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=% : NCAS 10·065 

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Report No. NCAS I 0-065 Page 4 of34 

Quality Assurance Statement 

Study Number: NCAS I 0-065 

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 of the 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 of the cell density 5/26,28/2010 5/28/2010 5/28/2010 

Measurement of the 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 

The original Quality Assurance Statement 

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

~ !ii:& ~ : O)~~ftlH.:xt-t-5 

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J:~~~O){;§'ii!FH!f*IDE(J)~1!i:j;t~1i~~~l@ffl GLP :toJ::U~{~JliJWt1*EiE~~F 1 (QAU) (J) SOP 

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• )i;j:}f~f'I·O)j*J& C j[IJ!JI 5/25, 28/2010 5/28/2010 5/28/2010 

· LC/MS/MS )i:j'Jf 5/25, 28/2010 5/28/2010 5/28/2010 

· li\Hf.!O)fll'.fH~~rol.~ (flt~~) 6/10, 14/2010 6/14/2010 6/14/2010 

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¥fli5&~~ 7/14-20/2010 7/20/2010 7/20/2010 

:&i ~~~ ¥& 15-if: 7/22/2010 7/22/2010 7/22/2010 

QAU fi, ::. 0)~~7J>~~Hl®1!:to J:: ~ SOP 1.:1.tt--J '"Ciibh., #Hs· ~ ht::.J5'¥t~-".:f.ffi~t.J>~~~.: 

13e:bht::. ~) 0) -c:· a:r; '? , *"~;;I!Hi~C.~ff< ~ h-1:::. 7·- ?' -2:.- .IE~rn.:.&:~ L. -cv' 0 ::. C: :3:-~'ffiM!, L. tc.o 

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Report No. NCAS 1 0-065 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 of test concentrations) 

Koji Baba (first pre-culture [first definitive test]) 

Shinpei Oonuki, Chiaki Urabe (analysis of test 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 

Immobilisation Test and Fish Acute toxicity test> IV Algal Growth Inhibition 

Test 
Report No. NCAS I 0-065 Page 7 of34 

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

10 years after the completion of the study, but the place for storage after that will be 

decided on discussion with the sponsor. The test substance will be retained in the 

test facility at least for I 0 years after the completion of the study, but only as long as 

the quality of the 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 0-065 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 of Contents ....................................................................................................................................... 8 

Su1n1nary ............ ... ....... ............... ...... .............................................. ....................... .......................... .... .... 1 0 

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

Materials and Methods .................................................... ....... .................................... .. ........................... 11 

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

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

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

4. Verification of solubility of test substance ................................. .................................................. 12 

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

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

7. Algal growth inhibition study ............... .... ................................................................................... 13 

8. Analysis of the test concentrations ............................................................................................... 15 

9. Calculation of the percent inhibition of growth rate ........................................ : ........ ................... 17 

10. Calculation of 50% inhibition of growth rate (ErC50) .............................................................. 17 

11. Determination of No Observed Effect Concentration (NOECr) .............................................. 18 

Results .. ................................................................................................................................................... 18 

1. Validation of the test substance .................................................................................................... 18 

2. Validation of the stability of the test substance ............................................................................ 18 

3. Validation of the analytical methods ................. ........................................................................... 18 

4. Test concentrations of the test substance in the test solutions ...................................................... 19 

5. Growth curve and growth rate ........................... .. ......................................................................... I 9 

6. ErC50, 95% confidence interval and NOECr ................................................................................ 19 

7. Temperature and light intensity in the incubator ........................................................... ............... 20 

8. pH ..................................................................................... ...... .. .. ............ ...................................... 20 

9. Observations of test solutions .................................... ... .......... .. ..... .. ............... ..... ............. ........... 20 

10. Microscopic observations of algae .. .... .................... ......................................................... .. ...... 20 

11. Validity of the test. .............. .. ........................... .................................................................. .. ..... 20 

Conclusions ........... ...................... .. .......... ........ ...... ................ ... .. ... ..... ... .... .............................................. 21 
Report No. NCAS 1 0-065 Page 9 of34 

