CiToxLAB Hungary Ltd.

Veszprém - Szabadságpuszta

FINAL REPORT

Acute immobilisation test with

Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] 

(hydrated form of ethanedioic acid [CAS RN 144-62-7])

on Daphnia (Daphnia magna)

Study code: 11/339-023DA

Study Director: Krisztina Sipos, M.Sc.

19 March 2013



Study code: 11/339-023DA Final Report Page 5 of 18

GENERAL INFORMATION

STUDY TITLE : Acute immobilisation test with Ethanedioic acid, 

hydrate (1:2) [CAS RN 6153-56-6] (hydrated form 

of ethanedioic acid [CAS RN 144-62-7])

on Daphnia (Daphnia magna)

TEST ITEM : Ethanedioic acid, hydrate (1:2) 

[CAS RN 6153-56-6] (hydrated form of 

ethanedioic acid [CAS RN 144-62-7])

SPONSORS : Univar USA Inc.

Address: 17425 NE Union Hill Road

Redmond, WA 98052

USA

Telephone: +1 425 889 3400

Facsimile: +1 425 889 3760

: WEGO Chemical and Mineral Corp.

Address: 239 Great Neck Road

Great Neck, NY 11021

USA

Telephone: +1 516 487 3510

Facsimile: +1 516 487 3794

STUDY MONITOR : Mr. John V. Massingale

Greenwich Chemical Consulting, Inc.

Address: 3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410

USA

Telephone: +1 561 578 8110

Facsimile: +1 561 578 8120

E-mail: john@grcci.com

TEST FACILITY : CiToxLAB Hungary Ltd.

Address: H-8200 Veszprém, Szabadságpuszta

Hungary

Phone: 36 88 545-300

Fax: 36 88 545-301
Study code: 11/339-023DA Final Report Page 6 of 18

STUDY DIRECTOR : Krisztina Sipos, M.Sc.

QUALITY ASSURANCE : Éva Makovi-Fábián, B.Sc.

Marietta Balassa, B.Sc.

RESPONSIBLE PERSON FOR

ANALYTICAL MEASUREMENTS : Zsolt Sárvári, M.Sc.

TECHNICAL STAFF : Ecotoxicological staff

START OF EXPERIMENT : 20 November 2012

END OF EXPERIMENT : 05 December 2012

BASIS OF STUDY:

- OECD Guidelines for Testing of Chemicals, No.:202 (Adopted: 13 April 2004) 

OECD: Paris

- Commission Regulation (EC) No 761/2009 of 23 July 2009 laying down test 

methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament 

and of the Council on the Registration, Evaluation, Authorisation and Restrictions 

of Chemicals (REACh), Annex Part C, C.2 (published in the Official Journal of the 

European Union L 142 of 31 May 2008)

- EPA Guideline 712-C-96-114: OPPTS 850.1010, “Aquatic Invertebrate Acute 

Toxicity Test, Freshwater Daphnids” April 1996

This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) 

Section 4 Test Rule and the regulations set forth at 40 CFR 799.5087.
Study code: 11/339-023DA Final Report Page 7 of 18

CONTENTS

Page

GENERAL INFORMATION 5

1. SUMMARY 8

2. INTRODUCTION 10

3. MATERIALS AND METHODS 10

3.1. TEST ITEM AND CONTROLS AND VEHICLE 10

3.2. EXPERIMENTAL ANIMALS 12

3.3. TEST CONDITIONS 12

3.4. DESCRIPTION OF THE TEST PROCEDURE 12

3.5. OBSERVATIONS 13

3.6. STATISTICAL EVALUATION 15

3.7. ANALYTICAL MEASUREMENTS 15

3.8. ARCHIVES 15

3.9. CONDITIONS FOR THE VALIDITY OF THE TEST 15

3.10.DISTRIBUTION OF THE FINAL REPORT 16

3.11.REFERENCES 16

4. RESULTS 17

4.1. VALIDITY 17

4.2. IMMOBILISATION 17

5. CONCLUSION 18

APPENDICES 19

Data of Test Conditions 1 - 4 of Appendix 1

Data of Immobilisation 1 - 2 of Appendix 2

Concentration/response curve (48h) 1 of Appendix 3

Analytical investigations of main study 1 - 4 of Appendix 4

Composition of ISO medium 1 of Appendix 5

Copy of the GLP Certificate 1 of Appendix 6

Copy of the Certificate of Analysis 1 of Appendix 7
Study code: 11/339-023DA Final Report Page 8 of 18

