### 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 

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## 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 ofGLP Compliance Statement appears on page 2.



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## 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 ofthe test solutions Exposure of daphnids to the test solutions Observation ofdaphnids 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 I0-064 Page 5 of27 

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# w )j-;j;JTt / ?'-Report No. NCAS I0-064 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 ofconcentration oftest substance) Chiho Nakamura (maintenance oftest 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 I0 Standard for the testing methods concerning new chemical substances. (Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, No.Il2I002, 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 ofthis study will be retained in the archives ofthe test facility for I0 years after the completion ofthe study, but the place for storage after that will be decided on discussion with the sponsor. The test substance will be retained in theReporiNo. NCAS 10-064 Page 7 of27 test facility at least for 10 years after the completion ofthe 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 ofthe 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 ofContents ............ ................... .. .... ....... ....... ........ .. ... .. .... . .. ......... ..... ...... ......... .. .... .... ...... .... . .. ......... . 8 Sun11nary ............................................. .. ....... .. ....... ...... ............................................. ..... .. .... ... .. ... ............. 1 0 Introduction ............................................... .. .............. .. .... ......... ........................................................... . ... 1 0 Materials and Methods ............................................................................................................................ 1 0 1. Test substance................................................. . ...... ........ ...... ........................... ..... . ..... .................. . 1 0 2. Verification ofthe test substance............... ................ .. ....... ...... ...... ....... .. ... ...... . ...... ..................... 11 3. Verification ofstability oftest substance . .. .... . ... .. ... ... ......... .... .... ... ... ... .. ... ... ................................ 11 

### 4. Verification of solubility oftest substance ...... ....................................... .... . ...................... .... ... .... 11 5. Reagents and apparatus .............................................................. . ................................................. 11 6. Test organisn1 ..... ..... ........... ...... .. .... .. .................................................................. .................. .. ... ... 12 7. Acute itntnobilization study ........................................................ .. ............. . ........... ...................... 13 Results .................................. ...... ........ .. ... ................ . .... ................ .................. ............. ... . ...... .............. .... 16 

### 1. Validation test ofthe test concentration analysis.......................................................................... I 6 2. The concentrations ofthe test substance in the test solutions ...................................................... 16 3. In1n1obility ...................................... .. ................ ........ . ........................... ...... ...... ... . ........................ 17 4. EC 50 

### and 95% confidence interval ............................................................................................... I7 5. Observations ofthe test solutions......................... ............... ... ........ .. ........... ................................ . 17 6. Temperature, pH and dissolved oxygen concentration in the test solutions .......... ......... .........,.... 17 7. Validity ofthe test ........................................................................................................................ 17 Conclusions ...... ... .. .. ........................ ..... .. .......... ....................................................................................... 18 Tables and Figures ................................................................................................................................... 19 Table 1 Result ofvalidation ofthe analysis .......... .................. ........ .......... .................... ........................ 19 Table 2 Concentration oftest substance in test solution ....................................................................... 19 Table 3 Number ofimmobilization and percentage ofimmobilization ................................................ 20 Table 4 · EC50 

### and 95% confidence interval .......................................................................... .. ............... 20 Table 5 Observations ofthe 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 ofthe test substance (before the exposure initiation) .................................... 22 Figure 3 NMR spectrum ofthe test substance (after the exposure termination) ................................ 23 Figure 4 Typical calibration curve . ........................... .... ........................................................................ 24 Figure 5 Typical chromatogram ofa standard solution (5 .05 mg/L, at the start ofthe exposure) ..... 24Repmt No. NCAS I0-064 Page 9 of27 Figure 6 Typical chromatogram of a repeatability test sample (nominal concentration; I00 mg/L) .... 25 Figure 7 Chromatogram of a control solution at the statt ofthe exposure.. .. .................... .. .................. 25 Figure 8 Chromatogram of a I00 mg/L solution at the stmt ofthe exposure ....................................... 26 Figure 9 Chromatogram of a I00 mg/L solution at the end ofthe exposure ........................................ 26 Figure IO Concentration ofthe test substance and percentage ofimmobilization.......... .... .... ........ ...... 27Repo1i No. NCAS 10-064 Page 10 of27 

