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Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 1 of 38 

Bioassay and Safety Assessment Lab 

SHANGHAI ACADEMY OF PUBLIC MEASUREMENT 

FINAL REPORT 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) 

Code: 

Study number of Dow: 

Study director: 

SPONSOR: 

The Dow Chemical Company 

Midland, MI 48674 USA 

pe~o '~;Q{~tory: ~\~ ~A': Bioassay and S . ~S~11l~V} Labor,atory, APM, 

1500# Zhanghe~O~~h~vang HI-tech Park, 

Sha~hina 

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Acute toxicity test of to Chinese Rare Minnow , 

(Gobiocypris rarus) Page 2 of 38 

Contents 

Title ............................................................................................................................. 1 

Contents ..................................................................................................................... 2 

Statement of GLP Compliance ................................................................................. 3 

Quality Assurance Statement .................................................................................. 4 

Laboratory Statement ............................................................................................... 5 

Signature Page .......................................................................................................... 6 

Key Study Personnel ................................................................................................ 7 

1 Abstract ............................................................................................................... 8 

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2 Study Sponsor .................................................................................................... 8 'l 

3 Study Mon itor ...................................................................................................... 9 

4 Study Director ..................................................................................................... 9 

5 Information of the Test Substance .................................................................... 9 

6 Date ...................................................................................................................... 9 

7 Objective ............................................................................................................ 10 

8 Guideline ........................................................................................................... 10 

9 Principle of the Test. ......................................................................................... 10 

10 Materials and Reagents ................................................................................. 1 0 

11 Apparatus ....................................................................................................... 1 0 

12 Species ........................................................................................................... 10 

13 Test Methods .................................................................................................. 11 

14 Test Validity .................................................................................................... 13 

15 Results and Discussion ................................................................................ 13 

16 Conclusions ................................................................................................... 14 

17 Reference ....................................................................................................... 14 

18 Archives .......................................................................................................... 15 

19 Departure from Protocol ............................................................................... 15 

20 Tables and Figures ........................................................................................ 16 

Appendix A .............................................................................................................. 19 

Information of the Test Substance (provided by the sponsor) ........................... 20 

Certificate of Analysis of the Test Substance (provided by the sponsor) ......... 21 

Appendix B. Analytical Report ............................................................................... 21 

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Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

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Statement of GLP Compliance 

The work to which this study refers was performed in compliant with The GECD 

fl I ; guideline of Good Laboratory Practices (ENVIMCICHEM (98)17), Requirements of L.C 

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conduct for good laboratory practice (G8fT 22275.1-2008) and The guidelines of 

chemical testing good laboratory practices (HJfT 155-2004). 

The report fully and accurately reflects the procedures used and data generated. 

Bioassay and Safety Assessment Lab, APM 

Date 

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Bioassay and Safety Assessment Lab, APM Code: ·l. 

Acute toxicity test of to Chinese Rare Minnow . 

(Gobiocypris rarus) Page 4 of 38 

Quality Assurance Statement 

The study conduct, raw data and report were inspected by the Quality Assurance Unit 

(QAU) at Bioassay and Safety Assessment Laboratory, APM to determine adherence 

with the study protocol and laboratory standard operating procedures. This report is 

an accurate representation of the raw data. Type and date of study inspections and 

dates of report to the Study Director and to Facility Management are listed below: 

Inspecting Type Inspection Date Report to study directorl 

Facility Management 

Study plan 2012.4.2 2012.4.2/2012.4.2 

Report 2012.7.23/24 2012.7.24/2012.7.24 

Validation of analytical method 2012.5.30 2012.5.30/2012.7.4 

Sample preparation, 

determinations and data record 2012.6.4/8 2012.6.15/2012.7.24 

in range-finding test 

Sample preparation, 

determinations and data record 2012.6.11/15 2012.6.15/2012.7.24 

in limit test 

Date 

(signed in original- 3F';Il if.) 

Bioassay and Safety Assessment Lab, APM 

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Laboratory Statement 

1, This test report would be invalid without the seal of the lab and the signatures of 

Study director, QA and Manager of Testing facility. 

2. Any unauthorized alteration of the test report would make the report invalid. 

3. The test report only reflects the test substance received from sponsor. 

4. The owner of the test report shall be responsible for any consequences caused by 

using incomplete information from the test report. 

- 
Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) . Page 6 of 38 

Signature Page 

Submitted by: Bioassay and Safety Assessment Lab, APM 

1500 Zhangheng Road, Zhangjiang Hi-tech Park, 

Shanghai, 201203, P.R. China 

Prepared by: 

Approved by: 

Bioassay and Safety Assessment Lab, APM 

(signed in original- )llUt-X) 

Bioassay and Safety Assessment Lab, APM 

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Key Study Personnel 

The following personnel were responsible for the conduct of the work and reporting of 

the study results 

Yang Jing Study Director 

Zhang Jingji Acute toxicity test and technical report writer 

Zhang Kun Acute toxicity test 

MaoWanlian Chemical analysis and technical report writer 

Liu Min Chemical analysis 

Shen Lu Chemical analysis 

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Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 8 of 38 

1 Abstract 

According to "OECD Guidelines for the Testing of Chemicals. 203 Fish, Acute Toxicity 

Test 1992", the acute toxicity of the test substance 

( to Gobiocypris rarus was determined by a static test. 

Based on the information provided by the sponsor, the test solution was prepared 

(nominal concentration of 100 mg/L) by adding a slurry of test substance with acetone 

(concentration of 0.1 ml acetone/L) directly into the test water. The solution was 

allowed to stir with a magnetic stirrer for 72 hours with the vortex height of 

approximately 1/5 of the solution. After stirring, the test solution (without removing 

insoluble material) was used directly for testing. Based on the results of the pre-test, a 

nominal concentration of 100 mg/L of the test substance was used to perform the limit 

test with a blank control and a solvent control. The test duration was 96h. 

There were no mortalities and abnormalities observed in treatment group during the 

test period. No mortality occurred in the blank control and solvent control during the 

test and the dissolved oxygen concentration was greater than 60.0% of the air 

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saturation value throughout the test duration. Hence, the test was considered to be n 

valid. During the test, the measured contents of the test substance determined by 

HPLC was not stable in test water since the solubility of the test substance in water is 

slight and dispersion of the insoluble fractions was inhomogeneous. The contents of 

the test substance in test solutions were between 129 - 1144 IJg/L. 