1. SUMMARY

Acute toxicity of Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form 

of ethanedioic acid [CAS RN 144-62-7]) was assessed with Acute immobilisation test 

on Daphnia magna, over an exposure period of 48 hours in a static system.

Due the analytical method validation since the test material was found to be stable in 

water over the test period static conditions were used during the experiment.

Based on the results of the preliminary range-finding test five test concentrations in a 

geometric series with a separation factor of 2.0 and one untreated control were tested 

in the main experiment (definitive test, Phase 1).

The nominal concentrations of test item used in the definitive test (Phase 1) were: 6.25; 

12.5; 25; 50 and 100 mg/L. The corresponding measured geometric mean test item 

concentrations were: 4.84; 8.26; 11.64; 21.94 and 73.65 mg/L.

Test concentrations was analytically determined at the beginning and end of the 

experiment. As the measured concentrations deviated more than 20 per cent from the 

nominal in most cases, biological results are based on the measured geometric mean

concentrations.

Because of the pH did not remain in the range 6-9 in the definitive test (Phase 1) at the 

two highest concentration levels of 50 and 100 mg/L, a complementary study (Phase 2) 

was carried out, adjusting the pH of the stock solution to that of the dilution water 

before addition of the test substance. We extended the study to provide more data to 

allow a better interpretation.

Twenty animals, divided into four groups (glass beaker) of five animals each were used 

at the test and for the control groups.

The 24 and 48h LC50 values of the test item were calculated using Probit analysis by 

SPSS PC+ software. The 48h LC100 values of the test item, NOEC and LOEC were 

determined from the raw data.

All validity criteria were met during this study.
Study code: 11/339-023DA Final Report Page 9 of 18

Under the conditions of this Daphnia magna acute immobilisation study the 

calculated and observed endpoints for the effect of Ethanedioic acid, hydrate 

(1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-

7]) were the following:

The 24h and 48h EC50 value: 38.89 mg/L (95 % conf. limits: 28.05 – 61.70 mg/L)

The 48h EC100 value: 73.65 mg/L

The 48h No-Observed Effect Concentration (NOEC): 21.94 mg/L

The 48h Lowest Observed Effect Concentration (LOEC): 73.65 mg/L

Based on the results of the performed definitive and complementary experiments 

the observed effect can be considered to be related to the acid character of the 

test item.
Study code: 11/339-023DA Final Report Page 10 of 18

2. INTRODUCTION

The objective of this study was to determine the effect of Ethanedioic acid on Daphnia

magna. Young Daphnia were exposed in a static test to ISO test medium containing 

the test item at different concentrations. The percentage of Daphnia being no longer 

capable of swimming at the end of the test or being dead was recorded for each test 

concentration.

The test method of application and the test species Daphnia magna are recommended 

by the test guidelines.

3. MATERIALS AND METHODS

3.1. TEST ITEM AND CONTROLS AND VEHICLE

3.1.1. Test Item

Name: Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] 

(hydrated form of ethanedioic acid [CAS RN 144-62-7])

Chemical name: Oxalic acid, hydrate

Synonym: Oxalic acid

Batch number: YP1-20120528C

CAS number: 6153-56-6

Molecular Formula: C2H2O4×2H2O

Molecular Weight: 126.1 g/mol (dihydrate form)

Purity: 99.69 %

Description of the test item: White crystals

Manufacture date: May 28, 2012

Retest date: May 27, 2013

Storage condition: controlled room temperature (15-25oC)

Safety Precautions: Routine safety precautions (lab coat, gloves, safety glasses 

and face mask) were applied to assure personnel health and 

safety.