Table of Contents (continued) 

Tables and Figures ....... ..... ... ... ..... ... ..... .... ....... ... ... ......... ... ........ .. ...... ............... ..... ... .... .... ..... ....... ....... ..... 22 

Table 1 Result of validation ofthe analysis ...................... .......... ........................................ .. ...... .. ......... 22 

Table 2 Concentration of test 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 ErC50, 95% confidence interval and NOECr ............................................................................ 25 

Table 6 Temperature and light intensity in the incubator ....................................................................... 25 

Table 7 pH of the test solutions ................................ ...... ............................................... ........................ 26 

Table 8 Observations of the 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 of the exposure) ... 29 

Figure 6 Typical chromatogram of a repeatability test sample .............................................................. 30 

Figure 7 Chromatogram of the control group (at the stmi of the exposure) ...................................... 30 

Figure 8 Chromatogram of the 100 mg/L group (at the start of the exposure) .................................. 3I 

Figure 9 Chromatogram of the 1 00 mg/L group (at the end of the exposure) ................................... 31 

Figure I 0 Growth curves of algae ...................................................... .. ................................................. 32 

Figure 11 Percentage inhibition of growth rate calculated for each test group ...................................... 33 

Appendix 1 OECD 1nediun1 .. ... ...... ........ .. .......... ....................................................................... ......... . 34 
Report 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 of the exposure and 99.6-103% ofthe nominal at the end ofthe exposure. Since the variability of 

the measured concentrations was within ± 20% of the nominal, the results were calculated on the basis of the 

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 

ErC50 was detennined > 1 00 mg/L. 

Introduction 

This study was conducted in accordance with the following requirement to determine the 50% inhibition 

growth rate (ErC50) 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 Test 
Report 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 of the 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 

(

19

F-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 of the test substance 

The storage stability of was verified by confirming identity between the two nuclear magnetic 
Report 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 of test 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/83 (Airtech Japan, Ltd.) 

Electronic particle counter: F-520P (Sysmex Corp.) 

Electrolytic solution for the particle counter: Cell puck (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: 1 0-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 were 
Repoti 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 of the algae was shown. 

Culture period; June 4-7,2010 

Temperature; 23.1 °C (in the incubator) 

Lightning; 72-76 f.!Ehn 2

1s 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 of the growth rates in the control group was 

out of the 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 of the test vessels in the incubator. In this repmi, only the second definitive test is described. 

7.1 Test conditions 

Type of culture: 

Exposure period: 

Volume of test 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 of the solutions in flasks) 

The pH values of the test solution were not adjusted 

OECD medium (see Appendix 1) 

A3 

7.2 Selection of the concentrations of the 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 1 0-065 Page 14 of34 

7.3 Preparation of the 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 of the 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 of the pre-culture cell density -7 Calculation of 

the inoculum volume -7 Exposure -7 Observation of test solutions, 

Measurement of pH, 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 of the cell density of the 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 were 
Report 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 of the stock culture 

2. Cell density of the 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 of the test solutions 

Aliquot of 

sample 

(~tL) 

100 

200 

5000 

200 

Volume of 

cell puck Dilution factor 

(~tL) 

9900 x100 

9800 x50 

5000 x2 

9800 X 50 

The observations (color and precipitate) of the test solutions were conducted visually at the start and 

after 24, 48 and 72 hours of the exposure. 

Microscopic observations of algae were conducted after 24, 48 and 72 hours of the 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 of the 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 

analyzed by using the LCIMCIMC conditions described in section 8.1. The Calibration curves were 
Report No. NCAS 1 0-065 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 of the test substance concentration in the test solutions 

8.3 .I Validation of the 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 of the 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 of the 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 of the 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 of the test substance on the chromatogram. 

