### Rep01i No. NCAS 10-066 Page 1 of26 

# Final Report

Study Title: Acute toxicity test in Oryzias latipes



### Author: Osamu Saika 

### Study Completed on: July 21,2010



### Test Facility: Nisso Chemical Analysis Service Co ., Ltd. (NCAS) Odawara Laboratory 345 Takada, Odawara, Kanagawa 250-0216, Japan 

### Sponsor:

Study Number: NCAS 10-066 

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## GLP Compliance Statement Study No.: NCAS 10-066 Study title: Acute toxicity test in 

### 01yzias latipes

This study was carried out in accordance with the following good laboratmy practice regulation; Standard for the test facility conducting tests concernmg new chemical substances, etc. (Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, No. 1121003, November 21, 2003; Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, No.3, November 17, 2003; Environmental Policy Bureau, Ministry of the Environment, No. 031121004) The latest amendment: July 4, 2008. The final repmi was prepared faithfully and consistently with the raw data obtained. Study Director: _('-'s'!.tigJ"-'1-"'a""tu""'r""'e.L)________ (seal) (July 21, 2010) Osamu Saika Nisso Chemical Analysis Service Co., Ltd. Odawara Laboratmy The original signature page ofGLP Compliance Statement follows on page 3.Report No. NCAS 10-066 Page 3 of26 

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## GLP Compliance Statement The English translation of GLP Compliance Statement appears on page 2.**W\~'i=%** 

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## Quality Assurance Statement Study Number: NCASl 0-066 Study Title: Acute toxicity test in 

### Oryzias latipes Quality Assurance inspections of the study referred above were conducted according to the appropriate GLP regulations and the standard operating procedures (SOPs) of the Quality Assurance Unit (QAU). The results of the inspections were repmied to the study director and the facility management on the following dates. Dates (Month/Day/Year) Items inspected Inspected Reported to Study Director Management Protocol 5/21/2010 5/21/2010 5/21/2010 Experimental procedures Acquisition and management offish 5/24/2010 5/24/2010 5/24/2010 Preparation ofthe test solutions 5/24/2010 5/24/2010 5/24/2010 Exposure offish to the test solutions 5/24/2010 5/24/2010 5/24/2010 Observation offish 5/24/2010 5/24/2010 5/24/2010 Analytical sampling and treatment 5/24/2010 5/24/2010 5/24/2010 Analysis ofthe concentrations 5/24/2010 5/24/2010 5/24/2010 (LC/MS/MS) Preparation ofthe standard solutions 5/24/2010 5/24/2010 5/24/2010 Measurement ofthe NMR spectrum 6/18,22/2010 6/22/2010 6/22/2010 Raw Data 6/23-24/2010 6/24/2010 6/24/2010 Draft Report 6/23-24/2010 6/24/2010 6/24/2010 Final Repmt 7/21/2010 7/21/2010 7/21/2010 The QAU found that the study was performed according to the protocol and SOPs, the reported methods and procedures were actually used, and the results accurately reflect the recorded data. QAU Manager: _ _,('""'s.:..::ig"""n=a=tu=r=e)"--________ (seal) (July 21, 2010) Ken Watabe Nissa Chemical Analysis Service Co., Ltd. The original signature page of Quality Assurance Statement follows on page 5.Repmi No. NCAS 10-066 Page 5 of26 

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# 2JBReport No. NCAS 10-066 Page 6 of26

Study No .: Study Title: Repmi No . : Sponsor:

Test Facility:

Study Director: Experimenter:

Study Initiation Date : Experimental Start Date: Exposure Period: 

## Study Information NCAS 10-066 NCAS 10-066

Nisso Chemical Analysis Service Co ., Ltd. Odawara Laboratory Acute toxicity test in 

### Ory z ias latipes

345 Takada, Odawara, Kanagawa 250-0216, Japan TEL 81-465-42-5268, FAX 81-465-42-3586 Osamu Saika Tomoko Yoshimura, Noboru Fujikake, Satoko Ogawa, Chiaki Urabe (management oftest organisms) Tomoko Yoshimura (test solution preparation, observation and analysis of water parameter) Shinpei Oonuki and Chiaki Urabe (analysis ofconcentration oftest substance) Shinpei Tsushima (Measurement of the NMR spectrum after the exposure termination)May 21,2010 May 24 , 2010 Experimental Completion Date: May 24.2010-May 28,2010 June 18,2010 Study Completion Date: Test Guideline:

