STATEMENT 


Study title: 

Partition Coefficient (I-0ctanoVWater) Study of 

I, the undersigned, hereby declare that this report provides a correct English translation of the 

Final Report 

August 3, 2004 

Study Director: 

000597 

CERTIFICATION 


Title of Test: 

Partition Coefficient (l-OctanollWater) Study of 

Identification code.: 

The above test was conducted in accordance with the Good Laboratory Practice Standards for industrial 

chemicals, "Basic standards to be observed by testing facilities in conducting tests stipulated in article 4 of 

the Order Prescribing Those Items of the Test Relating the New Chemical Substance and Study on 

Harmful Effects of Designated Chemical Substances" (March 31, 1984, Revised March 1, 2000, 

Kanpogyo No.39. Planning and Coordination Bureau, Environment Agency, Yakuhatu No.229. 

Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 59 Kikyoku No.85. Basic Industries 

Bureau, Ministry of International Trade and Industry, Japan) . 

April 4. 2001 

Study Director: __(,,:;S;..:Jign=e=d)<.-__ 

1 000598 
QUALITY ASSURANCE STATEMENT 


Type of Test Partition Coefficient (l-OctanollWater) Study 

Name of test substance 

This test was conducted in accordance with "Basic standards to be observed by testing facilities in 

conducting tests stipulated in article 4 of the Order Prescribing Those Items of the Test Relating the New 

Chemical Substance and Study on Hannful Effects of Designated Chemical Substances", 

We acknowledge that the Final Report describes the methods and procedures used in the test precisely and 

that the results reported herein reflect the raw data accurately, 

Aud'1t and'mspection were con ducted as fill 0 ows:

Audit or Inspection Items Date of Audit or 

Inspection Date of report 

Protocol March 2, 200I March 2, 200 1 

Conduct of test March 9,2001 March 9, 2001 

Draft Final Report April 3, 2001 April 3, 2001 

Draft Final Report Amendment April 3, 2001 April 3, 2001 

Verification of Draft Final Report 

Amendment April 4, 200 1 April 4, 2001 

Date of issue thereof April 4, 200 1 

Quality assurance personnel - _(Signed) -

I 

2 

000599 
Title of the test: Partition Coefficient (I-OctanollW ater) Study of 

Test Client: 

Name: 

Address: 

Test Facilities: 

Name: 

Address: 

Testing Personnel: 

Management: 

Study Director: 

Test Personnel: 

Test Period: Date of test initiation: 

Date of experiment initiation: 

Date of experiment termination: 

Date of test termination: 

March 2, 200 1 

March 9, 2001 

March 9,2001 

April 4, 200 1 

Final Report: 

Preparation: April 4, 2001 Study Director: 

APPROVAL OF FINAL REPORf 

Study Director: 

August 2, 1999 

(Signed) 

3 000600 
SUMMARY 

27~ I. Description of Test: Partition Coefficient (l-OctnnollWater) Study 

2. Test Substance: 

3. Test Method and Conditions 

3-1. Test Method 

The test was conducted in accordance with the "Commentary for the Measuring Method of the 

Partition Coefficient (l-OctanollWater) of Chemical Substances indicated" in the Order 

Prescribing the Interpretation and Application of the Law Concerning the Examination and 

Regulation of Manufacture, etc. of Chemical Substances (24th March. 1987, Pharmaceutical 

Affairs Bureau No. 291, and 62 kikyoku No.I71, Basic Industries Bureau (,87» and "Partition 

Coefficient (n-OctanollW ater): 107, Shake FI ask Method " in the OECD Guidelines for Testing of 

chemicals (July 27,1995). 

3-2. Testing Conditions 

Temperature: 23± I"C 

4. Test Results 

The log Pow value (fotal average) :2.18 

The standard deviation :0.109 

The difference between the maximum and the minimum value was 0.327, which was not 


significant. 


There was no environmental factor or abnonnal condition that was presumed to affect the 


reliability of test results. 


Therefore, the test results are judged to be fully reliable. 


4 

00060.1 
Contents '-77 

t. Purposc of the Tcst .............................................................................................................................. 6 


2. Test Method ......................................................................................................................................... 6 


3. Test Period ........................................................................................................................................... 6 


4. Test Substance ..................................................................................................................................... 6 


S. Conrumation of Stability under the Storage Conditions ..................................................................... 6 


6. Measurement of the Partition Coefficient (l-OctanoVW ater) ............................................................. 7 


7. Discussion and Conclusion ................................................................................................................ 11 


8. Environmental Factors Presumed to Mfect the Reliability of Test Results ...................................... 11 


9. Storage ofTset Substance and Raw Data etc .................................................................................... 12 


10. Compliance with GLP ..................................................................................................................... 12 


11. List of Attached Figures .................................................................................................................. 12 


12. List of Attached Tables .................................................................................................................... 12 


5 
 000602 
1. Purpose of the Test 

The purpose of this test is to conduct partition Coefficient (l-OctanoVWater) Study of 

to obtain infonnation on its 

degree of bioaccumulation as part of the program to verify the satety of a new chemical substance. 

2. Test Method 

The test was conducted in accordance with the "Conunentary for the Measuring Method of the 

Partition Coefficient (l-OctanoVWater) of Chemical Substances" indicated in the Order Prescribing the 

Interpretation and Application of the Law Concerning the Examination and Regulation of Manufacture. 

etc. of Chemical Substances (24th March. 1987, Pharmaceutical Affairs Bureau No. 291, , and 62 

kikyolru No.171, Basic IndustrIes Bureau ('87», and "Partition Coefficient (n-OctanoVWater): 107, 

Shake Flask Method" in the OECD Guidelines for Testing of chemicals (July 27,1995). 

3. Test Period 

Date of test initiation: March 2, 2001 

Date of experiment initiation: March 9, 2001 

Date of experiment termination: March 9, 200 I 

Date of test termination: April 4, 200 1 

4. Test Substance* 

Name: 

Abbreviation: 

Identification code: 

Structural fonnula: 

Lot No.: 

Purity: 100% 

Molecular formula: 

Molecular weight: 200.05 

CAS No.: 

Properties: 

Appearance: liquid 

Storage Stored at room temperature in a dark place 

conditions: 

*) Information provided by the test client (except for Identification code.) 

5. Confirmation of Stability under the Storage Conditions 

Infrared absorption spectra of the test substance were obtained at the beginning and at the end of the 

test, and the two spectra were found to show no difference. Based on this result. it was judged that the 

test substance remained stable throughout the test period. (Fig. 1) 

6 000603 

6. Measurement of the Partition Coefficient (l-OctanoVWater) 


6-1. Major Equipment and Reagents Used in the Test 


6-1-1. Reagents 

Pure water: 	 Prepared by means of pure water 

production equipment MiIIiQ-Labo and 

then distilled. Less than 1 Jl S/cm of 

electric conductivity and more than 60 

minutes of K2Mn04 color retention time 

were confmned prior to the use. 

1-0ctanol :( Partition Coefficient Grade) Mitsubishlkasei 


Methanol (lIS Guaranteed Grade): Iuosei Chemical 


Isopropyl alcohol (JIS Guaranteed Grade); Kishida Chemical 


6-1-2. Major Equipment 

Gas chromatograph HP-5890 Hewlet-Packard 


Mass Spectrometer IMS-AX505H IEOLLTD. 


Shaker Y A YOI YGG YAYOICO., LTD. 


Karl Fischer Moisture Titrator MKS-2IO Kyoto Electronics Manufacturing Co., Ltd. 


pH meter lIM-50V Toa Denpa Kogyo Co., Ltd. 


Electric conductivity meter CDM-83 RADIOMETER 


High Speed Refrigerated Centrifuges SCR-20BB Hitachi, Ltd. 


Water Purifier Mi1IiQ-Labo 	 Nihon Millipore Ltd. 


Water Stills WB-21 Yamato Scientific Inc. 


Chemical balance BP-210-D Sartorius Co., Ltd. 


6-2. Mutual Saturation of the Solvents 

Water and 1-0ctanol were mixed at the rate of 2:1, and the mixture was stirred using a magnetic 

stirrer for 24 hours. Next. all the quantity was shifted to a separatory funnel, allowed to settle, and 

separated. 

Water in l..Qctanol when it was mutually saturated with the water was analyzed using Karl Fischer 

Moisture Titrator. And, 1-0ctanol in Water when it was mutually saturated with the l-Octanol was 

analyzed using Gas chromatograph. Then, mutual saturation was confmned. (l-Octanol concentration 

in the water was 411mgIL, and it was beyond 400mglL. And. Water concentration in the l-Octanol 

was 4.94%. and it was beyond 4%.) 

6-3. Preparation of Stock Solution 

lOOmg of the test substance were weighed by use of Chemical balance into a IOO-mJ volumetric 

flask, dissolved in water-saturated l-octanol and diluted to the mark. (Test substance concentration: 

1000 mgIL) 

6-4. Determination Conditions and Method 

GC-MS Conditions 


Gas chromatograph: HP-5890 


Mass Spectrometer: JMS-AXSOSH 


Column: CP-PoraPLOT Q 0.53m q, x 25mm (Film thickness 20 Jl m) 

Carrier gas: He 7 psi SPLIT flow: 50mllmin 

Column oven Temp.: 60"C-(lO"C/min)-185"C 

Inj. Temp. , Separator Temp: 200"C 

Inj. Volume: 1 Jl L 

Measurement mode: SIM method (monitor ion: mle83(test substance), mle45i(internal 

standard» 

Data processor: DA-5000 

Determination method: Absolute calibration curve method, y = ax (3-point method), Decision 

7 

000604 
was made to carry out the quantitative determination of the test substance in the tested 

liquids by the one-point method, since the calibration curve was a straight line passing the 

origin. (see 6-S-3.) 

6-S. Measurement of Limit Value for Test Water Analysis, Calculation of Detection Limit Concentration, 

and Preparation of the Calibration Curve 

6-S-I. Preparation of Standard Solution for Preparation of the Calibration Curve 

A test substance solution with a concentration of 100 mglL was prepared by weighing 10 mg of 

the test substance by use of Chemical balance into a lOO-mL volumetric flask, dissolving it in 

methanol, and diluting to the mark. Then, 1 mL of the solution was transferred to a 20-mL 

volumetric flask by use of a volumetric pipet and diluted to the mark with distilled methanoL Thus 

a test substance solution with a concentration of 5 mglL was prepared. 

And, an internal standard substance solution with a concentration of 1000 mglL was prepared by 

weighing 10 mg of isopropyl alcohol into a 1O-mL volumetric flask, dissolving it in methanol, and 

diluting to the mark. 

Then, about 80% volume of 5-mglL test substance solution was transferred to a 10-mL 

volumetric flask, added 10 f.L L of the lOOO-mgIL internal standard substance solution by use of a 

micro syringe, dissolved in S-mgIL test substance solution and diluted to the mark. Thus, a solution 

of S mgIL test substance containing 1 mglL internal standard substance was prepared. 

Likewise, S mL and 1 mL of the S-mgIL test substance solution was transferred to 1O-mL 

volumetric flasks by use of a volumetric pipette and diluted to the mark with distilled methanol and 

10 f.L L of the lOOO-mgIL internal standard substance solution. Thus, solutions of 2.S or O..S mgIL 

test substance containing 1 mgIL internal standard substance were prepared. 

These three solutions containing internal standard were used as standards for the preparation of 

the calibration curve. 

6-S-2. Calculation of Determination Limit Value and Detection Limit Concentration 

The 5-mgIL test substance solution described in 6-5-1 was diluted with methanol 100 times, and 

internal standard substance solution was added so that internal standard substance concentration 

become ImgIL, and thus test substance solution with a concentration of O.lmgIL was prepared. 

This solution was analyzed using the determination conditions and method described in 6-4., and 

the measurement limit value and the detection limit concentration were calculated. The minimum 

detection limit concentration was 0.00536mgIL. And, the measurement limit concentration of 

analysis samples were calculated. 

Stock solution: 1.34 mg/L 

Partition eqUilibrium solution I-Octanol phase condition-I, 2, 3 : 1.34 mgIL 

water phase condition-I, 2, 3: 0.0179 mgIL 

6-5-3. Preparation of Calibration Curve 

Analysis was conducted using the determination conditions and method described in 6-4. 

The linear regression equation y =ax + b was found by the least square method, and the regression 

coefficient (a), intercept (b) and correlation efficient (r) were verified. 

y: peak area 

a: regression coefficient 

x: concentration 

b: intercept 

r: correlation coefficient 

As a result, b was not acknowledged to be significant. Consequently, calculation was made again 

using the regression equation y =ax, and the regression equation as shown below was obtained. 

From that point onwards, the one-point calibration curve method was adopted for determining the 

recovery and the test substance in the tested liquid. The calibration curve thus obtained is shown 

below. 

y= 1.6119x(r=0.99860) 

8 

000605 
Further, the calibration curve was also utilized for the analysis. 


(Fig. 2-1 Cnlihrntion Curve) 


(Fig. 2·2 Calibration Curve Chromatogram) 


6-5-4. Analysis of Test Substance Stock Solution 

1 ml of the stock solution with a concentration of 1000 mglL described in 6-3. above were 

transferred to a 25-ml volumetric flask by use of a volumetric pipette and diluted to the mark 

with methanol. 

Then, 1 mL of this solution was transferred to a lO-mL volumetric flask by use of a volumetric 

pipette, and after 10 J1. L of lOOO-mgIL internal standard substance solution was added, diluted to 

the mark with distilled methanol. 

Then analysis was conducted twice under the condition of 6-4. using this standard solution. 

Then. concentration of the test substance was determined by the one-point method. 

The results were as follows. Average test substance concentration in stock solution was 685 

mgIL, and the amount of the test substance in the partition equilibrium solution was 3.43mg. 

(Fig.3 Chromatogram for Analysis of Stock Solution, 

Table 1 Data table of Stock Solution) 

(F x H I G) x B x C x D x 100 

Concentration of stock solution(mglL) - .-------------­ AXE/1oo0 

A: Volume of test solution (mL) B: Volume of test solution (mL) 

C: Conversion coefficient* D: Transfer ratio* 

E: Recovery (%) F: Standard concentration (mgIL) 

G: Standard peak area ratio H:Test substance peak area ratio 

*)The term "conversion coefficient" means a coefficient that is used in quantitative 

determination of the test substance in terms of the non-test-substance components by 

subjecting the test substance to chemical decomposition. turning it into derivatives. etc. In 

this test, the conversion coefficient was set at 1. And. the recovery rate was made 100% 

because analysis operation such as extraction wasn't done. 

6-6. The Partition Equilibrium Test 

6-6-1. Testing condition 

1) Temperature: 23 + 1"C 

2) The number of repetition: 2 times per measurement condition 

3) Added amount of test substance: 3.43mg 

4) Volume of test substance solution and solvent, and volume ratio of solvents 

condition-l condition -2 condition -3 

Volume of test substance 

solution 5 5 5 

Volume of water-saturated 

l-octanol 0 5 15 

Volume of l-octanol-saturated 

water 35 30 20 

(mL) 

9 

000606 
6-6-2. Partition Equilibrium Flow 

1) 	 5 m1 aliquots of the stock solution with a concentration of 1000 mglL described in 6-3. above 

were transferred to 6 test vessels by use of a volumetric pipette. Furthermore, water-saturated 

I -octanol and l-octanol-saturated water were added in the exact volume set up in the 

above-mentioned examination conditions to the test vessels using volumetric pipettes. 

2) 	 The test vessels were set to the shaker and shaken for 5 minutes by 20rpm. 

3) 	 The test vessels were removed from the shaker and set in the rotor of the high speed 

refrigerated centrifuges maintained at examination temperature. The test vessels were 

centrifuged by 5000rpm for 20 minutes. 

4) 	 Two phases were confinned to be separate completely. A capillary pipette was inserted in the 

central part of l-octanol phase in each test vessel to extract l-octanol, and less than half amount 

of the phase was moved to a sample bottle. The remainder was carefully removed and 

discarded. 

5) 	 A capillary pipette was inserted in the lower part of the water phase in each vessel, and about 

10 mL of water phase was extracted. Then, after wiping off the outside of the capillary pipette 

with Kimwipe paper wipes, it was moved to a sample bottle. And, pH of remained water 

phase was measured. 

6) 	 According to the analysis method of 6-6-3., the test substance concentrations in I-octanol phase 

and water phase were measured. 

6-6-3. Analysis of Test Substance in Partition Equilibrium Solutions 

1) Analysis of test substance in I-octanol phase 

I-mL aliquots of l-octanol phase obtained by 6-6-2. were transferred to 25-mL volumetric 

flasks by use of a volumetric pipette, and after 10 /.t L of 1000-mglL internal standard substance 

solution was added, diluted to the mark with distilled methanol. Furthennore, I mL of each 

solution was transferred to a lO-mL volumetric flask by use of a volumetric pipette and diluted 

to the mark with distilled methanol. 

Next, they were analyzed under the condition of 6-4. repeatedly using the 2.5mgIL standard 

solution. Then, concentration of the test substance in the I-octanol phase was determined by the 

one-point method. 

(Figs.4-1--4-3 Chromatogram for Analysis of test sample, 

Table 2 data table of I-octanol phase) 

2) 	 Analysis of test substance in water phase 

3-mL aliquots of water phase obtained by 6-6-2. were transferred to lO-mL volumetric flasks 

by use of a volumetric pipette, and after 10/.t L of IOOO-mgIL internal standard substance 

solution was added, diluted to the mark with distilled methanol. 

Next, they were analyzed under the condition of 6-4. repeatedly using the 2.5mgIL standard 

solution. Then, concentration of the test substance in the I-octanol phase was determined by the 

one-point method. 

(Figs.4-1--4-3 Chromatogram for Analysis of test sample, 

- Table 3 data table of water phase) 

Concentration of Partition eqUilibrium = (F X H / G) X B X C X D X 100 

solution(mgIL) 	 AXE/lOOO 

A: Volume of test solution (mL) B: Volume of test solution (mL) 

C: Conversion coefficient* 0: Transfer ratio* 

E: Recovery (%) 	 F: Standard concentration (mgIL) 

G: Standard peak area ratio H:Test substance peak area ratio 

*)The tenn "conversion coefficient" means a coefficient that is used in quantitative 

determination of the test substance in tenns of the non-test-substance components by 

subjecting the test substance to chemical decomposition, turning it into derivatives, etc. In 

this test, the conversion coefficient was set at 1. And, the recovery rate was made 100% 

10 

000607 
because analysis operation such as -extraction wasn't done. 

3) The calculation method of Partition Coefficient 

According to the following formulas, the partition coefficient of test substance was calculated 

from the test substance concentration in I -octanol phase and water phase. LogPow means the 

logarithm to base 10. 

Co 

Partition Coefficient (Pow)=-C-wCo: Concentration of test substance in l-octanol phase (mgIL) 

Cw : Concentration of test substance in water phase(mgIL) 

6-7. Analytical Results 

The test substance concentrations'in each phase after Partition equilibrium analyzed according to 

6-6-3. are shown in the following table. pH of the water phase are also shown. 

concentration of 

l-octanol phase 

concentration of 

water phase pH of water phase 

condi

condit

condit

tion-I 

ion-2 

ion-3 

1 

2 

1 

2 

1 

2 

534 mg

625 

387 

302 

227 

203 

lL 4.76mgIL 

3.90 

2.75 

2.17 

1.56 

0.853 

6.97 

7.09 

7.15 

7.16 

7.84 

7.55 

7. Discussion and Conclusion 

7-I.Result of the Partition Coefficient 

From the test substance concentration in each phase after partition equilibrium acquired by 6-7., 

the Partition Coefficient was calculated and the Partition Coefficient result table was created. The 

result is shown below. (Table 4 Data table of Partition Coefficient) 

log Pow condition -1 : 2.05, 

condition -2 : 2.15. 

condition -3 : 2.16. 

2.20 

2.14 

2.38 

log Pow Total average: 2.18 

Standard deviation: 0

LogPow[max]-logPow

.109 

[min] : 0.327 

7-2. Discussion 

The LogPow value (total average) of test substance was 2.18 as a result of the study. The standard 

deviation"was 0.109 and the difference between the maximum and the minimum value was 0.327, 

which was not significant. 

Moreover, no functional group in the test substance is known as dissociable. And, pH of water phase 

after partition equilibrium was neutral. 

Therefore, these test results are judged to be fully reliable. 

8. Environmental Factors Presumed to Affect the Reliability ofTest Results 

The temperature change during the test examination period was less than ± 1 degree C. 

There was no environmental factor or abnormal condition that was presumed to affect the reliability 

of test results. 

11 

000608 
9. Storage of Test Substance and Raw Data etc. 

9-1. Test Substance 

The test substance for safekeeping was put in a storage container which was closed with a ground 

stopper. It is to be kept in storage in the storage room of this center for the period as stipulated in 

Article 32 of the Standards Concerning the Test Facilities As Set Forth in Article 4 of the Ordinance 

Stipulating the Items of Test Relating to New Chemical Substances (hereinafter referred to as "Test 

Facilities Standards"). 

9-2. Raw Data, Documents, Etc. 

Analytical data obtained from the test, results of measurement and observation raw data used 

for preparation of the final report, protocol, etc. are to be stored for the period provided by Test 

Facilities Standards in a storage room of the test substance in this center. 

l.Test Plan 

2.Final Report 

3.Records relating to the raw data, facilities, and performance of test that are used in the 

preparation of the fmal report (original or copy): 

-Records on professional career and training for individual employees 

-Records relating to administration 

-Records relating to the test substance 

-Records relating to guaranty for reliability 

-Records relating to performance of test 

-Raw data 

-Preliminary study memos, data, etc. 

4. Samples 

Samples for final analysis 

5. Other data deemed as necessary 

10. Compliance with GLP 

This test was conducted in accordance with the GLP standards (March 31, 1984, Revised March 1, 

2000, Kanpogyo No.39, Planning and Coordination Bureau, Environment Agency, Yakuhatu No.229, 

Pharmaceutical Affairs Bureau. Ministry of Health and Welfare. and 59 Kikyoku No.S5, Basic 

Industries Bureau, Ministry of International Trade and Industry, Japan) • and the statement of the 

Study Director and the written guaranty prepared by the person responsible for reliability guaranty are 

attached to the fmal report. 

11. List ofAttached Figures 

Fig. 1 IR Spectra 

Fig. 2-1 Calibration Curve 

Fig. 2-2 Calibration Curve Chromatogram 

Fig. 3 Chromatogram for Analysis of Stock Solution 

Figs. 4~.1 and 4-3 Chromatogram for Analysis of test sample 

12. List ofAttached Tables 

Table 1 Data table of stock solution 

Table 2 Data table of I-octanol phase 

Table 3 Data table of water phase 

Table 4 Data table of Partition Coefficient 

12 000609 
70 

Fig. 1 IR Spectra 

Identification code . Sample comment: initiation of the test Date: 200113/5 

Method: Liquid film Instrument: FfIIR-7000 Operator: Hirokazu Yamane 

w,---------------------------------------------------~ 

80 A ----rrv.~~\ . f\ 1\ 1I \ 

60 

96T II 

40 

l.~ f 

v·V 20 

1~0 4000 2000 

Wavenl..fllbericm-l] 

1000 400 

Identification code: 

Method: Liquid film 

Sample comment: termination of the test Date: 2001/3/23 

Instrument: FT/IR-7000 Operator: Hirokazu Yamane 

96T 


80 

4000 

\ /l 

1 

1000 400 2000 

Wavenumbericm-l] 

000610 

Test date : 2001/3/9 

Fig. 2-1 Calibration Curve 

Identification code. 

Type of test: Partition Coefficient 

Test item: Analysis of Stock Solution 

Operator: Hirokazu Ymane 10.00 

8.00 

0 

'.j:i 

ca 6.00 

Concentartion(mE/l) Area ratio 

0.540 0.9238 

2. 70 4.0881 

.- 5.40 1:J.j3306 - -

li.. 


(!) 


ca 4.00 

« 

li.. 

y = 1.6119x 2.00 

/~ Corre lat ion coefficient (R)

0.00 : 0.99860 

0.00 2.00 4.00 6.00 

Concentration (mg/L) 

o 

o 

o 

C'fJ 

~ 

~ 

kl 

~ 
Fig. 2-2 Calibration Curve Chromatogram 


QUANTITATION Data File: 9-MAR- 0 10:58 


SamPle: 010 

SamPle m/z: 83.000 R.T.: 11'30" Standard m/z· 45.000 R.T.: 11'00" 1. 0P9 

*0 01 R: 126 . 0482 C: " *002 A: 555.7351 C:

STD R: 136.4372 R:0.9238 O.540mglL STD R: 135.9386 R:4.0881 2.70mglL

I D: ID: 

10'59" 

200 

10'58" 

200­

~ ~ 3 100 100­ K2" 

A1'32" 


11 12 
 11 12 


*003 A: 1245.5172C: 


STD 5.40mglL 1=1: 141.0457 R:8.8306 

ID: 

10'58" 


200 


~11'32" 

~ ­ 100 

J ~ -. 
 11 12 
 -_ ......_­

"-' 

o 

(1) 

~ 

N 

o 

@~ 
Fig. 3 Chromatogram for Analysis of Stock Solution 

QUANTITATION Data rile: 9-M~R- 121 13:3121 

SamPle: 010 

SamP 1 eo m/z l 83.121121121 R. T . ~ 11/30" Standard m/z' 45.1211210 R. T.: 11/0121" 1.0Pg 

~12112I5 R: 718.5267 C: *006 ~: 795.9680 C: ! STD R: 169.121450 R: 4. 251215 STD ~: 196.5254 R:4.0502

ID: ID:

11'30" 10'57" 1121 ~11' 3130­ ' 57" 50

11210 \ 100 \.

J\ ) \

11 12 11 12

c 

c 

o 

(1'l .r-;: .'. 

. i ~ 

CJ '.-..>' ~ 

~ 
Fig. 4-1 Chromatogram for Analysis of test sample (Condition-I) 

QUANTITATION Data File: 9-MRR­ 0 14:46 

SamPlE.': 010 

SamplE.' m/z: 83.000 R. T.: 11/30" Standard m/z: 45.0013 R.T.: 11/00" 1. 0P~ 

1'007 A: 158.8915 C: *008 R: 148.6672 c: water phase 101.1 water phase lot.2 sTD A: 73.7662 R:2.1540 5TD R: 84.1788 R: 1. 7661 

ID: ID:

50 50 

100 100 

50 50 

11 12 11 12

1'009 A: 535.2990 c: *010 R: 634.6406 C: 1-0ctanol phase lot.l 1-0ctanol ph(lse 101.2 5TD R: 165.6289 R:3.2319 sTD R: 167.6723 R:3.7850 ID: ID: 

50 10 /57" 11/315'0

11/31" 100 100 

50 50 

11 12 11 12 

o 

~ 

~ '-..d ~ 

~ ~ 

c 
Fig. 4-2 Chromatogram for Analysis of test sample (Condition-2) 

QU~NTITATION Data File: 9-M~R- °15:54 

SamPle: 010 

SamP 1e m/2: 83.000 R. T.: 11'30" Standard m/2' 45.000 R.T.: 11'00" 1.0P9 

+011 A: 124.7745 C: #012 A: 140.7385 C: water phase lot.! STD ~: water phase 101.2 STD A: 100.0212 R:1.2475 142.9343 R:0.9846

ID: ID:

HI'57" 

50 50 

11 12 11 12 

*013 A: STD A: 

ID: 

60 

40 

20 

c 

c 

o 

r:n 

~ 

C/l 

313.6966 C: *014 ~: 271.8197 C: 

133.7744 R:2.3450 1-0etanol phase lot.l STD A: 148.5330 R:1.8300 1-0etanol phase lot.2 

ID: 

11'31" 10'S7" 

11'31" 50 

11 12 11 12 

,__ ~..J \... r-.J 

-S\ 

01 
Fig. 4-3 Chromatogram for Analysis of test sample (Condition-3) 

QURNTITRTION Data File: . 9-MRR- 0 17:30 

SamPle: 0H} SamPle m/z: 83.000 R.T.: 11'30" Standard m/z, 45.131313 R.T.: 11'1313" 1. 13PS 

*1315 

STD 

ID: 

R: 

R: 

31. 3358 

44. 1225 

c: 

R:13.71132 water phase lot.l 

40 

213 

*017 R: STn R: 

ID: 

11 

197.13522 

143.34313 

C: 

R:1.3747 

513 

113'57" 

1J 35" 

12 

l-Octanol phase lot.l 

40 11'31313 

20 

c 

c 

o 

C";

... 

en 

11 12 

*1315 R: 27.2835 c: 

STD R: 713.4418 R:13.3873 water phase lot2

ID: 


413 
 513 

20 

11'34" 

11 12 

*1318 R: 223.13757 c: 1-0ctanol phase lot.2 STD R: 181.13982 R:1.2318 

ID: 


11'313"
 413 513 

213 

11 12 

0ID 

--

~ 

..J\ 
Table 1 Data table of stock solution 

". 

