Day 28 No.2 100 1/10 8558 1/10 5860 1/10 5096 1/10 6776 1/10 3990 1/10 6267 1/10 5893 1/10 8049 1/10 3890 1/10 10446 D Concentration from 2-point calibration curve (mgL-1) 0.3541 0.2450 0.2069 0.2747 0.1571 0.2482 0.2343 0.3185 0.1561 0.4184 E Amount deposited in fish ( g) 3.541 2.450 2.069 2.747 1.571 2.482 2.343 3.185 1.561 4.148 F Fish weight (g) G H Recovery Concentration rate in fish (%) (gg-1) I Concentration in water (mgL-1) J K Concentration Date of factor measurement (BCF) 9.42 [4.68, 4.74] 83.2 9.18 [4.88, 4.30] 83.2 8.90 [4.36, 4.54] 83.2 8.85 [4.26, 4.59] 83.2 9.04 [4.62, 4.42] 83.2 9.38 [4.64, 4.74] 83.2 8.99 [4.52, 4.47] 83.2 9.21 [5.10, 4.11] 83.2 8.18 [4.51, 3.67] 83.2 8.45 [4.47, 3.98] 83.2 0.452 0.321 0.279 0.373 0.209 0.318 0.313 0.416 0.229 0.590 0.0201 0.0201 0.0203 0.0203 0.0181 0.0181 0.0202 0.0202 0.0198 0.0198 22 6.17.2015 16 14 6.24.2015 18 12 7.1.2015 18 15 7.6.2015 21 12 7.8.2015 30 Calculation methods: Amount deposited in fish Concentration in fish E =ABD H = E 100 F G F: total weight for 2 fish Concentration factor (BCF) J= H I (Note) Analysis was performed in duplicate (n=2), No.1 and No.2, respectively, where 2 fish were used as 1 sample. The weight of each two fish is shown in [ ] of column F. 26 26 27 [Level 2] Items Samples Day 7 No.1 Day 7 No.2 Day 14 No.1 Day 14 No.2 Day 21 No.1 Day 21 No.2 Day 26 No.1 Day 26 No.2 Day 28 No.1 Day 28 No.2 A Fixed volume (mL) 100 100 100 100 100 100 100 100 100 100 B Concentration Factor in preparation 1/10 1/10 1/10 1/10 1/10 1/10 1/10 1/10 1/10 1/10 C Peak area 524 633 463 467 499 370 1009 642 649 N.D. D Concentration from two-point calibration curve (mgL-1) 0.0292 0.0336 0.0199 0.0200 0.0175 0.0123 0.0436 0.0292 0.0283 < 0.0082 E Amount deposited in fish ( g) 0.292 0.336 0.199 0.200 0.175 0.123 0.436 0.292 0.283 < 0.082 F Fish weight (g) G H I J K Recovery rate (%) Concentration in fish (gg-1) Concentration in water (mgL-1) Concentration factor (BCF) Date of measurement 8.98 [4.97, 4.01] 83.2 8.91 [4.28, 4.63] 83.2 0.0391 0.0453 0.00200 0.00200 20 6.17. 2015 23 8.82 [4.48, 4.34] 83.2 8.65 [4.37, 4.28] 83.2 0.0271 0.0278 0.00198 0.00198 14 6.24. 2015 14 8.49 [4.12, 4.37] 83.2 0.0248 0.00175 14 7.1. 2015 8.45 [4.21, 4.24] 83.2 0.0175 0.00175 10 9.72 [4.66, 5.06] 83.2 9.51 [4.59,4.92] 83.2 0.0539 0.0369 0.00185 0.00185 29 7.6. 2015 20 8.29 [3.91, 4.38] 83.2 0.0410 0.00185 22 8.09 [3.81, 4.28] 83.2 < 0.0122 0.00185 < 7 7.8. 2015 Calculation methods: Amount deposited in fish Concentration in fish E =ABD H = E 100 F G F: total weight for 2 fish Concentration factor (BCF) J= H I (Note) Analysis was performed in duplicate (n=2), No.1 and No.2, respectively, where 2 fish were used as 1 sample. The weight of each two fish is shown in [ ] of column F. In column C, the peak area of 300 was set as the detection limit, and samples below the detection limit were indicated as N.D. E for N.D. was calculated by substituting the detection limit (0.0082 mgL-1) into D in the formula. 27 [Control] Items Samples Day 0 (at start of exposure) A Fixed volume (mL) 100 B Concentration Factor in preparation 1/10 C D E Concentration Amount Peak from 2-point deposited area calibration in fish (mcugrLve-1) (g) N.D. - - F Fish weight (g) 9.05 [4.02, 5.03] G H I J K Recovery rate (%) Concentration in fish (gg-1) Concentration in water (mgL-1) Concentration factor (BCF) Date of measurement - - - - 6.10.2015 Day 28 (at end of 100 1/10 N.D. - - 9.16 [4.35, 4.81] - - - - 7.8. 