DIETHYLTOLUENE DIAMINE (DETDA): A 48-HOUR STATIC ACUTE TOXICITY TEST

WITH THE CLADOCERAN ( Daphnia magna )

FINAL REPORT

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-122

OECD GUIDELINE 202 U.S. EPA OPPTS NUMBER 850.1010

AUTHORS:

Daniel S. Brougher, M.S. Timothy Z. Kendall, M.S.

Sean P. Gallagher, B.S. Henry O. Krueger, Ph.D.

STUDY INITIATION DATE: January 26, 2012

STUDY COMPLETION DATE: March 20, 2012

SUBMITTED TO:

Albemarle Corporation 451 Florida Street Baton Rouge, LA 70801 

# Wildlife International, Ltd.

8598 Commerce Drive Easton, Maryland 21601 USA 1-410-822-8600

Page 1 of 44

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STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality, on any basis whatsoever , is made for any information contained in this study. I acknowledge that the information not d esignated as within the scope of FIFRA section 10(d) (1)(A), (B) or (C) and which pertains to a registered or previously registered pesticide is not entitled to confidential treatment and may be released to the public, subject to the provisions regarding disclosure to multinational entities under FIFRA (10)g.

Company: ________________________________(Typed Name)

Company Agent: ________________________________(Typed Name)

Title: ___________

___________ __________

Signature:______________________________Date:______________________

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TABLE OF CONTENTS

Title/Cover Page .............................................................................................................. ....................... 1

Statement of No Data Confidentiality Claims ................................................................................... ..... 2

Good Laboratory Practice Compliance Statement ................................................................................. 3

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

Report Approval ............................................................................................................... ...................... 5

Table of Contents ............................................................................................................. ...................... 6

Summary ....................................................................................................................... ......................... 8

Introduction .................................................................................................................. .......................... 9

Objective ..................................................................................................................... ........................... 9

Experimental Design ........................................................................................................... ................... 9

Materials and Methods ......................................................................................................... .................. 9 Test and Reference Substances ............................................................................................... 10 Test Organism ................................................................................................................ ......... 10 Dilution Water ............................................................................................................... .......... 11 Test Apparatus ............................................................................................................... .......... 11 Preparation of Test Concentrations ......................................................................................... 1 2 Analytical Sampling .......................................................................................................... ...... 12 Analytical Method ............................................................................................................ ....... 12 Environmental Conditions ..................................................................................................... .. 13 Observations ................................................................................................................. ........... 14 Statistical Anal yses ......................................................................................................... ......... 14

Results and Discussion ........................................................................................................ ................. 15 Measurement of Test Concentrations ...................................................................................... 15 Observations and Measurements ............................................................................................. 15

Conclusions ................................................................................................................... ....................... 16

References .................................................................................................................... ........................ 17

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TABLE OF CONTENTS (Continued)

TABLES AND FIGURES

Table 1 . Measured Concentrations of Diethyltolunene Diamine (DETDA) in Test Water Samples... .................................................................................................. 18

Table 2. Temperature, Dissolved Oxygen and pH of Water in the Test Chambers ........................ 19

Table 3. Specific Conductance, Hardness, Alkalinity and Total Organic Carbon in the Dilution Water at Test Initiation ............................................................................. 20

Table 4 . Cumulative Immobility and Observations ........................................................................ 21

Table 5 . EC50 Values ................................................................................................................. .... 22

Figure 1. Concentration-Response Curve ( 48-Hour Immobility Data) ............................................ 23

APPENDICES

Appendix 1. Chemical Characterization ...................................................................................... 24

Appendix 2. Dilution Water Chemistry Parameters Measured During the 4-Week Period Immediatel y Preceding the Test ..................................................... 28

Appendix 3. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water ................................................................................ 29

Appendix 4. The Analysis of Diethyltoluene Diamine (DETDA) in Freshwater ........................ 31 4.1 Analytical Method Flowchart for the Analysis of Diethyltoluene Diamine (DETDA) in Freshwater ........................................................................... 32 4.2 Typical HPLC Operational Parameters ................................................................... 33 4.3 Analytical Stocks and Standards Preparation.......................................................... 34 4.4 Example Calculations for a Representative Sample ............................................... 35 4.5 Quality Control Samples of Diethyltoluene Diamine (DETDA) in Freshwater ..... 36 4.6 Representative Calibration Curve for Di ethyltoluene Diamine (DETDA) ............. 37 4.7 Representative Chromatogram of a Low-level Diethyltoluene Diamine (DETDA) Calibration Standard ................................................................ 38 4.8 Representative Chromatogram of a High-level Diethyltoluene Diamine (DETDA) Calibration Standard ................................................................ 39 4.9 Representative Chromatogram of a Matrix Blank Sample ..................................... 40 4.10 Representative Chromatogram of a Ma trix Fortification Sample ........................... 41 4.11 Representative Chromatogram of a Test Sample .................................................... 42

