DIETHYLTOLUENE DIAMINE (DETDA): A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FATHEAD MINNOW ( Pimephales promelas )

FINAL REPORT

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-123

OECD GUIDELINE 203 U.S. EPA OPPTS NUMBER 850.1075

AUTHORS:

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

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

STUDY INITIATION DATE: February 17, 2012

STUDY COMPLETION DATE: March 26, 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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TABLE OF CONTENTS

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

Good Laboratory Practice Compliance Statement ................................................................................. 2

Quality Assuran ce Statement ................................................................................................... .............. 3

Report Approval ............................................................................................................... ...................... 4

Table of Contents ............................................................................................................. ...................... 5

Summary ....................................................................................................................... ......................... 7

Introduction .................................................................................................................. .......................... 8

Objective ..................................................................................................................... ........................... 8

Experimental Design ........................................................................................................... ................... 8

Materials and Methods ......................................................................................................... .................. 9 Test Substance ............................................................................................................... ............ 9 Test Organism ................................................................................................................ ............ 9 Dilution Water ............................................................................................................... .......... 10 Test Apparatus ............................................................................................................... .......... 10 Preparation of Test Concentrations.......................................................................................... 1 1 Analytical Sampling .......................................................................................................... ...... 11 Analytical Method ............................................................................................................ ....... 11 Environmental Conditions ..................................................................................................... .. 12 Observations ................................................................................................................. ........... 13 Statistical Analyses ......................................................................................................... ......... 13

Results and Discussion ........................................................................................................ ................. 14 Measurement of Test Concentrations ...................................................................................... 14 Observations and Measurements ............................................................................................. 14

Conclusions ................................................................................................................... ....................... 15

Refere nces .................................................................................................................... ........................ 16

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TABLE OF CONTENTS (Continued)

TABLES AND FIGURES

Table 1. Measured Concentrations of Diethyltoluene Diamine (DETDA) in Freshwater Samples . 17

Table 2. Temperature, Dissolved Oxygen and pH of Water in the Test Chambers ......................... 18

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

Table 4. Cumulative Mortality and Observations ............................................................................. 21

Table 5. LC50 Values .................................................................................................................. ..... 23

APPENDICES

Appendix 1. Chemical characterization ....................................................................................... 24

Appendix 2. Dilution Water Chemistry Parameters Measured During the 4-Week Period Immediately 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 Operati onal 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 Matrix 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 96-Hour Static Acute Toxicity Test with the Fathead Minnow ( Pimephales promelas )

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471A-123

TEST DATES: Experimental Start (OECD): February 20, 2012 Experimental Start (EPA): February 20, 2012 Exposure Termination: February 24, 2012 Experimental Termination: February 24, 2012

LENGTH OF EXPOSURE: 96 Hours

TEST ORGANISM: Fathead Minnow ( Pimephales promelas )

SOURCE OF TEST ORGANISMS: Chesapeake Cultures Hayes, VA 23072

AGE OF TEST ORGANISMS: Juveniles

MEASUREMENTS OF 10 NEGATIVE CONTROL FISH: Mean Wet Weight: 0.27 g Range: 0.23 – 0.32 g Mean Total Length: 3.0 cm Range: 2.8 – 3.2 cm

TEST CONCENTRATIONS: Nominal Mean Measured Negative Control < LOQ Solvent Control < LOQ 7.5 mg a.i./L 7.2 mg a.i./L 15 mg a.i./L 15 mg a.i./L 30 mg a.i./L 29 mg a.i./L 60 mg a.i./L 70 mg a.i./L 120 mg a.i./L 106 mg a.i./L

RESULTS: Based on mean measured test concentrations: 96-Hour LC50: > 106 mg a.i./L No-Mortality Concentration: 15 mg a.i./L No-Observed-Effect Concentration: 15 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 February 20, 2012 to February 24, 2012. Raw data generated by Wildlife International, Ltd. and the final repor t are filed under Project Number 471A-123 in archives
located on the Wildlife International, Ltd. site.