## Summary The 

### Daphnia magna, acute immobilization study (static and open system) was performed to estimate the effect ofthe 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 ofthe exposure. Since the variability of concentrations was within ± 20% of the nominal, the median immobilization concentrations (EC 50) were calculated on the basis ofthe nominal concentrations as the test concentrations during the exposure period. The 24- and 48-hour EC 50 are presented below. The 95% confidence interval was not calculated since the EC 50 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 (EC 50) 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 ofEconomy, 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 MethodsRep01t 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 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 I and 2). This verification was conducted in another study, NCAS I0-022 "Determination of the Concentration of in Dosing Solutions". 3. Verification of stability oftest 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 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. Quality of theRep011 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: Illuminometer: 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 ofthe 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.) EppendorfResearch V (Eppendorf Co., Ltd.) 10-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 EC 50 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 ofparent 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 I0 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 offood. Exchange ofthe 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 oftest solutions: Static 48 hours 1 00-mL tall glass beaker I00 mL I vessel Replicates: 4 vessels I group Number ofdaplmids 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 exposureReport 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 ofthe 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 oftest 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 of31.3, 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 ofthe dechlorinated tap water were used. 7.4 Analysis oftest 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 solventReport No. NCAS I0-064 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 ofthe concentration ofthe 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 ofthe range ofthe calibration curve, and then analyzed by using the LC/MS/MS conditions as described in section 7.4.1. Since variations ofthe measured concentrations ofthe test substance at start and end ofthe 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 ofthe test substance on the chromatogram. 7 .5 Validation study ofthe analysis ofthe test substance concentration in the test solutions

7.5 .1 Validation ofthe 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 ofregression. 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 ofthe 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 hoursReport 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 (EC 50) The EC 50 were calculated from the number of immobilization and the number of daplmids used by graphical method. The values of EC 50 and 95% confidence limit were shown as 2 significant figures.



## Results 1. Validation test ofthe test concentration analysis

1.1 Validation ofthe linearity ofthe calibration cmve A typical calibration curve is shown in Figure 4 and a typical HPLC chromatogram ofthe standard solution (5.05 mg/L, at the start ofthe 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 ofthe 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 ofthe 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 ofthe test substance in the test solutions The analytical results of the test substance concentrations in the test solutions are presented in Table 2Report No . NCAS I0-064 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 I00%. 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 ofthe exposure. In the control group, no abnormality was observed during the exposure period. 4 . EC 50 and 95% confidence interval The values ofEC 50 are presented in Table 4. The 24-hour and 48-hour EC 50 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 ofthe test solutions The observations ofthe 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 ofthe test was confinned because there were no items met the following situation; More than I0% of daphnids in the control group was immobilized. Dissolved oxygen concentrations at the end ofthe 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 EC 50 

### values is 71 mg/L.Rep01t No. NCAS 10-064 Page 19 of27 

## Tables and Figures Table I Result ofvalidation ofthe analysis Nominal Measured concentration Mean measured Coefficient ofvariation 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 oftest 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 ofnominal 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

2.49Rep01i No. NCAS 10-064 Page 20 of27 

### Table 3 Number ofimmobilization and percentage ofimmobilization 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 EC 50 

### and 95% confidence interval ECso 95% confidence interval Exposure duration (rrig/L) (mg/L) 24 hours 89 

### -* 48 hours 71 

### -* *· Since the EC 50 were determined by graphical method, the 95% confidence interval was not calculated.



### Table 5 Observations ofthe 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 transparentRepm1 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.

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### Report No. NCAS I0-064 Page 23 of27

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### 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

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### 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 ofa 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 ofthe exposure

### Rep01i No. NCAS I0-064
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 ofa 100 mg/L solution at the start ofthe 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 ofthe exposureRepm1 No. NCAS 10-064

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## Authenticity ofTranslation

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