CiToxLAB Hungary Ltd.

Veszprém - Szabadságpuszta

FINAL REPORT

Acute toxicity test with

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

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

on Zebrafish (Brachydanio rerio)

Study code: 11/339-009H

Study Director: Krisztina Sipos, M.Sc.

19 March 2013



Study code: 11/339-009H Final Report Page 5 of 21

GENERAL INFORMATION

STUDY TITLE : Acute toxicity test with Ethanedioic acid, hydrate 

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

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

Zebrafish (Brachydanio rerio)

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

Phone: 36 88 545-300

Fax: 36 88 545-301
Study code: 11/339-009H Final Report Page 6 of 21

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 : 19 November 2012

END OF EXPERIMENT : 07 December 2012

BASIS OF STUDY:

– OECD Guideline for Testing of Chemicals, No. 203, "Fish, Acute Toxicity Test", 

adopted July 17, 1992

– Commission Regulation (EC) No 440/2008 of 30 May 2008 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.1 (published in the Official Journal of the 

European Union L 142 of 31 May 2008

– EPA Guideline 712-C-96-118: OPPTS 850.1075, "Fish Acute Toxicity Test, 

Freshwater and Marine" 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: 11/339-009H Final Report Page 7 of 21

CONTENTS

Page

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. ENVIRONMENTAL TEST CONDITIONS 12

3.4. DESCRIPTION OF THE TEST PROCEDURE 13

3.5. OBSERVATIONS 14

3.6. ANIMAL WELFARE 14

3.7. PERMISSION OF THE IACUC 14

3.8 STATISTICAL EVALUATION 15

3.9 ANALYTICAL MEASUREMENTS 15

3.10 ARCHIVES 15

3.11 VALIDITY CRITERIA OF THE STUDY 15

3.12 DISTRIBUTION OF THE FINAL REPORT 16

3.13 REFERENCES 16

3.14 DEVIATION FROM THE GUIDELINE 17

3.15 DEVIATIONS FROM THE STUDY PLAN 17

4. RESULTS 18

4.1. VALIDITY 18

4.2. MORTALITY DATA 18

4.3. SUB-LETHAL EFFECTS 18

4.4. BODY WEIGHT 19

5. CONCLUSION 20

APPENDICES 21

Data of Test conditions 1 - 2 of Appendix 1

Body Length of Test Fish 1 of Appendix 2

Graphical presentation of mortality 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: 11/339-009H Final Report Page 8 of 21

1. SUMMARY

The 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 fish toxicity test 

on Zebrafish (Brachydanio rerio), over an exposure period of 96 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: 4.48; 6.77; 14.62; 34.22 and 96.6 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 there was evidence of marked change in the pH of the test water after addition 

of the test substance, and the pH was below 6 in the definitive test (Phase 1) 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. We extended the study to provide more data to allow a better 

interpretation.

One aquarium was used in each test group and one in the control group. Each aquarium

comprised seven fish and five litre test solution.

The LC50 values of the test item were calculated using Probit analysis by SPSS PC+ 

software. The NOEC and LOEC and LC100 values were determined from the raw data.

All achievable validity criteria were met during this study.
Study code: 11/339-009H Final Report Page 9 of 21

Under the conditions of this acute fish toxicity study on zebrafish (Brachydanio 

rerio) the observed and calculated 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, 48h, 72h and 96h LC50 value: 56.0 mg/L (with 95 % conf. limits: 35.83 –105.46)

The 96h LC100 value: 96.60 mg/L

The 96h No-Observed Effect Concentration (NOEC): 34.22 mg/L

The 96h Lowest Observed Effect Concentration (LOEC): 96.60 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: 11/339-009H Final Report Page 10 of 21

2. INTRODUCTION

The objective of this study was to determine the median lethal concentration (LC50

value) of the test item in acute toxicity test on Zebrafish (Brachydanio rerio).

Fish were exposed in a static test to ISO test media, containing the test item at 

different concentration. 