OBJECTIVE The objective of this study was to determine the acute effects of Diethyltoluene Diamine (DETDA) on the fathead minnow ( Pimephales promelas ) during a 96-hour exposure period under static

test conditions.

EXPERIMENTAL DESIGN Fathead minnows were exposed to a geometric seri es of five test concentrations and a negative control (dilution water) for 96 hours under static c onditions. Two replicate test chambers were maintained in each treatment and control group, with 10 fish in each test chamber, for a total of 20 fish 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 7.5, 15, 30, 60 and 120 mg Diethyltoluene Diamine (DETDA) active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples of t est water collected from each treatment and control group at the beginning, the approximate mid-point and the end of the test.

Fathead minnows were impartially assigned to exposur e chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 2, 24, 48, 72 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups was used to determine LC50
values at 24, 48, 72 and 96 hours. The no-morta lity concentration and the no-observed-effect concentration (NOEC) were determined by visual interpretation of the mortality and biological

observation data.

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MATERIALS AND METHODS The study was conducted according to the proce dures outlined in the protocol, Diethyltoluene Diamine (DETDA): A 96-Hour Static Acute Toxicity Test with the Fathead Minnow ( Pimephales
promelas )”. The protocol was based on procedures outlin ed in the OECD Guidelines for Testing of
Chemicals, Guideline 203: Fish, Acute Toxicity Test (1); U.S. Environmental Protection Agency Series 850 – Ecological Effects Test Guidelines, OPPTS Number 850.1075: Fish Acute Toxicity Test,

Freshwater and Marine (2); and ASTM Standard E729-96: Standard Guide for Conducting Acute
Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians (3).

Test Substance The test substance used to prep are the test solutions and the analytical matrix fortification samples for the study was received from Albemarle Co rporation on September 21, 2011. It was assigned
Wildlife International, Ltd. identification num ber 10232 upon receipt and was stored under ambient conditions. The test substance, a liquid, was identified as: Diethyltoluene Diamine (DETDA); Lot
number 4500051816-011110; CAS number 68479-98-1. The t est substance contained 97.3% active ingredient and had an expiration da te of March 3, 2013. The test substance was characterized by the Sponsor under Good Laboratory Practices and the char acterization report is provided in Appendix I.

Test Organism The fathead minnow ( Pimephales promelas ) was selected as the test species for this study. This species is representative of an important group of aquatic vertebrates and was selected for use in the test based upon past history of use in the laboratory. Fathead minnows used in the test were received as juveniles from Chesapeake Cultures of Hayes, VA. Identification of the species was verified by the supplier.

All fish used in the test were juveniles from th e same source and year class, and the length of the longest fish measured was no more than twice the le ngth of the shortest. The average total length of 10 negative control fish measured at the end of the test was 3.0 cm, with a range of 2.8 to 3.2 cm. The average wet weight (blotted dry) of 10 negative c ontrol fish measured at the end of the test was 0.27 grams, with a range of 0.23 to 0.32 grams. Load ing was defined as the total wet weight of fish per liter of test water and was 0.18 g fish/L.

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The fish were held for at least 14 days prior to the test in water from the 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 21.4 to 23.5  C, measured with a
liquid-in-glass thermometer. The pH of the water ra nged from 8.3 to 8.5, measured with a Thermo Orion
Benchtop 4 Star Plus pH meter. Di ssolved oxygen concentrations were  8.1 mg/L (  93% of saturation),
measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter.

Daily during the holding period, except during periods of fasting prior to testing, the fish were fed a commercially-prepared diet supplied by Sera, North America, supplemented with brine shrimp nauplii ( Artemia sp.) supplied by Brine Shrimp Direct, Ogden, Utah. The fish were not fed for at least two days prior to the test or during the test.

During the 2-week period prior to the test the fish showed no signs of disease or stress. At test
initiation, fathead minnows were collected from the cu lture tank and impartially distributed one and two

at a time to the test chambers until each contained 10 fish.

