DETERMINATION OF THE n -OCTANOL/WATER PARTITION COEFFICIENT OF DIETHYLTOLUENE DIAMINE (DETDA) BY THE SHAKE FLASK METHOD

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471C-143

U.S. EPA Product Properties Test Guidelines OPPTS 830.7550, Partition Coefficient ( n -Octanol/Water), Shake Flask Method

and

OECD Guideline for the Testing of Chemicals OECD 107, Partition Coefficient ( n -octanol/water): Shake Flask Method

AUTHORS:

William R. Schutt, B.S. Willard B. Nixon, Ph.D.

STUDY INITIATION DATE: February 15, 2012

STUDY COMPLETION DATE: June 27, 2012

SUBMITTED TO:

Albemarle Corporation 451 Florida Street Baton Rouge, LA 70801 

# Wildlife International, Ltd.

8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600

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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 ......................................................................................................... .................. 8 Test Substance ............................................................................................................... ............ 9 Solvents and Reagents ........................................................................................................ ....... 9 Saturated Solvent Preparation ................................................................................................ ... 9 Stock and Standards Preparations ........................................................................................... 1 0 Partition Sample Preparation for Feasibility Trial ................................................................... 10 Partition Sample Preparation for Definitive Test .................................................................... 11 Analytical Methods ........................................................................................................... ...... 11 Calibration Curve and Limit of Quantitation .......................................................................... 12 Matrix Blank and Matrix Fortification Samples ..................................................................... 13 Example Calculations ......................................................................................................... ..... 13

Results and Discussion ........................................................................................................ ................. 14 Feasibility Trial ............................................................................................................ ........... 14 Stock Confirmation and pH Confirmation .............................................................................. 14 Mass Balance De termination ................................................................................................... 15 Partition Coefficient Determination ........................................................................................ 1 5

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

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

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TABLE OF CONTENTS - Continued -

TABLES

Table 1. HPLC Operational Parameters .......................................................................................... 17

Table 2. Quality Control Samples of DETDA in n -Octanol and Reagent Water Partition Phases ... 18

Table 3. Mass Balance Results for the n -Octanol/Water Partition Coefficient Definitive Test with DETDA ................................................................................................................... .. 19

Table 4. Measured Concentrations and Partition Coefficient Results for the n -Octanol/Water Partition Coefficient Definitive Test with DETDA .......................................................... 20

FIGURES

Figure 1. Analytical method flow-chart for the analysis of DETDA in

n -octanol/water partition samples ..................................................................................... 21

Figure 2. A representative calibration curve for the analysis of DETDA by HPLC/UV ..................................................................................................................... .... 22

Figure 3. Representative chromatograms of low-level and high-level DETDA calibration standards for HPLC/UV analyses ................................................................... 23

Figure 4. Chromatograms of the matrix blank samples.................................................................... 24

Figure 5. Chromatograms of the matrix fortification samples ......................................................... 25

Figure 6. Representative chromatograms of a partition test sample (471C-143-1) .......................... 26

APPENDICES

Appendix 1. Protocol ...................................................................................................................... 27

Appendix 2. Characterization Report .............................................................................................. 38

Appendix 3. Personnel Involved in the Study ................................................................................. 42

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SUMMARY

STUDY TITLE : Determination of the n -Octanol/Water Partition Coefficient of Diethyltoluene Diamine (DETDA) by the Shake Flask Method

WILDLIFE INTERNATIONAL , LTD

. PROJECT NUMBER : 471C-143

SPONSOR : Albemarle Corporation 451 Florida Street Baton Rouge, LA 70801

TESTING FACILITY : Wildlife International, Ltd. Easton, Maryland 21601

SPONSOR ’ S REPRESENTATIVE : Marcia L. Hardy, DVM, Ph.D.