The test method of application and the test species Zebrafish (Brachydanio rerio) 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: 11/339-009H Final Report Page 11 of 21

3.1.2. Dilution and Preparation of Testing Solutions

Prior to treatment, a test solution of 100 mg/L was prepared by diluting an appropriate 

amount of test item in ISO medium.

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 10000 –

50 2500 q.s. ad 5000

25 1250 q.s. ad 5000

12.5 625 q.s. ad 5000

6.25 312.5 q.s. ad 5000

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 10000 –

50 2500 q.s. ad 5000

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 fish.

3.1.3. Untreated Control

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

3.1.4. 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. 

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

Appendix 5.
Study code: 11/339-009H Final Report Page 12 of 21

3.2. EXPERIMENTAL ANIMALS

Species: Zebrafish (Brachydanio rerio)

Source: “Black Molly” Fish Farm; Ágnes Németh (self-employed), 

Veszprém, Hungary

Justification of species: Zebrafish (Brachydanio rerio) is one of the convenient 

species for acute fish toxicity test.

Number of animals: seven fish per test group

Body length of animals*: 2.6 – 3.4 cm (Phase 1) and 2.7 – 3.3 cm (Phase2)

Food and Feeding: The fish were not fed during the test

Acclimatisation: >12 days

Animal health: Fish were bred in a well-known fish farm, under diseaseand parasite-controlled conditions. Fish were observed 

during the acclimatisation and test periods, the fish were 

healthy. There was no mortality of the population for seven 

days before the test.

Holding: All fish were obtained and held in ISO medium for at least 

12 days acclimatisation before they are used for test. 

(Remark: Acclimatisation period before the preliminary test

was only 5 days.)

*Remark: Based on the recommendation of the Official Journal of the European Union, COUNCIL 

REGULATION (EC) No 440/2008.

3.3. ENVIRONMENTAL TEST CONDITIONS

Constant conditions were maintained throughout the study.

The water temperature, oxygen concentration and pH were measured daily in each test 

aquarium.

The test temperature was between 20.0 and 20.8 °C (Phase 1) and between 20.2 and 

20.7 °C (Phase 2).*

The dissolved oxygen concentration was in the range of 90 – 99 % (Phase 1) and in the 

range of 94 – 98 % (Phase 2) of the air saturation value at the temperature used.

The pH was in the range of 3.28 – 7.79 in the definitive test (Phase 1) and in the range 

of 7.00 – 7.55 in the complementary study (Phase 2).

The reconstituted water (ISO medium) had an approximate theoretical total hardness 

of 249 mg/L (as CaCO3).

The light-dark cycle during the test was 16 hours light and 8 hours darkness.

The test conditions above are detailed in Appendix 1.

*Remark: Based on the recommendation of the Official Journal of the European Union, COUNCIL 

REGULATION (EC) No 440/2008.
Study code: 11/339-009H Final Report Page 13 of 21

3.4. DESCRIPTION OF THE TEST PROCEDURE

The test duration was 96 hours. One aquarium was used for test groups and control 

group respectively. Each aquarium comprised 7 fish and 5 L test solution. The animals 

were not fed during the test. The loading of the test aquaria was less than 1.0 g fish/L 

test solution at the start of the experiment. 

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

preliminary range-finding test. 

3.4.1. Preliminary Range-Finding Test

A preliminary 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. Four fish were exposed to each concentration of 

the test item plus a control, for 96 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 (96 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 animals 4 4 4 4 4

Number of dead animals 0 0 0 0 4

3.4.2. Concentration Levels Investigated in the Definitive Test (Phase 1)

Because mortality was observed during the preliminary range-finding test, therefore 

five test concentrations in a geometric series with a separation factor of 2 and one 

control were used in the main static 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: 4.48; 6.77; 14.62; 34.22

and 96.6 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.

Concentrations were analytically determined at the beginning and end of the 

experiments.