Archiving : July 21 , 2010 Standard for the testing methods concerning new chemical substances. (Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, No.1121002, November 21 , 2003; Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, No .2, November 13 , 2003; Environmental Policy Bureau, Ministry of the Environment, No . 031121002 ) The latest amendment: November 20 , 2006, Fish Acute Toxicity Test All the documents ofthis study will be retained in the archives ofthe test facility forReport No. NCAS 10-066 Page 7 of26 10 years after the completion of the study, but the place for storage after that will be decided on discussion with the sponsor. The test substance will be retained in the test facility at least for 10 years after the completion ofthe study, but only as long as the quality ofthe substance affords evaluation. Deviation fi·om the SOPs and the protocol: Trifluoroacetic acid used as a chemical shift standard for NMR measurement was over the expiry date specified in the SOP. However, it was considered that the deviation did not influence the results because trifluoroacetic acid did not decompose from the result ofNMR measurement. Circumstance 

### I matter that may affect the reliability ofthe test results: None

### Report No. NCAS 10-066 Page 8 of26 

## Table of Contents Title Page . .. . .. . ..... .. . .......... ... . ... .. . . ...... . .. ... ........... . .... .. ............ .. ......... ... .. . .... .. .. ... . .... . ........ . ................ .... .. ... . I GLP Cornpliance Staternent . ................. .... .. . .. .. . .. ....... ... ...... ... . .... .... .. ........... . ..... ... ............. .... ... .... ....... . ..... 2 The original GLP Compliance Statement . .. .. .. . ............ ..... . .. .. . ......... .. .. ... ... .. .. .. .. ... ... . ... ... .. .. ... .. .. . .... ......... . . 3 Quality Assurance Staternent . . ... . ... . .... ............ . ...... . .. .. .. .................... . .... . ....... ...................... .......... .. .. . ...... . 4 The original Quality Assurance Statement ... . .. .. ...... .. .. ....... .... ... ....... . .. . . . ...... ............ ..... . ... . . ..... ... ....... ...... . 5 Study Information . ... ...... . .. . ..................... . ........ ..... . ....... .. ........ .. .............. ..... .. ........... . .. .. ................. .. ......... 6 Table of Contents .. .. .. ....................................... . .... .. .. ..... .. .. ... .......... .... ............ . ....... .. ............. .... ........ . ...... . 8 Surnrnary.... . . ...... .. ....... . ............. .. .. ................... .. . ......... ........ .. ............................................. ............. ........ IO Introduction ......... . .............................. .. .................................... .. ...... ... .. . ...... .. . . ........ .. .. . ...... ..... ........ .. ... .. I0 Materials and Methods ............. .... .. .. ............................................ . ....................................... . .................. 10 I . Test substance........................................ .. ........ .. .................................. . ........................................ I0 2. Verification ofthe test substance ....................... .. ................... .. .... .. .. .. ....... .. .. .. ...... .. ... ........... ... .... 11 3. Verification of stability ofthe test substance.. .. ........................................... .. ................... .. .. .. ...... 11 4. Verification of solubility oftest substance .... .. .. ........... .... .. ..... . ...... .. .......... .. ..................... .... ....... 11 5. Reagents and apparatus ......................................... . ...................................................................... 11 6. Test organism .. .... .. ............................... ..... ..... . ................. . .......... .. ........ .. .... .... .. .. ................... .. .... I2 7. Acclirnatization ... . ............................. .. ...... .. ............................. . ................................................... I3 8. Acute toxicity study ............................... . .. .. ........................................ .. ....... . ............................... 13 Results ...... .. . ... .. ..... .... ...................................................................................................... .... .................... 16 1. Validation ofthe stability ofthe test substance ............................................ .. .............................. 16 2 . Validation ofthe analysis procedure ofthe test concentrations in test solutions .... .. ................... 16 3. The concentrations ofthe test substance in test solutions ........... . ................................................ 16 4. The concentrations ofthe test substance and mortality ...... .......................... .... ............................ 16 5. The median lethal concentration (LC 50) . .. .. .. .. .. ............................................................................ 

