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## Bioassay and Safety Assessment Lab SHANGHAI ACADEMY OF PUBLIC MEASUREMENT FINAL REPORT
Acute toxicitytest of to Chinese Rare Minnow 

### (Gobiocypris rarus)

Code: Study number of Dow:

Study director:

SPONSOR: 

### The Dow Chemical Company Midland, MI 48674 USA

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l': •.' Acute toxicity test of to Chinese Rare Minnow , (Gobiocypris rarus) Page 2 of 38 

### Contents
Title
.............................................................................................................................1 Contents

.....................................................................................................................2 Statement ofGLP Compliance

.................................................................................3 QualityAssurance 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 Monitor
......................................................................................................9 4 Study Director
.....................................................................................................9 5 Information ofthe Test Substance

....................................................................9 6 Date
......................................................................................................................9 7 Objective

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

...........................................................................................................10 9 Principle ofthe Test.
.........................................................................................10 10 Materials and Reagents
.................................................................................10 11 Apparatus
.......................................................................................................10 12 Species

...........................................................................................................10 13 Test Methods
..................................................................................................11 14 TestValidity
....................................................................................................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 ofthe Test Substance (provided by the sponsor)

...........................20 Certificate ofAnalysis ofthe Test Substance (provided by the sponsor)

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

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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 

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### The report fully and accurately reflects 

### the procedures used and data generated.

Bioassay and SafetyAssessment Lab, APM Date: J 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 QualityAssurance Unit (QAU) at Bioassay and SafetyAssessment 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 ofanalytical 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- 

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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 ofTesting facility. 2. Any unauthorized alteration ofthe test report would make the report invalid. 3. The test report only reflects the test substance received from sponsor. 4. The owner ofthe 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 SafetyAssessment Lab, APM 1500 Zhangheng Road, Zhangjiang Hi-tech Park, Shanghai, 201203, P.R. China

Prepared by:

Approved by: Bioassay and SafetyAssessment Lab, APM

(signed in original- 

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### Key Study Personnel

The following personnel were responsible for the conduct ofthe 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'1 i

, ! Bioassay and Safety Assessment Lab, APM Code: 'l' 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 oftest 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 ofthe pre-test, a nominal concentration of 100 mg/L 

### ofthe 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 ;, J



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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 ofthe 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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. i**,~** Bioassay and Safety Assessment Lab, APM Code: Acute toxicity test of to Chinese Rare Minnow (Gobiocypris raros) Page 9 of 38 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 SafetyAssessment Laboratory, 1500 Zhangheng Road, Zhangjiang Hi-tech Park, Shanghai, 201203, P.R. China Tel: 86-21-50798123 Fax: 86-21-50798125 

### 5 Information ofthe Test Substance Name: Code: Receipt date: 2012-02-13 Email: yangj@apm.sh.cn

Conditions ofstorage: Store in dark at room temperature Full details oftesting 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 2012-09-18Bioassay 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 ofthe 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 ofthe Test The fish are exposed to the test substance solution preferably for a period of96 hours. Mortalities are recorded at 24, 48, 72 and 96 hours and the concentrations which kills 50 percent ofthe 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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## U Bioassay and Safety Assessment Lab, APM Code: Acute toxicity test of to Chinese Rare Minnow (Gobiocypris rarus) Page 11 of 38 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% ofair saturation value (ASV); Photoperiod: LightDark

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## I ! No diseases were observed during the acclimation period; No mortality was present within the 7 days before the start ofthe test. The Lengths and weights for fish used in the test and measured at the end ofthe test were 2.3 

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### 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% ofASV. 13.1.2 Test vessels 5L glass tanks were used containing 4L oftest solution. 13.1.3 Conditions ofexposure Duration: 96h;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 oftest substance with acetone (concentration of0.1 ml acetone/l) directly to 4 litres oftest 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 fortesting. 13.2.2 Pre-test One nominal concentration of 100 mg/l was used in the pre-test. In this test, a blank control oftest water and a solvent control ofacetone (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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### initiation and mortalities were recorded. Measurements of the pH, dissolved oxygen concentration and temperature in the test solutions were made atthe beginning and at the end of the test. The contents of the test solution were measured at Oh, 48h and 96h ofthe test. For determination oftest substance, six samples oftest 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 ofthe limit test were expressed as both nominal and measured geometric mean contents ofthe test solutions. 

### 14 TestValidity 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% ofthe 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 ofdetection (LOD) for the test substance using this method was calculated to be 5.00 IJg/L (based on a signal/noise of4.23). It was found thatthe 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 notBioassay 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 oftest 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 ofthe test sUbstance in water is slight and 

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### dispersion ofthe insoluble fractions was non-homogeneous. The contents of the test substance in test solutions were between 129 - 1144 J,Jg/L, with a measured 

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# l min), the contents ofthe 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 LCsofor 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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### 17 Reference 

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### OECD SERIES ON TESTING AND ASSESSMENT Number 23. GUIDANCE DOCUMENT ON AQUATIC TOXICITYTESTING OF DIFFICULT SUBSTANCESAND MIXTURES 2000. 

