### Report No. NCAS 10-065



# Final Repmi

Study Title: Algal Growth Inhibition Study



### Author: Koji Baba


### 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 10-065 Page 1 of34

### Rep01i No. NCAS I 

### 0-065 Page 2 of34


## GLP Compliance Statement Study No . : NCAS 10-065 Study title: Algal Growth Inhibition Study

This study was carried out in accordance with the following good laborat01y practice regulation; Standard for the test facility conducting tests conceming new chemical substances, etc. (Pharmaceutical and Food Safety Bureau, Minist1y 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 repOii was prepared faithfully and consistently with the raw data obtained. Study Director:**-----'(..,s~ig:>!.n~a~tu~r~e**)J__ ________ (

### seal) July 22, 2010 Koji Baba Date Nisso Chemical Analysis Service Co., Ltd. Odawara Laboratory The original signature page ofGLP Compliance Statement follows on page 3.

### Report No . NCAS 10-065 Page 3 of34 

## The original GLP Compliance Statement The English translation ofGLP Compliance Statement appears on page 2.**~~:1=%** 

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## Quality Assurance Statement Study Number: NCAS I0-065 Study Title: : Algal Growth Inhibition Study 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 reported 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 5/24/2010 5/24/2010 5/24/2010 Experimental procedures

Acquisition and cultivation of algae 617/2010 6/10/2010 6/10/2010 Preparation ofthe test solutions 5/25,28/2010 5/28/2010 5/28/2010 Exposure of algae to the test solutions 5/25,28/2010 5/28/2010 5/28/2010 Exposure of algae to the test solutions 617' 1 0/2010 6/10/2010 6/10/2010 (Re-experiment) Measurement ofthe cell density 5/26,28/2010 5/28/2010 5/28/2010 Measurement ofthe cell density 6/10, 14/2010 6/14/2010 6/14/2010

(Re-experiment) Analytical sampling and treatment 5/25, 28/2010 5/28/2010 5/28/2010 LC/MS/MS analysis 5/25, 28/2010 5/28/2010 5/28/2010 Microscopic observation(Re-experiment) 6/10, 14/2010 6/14/2010 6/14/2010 Raw Data 7114-20/2010 7/20/2010 7/20/2010 Draft Report 7/14-20/2010 7/20/2010 7/20/2010 Final Report 7/22/2010 7/22/2010 7/22/2010 The QAU found that the study was performed according to the protocol and SOPs, the repmied methods and procedures were actually used, and the results accurately reflect the recorded data. QAU Manager: _ _,(""'"s'""ig:..:.:n=at=u:..:.re=-)'--________ (seal) (July 22, 2010) Ken Watabe Nissa Chemical Analysis Service Co ., Ltd. The original signature page of Quality Assurance Statement follows on page 5.Report No. NCAS I 0-065 Page 5 of34


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### Report No. NCAS 10-065 Page 6 of 34

Study No. : Study Title: RepmiNo.: Sponsor:

Test Facility:

Study Director: Experimenter:

Study Initiation Date: 

## Study Information NCAS 10-065 NCAS 10-065

Nissa Chemical Analysis Service Co., Ltd. Odawara Laboratory Algal Growth Inhibition Study

345 Takada, Odawara, Kanagawa 250-0216, Japan TEL 81-465-42-5268, FAX 81-465-42-3586 Koji Baba Chiho Nakamura (management of test organisms and all experimental procedures except analysis oftest concentrations) Koji Baba (first pre-culture [first definitive test]) Shinpei Oonuki, Chiaki Urabe (analysis oftest concentrations) Experimental Sta1i Date: May 21 , 2010 May 25 , 2010 Exposure Period: Experimental Completion Date: May 25 , 2010- May 28 , 2010 (First definitive test, invalid) June 7 , 201 0 -June 1 0, 2010 (Second definitive test, valid) June 10 , 2010 Study Completion Date: Test Guideline : July 22, 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, Minist1y of Economy, Trade and Industry, No .2, November 13, 2003; Environmental Policy Bureau, Ministry of the Environment, No. 03 1121002 ) The latest amendment: November 20 , 2006 , <Algal Growth Inhibition Test, Daphnia sp. , Acute Immobilis ation Test and Fish Acute toxicity test> IV Algal Growth Inhibition TestReport No. NCAS I0-065 Page 7 of34
Archiving: All the documents ofthis study will be retained in the archives ofthe test facility for 10 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 the test facility at least for I0 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 I0-065 Page 8 of34 

## Table of Contents Title Page .................... .... .. .. ................................. . .... ...... ............ . ... . .... ..... . .... .... . . ..... . .... ... . .. . ... . ... . .......... . .. 

### 1 GLP Co1npliance Staten1ent ...................................... .... . ...... . ................... ... .................... . .............. . ........... 2 The original GLP Compliance Statement .. ......... ... ......... .................................................. .. ......... ........ ...... 3 Quality Assurance Statement ... ................................................. .. ............................... ........ ......... . ......... ..... 4 The original Quality Assurance Statement ......................... ....................................................................... 5 Study Inforn1ation . .. ... .... .......................................... .. . ... .... .......... ............................... ... ....... ....... ..... .... ..... 6 Table ofContents ....................................................................................................................................... 8 Su1n1nary .. ... ....... ... ....... . . . ............ .... .. .. ............................. .... ........... ..... . ................. ... .. ... .................. .... . .. . 

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

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

### 11 1. Test substance................................. ..... ............... ... .. .. ................................................................... 

### 11 2. Verification oftest substance............................................................................................. . .......... 

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

### 11 4. Verification of solubility oftest substance ......................... ........ .......... . .......... . ......... ................. .. 

### 12 5. Reagents and apparatus ... ..... .............. . ......... . .. .. ...... .. ................................................................... 

### 12 6. Test organis1ns ........................... . .................... .... .......................................................................... 

### 12 7. Algal growth inhibition study ............... .... . .................................................................................. 13 8. Analysis ofthe test concentrations ............................................................................................... 15 9. Calculation ofthe percent inhibition ofgrowth rate .................................... . ... :.... . ... .... ............... 

### 17 10. Calculation of 50% inhibition ofgrowth rate (ErC 50) .............................................................. 

