SUZHOU RESEARCH Study No. : 2014

- 139

- 01

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I 

# FINAL REPORT

Study Title: Fish

, Acute Toxicity Test for FRET 09

- 0536

Study No.: 2014

- 139

- 01

- 01

Test Facility

Suzhou Xishan Zhongke Drug R & D Co., Ltd.

Add:1336 Wuzhong Avenue, Suzhou City, Jiangsu Province, China, 215104

Tel: (86512) 6598 2476 Fax: (86512) 6627 1770



## Sponsor

International Flavors & Fragrances Inc.

Add: 800 Rose Lane, Union Beach NJ USA 07735

Tel: +1 732

- 203

- 8151SUZHOU RESEARCH Study No. : 2014

- 139

- 01

- 01

II 

# SIGNATURE PAGE

Signed in original



### John Chen Date
Study director

Signed in original



### Xia Zheng Date
QA management

Signed in original



### Yang Min Date
Test facility managementSUZHOU RESEARCH Study No. : 2014

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III 

## STATEMENT

Study No.: 2014

- 139

- 01

- 01

Study Title: Fish

, Acute Toxicity Test for FRET 09

- 0536

I, undersigned, hereby declare that this report provides an English translation of the Final Report

(Study No. 2014

- 139

- 01

- 01 , i ssued on ).

John Chen Date

TranslatorSUZHOU RESEARCH Study No. : 2014

- 139

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- 01

IV 

# GLP STATEMENT

The study described in this report was conducted in compliance with the following GLP principles:

HJ/T 155

- 2004 The Guidelines of Good Laboratory Practices on Chemical Testing. MEP. China.

This final report reflects the raw data accurately and it has been confirmed that the test data are valid.

Signed in original

John Chen Date Study DirectorSUZHOU RESEARCH Study No. : 2014

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V 

# QUALITY ASSURANCE ST ATEMENT

Study No.: 2014

- 139

- 01

- 01

Study Title: Fish, Acute Toxicity Test for FRET 09

- 0536

All inspections and review of QAU to this study were carried out independently in compliance

with The Guidelines of Good Laboratory Practices on Chemical Testing (HJ/T 155

- 2004, MEP,

China )

QAU reviewed the f

inal report and found this final report accurately describes the test materials

and methods, and that the reported results accurately reflect the raw data of the study.

QAU conducted the following inspections and review, and all findings were reported to the Study

Director and the Test Facility Management.

Items of inspection Date of inspection Date of report

Signed in original

Xia Zheng Date Quality Assurance ManagerSUZHOU RESEARCH Study No. : 2014

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Page 6 of 25 

# TABLE OF CONTENTS

GLP STATEMENT

................................ ................................ ................................ ....................... III

QUALITY ASSURANCE ST ATEMENT

................................ ................................ ...................... V

SU MMARY

................................ ................................ ................................ ................................ ..... 7

1. INTRODUCTION

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

1.1. Study Title (Study No.)

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

1.2. Objective

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

1.3. Regulatory Compliance and Test Guidelines

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

1.4. Test Facility

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

1.5. Spons or

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

1.6. Personnel

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

1.7. Dates

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

1.8. Animal Welfare

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

2. TEST SUBSTANCE

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

2.1. Information

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

3. MATERIAL S AND METHODS

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

3.1. Main apparatus

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

3.2. Main reagent s and materials

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

3.3. Test system

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

3.4. Test conditions

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

3.5. Tes t methods

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

3.6. Validity of the test

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

4. AMENDMENT AND DEVIAT ION

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

5. RESULTS AND CONCLUSI ON

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

5.1. Results

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

5.2. Conclusion

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

6. ARCHIVES

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

7. R EFERENCES

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

8. A PPENDIX

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

8.1. Attached tables

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

8.2. Figures

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

8.3. Attachment

................................ ................................ ................................ ....................... 24SUZHOU RESEARCH Study No. : 2014

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Page 7 of 25 

# SUMMARY

Objective

To evaluate the short

- term toxicity effects of the test substance FRET 09

- 0536 on z ebra

- fish species,

with the determination of a 96hr L C 50 and LL 50 in a semi

- static aquatic system.

Methods
According to the result of the preliminary test, five test concentrations of 1.0 mg/L, 1

. 5 mg/L, 2. 3

mg/L, 3.4 mg/L, 5

. 1 mg/L (nominal concentration

, in a geometric series with a factor 1. 5 ) and one

control group ( fish breeding water ) wer e used in the definitive test. Each group contained ten fish. The

duration of test was 96 hours

. The test fish were exposed in closed bottle filled with the test solution

under the semi

- static system with test solution renewed every 24 hours. T est was performed under the

conditions of 14 hour s photoperiod daily

, 2 3

. 6

~ 2 4. 6 °C of water temperature, 6. 53

~ 7. 75 of pH,

6 2.4 %

~ 101. 6 % a ir saturation value of d issolved oxygen concentration and with no feeding. The fish

were observed at 3 , 6, 24, 48, 72 and 96 hours after the start of the exposure. The concentrations of the

test substance were measured at the start and end of the exposure, before and after each renewal with

G C analysis

.