### 18 Archives The signed copies ofthe 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 ofthe 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 Measurementforfive years, and returned to the sponsor at designated address on agreed date. 

### 19 Departure from Protocol None.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 ofthe 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 

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SD 

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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, '1 

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### (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. 

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### - 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 noneBioassay 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 ofthe 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 

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SD 

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### - - - SD 

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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 

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SD 

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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

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### 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.

C' I 

# LBioassay and Safety Assessment Lab, APM Code: Acute toxicity test of to Chinese Rare Minnow (Gobiocypris rarus) Page 20 of 38 Information ofthe Test Substance (provided by the sponsor) Chemical name

Other name

Molecularformula 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 ofstorage 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 ofAnalysis ofthe Test Substance (provided by the sponsor)

# AnaClRDFJC6TEOfM'AAYSISfOBTUTIRIRBINCI'It9NTROL SUU'rANCq 1Tl'LEIOBJECTIVE: Characterizationof Lot forUse as aTest Substance TESTIREFERENCEfCONTROL SUBSTANCE: DESCRImON: CbemiCIIName:

CASNo.: STUDYINITIATION DATE: 01 Pebnwy2012 METHODS USED: MOLECULAR fORMULA MOL.ECUl.AJl WEIGHT:

PURITY: auchromatopaphywithflame ionizallOD detection (GC-FID) aDd KarlFilChertitratioll, MLAL-2011-0201182

IDBNTIF1CAnON: Fauriel'Tranlfonn-Infiarcd(Fr.m)IpcdroKOPY andauehromatoaraPhy·musIPCcttomctty (GC.MSl RESULTS AND CONCLUSIONS: Structural COnfirmatiOll of tile tcItmaterial wqperfOrmed byFT·IRandGC-MS. Ani_Percent: x(GC-FID) Intcmal Standard: N/A Other(explain): KarlFischer1itmion PURITY DETERMINATION Result: (95% CODfide.D.cc intaval)aftercorrcctin. forthe coulomctric Karl Fisehcrwatcr

....Iu. cmtBR.(SpocitY): 204

### = 13 ppm(95% coofidenceiolcrVal)walcrbyKarlFischertitratiOlL CALCULATIONS: Area Permrt; x(GC-FID) Internal Standard: NfA External Standard: NlA Othcr(explain): KadFillChcrtitration AUorisinalraw dataUIOclatodwith 1his study\VCRarchivedLathe SPOIllOff'acilityan:bive. OnlydelcriptiveItItiAticawere used unl_otberwiJcnoted inthereaalti. Thi$Jtud:ywas coMuctod in ICCOrdaDce withthe GoodubomoryPracticeStaDderd,40 CPR.Part792 and40 CPRPart 160, adtheJteaistratioll, EvaIUltioll, Autbori:rationand RescrlctiOll. ofChcmicaIJ (REACH)RcauhtioD wellotbc:rwiM DOled. Due to the montumaatwaoflhc study,tlJe stabilityof thetest.ublbulceUlldcl'Stoftge conditiOJllper40 CIRPart792.105(c) atthe testlitewunotverified.



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....... til STUDY DIRECfORANDTEST FACILITYADDRESS: Impa!:tAnalytical1910W. St. AnIiMws Rei. Midland,Ml48640 lA Project 2 ofl4pages lAReport SponsorNo, DOWCONFIDENTIAL**~**ReceIved from. third pllty IOtII'CIBioassay 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 ofthe test substance during the test process.. 2 Materials and methods 

### 2.1 Test samples The test samples were taken from test water offish 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 oftest substance 0.0100 g sample oftest 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. 

### 2.2.5Preparation of samples 0.5 ml ofthe sample were added to a 2ml vial and diluted with 0.5ml acetonitrile. Ifthe 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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# c 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 ofthe 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 oftest 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 ofthe 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 ofthe test substance in the sample; X= amount ofthe test substance calculated by equation 1;

F= factor (dilution factor or concentration factor). The recovery rate was calculated by equation (3): R= 

### CI Cnom

### x100% (3) 

### Where: R= recovery rate (%); C=concentration ofthe test substance in the sample calculated by equation 2; Cnom

### = nominal concentration ofthe 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 ofthe calibration curve used was 0.9993. Fig.2 gives the chromatogram of the solvent. Typical chromatograms ofsamples are shown in Figure 3 and Figure 4. The limit ofdetection (LOD) forthe test substance in test medium was calculated to be 5.00 1J9/L (based on a signall noise of4.23) showing in Figure 5. Before sample analysis, the recovery ofthe 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 '"l 

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Table 1 Calibration curve data oftest substance Concentration PeakArea (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 ofspiked 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 offish 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 

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### 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 

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

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## 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 

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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 

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### 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 

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### 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 NDBioassay 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 

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## ND 2 

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# I (Gobiocypris rarus) Page 32 of 38
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