Appendix 5. Changes to Protocol ................................................................................................ 43

Appendix 6. Personnel Involved in the Study ............................................................................. 44

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SUMMARY

SPONSOR: Albemarle Corporation

TITLE: Diethyltoluene Diamine (DETDA): A 48-Hour Static Acute Toxicity Test with the Cladoceran ( Daphnia magna )

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-122

TEST DATES: Experimental Start (OECD): February 8, 2012 Experimental Start (EPA): February 8, 2012 Biological Termination: February 10, 2012 Experimental Terminati on: February 10, 2012

LENGTH OF EXPOSURE: 48 Hours

TEST ORGANISM: Cladoceran, Daphnia magna

SOURCE OF TEST ORGANISMS: Wildlife International, Ltd. cultures Easton, Maryland 21601

AGE OF TEST ORGANISMS: Neonates <24 hours at test start

TEST CONCENTRATIONS: Nominal Mean Measured Negative Control < LOQ 0.25 mg a.i./L 0.24 mg a.i./L 0.50 mg a.i./L 0.49 mg a.i./L 1.0 mg a.i./L 0.98 mg a.i./L 2.0 mg a.i./L 1.9 mg a.i./L 4.0 mg a.i./L 3.9 mg a.i./L 8.0 mg a.i./L 7.8 mg a.i./L

RESULTS: Based on mean measured test concentrations: 48-Hour EC50: 5.8 mg a.i./L 95% Confidence Interval: 4.7 – 7.6 mg a.i./L No-Immobility Concentration: 1.9 mg a.i./L No-Observed-Effect Concentration: 0.98 mg a.i./L

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INTRODUCTION This study was conducted by Wildlife Internati onal, Ltd. for Albemarle Corporation at the Wildlife International, Ltd. aquatic toxicology fac ility in Easton, Maryland. The in-life phase of the definitive toxicity test was conducted from Februa ry 8, 2012 to February 10, 2012. Raw data generated by Wildlife International, Ltd. and the final report are filed under Project Number

471A-122 in archives located on the Wi ldlife International, Ltd. site.

OBJECTIVE The objective of this study was to determine the acute effects of Diethyltoluene Diamine
(DETDA) on the cladoceran, Daphnia magna , during a 48-hour exposure period under static test

conditions.

EXPERIMENTAL DESIGN Daphnids were exposed to a geometric series of six test concentrations and a negative control
(dilution water) for 48 hours under static conditions. Tw o replicate test chambers were maintained in each treatment and control group, with 10 daphnids in each test chamber, for a total of 20 daphnids per concentration. Nominal test concentrations we re selected in consultation with the Sponsor based on the results of exploratory range finding toxicity data. Nominal test concentrations selected were 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mg Diethyltoluene Diamine (DETDA) active ingredient (a.i.)/L. Mean

measured test concentrations we re determined from samples of test water collected from each treatment and control group at the beginning and end of the test.

Daphnids were impartially assigned to exposure ch ambers at test initiation. Observations of immobility and other signs of toxicity were made approximately 2.5, 24 and 48 hours after test initiation. Cumulative percent immobility observed in the treatment groups was used to determine

EC50 values at 24 and 48 hours. The no-immob ility concentration and the no-observed-effect concentration (NOEC) were determined by visual interpretation of the immobility and biological

observation data.

MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, “Diethyltoluene Diamine (DETDA): A 48-Hour Sta tic Acute Toxicity Test with the Cladoceran

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( Daphnia magna )”. The protocol was based on procedur es outlined in the OE CD Guidelines for Testing of Chemicals, Guideline 202: Daphnia sp., Acute Immobilization Test (1); U.S. Environmental Protection Agency Series 850 – Ecol ogical Effects Test Gu idelines, OPPTS Number

850.1010: Aquatic Invertebrate Acute Toxi city Test, Freshwater Daphnids (2); and ASTM Standard

E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates and Amphibians (3).

Test and Reference Substances The test substance used to prep are the test solutions, calibration standards, and the analytical matrix fortification samples for the study was r eceived from Albemarle Corporation on September 21, 2011. It was assigned Wildlife International, Lt d. identification number 10232 upon receipt and was stored under ambient conditions. The test substance, a liquid, was identified as: Diethyltolunene
Diamine (DETDA); Lot number - 4500051816-011110; CAS number – 68479-98. The test substance contained 97.27% active ingredient and had an expiration date of Marc h 3, 2013 (Appendix 1).

Test Organism
The cladoceran, Daphnia magna , was selected as the test species fo r this study. This species is representative of an important group of aquatic invertebrates and were selected for use in the test based upon past history of use in the laboratory. Daphnid neonates used in the test were less than

24 hours old and were obtained from cultures mainta ined by Wildlife International, Ltd., Easton, Maryland.

