THE DOW CHEMICAL COMPANY STUDY ID: PAGE 1 OF 43

- 

### STUDY TITLE

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER C LADOCERAN, Daphnia magna

Test Guidelines USEPA FIFRA, 72 -2, EPA 540/9-82-020 (1982) OECD, 202 (2004) EEC Directive 92/69/EEC, Part C.2 (1992)

USEPA TSCA, 40 CFR 7 97.1300 (1992)
Author(s)

Study Completion Date 22 December 2011


### Sponsor The Dow Chemical Company

Midland, Michigan 48674
Performing Laboratory Toxicology & Environmental Research and Consulting The Dow Chemical Company

Midland, Michigan 48674
Laboratory Project Study IDTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 2



### COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS Compound:
Title: AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia magna
All phases of this study were conducted in compliance with the following Good Laboratory Practice Standards: Organisation for Economic Co-Operation and Development (OECD)

-- OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, Number 1. OECD

Principles on Good Laboratory Practice (as revised in 1997) ENVIMC/CHEM(98) 17. US Environmental Protection Agency

-- TSCA GLPs Title 40 CFR, Part 792

- Toxic

Substances Control Act TSCA); Good Laboratory Practice Standards, Final Rule.

Study Director Manager Toxicology & 

### Environmental Research and Consulting

-THE DOW CHEMICAL COMPANY STUDY ID: 11 J 130 PAGE 3



### QUALITY ASSURANCE STATEMENT Compound:
Title: AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia magna
This study was examined for conformance with Good Laboratory Practices as published by the USEPA TSCA and OECD. The final report was determined to be an accurate reflection of the data obtained. The dates of Quality Assurance activities on this study
are listed below. Study Initiation Date: 16 June 2011

TYPE OF AUDIT: 

### Final protocol Study conduct

Protocol, data, and draft report DATE OF AUDIT: 

### 16 June 2011 10August 2011- 01 September 2011 07-10 November 2011 DATE FINDINGS REPORTED TO STUDY DIRECTOR/MANAGEMENT: 

### 16 June 2011 21 September 2011

19 December 2011

Final report The date of the signature below is the date of the final report audit.
The final report accurately reflects the raw data of the study.

Quality Assurance Toxicology & 

### Environmental Research and Consulting The Dow Chemical Company 1803 Building Midland, Michigan 48674

### SIGNATURE PAGE Compound: THE DOW CHEMICAL COMPANY STUDY ID: PAGE 4



### Title: AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia magna



### Lead Scientist/Study Director

Study Director In-Training

Analytical Chemist

Reviewed by:

(Date)

USEPA TSCA 8(e)/FIFRA 6(a)(2) ASSESSMENT This test material has been reviewed for possible TSCA 8(e)/FIFRA 6(a)(2) submission \- 

## 3-l 

## L InitialslDate

-THE DOW CHEMICAL COMPANY STUDY ID: PAGE 5



### TABLE OF CONTENTS Page

- 

### STUDY TITLE ....................................................................................................................1 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ..................2 QUALITY ASSURANCE STATEMENT ..........................................................................3 SIGNATURE PAGE ...........................................................................................................4 SUMMARY .........................................................................................................................7 INTRODUCTION ...............................................................................................................8 Purpose ............................................................................................................................8 Test Guidelines ................................................................................................................8 Quality Assurance ...........................................................................................................8 Archiving ........................................................................................................................8 Safety