2015 exposure) (Note) The peak area of 300 was set as the detection limit, and samples below the detection limit were indicated as N.D. 28 28 Reference 5 [Level 1] Items Samples Day 0 Day 7 Day 14 Day 21 Day 26 Day 28 Water analysis data A Fixed volume of the solution (mL) B Peak area C Concentration calculated by two-point calibration curve (mgL-1) 10 4063 10 4718 10 4758 10 4167 10 4625 10 4543 0.1644 0.1842 0.1864 0.1664 0.1857 0.1821 D Absolute amount in the fixed volume solution ( g) 1.644 1.842 1.864 1.664 1.857 1.821 E Amount of water sampled (mL) 100 100 100 100 100 100 Calculation methods: Absolute amount in the fixed volume of solution Concentration in the aquarium F Recovery rate (%) 91.8 91.8 91.8 91.8 91.8 91.8 D = AC G = DE 1F00 G Concentration in water tank (mgL-1) 0.0179 0.0201 0.0203 0.0181 0.0202 0.0198 H Date of measurement 6.10. 2015 6.17. 2015 6.24. 2015 7.1. 2015 7.6. 2015 7.8. 2015 29 29 [Level 2] Items Samples Day 0 Day 7 Day 14 Day 21 Day 23 Day 28 A Fixed volume of the solution (mL) B Peak area C Concentration calculated by two-point calibration curve (mgL-1) 2 4262 2 5004 2 4938 2 4276 2 4503 2 4522 0.1724 0.1959 0.1938 0.1707 0.1810 0.1813 D Absolute amount in the fixed volume solution ( g) 0.3448 0.3918 0.3876 0.3414 0.3620 0.3626 E Amount of water sampled (mL) 200 200 200 200 200 200 Calculation methods: Absolute amount in the fixed volume of solution Concentration in the aquarium F Recovery rate (%) 97.8 97.8 97.8 97.8 97.8 97.8 D = AC G = DE 1F00 G Concentration in water tank (mgL-1) 0.00176 0.00200 0.00198 0.00175 0.00185 0.00185 H Date of measurement 6.10. 2015 6.17. 2015 6.24. 2015 7.1. 2015 7.6. 2015 7.8. 2015 30 30 [Control Group] Items A Samples Fixed volume of the solution (mL) B Peak area C Concentration calculated by two-point calibration curve (mgL-1) D Absolute amount in the fixed volume solution ( g) E Amount of water sampled (mL) Day 0 2 N.D. - - 200 F Recovery rate (%) - G Concentration in water tank (mgL-1) - (Note) The peak area of 300 was set as the detection limit, and samples below the detection limit were indicated as N.D. H Date of measurement 6.10.2015 31 31 Reference 6 Chromatograms for preparing calibration curve 6-1 For fish analysis Calibration curve (fish analysis) Standard solution 0.0979 mgL-1 Calibration curve (fish analysis) Standard solution 0.6194 mgL-1 32 Calibration curve (fish analysis) Standard solution 0.9790 mgL-1 Calibration curve (fish analysis) Standard solution 6.1938 mgL-1 33 Calibration curve (fish analysis) Standard solution 9.7902 mgL-1 Calibration curve (fish analysis) Chromatogram of solvent for adjustment 34 6-2 For water analysis Calibration curve (water analysis) Standard solution 0.0963 mgL-1 Calibration curve (water analysis) Standard solution 0.6314 mgL-1 35 Calibration curve (water analysis) Standard solution 0.9630 mgL-1 Calibration curve (water analysis) Standard solution 6.3137 mgL-1 36 Calibration curve (water analysis) Standard solution 9.6304 mgL-1 Calibration curve (water analysis) Chromatogram of solvent for adjustment 37 7 Chromatograms for recovery rate of the test substance 7-1 