## FINAL REPORT

Study Title 

## DETERMINATION OF 'RE ADY' BIODEGRADABILIT Y: CARBON DIOXIDE (CO 2 

## ) EVOLUTION TEST (MO DIFIED STURM TEST) O F EXP1313100

Author

Test Facility WIL Research Europe B.V. Hambakenwetering 7 5231 DD ‘s - Hertogen bosch The Netherlands Laboratory Project Identification Project 505722 Substance 205622/A

- Page 1 of 23 -EXP1313100 Project 505722

- Page 2 - 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 CO 2 .................................................................................................... 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 CO 2 production ..................................................................................................... 14 7.2. Biodegradation ........................................................................................................................ 14 7.3. Monitoring of temperature and pH .......................................................................................... 16 8. CONCLUSION ................................................................................................................................ 16EXP1313100 Project 505722

- Page 3 - TABLES Table 1 Test set- up ................................................................ .................................................... 11 Table 2 pH values of different test media. ................................................................................. 16 Tabl e 3 HCl titrated in duplicate blank bottles ........................................................................... 17 Table 4 HCl titrated in Ba(OH) 2 solution .................................................................................... 17 Table 5 CO 2 production and percentage biodegradation of the positive control substance ...... 18 Table 6 CO 2 production in the blank .......................................................................................... 18 Table 7 CO 2 production and percentage biodegradation with direct addition of

EXP1313100 (bottle A). ................................................................................................ 18 Table 8 CO 2 production and percentage biodegradation with direct addition of

EXP1313100 (bottle B). ................................................................................................ 19 Table 9 CO 2 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 CO 2 production in the inert carrier blank. ................................................................ ...... 20 Table 13 CO 2 production and percentage biodegradation with EXP1313100 coated on

granular silica gel (bottle A). ......................................................................................... 21 Table 14 CO 2 production and percentage biodegradation with EXP1313100 coated on

granular silica gel (bottle B). ......................................................................................... 21 Table 15 CO 2 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 APP ENDICES APPENDIX 1 TABL ES ........................................................................................................................ 17 APPENDIX 2 CERTIFICATE OF ANALYSIS ...................................................................................... 23EXP1313100 Project 505722

- Page 6 - 4. SUMMARY Determination of ‘ready’ biodegradability: carbon dioxide (CO 2 ) 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 Commissi on 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 Carbo n (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 . ThCO 2 was calculated to be 2.8 0 mg CO 2 /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 direct ly 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 bottlescontaining 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 m ethano l 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 CO 2 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 P ositive control: sodium acetate Test series 1 (direct addition):

- 2 T est bottles: EXP1313100 applied by direct addition ,

- 1 T oxicity 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” granul ar silica gel,

- 2 T est bottles EXP1313100 coated granular silica gel,

- 1 T oxicity control EXP1313100 coated granular silica plus sodium acetate. The relative biodegradation values calculated from the measurements , for both test s ubstance application methods, per formed 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 wa s designated as not readily biodegradable.