page 1 of 23

Final Report (2nd Original)

Ready Biodegradability of

Ethanedioic acid, hydrate (1:2) 

[CAS RN 6153-56-6] (hydrated 

form of ethanedioic acid [CAS RN 

144-62-7])

in a Closed Bottle Test

(GLP compliant study based on the Commission Regulation 

440/2008/EC, C.4-E, 2008 and the OECD No. 301 D, 1992) 

Author: Dr. Ute Hammesfahr

Study Completion Date: January 10, 2013

CiToxLAB study code: 11/339-914AN

 (previous study code 11/339-322AN)

Sponsors

Univar USA Inc.

17425 NE Union Hill Road

Redmond, WA 98052

United States of America

WEGO Chemical and Mineral Corp.

239 Great Neck Road

Great Neck, NY 11021 

United States of America

Test Facility

Institut für Biologische Analytik Project 78591161

und Consulting IBACON GmbH

Arheilger Weg 17

64380 Rossdorf

Germany
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 2 of 23

Copy of GLP - Certificate

IBAC N 

lnsutut für Biologische Analytik und Consu~i g !BACON GmbH 

HESSEN 

Gute Laborpraxis/Good Laboratory Practice 

GLP-Bescbeinigung/Statcment of GLP Complianoo 

(&cml!31ao::cording 10 § 19b Abs. I Olemikalius.emz} 

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Chcllltlk~ll•n~c:sotz arid Directivc 2Q04./9mec M: 

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IMCh\.1 tllr Biologisdie Analytilc und ComultinG IBACOJII Omb}l 

A~llp We1 11 

64310 Rendorf 

(UnYerwedrselbarc Bczcidmong und Adrcsse/UnequiYQCa11111me and adrn•} 

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me genannte i>r11foinriclltong beftndcl sich im llllllonJien OL Obcn~hl.II1&1V.:rfShrc t Uftd wird ~clml.ßig aufl!inhollt1L~ <kr OLP·OI'\Jodsau:e Ob«waellt 

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MainurSimk80 0 65189 Wlubftdtn 

(Name Ufld Adreue der GLP-Obcrwachungsbellortlc/N'amc and addren of lltc GLP Mqnit(wing Aulhofit)' 
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 3 of 23

Contents

Copy of GLP - Certificate ..................................................................................................................2

1. Summary ........................................................................................................................................5

2. Survey of the Study........................................................................................................................7

2.1 General Information .................................................................................................................7

2.2 Good Laboratory Practice ........................................................................................................8

2.3 Archiving..................................................................................................................................8

2.4 Signatures.................................................................................................................................9

3. Quality Assurance Unit Statement .................................................................................................10

4. Statement of Compliance ...............................................................................................................11

5. Objectives of the Study ..................................................................................................................12

5.1 Title ..........................................................................................................................................12

5.2 Purpose.....................................................................................................................................12

5.3 Guidelines / Recommendations................................................................................................12

6. Material and Methods.....................................................................................................................13

6.1 Test Item and Reference Item ..................................................................................................13

6.2 Test System..............................................................................................................................14

6.3 Test Units.................................................................................................................................14

6.4 Test Conditions ........................................................................................................................14

6.5 Test Water................................................................................................................................14

6.6 Preparation of the Test Solutions.............................................................................................15

6.7 Course of the Test ....................................................................................................................15

6.8 Test Parameters........................................................................................................................16

6.9 Result Evaluation .....................................................................................................................17

6.10 Validity Criteria of the Study.................................................................................................17

6.11 Deviations to the Study Plan ..................................................................................................18

7. Results and Discussion...................................................................................................................19

7.1 Biodegradation of Test Item.....................................................................................................19

7.2 Biodegradation of Reference Item Sodium Benzoate ..............................................................19

7.3 Biodegradation of the Toxicity Control ...................................................................................19

8. References......................................................................................................................................20

9. Distribution of the Final Report .....................................................................................................20

Appendix ............................................................................................................................................21
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 4 of 23

List of Tables

Table 1. Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 

28 Days ................................................................................................................................................22

Table 2. Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days ..............22

Table 3. Biodegradation at Different Time Intervals during the Exposure Period of 28 Days...................23

List of Figures

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

ethanedioic acid [CAS RN 144-62-7]), of the Reference Item Sodium Benzoate and of the Toxicity 

Control during the Exposure Period of 28 Days..................................................................................23
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 5 of 23

1. Summary

Title: Ready Biodegradability of Ethanedioic acid, hydrate (1:2) 

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

[CAS RN 144-62-7]) in a Closed Bottle Test

Guidelines: − Commission Regulation 440/2008/EC, Method C.4-E 

of May 30, 2008: Closed Bottle Test (EEC Publication No. L 142/497-502, May 2008)

− OECD Guideline for Testing of Chemicals No. 301 

D: "Ready Biodegradability: Closed Bottle Test", 

adopted July 17, 1992.

