Report No. NCAS I 0-064 Page I of27 

Final Report 

Study Title: 

Daphnia magna, acute immobilization test 

Author: 

Osamu Saika 

Study Completed on: 

July 22,2010 

Test Facility: 

Nisso Chemical Analysis Service Co., Ltd. (NCAS) 

Odawara Laboratory 

345 Takada, Odawara, Kanagawa 250-0216, Japan 

Sponsor: 

Study Number: 

NCAS 10-064 

TRUE COPY OF 0~-.lGINAL 

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Repmi No. NCAS 1 0-064 Page 2 of27 

GLP Compliance Statement 

Study No.: NCAS 10-064 

Study title: : Daphnia magna, acute immobilization 

test 

This study was carried out in accordance with the following good laboratory 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.:.tign=a=tu=r=e),_ ________ (seal) (July 22. 201 0) 

Osamu Saika 

Nisso Chemical Analysis Service Co., Ltd. 

Odawara Laboratory 

The original signature page ofGLP Compliance Statement follows on page 3. 
Report No. NCAS 10-064 Page 3 of27 

The original GLP Compliance Statement 

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

W\!1ifri:i=% : NCAS 10-064 

.: O)W\IJI*!"i r~mft'¥:~1(~t::flf; Q Wt~ -8:-~Ji'tii-t .:s ~~:litl!fi9:t::!MJ-t Q £II!!:: "'Jv '-c 1 IJZPX: 15 fF. 

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Repo1i No. NCAS 10-064 Page 4 of27 

Quality Assurance Statement 

Study Number: NCAS 10-064 

Study Title: r:Japhnia magna, acute immobilization 

test 

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 

Protocol 

Experimental procedures 

Acquisition, breeding and keeping of 

daphnids 

Preparation of the test solutions 

Exposure of daphnids to the test 

solutions 

Observation of daphnids 

Analytical sampling and treatment 

Analysis ofthe concentrations 

Raw Data 

Draft Repmi 

Final Rep01t 

5/21/2010 

5/25/2010 

5/24, 25/2010 

5/24, 25/2010 

5/25/2010 

5/24, 26/2010 

5/24, 26/2010 

7/5-7/2010 

7/5-7/2010 

7/22/2010 

Study Director Management 

5/24/2010 5/24/2010 

5/27/2010 5/27/2010 

5/27/2010 5/27/2010 

5/27/2010 5/27/2010 

5/27/2010 5/27/2010 

5/27/2010 5/27/2010 

5/27/2010 5/27/2010 

7/7/2010 7/7/2010 

7/7/2010 7/7/2010 

7/22/2010 7/22/2010 

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: _ _.l(~s!..:ig"""n~a~tto!.!.lr~e )L-________ ( seal) (] uly 22, 201 0) 

Ken Watabe 

Nisso Chemical Analysis Service Co., Ltd. 

The original signature page of Quality Assurance Statement follows on page 5. 
Repoti No. NCAS I 0-064 Page 5 of27 

The original Quality Assurance Statement 

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

W\!!J~:i'% : NCAS 10-064 

J:BcW\~0)1~*j·I1:1*ID!:O)IIfBt~ t::J11lt~:a:-l@iJfJ GLP :B J:. t.H~J11'lJ~1'*~iE$F~ (QAU) 0) SOP 

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~~HiOO:f: 5/21/2010 5/24/2010 5/24/2010 

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. ~~~?~0)~~ 5/24, 25/2010 5/27/2010 5/27/2010 

. ~~~~?~-"O);J;~ 5/24, 25/2010 5/27/2010 5/27/2010 

• 3: ~/::I O)fj~~ 5/25/2010 5/27/2010 5/27/2010 

· )J-tff~f~·0)1*J&:B J:: V~f~J! 5/24, 26/20 10 5/27/2010 5/27/2010 

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1:7'-?7 7/5-7/2010 7/7/2010 7/7/2010 

¥~15-:f::&t~ 7/5-7/2010 717/2010 717/2010 

~~¥~15-:f: 7/22/2010 7/22/2010 7/22/2010 

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Report No. NCAS I 0-064 Page 6 of27 

Study No.: 

Study Title: 

Rep01tNo.: 

Sponsor: 

Test Facility: 

Study Director: 

Experimenter: 

Study Initiation Date: 

Experimental Strut Date: 

Exposure Period: 

Study Infonnation 

NCAS 10-064 

immobilization test 

NCAS 10-064 

Nisso Chemical Analysis Service Co., Ltd. 