Adult daphnids were cultured in water from th e same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 19.7 to 21 .1oC, measured with a hand-held, liquid-in-glass thermometer. The pH of the water ranged from 8.2 to 8.5, measured with a Thermo Orion Benchtop
4 Star Plus pH meter. Dissolved oxygen concentrations were  7.1 mg/L (  78% of saturation), measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter.

Daphnids in the cultures were fed daily a mixture of yeast, cereal grass media and trout chow (YCT), as well as a suspension of the freshwater green alga, Pseudokirchneriella subcapitata . The adults were fed prior to t est initiation, but neonates were not fed during the test.

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The five adult daphnids used to supply neonates for the test were held for at least 14 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. At test initiation, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 10 daphnids. Each group of dap hnids then was transferred to an indiscriminately assigned test chamber to initiate the test. All tr ansfers were made below the water surface using wide-bore pipettes.

Dilution Water The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well wa ter was passed through a
sand filter to remove particles greater than approximately 25  m, and pumped into a 37,800-L storage
tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45  m to remove fine particles and was passed through an ultraviolet (UV) sterilizer.

The well water is characterized as moderate ly-hard water. The specific conductance, hardness, alkalinity, and pH content of the we ll water during the four-week period immediately preceding the test are presented in Appendix 2. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the well water are presented in Appendix 3.

Test Apparatus Test chambers were 250-mL glass beakers filled w ith approximately 230 mL of test water. The depth of the test water in a representativ e chamber was 5.5 cm. The chambers were indiscriminately positioned by treatment group in a temperature-controlled environmental chamber to maintain the target water temperature throughout the t est period. All test chambers were labeled with the project number, test concentration and replicate designation.

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Preparation of Test Concentrations A primary stock solution was prepared by mixi ng a calculated amount of test substance in 2.0 L of dilution water at a nominal concentration of 8.0 mg a.i./L, the highest concentration tested. The stock solution was adjusted to 100% active in gredient during preparation, based on the test substance purity (97.3%). The stock solution was sonicated for approximately 2 minutes and mixed by inversion at least 20 times. Aliquots of th e primary stock solution were proportionally diluted with dilution water to prepare five additional test so lutions at nominal concentra tions of 0.25, 0.5, 1.0, 2.0, and 4.0 mg a.i./L. The solutions were mixed by inversion and approximately 250 mL of solution was placed in each of two replicate test chambers per treatment group. The negative control solution was dilution water only.

Analytical Sampling Samples were collected from the batches of test solution prepared for each treatment and control group at the beginning of the test and from each test chamber at 48 hours (± 1 hour) to

measure concentrations of the test substance. The samples (~20mL) were collected at mid-depth, placed in glass vials, and processed immediately for analysis.

Analytical Method The method used for the analysis of DETD A in the freshwater samples was based upon methodology developed by Wildlife International, Lt d. The analytical method consisted of diluting the samples with freshwater, as necessary, and an alyzing by high performance liquid chromatography with variable wavelength detection at 220 nm.

Concentrations of DETDA in the samples we re determined using an Agilent Series 1100/1200 High Performance Liquid Chromatograph (HPLC) equipped with an Agilent Series 1100 Variable Wavelength Detector. Chromatographi c separations were achieved using a YMC-PACK
ODS AM column (150 mm x 4.6 mm, 3  m particle size). A method flowchart is provided in Appendix 4.1, and typical instrumental pa rameters are summarized in Appendix 4.2.

Calibration standards of DETDA were prepared fo r the analysis of the test samples, ranging in concentration from 0.150 to 1.50 mg a.i./L, using a stock solution of DETDA in acetonitrile (Appendix 4.3). Fresh calibration standards were prepared and analyzed with each sample set.

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Linear regression equations were generated using the sum of the peak area responses versus the respective concentrations of the calibration standard s. The concentration of DETDA in the samples was determined by substituting the sum of the peak area responses of the samp les into the applicable linear regression equation. The peak areas of the 2 components of DETDA were summed in order to derive the concentration of DETDA expressed as "a.i.". An example of the calculations for a representative sample is included in Appendix 4.4.

The method limit of quantitation (LOQ) was defined as 0.150 mg a.i./L, calculated as the product of the concentration of the lowest calibra tion standard (0.150 mg a.i./L) and the dilution factor of the matrix blank samples (1.00). Two matrix blank samples were analyzed to determine possible interferences. No interferences were ob served at or above the LOQ during the sample analyses (Appendix 4.5).

Samples of freshwater were fortified at 0.250, 0.750 and 8.00 mg a.i./L using stock solutions of DETDA in acetonitrile (Appendix 4.3), and were anal yzed concurrently with the test samples. The

measured concentrations for the matrix fortifi cation samples ranged from 96.2 to 100% of fortified concentrations (Appendix 4.5).