Results

At the end of the test, the cumulative mortalities were 0% and visible abnormalities were no t found in

the blank group

. Some visible abnormalities were found in t he treatment groups, including swimming

with an upward flank, weakened swimming movements and swimming near the surface portion of th e

tank

. During the exposure period, the measured exposure concentrations of the freshly prepared test

solution remained within 8 3.0 %

- 9 7.0 % of nominal, which could be satisfactorily maintained within

80

- 120%

. The measured exposure concentrations of test solution s ranged from 3.5 % to 24.9 % of the

initi al ones before renewing the test solution , which could not be maintained within 80

- 120%.

According to the results of the stability test , stability was given: 9 5.5 % of the nominal concentration at

the 0 h an d 86.5 % at 96h without fish. A nd the estimated LogBCF value was 1.98 (moderate

enrichment) by EPIWEB 4.1 software

. So the loss of test item may be due to the adsorption or

bioconcentration of fish to the test substance. Therefore

, the geometric means of the measured

concentrations were used for the LC 50 calculation and loading rate s were used for the L L 50 calculation

. T he results are shown in the following table:

Recommended

o bservation times C alcu lated from the loading rates ( mg/L ) C alcu lated from the geometric means of

the measured concentrations ( mg/L )

LL 50 LC 50
24 h 4.164 1.067

48 h 3.785 0.884

72 h 3.785 0.975

96 h 3.525 0.913

C onclusion

Under the test conditions in this study, the LL 50 (96h) of test substance FRET 09

- 0536 on zebra

- fish

species was 3.525 mg/L based on no minal concentrations (loading rate) and LC 50 (96h) was 0. 913

mg/L based on the geometric means of measured concentrations.SUZHOU RESEARCH Study No. : 2014

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Page 8 of 25 

## 1. Introduction

1.1. S tudy Title ( Study No. )

Fish

, Acute Toxicity Test for FRET 09

- 0536 ( 2014

- 139

- 01

- 01 )



### 1.2. Objective

To evaluate the acute toxicity effects of the FRET 09

- 0536 on zebra

- fish species , with the

determination of a 96hr L C 50 and L L 50 in a semi

- static aquatic system.



### 1.3. Regulatory Compliance and Test Guidelines

1.3.1. GLP Compliance

T h is study had be en conducted in compliance with the following GLP principles:

HJ/T 155

- 2004 The Guidelines of Chemical Testing Good Laboratory Practices. MEP. China.

1.3.2. Test Guidelines

This study had been performed to the following guideline:

The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, Version 2, 203 Fish Acute

Toxicity Test, CRC

- MEP. China. 2013

OECD Guidelines for testing of chemicals, Guideline 203 on Fish, Acute Toxicity Test. Paris:

OECD, adopted 17 July 1992.



### 1.4. Test Facility

Name: Suzhou Xishan Zhongke Drug R&D Co., Ltd.

Address: 1336 Wuzhong Avenue, Suzhou City, Jiangsu Province, China, 215104

TFM: Professor Yang Min

Tel: (86512) 6637 0161

Fax: (86512) 6627 1770



### 1.5. Sponsor

Name: International Flavors & Fragrances Inc.

Add: 800 Rose Lane, Union Beach NJ USA 07735

Representative: Paul Sterchele

Tel: +1 732

- 203

- 8151

Fax: +1 732

- 203

- 8176

E

- mail: 

### paul.sterchele@iff.com

1.6. Personnel

Study Director : John Chen

Tel: (86512) 6504 2307

- 8269

Fax: (86512) 6627 1770

E

- mail : chenliang @szxszk.comSUZHOU RESEARCH Study No. : 2014

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Page 9 of 25 Add ress : 1336 Wuzhong Ave nue, Suzhou City, Jiangsu Province, China, 215104

Study Personnel :

T est t echnician: Baolong Xu, Qian Wu , Yibin Shen , Menglu Chen

Test Substance Control Management: Lai Su

, Qi Wu

Analyst: Jinliang Yu

, Mingzhen Zhou , Shan Chen

V eterinarian : Guoqiang Tang

Archivist: Hongmei Cao ,Yinyin Zhu



### 1.7. Dates

S tudy initiation date: 2014

- 0 9

- 17

E xperimental starting date: 2014

- 0 9

- 17

E xperimental completion date: 2014

- 0 9

- 28

Study completion date:



### 1.8. Animal Welfare

All test fish used in this study has been approved by IACUC unit. The study is designed to follow

rules described in the Guide for the Care and Use of Laboratory Animals (8 th ed., 2010)

.



## 2. Test substance

2.1. Information

Identification

Test substance name 4,7

- METHANO

- 1H

- INDENE

- 1

- PROPANAL,

OCTAHYDRO

Code FRET 09

- 0536

CAS No. 1464989

- 16

- 9

Molecular formula C 1 3 H 2 0 O

Formula weight 1 9 2

Physicochemical property

Appearance Liquid

Storage

Storage Stock at temperature 2

- 8 °C

Description

Source International Flavors & Fragrances Inc.