Odawara Laboratory 

Daphnia magna, acute 

345 Takada, Odawara, Kanagawa 250-0216, Japan 

TEL 81-465-42-5268, FAX 81-465-42-3586 

Osamu Saika 

Satoko Ogawa (test solution preparation, exposure, observation and analysis of 

water parameter) 

Shinpei Oonuki (analysis of concentration oftest substance) 

Chiho Nakamura (maintenance of test organisms) 

May 21,2010 

May 24,2010 

Experimental Completion Date: 

May 24, 2010-May 26,2010 

May 26,2010 

Study Completion Date: 

Test Guideline: 

Archiving: 

July 22, 20 I 0 

Standard for the testing methods concerning new chemical substances. 

(Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, 

No.Il2I 002, November 2I, 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, Daphnia sp., Acute Immobilization Test 

All the documents of this study will be retained in the archives of the test facility for 

I 0 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 
ReporiNo. NCAS 10-064 Page 7 of27 

test facility at least for 10 years after the completion of the study, but only as long as 

the quality ofthe substance affords evaluation. 

Deviations from the SOPs and the protocol: None 

Circumstance I matter that may affect the reliability of the test results: None 
Report No. NCAS I 0-064 Page 8 of27 

Table of Contents 

Title Page ..................... ......................................... ......... .. ..... .... ................................................................. 1 

GLP Cotnpliance Staten1ent ...... ..... ....................... ...................................................................... ............... 2 

The original GLP Compliance Statement .................................................................................................. 3 

Quality Assurance Statetnent .......................................................................................... .. ......................... 4 

The original Quality Assurance Statement ................................................................................................ 5 

Study Infonnation .............................................................................................. ........................................ 6 

Table of Contents .................................................................. ....... .............................................................. 8 

Sun11nary ............................................... .......................................................................... ......................... 1 0 

Introduction ................................................. ............................................................................................ 1 0 

Materials and Methods ............................................................................................................................ 1 0 

1. Test substance ............................................................................................................................... 1 0 

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

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

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

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

6. Test organisn1 ......................................................................................................................... ...... 12 

7. Acute itntnobilization study ......................................................................................................... 13 

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

1. Validation test of the test concentration analysis .......................................................................... I 6 

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

3. In1n1obility .................................................................................................................................... 17 

4. EC50 and 95% confidence interval ............................................................................................... I 7 

5. Observations of the test solutions ................................................................................................. 17 

6. Temperature, pH and dissolved oxygen concentration in the test solutions ............................ , .... 17 

7. Validity of the test ........................................................................................................................ 17 

Conclusions ........... .. ............................... ................................................................................................. 18 

Tables and Figures ................................................................................................................................... 19 

Table 1 Result of validation of the analysis .......................................................................................... 19 

Table 2 Concentration of test substance in test solution ....................................................................... 19 

Table 3 Number of immobilization and percentage of immobilization ................................................ 20 

Table 4 · EC50 and 95% confidence interval ........................................................................................... 20 

Table 5 Observations of the test solutions ................................................ ............................................ 20 

Table 6 Temperature, pH and dissolved oxygen concentration in the test solutions ............................. 21 

Figure 1 NMR spectrum of supplied by Sponsor ....................................................................... 22 

Figure 2 NMR spectrum of the test substance (before the exposure initiation) .................................... 22 

Figure 3 NMR spectrum of the test substance (after the exposure termination) ................................ 23 