The 96h LC50 for the test substance to Gobiocypris rarus was higher than 100mg/L, 

which exceeds the water solubility of the test substance under tested conditions 

(geometric mean measured concentration of non-centiruged and centrifuged samples 

was 270 1J9/L and 18 1J9/L, respectively). The measured contents in the sample 

solutions were not stable, and insoluble material was present in the these solutions 

during the entire exposure, which suggests fish were exposed to the maximum 

soluble concentration of test substance under the conditions tested throughout the 

entire exposure period. 

2 Study Sponsor 

Name: The Dow Chemical Company 

Address: Midland, MI 48674, USA 

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Acute toxicity test of to Chinese Rare Minnow 

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Contact person: Lorraine Francourt 

Tel: +49 7227912761 

Email: LFrancourt@dow.com 

3 Study Monitor 

Contact person: Summer Shen 

Address: No.936, Zhangheng Road, Zhangjiang Hi-Tech Park, Shanghai 201203, 

P.R.C. 

Tel: +86-21-38513187 Fax: +86-21-20249979 

Email: smshen2@dow.com 

4 Study Director 

Name: Yang Jing 

Address: Room 208, Bioassay and Safety Assessment Laboratory, 1500 Zhangheng 

Road, Zhangjiang Hi-tech Park, Shanghai, 201203, P.R. China 

Tel: 86-21-50798123 Fax: 86-21-50798125 

5 Information of the Test Substance 

Name: 

Code: 

Receipt date: 2012-02-13 

Email: yangj@apm.sh.cn 

Conditions of storage: Store in dark at room temperature 

Full details of testing substance are given in Appendix A. 

6 Date 

Study initiation date: 

Experimental starting date: 

Experimental completion date: 

Study completion date: 

2012-05-23 

2012-05-30 

2012-06-15 

2012-09-18 

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Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 10 of 38 

7 Objective 

The acute toxicity of the test substance, to fish was investigated 

in this study. 

8 Guideline 

OECD Guidelines for the Testing of Chemicals. 203 Fish, Acute Toxicity Test 1992. 

9 Principle of the Test 

The fish are exposed to the test substance solution preferably for a period of 96 hours. 

Mortalities are recorded at 24, 48, 72 and 96 hours and the concentrations which kills 

50 percent of the fish (LCso) are determined. 

10 Materials and Reagents 

10.1 Materials and Reagents 

Acetone (Riedel-De Haen). 

10.2 Reference Substance 

None. 

11 Apparatus 

Oxygen meter (Thermo 3 Star); 

Water hardness meter (HANNA HI93735); 

pH meter (HANNA HI98128); 

Analytical balance (Mettler Toledo AL204); 

HPLC (Waters 2695); 

A water tank with temperature control (Home-made device). 

12 Species 

The test was performed with Gobiocypris rarus which were obtained from the Institute 

of Hydrobiology, Chinese Academy of Sciences (10: GR-IHB-111212). This 

freshwater fish species is suitable for fish toxicity testing because of their ease of 

culturing and handling, sensitivity to a variety of chemical substances, and the 

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extensive data base for this species. This species complies with OECD Guidelines for 

the Testing of Chemicals. 203 Fish, Acute Toxicity Test 1992. 

Before being used in the test, all fish were held in the laboratory under the following 

conditions for more than 14 days: 

Water: the test water used was tap water dechlorinated by activated carbon and 

sterilized by UV lamp; 

Temperature: 21 - 25°C; 

pH: 6.0 - 8.5; 

Dissolved oxygen concentration: > 80% of air saturation value (ASV); 

Photoperiod: LightDark = 12 hours:12 hours; 

r' Feeding: fish were fed daily until 24 hours before the test was started. 

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No diseases were observed during the acclimation period; 

No mortality was present within the 7 days before the start of the test. 

The Lengths and weights for fish used in the test and measured at the end of the test 

were 2.3 ± 0.2 cm (Mean ± SO) and 0.20 ± 0.06 g (Mean ± SO), respectively. 

13 Test Methods 

13.1 Test Conditions 

13.1.1 water 

Tap water, dechlorinated by activated carbon and sterilized by UV lamp, was used. 

The water was aerated continuously over 24h. Water quality parameters were as 

follows: 

Total hardness: 165 mg CaC03l'L; 

Dissolved oxygen concentration: >90% of ASV. 

13.1.2 Test vessels 

5L glass tanks were used containing 4L of test solution. 

13.1.3 Conditions of exposure 

Duration: 96h; 

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Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 12 of 38 

Test method: static; 

loading: about 0.5 g fish/litre; 

Temperature: 22.8-23.6°C; 

Photoperiod: Light: Dark=12 hours:12 hours; 

Dissolved oxygen concentration: 74.4-90.3%A8V; 

Feeding: none. 

13.2 Procedures 

13.2.1 Test solutions preparation 

Based on the information provided by the sponsor, the test solution was prepared 

(nominal concentration of 100 mg/l) by adding a slurry of test substance with acetone 

(concentration of 0.1 ml acetone/l) directly to 4 litres of test water in a 5-l glass vessel. 

The solution was allowed to stir with a magnetic stirrer for 72 hours with the vortex 

height of approximately 1/5 of the solution. After stirring, the test solution (without 

removing insoluble material) was used directly for testing. 

13.2.2 Pre-test 

One nominal concentration of 100 mg/l was used in the pre-test. In this test, a blank 

control of test water and a solvent control of acetone (0.1 mill) were used. 

Five fish were introduced into each concentration and the test duration was 96 hours. 

Fish were observed and dead fish were recorded at 0, 24, 48, 72 and 96 hours after 

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the test. For determination of test substance, six samples of test solution were taken 

from each test vessel at each sampling interval. Three 2ml samples were collected 

and analyzed directly and three 20 ml samples were collected and analyzed after 

centrifugation (10000 rpm, 40 min). 

13.2.3 Limit test 

Based on the results of the pre-test, a limit test was performed. In this test, a blank 

control of test water and a solvent control of acetone (0.1 mill) as well as a nominal 

concentration of 100 mg/l were used. 

Ten fish were introduced into 1 replicate vessel at each concentration and the test 

duration was 96 hours. The fish were inspected at 0, 24, 48, 72 and 96 hours after test 

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initiation and mortalities were recorded. Measurements of the pH, dissolved oxygen 

concentration and temperature in the test solutions were made at the beginning and at 

the end of the test. The contents of the test solution were measured at Oh, 48h and 

96h of the test. For determination of test substance, six samples of test solution were 

taken from each test vessel at each sampling interval. Three 2ml samples were 

collected and analyzed directly and three 20ml samples were collected and analyzed 

after centrifugation (10000 rpm, 40 min). 