The analytically measured test item concentrations are detailed in Appendix 4.
Study code: 11/339-009H Final Report Page 14 of 21

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

Because there was evidence of marked change in the pH of the test water after addition 

of the test substance, and the pH was below 6 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: 15.99 and 

32.94 mg/L.

Concentrations were analytically determined at the beginning and end of the 

experiments.

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

3.5. OBSERVATIONS

The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 

and 96 hours. Mortality and any sub-lethal effect were determined in each observation 

period.

The test conditions (pH, temperature, oxygen saturation) were measured daily during 

the test.

The body weights of the fish were recorded before the introduction of fish. The body 

weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated 

on the basis of these body weights (g fish/litre testing liquid).

3.6. ANIMAL WELFARE

After the experiment, all fish were humanely killed. Euthanasia for all animals was 

applied by an overdose of an aqueous solution of 250 mg/L Tricaine 

Methanesulphonate (MS 222; CAS No.: 886-86-2). This technique is considered to 

provide a humane euthanasia by direct depression of the brain and vital centers. 

3.7. PERMISSION OF THE IACUC

The conduct of the study was permitted by Institutional Animal Care and Use 

Committee (IACUC) of CiToxLAB Hungary Ltd.
Study code: 11/339-009H Final Report Page 15 of 21

3.8. STATISTICAL EVALUATION

The 24, 48, 72 and 96 hours LC50 values were calculated by Probit analysis with 95 % 

confidence limits using using SPSS PC+ software (based on the measured

concentrations). 

The NOEC, LOEC and LC100 were determined directly from the raw data.

3.9. 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.

3.10. 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.

3.11. VALIDITY CRITERIA OF THE STUDY

- Constant conditions were maintained throughout the study.

- The mortality in the controls should not exceed 10 % at the end of the test (or one 

fish if less than ten are used). In case of limit test, no mortality should be occurred.

- The dissolved oxygen concentration (throughout the test) has to be at least

60 % of air saturation value.

- The concentration of the test item should be at least 80 % of the nominal 

concentration throughout the test. If the deviation from the nominal concentration is 

greater than 20%, results will be based on the measured concentration.
Study code: 11/339-009H Final Report Page 16 of 21

3.12. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound

1x copy, unbound

1x PDF file

Archive: 1x original, bound

3.13. 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 440/2008 of 30 May 2008 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.1 (published in the Official Journal of the 

European Union L 142 of 31 May 2008)

4. EPA Guideline 712-C-96-118: OPPTS 850.1075, "Fish Acute Toxicity Test, 

Freshwater and Marine" April 1996

5. OECD Guideline for Testing of Chemicals, Section 2, No. 203, "Fish, Acute 

Toxicity Test", adopted July 17, 1992.

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: 11/339-009H Final Report Page 17 of 21

3.14. DEVIATION FROM THE GUIDELINE

Due to the problem with analytical measurement of the test item in aquarium water 

(based on the analytical method development and validation) the study was performed 

using ISO medium.

This deviation is considered to have no impact on the outcome of the study and 

interpretation of the results.

3.15. DEVIATIONS FROM THE STUDY PLAN

Concerning: Holding, Temperature range

According to the Study Plan: 20 – 24 °C with a maximum deviation of ± 1 °C

Deviation: Temperature during the acclimatization was in the 

range of 19.2 – 22.8 °C

Reason: Technical

Presumed effect on the study: none

Concerning: Holding, Feeding

According to the Study Plan: Feeding daily, until 24 hours before the start of the 

test.

Deviation: three times per week or daily until 24 hours before 

the start of the test

Reason: Technical

Presumed effect on the study: none

Concerning: EXPERIMENTAL ANIMALS, Source

According to the Study Plan: “Black Molly” Fish Farm, Veszprém, Hungary

Deviation: “Black Molly” Fish Farm; Ágnes Németh (selfemployed), Veszprém, Hungary

Reason: Technical

Presumed effect on the study: none
Study code: 11/339-009H Final Report Page 18 of 21

4. RESULTS

4.1. VALIDITY

The achievable validity criteria were within acceptable limits and therefore the study 

can be considered as valid.