............................................................................................................................... 9 MATERIALS AND METHODS .........................................................................................9 Test Material ...................................................................................................................9 Culture and Test Water ...................................................................................................9 Test Organism .................................................................................................................9 Test Vessels ...................................................................................................................10 Selection of Test Concentration ....................................................................................10 Preparation of Test Solution .........................................................................................10 Definitive Test ...............................................................................................................11 Analytical Measurements ..............................................................................................12 Statisti cal Analysis ........................................................................................................12 RESULTS AND DISCUSSION ........................................................................................12 Analytical Results .........................................................................................................12 Test Conditions .............................................................................................................13 Biological Data ..............................................................................................................13 CONCLUSIONS ................................................................................................................13 ACCEPTANCE CRITERIA ..............................................................................................14 REFERENCES ..................................................................................................................15 TABLES ............................................................................................................................16 TABLE 1. Test Material Information ..........................................................................16 TABLE 2. Summary of Results from Analysis of Test Solutions for ................17 TABLE 3. Summary of Water Quality and Test Conditions .......................................18 TABLE 4. Summary of Biological Observations and Results .....................................19 APPENDICES ...................................................................................................................20 APPENDIX A. Certificate of Analysis ........................................................................20THE DOW CHEMICAL COMPANY STUDY ID: PAGE 6



### TABLE OF CONTENTS Page

- 

### APPENDIX B. Analytical Report ................................................................................21 APPENDIX C. Individual Dissolved Oxygen Measurements .....................................40 APPENDIX D. Individual pH Measurements .............................................................41 APPENDIX E. Individual Temperature Measurements ..............................................42 APPENDIX F. Individual Biological Observations by Replicate ...............................43THE DOW CHEMICAL COMPANY STUDY ID: PAGE 7

- 

### SUMMARY

TITLE: 

### AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia magna STUDY NUMBER: TEST SUBSTANCE: ( Lot No. Batch Identification No. purity of

TEST DATES: 30 August to 1 September 2011 TEST ORGANISM: Daphnia magna (< 24

- hours old neonates) from in

- house cultures. TEST CONDITIONS: Exposure Scenario: 48-hour (static ) Temperature Range: 20 -21 °C pH Range: 7.3-7.6 Dissolved Oxygen Range: 8.3 mg/L –9.2 mg/L ( 95– 106 % saturation) Light Intensity Range: 1850 –215 0 lux Photoperiod: 16 hour light/8 hour dark DILUTION WATER: Laboratory Dilution Water ( LDW) pH Range: 7.3 Alkalinity: 42 mg CaCO 3 

### /L Hardness: 56 mg CaCO 3 

### /L Conductivity: 175 **μ** mhos/cm NOMINAL TEST CONCENTRATIONS: 0 ( water control), 6.25, 12.5, 25.0, 50.0 and 100 mg L

MEASURED TEST CONCENTRATIONS: < LLQ (water control), 0.000923, 0.00379, 0.0163, 0.0394, and 0.130 mg L RESULTS ( Measured ): 24 and 48 hour EC 50 

### = > 0.130 mg L 24 and 48 hour NOEC = 0.130 mg L RESULTS ( Nominal): 24 and 48 hour EC 50 

### = > 100 mg L 24 and 48 hour NOEC = 100 mg LTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 8

- 

### INTRODUCTION Purpose The purpose of this study was to evaluate the potential acute toxicity of ( to the freshwater cladoceran, Daphnia magna, over a 48-hour static exposure period. Due to the limited solubility of this test substance in aqueous solution, this study was conducted as a limit test with daphn ids exposed to a water soluble fraction ( WS F) preparation of (OECD, 200 0). The data collected will be used to calculate the 24- and 48-hour EC 50 