For recovery from fish Recovery test (Fish analysis) Standard solution 0.0979 mgL-1 Recovery test (Fish analysis) Standard solution 0.6194 mgL-1 38 Recovery test (Fish analysis) Added Standard solution Recovery test (Fish analysis) Added group No.1 39 Recovery test (Fish analysis) Added group No.2 Recovery test (Fish analysis) Added group No.3 40 Recovery test (Fish analysis) Control group No.1 Recovery test (Fish analysis) Control group No.2 41 Recovery test (Fish analysis) Control group No.3 42 7-2 For recovery from water Recovery test (Water analysis) Standard solution 0.0963 mgL-1 Recovery test (Water analysis) Standard solution 0.6314 mgL-1 43 Recovery test (Water analysis) High exposure level Added Standard solution Recovery test (Water analysis) High exposure level Added group No.1 44 Recovery test (Water analysis) High exposure level Added group No.2 Recovery test (Water analysis) High exposure level Added group No.3 45 Recovery test (Water analysis) High exposure level Control group No.1 Recovery test (Water analysis) High exposure level Control group No.2 46 Recovery test (Water analysis) High exposure level Control group No.3 Recovery test (Water analysis) Standard solution 0.0963 mgL-1 47 Recovery test (Water analysis) Standard solution 0.6314 mgL-1 Recovery test (Water analysis) Low exposure level Added Standard solution 48 Recovery test (Water analysis) Low exposure level Added group No.1 Recovery test (Water analysis) Low exposure level Added group No.2 49 Recovery test (Water analysis) Low exposure level Added group No.3 Recovery test (Water analysis) Low exposure level Control group No.1 50 Recovery test (Water analysis) Low exposure level Control group No.2 Recovery test (Water analysis) Low exposure level Control group No.3 51 8 Chromatograms of fish analysis 8-1 Test group Fish analysis Day 7 Standard solution 0.0979 mgL-1 Fish analysis Day 7 Standard solution 0.6194 mgL-1 52 Fish analysis Day 7 High exposure level No.1 Fish analysis Day 7 High exposure level No.2 53 Fish analysis Day 7 Low exposure level No.1 Fish analysis Day 7 Low exposure level No.2 54 Fish analysis Day 14 Standard solution 0.0979 mgL-1 Fish analysis Day 14 Standard solution 0.9790 mgL-1 55 Fish analysis Day 14 High exposure level No.1 Fish analysis Day 14 High exposure level No.2 56 Fish analysis Day 14 Low exposure level No.1 Fish analysis Day 14 Low exposure level No.2 57 Fish analysis Day 21 Standard solution 0.0979 mgL-1 Fish analysis Day 21 Standard solution 0.9790 mgL-1 58 Fish analysis Day 21 High exposure level No.1 Fish analysis Day 21 High exposure level No.2 59 Fish analysis Day 21 Low exposure level No.1 Fish analysis Day 21 Low exposure level No.2 60 Fish analysis Day 26 Standard solution 0.0979 mgL-1 Fish analysis Day 26 Standard solution 0.9790 mgL-1 61 Fish analysis Day 26 High exposure level No.1 Fish analysis Day 26 High exposure level No.2 62 Fish analysis Day 26 Low exposure level No.1 Fish analysis Day 26 Low exposure level No.2 63 Fish analysis Day 28 Standard solution 0.0979 mgL-1 Fish analysis Day 28 Standard solution 0.9790 mgL-1 64 Fish analysis Day 28 High exposure level No.1 Fish analysis Day 28 High exposure level No.2 65 Fish analysis Day 28 Low exposure level No.1 Fish analysis Day 28 Low exposure level No.2 66 8-2 