TEST SUBSTANCE: 

DETERMINATION OF n-OCTANOL/WATER PARTITION COEFFICIENT 
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT 

SPONSOR: 

~ 
QUALITY ASSURANCE STATEMENT 

This study was examined for compliance with OECD Principles of Good Laboratory Practice, 

(ENVIMCICHEM (98)17) and Title 40 of the Code of Federal Regulations, Parts 160 and 792, Good 

Laboratory Practice Standards, 1989. The dates of all inspections and audits and the dates that any 

findings were reported to the Study Director and Laboratory Management were as follows: 

DATE REPORTED TO: 

ACTIVITY: DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT: 

Protocol August 9,2005 August 10,2005 August 22,2005 

Sample Preparation August 18,2005 August 18,2005 September 29,2005 

Raw Data and Draft Report September 27-28, 2005 September 28, 2005 September 29,2005 

Final Report October 3,2005 October 3, 2005 October 4,2005 

All inspections were study-based unless otherwise noted. 

Quality Assurance Representative 
REPORT APPROVAL 

SPONSOR: 

TITLE : 

STUDY DIRECTOR: 

,//~ / /& ,?J.(' 

~Koth? Z. Kendall, M.S. 

Laboratory Supervisor 

Wildlife International, Ltd. 

MANAGEMENT: 

Willard B. Nixon, P~.D. 

Director of Chemistry 

Wildlife International, Ltd. 

DATE 

/&6i' 

DATE 
- 5 - 

TABLE OF CONTENTS 

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

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

Quality Assurance Statement.................................................................................................................. 3 

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

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

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

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

Purpose ................................................................................................................................................... 8 

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

Materials and Methods............................................................................................................................ 9 

Test Substance................................................................................................................................ 9 

Reference Standards ....................................................................................................................... 9 

Solvents and Reagents.................................................................................................................. 11 

Mobile Phase Preparation............................................................................................................. 11 

Calibration Stocks and Standards Preparation .............................................................................. 11 

Test Substance Stock and Solution Preparation............................................................................ 11 

Test Procedure.............................................................................................................................. 11 

Data Analysis................................................................................................................................ 12 

Results .................................................................................................................................................. 12 

Conclusions .......................................................................................................................................... 13 

References ............................................................................................................................................ 14 
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TABLE OF CONTENTS 

- Continued - 

TABLES 

Table 1 - Preparation of Reference Stocks and Standard Solutions..................................................... 15 

Table 2 - HPLC Operational Parameters ............................................................................................. 16 

Table 3 - Reference Standards Retention Times, Capacity Factors, and Log KOW Values .................. 17 

Table 4 - n-Octanol/Water Partition Coefficients (Log KOW) for Test Substance................................ 18 

FIGURES 

Figure 1 - Reference standard calibration curve .................................................................................. 20 

Figure 2 - A representative chromatogram of a thiourea reference standard ....................................... 21 

Figure 3 - A representative chromatogram of an acetanilide reference standard ................................. 22 

Figure 4 - A representative chromatogram of a 2-nitrophenol reference standard............................... 23 

Figure 5 - A representative chromatogram of a toluene reference standard......................................... 24 

Figure 6 - A representative chromatogram of a naphthalene reference standard................................. 25 

Figure 7 - A representative chromatogram of a phenanthrene reference standard............................... 26 

Figure 8 - A representative chromatogram of a ............................................ 27 

Figure 9 - A representative chromatogram of the test substance ................................ 28 

APPENDICES 

Appendix 1 - Protocol..................................................................................................................... 29 

Appendix 2 - Example Calculation................................................................................................. 39 

Appendix 3 - Personnel Involved in the Study................................................................................ 40 
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SUMMARY 

Sponsor: 

Sponsor’s Representative: 

Location of study, raw 

data and a copy of the 

final report: 

Wildlife International, Ltd. 