### 17 11. Determination ofNo Observed Effect Concentration (NOECr) ........................................ . ..... 18 Results .. ...... .. ........................................................................................ . .................................................. 18 1. Validation ofthe test substance .................................................................................................... 18 2. Validation ofthe stability ofthe test substance .............................................. . ............................. 18 3. Validation ofthe analytical methods ............... .. ........................................................................... 18 4. Test concentrations ofthe test substance in the test solutions ...................................................... 19 5. Growth curve and growth rate ........................... .. ......................................................................... I 9 6. ErC 50 , 

### 95% confidence interval and NOECr ................................................................................ 19 7. Temperature and light intensity in the incubator ......................................................... .. ............... 20 

### 8. pH ........................... ... .... ..... ................................. . .... ...... .. .... .. .. .. .......... .. .... ............ .......... ...... ...... 20 9. Observations oftest solutions .................................... ... .......... .. ... .. .. ............... ..... ........... .. ........... 20 10. Microscopic observations of algae .. .... ................ .... ......................................................... .. ...... 20 

### 11. Validity ofthe test. .............. .. ........................... ..... ........................ . .................................. .. .. ..... 20 Conclusions ........... . . . . . ............. .... .. . ......... . ....... ...... ........... ..... . .. .. ... . .... ... .... . . ....................................... . .... 

### 21Report No. NCAS 10-065 Page 9 of34


## Table of Contents (continued) Tables and Figures . ...... . .... ... . .. ..... . .. . .... . . .. . ...... ... . .. . .. . ..... ... ........ .. . . . ... . .............. ..... ... . . .. .... . . ... . . ... .. . . ... .. ..... 22 Table 1 Result ofvalidation ofthe analysis ...................... .... .... .. .... .. ...................... .. ........ .. .. .. .... .. ...... .. .22 Table 2 Concentration oftest substance in test solution ...... .. .... .... .................................................. .. .... 22 Table 3 Cell density during the exposure period .......... .. .. .. .......... .. .. .. .......................................... .... .... .23 Table 4 Growth rate and percent inhibition of growth rate .............. .. .......... .. ...................... .. ............ .. .. 24 Table 5 ErC 50 , 95% confidence interval and NOECr .. ........ .. .... .. .............................................. .... ...... .. 25 Table 6 Temperature and light intensity in the incubator.... .. ................................................................. 25 Table 7 pH ofthe test solutions .... .. ...................... .. .. .. .. .. .... .. ............ . .................. .. ........ .. ...................... 26 Table 8 Observations ofthe test solutions ............ .. .......... .. ............ .. .. .. ................................................. 26 Table 9 Microscopic observations of algae.......... .. ...... .. ..... .. ....... .. ..... .. ................................................. 27 Figure I NMR spectrum of :supplied by Sponsor, 19F-NMR) .......................... .... ...................... 27 Figure 2 NMR spectrum of the test substance (Before the exposure initiation, Study No.: NCAS 10-022) .. .... . . .. . . ... . .. . ............ . .......................... ... .... .. ...... ... .. .... ... .... . . . . . . ............... . .......... .. ............... 28 Figure 3 NMR spectrum of the test substance (After the exposure termination, Study No.: NCAS 10-066) . ......... . ...... . ........ . ..... . .... . . . ............ .. ...... . ................. . .......... .. ... . .... . ............. . ... . .................... 28 Figure 4 Typical calibration curve . .......................... .. .. ... ..... .. ....... . ..................... . ... . .... . ...... . ..... . ... . . .. ..... 29 Figure 5 Typical chromatogram of a standard solution (5.05 mg/L, at the stmi ofthe exposure) ... 29 Figure 6 Typical chromatogram of a repeatability test sample .. .. ............................................ .... .......... 30 Figure 7 Chromatogram ofthe control group (at the stmi ofthe exposure) .... .. ................................ 30 Figure 8 Chromatogram ofthe 100 mg/L group (at the start ofthe exposure) .. .. ........................ .... .. 3I Figure 9 Chromatogram ofthe 100 mg/L group (at the end ofthe exposure) .. .. ............................... 31 Figure I0 Growth curves of algae .................................................... .. .. .. ........................................ .. ..... 32 Figure 11 Percentage inhibition ofgrowth rate calculated for each test group...................................... 33 Appendix 

### 1 OECD 1nediun1 .. ... . . . ... .. ...... .. .. . .... ... .................................. . .................................... . . . . . .... . 34Report No. NCAS 10-065 Page 10 of34 

## Summary A 72-hour growth inhibition study (open system, shaking culture) was performed by exposing the algae to test solutions containing ·- . A 24.0% aqueous solution of was used as a test substance and the concentrations of were expressed as itself unless otherwise specially stated. The measured concentrations of the test substance in the test solutions at 5 nominal concentrations of6.25, 12.5, 25.0, 50.0 and 100 mg/L (common ratio: 2.0) were 104-106% ofthe nominal at the start ofthe exposure and 99.6-103% ofthe nominal at the end ofthe exposure. Since the variability of the measured concentrations was within ± 20% ofthe nominal, the results were calculated on the basis ofthe nominal concentrations as the test concentrations during the exposure period. The concentration of the test substance that reduced algal growth by 50% (ErC50) and the No Observed Effect Concentration (NOECr) values based on the growth rate for the 0-72-hour exposure period are presented below: Exposure duration 0-72 hour ErCso (mg/L) > 100* 95% confidence interval (mg/L) NOECr (mg!L)

50.0 *:Since the% inhibition was under 50% even at the highest test concentration (100 mg/L), the ErC 50 was detennined > 1 00 mg/L.



## Introduction This study was conducted in accordance with the following requirement to determine the 50% inhibition growth rate (ErC 50) and the No Observed Effect Concentration (NOECr) by exposing the algae to the test solutions containing 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, <Algal Growth Inhibition Test, 

### Daphnia sp., Acute Immobilization Test and Fish Acute toxicity test> IV Algal Growth Inhibition TestReport No. NCAS I 0-065 Page II of34


## Materials and Methods 1. Test substance Name: Abbreviated name: Product name: Chemical name : Structure: CAS No.: Molecular formula: Molecular weight: Lot No.: NCAS retrieval No.: STD-1150 Purity: Solid content; 24.0% aqueous solution Impurities: 0.85% (as against solid content), Cl ion; 35 ppm In this rep01i, the concentrations ofthe test substance were expressed as those of Appearance: Obtained from: Received volume: Received date: Expiration date: Storage conditions: Water solubility: mless 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 Stability information: 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 oftest substance The identity of the test substance was verified by comparing the nuclear magnetic resonance spectrum ( 19F-NMR) which was measured before the exposure initiation with another spectrum which was supplied by the sponsor. This verification was conducted in another study, NCAS 10-022