Figure 4 Typical calibration curve ........................................................................................................ 24 

Figure 5 Typical chromatogram of a standard solution (5.05 mg/L, at the start of the exposure) ..... 24 
Repmt No. NCAS I 0-064 Page 9 of27 

Figure 6 Typical chromatogram of a repeatability test sample (nominal concentration; I 00 mg/L) .... 25 

Figure 7 Chromatogram of a control solution at the statt of the exposure .. ...................... .................... 25 

Figure 8 Chromatogram of a I 00 mg/L solution at the stmt of the exposure ....................................... 26 

Figure 9 Chromatogram of a I 00 mg/L solution at the end of the exposure ........................................ 26 

Figure IO Concentration ofthe test substance and percentage ofimmobilization ................................ 27 
Repo1i No. NCAS 1 0-064 Page 10 of27 

Summary 

The Daphnia magna, acute immobilization study (static and open system) was performed to estimate the 

effect of the test substance, A 24.0% aqueous solution of 

was used as the test substance. In this repmi, the concentrations of were expressed as , active 

ingredient, unless otherwise specially stated. The measured concentrations of the test substance in the test 

solutions at nominal concentrations of6.26, 12.5, 25.0, 50.0 and 100 mg/L were 102-106% ofthe nominal 

at the strui ofthe exposure and 97.8-101 % ofthe nominal at the end of the exposure. Since the variability of 

concentrations was within ± 20% of the nominal, the median immobilization concentrations (EC50) were 

calculated on the basis of the nominal concentrations as the test concentrations during the exposure period. 

The 24- and 48-hour EC50 are presented below. The 95% confidence interval was not calculated since the 

EC50 was determined by graphical method. 

Exposure duration EC;o (mg!L) 

24 hours 89 

48 hours 71 

Introduction 

This study was conducted in accordance with the following requirement to detennine the median 

immobilization concentrations (EC50) by exposing the Daphnia magna to the test solutions that contained 

for 48 hours. 

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, Daphnia sp., Acute Immobilization Test 

1. Test substance 

Name: 

Abbreviated name: 

Product name: 

Chemical name: 

Materials and Methods 
Rep01t No. NCAS 10-064 Page 11 of27 

Structure: 

CAS No.: 

Molecular formula: 

Molecular weight: 

Lot No.: 

NCAS retrieval No.: STD-1150 

Purity: Solid content; 24.0% aqueous solution 

Impurities: ).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. 

Appearance: 

Obtained from: 

Acquisition volume: 

Acquisition date: 

Expiration date: 

Storage conditions: 

Water solubility: 

Stability information: 

420 g 

January 18, 20IO 

January I8, 20I3 

Stored in a refrigerator with a polypropylene container 

Soluble 

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 I and 

2). This verification was conducted in another study, NCAS I 0-022 "Determination of the 

Concentration of in Dosing Solutions". 

3. Verification of stability of 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. The verification of stability of the test 

substance was conducted in another study, NCAS 10-066 · 

Acute toxicity Study in 01yzias latipes". 

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. Quality of the 
Rep011 No. NCAS 10-064 

Ion exchange water: 

Acetonitrile: 

Formic acid: 

Hardness measurement kit: 

Balances: 

Thermostat water bath: 

Cooling device: 

Thermo recorder: 

Dissolved oxygen meter: 

pH meter: 

Ill uminometer: 

Thermometer: 

Residual chloride meter: 

Centrifuge: 

Micropipette: 

Centrifuge tube: 

LC/MS/MS: 

6. Test organism 

6.1 Test organism 

Species: 

Obtained from: 

Growth stage 

Sensitivity: 

Page 12 of27 

water was confirmed to meet the standard criteria of 3rd-class fisheries 

water (Report 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 55 mg/L, within 

the prescribed range (total hardness as CaC03 was 40-100 mg/L), and 

the concentration ofthe residual chlorine was <0.01 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.) 

