FINAL REPORT

Study Title

DETERMINATION OF 'READY' BIODEGRADABILITY: CARBON 

DIOXIDE (CO2) EVOLUTION TEST (MODIFIED STURM TEST) OF 

EXP1313100

Author

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s-Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 505722

Substance 205622/A

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EXP1313100 Project 505722 

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1. CONTENTS 

1. CONTENTS ...................................................................................................................................... 2

2. STATEMENT OF GLP COMPLIANCE ............................................................................................. 4

3. QUALITY ASSURANCE STATEMENT ............................................................................................ 5

4. SUMMARY ........................................................................................................................................ 6

5. INTRODUCTION .............................................................................................................................. 7

5.1. Preface ...................................................................................................................................... 7

5.2. Aim of the study ........................................................................................................................ 7

5.3. Guidelines ................................................................................................................................. 7

5.4. Storage and retention of records and materials ........................................................................ 7

5.5. Definitions.................................................................................................................................. 8

6. MATERIALS AND METHODS .......................................................................................................... 8

6.1. Test Substance ......................................................................................................................... 8

6.1.1. Test substance information ............................................................................................. 8

6.1.2. Study specific test substance information ...................................................................... 8

6.1.3. Test concentration and preparation of test solutions ...................................................... 9

6.2. Reference substance ................................................................................................................ 9

6.2.1. Reference substance ...................................................................................................... 9

6.2.2. Reference substance concentration and preparation of test solutions.........................10

6.3. Test system .............................................................................................................................10

6.4. Test procedure and conditions ................................................................................................10

6.4.1. Preparation of bottles ....................................................................................................11

6.4.2. Determination of CO2....................................................................................................12

6.4.3. Measurements and recording .......................................................................................12

6.5. Electronic data capture ...........................................................................................................12

6.6. Interpretation ...........................................................................................................................13

6.6.1. Data evaluation .............................................................................................................13

6.6.2. Acceptability of the test .................................................................................................13

6.7. List of deviations .....................................................................................................................13

6.7.1. List of protocol deviations .............................................................................................13

6.7.2. List of standard operating procedures deviations .........................................................14

7. RESULTS .......................................................................................................................................14

7.1. Theoretical CO2 production .....................................................................................................14

7.2. Biodegradation ........................................................................................................................14

7.3. Monitoring of temperature and pH ..........................................................................................16

8. CONCLUSION ................................................................................................................................16

Final Report
EXP1313100 Project 505722 

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TABLES 

Table 1 Test set-up....................................................................................................................11

Table 2 pH values of different test media. .................................................................................16

Table 3 HCl titrated in duplicate blank bottles ...........................................................................17

Table 4 HCl titrated in Ba(OH)2 solution ....................................................................................17

Table 5 CO2 production and percentage biodegradation of the positive control substance ......18

Table 6 CO2 production in the blank ..........................................................................................18

Table 7 CO2 production and percentage biodegradation with direct addition of 

EXP1313100 (bottle A). ................................................................................................18

Table 8 CO2 production and percentage biodegradation with direct addition of 

EXP1313100 (bottle B). ................................................................................................19

Table 9 CO2 production and percentage biodegradation of the toxicity control with direct 

addition of EXP1313100. ..............................................................................................19

Table 10 Comparison of biodegradation in bottles A and B with direct addition of 

EXP1313100. ................................................................................................................19

Table 11 HCl titrated in duplicate inert carrier blank bottles ........................................................20

Table 12 CO2 production in the inert carrier blank. ......................................................................20

Table 13 CO2 production and percentage biodegradation with EXP1313100 coated on 

granular silica gel (bottle A). .........................................................................................21

Table 14 CO2 production and percentage biodegradation with EXP1313100 coated on 

granular silica gel (bottle B). .........................................................................................21

Table 15 CO2 production and percentage biodegradation of the toxicity control with 

EXP1313100 coated on granular silica gel. ..................................................................22

Table 16 Comparison of biodegradation of the test substance in bottles A and B with 

EXP1313100 coated on granular silica gel. ..................................................................22

FIGURES 

Figure 1 Biodegradation of EXP1313100 (direct addition) and sodium acetate in the 

modified Sturm test .......................................................................................................15

Figure 2 Biodegradation of EXP1313100 (coated on granular silica gel) and sodium 

acetate in the modified Sturm test ................................................................................15

APPENDICES 

APPENDIX 1 TABLES ........................................................................................................................17

APPENDIX 2 CERTIFICATE OF ANALYSIS ......................................................................................23

Final Report


EXP1313100 Project 505722 

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4. SUMMARY 

Determination of ‘ready’ biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) of

EXP1313100.

The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In 

addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) 

No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C, ISO 9439, 1999, ISO 10634, 1995 

and the OECD series on testing and assessment number 23, 2000. 

EXP1313100 was a brown solid and was not sufficiently soluble to allow preparation of an aqueous 

solution at a concentration of 1 g/L. Since the exact composition of the test substance was not known 

at the start of the test, the organic carbon content could not be calculated. Instead, the Total Organic 

Carbon (TOC) content of EXP1313100 was determined to be 76%. The test substance was tested in 

duplicate at approximately 15.7 mg/L, corresponding to 12 mg TOC/L. ThCO2 was calculated to be 

2.80 mg CO2/mg. 

Two parallel test series were performed applying different methods for the addition of the test 

substance. In the first series the test substance was added directly to the test medium and in the 

second series by the use of an inert carrier (granular silica gel). 

In the first series, weighed amounts of the test substance were added to the 2-litres test bottles 

containing the medium with microbial organisms and mineral components. To this end, 10 mL of MilliRO water was added to each weighing bottle containing the test substance. After vigorous mixing 

(vortex) with water the resulting suspension was added quantitatively to the inoculated test medium. 

In the second series, weighed amounts were dissolved in 50 mL of methanol. The clear slightly brown 

stock solutions were stirred and quantitatively added to the granular silica gel. The methanol was 

evaporated from granular silica gel and inoculated test medium was added. 

The test solutions were continuously stirred during the test, to ensure optimal contact between the test 

substance and the test organisms. Test duration was 28 days (last CO2 measurement on day 29). 

Test set-up: 

In total 11 test bottles were prepared containing the inoculated medium and if applicable additional 

component: 

 

- 2 Blanks: no test substance, 

- 1 Positive control: sodium acetate 

Test series 1 (direct addition): 

- 2 Test bottles: EXP1313100 applied by direct addition, 

- 1 Toxicity control EXP1313100 applied by direct addition plus sodium acetate. 

Test series 2 (coated on granular silica gel): 

- 2 Inert carrier blank: no test substance, methanol “coated” granular silica gel, 

- 2 Test bottles EXP1313100 coated granular silica gel, 

- 1 Toxicity control EXP1313100 coated granular silica plus sodium acetate. 

The relative biodegradation values calculated from the measurements, for both test substance 

application methods, performed during the test period revealed no significant biodegradation of 

EXP1313100. In the toxicity control, EXP1313100 was found not to inhibit microbial activity. 

Since all criteria for acceptability of the test were met, this study was considered to be valid. 

In conclusion, EXP1313100 was designated as not readily biodegradable. 

Final Report