" Determination of the Concentration of in Dosing Solutions " and the result was utilized in this test. 3. Verification of stability ofthe test substance The storage stability of was verified by confirming identity between the two nuclear magneticReport No. NCAS I 0-065 Page12of34

resonance spectra ( 19F-NMR) which were measured before and after the exposure. This verification was conducted in another study, NCAS 10-066 Acute Toxicity Study in 

### 01yzias latipes" and the result was utilized in this study. 4. Verification of solubility oftest substance Since the test substance was a 24% aqueous solution, its water solubility was not measured. 5. Reagents and apparatus Distilled water, acetonitrile: HPLC grade (Wako Pure Chemical Industries, Ltd.) Formic acid: Ion exchange water:

Balances: Sterilization filters: pH meter: Illuminometer: Thermometer: Incubator shaker: Clean bench: Special grade (Wako Pure Chemical Industries, Ltd .) 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.), or purified with an Ion exchange water system (SA2100E, Tokyo Rikakikai Co ., Ltd.) to prepare ion exchange water. AG285, AX205, AG204 and XP205 (Mettler Toledo) Nylon membrane filter units, 0.2**~un** (NALGENE) pH meter D-51(HORIBA, Ltd.) Ll-1400 (MEIWAFOSIS Co.) Standard thermometer; measuring temperature range from 0 to 50°C, minimum scale 0.1 

## ac (Ando Keiki Co., Ltd.) MR-1 OOLS (Takasaki Scientific Instruments Corp.) FG-1914L, BHC-1604 IIA/8 3 (Airtech Japan, Ltd.) Electronic particle counter: F-520P (Sysmex Corp.) Electrolytic solution for the particle counter: Cellpuck (Sysmex Corp.) Ultrasonic bath: UC-3 (Ikeda Scientific Co., Ltd.) Microscope: BX51 TRF (Olympus Corp.) Centrifuge: M200-IVD (Sakuma Co., Ltd.), KN-70 (Kubota Co.) Micropipette: EppendorfResearch V (EppendorfCo., Ltd.) Centrifuge tube and test tube: 10-mL glass test tube with ground-in stopper Liquid chromatograph mass spectrometer (LC/MS/MS): Acquity/Quattro micro API (Waters Corp.) 6. Test organisms Taxonomic group: Scientific name: Obtained from: Acquisition date: Strain: Sensitivity: unicellular green algae 

### Pseudokirchneriella subcapitata (former 

### capricornutum) National Institute for Environmental Studies July 3 , 2008 NIES-35 name; 

### Selenastrum

The background data of the 0-72 hour ErCso value of the reference substance (potassium dichromate) in 

### P subcapitata in our laboratory wereRepoti No. NCAS 10-065

Pre-culture conditions:

Test room used: Page 13 of34

1.4 and 1.2 mgiL (n=2 , conducted in June and December 2009 , NCAS No.; 09-059). The test organisms used in this study were cultured under the same conditions as those to be used in the study and in log growth phase at the stati of the exposure. After the pre-culture period, the cells were observed microscopically to confirm that neither modification nor abnormal appearance ofthe algae was shown. Culture period; June 4-7,2010 Temperature; 23.1 °C (in the incubator) Lightning; 72-76 f.!Ehn 21s under continuous illumination (cool white fluorescent lamp, measured at the level of the solutions in flasks) A4 7. Algal growth inhibition study The definitive tests were conducted twice. Since variability ofthe growth rates in the control group was out ofthe criteria in the first definitive test, the test was not accepted. Accordingly, the second definitive test was conducted under the same conditions as the first definitive test except changing the rotation position ofthe test vessels in the incubator. In this repmi, only the second definitive test is described. 7.1 Test conditions Type ofculture: Exposure period: Volume oftest solutions: Vessels: Replicates: Initial cell density: Test temperature: Lighting : pH: Medium: Test room used: Shaking culture (100 rpm) 72 hours 100 mL 

### I vessel 300-rnL conical glass vessels with aluminum caps (air-permeable) 3 vessels 

### I exposure group (6 vessels 

### I control group) 5000 cells 

### I mL (nominal) 23±2°C 65-100 f.!Eim21s under continuous illumination (cool white fluorescent lamp, measured at the level ofthe solutions in flasks) The pH values ofthe test solution were not adjusted OECD medium (see Appendix 1) A3 7.2 Selection ofthe concentrations ofthe test substance in the test solutions A range-finding test (Repmi No . ; NCAS 1 0-063NG) at nominal concentrations of 0.500 , 5.00 and 50.0 mgiL (as was conducted and% inhibition of growth rates during 0-72 hours were 0, 0 and 4.95% respectively. On the basis of the above results, the definitive test was conducted at 5 nominal concentrations (common ratio; 2.0 as LiFSl) of6 .25, 12.5, 25.0, 50.0 and 100 mgiL under open system.Report No. NCAS 10-065 Page 14 of34

7.3 Preparation ofthe test solutions An amount of the test substance (624.9 mg; 150 mg as [corrected for purity (24.0%)]) was weighed and transferred to a 300-mL volumetric flask and dissolved in the OECD medium and the volume was adjusted to prepare a 500 mg/L stock solution. This stock solutions of 6.25 , 12.5 , 25.0 50.0 and 100 mL were separately transferred to 500-mL flasks and filled to volume with OECD medium to prepare the test solutions of6.25 , 12.5, 25 .0, 50 .0 and 100 mg/L. Three vessels each containing 100 mL of the test medium were used for each exposure group. Six vessels each containing 100 mL of the OECD medium (without the test substance) were used for the control group. 7.4 Experimental procedures The cell density in the pre-culture was 5.25 x 105 cells/mL. An amount of the pre-culture (952 f..LL), which was calculated to provide the nominal cell density of 5000 cells/mL in each test solution, was aseptically introduced to each test vessel containing the test solution with a micropipette. After inoculation, the test vessels were placed in the incubator (nominal temperature ofthe incubator: 23.0°C) and the incubation started. The cell density in each test vessels was measured after 24, 48 and 72 hours of the exposure .The test vessels were repositioned daily during the exposure period. At the exposure initiation, pH in the remains of test solution was measured in each test group (each exposure and control group) after preparation. At the exposure termination, pH in one of the replicates was measured in each test group. Temperature and light intensity in the incubator were measured once daily during the exposure period. Working contents were shown below in chronological order.