Batch number YA266

- 82

Purity > 90%

Expiry date 2015

- 05

Remark: The information above was provided by the sponsor.SUZHOU RESEARCH Study No. : 2014

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Page 10 of 25 

## 3. M aterial s and Methods

3.1. Main apparatus

N ame Model M anufacturer

Electronic balance PL403 Mettler

Electronic balance AL204 Mettler

Electronic balance XS105DU Mettler

Multi

- parameter meter HQ40d HACH

Digital vernier calliper 0

- 150mm Shanghai Jiuliang

Photometer 7500 Palintest

GC 7890A A gilent

M agnetic stirrer RCT B S25 IKA



### 3.2. Main reagent s and materials

N ame G rade Batch No. Source

Potassium

permanganate AR 20110429 SCRC

Sea salt Not available 43LBS.80Z MEI

Sodium

bicarbonate Not available SC12 Aquatic Eco

- systems Inc.

Ethyl 4

- aminobenzoate Not available DJ28 1

- FE TCI

E thyl acetate HPLC SHBC6748 V S IGMA

Total hardness

reagent Not available 4808/5052 Palintest



### 3.3. Test system

3.3.1. Test species

Species Zebra

- fish (Brachydanio rerio)

S train AB

Supplier Hangzhou Minghong Aquarium

Receipt date 201 4

- 02

- 20

Generation No. FZA000

- 201 402 20

- 0 2

S ensitivity LC 50 ( 24 h ) of potassium dichromate was 2 25.187 mg/L

3.3.2. Justification for T est S pecies

Zebra

- fish is the recommended test species according to the test guidelines.SUZHOU RESEARCH Study No. : 2014

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Page 11 of 25 3.3.3. A cclimatization

T he f ish had be en taken from the f lowing w ater breeding system (which is a part of the testing

facility) and then follow ed a 7

- day a cclimatization period in the semi

- static test system.

D uring the acclimatization period, l ight was set as 1 4 hours photoperiod daily

. Temperature, pH

and dissolved oxygen concentration of water were daily measured and recorded, results are shown in

section 5.1. 4

. Fish wer e fed daily until 24 hours before the test was started. F ish not selected for

testing were returned to fish breeding system.

3.3.4. Feedstuff and w ater

Fairy shrimp eggs were purchased from from Tianjin . The eggs were hatched to larva as the food

for fish.

Reverse osmosis equipment was used to purify the water used in the test

. W ater samples are

taken annually and analyzed by an authorized testing organization

. T he analysis show ed the results to

meet the requirements of Water q uality standard for fisheries specified in GB

- 11607

- 89; the pH

(7.0±0. 5 ) and conductivity (500±100  s/cm) are verified by water quality automatic control system

.

3.3.5. D isposal of test system

At the end of the test, the fish alive in each group were put into ethyl 4

- aminobenzoate

(benzocaine) solution of 250 mg/L for euthanasia by non

- physical method.

All carcass es w ere put into the tagged plastic bag and temperately stored in low temperature

refrigerator

. The waste as well as the test substance w ere collected and delivered to the approved waste

disposal company for disposal

.



### 3.4. Test conditions

Duration 96 h ours

Loading ≤ 0. 564 g fish/litre

Light 14 hour s photoperiod daily

Temperature 2 3. 6 ~24. 6 °C

p H 6. 53 ~7. 75

Oxygen concentration 6 2.4

~ 101. 6 % ASV

Feeding N one

Test vessel W ide mouth reagent bottle (sealed with sealing film )



### 3.5. Test methods

3.5.1. Test methods

According to the stability test, test substance is stable in test water (the measured concentration of

test substance was within 80%

- 120% of the nominal concentration after 24 hours). The test fish were exposed in closed bottle filled with the test solution under the semi

- static system with test solution

renewed every 24 hours.SUZHOU RESEARCH Study No. : 2014

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Page 12 of 25 3.5.2. Exposure Concentrations and Justification

Semi

- static exposure method was performed in the preliminary test. The test fish was exposed in

the test solution with renewing test solution every 24 hours. Preliminary test showed that t here was no

mortality observed at the nominal concentration of 1 mg/L and 100% mortality was observed at the

nominal concentration of 5 mg/L within the test period of 96hr.

According to t he result of the preliminary test, five test concentrations of 1.0 mg/L, 1. 5 mg/L, 2. 3

mg/L, 3.4 mg/L, 5. 1 mg/L (nominal concentration, in a geometric series with a factor 1.5) and one

control group (fish breeding water) were used in the definitive test. E ach group contained ten fish.