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 water 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.

The well water is characterized as moderately-har d water. The specific conductance, hardness, alkalinity and pH of the well water during the four-week period immediately preceding the test are presented in Appendix 1. The results of periodic an alyses performed to measure the concentrations of selected organic and inorganic constituents in the well water are presented in Appendix 2.

Test Apparatus Test chambers were 25-L stainless steel aquaria f illed with approximately 15 L of test water. The depth of the test water in a representative test cham ber was 17 cm. The chambers were indiscriminately

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positioned by treatment group in two temperature-cont rolled water baths to maintain the target water temperature throughout the test period. All test cham bers were labeled with the project number, test concentration and replicate designation.

Preparation of Test Concentrations Individual test solutions were prepared directly in each test chamber. All test solutions were adjusted to 100% active ingredient during preparati on, based on the test substance purity (97.3%). Solutions were prepared at nominal concentrations of 7.5, 15, 30, 60 and 120 mg a.i./L by mixing

calculated amounts of test substance into dilution wate r with a final volume of 15 L. The test substances were weighed into tared 100 mL glass beakers. Appr oximately 60 mL of the pre-measured 15 L dilution water was added, and the beakers were sonicated for 2 – 40 minutes. Using a portion of the pre-measured
dilution water, each test solution was then rinsed into its appropriate 25 L test chamber, and stirred with a top down mixer for 2-3 minutes. The negative control solution was dilution water only.

Analytical Sampling Samples were collected from each test chambe r of each treatment and control group at the beginning of the test and at 48 and 96 hours (± 1 hour) to measure concentrations of the test substance. The samples were collected at mid-depth, placed in glass vials, and processed immediately for analysis.

Analytical Method The method used for the analysis of DET DA in the freshwater samples was based upon methodology developed by Wildlife International, Ltd. The analytical method consisted of diluting the samples with freshwater, as necessary, and analyz ing by high performance liquid chromatography with variable wavelength detection at 220 nm.

Concentrations of DETDA in the samples were determined using an Agilent Series 1100/1200 High Performance Liquid Chromatograph (HPLC) e quipped with an Agilent Series 1100 Variable Wavelength Detector. Chromatographic separa tions were achieved using a YMC-PACK ODS-AM
column (150 mm x 4.6 mm, 3  m particle size). A method flowchar t is provided in Appendix 3.1, and
typical instrumental parameters are summarized in Appendix 3.2.

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Calibration standards of DETDA were prepared for the analysis of the test samples, ranging in concentration from 3.00 to 30.0 mg a.i./L, using a st ock solution of DETDA in acetonitrile (Appendix 3.3). Fresh calibration standards were prepared and analyzed with each sample set. Linear regression equations were generated using the sum of the peak area res ponses versus the respective concentrations of the calibration standards. The concentration of DETDA in the samples was determined by substituting the sum of the peak area responses of the samples into the app licable 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 re presentative sample is included in Appendix 3.4.

The method limit of quantitation (LOQ) was defined as 3.00 mg a.i./L, calculated as the product of the concentration of the lowest calibration standard (3 .00 mg a.i./L) and the dilution factor of the matrix blank samples (1.00). Three matrix blank samples were analyzed to determine possible interferences. No

interferences were observed at or above the LOQ during the sample analyses (Appendix 3.5).

Samples of freshwater were fortified at 7.50, 20. 0, and 120 mg a.i./L using a stock solution of DETDA in acetonitrile (Appendix 3.3), and were analy zed concurrently with the test samples. The

measured concentrations for the matrix fortifi cation samples ranged from 81.7 to 102% of fortified concentrations (Appendix 3.5).

A representative calibration curve is presented in Appendix 3.6. Representative chromatograms of low and high-level calibration standards are pr esented in Appendices 3.7 and 3.8, respectively. Representative chromatograms of a matrix blank sample and a matrix fortification sample are presented in Appendices 3.9 and 3.10, respectively. A representativ e chromatogram of a test sample is presented in Appendix 3.11.