Exposure time (h) Working content 0 Preparation of test solutions -7 Measurement of temperature and light intensity in the incubator -7 Measurement ofthe pre-culture cell density -7 Calculation of the inoculum volume -7 Exposure -7 Observation of test solutions, Measurement ofpH, Microscopic observation ofthe cells in the pre-culture 24 , 48

72 Measurement of cell density, Observation of test solutions , Measurement of temperature and light intensity in the incubator, Reposition of test vessels , Microscopic observation Measurement of cell density, Observation of test solutions, Measurement of pH, Measurement of temperature and light intensity in the incubator, Microscopic observation 7.5 Method for measurement ofthe cell density ofthe test organism The test solutions (or cultures) were diluted as the following table and the cell densities (cells/mL) of those were measured with the electronic particle counter. At exposure initiation, the nominal cell density (5000 cells/mL) in the test solution was used as an initial cell density. The particle densities in the control and the highest concentration solutions without any algae wereReport No. NCAS 10-065 Page 15 of 34

1250 and 1180 countslmL respectively. Since the measured values were lower than 7 x 104 countslmL which was described in the protocol and sufficiently lower, the measured cell density with the electronic pmticle counter were recognized as cell density. Measurement item 1. Cell density ofthe stock culture 2. Cell density ofthe pre-culture Pmticle density of the OECD medium 3 . and the highest exposure solution (not containing algae) 4. Cell density at 24 hours 5. Cell density at 48 hours 6. Cell density at 72 hours 7 .6 Observations ofthe test solutions Aliquot of sample**(~tL)**

100 200 5000

200 Volume of cellpuck Dilution factor**(~tL)**

9900 x 100 9800 x50 5000 x2

9800 X50

The observations (color and precipitate) of the test solutions were conducted visually at the start and after 24, 48 and 72 hours ofthe exposure. Microscopic observations of algae were conducted after 24, 48 and 72 hours ofthe exposure. An aliquot (1.5 mL) of the culture was collected into an eppendorf tube with a micropipette and centrifuged (nominal: 23 

# oc, 16500 rpm, 5 minutes) . The supernatant was removed and the precipitation of alga was placed on a slide glass and observed microscopically. (1 vessel I group) 8. Analysis ofthe test concentrations

8.1 LCIMS/MS conditions Apparatus: Column: Mobile phase: Flow rate: Column temperature: Injection volume: Ionization mode: Monitoring ion: Acquity 

### I Quattro micro API (Waters) Acquity UPLC BEH C18 , 2.1 mm i.d. x 50 mm, particle diameter; 1.7**~m** (Waters) Acetonitrile+ 0.1% formic acid aqueous solution (vlv) = 50 +50 (vlv) 0.3 mL/min 40°C 

### 5**~L** ESI, Negative 180.33 

### > 96.90 (quantification) , 180.33 

### > 77 .84 (confinnation) 8.2 Preparation ofthe calibration solutions and the calibration curves An amount (5.05 mg) of the test substance was correctly weighed and transferred into a 50-mL volumetric flask and dissolved in 50% acetonitrile aqueous solution to prepare 101 mgiL calibration stock solution . This calibration stock solution was then flllther diluted with the same solvent to prepare 1.01 , 2.02 , 3.03 , 4.04 and 5 .05 mg!L calibration solutions. These calibration solutions were anal yzed by using the LCIMCIMC conditions described in section 8 . 1. The Calibration curves wereReport No. NCAS 10-065 Page 16 of34
generated by plotting the peak areas vs. the concentrations of and the linear regression equations and the correlation coefficients (r) were calculated by the least square method 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. 8.3 Validation ofthe test substance concentration in the test solutions

8.3 .I Validation ofthe calibration curve

The accuracy was calculated by finding the regression equation and correlation coefficient (r) 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%. 8.3 .2 Validation ofthe repeatability

Analytical method was validated by the repeatability test without any recovery tests, because extraction was not conducted. The measured concentrations of the test substance in the highest and lowest concentration were calculated by the same procedure as described in section 8.4 in the first definitive test. The analytical method was validated by the coefficient of variations of the measured test concentrations (n=3) which were within ±1 0%. 8.4 Measurement ofthe test substance concentration in the test solutions The measurement was conducted at the exposure initiation and termination. At the exposure initiation, I 0-mL samples were collected from each remainder ofthe test solutions from each group after preparation. At the exposure termination, 3-mL samples were collected from each triplicate test solution from the mid-depth ofthe test vessels and the three samples from the same group were mixed to be a 9-mL sample solution. (1.5-mL samples were collected from each six replicate test solution in the control group). The sample solutions were centrifuged (3000 rpm, 10 min) and the supernatants obtained were mixed with acetonitrile at the rate of 1:1 (v/v). These solutions were diluted with 50% acetonitrile aqueous solution to be an appropriate concentration that was in the middle of the range of the calibration curve, and then analyzed by using the LC/MS/MS conditions as described in section 8.1. The test substance concentration was calculated by substituting the peak area obtained into the calibration curve. The sample solutions from the control group were also analyzed by the same procedures to verify that any interfering peak did not appear at the retention time ofthe test substance on the chromatogram . Since the measured concentrations ofthe test substance at the sta1i and end of the exposure were within ±20% of the nominal, the nominal concentrations were regarded as the test concentrations during the exposure period.Rep01i No. NCAS 10-065 Page 17 of34

9 . Calculation ofthe percent inhibition of growth rate The growth curves (semilog graph) were prepared by plotting the time of measurement vs. the mean measured cell density in each test group (each exposure and control group). At this time, it was verified that the control culture was in log growth phase. Subsequently, the percent inhibition of growth rate was calculated as follows . The coefficient of variation for average specific growth rates and for section-by -section (daily) growth rates in replicate control cultures were calculated by using Microsoft®Excel 2003. 9.1 Calculation ofthe average specific growth rate 

### (p) The average specific growth rate (p) was calculated by using a natural logarithm : pi= OnN24-InNo) /24 fi2 

## = (In N4s- ln N24) /24 fi3 

## = On 

### Nn -In N4 s) /24 fi4 

## = On Nn-In 

### No) /72 where, No: cell density at the start ofthe exposure (nominal) 

### N24 : cell density at 24 hours N4s: cell density at 48 hours 

### Nn: cell density at 72 hours fil average growth rate from 0 to 24 hours fi2 average growth rate from 24 to 48 hours fi3 average growth rate from 48 to 72 hours fi4 average growth rate from 0 to 72 hours 9.2 Calculation ofthe percent inhibition of growth rate 

### (11 

## J The percent inhibition of growth rate (Ip) was calculated by comparison with the growth rates as follows: 

### I" 

## = (pc- fir) X 100 

### I fie Where, fi e : growth rate in the control group fir growth rate in the exposure group 10. Calculation of 50% inhibition of growth rate (ErC 50) Since the percent inhibition ofthe growth rate was less than 50% even at the highest test concentration (1 00 mg/L), ErC 50 (0-72 h) was not calculated statistically.Report No . NCAS 10-065 Page 18 of34

11. Determination ofNo Observed Effect Concentration (NOECr) The NOECr was determined as the highest test concentration that caused no significant growth reduction. Based on the result ofthe Brutlett test (a=O.O 1 ) , homogeneity of variances was verified. After 1-wa y ANOVA (a=O .Ol , 0.05) , Dunnett test (a=O.OI, 0.05 , 2-sided) was conducted as a method of multiple compari son . NOECr was defined as the concentration ofthe highest exposure group which was not significantly different from the control group. All statistical analyses were performed by using the Yukms software of Statlight 2000 (Yukms Corp. Tokyo). The value of NOEC was indicated in 3-digit number.