Specific information is shown below:

Group Test substance Vessel No. Nominal

concentrations ( mg /L ) N umber Test solution

v olume( L )

Blank None FE0

- 1 0 10 6

T reated group 1

FRET 09

-

0536 FE1

- 1 1.0 10 6

T reated group 2 FE2

- 1 1. 5 10 6

T reated group 3 FE3

- 1 2. 3 10 6

T reated group 4 FE4

- 1 3.4 10 6

T reated group 5 FE5

- 1 5. 1 10 6

3.5.3. Preparation of test substance solution

Due to the UVCB property of the test substance and its solubility lies below 100 mg/L, the water

accommodated fractions ( OECD SERIES ON TESTING AND ASSESSMENT Number 23 ) w as

prepared for the test. Desired amount of the test substance w as weighed and mixed with the test water

to produce test solutions with nominal concentration (loading rates) of 1.0 mg/L, 1.5 mg/L, 2.3

mg/L,3.4 mg/L and 5.1 mg/L. The mixed solutions w as stirred for about 20 hours. In preliminary

study

, the TOC concentrati on of te st solution were determined after a period of 6h,20h,24h

stirring .The results show that the TOC concentration was not significant difference after the stirring of

20h and 24 h, so the definitive test select stirring time for 20 h. T he aqueous phas e w as not separated

by separating funnel for non

- dissolved test substance was not appeared on the surface of test solutions

f ollowing a period of 1 hours settling.

T he test solution s w ere prepared e very 24 hours just before use.

3.5.4. Test substance analysis

Since the test substance is an UVCB, it is impossible to identify and determine all the

components. Therefore the substance w as used as a reference substance with 100% purity and the

component with the highest peak w as chosen as an indicative component to establish the calibration

curve by plotting the peak area of the indicative component in standard solutions as the vertical Y

versus the nominal concentration of test substance as the abscissa X. Then the peak area response

value of the indicative componen t in sample w as substituted into the regressed equation to calculate

the exposure concentration of test substance when doing sample analysis.SUZHOU RESEARCH Study No. : 2014

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Page 13 of 25 3.5.4.1. C hromatographic condition s

T emperature gradient 10 0 °C

, 5 °C /min to 15 0 °C

Detector FID

Detector temperature 250 °C

Inlet temperature 2 3 0 °C

Injection volume 1μL

Injection mode Split injection

split ratio 2 : 1

Gas flow N 2 , 2.5 mL/min

R un time 12min

Column Agilent 19091

- 413,

- 60~325 °C : 0.25 μm, 250 μm × 15m

3.5.4.2. Method Validation

 Calibration Curve

According to the results of measured concentrations of test samples in the preliminary test,

standard solutions were prepared as follows:

Solution

Code Stock solution/

Test substance Sample taken Solvent Final

Volume (

mL ) N

. C M. C R.D

S 1 Test substance 0.01086 g

Ethyl acetate 10 1086.00 N/A N/A

S 2 S 1 1 mL 10 108.60 N/A N/A

L 1 L4 1 mL 10 1.09 1.08

- 0.92

L 2 S 2 200 μ L 10 2.17 2.10

- 3.23

L 3 S 2 500 μ L 10 5.43 5.44 0.18

L4 S 2 1 mL 10 10.86 10.91 0.46

L 5 S 2 2mL 10 21.72 21.70

- 0.09

Remark: N.C= Nominal Concentration ( mg/L ); M.C= M easured Concentration ( mg/L );

R.D=R elative deviation (%)

.

L1

- L 5 w ere i nject ed in sequence. L inearity was established by a plot of peak area as the vertical

Y versus the nominal concentration (mg/L) as the abscissa X using least square fitting. According to

the results, the regression equation of calibration curve wa s Y = 4.04937X

- 0.17442

, with the

correlation coefficient r= 0.999986 > 0.99

. The relative deviation of measur ed concentration from

nominal concentration wa s

- 3.23%~0.46% , which was within the range of ± 1 0% . Result s met the

criteria of linearity , i.e., r ≥ 0.99 and relative deviations within ± 1 0%

.SUZHOU RESEARCH Study No. : 2014

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Page 14 of 25 QC samples with concentrations approximately equal to L2 and L5 were prepared every day to

check the ruggedness of the calibration curve before analysis. The relative deviation of measur ed

concentration from nominal concentration wa s

- 4.06%~0.60%

, which was within the required range of

± 1 0%

.

Solution Code N ominal concentration (mg/L) Measured concentration (mg/L) Relative deviation (%)

L2QC20140 924 2.01 1 .97

- 1.99

L 4 QC20140 924 10.05 10.10 0.50

L2QC20140 925 1.98 1.95

- 1.52

L 4 QC20140 925 9.92 9.98 0.60

L2QC20140 926 1.97 1.89

- 4.06

L 4 QC20140 926 9.87 9.71

- 1.62

L2QC20140 927 1.98 1.90

- 4.04

L 4 QC20140 927 9.90 9.79

- 1.11

 Specificity

Area of i nterfering GC peaks from fish breeding water and e thyl acetate in the retention time of

the test substance did n o t exceed 30% of the LOQ peak area

.

 Recovery

The test solutions were prepared in fish breeding water as follows then analyzed via GC. The

recovery of REL1~REL 5 were in the range of 92.62%~108.20% , and RSD= 6.23 %; the recovery of

REH1~REH 5 were in the range of 89.97%~108.80% , and RSD= 7.4 1 %. The results met the criteria for

recovery, i.e., between 80%~ 1 1 0% of nominal concentration with RSD less than 20%.