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 darkn ess. 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 two representativ e test chambers from each water bath at test initiation using a SPER Scientif ic Model 840006C light meter.

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The target test temperature during the study was 22 ± 1oC. Temperature was measured in each test chamber at the beginning and end of the test a nd at approximately 24-hour intervals during the test using a liquid-in-glass thermometer. Temperature also was monitored continuously in one negative control test chamber using a Fulscope ER/C Recorder , which was verified prior to test initiation with a liquid-in-glass thermometer.

Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at approximately 24-hour intervals during the test. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen mete r and measurements of pH were made using a Thermo Orion Model 525Aplus pH meter.

Hardness, alkalinity and specific conductance in the dilution water were measured at the beginning of the test. Hardness and alkalinity measure ments were made by titra tion 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.

Observations All organisms were observed periodically to determine the number of mortalities in each treatment group. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 2, 24, 48, 72 and 96 hours after test initiation.

Statistical Analyses In this study, there was less than 50% mortality in any of the Diethyltoluene Diamine (DETDA) treatment groups during the test which precluded the statistical calculation of LC50 values at 24, 48, 72

and 96 hours. Due to the method used to calcula te the 96-hour LC50 value, the slope of the dose response curve could not be calculated. Since ther e was <50% mortality at 24, 48, 72 and 96 hours, the 96-hour LC50 value, as well as the no-mortality concen tration and NOEC, were determined by visual interpretation of the mortality and observation data.

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RESULTS AND DISCUSSION

Measurement of Test Concentrations Nominal concentrations selected for use in this study were 7.5, 15, 30, 60 and 120 mg a.i./L. During the test, the negative control, 7.5, 15, 30 and 60 mg a.i./L test solutions were clear and colorless in appearance at test initiation. The 120 mg a.i./L test so lution was clear and had a slight purple color at test initiation. At test termination, the negative control, 7.5, and 15 mg a.i./L test solutions were clear and colorless in appearance, while the 30, 60 and 120 mg a. i./L were clear and purple in color with purple color intensity increasing with increasing concentration.

Results of analyses to measure concentrations of Diethyltoluene Diamine (DETDA) in the test solution samples collected during the test are presente d in Table 1. Measured concentrations of the samples ranged from approximately 74 to 139% of nom inal. When measured concentrations of the samples were averaged, the mean measu red test concentrations for this study were 7.2, 15, 29, 70 and 106 mg a.i./L, representing 96, 100, 97, 117 and 89% 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 w ithin the 22 ± 1oC range established for the test. Dissolved oxygen concentrations remained ≥ 6.1 mg/L ( ≥ 70% of saturation) throughout the test. Measurements of pH ranged from 8.2 to 8.4 during the test. The measurements of hardness, alkalinity and specific conductance in the dilution water at test initiation were typical of Wildlife International, Ltd. well water (Table 3). Light intensity at test in itiation was 695 lux at the surface of the water of one representative test chamber.

Daily observations of mortality and other signs of toxicity observed during the test are presented in Table 4. All fish in the negative control gr oup appeared normal throughout the test. All fathead minnows in the 7.2, and 15 mg a.i./L treatment groups also appeared normal throughout the test, with no mortalities or overt signs of toxicity observed. Signs of toxicity observed among fish in the 29, 70 and 106 mg a.i./L treatment groups included surfacing, loss of equilibrium and lethargy. Percent mortality in the 29, 70 and 106 mg a.i./L treatment groups at test termination was 5, 0 and 20%, respectively. While

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there was no mortality at the 70 mg a.i./L test concentration, signs of toxicity were noted in the 29 mg a.i./L treatment group and the single mortality at this concentration could not be precluded as treatment related. The no-mortality concentration and the NOEC were 15 mg a.i./L. LC50 values at 24, 48, 72 and 96 hours were determined from the mortality data and are shown in Table 5.