### values (the concentration estimated to immobilize 50 percent ofthe daphnids after 24 and 48 hours of exposure, respectively) and a 48-hour no-observedeffect concentration (NOEC).
Test Guidelines USEPA U.S. Environmental Protection Agency (1985). Hazard Evaluation Division: Standard Evaluation Procedure, Acute Toxicity Test for Freshwater Invertebrates. EPA -540/9-85-005. Washington D.C. U.S. Environmental Protection Agency (1982). Pesticide Assessment Guidelines, Subdivision E Hazard Evaluation: Wildlife and Aquatic Organisms, Guidelines 72-2. EPA 540/9-82-020, Washington, D.C. OECD Organisation for Economic Cooperation and Development (2004). OECD Guidelines for Testing of Chemicals, No. 202, Daphnia sp., Acute Immobilization Test. Adopted 13 April 2004. EC Official Journal of the European Communities (1992). Annex to Commission Directive 92/69/EEC, C.2 Acute Toxicity for Daphnia , Vol. 35, 29 December 1992. TSCA U.S. Environmental Protection Agency (1992). Toxic Substances Control Act Test Guidelines . 40 CFR 797.1300, Daphnid Acute Toxicity Test. Washington, D.C. 07/01/92 Edition. Quality Assurance The study conduct, data, protocol, protocol changes/revisions, and final report were inspected by the Quality Assurance Unit, Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Archiving The data, protocol, protocol changes/revisions, and final report were archived by the Toxicology & Environmental Research and Consulting archivist and stored at The Dow Chemical Company, Midland, Michigan.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 9

- 

### Safety Health and safety exposure data on the test material were reviewed prior to initiation of the study and appropriate safety precautions followed.
MATERIALS AND METHODS
Test Material The test material, ( was received on 22 March 2010 (Lot # Batch and stored under ambient conditions until use. The total purity

was as determined by gas chromatography with flame ionization dete Detailed information on the test chemical is presented in Table 1 , and the certificate analysis for the lot used in the study is presented in Appendix A

. Culture and Test Water The water used for daphnid testing and culturing (referred to as lab oratory dilution water or LDW) was Lake Huron water supplied to The Dow Chemical Company (TERC Laboratory) by the City of Midland Wate r Treatment Plant. The water was obtained from the upper Saginaw Bay of Lake Huron off Whitestone Point and was limed and flocculated with ferric chloride. The water was pumped to the laboratory prior to municipal treatment for human consumption. Before use in the laboratory, the water was sand

- filtered, pH -adjusted with gaseous CO 2 

### , carbon -filtered, and UV- irradiated. The water is typically monitored weekly for pH, alkalinity, hardness and conductivity. Periodically, the water is monitored for total organic carbon (TOC), total suspended solids (TSS), and selected inorganic and organic compounds. Test Organism The test organism was the freshwater daphnid, Daphnia magna , from in -house cultures initially obtained from Aquatic BioSystems, Inc., Fort Collins, Colorado. This species is widely accepted and recommended for toxicity testing by the test guidelines. Daphnid instars less than 24-hours old from a laboratory- rea red culture were used as the test organisms. The day before instars were needed for testing, culture vessels with daphnids, which have had at least three broods, were removed from the culturing incubator. The instars were separated from adults < 24 hours prior to test initiation by pipetting them from the cultureTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 10

- 

### vessel into a 1 -L beaker containing LDW. The instars were held in the incubator used for testing until test initiation.
Test Vessels
The test vessels were 30 mL glass beakers each containing approximately 2 0 mL of control or test solution and covered to reduce evaporation. Each test vessel was uniquely labeled for identification purposes.
Selection of Test Concentration The test concentrations selected for evaluating the effects of on Dap hnia magna were based on the results of a rangefinding test , which was set as a limit test . A 48-hour static probe test was conducted from 0 9 Aug ust 2011 to 11 August 2011. Three replicates of ten daphnids per test level was exposed to nominal test conce ntrations of 0 (water control) and 100 mg L. Following 48-hours of exposur e, immobility was observed in 17 % of the daphnids at the 100 mg L test level. No immobility was observed in the water control during the 48-hour test. Based on this preliminary information, the following target (nominal) concentrations for the definitive test were set at 0 (water control) 6.25, 12.5, 25.0, 50.0, and 100 mg L. Preparation of Test Solution Prior to testing, it was determined that the solubility of in LDW was low, approximately 0.04 mg L. Based on this low solubility, it was decide d to expose daphnids to a water soluble fraction (WSF), as outlined in the OECD guidance document on difficult- to -test substances (OECD, 2000 ). During the rangefinder, it was noted that particulate matter was suspended in the water column after mixing at 100 mg/L. To reduce the likelihood of physical toxicity to the daphnids, a trial test solution was filtered with a 0.2 μm nylon filter (Nalgene M75 Series Filter Unit) , then analyzed for content. Bec ause f iltration removed all of the ( i.e ., < LLQ, or < 0.000507 mg/L), the decision was made to centrifuge the stock solu tions prior to test initiation. Centrifugation separated most of the particulate matter, allowing the dissolved portion of the test material ( i.e ., the water soluble fraction) to remain. The bulk test solution was prepared as a WS F at the highest test nominal concentration 10 0 mg L . The test material was weighed out (exact weight = 0.1000 g) directlyTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 11