## Results 1. Validation ofthe test substance The 19 F-NMR spectrum of which was supplied by the sponsor, is shown in Figure I . The 19F-NMR spectrum ofthe test substance, which was measured before the exposure initiation (conducted in NCAS No: I0-022 " Determination of the Concentration of in Dosing Solution") is shown in Figure 2. On the spectrum which was supplied by the sponsor, 3,3,4,4,5,5,6,6,7,7 ,8, 8,8- Tridecafluoro-1-octanol was used as the chemical shift standard and its signal appeared at about 53 ppm. In this study, trifluoroacetic acid was used as the chemical shift standard and its signal appeared at about 51 ppm. It was considered that the difference in the chemical shift between the two spectrums was caused by using the different reference standard and the test substance was identified as 2. Validation ofthe stability ofthe test substance The 19F-NMR spectrum of the test substance, which was measured after the exposure termination (conducted in NCAS No: 10-066 : Acute toxicity test in 

### 0J

yzias latipes ") , is shown in Figure 3 . The stability during the storage period was verified by the consistency between the two spectrums which were measured at the strut and end ofthe exposure . 3. Validation ofthe analytical methods

3.1 Validation ofthe calibration curve A typical calibration curve is shown in Figure 4. A typical chromatogram of the standard solution (5 .05 mg/L, at the strut ofthe exposure) is shown in Figure 5. Each correlation coefficient (r) ofthe calibration curves was more than 0 .990 at the start and end ofthe exposure. The variations ofthe accuracy ofthe lowest standard samples were within ±20% and those of other standard samples were within ±15%. Thus , it was determined that the linearity was acceptable and the calibration curves were validated.Rep01i No. NCAS 10-065 Page 19 of34

3.2 Validation ofthe repeatability The results ofthe repeatability test are shown in Table 1. The typical chromatogram ofthe repeatability test (nominal concentration: 100 mg/L) is shown in Figure 6 . The coefficient of variation (n=3) of the test concentrations were within 10%, therefore the analytical method for the determination of the concentration of the test substance in the test solution was determined as valid in this study. 4 . Test concentrations ofthe test substance in the test solutions The analytical results of the test substance in the test solutions are presented in Table 2. The chromatogram ofthe control group at the start ofthe exposure is shown in Figure 7, and clu·omatograms of 100 mg/L group at the start and end ofthe exposure are shown in Figure 8 and 9, respectively. On the chromatogram ofthe control group, no interfering peak appeared at the retention time ofthe test substance. The concentrations of the test substance in the test solutions were 104-106% of the nominal at the exposure initiation and 99.6- 103% of the nominal at the exposure termination. The concentrations of the test substance in the test solutions were stable during the exposure period. 5. Growth curve and growth rate The cell densities during the exposure period are presented in Table 3 , the growth rates and the percent inhibitions ofthe growth rate are presented in Table 4 and the growth curves are presented in Figure 10. The cell density in the control cultures increased by a factor of 172 within the exposure of 72-hours which was satisfied the test criterion (more than 16-fold). The mean coefficient of variation (CV) for daily specific growth rates (days 0-1, 1-2, 2-3) in the control cultures was 8.54% which was satisfied the criterion (within 35%). The coefficient of the variation of average specific growth rates during the whole exposure period (0-72 h) in replicate control cultures was 3.62% which was satisfied the criterion (within 7%) . Since linearity of the growth curves in the control group was observed, it was confirmed that the cells were in the exponential growth phase during the exposure period. 6 . ErC 50 , 95% confidence interval and NOECr Results of Dunnett test (2-sided) are presented in Table 4, the values of ErC 50 , 95% confidence interval and NOECr are presented in Table 5 , and results of percent inhibition of growth rate in each test concentration are shown in Figure 11 . The ErC 50 (0-72 h) and NOECr were detennined on the basis of the nominal test concentrations. Since the percent inhibition was Jess than 50% even at the highest concentration tested , the ErC 50 (0-72h) was determined to be 

### > 1 00 mg/L. Based on the results of the Dunnett test (2-sidid) , the NOECr, which is defined as the highest concentration group that caused no significant different (p::::0.05 ) from the control data, was determined to be 50 .0 mg/L.Report No. NCAS I0-065 Page 20 of34

7. Temperature and light intensity in the incubator Measurements oftemperature and light intensity in the incubator are presented in Table 6. The temperatures ranged from 22.9 to 23.0°C which were within the acceptable range (23±2°C). Measurements of light intensity ranged from 69 to 86 11E /m2/s which were within the acceptable range (65-100 11E/m2 /s). 

### 8. pH The pH values ofthe test solutions are shown in Table 7. The pH values of the control group at the start and end of the exposure were 8.0 and 7 .9, respectively, and showed almost same value. 9. Observations oftest solutions The observations ofthe test solutions are presented in Table 8. All test solutions were clear and colorless, and no coloration or precipitate due to the test substance was observed. I 0. Microscopic observations of algae The observations ofthe algae are presented in Table 9. There were no morphological abnonnalities in cell shape of the control cultures throughout the exposure period. Morphological abnormality (distmied shape) was observed in the 100 mg/L from 48 hours after the exposure . 11. Validity ofthe test The validity ofthe test was confirmed because the following criteria were satisfied; The biomass (cell density) in the control cultures should have increased exponentially by a factor of at least 16 within 72 hours. The mean coefficient of variation for daily specific growth rates (days 0-1, 1-2, 2-3) in the control cultures must not exceed 35%. The coefficient of variation of average specific growth rates during the whole exposure period (0-3 day) in the control cultures must not exceed 7%.Repmi No. NCAS 10-065 Page 21 of34 

## Conclusions The 72-hour growth inhibition study (open system, shaking culture) was performed by exposing the algae to the test solutions which contained It is concluded that the 0-72 hour ErC 50 values is > I00 mg/L and correspondingly the No Observed Effect Concentration (NOECr) is 50.0 mg/L on the basis ofthe nominal concentrations.Report No. NCAS 10-065 Page 22 of34 