CONCLUSIONS Fathead minnows ( Pimephales promelas ) were exposed for 96 hours under static conditions to

five mean measured concentrations of Diet hyltoluene Diamine (DETDA) ranging from 7.2 to 106 mg a.i./L. The 96-hour LC50 value was >106 mg a.i./L, the highest concentration tested. Fathead minnows exposed to Diethyltoluene Diamine (DETDA) at concentrations ≤ 15 mg a.i./L appeared normal with no mortality or overt signs of toxicity. The 96-hour no-mortality concentration and the NOEC were 15 mg a.i./L.

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REFERENCES

1 Organisation for Economic Cooperation and Development . 1992. OECD Guidelines for Testing of Chemicals, Guideline 203: Fish, Acute Toxicity Test . Adopted 17 July 1992.

2 U.S. Environmental Protection Agency. 1996. Series 850 – Ecological Effects Test Guidelines ( draft ), OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine

.

3 American Society for Testing and Materials. 2007. ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Ma terials with Fishes, 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.

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Table 1

Measured Concentrations of Diethyltoluen e Diamine (DETDA) in Freshwater Samples

Nominal Test Concentration

(mg a.i./L) Sample Number (471A-123-) Sampling Time (Days) Measured Concentration

(mg a.i./L) 1 Percent of Nominal 2 Mean Measured Concentration

(mg a.i./L) Mean Measured Percent of Nominal Negative Control (0.0) 1 2 13 14 25 26 0 0 2 2 4 4 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ --

--

--

--

--

-- -- --

7.5 3 4 15 16 27 28 0 0 2 2 4 4 7.32 7.64 6.70 7.03 7.21 7.58 97.5 102 89.3 93.8 96.1 101 7.2 96

15 5 6 17 18 29 30 0 0 2 2 4 4 14.8 15.5 13.8 14.4 14.9 15.5 99.0 104 91.9 96.1 99.5 104 15 100

30 7 8 19 20 31 32 0 0 2 2 4 4 28.6 30.0 27.4 28.0 29.1 30.0 95.4 99.9 91.4 93.3 97.0 100 29 97

60 9 10 21 22 33 34 0 0 2 2 4 4 83.2 61.8 74.0 59.2 80.4 62.5 139 103 123 98.6 134 104 70 117

120 11 12 23 24 35 36 0 0 2 2 4 4 95.2 120 89.6 116 95.4 122 79.3 99.9 74.7 96.8 79.5 102 106 89