A representative calibration curve is presented in Appendix 4.6. Representative chromatograms of low and high-level calibration st andards are presented in Appendices 4.7 and 4.8, respectively. Representative chromatograms of a matrix blank sample and a matrix fortification sample are presented in Appendices 4.9 and 4.10, respectively. A representative chromatogram of a test sample is presented in Appendix 4.11.

Environmental Conditions Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006C light meter.

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The target test temperature during the study was 20 ± 1oC. Temperature was measured in each test chamber at the beginning and end of the test and at approximately 24 hours during the test using a liquid-in-glass thermometer. Temperature also was monitored continuously during the test in a container of water placed adjacent to the test chambers, using a Fulscope ER/C Recorder.

Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at approximately 24 hours during th e test. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter, and measurements of pH were made using a Thermo Orion Model 525Aplus meter.

Hardness, alkalinity, specific conductance a nd total organic carbon (TOC) in the dilution water at the beginning of the test were measured. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods for the Examination of Water and

Wastewater (4). Specific conductance was measured using an Acorn Series Model CON6 conductivity-temperature meter. TOC was measured using a Sh imadzu Model TOC-VCSH total organic carbon analyzer based on procedures in Standard Methods for the Examination of Water and

Wastewater (4).

Observations All organisms were observed periodically to de termine the number of immobile organisms in each treatment group. Immobility was defined as a lack of movement by the organism except for minor activity of the appendages. The numbers of individuals exhibiting signs of toxicity or

abnormal behavior also were evaluated. Observati ons were made approximately 2.5, 24 and 48 hours

after test initiation.

Statistical Analyses The immobility data were analyzed using the co mputer program of C. E. Stephan (5). The program was designed to calculate the EC50 valu e and the 95% confidence interval by probit analysis, the moving average method, and binomial proba bility with nonlinear interpolation (6, 7, 8). In this study, probit analysis was used to cal culate the 48-hour EC50 value and the 95% confidence interval. Since there was <50% immobility at 24 hours, the 24-hour EC50 value, as well as the

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no-immobility concentration and NOEC, were determined by visual interpretation of the immobility

and observation data.

RESULTS AND DISCUSSION

Measurement of Test Concentrations

Nominal concentrations selected for use in th is study were 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mg a.i./L. Test solutions in the test chambers at these nominal concentrations appeared clear and colorless during the test, with no evidence of precipitation observed.

Results of analyses to measure concentrations of Diethyltoluene Diamine (DETDA) in the test solution samples collected during the test are presented in Table 1. Measured concentrations of the samples ranged from approximately 95 to 98% of nominal. When measured concentrations of the samples were averaged, the mean measured test con centrations for this study were 0.24, 0.49, 0.98, 1.9, 3.9 and 7.8 mg a.i./L, representing 96, 98, 98, 95, 98 and 98% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

Observations and Measurements Measurements of temperature, dissolved oxygen a nd pH of the water in the test chambers are
presented in Table 2. Water temperatures were within the 20 ± 1  C range established for the test.
Dissolved oxygen concentrations remained  8.5 mg/L (  93% of saturation) throughout the test. Measurements of pH ranged from 8.1 to 8.5. The measurements of hardness, alkalinity, specific

conductance and TOC in the dilution water at test initiation were typical of Wildlife International, Ltd. well water (Table 3). Light intensity at test initiation was 617 lux at the surface of the water of one representative test chamber.

Daily observations for immobility and signs of toxicity during the test are presented in Table 4. Daphnia in the negative control groups appeared normal throughout the test. All daphnids in the 0.24, 0.49, and 0.98 mg a.i./L treatment groups also appeared normal throughout the test, with no immobility or overt signs of toxicity observed. Percent immobility at test termination in the 3.9 and 7.8 mg a.i./L treatment groups was 25 and 70%, respectively. Three daphnids in the 1.9 mg a.i./L treatment group appeared leth argic at test termination, while the remaining daphnids in this treatment group appeared normal throughout the te st. Lethargic daphnids were observed in the

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3.9 mg a.i./L treatment group at test termination, and lethargic daphnids were observed in the 7.8 mg a.i./L treatment group at both the 24-hour interval and at test termination. The no-immobility concentration was 1.9 mg a.i./L and the NOEC was 0.98 mg a.i./L. EC50 values at 24 and 48 hours were determined from the immobility data a nd are shown in Table 5. A graph of the concentration-response curve is included in Figure 1.

CONCLUSIONS
The cladoceran, Daphnia magna , was exposed for 48 hours under static conditions to six mean measured concentrations of Diethylto luene Diamine (DETDA) ranging from 0.24 to 7.8 mg a.i./L. The 48-hour EC50 value was 5.8 mg a.i./L, with a 95% confidence interval of 4.7 to 7.6 mg a.i./L. The slope of the concentra tion-response curve was 4.616. The no-immobility concentration was 1.9 mg a.i./L and the NOEC was 0.98 mg a.i./L.