Since the measured concentrations of the 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 of the 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 N4s) /24 

fi4 = On Nn-In No) /72 

where, 

No: cell density at the start of the 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 of the 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 (ErC50) 

Since the percent inhibition of the growth rate was less than 50% even at the highest test concentration 

(1 00 mg/L), ErC50 (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 of the Brutlett test (a=O.O 1 ), homogeneity of variances was verified. After 

1-way ANOVA (a=O.Ol , 0.05), Dunnett test (a=O.OI, 0.05, 2-sided) was conducted as a method of 

multiple comparison. NOECr was defined as the concentration of the 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 of the test substance 

The 19F-NMR spectrum of which was supplied by the sponsor, is shown in Figure I. The 

19F-NMR spectrum of the test substance, which was measured before the exposure initiation (conducted 

in NCAS No: I 0-022 "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 of the stability of the 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 

0Jyzias 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 of the exposure. 

3. Validation of the analytical methods 

3.1 Validation of the 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 of the exposure) is shown in Figure 5. 

Each correlation coefficient (r) of the calibration curves was more than 0.990 at the start and end of the 

exposure. The variations of the accuracy of the 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 of the repeatability test are shown in Table 1. The typical chromatogram of the 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 of the test substance in the test solutions 

The analytical results of the test substance in the test solutions are presented in Table 2. The 

chromatogram of the control group at the start of the exposure is shown in Figure 7, and clu·omatograms 

of 100 mg/L group at the start and end of the exposure are shown in Figure 8 and 9, respectively. 

On the chromatogram of the control group, no interfering peak appeared at the retention time of the 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 of the 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. ErC50, 95% confidence interval and NOECr 

Results of Dunnett test (2-sided) are presented in Table 4, the values of ErC50, 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 ErC50 (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 ErC50 (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 I 0-065 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/m

2

/s which were within the acceptable range 

(65-100 11E/m2/s). 

8. pH 

The pH values of the 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 of the 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 of the 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 of the 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 ErC50 values is > I 00 mg/L and correspondingly the No Observed Effect Concentration (NOECr) 

is 50.0 mg/L on the basis of the nominal concentrations. 
Report No. NCAS 10-065 Page 22 of34 

Tables and Figures 

Table 1 Result of validation of the 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 of test 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 of nominal 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 23,700 162,000 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 14 7,000 817,000 

6 5,000 24,300 165,000 922,000 .... /~ 

Average 5,000 24,350 152,167 860,167 172 

SD - *] 1,663 18,883 151,489 -

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 23,767 157,000 944,667 189 

SD - *1 1,007 20,664 314,301 -

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 25,967 175,667 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 25,200 182,000 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 I 0-065 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 of growth 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.o708 -

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 0.0785 -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 0.0824 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 0.00 156 -

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 18.5 

2 0.0561 0.0598 0.0490 0.0549 23.0 

3 0.0565 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 significant 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 0.00819 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 ErC50, 95% confidence interval and NOECr 

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, the ErC50 

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 of the 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 of the 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 transparent 
Report 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 ~:.._~~~~~~;,:,_\:)j,J)dd,.;_; ·~ ~ ~~~- W'-B~R "'.& 

~~ 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.OJOOO[}OO ne:::: 

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 

P112 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, 19F-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••tlC 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 of the test substance 

(Before the exposure initiation, Study No.: NCAS 1 0-022) 

- - --··-" 

!- r~- . 

i ~! 

q ~ !!: '! 

j 

; 

Figure 3 NMR spectrum of the test substance 

(After the exposure termination, Study No.: NCAS 10-066) 
Repmi No. NCAS I 0-065 

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 of34 
Repori No. NCAS I 0-065 

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 of the control group (at the start of the 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 2.00 

MRM of2 channels,ES180.33 > 96.90 

6.916e+004 

2.5 0 

Figure 9 Chromatogram of the 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 / 72111 

//g' 

,.....,_ 

-' 

E 

'-....... 

_...._lOOmg/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 of algae 
Report No. NCAS I 0-065 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 

14· 

I 

lJ]l~ I l~~ 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 of growth rate calculated for each test group 
Report No. NCAS 1 0-065 Page 34 of34 

Appendix 1 OECD medium 

Preparation of Algal 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 of the 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 · 6H20 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 of Translation 

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