WAD-TH (Kyoritsu Chemical-check Lab., Corp) 

AX205, AE240 and XP205 (Mettler Toledo) 

BF200 (Yamato Kagaku Co., Ltd.), 

LTC-450 (As One Co., Ltd.) 

SK.-L200T (SATO KEIRYOKI MTG Co., Ltd.) 

720A (Thermo Fisher Scientific Inc.), OM-51 (HORIBA, Ltd.) 

720A (Thermo Fisher Scientific Inc.), D-51(HORIBA, Ltd.) 

LX-13300 (Custom Co., Ltd.) 

Standard thermometer; measuring temperature range from 0 to 50°C, 

minimum scale 0.1 °C, (Ando Keiki Co., Ltd.) 

Digital residual chloride tester DCT-200, Digital residual chloride tester 

DCT-300 (Takumina Co., Ltd.) 

K.N-70 (Kubota Co.) 

Eppendorf Research V (Eppendorf Co., Ltd.) 

1 0-mL glass test tube with ground-in stopper 

Acquity/Quattro micro API (Waters Corp.) 

Daphnia magna 

National Institute for Environmental Studies (16-2, Onogawa, 

Tsukuba-City, Ibaraki, on June 20, 2008) 

Neonates (less than 24 hours old) were obtained from 17 days old 

parent animals 

The background data of the 48-hour EC50 value of the reference 

substance (potassium dichromate, special grade, Wako Pure Chemical 

Industries, Ltd.) in our laborat01y; 0.18-0.24 mg/L (n=4, July 

2008-February 201 0). 
Rep011 No. NCAS 10-064 Page 13 of27 

6.2 Breeding conditions of parent animals to obtain test organisms 

Young individuals were removed thoroughly from the stock cultures and then the neonates born by 

the next day were transferred to another vessel. These neonates (released on May 7, 20IO) were 

maintained as parent of the test organisms. After the parents started to breed, young individuals 

were removed at least twice a week. On the day before the exposure, young individuals were 

removed thoroughly from the breeding vessel, and the neonates released after that (less than 24 

hours old) were used for the test (exposure initiation date: May 24, 20IO). In breeding period, the 

parent animal mm1ality was 0%, and no abnormal signs such as the production of ephippia or males 

were shown. 

Breeding water: 

Culture density: 

Breeding container: 

Water temperature: 

pH: 

Dechlorinated tap water 

I 0 individuals (transferred at the start of breeding) I 500 mL breeding 

water 

500-mL glass container 

I9.8-19.9 oc 

7.4-7.8 

Total hardness: 55-65 mg/L (as CaC03) 

Dissolved oxygen concentration: 7.3-7.7 mg1L(2:82% of saturation) 

Lighting: fluorescent light (530-630 Lux), 16 hour light I 8 hours dark 

Feed: 

Feeding volume: 

unicellular green algae (Chiarella vulgaris, Raw Chiarella V12, 

Chiarella industry Co., Ltd) 

Foods were given at a rate of 0.1-0.2 mg-C per individual per day. 

Before the start of the production, the animals were fed with an 

adequate volume of food. 

Exchange of the breeding water: at least once a week 

Test room: B6 

7. Acute immobilization study 

7 .I Test conditions 

Exposure system: 

Exposure duration: 

Test vessels: 

Volume of test solutions: 

Static 

48 hours 

1 00-mL tall glass beaker 

I 00 mL I vessel 

Replicates: 4 vessels I group 

Number of daplmids used: 20 individuals I group (5 individuals I vessel) 

Dilution water: 

Test temperature: 

Dechlorinated tap water (the concentration of the residual 

chlorine was <0.01 mgiL, the total hardness were 55 mg/L (as 

CaC03)). 