LOCATION OF STUDY , RAW
DATA AND A COPY OF THE
FINAL REPORT : Wildlife International Ltd. Easton, Maryland 21601

TEST SUBSTANCE : Diethyltoluene Diamine (DETDA)

TEST DATES : Experimental OECD Start – February 16, 2012 Experimental EPA Start – February 16, 2012 Experimental Termination – February 23, 2012

TEST SYSTEM : HPLC-grade reagent water saturated with n -octanol, and n -octanol saturated with HPLC-grade reagent water.

S UMMARY : The n -octanol/water partition coefficient (P ow ) of DETDA at 25oC was determined by the shake flask method. The partition coefficient was evaluated as a function of solvent volume ratio. Three n -octanol/water volume ratios were evaluated in duplicate. After a 15-minute partition equilibration period (via gentle mechanical agitation) followed by centrifugation, sub-samples from each phase of each partition test vessel were prepared for analysis by volumetric dilution. Aliquots from the n -octanol phase samples were dilute d in 50:50 (v/v) acetonitrile:reagent water (ACN:H 2 O). An aliquot of each aqueous phase sample was diluted to a final solvent composition of 50:50 (v/v) ACN:H 2 O. The dilutions of both the n -octanol phase samples and the aqueous phase samples were analyzed by high performance liquid chromatography with ultraviole t absorbance detection (HPLC/UV). Mass balance for DETDA was obtained in all test vessels. At n -octanol/water solvent ratios of 1:1, 1:2 and 1:4, the mean P ow was 23.3, 24.9 and 24.0 respectively, corresponding to mean log P ow values of 1.37, 1.40 and 1.38, respectively. The overall mean log P ow was 1.38 ± 0.019 (n = 6, CV = 1.4%).

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INTRODUCTION

Tests were conducted to determine the n -octanol/water partition coefficient of diethyltoluene diamine (DETDA) at 25  C using the shake flask method. Wildlife International, Ltd. conducted this study for Albemarle Corporation at the Wildlife Interna tional, Ltd. analytical chemistry facility in Easton, Maryland. The experimental portion of this study was conducted between February 16, 2012 and February 23, 2012. Raw data generate d by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 471C-143 in archives located on the Wildlife International, Ltd. site. The original signed report will be maintained by Al bemarle Corporation, Baton Rouge, Louisiana.

OBJECTIVE The objective of this study was to experimentally determine the n -octanol/water partition coefficient of DETDA by equilibrating the te st substance in a two-phase system of n -octanol/water by the flask-shaking method and analyzing the two phases by appropriate analytical methodology.

EXPERIMENTAL DESIGN

The n -octanol/water partition coefficient of D ETDA was determined using the flask-shaking method. The method consisted of shaking the so lute (DETDA) with the two immiscible solvents ( n -octanol and HPLC-grade reagent water) at 25 ± 1° C over a period of time (15 minutes) and analyzing

the n -octanol and aqueous phases for the solute concen tration using HPLC/UV. The concentration of DETDA in each partition phase was maintained at less th an 0.01 M. A feasibility trial was performed at

an n -octanol/water volume ratio of 1:1 (v/v) to conf irm that the shake flask experimental design was appropriate for the test substance and to provide an estimate of appropriate solvent ratios for the definitive test. The definitive portion of the test was conducted at three n -octanol/water solvent ratios.

MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, “Determination of

the n -Octanol/Water Partition Coefficient of Diethy ltoluene Diamine (DETDA) by the Shake Flask Method” (Appendix 1). The study was also perfo rmed based on procedures in U.S. EPA Product Properties Test Guidelines, OPPTS 830.7550, Partition Coefficient (n-Octanol/Water), Shake Flask

Method (1) and OECD Guideline for the Testing of Chemicals, OECD 107, Partition Coefficient (n-octanol/water): Shake Flask Method (2).

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Test Substance

The test substance was received from Albemarl e Corporation on September 21, 2011, and was assigned Wildlife International, Ltd. identificati on number 10232. The test substance was a liquid identified as DETDA; Lot number – 4500051816-011110; CAS Number 68479-98-1. A purity of 97.27% was obtained from the characterization report provided by the Sponsor (Appendix 2). The test

substance was stored under ambient conditions. An expiration date of Ma rch 3, 2013 and molecular weight of the test substance, 178.28 g/mol, were provided by the Sponsor.

