## FINAL REPORT

Study Title



## ACUTE TOXICITY STUDY IN DAPHNIA MAGNA WITH

(STATIC)

Author

Test Facility

Laboratory Project Identi fication

- Page 1 of 28

-- Page 2 -

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

.................................................................................................................................. 8

6. MATERIALS AND METHODS

.......................................................................................................... 8

6.1. Test Substance

......................................................................................................................... 8

6.1.1. Test substance information

............................................................................................. 8

6.1.2. Study specific test substance information

...................................................................... 8

6.1.3. Reference substance

...................................................................................................... 9

6.2. Test system

............................................................................................................................... 9

6.3. Breeding

.................................................................................................................................... 9

6.4. Preparation of test solutions

.................................................................................................... 10

6.5. Combined limit/range-finding test

........................................................................................... 10

6.6. Final test

.................................................................................................................................. 10

6.6.1. Test concentrations

....................................................................................................... 10

6.6.2. Test procedure and conditions

..................................................................................... 11

6.6.3. Sampling for analysis of test concentrations

................................................................ 11

6.6.4. Measurements and recordings

..................................................................................... 11

6.7. Electronic data capture

........................................................................................................... 12

6.8. Interpretation

........................................................................................................................... 12

6.8.1. Data handling

................................................................................................................ 12

6.8.2. Acceptability of the test

................................................................................................ . 12

6.9. List of deviations

..................................................................................................................... 12

6.9.1. List of protocol deviations

............................................................................................. 12

6.9.2. List of standard operating procedures deviations

......................................................... 12

7. RESULTS

....................................................................................................................................... 12

7.1. Combined limit/range-finding test

........................................................................................... 12

7.2. Final test

.................................................................................................................................. 13

7.2.1. Measured concentrations

............................................................................................. 13

7.2.2. Immobility

...................................................................................................................... 13

7.2.3. Determination of effect concentrations

......................................................................... 14

7.2.4. Experimental conditions

................................................................................................ 15

8. CONCLUSION

................................................................................................................................ 15- Page 3 -

TABLES

Table 1 Incidence of immobility in the combined limit/range-finding test

.................................. 13

Table 2 Acute immobilisation of daphnids after 24 and 48 hours in the final test

..................... 14

Table 3 Effect parameters

.......................................................................................................... 14

Table 4 pH and oxygen concentrations during the final test

...................................................... 15

Table 5 EC 50 value at 24 hours and related parameters

........................................................... 16

Table 6 EC 50 value at 48 hours and related parameters

........................................................... 17

FIGURES

Figure 1 Percentage response (=immobility) of Daphnia magna as function of the log

concentration of at 24h

................................................................ . 16

Figure 2 Percentage response (=immobility) of Daphnia magna as function of the log

concentration of at 48h

................................................................ . 17

APPENDICES

APPENDIX 1 EC -VALUES

.................................................................................................................. 16

APPENDIX 2 REFERENCE TEST

...................................................................................................... 18

APPENDIX 3 GLP CERTIFICATE

...................................................................................................... 19

APPENDIX 4 ANALYTICAL REPORT

................................................................................................ 20- Page 6 -

4. SUMMARY

Acute Toxicity Study in Daphnia magna with

The study procedures described in this report were based on the OECD guideline No. 202

, 2004. In

addition, the procedures were designed to meet the test methods of the Commission Regulation (EC)

No 440/2008

, Part C.2, 2008

, the ISO International Standard 6341, 1996 and the OECD series on

testing and assessment number 23, 2000.

The batch of tested was a and not

completely soluble in test medium the loading rate initially prepared.

A final test was performed based on the results of a preceding combined limit/range-finding test.

Preparation of test solutions started with a loading rate of 100 mg/l applying two days of magnetic

stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous

mixture was left to stabilize for one hour, where after the colourless Water Soluble Fraction (WSF) that

contained small droplets was siphoned out and used as the highest test concentration. The lower test

concentrations were prepared by subsequent dilutions of the WSF in test medium.

Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to 1.0,

2.2, 4.6, 10, 22, 46 and 100% of the WSF and to a control. The total test period was 48 hours and

samples for analytical confirmation of actual exposure concentration were taken at the start and the

end of the test.