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REFERENCES

1 Organisation for Economic C ooperation and Development. 2004. OECD Guidelines for Testing of Chemicals, Guideline 202: Daphnia sp., Acute Immobilization Test . Adopted 13 April 2004.

2 U.S. Environmental Protection Agency. 1996. Series 850 – Ecological Effects Test Guidelines ( draft ), OPPTS Number 850.1010: Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids

.

3 American Society for Testing and Materials. 2007. ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fish es, Macroinvertebrates, and Amphibians

.

4 APHA, AWWA, WEF. 1998. Standard Methods for the Examination of Water and Wastewater . 20th Edition, American Public Health Association. American Water Works Association. Water Environment Federation, Washington, D.C.

5 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication.

6 Finney, D.J. 1971. Statistical Methods in Biological Assay . Second edition. Griffin Press, London.

7 Thompson, W.R. 1947. Bacteriological Reviews . Vol. II, No. 2. Pp. 115-145.

8 Stephan, C.E. 1977. “Methods for Calculating an LC50”, Aquatic Toxicology and Hazard Evaluations . American Society for Testing and Materials. Publication Number STP 634, pp 65-84.

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Table 1

Measured Concentrations of Diethyltoluene Diamine (DETDA) in Test Water Samples

Nominal Test Concentration

(m g a.i./L) Sample Number (471A-122-) Sampling Time (Da y s) Measured Concentration

(m g a.i./L) 1 Percent of N ominal 2 Mean Measured Concentration

(m g a.i./L) Mean Measured Percent of N ominal N egative Control (0.0) 1 0 2 2 < LOQ < LOQ < LOQ --

--

-- -- --

0.25 2 0 2 2 0.239 0.239 0.239 95.7 95.6 95.4 0.24 96

0.50 3 0 2 2 0.493 0.484 0.484 98.6 96.8 96.8 0.49 98

1.0 4 0 2 2 0.989 0.966 0.971 98.9 96.6 97.1 0.98 98

2.0 5 0 2 2 1.87 1.91 1.92 93.7 95.3 95.8 1.9 95

4.0 6 0 2 2 3.92 3.86 3.88 97.9 96.5 97.1 3.9 98

8.0 7 0 2 2 7.91 7.74 7.80 98.9 96.8 97.5 7.8 98

1 The limit of quantitation (LOQ) was 0.150 mg a.i./L, calculated as the p roduct of the concentration of the

lowest calibration standard (0.150 mg a.i./L) and the dilution factor of the matrix blank samples (1.00).
2 Results were g enerated usin g Excel 2000 in full precision mode. Manual calculations ma y differ sli g htl y

.

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Table 2

Temperature, Dissolved Oxygen and pH of Water in the Test Chambers

Mean Measured Concentration

(mg a.i./L) Replicate 0 Hour 24 Hours 48 Hours
Temp. 1

(°C) DO 2 (mg/L) pH Temp. 1 (°C) DO 2 (mg/L) pH Temp. 1 (°C) DO 2 (mg/L) pH

Negative A 19.3 8.7 8.1 20.4 8.7 8.4 20.4 8.6 8.5
Control B 19.2 8.7 8.1 20.3 8.8 8.4 20.4 8.6 8.5

0.24 A 19.1 8.8 8.2 20.3 8.8 8.4 20.3 8.6 8.5

B 19.2 8.8 8.2 20.2 8.8 8.4 20.2 8.6 8.5

0.49 A 19.3 8.7 8.2 20.3 8.8 8.4 20.3 8.6 8.5

B 19.3 8.8 8.2 20.3 8.8 8.4 20.3 8.6 8.5

0.98 A 19.2 8.8 8.2 20.3 8.8 8.4 20.3 8.5 8.5

B 19.2 8.8 8.2 20.3 8.8 8.4 20.3 8.6 8.5

1.9 A 19.4 8.7 8.2 20.3 8.8 8.4 20.3 8.5 8.5

B 19.3 8.8 8.2 20.3 8.8 8.4 20.3 8.7 8.5

3.9 A 19.4 8.9 8.2 20.3 8.7 8.4 20.3 8.7 8.5

B 19.3 8.9 8.2 20.3 8.7 8.4 20.3 8.7 8.5

7.8 A 19.4 8.8 8.2 20.4 8.7 8.4 20.2 8.8 8.5

B 19.3 8.8 8.2 20.3 8.7 8.4 20.3 8.8 8.5

1 Manual temperature measurements. Temperature monitored continuously during the test ranged from

approximately 20 to 20.5oC, m easured to the nearest 0.5oC.

