## FINAL DRAFT REPORT

- Version 2

-

Study Title



## FISH EARLY

- LIFE STAGE TOXICITY TEST WITH

EXP1313100

( SEMI

- STATIC )

Author

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD 's

- Hertogenbosch

The Netherla nds

Laboratory Project Identification

Project 505721

Substance 205622 /A

- Page 1 of 4 1

-EXP1313100 Project 505721

1. CO N TENTS

1. CONTENTS

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

2. STATEMENT OF GLP COM PLIANCE

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

3. QUALITY ASSURANCE ST ATEMENT

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

4. SUMMARY

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

5. PREFACE

................................................................................................ ......................................... 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

5.5.1. General Technical Terms

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

5.5.2. Toxicity Terms

................................................................................................................. 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

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

6.2. Test system

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

6.3. Holding of the brood stock

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

6.4. Acute toxicity

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

6.5. Preparation of stock and test solutions

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

6.6. Early Life Stage Test

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

6.6.1. Test concentrations

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

6.6.2. Test procedure and conditions

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

6.6.3. Sampling for measurement of test concentrations

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

6.6.4. Measurements and recordings

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

6.6.5. TOC analyses

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

6.7. Electronic data capture

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

6.8. Interpretation

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

6.8.1. Data ha ndling

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

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

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

7.1. Measured concentrations

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

7.2. Embryonic survival, development and hatching

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

7.3. Larval survival and development

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

7.4. Effects on larval growth

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

7.5. Experimental conditions

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

8. CONCLUSIONS

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

- Page 2 -EXP1313100 Project 505721

TABLES

Table 1 Mean concentrations calculated from the analytical results

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

Table 2 Egg survival and hatching success during the ELS test

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

Table 3 Survival of fish embryos and larvae of the control and the treated gr oups

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

Table 4 Effects on fish larvae of the control and the treated groups

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

Table 5 Individual body length (mm) of surviving larvae at the end of the test period

.............. 24

Table 6 Mean body weight (mg wet weight) of surviving larvae at the end of the test

period

............................................................................................................................ 25

Table 7 Measurements of the pH

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

Table 8 Measurements of dissolved oxygen (mg O 2 /L)

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

Table 9 Measurements of temperature (°C)

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

Table 10 Measurements of hardness (mg CaCO 3 /L)

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

FIGURES

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

Figur e 3 Hatching curves

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

Figure 4 Mean post hatch survival rates ± standard deviation

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

Figure 5 Mean body length ± standard deviation (SD) of the surviving larvae at the end of

the test (by replicate)

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

Figure 6 Mean body weight ± standard deviation (SD) of the surviving larvae at the end of

the test (by concentr ation)

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

APPENDICES

APPENDIX 1 SURVIVAL, APPEARANCE and OTHER EFFECTS

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

APPENDIX 2 BODY LENGTH OF LIVING LARVAE AT THE END OF THE STUDY

........................ 24

APPENDIX 3 BODY WEIGHT OF LIVING LARVAE AT THE END OF THE STUDY

........................ 25

APPENDIX 4 STATI STICAL ANALYSIS ON EMBRYONIC SURVIVAL

............................................ 26

APPENDIX 5 STATISTICAL ANALYSIS ON LARVAL SURVIVAL

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

APPENDIX 6 STATISTICAL ANALYSIS ON BODY LENGTH

........................................................... 28

APPENDIX 7 STATISTICAL ANALYSIS ON BODY WEIGHT

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

APPENDIX 8 TEST CONDITIONS

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

APPENDIX 9 CERTIFICATE OF ANALYSIS

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

APPENDIX 10 ANALYTICAL REPORT

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

- Page 3 -EXP1313100 Project 505721

2. STATEMENT OF GLP COM PLIANCE

WIL Research Europe B.V ., 's

- Hertogenbosch, The Netherlands

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

The Organization for Economic Co

- operation and Development (OECD) Principles of Good Laboratory

Practice (GLP) (as r evised 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 505721

3. QUALITY ASSURANCE ST ATEMENT

WIL Research Europe B.V., ‘s

- Hertogenbosch, The Netherlands

This report was inspected by the WIL Research Eu rope 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 given below.

Project 505721

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

Exposure 28

- Aug

- 2014 28

- Aug

- 2014 28

- Aug

- 2014

Report 11

- Dec

- 2014 11

- Dec

- 2014 11

- Dec

- 2014

Process Test Substance Receipt 24

- Jul

- 2014 31

- 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 Assurance

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

- Page 5 -EXP1313100 Project 505721

4. SUMMARY

Fish early

- life stage toxicity test with EXP1313100

.

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

Chemicals: Guideline No. 210, 2013 . In addition, the procedures were designed to meet the test

methods and validity criteria of the EPA Ecological Effects Test Guidelines, 'Public Dr aft', EPA 712 -C96

- 121, 1996 and the OECD series on testing and assessment number 23, 2000.

The batch of EXP1313100 tested was a brown 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

solutions. 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

magnetic 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

.

A 32

- day early life stage (ELS) test was performed under semi

- static conditions with renewal of test

solutions every 48 hours. The test was performed with four replicates of a co ntrol group and four

replicates for each WAF. Each test group contained 20 eggs per replicate. The test started by placing

fertilised eggs in Petri

- dishes

. On day s 6, 1 6 and 2 4 hatched larvae and/or juvenile fish were

transferred to larger vessels /volumes. During the embryonic and larval phases, the fish were observed

for effects on development, appearance and swimming behaviour. At the end of the test (day 3 2 ), the

surviving fish were measured and weighed.

During the test, samples for analyses were taken at the start and the end of five renewal intervals. At

the start of each renewal period the actual test concentrations ranged between 0.0046 and 0.078 mg/L

in the WAF prepared at 1.0 mg/L and between 0.017 and 0.46 mg/L in the WAF prepared at 10 mg/L.

Th ere 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 handle. 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 decreased to 6

- 71% of initial during the 48

- hour renewal periods. EXP1313100 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).

B ased on these results mean measured concentrations were calculated to be 0.015 and 0.14 mg/L for

the whole test period of 32 days. The coefficients of variation were 67 and 103%, respectively.

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

The results of the ELS test led to the following conclusion:

EXP1313100 did not induce any statistically significant effects on embryonic and larval survival and on

larval growth at a loading rate of 10 mg/L. Hence, the NOELR and LOELR for this study , based on

embryonic and larval survival and on larval growth, were 10 and >10 mg/L, respectively. The

concentration of EXP1313100 in the WAF prepared at a loading rate of 10 mg/L was determined to be

0.14 mg/L based on the mean exposure concentration. The coefficient of variation for this mean value

was 103%, reflecting the variability in attaining consistent concentrations.

- Page 6 -EXP1313100 Project 505721

Although it was the best option available, it was evident that the WAF preparation technique was not

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

evidenced by the variability in the measured concentrations throughout the study.

*NOELR and LOELR = No effect loading rate and lowest effect loading rate

- Page 7 -