Test substance 

Identification code 

lot. 1 lot. 2 

Volume of test solution 

Volume of analytical sample 

Injection volume 

Concentration of standard solution 

Peak area ratio of standard solution 

Peak area ratio of test substance in analytical sample 

Concentration of test substance in analytical sample 

Residual rate 

ml 

ml 

III 

mg/L 

mg/L 

" 

100 

10 

2. 70 

4.0881 

4.2505 

2.81 

100 

100 

10 

2. 70 

4.0881 

4.0502 

2.67 

100 

Conversion factor 1 1 

Ratio of portion used for analysis 2500 2500 

Absolute amount of test substance in analytical sample mg 0.0281 0.0267 

Residual amount of test substance in test solution mg 70.2 66.9 

Detection limited concentration mg/l 1.34 1.34 

Concentration of stock solution mg/l 702 669 _~~ ••• ••"~ •••••••_ ••••••• ",,_._._~_••••••_ ••,,,,_._.--,_.___.........__• _____._.___._......_ ••_ ................_ ......._ .............__._..........___••••_.___......_._••n ••___••_._......_______••___._....__.....__...""........___• 


Average concentration of stock solution mg/l 685 

,­

'-' 

c 

o 

a"J 

...... 

-J 

~ 

....1\

,., 
Table2 Data table of l-octanol phase 

" 

Test substance 

Identification code 

Test condition 1 condition-l condition-2 

-._.__.___._._....__._.__..._§!!!!Pl~l':Ip· ______..___...l''._...''...__1___._.._ .....__f.____._._..___.__1 ___._.__.....~_..._..___ 

Peak area ratio of test substance in analytical sample - i 3.2319 3.7850 2.3450 1.8300 

Concentration of standard solution mg/ll 2.70 2.10 2.70 2.70 

Peak area ratio of standard solution 1 4.0881 4.0881 4.0881 4.0881 

Ir\iection volume J.ll 1 1 1 1 

Concentration oftest substance in analytical sample mg/I i 2.13 2.50 1.55 1.21 

Volume oftest solution ml j 5 5 10 10 

Conversion factor ! 1 1 1 1 

Ratio of portion used for analysis j 125 125 250 250 

Volume of analytical sample ml! 10 10 10 10 

Detection limited concentration mg/l 1 1.34 1.34 1.34 1.34 

Residual rate "1 100 100 100 100 

Absolute amount of test substance in analytical sample mg 1 0.0213 0.0250 0.0155 0.0121 


Residual amount of test substance in test solution mg ~ 2.67 3.12 3.87 3.02 


Concentration oftest substance in l-octanol phase mg/l i 534 625 387 302 


o 

o 

(1) 

~ 

condition-3 

..._ . .1___......_.__ 2 

1.3747 1.2318 

2.70 2.70 

4.0881 4.0881 

1 

0.908 0.814 

20 20 

1 1 

500 500 

10 10 

1.34 1.34 

100 tOO 

0.00908 0.00814 

4.54 4.07 

227 203 

\...., 

~ 

W 


c 
Table 3 Data table of water phase 

Test substance 

Identification code 

Test condition ! condition-1 condition-2 condition-3 

2 _.....___...J_______..l_..___.... .__._..______.._2.~~~ No_.------.--~---............--.L--...-.J-.-...-.-..-.---.--?.._______--=1'--_. 


Peak area ratio oftest substance in analytical sample 1 2.1540 1.7661 1.2475 0.9846 0.7102 0.3873 


Concentration of standard solution mg/L ! 2.70 2.70 2.70 2.70 2.70 2.70 


Peak area ratio of standard solution ! 4.0881 4.0881 4.0881 4.0881 4.0881 4.0881 


lr1iection volume J.I. L I 1 1 1 1 1 1 


Concentration oHest substance in analytical sample mg/I! 1.42 1.17 0.824 0.650 0.469 0.256 


Volume of test solution mL 1 35 35 30 30 20 20 


Conversion factor ill 1 1 
 1


Ratio of portion used for analysis ! 11. 7 11.7 10.0 10.0 6.67 6.67 


Volume of analytical sample mL I 10 10 10 10 10 10 


Detection limited concentration mg/L 1 0.0179 0.0179 0.0179 0.0179 0.0179 0.0179 


Residual rate "1 100 100 100 100 100 100 


Absolute amount of test substance in analytical sample mg 1 0.0142 0.Q117 0.0082 0.0065 0.00469 0.00256 


Residual amount of test substance in test solution mg I 0.166 0.136 0.0824 0.0650 0.0313 0.0171 


Concentration of test substance in water phase mg/L ! 4.76 3.90 2.75 2.17 1.56 0.853 


-'-' 

o 

o \.,) 

0') -..,S:)

... ..J:: CD 
Table 4 Data table of Partition coefficient 

... 

Test substance 

Identification code 

Volume of test vessel 44 mL Proportion of solution in test vessel:90.9" 

Added amount of test substance 3.43 mg 

Test condition . conditiorr-1 condition-2 condition--:3 


-.-...-...------.--. San:a.P..t«:...t!4?~__.___._____.._.._._...._...._._..___________--...................---.-...-/...--..-.!..........-----.-.?.-.--__.._..!__.___.._.........?____......._L......_ .............g.........._. 


Volume of phase (mL) l-octanol phasel 5 5 10 10 20 20 


......~.!ter P.b.!!.~j 35 35 30 30 20 20 


i 


Amount of test substance (mg) l-octanol phasei 2.67 3.12 3.87 3.02 4.54 4.07 


._._!!..~.!.I!!:..p.has~J 0.166 0.136 0.0824 0.0650 0.0313 0.0171 


Concentration of test substance (mg/L) l-octanol phasel 534 625 387 302 227 203 


i 


water phase 1 4.76 3.90 2.75 2.17 1.56 0.853 


Material balance (%) I 82.7 95.2 115 90.1 133 119 


Ipow data I
 112 160 141 139 145 238 


! average data i 136 140 192 
 ~ i 


Residual amount of test substance in test solution lLogPow data i 2.05 2.20 2.15 2.14 2.16 2.38 

i 	 I 


Partition coefficient 	 ~-.--..--.-.---.~.Y.~~l~~~--L-._-...........--__2.:.'!~-.__--_...._.._....._..?.J.~....__.._........._____.._?..2L_._. 


ltotal average Pow i 156 


i 	 j

i LogPow 1 2.18 

lstandard deviation (LogPow) I 0.109 

1LogPow[max]-LogPow[min] i 0.327 

c

-(J") ­

N 

o 	 '" 

~\ 
P-04-635 

REPORT 

Study Title 

FRESH WATER ALGAL GROWTH INHIBITION TEST WITH 

Study completion date 

28 October 2005 

Test Facility 

,.- -, 

Laboratory Prolect Identification 

:~;):-T:~~ 

• Page 1 of 36 • ~::; '. >_! 

c.") 

CJ1 

111111111111111111111111111111111111111111111111111111111111111111111111111 

5 0 0 e 000 1 0 1 5 I S 

-rD f ~oi -(P3S 

1. CONTENTS 

1. CONTENTS ...........................................................................................................................2 


2. STATEMENT OF GLP COMPLIANCE .................................................................................. 4 


3. QUALITY ASSURANCE STATEMENT ................................................................................. 5 


4. SUMMARy.............................................................................................................................6 


5. INTRODUCTION....................................................................................................................7 


5.1. Preface ...........................................................................................................................7 


5.2. Aim of the study....................................................................................... , ...................... 7 


5.3. Guidelines.......................................................................................................................7 


5.4. Storaga and retention of records and materials .............................................................7 


5.5. Definitions.......................................................................................................................8 


6. MATERIALS AND METHODS ...............................................................................................8 


6.1.1. Test substance .....................................................................................................8 


6.1.2. Re1erenca substance ...........................................................................................8 


6.1..3. PrelimInary data ................................................................................................... 9 


6.2. Test System....................................................................................................................9 


6.3. Fresh water algae culture ...............................................................................................9 


6.4. Preparation of test solutions ......................................................................................... 10 


6.5. Range-finding test ........................................................................................................10 


6.6. First limit test ................................................................................................................ 11. 


6.7. Final limit 185t................................................................................................................ 11 


6.7.1. Test concentrations ............................................................................................ 11 


6.7.2. Test procedures and conditions......................................................................... 11 


6.7.3. Sampling for analysis of test concentrations ..................................................... 12 


6.7.4. Measurements ...................................................................................................12 


6.7.5. Recording of cell densities ................................................................................. 12 


6.8. Interpretation ................................................................................................................ 12 


6.8.1. Data handling .....................................................................................................12 


6.8.2. Acceptabilfty of the test ...................................................................................... 13 


6.9. Ust of protocol davialions ............................................................................................. 13 


7. RESULTS............................................................................................................................. 14 


7.1. Ranga-finding test ........................................................................................................ 14 


7.2. First limit test ................................................................................................................ 15 


7.2.1. Measured test substance concentrations .......................................................... 15 


7.2.2. Mean cell densities, inhibition of cell growth and reduction of growth rate........ 15 


7.3. Final limit test. .............................................................................................................. 16 


7.3.1 . Measured test substance concentrations .......................................................... 16 


7.3.2. Mean cell densities .............................................................................................16 


7.3.3. Inhibition of cell growth and reduction of growth rate ........................................ 17 


7.3.4. Determination of effect concentrations ..............................................................18 


7.3.5. Experimental conditions ..................................................................................... 18 


8. CONCLUSION .....................................................................................................................18 


TABLES 


Table 1 Mean cell densities (x10' cans/ml) during the range-finding test.. ..................... 14 


Table 2 Percentage reduction of growth rate and inhibition of total growth during 


the range-finding test .........................................................................................14 


Table 3 Mean cell densities (x 104 cells/ml) during the first limit te8t.............................. 15 


Table 4 Percentage Inhibition of cell growth during the first limit test.. ........................... 16 


Table 5 Percentage reduction of growth rate at different time intervals during the 


first limit test .......................................................................................................16 


Table 6 Mean cell densities (x 104 cells/ml) during the final limit test... .......................... 16 


Table 7 Percentage inhibition of cell growth during the limit test .................................... 17 


Table 8 Percentage reduction of growth rate at different time intervals during the 


limit test .............................................................................................................. 1 7 


Table 9 Effect parameters ............................................................................................... 18 


Table 10 pH levels recorded during tha limit test.. ............................................................ 18 


-Page2­
Table 11 


Table 12 


Table 13 


Table 14 


FIGURES 


Figure 1 


Figure 2 


APPENDICES 


APPENDIX I 


APPENDIX II 


APPENDIX III 


Individual celf densities (1 S1 limit test) ................................................................. 19 


Calculation of growth (area under growth curve) and growth rate (1 st limit 


test) ....................................................................................................................19 


Individual cell densities (final 11mit test) ..............................................................20 


Calculation of growth (area under growth curve) and growth rate (final limit 


test) ....................................................................................................................20 


Growth curves at different concentrations of during 


the 1S\ limit test ................................................................................................... 15 


Growth curves at different concentrations of during 


the final limit test ................................................................................................ 17 


WORKSHEET DATA ......................................................................................... 19 


REFEAENCETEST ...........................................................................................21 


ANALYTICAL REPORT 


- Page 3­
2. ST ATEMENT OF GLP COMPLIANCE 

The study described in this report has been correctly reported and was conducted In compliance 

with: 


The Organization for Economic Cooperation and Development (OECD) Good Laboratory 


Practice Guidelines (1997). 


Which essentially conform to: 


The United States Food and Drug Administration Good Laboratory Practice Regulations. 


The United States Environmental Protection Agency Good Laboratory Practice Regulations. 


The sponsor Is responsible for Good Laboratory Practice (GLP) compliance for all test 


substance information unless determined by 

Study~ Head of In Vitro &Environmental Toxicology 

Date: .7.9.\.O.da::;,~:e.q ...~~~............. Date: .........u:?JR}.tOOf::....................... 


-Page4­
3. QUALITY ASSURANCE STATEMENT 

This report was inspected by the Quality Assurance Unit to confirm that the methods 

and results accurately and completely reflect the raw data. 


The dates of Quality Assurance Inspections are given be[ow. 


During the on-site process Inspections procedures applicable to this type of study were 


Inspected. 


Type of 

inspecUons Phase/Process 

Start 

InspecUon 

date 

End 

Inspection 

date 

ReportIng 

date 

Study Protocol 03-Sep-04 03-Sep..()4 03-Sep-04 

Amendment 1 of protocol 03-May-05 03-May-05 03-May-OS 

Amendment 2 of protocol 30-Jun-OS 30-Jun-OS 3Q-Jun-05 

Amendment 3 of protocol 22-Jul-OS 22-Jul-05 22-Jul-05 

Amendment 4 of protocol OS-Sep-05 OB-Sap-05 OS-Sep-05 

Report 26-00t-05 26-0ct-05 26-0ct-05 

Process Environmental toxicology 1S-Apr-05 

OS-Aug-QS 

22-Apr-oS 

19-Aug-05 

22-Apr-OS 

19-Aug-oS 

Test substance handling 

Exposure 

Observalions/Measurements 

Analytical and phy6k:al 20-Jun-05 29-Jun·OS OS-Jul-06 

chemistry 20-Sep-QS 26-Sap-05 27-Sep-05 

Test substance handling 

Observations/Measuremants 

Head of Quality Assurance 

Date: 2.~..Q,.~~b~.200S 

- Page 5· 
4. SUMMARY 

Selensstrum capricornutlJm, Fresh Water Algal Growth Inhibition Test with 

The study procedures described in this report were based on the ISO Inlernational Standard 

8692, 1989. In addition. the procedures were designed to meet the 1est methods and validity 

criteria of the EEC directive 92169, Part C.3. 1992 and the OECD guideline No. 201. 1984. 

The batch of tested was a clear and colourless liquid with a purity of 

99.985%. was completely soluble in test medium at the ooncentrations 

tested and known to be extremely volatile. Consequently. measures were taken during this 

project to minimise loss of test substance by evaporation, which irlcluded but were not limited to 

short preparation times and the use of airtight closed vessels. 

The project started with a ranoe-findinQ test eXPOSing exponentially growing algal cultures to a 

blank-control and nominal concentrations of 0.1, 1.0, 10 and 100 mgll. No 

significant effects were observed on algal growth inhibition and growth rate reduction up to a 

nominal test concentration of 100 mg/l. Samples taken from nominal 10 and 100 mg/I were 

analysed, which showed mean start concentrations of 1.0 and 14.6 mgn, respectively. These 

concentrations decreased by approximately 80 to 90"10 below initial during the test period 

despite the fact that test vessels were air tightly closed. 

The project was continued with a limit test applying an adapted preparation method. 

Exponentially growing algal cultures were exposed to a blank-control and a nominal 

concentration of 147 mg/1. An amount of 100 pi was pipetted directly into 1 liter (nominal 

147 mg/!) of stirring algal growth medium. The vessel was immediately closed and the content 

magnetically stirred for 1 minute. Then, individual replicate vessels were filled with the solution. 

algae were added, samples were taken and the incubation started in completely closed vessels. 

The total test period was 72 hours and the initial algal cell density was 10" celis/mI. Analysis at 

the start of the limit test showed a mean measured concentration of 57 mgll in samples taken 

from nominally 147 mgII. The measured concentration decreased to 6.3 mgll after 24 hours and 

to 2.1 mgll after 72 hours of exposure. The Time Weight Average (1WA) concentration was 

calculated to correspond to 8.7 mgll. 

As the initial concentration was well below 100 mg/I in the first limit study it was decided to 

repeat this study. In the final limit study an amount of 175 pi was pipetted into 1 liter of stirring 

algal growth medium (nominal 257 mgII). Further test procedures were comparable to the first 

limit study, except that headspace in the test vessels was reduced by 50%. 

Analysis at the start of the final limit test showed a mean measured concentration of 213 mg/l in 

samples taken from nominally 257 mg/l. The measured concentration decreased to 13 mg/l after 

24 hours and to 6.3 mg/I after 72 hours of exposure. The Time Weight Average (TWA) 

concentration was calculated to correspond to 24 mglL 

The study met the acceptability criteria prescribed by the protocol and was considered valid. 

No significant Inhibition of cell growth Of reduction of growth rate was recorded at nominal 

concentrations up 10 257 mg/I, corresponding to a measured Initial 

concentration of 213 mg/I or a TWA concentration of 24 mgl1 (NOEC). 

The ECso for cell growth inhibition and growth rate reduction exceeded a measured initial 

concentration of 213 mgll or a TWA concentration of 24 mg/l. 

• Page 6· 
5. INTRODUCTION 

5.1. Preface 

Sponsor 

Study Monitor 

Test Facility 

Study Director 

Technical Coordinator 

Principal Scientist 

Study Plan 	 Start 23 May 2005 

Completion 16 September 2005 

5.2. Aim of the study 

The purpose of the study was to evaluate the test substance for Its ability to generate toxic 

effects in Selenastrum caprlcomutum during an exposure period of 72 hours and, it possible, to 

determine the ECso for both inhibition of cell growth and reduction of growth rate for the 72-hour 

period. 

5.3. Guidelines 

The study procedures described In this report were based on the ISO Intemational Standard 

8692: 'Water quality - Fresh water algal growth inhibition test with Scenooesmus subspicatus 

and Se/enastrum capricomututrl', First edition, 15 NOV9mber 1989. 

In addition, the procedures were designed to meet the test methods and validity criteria 

prescribed by the following guidelines: 

• 	 European Economic Community (EEC). EEC directive 92169, Part C: Methods for the 

determination of ecotoxlcity. Publication No. L3SS, December 1992, C-3: "Algal Inhibition 

Test". 

• 	 Organization for Economic Co-operation and Development (DECO), OECD guideline for 

Testing of Chemicals. guideline No. 201: "Algae, Growth Inhibition Test", Adopted June 7. 

1964. 

5.4. Storage and retention or records and materials 

Records and materials pertaining to the study including protocol. raw data, specimens (except 

specimens requiring refrigeration or freezing) and the final report are retained in the 

archives for a period of a11east 10 years after finalization of the report. After this period, the 

sponsor will be contacted to determine whether raw data and specimens should be returned to 

them. retained or destroyed on their behalf. 

Those specimens requiring refrigeration or freezing will be retained by for as long as 

U1e quality of the speoimens permits evaluation but no longer than three months after finalization 

of the report. . 

• Page 7· 
NOTOX will retain a test substance sample until the expiry date, but no longer than 10 years 

after finalization of the report. After this period the sample will be destroyed. 

5.5. Definitions 

Cell density is the number of cells per millilitre. 

Total growth or biomass Is defined as the increase in total cell density over the test period. It 

is derived from the area under the growth curve in a linear plot. The EBCSO is the concentration 

of test substance that results in a 50% inhibition of total cell growth relative to the control. 

Growth rate is the increase in cell density per unit time. It is derived from the slope of the 

growth curve in a logarithmic plot. Following from the mathematical nature of exponential 

growth, the measure of the specific growtl1 rate is preferable over biomass. The ERGsO is the 

concentration of test substance that results in a 50% reduction In growth rate relative to the 

control. 

No Observed Effect Concentration (HOEC) is the highest concentration tasted at which the 

measured parameter(s) show(s) no significant effect on algal growth relative to control values. 

6. MATERIALS AND METHODS 

6.1.1. Test substance 

Identification 

Structure 

Molecular formula 

Molecular weight 

CAS Number 

Description Clear colourless liquid 

Batch 

Purity 99.985% 

Test substance storage In refrigerator In the dark 

Stability under storage conditions Stable 

Expiry date 28 February 2006 

Specific Gravity 1.47 

Stability in water Not indicated 

6.1.2. Reference substance 

This report includes the results of the most recent reference test with potassium dichromate 

(Appendix II). 

- Page 8· 
6.1.3. Preliminary data 

The water solubilitv of 	 at 20.1 ± 0.5"C is 0.805 gil using the flask method 

Further essential information obtained from physico-chemical studies 

performed at were the vapour pressure being 31 kPa at 25°G. freezing point <-81 "C. 

boiling point 56°C and a log Koc of 2..54. The Log Pow of was 2.18 

according to information supplied by the sponsor. From these data it can be concluded that the . 

tast substance will evaporate rapidly at room temperature as pure substance but also from 

3 l aQueous solutions (calculated Henry coefficient is 7.24.103 Pa m mor ). 

concentrations showed to be stable during treezing and thawing of 

samples in a deep-freeze 

6.2. Test System 

Species 	 Se/enastrumcapricomurum, strain: range-finding test 

and 181 limit test NIVA CHL 1; final limit test: CCAP 

27814. 

Source 	 In-house laboratory culture. 

Reason for selection 	 This system is an unicellular algal species sensitive to 

toxic substances in the aquatiC ecosystem and has 

been selected as an internationally accepted species. 

6.S. Fresh water aJgae culture 

Stock culture 	 Algae stock cultures were started by inoculating 

growth medium with algal cells from a pure culture on 

agar. The suspensions were continuously aerated and 

exposed to light in a climate room at a temperature of 

23 ±2"C. 

Light intensity 	 60 to 120 pEtm'ls when measured in the 

photosynthetically effective wavelength range of 400 

to 700nm. 

Stock culture medium 	 M1; formulated using Milli·Ro water (tap-water purified 

by reverse osmosis; Mlilipore Corp.• Bedford, Mass., 

USA) with the following composition: 

NaNOa 

KzHPO. 

500 

40 

mgII

m\il 

MgSo•.7H~ 76 m\il 

NII2CO".10H.O 54 mgll 

CeHaOr.HaO 6 mgll 

NH~ 330 mgll 

CaCI2.HzO se mgll 

CeH5Feo,~O 6 mg/l 

H3BOa 2.9 mg/l 

t.AnClz.4HrO 1.81 mgll 

ZrICfz (1.11 mg/l 

CuSO..5H~O (I,OS mg/l 

(NH..)G~OM-4HtO 0.018 mgt1 

Pre-cuRure 	 3 days before the start of the test, cells from the algal • 

stock cuHure were inoculated in cuHure medium at a 

cell density of 2.10" celis/mi. The pre-culture was 

maintained under ttie same conditions as used in the 

test. The cell denSity was measured immediately 

before use. 

• Page 9· 
Pre-culture medium 	 M2; according to the ISO-Standard "Algal growth 

inhibition test" Nov. 1989; formulated using MiIIi..Q 

water (tap water purified by reverse osmosis (MiIIi-RO) 

and subsequently passed over activated carbon and 

ion-exchange cartridges: MiIIi-Q water; Millipore Corp., 

Bedford, Mass., USA) preventing precipitation and 

with ttle following composition: N~ 15 mgll 

MgC!••6H~ 

CaClr,.2HaO 

12 

18 

mgll 

mgll 

MgS04.7HJO 

I<liJ!P04 

15 

1.6 

mgll 

mgll 

F~6HP 80 pg/l 

NIbEDTA.2H~ 100 pgll 

~o. 185 pgll 

MnCb.4H~ 

ZnC~ 

415 

~ 

#0/1

pgII 

COCI:.6H.O 1.5 pgII 

CuClr,.2HP 0.01 pg/l 

Na,.1iAo04.2H~ 7 pgII 

NaHCOs 100 mgll 

Hardness (CIl+Mg) 0.24 mmof.ll (24 mg CaCOJl) 

pH 8.3:1;0.2 

6.4. Preparation of test solutions 

The standard test procedures required generation of test solutions. which contained completely 

dissolved test subs1ance concentrations or stable and homogeneous mixtures or dispersions. 

The testing of concentrations that would disturb the test system was prevented as much as 

possible (e.g. film of the test substance on the water surface). 

The batch of tested was a clear and colourless Ilquld with a purity of 

99.985%. was completely soluble in test medium at the concentrations 

tested and known to be extremely volatile. 

Preparation of test solutions for the range-finding test started with weighed amounts of test 

substance that were quantitatively added to volumetric flasks to provide test solutions of 

nominally 10 and 100 mg/l. The test solutions were magnetically stirred for 2-5 minutes. Care 

was taken that during preparation as little as possible evaporation occurred (weighing and 

preparation In closed vessels which already contained a small volume of test medium, short 

preparation period). The lower test concentrations were prepared by dilution of the test solution 

of 10 mgIJ. 

For the first limit test a target concentration of 100 mg/1 was prepared by pipetting an amount of 

100 pi directly into 1 liter (nominal 147 mgII) 0' stirring algal growth medium. The pipetted 

volume for the final limit test was 175 pi per liter (nominal 257 mgn). The vessels used for 

preparing the test solutions were immediately closed and the content magnetically stirred for 1 

minute (first limit) or 5 minutes (final test). Directly after the short stirring period. volumes of 50 

ml (first limit test) or 75 ml (final test) were added to each replicate of the respective test 

concentration. Hence. in the final study the headspace of the test vessels was reduced by 50% 

to further minimize loss of test substance ooncentration by evaporation. Subsequel'ltly, 1 mlof 

an algal suspension was added to each replicate providing a cell density of 1 Q4 cells/ml, 

samples were taken and the incubation started. The final test solutions were art clear and 

colourless. 

6.5. Range-finding test 

A range-finding test was performed to provide information about the range of ooncentrations to 

be used in the final test Test procedure and conditions were similar to those applied in the flnal 

test with the following exceptions: 

Exponentially growing algal cultures were exposed to a range of 0.1 to 100 mgllincreasing by a 

factor of 10 and to a blank-oontrol. Three replicates were tested per concentration and three 

• Page 10· 
replicates in the control group. One extra test vessel per concentration without algae was used 

as background for the determination of the algal cell density at each time interval. pH was only 

measured in the control and the highest test concentration. Duplicate samples for possible 

analysis were taken from all concentrations, but the control. The NaHCOsconcentration in the 

test medium was 50 mgll and the vessels were filled with 50 ml solution. 

6.6. First IImh test 

A first limit test was performed with algae exposed to a nominal concentration of 147 mgJI, 

prepared by direct addition of 100 pi of the pure test SubstaJ'1C9 to a liter test medium. A blankcontrol was included and test procedures and conditions were similar to those applied during 

the final test except for the fact that the NaHCOs concentratlon In the test medium was 50 mg/l 

and that vessels were filled with 50 ml test solution. 

6.7. Anal limit test 

6.7.1. Test concentrations 

175 JM, or nominal 257 mg/l, expected to result in a 

start conoentratlon of at least 100 mgJI, I.e. the 

regulatory limit concentration. 

Controls Test medium without test substance or other additives 

(blank-control) . 

Replicates 6 replicates of the blank-control and test 

concentration. 

2 replicates of the test ooncentration without algae. 

1 extra replicate of the blank-control and the test 

concentration for sampling after 24 hours of exposure. 