2 A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20.0oC in freshwater.

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Table 3

Specific Conductance, Hardness, Alkalinity and Total Organic Carbon in the Dilution Water at Test Initiation

Parameter Day 0

Specific Conductance 340 (μS/cm)

Hardness 140 (mg/L as CaCO 3 )

Alkalinity 180 (mg/L as CaCO 3 )

Total Organic Carbon <1 (mg C/L)Wildlife International, Ltd.Project Number 471A-122- 21 -Table 4Cumulative Immobility and ObservationsMean MeasuredConcentration(mg a.i./L)Rep.Daphnia/Replicate~ 2.5 Hours 24 Hours 48 HoursPercentImmobileNumberImmobile1Observations2Number Immobile1Observations2NumberImmobile1Observations2Negative Control A 10 0 5 AN; 5 Q, AN0 6 AN; 4 Q, AN 0 4 AN; 6 Q, AN 0B 10 0 5 AN; 5 Q, AN 0 7 AN; 3 Q, AN 0 10 AN0.24 A 10 0 3 AN; 7 Q, AN 0 10 AN 0 10 AN 0B 10 0 7 AN; 3 Q, AN 0 10 AN 0 10 AN0.49 A 10 0 10 Q, AN 0 10 AN 0 9 AN; 1 Q, AN 0B 10 0 7 AN; 3 Q, AN 0 10 AN 0 10 AN0.98 A 10 0 4 AN; 6 Q, AN 0 10 AN 0 10 AN 0B 10 0 4 AN; 6 Q, AN 0 9 AN; 1 Q, AN 0 10 AN1.9 A 10 0 6 AN; 4 Q, AN 0 10 AN 0 5 AN; 1 C; 4 Q, AN 0B 10 0 5 AN; 5 Q, AN 0 8 AN; 2 Q, AN 0 6 AN; 2 C; 2 Q, AN3.9 A 10 0 8 AN; 2 Q, AN 2 8 AN 2 1 AN; 2 C; 5 Q, C 25B 10 0 6 AN; 4 Q, AN 3 3 AN; 4 Q, AN 3 4 AN; 3 Q, C7.8 A 10 0 6 AN; 4 Q, AN 2 6 AN; 2 C 7 3 C 70B 10 0 4 AN; 6 Q, AN 3 4 AN; 3 C 7 3 C1Cumulative number of immobile daphnids.2Observed effects: AN = appear normal; C = lethargy; Q, AN = trapped at water surface but appear normal after gentle submersion; Q, C = trapped at water surface and appearlethargic after gentle submersion.

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Table 5

EC50 Values

Time EC50 (mg a.i./L) 95% Confidence Interval (mg a.i./L) Statistical Method

24 Hours > 7.8 -- 1 NA 2

48 Hours 5.8 4.7 – 7.6 Probit method 3

1 95% confidence limits could not be calcu lated with the immobility data obtained.

2 NA = not applicable; <50% immobility preclude d statistical calculation of an EC50 value.
3 The EC50 value and 95% confidence limits were determined by the Probit method.

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Figure 1

Concentration-Response Curve (48-Hour Immobility Data)



### Slope = 4.616

Mean, Measured Concentration (mg a.i./L) 0 2 4 6 8 10Percent Immobility



### 0 10 20 30 40 50 60 70 80 90 100

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Appendix 1

Chemical Characterization

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Appendix 2

Dilution Water Chemistry Parameters Measured During the 4-Week Period Immediately Preceding the Test

Parameter Mean Range

Specific Conductance 353 332 – 378 (μS/cm) (N = 4)

Hardness 147 144 – 156 (mg/L as CaCO 3 ) (N = 4)

Alkalinity 178 176 – 180 (mg/L as CaCO 3 ) (N = 4)

pH 8.2 8.0 – 8.7 (N = 4)

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Appendix 3

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water

Pesticides and Organics 1

Component Measured Concentration (μg/L) Component Measured Concentration (μg/L)

Aldrin < 0.0098 Gamma BHC – Lindane < 0.0098 Alpha BHC < 0.0098 Gamma Chlordane < 0.020 Alpha Chlordane < 0.0098 Guthion (Azinphos-methyl) < 5.9 Beta BHC < 0.0098 HCB < 0.0098 Bolstar < 2.0 Heptachlor < 0.0098 Chlordane < 0.49 Heptachlor Epoxide < 0.0098 Coumaphos < 2.9 Kepone < 0.20 Delta BHC < 0.0098 Malathion < 2.9 Demeton-O < 2.0 Merphos < 2.0 Demeton-S < 2.9 Methoxychlor < 0.098 Diazinon < 5.9 Methyl Parathion < 2.0 Dichlorvos < 2.9 Mevinphos < 4.9 Dieldrin < 0.020 Mirex < 0.25 Disulfoton < 2.0 Naled < 5.9 Dursban (Chlorpyrifos) < 2.9 o,p-DDD < 0.020 Endosulfan I < 0.0098 o,p-DDE < 0.74 Endosulfan II < 0.020 o,p-DDT < 0.020 Endosulfan Sulfate < 0.020 p,p-DDD < 0.020 Endrin < 0.020 p,p-DDE < 0.020 Endrin Aldehyde < 0.098 p,p-DDT < 0.020 Endrin Ketone < 0.020 Phorate < 2.0 EPN < 2.0 Ronnel < 2.0 Ethion < 2.0 Stirophos < 2.0 Ethoprop < 2.9 Telodrin < 0.0098 Ethyl Parathion < 2.0 Tokuthion < 2.0 Famphur < 2.9 Toxaphene < 2.9 Fensulfothion < 8.8 Trichloronate < 2.0 Fenthion < 2.0 Trithion < 2.0