Project Number: 

Test Substance: 

Study: 

Test Dates: Experimental Start (OECD): August 18, 2005 

Experimental Start (EPA): September 6, 2005 

Experimental Termination: September 7, 2005 

Summary: Under the chromatographic conditions specified in guidelines, the test substance eluted as 

15 resolved peaks. The corresponding partition coefficients (Log KOW) for the test substance 

were calculated as unretained, 1.06, 1.65, 2.20, 2.62, 3.76, 4.19, 5.33, 5.68, 5.79, 5.83, 

(6.38), (6.47), (6.68) and (7.21). The values in brackets were extrapolated. 
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INTRODUCTION

determined the n-octanol/water partition coefficient of the test substance 

using the high performance liquid chromatography estimation method. The study was conducted at the 

analytical chemistry facility in The study was performed 

following procedures in the Product Properties Test Guidelines, OPPTS 830.7570, Partition Coefficient 

(n-Octanol/Water), Estimation by Liquid Chromatography (1). This method is based on procedures 

presented in the OECD Guideline for the Testing of Chemicals, 117: Partition Coefficient 

(n-octanol/water), High Performance Liquid Chromatography (HPLC) Method (2); Official Journal of the 

European Communities No. L383 Method A.8: Partition Coefficient (3); and TSCA Title 40 of the 

Federal Code of Regulations, Part 796, Section 1570: Partition Coefficient (n-Octanol/Water) - 

Estimation by Liquid Chromatography (4). 

The n-octanol/water partition coefficient of the test substance was determined using high 

performance liquid chromatography (HPLC) with ultraviolet detection (UV). The study was identified as 

Project Number All raw data generated by and a copy of the final 

report are filed under Project Number in archives located on the 

PURPOSE 

The purpose of this study was to experimentally determine an estimate of the n-octanol/water 

partition coefficient (KOW) of the test substance, by a reverse-phase HPLC method. 

EXPERIMENTAL DESIGN 

The study was conducted according to procedures outlined in the corresponding protocol (Appendix 

1). The test substance and a series of reference substances bracketing the applicable Log KOW range of the 

HPLC estimation method were separately prepared in acetonitrile and diluted in 75% acetonitrile:25% 

HPLC grade bottled water (v/v). The reference and test substance solutions were sequentially injected into 

an HPLC system operating under standardized isocratic, reverse-phase operating conditions employing a 

C18-based stationary phase. Capacity factors were calculated for the reference standards and test substance 

using thiourea to estimate column dead time (i.e. the retention time of an unretained reference standard). 

The logarithms of the capacity factors were then plotted against published Log KOW values to establish a 
- 9 - 

linear regression calibration. The capacity factors of the test substance were used in conjunction with the 

linear regression equation to calculate the Log KOW range for the test substance. 

MATERIALS AND METHODS 

Test Substance 

 The test substance was received from on April 19, 2005 and was assigned 

 
- 10 - 

standard had a reported purity of 99.3%, was assigned an expiration date of March 19, 2006, and was 

stored under ambient conditions. 

Toluene was received from nd was assigned 

The reference standard, a liquid, was 

identified as: toluene; lot number 06238 LQ; The reference standard had a 

reported purity of 99.94%, was assigned an expiration date of March 19, 2006, and was stored under 

ambient conditions. 
Solvents and Reagents 

All solvents used in this study were HPLC grade or equivalent. All reagents were ACS reagent 

grade or equivalent. A reagent blank prepared and analyzed concurrently with the reference and test 

substances confirmed that the reagents were free of potentially interfering components. 

Mobile Phase Preparation 

The HPLC mobile phase was 75% acetonitrile : 25% water with 0.1% phosphoric acid (v/v). The 

mobile phase was degassed prior to use. 

Calibration Stocks and Standards Preparation 

Six reference standards were used to generate a Log KOw calibration curve. Calibration stock 

solutions of the individual reference standards were prepared separately by dissolving each in acetonitrile. 

Calibration standard solutions were prepared from these stock solutions of the reference standards by 

diluting appropriately in 75% acetonitrile: 25% HPLC grade water (v/v). The concentrations of the 

reference standards were adjusted, as necessary, to give a detector response of at least 900 milli-absorbance 

units for peak height. Details on the preparation of the calibration standards are provided in Table 1. 