Control group Fish analysis Day 0 Standard solution 0.0979 mgL-1 Fish analysis Day 0 Standard solution 0.6194 mgL-1 67 Fish analysis Day 0 (before starting exposure) Control group Fish analysis Day 28 Standard solution 0.0979 mgL-1 68 Fish analysis Day 28 Standard solution 0.9790 mgL-1 Fish analysis Day 28 (at the termination of exposure) Control group 69 9 Chromatograms of water analysis 9-1 Test group Water analysis Day 0 Standard solution 0.0963 mgL-1 Water analysis Day 0 Standard solution 0.6314 mgL-1 70 Water analysis Day 0 High exposure level Water analysis Day 0 Low exposure level 71 Water analysis Day 7 Standard solution 0.0963 mgL-1 Water analysis Day 7 Standard solution 0.6314 mgL-1 72 Water analysis Day 7 High exposure level Water analysis Day 7 Low exposure level 73 Water analysis Day 14 Standard solution 0.0963 mgL-1 Water analysis Day 14 Standard solution 0.6314 mgL-1 74 Water analysis Day 14 High exposure level Water analysis Day 14 Low exposure level 75 Water analysis Day 21 Standard solution 0.0963 mgL-1 Water analysis Day 21 Standard solution 0.6314 mgL-1 76 Water analysis Day 21 High exposure level Water analysis Day 21 Low exposure level 77 Water analysis Day 26 Standard solution 0.0963 mgL-1 Water analysis Day 26 Standard solution 0.6314 mgL-1 78 Water analysis Day 26 High exposure level Water analysis Day 26 Low exposure level 79 Water analysis Day 28 Standard solution 0.0963 mgL-1 Water analysis Day 28 Standard solution 0.6314 mgL-1 80 Water analysis Day 28 High exposure level Water analysis Day 28 Low exposure level 81 9-2 Control group Water analysis Day 0 Standard solution 0.0963 mgL-1 Water analysis Day 0 Standard solution 0.6314 mgL-1 82 Water analysis Day 0 Control group 83 Reference 10 Water quality test Analyzed at Institute of Ecotoxicology, Inc. <Quantitative analysis of the residual chlorine> The quantification of residual chlorine in water used in this study was performed according to JIS K0102 33.1 (o-Tolidine colorimetric method). The concentrations of the residual chlorine during the experimental period were determined to be 0.01 mgL-1 or less. Concentration of residual chlorine (mgL-1) <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Date of quantitative analysis 6. 3. 2015 6. 10. 2015 6. 17. 2015 6. 24. 2015 7. 1. 2015 7. 8. 2015 <Quantitative analysis of copper> The quantification of copper in water used in this study was performed according to JIS K0101 51.1 (Absorptiometry). The concentration of copper was confirmed to be 0.005 mgL-1 or less. Copper (mgL-1) < 0.005 Date of quantitative analysis 6. 30. 2015 <Quantitative analysis of dissolved organic carbon concentrations> The quantitative analysis was performed using a total organic carbon meter TOC - 5000A (Shimadzu Corporation) (SOP/INS/4.9). The dissolved organic carbon concentration was confirmed to be 2 mgL-1 or less. Dissolved organic carbon concentration (mgL-1) < 2 Date of quantitative analysis 6. 29. 