1 Analyses performed by Lancaster Laboratorie s on samples collected on December 29, 2010.

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Appendix 3 (Continued)

Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water

Metals 1

Component Measured Concentration (mg/L) Component Measured Concentration (mg/L)

Aluminum < 0.200 Magnesium 13.0 Antimony < 0.0200 Manganese < 0.0050 Arsenic < 0.0200 Mercury < 0.00020 Barium < 0.0050 Nickel < 0.0100 Beryllium < 0.0050 Nitrate Nitrogen 0.55 Bromide < 2.5 Nitrite Nitrogen < 0.50 Cadmium < 0.0050 Potassium 6.77 Calcium 35.1 Selenium < 0.0200 Chloride 3.5 Silver < 0.0050 Chromium < 0.0150 Sodium 18.5 Cobalt < 0.0050 Sulfate 5.6 Copper < 0.0100 Thallium < 0.0300 Fluoride < 0.50 Vanadium < 0.0050 Iron < 0.200 Zinc < 0.0200 Lead < 0.0150

1 Analyses performed by Lancaster Laboratorie s on samples collected on December 29, 2010.

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Appendix 4

The Analysis of Diethyltoluene Diamine (DETDA) in Freshwater

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Appendix 4.1

Analytical Method Flowchart for the Analysis of Diethyltoluene Diamine (DETDA) in Freshwater



### METHOD FLOWCHART FO R THE ANALYSIS OF DETDA IN FRESHWATER

Prepare calibration standards in freshwater usi ng volumetric flasks and gas-tight syringes.



Prepare QC samples in freshwater using volumetric flasks, volumetric pipettes, culture tubes and gas-tight syringes. The matrix blank will be freshwater.



Dilute samples with freshwater (as needed) using volumetric flasks, volumetric pipettes, culture tubes and gas-tight syringes.



Transfer to autosampler vials and submit for HPLC/UV analysis.

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Appendix 4.2

Typical HPLC Operational Parameters

INSTRUMENT: Agilent Model 1100/ 1200 High Performance Liquid Chromatograph (HPLC) with an Agilent Series 1100 Variable Wavelength Detector

ANALYTICAL COLUMN: YMC-PACK ODS-AM Column (150 mm x 4.6 mm, 3 μm particle size)

OVEN TEMPERATURE: 40  C

STOP TIME: 14 minutes

FLOW RATE: 1.000 mL/minute

SOLVENT A:

SOLVENT B: 0.1% H 3 PO 4

CH 3 CN

GRADIENT PROFILE: Time Flow (min) %A %B (mL/min) 0.01 90.0 10.0 1.000 1.00 90.0 10.0 1.000 9.00 50.0 50.0 1.000 10.00 50.0 50.0 1.000 10.10 90.0 10.0 1.000 14.00 90.0 10.0 1.000

INJECTION VOLUME: 100.00 μL

DETDA RETENTION TIME: Component 1: Approximately 5.3 minutes Component 2: Approximately 6.0 minutes PRIMARY ANALYTICAL WAVELENGTH: 220 nm

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Appendix 4.3

Analytical Stocks and Standards Preparation

A stock solution of DETDA test substance was prep ared by weighing 0.1030 grams (weight corrected for purity) of the test substance on an analytical balance. The test substance was transferred to a 100-mL class A volumetric flask, and brought to volume using acetonitrile. The primary stock solution (1.00 mg a.i./mL) was diluted in acetonitril e to prepare a 0.100 mg a.i./mL stock solution. The 1.00 and 0.100 mg a.i./mL stock solutions were used to prepare the fortification samples. The 0.100 mg a.i./mL stock solution was used to prep are the calibration standards in freshwater. The following shows the dilution scheme for a set of calibration standards for the test samples:

Stock Concentration (mg a.i./mL) Aliquot (  L ) Final Volume

(mL) Standard Concentration

(mg a.i./L) 0.100 0.100 0.100 0.100 0.100 15.0 45.0 80.0 120 150 10.0 10.0 10.0 10.0 10.0 0.150 0.450 0.800 1.20 1.50

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Appendix 4.4

Example Calculations for a Representative Sample

The analytical result and percent recovery for sample number 471A-122-4, with a nominal concentration of 1.0 mg a.i./L, were calculated using the following equations:

Concentration of DETDA in sample (mg a.i./L) = Sum of peak areas - (y-intercept) Slope x dilution factor