Test Substance Stock and Solution Preparation 

A stock solution of the test substance was prepared by accurately weighing 0.500 g of the test 

substance into a 20-mL glass scintillation vial. The test substance was then quantitatively transferred into 

a 100-mL volumetric flask. The stock solution was brought to volume using acetonitrile. This primary 

stock solution contained 5.00 mg/mL. Subsequent dilution of the acetonitrile stock solution yielded a 50.0 

μg /mL solution that was prepared using mobile phase. Each of the triplicate injections (100 μL) of this 

solution gave a detector response of at least 100 milli-absorbance units for the peak height of the most 

sensitive peak. 

Test Procedure 

Retention times of the test substance chromatographic components and reference standards were 

determined using an Agilent Series 1100 High Performance Liquid Chromatograph (HPLC) equipped with 

 an Agilent Series 1100 variable wavelength UV detector. Chromatographic separations were achieved 

using a YMC-Pack ODS-AM C18 column (150 mm x 4.6 mm, 3-μm particle size). The column 
temperature was maintained at 40°C. The flow rate was 1.0 mL/minute. Prior to the test substance 

analyses, the dead time was determined by injecting thiourea. The instrument parameters are summarized 

in Table 2. 

Reference standards (100 μL injection volume) were injected individually prior to and after the test 

substance to determine retention times. The test substance was injected, in triplicate, following the initial 

reference standard injections and was analyzed under the same chromatographic conditions. 

Data Analysis 

The capacity factor (k′), was calculated for the each of the test substance chromatographic 

components and each reference standard using the following equation: 

k′ = (tR – t0)/t0 

where tR was the retention time of the test substance or reference standard and t0 was the column dead time 

established with thiourea. A correlation graph of Log k′ versus Log KOW for the reference standards was 

plotted and fitted to a regression equation in the form y = mx + b. The calibration curve is presented in 

Figure 1. Log KOW for each component of the test substance was calculated by substituting the calculated 

logarithm of the capacity factor for each component into the linear regression equation for the calibration 

curve. An example calculation is presented in Appendix 2. 

RESULTS 

Along with analysis of reference standards, a standard of thiourea was injected in duplicate (once 

near the beginning and once near the end of the HPLC sequence) to determine the analytical column dead 

time (t0) for use in calculating capacity factors (k′) of the reference standards and the test substance. The 

mean retention time of the duplicate thiourea standard injections was 1.619 minutes. 

Six additional reference standards were analyzed with the test substance. The reference standard 

series was injected at the beginning and at the end of the HPLC sequence. The capacity factors of each 

standard were calculated based upon their retention times. Calculated Log k′ values for acetanilide, 2-

nitrophenol, toluene, naphthalene, phenanthrene, and DDT reference standards are presented in Table 3. 
The reference standard calibration curve is presented in Figure 1. Typical reference standard 

chromatograms are presented in Figures 2 through 8. 

The test substance was sequentially injected in triplicate. The test substance eluted as 15 resolved 

peaks. The peak retention times 

The capacity factors (k′) for the retained were then 

calculated based on the above retention time range. The corresponding partition coefficients (Log KOW) 

for the test substance were calculated to range from 1.06 to an extrapolated value of 7.21. The calculated 

mean retention times, capacity factors, and mean partition coefficients for the test substance are presented 

in Table 4. A representative chromatogram of the test substance is presented in Figure 9. 

CONCLUSIONS 

 Under the chromatographic conditions specified in guidelines, the test substance eluted as 

15 resolved peaks. The corresponding partition coefficients (Log KOW) for the test substance were 

calculated as unretained, 106, 1.65, 2.20, 2.62, 3.76, 4.19, 5.33, 5.68, 5.79, 5.83, (6.38), (6.47), (6.68) and 

(7.21). The values in brackets were extrapolated. 
REFERENCES

1. U.S. Environmental Protection Agency. 1996. Partition Coefficient (n-Octanol/Water), 

Estimation by the Liquid Chromatography. Series 830 – Product Properties Test Guidelines, 

OPPTS Number 830.7570. 

2. Organisation for Economic Cooperation and Development. 2001. Partition Coefficient (noctanol/water) High Performance Liquid Chromatography (HPLC) Method. OECD Guideline for 

Testing of Chemicals, Proposal for Updating Guideline 117. Revised draft document November 

2001. 

