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STUDY TITLE 

AN ACUTE TOXICITY STUDY WITH THE FRESH 

WATER CLADOCERAN, 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 797.1300 (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 ID 
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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 

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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 

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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

Statistical 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 ........................................................................20
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TABLE OF CONTENTS

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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 ...............................43
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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: 1850–2150 lux

Photoperiod: 16 hour light/8 hour dark 

DILUTION WATER: Laboratory Dilution Water (LDW) 

pH Range: 7.3 

Alkalinity: 42 mg CaCO3/L

Hardness: 56 mg CaCO3/L

Conductivity: 175 μmhos/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 EC50 = > 0.130 mg L 

24 and 48 hour NOEC = 0.130 mg L 

RESULTS (Nominal): 24 and 48 hour EC50 = > 100 mg L 

24 and 48 hour NOEC = 100 mg L

 
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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 daphnids exposed to a water soluble fraction 

(WSF) preparation of (OECD, 2000). The data collected will be used to calculate 

the 24- and 48-hour EC50 values (the concentration estimated to immobilize 50 percent of 

the 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. 
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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 laboratory dilution water 

or LDW) was Lake Huron water supplied to The Dow Chemical Company (TERC 

Laboratory) by the City of Midland Water 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 CO2, 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-reared 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 culture 
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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 20 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 Daphnia 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 09 August 2011 to 11 August 2011. Three 

replicates of ten daphnids per test level was exposed to nominal test concentrations of 0 

(water control) and 100 mg L. Following 48-hours of exposure, 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 decided 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. Because filtration removed all of the 

(i.e., < LLQ, or < 0.000507 mg/L), the decision was made to centrifuge the stock 

solutions 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 WSF at the highest test nominal concentration 

100 mg L. The test material was weighed out (exact weight = 0.1000 g) directly 
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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 by 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 plastic 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 vessels 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 temperature 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 test 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. 
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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 additional 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 operating in the multiple reaction monitoring 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 EC50 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 EC50 endpoints. The 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 and 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 level 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. 
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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 not possible to analyze these data to calculate 

an EC50 ̧ because only a single daphnid died at the highest concentration, it can reliably 

be reported that the 24- and 48-hour EC50s 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, the 24- and 48-hour EC50s were greater than the 

highest concentration tested (i.e., >100 mg L). The 24- and 48-hour EC50s 

based on mean measured concentrations 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. 
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Thus, it can be concluded that at the limit of solubility (i.e,. the concentration of 

bioavailable test material), is not 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 stress 

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 study 
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REFERENCES 

OECD (Organization for Economic 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. 
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AN ACUTE TOXICITY STUDY WITH THE FRESH WATER 

CLADOCERAN, Daphnia magna 

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TABLE 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 purity 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 Structure
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AN ACUTE TOXICITY STUDY WITH THE FRESH WATER 

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TABLE 2. Summary of Results from Analysis of Test Solutions for 

1111 
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AN ACUTE TOXICITY STUDY WITH THE FRESH WATER 

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TABLE 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 

Alkalinitya (mg/L CaCO3) 42, 74 

Hardnessa (mg/L CaCO3) 56, 60 

Conductivitya (μmhos/cm) 175, 158 

 a

Alkalinity, Hardness, and Conductivity measurements are listed as: control, highest 

treatment level (0.130 mg L)
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AN ACUTE TOXICITY STUDY WITH THE FRESH WATER 

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TABLE 4. Summary of Biological Observations and Results

Mean Measured Concentrations 

(mg L) 

Observations 

24-Hour 48-Hour 

Water Control (<LLQa) 18N, 2I 18N, 2I

0.000923 20N 20N

0.00379 20N 20N

0.0163 20N 20N

0.0394 20N 20N

0.130 20N 19N, 1I 

N = Normal, I = Immobile

 Mean Measured Concentration (mg L) 

(Nominal Concentration) 

 24-Hour 48-Hour 

EC50

>0.130

(> 100)

>0.130

(> 100)

48-Hour NOECb 100

a

Lowest Level quantified = 0.000591mg 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.
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APPENDIX A. Certificate of Analysis
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APPENDIX B. Analytical Report
• 

• 

Report Title : 

Toxicology Infomlation Collection (TJq Report 

The Dow Chemical Company 

Do o nfirl11. & Homoge neity ( 

Author(s) 

Julia A. F iting 

Nathan A. Malowinsk

No action required 

GeograRhic Location De paltment 

N. America TER 

Study number: 

Page Count 

18 

Databook N umbers 

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 samples: 

Number of samples or estimate: 

Date samples prepared for analysis: 

Analysis date: 

Numberofsamples analyzed: 

Analysis method: 

Method reference: 

TH E DOW CHEMICAL 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 

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• 

• 

• 

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: 

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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) 

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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 (<LLQb) 

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/L
THE DOW CHEMICAL COMPANY

STUDY ID: 

PAGE 41

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER 

CLADOCERAN, Daphnia magna

- 

APPENDIX D. Individual pH Measurements

Individual pH measurements

Mean Measured

Concentration of

(mg/L)

Vessel

Number

pH

0 hr a 48 hr

Water Control (<LLQb) 

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 L
THE DOW CHEMICAL COMPANY

STUDY ID: 

PAGE 42

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER 

CLADOCERAN, Daphnia magna

- 

APPENDIX E. Individual Temperature Measurements

Individual Temperature (°C) Measurements

Mean Measured

Concentration of

(mg/L)

Vessel

Number

Temperaturea 

(°C)

0 hr b 48 hr

Water Control (<LLQc) 

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 exposure period ranged from 19.6-21.0 °C.

b 0 hour measurements taken from bulk test solutions.

c Lowest level quantified= 0.000591mg L
THE DOW CHEMICAL COMPANY

STUDY ID: 

PAGE 43

AN ACUTE TOXICITY STUDY WITH THE FRESH WATER 

CLADOCERAN, Daphnia magna

- 

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 (<LLQa) 

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