Solution

No. Nominal

concentration (mg/L) Nominal concentration after pretreatment (mg/L) Measured

concentration (mg/L) Recovery (%) RSD(%)

REL1 0.61 1.22 1.32 108.20

6.23 REL2 0.61 1.22 1.31 107.38

REL3 0.61 1.22 1.22 100.00

REL4 0.61 1.22 1.13 92.62

REL5 0.61 1.22 1.27 104.10

REH1 6.08 12.16 12.76 104.93

7.41 REH2 6.08 12.16 11.96 98.36

REH3 6.08 12.16 10.94 89.97

REH4 6.08 12.16 12.87 105.84

REH5 6.08 12.16 13.23 108.80

 MDL and LOQ

A solution code d as MDL was p repare d with target concentration of 0. 1 mg/L in fish breeding

water and inject ed after pretreatment

. T he S/N was 9.62 , which met the criteria of S/N ≥ 3 MDL was

set as 0.1 mg/L A solution code d as LOQ was prepared wi th target concentration of 0. 3 mg/L in fish

breeding water and inject ed after pretreatment

. T he S/N was 27.16 , meeting the criteria of ≥ 10. LOQ

was set as 0. 3 mg/L.

 The results of m ethod v alidation indicate d that this analysis method wa s suitable for

concentration measurement of test substance in this z ebra

- fish acute toxicity test.SUZHOU RESEARCH Study No. : 2014

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Page 15 of 25 3.5.4.3. Concentration Analysis

The concentrations of test solutions were measured at the start and end of exposure, before and

after each renewal. About 2 0 mL sample w as taken from treated group s and inject ed after pretreatment

to analyze concentration

. The results are shown in section 5.1. 1. T ypical c hromatograph s are shown

in figure 2

~ 6

.

3.5.5. Measurement and observation

3.5.5.1. O bservation s

The fish were inspected at 3, 6, 24, 48, 72 and 96 hours after the start of the exposure , results are

shown in section 5.1.2

.

3.5.5.2. Measurement of water quality parameters

The total hardness and pH of test water were measured before exposure. pH, dissolved oxygen

and temperature were measured at the start and end of exposure, before and after each renewal during

the test. Results are shown in section 3.4 and section 5.1.4

.

3.5.5.3. Body length and body weight

F ish a live after the test were measured and recorded for body length (standard length) and body

weight (wet weight) ; results are shown in section 5.1. 5

.

3.5.6. Data processing

MS Excel 201 3 was used to analyze statistics and collected data (such as temperature, dissolved

oxygen, pH, etc.)

. Va lues following the decimal point in the report may differ from the raw data due to

rounding by spreadsheet.

Where the data obtained at 24h are inadequate for the use of standard methods of calculating the LC 50

and LL 50 , the highest concentration(loading rate) causing no mortality and the lowest concentration(loading

rate) producing 100 percent mortality was used to establish the approximate LC 50 and LL 50 (the geometric

mean of these two concentrations). SPSS 16. 0 w as em ployed to calculate the LC 50 and LL 50 values at

re commended observation times of 48 h, 72 h and 96 h after the start of exposure.



### 3.6. Validity of the test

T he mortality in the control group was 0% at the end of the test , less than 10 %, which met t he

validity requirements for the test;

T he dissolved oxygen concentration was within the range of 6 2.4 %~ 101. 6 % air saturation value

throughout the test

, more than the required 60% air saturation, which met t he validity requirements for

the test

.



## 4. Amendment and Deviation

This study had no amendment and deviation to the study plan in the study.SUZHOU RESEARCH Study No. : 2014

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Page 16 of 25 

## 5. Results and Conclusion

5.1. Results

5.1.1. C oncentration analysis of test substance

According to the contents about Substances toxic at low concentrations in OECD SERIES ON

TESTING AND ASSESSMENT Number 23 ( Guidance Document On Aquatic Toxicity Testing Of

Difficult Substances And Mixtures )

, the final concentration was taken as the limit of detection for

the method if the substance wa s not detected and use half of the limit of quantifi cation as the final

concentration when the substance wa s detected but not quantified in this study

. During the exposure period, the measured exposure concentrations of the freshly prepared test

solution remained within 83.0%

- 97.0% of nominal, which could b e satisfactorily maintained within

80

- 120%. The measured exposure concentrations of test solutions ranged from 3.5 % to 24.9 % of the

initial ones before renewing the test solution, which could not be maintained within 80

- 120%.

According to the results of the stability test, stability was given: 95.5% of the nominal concentration at

the 0 h and 86.5% at 96h without fish. And the estimated LogBCF value was 1.98 (moderate

enrichment) by EPIWEB 4.1 software. So the loss of test item may be due to the adsorptio n or

bioconcentration of fish to the test substance. Therefore, the geometric means of the measured

concentrations were used for the LC 50 calculation and loading rates were used for the LL 50 calculation.