The test item of a suitable chemical purity was supplied by the Sponsor. All precautions 

required in the handling and disposal of the test item were outlined by the Sponsor. 

These documents are part of the raw data. Identification of the test item was performed 

in the Central Dispensary of CiToxLAB Hungary Ltd. on the basis of the information 

provided by the Sponsor.
Study code: 11/339-023DA Final Report Page 11 of 18

3.1.2. Dilution and Preparation of Testing Solutions

An amount of 50 mg test substance was diluted in 500 mL ISO medium (stock solution 

concentration: 100 mg/L). The test solutions were prepared by appropriate dilution of 

this stock solution (see Table 1 below).

Table 1: Preparation of test solutions from stock solution (Phase 1)

Nominal concentration

[mg/L]

Amount of stock solution 

(mL)

Amount of ISO Medium 

(mL)

100 500 –

50 150 q.s. ad 300

25 75 q.s. ad 300

12.5 37.5 q.s. ad 300

6.25 18.75 q.s. ad 300

q.s. ad = quantum sufficiat ad (a sufficient quantity to make)

Table 2: Preparation of test solutions from stock solution (Phase 2)

Nominal concentration

[mg/L]

Amount of stock solution 

(mL)

Amount of ISO Medium 

(mL)

100 500 –

50 150 q.s. ad 300

q.s. ad = quantum sufficiat ad (a sufficient quantity to make)

In the complementary experiment the pH of the stock solution was adjusted and then 

the appropriate dilution was prepared and distributed into test vessels prior to 

introduction of the Daphnia.

3.1.3. Untreated Control

The dilution water (ISO-medium) was used without of addition of the test item.

3.1.4. Reference Control

For the evaluation of the quality of the Daphnia clone and the experimental conditions, 

Potassium dichromate is tested at least twice a year to demonstrate satisfactory test 

conditions.

The date of the last study (Study Code: 11/357-023DA) with reference item Potassium 

dichromate (batch no.: 0769128) is: 18-19 January 2012. 

The 24h EC50: 1.27 mg/L, (95 % confidence limits: 1.16 – 1.39 mg/L)
Study code: 11/339-023DA Final Report Page 12 of 18

3.1.5. Dilution Water

Reconstituted water (ISO medium, according to OECD 202) was used as dilution 

water for both the range finding and definitive tests. The same composition of 

reconstituted water was used for the tests and for breeding the test animals. Dilution 

water had a pH in the range of 6 to 9.

The composition of the reconstituted water and the chemicals used are given in 

Appendix 5.

3.2. EXPERIMENTAL ANIMALS

Species and strain: Daphnia magna

Source: National Institute of Public Health

1097 Budapest Gyáli u. 2-6. Hungary

Justification of strain: Daphnia magna is the standard species of the acute 

immobilisation test.

Number of animals: There were 20 animals in test and control groups

respectively, divided into 4 replicates

(5 animals / replicate)

Age of the animals: They were less than 24 h old at the beginning of the test

Acclimatization: There was no acclimatization because the water used 

was similar to the culture water

Test Daphnia were derived from healthy stock (showing no signs of stress such as high 

mortality, presence of males, ephippia, delay in the production of first brood, 

discolored animals.

3.3. TEST CONDITIONS

Temperature: The water temperature was measured at the start of the tests 

and 24-hour intervals thereafter in each test vessel. The test 

temperature was in the range of 20.4 – 21.1 °C (Phase 1) and 

20.5 – 20.8 °C (Phase 2) measured in the test vessels.

The additionally measured temperature in the climate chamber 

was between 20.5 and 21.4 °C (Phase 1) and between 20.1 and 

21.8 °C (Phase 2).

Oxygen concentration: The dissolved oxygen concentration was measured in each test 

vessel at the start and at the end of the tests and was in the 

range of 7.1 – 8.4 mg/L (Phase 1) and in the range of 

6.8 – 8.3 mg/L (Phase 2).

pH: The pH of the test solution was not adjusted and not varied by 

more than 1.5 units in the definitive test (Phase 1). The pH was 

measured at the start and at the end of the experiment in each 

test vessel and was in the range of 3.20 – 7.68.
Study code: 11/339-023DA Final Report Page 13 of 18

The pH of the stock solution was adjusted in the 

complementary study (Phase 2) and then the appropriate 

dilution was prepared. The pH was measured at the start and at 

the end of the experiment in each test vessel and was in the 

range of 7.62 – 7.91.