1 The limit of quantitation (LOQ) was 3.00 mg a.i./L, cal culated as the product of the concentration of the

lowest calibration standard (3.00 mg a.i./L) and the dilution factor of the matrix blank samples (1.00).
2 Results were generated using Excel 2000 in full preci sion mode. Manual calculations may differ slightly.Wildlife International, Ltd.Project Number 471A-123- 18 -Table 2Temperature, Dissolved Oxygen and pH of Water in the Test ChambersNominalConcentration(mg a.i./L)Replicate0 Hour 24 Hour 48 HourTemp.1(oC)DO2(mg/L)pHTemp.1(oC)DO2(mg/L)pHTemp.1(oC)DO2(mg/L)pHNegative Control A 21.5 8.7 8.321.6 7.6 8.2 21.7 7.4 8.2B 21.6 8.4 8.3 21.77.5 8.3 21.8 7.4 8.37.2 A 21.5 8.4 8.3 21.67.5 8.3 21.7 7.4 8.3B 21.6 8.5 8.3 21.77.7 8.3 21.7 7.4 8.315 A 21.5 8.3 8.3 21.77.7 8.3 21.7 7.5 8.3B 21.6 8.3 8.3 21.77.5 8.3 21.7 7.5 8.329 A 21.5 8.5 8.3 21.67.5 8.3 21.7 7.5 8.3B 21.6 8.5 8.3 21.87.4 8.3 21.7 7.5 8.370 A 21.5 8.5 8.3 21.67.4 8.3 21.6 7.5 8.3B 21.6 8.4 8.3 21.77.5 8.3 21.7 7.6 8.3106 A 21.5 8.6 8.3 21.67.6 8.3 21.6 7.4 8.3B 21.6 8.6 8.3 21.77.6 8.3 21.8 7.4 8.31Manual temperature measurements. Temperature monitored continuously during the test ranged from approximately 21.6 to 22.1°C,measured to the nearest0.1°C.2A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22oC in freshwater.Wildlife International, Ltd.Project Number 471A-123- 19 -Table 2 (Continued)Temperature, Dissolved Oxygen and pH of Water in the Test ChambersNominalConcentration(mg a.i./L)Replicate72 Hour 96 HourTemp.1(oC)DO2(mg/L)pHTemp.1(oC)DO2(mg/L)pHNegative Control A 21.7 7.2 8.2 21.7 6.8 8.3B 21.8 7.1 8.3 21.7 6.6 8.37.2 A 21.7 7.0 8.3 21.7 6.6 8.3B 21.7 7.2 8.3 21.7 6.7 8.315 A 21.7 7.3 8.3 21.7 6.7 8.3B 21.8 7.5 8.3 21.7 6.8 8.329 A 21.6 7.5 8.3 21.7 6.7 8.4B 21.7 7.5 8.3 21.7 6.7 8.470 A 21.6 7.4 8.3 21.7 6.7 8.3B 21.7 7.4 8.3 21.7 6.6 8.4106 A 21.6 6.8 8.3 21.6 6.4 8.3B 21.9 6.6 8.2 21.7 6.1 8.31Manual temperature measurements. Temperature monitored continuously during the test ranged from approximately 21.6 to 22.1°C,measured tothe nearest 0.1°C.2A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation at 22oC in freshwater.

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Table 3

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

Parameter Day 0

Specific Conductance 315 (μS/cm)

Hardness 136 (mg/L as CaCO 3 )

Alkalinity 172 (mg/L as CaCO 3 )Wildlife International, Ltd.Project Number 471A-123- 21 -Table 4Cumulative Mortality and ObservationsNominalConcentration(mg a.i./L)Rep.No.Exposed2 Hours 24 Hours 48 HoursNo. Dead1Observations2No. Dead1Observations2No. Dead1Observations2Negative Control A 10 0 10 AN 0 10 AN 0 10 ANB 10 0 10 AN 0 10 AN 0 10 AN7.2 A 10 0 10 AN 0 10 AN 0 10 ANB 10 0 10 AN 0 10 AN 0 10 AN15 A 10 0 10 AN 0 10 AN 0 10 ANB 10 0 10 AN 0 10 AN 0 10 AN29 A 10 0 10 AN 0 10 AN 0 10 ANB 10 0 10 AN 0 10 AN 1 9 AN70 A 10 0 10 AN 0 7 AN; 3 C 0 1 A; 4 AN; 5 CB 10 0 10 AN 0 9 AN; 1 C 0 8 AN; 2 C106 A 10 0 10 AN 1 2 AN; 7 C 1 4 A; 5 CB 10 0 10 AN 1 5 A; 4 C 2 6 A; 2 N1Cumulative number of dead fish.2Observations: A = surfacing; AN = appear normal; C = lethargy; N = loss of equilibrium.Wildlife International, Ltd.Project Number 471A-123- 22 -Table 4 (Continued)Cumulative Mortality and ObservationsNominalConcentration(mg a.i./L)Rep.No.Exposed72 Hours 96 HoursCumulativePercentMortalityNo. Dead1Observations2No. Dead1Observations2Negative Control A 10 0 10 AN 0 10 AN 0B 10 0 10 AN 0 10 AN7.2 A 10 0 10 AN 0 10 AN 0B 10 0 10 AN 0 10 AN15 A 10 0 10 AN 0 10 AN 0B 10 0 10 AN 0 10 AN29 A 10 0 10 AN 0 7 AN; 3 C 5B 10 1 9 AN 1 6 AN; 3 C70 A 10 0 3 A; 2 AN; 5 C 0 3 A; 1 AN; 6 C 0B 10 0 1 A; 5 AN; 4 C 0 1 A; 4 AN; 5 C106 A 10 1 5 A; 4 C 1 6 A; 3 C 20B 10 3 7 A 3 7 A1Cumulative number of dead fish.2Observations: A = surfacing; AN = appear normal; C = lethargy; N = loss of equilibrium.

