SUZHOU RESEARCH Study No.: 2014-139-01-01

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: +1 732-203-8151
SUZHOU 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 management 
SUZHOU RESEARCH Study No.: 2014-139-01-01

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, issued on ). 

John Chen Date

Translator 
SUZHOU RESEARCH Study No.: 2014-139-01-01

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 Director
SUZHOU RESEARCH Study No.: 2014-139-01-01

V

QUALITY ASSURANCE STATEMENT

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 final 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 Manager
SUZHOU RESEARCH Study No.: 2014-139-01-01

Page 6 of 25

TABLE OF CONTENTS

GLP STATEMENT .......................................................................................................................III

QUALITY ASSURANCE STATEMENT......................................................................................V

SUMMARY.....................................................................................................................................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. Sponsor..............................................................................................................................8

1.6. Personnel ...........................................................................................................................8

1.7. Dates..................................................................................................................................9

1.8. Animal Welfare .................................................................................................................9

2. TEST SUBSTANCE.................................................................................................................9

2.1. Information ........................................................................................................................9

3. MATERIALS AND METHODS............................................................................................10

3.1. Main apparatus.................................................................................................................10

3.2. Main reagents and materials............................................................................................10

3.3. Test system ......................................................................................................................10

3.4. Test conditions.................................................................................................................11

3.5. Test methods....................................................................................................................11

3.6. Validity of the test ...........................................................................................................15

4. AMENDMENT AND DEVIATION......................................................................................15

5. RESULTS AND CONCLUSION...........................................................................................16

5.1. Results .............................................................................................................................16

5.2. Conclusion.......................................................................................................................17

6. ARCHIVES.............................................................................................................................17

7. REFERENCES .......................................................................................................................17

8. APPENDIX.............................................................................................................................18

8.1. Attached tables.................................................................................................................18

8.2. Figures.............................................................................................................................21

8.3. Attachment.......................................................................................................................24
SUZHOU RESEARCH Study No.: 2014-139-01-01

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SUMMARY

Objective

To evaluate the short-term toxicity effects of the test substance FRET 09-0536 on zebra-fish species,

with the determination of a 96hr LC50 and LL50 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) were 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. Test was performed under the 

conditions of 14 hours photoperiod daily, 23.6~24.6 °C of water temperature, 6.53~7.75 of pH, 

62.4%~101.6% air saturation value of dissolved 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 

GC analysis.

Results

At the end of the test, the cumulative mortalities were 0% and visible abnormalities were not found in 

the blank group. Some visible abnormalities were found in the treatment groups, including swimming 

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

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

solution remained within 83.0%-97.0% of nominal, which could be 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 adsorption or 

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

concentrations were used for the LC50 calculation and loading rates were used for the LL50 calculation.

The results are shown in the following table:

Recommended

observation times

Calculated from the loading rates 

(mg/L)

Calculated from the geometric means of 

the measured concentrations (mg/L)

LL50 LC50

24 h 4.164 1.067

48 h 3.785 0.884

72 h 3.785 0.975

96 h 3.525 0.913

Conclusion

Under the test conditions in this study, the LL50 (96h) of test substance FRET 09-0536 on zebra-fish 

species was 3.525 mg/L based on nominal concentrations(loading rate) and LC50 (96h) was 0.913

mg/L based on the geometric means of measured concentrations.
SUZHOU RESEARCH Study No.: 2014-139-01-01

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1. Introduction

1.1. Study 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 LC50 and LL50 in a semi-static aquatic system.

1.3. Regulatory Compliance and Test Guidelines

1.3.1. GLP Compliance

This study had been 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.com
SUZHOU RESEARCH Study No.: 2014-139-01-01

Page 9 of 25

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

Study Personnel:

Test technician: Baolong Xu, Qian Wu, Yibin Shen, Menglu Chen

Test Substance Control Management: Lai Su, Qi Wu

Analyst: Jinliang Yu, Mingzhen Zhou, Shan Chen

Veterinarian: Guoqiang Tang

Archivist: Hongmei Cao,Yinyin Zhu

1.7. Dates

Study initiation date: 2014-09-17

Experimental starting date: 2014-09-17

Experimental completion date: 2014-09-28

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 (8th ed., 2010).