6.7.2. Test procedures and conditions 

Test duration 72 hours 

Test type Static 

Test vessels 100 ml, all-glass, containing 75 ml of test solution, 

airtight closed with a screw cap. 

Medium M2 


Cell density An Initial cell density of 1 x 1 ()4 celis/mi. 


Illumination Continuously using TLD-Iamps of the type 'Cool-white' 


of 30 Watt, with a Ught intensity within the range of 67 

to 82 J,LE.m·2.s·1

. 

Incubation 	 Vessels were distributed at random in the Incubator. 

During incubation the algal cells ware kept in 

suspension by continuous shaking • 

• Page 11 ­
6.7.3. Sampling for analysis of test concentrations 

During the final limit test duplicate samples were taken from the blank-control and the limit 

concentration. Samples were transferred to headspace-vlals that were immediately capped 

airtightty. The method of analysis Is described in the appended Analytical Report (Appendix III). . . Frequency at t=O h (without algae only). t=24 h and t=72 h 

Volume 10ml 

Storage Samples taken at the start and after 24 hours were 

stored in a freezer until analysis. 

Compliance with the QualHy crHena regarding maintenance of actual concentrations was 

demonstrated by running a test vessel without algae and duplicate samples for analysis were taken 

from the limit concentration at the start, after 24 hours of exposure and at the end of the test period. 

6.7.4. Measurements 

pH 	 At the beginning and at the end of the test. 

The pH of the solutions should pr9ferably not deviate 

by more than 1.5 units during the test. 

Temperature of medium 	 Continuously In a temperature control vessel. 

6.7.5. Recording of cell densities 

At the beginning of the test, cells were counted using a microscope and a counting chamber. 

Thereafter cell densities were determined by spectrophotometric measurement of samples at 

720 nm using a Varian CaIY 50 single beam spectrophotometer with immersion probe 

(pathlength =20 mm); Varian Nederland B.V., Houten, The Netherlands. applying Bio Pack 

version 2.00(26) software. Algal medium was used as blank. To prevent additional loss of test 

substance during spectrophotometric measurement, samples of -2 ml were taken from the 

closed test vessels through the septum in the screw cap using a syringe and needle. Samples 

ware transferred into a eweUe and then algal density was measured. 

6.8. Interpretation 

6.8.1. Data handling 

Calibration curve 


Quantification of cell densities was based on a calibration curve. Cell density was ploUed versus 


extinction using spectrophotometric measurements of a minimum of six dilutions of an algal 


suspension with different cell densities. The calibration curve was composed using linear 


regression. The software automatically calculates the cell densities based on this curve for the 


spectrophotometric measurements at the various points in time during the test period. 


Comparison of areas under the growth curves 


The area below the growth curve was calculated using the formula: 


Where: 	 A :;; area 

No=: nominal number of cellslml at the start of the test 

N1=measured number of cells/ml at t, 

Nn;; measured number of cells/ml at tn 

t,= time of first measurements after beginning of the test 

~= time of nih measurement after beginnIng ot the 1ast 

- Page 12­
The percentage inhibition of cell growth at each test concentration (~) was calculated using the 


following formula: 


IT = lie -A, x100 


Ac 


Where: 	 Ao =area below the growth curve obtained in the control 


AT= area below the growth curve at each test substance concentration 


Growth inhibition was calculated for the total period of 72h. 

Comoarison of growth rates 


The average specific growth rate (.ti) for exponentially growing cultures was calculated as: 


InNn-ln~ 

p= tn-~ 

The average growth rate at each test substance concentration was then compared to the control' 

value and the percentage reduction in growth rate was calculated. 

Determination of the average exposure concentrations 

The average exposure concentrations were calculated as: 

. 24X'/CI-oXC,.w;24ax.jC,.".XCtc72 I being the Time Weight Average (TWA) of the concentrations of 

measured in the samples taken at the start (Ct.o), after 24 hours (Cw.4) 


and the end of the test (C1,,72). 


Determination of the NOEC and calculation of the ECso 

For determination of the NOEC and the E~ the approaches recommended in the DECD 

guideline (201, adopted 7 June 1984) were used. Statistical analysis of the data was not needed 

as the effects recorded were not significant. No EC50-values could be calculated because the 

test substance proved to be non-toxic (ECso > maximum concentration tested). 

0.8.2. Acceptability of the test 

1. 	 In the controls. cell density increased by an average factor of > 16 within three days. 

2. 	Further, test conditions (temperature and pH) remaJned within the ranges prescribed by the 

guideline, except for the pH. The pH increased by more than 1.5 unit. but this was caused by 

the fact that test vessels were air tightly closed. 

6.9. List of protocol deviations 

1. 	The pH in the range-finding test and the two limit tests increased above a value of 9.0 and/or 

with more than 1.5 units. 

Evaluation: The observed increase was due to the fact that test vessels were completely 

closed and consequently gas exchange was reduced in comparison to norma! open 

systems. Increasing 1he NaHCO, concentration in the medium prevented an Increase In pH 

of more than 1.5 units but could not maintain the value below 9.0. 

The study Integrity was not adversely affected by the deviation. 

- Page 13­
7. RESULTS 

7.1. Rang&-nndlng test 

The mean cell densities measured during the range-finding test are presented in Table 1. Table. 

2 presents the percentages growth inhlbltJon and growth rate reduction perconcentrat[on. No 

biologically significant effects were observed on algal growth inhibition and growth rate 

reduction up to a nominal test concentration of 100 mgll. Samples taken from nominal 1 0 and 

100 mg/I were analysed. which showed mean start concentrations of 1.0 and 14.6 mg/l, 

respectively. These concentrations decreased by approximately 80 to 90% below initial during 

the test period despite the fact ,hat test vessels were air tightly closed (see Table 2 of the 

appended Analytical report). 

All test conditions were maintained within the limits prescribed by the protocol. except for the pH 

that increased above 9.0, which was likely to be caused by the fact that test vessels were 

closed. 

Table 1 Mean cell densities (x104 cellslml) during the range-finding test 

Concentration Exposure time (hours) 

~I(mg/l) 0 24 48 72 

ntro! 1.0 6.2 18.4 43.0 

0.1 1.0 5.5 13.5 37.9 

1.0 1.0 4.B 14.9 32.2 

10 1.0 3.0 13.5 41.5 

100 1.0 4.6 15.3 40.9 

Table 2 Percentage reduction of growth rate and inhibition of total growth during 

the range-finding test 

ConcentraUon Cell growth (0-72 hra) Mean growth rate 

Nominal (mgl'l) Mean area (A) Inhibition (~I II (0-72 hrs) Reduotion (%'1 

Blank-control 1047.24 0.05224 

0.1 851.24 18.7 0.05025 3.8 

1.0 798.80 23.7 0.04819 7.8 

10 834.48 20.3 0.05173 1.0 

100 907.64 13.3 0.06153 1.4 

Due to the extreme volatile charactar of the test substance, it was not possible to start at the 

intended nominal concentration with the method of preparation used in the present rangerlnding test (see paragraph 6.4) and to keep concentrations stable during exposure. 

Therefore, it was decided to adapt the preparation procedure for the final test as to be able to 

start at an actual exposure concentration close 10 100 mglL For this purpose, an amount of 

100 pi was plpetted directly into 1 liter (nominal 147 mg/l) ot stirring algal growth medium. The 

vessel was immediately closed and the content magnetically stirred for 1 minute. Immediately 

thereafter, individual replicate vessels were filled with test solution, algaewere added, samples 

were taken and the incubation started in completely closed vessels. All within the shortest time 

possible. 

- Page 14· 
7.2. First limn test 

7.2.1. Measured teat sub,lance concentrations 

The results of analysis of the samples taken during the first limit test are described in Table :3 of 

the appended Analytical Report. 

Analysis at the start of the test showed a mean measured concentration of 57 mg/l in samples 

taken from nominally 147 mgll. The measured concentration decreased to 6.3 mg/l after 24 

hours and to 2.1 mgJI after 72 hours of exposure. The Time Weight Average (TWA) 

concentration was calculated to correspond to 8.7 mgJI. 

7.2.2. Mean cell dens lUes, Inhibition 01 cell growth and reduction of growth rate 

Table 3 shows mean cell densities measured at 24-hour Intervals at the limit concentration of 

The respective growth curves are shown In Figure 1 (see Appendix I for 

the cell densities per replicate). Table 4 shows the calculation of !he percentages of inhibition 

and Table 5 shows the percentages 01 growth rate reduction at different time Intervals (see 

Appendix I for the areas of cell growth and values of growth rate per replicate), 

No significant differences were recorded between the values for cell growth or growth rate 

between the limit test concentration and the blank-control. However, as the initial measured 

concentration was only 57 mg/l. and thus stili not at the required 100 mgJI. it was decided to 

perform a second limit test In the second Imit test a higher nominal conoentretion was prepared 

as to be able to start the test at the required minimum concentration of 100 mgll. 

All test conditions were maintained within the limits prescribed by the protOCOl. except for the pH 

that inoreased with more than 1.5 units to a value of 10.4 at the end of the test. The pH increase 

was probably caused by the fact that test vessels were closed hampering the CO2 exchange. 

Table 3 Mean cell densities (x 104 cell'lml) during the first limit test 

ConcentraUon (mgII) 

Nominal/initial 

Expaaure time (hour.) 

0 24 48 72 

Blank-control 1.0 3.9 14.8 36.9 

147 I 57 1.0 4.5 12.5 30.9 

40 

jt' 

/x / ,--' .' 

/,./

/--,­

/.x" ./... .' 

--,-~ 5 

o 

o 24 

nomlnBl (mgll) 

-·.··147 

Figure 1 	 Growth curves at different concentrations of during 

the 1-' limit test 

• Page 15· 
Table 4 Percentage Inhibition of cell growth during the first limit test 

ConcenbaHon(mgn) Cell growth (0-72 hrs) 

Nominal/Initial Mean a.... (A) InhibHion (%) 

Blank-control 830.94 

147 I 57 717.80 13.6 

TableS Percentage reducUon of growth rate at different time Intervals during the 

first limit test 

Concentration (mgn) Mean growth rate 

Nominai/lnlDal 11(0-24 hra)· Reduction (%) II (0-48 hl'8) RedUction (%) II (0-72 hrs) Reduction (%) 

Blank-<:ontrol 0.05643 0.05612 0.05011 

147 I 57 0.06148 -9.0 0.05259 6.3 0.04765 4.9 

7.3. Final limit test 

7.3.1. Measured test substance concentrations 

The results of analysis of the samples taken during the final limit test are described in Table 4 of. 

1he appended Ana!ytical Report. 

Analysis at the start of the test showed a mean measured concentration of 213 mgll in samples 

taken from nominally 257 mg/l. Hence, this time a much higher recovery was achieved in the 

samples taken at the start of exposure. The measured concentration decreased to 13 mgll after 

24 hours and to 6.3 mgll after 72 hours of exposure. The Time Weight Average (TWA) 

concentration was calculated to correspond to 24 mgJI. 

7.3.2. Mean cell denslUes 

Tabla 6 shows mean cell densities measured at 24-hour intelVals at the limit ooncentration of 

The respective growth ourves are shown in Figure 2 (see Appendix I for 

the cell densities per replicate). 

Table 6 Mean cell densities (x 1rt cellS/ml) during the final limit test 

Concentration (mgn) Exposure time (hours) 

Nominal/Initial 0 24 48 72 

Blank-control 1.0 3.1 11.3 33.4 

257 J 213 1.0 3.3 12.0 34.7 

• Page 16­
40 

35 .? 


Concentration 

nomInal (mlJll) 

~f' 

/ 
 -+-Blankcontrol

/ 
 ~/ 

5 ~ 

o ~ 

o 24 48 72 

Exposure time (hours) 

Figure 2 	 Growth curves at different concentrations of during 

the finalllmH test 

7.3.3. Inhibition of cell growth and reduC1lon of growth rate 

Tabla 7 shows the calculation of the percentages of inhibition and Table 8 shows the 

percentages of growth rate reduction at different time intervals (see Appendix I for the areas of 

oell growth and values of growth rate per replicate). 

No significant differences were recorded between the values for cell growth or growth rate 

between the test concentration and the blank.-control. 

Table 7 	 Percen1age inhibition of cell growth during the final limit test 

Concentration (mgll) Cell growth (0-72 hrs) 

Nominal/Initial Mean area (A) Inhibition (%) 

Blank-control 686.70 

257 , 213 722.50 -52 

Table 8 Percentage reduction of growth rate at different time Intervals during the 

final limit test 

Concentration (mgll) Mean growth rate 

Nominal/Initial p(D-24 hrs) Reduction (%) p (0-48 hrs) Reduction (%) p (0-72 hrs) Reduction (%) 

Blank·control 0.04742 0.05044 0.04869 

257 I 213 0.04935 -4.1 0.05170 -2.5 0.04925 -1.1 

- Page 17­
7.3.4. Oetennlnatlon of effect concentrations 

Table 9 shows the effect parameters based on nominal and Initial measured concentrations. 

Table 9 Effect parameters 

Parameter conc.nlndion lmalll 

Nominal I Initial 

NOEse 257 I 213 

72h-EeC50 >257 I >213 

NOE"C 257 1213 

72h-ERC50 >257 I >213 

7.3.5. Experimental conditions 

Table 10 shows the pH recorded at the beglnning and the end of the test. The pH was within the 

limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit) at the 

start of exposure, but exceeded the upper limit al the end of the test due to the fact that test 

vessels were airtight closed. The temperature of the test medium was 22.B"C at the start of the 

test. During the exposure period the temperature measured in the incubator was maintained 

between 22.1 and 23.6°C. Temperature remained within the limits prescribed by the protocol 

(21-25°C, constant within 2°C). 

Table 10 pH levels recorded during 1I1e final limit test 

Concentration (mg/l) Exposur. time (hours) 

Nominal/Initial 0 72 

Blank-control 8.4 9.8 

257 I 213 8.4 9.9 

8. CONCLUSION 

Under the conditions of the.present study with Selenastrum csprlcornutum, no Significant 

Inhibition of cell growth or reduction of growth rate was recorded at nominal 

concentrations up to 257 mgJI, corresponding to a measured initial concentration of 

213 mwl or a TWA concentration of 24 mgJI (NOEC). 

The EC50 for cell growth inhibition and growth rata reduction exceeded a measured initial 

concentration of 213 mg/I or a TWA concentration of 24 mg/I. 

-PagelS­
APPENDIX I WORKSHEET DATA 


Table 11 Individual cell densities (1111 limit test) 


Number of Inoculated cells aU..o: 1 x 10 cells/ml 

Concentration 

Nominal (mgll) 

Vessel 

number 

Exposure Ome (hours) 

0 24 48 72 

Blank-corrtrol 1 1.00 3.94 14.44 35.53 

2 1.00 3.71 15.32 38.22 

3 1.00 4.14 14.82 38.44 

4 1.00 3.79 1421 36.38 

5 1.00 3.72 15.00 36.81 

IS 1.00 3.96 14.98 38.03 

147 1 1.00 4.36 12.63 31.29 

2 1.00 6.45 13.35 31.95 

3 1.00 4.08 11.94 32.27 

4 1.00 3.93 12.18 29.69 

S 1.00 4.17 13.30 31.09 

6 1.00 3.72 11.60 29.19 

Table 12 Calculation of growth (area under growth curve) and growth rate (1l11Umlt 

test) 

Concenntfon V8&&81 lvea(A) Qrowth rate Qrowth Inhlb. (%) Growth rate Alductlon (%) 

number , 

Nominal (mgll) O·72hra 0-2.4 hra Q.48hrs G-72hrs o.72hrs 0-24 hra Q.48brs

Blank-oontrol 1 8IJ7.48 0.05713 0.0S5S3 0.04959 

2 856.36 0.05463 0.05686 0.05060 

3 856.32 0.05920 0.05617 0.05068 

4 806.56 0.05552 0.05529 0.04992 . Ii 831.00 0.05474 0.0600S 

6 826.92 0.05734 ~:~~e: 0.04978 

Mean 831 0.05643 0.05612 0.05011 

147 1 0.04782 13 -9 6 5 

2 :!~ ~O~~ 0.077 0.05399 0.04811 4 ·38 4 4

3 711.72 O. 0.05166 0.04825 14 ·4 8 4 

4 682.92 O. 0.05208 0.04709 18 ·1 7 6 

5 732.36 O. 0.05391 0.04773 12 ·5 4 5 

6 657.96 0.0547 0.05105 0.04686 21 3 9 6 

- Page 19­
APPENDIX I WORKSHEET DATA (Continued) 


Table 13 Individual cell densities (final limit test) 


Concentration v..... Exposure time (hours) 

number 

Nominal (mgll) 0 24 48 72 

Blank-oontrol , 1.00 3.47 11.43 34.62 

2 1.00 3.40 12.77 37.09 

3 1.00 2.91 12.00 34.32 

4 1.00 2.85 10.23 30.34 

5 1.00 3.09 10.89 32.13 

6 1.00 2.95 10.44 31.79 

257 1 1.00 3.25 11.85 35.46 

2 1.00 2.79 11.38 34.02 

3 1.00 3.51 11.44 34.33 

4 1.00 3.79 11.60 33.13 

5 1.00 3.20 12.21 34.87 

6 1.00 3.16 13.39 36.50 

Table 14 Calculation of growth <area under growth curve) and growth rate (final 

limit test) 

Concentration 

Nominal (mgll) 

Vessel 

number 

Area (A) Growth rats Growth Inhlb. 

(%) 

GrowU1 rate reduction (%) 

O-72nra 0-24hrs 0-48 hre 0-72 hra 0-72hrs Q..24hrs 0-48hrs O-72hrs 

Blank-control 1 713.04 0.05184 0.05076 0.04923 

2 m.16 0.05099 0.05306 0.06019 

3 709.68 0.04451 0.05177 0.04911 

4 620.40 0.04508 0.04844 0.04740 

5 661.08 0.04701 0.04975 0.04819 

6 642.84 0.04508 0.04887 0.04804 

Mean 687 0.04742 0.05044 0.04869 

257 1 7ZT.92 0.04911 0.05151 0.04956 -6 -4 -2 ·2 

2 688.32 0.04275 0.05068 0.04899 0 10 a -1 

3 710.76 0.05232 0.05077 0.04911 -4 -10 ·1 ., 

4 708.92 0.05552 0.05100 0.04862 -3 ·17 ·1 0 

5 725.88 0.04846 0.05213 0.04925 -6 -2 -3 -1 

6 775.20 0.04794 0.05405 0.04998 -'3 -1 ·7 -3 

- Page 20­
APPENDIX II REFERENCE TEST 

Selanastrum caprlcomutum. strain: NIVA CHL 1. Fresh water algal growth inhibition test with 

potassium dichromate 

Start of first exposure: 14 March 2005 

Completion last exposure: 17 March 2005 

The study procedures described in this report were based on the EEC Directive 92169, 

Publication No. l383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted 

June 7, 1984; and ISO Standard 8692, First edition, 15 November 1989. 

This reference test was carried out to check the sensitivity of the test system used by to 

Potassium dichromate (Merck, Art. 4864, Batch K28974764). 

Algae were exposed for a period of 72 hours to K2Cr2()., (Potassium dichromate) concentrations 

of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and to a blank-control. The initial cell density was 1.0 x 

104 cells/ml. 

Results: 

Overview of % inhibition in cell growth and % reduction of growth rate in the reference test: 

Concentration 

K2Cr207 

(mgll) 

Total cell growth: 

Mean Area Inhibition 

O-72h % 

Growth rate: 

Interval Reduction 

0-72h % 

Blank-control 1508.80 0.06087 

0.18 1404.52 6.9 0.05964 2.0 

0.32 1376.44 8.8 0.05923 2.7 

0.56 1374.08 8.9 0.05936 2.5 

1.0 1127.88 25.2 0.05575 8.4 

1.8 560.24 62.9 0.04218 30.7 

3.2 217.20 85.6 0.02301 62.2 

Under the conditions of the reference study with $alanastrum capricomutum, potassium 

dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 

1.0 mgll and higher. 

The EC60 for cell growth inhibition (EeC50: 0-72h) was 1.5 mg/l with a 95% confidence interval 

ranging from 1.0 to 2.2 mgll. The historical ranges of the 72h EC50 for growth inhibition lie 

between 0.49 and 1.4 mg/l. Hence, the ESC50: 0-72h for the present batch corresponds with this 

range when taking into consideration the confidence interval. 

The EC50 for growth rate reduction (ERCSO: 0-72h) was 2.6 mgll with a 95% confidence interval 

ranging from 2.0 to 3.2 mgll. The historica! ranges for growth rate reduction lie between 0.82 

and 2.3 mgll. Hence, the ERC50: Q-72h for the present batch corresponds with this range when 

taking into consideration the confidence intervaL 

The protocol, raw data and report of this study are kept in the archives. The test 

described above was performed under GLP conditions with a QA-check. 

- Page 21 ­
APPENDIX III 


FRESH WATER ALGAL GROWTH INHIBITION TEST WITH 


DETERMINATION OF THE CONCENTRA"nONS 


Study completion date 


October 28, 2005 


Laboratory Profect Identification 

- Page 1 of 15 ­
4 

1. CONTENTS 

1. CONTENTS ...........................................................................................................................2 


2. REPORT APPROVAL............................................................................................................ 3 


3. INTRODUCTION....................................................................................................................4 


3.1. Preface ................................... : ....................................................................................... 4 


3.2. Aim of the study ................................................
n ...............................................< ............ 


4. MATERIALS AND METHODS ...............................................................................................4 . 


4.1. Reagents ........................................................................................................................4 


4.2. Samples..........................................................................................................................4 


4.3. Analysis ..........................................................................................................................5 


4.3.1. Analytical method.................................................................................................5 


4.3.2. Calibration ............................................................................................................5 


4.3.3. Injections ..............................................................................................................5 


4.3.4. Data acquisition and data processing .................................................................. 6 


4.3.5. Procedural recovery samples ..............................................................................6 


4.4. Interpretation ..................................................................................................................6 


4.4.1. Calculations..........................................................................................................6 


4:4.2. Formulas ..............................................................................................................6 


5. RESULTS...............................................................................................................................8 


5.1. Calibration curves...........................................................................................................8 


5.2. Samples..........................................................................................................................8 


5.2.1. Procedural recovery samples ..............................................................................8 


5.2.2. Test samples........................................................................................................8 


TABLES 

Table 1 Procedural recovery samples.................................................................................... 9 


Table 2 Concentrations of in test medium (range-finding test) ......... 10 


Table 3 Concentrations of in test medium (first limit test) ................. 11 


Table 4 Concentrations of in test medium (final test} ........................ 12 


FIGURES 

Figure 1 GC chromatograms of a 60.0 mgll calibration solution [top; 


res.ld. 3833J and blank M2-medlum Ibottom; res.id. 4031]..................................... 13 


[top; res.id. 35191. 100 mgll [middle; res.id. 3528] and 259 mgll [bottom; res.ld. 


Figure 2 GC chromatograms of procedural recovery samples at concentrations of 10.1 mg/l 


3531]..................................................................... , ................................................. 13 


Figure 3 GC chromatograms of blank control samples taken at t=O hours ftop; res.ld. 3497]. a1 


Figure 4 GC chromatograms of nominal 257 mgn samples taken at t=24 hours [top; res.id. 


t=24 hours [middle: res.id. 4032] and at t=72 hours [bottom: res.id. 4034]............ 14 


4036] and at t=72 hours [bottom; res.id. 4040]....................................................... 14 


Figure 5 GC chromatograms of nominal 257 mg/l without algae samples taken at t=Q hours 


[top; res.id. 3513]. at t=24 hours [middle; res.ld. 4038] and att=72 hours [bottom; 


res.id. 4042]............................................................................................................. 15 


- Page 2­
2. REPORT APPROVAL 

PRINCIPAL SCIENTIST: 

(Analytical Chemistry) 

-Page3· 
3. INTRODUCTION 

3.1. Preface 

Study plan Start : 30 May 2005 

(analytical study) Completion : 20 September 2005 

3.2. Aim of the study 

The purpose of the analytical study was to determine the actual concentrations in samples taken 

from the test solutions used during the ecotoxlchy test. 

4. MATERIALS AND METHODS 

4.1. Reagents 

Methanol 	 HPLC-grade. labscan, DubUn, Ireland 

MiIIi-Qwater 	 Tap water purified by reversed osmosis and 

subsequently passed over activated carbon and ionexchange cartridges; Millipore Corp, Bedford, MA, USA 

ISO-medium 	 Medium formulated using Milli-Ro water (tap water 

purified by reverse osmosis; Millipore Corp .• Bedford, 

Mass., USA) with the following composition: 

CaCI2.2HtO 293.8 mgll 

MgSO..7H:!O 123.3 mgll 

NaHCO~ 64.8 mg/l 

KCI 5.8mg11 

M2-medium see main report 

Tapwater 

4.2. Sample. 

All samples not analysed on the sampling day were stored frozen. Stability of the samples under 

these conditions was determined during flDevelopment and validation of 

an analytical method for the analysis of In MIlIi-Q water". On the day of 

analysis, the frozen samples were defrosted at room temperature. 

The samples (10 ml) were analysed using headspace (see 4.3.1 analytical method). 

-Page4­
4.3. Analysis 

4.3.1. Analytical method 

Quantitative analysis was based on the test substance peak in the GC chromatogram of 

Analytical conditions 

Column 

Slationary phase CP Porabond Q 

Dimensions 25 m x 0.25 mm, d,= 3 j.lm 

Brand Varian, Mulgrave, VICtoria, Australia 

Temperature program 

Initial temperature 40 DC 

Initial time 1 min 

Rate 1 20 ·C/mln 

Temperature 1 190 ·C 

Rate 2 50 ·C/min 

Final temperature 250·C 

Hold time 3.3 min 

Carrier gas Helium 

Column flow 1 mVmin 

Injection Splitless 

Temperature 250 ·C 

Splitless time 2 minutes 

Injection volume 500 ~I (Headspace) 

Injection speed 50 !-II/sec 

Headspace conditions 

Sample 10 ml of an aqueous sample in a 20 ml vial 

Incubation temperature 50·C 

Incubation time 30 minutes 

Syringe temperature 110·C 

Detection FlO (250 ·C; Hydrogen: 40 ml/min; Air: 450 mllmin) 

4.3.2. Calibration 

During the range finding test, calibration solutions in ISO-medium were made up from two 

standard solutions of in methanol. 

During the first limit test, calibration solutions in M2-medium were made up from two standard • 

solutions of in methanol. 

During the final test, calibration solutions in tapwater were made up from two or three standard 

solutions of in methanol. 

The differences in matrix between calibration and ecotoxlcity samples was tested and had no 

influence on the quantilBtive results. 

- Page 5· 
4.3.3. Injections 

Calibration solutions were injected in duplicate. Procedural recovery samples and test samples 

were analysed by single Injection. 

4.3.4. Data acquisition and data processing 

Data acquisition and data processing was perfonned using Empower software (Waters. Milford. • 

MA. USA) version 5.00. 

4.3.5. Procedural recovery samples 

Standard solutions of in methanol and dilutions of these solutions with 

methanol were used as spiking solutions. 

During the range-finding test. blank test medium {10 m~ was spiked at concentration levels of 

0.101 mgll and 101 mgtli using 10.1 mg/l and 10085 mgll spiking solutions in methanol, 

respectively. 

During the first limit test, blank test medium (10 m~ was spiked at concentration levels of 0.101 

mgll and 101 mgll' using 10.1 mg/l and 10115 mgtl spiking solutions in methanol. respectively. 

At 0.101 mgll one and at 101 mg/l five samples were prepared and treated as described in 

paragraph 4.2 'Samples' and analysed. The recovery was calculated for each sample. 

