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 

TRUE COPY OF CJ.LdL~lNAL 

bfs/r'-

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Repmi No. NCAS 1 0-066 Page 2 of 26 

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 

The original GLP Compliance Statement 

The English translation of GLP Compliance Statement appears on page 2. 

W\~'i=% : NCAS 10·066 

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Report No. NCAS 10-066 Page 4 of26 

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 of fish 5/24/2010 5/24/2010 5/24/2010 

Preparation of the 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 of fish 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 of the standard solutions 5/24/2010 5/24/2010 5/24/2010 

Measurement of the 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 

The original Quality Assurance Statement 

The English translation of Quality Assurance Statement appears on page 4. 

J::.!c~~~O)ffi!F.iHif*llifO)~~JU;:J:~~~JMiffl GLP :t:ld::LJ\1~wJi'lif*[i:EtJ'BF~ (QAU) (f) SOP t=: 

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· 7HJTaitN 0) I*Jr!Z:B J: LJ\9l1~ 5/24/2010 5/24/2010 5/24/2010 

• ¥lll/3t:7.t;fJT (LC/MS/MS) 5/24/2010 5/24/2010 5/24/2010 

~$~~0)Woj~ 5/24/2010 5/24/2010 5/24/2010 

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Report 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 Oryzias 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 

of test organisms) 

Tomoko Yoshimura (test solution preparation, observation and analysis of water 

parameter) 

Shinpei Oonuki and Chiaki Urabe (analysis of concentration 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 of this study will be retained in the archives of the test facility for 
Report 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 of the study, but only as long as 

the quality of the 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 of the 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 ................................................................................ ................................ ........................ ..... I 0 

Materials and Methods ............................................................................................................................ 10 

I. Test substance ............................................................................................................................... I 0 

2. Verification of the test substance ................................................................. ................................. 11 

3. Verification of stability of the test substance ................................................................................ 11 

4. Verification of solubility of test substance ...... ............................................................................. 11 

5. Reagents and apparatus ................................................................................................................ 11 

6. Test organism ............................................................................................................................... I2 

7. Acclirnatization ............................................................................................................................ I3 

8. Acute toxicity study .................................. ............................................ ....................................... 13 

Results ........ .... .. ....................................................................................................................................... 16 

1. Validation of the stability of the test substance ............................................................................ 16 

2. Validation ofthe analysis procedure ofthe test concentrations in test solutions ......................... 16 

3. The concentrations of the test substance in test solutions ............................................................ 16 

4. The concentrations ofthe test substance and mortality ................................................................ 16 

5. The median lethal concentration (LC50) . .. .. .. .. .. ............................................................................ 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 of the 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 .................... .......................................... .. 20 
Repmt No. NCAS 10-066 Page 9 of26 

Figure 1 NMR spectrum of supplied by Sponsor .......... ....... ... .............................. ...... ............ ... 21 

Figure 2 NMR spectrum of the test substance (before the exposure initiation) .... ................................ 21 

Figure 3 NMR spectrum of the 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 of the exposure) ..... 23 

Figure 6 Typical chromatogram of a repeatability test sample ........................... .................................. 24 

Figure 7 Chromatogram of the control group at the start of the exposure ............................... ............. 24 

Figure 8 Chromatogram of the exposure group (the 100 mg/L group) at the start of the exposure ...... 25 

Figure 9 Chromatogram of the exposure group (the I 00 mg/L group) 

after 48 hours of the exposure (before renewal) .................................................................... 25 

Figure IO Chromatogram ofthe exposure group (the IOO mg!L group) 

after 48 hours of the exposure (after renewal) ..................................................................... 26 

Figure II Chromatogram of the exposure group (the 100 mg/L group) at the end of the exposure .. 26 
Report No. NCAS 10-066 Page I 0 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 (LC50) 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 LC50 

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 (LC50) 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 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 

1. Test substance 

Name: 

Abbreviated name: 

Product name: 

Materials and Methods 
Report 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 of the test substance 

The identity of the 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 of test 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 1 0-066 

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 CaC03 was 60 mg/L, within the prescribed range (total 

hardness as CaC03 range from 40 to 100 mg/L), and the 

concentration of the 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: Eppendorf Research 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 LC50 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 

01yzias 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 of test 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 CaC03)). 

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 of the 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-hour 
Report 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 of test 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 of the concentration of the 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 of the 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) and 
Report 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 of the test substance on the chromatogram. 

8.5 Validation study of the analysis of the 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) of the 

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 if the coefficient of variations of the measured test concentrations (n=3) 

was within ± 1 0%. 

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 of the exposure. 

The test solutions were prepared as described in section 8.3 after 48 hours of the exposure initiation 

and surviving fish were transferred to them. 