At the the end of the test, the body length and body weight of the test fish were 

measured from the control group, The loading rate was also calculated with the body 

weight at the same time. 

13.3 Data analysis method 

The results of the limit test were expressed as both nominal and measured geometric 

mean contents of the test solutions. 

14 Test Validity 

No mortality was observed in the control during the test; 

Acceptable exposure conditions were maintained throughout the test; 

The dissolved oxygen concentration in test solutions were between 74.4-90.3% ASV, 

which was greater than 60.0% of the air saturation value, throughout the test. 

The test fulfilled the above requirements and was therefore considered to be valid. 

15 Results and Discussion 

The actual test substance contents of test solutions was determined using a HPLC 

analysis method. The routine limit of detection (LOD) for the test substance using this 

method was calculated to be 5.00 IJg/L (based on a signal/noise of 4.23). It was found 

that the average recovery was 101 % and RSD (relative standard deviation) valuewas 

3.19% for the spiked 100 IJg/L sample. Full details of analysis method are given in 

Appendix B. 

In the pre-test, there were no mortalities and abnormalities observed in fish of control 

groups and treatment group during the test. The results of the pre-test are shown in 

Table 20.1. During the pre-test, the measured contents of test substance was not 

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Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 14 of 38 

stable in test water. See 20.2 (analyzed directly without centrifugation) and 20.3 

(analyzed after centrifugation). 

In limit test, there were no mortalities and abnormalities observed in fish of control 

groups and treatment group during the test. See Table 20.4. In the limit test, 

the measured contents of test substance in 96h were shown in tables 20.5 and 20.6. 

During the test, the measured contents of test substance determined by HPLC was 

not stable in test water since the solubility of the test sUbstance in water is slight and 

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dispersion of the insoluble fractions was non-homogeneous. The contents of the test 

substance in test solutions were between 129 - 1144 J,Jg/L, with a measured l 

geometric mean concentration was 270 J,Jg/L. At the same time, although most of 

insoluble fractions in test solutions were removed after centrifugation (10000 rpm 40 l 

min), the contents of the test substance in test solutions were still not stable because 

of the residual insoluble fractions with contents between 11.3 - 29.0 J,Jg/L and a 

measured geometric mean concentration of 18 J,Jg/L. Though measured contents in 

the sample solutions were not stable, insoluble material was present in these 

solutions during the entire exposure, which suggests fish were exposed to the 

maximum soluble concentration of test substance under the conditions tested 

throughout the entire exposure period. 

During the test period, the range of the dissolved oxygen concentrations in the test 

solutions were between 74.4-90.3%A8V. The range of temperature in the test 

solutions were between 22.8-23.6°C. The range of pH value in the test solutions 

were between 7.09-7.69. The results of the pH, dissolved oxygen and temperature 

measurements are shown in Table 20.7. 

16 Conclusions 

The test results showed that the 96h LCso for the test substance, 

to Gobiocypris rarus was higher than 100 mg/L (geometric mean measured 

concentration of non-centiruged and centrifuged samples was 270 J,Jg/L and 18 J,Jg/L, 

respectively). Though measured contents in the sample solutions were not stable, 

insoluble material was present in these solutions during the entire exposure, which 

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under the conditions tested throughout the entire exposure period. 

17 Reference 

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OECD SERIES ON TESTING AND ASSESSMENT Number 23. GUIDANCE 

DOCUMENT ON AQUATIC TOXICITY TESTING OF DIFFICULT SUBSTANCES AND 

MIXTURES 2000. 

18 Archives 

The signed copies of the final reports (English and Chinese) and notarized copies of 

all raw data generated and recorded during the study will be sent to the sponsor at 

designated address after issuance of the final reports. All original raw data generated 

and recorded during the study and signed copies of the final reports (English and 

Chinese) will be stored in the Bioassay and Safety Assessment Laboratory of 

Shanghai Academy of Public Measurement for five years, and returned to the sponsor 

at designated address on agreed date. 

19 Departure from Protocol 

None. 

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Bioassay and Safety Assessment lab, APM Code: 

Acute toxicity' test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 16 of 38 

20 Tables and Figures 

20.1 The mortalities and abnormalities in pre-test (5 fish in each concentration) 

Group Duration (h) Mortalities Cumulative Abnormalities Mortalities 

Oh 0 0 none 

24h 0 0 none 

Blank Control 48h 0 0 none 

72h 0 0 none 

96h 0 0 none 

Oh 0 0 none 

Solvent Control 24h 0 0 none 

(0.1 mill acetone) 48h 0 0 none 

72h 0 0 none 

96h 0 0 none 

Oh 0 0 none 

Treatment Group 24h 0 0 none 

(Nominal Concentration 48h 0 0 none 

100 mg/l) 72h 0 0 none 

96h 0 0 none 

20.2 The measured contents of the test substance in pre-test (analyzed directly) 

Nominal Measured contents (!Jail) Measured 

Group concentration Replicates Geometric 

Oh 48h 96h Mean Content (mg/l) (!J9/l) 

1 N.D. N.D. N.D. 

Blank 2 N.D. N.D. N.D. 

Control 0 3 N.D. N.D. N.D. -

MEAN - - - SD - - -

Solvent 1 N.D. N.D. N.D. 

2 N.D. N.D. N.D. Control 0 3 N.D. N.D. N.D. (0.1 mill -

acetone) MEAN - - - SD - - - 1 1128 304 166.8 

Treatment 2 674 292 171.2 

Group 100 3 508 320 173.0 342 

MEAN 770 305 170.3 

SD 321 13 3.2 

lOD=5.00 (Jg/l, "-", not available, 

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Code: 

Page 17 of 38 

20.3 The measured contents of the test sUbstance in pre-test (analyzed after 

centrifugation with the supernate) 

Measured contents (~g/l) 

Nominal Measured 

Group concentration Replicates Geometric 

Oh 48h 96h Mean (mg/l) cont~)t 

--'J.l9/L 

1 N.D. N.D. N.D. 

Blank 2 N.D. N.D. N.D. 

Control 0 3 N.D. N.D. N.D. - MEAN - - - SD - - -

Solvent 1 N.D. N.D. N.D. 

Control 2 N.D. N.D. N.D. 

(0.1 mill 0 3 N.D. N.D. N.D. - MEAN - - - acetone) SD - - - 1 53.8 12.0 19.8 

Treatment 2 46.6 51.4 N.D. 