T he detailed concentration results are shown in Table 1

.

5.1.2. M ortalities and abnormalities

T he mortality during a cclimatization period was 0% . The results of accumulative total deaths and

m ortalities at 24 h, 48 h, 72 h and 96 h after the exposure of test fish are shown in a ttached Table 2

.

T he observed mor tality in the control group was 0%, and the highest concentration (loading rate) causing

no mortality was 2.3 mg/L

, and the lowest concentration (loading rate) producing 100 percent mortality

was 5. 1 mg/ L

.

There were no visib le abnormalities found in blank

. In the t reated groups, fis h had visible

abnormalities including swimming with an upward flank, weakened swimming movements and

swimming near the surface portion of the tank

. T he detailed results are shown in a ttached Table 3

.

5.1.3. LC 50 and Concentration

– mortality curv e

The LC 50 and LL 50 of FRET 09

- 0536 on zebra

- fish species are shown in the following table:

Recommended

o bservation times C alcu lated from the loading rates ( mg/L ) C alcu lated from the geometric means of

the measured concentrations ( mg/L )

LL 50 LC 50
24 h 4.164 1.067

48 h 3.785 0.884

72 h 3.785 0.975

96 h 3.525 0.913SUZHOU RESEARCH Study No. : 2014

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Page 17 of 25 The Concentration

– mortality curve is shown in F igure 1

.

5.1.4. W ater quality parameters

Photoperiod was set as light : dark= 14 h : 10 h during the test. The total hardness and pH of test

water were 45 mg CaCO 3 /L and 7. 34

, respectively

, which were measured before the exposure.

D issolved oxygen

, pH and temperature were measured daily during the test. Summarized results

are shown below, and detailed results are shown in Table 4

~ Table 7

.

Acclimatization E xposure duration

P roject T emperature

( °C ) pH DO( % ASV) T emperature ( °C ) pH DO( % ASV)

R ange 2 3.8

~ 2 4.2 6.55

~ 7. 30 89.1

~ 95.3 2 3.6

~ 2 4.6 6.53

~ 7. 75 62.4

~ 101.6

5.1.5. Body length and body weight

T he body length of test fish at the time of death or euthanasia were within the range of

2. 696

~ 2. 892 cm, which met the requirement of 2.0±1.0 cm of the test Guidelines. The maximum

loading was 0. 564 g fish/L during the test

. T he detailed results are shown in a ttached Table 8 and

Table 9

.



### 5.2. Conclusion

T he LL 50 (96h) of test substance FRET 09

- 0536 on zebra

- fish species was 3.525 mg/L based on

nominal concentrations(loading rate) and LC 50 (96h) was 0.913 mg/L based on the geometric means

of measured concentrati ons u nder the test conditions in this study,



## 6. Archives

The following documents will be stored in the archive room for ten years:

S tudy plan

Raw data and final report

The information of test substance

Moreover, the retained sample of the test substance will be stored in the archive room until the

expiry date.

I f the exten s ion of retention period is needed, specific details can be negotiated between test

facility and sponsor.



## 7. R eferences

1) HJ/T 155

- 2004 The Guidelines of Chemical Testing Good Laboratory Practices. MEP. 2004.SUZHOU RESEARCH Study No. : 2014

- 139

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Page 18 of 25 2) The Guidelines for the Testing of Chemicals, Effects on Biotic Systems, Version 2, 203 Fish

Acute Toxicity Test, CRC

- MEP. 2013

. 3) Guide for the Care and Use of Laboratory Animals: 8 th Edition. Committee for the Update of the

Guide. National Academies Press. 2010

. 4) OECD Guidelines for testing of chemicals, Guideline 203, Fish, Acute Toxicity Test. OECD,

adopted 17 July 1992

5) OECD SERIES ON TESTI NG AND ASSESSMENT Number 23, GUIDANCE DOCUMENT

ON AQUATIC TOXICITY TESTING O F DIFFICULT SUBSTANCES AND MIXTURES

.

2000.



## 8. A ppendix

8.1. Attached tables

T able 1 Concentration analysis of the test substance

G roups T reat ed group 1 T reat ed

group 2 T reat ed

group 3 T reat ed

group 4 T reat ed

group 5

N o minal c oncentration s ( mg/L ) 1.0 1

. 5 2.3 3.4 5.1

Measured

c oncentration s ( mg/L ) B eginning of the

exposure 0.83 1.30 1.98 2.87 4.26

B efore the first

renewal 0.10

( 12.0 %* ) 0.10

( 7.7 % ) 0.15

( 7.6 % ) 0.10

( 3.5 % ) 1.06

( 24.9 % )