19.6-19.TC (the temperature ofthe test solutions: test vessels were 

placed in a water bath installed a thermostat water bath) 

19.9-20.0°C (water temperature in water bath during the exposure 
Report No. NCAS 10-064 

pH: 

period) 

7.8-7.9 

Dissolved oxygen concentration: 7.3-9.1 mg/L (2:82% of saturation) 

Page 14 of27 

Lightning: fluorescent light (520-890 Lux), 16 hour light I 8 hours dark 

Feeding: None 

Test room: BJ 

7.2 Selection of the test concentrations and exposure systems. 

A 48-hour range-finding test (RepOii No.; NCAS 10-063NG) was conducted at nominal 

concentration of 10 and 100 mg/L under static condition, and the percentages of immobilization 

were 0 and 100% respectively. The nominal concentrations were maintained for 48 hours. Therefore, 

the definitive test was conducted at 5 nominal concentrations (common ratio; 2.0) of 6.26, 12.5, 

25.0, 50.0 and 100 mg/L. One control group using dechlorinated tap water was also made. 

7.3 Preparation of test solutions 

Test solutions were prepared before use on the day of exposure. An amount of test substance ( 417 

mg as aqueous solution; 100 mg as 1 was weighed and transferred to a 1 000-mL volumetric 

flask and dissolved in dechlorinated tap water (Two solutions were prepared, one was for stock 

solution and another was for the test solutions). These stock solutions of 31.3, 62.5, 125 and 250 mL 

were separately transferred to 500-mL flasks and brought to volume with dechlorinated tap water to 

prepare the test solutions of 6.26, 12.5, 25.0 and 50.0 mg/L respectively. After preparation, the 

conditions (appearance) of the test solutions were recorded. Four vessels containing 100 mL of 

solution were used for each exposure group. For the control group, four vessels containing 100 mL 

of the dechlorinated tap water were used. 

7.4 Analysis of test concentrations 

7.4.1 LCIMS/MS conditions 

Apparatus: Acquity I Quattro micro API (Waters) 

Column: Acquity UPLC BEH CIS, 2.1 mm i.d. x 50 mm, particle diameter; 1.7 

11m (Waters) 

Mobile Phase: Acetonitrile+ 0.1% formic acid aqueous solution (vlv) = 50 +50 (vlv) 

Flow rate: 0.3 mL!min 

Column temperature: 40°C 

Injection volume: 5 !J.L 

Ionization mode: ESI, Negative 

Monitoring ion: 180.33 > 96.90 (quantification), 180.33 > 77.84 (confirmation) 

7.4.2 Preparation ofthe standard solutions and the calibration curves 

An amount (5.05 mg) of the test substance (as 1 was correctly weighed and transferred into a 

50-mL volumetric flask and dissolved in 50% acetonitrile aqueous solution to prepare a 101 mgiL 

standard stock solution. This standard stock solution was then fllliher diluted with the same solvent 
Report No. NCAS I 0-064 Page 15 of27 

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 7.4.1. Calibration curves were 

prepared by plotting the peak areas vs. the concentrations of in the standard solutions 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. 

7.4.3 Analysis of the concentration of the test substance in the test solutions 

The test solutions were analyzed at the sta1i and end of the exposure. Samples were mixed with 

acetonitrile at the rate of 1:1 (v/v) and centrifuged (3000 rpm, 10 min). The supernatant 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 7 .4.1. 

Since variations of the measured concentrations of the test substance at start and end of the exposure 

were within ±20% of the nominal, the nominal concentrations were regarded as the test 

concentrations during the exposure period. 

The samples from the control group were also mixed with acetonitrile at the rate of 1:1 (v/v) and 

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. 

7.5 Validation study of the analysis of the test substance concentration in the test solutions 

7.5 .1 Validation of the calibration curve 

The accuracy was calculated by finding the regression equation and correlation coefficient (r) of 

calibration curve, and then determining the quantitative value using inverse estimation of regression. 

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%. 

7.5.2 Validation ofthe repeatability 

Analytical method was validated at the exposure initiation by a repeatability test without any 

recovery tests, because extract operation was not conducted. The concentrations of the test 

substance in the highest and lowest concentration were calculated by the same procedure as 

described in section 7.4.3 (n=3). The analytical method was validated by the coefficient of 

variations of the measured test concentrations (n=3) which was within ±10%. 