2. Test substance

2.1. Information

Identification

Test substance name 4,7-METHANO-1H-INDENE-1-PROPANAL, 

OCTAHYDROCode FRET 09-0536

CAS No. 1464989-16-9

Molecular formula C13H20O

Formula weight 192

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.
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3. Materials and Methods

3.1. Main apparatus

Name Model Manufacturer

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 Agilent

Magnetic stirrer RCT B S25 IKA

3.2. Main reagents and materials

Name Grade 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 DJ281-FE TCI

Ethyl acetate HPLC SHBC6748V SIGMA

Total hardness 

reagent

Not available 4808/5052 Palintest

3.3. Test system

3.3.1. Test species 

Species Zebra-fish (Brachydanio rerio)

Strain AB

Supplier Hangzhou Minghong Aquarium

Receipt date 2014-02-20

Generation No. FZA000-20140220-02

Sensitivity LC50(24 h) of potassium dichromate was 225.187 mg/L

3.3.2. Justification for Test Species

Zebra-fish is the recommended test species according to the test guidelines.
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3.3.3. Acclimatization

The fish had been taken from the flowing water breeding system (which is a part of the testing 

facility) and then followed a 7-day acclimatization period in the semi-static test system.

During the acclimatization period, light was set as 14 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 were fed daily until 24 hours before the test was started. Fish not selected for 

testing were returned to fish breeding system.

3.3.4. Feedstuff and water

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. Water samples are

taken annually and analyzed by an authorized testing organization. The analysis showed the results to 

meet the requirements of Water quality 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. Disposal 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 carcasses were put into the tagged plastic bag and temperately stored in low temperature

refrigerator. The waste as well as the test substance were collected and delivered to the approved waste 

disposal company for disposal.

3.4. Test conditions

Duration 96 hours

Loading ≤ 0.564 g fish/litre

Light 14 hours photoperiod daily

Temperature 23.6~24.6°C

pH 6.53~7.75

Oxygen concentration 62.4~101.6%ASV

Feeding None

Test vessel Wide 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. 
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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 there 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 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) were used in the definitive test. Each group contained ten fish. 

Specific information is shown below:

Group Test substance Vessel No. Nominal 

concentrations(mg/L)

Number Test solution 

volume(L)

Blank None FE0-1 0 10 6

Treated group 1

FRET 09-

0536

FE1-1 1.0 10 6

Treated group 2 FE2-1 1.5 10 6

Treated group 3 FE3-1 2.3 10 6

Treated group 4 FE4-1 3.4 10 6

Treated 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 wateraccommodated fractions(OECD SERIES ON TESTING AND ASSESSMENT Number 23) was

prepared for the test. Desired amount of the test substance was 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 was stirred for about 20 hours. In preliminary 

study , the TOC concentration of test 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. The aqueous phase was not separated 

by separating funnel for non-dissolved test substance was not appeared on the surface of test solutions 

following a period of 1 hours settling. 

The test solutions were prepared every 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 was used as a reference substance with 100% purity and the 

component with the highest peak was 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 component in sample was substituted into the regressed equation to calculate 

the exposure concentration of test substance when doing sample analysis. 
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3.5.4.1. Chromatographic conditions

Temperature gradient 100°C, 5°C/min to 150°C

Detector FID

Detector temperature 250°C

Inlet temperature 230°C

Injection volume 1μL

Injection mode Split injection

split ratio 2:1

Gas flow N2, 2.5 mL/min

Run 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

S1 Test substance 0.01086 g

Ethyl acetate

10 1086.00 N/A N/A

S2 S1 1 mL 10 108.60 N/A N/A

L1 L4 1 mL 10 1.09 1.08 -0.92

L2 S2 200 μL 10 2.17 2.10 -3.23

L3 S2 500 μL 10 5.43 5.44 0.18

L4 S2 1 mL 10 10.86 10.91 0.46

L5 S2 2mL 10 21.72 21.70 -0.09

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

R.D=Relative deviation(%).

L1-L5 were injected in sequence. Linearity 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 was Y=4.04937X -0.17442, with the 

correlation coefficient r=0.999986>0.99. The relative deviation of measured concentration from 

nominal concentration was -3.23%~0.46%, which was within the range of ±10%. Results met the 

criteria of linearity, i.e., r ≥0.99 and relative deviations within ±10%.
SUZHOU RESEARCH Study No.: 2014-139-01-01

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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 measured 

concentration from nominal concentration was -4.06%~0.60%, which was within the required range of 

±10%. 