Analysis of the samples taken at the start of the final test showed measured concentrations of 0.050,

0.14, 0.27, 0.91, 1.5, 2.8 and 6.1 mg/l at 1.0, 2.2, 4.6, 10, 22, 46 and 100% of the WSF, respectively

. During the exposure period the measured concentrations decreased to 33-44% of initial at the end of

the test. Based on these results, the average exposure concentrations were calculated to be: 0.032,

0.078, 0.18, 0.55, 0.96, 1.9 and 3.9 mg/l.

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

did not induce significant acute immobilisation of Daphnia magna at 0.18 mg/l after

48 hours of exposure (NOEC).

The 48h- EC 50 was 0.29 mg/l based on average exposure concentrations (95% confidence interval

between 0.25 and 0.37 mg/l).- Page 7 -

5. INTRODUCTION

5.1. Preface

Sponsor

Study Monitor

(on behalf of the sponsor)

Test F acility

Study Director

Technical Coordinator

Principal Scientist

Study Plan Start : 21 February 2012

Completi on : 15 March 2012

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 mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to

determine the EC 50 at 24 and 48 hours of exposure.

5.3. Guidelines

The study procedures described in this report were based on the Organization for Economic Cooperation and Development (OECD), OECD guidelines for Testing of Chemicals, guideline No. 202 :

" Daphnia sp ., Acute Immobilisation Test", Adopted April 13

, 2004

.

In addition, the procedures were designed to meet the test methods of the following guidelines and

guidance document:

Commission Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination

of ecotoxicity, Publication No. L142, C.2. "Daphnia Sp. Acute Immobilisation Test".

ISO International Standard 6341: "Water quality - Determination of the inhibition of the mobility of

Daphnia magna Straus - Acute toxicity test, Third edition, 1996- 04

- 01.

Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series

on testing and assessment number 23, December 14, 2000.

5.4. Storage and retention 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 2 years after finalization of the report. After this period, the sponsor will be

contacted to determine how the records and materials should be handled. will retain

information concerning decisions made.- Page 8 -

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.

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 within 15 seconds after gentle agitation of the test

vessel.

The EC 50 is the concentration of test substance estimated to immobilise 50% of the daphni ds after a

defined period of exposure.

No Observed Effect Concentration (NOEC) is the highest concentration tested at which no effect

(i.e. immobilisation) is recorded.

6. MATERIALS AND METHODS

6.1. Test Substance

6.1.1. Test substance information

Identification

Structure

Molecular formula

Molecular weight

CAS Number

Description

Batch

Purity

Test substance storage

Stability under storage conditions

Expiry date 17 October 2012

6.1.2. Study specific test substance information

Hygroscopic No

Volatile No

Test substance handling Flush con tainer with nitrogen after handling

Specific Gravity / Density

Stability in water At least 24 hours

Solubility in water No- Page 9 -

6.1.3. Reference substance

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

(APPENDIX 2 )

.

6.2. Test system

Species Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at

least third generation, obtained by acyclical 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.

Validity of batch Daphnids originated from a healthy stock, 2 nd to 5 th brood,

showing no signs of stress such as mortality >20%,

presence of males, ephippia or discoloured animals and

there was no delay in the production of the first brood.

Characteristics For the test selection of young daphni ds 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 all-glass

culture vessel.

Maximum age of the cultures 4 weeks

Renewal of the cultures After 7 days of cultivation half of the medium twice a week.

Temperature of medium 18 -22°C

Feeding Daily, a suspension of fresh water algae.

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).

Composition of medium M7:

Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (ROwater

, GEON Waterbehandeling

, Berkel-Enschot

, The Netherlands ):

Macro salts: CaCl 2 .2H 2 O 211.5 mg/l

MgSO 4 .7H 2 O 88.8 mg/l

NaHCO 3 46.7 mg/l

KCl 4.2 mg/l

Medium M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO medium to reach the following

concentrations:

Trace elements: B 0.125 mg/l

Fe 0.05 mg/l

Mn 0.025 mg/l

Li, Rb and Sr 0.0125 mg/l

Mo 0.0063 mg/l- Page 10

- Br 0.0025 mg/l

Cu 0.0016 mg/l

Zn 0.0063 mg/l

Co and I 0.0025 mg/l

Se 0.0010 mg/l

V 0.0003 mg/l

Na 2 EDTA.2H 2 O 2.5 mg/l

Macro nutrients: Na 2 SiO 3 . 9H 2 O 10.0 mg/l

NaNO 3 0.27 mg/l

KH 2 PO 4 0.14 mg/l

K 2 HPO 4 0.18 mg/l

Vitamins: Thiamine 75.0 μg/l

B 12 1.0 μg/l

Biotin 0.75 μg/l

The hardness: 180 mg/l expressed as CaCO 3 and the pH: 7.7 ± 0.3.