100 3 30.8 23.2 N.D. 22 Group MEAN 43.7 28.9 - SD 53.8 12.0 -

lOD=5.00 ~g/l, u_", not available. The measured contents not detected in test 

soluitons were calculated by 1/2 lOD. 

20.4 The mortalities and abnormalities in limit test (10 fish in each concentration) 

Group Duration (h) Mortalities Cumulative Abnormalities Mortalities 

0 0 0 none 

24 0 0 none 

Blank Control 48 0 0 none 

72 0 0 none 

96 0 0 none 

0 0 0 none 

24 0 0 none 

Solvent Control 48 0 0 none (0.1 mill acetone) 72 0 0 none 

96 0 0 none 

0 0 0 none 

Treatment Group 24 0 0 none 

(Nominal concentration 48 0 0 none 

100 mg/l) 72 0 0 none 

96 0 0 none 

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Bioassay and Safety Assessment lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 18 of 38 

20.5 The measured contents of the test substance in limit test (analyzed directly) 

Measured contents ~g/l) Measured 

Nominal Geometric 

Group Concentration Replicates Oh 48h 96h Mean 

(mg/l) Content 

(~g/l) 

1 N.D. N.D. N.D. 

Blank 2 N.D. N.D. N.D. 

Control 0 3 N.D. N.D. N.D. -

MEAN - - - SD - - -

Solvent 1 N.D. N.D. N.D. 

2 N.D. N.D. N.D. Control 0 3 N.D. N.D. N.D. (0.1 mill -

acetone) MEAN - - - SD - - - 1 1144 198 149 

Treatment 2 648 173 145 

100 3 512 172 129 270 Group MEAN 768 181 141 

SD 333 15 11 

lOD=5.001-19/l, "-", not available, 

20.6 The measured contents of the test substance in limit test (analyzed after 

centrifugation with the supernate) 

Measured contents (l-Ia/U 

Nominal Measured 

Group concentration Replicates Oh 48h 96h Geometric 

(mg/l) Mean Content 

(wall) 

1 N.D. N.D. N.D. 

Blank 2 N.D. N.D. N.D. 

Control 0 3 N.D. N.D. N.D. -

MEAN - - -

SD - - -

Solvent 1 N.D. N.D. N.D. 

2 N.D. N.D. N.D. Control 0 3 N.D. N.D. N.D. (0.1 mill -

acetone) MEAN - - -

SD - - -

1 23.2 15.7 17.9 

Treatment 2 29.0 11.3 11.9 

Group 100 3 21.6 16.4 21.0 18 

MEAN 24.6 14.5 16.9 

SD 3.9 2.8 4.6 

lOD=5.00 1-19/l, "-", not available, 

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Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 19 of 38 

20.7 Dissolved oxygen, temperature and pH in test solutions and the control of the 

limit test 

~ Treatment Group 

Blank Control Solvent Control (Nominal 

Parameter (0.1 mill acetone) Concentration 

100 mQ/l) 

Oxygen(%ASV) 81.1 79.0 90.3 

Oh TemperatureCC) 22.9 22.8 22.9 

pH 7.10 7.10 7.09 

Oxygen(%ASV) 74.4 79.3 76.2 

24h TemperatureCC) 23.2 23.2 23.1 

pH 7.27 7.22 7.14 

Oxygen(%ASV) 75.8 76.1 74.6 

48h Temperature(°C) 23.0 23.0 23.0 

pH 7.63 7.54 7.48 

Oxygen(%ASV) 79.2 80.5 77.4 

72h TemperatureCC) 23.1 23.1 23.1 

pH 7.69 7.64 7.56 

Oxygen(%ASV) 79.5 79.3 79.0 

96h TemperatureCC) 23.6 23.6 23.6 

pH 7.67 7.57 7.54 

L Appendix A. 

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- 
Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 20 of 38 

Information of the Test Substance (provided by the sponsor) 

Chemical name 

Other name 

Molecular formula 

CAS No 

Batch No 

Appearance 

Purity 

Odour Very slight 

Density n.a. 

Solubility in water 3.69 x10·6 gIL 

Melting point n.a. 

Boiling point (760mm Hg) n.a. 

Freezing point n.a. 

Vapour pressure 9.4 x 10.7 Pa (25°C) 

Absorption n.a. 

Flash point n.a. 

Ignition temperature n.a. 

Self-ignition temperature n.a. 

Oxidizing properties Predicted negative 

Explosion Predicted negative 

Log Pow 6.18 

pH n.a. 

Conditions of storage Store in dark at room temperature 

Stability Stable in water 

Expiry date 2014-02-01 

n.a.: not applicable 

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Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 21 of 38 

Certificate of Analysis of the Test Substance (provided by the sponsor) 

AnaClRDFJC6TE Of M'AAYSIS fOB TUTIRIRBINCI'It9NTROL SUU'rANCq 

1Tl'LEIOBJECTIVE: Characterization of Lot for Use as a Test 

Substance 

TESTIREFERENCEfCONTROL SUBSTANCE: 

DESCRImON: CbemiCII Name: 

CAS No.: 

STUDY INITIATION DATE: 01 Pebnwy2012 

METHODS USED: 

MOLECULAR fORMULA

MOL.ECUl.AJl WEIGHT: 

PURITY: au chromatopaphy with flame ionizallOD detection (GC-FID) aDd Karl FilCher titratioll, 

MLAL-2011-0201182 

IDBNTIF1CA nON: Fauriel' Tranlfonn-Infiarcd (Fr.m) IpcdroKOPY and au ehromatoaraPhy·mus IPCcttomctty 

(GC.MSl 

RESULTS AND CONCLUSIONS: 

Structural COnfirmatiOll of tile tcIt material wq perfOrmed by FT ·IR and GC-MS. 

Ani_ Percent: x (GC-FID) Intcmal Standard: N/A 

Other (explain): Karl Fischer 1itmion 

PURITY DETERMINATION 

Result: (95% CODfide.D.cc intaval) after corrcctin. for the coulomctric Karl Fisehcrwatcr .... Iu. 

cmtBR. (SpocitY): 204 = 13 ppm (95% coofidence iolcrVal) walcrby Karl FischertitratiOlL 

CALCULATIONS: 

Area Permrt; x (GC-FID) Internal Standard: NfA External Standard: NlA 

Othcr(explain): KadFillChcrtitration 

AU orisinal raw data UIOclatod with 1his study \VCR archived La the SPOIllOf f'acility an:bive. 