Solvents and Reagents All solvents were HPLC-grade or higher quality a nd reagents were ACS reagent-grade or better.

Solvent/Reagent Manufacturer/Lot Reagent Water: HPLC-Grade, Burdick & Jackson, Lot Nos. DE958-E (equivalent to ASTM Type II Designation D1193-06 (3)). Acetonitrile (ACN): HPLC-Grade, Burdick & Jackson, Lot Nos. DF154

n -Octanol: Sigma-Aldrich, Lot No. SHBB3592V

Saturated Solvent Preparation Mutually saturated partition phases were prepared. The solvents were mutually saturated by combining 500 milliliters of reagent water with 300 milliliters of n -octanol in a clean French-square bottle. The bottle was placed in an agitating water bath (Boekel 290400) maintained at 25oC and agitated for approximately 20 hours at 25 ± 0.2°C (temperature monitored using a min-max thermometer). After the saturation period, the contents of the bottle we re decanted into a separatory funnel and allowed to partition for a minimum of 10 minutes. The mutually saturated n -octanol and aqueous phases were separated by draining the lower aqueous phase and upper n -octanol phase into separate French-square bottles. The separated n -octanol and aqueous saturated solvents were stored under ambient conditions. Before use in an experimental trial, the saturated solvents were equilibrated at the test temperature (25 ± 1oC) in a temperature-controlled incubator (F isher Model 3724) with a data logger to continuously monitor temperature. Before use in the definitiv e test, the pH of the saturated reagent water was measured using the pH meter at the test temperature.

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Stock and Standards Preparations A primary stock solution of the test substance was prepared at a nominal concentration of 1.00 mg DETDA/mL by accurately weighing 0.1029 grams of the test substance in a scintillation vial (weight adjusted for 97.27% purity). The test substa nce aliquot was then quantitatively transferred into a 100-mL volumetric flask and was brought to final volume using ACN. The 1.00 mg DETDA/mL (1000 mg DETDA/L) primary stock solution, identified as 10232-001, was used to prepare a set of standard solutions in a final solvent composition of 50:50 (v/v) ACN: H 2 O, using the following dilution

scheme:

Stock Concentration (mg DETDA/mL) Aliquot ( μ L) Final Volume

(mL) Standard Concentration (mg DETDA/L)

1.00 1.00 1.00 1.00 1.00 50.0 100 200 300 500 10.0 10.0 10.0 10.0 10.0 5.00 10.0 20.0 30.0 50.0

A primary stock solution of the DETDA test substance in n -octanol saturated with reagent water was prepared by accurately weighing 0.4112 grams of the test substance directly into a 100-mL volumetric flask using an analytical balance. The volumetric flask was then brought to final volume with

saturated n -octanol. The nominal DETDA concentration in the resulting primary stock was 4.00 mg DETDA/mL. This primary stock solution in n -octanol was used to fortify all partition samples and the

n -octanol phase matrix fortification sample.

Partition Sample Preparation for Feasibility Trial An initial feasibility trial was conducted with equal volumes of saturated reagent water and

n -octanol solutions ( n -octanol:reagent water volume ratio of 1:1). Duplicate samples were prepared using volumetric pipettes to transfer 10.0 milliliters of reagent water (saturated with n -octanol) into each labeled 40-mL glass sample vial followed by 6.00 milliliters of n -octanol (saturated with reagent water). Each partition sample was then fortified with DETDA by transferring 4.00 milliliters of the 4.00-mg DETDA/mL primary stock solution in n -octanol saturated with reagent water. This brought the total

volume of n -octanol saturated with reagent water in each sample to 10.0 milliliters. The nominal fortified mass of DETDA was 16.0 milligrams in each partition sample. After fortification, the partition samples

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were sealed, gently swirled or agitated by hand for approximately thirty seconds and placed into a 25 ± 1oC agitating water bath for 15 minutes (temperatu re monitored using a min-max thermometer).