### 16 6. Observations on the fish ....................... .. .................... .. ................................ ... ............................. 17 7 . Observations on the test solutions .. ...... . .......... .... ..................................... .. .. .. .............................. 17 8. Water temperature, pH and dissolved oxygen concentration in test solutions .... .. ............. .... ...... I7 Conclusions ............... .... ..................... .. ............... .. ... .. .. .. ... .. ....... . . .... .. .... ....... . ......... .. .......... .... ................ 17 Tables and Figures . ........ . ..... . .... .. ... .. . ... . .. .. .. ...... ... ......... .. ...... .. .. .. ........ . ........ ...... .. .. ............. .. ........... .. ...... 18 Table 1 Result ofvalidation ofthe analysis .................................. .. ............ .. .............. .. ........................ 18 Table 2 Concentration oftest substance in test solution ................................................ .. ........ ....... ...... 18 Table 3 Number of deaths and mortality .......................... ................. .. .. .. ........ .. .. .. ...... .. .. . .. .. ........... .. .... 18 Table 4 Observations offish ............... .. ................. .... ........ .. ..... .. .. .. ....... .. ............ .. ..... .... ...................... 19 Table 5 Observations ofthe test solution .. .... ........... .. .. .. .... ... .... .............. .. .. .. .... .. .. .. .... .. .. .. ....... ...... ...... . 19 Table 6 Water temperature in the test solution .... .. .............................. .. ............ .. .................................. 19 Table 7 pH in the test solution .. .... ... .... .:.. .. ............ ........ .. .. .. .. .... .... .......... ......... .. .... ........ .... ...... .. .. .. .... .. 20 Table 8 Dissolved oxygen concentration in the test solution .................. .. ........ ...... ........ .. .. .. ............ .. .. 20Repmt No . NCAS 10-066 Page 9 of26 Figure 1 NMR spectrum of supplied by Sponsor ... .... ... . .. .... . .. .. . . . . . . .... ... .... .. .. . ... . .. .. .. .. .. . . ..... ... ... 21 Figure 2 NMR spectrum ofthe test substance (before the exposure initiation) .... .. . .......... . ........... . .... . .21 Figure 3 NMR spectrum ofthe test substance (after the exposure termination) . . .. ... . .. ... . . . . ... . ..... . ... . .22 Figure 4 Typical calibration curve ..... . ......... .. .................................... . ............................... .. .. .. ............. 23 Figure 5 Typical chromatogram of a standard solution (5 .05 mg/L, at the stmt ofthe exposure) . .... 23 Figure 6 Typical chromatogram of a repeatability test sample ......... .. .......... . .. ... ...... .... ... .. ................... 24 Figure 7 Chromatogram ofthe control group at the start ofthe exposure ....... .. .............. .. .. .... .......... .. .24 Figure 8 Chromatogram ofthe exposure group (the 100 mg/L group) at the start ofthe exposure...... 25 Figure 9 Chromatogram ofthe exposure group (the I00 mg/L group) after 48 hours ofthe exposure (before renewal) ................ . ............................. . ..................... 25 Figure IO Chromatogram ofthe exposure group (the IOO mg!L group) after 48 hours ofthe exposure (after renewal) ........................................... ......... .. .... .. .. ... .... 26 Figure II Chromatogram ofthe exposure group (the 100 mg/L group) at the end ofthe exposure .. 26Report No. NCAS 10-066 Page I0 of26 

## Summary The 96-hour acute toxicity study was performed to estimate the effect of the test substance, to 

### Oryzias latipes under semi-static (48-hour renewal cycle) and open system. A 24.0% aqueous solution of was used as the test substance. In this report, the concentrations of were expressed as , active ingredient, unless otherwise specially stated. On the basis of the analysis results of the concentrations of the test substance in the test solutions (LC/MS/MS), the variability was within ± 20% of the nominal concentrations, therefore the median lethal concentrations (LC 50) were calculated on the basis of the nominal concentrations as the test concentrations during the exposure period. No mo1tality or abnormality was observed during the exposure period. The 24-, 48-, 72- and 96-hour LC 50 are presented below. Exposure duration LCso (mg/L) 24 hours > 100 48 hours > 100 72 hours > 100 96 hours > 100



## Introduction This study was conducted in accordance with the following requirement to determine the median lethal concentrations (LC 50) by exposing the 

### Oryzias latipes to the test solutions that contained for 96 hours. Standard for the testing methods concerning new chemical substances. (Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, No.ll21002, November 21 , 2003; Manufacturing Industries Bureau, Ministry ofEconomy, Trade and Industry, No.2, November 13, 2003; Environmental Policy Bureau, Ministry of the Environment, No. 031121002) The latest amendment: November 20, 2006, Fish Acute Toxicity Test