- 

### into a tared scintillation vial. A volumetric flask was filled with 1 L of LDW, which was used to thoroughly rinse the contents of the scintillation vial into a 2- L Erlenmeyer flask. The water control was prepared b y pouring 1 L of LDW into a 2000- mL Erlenmeyer flask. Both flasks were covered with parafilm and placed on separate stir plates set to stir at approximately 350 rpm. After approximately 24 hours of stirring, ~250 mL of test solution was added to 250- mL p lastic bottles to be centrifuged (4 replicates of control and 2 replicates of the nominal 100 mg L). The vials were centrifuged at ~10,000 rpm for approximately 40 minutes. After centrifuging, the control solution was clear and colorless; the nominal 100 mg L was clear and colorless with some small white particulate suspended. The high-dose stock solution was diluted with LDW to create subsequent stock solutions for all lower doses in 250- mL glass beakers at the following nominal test concentrations: 50.0, 25.0, 12.5 and 6.25 mg L. All test solutions were clear and colorless

. Definitive Test The definitive test was conducted under static conditions for 48 hours from 30 August to 1 September 2011. Four replicate test vessels, each containing approximately 20 mL of the appropriate test solution, were prepared per test level. Twenty daphnids (five daphnids per replicate) were impartially added to replicate test vessel s within 30 minutes of sampling for analytical verification of test solution concentrations. Test vessels were placed in a walk -in environmental chamber according to a computer- generated randomization. The target test temperature was 20 ± 2°C. The photoperiod was set at 16 hours light: 8 hours dark with a target light intensity of 2050 ± 350 lux. Daphnids were not fed during the test. Daphnids were observed for immobility (inability to swim within 15 seconds after gentle agitation of the test vessel) at approximately 24 and 48 hours of exposure. Any sublethal effects (abnormal behavior or appearance) were recorded. Dissolved oxygen, pH, and te mperature data were recorded from each bulk test solution at test initiation and from all test vessels (spent test solutions) at test termination. Hardness, alkalinity and conductivity were measured from the bulk control and the 100 mg L stocks at te st initiation. Water temperature was continuously monitored with a minimum/maximum thermometer placed in a representative vessel. Light intensity was measured at test initiation with a light intensity meter at each position where replicate vessels were placed.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 12

- 

### Analytical Measurements Samples were collected from each bulk test solution at initiation (0 h). At termination (48 h) replicates from each test level were combined and samples were taken from the composite solution. To assess analytical method precision and solution homogeneity, three additiona l samples were collected at initiation from the lowest and highest concentration bulk treatment solutions. concentrations were then confirmed using liquid chromatography with positive ion electrospray ionization and tandem mass spectrometry detection operat ing in the multiple reaction mo nitoring mode (SPLC/ESIMS/MS). For a full description of the analytical sampling procedure and method refer to Appendix B

. Statistical Analysis An attempt was made to calculate 48 -hour EC 50 

### values (the concentration estimated to immobilize 50 percent of the daphnids after 48 hours of exposure) and corresponding 95% confidence intervals. Because only a single daphnid died during exposure, no data analyses were possible for EC 50 