7.6 Exposure procedure · 

The test was initiated when daphnids were assigned to the test vessels that contained the test 

solutions. 

The conditions (appearance) of the test solutions were recorded at the staJi, and at 24 and 48 hours 
Report No. NCAS 10-064 Page 16 of27 

after the beginning of the exposure. Water temperature, dissolved oxygen and pH were measured at 

the stari and end of the exposure. At the exposure initiation, one vessel which was prepared in 

addition to 4 replicate test solutions was measured. Temperature in the thermostat water bath, in 

which the test vessels ware placed, was continuously recorded during the exposure period with the 

temperature recorder. At the exposure termination, the measurement was conducted in one vessel in 

each exposure and control group. 

Daphnids were observed to check if there were immobilized daphnids, any abnormal behavior or 

appearance at 24 and 48 hours after the beginning of the exposure. Daphnids were considered to be 

immobilized when the daplmids did not swim within 15 seconds after gentle agitation of the test 

vessel. If daphnids were touched on the bottom of the vessel or trapped on water surface, it was 

indicated immobilization. 

7.7 Calculation ofthe median immobilization concentration (EC50) 

The EC50 were calculated from the number of immobilization and the number of daplmids used by 

graphical method. The values of EC50 and 95% confidence limit were shown as 2 significant 

figures. 

Results 

1. Validation test of the test concentration analysis 

1.1 Validation of the linearity of the calibration cmve 

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

solution (5.05 mg/L, at the start of the exposure) is shown in Figure 5 (measured in the study NCAS 

1 0-066). The coefficients of correlation (r) were more than 0.990 both at the start and end of the 

exposure. The all variations of the accuracy of the lowest standard samples were within ±20% and 

those of other standard samples were within ± 15%. Based on the above results, the linearity of the 

calibration curves were determined as acceptable. 

1.2 Validation of the repeatability 

The results of the repeatability test are shown in Table 1. A typical chromatogram of the 

repeatability test (nominal concentration: 100 mg/L) is shown in Figure 6. Chromatogram of the 

control group is shown in Figure 7. 

The Coefficients of variation (n=3) ofthe test solutions were all within 10%. On the chromatogram 

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. 

2. The concentrations of the test substance in the test solutions 

The analytical results of the test substance concentrations in the test solutions are presented in Table 2 
Report No. NCAS I 0-064 Page 17 of27 

and 9 respectively. 

The measured concentrations of the test substance in the test solutions were 102-106% of the nominal 

at the exposure initiation and 97.8-1 01% of the nominal at the exposure termination. The test substance 

concentrations in the test vessels were stable during the exposure period. Since the variability of the 

measured concentrations of the test substance was within ±20% of the nominal, the nominal 

concentrations were used as the test concentrations. 

3. Immobility 

The number and percentage of immobilized daphnids in each test group are shown in Table 3, and the 

relationship between the concentrations of the test substance and percentage of immobilization are 

shown in Figure 10. 

No immobility was observed in the 6.26, I2.5, 25.0 and 50.0 mgfL groups both after 24 and 48 hours of 

the exposure. Immobility in the 100 mg/L group after 24 hours of the exposure was 60% and after 48 

hours was I 00%. In addition, some abnormal behavior was observed among daphnids in the 50.0 and 

IOO mg/L group after 24 hours and in the 25.0 and 50.0 mg/L group after 48 hours of the exposure. 

In the control group, no abnormality was observed during the exposure period. 

4. EC50 and 95% confidence interval 

The values ofEC50 are presented in Table 4. 

The 24-hour and 48-hour EC50 value were determined to be 89 mg/L and 7I mgfL, respectively, by 

using graphical method. The 95% confidence interval was not calculated. 

5. Observations of the test solutions 

The observations of the test solutions were shown in Table 5. 

All solutions in exposure and control groups were colorless and transparent during the exposure period. 