Light: The test was carried out in 16-hour light and 8-hour dark cycle.

Hardness: The reconstituted water (ISO medium) had an approximate 

theoretical total hardness of 249 mg/L (as CaCO3). 

Test vessels were not aerated during the test.

3.4. DESCRIPTION OF THE TEST PROCEDURE

The test duration was 48 hours. Twenty animals, divided into four groups (glass 

beaker) of five animals each (at least 5 mL test solution/animal) were used at the test 

concentrations and for the control. The animals were not fed during the test.

The choice of the test concentrations was done on the basis of the results of a 

preliminary range-finding test. 

3.4.1. Preliminary Range Finding Test

A concentration range-finding test was conducted to determine the approximate 

toxicity of the test item so that appropriate test concentrations can be selected for use 

in the definitive test. Ten daphnids (divided into two replicates) in each test

concentration and control were exposed for 48 hours under static conditions.

During the formulation procedure the test solutions were prepared by the similar 

method described above (3.1.2.).

The concentration levels used and results (48 h) of the preliminary range-finding test 

are summarised in the following table.

Table 3: Results of the Preliminary Range-Finding Test

Nominal concentrations

[mg/L]

Untreated

control 0.1 1 10 100

Number of 

treated / immobilised animals 10 / 0 10 / 0 10 / 0 10 / 0 10 / 10
Study code: 11/339-023DA Final Report Page 14 of 18

3.4.2. Test item concentrations in the Definitive Test (Phase 1)

Because significant immobility was observed at the highest examined concentration 

level during the preliminary range-finding test, five test concentrations in a geometric 

series (factor 2.0) and one control were tested in the main test.

The following nominal concentrations were tested: 6.25, 12.5, 25, 50 and 100 mg/L.

The measured geometric mean test item concentrations were: 4.84; 8.26; 11.64; 21.94 

and 73.65 mg/L.

As the measured concentrations deviated more than 20 per cent from the nominal in 

most cases, biological results are based on the measured geometric mean 

concentrations.

The analytically measured test item concentrations are detailed in Appendix 4.

3.4.3. Test item concentrations in the Complementary Study (Phase 2)

Because of the pH did not remain in the range 6-9 in the definitive test at the two 

highest concentration levels of 50 and 100 mg/L, a complementary study was carried 

out, adjusting the pH of the stock solution to that of the dilution water before addition 

of the test substance. The pH was adjusted through the addition of NaOH.

The complementary study was carried out using only the two highest concentrations of 

50 and 100 mg/L, where the pH values were below the accepted range in the definitive 

test. The measured geometric mean test item concentrations were: 58.26 and 

109.4 mg/L.

One control was also tested in the experiment.

The analytically measured test item concentrations are detailed in Appendix 4.

3.5. OBSERVATIONS

The immobility or mortality of the Daphnia was determined by visual observation 24 

and 48 hours after the start of the test. Those animals not able to swim within 15 

seconds after gentle agitation of the test beaker are considered to be immobile.

The number of immobilised animals and the percentage of immobility were determined 

at 24 and 48 hours.

The water temperature was measured at the start of the test and 24-hour intervals

thereafter, oxygen concentrations and pH of the controls and the test solutions were 

measured at the beginning and at the end of the experiments.
Study code: 11/339-023DA Final Report Page 15 of 18

3.6. STATISTICAL EVALUATION

The 24 and 48 hours EC50 values were calculated by Probit analysis using SPSS PC+ 

software (based on the measured concentrations) (95 % confidence limits could not be 

determined).

The 48 hours EC100, the LOEC and NOEC values were determined directly from the 

raw data.

