## FINAL DRAFT REPORT

- Version 2

-

Study Title



## DAPHNIA MAGNA , REPRODUCTION TEST WITH

EXP1313100

(SEMI- STATIC)

Author

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 505720

Substance 205622/A

- Page 1 of 41

-EXP1313100 Project 505720

1. CONTENTS

1. CONTENTS

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

2. STATEMENT OF GLP COM PLIANCE

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

3. QUALITY ASSURANCE ST ATEMENT

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

4. SUMMARY

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

5. INTRODUCTION

.............................................................................................................................. 8

5.1. Preface

................................................................................................ ...................................... 8

5.2. Aim of the study

........................................................................................................................ 8

5.3. Guidelines

................................................................................................ ................................ . 8

5.4. Storage and retention of records and materials

........................................................................ 8

5.5. Definitions

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

6. MATERIALS AND METHOD S

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

6.1. Test Substance

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

6.1.1. Test substance information

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

6.1.2. Study specific test substance information

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

6.2. Test system

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

6.3. Breeding

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

6.4. Acute toxicity

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

6.5. Preparation of test solutions

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

6.6. Reproduction test

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

6.6.1. Test concentrations

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

6.6.2. Test procedure and conditions

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

6.6.3. Sampling for analysis of test concentrations

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

6.6.4. Measurements and recordings

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

6.7. Further investigations

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

6.8. Interpretation

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

6.8.1. Data handling

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

6.8.2. Electronic data capture

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

6.8.3. 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

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

7.1. Reproduction test

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

7.1.1. Measured concentrations

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

7.1.2. Condition of parental daphnids

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

7.1.3. Reproduction

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

7.1.4. Body length

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

7.1.5. Experimental conditions

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

7.2. Further investigation

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

8. CONCLUS ION

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

- Page 2 -EXP1313100 Project 505720

TABLES

Table 1 Mean concentrations calculated from the analytical results

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

Table 2 Mortality (immobility) of parental daphnids at the end of the test

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

Table 3 Group mean cumulative number of juveniles per surviving parent and reduction of

reproduction at the end of the test

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

Table 4 Incidence of immobilised offspring and aborted eggs

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

Table 5 Group mean body length (mm) and reduction of growth of parental daphnids at

the end of the test.

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

Table 6 Number of living newborn per parent per day

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

Table 7 Individual values for body length (mm) of the surviving parental daphnids

.................. 31

Table 8 pH values in freshly prepared media (F) and old media (O)

......................................... 33

Table 9 Dissolved oxygen concentrations (mg/L) in freshly prepared media (F) and old

media (O)

...................................................................................................................... 33

Table 10 Temperature (°C) in freshly prepared media (F) and old media (O)

............................ 33

Table 11 Hardness (mg/L CaCO 3 ) in freshly prepared media (F) and old media (O)

................. 33

FIGURE

Figure 1 Mean concentration during the refreshment periods (1.0 mg/L)

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

Figure 2 Mean concentration during the refreshment periods (10 mg/L)

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

Figure 3 Cumula tive mean number of living neonates per parent at different test groups of

EXP1313100 (in mg/L) after 21

- days.

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

APPENDICES

APPENDIX 1 NUMBER OF LIVING NEWBORN DAPHNIDS PER PARENT

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

APPENDIX 2 STATISTICS: PARENTAL MORTALITY

...................................................................... 29

APPENDIX 3 STATISTICS: REPRODUCTION

.................................................................................. 30

APPENDIX 4 PARENTAL BODY LENGTH

........................................................................................ 31

APPENDIX 5 STATISTICS: BODY LENGTH

..................................................................................... 32

APPENDIX 6 EXPERIMENTAL CONDITIONS

.................................................................................. 33

APPENDIX 7 CERTIFICATE OF ANALYSIS

...................................................................................... 34

APPENDIX 8 ANALYTICAL REPORT

................................................................................................ 35

- Page 3 -EXP1313100 Project 505720

2. STATEMENT OF GLP COM PLIANCE

WIL Research Europe B.V., 's

- Hertogenbosch, The Netherlands

Th e study described in this report has been correctly reported and was conducted in compliance with:

T he Organi z ation for Economic Co

- operation and Development (OECD) Principles of Good Laboratory

Practice (GLP) (as revised in 1997) ENV/MC/CHEM (98) 17

.

The sponsor is responsible for Good Laboratory Practice (GLP) compliance for all test substance

information unless determined by WIL Research Europe

.

The test performed to identify a suitable solvent for preparation of a stock solution of EXP1313100

was not performed under GLP and therefore excluded from this GLP statement.

WIL Research Europe B.V.

Date: ................................................... Date: ....................................................

- Page 4 -EXP1313100 Project 505720

3. QUALITY ASSURANCE ST ATEMENT

WIL Research Europe B.V., ‘s

- Hertogenbosch, The Netherlands

This report was inspected by the WIL Research Europe Quality Assurance Unit to confirm that the

methods and results accurately and completely reflect the raw data.

During the on

- site process inspections, procedures applicable to this type of study were inspected.

The dates of Quality Assurance inspections are give n below.

