page 1 of 23 

## Final Report ( 2 nd 

## 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)

Sponsor s

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 GermanyFinal Report ( 2 nd 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



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- Obcrwachungsbellortlc/N'amc and addren o f lltc GLP M qnit(wing Aulhofit )'Final Report ( 2 nd 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 S tudy

................................ ................................ ................................ ........................ 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 Preparatio n of the Test Solutions

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

................................ ................................ ................................ .................... 15 6.8 Test Parameters

................................ ................................ ................................ ........................ 16 6.9 Result Evaluation

................................ ................................ ................................ ..................... 17 6.10 V alidity 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

................................ ................................ ................................ ................................ ............ 21Final Report ( 2 nd 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. Biodegradatio n 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

................................ ................................ .................. 23Final Report ( 2 nd 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 Tes t
Guidelines:

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

- E of May 30, 2008: Closed Bottle Test (EEC Public ation 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 M ethods:

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

- 56

- 6] (h ydrated form of ethanedioic acid [CAS RN 144

- 62

- 7]) ; Batch/Lot Number: YP1

- 20120528C ; Purity: 99.69 % Test Species: Activated sludge ( micro organisms 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 bi odegradation was followed by the oxygen uptake of the micro organisms during exposure. A reference item was tested simultaneously under the same conditi ons 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 benzo ate 3.98 mg/L corr esponding to an oxygen demand of 6. 63 mg/L based on ThOD of 1.6 66 mg oxygen per mg test item.

Test Rate: 60.06 mg/L corresponding to an oxygen demand of 7. 6 3 mg/L based on ThOD of 0.127 mg oxygen per mg test item.

Test Conditions: 2 2°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] (h ydrated f orm of ethanedioic acid [CAS RN 144

- 62

- 7]) reached in the mean 4 1 % after 4 days of incubation and continuously increased to 80 % after 1 4 days and 98 % after 28 days. The percentage biodegradation exceeded 60Final Report ( 2 nd 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 t herefore be considered as ready biodegrada ble. Biodegradation of Sodium Benzo ate : The reference item sodium benzoate was sufficiently d egraded to a mea n 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 contain ing both, the test item and the reference item sodium benzoate , a mean of 5 7 % bi odegradation was noted within 14 days and 65 % biodegr adation after 28 days of incubation. Thus, the test item can be assumed to be not inhibitor y on the activa ted sludge micro organisms.Final Report ( 2 nd 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 Sponsor s : 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] (h ydrated 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 , 20 12

1 st Study Plan Amendment: December 10, 2012 Experimental Starting Date: October 1 7, 20 12 Experimental Completion Date: November 15 , 20 12 Study Completion Date: January 10 , 2013Final Report ( 2 nd 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 ME TI) on the basis of intergovernmental agreements. This study was asses sed 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. T he 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 (1 st original)

− any final report amendment or any revised final report (1 st 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 di scarded 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

I nstitut für Biologische An.Jiytik und Consulting I BACON G m bH

page 9 of 23Final Report ( 2"d Original) I BACON Project 7 8591161

- Ready Biodegradability of Ethanedioic 

# I BAC

acid

, hydrate (1 : 2) [CAS RN 6153-56-6] (hydrated form of ethanedioic acid [CAS RN 14 4- 62

- 7]) in

a Closed Bottle Test

3. Quality A s surance Unit Statement

Test Facility:

!BACON Project:

Title ofthe Study :

Test Item:

Study Director:

Study based Inspections

Phases inspected

Study Plan

I s t Study Plan Amendment:
Experimental Phase

Dosing

, Application

, Me as ur e ments

Measurements

Draft Repoti

Final Report Institut für Biologische Analytik und Consulting !BACON GmbH Arheilger Weg 17 64380 Rossdorf Germany

78591161 I nstitut für Biologische Analytik und Consul tin g I BACON G m bH

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 2 01 3

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 23Final 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 I nstitut für Biologisdle 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 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 23Final Report ( 2 nd 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 bi odegradability 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 th e oxyge n uptake of the micro organisms du ring 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 sol uble 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 biodegradatio n properties of the test item when inc ubated 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 Biode gradability: Closed Bottle Test", adopted July 17, 1992.Final Report ( 2 nd 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 S ponsor:

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

- 56

- 6] (h ydrated form of ethanedioic acid [CAS RN 144

- 62

- 7])

Chemical Name: Oxalic acid hydrate

Batch /Lot Number : YP 1

- 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: C 2 H 2 O 4 * 2H 2 O / 126.1 (dihydrate) Theoretical Oxygen Demand: 0.127 mg O 2 /mg test item (calculated) Storage: At room temperature (20 ± 5 °C), under dark and dry co nditions

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: C 7 H 5 O 2 Na / 144.10 g/mol Theoretical Oxygen Demand: ThOD NH4 : 1.666 mg oxygen per mg sodium benzoate (calculated) Storage: At room temperature (20 + 5°C), under dark and dry co nditionsFinal Report ( 2 nd 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 (micro organisms from a domestic waste water treatment plant) was supplied by sewage pl ant Ro s sdorf, 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