Material and Methods:

Test Item: Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]); 

Batch/Lot Number: YP1-20120528C;

Purity: 99.69 %

Test Species: Activated sludge (microorganisms from a domestic waste 

water treatment plant) was supplied by the sewage plant 

Rossdorf, Germany.

Test Design: The test item Ethanedioic acid, hydrate (1:2) [CAS RN 

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

144-62-7]) was investigated for its ready biodegradability 

in a Closed Bottle Test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the 

microorganisms during exposure. A reference item was 

tested simultaneously under the same conditions as the test 

item, and functioned as a procedure control.

Endpoints: Degradation rate of test item calculated by the oxygen 

consumption of the activated sludge microorganisms after 

28 days of incubation.

Reference Item: Sodium benzoate 

3.98 mg/L corresponding to an oxygen demand of 6.63

mg/L based on ThOD of 1.666 mg oxygen per mg test 

item.

Test Rate: 60.06 mg/L corresponding to an oxygen demand of 7.63

mg/L based on ThOD of 0.127 mg oxygen per mg test 

item.

Test Conditions: 22°C ± 1°C, darkness

Results:

Biodegradation of 

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

6153-56-6] (hydrated form of ethanedioic 

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

Under the test conditions the percentage biodegradation of 

Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7])

reached in the mean 41 % after 4 days of incubation and 

continuously increased to 80 % after 14 days and 98% 

after 28 days. The percentage biodegradation exceeded 60 
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 6 of 23

% within the 10-day window. The test item can therefore 

be considered as ready biodegradable.

Biodegradation of Sodium Benzoate:

The reference item sodium benzoate was sufficiently degraded to a mean of 88 % after 14 days, and to a mean of 

109 % after 28 days of incubation, thus confirming the 

suitability of the used activated sludge inoculum.

Biodegradation of the Toxicity Control:

In the toxicity control containing both, the test item and 

the reference item sodium benzoate, a mean of 57 % biodegradation was noted within 14 days and 65 % biodegradation after 28 days of incubation. Thus, the test item can 

be assumed to be not inhibitory on the activated sludge 

microorganisms.
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 7 of 23

2. Survey of the Study

2.1 General Information

Sponsors: Univar USA Inc.

17425 NE Union Hill Road

Redmond, WA 98052

United States of America

WEGO Chemical and Mineral Corp.

239 Great Neck Road

Great Neck, NY 11021

United States of America

Sponsor Representative: John V. Massingale

Greenwich Chemical Consulting, Inc.

3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410, USA

Monitoring: David Esdaile

CiToxLAB Hungary Ltd.

8200 Veszprém – Szabadságpuszta

Hungary

Test Item: Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7])

Test Facility: Institut für Biologische Analytik und 

Consulting IBACON GmbH

Arheilger Weg 17

64380 Rossdorf

Germany

IBACON-Project: 78591161

Project Staff:

Test Facility Management: Dr. Ralf Petto

Study Director: Dr. Ute Hammesfahr

Head of Quality Assurance Unit (QAU): Christiane Rutschmann-Fröhlich (Biologist)

Schedule:

Study Initiation Date: October 17, 2012

1st Study Plan Amendment: December 10, 2012

Experimental Starting Date: October 17, 2012

Experimental Completion Date: November 15, 2012

Study Completion Date: January 10, 2013
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 8 of 23

2.2 Good Laboratory Practice This study was performed conducted in compliance with 

the most recent edition of:

− The OECD Principles of Good Laboratory Practice 

− German Chemicals Act (ChemG), Annex 1 

− Directive 2004/10/EC 

These requirements are accepted by regulatory authorities 

throughout the European Community, the United States of 

America (FDA and EPA) and Japan (MHLW, MAFF and 

METI) on the basis of intergovernmental agreements.