During the final test, blank test medium (10 mil was spiked at concentration levels of 

approximately 10 mgll. 100 mgll and 260 mgJI using spiking solutions in methanol at 

concentration levels of approximately, 1000 mgll, 10000 mg/l and 26000 mgtl, respectively. 

4.4. Interpretation 

4.4.1. Calculations 

All calculations were performed using non-rounded values. Rounded values are reported in the 

tables. Therefore, some differences might be observed when calculating the parameters as 

mentioned in the tables. 

4.4.2. Formulas 

Genera! 

_ 1 n 

Mean: x=-~ Xi 

n 1-1 

where 

Xi = measured value 

n = number of measurements 

n 

L(Xi-x)2 

Standard deviation Sn-I= 11..:;;;"'... 1 __­

(n-I) 

Coefficient of variation 

, Concentrations were nol conected for spiking volume 

-page6­
Calibration 

Response: R = Peak area test substance [units] 

Calibration curve: The response was correlated with the test substance 

concentration, using linear regression analysis (least 

squares method; weighting factor (1/concentration2

)). 

R = response calibration solution [units] 

C = concentration of test substance in calibration 

solution [mgll) 

a = slope lunits*lImg] 

b = intercept [units] 

~amples 

Concentration of ASAHIKLlN AE-3OO0 analysed in the samples: 

C; (R-b}*d [mg"] 

a 

R ;;: response sample [units] 

d = dilution factor 

a = slope [units.VmgJ 

b = intercept [units] 

Recovery of procedural recovery samples: 

Concentration analysed .. 100 [%] 

Concentration prepared 

Concentration analysed Relative to nominal concentration: Concentration nominal • 100 [%] 

Concentration analysed at t =x hours .. 100 [%] Relative to initial concentration: Concentration analysed at t = 0 hours 

- Page 7· 
5. RESULTS 

5.1. Calibration curves 

On each day of analysis, a calibration curve was constructed using at least six concentration 

levels. At each concentration level, two responses were used. The coefficient of correlation was 

in excess of 0.99 for each calibration curve. 

Figure 1 shows chromatograms of a calibration solution and blank methanol. 

5.2. Sampies 

5.2.1. Procedural recovery samples 

Figure 2 shows chromatograms of procedural recovery samples. The results for the procedural 

recovery samples are summarized in Table 1. Mean recoveries of procedural recovery samples 

are required to be in the range 70-110%. 

(Mean) recoveries of the procedural recovery samples were between 86% and 113%. The reason 

for the relatively high recovery at a test substance level of 0.101 mgll determined on 16-06-05 

was unclear, but the result was considered acceptable. 

5.2.2. Testsamplas 

Figures 3 - 5 show chromatograms of blank control samples. nominal 257 mgll samples and 

nominal 257 mgl1 without algae samples from the final test The results for the test samples are 

summarized in Tables 2 - 4. 

During the range-finding test, the test substance concentration in the nominal 100 mgll test 

samples taken at t=24 and nominal 10 mg/l taken at t=72 were below the limit of quantification 

(lOQ) of the analytical method. 

During the first limit test, a small peak at the retention time of the test substance was observed in. 

the chromatograms of the blank test samples (nominal 0 mgll). Because a similar peak was found 

in blanks of methanol it was concluded that the compound did not derive from test medium. For 

the blank test samples, the response of the interference was below the limit of quantification 

(lOO) of the analytical method. For the nominal 147 mgll test samples with and without algae, 

the maximum contribution of the compound to the test sample with the smallest peak area was 

0.6%, and was therefore not considered to be a Significant interference. . 

During the final test. a small peak at the retention time of the test substance was observed. This 

peak was probably caused by a memory effect of the analytical system. For the test samples of 

nominal 257 mg/l samples with and without algae, the maximum contribution of the blank peak to 

the test sample with the smallest peak area was 2.2%, and was therefore not considered to be a 

significant interference. 

- Page 8­
TABLES 

Table 1 Procedural recovery samples 

Date of 

preparation 

[dd-mm-yy] 

Date of 

analysis 

[dd-mm-yy] 

Concentration 

nominal 

[mg/l] 

Concentration 

analysed 

[mgtn 

Recovery 

rib] 

Mean recovery 

(Coefficient of 

variation) 

r%1 

30-OS-05 30-05-05 0.101 0.103 102 

30-05-05 30-05-05 101 76.3 76 

101 75.0 74 86 

101 101 100 (13) 

101 96.1 95 

101 87.5 87 

16-06-05 16-06-05 0.101 0.115 113 

16-06-05 16-06-05 101 102 101 

101 99.5 98 100 

101 101 100 (1.0) 

101 101 99 

101 101 100 

13-09--05 13-09-05 10.1 11.0 109 107 

10.1 10.6 106 (2.0) 

10.1 10.6 105 

13·09-05 13-09...(15 100 103 103 106 

100 108 108 (2·n 

100 106 106 

13-09-05 13-09-05 259 261 108 

259 287 111 108 

259 281 108 (1.6) 

259 275 106 

259 280 108 

19-09-05 19-09-05 10.0 9.09 91 89 

10.0 9.04 90 (3.2) 

10.0 8.57 86 

19-09-05 19-09-05 100 78.2 78 76 

100 77.7 78 (5.6) 

100 70.7 71 

19-09-05 19-09-05 260 221 85 82 

260 206 79 (3.8) 

260 217 83 

. 


- Page 9 ­
Table 2 Coneentrations of In test medium (range-finding test) 

Time of 

sampling 

[hours) 

Date of 

sampling 

Idd-mm-yy] 

Date of 

analysis 

Idd-mm-yy] 1 

~. 

Nominal 

[mgll} 

Concentration 

Relative to Analysed nominal [mgll] [%] Relative to 

mean initial 

[0.41 

0 23-05~05 30-05-05 10 1.03 10 

10 1.02 10 

100 14.3 14 

100 14.9 15 

24 24-05-05 30-05-05 10 0.132 1 13 

10 0.132 1 13 

100 2.18 2 15 

100 <LOa n.a. n.a. . 72 26-05-05 30-05-05 10 <Loa n.a. n.a. 

10 0.255 3 25 I 100 1.n 2 12 

100 1.76 2 12 

I 

Samples were fro;ten until analysis. 

lOQ LImH of quantification was 0.100 mgn . "Dewlopmenland validation of an analytical 

method for the analysis of In MilII..Q water). 

n.a. Not applicable 

- Page 10­
Table 3 Concentrations of In t8St medium (first limit test) 

I Timeof 

. sampling 

[hours) 

Date of 

sampling 

[dd-mm-yy] 

Date of 

analysis 

[dd-mm-yy] 

Concentration 

Nominal 

[mgll] 

Analysed 

[mgll] 

Relative to 

nomina! 

[%] 

Relative to 

mean initial 

[%] 

0 13-06-05 16-06-05 1 0 <LOO 3 

0 <LOO3 

147 56.9 39 

147 57.9 39 

1472 n.i 

1472 56.9 39 

24 14-06-05 16-06-05 1 0 <LOa ~ 

0 <Loa 3 

147 6.11 4 11 

147 6.47 4 11 

1472 6.86 5 12 

1472 6.52 4 12 

72 16-06-05 16-06-05 0 <Loo3 

0 <LOQ' 

147 

147 

2.10 

2.04 

1 

1 

4 

4 . 1472 2.36 2 4 

1472 2.29 2 4 

Samples were frozen until analy&ls. 

Wrthoul algae. 

LOQ' A small peak was observed at the retention tine of test substance, but its respOJ'\M was not coosldeted to derIVe 

from test aample since It was also observed in blank methanol. Umit ofquantlfteatlon was 0.100 mgll (See 

"Development and validation of an analytical method for the analysis of 

In MiIIl-Q water"). 

n.l. NoIlnJected, due to a system error. 

- Page 11 ­
Table 4 Concentrations of in test medium (final test) 

Time of 

samp~ng 

[hours] 

Date of 

sampHng 

[dd-mm-yy] 

Date of 

analysis 

[dd-mm-yy] 

Concentration 

Nominal 

[mg/l] 

Analysed 

[mgll] 

Relative to 

nominal 

[%] 

Relative to 

mean initial 

[%1 

0 13-09-05 13-09-05 0 n.d. n.a. 

0 n.d. n.a. 

257 3 212 82 

257 3 214 83 

24 14-09-05 19-09-05 1 0 2 n.a. n.a. 

0 2 n.a. n.a. 

257 12.6 5 6 

257 13.3 5 6 

257 3 14.6 6 7 

257 3 13.4 5 6 

72 16-09-05 19-09-05 1 0 2 

n.a. n.a. 

0 2 

n.a. n.a. 

257 4.99 2 2 

257 7.61 3 4 

257 3 6.57 3 3 

257 3 5.83 2 3 

Samples were frozen until analysis. 

A smal peak was observed In these nominal 0 mgtl &les. this Wla probably II memory effect of the analytical 

system. The maximum contribution oflhls peak 10 the samples was 2.2% and this was not con.sldered to be a 

significan1interference. 

Without algae. 

n.d. Not detected 

n.a. Not applicable 

- Page 12­
FIGURES 

-

_ .... 
 _.. 


,'..... 

_.. 

..... 

'-

.... 

...... 

..­

,,--

.­

-.... .... ... ... ..... ..... -


Figure 1 GC chromatograms of a 80.0 mgn calibration solution 

[top: res.id. 3833] and blank M2-medium [bottom; res.ld.4031]. 

,~ 

,~ -.. 

'I.-" _... 

•... .... ,.. ,. - .... II." oj• 

­ Figure 2 GC chromatograms of procedural recovery samples at concentrations of 

10.1 man [top; res.ld. 3519], 100 mg/l [middle; res.id. 3528] and 259 mglJ 

[bottomj res.ld. 3531]. 

- Page 13­
.... 

.... 

t ".. 

.... 

.. 

... 

... 

,... t 

.....

,­

, 

-..... 

..... t 

..... 

..... 

.o. 

-

" ... ... 

J v 

--- II 

! ~ 

= 

Uo .... '.., 'lilt' , ..... T... • • flO ... ... .... .... FIgure 3 	 GC cbromatograms of blank control samples taken at tacO hours 

[top; res.ld. 349'1]. at t=24 hou... [middle; res.ld. 4032] and at t-72 hours 

[bottom; res.id. 40341. 

_.. 


-


,,"'''' 

-


"'. 

.... 

,..... 

.... 

"..... 

-

.­

.... ,. !---o~~~.r·~. ,.. 2...,--'~.~... .:.~'~. ... 	 ,-'~.~'-r-r.:.~~,---.-.~'~'-rl'J.a~'~....,.:."--;:;:""""~~"""",~,,,,, •••~,,,,,,,,,,,,,,,,~.......-l ....

...­

Figure 4 	 GC chromatograms of nominal 257 mgll samples taken at1-24 hours [top; 

res.ld. 4036] and at t-72 hours [bottomj res.ld. 4040]. 

- Page 14­
-


_. 

-~ 

_.. 

-..• 

_. 


_. 


_. 

....... 


..... 

'0. ,... 	 d. >lot ... .. obo .- ... ... .... ..... .... .... -


Figure 5 	 GC chromatograms of nominal 267 mg/I without algae samples taken at 

t=O hours [toPi res.ld. 3513], at t-24 hours [middle; res.ld. 4038] and at 

t-72 hours [bottom: res.ld.4042]. 

- Page 15­
P-04-635 


REPORT 


Study Title 

96-HOUR ACUTE TOXICITY STUDY IN CARP WITH 

(FLOW-THROUGH) 

Study completion date 

28 October 2005 

}i!st FacilHy 

Laboratory PrQject Identification 

. Pagel of 35 . 
1. CONTENTS 

1. CONTENTS ...................... ' ..................................................................................................... 2 


2. STATEMENT OF GLP COMPLIANCE ..................................................................................4 


3. QUALITY ASSURANCE STATEMENT .................................................................................5 


4. SUMMARy.............................................................................................................................6 


5. INTRODUCTION....................................................................................................................7 


5.1. Preface ...........................................................................................................................7 


5.2. Aim of the study..............................................................................................................7 


5.3. Guidelines.......................................................................................................................7 


5.4. Storage and retention of records and materials ............................................................. 7 


5.5. Definitions .......................................................................................................................a 


6. MATERIALS AND METHODS ...............................................................................................a 


6.1.1. Test substance .....................................................................................................8 


6.1.2. Reference substance ...........................................................................................9 


6.1.3. Preliminary data ...................................................................................................9 


6.2. Test System ....................................................................................................................9 


6.3. Holding ....................................................... : ................................................................... 9 


6.4. Range-finding test (static) ............................................................................................ 10 


6.5. Rrst and second limit flow-through test.. ...................................................................... 10 


6.5.1. Conoentrations ...................................................................................................10 


6.52. Dosing system .................................................................................................... 10 


6.5.3. Test procedure and conditions ........................................................................... 12 


6.5.4. Sampling for analysis of test concentrations ..................................................... 12 


6.5.5. Measurements and recordings ..........................................................................12 


6.6. Interpretation ...... , ......................................................................................................... 13 


6.6.1. Data handling .....................................................................................................13 


6.62. Acceptability of the test ...................................................................................... 13 


6.7. List of protocol deviations .............................................................................................13 


7. RESULTS.............................................................................................................................14 


7.1. Static range-finding test ................................................................................................ 14 


7.2. First flow-through test ................................................................................................... 14 


7.2.1. Measured ooncentrations ................................................................................... 14 


7.22. Mortality and other effects .................................................................................. 15 


7.2.3. Experimental conditions ..................................................................................... 15 


7.2.4. Discussion on results 1

st limit tesL ..................................................................... 15 


7.3. Second flow-through te8t.............................................................................................. 15 


7.3.1. Measured concentration8 ................................................................................... 15 


7.3.2. Mortality and other effects.................................................................................. '0. 


7.3.3. Experimental conditions ..................................................................................... 16 


7.3.4. Determination of effect concentrations .............................................................. 16 


8. DISCUSSION....................................................................................................................... '7 


9. CONCLUSION ..................................................................................................................... '7 


TABLES 


Table 1 DOSing program 1

81 limit test .............................................................................. 11 


Table 2 Dosing program limit test .............................................................................. 11 


Table 3 Incidence of mortality and total mortality during the range-finding test ............. 14 


Table 4 Mean measured collC9ntrations versus nominal concentration (1 S1 limit test) ... 14 


TableS Incidence of mortality and total mortality during the 1st limit test ....................... 15 


Table 6 Mean measured concentrations versus nominal concentration (2'" limit 


test) .................................................................................................................... 16 


Table 7 Incidence of mortality and total mortality during the 2nd limit test ...................... , 6 


Table 8 Effect parameters ............................................................................................... 16 


Table 9 pH-values and ,dissolved oxygen ooncentrations (mgll) during the 111 limit 


te6t...................................................................................................................... 18 


Table 10 Temperatures (OC) measured during the 1" limit test.. ...................................... 18 


Table 11 pH-values and dissolved oxygen concentrations (mgll) during the 2nd test....... 18 


Table 12 Temperatures (OC) measured during the 2nd test............................................... 18 


- Page 2· 
FIGURES 


Figure 1 Flow-through system, zt4l1mlt test..................................................................... 11 


APPENDICES 


APPENDIX I TEST CONDITIONS .......................................................................................... 18 


APPENDIX II REFERENCE TEST 

APPENDIX IV ANALYTICAL REPORT 


........................................................................................... 19 


APPENDIX III ANALYSES OF TAP WATER (Not performed under GLP) ............................... 20 


·Page3­
2. STATEMENT OF GLP COMPLIANCE 

The study described in this report has been correctly reported and was conducted In compliance 

with: 


The Organization for Economic Cooperation and Development (OECD) Good Laboratory 


Practice Guidelines (1997). 


Which essentially conform to: 


The United States Food and Drug Administration Good Laboratory Practice Regulations. 


The United States Environmental Protection Agency Good Laboratory Practice Regulations. 


The sponsor is responsible for Good laboratory Practice (GlP) compliance for all test 


substance Information unless determIned by 


Tap water analyses were not performed under GlP and therefore excluded from this GLP 


statement. 


Study Director Head of In Vitro & Environmental Toxicology 

~Dat9: .~s..I ..Od-.Q~.e..f..~.s:-.......... 
 Date: .......~¢'.ap..Q~............... ... . 


·Page4· 
3. QUALITY ASSURANCE STATEMENT 

This report was inspected by the Quality Assurance Unit to confirm that the methods 

and results accurately and completely reflect the raw data. 

The dates of Quality Assurance inspections are given below. 


During the on-site process Inspections procedures applicable to this type of study were 


Inspected. 

Type of 

Inspections 

Study 

Process 

PhaseJProcess 

Protocol 

Ameminent 1 of protocol 

Ameminent 2 of protocol 

Amendment 3 of protocol 

Amendment 4 of protocol 

Amendment 5 of protocol 

Amendment 6 of protocol 

Amendment 7 of protocol 

Amendment aof protocol 

Report 

Environmental toxicology 

Test substance handling 

Exposure 

ObservationsIMeasurements 

Analytical and physical 

chemistry 

Test substanqe handling 

Observations/Measurements 

Start 

Inspection 

date 

Q3-Sep-04 

Oa-May-05 

22-Jun-05 

30.Jun-05 

22-Jut-os 

08-Sep-OS 

3O-Sep-OS 

07-Oct-OS 

2a-Oct-OS 

28-Oct-oS 

08-Aug-OS 

20.Jun-05 

2D-Sep-05 

End 

Inspection 

date 

03-Sep-04 

03-May-05 

22-Jun-05 

3Q-Jun-05 

22-Jul-05 

OS-Sep-oS 

3O-Sep-05 

07-0ot-OS 

28-0c1-DS 

28-0ot-05 

19-Aug-05 

29.Jun-05 

26-Sep-OS 

Reporting· 

date 

03-Sep-04 

03-May-05 

22-Jun-oS 

30-Jun-05 

22.Jul-05 

OS-Sep-OS 

3O-Sep-05 

07-00t-05 

28-0c!-OS 

2a.oct-05 

19-Aug-05 

05.JuJ-05 

Z7-Sep-OS 

Head of Quality Assurance 

-Pages­
4. SUMMARY 

96-Hour Acute Toxicity Study in Carp with 

The study procedure described in this report was based on the ISO International Standard 7346:3: 

Flow-1hrough method. 1996. In addition. the procedures were designed to meet the test methods and 

validity cnterla of the EEC directive 92/69, Part C.1, 1992 and the DECO guideline No. 203, 1992. 

The batch of tested was a clear and colourless Ilqu1d with a purity of 

99.985%. was completely soluble in test medium at the concentrations 

tested and known to be extremely volatile. 

The project started with a static range-finding test exposing three carp per concentratIon to 

nominal • concentrations of 0.1, 1.0, 10 and 100 mgll. No mortality or clinIcal 

effects were observed at any of the test concentrations during the test period. Analytical results of 

samples taken from 100 mgll showed that the mean measured concentrations were 

. approximately 5 mg/l during the first 24 hours of exposure and 1 mgll at the end of the test period. 

Based on these low recoveries It was decided to continue testing applying a flow-through system. 

A limit test was performed in an airtight closed flow-through system with continuous renewal of 

test solutions. A volume of 150,pl pure test substance was closed every 5 minutes and mixed 

with tap water at a flow rate of 12 LIh. This flow resulted in 8 complete exchanges per day. 

Seven fish per concentration were exposed to a blank..control and a nominal concentration of 

221 mg/1 for a maximum of 96 hours. 

Analyses of samples taken one day before the start of the test showed a mean measured 

concentration of 84 mg/l, indicating that the system functioned sufficiently in reaching an actual 

concentration close to the target of 100 mgll. The mean measured exposure concentration 

during the 96-hour test period corresponded to 73 mgll. As this concentration was more than 

20% below the regulatory limit concentration of 100 mg/l and fish were significantly affected In 

their swimming behaviour at a mean concentration of 73 mgll, it was decided to continue with an 

additional limit test with improved mixing and increased renewal rate. 

The second limit test was also perfonned in an airtight closed flow-through system with 

continuous renewal of test solutions. A volume of 176 pi pure test substance was dosed every 5 

minutes applying two diluters and mixed with tap water at an increased flow rate of 14 LJh and 

smaller test vessels. This resulted in 34 complete exchanges per day. Seven fish per 

concentration were exposed to a blank..control and a nominal concentration of 221 mg/l for a 

maximum of 96 hours. Two fish died during the test period, while the surviving fish were 

discoloured and showed loss of righting reflex. The mean measured concentration was 76 mgll, 

which was only slightly higher than the mean concentration measured in the first study despite 

the improvements made to the test system. 

Other attempts were made to reach a higher exposure concentration (~1 00 mg/l) in test medium. 

ThIs Included a further increase of the flow rate, change of the nominal concentrations and even 

included extra mixing vessels. All of these did not result in measured concentrations that were 

higher than the concentratIons measured in the first two limit studies. The maximum achieved 

concentration again approximated 70 mg/l. On average. a recovery of 30% was found indicating a 

loss of 70%. In conclusion: with the present extremely volalDe substance it was not possible to 

test flsh In a stable flow-through test system at a concentration of at least 100 mg/l. 

The study met the acceptability criteria prescribed by the protocol and was considered valid. 

Under the flow-through conditions of the presenl test Induced less than 

50% mortality In carp at a mean measured test concentration of 76 mg/l, the highest 

concentration that could be achieved and maintained stable In tap water. Based on the 

observations of preCipitate in the form of droplets in both limit tests, it cannot be excluded that 

fish were, at least occaSionally. exposed to pure lest substance. In conclusion, the 96h-LCso 

exceeded a mean measured concentration of 76 mg/l. 

-Page6­
5. INTRODUCTION 

. 5.1 . Preface 

Sponsor 

Study Monitor 

Test Facility 

Study Director 

Technical Coordinator 

Principal Sclentlst 

Study Plan Start exposure 23 May 2005 

Completion exposure 17 September 2005 

Completion of the 

project 05 October 2005 

5.2. Aim of the study 

The purpose of the study was to evaluate the test substance for Its ability to generate acute 

toxic effects in Cyprinus carpio during an exposure period of 96 hours and, if posSible, to 

determine the LCso at ali observation times. 

5.3. GuIdelines 

The study procedure described in this report was based on the ISO International Standard 

7346-3: 'Water quality - Determination of the acute lethal toxicity of substances to a freshwater 

fish [Brachydsnfo ferlo Hamilton-Buchanan (Teleostel, Cyprlnldae)r - Part 3: Flow-through 

method, Second edition, 1996-06-15. 

In addition, the procedure is designed to meet the test methods and validity criteria prescribed 

by the following guidelines: 

• 	 European Economic Community (EEC), EEC directive 92169. Part C: Methods for the 

determination of ecotoxicity, Publication No. L383, December 1992, C.1. ·Acute toxicity • 

for fish". 

• 	 The OECD guidelines for Testing of Chemicals, guideline No. 203: "Rsh Acute ToxicHy 

Test", Adopted 17 July, 1992. 

5.4. Storage and retemlon of records and materials 

Records and materials pertaining to the study including protocol. raw data, specimens (except 

specimens requiring refrigeration or freezing) and the final report are retained in the 

archives for a period of at least 10 years after finalization of the report. After this period, the 

sponsor will be contacted to determine whether raw data and specimens should be returned to 

them, retained or destroyed on their behaH. 

Those specimens requiring refrigeration or freezing will be retained by for as long as 

the quality of the specimens permits evaluation but no longer than three months after finalization 

of the report. 

- Page 7 ­
will retain a test substance sample until the expiry date, but no longer than 10 years 

after finalization of the report. After this period the sample will be destroyed. 

5.5. Definitions· 

Fish were considered to be dead when no reaction was obserVed after touching the caudal 

peduncle and visible breathing movements were absent. In acklition. fish that were convulsing or 

showing other severe forms of distress not considered transient in nature and likely to become 

more severe before the exposure is terminated. will be sacrificed for humane reasons. These 

fish will be treated as having died In the test. based on OECD guldellne on humane endpoint 

(ENVlJMIMONO/(2000)7). 

The LCso is the concentration killing 50% of the fish after a defined period of exposure. 

A Flow-through system Is a system where test substance is dosed at fixed time points and 

mixed with a continuous flow of dilution water to provide continuous renewal of the test solutions. 

6. MATERIALS AND METHODS 

6.1.1. Test subslance 

Limit test 1 and 2 with fish; 

Identification 

Structure 

Molecular formula 

Molecular weight 

CAS Number 

Descriptlon Clear colourless liquid 

Batch 

Purity 99.985% 

Test substance storage In refrigerator In the dark 

Stability under storage conditions Stable 

expiry date 28 February 2006 

Specific Gravity 1.47 

Stability in water . Not indicated 

Extra attempts to reach higher exposure concentrations following the limit tes1s with fish: 

Identification 

Structure 

Molecular formula 

Molecular weight 

CAS Number 

Description Clear colourless liquid 

Batch 

Purity 99.989% 

Test substance storage In refrigerator (2-8·C) In the dark 

Stability under storage conditions Not indicated 

Expiry date 26 September 2006 (allocated by 1 year after • 

recerpt of the test substance) 

- PageS­
6.1.2. Reference substance 

This report includes the results'of a referen<:e test with pentachlorophenol (PCP) (Appendix I). 

6.1.3. Preliminary data 

The water solubility of 	 at 20.1 ± OSC is 0.805 gil using the flask method 

Further essential information obtained from physlco-chemical studies 

performed at were the vapour pressure being 31 kPa at 25"C, freezing point <-81 "C, 

boiling pOint 56"Cand a log ~of 2.54. The Log Pow of was 2.18 

according to infonnation supplied by the sponsor. From these data. it can be concluded that the 

test substance will evaporate rapidly at room temperature as pure substance but also from 

aQueous solutions (calculated Henry coefficient is 7.24.1 as Fa m3 mor'). 

concentrations showed to be stable during freezing and thawing of 

samples In a deep-freeze 

6.2. Test System 

Species 	 Carp (Cyprfnus carpio, Teleostel. Cyprinidae) 

Linnaeus, 1758. 

Source 

Mean length1 	 Range.finding test: 2.3 ± 0.1 cm 

First limit test 2.6 ± 0.2 cm 

Second limit test: 2.2 ± 0.1 em 

Mean weight' 	 Range-finding test: 0.33 ± 0.08 9 

First limit test: 0.56 :t: 0.14 9 

Second limit test: 0.29 ± 0.08 9 

Characteristics 	 F1 from a single parent-pair bred in UV-treated water. 

Reason for selection 	 This system has been selected as an internationally 

accepted species. 

Total fish used 	 40 

6.3. Holding 

Quarantine! Aoclimatisation 	 At least 12 days aftar delivery. 

Medium 	 ISO·medium, formulated using Mllli·RO water (tapwater purified by reverse osmosis; Mlllipore Corp., 

Bedford, Mass., USA) with the following composition: 

C8C1;.2H;O 293.8 mWI 

MgSO•.7HzO 123.3 IYI9'I 

NaHCo. 64.8 mo'I 

KCI 5.8 mgII 

Measurements pH, nitrate and nitrite concentraUon and ammonia 

concentration: once a week. Temperature: avery day. 

Feeding Daily with Nutra or Artemia. 

1 Ten fish of the batch used for Ihe lest, were weighed and measured prior to the start of the test. 

- Page 9­
Acclimatisation Following a 48-hour settling-in period the fish were 

allowed to acclimatise to the test medium (tap-water) 

wfthout test substance for at least seven days. 

Validity of batch In the batch of fish used for the test, mortality during the 

seven days prior to the start of the test was less than 5%. 