Project 505720

Type of

Inspections Phase/Process Start

Inspection

date End

Inspection

date Reporting

date

Study Protocol 24

- Jun

- 2014 24

- Jun

- 2014 24

- Jun

- 2014

Protocol Amendment 01 21

- Aug

- 2014 21

- Aug

- 2014 21

- Aug

- 2014

Test subst ance preparation 26

- Aug

- 2014 26

- Aug

- 2014 26

- Aug

- 2014

Exposure 26

- Aug

- 2014 26

- Aug

- 2014 26

- Aug

- 2014

Protocol Amendment 02 06

- Nov

- 2014 06

- Nov

- 2014 06

- Nov

- 2014

Report 15

- Jan

- 2015 16

- Jan

- 2015 16

- Jan

- 2015

Process Test Substance Receipt 24

- Jul

- 2014 3 1

- Jul

- 2014 01

- Aug

- 2014

Test Substance Handling

Analytical and physical

chemistry 05

- Aug

- 2014 12

- Aug

- 2014 13

- Aug

- 2014

Test Substance Handling

Exposure

Observations/Measurements

Specimen Handling

Environmental Science 11

- Aug

- 2014 15

- Aug

- 2014 15

- Aug

- 2014

Test Substance Handling

Exposure

Observations/Measurements

The review of the final report was completed on the date of signing this QA statement.

WIL Research Europe B.V.

Quality Assur ance

Date:.............................

- Page 5 -EXP1313100 Project 505720

4. SUMMARY

Daphnia magna , 21

- day reproduction study with EXP1313100

.

The study procedures described in this report were based on the OECD guidelines for Testing of

Chemicals: Guideline No. 211, 2012 . In addition, the procedures were designed to meet the test

methods and validity criteria of the ISO International Standard 10706

, 2000 , the Commission

Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000.

The batch of EXP1313100 tested was a brow n glassy solid that was not completely soluble in test

medium at the loading rates prepared

. A non

- GLP test was performed to identify a suitable solvent for

preparation of a stock solution of EXP1313100 that would subsequently be used to prepare the test

s olutions. EXP1313100 was not sufficiently soluble in any suitable solvent. Therefore, test solutions

were prepared as Water Accommodated Fractions (WAFs).

Preparation of test solutions started with loading rates of 1.0 and 10 mg/L applying two days of

m agnetic stirring in order to reach maximum solubility. The resulting aqueous mixtures were filtered

through a 0.45 μm membrane filter where after the clear and colourless WAFs were used for testing

.

The reproduction test was performed in a semi

- static sys tem and included 2 0 vessels per test

concentration and control group. Each of the vessels contained one neonate (<24h old) Daphnia

magna in 50 m L test medium. The study duration was 21 days and the test solutions were renewed

every 48 hours. The daphnids were fed on a daily basis with a Chlorella pyrenoidosa suspension.

Every workday the condition of the parental daphnids was recorded, and during the reproduction

phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs

was recorded. At the end of the test the lengths of the surviving parental daphnids were measured.

At the start of each renewal period the actual test concentrations ranged between 0.00037 and

0.0053 mg/L in the WAF prepared at 1.0 mg/L and between 0.00070 and 0.035 mg/L in the WAF

prepared at 10 mg/L There was considerable variability in the measured concentrations throughout the

study; this is likely to have been caused by two main factors. Firstly, the loading rates used in the

study were well in excess of the water solubility of EXP1313100 (0.02 mg/L

- Project 505730), which is

likely have contributed to the presence of undissolved test substance in the WAFs. Secondly,

EXP1313100 is a brown glassy solid and its physical nature makes it difficult to ha ndle. Specifically,

weighing out samples with uniform surface area is difficult. The surface area of the sample used to

prepare the WAFs is likely to have affected the dissolution rates of EXP1313100 during the

preparation of each renewal media. Thus, variable surface areas of EXP1313100 would be expected

to be present in each renewal media at a given loading rate, even though the mass was the same. The

measured concentrations generally decreased to 59

- 78% of initial during the 48

- hour renewal periods.

EX P1313100 does not contain any readily hydrolysable functional groups, so the decrease in the

measured concentrations during the 48

- hour renewal period was not attributed to hydrolysis. The

decrease may be due to precipitation and/or adsorption of the test substance (although precipitation

was not observed visually).

Based on these results mean concentrations were calculated to be 0.0019 and 0.011 mg/L for the

whole test period of 21 days. The coefficients of variation were 109 and 133%, respectively.

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

No significant mortality was observed in parental Daphnia. Statistically significant effects on

reproduction and growth were observed at loading rates of 1.0 and 10 mg/L. Hence, a NOELR for both

reproduction and growth of parental daphnids could no t be established in this study. The effects on

reproduction coincided with algal cells fed to the organisms adhering to the parental daphnids , a phenomenon noted from day 4 to 14 in both test treatments. Post day 14 this phenomenon ceased

and parental daphnids began to produce predominantly mobile offspring. This indicated that the

effects on reproduction were transient and related to parental daphnids being hampered by th e sticking of the algal cells. The algae stuck to the outside of the organisms would not have been able to

- Page 6 -EXP1313100 Project 505720

have been ingested and would have hampered the daphnids’ ability to access the remaining food in

the test media, reducing the ability of the parental daphnids to obtain sufficient food. Reduced feeding

ability would in turn account for the statistically significant reduction in growth and reproduction.

Although it was the best option available, the WAF preparation technique was concluded not to be

a dequate for generating test solutions of EXP1313100 with consistent exposure concentrations, as

evidenced by the variability in the measured concentrations throughout the reproduction study and the

additional investigations. Therefore, no reliable NOELR could be established in the study. It is

concluded that lower test concentrations could not be prepared, because the WAF preparation

technique is not adequate for generating consistent concentrations of EXP1313100 at set loading

rates

.

*NOELR = No effect loading rate

- Page 7 -