1. Test substance Name: Abbreviated name: Product name: 

## Materials and MethodsReport No. NCAS 10-066 Chemical name: Structure: CAS No.: Molecular formula: Molecular weight: Lot No . : NCAS retrieval No.: Purity: Impurities:

Appearance : Obtained from: Acquisition volume: Acquisition date: Expiration date: Storage conditions: Water solubility: Stability information: Page 11 of26

STD-1150 Solid content: 24.0% aqueous solution ( 0.85% (as against solid content), Cl ion; 35 ppm In this report, the concentrations of the test substance were expressed as those of active ingredient, unless otherwise specially stated. was defined as the solid contents except water in this study.

420 g January 18, 2010 January 18, 2013 Stored in a refrigerator with a polypropylene container Soluble in water Stable in water Summary of risk and harmfulness: Causes skin and eye irritation. Wear protective gloves, goggles and mask when it is used (from MSDS). 2. Verification ofthe test substance The identity ofthe test substance was verified by comparing a 19F-NMR spectrum which was measured before the exposure initiation with another spectrum which was supplied by the sponsor (Figure 1 and 2). This verification was conducted in another study, NCAS 10-022 "Determination of the Concentration of in Dosing Solutions". 3. Verification of stability ofthe test substance The stability under storage condition was verified by confirming identity between two 19F-NMR spectra which were measured before and after the exposure period. 4 . Verification of solubility oftest substance Since the test substance was a 24% aqueous solution , its solubility was not confirmed. 5. Reagents and apparatus Dechlorinated tap water: Odawara City tap water was dechlorinated by activated charcoal treatment and aerated sufficiently with an air pump before the test.Report No. NCAS 10-066

Ion exchange water:

Acetonitrile: Formic acid: Trifluoroacetic acid: Hardness measurement kit: Balances: Thermostat water bath: Thermo recorder: Dissolved oxygen meter: pH meter: Thermometer: Page 12 of26 Quality of the water was confirmed to meet the standard criteria of 3rd-class fisheries water (Repmi No.: NCAS 09-064NG, provided from Chiba Pharmacist Association Inspection Center Foundation, Sample No. : D9C-8079 , Sampling Day: December 8, 2009). The total hardness and the concentration of the residual chlorine were measured before the test and it was confirmed that the total hardness as CaC0 3 was 60 mg/L, within the prescribed range (total hardness as CaC0 3 range from 40 to 100 mg/L), and the concentration ofthe residual chlorine was <0.0 1 mg/L. Tap water was distilled with a Barnstead water distilling apparatus (WDA-15S, Isuzu Seisakusho Co ., Ltd.) and then purified with an ultrapure water system (LV-08, Toray Co., Ltd.) HPLC grade (Wako Pure Chemical Industries , Ltd.) Special grade (Wako Pure Chemical Industries, Ltd.) HPLC grade (Wako Pure Chemical Industries, Ltd.) WAD-TH (Kyoritsu Chemical-check Lab., Corp) AG285, AX205 (Mettler Toledo), BX-3200H (Shimadzu Co.) NTT-1200 (Tokyo Rikakikai Co., Ltd.), SK-L200T (Sato KEIRYOKI MTG Co. , Ltd.) OM-51 (HORIBA, Ltd.) D-51 (HORIBA, Ltd.) Standard thennometer; measuring temperature range from 0 to 50°C , minimum scale 0.1 

## oc, (Ando Keiki Co ., Ltd.) Residual chloride meter: Digital residual chloride tester DCT-300 (Takumina Co., Ltd.) Centrifuge : KN-70 (Kubota Co.) Micropipette: EppendorfResearch V (Eppendorf Co ., Ltd.) Centrifuge tube: 1 0-mL glass test tube with ground-in stopper Nuclear chromatograph resonance apparatus: JNM-ECASOO (JEOL Ltd .) LC/MSIMS: Acquity 

