## 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/ 3 39

- 009H

Study Director: Krisztina Sipos, M.Sc.

1 9 March 201 3Study code: 11/ 3 39

- 009H Final Report Page 5 of 2 1

GENERAL INFORMATION

STUD Y 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 o f

ethanedioic acid [CAS RN 144

- 62

- 7])

SPONSOR S : 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

S TUDY 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 : CiTox LAB Hungary Ltd.

Address: H

- 8200 Veszprém, Szabadságpuszta

Phone: 36 88 545

- 300

Fax: 36 88 545

- 301Study code: 11/ 3 39

- 009H Final Report Page 6 of 2 1

STUDY DIRECTOR : Krisztina Sipos, M.Sc.

QUALITY ASSUR ANCE : 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 Nov ember 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 P arliament

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 799.508 7

.Study code: 11/ 3 39

- 009H Final Report Page 7 of 2 1



### CONTENTS

Page

1. SUMMARY 8

2. INTRODUCTION 1 0

3. MATERIALS AND METHOD S 1 0

3.1. TEST ITEM AND CONTRO LS AND VEHICLE 1 0

3. 2

. EXPERIMENTAL ANIMALS 12

3. 3

. ENVIRONMENTAL TEST C ONDITIONS 12

3. 4

. DESCRIPTION OF THE T EST PROCEDURE 13

3.5. OBSERVATIONS 14

3.6. ANIMAL W ELFARE 14

3.7. PERMISSION OF THE IA CUC 14

3

. 8 STATISTICAL EVALUATI ON 15

3

. 9 ANALYTICAL MEASUREME NTS 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 G UIDELINE 1 7

3

. 15 DEVIATIONS FR OM THE STUDY PLAN 1 7

4. RESULTS 1 8

4. 1. VALIDITY 1 8

4. 2. MORTALITY DATA 1 8

4. 3. SUB

- LETHAL EFFECTS 1 8

4. 4

. BODY WEIGHT 19

5

. CONCLUSION 2 0

APPENDICES 2 1

Data of Test conditions 1

- 2 o f A ppendix 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

Cop y of the GLP Certificate 1 of Appendix 6

Cop y of the Certificate of Analysis 1 of Appendix 7Study code: 11/ 3 39

- 009H Final Report Page 8 of 2 1

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 t est material was found to be stable in

water over the test period s tatic 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 10 0 mg /L

. The corresponding measured geometric mean test it em

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 tha n 20 per cent from the

nominal in most cases, biolo gical 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 extend ed the study to provide more data to allow a better

interp retation.

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

comprised seven fish and five litre test solution

.

The LC 50 values of the test item were calculated using Probit analysis by SPSS PC+

software . The NOEC and LO EC and LC 100 values were determined from the raw data.

All achievable validity criteria were met during this study.Study code: 11/ 3 39

- 009H Final Report Page 9 of 2 1

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

rerio ) the observed and calculated endpoints for the ef fect 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 LC 50 value: 56

. 0 mg/L ( with 95 % conf. limits: 35.83

– 105.46 )

The 96h LC 100 value: 96.6 0 mg /L

The 96h No

- Observed Effect Concentration (NOEC) : 34.22 mg/L

The 96h Lowest Observed Effect Concentration (LOEC) : 96

. 6 0 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/ 3 39

- 009H Final Report Page 10 of 2 1

2. INTRODUCTION

The objective of this study was to determine the median lethal concentration (LC 50 value) of the test item in acute toxicity test on Z ebrafish ( Brachydanio rerio )

.

F ish 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 Z ebrafish ( Brachydanio rerio ) are

recommended by the test guidelines.

3. MATERIALS AND METHOD S

3.1. TEST ITEM AND CONTRO L S 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

Mol ecular Formula: C 2 H 2 O 4 ×2H 2 O

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/ 3 39

- 009H Final Report Page 11 of 2 1

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 sol ution (see

Table 1 below)

.