4.2. MORTALITY DATA

Cumulative mortality data from the exposure of zebrafish to the test material during the

definitive test and the complementary study are given below in Table 4-5.

Table 4: Cumulative mortality data in the Definitive test (Phase 1)

Concentration Cumulative mortality [number of fish]

(initial population = 7 fish)

Nominal

mg/L

3h 6h 24h 48h 72h 96h

Control 0 0 0 0 0 0

6.25 0 0 0 0 0 0

12.5 0 0 0 0 0 0

25 0 0 0 0 0 0

50 0 0 0 0 0 0

100 7 - - - - -

Table 5: Cumulative mortality data in the Complementary study (Phase 2)

Concentration Cumulative mortality [number of fish]

(initial population = 7 fish)

Nominal

mg/L

3h 6h 24h 48h 72h 96h

Control 0 0 0 0 0 0

50 0 0 0 0 0 0

100 0 0 0 0 0 0

4.3. SUB-LETHAL EFFECTS

Behaviour of fish was found as normal at the tested concentrations during the 

performed definitive and complementary experiments.
Study code: 11/339-009H Final Report Page 19 of 21

4.4. BODY WEIGHT

The body weight of 7 fish was weighed at the start of the test. The measured and 

calculated data are listed below in Tables 6-7.

Table 6: Measured and calculated data of bodyweight (Phase 1)

Concentration

Measured weight 

of 7 fish (g)

Calculated mean 

weight of 1 fish (g)

Loading of testing 

aquarium

(g fish/l testing liquid)

Nominal

mg/L

Control 2.17 0.31 0.43

6.25 1.57 0.22 0.31

12.5 2.43 0.35 0.49

25 1.99 0.28 0.40

50 2.04 0.29 0.41

100 2.32 0.33 0.46

There was no considerable difference observed concerning body weights between the 

groups.

Table 7: Measured and calculated data of bodyweight (Phase 2)

Concentration

Measured weight 

of 7 fish (g)

Calculated mean 

weight of 1 fish (g)

Loading of testing 

aquarium

(g fish/l testing liquid)

Nominal

mg/L

Control 2.38 0.34 0.48

50 2.52 0.36 0.50

100 2.59 0.37 0.52

There was no considerable difference observed concerning body weights between the 

groups.
Study code: 11/339-009H Final Report Page 20 of 21

5. CONCLUSION

The 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 fish toxicity test 

on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a static 

system.

Under the conditions of this acute fish toxicity study on zebrafish (Brachydanio 

rerio) the observed and calculated 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, 48h, 72h and 96h LC50 value: 56.0 mg/L (with 95 % conf. limits: 35.83 –105.46)

The 96h LC100 value: 96.60 mg/L

The 96h No-Observed Effect Concentration (NOEC): 34.22 mg/L

The 96h Lowest Observed Effect Concentration (LOEC): 96.60 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: 11/339-009H Final Report Page 21 of 21

APPENDICES
Study code: 11/339-009H Final Report Page 1 of Appendix 1

APPENDIX 1

DATA OF TEST CONDITIONS

Table 8: Temperature (Phase 1)

Concentration Measuring 

Nominal

mg/L

Measured

mg/L 0h 24h 48h 72h 96h

Control 0.0 20.2 20.4 20.0 20.1 20.7

6.25 4.48 20.2 20.4 20.0 20.1 20.8

12.5 6.77 20.2 20.4 20.0 20.1 20.8

25 14.62 20.2 20.4 20.0 20.1 20.8

50 34.22 20.2 20.4 20.0 20.1 20.8

100 96.60 20.2 - - - -

Table 9: Temperature (Phase 2)