### I Quattro micro API (Waters Corp .) 6. Test organism Species: Oryzias latipes Obtained from: Kitamura Fish Farm (obtained on May 11, 2010) Birth month and year: October 2009 Average total length and weight: 2.4 em (2 .3-2.5 em) , 0. 10 g (0.08-0.12 g) Sensitivity: The average value and range of 10 fish randomly selected at the exposure initiation. The background data of the 96-hour LC 50 value of the reference substance (copper (II) sulfate) in our laboratory; 0.59-2.0 mg/L (August 2008-February 2010, n=S)Repmi No. NCAS 10-066 Page 13 of26 7. Acclimatization 01y zias latipes were obtained on May 11, 2010 and treated with a medicated bath of ELEVAGE (Japan Pet Design Co ., Ltd) and salt for 6 hours on the same day. After 3 days of the holding, the fish were acclimatized for 10 days (May 14-24, 201 0) under the conditions described below. Mortality of 4.6% occurred for 7 days before the exposure initiation. Holding water: Dechlorinated tap water Holding chamber: 100-L glass chamber Water temperature : 22.7-23.4 

## oc Dissolved oxygen concentration : 95-99% (of saturation) pH: 7.3-7.5 Lighting: Feed: Feeding volume: Holding system: Test room: 8. Acute toxicity study

8.1 Test conditions Exposure system: Exposure duration: Test chamber: Volume oftest solutions: Replicates: Number oftest organism: Dilution water: Aeration: Test temperature: pH: fluorescent light, 16 hour light 

### I 8 hours dark MEDAKANOESA (Tetra Japan Co., Ltd) Foods were given at a rate of 1.5-1.8% of fish weight once a day. No feeds were supplied for 24 hours before the exposure initiation. Flow-through A6

Semi-static (48-hour renewal cycle, open system) 96 hours 3-L glass beaker 3 L 

### I test chamber 1 test chamber 

### I group 7 fish 

### I group Dechlorinated tap water (the concentration of the residual chlorine was <0.01 mgiL, the total hardness was 60 mg/L (as CaC0 3)). Not conducted 22.8-22.9°C (water temperature in the water bath during the exposure period; 23.1-23.3°C) 7.4-8.0 Dissolved oxygen concentration: 77-95% of saturation Lightning: Fluorescent light, 16 hour light 

### I 8 hours dark Feeding: No feed was supplied for 48 hours before the exposure. Test room: A8 8.2 Selection ofthe test concentrations and exposure systems Based on the results of a 96-hour semi-static range-finding test (Repmi No.: NCAS 10-063NG) , no mo1iality or abnormality was observed at the nominal concentration of 100 mgiL that was the limit concentration as active ingredient. The nominal concentrations were maintained during the 48-hourReport No. NCAS 10-066 Page 14 of26 static period. Therefore, the definitive test (semi-static system for 96 hours , 48-hour renewal cycle) was conducted at the highest concentration of 100 mg/L. 8.3 Preparation of test solutions Test solutions were prepared at the start and after 48 hours of the exposure. An amount of test substance (300 mg: 1249.8 mg as aqueous solution) was weighed and transferred to a 1 000-mL volumetric flask and dissolved in dechlorinated tap water. This solution was transferred into the test chamber and then added 2 L dechlorinated water to prepare the exposure solution. 8.4 Analysis oftest concentrations

8.4.1 LC/MS/MS conditions Apparatus: Acquity 

### I Quattro micro API (Waters) Column: Acquity UPLC BEH C18, 2.1 mm i.d. x 50 mm, pa1ticle diameter; 1.7 11m (Waters) Mobile Phase: Acetonitrile+ 0.1% formic acid aqueous solution (v/v) = 50 +50 (v/v) Flow rate: 0.3 mL!min Column temperature: 40°C Injection volume: 5 11L Ionization mode: ESI, Negative Monitoring ion: 180.33 > 96.90 (quantification), 180.33 > 77.84 (confirmation) 8.4 .2 Preparation of standard solutions and calibration curve An amount (5.05 mg, as ) of the test substance was correctly weighed and transferred into a 50-mL volumetric flask and dissolved in 50% acetonitrile aqueous solution to prepare a 101 mg/L standard stock solution. This standard stock solution was then fUJther diluted with the same solvent to prepare 1.01, 2.02, 3.03, 4.04 and 5.05 mg/L standard solutions. These standard solutions were analyzed by using the LC/MC/MC conditions described in section 8.4.1. Calibration curves were prepared by plotting the peak areas vs. the concentrations of and the linear regression equations and correlation coefficients (r) were calculated by using a computer program of LC/MS/MS (Mass Lynx v 4.1). The accuracy (%)for each calibration solution was calculated by inverse regression method. The weighting of 1/x was carried out. The standard solutions used to construct calibration curves were prepared before use. 8.4.3 Analysis ofthe concentration ofthe test substance in test solutions The test solutions were analyzed at the exposure initiation, before and after the 48-hour renewal, and the exposure termination. Samples were mixed with acetonitrile at the rate of 1:1 (v/v) and centrifuged (3000 rpm, 10 min). The supematant fluids obtained were diluted with 50% acetonitrile aqueous solution to be appropriate concentration which was approximated in the middle ofthe range of the calibration curve, and then analyzed by using the LC/MS/MS conditions as described in section 8.4.1. The samples from the control group were also mixed with acetonitrile at the rate of 1:1(v/v) andReport No. NCAS 10-066 Page 15 of26 centrifuged (3000 rpm, 10 min) and then analyzed by the same procedures to verify that any interfering peak did not appear at the retention time ofthe test substance on the chromatogram. 8.5 Validation study ofthe analysis ofthe test substance concentration in test solutions