This study was assessed in compliance with the study plan 

and the IBACON Standard Operating Procedures. This 

study and procedures are periodically inspected by the 

Quality Assurance Unit (QAU) and the dates and the 

phases of the inspections are included in the final report. 

The data presented in the final report were audited in 

comparison to the raw data. A quality assurance statement, 

signed by the Quality Assurance Unit, is included in the 

final report.

2.3 Archiving

The following documents and materials will be archived 

for the periods specified in the operative national GLP 

regulations of the Study Director:

− all raw data

− the study plan

− any study plan amendment

− the final report (1st original)

− any final report amendment or any revised final report 

(1st original)

− one sample of the test item and of any reference item

following the date on which the final report is audited by 

the Quality Assurance Unit at:

Institut für Biologische Analytik 

und Consulting IBACON GmbH 

Germany

No raw data or material relating to the study will be discarded without the sponsor's prior written consent.
Final Report (2"d Original) I BACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1 :2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test 

2.4 Signatores 

Study Director: Dr. Ute Hammesfahr 

Date: 

Test Facility Management: Dr. Ralf Petto 

Date: 

IBAC 

Institut für Biologische An.Jiytik und 

Consulting IBACON GmbH 

page 9 of 23 
Final Report (2"d Original) I BACON Project 78591161 - Ready Biodegradability of Ethanedioic I BAC 

acid, hydrate (1 :2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test 

3. Quality Assurance Unit Statement 

Test Facility: 

!BACON Project: 

Title ofthe Study: 

Test Item: 

Study Director: 

Study based Inspections 

Phases inspected 

Study Plan 

I st Study Plan Amendment: 

Experimental Phase 

Dosing, Application, Measurements 

Measurements 

Draft Repoti 

Final Report 

Institut für Biologische Analytik 

und Consulting !BACON GmbH 

Arheilger Weg 17 

64380 Rossdorf 

Germany 

78591161 

Institut für Biologische Analytik und 

Consulting I BACON GmbH 

Ready Biodegradability of Ethanedioic acid, hydrate (1 :2) 

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

[CAS RN 144-62-7]) in a Closed Bottle Test 

Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form ofethanedioic acid [CAS RN 144-62-7]) 

Dr. Ute Hammesfahr 

Dates ofQAU Inspections Dates ofRepmis to Study 

Director and to Test Facility 

Management 

October 15,2012 October 15, 2012 

December 10, 2012 December 10, 2012 

October 18,2012 

November 15, 2012 

November 28-29,2012 

January 10, 2013 

JAN 1 1 2013 

October 18, 2012 

November 15, 2012 

November 29, 2012 

January 10, 2013 

JAN 1 1 2013 

This statement confirms that the final repmi accurately reflects the raw data. 

Quality Assurance Unit: Biologist 

Dr. Steffen Schubert 

Date: J:z <t• 1..4."_., 1 ..AA , ZQtt l 

page 10 of 23 
Final Report (2"d Original) IBAGON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1 :2) [GAS RN 6153-56-6] (hydrated form of ethanedioic acid [GAS RN 144-62-7]) in 

a Glosed Bottle Test · 

4. Statement of Compliance 

IBACON Project: 

Title ofthe Study: 

Test Item: 

Study Director: 

GLP-Regulations: 

Integrity ofthe Study: 

Study Director: 

Date: 

78591161 

Institut für Biologisdle Analytik und 

Consulting IBACON GmbH 

Ready Biodegradability of Ethanedioic acid, hydrate (1 :2) 

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

[CAS RN 144-62-7]) in a Closed Bottle Test 

Ethanedioic acid, hydrate (1 :2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) 

Dr. Ute Hammesfahr 

- The OECD Principles of Good Labaratory Practice 

- German Chemieals Act (ChemG), Annex 1 

- Directive 2004/10/EC 

These requirements are accepted by regulatory authorities 

throughout the European Community, the United States of 

America (FDA and EPA) and Japan (MHLW, MAFF and 

METI) on the basis of intergovernmental agreements. 

This study, perfmmed in the test facility of IBACON, was 

conducted in compliance with the Good Labaratory Practice regulations. There were no circumstances that may 

have affected the quality or integrity ofthe study. 