6. Temperature, pH and dissolved oxygen concentration in the test solutions 

The measurements of temperature, pH and dissolved oxygen concentration in the test solutions are 

presented in Table 6. 

Throughout the exposure period, the temperatures in the test solutions ranged from 19.6 to 19.7°C and 

the pH ranged from 7.8 to 7.9. The dissolved oxygen concentrations ranged from 8.8 to 9.1 mg/L (~99% 

of saturation) at the exposure initiation and from 7.3 to 7.7 mg/L (~82% of saturation) at the exposure 

termination, therefore maintained within the prescribed range of~3 0 mg/L. 

7. Validity ofthe test 

The validity of the test was confinned because there were no items met the following situation; 

More than I 0% of daphnids in the control group was immobilized. 

Dissolved oxygen concentrations at the end of the exposure were less than 3 mg/L. 
Rep01i No. NCAS 10-064 Page 18 of27 

Conclusions 

The acute immobilization study (static and open system) was performed by exposing Daphnia magna to 

It is concluded that the 48-hour EC50 values is 71 mg/L. 
Rep01t No. NCAS 10-064 Page 19 of27 

Tables and Figures 

Table I Result of validation of the analysis 

Nominal Measured concentration Mean measured Coefficient of variation concentration concentration 

(mg!L) 

100 

6.26 

(mg/L) (mg/L) 

106 

104 106 

107 

6.34 

6.46 6.49 

6.66 

Table 2 Concentration of test substance in test solution 

Nominal Measured concentration (mg/L) 

concentration 

(mg/L) 0 hour 

Control N.D. 

6.26 6.48 (104) 

12.5 12.7 (102) 

25.0 25.5 (102) 

50.0 53.0 (1 06) 

100 105 (105) 

( ): Percent of nominal concentration(%) 

N.D.: Not detected 

72 hours 

N.D. 

6.12 (97.8) 

12.3 (98.4) 

25.3 (101) 

49.3 (98.6) 

98.0 (98.0) 

(%) 

1.44 

2.49 
Rep01i No. NCAS 10-064 Page 20 of27 

Table 3 Number of immobilization and percentage of immobilization 

n=20 

Nominal Number of immobilization 

concentration (Percentage ofthe immobilization(%)) 

(mg/L) 24 hours 48 hours 

Control 0 (0) 0 (0) 

6.26 0 (0) 0 (0) 

12.5 0 (0) 0 (0) 

25.0 0 (0) 0 (0) 

50.0 0 (0) 0 (0) 

100 12 (60) 20(100) 

Table 4 EC50 and 95% confidence interval 

ECso 95% confidence interval 

Exposure duration 

(rrig/L) (mg/L) 

24 hours 89 -* 

48 hours 71 -* 

*· Since the EC50 were determined by graphical method, the 95% confidence 

interval was not calculated. 

Table 5 Observations of the test solutions 

Nominal 

concentration 0 hour 24 hours 48 hours 

(mg!L) 

Colorless and Colorless and Colorless and 

Control transparent transparent transparent 

Colorless and Colorless and Colorless and 

6.26 transparent transparent transparent 

Colorless and Colorless and Colorless and 

12.5 transparent transparent transparent 

Colorless and Colorless and Colorless and 

25.0 transparent transparent transparent 

Colorless and Colorless and Colorless and 

50.0 transparent transparent transparent 

Colorless and Colorless and Colorless and 

100 transparent transparent transparent 
Repm1 No. NCAS 10-064 Page 21 of27 

Table 6 Temperature, pH and dissolved oxygen concentration in the test solutions 

Nominal Temperature CC) pH Dissolved oxygen 

concentration* (mg/L) 

concentration 

(mg!L) 0 hour 48 hours 0 hour 48 hours 0 hour 48 hours 

Control 19.7 19.7 7.8 7.8 8.9 (100) 7.4 (83) 

6.26 19.7 19.7 7.9 7.9 8.8 (99) 7.4 (83) 

12.5 19.6 19.7 7.9 7.8 9.0 (101) 7.3 (82) 