3.7. ANALYTICAL MEASUREMENTS

Analytical measurement was performed at the control and at the applied test 

concentration levels at the beginning and at the end of the tests (Phase 1 and 2).

The samples were analysed by HPLC system.

Methods and results of test item concentration analysis are described in Appendix 4.

3.8. ARCHIVES

The study documents:

- study plan and amendments,

- all raw data,

- sample of test item,

- study report, and any amendments,

- correspondence

are stored according to the Hungarian GLP and to the CiToxLAB Hungary Ltd. SOP-s 

in the archives of CiToxLAB Hungary Ltd. 8200 Veszprém, Szabadságpuszta, 

Hungary.

After the retention time has elapsed all the archived materials listed above will be 

returned to the Sponsor or retained for a further period if agreed by a contract. 

Otherwise the materials will be discarded.

3.9. CONDITIONS FOR THE VALIDITY OF THE TEST

- Constant conditions were maintained throughout the study.

- In the control, including any control containing the solubilising agent, not more than 

10 per cent of the control daphnids should show immobilization or other signs of 

disease or stress, for example, discoloration or unusual behavior such as trapping at 

surface of water.

- the dissolved oxygen concentration at the end of the test in control and test vessels 

should be ≥ 3 mg/L of the air saturation value at the temperature used.
Study code: 11/339-023DA Final Report Page 16 of 18

3.10. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound

1x copy, unbound

1x PDF file

Archive: 1x original, bound

3.11. REFERENCES

1. OECD Principles of Good Laboratory Practice, adopted by Council on 26th

November 1997 [C(97)186/Final], Environment Directorate, Organisation for 

Economic Co-operation and Development, Paris 1998.

2. Hungarian Good Laboratory Practice Regulations: 9/2001 (III. 30) EüM-FVM joint 

decree of the Minister of Health and the Minister of Agriculture and Regional 

Development which corresponds to the OECD GLP, 1997

3. Commission Regulation (EC) No 761/2009 of 23 July 2009 laying down test 

methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament 

and of the Council on the Registration, Evaluation, Authorisation and Restrictions of 

Chemicals (REACh), Annex Part C, C.2 (published in the Official Journal of the 

European Union L 142 of 31 May 2008)

4. EPA Guideline 712-C-96-114: OPPTS 850.1010, “Aquatic Invertebrate Acute 

Toxicity Test, Freshwater Daphnids” April 1996

5. OECD Guideline for Testing of Chemicals, Section 2, No. 202, “Daphnia sp., Acute 

Immobilisation Test”, updated 13th April 2004.

6. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 

Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series 

on Testing and Assessment No. 23, Paris September 2000.
Study code: 11/339-023DA Final Report Page 17 of 18

4. RESULTS

4.1. VALIDITY

There were no immobilized animal in the control group and the dissolved oxygen 

concentration at the end of the tests (Phase 1 and 2) in control and test vessels was 

more than 3 mg/L. All validity criteria were within acceptable limits and therefore the 

studies can be considered as valid.

4.2. IMMOBILISATION

4.2.1. Definitive test (Phase 1)

The number of immobilised animals and the percentage of immobility were determined 

at the 24th and 48th hour (Table 4).

Table 4: Number and percentage of immobilised animals

Concentration Number 

of treated 

animals

Immobilised animals

Nominal

mg/L

24 hours 48 hours

number percent number percent

Control 20 0 0 0 0

6.25 20 0 0 0 0

12.5 20 0 0 0 0

25 20 0 0 0 0

50 20 0 0 0 0

100 20 20 100 20 100

4.2.2. Complementary study (Phase 2)

The number of immobilised animals and the percentage of immobility were determined 

at the 24th and 48th hour (Table 5).

Table 5: Number and percentage of immobilised animals

Concentration Number 

of treated 

animals

Immobilised animals

Nominal

mg/L

24 hours 48 hours

number percent number percent

Control 20 0 0 0 0

50 20 0 0 0 0

100 20 0 0 0 0

Data of immobility in each test vessel (Phase 1 and 2) are detailed in Appendix 2.
Study code: 11/339-023DA Final Report Page 18 of 18

5. CONCLUSION

Acute toxicity of Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form 

of ethanedioic acid [CAS RN 144-62-7]) was assessed with Acute immobilisation test 

on Daphnia magna, over an exposure period of 48 hours in a static system.