### endpoints. Th e 48-hour NOEC was determined using a Fisher’s exact one

- tailed test, which indicated the highest concentration tested exhibiting daphnid immobility or sublethal effects was not significantly different from that of the controls.
RESULTS AND DISCUSSION Analytical Results Detailed results from the analytical confirmation of the test solutions, analytical method precision, solution homogeneity, and linearity, as well as supporting tables an d figures, can be found in Appendix B

. A summary of the results obtained from the analyses of test solutions is presented in Table 2

. Measured concentrations ranged from 0.00747 to 0.0235 % of nominal on day 0 and 0.0171 and 0.243% of nominal at 48 hours. The resulting mean measured concentrations, calculated for each treatment le vel by averaging the 0 and 48 hour measured concentrations, were 0.000923, 0.00379, 0.0163, 0.0394, and 0.130 mg L (corresponding to nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100 mg L, respectively ). None of the analyses of the controls exhibited a peak eluting at the retention time for at a concentration exceeding the lowest level quantified (LLQ) equivalent to 0.000591 mg L.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 13

- 

### No decrease in the recovery of test material was observed over the course of the study; therefore, the test material was assumed to be stable. Test Conditions
Test condition data are summarized in Table 3 and individual measurements for dissolved oxygen, pH, and temperature are reported in Appendices C , D , and E , respectively. Dissolved oxygen levels ranged from 8.3-9.2 mg/L ( 95-106% oxygen saturation), temperatures ranged from 20-21°C, and pH ranged from 7.3-7.6. Light intensity ranged from 1850-2150 lux. Biological Data All biological results are expressed in terms of mean measured concentrations of Summary results of the biological observations are presented in Table 4 and individual observations by replicate are presented in Appendix F . Following 24 hours of exposure, only two control daphnids were immobile. At test termination (48 -hours of exposure), immobility was observed in 0, 0, 0, 0, and 5%, of the daphnids in the 0.000923, 0.00379, 0.0163, 0.0394, and 0.130 mg L test levels, respectively. In the control treatment level, 10% immobility (mortality ) was seen at 24 and 48 hours of exposure. Other than the above reported immobility, no changes in daphnid behavior or appearance were observed at any test level. Although it was no t possible to analyze these data to calculate

an EC 50 

###  ̧ because only a single daphnid died at the high est concentration, it can reliably be reported that the 24- and 48-hour EC 50 

### s are greater than the highest concentration tested ( i.e., > 100 mg L, based on nominal concentrations) . A Fisher’s one- tailed test showed the NOEC to be 100 mg L.
CONCLUSIONS The acute toxicity values for Daphnia magna exposed to over a 48- hour static exposure period and based on both nominal and mean measured concentrations were as follows:

**•** Based on nominal concentrations, t he 24- and 48-hour EC 50 

### s were greater than the highest concentration tested ( i.e ., >100 mg L). The 24- and 48-hour EC 50 

### s based on mean measured concentration s were > 0. 130 mg L.

**•** Based on nominal concentrations, the 48-hour NOEC was 100 mg L. The NOEC based on mean measured concentrations was 0.130 mg L.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 14

- 

### Thus, it can be concluded that at the limit of solubility ( i.e ,. the concentration of bioavailable test material), is no t acutely toxic to Daphnia magna

. ACCEPTANCE CRITERIA The following acceptance criteria were met and the test is, therefore, considered valid :

**•** Less than 10% of the control animals were immobilized or showed signs of stres s
or disease ( e.g. , discoloration or being trapped at the surface of the water)

**•** Dissolved oxygen concentrations were maintained at ≥ 3 mg/L in the control and test vessels throughout the studyTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 15

- 

### REFERENCES OECD (Organization for Economi c Co -operation and Development) (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Series on Testing and Assessment (No. 23). ENF/JV/MONO(2000)6.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 16