6.4. Range-finding test (staUc) 

A static range-finding test was performed to provide infoll11ation about the range of 

oonoentrati0n8 to be used In the final test. Test procedure and conditions were similar to those 

applied in the limit testa wHh the following exceptions: a static test system was used in which 

three carp per concentration were exposed to nominal concentrations of 0.1 

to 100 mgllincreasing by a facfOr of 10. Dissolved oxygen concentrations, temperature and pH 

were only measured in the lowest and highest test concentration. Duplicate samples for possible 

analyses Were taken from all test solutions at the start, after 24 and 96 hours of exposure. 

Preparation for the range-finding test started with tast solutions prepared at 10 and 1 00 mg/l 

applying 1 and 13 minutes of magnetic stirring. respectively. Care was taken that during 

preparation as little as possible volatilisation occurred (weighing and preparation in clOsed 

vessels which already contained a sman volume of test medium. short preparation period). The 

lower test concentrations were prepared by dilution of the test solution of 10 mgll. The final test • 

solu1ions were all clear and colourless. 

Part of the test solutions was used for the simultaneously performed acute toxicity test with 

Daphnia magna 

8.5. First and second limit flow-through test 

6.5.1. Concentrations 

Nominal 221 mg/!, expected to coJTespond to an 

actual concentration close to 100 mgn. 

Controls 	 Test medium without test substance or other additives 

(blank-control). 

6.5.2. Dosing system 

First limit test; 

Approximately 75 ml 01 pure test substanoe was collected daily in an airtight sealed vial and 

used as 'stock' solution for dosing. The pure test substance was dosed via a computercontrolled system in combination with a dispenser (Gnson). VIa this system the dosed volumes 

entered a mixing flask separately from the tap water supply. The tap water was supplied via a 

flow meter with a flow rate of ca. 121111. In the mixing flask the dosed volumes and the tap water 

were mixed under continuous stirrinQ. The test solution coming from the mixing flask entered • 

first a vessel containing Daphnia and then a vessel containing the 

fish. The whole system was checked daily. The dosing program Is described In Table 1. 

The dosed volume of 1.8 ml per hour in combination with a tap-water flow of 12 I!tres per hour 

results in a nominal concentration of 221 mg/!. However, considering 1he rapid decrease of 

concentration in water due to the high volatility and the results obtained In a previously 

performed study with algae it was expected that this dosi1g regime 

would give an actual concentration close to 100 mgll. 

-Page 10­
Table 1 Dosing program 1st limit test 

Stock 

solution 

Dosing volumes stock 

(0.5 ml syringe) 

Tap water Nominal 

concentration 

Expeo1ed 

concentration 

Per cycle 

pL/Smin mUhour Llhour (mgIL) (mglL) 

Pure 150 1.8 12 221 100 

§econd limit testj 

Approximately 70 ml of pure test substance was collecred daily and dMded over two airtight 

sealed vials and used as 'stock' solution for dosing. The pure test substance was dosed via a 

computer-controlled system in combination with two dispensers (Gilson). Via this system the 

dosed volumes entered one mixing flask separately from the tap water supply. The tap water 

(14 I/h) was supplied via two flow meters, each with a flow rate of ca. 7 1Ih. In the mixing flask 

the dosed volumes and the tap water were mixed under continuous stirring. The whole system 

was checked dally. The dosing program Is described In Table 2 and a diagram of the system is 

presented in FlQure 1. 

Tabfe2 Dosing program 2nd limit test 

stock 

solution 

Pure 

Pure 

Dosing volumes stock 

(0.5 ml syringe) 

Percycl8 , 

pL/5 min mUhour 

88 

88 

Mixing Flow 

Yassel meter 

1.056 

1.058 

Tap water 

Llhour 

7 

7 

Nomina. Expected 

concentration concentraUon 

(mgIL) (mgIL) 

221 100 

Tes! vessel 

alrtlghC closed 

Figure 1 Flow-through system, 2nd limit test 

- Page 11 • 
8.s.3. Test procedure and conditions 

Test duration 96 hours 

Test type Flow-through, continuous renewal of test media. 

Test vessels 181 limit test: 35 litres; 2nd limit test: 10 Iltres. Both 

consisting of glass plates sealed with a thin fjlm of 

silicone and covered airtight by a removable Perspex 

plate. 

Exchange rates 1at limit test: 8 times per day. 

2nd limit test 34 times per day. 

Test medium Tap water (data of analysis are attached). 

Number of fish 7 fish per concentration and control. 

Loading 181 limit test ca. 0.014 g fishllitre/day. 

2nd limit test: ca. 0.006 9 flsMltreiday. 

Illumination 16 hours photoperiod daily. 

Aeration The test media were not aerated during the test. 

Feeding No feeding from 48 hours prior to the test and during 

the tolal test period. 

Introduction of fish 1 day (2"" limit) or 2 days (1 81 limit) after the stan of 

the dOSing system to allow stabilisation of the test 

concentrations. 

Euthanasia At the end of the test the surviving fish were rapidly 

killed by exposing them to ca. 1.2% ethylene glycol 

monophenylether in water. 

6.5.4. Sampling for Malysis of test concen'b'atlons 

During the limit tests duplicate samples for analysis were taken according to the schedule 

below. The method of analysis is described in the appended Analytical Aeport (Appendix IV). 

Sampling: Frequency 	 181 limit test: ca. 26 hours following the start of dosing, 

samples were taken from the 221 mgll-limit 

concentration to check the actual exposure 

concentration before the start of exposure. 

1st and 2nd limit test: at the start, after 48 hours and at 

the end of the 96-hour test period samples were taken 

from the blank-control and 221 mgll. 

Volume 	 10 ml from the approximate centre of the test vessel. 

Storage 	 Samples taken after 48 and 96 hours of exposure 

were stored In a deep-freeze. 

6.5.5. Measurements and recordings 

Mortality and other effects 	 At 2y'!-3, 24, 48, 72 and 96 hours following the start of 

exposure and every momlng from day 1 to observed 

for any dead fish. 

- Page 12­
Dissolved oxygen content, Daily in all vessels, beginning at the start of the test 

pH and temperature (day 0). 

8.6. Interpretation 

6.6.1. Data handling 

Calculation 01 LCeo: 

No LCso could be calculated because the test substance proved to be non-toxic (lCso > 

maximum concentration tested). 

Defining exposure concentrations: 

The results were based on the mean 01 the concentrations measured in samples taken at the 

V81lous tIme Intervals. 

8.6.2. Acceptability of the test 

1. No mortality or abnonnal behaving fish were observed in the control group. 

2. 	The oxygen concentration W!1S maintained at at least 60% 01 the air saturation value 

throughout the test (> 5 mgll at 22°C). Other test conditions (PH and temperature) were 

maintained within the limits prescribed by the guidelines. 

3. 	The analytical program provtded evidence that the actual test concentrations were generally 

maintained within ;l; 20% during the test period. 

4. 	The 96h-LCso of the reference chemical for .the stock of fish was in agreement with results 

obtained previously in our laboratory. 

6.7. Ust of protocol devlatlorts 

There were no deviations from the protocol. 

- Page 13· 
7. RESULTS 

7.1. Static range-finding test 

The results of the range-finding test are presented in Table 3. No mortality or clinical effects 

were observed at any of the t.concentrations during the test period. Based on these results 

samples taken from nominal 10 and 100 mgll were analysed. Analytical results of samples 

taken from 100 mg/Ishowed that the mean maasured concentrations were approximately 5 mgll 

during the first 24 hours of exposure and 1 mgJI at the end of the test period. The same low 

percentages of recovery were observed in samples taken from 10 mgll (see Table 2 of the 

appended Analytical report). Hence, with the test procedure applied actual exposure 

concentrations were far below nominal and they decreased significantly during the test period. 

To enable testing actual concentrations close to nominal and to maintain these stable Hwas 

decided to continue testing applying a flow-through system. 

All test conditions were maintained within the limits prescribed by the protocol. 

Table 3 Incidence of mortality and total mortality during the range-flndlng test 

Concentration Initial Cumulative mortality Total 

Mor1allty 

Nominal <rnsan) (%)

number 

offish 3,.h 24h 48h 72h 9611 

0.1 3 0 0 0 0 0 0 

1.0 3 0 0 0 0 0 0 

10 (0.2-<0.1) 3 0 0 0 0 0 0 

100 (5 ·1) 3 0 0 0 0 0 0 

BelWeen brack&ts: I'Bnge of actual m&l!IsunKi concentrations 

7.2. First flow-through test 

7.2.1. Measured concentratIOns 

The results of analysis of the samples taken during the first limit test are described In Table 3 of 

the appended Analytical Report. 

Analyses of the samples taken one day before the start of the test showed a mean measured 

concentration of 84 mgll, which Indicated that the system functioned as expected and that the 

actual concentration was close to the target of 100 mgll. 

The measured exposure concentrations in each of the duplicate samples taken at the various 

sampling times are shown in Table 4. The difference In duplicate values might be explained by 

variations In amounts of loss during sampling and the limited solubility in tap water. All results 

were used to determine the mean exposure concentration during the 96-hour test period, which 

corresponded to 73 mgII. 

Table 4 Mean measured concentrations versus nominal concentration (1 at limit 

test) 

NomInal concentration Measured Measured 

t=48h 

Measured 

t=96h 

Mean 

t=Oh measured

- Page 14­
7.2.2. Mortality and other effects 

Table 5 shows the mortality data recorded during the first flow-through test. No mortality was 

observed at the limit concentration tested. However. fish were affected in their swimming 

behaviour and appearance. Fish were observed to be discoloured during the entire test period. 

Further. all fish were observed swimming disorientated from 24 hours following exposure and 

droplets of test substance were observed on the bottom of the test tank. At the end of the test 

period only one fish was observed swimming disorientated. Hence. this effect diminished during 

the course of the test. 

TableS Incidence of mortality and total mortality during the 1-limn test 

COncentration Cumull1tlve mortality 

24h 72h 96h 

o o o 

o o o 

7.2.3. experimental conditions 

The results of measurement of pH. oxygen concentrations and temperature are presented in 

Appendix I. All test conditions remained within the ranges prescribed by the protocol (pH: 6.0­

B.S. constant within 1 unit; temperature 20-24"C, constant within 2°C; oxygen> 60% of air 


saturation value). 


7.2.4. Discussion on results,- limit test 

Based on the results obtained In the first limit test It was decided to continue with the project. as 

the mean measured concentration of 73 mgll was more than 20% below the regulatory limit 

concentration of 100 mgII. This decision was strengthened by the fact that significant effects on 

swimming behaviour were observed at 73 mgll and the water solubility was expected to exceed 

100mg./!. The rate of renewal of test solutions was Increased by a factor of four in the second 

test by decreasing the volume of the test tank from 35 to 10 Iltres and increasing the tap water 

flow rate from 12 to 14 Llhour. Hence, test solutions were renewed within one hour. 

7.3. Second flow-through test 

7.3.1. Measured concentrations 

The results of analysis of the samples taken during the second limit test are described in Table 

4 of the appended Analytical Report. 

Analyses of the samples taken one hour before introduction of the carp showed a mean 

measured concentration of 86 mg/l, which indicated that the system functioned as expected and 

that the actual concentration was within 20% of the target concentration of 100 mg./!. 

The measured exposure concentrations in each of the duplicate samples taken at the various 

sampling times are shown in Table 6. Again there were significant differences In dupUcate 

values. The difference In duplicate values might be explained by variations In amounts of loss 

during sampling, the limited solubillty In tap water and In this case especially the variation in 

recovery of the analytical method on the day of measurement (see Table 1 of the appended 

analytical report). All T9SUnS were used to determine the mean exposure concentration during 

the 96-hour test period. which corresponded to 76 mgll. 

- Page 15­
TableS Mean measured concentrations versus nominal concentration (2nd limit 

test) 

Nominal ooncentratfon MeasuNd 

t=-1h 

60.6/72.5 

Measured 

t:=96 h 

82.9/69.1 

Mean 

meuunKI 

7.3.2. Mortality and other effecta 

Table 7 shows the mortality data recorded during the second limit test. Two fish died during the 

course of the study. The surviving fish were observed to be discoloured during the entire test 

period. Further, all fish were observed swimming highly disorientated from 24 hours following 

exposure as was observed in the 1 It limit test. However, this effect did not diminish In time as 

was observed during the 1lit limit test. Again precipitation in the form of droplets was observed 

during the coorse of exposure. 

Table 7 Incidence of mortality and total mortality during the 2nd IImH test 

Concentration Initial Cumulative mortality Total 

(mgll) 

number 

offish 2Y.zh 24h 48h 72h 96h Mortality 

("') 

Blank-control 7 0 0 0 0 0 0 

76 7 0 1 1 1 2 29 

7.3.3. experimental conditions 

The results of measurement of pH. oxygen concentrations and temperature are presented In 

Appendix I. All test conditions remained within the ranges prescribed by the protocol (PH: B.OS.5, constant within 1 unit; temperature 20-24"C. constant within 2"0: oxygen> 60% of air 

saturation value). 

7.3.4. Determination of effect concentrations 

Table 8 shows the effect parameters basad on the results of both limit studies. The parameters • 

are based on the mean measured concentration. 

Table 8 Errect parameters 

I ParanMlter Concentration 

(mgll) 

NOEC <73 

24148f12196trLCso >76 

- Page 16­
8. DISCUSSION 

Two limit tests were performed with the intention of testing an actual exposure concentration of 

100 mg/l. the regulatory limit concentration. The mean measured concentration was however 73 

and 78 mgll, for the 11t and the 2nd limit test respectively. This despite the fact that a nominal 

concentration of 221 mgll was tested and that test solutions were refreshed >30 times per day 

In the tesl Fish were clearly affected in their swimming behaviour in both tests and two fish 

died in the 2nd limit. Hence, It could not be guaranteed that the LC50 was above 100 mgll. 

Therafore, various attempts with different procedures were tested to reach a higher exposure 

concentration (lt100 mgJ1) in test medium. The test system of the 2nd limit test applying 10-litre 

vessels was used. The flow-rate was increased to a maximum of.20 liters per hour to check 

whether an even higher refreshment rate (48 times/day) resulted in testing higher exposure 

concentrations. Further, nomin~ concentrations were Initially lowered to 176 and finally 

increased to 265 mgll to check whether any d~solutlon problems occurred at higher 

concentrations. AnaJly, the use of double mixing vessels (in series) to improve dissolution was 

tested. 

Dissolution appeared to be a problem as precipitate in the form of droplets was observed during 

the course of the two limit studies. In additIon to thls possible problem, sampling at various 

places In the test vessel was performed to check the homogeneity of the test solution. The 

results of this check showed rather large variations in test concentrations, which strengthens the 

anticipated dissolution problems, which are likely to be related to the relatively short mixing time 

which is again a consequence of the hIgh renewal rate. 

Analytical resuHs showed that the measured concentration remained clearly below 70 mgll 


when testing a lower nominal concentration of 176 mgll (221 mgllln the lImit tests). This did 


thus not Improve the results. At nominally 265 mgll, measured concentrations averaged those 


found in the two previous limit tests. Hence, even at this higher concentration a measured 


ooncentration of 100 mgll could not be reached. 


Finally, the use of double mixing vessels (In series) to improve dissolution was tested at a 

nominal concentratIon of 265 mgll. No precipitate in the test vessels was formed when applying 

. these exira mixing steps, but analytical results stili remained at the same level as was recorded 

during the two limit tests. 

Hence, all of these attempts did not result in measured concentrations Ihat were higher than the 

concentrations measured in the first two limit studies. The maxImum achIeved concentration 

approximated 70 mgll (see Table 5 of the appended Analytical report). On average, a recovery 

of 30% was found indicating a loss of 70%. In conclusion: with the present extremely volatlle 

substance It was not possible to test fish in a stable flow-through test system at a concentration 

of at least 100 mgll. 

9. CONCLUSION 

Under the flow~through conditions of the present test induced less than 


50% mortality in carp at a mean measured test concentration of 76 mg/l, the highest exposure 


concentration that could be achieved and maintained stable in tap water. 


Based on the observations of precipitate in the form of droplets in both limit tests, it cannot be 


excluded that fish were, at least occasionally, exposed to pure test substance. 


In conclusion, the 96h-LCM exceeded a mean measured concentration of 76 mg/l. 

- Page 17­
APPENDIX I TEST CONDmONS 

Elrst limit test 

Table 9 pH-yalues and dissolved oxygen concentrations (mg/f) during the 111t limit 

test 

Concentnltlon Day 2 Day 3 Day 4 

pH Oz pH Oz pH O:t 

8.0 8.6 7.9 8.4 8.0 8.5 

8.0 8.6 7.9 8.2 7.9 8.3 

Table 10 Temperatures (OC) measured during the 1lit limit test

Concentration 

(mg/'l) 

DIy 0 Dey 1 DIY 2 Day 3 Day 4 

Blank-oonb'Ol 21.2 21.1 21.0 21.4 21.1 

73 21.3 21.3 21.3 21.4 21.4 

Second limit test: 

Table 11 ptf,.yalu•• and dls.olved oxygen concentrations (mglJ) during the 2nd test 

Concentration Day 0 Day 1 Day 2 DayS Day 4 

(mgll) 

pH O:t pH O:t pH 02 pH O:t pH 02 

Blank-control 7.8 8.9 7.7 9.1 7.8 9.0 7.9 9.0 8.0 9.4 

75 7.1 8.7 7.7 B.S 7.8 10.1 7.8 8.9 7.9 9.1 

Table 12 Temperatures (OC) measured during the 2nd test 

COncentration 

(mg/'l) 

Day 0 Day 1 DIy 2 DayS Day 4 

Blank-control 21.4 21.6 21.3 21.3 21.4 

76 21.9 21.9 21.9 21.9 21.S 

-Page1S­
APPENDIX II REFERENCE TEST 

96-hour acute toxicity study in the carp with PCP; 

The study procedures described In this report were based on the EEC directive 92169, Part C.1, 

1992 "Acute toxicity for ·flsh·; and the OECO guideline No. 203: ·Flsh Acute Toxicity Test·, 

Adopted 17 July, 1992. 

Start: 17 January 2005 

End: 21 January 2005 

This referenoe test was carried O'-lt to check the sensitivity of the test system as used by 

The reference substance was pentachlorophenol (PCP. SIGMA, Art. P9441 , Batch 

103H3488). 

Concentrations: 0.10, 0.22 and 0.46 mgllin ISO-medium. 

(ncidence of mortality observed in the reference study: 

ConcentraOon 

PCP (mg/!) 


Nominal 


0.10 

0.22 

0.46 

Inl1lll1 

72h 9th 

0 0 

5 5 

5 5 

Tobll 

number Morbility

offish 

5 0 

5 100 

S 100 

During the test the pH, oxygen concentration and the temperature of the medium were within 

the opHmal ranges for fish. 

Under the conditions of the present test pcp induced no lethal effects in carp at 0.10 mgll. The 

96h-LC&o for carp exposed to pcp was 0.15 mg.ll (a 95% confidence interval between 0.10 and 

0..22 mgJl) and was already reached within 24 hours of exposure. The 96h-LCso for carp is 

generally between 0.10 and 0.46 mgll based on historical data of reference tests performed 

approximately every 3 months from AprR 1988 until the end 2000, and annually since then. 

Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of 

sensiHvlties generally observed during the past years. 

The raw data and report from this study are kept In the archives. The test descrIbed 

above was performed under GLP-conditions with a QA·check. 

·Page19­
APPENDIX III ANALYSES OF TAP WATER (Not performed under GLP) 

Date of sampling : 08-06-05 

Sample numbers : 2106017 (kamer 25) 

Laboratory 

ANALYSIS: ICOMPONENT: 

Metals: 

Selenium 

Arsenic 

C8dnium 

Chromium 

Copper 

Mercury 

Lead 

Zinc 

Manganese 

Calcium 

Magnesium 

Iron 

EOX (extractable organlo halogens) 

Polycyclic Aromatic Hydrocarbons: 

Naphtalene 

Acenaphtytene 

Acenaphtene 

Fluorene 

Phenanthrene 

Anthracene 

F1uoranthene 

Pyrena 

Benzo(a)antracene 

Chrysene 

Benzo(b)fluoranltlene 

Benzo(k)ftuoranthene 

Benzo(a)pyrene 

Dlbenzo(ah)antracene 

Benzo(ghl)perylene 

Indeno(123cd)pyrene 

PAC's Total EPA (16) 

PAC's Toeal VROM (10) 

<0.90 

<5.0 

<0.40 

<1.0 

29 

<0.050 

<5.0 

19 

<0.010 

53 

6.2 

<0.050 

<1.0 

<0.010 

<0.050 

<0.010 

<0.010 

<0.010 

<0.0050 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

Ilg/l 

1Ig/1 

1Ig/1 

Ilgll 

11911 ji) 

pgll 

!JgII 

jJgII 

mgll

mgllll) 

mgllll) 

mgll 

",gil 

pgII 

pgII 

pgII 

~ 

JIW'I 

IJg/I 

IJg/I 

IJg/I 

~ 

IJg/I 

JJgJ\ 

1Ig11 

JJgJ\ 

1Ig/1 

IJg/I 

IIg/I 

Total aerobic germ-count (22"C) 2 CFU/ml'" 

Total aerobio germ-counl (37"C) 2 CFU/m1 11 

Hardness: 1.6 mmolllji) 

Nitrate (expr. as N): 0.67 mgII 

Nitrite (elq)f. as N): 0.018 mgII 

Cyanide-totaal ( NEN 6655) <1.0 ugll 

Turbidity 0.049 FTE 

Colour intensity 20 mg Pt/I 

Dechlorination of tap water was not necessary since no chlorine was present. 

"'Tap WII&er was sampled from the copper-fnIe water supply system lhat supplied 1he water used for the present toxicity • 

B11Jdy. Among ethera, copper and badlJrfal activity were measured .,ltIe6e samples. The other oomponenta ware 

analysed In samples teken from Iha same waler supply but lIampted 11'1 the Animal House. 

- Page 20 ­
APPENDIX IV 


96-HOUR ACUTE TOXICITY STUDY IN CARP WITH 

(FLOW-THROUGH); 

DETERMINATION OF THE CONCENTRATIONS 

study comPletion date 

October 28. 2005 

Laboratory project Identification 

- Page 1 of 15 ­
1. CONTENTS 

1. CONTENTS ...........................................................................................................................2 


2. REPORT APPROVAL ............................................................................................................3 


3. INTRODUCTION....................................................................................................................4 


3.1. Preface ...........................................................................................................................4 


3.2. Aim of the study ..............................................................................................................4 


4. MATERIALS AND METHODS ...............................................................................................4 


4.1. Reagents ........................................................................................................................4 


4.2. Samples..........................................................................................................................4 


4.3. Analysis ..........................................................................................................................5 


4.3.1. Analytical method .................................................................................................5 


4.3.2. calibration ............................................................................................................5 


4.3.3. Injections ..............................................................................................................5 


4.3.4. Data acquisition and data processing .................................................................. 5 


4.3.5. Procedural recovery samples .............................................................................. 6 


4.4. Interpre1atlon ..................................................................................................................6 


4.4.1. calculations ..... ' .....................................................................................................6 


4.4.2. Fonnulas ..............................................................................................................6 


5. RESUlTS...............................................................................................................................8 


5.1. Calibration curves ...........................................................................................................8 


5.2. Samples..........................................................................................................................8 


5.2.1. Procedural recovery samples ..............................................................................8 


5.2.2. Test samples ........................................................................................................8 


TABLES 

Table 1 Procedural recovery samples .........................................................................................9 


Table 2 Concentrations of. In test medium (range-finding test) .............. 10 


Table 3 Concentrations of. in test medium (first limit test) ...................... 11 


Table 4 Concentrations of. in test medium (second limit test) ................ 12 


Table 5 Results of additional attempts with changes in the flow-through system Intended to 


reach a higher exposure concentration (~100 mgll) in test medium ............................ 13 


FIGURES 

Figure 1 GC chromatograms of a 0.0810 mgll calibration solution [lop; 


resJd. 2205] and blank methanol [bottom; resjd. 2217]............................................... 14 


Figure 2 GC chromatograms of procedural recovery samples at concentrations of 0.101 


Figure 3 GC chromatograms of blank control samples taken during the first limit test at t=O 


hours [top; res.ld. 1945], at t=4B hours [middle; res.ie!. 2248) and at t=96 hours 


Figure 4 GC chromatograms of target 221 mgn samples taken during the first limit test at t=­

24 hours [top; res.ld. 1888]. at t=O hours [second; res.id. 1913]. att=48 hours [third; 


mgn [top; res.ld. 2235] and 101 mgJl [bottom; res.id. 2240]......................................... 14 


[bottom; res.ld. 2250] .................................................................................................... 15 


res.id. 2228] and at t=96 hours [bottom; res.ld. 2230] ................................................. 15 


-Page2­
2. REPORT APPROVAL 

PRINCIPAL SCIENTIST: 

(Analytical Chemistry) .... 

Date: o(_:k~ l~ ,LGO;' 

-Page3­
3. INTRODUCTION 

3.1. Preface 

Study plan 

(analytical study) 

Start 

Completion 

: 30 May 2005 

: 05 October 2005 

3.2. Aim of the study 

The purpose of the analytical study was to determine the actual concentrations in samples taken 

from ·the test solutions used during the ecotoxicity test. 

4. MATERIALS AND METHODS 

4.1. Reagents 

Methanol HPlC-grade. labscan, Dublin, Ireland 

Milli-Qwater Tap water purified by reversed osmosis and 

subsequently passed over activated carbon and ionexchange cartridges; Mlillpore Corp, Bedford, MA. USA 

ISO-medium see main report 

Tap water see main report 

4.2. Samples 

All samples not analysed on the sampling day were stored frozen. Stability of the samples under 

these conditions was determined during "Development and validation of 

an analyttcal method for the analysis of in Milli-Q water". On the day of 

analysis, the frozen samples were defrosted at room temperature. 

The samples (10 ml) were analysed using headspace (see 4.3.1 analytical method). 

- Page 4 • 
4.3. Analysis 

4.3.1. Analytical method 

Quantitative analysis was based on the test substance peak in the GC chromatogram of 

Ana~k8lcondfflbns 

Column 

stationary phase 

Dimensions 

Brand 

Temperature program 

Initial temperature 

Initial time 

Rate 1 

Temperature 1 

Rate 2 

Final temperature 

Hold time 

Camer gas 

Column ftow 

Injection 

Temperature 

Splitless time 

Injection volume 

Injection speed 

Headspace conditions 

Sample 

Incubation temperature 

Incubation time 

Syringe temperature 

Detectfon 

4.3.2. Calibration 

CP Porabond Q 

25 m x 0.25 mm, dt =3 IJm 

Varian, Mulgrave, Vletorla, Australia 

40"C 

1 min 

20 "C/mln 

190 "c 

50 "C/mln 

250°C 

3.3 min 

Helium 

1 mllmin 

Splitress 

250"C 

2 minutes 

500 loll (Headspace) 

50 IJI/sec 

10 ml of an aqueous sample in a 20 ml vial 

5O"C 

30 minutes 

110°C 

FlO (250 "C; Hydrogen: 40 mVmin; Air. 450 mflmin) 

During the range finding test, calibration solutions in ISO-medium were made up from two 

standard solutions of In methanol. 

During the final test. callbratlon solutions in tap water were made up from two standard solutions 

of in methanol. 

4.3.3. Injections 

Calibration solutions were injected in duplicate. Procedural recovery samples and test samples 

were analysed by single InJectlon. 

4.3.4. Data acqulsltlon and data processing 

Data acquisition and data processing was perfOlmed using Empower software (Waters. Milford, 

MA, USA) version 5.00. 

-Page 5­
4.3.5. Procedural recovery sample. 

Standard solutions of In methanol and dilutions of these solutions with 

methanol were used as spiking solutions. 

Durin~ the range-finding test, ISO-medium (10 ml) was spiked at concentration levels of 

0.101 mgll and 101' mgll using 10.1 mgll and 10085 mg/l spiking solutions in methanol. 