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

6.25 6.04, 6 .02, 6.10 6.05 0.688
100 98 .5, 98.0, 99.0 98.5 0.508



### Table 2 Concentration oftest substance in test solution Nominal Measured concentration (mg!L) concentration (mg/L) 0 hour 72 hours Control - 

## -

6.25 6.60 6.34 (106) (1 01) 12.5 13.0 12.7 (104) (102) 25.0 26.2 25.4 (105) (102) 50.0 52.3 49 . 8 (1 05) (99 .6) 100 106 103 (1 06) (103) ( ) : Percent ofnominal concentration(%)

### Report No. NCAS 10-065 Page 23 of 34



### Table 3 Cell density during the exposure period

Nominal concentration Cell density (cells/mL) Multiplication Sample No. factor (mg/L) Oh 24 h 48 h 72 h (0-72 h) 1 5,000 26,100 159 ,000 782,000 2 5,000 23,700 16 2,000 1,040,000 3 5,000 24,700 116 ,000 622,000 0.000 (Control) 4 5,000 25,800 164,000 978,000 5 5,000 21,500 147,000 817,000

....

### /~ 6 5,000 24,300 165 ,000 922,000 Average 5,000 24,350 152,1 67 860,167 172 SD

- *] 1,663 1 8,883 151,489 -

1 5,000 24,700 176 ,000 1,090,000 ./ /' 2 5,000 22,700 135,000 584,000 /. / / / 6 .25 3 5,000 23,900 160,000 1,160,000 /" / Average 5 ,000 23,767 157,000 944,667 1 89 SD

- *1 1,007 20,664 314,30 1 

### -

1 5,000 27,300 168 ,000 1 ,360,000 

### // / 2 5 ,000 23,900 180,000 1,420,000 /"'/_./"' 12.5 3 5,000 26,700 179,000 963,000 / / Average 5,000 25 ,9 67 175 ,667 1,247 ,667 250 SD
- *] 1 ,815 6,658 248,347 -

1 5,000 29,000 175 ,000 1,090,000 / / / 5,000 21,900 / 2 181,000 902,000 / 25.0 3 5,000 25,200 1 82,000 1,110,000 _/,/ ' Average 5,000 25,367 179,333 1,034,000 207 SD
- * ] 3,553 3,786 114,752
-

1 5,000 22,700 146,000 971,000 // 2 5 ,000 25 ,700 155,000 799 ,000 

### ./ / 50 .0 3 5,000 21,400 157,000 915,000 / // Average 5,000 23,267 152,667 895,000 179 SD 

### - * ] 2,205 5,859 87,727 -

1 5,000 17,900 88,900 329,000**/~** / ' 2 5,000 19,200 80,700 26 1 ,000 / 100 3 5 ,000 19,400 57,000 124 ,000 /// /" Average 5,000 18 ,833 75 ,533 238,000 48 SD

- *] 814 16,566 104,417 - 

# * 1 : SD was not calculated smce the cell dens1ty at 0 hour was nominal initial density.

### Report No. NCAS I0-065 Page 24 of34

Nominal concentration (mg/L)

0.000 (Cantrall

6.25

12.5

25.0

50.0

100 

### Table 4 Growth rate and percent inhibition ofgrowth rate

Growth rate Growth rate Growth rate Growth rate % Inhibiton Sample No . j /J j/ 2 j/3 l].I.J (0-24 h) (24 · 48 h) (48-72 h) (0·72 h) fjJ, (%) 1 0.0689 0.0753 0.0664 0.0702
- 2 0.0648 0 .080 1 0 .0775 0.0741 - 3 0.0666 0.0644 0.0700 0.0670
- 4 0.0684 0.0771 0.0744 0.0733
- 5 0.0608 0.0801 0.0715 O.o70 8 - 6 0 .0659 0.0798 0.0717 0.0725 - Average 0.0659 0 .076 1 0.0719 0 .0713 -

SD 0 .00293 0.00607 0.00379 0.00258
- CV(%) 4.45 7.98 5.27 3 .62 - 1 0.0666 0.0818 0. 0760 0.0748 ·4.9 1 2 0.0630 0 .0743 0 .0610 0.0661 7 .29 3 0.0652 0 .0792 0.0825 0.0756 -6.03 Average 0.0649 0 .0784 0.0732 0.0722 n.s. -1.22

SD 0.00181 0.00381 0.0110 0 .00527 

## -

1 0.0707 0 .0757 0.0871 0.0779 -9.26 2 0.0652 0.0841 0. 0861 0.07 85 -10.1 3 0 .0698 0 .0793 0.0701 0 .0731 -2. 52 Average 0 .0686 0.0797 0.0811 0.0765 "·'· ·7. 29

SD 0.00295 0.00421 0.00954 0.00296
-

1 0.0732 0.0749 0.0762 0.0748 -4.91 2 0.0615 0.0880 0.0669 0.0722 -1.26 3 0.0674 0.08 24 0.0753 0 .0750 -5.19 Average 0.0674 0 .0818 0.0728 0.0740 n.s. · 3.79

SD 0.00585 0.00657 0 .00513 0 .00 1 56
-

1 0.0630 0.0776 0.0789 0.0732 ·2.66 2 0.0682 0. 0749 0.0683 O.o705 1.12 3 0.0606 0.0830 0.0734 0.0724 -1.54 Average 0.0639 0.0785 0.0735 0.0720 " '· · 1.03

SD 0.00389 0.00412 0 .00530 0.00139

-

1 0.0531 0 .0668 0.0545 0.0581 1 8.5 2 0.0561 0 .0598 0.0490 0.0549 23.0 3 0 .0 565 0.0449 0.0324 0.0446 37.4 Average 0.0552 0.0572 0.0453 0.0525 • 26.3

SD 0.00186 0.0112 0.0115 0.00705
- Results of Dunnett test (2-sided) '' : Indicates a significant defference from the cotrol group (P<0.05) n.s.: Indicates no s ignificant difference from the control group Growth rate in control group (JIJ. j/2_ j/3 ) Average SD CV(%) 0.0702 0 .00459 6.54 0.0741 0.008 19 11.1 0.0670 0.00282 4.2 1 0.0733 0.00445 6.07 O.D708 0.00967 13.7 0.0725 0 .00698 9.63