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia mag na

- T ABLE 1. Test Material Information



### Test Material Common Name

Chemical Name

CAS number
Molecular Formula Molecular Weight Supplier The Dow Chemical Company, Midland, Michigan (Lot # Batch Purity/Characterization The p urity of the test material was determined to be area by gas chromatography with identification by Fourier transform infrared spectroscopy and gas chromatography mass spectrometry Solubility 0.04 mg L

Chemical StructureTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 17

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia mag na

- T ABLE 2 . Summary of Results from Analysis of Test Solutions for



### 1111THE DOW CHEMICAL COMPANY STUDY ID: PAGE 18

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia mag na

- T ABLE 3 . Summary of Water Quality and Test Conditions



### Test Conditions : Light Intensity range (lux) 1850-2150 Temperature range (°C): 20-21 pH range 7.3-7.6 Dissolved oxygen range (mg/L) 8.3-9.2 Percent oxygen saturation range (%) 95-106 Alkalinity a 

### (mg/L CaCO 3 

### ) 42, 74 Hardness a 

### (mg/L CaCO 3 

### ) 56, 60 Conductivity a 

### (μmhos/cm) 175, 158

a 

### Alkalinity, Hardness, and Conductivity measurements are listed as: control, highest treatment level (0.130 mg L)THE DOW CHEMICAL COMPANY STUDY ID: PAGE 19

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia mag na

- T ABLE 4 . Summary of Biological Observations and Results

Mean Measured Concentrations (mg L) Observations
24

- Hour 48

- Hour
Water Control (<LLQ a ) 18 N , 2I 18N, 2I 0.000923 20N 20N 0.00379 20N 20N 0.0163 20N 20N 0.0394 20N 20N 0.130 20N 19N, 1 I N = Normal, I = Immobile

Mean Measured Concentration (mg L) (Nominal Concentration) 24

- Hour 48

- Hour

EC 50 >0.130 (> 100) >0.130 (> 100) 48

- Hour NOEC b 100

a Lowest Level quantified = 0.000591 mg L

b The 48

- hour NOEC was determined based on the highest nominal concentration tested exhibiting no

statistically significant immobility or change in behavior or appearance.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 20

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia mag na

- APPENDIX A. Certificate of AnalysisTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 21

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia mag na

- APPENDIX B . Analytical Report

# •

• Report T i t l e : Toxicology Infomlation Collection (TJq 

### Report The Dow Chemical Company

Do o n fi rl11. & Homog e neity ( 

### A u thor(s) Julia A. 

### F iting Nathan A. 

### Malowinsk

No ac tion r e quir e d 

### Geog r aRh i c Loca ti o n D e p a l tme nt
N. A m e r i ca TE R 

### Study number: Page Co unt 

### 18 D a t aboo k N um bers



### n

/

a 

### lD

Study Title: 

### AN ACUTE TOXICITY STUDY WITH THE FRESH WATERCLAOOCERAN, Daphnia magna File number: Project#: Task/rep: 

### 349-1 Project **I** 

### Sample Description (optional):

Test material(s): Test 

### material lot #: Report recipient(s): Analysis request date: Date submittor prepared sa m ples: Number of samples or estimate: Date samples prepared for analysis: Analysis date: Numberofsamples analyzed: Analysis method: Method reference:

TH E DOW CH E MICAL COMPANY 

### LOW

9 /

8/2011 8

/

30 / 2011 

### 6 08 / 30

- OYl02 / 20 

### 11 08/31

- 09/02/2011 

### 6 HPLC / ESI- MSIMS
see below

PAGE 1

# •

•

• INTRODUCTION

This report summarizes the analytical support provided for study The study was conducted to evaluate the acute toxicity effects of ( to the fresh water cladoceran, Daphnia magna. The test solutions were set at target concentrations of 0 (control lab dilution water), 6.25, 12.5,25,50 and 100 mg L 

### lab dilution water (LDW) in a static exposure scenario. The test solution concentrations were confirmed at 0 and 48-hours of the study. The samples were diluted with LDW as needed and then analyzed for using liquid chromatography with positive ion electrospray ionization and tandem mass spectrometry detection operating in the multiple reaction monitoring mode (HPLCIESI-MSIMS).