Partition Sample Preparation for Definitive Test Based on the findings of the feasibility trial, partition samples in the definitive tests were prepared at n -octanol/water volume ratios of 1:1, 1:2 and 1:4. The partition samples were prepared in duplicate at each volume ratio in 40-mL glass vials. Vo lumetric pipettes were used to transfer appropriate volumes of reagent water (saturated with n -octanol) to the sample vessels, followed by appropriate

volumes of n -octanol (saturated with reagent water). Th e samples were each fortified using a volumetric pipette to transfer 4.00 or 2.00 milliliters of the 4.00-mg DETDA/mL stock solution in n -octanol (saturated with reagent water). The nominal fortified mass of DETDA was 16.0, 16.0, and 8.00 milligrams for the 1:1, 1:2, and 1:4 partition samples, respectively. After fortification, the samples were sealed and gently agitated or swirled by hand for approximately 30 seconds. The samples were placed into a 25 ± 1oC agitating water bath for 15 mi nutes (temperature was monitored using a min-max thermometer).

Analytical Methods The sample preparation and instrumental determ ination procedures used for the analysis of

DETDA in the n -octanol and aqueous phases of the par tition samples were developed at Wildlife International, Ltd. A method flow-chart for the analysis of DETDA in the definitive test partition samples is presented in Figure 1.

The samples were centrifuged (Thermo Sorvall Legend XF) at a setting of 1500 rpm for 10 minutes to promote complete phase separation. A portion of the top ( n -octanol) phase of each partition sample was transferred into a labeled glass vi al. Using gas-tight syringes and volumetric flasks, a 0.100-mL aliquot of each n -octanol phase sample was diluted to a 10.0-mL final volume with 50:50 (v/v) ACN:H 2 O dilution solvent into the calibration range of the HPLC/UV methodology. An aliquot of

each diluted n -octanol phase sample was transferred to an autosampler vial and submitted for HPLC/UV analysis. Following n -octanol phase sampling, the aqueous ph ase was collected. Using a disposable syringe fitted with a detachable needle, th e needle was inserted through the remaining n -octanol phase in the 40-mL partition sample vial while gently and con tinuously expelling air. Once the tip of the needle

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was in the aqueous phase, a portion of the aqueous phase was drawn into the syringe. The syringe was removed from the sample and the needle was immediatel y detached. The contents of the syringe were expelled into a labeled glass vial. Using gas-tight syringes, a 1.00-mL aliquot of each aqueous phase sample was transferred into separate 5.00-mL volumetric flask preloaded with 2.50-mL of ACN. The volumetric flasks were brought to final volume with r eagent water to yield a final sample composition of 50:50 (v/v) ACN:H 2 O. An aliquot of each diluted aqueous phase sample was transferred to an autosampler vial and submitted for HPLC/UV analysis . The aqueous phase of one partition sample from the definitive test was selected and its pH was measured using a calibrated pH meter.

To confirm the DETDA concentration of the 4.00-mg DETDA/mL n -octanol stock solution used to fortify the partition samples, triplicate aliquots were volumetrically diluted with 50:50 (v/v) ACN:H 2 O dilution solvent (overall dilution factor of 250) using gas-tight syringes and volumetric flasks and an aliquot of each diluted n -octanol stock sample was submitted for HPLC/UV analysis concurrently with the feasibility trial samples.

Concentrations of DETDA were determined by Agilent Series 1100/1200 HPLC systems equipped with an Agilent Series 1100 and 1200 Variable Wavelength Detector (VWD) set at 216 nm for the feasibility trial and definitive test, respectively. Chromatographic separations were achieved using a YMC-Pack ODS-AM analytical column (150 mm x 4.6 mm, 3-  m particle size). The instrument parameters for analysis of DETDA in the definitive test are summarized in Table 1.