Percent of nominal concentration = Measured concentration of sample (mg a.i./L) Nominal concentration of sample (mg a.i./L) x 100

Sum of Peak Areas = 206.48758 Y-Intercept = -1.0083 Slope = 209.9061 Dilution Factor = 1.00

Concentration of DETDA in sample (mg a.i./L) = 206.48758 + 1.0083 209.9061 x 1.00

Concentration of DETDA in sample (mg a.i./L) = 0.989

Percent of nominal concentration = 0.989 mg a.i./L 1.0 mg a.i./L x 100

Percent of nominal concentration = 98.9%

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Appendix 4.5

Quality Control Samples of DETDA in Freshwater

Sample Number (471A-122-) Sample Type Fortified Concentration

(mg a.i./L) Measured Concentration

(mg a.i./L) 1 Percent Recovery 2

MAB-1 MAB-2

MAS-1 MAS-2 MAS-3

MAS-4 MAS-5 MAS-6 Matrix Blank Matrix Blank

Matrix Fortification Matrix Fortification Matrix Fortification

Matrix Fortification Matrix Fortification Matrix Fortification 0.0 0.0

0.250 0.750 8.00

0.250 0.750 8.00 < LOQ < LOQ

0.250 0.753 7.70

0.251 0.752 7.99 --

--

99.9 100 96.2

100 100 99.9 Mean = Standard Deviation = CV = 99.3 1.54 1.55%

1 The limit of quantitation (LOQ) was 0.150 mg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0 .150 mg a.i./L) and the dilution factor of the matrix blank samples (1.00).

2 Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly.

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Appendix 4.6

Representative Calibration Curve for DETDA

0 50 100 150 200 250 300 350

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 (mg a.i./L)Peak area counts

Slope = 209.9061; Y-Intercept = -1.0083; R 2 = 0.99996

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Appendix 4.7

Representative Chromatogram of a Low-level DETDA Calibration Standard

min 0 2 4 6 8 10 12 mAU

-10

0 10 20 30 40 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA\DETDA 2012-02-08 15-24-22\001-0101.D)5.3475.971

Nominal concentration: 0.150 mg a.i./L. The arro ws indicate the approximate retention times for the components of DETDA. Component 1 retention tim e is 5.3 minutes and Component 2 retention time is 6.0 minutes.

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Appendix 4.8

Representative Chromatogram of a Hi gh-level DETDA Calibration Standard

min 0 2 4 6 8 10 12 mAU

0 20 40 60 80 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA\DETDA 2012-02-08 15-24-22\005-0501.D)5.3475.980

Nominal concentration: 1.50 mg a.i./L. The arrows indicate the approximate retention times for the components of DETDA. Component 1 retention tim e is 5.3 minutes and Component 2 retention time is 6.0 minutes.

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Appendix 4.9

Representative Chromatogram of a Matrix Blank Sample

min 0 2 4 6 8 10 12 mAU

-10

0 10 20 30 40 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA\DETDA 2012-02-08 15-24-22\006-0601.D)

Sample number 471A-122-MAB-1. The arrows indi cate the approximate retention times for the components of DETA. Component 1 retention time is 5.3 minutes a nd Component 2 retention time is 6.0 minutes.

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Appendix 4.10

Representative Chromatogram of a Matrix Fortification Sample

min 0 2 4 6 8 10 12 mAU

-10

0 10 20 30 40 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA\DETDA 2012-02-08 15-24-22\007-0701.D)5.3615.986

Sample number 471A-122-MAS-1; 0.250 mg a.i./L nominal concentration. The arrows indicate the approximate retention times for the components of DETDA. Component 1 retention time is 5.3 minutes and Component 2 retention time is 6.0 minutes.

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Appendix 4.11

Representative Chromatogram of a Test Sample

min 0 2 4 6 8 10 12 mAU

-10

0 10 20 30 40 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA\DETDA 2012-02-08 15-24-22\013-1401.D)5.3565.985

Sample number 471A-122-4; Day 0; 1.0 mg a.i./L nominal concentration. The arrows indicate the approximate retention times for the components of DETDA. Component 1 retention time is 5.3 minutes and Component 2 retention time is 6.0 minutes.

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Appendix 5

Changes to Protocol

This study was conducted in accordan ce with the approved Protocol.

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Appendix 6

Personnel Involved in the Study

The following key Wildlife International, Ltd. pers onnel were involved in the conduct or management of this study:

1. Henry O. Krueger, Ph.D., Director of Aquatic, Plant and Insect Toxicology

2. Willard B. Nixon, Ph.D., Director of Chemistry

3. Sean P. Gallagher, B.S., Manager of Aquatic Toxicology

4. Timothy Ross, B.S., Laboratory Supervisor

5. Timothy Z. Kendall, M.S., Supervisor

6. Daniel S. Brougher, M.S., Senior Biologist

7. Jessica M. Griebel, B.S., Biologist