2015 84 Report of the test results No. 156016 April 21, 2015 To the Institute of Ecotoxicology Inc. 3-15-1 Komazawa, Setagaya-ku, Tokyo Shinnihon Environmental Research Corporation (Measurement Certification Industry Registration, Tokyo, No. 882) (Akira Kishi, Executive Director) The results of the test conducted on the sample as requested on April 1, 2010 were as follows. Subject: Analysis of water used for raising carp Organization which Institute of Ecotoxicology, Inc. collected the sample: Type of sample: Water quality Date when sample was April 7, 2015 collected: Sample Names Water used for raising Items analyzed carp Nitric nitrogen and (mg/L) 1.5 nitrous nitrogen Potassium (mg/L) 1.2 permanganate consumed Cyan (mg/L) Not detected* Total mercury (mg/L) <0.0005 Organic phosphorous (mg/L) Not detected* Iron (mg/L) 0.001 Manganese (mg/L) <0.001 Zinc (mg/L) 0.011 Lead (mg/L) <0.001 Hexavalent (mg/L) <0.005 chromium Cadmium (mg/L) <0.001 Arsenic (mg/L) <0.001 Fluorine (mg/L) 0.08 Total hardness (mg/L) 66 Phenols (mg/L) <0.005 Anionic surfactant (mg/L) <0.02 pH 7.1 (water temperature at time of analysis) (20.4) * "Not detected" means when measured by the quantifiable limit. Quantifiable lower limit Test methods 0.02 Service water test method (2001) VI-2 13.2 0.5 Service water test method (2001) VI-1 17.2 0.001 Service water test method (2001) VI-2 22.5 0.0005 0.05 0.001 0.001 0.001 0.001 0.005 0.001 0.001 0.05 0.02 0.005 0.02 0.1 Service water test method (2001) VI-3 25.2 1974 Environmental Agency Notification No. 64 Appendix 1 Service water test method (2001) VI-3 13.4 Service water test method (2001) VI-3 12.4 Service water test method (2001) VI-3 16.4 Service water test method (2001) VI-3 27.2 Service water test method (2001) VI-3 11. Reference 2 Service water test method (2001) VI-3 21.4 Service water test method (2001) VI-3 17.2.2 Service water test method (2001) VI-2 3.2 Service water test method (2001) VI-1 15.2.3 Service water test method (2001) VI-4 10.5 Service water test method (2001) VI-4 11.2 Service water test method (2001) VI-1 9.2 analytical method listed above, the result was less than the 85 Reference 11 Observation record of the test fish (carp) Observation record of the test fish No 1 Test Substance S510 Test No. C-277 Date 2015 6.10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Feeding period (days) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Swim status Concentration Level 1 Group (Water tank No. 4) Food Health Water Mortality intake flow Swim status Concentration Level 2 Group (Water tank No. 5) Food Health Water Mortality intake flow Swim status O.K. - O.K. O.K. 0 O.K. - O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. - O.K. O.K. 0 O.K. - O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. O.K. - O.K. O.K. 0 O.K. - O.K. O.K. 0 O.K. O.K. O.K. O.K. O.K. 0 O.K. O.K. O.K. O.K. 0 O.K. Flow rate: Level 1: 300 ml/min, Level 2: 300 ml/min, Control: 300 ml/min to be confirmed. Food intake: 0.06 g per fish Feed provided: Maruha-jirushi compound feed for juvenile carp Cumulative mortality at the end of the test: Level 1: 0% Level 2: 0%, Control: 0% Remarks (Situation at the time of abnormality and treatments taken): Not applicable. Control Group (Water tank No. 6) Food Health Water intake flow - O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. - O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. - O.K. O.K. O.K. O.K. O.K. C-2 (Hayashikane Sangyo Mortality 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Co., Ltd. Lot Temperature/C Level 1 24.5 24.4 24.2 24.2 24.5 24.5 24.9 24.8 24.5 24.6 24.5 24.2 24.6 25.3 25.3 Level 2 24.5 24.5 24.3 24.6 24.9 24.7 24.7 24.3 24.8 24.9 24.2 24.2 24.7 24.6 24.9 Control 24.5 24.4 24.5 24.2 24.4 24.4 24.4 24.4 24.6 24.9 24.6 24.7 24.9 25.1 24.9 01374) Observed by T.S T.S T.S T.S T.S T.S T.S T.S T.S T.S T.S T.S T.S T.S T.S 86 86 Observation record of the test fish No 2 Test Substance S510 Test No. C-277 Date 2015 6.25 26 27 28 29 30 7. 