Calibration Curve and Limit of Quantitation Five calibration standards of DETDA in 50:50 (v/v) ACN: H 2 O, ranging in concentration from 5.00 to 50.0 mg DETDA/L, were analyzed concurrently with each set of n -octanol and aqueous phase samples. The calibration set was inj ected at the beginning and end of the analytical sequence with one calibration standard injected after a maximum of every five test samples. Linear regression equations were generated for the analytical sequence using th e summed/total peak area response for retention times between approximately 7.4 and 7.5 minutes versus the respective DETDA concentrations of the

calibration standards. The concentration of DET DA in the samples was determined by substituting the total peak area responses for retention times between approximately 7.4 and 7.5 minutes of the samples into the applicable linear regression equation. The calibration curve for DETDA is presented in Figure 2.

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Representative chromatograms of low- and high-le vel DETDA calibration standards are presented in Figure 3.

For the definitive test, the method limits of quantitation (LOQ) for the determination of DETDA

in the n -octanol and aqueous phases were 500 mg DETDA/ L and 25.0 mg DETDA/L, respectively. Each LOQ was calculated as the product of the concentrati on of the low-level calibration standard and the dilution factor (DF) of the concurrently analyzed matrix blank samples.

Matrix Blank and Matrix Fortification Samples Matrix blank and matrix fortification samples were prepared and analyzed in each of the partition solvent phases. These quality control samples were processed in the same fashion as the definitive samples to determine possible interferences and to assess method efficiency. No chromatographic

interferences were observed in either the n -octanol or aqueous phase matrix blank samples. Chromatograms of the n -octanol and aqueous phase matrix blank sa mples are presented in Figure 4. The matrix fortification samples yielded procedural recoveries of 105% and 98.8% of the nominal

concentrations in n -octanol (1600 mg DETDA/L) and reagent water (60.0 mg DETDA/L), respectively (Table 2). Chromatograms of the n -octanol and reagent water matrix fortification samples are presented in Figure 5.

Example Calculations The analytical result for the c oncentration of DETDA in the n -octanol partition phase of sample

number 471C-143-1 was calculated using the following equations.

Test Substance (mg DETDA/L) in sample = Peak Area - (Y-intercept) Slope x Dilution Factor

Total Peak area = 13782.57471 Initial Volume (V i ) = 0.100 mL Y-intercept = -306.20822 Final Volume (V f ) = 10.0 mL Slope = 880.11029 Dilution Factor (V f /V i ) = 100 Molecular Weight DETDA = 178.28 g/mol Unit Conversion = 0.001 g/mg

Test Substance (mg DETDA/L) in sample = 13782.57471 - (-306.20822) 880.11029 x 100

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Test Substance (mg DETDA/L) in sample = 1600

DETDA (M) in sample = Test Substance in sample (mg DETDA/L) x Unit Conversion Molecular Weight DETDA

DETDA (M) in sample = 1600 mg DETDA/L x 0.001 g/mg 178.28 g/mol

DETDA (M) in sample = 8.98 x 10 -3

The molar concentration for the aqueous phase of this partition sample was calculated in the same manner and using the same equations as the molar concentration calculation for the n -octanol phase. The partition coefficient for this sample (sample number 471C-143-1) was calculated from the n -octanol and aqueous phase molar concentrations using the following equation:

P artition Coefficient = DETDA in n -octanol phase (M) DETDA in aqueous phase (M)

P ow = 8.98 x 10 -3 M 3.87 x 10 -4 M

P ow = 23.2

Log (P ow ) = 1.37

Calculations performed using Microsoft ® Excel 2000 in the full precision mode. Manual calculations may differ slightly.

RESULTS AND DISCUSSION Feasibility Trial The feasibility trial at a 1:1 n -octanol/water volume ratio yielded a mean estimated partition coefficient of 21.8 (log P ow = 1.34). Based on these results, it was concluded that the n -octanol to water volume ratio of 1:1would be appropriate for use in the definitive test.