EXPERIMENTAL

Test Material! Analytical Standard
Chemical Name: Synonyms:
Lot Number:
CAS Number:
Molecular Formula:
Molecular Weight: Purity:

Internal Standard (IS)
Chemical Name: Synonym:
Identification:
Molecular Formula: [Standards were not corrected for purity]

Nominal Concentration:
Molecular Weight:

THE DOW CHEMICAL COMPANY PAGE 2

### The internal standard was used tolacili/all! quantitaliol1 

### during mass spectral ana(ysis.

Solubilitv of ('1)13 in 

### tD\\, and Algal 

### (Jrowlh I'vledia 

### (AAP)

Prior to 

### study initiation. mixtures of 100 mg 

### L. in both LDW and AAP matrices \verc prepared and analyzed in a similar manner as described below to determine the solubility of C'DB 

### in LDW and 

### /\J\p.rhis \\<1S 

### performed for informational purposes to assist in study design only and the results, preparation and analysis details are not provided in tbis report, but. can be H)Lll1d in the study file. Initial results indicated the solubility ofCDB in AAP was approximately (1.004 ppm (mg'L) and 0.04 ppm in LD"V. Since test material was being added \velll.loove the solubility, centrifuging or filtering of the test solution was needed to

remove test material parlieulak's. Additional mixtures of 100 mg CDBiL in both LDW and AAP matrices were prepared and the concentrations were determined after centrifuging or filtering the test mixtures. Centrifuging resulted in a concentration of approximately 0.003
ppm in AAP and 0.07 

### ppn1 in LD\V. The tiltered samples were both lower than the lowest standard analyzed of 0.000507 ppm, indicating that filtering was also removing test material in solution. It \vas determined that the test solutions should be centrifuged prior to study in itiation.

Sample Collection 

### & 

### Preparation Samples were collected from the bulk dose solutions at study intitiation (O-hour) and the pooled test solutions at study termination (48-hours) 

### tt)!· analytical contirmation. Aliquots (3 mL) rronl each solution were transferred with disposable Eppendorr pipettes to glass vials. The samples were prepared in autosampler vials for analysis as follows:

• LD\V control samples:

<) 0 and 48-hour samples: lmL sample ..L 25**~lL** internal standard (No Dilution)



### • 6.25 andl mg/L (ppm) sarnplcs:

<) 0 and '+8-hour samples: I mL smnple 25 pI, internal standard (No Dilution)



### • 25 and 50 mglL (pprn) 

### samples: (i-hour samples: 1 mL sample 25 p L internal standard (No Dilution) 4g-hour samples: 0.100 mL sample 0.900 mL LDW + 

### 15 pL internal 

### standard (Dil 

### ution Fa;.::tor == 

### 10)THE DOW CHEMICAL COMPANY STUDY ID: PAGE 40

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia mag na

- APPENDIX C . Individual Dissolved Oxygen Measurements

Individual Dissolved Oxygen (D.O) (mg/L) measurements Mean Measured Concentration of (mg/L) Vessel Number D.O. (mg/L) 0 hr a 48 hr

Water Control (<LLQ b ) C-A
8.8 8.8 C

- B 8.8 C

- C 8.7 C

- D 8.8

0.000923 1-A
8.9 8.8 1-B 8.7 1-C 8.8 1

- D 8.7

0.00379 2-A
9.1 8.9 2-B 8.8 2-C 8.9 2

- D 8.8

0.0163 3

- A
8.4 8.4 3

- B 8.4 3-C 8.3 3-D 8.4

0.0394 4

- A
8.7 8.6 4

- B 8.6 4-C 8.6 4-D 8.6

0.130 5

- A
9.2 9.1 5

- B 9.1 5

- C 9.1 5-D 8.9 Min 8.4 8.3 Max 9.2 9.1

a 0 hour measurements taken from bulk test solutions. b Lowest level quantified= 0.000591 mg/LTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 41