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

Nominal c oncentration

[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. a d 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 c oncentration

[mg/L] Amount of stock solution

(mL) Amount of ISO medium

(mL)

10 0 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 prio r 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/ 3 39

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3.2. EXPERIMENTAL ANIMAL S

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 anim als: 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 diseas e

and 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 fo r at least

12 days acclimatisation before they are used for test.

(Remark: Acclimatisation period before the preliminary test

was only 5 days.)

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

REGULATION (EC) No 440/2008.

3.3. ENVIRONMENTAL TEST C ONDITIONS

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 2 0

. 0 and 2 0

. 8 °C (Pha se 1) and between 2 0.2 and

2 0.7 °C (Phase 2). *

The dissolved oxygen concentration was in the range of 9 0

– 9 9 % (Phase 1) and in the

range of 9 4

– 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

– 7.5 5 in the complemen tary study (Phase 2)

.

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

of 249 mg/L (as CaCO 3 ).

The light

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

The test conditions above are detailed in Appendix 1.

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

REGULATION (EC) No 440/2008.Study code: 11/ 3 39

- 009H Final Report Page 13 of 2 1

3 .4. DESCRIPTION OF THE T EST PROCEDURE

The t est duration was 96 hours. One aquarium was used for test group s and control

group respectively

. Each aquari um 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 cond ucted 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 con ditions.

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 we re 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 tha n 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/ 3 39

- 009H Final Report Page 14 of 2 1

3.4. 3

. Test item concentrati ons 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 t wo 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 beg inning 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 an y 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

. T he 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

applie d by an overdose of an aqueous solution of 2 50 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 IA CUC

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

Committee (IACUC) of CiTox LAB Hungary Ltd.Study code: 11/ 3 39

- 009H Final Report Page 15 of 2 1

3. 8

. STATISTICAL EVALUATI ON

The 24, 48, 72 and 96 hours LC 50 values were calculated by Probit analysis with 95 %

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

concentrations).

The NOEC

, LOEC and LC 100 were determined directly from the raw data.

3. 9

. ANALYTICAL MEASUREME NTS

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

concentra tion levels at the beginning and at the end of the test s (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 amendment s

,

- 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, Szabadság puszta,

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 dis solved 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 concentratio n is

greater than 20%, results will be based on the measured concentration.Study code: 11/ 3 39

- 009H Final Report Page 16 of 2 1

3

. 1 2

. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound

1x copy, unbound

1x PDF file

Archive: 1x original, bound

3. 1 3

. REFERENCES

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

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 o f 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 Europea n Parliament

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

of Chemicals (REAC H ), 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 850.107 5, "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/ 3 39

- 009H Final Report Page 17 of 2 1

3.14

. DEVIATION FROM THE G UIDELINE

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

(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.

3.1 5

. DEVIATION S 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 (self

employed), Veszprém, Hungary

Reason: Technical

Presumed effect on the study : noneStudy code: 11/ 3 39

- 009H Final Report Page 18 of 2 1

4. RESULTS

4.1. VALIDITY

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

can be considered as valid.

4. 2

. MORTALIT Y DATA

C umulative mortality data from the exposure of zebrafish to the test material d uring the

definitive test and the compl ementary study are given below in Table 4

- 5

.

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

Concentration Cumulative mortality [number of fish]

(initial population = 7 fish)

Nominal

mg/L 3h 6h 2 4h 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 complementa ry experiments.Study code: 11/ 3 39

- 009H Final Report Page 19 of 2 1

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 Table s 6

- 7

.

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

Concentration

Measured weig ht

of 7 fish (g) Calculated mean

weight of 1 fish (g) Loading of testing

aquarium

(g fish/l testin g liquid) Nominal

mg/L

Control 2. 17 0.31 0.43

6.25 1.57 0.22 0.31

12.5 2. 43 0.3 5 0.49

25 1.99 0.28 0.40

50 2.04 0.29 0.41

100 2.32 0.33 0.46

Ther e 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

aquar ium

(g fish/l testin g liquid) Nominal

mg/L

Control 2. 38 0.3 4 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/ 3 39

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5. CONCLUSION

The acute toxicity of Ethanedioic ac id, 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 a cute 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, 72 h and 96h LC 50 value: 56.0 mg/ L ( with 95 % conf. limits: 35.83