After the first

renewal 0.96 1.42 2.00 3.10

-

B efore the second

renewal 0.15

( 15.7 % ) 0.31

( 21.9 % ) 0.15

( 7.5 % ) 0.33

( 10.6 % ) -

After the second

renewal 0.84 1.32 2.02 3.12

-

B efore the thir d

renewal 0.10

( 12.0 % ) 0.31

( 23.5 % ) 0.15

( 7.4 % ) 0.32

( 10.3 % ) -

After the thir d

renewal 0.88 1.46 2.20 3.28

-

End of the exposure 0.10

( 11.4 %* ) 0.15

( 10.3 % ) 0.15

( 6.8 % ) 0.35

( 10.7 % ) -

Geometric mean value (mg/L) 0.31 0. 52 0.55 0.87 2.12

Remark:

- means the test solution had not been analyzed ; * ratio (%) =Measured concentration/Nominal

concentration

T able 2 Cumulative deaths and m ortalities

Nominal Conc.
( mg/L ) Vessel No. Exposure duration

24h 48h 72h 96h

0 FE0

- 1 0 ( 0% ) * 0 ( 0% ) 0 ( 0% ) 0 ( 0% )



### 1.0 FE 1

- 1 0 ( 0% ) 0 ( 0% ) 0 ( 0% ) 0 ( 0% )



### 1.5 FE 2

- 1 0 ( 0% ) 0 ( 0% ) 0 ( 0% ) 0 ( 0% )



### 2.3 FE 3

- 1 0 ( 0% ) 0 ( 0% ) 0 ( 0% ) 0 ( 0% )



### 3.4 FE 4

- 1 0 ( 0% ) 2 ( 2 0% ) 2 ( 2 0% ) 4 ( 4 0% )



### 5.1 FE 5

- 1 10 ( 10 0% ) 10 ( 10 0% ) 10 ( 10 0% ) 10 ( 10 0% )

Remark: * bracket outside is c umulative death ; bracket is c umulative mortality.SUZHOU RESEARCH Study No. : 2014

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Page 19 of 25 Table 3

. Clinical observations

Nom. Conc.
( mg/L ) Vessel

No. Exposure duration

3h 6h 24h 48h 72h 96h

0 FE0

- 1 A A A A A A



### 1.0 FE 1

- 1 A A A A A A



### 1.5 FE 2

- 1 A A A A A A



### 2.3 FE 3

- 1 A A A A A A



### 3.4 FE 4

- 1 A A A B B B



### 5.1 FE 5

- 1 C, D C, D

- - - -
Remark: A: normal ; B: swimming with an upward flank ;C: weakened swimming movements ; D : swimming near the surface portion of the tank ; "

- " : not obtained , all fish had died

.

Table 4

. Water quality parameters during the acclimatization

D ay N o. (a cclimatization ) Parameters of water quality

Temperature ( °C ) pH Dissolved Oxygen
( % ASV )

1 23.9 6.55 89.1

2 24.1 7.20 95.3

3 24.0 7.19 94.6

4 23.8 7.30 94.9

5 24.0 7.26 95.2

6 24.1 7.19 94.9

7 24.2 7.27 95.0

Minimum 23.8 6.55 89.1

Maximum 24.2 7.30 95.3

R emark: ASV = Air saturation value

Table 5

. Water quality parameters during the test (Temperature)

Nominal

Conc.
( mg/L ) Vessel

No. Exposure Day N o.

1 2 3 4

5 Before

renewal After

renewal Before

renewal After

renewal Before

renewal After

renewal

0 FE0

- 1 24.4 23.6 24.3 24.4 24.3 24.0 23.9 23.8

1.0 FE 1

- 1 24.6 24.0 24.1 24.0 23.8 23.9 23.8 23.9

1.5 FE 2

- 1 24.4 24.0 24.2 23.8 23.7 23.8 23.8 23.8

2.3 FE 3

- 1 24.1 23.6 24.2 23.7 23.8 23.8 23.9 23.8

3.4 FE 4

- 1 24.1 23.7 24.1 23.7 23.9 23.8 24.0 23.9

5.1 FE 5

- 1 24.0 23.9

- - - - - - Minimum 23.6

Maximum 24.6

Remark: "

- " = not obtained , all fish had died

.SUZHOU RESEARCH Study No. : 2014

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Page 20 of 25 Table 6

. Water quality parameters during the test (pH)

Nominal

Conc.
( mg/L ) Vessel

No. Exposure Day N o.

1 2 3 4

5 Before

renewal After

renewal Before

renewal After

renewal Before

renewal After

renewal

0 FE0

- 1 7.61 7.06 7.62 6.70 7.30 6.91 7.28 6.93

1.0 FE 1

- 1 7.72 6.68 7.68 6.77 7.27 6.88 7.25 7.01

1.5 FE 2

- 1 7.75 7.01 7.64 6.84 7.26 6.85 7.23 6.87

2.3 FE 3

- 1 7.63 6.81 7.60 6.59 7.25 6.62 7.24 6.69

3.4 FE 4

- 1 7.71 6.76 7.63 6.53 7.28 6.57 7.25 6.62

5.1 FE 5

- 1 7.70 6.65

- - - - - - Minimum 6.53

Maximum 7.75

Remark: "

- " = not obtained , all fish had died

.