Stock Confirmation and pH Confirmation

The stock solution of DETDA in saturated n -octanol used to fortify the partition samples had a mean measured concentration of 3.95 mg DETDA/mL (n = 3), which corresponded to 98.8% of the

nominal DETDA concentration.

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Prior to the definitive test, the measured pH of the reagent water (saturated with n -octanol) was 8.57. The measured pH value of the aqueous phase from a selected partition sample was 8.53.

Mass Balance Determination

The total mass recovered was calculated for each of the six definitive test vessels. Mass balance

was obtained for DETDA in all vessels. The overa ll mean percent recovery for DETDA for the six partition test vessels was 101 ± 2.4% (Table 3).

Partition Coefficient Determination

Partition coefficients were calculated from the meas ured concentrations of DETDA in each phase for each of six samples. Overall mean n -octanol/water partition coefficients and overall mean of the logarithm (base 10) of the n -octanol/water partition coefficients were calculated for each of the six samples.

The mean P ow values for DETDA at n -octanol/water solvent ratios of 1:1, 1:2 and 1:4 were determined to be 23.3, 24.9 and 24.0, respec tively (Table 4). The corresponding mean log P ow values were 1.37, 1.40 and 1.38, respectively. The overall mean log P ow was 1.38 ± 0.019 (n = 6). Representative chromatograms of the n -octanol and aqueous phase samples are presented in Figure 6.

CONCLUSIONS

The n -octanol/water partition coefficient of D ETDA at 25oC, expressed as the logarithm (base 10), was determined to be 1.38 ± 0.019 (n = 6, CV = 1.4%).

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REFERENCES

1. U.S. EPA Product Properties Test Guidelines. 1996. Product Properties Test Guidelines OPPTS 830.7550. Partition Coefficient (n-Octanol/Water), Shake Flask Method.

2. Organisation for Economic Cooperation and Development . 1995. Guideline for the Testing of Chemicals, 107: Partition Coefficient (n-octanol/water): Shake Flask Method.

3. American Society for Testing and Materials. 2006. Standard Specification for Reagent Water. D1193-06, ASTM Section II Water and Envi ronmental Technology, Vol. 11.01: 45-47.

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Table 1

HPLC Operational Parameters

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

ANALYTICAL COLUMN: YMC-Pack ODS-AM (150 mm x 4.6 mm, 3-  m particle size)

STOP TIME: 13.00 minutes

FLOW RATE: 1.000 mL/minute

OVEN TEMPERATURE: 40.0°C

MOBILE PHASE: Solvent A (80.0%): HPLC-grade Reagent Water Solvent B (20.0%): Acetonitrile

GRADIENT ELUTION PROFILE: TIME (min.) SOLVENT A (%) SOLVENT B (%) FLOW (mL/min.) 0.01 80.0 20.0 1.000 2.00 80.0 20.0 1.000 8.00 5.0 95.0 1.000 10.00 5.0 95.0 1.000 10.01 80.0 20.0 1.000 13.00 80.0 20.0 1.000

INJECTION VOLUME: 100.00 μL

DETDA RETENTION TIME RANGE: Peak area 1: 7.4 minutes Peak area 2: 7.5 minutes

UV WAVELENGTH: 216 nm

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Table 2

Quality Control Samples of DETDA in n -Octanol and Reagent Water Partition Phases

Sample Identification (471C-143-) Sample Type Partition Phase Concentration of DETD A Percent Recovery 3 Fortified (mg DETDA/L) Measured 3 (mg DETDA/L)

MAB-1 MAS-1

MAB-2 MAS-2 Matrix Blank Matrix Fortification

Matrix Blank Matrix Fortification n -octanol

n -octanol

aqueous aqueous 0.0 1600

0.0 60.0 < LOQ 1 1680

< LOQ 2

59.3 -- 105

-- 98.8

1 The LOQ for the n -octanol phase samples was 500 mg DETDA/L, calculated as the product of the

concentration of the lowest level calibration standard (5.00 mg DETDA/L) for n -octanol phase analysis and the dilution factor of the n -octanol matrix blank sample (100).