Dr. Ute Hammesfahr 

page 11 of 23 
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 12 of 23

5. Objectives of the Study

5.1 Title

Ready Biodegradability of Ethanedioic acid, hydrate (1:2) 

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

[CAS RN 144-62-7]) in a Closed Bottle Test

5.2 Purpose

The purpose of this study was to determine the ready biodegradability of the test item Ethanedioic acid, hydrate 

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

acid [CAS RN 144-62-7]). The test item was exposed to 

activated sludge from the aeration tank of a domestic waste 

water treatment plant for 28 days. The biodegradation was 

followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was 

tested simultaneously under the same conditions as the test 

item, and functioned as a procedure control.

A Closed Bottle Test was chosen for the determination of

the ready biodegradability since the test item is well soluble in test water at the recommended test concentration.

This type of study is recognized by the international test 

guidelines and should provide a rational basis to assess the 

ready biodegradation properties of the test item when incubated with activated sludge.

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.

5.3 Guidelines / Recommendations

This study was designed to comply with the following 

methods:

− Commission Regulation 440/2008/EC, Method C.4-E 

of May 30, 2008: Closed Bottle Test (EEC Publication 

No. L 142/497-502, May 2008)

− OECD Guideline for Testing of Chemicals No. 301 D: 

"Ready Biodegradability: Closed Bottle Test", adopted 

July 17, 1992.
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 13 of 23

6. Material and Methods

6.1 Test Item and Reference Item

Test Item

The test item and the information concerning the test item were provided by the Sponsor:

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 

Batch/Lot Number: YP1-20120528C

CAS Number: 6153-56-6

Colour / Appearance: White crystals

Purity: 99.69 %

Certificate of Analysis Date: June 01, 2012

Manufacture Date: May 28, 2012

Expiry (Retest) Date: May 27, 2013

Empirical Formula / Molecular Weight: C2H2O4 * 2H2O / 126.1 (dihydrate)

Theoretical Oxygen Demand: 0.127 mg O2/mg test item (calculated)

Storage: At room temperature (20 ± 5 °C), under dark and dry conditions

Reference Item

The information concerning the reference item were provided by the manufacturer:

Name: Sodium Benzoate

Batch No.: 110M1219V

Retest Date: November 2014

Purity: 100.4%

Physical State at Room Temperature: Solid

Colour: White

Quality Release Date: December 09, 2010

Empirical Formula / Molecular Weight: C7H5O2Na / 144.10 g/mol 

Theoretical Oxygen Demand: ThODNH4: 1.666 mg oxygen per mg sodium benzoate 

(calculated)

Storage: At room temperature (20 + 5°C), under dark and dry conditions
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 14 of 23

6.2 Test System

Species / Origin: Activated sludge (microorganisms from a domestic waste 

water treatment plant) was supplied by sewage plant 

Rossdorf, Germany.

Conditioning: The aerobic activated sludge used for this study was 

washed by centrifugation and the supernatant liquid phase 

was decanted. The solid material was re-suspended in tap 

water and centrifuged again. This procedure was done 

three times. The sediment was re-suspended in test water 

and aerated overnight. 

An aliquot of the final sludge suspension was weighed, 

dried and the ratio of wet sludge to its dry weight was 

determined. Based on this ratio, calculated aliquots of 

washed sludge suspension, corresponding to 4 g dry material per litre were mixed with test water (see 6.5) and then 

aerated until use. Before use the sludge was filtered 

through cotton wool.

6.3 Test Units

Type and Size: Karlsruher flasks of 268 mL volume with glass stoppers

Identification: Each test unit was uniquely identified with the study number and treatment.

6.4 Test Conditions

Surrounding Type: Controlled environment room

Temperature: 22+1°C

Light Conditions: Darkness

pH-Value of Test Water: 7.3 (measured at the start of the test)

Oxygen Concentration of Test Water: 8.9 – 9.0 mg O2/L (measured at the start of the test)

6.5 Test Water

Reconstituted Water: In deionised water analytical grade salts were added to 

prepare the following stock solutions:

a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 

H2O, 0.5 g NH4Cl filled up with deionised water to 

1000 mL volume

b) 22.5 g MgSO4 x 7H2O filled up with deionised water to 

1000 mL volume 

c) 36.4 g CaCl2 x 2H2O filled up with deionised water to 

1000 mL volume

d) 0.25 g FeCl3 x 6H2O filled up with deionised water to 

1000 mL volume

In order to avoid precipitation of iron hydroxide in the 

stock solution d) immediately before use, one drop of 

concentrated HCl per litre was added.
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 15 of 23

5 mL of the stock solutions a) - d) were combined and 

filled to a final volume of 5000 mL with deionised water. 