Table 7

. Water quality parameters during the test (Dissolved Oxygen in %ASV )

Nominal

Conc.
( mg/L ) Vessel

No. Exposure Day N o.

1 2 3 4

5 Before

renewa l After

renewal Before

renewal After

renewal Before

renewal After

renewal

0 FE0

- 1 101.6 72.4 98.8 68.9 99.9 69.3 99.2 71.4

1.0 FE 1

- 1 98.3 69.2 98.4 68.6 99.2 68.2 98.6 69.0

1.5 FE 2

- 1 98.2 67.4 98.5 65.8 99.2 65.9 98.2 66.1

2.3 FE 3

- 1 97.5 65.3 97.6 64.4 98.8 64.6 97.7 64.5

3.4 FE 4

- 1 97.9 64.5 97.3 62.7 98.4 62.4 97.8 62.7

5.1 FE 5

- 1 98.0 63.2

- - - - - - Minimum 6 2.4

Maximum 101. 6

Remark: "

- " = not obtained , all fish had died

.

Table 8

. Body length s (standard length) of test fish

Vessel No. Body length of test fish ( mm )

FE0

- 1 27.44 28.46 27.81 27.84 27.31 28.73 28.15 27.64 27.69 28.75

FE1

- 1 27.56 27.71 28.02 27.44 27.88 28.67 27.81 28.05 27.66 27.52

FE2

- 1 27.85 27.73 28.28 27.82 27.89 27.61 28.80 28.68 27.62 27.87

FE3

- 1 27.46 27.55 27.89 28.66 27.28 27.69 27.54 27.77 28.92 27.70

FE4

- 1 28.03 28.61 27.24 27.63 27.50 27.85 27.32 28.02 28.12 27.73

FE5

- 1 27.68 28.14 27.18 28.52 27.23 27.37 27.46 27.72 26.96 27.15

Maximum 28.92

Minimum 26.96

R emark: M inimum body length of test fish was 2. 696 cm , m aximum body length of test fish was 2. 892

cm

.SUZHOU RESEARCH Study No. : 2014

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Page 21 of 25 Table 9

. Body weight s ( wet weight) of test fish

Vessel No. Body weight of test fish (g) Loading ( g fish/litre )

FE0

- 1 0.331 0.356 0.329 0.325 0.308 0.366 0.348 0.317 0.322 0.359 0.560

FE1

- 1 0.317 0.336 0.343 0.311 0.327 0.360 0.324 0.330 0.316 0.309 0.546

FE2

- 1 0.342 0.321 0.351 0.333 0.329 0.315 0.367 0.362 0.322 0.340 0.564

FE3

- 1 0.306 0.317 0.343 0.359 0.310 0.339 0.308 0.344 0.369 0.318 0.552

FE4

- 1 0.346 0.355 0.303 0.318 0.314 0.327 0.309 0.345 0.345 0.328 0.548

FE5

- 1 0.312 0.324 0.297 0.341 0.308 0.296 0.295 0.304 0.287 0.291 0.509

Maximum 0.369 0.564

Minimum 0.287 0.509

R emark: The maximum loading was 0. 564 g fish/L during the test. Loading ( g fish/litre )= Total fish body weight(g)/test solution volum e (L).



### 8.2. F igure s

F igure 1 G raph of the concentration

- mortality curv e

F igure 2 C hromatogra m of specificity ( ethyl acetate )

-10% 10% 30% 50% 70% 90% 110%

0 1 2 3 4 5 6 Mortality (%)



### Concentration ( mg/L ) Concentration ( loading rate )

- mortality

curve(96h)SUZHOU RESEARCH Study No. : 2014

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Page 22 of 25 F igure 3

. C hromatogra m of LO Q (nominal concentration was 0. 3 mg/L

, fish breeding water as solvent )

F igure 4 C hromatogra m of recovery (nominal concentration of 1.22 mg/L FRET 09

- 0536 after

pretreatment

, fish breeding water as solvent )SUZHOU RESEARCH Study No. : 2014

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Page 23 of 25 F igure 5 C alibration cu r ve (linearity)

F igure 6 C hromatogra m of the test solution ( treated group 1 at 0 hr )SUZHOU RESEARCH Study No. : 2014

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Page 24 of 25 8.3. Attachment

A ttachment 1

. List of abbreviations

AR Analytical Reagent

ASV Air saturation value

IACUC Institutional Animal Care and Use Committee

L C 50 The concentration of the test substance which kills 50 per cent of test fish within a stated exposure period

L L 50 The loading rate of the test substance which kills 50 per cent of test fish within a stated exposure period

LOQ Limit Of Quantification

M D L Method Detection Limits

FID Flame Ionization Detector

QC Quality Control

REL Recovery of low concentration

REH Recovery of high concentration

RSD Relative Standard Deviation

SCRC Sinopharm Chemical Reagent Co., Ltd.

S/N Signal to noise ratio

SOPs S tandard operating procedures

TFM Test Facility Management

G C Gas Chromatography

A ttachment 2

. Substance informationSUZHOU RESEARCH Study No. : 2014

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Page 25 of 25