During the first limit test on 28-06-05 and 29-06-05, tap water (10 ml) was spiked at concentration 

levels of 0.101' mgll and 101' mglJ using 10.1 mglJ and 10150 mgll spiking solutions In methanol. 

Durin~ the first limit test on 04-07-05, tap water (10 ml) was spiked at concentration levels of 

0.101 mg/l and 101' mg/l using 10.1 mgll and 10119 mgll spiking solutions In methanol. 

During the second limit test on 13-09-05, tap water (10 ml) was spiked at concentration levels of 

10.1' mg/l and 100' mgll using 1013 mgll and 10011 mgll spiking solutions in methanol. 

During the second limit test on 19-09-05, tap water (10 ml) was spiked at concentration levels of 

10.0' mgll and 100' mgll using 1013 mgll and 10011 mgll spiking solutions In methanol. 

During the additional test, tap water (10 ml) was spiked at concentration levels of 10.0' mgll and 

100' mgll using 1001 mgll and 10009 mg/l spiking solutions in methanol. 

During the range finding test and first IimH test, at 0.101 mgll one and at 101 mg/l five samples • 

were prepared. During the second limit test and the additional test, three samples were prepared 

at each concentration level. All recovery samples were treated as described in paragraph 4.2 

'Samples' and analysed. The recovery was calculated for each sample 

4.4. Interpretation 

4.4.1. Calculations 

All calculations were performed using non-rounded values. Rounded values are reported In the 

tables. Therefore, some differences might be observed when calculating the parameters as 

mentioned In the tables. : 

4.4.2. Fonnulas 

General 

Mean: 

where 

Xl = measured value 

n = number of measurements 

n 

L{Xi-X)2 

Standard deviation Sn - 1 = U....:::1-1'-:-----:_ 

en-I) 

Coefficient of variation 

1 ConcenlraUons were not corrected for spiking volume 

- Page 6­
Calibration 

Response: 	 R = Peak area test substance [unlts1 

Calibralion curve: 	 The response was correlated wIIh the test substance 

concentration, using linear regression analysis Qeast 

squares method; weighting factor (1/concentration2)}. 

R = response calibration solution [units] 


C = concentration of test substance in calibration 


soiutlon [mg/l] 

a = slope [unlts*lImg] 

b = intercept [units] 

Samples 

Concentration of ASAHIKLIN AE-3000 analysed In the samples: 

C '" (R -b) *d [mgll} 

a 

R = response sample [units] 

d = dDution factor 

a = slope [unlts"'lfmg] 

b '" intercept [unHs) 

Recovery of procedural recovery samples: 

Concentration analysed ... 100 rk} 

Concentration prepared 

Concentration analysed '" 1 00 [%] Relative to nominal concentration: Concentration nominal 

Concentration analysed at t • x hours Relative to Initial concentration: 	 '" 100 [%J Concentration analysed at t == 0 hours 

-page7­
5. RESULTS 

5.1. Calibration curves 

On each day of analysis, a calibration curve was constructed using at least six concentration 

levels. At each concentration level, two responses were used. The coefficient of correlation was 

in excess of 0.99 for each calitXation curve. 

Figure 1 shows chromatograms of a calibration solution and blank methanol. 

5.2. Samples 

5.2.1. Procedul'IIl recovery samples 

Figure 2 shows chromatograms of procedural recovery samples. The results for the procedural 

recovery samples are summarized in Table 1. Mean recoveries of procedural recovery samples 

are required to be in the range 70-110%. 

Because the recoveries of the procedural recovery samples were met, the results of the test 

samples were considered accurate. 

5.2.2. Test samples 

Figures 3 and 4 show chromatograms of blank control samples and target 221 mgll samples 

taken during the first limit test. The results for the test samples are summarized in Tables 2 - 4. 

During the range-finding test, the test-substance concentrations if1 test samples of nominal 

10 mgll taken at t=0 hours were below the limit of quantlftcaUon (LOQ) of the analytical method, 

but withIn the calibrated concentration range olthe calibration solutions. Because the coeffICient 

of correlation of the calibration curve was in excess of 0.99 the results of the test samples were 

considered accurate. The concentrations of the test samples at t=96 hours were significantly 

below the concentration of the Ipwest calibration solution, therefore < LOQ was given. 

During the first limit test, a small peak at the retention time of the test substance was observed In 

the chromatograms of the blank test samples (nominal 0 mgll) at t=48 and F96 hours. Because a 

similar peak was found in blanks of methanol it was concluded that the compound did not derive 

from test medium. For the blank test samples, the response of the interference was smaller than 

the response of the lowest calibraUon solution, therefore < LOQ was given. For the test samples 

of target 221 mgll, the maximum contribution of Ifle compound to the peak area of the lowest test 

sample was 0.2%, and was therefore not considered to be a significant Interference. 

During the second limit test, 8 small peak at the retention time of the test substance was 

observed in the chromatograms of the blank test samples (nominal 0 mgll) at t=48 and t=96 

hours. This peak was probably caused by a carry-over effect of the analytical system. The 

maximum contribution of this peak to the samples of target 221 mgll was 0.08%, and was 

therefore not considered to be a significant interference. 

In Table 5, the results of additional attempts with changes in the fIow-through system intended to 

reach a higher exposure concentration (~100 mgll) in test medium are shown. 

-PageS. 
TABLES 


Table 1 Procedural recovery .ampl.. 


Date of 

preparation 

[dd-mm·yy] 

Date of 

analysis 

[dd-mIn*YY} 

Concentration 

nominal 

[mgJl) 

Concentration 

analysed 

[mgIIJ 

Recovery 

[%] 

Mean recovery 

(Coefficient of 

variatIon) 

r%1 

30·0S"()5 ' 30..()5..()5 0,101 0.109 108 

3Q..()5-05 1 30..()5..()5 101 

101 

101 

101 

101 

98.6 

79.1 

10S 

100 

74.2 

98 

78 

104 

99 

74 

91 

(1S) 

28..()6...(}S' 28..()6...(}5 , 0.101 0.109 108 

28..Q6...(}5' 28...(}6...(}5 101 

101 

101 

101 

101 

99.7 

99.4 

99.0 

102 

100 

98 

98 

98 

101 

99 

99 

(1.3) 

29...(}6...(}S ' 29...(}6...(}5 0.101 0.0813 2 80 

29...Q6..0S' 29...(}6...(}5 101 

101 

101 

101 

101 

97.6 

96.7 

96.3 

94.S 

94.8 

96 

95 

95 . 

93 

93 

95 

(1.3) 

04...Q7-05 ' 

04...Q7.05 ' 

04"'(}7..Q5 

04-07-05 

0.101 

101 

101 

101 

101 

101 

. 0.0989 2 

86.0 

92.S 

87.7 

91.4 

95.0 

98 

85 

92 

87 

90 

94 

90 

(4.1) 

. 

13-O9...(}5 13-09...(}S 

, 

10.1 

10.1 

10.1 

10.6 

10.4 

10.8 

104 

103 

107 

105 

(2.0) 

13-O9"()5 13-09-05 100 

100 

100 

110 

112 

111 

110 

112 

111 

111 

(0.79) 

19..Q9"()5 19..()9...(}5 10.0 

10.0 

10.0 

10.4 

8.06 

10.1 

104 

81 

101 

95 

(13.3) 

- Page 9· 
2 

Table 1 Continued 

19"()9-O5 19-09-05 100 95.0 95 82 

100 73.2 73 (13.7) 

100 79.0 79 

03-1Q..()5 03-1Q..05 10.0 10.0 100 100 

10.0 10.1 101 (0.46) 

10.0 10.1 101 

03-10-05 03·10-05 I 100 92.2 92 91 

100 89.9 90 (1.3) 

100 91.3 91 

04-10-05 04-1Q..05 10.0 10.1 101 100 

10.0 10.0 100 (0.56) 

10.0 10.0 100 

04-1Q..()5 04-1Q..05 100 97.5 97 96 

100 97.3 97 (2.5) . 100 93.2 93 

Combined with 


Concentration _ below the limit of quantification. but within the concentration range of the calibration solutions. 


Table 2 Concentrations of In test medium (range-finding test) 

Time of 

sampling 

[hours) 

Date of 

sampling 

[dd-mm-yy] 

Date of 

analysis 

[dd-mm-yy] t 

Concentration 

Nominal 

(mgll] 

Analysed 

[mgll] 

Relative to 

nominal 

[%] 

Relative to 

mean Initial 

[%1 

0 23-05-05 3Q..05-05 10 

10 

100 2 

100 2 

0.0822 3 

0.0871 3 

3.71 

6.58 

1 

1 

4 

7 

24 24"()S-OS 30-05-05 10 

10 

100 

100 

0.234 

0.245 

4.77 

5.34 

2 

2 

5 

5 

276 

290 

93 

104 

96 27-05-05 30-05-05 10 

10 

100 

100 

<LOO 

<LOa 

1.16 

0.969 

1 

1 

22 

19 

Samples were frw:en unlll analysis. 

Combined with 

Concentndion was below Ihe limit of quantification, but within the concentration range of the calbration solullons. 

LOO Limit of quanlOcatlon was 0.100 mgIl "Development and validation of an analytical 

method b" the ;marys,- of ' In MIlII-Q water"). 

- Page 10­
Table 3 Concentrations of In test medium (first limit test) 

Concentration Time of Date of ~ate of Relative to sampling sampling analysis Nominal Target Analysed nominal [hours] [dd-mm-yy] [dd-mm-yy1 [mgJI] (mgllJ [mgll] [%] 

~24 28-06-05 28-06-05 221 100 80.2 36 • 

221 100 87.9 40 

0 29-06-05 29~06-05 0 0 n.d. n.a.

0 0 n.d. n.a. 

221 100 81.0 37 , 221 100 87.7 40 

48 01-07·05 04-07-05 ' 0 <LOQ 2 0 n.a. 

0 <LOQ 2 0 n.B. 

221 100 84.2 29 i 

221 100 59.9 27 

96 03-07-05 04-07-05 0 1 <LOQ 2 0 n.a. 

0 <LOQ 2 0 n.s. 

221 100 SO.5 27 i 221 100 i 84.6 38 

i i 

Samples were fnIzen untiIlnllysis. 

lOO 2 AIImII peak was obseMICI at \tie retention lime of teet $Ub8t8nee, but lis reaponse was not considered to derive 

from test sample $Inee It WIIS also observed In blank methanol. Limit of quantlllciltlon WIll 0.100 mgII (See 

: ·Development and vandalion of an analytieal method fDr \tie anary. of 

In Mnll-Qwater1. 

n.d. Not~ 

n.a. Not applicable 

• Page 11 ­
Table 4 Concentrations of In test medium (second limit tell) 

Time of 

sampling 

[hours} 

Date of 

sampling 

[dd-mm-yy] 

Date of 

analysis 

{dd-mm-yy] 

Concentration 

Nominal 

[mgll] 

Target 

[mgll] 

Analysed 

{mgll} 

Relative to 

nominal 

[%] 

-1 

48 

96 

13-09-05 

15-09-05 

17-09-05 

13-09-05 

19-09-05 1 

19-09-05 1 

0 

0 

221 

221 

0 

0 

221 

221 

0 

0 

221 

221 

0 

0 

100 

100 

0 

0 

100 

100 

0 

0 

100 

100 

n.d. 

n.d. 

85.4 

85.6 

2 

z 

60.6 

72.5 

41 

2 

82.9 

69.1 

n.a. 

n.a. 

39 

39 

n.s. 

n.a. 

27 

33 

n.a. 

".a. 

38 . 31 

Samples were frozen until .nalysls. 

A sman peak was obs8M!Id in U- nominal 0 my/! samplet. This Mil probably caused by a C8I"1)'-OW! efI'ed of 

the analytical aystem•.The maximum conIIbJtIon ofthis peak to the samples was OJ16%. 

n.d. Not dtIteded 

n.a. Not applcable 

- Page 12­
Results of additional attempts with changes in thellow-through system 

intended to reach a higher exposure concentration ~100 rngIl) in test 

medium 

Table 5 

i Date of Date of 

sampling analysis 

[dd-mm-yy] [dcknm-yy) 

30-09-05 

moming 

30-09-05 

30-09-05 03-10-05'

afternoon 

04-10-05 

morning 

04-10-05 

04-10-05 04-10-05

afternoon 

Nominal 

[mgll] 

, 

0 


0 


0 


176 


176 


176 


176 e 

176 e 

176 7 

176 8 

176 s 

176 6 

176 7 

176 8 

0 


0 


0 


176 


176 


176 


265 


265 

265 

, 

Target 

[rng/l] 

0 


0 


0 


100 


100 


100 


100 

100 

100 

100 

100 

100 

100 

100 

0 


0 


0 


100 


100 


100 


100 

100 

100 

Concentration 

Analysed 

[mgll] 

2 

2 

3 

2 

2 

64.2 4 

20.1 

50.3 

56.7 

59.1 

45.1 

56.1 

36.6 

56.5 

n.d. 

n.d. 

n.d. 

46.1 

47.1 

47.5 

73.6 

72.9 

73.8 

Relative to 

~t 

n.l. 

n.s. 

n.a. 

n.a. 

n.a. 

36 

11 

29 

32 

34 

26 

32 

21 

32 

n.l. 

n.s. 

n.a. 

26 

27 

27 

28 

28 

28 

05-10-05 05-10-05 265 100 64.2 24

265 100 68.5 26

265 100 69.6 26

Samplea W8ra frozen until analy8is. 

Due to problems with the analytical system it was not P05llbie 10 determine an analysed concentration. 

Due 1.0 problems witt! !lie analytbll system this sample WItS not analysed. 

IndlcatM!ll8SUl. 


Bottom sample (from) 


Bottom sample (back) 


Top sample (front) 


Top 8IW11ple (back) 


n.d. Not detected 

n." Not applicable 

-Page 13­
FIGURES 

.... ... ... .... .... ... 


Figure 1 GC chromatograms of a 0.0810 mgll calibration 

solution [top; resold. 2205] and blank methanol [bottom; res.ld.2217]• 

.... ... ... I .. ... .... .... ..... 

Figure 2 GC chromatograms of procedural recovery samples at concentrations of 

0.101 mgn [top; res.ld. 2235] and 101 mg/l [bottom: res.ld. 2240], 

- Page 14­
! 


I 


·1


I 
 ... ...a

Figure 3 GC chromatograms of blank control samples taken during the first limit test 

at t-O hours [top: .....Id. 1943]. at t-48 hours [mlddla: .....Id. 2248] and at 

1-98 hours [bottom; ra• .ld. 2250] . 

.:~.~1-- ------------- k .~~------I-'-2r- L .~ut------~-t-':!r-- l 

.~~ ....~~~ , , 0 , i ilO ... ... .i. ,II ~ l.. .... ... 11,. "... i 

FIgure 4 	 GC chromatograms of target 221 mgn samples takan during the first limit 

test at (=-24 hours [top; res.id.1888], at t=o hours [second; res.ld. 19131. at 

t-48 hours [third; ras.ld. 2228] and at t=96 hours [bottom; res.id. 2230]. 

- Page 15­
P-04..635 


. REPORT 


Study Title 

ACUTE TOXICITY STUDY IN DAPHNIA MAGNA WITH 

(FLOW-THROUGH) 

Study completion elate 

16 September 2005 

Test Faclltlv 

Laboratory Project Identification 

• Page 1 of 32 ­
1. CONTENTS 

1. CONTENTS ...........................................................................................................................2 


2. STATEMENT OF GLP COMPLIANCE ..................................................................................3 


3. QUALITY ASSURANCE STATEMENT .................................................................................4 


4. SUMMARY.............................................................................................................................5 


5. INTRODUC·nON....................................................................................................................6 


5.1. Preface ...........................................................................................................................6 


5.2. Aim of the study ..............................................................................................................e 


5.3. Guidelines.......................................................................................................................6 


5.4. Storage and retention of records and materials .............................................................6 


5.5. Oeftnltlons.......................................................................................................................7 


6. MATERIALS AND METHODS ...............................................................................................7 


6.1.1. Test substance Information ..................................................................................7 


6.1.2. Reference tast...................................................................................................... 7 


6.1.3. Preliminary data ...................................................................................................7 


:I ~=r"s~.:::::::::::::::::,:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: 6.4. Range-finding test (static) ..............................................................................................9 


6.5. First flow-through test .....................................................................................................9 


6.6. Final flow-through test ....................................................................................................9 


6.6.1. Concentrations .....................................................................................................9 


6.6.2. Dosing system .................................................................................................... 10 


6.S.3. Test procedure and conditions........................................................................... 11 


6.6.4. Sampling for analysis of test concentrations ..................................................... 11 


6.6.5. Measurements and recordings .......................................................................... 11 


6.7. Interpretation ................................................................................................................11 


6.7.1. Data handling ..................................................................................................... 11 


6.7.2. Acceptability of the test ......................................................................................12 


6.S. List of protocol deviations .............................................................................................12 


7. RESULTS.............................................................................................................................12 


7.1. Range-finding test ........................................................................................................ 12 


7.2. First limit flow-through test ........................................................................................... 13 


7.3. Final flow-through test ..................................................................................................13 


7.3.1. Measured concenlrations ................................................................................... 13 


7.3.2. Immobility ........................................................................................................... 14 


7.3.3. Determination of effect concentrations .............................................................. 15 


7.3.4. Experimental conditions ..................................................................................... 15 


S. CONCLUSION .....................................................................................................................15 


TABLES 


Table 1 Dosing program.................................................................................................. 10 


Table 2 Incidence of immobility in the range-tlnding tesL.............................................. 12 


Table 3 Incldence of immobility In the limit test............................................................... 13 


Tabla 4 Mean measured concentrations versus nominal concentration ........................ 14 


TableS Acute immobilisation of daphnia after 24 and 48 hours In the final test ............ 14 


TableS Effect parameters ............................................................................................... 15 


Table 7 pH and oxygen concentrations during the final test. .......................................... 15 


FIGURES 


Figure 1 Row-through systElfn ..........................................................................................10 


APPENDICES 


APPENDIX I REFERENCE TEST...........................................................................................16 


APPENDIX II ANALYSIS OF TAP WATER (Not performed under GLP) 

APPENDIX III ANALYTICAL REPORT 


................................ 17 


-Page2· 
2. STATEMENT OF GLP COMPUANCE 

The study described in this report has been correctly reported and was conducted in compliance 

with: 


The Organization for Economic Cooperation and Development (DECO) Good Labormory Practice 


Guidelines (1997). 


Which essentially conform to: 


The United States Food and Drug AdmInistration Good Laboratory Practice Regulations. 


The United States Environmental Protection Agency Good Laboratory Practice Regulations. 


Analyses of tap water was excluded from this GlP statement. 


The sponsor is responsible for Good Laboratory Practice (GlP) compliance for all test substance 


Information unless determined by 

Study Director Head of In Vitro & Environmental Toxicology 

Tectftcal DIrector 

Date: ..19.I.O.S...~g5.......... -.......... . 


- Page 3 
3. QUALITY ASSURANCE STATEMENT 

This report was inspected by the Quality Assurance Unit to confirm that the methods and 


results accurately and completely reflect the raw data. 


The dates of Quality Assurance Inspections are given below. 


During the on-site process inspe<:1ions procedures applicable to this type of study were Inspected. 


Type 0' 

Inspections PhaselProcess ' 

Start 

Inspection 

date 

End 

Inspection 

date 

Reporting 

data 

Study Protocol ()3.-Sep-04 O~Sep4i 03-Sep-04 

Amendment 1 of protocol 03-May"()5 03-May-05 03-May-05 

Amendment 2 of protocol 27-May-OS 27-May-05 27-May-05 

Amendment 3 of protocol 22-Jun-05 22-Jun-05 22-Jun-05 

Amendment 4 of protocol 30-Jun-05 3O-Jun-05 30-Jun-05 

Amendment 5 of protocol 19-Jul-05 19-Jul-05 19-Jul-05 

Amendment 6 of protocol 22-Jul-05 22-Jul-05 22-Jul-05 

Report 01-Sep-05 01-8ep-OS 01-8ep-05 

Process Environmental toxicology 1B-Apr-05 22-Apr-oS 22-Apr-OS 

Test substance handling 

Exposure 

Observations/Measurements 

Analytical and physical chemistry 2O-Jun-05 29-Jun-OS 05-Jul-05 

Test substance handling 

ObservationslMeasurements 

Head of Quality Assurance 

-Page4­
4. SUMMARY 

Acute Toxicity Study in Daphnia magna with 

The study procedures described in this report were based on the ISO International Standard 

6341. 1996. In addition, the procedures were designed to meet the test methods Bnd validity 

criteria of the EEC directive 92169, Part C.2. 1992 and the OECD guideline No. 202 Part I, 1984. 

The batch of lested was a clear and coIoutless liquid with a purity of 

99.985%. was completely soluble in test medium at the concentrations 

tested and known to be extremely volatile. Consequently, measures were taken during this 

project to minimise loss of test substance by evaporation, which included but were not limited to 

short preparation times and the use of airtight closed vessels. 

The project started with a statiC range-flndlng test exposing ten daphnia per concentration (In 

duplicate, 5 per vessel) to a blank-control and nominal concentrations of 

0.1. 1.0, 10 and 100 mgll. No significant Immobilisation of daphnlds was observed at any of the 


concentrations tested. Analytical results of samples taken from 100 mg/l showed that the mean 


measured concentrations were stable at approximately 5 mgll during the 48-hour test period. 


The project was continued with a limit tes1 in an alr.tlght closed flow-through system with 

continuous renewal of test solutions. Twenty daphnia per concentration (four meshes, 5 daphnia 

per mesh) were exposed for 48 hours to a blank-control and a nominal concentration of 221 mg/!. 

Which was expected to correspond to an actual exposure concentration close to 100 mg/l. 

Precipitate in the form of droplets and floating daphnia Influenced the results of this test by 

mechanical disturbances. Therefore, the results were considered not representing the intrinsic 

toxicity of the lest substance. It was consequently decided to continue with a final flow-through 

study testing a range of concentrations. 

The final test was performed in an air-tight closed flow-through system with continuous renewal of 

test solutions. Exact volumes of pure test substance were dosed every 5 minutes and mixed with 

tap water al a flow rate of 12 LIh. Twenty daphnia per concentration (four meshes, 5 daphnia per 

mesh) were exposed for 48 hours to a blank-control and nominal 

concentrations of 22, 40, 71, 123 and 221 mgll. It was anticIpated that this nominal concentration 

range would correspond to a range of actual concentrations between 10 and 100 mgll. Samples 

for analytical confirmation of actual exposure concentrations were taken one day before onset, at 

the start and after 48 hours of exposure. 

Analyses of tt1e samples taken trom the lowes1 and highest test group one day before the start of 

the test showed that the system functioned property and thai the actual concentrations were close 

to the anticipated targets. 

The mean measured exposure concentrations were relatively stable at the two highest target 

concentrations and In agreement with what was expected. More variation was seen In the 

concentrations measured in the three lowest test groups. However, only the highest exposure 

concentration was essential for determination of the toxicfty parameters and corresponded to 

94 mg/l. 

The siudy met the acceptability criteria prescribed by the protocol and was considered valid. 

The 48h-ECso exceeded a mean !11easured concentration of 94 mgl1 under the conditions of the • 

present flow-through study with Daphnia magna exposed to 

-Page5­

;

! 

I 

~-.~--".------.--... 
5. 	 INTRODUCTION 

5.1. Preface 

Sponsor 

Study Monitor 

Test Facility 

Study Director 

Technical Coordinator 

Principal Scientist 

Study Plan Start 

Complstion 

: 23May2005 

: 22 July 2005 

5.2. Aim of the study 

The purpose of the study was to evaluate the test substance for its ability to generate acute toxic 

effects on the mobllity of Daphnia magna during an exposure period of 48 hours and, if possible, 

to determine the EC!IO at 24 and 48 hours of exposure. 

5.3. GuldellMs 

The study procedures described in this report were based on the ISO Intemational Standard 

6341: "Water quality· Determination of the Inhibition of the mobility of Daphnia magna StrausAcute toxicity test. Third edition, 1996-04-01. 

In addition, the procedures were designed to meet the test methods.and validity of the following 

guidelines: 	 . . 

• 	 European Economic Community (EEC), EEC directive 92169, Part C: Methods for the 

determination of ecotoxicity, Publication No. L383, December 1992, 0.2. "Acute Toxicity for 

Daphnia", 

• 	 Organization for Economic Co·operation and Development (OEOD), OECO guidelines for 

Testing of Chemicals, guideline No. 202 Part I: "Daphnia sp., Acute Immobilisation Test", 

Adopted April 4, 1984. 

5.4. Storage and retenUon of records and materials 

Records and materials pertaining to the study including protocol, raw data, specimens (except 

specimens requiring refrigeration or freezing) and the final report are retained in the 

archives for a period of at least 10 years after finalization of the report. After this period, the 

sponsor will be contacted to determine whether raw data and specimens should be returned to 

them. retained or destroyed on their behalf. 

Those specimens requiring refrigeration or freezing will be retained by for as long as the 

quality of the specimens permits evaluation but no longer than three mon1hs after finalization of 

the report. 

• PageS­
will retain a test substance sample until the expiry date, but no longer than 10 years after 

finalization of the report. After this period the sample will be destroyed. 

5.5. Definitions 

Immobile are those animals not able to swim wllt'lln 15 seconds after gentle agItation of the lest 

vessel. 

. The ECso Is the concentration of test substance estimated to immobllise 50% of the daphnia after 

a defined period of exposure. 

No Observed Effect Concentration (NOEC) Is the highest concentration tested at which no 

effect (I.e. immobllisatlon) Is recorded. 

A Flow-through test is a test In which the water containing the test substance is renewed 

continuously. 

6. MATERIALS AND METHODS 

6.1.1. Test substance InformaJion 

Identification 

Structure 

Molecular formula 

Molecular weight 

CAS Number 

Description Clear colourless liquid 

Batch 

Purity 99.985% 

Test substance storage In refrigerator in the daM< 

Stability under storage conditions Stable 

Expiry date 28 February 2006 

Specific Gravity 1.47 

Stability in water Not indicated 

6.1.2. Reference tasl 

This report includes the results of the most recent reference test with potassium dichromate 

(Appendix I). 

6.1.3. PrelimInary data 

The water solubility of at 20.1 ;I; OSC is 0.805 gil using the flask method 

. Further essential information obtained from physico--ohemlc:al studies 

performed at were the vapour pressure being 31 kPa at 25°C, freezing poInt <-81°e, 

boiling polnt'56"C and a log Koc of 2.54. The log Pow of was 2.18 accordlrlg 

to informatIon supplied by the sponsor. From these data it can be concluded that the test 

substance will evaporate rapidly at room temperature as pure substance but also from aqueous 3 1 solutions (calculated Henry coefficient is 7.24.103 Pa m mor ). 

concentrations showed to be stable during freezing and thawing of samples 

in a deep-freeze 

-Page7­
6.2. Test ayslem 

Species 	 Daphnia magna (Crustacea, Cladocera) (Straus, 1820), 

at least third generation, obtained by aeyclfcal 

parthenogenesis under specified breeding conditions. 

Source 	 In-house laboratory culture with a known history. 

Reason for selection 	 ThIs system has been selected as an internationally 


accepted Invertebrate species. 


to 5111 Validity of batch 	 Daphnids originated from a healthy stock. ~ 

brood. showing no signs of stress such 88 mortarlty 

>20%, presence of males, ephlppia or discoloured 

animals and there was no delay in the production of the 

first brood. 

Characteristics 	 For the test selection of young daphnia with an age of < 

24 hours, from parental daphnids of more than two 

weeks old. 

6.3. Breeding 

Start of each batch 	 With newborn daphnids, I.e. less than 3 days old, by 


placing about 250 of them Into 5 litres of medium In an 


alliJlass culture vessel. 