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia mag na

- APPENDIX D

. Individual pH Measurements

Individual pH measurements Mean Measured Concentration of (mg/L) Vessel Number pH
0 hr a 48 hr

Water Control (<LLQ b ) C

- A
7.3 7.3 C

- B 7.3 C

- C 7.3 C-D 7.3

0.000923 1

- A
7.4 7.3 1

- B 7.4 1

- C 7.4 1-D 7.4

0.00379 2

- A
7.4 7.4 2

- B 7.4 2

- C 7.4 2-D 7.4

0.0163 3

- A
7.4 7.5 3

- B 7.5 3

- C 7.5 3-D 7.5

0.0394 4-A
7.4 7.5 4-B 7.5 4

- C 7.5 4

- D 7.5

0.130 5-A
7.4 7.5 5-B 7.5 5

- C 7.6 5-D 7.6 Min 7.3 7.3 Max 7.4 7.6

a 0 hour measurements taken from bulk test solutions. b Lowest level quantified = 0.000591mg LTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 42

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia mag na

- APPENDIX E . Individual Temperature Measurements

Individual Temperature (°C) M easurements Mean Measured Concentration of (mg/L) Vessel Number Temperature a (°C) 0 hr b 48 hr

Water Control (<LLQ c ) C

- A
20.4 20.5 C

- B 20.7 C

- C 20.7 C-D 20.7

0.000923 1

- A
20.4 20.7 1

- B 20.8 1

- C 20.7 1-D 20.7

0.00379 2

- A
20.4 20.7 2

- B 20.8 2

- C 20.8 2-D 20.8

0.0163 3

- A
20.4 20.8 3

- B 20.8 3

- C 20.8 3-D 20.7

0.0394 4-A
20.5 20.6 4-B 20.6 4

- C 20.7 4

- D 20.7

0.130 5-A
20.5 20.8 5-B 20.8 5

- C 20.8 5

- D 20.8 Min 20.4 20.5 Max 20.5 20.8

a Temperatures from the continuous monitoring of a surrogate vessel during the ex posure period ranged from 19.6

- 21.0 ° C. b 0 hour measurements taken from bulk test solutions.

c Lowest level quantified = 0.000591mg LTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 43

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER CLADOCERAN, Daphnia mag na

- APPENDIX F . Individual Biological Observations by Replicate
Observed Immobility
Mean Measured Concentration of (mg/L) Vessel Number Observation Period 24 hr 48 hr Normal Immobile Normal Immobile

Water Control (<LLQ a ) C

- A 5 0 5 0 C

- B 5 0 5 0 C-C 3 2 3 2 C-D 5 0 5 0 Total 18 2 18 2

0.000923 1-A 5 0 5 0 1-B 5 0 5 0 1

- C 5 0 5 0 1-D 5 0 5 0 Total 20 0 20 0

0.00379 2-A 5 0 5 0 2

- B 5 0 5 0 2

- C 5 0 5 0 2-D 5 0 5 0 Total 20 0 20 0

0.0163 3

- A 5 0 5 0 3

- B 5 0 5 0 3

- C 5 0 5 0 3

- D 5 0 5 0 Total 20 0 20 0

0.0394 4

- A 5 0 5 0 4

- B 5 0 5 0 4-C 5 0 5 0 4-D 5 0 5 0 Total 20 0 20 0

0.130 5-A 5 0 5 0 5-B 5 0 4 1 5-C 5 0 5 0 5-D 5 0 5 0 Total 20 0 19 1

a Lowest level quantified = 0.000591mg L