Only delcriptive ItItiAtica were used unl_ otberwiJc noted in the reaalti. Thi$ Jtud:y was coMuctod in ICCOrdaDce with the 

Good ubomory Practice StaDderd, 40 CPR. Part 792 and 40 CPR Part 160, ad the Jteaistratioll, EvaIUltioll, Autbori:ration and 

RescrlctiOll. ofChcmicaIJ (REACH) RcauhtioD well otbc:rwiM DOled. Due to the montum aatwa oflhc study, tlJe stability of the test .ublbulce Ulldcl'Stoftge conditiOJll per 40 CIR Part 792.1 05(c) at the test lite wu not verified. 

8, 

-

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aq!M 9~ J<>ll. 

QA ....... til 

STUDY DIRECfORAND TEST FACILITY ADDRESS: 

Impa!:tAnalytical1910 W. St. AnIiMws Rei. Midland, Ml48640 

lA Project 2 ofl4 pages lA Report

Sponsor No,

DOWCONFIDENTIAL ~ ReceIved from. third pllty IOtII'CI 

- 
Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 22 of 38 

Appendix B. Analytical Report 

1 Objective 

HPLC analysis method of the test substance in test medium was developed to 

determine the contents of the test substance during the test process .. 

2 Materials and methods 

2.1 Test samples 

The test samples were taken from test water of fish acute toxicity test (Section 13.2). 

2.2 Analytical procedure 

2.2.1 Storage 

The samples were analyzed directly without storage. 

2.2.2 Reagents and solvents 

1) Acetonitrile (HPLC grade, Sigma); 

2) Water (MiIIiQ A10). 

2.2.3 Preparation the calibration solutions of test substance 

0.0100 g sample of test substance was dissolved in 10 ml acetonitrile to obtain 1000 

mg/L stock solution. Defined volumes of this stock solution were diluted with 

acetonitrile-water (1 :1) to prepare calibration solutions in the range of 5.00 ug/L to 200 

IJg/L. These solutions were used to plot the calibration curve. 

2.2.4 Preparation of spiked test water samples 

To demonstrate the validity of the method, a 50IJL solution with a concentration of 

1.00 mg/L was added to 450 IJI test water to obtain a 100.0 IJg/L spiked sample. Six 

replicates were prepared. At the same time, blank samples were prepared. These 

solutions were used to determine the recovery and valite the precision RSD(%) of 

method. 

2.2.5 Preparation of samples 

0.5 ml of the sample were added to a 2ml vial and diluted with 0.5ml acetonitrile. If the 

concentration of sample was too high, the sample could be diluted by 

acetonitrile-water (1 :1) again. Test samples after mixing were injected directly and 

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Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 23 of 38 

analyzed by HPLC. 

2.3 HPLC Conditions 

The high performance liquid chromatograhy (HPLC) method was developed at the 

Bioassay and Safety Assessment Laboratory Shanghai Academy of Public 

Measurement. The conditions of the method are as follows: 

Liquid chromatograph ( Waters 2695/PDA Waters 

Instrument 

Acquit/M ) 

Column Benetnach C18 (4.6x150 mm, 5 IJm ) 

Column temp. 40°C 

Flow rate 1.0 ml/min 

Injection colume 100 IJI 

Detection wave 230 nm 

A: water 

Mobile phase B: acetonitrile 

Gradient program Time (min) %A %B 

0.0 50 50 

2.0 50 50 

8.0 0 100 

9.0 0 100 

10.01 50 50 

12.0 50 50 

2.4 Data evaluation and quantification 

The standard curve for quantification was poltted with the concentration of test 

substance as X-axis and its corresponding peak area as Y-axis to obtain the linear 

equation (1). Unit of concentration of test substance is ng/ml or mg/L. 

From the calibration curve the concentration of the test substance in an injected 

sample was calculated by equation: 

Y=AxX+B (1) 

Where: Y = test substance peak area; 

X= test substance concentration. 

The concentration of the test substance in a sample was calculated by equation (2): 

- 
Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 24 of 38 

C=XxF (2) 

Where: C= concentration of the test substance in the sample; 

X= amount of the test substance calculated by equation 1; 

F= factor (dilution factor or concentration factor). 

The recovery rate was calculated by equation (3): 

R= CI Cnomx100% (3) 

Where: R= recovery rate (%); 

C=concentration of the test substance in the sample calculated by equation 2; 

Cnom= nominal concentration of the test substance in the sample. 

3 Results and discussion 

3.1 Test method 

The calibration data for the calibration solutions of the test substance was given in 

Table 1 and Figure 1. The linearity of the HPLC-system was within the calibration 

range of 5.00 - 200 1J9/L of the test substance. The resulting calibration curve 

equation was: Y = 4.92e+002 X + 3.23e+002. The correlation of the calibration curve 

used was 0.9993. Fig.2 gives the chromatogram of the solvent. Typical 

chromatograms of samples are shown in Figure 3 and Figure 4. The limit of detection 

(LOD) for the test substance in test medium was calculated to be 5.00 1J9/L (based on 

a signall noise of 4.23) showing in Figure 5. 

Before sample analysis, the recovery of the method was determined. It was found that 

the average recoveries were 101 % for the spiked 100 1J9/L sample with RSD value 

3.19% as shown in Table 2 .The chromatograms of blank and spiked samples are 

presented in Figure 6 and 7. 

3.2 Sample test 

The concentrations of the test samples collected from the fish acute toxicity test are 

presented in Table 3. Also, the typical chromatograms of the test samples are 

presented in Figure 8 to Figure 31. 

4 Tables and figure 

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Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 25 of 38 

Table 1 Calibration curve data of test substance 

Concentration Peak Area 

(lJg/L) 1 2 

5.00 2579 2848 

10.0 4906 4946 

20.0 9935 9861 

50.0 25550 25780 

80.0 40994 40981 

100 48057 48015 

200 98774 98789 

Table 2 The recoveries of spiked test water (blank control) 

Measured Dilution Sample Recovery Mean Precision Sample concentration factor Concentration recovery 

(lJg/L) IF] (1J9/L) % % RSD% 

Blank test ND 2 ND water - - -

47.5 2 95.0 95.0 

50.7 2 101 101 

100 IJg/L 51.1 2 102 102 

spiked 51.6 2 103 103 101 3.19 

sample 51.8 2 104 104 

51.5 2 103 103 

LOD: 5.001J9/L: ND: not detected; -: not applied 

Table 3 Data of fish acute toxicity test 

Measured Dilution Actual Contents Sample ConcentrationlX] factor IF] (lJg/L) IC] (ua/L) 

0390A-R-BLANK-1-0h ND 2 ND 

0390A-R-BLANK-2-0h ND 2 ND 

0390A-R-BLANK-3-0h ND 2 ND 

0390A-R-BLANK-4-C-Oh ND 2 ND 

0390A-R-BLANK-5-C-Oh ND 2 ND 

0390A-R-BLANK-6-C-Oh ND 2 ND 

0390A-R-AC-1-0h ND 2 ND 

0390A-R-AC-2-0h ND 2 ND 

0390A-R-AC-3-0h ND 2 ND 

- 
n 

Bioassay and Safety Assessment Lab, APM Code: l .... Acute toxicity test of to Chinese Rare Minnow . 