The test medium was aerated for 20 minutes and allowed 

to stand for about 20 hours at the test temperature. 

The dissolved oxygen concentration was 8.9-9.0 mg/L at 

about 22+2°C.

6.6 Preparation of the Test Solutions

Test Item (Flasks 1a and 1b): Based on the theoretical oxygen demand (ThODNH4) of 

0.127 mg O2/mg test item, 300.3 mg of Ethanedioic acid, 

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

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

mixed into 5 litres of aqueous test medium (corresponding 

to 60.06 mg/L test item and a ThODNH4 of about 7.63 mg 

O2/L, respectively). Then 2.5 mL of the filtered inoculum 

were added to the 5 litres test medium. The pH of the test 

item suspension was 3.4 and therefore adjusted to 7.2 with 

1n NaOH solution. 

The preparation was then filled into the test flasks, during 

this procedure the preparation was continuously stirred 

using a magnetic stirrer.

Procedure Control: Sodium Benzoate

(Flasks 2a and 2b):

Based on the theoretical oxygen demand (ThODNH4) of 

sodium benzoate (1.666 mg O2 per mg), 19.9 mg of sodium benzoate were dissolved in 5 litres of aqueous test 

medium (corresponding to 3.98 mg/L sodium benzoate 

and a ThODNH4 of 6.63 mg O2/L). Then 2.5 mL of the 

filtered inoculum were added to the 5 litres test medium.

Inoculum Control (Flasks 3a and 3b): Only 2.5 mL of the filtered inoculum were added to 5 

litres of aqueous test medium.

Toxicity Control (Flasks 4a and 4b): 300.5 mg of Ethanedioic acid, hydrate (1:2) [CAS RN 

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

144-62-7]) and 20.2 mg of sodium benzoate were thoroughly mixed into 5 litres of aqueous test medium (corresponding to a ThODNH4 of 14.36 mg O2/L). Then, 2.5 mL 

of the filtered inoculum were added to the 5 litres test 

medium. The pH of the toxicity control suspension was 

3.4 and therefore adjusted to 7.2 with 1n NaOH solution.

6.7 Course of the Test

Preparation of Test Flasks: A sufficient number of Karlsruher flasks was heated to

180°C for at least 6 hours. Then they were tightly closed 

by a glass stopper. 

The following bottles were prepared:

- 16 bottles containing the test item and inoculum

- 16 bottles containing the reference item sodium ben-
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 16 of 23

zoate and inoculum (procedure control)

- 16 bottles containing only inoculum (inoculum control)

- 10 bottles containing the test item, reference item sodium benzoate and inoculum (toxicity control)

Test Duration: 28 days

6.8 Test Parameters

Measurement of Oxygen: The oxygen concentrations were electrochemically measured in an airtight system with an O2-electrode (WTW Oxi 

340, Wissenschaftlich Technische Werkstätten GmbH, 

Weilheim, Germany) under constant stirring.

Oxygen measurements were performed in duplicate on 

days 0, 1, 4, 7, 12, 14, 21 and 28 (measurements of the 

toxicity control were done only on days 0, 7, 14, 21 and 

28).

Temperature: Temperature was measured each day of oxygen consumption measurement.
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 17 of 23

6.9 Result Evaluation

Definitions: ThODNH4: Theoretical Oxygen Demand

the total amount of oxygen required to oxidise a chemical 

completely. It is calculated from the molecular formula, 

assuming no nitrification occurs, and expressed as mg 

oxygen required per mg test item.

BOD: Biochemical Oxygen Demand

the amount of oxygen consumed by microorganisms when 

metabolising a test item; also expressed as mg oxygen 

uptake per mg test item.