6.4. Preparation of test solutions

The standard test procedures required generation of test solutions, which should contain completely

dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The

testing of concentrations that disturb the test system should be prevented (e.g. film of the test

substance on the water surface).

The batch of tested was a and not

completely soluble in test medium the loading rate initially prepared.

Preparation of test solutions started with a loading rate of 100 mg/l applying two days of magnetic

stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous

mixture was left to stabilize for one hour, where after the clear and colourless Water Soluble Fraction

(WSF) was siphoned out and used as the highest test concentration. Note that the WSF prepared for

the final test contained small droplets. The lower test concentrations were prepared by subsequent

dilutions of the WSF in test medium.

6.5. Combined limit/range-finding test

The study started with a combined limit/range-finding test. Twenty daphnids per concentration (four

replicates, 5 daphnids per vessel) were exposed to a control and a WSF prepared at a loading rate of

100 mg/l. Test procedure and conditions were similar to those applied in the final test with the

following exceptions:

Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to 0.10, 1.0 and

10 % of the WSF;

Dissolved oxygen concentrations and pH were only measured in the control and the highest

test concentration.

6.6. Final test

6.6.1. Test concentrations

1.0, 2.2, 4.6, 10, 22

, 46 and 100% of a WSF prepared at a

loading rate of 100 mg/l

Controls Test medium without test substance or other additives- Page 11

-

6.6.2. Test procedure and conditions

Test duration 48 hours

Test type Static

Test vessels 100 ml, all-glass

Medium Adjusted ISO medium

Number of daphni ds 20 per concentration

Loading 5 per vessel containing 80 ml of test solution

Light 16 hours photoperiod daily

Feeding No feeding

Aeration No aeration of the test solutions.

Introduction of daphni ds Within 30 minutes after preparation of the test solutions.

6.6.3. Sampling for analysis of test concentrations

Samples for analysis were taken from all test concentrations and the control according to the schedule

below. The method of analysis is described in the appended Analytical Report (APPENDIX 4)

.

Frequency at t=0 h and t= 48 h

Volume 4 ml from the approximate centre of the test vessels

Storage Not applicable, samples were analysed on the day of

sampling.

At the end of the exposure period, the replicates of the four lowest test concentrations were pooled at

each concentration before sampling. Due to the presence of a floating layer the samples from the

three highest test concentrations were taken from one replicate.

6.6.4. Measurements and recordings

Immobility (including mortality) At 24 hours and at 48 hours.

pH At the beginning and at the end of the test, for all

concentrations and the control.

Dissolved oxygen At the beginning and at the end of the test, for all

concentrations and the control. In addition after 24 hours,

immediately after counting the immobilized daphnids oxygen

levels were measured in the test container with the solution

of lowest concentration at which all the Daphnia magna were

immobilized, i.e. in the solution containing 22% of the WSF.

Temperature of medium Continuously in a temperature control vessel, beginning at

the start of the test.- Page 12

-

6.7. Electronic data capture

Observations/measurements in the study were recorded electronically using the following

programme(s):

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ,

USA): Temperature.

6.8. Interpretation

6.8.1. Data handling

Determination of the average exposure concentrations

The average exposure concentrations were calculated as , being the geometric means

of the concentrations of measured in the samples taken at the start (C t=0 ) and the

end of the test (C t=48 ).

Calculation of EC 50

The EC 50 -value was calculated at 24 and 48 hours of exposure from the probits of the percentages of

affected daphni ds and the logarithms of the corresponding test substance concentrations using the

maximum likelihood estimation method (Finney, D.J., 1971: Probit analysis, Cambridge University

Press, Cambridge, U.K., 3rd edition).