- - 8.54

### Report No. NCAS 10-065 Page 25 of 34



### Table 5 ErC 50 , 

### 95 % confidence interval and NOECr Exposure duration 

### 0-72 hour ErC so (mg/L) > 

### 100 * 95 % confidence interval (mg/L) 

### NOECr (mg/L)

50 .0 

## * · Since the inhibition was under 50% even at the highest test concentration, the ErC 50 

### was determined > 100 mg/L.


### Table 6 Temperature and light intensity in the incubator
Measurement Exposure duration (h) Parameter position Range 0 24* 48* 

### 72 Temperature (°C) - 

### 23 .0 23.0 23.0 22 .9 22.9-23.0 No.1 

### 72 - - 

### 73 No.2 70 71 73 73 Light intensity No.3 84 - - 

### 86 69-86 (flE/m2 

### /s) No.4 70 - - 

### 74 No.5 69 - - 

### 72 * :Light intensity was measured at only No.2 position.

### Report No. NCAS 10-065 Page 26 of 34



### Table 7 pH ofthe test solutions
Nominal pH concentration (mg/L) 0 hour 72 hours Control 8.0 

### 7.9

6.25 8.0 

### 7.9 12 .5 8.0 8.0 25.0 8.0 8.0 50.0 8.1 

### 7.9 100 8.I 

### 7.9



### Table 8 Observations ofthe test solutions Nominal Exposure duration Concentration (mg/L) 0 hour 24 hours 48 hours 72 hours Colorless and Colorless and Control Slightly green Green transparent transparent Colorless and Colorless and 6.25 Slightly green Green transparent transparent Colorless and Colorless and 12.5 Slightly green Green transparent transparent Colorless and Colorless and 25.0 Slightly green Green transparent transparent Colorless and Colorless and 50.0 Slightly green Green transparent transparent Colorless and Colorless and Colorless and 100 Slightly green transparent transparent transparentReport No. NCAS 10-065 Page 27 of 34



### Table 9 Microscopic observations of algae Nominal Exposure duration concentration (mg/L) 24 hours 48 hours 72 hours Control Normal Normal Normal

6.25 Normal Normal Normal 12.5 Nonnal Normal Normal 25.0 Nonnal Normal Normal 50.0 Normal Normal Nonnal Few distorted Distorted shape 100 Normal shape was observed was observed

# ;;g;~~~~~~~d~~~d~~~~~t:~~titi**B~R ~:.._~~~~~~;,:,_\:)j,J)dd,.;_;** **~~ ·~** **~ ~~~-**  W'- "' .&**LiFSHf~<48** E;XFIIO 10 FF.Ot:NO l DDte 20100222**T1J:~e-·** 10.06**I~STRUH** !.p<ICt:**PHO~HO** S =. ?ABBO DD/ r'l.l L.E'ROO**~g!higqn** 'l ' D 131012 SOLYZ:tl'T tt-ISO lUi :liS OS 0 5iil! 89.:!85. 7.11 II: FlORES 0.681196 H7. ArJ o.**7J 4.0~'l2**  aec RG ' 1030 nu 5.1100 u.sec ot &.so u:!et: 'rE 30J,'- K Ol 1.00000000**.se~** Dll Q .O JOOO[}OO n e:::: 01:? 0.00002000**r.e~** 'l'DO l ._.,."',.. CHAHNGL !J ,.__ __ NtiCl 19F' P:!. lJ . 50 u5ec Pl.l
- 1..20 dB Pl,]W l[i,9iH1113DO W !i:!:'Ol 376.49J30(il !"..3.::::

___.._..._.. CHA.nm::.L t2**-~ cr** **P~** ,..aUr:16 rrt1C2 lit PC?D2 80.00 U!ICC PL:? 0. 20 dS P1 12 15.53 tiD PL2 '.il 9. 4 99 37G 2C W 1**;~~~t '** **4~o:~~i~~;: ~1HZ**  

# lJL Sl 6D556

• 1**~~"** m.**<'B3 6~** **~** ''"''**~sa** o !.E O •. SC l!:: GB Q -----'--------- -·- ··--·---···-··----·-·· ---'----- -- -- PC l.Oil 60 40 20 0 -20 -60 -80 · 100 -120 ppm 

### Figure 1 NMR spectrum of (supplied by Sponsor, 19

### F-NMR)

### Report No. NCAS 10-065 

### Page 28 of34**~. ;**  ;; ; 

# dEOL&rl

···---- ··--· ···-········· · ··· ----'

••• •**l>e:JII~IUTIOII** rAAUL"T . lU
---- o.r.rJ¥•<1 !ro"' t.Hr.: \11"'2 . l l'il•**~:....... ~** **i:~rnf** ,, l.:H -"•ll:l>:>r • rlaha" " s•n;ol-**I~** • ;.1**•~1** t 'T:l- ll! Q) t,**Ox>l•ll~** • LJTI' I**IIM:D·H~b)-!!lP'"** Crtlltl0:1**Dl~t** • I1•UII.l••tl C**H•Ob:J~** ll•>'h !on**ll~tt ~** ...**~** Uooc •**nr ;.** **1:u _p)l~**  "1;: e:Y.l'U1 -lfr • H:" H • [ppll! •**~1."1Ul5 7JI** **TI** rs::tll!l

.. c**.:71~7:Ul[1}** •1'' • •n.;Jo4, 0U(r.Jhl**:i;~ '**  ., •**l.5~1l'710llllc]** : ;;;•t:UlUIUzl • t10.6'21:141Gttll!r.ai :**~~~r-Ill** K Ull.«lDUDH[1.1hl 

# :**~,.,.,**..

### , 

# .. ; J

_,o_ ..Jd:h • ":"•1**:~~!b.** 

# :**!~E~r~"'''** ; :C;:tuhc • 5\1;31 : trr .IDGII• • Of ! ;**~;~ r.!'':** **~u"t** :**~!~.** lr.lthlwaJr; • ll•l**~** llahu.t!an_dtllY • Hll l lhp•:.ltJcm tiM • 5.lH012Hr•J ; =----;>J•t- - ..**J!!~)**

Ia-**o~J** 0.-ffl**~.-;,:,.;;.** 

## r.r Figure 2 NMR spectrum ofthe test substance (Before the exposure initiation, Study No.: NCAS 1 0-022) - - -- ·· -"

!-**r~- .** 

## i**~!** q**~** !!: '!**j ;** 



### Figure 3 NMR spectrum ofthe test substance (After the exposure termination, Study No.: NCAS 10-066)

### Repmi No. NCAS I0-065

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

1.01 1894 0.948 2.02 3603 2.14 3.03 5041 3.13 4.04 6388 4.07 5.05 7527 4.86 Calibration: 07 Jun 2010 16:22:04 Compound name : Correlation coefficient: ; r**~** 0.. 9974:16 / r"2 = 0.994838 Calibration curve: 1438.82 • x + 530 .659 Response type: External Std, Area accuracy (%) 93 .8 106 103 101 96 .3