Solution Code Nominal concentration

(mg/L)

Measured concentration

(mg/L)

Relative deviation

(%)

L2QC20140924 2.01 1.97 -1.99

L4QC20140924 10.05 10.10 0.50

L2QC20140925 1.98 1.95 -1.52

L4QC20140925 9.92 9.98 0.60

L2QC20140926 1.97 1.89 -4.06

L4QC20140926 9.87 9.71 -1.62

L2QC20140927 1.98 1.90 -4.04

L4QC20140927 9.90 9.79 -1.11

 Specificity

Area of interfering GC peaks from fish breeding water and ethyl acetate in the retention time of 

the test substance did not 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~REL5 were in the range of 92.62%~108.20%, and RSD=6.23%; the recovery of 

REH1~REH5 were in the range of 89.97%~108.80%, and RSD=7.41%. The results met the criteria for 

recovery, i.e., between 80%~ 110% 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 coded as MDL was prepared with target concentration of 0.1 mg/L in fish breeding 

water and injected after pretreatment. The S/N was 9.62, which met the criteria of S/N≥3 MDL was 

set as 0.1 mg/L A solution coded as LOQ was prepared with target concentration of 0.3 mg/L in fish 

breeding water and injected after pretreatment. The S/N was 27.16, meeting the criteria of ≥10. LOQ 

was set as 0.3 mg/L.

 The results of method validation indicated that this analysis method was suitable for 

concentration measurement of test substance in this zebra-fish acute toxicity test.
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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 20 mL sample was taken from treated groups and injected after pretreatment

to analyze concentration. The results are shown in section 5.1.1. Typical chromatographs are shown 

in figure 2~6.

3.5.5. Measurement and observation

3.5.5.1. Observations

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

Fish alive 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 2013 was used to analyze statistics and collected data (such as temperature, dissolved 

oxygen, pH, etc.). Values 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 LC50 

and LL50, the highest concentration(loading rate) causing no mortality and the lowest concentration(loading 

rate) producing 100 percent mortality was used to establish the approximate LC50 and LL50 (the geometric 

mean of these two concentrations).SPSS 16.0 was employed to calculate the LC50 and LL50 values at 

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

3.6. Validity of the test

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

validity requirements for the test;

The dissolved oxygen concentration was within the range of 62.4%~101.6% air saturation value 

throughout the test, more than the required 60% air saturation, which met the validity requirements for 

the test.

4. Amendment and Deviation

This study had no amendment and deviation to the study plan in the study.
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5. Results and Conclusion

5.1. Results

5.1.1. Concentration 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 was not detected and use half of the limit of quantification as the final 

concentration when the substance was 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 be 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 adsorption or 

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

concentrations were used for the LC50 calculation and loading rates were used for the LL50 calculation. 

The detailed concentration results are shown in Table 1.

5.1.2. Mortalities and abnormalities

The mortality during acclimatization period was 0%. The results of accumulative total deaths and

mortalities at 24 h, 48 h, 72 h and 96 h after the exposure of test fish are shown in attached Table 2.

The observed mortality 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 visible abnormalities found in blank. In the treated groups, fish had visible 

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

swimming near the surface portion of the tank. The detailed results are shown in attached Table 3.

5.1.3. LC50 and Concentration–mortality curve

The LC50 and LL50 of FRET 09-0536 on zebra-fish species are shown in the following table:

Recommended

observation times

Calculated from the loading rates 

(mg/L)

Calculated from the geometric means of 

the measured concentrations (mg/L)

LL50 LC50

24 h 4.164 1.067

48 h 3.785 0.884

72 h 3.785 0.975

96 h 3.525 0.913
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The Concentration–mortality curve is shown in Figure 1.

5.1.4. Water 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 CaCO3/L and 7.34, respectively, which were measured before the exposure.

Dissolved 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 Exposure duration

Project Temperature

 (°C)

pH DO(% ASV) Temperature 

(°C)

pH DO(% ASV)

Range 23.8~24.2 6.55~7.30 89.1~95.3 23.6~24.6 6.53~7.75 62.4~101.6

5.1.5. Body length and body weight

The 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. The detailed results are shown in attached Table 8 and 

Table 9.

5.2. Conclusion

The LL50 (96h) of test substance FRET 09-0536 on zebra-fish species was 3.525 mg/L based on 

nominal concentrations(loading rate) and LC50 (96h) was 0.913 mg/L based on the geometric means 

of measured concentrations under the test conditions in this study,

6. Archives

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

Study 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.

If the extension of retention period is needed, specific details can be negotiated between test 

facility and sponsor.

7. References

1) HJ/T 155-2004 The Guidelines of Chemical Testing Good Laboratory Practices. MEP. 2004.
SUZHOU RESEARCH Study No.: 2014-139-01-01

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: 8th 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 TESTING AND ASSESSMENT Number 23, GUIDANCE DOCUMENT 

ON AQUATIC TOXICITY TESTING OF DIFFICULT SUBSTANCES AND MIXTURES.

2000.