6.8.2. Acceptability of the test

1. In the control, no daphni ds became immobilised.

2. The oxygen concentration at the end of the test was ≥3 mg/l in control and test vessels.

6.9. List of deviations

6.9.1. List of protocol deviations

There were no deviations from the protocol.

6.9.2. List of standard operating procedures deviations

Any deviations from standard operating procedures were evaluated and filed in the study file. There

were no deviations from standard operating procedures that affected the integrity of the study.

7. RESULTS

7.1. Combined limit/range-finding test

Table 1 shows the responses recorded during the combined limit/range-finding test. After 48 hours of

exposure 40 and 100% of the daphnids exposed to 10 and 100% of the WSF were immobilised. A

floating layer was observed at the highest test concentration but daphnids were not hampered by it.

No immobility was observed at any of the lower test concentrations during the test period. Note that a

maximum of 10% immobility is acceptable for the control and considered insignificant.

Analysis of the samples taken during the test showed that the initial concentrations were 0.0046,

0.033, 0.37 and 3.1 mg/l at 0.10, 1.0, 10 and 100% of the WSF, respectively. These concentrations

decreased to 31-50% of initial at the end of the test. Note that the lowest concentration seemed to

remain more stable (84% of initial) but this concentration was estimated and should therefore be

considered with care. S ee also Table 2 of the appended Analytical Report. The expected EC 50 was

between 0.37 and 3.1 mg/l.

All test conditions were maintained within the limits prescribed by the protocol.- Page 13

-

Table 1 Incidence of immobility in the combined limit/range-finding test

% of WSF

prep. at 100 mg/l Vessel

number Number

Daphnia

exposed Response at 24 h Response at 48 h

number Total

% number Total

%

control A

B

C

D 5

5

5

5 0

0

0

0 0 1

0

0

0 5

0.1 A

B 5

5 0

0 0 0

0 0

1.0 A

B 5

5 0

0 0 0

0 0

10 A

B 5

5 2 [2]

1 [1] 30 2

2 [1] 40

100 A

B

C

D 5

5

5

5 5

5

5

5 100 5

5

5

5 100

[ ] : number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the

respective solutions before recording of mobility.

7.2. Final test

7.2.1. Measured concentrations

The results of analysis of the samples taken during the final test are described in Table 3 of the

appended Analytical Report.

Analysis of the samples taken at the start of the final test showed measured concentrations of 0.050,

0.14, 0.27, 0.91, 1.5, 2.8 and 6.1 mg/l at 1.0, 2.2, 4.6, 10, 22, 46 and 100% of the WSF, respectively

. During the exposure period the measured concentrations decreased to 33-44% of initial at the end of

the test. Based on these results, the average exposure concentrations were calculated to be: 0.032,

0.078, 0.18, 0.55, 0.96, 1.9 and 3.9 mg/l.

7.2.2. Immobility

Table 2 shows the responses recorded during the final test. After 48 hours of exposure ≥90% of the

daphnids exposed to concentrations of 0.55 mg/l and higher were immobilised, while no (significant)

immobilisation was observed at any of the lower test concentrations. A floating layer was observed at

the three highest test concentrations. The responses recorded in this test allowed for reliable

determination of an EC 50

.- Page 14

-

Table 2 Acute immobilisation of daphni ds after 24 and 48 hours in the final test

Average c onc

.

(mg/l) Vessel

number Number

Daphnia

exposed Response at 24 h Response at 48 h

number Total

% number Total

%

control A

B

C

D 5

5

5

5 0

0

0

0 0 0

0

0

0 0

0.032 A

B

C

D 5

5

5

5 0

0

0

0 0 0

0

0

0 0

0.078 A

B

C

D 5

5

5

5 0

0

0

0 [2] 0 0

0

0

0 0

0.18 A

B

C

D 5

5

5

5 0 [2]

0

0 [1]

1 [1] 5 0

0 [1]

0

1 5

0.55 A

B

C

D 5

5

5

5 3 [5]

4 [4]

4 [4]

5 [4] 80 4

4

5

5 90

0.96 A

B

C

D 5

5

5

5 5 [3]

5 [2]

5 [2]

5 [4] 100 5

5

5

5 100

1.9 A

B

C

D 5

5

5

5 5 [3]

5

5 [2]

5 [3] 100 5

5

5

5 100

3.9 A

B

C

D 5

5

5

5 5

5 [1]

5 [1]

5 [1] 100 5

5

5

5 100

[ ] : number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the

respective solutions before recording of mobility.