1 2 3 4 5 6 7 8 Feeding period (days) 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Swim status O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. Concentration Level 1 Group (Water tank No. 4) Food Health Water Mortality intake flow O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 - O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. 0 O.K. O.K. O.K. 0 - O.K. O.K. 0 O.K O.K. O.K. 0 Swim status O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. Concentration Level 2 Group (Water tank No. 5) Food Health Water Mortality intake flow O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 - O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. O.K. 0 O.K. O.K. 0 O.K. O.K. O.K. 0 - O.K. O.K. 0 O.K O.K. O.K. 0 Swim status O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. Control Group (Water tank No. 6) Food Health Water intake flow O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. - O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. O.K. - O.K. O.K. O.K O.K. O.K. Mortality 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Temperature/C Level 1 25.1 24.6 24.9 24.4 24.9 24.9 24.8 24.6 24.6 24.7 24.5 25.2 24.8 24.6 Level 2 24.7 24.5 24.3 24.9 24.7 24.9 24.5 25.2 25.0 25.0 25.5 25.0 24.8 24.7 Control 24.5 24.4 24.5 24.7 24.9 24.4 24.4 24.2 24.5 24.4 24.5 24.7 24.4 24.6 Flow rate: Level 1: 300 ml/min, Level 2: 300 ml/min, Control: 300 ml/min to be confirmed. Food intake: 0.06 g per fish Feed provided: Maruha-jirushi compound feed for juvenile carp C-2 (Hayashikane Sangyo Co., Ltd. Lot 01374) Cumulative mortality at the end of the test: Level 1: 0% Level 2: 0%, Control: 0% Remarks (Situation at the time of abnormality and treatments taken): Not applicable. Observed by T.S T.S T.S T.S T.S T.S T.S T.S T.S M.T T.S M.T T.S T.S 87 87 Reference 12 UV/VIS absorption spectrum of the test substance UV (Acetonitrile) max (log ) 214.00 nm (3.95) 88 Reference 13 IR spectra for the test substance IR spectrum provided by the sponsor 89 File name: 3HHXFFCF3 Resolution: 4 cm-1 No. of scan: 4 Apodization: strong Date/time: 2015/04/16 11:23 (Standard time in Tokyo) Sample: S510 (3HHXFFCF3) Lot: 14NA007 Operator: Ichiro Sakabe IR spectrum obtained at our laboratory before the test 90 IR spectrum obtained at our laboratory after the test 91 On the following pages, the original (Japanese version) of the final report, excluding reference materials, is attached. 92  E4 -15022 C2 77 CH C -2 77 S510 Cyprinus carpio JNC 7 14 21 2015 6 2015 6 2015 7 1 2015 5 1l 2015 6 8 2015 7 13 2015 7 9 2015 7 15 2D 2015 7 1 2015 5 11 2015 6 8 2015 7 13 2015 7 9 2015 7 15 a 2015 7 15 A A 'llj 'I U 2015 7 15 r9 2015 7 15 11  L s510 Cyprinus carpio 2. JNC 100-8105 2-2-1 3 331-0811 2281 4 5 2333103317 2303295 110331009 0ECD ''No.305, Bioaccuniulationin Fish: Aqueous and Dietary Exposure"(2012102 6.GLP GLP: 2333103318 2303296110331010 0ECD-GLP : FOECD Principles of Good Laboratory Practicej(as revised in 1997)] l