8.5 . 1 Validation ofthe calibration curve The accuracy was calculated by finding the regression equation and correlation coefficient (r) ofthe calibration curves , and then determining the quantitative value with inverse estimation method. The criteria for the calibration curve were described below.

1. The correlation coefficient is more than 0.990. 2. The accuracy for the lowest concentration solution is within ±20% , and that for other solutions is within ±15%. 8.5.2 Validation ofthe repeatability Analytical method was validated by a repeatability test without any recovery tests, because extract operation was not conducted. The concentrations of the test substance in the exposure group were calculated by the same procedure as described in section 8.4.3 (n=3). The analytical method was detennined to be validated ifthe coefficient of variations ofthe measured test concentrations (n=3) was within ± 10%. 8.6 Exposure procedure At the stmt of the exposure, the weights and total lengths of 10 fish from the acclimated fish in this study were measured, and the total lengths were confirmed to be 2.2±1.0 em. After measurement of water temperature, dissolved oxygen concentration and pH in each test chmnber, 7 fish were added to each chmnber. Water temperature , dissolved oxygen and pH were measured at the exposure initiation, before m1d after the 48-hour renewal and at the end of the exposure. No feed was supplied during the exposure period. Temperature in the thermostat water bath, in which the test chambers were placed, was continuously recorded during the exposure period with a thermo recorder to monitor the change of the water temperature. Fish were observed and recorded just after exposure initiation, and after 3 , 6, 24, 48, 72 and 96 hours of the exposure initiation. The conditions of the test solutions were observed and recorded just after the exposure initiation, and after 24, 48 , 72 and 96 hours ofthe exposure . The test solutions were prepared as described in section 8.3 after 48 hours ofthe exposure initiation and surviving fish were transferred to them.

8.7 Calculation ofthe median lethal concentration (LC 50) The ranges of LC 50 were determined from the number of dead fish in each test group after 24, 48, 72 and 96 hours ofthe exposure.Report No. NCAS 10-066 Page 16 of26 

## Results 1. Validation ofthe stability ofthe test substance Since the spectrum which was measured before the exposure initiation (Figure 2) was corresponded to that was measured after the exposure termination (Figure 3), it was determined that the test substance was stable during the storage period. 2. Validation ofthe analysis procedure ofthe test concentrations in test solutions

2.I Validation ofthe calibration curve A typical calibration curve is shown in Figure 4 and a typical chromatogram ofthe standard solution (5.05 mg/L, at the strui ofthe exposure) is shown in Figure 5. The coefficients ofcorrelation (r) were more than 0.990 at the exposure initiation, after 48 hours and end of the exposure. The all variations of the accuracy of the lowest standard samples were within ±20% and those of other standard srunples were within ±I5%. Based on the results, the linearity of the calibration curves were determined as acceptable. 2.2 Validation ofthe repeatability The results of the repeatability test ru·e shown in Table 1. A typical chromatogram of the repeatability test (nominal concentration: I00 mg/L) is shown in Figure 6. A chromatogram of the control group is shown in Figure 7. The Coefficients ofvariation (n=3) ofthe measured concentrations ofthe test substance were within I0%. On the chromatogrrun of the control group, no interfering peak appeared at the retention time of the test substance. On the basis of these results, the analytical method of the test substance concentration in the test solution was determined to be valid in this study. 3. The concentrations ofthe test substance in test solutions The analytical results of the test substance concentrations in the test solutions are presented in Table 2 and chromatograms of the exposure solution at the exposure initiation, after 48 hours (before and after renewal) ofthe exposure and at the exposure termination are shown in Figure 