All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study the 

calculated and observed endpoints for the effect of Ethanedioic acid, hydrate 

(1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-

7]) were the following:

The 24h and 48h EC50 value: 38.89 mg/L (95 % conf. limits: 28.05 – 61.70 mg/L)

The 48h EC100 value: 73.65 mg/L

The 48h No-Observed Effect Concentration (NOEC): 21.94 mg/L

The 48h Lowest Observed Effect Concentration (LOEC): 73.65 mg/L

Based on the results of the performed definitive and complementary experiments 

the observed effect can be considered to be related to the acid character of the 

test item.
Study code: 11/339-023DA Final Report Page 19 of 18

APPENDICES
Study code: 11/339-023DA Final Report Page 1 of Appendix 1

APPENDIX 1

DATA OF TEST CONDITIONS

Table 6: Temperature measured in the test vessels (°C) (Phase 1)

Concentration

Replicate

Measuring

Nominal

mg/L

Measured 

mg/L 0 h 24 h 48 h

Control 0.0

1 20.9 20.8 20.7

2 20.9 20.8 20.8

3 20.9 20.8 20.8

4 20.9 20.8 20.8

6.25 4.84

1 21.0 20.8 20.8

2 21.0 20.8 20.8

3 20.9 20.8 20.7

4 20.9 20.8 20.7

12.5 8.26

1 21.0 20.7 20.7

2 21.0 20.7 20.7

3 21.0 20.6 20.7

4 21.0 20.6 20.7

25 11.64

1 21.0 20.7 20.7

2 21.0 20.7 20.7

3 21.0 20.7 20.7

4 21.0 20.7 20.7

50 21.94

1 21.0 20.7 20.7

2 21.1 20.7 20.7

3 21.0 20.7 20.7

4 21.0 20.7 20.7

100 73.65

1 20.9 20.4 20.6

2 20.9 20.4 20.6

3 20.9 20.4 20.6

4 20.9 20.4 20.6
Study code: 11/339-023DA Final Report Page 2 of Appendix 1

Table 7: Temperature measured in the test vessels (°C) (Phase 2)

Concentration

Replicate

Measuring

Nominal

mg/L

0 h 24 h 48 h

Control

1 20.7 20.6 20.5

2 20.7 20.6 20.6

3 20.7 20.6 20.5

4 20.7 20.5 20.5

50

1 20.7 20.5 20.6

2 20.7 20.6 20.6

3 20.7 20.5 20.6

4 20.8 20.6 20.6

100

1 20.8 20.6 20.6

2 20.8 20.6 20.5

3 20.8 20.6 20.5

4 20.8 20.5 20.6
Study code: 11/339-023DA Final Report Page 3 of Appendix 1

Table 8: Oxygen concentration measured in the test vessels (mg/L) (Phase 1)

Concentration

Replicate

Measuring

Nominal

mg/L

Measured 

mg/L 0 h 48 h

Control 0.0

1 8.4 7.1

2 8.4 7.1

3 8.4 7.2

4 8.4 7.1

6.25 4.84

1 8.3 7.1

2 8.3 7.1

3 8.3 7.1

4 8.3 7.1

12.5 8.26

1 8.3 7.1

2 8.3 7.1

3 8.3 7.1

4 8.3 7.1

25 11.64

1 8.3 7.1

2 8.3 7.1

3 8.3 7.1

4 8.3 7.1

50 21.94

1 8.3 7.1

2 8.3 7.1

3 8.3 7.1

4 8.3 7.1

100 73.65

1 8.3 7.1

2 8.3 7.1

3 8.3 7.1

4 8.3 7.1

Table 9: Oxygen concentration measured in the test vessels (mg/L) (Phase 2)