7.2.3. Determination of effect concentrations

Table 3 shows the effect parameters based on average exposure concentrations, see also

APPENDIX 1.

Table 3 Effect parameters

Parameter Concentration

(mg/l) 95%

-

confidence

interval

NOEC 0.18

24h

- EC 50 0.32 0.27

- 0.41

48h

- EC 50 0.29 0.25

- 0.37- Page 15

-

7.2.4. Experimental conditions

The results of measurement of pH and oxygen concentrations (mg/l) are presented in Table 4. These

test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more

than 1 .5 unit; oxygen: 3 mg/l at the end of the test).

The temperature of the test medium was 20.4 °C at the start of the test. The temperature continuously

measured in a temperature control vessel varied between 20.5 and 21.7°C during the test, and

complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

Table 4 pH and oxygen concentrations during the final test

Average c onc

.

(mg/l) Start (t=0 h) End (t=48 h)

pH O 2 pH O 2

c ontrol 7.8 9.3 7.8 9.2

0.032 7.8 9.3 7.9 9.1

0.078 7.8 9.3 7.9 9.1

0.18 7.8 9.3 7.9 9.1

0.55 7.8 9.3 7. 9 9.1

0.96 7.8 9.3 7.9 9.0

1.9 7.7 9.2 7.9 8.9

3.9 7.7 9.1 7.9 8.9

8. CONCLUSION

Under the conditions of the present study did not induce significant acute

immobilisation of Daphnia magna at 0.18 mg/l after 48 hours of exposure (NOEC ).

The 48h- EC 50 was 0.29 mg/l based on average exposure concentrations (95% confidence interval

between 0.25 and 0.37 mg/l).- Page 16

-

APPENDIX 1 EC -VALUES

Table 5 EC 50 value at 24 hours and related parameters

24h

- EC 50 = 324.4 μg/l

95 % fiducial limits: 270.5

- 413.6 μg/l

index of regression significance: g=0.02

chi

- squared=6.35, with 14 degrees of freedom

regression line: log10(conc.)=1.90+(probit

- 2.69)/3.81

conc. group response corrected expected chi2

μg/l size fraction fraction

------- ----- --------- --------- -------- -------

78 5 0 0.00 0.00 0.00

78 5 0 0.00 0.00 0.00

78 5 0 0.00 0.00 0.00

78 5 0 0.00 0.00 0.00

180 5 0 0.00 0.16 0.98

180 5 0 0.00 0.16 0.98

180 5 0 0.00 0.16 0.98

180 5 1 0.20 0.16 0.05

550 5 3 0.60 0.81 1.42

550 5 4 0.80 0.81 0.00

550 5 4 0.80 0.81 0.00

550 5 5 1.00 0.81 1.18

960 5 5 1.00 0.96 0.19

960 5 5 1.00 0.96 0.19

960 5 5 1.00 0.96 0.19

960 5 5 1.00 0.96 0.19

-------

6.35

Figure 1 Percentage response (=immobility) of Daphnia magna as function of the log

concentration of at 24h- Page 17

-

APPENDIX 1 EC -VALUES

– continued

–

Table 6 EC 50 value at 48 hours and related parameters

48h

- EC 50 = 292.7 μg/l

95 % fiducial limits: 245.2

- 372.8 μg/l

index of regression significance: g=0.02

chi

- squared=5.82, with 14 degrees of freedom

regression line: log10(conc.)=1.90+(probit

- 2.67)/4.13

conc. group response corrected expected chi2

μg/l size fraction fraction

------- ----- --------- --------- -------- -------

78 5 0 0.00 0.00 0.00

78 5 0 0.00 0.00 0.00

78 5 0 0.00 0.00 0.00

78 5 0 0.00 0.00 0.00

180 5 0 0.00 0.19 1.18

180 5 0 0.00 0.19 1.18

180 5 0 0.00 0.19 1.18

180 5 1 0.20 0.19 0.00

550 5 4 0.80 0.87 0.23

550 5 4 0.80 0.87 0.23

550 5 5 1.00 0.87 0.74

550 5 5 1.00 0.87 0.74

9 60 5 5 1.00 0.98 0.08

960 5 5 1.00 0.98 0.08

960 5 5 1.00 0.98 0.08

960 5 5 1.00 0.98 0.08

-------

5.82

Figure 2 Percentage response (=immobility) of Daphnia magna as function of the log

concentration of at 48h- Page 18

-

APPENDIX 2 REFERENCE TEST

Start: 29 November 2011

End: 01 December 2011

48 -hour Acute Toxicity Study in Daphnia magna with potassium dichromate (K 2 Cr 2 O 7 ) (

).