Maximum age of the cultures 	 4 weeks 

Renewal of the cuHu'res 	 After 7 days of cultivation half of the medium twice a 


week. 


Temperature of medium 

Feeding 	 Daily, a suspension of fresh water alga~. 

Medium 	 M7, as prescribed by Dr. Elendt-Schneider 

(Elendt, B.-P., 1990: Selenium deficiency in Crustacea. 

An ultrastructural approach to antennal damage In 

Daphnia magna Straus. Protoplasma 154,25-33). 

Qomposltfon of medium MZ: 


ISO medlm1: Ihe roIowtnv chemICal. (analytical grade) are dI1l8oIved In 18p water purified by R9V9rs8 OSl1'lOllis (Mllt-RO, 


MIIlipore Corp., Bedford. Mass., USA): 


Macro salts: 	 CaCI~ 293.8 mg/! 

MgSO••7HtO 123.3 mgll 

NaHC03 84.B mgll 

KCI 6.8 mg/! 

Medium M7: tmce C!lements. macro nuttienlll and vitamins are added to freshly prepared ISO medltm 10 reach the 

following concentJatlons: 

Trace elemenls: 	 B 0.125 mlf! 

Fe 0.05 TIl!o)1 

Mn 0.025 mgII 

LI. Rbend Sr 0.0125 ffi9II 

Me 0.0063 TIl!o)1 

Br 0.0025 mgII 

CU 0.0016 mgll 

-PageS­
Zn 0.0063 mlVl 

Co end I 0.0025 moll 

Be 0.0010 mgJ 

V 0.0003 mlil 

NaaEDTA.2H.o 2.5 mgll 

Macro nutrients: NaaSiOa. 9HtO 10.0 mgtl 

NaNOa Ozl mgtl 

KH.tPO. 0.14 m(VI 

KaHPO. 0.18 mlVl 

VItamins: Thiamine 75.0 pg/I 

Bu 1.0 WI 

Biotin 0.75 pg/I 

The hardr'IesB: 250 mgI1 expressed as Caeo, and the pH: 8.0:1: 0.2 after aemtlon. 

6.4. Rang..flndlng test (static) 

A static range-finding test was performed to provide information about the range of concentrations 

to be used In the final test. Test procedure and conditions were similar to those applied In the final 

test with the followlng exceptions: a static test system was used In which ten Daphnia per 

concentration (two vessels, five per vessel) were exposed to a blank-control and nominal 

concentrations of 0.1 to 100 mgJI increasing by a factor of 10. Dissolved 

oxygen concentrations, temperature and pH were only measured in the control and highest test 

concentration. Duplicate samples for possible analyses were taken from all test solutions at the 

start (in combination with and after 48 hours of exposure. 

Preparation for the range-finding test started with test solutions prepared at 10 and 100 mgll 

applying' or' 3 minutes of magnetic stirring, respectively. Care was taken that during preparation 

as little as possible volatinsation occurred (weighing and preparation in closed vessels which 

already contained a sman volume of test medium, short preparation period). The lower test 

concentrations were prepared by dilution of the test solution of 10 mgll. The tlnal test solutions 

were aU clear and colourless. 

The test solutions originated from those used for the simultaneously performed acute fish toxicity 

test 

6.5. First flow-through test 

The project was continued with a limit test under flow-through conditions. The nominal test 

concentration was 221 mg/l, which was expected to correspond to an actual exposure 

concentration close to 100 mgll. A blank-control was Included. Test procedure and conditions 

were similar to 1hos9 applied in the final flow-through test. 

6.8. Final flow-through test 

6.6.1. Concentrations 

Nominal 22, 40. 71, 123 and 221 mgJ1. expected to 

correspond to actual concentrations close to 10,18,32, 

56 and 100 mgJI. 

Controls 	 Test medium without test substance or other addi1iv9s 

(blank-control) • 

• Page 9­
6.6.2. Dosing system 

SuffICient amounts of pure test substance were collected daily in airtight sea/ed vials and used as 

'stock' solutions for dosing. The pure test substance was dosed vie a computer-controlled system 

in combination with 5 dispensers (GUson). Via this system the dosed volumes entered a mixing 

flask separately from the tap water supply. The tap water was supplied via a flow meter wfth a 

flow rate of ca. 12 1Ih. In the mixing flask the dosed volumes and \he tap water were mixed under 

continuous stirring. The whole system was checked dally. The dosing program is described In 

Table 1 and a diagram of the system is presented In Figure 1. 

The hourly dosed volumes in combination wfth a tap-water flow of 12 liters per hour resulted in a 

nominal concentration range between 22 and 221 mgll. However. anticipating the rapid loss of 

test substance, it was expected that this dosing regime would give an actual concentration range 

between 10 and 100 mgII (see the last column in table 1). 

Table 1 Dosing program 

STOCK SOLUTION DOSING VOLUMES 

(0.6 mt syrlngel 

NOMINAL 

CONCENTRATION 

EXPECTED 

CONCEHTRAnON 

PERCVCLE PER HOUR 

pl/5 min ml I hour (mg/I) (~) 

15 0:18 22 10 

27 0.32 40 18 

Pure 48 0.58 71 32 

84 1.0 123 58 

150 '.8 221 100 

Diluter 

1 +­

0

Computer 

1 ap-water 

.. 

Test ¥essel 

Figure 1 Flow-through system 

-Page10­
6.6.S. Test procedure and conditions 

Test duration 48 hours 

Test type Flow-through, continuous renewal of test media. 

Test vessels ca. 1.5 litres, stainless steel containing four stainless 

steel meshes and airtight closed with a Perspex lid. 

Medium Tap water (see Appendix II for data of analysis) 

Number of daphnia 20 per concentration 

Loading 5 per mesh 

Ught 16 hours photoperiod daily 

Feeding No feeding 

Aeration No aeration of the test solutions. 

Introduction of daphnia Two days after the start of the dosing to permit 

stabilisation of the system 

6.6.4. Sampling for analys's 01 test concentrations 

During the final test duplicate samples for analysis were taken according to the schedule below. 

The method of analysis is described in the appended Analytical Report (Appendix III). 

Sampling: Frequency 	 At ca 27 hours following the start of dosing, samples 

were taken from nominally 22 and 221 mgIJ to check the 

actual exposure concentrations. 

At the start and the end of the exposure period samples 

were taken from all concentrations and the cootrol. 

Volume 10 ml from the approximate centre of the test vessel. 

Storage Samples taken at the end of the test were stored in a 

deep-freeze until analysis. 

6.6.5. Measurements and ~rdlngs 

Immobility pnofudlng mortality) 	 At 24 hours and at 48 hours. 

pH and dissolved oxygen 	 At the beginnIng, after 24 hours and at the end of the 

test. for all concentrations and the control. 

Temperature of medium 	 Continuously in the vessel containing the blank-control, 

beginning at the start of the test. 

6.7. Interpretation 

6.7.1. Data handling 

Calculation of ECso: 

No ECeo could be calculated (EC60 > maximum concentration tested) . 

• Page 11 • 
Defining exposure cOllCentrations: 

The results were based on the mean of the concentrations measured in samples taken at the 

various time Intervals. 

6.7.2. AcceptabllHy ofthe test 

1. 	 In the control, no daphnia became immobilised or trapped at the surface of (he water. 

2. 	 The oxygen concentration was ;;e 5 mg/l at the end of the test. Other test conditions (pH and 

temperature) were maintained within the 1mits prescribed by the guidelines. 

3. 	 The 24h-ECso (based on the initial concentration) of potassium dichromate was within the 

range 0.6 mg/l to 1.7 mg/l. 

6.8. L1at of protocol deviations 

There were no deviations from the protocol. 

7. RESULTS 

7.1. Range-finding test 

Table 2 shows the responses recorded during the static range-finding test No significant 

immobHisation of daphnjds was observed at any of the concentrations tested. 

Analytical results of samples taken from 100 mgll showed that the mean measured 

COllCentrations W&l'e stable at approximately 5 mg/l during the 48-hour test period (see Table 2 of 

the appended Analytical report). Hence, with the current preparation procedure actual exposure 

collCentrations close to nominal could not be reached. To enable tasting higher actual 

collCentrations and to increase the security of maintaining these stable It was decided to continue 

testing applying a flow-through system. 

All further test conditions were maintained within the limits prescribed by the protocol. 

Table 2 Incidence of Immobility In the range-finding test 

Concentration v..... Number Response at 24 h Response at 48 h 

number Daphnia Total Total expoMd number % number % Nominal (mgll) 

Blank-conlrol A 5 1 5 1 5 

B 5 om 0 

0.1 A 5 0(3) 0 0(2) 5 

B 5 0(4) 1(5) I

1.0 A 5 0(5) 0 0(3) 0 I 

B 5 0 0 I 

10 A 5 0 0 0 0 I B 5 0 0 I 

100 A 5 0(3) 0 0(5) 0 , B 5 o(1) 0 I

Between bracI<8Ia: number d daphnlds observed trapped at 1I1e surface. These organisms __ relmmEu'sed InIO the 

1'86p&Ctive solutIonS before recording of mobility, 

-Page 12­
·7.2. First lIm1t flow-through test 

Table 3 shows the responses recorded during the limit flow-through test. A total of 80% of the 

daphnids exposed to the limit concentration were immobllised after 48 hours of exposure. 

Analyses of samples taken from nominal 221 mgll showed that the measured concentration was 

in agreement with what was anticipated and expected, i.e. 100 mgll (see Table 3 of the appended 

Analytical report). It should however be noted that during exposure droplets were present on the. 

bottom of the exposure vessels. This could have had a direct effect on the immobility If the 

droplets orlglnatetf from undissolved pure test substance, which was expected based on the test 

substances specific gravity of , .47. Consequently, the observed immobility averaging 30% after 

24 hours and 80% after 48 hours should be taken with care. Furthennore, relatively high numbers 

of daphnlds were trapped at the surface. Hence, the present test results were Influenced by 

mechanical disturbances and therefore considered nat representing the Intrinsic toxicity of the 

test substance. 

It was consequently decided to continue with a ffnal flow-through study testing a range of 

conoentratlons and to pay specIal attentton not to Include undissolved test substance droplets in 

the test vessels by increasing the stirring rate In the mixing vessels. 

The pH ranged between 7.9 and 8.0, the oxygen concentration was between 8.4 and 8.9 and Ihe 

temperature was kept between 21.1 and 21,SOC. Hence. all test conditions in the limit test were 

maintained within the limits prescribed by the protocol. 

Table 3 Incidence of immobility In the limit test 

Concentration Vea.el Number Response at 24 h Response at 48 h 

(mgll) 

number Daphnia 

exposed number 

Total 

% number 

Total 

% 

Blank-control A 

B 

C 

0 

5 

5 

5 

5 

0 

0 

0 

0 

0 0 

0 

0 

O. 

0 

100 A 

8 

C 

0 

5 

5 

! 5 

5 

0(5) 

0(4) 

5 

1(2) 

30 4 (2) 

4 (1) 

5 

3(1) 

80 

Between brackets: runbet' of daphnlds observed trapped at the swface. These Org&OI"'S were relmmeraed into the 


nrospectIve tdUllons before recording 01 mobility. . 


7.3. Final flow-through test 

7.3.1. Measured concentrations 

The results of analysis of the samples taken during the final test are described in Table 4 of the 

appended Analytical Report and summarised in Table 4 on Ihe next page. 

Analyses of the samples taken from nominal 22 and 221 mg/l one day before the start of the test 

showed mean measured concentrations of 7.7 and 95 mgll indicating that the system functioned 

properly and that the actual concentrations were close to the antlclpated targets of 10 and 100 mgll. 

All concentrations measured at the start of the experiment were In agreement with what was 

expected, exoept at the nominal concentration of 71 mgll. This concentration was lower than 

measured at nominal 40 mg/l. Concentrations remained stable at the two highest concemratlons, 

but decreased In the lower concentrations. However, only the highest exposure concentration 

was essential for determination of the toxicity parameters. The mean concentration measured In 

the highest test group corresponded to 94 mgll. 

-Page13­
Table 4 Mean measured concentrations versus nominal concet,tratlon 

IConcentration (mgn) Measured M..sured Mean

t=O h t:::48 h m_sured

Nominal I Expected' (mgll) (mg/I) (mgll)

1.6 4.7 2 22 10 7.8

40 18 20.4 10.4 15

71 8.4 12

Ht 

32 16.3 

78.3/36.7 3 123 51.7 55 

221 88.2 104 94

j Based on the expected loss 01 test suIlB1ance from water due to 8Y8pOIlltion. 

I Based on one meaau11IITlant 

aIndividual values gillen due to the high dlfler9l'lC8 In dupl'mte meuu11IITl9nts 

7.3.2. Immobility 

Table 5 shows the responses recorded during th~ final flow-through test Immobility percentages 

ranged between 5 and 30% In the different concentrations tested. Hence, a 50% Immobility rate 

was not reached In any of the concentrations tested. Including a highest tested concentration of 

94 mglt. There was no clear relation between the actual exposure concentrations and the 

observed Immobility. At termination of the study some droplets were observed in the four highest 

exposure concentrations indicating a possible disturbing effect on the mobility rates due to 

undissolved test substance. Comparison of the results of the highest exposure concentration with 

those obtained in the limit test showed that concentrations were comparable but effects were 

significantly lower in the final test. It can be argued that the higher effects In the limit. test were 

likely to be related to the presence of relatively large amounts of preclpftate (droplets) and the 

high numbers of floaters. Floaters were not observed in the final test. 

TableS Acute Immobillsation of daphnia after 24 and 48 hours in the final test 

Concentration 

-(man) 

Vessel 

number 

Number 

Daphnia 

exposed 

Respon..at 24 h Respon..at 48 h 

number 

Total 

% number 

Total 

% 

Blank-control A 5 0 0 0 5 

B 5 0 0 

C 5 0 0 

0 5 0 1 

4.7 A 5 1 10 1 10 

B 5 0 0 

C 5 1 1 

0 5 0 0 

15 A 5 0 0 0 5 

B 5 0 1 

C 5 0 0 

0 5 0 a 

12 A 5 0 0 1 15 

B 5 0 0 

C 5 , 1 1 

0 5 1 1 

55 A 5 1 20 1 30 

B 5 3 3 (1) 

c 5 0 0 

0 5 0 2 

94 A 

B 

C 

0 

5 

5 

5 

5 

1 

0 

0 

1 

10 1 

1 (1) 

0 

3 

25 

! 

Between bn:lckeIB: number 01 daptJnlda obseMld trapped at the surlaoe. These organIsms were reimmelse<f Into (he 

respective soIUIIons before rllCOfdtlg 01 mobBfty. 

- Page 14­
7.3.3. Determination of efl'ect concentrations 

Table 6 shows the effect parameters based on target concentrations, see also Appendix I. 

Table 6 Effect parameters 

Pal'llm8ter Concentration 

(mgll) 

NOEC Not determlnod 

24h-ECso >94 

48h·ECso >94 

7.3.4. Experimental conditione 

The results of measurement of pH and oxygen concentrations (mg/l) are presented in Table 7. 

These test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not 

varying by more than 1 unit; oxygen: >7 mg/1 at the start, ~5 mg/1 at the end of the test). 

The temperature of the test medium was 2O.5°C at the start of the test. The temperature 

continuously measured In a temperature control vessel varied between 20.3 and 20.7°C during 

the test, and complied with the requirements as laid down in the protocol (18-22DC, constant 

within 2OC). 

Table 7 pH and oxygen concentrations during the final test 

Concentration 

(mal'll 

Start 

pH 

bOh) 

02 

End (1:=24 h) EndCh=48 h) 

pH ~ pH 02 

Blank-control 8.1 8.8 8.1 8.8 8.1 9.0 

4.7 8.1 8.7 8.1 8.9 8.1 8.9 

15 8.0 8.7 8.1 8.8 8.0 8.8 

12 8.0 8.6 8.1 8.S 8.0 8.9 

55 7.9 8.6 7.9 8.4 8.0 8.7 

94 7.9 B.6 7.9 8.3 7.9 8.6 

8. CONCLUSION 

The 4Sh-ECso exceeded a mean measured concentration of 94 mg/l under the conditions of the 

present flow-through study with Daphnia magna exposed to 

- Page 15­
I 

APPENDIX 1 REFERENCE TEST 

Start: 21 June 2005 

End : 23 June 2005 

48-hour Acute ToxicIty Study In Daphnia magna with K2Crl!07 

The study procedures described in this report were based on the ISO Intemationel Standard 

8341, the EEC directive 92169, Part C.2. "Acute toxicity for Daphnia", December 1992, and the 

OECD guideline No. 202: "Daphnia sp., Acute Immobl1isation Test", Adopted April 4. 1984. 

The reference test was carried out to check the sensitivity of the test system as used by 

Daphnia were exposed for a maximum of 48 hours to KeCr2~concentrations of 0.10,0.18,0.32. 

0.56, 1.0 and 1.8 mgll and to a blank control. Twenty daphnia were exposed per concanlration. 

The reference substance, potassium dichromate O<2CrzO" art. 4864, batch no. K28974764) was 

obtained from Merck, Darmstadt, Germany. 

Acute Immobilization ofdaphnia after 24 and 48 hours In th9 reference test with porasslum 

dichromate: 

Concentration 

(mgn) 

Number 

Exposed 

% Immobile 

24h 48h 

ExpecIed respon•• (%) 

After 48 hoUrs ' 

Minimal Maxfmal 

Blank-eontrol 20 0 0 0 102 

0.10 20 0 0 0 10 

0.18 20 0 0 0 10 

0.32 20 10 5 0 30 

0.56 20 0 45 0 100 

1.0 20 80 I 100 40 100 

1.8 20 100 100 100 100 

Based on hIStorical data of the pl'e'lllOUS years (0)00). 


A maxhlum 1'8$pOI'I8& of 10% does not InYBIldale the n:lSUlt& of the test 


The actual responses in this reference test with KeCr207 were within the ranges of the expected • 

responses at the different concentralions. Hence, the sensitivity of this batch of Daphnia magna 

was in agreement with the historical data collected at 

The 24h-ECsa was 0.84 mgt! with a 95% confidence Interval between 0.78 and 0.94 mgll. 

The 48h-EC50 was 0.53 mgIJ with a 95% confidence interval between 0.46 and 0.64 mgll. 

The raw data from this study are f<ept in the archives. The fest described above was 

performed under GlP with a QA-check. 

• Page 16­
APPENDIX II ANALYSIS OF TAP WATER (Not performed under GLP) 

Date of sampling : 08-06-05 

Sample numbers : 2106017 (kamer 25) 

Laboratory 

jCOMPONENT: 

Metals: 

Selenium 

Arsenic 

Cadmium 

Chromium 

Copper 

Mercury 

Lead 

ZInc 

Manganese· 

CalCium 

Magnesium 

Iron 

EOX (extractable organic halogens) 

Polycyclic Aromat1c Hydrocarbons: 

. Naphtalene 

Acenaphtylene 

Acenaphtene 

Auorene 

Phenanthrene 

Anthrac::ene 

Auoranthane 

Pyrena 

Benzo(a)antraoene 

Chrysene 

Benzo(b)ftuoranthene 

Benzo(k)ftuoranthene 

Benzo(a)pyrene 

Dibenzo(ah)antracene 

Benzo(ghl)perylene 

Indeno(123cd)pyrene 

PAC's Total EPA (18) 

PAC's Total VROM (10) 

ANALYSIS: 

<0.90 

<5.0 

<0.40 

<1.0 

29 

<0.050 

<5.0 

19 

<0.010 

53 

5.2 

<0.050 

<1.0 

<0.010 

<0.050 

<0.010 

<0.010 

<0.010 

<0.0050 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

<0.010 

.. gil 

pgll 

.. gil 

pgll.,

pgll 

pg/l 

pgn 

IJg/I 

'~I)' 

mglll) 

mgI1 

IJg/I 

pgII 


pgl1 


pgl1 


IJg/I 


pgI1 


pgI1 


pgl1 

pgll 

JIgl1 

pgl1 

J.Ig/I 

pgI1 

IIQI! 

J.Ig/I 

J.Ig/I 

IJVIl 

Total aerobic gerrn-oount {22VC} 2 CFU/ml 1lJ 

Total aerobic germ-count {37!IC} 2 CFUlml1) 

Hardness: 1.6 mmoll1a) 

Nitrate (expr. as N); 0.67 mg/T 

Nitrite (expr. as N); 0.018 mgn 

Cyanlde-Iotasl (NEN 6655) <1.0 ugli 

Turbidity 0.049 FTE 

Colour Intensity 20 mgPt/1 

Dechlorination of tap water was not necessary since no chlorine was present. 


'Tap water was sampled from 1118 copper-free wat\M supply system 1I1at supplied the WIlIer used for the present toxlclly 


study. Among o1h$l8, oopper and baclBrlal activity __ maMCnd in theae samples. The alher comPOrnlflta __ 

analysed In II8/TIPkIG taken from 1he same waI8r supply but sampled In ttle Animal House . 

• Page 17­
P-04-635 


APPENDIX III 


ACUTE TOXICITY STUDY IN DAPHNIA MAGNA WITH 

(FLOW-THROUGH); 

DETERMINATION OF THE CONCENTRATIONS 

Study completion date 

September 13, 2005 

laboratory ProJect Identification 

- Page 1 of 15 • 
1. CONTENTS 

. 1. CONTENTS ...........................................................................................................................2 


2~ REPORT APPROVAL ............................................................................................................3 


3. INTRODUCTION....................................................................................................................4 


3.1. Preface ...........................................................................................................................4 


3.2. Aim of the study ..............................................................................................................4 


4. MATERIALS AND METHODS ...............................................................................................4 


4.1. Reagents ........................................................................................................................4 


4.2. Samples..........................................................................................................................4 


4.3. Analysis ..........................................................................................................................5 


4.3.1. Analytical method .................................................................................................5 


4.3.2. calibration ............................................................................................................5 


4.3.3. Injections ..............................................................................................................5 


4.3.4. Data acquisition and data processing .................................................................. 5 


4.3.5. Procedural recovery samples ..............................................................................6 


4.4. Interpretation ..................................................................................................................6 


4.4.1. Calculations.................................................................... ; ..................................... 6 


4.4.2. Formulas .................................................................................. : ........................... 6 


5. RESULTS...............................................................................................................................8 


5.1. calibration curves ...........................................................................................................8 


52. Samples..........................................................................................................................8 


5.2.1. Procedural recovery samples ..............................................................................8 


5.2.2. Test samples ........................................................................................................8 


TABLES 

Table 1 Procedural recovery samples .........................................................................................9 


Table 2 Concentrations of In test medium (range-finding test) .............. 11 


Table 3 Concentrations of in test medium (lIow·through limit test) ........ 11 


Table 4 Concentrations of In test medium (final flow-through test)........ 12 


FIGURES 

Figure 1 GC chromatograms of a 0.0780 mgll calibration solution [top; 


res.id. 3139] and blank methanol [bottom; res.ld. 3150] .............................................. 13 


Figure 2 GC chromatograms of procedural recovery samples at concentrations of 0.0975 


Figure 3 GC chrometograms of blank control samples taken at t=O hours [top; res.ld. 2884] 


Figure 4 GC chromatograms of target 10 mglJ samples taken at t=-24 hours [top; res.ld. 


Figure 5 GC chromatograms of target 100 mgll samples taken at t ....24 hours [top; res.ld. 


mgll [top; res.id. 3154) and 97.5 mglllboHom; res.ld. 3155] ........................................ 13 


and at t=48 hours [bottom; res.ld. 3234] ...................................................................... 14 


2534J. at t=O hours [middle; res.ld. 2885] and at t=48 hours [bottom; res.ld. 3235J.... 14 


2536J. at t=O hours [middle; res.ld. 2892] and at t=48 hours [bottom; res.ld. 32281.... 15 


- Page 2 ­
2. REPORT APPROVAL 

PRINCIPAl SCIENTIST: 

{Analytical Chemistry) 

--::: ...................................... . 


Date: ,.~; '1.£A)';­

-Page3­
3. INTRODUCTION 

3.1. Prmce 

Study plan 

(analytical study) 

Start 

Completion 

: 30 May 2005 

: 26 July 2005 

3.2. Aim oftheltudy 

The purpose of the analytical study was to determine the actual concentrations in samples taken 

from the test solutions used during the ecQtoxicily test. 

4. MATERIAlS AND METHODS 

4.1. Reagents 

Methanol HPLC-grade. Labscan, Dublin,. Ireland 

MiJli-Q water Tap water purified by reversed osmosis and 

subsequently passed over activated carbon and ionexchange cartridges; Millipore Corp, Bedford. MAo USA 

ISQ.-medium see main report 

Tapwster see main report 

4.2. Samples 

Ail samples not analysed on the sampling day were stored frozen. Stability of the samples under 

these conditions was determined during -Development and validation of 

an analytical method for the analysis of In MiIIi-Q water". On the day of 

analysis. the frozen samples were defrosted at room temperature. 

The samples (10 ml) were analysed using headspace (see 4.3.1 analytical method). 

-Page4- i 

j 

i

• 
4.3. AnalysIs 

4.3.1. Analytical method 

Quantitative analysis was based on the test substance peak In the GC chromatogram of 

Analyflca/condNlans 

Column 

Stationery phase 

Dimensions 

Brand 

Temperature program 

Initial temperature 

Inilial time 

Rate 1 

Temperature 1 

Rate 2 

Final temperature 

Hold time 

Carrier gas 

Column flow 

Injection 

Temperature 

Splitless time 

Injection volume 

Injection speed 

Headspace conditions 

Sample 

Incubation temperature 

Incubation time 

Syringe temperature 

Detection 

4.3.2. Calibration 

CP Porabond Q 

25 m x 0.25 mm, df= 3 I-Im 

Varian, Mulgrave, VIctoria, Australia 

40"C 

1 min 

20°Clrnin 

190"C 

50 GC/min 

250 ·C 

3.3 min 

Helium 

1 mVmln 

Splitless 

250 "c 

2 minutes 

500 IJI (Headspace) 

50 IJIIsec 

10 ml of an aqueous sample in a 20 ml vial 

SO"C 

30 minutes 

110°C 

FlO (250 GC; Hydrogen: 40 mllmin; Air: 450 ml/min) 

During the range finding test, calibration solutions in ISO-medium were made up from two 

standard solutions of in methanol. 

During the flow-through limit and final flow-through test. calibration solutions In tap weter were 

made up from two standard solutions of In methanol. 

4.3.3. Injections 

Calibration solutions were Injected In duplicate. Procedural recovery samples and test samples 

were analysed by single Injection. 

4.3.4. Data acquisition and data processing 

Data acqUisition and data processing was perfonned using Empower software (Waters. Milford, 

MA, USA) version 5.00. 

·Page5­
4.3.5. Procedural recovery samples 

Standard solutions of in methanol and dilutions of these solutJons with 

methanol were used as spiking solutions. 

During the range-finding test, ISO-medium (10 ml) was spiked at concentration levels of 

0.101 mg/l and 101 mgll using 10.1 mg/l and 10085 mgll spiking solutions In methanol. 

During the flow-through limit test on 28-06-05 and 29-06-05. tap water (10 ml) was spiked at 

concentration levels of 0.101 ' mgll and 101 ' mgll using 10.1 mg/l and 10150 mg/l spiking 

solutions in methanol. 

During the flow-through limit test on 04-07-05, tap water (10 ml) was spiked at ooncantration 

levels 010.101 ' mgII and 101 ' mgll using 10.1 mg/l and 10119 mgll spiking solutions In methanol. 

During the final flow-through tast from 19·07-05 and 20-07-05, tap water (10 ml) was spiked at 

concentration levels of 0:1 00' mgll and 97.4' mgll using 10.0 mglt and 9740 mgll spiking 

solutions in methanol. 