(Gobiocypris rarus) Page 26 of 38 

Measured 

Sample Concentration[X] 

(IJQ/L) 

0390A-R-AC-4-C-Oh ND 

0390A-R-AC-5-C-Oh ND 

0390A-R-AC-6-C-Oh ND 

0390A-R-1 OOmg/L -1-0h 56.4 

0390A-R-100mg/L-2-0h 33.7 

0390A-R-1 OOmg/L -3-0h 25.4 

0390A-R-1 OOmg/L -4-C-Oh 26.9 

0390A-R-100mg/L-5-C-Oh 23.3 

0390A-R-100mg/L-6-C-Oh 15.4 

0390A-R-BLANK-1-48h ND 

0390A-R-BLANK-2-48h ND 

0390A-R-BLANK-3-48h ND 

0390A-R-BLANK-4-C-48h ND 

0390A-R-BLANK-5-C-48h ND 

0390A-R-BLANK-6-C-48h ND 

0390A-R-AC-1-48h ND 

0390A-R-AC-2-48h ND 

0390A-R-AC-3-48h ND 

0390A-R-AC-4-C-48h ND 

0390A-R-AC-5-C-48h ND 

0390A-R-AC-6-C-48h ND 

0390A-R-100mg/L-1-48h 152 

0390A-R-100mg/L-2-48h 146 

0390A-R-100mg/L-3-48h 160 

0390A-R-100mg/L-4-C-48h 6.00 

0390A-R-100mg/L-5-C-48h 25.7 

0390A-R-100mg/L-6-C-48h 11.6 

0390A-R-BLANK-1-96h ND 

0390A-R-BLANK-2-96h ND 

0390A-R-BLANK-3-96h ND 

0390A-R-BLANK-4-C-96h ND 

0390A-R-BLANK-5-C-96h ND 

0390A-R-BLANK-6-C-96h ND 

0390A-R-AC-1-96h ND 

0390A-R-AC-2-96h ND 

0390A-R-AC-3-96h ND 

0390A-R-AC-4-C-96h ND 

Dilution 

factor [F] 

2 

2 

2 

20 

20 

20 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

2 

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2 

2 

2 

2 

2 

2 

2 

2 

2 

Actual Contents 

(lJg/L) [C] 

ND 

ND 

ND 

1128 

674 

508 

53.8 

46.6 

30.8 

ND 

ND 

ND 

ND 

ND 

ND 

ND 

ND 

ND 

ND 

ND 

ND 

304 

292 

320 

12.0 

51.4 

23.2 

ND 

ND 

ND 

ND 

ND 

ND 

ND 

ND 

ND 

ND 

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Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 27 of 38 

Measured Dilution Actual Contents Sample Concentration[X] 

(ua/l) factor [F] (1-I9/L) [C] 

0390A-R-AC-S-C-96h ND 2 ND 

0390A-R-AC-6-C-96h ND 2 ND 

0390A-R-1 OOmg/L -1-96h 83.4 2 166.8 

0390A-R-100mg/L-2-96h 8S.6 2 171.2 

0390A-R-100mg/L-3-96h 86.S 2 173.0 

0390A-R-100mg/L-4-C-96h 9.90 2 19.8 

0390A-R-1 OOmg/L -S-C-96h ND 2 ND 

0390A-R-1 OOmg/L -6-C-96h ND 2 ND 

0390A-L-BLANK-1-0h ND 2 ND 

0390A-L-BLANK-2-0h ND 2 ND 

0390A-L-BLANK-3-0h ND 2 ND 

0390A-L-BLANK-4-C-Oh ND 2 ND 

0390A-L-BLANK-S-C-Oh ND 2 ND 

0390A-L-BLANK-6-C-Oh ND 2 ND 

0390A-L -AC-1-0h ND 2 ND 

0390A-L-AC-2-0h ND 2 ND 

0390A-L-AC-3-0h ND 2 ND 

0390A-L-AC-4-C-Oh ND 2 ND 

0390A-L-AC-S-C-Oh ND 2 ND 

0390A-L-AC-6-C-Oh ND 2 ND 

0390A-L -1 OOmg/L -1-0h S7.2 20 1144 

0390A-L-100mg/L-2-0h 32.4 20 648 

0390A-L -1 OOmg/L -3-0h 2S.6 20 S12 

0390A-L -1 OOmg/L -4-C-Oh 11.6 2 23.2 

0390A-L-100mg/L-S-C-Oh 14.S 2 29.0 

0390A-L -1 OOmg/L -6-C-Oh 10.8 2 21.6 

0390A-L-BLANK-1-48h ND 2 ND 

0390A-L-BLANK-2-48h ND 2 ND 

0390A-L-BLANK-3-48h ND 2 ND 

0390A-L-BLANK-4-C-48h ND 2 ND 

0390A-L-BLANK-S-C-48h ND 2 ND 

0390A-L-BLANK-6-C-48h ND 2 ND 

0390A-L-AC-1-48h ND 2 ND 

0390A-L-AC-2-48h ND 2 ND 

0390A-L-AC-3-48h ND 2 ND 

0390A-L-AC-4-C-48h ND 2 ND 

0390A-L-AC-S-C-48h ND 2 ND 

- 
Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 28 of 38 

Measured Dilution Actual Contents Sample Concentration[X] factor [F] (lJg/L) [C] (lJg/L) 