The study procedures described in this report were based on the OECD guideline No. 202: "Daphnia

sp., Acute Immobilisation Test", Adopted April 13, 2004 and the ISO International Standard 6341.

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 K 2 Cr 2 O 7 concentrations of 0.10, 0.18, 0.32, 0.56,

1.0 and 1.8 mg/l and to a blank control. Twenty daphnia were exposed per concentration.

The reference substance, potassium dichromate (K 2 Cr 2 O 7 , art. 1.04864, batch no. K34869764 607)

was obtained from Merck, Darmstadt, Germany.

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium

dichromate:

Concentration

(mg/l) Number

Exposed % immobile

24h 48h Expected response (%)

After 48 hours 1

Minimal Maximal

control 20 0 0 0 10 2

0.10 20 0 0 0 10

0.18 20 0 0 0 10

0.32 20 5 70 0 30

0.56 20 50 100 0 100

1.0 20 100 100 40 100

1.8 20 100 100 100 100

1 Based on historical data of the previous years (n>60).

2 A maximum response of 10% does not invalidate the results of the test.

The actual responses in this reference test with K 2 Cr 2 O 7 were just outside (below) the ranges of the

expected historical responses at the different concentrations, i.e. a 48h- EC 50 between 0.3 and 1.0

mg/l. Hence, the sensitivity of this batch of D. magna was slightly higher when compared to the

historical data collected at

The 24h- EC 50 was 0.53 mg/l with a 95% confidence interval between 0. 47 and 0.62 mg/l.

The 48h- EC 50 was 0. 28 mg/l with a 95% confidence interval between 0. 26 and 0. 32 mg/l.

The raw data from this study are kept in the archives. The test described above was

performed under GLP with a QA-check.- Page 19

-

APPENDIX 3 GLP CERTIFICATE- Page 20

-

APPENDIX 4 ANALYTICAL REPORT



# DETERMINATION OF THE

CONCENTRATIONS

Author- Page 21

-

1. CONTENTS

1. CONTENTS

................................................................................................................................... 21

2. REPORT APPROVAL

.................................................................................................................... 22

3. INTRODUCTION

........................................................................................................................... 23

3.1. Preface

................................................................................................................................... 23

3.2. Aim of the study

..................................................................................................................... 23

4. MATERIALS AND METHODS

....................................................................................................... 23

4.1. Reagents

................................................................................................................................ 23

4.2. Samples

................................................................................................................................ . 23

4.3. Analytical method

................................................................................................................... 23

4.3.1. Analytical conditions

.................................................................................................... 23

4.3.2. Preparation of the calibration solutions

........................................................................ 24

4.3.3. Sample injections

......................................................................................................... 24

4.4. Electronic data capture

.......................................................................................................... 24

4.5. Formulas

................................................................................................................................ 25

5. RESULTS

...................................................................................................................................... 26

5.1. Calibration curves

.................................................................................................................. 26

5.2. Samples

................................................................................................................................ . 26

5.2.1. Procedural recovery samples

...................................................................................... 26

5.2.2. Test samples

................................................................................................................ 26

6. TABLES

......................................................................................................................................... 27

TABLES

Table 1 Procedural recovery samples

........................................................................................... 27

Table 2 Concentrations of the test substance in test medium - combined limit/range-finding

test

..................................................................................................................................... 28

Tabl e 3 Concentrations of the test substance in test medium - final test

...................................... 28- Page 23

-

3. INTRODUCTION

3.1. Preface

Study plan analytical phase Start : 21 February 2012

Completion : 15 March 2012

3.2. Aim of the study

The purpose of the analytical phase was to determine the actual concentrations in samples taken fro m

the test solutions used during the ecotoxicity test.

