## FINAL REPORT

Study Title



## FRESH WATER ALGAL GROWTH INHIBITION TEST

WITH

EXP1313100

Author

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identi fication

Project 505714

Substance 205622/A

- Page 1 of 32

-EXP1313100 Project 505714

- Page 2 - 1. CONTENTS

1. CONTENTS

................................................................................................ ................................ 2

2. STATEMENT OF GLP COM PLIAN CE

......................................................................................... 4

3. QUALITY ASSURANCE ST ATEMENT

........................................................................................ 5

4. SUMMARY

................................................................................................ .................................. 6

5. INTRODUCTI ON

......................................................................................................................... 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 METHOD S

..................................................................................................... 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. Reference substance

................................................................................................ . 9

6.2. Test System

......................................................................................................................... 9

6.3. Fresh water algae culture

..................................................................................................... 9

6.4. Preparation of test solutions

............................................................................................... 10

6.5. Combined limit/range

- finding test

....................................................................................... 10

6.5.1. Test concentrations

.................................................................................................. 10

6.5.2. Test procedures and conditions

................................................................................ 10

6.5.3. Sampling for analysis of test concentrations

............................................................. 11

6.5.4. Measurements

......................................................................................................... 11

6.5.5. Recording of cell densities

........................................................................................ 11

6.6. Electronic data capture

....................................................................................................... 11

6.7. Interpretation

...................................................................................................................... 11

6.7.1. Data handling

........................................................................................................... 11

6.7.2. Acceptability of the test

............................................................................................. 12

6.8. List of deviations

................................................................................................................ 13

6.8.1. List of protocol deviations

......................................................................................... 13

6.8.2. List of standard operating procedures deviations

...................................................... 13

7. RESULTS

................................................................................................ .................................. 13

7.1. Measured test substance concentrations

............................................................................ 13

7.2. Mean cell densities

............................................................................................................. 13

7.3. Inhibition of growth rate and inhibition of yield

..................................................................... 14

7.4. Determination of effect concentrations

................................................................................ 14

7.5. Experimental conditions

..................................................................................................... 15

8. CONCLUSION

.......................................................................................................................... 15EXP1313100 Project 505714

- Page 3 - TABLES

Table 1 Percentag e inhibition of growth rate (total test period)

............................................... 14

Table 2 Percentage inhibition of growth rate at different time intervals

................................... 14

T able 3 Percentage inhibition of yield

.................................................................................... 14

Table 4 Effect parameters

..................................................................................................... 14

Table 5 pH levels

.................................................................................................................. 15

Table 6 Individual cell densities (x10 4 cells/mL)

..................................................................... 16

Table 7 Individual growth rates (day -1 )

................................................................................... 17

Table 8 Section

- by

- section growth rate

................................................................................. 18

Table 9 Individual yields

........................................................................................................ 19

FIGURES

Figure 1 Growth curves at different concentrations of EXP1313100 (mg/L)

............................. 13

APPENDICES

APPENDIX 1 WORKSHEET DATA

................................................................................................ 16

APPENDIX 2 STATISTICS: GROWTH RATE

................................................................................ 20

APPENDIX 3 STATISTICS: YIELD

................................................................................................ 21

APPENDIX 4 REFERENCE TEST

................................................................................................ . 22

APPENDIX 5 CERTIFICATE OF ANALYSIS

.................................................................................. 23

APPENDIX 6 ANALYTICAL REPORT

............................................................................................ 24EXP1313100 Project 505714

- Page 6 - 4. SUMMARY

Pseudokirchneriella subcapitata , Fresh Water Algal Growth Inhibition Test with EXP1313100

.

The study procedures described in this report were based on the OECD guideline No. 201, 2006 ;

Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods

of the Commission Regulation (EC) No 440/200 8 , Part C .3, 2008; Amended by EC No. 761/2009 , the

ISO International S tandard 8692, 2012 and the OECD series on testing and assessment number 23,

2000.

The batch of EXP1313100 tested was a brown solid that was not completely soluble in test medium at

the loading rates initially prepared

.

All test solutions were prepared separately. Various loading rates of the test substance were added to

the test medium applying two days of magnetic stirring in order to reach maximum solubility. The

resulting aqueous mixtures were filtered through a 0.45 μm membrane filter where after the Water

Accommodated Fractions (WAFs) were used for testing

. WAFs prepared at loading rates of 1.0 and

10 mg/L were observed to be clear and colourless, while the WAF prepared at a loading rate of

100 mg/L was observed to be hazy.

A combined limit/range

- fin ding test was performed. Six replicates of exponentially growing algal

cultures per group were exposed to a WAF prepared at a loading rate of 100 mg/L and to a control. In

addition, three replicates per group were exposed to W AF s prepared at loading rate s of 1.0 and

10 mg/L

. The i nitial cell density was 10 4 cells/ml and the total test period was 72 hours . S amples for

analytical confirmation of exposure concentrations were taken at the start, after 24 hours of exposure

and at the end of the test.

T he init i al concentrations in the WAFs prepared at loading rates of 1.0, 10 and 100 mg/L were 0.37,

2.6 and 7.1 mg/L. The two highest loading rate remained stable during the test period (96

- 109% of

initial at the end of the test), while the lowest loading rate decreased to 52% of initial at the end of the

test. The concentration obtained in the WAF prepared at a loading rate of 100 mg/L was clearly above

the maximum solubility in test medium based on the observed haziness of the test solution. The

average exposure concentrations were calculated to be 0.27, 2.6 and 7.7 mg/L.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

N o inhibition of growth rate or inhibition of yield was recorded at any of the loading rates of

EXP1 313100 tested.

The 72h -E L* 50 for both growth rate and yield inhibition was above a loading rate of 100 mg

EXP1313100 per litre and correspondingly the No Observed Effect Loading rate was 100 mg/ L. The

concentration of EXP1313100 in the WAF prepared at a loading rate of 100 mg/L was determined to

be 7.7 mg/L based on the average exposure concentration. This concentration was considered to be

above the maximum solubility level of EXP1313100 in test medium

.

*EL = effective loading rate