25.0 19.7 19.7 7.8 7.8 9.0 (101) 7.6 (85) 

50.0 19.7 19.7 7.9 7.9 9.0 (101) 7.7 (87) 

100 19.7 19.7 7.9 7.9 9.1 (102) 7.6 (85) 

* · Figures in parentheses indicate the percentages of saturation. 
Report No. NCAS 10-064 Page 22 of27 

~~?~ ~co~ -··· ·~~ ~u~~ ~-

~~~:~~~~-~ ~~ ~~ ~::~~~~~~~~ - .. ." Vfoi o~~ ·· ·.j· --~•-· ~·Nr: o•~ '"'-'"''"'·"' o.c:."' 

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-20 -40 -60 -80 -100 -120 ppm 

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r;,.fi/ 

211.11 10.0 !:/:-!i· >-T 

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Figure 2 NMR spectrum of the test substance (before the exposure initiation) 
Report No. NCAS I 0-064 Page 23 of27 

dEOL; 

j·····-·--···------.. -- .......... ----------

1 

! 

I 

O. llOUOHI"l 

JOIO•ol 

f!dll 

,, ... , 

:it. 

ll•l 

11•1 

J.UOlODUteJ lSh~Cl 

Figure 3 NMR spectrum of the test substance (after the exposure tetmination) 
Rep01i No. NCAS l 0-064 

Concentration Quantitative Accuracy Peak area value (mg/L) (mg/L) (%) 

1.0 I 2006 0.956 94.7 

2.02 3728 2.13 106 

3.03 5151 3.11 103 

4.04 6471 4.01 99.3 

5.05 7830 4.94 97.8 

Method: C:\Masslym IMethDB\ .mdb 21 Apr 2010 18:23:08 

Calibration: 24 May 2010 16:38:14 

6000 

~ 5000 

~ 4000 . 

~ 3000 . 

~~~~~~~~~~~~~~~ppm 

0.00 1.00 2.00 3.00 4.00 5.00 

Figure 4 Typical calibration curve 

1000524-05 

STD-5-1 0.74 

7830 

MRM of2 channels,ES180.33 > 96.90 

8.199e+004 

100 

0.50 1.00 1.50 2.00 2.50 

Figure 5 Typical chromatogram of a standard solution 

(5.05 mg/L, at the stmi ofthe exposure) 

Page 24 of27 
Report No. NCAS 10-064 

1000524-11 

D-100-R1 

0.50 

0.75 

3699 

1.00 1.50 2.00 

Page 25 of27 

MRM of 2 channels,ES180.33 > 96.90 

8.199e+004 

2.50 

Figure 6 Typical chromatogram of a repeatability test sample 

(nominal concentration; 100 mg/L) 

1000524-17 

D-cont-0 

100 

% 

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 a control solution at the start of the exposure 
Rep01i No. NCAS I 0-064 

1000524-18 

D-1 00-0 

100 

% 

0.50 

0.75 

3 3 

1.00 1.50 2.00 

Page 26 of27 

MRM of2 channels ,ES180.33 > 96.90 

8.199e+004 

2.50 

Figure 8 Chromatogram of a 100 mg/L solution at the start of the exposure 

1000526-11 

D-100-48 

100 

% 

0.78 

3336 

MRM of 2 channels,ES180.33 > 96.90 

7.606e+004 

Figure 9 Chromatogram of a 100 mg/L solution at the end of the exposure 
Repm1 No. NCAS 10-064 

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100 

90 

80 

70 

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40 

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Concentration of the test substance (mg!L) 

Page 27 of27 

i 

i 

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I 

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1/ 

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r; 

# 

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100.00 

Figure 10 Concentration of the test substance and percentage of immobilization 
Authenticity of Translation 

I declare that the original Japanese final repmt (Repmt No. NCAS 1 0-064) is translated into English 

consistently. 

Translated by : 

Osamu 

~~ Saika / 

Nisso Chemical Analysis Service Co., Ltd. 

Odawara Laboratmy