During the final flow-through test on 25-07-05, tap water (10 ml) was spiked at concentration 

levels of 0.0975' mgII and 97.5' mgJl using 9.75 mg/l and 9746 mg/l spiking solutions In 

methanol. 

At approximately 0.1 mg/l one and at 100 mg/l five samples were prepared and treated as 

described In paragraph 4.2 'Samples' and analysed. The recovery was calculated for each 

semple 

4.4. Interpret.Uon 

4.4.1. calculations 

All calculations were performed using non-rounded values. Rounded values are reported in the 

tables. Therefore, some differences might be observed when calculating the parameters as 

mentioned In the tables. 

4.4.2. Fonnulas 

~eneral 

_ 1n 

Mean: x=-I: XI 

n 1-1 

where 

:xi = measured value 

n :: number of measurements 

" L(Xi-X}2 

Standard deviation Sn _I = U-,=I=~l--,-­

(n-l) 


Coefficient of variation 9n,:I.1oo% 

X 

I Concentrations were not corrected for spiking volume 

- Page 6 • 
Calibration 

Response: 	 R = Peak area test substance [units] 

Calibration curve: 	 The response was correlated with the test substance 

concentration, using linear regression analysis (least 

squares method; weighting factor (1!concentration2)). 

R = response calibration solution [units] 


C = concentration of test substance in calibration 


solution [mg/ll 

a = slope [unlts.Umg] 

b = Intercept [units] 

~amples 

Concentration of ASAHIKLIN AE-3000 analysed In the samples:. 

R = response sample [units) 

d = dUunon factor 

a ::z slope {unlts.Vmgl 

b = intercept [units) 

Recovery of procedural recovery samples: 

Concentration analysed • 100 [%] 

Concentration prepared 

Concentration analysed • 100 [%] Relative to nominal concentration: Concentration nominal 

Relative to mean initial concentration: Concentration analysed at t =x hours • 100 (%] 

Mean concentration analysed at t = 0 hours 

Concentration analysed II< 100 [%) Relative to target: Concentration target 

- Page 7­

i 

I 

I 
5. RESULTS 

5.1. Calibration curves 

On each day of analysis. a calibration curve was constructed using six concentration leve!s. At 

each concentration level, two responses were used. The coefficient of correlation was in excess 

of 0.99 for each calibration curve. 

Agure 1 shows chromatograms of a calibration solution and blank methanol. 

5.2. Sample. 

5.2.1. Procedural recovery samples 

Figure 2 shows chromatograms of procedural recovery samples. The results for the procedural 

recovery sampfes are summarized In Table 1. Mean recoveries of procedural recovery samples 

are required to be In the range 70-110%. 

Because the recoveries of the procedural recovery samples were met, the results of the test 

samples ware considered accurate. 

5.2.2. Test samples 

Figures 3 - 5 show chromatograms of blank control samples, nominal 1 0 mgn samples, and 

nominal 100 mg/l samples. The results for the test samples are summarized in Tables 2 - 4. 

During the flow-through tests, a small peak at the retention time of the test substance was 

observed in the chromatograms of several blank test samples (nominal 0 mgll). Because a similar 

peak was found In blanks of methanol It was concluded that the compound did not derive from 

test medium. For most of the blank test samples, the response of the interference was smaller 

than fhe response of the lowest calibration solution, therefore < LOQ was given. For one blank 

test sample a response within the concentration range of the calibratIon solutions was found and 

therefore an accurate value was given. The maximum contribution of the compound to the peak 

area of the lowest test sample was 0.6%, and was therefore not considered to be a significant 

interference. 

·PageS­
TABLES 

Table 1 Procedural recovery samples 

Concentration Recovery Mean recovery I 


(Coefficient of 


Date of Date of Concentration

preparation analysis nominal analysed r.41

[mgll] variation) : [dd.mm-yy] [mgll] [dd-mm-yy] 

0.101 
 0.109 108
 ~I 30-05-05 1 


30-05-05 30-05-05 1 


98 


101 


30-05-05 101
 98.6

78 


101 


79.1 

91 


101 


105 
 104


(15) 


101 


100 
 99


74 


108 


74.2 

0.109 


28-06-05 1 


28-06-05 28-06·05 0.101 1 


98 


101 


28-06-05 101
 99.7

99.4 98


99 


101 


101 
 99.0 98


(1.3) 


101 


102 
 101


100 
 99


0.0813 z 80
 29-06-05 ' 29-06-05 , 0.101

96 


101 


29-06-05 29-06-05 101
 97.6 1 


95 


101 


96.7 

95 


101 


96.3 95 


(1.3) 


101 


94.8 93


93 


0.0989 2 


94.8 

98 


04-07-05 1 


04-07-05 04-07-05 0.101 1 


86.0 85 


101 


04-07-05 101


92 


101 


92.8 

87.7 87 
 90


(4.1) 


101 


101 
 91.4 90


94 


19-07-05 


95.0 

104 


19-07-05 


19-07-05 0.100 0.104

19-07-05 97.4 101
 104


97.4 87.5 90


97.4 101 
 104
 101


97.4 104 
 106
 (6.5) 

97.4 98.6 101


-Page9­
Table 1 Continued 

Date of Date of Concentration Concentration Recovery !Mean recovery 

analysis nominal analysed [%J i (Coefficient of 

[dd-mm-yy] 

preparation 

[dd-mm-yy] [mgll] [mgll] variation)

r%l 

20-07-05 20-07-05 0.100 0.109 109

20-07-05 20-07-05 97.4 83.7 86

97.4 87.2 89 

97.4 88.2 91 89 , 97.4 85.9 88 (2.0) 

97.4 87.1 89 

25-07-05 25-07-05 0.0975 0.0866 89 

25-07-05 25-07-05 97.5 95.9 98 

97.5 98.4 101 

97.5 100 103 101

97.5 99.5 102 (2.0) 

97.5 96.3 99

Combined with 


ConcenlraHon was below \he Umlt ofquanllflcation. but withIn the concen1ration range ofthe ealibration solUuons. 


-Page 10­
Table 2 Concentrations of In test medium (range-finding test) 

I Tlmeof 

• sampling 

[hours] 

Date of 

sampling 

[dd-mm-yyJ 

Date of 

analysis 

[dd-mm-yy] 1 

Concentration 

Nominal 

[mgl\] 

Analysed 

[mgIJ] 

Relative to 

nominal 

[%1 

Relative to 

mean Initial 

r%l 

0 

48 

23-05-05 

25-05-05 

30-05-05 

30-05-05 

100 2 

100 2 

100 

100 

3.71 

6.58 

3.32 

6.86 

4 

7 

3 

7 

65 

133 

samples were frozen unlll analysis. 


Combined with 


Table 3 Concentrations of in test medium (flow-through limit test) 

1 Concentration i Time of Date of Date of Relative to sampling sampling analysis Target Analysed target [hours] [dd-mm-yy] [dd-mm-yy] [mgl\] [mgl\] f%] 

I -24 28-06-05 28-06-05 221 93.7 42 

221 102 46 I 

0 29-06-05 29-06-05 0 n.d. n.a. 

0 n.d. n.8. . 221 95.7 43 

221 100 45 

01-07-05 04-07-05 <LOQ2 1 48 0 n.a. 

0 <LOQ2 n.a. 

221 98.5 45 

221 99.7 45 

I 

Samples were frozen un1I ana1y.si5. 

LOQ 2 A small peak was obseM!d at the retenllon lime of lest lIub11ance, but Its response was not ccnsidered to derive 

from test sample since it was also obserwd In blank m9lt1aoo1. Umitof quantification 'ME 0.100 mgll (See 

"Development and validation of an analytical method for It. analysis of 

In MIIII-Q waler"~ 

n.d. Not detected 

n.8. Not applicable 

- Page 11 ­
in test medium (final flow-through test) Table 4 Concentrations of 

Concentration 

Time of 

sampling 

[hours] 

Date of 

sampling 

[dd-mm-yy] 

Date of 

analysis 

[dd-mm-yy] 

Target 

[mgll] 

Analysed 

[mgn] 

Relative to 

target 

f%1 

-24 19·.Q7-05 ,19-07-05 22 7.80 35 

22 7.64 35 

221 96.8 44 

221 93.7 42 

0 20-07-05 20-07-05 0 <LOQ2 n.a. 

0 0.130 n.a. 

22 7.60 35 

22 8.00 36 

40 20.3 51 

40 20.5 51 

71 15.3 22 

71 15.3 22 

123 54.0 44 

123 49.3 40 

221 90.2 41 

221 86.2 39 

48 22-07-05 26-07-051 0 

0 

<Loa 2 

<lOO2 

n.a. 

22 1.56 7 

22 n.1. n.a. 

40 10.4 26 

40 10.4 26 

71 8.55 12 

71 8.20 12 

123 78.3 :5 64 , 123 36.7 3 30 

221 105 48 

221 102 46 

Slwnples ware frozen unb1 analysla, 

Loa Z Asma. peak was observed at the I'8tentlon !me of lest substance. but Its response W8B not considered to <IerIYe 

from test sample sir1C8 it was also obselYBd in blank methanol. LImit of quanllf\caUon was 0.100 mgll (See 

: "Dllwloprnent and validatioo of an anCllytlca1 method lor the analysis of 

In MlnJ·Q wate!"). 

No analyUcaI explanaUon can be given for the dlfflll'8nflal results of the duplicate samples. 

n.a. Not applicable 

n.l. Not iJVectvC (sample viCiI we8 broken). 

- Page 12­
1 

FIGURES 

- Figure 1 	 GC chromatograms of a 0.0780 mgll calibration 

solution [top; ...Id. 3139} and blank methanol [bottom: resold. 3150]. 

1. 

..:.. ..... Figure 2 	 GC chromatograms of proceduraJ recovery sample. at concentrations of 

0.0975 mgtl [top; res.id. 3154] and 97.5 mglllbottom; res.ld. 3155]. 
_. ".. Figure 3 	 GC chromatograms of blank control samples taken at t=O hours 

[top; res.ld. 2884} and at t=48 hours [bottom; res.ld. 3234] . 

.~.~---~~... ~...­

.~ -~--.~ ·~*----1~-----~----.-&----.~----_-i--~_-i-----~~ ~ .-~----~-_----H!.----Q-~----1~ 

- Figure 4 	 GC chromatograms of target 10 mgll samples taken at f:lL.24 hours 

[top; res.ld. 2534]. at t-o hours [middle; resold. 2885] and at t=48 hours 

[bottom; res.ld. 3235J. 
.~,oJ---~~ 

.~~------------~.~,-----­

.:j~~~--~~~--.~-=~~e---~~~~:~==~.~,--~~~, -
 ..~&~~~.~-=~~.~~..~.~~_~~.~ Figure 5 	 GC chromatograms of target 100 mgllsamples taken at t=-24 hours 

[top; res.ld. 2536), at t=O hours [middle; res.ld. 2892) and at t=48 hours 

[bottom; .....Id. 3228]. 

- Page 15­
P-04·635 


LOCAL L ¥MPH NODE ASSAY 

REGULATORY/REPORT 

DATA REQUIREMENT: EPA OPPTS 870.2600 (2003) 

OECD 429 (2002) 

AUTHORS: 

STUDY COMPLETION DATE: 27 October 2005 

PERFORMING LABORATORY: 

LADORA TORY PROJECT ID: 	 Study Number: 

Report Number: 

Contract Number: 

SPONSOR: 

Report Number: 	 Page 1 of21 
GOODLABORATORYPRACTICECO~LMNCESTATEMENT 

I, the undersigned, declare that the objectives laid down in the protocol were achieved and that 

the data generated are valid. The report fully and accurately reflects the procedures used and the 

raw data generated in the above study. 

The study (GM7907) was conducted in compliance with the UK Principles ofGood Laboratory 

Practice (The United Kingdom GLP Regulations 1999, Statutory Instrument No. 3106, as 

amended 2004, Statutory Instrument No. 994) except for the deviation listed below. These 

Principles are in accordance with the OECD Principles ofGood Laboratory Practice, revised 

1997 (ENVlMClCHEM(98) 1 7). 

The following GLP deviation is considered not to affect the integrity of the study or the validity 

ofthe conclusions drawn: 

(i) 	 the stability and achieyed concentration of the test substance in the vehicle used were not 

determined by analysis. 

27 October 2005 

Study Director Date 

Research and Investigative Toxicology 

Performing Laboratory: 

Report Number: 	 Page 2 of 21 
Report Number: Page 3 of 21 
QUALITY ASSURANCE STATEMENT 

In accordance ",rith policy and QA procedw-es for Good Laboratory Practice, this report has 

been audited and the conduct ofthis study has been inspected as follows: 

Date AuditlInspection Date ofQA Report 

04 Aug 2005 Draft report 09 Aug 2005 

22 Oct 2005 Final report review 22 Oct 2005 

In addition, inspections associated with this type of study were made as follows: 

04 Apr 2005 Dose preparation 04 Apr 2005 

20 Apr 2005 Dose administration, bodyweights 22 Apr 2005 

16 May 2005 Protocol 16 May 2005 

23 May 2005 Radiolabelled preparation, injection of 23 May 2005 

radiolabel 

23 May 2005 Processing, removal ofJymph nodes 24 May 2005 

07 Jun 2005 Scintillation counting 14Jun 2005 

Facilities and process based procedures associated with this type ofstudy were inspected in 

accordance with QA Standard Operating Procedures. 


So far as can be reasonably established, the methods described and the results given in the final 


report accurately reflect the raw data produced during the study, GM7907. 


27 October 2005 

Quality Assurance Unit) 

Report Number: Page 3 of 19 

Report Number: Page 4 of 21 
Report Number: Page 5 of 21 
GENERAL INFORMATION 

Contributors 

The following contributed to this report in the capacities indicated: 

Name 	 Title 

Study Director 

Study Licensee 

Study Reviewer 

Study dates 

Study initiation date: 171h June 2005 

Experimental start date: 22nd June 2005 

Experimental termination date: 281h June 2005 

Deviations from the guidelines 

None 

Retention ofsamples 

An original report, the study protocol and all raw data, samples and specimens pertaining to this 

study are retained in the Archives, . 

for a minimum of 5 years from the date of issue ofthe final report. 

At the end ofthis period the Sponsor will be contacted regarding the further retention, return to 

the Sponsor or destruction ofthe archived materials. 

Report Number: 	 Page 6 of 21 
TABLE OF CONTENTS 

THE TITLE PAGE 1 


GOOD LADORATORY PRACTICE COMPLIANCE STATEMENT 


defined. 


QUALITY ASSURANCE STATEMENT 


GENERAL INFORMATION 6 


TABLE OF CONTENTS 7 


1.0 EXECUTIVE SUMMARY 9 


1.1 Study design' 9 


1.2 Reswts 9 


1.3 Conclusion 9 


2.0 INTRODUCTION 10 


2.1 Purpose 10 


2.2 Regulatory guidelines 10 


2.3 Justification for test system selection 10 


2.4 Positive control study 10 


2.5 Study dates 10 


2.6 Data storage 10 


3.0 MATERIALS AND METHODS 11 


3.1 Test substance 11 


3.2 Positive control substance 11 


3.3 Vehicles 11 


3.4 Dose preparations 11 


3.5 Experimental design 12 


3.5.1 Animals 12 


3.5.2 Accommodation and husbandry 12 


3.5.3 Acclimatisation 12 


3.5.4 Animal identification 13 


3.5.5 Test method for the evaluation of 13 


3.5.6 Clinical observations 13 


3.5.7 Bodyweights 13 


3.5.8 Positive control study 13 


3.6 Data evaluation 14 


Report Number: Page 7 of 21 

4.0 RESULTS 14 

4.1 Evaluation of test substance (Table 1) 14 


4.2 Positive control (Table 2) 14 


5.0 CONCLUSIONS 14 


6.0 REFERENCES 15 


TABLES SECTION 16 


TABLE 1 SKIN SENSITISA TION POTENTIAL OF 17 


TABLE 2 SKIN SENSITISA TION POTENTIAL OF THE POSITIVE CONTROL 


SUBSTANCE (HEXYLCINNAMALDEHYDE) 18 


APPENDICES SECTION 19 


APPENDIX 1 BODYWEIGHTS (g) 20 


Report Number: Page 8 or' 21 

1.0 EXECUTIVE SUMMARY 

1.1 Study design 

A sample of was assessed for its skin sensitisation potential using the mouse Local 

Lymph Node Assay. The assay determines the level ofT lymphocyte proliferation in the lymph 

nodes draining the site of chemical application, by measuring the ammmt of radiolabelled 

thymidine incorporated into the dividing cells. The test substance was applied as 25, 50 or 100% 

w/v preparations in acetone. A vehicle control group was similarly treated using acetone alone. 

1.2 Results 

• The test substance did not have the capacity to cause skin sensitisation when applied as 25 or 50 


%w/v preparations in acetone or when applied neat. 


In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin 


sensitisation when applied as 10% and 25% w/v preparations in acetone in olive oil (4: 1), 


confirming the validity ofthe protocol used for this study. 


1.3 Conclusion 

In conclusion, is unlikely to be a skin sensitiser under the conditions of the test. 

Report Number: Page 9 of 21 
2.0 INTRODUCTION 

2.1 Purpose 

The purpose of this study was to assess the skin sensitisalion potential of using the 

Local Lymph Node Assay (Kimber and Basketter 1992, Kimber et aI19Y4). 

2.2 Regulatory guidelines 

This study was conducted in accordance with the following Regulatory Guidelines: 

a) 	 OECD guideline reference 429 (2002): Skin sensitisation: Local Lymph Node Assay. 

b) 	 United States Environmental Protection Agency, Health Effects Test Guidelines, OPPTS 


870.2600 (2003): Skin Sensitisation. 


2.3 Justification for test system selection 

The CBAlCa mouse is the species and strain of choice because previous examination of strain 

difference in lymphocyte proliferation responses to skin sensitisers showed this strain ofmouse 

to exhibit the most vigorous response (Kimber et ai, 1994). 

2.4 Positive control study 

The reliability of the test system is assessed at approximately 6-monthly intervals using a known 

sensitiser (hexy1cinnamaldehyde, Dearman et ai, 2001). The positive control study closest in 

time to the main study is included in this report. 

2.5 Study dates 

The main study was initiated on 17th June 2005. The experimental phase started on 22nd June 

2005 and was completed on 28th June 2005. 

For the positive control study, the experimental phase started on 15th June 2005 and was 

completed on 21 st June 2005. 

2.6 Data storage 

An original report, the study protocol and all raw data, samples and specimens pertaining to this 

study are retained in the Archives, 

fQr a minimum of 5 years from the date of issue of the final report. 

At the end of this period the Sponsor will be contacted regarding the further retention, return to 

the sponsor or destruction of the archived materials. 

Report Number: 	 Page 10 of 21 
3.0 MATERIALS AND METHODS 

3.1 Test substance 

Name: 

Source: 

Colour: Colourless 

Physical state: Liquid 

Batch reference number: 

test substance reference number: 

Purity (% w/w): 99.99% 

Storage conditions: Ambient temperature in the dark 

Stability: 21th September 2005 

From information supplied by the Sponsor the test substance was used within the expiry date. A 

certificate of analysis (dated 21th September 2005) is retained in the Archives. The test 

substance characterisation was the responsibility of the Sponsor. 

3.2 Positive control substance 

Name: Hexylcinnamaldehyde 

Source: Aldrich Chemical Company 

Colour: Yellow 

Physical state: Liquid 

test substance reference ,number: 

Storage conditions: Refrigerated, under an inert gas, in the dark' 

3.3 Vehicles 

The vehicle for the test substance was acetone (I 

The vehicle for the positive control substance was acetone in olive oil (4: 1; 

3.4 Dose preparations 

All dose preparations were used within 24 hours of preparation. 

Report Number: Page 11 of 21 
No correction was made for the purity ofthe active ingredient in the test substance. Stability and 

achieved concentration were not determined. 

3.5 Experimental design 

3.5.1 Animals 

Species: 


Strain: 


Source: 


Sex: 


Number used: 


Specification: 


Mouse 

CBAlCa 

Charles River 

Female 

4 per group 

Young adults (8-12 weeks of age) 

3.5.2 Accommodation and husbandry 

A maximum of 4 mice was housed per cage, in cages suitable for animals ofthis strain and 

weight range. Environmental enrichment provided included tents, bases and nestlets. 

The animal room was designed to give the environmental conditions shown as follows. 

Temperature: 22±3°C 

Relative humidity: 30-70% 

Air: A minimum of 15 changes per hour 

Light cycle: Artificial, giving 12 hours light, 12 hours dark 

Both temperature and relative humidity were recorded daily. In general, the recorded values 

were within the specified ranges and any deviation is considered not to affect the integrity of the 

study. 

Diet , supplied by and mains water, 

supplied by an automatic system, were available ad libitum. 

Each batch of diet is routinely analysed for composition and for contaminants. Water is also 

periodically analysed for contaminants. No contaminants were found in the diet or water at 

levels considered likely to interfere with the purpose or outcome of the study. Certificates of 

analyses are retained in the 

3.5.3 Acdimatisation 

The animals were housed under the experimental conditions for at least 5 days, prior to th~ start 

of dosing. 

Report Number: Page 12 of 21 
3.5.4 Animal identification 

Animals were individually identified, within each cage, by a clipped area on the side(s). The first 

animal in each cage had a clipped area on the left side, the second on the right hand side, the 

third on both flanks and the fourth not clipped. 

3.5.5 Test method for the evaluation of 

Groups of four female mice were used for this study. Approximately 25JlI of a 25, 50 or 100% 

w/v preparation of the test substance in acetone was applied, using a variable volume micropipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using 

acetone alone. The procedure was repeated daily for 3 consecutive days. 

Three days after the third application, all the animals were injected, via the tail vein, with 

approximately 250JlI of phosphate buffered saline (PBS) containing 20J.1Ci of a 2.0Ci/mmol 

specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely 

killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular 

lymph nodes were removed from each animal and, together with the nodes from the other 

animals in the group, were placed in a container of PBS. 

A single cell suspension was prepared by mechanical disaggregation oflymph nodes through a 

200-mesh stainless steel gauze. The cell suspensions were then washed three times by 

centrifugation with approximately 10rnl of PBS. Approximately 3rnl of 5% w/v trichloroacetic 

acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by 

centrifugation and the supernatant was discarded. The cells were then resuspended in 

approximately 1 rnl of TCA. 

The lymph node suspensions were transferred to scintillation vials and 10rnl of scintillant 

(Optiphase) was added prior to J3-scintillation counting using a Packard Tri-Carb 31OOTR Liquid 

Scintillation Counter. 

3.5.6 Clinical observations 

Animals were checked at least once daily for signs of systemic toxicity. 

3.5.7 Bodyweights 

The bodyweight of each animal was recoded prior to dosing as day 1 and prior to injection of3

Hmethyl thymidine and day 6 bodyweights are given in Appendix 1. 

3.5.8 Positive control study 

The sensitisation potential of hexylcinnarnaldehyde was assessed using the method described 

above in 3.5.5. 

Report Number: Page 13 of 21 
Approximately 25/l1 of a 5%, 10% or 25% w/v preparation ofhexylcinnamaldehyde in acetone 

in olive oil (4: I) was applied, and a vehicle control group was similarly treated using acetone in 

olive oil alone (4:1). 

3.6 Data evaluation 

The results are expressed as a disintegrations per minute (dpm) value per lymph node for each 

group. The activity of each test group is then divided by the activity of the vehicle control group 

to give a testcontrol ratio known as the stimulation index (SI), for each concentration. 

The criterion for a positive response is that one or more concentrations ofthe test substance 

should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control 

group. The assay is able to identifY those materials that elicit responses in standard guinea pig 

tests for skin sensitisation (Kimber et aI1994). Consequently, a test substance which does not 

fulfil the above criterion is designated as unlikely to be a skin sensitiser. 

4.0 RESULTS 

4.1 Evaluation oftest substance (Table 1) 

The application of the test substance at concentrations of25, 50 and 100% w/v in acetone 

resulted in an isotope incorporation which was less than 3-fold at all concentrations. 

Consequently, the test substance is designated as unlikely to be a skin sensitiser under the 

conditions of the test. 

4.2 Positive control (Table 2) 

The application ofhexylcinnamaldehyde at concentrations of 5%, 10% and 25% w/v in acetone 

in olive oil (4: 1) resulted in a greater than 3-fold increase in isotope incorporation at the 10 and 

25 %w/v concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, 

confirming the validity of the protocol used for the study. 

5.0 CONCLUSIONS 

In conclusion, is unlikely to be a skin sensitiser under the conditions of the test. 

Report Number: Page 14 of 21 
6.0 REFERENCES 

Deannan R J, Wright Z M, Basketter D A, Ryan C A, Gerberick G F and Kimber I (2001), The 


suitability ofhexyl cinnamic aldehyde as a calibrant for the murine local lymph node assay. 


Contact Dermatitis, 44, 357-361. 


Kimber I and Basketter D A (1992). The murine local lymph node assay: a commentary on 


collaborative studies and new directions. Fd Chem Toxic 30, 165-169. 


Kimber I, Deannan RJ, Scholes EW and Basketter DA (1994). The Local Lymph Node Assay: 


Developments and Applications. Toxicology, 93, 13-31. 


Report Number: Page 15 of 21 

TABLES SECTION 


Report Number: Page 16 of 21 

TABLEt SKIN SENSITISATION POTENTIAL OF 

Concentration of 

test substance (% 

w/v) 

Number of lymph 

nodes assayed 

Disintegrations 

per minute 

(dpm) 

dpmper 

lymph node 

Test: control 

ratio (SI) 

o(vehicle only) 

25 

50 

100 

8 

8 

8 

8 

3352 

3748 

3500 

2813 

419 

469 

438 

352 

N/A 

1.1 

1.0 

0.8 

NtA - not applicable 

Report Number: Page 17 of 21 
TABLE 2 	 SKIN SENSITISATION POTENTIAL OF THE POSITIVE 

CONTROL SUBSTANCE (HEXYLCINNAMALDEHYDE) 

Current Study No.: 

Concentration of 

hexylcinnamaldehy de 

(% w/v) 

Number of 

lymph nodes 

assayed 

Disintegrations 

per minute 

(dpm) 

dpm per lymph 

node 

Testcontrol ratio 

(SI) 

o(vehicle only) 

5 

10 

25 

8 

8 

8 

8 

1636 

3793 

6821 

24807 

205 

474 

853 

3101 

N/A 

• 

2.3 

4.2 

15.1 

NIA - not applicable 

Report Number: Page 18 of 21 
APPENDICES SECTION 


Report Number: Page 19 of 21 

APPENDIX 1 BODYWEIGHTS (g) 

DOS&: 0 %w/w 

ANIMAL DAY DAY NOMBBR 1 6 

If'I!!Ml\.LES 

45 17.3 20.0 


46 l1L4 21.7 


47 lB.9 21.5 


4B 19.B 22.4 


DOSE: 25 "W/V 

ANIMAL DAY DAY 

NOMBBR 1 6 

1"I!lKII.I.ZS 

451 15.B lB.4 


50 19.2 21.1 


51 151.7 22.4 


52 lB.O lB.4 


Report Nwnber: Page 20 of 21 
APPENDIX 1 BODYWEIGHTS (g) 

OOS8: 50 ""IV 

ANlJ!oIIU. DAY DAY NUMII!.R 1 6 

l!'DlIlUJIIS 

53 lIL3 20.2 


54 19.0 20.7 


55 1S1.8 21.2 


56 20.4 22.1 


00511: 100 %W/W 

ANIMAL DAY DAY NtlMB!.R 1 6 

I'BMlUoII:S 

57 18.5 19.5 


58 18.3 19.7 


sg 18.0 lSI.7 


60 20.3 22.4 


Report Number: Page 21 of 21