3. Annex to Commission Directive 92/69/EEC. 1992. Partition Coefficient. Official Journal of the 

European Communities No. L383. Method A.8. 

4. TSCA Title 40 of the Code of Federal Regulations. 1994. Part 796, Section 1570; Partition 

Coefficient (n-Octanol/Water) – Estimation by Liquid Chromatography. 

5. Title 40 of the Code of Federal Regulations, Part 792. 1989. Toxic Substances Control Act; 

(TSCA); Good Laboratory Practice Standards; Final Rule.

6. Organisation for Economic Cooperation and Development. 1998. OECD Principles of Good 

Laboratory Practice. ENV/MC/CHEM (98) 17, Environment Directorate, Paris. 

7. Ministry of Agriculture, Forestry and Fisheries, Japan (MAFF). 1999. Good Laboratory 

Practice (GLP) for Agricultural Chemicals. 11 NohSan, Notification No. 6283, Agricultural 

Production Bureau, 1 October 1999. 
Table 1 

Preparation of Reference Stocks and Standard Solutions 

Reference 

Substance 

Amount 

Weighed 

(mg) 

Final 

Volume 

(mL)1

Stock 

Concentration 

(mg/mL) 

Initial 

Volume 

(mL)2

Final 

Volume 

(mL)3

Standard 

Concentration 

(μg/mL) 

1

 Solutions were brought to final volume with acetonitrile in volumetric flasks. 

2

 Initial volumes measured with a gas-tight syringe. 

3

 Solutions were brought to final volume with 75% acetonitrile:25% HPLC grade water (v/v) in 

graduated centrifuge tubes. 
Table 2 

HPLC Operational Parameters 

INSTRUMENT: Agilent Series 1100 High Performance Liquid Chromatograph 

(HPLC) with an Agilent Series 1100 Variable Wavelength (UV) 

Detector 

ANALYTICAL COLUMN: YMC-Pack ODS-AM C18 (150 mm x 4.6 mm, 3-μm) 

STOP TIME: 40 minutes 

FLOW RATE: 1.0 mL/minute 

OVEN TEMPERATURE: 40°C 

MOBILE PHASE: Acetonitrile:0.1% H3PO4; 75:25, v/v 

INJECTION VOLUME: 100 μL 

APPROXIMATE 

 

 

 

 
- 17 - 

Table 3 

Reference Standards Retention Times, Capacity Factors, and Log KOW Values 

Reference 

Standard 

Concentration 

(μg/mL) 

Retention 

Time 

(minutes) 

Capacity 

Factor 

(k′) 1,2

Log k′

2 Log KOW3

1

 Capacity factor (k′) is the standard retention time minus the mean column dead time (1.619 

minutes for thiourea) divided by the mean column dead time. 

2

 Values obtained utilizing Excel 2000 in full precision mode. Manual calculations may differ 

slightly. 

3 Log KOW values are based on literature values (see reference 1). 
- 18 - 

Table 4 

n-Octanol/Water Partition Coefficients (Log KOW) for Test Substance 

Sample 

Number 

 

 

 

 

 
- 19 - 

Table 4 (Continued) 

n-Octanol/Water Partition Coefficients (Log KOW) for Test Substance 

Sample 

Number 

Retention 

Time 

minutes) 

Mean (  ̄x ) 

Standard Deviation 

(SD)2

Capacity 

Factor 

(k′)

1,2

Log 

k′ 2

Log 

KOW

2,3

Mean (  ̄x )2,3 

Standard Deviation 

(SD) 

11.401 

11.387 

11.354 

 ̄x = 11.381 

SD = 0.02413 

6.044 

6.036 

6.015 

0.7813 

0.7807 

0.7792 

5.83 

5.83 

5.82 

 ̄x = 5.83 

SD = 0.00 

4 (Peak 12) 

5 (Peak 12) 

6 (Peak 12) 

15.633 

15.611 

15.565 

 ̄x = 15.603 

SD = 0.03470 

8.659 

8.645 

8.617 

0.9375 

0.9368 

0.9354 

(6.38) 

(6.38) 

(6.38) 

 ̄x = (6.38) 