8.7 Calculation ofthe median lethal concentration (LC50) 

The ranges of LC50 were determined from the number of dead fish in each test group after 24, 48, 72 

and 96 hours of the exposure. 
Report No. NCAS 10-066 Page 16 of26 

Results 

1. Validation of the 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 of the analysis procedure of the test concentrations in test solutions 

2.I Validation of the calibration curve 

A typical calibration curve is shown in Figure 4 and a typical chromatogram of the standard solution 

(5.05 mg/L, at the strui of the exposure) is shown in Figure 5. 

The coefficients of correlation (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 of the repeatability 

The results of the repeatability test ru·e shown in Table 1. A typical chromatogram of the 

repeatability test (nominal concentration: I 00 mg/L) is shown in Figure 6. A chromatogram of the 

control group is shown in Figure 7. 

The Coefficients of variation (n=3) ofthe measured concentrations ofthe test substance were within 

I 0%. 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 of the 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) of the 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 I 03% 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 of the 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 I 00 mg/L groups at any hours of exposure duration. 

5. The median lethal concentration (LC5o) 

The 24-, 48-, 72- and 96-hour LC50 are determined to be >I 00 mg/L since there were no dead fish at 

any hours of exposure duration. 
Report No. NCAS 10-066 Page 17 of26 

6. Observations on the fish 

The results of the observation were shown in Table 4. 

No abnormality was observed in the control or exposure groups at any hours of the exposure duration. 

7. Observations on the test solutions 

The observation results of the 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 (LC50) is >I 00 mg/L. 
Repo1t No. NCAS I 0-066 Page 18 of26 

Tables and Figures 

Table 1 Result of validation of the analysis 

Nominal Measured concentration Mean measured Coefficient of variation 

concentration concentration 

(mg/L) (mg/L) (mg/L) (%) 

100 102 105 105 104 1.67 

Table 2 Concentration of test substance in test solution 

Nominal concentration 

The measured concentration of test 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 of nominal concentration(%) 

N.D.: Not detected 

48 hours 

(after renewal) 

N.D. 

100 (100) 

Table 3 Number of deaths 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 of fish 

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 of the 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 22.9 
Report 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 40 20 0 -20 -40 -60 

Figure 1 NMR spectrum of 

:·······················-·············-···················································· ··················· · . ·· ···-· - -------·---···- · 

~ ·i 

Page 21 of26 

;~:t.~ 

c.f

3

-t (1=,.1_;. OI.,..CJi,.o\-1 

-80 

_....,. .... ., .. <..:!UUlf•.,J::.i. IG ~-~'="' 

<::PDPP.r;2 u el.i:o:1 6 

uuc~ Ui FCPD2 80 . 00 C:!IQC 

P12 0. 20 dB 

PL12 15.53 dB PL2!» 9.49937620 W 

I J'Ll2~ 0 .27!14!666 ~ 

sro:~ 400 ."!.316005 Hli: 

l SI 6!1~•36 

1 SF 316.!953650 HHZ WDW T.H 

!J~B a LoB O • .lli H; 

GB 0 

- ----<'"C 1.00 

-100 -120 ppm 

supplied by Sponsor 

dEOL4? 

' 'l , ti,U;I'I 

I 

! _.J 

I 

!::~~·r !~ :C&~:::~ 

lt- ~ .. 1 .. 

lrr_ .... .odt trl _ltnd t 

tla'OlHI_pra•~: 

ll:lti•l wd~ 

a tl .. ut!andolay hpl t!tJ<m- tir,. 

h=P •. 9"'t -

/o- o)J 

C.-&1 

Jlolll ) 

IJ J't~l"l'H tlt l 

Jl,>fd•;J 

6hl51 

~ 1:..:•! Ot! 

c ~ I r~;~.sc 

ll•i H al 

5.2 H7~H!•l 

1:ndel 

·····!· ·- -- ~., ....... r········. ~-·- ···~ ·· · ................ J -211.0 - JD.n - Ul.O - SIIJI -W.O - iii.O .-AA.n -'-10 1\-1110~ t/J./io r.f 1 :. .... ~n tlll.ll ;o.u (~:;; ·· f~.~ ' . .ttl.~ --;:;- -;~~ _, ., ~~ ~ .. . fl -1n.o 

I 

Figure 2 NMR spectrum of the test substance (before the exposure initiation) 
Report No. NCAS 1 0-066 

..... !j 

i sJ .:;l 

~ i 

------- - -·-------------

/o - u// 

Page 22 of26 

J \lllj 

O.llCl~GH\1 1 

JOIC•gl 

Ud•l 

::~ l 

'

r..t.~E " l l•l H•l 1-.UOUOJ, (ol 

U! C!.C l 

Figure 3 NMR spectrum of the 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 of the exposure) 

Page 23 of26 
Rep01i No. NCAS I 0-066 

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 of the control group at the stmi ofthe exposure 
Report No. NCAS I 0-066 

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 -1 00-48-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 of the 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 of the 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 of the exposure 
Authenticity of Translation 

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

consistently. 

S' J~. 2o/2 

' 

OsamuSaika 

Nisso Chemical Analysis Service Co., Ltd. 

Odawara Laboratory