10-day window: the 10 days immediately following the attainment of 10 % 

biodegradation

Calculation of BOD: The BOD (mg O2 per mg test item) exerted after each 

period was calculated as:

 mg O2/L of test item and/or Na-Benzoate - mg O2/L of inoculum control

BOD = —————————————————————————————

 mg test item and/or Na-Benzoate/L in flask

The percentage biodegradation of the test item and of the

reference item sodium benzoate was calculated as:

 BOD (mg O2/mg test item or Na-Benzoate)

% degradation = ———————————————————— x 100 %

 ThODNH4 (mg O2/mg test item or Na-Benzoate)

6.10 Validity Criteria of the Study

Inoculum Control: Oxygen depletion in the inoculum control did not exceed 

1.5 mg O2/L after 28 days.

Oxygen Concentration: The residual oxygen concentration in the test flasks did 

not drop below 1.6 mg O2/L at any time.

According to the test guideline, the residual oxygen concentration in the test flasks should not drop below 0.5 mg 

O2/L at any time except the analytical method is capable 

to detect these amounts.

The oxygen concentration dropped below 0.5 mg/L in the 

toxicity control due to the complete biodegradation of 

sodium benzoate. The oxygen measurement equipment 

used is able to detect an amount of 0.1 mg/L oxygen in 

aqueous solutions.

Test Item: The difference of duplicate values for the degradation of 
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 18 of 23

the test item at the plateau, at the end of the test and at the 

end of the 10-day window was less than 20 %.

Reference Item (Sodium Benzoate): The percentage degradation of the reference item sodium 

benzoate should reach the level for ready biodegradability 

(> 60 %) by exposure day 14. The degradation was 74 % 

after 4 days.

6.11 Deviations to the Study Plan

There were no deviations to the study plan
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 19 of 23

7. Results and Discussion

7.1 Biodegradation of Test Item

Percentage Biodegradation: Under the test conditions the percentage biodegradation of 

Ethanedioic acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7])

reached in the mean 41 % after 4 days of incubation and 

continuously increased to 80 % after 14 days and 98% after 

28 days. The results are represented in Tables 1 - 3 and 

Figure 1.

Conclusion: The percentage biodegradation exceeded 60 % within the 

10-day window. The test item can therefore be considered 

as ready biodegradable.

7.2 Biodegradation of Reference Item Sodium Benzoate

Percentage Biodegradation: The reference item sodium benzoate was sufficiently degraded to a mean of 88 % after 14 days, and to a mean of 

109 % after 28 days of incubation, thus confirming the 

suitability of the used activated sludge inoculum. The results are represented in Tables 1 - 3 and Figure 1.

Conclusion: The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.

7.3 Biodegradation of the Toxicity Control

Percentage Biodegradation: In the toxicity control containing both, the test item and the 

reference item sodium benzoate, a mean of 57 % biodegradation was noted within 14 days and 65 % biodegradation 

after 28 days of incubation. The results are represented in 

Tables 1 - 3 and Figure 1.

Conclusion: According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was > 25 % within 14 days.
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 20 of 23

8. References 

1. Chemikaliengesetz der Bundesrepublik Deutschland (ChemG), Anhang 1, in der Fassung der Bekanntmachung vom 2. Juli 2008 (BGBl. I S. 1146), zuletzt geändert durch Art. 5 Abs. 39 G v. 24. 

Februar 2012 (BGBl. I S. 212)

2. Commission Regulation 440/2008/EC, Method C.4-E of May 30, 2008: Closed Bottle Test (EEC 

Publication No. L 142/497-502, May 2008)

3. Directive 2004/10/EC of 11 February 2004 amending Council Directive 87/18/EEC, Official Journal 

of the European Union No. L 50: 44 - 59

4. OECD Guideline for Testing of Chemicals No. 301 D: "Ready Biodegradability: Closed Bottle Test", 

adopted July 17, 1992

5. OECD Principles of Good Laboratory Practice, adopted by Commission on 26th November 1997 

[C(97)186/Final], Environment Directorate, Organisation for Economic Co-operation and Development, ENV/MC/CHEM(98)17, Paris 1998

9. Distribution of the Final Report

Sponsor: 1x (2nd original)

IBACON: 1x (1st original)
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 21 of 23

Appendix
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 22 of 23

Table 1. Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period 

of 28 Days

Table 2. Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

Treatment Flask Conc.