2 The LOQ for the aqueous phase samples was 25.0 mg DETDA/L, calculated as the product of the

concentration of the lowest level calibration standard (5.00 mg DETDA/L) for aqueous phase analysis and the dilution factor of the aqueous matrix blank sample (5.00).

3 Results were generated using Microsoft ® Excel 2000 in full precision mode. Manual calculations may

differ slightly.

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Table 3

Mass Balance Results for the n -Octanol/Water Partition Coefficient Definitive Test with DETDA

Sample Identification (471C-143-) 1 Mass Fortified (mg DETDA) Measured Concentration (mg DETDA/L) 2 Phase Volume

(mL) Mass Recovered (mg DETDA) 2 Total Mass Recovered (mg DETDA) 2 Percent Recovery

(%) 2

1-oct 1-H2O

2-oct 2-H2O

3-oct 3-H2O

4-oct 4-H2O

5-oct 5-H2O

6-oct 6-H2O 16.0

16.0

16.0

16.0

8.00

8.00 1.60 x 10 3 68.9

1.59 x 10 3 67.7

1.49 x 10 3 63.1

1.49 x 10 3 56.9

1.35 x 10 3 56.4

1.35 x 10 3 56.5 10.0 10.0

10.0 10.0

10.0 20.0

10.0 20.0

5.00 20.0

5.00 20.0 16.0 0.689

15.9 0.677

14.9 1.26

14.9 1.14

6.77 1.13

6.75 1.13 16.7

16.5

16.1

16.0

7.90

7.88 104

103

101

100

98.8

98.5

Overall Mean: Standard Deviation: CV: 101 2.4 2.4%

1 The sample identification suffix “oct” = n -octanol phase and “H2O” = aqueous phase of a given test system.

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

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Table 4

Measured Concentrations and Partition Coefficient Results for the n -Octanol/Water Partition Coefficient Definitive Test with DETDA

Sample Identification (471C-143-) 1 Solvent Ratio 2 Measured Concentration (M) 3 P ow 3 Mean P ow 3 Log P ow 3 Mean Log P ow 3

1-oct 1-H2O

2-oct 2-H2O 1:1

1:1 8.98 x 10 -3 3.87 x 10 -4

8.90 x 10 -3 3.80 x 10 -4 23.2

23.4 23.3 1.37

1.37 1.37

3-oct 3-H2O

4-oct 4-H2O 1:2

1:2 8.34 x 10 -3 3.54 x 10 -4

8.36 x 10 -3 3.19 x 10 -4 23.6

26.2 24.9 1.37

1.42 1.40

5-oct 5-H2O

6-oct 6-H2O 1:4

1:4 7.60 x 10 -3 3.16 x 10 -4

7.58 x 10 -3 3.17 x 10 -4 24.0

23.9 24.0 1.38

1.38 1.38

Overall Mean: Standard Deviation: CV (%): 24.1 1.1 4.5 1.38 0.019

1.4

1 The sample identification suffix “oct” = n -octanol phase and “H2O” = aqueous phase of a given test system.

2 Expressed as the volume ratio of n -octanol to water.
3 Results were generated using Microsoft ® Excel 2000 in full precision mode. Manual calculations may differ slightly.

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Octanol Phase Analysis:

Transfer an aliquot from the top (octanol) phase of each sample into a clean glass vial and label the vial, appending the sample ID with '-oct'. Prepare a ma trix blank and a matrix fortification sample in n -octanol saturated with water.



Dilute an appropriate aliquot of each n -octanol sample in 50:50 ACN:H 2 O (v/v). Transfer an aliquot of the

dilution of each n -octanol sample to an autosampler vial and submit for HPLC/UV analysis. Place any remaining n -octanol samples and n -octanol sample dilutions in refrigerated storage.

 Water Phase Analysis:

Using a disposable plastic syringe with a detachab le needle, insert the needle through the remaining n -octanol

phase in the original sample vessel while gently expe lling air. Once into the water phase, draw a sufficient volume of water into the syringe.