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Table 5

LC50 Values

Time LC50 (mg a.i./L) 95% Confidence Interval (mg a.i./L) Statistical Method

24 Hours > 106 -- 1 NA 2

48 Hours > 106 -- 1 NA 2

72 Hours > 106 -- 1 NA 2

96 Hours > 106 -- 1 Binomial test 3

1 95% confidence limits could not be calcula ted with the mortality data obtained.

2 NA = not applicable; <50% mortality preclude d statistical calculation of an LC50 value.

3 An upper 95% confidence limit could not be calcu lated. At a confidence level of 95%, the

binomial test shows that the LC50 is above 106 mg a.i./L.

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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 362 347 – 378 (μS/cm) (N = 4)

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

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

pH 8.3 8.1 – 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 Laborator ies 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 Laborator ies 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 FOR THE ANALYSIS OF DIETHYLTOLUENE DIAMINE ( 

### DETDA) IN FRESHWATER

Prepare calibration standards in freshwater us ing volumetric flasks and gas-tight syringes.



Prepare QC samples in freshwater using volumetri c 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: 25.00 μL

APPROXIMATE DETDA RETENTION TIMES: Component 1: 5.0 minutes Component 2: 5.7 minutes

PRIMARY ANALYTICAL WAVELENGTH: 220 nm

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Appendix 4.3

Analytical Stocks and Standards Preparation

A stock solution of Diethyltoluene Diamine (DET DA) test substance was prepared 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 volum etric flask, and brought to volume using acetonitrile. The primary stock solution (1.00 mg a.i./mL) was used to prepare the fortification samples and to prepare

the calibration standards in freshwater. The followi ng 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) 1.00 1.00 1.00 1.00 1.00 30.0 50.0 100 200 300 10.0 10.0 10.0 10.0 10.0 3.00 5.00 10.0 20.0 30.0

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Appendix 4.4

Example Calculations for a Representative Sample

The analytical result and percent recovery for sample number 471A-123-5, with a nominal concentration of 15 mg a.i./L, were calculated using the following equations:

Concentration of 

### Diethyltoluene Diamine (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 = 720.71421 Y-Intercept = 2.8258 Slope = 48.3656 Dilution Factor = 1.00

Concentration of 

### Diethyltoluene Diamine (DETDA) in sample (mg a.i./L)

= 720.71421 - 2.8258 48.3656 x 1.00

Concentration of 

### Diethyltoluene Diamine (DETDA) in sample (mg a.i./L) = 14.8

Percent of nominal concentration = 14.8 mg a.i./L 15 mg a.i./L x 100

Percent of nominal concentration = 99.0%*

* Results were generated using Excel 2000 in fu ll precision mode. Manual calculations may differ slightly.