0390A-L-AC-6-C-48h ND 2 ND 

0390A-L-100mg/L-1-48h 99.0 2 198 

0390A-L-100mg/L-2-48h 86.7 2 173 

0390A-L-100mg/L-3-48h 86.0 2 172 

0390A-L-100mg/L-4-C-48h 7.86 2 15.7 

0390A-L-100mg/L-5-C-48h 5.65 2 11.3 

0390A-L-100mg/L-6-C-48h 8.19 2 16.4 

0390A-L-BLANK-1-96h ND 2 ND 

0390A-L-BLANK-2-96h ND 2 ND 

0390A-L-BLANK-3-96h ND 2 ND 

0390A-L-BLANK-4-C-96h ND 2 ND 

0390A-L-BLANK-5-C-96h ND 2 ND 

0390A-L-BLANK-6-C-96h ND 2 ND 

0390A-L -AC-1-96h ND 2 ND 

0390A-L-AC-2-96h ND 2 ND 

0390A-L-AC-3-96h ND 2 ND 

0390A-L-AC-4-C-96h ND 2 ND 

0390A-L-AC-5-C-96h ND 2 ND 

0390A-L-AC-6-C-96h ND 2 ND 

0390A-L-100mg/L-1-96h 74.7 2 149 

0390A-L-100mg/L-2-96h 72.6 2 145 

0390A-L-100mg/L-3-96h 64.5 2 129 

0390A-L-100mg/L-4-C-96h 8.93 2 17.9 

0390A-L-100mg/L-5-C-96h 5.95 2 11.9 

0390A-L-100mg/L-6-C-96h 10.5 2 21.0 

LOD.5.00~g/L 

10000 

rn .l( 50000 

a 

I I I I I I I I I I I I I I , I I I I I I I I I I I I I I I I I I I I I I I I I I I 

0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00 200.00 

Amount 

Figure 1 Calibration curve of test substance 

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Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) . Page 29 of 38 

0.006 

~ 0.004 

0.0022l 

O.OO~ I 

d~~~~=]\ N~ I I I I I I I I 

0.00 2.00 4.00 6.00 8.00 10.00 12.00 

0.00 6 

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Mnutes 

Figure 4 Chromtogram of 200 ~g/L test substance 

- 
Bioassay and Safety Assessment Lab, APM 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) 

0.005-

Code: 

Page 30 of 38 

\ 

1 

ill 

0.004=;=:;-=:-:;=~:::=~:;:::::;::::;:=;:::=::;==;::~=~~==-'_,-----,-----,-----,--=~-,------,--~ 1 1 1 1 1 1 

0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Mnutes 

Figure 5 Chromtogram of LOD (5.00 iJg/L; s/n=4.23) 

0.04-

=> 

<>: 0.02-

.A. 

1 

0.010{, =;=~~=;I J~ ::::::~:::;=::::;:=;'=:;=::::;=:::::;:=;=I =;:=;=:::;==;::, =;=:;:==;:::=;:1 =;=:;:::=;==1 

0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Mnutes 

Figure 6 Chromtogram of blank test water 

0.04- m 

'" ... 

ci 

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=> <>: 0 0.02 m 

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0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Mnutes 

Figure 7 Chromtogram of 1 OOiJg/L spiked sample 

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Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rams) Page 31 of 38 

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o,ootl =;=J~'---~I,=A~==;:=;:::=:;:I =;=;::::::;:::::::;I=;:::::::;=::;::::=;:I =:;=;:=:~::;,=:;=::;=:l~'---::=11 0,00 2,00 4,00 6,00 8,00 10,00 12,00 

Mnutes 

Figure 8 Chromatogram of sample 0390A-R-BLANK-1-0h 

0,04-

:::l 

« 0,02-

:::l 

« 

o,ooll,=:;=:::,:~;;":::;::, :::::;=:;=:;:::::::;1=:;::=::;::::::;==;:1 ::::::;=;:=:;=::;,=:;=::;=:::;==;:, =;=:;=~~I 0,00 2,00 4,00 6,00 8,00 10,00 12,00 

0,06-

0,04-

0,02-

0,00 1 

0,00 

Mnutes 

Figure 9 Chromatogram of sample 0390A-R-BLANK-4-C-Oh 

, 

2,00 1 

4,00 1 

6,00 

Mnutes 

, 

8,00 

, 

10,00 

Figure 10 Chromatogram of sample 0390A-R-AC-1-0h 

1 

12,00 

- 
Bioassay and Safety Assessment Lab, APM Code: -

Acute toxicity test of to Chinese Rare Minnow '. I 

(Gobiocypris rarus) Page 32 of 38 

0.06 

~ 0.04 

0.02 

o.o.vat, =;:=~:;::::::;,==;=::;=:::;==;::, =;:=;=:;::::::;,=;=::;=='=:::::;::, =::=;:::::;=::;,=;=::;=~=1, 0.00 2.00 4.00 6.00 8.00 10.00 12.00 

0.01 0-

:::> 

« 0.00 5 

0.00 

0.08 

v , 

0.00 

0.06-

~ 0.04 

0.02 

0.00 

I 

0.00 

, 

Minutes 

Figure 11 Chromatogram of sample 0390A-R-AC-4-C-Oh 

\ , , , 

2.00 

, , , , 

4.00 

, , , , 

6.00 

Minutes 

, , , , 

8.00 

, , , 

t.'..!::' 

, 

10.00 

, 

Figure 12 Chromatogram of sample 0390A-R-100mg/L-1-0h 

co 

'" "= 0 

0 

'" '" j 

I , , I I 

2.00 4.00 6.00 8.00 10.00 

Mnutes 

Figure 13 Chromatogram of sample 0390A-R-100mg/L-4-C-Oh 

, 

~ , , 

12.00 

I 

12.00 

-I 

I 

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~ 

i 

, I 

n 

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l 

l 

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l 

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l 

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Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 33 of 38 

0.04 

~ 0.02-

O.Ov .~~~~~~~~~~~~~~~~~~~~~~~~~ , , , , , , -, 

0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Mnutes 

Figure 14 Chromatogram of sample0390A-R-BLANK-1-96h 

:::> 0.02 

« 

o.ool~ , ~ , 0.00 2.00 

, 

4.00 

, 

6.00 

Mnutes 

, 

8.00 

, 

10.00 

Figure 15 Chromatogram of sample0390A-R-BLANK-4-C-96h 

0.06 

:::> 0.04-

« 

0.02-

I~ 

, 

12.00 

f... 