Remove the syringe from the sample and immediately detach the needle. Expel the contents of the syringe into a clean glass vial and label the vial, appending the sample ID with '-H2O'. Prepare a matrix blank and a matrix fortification sample in water saturated with n -octanol.



Transfer an appropriate amount of each water sample in to the appropriate number of 5.00-mL volumetric flasks preloaded with an appropriate amount of acetonitrile (A CN) and bring volumetric flasks to a final volume by adding an appropriate volume of reagent water to each vol umetric flask to yield a final sample composition of

50:50 ACN:H 2 O (v/v). Transfer an aliquot of the dilution of each water sample to an autosampler vial and submit for HPLC/UV analysis. Place any remaining water samples and water sample dilutions in refrigerated storage.

Figure 1 . Analytical method flow-chart for the analysis of DETDA in n -octanol/water partition samples.

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y = 880.11029x - 306.20822

R 2 = 0.98935

0 5000 10000 15000 20000 25000 30000

0.0 5.0 10.0 15.0 20.0 25.0 30.0 Concentration (mg a.i./L)Total Peak Area (216 nm)

Figure 2. A representative calibration curve for the analysis of DETDA by HPLC/UV

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min 0 2 4 6 8 10 12 mAU

0 500 1000 1500 2000 2500 3000 3500 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\002-0201.D)7.3777.543

(A)

min 0 2 4 6 8 10 12 mAU

0 500 1000 1500 2000 2500 3000 3500 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\005-0501.D)7.3757.540

(B)

Figure 3. Representative chromatograms of low-level and high-level DETDA calibration standards for HPLC/UV analyses.

(A): Low-level calibration standard (5.00 mg DETDA/L) (B): High-level calibration standard (30.0 mg DETDA/L)

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min 0 2 4 6 8 10 12 mAU

0 500 1000 1500 2000 2500 3000 3500 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\007-0801.D)

(A)

min 0 2 4 6 8 10 12 mAU

0 500 1000 1500 2000 2500 3000 3500 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\015-2301.D)

(B)

Figure 4. Chromatograms of the matrix blank samples

(A): n -Octanol phase (Sample ID: 471C-143-MAB-1), dilution factor (DF) = 100 (B): Aqueous phase (Sample ID: 471C-143-MAB-2), DF = 5.00

The arrows indicate the approximate retention times of the peaks for DETDA.

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min 0 2 4 6 8 10 12 mAU

0 500 1000 1500 2000 2500 3000 3500 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\008-0901.D)7.3717.537

(A)

min 0 2 4 6 8 10 12 mAU

0 500 1000 1500 2000 2500 3000 3500 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\016-2401.D)7.3727.536

(B)

Figure 5. Chromatograms of the matrix fortification samples

(A): n -Octanol phase (Sample ID: 471C-143-MAS- 1), nominal conc. = 1600 mg DETDA/L, DF = 100 (B): Aqueous phase (Sample ID: 471C-143-MAS- 2), nominal conc. = 60.0 mg DETDA/L, DF = 5.00

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min 0 2 4 6 8 10 12 mAU

0 500 1000 1500 2000 2500 3000 3500 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\009-1101.D)7.3687.534

(A)

min 0 2 4 6 8 10 12 mAU

0 500 1000 1500 2000 2500 3000 3500 VWD1 A, Wavelength=216 nm (X:\HPLC20\1\DATA\DETDAPOW002\DETDAPOW002 2012-02-22 19-09-28\017-2601.D)7.3747.539

(B)

Figure 6. Representative chromatograms of a partition test sample (471C-143-1)

(A): n -Octanol phase (-oct), DF = 100 (B): Aqueous phase (-H2O), DF = 5.00

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Appendix 1

Protocol

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Appendix 2

Characterization Report

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Appendix 3

Personnel Involved in the Study

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

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

2. Raymond L. Van Hoven, Ph.D., Scientist

3. William R. Schutt, B.S., Chemist

4. John R. Murrell, B.S., Senior Chemist