4. MATERIALS AND METHODS

4.1. Reagents

Acetone Merck, Darmstadt, Germany

Cyclohexane Merck

ISO-medium see main report

All reagents were of analytical grade, unless specified otherwise.

4.2. Samples

On the day of sampling

, the test samples (4 ml) were pipetted into 4 ml cyclohexane. After shaking the

mixture for 30 seconds by hand, the cyclohexane phase was analysed. If necessary, the cyclohexane

phase was further diluted with cyclohexane to obtain concentrations within the calibration range.

4.3. Analytical method

4.3.1. Analytical conditions

Quantitative analysis was based on the analytical method validated for the test substance in

project 498188.

Instrument Gaschromatograph 6890N

(Agilent Technologies, Palo Alto, CA, USA)

Injector CombiPal (CTC analytics, Zwingen, Switzerland)

Detector Mass selective detector (MSD) 5973N (Agilent Technologies)

Column DB -5MS; 25 m  250 μm i.d., d f = 0.25 μm

(J&W Scientific, Folsom, CA, USA)

Carrier gas helium

Carrier gas flow 1 ml/min

Injection Programmed temperature vaporizer (PTV)

PTV Mode Splitless

Splitless time 60 sec

Injection volume 1 μl

PTV temperature program

Initial temperature 70°C

Initial time 0.1 min

Rate 720°C/min

Final temperature 250°C

Hold time 5 min- Page 24

-

Oven temperature program

Initial temperature 70°C

Initial time 1 min

Rate 1 20°C/min

Temperature 1 210°C

Hold time 0 min

Rate 2 40°C/min

Final temperature 300°C

Hold time 2 min

MS Detection

Ionisation source EI +

MS interface temperature 280 ° C

Solvent delay 3 min

Acquisition m/z 165.1, m/z

181.2 and m/z

196.2 amu

Quantitation m/z 181.2 amu

Qualifier ions m/z 165.1 and 196.2 amu

4.3.2. Preparation of the calibration solutions

Stock and spiking solutions

Stock solutions of the test substance were prepared in cyclohexane at concentrations of 1955

–

3685 mg/l.

Spiking solutions of the test substance were prepared in acetone at concentrations of 0.617

–

3085 mg/l.

Calibration solutions

Six calibration solutions in the concentration range of 0.00805

– 4.20 mg/l were prepared from two

stock solutions. The end solution of the calibration solutions was cyclohexane

.

Procedural recovery samples

4 ml blank medium was spiked with the test substance at a target concentration of 0.01, 0.1

, 1 or

10 mg/l. The accuracy samples were treated similarly as the test samples (see paragraph 4.2

‘Samples’).

4.3.3. Sample injections

Calibration solutions were injected in duplicate. Test samples and procedural recovery samples wer e

analysed by single injection.

4.4. Electronic data capture

System control, data acquisition and data processing were performed using the following programme:

- Enhanced Chemstation version D.00.01.27 (Agilent Technologies, Wilmington, DE, USA)

Temperature, relative humidity and/or atmospheric pressure during sample storage and/or

performance of the studies was monitored continuously using the following programme:

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ,

USA).- Page 25

-

4.5. Formulas

Response (R) Peak area test substance [units]

Calibration curve

where:

C N = nominal concentration [mg/l]

a = slope [units  l/mg]

b = intercept [units]

Analysed concentration (C A )

where:

d = dilution factor

Calibration curve final test

where:

a = quadratic regression factor [units  (l/mg) 2 ]

b = linear regression factor [units  l/mg]

c = intercept [units]

Analysed concentration (C A )

where:

d = dilution factor

Recovery

Relative to initial concentration- Page 26

-

5. RESULTS

5.1. Calibration curves

Combined limit/Range-finding test

A calibration curve was constructed excluding two data points since the back calculated accuracy was

> 15% from the nominal concentration. Linear regression analysis was performed using the least

squares method with a 1/concentration 2 weighting factor. The coefficient of correlation (r) was > 0.99.

Final test

A calibration curve was constructed using all data points. For each concentration, two responses were

used. Quadratic regression analysis was performed using the least squares method with a

1/concentration 2 weighting factor. The coefficient of correlation (r) was > 0.99.