O.Olat, ::::::;=~~=,;=:;=:::;=::::;:::::::;:, ::::::;=:;=:::;==,;=:;=::;:::::::;::=;::, ===:;=::;=:::;::, =;:===~, 0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Mnutes 

Figure 16 Chromatogram of sample 0390A-R-AC-1-96h 

- 
i 

i J 

Bioassay and Safety Assessment Lab, APM Code: 'I 

Acute toxicity test of to Chinese Rare Minnow , I 

(Gobiocypris raros) Page 34 of 38 

0,08-

0,06-

~ 0,04-

0,02 

1- o.OOi,=;==;'--'=;::=;,=;==;==;::=;,=;==;==;::=;,=;==;==;::=;,==:;::::::::;=::::;::=;,=:;::::::::;==;-'::::j, 0,00 2,00 4,00 6,00 8,00 10,00 12,00 

0,1lr 

~ 0,05-

Mnutes 

Figure 17 Chromatogram of sample 0390A-R-AC-4-C-96h 

a 

en 

1 l 0,00-1=, =;=~::;==;::=;=;==;==;::=;=;==;==;::=;=;=:::;:::=;::=;=;=::;==;::, I I I I - I I ~~;=:¢:::j, 0,00 2,00 4,00 6,00 8,00 10,00 12,00 

Mnutes 

Figure 18 Chromatogram of sample 0390A-R-1 OOmg/L-1-96h 

0,08,-r----:---------------------------, 

0,06 

~ 0,04 

a 0,02 ~ 

1 J-. 0,001,=;=~~=;,=;=:::;:::=;::=;,=;==;==;::=;,=;=:::;:::=;::=;,=;=:::;=::::;::=;,:::::zj';=::z;;:=;-'::::j, 

0,00 2,00 4,00 6,00 8.00 10,00 12,00 

Mnutes 

Figure 19 Chromatogram of sample 0390A-R-100mg/L-4-C-96h 

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1 , 

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j 

1 

: i 

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Bioassay and Safety Assessment Lab, APM Code: 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 35 of 38 

0.04-

=> 

..: 0.02-

0.00 J=~~~~~~~~~~=;=;~==~~~~~~::~==;=~~~~ f , , , 1 , 1 

0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Mnutes 

Figure 20 Chromatogram of sample 0390A-L-BLANK-1-0h 

0.06 

0.04 

0.02 

0.001,=:;=~~:::::;I=:;=::;::=;:::::;,=:;=:::;==;:::::::;,=;=:::;=:::::;:=;,=::=::;:::::;::=;,=:;::::::'=~:=1, 0.00 2.00 4.00 6.00 8.00 10.00 12.00 

0.06~ 

=> 0.04 

..: 

0.02 

Mnutes 

Figure 21 Chromatogram of sample 0390A-L-BLANK-4-C-Oh 

0.00-1=, :::::;=~~:::;::, =;=:;=:::;==;:, ==;=:;=:::;==;:, =;=;=::;::::=;:, =;=::=::;:::=;:, =;~:;:::::;::=j, 0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Mnutes 

Figure 22 Chromatogram of sample 0390A-L-AC-1-0h 

- 
Bioassay and Safety Assessment Lab, APM Code: ·l .. Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 36 of 38 

0.06 

::::J 0.04-

« 

~ 

0.02 

o.oo,J=:::;=~~:::;:::::::;::::=;=::::;=:::::;==;==;==;==;==;==;::=;::=;::=;:=;:=.::;:=;:=;:=;:=;~ , ~ I 1 I I 1 I 

0.00 2.00 4.00 6.00 8.00 10.00 12.00 

0.01 

0.01 

0.00 

0.00 

0.06 

5-

0-= 

5-

I 

0.00 

0.04-

0.02-

0.00 

I 

0.00 

, 

Mnutes 

Figure 23 Chromatogram of sample 0390A-L-AC-4-C-Oh 

, , I 

2.00 

, , , I 

4.00 

, , , I 

6.00 

Minutes 

, , , I 

8.00 

, , , 

Lj 

I 

10.00 

~ , 

Figure 24 Chromatogram of sample 0390A-L-100mg/L-1-0h 

on 

0 

.... 

ci 

0 

'" '" I 

I I I I I 

2.00 4.00 6.00 8.00 10.00 

Mnutes 

Figure 25 Chromatogram of sample 0390A-L-100mg/L-4-C-Oh 

, ~ , I 

12.00 

I 

12.00 

• J 

, 

, j 

, · , 

" , 

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I · , 

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Bioassay and Safety Assessment Lab, APM Code: R 1202130390A 

Acute toxicity test of to Chinese Rare Minnow 

(Gobiocypris rarus) Page 37 of 38 

0.08-

0.06 

;j! 0.04 

0.02 

O.OO'l,=;=~-,=;:==;,=;==;==;:==;,=;==;==;:==;,=:;=::;::::::;::=;,=:;=::;::=;::::::;,~:;:==;::=;:::::J, 0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Mnutes 

Figure 26 Chromatogram of sample0390A-L-BLANK-1-96h 

0.06 

;j! 0.04 

0.02-

O.Olol:, =;:=~~::::::;:, =;:=::;=:::;=::::::;:, =;:=::;=:::;=::::::;:, =;:::::::;:::=;:::::::;:, =;=::;=:::::;:::::::;:, =;=::;==;:==1, 

0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Mnutes 

Figure 27 Chromatogram of sample0390A-L-BLANK-4-C-96h 

0.06 

;j! 0.04 

0.02 

O.Ouo-t, =;=:!~::::;:, ::::;;:=:;:::::;:::::::;:, ::::;;:=:;:::::;:::::::;:, =;::::::;=::;::=;:, =~:;:=:::::;:::::::;:, =;=::;==;:~, 0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Minutes 

Figure 28 Chromatogram of sample 0390A-L-AC-1-96h 

- 
Bioassay and Safety Assessment Lab, APM Code: l. 

Acute toxicity test of to Chinese Rare Minnow . 

(Gobiocypris raros) Page 38 of 38 

0.08 

0.06-

~ 0.04 

0.02 

0.00111=:;=~~::::::::;1c=:;==;==;:=1c=:;==;==;:=1c=:;=::::;:::::;:=1;=:;=:::;::::=;:=,;=:;=:::;::=;:==j, 0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Mnutes 

Figure 29 Chromatogram of sample 0390A-L-AC-4-C-96h 

0.06 

0.04 

::::l '" N 

« ... 

ci 

0.02 

, I 

0.0 

I I I I I I I 

0.00 2.00 4.00 6.00 8.00 10.00 12.00 

Mnutes 

Figure 30 Chromatogram of sample 0390A-L-100mg/L-1-96h 

0.06 

... 0.04 :;;: 

::::l ci 

« 

0.02 0 

m 

'" 'j' 

0.00 

I I I I I I I 

0.00 2.00 4.00 6.00 8.00 10.00 12.00 

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Figure 31 Chromatogram of sample 0390A-L-100mg/L-4-C-96h 

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