No. [mg/L] 0 1 4 7 12 14 21 28

Test Item 1a 60.06 8.8 9.0 5.6 3.6 2.5 2.2 1.7 1.9

1b 60.06 8.8 8.9 5.6 3.5 2.6 2.3 1.6 1.9

mean 60.06 8.8 9.0 5.6 3.6 2.6 2.3 1.7 1.9

Procedure Control 2a 3.98 8.9 8.3 3.8 3.2 2.7 2.5 2.2 2.2

Na-Benzoate 2b 3.98 8.8 8.4 3.9 3.2 2.7 2.5 2.1 2.3

mean 3.98 8.9 8.4 3.9 3.2 2.7 2.5 2.2 2.3

Inoculum Control 3a --- 8.8 9.0 8.8 8.7 8.6 8.4 7.8 9.4

3b --- 8.9 9.0 8.7 8.7 8.5 8.3 7.9 9.6

mean --- 8.9 9.0 8.8 8.7 8.6 8.4 7.9 9.5

Toxicity Control 4a TI: 60.10 8.8 n.d. n.d. 3.2 n.d. 0.1 0.1 0.1

Ref: 4.04

4b TI: 60.10 8.8 n.d. n.d. 3.0 n.d. 0.1 0.1 0.1

Ref: 4.04

mean 8.8 --- --- 3.1 --- 0.1 0.1 0.1

n.d.: not determined

TI: test item

Ref: reference item Na-Benzoate

mg O2/L after n Days of Exposure

Treatment Flask Conc.

No. [mg/L] 1 4 7 12 14 21 28

Test Item 1a 60.06 -0.1 3.1 5.0 6.0 6.1 6.1 7.5

1b 60.06 0.0 3.1 5.1 5.9 6.0 6.2 7.5

Procedure Control 2a 3.98 0.7 5.0 5.5 5.9 5.9 5.7 7.3

Na-Benzoate 2b 3.98 0.5 4.8 5.4 5.8 5.8 5.7 7.1

Inoculum Control mean --- -0.1 0.1 0.2 0.3 0.5 1.0 -0.6

Toxicity Control 4a TI: 60.10 n.d. n.d. 5.4 n.d. 8.2 7.7 9.3

4b Ref: 4.04 n.d. n.d. 5.6 n.d. 8.2 7.7 9.3

n.d.: not determined

TI: test item

Ref: reference item Na-Benzoate

oxygen depletion : (mt0 - mtx) - (mbo - mbx), where:

mt0 : oxygen concentration (mg/L) of test item on day 0 (1a and 1b, 2a and 2b or 4a and 4b from Table 1)

mtx: oxygen concentration (mg/L) of test item on day x (1a and 1b, 2a and 2b or 4a and 4b from Table 1)

mb0: oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b)

mbx: oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b)

O2 Depletion (mg/L) after n Days of Exposure
Final Report (2nd Original) IBACON Project 78591161 - Ready Biodegradability of Ethanedioic 

acid, hydrate (1:2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 144-62-7]) in 

a Closed Bottle Test

page 23 of 23

Table 3. Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

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

ethanedioic acid [CAS RN 144-62-7]), of the Reference Item Sodium Benzoate and of the Toxicity 

Control during the Exposure Period of 28 Days

Treatment Flask Conc.

No. [mg/L] 1 4 7 12 14 21 28

Test Item 1a 60.06 -1 41 66 79 80 80 98

1b 0 41 67 77 79 81 98

mean -1 41 67 78 80 81 98

Procedure Control 2a 3.98 11 75 83 89 89 86 110

Na-Benzoate 2b 8 72 81 87 87 86 107

mean 10 74 82 88 88 86 109

Toxicity Control 4a TI: 60.10 n.d. n.d. 38 n.d. 57 54 65

4b Ref: 4.04 n.d. n.d. 39 n.d. 57 54 65

mean n.d. n.d. 39 n.d. 57 54 65

n.d.: not determined

TI: test item

Ref: reference item Na-Benzoate

BOD = ( mg O2/L of test item and/or reference item - mg O2/L of inoculum control) / (mg test item and/or reference item/L in flask)

% biodegradation = BOD x 100 % / ThODNH4 of reference item and/or test item

where: ThODNH4 of test item = 0.127 mg O2/mg test item

ThODNH4 of reference = 1.666 mg O2/mg reference item

% Biodegradation after n Days of Exposure

-20

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

% Biodegradation (ThODNH4)

Days

Test Item Flask 1 Test Item Flask 2 Procedure Control Toxicity Control

10-days-window