8. Appendix

8.1. Attached tables

Table 1 Concentration analysis of the test substance 

Groups

Treated

group 1

Treated

group 2

Treated

group 3

Treated

group 4

Treated

group 5

Nominal concentrations (mg/L) 1.0 1.5 2.3 3.4 5.1

Measured 

concentrations

(mg/L)

Beginning of the 

exposure

0.83 1.30 1.98 2.87 4.26

Before 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 -

Before 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 -

Before the third 

renewal

0.10

(12.0%)

0.31

(23.5%)

0.15

(7.4%)

0.32

(10.3%)

-

After the third 

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

Table 2 Cumulative deaths and mortalities

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 FE1-1 0(0%) 0(0%) 0(0%) 0(0%)

1.5 FE2-1 0(0%) 0(0%) 0(0%) 0(0%)

2.3 FE3-1 0(0%) 0(0%) 0(0%) 0(0%)

3.4 FE4-1 0(0%) 2(20%) 2(20%) 4(40%)

5.1 FE5-1 10(100%) 10(100%) 10(100%) 10(100%)

Remark: * bracket outside is cumulative death; bracket is cumulative mortality. 
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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 FE1-1 A A A A A A

1.5 FE2-1 A A A A A A

2.3 FE3-1 A A A A A A

3.4 FE4-1 A A A B B B

5.1 FE5-1 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

Day No.

(acclimatization)

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

Remark: ASV = Air saturation value

Table 5. Water quality parameters during the test (Temperature)

Nominal 

Conc.

(mg/L)

Vessel 

No.

Exposure Day No.

1 

2 3 4 

Before 5 

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 FE1-1 24.6 24.0 24.1 24.0 23.8 23.9 23.8 23.9 

1.5 FE2-1 24.4 24.0 24.2 23.8 23.7 23.8 23.8 23.8 

2.3 FE3-1 24.1 23.6 24.2 23.7 23.8 23.8 23.9 23.8 

3.4 FE4-1 24.1 23.7 24.1 23.7 23.9 23.8 24.0 23.9 

5.1 FE5-1 24.0 23.9 - - - - - -

Minimum 23.6

Maximum 24.6

Remark: "-"=not obtained, all fish had died.
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Table 6. Water quality parameters during the test (pH)

Nominal 

Conc.

(mg/L)

Vessel 

No.

Exposure Day No.

1 

2 3 4 

Before 5 

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 FE1-1 7.72 6.68 7.68 6.77 7.27 6.88 7.25 7.01 

1.5 FE2-1 7.75 7.01 7.64 6.84 7.26 6.85 7.23 6.87 

2.3 FE3-1 7.63 6.81 7.60 6.59 7.25 6.62 7.24 6.69 

3.4 FE4-1 7.71 6.76 7.63 6.53 7.28 6.57 7.25 6.62 

5.1 FE5-1 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 No.

1 

2 3 4 

Before 5 

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 FE1-1 98.3 69.2 98.4 68.6 99.2 68.2 98.6 69.0 

1.5 FE2-1 98.2 67.4 98.5 65.8 99.2 65.9 98.2 66.1 

2.3 FE3-1 97.5 65.3 97.6 64.4 98.8 64.6 97.7 64.5 

3.4 FE4-1 97.9 64.5 97.3 62.7 98.4 62.4 97.8 62.7 

5.1 FE5-1 98.0 63.2 - - - - - -

Minimum 62.4

Maximum 101.6

Remark: "-"=not obtained, all fish had died.

Table 8. Body lengths (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 

Remark: Minimum body length of test fish was 2.696 cm, maximum body length of test fish was 2.892

cm.
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Table 9. Body weights (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 

Remark: The maximum loading was 0.564 g fish/L during the test. Loading (g fish/litre)= Total fish 

body weight(g)/test solution volume(L).

8.2. Figures

Figure 1 Graph of the concentration-mortality curve

Figure 2 Chromatogram 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)
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Figure 3. Chromatogram of LOQ (nominal concentration was 0.3 mg/L, fish breeding water as 

solvent)

Figure 4 Chromatogram of recovery (nominal concentration of 1.22 mg/L FRET 09-0536 after 

pretreatment, fish breeding water as solvent)
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Figure 5 Calibration curve (linearity)

Figure 6 Chromatogram of the test solution (treated group 1 at 0hr)
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8.3. Attachment

Attachment 1. List of abbreviations

AR Analytical Reagent

ASV Air saturation value

IACUC Institutional Animal Care and Use Committee

LC50

The concentration of the test substance which kills 50 per cent of test 

fish within a stated exposure period

LL50

The loading rate of the test substance which kills 50 per cent of test fish 

within a stated exposure period

LOQ Limit Of Quantification

MDL 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 Standard operating procedures

TFM Test Facility Management

GC Gas Chromatography

Attachment 2. Substance information
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