### 8-11 respectively. The test substance concentrations in the test solutions ranged from 99.0 to I03% of the nominal during the exposure period. Since the variability of the measured concentrations was within ±20% of the nominal, the nominal concentrations were considered as the test concentrations. 4. The concentrations ofthe test substance and mortality The number and percentage of deaths in each test group are shown in Table 3 . No moiiality occurred both in control and the I00 mg/L groups at any hours of exposure duration. 5. The median lethal concentration (LC 5o) The 24-, 48-, 72- and 96-hour LC 50 are determined to be >I00 mg/L since there were no dead fish at any hours ofexposure duration.Report No. NCAS 10-066 Page 17 of26 6 . Observations on the fish The results ofthe observation were shown in Table 4. No abnormality was observed in the control or exposure groups at any hours ofthe exposure duration. 7 . Observations on the test solutions The observation results ofthe test solutions were shown in Table 5. Throughout the exposure period, no turbidity or precipitate was observed and the color was clear and transparent in any groups. 8. Water temperature, pH and dissolved oxygen concentration in test solutions The measurements of temperature, pH and dissolved oxygen concentration in the test solutions are presented in Table 6, 7 and 8 respectively. The water temperatures ranged from 22.8 to 22.9 °C and the pH ranged from 7.4 to 8.0 during the exposure period. The dissolved oxygen concentrations ranged from 77 to 94% which were maintained 2::60% of saturation.



## Conclusions The 96-hour acute toxicity study under semi-static conditions (48-hour renewal cycle and open system) was performed by exposing 

### Oryzias latipes to It is concluded that the 96-hour median lethal concentration (LC 50) is >I 00 mg/L.Repo1t No . NCAS I0-066 Page 18 of26 

## Tables and Figures Table 1 Result ofvalidation ofthe analysis Nominal Measured concentration Mean measured Coefficient ofvariation concentration concentration (mg/L) (mg/L) (mg/L) 

### (%)

100 102 105 105 104 1.67



### Table 2 Concentration oftest substance in test solution

Nominal concentration The measured concentration oftest substance at each exposure duration (mg/L) (mg/L) Control

100

Nominal 0 hour 48 hours (before renewal) N.D. N .D. 103 (103) 101 (101) ( ): Percent ofnominal concentration(%) N.D.: Not detected 48 hours (after renewal) N.D. 100 (100)



### Table 3 Number ofdeaths and mortality Cumulative number of deaths (Mortality (%)) concentration (mg/L) 3 hours 6 hours 24 hours 48 hours 72 hours Control 0(0) 0 ( 0) 0 ( 0) 0 ( 0) 0 ( 0)

100 0(0) 0 ( 0) 0 ( 0) 0(0) 0(0) 96 hours N.D. 99.0 (99.0)

96 hours

0 ( 0) 0 ( 0)Repo1iNo. NCAS 10-066 Page 19 of26 

### Table 4 Observations offish Nominal Observations at each exposure duration concentration (mg/L) 0 hour 3 hours 6 hours 24 hours 48 hours* 1 48 hours* 2 72 hours 96 hours Control N-7 N-7 N-7 N-7 N-7 N-7 N-7 N-7

100 N-7 N-7 N-7 N-7 N-7 N-7 N-7 N-7 N: Normal *1 · Before renewal *2 • After renewal



### Table 5 Observations ofthe test solution Nominal Observations at each exposure duration concentration (mg/L) 0 hour 24 hours 48 hours* 1 48 hours* 2 72 hours 96 hours Clear and Clear and Clear and Clear and Clear and Clear and Control transparent transparent transparent transparent transparent transparent Clear and Clear and Clear and Clear and Clear and Clear and 100 transparent transparent transparent transparent transparent transparent *1 · Before renewal 

## *2 · After renewal



### Table 6 Water temperature in the test solution Nominal Water temperature at each exposure duration (0 C) concentration 0 hour 48 hours 48 hours 96 hours (mg/L) (before renewal) (after renewal) Control 22.8 22.9 22.9 22.9

