## FINAL REPORT

Study Title



## ACTIVATED SLUDGE RES PIRATION INHIBITION TEST (CARBON AND AMMONIUM OXIDATION)

WITH

EXP1313100

Author

Test Facility

WIL Research Europe B.V. Hambakenwetering 7 5231 DD ‘s

- Hertogenbosch The Netherlan ds

Laboratory Project Identification

Project 505719 Substance 205622/A

- Page 1 of 29

-EXP1313100 Project 505719

- 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 substance – preparation of test solutions

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

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

...................................................................................................... 9 6.2.2. Reference substance – preparation of stock and test concentrations

............................ 9 6.3. Combined limit/range-finding test

........................................................................................... 10 6.4. Limit test

.................................................................................................................................. 10 6.5. Test system

............................................................................................................................. 10 6.6. Test procedure and conditions

................................................................................................ 11 6.7. Electronic data capture

................................................................ ........................................... 12 6.8. Interpretation

........................................................................................................................... 12 6.8.1. Calculations

................................................................ .................................................. 12 6.8.2. Acceptability of the test

................................................................................................ . 14 6.9. List of deviations

................................................................ ..................................................... 14 6.9.1. List of protocol deviations

............................................................................................. 14 6.9.2. List of standard operating procedures deviations

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

....................................................................................................................................... 14 7.1. Study results

........................................................................................................................... 14 7.2. Determination of effect parameters

......................................................................................... 15 7.3. Experimental conditions

................................................................ .......................................... 15 8. CONCLUSION

................................................................................................................................ 15EXP1313100 Project 505719

- Page 3 - 1. CONTENTS, continued

TABLES Table 1 Combined limit/range-finding test

................................................................................. 10 Table 2 Li mit test

........................................................................................................................ 10 Table 3 Effect parameters for the inhibition of the respiration rate

............................................ 15 Table 4 Combined limit range/finding results

............................................................................. 16 Table 5 Li mit test results

................................................................ ............................................ 17 Table 6 Combined limit/range-finding test: Probit analysis (respiration inhibition) 3,5-

dichlorophenol

................................................................ ............................................... 26 Table 7 Limit test: Probit analysis (respiration inhibition) 3,5 -dichlorophenol

............................ 27

FIGURES Figure 1 Combined limit/range-finding test - Recording of oxygen consumption (11:00-

11:11)

............................................................................................................................ 19 Figure 2 Combined limit/range-finding test -Recording of oxygen consumption (11:20-

11:31)

............................................................................................................................ 20 Figure 3 Combined limit/range-finding test - Recording of oxygen consumption (13:30-

13:41)

............................................................................................................................ 21 Figure 4 Limit test - Recording of oxygen consumption (13:50-14:01)

....................................... 22 Figure 5 Limit test - Recording of oxygen consumption (11:00-11:12)

....................................... 23 Figure 6 Limit test -Recording of oxygen consumption (11:20-11:31)

........................................ 24 Figure 7 Limit test -Recording of oxygen consumption (14:50-15:01)

........................................ 25 Figure 8 Combined limit/range-finding test: Percentage inhibition of the respiration as

function of the log concentration (mg/l) of 3,5-dichlorophenol

...................................... 26 Figure 9 Limit test: Percentage inhibition of the respiration as function of the log

concentration (mg/l) of 3,5-dichlorophenol

................................................................ ... 27

APPENDICES APPENDIX 1 STUDY RESULTS

........................................................................................................ 16 APPENDIX 2 EC -VALUE OF REFERENCE SUBSTANCE

............................................................... 26 APPENDIX 3 DETERMINATION OF NOEC

....................................................................................... 28 APPENDIX 4 CERTIFICATE OF ANALYSIS

...................................................................................... 29EXP1313100 Project 505719

- Page 6 - 4. SUMMARY

The influence of EXP1313100 on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based on the OECD guideline No. 209, 2010

. In add ition, the procedures were designed to meet the test methods of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

The batch of EXP1313100 tested was a brown solid. No correction was made for the purity/composition of the test substance.

The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 24 to 243⁄4 hours. Subsequently, 16 ml synthetic medium (= sewage feed), 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring. Thereafter, oxygen consumption was recorded for 10 to 12 minutes.

In the combined limit/range-finding test loading rates of 10, 100 and 1000 mg/l were tested. The highest loading rate was tested in triplicate; lower concentrations consisted of one replicate. Furthermore, at 1000 mg/l an abiotic control (1 replicate) and three replicates with a nitrification inhibitor were tested.

However, the respiration rate of one replicate of the highest loading rate could not be determined, therefore a limit test was performed. The highest loading rate was tested in triplicate with and without a nitrification inhibitor.

In both tests the responses were compared to the blank and nitrification controls.

In the limit test n o statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg EXP1313100 per litre. Therefore, was the EC 50 above the highest loading rate tested (1000 mg/l).

The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5 -dichlorophenol, which showed normal sensitivity in both tests.

Both tests met the acceptability criteria prescribed by the protocol and were considered valid. However, since the effects at the limit concentration could not be determined in the combined limit/range-finding test the results at 1000 mg/l of the limit test w as used for evaluation.

In conclusion, EXP1313100 was not toxic to waste water (activated sludge) bacteria at a loading rate of 1 000 mg/l (NOEC).

The EC 50 was above 1000 mg/l.