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Appendix 4.5

Quality Control Samples of 

### Diethyltoluene Diamine (DETDA) in Freshwater

Sample Number (471A-123-) Sample Type Fortified Concentration

(mg a.i./L) Measured Concentration

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

MAB-1 MAB-2 MAB-3

MAS-1 MAS-2 MAS-3

MAS-4 MAS-5 MAS-6

MAS-7 MAS-8 MAS-9 Matrix Blank Matrix Blank Matrix Blank

Matrix Fortification Matrix Fortification Matrix Fortification

Matrix Fortification Matrix Fortification Matrix Fortification

Matrix Fortification Matrix Fortification Matrix Fortification 0.0 0.0 0.0

7.50 20.0 120

7.50 20.0 120

7.50 20.0 120 < LOQ < LOQ < LOQ

7.50 20.1 119

6.89 16.3 110

7.50 20.4 122 --

--

--

99.9 100 99.0

91.9 81.7 91.4

99.9 102 102
Mean = Standard Deviation = CV = 96.4 6.78 7.03%

1 The limit of quantitation (LOQ) was 3.00 mg a.i./L, calculated as the product of the concentration of the lowest calibration standard (3.00 mg a.i./L) and the dilution fact or of the matrix blank samples (1.00).

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

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Appendix 4.6

Representative Calibration Curve for 

### Diethyltoluene Diamine (DETDA)

0 200 400 600 800 1000 1200 1400 1600

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 (mg a.i./L)Peak area counts

Slope = 48.3656; Y-Intercept = 2.8258; R 2 = 0.99981

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Appendix 4.7

Representative Chromatogram of a Low-level Diethyltoluene Diamine (DETDA) Calibration Standard

min 0 2 4 6 8 10 12 mAU

0 25 50 75 100 125 150 175 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA3\DETDA3 2012-02-20 15-49-12\001-0101.D)4.9895.694

Nominal concentration: 3.00 mg a.i./L. The arrows indicate the approximate retention times for the components of 

### Diethyltoluene Diamine (DETDA) . Component 1 retention time is 5.0 minutes and Component 2 retention time is 5.7 minutes.

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Appendix 4.8

Representative Chromatogram of a High-level Diethyltoluene Diamine (DETDA) Calibration Standard

min 0 2 4 6 8 10 12 mAU

0 50 100 150 200 250 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA3\DETDA3 2012-02-20 15-49-12\005-0501.D)4.9715.6996.553

Nominal concentration: 30.0 mg a.i./L. The arrows indicate the approximate retention times for the components of 

### Diethyltoluene Diamine (DETDA) . Component 1 retention time is 5.0 minutes and Component 2 retention time is 5.7 minutes.

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Appendix 4.9

Representative Chromatogram of a Matrix Blank Sample

min 0 2 4 6 8 10 12 mAU

0 20 40 60 80 100 120 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA3\DETDA3 2012-02-20 15-49-12\006-0601.D)

Sample number 471A-123-MAB-1. The arrows i ndicate the approximate retention times for the components of Diethyltoluene Diamine (DETDA). Component 1 retention time is 5.0 minutes and Component 2 retention time is 5.7 minutes.

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Appendix 4.10

Representative Chromatogram of a Matrix Fortification Sample

min 0 2 4 6 8 10 12 mAU

0 20 40 60 80 100 120 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA3\DETDA3 2012-02-20 15-49-12\007-0701.D)5.0045.711

Sample number 471A-123-MAS-1; 7.50 mg a.i./L nom inal concentration. The arrows indicate the approximate retention times for the components of Diethyltoluene Diamine (DETDA). Component 1 retention time is 5.0 minutes and Component 2 retention time is 5.7 minutes.

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Appendix 4.11

Representative Chromatogram of a Test Sample

min 0 2 4 6 8 10 12 mAU

0 20 40 60 80 100 120 VWD1 A, Wavelength=220 nm (X:\HPLC14\1\DATA\DETDA3\DETDA3 2012-02-20 15-49-12\014-1501.D)5.0225.7316.569

Sample number 471A-123-5; Day 0; 15 mg a.i./L nominal concentration. The arrows indicate the approximate retention times for the components of Diethyltoluene Diamine (DETDA). Component 1 retention time is 5.0 minutes and Component 2 retention time is 5.7 minutes.

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Appendix 5

Changes to Protocol

This study was conducted in accordance with the approved protocol.

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Appendix 6

Personnel Involved in the Study

The following key Wildlife International, Ltd. personne l 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. Christian Disque, B.S., Biologist