FINAL REPORT

Study Title

ACUTE TOXICITY STUDY IN DAPHNIA MAGNA WITH

(STATIC)

Author

Test Facility

Laboratory Project Identification

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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

Final Report
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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 EC50 value at 24 hours and related parameters ...........................................................16

Table 6 EC50 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

Final Report


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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-EC50 was 0.29 mg/l based on average exposure concentrations (95% confidence interval 

between 0.25 and 0.37 mg/l). 

Final Report
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5. INTRODUCTION 

5.1. Preface 

Sponsor

Study Monitor

(on behalf of the sponsor)

Test Facility

Study Director

Technical Coordinator

Principal Scientist

Study Plan Start : 21 February 2012

Completion : 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 EC50 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. 

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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 EC50 is the concentration of test substance estimated to immobilise 50% of the daphnids 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 container with nitrogen after handling

Specific Gravity / Density

Stability in water At least 24 hours

Solubility in water No

Final Report
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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, 2nd to 5th 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 daphnids 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: CaCl2.2H2O 211.5 mg/l 

 MgSO4.7H2O 88.8 mg/l 

 NaHCO3 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 

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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 

 Na2EDTA.2H2O 2.5 mg/l 

 Macro nutrients: Na2SiO3. 9H2O 10.0 mg/l 

NaNO3 0.27 mg/l 

 KH2PO4 0.14 mg/l 

 K2HPO4 0.18 mg/l 

 Vitamins: Thiamine 75.0 μg/l 

 B12 1.0 μg/l 

 Biotin 0.75 μg/l 

The hardness: 180 mg/l expressed as CaCO3 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 

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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 daphnids 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 daphnids 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. 

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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 

0 CC tt 48

, being the geometric means 

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

end of the test (Ct=48).

Calculation of EC50

The EC50-value was calculated at 24 and 48 hours of exposure from the probits of the percentages of 

affected daphnids 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 daphnids 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. See also Table 2 of the appended Analytical Report. The expected EC50 was 

between 0.37 and 3.1 mg/l. 

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

Final Report
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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 22 [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 EC50. 

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

Average conc.

(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-EC50 0.32 0.27 - 0.41

48h-EC50 0.29 0.25 - 0.37

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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 conc.

(mg/l)

Start (t=0 h) End (t=48 h)

pH O2 pH O2

control 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-EC50 was 0.29 mg/l based on average exposure concentrations (95% confidence interval 

between 0.25 and 0.37 mg/l). 

Final Report
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APPENDIX 1 EC-VALUES 

Table 5 EC50 value at 24 hours and related parameters 

24h-EC50 = 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 

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APPENDIX 1 EC-VALUES – continued – 

Table 6 EC50 value at 48 hours and related parameters 

48h-EC50 = 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

 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

 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 

Final Report
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APPENDIX 2 REFERENCE TEST 

Start: 29 November 2011 

End: 01 December 2011 

48-hour Acute Toxicity Study in Daphnia magna with potassium dichromate (K2Cr2O7) ( 

). 

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 K2Cr2O7 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 (K2Cr2O7, 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 0 102

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 K2Cr2O7 were just outside (below) the ranges of the 

expected historical responses at the different concentrations, i.e. a 48h-EC50 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-EC50 was 0.53 mg/l with a 95% confidence interval between 0.47 and 0.62 mg/l. 

The 48h-EC50 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. 

Final Report
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APPENDIX 3 GLP CERTIFICATE 

Final Report
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APPENDIX 4 ANALYTICAL REPORT 

DETERMINATION OF THE

CONCENTRATIONS

Author

Final Report
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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

Table 3 Concentrations of the test substance in test medium - final test ...................................... 28

Final Report

- 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 from 

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., df = 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 

Final Report
- 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 were 

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). 

Final Report
- Page 25 - 

4.5. Formulas 

Response (R) Peak area test substance [units] 

Calibration curve 

N  b C a R

where: 

CN = nominal concentration [mg/l] 

a = slope [units  l/mg] 

b = intercept [units] 

Analysed concentration (CA) 

d [mg/l]

a

(R b) CA 



where: 

d = dilution factor 

Calibration curve final test 

c C bC a R N

2

N

 

where: 

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

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

c = intercept [units] 

Analysed concentration (CA) 

 

d

a 2

Rca 4bb

C

2

A 





where: 

d = dilution factor 

Recovery 

100 [%]

C

C

N

A



Relative to initial concentration 

100 [%]

 C (t hours) 0

 C (t x hours)

A

A







Final Report
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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/concentration2 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/concentration2 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.

Final Report
- 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

[%]

Mean 

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 179 78

0.0100 0.00776 177

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. 

Final Report
- 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 284

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 

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 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. 

Final Report