5.2. Samples

5.2.1. Procedural recovery samples

The results for the procedural recovery samples are given in Table 1

.

The mean recoveries of the procedural recovery samples fell within the criterion of 70-110%. It

demonstrated that the analytical method was adequate for the determination of the test substance in

the test samples.

5.2.2. Test samples

The results for the test samples are given in Table 2 and Table 3

.- Page 27

-

6. TABLES

Table 1 Procedural recovery samples

Date of

preparation

[dd

- mm

- yy] Date of

analysis

[dd

- mm

- yy] Target

concentration

[mg/l] Nominal

concentration

[mg/l] Analysed

concentration

[mg/l] Recovery

[%] Mea n

recovery

[%]

21

- 02

- 12 21

- 02

- 12 0.01 0.0100 0.00985 98 98

0.0100 0.00985 98

21

- 02

- 12 21

- 02

- 12 0.1 0.100 0.0897 89 91

0.100 0.0935 93

21

- 02

- 12 21

- 02

- 12 1 1.00 0.973 97 97

1.00 0.963 96

23

- 02

- 12 23

- 02

- 12 0. 01 0.0100 0.00826 82 83

0.0100 0.00848 85

23

- 02

- 12 23

- 02

- 12 0.1 0.100 0.0796 79 77

0.100 0.0742 74

23

- 02

- 12 23

- 02

- 12 1 1.00 0.855 85 85

1.00 0.854 85

23

- 02

- 12 23

- 02

- 12 10 10.0 9.34 93 93

10.0 9.32 93

13

- 03

- 12 13

- 03

- 12 0.01 0.0100 0.00835 83 83

0.0100 0.00825 82

13

- 03

- 12 13

- 03

- 12 0.1 0.100 0.0946 94 94

0.100 0.0943 94

13

- 03

- 12 13

- 03

- 12 1 1.00 1.07 106 105

1.00 1.03 103

13

- 03

- 12 13

- 03

- 12 10 10.0 9.87 98 96

10.0 9.30 93

15

- 03

- 12 15

- 03

- 12 0.01 0.0100 0.00789 1 79 78

0.0100 0.00776 1 77

15

- 03

- 12 15

- 03

- 12 0.1 0.100 0.0866 86 84

0.100 0.0828 83

15

- 03

- 12 15

- 03

- 12 1 1.00 0.980 98 95

1.00 0.931 93

15

- 03

- 12 15

- 03

- 12 10 10.0 8.62 86 88

10.0 9.01 90

1 Obtained by slight extrapolation of the calibration curve.- Page 28

-

Table 2 Concentrations of the test substance in test medium - combined limit/range-finding

test

Time of

sampling

[hours] Date of

sampling

[dd

- mm

- yy] Date of

analysis

[dd

- mm

- yy] Percentage of

WSF 1

[%] Analysed

concentration

[mg/l] Relative to

initial

[%]

0 21

- 02

- 12 21

- 02

- 12 0 0.00210 2

0.1 0.00463 2

1 0.0325

10 0.366

100 3.05

48 23

- 02

- 12 23

- 02

- 12 0 n.d.

0.1 0.00391 2 84

1 0.0134 41

10 0.115 31

100 1.53 50

1 Percentage of a water soluble fraction (WSF) prepared at a loading rate of 100 mg/l.

2 Obtained by extrapolation of the calibration curve.

n.d. Not detected.

Table 3 Concentrations of the test substance in test medium - final test

T ime of

sampling

[hours] Date of

sampling

[dd

- mm

- yy] Date of

analysis

[dd

- mm

- yy] Percentage of

WSF 1

[%] Analysed

concentration

[mg/l] Relative to

initial

[%]

0 13

- 03

- 12 13

- 03

- 12 0 n.d.

1 0.0502

2.2 0.136

4.6 0.269

10 0.910

22 1.51

46 2.84

100 6.06

48 15

- 03

- 12 15

- 03

- 12 0 n.d.

1 0.0207 41

2.2 0.0451 33

4.6 0.117 44

10 0.331 36

22 0.614 41

46 1.26 44

100 2.46 41

1 Percentage of a water soluble fraction (WSF) prepared at a loading rate of 100 mg/l.

n.d. Not detected.