– 105.46 )

The 96h LC 100 value: 96

. 6 0 mg/L

The 96h No

- Observed Effect Concentration (NOEC) : 34

. 22 mg/L

The 96h Lowest Observed Effect Concentration (LOEC) : 96

. 6 0 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/ 3 39

- 009H Final Report Page 21 of 2 1



## APPENDICESStudy code: 11/ 3 39

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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 2 0

. 2

- - - -

Ta ble 9 : Temperature (Phase 2)

Test Group Measuring

0h 24h 48h 72h 96h

Control 20.2 20.4 20.4 20.5 20.7

5 0 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 20.7Study code: 11/ 3 39

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Table 10 : Dissolved oxygen concentration (% of the air sa turation 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

- - - -

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

Test Group Measuring

0h 24h 48h 72h 96h

Control 9 8 96 95 95 94

5 0 mg/L (nominal) 98 96 95 94 94

100 mg/L (nominal) 9 8 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 7.4 5 7.42 7.43 7.42

5 0 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 7.50Study code: 11/ 3 39

- 009H Final Report Page 1 of Appendix 2

APPENDIX 2

BODY LENGTH OF TEST FISH

Table 1 4 : B ody 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 1 5 : 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 gui deline [ Commission Regulation (EC) No 440/2008; Annex Part C, C.1]Study code: 11/ 3 39

- 009H Final Report Page 1 of Appendix 3

APPENDIX 3

GRAPHICAL PRESENTATION OF MORTALITY

Figure 1 : The Concentration / Response curve (96h)Study code: 11/ 3 39

- 009H Final Report Page 1 of Appendix 4

APPENDIX 4

ANALYTICAL INVESTIGA TIONS 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 begin ning

and at the end of the test.

The samp les 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

Balance s : BP221S Sartorius, No.: 11809117

L2200P Sartorius, No.:38100037

Ultrasonic bath: Elmasonic S300H, ELMA, N o.: 010890105

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

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

Refrigerator: Zanussi, No.: ZLKI

- 262Study code: 11/ 3 39

- 009H Final Report Page 2 of Appendix 4

2.2. Test Item

Name: Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated

fo rm 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: C 2 H 2 O 4 ×2H 2 O

Molecular weight: 126.1 (dihydrate form)

Appearance: White crystals

Purity: 99.69 %

Manufact ure date: May 28, 2012

Expiry date: May 27, 2013

Storage conditions: Controlled room temperature (15

- 25 o 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: 11Z 3738

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 c oncentration

Samples were analyse d by the following HPLC

- UV method :

Column: Zorbax SB

- C 8 2 50 × 4.6 mm, 5μm

Mobil Phase : 50 mM KH 2 PO 4 (pH:2.1) : ACN 9 : 1

Flow: 1.0 ml/min.

Injection volume: 10 μl

Temperature: 25 °C

Detector: UV at 2 2 0 nm

Retention time: 2

. 5 min ± 10%Study code: 11/ 3 39

- 009H Final Report Page 3 of Appendix 4

4. Results of the Method Validation

Table 1 : Method va lidation 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. Mea sured data

5.1. Calibration

Table 2 : Data of the regression lines

Date of measurement Slope Intercept Correlation

coefficient (r 2 )

19 November 2012 2772 564 1.000

23 November 2012 2791 644 1.000Study code: 11/339

- 0 09H 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 (com bined solution).

Stock solutions Amount added Stock solutions

(single substance) to deionised added to deionised water

water to prepare media

(g/L) (ml/L)

CaCl 2 ×2 H 2 O 11.76 25

MgSO 4 ×7 H 2 O 4.93 25

KCl 0.23 25

NaHCO 3 2.59 25Study code: 11/ 3 39

- 009H Final Report Page 1 of Appendix 6

APPENDIX 6

COPY O F THE GLP CERTIFICAT EStudy code: 11/339

- 009H Final Report Page 1 of Appendix 7

APPENDIX 7

COPY OF THE CERTIFIC ATE OF ANALYSIS