Test Group Measuring 

0h 24h 48h 72h 96h

Control 20.2 20.4 20.4 20.5 20.7

50 mg/L (nominal) 20.2 20.4 20.4 20.5 20.7

100 mg/L (nominal) 20.2 20.4 20.3 20.5 20.7
Study code: 11/339-009H Final Report Page 2 of Appendix 1

Table 10: Dissolved oxygen concentration (% of the air saturation value) (Phase 1)

Concentration Measuring 

Nominal

mg/L

Measured

mg/L 0h 24h 48h 72h 96h

Control 0.0 98 98 97 95 94

6.25 4.48 97 97 96 95 94

12.5 6.77 97 97 96 94 94

25 14.62 96 94 91 90 90

50 34.22 96 99 99 96 95

100 96.60 96 - - - -

Table 11: Dissolved oxygen concentration (% of the air saturation value) (Phase 2)

Test Group Measuring 

0h 24h 48h 72h 96h

Control 98 96 95 95 94

50 mg/L (nominal) 98 96 95 94 94

100 mg/L (nominal) 98 96 95 94 94

Table 12: pH (Phase 1)

Concentration Measuring 

Nominal

mg/L

Measured

mg/L 0h 24h 48h 72h 96h

Control 0.0 7.70 7.69 7.62 7.61 7.79

6.25 4.48 7.29 7.76 7.65 7.55 7.62

12.5 6.77 7.06 7.71 7.60 7.50 7.65

25 14.62 6.61 7.34 7.47 7.39 7.60

50 34.22 4.93 5.36 5.81 7.29 7.45

100 96.60 3.28 - - - -

Table 13: pH (Phase 2)

Test Group Measuring 

0h 24h 48h 72h 96h

Control 7.43 7.45 7.42 7.43 7.42

50 mg/L (nominal) 7.43 7.44 7.45 7.44 7.43

100 mg/L (nominal) 7.00 7.53 7.55 7.51 7.50
Study code: 11/339-009H Final Report Page 1 of Appendix 2

APPENDIX 2

BODY LENGTH OF TEST FISH 

Table 14: Body length of test fish at the end of the test (Phase 1)

Concentration

Concentration

Nominal

mg/L

Measured

mg/L

Control 0.0 3.3 2.7 2.9 2.7 2.7 3.2 3.1

6.25 4.48 3.1 3.0 2.7 2.8 2.8 3.0 3.0

12.5 6.77 2.8 3.4 3.1 3.2 2.8 2.9 2.9

25 14.62 3.2 2.7 3.1 2.8 3.3 3.4 2.6

50 34.22 3.0 3.3 3.4 2.7 2.8 2.6 3.0

100 96.60 3.1 3.2 2.7 2.9 3.3 2.7 3.2

Table 15: Body length of test fish at the end of the test (Phase 2)

Concentration

Nominal

(mg/L)

Body length [cm]

Control 3.1 3.0 3.0 2.9 3.2 3.3 2.8

50 2.9 3.1 3.2 3.3 2.8 2.7 3.0

100 2.8 3.0 3.1 3.2 2.9 2.8 3.2

Remark: At the end of the test (or when mortality occurred), body length of test fish were 

measured in order to check their compliance with the size range recommended for the species by 

the test guideline [Commission Regulation (EC) No 440/2008; Annex Part C, C.1]
Study code: 11/339-009H Final Report Page 1 of Appendix 3

APPENDIX 3

GRAPHICAL PRESENTATION OF MORTALITY

Figure 1: The Concentration / Response curve (96h)
Study code: 11/339-009H 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: 11/339-009H 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: 11/339-009H 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 (4 days) for fish test 112 – 115 %

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

)

19 November 2012 2772 564 1.000

23 November 2012 2791 644 1.000

Study code: 11/339-009H 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: 11/339-009H Final Report Page 1 of Appendix 6

APPENDIX 6

COPY OF THE GLP CERTIFICATE
Study code: 11/339-009H Final Report Page 1 of Appendix 7

APPENDIX 7

COPY OF THE CERTIFICATE OF ANALYSIS