100 22.8 22.9 22.9 22 .9Report No . NCAS 10-066 Page 20 of 26 

### Table 7 pH in the test solution Nominal pH at each exposure duration concentration 0 hour 48 hours 48 hours 96 hours (mg/L) (before renewal) (after renewal) Control 7.7 7.5 7.9 7.5

100 7 .8 7.7 8.0 7.4



### Table 8 Dissolved oxygen concentration in the test solutions Nominal Dissolved oxygen concentration at each exposure duration(%) concentration 0 hour 48 hours 48 hours 96 hours (mg/L) (before renewal) (after renewal) Control 94 (7.9) 77 (6.5) 93 (7.8) 83 (7.0)

100 94 (7.9) 79 (6.6) 95 (8.0) 77 (6.5) Figures in parentheses indicate the concentration.

### Report No . NCAS 10-066

60 4 0 20 0 -20 -40 -60 

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### Report No . NCAS 1 0-066

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### Figure 3 NMR spectrum ofthe test substance (after the exposure teimination)Rep01i No. NCAS 10-066

Concentration Peak area (mg/L)

1.0 I 2006 2.02 3728 3.03 5151 4.04 6471 5.05 7830 Method: C:\MassLynx' \MethDB ' Calibration: 24 May 2010 16:38:14

6000. 70001.**~** 5000 " g_ 4000-i :G -l a:**3000~** -l 2000-j -l 10Q0cJ Quantitative Accuracy value (mg/L) (%) 0.956 94.7 2.13 106 3.11 103 4 .01 99.3 4.94 97.8

:1 Apr 2010 18:23 : 08



### Figure 4 Typical calibration curve 1000524-05 STD-5-1 100

0 .50 0.74 7830

1.00 1.50 2.00 MRM of2 channels,ES180.33 > 96 .90

8.199e+004

2.50 

### Figure 5 Typical clu·omatogram of a standard solution (5.05 mg/L, at the start ofthe exposure) Page 23 of26Rep01i 

### No. NCAS I0-066

1000524-06 0-1 OO-R1 100

%



### Figure 6

1000524-10 O-cont-O 100

% 0.75 3595

2 .00 

### Page 24 of26

MRM of2 channels,ES180 .33 > 96.90 8.199e+004



### Typical chromatogram of a repeatability test sample

0.50 1.00 1.50 2 .00 MRM of 2 channels , ES180.33 > 96.90 8.199e+004

2 .50 

### Figure 7 Chromatogram ofthe control group at the stmi ofthe exposure

### Report No. NCAS I0-066 1000524-09 0 - 100-0 100

%

0 .50 0 .75 3 15

1 .00 1.50 2 .00 

### Page 25 of26 MRM of 2 channels , ES180 .33 > 96 .90 8 . 199e+004

min 2.50 

### Figure 8 Chromatogram ofthe exposure group (the 100 mg/L group) at the start ofthe exposure 1000526-07 0 - 100-48-before 100

%

0 .50 0 .78 3415 MRM of2 channels , ES180 .33 > 96.90 7.606e+004**:::;::r;=;=;=~=;=;=.......-~=;=;=;-=;=;=.-.-.='FF"'""""'i,.....-,'"""'f''i""i=T'-.=** min 1 .00 1 .50 2 .00 2 .50 

### Figure 9 Chromatogram ofthe exposure group (the 100 mg/L group) after 48 hours ofthe exposure (before renewal)

### Rep01i No. NCAS 10-066 1000526-09 0-1 00-48-after

%

0.50 0 .78 3392

1 .00 1 .50 2.00 MRM of 2 channels , ES180.33 > 96 .90 7 .606e+004

2.50 

### Page 26 of 26



### Figure 10 Chromatogram ofthe exposure group (the 100 mg/L group) after 48 hours ofthe exposure (after renewal) 1000528-07 0-100-96 100

%

0.50 0 . 78 3 13

1 .00 1 .50 2.00 MRM of 2 channels , ES180.33 > 96.90 8.029e+004

2.50 

### Figure 11 Chromatogram ofthe exposure group (the 100 mg/L group) at the end ofthe exposure

## Authenticity of Translation

I declare that the original Japanese final report (Report No. NCAS 1 0-066) is translated into English consistently.



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### OsamuSaika Nisso Chemical Analysis Service Co., Ltd. Odawara Laboratory