Curve type: Linear, Origin : Exclude, Weighting : 1/x, Axis trans: None

6000
- Q) VJ c 0 4000 0. VJ Q) 0: 2000 0 ' ppm 0 .00 1.00 2.00 3 .00 4.00 5.00 

### Figure 4 Typical calibration curve 100607-05 STD-5-5 0.82 7527 MRM of2 channels,ES180.33 > 96.90 7.425e+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 start ofthe exposure) Page 29 of34Repori No . NCAS I0-065

1000525-06 P-1 OO-R1 10

0 .50 

### Figure 6 100607-06 P-cont-0-2 100

%

0 .50 0 .77 3524

1.00 1.50 2.00 Page 30 of34

MRM of2 channels,ES180.33 > 96.90 7 .955e+004

2.50 

### Typical chromatogram of a repeatability test sample

1 .00 1 .50 2.00 MRM of 2 channels,ES180.33 > 96.90 7.425e+004

2 .50 

### Figure 7 Chromatogram ofthe control group (at the start ofthe exposure)

### Report No. NCAS 10-065

100607-07 P-1 00-0-2 100

0.50 0 .83 3584

1.00 1.50 2.00 

### Page 31 of34

MRM of 2 channels,ES180 .33 > 96.90 7.425e+004

2 .50 

### Figure 8 Chromatogram ofthe 100 mg/L group (at the start ofthe exposure)
1 0 0061 0-07 P-1 00-72-2 100

%

0.50 0 . 80 3309
I

### ~ 

# ..

1 . 00 1 .50 2.00MRM of2 channels,ES180.33 > 96.90 6.916e+004

2.5 0 

### Figure 9 Chromatogram ofthe 100 mg/L group (at the end ofthe exposure)

### Report No. NCAS 1

### 0-065 Page 32 of34

Nominal concentration Mean cell density (cells/mL) (mg/L) 0 hour 24 hours 48 hours 72 hours 0.000 (Control) 5,000 24,350 152,167 860,167 6.25 5,000 23,767 157,000 944,667 12.5 5,000 25,967 175 ,667 1,247,667 25.0 5,000 25,367 179,333 1,034,000 50.0 5,000 23,267 152,667 895,000 100 5,000 18,833 75,533 238,000 10,000,000


## -e- 0.000 mg/L (Control) 

### -8- 6.25 mg/L ---tr- 

### 12 .5 mg!L 1,000,000 

## -e-- 25.0 mg/L 

# Ai 50.0 mg/L 

## / 7211 1 //g'
,.....,_ 

## -' E '-....... 

### _...._l OOmg/L**/.,:'~**

- · //.#' #L_ 

# /7 C/) Q) 

# ff**__.-A ~**  (.)

..........



### >- :t= C/) 100,000 

### c 

# / /

/L / Q) /, 

## ...... '"0 // / // / Q) (.) 

### c ro Q)**~** 10,000 

# ///

-- 

# f/ 

# £**~** -- .L'_

1 ,000**I! ~**  

# ....

# F-- - --j

-"---------L . __

# j

0 24 48 72 

### Exposure duration (h) 

### Figure 10 Growth curves ofalgae

### Report No. NCAS I0-065 

### Page 33 

### of 34

Nominal concentration Inhibition of growth rate(%) (mg/L) 0-72 h

6.25 0.00* 12.5 0. 00* 25 .0**o.oo -~.-**  50.0 0.00* 100 26.3

. .. *: Values were regarded as 0 .00 smce 

### % mhlb1t1on was <0

100
90
80 

### -e- o-72 h

,........ 70**~** 0 ...._..-

<D .j-J 

### ro 60 l.... 1- ..!: .j-J**~** 0 l.... 50 b.O 4- 0 

### s:: 0 40 

# :-e - ..0 ..!: 

### s::**~** 30 

## I 

# 1 4· I

# l~~**lJ]l~** I 

# I I

# -~~~~ I I I

# T-1-1-n I 

# li1 1 iI I I 

## I I 

### I

.. I i I 

# L I I - 

# - I 

# - 

# - 

# - - 20
10
0 1 .0 10.0 100.0 

## Nominal concentration (mg/L) Figure 11 Percentage inhibition ofgrowth rate calculated for each test groupReport No. NCAS 10-065 Page 34 of34 

## Appendix 1 OECD medium Preparation ofAlgal Medium 

### A series of stock solutions of A 

### (1 0 mL), 

### B (1 .0 mL) and C (1.0 mL) which had been sterilized with a filter (0.2**~un)** were transferred to a 1-L volumetric flask in order and the volume was adjusted with Ion exchange water and then sterilized with the filter again to prepare the algal culture medium. The pH ofthe medium after preparation was confirmed to be within 8.1 ± 0.2 by measurements. Stock solution

A Final concentration in test solution Reagent (mg/L) NH4Cl 15

- -·--·--·-·---··-··-·--··----··--·-----··-----·---·-··-·----------··---···----··-·-·-----··--·-·····-··--·

## --···------**~&S::!?.:. ?H?.Q**  

## __ ___·----------- ·-----···------·----- _g_ -- ····------ ·-----·----- - 18

## ···-**-··-~~~?.1 :7..Ji?.2** 

# .. - ..- - ---- ······· ·---····-· · ----·· -·---- ------·- . .....**!~- ·-·----·---·-····-·---·---··------**  KH2P04 1.6 FeCh · 6H2 0 0.064 B ---··--··--··-··----···--··-·--·-·-··---- ·--····------·--·-···--·-··· -·--·--·--- -·---·-----···-- Na2EDTA-2H20 0.1 0.185 0.415 ZnCh 0.003 

# c CoCh-6H,O 0.0015 -··-·--------··--···=··-······--=-· ·---· -- ·-··--·-··----·--·-·-····---···---···--··-··---··-··-·-·-·--·-··--·----··-·-·--·---··--·--·--·--···--· · --··--··- 0.00001 0.007 D 50 All reagents used were special grade, obtained from Wako Pure Chemical Industries, Ltd. All solutions described above were diluted with ion exchange water.

## Authenticity ofTranslation

I declare that the original Japanese fmal rep01t (Rep01t No . NCAS 1 0-065) is translated into English consistently.

Nissa Chemical Analysis Service Co. , Ltd. Odawara Laboratory