- su spended 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 mat erial 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 nu mber 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 O 2 /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 KH 2 PO 4 , 21.75 g K 2 HPO 4 , 33.4 g Na 2 HPO 4 x 2 H 2 O, 0.5 g NH 4 Cl filled up with deionised water to 1000 mL volume b) 22.5 g MgSO 4 x 7H 2 O filled up with deionised water to 1000 mL volume c) 36.4 g CaCl 2 x 2H 2 O filled up with deionised water to 1000 mL volume d) 0.25 g FeCl 3 x 6H 2 O 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 ( 2 nd 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 soluti ons a)

- d) were combined and filled to a final volume of 5 000 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 2 2 + 2 ° C. 6.6 Preparation of the Test Solutions
Test Item (Flasks 1a and 1b): Based on the theoretical oxygen demand (ThOD NH4 ) of 0.12 7 mg O 2 /mg test item

, 300.3 mg of Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated form of ethanedioic ac id [CAS RN 144

- 62

- 7]) were thoroughly mixed into 5 litres of aqu eous test medium (corresponding
to 60.06 mg/L test item and a ThOD NH4 of about 7.63 mg O 2 /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 (ThOD NH4 ) of sodium benzoate (1.666 mg O 2 per mg), 19.9 mg of sod ium benzoate were dissolved in 5 litres of aqueous test medium (corresponding to 3.98 mg/L sodium benzoate and a ThOD NH4 of 6. 63 mg O 2 / 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 tho roughly mixed into 5 litres of aqueous test medium (corr esponding to a ThOD NH4 of 14. 36 mg O 2 /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 refe rence item sodium be n-Final Report ( 2 nd 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 co ntrol)

- 10 bottles containing the test item, reference item s odium ben zoate and inoculum (toxicity control)

Test Duration: 28 days

6.8 Test Parameters Measurement of Oxygen: The oxygen concentrations were electrochemically mea sured in an airtight system with an O 2 - 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 ( 2 nd 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: ThOD NH4 : 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 p er mg test item.

BOD: Biochemical Oxygen Demand
the amo unt of oxygen consumed by micro organisms when metabolising a test item; also expressed as mg oxygen uptake per mg test item.

10

- day wi n dow: the 10 days immediately following the attainment of 10 % biodegradation

Calculation of BOD: The BOD (mg O 2 per mg test item) exerted after each period was calculated as:

mg O 2 /L of test item and/or Na

- Benzoate

- mg O 2 /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 O 2 /mg test item or Na

- Benzoate) % degradation =

———————————————————— x 100 %
ThOD NH4 (mg O 2 /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 O 2 /L a fter 28 days. Oxygen Concentration: The residual oxygen concentration in the test flasks did not drop bel ow 1.6 mg O 2 /L at any time. According to the test guideline, the residual oxygen co ncentration in the test flasks should not drop below 0.5 mg O 2 /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 ofFinal Report ( 2 nd 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 %.

Referen ce Item (Sodium Benzoate ): The percentage degradation of the reference item sodium benzoate should reach the level for ready biodegradabil ity (> 60 %) by e xposure day 14. The degradation w as 7 4 % after 4 day s

. 6.11 Deviations to the Study Plan

There were no deviations to the study planFinal Report ( 2 nd 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] (h ydrated form of ethanedioic acid [CAS RN 144

- 62

- 7]) reached in the mean 4 1 % 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 d egraded to a mean of 88 % after 14 days, and to a mean of 109 % after 28 days o f incubation, thus confirming the suitability of the used activated sludge inoculum. The r esults are represented in Tables 1

- 3 and Figure 1.

Conclusion: The percentage biodegradation of the reference item co nfirms 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 me a n of 5 7 % biodegr adation was noted within 14 days and 65 % biodegradation after 28 days of incubation. The results a re represented in Tables 1

- 3 and Figure 1.

Conclusion: According to the test guidelines the test item can be a ssumed to be not inhibitor y on the activated sludge microo rganisms because degradation was > 25 % within 14 days.Final Report ( 2 nd 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 B ekanntmachung 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 Regu lation 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 Co mmission on 26 th November 1997 [C(97)186/Final], Environment Directorate, Organisation for Economic Co

- operation and Develo pment, ENV/MC/CHEM(98)17, Paris 1998

9. Distribution of the Final Report

Sponsor: 1x (2 nd original)

IBACON: 1x (1 st original)Final Report ( 2 nd 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 

## AppendixFinal Report ( 2 nd 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 d uring the Exposure Period of 28 Days

Table 2

. Oxygen Depletion at Different Time Intervals d uring 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 O 2 /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 : (m t0 - m tx ) - (m bo - m bx ), where:

m

t0 : oxygen concentration (mg/L) of test item on day 0 (1a and 1b, 2a and 2b or 4a and 4b from Table 1)

m

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

m

b0 : oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b)

m

bx : oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b) O 2 Depletion (mg/L) after n Days of ExposureFinal Report ( 2 nd 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 d uring 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 d uring 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 O 2 /L of test item and/or reference item - mg O 2 /L of inoculum control) / (mg test item and/or reference item/L in flask) % biodegradation = BOD x 100 % / ThOD NH4 of reference item and/or test item

where: ThOD NH4 of test item = 0.127 mg O 2 /mg test item
ThOD NH4 of reference = 1.666 mg O 2 /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