SD = 0.00 

4 (Peak 13) 

5 (Peak 13) 

6 (Peak 13) 

16.493 

16.465 

16.416 

 ̄x = 16.458 

SD = 0.03897 

9.190 

9.173 

9.143 

0.9633 

0.9625 

0.9611 

(6.47) 

(6.47) 

(6.47) 

 ̄x = (6.47) 

SD = 0.00 

4 (Peak 14) 

5 (Peak 14) 

6 (Peak 14) 

18.636 

18.609 

18.554 

 ̄x = 18.600 

SD = 0.04179 

10.514 

10.498 

10.464 

1.0218 

1.0211 

1.0197 

(6.68) 

(6.68) 

(6.67) 

 ̄x = (6.68) 

SD = 0.00 

4 (Peak 15) 

5 (Peak 15) 

6 (Peak 15) 

25.665 

25.624 

25.548 

 ̄x = 25.612 

SD = 0.05937 

14.857 

14.832 

14.785 

1.1719 

1.1712 

1.1698 

(7.21) 

(7.21) 

(7.20) 

 ̄x = (7.21) 

SD = 0.00 

1 Capacity factor (k’) is the sample retention time minus the mean column dead time (1.619 minutes for thiourea) divided 

by the mean column dead time. 

2 Values obtained utilizing Excel 2000 in full precision mode. Manual calculations may differ slightly. 

3 Values in parenthesis are extrapolated values. 
- 20 - 

-1.0

-0.5

0.0

0.5

1.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

Log Kow

Log k

Figure 1. Reference standard calibration curve. 
0 5 10 15 20 25 30 35 min

mAU 

0

500

1000

1500

2000

 VWD1 A, Wavelength=210 nm (POW002\002-0201.D)

Figure 2. A representative chromatogram of a thiourea reference standard. 
- 22 - 

0 5 10 15 20 25 30 35 min

mAU 

0

500

1000

1500

2000

 VWD1 A, Wavelength=210 nm (POW002\003-0301.D)

Figure 3. A representative chromatogram of an acetanilide reference standard. 
- 23 - 

0 5 10 15 20 25 30 35 min

mAU 

0

500

1000

1500

2000

 VWD1 A, Wavelength=210 nm (POW002\004-0401.D)

Figure 4. A representative chromatogram of a 
- 24 - 

0 5 10 15 20 25 30 35 min

mAU 

0

500

1000

1500

2000

 VWD1 A, Wavelength=210 nm (POW002\005-0501.D)

Figure 5. A representative chromatogram of a toluene reference standard. 
- 25 - 

0 5 10 15 20 25 30 35 min

mAU 

0

500

1000

1500

2000

 VWD1 A, Wavelength=210 nm (POW002\006-0601.D)

Figure 6. A representative chromatogram of a naphthalene reference standard. 
- 26 - 

0 5 10 15 20 25 30 35 min

mAU 

0

500

1000

1500

2000

 VWD1 A, Wavelength=210 nm (POW002\007-0701.D)

Figure 7. A representative chromatogram of a phenanthrene reference standard. 
0 5 10 15 20 25 30 35 min

mAU 

0

200

400

600

800

1000

 VWD1 A, Wavelength=210 nm (POW002\008-0801.D) 12.168

Figure 8. A representative chromatogram of a DDT reference standard. 
0 5 10 15 20 25 30 35 min

mAU 

0

50

100

150

200

 VWD1 A, Wavelength=210 nm (POW002\009-0901.D) 1.449 1.895 2.058 2.261 2.538 2.830 4.168 4.989 8.679 10.490 11.142 11.401

 15.633

 16.493

 18.636

 25.665

Figure 9. A representative chromatogram of the test substance 
- 29 - 

Appendix 1 

Protocol 
- 30 - 

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- 38 - 
- 39 - 

Appendix 2 

Example Calculation 

An example calculation is presented for the first injection of 

 

 

 
- 40 - 

Appendix 3 

Personnel Involved in the Study 

The following key . personnel were involved in the conduct or 

management of this study: 

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

2. Timothy Z. Kendall, Laboratory Supervisor 

3. Frank J. Lezotte, B.S., Chemist