Concentration

Replicate

Measuring

Nominal

mg/L

0 h 48 h

Control

1 8.3 8.3

2 8.3 8.3

3 8.3 8.3

4 8.3 8.2

50

1 8.3 7.1

2 8.3 7.2

3 8.3 6.8

4 8.3 7.0

100

1 8.2 7.4

2 8.2 7.2

3 8.2 7.4

4 8.2 6.9
Study code: 11/339-023DA Final Report Page 4 of Appendix 1

Table 10: pH measured in the test vessels (Phase 1)

Concentration

Replicate

Measuring

Nominal

mg/L

Measured 

mg/L 0 h 48 h

Control 0.0

1 7.61 7.48

2 7.60 7.44

3 7.61 7.52

4 7.61 7.41

6.25 4.84

1 7.68 7.21

2 7.65 7.20

3 7.62 7.19

4 7.67 7.22

12.5 8.26

1 7.53 7.12

2 7.50 7.14

3 7.56 7.18

4 7.54 7.12

25 11.64

1 7.26 7.06

2 7.26 7.08

3 7.24 7.12

4 7.22 7.01

50 21.94

1 5.48 5.97

2 5.46 5.98

3 5.48 5.97

4 5.47 5.96

100 73.65

1 3.21 3.29

2 3.21 3.28

3 3.20 3.26

4 3.23 3.25

Table 11: pH measured in the test vessels (Phase 2)

Concentration

Replicate

Measuring

Nominal

mg/L

0 h 48 h

Control

1 7.78 7.82

2 7.78 7.85

3 7.78 7.89

4 7.78 7.91

50

1 7.72 7.69

2 7.72 7.66

3 7.72 7.65

4 7.72 7.66

100

1 7.74 7.68

2 7.74 7.64

3 7.74 7.62

4 7.74 7.62
Study code: 11/339-023DA Final Report Page 1 of Appendix 2

APPENDIX 2

DATA OF IMMOBILISATION

Table 12: Immobilisation of the test animals (Phase 1)

Concentration

Replicate

Number of

treated 

animals

Number of immobilised 

animals

Nominal

mg/L

Measured 

mg/L

24 h 48 h

Control 0.0

1 5 0 0

2 5 0 0

3 5 0 0

4 5 0 0

6.25 4.84

1 5 0 0

2 5 0 0

3 5 0 0

4 5 0 0

12.5 8.26

1 5 0 0

2 5 0 0

3 5 0 0

4 5 0 0

25 11.64

1 5 0 0

2 5 0 0

3 5 0 0

4 5 0 0

50 21.94

1 5 0 0

2 5 0 0

3 5 0 0

4 5 0 0

100 73.65

1 5 5 5

2 5 5 5

3 5 5 5

4 5 5 5
Study code: 11/339-023DA Final Report Page 2 of Appendix 2

Table 13: Immobilisation of the test animals (Phase 2)

Concentration

Replicate

Number of

treated 

animals

Number of immobilised 

animals

Nominal

mg/L

24 h 48 h

Control

1 5 0 0

2 5 0 0

3 5 0 0

4 5 0 0

50

1 5 0 0

2 5 0 0

3 5 0 0

4 5 0 0

100

1 5 0 0

2 5 0 0

3 5 0 0

4 5 0 0
Study code: 11/339-023DA Final Report Page 1 of Appendix 3

APPENDIX 3

CONCENTRATION / RESPONSE CURVE (48 h)

Figure 1: Effect of Acetonoxime on immobilisation
Study code: 11/339-023DA Final Report Page 1 of Appendix 4

APPENDIX 4

ANALYTICAL INVESTIGATIONS OF MAIN STUDY

(METHODS AND RESULTS)

1. Principle of the Analytical Method

Concentration of Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of 

ethanedioic acid [CAS RN 144-62-7]) in the test solutions was determined at the beginning 

and at the end of the test.

The samples were taken from the test solutions containing the test item and one sample from 

the control at both sampling time. 

The samples were analysed by an HPLC-UV method described in details in the analytical 

method validation of Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form 

of ethanedioic acid [CAS RN 144-62-7]) (11/339-316AN). 

2. Equipment and Chemicals

2.1. Apparatus

HPLC system: Merck-Hitachi LaChrom HPLC system

D-7000 Interface, No.: 1126-175

L-7100 HPLC pump, No. : 1388-056

L-7200 Autosampler, No.: 1380-033

L-7400 UV Detector, No.: 1374-025

L-7614 Degasser, No.: 144116A0094

L-7360 Column Oven, No.: 00118060

Balances: BP221S Sartorius, No.: 11809117

L2200P Sartorius, No.:38100037

Ultrasonic bath: Elmasonic S300H, ELMA, No.: 010890105

Water purification system: MILLIPORE, DIRECT Q3, FOMNO 7334I

pH meter: SevenEasy S20 Mettler Toledo, No.:1231025119

Refrigerator: Zanussi, No.: ZLKI-262
Study code: 11/339-023DA Final Report Page 2 of Appendix 4

2.2. Test Item

Name: Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated 

form of ethanedioic acid [CAS RN 144-62-7])

Chemical name: Oxalic acid, hydrate

CAS number: 6153-56-6

Batch/Lot number: YP1-20120528C

Molecular formula: C2H2O4×2H2O

Molecular weight: 126.1 (dihydrate form)

Appearance: White crystals

Purity: 99.69 %

Manufacture date: May 28, 2012

Expiry date: May 27, 2013

Storage conditions: Controlled room temperature (15-25o

C, < 70 RH%)

2.3. Other materials

Ultra pure water (ASTM Type I): prepared by Direct-Q 3 system, Millipore

Acetonitrile: HPLC grade, VWR, Batch: 11Z3738

HPLC grade, Sigma-Aldrich, Batch: SZBC300SV

Potassium dihydrogen phosphate: reagent, Lach-Ner, Batch: PP/2010/02241

Phosphoric acid 85%: reagent, Lach-Ner, Batch: PP/2011/12163

3. Method of analysis of test item concentration

Samples were analysed by the following HPLC-UV method:

Column: Zorbax SB-C8 250 × 4.6 mm, 5μm

Mobil Phase: 50 mM KH2PO4 (pH:2.1) : ACN 9:1

Flow: 1.0 ml/min.

Injection volume: 10 μl

Temperature: 25 °C

Detector: UV at 220 nm 

Retention time: 2.5 min ± 10%
Study code: 11/339-023DA Final Report Page 3 of Appendix 4

4. Results of the Method Validation

Table 1: Method validation results (11/339-316AN)

Selectivity No interfering component was 

observed

Reinjection repeatability

(7 injections) CV% ≤ 1.6 %

Linear range 1 – 100 μg/ml

Limit of Quantification 1 μg/ml

Recovery 100 - 106 %

Accuracy 0 - 6 %

Precision 1 – 4 %

Stability of the test item in Daphnia Test 

Medium (2 days) 89 – 100 %

Stability of the samples At least 19 hours in the autosampler

Stock solution stability At least 4 days at 5 ± 3 °C

5. Measured data

5.1. Calibration 

Table 2: Data of the regression lines

Date of measurement Slope Intercept Correlation 

coefficient (r2

)

20 November 2012 2856 498 1.000

22 November 2012 2964 486 0.9998

Study code: 11/339-023DA Final Report Page 1 of Appendix 5

APPENDIX 5

COMPOSITION OF ISO MEDIUM

Separate stock solutions of individual trace elements were first prepared in deionised 

water. The medium was prepared of these different stock solutions, so it contained all 

trace elements (combined solution).

Stock solutions Amount added Stock solutions

(single substance) to deionised added to deionised water

water to prepare media

(g/L) (ml/L)

CaCl2×2 H2O 11.76 25

MgSO4×7 H2O 4.93 25

KCl 0.23 25

NaHCO3 2.59 25
Study code: 11/339-023DA Final Report Page 1 of Appendix 6

APPENDIX 6

COPY OF THE GLP CERTIFICATE
Study code: 11/339-023DA Final Report Page 1 of Appendix 7

APPENDIX 7

COPY OF THE CERTIFICATE OF ANALYSIS